Crosslinking Density Influences Chondrocyte Metabolism in Dynamically Loaded Photocrosslinked Poly(ethylene glycol) Hydrogels

In approaches to tissue engineer articular cartilage, an important consideration for in situ forming cell carriers is the impact of mechanical loading on the cell composite structure and function. Photopolymerized hydrogel scaffolds based on poly(ethylene glycol) (PEG) may be synthesized with a range of crosslinking densities and corresponding macroscopic properties. This study tests the hypothesis that changes in the hydrogel crosslinking density influences the metabolic response of encapsulated chondrocytes to an applied load. PEG hydrogels were formulated with two crosslinking densities that resulted in gel compressive moduli ranging from 60 to 670 kPa. When chondrocytes were encapsulated in these PEG gels, an increase in crosslinking density resulted in an inhibition in cell proliferation and proteoglycan synthesis. Moreover, when the gels were dynamically loaded for 48 h in unconfined compression with compressive strains oscillating from 0 to 15% at a frequency of 1 Hz, cell proliferation and proteoglycan synthesis were affected in a crosslinking-density-dependent manner. Cell proliferation was inhibited in both crosslinked gels, but was greater in the highly crosslinked gel. In contrast, dynamic loading did not influence proteoglycan synthesis in the loosely crosslinked gel, but a marked decrease in proteoglycan production was observed in the highly crosslinked gel. In summary, changes in PEG hydrogel properties greatly affect how chondrocytes respond to an applied dynamic load.     

Cell–matrix interactions and dynamic mechanical loading influence chondrocyte gene expression and bioactivity in PEG-RGD hydrogels

The pericellular matrix (PCM) surrounding chondrocytes is thought to play an important role in transmitting biochemical and biomechanical signals to the cells, which regulates many cellular functions including tissue homeostasis. To better understand chondrocytes interactions with their PCM, three-dimensional poly(ethylene glycol) (PEG) hydrogels containing Arg–Gly–Asp (RGD), the cell-adhesion sequence found in fibronectin and which is present in the PCM of cartilage, were employed. RGD was incorporated into PEG hydrogels via tethers at 0.1, 0.4 and 0.8 mM concentrations. Bovine chondrocytes were encapsulated in the hydrogels and subjected to dynamic compressive strains (0.3 Hz, 18% amplitude strain) for 48 h, and their response assessed by cell morphology, ECM gene expression, cell proliferation and matrix synthesis. Incorporation of RGD did not influence cell morphology under free swelling conditions. However, the level of cell deformation upon an applied strain was greater in the presence of RGD. In the absence of dynamic loading, RGD appears to have a negative effect on chondrocyte phenotype, as seen by a 4.7-fold decrease in collagen II/collagen I expressions in 0.8 mM RGD constructs. However, RGD had little effect on early responses of chondrocytes (i.e. cell proliferation and matrix synthesis/deposition). When isolating RGD as a biomechanical cue, cellular response was very different. Chondrocyte phenotype (collagen II/collagen I ratio) and proteoglycan synthesis were enhanced with higher concentrations of RGD. Overall, our findings demonstrate that RGD ligands enhance cartilage-specific gene expression and matrix synthesis, but only when mechanically stimulated, suggesting that cell–matrix interactions mediate chondrocyte response to mechanical stimulation.     

Temporomandibular joint formation and condyle growth require Indian hedgehog signaling.

