Association between blood markers and the progression of osseointegration in percutaneous prostheses patients-A pilot study.

Patients implanted with osseointegrated (OI) prosthetic systems have reported vastly improved upper and lower extremity prosthetic function compared with their previous experience with socket-suspension systems. However, OI systems have been associated with superficial and deep-bone infections and implant loosening due, in part, to a failure of the osseointegration process. Although monitoring the osseointegration using circulating biomarkers has clinical relevance for understanding the progression of osseointegration with these devices, it has yet to be established. Ten patients were enrolled in this study. Blood samples were collected at pre-selected times, starting before implantation surgery, and continuing to 12 months after the second surgery. Bone formation markers, bone resorption markers, and circulating amino acids were measured from blood samples. A linear mixed model was generated for each marker, incorporating patient ID and age with the normalized marker value as the response variable. Post hoc comparisons were made between 1 week before Stage 1 Surgery and all subsequent time points for each marker, followed by multiple testing corrections. Serial radiographic imaging of the residual limb containing the implant was obtained during follow-up, and the cortical index (CI) was calculated for the bone at the porous region of the device. Two markers of bone formation, specifically bone-specific alkaline phosphatase (Bone-ALP) and amino-terminal propeptide of type I procollagen (PINP), exhibited significant increases when compared with the baseline levels of unloaded residual bone prior to the initial surgery, and they subsequently returned to their baseline levels by the 12-month mark. Patients who experienced clinically robust osseointegration experienced increased cortical bone thickness at the porous coated region of the device. A medium correlation was observed between Bone-ALP and the porous CI values up to PoS2-M1 (p = .056), while no correlation was observed for PINP. An increase in bone formation markers and the lack of change observed in bone resorption markers likely reflect increased cortical bone formation induced by the end-loading design of the Utah OI device used in this study. A more extensive study is required to validate the correlation observed between Bone-ALP and porous CI values.

Serum procalcitonin level is independently associated with mechanical ventilation and case-fatality in hospitalized COVID-19-positive US veterans-A potential marker for disease severity.

The Coronavirus-19 disease (COVID-19) has claimed over 6.8 million lives since first being reported in late 2019. The virus that causes COVID-19 disease is highly contagious and spreads rapidly. To date, there are no approved prognostic tools that could predict why some patients develop severe or fatal disease outcomes. Early COVID-19 studies found an association between procalcitonin (PCT) and hospitalization or duration of mechanical ventilation and death but were limited by the cohort sizes. Therefore, this study was designed to confirm the associations of PCT with COVID-19 disease severity outcomes in a large cohort. For this retrospective data analysis study, 27,154 COVID-19-positive US veterans with post-infection PCT laboratory test data and their disease severity outcomes were accessed using the VA electronic healthcare data. Cox regression models were used to test the association between serum PCT levels and disease outcomes while controlling for demographics and relevant confounding variables. The models demonstrated increasing disease severity (ventilation and death) with increasing PCT levels. For PCT serum levels above 0.20 ng/ml, the unadjusted risk increased nearly 2.3-fold for mechanical ventilation (hazard ratio, HR, 2.26, 95%CI: 2.11-2.42) and in-hospital death (HR, 2.28, 95%CI: 2.16-2.41). Even when adjusted for demographics, diabetes, pneumonia, antibiotic use, white blood cell count, and serum C-reactive protein levels, the risks remained relatively high for mechanical ventilation (HR, 1.80, 95%CI: 1.67-1.94) and death (HR, 1.76, 95%CI: 1.66-1.87). These data suggest that higher PCT levels have independent associations with ventilation and in-hospital death in veterans with COVID-19 disease, validating previous findings. The data suggested that serum PCT level may be a promising prognostic tool for COVID-19 severity assessment and should be further evaluated in a prospective clinical trial.

A preliminary, observational study using whole-blood RNA sequencing reveals differential expression of inflammatory and bone markers post-implantation of percutaneous osseointegrated prostheses.

