Papers by Author: Yan Fei Tan

Abstract: With good biocompatibility, collagen is often used in cartilage tissue engineering. Collagen/alginate composite was hoped to improve the poor mechanical property of pure collagen but the biocompatibity was decreased. In this study, hydroxyapatite (HA) particles were used to get collagen/alginate/HA (CAHA) composite film to enhance the bioactivity properties. The bioactivity of the composite was investigated by in vitro co-culture with chondrocytes. During the 6-day cell culture in vitro, the composite showed a significant improvement in promoting proliferation and maintaining morphology/phenotype of the chondrocytes over collagen/alginate composite by MTT, SEM, fluorescent and immunohistochemical assays. Cytocompatibility and cytoviablility of CAHA even come up to that of collagen film alone. The results indicated that the composite film may provide an appropriate environment for the proliferation and maintaining the morphology and phenotype of chondrocytes and have a potential clinical application in the cartilage tissue engineering field.

Abstract: Machinable ZrO2/CePO4 composite was fabricated. ZrO2/CePO4 and mild steel materials were machined with tungsten-cobalt carbide twist drills. The material removal characteristic was analyzed by consideration of tool wear in different stock removals. Wear of twist drills was observed with SEM. Wear widths on major flank were measured with microscope. The experimental results show when ZrO2/CePO4 was drilled, wear widths on flank were much bigger comparing to mild steel drilling processes. The wear of twist drills occurred on major flank, minor flank and on chisel edge. Wear on minor flank is a unique characteristic in drilling of ZrO2/CePO4 ceramics. There exist obvious scratch remarks on tool wear. The tool wear results from abrasive wear, adhesive wear and oxidation wear.

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 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: The purpose of this study was to develop a feasible technique for bone reparation and further explore the possible applications of Ca-P ceramics in segmental load-bearing bone reparation. HA/TCP ceramics sintered at 1250oC were fabricated into tube-like columns of Φ15mm×30mm with a central canal of Φ4mm. Bone-like apatite was precipitated on the ceramics before implantation. 12 male dogs were used in this study, and a 30mm long segmental bone defect was made in the middle of one femur of each dog. Supported by the fixation of net-cage-structured TC4, the osteoinductive Ca-P ceramic cylinder was used to repair the segmental defect in dog femur. Stress was analyzed by ANSYS. The morphology recovery, function restoration, gait analyses and bone regeneration were evaluated. After implantation at 2, 4 and 8 months, the specimens were harvested respectively. The specimens were evaluated with morphological observation and mechanical testing. Stress analysis showed that the thickness of TC4 net cage was 0.3mm. The morphology recovery of the experimental animal was good and function was restored after 2 months gradually. Aided by stress analysis and by optimizing the design and fixation of implants, Ca-P materials with excellent osteoinductivity could be applied in repairing segmental bone defects.

Abstract: The purpose of this study was to evaluate the response of osteoblasts to calcium phosphate with different surface modification, and to evaluate the osteoinductive capabilities of these biomaterials. 60HA/40α-TCP ceramics sintered at 1250oC was applied in this study. A ceramic cylinder with F5mm×8mm and slice with F10mm×1mm were prepared respectively. One third of the ceramics was used to form bone-like apatite (BLA), and the surface of another one third was modified with collagen. Osteoblasts (1×106/ml) were co-cultured with the three kinds of thin slices for 12h, 24h and 48h. SEM observation was applied to evaluate whether the surface modification and BLA formation could affect the attachment and proliferation of osteoblast in vitro. The three kinds of cylinder samples were implanted in dog muscle to evaluate their differences in osteoinduction. Cells grew in multi-layers and attached to the surface and proliferated well in the collagen and HA/TCP group. In the untreated and BLA precipitated groups, cells did not attach to the surface well. Osteoinduction was good in the BLA precipitated group and the amount of bone formed was higher; in the untreated group and collagen-treated group, no bone formation was observed in the tested period. This result indicated that the scaffold used in cell-materials composites in vitro and that in osteoinductive material based tissue engineering in vivo was not same.

Abstract: The osteoinduction of Calcium Phosphate (CaP) had been proved and generally been investigated by in vivo implantation. However, the mechanism of the osteoinductivity was not clear and it was difficult to judge the osteoinductivity in vitro. In this study, Mouse C2C12 cell line, a kind of myoblast precursor cell, was employed to co-culture with CaP. The induction of cell differentiation by materials was tested by MTT method, fluorescence observation, especially the mRNA expression of Osteocalcin, Type I collagen and Fibronectin by RT-PCR. It was founded that C2C12 cells could be induced to expression osteocalcin when growth on the surface of the HA/TCP ceramics. At the same time, the ceramics with different composition and sintering temperature seemed to induce difference expression level of the related genes. The results proved that phase composition was one of the most important factors in the regulation of bone-related genes. This study provided a potential model to evaluate the osteoinductivity of CaP ceramics in vitro.

Abstract: Glutaraldehyde was increasingly used to improve the stability of the collagen-based biomaterials as cross-linking. To investigate in vitro cytotoxicity of Hydroxyapatite/Collagen (HA/COL) composite with glutaraldehyde (GA) as cross-link reagent, which was abbreviated as cHA/COL, the mouse fibroblasts L929 cell was selected. The method of MTT colorimetric assay was adopted to evaluate the availability of cells. After cultured for 24h、 48h, the attachment and morphology of L929 cell on the surface of this material were observed by scanning electron microscopy (SEM) and fluorescent microscopy. MTT analysis indicated L929 cells cultured on cHA/COL had a perfect growth capacity. Cells proliferated well and cell colonization was observed on the materials by scanning electron microscopy and fluorescent microscopy. It demonstrated that cHA/COL composite exhibited good cytocompatibility and might be suitable as bone substitutes. The result provided the evidence for further animal experiment in vivo.

Abstract: A nano-grade hydroxyapatite/collagen composite was prepared by an in situ synthesis technique from calcium nitrate, diammoniun hydrogen phosphate, and a cowhide collagen sol at low temperature. XRD and TEM analyses of the composite indicated that crystals formed in the collagen fibril matrix were nanohydroxyapatite with low crystallinity. Biocompatibility of the composite was evaluated by in vitro cytotoxicity test and in vivo genotoxicity and sensitization test. No mutagenic activity of the composite was observed in mouse micronucleus tests. No evidence of dermal sensitization of the composite was found in guinea pig maximization tests. The results from a filter diffusion test indicated that the composite did not induce a cytotoxic behavior. All these results suggest that the composite has excellent biocompatibility.

Abstract: A electrospinning process to prepare soft tissue engineering scaffold was introduced in this study. This kind of scaffold was composed with ultrathin fiber and characterized with high porosity, well-interconnected pores and high surface-to-volume ratio. Biodegradable polylaticacid (PLA) was used to spin the scaffold and the scaffold was evaluated in vitro by analysis the microscopic structure, porosity, mechanical property, especially cytocompatibility. The results indicated that the electrospun PLA scaffold showed good cytocompatibility and the tensile property of electrospun scaffold was similar to human’s soft tissue. It could be expected that the electrospun scaffold would be potential in soft tissue engineering or soft tissue repair.