Prospective, Randomized, Single Blind Clinical Trial to Investigate the Impact of Autologous Bone Marrow Concentrate in Knee Osteochondral Allograft Transplantation
Phase: NA
Conditions: Cartilage Injury
Sponsor: Rush University Medical Center

Prospective, Randomized, Single Blind Clinical Trial to Investigate the Impact
of Autologous Bone Marrow Concentrate in Knee Osteochondral Allograft
Transplantation
Document Date 01/31/2021
Study Protocol:

Current Version: 8/3/19
Prospective, Randomized, Single Blind Clinical Trial to Investigate the Impact 
of Autologous Bone Marrow Concentrate in Knee Osteochondral Allograft 
Transplantation
Principal Investigators: Brian J. Cole, MD, MBA, and Adam B. Yanke, MD
Co-Investigators: Susan Chubinskaya, PhD
Study Coordinator: Mukesh Ahuja
Abstract:
Articular cartilage injuries in the knee continue to grow in number as detection and 
treatment options have advanced. Treatment options, including microfracture, 
autologous chondrocyte implantation, osteochondral grafting and meniscus 
transplantation can hopefully deter the progression of degeneration and have 
promise to function as disease modifying solutions.  Osteochondral allograft 
transplantation (OCA) has emerged as a preferred method of treating large focal 
chondral defects as it structurally replaces the cartilage and often involved 
subchondral bone with native hyaline cartilage and bone. The results of OCA are 
successful with greater than 85% survival at 5-year follow-up
1. Failure can occur 
due to a lack of boney integration or low chondrocyte viability2,3. Therefore, 
anything to enhance the graft augmentation process may be useful in preventing 
failure. The purpose of this prospective, randomized study is to determine the effect 
of bone marrow aspirate concentrate (BMAC) containing bone marrow derived 
mesenchymal stem cells on improving graft incorporation and preventing failure. 
The effect of BMAC on the graft and intraarticular knee environment will be 
evaluated using 3 techniques: CT imaging, synovial fluid cytokine analysis, and 
serum biomarker analysis.  Information learned from this study can be used to 
biochemically compare treatment response and to assess emerging therapeutic 
options that may positively alter the biochemical environment in patients 
undergoing osteochondral allograft transplantation.  
Statement of Clinical Relevance:
ORA: 17121805-IRB01   Date IRB Approved: 3/13/2019   Amendment Date: 8/22/2019
Treatment of articular cartilage injuries in the knee is continuing to evolve as we 
recognize systemic and local factors that promote growth of cartilage. However, 
given the varying nature of these injuries, with significant differences in depth, size, 
and location, determination of the best treatment can be a difficult decision for the 
surgeon. Based on these factors, treating physicians use varying techniques 
including microfracture, autologous chondrocyte implantation, and osteochondral 
grafts as treatments for cartilage defect.  These treatment options all aim to restore 
cartilage and healing. Osteochondral allograft transplantation involves the 
implantation of an allograft plug containing native hyaline cartilage and underlying 
bone. The cartilage survival relies on chondrocyte viability and, over time, bone 
integration occurs by a process of creeping substitution. Failure can occur either to a 
lack of chondrocyte viability or failed bone incorporation. Anything to augment 
these two factors, may help prevent failure and improve patient outcomes. 

