组织工程化软骨修复运动性软骨损伤

BACKGROUND: Sports-induced cartilage injury is very common; due to the poor self-healing capacity of the cartilage, cartilage repair has always been a difficult problem. OBJECTIVE: To review the features of different seed cells in tissue-engineered cartilage construction and to explore the application of tissue-engineered cartilage construction in the repair of sports-induced cartilage injury in vitro. METHODS: We searched PubMed database, Wanfang database and CNKI database for articles related to tissue-engineered cartilage repair of sports-induced cartilage injuries, as well as stem cells and scaffold materials used in tissue-engineered cartilage construction. Totally 190 articles were retrieved, and finally 47 articles were included in result analysis after repetitive studies were excluded. RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells under different conditions can differentiate into chondrocytes, and have better potential of chondrogenic differentiation compared with adipose-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells. But, their safety still needs to be further studied. Good scaffolds cannot only induce stem cell differentiation, but also be the key to cartilage construction. Composite materials are the future direction of the scaffold research.      

Effects of cell-to-collagen ratio in stem cell-seeded constructs for Achilles tendon repair

The objective of the present study was to test the hypotheses that implantation of cell-seeded constructs in a rabbit Achilles tendon defect model would 1) improve repair biomechanics and matrix organization and 2) result in higher failure forces than measured in vivo forces in normal rabbit Achilles tendon (AT) during an inclined hopping activity. Autogenous tissue-engineered constructs were fabricated in culture between posts in the wells of silicone dishes at four cell-to-collagen ratios by seeding mesenchymal stem cells (MSC) from 18 adult rabbits at each of two seeding densities (0.1 x 10(6) and 1 x 10(6) cell/mL) in each of two collagen concentrations (1.3 and 2.6 mg/mL). After 5 days of contraction, constructs having the two highest ratios (0.4 and 0.8 M/mg) were damaged by excessive cell traction forces and could not be used in subsequent in vivo studies. Constructs at the lower ratios (0.04 and 0.08 M/mg) were implanted in bilateral, 2 cm long gap defects in the rabbit's lateral Achilles tendon. At 12 weeks after surgery, both repair tissues were isolated and either failed in tension (n = 13) to determine their biomechanical properties or submitted for histological analysis (n = 5). No significant differences were observed in any structural or mechanical properties or in histological appearance between the two repair conditions. However, the average maximum force and maximum stress of these repairs achieved 50 and 85% of corresponding values for the normal AT and exceeded the largest peak in vivo forces (19% of failure) previously recorded in the rabbit AT. Average stiffness and modulus were 60 and 85% of normal values, respectively. New constructs with lower cell densities and higher scaffold stiffness that do not excessively contract and tear in culture and that further improve the repair stiffness needed to withstand various levels of expected in vivo loading are currently being investigated.     

A three-dimensional osteochondral composite scaffold for articular cartilage repair

There is a recognized and urgent need for improved treatment of articular cartilage defects. Tissue engineering of cartilage using a cell-scaffold approach has demonstrated potential to offer an alternative and effective method for treating articular defects. We have developed a unique, heterogeneous, osteochondral scaffold using the TheriForm three-dimensional printing process. The material composition, porosity, macroarchitecture, and mechanical properties varied throughout the scaffold structure. The upper, cartilage region was 90% porous and composed of D,L-PLGA/L-PLA, with macroscopic staggered channels to facilitate homogenous cell seeding. The lower, cloverleaf-shaped bone portion was 55% porous and consisted of a L-PLGA/TCP composite, designed to maximize bone ingrowth while maintaining critical mechanical properties. The transition region between these two sections contained a gradient of materials and porosity to prevent delamination. Chondrocytes preferentially attached to the cartilage portion of the device, and biochemical and histological analyses showed that cartilage formed during a 6-week in vitro culture period. The tensile strength of the bone region was similar in magnitude to fresh cancellous human bone, suggesting that these scaffolds have desirable mechanical properties for in vivo applications, including full joint replacement.     

