Preparation and characterization of bionic bone structure chitosan/hydroxyapatite scaffold for bone tissue engineering

Three-dimensional oriented chitosan (CS)/hydroxyapatite (HA) scaffolds were prepared via in situ precipitation method in this research. Scanning electron microscopy (SEM) images indicated that the scaffolds with acicular nano-HA had the spoke-like, multilayer and porous structure. The SEM of osteoblasts which were polygonal or spindle-shaped on the composite scaffolds after seven-day cell culture showed that the cells grew, adhered, and spread well. The results of X-ray powder diffractometer and Fourier transform infrared spectrometer showed that the mineral particles deposited in the scaffold had phase structure similar to natural bone and confirmed that particles were exactly HA. In vitro biocompatibility evaluation indicated the composite scaffolds showed a higher degree of proliferation of MC3T3-E1 cell compared with the pure CS scaffolds and the CS/HA10 scaffold was the highest one. The CS/HA scaffold also had a higher ratio of adhesion and alkaline phosphate activity value of osteoblasts compared with the pure CS scaffold, and the ratio increased with the increase of HA content. The ALP activity value of composite scaffolds was at least six times of the pure CS scaffolds. The results suggested that the composite scaffolds possessed good biocompatibility. The compressive strength of CS/HA15 increased by 33.07% compared with the pure CS scaffold. This novel porous scaffold with three-dimensional oriented structure might have a potential application in bone tissue engineering.     

生物活性玻璃与壳聚糖复合的骨修复材料

背景：生物活性玻璃是一种多相复合材料,具有良好的生物活性、骨传导性及生物相容性,但作为骨修复材料仍然存在不能完全降解、机械强度较低等不足。 目的：设计生物活性玻璃/壳聚糖复合材料骨组织工程支架,并检测其理化性能。 方法：将2.0%壳聚糖盐酸溶液与β-甘油磷酸钠以7∶1的体积比混合制备壳聚糖溶液。称取0.5,1.0,1.5 g生物活性玻璃分别加入上述壳聚糖溶液中,使得壳聚糖与生物活性玻璃的质量比为2∶1,1∶1及1∶1.5。将复合材料浸泡于模拟生理体液中7 d进行体外矿化。 结果与结论：扫描电镜见复合支架具有相互贯通的多孔结构,孔隙率最高可达89%,孔径大小合适,为100-  300 µm,生物活性玻璃以针状形式分散在壳聚糖支架之间,均匀排列,被壳聚糖支架充分包裹结合紧密。随生物活性玻璃含量的增加,复合材料的孔隙率逐渐下降,断裂强度逐渐升高,他们之间呈正相关性。X射线衍射图及傅里叶变换红外光谱证实复合支架中的单一材料未发生性质改变,示差扫描量热法分析显示正常体温情况下材料无质量丢失。矿化3 d后材料表面形成的羟基磷灰石逐渐长大为绒毛状,数量也明显增多;矿化7 d后绒毛状的羟基磷灰石长成为针状,数量进一步增多,且众多的矿化物结成球状。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;组织工程全文链接：     

Comparison of the properties of collagen–chitosan scaffolds after γ-ray irradiation and carbodiimide cross-linking

The property of collagen–chitosan porous scaffold varies according to cross-linking density and scaffold composition. This study was designed to compare the properties of collagen–chitosan porous scaffolds cross-linked with γ-irradiation and carbodiimide (CAR) for the first time. Eleven sets of collagen–chitosan scaffolds containing different concentrations of chitosan at a 5% increasing gradient were fabricated. Fourier transform infrared spectroscopy was performed to confirm the success of cross-linking in the scaffolds. The scaffold morphology was evaluated under scanning electron microscope (SEM). SEM revealed that chitosan was an indispensable material for the fabrication of γ-ray irradiation scaffold. The microstructure of γ-ray irradiation scaffold was less stable than those of alternative scaffolds. Based upon swelling ratio, porosity factor, and collagenase degradation, γ-ray irradiation scaffold was less stable than CAR and 25% proportion of chitosan scaffolds. Mechanical property determines the orientation in γ-irradiation and CAR scaffold. In vitro degradation test indicated that γ-irradiation and CAR cross-linking can elevate the scaffold biocompatibility. Compared with γ-ray irradiation, CAR cross-linked scaffold containing 25% chitosan can more significantly enhance the bio-stability and biocompatibility of collagen–chitosan scaffolds. CAR cross-linked scaffold may be the best choice for future tissue engineering.     

