Regeneration of whole meniscus using meniscal cells and polymer scaffolds in a rabbit total meniscectomy model

The current treatments of meniscal lesion in knee joint are not perfect to prevent adverse effects of meniscus injury. Tissue engineering of meniscus using meniscal cells and polymer scaffolds could be an alternative option to treat meniscus injury. This study reports on the regeneration of whole medial meniscus in a rabbit total meniscectomy model using the tissue engineering technique. Biodegradable scaffolds in a meniscal shape were fabricated from polyglycolic acid (PGA) fiber meshes that were mechanically reinforced by bonding PGA fibers at cross points with 75:25 poly(lactic-co-glycolic acid). The compressive modulus of the bonded PGA scaffold was 28-fold higher than that of nonbonded scaffold. Allogeneic meniscal cells were isolated from rabbit meniscus biopsy and cultured in vitro. The expanded meniscal cells were seeded onto the polymer scaffolds, cultured in vitro for 1 week, and transplanted to rabbit knee joints from which medial menisci were removed. Ten or 36 weeks after transplantation, the implants formed neomenisci with the original scaffold shape maintained approximately. Hematoxylin and eosin staining of the sections of the neomenisci at 6 and 10 weeks revealed the regeneration of fibrocartilage. Safranin-O staining showed that abundant proteoglycan was present in the neomenisci at 10 weeks. Masson's trichrome staining indicated the presence of collagen. Immunohistochemical analysis showed that the presence of type I and II collagen in neomenisci at 10 weeks was similar to that of normal meniscal tissue. Biochemical and biomechanical analyses of the tissue-engineered menisci at 36 weeks were performed to determine the quality of the tissue-engineered menisci. Tissue-engineered meniscus showed differences in collagen content and aggregate modulus in comparison with native meniscus. This study demonstrates, for the first time, the feasibility of regenerating whole meniscal cartilage in a rabbit total meniscectomy model using the tissue engineering method.     

半月板组织工程化修复实验研究及其生物力学问题的讨论

目的 应用组织工程方法在具完全免疫功能的哺乳动物体内修复半月板缺损并讨论其生物力学问题。方法 15只45天龄的长枫杂交仔猪为实验动物，以改良的Klaflsbrun酶消化法从左膝半月板获得的自体纤维软骨细胞在体外扩增至一定数量，在右膝内侧副韧带前方的内侧半月板造成长1cm的全层缺损，分别将PGA-纤维软骨细胞-pluronic复合物、纤维软骨细胞-pluronic复合物和单纯PGA植入缺损，以正常半月板和旷置缺损作为对照，分别于第9周、16周、25周和36周取材。标本采用大体观察、组织学、生物化学和生物力学作为评估指标。结果 PGA-细胞-pluronic复合物在大体形态、组织学结构和压弹性模量(36周为正常组的69．5％)方面均显示形成了最佳的修复组织，并使对应股骨髁软骨的GAG含量(36周为正常组的58．3％)保持相对稳定。结论自体组织工程化纤维软骨在适宜的力学条件下能够修复乃至再造半月板，并且可以防止膝关节的创伤性退变。     

Comparison of scaffolds and culture conditions for tissue engineering of the knee meniscus

The menisci of the knee are semilunar fibrocartilaginous structures critical in load bearing, shock absorption, stability, and lubrication. In this study, two commonly used biomaterials, a hydrogel (agarose) and a nonwoven mesh polymer [poly(glycolic acid); PGA], were compared for suitability as scaffold materials for tissue engineering the knee meniscus. In addition, a rotating wall bioreactor culture of both scaffold materials was compared with static cultures. Constructs were cultured for up to 7 weeks in static and rotating wall bioreactor culture. Cell numbers were 22 times higher in PGA than agarose after 7 weeks in culture. Static PGA scaffolds had more than twice the amount of sulfated glycosaminoglycans and three times the amount of collagen compared to static agarose constructs at week 7. The rotating wall bioreactor was not found with increase matrix production or cell proliferation significantly over static cultures.     

Design and mechanical evaluation of a novel fiber-reinforced scaffold for meniscus replacement

A fiber-reinforced degradable scaffold for replacement of meniscal tissue was designed, fabricated, and mechanically evaluated. The hypotheses were that (1) the fiber network design would share a portion of compressive loads via the generation of circumferential tensile loads, and (2) the scaffold tensile properties would be similar to those of the meniscus. Two meniscus scaffold designs varying in fiber content (1000 or 500 fibers: MS1000, MS500) underwent cyclic compressive loading up to 100 and 250N, with resultant tensile loads measured at the anterior and posterior anchors. Standard tensile testing was also performed on each device and ovine menisci. Both scaffolds generated tensile loads directly proportional to the applied compressive loads, with MS1000 scaffolds generating approximately twice the tensile loads of MS500 scaffolds. The tensile strength of MS1000 scaffolds was significantly higher than that of the medial and lateral ovine menisci, and approximately twice that of the MS500 scaffolds. The stiffness of MS1000 scaffolds was lower than that of the lateral meniscus, but not statistically different from that of the medial meniscus. These results support our hypotheses that this novel fiber-reinforced scaffold can mimic the tensile and hoop stress behavior of normal meniscal tissue under compressive loading. The circumferential tensile strength and stiffness are appropriate for a meniscus replacement device.     

Meniscal regeneration using tissue engineering with a scaffold derived from a rat meniscus and mesenchymal stromal cells derived from rat bone marrow

The purpose of this study was to regenerate a meniscus using a scaffold from a normal meniscus and mesenchymal stromal cells derived from bone marrow (BM-MSCs). Thirty Sprague-Dawley rat menisci were excised and freeze-thawed three times with liquid nitrogen to kill the original meniscal cells. Bone marrow was aspirated from enhanced green fluorescent protein transgenic Sprague-Dawley rats. BM-MSCs were isolated, cultured for 2 weeks, and 2 x 10(5) cells were then seeded onto the meniscal scaffolds. Using a fluorescent microscope and immunohistochemical staining, repopulation of enhanced green fluorescent protein positive cells was observed in the superficial zone of the scaffold after 1 week of culture, and then in the deep zone after 2 weeks. At 4 weeks, expression of extracellular matrices was detected histologically and expression of mRNA for aggrecan and type X collagen was detected. Stiffness of the cultured tissue, assessed by the indentation stiffness test, had increased significantly after 2 weeks in culture, and approximated the stiffness of a normal meniscus. From this study, we conclude that a scaffold derived from a normal meniscus seeded with BM-MSCs can form a meniscus approximating a normal meniscus.     

Stem cell based tissue engineering for meniscus repair

Defects of the meniscus greatly alter knee function and predispose the joint to degenerative changes. The purpose of this study was to test a recently developed cell-scaffold combination for the repair of a critical-size defect of the rabbit medial meniscus. A bilateral, complete resection of the pars intermedia of the medial meniscus was performed in 18 New Zealand White rabbits. A hyaluronan/gelatin composite scaffold was implanted into the defect of one knee of 6 rabbits and the contralateral defect was left untreated. Scaffolds loaded with autologous marrow-derived mesenchymal stem cells and cultured in a chondrogenic medium for 14 days were implanted in a second series of 12 rabbits. Empty scaffolds were implanted in the contralateral knees. Meniscii were harvested at 12 weeks. Untreated defects had a muted fibrous healing response. Defects treated with cell-free implants showed also predominantly fibrous tissue whereas fibrocartilage was present in some scaffolds. The cross-sectional width of the repair tissue after treatment with cell-free scaffolds was significantly greater than controls (p < 0.05). Pre-cultured implants integrated with the host tissue and 8 of 11 contained meniscus-like fibrocartilage, compared with 2 of 11 controls (p < 0.03). The mean cross-sectional width of the pre-cultured implant repair tissue was greater than controls (p < 0.004). This study demonstrates the repair of a critical size meniscal defect with a stem cell and scaffold based tissue engineering approach.     

构建组织工程化半月板：细胞、支架及生物因子

背景：自体或异体移植修复损伤的半月板治疗效果并不理想,应用组织工程化技术重建半月板的研究成为目前的研究热点。 目的：探讨组织工程化技术修复重建损伤半月板的可行性。 方法：对体外构建组织工程化半月板的种子细胞进行培养,并制备半月板支架材料,将种子细胞依附于支架材料上,利用细胞因子调控种子细胞的黏附、生长、分化和迁移,组织学检查细胞与支架的结合情况以及细胞的数量等。 结果与结论：组织工程化半月板修复研究主要包括种子细胞、支架材料和细胞因子等方面。构建需要的种子细胞有骨髓间充质干细胞和半月板纤维软骨细胞,半月板纤维软骨细胞传至第3代可得出最佳效应浓度。对组织工程化半月板支架材料进行表面修饰,由多材料组成的复合材料具有更好的生物相容性。应用组织工程化技术修复重建损伤的半月板,是今后半月板损伤修复研究一种新的治疗方法。     

组织工程技术修复半月板损伤：从基础到临床的差距

BACKGROUND: The use of tissue engineering technology to build a functional meniscus is a new idea for repair of meniscus injury. OBJECTIVE: To analyze the research progress of seed cells and scaffold materials in tissue-engineered meniscus repair. METHODS: A computer-based search of CNKI and PubMed was performed for articles related to tissue-engineered meniscus repair published from 1996 to 2015. The keywords were "meniscal repair, meniscal injury, tissue engineering, tissue-engineered meniscus, biomaterials, stem cells" in Chinese and English, respectively. RESULTS AND CONCLUSION: Tissue-engineered meniscus reconstruction is a more viable method for repair of meniscus injury. Mesenchymal stem cells are pluripotent cells that are ideal seed cells for tissue-engineered meniscus reconstruction. Scaffolds are one of important factors for meniscus repair, and natural meniscal scaffolds play an important role. Selection and development of scaffold materials for meniscus tissue engineering have experienced a rapid development period from a single material to composite materials. Composite materials make up a lot of shortcomings and deficiencies that a single material has, and open up new ideas for developing new materials. Meniscal tissues with geometric shapes can be constructed using tissue engineering technology. However, the long-term observation of the biological properties of meniscal tissues is necessary, and from basic to clinic, there is still a lack of reliable data to prove the effect of tissue engineering technology in the meniscus repair.      

Expanded human meniscus-derived cells in 3-D polymer-hyaluronan scaffolds for meniscus repair

Treatment options for lesions of the avascular region of the meniscus using regenerative medicine approaches based on resorbable scaffolds are rare. Recent approaches using scaffold-based techniques for tissue regeneration known from cartilage repair may be a promising treatment option for meniscal tears. The aim of the study was the investigation of meniscus matrix formation of in vitro expanded human meniscus-derived cells in a three-dimensional (3-D) bioresorbable polymer graft for meniscal repair approaches. Cultivation of the human meniscus cells was performed in a resorbable scaffold material made of polyglycolic acid (PGA) and hyaluronic acid, stabilized with fibrin glue. Cell viability and distribution of human meniscus cells in PGA-hyaluronan scaffolds were evaluated by fluorescein diacetate and propidium iodide staining. Verification of typical meniscal extracellular matrix molecules like type I and type III collagen was performed histologically, immunohistochemically and by gene expression analysis. In results, 3-D scaffold-based meniscus cultures showed high cell viability over an observational period of 21 days in PGA-hyaluronan scaffolds. On the protein level, type I collagen and proteoglycans were evident. Gene expression analysis confirmed the re-expression of meniscus-specific markers in PGA-hyaluronan scaffolds. This study demonstrated that in vitro expanded human meniscus cells allow for formation of meniscal matrix components when cultured in 3-D PGA-hyaluronan scaffolds stabilized with fibrin. These results encourage scaffold-based approaches for the treatment of meniscal lesions.     

The structural and compositional transition of the meniscal roots into the fibrocartilage of the menisci

