Structure and properties of nano-hydroxyapatite/polymer composite scaffolds for bone tissue engineering

To better mimic the mineral component and the microstructure of natural bone, novel nano-hydroxyapatite (NHAP)/polymer composite scaffolds with high porosity and well-controlled pore architectures were prepared using thermally induced phase separation (TIPS) techniques. The morphologies, mechanical properties and protein adsorption capacities of the composite scaffolds were investigated. The high porosity (90% and above) was easily achieved and the pore size was adjusted by varying phase separation parameters. The NHAP particles were dispersed in the pore walls of the scaffolds and bound to the polymer very well. NHAP/polymer scaffolds prepared using pure solvent system had a regular anisotropic but open 3D pore structure similar to plain polymer scaffolds while micro-hydroxyapatite (MHAP)/polymer scaffolds had a random irregular pore structure. The introduction of HAP greatly increased the mechanical properties and improved the protein adsorption capacity. In a dioxane/water mixture solvent system, NHAP-incorporated poly(L-lactic acid) (PLLA) scaffolds developed a fibrous morphology which in turn increased the protein adsorption three fold over non fibrous scaffolds. The results suggest that the newly developed NHAP/polymer composite scaffolds may serve as an excellent 3D substrate for cell attachment and migration in bone tissue engineering.     

Biocompatibility and osteogenesis of biomimetic nano-hydroxyapatite/polyamide composite scaffolds for bone tissue engineering

In this study, we prepared nano-hydroxyapatite/polyamide (n-HA/PA) composite scaffolds utilizing thermally induced phase inversion processing technique. The macrostructure and morphology as well as mechanical strength of the scaffolds were characterized. Mesenchymal stem cells (MSCs) derived from bone marrow of neonatal rabbits were cultured, expanded and seeded on n-HA/PA scaffolds. The MSC/scaffold constructs were cultured for up to 7 days and the adhesion, proliferation and differentiation of MSCs into osteoblastic phenotype were determined using MTT assay, alkaline phosphatase (ALP) activity and collagen type I (COL I) immunohistochemical staining and scanning electronic microscopy (SEM). The results confirm that n-HA/PA scaffolds are biocompatible and have no negative effects on the MSCs in vitro. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both pure n-HA/PA scaffolds and MSC/scaffold constructs were implanted in rabbit mandibles and studied histologically and microradiographically. The results show that n-HA/PA composite scaffolds exhibit good biocompatibility and extensive osteoconductivity with host bone. Moreover, the introduction of MSCs to the scaffolds dramatically enhanced the efficiency of new bone formation, especially at the initial stage after implantation. In long term (more than 12 weeks implantation), however, the pure scaffolds show as good biocompatibility and osteogenesis as the hybrid ones. All these results indicate that the scaffolds fulfill the basic requirements of bone tissue engineering scaffold, and have the potential to be applied in orthopedic, reconstructive and maxillofacial surgery.     

Preparation of gamma-PGA/chitosan composite tissue engineering matrices

Gamma-poly(glutamic acid) (gamma-PGA), a hydrophilic and biodegradable polymer, was chosen to modify chitosan matrices to produce a gamma-PGA/chitosan composite biomaterial. Three types of both dense and porous composite matrices containing different amounts of gamma-PGA were fabricated. Chitosan and gamma-PGA matrices were also prepared as controls. Fluorescence staining indicated that chitosan and gamma-PGA were evenly distributed in the composite matrices. SEM micrographs showed that an interconnected porous structure with a pore size of 30-100 microm was present in all porous matrices except the gamma-PGA ones. By increasing the percentage of gamma-PGA from 0% to 20%, the swelling ratio of the matrices was enhanced from 1.6 to 3.2. Similarly, the contact angle of the matrices decreased from 113 degrees to 94 degrees . These data suggested that the surface hydrophilicity, water absorption rate, and swelling ratio were improved by adding gamma-PGA to the matrices. Additionally, the mechanical strength of the porous gamma-PGA/chitosan matrices was about 25-50%, higher than that of the unmodified chitosan matrices. The composite matrices were also examined and found to be an appropriate environment for cell attachment and proliferation. The cell density on the 20% gamma-PGA-modified matrices was almost triple that on the unmodified chitosan matrices on day 5. In summary, the gamma-PGA/chitosan composite matrices, due to their better hydrophilic, cytocompatible, and mechanical properties, are very promising biomaterials for tissue engineering applications.     