The temporomandibular joint (TMJ) is essential for jaw function, but the mechanisms regulating its development remain poorly understood. Because Indian hedgehog (Ihh) regulates trunk and limb skeletogenesis, we studied its possible roles in TMJ development. In wild-type mouse embryos, Ihh expression was already strong in condylar cartilage by embryonic day (E) 15.5, and expression of Ihh receptors and effector genes (Gli1, Gli2, Gli3, and PTHrP) indicated that Ihh range of action normally reached apical condylar tissue layers, including polymorphic chondroprogenitor layer and articular disc primordia. In Ihh(-/-) embryos, TMJ development was severely compromised. Condylar cartilage growth, polymorphic cell proliferation, and PTHrP expression were all inhibited, and growth plate organization and chondrocyte gene expression patterns were abnormal. These severe defects were partially corrected in double Ihh(-/-)/Gli3(-/-) mutants, signifying that Ihh action is normally modulated and delimited by Gli3 and Gli3(R) in particular. Both single and double mutants, however, failed to form an articular disc primordium, normally appreciable as an independent condensation between condylar apex and neighboring developing temporal bone in wild-type. This failure persisted at later stages, leading to complete absence of a normal functional disc and lubricin-expressing joint cavities. In summary, Ihh is very important for TMJ development, where it appears to regulate growth and elongation events, condylar cartilage phenotype, and chondroprogenitor cell function. Absence of articular disc and joint cavities in single and double mutants points to irreplaceable Ihh roles in formation of those critical TMJ components.     

A Paradigm for Functional Tissue Engineering of Articular Cartilage via Applied Physiologic Deformational Loading

Deformational loading represents a primary component of the chondrocyte physical environment in vivo. This review summarizes our experience with physiologic deformational loading of chondrocyte-seeded agarose hydrogels to promote development of cartilage constructs having mechanical properties matching that of the parent calf tissue, which has a Young's modulus E(Y) = 277 kPa and unconfined dynamic modulus at 1 Hz G* = 7 MPa. Over an 8-week culture period, cartilage-like properties have been achieved for 60 x 10(6) cells/ml seeding density agarose constructs, with E(Y) = 186 kPa, G* = 1.64 MPa. For these constructs, the GAG content reached 1.74% ww and collagen content 2.64% ww compared to 2.4% ww and 21.5% ww for the parent tissue, respectively. Issues regarding the deformational loading protocol, cell-seeding density, nutrient supply, growth factor addition, and construct mechanical characterization are discussed. In anticipation of cartilage repair studies, we also describe early efforts to engineer cylindrical and anatomically shaped bilayered constructs of agarose hydrogel and bone (i.e., osteochondral constructs). The presence of a bony substrate may facilitate integration upon implantation. These efforts will provide an underlying framework from which a functional tissue-engineering approach, as described by Butler and coworkers (2000), may be applied to general cell-scaffold systems adopted for cartilage tissue engineering.     

Novel poly(ethylene glycol) scaffolds crosslinked by hydrolyzable polyrotaxane for cartilage tissue engineering

Highly porous poly(ethylene glycol) (PEG) hydrogel scaffolds crosslinked with hydrolyzable polyrotaxane for cartilage tissue engineering were prepared by a solvent casting/salt leaching technique. The resultant scaffolds have well interconnected microporous structures ranging from 87 to 90%. Pore sizes ranging from 115.5-220.9 microm appeared to be dependent on the size of the sieved sodium chloride particulates. Moreover, a dense surface skin layer was not found on either side of the scaffold surfaces. Using microscopic Alcian blue staining of the chondrocyte-seeded scaffolds, well adhered cells and newly produced glycosaminoglycans (GAG) were confirmed. Following the initial chondrocyte seeding onto the hydrogel scaffolds, the cell number was significantly increased, reaching 149, 877, and 1228 cells/mg of tissue at 8, 15, and 21 days in culture, respectively. The micrograph shows well adhered and spread chondrocytes in the interior pores and a cartilaginous extracellular matrix with a GAG fraction produced from the chondrocytes. Results suggest that the PEG hydrogel scaffolds crosslinked with the hydrolyzable polyrotaxane are a promising candidate for chondrocyte culture.     

