Osteochondral tissue engineering: scaffolds, stem cells and applications

Osteochondral tissue engineering has shown an increasing development to provide suitable strategies for the regeneration of damaged cartilage and underlying subchondral bone tissue. For reasons of the limitation in the capacity of articular cartilage to self-repair, it is essential to develop approaches based on suitable scaffolds made of appropriate engineered biomaterials. The combination of biodegradable polymers and bioactive ceramics in a variety of composite structures is promising in this area, whereby the fabrication methods, associated cells and signalling factors determine the success of the strategies. The objective of this review is to present and discuss approaches being proposed in osteochondral tissue engineering, which are focused on the application of various materials forming bilayered composite scaffolds, including polymers and ceramics, discussing the variety of scaffold designs and fabrication methods being developed. Additionally, cell sources and biological protein incorporation methods are discussed, addressing their interaction with scaffolds and highlighting the potential for creating a new generation of bilayered composite scaffolds that can mimic the native interfacial tissue properties, and are able to adapt to the biological environment.     

Biomechanics and mechanobiology in functional tissue engineering

The field of tissue engineering continues to expand and mature, and several products are now in clinical use, with numerous other preclinical and clinical studies underway. However, specific challenges still remain in the repair or regeneration of tissues that serve a predominantly biomechanical function. Furthermore, it is now clear that mechanobiological interactions between cells and scaffolds can critically influence cell behavior, even in tissues and organs that do not serve an overt biomechanical role. Over the past decade, the field of “functional tissue engineering” has grown as a subfield of tissue engineering to address the challenges and questions on the role of biomechanics and mechanobiology in tissue engineering. Originally posed as a set of principles and guidelines for engineering of load-bearing tissues, functional tissue engineering has grown to encompass several related areas that have proven to have important implications for tissue repair and regeneration. These topics include measurement and modeling of the in vivo biomechanical environment; quantitative analysis of the mechanical properties of native tissues, scaffolds, and repair tissues; development of rationale criteria for the design and assessment of engineered tissues; investigation of the effects biomechanical factors on native and repair tissues, in vivo and in vitro; and development and application of computational models of tissue growth and remodeling. Here we further expand this paradigm and provide examples of the numerous advances in the field over the past decade. Consideration of these principles in the design process will hopefully improve the safety, efficacy, and overall success of engineered tissue replacements.     

Biodegradable zinc oxide composite scaffolds promote osteochondral differentiation of mesenchymal stem cells

Osteoarthritis (OA) involves the degeneration of articular cartilage and subchondral bone. The capacity of articular cartilage to repair and regenerate is limited. A biodegradable, fibrous scaffold containing zinc oxide (ZnO) was fabricated and evaluated for osteochondral tissue engineering applications. ZnO has shown promise for a variety of biomedical applications but has had limited use in tissue engineering. Composite scaffolds consisted of ZnO nanoparticles embedded in slow degrading, polycaprolactone to allow for dissolution of zinc ions over time. Zinc has well-known insulin-mimetic properties and can be beneficial for cartilage and bone regeneration. Fibrous ZnO composite scaffolds, having varying concentrations of 1–10 wt.% ZnO, were fabricated using the electrospinning technique and evaluated for human mesenchymal stem cell (MSC) differentiation along chondrocyte and osteoblast lineages. Slow release of the zinc was observed for all ZnO composite scaffolds. MSC chondrogenic differentiation was promoted on low percentage ZnO composite scaffolds as indicated by the highest collagen type II production and expression of cartilage-specific genes, while osteogenic differentiation was promoted on high percentage ZnO composite scaffolds as indicated by the highest alkaline phosphatase activity, collagen production, and expression of bone-specific genes. This study demonstrates the feasibility of ZnO-containing composites as a potential scaffold for osteochondral tissue engineering.     

Improvement of PHBV Scaffolds with Bioglass for Cartilage Tissue Engineering

Polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) have proven to be possible matrices for the three-dimensional growth of chondrocyte cultures. However, the engineered cartilage grown on these PHBV scaffolds is currently unsatisfactory for clinical applications due to PHBV’s poor hydrophilicity, resulting in inadequate thickness and poor biomechanical properties of the engineered cartilage. It has been reported that the incorporation of Bioglass (BG) into PHBV can improve the hydrophilicity of the composites. In this study, we compared the effects of PHBV scaffolds and PHBV/BG composite scaffolds on the properties of engineered cartilage in vivo. Rabbit articular chondrocytes were seeded into PHBV scaffolds and PHBV/BG scaffolds. Short-term in vitro culture followed by long-term in vivo transplantation was performed to evaluate the difference in cartilage regeneration between the cartilage layers grown on PHBV and PHBV/BG scaffolds. The results show that the incorporation of BG into PHBV efficiently improved both the hydrophilicity of the composites and the percentage of adhered cells and promoted cell migration into the inner part the constructs. With prolonged incubation time in vivo, the chondrocyte-scaffold constructs in the PHBV/BG group formed thicker cartilage-like tissue with better biomechanical properties and a higher cartilage matrix content than the constructs in the PHBV/BG group. These results indicate that PHBV/BG scaffolds can be used to prepare better engineered cartilage than pure PHBV.     

Functional tissue engineering: Ten more years of progress

“Functional tissue engineering” is a subset of the field of tissue engineering that was proposed by the United States National Committee on Biomechanics over a decade ago in order to place more emphasis on the roles of biomechanics and mechanobiology in tissue repair and regeneration. Over the past decade, there have been tremendous advances in this area, pointing out the critical role that biomechanical factors can play in the engineered repair of virtually all tissue and organ systems. In this special issue of the Journal of Biomechanics, we present a series of articles that address a broad array of the fundamental topics of functional tissue engineering, including: (1) measurement and modeling of the in vivo biomechanical environment and history in native and repair tissues; (2) further understanding of the biomechanical properties of native tissues across all geometric scales, in the context of repair or regeneration; (3) prioritization of specific biomechanical properties as design criteria; (4) development of biomaterials, scaffolds, and engineered tissues with prescribed biomechanical properties; (5) development of success criteria based on appropriate outcome measures; (6) investigation of the effects of mechanical factors on tissue repair in vivo; (7) investigation of the mechanisms by which physical factors may enhance tissue regeneration in vitro; and (8) development and validation of computational models of tissue growth and remodeling. These articles represent the tremendous expansion of this field in recent years, and emphasize the critical roles that biomechanics and mechanobiology play in controlling tissue repair and regeneration.     

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.     

Temporal assessment of ribose treatment on self-assembled articular cartilage constructs

Articular cartilage cannot repair itself in response to degradation from injury or osteoarthritis. As such, there is a substantial clinical need for replacements of damaged cartilage. Tissue engineering aims to fulfill this need by developing replacement tissues in vitro. A major goal of cartilage tissue engineering is to produce tissues with robust biochemical and biomechanical properties. One technique that has been proposed to improve these properties in engineered tissue is the use of non-enzymatic glycation to induce collagen crosslinking, an attractive solution that may avoid the risks of cytotoxicity posed by conventional crosslinking agents such as glutaraldehyde. The objectives of this study were (1) to determine whether continuous application of ribose would enhance biochemical and biomechanical properties of self-assembled articular cartilage constructs, and (2) to identify an optimal time window for continuous ribose treatment. Self-assembled constructs were grown for 4 weeks using a previously established method and were subjected to continuous 7-day treatment with 30 mM ribose during culture weeks 1, 2, 3, or 4, or for the entire 4-week culture. Control constructs were grown in parallel, and all groups were evaluated for gross morphology, histology, cellularity, collagen and sulfated glycosaminoglycan (GAG) content, and compressive and tensile mechanical properties. Compared to control constructs, it was found that treatment with ribose during week 2 and for the entire duration of culture resulted in significant 62% and 40% increases in compressive stiffness, respectively; significant 66% and 44% increases in tensile stiffness; and significant 50% and 126% increases in tensile strength. Similar statistically significant trends were observed for collagen and GAG. In contrast, constructs treated with ribose during week 1 had poorer biochemical and biomechanical properties, although they were significantly larger and more cellular than all other groups. We conclude that non-enzymatic glycation with ribose is an effective method for improving tissue engineered cartilage and that specific temporal intervention windows exist to achieve optimal functional properties.     

A Phase-Field Model for Articular Cartilage Regeneration in Degradable Scaffolds

Degradable scaffolds represent a promising solution for tissue engineering of damaged or degenerated articular cartilage which due to its avascular nature, is characterized by a low self-repair capacity. To estimate the articular cartilage regeneration process employing degradable scaffolds, we propose a mathematical model as the extension of Olson and Haider’s work (Int. J. Pure Appl. Math. 53:333–353, 2009). The simulated tissue engineering procedure consists in (i) the explant of a cylindrical sample, (ii) the removal of the inner core region, and (iii) the filling of the inner region with hydrogels, degradable scaffolds enriched with nutrients, such as oxygen and glucose. The phase-field model simulates the cartilage regeneration process at the scaffold-cartilage interface. It embeds reaction-diffusion equations, which are used to model the nutrient and regenerated extracellular matrix. The equations are solved using an unconditionally stable hybrid numerical scheme. Cartilage repair processes with full-thickness defects, which are controlled by properties of hydrogel materials and cartilage explant culture based on biological interest are observed. The implemented mathematical model shows the capability to simulate cartilage repairing processes, which can be virtually controlled evaluating hydrogel and cartilage material properties including nutrient supply and defected magnitude. In particular, the adopted methodology is able to explain the regeneration time of cartilage within hydrogel environments. With the numerical scheme, the numerical simulations are demonstrated for the potential improvement of hydrogel structures.     

A modular approach to creating large engineered cartilage surfaces

Native articular cartilage has limited capacity to repair itself from focal defects or osteoarthritis. Tissue engineering has provided a promising biological treatment strategy that is currently being evaluated in clinical trials. However, current approaches in translating these techniques to developing large engineered tissues remains a significant challenge. In this study, we present a method for developing large-scale engineered cartilage surfaces through modular fabrication. Modular Engineered Tissue Surfaces (METS) uses the well-known, but largely under-utilized self-adhesion properties of de novo tissue to create large scaffolds with nutrient channels. Compressive mechanical properties were evaluated throughout METS specimens, and the tensile mechanical strength of the bonds between attached constructs was evaluated over time. Raman spectroscopy, biochemical assays, and histology were performed to investigate matrix distribution. Results showed that by Day 14, stable connections had formed between the constructs in the METS samples. By Day 21, bonds were robust enough to form a rigid sheet and continued to increase in size and strength over time. Compressive mechanical properties and glycosaminoglycan (GAG) content of METS and individual constructs increased significantly over time. The METS technique builds on established tissue engineering accomplishments of developing constructs with GAG composition and compressive properties approaching native cartilage. This study demonstrated that modular fabrication is a viable technique for creating large-scale engineered cartilage, which can be broadly applied to many tissue engineering applications and construct geometries.     

New materials for tissue engineering: towards greater control over the biological response

One goal of tissue engineering is to replace lost or compromised tissue function, and an approach to this is to control the interplay between materials (scaffolds), cells and growth factors to create environments that promote the regeneration of functional tissues and organs. An increased understanding of the chemical signals that direct cell differentiation, migration and proliferation, advances in scaffold design and peptide engineering that allow this signaling to be recapitulated and the development of new materials, such as DNA-based and stimuli-sensitive polymers, have recently given engineers enhanced control over the chemical properties of a material and cell fate. Additionally, the immune system, which is often overlooked, has been shown to play a beneficial role in tissue repair, and future endeavors in material design will potentially expand to include immunomodulation.     

Advances in 3D printing of composite scaffolds for the repairment of bone tissue associated defects

The conventional methods of using autografts and allografts for repairing defects in bone, the osteochondral bone, and the cartilage tissue have many disadvantages, like donor site morbidity and shortage of donors. Moreover, only 30% of the implanted grafts are shown to be successful in treating the defects. Hence, exploring alternative techniques such as tissue engineering to treat bone tissue associated defects is promising as it eliminates the above-mentioned limitations. To enhance the mechanical and biological properties of the tissue engineered product, it is essential to fabricate the scaffold used in tissue engineering by the combination of various biomaterials. Three-dimensional (3D) printing, with its ability to print composite materials and with complex geometry seems to have a huge potential in scaffold fabrication technique for engineering bone associated tissues. This review summarizes the recent applications and future perspectives of 3D printing technologies in the fabrication of composite scaffolds used in bone, osteochondral, and cartilage tissue engineering. Key developments in the field of 3D printing technologies involves the incorporation of various biomaterials and cells in printing composite scaffolds mimicking physiologically relevant complex geometry and gradient porosity. Much recently, the emerging trend of printing smart scaffolds which can respond to external stimulus such as temperature, pH and magnetic field, known as 4D printing is gaining immense popularity and can be considered as the future of 3D printing applications in the field of tissue engineering.     

The osteochondral interface as a gradient tissue: From development to the fabrication of gradient scaffolds for regenerative medicine

The osteochondral (OC) interface is not only the interface between two tissues, but also the evolution of hard and stiff bone tissue to the softer and viscoelastic articular cartilage covering the joint surface. To generate a smooth transition between two tissues with such differences in many of their characteristics, several gradients are recognizable when moving from the bone side to the joint surface. It is, therefore, necessary to implement such gradients in the design of scaffolds to regenerate the OC interface, so to mimic the anatomical, biological, and physicochemical properties of bone and cartilage as closely as possible. In the past years, several scaffolds were developed for OC regeneration: biphasic, triphasic, and multilayered scaffolds were used to mimic the compartmental nature of this tissue. The structure of these scaffolds presented gradients in mechanical, physicochemical, or biological properties. The use of gradient scaffolds with already differentiated or progenitor cells has been recently proposed. Some of these approaches have also been translated in clinical trials, yet without the expected satisfactory results, thus suggesting that further efforts in the development of constructs, which can lead to a functional regeneration of the OC interface by presenting gradients more closely resembling its native environment, will be needed in the near future. The aim of this review is to analyze the gradients present in the OC interface from the early stage of embryonic life up to the adult organism, and give an overview of the studies, which involved gradient scaffolds for its regeneration. Birth Defects Research (Part C) 105:34–52, 2015. © 2015 Wiley Periodicals, Inc.     

Biochemical and biophysical aspects of collagen nanostructure in the extracellular matrix

ECM is composed of different collagenous and non-collagenous proteins. Collagen nanofibers play a dominant role in maintaining the biological and structural integrity of various tissues and organs, including bone, skin, tendon, blood vessels, and cartilage. Artificial collagen nanofibers are increasingly significant in numerous tissue engineering applications and seem to be ideal scaffolds for cell growth and proliferation. The modern tissue engineering task is to develop three-dimensional scaffolds of appropriate biological and biomechanical properties, at the same time mimicking the natural extracellular matrix and promoting tissue regeneration. Furthermore, it should be biodegradable, bioresorbable and non-inflammatory, should provide sufficient nutrient supply and have appropriate viscoelasticity and strength. Attributed to collagen features mentioned above, collagen fibers represent an obvious appropriate material for tissue engineering scaffolds. The aim of this minireview is, besides encapsulation of the basic biochemical and biophysical properties of collagen, to summarize the most promising modern methods and technologies for production of collagen nanofibers and scaffolds for artificial tissue development.     

生物可降解材料构建组织工程软骨的研究进展

关节软骨修复困难,目前临床上治疗关节软骨损伤难以达到满意的效果。组织工程学的兴起为其提供了新的选择。本文介绍了组织工程软骨的发展历史,重点叙述了各种天然支架材料、人工合成材料、复合材料及纳米材料在软骨组织工程中的应用及其优势。目前应用的天然材料存在力学强度差及免疫源性的不足;人工合成材料降解速率快,降解产物具有细胞毒性,有待进一步完善。表面修饰等技术的应用在一定程度上克服了某些材料的不足;复合材料综合了数种材料的优点,是今后材料技术发展的方向;纳米技术的出现使新合成的材料成为纳米量级,具有了普通材料无可比拟的优势,这为组织工程材料的发展提供了新的思路。本文还对组织工程支架材料存在的问题、下一步的发展方向和前瞻性研究做了介绍。     

Cartilage biomechanics: A key factor for osteoarthritis regenerative medicine

Osteoarthritis (OA) is a joint disorder that is highly extended in the global population. Several researches and therapeutic strategies have been probed on OA but without satisfactory long-term results in joint replacement. Recent evidences show how the cartilage biomechanics plays a crucial role in tissue development. This review describes how physics alters cartilage and its extracellular matrix (ECM); and its role in OA development. The ECM of the articular cartilage (AC) is widely involved in cartilage turnover processes being crucial in regeneration and joint diseases. We also review the importance of physicochemical pathways following the external forces in AC. Moreover, new techniques probed in cartilage tissue engineering for biomechanical stimulation are reviewed. The final objective of these novel approaches is to create a cellular implant that maintains all the biochemical and biomechanical properties of the original tissue for long-term replacements in patients with OA.     

Osteochondral tissue engineering

Osteochondral defects (i.e., defects which affect both the articular cartilage and underlying subchondral bone) are often associated with mechanical instability of the joint, and therefore with the risk of inducing osteoarthritic degenerative changes. Current surgical limits in the treatment of complex joint lesions could be overcome by grafting osteochondral composite tissues, engineered by combining the patient's own cells with three-dimensional (3D) porous biomaterials of pre-defined size and shape. Various strategies have been reported for the engineering of osteochondral composites, which result from the use of one or more cell types cultured into single-component or composite scaffolds in a broad spectrum of compositions and biomechanical properties. The variety of concepts and models proposed by different groups for the generation of osteochondral grafts reflects that understanding of the requirements to restore a normal joint function is still poor. In order to introduce the use of engineered osteochondral composites in the routine clinical practice, it will be necessary to comprehensively address a number of critical issues, including those related to the size and shape of the graft to be generated, the cell type(s) and properties of the scaffold(s) to be used, the potential physical conditioning to be applied, the degree of functionality required, and the strategy for a cost-effective manufacturing. The progress made in material science, cell biology, mechanobiology and bioreactor technology will be key to support advances in this challenging field.     

微载体技术在软骨损伤修复中的应用与展望北大核心CSCD

软骨内部无血管结构、细胞外基质含量高的特点,使软骨组织的自我恢复能力很差。在临床治疗中,轻度的软骨缺损通常采用物理治疗或药物治疗方式,严重者需进行手术治疗。近年来,软骨组织工程技术为治疗软骨缺损提供了新的思路,与传统的手术治疗方式相比,结合软骨组织工程技术进行治疗具有创口小、恢复佳的优点。将微载体技术融入组织工程支架的设计中,可以利用微载体直径小、能够负载多种生长因子的特点,进一步扩展支架功能、促进软骨组织再生。文中首先对微载体技术进行介绍,对近年来微载体的主要制备方式和创新内容进行了概括总结,作为后续介绍的基础内容。然后对应用于软骨修复中的微载体进行了材料和功能上的划分,介绍了不同材料、不同功能微载体的属性特征和在软骨修复方面的具体应用,最后结合该领域发展历程对其今后发展趋势及方向进行展望,并基于笔者团队关于骨软骨一体化层状支架的研究,提出了通过微载体优化层状支架性能的思路,有望制备出更贴合天然软骨结构特征的仿生支架。     

Effect of a mechanical stimulation bioreactor on tissue engineered, scaffold-free cartilage

Achieving sufficient functional properties prior to implantation remains a significant challenge for the development of tissue engineered cartilage. Many studies have shown chondrocytes respond well to various mechanical stimuli, resulting in the development of bioreactors capable of transmitting forces to articular cartilage in vitro. In this study, we describe the production of sizeable, tissue engineered cartilage using a novel scaffold-free approach, and determine the effect of perfusion and mechanical stimulation from a C9-x Cartigen bioreactor on the properties of the tissue engineered cartilage. We created sizable tissue engineered cartilage from porcine chondrocytes using a scaffold-free approach by centrifuging a high-density chondrocyte cell-suspension onto an agarose layer in a 50 mL tube. The gross and histological appearances, biochemical content, and mechanical properties of constructs cultured in the bioreactor for 4 weeks were compared to constructs cultured statically. Mechanical properties were determined from unconfined uniaxial compression tests. Constructs cultured in the bioreactor exhibited an increase in total GAG content, equilibrium compressive modulus, and dynamic modulus versus static constructs. Our study demonstrates the C9-x CartiGen bioreactor is able to enhance the biomechanical and biochemical properties of scaffold-free tissue engineered cartilage; however, no additional enhancement was seen between loaded and perfused groups.     

Promoting increased mechanical properties of tissue engineered neocartilage via the application of hyperosmolarity and 4α-phorbol 12,13-didecanoate (4αPDD)

Osteoarthritis, a degenerative disease of the load-bearing joints, greatly reduces quality of life for millions of Americans and places a tremendous cost on the American healthcare system. Due to limitations of current treatments, tissue engineering of articular cartilage may provide a promising therapeutic option to treat cartilage defects. However, cartilage tissue engineering has yet to recapitulate the functional properties of native tissue. During normal joint loading, cartilage tissue experiences variations in osmolarity and subsequent changes in ionic concentrations. Motivated by these known variations in the cellular microenvironment, this study sought to improve the mechanical properties of neocartilage constructs via the application of hyperosmolarity and transient receptor potential vanilloid 4 (TRPV4) channel activator 4α-phorbol 12,13-didecanoate (4αPDD). It was shown that 4αPDD elicited significant increases in compressive properties. Importantly, when combined, 4αPDD positively interacted with hyperosmolarity to modulate its effects on tensile stiffness and collagen content. Thus, this study supports 4αPDD-activated channel TRPV4 as a purported mechanosensor and osmosensor that can facilitate the cell and tissue level responses to improve the mechanical properties of engineered cartilage. To our knowledge, this study is the first to systematically evaluate the roles of hyperosmolarity and 4αPDD on the functional (i.e., mechanical and biochemical) properties of self-assembled neotissue. Future work may combine 4αPDD-induced channel activation with other chemical and mechanical stimuli to create robust neocartilages suitable for treatment of articular cartilage defects.     

Functional tissue engineering of chondral and osteochondral constructs

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.