Key Engineering Materials Vols. 330-332

Abstract: The mRNA expression of Cbfa1 and osteocalcin gene induced by calcium phosphate ceramics (Ca/P) were quantitative analyzed according to real-time RT-PCR method in this work. C2C12 cells were co-culture with four kinds of porous Ca/P ceramics for 2 and 5d without adding other growth factors. The four kinds of Ca/P ceramics were pure hydroxyapatite (HA) sintered at 1250°C and HA/TCP with a ratio of 60/40 sintered at 1100°C (HT1), 1200°C (HT2) and 1250°C (HT3) respectively. Real-time RT-PCR analysis found the Ca/P ceramics induced positive expression of Cbfa1 and osteocalcin in C2C12 cells, After 5 days culture, Cbfa1 and osteocalcin showed obvious higher expression compared with that in 2 days. Cbfa1 and osteocalcin expression in BCP was much higher than HA, and the expression level of osteocalcin was HT1>HT2>HT3>HA. Our results showed that Ca/P ceramics alone were sufficient to induce C2C12 cells to osteoblastic differentiation and the sinter temperature and phase composition of Ca/P ceramics could affect their osteoindctive capacity significantly.

Abstract: Osteoclasts isolated from rabbits were cultured on zinc-containing tricalcium phosphate (ZnTCP) disks with zinc contents of 0.316 and 0.633 wt%, and on β-tricalcium phosphate (TCP) disks with nearly identical porosities, grain sizes and surface roughnesses. ZnTCPs directly suppressed the resorption activity of mature osteoclasts by enhancing apoptosis. We hypothesized that resorbing osteoclasts attached to ZnTCP locally accumulate zinc ions within the space defined by the clear zone during resorption, which in turn leads to apoptosis induction, even though the change in chemical composition of the culture medium is very small.

Abstract: The purpose of this study was to examine if the application of titanium-reinforced expanded polytetrafluoroethylene(TR-ePTFE) membrane combined with bovine bone mineral(BBM) soaked in inorganic polyphosphate promotes exophytic bone formation in rabbit calvaria. For this purpose, a total of 8 rabbits were used, and rectangular decorticated calvaria sites were created using a round carbide bur. In the control group, rectangular parallelepiped-shaped TR-ePTFE membranes (RPTPMs) were filled with BBM soaked in saline and placed on the decorticated sites and fixed with metal pins. In the experimental groups, RPTPMs were filled with BBM soaked in 4%, 8% and 16% inorganic polyphosphate prior to fixing with metal pins. Animals were sacrificed at 4 and 8 weeks after surgery, and new bone formation was assessed by histomorphometric as well as statistical analysis. The results indicated that at 8 weeks, all the experimental groups demonstrated exophytic bone formation. At 8 weeks, the 8% polyphosphate group revealed the most new bone formation (p<0.05). On the basis of these findings, we conclude that inorganic polyphosphate has a promoting effect on bone regeneration, possibly by enhancing osteoinductivity of the decorticated wound area and osteoconductivity of the carrier material, but not much as we expected.

Abstract: This study investigated the utility of genetically modified cell line for fast and non-destructive cytotoxicity evaluation of biomaterials. The EGFP(enhanced green fluorescence protein)-expressing plasmid pcDNA-EGFP was constructed, and electroporated into ROS 17/28 osteoblastic cells to generate an EGFP-labeled stable cell line, ROS-EGFP. This genetically modified cell line provided two unique opportunities to qualitative and semi-quantitative evaluation of cell growth on biomaterials without destruction of samples. Firstly, utilizing the fluorescence of EGFP expressed in the cells, the viability state of cells on biomaterials was evaluated using a fluorescent light microscope. Secondly, the proliferation of cells on biomaterials, which was identified by MTT assay，was demonstrated according to the microscopically counted fluorescent cell numbers. From the results, it could be concluded that the ROS-EGFP cell line was an effective tool to trace the fate of cells on biomaterials and to evaluate the biocompatibility of biomaterials to cell growth in vitro.

Abstract: The purpose of this study was to evaluate the effect of HA/TCP biphasic ceramic on osteoinduction. HA/TCP ceramic cylinders with Φ8mm*10mm were prepared. The cylinder materials were stained by the calcein and alizarin red respectively, which were implanted into pigs’ dorsal muscle. The samples were harvested at 1, 2 and 3 month post-implantation. At 3 month post-implantation, the new bone tissues were observed in the inner pores of cylinders, which were stained by calcein or alizarin red at the same time, it was also found that the osteoblast in the margin of new bony tissue were stained by calcein and alizarin red. This result indicated that HA/TCP biphasic ceramic are osteoinductive in muscles of pigs, and participate in the process of new bone formation and calcification.

