Cartilage regeneration with highly-elastic three-dimensional scaffolds prepared from biodegradable poly(L-lactide-co-epsilon-caprolactone)

Compressive mechanical stimuli are crucial in regenerating cartilage with tissue engineering, which creates a need for scaffolds that can maintain their mechanical integrity while delivering mechanical signals to adherent cells during strain applications. With these goals in mind, the aim of this study was to develop a mechano-active scaffold that facilitated effective cartilaginous tissue formation under dynamic physiological environments. Using a gel-pressing method, we fabricated a biodegradable and highly-elastic scaffold from poly(L-lactide-co-epsilon-caprolactone) (PLCL; 5:5), with 85% porosity and a 300-500-microm pore size, and we compared it to control scaffolds made of rigid polylactide (PLA) or poly(lactide-co-glycolide) (PLGA). After tensile mechanical tests and recovery tests confirmed the elasticity of the PLCL scaffolds, we seeded them with rabbit chondrocytes, cultured them in vitro, and subcutaneously implanted them into nude mice for up to eight weeks. The PLCL scaffolds possessed a completely rubber-like elasticity, were easily twisted and bent, and exhibited an almost complete (over 97%) recovery from applied strain (up to 500%); the control PLA scaffolds showed little recovery. In vitro and in vivo accumulations of extracellular matrix on the cell-PLCL constructs demonstrated that they could not only sustain but also significantly enhance chondrogenic differentiation. Moreover, the mechanical stimulation of the dynamic in vivo environment promoted deposition of the chondral extracellular matrix onto the PLCL. In contrast, on the PLA scaffolds, most of the chondrocytes had de-differentiated and formed fibrous tissues. In a rabbit defect model, the groups treated with PLCL scaffolds exhibited significantly enhanced cartilage regeneration compared to groups harboring an empty control or PLGA scaffolds. These results indicated that the mechano-active PLCL scaffolds effectively delivered mechanical signals associated with biological environments to adherent chondrocytes, suggesting that these elastic PLCL scaffolds could successfully be used for cartilage regeneration.     

The Effect of Hybridization of Hydrogels and Poly(L-lactide-co-ε-caprolactone) Scaffolds on Cartilage Tissue Engineering

For repairing cartilage defects by cartilage tissue engineering, it is important that engineered cartilage that is fabricated with scaffolds and cells can maintain the biological and physiological functions of cartilage, and also can induce three-dimensional spatial organization of chondrocytes. In this sense, hydrogels such as fibrin gels (FG) and hyaluronan (HA) are widely used for application in cartilage treatment. However, the use of hydrogels alone as a scaffold has a physical weakness; the mechanical properties of hydrogels are too weak to endure complex loading in the body. In this study, for mimicking a native cartilage microenvironment, we made cell–hybrid scaffold constructs with poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds and hydrogels to guide three-dimensional spatial organization of cells and extracellular matrix. A highly elastic scaffold was fabricated from PLCL with 85% porosity and 300–500 μm pore size using a gel-pressing method. The mixture of rabbit chondrocytes and hydrogels was seeded on PLCL scaffolds, and was subcutaneously implanted into nude mice for up to eight weeks. The cell seeding efficiency of the hybrid scaffolds with FG or HA was higher than that of the PLCL scaffolds. From in vivo studies, the accumulation of cartilaginous extracellular matrices of constructs, which was increased by hybridization of hydrogels and PLCL scaffolds, showed that the cell–hybrid scaffold constructs formed mature and well-developed cartilaginous tissue. In conclusion, the hybridization of hydrogels and PLCL scaffold for three-dimensional spatial organization of cells would provide a biomimetic environment where cartilage tissue growth is enhanced and facilitated. It can enhance the production of cartilaginous extracellular matrices and, consequently, improve the quality of the cartilaginous tissue formed.     

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.     

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.     

