韧带组织工程研究进展

韧带组织血供少 ,损伤后很难完全愈合 ,造成关节功能严重障碍。组织工程学的发展为韧带损伤的治疗提供了新的可能途径。本文对组织工程韧带领域中目前研究的主要成果进行综述 ,着重就组织工程韧带种子细胞的选择、生长因子的应用、支架材料的选择以及细胞与支架材料之间的相互作用四个方面进行了阐述。目前的研究虽取得了一些进展 ,但仍需在构建有良好力学性能和降解性能支架材料、设计制造能对韧带细胞进行三维培养的生物反应器等方面进行大量研究。     

组织工程韧带支架材料的选择及其生物相容性

目的:总结修复韧带损伤的主要生物材料及研究进展,强调韧带的损伤类型,评价修复运动性韧带损伤中所应用的组织工程韧带支架的各种生物材料的性能和应用,寻找合理的组织工程韧带支架材料。方法:由第一作者采用电子检索的方式,在PubMed数据库及万方数据库中检索1999-01/2010-07有关生物材料应用于组织工程韧带支架的研究文章,中文关键词为"重建韧带,生物材料,人工韧带,组织工程,支架材料";英文关键词为"cruciate ligamen,LARS artificial ligament,ligament reconstruction"。排除重复研究、普通综述或Meta分析类文章,筛选纳入19篇文献进行评价。结果:论述了组织工程支架材料的选择及其生物相容性,虽然目前的研究取得了较大的进步,但仅处在起步阶段,距离大规模的临床应用还很远。结论:天然及合成高分子材料作为组织工程支架材料都有各自的优缺点,绝大多数还处于研究阶段,尚未应用至临床,因此改进他们作为支架材料的性能是目前研究的主要方向之一。     

组织工程韧带支架材料在体内的转归

背景:组织工程韧带由于具有自我更新和改建能力,生物力学性能优良,从而克服了现有人造韧带的不足,有望成为理想的韧带替代物,且已成为目前韧带修复重建外科领域关注的热点。目的:通过分析和总结1998年以来采用复合培养支架移植修复损伤韧带的研究,以寻求适合组织工程韧带的新型支架材料及适用标准。方法:分别以"韧带、组织工程、支架材料、细胞外基质","Ligament、Tissue Engineering,、Scaffold material、ECM"为检索词,应用计算机检索Pubmed数据库及重庆维普期刊全文数据库1998-01/2009-12有关文章。纳入有关组织工程韧带的文献。排除与研究目的无关和内容重复者。保留31篇文献做进一步分析。结果与结论:韧带和肌腱损伤的治疗已研究较多,但对其修复重建以至完全恢复到受伤前的功能水平,迄今尚未实现。组织工程韧带的出现可能给韧带和肌腱损伤提供理想的治疗途径。组织工程支架材料研究结果报道越来越多,然而均各有利弊,绝大多数还处于研究阶段。目前仍在寻找一种完全合适的支架材料来治疗韧带损伤。     

膝关节前交叉韧带修复的组织工程研究进展

本文介绍了前交叉韧带组织工程(即利用组织工程的方法重建具有生物性能的韧带,恢复损伤韧带的生理功能)的研究现状,展望了其发展前景,并分析了该领域有待解决的关键问题,重点论述了前交叉韧带组织工程中种子细胞、生物支架、材料表面修饰、生长因子等的选择和应用。     

人工韧带材料与膝关节韧带损伤

背景：目前临床重建膝关节交叉韧带使用的材料包括自体移植物、异体移植物和人工合成材料。自体和异体移植物重建交叉韧带依然是目前的主流选择,但均存在各自的缺点。 目的：探讨膝关节韧带损伤人工合成材料的优化选择策略。 方法：由作者采用电子检索的方式,在万方数据库(http://www.wanfangdata.com.cn/)及PubMed数据库(http://www.ncbi.nlm.nih.gov/PubMed)中检索1988-01/2010-12有关人工韧带生物工程学设计及临床应用的研究文章,检索词为“人工韧带,材料,组织工程,重建”。经检索排除内容重复、Meta分析类文章后筛选纳入20篇文献进行评价。 结果与结论：目前组织工程人工韧带在某些方面取得了一定的进展,但在很多方面仍需进一步研究。种子细胞的选择支架材料的组织相容性和力学性质的统一,细胞和支架材料界面生物相容性的不确定性等是组织工程韧带的研究热点和趋势。随着研究的深入,组织工程人工韧带有望尽早得到临床使用。 关键词：人工韧带;膝关节;材料;组织相容性;组织工程 doi:10.3969/j.issn.1673-8225.2012.12.035     

