羟基磷灰石/纤维素骨仿生支架的制备及性能研究

为探究制备方法对羟基磷灰石/纤维素支架材料性能的影响,采用仿生矿化法、原位复合法和溶液共混法制备了3种羟基磷灰石(HA)/纤维素支架材料,表征了3种支架材料的结构、形貌和热稳定性,分析了制备方法对支架材料生物相容性的影响。结果表明：3种方法获得的复合支架中,HA和纤维素气凝胶(CAS)之间仅存在物理作用,且HA的引入提高了复合支架的稳定性,但仿生矿化法和原位复合法因CAS的引入限制了HA的生长,结晶度较低。3种复合材料均无细胞毒性且具有生物相容性,但仿生矿化法制备的复合支架材料具有较大的孔径、较高的比表面积,更有利于细胞的粘附及增殖。因此,仿生矿化法是制备医用骨支架材料的最佳途径。     

羟基磷灰石基多孔复合支架的制备及其表征

研究利用造孔剂法制备高度贯通的多孔羟基磷灰石(HA)支架,孔隙率约为78%,并利用聚己内酯(PCL)分别复合纳米HA(nHA)或微纳米生物玻璃(nBG)粉末对其进行涂覆改性,粉末的添加量均为10%~40%(质量分数)。4种类型支架分别记为HA、PCL/HA、nHA-PCL/HA和nBG-PCL/HA。实验结果发现,nHA-PCL/HA和nBG-PCL/HA复合支架最大抗压强度分别为1.41~1.98 MPa和1.35~1.78MPa。4类支架矿化实验显示,浸泡21d后nBG-PCL/HA表面促进生成较多的磷灰石矿化物;细胞实验结果显示细胞在4类支架上均生长良好,说明支架具有良好的生物相容性。支架在实验犬背部肌肉组织内植入2个月的组织学检测显示,4种支架内均有新骨形成,尤其是nHA-PCL/HA和nBG-PCL/HA孔内有更多的新生骨组织,说明这两种支架表面复合涂层中的生物活性纳米颗粒对诱导新骨生成具有积极的促进作用。     

生物陶瓷及其复合材料表面诱导类骨磷灰石层形成的研究

钙磷生物材料表面类骨磷灰石层的形成对其植入体内诱导新骨生成起非常重要的作用.本实验采用2倍模拟体液(2×SBF)为介质,通过仿生浸泡的方法研究了羟基磷灰石(HA)陶瓷及其复合材料羟基磷灰石/聚乳酸(HA/PLA)、羟基磷灰石/聚己內脂(HA/PCL)、羟基磷灰石/丝素(HA/SF)和羟基磷灰石/壳聚糖(HA/CS)表面诱导类骨磷灰石层形成的差异.实验结果表明,HA陶瓷及其复合材料在2×SBF溶液中仿生浸泡7d后,各样品表面均有一层厚度不同的类骨磷灰石层生成.并且该类骨磷灰石层的结晶度较低,晶粒较细(15～30nm),与人体自然骨无机物的结构非常类似.其中,在这4种复合材料中,HA/CS和HA/SF复合材料中因丝素蛋白和壳聚糖富含多种功能基团,能有效促进类骨磷灰石晶体的形核和生长,因而诱导类骨磷灰石生成的能力最强,显示其良好的生物活性.     

羟基磷灰石接枝壳聚糖表面改性及其复合水凝胶的生物相容性

用壳聚糖修饰HA表面制备HA-接枝-壳聚糖纳米羟基磷灰石(HA-g-CS),然后将其与壳聚糖共混制成CS/HA-g-CS复合水凝胶。FTIR、TGA、XRD的测试结果表明,CS已经成功地接枝到HA的表面,接枝率为15.8%;SEM结果表明,HA-g-CS在CS基体的分散性相对于HA得到明显的改善,且CS/HA-g-CS比CS/HA复合水凝胶的抗压强度提高了43%。CS/HA-gCS生物相容性评价的结果表明,材料的细胞毒性和植入安全性均达到了国家标准要求。这表明,CS/HA-g-CS复合水凝胶可作为一种优良的支架材料应用于组织工程领域。     