The meniscal roots, or insertional ligaments, firmly attach the menisci to tibial plateau. These strong attachments anchor the menisci and allow for the generation of hoop stress in the tissue. The meniscal roots have a ligament-like structure that transitions into the fibrocartilagenous structure of the meniscal body. The purpose of this study was to carry out a complete analysis of the structure and tissue organization from the body of the meniscus through the transition region and into the insertional roots. Serial sections were obtained from the meniscal roots into the meniscal body in fixed juvenile bovine menisci. Sections were stained for collagen and proteoglycans (PG) using fast green and safranin-o staining protocols. Unstained sections were imaged used a backlit stereo microscope. Optical projection tomography (OPT) was employed to evaluate the three-dimensional collagen architecture of the root–meniscus transition in lapine menisci. Tie-fibres were observed in the sections of the ligaments furthest from the bovine meniscal body. Blood vessels were observed to be surrounded by these tie-fibres and a PG-rich region within the ligaments. Near the tibial insertion, the roots contained large ligament-like collagen fascicles. In sections approaching the meniscus, there was an increase in tie-fibre size and density. Small tie-fibres extended into the ligament from the epiligamentous structure in the outermost sections of the meniscal roots, while large tie-fibre bundles were apparent at the meniscus transition. The staining pattern indicates that the root may continue into the outer portion of the meniscus where it then blends with the more fibrocartilage-like inner portions of the tissue. In unstained sections it was observed that the femoral side of the epiligamentous structure surrounding the root becomes more fibrous and thickens in the inferior inner portion of the posterior medial root. This thickening changes the shape of the root to more closely resemble the meniscus wedge shape. These observations support the concept of root continuity with the outer portion of the meniscus, thereby connecting with the hoop-like structure of the peripheral meniscus. OPT identified continuous collagen organization from the root into the meniscal body in longitudinal sections. In the radial direction, the morphology of the root continues into the meniscal body consistent with the serially sectioned bovine menisci. Blood vessels were prevalent on the periphery of the root. These blood vessels then arborized to cover the anterior femoral surface of the meniscus. This is the first study of the structural transition between the insertional ligaments (roots) and the fibrocartilagenous body of the menisci. These new structural details are important to understanding the meniscal load-bearing mechanism in the knee.     

运动性半月板损伤及组织工程半月板的应用

背景：半月板损伤是常见的膝关节运动损伤之一,常伴有关节软骨的损伤,其原因是膝关节内环境的紊乱。 目的：评价组织工程半月板在半月板构建中的生物支架材料的性能,以寻求合理的半月板替代物。 方法：以“组织工程;半月板修复;生物材料支架;运动性半月板损伤”为中文关键词,以“tissue engineering, the meniscus repair,biological material scaffold; athletic meniscal injury”为英文关键词,应用计算机检索维普数据库(1994-01/2009-12)和Pubmed数据库(1994-01/2009-12),纳入30篇运动性半月板损伤和组织工程半月板相关的文献。对半月板组织的特征、半月板组织工程种子细胞的来源、组织工程半月板支架材料生物相容性和可降解性以及细胞因子在组织工程半月板构建过程中的作用进行综合评价。 结果与结论：与传统半月板修复相比,组织工程化半月板具有无抗原性,来源不受限制,可按预先设计塑型,具有生命力等许多优点。但是组织工程化半月板仍有许多问题有待研究和解决,如何模拟体内环境,在体外成功构建半月板组织,如何提高支架材料的应用性,研制具有与正常人体半月板组织相接近的力学性能的支架材料是一个半月板修复的关键性问题。     

天然胶原半月板组织工程支架修复运动半月板损伤

背景：可吸收天然胶原支架材料是成熟和理想的半月板替代物。 目的：总结半月板组织工程学研究的现状。 方法：以“组织工程学、运动性半月板损伤、种子细胞、可吸收天然胶原、生物支架材料、应力刺激、力学因素”为中文关键词,以“Tissue engineering、Movement of the meniscus、Seed cells、Natural collagen can be absorbed、Biological scaffolds、Stress stimulation、Mechanical factor”为英文关键词,采用计算机检索PubMed数据库和维普数据库中1994年1月至2011年12月与运动性半月板损伤及半月板组织工程研究相关的文章。 结果与结论：目前的研究重点包括半月板损伤机制、可吸收天然胶原作为半月板组织工程支架的可行性分析、应力刺激、半月板恢复力学因素4个方面。研究表明半月板组织工程修复运动性半月板损伤具有良好的应用前景和广阔的使用空间,但在实际应用中,半月板组织工程支架的构建、细胞外基质复合材料的研究及其与组织的相容性,修复后组织工程半月板的应力刺激和所能承受的力学因素问题仍是半月板组织工程学方面的难点问题。     