Current strategies for integrative cartilage repair

Osteoarthritis (OA) is a degenerative joint condition characterized by painful cartilage lesions that impair joint mobility. Current treatments such as lavage, microfracture, and osteochondral implantation fail to integrate newly formed tissue with host tissues and establish a stable transition to subchondral bone. Similarly, tissue-engineered grafts that facilitate cartilage and bone regeneration are challenged by how to integrate the graft seamlessly with surrounding host cartilage and/or bone. This review centers on current approaches to promote cartilage graft integration. It begins with an overview of articular cartilage structure and function, as well as degenerative changes to this relationship attributed to aging, disease, and trauma. A discussion of the current progress in integrative cartilage repair follows, focusing on graft or scaffold design strategies targeting cartilage–cartilage and/or cartilage–bone integration. It is emphasized that integrative repair is required to ensure long-term success of the cartilage graft and preserve the integrity of the newly engineered articular cartilage. Studies involving the use of enzymes, choice of cell source, biomaterial selection, growth factor incorporation, and stratified versus gradient scaffolds are therefore highlighted. Moreover, models that accurately evaluate the ability of cartilage grafts to enhance tissue integrity and prevent ectopic calcification are also discussed. A summary and future directions section concludes the review.     

组织工程支架材料修复关节软骨缺损

BACKGROUND: Articular cartilage repair has been a difficulty in the clinical setting, which is mainly treated with autologous or allogeneic osteochondral grafts, and cartilage periosteum or periosteum grafts. However, the limited source, secondary lesion and immunological rejection force some researchers to search for a novel treatment strategy, cartilage tissue engineering, that is of great significance for cartilage regeneration and repair. OBJECTIVE: To investigate the tissue-engineered scaffolds for the repair of articular cartilage defects. METHODS: The first author searched the PubMed and WanFang databases for the articles addressing tissue-engineered cartilage for articular cartilage defects published between 1991 and 2015 using the keywords “articular cartilage defect, scaffold, tissue engineered cartilage” in English and Chinese, respectively. The irrelative and repetitive literatures were excluded. RESULTS AND CONCLUSION: Finally 48 eligible literatures were enrolled based on the inclusion and exclusion criteria. Cartilage tissue engineering possesses the advantages of controllability, little damage to tissue itself, and biological repair of injured cartilage. Tissue-engineered scaffold material is a critical factor in tissue engineering construction; therefore, it should hold biodegradability and histocompatibility. The commonly used scaffold materials include natural macromolecule materials (collagen, silk fibroin and chitosan), and synthetic polymer materials (polylactic acid and tricalcium phosphate). It is necessary to prepare composite scaffolds with high bioactivity integrate advantages of each material. The tissue engineering is bound to be a hotspot in the field of articular cartilage repair.      

Cell-based tissue-engineered allogeneic implant for cartilage repair

The potential for using of allogeneic cartilage chips, transplanted in a biologic polymer with articular chondrocytes, as a tool for articular cartilage repair was studied. Small lyophilized articular cartilage chips were mixed with a cell/fibrinogen solution and thrombin to obtain implantable constructs made of fibrin glue, chondrocytes, and cartilage chips. Specimens were implanted in the subcutaneous tissue on the backs of nude mice (experimental group A). Three groups of controls (groups B, C, and D) were also prepared. Group B consisted of fibrin glue and cartilage chips without chondrocytes. Group C consisted of fibrin glue and chondrocytes without cartilage chips, and group D was composed solely of fibrin glue. All samples were carefully weighed before implantation in the mice. The constructs were harvested from the animals at 6, 9, and 12 weeks, examined grossly, and weighed. The samples were then processed and stained with hematoxylin and eosin for histological examination. Gross evaluation and weight analysis of the constructs at the time of retrieval showed retention of the original mass in the samples made of fibrin glue, chondrocytes, and cartilage chips (group A) and demonstrated a cartilaginous consistency upon probing. Specimens from constructs of fibrin glue and cartilage chips without chondrocytes (control group B) retained most of their volume, but were statistically lighter than specimens from group A and were much softer and more pliable than those in group A. Samples of specimens from constructs of fibrin glue and chondrocytes (groups C) and fibrin glue alone (group D) both showed a substantial reduction of their original masses over the experimental time periods when compared to the samples in groups A and B, although specimens from group C demonstrated new cartilage matrix formation. Histological analysis of specimens in experimental group A demonstrated the presence of cartilage chips surrounded by newly formed cartilaginous matrix, while specimens of control group B showed only fibrotic tissue surrounding the devitalized cartilage pieces. Cartilaginous matrix was also observed in control group C, in which cartilage chips were absent, whereas only fibrin glue debris was observed in control group D. This study demonstrated that a composite of fibrin glue and devitalized cartilage can serve as a scaffold for chondrocyte transplantation, preserve the original phenotype of the chondrocytes, and maintain the original mass of the implant. This may represent a valid option for addressing the problem of articular cartilage repair.     

Mechanical conditioning of cell-seeded small intestine submucosa: a potential tissue-engineering strategy for tendon repair

Our long-term objective is to enhance tendon repair by delivering cells on natural biologic scaffolds to the repair site. Clinical outcomes may be improved by first preconditioning these cell-seeded constructs in bioreactors to enhance their properties at implantation and to deliver cells expressing a desired phenotype. In this work, we have investigated the effect of in vitro mechanical conditioning on small-intestine submucosa (SIS) scaffolds seeded with primary tendon cells (tenocytes). SIS scaffolds (with and without cells) were conditioned under various loading regimes over a 2-week period. In vitro cyclic loading significantly increased the biomechanical properties (e.g., stiffness) of cell-seeded SIS constructs (129.1 +/- 10.2%) from time 0. The stiffness change of cyclically loaded constructs without cells was 33.9 +/- 13.8% and of statically loaded constructs with cells was 34.0 +/- 15.2% and without cells was 33.4 +/- 10.7%. In the cell-seeded groups, our data demonstrate a direct role (e.g., cell tensioning) for cells in construct stiffening. In addition, the initial stiffness of the cell-seeded, cyclically loaded constructs was found to be a strong predictor of the change in construct stiffness. Despite the mechanical integrity of these constructs being significantly less than native tendon, our data show that structural properties can be improved with in vitro mechanical conditioning. These data provide the basis for future studies investigating in vitro conditioning (mechanical, chemical) of cell-seeded ECM scaffolds and the use of such constructs for enhancing tendon repair in vivo.     

Development of gene-based therapies for cartilage repair

Articular cartilage is particularly vulnerable to injury and degenerative conditions, and has a limited capacity for self-repair. Although current clinical procedures cannot restore a normal articular surface, there are a growing number of proteins that may be used to augment a repair process, or protect cartilage from degeneration. Because proteins are often difficult to administer effectively, gene therapy approaches are being developed to provide their sustained synthesis at sites of injury or disease. To promote cartilage repair, cDNAs can be targeted to synovium, or cartilage. Gene transfer to the synovium is generally considered more suitable for chondroprotective therapies that rely on expression of large amounts of anti-inflammatory mediators. The delivery of genes to cartilage defects to promote enhanced repair can be performed by either direct administration of gene delivery vectors, or by implantation of genetically modified chondrogenic cells. Variations of these methods have been used to demonstrate that exogenous cDNAs encoding growth factors can be delivered locally to sites of cartilage damage where they are expressed at physiologically relevant levels. Data is beginning to emerge that suggests that delivery and expression of these genescan influence a repair response toward the synthesis of normal articular cartilage in vivo. This article reviews the current status of gene delivery for cartilage healing and presents some of the remaining challenges.     

Engineered mesenchymal stem cells for cartilage repair

Healthy cartilage is a highly robust tissue, and is resilient against the stringent mechanical and biological constraints imposed upon it. Cartilage defects are common features of joint diseases, but current treatments can rarely restore the full function of native cartilage. Recent studies have provided new perspectives for cartilage engineering using mesenchymal stem cells (MSCs). However, the sequential events occurring during chondrogenesis must be fully understood before we are able to reproduce faithfully the complex molecular events that lead to MSC differentiation and long-term maintenance of cartilage characteristics. Here, we focus on the potential of MSCs to repair cartilage with an emphasis on the factors that are known to be required in inducing chondrogenesis.     

软骨修复材料在运动性膝关节软骨损伤修复中的作用

背景：评价软骨修复材料在膝关节软骨损伤修复中的效果,为医务、科研工作者的研究提供一定的借鉴。 方法：采用电子检索的方式,在万方数据库(http://www.wanfangdata.com.cn/)中检索2000-01/2011-03关于修复材料在膝关节软骨损伤研究的文章,关键词为“生物材料;关节软骨;缺损;修复”。排除重复研究、普通综述或Meta分析类文章,筛选纳入26篇文献进行评价。 结果：膝关节软骨损伤在运动性损伤中较为常见,现在主要的治疗方法是自体骨软骨移植修复膝关节软骨缺损。新型的软骨替代材料研究仍处于动物试验阶段,且在动物体内长期疗效及远期的生物力学变化还未有进一步的证实,进入临床试验更需要一个过程。 结论：关节软骨损伤修复的基础研究与临床治疗,虽仍存在许多重点和难点问题亟待探索,但关节软骨损伤修复正从生物材料移植向人工再造活性软骨的崭新阶段迈进。随着各种新型材料的研制和开发,关节软骨损伤修复研究将日益完善,并为临床奠定坚实的基础,应用前景十分广阔。     

Chondrocyte-seeded hydroxyapatite for repair of large articular cartilage defects. A pilot study in the goat

The aim of this study was to evaluate the potential for restoration of a large cartilage defect in the goat knee with hydroxyapatite (HA) loaded with chondrocytes. Isolated chondrocytes were suspended in fibrin glue, seeded on top of the HA, and then the composite graft was implanted in the defect. After transplantation, cell behaviour, newly synthesised matrix and the HA–glue interface were assessed histologically after 2, 4, 12, 26 and 52 weeks. Special attention was paid to the incorporation process of HA in the subchondral bone and interactions between this biomaterial and the fibrin-glue–chondrocyte suspension.

Chondrocytes in the glue proved to survive the transplantation procedure and produced new metachromatically stained matrix two weeks after implantation. The glue–cell suspension had penetrated the superficial porous structure of the HA. Four weeks after surgery, islands of hyaline-like cartilage were observed at the HA–glue interface. A layer of fibrous tissue was formed surrounding the HA graft, resulting in a relatively instable fixation of the HA in the defect. This instability of the graft in the defect, possibly together with early weight bearing, resulted in a gradual loss of the newly formed hyaline cartilage-like repair tissue. Progressive resorption of the HA occurred without any sign of active bone remodelling from the host site. One year after surgery part of the defect which extended down to the cancellous bone had been predominantly restored with newly formed lamellar bone. Only small HA remnants were still present at the bottom of the original defect. Resurfacing of the joint had occurred with fibrocartilaginous repair tissue.

The absence of adequate fixation capacity of the HA near the joint space resulted in a relative instability of the graft with progressive resorption. Therefore, HA is not a suitable biomaterial to facilitate the repair of large articular cartilage defects.     

新型可塑形同种骨修复材料修复运动性关节软骨损伤

BACKGROUND: Currently, the components and preparation methods of plastic bone repair materials remain controversial, and rare studies focus on their repair effects on sport-related articular cartilage injury. OBJECTIVE: To analyze the effect of a novel plastic homogeneous bone repair material to repair sport-related articular cartilage injury. METHODS: Thirty-six New Zealand rabbits were selected to prepare articular cartilage injury models, and were randomized into two groups. Model rabbits were repaired with the novel plastic homogeneous bone material (a composite of demineralized bone matrix and collagen) as experimental group, while the others repaired with sodium hyaluronate gel as control group. At 3 weeks after repair, treadmill system was utilized to stimulate sports training after athlete injury, for five consecutive sessions of 30 minutes each. At 16, 18 and 20 weeks after repair, the defect region was observed histologically. RESULTS AND CONCLUSION: At 16 weeks after repair, the defect region healed well and integrated with the surrounding tissues, and no significant inflammatory reaction appeared around the material in the experimental group; in the control group, a fissure appeared at the defect region but with no inflammatory reaction, and the material integrated with the interstitial site. At 18 weeks after repair, closely arranged acellular bone matrix could be found in the defect region, fibrous connective tissues were fewer, and there was a new bone formation around the edge of host bone in the experimental group; the defect region healed, but new bones were fewer in the control group. At 20 weeks after repair, the defect region in the experimental group was filled with fibrous connective tissues, in which there were numerous new blood vessels, and a few of new tissues appeared around the edge of host bone; the defect region in the control group was improved and no neovascularization occurred. These findings suggest that the novel plastic homogeneous bone material can promote the repair of sport-related articular cartilage injury with less rejection reactions.     

Porous, resorbable, fiber-reinforced scaffolds tailored for articular cartilage repair.

Porous 75:25 poly(D,L-lactide-co-glycolide) scaffolds reinforced with polyglycolide fibers were prepared with mechanical properties tailored for use in articular cartilage repair. Compression testing was performed to investigate the influence of physiological testing conditions, manufacturing method, anisotropic properties due to predominant fiber orientation, amounts of fiber reinforcement (0 to 20 wt, %), and viscoelasticity via a range of strain rates. Using the same testing modality, the mechanical properties of the scaffolds were compared with pig and goat articular cartilage. Results showed that mechanical properties of the scaffolds under physiological conditions (aqueous, 37 degrees C) were much lower than when tested under ambient conditions. The manufacturing method and anisotropy of the scaffolds significantly influenced the mechanical properties. The compressive modulus and yield strength proportionally increased with increasing fiber reinforcement up to 20%. From 0.01 to 10 mm/mm/min strain rate, the compressive modulus increased in a logarithmic fashion, and the yield strength increased in a semi-log fashion. The compressive modulus of the non-reinforced scaffolds was most similar to the pig and goat articular cartilage when compared using similar testing conditions and modality, but the improvement in yield strength using the stiffer scaffolds with fiber reinforcement could provide needed structural support for in vivo loads.     

Processing cell-seeded polyester scaffolds for histology

Biodegradable 3-dimensional scaffolds of various morphologies are currently being developed for tissue engineering. Poly(lactide-co-glycolide)s (PLGAs) of various lactide to glycolide ratios are frequently used for such applications. Tissue engineering involves an in vitro stage during which cells are seeded onto scaffolds and allowed to settle and/or grow for various time periods. To assess cell distribution and/or tissue formation throughout the scaffolds during this in vitro stage, techniques such as confocal microscopy and magnetic resonance imaging have been applied. However, such cultured scaffolds have been refractory to histological evaluation because of numerous technical difficulties. We describe a method to prepare histological sections of cell cultured PLGA scaffolds for tissue engineering. The technique involves in situ labeling of cultured scaffolds, infiltration of the scaffolds with a 10% poly(vinyl alcohol) solution under a low vacuum, and cryosectioning of samples onto acid-treated glass coverslips. Sections obtained with this technique show cell distribution and cell-tissue morphology on the pore wall structures of entire centimeter-thick scaffolds. This rapid and easy technique allows for fast evaluation of tissues grown on biodegradable scaffolds.     

Hydrogels for the repair of articular cartilage defects

The repair of articular cartilage defects remains a significant challenge in orthopedic medicine. Hydrogels, three-dimensional polymer networks swollen in water, offer a unique opportunity to generate a functional cartilage substitute. Hydrogels can exhibit similar mechanical, swelling, and lubricating behavior to articular cartilage, and promote the chondrogenic phenotype by encapsulated cells. Hydrogels have been prepared from naturally derived and synthetic polymers, as cell-free implants and as tissue engineering scaffolds, and with controlled degradation profiles and release of stimulatory growth factors. Using hydrogels, cartilage tissue has been engineered in vitro that has similar mechanical properties to native cartilage. This review summarizes the advancements that have been made in determining the potential of hydrogels to replace damaged cartilage or support new tissue formation as a function of specific design parameters, such as the type of polymer, degradation profile, mechanical properties and loading regimen, source of cells, cell-seeding density, controlled release of growth factors, and strategies to cause integration with surrounding tissue. Some key challenges for clinical translation remain, including limited information on the mechanical properties of hydrogel implants or engineered tissue that are necessary to restore joint function, and the lack of emphasis on the ability of an implant to integrate in a stable way with the surrounding tissue. Future studies should address the factors that affect these issues, while using clinically relevant cell sources and rigorous models of repair.     

关节软骨的修复与修复失败

文题释义： 自体软骨细胞移植：对于3.5-10 cm2的软骨缺损或多个缺损来说,自体软骨细胞移植是一种有效的软骨修复措施,取少量患者自体软骨于体外培养软骨细胞,并增殖到一定数量后植入软骨缺损处,从而达到修复缺损的目的。 基质诱导的自体软骨细胞移植：把经培养增殖后的软骨细胞接种到Ⅰ/Ⅲ型双层胶原膜上,继续培养数日,细胞与支架结合紧密之后,使用生物蛋白胶粘贴到关节软骨缺损病灶底部。术后,软骨细胞从胶原膜上游离并穿过生物胶,迁徙到软骨缺损的基底部。胶原膜和生物胶逐步降解并被吸收。接种的软骨细胞在局部生长、繁殖,并分泌基质,形成新的软骨组织修复缺损。背景：由于关节软骨具有复杂的生物学特性和高度的耐用性,自然退变或创伤引起的缺损都可能导致其结构和功能上不可逆的损害,因此关节软骨损伤后的修复治疗是临床上急需解决的问题。 目的：报告关节软骨修复技术失败最常见的危险因素及其发生率,分析影响选择特定手术治疗方法来处理软骨修复失败最重要的因素。 方法：以“articular cartilage, repair, clinic/clinical failure, surgery”为检索词,检索 PubMed和MEDLINE数据库,时限为2007至2019年,语言限制为英文。初检得到文献约343篇,根据纳入排出标准筛选,共纳入38篇文章进行分析。 结果与结论：①微骨折术和软骨镶嵌成形术在关节软骨修复后的前期和中期显示出不可忽视的失败率,而使用自体软骨细胞移植和异体骨软骨移植修复关节软骨的效果更好。②对于软骨修复失败的治疗：在以往软骨修复失败的患者中应用异体骨软骨移植可能是一个安全的选择,但对于失败的异体骨软骨移植的修复则有更高的失败率;而既往自体软骨细胞移植或基质诱导的自体软骨细胞移植失败的患者,经进一步的自体软骨细胞移植或基质诱导的自体软骨细胞移植治疗后,其治疗效果是可以接受的。此外,有软骨下骨髓刺激病史的患者,自体软骨细胞移植的失败率更高。③软骨修复失败的处理取决于手术治疗失败的类型以及软骨缺损的面积、部位的不同,异体骨软骨移植是治疗软骨下骨髓刺激患者软骨修复失败的最可靠的方法,而自体软骨细胞移植或基质诱导的自体软骨细胞移植在既往软骨修复失败的患者中显示出可以接受的治疗效果,在处理软骨修复失败的患者时,应该特别注意软骨下骨质的情况。ORCID: 0000-0002-3907-9145(张宇) 中国组织工程研究杂志出版内容重点：人工关节;骨植入物;脊柱;骨折;内固定;数字化骨科;组织工程     

软骨修复材料种类的比较

背景：软骨修复材料要求具有特定的生化、物理性质,如极强的生物相容性、合适的生物降解性、可控的孔径大小、足够的孔隙率等。 目的：对比分析各种软骨修复材料的特点。 方法：应用计算机检索万方数据库、CNKI数据库2001/2010与软骨修复材料相关的文章,检索关键词为“软骨,修复,支架材料,组织工程,生物材料”。 结果与结论：软骨组织工程支架材料分为天然支架材料、复合支架材料、可注射支架材料、仿生支架材料等。但是各种材料具有各自的优势与不足,目前多采用复合支架材料或利用仿生原理制备仿生支架材料或是可注射型支架材料,以充分发挥材料的优势,克服不足,使其生物力学特性更加接近天然骨组织。尽管骨组织工程研究已经取得了相当快速的进展和成果,但仍有许多问题需要解决：支架的免疫原性即降解转归及对机体的影响;支架是否可与软骨下骨有效结合;支架材料的降解速度是否可与组织形成相匹配;支架的生物力学性能是否与软骨组织相同或接近等。     

A fiber-optic-based imaging system for nondestructive assessment of cell-seeded tissue-engineered scaffolds

A major limitation in tissue engineering is the lack of nondestructive methods that assess the development of tissue scaffolds undergoing preconditioning in bioreactors. Due to significant optical scattering in most scaffolding materials, current microscope-based imaging methods cannot "see" through thick and optically opaque tissue constructs. To address this deficiency, we developed a fiber-optic-based imaging method that is capable of nondestructive imaging of fluorescently labeled cells through a thick and optically opaque scaffold, contained in a bioreactor. This imaging modality is based on the local excitation of fluorescent cells, the acquisition of fluorescence through the scaffold, and fluorescence mapping based on the position of the excitation light. To evaluate the capability and accuracy of the imaging system, human endothelial cells (ECs), stably expressing green fluorescent protein (GFP), were imaged through a fibrous scaffold. Without sacrificing the scaffolds, we nondestructively visualized the distribution of GFP-labeled cells through a ～500?μm thick scaffold with cell-level resolution and distinct localization. These results were similar to control images obtained using an optical microscope with direct line-of-sight access. Through a detailed quantitative analysis, we demonstrated that this method achieved a resolution on the order of 20-30?μm, with 10% or less deviation from standard optical microscopy. Furthermore, we demonstrated that the penetration depth of the imaging method exceeded that of confocal laser scanning microscopy by more than a factor of 2. Our imaging method also possesses a working distance (up to 8?cm) much longer than that of a standard confocal microscopy system, which can significantly facilitate bioreactor integration. This method will enable the nondestructive monitoring of ECs seeded on the lumen of a tissue-engineered vascular graft during preconditioning in vitro, as well as for other tissue-engineered constructs in the future.     

Grafting of mesenchymal stem cell-seeded small intestinal submucosa to repair the deep partial-thickness burns

Purpose: Regenerative medicine provides many treatments for burn wounds, of which cell-seeded substitutes are encouraging for large and deep burns. To assess the feasibility of mesenchymal stem cell (MSC)-seeded small intestinal submucosa (SIS) to repair the deep partial-thickness burns, a rat study was performed. Materials & Methods: The burn model was created by contacting the dorsal surface directly with boiled water for 10 seconds. MSCs at passage 3 were seeded on the SIS before implantation. Three days after burn injury, the grafts were implanted onto the burn area. At 3, 7, 14 and 21 days post implantation, gross observation and histological assessments were performed. Results: SIS alone and MSC-seeded SIS were able to accelerate the burn wound closure by enhancing granulation tissue formation, increasing wound maturity, improving revascularization, and inducing the proliferation of neo-epidermal cells. Additionally, MSC-seeded SIS was much more effective than SIS alone for the repair of deep partial-thickness burns. Conclusion: Both SIS and MSC-seeded SIS were able to repair the large and deep burn wounds and the loaded MSCs possessed positive effects to accelerate the wound closure in a rat model.     

Novel strategies for enhancing tissue integration in cartilage repair

Introduction Articular cartilage is unable to initiate a spontaneous repair response when injured due to its avascular and aneural properties. Within adult cartilage, chondrocytes are entrapped within an extensive extracellular matrix and are unable to migrate to sights of injury to regulate tissue repair. Injury to this tissue therefore inevitably leads to degeneration of the cartilage and the development of degenerative diseases such as osteoarthritis. The surgical technique of autologous chondrocyte transplantation (ACT) was developed for the treatment of full‐thickness cartilage defects ( Brittberg et al. 1994 ). Implantation of chondrocytes into the defect site repairs the injury site with a mixture of fibrocartilaginous and hyaline‐like tissue that poorly integrates with the existing cartilage and frequently degenerates with time. In this current study, we have developed an in vitro model to investigate methods for enhancing this integration and the development of a more biomechanically stable repair tissue. Materials and methods Bovine articular cartilage explants from the metacarpalphalangeal joint were experimentally injured using a stainless steel trephine and cultured for a period of 28 days. Autologous chondrocytes in an agarose suspension were injected into the interface region at the injury site. Media was collected and analysed for proteoglycan and collagen content using the DMMB and hydroxyproline assays, respectively. Matrix metalloproteinase (MMP) expression was also analysed using zymography and an adapted collagen fibril assay. Results Morphological analyses indicate attempts at repair and integration within both control and experimental treatment groups, although the presence of autologous chondrocytes appeared to amplify this repair response. Although not statistically significant, considerable differences in proteoglycan release between injured explants and the intact control group were seen. Collagen release into the media was only seen at day 28 within experimental cultures. An up‐regulation of MMP‐2 and MMP‐9 was seen within the experimental cultures compared to the controls. Preliminary data also suggest up‐regulation of collagenases in the experimental group when compared to controls. Discussion As seen with clinical ACT treatment, the presence of autologous chondrocytes appears to enhance repair and integration attempts; however, morphologically, this repair tissue appears to be fibrocartilaginous. Further analysis will establish whether the repair tissue is true hyaline cartilage and monitor the synthesis and turnover of macromolecules within the established culture system.