Porous methacrylate scaffolds: supercritical fluid fabrication and in vitro chondrocyte responses

This paper describes a method of foaming a polymer system comprising poly(ethyl methacrylate)/tetrahydrofurfuryl methacrylate (PEMA/THFMA), characterisation of the resulting porosity and use of the foam for chondrocyte culture. The potential for this polymer system to support cartilage repair has been investigated previously, both in vivo and in vitro. PEMA/THFMA foamed created using supercritical carbon dioxide were characterised using scanning electron microscopy, mercury intrusion porosimetry and helium pycnometry. Foams were found to be 82% porous with open porosities of 57%. The mean pore diameter was found to be 99+60 microm. Bovine chondrocytes seeded directly onto the surface of the foamed and unfoamed PEMA/THFMA demonstrated lower proliferation on the foamed material, greater retention of the rounded cell morphology and increased glycosaminoglycan synthesis. In conclusion, this study has shown that a porous PEMA/THFMA system can further enhance the ability of the material to support chondrocytes in vitro. However, further modifications in processing are necessary to determine optimum conditions for cartilage tissue formation.     

The role of protein adsorption on chondrocyte adhesion to a heterocyclic methacrylate polymer system.

Chondrocyte adhesion to a polymer system consisting of poly(ethyl methacrylate) and tetrahydrofurfuryl methacrylate (PEMA/THFMA) has been investigated in vitro. The adhesive glycoproteins, fibronectin and vitronectin were studied for their role in promoting cell attachment. Fibronectin was the best substrate for chondrocyte attachment, if it was pre-adsorbed and did not have to compete with other proteins for attachment sites. Chondrocytes began to spread on fibronectin coated glass although they remained rounded on the libronectin coated PEMA/THFMA system. Vitronectin was better at competing with the other proteins in serum and was the main adhesive protein for chondrocyte attachment to TCP and the PEMA/THFMA system in normal serum medium. Serum contains non-adhesive proteins that compete for binding sites and hence reduce cell attachment. The alpha5beta1 and alpha(v)beta3/beta5 integrins were detected on the chondrocytes although there may be a difference in expression between different material surfaces.     

Behavior of human articular chondrocytes derived from nonarthritic and osteoarthritic cartilage in a collagen matrix

The purpose of this study was to evaluate the morphologic and biochemical behavior and activity of human chondrocytes taken from nonarthritic and osteoarthritic cartilage and seeded on a three-dimensional matrix consisting of collagen types I, II, and III. Human articular chondrocytes were isolated from either nonarthritic or osteoarthritic cartilage of elderly subjects, and from nonarthritic cartilage of an adolescent subject, seeded on collagen matrices, and cultured for 12 h, 7 days, and 14 days. Histological analysis, immunohistochemistry, and biochemical assays for glycosaminoglycans (GAGs) and DNA content were performed for cell-seeded and unseeded matrices. Chondrocytes of nonarthritic cartilage revealed a larger number of spherical cells, consistent with a chondrocytic phenotype. The biochemical assay showed a net increase in GAG content in nonarthritic chondrocytes, whereas almost no GAGs were seen in osteoarthritic cells. The DNA results suggest that more osteoarthritic cells than chondrocytes from nonarthritic cartilage attached to the matrix within the first week. Human articular chondrocytes isolated from osteoarthritic cartilage seem to have less bioactivity after expansion and culture in a sponge consisting of type I, II, and III collagen compared with chondrocytes from nonarthritic cartilage.     