组织工程半月板支架材料研究进展

组织工程半月板为临床严重的半月板损伤提供了一种新的治疗手段,而半月板支架材料是组织工程半月板的基本要素,也是近年来组织工程半月板研究的热点领域。近年来组织工程半月板支架材料无论是天然生物材料还是合成高聚物材料的研究都取得了可喜的进展。目前胶原半月板移植物(collagen meniscus implant,CMI)已经开始应用于临床。制成的水凝胶支架材料由于含水量高,结构与体内细胞外基质类似,生物相容性好,还为临床微创手术提供可能,因此在近年来成为研究热点。同时,综合各种支架材料优点的生物复合材料更是组织工程半月板支架材料研究的重点。本文通过广泛查阅近年有关组织工程半月板研究的文献并从支架材料的角度进行综述,为组织工程半月板研究提供参考。     

组织工程化前交叉韧带在运动损伤修复中应用

背景：运动员前交叉韧带的损伤极难彻底恢复,降低运动寿命。 目的：总结组织工程化前交叉韧带在运动损伤修复中的研究现状和最新进展。 方法：采用计算机检索维普数据库和PubMed数据库1994-01/2010-12相关文章,纳入28篇与运动性前交叉韧带损伤及组织工程韧带相关的文章,重点对组织工程前交叉韧带重建的研究进展、前交叉韧带种子细胞的来源、细胞因子在前交叉韧带重建中的应用及组织工程前交叉韧带附丽的基础研究4个方面进行探讨。 结果与结论：运动中前交叉韧带损伤后自体愈合能力极差,对于运动员伤后痊愈造成极大困难。组织工程前交叉韧带近些年来飞速发展,具有良好的应用和发展前景。但在实际应用过程中,组织工程化种子细胞的选择、支架材料的构建和组织工程前交叉韧带附丽的基础研究三者有机相互融合,并合理应用生长因子,才能在临床上起到最佳治疗效果,最终达到修复和重建前交叉韧带的目的。     

可降解聚氨酯构建组织工程前交叉韧带支架材料的可行性

背景：前交叉韧带是膝关节常见损伤部位,组织工程前交叉韧带研究在过去的10年内取得了长足的进展,但人们尚未找到理想的支架材料。 目的：探讨使用可降解聚氨酯材料构建组织工程交叉韧带支架的可行性。 方法：将可降解聚氨酯纤维丝编织的聚氨酯材料于含胎牛血清的DMEM培养基制备浸提液,分别以体积分数100%,50%,25%,12.5%的浸提液培养NIH3T3细胞,CCK-8实验检测聚氨酯材料浸提液的细胞毒性;然后将体外分离培养的兔前交叉韧带细胞和NIH3T3细胞分别种植在支架材料上进行立体培养,观察细胞在支架材料上的生长情况;使用电子拉力机测试支架材料的力学性能并分析。 结果与结论：构建组织工程交叉韧带支架的可降解聚氨酯材料没有细胞毒性;前交叉韧带和NIH3T3细胞贴附于该材料生长,生长状态良好;支架材料的杨氏模量,极限抗张强度和断裂伸长率分别为(41.2±2.1) MPa,(51.0±1.5) MPa和(600±60)%。提示聚氨酯支架材料具有良好的生物相容性和生物安全性。     

不同支架材料与间充质干细胞构建组织工程韧带在前交叉韧带损伤中的应用

背景：前交叉韧带是连接于股骨和胫骨之间的致密结缔组织,是能维持膝关节前向稳定性的重要结构,前交叉韧带损伤会导致膝关节前向不稳,若不及时治疗,将继发软骨、半月板等结构损伤。 目的：综述间充质干细胞与不同支架材料构建组织工程韧带在前交叉韧带损伤中的应用。 方法：应用计算机检索1950-01/2010-11 PubMed数据库相关文章,检索词“ACL,stem cells,MSC,tendon,ligament”。共检索到文献103篇,最终纳入符合标准的文献35篇。 结果与结论：应用间充质干细胞和支架构成的组织工程韧带目前已成功在动物实验中应用,间充质干细胞能明显加快韧带损伤修复,加强组织愈合的生物力学性能,但是再生组织的生物力学性能仍未达到正常肌腱水平。同时,间充质干细胞能促进移植物与骨的愈合,防止前交叉韧带重建后腱骨不愈合导致的失败。选择合适的间充质干细胞,支架,适当的机械刺激和必要的细胞因子是构建良好组织工程韧带的关键。随着组织工程科学及支架材料工程学的发展,应用间充质干细胞构建组织工程韧带不久将从实验室走向临床应用。     

