Regulation of growth plate chondrocytes by 1,25-dihydroxyvitamin D3 requires caveolae and caveolin-1.

Unlabelled: We examined the role of caveolae and caveolin-1 in the mechanism of 1alpha,25(OH)(2)D(3) action in growth plate chondrocytes. We found that caveolae are required for rapid 1alpha,25(OH)(2)D(3)-dependent PKC signaling, and caveolin-1 must be present based on studies using chondrocytes from Cav-1(-/-) mice.

Introduction: 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] regulates endochondral ossification in part through membrane-associated mechanisms, including protein kinase C (PKC) signaling activated by a membrane-associated 1alpha,25(OH)(2)D(3)-binding protein, ERp60.

Mineralization capacity of Runx2/Cbfa1-genetically engineered fibroblasts is scaffold dependent.

Development of tissue-engineered constructs for skeletal regeneration of large critical-sized defects requires the identification of a sustained mineralizing cell source and careful optimization of scaffold architecture and surface properties. We have recently reported that Runx2-genetically engineered primary dermal fibroblasts express a mineralizing phenotype in monolayer culture, highlighting their potential as an autologous osteoblastic cell source which can be easily obtained in large quantities. The objective of the present study was to evaluate the osteogenic potential of Runx2-expressing fibroblasts when cultured in vitro on three commercially available scaffolds with divergent properties: fused deposition-modeled polycaprolactone (PCL), gas-foamed polylactide-co-glycolide (PLGA), and fibrous collagen disks.

Alteration of femoral bone morphology and density in COX-2-/- mice.

A role of COX-2 in pathological bone destruction and fracture repair has been established; however, few studies have been conducted to examine the involvement of COX-2 in maintaining bone mineral density and bone micro-architecture. In this study, we examined bone morphology in multiple trabecular and cortical regions within the distal and diaphyseal femur of 4-month-old wild-type and COX-2-/- mice using micro-computed tomography. Our results demonstrated that while COX-2-/- female mice had normal bone geometry and trabecular microarchitecture at 4 months of age, the male knockout mice displayed reduced bone volume fraction within the distal femoral metaphysis.

Fluid flow increases type II collagen deposition and tensile mechanical properties in bioreactor-grown tissue-engineered cartilage.

A novel parallel-plate bioreactor has been designed to apply a consistent level of fluid flow-induced shear stress to tissue-engineered articular cartilage in order to improve the matrix composition and mechanical properties and more nearly approximate to that of native tissue. Primary bovine articular chondrocytes were seeded into the bioreactor at high densities (1.7 x 10(6) cell/cm2) without a scaffold and cultured for two weeks under static, no-flow conditions.

Inducible regulation of Runx2-stimulated osteogenesis.

Ex vivo gene therapy is a promising approach to orthopedic regenerative medicine. These strategies typically focus on the constitutive overexpression of osteogenic factors to induce osteoblastic differentiation and matrix mineralization. However, the unregulated production of osteoinductive molecules has also resulted in abnormal bone formation and tumorigenesis.

Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.

Unlabelled: A murine segmental femoral bone graft model was used to show the essential role of donor periosteal progenitor cells in bone graft healing. Transplantation of live bone graft harvested from Rosa 26A mice showed that approximately 70% of osteogenesis on the graft was attributed to the expansion and differentiation of donor periosteal progenitor cells. Furthermore, engraftment of BMP-2-producing bone marrow stromal cells on nonvital allografts showed marked increases in cortical graft incorporation and neovascularization, suggesting that gene-enhanced, tissue engineered functional periosteum may improve allograft incorporation and repair.

Effects of Runx2 genetic engineering and in vitro maturation of tissue-engineered constructs on the repair of critical size bone defects.

Genetic and tissue engineering strategies are being pursued to address the clinical limitations of current bone grafting materials. Based on our previous work demonstrating that overexpression of the Runx2 osteoblastic transcription factor and in vitro construct maturation synergistically enhanced in vivo mineralization in an ectopic site (Byers et al., Tissue Eng 2004;10:1757-1766), we examined the effects of these two parameters on the repair of critical size bone defects.

Biological effects of rAAV-caAlk2 coating on structural allograft healing.

Structural bone allografts often fracture due to their lack of osteogenic and remodeling potential. To overcome these limitations, we utilized allografts coated with recombinant adeno-associated virus (rAAV) that mediate in vivo gene transfer. Using beta-galactosidase as a reporter gene, we show that 4-mm murine femoral allografts coated with rAAV-LacZ are capable of transducing adjacent inflammatory cells and osteoblasts in the fracture callus following transplantation.

Variations in matrix composition and GAG fine structure among scaffolds for cartilage tissue engineering.

Objective: To compare matrix composition and glycosaminoglycan (GAG) fine structure among five scaffolds commonly used for in vitro chondrocyte culture and cartilage tissue engineering.

Design: Bovine articular chondrocytes were seeded into agarose, alginate, collagen I, fibrin and polyglycolic acid (PGA) constructs and cultured for 20 or 40 days. In addition to construct DNA and sulfated GAG (sGAG) contents, the delta-disaccharide compositions of the chondroitin/dermatan sulfate GAGs were determined for each scaffold group via fluorophore-assisted carbohydrate electrophoresis (FACE).

Trabecular bone microdamage and microstructural stresses under uniaxial compression.

The balance between local remodeling and accumulation of trabecular bone microdamage is believed to play an important role in the maintenance of skeletal integrity. However, the local mechanical parameters associated with microdamage initiation are not well understood. Using histological damage labeling, micro-CT imaging, and image-based finite element analysis, regions of trabecular bone microdamage were detected and registered to estimated microstructural von Mises effective stresses and strains, maximum principal stresses and strains, and strain energy density (SED).

