3D scaffolds for bone marrow stem cell support in bone repair

Bone tissue repair is one of the major concerns of regenerative medicine. The current need for tissue replacements has necessitated the development of a new science termed 'bone tissue engineering'. The basic organization of bone tissue requires the design and fabrication of a porous 3D structure or 'scaffold' to contain the bone-forming cells. This scaffold should be formulated from biocompatible, osteoconductive materials that are not immunoreactive. 3D scaffolds provide the necessary support for cells to proliferate and maintain their capacity to differentiate and scaffolds containing bone marrow-derived osteoprogenitors can be employed within implants to enhance bone repair. The complex construct is intended to mimic the native in vivo microenvironment and this demands construction of bioactive scaffolds that are also capable of supporting vascularization as well as cell proliferation and osteogenic differentiation. 3D bioactive scaffolds containing committed osteoprogenitors can provide a promising surgical tool for bone tissue engineering directed at orthopedic and cranio-maxillofacial clinical applications.     

In vivo efficacy of bone-marrow-coated polycaprolactone scaffolds for the reconstruction of orbital defects in the pig

Alloplastic materials offer a number of advantages over bone autografts in the reconstruction of craniofacial defects. These include: lack of donor site morbidity, unlimited quantities of available material, and the possibility to conform exactly to the defect. An ideal bioresorbable material would degrade slowly, and have osteoconductive properties to allow replacement and remodeling by osseous tissue. This is seldom observed, the materials instead being replaced by fibrous tissue. Polycaprolactone (PCL), an FDA-approved bioresorbable polymer, has several properties that might make it suitable for reconstruction of craniofacial defects. The technique of fused deposition modeling (FDM) allows for the fabrication of highly reproducible bioresorbable 3D scaffolds. The nature of the fully interconnected pore network might enhance vascular ingrowth and osteoconductive properties. It was hypothesized that coating the scaffolds in bone marrow might enhance bone formation due to the osteoinductive nature of the bone-marrow mesenchymal cells. This study aimed to test these hypotheses in the pig model. Defects measuring 2 x 2 cm were surgically created in each orbit of eight Yorkshire pigs. The orbits were divided into three groups: Group 1 (n=4), no reconstruction (control); Group 2 (n=6), reconstruction with no coated PCL scaffolds; and Group 3 (n=6) reconstruction with bone-marrow-coated PCL scaffolds. The results were evaluated at 3 months by histological and histomorphometric analyses. The defects in Group 1 were covered with fibrous scar tissue. The shape of the reconstructed area was insufficient. The defects in Groups 2 and 3 were reconstructed correctly. In Group 2 the noncoated scaffolds showed 4.5% of new bone formation compared with 14.1% in Group 3, which is statistically significant (p<0.05). The entirely interconnected 3D polycaprolactone scaffold seems to be a promising material. It induces the bone ingrowth required for reconstructing craniofacial and orbital defects. Further long-term evaluations of these PCL scaffolds must be made in order to confirm these conclusions.     

利用同轴3D打印技术构建促内皮细胞生长类血管组织工程支架

体外构建血管网络对组织工程领域厚组织与器官再生至关重要。利用同轴3D打印技术,以海藻酸钠/丝素蛋白为生物墨水,可快速制备含人脐静脉内皮细胞(HUVECs)的类血管组织工程支架。首先通过材料压缩模量和可打印性测试,优化适用于同轴系统的材料浓度;然后通过光学相干层析成像技术,研究打印参数对中空纤维丝形状的影响,优化同轴打印参数;结合模拟灌流实验,对支架内部类血管结构进行表征;最后通过细胞活、死染色和Alamar Blue法,检测支架中HUVECs生长情况。结果表明,经优化的生物墨水及打印参数能顺利制备具有内部联通性完整的类血管组织工程支架;HUVECs在体外培养时存在团聚生长现象,类血管通道的存在有利于维持组织整体活性,一周存活率在97%以上,且相比对照组能够维持较高的增殖速率。研究证明,利用同轴3D打印技术能成功构建促内皮细胞生长的类血管组织工程支架,可为厚组织及器官再生提供新的可能。     

Structural and material approaches to bone tissue engineering in powder-based three-dimensional printing

This article reviews the current state of knowledge concerning the use of powder-based three-dimensional printing (3DP) for the synthesis of bone tissue engineering scaffolds. 3DP is a solid free-form fabrication (SFF) technique building up complex open porous 3D structures layer by layer (a bottom-up approach). In contrast to traditional fabrication techniques generally subtracting material step by step (a top-down approach), SFF approaches allow nearly unlimited designs and a large variety of materials to be used for scaffold engineering. Today's state of the art materials, as well as the mechanical and structural requirements for bone scaffolds, are summarized and discussed in relation to the technical feasibility of their use in 3DP. Advances in the field of 3DP are presented and compared with other SFF methods. Existing strategies on material and design control of scaffolds are reviewed. Finally, the possibilities and limiting factors are addressed and potential strategies to improve 3DP for scaffold engineering are proposed.     

