We compared the biological characteristics of extrinsic fibroblasts infiltrating the patellar tendon with those of normal, intrinsic fibroblasts in the normal tendon in vitro. Infiltrative fibroblasts were isolated from the patellar tendons of rabbits six weeks after an in situ freeze-thaw treatment which killed the intrinsic fibroblasts. These intrinsic cells were also isolated from the patellar tendons of rabbits which had not been so treated. Proliferation and invasive migration into the patellar tendon was significantly slower for infiltrative fibroblasts than for normal tendon fibroblasts. Flow-cytometric analysis indicated that expression of α5β1 integrin at the cell surface was significantly lower in infiltrative fibroblasts than in normal tendon fibroblasts. The findings suggest that cellular proliferation and invasive migration of fibroblasts into the patellar tendon after necrosis are inferior to those of the normal fibroblasts. The inferior intrinsic properties of infiltrative fibroblasts may contribute to a slow remodelling process in the grafted tendon after ligament reconstruction

In order to clarify the role of cytokines in the remodelling of the grafted tendon for ligament reconstruction we compared the responses to interleukin (IL)-1β, platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-β1 of extrinsic fibroblasts infiltrating the frozen-thawed patellar tendon in rats with that of the normal tendon fibroblasts, in regard to the gene expression of matrix metalloproteinase (MMP)-13, using Northern blot analysis. We also examined, immunohistologically, the local expression of IL-1β, PDGF-BB, and TGF-β1 in fibroblasts infiltrating the frozen-thawed patellar tendon. Northern blot analysis showed that fibroblasts derived from the patellar tendon six weeks after the freeze-thaw procedure in situ showed less response to IL-1β than normal tendon fibroblasts with respect to MMP-13 mRNA gene expression. The immunohistological findings revealed that IL-1β was over-expressed in extrinsic fibroblasts which infiltrated the patellar tendon two and six weeks after the freeze-thaw procedure in situ, but neither PDGF-BB nor TGF-β1 was over-expressed in these extrinsic fibroblasts. Our findings indicated that IL-1β had a close relationship to matrix remodelling of the grafted tendon for ligament reconstruction, in addition to the commencement of inflammation during the tissue-healing process

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

The interactions between the different cell types in periprosthetic tissue are still unclear. We used a non-contact coculture model to investigate the effects of polymethylmethacrylate (PMMA) particles and human macrophage-derived soluble mediators on fibroblast activation. Macrophages were either exposed or not exposed to phagocytosable PMMA particles, but fibroblasts were not. Increasing numbers of macrophages were tested in cocultures in which the fibroblast cell number was held constant and cultures of macrophages alone were used for comparison of cytokine release. We used the release of interleukin-1 beta (IL-1β), interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-α), lysosomal enzyme and metalloproteinase activity to assess the cultivation of macrophages and fibroblasts. In cocultures, IL-6 release was increased 100-fold for both unchallenged and particle-challenged cultures when compared with macrophage cultures alone. Furthermore, particle-challenged cocultures had threefold higher IL-6 levels than unchallenged cocultures. Release of TNF-α was similar in cocultures and in macrophage cultures. IL-1β release in cocultures was independent of the macrophage-fibroblast ratio. Lysosomal enzyme activity and metalloproteinase activity were increased in cocultures. Our data show that macrophages and fibroblasts in coculture significantly increase the release of IL-6 and to a less degree other inflammatory mediators; particle exposure accentuates this effect. This suggests that macrophage accumulation in fibrous tissue may lead to elevated IL-6 levels that are much higher than those caused by particle activation of macrophages alone. This macrophage-fibroblast interaction represents a novel concept for the initiation and maintenance of the inflammatory process in periprosthetic membranes

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

We have investigated in vitro the release kinetics and bioactivity of fibroblast growth factor-2 (FGF-2) released from a carrier of fibrin sealant. In order to evaluate the effects of the FGF-2 delivery mechanism on the repair of articular cartilage, full-thickness cylindrical defects, 5 mm in diameter and 4 mm in depth, which were too large to undergo spontaneous repair, were created in the femoral trochlea of rabbit knees. These defects were then filled with the sealant. Approximately 50% of the FGF-2 was released from the sealant within 24 hours while its original bioactivity was maintained. The implantation of the fibrin sealant incorporating FGF-2 successfully induced healing of the surface with hyaline cartilage and concomitant repair of the subchondral bone at eight weeks after the creation of the defect. Our findings suggest that this delivery method for FGF-2 may be useful for promoting regenerative repair of full-thickness defects of articular cartilage in humans

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

We have studied the formation of collagen fibrils in ‘activated fibroblasts’ of tendo Achillis of rabbits. The tendon was in the process of regeneration after experimental partial tenotomy. Samples were taken from the peri-incisional region and analysed by transmission electron microscopy. Ultrastructural examination showed the presence of a ‘fine dense granular substance’ inside the rough endoplasmic reticulum and procollagen filaments. These come together to form collagen fibrils in the dilated vacuoles of the rough endoplasmic reticulum. The possible intra- and extracellular origin of collagen fibrils is suggested. Within the cell biosynthesis of collagen fibrils take place with the formation of collagen substance which gives rise to procollagen filaments. These make contact in parallel apposition to produce striated ‘spindle-shaped bodies’ which elongate by the longitudinal attachment of more procollagen filaments and form intracellular nascent collagen fibrils

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