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Category: Basic Sciences/Biologics; Arthroscopy Introduction/Purpose: Osteochondral lesions of the talus (OLT) are frequently treated with microfracture and may be augmented with decellularized extracellular matrix or particulated juvenile cartilage. The damaged OLT cartilage is often discarded. Infrequently it is used as a source for autologous chondrocyte expansion and implantation in a subsequent procedure. This resected cartilage has demonstrated variable chondrocyte viability. The influence of cartilage type on chondrocyte viability, whether frayed fibrocartilage from in vivo repair or delaminated hyaline cartilage, remains unknown. Also, little has been reported on the use of the OLT cartilage as a source for immediate cartilage autograft. The purpose of this study is to evaluate chondrocyte quantity and viability from the excised cartilage of OLTs at the time of arthroscopic debridement and microfracture. Methods: Patients aged 18 to 65 undergoing arthroscopic debridement and microfracture of talar OLTs were included. IRB approval of the study protocol was obtained. At the time of ankle arthroscopy, the cartilage from the OLT was collected either by GraftNet™ Autologous tissue collector (GraftNet™ Autologous Tissue Collector, Arthrex; Naples, FL) or manual collection and mincing of the specimen with a scalpel. Chondrocytes were isolated by overnight digestion with 2% collagenase in tissue culture medium at 37°C. The isolated cells were stained with Trypan Blue and counted for total cell number and viability (%), using an automated cell counter. Data collection included patient age, size and location of the OLT, intraoperative surgeon assessment of cartilage type (hyaline cartilage, fibrocartilage, or mixed fibro-hyaline cartilage), histologic determination of cartilage type, weight of the specimen, and chondrocyte cell count and viability. Results: Fourteen OLT specimens were collected. The average size was 0.831 (SD ± 1.10) cm 2 , and average weight 0.19 (SD ± 0.17) grams. Overall, the average number of chondrocytes was 1.12 x106 (SD ± 1.13 x106) cells/mm 2 and 3.99 x106 (SD ± 3.28 x106) cells/gram. The average viability was 72.36% (SD ± 12.25%). Using histologic determination of cartilage type, in hyaline specimens (n = 8, 57.14%) we found 1.08 x106 (SD ± 0.85 x106) chondrocytes/cm 2 , 70.73% (SD ± 11.73 %) viability. In fibrocartilage specimens (n = 2, 14.29%) we found 0.37 x106 (SD ± 0.10 x106) chondrocytes/cm 2 , 78.08% (SD ± 22.03%) viability. In mixed cartilage specimens (n = 4, 28.57%) we found 1.58 x106 (SD ± 1.80 x106) cells/cm 2 , 72.78% (SD ± 11.79%) viability. (Table 1) Conclusion: We found OLT chondrocyte viability of 72.36%, with a trend toward greater chondrocyte concentration and similar viability comparing hyaline and fibro cartilage, respectively. Cartilage type was consistent between intraoperative and histologic assessment in all but four specimens. OLT cartilage has the potential to serve as an immediate intraoperative chondrocyte autograft source for enhancement of talus cartilage repair procedures, eliminating the risk of donor site morbidity and additional surgeries as in culture-based methods. Further study with more patients may shed light on the accuracy of intraoperative cartilage classification, and differences in chondrocyte quantity and viability between cartilage types.

