Current approaches to the development of regenerative therapies have been influenced by our understanding of embryonic development, stem cell biology, and tissue engineering technology. The ultimate goal of regenerative therapy is to develop fully functioning bioengineered organs which work in cooperation with surrounding tissues to replace organs that were lost or damaged as a result of disease, injury, or aging. Here, we report a successful fully functioning tooth replacement in an adult mouse achieved through the transplantation of bioengineered tooth germ into the alveolar bone in the lost tooth region. We propose this technology as a model for future organ replacement therapies. The bioengineered tooth, which was erupted and occluded, had the correct tooth structure, hardness of mineralized tissues for mastication, and response to noxious stimulations such as mechanical stress and pain in cooperation with other oral and maxillofacial tissues. This study represents a substantial advance and emphasizes the potential for bioengineered organ replacement in future regenerative therapies.regenerative therapy ͉ transplantation

Donor organ transplantation is currently an essential therapeutic approach to the replacement of a dysfunctional organ as a result of disease, injury or aging in vivo. Recent progress in the area of regenerative therapy has the potential to lead to bioengineered mature organ replacement in the future. In this proof of concept study, we here report a further development in this regard in which a bioengineered tooth unit comprising mature tooth, periodontal ligament and alveolar bone, was successfully transplanted into a properly-sized bony hole in the alveolar bone through bone integration by recipient bone remodeling in a murine transplantation model system. The bioengineered tooth unit restored enough the alveolar bone in a vertical direction into an extensive bone defect of murine lower jaw. Engrafted bioengineered tooth displayed physiological tooth functions such as mastication, periodontal ligament function for bone remodeling and responsiveness to noxious stimulations. This study thus represents a substantial advance and demonstrates the real potential for bioengineered mature organ replacement as a next generation regenerative therapy.

The purpose of this study is to evaluate the osteoconductivity of three different bone substitute materials: α-tricalcium phosphate (α-TCP), (β-TCP), and hydroxyapatite (HA), combined with or without simvastatin, which is a cholesterol synthesis inhibitor stimulating BMP-2 expression in osteoblasts. We used 72 Wistar rats and prepared two calvarial bone defects of 5 mm diameter in each rat. Defects were filled with the particles of 500-750 μm diameter combined with or without simvastatin at 0.1 mg dose for each defect. In the control group, defects were left empty. Animals were divided into seven groups: α-TCP, β-TCP, HA, α-TCP with simvastatin, β-TCP with simvastatin, HA with simvastatin, and control. The animals were sacrificed at 6 and 8 weeks. The calvariae were dissected out and analyzed with micro CT. The specimens were evaluated histologically and histomorphometrically. In α-TCP group, the amount of newly formed bone was significantly more than both HA and control groups but not significantly yet more than β-TCP group. Degradation of α-TCP was prominent and β-TCP showed slower rate while HA showed the least degradation. Combining the materials with Simvastatin led to increasing in the amount of newly formed bone. These results confirmed that α-TCP, β-TCP, and HA are osteoconductive materials acting as space maintainer for bone formation and that combining these materials with simvastatin stimulates bone regeneration and it also affects degradability of α-TCP and β-TCP. Conclusively, α-TCP has the advantage of higher rate of degradation allowing the more bone formation and combining α-TCP with simvastatin enhances this property.

The nature and tissue distribution of non-collagenous bone proteins synthesized by adult rat bone marrow cells, induced to differentiate in the presence of dexamethasone (DEX) and beta-glycerophosphate (beta-GP), was studied in vitro to determine the potential role of these proteins in bone formation. Northern hybridization analysis revealed a strong induction of bone sialoprotein (BSP) and osteocalcin in DEX-treated cultures, whereas the constitutive expression of secreted phosphoprotein I (SPP-1), type I collagen, SPARC, and alkaline phosphatase was stimulated 6-, 5-, 3-, and 2.5-told, respectively. Metabolic labeling of proteins showed that the sialoproteins (SPP-1 and BSP) were mostly secreted into the culture medium in the non-mineralizing (-beta-GP) cultures, but were the predominant non-collagenous proteins associated with the hydroxyapatite of the bone nodules in mineralizing cultures (+ beta-GP). Extraction of the tissue matrix with 4 M GuHCl and digestion of the demineralized tissue matrix with bacterial collagenase revealed that some BSP was also associated non-covalently and covalently with the collagenous matrix. SPP-1 was present in two distinct, 44 kDa and 55 kDa, forms in the conditioned medium of all cultures and was preferentially associated with the hydroxyapatite in the mineralizing cultures. In comparison, SPARC was abundant in culture media but could not be detected in de-mineralizing extracts of the mineralized tissue. Radiolabeling with [35SO4] demonstrated that both SPP-1 and BSP synthesized by bone cells are sulfated, and that a 35 kDa protein and some proteoglycan were covalently associated with the collagenous matrix in +DEX cultures. Labeling with [32PO4] was essentially confined to the sialoproteins; the 44 kDa SPP-1 incorporating significantly more [32PO4] than the 55 kDa SPP-1 and the BSP. These studies demonstrate that BSP and osteocalcin are only expressed in differentiated osteoblasts and that most of the major non-collagenous bone proteins associate with the bone mineral. However, some novel proteins together with some of the BSP are associated with the collagenous matrix where they can influence hydroxyapatite formation.

