Simultaneous electrospin–electrosprayed biocomposite nanofibrous scaffolds for bone tissue regeneration

Currently, the application of nanotechnology in bone tissue regeneration is a challenge for the fabrication of novel bioartificial bone grafts. These nanostructures are capable of mimicking natural extracellular matrix with effective mineralization for successful regeneration of damaged tissues. The simultaneous electrospraying of nanohydroxyapatite (HA) on electrospun polymeric nanofibrous scaffolds might be more promising for bone tissue regeneration. In the current study, nanofibrous scaffolds of gelatin (Gel), Gel/HA (4:1 blend), Gel/HA (2:1 blend) and Gel/HA (electrospin–electrospray) were fabricated for this purpose. The morphology, chemical and mechanical stability of nanofibres were evaluated by means of field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy and with a universal tensile machine, respectively. The in vitro biocompatibility of different nanofibrous scaffolds was determined by culturing human foetal osteoblasts and investigating the proliferation, alkaline phosphatase (ALP) activity and mineralization of cells. The results of cell proliferation, ALP activity and FESEM studies revealed that the combination of electrospinning of gelatin and electrospraying of HA yielded biocomposite nanofibrous scaffolds with enhanced performances in terms of better cell proliferation, increased ALP activity and enhanced mineralization, making them potential substrates for bone tissue regeneration.     

The effects of collagen concentration and crosslink density on the biological,structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering

In this study, we examined the effects of varying collagen concentration and crosslink density on the biological, structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering. Three different collagen contents (0.25%, 0.5% and 1% collagen) and two different dehydrothermal (DHT) crosslinking processes [1] 105 ^°C for 24 h and [2] 150 ^°C for 48 h were investigated. These scaffolds were assessed for (1) pore size, (2) permeability (3) compressive strength and (4) cell viability. The largest pore size, permeability rate, compressive modulus, cell number and cell metabolic activity was all found to occur on the 1% collagen scaffold due to its increased collagen composition and the DHT treatment at 150 ^°C was found to significantly improve the mechanical properties and not to affect cellular number or metabolic activity. These results indicate that doubling the collagen content to 1% and dehydrothermally crosslinking the scaffold at 150 ^°C for 48 h has enhanced mechanical and biological properties of the scaffold making it highly attractive for use in bone tissue engineering.     

Electrospun nanostructured scaffolds for bone tissue engineering

The current challenge in bone tissue engineering is to fabricate a bioartificial bone graft mimicking the extracellular matrix (ECM) with effective bone mineralization, resulting in the regeneration of fractured or diseased bones. Biocomposite polymeric nanofibers containing nanohydroxyapatite (HA) fabricated by electrospinning could be promising scaffolds for bone tissue engineering. Nanofibrous scaffolds of poly-l-lactide (PLLA, 860 ± 110 nm), PLLA/HA (845 ± 140 nm) and PLLA/collagen/HA (310 ± 125 nm) were fabricated, and the morphology, chemical and mechanical characterization of the nanofibers were evaluated using scanning electron microscopy, Fourier transform infrared spectroscopy and tensile testing, respectively. The in vitro biocompatibility of different nanofibrous scaffolds was also assessed by growing human fetal osteoblasts (hFOB), and investigating the proliferation, alkaline phosphatase activity (ALP) and mineralization of cells on different nanofibrous scaffolds. Osteoblasts were found to adhere and grow actively on PLLA/collagen/HA nanofibers with enhanced mineral deposition of 57% higher than the PLLA/HA nanofibers. The synergistic effect of the presence of an ECM protein, collagen and HA in PLLA/collagen/HA nanofibers provided cell recognition sites together with apatite for cell proliferation and osteoconduction necessary for mineralization and bone formation. The results of our study showed that the biocomposite PLLA/collagen/HA nanofibrous scaffold could be a potential substrate for the proliferation and mineralization of osteoblasts, enhancing bone regeneration.     

Fabrication and characterization of three-dimensional electrospun scaffolds for bone tissue engineering

When traumatic injury, tumor removal, or disease results in significant bone loss, reconstructive surgery is required. Bone grafts are used in orthopedic reconstructive procedures to provide mechanical support and promote bone regeneration. In this study, we applied a heat sintering technique to fabricate 3D electrospun scaffolds that were used to evaluate effects of mineralization and fiber orientation on scaffold strength. We electrospun PLLA/gelatin scaffolds with a layer of PDLA and heat sintered them into three-dimensional cylindrical scaffolds. Scaffolds were mineralized by incubation in 10× simulated body fluid for 6, 24, and 48 h to evaluate the effect of mineralization on scaffolds compressive mechanical properties. The effects of heat sintering hydroxyapatite (HA) microparticles directly to the scaffolds on mineral deposition, distribution and mechanical properties of the scaffolds were also evaluated. We found that orientation of the fibers had little effect on the compressive mechanical properties of the scaffolds. However, increasing the mineralization times resulted in an increase in compressive mechanical properties. Also, the direct addition of HA microparticles had no effect on the scaffold mechanical properties, but had a significant effect on the mineral deposition on PLLA/gelatin scaffolds.     

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.     

骨髓间充质干细胞与不同基质修饰的纳米晶胶原基骨体外生物相容性研究

研究大鼠骨髓间充质干细胞(MSCs)诱导培养后与不同基质修饰的纳米晶胶原基骨(nanoHydroxyapatite/collagen,nHAC)的生物相容性,为骨组织工程提供一种新型复合支架材料。SD大鼠MSCs经成骨诱导培养、扩增,进行成骨细胞表征后,种植与支架材料体外复合培养。实验分为4组:实验组A,纤维蛋白(FB)和纤维连接蛋白(FN)修饰的纳米晶胶原基骨((FB FN)-nHAC);实验组B,纤维蛋白修饰的纳米晶胶原基骨(FB-nHAC);实验组C,纤维连接蛋白修饰的纳米晶胶原基骨(FN-nHAC);对照组D,单纯的纳米晶胶原基骨(nHAC)。通过检测支架材料的细胞黏附率、不同时间点(3、7、10、14d)支架材料中细胞数、碱性磷酸酶活性以及扫描电镜观察细胞在材料上的生长状况,比较分析不同支架材料与细胞生物相容性差异。大鼠MSCs经诱导培养14d后,碱性磷酸酶细胞化学染色、I型胶原免疫荧光染色及矿化沉积茜素红染色均为阳性;细胞与支架材料黏附率A组最高为74.4%;支架材料中细胞数量均随培养时间延长而增长,且A组细胞数增加较快,与相同时相点其他各组材料中细胞数差异有显著性(P<0.05);各时相点细胞碱性磷酸酶活性表达A组最高,差异亦有显著性(P<0.05)。电镜观察发现4组材料上均有细胞生长,但A组的细胞生长状况明显好于其他组。大鼠MSCs经成骨诱导培养,可表达成骨细胞表型,(FB FN)-nHAC在体外实验中表现出与细胞优良的生物相容性,可作为较理想的新型复合支架应用于骨组织工程。     

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。     

Enhanced bone tissue regeneration by antibacterial and osteoinductive silica-HACC-zein composite scaffolds loaded with rhBMP-2

Next-generation orthopedic implants with both osteoinductivity and antibacterial ability are greatly needed. In the present study, biodegradable rhBMP-2 loaded zein-based scaffolds with a macroporous structure were synthesized, and SBA-15 nanoparticles and hydroxypropyltrimethyl ammonium chloride chitosan (HACC) were incorporated into the scaffolds to produce an anti-infective composite scaffold for delivery of osteogenic factors that facilitate the functional repair of bone defects. The silica/HACC/zein scaffolds developed here showed bioactivity, biocompatibility, and effective antibacterial activity. Confocal laser scanning microscopy (CLSM) was used to quantitatively measure the bactericidal efficacy with respect to bacterial adhesion. Results showed that the sample zein-HACC-S20 exhibited long-lasting antibacterial activity against Escherichia coli and Staphylococcus aureus up to 5 d. At a low dosage of rhBMP-2 (ca. 80 μg), the scaffolds released rhBMP-2 protein efficiently at a relatively slow rate, even after 27 d. An ALP activity and ECM mineralization assay showed that the zein-HACC-S20 scaffolds exhibited significant early osteogenic differentiation by generating enhanced ALP product on day 14 and ECM mineralization on day 21. In a mouse model of thigh muscle pouches, zein-S20 and zein-HACC-S20 groups resulted in obvious bone formation and gave more extensive mineralization to the implants than silica free groups, indicating effective bone induction in vivo. In a rabbit model of critical-sized radius bone defects (20 mm in length and 5 mm in diameter), the bone defects were almost fully repaired and bone marrow cavity recanalization was detectable by 3D micro-CT technique and histological analysis after 12 weeks. In this way, the zein-HACC-S20 scaffolds were proven to significantly promote the bone repair. They also demonstrated considerable promise for tissue engineering. Silica/HACC/zein scaffolds with both antibacterial activity and the ability to induce osteogenesis have immense potential in orthopedics and other biomedical applications.     

Biocompatibility of anionic collagen matrix as scaffold for bone healing.

The basic approach to the treatment of bone defects involves the use of scaffolds to favor tissue growth. Although several bioscaffolds have been proposed for this purpose, the search for new and enhanced materials continues in an attempt to address the drawbacks of the present ones. Modifying current materials can be a fast and cheap way to develop new ones. Among them, type I collagen allows its structure to be modified using relatively simple techniques. By means of an alkaline treatment, anionic collagen with enhanced piezoelectric properties can be obtained through hydrolysis of carboxyamides groups of asparagine and glutamine residues from collagen in carboxylic. The process applied to a raw source of collagen, bovine pericardium, provided a sponge-like structure, with heterogeneous pore size, and, moreover, the complete removal of interstitial cells. For the evaluation of the biocompatibility of such matrices, they were implanted in surgically created bone defects in rat tibias. Empty defects served as controls. This experimental model allowed a preliminary evaluation of the osteoconductiveness of the matrices. The histological results presented a low inflammatory response and bone formation within a short period of time, similar to that of controls. The low cost of production associated to the biocompatibility and osteoconductivity performance make the anionic collagen matrices promising alternatives for bone defects treatment.     

胶原/生物活性玻璃/壳聚糖增强型复合支架

背景：生物活性玻璃/胶原复合材料具有优良的成骨活性和的生物学性能,然而其在人体环境中易降解而导致支架溃散、力学性能下降。 目的：构建具有良好力学性能、抗降解性能和骨修复特性的胶原/生物活性玻璃/壳聚糖增强型复合支架。 方法：以壳聚糖作为分散剂,将生物活性玻璃粉体预先在壳聚糖溶液中均匀分散,然后与胶原溶液混合,结合冷冻干燥法制备多孔胶原/生物活性玻璃/壳聚糖增强型复合骨修复支架。采用傅里叶变换红外光谱仪、场发射扫描电子显微镜、X射线衍射仪、动态生物力学试验机等对复合支架的结构和性能进行表征。 结果与结论：由于壳聚糖和生物活性玻璃粉体在微酸性环境下的电荷吸引,使在壳聚糖中预分散的生物活性玻璃颗粒在复合支架中分散更均匀;壳聚糖的引入大量增加了机体中的羟基和氨基,使分子间的相互作用增强,显著提高了材料的抗压模量和强度;壳聚糖和胶原在分子尺度的混合,使胶原分子被壳聚糖包裹,降低了胶原酶对胶原分子的酶切能力,显著提高了复合支架的抗胶原酶解性;壳聚糖分子使生物活性玻璃颗粒更均匀的包裹在大分子基相中,减少了生物活性玻璃颗粒的团聚和暴露,导致复合支架在模拟体液中的矿化活性略微降低。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：     

Immobilization of alkaline phosphatase on microporous nanofibrous fibrin scaffolds for bone tissue engineering

Alkaline phosphatase (ALP) promotes bone formation by degrading inorganic pyrophosphate (PP_i), an inhibitor of hydroxyapatite formation, and generating inorganic phosphate (P_i), an inducer of hydroxyapatite formation. P_i is a crucial molecule in differentiation and mineralization of osteoblasts. In this study, a method to immobilize ALP on fibrin scaffolds with tightly controllable pore size and pore interconnection was developed, and the biological properties of these scaffolds were characterized both in vitro and in vivo. Microporous, nanofibrous fibrin scaffolds (FS) were fabricated using a sphere-templating method. ALP was covalently immobilized on the fibrin scaffolds using 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC). Scanning electron microscopic observation (SEM) showed that mineral was deposited on immobilized alkaline phosphatase fibrin scaffolds (immobilized ALP/FS) when incubated in medium supplemented with β-glycerophosphate, suggesting that the immobilized ALP was active. Primary calvarial cells attached, spread and formed multiple layers on the surface of the scaffolds. Mineral deposition was also observed when calvarial cells were seeded on immobilized ALP/FS. Furthermore, cells seeded on immobilized ALP/FS exhibited higher osteoblast marker gene expression compared to control FS. Upon implantation in mouse calvarial defects, both the immobilized ALP/FS and FS alone treated group had higher bone volume in the defect compared to the empty defect control. Furthermore, bone formation in the immobilized ALP/FS treated group was statistically significant compared to FS alone group. However, the response was not robust.     

动物血管脱细胞方法及细胞外基质材料评价研究

采用独立设计的反复冻融方法,去除兔颈总动脉中的血管细胞,对所获得的细胞外基质进行组织、生化、力学等分析。在分离兔颈总动脉后,首先用低渗缓冲液处理,然后经低温反复冻融,最后用非离子型去污剂处理。所得的支架行组织染色和扫描电镜分析,显示基质的微观结构;荧光染色和基因组DNAPCR分析,检测细胞DNA残留;分别行羟脯氨酸定量分析和轴向拉伸强度分析,检测胶原蛋白和血管生物力学性能的变化;支架没有明显的细胞毒性,溶血率低于医用材料的标准;支架随时间缓慢降解。种植兔的骨髓干细胞,研究细胞在支架上的粘附生长能力。应用本方法能彻底去除血管细胞,没有细胞碎片和DNA的残留,并保留了较完整的细胞外基质和力学性能,具有开放的大孔径结构,细胞生物相容性好,是一种理想的组织工程血管支架材料。     

The osteogenesis of bacterial cellulose scaffold loaded with bone morphogenetic protein-2

Bacterial cellulose (BC) is a nanofibrous biological material with attractive physicochemical properties and biocompatibility. Its fiber is similar to the collagenous fiber of bone. To explore if BC could be utilized as a localized delivery system to increase the local concentration of cytokines for tissue engineering, we prepared the BC scaffold from Acetobacter xylinum X-2 (A.?xylinum X-2) and investigated the osteogenic potential of the BC scaffold coated with bone morphogenetic protein-2 (BMP-2). The data showed that BC had a good biocompatibility and induced differentiation of mouse fibroblast-like C2C12 cells into osteoblasts in the presence of BMP-2 in?vitro, as demonstrated by alkaline phosphatase (ALP) activity assays. Within a certain range (0?～?3?μg/scaffold), the osteogenic activity of induced osteoblasts was positively correlated to the concentrations of BMP-2. In in?vivo subcutaneous implantation studies, BC scaffolds carrying BMP-2 showed more bone formation and higher calcium concentration than the BC scaffolds alone at 2 and 4 weeks, respectively. The ALP activity assay and the measurement of calcium concentration of BC scaffolds also showed that more new bone was developed in the BC scaffolds carrying BMP-2 than in the BC scaffolds alone. Our studies suggest that BC is a good localized delivery system for BMPs and would be a potential candidate in bone tissue engineering.     