A review of materials, fabrication methods, and strategies used to enhance bone regeneration in engineered bone tissues

Over the last decade, bone engineered tissues have been developed as alternatives to autografts and allografts to repair and reconstruct bone defects. This article provides a review of the current technologies in bone tissue engineering. Factors used for fabrication of three-dimensional bone scaffolds such as materials, cells, and biomolecular signals, as well as required properties for ideal bone scaffolds, are reviewed. In addition, current fabrication techniques including rapid prototyping are elaborated upon. Finally, this review article further discusses some effective strategies to enhance cell ingrowth in bone engineered tissues; for example, nanotopography, biomimetic materials, embedded growth factors, mineralization, and bioreactors. In doing so, it suggests that there is a possibility to develop bone substitutes that can repair bone defects and promote new bone formation for orthopedic applications.     

Development of arginine-glycine-aspartate-immobilized 3D printed poly(propylene fumarate) scaffolds for cartilage tissue engineering

Abstract

Poly(propylene fumarate) (PPF) has known to be a good candidate material for cartilage tissue regeneration because of its excellent mechanical properties during its degradation processes. Here, we describe the potential application of PPF-based materials as 3D printing bioinks to create macroporous cell scaffolds using micro-stereolithography. To improve cell-matrix interaction of seeded human chondrocytes within the PPF-based 3D scaffolds, we immobilized arginine-glycine-aspartate (RGD) peptide onto the PPF scaffolds. We also evaluated various cellular behaviors of the seeded chondrocytes using MTS assay, microscopic and histological analyses. The results indicated that PPF-based biocompatible scaffolds with immobilized RGD peptide could effectively support initial adhesion and proliferation of human chondrocytes. Such a 3D bio-printable scaffold can offer an opportunity to promote cartilage tissue regeneration.     

生物支架材料修复感染性骨缺损的研究进展

骨髓炎所致的感染性骨缺损复发频繁，难以治愈。各种生物支架材料作为极具潜力的新型骨植入材料，有效弥补了现今感染性骨缺损修复材料的缺陷。其中天然生物衍生材料具有良好生物相容性，人工合成无机材料和有机高分子材料抗感染能力显著，复合材料结合3D打印和表面涂层技术，改善了常规植入物机械性能差、抗菌能力差、缺乏骨诱导功能等缺点。生物支架材料已在修复感染性骨缺损、促进骨再生等方面展现出良好前景。本文就生物支架材料修复感染性骨缺损的研究进展作一综述。     

3D生物打印技术及其在组织工程中的应用

3D生物打印是在3D打印(three-dimensional printing,3DP)和组织工程等多学科融合的基础上发展出来的一种新兴应用技术,该技术能够根据数字化的模型设计将生物材料和/或细胞打印成三维生物功能体,被广泛应用于血管、骨骼等组织的再生与重建领域.     

骨软骨组织工程支架的研究现状及发展趋势

目前,随着骨软骨组织工程的发展,为临床上骨软骨缺损的修复带来了新希望。应用自体细胞、支架、生长因子可以修复骨、软骨的缺损;选取具有生物相容性和可吸收性的复合支架可为细胞提供暂时的支持、黏附、生长环境,促进骨软骨缺损的修复。就骨软骨组织工程支架的分类、特性、应用以及存在的问题和发展趋势作一综述。     

骨髓间充质干细胞联合3D生物打印技术治疗骨缺损的研究进展

骨缺损一直以来都是临床治疗中的难题,目前主要是行自体骨或人工骨移植治疗。但自体骨取骨的损伤和人工骨资源有限且价格昂贵使得骨移植手术在临床广泛应用受到限制。近年来国内外学者对骨髓间充质干细胞诱导分化成骨的研究愈加重视,对其分离提取和定向分化的研究取得了长足的进步。骨髓间充质干细胞可向成骨细胞、软骨细胞等分化且不存在排斥反应及伦理问题,其结合3D生物打印技术修复骨缺损具有精准化和可控性的优势,是一种极具潜力和应用前景的新型骨缺损修复技术。文章对骨髓间充质干细胞的生物学特性、成骨诱导及结合载体支架治疗骨缺损的研究进行阐述,为今后在骨缺损的临床治疗中提供理论依据。     

组织工程技术应用于创面修复的现状与未来

组织工程技术已在创面修复领域应用数十年，先后研制出了不同类型的组织工程皮肤替代物。随着3D生物打印技术的发展，为组织工程皮肤产品的研制提供了最理想的方法。目前，组织工程技术虽取得了不少进展，同时也呈现出诸多发展机遇。本文回顾和分析了组织工程在创面修复中的关键要素，包括细胞疗法、支架材料及其生长因子的调节，并提出了组织工程技术在创面修复中的意义、难点、热点和今后发展的策略与方向。     

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       

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。     

骨组织工程支架材料的研究现状与应用前景

背景：目前组织工程骨修复骨缺损在临床应用中较为关键的问题是建立血管网,为新骨的形成提供氧气及营养物质,并为机体提供代谢途径。 目的：综述近年组织工程骨支架材料的特点,并着重介绍复合支架材料的研究现状。 方法：以“骨组织工程,血管化,支架材料,复合支架材料”为中文检索词,以“bone tissue engineering, vascularization,scaffold,composite scaffold”英文检索词,应用计算机在中国期刊全文数据库和PubMed数据库检索2001年1月至2014年1月的相关文章,将所有文章进行初步筛选后,对保留的文章进一步详细分析、归纳并总结。 结果与结论：按照组织工程骨支架材料的来源不同,可将其分为人工合成材料、天然衍生材料和复合支架材料,单一支架材料难以作为最理想的材料修复骨缺损,复合支架材料能在不同程度上弥补单一支架材料的缺陷,因此近年来组织工程支架材料的发展由单一材料发展为复合材料,并呈现人工合成材料与天然材料有机结合的趋势。但复合支架材料在临床应用中仍然有许多尚待解决的问题,主要有控制复合材料比例,使材料降解速率与组织细胞的生长速率相适应,保持复合材料的多孔隙和高机械强度。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：     

面向组织工程化软组织的制造技术及增材制造

背景：近年来,组织工程支架的制造方法众多,特别是增材制造技术因其独特的累积成型原理,为复杂软组织支架的高精度制造提供了高效、可靠的制造技术,也推动了大缺损软组织修复研究。 目的：总结近年来关于面向软组织支架的制造方法,对其进行简要的综述,并探讨其存在的问题与前景。 方法：应用计算机检索PubMed数据库及中国知网数据库2010年1月至2013年9月关于软组织支架的制造方法的文章,英文检索词为“additive manufacturing,microfabrication,vascular tissue engineering,muscle tissue engineering,cartilage tissue engineering,stereolithography,3D printing,biodegradable hydrogel”, 中文检索词为“增材制造,微制造,血管组织工程,肌肉组织工程,软骨组织工程,光固化快速成型,三维打印技术,可降解水凝胶”。 结果与结论：软组织大块缺损支架的制造,已由简单平面结构向复杂三维转变,并考虑到软组织内部血管的作用,在制造过程中将软组织支架材料与细胞、生长因子结合,达到解决支架内部血管化的问题。增材制造技术为复杂形状的软组织活性支架的高精度制造提供了新的方法。水凝胶/细胞的构建是软组织支架的关键,而与之相关的高精度增材制造技术原理和制造工艺,以及水凝胶、细胞与生长因子的组装方法则是突破这一关键的核心技术。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：     

三维打印技术在骨组织工程领域的应用研究进展

综述了三维(3D)打印技术的出现、分类与优势等.介绍了该技术在骨组织工程领域的应用,包括光固化立体印刷、熔融沉积成型、选择性激光烧结和3D喷印的工作原理、存在的优缺点以及国内外学者在该领域的研究进展.目前骨组织工程支架的制备大多应用了3D打印技术,以生物可降解的活性材料为原料制备而成.在我国该领域虽然发展迅速,利用3D打印技术进行人工骨合成、骨科术前模拟等已经越来越普遍,亦取得了令人满意的效果,但要研发出合适的生物材料以及设备精度的改进仍是亟待解决的问题.目前,仿生器官的功能化已成为3D打印技术领域的一大困难,其中多细胞共培养、血管化及支架的制备是实现功能化必须克服的问题,相信通过努力,该项技术将会为器官的再生与修复带来更多令人瞩目的成果.     

水凝胶材料和间充质干细胞在组织工程中的应用进展

随着组织工程学的发展, 人们越来越关注将水凝胶作为支架材料并与细胞3D培养相结合用于组织器官再生与修复。水凝胶由亲水性聚合物、共聚物或可以形成大分子链的单体大分子交联而成, 可吸收大量水分并保持3D结构, 具有良好的生物相容性、可包埋细胞和有效的递送生物活性分子等特点, 因而被广泛用于生物医药领域的药物输送和组织工程等领域。间充质干细胞可以从骨髓、脂肪、脐带等多种组织中获取, 具有低免疫原性及多向分化潜能, 是细胞3D培养以及细胞治疗的首选。目前间充质干细胞主要是2D培养模式, 该培养模式下的间充质干细胞繁殖率低, 且无法模拟体内的生长环境。水凝胶材料作为3D细胞培养支架具有良好的相容性, 可以模拟体内的生长环境, 在修复受损软骨、骨、皮肤和心脏等组织中有巨大潜力。概述水凝胶、间充质干细胞以及间充质干细胞和水凝胶材料在组织工程中的应用, 展示水凝胶材料与间充质干细胞的3D培养在不同组织再生和修复中的发展趋势和可能性, 以期为后续水凝胶和干细胞的深入应用研究提供参考。     

Biomimetic porous scaffolds with high elasticity made from mineralized collagen--an animal study

Histological investigations of a new hydroxyapatite-collagen composite material were carried out to evaluate its possible suitability as a bone substitute. The three-dimensional scaffolds made from biomimetically mineralized collagen exhibit an interconnecting pore structure and elastic mechanical properties. They were implanted into the subcutaneous tissue and bone defects made in the femur of rats and harvested with the surrounding tissue at 1, 2, 4, 8, and 12 weeks after surgery. The materials implanted in the subcutaneous tissue were covered by fibrous connective tissue with a slight inflammatory response, and many foreign-body giant cells were observed on the surface of the scaffolds. Most of the material implanted in the subcutaneous tissue was resorbed at 8 weeks by phagocytosis. In the bone defects, new bone formation was observed on the surface of the material at 1 week. New bone increased with time, and osteoclasts were seen on the surface of the scaffolds at 2 weeks. Resorption and replacement by new bone of many parts of the materials implanted in the femur were observed by 12 weeks. These responses occurred faster than those of other hydroxyapatite-collagen composites. The results suggested that the new biomimetically mineralized collagen scaffolds were suitable as an implant material for bone-tissue reconstruction.     

基于同轴流技术的肝组织生物3D打印研究

生物三维打印为医疗领域提供全新的技术可能,可广泛应用于制造人工组织和器官。人工组织的功能和尺寸大小受限于组织的血管化,可利用同轴流挤出系统制造封装肝细胞的中空细丝,结合生物3D打印系统,叠层制造含微通道网络的肝组织。首先搭建集成化的同轴流生物3D打印系统,研究材料挤出速率、材料浓度等参数对中空细丝尺寸及出丝速度的影响;然后以肝细胞株C3A为材料,打印含多层管网机构的仿生肝组织;最后,对含微通道的肝组织进行分组培养,利用细胞活死染色法检测第24、48、72 h肝细胞在灌流组和非灌流组中的细胞存活率。实验表明,同轴流3D打印的组织,中空细丝之间有效融合,支架内部的立体微通道网络完整;打印过程对肝细胞损伤较小,中空细丝中的肝细胞存活率达90%以上;灌流组和非灌流组在培养72 h后,细胞存活率有显著的差异,证明对微通道灌流可以促进组织内部的物质交换,提高微通道周围肝细胞的存活率。研究提出打印方法和灌流系统,为人工组织的血管化以及培养方式提供全新的思路。     

Double protein-coated poly-ε-caprolactone scaffolds: successful 2D to 3D transfer

In the past decade, tissue engineering has evolved from a promising technology to an established scientific field. Large attention has focussed on developing scaffolds from both biodegradable and nondegradable polymers to be cultivated with cells, to replace human body defects. The major drawback of most polymers is however their limited cell-interactive properties. An additional complication when developing a surface modification protocol for those materials is the transferability of protocols from 2D substrates to 3D scaffolds. In the present work, we therefore report on possible biological effects originating from the transfer of a double protein coating protocol, involving gelatin type B and fibronectin, from 2D poly-ε-caprolactone (PCL) films to 3D PCL scaffolds produced by rapid prototyping. A variety of techniques including scanning electron microscopy, X-ray photoelectron spectroscopy and confocal fluorescence microscopy confirmed a successful and homogeneous protein-coating on both 2D and 3D substrates. Interestingly, the biological performance of the double protein-coated PCL substrates, reflected by the initial cell adhesion, proliferation, and colonization was superior compared to the other surface modification steps, independent of the material dimension.