Improving pore interconnectivity in polymeric scaffolds for tissue engineering

A new scaffold fabrication technique aiming to enhance pore interconnectivity for tissue engineering has been developed. Medical grade poly(lactic acid) was utilized to generate scaffolds by a solvent‐evaporating/particulate‐leaching technique, using a new dual‐porogen system. Water‐soluble sodium chloride particles were used to control macro‐pore size in the range 106–255 µm, while organic naphthalene was utilized as a porogen to increase pore interconnections. The three‐dimensional (3D) morphology of the scaffolds manufactured with and without naphthalene was examined by optical coherence tomography and scanning electron microscopy. The mechanical properties of the scaffolds were characterized by compression tests. MG63 osteoblast cells were seeded in the scaffolds to study the cell attachment and viability evaluated by confocal microscopy. It was revealed that introducing naphthalene as the second porogen in the solvent‐evaporating/particulate‐leaching process resulted in improvement of the pore interconnectivity. Cells grew in both scaffolds fabricated with and without naphthalene. They exhibited strong green fluorescence when using a live/dead fluorescent dye kit, indicating that the naphthalene in the scaffold process did not affect cell viability. Copyright © 2009 John Wiley & Sons, Ltd.     

Feasibility of ceramic-polymer composite cryogels as scaffolds for bone tissue engineering

The purpose of the current study was to investigate whether the cryopolymerization technique is capable of producing suitable scaffolds for bone tissue engineering. Cryopolymers made of 2-hydroxyethyl methacrylate and acrylic acid with (W1 and W20) and without (W0) wollastonite particles were prepared. The elastic modulus of the specimens rose one order of magnitude from W1 to W20. Total porosity reached 56% for W0, 72% for W1 and 36% for W20, with pore sizes of up to 2 mm, large interconnection sizes of up to 1 mm and small interconnection sizes of 50-80 μm on dry specimens. Cryogels swell up to 224 ± 17% for W0, 315 ± 18% for W1 and 231 ± 27% for W20 specimens, while maintaining the integrity of the bodies. Pore sizes > 5 mm can be observed for swollen specimens. The biocompatibility of the samples was tested using human mesenchymal stem cells isolated from bone marrow and adipose tissues. Both types of cells attached and grew on the three tested substrates, colonized their inner regions and organized an extracellular cell matrix. Fibronectin and osteopontin levels decreased in the media from cells cultured on W20 samples, likely due to increased binding on the ECM deposited by cells. The osteoprotegerin-to-receptor activator of nuclear factor-κB ligand secretion ratios increased with increasing wollastonite content. Altogether, these results indicate that an appropriate balance of surface properties and structure that favours stromal cell colonization in the porous cryogels can be achieved by modulating the amount of wollastonite.     

聚乙烯醇及其复合材料在组织工程支架中的应用

背景：聚乙烯醇是具有良好生物相容性和生物降解特性的聚合物,因其水溶性、成膜性、乳化性、胶黏性,而且无味无毒,被广泛用于临床领域。
　　目的：综述聚乙烯醇及其复合材料在骨、软骨、皮肤、血管等组织工程支架中的应用。
　　方法：由第一作者检索2000年1月至2011年12月中国知网数据库、1980年1月至2012年12月Pubmed数据库及 Elsevier数据库中,有关聚乙烯醇及其复合材料在骨、软骨、皮肤、血管等组织工程支架中应用的文章,中文关键词为“聚乙烯醇,复合材料,组织工程支架”,英文关键词为“Poly (vinyl alcohol),composite material, tissue engineering scaffold”。
　　结果与结论：虽然聚乙烯醇及其复合材料还存在强度不够高、植入后有并发症等缺点,但这类材料具有良好的生物相容性和生物可降解特性,在组织工程中的应用从实验室到临床前研究都有很大的进展。对于其修复的长期效果还需要进一步深入研究。通过对材料表面进行修饰,改善细胞与支架材料的相互作用;通过模拟细胞生长微环境,制备仿生材料,提高材料的亲水性、对细胞的黏附性,促进细胞的分化增殖;构建具有可控三维多孔结构的支架,并赋予其控制释放细胞生长因子等功能,更好地仿生天然细胞外基质的结构和功能;制备出降解速度与机械强度能够完全适应组织再生需要的支架,研制复合、仿生材料是今后支架材料研究的主要方向。     