AIMS: While the benefits of direct skeletal attachment of artificial limbs are well recognized, device failure due to infection and insufficient osseointegration remain obstacles to obtaining consistently successful outcomes. Currently, the potential for device failure is assessed by subjective pain, clinical function scores, radiographic evidence of bone atrophy, and the presence of radiolucent lines at the bone-implant interface, and subjective pain and function scores. Our hypothesis is that measurable biological indices might add another objective means to assess trends toward bone and stomal healing. This longitudinal cohort study was undertaken to identify potential serological biomarkers suggestive of bone remodeling and the presence of stomal tissue inflammation. METHODS: Ten unilateral transfemoral amputee veterans, who were implanted with a percutaneous osseointegrated (OI) skeletal limb docking system, were recruited to participate in this IRB-approved study. Venous blood samples were obtained from before the Stage 1 Surgery up to 1 year following the Stage 2 Surgery. Whole-blood RNA was extracted, sequenced, mapped, and analyzed. Of the significant differentially expressed (DEGs) genes (p<0.05) identified, four genes of interest (IL12B, IL33, COL2A1, and SOST) were validated using qPCR. Enrichment analysis was performed to identify significant (p<0.01) Gene Ontology (GO) terms. RESULTS: Most differentially expressed genes were only detected at PoS1 immediately after the first surgery. Of the significant genes identified, IL12B and IL33 were related to inflammation, and COL2A1 and SOST were associated with bone remodeling. These four genes were identified with greater than 20 log fold-change. CONCLUSION: Whole-blood RNA-seq data from 10 patients who previously underwent percutaneous osseointegrated lower limb implantation revealed four genes of interest that are known to be involved in inflammation or bone remodeling. If verified in future studies, these genes may serve as markers for predicting optimal bone remodeling and stomal tissue healing following OI device implantation.

Evaluation of soft-tissue response around laser microgrooved titanium percutaneous devices.

Percutaneous devices are prone to epidermal downgrowth and sinus tract formation, which can serve as a nidus for bacterial colonization and increase the risk of peri-prosthetic infection. A laser microgrooved topography has been shown to limit gingival epidermal downgrowth around dental implants. However, the efficacy of this laser microgrooved topography to limit epidermal downgrowth around nongingival percutaneous devices is yet to be investigated. In this study, devices with a porous-coated subdermal component and a percutaneous post were designed and manufactured. The proximal 2 mm section of the percutaneous post were left smooth, or were textured with either a porous coating, or with the laser microgrooved topography. The smooth and porous topographies served as controls. The devices were tested in a hairless guinea pig back model, where 18 animals were randomly assigned into three groups, with each group receiving one implant type (n = 6/group). Four weeks postimplantation, the devices with surrounding soft-tissues were harvested and processed for histological analyses. Results indicated that the laser microgrooved topography failed to prevent epidermal downgrowth (23 ± 4%) around percutaneous posts in this model. Furthermore, no significant differences (p = 0.70) in epidermal downgrowth were present between the three topographies, with all the groups exhibiting similar measures of downgrowth. Overall, these findings suggest that the laser microgrooved topography may not halt downgrowth around percutaneous devices for dermal applications.

Epidermal growth factor receptor genes are overexpressed within the periprosthetic soft-tissue around percutaneous devices: A pilot study.

Epidermal downgrowth around percutaneous devices produce sinus tracts, which then accumulate bacteria becoming foci of infection. This mode to failure is epidermal-centric, and is accelerated by changes in the chemokines and cytokines of the underlying periprosthetic granulation tissue (GT). In order to more fully comprehend the mechanism of downgrowth, in this 28-day study, percutaneous devices were placed in 10 Zucker diabetic fatty rats; 5 animals were induced with diabetes mellitus II (DM II) prior to the surgery and 5 animals served as a healthy, nondiabetic cohort. At necropsy, periprosthetic tissues were harvested, and underwent histological and polymerase chain reaction (PCR) studies. After isolating GTs from the surrounding tissue and extracting ribonucleic acids, PCR array and quantitative-PCR (qPCR) analyses were carried-out. The PCR array for 84 key wound-healing associated genes showed a five-fold or greater change in 31 genes in the GTs of healthy animals compared to uninjured healthy typical skin tissues. Eighteen genes were overexpressed and these included epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR). Thirteen genes were underexpressed. When GTs of DM II animals were compared to healthy animals, there were 8 genes overexpressed and 25 genes underexpressed; under expressed genes included EGF and EGFR. The qPCR and immunohistochemistry data further validated these observations. Pathway analysis of genes up-regulated 15-fold or more indicated two, EGFR and interleukin-10, centric clustering effects. It was concluded that EGFR could be a key player in exacerbating the epidermal downgrowth, and might be an effective target for preventing downgrowth.

Analysis of the Stomal Microbiota of a Percutaneous Osseointegrated Prosthesis: A Longitudinal Prospective Cohort Study.