Preparation and characterization of nano-hydroxyapatite/chitosan composite scaffolds

A novel nano-hydroxyapatite (HA)/chitosan composite scaffold with high porosity was developed. The nano-HA particles were made in situ through a chemical method and dispersed well on the porous scaffold. They bound to the chitosan scaffolds very well. This method prevents the migration of nano-HA particles into surrounding tissues to a certain extent. The morphologies, components, and biocompatibility of the composite scaffolds were investigated. Scanning electron microscopy, porosity measurement, thermogravimetric analysis, X-ray diffraction, X-ray photoelectron spectroscopy, and Fourier transformed infrared spectroscopy were used to analyze the physical and chemical properties of the composite scaffolds. The biocompatibility was assessed by examining the proliferation and morphology of MC 3T3-E1 cells seeded on the scaffolds. The composite scaffolds showed better biocompatibility than pure chitosan scaffolds. The results suggest that the newly developed nano-HA/chitosan composite scaffolds may serve as a good three-dimensional substrate for cell attachment and migration in bone tissue engineering.     

纳米羟基磷灰石/聚羟基丁酸酯骨组织工程支架的制备及性能表征

背景：高分子聚合物聚羟基丁酸酯具有优良的生物相容性、生物降解性及压电性,但也存在脆性大、亲水性较差等不足。 目的：制备不同组成比例的电纺纳米羟基磷灰石/聚羟基丁酸酯纤维支架材料,分析其结构及性能。 方法：通过气流-高压静电纺丝技术制备纳米羟基磷灰石质量百分比分别为0、10%、20%、30%的电纺纳米羟基磷灰石/聚羟基丁酸酯纤维支架,检测支架材料的微观结构、基团组成、晶相分布、热学性能及表面润湿性。 结果与结论：扫描电镜观察显示,随着纳米羟基磷灰石含量的增大,越来越多的纳米羟基磷灰石颗粒分布于复合纤维表面且分布趋于均匀,到含量达到30%时,纳米羟基磷灰石已基本布满纤维表面,纤维表面的粗糙度也随之增加;差示扫描量热法及X射线衍射结果表明,纳米羟基磷灰石的加入可以降低复合纤维中聚羟基丁酸酯的结晶度及结晶规整程度,且纳米羟基磷灰石含量越大效果越明显;随着纳米羟基磷灰石含量的增大,复合支架表面的接触角逐渐降低,亲水性有所提高。表明将纳米羟基磷灰石与聚羟基丁酸酯复合进行电纺可以有效提高材料的表面润湿性及结晶度,改善材料的亲水性及脆性,且纳米羟基磷灰石含量越高作用越明显。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Preparation and characterization of nano-hydroxyapatite/chitosan/konjac glucomannan composite

Nano-hydroxyapatite (n-HA)/chitosan (CS)/konjac glucomannan (KGM) composite was prepared by coprecipitation method and investigated by thermal gravitivity/differentiate thermal analysis, Fourier transform infrared spectroscopy, X-ray diffraction, inductively coupled plasma emission spectroscopy, scanning electron microscopy, and energy dispersive X-ray analyzer. The analyses showed that the three phases of n-HA, CS, and KGM combined closely to each other. Further, in vitro tests were conducted to investigate the degradation and bioactivity of the composite. During immersion in simulated body fluid (SBF), pores appeared and a new substance containing Ca and P formed on the surface of the composite. Also, the concentration of Ca and P in SBF changed and weight loss of the composite was observed during time. The composite revealed a high degradation in SBF. Evidently, the new composite has a potential to be used as a carrier of implantable drug delivery system. The biodegradation rate and route could be different from CS and KGM, which will provide an opportunity to control the degradation rate or drug releasing rate by simply adjusting the ratio of CS and KGM.     

Design of a composite biomaterial system for tissue engineering applications

Biomaterials that regulate vascularized tissue formation have the potential to contribute to new methods of tissue replacement and reconstruction. The goal of this study was to develop a porous, degradable tissue engineering scaffold that could deliver multiple growth factors and regulate vessel assembly within the porous structure of the material. Porous hydrogels of poly(ethylene glycol)-co-(l-lactic acid) (PEG–PLLA) were prepared via salt leaching. The degradation time of the hydrogels could be controlled between 1 and 7 weeks, based on hydrogel composition. Fibrin was incorporated into the interconnected pores of the hydrogels to promote neovascularization and as a reservoir for rapid (<5 days) growth factor delivery. Poly(lactic-co-glycolic acid) (PLGA) microspheres were incorporated into the degradable polymeric hydrogel scaffold to allow sustained (>30 days) growth factor delivery. Fibroblast growth factor-1 (FGF-1) and platelet-derived growth factor-BB (PDGF-BB) were delivered from the system owing to their roles in the promotion of angiogenesis and vascular stabilization, respectively. Hydrogels tested in vivo with a subcutaneous implantation model were selected based on the results from in vitro degradation and growth factor release kinetics. Dual growth factor delivery promoted significantly more tissue ingrowth in the scaffold compared with blank or single growth factor delivery. The sequential delivery of FGF-1 following PDGF-BB promoted more persistent and mature blood vessels. In conclusion, a biomaterials system was developed to provide structural support for tissue regeneration, as well as delivery of growth factors that stimulate neovascularization within the structure prior to complete degradation.     

天冬氨酸修饰纳米羟基磷灰石/聚乳酸复合物制备软骨组织工程支架及其生物性能研究

目的:通过天冬氨酸修饰的纳米羟基磷灰石（Asp-nHA）和左旋聚乳酸（PLLA）复合,制备软骨组织工程支架,并进行体外实验研究,探索其作为软骨组织工程支架的可行性。 方法:以碳二亚胺盐酸盐/N-羟基琥珀酰亚胺偶联法制备Asp-nHA,并与PLLA复合,获得新型Asp-nHA/PLLA纳米复合支架。比较成骨细胞在该新型材料表面的粘附、生长和增殖的情况。 结果:加入nHA后,Asp-nHA/PLLA复合材料可显著增强细胞的粘附、生长、增殖;Asp-nHA/PLLA的生物学性能明显优于PLLA及HA/PLLA,且Asp-nHA/PLLA具有最强的体外诱导成骨细胞分化能力。 结论:Asp-nHA/PLLA制备简单,具有良好的生物相容性和成骨活性,是一种性能良好的骨组织工程支架材料。     

Tensile properties of bioactive fibers for tissue engineering applications

Cell transplantation using biocompatible, biodegradable scaffolds offers the possibility of creating or regenerating tissue to replace organ function when deficiency arises. The role of these temporary substrates is to support and guide the expanding cell culture until it becomes structurally integrated with the host tissue. 45S5 Bioglass(R) is a 4-component, melt-derived bioactive glass, which has been approved for human clinical use by the Food and Drug Administration. The biocompatibility and biodegradability of 45S5 Bioglass(R) are long established, whereas research into its performance as an extracellular scaffold is currently underway. In this study the tensile strengths (93 +/- 8 and 82 +/- 14 MPa), elongation to fracture (0.7 +/- 0.05%) and Weibull's moduli (3.0 and 3.5) of 45S5 Bioglass(R) fibers (mean diameters 193 and 280 microm) for tissue engineering applications are reported. The tensile strengths of the fibers are compared with those of bulk 45S5 Bioglass(R) and a range of biodegradable polymer materials currently used in the field of tissue engineering. Aspects of glass and fiber technology relevant to the design and manufacture of extracellular ceramic scaffolds are also discussed.     

Synthesis and characterization of polypyrrole-hyaluronic acid composite biomaterials for tissue engineering applications

New tissue engineering technologies will rely on biomaterials that physically support tissue growth and stimulate specific cell functions. The goal of this study was to create a biomaterial that combines inherent biological properties which can specifically trigger desired cellular responses (e.g., angiogenesis) with electrical properties which have been shown to improve the regeneration of several tissues including bone and nerve. To this end, composites of the biologically active polysaccharide hyaluronic acid (HA) and the electrically conducting polymer polypyrrole (PP) were synthesized and characterized. Electrical conductivity of the composite biomaterial (PP/HA) was measured by a four-point probe technique, scanning electron microscopy was used to characterize surface topography, X-ray photoelectron spectroscopy and reflectance infrared spectroscopy were used to evaluate surface and bulk chemistry, and an assay with biotinylated hyaluronic acid binding protein was used to determine surface HA content. PP/HA materials were also evaluated for in vitro cell compatibility and tissue response in rats. Smooth, conductive, HA-containing PP films were produced; these films retained HA on their surfaces for several days in vitro and promoted vascularization in vivo. PP/HA composite biomaterials are promising candidates for tissue engineering and wound-healing applications that may benefit from both electrical stimulation and enhanced vascularization.     

Preparation of porcine carotid arteries for vascular tissue engineering applications

Biomaterials derived from tissue continue to offer viable alternatives to synthetic materials when autologous materials are unavailable for transplantation due to their unique chemical and mechanical properties. Tissue processing aims to stabilize the material against host degradation and render it immunologically inert by removing cellular material and crosslinking the structural proteins. It is clear that different approaches taken to achieve these goals have very different chemical and mechanical effects on the material. We describe herein the development of a tissue processing methodology to generate acellular scaffolds for tissue engineering small-diameter vascular grafts. Carotid arteries were isolated from Great White pigs and exposed to various solvent treatments, xylene, butanol, and ethanol to determine optimal parameters for the extraction of host lipids. The tissue was then exposed to a limited proteolysis with trypsin to disrupt cellular protein. This resulted in a controlled digestion that disrupted porcine nuclear DNA and cleared bulk cellular protein, leaving the more resistant structural proteins largely intact and retaining the bulk mechanical properties of the matrix. Histological analysis and scanning electron microscopy illustrated the complete removal of intact cells and nuclear material. The decellularized graft was stabilized by crosslinking with the photooxidative dye methylene green in the presence of 30,000 LUX of broad-band light energy. High-performance liquid chromatography analysis showed that the crosslinked tissue yielded 78.6% less hydroxyproline, compared with control tissue, after 20 h incubation with pepsin. Analysis of the crosslinked vessels' burst-pressure and stress-strain characteristics have shown comparable mechanical properties to those of control vessels. Assessment of in vitro cell adhesion and compatibility was conducted by seeding primary human umbilical vein endothelial cells and adult human vascular smooth muscle cells onto the lumenal and ablumenal surfaces, respectively; these cells were shown to adhere and proliferate under traditional static culture conditions.     

Preparation and characterization of macroporous chitosan-gelatin/beta-tricalcium phosphate composite scaffolds for bone tissue engineering

A biodegradable composite scaffold was developed using beta-tricalcium phosphate (beta-TCP) with chitosan (CS) and gelatin (Gel) in the form of a hybrid polymer network (HPN) via co-crosslinking with glutaraldehyde. Various types of scaffolds were prepared by freezing and lyophilizing. These scaffolds were characterized by Fourier transform infrared, X-ray diffractometer, and scanning electron microscopy. The macroporous composite scaffolds exhibited different pore structures. Compressive properties were improved, especially compressive modulus from 3.9-10.9 MPa. Biocompatibility was evaluated subcutaneously on rabbits. A mild inflammatory response was observed over 12 weeks. The results suggest that the scaffolds can be utilized in nonloading bone regeneration.     

Preparation and characterization of collagen-hydroxyapatite composite used for bone tissue engineering scaffold

In this study, highly porous collagen-HA scaffolds were prepared by solid-liquid phase separation method. Microstructure of the composites was characterized by SEM, TEM and XRD. The results show that collagen-HA scaffolds are porous with three-dimension interconnected fiber microstructure, pore sizes are 50-150 microm, and HA particles are dispersed evenly among collagen fiber. Compared with pure collagen, the mechanical property of collagen-HA composite improves significantly. To gain further insight into cell growth throughout 3D scaffolds, the cell proliferation and attachment on the scaffold in vitro was investigated. The collagen-HA composite has good biocompatibility, and adding HA does not affect the histocompatibility of the scaffold materials. The porous collagen-HA composite is suitable as scaffold used for bone tissue engineering.     

纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的性能

背景：目前普遍使用的黏合剂对粉碎骨折块进行黏合复位或多或少都存在一些缺陷。 目的：研制具有黏接骨骼作用的生物活性骨水泥。 方法：应用共沉淀法制备纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合材料作为骨水泥的固相粉体,将柠檬酸衍生物配制成溶液作为液相。通过优化实验,从骨水泥的固化时间、抗压强度、抗拉强度、抗稀散性等方面确定最佳配比。 结果与结论：纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠质量比为65/35,其中羧甲基壳聚糖和海藻酸钠质量比为4∶1时复合成粉体,并按固液比为1.0∶0.5(g∶mL)调拌后形成的骨水泥呈膏状,塑形性和抗稀散性能良好,固化时间12~18 min,抗压强度为(4.5±2.1) MPa。体外黏接猪股骨头抗拉强度在不同室温下无显著性差异无显著性意义(P > 0.05),固化后2 h的抗拉强度达到24 h的94%。骨水泥为多孔状结构,孔径为100~300 μm,纳米羟基磷灰石分布较均匀。提示制备的纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥具有良好的生物活性、适当的力学强度以及较好的黏合强度。     

聚磷酸钙纤维/羟基磷灰石/明胶软骨组织工程支架材料的制备及性能

背景：目前明胶基组织工程支架材料存在力学性能低、生物相容性差、降解速率难以控制等缺陷。 目的：通过添加聚磷酸钙纤维和羟基磷灰石改善明胶支架材料的性能。 方法：以自制聚磷酸钙纤维和羟基磷灰石为添加材料,明胶为基体材料,以戊二醛为交联剂,采用溶媒浇铸/粒子滤取技术制备配比为50/10/40的聚磷酸钙纤维/羟基磷灰石/明胶软骨组织工程支架复合材料。测试支架材料的物理力学性能,并观察其微观结构。 结果与结论：采用溶胶凝胶法制得的羟基磷灰石粉末结晶程度较差,经900 ℃下煅烧0.5 h后,可制得结晶程度较高的羟基磷灰石粉末。聚磷酸钙纤维/羟基磷灰石/明胶软骨组织工程支架材料具有三维、连通、微孔网状空间结构,孔隙率在65%-90%之间,满足软骨组织工程对其支架材料孔隙的要求。戊二醛的交联作用和聚磷酸钙纤维的增强作用,克服了明胶在制备多孔支架时容易收缩的缺点,制得高孔隙率三维连通的支架材料。     

Architecture and properties of anisotropic polymer composite scaffolds for bone tissue engineering

Bone is a complex porous composite structure with specific characteristics such as viscoelasticity and anisotropy, both in morphology and mechanical properties. Bone defects are regularly filled with artificial tissue grafts, which should ideally have properties similar to those of natural bone. Open cell composite foams made of bioresorbable poly(L-lactic acid) (PLA) and ceramic fillers, hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP), were processed by supercritical CO2 foaming. Their internal 3D-structure was then analysed by micro-computed tomography (microCT), which evidenced anisotropy in morphology with pores oriented in the foaming direction. Furthermore compressive tests demonstrated anisotropy in mechanical behaviour, with an axial modulus up to 1.5 times greater than the transverse modulus. Composite scaffolds also showed viscoelastic behaviour with increased modulus for higher strain rates. Such scaffolds prepared by gas foaming of polymer composite materials therefore possess suitable architecture and properties for bone tissue engineering applications.     

Preparation and evaluation of the electrospun chitosan/PEO fibers for potential applications in cartilage tissue engineering

Fibrous materials have morphological similarities to natural cartilage extracellular matrix and have been considered as candidate for bone tissue engineering scaffolds. In this study, we have evaluated a novel electrospun chitosan mat composed of oriented sub-micron fibers for its tensile property and biocompatibility with chondrocytes (cell attachment, proliferation and viability). Scanning electronic microscope images showed the fibers in the electrospun chitosan mats were indeed aligned and there was a slight cross-linking between the parent fibers. The electrospun mats have significantly higher elastic modulus (2.25 MPa) than the cast films (1.19 MPa). Viability of cells on the electrospun mat was 69% of the cells on tissue-culture polystyrene (TCP control) after three days in culture, which was slightly higher than that on the cast films (63% of the TCP control). Cells on the electrospun mat grew slowly the first week but the growth rate increased after that. By day 10, cell number on the electrospun mat was almost 82% that of TCP control, which was higher than that of cast films (56% of TCP). The electrospun chitosan mats have a higher Young's modulus (P < 0.01) than cast films and provide good chondrocyte biocompatibility. The electrospun chitosan mats, thus, have the potential to be further processed into three-dimensional scaffolds for cartilage tissue repair.     

Mechanical properties of highly porous PDLLA/Bioglass composite foams as scaffolds for bone tissue engineering

This study developed highly porous degradable composites as potential scaffolds for bone tissue engineering. These scaffolds consisted of poly-d,l-lactic acid filled with 2 and 15 vol.% of 45S5 Bioglass^® particles and were produced via thermally induced solid–liquid phase separation and subsequent solvent sublimation. The scaffolds had a bimodal and anisotropic pore structure, with tubular macro-pores of 100 μm in diameter, and with interconnected micro-pores of 10–50 μm in diameter. Quasi-static and thermal dynamic mechanical analysis carried out in compression along with thermogravimetric analysis was used to investigate the effect of Bioglass^® on the properties of the foams. Quasi-static compression testing demonstrated mechanical anisotropy concomitant with the direction of the macro-pores. An analytical modelling approach was applied, which demonstrated that the presence of Bioglass^® did not significantly alter the porous architecture of these foams and reflected the mechanical anisotropy which was congruent with the scanning electron microscopy investigation. This study found that the Ishai–Cohen and Gibson–Ashby models can be combined to predict the compressive modulus of the composite foams. The modulus and density of these complex foams are related by a power-law function with an exponent between 2 and 3.     

Electrospun PHBV/collagen composite nanofibrous scaffolds for tissue engineering

Electrospinning has recently emerged as a leading technique for the formation of nanofibrous structures made of synthetic and natural extracellular matrix components. In this study, nanofibrous scaffolds were obtained by electrospinning a combination of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and type-I collagen in 1,1,1,3,3,3-hexafluoro-2-isopropanol (HIFP). The resulting fibers ranged from 300 to 600 nm in diameter. Their surfaces were characterized by attenuated total reflection Fourier transform infrared spectroscopy, electron spectroscopy for chemical analysis and atomic force microscopy. The PHBV and collagen components of the PHBV/collagen nanofibrous scaffold were biodegraded by PHB depolymerase and a type-I collagenase aqueous solution, respectively. The cell culture experiments indicated that the PHBV/collagen nanofibrous scaffold accelerated the adhesion and growth of NIH3T3 cells more effectively than the PHBV nanofibrous scaffold, thus making the former a good scaffold for tissue engineering.     

Carbon nanotube applications for tissue engineering

As the field of tissue engineering advances, new tools for better monitoring and evaluating of engineered tissues along with new biomaterials to direct tissue growth are needed. Carbon nanotubes may be an important tissue engineering material for improved tracking of cells, sensing of microenvironments, delivering of transfection agents, and scaffolding for incorporating with the host's body. Using carbon nanotubes for optical, magnetic resonance and radiotracer contrast agents would provide better means of evaluating tissue formation. In addition, monitoring and altering intra and intercellular processes would be useful for design of better engineered tissues. Carbon nanotubes can also be incorporated into scaffolds providing structural reinforcement as well as imparting novel properties such as electrical conductivity into the scaffolds may aid in directing cell growth. Potential cytotoxic effects associated with carbon nanotubes may be mitigated by chemically functionalizing the surface. Overall, carbon nanotubes may play an integral role as unique biomaterial for creating and monitoring engineered tissue.