A Pilot Trial to Assess Low-Intensity Ultrasound in Osteoarthritis
Phase: PHASE1
Conditions: Osteoarthritis, Degenerative
Sponsor: VA Office of Research and Development

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A Pilot Trial to Assess Low-intensity Ultrasound in Osteoarthritis 
Cooperative Clinical Trial Award #0008 
PROTOCOL  
 
 
 
 
 
 
 
 
  
NCT02034409 
October 1, 2018 
Version 5.0 
 
 

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Table of Contents 
I. SPECIFIC AIMS........................................................................................................................................... 4 
 Objectives .........................................................................................................................................................4 
 Specific Aims: ....................................................................................................................................................4 
 Expected Outcome and Impact: .........................................................................................................................6 
II. RESEARCH PLAN ....................................................................................................................................... 6 
 Background and Significance ............................................................................................................................6 
 Overview ................................................................................................................................................................. 6 
 Effects of PLIUS on Cartilage Repair ....................................................................................................................... 7 
 Challenges and Innovations in Clinical Trials in OA ................................................................................................ 8 
 The Osteoarthritis Initiative (OAI) and the Development of MRI Measures, Including Cartilage Thickness, as  
Sensitive Modalities for OA Assessment ......................................................................................................................... 9 
 Impact on Patient Care of Disease Modification in OA ........................................................................................ 10 
 Implications of Potential Study Outcomes ........................................................................................................... 11 
 Preliminary Studies ........................................................................................................................................ 11 
 PLIUS in OA ........................................................................................................................................................... 11 
 MRI as a Measure for Cartilage Health ................................................................................................................. 16 
 Experience of the Investigative Team .................................................................................................................. 17 
 Research Design and Methods ........................................................................................................................ 17 
 Overview of Study Design ..................................................................................................................................... 17 
 Participant Recruitment & Retention ................................................................................................................... 17 
 Schedule of Assessments ...................................................................................................................................... 18 
 Screening Period 1 ................................................................................................................................................ 20 
 Inclusion and Exclusion Criteria ............................................................................................................................ 21 
 4-Week Prerandomization Sham Run-in Period 2 ................................................................................................ 24 
 48-Week Sham controlled Treatment Period 3 .................................................................................................... 26 
 Post End of Treatment Period 4 Safety Follow-up ............................................................................................... 28 
 Interim Visits ......................................................................................................................................................... 29 
 Compensation of Study Participants .................................................................................................................. 29 
 Outcome Measures ............................................................................................................................................ 29 
 Biostatistical Considerations ........................................................................................................................... 33 
 Primary Outcome Measures................................................................................................................................. 33 
 Hypothesized Treatment Effects .......................................................................................................................... 34 
 Ranking and Selection Procedures ....................................................................................................................... 34 
 Sample Size Calculation ........................................................................................................................................ 35 
 Statistical Analysis Plan ................................................................................................................................... 36 
 Baseline Characteristics ........................................................................................................................................ 36 
 Analyses of Co-Primary Outcomes........................................................................................................................ 36 
 Analyses of Secondary Outcome Measures and Secondary Analyses of Primary Outcome Measures ............... 38 
 Stratified Analyses .......................................................................................................................................... 39 
 Site Performance Monitoring ......................................................................................................................... 39 
 Data Collection  and Quality Control Measures ............................................................................................... 39 

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 MRI Acquisition and Quality Control Measures ................................................................................................. 40 
 Bilateral PA View Fixed-Flexion Knee Radiograph Acquisition and Quality Control Measures............................. 41 
 General Guideline for Administration of Study Participant Questionnaires ....................................................... 42 
 Administration of the WOMAC Questionnaire ................................................................................................. 43 
 Device Quality Assurance .............................................................................................................................. 43 
 Study Organizational Structure ....................................................................................................................... 43 
III. Human Subjects .................................................................................................................................... 44 
 Risk to Participants ........................................................................................................................................ 44 
 Human Participant Involvement and Characteristics .......................................................................................... 44 
 Potential Risk ....................................................................................................................................................... 45 
 Sources of Materials ............................................................................................................................................ 46 
 Therapeutic Risks ................................................................................................................................................. 46 
 Research Risk ....................................................................................................................................................... 47 
 Adequacy of Protection Risk .......................................................................................................................... 47 
 Recruitment and informed consent .................................................................................................................... 47 
 Protection Against Risk ........................................................................................................................................ 48 
 Potential Benefits of Research to Participants ................................................................................................ 51 
 Importance of Knowledge to be Gained ......................................................................................................... 51 
 Data Monitoring and Reporting ...................................................................................................................... 51 
 Data Monitoring ................................................................................................................................................... 51 
 Data Reporting ..................................................................................................................................................... 52 
 Safety Monitoring and Reporting ................................................................................................................... 52 
 Safety Monitoring ................................................................................................................................................ 52 
 AE, SAE, ADE, and UADE Monitoring and Reporting Requirements .................................................................... 55 
 Reporting of Events of Special Concerns ............................................................................................................. 56 
 Data Monitoring Committee .......................................................................................................................... 56 
 Reporting AEs, UADEs and SAEs to the Data Monitoring Committee (DMC) ..................................................... 56 
 Females and Pregnancy Testing ..................................................................................................................... 56 
 Women, Minorities and Children .................................................................................................................... 57 
IV. REFERENCES ......................................................................................................................................... 58 
 
 
 
 

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I. SPECIFIC AIMS 
 Objectives  
The objective of this Phase IIa clinical trial is to determine whether pulsed low-intensity ultrasound (PLIUS) may provide 
a therapeutic benefit for idiopathic knee osteoarthritis (OA). If so, this would strongly motivate a subsequent Phase IIb 
or III trial.  The trial incorporates two co-primary outcomes: symptomatic improvement, assessed by OMERACT-OARSI 
Responder Criteria[11], and disease-modification, assessed by MRI-determined cartilage thickness. 
 
Knee OA is a leading cause of age-associated disability with still greater prevalence in people with military service [12]; it 
is the most common indication for knee arthroplasty in the US.  Therapies for pain are only modestly effective, and 
disease-modifying interventions for the cartilage degeneration accompanying OA are entirely lacking [13]. 
 
The proposed intervention is based upon the established fact that mechanical stimulation increases chondrocyte matrix 
production [14, 15].  PLIUS is a clinically feasible, easily-applied form of mechanical stimulation with longstanding use in 
orthopedics for fracture healing [16, 17].  The anabolic effects of PLIUS on chondrocytes have been established in basic 
science studies of cellular systems [18-24], cartilage explants [25, 26] and engineered cartilage constructs [27].  These 
anabolic effects have also been consistently observed in preclinical studies [5, 6, 28-30], including and perhaps most 
importantly, in the marked attenuation of cartilage degeneration in the Hartley guinea pig model of human OA [5].  
 
Based on this substantial body of literature, we propose a Phase IIa trial of PLIUS as a potential therapeutic modality for 
knee OA in humans. PLIUS will be applied using a light and portable FDA-approved device, the Sonic Accelerated 
Fracture Healing System (SAFHS) [31] currently in clinical use for fracture healing.  We will file an Investigational Device 
Exemption (IDE) for approval to use the SAFHS in this trial.  This is a truly translational bench-to-bedside proposal 
targeting a very debilitating and highly prevalent disease with no disease-modifying and only marginally effective 
symptom-modifying therapies currently available.  If this intervention is successful and subsequently confirmed in a 
pivotal trial, it would radically change the standard of care for this highly prevalent and debilitating disease. 
 
 Specific Aims:   
Primary Hypothesis: 
1. PLIUS is potentially more effective than sham treatment as a disease- and symptom-modifying intervention for OA of 
the knee.  
 

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Aim 1A.  Measure the change in cartilage thickness of the central, weight bearing, region of the medial femoral condyle 
after 48 weeks of PLIUS treatment compared to 48 weeks of sham-treatment in OA patients using MRI morphologic 
measures [32].  The 48-week time point is selected due to the expected time course of cartilage regeneration. 
 
Aim 1B.  In the same individuals, assess symptomatic response using the OMERACT-OARSI Responder Criteria [11].  
OMERACT-OARSI is widely-used to assess response to OA treatment; response will be evaluated throughout the study.   
Note that the above hypothesis and associated aims form the basis of this Phase IIa clinical trial. 
 
Secondary Hypotheses:    
2. PLIUS is more effective than sham in treating knee pain and improving knee function associated with OA. 
 
Aim 2.  Assess the change in knee pain and function using the corresponding validated WOMAC scales [33] after 48 
weeks of PLIUS treatment compared to sham-treatment in OA patients. 
 
3. PLIUS is more effective than sham in decreasing severity of bone marrow lesions (BMLs) associated with OA as 
measured by BML maximal size assessed with MRI. 
 
Aim 3.  MRI analysis of subchondral BMLs using images obtained as part of the morphologic study of cartilage thickness 
will be performed.  Bone marrow lesions have been consistently found to be associated with OA pain. 
 
4.  PLIUS is more effective than sham in decreasing serum and urine biomarkers associated with OA progression. 
 
Aim 4.  Soluble OA biomarkers will be collected concurrent with knee MRIs to evaluate the correlation between these 
outcomes.  In addition, the change in these biomarkers at 48 weeks will be compared between treatment groups. 
 
5.  PLIUS is more effective than sham as a disease-modifying intervention for structural progression of knee OA as 
assessed by radiographic joint space narrowing (JSN). 
 
Aim 5.  JSN in the medial compartment of the treated knee will be assessed on a Fixed-Flexion (FF) radiograph [8] after 
48 weeks of PLIUS treatment compared to sham.  Radiographic JSN remains the FDA gold standard for disease 
modification.   
Note that the above Secondary Hypotheses and associated Aims are purely exploratory in nature, and while they may 
guide further studies, they will not be incorporated as primary outcomes in the present study. 

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 Expected Outcome and Impact: 
As discussed, there is a great deal of mechanistic and basic science support for the proposed intervention PLIUS. In 
addition, Loyola-Sanchez et al. have shown the feasibility of applying PLIUS to OA patients [34].  Further, OA is 
increasingly prevalent [35], particularly in veterans [12], and lacks both effective long-term symptom-modifying 
therapies and any disease-modifying intervention [13].  Demonstration of the hypothesized effects would strongly 
motivate a pivotal trial that could establish a major advance in OA treatment. 
 
II. RESEARCH PLAN 
 Background and Significance 
 Overview 
Osteoarthritis (OA) affects almost 27 million Americans with an estimated net cost of over $80 billion/year [36].  In 
addition, the prevalence of OA is increasing rapidly, and it is estimated that by 2030 some 60 million Americans will be 
affected [37].  Further, OA is the leading cause of lower extremity disability in older age [35] and is the leading indication 
for total knee replacement (TKR).  The number of TKR procedures has more than doubled over the last decade.  OA is 
known to occur disproportionally in members of the armed services [12].  Within the VA system, 3351 TKRs were 
performed in FY02, while in the year ending March 31, 2012, 6698 TKRs were performed.  Aside from phakectomy, TKR 
is the most common elective surgical procedure within the VA system.  The immensity of the burden of OA is due largely 
to the fact that available medical management addresses only symptoms [13] and is of marginal long-term efficacy at 
best. In spite of significant progress in understanding the pathogenesis of OA, no disease-modifying interventions have 
been established.  A number of surgical techniques to repair or replace damaged cartilage have been attempted, such as 
microfracture [38], autograft and allograft transplants [39], and autologous chondrocyte implantation [40] with 
disappointing long-term outcomes.  Use of tissue engineered replacement cartilage is another approach that has 
generated a great deal of research interest, but to date has failed to yield any actual therapeutic products.   
 
Degenerative articular cartilage is thought to be central to the pathogenesis of OA.  However, articular cartilage has 
limited capacity for repair [41], and so the development of new cartilage regeneration and repair strategies is of great 
importance.  Therefore, based on a large body of preclinical work demonstrating the anabolic effect of mechanical 
stimulation on chondrocyte metabolism, we propose to investigate the potential of PLIUS as a symptom and structure-
modifying intervention in human subjects with early OA.   

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 Effects of PLIUS on Cartilage Repair  
Cyclic mechanical stimulation increases cartilage matrix production under a wide variety of circumstances [14], which is 
analogous to the anabolic effects of loading on bone.  Ultrasound is a particularly convenient mode of delivering 
mechanical stimulation to human subjects. A large number of studies demonstrated the anabolic effects of PLIUS on 
bone, which led to the development of the Sonic Accelerated Fracture Healing System (SAFHS;) introduced into clinical 
orthopedic practice in the United States for promoting the healing of certain fractures [42].  In addition to clinical 
results, there is a growing body of investigational literature on the mechanism by which ultrasound may exert its effects.  
Up-regulation of cycoloxygenase-2 via integrin signaling is thought to play an important role [43], while other proposed 
mechanisms include the exquisite sensitivity of certain enzymes (MMP1, collagenase) involved in cartilage homeostasis 
to the imperceptible degree of tissue heating induced by PLIUS [42].   
 
A number of studies have demonstrated an ultrasound-induced increase in aggrecan gene expression and chondroitin 
sulfate synthesis for cartilage.  Systems studied include chondrocytes harvested from embryonic chick sterna [18] and 
from the rabbit [19] and rat [20]; the healing fracture callus in the rat [21]; cells harvested from bovine [22]; rabbit [23] 
intervertebral disc; chondrocytes obtained from human OA patients [24]; and cultured explants from embryonic chick 
sterna [26] and from human joints [25].  Increased production of the two major macromolecular components of 
cartilage, proteoglycans and type II collagen, have been found in several of these ultrasound-stimulated systems [18, 22, 
24-26].  It is important to emphasize that the energy of ultrasound used in these studies, as with the fracture healing 
device discussed above, is considerably lower than that used in physical therapy applications.  In further work of 
particular relevance to tissue regeneration in situ, the up-regulation of appropriate matrix molecules in response to 
PLIUS was demonstrated in engineered cartilage over a time course [44].  This represents application of mechanical 
input as a therapeutic modality to an actual model of repair. 
 
The mechanism underlying the increased matrix production by chondrocytes exposed to ultrasound is of substantial 
interest but remains unclear.  Recent studies indicate that integrin signaling may play a central role in cartilage 
metabolism [45].  One report has indicated a difference in mechanotransduction between normal and OA chondrocytes 
and attributed that difference to altered integrin signaling [46].  Transduction pathways acting directly through integrin 
signaling may therefore represent major mediators of the effect of ultrasound on cartilage metabolism.   
 
Ultrasound has also been applied to animal models of degenerative cartilage lesions.  In two studies [28, 47] knee 
cartilage degeneration was induced in the rat by injection of papain.  It was found that subsequent cartilage repair was 

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enhanced by ultrasound exposure in cases of mild pathology, while progression of the disease was attenuated.  In 
another study, an acute chemical arthritis was induced in the radiocarpal joint of calves by intraarticular injections of 
turpentine oil [29]. Again, the effect of ultrasound applied over the joint was to improve the histologic state of the 
cartilage.  Salutary effects of ultrasound were similarly observed in donkeys [48].  It was found that application of 
ultrasound to surgically induced full-thickness osteochondral lesions in the rabbit knee resulted in a statistically 
significant improvement in joint pathology and in the histology of repair cartilage [6].  Ultrasound in conjunction with 
high-molecular weight hyaluronate was found to be effective in reducing OA pathology in a rabbit model [49].  Finally, 
ultrasound has been successfully implemented as a therapeutic modality for the prevention and attenuation of the OA 
disease process in the guinea pig [5].  This is of substantial importance since the Hartley guinea pig develops 
spontaneous age-associated OA and is a particularly good model for idiopathic OA in humans [50]. The evidence from 
these preclinical studies set the stage for the conduct of the proposed Phase IIa trial using a device that has already 
undergone Phase I testing for promoting bone healing. 
 
