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Background: Particulated autograft cartilage implantation is a surgical technique that has been previously described for the repair of osteochondral lesions of the talus (OLT). It uses cartilage fragments harvested from the OLT that are minced into 1-2-mm 3 fragments and then immediately reimplanted back into the chondral defect and sealed with fibrin glue during a single-stage surgery. The purpose of this study was to characterize the suitability of these minced cartilage fragments as immediate autograft for the treatment of OLTs. Methods: Thirty-one patients undergoing primary arthroscopic surgery for their OLT consented to have their loose or damaged cartilage fragments removed and analyzed in the laboratory. Harvested specimens were minced into 1- to 2-mm 3 fragments and cell count, cell density, and cell viability were determined. In addition, physical characteristics of the OLT lesion were recorded intraoperatively and analyzed including size, location, Outerbridge chondromalacia grade of the surrounding cartilage, density of underlying bone, and whether the surgeon thought the OLT was primarily hyaline or fibrocartilage. Results: An average of 419 000 cells was able to be obtained from the harvested OLT fragments. The cells were 71.2% viable after mincing. Specimens from younger patients and from lesions with worse chondromalacia adjacent to the OLT had significantly higher cell numbers. Those from lateral lesions and with worse neighboring chondromalacia had a significantly higher cell density. None of the remaining physical OLT characteristics studied seemed to significantly affect cell number or viability. Conclusion: A large number of viable cells are available for immediate autografting by removing the loose or damaged cartilage from an OLT and mincing it into 1- to 2-mm 3 fragments. These can be reimplanted into the chondral defect in a single-stage surgery. Future clinical studies are needed to determine if the addition of these live autologous cells either alone or in conjunction with other techniques significantly improves the quality of the repair tissue and clinical outcomes. Level of Evidence: Level IV, case series.

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.

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.