Category: Ankle Arthritis; Ankle Introduction/Purpose: Total ankle arthroplasty (TAA) has surpassed arthrodesis as the gold standard for treatment of end-stage ankle arthritis. The Zimmer Trabecular Metal Total Ankle Implant (Zimmer-Biomet, Warsaw, IN) differs from other newer- generation implants in the transfibular approach, non-mobile-bearing prosthesis, high-molecular-weight polyethylene, curved surface at every interface in the sagittal plane, and shallow resection depth. There is limited mid-term follow-up for this system. Therefore, we sought to report mid-term clinical, functional, and radiographic results we well as complications and survival for patients treated with the transfibular TAA at a minimum of 5-years follow-up. Methods: A retrospective review was performed on patients who underwent primary transfibular TAA by a single surgeon from October 2012 to January 2018. Inclusion criteria was a minimum of 5-years of follow-up with a completed consent for participation in the study and patient-reported outcome measures (PROM). Patient demographics and perioperative data were collected. PROMS included: 12-item Short Form Health Survey (SF-12) physical (PCS) and mental (MCS) component scores; Ankle Osteoarthritis Scale (OAS); and Visual Analog Scale (VAS). Functional outcomes included range of motion (ROM) measured on weightbearing ROM radiographs. Radiographic outcomes included coronal and sagittal alignment. Adverse events and reoperations were reported using the Canadian Orthopedic Foot and Ankle Society (COFAS) Reoperation Coding System (CROCS). Of the consecutive 151 TAAs, 83 (55.0%) ankles are included in this study, evaluated at a mean of 5.8 years clinical and 6.3 years radiographic follow-up. Results: The average age was 60.6 years-old. Preoperative tibiotalar coronal deformity included 27 valgus (10°, range 2-20°) and 25 varus ankles (-9°, range -2--25°), successfully corrected to neutral postoperatively. The postoperative PROMs were: SF-12 PCS: 40.4, SF-12 MCS: 56.0, VAS: 2.3, AOS Pain: 17.0, and AOS Disability: 24.9. Radiographic postoperative tibiopedal ROM was 17.8° dorsiflexion and 21.8° degrees plantarflexion. The overall reoperation rate was 38.6%(n=32) at an average of 28.7 months postoperative. Most common were removal of the fibula hardware (n=21,25.3%) and medial gutter debridement (n=15, 18.1%). There were 3 (3.6%) cases of acute postoperative infection (< 3 months) treated with operative irrigation and debridement but retention of the metal components. There were no cases of septic or aseptic loosening or subsidence. The overall implant survival, defined by retention of the metal components, was 100%. Conclusion: Transfibular TAA is a safe and effective treatment for end-stage ankle arthritis at 5-year mid-term follow-up. This study confirms excellent clinical results including PROMs and ROM as well as radiographic alignment correction and maintenance. The survival rate was 100% for retention of the trabecular metal components. The survival rate to reoperation was 60.7% with patients commonly requiring removal of the fibula hardware or medial gutter debridement. Patients should be counseled on the risk of reoperation preoperatively for postoperative expectation management.

Category: Ankle; Other Introduction/Purpose: Periprosthetic joint infection (PJI) is a devastating complication of arthroplasty. The well known PJI risk factors are useful for identifying high-risk patients but would be even more helpful for clinical decision-making if they are converted to a risk assessment scores. The Mayo Prosthetic Joint Infection Risk Score was based on the data of total hip and knee arthroplasty and has not been validated for application for total ankle arthroplasty. Methods: A series of 398 consecutive cases of total ankle arthroplasty, with minimal follow-up of 6 months, was reviewed for Mayo Periprosthetic Joint Infection Risk Score (Mayo score) and PJI. The patients' Mayo scores and PJI was examined by logistic regression. T-test was performed to compare the Mayo score between the non-infected TAA cases and infected cases. Receiver Operating Characteristic (ROC) was used to identify the critical value of Mayo score for total ankle arthroplasty. Results: There were 12 cases of PJI or 3.0% in the series. Of the 398 patients, the Mayo scores were in the range from -4 to 13 (median 2; interquartile range (IQR) 0-4). Preliminary analysis showed that, by logistic regression, the probability of PJI was increased as increases of the Mayo scores (Fig A). The mean Mayo score of the PJI patients (8.6+-1.8) was significantly greater than the rest of the patients (mean Mayo score 1.9+-3.4; p < .0001). ROC analysis showed that, when a Mayo score was greater than 5, the patient has a high probability of PJI (sensitivity = 100%; specificity = 86.9%; Fig B). Conclusion: This study showed a high correlation between the Mayo score and PJI in total ankle arthroplasty, which is the same trend as in total knee and hip arthroplasty. The preliminary results suggest that strategically focusing on the patients, who have a Mayo score > 5, could be a more efficient approach to prevent PJI after total ankle arthroplasty.

Category: Basic Sciences/Biologics; Diabetes Introduction/Purpose: Charcot neuroarthropathy (CNA) is a destructive joint condition secondary to neuropathic deficiency. For more than a century, the pathogenesis of CNA has been interpreted as unprotected injury and/or uncontrollable inflammation in a neuropathic joint, with little understanding its cellular and molecular pathology. Histologically, a hallmark of CNA is bone and cartilage fragments engulfed by hyperplastic synovium. Synovium is richly innervated. Neuropeptides, such as calcitonin gene- related peptide (CGRP), are secreted by neuronal and non-neuronal cells of the nervous, endocrine, and immune systems, and regulate inflammation. This study investigated the expression of CGRP in the synovial samples collected from CNA and non-CNA joint conditions, and the effects of CGRP on the proliferation and collagenolysis of fibroblast-like synoviocytes (FLS) isolated from CNA synovium in vitro. Methods: For this study, six CNA and 14 non-CNA synovial samples were collected during foot and ankle surgery. The donors included 10 male and 10 female, age from 14 to 79 years (mean 48). The non-CNA conditions consisted of osteoarthritis, ankle instability, Os Trigonum and osteochondritis dissecans. Western blot and immunohistochemistry of CGRP were performed to detect and localize CGRP expression in the CNA and non- CNA synovium. FLS was isolated from CNA synovium and cultured with, or without, human CGRP (10nM) in the culture media, for comparison of cell proliferation. Additionally, FLS were seeded on the collagen-coated 24-well plate and simulated with CGRP (10nM) and recombinant human tumor necrosis factor-alpha (TNF-α). After 7 days, the residual collagen on the bottom of the culture plate were stained and imaged for area measurements. Data were analyzed with t test or one-way Analysis of Variance, followed with post hoc Tukey's test. Results: By western blot, there was significant CGRP expression in the CNA synovium (5/6; Fig1). Except of an intense CGRP band in one of the osteoarthritis samples, only recognizable CGRP bands were shown in the samples of other non-CNA conditions. The average density of CGRP (in greyscale) in the CNA group was about 2-fold of that in the non-CNA group (10126 +- 5346 vs. 5377+-3734; p < 0.05). Immunohistochemistry demonstrated intense CGRP staining in the intimal layer of the CNA synovium (arrows) but not in the non-CNA synovium (Fig1). Treated with CGRP, the number of FLS in tissue culture increased. Cell number doubling time was 1.0 day (+-0.4) for the CGRP treated FLS and 2.2 days (+-0.5) for the control (p < 0.05). When the culture media were supplemented with CGRP and TNF-α, FLS eroded a larger area of collagen coating comparing with TNF-α alone in the media (p < 0.05). Conclusion: A knowledge gap in understanding the molecular and cellular pathology of CNA hampers the development of disease-modifying therapies. This study showed an increased CGRP, with a concentration in the intimal layer, in the CNA synovium as compared with the synovium in the non-CNA foot conditions. The increased CGRP expression in CNA synovium may be part of the unbalanced neuropeptide signaling in the neuropathic pathology. Functionally, CGRP stimulated FLS proliferation and degrading collagen in vitro. These effects suggest that CGRP may play a role in bone and joint destruction in the Charcot foot.