Abstract: Gene expression profile of osteoblast-like cells cultured on dense disk materials and porous materials of calcium phosphate ceramics was constructed from DNA microarray analyses. The profile revealed that gene expression patterns of porous materials were significantly different from those of dense disk materials. The porous materials had a capacity to induce expressions of genes involved in osteoblast differentiation, while dense disk materials regulated gene expressions related to osteoclastogenesis.

Abstract: Demineralized bone matrix (DBM)-calcium phosphate cement (CPC) composites were subjected to cellular test of osteogenic potentials and implantation in animal model. The expression of osteogenic marker gene from mouse preosteoblast cell line MC3T3-E1 adhered to the DBM-CPC composite was much higher than plain CPC. In addition, the DBM-CPC composite implanted nude mice revealed osteoinduction between the implanted composite and adjacent tissues, whereas the plain CPC induced osteoconduction.

Abstract: Hydroxyapatite (HA) ceramics is one of the most widely used bone substitute in clinics. Limited information is available concerning how HA ceramics may affect osteoprotegerin (OPG) and RANKL expression. In this study, osteoblastic-like SaOS-2 cells were grown on HA ceramics sintered at different temperature for 3 and 6 days, RANKL and OPG mRNA expression were analyzed with quantitative in situ hybridization (QISH) technique. Result showed that SaOS-2 grown on HA ceramics sintered at 800°C expressed higher RANKL mRNA than on other two HA ceramics after 3 days’ culture. No significant difference in OPG expression on different surfaces was detected after 3 days and 6 days culture. This result suggests that HA sintered at low temperature tend to induce more bone remodeling after implantation.

Abstract: A novel collagen sponge that can protect cell leakage during cell seeding was developed by wrapping all the surfaces except the upside of a collagen sponge with membrane that has pores smaller than cell. The collagen sponge was used for three-dimensional culture of human bone marrow-derived mesenchymal stem cells (MSCs). The cells adhered to the collagen, and proliferated to fill the spaces in the sponge. The cell seeding efficiency was higher than 95%. The MSCs cultured in the collagen sponge in the chondrogenic induction medium supplemented with TGF-β3 and BMP6 expressed genes encoding type II collagen, SOX9 and aggrecan. HE staining indicated the round morphology of differentiated cells and the extracelluler matrices were positively stained by safranin O and toluidine blue. Type II collagen and cartilage proteoglycan were detected by immunostaining with anti-type II collagen and anti-cartilage proteoglycan. These results suggest the chondrogenic differentiation of MSCs. The collagen sponge facilitated cell seeding and chondrogenic differentiation of MSCs, and will be useful for cartilage tissue engineering.

Abstract: Cranial sutures produce new bone at the sutural edges of the bone fronts in response to external stimuli. Little is known regarding the mechanism of osteogenesis in cranial sutures. Ets1 and Cbfa1 are two important osteogenic transcription factors regulating the differentiation and maturation of osteoblasts. But their function in cranial sutures is not still elucidated. We have investigated the gene expression of Ets1 and Cbfa1 in rat’s calvarial sutural osteoblast-like cells under a single period of mechanical strain. The cells were isolated from the cranial suture of SD rats and cultured in vitro, and subjected to a single 40 minutes mechanical strain using a four-point bending apparatus. The gene expression patterns of Ets1 and Cbfa1 were examined by RT-PCR. Both mRNA levels of Ets1 and Cbfa1 have increased significantly within 6 and 12 hours respectively after mechanical strain were applied, and the increase returned to control level thereafter. However, Ets1 and Cbfa1 exhibited different temporal expression patterns: Ets1 expressed immediately after the mechanical loading and reached the maximum transcription at 0.5h; whereas Cbfa1 experienced a latency period first, then increased slowly within 2 hours, and reached the maximum transcription at 6 h. The maximum transcription of Cbfa1 was about 2.58 fold of that of Ets1. Ets1and Cbfa1 may play different roles in regulating bone matrix protein expressions in osteoblast-like cells during suture distraction and their function is time-dependent. High frequency distraction (>2times/24h) is favourable to the maximal expression of the two genes.