Objectives. Many in vitro studies have investigated the mechanism by which mechanical signals are transduced into biological signals that regulate bone homeostasis via periodontal ligament fibroblasts during orthodontic treatment, but the results have not been systematically reviewed. This review aims to do this, considering the parameters of various in vitro mechanical loading approaches and their effects on osteogenic and osteoclastogenic properties of periodontal ligament fibroblasts. Methods. Specific keywords were used to search electronic databases (EMBASE, PubMed, and Web of Science) for English-language literature published between 1995 and 2017. Results. A total of 26 studies from the 555 articles obtained via the database search were ultimately included, and four main types of biomechanical approach were identified. Compressive force is characterized by static and continuous application, whereas tensile force is mainly cyclic. Only nine studies investigated the mechanisms by which periodontal ligament fibroblasts transduce mechanical stimulus. The studies provided evidence from in vitro mechanical loading regimens that periodontal ligament fibroblasts play a unique and dominant role in the regulation of bone remodelling during orthodontic tooth movement. Conclusion. Evidence from the reviewed studies described the characteristics of periodontal ligament fibroblasts exposed to mechanical force. This is expected to benefit subsequent research into periodontal ligament fibroblasts and to provide indirectly evidence-based insights regarding orthodontic treatment. Further studies should be performed to explore the effects of static tension on cytomechanical properties, better techniques for static compressive force loading, and deeper analysis of underlying regulatory systems. Cite this article: M. Li, C. Zhang, Y. Yang. Effects of mechanical forces on osteogenesis and osteoclastogenesis in human periodontal ligament fibroblasts: A systematic review of in vitro studies. Bone Joint Res 2019;8:19–31. DOI: 10.1302/2046-3758.81.BJR-2018-0060.R1

Aims. Dupuytren’s contracture is characterized by increased fibrosis of the palmar aponeurosis, with eventual replacement of the surrounding fatty tissue with palmar fascial fibromatosis. We hypothesized that adipocytokines produced by adipose tissue in contact with the palmar aponeurosis might promote fibrosis of the palmar aponeurosis. Methods. We compared the expression of the adipocytokines adiponectin and leptin in the adipose tissue surrounding the palmar aponeurosis of male patients with Dupuytren’s contracture, and of male patients with carpal tunnel syndrome (CTS) as the control group. We also examined the effects of adiponectin on fibrosis-related genes and proteins expressed by fibroblasts in the palmar aponeurosis of patients with Dupuytren’s contracture. Results. Adiponectin expression in the adipose tissue surrounding the palmar aponeurosis was significantly lower in patients with Dupuytren’s contracture than in those with CTS. The expression of fibrosis-related genes and proteins, such as types 1 and 3 collagen and α-smooth muscle actin, was suppressed in a concentration-dependent manner by adding AdipoRon, an adiponectin receptor agonist. The expression of fibrosis-related genes and proteins was also suppressed by AdipoRon in the in vitro model of Dupuytren’s contracture created by adding TGF-β to normal fibroblasts collected from patients with CTS. Conclusion. Fibrosis of the palmar aponeurosis in Dupuytren’s contracture in males may be associated with adiponectin expression in the adipose tissue surrounding the palmar aponeurosis. Although fibroblasts within the palmar aponeurosis are often the focus of attention when elucidating the pathogenesis of Dupuytren’s contracture, adiponectin expression in adipose tissues warrants closer attention in future research. Cite this article: Bone Joint Res 2023;12(8):486–493

Objectives. The aims of this study were to determine whether the administration of anti-inflammatory and antifibrotic agents affect the proliferation, viability, and expression of markers involved in the fibrotic development of the fibroblasts obtained from arthrofibrotic tissue in vitro, and to evaluate the effect of the agents on arthrofibrosis prevention in vivo. Methods. Dexamethasone, diclofenac, and decorin, in different concentrations, were employed to treat fibroblasts from arthrofibrotic tissue (AFib). Cell proliferation was measured by DNA quantitation, and viability was analyzed by Live/Dead staining. The levels of procollagen type I N-terminal propeptide (PINP) and procollagen type III N-terminal propeptide (PIIINP) were evaluated with enzyme-linked immunosorbent assay (ELISA) kits. In addition, the expressions of fibrotic markers were detected by real-time polymerase chain reaction (PCR). Fibroblasts isolated from healthy tissue (Fib) served as control. Further, a rabbit model of joint contracture was used to evaluate the antifibrotic effect of the three different agents. Results. Dexamethasone maintained the viability and promoted the proliferation of AFib. Diclofenac decreased the viability and inhibited the cell proliferation during the first week of cultivation. However, decorin inhibited AFib proliferation and downregulated the expressions of fibrotic markers. Additionally, decorin could improve the flexion contracture angle and inhibit the deposition of interstitial matrix components in the rabbit joint model. Conclusion. Decorin decreased the expression of myofibroblast markers in AFib, inhibited the proliferation of AFib, and prevented the initial procedure of arthrofibrosis in vivo, suggesting that decorin could be a promising treatment to inhibit the development of arthrofibrosis. Cite this article: X. Tang, S. Teng, M. Petri, C. Krettek, C. Liu, M. Jagodzinski. The effect of anti-inflammatory and antifibrotic agents on fibroblasts obtained from arthrofibrotic tissue: An in vitro and in vivo study. Bone Joint Res 2018;7:213–222. DOI: 10.1302/2046-3758.73.BJR-2017-0219.R2