Mechano-active scaffold design based on microporous poly(L-lactide-co-epsilon-caprolactone) for articular cartilage tissue engineering: dependence of porosity on compression force-applied mechanical behaviors

An essential component of functional articular cartilage tissue engineering is a mechano-active scaffold, which responds to applied compression stress and causes little permanent deformation. As the first paper of a series on mechano-active scaffold-based cartilage tissue engineering, this study focused on mechanical responses to various modes of loading of compression forces and subsequent selection of mechano-active scaffolds from the biomechanical viewpoint. Scaffolds made of elastomeric microporous poly(L-lactide-co-epsilon-caprolactone) (PLCL) with open-cell structured pores (300 approximately 500 microm) and with different porosities ranging from 71 to 86% were used. The PLCL sponges and rabbit articular cartilage tissue were subjected to compression/unloading tests (0.1 and 0.005 Hz) at 5 kPa, and stress relaxation tests at 10, 30, and 50% strain. The measurements of the maximum strain under loading and residual strain under unloading for compression tests and the maximum stress and equilibrium stress in the stress relaxation test showed that the lower the porosity, the closer the mechanical properties are to those of native cartilage tissue. Among the PLCL sponges, the sponge with 71% porosity appears to be a suitable cartilage scaffold.     

Elastic cartilage engineering using novel scaffold architectures in combination with a biomimetic cell carrier

Tissue engineering of an elastic cartilage graft that meets the criterion for both structural and functional integration into host tissue, as well as allowing for a clinically tolerable immune response, is a challenging endeavour. Conventional scaffold technologies have limitations in their ability to design and fabricate complex-shaped matrix architectures of structural and mechanical equivalence to elastic cartilage found in the body. We attempted to investigate the potential of conventionally isolated and passaged chondrocytes (2D environment) when seeded and cultured in combination with a biomimetic hydrogel in a mechanically stable and biomimetic composite matrix to form elastic cartilage within ectopic implantation sites. In vitro cultured scaffold/hydrogel/chondrocytes constructs showed islets of cartilage and mineralized tissue formation within the cell-seeded specimens in both pig and rabbit models. Specimens with no cells seeded showed only vascularized fibrous tissue ingrowth. These studies demonstrated the potential of such scaffold/hydrogel/cell constructs to support chondrogenesis in vivo. However, it also showed that even mechanically stable scaffolds do not allow regeneration of a large mass of structural and functional cartilage within a matrix architecture seeded with 2D passaged chondrocytes in combination with a cell biomimetic carrier. Hence, future experiments will be designed to evaluate an initial 3D culture of chondrocytes, effect on cell phenotype and their subsequent culture within biomimetic 3D scaffold/cell constructs.     

Photocrosslinkable Hyaluronan as a Scaffold for Articular Cartilage Repair

Hyaluronan-based scaffolds are of interest for tissue-engineered cartilage repair due to an important role for hyaluronan in cartilage development and function. In this study, an in situ photocrosslinkable hyaluronan (HA-MA) was developed and evaluated as a scaffold for articular cartilage repair. Chondrocytes were encapsulated in crosslinked HA-MA and evaluated for their ability to synthesize cartilaginous matrix in vitro. The mechanical and physical properties of the crosslinked HA-MA hydrogels were similar to that of other hydrogels, with compressive and dynamic shear moduli of 0.6 and 0.3 kPa, respectively, and diffusion coefficients of 600-8000 microm2/s depending on molecular weight. Chondrocytes remained rounded in the HA-MA hydrogels in vitro, and accumulated significant amounts of cartilaginous matrix. Osteochondral defects filled with HA-MA were infiltrated with cells, appeared to integrate well with native tissue, and also accumulated substantial cartilaginous matrix by 2 weeks after surgery. In summary, photocrosslinkable HA-MA promoted the retention of the chondrocytic phenotype and cartilage matrix synthesis for encapsulated chondrocytes in vitro and accelerated healing in an in vivo osteochondral defect model.     

Improved mesenchymal stem cells attachment and in vitro cartilage tissue formation on chitosan-modified poly(L-lactide-co-epsilon-caprolactone) scaffold

Considering the load-bearing physiological requirement of articular cartilage, scaffold for cartilage tissue engineering should exhibit appropriate mechanical responses as natural cartilage undergoing temporary deformation on loading with little structural collapse, and recovering to the original geometry on unloading. A porous elastomeric poly l-lactide-co-?-caprolactone (PLCL) was generated and crosslinked at the surface to chitosan to improve its wettability. Human bone marrow derived mesenchymal stem cells (MSC) attachment, morphological change, proliferation and in vitro cartilage tissue formation on the chitosan-modified PLCL scaffold were compared with the unmodified PLCL scaffold. Chitosan surface promoted more consistent and even distribution of the seeded MSC within the scaffold. MSC rapidly adopted a distinct spread-up morphology on attachment on the chitosan-modified PLCL scaffold with the formation of F-actin stress fiber which proceeded to cell aggregation; an event much delayed in the unmodified PLCL. Enhanced cartilage formation on the chitosan-modified PLCL was shown by real-time PCR analysis, histological and immunochemistry staining and biochemical assays of the cartilage extracellular matrix components. The Young's modulus of the derived cartilage tissues on the chitosan-modified PLCL scaffold was significantly increased and doubled that of the unmodified PLCL. Our results show that chitosan modification of the PLCL scaffold improved the cell compatibility of the PLCL scaffold without significant alteration of the physical elastomeric properties of PLCL and resulted in the formation of cartilage tissue of better quality.     

The effect of cyclic strain on embryonic stem cell-derived cardiomyocytes

Cardiomyocytes in the body are subjected to cyclic mechanical strain induced by the rhythmic heart beating. In this study, we tested the hypothesis that cyclic strain promotes cardiomyogenesis of embryonic stem cell-derived cardiomyocytes (ESCs). ESCs cultured on elastic polymer [poly(lactide-co-caprolactone), PLCL] scaffolds subjected to cyclic strain in vitro displayed elevated cardiac gene expression compared to unstrained controls. Six weeks after implantation into infarcted rat myocardium, the elastic cardiac patches (ESC-seeded PLCL scaffolds) showed reduced fibrotic tissue formation, likely due to a combination of lower apoptotic activity, higher vascular endothelial growth factor (VEGF) expression, and more extensive angiogenesis in the strained versus unstrained control [ESC-seeded, non-elastic poly(lactide-co-glycolide) scaffolds] patches. Importantly, cardiac gene expression was upregulated in the elastic patches compared to control, with evidence for cardiomyocyte-specific microstructures including myofibrillar bundles and Z-lines. This study shows that the use of an elastic polymer scaffold designed to permit mechanical strain transduction as a cell transplantation vehicle significantly increases cardiomyogenesis of the implanted ESCs.     

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.     

A Collagen/Smooth Muscle Cell-Incorporated Elastic Scaffold for Tissue-Engineered Vascular Grafts

Biodegradable tubular scaffolds have been developed for vascular graft application. This study was focused to improve the adhesion and proliferation of vascular smooth muscle cells (SMCs) in a tubular scaffold. Tubular scaffolds (ID 4 mm, OD 6 mm) were fabricated from a biodegradable elastic polymer, poly(L-lactide-co-ε-caprolactone) (PLCL) (50:50, M _n 1.58 × 10⁵), by an extrusion/particulate leaching method. SMCs suspended in a collagen solution were infiltrated in tubular PLCL scaffolds under vacuum and incubated for 1 h at 37°C to form a collagenous gel. Results from SEM image analysis showed that collagen was infiltrated into the inside of the scaffolds. Cell adhesion and proliferation rate increased in collagen/SMC-incorporated tubular PLCL scaffolds as compared with the scaffolds in which only SMCs were seeded. From SEM image and histological analysis, we further found that SMCs grew on the inside as well as on the surface of collagen/SMCs-incorporated scaffolds and the cells continued to grow as a monolayer on collagen fibers. In particular, cell proliferation and elastin contents were the highest in a PLCL scaffold with 50–100 μm pore size than any other scaffolds used in this experiment. A collagen/SMC-incorporated PLCL scaffold may support SMC growth and functions and can be used as a scaffold for tissue engineering to facilitate small-diameter vascular-tissue formation.     

Regeneration of Achilles' Tendon: The Role of Dynamic Stimulation for Enhanced Cell Proliferation and Mechanical Properties

The tissue engineering of tendon was studied using highly elastic poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds and focusing on the effect of dynamic tensile stimulation. Tenocytes from rabbit Achilles tendon were seeded (1.0 × 10⁶ cells/scaffold) onto porous PLCL scaffolds and cultured for periods of 2 weeks and 4 weeks. This was performed in a static system and also in a bioreactor equipped with tensile modulation which mimicked the environmental surroundings of tendons with respect to tensile extension. The degradation of the polymeric scaffolds during the culture was relatively slow. However, there was an indication that cells accelerated the degradation of PLCL scaffolds. The scaffold/cell adducts from the static culture exhibited inferior strength (at 2 weeks 350 kPa, 4 weeks 300 kPa) compared to the control without cells (at 2 weeks 460 kPa, 4 weeks 340 kPa), indicating that the cells contributed to the enhanced degradation. On the contrary, the corresponding values of the adducts from the dynamic culture (at 2 weeks 430 kPa, 4 weeks 370 kPa) were similar to, or higher than, those from the control. This could be explained by the increased quantity of cells and neo-tissues in the case of dynamic culture compensating for the loss in tensile strength. Compared with static and dynamic culture conditions, mechanical stimulation played a crucial role in the regeneration of tendon tissue. In the case of the dynamic culture system, cell proliferation was enhanced and secretion of collagen type I was increased, as evidenced by DNA assay and histological and immunofluorescence analysis. Thus, tendon regeneration, indicated by improved mechanical and biological properties, was demonstrated, confirming the effect of mechanical stimulation. It could be concluded that the dynamic tensile stimulation appeared to be an essential factor in tendon/ligament tissue engineering, and that elastic PLCL co-polymers could be very beneficial in this process.     

TGF-beta1 immobilized tri-co-polymer for articular cartilage tissue engineering

Tri-co-polymer with composition of gelatin, hyaluronic acid and chondroitin-6-sulfate has been used to mimic the cartilage extracellular matrix as scaffold for cartilage tissue engineering. In this study, we try to immobilize TGF-beta1 onto the surface of the tri-co-polymer sponge to suppress the undesired differentiation during the cartilage growth in vitro. The scaffold was synthesized with a pore size in a range of 300-500 microm. TGF-beta1 was immobilized on the surface of the tri-co-polymer scaffold with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent. Tri-co-polymer scaffolds with and without TGF-beta1 were seeded with porcine chondrocytes and cultured in a spinner flask for 2, 4, and 6 weeks. The chondrocytes were characterized by the methods of immunohistochemical staining with anti-type II collagen and anti-S-100 protein monoclonal antibody, and RT-PCR. After culturing for 4 weeks, chondrocytes showed positive in S-100 protein, Alcian blue, and type II collagen for the scaffold with TGF-beta1 immobilization. There is no observed type I and type X collagen expression in the scaffolds from the observation of RT-PCR. In addition, the scaffold without TGF-beta1 immobilization, type X collagen, can be detected after cultured for 2 weeks. Type I collagen was progressively expressed after 4 weeks. These results can conclude that TGF-beta1 immobilized scaffold can suppress chondrocytes toward prehypertrophic chondrocytes and osteolineage cells. The tri-co-polymer sponge with TGF-beta1 immobilization should have a great potential in cartilage tissue engineering in the future.     

Chondrocyte aggregation and reorganization into three-dimensional scaffolds.

Articular cartilage has a very limited self-repairing capacity; thus, chondral lesions normally result in chronic degeneration and, eventually, osteoarthritis development. Currently, tissue engineering offers a new tool for the clinical treatment of osteochondral defects. The present investigation aimed to develop an in vitro engineered cartilage using a new class of semisynthetic scaffolds. Two nonwoven meshes of hyaluronan esters (Hyaff(R) derivatives) were seeded with sternal chick embryo chondrocytes cultured for up to 21 days, after which time they were assessed for both the cellular growth profile and histological features. Avian chondrocytes easily adhered and proliferated onto hyaluronan-based scaffolds, demonstrating a significant preference for the fully esterified benzylic form. Histochemical staining revealed the presence of a neosynthesized glycosaminoglycan-rich extracellular matrix, and immunohistochemistry confirmed the deposition of collagen type II. Moreover, ultrastructural observations supported evidence that chondrocytes grown onto a hyaluronan-derived three-dimensional scaffold maintained their unique phenotype and organization in a cartilage-like extracellular matrix. These findings support the further pursuit of a transplantable engineered cartilage using human chondrocytes for the regeneration of chondral lesions.     