The evolution of simulation techniques for dynamic bone tissue engineering in bioreactors

Bone tissue engineering aims to overcome the drawbacks of current bone regeneration techniques in orthopaedics. Bioreactors are widely used in the field of bone tissue engineering, as they help support efficient nutrition of cultured cells with the possible combination of applying mechanical stimuli. Beneficial influencing parameters of in vitro cultures are difficult to find and are mostly determined by trial and error, which is associated with significant time and money spent. Mathematical simulations can support the finding of optimal parameters. Simulations have evolved over the last 20 years from simple analytical models to complex and detailed computational models. They allow researchers to simulate the mechanical as well as the biological environment experienced by cells seeded on scaffolds in a bioreactor. Based on the simulation results, it is possible to give recommendations about specific parameters for bone bioreactor cultures, such as scaffold geometries, scaffold mechanical properties, the level of applied mechanical loading or nutrient concentrations. This article reviews the evolution in simulating various aspects of dynamic bone culture in bioreactors and reveals future research directions. Copyright © 2013 John Wiley & Sons, Ltd.     

Microfabrication of PDLLA scaffolds

This study aimed to comprehend the potentialities of the microfabrication to produce tissue-engineering scaffolds. Structures presenting homogeneously distributed pores of size 100 and 200 μm were fabricated through layer-by-layer deposition of filaments of poly(D,L-lactic acid) (PDLLA) prepared from dichloromethane/dimethylformamide solutions. Rheological tests on the solution and molecular weight distributions of PDLLA, solvent cast films and microfabricated scaffolds were performed to determine which material conditions are optimal for the microfabricated system and to identify any possible material modification induced by the process. In vitro qualitative preliminary cell culture studies were conducted using MG63 osteoblast cell lines after assuring the non-cytotoxicity of the scaffold material by the lactate dehydrogenase in vitro toxicology assay; biological evaluations were initially performed using scaffolds with the smaller (100 μm) pore size. Scanning electron microscopy imaging was used to determine cell morphology distribution. A second cell culture test was performed, using the scaffold with the higher (200 μm) porosity. Confocal laser microscopy (CLM) was utilized to examine cell morphology and growth behaviour. Cellular metabolic activity and viability were also examined using Alamar Blue assay and further verifications were performed using CLM. Cell culture studies indicated homogeneous distribution, high viability and metabolic activity. Pore dimension affects cell distribution: pores < 100 μm acted as barrier structures for the MG63 osteoblast cell line; penetration inside the matrix was hindered and cells grew on the outer part. Increasing pore size resulted in a more homogeneous cell distribution and penetration of cells inside the structure was achieved.     

骨组织工程的研究进展

本文对组织工程骨的研究作了概述,介绍了用于骨组织工程的种子细胞、支架材料和骨组织构建的基本做法.介绍了用珊瑚、珊瑚-羟基磷灰石、松质骨基质和其他一些天然生物材料,用作成骨细胞培养的支架材料,研究证实除了人工合成的聚合物类材料之外,一些天然生物材料也可作为骨组织工程理想的支架材料.     

Channelled scaffolds for engineering myocardium with mechanical stimulation

The characteristics of the matrix (composition, structure, mechanical properties) and external culture environment (pulsatile perfusion, physical stimulation) of the heart are important characteristics in the engineering of functional myocardial tissue. This study reports on the development of chitosan‐collagen scaffolds with micropores and an array of parallel channels (~ 200 µm in diameter) that were specifically designed for cardiac tissue engineering using mechanical stimulation. The scaffolds were designed to have similar structural and mechanical properties of those of native heart matrix. Scaffolds were seeded with neonatal rat heart cells and subjected to dynamic tensile stretch using a custom designed bioreactor. The channels enhanced oxygen transport and facilitated the establishment of cell connections within the construct. The myocardial patches (14 mm in diameter, 1–2 mm thick) consisted of metabolically active cells that began to contract synchronously after 3 days of culture. Mechanical stimulation with high tensile stress promoted cell alignment, elongation, and expression of connexin‐43 (Cx‐43). This study confirms the importance of scaffold design and mechanical stimulation for the formation of contractile cardiac constructs. Copyright © 2011 John Wiley & Sons, Ltd.     

β-磷酸三钙/聚乳酸叠层复合骨组织工程支架的制备、性能及其应用评价

目的进行β-磷酸三钙/聚乳酸叠层复合支架的应用评价。方法在体外37℃生理盐水中降解过程中,观察该材料的重量、力学强度、聚乳酸分子量、材料周围环境的pH值、Ca2+浓度等参数随时间的变化情况,以及将该支架材料和骨髓基质细胞复合后,植入中国青山羊胫骨段缺损处进行修复实验。结果对修复效果进行了观察,发现其成骨能力较强。结论此种材料在骨组织工程领域有一定应用前景。     

Gelatin cryogels crosslinked with oxidized dextran and containing freshly formed hydroxyapatite as potential bone tissue‐engineering scaffolds

Gelatin‐based cryogels were prepared by using a novel crosslinker, oxidized dextran, which was synthesized and used in the study. The cryogels were also loaded with freshly formed hydroxyapatite (HA) particles. These cryogels are opaque, spongy and highly elastic and have a pore structure with large interconnected pores. They swell about 500% in aqueous media and within a few minutes reach their final swollen forms. The elastic moduli of HA‐containing cryogels were 18.5 ± 3.0 kPa, which is suitable for non‐load‐bearing bone tissue‐engineering (TE) applications, especially for the craniofacial area. Copyright © 2012 John Wiley & Sons, Ltd.     

骨组织工程研究中的血管、神经化问题

血管与神经组织在骨组织发生、发育及损伤后再生中起着重要作用.本文结合最近国内外研究进展,对组织工程骨组织再生中血管、神经的作用进行综述,介绍血管神经化组织工程骨组织的构建方法.     

The effect of wicking fibres in tissue‐engineered bone scaffolds

The major limitation of large tissue‐engineered constructs used for bone regeneration is the lack of vasculature and, therefore, lack of transport of essential nutrients, chemical factors and progenitor cells. Research approaches to improve the transport properties of large scaffolds focus on using angiogenic factors and vasculogenic cells to create new vasculature; however, the slow rate of vessel formation and reliance on vessel self‐assembly in these approaches is problematic. In this study, a novel approach has been proposed, using proprietary engineered ‘wicking’ fibres of non‐circular cross‐section that provide highly efficient transport for fluid and cells. The effect of wicking fibres on the movement of fluorescein isothiocyanate (FITC)‐conjugated protein in a three‐dimensional (3D) hydrogel system was analysed. The results indicated that the rate of diffusion of the fluorescent protein was greatly enhanced in hydrogels that contained wicking fibres in comparison to those that did not. The movement of progenitor cells along wicking fibres and round fibres was assessed. This study demonstrated that wicking fibres enhance the movement of critical growth factors and progenitor cells central for bone regeneration. The results suggested that the incorporation of wicking fibres into large tissue‐engineered constructs may improve the transport of growth factors and progenitor cells essential for bone formation. Copyright © 2014 John Wiley & Sons, Ltd.     