Current Version: 8/3/19
Additionally, the biochemical and inflammatory environment has a major effect on 
the development, maturation, and healing in cartilage repair. Our goal is to establish 
if mesenchymal stem cell augmentation improves graft incorporation and to analyze 
the cytokine environment of the joint after OCA with and without intraarticular 
BMAC injection. Information learned from this study can be used to biochemically 
compare treatment response and to assess emerging therapeutic options that may 
positively alter the biochemical environment in patients who suffer from articular 
cartilage disorders.  
Specific Aims:
1. To evaluate the effects of BMAC on graft incorporation in patients 
undergoing osteochondral allograft transplantation in the knee
2. To measure the amount of cytokines and cartilage biomarkers in the synovial 
fluid of patients undergoing osteochondral allograft transplantation with and 
without BMAC augmentation at different time points, including BMP-2, BMP-
7, FGF-2, FGF-18, IGF-1, PDGF, TGF-β, IL-1a, IL-1β, IL-4, IL-6, IL-8, IL-10, TNF-
α, Bcl-2, Bcl-XL, iNOS, SOX9, SMADs, EGF, IFN, MIP, MCP, TIMP-1, OPN, MMP-
1, MMP-3, VEGF, PDGF, and RANTES .
3. To determine changes in serum biomarkers of cartilage turnover over the 
course of osteochondral allograft transplantation with and without BMAC 
augmentation
4. To evaluate patient reported outcomes after osteochondral allograft 
transplantation with and without BMAC augmentation
5. To correlate changes in inflammatory and cartilage biomarkers to clinical 
patient reported outcomes
The goal of this study is to investigate the effect of BMAC augmentation on graft 
incorporation after osteochondral allograft transplantation in the knee.  
Additionally, using serum analysis of a cartilage biomarker, COMP, we can 
determine if this marker can be used in the future to assess patients following the 
treatment of articular cartilage disease.  We hypothesize that BMAC augmentation 
will improve graft incorporation
Research Strategy:
Significance:
Procedures for articular cartilage lesions in the knee continue to grow in number.  
While the true incidence of full-thickness articular cartilage lesions is difficult to 
define, in athletes, studies have reported an average incidence of 14%4.  Impaction 
injuries or significant injuries increasing the force in the joint may cause injury to 
the cartilage, leading to gross disruption or injury at the cellular level that can later 
lead to macroscopic change
5
ORA: 17121805-IRB01   Date IRB Approved: 3/13/2019   Amendment Date: 8/22/2019
.  For some patients, chondral injuries cause significant 
pain, especially with weight bearing, and swelling.  
Chondral injuries vary widely in their depth, size, and location with several 
treatment options available based upon various evidence-based treatment 

Current Version: 8/3/19
algorithms.  Osteochondral allograft transplantation is typically performed for large, 
symptomatic chondral defects. The success of this procedure is dependent on 
integration of the graft into the recipient femur, which may be able to be augmented 
by the use of autologous bone marrow derived stem cells. Additionally, little is 
known about the biochemical mediators that influence cartilage healing.  The 
biochemical and inflammatory milleu has a major effect on the development, 
maturation, healing, and chemical environment in the joints of patients undergoing 
cartilage repair. Chrondrocytes and synoviocytes in the joint both secrete 
substances and cytokines, which influence each other and affect the overall joint 
environment. 
Innovation:
Bone marrow aspirate concentrate is known to contain osteoprogenitor cells and 
osteoconductive proteins which may augment bone healing. Previous animal studies 
have shown that BMAC accelerates bone integration as an adjuvant therapy6,7. 
Additionally, a recent study of a retrospective cohort used radiographs to evaluate 
graft incorporation and found a significant improvement at 3 months post-
operatively OCA patients who received a BMAC injection8. Augmentation of OCA 
with BAMC may help prevent failure due to a lack of graft incorporation.
Numerous studies have analyzed synovial fluid after anterior cruciate ligament 
(ACL) injuries focusing on the cytokine environment after injury and following 
surgery
9-11. Synovial fluid has also been analyzed during diagnostic arthroscopy 
before any cartilage intervention. Vasara et al. found an increase in matrix 
metalloproteinase-3 (MMP-3) and insulin like growth factor-I (IGF-I) associated 
with cartilage lesions12. Within 1 year after ACI, levels of MMP-3 and IGF-I remained 
elevated, even though the lesions were filled with repair tissue12. Cuellar et al. 
similarly evaluated the correlation of synovial fluid markers with symptoms in 
patients undergoing knee arthroscopy13.  They found a strong correlation between 
MCP-1 and IL-6 in patients with severe cartilage lesions, whereas IL-1Ra was 
inversely related
13.  Schneider et al. assessed the presence of molecular markers 
following ACI surgery including pyridinium crosslink (PY), deoxypyridinolin (DPD), 
n-telopeptide (NTX) from type I collage, MMP-1, MMP-3, TIMP-1, PICP, 
proteoglycan, and YKL-40 at 4 times points14.  They found that DPD continuously 
increase after surgery, however all molecular markers for cartilage degradation 
increased initially then dropped off around 3 months
14
ORA: 17121805-IRB01   Date IRB Approved: 3/13/2019   Amendment Date: 8/22/2019
. To our knowledge, the 
impact of BMAC in conjunction with cartilage restoration procedures on synovial 
fluid contents has not been investigated.
Our goal is to investigate the enhancement on osteochondral allograft incorporation 
with the use of BMAC. Additionally, we want to define the cytokine environment of 
the joint before and after OCA with and without BMAC augmentation.  With this 
information, consideration for therapeutic options such as BMAC augmentation or 
the exogenous addition of hyaluronic acid, IL-1Ra, IL-4, or IL-10 might positively 
impact the post-operative outcome of patients undergoing osteochondral allograft 
transplantation.