Osteopontin (OPN) is a prominent bone matrix protein that is synthesized by osteoblastic cells. To elucidate the function of OPN in bone we studied the regulated expression of the rat OPN protein during bone formation in vivo and in vitro. OPN mRNA is expressed by preosteoblastic cells early in bone formation, but the highest expression is observed in mature osteoblasts at sites of bone remodelling. A low-phosphorylated, 55-kDa form of OPN is produced by the preosteoblastic cells, whereas osteoblasts produce a highly phosphorylated, 44-kDa protein; the two forms of OPN corresponding to pp69 and pp62 in transformed rat cells. The synthesis of the 55-kDa OPN correlates with the formation of a 'cement' matrix that is synthesized prior to bone deposition, whereas the 44-kDa OPN synthesized by osteoblasts associates rapidly with hydroxyapatite, possibly regulating crystal growth, and may also provide a substratum for osteoclast attachment. Expression of OPN mRNA is upregulated by growth and differentiation factors (PDGF, EGF, TGF-beta and BMP-7/OP-1) and by mechanical stress, which promote bone formation, as well as by osteotropic hormones (retinoic acid and vitamin D3), which can promote bone resorption and remodelling. However, OPN mRNA is down-regulated by bisphosphonates, which abrogate bone resorption. Regulation of OPN expression is, therefore, consistent with a multiplicity of functions for OPN that involve specific structural motifs in both the synthesis and resorption of bone.

Abstract. Simvastatin, a cholesterol synthesis inhibitor, enhances BMP2 expression in osteoblasts. The purpose of the present study was to examine whether simvastatin stimulates bone regeneration when combined with calcium sulfate as a carrier. Critical-sized bone defects in rat calvaria were treated with calcium sulfate or with combination of 1 mg simvastatin and calcium sulfate. In the combination group, although the least amount of bone formation with intense soft tissue inflammation was observed at 2 and 4 weeks, remarkable bone formation was evident at 8 weeks. Conclusively, the combination of simvastatin and calcium sulfate stimulated bone regeneration in spite of the inflammatory response.

Abstract. Periosteum has been demonstrated to contain mesenchymal progenitor cells differentiating to osteoblasts, and both bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) may play important roles in cell-based approaches to bone regeneration. The purpose of this study was to evaluate the feasibility and efficacy of BMP-2 and/ or VEGF on periosteal cell differentiation to osteoblasts in vitro and ectopic bone formation in vivo. Human periosteum-derived cells were transfected with BMP-2, VEGF, BMP-2 + VEGF, or vehicle as a control by non-viral gene transfer and then cultured and implanted to nude mice intramuscularly. Real-time polymerase chain reaction analysis of the culture revealed that transgenes for BMP-2 and BMP-2 + VEGF induced more mRNA expression of alkaline phosphatase, collagen type I, and osteocalcin than VEGF and vehicle treatments; additionally, alizarin red S staining, alkaline phosphatase staining, and alkaline phosphatase activity were significantly higher in the BMP-2 + VEGF transgene than in the other versions. After implantation, ectopic bone was observed at 4 weeks and greatly increased at 8 weeks in all groups. In particular, the combination of BMP-2 and VEGF formed significantly more bone at 4 weeks, and VEGF transfection resulted in more blood vessels relative to the conditions without VEGF. Thus, VEGF might enhance BMP2-induced bone formation through modulation of angiogenesis.

When combined with alpha-TCP particles, 0.1 mg simvastatin is the optimal dose for stimulation of the maximum bone regeneration in rat calvarial defects without inducing inflammation and it could be applied as an effective bone graft material.