Tissue engineering of autologous cartilage grafts in three-dimensional in vitro macroaggregate culture system

In the field of tissue engineering, techniques have been described to generate cartilage tissue with isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of biomaterial, and unforeseeable cell-biomaterial interactions remain. This study represents a novel technique to engineer cartilage by an in vitro macroaggregate culture system without the use of biomaterials. Human nasoseptal or auricular chondrocytes were enzymatically isolated and amplified in conventional monolayer culture before the cells were seeded into a cell culture insert with a track-etched membrane and cultured in vitro for 3 weeks. The new cartilage formed within the in vitro macroaggregates was analyzed by histology (toluidine blue, von Kossa-safranin O staining), and immunohistochemistry (collagen types I, II, V, VI, and X and elastin). The total glycosaminoglycan (GAG) content of native and engineered auricular as well as nasal cartilage was assayed colorimetrically in a safranin O assay. The biomechanical properties of engineered cartilage were determined by biphasic indentation assay. After 3 weeks of in vitro culture, nasoseptal and auricular chondrocytes synthesized new cartilage with the typical appearance of hyaline nasal cartilage and elastic auricular cartilage. Immunohistochemical staining of cartilage samples showed a characteristic pattern of staining for collagen antibodies that varied in location and intensity. In all samples, intense staining for cartilage-specific collagen types I, II, and X was observed. By the use of von Kossa-safranin O staining a few positive patches-a possible sign of beginning mineralization within the engineered cartilages-were detected. The unique pattern for nasoseptal cartilage is intense staining for type V collagen, whereas auricular cartilage is only weakly positive for collagen types V and VI. Engineered nasal and auricular macroaggregates were negative for anti-elastin antibody (interterritorially). The measurement of total GAG content demonstrated higher GAG content for reformed nasoseptal cartilage compared with elastic auricular cartilage. However, the total GAG content of engineered macroaggregates was lower than that of native cartilage. In spite of the mechanical stability of the auricular macroaggregates, there was no equilibrium of indentation. The histomorphological and immunohistochemical results demonstrate successful cartilage engineering without the use of biomaterials, and identify characteristics unique to hyaline as well as elastic cartilage. The GAG content of engineered cartilage was lower than in native cartilage and the biomechanical properties were not determinable by indentation assay. This study illustrates a novel in vitro macroaggregate culture system as a promising technique for tissue engineering of cartilage grafts. Further long-term in vitro and in vivo studies must be done before this method can be applied to reconstructive surgery of the nose or auricle.     

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.     

Water absorption and surface properties of novel poly(ethylmethacrylate) polymer systems for use in bone and cartilage repair

The surface and bulk properties of novel methacrylate polymers prepared by gelling poly(ethyl methacrylate) (PEMA) powder with different ratios of tetrahydrofurfuryl methacrylate (THFMA) and hydroxyethyl methacrylate (HEMA) monomers were investigated. The water adsorption and desorption characteristics of these polymers were measured in water and phosphate buffered saline (PBS). The desorption diffusion coefficients were higher than the adsorption coefficients in both water and PBS. Linear relationships between the equilibrium mass of water taken up and the mass of water desorbed with the concentration of HEMA in the polymer were established. Polymer surfaces were analysed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Surface features varied with polymer composition; during hydration only selective areas of the surface hydrated indicating a heterogeneous surface. Contact angle data showed no trend between the different polymers indicating that contact angles are not an acceptable method of assessing hydrophobicity/wettability of a material which does not have a homogeneous surface. The effect of these bulk and surface characteristics on biological interactions were examined using bovine chondrocytes and human osteoblast (HOB) cell cultures. Cell attachment decreased when HEMA was present in the copolymer.     

Collagen-PVA aligned nanofiber on collagen sponge as bi-layered scaffold for surface cartilage repair

Researchers have made bi-layered scaffolds but mostly for osteochondral repairs. The anatomic structure of human cartilage has different zones and that each has varying matrix morphology and mechanical properties is often overlooked. Two bi-layered collagen-based composites were made to replicate the superficial and transitional zones of an articular cartilage. Aligned and random collagen-PVA nanofibers were electrospun onto a freeze-dried collagen sponge to make the aligned and random composites, respectively. The morphology, swelling ratio, degradation and tensile properties of the two composites were examined. Primary porcine chondrocytes were cultured on the composites for three weeks and their proliferation and secretion of glycosaminoglycan (GAG) and type II collagen were measured. The influences of the cell culture on the tensile properties of the composites were studied. The nanofiber layer remained adhered to the sponge after three weeks of cell culture. Both composites lost 30–35% of their total weight in a saline buffer after three weeks. The tensile strength and Young’s modulus of both composites increased after three weeks of chondrocyte culture (p < 0.05). The aligned composite with extracellular matrix deposition had a Young’s modulus (0.35 MPa) similar to that of articular cartilage reported in literature (0.36–0.8 MPa). The chondrocytes on both aligned and random composites proliferated and secreted similar amounts of GAG and type II collagen. They were seen embedded in lacunae after three weeks. The aligned composite may be more suitable for articular cartilage repair because of the higher tensile strength from the aligned nanofibers on the surface that can better resist wear.     