Current Version: 8/3/19
Approach:
Inclusion Criteria:
1. Patients aged 18-50 with a cartilage defect indicated for treatment with 
osteochondral allograft 
Exclusion Criteria: 
ORA: 17121805-IRB01   Date IRB Approved: 3/13/2019   Amendment Date: 8/22/2019
1. Patients with known rheumatoid arthritis, any other inflammatory 
arthropathy or synovial tissue disorder.
2. Patients with known bipolar osteoarthritis of the knee as determined by the 
treating physician, greater than Kellegren-Lawrence Grade 3 on xray imaging
3. Patient with a known infection or history of infection in the affected knee
All patients meeting inclusion and exclusion criteria will be offered enrollment. This 
study will include patients undergoing surgery with Drs. Brian Cole and Adam 
Yanke.  All patients will be evaluated in the office setting.  Upon clinical evaluation 
and MRI suspicion of a cartilage defect, patients will be offered enrollment in the 
study. Patients will then undergo routine care, including a diagnostic arthroscopy 
followed by a cartilage restoration procedure at a later date.  A synovial fluid 
aspiration will be performed prior to knee arthroscopy.
The patient will then undergo their definitive osteochondral allograft treatment 
based on standard clinical protocol.  At that time, a synovial fluid aspiration will be 
performed prior to beginning the surgical procedure. Defect size and location will be 
recorded. Patients will be randomized to the BMAC or control group. If in the BMAC 
group, a bone marrow aspiration will be performed from the iliac crest or proximal 
tibia depending on surgeon preference. The bone marrow aspirate will be processed 
using a BMAC harvesting system which concentrates the aspirate using flow 
cytometry to isolate the mesenchymal stem cells and growth factors. The 
osteochondral allograft plug will then be soaked in the BMAC for a minimum 2 
minutes prior to implantation. The remaining BMAC will be placed in the defect site 
prior to plug implantation. The control group will receive a 0.5cm sham incision 
over the iliac crest, but bone marrow aspiration will not be performed.
Bone marrow aspirate and bone marrow aspirate concentrate samples will be 
collected for analysis by flow cytometry to evaluate stem cell concentrations.
Patients will then be seen in the office setting at the normally scheduled follow-up 
visits at 2 weeks, 6 weeks, 6 months, and 12 months and routine radiographs will be 
obtained at those time points.  Synovial fluid aspirations of the knee will be repeated 
at the following time points: 2 weeks, 6 weeks, 6 months, and 12 months after 
surgery. 
Patients will also be asked to have a computed tomography (CT) scan of their 
operative knee 6 months post-operatively to evaluate graft integration.

