Modulation of the proliferation and matrix synthesis of chondrocytes by dynamic compression on genipin-crosslinked chitosan/collagen scaffolds

Dynamic compression is an important physical stimulus for the physiology of chondrocyte and articular cartilage tissue engineering. In this study, modulation of chondrocyte behaviors in chitosan/collagen scaffolds with different mechanical properties under free-swelling or dynamic compression conditions was investigated. Rabbit chondrocytes were seeded in chitosan/collagen scaffolds crosslinked by genipin (GP) with different concentrations, and then cultured for 3?days prior to cyclic compression of 40% strain, 0.1?Hz, and 30?min/day for 2?weeks. The results showed that the cell proliferation was increased with increasing genipin concentrations and dynamic compression. On the other hand, although total glycosaminoglycans (GAGs) deposition was enhanced by dynamic compression under certain conditions, e.g. the GP0.5 chitosan/collagen scaffolds for 1?week of compression culture, normalized GAGs deposition per cell was decreased by dynamic compression. Our results suggest that while several studies suggest that dynamic compression benefits articular cartilage tissue engineering, many factors including scaffold types and compression conditions determine the outcome of dynamic compression culture.     

Alginate/lactose-modified chitosan hydrogels: a bioactive biomaterial for chondrocyte encapsulation

A new bioactive scaffold was prepared from a binary polysaccharide mixture composed of a polyanion (alginate) and a polycation (a lactose-modified chitosan, chitlac). Its potential use for articular chondrocytes encapsulation and cartilage reconstructive surgery applications has been studied. The hydrogel combines the ability of alginate to act as a 3D supporting structure with the capability of the second component (chitlac) to provide interactions with porcine articular chondrocytes. Physico-chemical characterization of the scaffold was accomplished by gel kinetics and compression measurements and demonstrated that alginate-chitlac mixture (AC-mixture) hydrogels exhibit better mechanical properties when compared with sole alginate hydrogels. Furthermore, biochemical and biological studies showed that these 3D scaffolds are able to maintain chondrocyte phenotype and particularly to significantly stimulate and promote chondrocyte growth and proliferation. In conclusion, the present study can be considered as a first step towards an engineered, biologically active scaffold for chondrocyte in vitro cultivation, expansion, and cell delivery.     

Mechanical properties and cellular proliferation of electrospun collagen type II

A suitable technique for articular cartilage repair and replacement is necessitated by inadequacies of current methods. Electrospinning has potential in cartilage repair by producing scaffolds with fiber diameters in the range of native extracellular matrix. Chondrocytes seeded onto such scaffolds may prefer this environment for differentiation and proliferation, thus approaching functional cartilage replacement tissue. Scaffolds of collagen type II were created by an electrospinning technique. Individual scaffold specimens were prepared and evaluated as uncross-linked, cross-linked, or crosslinked/seeded. Uncross-linked scaffolds contained a minimum and average fiber diameter of 70 and 496 nm, respectively, whereas cross-linked scaffolds possessed diameters of 140 nm and 1.46 microm. The average thickness for uncross-linked scaffolds was 0.20 +/- 0.02 mm and 0.52 +/- 0.07 mm for cross-linked scaffolds. Uniaxial tensile tests of uncross-linked scaffolds revealed an average tangent modulus, ultimate tensile strength, and ultimate strain of 172.5 +/- 36.1 MPa, 3.3 +/- 0.3 MPa, and 0.026 +/- 0.005 mm/mm, respectively. Scanning electron microscopy of cross-linked scaffolds cultured with chondrocytes demonstrated the ability of the cells to infiltrate the scaffold surface and interior. Electrospun collagen type II scaffolds produce a suitable environment for chondrocyte growth, which potentially establishes the foundation for the development of articular cartilage repair.     

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

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

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.     

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.     

