Key Engineering Materials Vols. 361-363

Abstract: Osteoinductivity of hydroxyapatite (HA) was investigated using uncommitted pluripotent mouse stem cells, C3H10T1/2 in an in vitro differentiation assay. HA exhibited impressive ability to induce expression of osteo-specific genes in C3H10T1/2, including alkaline phosphatase (ALP), type I collagen (COL1) and osteocalcin (OCN); compared with its insignificant up-regulation of the same genes in osteoblast-like cells, Saos-2. HA osteoinductivity exhibited in C3H10T1/2 was comparable to that of a bone morphogenetic protein (BMP) with reference to up-regulating osteo-specific genes except the core binding factor 1 (Cbfa1, Runx). This result implies a difference in osteogenic induction pathway initiated by HA and BMP. HA osteoinductivity was also demonstrated in the stem cells culture using conditioned medium derived from cells cultured on HA substrates. The medium exhibited excellent ability to up-regulate ALP without the presence of HA and BMP. The result suggests that the HA can interact with the cells and generate potent inductive substance released into the medium. Such substance in turn is able to induce uncommitted cells to differentiate into the osteolineage.

Abstract: According to the gene repertoire, distinct morphology and the organisation of extracellular matrix, osteoblast development was identified as a series of stages, proliferation, differentiation, matrix deposition, matrix maturation and mineralization. Each of these stages required tightly regulated and functionally coupled expression of genes related to the transcription factors and bone matrix. In this paper, we identify the effects of OCP to the differentiation of osteoblasts from the point of view of differentiation sequence development. Osteogenic medium (Ost MEM) mainly regulated the osteocalcin (OC) mRNA expression in the first week of culture. As culture continued to 24 days, OCP crystal assemblies became the main regulator. This shift in the role that OCP and Ost MEM played in regulation may reflect different biological functions of OC in Ost MEM induced regulation and OCP crystals induced regulation. The up-regulated OC mRNA expression by OCP crystal assemblies may function as a signal to coordinate the activities of osteoblasts and osteoclasts instead of inducing mineralisation at the end of the differentiation sequence of osteoblasts. By comparing the modified expression pattern observed on the OCP crystal particles with the patterns of differentiation sequences, it was found that BMSCs colonising OCP crystal assemblies from day 7 to day 24 matched the process of differentiation in the early stages of matrix deposition. The gene expressions of BMSC cultured in the osteogenic medium (positive control) corresponded with the process from matrix deposition to mineralisation. Hence, the differentiation process of BMSCs on OCP crystal assemblies was different from that found on the positive control. BMSCs could differentiate to osteoblasts that would function as a regulator for osteoclast activities.

Abstract: Globular-shaped beta-tricalcium phosphate (β-TCP) granules were synthesized using a unique dropping slurry method and the biological response was analyzed by implantation into the rat femur. Two kinds of globular-shaped β-TCP granules, composed of either rod-shaped particles or conventional non-rod-shaped particles were synthesized, implanted into the left femurs of 8-week-old female Wistar rats, and analyzed histologically. Commercially available β-TCP granules composed of conventional non-rod-shaped particles were also implanted as a control experiment. Four weeks after the operation, part of each implant was already resorbed and the resorbed area was replaced by newly formed bone tissue and bone marrow. Eight weeks after the operation, the resorption and replacement were advanced in each implant. At 12 weeks after the operation, residual globular-shaped β-TCP granules composed of rod-shaped particles were resorbed into compact petrotic bone tissue almost completely. The formation of less compact petrotic bone tissue was observed in specimens implanted with globular-shaped β-TCP granules composed of conventional non-rod-shaped particles. Commercially available β-TCP granules composed of conventional non-rod-shaped particles were mostly resorbed into trabecular bone and the formation of compact petrotic bone tissue was rarely observed. These data suggested that both the unique spherical shape and microstructure of β-TCP particles affected bone-forming activity after the operation.

Abstract: The nacre layer from the pearl oyster shell is considered as a promising osteoinductive biomaterial. Nacre contains one or more signal molecules capable of stimulating bone formation. The identity and the mode of action of these molecules on the osteoblast differentiation were analyzed. Water-soluble molecules from nacre were fractionated according to dialysis, solvent extraction and reversed-phase HPLC. The activity of a fraction composed of low molecular weight molecules in the mineralization of the MC3T3-E1 extracellular matrix was investigated. Mineralization of the preosteoblast cells was monitored according to alizarin red staining, Raman spectroscopy, scanning electron microscopy and quantitative RT-PCR. Molecules isolated from nacre, ranging from 50 to 235 Da, induced a red alizarin staining of the preosteoblasts extracellular matrix after 16 days of culture. Raman spectroscopy demonstrated the presence of hydroxyapatite in samples treated with these molecules. Scanning electron microscopy pictures showed at the surface of the treated cells the occurence of clusters of spherical particles ressembling to hydroxyapatite. The treatment of cells with nacre molecules accelerated expression of collagen I and increased the mRNA expression of Runx2 and osteopontin. This study indicated that the nacre molecules efficient in bone cell differentiation are certainly different from proteins, and could be useful for in vivo bone repair.

Abstract: Several kinds of biomaterials are known to possess osteoinductive ability without bone inductive substances in ectopic implantation. β-TCP is one such material that has been reported to exhibit this ability in a canine model. In addition, prostaglandin E2 has been proved to accelerate osteogenesis in a rodent model, and one of its receptors EP4 has been considered to play a particularly important role. We examined that the EP4 agonist accelerates β-TCP-induced osteoinduction in a canine model. The results suggested that the EP4 agonist accelerated not only osteoinduction but also osteoclastogenesis prior to bone formation.

Abstract: The present study was designed to investigate whether bone morphogenetic protein-2 (BMP-2) adsorbed onto octacalcium phosphate (OCP) and hydroxyapatite (HA) surfaces influences osteoblastic cell differentiation. Osteoblastic cell line, UAMS32, were cultured on plastic plate that was coated of synthetic OCP and HA. BMP-2 was pre-adsorbed on these calcium phosphates at 4°C or room temperature for 24h. The cells more proliferated on the BMP-2 pre-adsorbed OCP and HA than that of the controls (OCP and HA coating without BMP-2). ALP activities were higher on HA than on OCP when BMP-2 were pre-adsorbed at 4°C than at room temperature. The results suggest that pre-adsorption of BMP-2 in different temperature affects osteoblastic cell differentiation, most probably through different adsorption state of BMP-2 on this calcium phosphate.

Abstract: Surfaces of sintered hydroxyapatite (HAp) were treated with four kinds of acids, hydrochloric acid (HCl), nitric acid (HNO3), sulfuric acid (H2SO4) and hydrogen peroxide (H2O2). On these acid-treated HAp surfaces, osteoblast-like MC3T3-E1 cells were cultured for 20 days. The matrix mineralization was observed in HCl-treated HAp and HNO3-treated HAp, but not in non-treated HAp, H2SO4-treated HAp and H2O2-treated HAp. Gene expressions of Bglap2 encoding osteocalcin and Akp2 encoding alkaline phosphatase were higher in HCl-treated HAp and HNO3-treated HAp than those in the others. These results indicate that not only hydrochloric acid but also nitric acid improves performance of HAp to mature osteoblast.

Abstract: The aim of this study is to investigate in vivo local BMP-2 PK and bone induction in two bioceramics blocks (HAp, β-TCP), based on different composition and surface structures. We estimated the in vivo release profile of 125I–labeled BMP-2 and bone induction of hard tissues histologically. β-TCP is more effective for both BMP-2 retention and bone induction, compared to HAp, in the ectopic model.

Abstract: Various bioactive calcium phosphates such as hydroxyapatite (HA) and carbonate apatite (CA) bone substitutes have been studied because of the biocompatibility and osteoconductivity when implanted into bone defects. In this study, the interaction between bioceramics and osteoclast-like cell using the cell-line such a RAW264 was examined for the investigation of the important factors of the osteoclastic responses. From the results, the possibility of effectiveness by surface geometry and chemical property means solubility was suggested. Moreover, it was considerable that the CA induced much stronger responses to osteoclast-like cells than the HA.

Abstract: In this study the degradation behaviour of pure hydroxyapatite (HA), pure tricalcium phosphate (β-TCP) and four biphasic calcium phosphate ceramics was studied to gain information about the influence of the phase composition on this property with the aim to tailor individually adapted bone substitute materials. The chemical dissolution of each ceramic composition was measured by its release of calcium ions into a buffered solution. With decreasing HA content in the ceramics the degradation rate increased. Cell experiments were carried out with stimulated osteoclast-like RAW 264.7 cells. Using biochemical, genetic and microscopic analysis, the differentiation of the cells on the ceramic samples was studied. The monocytic precursor cells differentiated into osteoclast-like cells on all ceramics. The strongest cell differentiation into osteoclast-like cells was found on ceramics with HA/β-TCP ratios of 80/20, 60/40 and 40/60. Cells on these ceramics had many nuclei and the largest cell size. As a result of resorption, lacunas were found on all ceramics except β-TCP. All these experimental results proved the influence of the phase composition on degradation and resorption of calcium phosphate ceramics. Biphasic calcium phosphate ceramics with HA/β-TCP ratios of 80/20 and 60/40 exhibited the most promising properties to serve as synthetic bone substitute materials because for integration in the physiological bone remodeling process the implanted bone substitute materials should have optimized dissolution and resorption properties.