Current Version: 8/3/19
Data collected preoperatively will include age, gender, laterality, inciting event, body 
mass index, smoking status, operative history on the operative extremity, and any 
history of trauma to the affected knee. The synovial fluid aspirates taken from the 
times points (during staging knee arthroscopy, pre-cartilage restoration procedure, 
2 weeks postoperatively, 6 weeks postoperatively,  6 months postoperatively, and 
12 months postoperatively) will then be analyzed for approximately 20 cytokines 
and cartilage specific biomarkers using biomarker assays.  These cytokines and 
biomarkers will include: BMP-2, BMP-7, FGF-2, FGF-18, IGF-1, PDGF, TGF-β, IL-1a, 
IL-1β, IL-4, IL-6, IL-8, IL-10, TNF-α, Bcl-2, Bcl-XL, iNOS, SOX9, SMADs, EGF, IFN, MIP, 
MCP, TIMP-1, OPN, MMP-1, MMP-3, VEGF, PDGF, and RANTES15. 
Additionally, at 2 time points (cartilage restoration procedure and 6 weeks 
postoperatively) serum will be collected for analysis of cartilage oligomeric matrix 
protein (COMP)16-18.  COMP is a component of the articular cartilage extracellular 
matrix that is synthesized by chondrocytes.  It has been suggested to be associated 
with cartilage turnover and may be sensitive biomarker of chondrocyte 
differentiation and cell turnover. 
Patients will be administered validated clinical questionnaires including a visual 
analog scale (VAS), Knee Injury and Osteoarthritis Outcome Score (KOOS), and 
Lysholm scale at 7 clinical time points: diagnostic staging knee arthroscopy, 
cartilage restoration procedure, 2 weeks postoperatively, 6 weeks postoperatively, 
6 months postoperatively, 12 months postoperatively, and 24 months 
postoperatively. Patients will also be administered IKDC, Marx activity scale, and 
VR/SF12 at 4 clinical time points: cartilage restoration procedure, 6 months 
postoperatively, 12 months postoperatively, and 24 months postoperatively.
All portions of this study will be part of conventional care with the following 
exceptions: (1) Synovial fluid aspirations at 6 time points, 2 intraoperatively while 
the patient in under anesthesia and 4 in the outpatient office setting, (2) serum 
collection at 2 time points, (3) and CT imaging at 6 months post-operatively.  Knee 
aspirations and injections are routinely performed in the outpatient office setting 
using sterile technique. 
Schedule of Events
Table 1. Outline of pre- and postoperative timing of events and schedule of data 
collection.
Diagnostic 
Arthroscopy
Post-Operative Week
Event
Pre-
Procedure 
≤ 21 days
Cartilage 
Procedure 
Day
2
6 12 24 52 104
Informed Consent
x
ORA: 17121805-IRB01   Date IRB Approved: 3/13/2019   Amendment Date: 8/22/2019

Current Version: 8/3/19
*Note: The grace period for clinic visits for week 2 will be 4 days, for weeks 6-12 the 
period will be 7 days, and from 24 weeks it will be 14 days.
Sample Size Determination:
Sample size determination will be based off past retrospective studies that 
osteochondral allograft integration. Oladeji et al. used radiographs to compare 
retrospective sample populations of 17 osteochondral allografts without BMAC and 
29 osteochondral allografts with BMAC8.  Brown et al. used CT 6 months post-
operatively to evaluate graft incorporation in 34 patients receiving osteochondral 
allografts.19 As this is a pilot study looking at the effect of BMAC on graft 
incorporation evaluated by CT, we will aim for a target population of 20 patients in 
each group.
Timelines:
Demographics
x
Confirm 
Inclusion/Exclusion 
Criteria
x
x
Medical History
x
x
Physical Examination
x
x
x
x
x
x
x
x
Vital Signs
x
x
x
x
x
x
x
x
Radiographs Standard 
Views (4 views)
x
x
x
x
x
x
Radiographs 
Mechanical Axis
x
CT
x
MRI
x
Questionnaires
(KOOS, VAS, 
Lysholm)
x
x
x
x
x
x
x
x
Questionnaires (IKDC, 
Marx, VR/SF-12)
x
x
x
Randomization
x
Synovial Fluid Testing
x
x
x
x
x
x
Serum Testing
x
x
BMAC Harvest
x
Injection
x
ORA: 17121805-IRB01   Date IRB Approved: 3/13/2019   Amendment Date: 8/22/2019

