Purpose: A preliminary biomechanical test conducted on cadaver specimens validated a new technique for vertebral bone harvesting for anterior intervertebral grafting of the lumbar spine. A cylinder of autologous bone harvested from a neighboring vertebra was used for the intervertebralimplant. The harvesting site was filled with a bone substitute. The biomechanical tests confirmed good restoration of the vertebral body structure. An in vivo study was conducted in the baboon. A block of tricalcium-phosphate (beta-TCP) impregnated with transforming growth factor beta3 (TGF-beta3) was used to fill the bone gap. The purpose of the present study was: 1) to assess the efficacy of this in vivo technique on a primate model, 2) to validate the surgical technique.

Material and methods: The retroperitoneal approach was used to operate nine baboons. Eighteen bone cylinders were harvested. The harvesting hole was left empty or filled with a 15 mm diameter beta-TCP cylinder, or with a beta-TCP cylinder impregnated with TGR-beta3. Control scans were obtained at three and six months postoperatively. The baboons were sacrificed at 6 months and the vertebral bodies were removed for histology study.

Results: There was no evidence of fracture or loss of vertebral body height. The harvesting holes left empty did not fill, while osteointegration and substantial resorption of the bone substitute was observed in the two other groups. In the group with beta-TCP impregnated with TGF-beta3 the resorption of the cylinder was more complete and signs of prevertebral neoformation of subperiosteal bone, not observed in the beta-TCP group, was observed. There was no neoformation of bone in the spinal canal or in the foramens. The scans showed progressive resorption starting three months postoperatively in the beta-TCP group impregnated with TGF-beta3.

Conclusion: The beta-TCP block used is a very good bone substitute for the primate spine. TGF-beta3 accelerates bone resorption and induces neoformation of subperiosteal bone. The new surgical technique for local harvesting of vertebral bone was validated.

Studies have demonstrated that use of peptides including bone morphogenetic proteins, fibroblast growth factors, insulin-like growth factor (IGF), and transforming growth factor-beta (TGF-beta), may be pivotal in promoting chondrogenesis and matrix development. As a prelude to studies, it is necessary to determine which gene or combination of genes gives the best result to improve proliferation of chondrocytes and synthesis of extracellar matrix. We investigate the effect of transfec-tion of recombined rat TGF-beta1 and recombined rat IGF-1 on rabbit chondrocytes ex vivo.

Chondrocytes were isolated from articular cartilage of knee joint of mature New Zealand White rabbits. Cells were seeded at a density of 1×105 cells/ml into 6-well plates. Monolayer cultures were infected respectively with recombinant rat gene pcDNA3+TGF-beta 1, pAT153+IGF-1 and lac Z reporter gene by using lipo-fectamine, and were co-transfected by pcDNA3+TGF-beta 1, pAT153+IGF-1. The control group remained uninfected. To determine whether the genes transcript were translated and the gene products were released, the synthesis of TGF-beta 1, IGF-1,and type II collagen were measured by in situ hybridization, immunohisto-chemistry and immunofluoroscopy. The proliferation of chondrocytes was detected by flow cytometer and 3H-TdR radiolabeling.

The expression of TGF-beta1,IGF-1 and type II collagen in recombinant rat gene transfection groups was high beyond control levels and the lac Z gene levels (P< 0.05). The co-transfection elevated these factors synthesis beyond the levels of single gene transfection (P< 0.05). In pcDNA3 +TGF-beta1 transfection group, the level of TGF-beta1 and type II collagen were higher than the levels of pAT153+IGF-1 group (P< 0.05), while the content of IGF-1 has no significant difference with pAT153+IGF-1 group. By using flow cytometer, the chondrocytes ratio of S stage in pcDNA3+TGF-beta 1 group, pAT153+IGF-1 group and co-transfection group was 33.4%,28.7% and 40.1% respectively, which was higher than 5.6% and 4.8% of the control group and the lac Z gene group (P< 0.05). The 3H-TdR radiolabeling detection also indicated that the recombinant rat gene transfection groups improved the chondrocytes proliferation, and co-transfection group has the best effect.

The data presented support that transfection of genes of TGF-beta1 and IGF-1 into chondrocytes ex vivo can greatly increase cell proliferation and matrix synthesis, and the co-transfection can provoke more increase in the synthesis of TGF-beta1, IGF-1 and type II collagen, which encourages the further research of gene potential therapeutic use for osteoarthritis.

