Introduction. Matrix metalloproteinases (MMP) play a key role in cartilage degradation in osteoarthritis. Statins are a potential suppressor of MMPs. The aim of this research was to assess the efficacy of Pravastatin in suppressing MMP gene and protein expression in an in vitro model. Methods. We stimulated normal human chondrocytes with IL-1b for 6 hours to induce MMP expression and then treated with Pravastatin (1, 5 & 10 mM) for a further 18 hours. Cells stimulated with IL-1b but not treated with Pravastatin served as controls. Real-time PCR was used to assess expression of MMP-3 and MMP-9 mRNA. MMP enzyme activity was assessed using a fluorescent MMP-specific substrate. Staistical analysis was performed using ANOVA. Results. MMP-3 and -9 mRNA expression was reduced at all concentrations tested with a statistically significant trends in reduction (p=0.002 and < 0.001 respectively). Analaysis of culture supernatants revealed that Pravastatin treatment led to a reduction in total MMP activity but not to a statistically significant degree (p=0.07). Conclusion. We conclude that treatment with Pravastatin of stimulated human chondrocytes leads to a down regulation of selected MMP genes and a reduction in MMP enzyme activity. Our results are further evidence that statins may have a role to play in the treatment of osteoarthritis and other disorders of cartilage degradation

Introduction: After a meniscus trauma, preservation of the meniscus is the most important surgical goal. The use of scaffolds colonized with meniscus cells (fibrochondrocytes) to reconstruct meniscal defects seem to be a promising way for the treatment of a meniscus trauma. The goal of our investigations was the analysis of expression of different anabolic and catabolic factors in human fibrochondrocytes after seeding these cells onto a collagen I scaffold to investigate the regenerative potential of such a construct for the treatment of meniscus tears.

Material and Methods: Human meniscus tissue was digested in collagenase and dispase and cells were characterized by immunohistochemistry. To test scaffolds, we used a commercially available bovine collagen I matrix approved for surgical purposes. The scaffold was colonized with human fibrochondrocytes in a density of 106 cells per cm2. Cells expanded at the same ínoculation density w/o scaffold served as mock-controls. After 14 and 28 days in culture, the cells were extracted from the scaffold by aid of collagenase (Sigma, Deisenhofen, FGR) and analyzed for the expression of different factors, including IL-1β, IL-6, TGF-β, TIMP-1, TIMP-3, MMP-1, and MMP-3 using a quantitative RT-PCR-technology.

Results: Bovine collagen I matrices could be colonized with human fibrochondrocytes. After 14 and 28 days of incubation on the scaffolds, the cells show the same mRNA expression levels of IL-1β, TIMP-1, TIMP-3, and TGF-β when compared to controls. In contrast, after 14 days IL-6 (12.7-fold ± 4.4, p< 0.001), MMP-1 (11.3-fold ± 2.4, p< 0.001), and MMP-3 (13.7-fold ± 6.8, p< 0.031) were upregulated on transcription levels in the scaffold when compared to controls after the same period of culture. After 28 days of culture in scaffold the expression of MMP-3 was upregulated 78.2-fold (± 7.4, p< 0.0001), MMP-1 (71.3-fold ± 5.9, p< 0.0001) and IL-6 was elevated 98.9-fold (± 9.1, p< 0.0001) compared to controls.

Discussion/Conclusion: We were able to cultivate and characterize human fibrochondrocytes from menisci of the knee joint colonized onto a bovine collagen I matrix. We could show that meniscus cells revealed a significantly increased expression of MMP-1 and MMP-3, and also a significant elevation of IL-6 mRNA after 14 and 28 days of culture. No changes were found in the expression levels of IL-1β, TGF-β, and the TIMPs. This suggests that the meniscus cells colonized onto a bovine collagen I scaffold produce a considerable amount of catabolic or inflammatory factors. This may lead to a destruction of the scaffold-matrix itself and the extracellular matrix of the meniscus. Secondly, IL-6 could induce a global inflammation around the scaffold by activating the IL-6 inflammation cascade.

*Winner of ISFR Young Investigator Award

Purpose: The aim of this study was to compare the temporal expression pattern of factors related to cartilage and bone formation and endochondral ossification during standard and delayed bone healing for a more in-depth understanding of the molecular basis of disturbed bone healing and to elucidate suitable timing for substitution of factors to stimulate the healing process.

Methods: A tibial osteotomy was performed in two groups of sheep (n=30 each) and stabilized with either a rigid external fixator leading to standard healing or with a mechanically critical one leading to delayed healing. Hematoma/callus tissue was harvested 4, 7, 14, 21 and 42 days postop. qPCR was employed to determine the expression patterns of BMPs and other molecules.

Results: Gene expressions of BMP2, BMP4, BMP7, Noggin, MMP9 and MMP13 were distinctly lower in the delayed compared to the standard healing group at several time points from day 14, whilst no differential gene expression of Coll II and Coll X was found between both groups. Among the BMPs, BMP7 showed the most markedly differential expression. The first evident difference in BMP7 expression between both groups was found at day 14 suggesting that exogen substitution in the context of a therapeutic approach should be postponed. The differential expression pattern of both MMP9 and MMP13 suggests that there might be a failure or delay in endochondral ossification in delayed bone healing.