Synergy between genetic and tissue engineering: Runx2 overexpression and in vitro construct development enhance in vivo mineralization.

Tissue engineering has emerged as a promising strategy to generate bone-grafting substrates. These approaches, however, are limited by an insufficient supply of committed osteoprogenitor cells and dedifferentiation of osteogenic cells during in vitro culture. To address these limitations, we engineered bone marrow stromal cells to constitutively express the osteoblastic transcription factor Runx2/Cbfa1, using retroviral gene delivery.

3-D computational modeling of media flow through scaffolds in a perfusion bioreactor.

Media perfusion bioreactor systems have been developed to improve mass transport throughout three-dimensional (3-D) tissue-engineered constructs cultured in vitro. In addition to enhancing the exchange of nutrients and wastes, these systems simultaneously deliver flow-mediated shear stresses to cells seeded within the constructs. Local shear stresses are a function of media flow rate and dynamic viscosity, bioreactor configuration, and porous scaffold microarchitecture.

Estrogen prevents bone loss through transforming growth factor beta signaling in T cells.

Estrogen (E) deficiency leads to an expansion of the pool of tumor necrosis factor (TNF)-producing T cells through an IFN-gamma-dependent pathway that results in increased levels of the osteoclastogenic cytokine TNF in the bone marrow. Disregulated IFN-gamma production is instrumental for the bone loss induced by ovariectomy (ovx), but the responsible mechanism is unknown. We now show that mice with T cell-specific blockade of type beta transforming growth factor (TGFbeta) signaling are completely insensitive to the bone-sparing effect of E.

Microcomputed tomography imaging of skeletal development and growth.

Skeletogenesis is an exquisitely orchestrated and dynamic process, culminating in the formation of highly variable and complex mineralized structures that are optimized for their function. While cellular and molecular biology studies have provided tremendous recent progress toward understanding how patterns of bone formation are regulated, high resolution imaging techniques such as microcomputed tomography (micro-CT) can provide complementary quantitative information about the progressive changes in three-dimensional (3-D) skeletal morphology and density that occur during early skeletal development and postnatal growth. Furthermore, recently developed in vivo micro-CT systems promise to be a powerful and efficient tool for noninvasively monitoring normal skeletogenesis, as well as for evaluating the effects of genetic or environmental manipulation.

Runx2/Cbfa1-genetically engineered skeletal myoblasts mineralize collagen scaffolds in vitro.

Genetic engineering of progenitor and stem cells is an attractive approach to address cell sourcing limitations associated with tissue engineering applications. Bone tissue engineering represents a promising strategy to repair large bone defects, but has been limited in part by the availability of a sustained, mineralizing cell source. This study examined the in vitro mineralization potential of primary skeletal myoblasts genetically engineered to overexpress Runx2/Cbfa1, an osteoblastic transcriptional regulator essential to bone formation.

Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury.

Transgenic mouse models are increasingly being used to investigate the functions of specific growth factors or matrix proteins to design therapeutic strategies for controlling blood vessel growth. However, the available methodologies for evaluating angiogenesis and arteriogenesis in these models are limited by animal size, user subjectivity, the power to visualize the three-dimensional vessel networks, or the capability to employ a vigorous quantitative analysis. In this study, we employed contrast-enhanced microcomputed tomography imaging to assess collateral development after induction of hindlimb ischemia in the mouse.

Quantitative microcomputed tomography analysis of mineralization within three-dimensional scaffolds in vitro.

Synthetic and naturally derived scaffold biomaterials in combination with osteogenic cells or bioactive factors have the potential to serve as bone graft substitutes. Porous poly(l-lactide-co-dl-lactide) (PLDL) scaffolds with mechanical properties comparable to trabecular bone and an oriented, interconnected porosity designed to enhance internal mass transport were recently developed. In this study, PLDL scaffolds were seeded with rat calvarial or rat stromal cells and cultured up to 8 weeks in media containing osteogenic supplements.

Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro.

Cellular activity at the center of tissue-engineered constructs in static culture is typically decreased relative to the construct periphery because of transport limitations. We have designed a tissue culture system that perfuses culture medium through three-dimensional (3D) porous cellular constructs to improve nutrient delivery and waste removal within the constructs. This study examined the effects of medium perfusion rate on cell viability, proliferation, and gene expression within cell-seeded 3D bone scaffolds.

Repair of calvarial defects with customised tissue-engineered bone grafts II. Evaluation of cellular efficiency and efficacy in vivo.

We have demonstrated in Part I of this study [see Schantz, J.-T., et al.

Bone formation on tissue-engineered cartilage constructs in vivo: effects of chondrocyte viability and mechanical loading.

Interactions between bone and cartilage formation are critical during growth and fracture healing and may influence the functional integration of osteochondral repair constructs. In this study, the ability of tissue-engineered cartilage constructs to support bone formation under controlled mechanical loading conditions was evaluated using a lapine hydraulic bone chamber model. Articular chondrocytes were seeded onto polymer disks, cultured for 4 weeks in vitro, and then transferred to empty bone chambers previously implanted into rabbit femoral metaphyses.

Microarchitectural and mechanical characterization of oriented porous polymer scaffolds.

Biodegradable porous polymer scaffolds are widely used in tissue engineering to provide a structural template for cell seeding and extracellular matrix formation. Scaffolds must often possess sufficient structural integrity to temporarily withstand functional loading in vivo or cell traction forces in vitro. Both the mechanical and biological properties of porous scaffolds are determined in part by the local microarchitecture.