Objective To decide whether recombined rat transforming growth factor beta-1 gene and insulin-like growth factor-1 gene have positive influences on ACLT-induced osteoarthritis-like changes in NZW rabbit articular cartilage.

Methods Twenty-four NZW rabbits, with osteoarthritis caused by anterior cruciate ligament transection£^..ACLT£©, were distributed to 4 groups randomly and another six rabbits were taken as normal control group (group 1). Chondrocytes which had been transfected with TGF-¦Â1 gene, IGF-1 gene (group 3–5) were injected into the knee of these NZW rabbits. Experimental control group (group 2) was only suffered ACLT but nothing injected. After 4, 8 weeks, rabbits were sacrificed and evaluated by morphological grades, histological examination, examination of in situ hybridization, immunohistochemistry, and transmission electron microscopy (TEM).

Results The data of morphological grades showed that the normal control showed a significant difference compared with experimental control group (P< 0.01). The groups with injected chondrocytes carring TGF-¦Â1 gene and double genes (group 3,5) had a significant difference compared with experimental control group (P< 0.05). The in situ hybridization and immunohis-tochemistry examination showed the same results as above, and the group carring double genes (group 5) had a significant difference with that single gene (group 3,4) (P< 0.05). After 8 weeks, the examination data showed that all groups lower than the data of 4 weeks except the normal control group and experimental control group (P< 0.05). Ultrastructural examination indicated that the ultrastructure of experimental control group was more turbulent than that of normal control group. The ultra-structure of the gene therapy groups was more normal than that of experimental control group after gene therapy, but it turned to be turbulent again after 8 weeks.

Conclusion It is effectual on osteoarthritis to inject chondrocytes carring recombined TGF-¦Â1,IGF-1 genes into NZW rabbits knee joints. It was obvious that the therapy effect of double genes was better than single gene. The fact that gene expression was decreased gradually after 4 weeks makes out that gene therapy is limited by time. These results suggest that therapeutic TGF-¦Â1 and IGF-1 gene transfer may be applicable for the treatment of OA.

Tendon and ligament injuries represent highly prevalent and unmet clinical challenge that may significantly benefit from tissue engineering therapeutic strategies, once optimal cell source and biomolecules regulating tendon homeostasis are properly defined. Herein, we aimed to evaluate the expression of tendon/ligament markers in two novel cell populations, namely human dental pulp stem cells (DPSCs) and periodontal ligament cells (PDLCs), in response to supplementation with TGF-β ligands relevant for tendon development and healing, as well as under standard tri-lineage differentiation conditions. DPSCs and PDLCs were isolated from sound human permanent molars removed for orthodontic reasons. Pulp tissue and periodontal ligament were minced and digested with collagenase (3mg/mL) and cells were expanded in α-MEM supplemented with 10% fetal bovine serum (basal medium). To evaluate the susceptibility of DPSCs and PDLCs to tenogenic induction, cells were seeded at density of 1000 cells/cm² and cultured up to 21 days in basal medium or media supplemented with TGF-β3 (10ng/ml), or GDF-5 (50 ng/ml). Cell response was evaluated weakly by analysis of expression of tendon, bone and cartilage markers, employing real time RT-PCR and immunocytochemistry. A significant increase in collagen I and collagen III expression was observed with the culture progression in all conditions, with abundant matrix being deposited by day 14. A significant upregulation of scleraxis expression was demonstrated in response to supplementation with TGF-β3 in both cell populations, when compared to basal medium and medium with GDF-5. It was concluded that TGF-β3 may represent an effective inducer of stem cell tenogenic differentiation.

The incidence of acute and chronic conditions of the tendo Achillis appear to be increasing. Causation is multifactorial but the role of inherited genetic elements and the influence of environmental factors altering gene expression are increasingly being recognised. Certain individuals’ tendons carry specific variations of genetic sequence that may make them more susceptible to injury. Alterations in the structure or relative amounts of the components of tendon and fine control of activity within the extracellular matrix affect the response of the tendon to loading with failure in certain cases.

This review summarises present knowledge of the influence of genetic patterns on the pathology of the tendo Achillis, with a focus on the possible biological mechanisms by which genetic factors are involved in the aetiology of tendon pathology. Finally, we assess potential future developments with both the opportunities and risks that they may carry.

Cite this article: Bone Joint J 2013;95-B:305–13.

Vascular inflammation and activation of myofibroblasts are significant contributors to the progression of fibrosis, which can severely impair tissue function. In various tissues, including tendons, Transforming growth factor beta 1 (TGF-β1) has been identified as a critical driver of adhesion and scar formation. Nevertheless, the mechanisms that underlie fibrotic peritendinous adhesions are still not well comprehended, and human microphysiological systems to help identify effective therapies remain scarce. To address this issue, we developed a novel human Tendon-on-a-Chip (hToC), comprised of an endothelialized vascular compartment harboring circulating monocytes and separated by a 5 μm/100 nm dual-scale ultrathin porous membrane from a type I/III collagen hydrogel with primary tendon fibroblasts and tissue-resident macrophages, all under defined serum-free conditions. The hToC models the crosstalk of the various cells in the system leading to the induction of inflammatory and fibrotic pathways including the activation of mTOR signaling. Consistent with phenotypes observed in vivo in mouse models and clinical human samples, we observed myofibroblast differentiation and senescence, tissue contraction, excessive extracellular matrix deposition, and monocytes’ transmigration and macrophages’ secretion of inflammatory cytokines, which were dependent on the presence of the endothelial barrier. This model offers novel insights on the role of vasculature in the pathophysiology of adhesions, which were previously underappreciated. Moreover, in testing whether the hToC could be used to evaluate efficacy of therapeutics, we were able to capture donor-specific variability in the response to Rapamycin treatment, which reduced myofibroblast activation regardless. Thus, our findings demonstrate the value of the hToC as a human microphysiological system for investigating the pathophysiology of fibrotic conditions in the context of peritendinous injury and similar fibrotic conditions, providing an alternative to animal testing

Introduction and Objective. Chronic tendinopathy is a multifactorial disease and a common problem in both, athletes and the general population. Mechanical overload and in addition old age, adiposity, and metabolic disorders are among the risk factors for chronic tendinopathy but their role in the pathogenesis is not yet unequivocally clarified. Materials and Methods. Achilles tendons of young (10 weeks) and old (100 weeks) female rats bred for high (HCR) and low (LCR) intrinsic aerobic exercise capacity were investigated. Both Achilles tendons of 28 rats were included and groups were young HCR, young LCR, old HCR, and old LCR (n = 7 tendons per group/method). In this rat model, genetically determined aerobic exercise capacity is associated with a certain phenotype as LCR show higher body weight and metabolic dysfunctions in comparison to HCR. Quantitative real-time PCR (qPCR) was used to evaluate alterations in gene expression. For histological analysis, semi-automated image analysis and histological scoring were performed. Results. Age-related downregulation of tenocyte marker genes (Tenomodulin), genes related to matrix modelling and remodeling (Collagen type 1, Collagen type 3, Elastin, Biglycan, Fibronectin, Tenascin C), and Transforming growth factor beta 3 (Tgfb3) were detected in tendons from HCR and LCR. Furthermore, inflammatory marker Cyclooxygenase 2 (Cox2) was downregulated, while Microsomal prostaglandin E synthase 2 (Ptges2) was upregulated in tendons from old HCR and old LCR. No significant alteration was seen in Interleukin 6 (Il6), Interleukin 1 beta (Il1b), and Tumor necrosis factor alpha (Tnfa). Histological analysis revealed that Achilles tendons of old rats had fewer and more elongated tenocyte nuclei compared to young rats, indicating a reduced metabolic activity. Even though higher content of glycosaminoglycans as a sign of degeneration was found in tendons of old HCR and LCR, no further signs of tendinopathy were detectable in histological evaluation. Conclusions. Overall, aging seems to play a prominent role in molecular and structural alterations of Achilles tendon tissue, while low intrinsic exercise capacity did not cause any changes. Even though tendinopathy was not present in any of the groups, some of the shown age-related changes correspond to single characteristics of chronic tendon disease. This study gives an insight into tendon aging and its contribution to molecular and cellular changes in Achilles tendon tissue

Introduction and Objective. Bone is a tissue which continually regenerates and also having the ability to heal after injuries however, healing of large defects requires intensive surgical treatment. Bioactive glasses are unique materials that can be utilized in both bone and skin regeneration and repair. They are degradable in physiological fluids and have osteoconductive, osteoinductive and osteostimulative properties. Osteoinductive growth factors such as Bone Morphogenetic Proteins (BMP), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Transforming Growth Factor (TGF) are well known to stimulate new bone formation and regeneration. Unfortunately, the synthesis of these factors is not cost- effective and, the broad application of growth factors is limited by their poor stability in the scaffolds. Instead, it is wise to incorporate osteoinductive nanomaterials such as graphene nanoplatelets into the structures of synthetic scaffolds. In this study, borate-based 13-93B3 bioactive glass scaffolds were prepared by polymer foam replication method and they were coated with graphene-containing poly (ε-caprolactone) layer to support the bone repair and regeneration. Materials and Methods. Effects of graphene concentration (1, 3, 5, 10 wt%) on the healing of rat segmental femur defects were investigated in vivo using male Sprague–Dawley rats. Fabricated porous bioactive glass scaffolds were coated by graphene- containing polycaprolactone solution using dip coating method. The prepared 0, 1, 3, 5 and 10 wt% graphene nanoparticle-containing PCL-coated composite scaffolds were designated as BG, 1G-P-BG, 3G-P-BG, 5G-P-BG and 10G-P-BG, for each group (n: 4) respectively. Histopathological and immunohistochemical (bone morphogenetic protein, BMP-2; smooth muscle actin, SMA and alkaline phosphatase, ALP) examinations were made after 4 and 8 weeks of implantation. Results. Results showed that after 8-weeks of implantation both cartilage and bone formation were observed in all animal groups. After 4 and 8 weeks of implantation the both osteoblast and osteoclast numbers were significantly higher in the group 4 compared to the control group. Bone formation was significant starting from 1 wt% graphene-coated bioactive glass implanted group and highest amount of bone formation was obtained in group containing 10 wt% graphene (p<0.001). Newly formed vessels expressed this marker and increased vascularization was observed in 8- weeks period compared to the 4-weeks period. In addition, an increase in new vessel formation were observed in graphene-coated scaffold implanted groups compared to the control group. While cartilage tissue was observed in control group, bone formation percentages were significant in graphene-coated scaffold implanted groups. Highest amount of bone formation occurred in group 4 (10 % wt G-C). Conclusions. Additionally, the presence of graphene nanoplatelets enhanced the BMP-2, SMA and ALP levels compared to the bare bioactive glass scaffolds. It was concluded that pristine graphene-coated bioactive glass scaffolds improve osteointegration and bone formation in rat femur defect when compared to bare bioglass scaffolds

As cartilage has poor intrinsic repair capacity, in vitroexpansion of human articular chondrocytes (HACs) is required for cell-based therapies to treat cartilage pathologies. During standard expansion culture (i.e. plasma osmolarity, 280 mOsm) chondrocytes inevitably lose their specific phenotype and de-differentiate, which makes them inappropriate for autologous chondrocyte implantation. It has been shown that physiological osmolarity (i.e. 380 mOsm) increases collagen type II (COL2) expression in vitro, but the underlying molecular mechanism is unknown. Transforming growth factor beta (TGFβ) super family members are accepted key regulators of chondrocyte differentiation and known to stimulate COL2 production. In this study we aimed to elucidate the role of TGFβ superfamily member signalling as a molecular mechanism potentially driving the COL2 expression under physiological (380 mOsm) culture conditions. HACs from OA patients (p1) were cultured in cytokine-free medium of 280 or 380 mOsm, under standard 2D in vitroconditions, with or without lentiviral TGFβ2 knockdown (RNAi). Expression of TGFβ isoforms, BMPs and chondrocyte marker genes was evaluated by QPCR. TGFβ2 protein secretion was evaluated using ELISA and bioactivity was determined using an established reporter cell line. Involvement of BMP signaling was investigated by culturing OA HACs (p1) in the presence or absence of dorsomorphin (10 µM). Physiological osmolarity increased TGFβ2 and TGFβ3 mRNA expression, TGFβ2 protein secretion as well as general TGFβ activity by 380 mOsm. Upon TGFβ2 isoform-specific knockdown COL2 mRNA expression was induced. TGFβ2 RNAi induced expression of several BMPs (e.g. BMP2,-4,-6) and this induction was enhanced in culture conditions with physiological osmolarity. Dorsomorphin inhibited physiological osmolarity induced COL2 mRNA expression. TGFβ2 knockdown under 380 mOsm increases COL2 expression in human osteoarthritic chondrocytes in vitromost likely through a regulatory feedback loop via BMP signaling, which is involved in osmolarity-induced COL2 expression. Future studies will further elucidate the BMP-mediated regulatory feedback loop after TGF β2 knockdown and its influence on COL2 expression

Injured skeletal muscle repairs spontaneously via regeneration, however, this process is often incomplete because of fibrotic tissue formation. In our study we wanted to show improved efficiency of regeneration process induced by antifibrotic agent decorin in a combination with Platelet Rich Plasma (PRP)-derived growth factors. A novel human myoblast cell (hMC) culture, defined as CD56 (NCAM)+ developed in our laboratory, was used for evaluation of potential bioactivity of PRP and decorin. To determine the their effect on the viability of hMC we performed a MTT assay. To perform the cell proliferation assay, hMCs were separately seeded on plates at a concentration of 30 viable cells per well. Cell growth medium prepared with different concentrations of PRP exudates (5%, 10%, and 20%) and decorin (10 ng/mL, 25 ng/mL, and 50 ng/mL) were added and incubated for 7 days. After incubation we stained the cells with crystal-violet and measured the absorbance. To study the expression of Transforming Growth Factor Beta (TGF-β) and myostatin (MSTN), two main fibrotic factors in the process of muscle regeneration we performed several ELISA assays in groups treated with all therapeutic agents (PRP, decorin and their combination). Further, we have studied the ability of these agents to influence the differential cascade of dormant myoblasts towards fully differentiated myotubes by monitoring step wise activation of single nuclear factors like MyoD and Myogenin via multicolor flow cytometry. We stained the cells simultaneously with antibodies against CD56, MyoD and myogenin. We acquired cell images of 5,000 events per sample at 40 x magnification using 488 nm and 658 nm lasers and fluorescence was collected using three spectral detection channels. We analysed the cells populations according to expression of single or multiple markers and their ratios. Finally, we examined the treated cell populations using a multicolour laser microscope after staining for desmin (a key marker of myogenic differentiation of hMC), α-tubulin, and nuclei. Optical images were acquired at the center of chamber slides where the cell density is at its highest using a Leica TCS SP5 II confocal microscope and analysed using Photoshop CS6, where a “Color Range” tool was used in combination with a histogram palette to count the pixels that correspond to desmin-positive areas in an image. The mitochondrial activity of cells, as determined by the MTT assay, was significantly increased (p < 0 .001) after exposure to tested concentrations of PRP exudate. Similarly, viability was elevated in all tested concentrations of decorin. PRP exudate enhanced the viability of cells to more than 400% when compared to the control (p < 0 .001). The viability of cells treated with PRP exudates was also significantly higher when compared to decorin (p < 0 .001). Decorin did not show a significant effect on cell proliferation compared to the control, however, cultivation with PRP exudate leads to a 5-fold increase in cell proliferation (p < 0 .001). Decorin was shown to down-regulate the expression of TGF-β when compared to the control by more than 15% (p < 0 .001) but significantly less than PRP exudate p < 0 .005). PRP significantly down-regulated TGF-β expression by more than 30% (p < 0 .001). Similarly, the MSTN expression levels were significantly down-regulated by decorin and PRP. MSTN levels of cells treated with decorin were decreased by 28.4% (p < 0 .001) and 23.1% by PRP (p < 0 .001) when compared to the control group. Using flow cytometry we detected a 39.1% increase in count of myogenin positive cells in the PRP-treated group compared to the control. Moreover, there was a 3.09% increase in cells positive only for myogenin, whereas no such cells were found in the control cell population. The population of cells positive only for myogenin is considered as fully differentiated and capable of fusion into myotubes as well as future mucle fibers and is thus of great importance for muscle regeneration. At the same time 20.6% fewer cells remained quiescent (positive only for CD56). Cells positive for both MyoD and myogenin represent the population that shifted significantly towards mature myocites during myogenesis but are not yet fully committed. Finally, a statistically significant up-regulation of desmin expression (p < 0 .01 for the PRP treated group, p < 0 .005 for the decorin and PRP + decorin treated groups) was present in all therapeutic groups when compared to the control. While no significant difference was found between the PRP and decorin-treated groups, their combination led to a more than 3-fold increase (p < 0 .005) of desmin expression when compared to single bioactives. PRP can be a highly potential therapeutic agent for skeletal muscle regeneration and repair, especially if in combination with a TGF-β antagonis decorin. Achieving better healing could likely result in faster return to play and lower reinjury rate

