The biology and function of orthotopic transplantation of Achilles tendon allograft are unknown. Particularly, the revitalization of Achilles allograft is a clinical concern. Achilles allografts were harvested from donor rats and stored at −80 °C. Subcutaneous adipose tissue was harvested from the would-be allograft recipient rats for isolation of mesenchymal stem cells (MSCs). MSCs were cultured with growth differentiation factor-5 (GDF-5) and applied onto Achilles allografts on the day of transplantation. After the native Achilles tendon was resected from the left hind limb of the rats, Achilles allograft, with or without autologous MSCs, was implanted and sutured with calf muscles proximally and calcaneus distally. Animal gait was recorded presurgery and postsurgery weekly. The animals were sacrificed at week 4, and the transplanted Achilles allografts were collected for biomechanical testing and histology. The operated limbs had altered gait. By week 4, the paw print intensity, stance time, and duty cycle (percentage of the stance phase in a step cycle) of the reconstructed limbs were mostly recovered to the baselines recorded before surgery. Maximum load of failure was not different between Achilles allografts, with or without MSCs, and the native tendons. The Achilles allograft supplemented with MSCs had higher cellularity than the Achilles allograft without MSCs. Deposition of fine collagen (type III) fibers was active in Achilles allograft, with or without MSCs, but it was more evenly distributed in the allografts that were incubated with MSCs. In conclusion, orthotopically transplanted Achilles allograft healed with host tissues, regained strength, and largely restored Achilles function in 4 wk in rats. It is therefore a viable option for the reconstruction of a large Achilles tendon defect. Supplementation of MSCs improved repopulation of Achilles allograft, but large animal models, with long-term follow up and cell tracking, may be required to fully appreciate the functional benefits of MSCs.
Aims Femoroacetabular impingement (FAI) is a potential cause of hip osteoarthritis (OA). The purpose of this study was to investigate the expression profile of matrix metalloproteinases (MMPs) in the labral tissue with FAI pathology. Methods In this study, labral tissues were collected from four FAI patients arthroscopically and from three normal hips of deceased donors. Proteins extracted from the FAI and normal labrums were separately applied for MMP array to screen the expression of seven MMPs and three tissue inhibitors of metalloproteinases (TIMPs). The expression of individual MMPs and TIMPs was quantified by densitometry and compared between the FAI and normal labral groups. The expression of selected MMPs and TIMPs was validated and localized in the labrum with immunohistochemistry. Results On MMP arrays, most of the targeted MMPs and TIMPs were detected in the FAI and normal labral proteins. After data normalization, in comparison with the normal labral proteins, expression of MMP-1 and MMP-2 in the FAI group was increased and expression of TIMP-1 reduced. The histology of the FAI labrum showed disorderly cell distribution and altered composition of thick and thin collagen fibres. The labral cells expressing MMP-1 and MMP-2 were localized and their percentages were increased in the FAI labrum. Immunohistochemistry confirmed that the percentage of TIMP-1 positive cells was reduced in the FAI labrum. Conclusion This study established an expression profile of MMPs and TIMPs in the FAI labrum. The increased expression of MMP-1 and MMP-2 and reduced expression of TIMP-1 in the FAI labrum are indicative of a pathogenic role of FAI in hip OA development. Cite this article: Bone Joint Res. 2020;9(4):173–181.
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.
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