Advanced Biomaterials VI

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Authors: Hong In Shin, K.H. Kim, Inn Kyu Kang, Kyung Sik Oh
Abstract: To improve the potential of osteogenic repair, we developed macroporous biphasic hydroxyapatite-tricalcium phosphate (HA-TCP) ceramic and evaluated its efficiency as a scaffold for tissue engineered bone regeneration, which allows for appropriate cellular attachment and proliferation with osteogenic differentiation by evaluating ectopic bone formation ability after the implantation of cell-matrix construct in the skid mice subcutaneous pouches for 3 weeks. The macroporous biphasic HA-TCP ceramic matrix, with an average porosity of 86% and 200 µm mean pore size, provided favorable conditions for the attachment of cultured bone marrow derived osteoblastic cells along its inner surfaces in a filed up pattern and the active proliferation of them. The implanted cell-matrix constructs in the subcutaneous pouches induced favorable ectopic bone formation without any remarkable inflammatory reactions. These findings suggest that the biphasic HA-TCP ceramic matrix with macroporous structure has excellent biocompatibility, and that it allows for favorable cellular attachment with the acceleration of cellular proliferation and osteogenic differentiation support as well. Thus, with the controlled biodegradability, the biphasic HA-TCP ceramic may be a promising scaffold for tissue engineered bone regeneration technology.
Authors: Hong In Shin, H.M. Ryoo, Suk Young Kim
Abstract: Bone regeneration with demineralized bone preparations has demonstrated its potential in grafting procedures in surgical disciplines of both medicine and dentistry. To improve the effectiveness in preparation and osteogenic regeneration, we developed a simple and rapid demineralizing method for osteogenic allograft preparation and evaluated its osteogenic effect using an ectopic bone formation assay. The rat diaphyseal cortical bones were demineralized in heated 0.6N HCl at 60° C for 10 to 30 mins using a controlled-heat ultrasonic cleaner, washed with sterilized distill water, and dehydrated with graded alcohol after 30% H2O2 treatment. The prepared grafts were implanted in rat dorsal subcutaneous pouches for 1-3 weeks and then the harvested tissue samples were prepared for routine light microscopic observation. The allografts demineralized with 60°C HCl for more than 20 mins in an ultrasonic condition were completely demineralized and effectively induced ectopic formation without specific pathologic findings. These findings suggest that demineralization with 60°C HCl for around 20 mins using a controlled-heat ultrasonic cleaner and dehydration with graded alcohol after short treatment with 30% H2O2 is a very simple but effective osteogenic allograft preparation method with minimal antigenicity and sterilizing effects.
Authors: Masaya Yamamoto, Yoshitake Takahashi, Yasuhiko Tabata
Abstract: The objective of this study is to examine feasibility of biodegradable gelatin hydrogels incorporating bone morphogenetic protein (BMP)-2 in inducing bone regeneration at a bone defect of non-human primates and rabbits considering their BMP-2 release profiles. As a result, controlled release by the hydrogel system enabled BMP-2 to induce successful bone regeneration in non-human primates even at the BMP-2 dose as low as that for rabbit case (0.034 mg of BMP-2/cm3 of hydrogel).
Authors: Dong Lim Seol, Won Hee Jang, Sung Jae Lee, Young Il Yang
Abstract: The goal of this study was to investigate effects of fibrin reinforcement of collagen sponges on fibroblasts-mediated contraction and in vivo tissue regeneration, especially angiogenesis. Human dermal fibroblasts (HDFs)-populated collagen sponges reinforced with or without fibrin were cultivated via the free-floating method in vitro. They were then evaluated using xenogeneic implantation into nude mice. The HDFs-populated collagen sponges reinforced with fibrin exhibited significantly decreased HDFs-mediated contraction in vitro (p<0.05). Microvascular and cellular densities of the collagen sponges were significantly higher with fibrin than without (p<0.01). Cell ingrowths, neovascularization, and deposition of ECM matrix were more evenly distributed in the fibrin-reinforced collagen matrices. The results demonstrated that fibrin reinforcement of porous collagen sponges can reduce cell-mediated contraction in vitro while enhancing functional integration with surrounding tissue in vivo.
