Microbiome and Innate Immunity With Percutaneous Osseointegrated Prostheses
Conditions: Transfemoral Amputation
Sponsor: VA Office of Research and Development

 
Protocol Summary (ERICA) 
 
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IRB Approval 7/12/2019 
 
 
Microbiome and Innate Immunity with Percutaneous 
Osseointegrated Prostheses 
 
Protocol Summary 
 
 
 
IRB Approval 
Date of 
Current 
Version: 
7/12/2019 
University of 
Utah IRB #: 
IRB_00073178 
Sponsor: VA REHABILITATION RESEARCH AND DVLPMNT 
  
Principal 
Investigator: 
James Beck 
Internal Staff 
and  Sub-
Investigators: 
Site Name 
Staff Names 
Veterans Affairs SLC Health Care 
System (VAMC) 
James Beck 
 
Jayant Agarwal 
 
Sujeevini Jeyapalina 
 
Brian Bennett 
 
This document was created using the ERICA Online System at the University of Utah. The document is created 
from study information approved by the IRB on the date listed above.  Any alteration to the original content of this 
document may not be considered to represent the study as approved by the IRB.  
 
 

 
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Background and Introduction 
 
Percutaneous osseointegrated prosthetic attachment (i.e., the direct skeletal attachment of 
artificial limbs) is a rapidly evolving technology that is now ready to be introduced into the 
United States. This follows over a decade of European trials that largely involved transfemoral 
amputees. Translational animal research carried out over the past seven years at the VA , Salt 
Lake City, Utah, has clarified principles of osseointegration, and bone and skin interface 
responses to chronic weight bearing on a skeletally attached artificial limb. This information 
has produced new prosthetic designs that make the technique safer and more likely to succeed 
over the long-term. This translational animal data, gathered in parallel to the European human 
data, has made it possible to commence an Early Feasibility Device Exemption (IDE) Pilot 
Program under the direction of the FDA. This trial is anticipated to begin October 19, 2015, at 
the VAH in Salt Lake City, Utah, and is funded by VA RR&D grant (RX001208-01). Ten 
transfemoral amputees, selected from the veteran and active military populations will receive a 
PODS device. 
The critical limiting factor to the prior U.S. introduction of this technology has been an up to 
30% infection rate at the implant/skin interface in European patients. Evolving designs and 
surgical techniques have lowered this incidence, but it remains remarkable that 70% to perhaps 
95% of European patients now remain infection free by simply using mild soap and shower 
hygiene at the stoma, and avoiding the use of antibiotics. In fact antibiotics and disinfectants 
have been found to be counter productive and lead to antibiotic resistance. It is increasingly 
evident that the key to this improbable success is the biology and presence of a mutualistic-
commensal microbiota at the skin/implant interface. These “good bacteria”  are the “guard 
dogs at the gate”. 
Microbiome studies: Traditionally, wound microbiotas have been characterized by culture 
based assays. Cultures, however, account for only 3% of the bacterial strains actually present. 
A recent study, reported that, on average, culturing techniques underestimated microbial load 
by 2.34 logs (P<0.0001), and in some cases greater than 6 logs. Bacterial cultures, because 
they are designed to select for specific pathogens, are unable to grow and recognize most of 
the other resident skin microbiota. These non-molecular techniques do not allow a full 
understanding of the complexity of stomal colonization in both the diseased and healthy states. 
The relationships between the bacterial subsets within an ecological community are complex. 
There is strong evidence to suggest that microbes that are commonly thought of as pathogens 
can live commensally on the skin. In fact, bacteria from the genera Staphylococcus, 
Corynebacterium, Propionibacterium, Streptococcus and Pseudomonas, may directly benefit 
the host (mutualism) and only rarely will they become pathogens. Recent developments in 
molecular based technology have now paved the way to more advanced genome based 
techniques for identifying bacterial ecologies (microbiome). Techniques based upon distinct 
bacterial molecular markers, such as 16S rRNA are now being employed to truly understand 
the complexity and complicity of the human microbiome. 
Inflammatory biomarkers: Wound healing involves a complex process with many 
overlapping stages: coagulation, inflammation, cell proliferation and migration, and finally 
tissue remodeling with wound closure within 3-14 days. However, as a result of our 
percutaneously implanted device, we expect the area around the stoma of our patients to 