Aims. Developmental dysplasia of the hip (DDH) is a complex musculoskeletal disease that occurs mostly in children. This study aimed to investigate the molecular changes in the hip joint capsule of patients with DDH. Methods. High-throughput sequencing was used to identify genes that were differentially expressed in hip joint capsules between healthy controls and DDH patients. Biological assays including cell cycle, viability, apoptosis, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were performed to determine the roles of the differentially expressed genes in DDH pathology. Results. More than 1,000 genes were differentially expressed in hip joint capsules between healthy controls and DDH. Both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that extracellular matrix (ECM) modifications, muscle system processes, and cell proliferation were markedly influenced by the differentially expressed genes. Expression of Collagen Type I Alpha 1 Chain (COL1A1), COL3A1, matrix metalloproteinase-1 (MMP1), MMP3, MMP9, and MMP13 was downregulated in DDH, with the loss of collagen fibres in the joint capsule. Expression of transforming growth factor beta 1 (TGF-β1) was downregulated, while that of TGF-β2, Mothers against decapentaplegic homolog 3 (SMAD3), and WNT11 were upregulated in DDH, and alpha smooth muscle actin (αSMA), a key myofibroblast marker, showed marginal increase. In vitro studies showed that fibroblast proliferation was suppressed in DDH, which was associated with cell cycle arrest in G0/G1 and G2/M phases. Cell cycle regulators including Cyclin B1 (CCNB1), Cyclin E2 (CCNE2), Cyclin A2 (CCNA2), Cyclin-dependent kinase 1 (CDK1), E2F1, cell division cycle 6 (CDC6), and CDC7 were downregulated in DDH. Conclusion. DDH is associated with the loss of collagen fibres and fibroblasts, which may cause loose joint capsule formation. However, the degree of differentiation of fibroblasts to myofibroblasts needs further study. Cite this article: Bone Joint Res 2021;10(9):558–570

Summary. Previous work in a rabbit model of post-traumatic joint contractures shows that the mast cell stabilizer ketotifen decreases contracture severity. We show here that ketotifen decreases collagen gel contraction mediated by rabbit joint capsule fibroblasts when mast cells are present. Introduction. Ketotifen was shown to decrease contracture severity and associated joint capsule fibrosis in an animal model of post-traumatic joint contractures. Ketotifen prevents the release of profibrotic growth factors from mast cells (MC). An in vitro collagen gel contraction assay is used to examine the effect of ketotifen on joint capsule fibroblasts obtained from this animal model. Methods. Six New Zealand White rabbits had a standardised procedure to induce post-traumatic joint contractures and the joint capsule was harvested 4 weeks later. The capsules were minced, placed into T75 culture flasks and incubated at 37. 0. C in a humidified atmosphere containing 5% CO. 2. The Joint Capsule fibroblasts (JC, 2.5 × 10. 5. cells/mL) were mixed with neutralised collagen solution composed of 59% neutralised PureCol collagen I, serum free DMEM/F12 with 1x serum replacement and 1x antibiotic-antimycotic. Aliquots of solution were then cast into wells of a tissue culture plate. Gelation occurred over 3h at 37°C in a humidified incubator. The collagen gel/cells were maintained with DMEM/F-12 plus 1% serum replacement and 1% antibiotic-antimycotic and incubated at 37°C for 12 h. The gels were released and gel area was calculated up to 72h post-release. Different experiments were conducted with various combinations of a human mast cell line (HMC-1, 7.5 × 10. 5. cells/mL), the neuropeptide Substance P (SP, 10. −6. M) and Ketotifen fumurate at 10. −4. , 10. −6. , 10. −8. and 10. −10. M. The various interventions were combined with the JC and collagen gel during the gelation step. Statistical comparisons used a two way ANOVA with a Posthoc Tukey test. Significance was set at p < 0.05. Results. The JC contracted the collagen gels in all conditions, with statistically significant differences between time intervals from 6 h to 72 h. When ketotifen alone was added to JC, there was no effect on collagen gel contraction in the range of doses tested. Adding MC to JC led to a significantly increased rate of gel contraction that was inhibited by ketotifen in a dose-dependent manner. The effect was maximal with a concentration of 10. −4. M while the effect was absent by the dose of 10. −10. M. There were statistically significant differences amongst different doses except for comparisons between doses closest to each other (10. −4. vs 10. −6. , 10. −6. vs 10. −8. , 10. −8. vs 10. −10. M). Including SP with MC and JC further increased the rate of gel contraction, which was also significantly inhibited by ketotifen in a similar dose-dependent fashion. Discussion/Conclusion. Fibroblasts from rabbit joint capsules contract collagen gels with the effect enhanced by the addition of mast cells. Ketotifen prevents the release of mediators by mast cells, and ketotifen modified the collagen gel assay. It appears that the inhibition of the gel contraction by the fibroblasts is via mast cell stabilization since ketotifen had no direct affect on the fibroblasts in the concentrations evaluated. Ketotifen is a medication used in the chronic treatment of asthma. It has a wide safety profile, it is already approved for human use and it is available in oral preparations