Cultured chondrocytes produce injectable tissue-engineered cartilage in hydrogel polymer.

The purpose of this study was to determine if chondrocytes cultured through several subcultures at very low plating density would produce new cartilage matrix after being reimplanted in vivo with or without a hydrogel polymer scaffold. Chondrocytes were initially plated in low-density monolayer culture, grown to confluence, and passaged four times. After each passage cells were suspended in purified porcine fibrinogen and injected into the subcutaneous space of nude mice while simultaneously polymerizing with thrombin to reach a final concentration of 40 million cells/cc. Controls were made by injecting fresh, uncultured cells with fibrin polymer and by injecting the cultured cells in saline (without polymer). All samples were harvested at 6 weeks. When injected in polymer, both fresh cells and cells that had undergone only one passage in culture produced cartilaginous nodules. Cultured cells did not produce cartilage, regardless of length of time spent in culture, when injected without polymer. Cartilage was also not recovered from samples with cells kept in culture for longer than one passage, even when provided with a polymer matrix. All samples harvested were subjected to histological analysis and assayed for total DNA, glycosaminoglycan (GAG), and type II collagen. There was histological evidence of cartilage in the groups that used fresh cells and cultured cells suspended in fibrin polymer that only underwent one passage. No other group contained areas that would be consistent with cartilage histologically. All experimental samples had a higher percent of DNA than native swine cartilage, and there was no statistical difference between the DNA content of the groups containing cultured or fresh cells in fibrin polymer. Whereas the GAG content of native cartilage was 8.39% of dry weight and fresh cells in fibrin polymer was 12.85%, cultured cells in fibrin polymer never exceded the 2.48% noted from first passage cells. In conclusion, this study demonstrates that porcine chondrocytes that have been cultured in monolayer for one passage will produce cartilage in vivo when suspended in fibrin polymer.     

Chondrogenesis and Integration of Mesenchymal Stem Cells Within an <Emphasis Type="Italic">In</Emphasis> <Emphasis Type="Italic">Vitro</Emphasis> Cartilage Defect Repair Model

Integration of repair tissue is a key indicator of the long-term success of cell-based therapies for cartilage repair. The objective of this study was to compare the in vitro chondrogenic differentiation and integration of agarose hydrogels seeded with either chondrocytes or bone marrow-derived mesenchymal stem cells (MSCs) in defects created in cartilage explants. Chondrocytes and MSCs were isolated from porcine donors, suspended in 2% agarose and then injected into cylindrical defects within the explants. These constructs were maintained in a chemically defined medium supplemented with 10 ng/mL of TGF-β3. Cartilage integration was assessed by histology and mechanical push-out tests. After 6 weeks in culture, chondrocyte-seeded constructs demonstrated a higher integration strength (64.4 ± 8.3 kPa) compared to MSC-seeded constructs (22.7 ± 5.9 kPa). Glycosaminoglycan (GAG) (1.27 ± 0.3 vs. 0.19 ± 0.03 kPa) and collagen (0.31 ± 0.08 vs. 0.09 ± 0.01 kPa) accumulation in chondrocyte-seeded constructs was greater than that measured in the MSC-seeded group. The GAG, collagen, and DNA content of both chondrocyte- and MSC-seeded hydrogels cultured in cartilage explants was significantly lower than control constructs cultured in free swelling conditions. The results of this study suggest that the explant model may constitute a more rigorous in vitro test to assess MSC therapies for cartilage defect repair.     