Conclusion: Downregulation in gene expression of osteogenic BMPs and cartilage matrix degrading MMPs may account for a considerable delay of bone healing.

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

Introduction. Within articular cartilage, chondrocytes reside within the pericellular matrix (PCM), collectively constituting the microanatomical entity known as a chondron. The PCM functions as a pivotal protective shield and mediator of biomechanical and biochemical cues. In the context of Osteoarthritis (OA), enzymatic degradation of the PCM is facilitated by matrix metalloproteinases (MMPs). This study delves into the functional implications of PCM structural integrity decline on the biomechanical properties of chondrons and impact on Ca. 2+. signaling dynamics. Method. Chondrons isolated from human cartilage explants were incubated with activated MMP-2, -3, or -7. Structural degradation of the pericellular matrix (PCM) was assessed by immunolabelling (collagen type VI and perlecan, n=5). Biomechanical properties of chondrons (i.e. elastic modulus (EM)) were analyzed using atomic force microscopy (AFM). A fluorescent calcium indicator (Fluo-4-AM) was used to record and quantify the intracellular Ca. 2+. influx of chondrons subjected to single cell mechanical loading (500nN) with AFM (n=7). Result. Each of the three MMPs disrupted the structural integrity of the PCM, leading to attenuated fluorescence intensity for both perlecan and collagen VI. A significant decrease of EM was observed for all MMP groups (p<0.005) with the most notable decrease observed for MMP-2 and MMP-7 (p<0.001). In alignment with the AFM results, there was a significant alteration in Ca. 2+. influx observed for all MMP groups (p<0.05), in particular for MMP-2 and MMP-7 (p<0.001). Conclusion. Proteolysis of the PCM by MMP-2, -3, and -7 not only significantly alters the biomechanical properties of articular chondrons but also affects their mechanotransduction profile and response to mechanical loading, indicating a close interconnection between these processes. These findings underscore the influence of an intact pericellular matrix (PCM) in protecting cells from high stress profiles and carry implications for the transmission of mechanical signaling during OA onset and progression

Purpose of the study: The purpose of this study was to compare two types of treatment for fractures of the distal radius with posterior shift: the volar locking plate (c) or mixed multiple pinning (MMP). We conducted a prospective randomised trial. Material and methods: One hundred ten patients aged over 50 years victims of an articular or extra-articular fracture of the distal radius with posterior shift were included in this study. Mean age was 74 years. Patients were recruited via our emergency unit. After obtaining the written informed consent of the patients, patients were assigned to a treatment group using the nQuery Advisor 6.01 available on the internet, 24 hours/d 7d/7. Patients were treated by one of the two surgical techniques according to the randomisation. Patients were reviewed at 3 and 6 weeks and at 3 and 6 months. The DASH and Herzberg scores were noted and plain x-rays of the wrist (ap and lateral views) were obtained at each visit. Results: Fifty-two patients were treated with MMP and 50 with VLP. Postoperative anteversion of he radial glenoid was significantly better in patients treated with MMP. At six months, the DASH and Herzberg score were significantly better in the LAP group. Discussion: MMP allows better anteversion of the glenoid than VLP. However, with MMP there is a risk of over reduction (15% of patients in our series). Treatment with VLP should enable restoration of better radius length with a lesser loss at three months than with MMP. All studies reported, irrespective of the function score used, have found better functional outcome with plating than with pinning. Conclusion: MMP offers a less costly alternative for the treatment of most all distal fractures of the radius with posterior shift. This option provides quite satisfactory clinical and radiographic outcomes. There is a risk of postoperative defect in reduction or stability with MMP, suggesting surgeons should opt for another technique, VLP for example

The purpose of this study was to evaluate whether AGEs induce annulus fibrosus (AF) cell apoptosis and to further explore the mechanism by which this process occurs. AF cells were treated with various concentrations of AGEs for 3 days. Cell proliferation was measured by the Cell Counting Kit-8 (CCK-8) and EdU incorporation assays. Cell apoptosis was examined by the Annexin V/PI apoptosis detection kit and Hoechst 33342. The expression of apoptosis-related proteins, including Bax, Bcl-2, cytochrome c, caspase-3 and caspase-9, was detected by western blotting. In addition, Bax and Bcl-2 mRNA expression levels were detected by RT-PCR. Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) production of AF cell were examined by JC-1 staining and DCFH-DA fluorescent probes, respectively. Our results indicated that AGEs had inhibitory effects on AF cell proliferation and induced AF cell apoptosis. The molecular data showed that AGEs significantly up-regulated Bax expression and inhibited Bcl-2 expression. In addition, AGEs increased the release of cytochrome c into the cytosol and enhanced caspase-9 and caspase-3 activation. Moreover, treatment with AGEs resulted in a decrease in MMP and the accumulation of intracellular ROS in AF cells. The antioxidant N-acetyl-L-cysteine significantly reversed AGE-induced MMP decrease and AF cell apoptosis. These results suggest that AGEs induce rabbit AF cell apoptosis and mitochondrial pathways may be involved in AGE-mediated cell apoptosis, which may provide a theoretical basis for diabetic IVD degeneration

