Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue^1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz^1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue^4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.     

Low intensity ultrasound as a supporter of cartilage regeneration and its engineering

Ultrasound (US) is being used widely in clinic for diagnostic and therapeutic purposes, but clinical utilization of low intensity ultrasound (LIUS) has been very limited. However, therapeutic potential of LIUS has been reported in animal models of musculoskeletal system disorders, and its application is being expanded in various fields. This review will focus on the application of LIUS on the cartilage tissue engineering and repair of cartilage disorder such as osteoarthritis (OA). We will introduce our experimental results showing the LIUS effects on the chondrocyte viability, proliferation and matrix protein synthesisin vitro, and its application in the cartilage tissue engineering using mesenchymal stem cells (MSCs)in vivo. Also the current status on the issues will be discussed by comparing our results with those of other laboratories. In conclusion, we suggest that LIUS is an efficient and clinically applicable method for cartilage tissue engineering and cartilage repair.     

Mesenchymal stem cells as a potent cell source for articular cartilage regeneration

Since articular cartilage possesses only a weak capac-ity for repair, its regeneration potential is considered one of the most important challenges for orthopedic surgeons. The treatment options, such as marrow stimulation techniques, fail to induce a repair tissue with the same functional and mechanical properties of native hyaline cartilage. Osteochondral transplantation is considered an effective treatment option but is as-sociated with some disadvantages, including donor-site morbidity, tissue supply limitation, unsuitable mechani-cal properties and thickness of the obtained tissue. Although autologous chondrocyte implantation results in reasonable repair, it requires a two-step surgical pro-cedure. Moreover, chondrocytes expanded in culture gradually undergo dedifferentiation, so lose morpho-logical features and specialized functions. In the search for alternative cells, scientists have found mesenchymal stem cells(MSCs) to be an appropriate cellular mate-rial for articular cartilage repair. These cells were origi-nally isolated from bone marrow samples and further investigations have revealed the presence of the cells in many other tissues. Furthermore, chondrogenic dif-ferentiation is an inherent property of MSCs noticedat the time of the cell discovery. MSCs are known to exhibit homing potential to the damaged site at which they differentiate into the tissue cells or secrete a wide spectrum of bioactive factors with regenerative proper-ties. Moreover, these cells possess a considerable im-munomodulatory potential that make them the general donor for therapeutic applications. All of these topics will be discussed in this review.     

Development of large engineered cartilage constructs from a small population of cells

Confronted with articular cartilage's limited capacity for self‐repair, joint resurfacing techniques offer an attractive treatment for damaged or diseased tissue. Although tissue engineered cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for the repair of large defects. As routine cell expansion methods tend to elicit negative effects on chondrocyte function, we have developed an approach to generate phenotypically stable, large‐sized engineered constructs (≥3 cm²) directly from a small amount of donor tissue or cells (as little as 20,000 cells to generate a 3 cm² tissue construct). Using rabbit donor tissue, the bioreactor‐cultivated constructs were hyaline‐like in appearance and possessed a biochemical composition similar to native articular cartilage. Longer bioreactor cultivation times resulted in increased matrix deposition and improved mechanical properties determined over a 4 week period. Additionally, as the anatomy of the joint will need to be taken in account to effectively resurface large affected areas, we have also explored the possibility of generating constructs matched to the shape and surface geometry of a defect site through the use of rapid‐prototyped defect tissue culture molds. Similar hyaline‐like tissue constructs were developed that also possessed a high degree of shape correlation to the original defect mold. Future studies will be aimed at determining the effectiveness of this approach to the repair of cartilage defects in an animal model and the creation of large‐sized osteochondral constructs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Cartilage tissue engineering: Molecular control of chondrocyte differentiation for proper cartilage matrix reconstruction

Background

Articular cartilage defects are a veritable therapeutic problem because therapeutic options are very scarce. Due to the poor self-regeneration capacity of cartilage, minor cartilage defects often lead to osteoarthritis. Several surgical strategies have been developed to repair damaged cartilage. Autologous chondrocyte implantation (ACI) gives encouraging results, but this cell-based therapy involves a step of chondrocyte expansion in a monolayer, which results in the loss in the differentiated phenotype. Thus, despite improvement in the quality of life for patients, reconstructed cartilage is in fact fibrocartilage. Successful ACI, according to the particular physiology of chondrocytes in vitro, requires active and phenotypically stabilized chondrocytes.

Scope of review

This review describes the unique physiology of cartilage, with the factors involved in its formation, stabilization and degradation. Then, we focus on some of the most recent advances in cell therapy and tissue engineering that open up interesting perspectives for maintaining or obtaining the chondrogenic character of cells in order to treat cartilage lesions.

