Introduction. Modulation of signaling pathways, which involves tendon development, regeneration, or homeostasis, is one of the potential modalities to facilitate proper regeneration of the injured tendon. Authors have previously reported that activation of Wnt/beta-catenin signaling suppressed the expression of tenogenic genes (i.e. Scleraxis (Scx), Mohawk (Mkx), Tenomodulin (Tnmd)) in rat primary tendon-derived cells (TDCs) and SCX-transduced human mesenchymal stem cells (hMSC-Scx cells), as a tendon progenitor cell line (kindly provided Dr. Docheva). The roles of TGF-beta signaling in tenogenesis have been elucidated. The purpose of the study was to evaluate the effect of TGF-beta signaling on tenogenic genes and relationship between both two signalings in rat TDCs and hMSC-Scx cells. Materials and Methods. Cell cultures. TDCs were harvested from the Achilles tendons of 6 week-old SD rats and the 3rd passage TDCs were used. To evaluate the effect of TGF-beta signaling, TGF-beta 1 protein and SD208 (TGF-beta receptor inhibitor) were utilized. To assess the effect of Wnt/beta-catenin signaling on TGF-beta signaling, BIO (Wnt/b-catenin activator) was utilized. Quantative RT-PCR. TDCs and hMSC-Scx cells were cultured with TGF-beta1 or SD208. Total RNA was isolated from the cells and cDNA was synthesized. Expression of Axin2, as a target gene for Wnt/beta-catenin signaling, as well as tenogenic genes was quantified and normalized by Gapdh. Western blotting. Each cells were cultured with BIO. Whole cell lysates were used for immunoblotting with antibodies against beta-actin and phosphorylated Smad2/Smad3 (p-Smad2/3), which indicates activation of TGF-beta signaling. Band intensity was quantified and normalized by beta-actin. Statistical analysis. Two groups were compared by unpaired Student”s t-test. Multiple groups were analyzed by one-way analysis of variance (ANOVA). (P <0.05). Results. Scx expression was increased by TGF-beta1 and decreased by SD208 in a dose-dependent manner in TDCs. However, TGF-beta1 and SD208 showed no significant effect on Mkx and Tnmd expression in TDCs and hMSC-Scx cells. BIO treatment decreased p-Smad2/3 proteins, while TGF-beta1 and SD208 had no effect on Axin2 expression in both cells. Discussion. Activation of TGF-b signaling induced Scx expression, independent of Wnt/beta-catenin signaling. Activation of Wnt/beta-catenin signaling suppressed p-Smad2/3 amounts for TGF-beta signaling and further Scx expressions. Taken together, activation of Wnt/beta-catenin signaling antagonizes Scx expression induced by TGF-beta signaling. Identification of compounds, which control Wnt/beta-catenin and/or TGF-beta signaling, may lead to developments of novel therapeutic options to facilitate regeneration of injured tendons

Osteoprogenitors on the inner layer of periosteum are the major cellular contributors to appositional bone growth and bone repair by callus formation. Previous work showed that periosteal-derived cells have little or no osteogenic activity under standard in vitro osteogenic culture conditions. This study was conducted to determine what growth factor(s) can activate periosteal osteogenic capacity.

This study was conducted with IACUC approval. Periosteum from five equine donors was digested in collagenase for 3-4 hours at 37C. Isolated periosteal cells were maintained in DMEM/10% FBS medium and exposed to PDGF, Prostaglandin E2, BMP-2 and TGF-b3 at a range of concentrations for 72 hours. Changes in osteogenic gene expression (Runx2, OSX and ALP) were measured by qPCR. Periosteal cells were pre-treated with TGF-b3 or maintained in control medium were transferred into basal or osteogenic medium. Osteogenic status was assessed by Alizarin Red staining for mineralized matrix, ALP enzymatic activity and induction of osteogenic genes.

PDGF, PgE2 and BMP-2 had little impact on expression of osteogenic markers by periosteal cells. In contrast, TGF-b3 stimulated significant increases in Osterix (over 100-fold) ALP expression (over 70-fold). Pre-treating periosteal cells with TGF-b3 for 72 hours stimulated rapid cell aggregation and aggregate mineralization once cells were transferred to osteogenic medium, while cells not exposed to TGF-b3 exhibited minimal evidence of osteogenic activity.

This study indicate that TGF-b signaling is vital for periosteal osteogenic activity. Transient ‘priming’ of periosteal cells through TGF-b exposure was sufficient to activate subsequent osteogenesis without requiring ongoing growth factor stimulation. TGF beta ligands are secreted by many cell types, including periosteal progenitors and osteocytes, providing opportunities for both autocrine and paracrine pathways to regulate periosteal bone formation under homeostatic and reparative conditions.

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.

A variety of scaffolds, including collagen-based membranes, fleeces and gels are seeded with osteoblasts and applied for the regeneration of bone defects. However, different materials yield different outcomes, despite the fact that they are generated from the same matrix protein, i.e. type I collagen. Recently we showed that in fibroblasts MMP-3 is induced upon attachment to matrix proteins in the presence of TGFbeta.

Aim: To investigate the regulation of matrix metalloproteinases (MMPs) and interleukins (IL) in osteoblasts upon attachment to type I collagen (col-1) in comparison to laminin -1 (LM-111) in the presence or absence of costimulatory signals provided by transforming growth factor beta (TGFbeta).

Methods: Osteoblasts were seeded in col-1–and LM-111-coated flasks and activated by the addition of TGFbeta. Mock-treated cells served as controls. The expression of genes was investigated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunocytochemistry and ELISA.

Results: Attachment of osteoblasts to col-1 or LM-111 failed to activate the expression of MMPs or ILs. In contrast, TGFbeta induced the expression of MMP-3, MMP-9, and MMP-13, IL-6 and IL-16 mRNAs. MMP-3 was found to be elevated in supernatants of activated cells. No difference was found in the expression of MMP-1, IL-8 and IL–18. Interestingly, the expression of IL-1beta mRNA was not activated by TGFbeta alone, but it was activated by attachment of osteoblasts to LM-111 in the presence of TGFbeta.

Conclusion: In contrast to fibroblasts, attachment of osteoblasts to col-1 or LM-111 had no effect on the induction of MMPs and ILs. TGFbeta induced the expression of MMPs and ILs in these cells but only MMP-3 was released. The results show significant differences between osteoblasts and fibroblasts in the effects of attachment to scaffold materials. This may have important consequences for tissue engineering of bone and for wound healing after surgery.

The aim of this study was to assess the effect of injecting genetically engineered chondrocytes expressing transforming growth factor beta 1 (TGF-β1) into the knees of patients with osteoarthritis. We assessed the resultant function, pain and quality of life.

A total of 54 patients (20 men, 34 women) who had a mean age of 58 years (50 to 66) were blinded and randomised (1:1) to receive a single injection of the active treatment or a placebo. We assessed post-treatment function, pain severity, physical function, quality of life and the incidence of treatment-associated adverse events. Patients were followed at four, 12 and 24 weeks after injection.

At final follow-up the treatment group had a significantly greater improvement in the mean International Knee Documentation Committee score than the placebo group (16 points; -18 to 49, vs 8 points; -4 to 37, respectively; p = 0.03). The treatment group also had a significantly improved mean visual analogue score at final follow-up (-25; -85 to 34, vs -11 points; -51 to 25, respectively; p = 0.032). Both cohorts showed an improvement in Western Ontario and McMaster Osteoarthritis Index and Knee Injury and Osteoarthritis Outcome Scores, but these differences were not statistically significant. One patient had an anaphylactic reaction to the preservation medium, but recovered within 24 hours. All other adverse events were localised and resolved without further action.

This technique may result in improved clinical outcomes, with the aim of slowing the degenerative process, leading to improvements in pain and function. However, imaging and direct observational studies are needed to verify cartilage regeneration. Nevertheless, this study provided a sufficient basis to proceed to further clinical testing.

Cite this article: Bone Joint J 2015;97-B:924–32.

