Introduction. Stem cells are widely known in the state of the art of cell-based therapies. Recently, ADSCs are becoming a popular resource of adult stem cells across different fields, and latest publications show its wide application for the treatment of soft tissue injuries like tendon injuries, which represent a high percentage of the consultations in orthopaedic practitioners. Molecular-based therapies and local deliveries are necessary for an effective treatment of chronic tendon injuries. In this study, human ADSCs were selected to investigate its differentiation potential into the tendon phenotype. Customised cell culture media was used as the differentiation factor. Materials and Methods. In the present study, ADSCs were used in passage 3 to ensure pluripotency in vitro. Using the customised cell culture media, its time, concentration and frequency of refreshment effects were investigated. On the selected time points different techniques were performed: 1,) cells were harvested, and messenger RNA (mRNA) was examined by Real Time Polymerase Chain Reaction (RT-PCR), analysing the expression of common tendon and extracellular matrix (ECM) markers. Protein expression was determined by Western Blotting. 2) Collagen content was analysed by tissue digestion and colorimetric techniques. 3) Deoxyribonucleic Acid (DNA) was stained, and fluorescent imaging was used to characterise nuclear roundness. 4) Metabolic activity of the cultures was assessed using CellTiter 96® Aqueous One Solution (MTS). 5) Cell proliferation was evaluated using CyQuant® Cell Proliferation Assay. Results. In this work, we systematically evaluated the doses and time effect of the customised media on the differentiation potential of ADSCs. Our results showed significant differences in the cell performance between the conditions investigated. Interestingly, ADSCs presented enhanced tendon marker expression (mRNA and protein level) and collagen content. The different tendon and ECM markers analysed by RT-PCR showed doses and time-dependent effect, establishing a connection with. its role in the tissue. We believe this could offer a possible regenerative treatment without overstimulation. Despite the condition, ADSCs presented 95%–100% viability and proliferation values, demonstrating the non-toxic effect of the media. Conclusion. This study contributes to the knowledge of differentiation potential of ADSCs in tendon repair. Furthermore, the tendon phenotype generated in the 2D cultures changes when different variables are investigated. Knowing the molecular basis and conformations of the tendon phenotype is key in tendon research. Hence we believe these results can show a new paradigm in tendon repair, making possible to select more suitable treatments depending on the status of the injury on the patients. Acknowledgements. This work was supported by Rosetrees Trust, Arthritis Research UK and the Universityof East Anglia

Mesenchymal stromal cells (MSC) are multipotent, self-renewing cells that are an attractive cell source for cartilage regeneration strategies. While articular chondrocytes form stable cartilage-like tissue under chondrogenic in vitro conditions, a still unsolved problem of chondrocyte production from MSC is their endochondrol development leading to the formation of transient instead of stable articular cartilage. In order to identify relevant molecular determinants of chondrocyte redifferentiation versus MSC chondrogenesis and hypertrophy, this study assessed the differential expression of members of the transforming growth factor β (TGF-β) -superfamily, their receptors and antagonists between differentiating MSC and human articular chondrocytes (HAC). Chondrogenesis of human MSC and redifferentiation of HAC was induced in micromass pellet culture. Gene expression of MSC (n=5) and HAC (n=5) was compared using a transcriptome analysis on Illumina platform. Functional regulation of relevant candidate molecules was assessed in independent MSC and HAC populations by qRT-PCR. Smad signalling during chondrogenic differentiation was analysed by immunohistochemistry and Western Blotting. BMP signalling in both populations was modulated by co-treatment with BMP-4/7 or an inhibitor of Smad1/5/9 signalling. Proteoglycan and DNA content, collagen type II and -X deposition, gene expression of chondrogenic and hypertrophic markers as well as alkaline phosphatase (ALP) activity were quantitatively assessed at different time points. In HAC, TGF-β receptor 