Major conclusions

Current research involves the use of chondrocytes or progenitor stem cells, associated with “smart” biomaterials and growth factors. Other influential factors, such as cell sources, oxygen pressure and mechanical strain are considered, as are recent developments in gene therapy to control the chondrocyte differentiation/dedifferentiation process.

General significance

This review provides new information on the mechanisms regulating the state of differentiation of chondrocytes and the chondrogenesis of mesenchymal stem cells that will lead to the development of new restorative cell therapy approaches in humans. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Hypoxia. HIF-mediated articular chondrocyte function: prospects for cartilage repair

In a chronically hypoxic tissue such as cartilage, adaptations to hypoxia do not merely include cell survival responses, but also promotion of its specific function. This review will focus on describing such hypoxia-mediated chondrocyte function, in particular in the permanent articular cartilage. The molecular details of how chondrocytes sense and respond to hypoxia and how this promotes matrix synthesis have recently been examined, and specific manipulation of hypoxia-induced pathways is now considered to have potential therapeutic application to maintenance and repair of articular cartilage.     

Emergence of chondrogenic progenitor stem cells in transplantation biology-prospects and drawbacks

Avascular tissues such as a cartilage contains a unique type of cell called as the chondrocyte. We, however, have not understood the origin of the chondrocyte population and how this population is maintained in the normal tissue. In spite of being considered to be a simple tissue, scientist had always faced difficulties to engineer this tissue. This is because different structural regions of the articular cartilage were never understood. In addition to this, the limited self-repair potential of cartilage tissue and lack of effective therapeutic options for the treatment of damaged cartilage has remained an unsolved problem. Mesenchymal stem cell based therapy may provide a solution for cartilage regeneration. This is due to their ability to differentiate into chondrogenic lineage when appropriate conditions are provided. An ideal cell source, a three-dimensional cell culture, a suitable scaffold material that accomplishes all the necessary properties and bioactive factors in specific amounts are required to induce chondrocyte differentiation and proliferation. Cartilage tissue engineering is a promising and rapidly expanding area of research that assures cartilage regeneration. However, many unsolved questions concerning the mechanism of engraftment of chondrocytes following transplantation in vivo, biological safety after transplantation and the retention of these cells for lifetime remain to be addressed that is possible only through years of extensive research. Further studies are therefore required to estimate the long-term sustainability of these cells in the native tissue, to identify well suited delivery materials and to have a thorough understanding of the mechanism of interaction between the chondrocytes and extracellular matrix and time is not far when this cell based therapy will provide a comprehensive cure to cartilage disease.     

3D生物打印在软骨修复中的应用*

关节软骨损伤后的自我修复是医学界一直在研究和探讨的难题。3D生物打印技术可以精准的分配载细胞生物材料,构建复杂的三维活体组织,在优化软骨缺损修复组织的内部结构、机械性能以及生物相容性上有很大优势,因此近年来成为软骨修复组织工程领域的研究热点。重点介绍了软骨生物3D生物打印的最新进展,包括软骨生物打印“墨水”材料的选择、种子细胞的来源以及3D生物打印技术的发展。此外,还阐述了3D生物打印技术在组织工程学应用上的部分局限性,并对其在软骨修复领域的发展与应用进行了预测。     

Mini-review: Mechanical factors affecting cartilage regeneration in vitro

In the last 5 to 10 years, tissue engineering has revolutionized the way in which medical researchers and clinicians are thinking of and, in some cases, actually treating diseases involving tissue damage and destruction. One such disease, osteoarthritis, results from progressive degeneration of articular cartilage, which has a limited ability to repair itself. With tissue engineering, scientists are now able to regenerate cartilage in vitro from isolated mature chondrocytes. While the regeneration process is still not fully understood, enough has been learned that physicians are already implanting cultured chondrocytes into humans and other animals in the hopes of effecting joint repair. One aspect which has not been fully explored is the effect of mechanical stress on developing and implanted cartilage, especially over the long term. This article will review in brief what is now known about the mechanical factors affecting cartilage regeneration in vitro and what still remains to be determined for optimum tissue engineering of cartilage constructs. (c) 1996 John Wiley & Sons, Inc.     