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

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

Tissue-engineered polyethylene oxide/chitosan scaffolds as potential substitutes for articular cartilage

Applications of composite scaffolds comprising polyethylene oxide (PEO) and chitosan to the culture of bovine knee chondrocytes (BKC) were investigated. Here, PEO and chitosan with various weight ratios were crosslinked, refrigerated at -80 degrees C, and lyophilized. Pore surfaces of the PEO/chitosan scaffolds were chemically modified by human fibronectin for accelerating BKC adhesion and growth. The results revealed that the range of pore diameters was between 200 and 400 mum. A high content of PEO in scaffolds generated high porosity, moisture content, physical ductility, biodegradation rate, and BKC viability, as well as low Young's and compression moduli. High levels of PEO, human fibronectin, and extracellular calcium were favorable to the BKC culture, as indicated by the enhanced amounts of BKC, glycosaminoglycans, and collagen. However, a high concentration of medium potassium caused detrimental influences on the proliferation of BKC and the secretion of extracellular matrices. The present PEO/chitosan scaffolds showed enhancements in biomedical characteristics for the formation of tissue-engineered cartilage toward clinical prosthesis.     

Chitosan scaffolds: interconnective pore size and cartilage engineering

This study was designed to determine the effect of interconnective pore size on chondrocyte proliferation and function within chitosan sponges, and compare the potential of chitosan and polyglycolic acid (PGA) matrices for chondrogenesis. Six million porcine chondrocytes were seeded on each of 52 prewetted scaffolds consisting of chitosan sponges with (1) pores 10 microm in diameter (n=10, where n is the number of samples); (2) pores measuring 10-50 microm in diameter (n=10); and (3) pores measuring 70-120 microm in diameter (n=10), versus (4) polyglycolic acid mesh (n=22), as a positive control. Constructs were cultured for 28 days in a rotating bioreactor prior to scanning electron microscopy (SEM), histology, and determination of their water, DNA, glycosaminoglycan (GAG) and collagen II contents. Parametric data was compared (p=0.05) with an ANOVA and Tukey's Studentized range test. PGA constructs consisted essentially of a matrix containing more cells than normal cartilage. Whereas very few remnants of PGA remained, chitosan scaffolds appeared intact. DNA and GAG concentrations were greater in PGA scaffolds than in any of the chitosan groups. However, chitosan sponges with the largest pores contained more chondrocytes, collagen II and GAG than the matrix with the smallest pores. Constructs produced with PGA contained less water and more GAG than all chitosan groups. Chondrocyte proliferation and metabolic activity improved with increasing interconnective pore size of chitosan matrices. In vitro chondrogenesis is possible with chitosan but the composition of constructs produced on PGA more closely approaches that of natural cartilage.     

The differential effect of scaffold composition and architecture on chondrocyte response to mechanical stimulation

This study aims to explore the differential effect of scaffold composition and architecture on chondrogenic response to dynamic strain stimulation using encapsulating PEG-based hydrogels and primary bovine chondrocytes. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic materials to be used as scaffolds. Four different compositions were tested, including: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only. Primary articular chondrocytes were encapsulated in the hydrogel scaffolds and subjected to 15% dynamic compressive strain stimulation at 1-Hz frequency for 28 days. Stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the unconfined true compressive modulus by 32%, 45.4%, 33.6%, and 28.2%, compared to their static controls. The PF showed a significantly larger relative increase in the modulus in comparison to all other scaffolds tested. These results support the hypothesis that mechanical stimulation and material bioactivity have a significant effect on the reported chondrocyte response. Similar trends were observed with the swelling ratio of the constructs. These findings indicate that while stimulation causes metabolic changes in chondrocytes seeded in PEG hydrogels, the matrix bioactivity has a significant role in enhancing chondrocyte mechanotransduction in encapsulating scaffolds subjected to physical deformations.     

