原位自发泡制备磷酸钙/聚氨酯复合骨修复支架

磷酸钙/聚氨酯(CaP/PU)复合骨修复支架制备过程中随着材料体系粘度逐渐增大, 后期加入的发泡剂难于均匀分散, 影响支架孔隙率及孔结构均匀性。本研究在CaP/PU材料合成过程中将发泡剂水以磷酸氢钙结晶水合物(DCPD)的形式均匀复合在材料中, 在一定条件下释放结晶水与聚氨酯(PU)中的异氰酸根反应生成CO₂, 实现自发泡成型。实验结果显示, 90 ℃条件下自发泡制备的CaP/PU支架孔隙率高、孔结构均匀、贯通性好。将90 ℃发泡成型的CaP/PU多孔支架在110 ℃再熟化处理, 可提高支架的力学性能高达1倍以上。该方法简便易行, 为聚氨酯基多孔支架的制备提供了新思路。     

新型可降解钙磷骨水泥多孔支架研究

采用一种特殊的方法制备了孔径、孔隙率和孔形状可控的多孔羟基磷灰石骨水泥支架. 材料的抗压强度可达4MPa, 孔隙率可达70%, 孔与孔之间互相贯通, 大孔壁富含微孔. 细胞在材料表面黏附铺展且增殖良好, 体外模拟实验显示材料的降解速度随孔隙率的增加和Ca/P比的降低而加快, 多孔支架有优良的生物降解性和生物相容性. 该材料可用于修复骨组织缺损和作为支架材料用于组织工程.     

Sol–gel derived composite from bioactive glass–polyvinyl alcohol

Investigation of novel biomaterials for bone engineering is based on the development of porous scaffolds, which should match the properties of the tissue that is to be replaced. These materials need to be biocompatible, ideally osteoinductive, osteoconductive, and mechanically well-matched. In the present paper, we report the preparation and characterization of hybrid macroporous scaffold of polyvinyl alcohol (PVA)/bioactive glass through the sol–gel route. Hybrids containing PVA (80, 70 and 60 wt%) and bioactive glass with composition 58SiO₂–33CaO–9P₂O₅ were synthesized by foaming a mixture of polymer solution and bioactive glass via sol–gel precursor solution. PVA with two different degree of hydrolysis (DH), 98.5% (high degree) and 80% (low degree) were also investigated, in order to evaluate the influence of residual acetate group present in polymer chain on the final structure and properties of 3D porous composite produced. The microstructure, morphology and crystallinity of the hybrid porous scaffolds were characterized by X-ray diffraction (XRD), Infrared Fourier Transform spectrometry (FTIR) and Scanning electron microscopy (SEM/EDX) analysis. In addition, specific surface area was assessed by B.E.T. nitrogen adsorption method and mechanical behavior was evaluated by compression tests. Preliminary cytotoxicity and cell viability were also performed by the MTT assay. VERO cell monolayers were grown in 96-well microtiter plates. The results have clearly showed that hybrid foams of polyvinyl alcohol/bioactive glass (PVA/BG) with interconnected macroporous 3D structure were successfully produced. All the tested hybrids of PVA/BG have showed adequate cell viability properties for potential biological applications.     

Elaboration of self-coating alumina-based porous ceramics

Materials used for bone substitution (i.e. hydroxyapatite and calcium phosphate) are highly successful, since when implanted they provide an efficient scaffold that can be colonized by the patient’s bone. However, their poor mechanical properties impede their use for load-bearing applications. In contrast, no material with high mechanical properties also presents a high bioactivity. A possible way of finding a material both strong and bioactive is to use a composite. We propose here a composite deriving its strength from its alumina core and its bioactivity from a calcium phosphate surface. Ceramic scaffolds have been produced by infiltration of polymer open-celled foams. Several compositions of the slurries have been tested, leading to the realization of porous pieces with a biocompatibility gradient at a micrometric scale. The mechanical properties of several new materials are presented and correlated to their microstructure.     