三维打印羟基磷灰石晶须增强复合骨修复支架

利用三维打印技术成功制备羟基磷灰石晶须(HAPw)增强的聚己内酯(PCL)复合骨修复支架。通过改变三维打印的挤出速度和挤出气压, 使不同含量HAPw均能在PCL基材中一致排列并均匀分布。PCL支架的机械强度随HAPw含量增加显著提高, 添加33wt%HAPw使PCL支架强度提升了高达3倍。此外, HAPw使PCL支架表面与水的接触角从近100º降低至约50º, 有效改善了细胞表面粘附。经过体外人类骨髓间充质干细胞(hBMSC)在支架上的培养实验, 发现添加HAPw的复合支架具有更好的生物相容性, 能够有效促进hBMSC的增殖生长, 且HAPw-PCL复合支架上细胞具有更高的碱性磷酸酶(ALP)活性和OCN、RUNX2等相关成骨基因表达, 显示出hBMSCs向成骨方向更好的分化及成骨活性。     

纳米非计量磷灰石/聚合物复合支架的制备和性能研究

采用快速成形法制备了孔径和孔隙率可控、大孔互相贯通的纳米缺钙羟基磷灰石(cd-HA)与聚己内酯(PCL)复合材料多孔支架,并对复合支架的微结构进行了表征.通过细胞培养和体内动物实验研究了该支架的生物学性能.结果表明:复合材料的亲水性和细胞粘附率随磷灰石含量增加而提高;成骨细胞在复合支架上的增殖明显高于纯PCL;μ-CT和组织学分析结果显示,新骨在支架的表面形成并长入其中.相互贯通的多孔支架促进了细胞的增殖和新骨长入支架内部.cd-HA/PCL复合材料支架具有很好的生物相容性,在组织工程领域中有潜在的应用前景.     

仿生壳聚糖棒材表面的生物矿化

首先采用原位沉析法制得仿木年轮结构的壳聚糖(Chitosan,CS)凝胶棒材,然后在此表面进行生物矿化交替沉积,得到羟基磷灰石(Hydroxyapatite,HA)涂层.经XRD测试,结果证明棒材表面确实生成了HA.SEM测试表明,HA层为多孔蜂窝状结构,孔径均一为2μm左右.CS棒材的水相接触角从沉积前的87°士5°提高到沉积后的105°士4°,棒材吸水率沉积HA后也有所降低,矿化后棒材仍然保持较高力学强度.上述结果表明,生物矿化后的CS棒材有望在骨移植和骨替代方面具有一定的应用价值.     

原位增强羟基磷灰石/壳聚糖复合棒材

利用低温水溶液均相沉积法制备了磷酸钙盐微纤维; 应用原位沉析法制备了壳聚糖(CS)三维棒材及羟基磷灰石(HA*)/CS复合棒材。XRD证实应用原位沉析法制备HA*/CS复合棒材过程中, 磷酸钙盐转化为羟基磷灰石结构, 尺寸为10~60 μm, 并用SEM对晶体形貌进行了表征, 分析了转化机制。HA*/CS复合材料的微观形貌表明, HA*晶体在CS凝胶棒原位沉析的过程中析出而与CS基体形成镶嵌、 相互咬合结构, 且在基体中分散均匀, 有效地提高了HA*与CS基体的界面连接作用, 使力学性能显著提高。所制备的HA*/CS棒材随HA*含量的增大(在其饱和溶解度3.3 wt%范围内), 复合材料的弯曲性能逐渐提高, 当羟基磷灰石质量分数为3.3%时, 复合材料的弯曲强度达到159.6 MPa, 弯曲模量达到5.1 GPa, 比CS基体分别提高85.6%和54.5%。HA*/CS复合棒材的弯曲强度和弯曲模量远高于松质骨, 弯曲强度也比密质骨高。      

羟基磷灰石/聚醚酯聚氨酯支架的体外降解行为和细胞相容性研究

采用XRD、DSC、体外降解实验和细胞相容性实验等方法对羟基磷灰石/聚醚酯聚氨酯(HA/PU)多孔支架的结构和性能进行了研究。结果表明,HA粒子添加到聚醚酯聚氨酯基体中,在一定程度上降低了聚氨酯软段的结晶,提高了聚氨酯基体的力学性能。体外降解实验表明HA/PU复合支架的降解不会引起浸泡液pH值较大的波动,且降解初期的力学性能衰减缓慢。MG63细胞与HA/PU复合支架共培养的实验表明,细胞生长良好,牢固地黏附在支架表面,并在支架表面充分伸展,复合支架具有良好的细胞相容性。这些结果表明HA/PU支架有望用于骨组织工程修复。     