Starch-poly(epsilon-caprolactone) and starch-poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering applications: structure, mechanical properties and degradation behaviour

In scaffold-based tissue engineering strategies, the successful regeneration of tissues from matrix-producing connective tissue cells or anchorage-dependent cells (e.g. osteoblasts) relies on the use of a suitable scaffold. This study describes the development and characterization of SPCL (starch with epsilon-polycaprolactone, 30:70%) and SPLA [starch with poly(lactic acid), 30:70%] fibre-meshes, aimed at application in bone tissue-engineering strategies. Scaffolds based on SPCL and SPLA were prepared from fibres obtained by melt-spinning by a fibre-bonding process. The porosity of the scaffolds was characterized by microcomputerized tomography (microCT) and scanning electron microscopy (SEM). Scaffold degradation behaviour was assessed in solutions containing hydrolytic enzymes (alpha-amylase and lipase) in physiological concentrations, in order to simulate in vivo conditions. Mechanical properties were also evaluated in compression tests. The results show that these scaffolds exhibit adequate porosity and mechanical properties to support cell adhesion and proliferation and also tissue ingrowth upon implantation of the construct. The results of the degradation studies showed that these starch-based scaffolds are susceptible to enzymatic degradation, as detected by increased weight loss (within 2 weeks, weight loss in the SPCL samples reached 20%). With increasing degradation time, the diameter of the SPCL and SPLA fibres decreases significantly, increasing the porosity and consequently the available space for cells and tissue ingrowth during implantation time. These results, in combination with previous cell culture studies showing the ability of these scaffolds to induce cell adhesion and proliferation, clearly demonstrate the potential of these scaffolds to be used in tissue engineering strategies to regenerate bone tissue defects.     

A new composite hydrogel combining the biological properties of collagen with the mechanical properties of a supramolecular scaffold for bone tissue engineering

Tissue engineering is a promising alternative to autografts, allografts, or biomaterials to address the treatment of severe and large bone lesions. Classically, tissue engineering products associate a scaffold and cells and are implanted or injected into the lesion. These cells must be embedded in an appropriate biocompatible scaffold, which offers a favourable environment for their survival and differentiation. Here, we designed a composite hydrogel composed of collagen I, an extracellular matrix protein widely used in several therapeutic applications, which we associated with a physical hydrogel generated from a synthetic small amphiphilic molecule. This composite showed improved mechanical and biological characteristics as compared with gels obtained from each separate compound. Incorporation of the physical hydrogel prevented shrinkage of collagen and cell diffusion out of the gel and yielded a gel with a higher elastic modulus than those of gels obtained with each component alone. The composite hydrogel allowed cell adhesion and proliferation in vitro and long‐term cell survival in vivo. Moreover, it promoted the differentiation of human adipose‐derived stem cells in the absence of any osteogenic factors. In vivo, cells embedded in the composite gel and injected subcutaneously in immunodeficient mice produced lamellar osteoid tissue and differentiated into osteoblasts. This study points this new composite hydrogel as a promising scaffold for bone tissue engineering applications.     

牙髓牙本质组织工程生物支架材料的研究进展

背景：组织工程方法中选择合适的支架是关键性的步骤。目的：回顾分析牙髓牙本质组织工程中支架材料的应用研究。方法：由第一作者检索1993至2012年PubMed数据及万方数据库有关牙髓牙本质组织工程中支架材料应用研究等方面的文献。结果与结论：在牙髓牙本质组织工程中有包括天然生物、人工合成材料和复合材料在内的大量生物材料可供选择,每一种材料都有各自的生物学特点。其中胶原、聚酯、羟基磷灰石等是研究较多的支架材料。自组装多肽水凝胶是由氨基酸制成的新型支架材料,满足理想牙髓牙本质组织工程支架材料的大部分要求,是一种前景广阔的牙髓牙本质组织工程支架材料。     

壳聚糖支架应用在组织工程中：组成复合支架及改性支架的前景

背景：壳聚糖具有无毒性、无刺激性、生物相容性、生物可降解性等优良性能,对多种组织细胞的黏附和增殖具有促进作用,目前已成为组织工程学支架研究方面的一个热点。目的：综述壳聚糖及其衍生物支架在组织工程上的应用进展。方法：应用计算机检索中国生物文献数据库及PubMed数据库1989至2013年,有关壳聚糖及其衍生物支架在组织工程中应用的文献,关键词为“壳聚糖,支架,组织工程;Chitosan,scaffold, tissue engineering”。结果与结论：壳聚糖及其衍生物支架在组织工程中的应用主要集中在几方面：作为细胞培养支架;与各种材料形成复合支架使用;改性壳聚糖支架。壳聚糖及其衍生物是一类有独特生物活性的天然高分子材料,在组织工程研究中有广阔的应用前景,与其他生物材料组成复合材料及制成改性支架将是它今后的研究重点。     