Introduction: Hydrogenated (acetylene:C2H2) and silanized (tetra methyl silane:TMS) diamond-like-carbon coatings (DLC) are applied to titanium alloy to reduce surface energy, cell adhesion and hydrophilicity. The incorporation of silicon into DLC reduces its surface energy. It was hypothesized that surfaces that have high surface energy and high hydrophilicity favoured the adhesion and maturation of fibroblasts when compared with C2H2 and TMS coated substrates in vitro. This would help in achieving a seal at the prosthesis – soft tissue interface, thereby helping in reducing infection. Methods: and Materials: Fibroblasts were cultured on 10 mm diameter titanium alloy, C2H2 and TMS coated titanium alloy discs for 4 hours and 24 hours (2500 cells per disc). Cell area, adhesion plaque numbers, number of plaques per unit area (plaque density) and the total area of adhesion plaques per cell were analysed. The results were compared between experimental groups and controls at 4 and 24 hours. In order to measure the strength of adhesion of cells fibroblasts were cultured on discs (30 mm diameter)[machine finished and polished(Ra = 0.031)](density-300,000 cells per disc) for 4 and 24 hours with similar coatings and exposed to radial shear by flow (100 mls/min) of culture media over their surface. These discs were then stained and analysed using Photoshop (ver.5.5) and SPSS (ver.16). Mann-Whitney tests were used to calculate significance (p< 0.05). Results: At 4 and 24 hours, the number of adhesion plaques was significantly greater on control and C2H2 compared with TMS. At 4 hours, cell area on control discs was significantly greater than C2H2 and TMS. At 24 hours, cell area on control and C2H2 was significantly greater than TMS. Between 4 and 24 hours, the number of adhesion plaques increased significantly on all the surfaces. Cell area increased significantly on C2H2 and TMS between 4 and 24 hours. At 4 hours, shear stress needed to dislodge the cells was highest for polished C2H2 and least for titanium unpolished surface. Cells on polished surfaces in corresponding groups required higher shear stress to remove the cells than cells on unpolished surfaces. At 24 hours, cells on polished C2H2 required significantly higher shear stresses to detach them than cells on unpolished C2H2 and TMS (polished and unpolished). Cells on unpolished Ti required higher stress to dislodge than cells on unpolished TMS. From 4 to 24 hours, a significant increase in shear stress to remove the cells was required on all unpolished surfaces and polished C2H2. A significant correlation was seen between adhesion plaque density at 4 hours and shear stress. Discussion: This work supports the hypothesis that surfaces with high surface energy and high hydrophilicity lead to increased cell attachment and cell area. It also shows the correlation between adhesion plaque density and the shear stress needed to dislodge fibroblasts from bioactive surfaces

