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.     

Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.     

Chitosan scaffolds containing hyaluronic acid for cartilage tissue engineering

Scaffolds derived from natural polysaccharides are very promising in tissue engineering applications and regenerative medicine, as they resemble glycosaminoglycans in the extracellular matrix (ECM). In this study, we have prepared freeze-dried composite scaffolds of chitosan (CHT) and hyaluronic acid (HA) in different weight ratios containing either no HA (control) or 1%, 5%, or 10% of HA. We hypothesized that HA could enhance structural and biological properties of CHT scaffolds. To test this hypothesis, physicochemical and biological properties of CHT/HA scaffolds were evaluated. Scanning electron microscopy micrographs, mechanical properties, swelling tests, enzymatic degradation, and Fourier transform infrared (FTIR) chemical maps were performed. To test the ability of the CHT/HA scaffolds to support chondrocyte adhesion and proliferation, live-dead and MTT assays were performed. Results showed that CHT/HA composite scaffolds are noncytotoxic and promote cell adhesion. ECM formation was further evaluated with safranin-O and alcian blue staining methods, and glycosaminoglycan and DNA quantifications were performed. The incorporation of HA enhanced cartilage ECM production. CHT/5HA had a better pore network configuration and exhibited enhanced ECM cartilage formation. On the basis of our results, we believe that CHT/HA composite matrixes have potential use in cartilage repair.     

Tissue engineering of bovine articular cartilage within porous poly(ether ester) copolymer scaffolds with different structures

The potential of porous poly(ether ester) scaffolds made from poly(ethylene glycol) terephthalate: poly(butylene terephthalate) (PEGT:PBT) block copolymers produced by various methods to enable cartilaginous tissue formation in vitro was studied. Scaffolds were fabricated by two different processes: paraffin templating (PT) and compression molding (CM). To determine whether PEGT:PBT scaffolds are able to support chondrogenesis, primary bovine chondrocytes were seeded within cylindrical scaffolds under dynamic seeding conditions. On day 3, constructs were transferred to six-well plates and evaluated for glycosaminoglycan (GAG) distribution (3, 10, and 24 days), type II collagen distribution, cellularity, and total collagen and GAG content (10 and 24 days). It was observed that better cell distribution during infiltration within PT scaffolds allowed greater chondrogenesis, and at later time points, than in CM scaffolds. The amount of GAG remained constant for all groups from 10 to 24 days, whereas collagen content increased significantly. These data suggest that PEGT:PBT scaffolds are suitable for cartilage tissue engineering, with the PT process enabling greater chondrogenesis than CM.     

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.     

Chitosan/polyester-based scaffolds for cartilage tissue engineering: Assessment of extracellular matrix formation

Naturally derived polymers have been extensively used in scaffold production for cartilage tissue engineering. The present work aims to evaluate and characterize extracellular matrix (ECM) formation in two types of chitosan-based scaffolds, using bovine articular chondrocytes (BACs). The influence of these scaffolds’ porosity, as well as pore size and geometry, on the formation of cartilagineous tissue was studied. The effect of stirred conditions on ECM formation was also assessed. Chitosan-poly(butylene succinate) (CPBS) scaffolds were produced by compression moulding and salt leaching, using a blend of 50% of each material. Different porosities and pore size structures were obtained. BACs were seeded onto CPBS scaffolds using spinner flasks. Constructs were then transferred to the incubator, where half were cultured under stirred conditions, and the other half under static conditions for 4 weeks. Constructs were characterized by scanning electron microscopy, histology procedures, immunolocalization of collagen type I and collagen type II, and dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Both materials showed good affinity for cell attachment. Cells colonized the entire scaffolds and were able to produce ECM. Large pores with random geometry improved proteoglycans and collagen type II production. However, that structure has the opposite effect on GAG production. Stirred culture conditions indicate enhancement of GAG production in both types of scaffold.     