Breast and other cancers commonly metastasize to bone to cause bone destruction, pain, fractures hypercalcemia and muscle weakness. Recently, we described a specific molecular mechanism by which bone-derived transforming growth factor (TGF)-beta, released as a consequence of tumor-induced bone destruction causes muscle dysfunction, before the loss of muscle mass. Circulating TGF-beta induces oxidation of the ryanodine receptor (RYR1) on the sarcoplasmic reticulum of skeletal muscle to induce calcium leak and muscle weakness. Blocking TGF-beta, or its release from bone (with bisphosphonates), preventing oxidation of or stabilizing RyR1 all prevented muscle weakness in mouse models of breast cancer bone metastases. In addition to these effects on skeletal muscle, circulating TGF-beta may act on beta cells of the pancreas to impair insulin secretion and result in glucose intolerance. These and other potential systemic effects of TGF-beta released from the tumor-bone microenvironment or from cancer treatment-induced bone destruction implicate bone as a major source of systemic effects of cancer and cancer treatment. Therapy to block the systemic effects of the bone microenvironment will improve morbidity associated with bone metastases and cancer treatment

Abstract. Introduction. Synovitis impacts osteoarthritis symptomatology and progression. The transcription factors controlling synovial gene expression have not been described. This study analyses gene expression in synovium samples from 16 patients with osteoarthritis with 9 undergoing arthroscopic and 8 knee trauma surgery for non-arthritic pathologies. Methodology. Intra-operative synovial biopsies were immersed in RNAlater at 4oC before storage at -80oC. Total RNA was extracted using RNAeasy. After purification, RT-PCR and quality assessment, cDNA was applied to Affymetrix Clariom D microarray gene chips. Bioinformatics analyses were performed. Linear models were prepared in limma with gender and BMI factors incorporated sequentially for each pathology comparison, generating 12 models of probes differentially expressed at FDR p<0.05 and Bayes number, B>0. Data analysis of differently expressed genes utilized Ingenuity Pathway Analysis and Cytoscape with Cluego and Cytohubba plug-ins. Results. Amongst the 2084 genes with significantly differential expression (DEG), 135 had transcription regulator capabilities and 121 a nuclear location. IPA analysis of OATKR and arthroscopic tissue comparison DEG identified 12 nuclear transcription factors linked to 31 DEG whose encoded proteins located within cytoplasmic and cell membrane compartments. All 12 were significantly up-regulated and acting in pathways up-regulating transcription of DNA and RNA, cell survival and angiogenesis while down-regulating senescence and apoptosis. NFE2L2, integral to the TGF-beta signalling pathway, was identified as a bottleneck gene. Conclusion. This analysis indicates the complexity of synovial gene expression regulation and offers target genes and pathways for evaluation during osteoarthritis pathogenesis

To overcome the severely limited regenerative capacity of cartilage, bone marrow mesenchymal stromal cells (MSCs) are an attractive cell source that is accessible less invasively and in higher quantity than articular chondrocytes (ACs). However, current in vitro chondrogenic protocols induce MSCs to form transient cartilage reminiscent of growth plate cartilage that becomes hypertrophic and is remodeled into bone. In contrast, under the same conditions, ACs form stable articular-like cartilage. Developmental studies in mice have revealed that TGF-beta/BMP, Wnt, and Hedghog/PTHrP signaling are the major regulators of both, articular cartilage and endochondral bone formation. While the differential regulation of TGF-beta/BMP and Hedgehog/PTHrP in endochondral MSC versus AC chondral differentiation is established knowledge, little is known about Wnt in these cells. Aim of this study was therefore to compare in vitro levels of Wnt network components in MSC-derived endochondral versus AC-derived articular cartilage. Whole genome expression data comparing human MSCs and ACs at days 0 and 28 of in vitro chondrogenesis were screened for differential expression of Wnt ligands, receptors, co-receptors, activators/inhibitors and signaling molecules. Expression of the most strongly differentially regulated Wnt network genes was studied in detail during in vitro chondrogenesis of MSCs vs ACs via qPCR at days 0, 7, 14, 21, 35, and 42. During early chondrogenesis, most Wnt components were expressed at low levels in both MSCs and ACs, with two exceptions. MSCs started into chondrogenesis with significantly higher levels of the non-canonical ligand WNT5A. ACs on the other hand expressed significantly higher levels of the canonical antagonist FRZB on day 0. During advancing and late chondrogenesis, MSCs downregulated WNT5A but still expressed it at significantly higher levels at day 42 than ACs. Strong regulation was also evident for WNT11 and the receptor PTK7 which were both strongly upregulated in MSCs. Unlike MSCs, ACs barely regulated these non-canonical Wnt genes. With regard to canonical signaling, only the transcription factor LEF1 showed strong upregulation in MSCs, while FZD9 and FRZB were only slightly upregulated in late MSC chondrogenesis. Again, these genes remained unregulated in ACs. Our data suggest that a dynamic Wnt network regulation may be a unique characteristic of endochondral MSC differentiation while during AC chondral differentiation Wnt expression remained rather low and stable. Overall, mRNA of the non-canonical Wnt network components were stronger regulated than canonical factors which may indicate that primarily non-canonical signaling is dynamic in endochondral differentiation. Next step is to assess levels of active and total beta-catenin, the canonical Wnt mediator, and to use Wnt antagonists to establish a causal relationship between Wnt signaling and endochondral differentiation