 Challenges and Innovations in Clinical Trials in OA 
Clinical trials in OA are rendered particularly problematic by the relatively subjective nature of pain and function as 
outcome measures used to assess efficacy, varied manifestations from patient to patient, and insensitivity and lack of 
specificity of knee radiographs which are used to assess structural progression.  OA clinical trials generally address the 
dual objectives of improvement in OA pain and functional impairment.  Generally these trials are complicated by high 
rates of placebo response.  One method of addressing this is to implement a flare design in which the patient is 
withdrawn from background medication, experiences the expected increase in pain, and is then assessed over a 
relatively short time (2-6 weeks) for improvement in pain following application of an agent or placebo.  Several studies 
have evaluated pain and function over longer periods of time or without flare design and have generally shown higher 
placebo response rates and less dramatic improvement in pain and function.    
 
An advance of potentially great significance would be the development of an intervention that alters OA disease 
progression through attenuating the progression of anatomic findings seen with worsening disease.   This is entirely 
analogous to the case of rheumatoid arthritis, in which the development of disease-modifying agents has revolutionized 
treatment.  For OA, evaluation of disease-modifying agents is greatly complicated by the use of plain radiographs as 
outcome measures.  The primary radiographic outcome of joint space narrowing (JSN) is an indirect and relatively 
insensitive marker of articular cartilage status, and in addition depicts only a two-dimensional representation of the 
gross bony changes associated with late-stage OA.  There is increasing evidence that for knee OA, direct assessment of 
cartilage thickness in three dimensions by MRI is a more precise structural outcome measure[51] than radiography.  In 

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addition, bone marrow lesions (BMLs) can be identified on MRI and are associated with articular pain in OA [52]. This 
proposed trial will evaluate symptoms and structure using current standard outcome measures for both pain and 
function, and structural improvement.  In addition, outcomes will include important state-of-the-art quantitative 
measurements of cartilage thickness in a targeted region, and of the maximal size of BMLs by MRI. BMLs in particular 
have been shown to be associated with both cartilage loss and knee pain [52, 53].   
 
Finally, in terms of the practicality of the proposed treatment, Loyola-Sanchez and colleagues recently reported 
application of PLIUS in 27 patients with knee OA.  Their study, while too small to detect a treatment response, directly 
demonstrated the feasibility of this therapeutic approach [34]. 
  
 The Osteoarthritis Initiative (OAI) and the Development of MRI Measures, Including Cartilage Thickness, as  
Sensitive Modalities for OA Assessment 
There has been considerable activity over the past decade in the development of robust imaging-based biomarkers of 
disease activity for OA.  In particular, morphometric measurement of cartilage volume and thickness using magnetic 
resonance imaging (MRI) is increasingly used as a structural endpoint for progression of OA, [54, 55], [56].  Cartilage 
thickness in particular has been shown to be very sensitive to change and progression of OA, notwithstanding the fact 
that OA is a disease of the entire joint [57]. High-resolution 3D MRI techniques such as double-echo steady-state (DESS) 
imaging at 3 Tesla are well suited for quantitative assessment and monitoring disease progression of knee OA [58] and 
have been used for assessing longitudinal changes in the OAI-and the OAI ancillary-Pivotal OAI MRI Analyses (POMA: 
HHSN26820100002, PI: Kwoh CK)[59]. 
 
The OAI (http://oai.ucsf.edu/) is a nationwide multi-center longitudinal cohort study sponsored by the National 
Institutes of Health.  Launched in 2004, the overall objective of the OAI is to establish a public database resource to aid 
in the identification of biological and behavioral biomarkers, including MRI, for the development or progression of knee 
OA for use in epidemiologic studies and to evaluate therapeutic strategies in clinical trials. (http://oai.epi-
ucsf.org/datarelease/docs/StudyDesignProtocol.pdf). The OAI collects extensive annual clinical, radiographic and MRI 
data on a cohort of 4,796 men and women ages 45 to 79 with a planned eight years of follow-up. MRI protocols of the 
OAI include high-resolution (3T) proton sagittal DESS imaging with water-excitation sequences on both knees to assess 
joint and cartilage morphology. The OAI cohort is divided into subcohorts (i.e., ~28% progression subcohort, ~68% 
incidence subcohort, and ~4% reference subcohort).  MRI data are also available for a subset of the progression cohort 
at 18 and 30 months.  These images form the basis for many studies of MRI volume and thickness measurements, and 
define the sensitivity of cartilage thickness as a biomarker of OA progression.  Many of the recent studies extend the 

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analysis to femorotibial subregions in the cartilage, using sophisticated computer-based segmentation tools, [60], [56, 
61].   
 
In addition to cartilage morphometric measurements, the OAI MRI dataset permits whole-joint evaluation according to 
several semi-quantitative OA scales that combine features relevant to functional integrity of the knee.  These features 
include cartilage morphology, subchondral BMLs and cysts, osteophytes, meniscal and ligamentous integrity, synovial 
properties, joint effusion, bone attrition, intra-articular loose bodies, and periarticular cysts or bursitis [62].  These semi-
quantitative knee scores include the Whole-Organ MRI Score (WORMS) [63, 64].  Several variations and improvements 
on WORMS have been proposed, including the Boston-Leeds OA Knee Score (BLOKS) [64], and most recently the MRI 
Osteoarthritis Knee Score (MOAKS) [62]. 
 
Accurate and repeatable measurement of cartilage thickness requires consistency and careful control of both MR image 
acquisition and subsequent segmentation and analysis.  Despite these challenges, cartilage thickness by MRI is emerging 
as a robust indicator of disease modification in OA.  The OAI has established a detailed and highly repeatable protocol 
for image acquisition, and other studies [60, 61] have quantified the efficacy of tracking OA progression using cartilage 
thickness measurement from OAI data.  Our group of investigators includes experts with extensive experience in 
quantitative MRI of articular cartilage and experienced statisticians.  The MRI acquisition protocol and analysis approach 
proposed in this study are the result of careful analysis of the OAI data and published analyses.  Our MRI acquisition 
mirrors that used on the 4,796 subjects in the OAI, and we are adopting best available practices in image segmentation 
and analysis.   
 
 Impact on Patient Care of Disease Modification in OA  
OA is the most common rheumatologic condition.  Its incidence is increasing with the aging of the population and with 
the epidemic of obesity.  It is particularly prevalent in individuals with military service [12]; by age 75 the vast majority of 
veterans will have symptomatic OA.  Current therapies are only marginally effective and are aimed exclusively at 
managing symptoms.  In addition, the analgesics and anti-inflammatory drugs used for OA are frequently not tolerated 
by the target population, which is comprised disproportionately of individuals of advanced age and with common 
comorbidities such as hypertension, diabetes mellitus and renal insufficiency.  Along with these limitations on 
medications that relieve OA symptoms, there are no FDA-approved interventions for OA that alter disease progression.  
Indeed, OA is the single most common indication for total joint replacement.  If data from this Phase IIa study suggest 
that PLIUS is effective in treating OA symptoms and has the potential to ameliorate disease progression, it would 
strongly motivate a definitive follow-up trial.  If confirmatory in a follow-up pivotal trial, this would then have a major 

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impact on clinical management of OA.  If disease modification in particular is established, PLIUS could greatly impact the 
now spiraling costs of surgical OA management in the US [65] and within the VA.  This burden will continue to increase 
along with the increasing prevalence of OA.   
 
 Implications of Potential Study Outcomes 
As detailed above, knee OA is a highly prevalent disease with only modestly effective long-term medical options for 
symptomatic therapy and no FDA-approved structure-modifying interventions.  Note that while PLIUS is an extremely 
promising therapy, there are at present insufficient data on the application of this modality to human OA patients to 
justify a full, pivotal, clinical trial.  We are instead seeking a signal of efficacy for symptomatic improvement and for 
disease modification. Thus, there are four possible outcomes of the proposed preliminary trial, depending on the signal 
found for these two primary endpoints:  1) If our results suggest that PLIUS may be effective both for symptom relief and 
to ameliorate disease progression, it would strongly motivate a subsequent definitive clinical trial to further substantiate 
these effects;  2) If the results suggest that PLIUS is effective for symptoms but not for structure modification, a 
definitive trial to more clearly establish and quantify the extent of symptomatic improvement and further affirm safety 
issues would be indicated.  This would help to determine the role of PLIUS in the symptomatic management of OA; 3) 
Results suggesting that PLIUS has efficacy in structure modification but not for symptomatic improvement would 
suggest that OA pain and functional limitations on the one hand, and structural progression on the other, may not be 
linked pathophysiologically.  This would suggest a potential role for PLIUS in disease prevention or prophylaxis, for 
example, following knee injury, to preserve cartilage health.  A thoughtfully designed follow-up study would therefore 
be of substantial interest; and 4) Results demonstrating no improvement in symptoms or structure would suggest that, 
in spite of substantial basic science evidence, there may be no PLIUS effect in human OA or that the selected dose, 
duration or mode of administration may need to be reconsidered. 
 
 Preliminary Studies 
 PLIUS in OA 
 Anabolic Effects of Ultrasound on Chondrocytes and Cartilage 
We have applied ultrasound to chondrocytes [18], cartilage explants [26], engineered cartilage tissue constructs [44], 
and OA in an animal model [5].  In all cases, the application of ultrasound was through use of the sonic accelerated 
fracture healing system (SAFHS), an FDA-approved device in clinical use for fracture healing.  This is the same device that 
we will use in this Phase IIa trial of PLIUS in human subjects. 

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Type II Collagen, distal sternum
Control, day 5
PLIUS, day 5
Aggrecan, distal sternum
Control, day 1     PLIUS, day 1
Chondrocyte study [18]:  PLIUS at an FDA-approved level for fracture healing, 30 mW/cm2, and at 2 mW/cm2, was 
applied to chondrocytes in culture in a single application.  The effect on cell proliferation and on expression of mRNA for 
collagen II, collagen X, Sox9 (a gene associated with collagen II synthesis), and aggrecan was followed for a period of one 
week in addition to immunostaining for collagen II and aggrecan.  Overall anabolic effects of PLIUS were documented, 
with the lower power resulting in effects that were at least as large as those seen with the higher power.  This is in 
agreement with literature results indicating that the ultrasound effect may require a small threshold signal, rather than 
following a usual dose-response profile.  
 
Cartilage explant study [26]:  PLIUS was applied to chick sternal explants.  Quantitative immunohistochemical staining 
was used to evaluate regional production of collagen II, collagen X (as a marker for chondrocyte hypertrophy, indicating 
a pre-ossification state), and aggrecan.  The proximal and distal portions were investigated separately, due to their 
different developmental fates (ossifying and non-ossifying, respectively).  Immunohistochemistry demonstrated 
increased aggrecan and collagen II (below). Type X collagen was not produced in the distal part of the sternum, 
indicating that ultrasound did not induce a tendency towards ossification in that region. 
 
 
         
 
 
 
 
 
 
 
Tissue regeneration study [44]:  We tested the hypothesis that cartilage regeneration would be augmented by 
application of PLIUS, and that matrix-sensitive MR parameters could be used for non-invasive assessment of this 
phenomenon.  As a model, we used bovine stifle joint chondrocytes and a type I collagen scaffold, which has been 
shown to support the chondrocyte phenotype.  In order to interpret the response seen in the standard MR outcome 
measures T1, T2, and km, we performed correlative histologic, immunohistochemical, biochemical, and FT-IR 
measurements.  Biochemical data showed increased PG at 3 and 5 weeks of growth in PLIUS-treated as compared to 
untreated constructs (left hand panel below), and increased collagen at 3 weeks (right hand panel below).  A decrease in 
total collagen at 5 weeks reflects breakdown of the biodegradable type I collagen scaffold. 
 
 
 
 
 
Immunohistochemical staining of 
control and PLIUS-treated chick 
sternal explants.  The increase in 
production of type II collagen and 
aggrecan in the distal, non-ossifying, 
part of the sternum is illustrated. 
Effect of PLIUS application to 
tissue engineered cartilage 
constructs (see text), showing 
the resulting increase in matrix 
production.  The apparent loss 
of collagen at 5 weeks is due to 
collagen scaffold breakdown.   

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Immunohistochemical results indicated more precisely the composition of the construct.  As shown in the panel below, 
overall staining for collagen types I and X was weak, as was staining for MMP-13.  Collagen type X and MMP-13 staining 
intensities were further decreased with PLIUS.  In contrast, collagen type II staining was strong for all groups, and 
increased with time in culture.  More intense pericellular collagen type II staining was seen in the 5-week PLIUS-treated 
group, with significant augmentation by PLIUS at that time point. 
 
 
 
 
 
 
 
Corresponding results were obtained using Fourier transform infrared spectroscopic analysis, evaluating the 
absorbances at wave numbers specific to particular macromolecular components.  Finally, MRI experiments indicated 
consistency with the above interpretation of increasing macromolecular content, and hence decreased molecular 
mobility, as reflected by decreased T2 and increased km; again, the decrease in km and trend to increase in T2 at 5 weeks 
is due to the breakdown of the collagen scaffold.     
 
 
Immunostaining results for 3 weeks (upper row) and 5 weeks (lower row) of tissue development under PLIUS as 
compared to control constructs.  A:  Collagen I; B:  Collagen II; C:  Collagen X; D:  MMP-13. 

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Together, these results strongly support our hypothesis that PLIUS induced anabolic effects in engineered cartilage 
constructs, and that these effects could be monitored noninvasively by MRI.  
 
Animal model study [5]: The Hartley Guinea pig develops spontaneous age-associated OA and is a particularly good 
model for idiopathic OA in humans [50].  We used this to test the efficacy of PLIUS in the treatment of OA.  Four groups 
were studied:  i) treatment initiated at two months of age, treatment duration of four months (E2/T4); ii) treatment 
initiated at two months of age, duration ten months (E2/T10); iii) treatment initiated at twelve months of age, duration 
three months (E12/T3); iv) treatment initiated at twelve months of age, duration six months (E12/T6).  Groups i) and ii) 
test efficacy in preventing pathology, while groups iii) and iv) provide results related to retarding progression of, or 
reversing, established pathology.  Treatment was applied to one limb, with the contralateral limb serving as a control.  
Overall, the cartilage from the treated limbs exhibited less staining loss and greater uniformity, less fibrillation, and less 
deep fissuring than did cartilage from control limbs.  These effects were seen in all joint compartments.  Representative 
histology is shown below. 
 
 
MRI analysis of the effect of PLIUS treatment on (A) T1, (B) km, and (C) T2 measurements of collagen constructs (n=10 per group). 
Although there were no significant changes in T1 with PLIUS, km increased while T2 decreased at 3 weeks, reflecting matrix 
maturation. The decreases at 5 weeks are due to scaffold breakdown. *p<0.05 versus 3-week control, #p<0.05 versus 5-week 
control. 

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Illustrative results for treatment of animals initiated 
at 2 months of age, with treatment duration of 4 
months.  Left:  PLIUS-treated.  The medial tibial 
plateau (MTP) shows an intact surface with mild loss 
of matrix staining.  The medial femoral condyle 
(MFC) shows an intact surface with uniform matrix 
staining.   Right:  Untreated control animal.  The MTP 
shows extensive surface fibrillation and deeper 
fissuring, with greater loss of staining.  Staining is 
with thionin. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Quantification of these effects was performed using a modified Mankin scale [66] assessing fibrillation (1 - 5), matrix 
distribution (1 - 4), chondrocyte loss (1 - 3), and chondrocyte cloning (1 - 4), with a minimum (best) score of 4 and a 
maximum (worst) score of 16.  Scoring results are shown in the table below.  Note that the intention of this study was to 
evaluate treatment at earlier as compared to later stages of disease.  Therefore, we compared disease treatment 
initiated at 2 months of age with treatment initiated at 12 months of age.  We were secondarily interested in treatment 
duration, and therefore divided each of these two groups into two treatment duration arms.  While data were presented 
for all subgroups in the original publication, the prevention and treatment groups each in fact consisted of two 
treatment durations, as shown in the Table below: 
 
 
 
 
 
 
 
 
 
 
 
 
Statistical comparisons are between control and treated knees. Bold indicates statistically significant differences (**P< 
0.01; *P< 0.05) or trends towards significant differences (&P < 0.10).   In addition to favorable trends throughout nearly 
all outcomes, statistically significant results are especially pronounced in the tibial plateau for the prevention group.  
Overall, PLIUS demonstrated substantial efficacy. 
 