Current Version: 8/3/19
The primary outcome of this study will by synovial fluid analysis of cytokines and 
biomarkers at 5 time points. Our target sample size will be 40 patients (20 in each 
group). At our institution, approximately 5 cartilage restoration procedures are 
performed monthly. Given a 75% recruitment into the study, we would be able to 
achieve the target sample size within 12 months.
Specific Aim 1: To establish the effect of graft incorporation of BMAC on 
osteochondral allograft transplantation in the knee. 
Specific Aim 2: To assess the level of inflammatory cytokines and cartilage 
biomarkers in the synovial fluid of patients undergoing osteochondral allograft 
transplantation with and without intraarticular BMAC injection
6 months: Recruit and collect data from 20 patients, half the target 
population
12 months: Recruit and collect data from an additional 20 patients, 
completing the study
Specific Aim 3: To assess changes in serum biomarkers of cartilage turnover in the 
perioperative period in patients undergoing osteochondral allograft transplantation 
with and without intraarticular BMAC injection
6 months: Recruit and collect data from 20 patients, half the target 
population.
12 months: Recruit and collect data from 20 patients, completing the study
Specific Aim 4: To evaluate patient reported outcomes after osteochondral allograft 
transplantation with and without BMAC augmentation
6 months: Recruit and collect preoperative data from 20 patients, half the 
target population
12 months: Recruit and collect preoperative data from 20 patients, 
completing the study target population
Specific Aim 5: To correlate changes in inflammatory and cartilage biomarkers to 
clinical patient reported outcomes in patients undergoing osteochondral allograft 
transplantation with and without intraarticular BMAC injection

ORA: 17121805-IRB01   Date IRB Approved: 3/13/2019   Amendment Date: 8/22/2019
12 months: Once all patients have been recruited and synovial samples 
analyzed, the final portion of the project will be to correlate changes in 
inflammatory and biomarkers to patient reported outcomes collected at the 
5 time points. 