Immunohistological study on collagen in cartilage-bone metamorphosis and degenerative osteoarthrosis

Summary Synthesis of collagen by chondrocytes was studied by immunofluorescence using antibodies specific for type I, II and III collagen. The following tissues and culture conditions were chosen for this immunohistological study: normal articular cartilage, epiphyseal growth cartilage, cartilage undergoing osteoarthrotic degeneration, suspension culture and monolayer culture. While type II collagen is the unique collagen all over hyaline cartilage, type I collagen is produced by hypertrophic chondrocytes in the growth plate. In addition, chondrocytes in osteoarthrotic areas of articular cartilage synthesize type I collagen. Under in vitro culture conditions, chondrocytes initially produce type II collagen and synthesize later on type I collagen. The change of synthesis from type II to type I collagen is more rapid in monolayer than in suspension culture. It is concluded that the presence of matrix compounds and the cellmatrix interaction as well are necessary to maintain synthesis of type II collagen in chondrocytes. Alterations in the cell-matrix interactions are shown to occur in the hypertrophic zone of the epiphyseal growth plate, in cartilage undergoing osteoarthrotic degeneration as well as in chondrocytes grown in culture. Thus, change in the control of gene activity may subsequently lead to change in collagen synthesis. It is possible that the synthesis of type I collagen, which cannot fulfil the physiological function of a structural element in cartilageneous tissue, is a crucial factor in the process of osteoarthrosis.

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Abbreviations EDTA Ethylendiaminetetraacetate - FITC Fluoresceine isothiocyanate This investigation was supported by grants of the Deutsche Forschungsgemeinschaft, Mu 378/4, Re 388/1 and SFB 51     

Functionalization of dynamic culture surfaces with a cartilage extracellular matrix extract enhances chondrocyte phenotype against dedifferentiation

Culture on silicone rubber surfaces has been shown to partially overcome the chondrocyte dedifferentiation characteristic of standard culture on rigid polystyrene. These methods typically involve functionalization of culture surfaces with proteins. Collagen type I is often used, but more cartilage-specific proteins may be more appropriate for chondrocytes. To explore this hypothesis, a twofold experimental design was applied. First, chondrocytes were cultured in rigid Petri dishes coated with silicone rubber ("static silicone" or SS culture) functionalized with either cartilage extracellular matrix (ECM) extract or collagen type I. Second, chondrocytes were cultured on monotonically expanded high extension silicone rubber dishes ("continuous expansion" or CE culture) functionalized with ECM extract and compared to cells grown in SS culture. There were no differential effects of surface functionalization with the ECM extract vs. collagen type I on chondrocyte morphology, viability, proliferation or apoptosis in SS culture. However, chondrocyte growth on the ECM extract was associated with significantly reduced collagen types I and X gene expression and significantly increased glycosaminoglycan (GAG) secretion. After 3 passages (P3) on ECM-coated SS culture, chondrocyte phenotype and GAG secretion was enhanced compared to cells passaged on collagen type I. Pellet cultures from P3 SS culture displayed enhanced collagen type II content when ECM extract was used for functionalization rather than collagen type I. In CE culture with ECM functionalization, chondrocyte dedifferentiation was significantly inhibited vs. SS cultures, as evidenced by both gene expression and pellet cultures. Functionalization of extendable culture surfaces with cartilage ECM extract therefore supports enhanced preservation of chondrocyte phenotype.     

Bovine chondrocyte behaviour in three-dimensional type I collagen gel in terms of gel contraction, proliferation and gene expression

This study evaluated the in vitro behaviour of bovine chondrocytes seeded in collagen gels, promising recently reported scaffolds for the treatment of full-thickness cartilage defects. To determine how chondrocytes respond to a collagen gel environment, 2 x 10(6) chondrocytes isolated from fetal, calf and adult bovine cartilage were seeded within type I collagen gels and grown for 12 days in both attached and floating (detached from the culture dish after polymerisation) conditions. Monolayer cultures were performed in parallel. All chondrocytes contracted floating gels to 55% of the initial size, by day 12. Contraction was dependent on initial cell density and inhibited by the presence of dihydrocytochalasin B as previously observed with fibroblasts. Gene expression was determined using conventional and real-time PCR. The chondrocyte phenotype was better maintained in floating gels compared to attached gels and monolayers. This was demonstrated by comparing the ratio of COL2A1/ COL1A2 mRNA and also of alpha10/alpha11 integrin mRNA. A strong up-regulation of MMP13 expression was measured at day 12 in floating gels. The composition of cartilage-like tissue obtained by growing chondrocytes in a collagen gel varied depending on the floating or attached conditions and initial cell density. It is thus important to consider these parameters when using this culture system in order to prepare a well-defined implant for cartilage repair.     

BMP-2 embedded atelocollagen scaffold for tissue-engineered cartilage cultured in the medium containing insulin and triiodothyronine--a new protocol for three-dimensional in vitro culture of human chondrocytes

When the chondrocytes are isolated from the native cartilage and proliferate in vitro, they soon lose their original ability to express glycosaminoglycan (GAG) and type II collagen, which is termed dedifferentiation, or decrease cell viability. We first examined in vitro cartilage regeneration of tissue-engineered pellets that consisted of human auricular chondrocytes and atelocollagen and that were incubated in vitro under stimulation with bone morphogenetic protein-2 (BMP-2), insulin, and T(3). We then examined the administration of those growth factors into the scaffold or in the medium and explored the possibility that the atelocollagen, the hydrogel scaffold of the chondrocytes, may function for drug delivery of the factors. BMP-2 in the atelocollagen with the supplement of insulin and T3 in the medium could not only produce a greater GAG matrix in a shorter period but also sustain cell viability with lower mortality. The insulin in the medium could be better administered only for 2 weeks, rather than 3 weeks, which would save time and cost, hence shortening the in vitro culture of chondrocytes. Our protocol of mixing BMP-2 into the atelocollagen with the supplement of insulin and T3 hormone might provide a new insight into the development of tissue engineering in chondrogenesis.     

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

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

In vivo cultivation of human articular chondrocytes in a nude mouse-based contained defect organ culture model

The nude mouse model is an established method to cultivate and investigate tissue engineered cartilage analogues under in vivo conditions. One limitation of this common approach is the lack of appropriate surrounding articular tissues. Thus the bonding capacity of cartilage repair tissue cannot be evaluated. Widely applied surgical techniques in cartilage repair such as conventional and three-dimensional autologous chondrocyte implantation (ACI) based on a collagen gel matrix cannot be included into nude mouse studies, since their application require a contained defect. The aim of this study is to apply an organ culture defect model for the in vivo cultivation of different cell-matrix-constructs.Cartilage defects were created on osteochondral specimens which had been harvested from 10 human knee joints during total knee replacement. Autologous chondrocytes were isolated from the cartilage samples and cultivated in monolayer until passage 2. On each osteochondral block defects were treated either by conventional ACI or a collagen gel seeded with autologous chondrocytes, including a defect left empty as a control. The samples were implanted into the subcutaneous pouches of nude mice and cultivated for six weeks. After retrieval, the specimens were examined histologically, immunohistochemically and by cell morphology quantification.In both, ACI and collagen gel based defect treatment, a repair tissue was formed, which filled the defect and bonded to the adjacent tissues. The repair tissue was immature with low production of collagen type II. In both groups redifferentiation of chondrocytes remained incomplete. Different appearances of interface zones between the repair tissue and the adjacent cartilage were found.The presented contained defect organ culture model offers the possibility to directly compare different types of clinically applied biologic cartilage repair techniques using human articular tissues in a nude mouse model.     

