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

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

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

Purpose: To determine if administration of recombinant bFGF in an alginate gel would increase early healing mechanical parameters in acutely injured rat rotator cuff tendon at specific time points.

Method: Sprague Dawley rats were randomly divided into 2 groups and had surgically created 1mm (half tendon width) full thickness injuries at exactly 2mm from insertion site of Infraspinatus on the humerus. 200ng of bFGF or vehicle control was administered to randomly chosen rats. Tendons were harvested at 1 week, 2 weeks and 4 weeks. In both groups, the Infraspinatus tendon was dissected, and left attached to the humerus. At the time of testing, the intact portion of the injured tendon was divided sharply across tendon fibers at the level of the injury leaving only the healing tissue callus in continuity with the remaining proximal and distal portions of the tendon and loaded to failure.

Results: At 1 week the injury group’s average load to failure was 0.60N versus 0.61N in the bFGF injury group P = 1.000. At 2 weeks the injury group’s average load to failure increased to 1.03N versus 2.08N in the bFGF injury group P = 0.440 At 4 weeks the injury group’s average load to failure increased to 3.93N versus 5.56N in the bFGF injury group P = 0.008 representing a 41% increase in ultimate load. At 4 weeks, callus size of the injury group was 0.4mm2 versus 2.7mm2 in the bFGF injury group P < 0.001. Stiffness at 4 weeks for the injury tendons was 2.15 N/mm versus 3.54 N/mm in the bFGF group P = 0.008.

Conclusion: At 4 weeks healing tissue of acutely injured rotator cuff exposed to bFGF has an increase in ultimate load to failure (41% compared to control), increase in tendon callus size and stiffness. Our findings suggest a role of bFGF or similar growth factors in accelerating the healing of injured rotator cuff tendon.

The aim of the study is to determine the histological, biochemical, and biomechanical efficacy of fibrin clot and vitamin C in the healing of Achilles tendon ruptures (ATR) in a rat model.52 adult Wistar Albino rats (300–450 g) were used in the study. 12 groups were divided into four groups as Monitor (Group I), Control (Group II), Fibrin Clot (Group III), Fibrin Clot with vitamin C (Group IV). Four rats were used to obtain fibrin clots. Fibroblast Growth Factor (FGF) and Vascular Endothelial Growth Factor (VEGF) were measured in the blood of tail vein (1 cc) on the 3rd, 7th, 14th, and 21st day. Four rats were sacrificed on the 21st day from each group for histological evaluation. The rest of the rats were sacrificed at 42nd day, half for biomechanical and a half for histological evaluation. The 42nd-day HSS scores in group III and group IV were significantly lower than those of group I and group II (p =0.036 and 0.019; respectively). The 42nd-day HSS score of group IV was significantly lower than group III (p =0.036). The Maximum force N value of group III and group IV was significantly higher than those of group I and group II (p <0.05). Group IV showed a significantly higher Maximum force N value than group III (p =0.025). The blood FGF and VEGF levels of group III and group IV on the 3rd, 7th, 14th, and 21st days were higher than those of group I and group II (p <0.05). In the experimentally formed ATR model, fibrin clot and vitamin C produced a stronger tendon structure in terms of biomechanics while providing histological and biochemically better quality tendon healing in the surgical treatment of ATR. We believe that this model can be used to accelerate high-quality tendon healing after ATR

X-Linked Hypophosphataemia (XLH) is a rare, progressive, hereditary phosphate-wasting disorder characterised by excessive activity of fibroblast growth factor 23. The International XLH Registry was established to provide information on the natural history of XLH and impact of treatment on patient outcomes. The cross-sectional orthopaedic data presented are from the first interim analysis. The XLH Registry (NCT03193476) was initiated in August 2017, aims to recruit 1,200 children and adults with XLH, and will run for 10 years. At the time of analysis (Last Patient In: 30/11/2020; Database Lock: 29/03/2021) 579 subjects diagnosed with XLH were enrolled from 81 hospital sites in 16 countries (360 (62.2%) children, 217 (37.5%) adults, and 2 subjects of unknown age). Of subjects with retrospective clinical data available, skeletal deficits were the most frequently self-reported clinical problems for children (223/239, 93.3%) and adults (79/110, 71.8%). Retrospective fracture data were available for 183 subjects (72 children, 111 adults); 50 had a fracture (9 children, 41 adults). In children, fractures tended to occur in tibia/fibula and/or wrist; only adults reported large bone fractures. Joint conditions were noted for 46 subjects (6 children, 40 adults). For adults reporting osteoarthritis, knees (60%), hips (42.5%), and shoulders (22.5%) were the most frequently affected joints. Retrospective orthopaedic surgery data were collected for 151 subjects (52 children, 99 adults). Osteotomy was the most frequent surgery reported (n=108); joint replacements were recorded for adults only. This is the largest set of orthopaedic data from XLH subjects collected to date. Longitudinal information collected during the 10-year Registry duration will generate real-world evidence which will help to inform clinical practice. Authors acknowledge the contribution of all International XLH Registry Steering Committee members

Summary:. Hamstring tendons (HT) represent a widely used autograft for ACL reconstruction. Harvesting, processing and pretensioning procedures together with the time out of the joint could theoretically hamper tendon cells (TCs) viability. The authors hypothesize that HT cells are not impaired at the end of the surgical procedures and their tenogenic phenotype may be strongly improved by exposure to PEMF. Methods. Remnants of semitendinosus and gracilis tendons were collected at the end of the surgical procedures before skin closure from 15 healthy donors who underwent ACL reconstruction with autologous hamstring tendons. To isolate TCs, the tendon was minced and digested with 0.3 % type I collagenase and the nucleated cells were plated at a density 5x10E3 cells/cm2 and cultured in chamber slides in differentiation medium composed of DMEM + 5ng/ml basic fibroblast growth factor (b-FGF) for 7, 14, 21 days. The following cell cultures were set up:. -. TCs cultured with differentiation medium + exposure to PEMF 8 h/day (PEMF generator system IGEA, intensity of magnetic field = 1.5 mT, frequency = 75 Hz). -. TCs cultured with differentiation medium without exposure to PEMF. At day 0, day 7, day 14 and day 21, immunofluorescence analysis was performed to evaluate the expression of collagen type I, collagen type VI, scleraxis and PCNA (proliferative marker). Subsequently, tendon explant cultures were set up to verify, at day 21, explant viability and the expression of collagen type I, collagen type VI, beta-catenin and PCNA. Results. The TCs from the tendon fragments at the end of the ACL reconstruction were alive and they expressed markers of proliferation and tendon phenotype at the end of the culture periods. The TCs in the presence PEMF 8h/day showed greater production of collagen type I, collagen type VI and scleraxis than that of TCs cultured without PEMF (p<0,05): the expression of this markers increased from 7 to 21 days of culture. The expression of PCNA, in the presence of PEMF stimulus, was significantly lower (p<0,05) than that of TCs cultured without PEMF. A similar behavior was surprisingly observed in tendon explant cultures. Conclusions. Hamstring tendons used for ACL reconstruction are not simple autologous tenoconductive scaffold but are a biologic structure rich in progenitor cells that show tenogenic behavior. Their tenogenic phenotype may be strongly improved by exposure to PEMF. In a future clinical perspective, the postoperative use of PEMF could be used to enhance the ligamentization processes of autologous hamstring tendons, when used as autografts for ACL reconstructions