Percutaneous osseointegrated (OI) prostheses (POPs) are used to skeletally attach artificial limbs in amputees. While any permanent percutaneous interface is at risk of becoming infected by the resident microbiota colonizing the stoma, most of these patients remain infection-free. Avoidance of infection likely depends upon a mechanically and/or biologically stable skin-to-implant interface. The ultimate question remains, "why do some stomata become infected while others do not?" The answer might be found in the dynamic bacterial communities of the patient and within the stomal site itself. This study is an appendix to the first Food and Drug Administration approved prospective early feasibility study of OI prosthetic docking, in which, 10 transfemoral amputees were implanted with a unique POP device. In this analytical, longitudinal cohort study, each patient's skin and stomal microbiota were analyzed from the initial surgery to 1 year following the second-stage surgery. During each follow-up visit, three swab samples-stomal, device thigh skin and contralateral thigh skin-were obtained. DNA was extracted, and bacterial 16S ribosomal RNA (rRNA) genes were amplified and sequenced to profile microbial communities. The stomal microbiota were distinct from the microbiota on the adjacent thigh skin and the skin of the contralateral thigh, with a significantly increased abundance of Staphylococcus aureus within the stoma. Early on stomal microbiota were characterized by high diversity and high relative abundance of obligate anaerobes. Over time, the stomal microbiota shifted and stabilized in communities of lower diversity dominated by Streptococcus, Corynebacterium, and/or Staphylococcus spp. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2645-2654, 2019.

Influence of negative pressure wound therapy on peri-prosthetic tissue vascularization and inflammation around porous titanium percutaneous devices.

Negative Pressure Wound Therapy (NPWT) has been shown to limit downgrowth around percutaneous devices in a guinea pig model. However, the influence of NPWT on peri-prosthetic tissue characteristics leading to limited downgrowth is still unclear. In order to investigate this, 12 CD hairless rats were assigned into two groups, NPWT and Untreated (n = 6/group). Each animal was implanted with a porous coated titanium percutaneous device and was dressed with a gauze and semi-occlusive base dressing. Post-surgery, animals in the NPWT Group received a regimen of NPWT treatment (-70 to -90 mmHg). After 4 weeks, tissue was collected over the device and stained with CD31 and CD68 to quantify blood vessel density and inflammation, respectively. The device with the surrounding tissue was also collected to quantify downgrowth. NPWT treatment led to a 1.6-fold increase in blood vessel densities compared to untreated tissues (p < 0.05). NPWT treatment also resulted in half the downgrowth as the Untreated Group, although not statistically significant (p = 0.19). Additionally, the results showed a trend toward increased CD68 cell densities in the NPWT Group compared to the Untreated Group (p = 0.09). These findings suggest that NPWT may influence wound healing responses in percutaneous devices by increasing blood vessel densities, limiting downgrowth and potentially increasing inflammation. Overall, NPWT may enhance tissue vascularity around percutaneous devices, especially in patients with impaired wound healing. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2091-2101, 2019.

Peri-prosthetic tissue reaction to discontinuation of negative pressure wound therapy around porous titanium percutaneous devices.

Negative pressure wound therapy (NPWT) has been reported to limit epithelial downgrowth, one of the failure mechanisms of percutaneous devices. In a previous study, when NPWT was applied for 4 weeks (NPWT Group) to porous coated titanium percutaneous devices, downgrowth (5 ± 4%; mean ± one SD) was significantly reduced compared to untreated controls (Untreated Group) (16 ± 6%; p ≤ 0.01). However, it was unclear whether this beneficial effect was sustained when NPWT was discontinued. In order to test this, porous coated titanium percutaneous devices were implanted into 6 hairless guinea pigs. Post-surgery, animals received 4 weeks of NPWT treatment followed by 4 weeks of no treatment (Discontinued Group). At necropsy, the devices and surrounding tissues were harvested and processed. Quantitative downgrowth measurements and qualitative analyses of tissue characteristics were performed, and compared to historical controls (NPWT and Untreated Groups). The Discontinued Group, at 8 weeks, had significantly more downgrowth than the NPWT Group at 4 weeks (23 ± 3% vs. 5 ± 4%; p ≤ 0.01). At 8 weeks, the Discontinued Group qualitatively appeared to exhibit reduced numbers of blood vessels and increased degree of fibrosis compared to the NPWT Group at 4 weeks. This study suggests that NPWT will only be an effective treatment for limiting downgrowth if used continuously. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 564-572, 2019.

Negative pressure wound therapy limits downgrowth in percutaneous devices.

Maintenance of a soft tissue seal around percutaneous devices is challenged by the downgrowth of periprosthetic tissues-a gateway to potential infection. As negative pressure wound therapy (NPWT) is used clinically to facilitate healing of complex soft tissue pathologies, it was hypothesized that NPWT could limit downgrowth of periprosthetic tissues. To test this hypothesis, 20 hairless guinea pigs were randomly assigned into four groups (n = 5/group). Using a One-Stage (Groups 1 and 3) or a Two-Stage (Groups 2 and 4) surgical procedure, each animal was implanted with a titanium-alloy subdermal device porous-coated with commercially pure, medical grade titanium. Each subdermal device had a smooth titanium-alloy percutaneous post. The One-Stage procedure encompassed insertion of a fully assembled device during a single surgery. The Two-Stage procedure involved the implantation of a subdermal device during the first surgery, and then three weeks later, insertion of a percutaneous post. Groups 1 and 2 served as untreated controls and Groups 3 and 4 received NPWT. Four weeks postimplantation of the post, the devices and surrounding tissues were harvested, and histologically evaluated for downgrowth. Within the untreated control groups, the Two-Stage surgical procedure significantly decreased downgrowth (p = 0.027) when compared with the One-Stage procedure. Independent of the surgical procedures performed, NPWT significantly limited downgrowth (p ≤ 0.05) when compared with the untreated controls.