Development of arginine-glycine-aspartate-immobilized 3D printed poly(propylene fumarate) scaffolds for cartilage tissue engineering

Abstract

Poly(propylene fumarate) (PPF) has known to be a good candidate material for cartilage tissue regeneration because of its excellent mechanical properties during its degradation processes. Here, we describe the potential application of PPF-based materials as 3D printing bioinks to create macroporous cell scaffolds using micro-stereolithography. To improve cell-matrix interaction of seeded human chondrocytes within the PPF-based 3D scaffolds, we immobilized arginine-glycine-aspartate (RGD) peptide onto the PPF scaffolds. We also evaluated various cellular behaviors of the seeded chondrocytes using MTS assay, microscopic and histological analyses. The results indicated that PPF-based biocompatible scaffolds with immobilized RGD peptide could effectively support initial adhesion and proliferation of human chondrocytes. Such a 3D bio-printable scaffold can offer an opportunity to promote cartilage tissue regeneration.     

以壳聚糖-胶原共混膜为三维支架同种异体工程化软骨的构建

目的探讨以壳聚糖-胶原共混膜为三维支架材料的同种异体软骨细胞构建组织工程化软骨的能力。方法将分离、培养、扩传兔软骨细胞，接种在壳聚糖-胶原共混膜上，倒置显微镜下观察细胞在共混膜上的生长情况。体外培养7d后，将细胞-材料复合物种植在新西兰兔皮下，6周取材，对获得的同种异体工程化软骨进行组织学评价。结果兔软骨细胞接种于壳聚糖-胶原共混膜上4h后有贴壁现象出现，细胞呈梭形。培养48h后，软骨细胞分裂增殖越来越多并向周围延伸，培养第7天取材，HE染色示细胞生长良好，呈梭形。体内培养6周取材，HE染色、Masson染色为均一的成熟软骨组织，且共混膜已降解。结论以壳聚糖-胶原共混膜为支架材料同种异体软骨细胞在有免疫力的动物体内可形成工程化软骨。     

Synthesis and in vitro evaluation of enzymatically cross-linked elastin-like polypeptide gels for cartilaginous tissue repair

Genetically engineered elastin-like polypeptide (ELP) hydrogels offer unique promise as scaffolds for cartilage tissue engineering because of the potential to promote chondrogenesis and to control mechanical properties. In this study, we designed and synthesized ELPs capable of undergoing enzyme-initiated gelation via tissue transglutaminase, with the ultimate goal of creating an injectable, in situ cross-linking scaffold to promote functional cartilage repair. Addition of the enzyme promoted ELP gel formation and chondrocyte encapsulation in a biocompatible process, which resulted in cartilage matrix synthesis in vitro and the potential to contribute to cartilage mechanical function in vivo. A significant increase in the accumulation of sulfated glycosaminoglycans was observed, and histological sections revealed the accumulation of a cartilaginous matrix rich in type II collagen and lacking in type I collagen, indicative of hyaline cartilage formation. These results provide evidence of chondrocytic phenotype maintenance for cells in the ELP hydrogels in vitro. In addition, the dynamic shear moduli of ELP hydrogels seeded with chondrocytes increased from 0.28 to 1.7 kPa during a 4-week culture period. This increase in the mechanical integrity of cross-linked ELP hydrogels suggests restructuring of the ELP matrix by deposition of functional cartilage extracellular matrix components.     

Poly-L-D-lactic acid scaffold in the repair of porcine knee cartilage lesions

Articular cartilage injuries cause a major clinical problem because of the negligible repair capacity of cartilage. Autologous chondrocyte transplantation is a surgical method developed to repair cartilage lesions. In the operation, cartilage defect is covered with a periosteal patch and the suspension of cultured autologous chondrocytes is injected into the lesion site. The method can form good repair tissue, but new techniques are needed to make the operation easier and to increase the postoperative biomechanical properties of the repair tissue. In this study, we investigated poly-L,D-lactic acid (PLDLA) scaffolds alone or seeded with autologous chondrocytes in the repair of circular 6-mm cartilage lesions in immature porcine knee joints. Spontaneous repair was used as a reference. Histologic evaluation of the repair tissue showed that spontaneous repair exhibited higher scores than either PLDLA scaffold group (with or without seeded chondrocytes). The scaffold material was most often seen embedded in the subchondral bone underneath the defect area, probably because of the hardness of the PLDLA material. However, some of the cell-seeded and nonseeded scaffolds contained cartilaginous tissue, suggesting that invasion of mesenchymal cells inside nonseeded scaffolds had occurred. Hyaluronan deposited in the scaffold had possibly acted as a chemoattractant for the cell recruitment. In conclusion, the PLDLA scaffold material used in this study was obviously mechanically too hard to be used for cartilage repair in immature animals.     

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.