In Vitro Characterizations of PLLA/β-TCP Porous Matrix Materials and RMSC-PLLA-β-TCP Composite Scaffolds

To develop a novel degradable poly (L-lactic acid)/β-tricalcium phosphate (PLLA/β-TCP) bioactive materials for bone tissue engineering, β-TCP powder was produced by a new wet process. Porous scaffolds were prepared by three steps, I.e. Solvent casting, compression molding and leaching stage. Factors influencing the compressive strength and the degradation behavior of the porous scaffold, e.g. Weight fraction of pore forming agent-sodium chloride (NaCl), weight ratio of PLLA: β-TCP, the particle size ofβ-TCP and the porosity, were discussed in details. Rat marrow stromal cells (RMSC) were incorporated into the composite by tissue engineering approach. Biological and osteogenesis potential of the composite scaffold were determined with MTT assay, alkaline phosphatase (ALP) activity and bone osteocalcin (OCN) content evaluation. Results show that PLLA/β-TCP bioactive porous scaffold has good mechanical and pore structure with adjustable compressive strength needed for surgery. RMSCs seeding on porous PLLA/β-TCP composite behaves good seeding efficacy, biocompatibility and osteoinductive potential. Osteoprogenitor cells could well penetrate into the material matrix and begin cell proliferation and osteogenic differentiation. Osseous matrix could be formed on the surface of the composite after culturing in vitro. It is expected that the PLLA/β-TCP porous composites are promising scaffolds for bone tissue engineering in prosthesis surgery.     

Osteogenesis and Degradation Behavior of rhBMP-2/β-Tricalcium Phosphate Porous Composite Materials

Ultrafine β-tricalcium phosphate (β-TCP) powders with good crystalline structure were produced by a new wet process. Through bone tissue engineering approach, porous β-TCP ceramic was combined with recombined human bone morphogenetic proteins-2 (rhBMP-2) to develop a novel composite material. Osteogenesis capacity of the composite was investigated intramuscularly in rat with histological analyses and SEM examination. Pure β-TCP porous ceramic was investigated as the control. Results show that the composite materials possess good biocompatibility, biodegradation and strong osteogenesis capacity through inductive process after implantation. Material degradation began from 2 weeks post-implantation accompanying with the changing of pore structure, with the enwrapping and separation of materials by hyperplatic mesenchymal cells and fibroblast, and with the phagocytose reaction of multinucleated giant cells. Early in 72 h, immature cartilage could be found within novel composite; mature lamellar bone was induced to generate after 3 weeks. With strong osteoinduction capacity and controllable biodegradation, the novel rhBMP-2/β-TCP porous ceramic is expected to be a promising bone grafting substitute for bone tissue engineering.     

Hierarchical Micropore/Nanorod Apatite Hybrids In-Situ Grown from 3-D Printed Macroporous Ti6Al4V Implants with Improved Bioactivity and Osseointegration

The advent of three-dimensional(3-D) printed technique provides great possibility in the fabrication of customized porous titanium(Ti) implant. However, the bioinert property of the printed Ti poses an outstanding problem. Hybrid micro-arc oxidation and hydrothermal(MAO–HT) treatment on porous metals is able to produce multi-scaled hierarchical orthopedic implant, showing great potential for surface modification of 3-D printed implant. In this study, cylindrical porous Ti6Al4V(Ti64) scaffolds with pore size of 640 μm, porosity of 73% were 3-D printed by electron beam melting process, and their surfaces were left untreated or treated by a combined MAO–HT procedure. In vitro bioactivity was tested by immersion in simulated body fluid for different time points. Then, 12 scaffolds in each group were implanted into the femoral condyles of New Zealand rabbit for 8 weeks. Osseointegration was evaluated by qualitative and quantitative histological analysis, and the bone ingrowth features were probed by sequential fluorescent labeling at 3 and 6 weeks post-surgery. Following the MAO–HT treatment, the porous Ti64 scaffold was endowed with multi-scaled micro/nano-topographies and high amounts of Ca P on its surface.The treated scaffold exhibited drastically enhanced apatite forming ability compared with the untreated one. In vivo test revealed significantly that a higher amount of bone ingrowth and bone implant contact at the treated scaffold. The 2 types of scaffolds had different patterns of bone ingrowth: the treated scaffold exhibited a pattern of contact osteogenesis, by which bone formed directly on the treated implant surface, whereas bone formed distal to the implant surface of the untreated scaffold. MAO–HT treatment can significantly enhance the in vitro apatite-inducing ability and in vivo osseointegration capacity of 3-D porous Ti64 scaffold and may provide as a viable approach for the fabrication of bioactive 3-D printed porous implant for orthopedic applications.     