 
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evidence a chronic/continuous wound healing process. Understanding the relationship between 
the microbiome and modulating the cycle of inflammation produced by microbial colonization 
and/or infection is a critical step towards improving treatment strategies. 
We postulate that comparing the profiles of local and systemic inflammatory biomarkers can 
provide valuable information regarding the state of health of the skin surrounding the implant. 
Because different cytokines can have overlapping biological functions, as well as regulate 
production of other cytokines, the ability to simultaneously analyze a large set of cytokines 
within a localized environment (such as a site of inflammation) can be more valuable than 
detecting the levels of individual cytokines (particularly if only small volume test samples are 
obtained). Collecting multiple samples over time will also allow for longitudinal studies of 
cytokine profiles within a single patient. 
Biomarkers (e.g., cytokines and chemokines) are proteins that act as effectors or modulators of 
the inflammatory response. Custom TaqMan microfluid array cards (assaying 192 genes) will 
be able to detect mRNA expression patterns of systemic inflammatory proteins. These can 
then be compared to native secreted proteins locally present at the implant site: the latter 
detected using the Bio-Plex multiplex immunoassay system measuring 27 cytokines. 
The biomarkers detected in this study (using the Bio-Plex system) outperformed the 
erythrocyte sedimentation rate (ESR) and the C-reactive protein (CRP); tests that are 
commonly used in standard clinical practice to detect the presence of infection. We therefore 
believe that in addition to having a better understanding of the cytokine profile expected of a 
chronic wound in a stable state (when compared to an infected state) it is possible that a 
diagnostic cytokine profile may be determined that will predict the onset or detect the presence 
of a subclinical infection. 
 
 
 
Purpose and Objectives 
 
Purpose 
The purpose of this study is to investigate the clinical implementation of a new percutaneous 
prosthetic attachment system (Safety Study of Percutaneous Osseointegrated Implants for 
Prosthetic Attachment; separate IRB # 00082763 ) by determining the resident microbial 
ecology of the implant exit site and to simultaneously study the systemic and local stomal 
immune responses. We will use two, state-of-the-art, pre- and post-surgery bacterial 
monitoring technologies: these procedures are intended to facilitate the early prediction, 
detection, and treatment of infection, as well as to provide follow-up data that can potentially 
be used to advantageously manipulate the stomal microbial environment in future clinical 
trials. 
Objectives 
The objective of this study is to follow 10 patients implanted with a percutaneous 
osseointegrated exoprosthetic docking system (PODS) for a period of one year. This PODS 

 
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technology requires invasive surgery to skeletally attach a metal implant, which protrudes 
from the long bone and exits the stump skin of the amputee patient (IRB# 00082763, as 
above). All stomas are colonized by local skin bacteria; colonization does not necessarily 
result in infection. Over time, the presence of this skin penetrating foreign object (implant) 
will cause measurable changes in the bacterial population (microbiota) at and around the 
PODS exit site. 
It is anticipated that the evolving microbiota, in concert with measurable changes in the local 
and systemic cytokine responses, will reveal patterns associated with mutualistic-commensal 
bacteria and/or pathogenic bacteria related to the stages of chronic wound healing. These 
patterns could be used to determine the presence of a stable uninfected stoma or the 
progression of a stomal infection. Hopefully, this information will allow timely intervention to 
prevent infection, i.e. by detecting early stages of infection or discerning common patterns of 
stable mutualistic-commensal bacterial strains, effective intervention protocols (antibiotics, 
probiotics or manipulation of the stomal and skin microbiota) may be developed to avoid 
patient morbidity and assure implant survival. 
The study aims will test the following: 
Aim 1: Determine and characterize the microbiota in the region surrounding the 
skin/implant interface. Sampling will take place over all stages of wound healing and 
stomal maturation and will begin with Stage 1 and Stage 2 surgeries, as well as at 
defined time points, and collection sites (i.e., the stoma, ipsilateral and contralateral 
thigh skin) for up to one year post surgery. This will be carried-out by using a specific 
swabbing technique to collect bacterial DNA and to amplify and sequence bacterial 
16S rRNA genes. 
AIM 2: Compare the expression patterns of the local and systemic inflammatory 
biomarkers over time and determine if there is a correlation with the microbiota pattern 
to diagnose the state of wound healing at the skin/implant interface and the systemic 
response to a potentially life-long chronic wound. The measurements of the pro-
inflammatory cytokines, found in the stomal exudate (local biomarkers) and blood 
serum (systemic biomarkers), along with evolving microbiota profiles (Aim #1) will 
help to better characterize the homeostatic state of the stoma and subsequent optimum 
wound care therapies. 
The ability to predict infection and to avoid it without the use of antibiotics would be of great 
value to future clinical trials. Assuming the success of this feasibility pilot trial, it is 
anticipated that the trial will be expanded to include 200 patients. 
 
 
 
Study Population 
 
Age of Participants: 18-70 years old 
 
Sample Size: 

 
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At Utah: 
10  
All Centers: 
10 
 
Inclusion Criteria: 
Only participants enrolled in IRB #00082763 study will be enrolled in this study.  For the 
IRB study #00082763 study, participants come from a pool of subjects who are veterans and 
have transfemoral limb loss that is not a result of dysvascular disease. These patients are 
considered to be capable of functioning with socket suspension transfemoral prostheses but 
are also suitable for fitting with a percutaneous implant system. 
Inclusion criteria for # IRB 00082763 are: 
A potential subject will be included in the study if he or she meets all of the following 
inclusion criteria: 
1.   Is a US military veteran with transfemoral limb loss, that occurred at least 6 
months prior to consent, and that the amputation is not a result of dysvascular 
disease. 
2.   Is at least 18 years of age or older. 
3.   Has previously used or is currently using a “socket suspension technology” 
prosthesis 
4.   Has, in the opinion of the investigator, normal cognitive function and no physical 
limitations, addictive diseases, or underlying medical conditions including tobacco 
use (continued testing for tobacco use will be performed at screening) that may 
prevent the subject from being an appropriate study candidate. 
5.   Is willing, able, and committed to participation in baseline and follow-up 
evaluations for the entire duration of the study. 
6.   Can provide written informed consent to participate. 
 
 
Exclusion Criteria: 
A potential subject will be excluded from study participation if he/she meets any of the 
following criteria (the IRB 00082763 study criteria) 
1. Is currently on active or reserve military duty 
2. Has experienced systemic bacterial infection or localized infection at the stump site 
within the previous 6 months 
3. Has had more than 1 limb amputated 
4. Has a body mass index (BMI) ≥ 30 
5. Has insulin dependent diabetes mellitus (IDDM) or has adult onset DM with a 
glycated hemoglobin (HbA1c) > 53 mmol/mol (7.0%) at screening 

 
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6. Has residual femur bone length of less than 25% of the length of the contralateral 
femur. 
7. Has clinically diagnosed vascular compromise proximal to the surgical site 
8. Is pregnant at the time of surgery or plans to become pregnant within the first year of 
follow-up 
9. Has evidence of recent tobacco use (urine cotinine test > 300 ng/mL [1703 nmol/L]) 
and is not committed to a smoking-cessation program 
10. Has renal insufficiency (defined as serum creatinine of ≥ 1.8 mg/dL) or is currently 
receiving renal dialysis 
11. Is currently involved in or plans to be involved in high levels of physical activity 
(competitive sports, heavy physical labor, etc) during the first 12 months of the 
rehabilitation stage 
12. Has muscular, neurologic or vascular deficiencies that may compromise the bone or 
soft tissue healing of the affected extremity 
13. Has anemia characterized by a hemoglobin of ≤ 11 g/dL at the time of surgery 
14. Is currently on oral anticoagulation (excluding low-dose aspirin for cardiac 
prophylaxis) 
 
 
Design  
 
Other 
 
 
Retrospective sample analyses 
 
Study Procedures 
 
Recruitment/Participant Identification Process: 
Only patients, who will be recruited and consented to participate in IRB#00082763 study, will 
participate in this "Microbiome" study to retrospectively assess the progression of wound and 
stomal healing and/or the occurrence of infection, as a safety survey and previously described. 
  