TGF-beta signaling has a well established role not only in adult organ homeostasis but also in skeletal development. Follistatin-like 3 (FSTL3), related to follistatin, is an inhibitor of TGF-beta ligands, with an established role in glucose and fat metabolism. However it has not previously been studied in skeletal development. Using a FSTL3 knock-out (KO) mouse model we have studied both embryonic skeletal development and adult bone phenotypes. Staining for skeletal and cartilage markers during development shows acceleration of skeletal tissue differentiation, with an eventual normalization at E18.5 (which is just prior to birth). Acceleration of bone mineralization occurs during both endochondral and intramembranous ossification. Use of micro-CT imaging highlighted the development of a scoliosis in the KO animals, along with abnormal shape of cranium and cranial sutures. Further investigation of the cranial phenotype in adult KO mice reveals craniosynastosis, with atypical fusion of the frontal suture. These mice have a change in overall cranial shape with shortening of the anterior head and a compensatory expansion of the posterior cranial bones, in a similar fashion to brachyencephaly. Our study therefore highlights a significant role of FSTL3 in skeletal tissue development and mineralization, as well as the development of clinically significant skeletal developmental disorders such as scoliosis, craniosynastosis and brachyencephaly

Autologous growth factors or AGF is a technology that uses the patient’s own platelets as a source of growth factors. The platelets are super concentrated and then de-granulated to release mitogens, such as TGF-beta, PDGF, IGF, FGF and VEGF. AGF can be used alone as a source of signaling factors or it can be used with a variety of bone grafting materials, including autograft, allograft or porous ceramics. AGF is advantageous because it is completely autologous and obtained at the point of care in the operating room. AGF has been shown to be successfully collected by pheresing or separating the whole blood into its components to capture the buffy coat (i.e., platelets and white cells) using a conventional cell washer. To assure a predictable two to four fold increase in platelets and white cells over the peripheral blood concentration, the operator of the cell washer must follow a defined protocol. The buffy coat is then transferred to a processor containing an ultra concentrator. This achieves a further three fold concentration of platelets and white cells, as well as a three fold concentration of fibrinogen. This process can be effectively achieved using a single unit (approximately 450cc) of blood which produces approximately 60cc of autologous growth factors (AGF). The excess plasma and red cells can be returned to the patient. We have developed as self-contained, electromechanically, software driven, turnkey device that separates and concentrates to produce AGF from as little as 100 cc of the patient’s whole blood. The device is the size and weight of a portable, tabletop clinical centrifuge. A single disposable, pre-sterilized cartridge is inserted into a centrifuge and automatic pumps. After the operator attaches the blood bag and provides minimal input, the machine automatically provides 10–15 ml of AGF. Total processing time is less than 15 minutes. Multiple, simultaneous cycling can process up to 450 ml of blood with operating times comparable to our contemporary system. Using human blood, the concentrations of platelets, white cells, and fibrinogen was comparable to the contemporary system. The AGF platelets were then de-granulated into a fibrin gel using either xenogenic or autologous thrombin. Consequently, the concentrations of TGF-beta, PDGF, IGF, FGF and VEGF were approximately 10 fold greater than blood levels and comparable to the separate component system. We believe that we have developed a method and device that safely, simply, cost-effectively produces intra-operatively clinically relevant levels of autologous growth factors from 100 ml of autologous blood