Ultrastructural Analysis on the Osteogenesis and Transformation of Calcium Phosphate Ceramics in Vivo

To study the osteogenesis and transformation process of calcium phosphate bioceramic in vivo, biodegradable porous β-tricalcium phosphate ceramics (β-TCP, φ5×8 mm) were implanted in the tibia of rabbits. β-TCP ceramics with surrounding bone tissue were retrieved and observed by SEM, TEM and EPMA every month after implantation.The results showed that osteogenesis was active and β-TCP ceramics bonded to bones directly. The new bones were forming and maturing as materials were continuously degrading, and materials were finally replaced by new bone. Parts of the materials were degraded, absorbed and recrystallized, while the rest were dispersed to the spongy bone and the Haversian lamella in an irregular arrangement, becoming incorporated into bone formation directly by remodeling the structure. Some β-TCP crystals cleaved along its (001) rhombohedral plane and formed lath-like crystals in vivo.     

Preparation and application of highly porous aerogel-based bioactive materials in dentistry

In this study, the possibility of preparation and application of highly porous silica aerogel-based bioactive materials are presented. The aerogel was combined with hydroxyapatite and p.tricalcium phosphate as bioactive and osteoinductive agents. The porosity of aerogels was in themesoporous region with a maximum pore diameter of 7,4 and 12.7 nm for the composite materials. The newly developed bioactive materials were characterized by scant electron microscopy. The in vitro biological effect of these modified surfaces was also tested on SAOS-2 osteogenic sarcoma cells by confocal laser scanning microscopy.     

EPMA Study of the Interface of β-Tricalcium Phosphate Ceramies in Vivo

Electron probe and X-ray energy spectrum were used to investigate the chemical composition of the interface between material and new bone after porous tricalcium phosphate ceramie implanted in tibia of rabbits. The element changes of the interface, the materials transformation and the situation of new bone formation at different implantation period were observed. The results showed that the carbon element content decreased gradually in new bone tissue, and the content of calcium and phosphor element increased by degrees with the implantation time. At the same time, calcium-phosphor ratio in the new bone kept a higher Ievel. New bone grew into the materials interior, material dispersed and degraded simultaneously. Both composition of materials and new bone tended to be consentient. Finally, the materials were substituted by new bone. After implantation, not only the materials itself dissolved and degraded partially, but also new bone formed on the outer and pore surface of β-TCP porous bioceramics, which     

EPMA Study of the Interface of β-Tricalcium Phosphate Ceramics in Vivo

Electron probe and X-ray energy spectrum were used to investigate the chemical composition of the interface between material and new bone after porous tricalcium phosphate ceramic implanted in tibia of rabbits. The element changes of the interface, the materials transformation and the situation of new bone formation at different implantation period were observed. The results showed that the carbon element content decreased gradually in new bone tissue, and the content of calcium and phosphor element increased by degrees with the implantation time. At the same time, calcium-phosphor ratio in the new bone kept a higher level. New bone grew into the materials interior, material dispersed and degraded simultaneously. Both composition of materials and new bone tended to be consentient. Finally, the materials were substituted by new bone. After implantation, not only the materials itself dissolved and degraded partially, but also new bone formed on the outer and pore surface of β-TCP porous bioceramics, which showed that the degradation products of lifeless calcium phosphate inorganic materials took part in constituting of new bone tissue.     

Improvement on compressive properties of lotus-type porous copper by a nickel coating on pore walls

The aim of this work is to understand the effect of a thin coating on the compressive properties of the porous metal. In our work, the uniaxial compressive behavior and the energy absorption properties of the lotus-type porous copper deposited with Ni coatings with thickness from 3.9 to 4.8 μm on pore walls were investigated. It is found that the Ni coating on pore walls shows a clear enhancement effect on compressive properties of the lotus-type porous copper, in which the specific yield strength and the energy absorption per unit mass at densification strain increase from 5.27 to 7.31 MPa cm~3 g~(-1) and from 11.50 to 18.21 J g~(-1) with the Ni coating, respectively. Furthermore, the enhancement appears to be insensitive to the coating thickness. It is considered that the resistance of the interface between the nickel coating and the pore walls to the dislocation slip plays an important role in the improvement on compressive properties of the lotus-type porous copper.     