Aims. This study aimed to define the histopathology of degenerated humeral head cartilage and synovial inflammation of the glenohumeral joint in patients with omarthrosis (OmA) and cuff tear arthropathy (CTA). Additionally, the potential of immunohistochemical tissue biomarkers in reflecting the degeneration status of humeral head cartilage was evaluated. Methods. Specimens of the humeral head and synovial tissue from 12 patients with OmA, seven patients with CTA, and four body donors were processed histologically for examination using different histopathological scores. Osteochondral sections were immunohistochemically stained for collagen type I, collagen type II, collagen neoepitope C1,2C, collagen type X, and osteocalcin, prior to semiquantitative analysis. Matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 levels were analyzed in synovial fluid using enzyme-linked immunosorbent assay (ELISA). Results. Cartilage degeneration of the humeral head was associated with the histological presentation of: 1) pannus overgrowing the cartilage surface; 2) pores in the subchondral bone plate; and 3) chondrocyte clusters in OmA patients. In contrast, hyperplasia of the synovial lining layer was revealed as a significant indicator of inflammatory processes predominantly in CTA. The abundancy of collagen I, collagen II, and the C1,2C neoepitope correlated significantly with the histopathological degeneration of humeral head cartilage. No evidence for differences in MMP levels between OmA and CTA patients was found. Conclusion. This study provides a comprehensive histological characterization of humeral cartilage and synovial tissue within the glenohumeral joint, both in normal and diseased states. It highlights synovitis and pannus formation as histopathological hallmarks of OmA and CTA, indicating their roles as drivers of joint inflammation and cartilage degradation, and as targets for therapeutic strategies such as rotator cuff reconstruction and synovectomy. Cite this article: Bone Joint Res 2024;13(10):596–610

Matrix metalloproteinase enzymes (MMPs) play a crucial role in the remodeling of articular cartilage, contributing also to osteoarthritis (OA) progression. The pericellular matrix (PCM) is a specialized space surrounding each chondrocyte, containing collagen type VI and perlecan. It acts as a transducer of biomechanical and biochemical signals for the chondrocyte. This study investigates the impact of MMP-2, -3, and -7 on the integrity and biomechanical characteristics of the PCM. Human articular cartilage explants (n=10 patients, ethical-nr.:674/2016BO2) were incubated with activated MMP-2, -3, or -7 as well as combinations of these enzymes. The structural degradative effect on the PCM was assessed by immunolabelling of the PCM's main components: collagen type VI and perlecan. Biomechanical properties of the PCM in form of the elastic moduli (EM) were determined by means of atomic force microscopy (AFM), using a spherical cantilever tip (2.5µm). MMPs disrupted the PCM-integrity, resulting in altered collagen type VI and perlecan structure and dispersed pericellular arrangement. A total of 3600 AFM-measurements revealed that incubation with single MMPs resulted in decreased PCM stiffness (p<0.001) when compared to the untreated group. The overall EM were reduced by ∼36% for all the 3 individual enzymes. The enzyme combinations altered the biomechanical properties at a comparable level (∼36%, p<0.001), except for MMP-2/-7 (p=0.202). MMP-induced changes in the PCM composition have a significant impact on the biomechanical properties of the PCM, similar to those observed in early OA. Each individual MMP was shown to be highly capable of selectively degrading the PCM microenvironment. The combination of MMP-2 and -7 showed a lower potency in reducing the PCM stiffness, suggesting a possible interplay between the two enzymes. Our study showed that MMP-2, -3, and -7 play a direct role in the functional and structural remodeling of the PCM. Acknowledgements: This work was supported by the Faculty of Medicine of the University of Tübingen (grant number.: 2650-0-0)

Early identification of patients at risk for impaired tendon healing and corresponding novel therapeutic approaches are urgent medical needs. This study aimed to clarify the role of CD3+ T-cells during acute Achilles tendon (AT) healing. Blood and hematoma aspirate were taken from 26 patients during AT reconstruction, and additional blood samples were obtained during clinical follow-up at 6, 26 and 52 weeks after surgery. T-cell subsets were analyzed by flow cytometry using CD3, CD4, CD8, CD11a, CD57 and CD28 antibodies. Clinical follow-up included functional tests, MRI assessments, and subjective questionnaires. In vitro, the functional behavior of patient-derived tenocytes was investigated in co-cultures with autologous unpolarized CD4+ or CD8+ T-cells, or IFNy-polarized CD8+ or IL17-polarized CD4+ Tcells (n=5-6). This included alterations in gene expression (qPCR), MMP secretion (ELISA), migration rate (scratch wound healing assay) or contractility (collagen gels). Analysis revealed that elevated CD4+ T-cell levels and reduced CD8+ T-cell levels (increased CD4/CD8 ratio) in hematoma aspirate and pre-operative blood were associated with inferior clinical outcomes regarding pain and function at 26 and 52 weeks. Increased levels of CD8+ -memory T-cell subpopulations in blood 6 weeks after surgery were associated with less tendon elongation. In vitro, tenocytes showed increased MMP1/2/3 levels and collagen III/I ratio in co-culture with unpolarized and/or IL17-polarized CD4+ T-cells compared to unpolarized CD8+ T-cells. This coincided with increased IL17 receptor expression in tenocytes co-cultured with CD4+ T-cells. Exposure of tenocytes to IL17-polarized CD4+ T-cells decreased their migration rate and increased their matrix contractility, especially compared to IFNy-polarized CD8+ T-cells. The CD4+ /CD8+ T-cell ratio could serve as prognostic marker for early identification of patients with impaired AT healing potential. Local reduction of CD4+ T-cell levels or their IL17 secretion represent a potential therapeutic approach to improve AT healing and to prevent weakening of the tendon ECM

Introduction. Chondrocytes are enveloped within the pericellular matrix (PCM), a structurally intricate network primarily demarcated by the presence of collagen type VI microfibrils and perlecan, resembling a protective cocoon. The PCM serves pivotal functions in facilitating cell mechanoprotection and mechanotransduction. The progression of osteoarthritis (OA) is associated with alterations in the spatial arrangement of chondrocytes, transitioning from single strings to double strings, small clusters, and eventually coalescing into large clusters in advanced OA stages. Changes in cellular patters coincide with structural degradation of the PCM and loss of biomechanical properties. Here, we systematically studied matrix metalloproteinases (MMPs), their distribution, activity, and involvement in PCM destruction, utilizing chondrocyte arrangement as an OA biomarker. Methods. Cartilage specimens were obtained from 149 osteoarthritis (OA) patients, and selected based on the predominant spatial pattern of chondrocytes. Immunoassays were employed to screen for the presence of various MMPs (-1, -2, -3, -7, -8, -9, -10, -12, -13). Subsequently, the presence and activity of elevated MMPs were further investigated through immunolabeling, western blots and zymograms. Enzymatic assays were utilized to demonstrate the direct involvement of the targeted MMPs in the PCM destruction. Results. Screening revealed increased levels of MMP-1, -2, -3, -7, and -13, with their expression profile demonstrating a distinct dependency on the stage of degeneration. We found that MMP-2 and -3 can directly compromise the integrity of collagen type VI, whereas MMP-3 and MMP-7 disrupt perlecan. Conclusions. Presence of both pro- and active forms of MMP-2, -3, and -7 in OA-induced patterns, along with their direct involvement in collagen type VI and perlecan degradation, underscores their crucial role in early PCM destruction. Given the early stages of the disease already exhibit heightened MMP expression, this understanding could inform early targeted therapies aimed at arresting abnormal PCM remodelling. Acknowledgments. Faculty of Medicine of the University of Tübingen (grant: 2650-0-0)

Introduction. We studied the safety and efficacy of multimodal thromboprophylaxis (MMP) in patients with a history of venous thromboembolism (VTE) undergoing total hip arthroplasty (THA). MMP includes discontinuation of procoagulant medications, VTE risk stratification, regional anesthesia, an intravenous bolus of unfractionated heparin before femoral work, rapid mobilization, the use of pneumatic compression devices, and chemoprophylaxis tailored to the patient's risk. Material and methods. From 2004 to 2018, 257 patients (mean age: 67 years; range: 26–95) with a history of VTE underwent 277 primary, elective THAs procedures (128 right, 100 left, 9 single-stage bilateral, 20 staged bilateral) by two orthopaedic surgeons at a single institution. The patients had a history deep vein thrombosis (DVT) 186 (67%), pulmonary embolism (PE) 43 (15.5%), or both 48 (17.5%). Chemoprophylaxis included aspirin (38 patients) and anticoagulation (239 patients; Coumadin: 182, low-molecular-weight heparin: 3, clopidogrel: 1, rivaroxaban: 3, and a combination: 50). Forty eight patients (17.3%) had a vena cava filter at the time of surgery. Patients were followed for 120 days to detect complications, and for a year to detect mortality. Results. Postoperative VTE was diagnosed in seven patients (2.5%): DVT in five, and PE with and without DVT in one patient each. Bleeding complications occurred in 2 patients, one requiring surgical evacuation of a hematoma. Seven patients died during the first year (2.5%). One patient died 5 months postoperatively of a fatal PE during open thrombectomy, and one patient died of a hemorrhagic stroke while receiving Coumadin. PE or bleeding was not suspected in any of the remaining 5 fatalities. Conclusions. The result of this study spanning over 13 years, suggests that MMP is safe and effective. Postoperative anticoagulation should be prudent as very few patients developed postoperative VTE (2.5%) or died of suspected or confirmed PE. Mortality during the first year was mostly unrelated to VTE or bleeding. For any tables or figures, please contact the authors directly

Degenerative joint disease (DJD) involves the proteolysis of many extracellular matrix molecules (ECM) present in articular cartilage and other joint tissues such as tendon, meniscus and ligaments. Recent research has identified key enzymes involved in the catabolism of ECM. Two classes of enzyme the Matrix Metalloproteinases (MMP’s) MMP-2, MMP-3, MMP-13 and the ADAMTS family (a disintegrin and metalloproteinase with thrombospondin motifs) of proteinases most notably, ADAMTS-1, -4 and −5, have been shown to be involved in the catabolism of ECM (such as type II collagen and cartilage aggrecan). The presence of several MMPs in the synovial fluid has been reported; however, little data has yet been gathered on the presence of ADAMTS-1, -4 or −5 (the aggrecanases) in synovial fluids. In this study we have used a recombinant artificial substrate and specific neoepitope antibodies that recognise either MMP- generated or aggrecanase -generated degradation products to measure the relative activity of these two enzyme families in the synovial fluid from human patients. Methods: A recombinant substrate containing the interglobular domain of cartilage aggrecan , flanked by a complement regulator and the Fc region of IgG has been stably transfected into CHO cells. The recombinant protein has been purified from the medium using a Protein A column followed by gel chromatography using a Superose 12 column. Synovial fluid samples were depleted of serum immunoglobulin by pre-absorption with ProSepA. The recombinant substrate was then added to synovial fluid samples and incubated overnight as 37?C. The recombinant substrate was recovered from samples using ProsepA and then separated by SDS-PAGE (10% gels). Gels were transferred to nitrocellulose membranes and immunoblotted with antibodies recognising the undigested substrate and using neoeptiope antibodies specifically recognising MMP or aggrecanase –generated catabolites. Results: Preliminary analysis by Western blot using the anti IGD neoepitopes BC-14 (detecting cleavage at the major MMP site) and BC-3 (detecting cleavage at the aggrecanase site) demonstrated that enzymes in human synovial fluid collected from patients diagnosed with rheumatoid arthritis cleaved the pro-drug at the MMP site with little or no evidence of aggrecanase catabolism. In contrast, synovial fluid collected from patients diagnosed with osteoarthritis indicted that there was cleavage at the aggrecanase site. In these preliminary studies we have also examined the enzyme activity in a set of clinical samples collected from patients that have undergone knee replacement surgery having been given either n-3 fatty acids or a placebo 10 weeks prior to surgery. Results indicate that aggrecanase generated fragments were found in synovial fluid from placebo patients, and reduced levels of enzyme activity were apparent in fluids tested from patients that had received n-3 fatty acids prior to surgery. Discussion: This data suggests that the recombinant substrate will aid in the detection of MMP or aggrecanase activities in synovial fluid samples. The ratio of MMP to aggrecanase activity has potential as a biomarker for the severity of cartilage degeneration in degenerative joint diseases

Objectives. Re-rupture is common after primary flexor tendon repair. Characterization of the biological changes in the ruptured tendon stumps would be helpful, not only to understand the biological responses to the failed tendon repair, but also to investigate if the tendon stumps could be used as a recycling biomaterial for tendon regeneration in the secondary grafting surgery. Methods. A canine flexor tendon repair and failure model was used. Following six weeks of repair failure, the tendon stumps were analyzed and characterized as isolated tendon-derived stem cells (TDSCs). Results. Failed-repair stump tissue showed cellular accumulation of crumpled and disoriented collagen fibres. Compared with normal tendon, stump tissue had significantly higher gene expression of collagens I and III, matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF). The stump TDSCs presented both mesenchymal stem and haematopoietic cell markers with significantly increased expression of CD34, CD44, and CD90 markers. Stump TDSCs exhibited similar migration but a lower proliferation rate, as well as similar osteogenic differentiation but a lower chondrogenic/adipogenic differentiation capability, compared with normal TDSCs. Stump TDSCs also showed increasing levels of SRY-box 2 (Sox2), octamer-binding transcription factor 4 (Oct4), tenomodulin (TNMD), and scleraxis (Scx) protein and gene expression. Conclusion. We found that a failed repair stump had increased cellularity that preserved both mesenchymal and haematopoietic stem cell characteristics, with higher collagen synthesis, MMP, and growth factor gene expression. This study provides evidence that tendon stump tissue has regenerative potential. Cite this article: C-C. Lu, T. Zhang, R. L. Reisdorf, P. C. Amadio, K-N. An, S. L. Moran, A. Gingery, C. Zhao. Biological analysis of flexor tendon repair-failure stump tissue: A potential recycling of tissue for tendon regeneration. Bone Joint Res 2019;8:232–245. DOI: 10.1302/2046-3758.86.BJR-2018-0239.R1

Introduction: Chondro-epiphyseal cartilage is generally resistant to vascular invasion. At the time of formation of the secondary ossification center in skeletal ‘long’ bones, the anti-angiogenic nature of cartilage is altered in favor of angiogenesis and vascular invasion takes place. We studied the control of this angiogenic ‘switch’ by experimentally investigating two factors which might influence vascular invasion. MMP 9 is a 92Kda gelatinase which degrades collagen types IV, V and X and gelatin (denatured collagen). It has been implicated in the control of endochondral ossification at the growth plate and has been shown to modulate endothelial cell morphogenesis. Basic Fibro-blast Growth Factor (b-FGF) is a cytokine with well established angiogenic capability and has also been implicated in the development of the growth plate. We investigated whether MMP-9 caused an effect on the development of the vasculature of the chondro-epiphysis of neo-natal rabbits and compared this to the effects of b-FGF. Materials and Methods: The CAM Culture consists of placing a small tissue explant onto the the chorioallantoic membrane of 10 day-old chick embryos and continuing culture for a further 10 days. CAM derived vessels will invade the tissue, unless anti-angiogenic factors are present. Hence, CAM culture is used as an assay system for angiogenesis and factors that will influence it. We utilized the CAM culture model to investigate vascular in-growth into explants of femoral and humeral heads from 4 day old postnatal rabbits to test the influence of MMP-9 and b-FGF. A small nylon membrane, pre-soaked in a solution containing the factor, was placed on to a tangential cut across the perichondrium. The explant was then cultured on the CAM for 3–10 days. Results: In control epiphyses, the in-growth of CAM derived blood vessels was rare and invasion of cartilage canals through the perichondrium seldom occurred, thus confirming the anti-angiogenic nature of epiphyseal cartilage. The initial presence of MMP 9 caused a tremendous increase in the de novo vascular invasion. MMP 9 treated epiphyses contained numerous large cartilage canals. In b-FGF treated epiphyses, a greater level of vascular in-growth was seen compared with controls, but this was not as marked as with MMP 9. Our findings indicate that b-FGF and perhaps, more interestingly, MMP-9 are implicated in the activation of the angiogenic ‘switch’ at the chondroepiphysis leading to vascular invasion. The fact that MMP-9 can act as a stimulator to angiogenesis is a novel finding. The mechanism of action remains unclear although it is possible that it is involved in the deactivation of inhibitors of vasculogenesis or the activation of angiogenic factors, or both

Purpose: The inflammatory response around herniated tissue in the epidural space is believed to play a major role in the spontaneous regression of herniated lumbar disc. Numerous macrophages invade the herniated tissue along with newly formed blood vessels which influence oxygen gradient. Inflammatory cytokines such as interleukin-1 are produced by macrophages. These chemical mediators could stimulate disc cells to produce proteases such as MMPs which degrade the intervertebral disc matrix and could hence influence regression of the herniation. Here we have examined the influence of IL-1β and oxygen tension on proteoglycan turnover using a three-dimensional disc-cell culture system. Methods: Cells were isolated from the nucleus pulposus of 18–24 month bovine caudal discs by enzyme digestion. They were initially cultured for 14 days in alginate beads in DMEM containing 6% FBS at 4.10. 6. cells/ml under 21% oxygen to accumulate matrix. They were then cultured for 6 days under 0% or 21% oxygen and with or without IL-1β. Glycosaminoglycan (GAG) accumulation (as a measure of proteoglycan content) was measured using a DMB assay. Lactate and glucose production were measured using a standard enzymatic method. Rates of sulfated GAG synthesis was measured from rates of . 35. S-sulfate accumulation. MMP activity was measured using coumarin fluorescent assay. Results: The results showed that IL-1β had a significant effect on GAG accumulation and production and that its effect was dependent on oxygen tension. GAG production and sulfate incorporation rates decreased in the presence of IL-1β at high oxygen but low oxygen inhibited the effects of this cytokine. MMP activity increased with IL-1β under 21% oxygen, but not at low oxygen. Conclusion: Exogenous IL-1β can activate MMP activity and digest the extracellular matrix of the disc but only at high oxygen tensions. Angiogenesis as well as inflammation is thus required for resorption of herniations

In patients with DM (Diabetes Mellitus types I & II), primary frozen shoulders tend to be refractory to all forms of treatment. We collected tissue from the joint capsule of shoulder joints from a variety of patients undergoing surgery as follows:. Diabetic Group (DFS): patients with DM who have primary frozen shoulders. Other patients suffering from primary frozen shoulders (FS). Control group (NS). Patients undergoing shoulder surgery that does not involve stiffness of the gleno-humeral joint. Tissue was collected from near to the rotator interval under arthroscopic control. Fibroblast lines were established by serial passage. Thereafter they were exposed to graded concentrations of insulin in vitro for 24 hours and the supernatant retained for assay. Fibroblast lines were analysed from 3 subjects in each group (n=9). Luminex multiplex analysis was performed for MMPs (Matrix Metalloproteinases). TIMP-1 (Tissue Inhibitor of MetalloProteinases) expression. Informed consent was obtained from all subjects. Results: Production of MMP 1,2,3 and 8 by fibroblast lines were distinct between patient groups. MMP-1 production in DFS (mean 716pg/mL) was significantly reduced compared to FS derived patient cells (mean 972pg/mL) (p=0.0138, Mann-Whitney Test). Moreover, striking differences were observed when fibroblasts from DFS patients were compared with those from NS controls (mean 5898pg/mL) (p< 0.000). Calculating MMP-1/TIMP-1 ratios revealed significantly lower ratios in DFS (2597), or FS (2860) compared with NS (24,326) (p < 0.001). There was no significant difference between ratios of MMP1/TIMP1 in DFS and FS (p=0.977). MMPs 7,9,12 and 13 were not detected in any of the samples. This is the first time these enzymes have been measured and quantified in cells derived from shoulder tissues. Primary Frozen Shoulders produce less MMPs and have a smaller MMP/TIMP ratio than controls. Similarly the diabetic patient derived cells produce less MMP-1, at an even lower level. These deficiencies in MMP1 production may reflect an altered capacity for local tissue re-modelling. MMP modulation may allow therapeutic intervention in the diabetic and frozen shoulder group of patients

Introduction: Energy storing tendons, such as the human Achilles tendon, suffer a much higher incidence of rupture than non- energy storing positional tendons, such as the anterior tibialis tendon. Similarly, in the horse partial rupture of the energy storing superficial digital flexor tendon (SDFT) and suspensory ligament (SL) occurs much more frequently than to the deep digital flexor tendon (DDFT) and common digital extensor tendon (CDET) which are not involved in energy storage. In order to function effectively, energy storing tendons experience strains during high speed locomotion which are much closer to failure strain than non-energy storing tendons. Therefore, these tendons are likely to sustain high levels of microdamage, hence cell metabolism may also be higher in order to repair damage and maintain matrix integrity. Maintenance of the matrix requires not only synthesis of new matrix components but also degradation of matrix macromolecules which is achieved, in part, by a family of matrix metalloproteinase enzymes (MMPs). In this study we test the hypothesis that the energy storing equine SDFT and SL which are prone to degenerative changes have higher levels of MMP2 and MMP9 than the positional DDFT and CDET that are rarely injured. Methods: Tendons (SDFT, DDFT, SL, CDET) were harvested from the distal part of the forelimbs of 18 month old Thoroughbred horses (n = 12). Tissue from the mid-metacarpal region of each tendon was snap frozen, lyophilised, powdered and MMPs extracted. Gelatin zymography was used to determine levels of the pro and active forms of the gelatinase enzymes, MMP2 and MMP 9. Proteolytic activity (units per mg dry weight tissue) was quantified based on densitometry measurements and standardised between gels using an equine neutrophil MMP extract. Statistical significance was evaluated using a general linear model (SPSS software). Results: The main activity observed in all tendon samples was that of proMMP2. Quantification showed that the energy storing SDFT (23.4 ± 10.95) and SL (18.9 ± 5.3) had significantly higher levels than the non-energy storing DDFT (2.90 ± 0.99) and CDET (4.06 ± 2.06). Active MMP2 levels were lower than the pro form and were not sufficient to quantify. However, there appeared to be more in the energy storing structures compared with the non energy storing structures. MMP9 activity was detected in some samples. A higher number of the CDET extracts contained MMP9 activity compared to extracts from the other structures. Discussion: The results of this study show higher levels of MMPs in energy storing structures than in non-energy storing structures. This suggests that there may be an increased demand for repair of micro-damage in these tendons and hence an increased capacity for matrix degradation. Previous studies on energy storing structures in the horse have shown that they do not undergo adaptive hypertrophy or a change in structural architecture in response to mechanical demand, unlike non-energy storing structures. The results of this study indicate that this lack of adaptation in energy storing structures is not due to a general deficiency in cell activity but may be a means of preventing increase in tendon stiffness and a subsequent decrease in efficiency. In order to maintain tendon integrity MMP activity must be matched by mechanisms to inhibit activity and/or to synthesize new matrix components. Degeneration may therefore occur when there is an imbalance between these processes

Osteoarthritis (OA) is a chronic degenerative joint disorder that affects millions of people. There are currently no therapies that reverse or repair cartilage degradation in OA patients. Link N (DHLSDNYTLDHDRAIH) is a naturally occurring peptide that has been shown to increase both collagen and proteoglycan synthesis in chondrocytes and intervertebral disc cells [1,2]. Recent evidence indicates that Link N activates Smad1/5 signaling in cultured rabbit IVD cells presumably by interacting with the bone morphogenetic protein (BMP) type II receptor [3], however, whether a similar mechanism exists in chondrocytes remains unknown. In this study we determined whether Link N can stimulate matrix production and reverse degradation of human OA cartilage under inflammatory conditions. OA cartilage was obtained from donors undergoing total knee arthroplasty with informed consent. OA cartilage/bone explants and OA chondrocytes were prepared from each donor. Cells were prepared in alginate beads (2×106 cells/mL) for gene expression analysis using qPCR. Cells and cartilage explants were exposed to IL-1β (10ng/ml), human Link N (hLN) (1μg/ml) or co-incubated with IL-1β+hLN for 7 and 21 days, respectively. Media was supplemented every three days. Cartilage/bone explants were measured for total glycosaminoglycan (GAG) content (retained and released) using the dimethylmethylene blue (DMMB) assay. Western blotting was performed to determine aggrecan and collagen expression in cartilage tissue. To determine NFκB activation, Western blotting was performed for detection of P-p65 in chondrocytes cultured in 2D following 10 min exposure of IL-1β in the presence of 10, 100, or 1000 ng/mL hLN. Link N significantly decreased in a dose-dependent manner IL-1β-induced NFκB activation in chondrocytes. Gene expression profiling of matrix proteins indicated that there was a trend towards increased aggrecan and decreased collagen type I expression following hLN and IL-1β co-incubation. HLN significantly decreased the IL-1β-induced expression of catabolic enzymes MMP3 and MMP13, and the neuronal growth factor NGF (p < 0 .0001, n=3). In OA cartilage/bone explants, hLN reversed the loss of proteoglycan in cartilage tissue and significantly increased its synthesis whilst in the presence of IL-1β. Link N stimulated proteoglycan synthesis and decreased MMP expression in OA chondrocytes under inflammatory conditions. One mechanism for Link N in preserving matrix protein synthesis may, in part, be due to its ability in rapidly suppressing IL-1β-induced activation of NF-κB. Further work is needed to determine whether Link N directly inhibits the IL-1β receptor or interferes with NFκB activation through an independent pathway(s)

Summary. PCA-III, a phosphocitrate analog, acts not only as a potent calcification inhibitor but also as a protective agent for extracellular matrices. PCA-III has potential as a disease-modifying drug in the treatment of primary osteoarthritis and posttraumatic osteoarthritis in humans. Introduction. Phosphocitrate (PC) inhibits the development of primary osteoarthritis (OA) in Hartley guineas pigs but not menisectomy-induced OA in rabbits (1). We sought to examine the molecular mechanisms underlying the disease-modifying activity of PC, and evaluate the effect of PCA-III, a PC analog (PCA), on the development of primary and secondary OA. Patients & Methods. Meniscal explant and microarray. OA menisci were obtained from OA patients undergoing joint replacement surgery. OA meniscal explants were cultured in medium containing PC (three wells) and medium without PC (three wells). Total RNA was extracted from the explant, and subjected to microarray analysis. RT-PCR. OA fibroblast-like synoviocytes were treated with basic calcium phosphate (BCP) crystals in the absence or presence of PCA-III. RNAs were extracted, and subjected to semi-quantitative RT-PCR to examine the expression of MMP1 and IL-1b. Micromass culture. A droplet of OA chondrocyte suspension was placed in each well of a 24-well plate. After placing all droplets, the wells were fed with chondrogenesis medium with PCA-III (five wells) and without PCA-III (five wells). The production of proteoglycans was examined by alcian blue staining. Animal treatment. The first group of Hartley guinea pigs (n=5) received injections of PCA-III and the second group received injections of saline as control. Two months later, partial-menisectomy surgery was performed on the right knee of all guinea pigs. After the surgery, injections of PCA-III and saline were resumed. All animal were euthanatised four months later, and both knees were examined. Results. PC inhibited the expression of many genes classified into the molecular function group of MMP activity. Of the 23 genes classified into MMP activity, the expression of 16 genes, including CPM, ADAM28, MMP7, MMP10, MMP1, MMP3, ADAMTS5, ADAMTS1, and ADAMTS9, was inhibited. In contrast, the expression of many genes classified into the molecular function group of extracellular matrix structural constituents, was induced by PC, including COL2A1, COL11A1, COL1A1 and ACAN. PC also inhibited the expression of numerous genes classified into the biological process of inflammatory response (data not shown). PCA-III, similar to PC, inhibited BCP crystals-induced expression of MMP1 and IL-1b). In addition, PC-III strongly stimulated the production of proteoglycans by OA chondrocytes while inhibiting calcium deposition (not shown). Microscopic examination of the Indian ink stained medial tibia plateau of the left knees (non-surgery knee) of the guinea pigs indicated that PCA-III inhibited the development of primary OA in the Hartley guinea pigs. Microscopic examination also indicated that PCA-III inhibited the development of partial-menisectomy-induced OA or posttraumatic OA in the post-operative knees. Discussion/Conclusion. PC is thought to act as a potential structural disease-modifying drug for crystal-associated OA by inhibiting crystal deposition within the OA joints. However, PC and its analogs are not only potent calcification inhibitors, but also protective agents for extracellular matrices. Our findings indicate that PCA-III has potential as a disease-modifying drug for both human crystal-associated OA and posttraumatic OA