Purpose. The aim of this study was to investigate whether growth factors essential for fracture healing are released in the immediate aftermath following fracture and whether reaming of IM cavity causes increased liberation of these autocoids. Methods. Consecutive adult patients with femoral shaft fractures forming two groups (a group who received unreamed nail (n=10) and a second group who received reamed nail (n=10) were recruited for this study. Peripheral blood samples and samples from the femoral canal before and after reaming and before and after the solid nail insertion were collected. Serum was extracted and using Elisa colorimetric assays the concentration of Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor I (IGF-I) Transforming Growth Factor beta 1 (TGF-. 2. 1) and BMP-2 levels was measured. Results. In total 20 patients were studied. The mean age was 38 years (range 20-63). Reaming substantially increased all studied growth factors locally in the femoral canal. VEGF and PDGF were increased after reaming by 111.2% and 115.6% respectively. IGF-1 was increased by 31.5% and TGF-b1 was increased by 54.2%. In the unreamed group the levels of PDGF-BB, VEGF and TGF-. 2. 1 were not changed while the levels of IGF-I were decreased by 10%. The levels of these factors in peripheral circulation were not altered despite the technique used. BMP-2 levels during all time points were below the detection limit of the immunoassay. Conclusion and significance. This study indicates that reaming of IM Canal is associated with increased liberation of growth factors. The osteogenic effect of reaming could be secondary not only to grafting debris but also to the increased liberation of these molecules

Dupuytren’s disease is a benign fibroproliferative disease of unknown aetiology. It is often familial and commonly affects Northern European Caucasian men, but genetic studies have yet to identify the relevant genes. Transforming growth factor beta one (TGF-β1) is a multifunctional cytokine which plays a central role in wound healing and fibrosis. It stimulates the proliferation of fibroblasts and the deposition of extracellular matrix. Previous studies have implicated TGF-β1 in Dupuytren’s disease, suggesting that it may represent a candidate susceptibility gene for this condition. We have investigated the association of four common single nucleotide polymorphisms in TGF-β1 with the risk of developing Dupuytren’s disease. A polymerase chain reaction-restriction fragment length polymorphism method was used for genotyping TGF-β1 polymorphisms. DNA samples from 135 patients with Dupuytren’s disease and 200 control subjects were examined. There was no statistically significant difference in TGF-β1 genotype or allele frequency distributions between the patients and controls for the codons 10, 25, −509 and −800 polymorphisms. Our observations suggest that common TGF-β1 polymorphisms are not associated with a risk of developing Dupuytren’s disease. These data should be interpreted with caution since the lack of association was shown in only one series of patients with only known, common polymorphisms of TGF-β1. To our knowledge, this is the first report of a case-control association study in Dupuytren’s disease using single nucleotide polymorphisms in TGF-β1

Introduction. Tendons are critical to mobility, and are susceptible to degeneration through injury and ageing. Type I collagen is the most abundant protein in vertebrates; it is the main structural protein of the extracellular matrix in numerous musculoskeletal tissues, including tendons. Type I collagen predominantly is a heterotrimer, which consists of two alpha-1 chains and one alpha-2 chain (α1). 2. (α2) encoded by the COL1A1 and COL1A2 genes, respectively. However, type I collagen can form homotrimers (α1). 3. which are protease-resistant, and are associated with age-related musculoskeletal diseases, fibrotic and connective tissue pathologies. Transforming growth factor beta (TGFβ) enhances collagen (I) gene expression, is involved in tendon mechanobiology and repair processes, while its effect on homotrimer formation is unknown. Our aim is to investigate the relative expressions of collagen (I) α1 and α2 polypeptide chains in tenocytes (tendon fibroblasts) stimulated with TGFβ. Materials and Methods. Included RT-qPCR to measure the relative expression of COL1A1 and COL1A2 genes. [. 14. C]-proline metabolic labelling was used to measure the expression of the collagen (I) α1 and α2 polypeptide chains. These techniques were performed in equine superficial digital flexor tendon (SDFT) tenocytes (n=3) and murine tail tendon tenocytes (n=3) with different concentrations of TGFβ (0.01 ng/ml-100 ng/ml). Results. There was an increase in both COL1A1 and COL1A2 gene expression when stimulated with TGFβ in both cell types. In equine tenocytes the gene expression ratio of COL1A1:COL1A2 increased from 1.73 ± 0.75 to 7.87 ± 2.9 (p=0.003) when stimulated with 100 ng/ml of TGFβ3. TGFβ upregulated collagen (I) protein in both cell types. In equine tenocytes (n=3) when stimulated with 100 ng/ml of TGFβ3, the α1:α2 protein chain ratio increased from 1.93 ± 0.54 to 3.02 ± 0.32 (p=0.059) in comparison with serum-starved cells, which alongside the changes in gene expression, may be indicative of collagen (I) homotrimer production. Discussion. There were biosynthetic alterations in collagen production, and putative collagen (I) homotrimer when equine tenocytes were stimulated with 100 ng/ml TGFβ3. Future work will focus isolating different collagens by repeated differential salt precipitation. The level of TGFβ receptors and Smad signaling molecules will be also analysed using RT-qPCR and western blotting