Is the repair of articular cartilage lesion by costal chondrocyte transplantation donor age-dependent? An experimental study in rabbits

The repair of chondral injuries is a very important problem and a subject of many experimental and clinical studies. Different techniques to induce articular cartilage repair are under investigation. In the present study, we have investigated whether the repair of articular cartilage folowing costal chondrocyte transplantation is donor age-dependent. Transplantation of costal chondrocytes from 4- and 24-week old donors, with artificially induced femoral cartilage lesion, was performed on fourteen 20-week-old New Zealand White male rabbits. In the control group, the lesion was left without chondrocyte transplantation. The evaluation of the cartilage repair was performed after 12 weeks of transplantation. We analyzed the macroscopic and histological appearance of the newly formed tissue. Immunohistochemistry was also performed using monoclonal antibodies against rabbit collagen type II. The newly formed tissue had a hyaline-like appearance in most of the lesions after chondrocyte transplantation. Positive immunohistochemical reaction for collagen II was also observed in both groups with transplanted chondrocytes. Cartilage from adult donors required longer isolation time and induced slightly poorer repair. However, hyaline-like cartilage was observed in most specimens from this group, in contrast to the control group, where fibrous connective tissue filled the lesions. Rabbit costal chondrocytes seem to be a potentially useful material for inducing articular cartilage repair and, even more important, they can also be derived from adult, sexually mature animals.     

Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage

Background

Articular cartilage displays a poor repair capacity. The aim of cell-based therapies for cartilage defects is to repair damaged joint surfaces with a functional replacement tissue. Currently, chondrocytes removed from a healthy region of the cartilage are used but they are unable to retain their phenotype in expanded culture. The resulting repair tissue is fibrocartilaginous rather than hyaline, potentially compromising long-term repair. Mesenchymal stem cells, particularly bone marrow stromal cells (BMSC), are of interest for cartilage repair due to their inherent replicative potential. However, chondrocyte differentiated BMSCs display an endochondral phenotype, that is, can terminally differentiate and form a calcified matrix, leading to failure in long-term defect repair. Here, we investigate the isolation and characterisation of a human cartilage progenitor population that is resident within permanent adult articular cartilage.

Methods and Findings

Human articular cartilage samples were digested and clonal populations isolated using a differential adhesion assay to fibronectin. Clonal cell lines were expanded in growth media to high population doublings and karyotype analysis performed. We present data to show that this cell population demonstrates a restricted differential potential during chondrogenic induction in a 3D pellet culture system. Furthermore, evidence of high telomerase activity and maintenance of telomere length, characteristic of a mesenchymal stem cell population, were observed in this clonal cell population. Lastly, as proof of principle, we carried out a pilot repair study in a goat in vivo model demonstrating the ability of goat cartilage progenitors to form a cartilage-like repair tissue in a chondral defect.

Conclusions

In conclusion, we propose that we have identified and characterised a novel cartilage progenitor population resident in human articular cartilage which will greatly benefit future cell-based cartilage repair therapies due to its ability to maintain chondrogenicity upon extensive expansion unlike full-depth chondrocytes that lose this ability at only seven population doublings.     

Cartilage tissue engineering and bioreactor systems for the cultivation and stimulation of chondrocytes

Damage to and degeneration of articular cartilage is a major health issue in industrialized nations. Articular cartilage has a particularly limited capacity for auto regeneration. At present, there is no established therapy for a sufficiently reliable and durable replacement of damaged articular cartilage. In this, as well as in other areas of regenerative medicine, tissue engineering methods are considered to be a promising therapeutic component. Nevertheless, there remain obstacles to the establishment of tissue-engineered cartilage as a part of the routine therapy for cartilage defects. One necessary aspect of potential tissue engineering-based therapies for cartilage damage that requires both elucidation and progress toward practical solutions is the reliable, cost effective cultivation of suitable tissue. Bioreactors and associated methods and equipment are the tools with which it is hoped that such a supply of tissue-engineered cartilage can be provided. The fact that in vivo adaptive physical stimulation influences chondrocyte function by affecting mechanotransduction leads to the development of specifically designed bioreactor devices that transmit forces like shear, hydrostatic pressure, compression, and combinations thereof to articular and artificial cartilage in vitro. This review summarizes the basic knowledge of chondrocyte biology and cartilage dynamics together with the exploration of the various biophysical principles of cause and effect that have been integrated into bioreactor systems for the cultivation and stimulation of chondrocytes. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Biomechanical assessment of tissue retrieved after in vivo cartilage defect repair: tensile modulus of repair tissue and integration with host cartilage