Fabrication and characterization of gelatin-based biocompatible porous composite scaffold for bone tissue engineering

In this study, composite scaffolds were prepared with polyethylene oxide (PEO)-linked gelatin and tricalcium phosphate (TCP). Chitosan, a positively charged polysaccharide, was introduced into the scaffolds to improve the properties of the artificial bone matrix. The chemical and thermal properties of composite scaffolds were investigated by Fourier transform infrared spectroscopy, thermogravimetric analyzer, differential thermal analyzer. In vitro cytotoxicity of the composite scaffold was also evaluated and the sample showed no cytotoxic effect. The morphology was studied by SEM and light microscopy. It was observed that the prepared scaffold had an open interconnected porous structure with pore size of 230-354 μm, which is suitable for osteoblast cell proliferation. The mechanical properties were assessed and it was found that the composite had compressive modulus of 1200 MPa with a strength of 5.2 MPa and bending modulus of 250 MPa having strength of 12.3 MPa. The porosity and apparent density were calculated and it was found that the incorporation of TCP can reduce the porosity and water absorption. It was revealed from the study that the composite had a 3D porous microstructure and TCP particles were dispersed evenly among the crosslinked gelatin/chitosan scaffold. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3020-3028, 2012.     

Fabrication and characterization of novel nano- and micro-HA/PCL composite scaffolds using a modified rapid prototyping process

Novel three-dimensional scaffolds consisting of nano- and microsized hydroxyapatite (HA)/poly(epsilon-caprolactone) (PCL) composite were fabricated using a modified rapid-prototyping (RP) technique for bone tissue engineering applications. The size of the nano-HA ranged from 20 to 90 nm, whereas that of the micro-HA ranged from 20 to 80 microm. The scaffold macropores were well interconnected, with a porosity of 72-73% and a pore size of 500 microm. The compressive modulus of the nano-HA/PCL and micro-HA/PCL scaffolds was 3.187 +/- 0.06 and 1.345 +/- 0.05 MPa, respectively. The higher modulus of the nano-HA/PCL composite (n-HPC) was to be likely caused by a dispersion strengthening effect. The attachment and proliferation of MG-63 cells on n-HPC were better than that on the micro-HA/PCL composite (m-HPC) scaffold. The n-HPC was more hydrophilic than the m-HPC because of the greater surface area of HA exposed to the scaffold surface. This may give rise to better cell attachment and proliferation. Bioactive n-HA/PCL composite scaffold prepared using a modified RP technique has a potential application in bone tissue engineering.     

壳聚糖/磷酸三钙复合组织工程支架材料的制备及其性能*

背景：通过适当的工艺混合、加工来制备复合支架材料,可以弥补单一材料的不足,最大限度地满足组织工程的需要。 目的：制备壳聚糖/磷酸三钙复合支架,探讨其作为牙髓组织工程支架材料的可行性。 方法：壳聚糖粉末溶于微量冰醋酸溶液中,搅拌均匀,静置脱泡,预冷冻,交联,再次冷冻制成海绵状多孔壳聚糖/磷酸三钙支架。 结果与结论：冻干法制备的壳聚糖/磷酸三钙多孔支架平均孔隙率达85.78%,最高孔隙率达90%以上,孔径在100~300 μm,复合后的支架材料具有良好的韧性,当轴向压缩变形量超过5 mm时,材料仍然没有发生破坏。材料浸提液与牙髓细胞复合培养后,细胞毒性均为0级,由此可见壳聚糖/磷酸三钙复合材料具有良好的生物相容性、细胞亲和性和一定的力学性能,满足生物材料基本要求。     

In vitro and animal study of novel nano-hydroxyapatite/poly(epsilon-caprolactone) composite scaffolds fabricated by layer manufacturing process

The purpose of this study was to propose a computer-controllable scaffold structure made by a layer manufacturing process (LMP) with addition of nano- or micro-sized particles and to investigate the effects of particle size in vitro. In addition, the superiority of this LMP method over the conventional scaffolds made by salt leaching and gas forming process was investigated through animal study. Using the LMP, we have created a new nano-sized hydroxyapatite/poly(epsilon-caprolactone) composite (n-HPC) scaffold and a micro-sized hydroxyapatite/poly(epsilon-caprolactone) composite (m-HPC) scaffold for bone tissue engineering applications. The scaffold macropores were well interconnected, with a porosity of 73% and a pore size of 500 microm. The compressive modulus of the n-HPC and m-HPC scaffolds was 6.76 and 3.18 MPa, respectively. We compared the cellular responses to the two kinds of scaffolds. Both n-HPC and m-HPC exhibited good in vitro biocompatibility. Attachment and proliferation of mesenchymal stem cells were better on the n-HPC than on the m-HPC scaffold. Moreover, significantly higher alkaline phosphatase activity and calcium content were observed on the n-HPC than on the m-HPC scaffold. In an animal study, the LMP scaffolds enhanced bone formation, owing to their well-interconnected pores. Radiological and histological examinations confirmed that the new bony tissue had grown easily into the entire n-HPC scaffold fabricated by LMP. We suggest that the well-interconnected pores in the LMP scaffolds might encourage cell attachment, proliferation, and migration to stimulate cell functions, thus enhancing bone formation in the LMP scaffolds. This study shows that bioactive and biocompatible n-HPC composite scaffolds prepared using an LMP have potential applications in bone tissue engineering.     

Design and characterization of a novel chitosan/nanocrystalline calcium phosphate composite scaffold for bone regeneration

To meet the challenge of regenerating bone lost to disease or trauma, biodegradable scaffolds are being investigated as a way to regenerate bone without the need for an auto- or allograft. Here, we have developed a novel microsphere-based chitosan/nanocrystalline calcium phosphate (CaP) composite scaffold and investigated its potential compared to plain chitosan scaffolds to be used as a bone graft substitute. Composite and chitosan scaffolds were prepared by fusing microspheres of 500-900 microm in diameter, and porosity, degradation, compressive strength, and cell growth were examined. Both scaffolds had porosities of 33-35% and pore sizes between 100 and 800 . However, composite scaffolds were much rougher and, as a result, had 20 times more surface area/unit mass than chitosan scaffolds. The compressive modulus of hydrated composite scaffolds was significantly higher than chitosan scaffolds (9.29 +/- 0.8 MPa vs. 3.26 +/- 2.5 MPa), and composite scaffolds were tougher and more flexible than what has been reported for other chitosan-CaP composites or CaP scaffolds alone. Using X-ray diffraction, scaffolds were shown to contain partially crystalline hydroxyapatite with a crystallinity of 16.7% +/- 6.8% and crystallite size of 128 +/- 55 nm. Fibronection adsorption was increased on composite scaffolds, and cell attachment was higher on composite scaffolds after 30 min, although attachment rates were similar after 1 h. Osteoblast proliferation (based on dsDNA measurements) was significantly increased after 1 week of culture. These studies have demonstrated that composite scaffolds have mechanical properties and porosity sufficient to support ingrowth of new bone tissue, and cell attachment and proliferation data indicate composite scaffolds are promising for bone regeneration.     

聚乳酸-羟基乙酸共聚物/硅酸钙三维多孔骨组织工程支架的构建与性能

BACKGROUND: Some disadvantages exsist in commonly used poly(lactic-co-glycolic acid) (PLGA) scaffolds, including acidic degradation products, suboptimal mechanical properties, low pore size, poor porosity and pore connectivity rate and uncontrollable shape. OBJECTIVE: To construct a scaffold with three-dimensional (3D) pores by adding calcium silicate to improve the properties of PLGA, and then detect its degradability, mechanical properties and biocompatibility. METHODS: PLGA/calcium silicate porous composite microspheres were prepared by the emulsion-solvent evaporation method, and PLGA 3D porous scaffold was established by 3D-Bioplotter, and then PLGA/calcium silicate composite porous scaffolds were constructed by combining the microspheres with the scaffold using low temperature fusion technology. The compositions, morphology and degradability of the PLGA/calcium silicate porous composite microspheres and PLGA microspheres, as well as the morphology, pore properties and compression strength of the PLGA 3D scaffolds and PLGA/calcium silicate composite porous scaffolds were measured, respectively. Mouse bone marrow mesenchymal stem cells were respectively cultivated in the extracts of PLGA/calcium silicate porous composite microspheres and PLGA microspheres, and then were respectively seeded onto the PLGA 3D scaffolds and PLGA/calcium silicate composite porous scaffolds. Thereafter, the cell proliferation activity was detected at 1, 3 and 5 days. RESULTS AND CONCLUSION: Regular pores on the PLGA microspheres and internal cavities were formed, and the PH values of the degradation products were improved after adding calcium silicate. The fiber diameter, pore, porosity and average pore size of the composite porous scaffolds were all smaller than those of the PLGA scaffolds. The compression strength and elasticity modulus of the composite porous scaffolds were both higher than those of the PLGA scaffolds (P < 0.05). Bone marrow mesenchymal stem cells grew well in above microsphere extracts and scaffolds. These results indicate that PLGA/calcium silicate composite porous scaffolds exhibit good degradability in vitro, mechanical properties and biocompatibility.     

丝素蛋白/壳聚糖三维多孔支架的构建及结构表征**

背景：丝素蛋白和壳聚糖均无毒性且具有良好的生物相容性,但是单一成分作为生物支架时都不能满足支架材料的需求。 目的：制备各种不同组分的丝素蛋白及壳聚糖复合支架材料,观察其微观结构及相关性能,筛选出适合成骨细胞生长的理想支架材料。 方法：通过CaCl2∶C2H5OH∶H2O=1∶2∶8(摩尔比)溶解体系溶解、过滤、浓缩提纯,制备出2%的丝素蛋白溶液,壳聚糖溶解于乙酸溶液配制成的3%壳聚糖溶液,将两者以不同的比例相混合,经数次冷冻干燥后,得到成品支架材料。采用电镜观察形貌,计算孔隙率并对支架的结构进行红外、X射线衍射、电子能谱分析观察。 结果与结论：将壳聚糖和丝素蛋白共混后,互为改性,制备出了结构较稳定的支架材料。其中40%丝素蛋白-60%壳聚糖组具有适合成骨细胞生长的较佳孔径,可作为细胞支架的首选配比。 关键词：丝素蛋白;壳聚糖;骨组织工程;支架;生物材料 doi:10.3969/j.issn.1673-8225.2012.12.025     