Category: Ankle; Ankle Arthritis; Other Introduction/Purpose: Periprosthetic infection is a devastating complication in total ankle arthroplasty (TAA). Measures that can reduce or eliminate infection occurrence are warranted for further investigation, particularly if they also minimize added costs, operative time or potential adverse effects. There remains insufficient evidence to show definitive benefits or risks associated with use of local antibiotics such as vancomycin for infection prevention. The purpose of this study was to determine the effects of topical vancomycin powder application in TAA and whether it may decrease infection rate in the subacute postoperative period. To our knowledge, this is the first comparative study in a general population undergoing TAA. Methods: A retrospective study was conducted based on a consecutive series of patients who underwent TAA performed by a single senior surgeon. The control group without vancomycin powder was matched to the study group that received application over the implants prior to closure, which was based on when the surgeon transitioned to usage in all patients. Various demographic data for the patients was collected. Only patients with a minimal follow up of 80 days were included in order to capture the perioperative period of infection. Infection rate was based on a definition of deep infection necessitating inpatient hospitalization for IV antibiotics and/or surgical intervention. The associations of various demographic factors and vancomycin application with infection rate were calculated with chi square and odds ratio. Significance level was set to 0.05. Results: A total of 252 patients (n=103 without, and 149 with vancomycin powder) were included in the study. The overall infection rate was 3.2%, which was not correlated with a history of diabetes, BMI, additional concurrent surgery, or age, but was associated with current positive smoking status (p=0.03). The infection rates between the groups that received or did not receive vancomycin powder were not statistically significantly different (2.7% vs 3.9%, respectively, p=0.59). The odds ratio (OR) of infection with vancomycin powder was not statistically significant either, at OR=0.68 (95% CI 0.17-2.81). However, the vancomycin group did have a significantly higher number of patients with previous wound issues (12 vs. 2; p=0.04) and infection (7 vs. 0; p=0.04) as well as concomitant surgeries (81 vs. 41; p=.02). Conclusion: The consequences of periprosthetic TAA infection are substantial, and the risks should be mitigated. Although this study did not detect a statistically significant difference in infection rates between patients who received or did not receive vancomycin powder, there was a trend towards risk reduction by about 30%. In addition, current smoking was found to be significantly associated with higher rates of infection and smoking cessation can serve as another point of intervention. As the complexity of patient factors and indications for performing total ankle arthroplasty expand, vancomycin powder offers a relatively cost-effective, safe, and convenient option to potentially reduce infection.

Category: Other; Ankle; Arthroscopy; Bunion; Hindfoot; Lesser Toes; Midfoot/Forefoot; Trauma Introduction/Purpose: The COVID-19 pandemic has brought a major increase in telemedicine utilization to conserve resources and maintain social distancing. The rapid transition over a short period of time has exposed challenges and limitations of virtual visits, especially with regards to performing an effective and efficient physical examination. Within months, orthopedic telemedicine guides were published for patient education and facilitating the musculoskeletal examination. These publications, however, included only a short video clip to explain to the patient how to participate in the examination during a telemedicine visit. In the foot and ankle subspecialty, the closely adjacent complex anatomy compared to other areas of the body presents unique challenges for physical exam. Methods: To optimize the efficiency and efficacy of the examination portion of the visit for both patients and providers, we designed and recorded an instructional video that is sent to all patients by email a minimum of 1 week prior to their appointment. This video consists of a step-by-step guide on how to take suitable photos and videos of their feet and ankles for the physical examination portion of their visit. These media files are then directly uploaded by the patients into their EMR or emailed to the physician's office to be uploaded for access by the provider before and during the virtual visit. Along with the video, patients are given a link to test their microphone and camera in advance of the visit to ensure adequate functionality and positioning. Results: Our pre-appointment instructional video has been effective to methodically replicate the most important aspects of an in-person foot and ankle evaluation. Both physicians and patients appear to benefit from the tutorial video by having more time during the telemedicine visit to focus on history, assessment, and plan. Patients are more actively involved in the clinical data collection process because they are educated on what to expect and how to best prepare and participate in telemedicine visits. Moreover, having a set of systematic and detailed photos and recordings of the patient's foot and ankle beforehand better focuses interaction and discussion with the patient during the telemedicine encounter. Additionally, these are saved into the patient's electronic medical record, available for reference and later review. Conclusion: Physical examination plays a critical role in diagnosis and treatment in the orthopedic foot and ankle subspecialty. More research is needed to discern how best to utilize virtual modalities as they become more commonplace, not just from the imposition from the COVID pandemic but also from the standpoint of patient convenience and time efficiency. Here we have described our creation of a pre-visit educational video to guide and engage patients on acquiring invaluable physical examination information for the orthopedic foot and ankle telemedicine encounter.