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。     

In situ synthesis and characterization of porous polymer-ceramic composites as scaffolds for gene delivery

The use of three-dimensional scaffolds in gene delivery has emerged as a popular and necessary delivery vehicle for obtaining controlled gene delivery. In this report, techniques to synthesize composite scaffolds by combining natural polymers such as agarose and alginate with calcium phosphate (CaP) are described. The incorporation of CaP into the agarose or alginate hydrogels was performed in situ and the presence of CaP was confirmed by X-ray diffraction (XRD). The crystallite size of the CaP particles was determined to be 7.20 nm. Lyophilized, porous composites were examined under scanning electron microscopy (SEM) to estimate the size of the pores, an essential requirement for an ideal scaffold. The swelling properties of the composite samples were also investigated to study the effect of CaP incorporation on the behavior of the hydrogels. By incorporating CaP into the hydrogel, the aim is to synthesize a scaffold that is mechanically strong and chemically suitable for use as a gene delivery vehicle in tissue engineering.     

Sol–gel method to fabricate CaP scaffolds by robocasting for tissue engineering

Highly porous calcium phosphate (CaP) scaffolds for bone-tissue engineering were fabricated by combining a robocasting process with a sol–gel synthesis that mixed Calcium Nitrate Tetrahydrate and Triethyl Phosphite precursors in an aqueous medium. The resulting gels were used to print scaffolds by robocasting without the use of binder to increase the viscosity of the paste. X-ray diffraction analysis confirmed that the process yielded hydroxyapatite and β-tricalcium phosphate biphasic composite powders. Thus, the scaffold composition after crystallization of the amorphous structure could be easily modified by varying the initial Ca/P ratio during synthesis. The compressive strengths of the scaffolds are ~6 MPa, which is in the range of human cancellous bone (2–12 MPa). These highly porous scaffolds (~73 vol% porosity) are composed of macro-pores of ~260 μm in size; such porosity is expected to enable bone ingrowth into the scaffold for bone repair applications. The chemistry, porosity, and surface topography of such scaffolds can also be modified by the process parameters to favor bone formation. The studied sol–gel process can be used to coat these scaffolds by dip-coating, which induces a significant enhancement of mechanical properties. This can adjust scaffold properties such as composition and surface morphology, which consequently may improve their performances.     

Feasibility of three-dimensional macroporous scaffold using calcium phosphate glass and polyurethane sponge

Tissue engineering presents an alternative approach to the repair of a damaged tissue by avoiding the need for a permanent implant made of an engineered artificial material. A suitable temporary scaffold material that exhibits adequate mechanical and biological properties is required to enable tissue regeneration by exploiting the body’s inherent repair mechanism, i.e. a regenerative allograft. Synthetic bioresorbable polymers have been attracting attention as tissue engineering scaffolds. However, a number of problems have been encountered such as inflammatory responses and lack of bioactivity. Another good candidate for a tissue engineering scaffold is the calcium phosphates because of their good biocompatibility and osteointegrative properties. Their slow biodegradation is still remains problem, especially for the filling of large bony defects. In this study, we investigated the fabrication method of a three-dimensional reticulated scaffold with interconnected pores of several hundred micrometers using calcium phosphate glass in the system of CaO-CaF₂-P₂O₅-MgO-ZnO and a polyurethane sponge as a template. Calcium phosphate glass slurry was homogenously thick coated when the weight percentage of the calcium phosphate glass powder was 40% with 8 wt% of polyvinyl alcohol as a binder. Addition of 10 wt% dimethyl formamide as a drying control chemical additive into a slurry almost prevented the crack formation during drying. Sintering of the dried porous block at 850°C exhibited the densest microstructure as well as the entire elimination of the organic additives. Repeating the process significantly increased compressive strength of sintered porous body due to the thickening of the struts. To summarize, macroporous calcium phosphate glass can be fabricated with 500∼800 μm of pore size and a three-dimensionally interconnected open pore system. It is thought that this kind of biodegradable glass scaffold combined with osteogenic cells has potential to be studied further as a tissue-engineered bone substitute.     