组织工程材料修复半月板运动损伤的可行性及特点

背景：组织工程技术的发展为半月板损伤的修复和再造开辟了新的途径,利用该技术构建有功能的半月板在防治半月板切除后的并发症中有重要意义。 目的：综述了组织工程材料在半月板运动损伤修复中的可行性及特点。 方法：由第一作者检索1990/2010 PubMed数据库及中国知网数据库有关天然生物材料、人工合成材料、纳米材料修复半月板损伤的文章。英文检索词为“meniscus,sports injuries,repair,tissue engineering,material ”,中文检索词为“半月板,运动损伤,修复,组织工程,支架材料”。 结果与结论：由于半月板的血供特点,致使半月板无血运区损伤不具备愈合能力。组织工程技术的发展为半月板损伤的修复和再造开辟了新途径。目前报道较多的修复半月板支架材料主要有天然生物材料、人工合成材料、纳米材料等。组织工程化半月板的研究已取得了阶段性的成果,但对支架材料正处于研发实验阶段,还没确定出一种最理想的材料,因此寻求一种具有良好的细胞相容性,可控制的降解率并具有一定力学强度的支架材料仍是半月板组织工程的研究热点。     

Indentation properties and glycosaminoglycan content of human menisci in the deep zone

Menisci are two crescent shaped fibrocartilaginous structures that provide fundamental load distribution and support within the knee joint. Their unique shape transmits axial stresses (i.e. “body force”) into hoop or radial stresses. The menisci are primarily an inhomogeneous aggregate of glycosaminoglycans (GAGs) supporting bulk compression and type I collagen fibrils sustaining tension. It has been shown that the superficial meniscal layers are functionally homogeneous throughout the three distinct regions (anterior, central and posterior) using a 300 μm diameter spherical indenter tip, but the deep zone of the meniscus has yet to be mechanically characterized at this scale. Furthermore, the distribution and content of GAG throughout the human meniscal cross-section have not been examined. This study investigated the mechanical properties, via indentation, of the human deep zone meniscus among three regions of the lateral and medial menisci. The distribution of GAGs through the cross-section was also documented. Results for the deep zone of the meniscus showed the medial posterior region to have a significantly greater instantaneous elastic modulus than the central region. No significant differences in the equilibrium modulus were seen when comparing regions or the hemijoint. Histological results revealed that GAGs are not present until at least ～600 μm from the meniscal surface. Understanding the role and distribution of GAG within the human meniscus in conjunction with the material properties of the meniscus will aid in the design of tissue engineered meniscal replacements.     

Biomechanical Properties of Transplanted Xenogenic Meniscal Tissue

The clinical repair approaches to meniscal lesions include total or subtotal meniscectomy, transplantation, and tissue engineering. The investigations found that the transplanted xenogenic meniscal tissues, which were treated by60 Co irradiation and deep freezing, maybe one of the effective approaches. In this paper, we evaluated the biomechanical properties of the transplanted xenogenic meniscal tissues at postoperative 1 year. In vitro tensile and compressive tests are performed to compare the properties of tensile elasticity, tensile strength, and compressive elasticcity,between three groups: RAB group of normal rabbit meniscus tissue, Allo group of transplanted allograft meniscal tissue, and Xeno group of transplanted xenogenic meniscal tissue. The meniscus of the Xeno group showed similar tension and compression modulus as the native rabbit meniscus without significant difference(p0.05),and the tension strength of both the Xeno group and the Allo group were less than that of the native rabbit meniscus(0.05p0.1). No significant difference was found between the Xeno group and the Allo group with regard to each biomechanical parameter(p0.05). These base studies will be helpful in future transplantation and tissue engineering efforts.     

The effect of nanofiber alignment on the maturation of engineered meniscus constructs

The fibrocartilaginous menisci are load-bearing tissues vital to the normal functioning of the knee. Removal of damaged regions of the meniscus subsequent to injury impairs knee function and predisposes patients to osteoarthritis. In this study, we employed biodegradable nanofibrous scaffolds for the tissue engineering of the meniscus. Non-aligned (NA) or fiber-aligned (AL) nanofibrous scaffolds were seeded with meniscal fibrochondrocytes (MFCs) or mesenchymal stem cells (MSCs) to test the hypothesis that fiber-alignment would augment matrix content and organization, resulting in improved mechanical properties. Additionally, we proposed that MSCs could serve as an alternative to MFCs. With time in culture, MSC- and MFC-seeded NA and AL constructs increased in cellularity and extracellular matrix (ECM) content. Counter our initial hypothesis, NA and AL constructs contained comparable amounts of ECM, although a significantly larger increase in mechanical properties was observed for AL compared to NA constructs seeded with either cell type. Cell-seeded NA constructs increased in modulus by approximately 1MPa over 10 weeks while cell-seeded AL construct increased by >7MPa. Additionally, MSC-constructs yielded greater amounts of ECM and demonstrated comparable increases in mechanical properties, thereby confirming the utility of MSCs for meniscus tissue engineering. These results demonstrate that cell-seeded fiber-aligned nanofibrous scaffolds may serve as an instructive micro-pattern for directed tissue growth, reconstituting both the form and function of the native tissue.     

Regional and Zonal Histo‐Morphological Characteristics of the Lapine Menisci

The menisci have crucial weight‐bearing roles in the knee. Regional variations in structure and cellularity of the meniscus have only been minimally investigated. Therefore, the goal of this study was to illustrate the regional cell density, tissue area, and structure of healthy lapine menisci. Skeletally mature Flemish Giant rabbits were used for this study. Upon sacrifice, menisci were removed, fixed in formalin, and cryosectioned. Histological analysis was performed for the detection of sulfated glycosaminoglycans (GAG), collagen Types I and II, cellular density, and tissue area. ANOVA and paired t tests were used for testing of statistical significance. Glycosaminoglycan coverage of the medial meniscus significantly varied between regions, with the anterior region demonstrating significantly more GAG coverage than the posterior region. Inter‐ and intra‐meniscal comparisons revealed variations between zones, with trends that outer zones of the medial menisci had less GAG coverage. Collagen Types I and II had marked characteristics and varying degrees of coverage across regions. Tissue area varied between regions for both medial and lateral menisci. Cellular density was dependent on region in the lateral meniscus. This is the first study to illustrate regional and zonal variation in glycosaminoglycan coverage, size, and cellular density for healthy lapine meniscal tissue. This data provides baseline information for future investigations in meniscal injury models in rabbits. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Multilayered silk scaffolds for meniscus tissue engineering

Removal of injured/damaged meniscus, a vital fibrocartilaginous load-bearing tissue, impairs normal knee function and predisposes patients to osteoarthritis. Meniscus tissue engineering solution is one option to improve outcomes and relieve pain. In an attempt to fabricate knee meniscus grafts three layered wedge shaped silk meniscal scaffold system was engineered to mimic native meniscus architecture. The scaffolds were seeded with human fibroblasts (outside) and chondrocytes (inside) in a spatial separated mode similar to native tissue, in order to generate meniscus-like tissue in vitro. In chondrogenic culture in the presence of TGF-b3, cell-seeded constructs increased in cellularity and extracellular matrix (ECM) content. Histology and Immunohistochemistry confirmed maintenance of chondrocytic phenotype with higher levels of sulfated glycosaminoglycans (sGAG) and collagen types I and II. Improved scaffold mechanical properties along with ECM alignment with time in culture suggest this multiporous silk construct as a useful micro-patterned template for directed tissue growth with respect to form and function of meniscus-like tissue.     

Generation and characterization of a human acellular meniscus scaffold for tissue engineering

Meniscus tears are frequent indications for arthroscopic evaluation which can result in partial or total meniscectomy. Allografts or synthetic meniscus scaffolds have been used with varying success to prevent early degenerative joint disease in these cases. Problems related to reduced initial and long-term stability, as well as immunological reactions prevent widespread clinical use so far. Therefore, the aim of this study was to develop a new construct for tissue engineering of the human meniscus based on an acellular meniscus allograft. Human menisci (n = 16) were collected and acellularized using the detergent sodium dodecyl sulfate as the main ingredient or left untreated as control group. These acellularized menisci were characterized biomechanically using a repetitive ball indentation test (Stiffness N/mm, residual force N, relative compression force N) and by histological (hematoxylin-eosin, phase-contrast) as well as immunohistochemical (collagen I, II, VI) investigation. The processed menisci histologically appeared cell-free and had biomechanical properties similar to the intact meniscus samples (p > 0.05). The collagen fiber arrangement was not altered, according to phase-contrast microscopy and immunohistochemical labeling. The removal of the immunogenic cell components combined with the preservation of the mechanically relevant parts of the extracellular matrix could make these scaffolds ideal implants for future tissue engineering of the meniscus.