Level of Evidence: Level V, clinical tips.

Category: Basic Sciences/Biologics; Hindfoot; Sports Introduction/Purpose: Different animal models of Achilles tendinopathy have been proposed in the literature. They usually involve the induction of tendinopathic findings by either chemical stress (most commonly with one or more injections of collagenase, mimicking intrinsic factors) or mechanical stress (by repetitive exercise-induced stress with treadmill running exercises, simulating extrinsic risk factors). To date, no study has evaluated the combination of a mechanical trigger followed by collagenase injections, replicating the logical and sequential steps involved in the development the human pathology. Our goal was to develop this novel animal model of Achilles tendinopathy and to compare histological and functional findings with animals subjected to isolated mechanical or chemical stress, as well as to controls. Methods: Sixty-four Sprague-Dawley rats were divided into four groups (n=16): isolated treadmill running protocol (15o uphill running, 20meters/minute, 1hour/day, 3 weeks duration, weeks 2-4); isolated injections of collagenase (0.1mg each, 3 injections total, weeks 5-7); treadmill protocol (weeks 2-4) followed by three consecutive collagenase injections (weeks 5-7); and controls, no running and three injections of normal saline (weeks 5-7). Five animals from each group were sacrificed at weeks 8 and 10. Six animals by group were sacrificed at week 12. Gait analysis was performed at weeks one (after acclimation), five (following running protocol), eight (following injection protocol) and twelve (just before latest sacrifice time-point). Histological findings were assessed by the Movin Tendinopathy Score (eight parameters, scored from 0-3, total score 0-24), assessing collagen arrangement, structure, and stainability, cellularity, vascularity, nuclear rounding, hyalinization and presence of glycosaminoglycans. Gait parameters included stand and swing times, stride length, duty cycle and swing length. Results: After 8 weeks, significantly increased tendinopathic scores (p

Category: Basic Sciences/Biologics Introduction/Purpose: Tendinopathy causes pain and dysfunction. Conservative treatments of tendinopathy include physical therapy, rest, training modification, splintage, taping, cryotherapy, electrotherapy, shock wave therapy, hyperthermia and local injections. These therapies, however, do not target specific pathology and are unable to modify the progression of the disease. The pathology of tendinopathy is considered as a continuum of tendon degeneration but the precise molecular pathology of tendinopathy is still lacking. Accumulation of advanced glycation end-products (AGEs) happens during connective tissue aging and affects tendon viscoelasticity. Estrogen, which functions via estrogen receptor (ER), is essential for metabolism and plays a role in tendon health and aging. This preliminary study investigated the expression of AGEs and ER in tendinopathy. Methods: This study included six patients with posterior tibial tendon (PTT) tendinopathy (age from 20 to 72 years, 5 male and 1 female). Normal flexor digitorum longus (FDL) tendon samples were collected from three donors (female, age from 42 to 51 years, approved by IRB). Tissue samples of tendinopathy and normal tendon were fixed with 4% paraformaldehyde and sectioned with a cryostat. Picrosirius Red stain was used for collagen structure. Immunohistochemistry of AGEs, AGEs receptor (RAGEs) and ER was performed on separate tissue sections. The primary antibody of AGEs, RAGEs and ER was separately applied onto the tissue sections for one hour. The proper secondary antibody that conjugated with peroxidase was applied and incubated for 30 min, followed by the application of 3,3’-diaminobenzidine for chromogenic detection of protein expression. Cell nuclei were conterstained with hematoxylin. Tissue sections omitted application of primary antibody were used as negative controls. Results: Tissue sections stained with Picosirius Red were examined under a circular polarizing microscope. Tendinopathy of PTT was confirmed by a disorderly organized collagen network and highly heterogeneous collagen contents. Cellularity was greatly increased in the pathological region. ER was uniformly expressed by tenocytes in the PTT pathologic tissues. The ER-positive cells were particularly located in areas featured disorganized collagen fibers and dense cellularity. After antigen retrieval with sodium citrate buffer, enhanced expression of AGEs was detected in the tendon tissue of PTT. RAGEs was expressed by a few tenocytes in the pathological region of PTT sections but not in normal FDL sections. Conclusion: The long-term goal of this study is to identify key molecules involved in tendinopathy, for developing disease- modifying therapies. This preliminary study revealed the expression of several molecules, which are associated with connective tissue aging process, in tendinopathy. The accumulation of AGEs and increased expression of ER in the pathological tendon add more molecular features to tendinopathy. Future study will focus on the pathological role of these molecules in regulation of tenocytes.