钛表面制备羟基磷灰石/壳聚糖复合涂层研究

通过原位水热合成和溶胶-凝胶浸提涂敷法在碱处理的钛表面制备了HA/CS复合涂层. 接触角检测表明碱处理使钛表面具有超亲水性.X射线衍射分析表明复合涂层成分为HA和CS, 各组分含量由热重分析确定. 用扫描电镜对复合涂层的形貌进行观察,发现不同HA含量的复合涂层具有不同的形貌. 通过培养成骨细胞考察了复合涂层的细胞相容性.Alamar Blue检测表明HA/CS复合涂层表面细胞粘附及增殖能力较好. ALP检测表明HA/CS复合涂层表面的细胞分化能力较好. 综合研究结果表明, 复合涂层有较好的细胞相容性.     

In vitro study of hydroxyapatite/polycaprolactone (HA/PCL) nanocomposite synthesized by an in situ sol–gel process

Hydroxyapatite (HA) is the most substantial mineral constituent of a bone which has been extensively used in medicine as implantable materials, owing to its good biocompatibility, bioactivity high osteoconductive, and/or osteoinductive properties. Nevertheless, its mechanical property is not utmost appropriate for a bone substitution. Therefore, a composite consist of HA and a biodegradable polymer is usually prepared to generate an apt bone scaffold. In the present work polycaprolactone (PCL), a newly remarkable biocompatible and biodegradable polymer, was employed as a matrix and hydroxyapatite nanoparticles were used as a reinforcement element of the composite. HA/PCL nanocomposites were synthesized by a new in situ sol–gel process using calcium hydroxide and phosphoric acid precursors in the presence of Tetrahydrofuran (THF) as a solvent. Chemical and physical characteristics of the nanocomposite were studied by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM) and Fourier transform infrared (FTIR) analyses. The results indicated that pure HA nanoparticles were well-incorporated and homogenously dispersed in the PCL matrix. It was found that the mechanical property of PCL was improved by addition of 20 wt.% HA nanoparticles. Furthermore, the biological property of nanocomposites was investigated under in vitro condition. For this purpose, HA/PCL scaffolds were prepared through a salt leaching process and immersed in a saturated simulated body fluid (SBF) after 3 and 7 days. It was found that a uniform layer of biomimetic HA could be deposited on the surface of HA/PCL scaffolds. Therefore, the prepared HA/PCL scaffolds showed good potential for bone tissue engineering and could be used for many clinical applications in orthopedic and maxillofacial surgery.     

纳米羟基磷灰石/聚己内酯复合生物活性多孔支架研究

采用水热法制备了纳米羟基磷灰石（n-HA）及其与聚己内酯（PCL）的复合材料. 用熔融浇铸/食盐微粒浸出法制备了孔径在200～400μｍ、大孔互相贯通的复合材料支架. 通过细胞培养和体内动物实验研究了该支架的生物学性能. 结果表明,复合支架的孔隙率随致孔剂用量的增加而增加,而抗压强度随之而减小;支架的最大孔隙率可达86%,相应的抗压强度为2.4MPa. 成骨细胞在支架上的细胞粘附率和增殖随磷灰石含量增加而提高,复合材料明显高于单纯的PCL支架. 组织学观察显示,新生骨长入多孔支架和复合材料形成了直接的骨性结合. n-HA/PCL复合材料支架有很好的生物相容性和生物活性.     

Hybrid composite scaffolds prepared by sol–gel method for bone regeneration

This study reports the synthesis and characterization of hydroxyapatite (HA)/polycaprolactone (PCL) hybrid composite materials synthesized by sol–gel method. The fabrication of scaffolds was performed by salt-leaching technique using NaCl as porogen agent. In the first step, the physico-chemical characterization of composite material was performed to evaluate the composition and the interaction between the organic/inorganic phases. In the second step, optimized scaffolds were bioactivated on the surface. The combined effect of scaffold morphology and surface bioactivity is ideal for bone tissue engineering, supporting bone cells adhesion, proliferation and differentiation. Here, a combined strategy involving biomimetic approach, using a supersaturated Simulated Body Fluid (SBF), and salt-leaching technique has been developed to grow hydroxyapatite in composite scaffolds able to regenerate the natural bone.     

Electrospun PCL/PLA/HA based nanofibers as scaffold for osteoblast-like cells

Polycaprolactone (PCL), poly (lactic acid) (PLA) and hydroxyapatite (HA) are frequently used as materials for tissue engineering. In this study, PCL/PLA/HA nanofiber mats with different weight ratio were prepared using electrospinning. Their structure and morphology were studied by FTIR and FESEM. FTIR results demonstrated that the HA particles were successfully incorporated into the PCL/PLA nanofibers. The FESEM images showed that the surface of fibers became coarser with the introduction of HA nanoparticles into PCL/PLA system. Furthermore, the addition of HA led to the decreasing of fiber diameter. The average diameters of PCL/PLA/HA nanofiber were in the range of 300-600 nm, while that of PCL/PLA was 776 +/- 15.4 nm. The effect of nanofiber composition on the osteoblast-like MC3T3-E1 cell adhesion and proliferation were investigated as the preliminary biological evaluation of the scaffold. The MC3T3-E1 cell could be attached actively on all the scaffolds. The MTT assay revealed that PCL/PLA/HA scaffold shows significantly higher cell proliferation than PCL/PLA scaffolds. After 15 days of culture, mineral particles on the surface of the cells was appeared on PCL/PLA/HA nanofibers while normal cell spreading morphology on PCL/PLA nanofibers. These results manifested that electrospun PCL/PLA/HA scaffolds could enhance bone regeneration, showing their marvelous prospect as scaffolds for bone tissue engineering.     

Electrospun fibrous scaffold of hydroxyapatite/poly (ε-caprolactone) for bone regeneration

Development of fibrous scaffold of hydroxyapatite/biopolymer nanocomposite offers great potential in the field of bone regeneration and tissue engineering. Hydroxyapatite (HA)/poly (ε-caprolactone) (PCL) fibrous scaffolds were successfully prepared by electrospinning dopes containing HA and PCL in this work. It was found that pre-treating HA with γ-glycioxypropyltrimethoxysilane (A-187) was effective in improving HA dispersion both in solutions and in a PCL matrix. Mechanical properties of the scaffolds were greatly enhanced by the filling of A187-HA. The bioactivity of PCL was remarkably improved by the addition of HA and A187-HA. Fibroblasts and osteoblasts were seeded on scaffolds to evaluate the effect of A-187 on biocompatibility of HA/PCL composites. Based on this study, good dispersion of HA in PCL matrix was granted by pretreatment of HA with A-187 and A187-HA/PCL fibrous scaffolds were obtained by electrospinning. These results demonstrated that the scaffolds may possess improved mechanical performance and good bioactivity due to A187-HA incorporation.     

Hardystonite improves biocompatibility and strength of electrospun polycaprolactone nanofibers over hydroxyapatite: A comparative study

The aim of this study was to compare physico-chemical and biological properties of hydroxyapatite (HA) and hardystonite (HS) based composite scaffolds. Hardystonite (Ca₂ZnSi₂O₇) powders were synthesized by a sol–gel method while polycaprolactone–hardystonite (PCL–HS) and polycaprolactone–hydroxyapatite (PCL–HA) were fabricated in nanofibrous form by electrospinning. The physico-chemical and biological properties such as tensile strength, cell proliferation, cell infiltration and alkaline phosphatase activity were determined on both kinds of scaffolds. We found that PCL–HS scaffolds had better mechanical strength compared to PCL–HA scaffolds. Addition of HA and HS particles to PCL did not show any inhibitory effect on blood biocompatibility of scaffolds when assessed by hemolysis assay. The in vitro cellular behavior was evaluated by growing murine adipose-tissue-derived stem cells (mE-ASCs) over the scaffolds. Enhanced cell proliferation and improved cellular infiltrations on PCL–HS scaffolds were observed when compared to HA containing scaffolds. PCL–HS scaffolds exhibited a significant increase in alkaline phosphatase (ALP) activity and better mineralization of the matrix in comparison to PCL–HA scaffolds. These results clearly demonstrate the stimulatory role of Zn and Si present in HS based composite scaffolds, suggesting their potential application for bone tissue engineering.     

Electrospun chitosan/hydroxyapatite nanofibers for bone tissue engineering

Chitosan (CS) nanofibers were prepared by an electrospinning technique and then treated with simulated body fluid (SBF) to encourage hydroxyapatite (HA) formation on their surface. The CS/HA nanofibers were subjected to scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), Fourier transform infrared spectroscopy, and X-ray diffraction (XRD) to confirm HA formation as well as determine the morphology of the nanofibrous scaffolds. The SEM image indicated that the distribution of HA on the CS nanofibers was homogeneous. The results from EDS and XRD indicated that HA was formed on the nanofibrous surfaces after 6-day incubation in the SBF. The calcium/phosphorus ratio of deposited HA was close to that of natural bone. To determine biocompatibility, the CS/HA scaffolds were applied to the culture of rat osteosarcoma cell lines (UMR-106). The cell densities on the CS/HA nanofibers were higher than those on the CS nanofibers, the CS/HA film, and the CS film, indicating that cell proliferation on CS/HA nanofibers was enhanced. Moreover, the early osteogenic differentiation on CS/HA was also more significant, due to the differences in chemical composition and the surface area of CS/HA nanofibers. The biocompatibility and the cell affinity were enhanced using the CS/HA nanofibers. This indicates that electrospun CS/HA scaffolds would be a potential material in bone tissue engineering.     

Porous scaffolds of polycaprolactone reinforced with in situ generated hydroxyapatite for bone tissue engineering

Polycaprolactone/hydroxyapatite (PCL/HA) composites were prepared by in situ generation of HA in the polymer solution starting from the precursors calcium nitrate tetrahydrate and ammonium dihydrogen phosphate via sol–gel process. Highly interconnected porosity was achieved by means of the salt-leaching technique using a mixture of sodium chloride and sodium bicarbonate as porogens. Structure and morphology of the PCL/HA composites were investigated by scanning electron microscopy, and mechanical properties were determined by means of tensile and compression tests. The possibility to employ the developed composites as scaffolds for bone tissue regeneration was assessed by cytotoxicity test of the PCL/HA composites extracts and cell adhesion and proliferation in vitro studies.     

Design of novel three-phase PCL/TZ–HA biomaterials for use in bone regeneration applications

The design of bioactive scaffold materials able to guide cellular processes involved in new-tissue genesis is key determinant in bone tissue engineering. The aim of this study was the design and characterization of novel multi-phase biomaterials to be processed for the fabrication of 3D porous scaffolds able to provide a temporary biocompatible substrate for mesenchymal stem cells (MSCs) adhesion, proliferation and osteogenic differentiation. The biomaterials were prepared by blending poly(ε-caprolactone) (PCL) with thermoplastic zein (TZ), a thermoplastic material obtained by de novo thermoplasticization of zein. Furthermore, to bioactivate the scaffolds, microparticles of osteoconductive hydroxyapatite (HA) were dispersed within the organic phases. Results demonstrated that materials and formulations strongly affected the micro-structural properties and hydrophilicity of the scaffolds and, therefore, had a pivotal role in guiding cell/scaffold interaction. In particular, if compared to neat PCL, PCL–HA composite and PCL/TZ blend, the three-phase PCL/TZ–HA showed improved MSCs adhesion, proliferation and osteogenic differentiation capability, thus demonstrating potential for bone regeneration.     

Robotic dispensing of composite scaffolds and in vitro responses of bone marrow stromal cells

The development of bioactive scaffolds with a designed pore configuration is of particular importance in bone tissue engineering. In this study, bone scaffolds with a controlled pore structure and a bioactive composition were produced using a robotic dispensing technique. A poly(ε-caprolactone) (PCL) and hydroxyapatite (HA) composite solution (PCL/HA = 1) was constructed into a 3-dimensional (3D) porous scaffold by fiber deposition and layer-by-layer assembly using a computer-aided robocasting machine. The in vitro tissue cell compatibility was examined using rat bone marrow stromal cells (rBMSCs). The adhesion and growth of cells onto the robotic dispensed scaffolds were observed to be limited by applying the conventional cell seeding technique. However, the initially adhered cells were viable on the scaffold surface. The alkaline phosphatase activity of the cells was significantly higher on the HA–PCL than on the PCL and control culture dish, suggesting that the robotic dispensed HA–PCL scaffold should stimulate the osteogenic differentiation of rBMSCs. Moreover, the expression of a series of bone-associated genes, including alkaline phosphatase and collagen type I, was highly up-regulated on the HA–PCL scaffold as compared to that on the pure PCL scaffold. Overall, the robotic dispensed HA–PCL is considered to find potential use as a bioactive 3D scaffold for bone tissue engineering. Seok-Jung Hong and Ishik Jeong contributed equally.