Failure to restore the mechanical properties of tissue at the repair site and its interface with host cartilage is a common problem in tissue engineering procedures to repair cartilage defects. Quantitative in vitro studies have helped elucidate mechanisms underlying processes leading to functional biomechanical changes. However, biomechanical assessment of tissue retrieved from in vivo studies of cartilage defect repair has been limited to compressive tests. Analysis of integration following in vivo repair has relied on qualitative histological methods. The objectives of this study were to develop a quantitative biomechanical method to assess (1) the tensile modulus of repair tissue and (2) its integration in vivo, as well as determine whether supplementation of transplanted chondrocytes with IGF-I affected these mechanical properties. Osteochondral blocks were obtained from a previous 8 month study on the effects of IGF-I on chondrocyte transplantation in the equine model. Tapered test specimens were prepared from osteochondral blocks containing the repair/native tissue interface and adjacently located blocks of intact native tissue. Specimens were then tested in uniaxial tension. The tensile modulus of repair tissue averaged 0.65 MPa, compared to the average of 5.2 MPa measured in intact control samples. Integration strength averaged 1.2 MPa, nearly half the failure strength of intact cartilage samples, 2.7 MPa. IGF-I treatment had no detectable effects on these mechanical properties. This represents the first quantitative biomechanical investigation of the tensile properties of repair tissue and its integration strength in an in vivo joint defect environment.     

A high throughput mechanical screening device for cartilage tissue engineering

Articular cartilage enables efficient and near-frictionless load transmission, but suffers from poor inherent healing capacity. As such, cartilage tissue engineering strategies have focused on mimicking both compositional and mechanical properties of native tissue in order to provide effective repair materials for the treatment of damaged or degenerated joint surfaces. However, given the large number design parameters available (e.g. cell sources, scaffold designs, and growth factors), it is difficult to conduct combinatorial experiments of engineered cartilage. This is particularly exacerbated when mechanical properties are a primary outcome, given the long time required for testing of individual samples. High throughput screening is utilized widely in the pharmaceutical industry to rapidly and cost-effectively assess the effects of thousands of compounds for therapeutic discovery. Here we adapted this approach to develop a high throughput mechanical screening (HTMS) system capable of measuring the mechanical properties of up to 48 materials simultaneously. The HTMS device was validated by testing various biomaterials and engineered cartilage constructs and by comparing the HTMS results to those derived from conventional single sample compression tests. Further evaluation showed that the HTMS system was capable of distinguishing and identifying ‘hits’, or factors that influence the degree of tissue maturation. Future iterations of this device will focus on reducing data variability, increasing force sensitivity and range, as well as scaling-up to even larger (96-well) formats. This HTMS device provides a novel tool for cartilage tissue engineering, freeing experimental design from the limitations of mechanical testing throughput.     

Osteoarthritis

Osteoarthritis (OA) is characterized by degeneration of articular cartilage, limited intraarticular inflammation with synovitis, and changes in peri-articular and subchondral bone. Multiple factors are involved in the pathogenesis of OA, including mechanical influences, the effects of aging on cartilage matrix composition and structure, and genetic factors. Since the initial stages of OA involve increased cell proliferation and synthesis of matrix proteins, proteinases, growth factors, cytokines, and other inflammatory mediators by chondrocytes, research has focused on the chondrocyte as the cellular mediator of OA pathogenesis. The other cells and tissues of the joint, including the synovium and subchondral bone, also contribute to pathogenesis. The adult articular chondrocyte, which normally maintains the cartilage with a low turnover of matrix constituents, has limited capacity to regenerate the original cartilage matrix architecture. It may attempt to recapitulate phenotypes of early stages of cartilage development, but the precise zonal variations of the original cartilage cannot be replicated. Current pharmacological interventions that address chronic pain are insufficient, and no proven structure-modifying therapy is available. Cartilage tissue engineering with or without gene therapy is the subject of intense investigation. There are multiple animal models of OA, but there is no single model that faithfully replicates the human disease. This review will focus on questions currently under study that may lead to better understanding of mechanisms of OA pathogenesis and elucidation of effective strategies for therapy, with emphasis on mechanisms that affect the function of chondrocytes and interactions with surrounding tissues.     

Current clinical therapies for cartilage repair, their limitation and the role of stem cells

The management of osteochondral defects of articular cartilage, whether from trauma or degenerative disease, continues to be a significant challenge for Orthopaedic surgeons. Current treatment options such as abrasion arthroplasty procedures, osteochondral transplantation and autologous chondrocyte implantation fail to produce repair tissue exhibiting the same mechanical and functional properties of native articular cartilage. This results in repair tissue that inevitably fails as it is unable to deal with the mechanical demands of articular cartilage, and does not prevent further degeneration of the native cartilage. Mesenchymal stem cells have been proposed as a potential source of cells for cell-based cartilage repair due to their ability to self-renew and undergo multi-lineage differentiation. This proposed procedure has the advantage of not requiring harvesting of cells from the joint surface, and its associated donor site morbidity, as well as having multiple possible adult donor tissues such as bone marrow, adipose tissue and synovium. Mesenchymal stem cells have multi-lineage potential, but can be stimulated to undergo chondrogenesis in the appropriate culture medium. As the majority of work with mesenchymal stem cell-derived articular cartilage repair has been carried out in vitro and in animal studies, more work still has to be done before this technique can be used for clinical purposes. This includes realizing the ideal method of harvesting mesenchymal stem cells, the culture medium to stimulate proliferation and differentiation, appropriate choice of scaffold incorporating growth factors directly or with gene therapy and integration of repair tissue with native tissue.     