Plasmid DNA-loaded asymmetrically porous membrane for guided bone regeneration

Although bone defects can be restored spontaneously,bone reconstruction with sufficient strength and volume continues to be a challenge in clinical practices.In recent years,the use of a variety of biomaterials with bioactivity has been attempted to compensate for this limitation.Herein,we fabricated a pDNA(encoding for BMP-2)-loaded asymmetrically porous polycaprolactone(PCL)/Pluronic F127 membrane as a bioactive guided bone regeneration(GBR)membrane,using a modified immersion-precipitation method.It was observed that the GBR membrane allows continuous release of pDNA for more than20 weeks.The pDNA was sufficiently transfected into human bone marrow stem cells(h BMSCs)without significant cytotoxicity and the gene-transfected cells showed prolonged synthesis of BMP-2.From in vitro osteogenic differentiation and in vivo animal studies,the effective induction of osteogenic differentiation of h BMSCs and enhanced bone regeneration by the pDNA-loaded asymmetrically porous PCL/Pluronic F127 membrane was observed,suggesting that the pDNA-loaded membrane as a bioactive GBR membrane can be an alternative therapeutic technique for effective bone regeneration.     

Anisotropic Porous Ti6Al4V Alloys Fabricated by Diffusion Bonding:Adaption of Compressive Behavior to Cortical Bone Implant Applications

In this work, porous Ti6Al4V alloys with 30%–70% porosity for biomedical applications were fabricated by diffusion bonding of alloy meshes. Pore structure was characterized by Micro-CT and SEM. Compressive behavior in the out-of-plane direction and biocompatibility with cortical bone were studied. The results reveal that the fabricated porous Ti6Al4V alloys possess anisotropic structure with square pores in the in-plane direction and elongated pores in the out-of-plane direction. The average pore size of porous Ti6Al4V alloys with 30%–70% porosity is in the range of 240–360 μm. By tailoring diffusion bonding temperature, aspect ratio of alloy meshes and porosity, porous Ti6Al4V alloys with different compressive properties can be obtained, for instance, Young's modulus and yield stress in the ranges of 4–40 GPa and70–500 MPa, respectively. Yield stress of porous Ti6Al4V alloys fabricated by diffusion bonding is close to that of alloys fabricated by rapid prototyping, but higher than that of fabricated by powder sintering and space-holder method. Diffusion bonding temperature has some effects on the yield stress of porous Ti6Al4V alloys, but has a minor effect on the Young's modulus. The relationship between compressive properties and relative density conforms well to the Gibson–Ashby model. The Young's modulus is linear with the aspect ratio, while the yield stress is linear with the square of aspect ratio of alloy meshes. Porous Ti6Al4V alloys with 60%–70% porosity have potential for cortical bone implant applications.     

Study on β-TCP Coated Porous Mg as a Bone Tissue Engineering Scaffold Material

Three-dimensional honeycomb-structured magnesium (Mg) scaffolds with interconnected pores of accurately controlled pore size and porosity were fabricated by laser perforation technique. Biodegradable and bioactive β-tricalcium phosphate (β-TCP) coatings were prepared on the porous Mg to further improve its biocompatibility, and the biodegradation mechanism was simply evaluated in vitro. It was found that the mechanical properties of this type of porous Mg significantly depended on its porosity. Elastic modulus and compressive strength similar to human bones could be obtained for the porous Mg with porosity of 42.6%-51%. It was observed that the human osteosarcoma cells (UMR106) were well adhered and proliferated on the surface of the β-TCP coated porous Mg, which indicates that the β-TCP coated porous Mg is promising to be a bone tissue engineering scaffold material.     

Study on β-TCP Coated Porous Mg as a Bone Tissue Engineering Scaffold Material

Three-dimensional honeycomb-structured magnesium (Mg) scaffolds with interconnected pores of accurately controlled pore size and porosity were fabricated by laser perforation technique. Biodegradable and bioactive β- tricalcium phosphate (β-TCP) coatings were prepared on the porous Mg to further improve its biocompatibility, and the biodegradation mechanism was simply evaluated in vitro. It was found that the mechanical properties of this type of porous Mg significantly depended on its porosity. Elastic modulus and compressive strength similar to human bones could be obtained for the porous Mg with porosity of 42.6%-51%. It was observed that the human osteosarcoma cells (UMR106) were well adhered and proliferated on the surface of the β- TCP coated porous Mg, which indicates that the β-TCP coated porous Mg is promising to be a bone tissue engineering scaffold material.     

