Chun Lin Deng

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

Bone-Like Apatite Formation on Modified PCL Surfaces under Different Conditions

The PCL plates hydrolyzed by NaOH aqueous solutions and carboxylate groups were introduced onto the surfaces of specimen. Specimens were treated by CaCl2 and K2HPO4⋅3H2O under the normal-pressure condition and low-pressure of 103 Pa condition for 30min separately. Dense and uniform bone-like layers could be formed on the surface of specimens after mineralizing for less than 24h in simulated body fluids (SBF). The low-pressure condition could accelerate the formation of apatite layer.

The Effect of Organisms on Formation of Bone-Like Apatite on Porous Calcium Phosphate in Simulated Body Fluid

This research evaluated the effect of organisms on formation of bonel ike apatite on porous calcium phosphate, in simulated body fluid(SBF) at 37°C. Organisms such as chondroitin sulfate or collagen or citric acid or ATP were joined in SBF at certain concentration. The spectra of IR showed that characteristic peaks of CO 3 2appeared on the surface of ceramics. The SEM results indicated that the morphologies of the bonelike apatite formed in S BF with and without organism were: 1) different each other, and 2) the morphologies of the bonelike apa tite formed in SBF with different organism were also in contrast to each other. Introduction In vivo osteoinduction of bioceramics has attracted great interests in recent years. The formation of bonelike apatite is one of the important subjects in osteoinduction of bioc eramics. Chondroitin sulfate, collagen, citric acid and ATP are important organisms e xisting in plasma [1]. They play an important role in ossification through forming dissociated organisms a nd c lcium complexes in the surrounding body fluid. In vivo organic polymer, for example, chondroitin sulfat e, collagen, citric acid, and ATP usually controls the nucleation and crystal growth [2,3]. In order to investigate the effect of organisms on the formation of bone like apatite on porous calcium phosphate, we developed a methodology that utilises simulated body fluid. Materials and Methods Porous hydroxyapatite /tricalcium phosphate (HA/TCP:70/30) ceramics , the samples sized is 4 8mm, cylinder, cleaning out by ultrasonic. Experiments on formation of bonelike apatite in conventional Kokubos simula ted body fluid (SBF) were performed at 36.5°C and controlled pH7.4 [4]. Organisms, such as chondroitin sulfate, collagen, citric acid and ATP were joined in SBF according to 200mg .L. HA/TCP ceramics were immersed in SBF containing the organism and the SBF was changed eve ry two days. HA/TCP ceramics were taken out after 14 days. The microstructures of materials were analyzed by SEM. T he formation of hydroxycarbonate apatite layer on the surface was observed by Infrared spectroscopy (IR). Results and Discussion After HA/TCP ceramics were immersed in SBF for 14 days, the IR results showed that characteristic peaks of CO 3 , at 1416~1458cm approximately, appeared on the surface of the ceramics besides characteristic peaks of PO 4 3(1044~1090cm, 473 cm approximately) and OH Key Engineering Materials Online: 2003-05-15 ISSN: 1662-9795, Vols. 240-242, pp 81-84 doi:10.4028/www.scientific.net/KEM.240-242.81

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

Growth of Apatite in Bovine Serums on Porous HA/TCP Ceramics

Porous HA/TCP bioceramics were immersed in pure bovine serum to observe the growth and formation of apatite. HRTEM, FTIR, and SEM coupled with EDS were used for the characterization of immersed samples. SEM results showed that some beamed crystals formed on the surface of ceramics granules, and with postponement of immersion time, crystals extended and became bigger, strap-like crystals became sheet-like crystals. HRTEM observations indicated that new-formed crystals developed along axes direction according to parallel layers. IR spectrum showed CO3 2- characteristic peaks existed besides O-P-O and OH- characteristic peaks. EDS results showed that calcium and phosphor ratio was 1.95 (mol ratio). The results indicated that bovine serums were advantaged to bone-like apatite formation.

Apatite Formation on Porous HA/TCP in Animals’ Serums In Vitro

Porous HA/TCP bioceramics were immersed in pure dog serum to observe apatite formation. Deposited crystals were examined using SEM. Results showed that beamed sheet-like crystals formed on the surface of ceramics granules, and after postponement immersion time, crystals extended and became bigger. EDS and IR results suggested formed crystals were defect-calcium type carbonated hydroxyapatite. HRTEM photograph suggested formation process of new-formed crystals from non-crystal to crystal in serum. Directional organisms acted maybe as a template in process of crystals formation, so new crystals developed along certain direction.

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.

A Comparative Study Between Dynamic and Static Simulated Body Fluid Methods

In vitro method has often been used in the biodegradation/bioactivity evaluation of bioactive ceramics for its convenience and saving in time and outlay. The simulated body fluid (SBF) suggested by Kokubo was a good simulation of the osteoproduction environment in osseous tissue and has been proved to be a good method to study the bioactivity of biomaterials and the mechanism of bone bonding. But SBF is not a suitable method to research the osteoinduction of biomaterials. The results from SBF were not consistent with that from in vivo in muscle. The local ion concentration is the key factors to affect the nucleation and growth of apatite. In muscle the effect of body fluid flowing on local ion concentration cannot be ignored. A dynamic SBF suggested by these authors of this paper not only simulated the ion concentration of body fluid, but also simulated the effect of body fluid flowing on the local ion concentration near the surface or in biomaterials in muscle. The results from the dynamic SBF were in good agreement with that of the implantation experiments in muscle. The results from dynamic SBF showed that apatite only formed on the walls of macropores of the porous CaP, no apatite formed on the surface of both dense and porous CaP. The new bone only formed on the walls of macropores of porous CaP implanted in muscles, no apatite or osseous tissue could be found on the surfaces of both porous and dense CaP. The dynamic SBF preferably simulated the osteoinduction environment in non-osseous tissue and can be used in osteoinductivity evaluation of bioceramics.

Effect of Phase Components on Apatite Formation on the Surface of Ca-P Bioceramics

Dense hydroxyapatite (HA) and hydroxyapatite/tricalcium (HA/TCP) were immersed in Fast calcified solution (FCS) at 37°C in constant temperature culture box for 7 days. It was observed that the phase components of dense ceramics affected the composition, crystallinity and phase components of formed apatites on the surface of ceramics. It is a mixture of hydroxyapatite with a strong preferential crystallographic direction of 002 and octacalcium phosphate (OCP) on the surface of HA/TCP ceramics, whereas only an HA phase was detected on the surface of HA ceramics.

Influence of Dynamic Flow Speed on Bonelike Apatite Formation in Porous Calcium Phosphate Ceramic in RSBF

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).