A comparison of human umbilical cord matrix stem cells and temporomandibular joint condylar chondrocytes for tissue engineering temporomandibular joint condylar cartilage

The temporomandibular joint (TMJ) presents many problems in modern musculoskeletal medicine. Patients who suffer from TMJ disorders often experience a major loss in quality of life due to the debilitating effects that TMJ disorders can have on everyday activities. Cartilage tissue engineering can lead to replacement tissues that could be used to treat TMJ disorders. In this study, a spinner flask was used for a period of 6 days to seed polyglycolic acid (PGA) scaffolds with either TMJ condylar chondrocytes or mesenchymal-like stem cells derived from human umbilical cord matrix (HUCM). Samples were then statically cultured for 4 weeks either in growth medium containing chondrogenic factors or in control medium. Immunohistochemical staining of HUCM constructs after 4 weeks revealed a strong presence of collagen I and minute amounts of collagen II, whereas TMJ constructs revealed little collagen I and no collagen II. The HUCM constructs were shown to contain more GAGs than the TMJ constructs quantitatively at week 0 and histologically at week 4. Moreover, the cellularity of HUCM constructs was 55% higher at week 0 and nearly twice as high after 4 weeks, despite being seeded at the same density. The increased level of biosynthesis and higher cellularity of HUCM constructs clearly demonstrates that the HUCM stem cells outperformed the TMJ condylar cartilage cells under the prescribed conditions. HUCM stem cells may therefore be an attractive alternative to condylar cartilage cells for TMJ tissue engineering applications. Further, given the availability and ease of obtaining HUCM stem cells, these findings may have far-reaching implications, leading to novel developments in both craniofacial and orthopaedic tissue replacement therapies.     

Transient exposure to transforming growth factor beta 3 under serum-free conditions enhances the biomechanical and biochemical maturation of tissue-engineered cartilage

A goal of cartilage tissue engineering is the production of cell-laden constructs possessing sufficient mechanical and biochemical features to enable native tissue function. This study details a systematic characterization of a serum-free (SF) culture methodology employing transient growth factor supplementation to promote robust maturation of tissue-engineered cartilage. Bovine chondrocyte agarose hydrogel constructs were cultured under free-swelling conditions in serum-containing or SF medium supplemented continuously or transiently with varying doses of transforming growth factor beta 3 (TGF-beta3). Constructs were harvested weekly or bi-weekly and assessed for mechanical and biochemical properties. Transient exposure (2 weeks) to low concentrations (2.5-5 ng/mL) of TGF-beta3 in chemically defined medium facilitated robust and highly reproducible construct maturation. Constructs receiving transient TGF-beta3 exposure achieved native tissue levels of compressive modulus (0.8 MPa) and proteoglycan content (6-7% of wet weight) after less than 2 months of in vitro culture. This maturation response was far superior to that observed after continuous growth factor supplementation or transient TGF-beta3 treatment in the presence of serum. These findings represent a significant advance in developing an ex vivo culture methodology to promote production of clinically relevant and mechanically competent tissue-engineered cartilage constructs for implantation to repair damaged articular surfaces.     

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 the mandibular condyle

Tissue engineering provides the revolutionary possibility for curing temporomandibular joint (TMJ) disorders. Although characterization of the mandibular condyle has been extensively studied, tissue engineering of the mandibular condyle is still in an inchoate stage. The purpose of this review is to provide a summary of advances relevant to tissue engineering of mandibular cartilage and bone, and to serve as a reference for future research in this field. A concise anatomical overview of the mandibular condyle is provided, and the structure and function of the mandibular condyle are reviewed, including the cell types, extracellular matrix (ECM) composition, and biomechanical properties. Collagens and proteoglycans are distributed heterogeneously (topographically and zonally). The complexity of collagen types (including types I, II, III, and X) and cell types (including fibroblast-like cells, mesenchymal cells, and differentiated chondrocytes) indicates that mandibular cartilage is an intermediate between fibrocartilage and hyaline cartilage. The fibrocartilaginous fibrous zone at the surface is separated from hyaline-like mature and hypertrophic zones below by a thin and highly cellular proliferative zone. Mechanically, the mandibular condylar cartilage is anisotropic under tension (stiffer anteroposteriorly) and heterogeneous under compression (anterior region stiffer than posterior). Tissue engineering of mandibular condylar cartilage and bone is reviewed, consisting of cell culture, growth factors, scaffolds, and bioreactors. Ideal engineered constructs for mandibular condyle regeneration must involve two distinct yet integrated stratified layers in a single osteochondral construct to meet the different demands for the regeneration of cartilage and bone tissues. We conclude this review with a brief discussion of tissue engineering strategies, along with future directions for tissue engineering the mandibular condyle.     

Effect of low oxygen tension on tissue-engineered cartilage construct development in the concentric cylinder bioreactor

Cartilage is exposed to low oxygen tension in vivo, suggesting culture in a low-oxygen environment as a strategy to enhance matrix deposition in tissue-engineered cartilage in vitro. To assess the effects of oxygen tension on cartilage matrix accumulation, porous polylactic acid constructs were dynamically seeded in a concentric cylinder bioreactor with bovine chondrocytes and cultured for 3 weeks at either 20 or 5% oxygen tension. Robust chondrocyte proliferation and matrix deposition were achieved. After 22 days in culture, constructs from bioreactors operated at either 20 or 5% oxygen saturation had similar chondrocyte densities and collagen content. During the first 12 days of culture, the matrix glycosaminoglycan (GAG) deposition rate was 19.5 x 10(-9) mg/cell per day at 5% oxygen tension and 65% greater than the matrix GAG deposition rate at 20% oxygen tension. After 22 days of bioreactor culture, constructs at 5% oxygen contained 4.5 +/- 0.3 mg of GAG per construct, nearly double the 2.5 +/- 0.2 mg of GAG per construct at 20% oxygen tension. These data demonstrate that culture in bioreactors at low oxygen tension favors the production and retention of GAG within cartilage matrix without adversely affecting chondrocyte proliferation or collagen deposition. Bioreactor studies such as these can identify conditions that enhance matrix accumulation and construct development for cartilage tissue engineering.     

Differential behavior of auricular and articular chondrocytes in hyaluronic acid hydrogels

Chondrocytes isolated from a variety of sources, including auricular (AU) and articular (AR) cartilage, can differ in cell behavior, growth, and extracellular matrix (ECM) production, which can impact neocartilage properties in tissue engineering approaches. This behavior is also affected by the surrounding microenvironment, including soluble factors, biomaterials, and mechanical loading. The objective of this study was to investigate differences in juvenile AU and AR chondrocyte behavior when encapsulated in radically polymerized hyaluronic acid hydrogels. When implanted in vivo, differences in macroscopic appearance, mechanical properties, glycosaminoglycan content, and collagen content were observed depending on the chondrocyte type encapsulated. Specifically, AU constructs exhibited construct growth and neocartilage formation with increases in aggregate modulus and ECM accumulation with culture, whereas AR constructs retained their construct size and remained translucent with only a minimal increase in the compressive modulus. When cultured in vitro, both cell types remained viable and differences in gene expression were observed for type I and II collagens. Likewise, differences in gene expression were noted after dynamic mechanical loading, where stimulated AR constructs exhibited 2.3- and 1.5-fold increases in type II collagen and aggrecan over free-swelling controls, while AU samples exhibited smaller fold increases of 1.4- and 1.3-fold, respectively. Thus, these data indicate that the specific cell source, cell/material interactions, and loading environment are important in the final properties of tissue-engineered products.     

Cotransplantation of autologous bone marrow stromal cells and chondrocytes as a novel therapy for reconstruction of condylar cartilage

Condylar cartilage is absolutely necessary for the normal function of temporomandibular joint (TMJ). Unfortunately, condylar cartilage defect or missing is also one of the common clinical problems. Repair or reconstruction of cartilage is always a hot topic. Cell based cartilage regeneration is suggested as novel therapies in cartilage tissue engineering, and autologous chondrocytes were initially regarded as the ideal cell source. However, there are some disadvantages such as its limited augmentation capability for culture in vitro and may differentiate to other types of cells. On the other hand, bone marrow stromal cells (BMSCs) have gained special interest in tissue engineering. Because they can be obtained easily, cause relatively minor trauma and show the potential of long-run ex vivo expansion capacity. What most important is their capacity of multi-directional differentiation. They can differentiate into a variety of other types of cells when there are supplement exogenous factors or genes, but their clinical use is limited by safety concerns such as toxicity, insertional teratogenic, uncontrollable gene expression. Fortunately, the chondrocytes microenvironment has been demonstrated that could induce BMSCs to structure cartilage when culture in vitro or reimplanted in nude mice subcutaneously area. So in this article, we hypothesize that cotransplantation of autologous BMSCs and chondrocytes, which coculture with extracellular scaffolds, is a novel therapy for reconstruction of TMJ condylar cartilage. In our strategy, advantages of two types of cells are utilized and shortcomings are avoided, which strongly improve the feasibility and clinical safety, finally bring great hope to the patients with TMJ disease.     

Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel

Tissue engineering strategies, based on developing three-dimensional scaffolds capable of transferring autologous chondrogenic cells, holds promise for the restoration of damaged cartilage. In this study, the authors aimed at determining whether a recently developed silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel can be a suitable scaffold for human nasal chondrocytes (HNC)-based cartilage engineering. Methyltetrazolium salt assay and cell counting experiments first revealed that Si-HPMC enabled the proliferation of HNC. Cell tracker green staining further demonstrated that HNC were able to form nodular structures in this three-dimensional scaffold. HNC phenotype was then assessed by RT-PCR analysis of type II collagen and aggrecan expression as well as alcian blue staining of extracellular matrix. Our data indicated that Si-HPMC allowed the maintenance and the recovery of a chondrocytic phenotype. The ability of constructs HNC/Si-HPMC to form a cartilaginous tissue in vivo was finally investigated after 3 weeks of implantation in subcutaneous pockets of nude mice. Histological examination of the engineered constructs revealed the formation of a cartilage-like tissue with an extracellular matrix containing glycosaminoglycans and type II collagen. The whole of these results demonstrate that Si-HPMC hydrogel associated to HNC is a convenient approach for cartilage tissue engineering.     

Delivery of TGF-beta1 and chondrocytes via injectable, biodegradable hydrogels for cartilage tissue engineering applications

In this work, novel hydrogel composites, based on the biodegradable polymer, oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) were utilized as injectable cell and growth factor carriers for cartilage tissue engineering applications. Specifically, bovine chondrocytes were embedded in composite hydrogels co-encapsulating gelatin MPs loaded with transforming growth factor-beta1 (TGF-beta1). Hydrogels with embedded cells co-encapsulating unloaded MPs and those with no MPs served as controls in order to assess the effects of MPs and TGF-beta1 on chondrocyte function. Samples were cultured up to 28 days in vitro. By 14 days, cell attachment to embedded gelatin MPs within the constructs was observed via light microscopy. Bioassay results showed that, over the 21 day period, there was a statistically significant increase in cellular proliferation for samples containing gelatin MPs, but no increase was exhibited in samples without MPs over the culture period. The release of TGF-beta1 further increased cell construct cellularity. Over the same time period, glycosaminoglycan content per cell remained constant for all formulations, suggesting that the dramatic increase in cell number for samples with TGF-beta1-loaded MPs was accompanied by maintenance of the cell phenotype. Overall, these data indicate the potential of OPF hydrogel composites containing embedded chondrocytes and TGF-beta1-loaded gelatin MPs as a novel strategy for cartilage tissue engineering.     

