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.

Biological responses of silver-coated thermosets: an in vitro and in vivo study.

Bisphenol A glycidylmethacrylate (BisGMA)/triethyleneglycol dimethacrylate (TEGDMA) thermosets are biomaterials commonly employed for orthopedic and dental applications; for both these fields, bacterial adhesion to the surface of the implant represents a major issue for the outcome of the surgical procedures. In this study, the antimicrobial properties of a nanocomposite coating formed by polysaccharide 1-deoxylactit-1-yl chitosan (Chitlac) and silver nanoparticles (nAg) on methacrylate thermosets were studied. The Chitlac-nAg system showed good anti-bacterial and anti-biofilm activity although its biocidal properties can be moderately, albeit significantly, inhibited by serum proteins. In vitro studies on the silver release kinetic in physiological conditions showed a steady metal release associated with a gradual loss of antimicrobial activity. However, after 3weeks there was still effective protection against bacterial colonization which could be accounted for by the residual silver. This time-span could be considered adequate to confer short-term protection from early peri-implant infections. Preliminary in vivo tests in a mini-pig animal model showed good biological compatibility of Chitlac-nAg-coated materials when implanted in bony tissue. The comparison was made with implants of titanium Ti6Al4V alloy and with a Chitlac-coated thermoset. Bone healing patterns and biocompatibility parameters observed for nAg-treated material were comparable with those observed for control implants.

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.

Efficacy of bioabsorbable antibiotic containing bone screw in the prevention of biomaterial-related infection due to Staphylococcus aureus.

Impregnation of antimicrobial agents within biodegradable orthopedic implants provides a possibility for local antimicrobial prophylaxis of biomaterial-related infections. The objective of this study was to evaluate the efficacy of a bioabsorbable ciprofloxacin containing bone screw (Ab-PLGA) in the prevention of biomaterial-related infection due to Staphylococcus aureus in a rabbit model. Animals in Group I (n=8) received a Ab-PLGA screw contaminated with S. aureus, while animals in Group II (n=8) received a stainless steel (SS) screw contaminated with S. aureus. In two negative control groups, the animals received a Ab-PLGA screw (Group III, n=4) or a SS screw (Group IV, n=4) without bacterial contamination. 18F-FDG-PET imaging, performed at 6 weeks, was applied as a novel quantitative in vivo imaging modality of implant-related infection. Infection was verified by swab cultures, direct cultures of the retrieved implant, and quantitative cultures of pulverized bone. The concentrations of ciprofloxacin in serum and local bone tissue were determined by a high performance liquid chromatographic (HPLC) method with fluorescence (FLD) detection. In the group of contaminated Ab-PLGA screws, all cultures were negative. In the group of contaminated SS screws, all cultures of retrieved implants and six cultures out of eight of pulverized bone were positive for inoculated S. aureus. In negative control groups, all cultures were negative except one contaminant (S. cohnii) found in a SS screw culture. Verified infection of contaminated SS screws was collaborated by the increased 18F-FDG-PET uptake (P=0.004 compared with the group of contaminated Ab-PLGA screws). The mean bone tissue concentration of ciprofloxacin varied from 2.54 to 0.83 microg/g bone as a function of distance from the implantation site. The serum concentration of ciprofloxacin remained undetectable and below the resolution of the analytic method (<5.0 ng/ml). This study confirmed the in vivo efficacy of bioabsorbable antibiotic containing bone screw in the prevention of biomaterial-related infection due to S. aureus.

Bone defect repair in immobilization-induced osteopenia: a pQCT, biomechanical, and molecular biologic study in the mouse femur.

The present study was carried out to determine whether immobilization-induced (Im) osteopenic bone possesses the same reparative capacity as normal healthy bone. Furthermore, the effects of mechanical loading versus immobilization on bone defect healing were studied. Three-week cast-immobilization was used to induce local osteopenia in mice. A standardized metaphyseal bone defect of the distal femur was created unilaterally both in immobilization-induced (Im) osteopenic mice and in nonimmobilized (Mo) age-matched control animals. After creation of the bone defect, the animals in both groups were further divided into two groups: 3-week cast-immobilization (Im-Im and Mo-Im) groups, and unrestricted weight-bearing (Im-Mo and Mo-Mo) groups. The healing process was followed up to 3 weeks using RNA analysis, histomorphometry, biomechanical testing, and pQCT measurements. At 3 weeks of healing without immobilization, bone mineral density (BMD), as well as bone bending stiffness and strength were higher in normal (Mo-Mo) than in osteopenic (Im-Mo) bone. Although the levels of mRNAs characteristic to chondrocytes (Sox9 and type II collagen), hypertrophic chondrocytes (Type X collagen), osteoblasts (type I collagen and osteocalcin), and osteoclasts (cathepsin K) during the bone defect healing exhibited similarities in their expression profiles, mechanical loading conditions also caused characteristic differences. Mechanical loading during healing (Mo-Mo group) induced stronger expression of cartilage- and bone-specific genes and resulted in higher BMD than that seen in the cast-immobilized group (Mo-Im). In biomechanical analysis, increased bending stiffness and strength were also observed in animals that were allowed weight-bearing during healing. Thus, our study shows that bone healing follows the same molecular pathway both in osteopenic and normal bones and presents evidence for reduced or delayed regeneration of noncritical size defects in immobilization-induced osteopenic bone.

Molecular biologic comparison of new bone formation and resorption on microrough and smooth bioactive glass microspheres.

In a recent in vitro study, chemical microroughening of a bioactive glass surface was shown to enhance attachment of MG-63 osteoblastic cells to glass. The current study was designed to delineate the effects of microroughening on the gene expression patterns of bone markers during osteogenesis and new bone remodeling on bioactive glass surface in vivo. With the use of a rat model of paired comparison, a portion of the medullary canal in the proximal tibia was evacuated through cortical windows and filled with microroughened or smooth bioactive glass microspheres. The primary bone-healing response and subsequent remodeling were analyzed at 1, 2, and 8 weeks, respectively, by radiography, pQCT, histomorphometry, BEI-SEM, and molecular biologic analyses. The expression of various genes for bone matrix components (type I collagen, osteocalcin, osteopontin, osteonectin) and proteolytic enzymes (cathepsin K, MMP-9) were determined by Northern analysis of the respective mRNAs. Paired comparison showed significant differences in the mRNAs levels for specific bone matrix components at 2 weeks: osteopontin was significantly higher (p =.01) and osteonectin significantly lower (p =.05) in bones filled with microroughened microspheres than in those filled with smooth microspheres. Bones filled with microrough microspheres also showed significantly increased ratios of cathepsin K and MMP-9 (both markers of osteoclastic resorption) to type I collagen (p =.02 and p =.02, respectively) at 2 weeks and a significantly increased expression of MMP-9 at 8 weeks (p =.05). The pQCT, histomorphometric, and BEI-SEM analyses revealed no significant differences in the pattern of bone-healing response. Based on these results, microroughening of a bioactive glass surface could trigger temporal changes in the expression of specific genes especially by promoting the resorption part of new bone-remodeling processes. Future studies are needed to evaluate if the observed changes of gene expression are directly related to the microrough surface of any biomaterial or are biomaterial specific.

Characterization of microrough bioactive glass surface: surface reactions and osteoblast responses in vitro.

The current study characterized the in vitro surface reactions of microroughened bioactive glasses and compared osteoblast cell responses between smooth and microrough surfaces. Three different bioactive glass compositions were used and surface microroughening was obtained using a novel chemical etching method. Porous bioactive glass specimens made of sintered microspheres were immersed in simulated body fluid (SBF) or Tris solutions for 1, 6, 24, 48, or 72 h, and the formation of reaction layers was studied by means of a scanning electron microscope/energy dispersive X-ray analysis (SEM/EDXA). Cell culture studies were performed on bioactive glass disks to examine the influence of surface microroughness on the attachment and proliferation of human osteoblast-like cells (MG-63). Cell attachment was evaluated by means of microscopic counting of in situ stained cells. Cell proliferation was analyzed with a nonradioactive cell proliferation assay combined with in situ staining and laser confocal microscopy. The microroughening of the bioactive glass surface increased the rate of the silica gel layer formation during the first hours of the immersion. The formation of calcium phosphate layer was equal between control and microroughened glass surfaces. In cell cultures on bioactive glass, the microrough surface enhanced the attachment of osteoblast-like cells but did not have an effect on the proliferation rate or morphology of the cells as compared with smooth glass surface. In conclusion, microroughening significantly accelerated the early formation of surface reactions on three bioactive glasses and had a positive effect on initial cell attachment.

Pore diameter of more than 100 microm is not requisite for bone ingrowth in rabbits.

The optimal pore size for bone ingrowth is claimed to be 100-400 microm. With the use of a highly standardized experimental model, the present study reevaluated whether a pore size of 100 microm is the threshold value for bone ingrowth into porous structures under non-load-bearing conditions. Titanium triangle-shaped plates 250 or 500 microm thick were perforated with the use of a laser in order to create standard-sized holes ( 50, 75, 100, and 125 microm) in multiple rows. The amount of bone ingrowth through the implant holes was studied in the cancellous bone of the distal rabbit femur. Twelve weeks after implantation, detailed analysis of bone ingrowth was performed with computerized image analysis of backscattered electron imaging techniques of scanning electron microscopy. The results showed that the amount of ingrown new bone was independent of the pore size and implant thickness. The median value for bone ingrowth varied between 64 and 78%. A striking feature was the formation of secondary osteonal structures even in the smallest holes. Based on these results, there is no threshold value for new bone ingrowth in pore sizes ranging from 50 to 125 microm under non-load-bearing conditions.