前交叉韧带重建修复运动性膝关节损伤:临床现状与未来

背景:膝关节交叉韧带损伤后自愈能力较差,治疗上以移植物植入重建交叉韧带为主。目的:总结膝关节前交叉韧带的功能、结构,力学特点,及其缺损后人工韧带重建研究的进展,为人工韧带的临床应用提供依据。方法:作者应用计算机检索数据库,检索关键词"人工合成材料;膝交叉韧带,韧带修复,运动,韧带重建。选择的文献内容与材料学特点、生物相容性及其膝关节韧带损伤相关领域的文章,共引用25篇文献,重点讨论重建膝关节交叉韧带的性能,及其生物材料的种类。结果与结论:前交叉韧带重建作为治疗前交叉韧带损伤的有效治疗手段,近年来发展快速。目前国内外修复前交叉韧带损伤可供选择的移植物有自体组织替代物、同种异体韧带、生物组织工程韧带以及人工韧带等。人工合成材料与组织工程韧带近几年研究较多,生物型人工韧带对膝关节韧带损伤的修复取得了很好的效果。分子生物学技术以及基因学和细胞学在韧带中的运用,为未来膝关节韧带损伤的康复及治疗提供新的研究方向。     

ACL reconstruction using a novel hybrid scaffold composed of polyarylate fibers and collagen fibers

The objective was to perform an initial in vivo evaluation of a novel braided hybrid polyarylate and collagen fiber scaffold for the reconstruction of the anterior cruciate ligament (ACL). The braided hybrid scaffold is composed of 75% poly(desaminotyrosyl-tyrosine dodecyl dodecanedioate)(12,10), [p(DTD DD)] fibers and 25% type I bovine collagen fibers. The scaffold is designed to temporarily bear mechanical loads and gradually degrade as neoligament tissue is deposited. Scaffolds were electron beam sterilized and used to reconstruct the ACL in five Finnish Dorset crossed-bred sheep in this feasibility study. At 4 (n = 1) and 12 (n = 4) weeks post-op, scaffolds were retrieved and analyzed for cellular ingrowth and strength retention. There was extensive cell infiltration and vascularity, which increased with time. Tissue ingrowth occurred throughout the cross section in the midsubstance of the scaffolds. After 12 weeks all scaffolds were intact. Femur-scaffold-tibia complex (FSTC) explanted at 12 weeks had a yield load of 42 ± 22 N and a stiffness of 9 ± 3 N mm(-1) . All scaffolds were well tolerated in the intraarticular space and induced tissue ingrowth, including new blood vessels, fibroblasts, inflammatory cells, and newly deposited collagen, throughout the cross section of the scaffold. Tissue ingrowth is critical to the success of a degradable scaffold for ACL reconstruction. Long-term studies in a large animal model are required to determine the efficacy of these novel hybrid scaffolds for ACL reconstruction. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:2913-2920, 2012.     

Assessment of tissue ingrowth rates in polyurethane scaffolds for tissue engineering

The continuous development of new biomaterials for tissue engineering and the enhancement of tissue ingrowth into existing scaffolds, using growth factors, create the necessity for developing adequate tools to assess tissue ingrowth rates into porous biomaterials. Current histomorphometric techniques evaluating rates of tissue ingrowth tend either to measure the overall tissue content in an entire sample or to depend on the user to indicate a front of tissue ingrowth. Neither method is particularly suitable for the assessment of tissue ingrowth rates, as these methods either lack the sensitivity required or are problematic when there is a tissue ingrowth gradient rather than an obvious tissue ingrowth front. This study describes a histomorphometric method that requires little observer input, is sensitive, and renders detailed information for the assessment of tissue ingrowth rates into porous biomaterials. This is achieved by examining a number of computer-defined concentric zones, which are based on the distance of a pixel from the scaffold edge. Each zone is automatically analyzed for tissue content, eliminating the need for user definition of a tissue ingrowth front and thus reducing errors and observer dependence. Tissue ingrowth rates in two biodegradable polyurethane scaffolds (Estane and polycaprolactone-polyurethane [PCLPU]) specifically designed for tissue engineering of the knee meniscus were assessed. Samples were subcutaneously implanted in rats with follow-up until 6 months. Especially at the earlier follow-up points, PCLPU scaffolds showed significantly higher tissue ingrowth rates than Estane scaffolds, making the PCLPU scaffold a promising candidate for further studies investigating meniscus tissue engineering.     