 
 
Informed Consent: 
Description of location(s) where consent will be obtained:  
George E Wahlen Department of Veterans Affairs Hospital, Salt Lake City Health Care 
System. 
 
Description of the consent process(es), including the timing of consent:  
Consent process is now part of the IRB study # 00082763. No separate consent process will be 
used. 
 
Requested Waivers/Alterations of Consent: 
Waiver of Informed 
Consent 
VA Waiver of Consent for Recruitment Purposes Only 

 
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Procedures: 
-Patient selection 
All patients selected to participate in the "Safety Study of Percutaneous Osseointegrated 
Implants for Prosthetic Attachment (IRB #00082763)" will  participate in this study. As this is 
safety evaluation study, subjects will not be randomized. Follow-up end point will be 1 year 
post 2nd stage surgery. 
Patients will be consented through IRB study # 00082763.  
- Obtaining consent 
Patients are consented as part of the IRB #00082763 consenting process 
- Sample collection 
Samples (skin swabs, tissue and blood) for this study will come from the samples collected 
and tissue-banked as part of the IRB # 00082763. 
Collection process is described in IRB # 00082763. Briefly, samples will be collected from 
each patients at different pre- and post-operative time points (see below). 
Pre-op stage 1/day of the procedure, 3+/-7 days of stage 1 post-op, pre-op of stage 2/day of 
procedure, day 7 +/- 7 days of stage 2, 2 wk  +/- 14 days of Stage 2, and 6wk +/- 14 days, 3, 
6,9, 12 mo +/- 30 Days of stage 2.  
-Analysis 
-Reporting 
 
 
Procedures performed for research purposes only:  
 
 
Statistical Methods, Data Analysis and Interpretation 
 
Statistical analysis: A sample size of n=10 patients is planned to be used. 
Sample Size Justification. A sample size of n=10 patients will be used. Even if the 
specific comparisons, which involve a single predictor variable, described for Aims 
1 to 3 reveal trends that are not statistically significant, it would be sufficient to 
show that overall model fit is significant. That is, a significant multivariable 
curvilinear correlation with pro-inflammatory cytokines, for example, would 
demonstrate that Bio-Plex array is potentially a sensitive indicator of inflammation 
and/or infection, provided that a visible shift in the graphed growth curve occurs 