Biosynthetic response of passaged chondrocytes in a type II collagen scaffold to mechanical compression

To investigate the potential utility of mechanical loading in articular cartilage tissue engineering, porous type II collagen scaffolds seeded with adult canine passaged chondrocytes were subjected to static and dynamic compressions of varying magnitudes (0-50% static strain) and durations (1-24 h), and at different times during culture (2-30 days postseeding). The effects of mechanical compression on the biosynthetic activity of the chondrocytes were evaluated by measuring the amount of (3)H-proline-labeled proteins and (35)S-sulfate-labeled proteoglycans that accumulated in the cell-scaffold construct and was released to the medium during the loading period. Similar to published results on loading of articular cartilage explants, static compression decreased protein and proteoglycan biosynthesis in a time- and dose-dependent manner (each p < 0.005), and selected dynamic compression protocols were able to increase rates of biosynthesis (p < 0.05). The main difference between the results seen for this tissue engineering system and cartilage explants was in the amount of newly synthesized matrix molecules that accumulated within the construct under dynamic loading, with less accumulating in the type II collagen scaffold. In summary, the general biosynthetic response of passaged chondrocytes in the porous type II collagen scaffolds is similar to that seen for chondrocytes in their native environment. Future work needs to be directed to modifications of the cell-seeded construct to allow for the capture of the newly synthesized matrix molecules by the scaffold.     

Single-step mineralization of woodpile chitosan scaffolds with improved cell compatibility

A facile and efficient single-step mineralization approach was exploited for achieving nanoscopic hydroxyapatite (HAP) crystal layer in chitosan porous matrix, wherein a mixed water-ethanol solvent was used to control the growth of minerals. The crystallographic structure, morphology, and mechanical properties of the scaffold were analyzed with XRD, FTIR, environmental scanning electric microscopy (ESEM), TEM, and compression tests. The behaviors and responses of MC3T3-E1 pre-osteoblast cells on the scaffolds were studied as well. The results showed that the scaffolds kept woodpile structure with predefined and controlled hierarchical structure after mineralization. The inorganic phase in the mineralized chitosan scaffolds was determined as pure rod-like HAP, which settled densely on the matrix. The compression strength and compressive modulus of the scaffolds increased dramatically to 0.54 ± 0.005 MPa and 5.47 ± 0.65 MPa, respectively. During a culture period of 2 and 3 weeks, cell proliferation and in-growth were observed by phase contrast light microscopy and SEM. The alkaline phosphatase (ALP) activity increased after 1 week. Cell viability and cell proliferation index (PI) obtained higher values than that of the chitosan scaffolds. The novel single-step mineralization approach and the porous hybrid scaffolds would be a promising method for designing hybrid bone graft.     

Study on the three-dimensional proliferation of rabbit articular cartilage-derived chondrocytes on polyhydroxyalkanoate scaffolds

Polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx)/polyhydroxybutyrate (PHB) (PHBHHx/PHB) were investigated for possible application as a matrix for the three-dimensional growth of chondrocyte culture. Blend polymers of PHBHHx/PHB were fabricated into three-dimensional porous scaffolds by the salt-leaching method. Chondrocytes isolated from rabbit articular cartilage (RAC) were seeded on the scaffolds and incubated over 28 days, with change of the culture medium every 4 days. PHB scaffold was taken as a control. Methylthiazol tetrazolium (MTT) (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertra-zolium bromide) assay was used to quantitatively examine the proliferation of chondrocytes. Results showed that chondrocytes proliferated better on the PHBHHx/PHB scaffolds than on PHB one. The maximal cell densities were all observed after 7 days of incubation. As for the blend polymers, cells grew better on scaffolds consisting of PHBHHx/PHB in ratios of 2:1 and 1:2 than they did on PHBHHx/PHB of 1:1. Scanning electron microscopy (SEM) also showed that large quantities of chondrocytes grew initially on the surface of the scaffold. After 7 days, they further grew into the open pores of the blend polymer scaffolds. Morphologically, cells found on the surface of the scaffold exhibited a flat appearance and slowly form confluent cell multilayers starting from 14 to 28 days of the growth. In contrast, cells showed rounded morphology, formed aggregates and islets inside the scaffolds. In addition, chondrocytes proliferated on the scaffold and preserved their phenotype for up to 28 days.     

