Enhanced osteoblastic differentiation and bone formation in co-culture of human bone marrow mesenchymal stromal cells and peripheral blood mononuclear cells with exogenous VEGF.

BACKGROUND: Despite recent advances in bone tissue engineering, efficient bone formation and vascularization remains a challenge for clinical applications. HYPOTHESIS: The aim of this study was to investigate if the osteoblastic differentiation of human mesenchymal stromal cells (MSCs) can be enhanced by co-culturing them with peripheral blood (PB) mononuclear cells (MNCs), with and without vascular endothelial growth factor (VEGF), a coupling factor of bone formation and angiogenesis. MATERIALS AND METHODS: Human bone marrow (BM) derived MSCs were co-cultured with PB-MNCs in osteogenic medium with or without VEGF. Osteoblastic differentiation and mineral deposition were studied by staining for alkaline phosphatase (ALP), and von Kossa, respectively, and measurements for ALP activity and calcium concentration (Ca). Cell proliferation was assayed with Alamar blue. The mechanism(s) were further studied by Transwell(®) cell culture experiments. RESULTS: Both ALP and mineralization (von Kossa and Ca) were significantly higher in the MSC-MNC co-cultures compared to plain MSC cultures. VEGF alone had no effect on osteoblastic differentiation of MSCs, but further enhanced differentiation in co-culture settings. The mechanism was shown to require cell-cell contact between MSCs and MNCs and the factors contributing to further differentiation appear to be soluble. No differences were observed in cell proliferation. CONCLUSION: Our study demonstrates that the in vitro ALP activity and mineralization of human BM-MSCs is more efficient in the presence of PB-MNCs, and exogenously added VEGF further enhances the stimulatory effect. This indicates that PB-MNCs could be a potential cell source in development of co-culture systems for novel tissue engineering applications for enhanced bone healing.

Mechanical properties and in vivo performance of load-bearing fiber-reinforced composite intramedullary nails with improved torsional strength.

Fiber-reinforced composites (FRC) could be feasible materials for fracture fixation devices if the mechanical properties of the composites are congruent with the local structural properties of bone. In a recently developed FRC implant, bisphenol A dimethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) resin was reinforced with unidirectional E-glass fibers. The addition of a braided glass fiber sleeving to the unidirectional fibers increased the torsional strength (99.5MPa) of the FRC implants at the expense of the flexural strength (602.0MPa). The flexural modulus was 15.3GPa. Two types of FRC intramedullary nails were prepared; first type was FRC as such, second type was FRC with a surface layer of bioactive glass (BG) granules. Experimental oblong subtrochanteric defect was created in 14 rabbits. The defect, which reduced the torsional strength of the bones by 66%, was fixed with an FRC intramedullary nail of either type. The contralateral intact femur served as the control. This model simulated surgical stabilization of bone metastasis. After 12 weeks of follow-up, the femurs were harvested and analyzed by torsional testing, micro-CT and hard tissue histology. Healed undisplaced peri-implant fractures were noticed in half of the animals irrespective of the type of FRC implant. Torsional testing showed no significant differences between the implantation groups. The torsional strength of the bones stabilized by either type of FRC implant was 83% of that of the contralateral femurs. In histological analysis, no implant debris and no adverse tissue reactions were observed. While the mechanical properties of the modified FRCs were suboptimal, the FRC intramedullary nails supported the femurs without structural failure, even in the cases of peri-implant fractures.

Interaction between marrow-derived human mesenchymal stem cells and peripheral blood mononuclear cells in endothelial cell differentiation.

BACKGROUND AND AIMS: In adult connective tissues, mesenchymal stem cells (MSCs) play a key role in normal tissue turnover and repair. MSCs can participate in these processes not only through proliferation and differentiation but also through paracrine/autocrine functions. These characteristics make MSCs the optimal target in the development of cell-based therapies. This study describes a novel interaction between human MSC and blood mononuclear cells (MNCs), resulting in formation of blood vessel-like structures. MATERIALS AND METHODS: Human marrow-derived MSCs and peripheral blood MNCs were co-cultured in monolayer cultures as well as in bovine collagen sponge up to 20 days. No exogenously supplied growth factors were applied. Morphological changes and formations of three dimensional structures were detected by light microscopy. The process was further stu-died for the expression of different endothelial cell markers. The expression of PECAM-1 and endoglin was studied by immunohistochemistry and the expression of vascular endothelial growth factor receptors 1 and 2 using quantitative real time PCR. RESULTS: In co-cultures of human MSCs and MNCs, the previously nonadherent cells attached and started to elongate and formed tube-like structures within one week. At day 10, elongated PECAM-1 and endoglin expressing cells were detected in co-cultures. At day 20, PECAM-1 and endoglin-positive vessel-like structures were observed. VEGFR1 was up-regulated in co-cultures after 10 days, and expression levels increased with time. No PECAM-1, endoglin or VEGFR1 expressing cells were discovered in MSC-cultures without MNCs at any time point. CONCLUSIONS: This study demonstrates induction of endothelial differentiation in co-cultures of human MSCs and MNCs, indicating a mechanism by which local application of MSCs could induce angiogenesis in vivo.

Development of a multi-component fiber-reinforced composite implant for load-sharing conditions.

Fiber-reinforced composites (FRC) have the potential for use as load-bearing orthopaedic implants if the high strength and elastic modulus of FRC implant can be matched with local requirements. This study tested the in vivo performance of novel FRC implants made of unidirectional glass fibers (E-glass fibers in Bis-GMA and TEGDMA polymeric matrix). The implant surface was covered with bioactive glass granules. Control implants were made of surface-roughened titanium. Stress-shielding effects of the implants were predicted by finite element modelling (FEM). Surgical stabilization of bone metastasis in the subtrochanteric region of the femur was simulated in 12 rabbits. An oblong subtrochanteric defect of a standardized size (reducing the torsional strength of the bones approximately by 66%) was created and an intramedullary implant made of titanium or the FRC composite was inserted. The contralateral femur served as the intact control. At 12 weeks of healing, the femurs were harvested and analyzed by radiography, torsional testing, micro-CT imaging and hard tissue histology. The functional recovery was unremarkable in both groups, although the final analysis revealed two healed undisplaced peri-implant fractures in the group of FRC implants. FEM studies demonstrated differences in stress-shielding effects of the titanium and FRC implants, but the expected biological consequences did not become evident during the follow-up time of the animal study. Biomechanical testing of the retrieved femurs showed no significant differences between the groups. The torsional strength of the fixed bones had returned the level of contralateral intact femurs. Both implants showed ongrowth of intramedullary new bone. No adverse tissue reactions were observed. Based on these favorable results, a large-scale EU-project (NewBone, www.hb.se/ih/polymer/newbone) has been launched for development of orthopaedic FRC implants.

Bioactive glass granules as extender of autogenous bone grafting in cementless intercalary implant of the canine femur.

BACKGROUND AND AIMS: Ceramic bone graft substitutes have a potential to be used as replacement of allogeneic bone grafting and, under optimal distribution of particle size, they may even provide mechanical support. The current study examined the efficacy of bioactive glass granules as an extender of autogenous bone grafting in a segmental bone replacement model of the canine femur. MATERIAL AND METHOD: A 16 mm long segment of the femur shaft was bilaterally replaced with an intercalary titanium implant in eight animals. The implant had cementless grooved proximal and distal stems. In one leg, the peri-implant space was packed with composite graft consisting of a mixture of bioactive glass granules and autogenous bone graft in proportion of 50:50. In the opposite leg, the peri-implant space was treated with autogenous bone graft alone. After surgery, unlimited functional loading was allowed. The outcome was evaluated at three months. RESULTS: Eight out of sixteen autografted implants and seven out of sixteen composite-grafted implants were radiographically incorporated and clinically stable at three months. In the paired comparison, the proximal components of composite-grafted implants showed lower maximum load under torsional testing (p = 0.068), less new bone in the longitudinal grooves of the stems (p = 0.036) and lower affinity of new bone to implant surface (p = 0.046). The distal components of the two sides showed a similar trend for less new bone in the grooves and lower bone affinity of new bone in the distal composite-grafted components. CONCLUSIONS: The current study suggests that supplementation of periprosthetic bone graft with bioactive ceramic particles may not help to promote healing of cementless implants under high dynamic loading conditions.

Molecular basis for action of bioactive glasses as bone graft substitute.

Bone grafting procedures are undergoing a major shift from autologous and allogeneic bone grafts to synthetic bone graft substitutes. Bioactive glasses are a group of synthetic silica-based bioactive materials with bone bonding properties first discovered by Larry Hench. They have several unique properties compared with other synthetic bioresorbable bioactive ceramics, such as calcium phosphates, hydroxyapatite (HA) and tricalcium phosphate (TCP). Bioactive glasses have different rates of bioactivity and resorption rates depending on their chemical compositions. The critical feature for the rate of bioactivity is a SiO2 content < 60% in weight. In vivo, the material is highly osteoconductive and it seems to promote the growth of new bone on its surface. In a recent study, the activity of the material was found even to overshadow the effect of BMP-2 gene therapy. In vivo, there is a dynamic balance between intramedullary bone formation and bioactive glass resorption. Recent studies of molecular biology have shown that bioactive glass induces a high local turnover of bone formation and resorption. Many osteoporotic fracture patients are candidates for concurrent treatment with bisphosphonates and bioceramic bone graft substitutes. Since osteopromotive silica-based bioactive glasses induce accelerated local bone turnover, adjunct antiresorptive agents may affect the process. However, a recent study showed that an adjunct antiresorptive therapy (zoledronic acid) is even beneficial for bone incorporation of bioactive glass. Based on these observations, bioactive glasses are a promising group of unique biomaterials to act as bone graft substitutes.

RSA applications in monitoring of fracture healing in clinical trials.

Radiostereometric analysis (RSA) was originally developed as a method for performing highly accurate three-dimensional measurements in vivo over time from sequential radiographs. Since its introduction over twenty years ago, the RSA method has proven itself as a powerful tool with numerous orthopaedic applications. RSA has been used extensively in studies of prosthetic fixation and has been shown to be the method of choice for these studies. RSA has, however, also been successfully applied to a limited number of studies examining fracture healing, namely in fractures of the radius, ankle, tibial plateau, trochanter and femoral neck, as well as studies of bone healing following spinal fusion and tibial osteotomies. RSA follow-up of a fracture will provide definitive demonstration of the exact time of union, i.e. the achievement of fracture stability. This information can be invaluable in randomized clinical trials of fracture treatment. Phantom model studies have proven useful for effective preoperative planning and interpretation of RSA results. The RSA method is a highly accurate, precise and safe objective method for studying fracture healing in clinical trials. The RSA method may serve as a scientific tool to accurately evaluate the significance of supporting novel biomaterials for the early stability and the rate of healing in fractures.

Comparison of digital and conventional radiostereometric image analysis in an ankle phantom model.

BACKGROUND AND AIMS: Radiostereometric analysis (RSA) allows accurate three-dimensional measurements of micromotion in skeletal structures. The current RSA techniques are based on the analysis of scanned plain films. This study was undertaken to compare digital filmless RSA technique to conventional scanning technique using a phantom model of the ankle mortise. MATERIAL AND METHODS: In the first experiment, the relative displacement of the markers inserted to the fibula in relation to the markers inserted to the tibia was studied by means of double examinations and the precision of DICOM images were compared to scanned images of printed radiographs. In the second experiment, the film pair of double examination was re-imported or re-scanned and self-compared in order to show merely the error related to the image processing. RESULTS: The precision of RSA using scanned images of printed radiographs was compatible to DICOM images. However, the mean error of rigid body fitting (ME) values were significantly lower in use of DICOM images compared with scanned radiographs, indicating less deformation of rigid body segments in filmless analysis. CONCLUSIONS: Precision of the RSA method was improved under the completely filmless environment. Therefore, this technique can be recommended for clinical studies of radiostereometric analysis.

Combined effect of BMP-2 gene transfer and bioactive glass microspheres on enhancement of new bone formation.

Adenovirus-mediated recombinant human BMP-2 (RAdBMP-2) gene transfer has been found to have significant osteoinductive properties. The hypothesis of the current study was that bioactive glass surface could provide favorable osteoconductive conditions for cellular action of osteoinductive RAdBMP-2 gene transfer. In the rat proximal tibia, a portion of the medullary cavity was evacuated and filled with bioactive glass microspheres and injected with adenovirus carrying the human BMP-2 gene (BG/RAdBMP-2). Control defects filled with BG microspheres were injected with adenovirus carrying the LacZ reporter gene (BG/RAdLacZ) or saline (BG). Empty control defects were also used. Bone healing response was analyzed at 4 days, and at 2 and 8 weeks by radiography, peripheral quantitative computed tomography (pQCT), histomorphometry, and backscattered electron imaging of scanning electron microscopy (BEI-SEM) equipped with energy dispersive X-ray analysis (EDXA). In empty controls, the amount of intramedullary new bone peaked at 2 weeks, whereas defects filled with bioactive glass with and without RAdBMP-2 gene transfer showed a constant time-related increase of intramedullary new bone. At 8 weeks, there was significantly more new bone in defects treated with BG and RAdBMP-2 than in defects left to heal without filling (p < 0.001). Compared with the other controls (BG only or BG/RAdLacZ), the difference was not significant. In the current model, the osteopromotive effect of bioactive glass microspheres appears synergistic with the osteoinductive action of BMP-2 gene transfer, or one overshadows the other, as no additive effect was observed.

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human chondrosarcomas.

The aim of the present study was to characterise the ability of malignant chondrosarcomas to invade normal bone by analysing their production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). For this purpose 12 chondrosarcomas were investigated for the expression of mRNAs for several MMPs and all 4 TIMPs by Northern hybridisation, and for immunohistochemical localisation of the proteins. A characteristic finding of these analyses was increased expression of MMP-13, MMP-14 and TIMP-2 mRNAs in chondrosarcomas when compared with nonmalignant control samples. Individual chondrosarcomas also exhibited elevated levels of MMP-1, MMP-7 and MMP-9 mRNAs. The results of Northern hybridisations were supported by immunohistochemical stainings of the corresponding tumour areas for MMP-2, MMP-14 and TIMP-2, further suggesting that these may have prognostic value for determining whether individual chondrosarcomas are locally aggressive or have a probability of recurrence. Another finding of the present study was a marked heterogeneity in histologic appearance and gene expression of the chondrosarcomas, emphasising the importance of analysing several areas of these tumours to get representative results. These findings suggest that analysis of MMPs could be a useful diagnostic indicator in patients with cartilaginous tumours and could help in differentiating between a low-grade malignant chondrosarcoma and a benign growing enchondroma.

MR-guided core biopsies of soft tissue tumours on an open 0.23 T imager.

PURPOSE: To assess the feasibility of MR-guided soft tissue core biopsies on an open 0.23 T magnet. MATERIAL AND METHODS: Twenty-nine consecutive patients with known or suspected benign or malignant soft tissue tumours underwent MR imaging. A one-slice dynamic enhancement sequence was used to obtain an enhancement curve of the tumour. MR-guided core biopsy of the tumour was performed in the same session. RESULTS: All biopsies could be performed on an open 0.23 T magnet. Standard MR images and dynamic enhancement curves were used in deciding biopsy route and target. The MR-guided core biopsy specimens were sufficient for histopathological diagnosis in 27 of 29 cases. CONCLUSION: Open magnet configuration allows easy access to the patient and near real-time imaging guidance of soft tissue tumours. Minimally invasive MR-guided core biopsies of soft tissue tumours are feasible and help to avoid open surgical biopsies.

A metaphyseal defect model of the femur for studies of murine bone healing.

A bone defect model was developed in the distal metaphysis of the femur for studies on bone healing in the mouse. The circular defect involving 20% of the bone circumference resulted in a 34% reduction in the bending moment compared to intact bone. The healing process was followed using histomorphometry, peripheral quantitative computed tomography (pQCT), biomechanical testing, and molecular biological analyses. Histologically, healing of the defect was characterized by filling of the medullary cavity with trabecular new bone during the first week of healing, and by closing of the cortical window by 6 weeks. Small areas of periosteal chondrogenesis were frequently observed during defect healing. In pQCT, bone mineral content (BMC) of the defect area approached that of intact control bone already by 3 weeks, reflecting the production of trabecular bone. Similarly, the bending strength and stiffness of the healing femur reached the level of intact control femur already at 3 weeks. Bone formation and remodeling was followed by Northern analyses, which demonstrated elevated mRNA levels for bone components (type I collagen and osteocalcin), and for osteoclastic enzymes (cathepsin K, matrix metalloproteinase-9, and tartrate-resistant acid phosphatase) throughout the healing period. Finally, the applicability of the defect model for gene therapy experiments was tested using adenovirus-mediated transfer of the LacZ reporter gene. Both histochemistry and mRNA analyses demonstrated that the gene was expressed in the repair tissue with the highest expression during the first week of healing. The present model thus provides a standardized environment for studies on induction and remodeling of trabecular new bone in normal and genetically engineered mice.

Silica-based bioactive glasses modulate expression of bone morphogenetic protein-2 mRNA in Saos-2 osteoblasts in vitro.

A chemical exchange of the silica gel layer forming on the surface of bioactive glasses is thought to be the principal reaction for bone-bioactive glass bonding. The contribution of biological molecules on cell-bioactive glass interaction is largely unknown. To further analyze the mechanisms involved in efficient bone bonding to bioactive glass, Saos-2 osteoblastic cells with proven osteogenic phenotype were cultured for 4, 7 and 14 days on two bioactive glasses with different Si contents. Culture plates and dishes made of bioactive (BAG, 53 % SiO2), biocompatible (BCG, 58% SiO2) and control (GO) glasses were extensively conditioned with phosphate buffer and DMEM medium before seeding the cells. Northern hybridization was used for analysis of mRNA levels of collagen type I (Col-I), alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2). A significant increase was observed in Col-I mRNA levels in cells grown on the two bioactive glasses when compared with those grown on controls at 4 and 7 days (p < 0.04). The mRNA level for ALP in the cultures of bioactive glasses-made plates and dishes was also increased over control at 7 days (p < 0.02) and remained this way between BAG and G0 at 14 days. Striking differences in BMP-2 mRNA levels existed between BAG and G0 plates and dishes at 7 days (p < 0.05). BMP-2 mRNA level in BAG group was higher than in BCG group at 4, 7 and 14 days, but without statistical significance. Saos-2 osteoblastic cells with strong ALP staining were mostly seen on BAG plates under a light microscope. In confocal microscopy, a bright FITC-stained F-actin ring was present in the cytoplasm of cells grown on BAG dish, demonstrating an active functional status. Stimulation of the expression of BMP-2 and other bone mRNAs by bioactive glasses in osteoblastic cells suggests biological involvement of bone related growth factors, peptides and cytokines in bone-bioactive glass bonding.

Cysteine proteinases in chondrosarcomas.

The aim of the present study was to define the role of cathepsins B, H, K, L and S in the pathogenesis of human chondrosarcomas. For this purpose 40 tumour samples obtained from 12 patients with the diagnosis of conventional chondrosarcoma were systematically investigated for the expression of cathepsin mRNAs by Northern hybridisation, and for immunohistochemical localisation of the proteins. Northern analysis demonstrated the highest levels of cathepsins B and L in a recurring grade 1 chondrosarcoma, and in a grade 3 chondrosarcoma and in fibrous histiocytomas. Increased expression of cathepsin K mRNA was seen in seven chondrosarcomas, as well as in control tumours; fibrous histiocytomas, osteosarcomas, enchondromas and a giant cell tumour of bone. Cathepsin L was immunolocalised within the large chondrocytes, while cathepsin K was predominantly localised in large multinucleated osteoclastic cells and in some hypertrophic chondrocytes. These results suggest that chondrosarcoma can be included in the growing list of tumours, where cathepsins may well be involved in tumour progression. The simultaneous upregulation of cathepsins B and L, together with matrix metalloproteinase-13, and the association of cathepsin K with negative prognostic parameters suggests that an aggressive biological behaviour of chondrosarcoma may be related to the synthesis of cysteine proteinases and activation of other proteolytic enzymes. If this turns out to be the case, cathepsin inhibitors could provide the much needed adjuvant therapy for chondrosarcomas.

Dynamic contrast-enhanced MR imaging and MR-guided bone biopsy on a 0.23 T open imager.

OBJECTIVE: To assess the feasibility of MR (magnetic resonance)-guided bone biopsies. DESIGN AND PATIENTS: Thirty-six consecutive patients with known or suspected benign or malignant bone lesions underwent comprehensive MR imaging. A dynamic contrast-enhanced sequence followed by stationary T1-weighted sequences were obtained and MR-guided bone biopsy of the tumor at the site with fastest enhancement was performed using an open 0.23 T MR imager. RESULTS: All MR-guided bone biopsies samples were estimated to be sufficient by the pathologists. The biopsy specimens were diagnostic in 34 of 36 cases. CONCLUSION: MR-guided bone biopsies combined with dynamic contrast-enhanced imaging are feasible and safe for the diagnostic investigation of equivocal bone lesions.