Current Version: 8/3/19
References
1. 
Frank RM, Lee S, Levy D, et al. Osteochondral Allograft Transplantation of the 
Knee: Analysis of Failures at 5 Years. Am J Sports Med
. 2017;45(4):864-874.
2. 
Nuelle CW, Nuelle JA, Cook JL, Stannard JP. Patient Factors, Donor Age, and 
Graft Storage Duration Affect Osteochondral Allograft Outcomes in Knees 
with or without Comorbidities. J Knee Surg
. 2017;30(2):179-184.
3. 
Cook JL, Stannard JP, Stoker AM, et al. Importance of Donor Chondrocyte 
Viability for Osteochondral Allografts. Am J Sports Med
. 2016;44(5):1260-
1268.
4. 
Flanigan DC, Harris JD, Trinh TQ, Siston RA, Brophy RH. Prevalence of 
chondral defects in athletes' knees: a systematic review. Med Sci Sports Exerc
. 
2010;42(10):1795-1801.
5. 
Mall NA, Harris JD, Cole BJ. Clinical Evaluation and Preoperative Planning of 
Articular Cartilage Lesions of the Knee. J Am Acad Orthop Surg
. 
2015;23(10):633-640.
6. 
Enea D, Cecconi S, Calcagno S, et al. Single-stage cartilage repair in the knee 
with microfracture covered with a resorbable polymer-based matrix and 
autologous bone marrow concentrate. Knee
. 2013;20(6):562-569.
7. 
Fortier LA, Potter HG, Rickey EJ, et al. Concentrated bone marrow aspirate 
improves full-thickness cartilage repair compared with microfracture in the 
equine model. J Bone Joint Surg Am
. 2010;92(10):1927-1937.
8. 
Oladeji LO, Stannard JP, Cook CR, et al. Effects of Autogenous Bone Marrow 
Aspirate Concentrate on Radiographic Integration of Femoral Condylar 
Osteochondral Allografts. Am J Sports Med
. 2017:363546517715725.
9. 
Tourville TW, Poynter ME, DeSarno MJ, Struglics A, Beynnon BD. 
Relationship between synovial fluid ARGS-aggrecan fragments, cytokines, 
MMPs, and TIMPs following acute ACL injury: A cross-sectional study. J 
Orthop Res
. 2015;33(12):1796-1803.
10. 
Yamaga M, Tsuji K, Miyatake K, et al. Osteopontin level in synovial fluid is 
associated with the severity of joint pain and cartilage degradation after 
anterior cruciate ligament rupture. PLoS One
. 2012;7(11):e49014.
11. 
Swärd P, Frobell R, Englund M, Roos H, Struglics A. Cartilage and bone 
markers and inflammatory cytokines are increased in synovial fluid in the 
acute phase of knee injury (hemarthrosis)--a cross-sectional analysis. 
Osteoarthritis Cartilage
. 2012;20(11):1302-1308.
12. 
Vasara AI, Konttinen YT, Peterson L, Lindahl A, Kiviranta I. Persisting high 
levels of synovial fluid markers after cartilage repair: a pilot study. Clin 
Orthop Relat Res
. 2009;467(1):267-272.
13. 
Cuéllar VG, Cuéllar JM, Kirsch T, Strauss EJ. Correlation of Synovial Fluid 
Biomarkers With Cartilage Pathology and Associated Outcomes in Knee 
Arthroscopy. Arthroscopy
. 
ORA: 17121805-IRB01   Date IRB Approved: 3/13/2019   Amendment Date: 8/22/2019
2016;32(3):475-485.

Current Version: 8/3/19
14. 
Schneider U, Schlegel U, Bauer S, Siebert CH. Molecular markers in the 
evaluation of autologous chondrocyte implantation. Arthroscopy
. 
2003;19(4):397-403.
15. 
Tuan RS, Chen AF, Klatt BA. Cartilage regeneration. J Am Acad Orthop Surg
. 
2013;21(5):303-311.
16. 
Palmieri-Smith RM, Wojtys EM, Potter HG. Early Cartilage Changes After 
Anterior Cruciate Ligament Injury: Evaluation With Imaging and Serum 
Biomarkers-A Pilot Study. Arthroscopy
. 2016;32(7):1309-1318.
17. 
Mendias CL, Lynch EB, Davis ME, et al. Changes in circulating biomarkers of 
muscle atrophy, inflammation, and cartilage turnover in patients undergoing 
anterior cruciate ligament reconstruction and rehabilitation. Am J Sports Med
. 
2013;41(8):1819-1826.
18. 
Catterall JB, Stabler TV, Flannery CR, Kraus VB. Changes in serum and 
synovial fluid biomarkers after acute injury (NCT00332254). Arthritis Res 
Ther
. 2010;12(6):R229.
19. 
Brown D, Shirzad K, Lavigne SA, Crawford DC. Osseous Integration after 
Fresh Osteochondral Allograft Transplantation to the Distal Femur: A 
Prospective Evaluation Using Computed Tomography. Cartilage
. 
ORA: 17121805-IRB01   Date IRB Approved: 3/13/2019   Amendment Date: 8/22/2019
2011;2(4):337-345.