Introduction: Autologous chondrocyte implantation (ACI) has been developed in order to repair cartilage successfully. Experimental models are based on osteochondral defects with potentially triphasic chondrogenic system: periosteal flaps, bone marrow cells and transplanted chondrogenic cells. All these three have chondrogenic activity so it is difficult to determinate the role of the implanted cells unless appropriate control is set up. The purpose of this study is to determinate if the inoculation of chondrocytes under periosteal flaps does improve the chondrogenic potential of periosteal flaps. MATERIALS AND Methods: 10 New Zealand rabbits, 8 months old were used. Right knees served as study group (ACI Group; N5: Chondrocytes + Periosteal Flap) – (Fibroblast Group: N5 Fibroblast + Periosteal Flap) and left knees as control group (N: 10: osteochondral defect alone). During the first procedure dermal fibroblast cells were isolated from skin biopsy and chondrocytes were isolated from the medial femoral condyle as a full thickness of the right and left knee were done. Chondrocytes and dermal fibroblasts cells were incubated for 4 weeks. Then they were implanted under periostel flap according to study group. Chondrocyte and Fibroblast Implantation:. A parapatellar incision was performed on both knees. Defect was cleaned and on study group the periosteum taken from the tibia was sutured leaving one edge free to inoculate the chondrocytes or fibroblast according to group using a needle Then the defect was closed using fibrin glue. The animals were euthanatized 8 months postoperative. Analysis: Specimens were evaluated using Hematoxylin and Eosin. Safranine and inmunohistochemistry for Collagen Type 2 using the ICRS score system. Statistical Analysis: T student, Fisher and confidence interval were used. A p value < 0,05 was considered significant. Results: Control non treated group presented a histological score grade mean IV (95% CI: 44–97). The ACI group showed a tissue type means II (ICRS) (95% CI: 28–99%) Collagen type 2 was evident only in the deep layers. The fibroblast group did show a reparative tissue, tissue type mean II (95% CI: 28–99%) Collagen type 2 was evident in deep layers. DISCUSSION: According to this study the inoculation of chondrocytes under periosteal flaps does not improve significally the chondrogenic potential of periosteal flaps.(p: 0,77). Comparing the same procedure with chondrogenic and non chondrogenic cell lines could determinate the role of different chondrogenic components (periosteum and chondrocytes). Probably the chondrogenic capacity of the periosteum is sufficient to stimulate a reparative tissue. However none of these procedures could establish an adult normal cartilage hyaline tissue

Introduction. Intraosseous transcutaneous amputation prostheses (ITAP) provide an alternative means of attaching artificial limbs for amputees. Conventional stump-socket devices are associated with soft tissue complications including; pressure sores and tissue necrosis. ITAP resolves these problems by attaching the exo-prosthesis transcutaneously to the skeleton. The aim of this study is to increase the attachment of dermal fibroblasts to titanium alloy in vitro. Fibronectin (Fn) and laminin 332 (Ln) enhance early cell growth and adhesion. We hypothesize that silanized dual coatings of fibronectin and laminin (SiFnLn) will be more durable when compared with adsorbed dual coating (AdFnLn), and will enhance early fibroblast growth and adhesion compared to single coatings. Methods. The kinetics of dual single and dual protein coating attachment onto titanium alloy was quantified on silanized 10mm diameter discs using radiolabelled Fn (125I-Fn) and Ln (125I-Ln). Sixty discs were polished, sterilized and silanized. Coating durability was assessed when soaked in fetal calf serum (FCS) for 0, 1, 24, 48 and 72hrs. Data was compared to un-silanized Ti discs with the same coatings. Five thousand human dermal fibroblasts were seeded on discs (n = 6) of Ti polished alone (Pol), Ti with adsorbed fibronectin (AdFn), Ti with adsorbed laminin (AdLn), Ti adsorbed dual coating (AdFnLn), Ti silanized (Si), Ti silanized with fibronectin (SiFn), Ti silanized with laminin (SiLn), Ti silanized with a dual coating (SiFnLn) for 24hrs. In order to measure cell adhesion fibroblasts were fixed, vinculin stained using mouse vinculin antibody and alexa fluor. Axiovision Image Analysis software was used to measure cell area, vinculin focal adhesion markers per cell and per unit cell area. Data was analysed in SPSS and significance was assumed at the 0.05 level. Results. Silanized dual coatings bonded to Ti alloy in significantly larger quantities compared with adsorbed coatings at all time points (all p values < 0.05). Fibroblasts cultured on dual coatings were significantly larger, produced more vinculin markers per cell, and per unit cell area compared with single coatings. Cells on SiFnLn were larger with more numerous vinculin markers per cell, and per unit cell area compared with AdFnLn (p<0.05). Conclusion. This study has demonstrated that covalently bonding both fibronectin and laminin to Ti alloy provides a durable, dual coating that enhances early fibroblast growth and attachment compared with either protein coating alone in vitro. Our study showed that there is non-competitive binding of laminin on Ti surfaces in the presence of fibronectin. Dual coatings may be applied to the skin-penetrating region of transcutaneous devices to improve the skin seal and this may have positive implications for the development of ITAP

Rheumatoid arthritis (RA) is an autoimmune disease that involves T and B cells and their reciprocal immune interactions with proinflammatory cytokines. T cells, an essential part of the immune system, play an important role in RA. T helper 1 (Th1) cells induce interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α), and interleukin (IL)-2, which are proinflammatory cytokines, leading to cartilage destruction and bone erosion. Th2 cells primarily secrete IL-4, IL-5, and IL-13, which exert anti-inflammatory and anti-osteoclastogenic effects in inflammatory arthritis models. IL-22 secreted by Th17 cells promotes the proliferation of synovial fibroblasts through induction of the chemokine C-C chemokine ligand 2 (CCL2). T follicular helper (Tfh) cells produce IL-21, which is key for B cell stimulation by the C-X-C chemokine receptor 5 (CXCR5) and coexpression with programmed cell death-1 (PD-1) and/or inducible T cell costimulator (ICOS). PD-1 inhibits T cell proliferation and cytokine production. In addition, there are many immunomodulatory agents that promote or inhibit the immunomodulatory role of T helper cells in RA to alleviate disease progression. These findings help to elucidate the aetiology and treatment of RA and point us toward the next steps. Cite this article: Bone Joint Res 2022;11(7):426–438