Finally, immunostaining was performed on cartilage from animals in which PLIUS was initiated at eight weeks of age, for 
a duration of 4 months.  Chondrocyte expression of MMP-3, MMP-13, IL-1ra, and TGF-1 in the femoral condyles was 

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D. 
A. 
C 
B 
. 
quantified using a Bioquant Image Analysis System.  PLIUS had a striking effect on the expression level of TGF-1, which 
was expressed by chondrocytes throughout the full extent of the articular cartilage of the femoral condyle.  Panel A 
below shows a typical example of the widespread expression of TGF−1 throughout the articular surface of a non-
treated joint.  The expression of TGF−1 was often observed to be associated with chondrocytes found in colonies, 
which we interpret as part of a widespread repair response (Panel B).  Panel C shows the widespread expression of 
TGF−1 in a non-treated joint from another representative animal. In contrast, the expression of TGF−1 was 
dramatically reduced in articular cartilage exposed to the PLIUS treatment (Panel D). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 MRI as a Measure for Cartilage Health 
It is well-established that disease progression in OA is typically accompanied by structural changes in the joint, including 
loss of cartilage volume and thickness.  In human studies, the standing anteroposterior (AP) knee radiograph has been 
the standard for measurement of joint space narrowing (JSN) for more than 30 years, but this technique is severely 
limited as a means to reproducibly visualize the thickness of articular cartilage [67] for a number of reasons.   The 
intrinsically two-dimensional nature of the measurement requires particularly consistent joint positioning to obtain 
reproducible results.  However, precisely reproducible positioning of the knee in serial examinations is extremely 
difficult; for example, differences in joint pain from examination to examination have been shown to affect positioning 
[68].  The Fixed-Flexion (FF) [8]views address some of these alignment issues and are superior to the standing AP view, 
but repeatability and accuracy still suffer from variations in positioning.  Further difficulties arise from the fact that the x-
ray measurement of cartilage thickness is based on hard tissue outlines, while cartilage itself is not visualized.  
Therefore, the status of other soft tissue structures, especially the menisci, affects the measurement of cartilage 
thickness.   
 
Quantitative measurement of cartilage thickness from 3 Tesla MRI is emerging as an excellent and highly stable 
alternative to radiographic measurements [69].  Furthermore, data from more than 4700 subjects in the OAI has 
TGF-1 expression.  A) Femoral condyle from control 
knee joint.  B) Higher magnification of joint surface 
shown in A.  Arrowheads indicate enhanced expression 
of TGF-1 in areas where chondrocyte proliferation, 
indicative of a repair response, is occurring.  C) 
Femoral condyle from the non-ultrasound treated joint 
of another animal.  D) The ultrasound−treated joint 
contralateral to the joint pictured in A.   

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established a clear relation between cartilage thickness changes as assessed by MRI and radiographic disease status 
[70].  A recent study by Wirth et al. [56] compared the standardized response mean (SRM), a measure of the sensitivity 
to change, and annualized rates of change in femorotibial cartilage plates and subregions for a 2-year vs. a 1-year study 
period using a subset of the progression cohort of the OAI.  Significant structural progression was observed even over a 
1-year observation period.  In the central portion of the central medial femoral condyle, Wirth et al. measured baseline 
to 1-year mean change of -2.5% and an SRM of -0.31. 
 
 Experience of the Investigative Team 
Inception, design, execution and reporting of federally funded controlled clinical trials in rheumatic diseases have been 
the career-long academic focus of the PI.      
 
 Research Design and Methods  
 Overview of Study Design  
This is a Phase IIa, multi-center, randomized, sham-controlled, parallel, double-blind trial with a prerandomization four-
week sham run-in period to determine if PLIUS is potentially more effective than sham as a disease and symptom 
modifying intervention in patients with early OA of the knee. It is recognized that this proposed trial is shorter in 
duration than the current standard of 2 years for Phase III trials based on radiographic outcomes. Our objective at this 
early stage is to determine whether there is sufficient evidence of efficacy to warrant further investigation in a Phase IIb 
or III trial.  If a signal were detected, it would also provide data to calculate the sample size required for the next trial.  
The study consists of four periods:  a Screening Period 1, a 4-Week Prerandomization Sham Run-in Period 2, a 48-Week 
Sham-controlled Treatment Period 3, and a 30-day Post End of Treatment Safety Follow-up Period 4 for participants with 
unresolved Serious Adverse Event(s) (SAE) at study completion.  A total of 180 patients will be randomized.  All patients 
will be followed during the Treatment Period for 48 weeks, which is the time point for assessment of the primary 
outcome measures.   
 
 
 
 
 Participant Recruitment & Retention 
Study participant recruitment will be the responsibility of the local principal investigator (PI) and the research staff. 

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Potential study participants will be identified and recruited using a number of strategies. Research staff will educate 
their clinical colleagues at staff meetings regarding this study opportunity and will maintain frequent contact with them 
so potentially eligible patients seen during routine outpatient care visits will be referred to the study.  Clinical staff, 
including rheumatologists, orthopedists, family practitioners, internists and musculoskeletal radiologists, will also be 
continually informed about the study through IRB approved print or web media, such as internal hospital 
communications (e.g., newsletters).  IRB approved advertising (e.g., flyers or brochures) will be placed in clinic rooms 
and patient care areas as allowed by each center’s policies.   
 
As permitted by each study site, study participants with a diagnosis of OA of the knee will be identified through a search 
of patient databases. Study participants identified through this search will have their medical history pre-screened by 
study staff to determine potential eligibility per inclusion and exclusion criteria.  Potentially eligible study participants 
will be told about this study by their primary or specialty care providers at upcoming appointments or by mail and 
instructed how to contact study site staff if interested in participating.  Study participants expressing interest in 
participating after having an opportunity to learn more about the study will be pre-screened by phone or in person by 
study site personnel. If eligible based on the pre-screening information, they will be scheduled to come in for the 
informed consent process and the Screening visit.  
 Schedule of Assessments 
The table on the page below contains the chronological sequence of the assessments and procedures to be performed:   
 
 

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NAM E
Screening
/ Period 1
Post End of 
Treatment / 
Period 4
DURATION
Up to 4 
weeks
NUM BER
1
2
3
4
6
7
8 (6th)
9
10
11
12 (7th)
13 (8th)
14 (9th) 15 (10th) 16 (11th) 17 (12th) 18 (13th)
Screen
Begin 
Period 2
TC1
Clinic5
Clinic
(End of 
Period 2)
Begin 
Period 3
TC
Clinic
Clinic
TC
Clinic
TC
Clinic
TC
Clinic
TC
Clinic
TC
Clinic
(End of 
Period 
3)
Safety 
Follow-up2
Day 0
Period 2
 Wk 1 
 Wk 2
Wk 4
Day 0
Period 3
Wk 1
 [+/-3d]
Wk 2
 [+/-
3d]
Wk 4
 [+/-1wk]
Wk 6
 [+/-1wk]
Wk 8
 [+/-1wk]
Wk 10
 [+/-1wk]
Wk 12
 [+/-1wk]
Wk 18
 [+/-1wk]
Wk 24
 [+/-1wk]
Wk 30
 [+/-1wk]
Wk 36
 [+/-1wk]
Wk 42
 [+/-1wk]
Wk 48
 [+/-1wk]
30 days post 
Period 3
[+/-1wk]
X
X
Re-Confirm
Re-Confirm
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X2
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
V6.2
10/1/2018
1 TC = Telephone Call; 2 Post end of treatment safety follow-up by telephone for unresolved ADEs and SAEs; 3 Height performed at Visit 1 only;
 4Only for females of ; childbearing potential; 5 V4 may be conducted by telephone if approved by site investigator. 6 MRI should be completed within 2 weeks of completed visit.
Urine Pregnacy Test4
Adverse Events, Adverse Device Events & Serious 
Adverse Review
Follow-up Telephone Call
Study Device Dispense / Return; Compliance Review 
OA Biomarkers
Visit removed in Protocol V4 and is not longer required 
Visit removed in Protocol V4 and is not longer required 
Visit removed in Protocol V4 and is not longer required 
Visit removed in Protocol V4 and is not longer required 
Visit removed in Protocol V4 and is not longer required 
Fixed Flexion Knee Radiograph
M RI of Index Knee6
Physical Examination
Vital Signs (BP, HR, Temperature)
Weight, Height
3
ARA Functional Class Assesment
Patient Global Assessment of Disease Status
Investigator Knee Examination
Investigator Global Assessment of Disease Status
 Sham-controlled Treatment / Period 3 
48 Weeks
Informed consent
Inclusion/Exclusion Criteria Review
M edical History Review
PERIOD
5
4 Weeks
 Sham Run-In / Period 2
VISIT
Dispense & Review of Rescue M edication Diary 
Patient & Investigator Global Assessment of Response to 
Therapy
Review Analgesic Therapy Use
ICOAP Questionnaire
WOM AC Osteoarthritis Index

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The length of study, the potentially delayed or slow rate of treatment response, daily frequency of treatment sessions, 
or visit schedule, to name a few, can potentially adversely affect study participant retention.  The research staff will 
strive to minimize withdrawals from the study and will take measures to mitigate withdrawals by, for example, 
maintaining regular contact with study participants through clinic and telephone contact and encouraging study 
participants to voice any concerns or issues that arise as a result of their participation so that the research staff can 
discuss their concerns and as appropriate offer potential solutions that may prevent unnecessary withdrawals.  While 
the research teams will strive to minimize withdrawals, study participants will not be coerced or intimidated to remain in 
the trial and will be advised that they always have the option to withdraw for any or no reason at all. However, if a 
patient elects to withdraw from treatment, they will be asked whether they would be willing to remain in the study for 
continued data collection as per the protocol schedule. 
 
 Screening Period 1 
The Screening visit will occur prior to the Prerandomization Run-in Day 0 visit. At the Screening visit patients will 
undergo the following according to the schedule of assessments:  Western Ontario and McMaster (WOMAC®) Index 
patient questionnaire; patient global evaluation of disease status; standardized physical examination of the knees; 
physical exam; Intermittent and Constant Osteoarthritis Pain (ICOAP) patient questionnaire; height, weight, vital signs 
(e.g., temperature, sitting blood pressure, sitting pulse rate & respiratory rate) and demographics; analgesic medication 
and other therapies for osteoarthritis use; urine pregnancy test for women of childbearing potential. Descriptions of 
questionnaires completed by patients can be found in SectionII.C.11.b.  Sample WOMAC and ICOAP questionnaires can 
be found with the Case Report Forms. Assessment of Adverse Events (AEs), Adverse Device Events (ADEs) and Serious 
Adverse Events will begin after informed consent is obtained.  The investigator and participant will decide which knee is 
the more symptomatic knee and designate that knee as the index joint. In the event that both knees are equally 
symptomatic, the right knee will be designated the index knee. Only the index knee will receive treatments with the 
study device. 
 
At the Screening visit study participants will also have bilateral Fixed-Flexion knee radiographs [8]. From the Fixed-
Flexion radiographs, K-L Grade and joint space width will be determined by the central reader to verify the diagnosis of 
OA of the knee and establish the potential for response. Radiograph acquisition and interpretation is discussed in 
Section II.J ..  
 
Study participants will be allowed to take non-steroidal anti-inflammatory drugs (NSAIDs) , and will also be allowed to 
take  acetaminophen (APAP) up to 3000mg/day and/or tramadol immediate release (IR) up to 200 mg/day as rescue 

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analgesia for severe knee pain throughout the trial. Participants should be on a stable dose of NSAID for at least 7 days 
prior to visit 2 (Day 1 of run-in period) and avoid changes in dose and type of NSAID throughout the trial.  The 
investigators selected APAP and tramadol as rescue  medication because they would be the best tolerated, least toxic 
and least likely to cause problems that might lead to early withdrawal from the study due to adverse events. Rescue 
analgesics or NSAIDs will not be provided to the patients by the study.  If not prescribed by the study participant’s 
primary care physician, directions for use and appropriate prescriptions for NSAIDs and rescue medication in accordance 
with the protocol and standard of care will be provided by the site investigator. Study participants will be instructed to 
use as little rescue analgesia as possible by reserving its use for the relief of uncontrolled pain and will be given a diary to 
record date, name and amount of rescue medication used.  It should take patients about five minutes per day to record 
daily rescue medication (i.e., acetaminophen, tramadol) in a diary.  While reliability and data quality are often cited as 
drawbacks to diary use, they offer a systematic method of collecting data on rescue medication (RM) use and may also 
provide for a systematic assessment of a patient’s perspective on symptoms and efficacy. To address reliability and data 
quality concerns, study staff will explain at the beginning and throughout the trial the importance of the RM diary, how 
it is to be used and, when appropriate, make attempts to correct deficiencies in RM diary completion and use. A similar 
diary was effectively used in GAIT [75].  Study participants must discontinue NSAIDs and rescue analgesia 24 hours prior 
to assessment of outcome measures and will be contacted prior to each study visit and reminded to avoid NSAID and 
analgesia use for 24 hours preceding each visit.   
 
If at the Screening visit a study participant is taking an  excluded concomitant treatment, it must be discontinued and 
the Run-in Day 0 visit should not occur until the applicable washout period(s) has been completed.  A list of  excluded 
therapies with their minimal washout periods will be provided to study sites Patients taking low-dose aspirin (325 
mg/day or less) for vascular indications may continue to do so throughout the study.  Patients taking excluded 
analgesics, including all narcotic analgesics, must discontinue use at least two weeks prior to the Run-in Day 0 visit.   
 
 Inclusion and Exclusion Criteria 
Inclusion Criteria 
Eligible patients must meet all of the following inclusion criteria: 
1. Study participant gives voluntarily written informed consent to participate in the study prior to completion of any 
study-related procedures. 
2. Male or female, at least 40 years of age, with a clinical diagnosis of primary OA of the index knee (tibiofemoral joint) 
based on clinical and radiographic criteria (Kellgren and Lawrence Grades 1, 2 or3) [80] as specified below at the 
Run-in Day 0 visit: 

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i) Pain in the index knee on motion or weight bearing for the majority of days during the previous month, at 
least partially relieved by rest;  
AND 
ii) Kellgren and Lawrence Grades 1, 2 or 3 in the index knee as determined by the central radiology review. 
3. Clinical symptoms of OA for at least 6 months prior to the Run-in Day 0 visit. 
4. Sum of WOMAC pain subscale (Section A)  between 125 and 400 mm inclusive in the index knee at the Day 0 Period 
3 visit.   
5. American Rheumatism Association (ARA) Functional Class I, II, or III [81] at the Screening visit.  
Class I 
Completely able to perform usual activities of daily living (self-care, vocational, and 
avocational) 
Class II 
Able to perform usual self-care and vocational activities, but limited in avocational 
activities 
Class III 
Able to perform usual self-care activities, but limited in vocational and avocational 
activities 
Class IV Limited in ability to perform usual self-care, vocational, and avocational activities 
 
6. Females of childbearing potential must be willing to use a reliable form of medically acceptable contraception 
(Examples of acceptable contraception include, but may not be limited to: oral/parenteral/implantable hormonal 
contraceptives, intrauterine device or barrier and spermicide). Women who are surgically sterile or have been post-
menopausal for at least 2 years are not considered to be of child-bearing potential.  Abstinence only is not an 
acceptable method.     
 