Category: Basic Sciences/Biologics Introduction/Purpose: Diabetic foot infection (DFI), including skin infection and osteomyelitis, is a severe complication of late- stage diabetes. Mesenchymal stem cells (MSCs) facilitate bacterial clearance. In bacterial infection, MSCs, via paracrine mediators, regulate the host cell metabolism and inflammatory response. Particularly, MSCs augment the antibacterial function of neutrophils. It is generally believed that hyperglycemia in diabetes is toxic to MSCs/progenitors and detrimental to their regenerative function. It is unknown, however, whether the antibacterial function of MSCs is compromised in diabetes. Methods: Bone marrow samples from 6 diabetic and 4 non-diabetic patients (approved by IRB) were used for MSC isolation. 1. MSCs from both diabetic and non-diabetic patients were treated with lipopolysaccharides (LPS), a bacterial wall component, for 6 hours. The tissue culture media were collected as conditioned medium. E. coli from a single colony were cultured with addition of the conditioned medium generated by either diabetic or non-diabetic MSCs and inoculated on LB-agar plates overnight. Bacterial colonies were counted. 2. Human macrophages were isolated from umbilical cord blood and co-cultured with either diabetic or non-diabetic MSCs, in a trans-well system, for 24 hours. The macrophages were then cultured with heat-inactivated E. coli for one hour. After extensive washing, macrophage and bacteria were stained with Pappenheim method. Bacterial phagocytosis of macrophages, after co- cultured with diabetic or non-diabetic MSCs, was assessed under a microscope. Results: There was no statistical difference in the number of E. coli colonies when regular medium produced by diabetic and non- diabetic MSCs was added into the bacterial culture. When the diabetic and non-diabetic MSCs were treated with LPS and the conditioned medium was collected and added into bacterial cultures, E. coli colonies increased in the diabetic group, about 3 fold, as compared with the non-diabetic group (p < 0.05). Macrophages were counted in defined areas of the Petri dishes and designated as infected or uninfected, according to the presentation of bacterial bodies or not. While the infection rate of macrophages co-cultured with non-diabetic MSCs was 85% (±5.5%), it was 70% (±6.6%) when macrophages were co-cultured with diabetic MSCs (p = 0.006). Conclusion: MSCs-produced paracrine factors suppressed the growth of E. coli but diabetic and non-diabetic MSCs had no difference in such a function. Activation with LPS did not augment the non-diabetic MSCs but weakened diabetic MSCs in suppression of bacterial growth. MSCs regulate macrophages in bacterial phagocytosis. Diabetic MSCs, however, had a limited role in regulation of macrophages. This study demonstrated that MSCs in diabetic patients are compromised in anti-bacterial infection. The results not only deepen the understanding of bacterial infection in diabetes but also open up new strategy to control bacterial infection in diabetic patients.

Category: Basic Sciences/Biologics Introduction/Purpose: Achilles tendinopathy and rupture are common and generally can heal without aggressive intervention. When a large defect of Achilles tendon is left by trauma or severe tendinopathy, however, it is a challenge to restore the function of Achilles tendon, because of the size of the tendon and the limitation in Achilles autograft. In contrast, allograft of Achilles tendon does not have restrain of supply. The biology, biomechanics and function of the transplanted Achilles allograft, however, are unknown. Particularly, the revitalization of Achilles allograft is a concern. Mesenchymal stem cells (MSCs) are known for their capability of multi-lineage differentiation and regenerative potentials. Methods: Achilles allografts were harvested from donor rats (approved by Institutional Animal Care and Usage Committee) and kept at -80ºC before transplantation. Subcutaneous adipose tissue was harvested from the would-be allograft recipient rats for isolation of MSCs. MSCs were culture expanded and characterized. On the day of allograft transplantation, adipose tissue derived MSCs were collected and applied onto allografts (1x105 per allograft). Achilles tendon was resected from the left hind limb of the adipose tissue donor rats. Achilles allograft, with or without autologous MSCs, was implanted and sutured with calf muscles proximately and calcaneus distally. Animal gait was recorded in the week prior to Achilles allograft transplantation (week 0) and every week postoperatively, using a CatWalk system. The transplanted Achilles allografts, with or without MSC incorporation, as well as the normal Achilles tendon in the opposite limbs were harvested at 4 weeks for biomechanical testing and histology. Results: The operated limbs altered gait significantly. By week 4, the recoveries of stand index (speed at which the paw loses contact with ground) and duty cycle (percentage of the stance phase in a step cycle) of the reconstructed limbs were not statistically different between the Achilles allograft group and Achilles allograft+MSCs group. Maximum load of failure among Achilles allograft group (27.2±11.5 N), Achilles allograft+MSCs group (27.6±6.4 N) and normal Achilles tendon group (19.9±9.9 N) was not significantly different. On histology, cellularity was generally higher in Achilles allograft+MSCs group than Achilles allograft group (average cellularity grade 2.7±0.5 vs 1.7±0.5). Type III collagen staining was more intense in the Achilles allograft+MSCs group than in the Achilles allograft group. Conclusion: In this pilot study, the allograft incorporated with autologous MSCs demonstrated no significant differences from Achilles allograft alone in maximum failure load and gait analysis. Supplementation of MSCs increased the cellularity and led to more active matrix remodeling in the allograft but these biological improvements did not translate into rat gait and the strength of the implanted allograft. It may be necessary to follow up the animals for an extended period because the remodeling of Achilles allograft takes longer than 4 weeks. In addition, the small size of the rat Achilles allograft might falsely show an accelerated revitalization.

Category: Basic Sciences/Biologics Introduction/Purpose: Managing Charcot Neuroarthropathy (CNA) remains a challenge facing the foot and ankle surgeons. Lack of understanding the pathogenesis of the disease prevents the development of novel therapies. Our previous studies revealed the involvement of fibroblast-like synoviocytes in the pathology of CNA. Residing in the synovium are also mesenchymal stem cells (MSCs), which maintain tissue homeostasis. In this study, MSCs isolated from the synovium of CNA and non-CNA patients were comparatively analyzed for their potential role in the pathology of CNA. Methods: Synovial samples were collected from CNA patients (n=7) and patients with osteoarthritis or intraarticular fracture (non-CNA; n=7), during foot and ankle procedures (approved by IRB). The synovial samples were minced and digested with 0.1% collagenase to isolate MSCs. The cells were plated and cultured in low density for counting colony-forming units-fibroblast (CFU- F), which is indicative of the number of MSCs. The colonies formed by CNA-MSCs and non-CNA MSCs were further characterized by size and imaging density. CNA-MSCs and non-CNA MSCs were cultured in adipogenic, osteogenic and chondrogenic differentiation media. The differentiation potentials of CNA-MSCs and non-CNA MSCs were assessed with histochemical staining and quantitative gene expression. Results: CFU-F: The number of colonies formed by CNA-MSCs was 6 ± 3.5 per (1/2) plate, while it was 43 ± 21.6 by the non- CNA MSCs (p < 0.05; Fig 1). The average size (pixels) of colonies was smaller in the CNA-MSCs than the non-CNA MSCs (4780 vs. 7960). When the colonies were stratified into high, medium and low density subgroups, colonies formed by the CNA-MSCs had a reduced density compared with the non-CNA MSCs (30±6 vs. 35±10) in the high density subgroup. Differentiation: Expression of adipogenic marker gene PPAR-γ by CNA-MSCs was less than half of that by non-CNA MSCs. The expression of osteogenic marker gene RUNX2 by CNA-MSCs was a third of that by the non-CNA MSCs. Similarly, the expression of chondrogenic marker genes, including SOX9 and type II collagen, by the CNA-MSCs declined as compared with the non- CNA MSCs. Conclusion: The synovium in CNA joints had fewer MSCs and that may impact joint’s response to inflammation and tissue repair. Furthermore, CNA-MSCs were declined in differentiation potentials. Analyses of the size and density of the colonies formed by CNA-MSCs showed that a subpopulation of MSCs was particularly affected. Further study will focus on the pathological role of the high-density subpopulation of MSCs in the development of CNA.

Category: Basic Sciences/Biologics Introduction/Purpose: The pathology of Charcot neuroarthropathy (CNA) presents inflammation, and bone and cartilage destruction. It is still unclear how the neuropathic signals lead to joint inflammation and destruction. Neuropeptides are secreted by neurons and involve in the regulation of inflammation. Synovium is richly innervated and known to play a critical role in the pathology of inflammatory arthritis. This study is designed to investigate the distribution of several neuropeptides, such as vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP), in CNA synovium, in comparison with non-CNA synovium, with immunohistochemistry. Methods: Synovial samples were collected from CNA patients (n=3) and patients with osteoarthritis and intraarticular fracture (non-CNA; n=3), during foot and ankle surgery (approved by IRB). The tissue samples were fixed with 4% paraformaldehyde, embedded in O.T.C media and sectioned with a cryostat. From each patient, 6-9 sections were randomly selected for immunohistochemistry. After blocking the tissue sections with normal serum, primary antibody of SP, VIP and CGRP was separately applied and incubated at 4ºC overnight. The biotinylated secondary antibody and ABC kit (Victor Laboratories) were applied subsequently. DAB (3,3’-diaminobenzidine) was used for colorimetric detection (brown) of immunohistochemical reactions. The cell nuclei were stained with hematoxylin. The staining was viewed under a microscope and images were taken with a digital camera. Results: In the non-CNA synovium, SP was stained around neuro-vascular bundles and intensely stained on the surface of the synovium, i.e. the intimal layer. In the CNA synovium, SP staining was increased and more diffuse into the subintimal layer of the synovium (Fig 1). In non-CNA synovium, VIP was detected along the surface area of the synovium but not limited in the intimal layer. In CNA synovium, VIP was a similar staining pattern with a reduced staining intensity. The CGRP staining was primarily limited in the intimal layer in the non-CNA synovium. In CNA synovium, CGRP was stained in the intimal layer with an increased intensity. Conclusion: SP, VIP and CGRP are well-studied neuropeptides and have broad biological and pathological functions. All of them were detected in the intimal layer, where fibroblast-like synoviocytes are located. Fibroblast-like synoviocytes are fundamental elements of joint inflammation and are capable of degradation of bone and cartilage directly and indirectly. The changed patterns of SP, VIP and CGRP distributions in CNA synovium revealed by immunohistochemistry indicate a possible pathological pathway in CNA development: the unbalanced neuropathic signals direct fibroblast-like synoviocytes to an inflammatory state, which trigger the cascade of bone and cartilage destruction in CNA.