Zonal Uniformity in Mechanical Properties of the Chondrocyte Pericellular Matrix: Micropipette Aspiration of Canine Chondrons Isolated by Cartilage Homogenization

The pericellular matrix (PCM) is a region of tissue that surrounds chondrocytes in articular cartilage and together with the enclosed cells is termed the chondron. Previous studies suggest that the mechanical properties of the PCM, relative to those of the chondrocyte and the extracellular matrix (ECM), may significantly influence the stress–strain, physicochemical, and fluid-flow environments of the cell. The aim of this study was to measure the biomechanical properties of the PCM of mechanically isolated chondrons and to test the hypothesis that the Young's modulus of the PCM varies with zone of origin in articular cartilage (surface vs. middle/deep). Chondrons were extracted from articular cartilage of the canine knee using mechanical homogenization, and the elastic properties of the PCM were determined using micropipette aspiration in combination with theoretical models of the chondron as an elastic incompressible half-space, an elastic compressible bilayer, or an elastic compressible shell. The Young's modulus of the PCM was significantly higher than that reported for isolated chondrocytes but over an order of magnitude lower than that of the cartilage ECM. No significant differences were observed in the Young's modulus of the PCM between surface zone (24.0 ± 8.9 kPa) and middle/deep zone cartilage (23.2 ± 7.1 kPa). In combination with previous theoretical biomechanical models of the chondron, these findings suggest that the PCM significantly influences the mechanical environment of the chondrocyte in articular cartilage and therefore may play a role in modulating cellular responses to micromechanical factors.     

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.     

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.     

Preparation and evaluation of the electrospun chitosan/PEO fibers for potential applications in cartilage tissue engineering

Fibrous materials have morphological similarities to natural cartilage extracellular matrix and have been considered as candidate for bone tissue engineering scaffolds. In this study, we have evaluated a novel electrospun chitosan mat composed of oriented sub-micron fibers for its tensile property and biocompatibility with chondrocytes (cell attachment, proliferation and viability). Scanning electronic microscope images showed the fibers in the electrospun chitosan mats were indeed aligned and there was a slight cross-linking between the parent fibers. The electrospun mats have significantly higher elastic modulus (2.25 MPa) than the cast films (1.19 MPa). Viability of cells on the electrospun mat was 69% of the cells on tissue-culture polystyrene (TCP control) after three days in culture, which was slightly higher than that on the cast films (63% of the TCP control). Cells on the electrospun mat grew slowly the first week but the growth rate increased after that. By day 10, cell number on the electrospun mat was almost 82% that of TCP control, which was higher than that of cast films (56% of TCP). The electrospun chitosan mats have a higher Young's modulus (P < 0.01) than cast films and provide good chondrocyte biocompatibility. The electrospun chitosan mats, thus, have the potential to be further processed into three-dimensional scaffolds for cartilage tissue repair.     

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.     

Comparison of different chondrocytes for use in tissue engineering of cartilage model structures

This study compares bovine chondrocytes harvested from four different animal locations--nasoseptal, articular, costal, and auricular--for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue- engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue-engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model.     

Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels.

Articular cartilage responds to its mechanical environment through altered cell metabolism and matrix synthesis. In this study, isolated articular chondrocytes were cultured in collagen type I gels and exposed to uniaxial static compression of 0%, 25%, or 50% of original thickness for 0.5, 4, and 24 h, and to oscillatory (25 +/- 4%, 1 Hz) compression for 24 h. The cellular response was assessed through competitive and real-time RT-PCR to quantify expression of genes for collagen type I, collagen type II, and aggrecan core protein, and through radiolabelled proline and sulfate incorporation to quantify protein and proteoglycan synthesis rates. Static compression for 24 h inhibited expression of collagen I and II mRNAs and inhibited 3H-proline and 35S-sulfate incorporation. The mRNA expression exhibited transient fluctuations at intermediate time points. Oscillatory compression had no effect upon mRNA expression, and 24 h after release from static compression, there was no difference in collagen II or aggrecan mRNA, while there was an inhibition of collagen I. We conclude that the chondrocytes maintained some aspects of their ability to sense and respond to static compression, despite a biochemical and mechanical environment which is different from that in tissue. This suggests that mechanical stimuli may be useful in modulating chondrocyte metabolism in tissue engineering systems using fibrillar protein scaffolds.