海藻酸钙水凝胶/聚乳酸复合材料的制备与性能

通过化学发泡-冷冻干燥-粒子滤出复合法制备聚乳酸(PLLA)大孔支架, 然后在大孔内以海藻酸钠(SA)、碳酸钙、葡萄糖酸内酯(GDL)为原料, 通过原位相转变制备海藻酸钙水凝胶/聚乳酸复合材料(CA/PLLA); 分别利用SEM、压缩强度测试和细胞培养对CA/PLLA支架的形貌、力学性能及生物相容性进行了研究。结果表明: PLLA具有直径小于2 mm、孔道相互连通的孔洞, 且在大孔中能够形成均匀的CA。CA/PLLA复合材料的压缩强度(2.74 MPa)远大于单一的海藻酸钙水凝胶的压缩强度(0.10 MPa)。在CA/PLLA复合支架中, 软骨细胞呈簇状圆形生长状态, 与其在天然软骨陷窝里生长状态一致。这种软硬结合、天然与合成高分子杂化的CA/PLLA复合材料的力学强度和生物相容性同时得到提高, 可进一步作为骨和软骨修复材料研究。     

磷灰石-硅灰石/β-磷酸三钙复合多孔支架材料的制备和表征

以磷灰石-硅灰石玻璃陶瓷(AW)粉和β-磷酸三钙(β-TCP)粉为原料. 以硬脂酸为致孔剂. 经模压成型、1170℃烧结制备磷灰石-硅灰石/β-磷酸三钙复合多孔支架材料(AW/βTCP). 采用X射线衍射(XRD)、扫描电镜(SEM)、能谱(EDS)、诱导耦合等离子体原子发射光谱(ICP-AES)等方法分析支架的晶相组成、显微结构、物理性能、生物活性和降解性. 将大鼠骨髓间充质干细胞(rMSCs)与支架体外复合培养评价支架的生物相容性. 结果表明: 所制备的AW/β-TCP支架材料的抗压强度达14.3MPa. 孔隙率达66.9%. 孔径为100～700μm. 具有良好的生物相容性、生物活性和降解性. 可作为骨组织工程支架的候选材料.     

The fabrication of biomineralized fiber-aligned PLGA scaffolds and their effect on enhancing osteogenic differentiation of UCMSC cells

The key factor of scaffold design for bone tissue engineering is to mimic the microenvironment of natural bone extracellular matrix (ECM) and guide cell osteogenic differentiation. The biomineralized fiber-aligned PLGA scaffolds (a-PLGA/CaPs) was developed in this study by mimicking the structure and composition of native bone ECM. The aligned PLGA fibers was prepared by wet spinning and then biomineralized via an alternate immersion method. Introduction of a bioceramic component CaP onto the PLGA fibers led to changes in surface roughness and hydrophilicity, which showed to modulate cell adhesion and cell morphology of umbilical cord mesenchymal stem cells (UCMSCs). It was found that organized actin filaments of UCMSCs cultured on both a-PLGA and a-PLGA/CaP scaffolds appeared to follow contact guidance along the aligned fibers, and those cells grown on a-PLGA/CaP scaffolds exhibited a more polarized cellular morphology. The a-PLGA/CaP scaffold with multicycles of mineralization facilitated the cell attachment on the fiber surfaces and then supported better cell adhesion and contact guidance, leading to enhancement in following proliferation and osteogenic differentiation of UCMSCs. Our results give some insights into the regulation of cell behaviors through design of ECM-mimicking structure and composition and provide an alternative wet-spun fiber-aligned scaffold with HA-mineralized layer for bone tissue engineering application.     

Fabrication of mineralized electrospun PLGA and PLGA/gelatin nanofibers and their potential in bone tissue engineering

Surface mineralization is an effective method to produce calcium phosphate apatite coating on the surface of bone tissue scaffold which could create an osteophilic environment similar to the natural extracellular matrix for bone cells. In this study, we prepared mineralized poly(d,l-lactide-co-glycolide) (PLGA) and PLGA/gelatin electrospun nanofibers via depositing calcium phosphate apatite coating on the surface of these nanofibers to fabricate bone tissue engineering scaffolds by concentrated simulated body fluid method, supersaturated calcification solution method and alternate soaking method. The apatite products were characterized by the scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), and X-ray diffractometry (XRD) methods. A large amount of calcium phosphate apatite composed of dicalcium phosphate dihydrate (DCPD), hydroxyapatite (HA) and octacalcium phosphate (OCP) was deposited on the surface of resulting nanofibers in short times via three mineralizing methods. A larger amount of calcium phosphate was deposited on the surface of PLGA/gelatin nanofibers rather than PLGA nanofibers because gelatin acted as nucleation center for the formation of calcium phosphate. The cell culture experiments revealed that the difference of morphology and components of calcium phosphate apatite did not show much influence on the cell adhesion, proliferation and activity.     

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 tissueengineering, β-TCP powder was produced by a new wet process. Porous scaffolds were prepared by three steps, i.e. solventcasting, compression molding and leaching stage. Factors influencing the compressive strength and the degradation behaviorof the porous scaffold, e.g. weight fraction of pore forming agent-sodium chloride (NaCl), weight ratio of PLLA: β-TCP,the particle size of β-TCP and the porosity, were discussed in details. Rat marrow stromal cells (RMSC) were incorporatedinto the composite by tissue engineering approach. Biological and osteogenesis potential of the composite scaffold weredetermined with MTT assay, alkaline phosphatase (ALP) activity and bone osteocalcin (OCN) content evaluation. Resultsshow that PLLA/β-TCP bioactive porous scaffold has good mechanical and pore structure with adjustable compressive strengthneeded for surgery. RMSCs seeding on porous PLLA/     

多孔磷酸钙骨水泥组织工程支架的高分子灌注增强

利用棒状谷氨酸钠晶体作为造孔粒子,采用可溶盐造孔法,制备了三维连通的大孔径多孔磷酸钙骨水泥支架,分别将明胶(Gelatin) 、聚乳酸2羟基乙酸共聚物(PLGA) 、聚乳酸(PLA) 、聚己内酯(PCL) 、聚羟基丁酸戊酸酯(PHBV)灌注到多孔磷酸钙骨水泥(CPC)支架的孔隙中以改善支架材料的力学性能。结果表明,5 种高分子材料与水的接触角大小顺序为PHBV > PCL > PLA > PL GA > Gelatin , 复合支架材料的强度随高分子材料与水接触角的减小而增大;除PHBV外,其余4种均有明显的增强效果,其中Gelatin/CPC复合支架增强效果最好,强度达到2. 25 MPa±0. 02 MPa ,是CPC支架强度的25倍。经过增强的大孔径多孔磷酸钙骨水泥复合支架可用作骨组织工程支架材料。      

磷酸钙/纤维蛋白胶复合支架材料的结构及力学性能分析

用可吸收磷酸钙骨水泥和纤维蛋白胶按一定比例体外构建复合支架材料,通过XRD、SEM、抗压实验和空隙率测试等方法对其结构及力学性能进行分析.结果发现:由于加入纤维蛋白胶,复合支架材料在一定程度上延长了磷酸钙骨水泥的初凝时间,但并不影响磷酸钙骨水泥的终凝时间;同时,加入纤维蛋白胶改变了骨水泥固化体的微观结构,提高了骨水泥的抗压强度,其最大抗压强度达到14MPa,弹性模量在96.64～269.39MPa之间,空隙率为38.8%.与在同样条件下制备的磷酸钙骨水泥比较,复合支架材料的抗压强度增强了55.6%,而空隙率仅仅下降了6.9%;XRD分析显示,复合支架材料并不影响磷酸钙骨水泥的最终的转化,其结晶结构仍是羟基磷灰石结构,是更好的骨组织工程支架材料.     