Dynamic compressive loading enhances cartilage matrix synthesis and distribution and suppresses hypertrophy in hMSC-laden hyaluronic acid hydrogels

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair, and there is growing evidence that mechanical signals play a critical role in the regulation of stem cell chondrogenesis and in cartilage development. In this study we investigated the effect of dynamic compressive loading on chondrogenesis, the production and distribution of cartilage specific matrix, and the hypertrophic differentiation of human MSCs encapsulated in hyaluronic acid (HA) hydrogels during long term culture. After 70 days of culture, dynamic compressive loading increased the mechanical properties, as well as the glycosaminoglycan (GAG) and collagen contents of HA hydrogel constructs in a seeding density dependent manner. The impact of loading on HA hydrogel construct properties was delayed when applied to lower density (20 million MSCs/ml) compared to higher seeding density (60 million MSCs/ml) constructs. Furthermore, loading promoted a more uniform spatial distribution of cartilage matrix in HA hydrogels with both seeding densities, leading to significantly improved mechanical properties as compared to free swelling constructs. Using a previously developed in vitro hypertrophy model, dynamic compressive loading was also shown to significantly reduce the expression of hypertrophic markers by human MSCs and to suppress the degree of calcification in MSC-seeded HA hydrogels. Findings from this study highlight the importance of mechanical loading in stem cell based therapy for cartilage repair in improving neocartilage properties and in potentially maintaining the cartilage phenotype.     

Engineering endochondral bone: in vitro studies

Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification.     

The effects of intermittent hydrostatic pressure on self-assembled articular cartilage constructs

To date, static culture for the tissue engineering of articular cartilage has shown to be inadequate in conferring functionality to constructs. Various forms of mechanical stimuli accompany articular cartilage development in vivo, and one of these is hydrostatic pressure. This study used histology, biochemistry, and biomechanics to examine the effects of intermittent hydrostatic pressure, applied at 10 MPa and 1 Hz for 4 h per day for 5 days per week for up to 8 weeks on self-assembled chondrocyte constructs. The self-assembling process is a novel approach that allows engineering of articular cartilage constructs without the use of exogenous scaffolds. The self-assembled constructs were found to be capable of enduring this loading regimen. Significant increases in collagen production were only observed in pressurized samples. Intermittent hydrostatic pressure prevented a significant decrease in total GAG, which was significant in controls. Aside from the beneficial effects intermittent hydrostatic pressure may have on ECM synthesis, its effects on mechanical properties may require longer culture periods to manifest. This study demonstrates the successful use of the self-assembling process to produce articular cartilage constructs. It also shows for the first time that long-term culture of tissue-engineered articular cartilage construct benefits from intermittent hydrostatic pressure.     

Cell-nanofiber-based cartilage tissue engineering using improved cell seeding, growth factor, and bioreactor technologies

Biodegradable nanofibrous scaffolds serving as an extracellular matrix substitute have been shown to be applicable for cartilage tissue engineering. However, a key challenge in using nanofibrous scaffolds for tissue engineering is that the small pore size limits the infiltration of cells, which may result in uneven cell distribution throughout the scaffold. This study describes an effective method of chondrocyte loading into nanofibrous scaffolds, which combines cell seeding, mixing, and centrifugation to form homogeneous, packed cell-nanofiber composites (CNCs). When the effects of different growth factors are compared, CNCs cultured in medium containing a combination of insulin-like growth factor-1 and transforming growth factor-beta1 express the highest mRNA levels of collagen type II and aggrecan. Radiolabeling analyses confirm the effect on collagen and sulfated-glycosaminoglycans (sGAG) production. Histology reveals chondrocytes with typical morphology embedded in lacuna-like space throughout the entire structure of the CNC. Upon culturing using a rotary wall vessel bioreactor, CNCs develop into a smooth, glossy cartilage-like tissue, compared to a rough-surface tissue when maintained in a static environment. Bioreactor-grown cartilage constructs produce more total collagen and sGAG, resulting in greater gain in net tissue weight, as well as express cartilage-associated genes, including collagen types II and IX, cartilage oligomeric matrix protein, and aggrecan. In addition, dynamic culture enhances the mechanical property of the engineered cartilage. Taken together, these results indicate the applicability of nanofibrous scaffolds, combined with efficient cell loading and bioreactor technology, for cell-based cartilage tissue engineering.     

Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia

The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering.