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

Aims. This study investigated the effects of β-caryophyllene (BCP) on protecting bone from vitamin D deficiency in mice fed on a diet either lacking (D-) or containing (D+) vitamin D. Methods. A total of 40 female mice were assigned to four treatment groups (n = 10/group): D+ diet with propylene glycol control, D+ diet with BCP, D-deficient diet with control, and D-deficient diet with BCP. The D+ diet is a commercial basal diet, while the D-deficient diet contains 0.47% calcium, 0.3% phosphorus, and no vitamin D. All the mice were housed in conditions without ultraviolet light. Bone properties were evaluated by X-ray micro-CT. Serum levels of klotho were measured by enzyme-linked immunosorbent assay. Results. Under these conditions, the D-deficient diet enhanced the length of femur and tibia bones (p < 0.050), and increased bone volume (BV; p < 0.010) and trabecular bone volume fraction (BV/TV; p < 0.010) compared to D+ diet. With a diet containing BCP, the mice exhibited higher BV and bone mineral density (BMD; p < 0.050) than control group. The trabecular and cortical bone were also affected by vitamin D and BCP. In addition, inclusion of dietary BCP improved the serum concentrations of klotho (p < 0.050). In mice, klotho regulates the expression level of cannabinoid type 2 receptor (Cnr2) and fibroblast growth factor 23 (Fgf23) through CD300a. In humans, data suggest that klotho is connected to BMD. The expression of klotho is also associated with bone markers. Conclusion. These data indicate that BCP enhances the serum level of klotho, leading to improved bone properties and mineralization in an experimental mouse model. Cite this article: Bone Joint Res 2022;11(8):528–540

Aims. There is an increasing concern of osteoporotic fractures in the ageing population. Low-magnitude high-frequency vibration (LMHFV) was shown to significantly enhance osteoporotic fracture healing through alteration of osteocyte lacuno-canalicular network (LCN). Dentin matrix protein 1 (DMP1) in osteocytes is known to be responsible for maintaining the LCN and mineralization. This study aimed to investigate the role of osteocyte-specific DMP1 during osteoporotic fracture healing augmented by LMHFV. Methods. A metaphyseal fracture was created in the distal femur of ovariectomy-induced osteoporotic Sprague Dawley rats. Rats were randomized to five different groups: 1) DMP1 knockdown (KD), 2) DMP1 KD + vibration (VT), 3) Scramble + VT, 4) VT, and 5) control (CT), where KD was performed by injection of short hairpin RNA (shRNA) into marrow cavity; vibration treatment was conducted at 35 Hz, 0.3 g; 20 minutes/day, five days/week). Assessments included radiography, micro-CT, dynamic histomorphometry and immunohistochemistry on DMP1, sclerostin, E11, and fibroblast growth factor 23 (FGF23). In vitro, murine long bone osteocyte-Y4 (MLO-Y4) osteocyte-like cells were randomized as in vivo groupings. DMP1 KD was performed by transfecting cells with shRNA plasmid. Assessments included immunocytochemistry on osteocyte-specific markers as above, and mineralized nodule staining. Results. Healing capacities in DMP1 KD groups were impaired. Results showed that DMP1 KD significantly abolished vibration-enhanced fracture healing at week 6. DMP1 KD significantly altered the expression of osteocyte-specific markers. The lower mineralization rate in DMP1 KD groups indicated that DMP1 knockdown was associated with poor fracture healing process. Conclusion. The blockage of DMP1 would impair healing outcomes and negate LMHFV-induced enhancement on fracture healing. These findings reveal the importance of DMP1 in response to the mechanical signal during osteoporotic fracture healing. Cite this article: Bone Joint Res 2022;11(7):465–476

Aims. Here we introduce a wide and complex study comparing effects of growth factors used alone and in combinations on human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation. Certain ways of cell behaviour can be triggered by specific peptides – growth factors, influencing cell fate through surface cellular receptors. Methods. In our study transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were used in order to induce osteogenesis and proliferation of hMSCs from bone marrow. These cells are naturally able to differentiate into various mesodermal cell lines. Effect of each factor itself is pretty well known. We designed experimental groups where two and more growth factors were combined. We supposed cumulative effect would appear when more growth factors with the same effect were combined. The cellular metabolism was evaluated using MTS assay and double-stranded DNA (dsDNA) amount using PicoGreen assay. Alkaline phosphatase (ALP) activity, as early osteogenesis marker, was observed. Phase contrast microscopy was used for cell morphology evaluation. Results. TGF-β and bFGF were shown to significantly enhance cell proliferation. VEGF and IGF-1 supported ALP activity. Light microscopy showed initial extracellular matrix mineralization after VEGF/IGF-1 supply. Conclusion. A combination of more than two growth factors did not support the cellular metabolism level and ALP activity even though the growth factor itself had a positive effect. This is probably caused by interplay of various messengers shared by more growth factor signalling cascades. Cite this article: Bone Joint Res 2020;9(7):412–420

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

The August 2012 Research Roundup. 360. looks at: PRP and chondrogenic differentiation; basic fibroblast growth factor; whether glucosamine works; randomised trials; ossification of the ligamentum flavum; treadmill running; inhibiting BMP antagonists; and whether NSAIDs delay union after all

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

Objectives. Regenerative medicine is an emerging field aimed at the repair and regeneration of various tissues. To this end, cytokines (CKs), growth factors (GFs), and stem/progenitor cells have been applied in this field. However, obtaining and preparing these candidates requires invasive, costly, and time-consuming procedures. We hypothesised that skeletal muscle could be a favorable candidate tissue for the concept of a point-of-care approach. The purpose of this study was to characterize and confirm the biological potential of skeletal muscle supernatant for use in regenerative medicine. Methods. Semitendinosus muscle was used after harvesting tendon from patients who underwent anterior cruciate ligament reconstructions. A total of 500 milligrams of stripped muscle was minced and mixed with 1 mL of saline. The collected supernatant was analysed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The biological effects of the supernatant on cell proliferation, osteogenesis, and angiogenesis in vitro were evaluated using human mesenchymal stem cells (hMSCs) and human umbilical cord vein endothelial cells (HUVECs). Results. The supernatant contained several GFs/CKs, with especially high levels of basic fibroblast growth factor, and CD34+ cells as the stem/progenitor cell fraction. With regard to biological potential, we confirmed that cell proliferation, osteoinduction, and angiogenesis in hMSCs and HUVECs were enhanced by the supernatant. Conclusions. The current study demonstrates the potential of a new point-of-care strategy for regenerative medicine using skeletal muscle supernatant. This attractive approach and readily-available material could be a promising option for tissue repair/regeneration in the clinical setting. Cite this article: M. Yoshikawa, T. Nakasa, M. Ishikawa, N. Adachi, M. Ochi. Evaluation of autologous skeletal muscle-derived factors for regenerative medicine applications. Bone Joint Res 2017;6:277–283. DOI: 10.1302/2046-3758.65.BJR-2016-0187.R1

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