Authors: L.R. Liu, L.H. Zhang, F.J. Wang, Q.Q. Zhang
Abstract: Collagen-HA scaffolds were prepared by a phase-separation technique, cross-linked by formaldehyde or carbodiimide(EDC)/N-hydroxysuccinimide(NHS), which is also used to covalently attach chondroitin sulfate(CS) to the scaffold. BMP was incorporated into the scaffold by adsorption. The scaffolds were characterized by SEM and ESCA. Rat mesenchymal stem cell(MSCs) was seeded into the scaffold and cultured to form a cell/scaffold construct. The ectopic osteoinduction of the scaffolds were evaluated in vivo. The results show that the scaffolds are porous with three-dimensionality, have good cell compatibility and osteoinduction. Attachment of CS can improve the adhesion and differentiation of cells.
Authors: Yan Fei Tan, Ling Li Zhang, Xin Lai He, Wei Qiang Xiao, Hong Song Fan, Xing Dong Zhang
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.
Authors: Y. Yu, Jin Biao Chen, J.L. Yang, D.A.F. Morgan, W.R. Walsh
Abstract: Deep-frozen morselized human bone grafts showed osteoconductivity and osteoinductivity when implanted into tibial window defects of nude rats. The osteoconductivity was assessed by measuring the total area of newly formed bone bridged by the implanted bone grafts in the entire defect area. The osteoinductivity was evidenced by the presence of active osteoblast-like cells and new bone formation around the implanted bone grafts, which were surrounded by soft tissues distant from the host cortex. Gamma irradiation at the doses of 15 or 25 kGray reduced the osteoconductivity (ANOVA and LSD tests, p<0.05) at 3 weeks post operation. The 25 kGray group had a significantly lower level of new bone formation compared with the 0 and 15 kGray groups. The evidences of osteoinductivity were only noted in the 0 and 15 kGray groups. Our data indicate that 25 kGray gamma irradiation reduces the osteoconductive and osteoinductive properties of the morselized human bone graft.
Authors: Chun Lin Deng, Ji Yong Chen, Hong Song Fan, Xing Dong Zhang
Abstract: A revised dynamic immersion device was designed, and the effects of flowing speed of SBF in inner pores of ceramics on the formation of apatite in the pores were investigated in this study. The results showed some crystals were overlaid on the walls of inner pores. The crystal shape changed with flow rate of RSBF. When flow rates of RSBF were 2 ml/min, some stamen-like crystals formed on the inner walls of ceramics. In the ceramics in RSBF with flow rate of 5ml/min, besides more stamen -like crystals, a thin layer of fine deposits was overlaid on the walls of inner holes and the surface of the stamen -like crystals. But when the flow rate was 8 ml/min, only a thick layer of fine deposits on the walls of inner pores could be found. Energy dispersive spectroscopy (EDS) and Fourier Transform Infrared spectroscopy (FTIR) results revealed the deposits obtained in this study are carbonate hydroxyapatite (CHA).
Authors: Jie Weng, Bo Feng, Min Wang, Xing Dong Zhang
Abstract: The biomimetic approach of mineralization in vitro is adopted to investigate systematically the nucleation and growth of bone-like apatite on the surface of biomaterials such as bioceramics, metals and polymers, and those chemically surface-treated. The simulated environment is kept isothermic at the human body temperature of 36.5C with three kinds of simulated physiological fluids. The experimental results show that (1) inherent properties of biomaterials determine their bioactivity and the different crystalline structure of same materials results in the difference in bioactivity; (2) the bioactivity can effectively be improved by the surface treatment of biomaterials via chemical methods and by the addition of bioactive particles in a polymer matrix; (3) the bone-like apatite, nucleated and grown in the simulated body fluid with the same ion concentrations to that of the human plasma, possesses the same composition, structure and morphology despite of matrixes; (4) the difference in bioactivity with biomaterials is indicated by the different time for bone-like apatite to nucleate and to grow on their surfaces.
Authors: C.Y. Bao, P. Li, Yan Fei Tan, Y. Cao, Xiao Yan Lin, Chun Lin Deng, Xu Dong Li, Guo Min Ou, W.C. Chen, P. Gong, Hong Song Fan, Xing Dong Zhang
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.

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