Yan Fei Tan

Preparation of Electrospun PLA Nanofiber Scaffold and the Evaluation In Vitro

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.

Compare of Electrospinning PLA and PLA/β-TCP Scaffold in Vitro

In recent years, electrospinning process is gradually applied in producing tissue-engineering scaffold. In this study, we chose polylacticacid(PLA) and β-tertiary calcium phosphate(β-TCP) as raw materials to fabricate PLA/β-TCP biodegradable composite scaffold by electrospinning process. The characteristics of the scaffold and effect of the scaffolds to cell proliferation and cell adhesion was studied. Compare with pure PLA scaffold, blendingβ-TCP in the spinning process of the scaffold could improve the properties of the scaffold, especially the hydrophilicity and the proliferation and adhesion of cells, this means that the material is more potential to be used as tissue engineering scaffolds.

Experimental Study on Construction of Vascularized Bone Graft with Osteoinductive Calcium Phosphate Ceramics In Vivo

This study was to aimed to explore a new method for fabricating a bone substitute, which is constructed of vascularized bone graft with osteoinductive calcium phosphat e cer mics in vivo. A total of 32 small and 16 big ceramic cylinders were prepared, and bone-like apatite l yer was formed on the surface and the pore walls of ceramics in revised simulate d body fluid (RSBF). The smaller ceramics cylinders were implanted in dorsal muscles of dogs to evaluate the osteoinductive capacity. The bigger cylinders were mixed with fresh bone tissue and were implanted near a branch of femoral blood vessel in the dog’s leg muscles to construct vascularized bone gr afts. After implantation for 6 and 12 weeks, the specimens were harvested. The specimens were evalua t d by blood vessel staining, histological observation, tetracycline fluorescence labeling, Tc–MDP SPECT and mechanical tests. The results showed that bone formation was found in all the samples a nd bone grafts had good blood supply. This result indicated that this kind of calcium phosphate cera mi s had a good osteoinductivity and that vascularized bone grafts could be constructed wi th osteoinductive calcium phosphate ceramics in vivo. Introduction Bone defect is a common disease in clinic and to date, there is no s atisfactory method to treat the segmental bone defect. At present, autograft, allograft, xenograft and alloplasm materials could be used for repairing bone defects. Autograft is the best bone substitute, but it causes a secondary wound. Allograft and xenograft may bring immune response and infectious dise ase. Alloplasm material have been developed as a major bone substitute [1]. An ideal bone substitute should possess good biological and mechanical properties. The biological properties of an i deal bone substitute include biocompatibility, osteoconductivity, osteoinductivity, osteogenesis capabilit y. In 1980s, the appearance of tissue engineering brought about opportunities for repairing bone defects. Great achievements have been obtained in this field in the last 20 years [2] . But there are still some challenges ahead for bone tissue engineering [3]. Until now, no tissue e ngine ring bone composed of scaffold and cultured cells in vitro has been permitted to be applied in clinic [4]. Calcium phosphates ceramics are frequently used as bone substitute m a erials because of their similarity to the mineral phase of bone, absence of antigenicity, a nd excellent osteoconductivity. Some studies have proved that calcium phosphate ceramics with special str u ture could induce bone formation in soft tissue [5]. Based on the exploration of the osteoinducti ve mechanism, Zhang [6] proposed a new concept of bone tissue engineering in vivo, i.e., bone grafts could be constructed directly with osteoinductive biomaterials singly or osteoinductive biom aterials mixed with fresh bone tissue in some areas of the body. This study was conducted to explore a new method for fabricating bone substitute, which constructed vascularized bone graft with osteoinductive c alcium phosphate in vivo. Materials and Methods Preparation of the ceramics Calcium phosphate ceramics was prepared by H 2O2 foaming method and sintered at 1250oC for 3 hours with wet-synthetic calcium phosphate powde r. The chemistry of Key Engineering Materials Online: 2003-12-15 ISSN: 1662-9795, Vols. 254-256, pp 801-804 doi:10.4028/www.scientific.net/KEM.254-256.801

In Vitro and In Vivo Biocompatibility of Nano-Hydroxyapatite/Collagen Composites

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.

