Onset Of Bone Formation Research Articles

Spatiotemporal Control Strategies for Bone Formation through Tissue Engineering and Regenerative Medicine Approaches.

Global increases in life expectancy drive increasing demands for bone regeneration. The gold standard for surgical bone repair is autografting, which enjoys excellent clinical outcomes; however, it possesses significant drawbacks including donor site morbidity and limited availability. Although collagen sponges delivered with bone morphogenetic protein, type 2 (BMP2) are a common alternative or supplement, they do not efficiently retain BMP2, necessitating extremely high doses to elicit bone formation. Hence, reports of BMP2 complications are rising, including cancer promotion and ectopic bone formation, the latter inducing complications such as breathing difficulties and neurologic impairments. Thus, efforts to exert spatial control over bone formation are increasing. Several tissue engineering approaches have demonstrated the potential for targeted and controlled bone formation. These approaches include biomaterial scaffolds derived from synthetic sources, e.g., calcium phosphates or polymers; natural sources, e.g., bone or seashell; and immobilized biofactors, e.g., BMP2. Although BMP2 is the only protein clinically approved for use in a surgical device, there are several proteins, small molecules, and growth factors that show promise in tissue engineering applications. This review profiles the tissue engineering advances in achieving control over the location and onset of bone formation (spatiotemporal control) toward avoiding the complications associated with BMP2.

Combination of bone morphogenetic protein-2 plasmid DNA with chemokine CXCL12 creates an additive effect on bone formation onset and volume

Bone morphogenetic protein-2 (BMP-2) gene delivery has shown to induce bone formation in vivo in cell-based tissue engineering. In addition, the chemoattractant stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is known to recruit multipotent stromal cells towards its release site where it enhances vascularisation and possibly contributes to osteogenic differentiation. To investigate potential cooperative behaviour for bone formation, we investigated combined release of BMP-2 and SDF-1α on ectopic bone formation in mice. Multipotent stromal cell-seeded and cell-free constructs with BMP-2 plasmid DNA and /or SDF-1α loaded onto gelatin microparticles, were implanted subcutaneously in mice for a period of 6 weeks. Histological analysis and histomorphometry revealed that the onset of bone formation and the formed bone volume were both enhanced by the combination of BMP-2 and SDF-1α compared to controls in cell-seeded constructs. Samples without seeded multipotent stromal cells failed to induce any bone formation. We conclude that the addition of stromal cell-derived factor-1α to a cell-seeded alginate based bone morphogenetic protein-2 plasmid DNA construct has an additive effect on bone formation and can be considered a promising combination for bone regeneration.

Runx2 Protein Expression Utilizes the Runx2 P1 Promoter to Establish Osteoprogenitor Cell Number for Normal Bone Formation

The Runt-related transcription factor, Runx2, is essential for osteogenesis and is controlled by both distal (P1) and proximal (P2) promoters. To understand Runx2 function requires determination of the spatiotemporal activity of P1 and P2 to Runx2 protein production. We generated a mouse model in which the P1-derived transcript was replaced with a lacZ reporter allele, resulting in loss of P1-derived protein while simultaneously allowing discrimination between the activities of the two promoters. Loss of P1-driven expression causes developmental defects with cleidocranial dysplasia-like syndromes that persist in the postnatal skeleton. P1 activity is robust in preosteogenic mesenchyme and at the onset of bone formation but decreases as bone matures. Homozygous Runx2-P1(lacZ/lacZ) mice have a normal life span but exhibit severe osteopenia and compromised bone repair in adult mice because of osteoblastic defects and not increased osteoclastic resorption. Gene expression profiles of bone, immunohistochemical studies, and ex vivo differentiation using calvarial osteoblasts and marrow stromal cells identified mechanisms for the skeletal phenotype. The findings indicate that P1 promoter activity is necessary for generating a threshold level of Runx2 protein to commit sufficient osteoprogenitor numbers for normal bone formation. P1 promoter function is not compensated via the P2 promoter. However, the P2 transcript with compensatory mechanisms from bone morphogenetic protein (BMP) and Wnt signaling is adequate for mineralization of the bone tissue that does form. We conclude that selective utilization of the P1 and P2 promoters enables the precise spatiotemporal expression of Runx2 necessary for normal skeletogenesis and the maintenance of bone mass in the adult.

Orthotopic Location Has Limited Benefit from Allogeneic or Autologous Multipotent Stromal Cells Seeded on Ceramic Scaffolds

Improvement of tissue-engineered grafts is still a challenge in the field of regenerative medicine. Using multipotent stromal cells (MSCs), which have immunosuppressive qualities in an allogeneic situation, off-the-shelf implants can be created. This study compared allogeneic and autologous MSCs at an orthotopic (L1 transverse process model) and ectopic (intramuscular) implantation location in 2-year-old goats. Further, the possible additional effect of platelet-leukocyte gel (PLG) as a source of growth factors on bone formation was investigated. For the orthotopic implantation, cassettes were implanted in nine goats bilaterally on the lumbar transverse processes, either with PLG-seeded or with plasma-seeded constructs. To assess the onset of bone formation, fluorochromes were administered at weeks 3, 5, and 9. Their incorporation in newly formed bone indicated that seeded cells enhanced bone formation in the first weeks. Nevertheless, after 16 weeks no beneficial effects of cells were found in the cassettes in contrast to the ectopic location. No effect of PLG on bone formation was shown at either location. Finally, we show no significant difference in bone formation between autologous and allogeneic MSCs, an important finding when considering the use of allogeneic cells as an off-the-shelf component in tissue-engineered bone in goats.

Bone neogenesis in domes made of expanded polytetrafluoroethylene: efficacy of rhBMP-2 to enhance the amount of achievable bone in rats.

For bone reconstructive purposes, it would be a great advantage to be able to gain bone without grafting. In experimental studies, barrier membranes have been used to accomplish this, however, with limited efficacy. In this study, the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the early onset of bone formation, as well as on the final amount of achievable bone, was investigated in an experimental osteo-neogenesis model. In 60 adult rats, dome-shaped barrier membranes made of expanded polytetrafluoroethylene (Gore-Tex membrane), with an inside volume of approximately 60 mm3, were placed on the left parietal bone. The domes were pretreated according to four different alternatives: (1) filled with autogenous blood only (n = 15); (2) filled with 5 microg of rhBMP-2 in an absorbable collagen sponge carrier (n = 15); (3) filled with 15 microg of rhBMP-2 in absorbable collagen sponge carrier (n = 15); or (4) filled with the absorbable collagen sponge carrier only (n = 15). The animals treated according to each alternative were then divided into three equal groups with five rats in each, and subsequently killed after 3, 6, or 12 weeks. The amount of bone formed within the domes was evaluated by light microscopy and computer-assisted image analysis. It was found that the amount of newly formed bone could be enhanced by approximately 100 percent by simultaneous implantation of rhBMP-2, irrespective of dose. The early onset of bone formation was, however, not affected by the rhBMP-2 supplementation. This finding was interpreted as being due to the delivery system used, because as long as the carrier was still present, no significant difference between the treatment groups was observed. The bone formed in domes with carrier implantation, with or without rhBMP-2, displayed more marrow spaces in comparison to controls. The combined treatment with barrier membranes and local delivery of rhBMP-2 may be a useful tool in reconstructive surgery, for instance replacing onlay grafting, especially when a more delicate anatomy is necessary, because membranes can be shaped in multiple ways.

Ultrastructural localization and biochemical characterization of vitronectin in developing rat bone.

This study has used light and electron microscope immunohistochemical and biochemical methods to localize and characterize vitronectin in early bone formation of developing rat mandible with rabbit antimurine vitronectin IgG. Developing jaws of foetuses were collected at embryonic day 15 (day 15) to day 18 from pregnant Wistar rats. After aldehyde fixation, specimens with and without osmium post-fixation were dehydrated and embedded in paraffin, Spurr's resin or LR gold resin for morphological and immunohistochemical examinations. At the light microscope level, in day 15 samples, positive vitronectin immunostaining was observed in small elongated areas of intercellular matrix and osteoblasts. Concomitant with initiation of matrix mineralization at day 16, vitronectin staining was similarly observed in small elongated areas containing intercellular matrix and osteoblasts but not clearly detected in fully mineralized bone matrix. The same staining profile was observed at days 17 and 18. At the ultrastructural level, immunogold particles were clearly detected over unmineralized matrix and cisterns of the rough-surfaced endoplasmic reticulum and the Golgi apparatus of osteoblasts as well as over demineralized bone matrix at day 16-18. In order to assess the presence of vitronectin in the mineral phase, mineral-binding bone proteins were extracted from fresh day 18 specimens using a three-step technique: 4M guanidine HCl (G1 extract), aqueous EDTA without guanidine HCl (E extract), followed by guanidine HCl. Subsequent Western blot analysis of sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis revealed that the antibodies produced only a single band at an M(r) of approximately 73000 in both G1 and E extracts, indicating the presence of vitronectin in the mineralized bone matrix. These results indicate that, at the onset of bone formation, osteoblasts synthesize and release vitronectin, which is subsequently incorporated into the bone matrix and becomes a specific component of bone tissues. The observation of vitronectin in these critical stages of bone formation suggests that it may be involved in the regulation of bone formation.

Expression of interstitial collagenase is restricted to skeletal tissue during mouse embryogenesis.

Collagenases are thought to be involved in physiological and pathological processes that require extracellular matrix remodeling. Using the in situ hybridization technique, we describe the expression of interstitial collagenase gene during mouse embryogenesis between E6.5 and E17. We demonstrate that interstitial collagenase expression is exclusively detected in one event, namely the onset of bone formation. Transcripts accumulate in hypertrophied chondrocytes, found in the mature cartilaginous matrix of long-bone growth plates or ribs, and in osteoblasts and/or in endothelial cells that have migrated into the shafts of developing long bones. The expression of the tissue inhibitor of metalloproteinases (TIMP-2) gene precedes the expression of interstitial collagenase in developing bones. These data suggest that interstitial collagenase plays a specific role in bone development and that the tight regulation of its activity during development is achieved not only by post-translational mechanisms with TIMPs, as previously suggested, but also at the transcriptional level.

Vertebral development of the chick embryo during days 3-19 of incubation.

Chick embryo vertebral development has been studied during the period from 3 to 19 days of incubation. Whole mount preparations stained with alcian blue for cartilage and alizarin red for bone show the vertebral bodies as cartilaginous at 5 days, with dorsal spinous processes at 6 1/2 days, the onset of bone formation at the ventral and dorsal surfaces of the bodies at 13 days, ossification extending dorsally within the neural arches at 13 1/2 days, and bone development occurring throughout the vertebrae at 16 days. Descriptions at each time period center on the thoracic vertebrae with occasional inclusion of adjacent lower cervical and upper lumbar vertebrae. Histologic development is correlated with the Hamburger-Hamilton stages, which are based on external characteristics. The notochord and neural tube are well developed by 3 days and surrounded by sclerotome, myotome, and dermatome cells. Cartilage formation in the perinotochordal region of the vertebral body is seen at 5 days and precedes development of the neural arches and spinous processes. Separate centers of chondrocyte hypertrophy occur in the body (9 days), the lateral neural arches and the dorsal spinous process. Bone formation is under way at 13 days, beginning in the vertebral bodies. Intramembranous periosteal bone formation is seen adjacent to internal regions of chondrocyte hypertrophy. Vascular invasion of hypertrophic chondrocyte regions occurs, but the mechanism of endochondral ossification differs from that of mammals. The cartilage is resorbed by multinucleated chondroclasts and marrow round cells. Clumps of growth plate cartilage cells and matrix are surrounded occasionally by newly synthesized bone, but invasion of individual hypertrophic chondrocyte lacunae by vessels with bone deposition by accompanying osteoblasts on single trabeculae of cartilage does not occur. 3H-thymidine autoradiography shows high uptake at 3 and 5 days in the germinal neuroepithelial cells of the neural tube (spinal cord) and notochord. By 7 days, notochordal uptake is markedly diminished, and no uptake of isotope occurs from 8 days onward. Spinal cord uptake is highest in the first 8 days but persists in lessened amounts to 19 days. From 5 days onward, both undifferentiated mesenchymal cells and differentiated chondrocytes show positive 3H-thymidine uptake, but labeling is never seen in hypertrophic chondrocytes.

Gene expression during endochondral bone development: Evidence for coordinate expression of transforming growth factor β1 and collagen type I

Subcutaneous implantation of demineralized bone particles (DBP) into rats induces the formation of a bone ossicle by a tightly controlled sequence of chondro- and osteo-inductive events which are directly comparable to those which occur in normal endochondral bone development. Although the morphological and biochemical sequence associated with endochondral bone formation in this model has been well characterized, to date little information is available as to the gene regulation by which these events occur. To examine the expression of genes in this system, RNA was isolated from implants every 2 days over a time course spanning 3 to 19 days after implantation of DBP into rats. Cellular levels of mRNA transcripts of cell-growth-regulated and tissue-specific genes were examined by slot blot analysis and compared to the morphological changes occurring during formation of the ossicle. Analysis of the mRNA levels of histone H4 and c-myc, markers of proliferative activity, revealed several periods of actively proliferating cells, corresponding to 1) production of fibroprogenitor cells (day 3), 2) onset of bone formation (day 9), and 3) formation of bone marrow (day 19). The mRNA levels of collagen type II, a phenotypic marker of cartilage, peaked between days 7 and 9 post-implantation, corresponding to the appearance of chondrocytes in the implant, and rapidly declined on day 11 (to 5% of maximum value) when bone formation was observed. The peak mRNA levels of collagen type I, found in fibroblasts and osteoblasts, occurred first with the onset of bone formation (days 7-10) and again during formation of bone marrow (day 19). This study has demonstrated that the temporal patterns of mRNA expression of cartilage type II and bone type I collagens coincide with the morphological sequence in this model of endochondral bone formation. Further, the mRNA levels of transforming growth factor beta 1 (TGF beta) were compared to those of collagen types I and II; a direct temporal correlation of TGF beta mRNA levels with that of collagen type I was found throughout the developmental time course. This observation of a tightly coupled relationship between TGF beta and type I collagen mRNA levels is consistent with a functional role for TGF beta in extracellular matrix production during in vivo bone formation.