Exclusion Criteria    
Eligible patients must meet none of the following exclusion criteria: 
1. Concurrent medical/arthritic disease that could confound or interfere with evaluation of pain or efficacy including: 
Inflammatory arthritis (e.g., rheumatoid arthritis, systemic lupus, spondyloarthropathy, psoriatic arthritis, 
polymyalgia rheumatica), gout, episodes of acute monarticular arthritis clinically consistent with pseudogout, 
Paget's disease affecting the index knee, a history of septic arthritis or avascular necrosis or intra-articular fracture 
of the index knee, Wilson's disease, hemochromatosis, alkaptonuria, or primary osteochondromatosis.  
2. Spine or hip pain of sufficient magnitude to interfere with the evaluation of the index knee. 
3. Isolated patellofemoral disease manifested by primarily anterior knee pain in the absence of tibiofemoral 
radiographic finding.  
4. History of significant collateral ligament, anterior cruciate ligament or meniscal injury to the index joint requiring 
surgical repair.  
5.   History of arthroscopy of the index knee within 6 months prior to the Run-in Day 0 visit. 
6. Participant has a valgus deformity of greater than 15 degrees in the index knee per the investigator’s knee 

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examination or has a valgus deformity of greater than 10 degrees in the index knee as determined by the central 
radiographer. 
7. History of any illness that in the opinion of the investigator might confound the results of the study or pose 
additional risk to the patient. 
8. Female study participants who are breastfeeding, pregnant or plan to become pregnant during the study or within 3 
months following the last study treatment. 
9. Corticosteroid treatment as follows: 
a. Use of oral corticosteroids within the previous four weeks. 
b. Exposure to intramuscular corticosteroids within one month prior to the Run-in Day 0 visit. 
c. Administration of intra-articular steroids to the index knee, within 3 months of the Run-in Day 0 visit.  
d. Administration of intra-articular steroids to any other joint, within 1 month of the Run-in Day 0 visit.  
10. Patients taking excluded therapy must discontinue use in accordance with the washout schedule before the Run-in 
Day 0 visit. Aspirin (up to 325 mg/day) for cardiovascular reasons may be continued. Excluded analgesics or aspirin 
for analgesic purposes, may be used during the trial for treatment of acute pain unrelated to knee OA, such as for 
treatment of pain related to a dental procedure, only if such use is no more than 14 consecutive days and is 
discontinued in accordance with the washout schedule prior to a clinic study visit. 
11. Known contraindications to MRI scanning (e.g., pacemaker, metal fragments, spinal nerve stimulators and/or a body 
habitus that would preclude a patient from having an MRI). 
12. Intra-articular injection of hyaluronic acid or congeners into the index knee within 6 months of the Run-in Day 0 visit. 
13. Topical analgesics (e.g., capsaicin preparations) to the index knee or any oral analgesics (e.g., opiates containing 
analgesics) with the exception of permitted NSAIDs,  acetaminophen and tramadol within 2 weeks of the Run-in Day 
0 visit or during the study. 
14. Implementation of any medical therapy, complementary or alternative regimens for the treatment of osteoarthritis 
within 7 days prior to the Run-in Day 0 visit. 
15. Participation in another clinical study with an investigational agent within 4 weeks of the Run-in Day 0 visit. 
16. Exposure to glucosamine or chondroitin sulfate within one month of the Run-in Day 0 visit. 
17. Initiation of physical therapy or muscle conditioning program to the lower extremities within 2 months of the Run-in 
Day 0 visit. 
18. Concurrent use of the following medications or interventions: 
a. Disease modifying antirheumatic medications including methotrexate, leflunomide, sulfasalazine and 
hydroxychloroquine. 
b. Chronic therapy with tetracycline or tetracycline derivatives. 

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19. Inability to understand and complete study questionnaires including questions requiring a visual analog scale (VAS) 
response. 
20. Any condition that in the opinion of the investigator prevents adherence with the protocol or compliance to study 
treatment. 
 
Study participants meeting entry criteria at screening will be eligible for the Run-in Day 0 visit.  
 
 4-Week Prerandomization Sham Run-in Period 2 
It is anticipated that a 4-week Prerandomization Sham Run-in Period will enrich the patient population with those more 
likely to be compliant with the treatment regimen and increase the likelihood of demonstrating a positive result. The 
doxycycline in OA trial [10] successfully implemented a run-in period. In this trial’s run-in period, 463 patients were 
enrolled and 32 patients failed to complete the run-in period (29 patients missed appointments, 3 patients had <80% 
medication compliance) yielding a 7% run-in period dropout rate.  We project a slightly higher run-in period dropout 
rate of 10% for the proposed trial given that adherence to a daily treatment regimen involving a 20 minute treatment 
session is likely more demanding than taking oral medication. Therefore, we plan to enroll at least 200 patients in order 
to randomize a minimum of 180 patients. 
 
The Run-in Period will consist of a follow-up telephone call, a follow-up clinic visit (if deemed appropriate by the site 
investigator this clinic visit may be conducted by telephone to reduce the burden for select participants who have 
significant difficulty traveling to clinic), and an End of Run-in clinic visit at weeks 1, 2 and 4 respectively after the Run-in 
Day 0 visit.  
 
Run-in Period 2 - Day 0 Visit 
The Run-in Day 0 visit is the beginning of the 4-week Prerandomization Sham Run-in Period and will occur after 
completion of the Screening visit. Prior to this visit, participants will be contacted and reminded to return their 
completed rescue medication diary and to discontinue NSAID and rescue analgesia 24 hours prior to their scheduled Day 
0 Run-in visit.  At this visit study participants will be re-evaluated  to confirm that they meet eligibility criteria as 
previously described.NSAID andrescue medication use and diary will be reviewed, and participants will be reminded to 
use as little rescue analgesia as possible by reserving its use for the relief of uncontrolled pain. Study participants will 
also undergo all other assessments and procedures as detailed in the Schedule of Assessments.  
 

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Eligible patients will be enrolled in the Run-in Period and given a sham device. Patients will not know this is a sham 
device, however, as they will only be informed that all participants will receive sham device treatments during some 
portion of their participation in the study. Participants and research staff will be informed that there is no detectable 
difference between the sham and PLIUS devices. The devices appear and operate similarly, except the sham device does 
not generate an ultrasound signal. The PLIUS device does not generate vibration, warmth or other sensations that may 
un-blind research staff or participants. Patients will be shown how to properly operate and correctly position the device 
over the medial compartment of the index knee. They will then be asked to demonstrate proper treatment 
administration technique. Once the participant has verbalized and demonstrated understanding, they will be asked at 
this visit to self-administer their first 20-minute daily treatment. The device is intended for use in environments in which 
radio frequency interference (RFI) is controlled.  At each visit addressing adherence to the treatment regimen, 
participants will be instructed on how to help prevent RFI with the device to ensure the device works at full capacity 
during treatments.  Participants will receive a Participant Device Brochure for reference on the device, treatment 
administration process and preventing RFI. 
 
Run-in Period 2 - Week 1 Follow-up Telephone Call 
To ensure that study patients are not experiencing difficulty properly administering daily treatments and are compliant 
with the treatment regimen, patients will receive a follow-up telephone call at visit 3.0 approximately 1 week after the 
Run-in Day 0 visit. Adverse Events (AEs) and other analgesic therapy use will also be assessed at this visit. 
 
Run-in Period 2 - Week 2 Follow-up Clinic Visit 
Participants will have a follow-up clinic visit approximately 2 weeks from completion of the Run-in Day 0 visit. This visit 
may be conducted by telephone if deemed appropriate by the site investigator to reduce the burden for select 
participants who have significant difficulty traveling to clinic. Prior to presenting for this visit, study participants will be 
contacted and reminded to discontinue NSAID and rescue analgesia 24 hours prior to their scheduled visit, and to bring 
with them to the visit their rescue medication diary and study device. Treatment compliance data stored in the device 
will be reviewed and documented by the study staff. Participants will be withdrawn if they are less than 60% compliant 
[34] with the daily treatment regimen. NSAID use, rescue medication use and diary, other analgesic therapy use, and 
Adverse Events (AEs) will be reviewed. At this visit participants will be asked to review and demonstrate the treatment 
administration procedures they follow to administer a treatment using their assigned study device. Participants will not, 
however, actually turn on the device to administer a treatment.  The importance of preventing RFI will also be reviewed. 
The study staff will observe their administration technique and make recommendations as appropriate to ensure proper 
administration of study treatment. Participants will be reminded to use as little rescue analgesia as possible by reserving 
its use for the relief of uncontrolled pain.   

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Run-in Period 2 - Week 4 End of Run-in Clinic Visit 
The Week 4 End of Run-in clinic visit (named in the Schedule of Assessments as the Week 4, end of Period 2 part of visit 
5) marks the end of the Run-in Period and will occur approximately 4 weeks from completion of the Run-in Day 0 visit.  
Prior to presenting for this visit, participants will be contacted and reminded to discontinue NSAID and rescue analgesia 
24 hours prior to their scheduled visit, and to bring with them to the visit their rescue medication diary and study device. 
Treatment compliance data stored in the device will be reviewed and documented by the study staff. Participants with 
less than 80% treatment compliance [34] will be withdrawn from the study. Participants who successfully complete the 
Run-in Period will then at this same clinic visit undergo baseline assessments and procedures per the Schedule of 
Assessments.   
 
 48-Week Sham controlled Treatment Period 3  
 Period 3 - Baseline Assessment  
Participants who successfully complete the Run-in Period as determined by assessments performed at the Week 4, End 
of period 2 part of visit 5, will undergo baseline assessments at the Day 0, Beginning of Period 3 part of visit 5 to confirm 
that they meet randomization criteria as previously described and are eligible to be randomized into the 48-Week Sham-
controlled Treatment Period. Participants at baseline must have a summed score of 125-400 mm inclusive in Pain 
Section A of the WOMAC questionnaire to qualify for randomization into the study.  The investigator will perform an 
examination of the index knee and global assessment of disease status. NSAID, other analgesic therapy and rescue 
medication use and diary will be reviewed, and biomarker samples will be collected.  Adverse Events (AEs) will be 
assessed and recorded when applicable.  A urine pregnancy test will be performed on all females of childbearing 
potential prior to randomization into the study. Study participants will also undergo all other assessments and 
procedures as detailed in the Schedule of Assessments and will be reminded to use as little rescue analgesia as possible 
by reserving its use for the relief of uncontrolled pain. 
 
Study participants randomized into the study will have a baseline MRI to evaluate cartilage thickness of the medial 
condyle of the medial segment of the index knee [32, 56], and a bilateral PA view Fixed-Flexion [8] radiograph of the 
knees to establish the baseline radiographic status of the medial compartment of the knee. Knee MR and radiographic 
imaging protocols are discussed later in this proposal. The MRI should be performed within 2 weeks of the completed  
baseline visit. 
 
 

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Eligible participants will be randomized to the PLIUS or sham device and as in the Run-in Period will apply the study 
device for 20 minutes daily over the medial compartment of the index knee. Participants will review and be expected to 
demonstrate proper treatment administration technique.  The importance of preventing RFI will also be reviewed. 
 
 Period 3 - Randomization Methods  
The Hines CSPCC will develop the randomization scheme and associated codes. Permuted block randomization will be 
employed to assign patients to PLIUS or sham device. Randomization will be stratified by study site and Kellgren-
Lawrence grade (KLG) 1, 2 and. 3.  
 
Eligible patients will be randomly assigned to PLIUS or sham device using a web-based central randomization system 
with a telephone call randomization system as back-up. The Study Coordinator at the participating facility will be 
required to sign into this password-protected site. The Study Coordinator will enter the patient study number and will be 
asked to answer a few questions about eligibility in order to complete the randomization procedure. The website will 
then assign a randomization number to a study participant. The site will select the double-blind device with that 
randomization number and dispense the device to the study participant.  A copy of appropriately executed and 
completed consent and Health Insurance Portability and Accountability Act (HIPAA) authorization documents must be 
on file at the Hines CSPCC, preferably before the patient is randomized or within 24 hours of randomization. 
 
 Period 3 – Follow-up Clinic Visits 
During the Treatment Period, study participants will have follow-up clinic visits scheduled from the day of randomization 
at Weeks 4, 12, 24, 36 and 48.  Prior to presenting to the clinic for each follow-up clinic visit, study participants will be 
contacted and reminded to discontinue NSAID and rescue analgesia 24 hours prior to their scheduled visit, and to bring 
their rescue medication diary and study device to the visit. Patients will have an MRI of the index knee at Weeks 24 and 
48. The MRI should be performed within 2 weeks of the completed Week 24 and 48 visits.  A Fixed-Flexion [8] 
radiograph of the knees will be performed at Week 48. Urine and blood samples will be collected at Weeks 12, 24 and 
48 for OA biomarkers analyses. Treatment compliance data stored in the device will be reviewed and documented by 
the study staff. Study participants with less than 80% [34] or greater than 125% treatment compliance will be counseled 
as appropriate.  Device surveillance will occur at each visit to ensure study devices are operating properly. At each 
follow-up clinic visit study participants will be asked to review and demonstrate the treatment administration 
procedures they follow to administer a treatment using their assigned study device. Participants will not, however, 
actually turn on the device to administer a treatment. The study staff will observe their administration technique and 

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make recommendations as appropriate to ensure proper administration of study treatment.  The importance of 
preventing RFI will also be reviewed. During the Treatment Period study participants will also undergo all other 
assessments and procedures per the Schedule of Assessments including:  WOMAC; patient and investigator global 
evaluations of disease status and response to study treatment; examination of the index knee; collection of urine and 
blood samples for biomarker analyses; ICOAP; weight and vital signs (temperature, sitting blood pressure, sitting pulse 
rate & respiratory rate will only be collected at Weeks 24 & 48); Adverse Events (AEs) assessment and collection when 
applicable; NSAID, other analgesic therapy and rescue medication use, and diary review. Study participants will also be 
reminded to use as little rescue analgesia as possible by reserving its use for the relief of uncontrolled pain.  
  
 Period 3 – Follow-up Telephone Calls 
To ensure study participants are not experiencing difficulty properly administering their daily treatments and are 
compliant with the treatment regimen and preventing RFI, follow-up telephone calls will be conducted at Weeks 18, 30 
and 42, which are the midpoints between clinic visits at weeks 12, 24, 36 and 48. Adverse Events and use of NSAIDs and 
other analgesic therapies will also be assessed during each follow-up telephone call. 
 Period 3 – Additional Safety Follow-up 
Study participants will be monitored per the Schedule of Assessments at each visit for Adverse Events (AEs), which 
includes Adverse Device Events (ADEs) and Serious Adverse Events (SAEs) that occur after informed consent is obtained.     
Safety follow-up monitoring for unresolved SAEs and ADEs will occur every 30 days by phone or during a routine clinic 
visit, until resolved or resolved with sequelae and the participant is clinically stable.   
 End of Study/Early Termination 
The End of Study will occur when the last randomized study participant, has completed the Week 48 visit. In the event a 
study participant prematurely and permanently discontinues treatment (for any reason) during the Treatment Period 3 
all assessments planned for the Week 48 visit per the Schedule of Assessments must be performed no later than 2 
weeks following the date of the last administration of study device.  
 Post End of Treatment Period 4 Safety Follow-up 
Participant’s with unresolved Adverse Device Events (ADEs) and Serious Adverse Events (SAEs) at completion or early 
termination will be contacted by phone 30 days after their completion of, or early termination from, the study. This 
follow-up assessment will be documented in the appropriate study case report form. In addition, each investigator will 
record and report all ADEs and SAEs they are made aware of that occur within 30 days of a participant’s completion of or 

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early termination from the study. Investigators will ensure that participants with unresolved ADEs and SAEs requiring 
follow-up care after study completion or early termination are referred for appropriate follow-up care.  
 Interim Visits 
An Interim visit may occur between scheduled study visit(s) when a study participant, for reasons related to the study, 
requires evaluation for safety or is asked to return to clinic for other reasons, such as to complete a missed study 
procedure or to repeat a study procedure.  
The Interim visit will be numbered from a study participant’s most recent study visit.  For example, an Interim visit 
following study visit 3.0 would be visit 3.1.  If a second visit was necessary before the next scheduled Visit 4, it would be 
visit 3.2 and so forth.  A case report form will be completed for each patient at each Interim visit documenting the 
reason for the interim visit.  All patients will be screened for Adverse Events (AEs) at Interim visits.  
 Compensation of Study Participants 
Compensation for time and effort put forth by study participants will be offered since the study visit schedule and the 
assessments and procedures performed for this research will not be integrated with a patient’s medical care and will 
place special demands on a patient beyond those of usual medical care.  
 