Gene therapy for cartilage defects

Focal defects of articular cartilage are an unsolved problem in clinical orthopaedics. These lesions do not heal spontaneously and no treatment leads to complete and durable cartilage regeneration. Although the concept of gene therapy for cartilage damage appears elegant and straightforward, current research indicates that an adaptation of gene transfer techniques to the problem of a circumscribed cartilage defect is required in order to successfully implement this approach. In particular, the localised delivery into the defect of therapeutic gene constructs is desirable. Current strategies aim at inducing chondrogenic pathways in the repair tissue that fills such defects. These include the stimulation of chondrocyte proliferation, maturation, and matrix synthesis via direct or cell transplantation-mediated approaches. Among the most studied candidates, polypeptide growth factors have shown promise to enhance the structural quality of the repair tissue. A better understanding of the basic scientific aspects of cartilage defect repair, together with the identification of additional molecular targets and the development of improved gene-delivery techniques, may allow a clinical translation of gene therapy for cartilage defects. The first experimental steps provide reason for cautious optimism.     

三维支架材料中软骨细胞生长过程的数值模拟

组织工程是临床上用于修复以及重建受损软骨的一项有广泛应用前景的方法。但是在支架材料内部物质传递仅仅依赖于扩散,这是支架材料中细胞生长的主要限制因素。利用氧扩散-反应基本原理建立了软骨细胞生长过程的数学模型,同时考虑了由于细胞生长引起的扩散系数下降以及空间抑制因素对细胞生长的影响。模拟结果与实验数据吻合良好,表明该模型对材料内部的细胞生长的分析具有较高的可靠性,可用于组织工程生物反应器以及三维多孔支架材料的优化设计。     

三维支架材料中软骨细胞生长过程的数值模拟

组织工程是临床上用于修复以及重建受损软骨的一项有广泛应用前景的方法。但是在支架材料内部物质传递仅仅依赖于扩散,这是支架材料中细胞生长的主要限制因素。利用氧扩散-反应基本原理建立了软骨细胞生长过程的数学模型,同时考虑了由于细胞生长引起的扩散系数下降以及空间抑制因素对细胞生长的影响。模拟结果与实验数据吻合良好,表明该模型对材料内部的细胞生长的分析具有较高的可靠性,可用于组织工程生物反应器以及三维多孔支架材料的优化设计。     

Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy

Injuries to the articular cartilage and growth plate are significant clinical problems due to their limited ability to regenerate themselves. Despite progress in orthopedic surgery and some success in development of chondrocyte transplantation treatment and in early tissue-engineering work, cartilage regeneration using a biological approach still remains a great challenge. In the last 15 years, researchers have made significant advances and tremendous progress in exploring the potentials of mesenchymal stem cells (MSCs) in cartilage repair. These include (a) identifying readily available sources of and devising appropriate techniques for isolation and culture expansion of MSCs that have good chondrogenic differentiation capability, (b) discovering appropriate growth factors (such as TGF-beta, IGF-I, BMPs, and FGF-2) that promote MSC chondrogenic differentiation, (c) identifying or engineering biological or artificial matrix scaffolds as carriers for MSCs and growth factors for their transplantation and defect filling. In addition, representing another new perspective for cartilage repair is the successful demonstration of gene therapy with chondrogenic growth factors or inflammatory inhibitors (either individually or in combination), either directly to the cartilage tissue or mediated through transducing and transplanting cultured chondrocytes, MSCs or other mesenchymal cells. However, despite these rapid pre-clinical advances and some success in engineering cartilage-like tissue and in repairing articular and growth plate cartilage, challenges of their clinical translation remain. To achieve clinical effectiveness, safety, and practicality of using MSCs for cartilage repair, one critical investigation will be to examine the optimal combination of MSC sources, growth factor cocktails, and supporting carrier matrixes. As more insights are acquired into the critical factors regulating MSC migration, proliferation and chondrogenic differentiation both ex vivo and in vivo, it will be possible clinically to orchestrate desirable repair of injured articular and growth plate cartilage, either by transplanting ex vivo expanded MSCs or MSCs with genetic modifications, or by mobilising endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone.