Human Mesenchymal stem cells (hMSCs) are a promising source for articular cartilage repair. Unfortunately, under Pellets of passage 2 hMSCs were formed in V-bottom well plates by centrifugation and pre-differentiated in a chemically defined medium containing 10ng/mL TGFß (CM+) for 14 days. Thereafter, pellets were cultured for an additional 14 days under 6 conditions: CM+, CM- (w/out TGFß), and hypertrophic medium (CM- with 25 ng/ml BMP 4, w/out dexamethasone). Each of these first three conditions was additionally supplemented with the RA receptor (RAR) inverse agonist BMS493 (BMS) at 2μM after 14 days of chondrogenic pre-differentiation. One additional BMP4 group was supplemented with BMS from the beginning of chondrogenic differentiation until day 14. The pellets were assessed for gene expression (Col 2, Col 10, Col 1 and MMP13) and histologically using dimethyl methylene blue (DMMB), alkaline phosphatase staining (ALP) and collagen II and X immunohistochemistry.Introduction
Methods
Osteoarthritis is a degenerative disease mainly caused by aging, although in younger patients (aged 25 – 50) it can be a consequence of sports-related injuries or trauma. This results in early osteoarthritis with subsequent changes in cartilage extracellular matrix. Cell-based tissue engineering approaches using mesenchymal stem cells (MSCs) are an ideal cell type for the treatment of early osteoarthritc defects. Our group has demonstrated in a clinical study, that interleukin-1β (IL-1β) was expressed in cartilage plugs from patients with early osteoarthritis. Human MSCs (Male donors; aged 18–60 years, n = 6) were isolated from bone marrow and expanded in culture for one passage. 2 × 105 MSCs were aliquoted into wells of a 96-well cell culture plate in the presence of 10ng/ml TGF-β1 or in combination with IL-1β administered at a range of concentrations (0.1, 0.5, 1 and 10ng/ml) and centrifuged to form pellets. Pellets were removed from culture on days 7, 14 and 21. Pellets were evaluated for wet weight, pellet area, histological (DMMB staining, collagen type I, II, MMP-13 and TGF-β receptor II) and collagen type II ELISA analysis. Chondrogenic pellets in the presence of IL-1β demonstrated a dose-dependant inhibition in chondrogenesis. Concentrations equal or greater than 0.5ng/ml IL-1β showed significant reduction (p < 0.05) in pellet area and wet weight, with no positive staining for collagen type I, II (including ELISA analysis) and DMMB. However, at 0.1ng/ml IL-1β, despite a slight reduction in pellet area, positive staining for collagen type I, II and DMMB was observed. Furthermore, MMP-13 matrix staining was increased and TGF-b receptor II staining was decreased in pellets at IL-1β concentrations above 0.5ng/ml.Method
Results
Osteoarthritis is a degenerative disease that results in changes in cartilage extracellular matrix. Human MSCs (Male donors; aged 18–60 years, n = 6) were isolated from bone marrow and expanded for one passage and split into hyperoxic and physioxic MSC cultures, the latter conditions were isolated and expanded using a hypoxia controlled incubator. MSCs with or without physioxic preconditioning were aliquoted into wells of a 96-well cell culture plate in the presence of 10ng/ml TGF-β1 or in combination with either 0.1 or 0.5ng/ml IL-1ß and centrifuged to form pellets. Pellets were then differentiated under their isolation conditions. Pellets removed from culture on days 7, 14 and 21, were evaluated for wet weight, histological (DMMB staining, collagen type I, II, MMP-13 and TGF-β receptor II) and collagen type II ELISA analysis. Preconditioned MSCs demonstrated an enhanced collagen type II and GAG production undergoing chondrogenesis compared to hyperoxic pellets. In the presence of IL-1β, preconditioned MSCs reduced the inhibitory effect of IL-1ß compared to the equivalent conditions under hyperoxic, whereby there was a significant increase in wet weight, GAG and collagen type II production (p < 0.05). Furthermore, preconditioning MSCs had reduced collagen type X expression compared to hyperoxic cultures.Methods
Results
Modulation of signaling pathways, which involves tendon development, regeneration, or homeostasis, is one of the potential modalities to facilitate proper regeneration of the injured tendon. Authors have previously reported that activation of Wnt/beta-catenin signaling suppressed the expression of tenogenic genes (i.e. Scleraxis (Introduction
Materials and Methods
The exact pathways of collagen remodeling in tendon tissue are not well understood. Therefore, we have established an ex vivo 3D collagen gel-based system and we studied the remodeling capacity of two different TSPC lines from young, Y-TSPC and aged/degenerative, A-TSPC donors. Here, we specifically focused on investigating the involvement of integrin receptors in the remodeling process. Integrins are transmembrane receptors consisting of alpha (a) and beta (b) subunits, which form cell-to-matrix bonds, activate various pathways and thereby control cell proliferation, differentiation and survival. Y- and A-TSPC were derived from human Achilles tendons and are fully described in Kohler et al. 2013. RT-PCR was used to assess the expression of collagen-binding integrins in the TSPC cultivated in collagen gels. Next, a1 and a11 integrins were silenced by stable lentiviral delivery of target-specific shRNA in the Y-TSPC. Control (con-shRNA), integrin (a1-shRNA) and integrin a11 (a11-shRNA) virus-containing supernatant was given for 24h and then cells were selected with 50 microg./ml zeocin for 10 days. The integrin knockdown (KD) efficiency was assessed by quantitative PCR and western blotting. Last, functional tests were carried out by time-lapse recording gel contraction of four cell groups (Y-TSPC+con, Y-TSPC+a1KD, Y-TSPC+a11KD, and A-TSPC). Among the screened integrins we found that integrin a1 and a11 were significantly downregulated in A-TSPC with 3.8 and 5.6 folds, correspondingly. Therefore, to mimic the A-TSPC we carried out a gene KD of a1 and a11 in Y-TSPC. PCR and western blot clearly validated the efficient KD. Analyses of collagen contraction, revealed that Y-TSPC+a11KD significantly reduced collagen contractability comparable to A-TSPC. This indicated the indispensable role of this integrin in the signaling pathway of collagen matrix remodeling. In respect to integrin a1, we found that this receptor did not affect the contraction rate of Y-TSPC, which was similar to Y-TSPC+con. To our knowledge we have now identified for the first time the critical role of a11 integrin receptor in tendon collagen remodeling, and a follow up analysis of its exact downstream cascade is on the way. Future efforts in deciphering how tendon matrix makeover is regulated can lead to innovation in preventive strategies for tendon degeneration.
Previous studies have shown that Tnmd is important for tendon maturation and has key implications for the residing tendon stem/progenitor cells. The putative signaling in which Tnmd participates is just starting to be better understood (Dex et al. 2016). However, its exact functions during tendon healing process still remain elusive. Therefore, the aims of this study were to perform systematic review of the literature on Tnmd-related research and to investigate the role of Tnmd in early tendon healing by applying a tendon rupture model in Tnmd-deficient mice. First, we searched in the PubMed database for articles containing “tenomodulin” or its alternative names and abbreviations. After exclusion of papers only available in abstract form and foreign language, we grouped the remaining 128 full-text publications into four study types: 1) looking into functions of Tnmd; 2) using Tnmd as a tendon marker; 3) correlating Tnmd mutations to a variety of diseases; and 4) reviews. Following literature analysis, we carried out a pilot Achilles tendon injury model with Tnmd-knockout (KO) mouse strain. Adult Tnmd-KO (n = 8) and wild-type (WT) (n = 8) mice underwent unilateral surgery of Achilles tendon based on Palmes et al. 2002 and were compared at day 8 postoperatively by: 1) H&E staining for overall assessment; 2) immunohistochemical BrdU analysis for cell proliferation; and 3) Safranin O staining for endochondral formation. Our literature screen revealed that Tnmd has been strongly justified as the best tendon and ligament marker in more than 90 different studies. Moreover, in vivo and in vitro investigations have demonstrated its positive role on tendon cell proliferation and tissue functions. Our follow up surgical study showed a very different scar organization in Tnmd-KO with a clearly reduced cell density. BrdU analysis confirmed a lower number of proliferating cells in Tnmd-KO scar area. Interestingly, endochondral formation was not observed in the scar tissues in either of the genotypes at day 8. Taken together, we systematically summarized the current knowledge on Tnmd gene and highlighted several future research perspectives. Lack of studies on the role of Tnmd in tissue healing, motivated our pilot investigation on Achilles tendon rupture, which in turn suggested that loss of Tnmd results in inferior repair process.
Progenitor cells from the periosteal niche are of great clinical interest due to their remarkable regenerative capacity. Here we report on progenitor cells from arthritic patients whose femoral neck periosteum was resected over the course of hip replacement. This study aims to determine whether periosteum derived cells (PDCs) can be isolated from tissue resected in the normal course of hip arthroplasty. Further, it aims to determine how different isolation protocols affect PDC behavior (surface marker expression, proliferation, and differentiation). In addition, the study aims to characterise the populations of PDCs, isolated through either enzymatic digestion or migration, and their relative capacity to differentiate down multiple capacities; direct comparison with commercially available human marrow-derived stromal cells cultured under identical conditions will enable the placement of the PDC data in context of the current state of the field.Summary Statement
Introduction
Irreparable tendon ruptures constitute a grave clinical problem. Especially for large rotator cuff tears, there often is no primary causal therapy available. As a sad result, the development of a rotator cuff tear arthropathy is more often than not inevitable. Our study investigates the effects of scaffold based tendon regeneration with special focus on mesenchymal stem cells in a rat model. We used ‘native’ bone marrow stromal cells and cultivated mesenchymal stem cells from male rats that were implanted into female rats. As scaffolds polyglycol acid (PGA) and a collagen I were used. A full-thickness-defect of 2–3 mm in the middle third of the rats achilles tendon was created, which was then filled, with either cell-seeded or not cell-seeded scaffolds and, due to the low primary stability of the scaffolds, fixed with a 4-0 suture. After 12 weeks, a DNA PCR was conducted to verify the existence of male Y-chromosomes in the female regenerated tissue. We determined the maximum tensile load of the regenerated tissue and also did a histological evaluation. Macroscopically the regenerated tendons were much bigger in diameter, much firmer and also much less elastic than a normal tendon. In the ‘mesenchymal stem cells’ group the implanted cells could be clearly identified after 12 weeks by DNA PCR. The collagen I scaffold yielded better results in the biomechanical study than the PGA scaffold. No evidence of positive influence of the cells on the mechanical stability of the regenerated tissue was found. Collagen I and the use of BMSC histologically lead to increased ossification of the regenerated tissue. In the PGA scaffold group a significant inflammatory reaction was found. Both scaffold/cell combination seem to be unsuitable for tendon replacement. in-vitro studies on the influence of scaffold material on cell differentiation needs to be done.
