Design of porous scaffolds for cartilage tissue engineering using a three-dimensional fiber-deposition technique

In this study, we present and characterize a fiber deposition technique for producing three-dimensional poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT/PBT) block co-polymer scaffolds with a 100% interconnecting pore network for engineering of articular cartilage. The technique allowed us to "design-in" desired scaffold characteristics layer by layer by accurately controlling the deposition of molten co-polymer fibers from a pressure-driven syringe onto a computer controlled x-y-z table. By varying PEGT/PBT composition, porosity and pore geometry, 3D-deposited scaffolds were produced with a range of mechanical properties. The equilibrium modulus and dynamic stiffness ranged between 0.05-2.5 and 0.16-4.33 MPa, respectively, and were similar to native articular cartilage explants (0.27 and 4.10 MPa, respectively). 3D-deposited scaffolds seeded with bovine articular chondrocytes supported a homogeneous cell distribution and subsequent cartilage-like tissue formation following in vitro culture as well as subcutaneous implantation in nude mice. This was demonstrated by the presence of articular cartilage extra cellular matrix constituents (glycosaminoglycan and type II collagen) throughout the interconnected pore volume. Similar results were achieved with respect to the attachment of expanded human articular chondrocytes, resulting in a homogeneous distribution of viable cells after 5 days dynamic seeding. The processing methods and model scaffolds developed in this study provide a useful method to further investigate the effects of scaffold composition and pore architecture on articular cartilage tissue formation.     

Evaluation of three-dimensional scaffolds prepared from poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for growth of allogeneic chondrocytes for cartilage repair in rabbits

Articular cartilage repair using tissue engineering approach generally requires the use of an appropriate scaffold architecture that can support the formation of cartilage tissue. In this investigation, the potential of three-dimensional scaffolds made of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was evaluated in rabbit articular cartilage defect model. Engineered PHBHHx cartilage constructs inoculated in vitro with rabbit chondrocytes for 30 days were examined. Subsequently the constructs inoculated with chondrocytes for 10 days were selected for transplantation into rabbits. After 16 weeks of in vivo implantation, both the engineered cartilage constructs and the bare scaffolds were found to be filled the defects with white cartilaginous tissue, with the engineered constructs showing histologically good subchondral bone connection and better surrounding cartilage infusion. Owing to pre-seeded chondrocytes in the PHBHHx scaffolds, better surface integrality and more accumulation of extracellular matrix (ECM) including type II collagen and sGAG were achieved in the engineered cartilage constructs. The repaired tissues possessed an average compressive modulus of 1.58MPa. For comparison, the defects without repair treatments still showed defects with fibrous tissues. These results demonstrated that PHBHHx is a useful material for cartilage tissue engineering.     

Gene expression profiling of human articular cartilage grafts generated by tissue engineering

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. In this study autologous human cartilage tissue engineering grafts based on bioresorbable polyglactin/polydioxanone scaffolds were analyzed on the broad molecular level. RNA from freshly isolated, primary and expanded adult articular chondrocytes and from three-dimensional cartilage grafts were used for gene expression profiling using oligonucleotide microarrays. The capacity of cartilage grafts to form cartilage matrix was evaluated after subcutaneous transplantation into nude mice. Gene expression profiling showed reproducibly the regulation of 905 genes and documented that chondrocytes undergo fundamental changes during cartilage tissue engineering regarding chondrocyte metabolism, growth, and differentiation. Three-dimensional assembly of expanded, dedifferentiated chondrocytes initiated the re-differentiation of cells that was accompanied by the reversal of the expression profile of multiple players of the transforming growth factor (TGF) signaling pathway including growth and differentiation factor-5 and inhibitor of differentiation-1 as well as by the induction of typical cartilage-related matrix genes such as type II collagen and cartilage oligomeric matrix protein. Cartilage grafts formed a cartilaginous matrix after transplantation into nude mice. Three-dimensional tissue culture of expanded articular chondrocytes initiates chondrocyte re-differentiation in vitro and leads to the maturation of cartilage grafts towards hyaline cartilage in vivo.     

Application of microstereolithography in the development of three-dimensional cartilage regeneration scaffolds

Conventional methods for fabricating three-dimensional (3-D) tissue engineering scaffolds have substantial limitations. In this paper, we present a method for applying microstereolithography in the construction of 3-D cartilage scaffolds. The system provides the ability to fabricate scaffolds having a pre-designed internal structure, such as pore size and porosity, by stacking photopolymerized materials. To control scaffold structure, CAD/CAM technology was used to generate a scaffold pattern algorithm. Since tissue scaffolds must be constructed using a biocompatible, biodegradable material, scaffolds were synthesized using liquid photocurable TMC/TMP, followed by acrylation at the terminal ends, and photocured under UV light irradiation. The solidification properties of the TMC/TMP polymer were also assessed. To assess scaffold functionality, chondrocytes were seeded on two types of 3-D scaffold and characterized for cell adhesion. Results indicate that scaffold geometry plays a critical role in chondrocyte adhesion, ultimately affecting the tissue regeneration utility of the scaffolds. These 3-D scaffolds could eventually lead to optimally designed constructs for the regeneration of various tissues, such as cartilage and bone.     

The influence of scaffold material on chondrocytes under inflammatory conditions

Cartilage tissue engineering aims to repair damaged cartilage tissue in arthritic joints. As arthritic joints have significantly higher levels of pro-inflammatory cytokines (such as IL-1β and TNFα that cause cartilage destruction, it is critical to engineer stable cartilage in an inflammatory environment. Biomaterial scaffolds constitute an important component of the microenvironment for chondrocytes in engineered cartilage. However, it remains unclear how the scaffold material influences the response of chondrocytes seeded in these scaffolds under inflammatory stimuli. Here we have compared the responses of articular chondrocytes seeded within three different polymeric scaffolding materials (silk, collagen and polylactic acid (PLA)) to IL-1β and TNFα. These scaffolds have different physical characteristics and yielded significant differences in the expression of genes associated with cartilage matrix production and degradation, cell adhesion and cell death. The silk and collagen scaffolds released pro-inflammatory cytokines faster and had higher uptake water abilities than PLA scaffolds. Correspondingly, chondrocytes cultured in silk and collagen scaffolds maintained higher levels of cartilage matrix than those in PLA, suggesting that these biophysical properties of scaffolds may regulate gene expression and the response to inflammatory stimuli in chondrocytes. Based on this study we conclude that selecting the proper scaffold material will aid in the engineering of more stable cartilage tissues for cartilage repair, and that silk and collagen are better scaffolds in terms of supporting the stability of three-dimensional cartilage under inflammatory conditions.     

Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model

This work investigated the ability of co-cultures of articular chondrocytes and mesenchymal stem cells (MSCs) to repair articular cartilage in osteochondral defects. Bovine articular chondrocytes and rat MSCs were seeded in isolation or in co-culture onto electrospun poly(?-caprolactone) (PCL) scaffolds and implanted into an osteochondral defect in the trochlear groove of 12-week old Lewis rats. Additionally, a blank PCL scaffold and untreated defect were investigated. After 12 weeks, the extent of cartilage repair was analyzed through histological analysis, and the extent of bone healing was assessed by quantifying the total volume of mineralized bone in the defect through microcomputed tomography. Histological analysis revealed that the articular chondrocytes and co-cultures led to repair tissue that consisted of more hyaline-like cartilage tissue that was thicker and possessed more intense Safranin O staining. The MSC, blank PCL scaffold, and empty treatment groups generally led to the formation of fibrocartilage repair tissue. Microcomputed tomography revealed that while there was an equivalent amount of mineralized bone formation in the MSC, blank PCL, and empty treatment groups, the defects treated with chondrocytes or co-cultures had negligible mineralized bone formation. Overall, even with a reduced number of chondrocytes, co-cultures led to an equal level of cartilage repair compared to the chondrocyte samples, thus demonstrating the potential for the use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.     

Cartilaginous tissue formation using a mechano-active scaffold and dynamic compressive stimulation

It is known that complex loading is involved in the development and maintenance of articular cartilage in the body. It means the compressive mechanical stimulation is a very important factor for formation of articular cartilage using a tissue-engineering technique. The objective of this study is to engineer cartilaginous constructs with mechano-active scaffolds and to evaluate the effect of dynamic compression for regeneration of cartilage. The mechano-active scaffolds were prepared from a very elastic poly(L-lactide-co-epsilon-caprolactone) (PLCL) with 85% porosity and 300-500 mum pore size using a gel-pressing method. The scaffold was seeded with 2 x 10(6) chondrocytes and the continuous compressive deformation of 5% strain was applied with 0.1 Hz for 10 days and 24 days, respectively. Then, the chondrocytes-seeded constructs were implanted subcutaneously into nude mice. Mechano-active scaffolds with complete rubber-like elasticity showed almost complete (over 97%) recovery at an applied strain of up to 500%. The amount of chondral extracellular matrix was increased significantly by mechanical stimulation on the highly elastic mechano-active scaffolds. Histological analysis showed the mechanically stimulated implants formed mature and well-developed cartilaginous tissue, as evidenced by the chondrocytes within lacunae and the abundant accumulation of sulfated GAGs. However, unhealthy lacunae shapes and hypertrophy forms were observed in the implants stimulated mechanically for 24 days, compared with those stimulated for 10 days. In conclusion, the proper periodical application of dynamic compression can encourage chondrocytes to maintain their phenotypes and enhance the production of GAGs, which would improve the quality of cartilaginous tissue formed both in vitro and in vivo.     

Design of biphasic polymeric 3-dimensional fiber deposited scaffolds for cartilage tissue engineering applications

This report describes a novel system to create rapid prototyped 3-dimensional (3D) fibrous scaffolds with a shell-core fiber architecture in which the core polymer supplies the mechanical properties and the shell polymer acts as a coating providing the desired physicochemical surface properties. Poly[(ethylene oxide) terephthalate-co-poly(butylene) terephthalate] (PEOT/PBT) 3D fiber deposited (3DF) scaffolds were fabricated and examined for articular cartilage tissue regeneration. The shell polymer contained a higher molecular weight of the initial poly(ethylene glycol) (PEG) segments used in the copolymerization and a higher weight percentage of the PEOT domains compared with the core polymer. The 3DF scaffolds entirely produced with the shell or with the core polymers were also considered. After 3 weeks of culture, scaffolds were homogeneously filled with cartilage tissue, as assessed by scanning electron microscopy. Although comparable amounts of entrapped chondrocytes and of extracellular matrix formation were found for all analyzed scaffolds, chondrocytes maintained their rounded shape and aggregated during the culture period on shell-core 3DF scaffolds, suggesting a proper cell differentiation into articular cartilage. This finding was also observed in the 3DF scaffolds fabricated with the shell composition only. In contrast, cells spread and attached on scaffolds made simply with the core polymer, implying a lower degree of differentiation into articular cartilaginous tissue. Furthermore, the shell-core scaffolds displayed an improved dynamic stiffness as a result of a "prestress" action of the shell polymer on the core one. In addition, the dynamic stiffness of the constructs increased compared with the stiffness of the bare scaffolds before culture. These findings suggest that shell-core 3DF PEOT/PBT scaffolds with desired mechanical and surface properties are a promising solution for improved cartilage tissue engineering.     

Cyclooxygenases (COX-1 and COX-2) for tissue engineering of articular cartilage--from a developmental model to first results of tissue and scaffold expression

Tissue engineering of articular cartilage remains an ongoing challenge. Since tissue regeneration recapitulates ontogenetic processes the growth plate can be regarded as an innovative model to target suitable signalling molecules and growth factors for the tissue engineering of cartilage. In the present study we analysed the expression of cyclooxygenases (COX) in a short-term chondrocyte culture in gelatin-based scaffolds and in articular cartilage of rats and compared it with that in the growth plate. Our results demonstrate the strong cellular expression of COX-1 but only a focal weak expression of COX-2 in the seeded scaffolds. Articular cartilage of rats expresses homogeneously COX-1 and COX-2 with the exception of the apical cell layer. Our findings indicate a functional role of COX in the metabolism of articular chondrocytes. The expression of COX in articular cartilage and in the seeded scaffolds opens interesting perspectives to improve the proliferation and differentiation of chondrocytes in scaffold materials by addition of specific receptor ligands of the COX system.     

Thermoreversible hydrogel scaffolds for articular cartilage engineering

Articular cartilage has limited potential for repair. Current clinical treatments for articular cartilage damage often result in fibrocartilage and are associated with joint pain and stiffness. To address these concerns, researchers have turned to the engineering of cartilage grafts. Tissue engineering, an emerging field for the functional restoration of articular cartilage and other tissues, is based on the utilization of morphogens, scaffolds, and responding progenitor/stem cells. Because articular cartilage is a water-laden tissue and contains within its matrix hydrophilic proteoglycans, an engineered cartilage graft may be based on synthetic hydrogels to mimic these properties. To this end, we have developed a polymer system based on the hydrophilic copolymer poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)]. Solutions of this polymer are liquid below 25 degrees C and gel above 35 degrees C, allowing an aqueous solution containing cells at room temperature to form a hydrogel with encapsulated cells at physiological body temperature. The objective of this work was to determine the effects of the hydrogel components on the phenotype of encapsulated chondrocytes. Bovine articular chondrocytes were used as an experimental model. Results demonstrated that the components required for hydrogel fabrication did not significantly reduce the proteoglycan synthesis of chondrocytes, a phenotypic marker of chondrocyte function. In addition, chondrocyte viability, proteoglycan synthesis, and type II collagen synthesis within P(PF-co-EG) hydrogels were investigated. The addition of bone morphogenetic protein-7 increased chondrocyte proliferation with the P(PF-co-EG) hydrogels, but did not increase proteoglycan synthesis by the chondrocytes. These results indicate that the temperature-responsive P(PF-co-EG) hydrogels are suitable for chondrocyte delivery for articular cartilage repair.     

Tissue engineering of articular cartilage using an allograft of cultured chondrocytes in a membrane-sealed atelocollagen honeycomb-shaped scaffold (ACHMS scaffold)

The aim of this study was to investigate with tissue engineering procedures the possibility of using atelocollagen honeycomb-shaped scaffolds sealed with a membrane (ACHMS scaffold) for the culturing of chondrocytes to repair articular cartilage defects. Chondrocytes from the articular cartilage of Japanese white rabbits were cultured in ACHMS scaffolds to allow a high-density, three-dimensional culturing for up to 21 days. Although the DNA content in the scaffold increased at a lower rate than monolayer culturing, scanning electron microscopy data showed that the scaffold was filled with grown chondrocytes and their produced extracellular matrix after 21 days. In addition, glycosaminoglycan (GAG) accumulation in the scaffold culture was at a higher level than the monolayer culture. Cultured cartilage in vitro for 14 days showed enough elasticity and stiffness to be handled in vivo. An articular cartilage defect was initiated in the patellar groove of the femur of rabbits and was subsequently filled with the chondrocyte-cultured ACHMS scaffold, ACHMS scaffold alone, or non-filled (control). Three months after the operations, histological analysis showed that only defects inserted with chondrocytes being cultured in ACHMS scaffolds were filled with reparative hyaline cartilage, and thereby highly expressing type II collagen. These results indicate that implantation of allogenic chondrocytes cultured in ACHMS scaffolds may be effective in repairing articular cartilage defects.     

Cartilaginous tissue formation using a mechano-active scaffold and dynamic compressive stimulation

It is known that complex loading is involved in the development and maintenance of articular cartilage in the body. It means the compressive mechanical stimulation is a very important factor for formation of articular cartilage using a tissue-engineering technique. The objective of this study is to engineer cartilaginous constructs with mechano-active scaffolds and to evaluate the effect of dynamic compression for regeneration of cartilage. The mechano-active scaffolds were prepared from a very elastic poly(L-lactide-co-ε-caprolactone) (PLCL) with 85% porosity and 300–500 μm pore size using a gel-pressing method. The scaffold was seeded with 2 × 10⁶ chondrocytes and the continuous compressive deformation of 5% strain was applied with 0.1 Hz for 10 days and 24 days, respectively. Then, the chondrocytes-seeded constructs were implanted subcutaneously into nude mice. Mechano-active scaffolds with complete rubber-like elasticity showed almost complete (over 97%) recovery at an applied strain of up to 500%. The amount of chondral extracellular matrix was increased significantly by mechanical stimulation on the highly elastic mechano-active scaffolds. Histological analysis showed the mechanically stimulated implants formed mature and well-developed cartilaginous tissue, as evidenced by the chondrocytes within lacunae and the abundant accumulation of sulfated GAGs. However, unhealthy lacunae shapes and hypertrophy forms were observed in the implants stimulated mechanically for 24 days, compared with those stimulated for 10 days. In conclusion, the proper periodical application of dynamic compression can encourage chondrocytes to maintain their phenotypes and enhance the production of GAGs, which would improve the quality of cartilaginous tissue formed both in vitro and in vivo.     

Accelerated chondrocyte functions on NaOH-treated PLGA scaffolds

Compared to conventional poly(lactic-co-glycolic acid) (PLGA), previous studies have shown that NaOH-treated PLGA two-dimensional substrates enhanced functions of osteoblasts (bone-forming cells), vascular and bladder smooth muscle cells, and chondrocytes (cartilage-synthesizing cells). In this same spirit, the purpose of this in vitro study was to fabricate three-dimensional NaOH-treated PLGA scaffolds and determine their efficacy toward articular cartilage applications. To improve functions of chondrocytes including their adhesion, growth, differentiation, and extracellular matrix synthesis, PLGA scaffolds were modified via chemical etching techniques using 1N NaOH for 10 min. Results demonstrated that NaOH-treated PLGA three-dimensional scaffolds enhanced chondrocyte functions compared to non-treated scaffolds. Specifically, chondrocyte numbers, total intracellular protein content, and the amount of extracellular matrix components (such as glycosaminoglycans and collagens) were significantly greater on NaOH-treated than on non-treated PLGA scaffolds. Underlying material properties that may have enhanced chondrocyte functions include a more hydrophilic surface (due to hydrolytic degradation of PLGA by NaOH), increased surface area, altered porosity (both percent and diameter of individual pores), and a greater degree of nanometer roughness. For these reasons, this study adds a novel tissue-engineering scaffold to the cartilage biomaterial community: NaOH-treated PLGA. Clearly, such modifications to PLGA may ultimately enhance the efficacy of tissue-engineering scaffolds for articular cartilage repair.     

Proliferation of chondrocytes on porous poly(DL-lactide)/chitosan scaffolds

Porous poly(DL-lactide)(PDLLA)/chitosan scaffolds with well-controlled pore structures and desirable mechanical characteristics were fabricated via a combination of solvent extraction, phase separation and freeze-drying. These scaffolds were further evaluated for the proliferation of isolated rabbit chondrocytes in vitro for various incubation periods up to 4 weeks in order to finally use them for the cartilage tissue engineering. MTT assay data revealed that the number of cells grown on PDLLA/chitosan scaffolds measurably increased with the weight ratio of the chitosan component and was significantly higher than those collected from pure PDLLA scaffolds for the entire incubation period. Scanning electron microscopy examinations, histological observations and proteoglycan measurements indicated that the resulting PDLLA/chitosan scaffolds exhibited increasing ability to promote the attachment and proliferation of chondrocytes, and also helped seeded chondrocytes spread through the scaffolds and distribute homogeneously inside compared to pure PDLLA scaffolds. Immunohistochemical staining verified that these PDLLA/chitosan scaffolds could preserve the phenotype of chondrocyte and effectively support the production of type II collagen.     

Fabrication and characterization of poly(γ-glutamic acid)-graft-chondroitin sulfate/polycaprolactone porous scaffolds for cartilage tissue engineering

The development of blended biomacromolecule and polyester scaffolds can potentially be used in many tissue engineering applications. This study was to develop a poly(γ-glutamic acid)-graft-chondroitin sulfate-blend-poly(ε-caprolactone) (γ-PGA-g-CS/PCL) composite biomaterial as a scaffold for cartilage tissue engineering. Chondroitin sulfate (CS) was grafted to γ-PGA, forming a γ-PGA-g-CS copolymer with 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) system. The γ-PGA-g-CS copolymers were then blended with PCL to yield a porous γ-PGA-g-CS/PCL scaffold by salt leaching. These blended scaffolds were characterized by ¹H NMR, ESCA, water-binding capacity, mechanical test, degradation rate and CS assay. The results showed that with γ-PGA-g-CS as a component, the water-binding capacity and the degradation rate of the scaffolds would substantially increase. During a 4 week period of culture, the mechanical stability of γ-PGA-g-CS/PCL scaffolds was raised gradually and chondrocytes were induced to function normally in vitro. Furthermore, a larger amount of secreted GAGs was present in the γ-PGA-g-CS/PCL matrices than in the control (PCL), as revealed by Alcian blue staining of the histochemical sections. Thus, γ-PGA-g-CS/PCL matrices exhibit excellent biodegradation and biocompatibility for chondrocytes and have potential in tissue engineering as temporary substitutes for articular cartilage regeneration.     

The effect of PEGT/PBT scaffold architecture on the composition of tissue engineered cartilage

A highly interconnecting and accessible pore network has been suggested as one of a number of prerequisites in the design of scaffolds for tissue engineering. In the present study, two processing techniques, compression-molding/particulate-leaching (CM), and 3D fiber deposition (3DF), were used to develop porous scaffolds from biodegradable poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) co-polymers with varying pore architectures. Three-dimensional micro-computed tomography (microCT) was used to characterize scaffold architectures and scaffolds were seeded with articular chondrocytes to evaluate tissue formation. Scaffold porosity ranged between 75% and 80%. Average pore size of tortuous CM scaffolds (182 microm) was lower than those of organized 3DF scaffolds (525 microm). The weight ratio of glycosaminoglycans (GAG)/DNA, as a measure of cartilage-like tissue formation, did not change after 14 days of culture whereas, following subcutaneous implantation, GAG/DNA increased significantly and was significantly higher in 3DF constructs than in CM constructs, whilst collagen type II was present within both constructs. In conclusion, 3DF PEGT/PBT scaffolds create an environment in vivo that enhances cartilaginous matrix deposition and hold particular promise for treatment of articular cartilage defects.     

软骨组织工程学方法修复关节软骨缺损

背景：关节软骨缺损在临床上十分常见,随着分子生物学和组织工程学等学科的不断发展,为关节软骨缺损的修复提供了大量新的方法和思路,利用软骨组织工程学方法修复关节软骨缺损成为目前骨科领域研究的热点。 目的：总结并讨论目前软骨组织工程学方法修复关节软骨缺损的研究现状,综述应用软骨组织工程学方法修复关节软骨缺损的研究进展。 方法：由第一作者应用计算机检索中国期刊全文数据库(CNKI：2000/2010)和Medline(1990/2010)数据库中的相关文章,检索词分别为“关节软骨缺损,软骨组织工程”和“articular cartilage defects (ACD),cartilage tissue engineering”,语言分别设定为中文和英文。共检索得文章786篇,从中选取相关文章44篇,从软骨组织工程学方法修复关节软骨缺损过程中的种子细胞、支架材料和生物因子3个具体方面的研究进展进行归纳及总结。 结果与结论：支架、种子细胞和可调控细胞生长、增殖及分化的生物因子是软骨组织工程的3大要素。软骨组织工程方法主要包括利用体外培养、扩增后的种子细胞,将其种植于支架材料中,在相关调节因素的作用下形成组织工程化软骨,此方法已成为目前治疗关节软骨缺损的重要方法之一,并取得不错的疗效,但迄今为止尚未出现一种法被广泛认可的治疗方案。利用不同新型复合支架材料如修复治疗关节软骨缺损将成为今后研究的主要方向。