 
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with the onset of inflammation and/or infection. With a sample size of 10, there is 
80% power using a two-sided alpha 0.05 comparison, to detect a correlation of 
r=0.71 or greater. Converting this to a coefficient of determination, r2 = 0.50, the 
multivariable model would have to account for 50% of the variability in pro-
inflammatory cytokines concentration. Since cytokines is measurable, going up or 
down across healing time in an anticipated fashion, with time being one of the 
predictor variables making up the multivariable curvilinear correlation, it is 
reasonable to assume that this magnitude of correlation will be observed in this 
study. Furthermore, the repeated measurements increase the statistical power 
somewhat. The study, then, is adequately powered to demonstrate the feasibility of 
microbiota, and pro-inflammatory cytokines/markers as indicators of inflammation 
and/or infection. 
Microbiota analysis 
In this microbiota study, the temporal dynamics of the stomal bacterial ecology of 
these ten patients were investigated over the duration of one-year following the 
second surgery. skin swab samples were collected at regular intervals using swabs 
obtained from 3 different sites. Pre-surgery data was used as a baseline, thus, each 
patient had their internal control, and the microbiota changes to the individual 
patient’s per- and post- skin swab samples were compared. The DNA extraction 
were performed using the standard procedure. The 16S ribosomal RNA genes were 
amplified using broad range PCR primers targeted to the V1-V3 hypervariable 
region (V1V3 Forward primer     5’-AGAGTTTGATCCTGGCTCAG-3’ and 
V1V3 Reverse primer 5-ATTACCGCGGCTGCTGG-3’), with barcodes unique to 
each sample. PCR reactions were carried out in quadruplicate using high fidelity 
Accuprime Taq. The resulting 16S rDNA amplicons were purified using a 1:1 
volume of SPRI beads, quantified using PicoGreen, pooled in equal amounts, and 
sequenced on the Illumina MiSeq using 2x300 bp chemistry.  Extraction blanks and 
DNA-free water were subjected to the same amplification and purification 
procedure to allow for empirical assessment of environmental and reagent 
contamination. Sequencing was performed by the PennCHOP Microbiome Center 
on the Illumina MiSeq instrument. Sequencing results were then processed using 
the hmmUFOtu workflow. Reads were demultiplexed using the prior mentioned 
barcodes. Individual reads (with ≥97% identity) were treated as same species 
sequences, and were clustered and assigned to operational taxonomic units (OTUs) 
using the Greengenes 13_8 database. OTUs that were found to represent just one 
sequence in one sample were also removed. Any reads assigned to Cyanobacteria, a 
common contaminant, were also removed. OTU tables and phylogenetic trees 
output from hmmUFOtu were then further analyzed using the phyloseq package in 
R. This analysis included rarefaction of samples down to an even depth of ~1000 
sequences per sample. Alpha and beta diversity metrics were computed using the 
vegan package. 
All statistical analysis was performed in R. For testing statistically significant 
differences between populations, the nonparametric Wilcoxon rank-sum test was 
used (wilcox.test function in R). Due to the sample losses from rarefaction, the 

 
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unpaired test was used. Multiple testing correction was also applied with the 
p.adjust function, using the false discovery rate correction. For differential 
abundance testing, the DESeq2 and phyloseq packages were used with default 
settings. Sample similarity was calculated using the Weighted UniFrac, 
Unweighted UniFrac, and Bray Curtis Beta diversity metrics, and/or statistical 
comparisons of community composition. The Adonis function (PERMANOVA 
test), from the vegan package was applied to each of the aforementioned metrics. 
Dirichlet multinomial mixture modelling was performed using the 
DirichletMultinomial package. Ordination was performed using the labdsv 
package. 
 
Whole Blood RNA expression data 
Whole-blood samples were collected from these patients at the following time 
points: pre-surgery 1 (PrS1), post-surgery 1 (PoS1), pre-surgery 2 (PrS2), post-
surgery 2 week 1 (PoS2-W1), post-surgery 2 week 2 (PoS2-W2), post-surgery 2 
month 1 (PoS2-M1), post-surgery 2 month 3 (PoS2-M3), post-surgery 2 month 6 
(PoS2-M6), post-surgery 2 month 9 (PoS2-M9), and post-surgery 2 month 12 
(PoS2-M12). Whole-blood RNAs were isolated and undergone complete RNA 
sequencing, where single-end base pair sequencing was performed at the University 
of Utah Core genomic  facility. Gene read counts were quantified via featureCounts 
and differential gene expression (DEG) analysis was performed using DESeq2.  
For the DEG analysis, pair-wise comparisons were made in two sets: first, between 
PrS1 and all subsequent time points, second between PrS2 and all subsequent time 
points. Results from the DEG analysis were used to perform an enrichment 
analysis, which implemented topGO’s elim graph algorithm paired with the 
Kolmogorov-Smirnov test for significance. Enrichment analysis was run on each of 
the Gene Ontology’s (GO) sub ontologies: biological process (bp), cellular 
component (cc) and molecular function (mf). 
 
References: 
1. Bland, J.M. and D.G. Altman, Calculating correlation coefficients with repeated 
observations: Part 1--Correlation within subjects. BMJ, 1995. 310(6977): p. 446. 
2. Bland, J.M. and D.G. Altman, Calculating correlation coefficients with repeated 
observations: Part 2--Correlation between subjects. BMJ, 1995. 310(6980): p. 633.