 
 Outcome Measures   
 Co- Primary Outcome Measure 
a.  Cartilage Thickness by MRI   
The study has been designed to determine if PLIUS potentially diminishes articular cartilage thinning by measuring 
cartilage thickness in the tibial femoral joint.  The measure selected to best evaluate this is cartilage thickness of the 
central medial femoral condyle because studies of the OAI data have shown this region to be among those most 
sensitive to change [32, 56] and, due to the anatomical positioning of the device, the most likely to be affected by 
treatment. 
 
The OAI MRI protocol is considered the current standard for MR imaging-based assessment of cartilage OA.  We will use 
a variation of the OAI MRI protocol that eliminates scans of the contralateral knee, eliminates the redundant 3D FLASH 
acquisition, and adds a secondary DESS acquisition.  The secondary DESS acquisition, when averaged with the primary 
acquisition, will improve SNR and accuracy of cartilage segmentation for measurement of cartilage thickness.  Additional 

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physiological data can also be derived from the two DESS acquisitions. Each participating center will be provided with a 
detailed MRI protocol. A summary of the MRI protocol is summarized in the table below: 
 
Order 
Scan 
Time 
1 
3-plane Localizer 
1.0 
2 
Sagittal 3D DESS (water excitation) 
10.6 
3 
Sagittal 3D DESS (water excitation) – modified gradient 
10.6 
4 
Coronal IW TSE FS (3200 29) 
3.4 
5 
Sagittal IW TSE FS (3200 30) 
4.7 
6 
Sagittal T2 Map (120 mm FOV) 
10.6 
 
Total (minutes) 40.9 
 
The protocol as proposed will allow full cartilage morphometry measurements in support of the primary outcome 
measure of cartilage thickness in the central portion of the central medial femoral condyle, in addition to allowing 
assessment of maximal size of bone marrow lesions (a secondary outcome measure). 
 
b. OMERACT-OARSI Response 
Symptom reduction is the second primary aim of the study.  Outcome Measures in Rheumatology Clinical Trials- 
Osteoarthritis Research Society International (OMERACT-OARSI) Response [11] incorporates changes in pain and global 
assessment to define response in OA clinical trials.  A response is classified as: 
1) An improvement in either pain or function of at least 50 percent accompanied by an absolute decrease of at least 20 
mm on the visual-analogue scale for pain or function, OR   
2) The occurrence of at least two of the following: a) a decrease in pain of at least 20 percent accompanied by a 
decrease of at least 10 mm on the visual-analogue scale; b)  an improvement in function of at least 20 percent and a 
decrease of at least 10 mm on the visual-analogue scale; c) an increase in the patient’s global assessment score by at 
least 20 percent accompanied by a decrease of at least 10 mm on the visual-analogue scale 
 
 Secondary Outcome Measures 
Secondary outcome measures were selected in accordance with the preliminary recommendations of the Osteoarthritis 
Research Society task force[82] and include the following: 
 
a. WOMAC Pain, Function Subscales and Total WOMAC Score 

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The WOMAC® Index questionnaire [33] is a tri-dimensional, disease-specific, self-administered, health status measure.  
It probes clinically important, patient-relevant symptoms in the areas of pain, stiffness and physical function in patients 
with osteoarthritis of the hip and/or knee.  The WOMAC has been linguistically validated and is a reliable and responsive 
measure of outcome, and has been used in diverse clinical and interventional environments.  The index can be 
completed in less than five minutes and consists of 24 questions divided into 3 subscales: 1) Pain (5 items): during 
walking, using stairs, in bed, sitting or lying, and standing; 2) Stiffness (2 items): after first waking and later in the day; 
and 3) Physical Function (17 items): stair use, rising from sitting, standing, bending, walking, getting in / out of a car, 
shopping, putting on / taking off socks, rising from bed, lying in bed, getting in / out of bath, sitting, getting on / off 
toilet, heavy household duties, light household duties.  
 
The Total WOMAC Score, along with the Pain and Function subscales, are secondary outcome measures.  The subscale 
results will be obtained to allow for the possibility that PLIUS may differentially impact measures of OA pain and 
function.  Participants will complete this questionnaire at each clinic visit. 
 
b. Patient Global Assessment of Disease Status  
A patient global assessment of disease status on a Visual Analog Scale (VAS) will be measured at each clinic visit. Patients 
will be asked to quantify their disease status on a 100 mm VAS as follows: “Considering all the ways your arthritis of the 
knee affects you, mark ‘X’ on the scale for how well you are doing.”  The scale shows the left hand marker “Very Well”, 
and the right hand marker “Very Poor.” 
 
c. Patient Global Assessment of Response to Therapy  
A patient global assessment of response to therapy will be assessed at each post randomization visit.  The patient will be 
asked to rate the overall response of the arthritis in the index knee to the study intervention on a 100 mm VAS as 
follows: Left hand marker “Excellent-Ideal response, virtually pain free”, right hand marker “None-No good at all-
ineffective.” 
 
d. Investigator Global Assessment of Disease Status  
An investigator global assessment of disease status on a 100 mm VAS will be measured starting at Visit 2 and at each 
clinic visit thereafter as follows: “Make a global assessment of the patient's disease status, with regard to the index 
knee, by marking an ‘X' on the scale below.”, with left hand marker “Very Well” and right hand marker “Very Poor.” 
 
e. Investigator Global Assessment of Response to Therapy 

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An investigator global assessment of response to therapy will be assessed at each post randomization clinic visit.  The 
investigator will evaluate the patient's response to therapy of the index knee on a 100 mm VAS as follows:  Left hand 
marker “Excellent-Best possible anticipated response, considering the severity and stage of disease”, right hand marker 
“None-No response, absence of intervention effect.” 
 
f. Knee Evaluation 
The site investigator will perform a standardized physical examination of both knees at visit 1 and on the index knee only 
at visit 2 and thereafter on all patients as modified from the OAI Protocol.  The examination will include the following 
components: visual assessment of knee alignment, anserine bursa tenderness, patellar quadriceps 
tendonitis/tenderness, crepitus, knee flexion pain, presence of flexion contracture, knee effusions, tibiofemoral joint line 
tenderness, and patellar tenderness.   The objectives of the knee exam are to characterize possible sources of knee pain, 
assess the severity of selected OA-related knee impairments, identify findings that may correlate with abnormalities 
detected by MRI (such as knee effusions with synovial enlargement), and evaluate the prognostic value of standard 
exam findings (i.e., effusion, malalignment and crepitus).  Knee exam components were selected based on data 
demonstrating the potential for reproducibility across examiners [83].  Site investigators will be trained to perform the 
knee examination.  
 
g. Measurement of Intermittent and Constant Osteoarthritis Pain (ICOAP) 
This 11-item tool is designed to assess pain in individuals with knee osteoarthritis taking into account both constant and 
intermittent pain experiences. This tool is designed to be interviewer-administered and takes less than 10 minutes to 
complete. Study participants will be asked to respond to the questionnaire items based on index knee in the past week 
(i.e., past 7-day period). Study participants will respond to all questions for the same joint.  This tool is intended to be 
responsive to change in OA pain over time or with treatment.  
 
h. Use of Rescue Analgesic    
At each clinic visit patients will return the diary recording all rescue medication (e.g., acetaminophen, tramadol) usage, 
including name, date, and amount taken daily.   
 
i. Discontinuation of Study Device Due to Adverse Device Event 
Safety will be measured by assessing the number of patients who require discontinuation of the study device because of 
Adverse Device Events (ADE).   
 
j. Bilateral Fixed-Flexion Knee Radiograph for Joint Space Narrowing 

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At the Day 0 Beginning of Period 3 and the 48-week follow-up visits patients will have a bilateral Fixed-Flexion knee 
radiograph [8] to assess the status of the medial compartment of the knee. 
 
k. Assessment of BML Maximal Size from MRI 
Bone marrow lesions (BMLs), as identified on MRI, are associated with articular pain in OA [52].  BML maximal size will 
be determined from two-dimensional fat saturated fast spin echo MRI sequences acquired in the sagittal plane, as 
previously described in [84].  Measurements will be made at each MRI scan performed at the Day 0 Beginning of Period 
3, and the Week 24 and 48 visits.  
 
l. Biomarkers 
OA Biomarker determination provides a potential independent outcome measure.  The use of biomarkers to follow 
patients with OA is rapidly developing [85] and will soon become a standard assessment tool.  The combination of MRI 
and biomarker results has been shown to be effective in identification of OA progressors [86], and urinary Coll 2-1 levels 
over 1 year were highly predictive of radiographic progression [87].  The ratio of biomarkers associated with cartilage 
degradation and synthesis has been proposed to differentiate OA stages [88], as has area-under-the-curve (AUC) 
analysis of serum and urinary markers in combination with imaging, demographic and baseline data.  Therefore, 
collection of soluble biomarkers, especially in combination with our MRI outcomes, may provide important 
complementary information in the proposed trial.  Urine and blood samples will be collected for analysis of:  1) serum 
cartilage oligomeric matrix (sCOMP); 2) urine C-terminal telopeptide of type II collagen (uCTXII); 3) a peptide of the 
alpha-helical region of type II collagen 108HRGYPGLDG116 (Coll 2-1); and 4) a peptide of 9 amino acids nitrated on its 
tyrosine residue [HRGY(NO2)PGLDG] specific for the type II collagen a1 chain (Coll2-1NO2). 
 
The levels and AUC responses of these metabolites will be analyzed to indicate correspondence with diagnostic severity, 
prognosis and clinical response.   Perhaps most importantly, biomarker response may precede symptomatic, 
radiographic, and even MRI response to PLIUS.   The proposed trial provides a unique opportunity to test the leading 
soluble biomarkers individually and in combination with imaging outcomes in a prospective fashion as measures of 
disease modification or progression, adding importantly to the development of biomarkers for OA. 
 
 Biostatistical Considerations 
 Primary Outcome Measures 
Since structural modification and symptomatic improvement are considered equally important in OA, two co-primary 
outcome measures are proposed for this Phase IIa trial: central medial femoral condyle cartilage thickness (cMFCtTh) 

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change from baseline (Day 0, Beginning of Period 3 part of visit 5) to 48-weeks and OMERACT-OARSI response rate 
(OORR) at 48-weeks. Justification to proceed to a Phase III trial would include demonstrating potential benefit for either 
outcome measure or both.  
 Hypothesized Treatment Effects 
48-Week cMFCtTh Change Treatment Effect 
We will consider PLIUS successful for the cartilage thickness measure if we observe a difference in 48-week cMFCtTh 
change between sham and PLIUS groups of 33 μm, as realized by a 48-week cMFCtTh change of -14 μm (SD 152 μm) in 
the PLIUS group (47 μm -14 μm =33 μm) and a 48-week cMFCtTh change of -47 μm  (SD 152 μm) in the Sham group.  
While the treatment attenuated the progression of cartilage degeneration in Guinea pigs in a laboratory setting[5], the 
effect may be reduced for humans in a clinical trial setting due to less than complete compliance with using the study 
device or other factors that are less likely to be controlled in a human cohort.  We do not expect to reverse progression, 
but would consider a slowing of progression to be a clinically meaningful result.  We assume that the sham group will 
progress naturally. The assumptions of -47 μm  (SD 152 μm) change in the sham group is based on observational data of 
the natural history of knee OA from the OAI [56] of 1-year cartilage thickness change in the medial femoral condyle 
region as measured in the sagittal plane using the DESS MRI pulse sequence, with a sample size (n=104) of 
predominantly KLG 2/3. 
 
48-Week OMERACT-OARSI Response Treatment Effect 
We will consider PLIUS successful for the OMERACT-OARSI measure if there is a 10% greater response rate in the 48-
week OMERACT-OARSI in the PLIUS group compared to the sham group. In a meta-analysis of 11 placebo-controlled 
trials of osteoarthritis the average difference in percentage points between the placebo and treatment groups in terms 
of OMERACT-OARSI responders was 12.7 (95% CI 7.8 – 17.6)[89].  In GAIT, the OMERACT-OARSI response in the placebo 
and celecoxib groups was 56.9% and 67.3%, respectively, a difference of 10.4% [75].   
 
 Ranking and Selection Procedures 
Simon et al. [90] first proposed ranking and selection procedures for Phase II trials.  The procedure and sample size 
calculations for continuous normal and binomial outcomes are detailed in Chapters 2 and 4 respectively of Gibbons et al. 
[91].  A concise summary of the important concepts in this textbook as well as all formulae and tables necessary for 
sample size calculations are presented in a journal article by the same authors and have been provided [92].  The 
purpose of the procedure as applied to this study is to identify whether PLIUS has a high probability of being more 

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effective than sham.  This is accomplished by obtaining sample estimates of the outcome for the PLIUS and sham groups 
and determining whether the PLIUS group outcome is better by the pre-specified margin of difference discussed above. 
The sample sizes accompanying ranking and selection procedures are generally smaller than traditional hypothesis 
testing procedures. They are established to ensure that if PLIUS is better than sham by amount D then there is a high 
probability P it is more effective.  The magnitude of difference in the sample estimates between the two groups will be 
considered in the decision of whether or not to recommend a Phase III trial.  This process will be used for the structural 
modification outcome and separately for the OMERACT-OARSI outcome. 
 
Since the ranking and selection procedures are fundamentally different from traditional hypothesis testing, concepts of 
statistical significance and power have no direct analogue [92].  Furthermore, for the purposes of this Phase II trial, 
demonstrating potential superiority in either or both primary outcomes would be important.  Thus no multiplicity 
adjustments for co-primary outcomes will be done.   
 
 Sample Size Calculation  
cMFCtTh Change at 48-Weeks 
For the 48-week cMFCtTH change outcome measure the minimally clinically significant difference is 33 μm.  With a 
difference in cMFCtTh measurement of 33 μm, σ=152 μm standard deviation, k=2 groups, and P=0.90 probability of 
correct selection we use Table 1 of Gibbons et al. 1979 [92] to obtain τ=1.8124, which we use in the equation to 
estimate the per-group sample size: n=σ2(τ/δ*)2=72 or a total of 144.  With a total sample size of 144 the probability of 
correctly selecting the group with the greatest true cMFCtTh change at 48-weeks is slightly above 0.90 if the true group 
difference in cMFCtTh change at 48-weeks is at least 33 μm.  If the assumed standard deviation of 152 μm turns out to 
be conservative, realized standard deviations of 196 μm and 237 μm will provide us with probabilities of correct 
selection of 0.85 and 0.80 respectively. 
 
OMERACT-OARSI Response Rate Difference 
For the 48-week OMERACT-OARSI response the minimally clinically significant difference is an absolute rate difference of 
10%. We assume a 10% difference in 48-week OORR between sham and PLIUS groups.  Using linear interpolation as 
described in Ranking and Selection (Gibbons el al. 1977)[91] and Table 2 of Gibbons et al. 1979[92], with a total sample 
size of 144 the probability of correctly selecting the group with the greatest OORR is at least 0.885 assuming (δ=10%) 
that the sham group is drawn from a population with a 50% response rate and the PLIUS group is drawn from a 
population with response rate of at least 60% or at most 40% (absolute difference of 10%).  The 50% response rate 
assumption is conservative in that it requires the largest sample size among all possible rates p where 0%<p<100%.  In 

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other words, with a total sample size of 144 we can assert with probability at least 0.885 that the treatment effect will 
be detected if the true difference in OORR is at least 10%. 
 
Adjustment for Withdrawal 
We assume an overall withdrawal rate of 20% for the proposed study and will increase the final sample size from 144 to 
180 to adjust for this.  The strontium renelate OA trial , a 3-year drug intervention trial, reported a withdrawal/exclusion 
rate of 19% [93].    The Doxycycline OA Trial [10] lasted 30 months and  reported withdrawal rate of 29%. In addition to 
the proposed trial being shorter, it is not a drug trial so we expect fewer withdrawals due to adverse effects.  Therefore, 
we expect the overall withdrawal rate to be lower than the above mentioned trials.  144 subjects is a lower bound 
required to obtain the probabilities of correct selection stated above, but because we will be using multiple imputation 
to use some of the withdrawn subjects in the final analysis we will in reality have the ability to detect even smaller 
treatment effects than previously stated. 
 
Under the current assumptions, the sample size for a Phase III trial to detect a 10% difference in OMERACT-OARSI 
response rate with a 5% type-I error and 85% power is estimated to be 886.  A definitive trial for a cartilage thickness 
outcome would require a sample size of 816 patients with the same assumptions. Therefore, we do not believe that a 
definitive pain study could be sufficiently powered without dramatically increasing the sample size.  Most importantly, 
there is at present insufficient evidence regarding PLIUS treatment in humans in a clinical setting to justify the 
expenditure required to support a large Phase III trial. 
 
 Statistical Analysis Plan 
 Baseline Characteristics 
The randomization will be assessed by comparing the distribution of all baseline characteristics including WOMAC Pain, 
WOMAC Function, cMFCtTh, JSW, BML maximal size, KLG, BMI, age, race, marital status, education, duration of 
symptoms, military cohort, and site across the two randomized groups with Wilcoxon rank tests or chi-square tests. 
 
 Analyses of Co-Primary Outcomes 
cMFCtTh & OORR 

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Both 48-week cMFCtTh change and 48-week OORRs will be estimated independently and compared in the PLIUS and 
sham groups.   PLIUS-treated will be identified as the group showing a treatment effect if the estimate in the PLIUS 
group is the larger of the two by the previously specified margins.  
  
Handling of Missing Data 
Statistical analyses will be conducted under intent-to-treat principles; specifically, all patients randomized will be used in 
the final analysis.  We estimate about 10% of patients to have no follow-up cMFCtTh data, and about 5% of patients to 
have less than 12 weeks of OORR data. For these scenarios, group estimates will be obtained using multiple imputation 
techniques as outlined in Molenberghs and Kenward 2007 Chapter 9 [94].   
 
Multiple imputation will be implemented in SAS PROC MI in two stages. The first imputation stage will use the 
Markov chain Monte Carlo method (multivariate normal assumption) to produce an imputed dataset with a monotone 
missing data pattern.  The second stage will use the propensity score (nonparametric assumption) method to produce a 
fully imputed dataset from the monotone-patterned missing data of the first stage.  We will create n=5 imputed 
datasets for analysis and combine the results with PROC MIANALYZE.  The 48-week cMFCtTh change will be imputed 
directly, while the OORR will be calculated from imputed values of 48-week WOMAC pain/stiffness and patient global 
assessment changes.  If a patient elects to receive a total joint replacement of the index knee, their 48-week OORR 
outcome will be analyzed as “non-responder”. 
  
Multiple imputation procedures produce unbiased estimates under the missing at random (MAR) assumption.  MAR 
means that the dropout can depend on observed data but not unobserved data.  Therefore if the chosen imputation 
model is structurally accurate and includes observed data on all covariates that are relevant to both the outcomes and 
dropout, then estimates calculated from the imputed datasets are unbiased.  Since all randomized patients will have 
baseline and follow-up data, we can reasonably assume that the MAR assumption will be met if we include all available 
information in the imputation model.  The model used to impute missing data will include baseline covariates listed in 
Section II.E.1 as well as any WOMAC pain/stiffness, patient global assessment and cMFCtTh outcome data available 
through 48-weeks.  Compliance data will also be included in the imputation model as it will likely be predictive of 
dropout, and therefore important in meeting the MAR assumption [94].  However, to provide additional assurance we 
will perform a sensitivity analysis for the primary outcomes in which we will vary the relationship between the mean of 
the imputed missing outcome data and dropout and compare the resulting estimates to assess the MAR assumption. 
 

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 Analyses of Secondary Outcome Measures and Secondary Analyses of Primary Outcome Measures   
The continuous outcome measures cMFCtTh, Fixed-Flexion view JSW, BML maximal size,  WOMAC Total, WOMAC Pain 
subscale, WOMAC Function subscale, ICOAP total pain score, Patient/Physician Global Assessment of Disease Status and 
Response to therapy will be analyzed as follows:  1.) A mixed-effects linear regression model, as outlined in Hedeker and 
Gibbons Chapter 9 [95], using all available data over the 48-week study period will be fit to determine group differences 
in rate of change over time.†  2.) In addition, mean change from baseline to 24 and 48 weeks and the associated 
standard deviation will be calculated for each group. 95% confidence intervals will be calculated and a t-test (α=0.05) for 
statistical significance will be performed.  3.)  ANCOVA will also be performed (α=0.05) on continuous measures at 48-
weeks, adjusting for baseline measures mentioned in Section II.E.1.  4.) For the WOMAC pain and function subscale 
mean change from baseline to 48-weeks, as well as a 1-way MANCOVA with and without adjusting for baseline 
measures as described in Section II.E.1 will be performed, as will a test for overall group differences (α=0.05).  
   
The categorical outcome measures OORR and standardized physical examination measures will be analyzed as follows:  
1.) A mixed-effects logistic regression model, as outlined in Hedeker and Gibbons 2007 Chapter 4 [95], using all available 
data over the 48-week study period will be fit to determine group differences in response rate over time.†  2.) A Chi-
square (α=0.05) test of the 48-week response rates will be performed.  Proportions, standard deviations, and 95% 
confidence intervals will be reported at each time point.  3.) Logistic regression will also be performed (α=0.05) on the 
above measures at 48-weeks, adjusting for baseline measurements as described in Section II.E.1.  4.) Longitudinal mixed-
effects logistic regression analysis will be performed using outcomes at 4, 8, 12, 24, 36 and 48 weeks as per Hedeker and 
Gibbons 2007, chapter 9 [95] for binary OORR.   
 
Device Usage Compliance 
Descriptive statistics of the percent compliant over time will be calculated as the total number of treatments 
administered (as reported by the device) divided by the number of days the device was available in the patient’s home 
since the last clinical visit X 100.  This measure, collected every visit over 48 weeks, will be treated as continuous data 
and as such point estimates and confidence intervals will be reported for each time interval as well as cumulatively up to 
the end of the study for both groups separately and combined.  A mixed-effects linear regression model, as outlined in 
Hedeker and Gibbons 2007 Chapter 4 [95], using all available data over the randomized 48 week study period will be fit 
to determine group differences in compliance rate over time.†   
† (H0:β(treatment x time interaction)=0) Fixed-effects will include: intercept, time, treatment, treatment x time.  
Random-effects will include: intercept and time.   
 

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 Stratified Analyses 
To account for potential confounding due to intermittent use of NSAIDs or site-to-site variability we propose two 
stratified analyses based on (1) intermittent use of NSAIDs and (2) study site. Intermittent NSAID use will be defined as 
less than 30 days of continuous use at a stable dose. Stratified analyses will be performed for each of the co-primary 
outcomes.  As discussed in Section II.D.3, the concepts of statistical significance and power have no direct analogue; 
subsequently, attempts to formally test heterogeneity (e.g., examining model-based interactions, Cochran-Mantel-
Haenszel) may suffer from low statistical power. Therefore, descriptive statistics (e.g., mean, median, standard deviation 
for continuous outcomes and proportions for categorical outcomes) and plots (e.g., mean for continuous outcomes and 
proportions for categorical outcomes) of the co-primary outcomes by treatment group will be reported for each strata. 
If heterogeneity in primary outcomes is suspected across the strata, further assessment of sources of variability, 
including, sampling factors and study protocol characteristics, will be conducted [99].    
 
 Site Performance Monitoring 
There  are the three participating centers for this study. The Hines VA CSPCC will evaluate each center’s recruitment and 
retention performance monthly.  Other performance problems such as protocol deviations, poor data quality, missing or 
overdue data, and reasons for withdrawal from study will also be tracked.  A study conference call will be held monthly 
for study personnel including Hines VA CSPCC, CSPCRPCC, the Study Chair’s office, and all sites to review study 
recruitment, data quality, and protocol adherence until the last enrolled study participant has completed the follow-up 
visit.  Conference calls may be more frequent if study leadership decides it would be advantageous.  Study sites will be 
put on probation for poor performance, including under-recruitment.  Typically the probationary period is three months, 
at which time the site may be taken off probation, have probation continued, or have funding reduced or stopped, 
depending on its performance during the probationary period.  The Hines CSPCC Director is authorized to make those 
recommendations to the CSR&D Director and seek approval.  Should the site have funding stopped, an alternate site will 
be selected.  
 Data Collection  and Quality Control Measures  
The Hines CSPCC will be responsible for the management and the quality control of the data. DataFax, a clinical trial data 
management system (by Clinical DataFax Systems, Inc.), will be used for data collection and management.  DataFax 
allows for paper data form collection as well as electronic data capture (EDC). After a patient consents to participate in 
the study, the site coordinator will create a patient casebook, which will contain the consent forms, all relevant source 
documents, and any other information pertinent to the study. The Study Coordinators from the sites will complete case 

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report forms (CRFs) on a daily basis and fax them directly to the DataFax computer server, where data images of the 
CRFs are stored as files.  The original forms will be kept in the investigator’s study files. The DataFax system uses an 
optical character recognition (OCR) paradigm to automatically process and store the information from the image as data 
into the study database. The original fax image is also stored. Data management staff at CSPCC will review each CRF by 
comparing the faxed image with the OCR data and ensure that the two match.  If in the future the data capture process 
changes to electronic CRFs, the Study Coordinators will log into the web-based DataFax system and enter study data 
directly, rather than completing and faxing a paper CRF. 
 
Data management staff at CSPCC will review CRFs for protocol adherence data consistency, and add data queries to 
items that fail these checks.  Checks will be performed manually and programmatically.  On a regular basis, data 
management staff will produce site-specific Quality Control reports that list all unresolved data queries.  Data 
management staff will make the reports available to each site and work with the Study Coordinators to help them 
resolve queries.  Queries will be resolved when the appropriate corrections to the CRF are made and data resent, or 
when an explanation is provided that allows for data management staff to resolve the query.  All corrections and 
changes to the data will be reviewed by data management staff.  In addition to the Quality Control report, CSPCC may 
generate and distribute targeted data edit reports on an as needed basis.  
 
 Study site personnel will be provided with an Operations Manual to guide them through the operation and 
management of the study and data collection tools. Study personnel will receive training at a kick-off meeting to assure 
uniformity in patient management, data collection, study procedures, and Good Clinical Practices.  All participating 
investigators, site research coordinators, and CSP coordinating center personnel will be in attendance. The Study Chair’s 
office and the data (Hines VA CSPCC) and pharmacy coordinating (CRPCSPCC) centers will provide the training.  The 
general training will include the study device, patient screening and consent, baseline evaluation, follow-up procedures, 
proper collection and maintenance of data, and ADE/AE/SAE safety reporting. 
  
At this training, the Study Coordinators will be provided with instruction and reference materials on the case report form 
completion. Formal training on case report forms for clinical study management will also be provided.  Training will also 
be held at annual meetings and on an as-needed basis for new study personnel.  
 MRI Acquisition and Quality Control Measures 
The use of a quantitative cartilage morphometry measure in MRI such as the cartilage thickness outcome requires close 
attention to image acquisition consistency, image quality, and analysis consistency.  Standardization of the MRI 
acquisition procedures and analysis across sites is critical to the study’s success, and has been carefully considered in the 

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study design.  We have assembled a team of OA MR imaging experts to develop and execute the MR protocol.  An MR 
image team will perform all MRI analyses (including image segmentation, quantitative measurements, and semi-
quantitative measures).  The study is based on identifying longitudinal changes across the treatment period in individual 
study participants who will be imaged on the same MRI hardware at each imaging point, mitigating problems arising 
from inconsistencies across hardware platforms.  The chance of any such difficulties arising is greatly minimized by the 
fact that each participating site uses a Siemens 3 Tesla MRI system, similar to those extensively validated in the OAI.  
Analyses will be performed at the end of each patient’s participation in the study. The reader will be blinded to time 
point and the participant’s images will be segmented simultaneously to ensure intrasubject consistency.  Test-retest 
reliability will be calculated at each time point by sampling 20 images.  The reader will analyze each image twice, 7 days 
apart, followed by calculation of Pearson’s correlation coefficient.  . Images will be sent electronically to the study’s MR 
imaging team using a VA-approved transmission process.  Site personnel will be trained in image acquisition, MR coil 
usage, patient positioning, QA and procedures at each site.  Th imaging team will also monitor QA phantom and patient 
images monthly from each site to ensure suitability for analysis. 
 
The complete MR imaging protocol will also be validated at each of the recruitment/imaging sites, both on phantoms to 
assess image quality across sites and in vivo, prior to commencement of the study.  Scan operators at each site will be 
trained to exactly follow the prescribed protocol, ensure consistency of study participant positioning in the scanner, and 
visually inspect images as they become available during the scan for image quality and identification of potentially 
problematic aliasing or other image artifacts.  Scan operators at each site will provide the sample MR images monthly 
for evaluation and control by the imaging team.   
 
As mentioned, all participating centers have Siemens 3 Tesla MRI systems.  This platform has been extensively validated 
by the OAI for the MRI-based outcome measures proposed in this study.  The DESS contrast needed for morphologic 
measurements of cartilage is easily and consistently achieved on this platform, and the proposed study will further 
analyze longitudinal changes within individuals, each of whom will be scanned at all time points on a single scanner.  We 
have considered this point very carefully in the study design, and feel the team is well qualified and equipped to ensure 
that each image acquisition site will yield consistent data of sufficient quality for the proposed analysis. 
 
 Bilateral PA View Fixed-Flexion Knee Radiograph Acquisition and Quality Control Measures  
Bilateral Fixed-Flexion knee radiographs will be obtained at the Screening, Day 0 of Period 3 and Week 48 visits. A 
radiographic imaging protocol will be provided to each center.  In summary, the radiographs will be obtained in PA 
projection and 20-30 degrees of flexion with feet externally rotated 10 degrees; right and left knees will be imaged 

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together on 14/17 inch film using a focus-to-film-distance of 72 inches, and knee flexion and root rotation will be 
determined for each study participant using a plexiglass positioning frame (Synaflexer™) provided to each center.  Each 
participating center will need to dedicate at a minimum a primary and a back-up radiology technician who have been 
certified through training to perform the radiographic imaging protocol. Any new personnel will be required to complete 
training before performing radiographs.  
 
Digital radiographic equipment will be used and will need to meet the requirements of the imaging protocol and pass an 
initial evaluation of image quality.  All radiographic equipment used in this study must be maintained on an ongoing 
basis and according to a regular quality assurance program. Each study center will attempt to identify a single x-ray unit 
that can be used for all study radiographs for the duration of the study to avoid an unnecessary source of variability. 
 
Knee radiographs will be sent electronically using a VA-approved transmission process to the study’s Radiology 
Coordinating Center and will be read by one musculoskeletal radiologist blinded to identifying information about the 
study participants.  The central radiologist will also grade X-rays and make the final determination on X-ray eligibility 
based on the Screening Fixed-Flexion X-ray[8]. The central radiologist will record the presence or absence of each of the 
following radiographic features: tibiofemoral joint space narrowing, osteophytes, subchondral bony sclerosis, and valgus 
deformity of greater than 10 degrees. Joint space width will be determined by the central reader blinded to study 
participant identity and order of images.  
 
To avoid drift in radiology scoring, the primary reviewer will annually rescore a random sample (approximately 20) of 
radiographs obtained during the previous year.  Agreement with the previous reading of these teaching radiographs 
must be 85% before new radiographs are scored. Joint space width will be determined by the central reader blinded to 
study participant identity and order of images.   
 
It is expected that the majority of the radiographs will be of acceptable quality. Ideally, radiographs of insufficient 
quality should be identified by the radiology technologist at the time of acquisition and repeated immediately. If any 
problems are detected, however, the Radiology Coordinating Center will notify both the responsible site and the Hines 
CSPCC, suggest possible causes of the problem, and offer potential solutions to assist the site in producing a good quality 
repeat exam.  
 General Guideline for Administration of Study Participant Questionnaires  
All participant questionnaires are self-administered and will be done at the start of each clinic visit before any other 
procedures are performed. Study participants will be instructed to complete all the self-administered questionnaires 

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without any assistance from study staff. Study staff should not assist study participants in answering any questions. 
Study staff will check that the patient has completed all questions before they leave the clinic.  
 Administration of the WOMAC Questionnaire 
The WOMAC index is self-administered and does not require the presence of a study coordinator.  Study staff will read 
instructions for completing the questionnaire to each study participant before they answer any questions at Screening 
Visit 1.0.  Study participants will be instructed to answer WOMAC questions considering their symptoms over the 
preceding 24 hours.  Patients may hear instructions as frequently as needed.  
 Device Quality Assurance 
The CRPCC and study sites will maintain the PLIUS devices supplied provided under the manufacturer’s recommended 
temperature and humidity storage conditions (32o to 122o Fahrenheit and 30% to 75% relative humidity). These 
conditions are achievable at the selected study sites and participants’ homes. The device does not have any user 
serviceable or internally accessible parts, which minimizes the risk for output configuration changes. Importantly, the 
FDA classified the device during its development as a class III device, which required a Pre-Market Approval application 
for marketing clearance. Manufacturers of approved class III devices must establish and follow quality systems to help 
ensure that their products consistently meet applicable requirements and specifications. Consequently, class III devices 
are subject to the strictest level of post-marketing regulatory control and quality. Accordingly, the device supplier 
maintains the device under a full-scope Quality Management System. This system meets the requirements of U.S. 21 
CFR part 820 “Quality System Regulation,” as well as certification to ISO 13485:2003 “Comprehensive Quality System for 
the Design and Manufacture of Medical Devices.” 
 Study Organizational Structure  
The groups charged with centrally monitoring the various aspects of the study will be: the Executive Committee (EC) and 
the independent Data Monitoring Committee (DMC).  For information on the DMC, refer to section III.G. Both 
committees meet at study start-up and annually thereafter.  The EC will also have quarterly conference calls.  The EC and 
DMC may elect to meet more frequently if deemed necessary.   
 
The EC is the management and decision-making group for the operational aspects of the study.  One of its major 
responsibilities is to monitor the performance of the participating medical centers.  The EC considers the need for 
protocol modifications.  The EC also reviews and approves all manuscripts and abstracts emerging from the study.  The 
EC will comprise the original study Planning Committee and will be chaired by the Study Chairman.  

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The Study Group, which consists of all participating investigators and study personnel at the Hines CSPCC and the 
Albuquerque CSPCRPCC, will meet annually to discuss the progress of the study and any problems encountered during 
the conduct of the trial.  Finally, the study protocol and progress must be reviewed annually by the local IRB and the 
local R&D Committees at each participating study site. 
III. Human Subjects 
 Risk to Participants 
 Human Participant Involvement and Characteristics  
Eligible male or female participants will be at least 40 years of age and have clinical and radiographic evidence of 
osteoarthritis of the knee. Participants will need to have a summed pain score of 125mm to 400mm on the index (more 
symptomatic) knee according to the Western Ontario and McMaster Universities Osteoarthritis Index and be in 
American Rheumatism Association Functional Class I, II, or III. Patients will be excluded if they have concurrent medical 
or arthritic conditions that could confound evaluation of the index joint, predominant patellofemoral disease, a history 
of clinically significant trauma or injury to the index knee requiring surgical repair, or coexisting disease that could 
preclude successful completion of the trial.  All participants will be evaluated at the Screening visit to determine whether 
they meet entry criteria and are eligible to participate in the Prerandomziation 4 Week Sham Run-in Period.  Complete 
inclusion and exclusion criteria are contained in Section II.C.5. Subjects not meeting entry criteria will be excluded.  The 
Run-in Period will consist of a follow-up telephone call for treatment compliance, a follow-up clinic visit (may be 
conducted by telephone, refer to section II.C.6 for details), and an End of Run-in clinic visit at weeks 1, 2 and 4, 
respectively, after the Run-in Day 0 visit. Participants will be withdrawn from the Run-in Period if they are less than 60% 
[28] compliant with the daily treatment regimen at week 2 and less than 80% compliant at week 4. Participants who 
successfully complete the Run-in Period will be evaluated at the Day 0 Period 3 part of visit 5 to confirm they are eligible 
to be randomized into the Sham-controlled Treatment Period 3 of the study. Eligible participants will be randomly 
assigned to the PLIUS or sham device. During the Run-in and Treatment Periods participants will apply the study device 
for one treatment period of twenty-minutes each day. Participants will be allowed to take a stable dose of NSAID, and 
acetaminophen or tramadol as rescue analgesia for the relief of uncontrolled knee pain. Participants will be asked to 
avoid NSAID and rescue medication use  during the 24 hours before a clinical evaluation for joint pain.  
 
During the 48-week Sham-controlled Treatment Period 3, participants will be evaluated at Day 0, and at  4, 12, 24, 36 
and 48 weeks, and will receive follow-up phone calls for treatment compliance, other analgesic use, and AE/ADE review 
at 18, 30 and 42 weeks after randomization.  
 
Pregnant women will not be eligible to participate since the safety and effectiveness of the use of this device for 
pregnant women is unknown. Economically and educationally disadvantaged, homeless, employees, and students will 

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not be directly recruited but will not be excluded if they meet entry criteria and wish to participate.  Other vulnerable 
populations, such as children, prisoners, institutionalized individuals or individuals with decisional impairment will not 
be allowed to participate. 
 
Compensation for time and effort expended by study participants will be offered since the study visit schedule and the 
assessments and procedures performed for this research will not be integrated with a participant’s medical care and will 
place special demands on a participant beyond those of usual medical care.. Participants will not receive compensation 
for interim study visit(s). .  
 
 Potential Risk 
Participants will be at minimal risk. The PLIUS device that will be used in this study is a commercially available FDA-
approved device indicated for the non-invasive treatment of non-union bone fractures and for accelerating healing time 
of fresh fractures.  The PLIUS system consists of one main operating unit, gel bottles and strap. The main operating unit 
provides the treatment control circuitry, the primary battery supply, and monitors the operation of the transducer at the 
treatment site. The signal specifications cannot be altered. The sham and PLIUS devices will appear and operate 
similarly, except no ultrasound signal will be generated by the main operating unit of the sham devices. The PLIUS device 
is incapable of producing harmful temperature increases in body tissue.. The ultrasound intensity is comparable to 
diagnostic ultrasound (1 to 50 mW/cm2), such as the intensities used in obstetrical sonogram procedures (fetal 
monitoring). In addition, there is no evidence of non-thermal adverse effects (cavitation).  
 
The device (called the Sonic Accelerated Fracture Healing System or SAFHS) selected for use in this study to deliver the 
low intensity ultrasound dose is FDA-approved for the treatment of non-union bone fractures.  Therefore, the 
Albuquerque Clinical Research Pharmacy Coordinating Center (CRPCC) filed an Investigational Device Exemption (IDE) 
application with the FDA on the basis that promotion of cartilage growth via PLIUS is currently not an approved 
indication for the device in the U.S. The FDA granted approval of the IDE application. The company that manufacturers 
the SAFHS, follows U.S. 21 CFR part 820  Subpart C “Design Controls & Quality System Regulation” and ISO certification 
13485:2003 “Comprehensive Quality System” for the design and manufacture of medical devices. This system ensures 
the device remains safe and effective during its intended use and includes broad-scope activities such as design, 
specification and supplier controls, training, complaints and risk management. The FDA classified the device as “Class III” 
device, which means the device must undergo a pre-market FDA approval and is subject to the strictest level of 
regulatory controls both during development and following market release. 
 

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Participants will administer treatment once daily for 20 minutes. The operation of active, implantable devices, such as 
cardiac pacemakers, may be adversely altered by close exposure to the device.  Participants with a cardiac pacemaker 
will not be eligible to participate in the study. There are no other known contraindications to the use of the PLIUS device 
and no device related adverse reactions or medical complications related to the use of the PLIUS device have been 
reported during non-union bone fracture clinical trials. Some participants have experienced mild skin irritation caused by 
skin sensitivity to the coupling gel. Resolution can be obtained by a change of coupling medium to mineral oil or glycerin.   
While participants are expected to be at minimal risk in this trial and the safety of the use of this device has been 
demonstrated in non-union bone fracture clinical trials in subjects followed over a period of 6.5 years, participants in 
this trial will be carefully monitored for Adverse Device Events (ADEs). 
 
There is a small risk that by participating in the study the participant’s confidentiality may be breached or the 
participant’s protected health information, whether electronic or paper based, may be stolen.  There may be risks that 
are currently unforeseeable. 
 
 Sources of Materials 
Research material will be obtained from participants’ existing medical records and data collected from assessments, 
questionnaires, knee MRIs and X-rays, urine and blood specimens, and all other procedures performed as described in 
Section II.C of this application. Information from existing medical records and data collected during this trial will be 
obtained specifically for research purposes.   
 
We plan to collect urine and blood samples from all participants for OA biomarkers analyses. OA biomarkers have 
potential to assist in the clinical management of patients with OA for diagnostic, prognostic, and response-to-treatment 
assessment purposes. Planned biomarker analyses in this research are: cartilage oligomeric matrix protein (COMP), 
collagen fragments in urine (uCTX-II), a peptide of the alpha-helical region of type II collagen 108HRGYPGLDG116 (Coll 2-
1), and a peptide of 9 amino acids nitrated on its tyrosine residue [HRGY(NO2)PGLDG] specific for the type II collagen a1 
chain (Coll2-1NO2).  
 
 Therapeutic Risks 
No therapeutic risks are associated with this investigational device intervention as all procedures for this trial will be 
performed for the purpose of this research project and would not normally be performed as part of a person’s routine 
medical care. 

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 Research Risk 
Several interventions will be performed for the purposes of this research project and all interventions are necessary to 
achieve the aims of the research. There are no alternative procedures available that will provide the needed data. 
Participants would not normally have these interventions as part of their routine medical care. Participants who 
complete all visits in this trial will have these research interventions: MRI of index knee at the Day 0 Period 3, Week 24 
and Week 48 visits; Fixed-Flexion x-ray of the knees at the Screening visit, Day 0 Period 3 and Week 48 visits.   
 
The risk associated with an MRI of the knee is very minimal. MRI has no known long-term risks. The MRI machine is a 
large magnet and could move metal-containing objects in the body.  As part of the screening process and prior to 
enrollment in the trial, participants will be screened for contraindications to MRI scanning and will be asked questions 
about their medical history. If a participant has a piece of metal in their body, such as a fragment in the eye, aneurysm 
clips, ear implants, spinal nerve stimulators, or a pacemaker, they will not be able to receive an MRI.  Participants with 
known contraindications to MRI scanning will not be eligible to participating in the trial. Ear protection will be provided 
to participants since the MRI produces repetitive tapping noises resulting from normal operation of the scanner.  
 
Some participants may be claustrophobic and experience fear when confined to a small space. Participants will be asked 
if they have had an MRI and were given pre-medication to help with anxiety or claustrophobia. Arrangements for pre-
medication will be made and administered by the radiologist prior to the MRI.  
 
This research study involves X-rays of the knees and exposure to ionizing radiation. The risk of these procedures is small. 
The amount of radiation exposure participants will receive from these procedures is very low. 
 
Participants will have their blood drawn for collection of biomarker samples at Day 0 Period 3, and at 12, 24 and 48 
weeks after randomization. Needle punctures carry some risk such as fainting, bleeding, bruising, discomfort, dizziness, 
infection and pain at the point of puncture site.  
 
 Adequacy of Protection Risk 
 Recruitment and informed consent 
Participant recruitment will occur at each participating center.  The recruitment period will last approximately 23-
months. Potential participants will be identified and recruited using a number of strategies as described in Section II.C.2. 
Participants identified through the aforementioned recruitment methods will have their medical history pre-screened by 
study staff to determine potential eligibility per inclusion and exclusion criteria.  Potentially eligible participants will be 

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informed of the study by their primary or specialty care providers and instructed on how to contact study staff if 
interested in participating.  Interested individuals will be contacted by phone to discuss the research project, what 
participation will involve, and to review their eligibility to participate in the study.  If potentially eligible based on the 
phone screen, the patient will be scheduled for a Screening visit at the clinic and will be mailed a consent document. 
Patients will be asked to read the consent document prior to appearing for their Screening visit, to discuss the study 
with family or friends if they wish, and to take time to decide whether they want to participate. They will also be 
encouraged to contact study staff if they have any questions.  
 
The investigators or their delegated research staff will be responsible for obtaining consent. The purpose of the consent 
process will be explained to the patient at the onset of the Screening visit and the consent document will serve as the 
basis for a meaningful exchange between the research staff and the patient. Consent will be obtained before any study-
related procedure is performed. The patient will have a copy of the consent document to follow and reference during 
the consent process.  All elements of the consent document will be reviewed with the patient and they will be 
encouraged to ask questions. They will also be asked questions to verbalize understanding of the risks, why the study is 
being done and what it will involve. To minimize the possibility of coercion, patients will be encouraged to take as much 
time as they like to consider participating and will be told no matter what they decide to do their decision will not affect 
their medical care.  
 
A signed copy of the consent document will be provided to the participant and the original signed consent document will 
be retained in the study records. The original consent form will be kept in locked file cabinets within locked offices and 
referenced by participant ID number.  
 
 Protection Against Risk  
The risks of the investigational intervention, procedures and participation in this trial are minimal. Nonetheless, each 
participant’s health and well-being will be the primary concern of the research team. Participants will be monitored for 
any adverse experiences and unanticipated problems involving risks to participants, whether or not considered related 
to a study procedure.  All participants presenting to the VA medical center will be provided treatment when appropriate 
for all AEs, UADEs and SAEs that occur during the study period.  Participant privacy and confidentiality and data security 
risks are minimal in this research project. Protections and precautions will be taken to ensure risks are minimized. The 
following precautions will be used to ensure privacy is maintained: research procedures will be conducted in private 
clinic rooms or appropriate ancillary care facilities; only those authorized to conduct study-related procedures will be 
permitted in patient care areas unless express permission is given from the participant for other persons to be present; 

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the study will be discussed with participants individually instead of in front of a group; and the collection of information 
about participants will be limited to the amount necessary to achieve the aims of the research so that no unneeded 
information is being collected. 
 
Handling and storage of study data will adhere to current VA policies.  The database will not contain information that 
can directly identify the study participant (such as name, address, etc.); however, it will not be a completely de-
identified database since age and study visit dates will be collected.  The Hines CSPCC requires that a copy of the signed 
consent form, HIPAA waiver, and a participant contact sheet be on file at CSPCC.  The consent form is required by CSP 
policy in order for the coordinating center to independently certify that all study participants have provided written 
informed consent.  The consent process is needed in order for participants to be screened and participate in the study.  
Because Hines VA CSPCC is the final data repository for the study, participant contact sheets are collected in the event 
study participants need to be contacted (such as safety notices) after study sites have completed the study.  The CSPCC 
is the final repository of all study data and the only component that remains open and funded after the study ends.  In 
the event that a study participant should need to be contacted well after the end of the study, his or her name, address, 
and phone number will be vital to the CSP center.  Each participant’s SSN is also necessary in the event that the 
Coordinating Center should have to access the Beneficiary Identification Records Locator (BIRLS) files to locate a 
participant.  The electronic information from the contact sheets is stored separately from the main study database in a 
password-protected file.  Consent forms and contact form information will be uploaded or faxed into the electronic 
system as password-protected files. 
 
Two mirror-images of the study database will be housed on separate servers located at different secure VA facilities that 
support round-the-clock web services and monitoring within a secure VA environment in order to provide an optimal 
infrastructure for the protection of sensitive information.  Data entered by the site coordinator is sent to the production 
server which is then copied almost continuously to a back-up server at a different location.  The clinical database with all 
research data will be housed behind the VA firewall on VA-owned and maintained servers.  Accordingly, the information 
housed within the DataFax system will be afforded the same level of security as all forms of VA protected and/or highly 
sensitive information.  Additionally, the system will be monitored by the Hines VA CSPCC Quality Assurance and 
Information Technology teams to ensure that all applicable VA regulations and directives are strictly followed. 
 