Progression of bone ingrowth and attachment strength for stability of percutaneous osseointegrated prostheses.

BACKGROUND: Percutaneous osseointegrated prosthetic (POP) devices have been used clinically in Europe for decades. Unfortunately, their introduction into the United States has been delayed, in part due to the lack of data documenting the progression of osseointegration and mechanical stability. QUESTIONS/PURPOSES: We determined the progression of bone ingrowth into porous-coated POP devices and established the interrelationship with mechanical stability. METHODS: After amputation, 64 skeletally mature sheep received a custom porous-coated POP device and were then randomized into five time groups, with subsequent measurement of percentage of bone ingrowth into the available pore spaces (n = 32) and the mechanical pullout force (n = 32). RESULTS: Postimplantation, there was an accelerated progression of bone ingrowth (~48% from 0 to 3 months) producing a mean pullout force of 5066 ± 1543 N. Subsequently, there was a slower but continued progression of bone ingrowth (~23% from 3 to 12 months) culminating with a mean pullout force of 13,485 ± 1855 N at 12 months postimplantation. There was a high linear correlation (R = 0.94) between the bone ingrowth and mechanical pullout stability. CONCLUSIONS: This weightbearing model shows an accelerated progression of bone ingrowth into the porous coating; the amount of ingrowth observed at 3 months after surgery within the porous-coated POP devices was sufficient to generate mechanical stability. CLINICAL RELEVANCE: The data document progression of bone ingrowth into porous-coated POP devices and establish a strong interrelationship between ingrowth and pullout strength. Further human data are needed to validate these findings.

Cortical bone response to the presence of load-bearing percutaneous osseointegrated prostheses.

Although the current percutaneous osseointegrated (OI) prosthetic attachment systems are novel clinical treatments for patients with limb loss, there have only been limited translational studies undertaken to date. To bridge this knowledge gap, from a larger study group of 86 animals that were implanted with a novel percutaneous OI implant construct, 33 sheep were randomly selected from the 0-, 3-, 6-, 9- and 12-month groups for histomorphometric analyses of periprosthetic cortical bone tissue. At necropsy, implanted and nonimplanted limbs were harvested and processed for the evaluation of cortical bone porosity and mineral apposition rate (MAR). The data showed a maximum increase in bone porosity within the first 3 months following implantation and then a progressive reduction in porosity to the baseline steady-state ("Time 0") value by 12 months. The data further verified that the MAR increased during the first 6 months of implantation, reaching a plateau between 6 and 9 months, followed by a progressive decline to the baseline steady state. It was concluded that clinical load bearing and falls precautions, taken during the first 3-6 months following percutaneous OI device implantation surgery, could greatly limit bone fractures during this vulnerable time of increasing cortical bone porosity.

Efficacy of a porous-structured titanium subdermal barrier for preventing infection in percutaneous osseointegrated prostheses.

Infections of percutaneous osseointegrated prostheses (POP) cause prolonged morbidity and device failure because once established, they are refractory to antibiotic therapy. To date, only limited translational animal studies have investigated the efficacy of POP designs in preventing infections. We developed an animal model to evaluate the efficacy of a porous-coated titanium (Ti) subdermal barrier to achieve skin-implant integration and to prevent periprosthetic infection. In a single-stage "amputation and implantation" surgery, 14 sheep were fitted with percutaneous devices with an attached porous-coated Ti subdermal barrier. Nine sheep were implanted with a smooth Ti subdermal barrier construct and served as controls, with one control sheep removed from the study due to a fractured bone. Clinical, microbiological, and histopathological data showed that the porous Ti barrier prevented superficial and deep tissue infections in all animals (14/14, 100%) at the 9-month endpoint. In contrast, animals with the smooth Ti implant construct had a 25% (2/8) infection rate. Survival analysis indicated a significant difference between the groups (log-rank test, p = 0.018). Data also indicated that although skin marsupialization was evident in both implant types, animals in the control group had a four times greater marsupialization rate. We concluded that osseointegrated implants incorporating porous-coated Ti subdermal barriers may have the ability to prevent infection by maintaining a healthy, biologically attached epithelial barrier at the skin-implant interface in load-bearing animals up to a 9-month terminus.