Purpose: The aim of this study was to investigate whether growth factors essential for fracture healing are released in the immediate aftermath following fracture and whether reaming of IM cavity causes increased liberation of these autocoids. Methods: Consecutive adult patients with femoral shaft fractures forming two groups (a group who received unreamed nail (n=10) and a second group who received reamed nail (n=10) were recruited for this study. Peripheral blood samples and samples from the femoral canal before and after reaming and before and after the solid nail insertion were collected. Serum was extracted and using Elisa colorimetric assays the concentration of Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor I (IGF-I) Transforming Growth Factor beta 1 (TGF-. 2. 1) and BMP-2 levels was measured. Results: In total 20 patients were studied. The mean age was 38 years (range 20–63). Reaming substantially increased all studied growth factors locally in the femoral canal. VEGF and PDGF were increased after reaming by 111.2% and 115.6% respectively. IGF-1 was increased by 31.5% and TGF-b1 was increased by 54.2%. In the unreamed group the levels of PDGF-BB, VEGF and TGF-. 2. 1 were not changed while the levels of IGF-I were decreased by 10%. The levels of these factors in peripheral circulation were not altered despite the technique used. BMP-2 levels during all time points were below the detection limit of the immunoassay. Conclusion and Significance: This study indicates that reaming of IM Canal is associated with increased liberation of growth factors. The osteogenic effect of reaming could be secondary not only to grafting debris but also to the increased liberation of these molecules

Purpose: Growth factors are released and circulate in peripheral blood after fracture. The purpose of this study was to characterize the early systemic release of several growth factors following accidental fractures and surgery. Methods: 14 patients (8 male and 6 female) suffering from lower limb long bone fractures were prospectively included in the study. The mean age was 34 years (range 18-61). In all patients the time from fracture occurrence till operation was less than 24 hours. Peripheral blood samples were collected on patients’ admission, induction, and postoperatively at 1, 3 and 5 days. Serum was extracted and using Elisa colorimetric assays the concentration of Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor I (IGF-1) and Transforming Growth Factor beta 1 (TGF-b1) was measured. Results: From fracture occurrence till induction for surgery a substantial decreased was observed (VEGF concentration was decreased by 189%, PDGF was decreased by 363%, TGF-b1 was decreased by 247 % and IGF-1 was decreased only by 25%. Surgery itself decreased VEGF peripheral levels by a further 50% and PDGF by 40 % while IGF and TGF-b1 levels remained unchanged. During the first post-operative day the levels of VEGF were increased by 82%, TGF-b1 and IGF-1 remained constant and PDGF was further decreased by 20%. Between the 1st and 3rd postoperative days VEGF was increased by 132%, PDGF by 220% and TGF-b1 by 230 %. During this period, IGF-1 was decreased by 20 %. Finally, during the 3rd to 5th postoperative day, the levels of all growth factors continue to increase. Conclusion: This study illustrates the early pattern of release of 4 growth factors following fractures and surgery. A substantial decreased during the first 24 hours was noted but thereafter an upward trend was observed. This data provide insight into the levels and kinetics of growth factors before and after surgery of fractures