Introduction. Elevated remodelling of subchondral bone and marrow tissues has been firmly established as diagnostic and prognostic radiological imaging marker for human osteoarthritis. While these tissues are considered as promising targets for disease-modifying OA drugs, the development of novel treatment approaches is complicated by the lack of knowledge whether similar tissue changes occur in rodent OA models and poor understanding of joint-specific molecular and cellular pathomechanisms in human OA. Here, we describe the establishment of a human OA explant model to address this crucial niche in translational preclinical OA research. Methods. Osteochondral (knee, spine) and bone (iliac crest) clinical specimens were acquired from patients undergoing total knee arthroplasty (n=4) or lumbar spine fusion using bone autografts (n=6). Fresh specimens were immediately cut in equal-sized samples (50–500 mg wet weight) and cultured in 8 mL osteogenic medium for one week. Samples were either left untreated (control) or stimulated with lipopolysaccharide (LPS, 100 ng/mL) in the absence and presence of transforming growth factor-beta inhibitor (SB-505124, 10 μm). Pro-collagen-I (Col-I), interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1) secretion was determined in conditioned medium by ELISA. Tissue viability was assessed using MTT and alkaline phosphatase (ALP) activity staining. Results. Explanted tissues remained viable after one week culture in control and treatment conditions. Osteocytes, subchondral marrow spaces and calcified cartilage stained positive for ALP activity without gross morphological differences between groups. Median basal secretion levels were Col-I (2.3 ng/mg), IL-6 (90 pg/mg) and MCP-1 (25 pg/mg). LPS treatment led to a significant increase of IL-6 (330 pg/mg) and MCP-1 (70 pg/mg), but not Col-I secretion. Interestingly, inhibition of TGF-beta signalling in osteochondral tissues specifically reduced Col-I levels (0.4 ng/mg) compared to controls and LPS-treated samples. LPS-induced IL-6 and MCP-1 levels were slightly reduced (−120 pg/mg, p=0.03) and increased (+50 pg/mg) by SB-505124 treatment, respectively. IL-6 and MCP-1 levels were strongly correlated under basal (r=0.80) and treatment conditions (r=0.62). Conclusion. In this study, we provided proof of concept for the first ex vivo explant model of human osteoarthritis. Osteochondral tissue specimens can readily be cultured without loss of tissue viability and mount a robust inflammatory response upon LPS challenge. Treatment with a potential disease-modifying agent (TGF-beta signalling inhibitor) reduced collagen metabolism in bone and marrow and modified cytokine and chemokine expression. The osteochondral explant model might be highly valuable to evaluate disease-modifying OA drugs

There is believed to be a correlation between congenital idiopathic scoliosis and congenital heart disease (CHD). Clinical and cardiological data was recorded for 3538 adolescents suffering from CHD. Data collected included the type of scoliosis; the direction of the curve; the Cobb angle; the number of curves and the presence or not of previous corrective cardiac surgery. Over 30% of the study group were found to suffer from scoliosis and a positive correlation with specific syndromes was also identified. The mean age of the patients was 34.0 +/− 14.0 years. The maximum Cobb angle was 107 degrees while the median was 7.6 degreees. Scoliosis was present in 37/188 (19.7%) was Eisenmenger syndrome (with R-L shunt) and 60/158= 38% with complex cardiac anatomy. There were also 20/103= 19.4% patients with univentricular (Fontan) circulation. Scoliosis was not necessarily related to previous corrective cardiac surgery, contrary to the current assumption in the literature. The hypothesis of common genetic pathway defects expressed both in cardiovascular and musculoskeletal organogenesis was raised and the TGF-beta pathway involvement is speculated

Introduction. Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily of growth factors and are known to regulate proliferation and expression of the differentiated phenotype of chondrocytes, osteoblasts, and osteoclasts. To investigate the osteoblastic differentiation gene expressions that contribute to BMP-7 dependent ostogenesis, we performed gene expression profiling of BMP-7-treated mouse bone marrow stromal cells. Methods. D1 cells (mouse bone marrow stromal cells) were cultured in osteogenic differentiation medium (ODM) for 3 days, and then treated with BMP-7 for 24 hr. Total RNA was extracted using Trizol, purified using RNeasy columns. Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit to yield biotinylated cRNA. The data analysis up- and down-regulation developmental processes (anterior/posterior patterning, ectoderm development, embryogenesis, gametogenesis, mesoderm development, other development process, and segment specification) genes expression fold. Results. We detected 18 mRNAs (Id2, Igf2, Pparg, S100a10, Foxn3, Tulp3, Mycbp2, Notch3, Ptk7, Lrp4, Tnfrsf11b, Ogn, Cyr61, Mglap, Akp2, Ltbp4, Ibsp, and Thbs1) that were differentially up-regulated after BMP-7 stimulation. 3 mRNAs (Wars, Adss and Trim35) were differentially down-regulated after BMP-7 stimulation. Conclusion. The data indicate that BMP-7 regulate various developmental processes genes expression during osteoblastic differentiation. Though further studies are needed in relation to each expression gene profiles and osteoblastic differentiation, this information may serve as a point of comparison for osteoblastic differentiation of BMP-7. Furthermore, the data should facilitate the informed use of BMP-7 as a therapeutic agent and tissue engineering tool. Acknowledgement. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R01-2008-000-10089-0)