Objective. Excessive mechanical stress on synovial joints causes osteoarthritis (OA) and results in the production of prostaglandin E2 (PGE2), a key molecule in arthritis, by synovial fibroblasts. However, the relationship between arthritis-related molecules and mechanical stress is still unclear. The purpose of this study was to examine the synovial fibroblast response to cyclic mechanical stress using an in vitro osteoarthritis model. Method. Human synovial fibroblasts were cultured on collagen scaffolds to produce three-dimensional constructs. A cyclic compressive loading of 40 kPa at 0.5 Hz was applied to the constructs, with or without the administration of a cyclooxygenase-2 (COX-2) selective inhibitor or dexamethasone, and then the concentrations of PGE2, interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-6, IL-8 and COX-2 were measured. Results. The concentrations of PGE2, IL-6 and IL-8 in the loaded samples were significantly higher than those of unloaded samples; however, the concentrations of IL-1β and TNF-α were the same as the unloaded samples. After the administration of a COX-2 selective inhibitor, the increased concentration of PGE2 by cyclic compressive loading was impeded, but the concentrations of IL-6 and IL-8 remained high. With dexamethasone, upregulation of PGE2, IL-6 and IL-8 was suppressed. Conclusion. These results could be useful in revealing the molecular mechanism of mechanical stress in vivo for a better understanding of the pathology and therapy of OA. Cite this article: Bone Joint Res 2014;3:280–8

Introduction. Cell-based tendon engineering is an attractive alternative therapeutic approach to established treatments of tendon injuries. Numerous cell types are promising source of tendon engineering; however, there are certain disadvantages for each cell type. Interestingly, dermal fibroblasts (DFs) are able to transdifferentiate into other cell types, they are widely distributed in dermis and easy to harvest and isolate. Furthermore, pilot clinical studies suggested a promising therapeutic potential of autologous DFs for discorded tendons (Connell et al., 2009&2011), but the underlining repair mechanisms remain unclarified. To investigate tenogenic differentiation process in great detail, we have previously established a three-dimensional (3D) cell sheet model, comprising of three consecutive step (expansion, stimulation and maturation) leading to the formation of 3D tendon-like tube (Hsieh et al., 2018; Yan et al., 2020). Hence, the aim of this study was to carry out pilot examination of the tenogenic potential of human DFs (hDFs) by implementing the 3D cell sheet model. Methods. hDFs (company purchased, n=2), hBMSCs (human bone marrow mesenchymal stem cells, n=1) and hTSPCs (human tendon stem/progenitor cells, n=1) were used and subjected to the 3D model. In 2D culture, semi-qPCR was performed to validate the expression of DF markers in hDFs, namely NTN1, PDPN and CD26 for papillary dermis layer, and PPARG, ACTA2 and CD36 for reticular dermis layer). FACS analysis and immunofluorescence were employed to validate expression of CD73, CD90, CD105 and vimentin (mesenchyme marker), respectively. After harvesting the 3D cell sheets, wet weigh measurements, H&E and collagen type I stainings, and semi-qPCR for Scleraxis and tenomodulin were executed. Results. Semi-qPCR of DF markers validated the dermal origin of both donor-derived hDFs; however, the data suggested that donor 1 was mixed cell pool of papillary and reticular dermal cells, whilst donor 2 was reticular dermal cells. In FACS analysis, the expression levels of CD73 and CD90 were comparable among all cell types. For CD105, ca. 20% of the cells were negative in both hDF and hTSPC cultures, but only 2% in hBMSCs. As expected, all three cell types were vimentin-positive. 3D cell sheet formation was successful for all cell type. Interestingly, the hDF cell sheets were thicker and ca. 2-fold heavier than that of hBMSCs and hTSPCs. Next, H&E and collagen I analyses revealed higher cellularity as well as higher collagen I deposition in the hDF sheets compared to the other two cell types. Last, semi-qPCR for Scleraxis and tenomodulin suggested upregulation of both genes in hDF and hTSPC sheets versus 2D culture. Discussion. Our pilot data suggests that hDFs perform well and even exceed hBMSCs and hTSPCs in the 3D model in terms of size, cellularity and collagen I expression. However, increase of cohort size and further detailed molecular and histomorphometric analyses are necessary to conclude on the promising tenogenic potential of hDFs