Enhancement of neurite outgrowth using nano-structured scaffolds coupled with laminin

Cell interactions with scaffolds are important for cell and tissue development in the process of repairing and regeneration of damaged tissue. Scaffolds that mimic extracellular matrix (ECM) surface topography, mechanical stiffness, and chemical composition will be advantageous to promote enhanced cell interactions. Electrospinning can easily produce nano-structured synthetic polymer mats with architecture that structurally resembles the ECM of tissue. Although electrospinning can produce sub-micron fibrous scaffolds, modification of electrospun scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation and differentiation. Incorporation of laminin, a neurite promoting ECM protein, onto the nanofibers is an alternative to further mimic the biochemical properties of the nervous tissue to create a biomimetic scaffold. In this study, we investigated the feasibility to functionalize scaffolds by coupling laminin onto poly(L-lactic acid) (PLLA) nanofibers. Laminin was successfully added to nanofibers using covalent binding, physical adsorption or blended electrospinning procedures. PC12 cell viability and neurite outgrowth assays confirmed that the functionalized nanofibers were able to enhance axonal extensions. Significantly, compared to covalent immobilization and physical adsorption, blended electrospinning of laminin and synthetic polymer is a facile and efficient method to modify nanofibers for the fabrication of a biomimetic scaffold. Using these functionalization techniques, nanofibers can be effectively modified with laminin for potential use in peripheral nerve regeneration applications.     

Engineering of fibrillar decorin matrices for a tissue-engineered trachea

Decorin is a structural and functional proteoglycan (PG) residing in the complex network of extracellular matrix (ECM) proteins in many connective tissues. Depending on the protein core and the glycosaminoglycan chain, PGs support cell adhesion, migration, proliferation, differentiation, ECM assembly and growth factor binding. For applications in tissue engineering, it is crucial to develop reliable, ECM-mimicking biomaterials. Electrospinning is a suitable method for creating three-dimensional (3D), fibrillar scaffolds. While there are numerous reports on the electrospinning of proteins including collagen, to date, there are no reports on the electrospinning of PGs. In the following study, we used electrospinning to generate decorin-containing matrices for tracheal tissue engineering applications. The electrospun scaffolds were analyzed using scanning electron microscopy, atomic force microscopy, contact angle measurements and dynamic mechanical analysis. Additionally, we confirmed PG functionality with immunostaining and 1,9-dimethylmethylene blue. To determine cell-matrix-interactions, tracheal cells (hPAECs) were seeded and analyzed using an FOXJ1-antibody. Moreover, interactions of the electrospun scaffolds with immune-mediated mechanisms were analyzed in detail. To conclude, we demonstrated the feasibility of electrospinning of decorin to generate functional 3D scaffolds with low immunogenicity for hPAEC expansion. Our data suggest that these hybrid materials may be suitable as a substrate for tracheal tissue engineering.     

In vitro chondrocyte behavior on porous biodegradable poly (e-caprolactone)/polyglycolic acid scaffolds for articular chondrocyte adhesion and proliferation

In this study, poly(e-caprolactone)/polyglycolic acid (PCL/PGA) scaffolds for repairing articular cartilage were fabricated via solid-state cryomilling along with compression molding and porogen leaching. Four distinct scaffolds were fabricated using this approach by four independent cryomilling times. These scaffolds were assessed for their suitability to promote articular cartilage regeneration with in vitro chondrocyte cell culture studies. The scaffolds were characterized for pore size, porosity, swelling ratio, compressive, and thermal properties. Cryomilling time proved to significantly affect the physical, mechanical, and morphological properties of the scaffolds. In vitro bovine chondrocyte culture was performed dynamically for 1, 7, 14, 28, and 35 days. Chondrocyte viability and adhesion were tested using MTT assay and scanning electron microscopy micrographs. Glycosaminoglycan (GAG) and DNA assays were performed to investigate the extracellular matrix (ECM) formation and cell proliferation, respectively. PCL/PGA scaffolds demonstrated high porosity for all scaffold types. Morphological analysis and poly(ethylene oxide) continuity demonstrated the existence of a co-continuous network of interconnected pores with pore sizes appropriate for tissue engineering and chondrocyte ingrowth. While mean pore size decreased, water uptake and compressive properties increased with increasing cryomilling times. Compressive modulus of 12, 30, and 60 min scaffolds matched the compressive modulus of human articular cartilage. Viable cells increased besides increase in cell proliferation and ECM formation with progress in culture period. Chondrocytes exhibited spherical morphology on all scaffold types. The pore size of the scaffold affected chondrocyte adhesion, proliferation, and GAG secretion. The results indicated that the 12 min scaffolds delivered promising results for applications in articular cartilage repair.     

Composite chondroitin-6-sulfate/dermatan sulfate/chitosan scaffolds for cartilage tissue engineering

Conjugating a single glycosaminoglycan (GAG) species such as chondroitin-6-sulfate (CSC) to chitosan is beneficial to chondrocyte culture and extracellular matrix (ECM) production, but whether fabrication of 3D chitosan scaffolds with additional minor GAG species such as dermatan sulfate (DS) further improves the ECM production is unknown. In this study, Response Surface Methodology (RSM) was employed to design CSC/DS/chitosan scaffolds of various formulations for cartilage engineering and to investigate the roles of individual GAG species in cartilage formation. The CSC/DS formulation affected neither the physical properties of scaffolds nor cell adhesion, but influenced cell morphology, GAGs and collagen production and chondrocytic gene expression. The linear effects elucidated by RSM analysis suggested that within the level range higher CSC levels favored GAGs and collagen production, whereas lower DS levels were desired for these responses. Nonetheless, the quadratic effects of DS and two-way interactions between CSC and DS also contributed to the GAGs and collagen production. Accordingly, the optimal formulation, as predicted by RSM and validated by experiments, comprised 2.8 mg CSC and 0.01 mg DS per scaffold. This study confirmed the importance of DS in cartilage tissue engineering and implicated the feasibility of rational CSC/DS/chitosan scaffold design with the aid of RSM.     

3D fiber-deposited scaffolds for tissue engineering: influence of pores geometry and architecture on dynamic mechanical properties

One of the main issues in tissue engineering is the fabrication of scaffolds that closely mimic the biomechanical properties of the tissues to be regenerated. Conventional fabrication techniques are not sufficiently suitable to control scaffold structure to modulate mechanical properties. Within novel scaffold fabrication processes 3D fiber deposition (3DF) showed great potential for tissue engineering applications because of the precision in making reproducible 3D scaffolds, characterized by 100% interconnected pores with different shapes and sizes. Evidently, these features also affect mechanical properties. Therefore, in this study we considered the influence of different structures on dynamic mechanical properties of 3DF scaffolds. Pores were varied in size and shape, by changing fibre diameter, spacing and orientation, and layer thickness. With increasing porosity, dynamic mechanical analysis (DMA) revealed a decrease in elastic properties such as dynamic stiffness and equilibrium modulus, and an increase of the viscous parameters like damping factor and creep unrecovered strain. Furthermore, the Poisson's ratio was measured, and the shear modulus computed from it. Scaffolds showed an adaptable degree of compressibility between sponges and incompressible materials. As comparison, bovine cartilage was tested and its properties fell in the fabricated scaffolds range. This investigation showed that viscoelastic properties of 3DF scaffolds could be modulated to accomplish mechanical requirements for tailored tissue engineered applications.     

The effect of source animal age upon the in vivo remodeling characteristics of an extracellular matrix scaffold

Biologic scaffolds composed of mammalian extracellular matrix (ECM) are routinely used for the repair and reconstruction of injured or missing tissues in a variety of pre-clinical and clinical applications. However, the structural and functional outcomes have varied considerably. An important variable of xenogeneic biologic scaffolds is the age of the animal from which the ECM is derived. The present study compared the in?vivo host response and remodeling outcomes of biologic scaffolds composed of small intestinal submucosa (SIS)-ECM harvested from pigs that differed only in age. Results showed that there are distinct differences in the remodeling characteristics as a consequence of source animal age. Scaffolds derived from younger animals were associated with a more constructive, site appropriate, tissue remodeling response than scaffolds derived from older animals. Furthermore, the constructive remodeling response was associated with a dominant M2 macrophage response.     

Bone response to 3D periodic hydroxyapatite scaffolds with and without tailored microporosity to deliver bone morphogenetic protein 2

Three types of model hydroxyapatite (HA) scaffolds were implanted in the metacarpal and metatarsal bones of goats. Scaffolds, consisting of a latticed pattern of rods, were fabricated with a solid freeform fabrication (SFF) technique. All scaffolds contained macropores; some were also fabricated with micropores (5.2 +/- 2.0 microm). Recombinant human bone morphogenetic protein-2 (rhBMP-2) was added to some microporous scaffolds. rhBMP-2 caused increased percent filled with bone tissue compared to microporous scaffolds without rhBMP-2. Lamellar bone in the scaffolds was aligned perpendicular to the long axis of the bone near the junctions of the rods that make up the scaffold but was more random away from the junctions of rods. Microporous scaffolds stained beneath areas of contact with new bone. This staining might indicate either extracellular matrix (ECM) in the rods, byproducts of ECM production, or reaction of cellular products with the scaffold.     

Effects of scaffold composition and architecture on human nasal chondrocyte redifferentiation and cartilaginous matrix deposition

We investigated whether the post-expansion redifferentiation and cartilage tissue formation capacity of adult human nasal chondrocytes can be regulated by controlled modifications of scaffold composition and architecture. As a model system, we used poly(ethylene glycol)-terephthalate-poly(butylene)-terephthalate block copolymer scaffolds from two compositions (low or high PEG content, resulting in different wettability) and two architectures (generated by compression molding or three-dimensional (3D) fiber deposition) with similar porosity and mechanical properties, but different interconnecting pore architectures. Scaffolds were seeded with expanded human chondrocytes and the resulting constructs assessed immunohistochemically, biochemically and at the mRNA expression level following up to 4 weeks of static culture. For a given 3D architecture, the more hydrophilic scaffold enhanced cell redifferentiation and cartilaginous tissue formation after 4 weeks culture, as assessed by higher mRNA expression of collagen type II, increased deposition of glycosaminoglycan (GAG) and predominance of type II over type I collagen immunostain. The fiber-deposited scaffolds, with a more accessible pore volume and larger interconnecting pores, supported increased GAG deposition, but only if a more hydrophilic composition was used. By applying controlled and selective modifications of chemico-physical scaffold parameters, we demonstrate that both scaffold composition and architecture are instructive for expanded human chondrocytes in the generation of 3D cartilaginous tissues. The observed effects of composition and architecture were likely to have been mediated, respectively, by differential serum protein adsorption and efficiency of nutrient/waste exchange.     

The performance of human dental pulp stem cells on different three-dimensional scaffold materials

The aim of this study was to investigate the in vitro and in vivo behavior of human dental pulp stem cells (DPSCs) isolated from impacted third molars, when seeded onto different 3-dimensional (3-D) scaffold materials: i.e. a spongeous collagen, a porous ceramic, and a fibrous titanium mesh. Scaffolds were loaded with DPSC, and subsequently divided into two groups. The first group was cultured in osteogenic differentiation medium in vitro for 4 weeks. The second group of samples was implanted subcutaneously in nude mice for 6 or 12 weeks. Samples cultured in vitro were analyzed by scanning electron microscopy and RT-PCR for dentin sialophosphoprotein (DSPP) expression. In vivo samples were evaluated by histology, RT-PCR and immunohistochemistry. The results indicated that in vitro, cells developed abundant deposition of mineralized extracellular matrix (ECM) with expression of DSPP in all 3-D materials. The simultaneous implantation experiment showed formation of tissue that was DSPP positive in all three scaffolds materials. However, the aspect of the formed tissues in all scaffolds resembled more connective tissue than a dentin-like tissue. Limited calcification of the ECM was only seen in the ceramic scaffold. In both experiments, no other differences could be attributed to the different materials used. In conclusion, the in vivo behavior of DPSC and their relations with 3-D scaffold materials should be further studied before clinical use can be considered.     

Facile surface functionalization with glycosaminoglycans by direct coating with mussel adhesive protein

The use of mussel adhesive proteins (MAPs) as a surface coating for cell adhesion has been suggested due to their unique properties of biocompatibility and effective adhesion on diverse inorganic and organic surfaces. The surface functionalization of scaffolds or implants using extracellular matrix (ECM) molecules is important for the enhancement of target cell behaviors such as proliferation and differentiation. In the present work, we suggest a new, simple surface functionalization platform based on the charge interactions between the positively charged MAP linker and negatively charged ECM molecules, such as glycosaminoglycans (GAGs). MAP was efficiently coated onto a titanium model surface using its adhesion ability. Then, several GAG molecules, including hyaluronic acid (HA), heparin sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS), were effectively immobilized on the MAP-coated surfaces by charge interactions. Using HA as a model GAG molecule, we found that the proliferation, spreading, and differentiation behaviors of mouse preosteoblast cells were all significantly improved on MAP/HA-layered titanium. In addition, we successfully constructed a multilayer film on a titanium surface with oppositely charged layer-by-layer coatings of MAP and HA. Collectively, our simple MAP-based surface functionalization strategy can be successfully used for the efficient surface immobilization of negatively charged ECM molecules in various tissue engineering and medical implantation applications.     

Biologic scaffolds composed of central nervous system extracellular matrix

Acellular biologic scaffolds are commonly used to facilitate the constructive remodeling of three of the four traditional tissue types: connective, epithelial, and muscle tissues. However, the application of extracellular matrix (ECM) scaffolds to neural tissue has been limited, particularly in the central nervous system (CNS) where intrinsic regenerative potential is low. The ability of decellularized liver, lung, muscle, and other tissues to support tissue-specific cell phenotype and function suggests that CNS-derived biologic scaffolds may help to overcome barriers to mammalian CNS repair. A method was developed to create CNS ECM scaffolds from porcine optic nerve, spinal cord, and brain, with decellularization verified against established criteria. CNS ECM scaffolds retained neurosupportive proteins and growth factors and, when tested with the PC12 cell line in vitro, were cytocompatible and stimulated proliferation, migration, and differentiation. Urinary bladder ECM (a non-CNS ECM scaffold) was also cytocompatible and stimulated PC12 proliferation but inhibited migration rather than acting as a chemoattractant over the same concentration range while inducing greater rates of PC12 differentiation compared to CNS ECM. These results suggest that CNS ECM may provide tissue-specific advantages in CNS regenerative medicine applications and that ECM scaffolds in general may aid functional recovery after CNS injury.     

Osteoblast activity on collagen-GAG scaffolds is affected by collagen and GAG concentrations

Optimization of a tissue engineering scaffold for use in bone tissue engineering requires control of many factors such as pore size, porosity, permeability and, as this study shows, the composition of the matrix. The collagen-glycosaminoglycan (GAG) scaffold variants were fabricated by varying the collagen and GAG content of the scaffold. Scaffolds were seeded with MC3T3 osteoblasts and cultured for up to 7 days. During the culture period, osteoblastic activity was evaluated by measuring metabolic activity, cell number, and spatial distribution. Collagen and GAG concentrations both affected osteoblast viability, proliferation, and spatial distribution within the scaffold. Scaffolds containing 1% collagen (w/v) and 0.088% GAG (w/v) were found to have a porosity of approximately 99%, high cell metabolic activity and cell number, and good cell infiltration over the 7 days in culture. Taken together, these results indicate the need to tailor the parameters of a biological substrate for use in a specific tissue application, in this case bone tissue engineering.     

Hydrogel-filled polylactide porous scaffolds for cartilage tissue engineering

Polymer porous scaffolds and hydrogels have been separately employed as analogues of the native extra-cellular matrix (ECM). However, both of these two kinds of materials have their own advantages and shortcomings. In this work, an attempt to combine the advantages of these two kinds of materials is carried out. Poly-L-lactide (PLLA) scaffolds with good mechanical properties were prepared by thermally induced phase separation, which were then filled with hydrogel aiming at entrapment of cells within a support of predefined shape. Agar, which has a function to promote chondrogenesis, was selected to entrap chondrocytes, acting as analogues of native ECM. A straight forward merit of this construct is that both mechanical strength and macroscopic shape, and analogous ECM can be simultaneously achieved. The morphology and distribution of the chondrocytes were studied by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The cell growth behaviors were determined by MTT assay and collagen and glycosaminoglycan (GAG) secretion. After culture for 7 and 14 days, the cells in the construct were round and surrounded by the hydrogel. The MTT viability and the cell secretion in the chondrocytes/agar/scaffold construct were also higher than that of the chondrocytes/scaffold construct (control). Gelatin was further introduced into the construct, yielding improved GAG secretion and cytoviability. After implantation in the subcutaneous dorsum of nude mice for 4 weeks, cartilage-like specimens maintaining their original rectangular shapes were harvested. Histological examination showed that new cartilage was regenerated and a large quantity of collagen and GAG were secreted, while the cells in the control PLLA scaffold turned to be fibroblast-like with less secretion of extracellular matrices. The method provides a useful pathway of scaffold preparation and cell transplantation, which can achieve suitable mechanical properties and good cell performance simultaneously.