丝素/壳聚糖/纳米羟基磷灰石构建的骨组织工程支架***

背景：丝素蛋白、壳聚糖及纳米羟基磷灰石均是天然材料,具有良好的生物活性和理化特性,作为人体组织工程材料已取得了一定的成果,但3种材料在单独应用的研究中还存在一定的缺陷。目的：制作丝素蛋白/壳聚糖/纳米羟基磷灰石三维支架材料,分析其特性。方法：将丝素蛋白、壳聚糖、纳米羟基磷灰石分别配制成2%的溶液后,分别按照1∶1∶0.5,1∶1∶1,1∶1∶1.5的体积比混合,采用冷冻干燥与化学交联技术制备成三维复合支架材料。检测三维复合支架的孔隙率、吸水膨胀率及热水溶失率,采用材料力学测验机测试干燥三维复合支架材料的拉伸和压缩弹性模量,采用扫描电镜检测三维复合支架的孔径。结果与结论：丝素蛋白/壳聚糖/纳米羟基磷灰石三维复合支架在干燥状态下呈白色,无特殊气味,为稳定固态的圆柱体,触之有明显的抗压能力和弹性。随着复合支架材料中纳米羟基磷灰石含量的增高,支架材料的孔隙率、吸水膨胀率、平均孔径呈逐渐减小趋势,热水溶失率及抗压能力表现出相反的趋势,结果显示以1∶1∶1体积比制作的支架更符合骨替代材料要求,其平均孔径为85.67μm、吸水膨胀率的为(135.65±4.56)%、热水溶失率为(22.84±1.06)%,支架材料内部孔隙均匀,呈现网状结构,孔隙之间交通发达,网状结构本身约10μm。     

高性能多孔β-磷酸三钙骨组织工程支架的3D打印

背景：虽然采用溶液浇铸/离子洗出法、原位成型法、静电纺丝法、相分离/冻干法、气体成孔法等制备骨组织工程支架可以获得比较满意的效果,但在精确性、孔隙均匀性、空间结构复杂性、支架个性化等方面略显不足。 目的：利用3D打印制备β-磷酸三钙骨组织工程支架。 方法：利用3D打印制备载药β-磷酸三钙支架,观察其结构,测量其孔隙率和力学强度。将载药β-磷酸三钙支架置入模拟体液中15周,观察其质量变化。将载药β-磷酸三钙支架与大鼠骨髓间充质干细胞共培养7 d,观察细胞黏附与形态变化。分别采用载药β-磷酸三钙支架浸提液与含体积分数15%胎牛血清的低糖 DMEM培养基培养大鼠骨髓间充质干细胞,培养24,48,72 h检测细胞A值,并确定细胞毒性分级;同时成骨诱导培养1周,检测两组细胞碱性磷酸酶活性。 结果与结论：实验制备的支架微观孔隙呈不规则形,孔隙率高,孔隙分布均匀,孔隙连通率高,抗压强度大。载药β-磷酸三钙支架在15周内基本降解完全,与松质骨缺损修复时间相当。大鼠骨髓间充质干细胞黏附于载药β-磷酸三钙支架表面,并深入支架内部,生长良好,增殖活跃,细胞碱性磷酸酶活性有提高,说明载药β-磷酸三钙支架具有良好的细胞相容性。     

Novel cellulose/hydroxyapatite scaffolds for bone tissue regeneration: In vitro and in vivo study

Cellulose scaffolds containing nano‐ or micro‐hydroxyapatite (nHA or μHA) were prepared by the regeneration of cellulose from its acetylated derivative and the mechanical immobilization of inorganic particles, followed by freeze‐drying. Microtomographic (micro‐computed tomography) evaluation revealed that both scaffolds presented a highly interconnected porous structure, with a mean pore diameter of 490 ± 94 and 540 ± 132 μm for cellulose/nHA and cellulose/μHA, respectively. In vitro and in vivo characterizations of the developed scaffolds were investigated. Commercially available bone allograft was used as a control material. For the in vitro characterization, osteoblastic cell cultures were used and characterized over time to evaluate cell adhesion, metabolic activity, and functional output (alkaline phosphatase activity and osteoblastic gene expression). The results revealed greater spreading cell distribution alongside an increased number of filopodia, higher MTT values, and significantly increased expression of osteoblastic genes (Runx‐2, alkaline phosphatase, and BMP‐2) for cellulose/nHA, compared with cellulose/μHA and the control. The in vivo biocompatibility was evaluated in a rabbit calvarial defect model. The investigated scaffolds were implanted in circular rabbit calvaria defects. Four‐ and 12‐week bone biopsies were investigated using micro‐computed tomography and histological analysis. Although both cellulose/HA scaffolds outperformed the assayed control, a significantly higher amount of newly formed mineralized tissue was found within the defects loaded with cellulose/nHA. Within the limitations of this study, the developed cellulose/HA scaffolds showed promising results for bone regeneration applications. The biological response to the scaffold seems to be greatly dependent on the HA particles' characteristics, with cellulose scaffolds loaded with nHA eliciting an enhanced bone response.     

骨组织工程支架材料及其血管化的研究进程

背景：随着骨组织工程学技术的不断发展,利用组织工程骨修复大面积骨缺损成为当今研究的热点。 目的：介绍骨组织工程中的种子细胞、细胞因子、支架材料的特性及材料血管化情况。 方法：以“骨组织工程,支架材料,血管化”为中文关键词,以“bone tissue engineering,scafold, vascularization”为英文关键词,采用计算机检索2000年1月至2012年1月CNKI数据库和PubMed数据库相关文章,选择与骨组织工程学概述、支架材料和血管化方面相关的文章进行分析。 结果与结论：种子细胞的选择、细胞因子的应用、支架材料的性能及血管化程度均对组织工程骨成功修复骨损伤产生着重要影响。适宜的种子细胞是骨组织工程的研究基础,细胞因子是骨组织工程研究的催化剂,具有良好三维结构的支架材料对于促进细胞的生长增殖、组织长入、成骨方式和血管化等方面均有积极的促进作用。但每种支架材料都有其不足之处,所以可以通过将多种材料进行复合达到综合效应来满足临床需求。另外也要积极寻求新的材料制备工艺和对已有方法进行改进,以制造出更加优良的支架材料。但血管化仍然是骨组织工程要面对的重大考验。目前所应用的促进组织工程骨血管化的方法均存在一定缺陷,如利用生长因子促进血管化时,易造成代谢异常患者病情恶化等情况发生;利用显微外科技术促进组织工程骨血管化,易导致其他部位形成创伤和畸形,不利于患者的身体康复等。     

Photopolymerizable chitosan–collagen hydrogels for bone tissue engineering

Photopolymerizable hydrogels derived from naturally occurring polymers have attracted significant interest in tissue‐engineering applications due to their excellent biocompatibility, hydrophilic nature favourable for cell ingrowth and ability to be cured in situ through a minimally invasive procedure. In this study, we developed a composite hydrogel consisting of photocrosslinkable methacrylated glycol chitosan (MeGC) and semi‐interpenetrating collagen (Col) with a riboflavin photoinitiator under blue light. The incorporation of Col in MeGC hydrogels enhanced the compressive modulus and slowed the degradation rate of the hydrogels. MeGC–Col composite hydrogels significantly enhanced cellular attachment, spreading, proliferation and osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) seeded on the hydrogels compared with pure MeGC hydrogels, as observed by upregulated alkaline phosphatase (ALP) activity as well as increased mineralization. Similarly, when cells were encapsulated within hydrogels, BMSCs exhibited greater proliferation, ALP activity and mineral deposits in the presence of Col. These findings demonstrate that MeGC–Col composite hydrogels may be useful in promoting bone regeneration. Copyright © 2014 John Wiley & Sons, Ltd.