Cartilage calcification induces the synthesis of degrading enzymes, such as matrix metalloproteinases (MMPs) and prostaglandin E2 leading to tissue degeneration. The aim of the study was to investigate the effect of vitamin D on the calcification process in osteoarthritic cartilage. We evaluated the effect of vitamin D on klotho (KL), Fibroblast Growth Factor 23 (FGF23) and Fibroblast Growth Factor Receptor 1c (FGFR1c) mRNA and protein expression levels by real-time PCR and western blot analysis, respectively. Possible interactions between klotho and FGF23 on the receptor FGFR1c in normal chondrocytes were investigated using immunoprecipitation assay. The direct effect of 1,25 dihydroxyvitamin D3 (1,25D) on KL, FGF23 and FGFR1c promoter was also evaluated. We found that FGF23 and FGFR1c mRNA expression levels were significantly increased in osteoarthritic chondrocytes compared to normal, while KL mRNA levels were decreased (p=0.001 for all genes). We showed that klotho-FGF23-FGFR1c form complexes in normal chondrocytes and confirmed the participation of klotho in the initiation of FGF23-FGFR1c signalling. Treatment of normal chondrocytes with 1,25D resulted in a significant dose and time dependent increase of FGF23 and FGFR1c mRNA levels and in an increase of KL mRNA levels in osteoarthritic chondrocytes compared to untreated (p=0.001). We revealed, for the fist time, the presence of conserved, canonical VDREs in the proximal promoters of KL, FGF23 and FGFR1c. We propose a common regulatory scheme of mineral homeostasis and aging in osteoarthritic chondrocytes evidenced by the positive/negative feedback actions by KL, FGF23, FGFR1c and 1,25D, through binding of vitamin D receptor (VDR) on the promoters of the above mentioned genes

Although multifunctional delivery systems can potentially improve safety and efficacy of therapeutic protein delivery in the biological treatment of injured tissues, ability to track and manipulate protein delivery systems in vivo to ensure localization at the treatment site is still a concern. We hypothesized that incorporating superparamagnetic iron oxide (SPIO) into calcium phosphate (CaP) coated β-tricalcium phosphate (β-TCP) microparticles would allow for Magnetic Resonance Imaging (MRI) based tracking in vivo and SPIO incorporation would not impact the biological activity of proteins delivered with these microparticles. To address the efficacy and limitations in therapeutic protein delivery, a CaP coated microparticle which incorporates superparamagnetic iron oxide (SPIO-CaP-MP) was created and used in a rat knee medial collateral ligament. The system has trifunctional properties: (1) it is trackable using magnetic resonance imaging (MRI), (2) it can be manipulated with a magnetic field, (3) it can release active proteins in the injury site. SPIO-Ca-MPs were formed on β-tricalcium phosphate cores. Using MRI, SPIO-CaP-MPs were visible in T2 weighted sequences as an area of hypointesive signal. SPIO-CaP-MPs could be visualized and remained localized for at least 15 days after injection into the medial collateral ligament. Recombinant human basic fibroblast growth factor delivered with SPIO-CaP-MPs stimulated the proliferation of human dermal fibroblasts. Finally, SPIO-CaP-MPs could be localized to a bar magnet when suspended in solution. Taken together, these results suggest that SPIO-CaPMPs could be useful for protein delivery applications in the treatment of ligament injury that may benefit from externally controlled localization and MRI-based tracking

Objective. Early cell loss of up to 50% is common to in vitro chondrogenesis of mesenchymal stromal cells (MSC) and stimulation of cell proliferation could compensate for this unwanted effect and improve efficacy and tissue yield for cartilage tissue engineering. We recently demonstrated that proliferation is an essential requirement for successful chondrogenesis of MSC, however, how it is regulated is still completely unknown. We therefore aimed to identify signaling pathways involved in the regulation of proliferation during in vitro chondrogenesis and investigated, whether activation of relevant pathways could stimulate proliferation. Design. Human MSC were subjected to in vitro chondrogenesis for up to 42 days under standard conditions in the presence of 10 ng/ml TGF-β. Cells were or were not additionally treated with inhibitors of bone morphogenetic protein (BMP), insulin-like growth factor (IGF) IGF/PI3K, fibroblast growth factor (FGF) or indian hedgehog (IHH) pathways for two or four weeks. To investigate the stimulation of proliferation by exogenous factors, cells were treated with BMP-4, IGF-1, FGF-18 or purmorphamine (small molecule hedgehog agonist). Proliferation was determined by [3H]-thymidine incorporation. Results and Discussion. Quantitative assessment of proliferation revealed that proliferation arrest occurred during condensation up to day 3 and cell division was re-initiated thereafter with a peak on day 28. To test which pathways are relevant for the restart of proliferation, BMP, IGF/PI3K, FGF or IHH signaling was inhibited up to day 14. All treatments significantly reduced proliferation > 50% and, thus, seemed to participate in the re-entry into the cell cycle. To study whether the same pathways are relevant to maintain cells in a proliferative state later on, inhibitors were supplemented from day 14–28. This resulted in a significant decrease of proliferation in the groups treated with inhibitors of BMP (67% decrease), FGF (70%) and IHH (30%) signaling, while inhibition of IGF/PI3K did not influence late proliferation. Although BMP-4, IGF-1 or FGF-18 are known mitogenic factors in the growth plate, stimulation of cells by exogenous addition of these factors did not enhance proliferation in any differentiation phase. In contrast, stimulation of IHH signaling from day 14–28 significantly increased proliferation by 44%. This is in line with the documented strong mitogenic activity of hedgehog signaling in the proliferative zone of the growth plate. Thus, our data demonstrated that BMP, IGF/PI3K, FGF and IHH essentially participate in the regulation of proliferation during in vitro chondrogenesis. Early or late activation of single pathways by exogenous factors was, however, not sufficient to increase proliferation significantly with the exception of late activation of hedgehog signaling. Optimization of stimulation of the hedgehog pathway with a focus on increased tissue yield will now be the next step

Infection and skin ulcer are major problems in Total Knee Arthroplasty (TKA) and Bipolar Hip Prosthesis (BHP). Sugar (sucrose) has been used for wound care in many countries because it absorbs fluid, stimulates granulation, and suppress growth of bacteria. Trafermin ∗∗∗∗∗ recombinant human basic fibroblast growth factor ∗∗∗∗∗ FGF ∗∗∗∗∗ accelerates granulation process and improves quality of wound healing. We have used sucrose and trafermin for treatment of infection after TKA and BHP. Six infected TKA with skin ulcer and one infected BHP with fistula were treated with Trafermin and sugar. TKA were performed in four osteoarthritis and two rheumatoid arthritis, and BHP was for femoral neck fracture. Implants were removed in three cases because of deep infection. One was male and six were female, average age were 60.8 years old ranged 43 to 77. Follow-up period were one to 5 years. Four cases were related to MRSA. Sugar treatment were performed for two to 23 weeks, and Trafermin was sprayed once a day for two to 16weeks. In BHP case, sugar therapy was performed intermittently. In two deep infected TKA cases, infection ceased in one to 4 month and revision TKA were performed. In other four TKA cases, infection were ceased in two to 16 weeks. In BHP case, fistula closed in three years. Combination of Trafermin and sugar is useful for management of infection and skin ulcer after TKA and BHP

The fixation of titanium or titanium alloy implants is related to their surface composition and topography. Osteoconductive calcium phosphate coatings promote bone healing and apposition, leading to the rapid biological fixation of implants. It’s no doubt that the addition of certain biologically active protein with biomaterial will improve the bioactivity of the material. Previously, we examined the biocompatibility of basic fibroblast growth factor (bFGF) incorporation with titanium implants. Now we investigate the effect of fibronectin (FN) incorporation with thin calcium phosphate film deposited on titanium by electron-beam evaporation since fibronectin is actively involved in cell adhesion, spreading, would healing, cytoskeletal reorganization, and bone tissue formation. A FN-apatite composite layer was formed on the surface of titanium by biomimetic process. The coating process was carried out by immersing thin calcium phosphate film coated Ti in Dulbecco’s Phosphate buffered saline containing FN (20 ug/ml). The surfaces of samples were examined with FESEM, Fourier transform infrared spectroscopy and X-ray diffraction. The quantity of FN taken up and the kinetics of protein release were monitored by BCA method and Elisa. The fibronectin was immobilized in the newly formed apatite layer. The adhesion of osteoblast cells to the FN-apatite composite layer was to show the biocompatibility of implants, and FN-apatite composite layer could enhance osseintegration of implants in vivo. This research was supported by a grant (code #: 08K1501-01220) from Center for Nanostructured Materials Technology under 21st Century Frontier R& D Program of the Ministry of Education, Science and Technology, Korea

Background. The process of osteolysis is well studied both in vivo and in vitro. Although multiple pathways have been implicated in osteolytic change and animal models have been developed there are few human tissue studies. There are no extensive human tissue studies comparing osteoarthritic hips to well fixed and loose prostheses. Methods. We have investigated 96 genes previously implicated in the osteolytic pathway. Genes were included based on previous implication in osteolysis in basic science studies. Candidates included cytokines, growth factors, apoptotic factors, matrix proteinases, interleukins, apoptotic proteins and macrophage activators. Results. One hundred patients were enrolled into the study and had intraoperative hip tissue removed after ethics approval. Patients were recruited from three cohorts, those undergoing primary hip replacement, revision of a well fixed prosthesis and revision for osteolytic change. A low density Taqman array method was used to determine gene expression for the 96 candidate genes. Expression of five housekeeping genes was measured and expression normalised between the samples. Statistical analysis was undertaken using significance testing and ROC analysis. There were seven candidate genes that were statistically significantly linked to aseptic loosening (p< 0.05) and strongly associated (AUC >0.77); these were BMP4, Frizzled related protein, fibroblast growth factor 18, IL8, IRAK 3, osteoprotegrin and PTGS2. There were a further nine genes which were highly predictive of osteolytic change (AUC >0.77), but did not reach significance (p>0.05): VEGFB, SFRP, TLR3, TLR5, TP53, IGF1, CTSK, CHIT 1 and CCL 18. Conclusion. We have been able to distinguish for the first time between factors which are associated with osteolytic change, those associated with exposure to wear debris in a well fixed prosthesis and those associated with the process of osteoarthritis

Autologous chondrocytes transplantation (ACT) was first used in humans in 1987 and is based on a surgical technique where cells are injected under a periosteal flap. Due to the sometimes tricky surgical isolation and suture of the periosteum and complications with hypertrophy of periosteal tissue (5 – 10% of the cases) that in some cases requires a second arthroscopic trimming ‘easier’ transplantation techniques based on cells cultured on scaffolds and membranes have been suggested. However, the standard ACT technique creates a unique in vivo bioreactor where chondrocytes and periosteum form a unique local environment. If live periosteum and chondrocytes are transplanted to a defect in the rabbit patellae a cartilage repair tissue is formed in contrast to treatment with ‘dead’ periosteum and live chondrocytes were no repair tissue is demonstrated. The unique environment formed by the periosteum and chondrocytes might be responsible for the unique in vivo induction of early embryological development patterns seen in limb formation in the foetus: We have found that the transplanted chondrocytes are expressing early developmental genes e.g Sox 9 and wnt14 and fibroblast growth factor 3 receptors (FGFR3), a marker of chondrocytes progenitor cells. Furthermore, we have found that the articular chondrocytes are able to demonstrate a phenotypic expressivity with an additional ability of bone and adipose tissue formation. Changes to the transplantation procedure must address these unique features of the ACT technology in order to maintain the long term clinical outcome

Objective: Bone marrow stromal cells (BMSC) represent an interesting target for novel strategies in the gene and cell therapy of skeletal pathologies, involving BMSC in vitro expansion/transfection and reinfusion. Materials and Methods: Stromal cells were obtained from healthy donors. For the first 2 weeks, culture medium was supplemented only with human recombinant fibroblast growth factor 2 (FGF-2) to promote cell proliferation and maintain cells in a more immature state. Confluent cultures were detached with trypsin-EDTA. Cells were replated for the in vitro differentiation experiments and for determination of BMSC growth kinetics. Cultures were stimulated with appropriate inductive media and the chondro-/osteo-/adipo-diferentiations were tested by staining with alizarin red, alcian blue, Sudan black and by immunostaining for osteocalcina or collagen II. Results: After the first passage, BMSC had a markedly diminish proliferation rate and gradually lost their multiple differentiation potential. Their bone-forming efficiency in vivo was reduced by about 36 times at first confluence as compared to fresh bone marrow. Conclusion: Culture expansion causes BMSC gradually to lose their early progenitor properties. Both the duration and the conditions of culture could be crucial to successful clinical use of these cells and must be considered when designing novel therapeutic strategies involving stromal mesenchymal progenitor manipulation and reinfusion. There are numerous potential applications of this novel strategy, for example: reconstruction of extensive long-bone defects, osteochondral defect repair, treatment of bone cyst, bioactivable scaffolds, etc

Summary. Based upon genetic analysis, decorin is an exciting pharmacologic agent of potential anti-fibrogenic effect on arthrofibrosis in our animal model. Introduction. While the pathophysiology of arthrofibrosis is not fully understood, some anti-fibrotic molecules such as decorin could potentially be used for the prevention or treatment of joint stiffness. The goal of this study was to determine whether intra-articular administration of decorin influences the expression of genes involved in the fibrotic cascade ultimately leading to less contracture in an animal model. Material and Methods. Eighteen rabbits had their right knees operated on to form contractures. The left knees served as controls. The 6 right limbs in the experimental group (Group 1) received four 500 ug/ml intra-articular injections of decorin over 8 days starting at 8 week, for a total of 2 mg. The 6 right limbs in the first control group (Group 2) received four intra-articular injections of bovine serum albumin (BSA) over 8 days starting at 8 weeks as well. The 6 six right limbs in the second control group (Group 3) received no injections. The contracted limbs of rabbits in Group 1 were biomechanically and genetically compared to the contracted limbs of rabbits in Groups 2 and 3 with the use of a calibrated joint measuring device and custom microarray, respectively. Results. There was no statistical difference in the flexion contracture angles between those right limbs that received intra-articular decorin versus those that received intra-articular BSA (66° vs. 69°; p = 0.41). Likewise, there was no statistical difference between those right limbs that received intra-articular decorin as opposed to those who had no injection (66° vs. 72°; p = 0.27). The lack of significance remained when the control left limbs were taken into account (p > 0.40). When compared to bovine serum albumin (BSA), decorin led to a statistically significant increase in the mRNA expression of 5 genes: substance P, neuropeptide γ, and neurokinin A, cyclin E2, and MMP-9 (p < 0.001). In addition, there was a statistically significant decrease in fibroblast growth factor receptor-2 (FGFR-2), rho-associated coiled-coil containing protein kinase-1 (ROCK-1), and vascular cell adhesion molecule-1 (VCAM-1) genes when intra-articular decorin was compared to no injection (p < 0.001). Conclusions. In this model, when administered intra-articularly at 8 weeks, 2 mg of decorin had no significant effect on joint contractures. However, our genetic analysis revealed a significant alteration in the expression of several fibrotic genes. Further studies investigating the route of administration, dosing, frequency, and timing are required before definitive conclusions may be drawn on the effects of decorin on joint contractures

These studies are indicative of the potential utility of resorbable and nonresorbable inorganic materials as bone graft substitutes. Bone transplants and bone substitute materials are necessary in +/−10% of all skeletal reconstructive operations. The higher osteogenic potential of autografts compared to allogenic transplants is undisputed, but restricted by limited availability and necessity of secondary operations. Commercial bone graft materials show variety of compositions and properties, many very different from those of autologous bone. Physicochemical properties of these materials were compared using x-ray diffraction, scanning and transmission electron microscopy. Biological reactivity of different materials was also compared in histological evaluations in animal models. Experimental and clinical studies have been encouraging, especially in metaphyseal defects. Bone substituting the artificial material should be able to bear weight and, if possible, be lamellar bone. Since fundamental examinations of osteoinduction and affiliated isolation of growth factors (Urist 1965), extensive scientific research on growth factors contained in bone matrix has been performed. Proteins of the TGF-β family play a key role in regulation of bone regeneration. In past years, alkaline fibroblast growth factor has raised increased interest among researchers. Its presence implies that it plays an important role in the development of bone substance. One best known effect is significant augmentation of microangiogenesis, which could be demonstrated among others in experimental wound healing investigations. Further experimental examinations showed significant increase of callus formation in rats and miniature pigs, in which FGF had been injected into the fracture site. Current bone substitute materials are only to be used in clearly defined indications, as they do not currently meet the biological or mechanical properties of autogenous bone. Our knowledge is grounded on various experimental models, which are not always comparable. Therefore many aspects have to be considered as a working understanding

Introduction: In hereditary multiple exostosis (HME) the synthesis of the polysaccharide heparan sulphate (HS) is disrupted. HS-proteoglycans are low affinity receptors involved in fibroblast growth factor signaling. Activation of FGF receptor 3 (FGFr3) on mature chondrocytes leads to growth attenuation rather than stimulation. We tested the hypothesis that in HME chondrocytes with absent or reduced HS-PG synthesis there is impaired response to the FGFr3 ligand and loss of control of chondrocyte proliferation. Materials and methods: Chondrocytes were harvested from normal growth plate (epiphyseodesis) or HME osteochondroma cartilage cap obtained as surgical discard and cultured to 70% confluence in growth media. Cells were re-plated for experimentation. Growth curves were obtained for cells over a period of 5 days. In addition proliferative responses of healthy and HME chondrocytes were determined after low serum synchronization followed by challenge with FGF 9 (10 and 100ng/ml) and incorporation of BrdU for 2hours every two hours over a twenty eight hour period. Using these techniques it is possible to describe in detail the time dependent entry of cells into S-phase of the cell cycle and compare cell lines and treatment. Results: Significant differences were observed in the growth characteristics over a five-day period (p< 0.05). Under baseline growing conditions the chondrocytes derived from osteochondroma had a more rapid doubling time when compared with the normal growth plate chondrocyte (2.6+/− 0.6 vs 4.9+/−1.0, p< 0.05). In response to incubation with FGF-9 cells from normal growth plate have a lower peak proportion of cells entering the s-phase than with media alone (7% vs 25%). This inhibition is not observed in chondrocytes from osteochondroma. Conclusions: It would appear that osteochondroma chondrocytes are resistant to the normal regulatory effect of FGF-9 on cell proliferation. The differential response to FGF may be responsible for the growth differences observed both in-vitro and in-vivo

Introduction and Aims: There has been a lack of studies investigating the effect of cytokines on human meniscal cartilage. We investigated the regenerative potential of meniscal cartilage at different zones of human and sheep menisci under the effect of platelet-derived growth factor AB (PDGF-AB), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (bFGF) in a dose-dependent manner. Method: Monolayer cell cultures were prepared from the inner (avascular), middle, and outer (vascular) zones of human and sheep lateral menisci. Fibrochondrocytes were exposed to various concentrations of each of the three growth factors. To assess cell proliferation, 3H-Thymidine uptake assay was used. For assessment of matrix formation, radioactive 35S uptake assay and Blyscan assay were used to assess newly formed glycosaminoglycans (GAGs), 14C-Proline uptake assay and ELISA were used to assess newly formed collagen. Results: The growth factors under investigation stimulated the fibrochondrocytes isolated from all meniscal zones in a similar dose-dependent fashion. In human meniscal cells, PDGF-AB at a concentration of 200 ng/ml stimulated proliferation by up to four-fold and GAGs synthesis by up to 12-fold (p < 0.001). IGF-I and to a lesser extent bFGF, at concentrations of 100 and 200 ng/ml, also stimulated both cell proliferation and matrix formation in all three meniscal zones (p < 0.001). PDGF and to a lesser degree IGF at concentrations of 100 and 200 ng/ml have stimulated collagen type I synthesis by up to two-fold. Conclusion: The results of this study indicate that fibro-chondrocytes originating from the avascular zone of the meniscus have the ability to proliferate and to regenerate their surrounding extracellular matrix when exposed to growth factors. This is an encouraging observation that will form the basis for in vivo research, aimed at enhancing meniscal repair, even within the avascular zone, following surgical repair

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