Preparation and properties of poly(L-lactide)/hydroxyapatite composites

In this study, two different viscosity-average molecular weight (η = 4.0 and 7.8) poly(L-lactide) (PLLA) were synthesized by ring-opening polymerization and the poly(L-lactide)/hydroxyapatite composites (PLLA/HA) were prepared by blending HA particles (size range: 25-45 μm and Ca/P = 1.69) with a content of 10, 30, and 50 wt% in PLLA solution with further evaporation of the solvent. The plain PLLA polymers and PLLA/HA composites were compressionmolded and machined to yield 25×3×2 mm3 specimens. The molar mass of resulting specimens was decreased drastically due to the hydrolytic and thermal degradation of ester bonds. Scanning electron microscopy and thermal gravimetric results indicated that the compositions of HA in PLLA were well dispersed. With increasing HA content, the crystallinity of PLLA/HA composites are slightly increased due to the effect of HA as a nucleating agent. The dynamic mechanical analysis is useful in studying the viscoelastic behaviour of the PLLA/HA composites and no secondary relaxation was observed below the glass-to-rubber transition (60°C). The mechanical properties of the PLLA/HA composites were found to vary with HA content. Increased levels of HA resulted in increased bending modulus and strength.     

Compressive properties and degradability of poly(epsilon-caprolatone)/hydroxyapatite composites under accelerated hydrolytic degradation

Hydroxyapatite (HA) was incorporated as filler into polycaprolactone (PCL) matrix to improve the bioactivity as well as the compressive properties of the polymer composites that can be typically used in tissue engineering scaffolds. The compressive properties of five PCL/HA composites of different compositions were investigated in conjunction with the study of their rate of degradation. As PCL has a slow degradation rate, the experiment was conducted in a concentrated 5M sodium hydroxide medium to accelerate the degradation process. The compressive strength and modulus of all PCL/HA compositions were observed to decrease as the degradation experiment progressed, with samples having high HA content degraded most significantly as compared with samples with lower HA content. Pure PCL samples, however, were found to retain their mechanical properties comparatively well in the same degradation experiments. Although the addition of HA as filler into the PCL matrix was shown to have improved mechanical properties and bioactivity initially, these results do raise concerns of material properties being compromise during hydrolytic degradation.     

HA/PDLLA复合材料的体内成骨过程研究

目的研究HA／PDLLA复合材料植入体内后与细胞、组织的相互作用，探讨HA／PDLLA复合材料在体内的成骨过程，为其临床应用及设计具有生物功能的人工骨替换材料和骨组织工程支架材料提供依据。方法采用液相吸附法制备了HA／PDLLA复合材料，以纯PDLLA和空白组进行对照，进行体内植入实验，通过组织学观察和四环素标记考察其成骨过程。结果HA／PDLLA复合材料植入机体后，体内的无菌性炎症轻微，新骨形成速率高于PDLLA材料。HA微粒的存在，加强了复合材料的机械强度，使之可以避免过早的丧失力学强度。第24w时，材料被组织分隔包裹，新生骨组织长入材料，骨愈合情况良好。结论HA／PDLLA复合材料具有良好的生物相容性、生物降解性能、生物学活性和骨传导性能。     

Porous scaffold of gelatin-starch with nanohydroxyapatite composite processed via novel microwave vacuum drying

Hydroxyapatite (HA) is a fundamental mineral-based biomaterial, used for preparing composites for bone repair and regeneration. Gelatin blended with starch results in scaffold composites with enhanced mechanical properties. A gelatin-starch blend reinforced with HA nanocrystals (nHA) gave biocompatible composites with enhanced mechanical properties. In this study, a porous scaffold of gelatin-starch-nHA composites was fabricated through microwave vacuum drying and crosslinking using trisodium citrate. Three different composite scaffolds were prepared at three different percentages of nHA: 20%, 30% and 40%. The microstructures and compositions of the composites were analyzed. Within the porous structure, the nHA crystals were observed to precipitate. The interaction between the gelatin-starch network film and nHA crystalline material was studied using Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction analysis (XRD). XRD reflections showed that there are two different minerals present in the scaffold composite. There were strong reflection peaks close to the 26 degrees and 32 degrees 2theta angles of HA, and close to the 8 degrees and 49 degrees 2theta angles for sodium citrate minerals. The FTIR result suggested that carboxyl groups, C=O and amino groups play crucial roles in HA formation on the surface of a gelatin network.     

Carbon Nanotube Reinforced Collagen/Hydroxyapatite Scaffolds Improve Bone Tissue Formation <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis>

Current bone regeneration strategies faced major challenges in fabricating the bionic scaffolds with nano-structure, constituents and mechanical features of native bone. In this study, we developed a new porous scaffold by adding the multi-walled carbon nanotube (MWCNT) into collagen (Col)/hydroxyapatite (HA) composites. Data showed that 0.5%CNT/Col/HA (0.5%CNT) group was approximately tenfolds stiffer than Col–HA, and it was superior in promoting bone marrow mesenchymal stem proliferation and spreading, mRNA and protein expressions of bone sialoprotein (BSP) and osteocalcin (OCN) than Col–HA group. Moreover, we utilized 0.5%CNT composite to repair the rat calvarial defects (8 mm diameter) in vivo, and observed the new bone formation by 3D reconstruction of micro CT, HE and Masson staining, and BSP, OCN by immunohistochemical analysis. Results showed that newly formed bone in 0.5%CNT group was significantly higher than that in Col–HA group at 12 weeks. These findings highlighted a promising strategy in healing of large area bone defect with MWCNT added into the Col–HA scaffold as they possessed the combined effects of mechanical strength and osteogenicity.     

羟基磷灰石/单壁碳纳米管复合材料的表征

背景：羟基磷灰石具有接近自然人骨的强韧度和优越的生物相容性,但其力学性能却较差。 目的：制成并研究一种新型生物活性材料(羟基磷灰石/单壁碳纳米管复合材料)的各种性质。 方法：利用原位合成法制备羟基磷灰石单壁碳纳米管复合材料,并对其红外光谱、微观结构及XDR衍射分析,力学性能进行测试,对不同SWNT含量的SWNT/HAp复合材料弯曲强度与断裂韧性比较分析。 结果与结论：成功制备出的纳米羟基磷灰石单壁碳纳米管复合材料,其抗弯强度最大增幅将近50%,达到73 MPa;而断裂韧性的最大提高幅度为3倍,达到2.6 MPa•m^1/2。随着单壁碳纳米管复合材料含量的增加,复合材料的弯曲强度与断裂韧性呈现出缓慢上升的趋势。提示,纳米羟基磷灰石单壁碳纳米管复合材料显著提高了接近自然人骨的纳米级磷灰石骨材料的抗弯强度和断裂韧性,从而克服传统支撑骨材料的力学性能缺陷。     

Novel porous hydroxyapatite prepared by combining H2O2 foaming with PU sponge and modified with PLGA and bioactive glass

Porous hydroxyapatite (HA) scaffolds have been intensively studied and developed for bone tissue engineering, but their mechanical properties remain to be improved. The aim of this study is to prepare HA-based composite scaffolds that have a unique macroporous structure and special struts of a polymer/ceramic interpenetrating composite and a bioactive coating. A novel combination of a polyurethane (PU) foam method and a hydrogen peroxide (H(2)O( 2)) foaming method is used to fabricate the macroporous HA scaffolds. Micropores are present in the resulting porous HA ceramics after the unusual sintering of a common calcium phosphate cement and are infiltrated with the poly(D,L-lactic-co-glycolic acid) (PLGA) polymer. The internal surfaces of the macropores are further coated with a PLGA-bioactive glass composite coating. The porous composite scaffolds are characterized in terms of microstructure, mechanical properties, and bioactivity. It is found that the HA scaffolds fabricated by the combined method show high porosities of 61-65% and proper macropore sizes of 200-600 microm. The PLGA infiltration improved the compressive strengths of the scaffolds from 1.5-1.8 to 4.0-5.8 MPa. Furthermore, the bioactive glass-PLGA coating rendered a good bioactivity to the composites, evidenced by the formation of an apatite layer on the sample surfaces immersed in the simulated body fluid (SBF) for 5 days. The porous HA-based composites obtained from this study have suitable porous structures, proper mechanical properties, and a high bioactivity, and thus finds potential application as scaffolds for bone tissue engineering.     

Recombinant growth/differentiation factor-5 (GDF-5) stimulates osteogenic differentiation of marrow mesenchymal stem cells in porous hydroxyapatite ceramic

To evaluate the growth/differentiation factor-5 (GDF-5) in the in vivo osteogenic potential of bone marrow mesenchymal stem cells (MSCs), we subcutaneously implanted five different kinds of hydroxyapatite (HA) ceramic implants: HA alone, GDF-5/HA composites (GDF/HA), MSCs/HA composites, the MSCs/HA composites supplemented with GDF-5 (GDF/MSCs/HA), and recombinant bone morphogenetic protein-2 (BMP/MSCs/HA). Neither the HA alone nor the GDF/HA composites exhibited any bone formation at any time after implantation. At 4 weeks, the MSCs/HA composites exhibited a certain amount of bone formation in some pore areas. In contrast, at 2 weeks, the GDF/MSCs/HA composites exhibited histologically obvious de novo bone formation together with active osteoblasts in many pore areas and additional bone formation at 4 weeks. In the de novo formed bone, neither chondrocytes nor endochondral bone was detected. The GDF/MSCs/HA composites also showed high alkaline phosphatase (ALP) and osteocalcin expression determined at both the protein and gene levels and the high level of expression was well maintained even at 4 weeks. Compared with GDF/MSCs/HA, the BMP/MSCs/HA composites exhibited excellent osteogenesis with relatively early osteoblastic phenotype expression. The results indicate that GDF-5 synergistically enhances de novo bone formation capability of MSCs/HA composite and suggest that tissue-engineered GDF/MSCs/HA composites could be used as bone graft substitutes.     

Preparation and properties of poly(L-lactide)/hydroxyapatite composites

In this study, two different viscosity-average molecular weight (eta = 4.0 and 7.8) poly(L-lactide) (PLLA) were synthesized by ring-opening polymerization and the poly(L-lactide)/hydroxyapatite composites (PLLA/HA) were prepared by blending HA particles (size range: 25-45 microm and Ca/P = 1.69) with a content of 10, 30, and 50 wt% in PLLA solution with further evaporation of the solvent. The plain PLLA polymers and PLLA/HA composites were compression-molded and machined to yield 25 x 3 x 2 mm3 specimens. The molar mass of resulting specimens was decreased drastically due to the hydrolytic and thermal degradation of ester bonds. Scanning electron microscopy and thermal gravimetric results indicated that the compositions of HA in PLLA were well dispersed. With increasing HA content, the crystallinity of PLLA/HA composites are slightly increased due to the effect of HA as a nucleating agent. The dynamic mechanical analysis is useful in studying the viscoelastic behaviour of the PLLA/HA composites and no secondary relaxation was observed below the glass-to-rubber transition (60 degrees C). The mechanical properties of the PLLA/HA composites were found to vary with HA content. Increased levels of HA resulted in increased bending modulus and strength.     

Development of a bioactive glass fiber reinforced starch-polycaprolactone composite

For bone regeneration and repair, combinations of different materials are often needed. Biodegradable polymers are often combined with osteoconductive materials, such as bioactive glass (BaG), which can also improve the mechanical properties of the composite. The aim of this work was to develop and characterize BaG fiber reinforced starch-poly-epsilon-caprolactone (SPCL) composite. Sheets of SPCL (30/70 wt %) were produced using single-screw extrusion. They were then cut and compression-molded in layers with BaG fibers to form composite structures with different combinations. Mechanical and degradation properties of the composites were studied. The actual amount of BaG in the composites was determined using combustion tests. Initial mechanical properties of the reinforced composites were at least 50% better than the properties of the nonreinforced specimens. However, the mechanical properties of the composites after 2 weeks of hydrolysis were comparable to those of the nonreinforced samples. During the 6 weeks hydrolysis the mass of the composites had decreased only by about 5%. The amount of glass in the composites remained as initial for the 6-week period of hydrolysis. In conclusion, it is possible to enhance initial mechanical properties of SPCL by reinforcing it with BaG fibers. However, mechanical properties of the composites are typical for bone fillers and strength properties need to be further improved for allowing more demanding bone applications.     

Hydroxyapatite-based composite for dental implants: an in vivo removal torque experiment

Screw-shaped dental implants were fabricated from commercially pure Ti (c.p. Ti) and HA-based composites. The HA-based composites were fabricated by mixing HA with Al(2)O(3)-coated ZrO(2) powders. The mechanical properties of these composites were enhanced by a factor of 3. These were implanted into the rabbit tibiae and the removal torque to loosen the implants in vivo was measured in order to investigate the osteointegration. After a healing period of 6 weeks, the implants were retrieved with a torque gauge instrument. The HA-based composite implants showed an almost 2-times-higher removal torque when compared to the Ti implants (ANOVA, p < 0.05), indicating excellent biocompatibility to bone. Thus, HA-based composites had not only better mechanical properties but also similar bioactivity as HA itself. It is believed that a HA-based composite is suitable for artificial dental implants.     

Long-term durability of porous hydroxyapatite with low-pressure system to support osteogenesis of mesenchymal stem cells

In this study, we hypothesize that loading more marrow-derived mesenchymal stem cells (MSCs) into porous material by using a low-pressure system during subculture, creating a composite which combines MSCs and a novel mechanical reinforced porous hydroxyapatite, can result in more bone tissue formation in vivo. Within 26 weeks postimplantation, we examined in vivo bone formation of the experimental group with 100 mmHg pressure applied to porous HA blocks loaded with MSCs. For in vivo testing, the 2-week subcultured HA/MSC composites were implanted into subcutaneous sites of syngeneic rats. These implants were harvested at 13 and 26 weeks after implantation. SEM showed that the pore surface is covered by osteoblasts as well as collagenous extracellular matrix at 13 weeks. Light microscopy revealed the quantity of bone at 26 weeks was greater than at 13 weeks. These results showed that the novel mechanical reinforced porous HA combined with MSC has more potential for bone formation at 100 mmHg, making this method very efficient for bone reconstruction.     

Synthesis, characterization of calcium phosphates/polyurethane composites for weight-bearing implants

Calcium phosphate (CaP)/polymer composites have been studied as an alternative graft material for the treatment of bone defects. In this study, lysine-triisocyanate-based polyurethane (PUR) composites were synthesized from both hydroxyapatite (HA) and β-tricalcium phosphate (TCP) to reduce the brittleness of CaP and increase the bioactivity of the polymer. The mechanical properties and in vitro cellular response were investigated for both HA/PUR and TCP/PUR composites. The composites were implanted in femoral defects in rats, and in vivo bioactivity was evaluated by X-rays, micro-computed tomography (μCT), and histological sections. In biomechanical testing, PUR improved the mechanical properties of the CaP, thus rendering it potentially suitable for weight-bearing applications. In vitro cell culture studies showed that CaP/PUR composites are biocompatible, with β-TCP enhancing the cell viability and proliferation relative to HA. CaP/PUR composites also supported the differentiation of osteoblastic cells on the materials. When implanted in rat femoral defects, the CaP/PUR composites were biocompatible and osteoconductive with no adverse inflammatory response, as evidenced by X-rays, μCT images, and histological sections. Additionally, a histological examination showed evidence of cellular infiltration and appositional remodeling. These results suggest that CaP/PUR composites could be potentially useful biomaterials for weight-bearing orthopaedic implants.     

硬组织替换用羟基磷灰石复合材料的研究进展

羟基磷灰(HA)石具有与人骨无机质相似的化学成分和晶体结构，被认为是一种很有潜力的人体硬组织替换材料，但脆性太大限制了其在承载部位骨替换中的应用。因而各种羟基磷灰石复合材料受到了极大的关注。本文按照增强体的种类对羟基磷灰石复合材料进行了分类介绍。生物活性陶瓷、生物活性玻璃及玻璃陶瓷、生物惰性陶瓷、聚合物及金属等都被用来制备羟基磷灰石复合材料，但仍没有一种材料能够很好的满足硬组织替换的需要。现有HA复合材料存在的关键问题是生物性能与力学性能之间不能很好地匹配。     

Coating nanothickness degradable films on nanocrystalline hydroxyapatite particles to improve the bonding strength between nanohydroxyapatite and degradable polymer matrix

Hydroxyapatite (HA) nanoparticles are similar to bone apatite in size, phase composition, and crystal structure. When compared with micron-size HA particles, nano-HA possesses improved mechanical properties and superior bioactivity for promoting bone growth and regeneration. However, scaffolds fabricated from nano-HA alone cannot meet the mechanical requirements for direct-loading applications. A number of studies suggest that nanostructured composites may offer surface and/or chemical properties of native bone, and therefore represent ideal substrates to support bone regeneration. However, a common problem with nanohydroxyapatite (nano-HA)-polymer composites is the weak binding strength between the nano-HA filler and the polymer matrix since they are two different categories of materials and cannot form covalent bonds between them during the mixing process. Often, the mechanical strength of the composite is compromised due to the phase separation of the HA filler from the polymer matrix during the tissue repair process. To overcome this problem, an ultrathin degradable polymer film was grafted onto the surface of nano-HA using a radio-frequency plasma polymerization technology from acrylic acid monomers. The treated nano-HA powders are expected to bind to the polymer matrix via covalent bonds, thus enhancing the mechanical properties of the resultant composites. High-resolution transmission electron microscopy (HRTEM) experiments showed that an extremely thin polymer film (2 nm) was uniformly deposited on the surfaces of the nanoparticles. The HRTEM results were confirmed by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectroscopy (TOFSIMS). Tensile tests performed on the specimens revealed that the degradable coating had improved elastic and strength properties when compared with the nondegradable and uncoated controls. XPS and TOSIMS data revealed that more functional carboxyl groups were formed on degradable coatings than cross-linked nondegradable coatings. Cytocompatibility assay demonstrated that both the degradable and nondegradable coatings are cytocompatible.     

Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites by bovine bone morphogenetic protein

A composite of marrow mesenchymal stem cells and porous hydroxyapatite (HA) has in vivo osteogenic potential. To investigate factors enhancing the osteogenic potential of marrow/HA composites, we prepared a bone morphogenetic protein (BMP) fraction from the 4M guanidine extract of bovine bone by heparin-sepharose affinity chromatography. Marrow/HA composites or composites containing marrow mesenchymal stem cells, BMP, and HA (marrow/BMP/HA composites) were implanted subcutaneously in 7-week-old male Fischer rats. BMP/HA composites and HA alone were also implanted. The implants were harvested after 2, 4, or 8 weeks and were prepared for histological and biochemical studies. Histological examination showed obvious de novo bone formation together with active osteoblasts at 2 weeks, as well as more extensive bone formation at 4 and 8 weeks in many pores of the marrow/BMP/HA composites. The marrow/HA composites did not induce bone formation at 2 weeks, but there was moderate bone formation at 4 weeks. At 2 weeks, only marrow/BMP/HA composites resulted in intensive osteogenic activity, judging from alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of marrow mesenchymal stem cells, porous HA, and BMP synergistically enhances osteogenic potential, and may provide a rational basis for their clinical application, although further in vivo experiment is needed.     

Hydroxyapatite/PMMA composites as bone cements

Currently PMMA is the polymer most commonly used as a bone cement for the fixation of total hip prostheses. Ideally, a bone cement material should be easy to handle, biologically compatible, nonsupporting of oral microbial growth, available in the particulate and molded forms, easy to obtain, nonallergenic, adaptable to a broad range of dental and medical applications, in possession of high compressive strength, and effective in guided tissue regenerative procedures. One of the problems associated with the conventional types of bone cement used is their unsatisfactory mechanical and exothermic reaction properties. The purpose of this in vitro study was to investigate and compare the mechanical properties (three-point bending strength, energy-to-break, and modulus of elasticity) and physical properties (setting time, water sorption, and exothermic heat) of HA/PMMA (HA group) and bovine-bone originated HA/PMMA (BB group) composites. Composites samples were fabricated by admixing method. It was found that the addition of HA and BB particles increased the water sorption. Generally 10 v/o 20 v/o HA and 0 v/o to 10 v/o BB ratio combinations had significant beneficial effects on the mechanical properties. The heat generated during polymerization was influenced by the different admixtures. More than 40 v/o HA and 40 v/o BB should be mixed into PMMA to reduce the peak temperature. Overall evaluation indicated that the BB group had better properties than the HA group.     

Bone response and mechanical strength of rabbit femoral defects filled with injectable CaP cements containing TGF-beta 1 loaded gelatin microparticles

This study focused at the potential of transforming growth factor beta 1 (TGF-beta 1) loaded gelatin microparticles to enhance the bone response and mechanical strength of rabbit femoral defects filled with injectable calcium phosphate (CaP)/gelatin microparticle composites. Therefore, TGF-beta1 loaded composites and non-loaded controls were injected in circular defects as created in the femoral condyles of rabbits and were left in place for 4, 8 and 12 weeks. The specimens were evaluated mechanically (push-out test), and morphologically (scanning electron microscopy (SEM), histology, and histomorphometry). The results showed a gradual increase in mechanical strength with increasing implantation periods. Histological and histomorphometrical evaluation showed similar results for both composite formulations regarding histological aspect, new bone formation and bone/implant contact. However, TGF-beta1 loading of the composites demonstrated a significant effect on composite degradation after twelve weeks of implantation. The results of this study showed that CaP/gelatin composites show excellent osteogenic properties and a rapid increase in mechanical strength. The addition of TGF-beta1 significantly enhances the bone remodeling process.     

可吸收羟基磷灰石/聚DL-乳酸骨折内固定材料机械强度和生物降解性研究

目的评价自行研制的可吸收羟基磷灰石/聚DL-乳酸(HA/PDLLA)复合骨折内固定材料的机械强度和生物降解性。方法体外降解实验是把相同分子量的HA/PDLLA和单纯PDLLA试件分别置于PBS缓冲液中,于2、4、6、8、10、12周取材,测试生物降解率、吸水率、失重率、机械强度及降解液pH值,并作扫描电镜(SEM)观察;体内实验是用一枚HA/PDLLA棒内固定兔股骨髁松质骨部横形截骨,作X线摄片、组织学、机械强度及材料骨界面SEM观察。结果HA/PDLLA复合材料较单纯PDLLA材料降解速度减慢,机械强度提高,骨折正常愈合。结论HA/PDLLA材料具有足够的强度保证实验性松质骨骨折正常愈合。     

Promotion of bone formation using highly pure porous beta-TCP combined with bone marrow-derived osteoprogenitor cells

Beta-tricalcium phosphate (TCP) exhibits rapid degradation and weak mechanical properties, which has limited its application as bone graft substitutes, though it has good biocompatibility and osteoconductivity. We hypothesized that a composite of highly pure porous beta-TCP and bone marrow-derived osteoprogenitor cells (BMO) could improve bone formation, and slow down the degradation of beta-TCP. A highly pure porous beta-TCP with 75% porosity was fabricated. The pores averaged 200-400 microm in diameter, with interconnecting paths 100-200 microm. Blocks of beta-TCP 5 mm3 were combined with BMO, and incubated 2 weeks with (+) or without (-) osteogenic medium. They were then implanted into subcutaneous sites of syngeneic rats for 24 weeks. These composites were harvested at different time points. The alkaline phosphatase activity and bone osteocalcin content of the composites (+) were much higher than corresponding values in the composites (-) of the control group (p<0.01). Light microscopy revealed mature bone and lots of blood vessels only in the TCP/BMO composite (+). The amount of newly formed bone increased until week 24. Slow resorptive activity could be found. The mechanical parameters of the composites were much improved over those of dry beta-TCP blocks. These results showed that tissue engineering treatment on incubating the composites of beta-TCP and BMO cells in osteogenic medium results in a good osteogenic activity.