Purpose: Vascular Endothelial Growth Factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy accelerates bone healing of a rabbit tibia segmental bone defect in-vivo, and increases osteoblast proliferation and mineralization in-vitro. The aim of this project was to examine the effect of exogenous human VEGF (hVEGF) on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Method: The osteoblasts were obtained from the rat periosteum. The fibroblasts were obtained from the rat dermal tissue. The cells were then cultured to reach 60% confluence and transfected with hVEGF using Superfect. Four groups were:. osteoblast-hVEGF,. fibroblast-hVEGF,. Osteoblasts alone, and. Fibroblasts only. The cultured cells were harvested at 1, 3 and 7 days after the transfection. The total mRNA was extracted (TRIZOL); both hVEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantified by VisionWorksLS. Results: The hVEGF mRNA was detected by RT-PCR from transfected osteoblasts after three days of gene transfection. The hVEGF mRNA expression in transfected fibroblasts increased exponentially at days 1, 3 and 7 after the transfection. We compared the endogenous rat VEGF mRNA expression level of the osteoblasts or fibroblasts that were transfected with hVEGF with the cells without the transfection. The hVEGF transfected osteoblasts had a greater rat VEGF mRNA expression than the non-transfected osteoblasts. Furthermore, when hVEGF was transfected to the rat fibroblasts, the endogenous mRNA expression level measured was also greater than that from the non-transfected fibroblasts. Rat VEGF mRNA expression increased in the first three days of the hVEGF transfection, but the expression level was reduced at Day 7. Conclusion: These results suggest that cell-based hVEGF gene therapy enhances endogenous rat VEGF mRNA expression in both osteoblasts and fibroblasts

Objective: To determine if the anatomical location of a tendon (hand or forearm) influences fibroblast function in the presence of physical forces. Introduction Tendons are anatomical structures specialized to transmit high tensile loads from muscle to bone. When damaged, clinical recovery is slow and incomplete. Various authors have shown that application of tensile loading during recovery (such as in early active motion following hand flexor tendon repair) will accelerate the recovery of tensile strength. The mechanism is unknown and the optimum loading regime has not been quantitated. It is likely that similar influences are working in rheumatoid arthritis but there is clinical evidence that the response to applied load is very different. In this study a commercial system (Bio stretch) was used to apply different strain regimes to cells in culture, and then to assess the response by a series of quantitative methodologies. Materials Cells were obtained by the explant technique from tendons of the hand and forearm to generate confluent cultures. In this experiment fibroblasts cultured from intra-synovial tendons (Group 1)were compared with cultured fibroblasts of forearm tendons (Group II). We used the Biostretch Apparatus (ICCT Technologies Canada), to stretch fibroblasts in a gel foam (Helistat, Integra TM ) construct. The Biostretch apparatus uses a magnetic field to stretch cells within the gel foam. After seeding the gel foam pieces (1cm2) with a concentrated cell suspension (4 x105 cells/100 μlitre) , the apparatus was used at 40% stretch, with a burst time of 15 minutes and a rest time of 45 minutes at 37° C and 60 cycles a second for 24 hours. The experiment was performed in triplicate for both type of cells (Group I & II), with another group of cells serving as controls. At the end of 24 hours the BCA method was used to estimate Total Protein content while the Sircol method was used to determine Type 1 Collagen levels. Results: Preliminary results indicate that there is a trend towards increased secretion of proteins and collagen in the stretched samples compared to the controls. Similarly the fibroblasts obtained from intra-synovial tendons seemed to produce more total protein and collagen as compared to the forearm. However both these observations failed to reach statistical significance. Conclusions: Previous work (. Evans CE et al. 2001. ) has shown no difference between collagen and protein production between flexor and extensor tendon, even under strain,. In this study the increased production of matrix proteins and collagen under the influence of physical strain may explain why flexor tendon injuries in the hand tend to heal with the formation of adhesions and poor functional results as compared with the forearm where the results tend to be uniformly better. However it must be stressed that these are preliminary results and further work will be required to provide definitive data

Introduction and Aims: To establish whether basic fibroblast growth factor (bFGF) plays a role in the changes in chondrocyte metabolism exhibited in human osteoarthritis (OA). Method: BFGF and its receptor was localised by immunohistochemistry within human OA. The results from OA tissue graded ‘early’ and ‘advanced’ were compared. This was correlated with the identification of proliferating chondrocytes (using by localising PCNA) and dead/dying chondrocytes (using the TUNEL technique). Results: Results showed that bFGF and its receptor were strongly localised around chondrocytes in proliferating clusters in ‘early’ OA, whereas no bFGF was detected in ‘advanced’ OA. In addition, a loss of bFGF activity in ‘advanced’ OA correlated with the identification of large numbers of dead/dying chondrocytes. Conclusion: Results suggest that high levels of bFGF activity in OA play an important role in chondrocyte proliferation and the formation of chondrocyte clusters. In addition, the loss of this activity appears to be directly related to an increase in cell death in ‘advanced’ OA, suggesting that bFGF acts as a ‘survival’ factor in this tissue. The more we understand about the metabolic changes in chondrocytes during OA, the closer we come to delaying or preventing this debilitating joint disease

Background. Mechanical trauma to articular cartilage is a known risk factor for Osteoarthritis (OA). The application of single impact load (SIL) to equine articular cartilage is described as a model of early OA changes and shown to induce a damage/repair response. Recombinant Human Fibroblast Growth Factor-18 (rhFGF-18) has been previously shown to have anabolic effects on chondrocytes in vitro. The aim of this in vitro study was to ascertain the effect of rhFGF-18 on the repair response of mechanically damaged articular cartilage. Methods. Articular cartilage discs were harvested from healthy mature horses (n=4) and subjected to single impact load using a drop tower device. The impacted explants, together with unimpacted controls were cultured in modified DMEM +/− 200ng/ml rhFGF-18 for up to 30 days. Glycosaminoglycan (GAG) release into the media was measured using the dimethylmethylene blue (DMMB) assay, aggrecan neopepitope CS846 and Collagen Propeptide II (CPII) were measured by ELISA. Histological analysis, immunohistochemistry and TUNEL staining were used to assess proteoglycan content, type II and type VI collagen localisation, cell morphology, repair cell number and cell death. Results. Impacted explants treated with rhFGF-18 showed significantly more GAG release and CS846 release into the media compared to other experimental groups (p<0.05), but no significant increase in CPII levels. Loaded sections treated with rhFGF-18 had increased type II and VI collagen immunohistochemistry scores, more repair cells on the tissue surface and significantly less cell death (p<0.001) compared to other experimental groups at day 30 in culture. Conclusion. In an in vitro damage/repair model, rhFGF-18 increases the proteoglycan synthesis, collagen type II and VI protein within sections and the repair cell number and prevents apoptosis at Day 30. This suggests that rhFGF18 may be a good candidate for enhancement of cartilage repair following mechanical damage

Aims

The antidiabetic agent metformin inhibits fibrosis in various organs. This study aims to elucidate the effects of hyperglycaemia and metformin on knee joint capsule fibrosis in mice.

Introduction: In this study infrapatellar fat pad (IPFP) derived stem cells were expanded with and without Fibroblast Growth Factor-2 (FGF-2) supplementation and were compared with regards to their ability to proliferate and differentiate into chondrocytes. Materials and Methods: Cells were isolated from the IPFP tissue and expanded in monolayer culture with and without rhFGF-2 supplementation (final concentration 10ng/ ml). Cell aggregates were placed in chondrogenic media for two weeks. Gene expression studies were carried out using quantitative real time PCR. Immunohistochemical labelling was performed with antibody localisation determined by an immunoperoxidase procedure. The pellets were also weighed and digested in papain for DNA and glycosoaminoglycan (GAG) analysis. Results: Cells expanded in FGF-2 supplemented media were smaller and proliferated more rapidly. The FGF-2 supplemented cell aggregates also showed 100 times higher expression of collagen type II (COL2A1). Immunohistochemical studies showed that pellets made from FGF-2 treated cells stained more strongly for collagen II and more weakly for collagen I. Pellets made with FGF-2 treated cells were larger, continued with enhanced proliferation and contained more proteoglycan. Conclusion: Our findings show enhanced proliferation and chondrogenic differentiation in IPFP derived stem cells expanded in FGF-2 supplemented media

Mesenchymal stem/stromal cells (MSC) have the ability to home and migrate towards injured and inflamed tissues which can be useful as a minimally invasive systemic approach to deliver MSC to the site of damaged articular surface in arthritis in human and veterinary patients. From a molecular point of view, the CXCR4/SDF-1 plays an important role in this phenomenon and can be used as a target to enhance the therapeutic efficacy of culture expanded MSC. It has been demonstrated that extensive in vitro expansion down-regulates CXCR4 expression in human, murine and canine MSCs hindering their therapeutic efficacy. Therefore, the aim of the present study was to assess the effect of hypoxia and basic fibroblast growth factor (bFGF) pre-conditioning on CXCR4 and SDF-1 expression in canine adipose derived MSC (cAT-MSC). MSC were isolated from subcutaneous adipose tissue of two adult Beagle dogs (n=2; 3–5 years old, 9–12kg) and cultured under standard conditions (5%CO. 2. , 37°C). Cells at passage 3 were then cultured in hypoxia (2%O. 2. ) and normoxia, with supplementation of 1 and 5 ng/ml bFGF for 24h. MTT assay, flow cytometry, immunohistochemistry and qRT-PCR analysis were conducted to assess respectively the modulation effect on cell proliferation, CXCR4 protein expression and CXCR4 and SDF-1 gene expression. Cell proliferation increased proportionally with the increasing bFGF concentrations, with a statistically significant higher proliferative rate in normoxic conditions (p<0.05). The gene expression of CXCR4 and SDF-1 increased in hypoxic conditions with bFGF supplementation (p<0.05). bFGF supplementation increased cytoplasmatic expression of CXCR4 in hypoxic conditions (p<0.05), however the surface expression remained low in all culture conditions. The described pre-conditioning method can be used for the enhancement of the therapeutic potential of systemically administered canine AT-MSC and can have a relevant translational character for the optimization of culturing protocols of human adipose derived MSC