Flock technology is well known from textile industry. Short fibres are applied vertically on a substrate, coated with a flocking adhesive. Until now this technology has not been used in the field of biomaterials although it offers the possibility to create anisotrophic matrices with a high compressive strength despite of high porosity. Matrices presently used in matrix assisted autologous chondrocyte implantation do not show any orientation of the embedded chondrocytes. However column orientation and anisotropic direction of embedded cells and collagen fibers are thought to be necessary for proper cartilage matrix biomechanics. Combination of matrices as a guiding structure and chondrogenically differentiated mesenchymal stem cells (MSC) could offer new possibilities in the treatment of cartilage defects. Our aim was to evaluate whether anisotropic scaffolds are capable to support a cellular cartilaginous phenotype in vitro. Electrostatically flocked matrices consisted of a collagen substrate, gelatine as adhesive and polyamide flock fibres. Chondrogenic cells and MSC were embedded in the scaffolds. Adherence, vitality and proliferation was assessed using confocal laser-scan microscopy (cLSM). Chondrogenic induction was performed in the presence of TGF-beta 3. Accumulation of proteoglycans was quantified by alcian-blue stain and collagen type II synthesis after extraction of the newly synthesized matrix. cLSM showed proliferation of embedded MSC as evidenced by DAPI/Phalloidin stain. Vitality of embedded cells remained high over time. Articular chondrocytes and nucleus pulposus cells synthesized proteoglycans and collagen type II in the scaffolds. Also MSC embedded in the flock scaffolds differentiated and increased their chondrogenic phenotype over time. Using cLSM and biochemical analyses we demonstrated that cells adhered and proliferated well in the new scaffolds. Furthermore we showed that the scaffolds are capable to support induction and maintenance of the chondrogenic phenotype. We conclude that flocking technology is suitable for fabrication of scaffolds for cell cultivation and cartilage tissue engineering

Introduction: A number of surgical techniques have been described for the operative treatment of late stage osteochondritis dissecans (OCD) in the knee and ankle that have failed nonoperative management. However, no particular technique has been universally successful. We report the results of a new technique using retrograde drilling combined with the use of a novel collagen based bone void filler to prevent mechanical failure of the joint surface. The purpose of this study was to evaluate the results of this new technique and analyze the efficacy of both collagen Type-1 based osteoinductive bone void fillers Colloss and Colloss E with and without additional bone grafting. Methods: The osteoinductive bone void fillers Colloss (bovine) and Colloss E (equine) are bone inducing collagenous sponges. The osteoinductive properties are due to the interactive release of BMP-2, BMP-7, IGF-1 and TGF-beta from the implant and the surrounding host tissue by osteoclastic and osteoblastic action. All surgeries performed in the present series included retrograde drilling procedures for OCD in the talar dome and the femoral condyles. Between 2000 and 2006 eight patients were treated by retrograde drilling or trephine drilling under arthroscopic and fluoroscopic control preventing injury to the cartilage surface. The subchondral cavity was filled with a mixture of 20–40 mg bone void filler and morsellized bone graft. The bone void filler is tamped through the drill guide and into position with a Steinmann pin. Thus, only the subchondral defect was filled but the peripheral area of the drill hole remained empty. Evaluation was achieved by clinical assessment, radiographic, and magnetic resonance imaging examination. The follow-up averaged 24 months up to 48 months. Results: In all cases, osseous density increased in the Colloss filled subchondral area and mechanical impression of the joint surface could be prevented. Interestingly, clinical examination and follow-up MRI exams demonstrated moderate swelling and joint effusion in 5 of 8 cases for a period of 4 to 10 weeks postoperatively. This may be in part due to the augmentation technique. Nevertheless, good clinical (range of motion, pain) and radiographic results (bony healing) were obtained after this new treatment modality. Discussion: The subchondral application of Colloss in OCD bone cysts or osteonecrosis induced solid osseous formation at the implantation site. The results of persisting joint reaction such as swelling, pain, and prolonged bone edema in MRI scans may be due to mechanical bearing indicating that augmentation of the defect has to be improved to ensure a solid bony reconstruction. Major advantages of this technique include the ease of performing this procedure, the one-step nature of the procedure, and the ability to avoid violation of stable articular cartilage. In addition, this technique may be repeated according to the size of the lesion

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

Objectives: The replacement tissue used for anterior cruciate ligament reconstruction undergoes extensive biologic remodelling and incorporation after implantation. These changes, in which the tendon loses some of its characteristic features and adopts those typically associated with ligaments, has been referred to as ligamentization. The purpose of this study was to identify the proinflammatory response in the healing graft in the early phase. Methodes: Twenty New Zealand White Rabbits underwent ACL reconstruction with a semitendinosus tendon. Animals were sacrificed at 3 and 6 weeks. The harvested tissue including parts of remaining grafted tendon and genuine anterior cruciate ligament at time of the surgery as well as the tendon graft withdrawn at sacrification were prepared for immunohistochemical, histomorphometry and electromicroscopical analysis; synovia samples were taken at the sacrification as well. The tissues were immunostained for IL-1beta, TGF-beta, TNF-alpha (induction of inflammatory cascade), COX-2 (mediator of inflammatory response), Matrix Metalloproteinases (MMP-1, MMP-3, MMP-13, matrix destructive enzymes), TIMP-2 (Tissue Inhibitor of MMPs); the PGE2 (mediator of inflammatory response) content in the synovia was quantified by ELISA. Results: At 3 weeks after surgery the COX-2+ cells accounted for 70% of all cells present in the graft tissue, and decreased to 28% at 6 weeks. Similar, IL-1beta+ cells within the tendon decreased from week 3 to week 6. Controversly, there was an increase of COX-2, IL-1beta and MMP-1 in the intercellular tissue. The numbers of COX-2+ cells and IL-1beta+ cells at 3 weeks as well as the intercellular area stained positiv for COX-2, IL-1beta and MMP-1 at 6 weeks were significantly larger compared to the genuine ACL (p =< 0.05). At 3 weeks some cells stained positiv for MMP-3 and MMP-13, but not at 6 weeks. There was a slight pericellular staining for TIMP-2 at 3 weeks. TGF-beta+ cells and TNF-alpha+ cells were almost not detectable at every time point. Thus, proinflammatory cytokines and MMPs were synthesized in the early phase after ACL reconstruction by the tendon cells and accumulated at 6 weeks in the intercellular tissue. Conclusions: In the early phase of the graft healing after ACL reconstruction, there was a signifikant increase in proinflammatory cytokines and matrix destructive enzymes in the tendon graft. With the capability of synthesizing cytokines, tendon cells may play a critical role in tendon healing at early time points. Facing the widespread use the bias of cox-2 inhibitors on these immunologic processes has to be checked. Activating matrix destructive enzymes, cytokines appear to be crucial for connective tissue remodelling and graft stability after ACL reconstruction

Aims

The aim of this study was to screen the entire genome for genetic markers associated with risk for anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) injury.

Angiogenesis is the formation of new blood vessels occurring in an adult through migration and proliferation of endothelial cells, and tubular structures formation. Angiogenesis is modulated by growth factors, cytokines, adhesion molecules, integrins, and enzymes. Angiogenesis plays a role in many physiological processes (i.e. remodeling of ischemic muscle, woumd healing, fracture repair) as well as in pathological process such as rheumathoid arthritis and metastases. In bone, vasculature is essential for cartilage resorption and angiogenesis temporally precedes osteogenesis: the origin of bone is the artery carrying calcium and phosphate ions. Osteogenesis takes place near newly formed vessels, that mediate delivery of osteoprogenitor cells, secrete mitogens for osteoblasts, and transport nutrients and oxygen. Inadequate bone vascularity is associated with decreased bone formation and bone mass. In animals, inhibition of angiogenesis during fracture repair results in the formation of fibrous tissue. A poor blood supply is therefore considered as a risk factor for an impaired bone healing. Angiogenesis is vital in tissue engineering, especially when matrices are colonized by cells with an aerobic metabolism. The scaffold must not only support the growth of the cells making up the organ which should be replaced in vivo (i.e. osteoblasts); it must also support the growth of endothelial cells and develop an effectively functioning vasculature to supply the cells with oxygen. Osteogenesis of tissue engineered materials could be limited by a lack of vascularization, and the bioengineered graft may be potentially resorbed in the same way as a conventional bone graft. In rats, angiogenesis in coralline materials implanted in ectopic muscular sites, was higher when the biomaterial was combined with a vascular pedicle or was coated with bone marrow stromal cells. A combination of both enhanced vascularization and osteogenesis to a greater extent. Endothelial cells release growth factors and cytokines promoting bone deposition: PDGF-AB, TGF-beta 1 and 2, FGF-2, EGF, BMP. However, under inflammatory stimula, endothelial cells release bone resorbing cytokines: IL-6, M-CSF, GM-CSF. Bone marrow stromal cells release angiogenetic proteins such as VEGF, FGF-2, PDGF, TGF, and, after induction with BMP, PlGF. A conversation between bone marrow stromal cells and endothelial cells may therefore be hypothesized. Cultures of bone marrow stromal cells with endothelial cell conditioned medium showed significantly higher phosphatase alkaline activity and osteocalcin production. It was also be hypothesized that stromal cells may acquire immunophenotypic characteristics consistent with endothelial cells. Therefore scaffold requirements are also the ability to favour angiogenesis; endothelial cells growing on the artificial scaffold should mantain a normal phenotype and should not exhibit a pro-inflammatory and bone.resorbing phenotype. Endothelial cell cultures are useful supplementary in vitro tests for the evaluation of scaffolds for bone tissue engineering. Endothelial cell cultures are derived both from animals (usually ox, calf or pig vessels) and from human tissues, mainly the human umbilical vein and the vessels of microcirculation (derma or subcutaneous fat). Endothelial cells in non-human species show different reactions: they have usually a faster replication rate and grow better on the artificial substrata. Endothelial cells from different organs are intrinsically different and exhibit different responses to stimula. if the use of endothelial cells from bone microcirculation should be desirable, they require transfection with viral vectors to be immortalized. To study the response of endothelial cells cultured in vitro on artificial scaffolds, their adhesion, growth, viability and production of metabolites should be evaluated. Adhesion and growth on the materials may be evaluated indirectly by the uptake of Alamar Blue, which measures the amount of oxido-reduction reactions in the cell. A direct evaluation may be obtained by fluorescence microscopy using specific staining for the different cell structures. By studying the expression of adhesins and integrins, the interference of the scaffold with the cell/cell and cell/substrate adhesion should be verified. The release of substances in conditioned medium, as well as the evaluation of specific mRNAs in cells, should be assayed. Among the metabolites released by endothelial cells, the substances promoting bone deposition or favouring resorption, should be investigated. In particular, the release of growth factors may be explored, as they favour cell proliferation and the incorporation of the engineered scaffold within tissues. For the enhancement of bone formation, growth factors may be delivered in different ways: through incorporation on the scaffold, through transfection of bone marrow stromal cells, through platelet gel. Angiogenic growth factors are stored in platelet alpha granules and released during activation. A significant increase in the proliferation of bovine bone endothelial cells was demonstrated after 72 hour incubation with platelet gel in comparison with serum free conditions; the proliferation was similar to the growth induced by the fetal calf serum supplementation (platelet gel: 82.2B18.1x103 cells; serum free: 19.5B11.1x103 cells; fetal calf serum: 72.4B12.4x103 cells). However, the platelet gel inhibited the formation of tubular structures on Matrigel. In conclusion, the development of newly formed vessels on the bone cell engineered scaffold improves the incorporation in the host tissues and the success of the device. The use of exogenous growth factors or of platelet gel favours angiogenesis, besides osteoblast differentiation. The in vitro evaluation of the scaffold should be supplemented by tests on the adhesion, growth and functionality of endothelial cells

Objectives

The aim of this study was to provide a comprehensive understanding of alterations in messenger RNAs (mRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs) in cartilage affected by osteoarthritis (OA).