Backup copies of the database will be transferred behind the VA firewall to the Hines CSPCC on a frequent basis 
depending on the study need (at least once per day).  These backup copies will be transferred and stored across secure 
connections according to VA regulations and Hines CSPCC operating procedures.  Periodic off-site back-ups will be made 
as part of a comprehensive disaster recovery plan.  The Data Management group will ensure that backup media are 

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stored in compliance with all federal and VA regulations on the storage of potentially sensitive information.  The Data 
Management group will also ensure that all backup media is encrypted in compliance with the current best practices 
established and approved by the Center Director(s).  Encrypted backup media will be stored in a physically secure 
location with access restricted to essential personnel.  Access to back-ups may be at the discretion of the Data 
Management group. 
 
Access to the study data is heavily restricted to individuals with CSP approval.  Individuals must be properly credentialed 
research staffs who are compliant with VA security trainings (i.e., Research Data Security, HIPAA and VA Privacy Training, 
Information Security Awareness, and Good Clinical Practices).  In addition, research data will be stored on VA secure 
servers with restricted permissions for copying and exporting data.  Only properly approved coordinating center 
personnel will have the ability to copy and export data.  These individuals have received training on the local standard 
operating procedures (SOP) governing their permissions and will not access or export data without approval from the 
Hines CSPCC Center Director.  Furthermore, the permissions of the electronic systems are structured such that individual 
sites can only see the data for their study participants.  They cannot see or access the data for another clinical site or for 
another participant. 
 
Access to protected health information (PHI) will be heavily restricted to individuals approved by CSP to have access to 
the data.   
 
At the local clinical sites, the staff positions that have access to PHI include: Site Principal Investigator, Site Co-
Investigator, and Study Coordinators.  Individuals in these positions will be able to access all forms of PHI. 
 
At the Hines CSPCC, the staff positions that have access to PHI include: Project Manager, Biostatistician, Data 
Management Programmer, Quality Assurance Officer, Quality Assurance Nurse Specialist, Data Coordinator and 
Statistical Programmer.  Individuals in these positions will be able to access all forms of PHI. 
 
At the CSPCRPCC, the staff positions that have access to PHI include: clinical monitors and Adverse Event Specialist 
(Regulatory Affairs and Safety Officer).  Individuals in these positions will be able to access de-identified forms of PHI. 
So that research staff understand and are able to implement risk minimizing measures, VA and VA-WOC employees will 
be required to completed regular training such as Data and Information Security, Privacy, HIPAA, Human Subjects 
Research Protection, and Good Clinical Practices. 
 

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 Potential Benefits of Research to Participants 
 The risks to participants are minimal and are reasonable compared to the potential benefits of the knowledge that will 
be gained from this research project.  We hope PLIUS demonstrates potential as a therapeutic modality for knee OA in 
humans.  While there may be no direct benefit to participants taking part in this study, we hope that our improved 
understanding of the biology of OA gained through this research will benefit others in the future. 
  
 Importance of Knowledge to be Gained 
The importance of knowledge to be gained is significant and the minimal risks to participants are reasonable in relation 
to the importance of the knowledge that reasonably may be expected to result.  Knee OA is a leading cause of disability 
[1] in the US and is seen in even higher frequency among individuals with military service [2].  Current therapies are 
modestly effective at best and are primarily directed at pain relief. There are no FDA-approved interventions that have 
been shown to impact the progression of OA[3].  
 
In this protocol we have presented consistent and striking evidence for the salutary effects of PLIUS on chondrocyte 
metabolism and on cartilage, including its effect to forestall the onset of OA and to limit its progression after 
establishment of disease in an animal model of human idiopathic OA.  This research project is an exploratory evaluation 
of PLIUS as a potential therapeutic modality for knee OA in humans.  If PLIUS demonstrates potential as a disease and 
symptom modifying intervention for idiopathic OA of the knee, it would remarkably advance the understanding of the 
biology of OA and serve as strong endorsement for a pivotal trial designed to rigorously investigate PLIUS as a treatment 
that could delay the progression of a debilitating and ultimately incapacitating disease. 
  
 Data Monitoring and Reporting  
 Data Monitoring 
The Quality Assurance section at Hines CSPCC will assist in centralized monitoring and remote-based methods of 
monitoring of study sites to ensure compliance with the protocol, Good Clinical Practice (ICH E6) and VA 
regulations.   Monitoring may include, but not be limited to; the informed consent process, regulatory documentation 
review, source document verification, and safety reporting.  Additionally, study and site metrics will be used as triggered 
monitoring actions, as appropriate.   Site performance concerns may result in on-site visits by the CSP S.M.A.R.T. group 
and/or the QA Nurse Specialist to evaluate the sites’ need for additional training to remedy any compliance concerns. 

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SMART (Site Monitoring, Auditing, and Resource Team), a division of the CSP Clinical Research Pharmacy Coordinating 
Center (CSPCRPCC), may conduct full audits of participating sites if requested by the sponsor (VA CSRD). 
 
VA study personnel will adhere to all ORD policies on human subjects’ protections by fully completing approved required 
Good Clinical Practices (GCP) training per VHA Handbook 1200.05.  Additionally, completed training certificates will be 
provided by study personnel to the Hines Coordinating Center to maintain on file throughout the study.   
 
The Data Monitoring Committee (DMC) will also review the progress of the study, including patient recruitment, 
participant compliance,completeness of follow-up, data quality, protocol deviations, and safety. For more information 
on the DMC, refer to section III.G.  
 Data Reporting 
The Study Co-Chairs, the Site Investigators and Site Coordinators will receive biannual reports regarding the quality and 
quantity of data submitted to the CSPCC. Other quality control measures include periodic reports containing participant 
recruitment information and relevant medical data for review by the Study Co-Chairs.  The CSPCC will also prepare 
summary reports for the Study Co-Chairs, the Data Monitoring Committee, and other monitoring groups to track 
progress, and conduct final analyses of the study data. 
Study reports will be generated using DataFax, SAS, and other tools (e.g., Microsoft Excel and Access).  SAS and other 
statistical software packages will be used to conduct data analysis for the study.  The CSPCC is using SAS Version 9.3 and 
will upgrade to newer versions once they are purchased and validated. 
 
 Safety Monitoring and Reporting 
 Safety Monitoring 
Study participants will be monitored at each visit for Adverse Events (AEs). Adverse Events include Adverse Device 
Events (ADEs), non-device related Adverse Events and Serious Adverse Events (SAEs). Adverse Device Events are further 
categorized into Adverse Reactions (ARs), Suspected Adverse Reactions (SARs), and Unanticipated Adverse Device 
Events (UADEs). Monitoring and collection of AEs will begin at the time  informed consent is obtained.  All AEs (including 
SAEs) regardless of their relationship to the study and ADEs  will be collected and recorded on the appropriate study 
event forms.  New AEs and ADEs will be assessed at follow-up and interim visits. In addition, all unresolved ADEs and 
SAEs will be followed at a minimum every 30 days until 1) resolution of the event or 2) the event resolves with sequalea 
and the participant is clinically stable. The every-30 day follow up will be conducted by phone or routine clinic visit, 
whichever permits adherence to an every-30-day follow up schedule. Participants with unresolved ADEs or SAEs at study 

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completion or early termination will be contacted by phone 30 days after their termination from, or completion of, the 
study. Investigators will ensure that participants with unresolved ADEs or SAEs requiring follow-up care 30 days past 
study completion or early termination are referred for appropriate follow-up care. Each investigator will record and 
report all SAEs they are made aware of that occur within 30 days of a participant’s completion of or early termination 
from the study.  
 
The study intervention is the PLIUS ultrasound device as described in this proposal. The operation of active, implantable 
devices, such as cardiac pacemakers, may be adversely altered by close exposure to the device.  Participants with a 
cardiac pacemaker will not be eligible to participate in the study. There are no other known contraindications to the use 
of the PLIUS device and no device related adverse reactions or medical complications related to the use of the PLIUS 
device have been reported during non-union bone fracture clinical trials. Based on this we do not anticipate an 
investigator would withdraw a study participant due to an Adverse Device Event.  
 Role of the Local Site Investigator in Safety Monitoring 
The local site investigator will be responsible for following these safety reporting requirements: 
a. Reviewing the accuracy and completeness of all reported AEs, UADEs and SAEs prior to submission in the 
study database 
b. Complying with study/IRB policies as well as the Operations Manual  for the reporting of AEs, UADEs, SAEs, 
Events of Special Concerns and protocol deviations 
c. Reporting to the IRB safety issues reported to the site by the sponsor. 
d. Reporting UADEs, unanticipated SAEs and unanticipated problems to the local IRB 
e. Grading AEs in terms of their severity and attributability using Operations Manual definitions 
f. Closely monitoring study participants for any new AEs  
g. Following unresolved ADEs and SAEs every 30 days until resolution or resolution with sequaleae 
h. Transitioning participants whose ADEs or SAEs have not resolved within 30 days of study completion or early 
termination to local clinical care 
i. 
Promptly terminating study treatment in female participants who would become pregnant during the trial. 
 Definitions   
An Adverse Event (AE) is any untoward physical or psychological occurrence in a human subject participating in research.  
An AE can be any unfavorable and unintended event, including an abnormal laboratory finding, symptom, or disease 
associated with the research or the use of a medical investigational test article.  An AE does not necessarily have to have 
a causal relationship with the research (Ref: VHA Handbook 1200.05, May 2, 1012).  

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In this trial, an Adverse Reaction (AR) is defined as an AE for which there is evidence the AE is clearly attributed to the 
device. A Suspected Adverse Reaction (SAR) is defined as an AE for which there is evidence to suggest the AE is possibly 
attributed to use of the device.  
 
Treatment-emergent Adverse Events will be any event not present prior to the initiation of the study treatment, or any 
event already present that worsens in either intensity or frequency following exposure to the study treatment.  
 
The severity of each Adverse Event will be graded according to the following scale:  
• 
Mild (Grade 1): Asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not 
indicated. 
• 
Moderate (Grade 2): Minimal, local, or noninvasive intervention (e.g., packing, cautery) indicated; limiting age-
appropriate instrumental activities of daily living (ADL). 
• 
Severe (Grade 3): Medically significant but not immediately life-threatening; hospitalization or prolongation of 
hospitalization may be indicated; disabling; limiting self-care ADL. 
• 
Life Threatening (Grade 4): In view of investigator or sponsor, participant is at an immediate risk of death; 
urgent intervention indicated. 
• 
Death Related (Grade 5) 
 
An Unanticipated Adverse Device Effect (UADE) means any serious adverse effect on health or safety or any life-
threatening problem or death caused by, or associated with, a device, if that effect, problem or death was not previously 
identified in nature, severity, or degree of incidence in the investigational plan or application, or any other or any other 
unanticipated serious problem associated with a device that relates to the rights, safety, or welfare of subjects.  
 
All AEs/UADEs with a reasonable causal relationship to the investigative test article should be considered at least 
“reasonably attributable.”  A definite relationship does not need to be established.   
 
For the purpose of this study Serious Adverse Events (SAE) are a subset of AEs that: 
a. Results in death;  
b. Are life-threatening;  
c. Requires inpatient hospitalization or prolongation of existing hospitalization;  
d. Results in persistent or significant incapacity or substantial disruption of the ability to conduct normal 
functions;  

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e. Results in a congenital anomaly/birth defect.  
f. Note: Important medical events that may not result in death, be life-threatening, or require hospitalization 
may be considered serious when, based upon appropriate medical judgment, they may jeopardize the 
patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in 
the SAE definition. 
Events of Special Concerns include 1) suicide, 2) death due to an unusual circumstance, 3) death, great bodily harm, or 
significant property damage by a study participant, and 4) medical errors. The following table summarizes reporting 
requirements for site investigators. 
 AE, SAE, ADE, and UADE Monitoring and Reporting Requirements  
 
Event Type 
Every 30-day Follow-
up 
Expedited Reporting 
to Sponsor 
Review of Study CRF 
Local IRB Reporting 
Adverse Reactions 
(ARs) 
Yes 
No 
Yes 
No 
Suspected Adverse 
Reactions (SAR) 
Yes 
No 
Yes 
No 
Unanticipated 
Adverse Device 
Effects (UADEs)† 
Yes 
Yes, 72 hours 
Yes 
Yes 
Non-Device-Related 
Adverse Events 
No 
No 
Yes 
No 
Events of Special 
Concerns 
Sponsor may direct 
follow up frequency 
when applicable 
Yes, immediately 
Yes 
Yes 
Serious Adverse 
Events (SAEs)† 
Yes 
Yes, 72 hours 
Yes 
Yes, if unanticipated 
†Note: UADEs and SAEs will be followed up to 30 days past study completion or early termination  
 
UADEs and SAEs require expedited reporting by site investigators.  Expedited reporting is defined as the completion and 
submission of the UADE or SAE form to the study’s SharePoint website within 72 clock-hours of the site investigator or 
site coordinator becoming aware of either an SAE or UADE.   
 
The site investigator is responsible for grading AEs/SAEs/ADEs/UADEs’ severity, determining attributability to the study 
intervention, and determining whether any event is anticipated.  The CSPCRPCC is responsible for evaluating all UADE 
and SAE reports in a timely manner.  In addition, a Clinical Events Committee (CEC) will be formed to adjudicate UADEs 
and SAEs that occur in the study.  The committee involves the study Chair, a physician from the CSPCC, a GCP and safety 

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monitor from the CSPCC, and the AE Specialist from the CSPCRPCC.  This committee is independent from the site 
investigators. 
 
UADEs and SAEs that are related to the investigative treatment and determined to be unexpected will be reported to 
the Food and Drug Administration (FDA), the CSPCC Director, and the site investigators after review by the Study Chair, 
the CSPCC Director, and the AE Specialist.  Site investigators should follow local regulations to inform their IRB of Record. 
All participants presenting to the VA medical center will be provided treatment for all AEs/SAEs/ADEs that occur during 
the study period.   
 Reporting of Events of Special Concerns 
Site investigators will generate a written report to describe Events of Special Concerns immediately upon becoming 
aware of such events. The report will be promptly forwarded to the CEC for review and notification of CSP Central Office.  
Events of Special Concerns are defined in section III.F.1.b. 
 Data Monitoring Committee  
This study will assemble a Data Monitoring Committee (DMC) who will review the progress of the study, including 
patient recruitment, participant compliance,completeness of follow-up, data quality, protocol deviations, and safety.  
The DMC may also choose to implement a formal interim monitoring rule to monitor efficacy.  The DMC will review any 
protocol modifications recommended by the Executive Committee. The DMC will establish criteria for study termination 
and make recommendations to the Director, Clinical Sciences Research and Development (CSR&D), through the Director, 
Hines VA CSPCC, as to whether the study should continue or be terminated.  Should the DMC recommend study 
termination, the CSPCRPCC will promptly notify the FDA. Interim unblinded progress reports will be provided to the DMC 
by the study biostatistician.  The DMC shall be composed of four experts in the study’s subject matter fields, specifically, 
rheumatic diseases, clinical trials, biostatistics, and ethics. 
 Reporting AEs, UADEs and SAEs to the Data Monitoring Committee (DMC)   
The Hines CSPCC will generate tabulations of all AEs, UADEs and SAEs for the DMC on an annual basis or on a more 
frequent schedule if requested by the DMC. 
 
 Females and Pregnancy Testing 
Females of childbearing potential who are breastfeeding, pregnant or plan to become pregnant during the study or 
within 3 months following the last study treatment will be excluded from participating in the study for safety reasons. 

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Women who are surgically sterile or have been post-menopausal for at least 2 years are not considered to be of 
childbearing potential. Females of childbearing potential will have a urine pregnancy test performed at the Screening 
visit 1.0 and the Day 0, Beginning of Period 3 part of visit 5.0, and must be willing to use a reliable form of medically 
acceptable contraception throughout their participation in the study.  
 Women, Minorities and Children 
Male and female individuals of all races will be recruited and no minorities will be excluded. To maximize the 
homogeneity of the participant group in terms of speech/language characteristics, participants must have spoken 
English as their primary language.  Children will be excluded from participating. 
 
 
 
 
 

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