Development of macroporous calcium phosphate scaffold processed via microwave rapid drying

Porous hydroxyapatite (HA) scaffold has great potential in bone tissue engineering applications. A new method to fabricate macroporous calcium phosphate (CP) scaffold via microwave irradiation, followed by conventional sintering to form HA scaffold was developed. Incorporation of trisodium citrate dihydrate and citric acid in the CP mixture gave macroporous scaffolds upon microwave rapid drying. In this work, a mixture of β-tricalcium phosphate (β-TCP), calcium carbonate (CaCO₃), trisodium citrate dihydrate, citric acid and double distilled de-ionised water (DDI) was exposed to microwave radiation to form a macroporous structure. Based on gross eye examinations, addition of trisodium citrate at 30 and 40 wt.% in the CP mixture (β-TCP and CaCO₃) without citric acid indicates increasing order of pore volume where the highest porosity yield was observed at 40 wt.% of trisodium citrate addition and the pore size was detected at several millimeters. Therefore, optimization of pore size was performed by adding 3–7 wt.% of citric acid in the CP mixture which was separately mixed with 30 and 40 wt.% of trisodium citrate for comparison purposes. Fabricated scaffolds were calcined at 600 °C and washed with DDI water to remove the sodium hydroxycarbonate and sintered at 1250 °C to form HA phase as confirmed in the X-ray diffraction (XRD) results. Based on Archimedes method, HA scaffolds prepared from 40 wt.% of trisodium citrate with 3–7 wt.% of citric acid added CP mixture have an open and interconnected porous structure ranging from 51 to 53 vol.% and observation using Scanning electron microscope (SEM) showed the pore size distribution between 100 and 500 μm. The cytotoxicity tests revealed that the porous HA scaffolds have no cytotoxic potential on MG63 osteoblast-like cells which might allow for their use as biomaterials.     

Promotion of osteogenic differentiation of stem cells and increase of bone-bonding ability in vivo using urease-treated titanium coated with calcium phosphate and gelatin

Abstract

Because of its excellent biocompatibility and low allergenicity, titanium has been widely used for bone replacement and tissue engineering. To produce a desirable composite with enhanced bone response and mechanical strength, in this study bioactive calcium phosphate (CaP) and gelatin composites were coated onto titanium (Ti) via a novel urease technique. The cellular responses to the CaP/gelatin/Ti (CaP/gel/Ti) and bone bonding ability were evaluated with proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) on CaP/gel/Ti and CaP/Ti in vitro. The results showed that the optical density values, alkaline phosphatase expression and genes expression of MSCs on CaP/gel/Ti were similar to those on CaP/Ti, yet significantly higher than those on pure Ti (p < 0.05). CaP/gel/Ti and CaP/Ti rods (2 mm in diameter, 10 mm in length) were also implanted into femoral shaft of rabbits and pure Ti rods served as control (n = 10). Histological examination, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) measurements were performed at 4 and 8 weeks after the operation. The histological and SEM observations demonstrated clearly that more new bone formed on the surface of CaP/gel/Ti than in the other two groups at each time point. The CaP/gel/Ti bonded to the surrounding bone directly with no intervening soft tissue layer. An interfacial layer, containing Ti, Ca and P, was found to form at the interface between bone and the implant on all three groups by EDS analysis. However, the content of Ca, P in the surface of CaP/gel/Ti implants was more than in the other two groups at each time point. The CaP/gel/Ti modified by the urease method was not only beneficial for MSCs proliferation and osteogenic differentiation, but also favorable for bone bonding ability on Ti implants in vivo, suggesting that Ti functionalized with CaP and gelatin might have a great potential in clinical joint replacement or dental implants.     

In situ synthesized ceramic–polymer composites for bone tissue engineering: bioactivity and degradation studies

As an alternative to current bone grafting strategies, a poly-lactide-co-glycolide/calcium phosphate composite microsphere-based scaffold has been synthesized by the direct formation of calcium phosphate within forming microspheres. It was hypothesized that the synthesis of low crystalline calcium phosphate within forming microspheres would provide a site-specific delivery of calcium ions to enhance calcium phosphate reprecipitation onto the scaffold. Both polymeric and composite scaffolds were incubated in simulated body fluid (SBF) for 8 weeks, during which time polymer molecular weight, scaffold mass, calcium ion concentration of SBF, pH of SBF, and calcium phosphate reprecipitation was monitored. Results showed a 20% decrease in polymeric scaffold molecular weight compared to 11–14% decrease for composite scaffolds over 8 weeks. Composite scaffold mass and SBF pH decreased for the first 2 weeks but began increasing after 2 weeks and continued to do so up to 8 weeks, suggesting interplay between pH changes and calcium phosphate dissolution/reprecipitation. Free calcium ion concentration of SBF containing composite scaffolds increased 20–40% over control values within 4 h of incubation but then dropped as low as 40% below control values, suggesting an initial burst release of calcium ions followed by a reprecipitation onto the scaffold surface. Scanning electron micrographs confirm calcium phosphate reprecipitation on the scaffold surface after only 3 days of incubation. Results suggest the composite scaffold is capable of initiating calcium phosphate reprecipitation which may aid in bone/implant integration.     

How Degradation of Calcium Phosphate Bone Substitute Materials is influenced by Phase Composition and Porosity

The chemical composition of calcium phosphate (CaP) materials for the regenerative therapy of large bone defects is similar to that of bone. Additionally, calcium phosphates show an excellent biocompatibility. Besides the support of defect healing calcium phosphate implants should be completely degraded within an adequate time period to be replaced by newly formed bone. Although degradation of CaP‐implants occurs mainly by dissolution of the material, it is important to characterize the osteoclastic resorption as well, which is involved in native bone remodeling. The degradation of bone substitutes made of calcium phosphate ceramics is influenced by various parameters, such as defect size and localization, the general health situation, and age of the patient, but also material properties are important. Especially, the calcium phosphate composition is crucial for the degradation behavior of a calcium phosphate material. Additionally, at the cellular level the micro‐ and macroporosity, including interconnecting pores, influences both, the dissolution and the osteoclastic resorption. In our study, three different calcium phosphate materials (hydroxyapatite, tricalcium phosphate, and a biphasic calcium phosphate) and two different geometries (dense 2D samples and porous 3D scaffolds) are compared regarding their dissolution and resorption behavior. The results show, that the dissolution of CaP‐ceramics, as examined by the incubation in a degradation solution, depends mainly on the calcium phosphate phase but also on the porosity of the implant. Regarding the resorption, cell proliferation and differentiation of a monocytic cell line as well as the formation of resorption lacunas are analyzed. Cell proliferation is comparable on all phase compositions. Cell differentiation and resorption, however, are influenced by the calcium phosphate phase composition and by the implant porosity as well. By understanding these two mechanisms of degradation, bone substitute materials and, as a result, the bone regeneration of large bone defects using CaP‐ceramics can be improved.     

Fabrication and in vitro evaluations with osteoblast-like MG-63 cells of porous hyaluronic acid-gelatin blend scaffold for bone tissue engineering applications

In this study, hyaluronic acid–gelatin (HyA–Gel) scaffolds were prepared with HyA:Gel ratios of 15:85, 50:50, and 85:15 with the goal of obtaining a porous biocompatible scaffold for bone tissue engineering applications. Scanning electron microscopy and Fourier-transform infrared spectroscopy were done to characterize the morphological orientations of the scaffolds. The biocomposite structure was highly porous and the pores in the scaffolds were interconnected. The compressive strength of the scaffold was 7.39 ± 0.2 MPa for the HyA–Gel when fabricated at a ratio of 15:85. To assess the biocompatibility and cell behavior on the HyA–Gel biocomposite, the proliferation of MG-63 osteoblast cell on the scaffolds was examined using the MTT assay, optical microscopy, and confocal microscopy. Collagen type I and osteopontin expression of cells cultured on the scaffolds were examined using immunoblotting. The scaffolds fabricated with a 15:85—HyA:Gel ratio showed excellent biocompatibility, good mechanical properties, and high porosity, which suggest that the highly porous scaffold holds great promise for use in bone tissue engineering applications.