2 and 3 (TGFBR2/3) were up-regulated to significantly higher levels than in MSC. BMP4, expressed during HAC expansion, was suppressed while CHL2 and CHRD levels raised. In MSC, BMP4 and BMP7 were induced while TGFBR2 and TGFBR3 were down-regulated. Staining for pSmad1/5/9 in HAC demonstrated positive cells dispersed throughout the pellets at day 3 and 5 while lower pSmad1/5/9 immunostaining was observed in MSC. In HAC and MSC pellets pSmad staining decreased during chondrogenesis, in line with Western Blot results. Medium supplementation with BMP-4/7 did not improve cartilaginous matrix deposition by MSC but raised ALP-activity. When Smad1/5/9 phosphorylation was blocked in MSC culture by dorsomorphin treatment (day 14–42) COL2A1 and COL10A1 expression decreased significantly and collagen type II and type X deposition were reduced. ALP activity dropped to 12 % of control levels. Inhibition of pSmad1/5/9 signalling was unattractive to shift chondrogenesis of MSC away from endochondral development since it unpaired SOX9 expression and strongly reduced cartilaginous matrix deposition along with hypertrophy. Thus no simple correlation exists between beneficial pSmad2/3 versus unwanted pSmad1/5/9 signalling during MSC chondrogenesis

Fragmentation of SLRPs, including decorin, biglycan, lumican, keratocan and fibromodulin, has been shown to occur in osteoarthritic articular cartilage. We have previously shown an increased expression of lumican and keratocan, in osteoarthritic articular cartilage. The long-term aim of this project is to develop ELISAs for the detection of SLRP metabolites, and validate these potential biomarkers with synovial fluid and serum samples from a large cohort of normal and osteoarthritic patients. Initially, we aimed to determine whether SLRPs could be detected in synovial fluid and whether they were post-translationally modified with glycosaminoglycan (GAG) attachments; and whether bovine nasal cartilage (BNC) would be a plentiful source of native SLRP for ELISA development. Proteoglycans were extracted from BNC in guanidine hydrochloride. BNC extract and bovine synovial fluid was separated on an associative CsCl gradient. BNC CsCl cuts containing sulphated GAG were further purified using anion exchange chromatography. SLRPs in each fraction were detected using Western Blotting. Human recombinant lumican was expressed in Chinese hamster ovary (CHO) cells. Monoclonal antibodies that recognise epitopes on the core protein of human and bovine lumican and decorin were purified from hybridoma media using Protein G and Protein A affinity chromatography respectively. Monoclonal antibody activity against native and recombinant SLRPs was then determined using a direct ELISA. Preliminary tests showed that bovine synovial fluid contains keratocan and lumican with GAG attachments. BNC is a good source of post-translationally modified decorin, keratocan and biglycan but lumican was present predominantly without GAG attachments. Human recombinant lumican was successfully expressed with GAG attachments by CHO cells. Initial tests showed that the mAb against decorin was able to detect native decorin, with GAG attachments, in direct ELISA conditions. We have identified a plentiful source of native SLRP and begun ELISA development to ascertain whether these proteoglycans are potential biomarkers of OA

Introduction. Novel chondroitin sulphate (CS) sulphation motifs on cell-associated proteoglycans (PGs) have been shown to be putative biomarkers of progenitor/stem cell sub-populations (Hayes et al., 2007; Dowthwaite et al., 2005). Also, recent studies show that unique CS sulphation motifs are localized in putative stem/progenitor cell niches at sites of incipient articular cartilage & other musculoskeletal tissues (Hayes et al., 2011), which indicates their potential importance in cell differentiation during development. In this study, we investigated the importance of CS in the differentiation of bone marrow stem cells to the chondrogenic phenotype in vitro using p-nitrophenyl xyloside (PNPX) as a competitive inhibitor of CS substitution on matrix PGs. Methods. Bovine bone marrow stem cells (BMSCs) were isolated from 7-day-old cow hock joints and cultured as monolayer for 4 weeks with chondrogenic medium ± 0.25mM PNPX. DMMB assay, real-time PCR, Western Blotting & immunohistochemistry (IHC) were used to analysis the chondrogenic markers. The expression and distribution of structural CS proteoglycans (CS-PGs) were analysed by immunofluorescent staining combined with confocal microscopy scanning. Results. BMSCs cultured in chondrogenic medium started to aggregate and form mini-cell beads in 3 days and these mini cell beads clustered together to form a large single alcian blue positive cartilaginous cell bead in 2-4 weeks, indicative of the chondrogenesis. In contrast, there was an apparent delay in the cell bead formation in the BMSCs cultured with PNPX. Moreover, PNPX significantly inhibited or delayed the expression of chondrogenic markers including aggrecan, SOX-9 & type II collagen gene and/or protein expression. Furthermore, IHC analyses showed that a decreased expression of native CS sulphation epitopes in chondrogenic media + PNPX, suggesting the importance of their role in allowing the chondrogenic differentiation to occur. Discussion. These results suggest that CS sulphation motifs play an important role in the differentiation of BMSCs into chondrocytes. The precise mechanism is not known, but CS sulphation motifs may be involved in the growth factor presentation needed for cell differentiation that leads to cell aggregation and extracellular matrix-cell interactions during chondrogenesis

Summary Statement. CXCR4 gene and protein expression is regulated in a dose and time-dependent manner by metallic wear debris but not polyethylene wear debris in vitro and in vivo. Introduction. Progressive osteolysis leading to aseptic loosening among metal-on-metal (MoM) total hip arthroplasties (THA's), and adverse reactions to metallic debris (ARMD) are increasing causes for concern among existing patients who have been implanted with MoM hip replacements. Close surveillance of these patients is necessary and difficulties lie in early detection as well as differentiating low-grade infection from ARMD in the early stages. Several inflammatory markers have been investigated in this context, but to date, none is specific with regards to the offending material. In earlier studies, it has been shown that osteoblastic phenotypes and differentiation are regulated by different types of wear particles. Methods. In vitro experiments were performed using MG63 and SaOs-2 osteoblast-like cells co-cultured with increasing concentrations of metallic (Co-35Ni-20Cr-10Mo and Co-28Cr-6Mo) and polyethylene (UHMWPE-GUR1020) particles simulating periprosthetic wear debris. Real-time Polymerase Chain Reaction (RT-PCR) and Western Blotting were used to quantify gene and protein expression of CXCR4. The expression of TNF-a and the effects of AMD3100 on both CXCR4 and TNF-a expression among these cells was also investigated. Immunohistochemical techniques were used to investigate the in-vivo expression of CXCR4 in retrieval tissues obtained from 2 cohorts of failed metal-on-metal and ceramic-on-polyethylene THA's. Results. In-vitro RT-PCR and experiments demonstrated a dose-dependent increase in CXCR4 mRNA (7.5 fold for MG63 and 4.0 fold for SaOs-2 cells) among cells co-cultured with metal alloy particles. Western blotting also showed a time-dependent increase in protein expression of CXCR4. No regulatory effects on CXCR4 gene expression were seen among cells co-cultured with UHMWPE particles. The attempted blockade of CXCR4 by it's known competitive receptor agonist AMD3100 (bicyclam) led to a significant inhibition of metal particle induced TNF-a mRNA expression. In-vivo immunohistochemical data from the 2 cohorts of patients with failed THA's showed CXCR4 positivity among 83% of patients with metal-on-metal hip replacements but none among ceramic-on-polyethylene hip replacements. Discussion/Conclusion. CXCR4, the chemokine receptor for the chemokine SDF-1 (stromal cell derived factor-1), has been shown to play a pivotal role in bone metastasis, inflammatory and autoimmune conditions but has not been investigated in the context of periprosthetic osteolysis in failed joint replacements. Our in-vivo and in-vitro findings collectively suggest that the CXCR4 chemokine is specifically upregulated in a dose and time-dependent manner in the presence of metallic (cobalt-chrome) wear debris but not by polyethylene wear debris. The CXCR4 chemokine receptor may be a selective and specific biomarker for progressive osteolysis seen in failed MoM hip replacements and this phenomenon could potentially have a translational effect on the practice of orthopaedic surgery. Further research is needed to evaluate the interactions of CXCR4 with osteoclast activation and signalling pathways