丝素蛋白/壳聚糖复合支架有良好的细胞相容性与渗透性

文题释义：丝素蛋白/壳聚糖复合支架：将壳聚糖溶于浓度为1%的冰乙酸,制备成质量浓度为35 g/L的壳聚糖溶液并与丝素蛋白溶液融合,将素蛋白与羧化壳聚糖按照体积比8∶2的比例混合,再将混合溶液注入48孔板中,每孔注入1 mL,最后通过冷冻干燥得到丝素蛋白/壳聚糖复合支架。 热重分析：是指在程序控制温度下测量待测样品的质量与温度变化关系的一种热分析技术,用来研究材料的热稳定性和组分。实验中将10 mg待检验的样品放于氮气环境下进行检测,测试温度升高控制范围为30-800 ℃,温度上升速度为10 ℃/min。 背景：丝素蛋白与壳聚糖为组织工程常用的支架材料,但二者单独应用均存在一定的不足,将两者混合使用可以互为改性,充分发挥优点,获取理想的复合支架材料。 目的：制备丝素蛋白/壳聚糖复合支架并对其进行性能测定。 方法：通过冷冻干燥方法制备丝素蛋白/壳聚糖复合支架,采用电镜扫描检测复合支架的形态结构,并进行热重分析、力学性能及细胞毒性检测。制备季铵化壳聚糖,利用核磁共振仪表征其核磁氢谱,Zeta电位仪检测其电位和粒径分布,凝胶电泳实验检测其保护DNA的情况,透射电镜观察其与DNA结合情况。 结果与结论：①扫描电镜显示丝素蛋白/壳聚糖复合支架具体良好的三维孔洞结构,孔径为50-100 μm;②热重分析显示当温度小于200 ℃时,丝素蛋白/壳聚糖复合支架的质量损失下降速度较低;当温度上升至200-500 ℃时,支架质量损失速度开始加快,损失量增多;在800 ℃时,复合支架的残余质量为38%;③丝素蛋白/壳聚糖复合支架的最大应变可以达到94.94%,最大承受应力为7.01 MPa;④CCK-8实验显示,丝素蛋白/壳聚糖复合支架对兔骨髓间充质干细胞没有细胞毒性,具有良好的细胞相容性;⑤核磁氢谱检测显示,季铵化壳聚糖的季铵化程度约为20%;⑥季铵化壳聚糖的粒径分布为(588.56±52.39) nm,季铵化壳聚糖颗粒的表面带正电荷,电位为(16.3±3.92) mV,有利于与DNA结合;⑦凝胶电泳实验显示,季铵化壳聚糖材料的比例越高,对DNA的包裹越好,当其与DNA的比例为1∶3时,对DNA达到包裹作用;⑧透射电镜显示,季铵化壳聚糖/DNA大部分的微粒呈实心圆形,微粒粒径差别较小,平均粒径约为200 nm;⑨结果表明,丝素蛋白/壳聚糖复合支架有良好的细胞相容性与细胞渗透性,利于细胞在支架间的生长。 ORCID: 0000-0002-2572-0229(章晓云) 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     

京尼平交联3D打印胶原/壳聚糖支架的表征北大核心

背景:胶原/壳聚糖支架需交联才能达到相应力学性能,有研究表示调节交联剂浓度可以在一定范围内调控支架的理化性能。目的:探究京尼平浓度对胶原/壳聚糖支架理化性能的影响,制备理化性能可调节的组织工程支架。方法:将胶原和壳聚糖粉末分别溶于弱酸后混合均匀,作为打印墨水,利用生物3D打印机低温打印胶原支架与胶原/壳聚糖支架,经冻干、中和处理后分别以1,3,5 mmol/L的京尼平进行交联。检测各组支架的表观结构稳定性、抗拉能力、溶胀性能、降解性能与生物相容性。结果与结论:①将支架在PBS中浸泡3 d后,对比未交联的冻干支架,交联后胶原支架表面维持规则的孔结构,但是支架出现明显变形;交联后胶原/壳聚糖支架表面结构规则,仅1 mmol/L京尼平交联的胶原/壳聚糖支架存在轻微变形。②随着京尼平浓度的增加,各组支架的力学性能增加,并且对应交联浓度下的胶原/壳聚糖支架力学性能好于胶原支架。③随着京尼平浓度的增加,胶原支架的溶胀率下降,胶原/壳聚糖支架的溶胀率无明显变化。④浸泡于胶原酶溶液中后,不同浓度京尼平交联的胶原支架在1 h内被完全降解,胶原/壳聚糖支架的降解速率随京尼平浓度的增加而降低,均呈现先快速后平缓的趋势。⑤将骨髓间充质干细胞接种于各组交联支架3 d后,1,3 mmol/L京尼平交联的胶原/壳聚糖支架(或胶原支架)上的细胞数量明显多于5 mmol/L京尼平交联的胶原/壳聚糖支架(P<0.05)。⑥结果表明,京尼平可在一定范围调节胶原/壳聚糖支架理化性能,其中3 mmol/L京尼平交联的胶原/壳聚糖支架具有较好的力学性能、抗酶解能力与生物相容性。     

Biomimetic chitosan–nanohydroxyapatite composite scaffolds for bone tissue engineering

We describe a comparative assessment of the structure–property–process relationship of three-dimensional chitosan–nanohydroxyapatite (nHA) and pure chitosan scaffolds in conjunction with their respective biological response with the aim of advancing our insight into aspects that concern bone tissue engineering. High- and medium-molecular-weight (MW) chitosan scaffolds with 0.5, 1 and 2 wt.% fraction of nHA were fabricated by freezing and lyophilization. The nanocomposites were characterized by a highly porous structure and the pore size (～50 to 120 μm) was in a similar range for the scaffolds with different content of nHA. A combination of X-ray diffraction, Fourier transform infrared spectroscopy and electron microscopy indicated that nHA particles were uniformly dispersed in chitosan matrix and there was a chemical interaction between chitosan and nHA. The compression modulus of hydrated chitosan scaffolds was increased on the addition of 1 wt.% nHA from 6.0 to 9.2 kPa in high-MW scaffold. The water uptake ability of composites decreased with an increase in the amount of nHA, while the water retention ability was similar to pure chitosan scaffold. After 28 days in physiological condition, nanocomposites indicated about 10% lower degree of degradation in comparison to chitosan scaffold. The biological response of pre-osteoblasts (MC 3T3-E1) on nanocomposite scaffolds was superior in terms of improved cell attachment, higher proliferation, and well-spread morphology in relation to chitosan scaffold. In composite scaffolds, cell proliferation was about 1.5 times greater than pure chitosan after 7 days of culture and beyond, as implied by qualitative analysis via fluorescence microscopy and quantitative study through MTT assay. The observations related to well-developed structure morphology, physicochemical properties and superior cytocompatibility suggest that chitosan–nHA porous scaffolds are potential candidate materials for bone regeneration although it is necessary to further enhance the mechanical properties of the nanocomposite.     

多孔聚乙烯醇/明胶软骨组织工程支架复合材料的制备及其生物相容性

背景：以明胶为基体制备的组织工程支架材料具有良好的生物相容性和生物降解性能,但存在力学性能低,降解速率难以控制的缺陷。 目的：制备一种软骨组织工程支架材料多孔聚乙烯醇/明胶复合物,并检测其理化性能和生物相容性。 方法：采用乳化发泡法制备聚乙烯醇/明胶多孔支架,并通过电镜分析、力学测试、皮下植入实验,检测材料孔径和孔隙率、IR光谱、力学性能和生物相容性。 结果与结论：多孔材料内部呈三维网状多孔结构,孔径均匀,有相似的孔隙率61.8%,含水率44.6%,抗拉强度为(5.01±0.03) MPa,抗压强度为(1.47±0.36) MPa,有较好的力学性能,IR光谱分析表明材料内部结构均匀。皮下植入后,炎症反应逐渐减轻,囊壁逐渐变薄,并趋于稳定,提示多孔聚乙烯醇/明胶支架材料具有较好的生物相容性和力学性能。     

壳聚糖-聚乳酸软骨与软骨下骨复合支架的制备与表征*

目的制备羟乙基壳聚糖-g-左旋聚乳酸（HECS-g-PLLA）和羟乙基壳聚糖-g-左旋聚乳酸＋Ⅱ型胶原蛋白（HECS-g-PLLA＋II型胶原蛋白）复合支架,并进行表征。方法采用热致相分离法制备HECS-g-PLLA、HECS-g-PLLA＋II型胶原蛋白聚合物,再用压片机,设定不同大气压,将聚合物压模成形具有不同性能的复合支架,测定复合支架的微观形貌、红外光谱、压缩模量、孔隙直径及生物相容性。结果复合支架具有纳米微米共存的高孔隙直径的亚微观结构。红外光谱显示壳聚糖成功羟乙基化,羟乙基壳聚糖与左旋聚乳酸聚合成功。随着压片机设定的压模成形的大气压力增大,样品的压缩模量增强,但空隙直径逐渐减少,可以根据需要制作各种不同性能的支架。结论热源实验和全身急性毒性实验提示复合支架浸提液注入动物体内不会引起发热反应和毒性反应,具有良好的生物相容性。     

Three-dimensional macroporous calcium phosphate bioceramics with nested chitosan sponges for load-bearing bone implants

Three-dimensional macroporous calcium phosphate bioceramics embedded with porous chitosan sponges were synthesized to produce composite scaffolds with high mechanical strength and a large surface/volume ratio for load-bearing bone repairing and substitutes. The macroporous calcium phosphate bioceramics with pore diameters of 300 microm to 600 microm were developed using a porogen burnout technique, and the chitosan sponges were formed inside the pores of the bioceramics by first introducing chiosan solution into the pores followed by a freeze-drying process. Our scanning electron microscopy results showed that the pore size of chitosan sponges formed inside the macroporous structure of bioceramics was approximately 100 microm, a structure favorable for bone tissue in-growth. The compressive modulus and yield stress of the composite scaffolds were both greatly improved in comparison with that of HA/beta-TCP scaffolds. The simulated body fluid (SBF) and cell culture experiments were conducted to assess the bioactivity and biocompatibility of the scaffolds. In the SBF tests, a layer of randomly oriented needle-like apatite crystals formed on the scaffold surface after sample immersion in SBF, which suggested that the composite material has good bioactivity. The cell culture experiments showed that MG63 osteoblast cells attached to the composite scaffolds, proliferated on the scaffold surface, and migrated onto the pore walls, indicating good cell biocompatibility of the scaffold. The cell differentiation on the composite scaffolds was evaluated by alkaline phosphatase (ALP) assay. Compared with the control in tissue culture dishes, the cells had almost the same ALP activity on the composite scaffolds during the first 11 days of culture.     

The fabrication and characterization of biodegradable HA/PHBV nanoparticle–polymer composite scaffolds

This study reports the fabrication and characterization of nano-sized hydroxyapatite (HA)/poly(hydroxyabutyrate-co-hydroxyvalerate) (PHBV) polymer composite scaffolds with high porosity and controlled pore architectures. These scaffolds were prepared using a modified thermally induced phase-separation technique. This investigation focuses on the effect of fabrication conditions on the overall pore architecture of the scaffolds and the dispersion of HA nanocrystals within the composite scaffolds. The morphologies, mechanical properties and in vitro bioactivity of the composite scaffolds were investigated. It was noted that the pore architectures could be manipulated by varying phase-separation parameters. The HA particles were dispersed in the pore walls of the scaffolds and were well bonded to the polymer. The introduction of HA greatly increased the stiffness and strength, and improved the in vitro bioactivity of the scaffolds. The results suggest these newly developed nano-HA/PHBV composite scaffolds may serve as an effective three-dimensional substrate in bone tissue engineering.     

Preparation,characterization and bioactivities of nano anhydrous calcium phosphate added gelatin–chitosan scaffolds for bone tissue engineering

Abstract

Gelatin, chitosan and nano calcium phosphate based composite scaffold with tailored architectures and properties has great potential for bone regeneration. Herein, we aimed to improve the physico chemical, mechanical and osteogenic properties of 3D porous scaffold by incorporation of dihydrogen calcium phosphate anhydrous (DCPA) nanoparticles into biopolymer matrix with variation in composition in the prepared scaffolds. Scaffolds were prepared from the slurry containing gelatin, chitosan and synthesized nano DCPA particle using lyophilization technique. DCPA nano particles were synthesized using calcium carbonate and phosphoric acid in water–ethanol medium. XRD pattern showed phase pure DCPA in synthesized nanopowder. Scaffolds were prepared by addition of DCPA nanoparticles to the extent of 5–10?wt% of total polymer into gelatin–chitosan solution with solid loading varying between 2.5 and 2.75?wt%. The prepared scaffold showed interconnected porosity with pore size varying between 110 and 200 micrometer. With addition of DCPA nanoparticles, average pore size of the prepared scaffolds decreased. With increase in nano ceramic phase content from 5?wt% to 10?wt% of total polymer, the compressive strength of the scaffold increased. Scaffold containing 10?wt% DCPA showed the highest average compressive strength of 2.2?MPa. Higher cellular activities were observed in DCPA containing scaffolds as compared to pure gelatin chitosan scaffold suggesting the fact that nano DCPA addition into the scaffold promoted better osteoblast adhesion and proliferation as evident from MTT assay and scanning electron microscopic (SEM) investigation of osteoblast cultured scaffolds. A higher degree of lamellopodia and filopodia extensions and better spreading behavior of osteoblasts were observed in FESEM micrographs of MG 63 cultured DCPA containing scaffold. The results demonstrated that both mechanical strength and osteogenic properties of gelatin–chitosan scaffold could be improved by addition of anhydrous dihydrogen calcium phosphate nanoparticles into it.     

基于丝素/胶原/羟基磷灰石仿生骨材料的多维结构及其性能

目的 体外构建丝素蛋白（silk fibroin,SF）、I型胶原(type I collagen,Col-I)和羟基磷灰石(hydroxyapatite, HA)共混体系制备二维复合膜和三维仿生支架,研究其理化性质和生物相容性,探讨其在组织工程支架材料中应用的可行性。方法 通过在细胞培养小室底部共混SF/Col-I/HA以及低温3D打印结合真空冷冻干燥法制备二维复合膜及三维支架。通过机械性能测试、电子显微镜和Micro-CT检测材料的理化性质,检测细胞的增殖评估其生物相容性。结果 通过共混和低温3D打印获得稳定的二维复合膜及三维多孔结构支架;力学性能具有较好的一致性,孔径、吸水率、孔隙率和弹性模量均符合构建组织工程骨的要求;支架为网格状的白色立方体,内部孔隙连通性较好; HA均匀分布在复合膜中,细胞黏附在复合膜上,呈扁平状;细胞分布在支架孔壁周围,呈梭形状,生长及增殖良好。结论 利用SF/Col-I/HA共混体系成功制备复合膜及三维支架,具有较好的孔连通性与孔结构,有利于细胞和组织的生长以及营养输送,其理化性能以及生物相容性符合骨组织工程生物材料的要求。     

3D打印成型纳米羟基磷灰石/壳聚糖/聚己内酯三元复合支架材料的构建及表征

文题释义： 组织工程骨：将体外培养的功能相关的种子细胞种植于天然的或人工合成的支架材料内,加入生长因子体外培养一段时间,将他们移植到体内,促进组织修复和骨再生的人工骨。组织工程骨形成的3要素为：支架材料、成骨细胞、生长因子。 生物陶瓷：生物表面活性陶瓷通常含有羟基,还可做成多孔性,生物组织可长入并同其表面发生牢固的键合;生物吸收性陶瓷的特点是能部分吸收或者全部吸收,在生物体内能诱发新生骨的生长。生物活性陶瓷具有骨传导性,它作为一个支架,成骨在其表面进行;还可作为多种物质的外壳或填充骨缺损。生物陶瓷有羟基磷灰石陶瓷、磷酸三钙陶瓷等。  背景：目前常用的骨缺损修复支架材料种类较多,但单一类型材料难以满足骨组织工程支架材料的要求,通过合适的方法将几种单一材料组合形成复合型材料,综合考虑各种材料优缺点,是近年来学者们的研究重点。 目的：构建纳米羟基磷灰石/壳聚糖/聚己内酯三元复合支架材料,并作表征分析研究。 方法：采用3D打印成型技术制备纳米羟基磷灰石/壳聚糖/聚己内酯多孔三元复合支架材料,从X射线衍射分析、吸水率、抗压强度、体外降解性能、孔径分析、扫描电镜分析等多个维度对支架材料进行表征研究。 结果与结论：①X射线衍射分析显示,纳米羟基磷灰石/壳聚糖/聚己内酯多孔三元复合支架的晶型峰图与羟基磷灰石粉末衍射标准卡片类似,表明该三元复合支架是通过物理作用相互结合的,不影响羟基磷灰石的生物学功能;②三元复合支架的吸水率为18.28%,亲水性好,支架可承受的最大压力为1 415 N,其体外降解速率与成骨速率相当;③显微镜下可见三元复合支架的内孔为方形,孔径250 µm,孔径大小均匀、分布有致;④扫描电镜下三元复合支架可见,壳聚糖和聚己内酯组成的纤维排列整齐有序,成网格状, 羟基磷灰石呈颗粒状在纤维表面均匀分布,三元复合材料呈现均匀、疏松的微孔结构;⑤结果表明,通过3D打印成型技术可成功制备纳米羟基磷灰石/壳聚糖/聚己内酯三元复合支架材料,其具有适度的抗压强度、一定的孔隙率、适宜的降解速度和吸水率,能为修复骨缺损的奠定基础。 ORCID: 0000-0002-6321-9160(余和东) 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