Radiation synthesis of gelatin/CM-chitosan/β-tricalcium phosphate composite scaffold for bone tissue engineering

A series of biodegradable composite scaffolds was fabricated from an aqueous solution of gelatin, carboxymethyl chitosan (CM-chitosan) and β-tricalcium phosphate (β-TCP) by radiation-induced crosslinking at ambient temperature. Ultrasonic treatment on the polymer solutions significantly influenced the distribution of β-TCP particles. An ultrasonic time of 20 min, followed by 30 kGy irradiation induced a crosslinked scaffold with homogeneous distribution of β-TCP particles, interconnected porous structure, sound swelling capacity and mechanical strength. Fourier Transform Infrared Spectroscopy and X-ray Diffraction analysis indicated that β-TCP successfully incorporated with the network of gelatin and CM-chitosan. In vivo implantation of the scaffold into the mandible of beagle dog revealed that the scaffolds had excellent biocompatibility and the presence of β-TCP can accelerate bone regeneration. The comprehensive results of this study paved way for the application of gelatin/CM-chitosan/β-TCP composite scaffolds as candidate of bone tissue engineering material.     

Microstructure,mechanical property and corrosion behavior of co-continuous β-TCP/MgCa composite manufactured by suction casting

The co-continuous β-TCP/MgCa composite was fabricated by infiltrating MgCa alloy into porous β-TCP using suction casting technique. The microstructure, mechanical property and corrosion behaviors of the composite have been evaluated by means of scanning electron microscopy (SEM), X-ray diffraction (XRD), mechanical testing, electrochemical and immersion tests. It was shown that the composite structure was compact and the interfacial combination between MgCa alloy and β-TCP scaffold was very well. The composite had an ultimate compressive strength of (147 ± 13) MPa, which was near with the natural bone (2–180 MPa) and about 1000-fold higher than that of the original porous β-TCP scaffold, but it still retained over half of the strength of the MgCa bulk alloy. The electrochemical and immersion tests indicated that the corrosion resistance of the composite was better than that of the MgCa bulk alloy, and the corrosion rate of the MgCa matrix alloy was quicker than that of the porous scaffold for the composite. The corrosion products of the composite surface were mainly Mg(OH)₂, hydroxyapatite (HA) and Ca₃(PO₄)₂.     

骨组织工程用硅胶/掺锶β-磷酸三钙/硫酸钙复合多孔支架的制备与性能研究

以掺锶β-磷酸三钙/硫酸钙为原料,利用搅拌喷雾干燥法制备出掺锶β-磷酸三钙/硫酸钙复合小球,再将硅胶与制备的复合小球复合,通过在模具中堆垛聚集的方法,制备出硅胶/掺锶β-磷酸三钙/硫酸钙复合生物支架。采用XRD,SEM,FT-IR等方法分析制得复合多孔支架的成分、形貌以及结构特征,并研究复合生物支架的降解性、孔隙率、力学性能和细胞毒性等。结果表明:该复合多孔生物支架具有一定的不规则孔洞结构,小球与小球之间的孔隙约为0.2~1mm,而每个小球上也有大量的微孔,孔径在50~200μm之间,且平均孔隙率达到62%,基本能满足骨组织工程支架对孔隙率的要求;该复合多孔支架无细胞毒性,其降解周期约为80天,抗压强度约为0.1MPa,因此该支架在非承重骨组织修复方面具有良好的应用前景。     

β-CaSiO_3/丝素蛋白复合支架材料结构与性能的研究

利用冷冻干燥法制备了β-CaSiO_3/丝素蛋白复合支架材料,经XRD和FTIR分析表明复合支架中丝素的结构主要以β-折叠为主;SEM分析显示材料孔隙分布均匀,孔连通性较好,孔径尺寸约为100～300μm.对支架的孔隙率和机械强度等性能进行了表征,研究表明复合支架的孔隙率为83%～87%,机械强度有较大提高.应用模拟体液浸泡实验研究了复合支架的体外生物活性,并用XRD、FESEM和EDS对试样表面进行了表征;结果显示,样品经模拟体液浸泡3天后,表面都能沉积出类骨羟基磷灰石(HA)层,β-CaSiO_3的加入能加快复合支架表面沉积类骨HA的速度.研究结果显示β-CaSiO_3/丝素蛋白复合支架材料有望作为强度较好的生物活性硬组织修复材料.     

分级多孔壳聚糖/聚乳酸复合支架的制备及骨修复性能

为了仿生莲藕内部的贯穿大孔结构,以生物相容性好的壳聚糖(CS)作为基质材料,利用冰粒致孔、石蜡模具和冰模具成型3种成型方法制备了分级多孔CS支架材料,然后与力学强度较高的聚乳酸(PLLA)复合,制备网络互穿CS/PLLA复合支架。通过SEM、压缩强度测试和兔股骨髁骨缺损模型对CS/PLLA复合材料的形貌、力学强度和骨修复性能进行了表征。结果表明:利用冰模具制备的CS/PLLA复合支架能可控、批量制备,具有微米-毫米分级多孔结构,大孔孔径约为2mm,内部均匀分布着孔径约为60μm的贯穿微孔,并在微孔内形成密集的PLLA絮状网络结构。干态复合材料的压缩强度和模量分别比纯CS支架的提高了6倍和15倍。体内植入实验结果表明,CS/PLLA复合材料能够促进骨缺损的愈合,并随着新骨的形成,复合材料逐渐被降解吸收。     

Biocompatibility and biodegradation studies of PCL/β-TCP bone tissue scaffold fabricated by structural porogen method

Three-dimensional printer (3DP) (Z-Corp) is a solid freeform fabrication system capable of generating sub-millimeter physical features required for tissue engineering scaffolds. By using plaster composite materials, 3DP can fabricate a universal porogen which can be injected with a wide range of high melting temperature biomaterials. Here we report results toward the manufacture of either pure polycaprolactone (PCL) or homogeneous composites of 90/10 or 80/20 (w/w) PCL/beta-tricalcium phosphate (β-TCP) by injection molding into plaster composite porogens fabricated by 3DP. The resolution of printed plaster porogens and produced scaffolds was studied by scanning electron microscopy. Cytotoxicity test on scaffold extracts and biocompatibility test on the scaffolds as a matrix supporting murine osteoblast (7F2) and endothelial hybridoma (EAhy 926) cells growth for up to 4?days showed that the porogens removal process had only negligible effects on cell proliferation. The biodegradation tests of pure PCL and PCL/β-TCP composites were performed in DMEM with 10?% (v/v) FBS for up to 6?weeks. The PCL/β-TCP composites show faster degradation rate than that of pure PCL due to the addition of β-TCP, and the strength of 80/20 PCL/β-TCP composite is still suitable for human cancellous bone healing support after 6?weeks degradation. Combining precisely controlled porogen fabrication structure, good biocompatibility, and suitable mechanical properties after biodegradation, PCL/β-TCP scaffolds fabricated by 3DP porogen method provide essential capability for bone tissue engineering.     

Three dimensional printed macroporous polylactic acid/hydroxyapatite composite scaffolds for promoting bone formation in a critical-size rat calvarial defect model

We have explored the applicability of printed scaffold by comparing osteogenic ability and biodegradation property of three resorbable biomaterials. A polylactic acid/hydroxyapatite (PLA/HA) composite with a pore size of 500 μm and 60% porosity was fabricated by three-dimensional printing. Three-dimensional printed PLA/HA, β-tricalcium phosphate (β-TCP) and partially demineralized bone matrix (DBM) seeded with bone marrow stromal cells (BMSCs) were evaluated by cell adhesion, proliferation, alkaline phosphatase activity and osteogenic gene expression of osteopontin (OPN) and collagen type I (COL-1). Moreover, the biocompatibility, bone repairing capacity and degradation in three different bone substitute materials were estimated using a critical-size rat calvarial defect model in vivo. The defects were evaluated by micro-computed tomography and histological analysis at four and eight weeks after surgery, respectively. The results showed that each of the studied scaffolds had its own specific merits and drawbacks. Three-dimensional printed PLA/HA scaffolds possessed good biocompatibility and stimulated BMSC cell proliferation and differentiation to osteogenic cells. The outcomes in vivo revealed that 3D printed PLA/HA scaffolds had good osteogenic capability and biodegradation activity with no difference in inflammation reaction. Therefore, 3D printed PLA/HA scaffolds have potential applications in bone tissue engineering and may be used as graft substitutes in reconstructive surgery.     

硬脂酸改性β-磷酸三钙及其复合材料机械性能研究

为了提高β-磷酸三钙(β-TCP)复合材料的机械性能,采用硬脂酸(C17H25COOH)对β-TCP表面进行改性处理,研究了β-TCP与C17H25COOH的界面作用机理.利用透射电镜、傅里叶红外光谱、热重分析等技术分别对改性前后β-TCP的颗粒形貌、组分和表面—OH基团进行了表征,研究了改性β-TCP/聚左旋乳酸(PLLA)复合材料的机械性能,并利用扫描电镜观察了复合材料断面形貌.研究表明:硬脂酸包覆在β-TCP表面,改性后β-TCP粉末具有一定的疏水性,硬脂酸的H+可以与β-TCP中的PO43-的一个O发生质子化反应形成—OH.改性β-TCP/PLLA复合材料的机械性能相比改性前有明显提高,改性后的β-TCP微粒在PLLA中分散均匀,两者结合紧密.