In Vitro Simulation of Calcium Phosphate Crystallization from Dynamic Revised Simulated Body Fluid

A dynamic device was designed to study the formation of bonelike apatite on the inner surface of biphasic porous calcium phosphate in RSBF in vitro. The ceramics were examined with scanning electron microscopy (SEM), energy dispersive spectroscop y (EDS) and Infrared spectroscopy (IR). Spherical amorphous crystal on the surface pore walls and sheet-shaped crystals on the inner pore walls of the ceramics were found. According to co -cultured with bone marrow stromal cell (BMSCs), the cell response to the samples was investigated with SEM and MTT assay. Introduction Formation of bone-like apatite is one of the important subjects in os teoinductivity of bioceramics. The in vivo process of apatite formation is usually simulated in vitro by precipitation of calcium phosphates from supersaturated calcium and phosphate-containing solutions. D ynamic method, introduced by Duan et al. [1, 2] recently, is closer to physiological condition than static imme rsion assay. However, the pH value of simulated body fluid (SBF) changes with time after immersion, in which calcium phosphates precipitate to form non-stoichiometric hyd yoxyapatite. In addition, the simulation in vitro is different from physiological condition in vivo in that CO2 is produced in the process of metabolism in vivo . To simulate the behavior of calcium phosphate ceramics veritably, a modified CO2 supplying device was designed to adjust the pH value and to enhance bu ffer a ility of solution and porous biphase calcium phosphate ceramics were immersed in a revised body fluid (R-SBF) introduced by Kim et al. . Subsequently, cell biocompatibility was investigated by MTT test. Materials and Methods Biphasic porous calcium-phosphate ceramics (HA/ α-TCP=60/40) were prepared by foaming with H2O2. The cylindrical-shaped samples ( 4×8mm) were cleaned by ultrasonication. 4–4,5–dimethylthiazol-2-y1-2,-diphenyltetrazolium bromide (MTT) was purc hased from Sigma Chemical Co. (USA), and DMEM/F12 (1:1) medium was purchased from Hyclone Co (USA ). Experiments on the formation of bone-like apatite in revised simulate d body fluid (R-SBF) were performed at 37°C and a stable pH (7.4), which was controlled by filling CO2 gas into R-SBF storage tank at a suitable rate. R-SBF flowed at a rate of 2 m l/min by a peristaltic pump (Fig. 1). HA/α-TCP was immersed for 2 to 7 days in R-SBF. After that, sample s were removed, washed with distilled water for several times, then, dried at 50°C and split in halves. The microstructures of the samples were analyzed with scanning electron microscopy (SEM). The Key Engineering Materials Online: 2003-12-15 ISSN: 1662-9795, Vols. 254-256, pp 7-10 doi:10.4028/www.scientific.net/KEM.254-256.7

Quantitative Analysis of Core Binding Factor 1 (Cbfa1) and Osteocalcin in C2C12 Cells Induced by Ca/P Ceramics In Vitro

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.

Stress Analysis and Optimizing of Osteoinductive Ca-P Ceramics and Net-Cage-Structured Titanium Alloy in Dog Segmental Femoral Defect Reparation

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.

Experimental Study on the Osteoinduction of Calcium Phosphate Biomaterials In Vivo and the Capability of Supporting Osteoblast Proliferation In Vitro

The purpose of this study was to evaluate the response of osteoblasts to the 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. The ceramic cylinder with F5mm×8mm and slice with F10mm×1mm were prepared respectively. One thirds of the ceramics were formed bone-like apatite (BLA), and the surface of another one thirds 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 if 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 well attached to the surface and proliferated well in the group of collagen and HA/TCP. In untreated and BLA precipitated group, cells did not attach to the surface well. Osteoinduction was good in BLA precipitated group and the amount of bone formed was higher; in 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.

Wear of Twist Drills in ZrO2/CePO4 Composite Machining

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.

Gene Expression of C2C12 Cells on Calcium Phosphate Ceramic In Vitro

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.