Introduction: Fibroblast growth factor receptor 3 (FGFR3) is a tyrosine kinase membrane-spanning protein whose function is to regulate chondrocyte proliferation, differentiation and matrix production during cartilage development. Several mutations in FGFR3 have now been documented to link to human diseases. A number of these mutations result in constitutive activation of the FGFR3, leading to proliferation and premature differentiation of chondrocytes. Depending on the mutation and the resultant level of FGFR3 activation, mild to severe skeletal dysplasias such as achondroplasia (ACH), hypochondroplasia (HCH), thanatophoric dysplasia type I (TDI) and type II (TDII), and severe achondroplasia with developmental delay and acanthosis nigrans (SADDAN) may result. It has been postulated that the signalling pathways downstream of FGFR3 may be responsible for activating transcription factors, leading to up-regulation of cell cycle inhibitors and causing abnormal suppression of chondrocyte cell proliferation. However, the precise signalling pathways involved in FGFR3 mutation have as yet to be elucidated. The aim of this study was to investigate and compare the differences in the downstream signalling pathways between FGFR3 mutants. Methods and Results: Wild type FGFR3 has been cloned into expression vector pcDNA3 and the construct has been used to generate four different FGFR3 mutants using site-directed mutagenesis. The mutations which have been introduced and the types of dysplasia they correspond to were as follows: K380R (ACH), N540K (HCH) and K650E (TDII). A kinase dead form of the receptor, K504R has also been generated. Wild type and each of the four mutant FGFR3 proteins in pcDNA3 vector have been successfully transfected into 293T cells using the calcium phosphate method. Immunoprecipitation and Western Blot analysis of cell lysates revealed expression of wild type protein in three isoforms of size 135kDa (mature), 120kDa (intermediate) and 98kDa (immature). The mutant proteins all followed a similar pattern of expression with the exception of the TDII mutant that did not express the mature form of the FGFR3. Changes in MAPK, PLCã and STAT 1 signalling pathways in response to FGFs-1, 2, 9 and 18 in the 293-cells of wild type and mutant forms of FGFR3 are now under investigation, in an attempt to define which pathways are mostly responsible for the resultant abnormal phenotypes. Discussion: Genomics studies have demonstrated that FGFR3 expression is significantly upregulated during the osteoblastic differentiation of mesenchymal stem cells (MSCs) under BMP-2 stimulation in vitro. Subsequent functional studies have demonstrated that a selective ligand for FGFR3, FGF9, is able to induce tyrosine kinase signalling, and the osteoblastic differentiation of MSCs in vitro. Further understanding the signalling mechanisms of FGFR3 activation in normal and mutant forms may lead to discover potential anabolic agents that are based on FGFR3-FGFs pathways

To try and aid the formation of a soft tissue seal to promote dermal and epidermal attachment to Intraosseous Transcutaneous Amputation Prostheses we compared the effect of titanium surfaces functionalised with fibronectin (fn) or YRGD peptide sequences on human dermal cell (HDF) attachment. We hypothesise that YRGD and fn coatings will significantly increase HDF attachment to titanium alloy substrates. Titanium alloy 10mm discs were polished and acted as control substrates, functionalised surfaces had YRGD or fn adsorbed or silanised onto the polished surface. HDFs were seeded at 10,000/disc and cultured for 1, 4, 24 and 96 hours, fixed and fluorescent immnolocalisation for vinculin was performed. Individual vinculin markers were counted and density calculated as a measure of cell attachment. All assays were performed in triplicate and data were analysed in SPSS 19.0 and results were considered significant at the 0.05 level. Results showed an up-regulation of Focal adhesion density (FA) against controls at all time-points (excluding ad-fn at 4 hours, p=0.057), p values < 0.05, the use of functionalised titanium surfaces may lead to long-term clinical success of ITAP. We have shown a significant positive effect on cell attachment when a synthetic peptide sequence is used. Using synthetic peptide sequence may also be more beneficial from a regulatory stand-point compared with using isolated proteins.