Microstructure design of biodegradable scaffold and its effect on tissue regeneration

Biodegradable scaffolds play a critical role in therapeutic tissue engineering, in which the matrix degradation and tissue ingrowth are of particular importance for determining the ongoing performance of tissue-scaffold system during regenerative process. This paper aims to explore the mechanobiological process within biodegradable scaffolds, where the representative volume element (RVE) is extracted from periodic scaffold micro-architectures as a base-cell design model. The degradation of scaffold matrix is modeled in terms of a stochastic hydrolysis process enhanced by diffusion-controlled autocatalysis; and the tissue ingrowth is modeled through the mechano-regulatory theory. By using the finite element based homogenization technique and topology optimization approach, the effective properties of various periodic scaffold structures are obtained. To explore the effect of scaffold design on the mechanobiological evolutions of tissue-scaffold systems, different scaffold architectures are considered for polymer degradation and tissue regeneration. It is found that the different tissues can grow into the degraded voids inside the polymer matrix. It is demonstrated that the design of scaffold architecture has a considerable impact on the tissue regeneration outcome, which exhibits the importance of implementing different criteria in scaffold micro-structural design, before being fabricated via rapid prototyping technique, e.g. solid free-form fabrication (SFF). This study models such an interactive process of scaffold degradation and tissue growth, thereby providing some new insights into design of biodegradable scaffold micro-architecture for tissue engineering.     

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：     

Image analysis of the axonal ingrowth into poly(D,L-lactide) porous scaffolds in relation to the 3-D porous structure

Porous polymer scaffolds are promising materials for neural tissue engineering because they offer valuable three-dimensional (3-D) supports for the in vitro and in vivo axonal growth and tissue expansion. At the time being, how the in vivo neuronal cell development depends on the scaffold 3-D architecture is unknown. Therefore, scanning electron micrographs of longitudinal sections of porous polylactide scaffolds and immunohistological sections of these scaffolds after implantation and neurofilament staining have been studied by image analysis. Pore orientation and axonal ingrowth have been investigated by spectral analysis on gray level SEM images. Binary image processing has been carried out and the binary images have been studied by spectral analysis in order to estimate the possible effect of the image noise on the real pattern. In addition to axonal orientation, density and length distribution of the regenerated axons into the polymer scaffold have been measured. Dependence of the axonal ingrowth on the 3D-polymer scaffold has been discussed on the basis of the collected data.     

Assessment of bone ingrowth into porous biomaterials using MICRO-CT

The three-dimensional (3D) structure and architecture of biomaterial scaffolds play a critical role in bone formation as they affect the functionality of the tissue-engineered constructs. Assessment techniques for scaffold design and their efficacy in bone ingrowth studies require an ability to accurately quantify the 3D structure of the scaffold and an ability to visualize the bone regenerative processes within the scaffold structure. In this paper, a 3D micro-CT imaging and analysis study of bone ingrowth into tissue-engineered scaffold materials is described. Seven specimens are studied in this paper; a set of three specimens with a cellular structure, varying pore size and implant material, and a set of four scaffolds with two different scaffold designs investigated at early (4 weeks) and late (12 weeks) explantation times. The difficulty in accurately phase separating the multiple phases within a scaffold undergoing bone regeneration is first highlighted. A sophisticated three-phase segmentation approach is implemented to develop high-quality phase separation with minimal artifacts. A number of structural characteristics and bone ingrowth characteristics of the scaffolds are quantitatively measured on the phase separated images. Porosity, pore size distributions, pore constriction sizes, and pore topology are measured on the original pore phase of the scaffold volumes. The distribution of bone ingrowth into the scaffold pore volume is also measured. For early explanted specimens we observe that bone ingrowth occurs primarily at the periphery of the scaffold with a constant decrease in bone mineralization into the scaffold volume. Pore size distributions defined by both the local pore geometry and by the largest accessible pore show distinctly different behavior. The accessible pore size is strongly correlated to bone ingrowth. In the specimens studied a strong enhancement of bone ingrowth is observed for pore diameters>100 microm. Little difference in bone ingrowth is measured with different scaffold design. This result illustrates the benefits of microtomography for analyzing the 3D structure of scaffolds and the resultant bone ingrowth.     

VEGF-mediated angiogenesis and vascularization of a fumarate-crosslinked polycaprolactone (PCLF) scaffold

Purpose: Revascularization of natural and synthetic scaffolds is a critical part of the scaffold’s incorporation and tissue ingrowth. Our goals were to create a biocompatible polymer scaffold with 3D-printing technology, capable of sustaining vascularization and tissue ingrowth. Methods: We synthesized biodegradable polycaprolactone fumarate (PCLF) scaffolds to allow tissue ingrowth via large interconnected pores. The scaffolds were prepared with Poly(lactic-co-glycolic acid)(PLGA) microspheres seeded with or without different growth factors including VEGF,FGF-2, and/or BMP-2. Scaffolds were implanted into the subcutaneous tissues of rats before undergoing histologic and microCT angiographic analysis.

Results: At harvest after 12 weeks, scaffolds had tissue infiltrating into their pores without signs of scar tissue formation, fibrous capsule formation, or immune responses against PCLF. Histology for M1/M2 macrophage phenotypes confirmed that there were no overt signs of immune responses. Both microCT angiography and histologic analysis demonstrated marked tissue and vessel ingrowth throughout the pores traversing the body of the scaffolds. Scaffolds seeded with microspheres containing VEGF or VEGF with either BMP-2 or FGF-2 had significantly higher vascular ingrowth and vessel penetration than controls. All VEGF-augmented scaffolds were positive for Factor-VIII and exhibited collagen tissue infiltration throughout the pores. Furthermore, scaffolds with VEGF and BMP-2 had high levels of mineral deposition throughout the scaffold that are attributable to BMP-2.

Conclusions: PCLF polymer scaffold can be utilized as a framework for vascular ingrowth and regeneration of multiple types of tissues. This novel scaffold material has promise in tissue regeneration across all types of tissues from soft tissue to bone.     

Combination of platelet-rich plasma with polycaprolactone-tricalcium phosphate scaffolds for segmental bone defect repair

Porous scaffold biomaterials may offer a clinical alternative to bone grafts; however, scaffolds alone are typically insufficient to heal large bone defects. Numerous studies have demonstrated that osteoinductive growth factor or gene delivery significantly improves bone repair. However, given the important role of vascularization during bone regeneration, it may also be beneficial to incorporate factors that promote vascular ingrowth into constructs. In this study, a strategy combining structural polycaprolactone-20% tricalcium phosphate (PCL-TCP) composite scaffolds with platelet-rich plasma (PRP) was tested. Following bilateral implantation of constructs into 8 mm rat nonunion femoral defects, 3D vascular and bone ingrowth were quantified at 3 and 12 weeks using contrast-enhanced microcomputed tomography (micro-CT) imaging. At week 3, PRP-treated femurs displayed 70.3% higher vascular volume fraction than control femurs. Interestingly, bone volume fraction (BVF) was significantly higher for the empty scaffold group at the early time point. At 12 weeks, BVF measurements between the two groups were statistically equivalent. However, a greater proportion of PRP-treated femurs (83%) achieved bone union as compared to empty scaffold controls (33%). Consistent with this observation, biomechanical evaluation of functional integration also revealed a significantly higher torsional stiffness observed for PRP-treated defects compared to empty scaffolds. Ultimate torque at failure was not improved, however, perhaps due to the slow resorption profile of the scaffold material. Histological evaluation illustrated infiltration of vascularized connective tissue and bone in both groups. Given that bone ingrowth into untreated defects in this model is minimal, PCL-TCP scaffolds were clearly able to promote bone ingrowth but failed to consistently bridge the defect. The addition of PRP to PCL-TCP scaffolds accelerated early vascular ingrowth and improved longer-term functional integration. Taken together, the results of this study suggest that the use of PRP, alone or in combination with other bioactive components, may be an effective approach to augment the ability of porous biomaterial scaffolds to repair orthotopic defects.