Current issues being debated in ACL reconstruction include injury prevention, graft choice, graft positioning, graft fixation, graft remodelling and rehabilitation. Tissue engineering, the alteration of biological mechanisms by application of novel proteins, enzymes and hormones, is rapidly changing the way we approach all aspects of surgery. Tissue engineering techniques in ACL/PCL reconstruction focus on new biosynthetic ACL material, fixation of soft tissue grafts to bony tunnels and graft remodelling. OP-1 is recombinant human Osteogenic Protein 1 (BMP-7). It is a member of the Transforming Growth Factor β (TGFβ) super family. OP-1 promotes the recruitment, attachment, proliferation and differentiation of pluripotential mesenchymal stem cells. It promotes both osteogenesis and chondrogenesis. The carrier is highly purified bovine bone type 1 collagen, which provides an osteoconductive matrix. We have completed a study assessing the use of OP-1 as a means of enhancing early biological fixation of soft tissue grafts within bone tunnels in a sheep ACL model. We have commenced a clinical trial using OP-1 in adult ACL reconstruction, believing that OP-1 will enhance early biological graft fixation, and hence, improve clinical results, speed up rehabilitation and prevent tunnel widening. Other studies have shown the beneficial effects of BMP-2 on an extraarticular bone tendon fixation model, the use of TGF-B to enhance graft remodelling and the application of gene therapy to deliver BMP’s for enhanced graft fixation. Several projects are underway looking at creating biosynthetic ACL grafts using tissue engineering techniques. As opposed to purely synthhetic grafts, bioACL grafts are made of a collagen scaffold, allowing for remodelling and revascularisation. ACL reconstructive surgery is constantly evolving. Tissue engineering may provide us with a means of minimising morbidity, accelerating rehabilitation and improving the clinical outcome following this common surgery

During fracture repair, a number of growth factors and cytokines are present at elevated levels at the fracture site such as Transforming Growth Factor Beta (TGF-), Fibroblast Growth Factor (FGF) and Platelet Derived Growth Factor (PDGF). The aim of the study was to investigate the presence of these growth factors in healing fractures and fracture non-unions, in order to test the hypothesis that atrophic non-unions express a lower level of growth factors than hypertrophic non-unions and healing fractures. Biopsies were taken from the fracture site of 23 patients (mean age 46) with uninfected non-unions, 12 patients with hypertrophic (mean 13.8 months after fracture) and 11 patients with atrophic (mean 16.5 months after fracture). A comparison group of biopsies from early fracture callus (one to four weeks after fracture) in five patients with healing fractures was also included. Five-micron paraffin sections were immunohistochemically stained for TGF-, FGF-II and PDGF. Growth factors were then assessed in six different cell types. Fibroblasts, endothelial cells and macrophages were found to express TGF-, FGF-II and PDGF in all three-fracture groups. Osteoblasts, osteoclasts and chondrocytes were not present in the healing fracture group. The growth factor expression in osteoblasts, osteoclasts and chondrocytes in the non-union groups were found to be variable, however, the expression of these growth factors appeared to be less in the atrophic non-unions than hypertrophic non-unions. The expression of these growth factors was found to be less in the atrophic non-union group than the hypertrophic non-union group in osteoblasts, osteoclasts and chondrocytes. These results may have relevance for new therapies that can be aimed at delivering growth factors to treat fracture non-unions. By further investigation of the differential expression of these growth factors it may be possible to determine which factors are likely to stimulate fracture healing

Aims

It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth.

Aims

MicroRNA-183 (miR-183) is known to play important roles in osteoarthritis (OA) pain. The aims of this study were to explore the specific functions of miR-183 in OA pain and to investigate the underlying mechanisms.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by symmetrical and chronic polyarthritis. Fibroblast-like synoviocytes are mainly involved in joint inflammation and cartilage and bone destruction by inflammatory cytokines and matrix-degrading enzymes in RA. Approaches that induce various cellular growth alterations of synoviocytes are considered as potential strategies for treating RA. However, since synoviocytes play a critical role in RA, the mechanism and hyperplastic modulation of synoviocytes and their motility need to be addressed. In this review, we focus on the alteration of synoviocyte signalling and cell fate provided by signalling proteins, various antioxidant molecules, enzymes, compounds, clinical candidates, to understand the pathology of the synoviocytes, and finally to achieve developed therapeutic strategies of RA.

Cite this article: Bone Joint Res 2021;10(4):285–297.