In vitro osteogenic potential of human bone marrow stromal cells cultivated in porous scaffolds from mineralized collagen

Porous 3D structures from mineralized collagen were fabricated applying a procedure in which collagen fibril reassembly and precipitation of nanocrystalline hydroxyapatite (HA) occur simultaneously. The resulting matrices were evaluated in vitro with respect to their suitability as scaffolds for bone tissue engineering. We found a high capacity of the material to bind serum proteins as well as to absorb Ca2+ ions, which could be advantageous to promote cell attachment, growth, and differentiation. Human bone marrow stromal cells (hBMSCs) were seeded onto the 3D scaffolds and cultivated for 4 weeks in the presence and absence of osteogenic supplements. We studied viability, proliferation, and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content, and alkaline phosphatase (ALP) activity. Furthermore, the expression for bone-related genes (ALP, bone sialo protein II (BSP II), and osteocalcin) was analyzed. In our investigation we found a 2.5-fold to 5-fold raise in DNA content and an increase of ALP activity for osteogenic induced hBMSC on collagen HA scaffolds. The expression of ALP and BSP II in these cells was also stimulated in the course of cultivation; however, we did not detect an upregulation of osteocalcin gene expression. These data suggest, that porous collagen HA scaffolds are suitable for the expansion and osteogenic differentiation of hBMSC and are therefore promising candidates for application as bone grafts.     

Accelerated mineralization of dense collagen-nano bioactive glass hybrid gels increases scaffold stiffness and regulates osteoblastic function

Plastically compressed dense collagen (DC) gels mimic the microstructural, mechanical, and biological properties of native osteoid. This study investigated the effect of hybridizing DC with osteoinductive nano-sized bioactive glass (nBG) particles in order to potentially produce readily implantable, and mineralizable, cell seeded hydrogel scaffolds for bone tissue engineering. Due to the high surface area of nBG and increased reactivity, calcium phosphate formation was immediately detected within as processed DC-nGB hybrid gel scaffolds. By day 3 in simulated body fluid, accelerated mineralization was confirmed through the homogeneous growth of carbonated hydroxylapatite on the nanofibrillar collagen framework. At day 7, there was a 13 fold increase in the hybrid gel scaffold compressive modulus. MC3T3-E1 pre-osteoblasts, three-dimensionally seeded at the point of nanocomposite self-assembly, were viable up to day 28 in culture. In the absence of osteogenic supplements, MC3T3-E1 metabolic activity and alkaline phosphatase production were affected by the presence of nBG, indicating accelerated osteogenic differentiation. Additionally, no cell-induced contraction of DC-nBG gel scaffolds was detected. The accelerated mineralization of rapidly produced DC-nBG hybrid gels indicates their potential suitability as osteoinductive cell delivery scaffolds for bone regenerative therapy.     

Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts

Nanotechnology has enabled the engineering of nanostructured materials to meet current challenges in bone replacement therapies. Biocomposite nanofibrous scaffolds of poly(l-lactic acid)-co-poly(?-caprolactone), gelatin and hydroxyapatite (HA) were fabricated by combining the electrospinning and electrospraying techniques in order to create a better osteophilic environment for the growth and mineralization of osteoblasts. Electrospraying of HA nanoparticles on electrospun nanofibers helped to attain rough surface morphology ideal for cell attachment and proliferation and also achieve improved mechanical properties than HA blended nanofibers. Nanofibrous scaffolds showed high pore size and porosity up to 90% with fiber diameter in the range of 200–700 nm. Nanofibrous scaffolds were characterized for their functional groups and chemical structure by FTIR and XRD analysis. Studies on cell–scaffold interaction were carried out by culturing human fetal osteoblast cells (hFOB) on both HA blended and sprayed PLACL/Gel scaffolds and assessing their growth, proliferation, mineralization and enzyme activity. The results of MTS, ALP, SEM and ARS studies confirmed, not only did HA sprayed biocomposite scaffolds showed better cell proliferation but also enhanced mineralization and alkaline phosphatase activity (ALP) proving that electrospraying in combination with electrospinning produced superior and more suitable biocomposite nanofibrous scaffolds for bone tissue regeneration.     

PCL基复合骨组织工程支架研究现状及发展

三维骨组织工程支架已成为成骨研究领域的热点。聚己内酯(PCL)具有良好的生物相容性,在骨组织工程研究中被广泛应用于三维支架的制备。但纯PCL支架亲水性差、生物活性低,限制了其在生物医学领域的应用。随着骨组织工程材料研究的发展,大量研究者将PCL与各种无机物、金属元素或胶原等活性材料进行复合,以改善支架性能或引入新的性能。针对PCL基骨组织工程复合支架的材料选择,从PCL复合无机材料、PCL复合水凝胶材料、PCL复合金属元素、PCL复合小分子药物以及PCL复合生物活性分子等5个方面,对各类复合支架的性能及体内外成骨效果等方面进行综述,希望对PCL在骨组织工程中的研究及临床应用提供一定的帮助。     

丝素蛋白/壳聚糖复合支架材料的细胞相容性**

背景：大量研究表明丝素蛋白、壳聚糖为天然高分子材料,具有良好的细胞生物相容性。 目的：探讨丝素蛋白/壳聚糖复合支架材料与诱导的兔骨髓间充质干细胞的生物相容性。 方法：将兔骨髓间充质干细胞分离培养、诱导后,与丝素蛋白/壳聚糖三维支架材料体外共培养,以材料的细胞毒性、细胞增殖活力、材料细胞黏附率及扫描电镜等检测评价材料的细胞相容性。 结果与结论：经诱导后的骨髓间充质干细胞在支架材料上黏附、生长良好,保持正常的分裂增殖速度;随时间的增加,细胞黏附率增加,材料组较对照组黏附率强,差异有显著性意义(P < 0.05)。扫描电镜观察发现细胞接种48 h后细胞生长良好,与支架黏附紧密,增殖分裂活跃。说明丝素蛋白/壳聚糖三维支架材料具有良好的细胞相容性。     

Single-step mineralization of woodpile chitosan scaffolds with improved cell compatibility

A facile and efficient single-step mineralization approach was exploited for achieving nanoscopic hydroxyapatite (HAP) crystal layer in chitosan porous matrix, wherein a mixed water-ethanol solvent was used to control the growth of minerals. The crystallographic structure, morphology, and mechanical properties of the scaffold were analyzed with XRD, FTIR, environmental scanning electric microscopy (ESEM), TEM, and compression tests. The behaviors and responses of MC3T3-E1 pre-osteoblast cells on the scaffolds were studied as well. The results showed that the scaffolds kept woodpile structure with predefined and controlled hierarchical structure after mineralization. The inorganic phase in the mineralized chitosan scaffolds was determined as pure rod-like HAP, which settled densely on the matrix. The compression strength and compressive modulus of the scaffolds increased dramatically to 0.54 ± 0.005 MPa and 5.47 ± 0.65 MPa, respectively. During a culture period of 2 and 3 weeks, cell proliferation and in-growth were observed by phase contrast light microscopy and SEM. The alkaline phosphatase (ALP) activity increased after 1 week. Cell viability and cell proliferation index (PI) obtained higher values than that of the chitosan scaffolds. The novel single-step mineralization approach and the porous hybrid scaffolds would be a promising method for designing hybrid bone graft.     

In vitro cell performance on hydroxyapatite particles/poly(L-lactic acid) nanofibrous scaffolds with an excellent particle along nanofiber orientation

Highly porous hydroxyapatite (HA)/poly(L-lactide) (PLLA) nanofibrous scaffolds were prepared by incorporating needle-shaped nano- or micro-sized HA particles into PLLA nanofibers using electrospinning. The scaffolds had random or aligned fibrous assemblies and both types of HA particles were perfectly oriented along the fiber long axes. The biocompatibility and cell signaling properties of these scaffolds were evaluated by in vitro culture of rat osteosarcoma ROS17/2.8 cells on the scaffold surface. Cell morphology, viability and alkaline phosphatase (ALP) activity on each scaffold were examined at different time points. The HA/PLLA scaffolds exhibited higher cell viability and ALP activity than a pure PLLA scaffold. In addition, micro-sized HA particles supported cell proliferation and differentiation better than nano-sized ones in random scaffolds through a 10 day culture period and in aligned scaffolds at an early culture stage. The fibrous assembly of the scaffold had a pronounced impact on the morphology of the cells in direct contact with the scaffold surface, but not on cell proliferation and differentiation. Thus, HA/PLLA nanofibrous scaffolds could be good candidates for bone tissue engineering.