Novel strategies for enhancing tissue integration in cartilage repair

Introduction Articular cartilage is unable to initiate a spontaneous repair response when injured due to its avascular and aneural properties. Within adult cartilage, chondrocytes are entrapped within an extensive extracellular matrix and are unable to migrate to sights of injury to regulate tissue repair. Injury to this tissue therefore inevitably leads to degeneration of the cartilage and the development of degenerative diseases such as osteoarthritis. The surgical technique of autologous chondrocyte transplantation (ACT) was developed for the treatment of full‐thickness cartilage defects ( Brittberg et al. 1994 ). Implantation of chondrocytes into the defect site repairs the injury site with a mixture of fibrocartilaginous and hyaline‐like tissue that poorly integrates with the existing cartilage and frequently degenerates with time. In this current study, we have developed an in vitro model to investigate methods for enhancing this integration and the development of a more biomechanically stable repair tissue. Materials and methods Bovine articular cartilage explants from the metacarpalphalangeal joint were experimentally injured using a stainless steel trephine and cultured for a period of 28 days. Autologous chondrocytes in an agarose suspension were injected into the interface region at the injury site. Media was collected and analysed for proteoglycan and collagen content using the DMMB and hydroxyproline assays, respectively. Matrix metalloproteinase (MMP) expression was also analysed using zymography and an adapted collagen fibril assay. Results Morphological analyses indicate attempts at repair and integration within both control and experimental treatment groups, although the presence of autologous chondrocytes appeared to amplify this repair response. Although not statistically significant, considerable differences in proteoglycan release between injured explants and the intact control group were seen. Collagen release into the media was only seen at day 28 within experimental cultures. An up‐regulation of MMP‐2 and MMP‐9 was seen within the experimental cultures compared to the controls. Preliminary data also suggest up‐regulation of collagenases in the experimental group when compared to controls. Discussion As seen with clinical ACT treatment, the presence of autologous chondrocytes appears to enhance repair and integration attempts; however, morphologically, this repair tissue appears to be fibrocartilaginous. Further analysis will establish whether the repair tissue is true hyaline cartilage and monitor the synthesis and turnover of macromolecules within the established culture system.     

The use of absorbable co-polymer pads with alginate and cells for articular cartilage repair in rabbits

PURPOSE: To determine the effect of polyglycolic acid (PGA)-polylactic acid (PLA) co-polymer pads with calcium alginate on chondrogenic gene expression for chondrocytes cultured in vitro. We also evaluated the ability of these absorbable pads with alginate to deliver chondrocytes and influence osteochondral defect repair in vivo in immature rabbit knees. METHODS: Rabbit rib chondrocytes were suspended in calcium alginate and co-polymer pads composed of either 47.5/52.5 PGA-PLA or 90/10 PGA-PLA at two different cell concentrations and cultured in vitro for 1, 3, and 5 days. Analysis was performed using RT-PCR for chondrogenic gene expression of aggrecan, type II collagen, and type I collagen. Cells labeled with a traceable green fluorescent protein (GFP) marker in vitro were suspended within the pads to analyze for dispersion and attachment to the pad. An in vivo study was performed in which full-thickness (3x4mm(2)) osteochondral defects were made in 60 rabbit knees. The study comprised four treatment groups based on the type of implant into the defect (empty, alginate alone, or either type of co-polymer pad) and harvested at either 6 or 12 weeks. Two independent blinded observers analyzed and scored the defects grossly and histologically. RESULTS: In vitro analysis of the chondrocytes after 1, 3, and 5 days in culture showed no statistical differences between the types of PGA/PLA co-polymer pad with regard to expression of aggrecan, type II collagen, or type I collagen. However, although statistically insignificant, the expression of aggrecan and type II collagen was found to be greater than that for type I collagen in both types of pads, confirming the chondrogenic effect of suspension culture for this system. The addition of alginate to polymer pads allowed costal chondrocytes to be implanted in vivo, as evidenced by the attachment of the cells to the fibers and the uniform dispersion of the GFP-labeled cells through the pad as seen on fluorescent microscopy. Histologic results showed improved scores for the 47.5/52.5 PGA-PLA group (21.3) and the 90/10 PGA-PLA group (18.3) when compared to empty (15.3) or alginate alone (15.1) defects at 12 weeks. CONCLUSION: The addition of calcium alginate to the co-polymer pads offers a new approach to deliver cells to an osteochondral defect and may enhance cartilage regeneration. Future application of this model may allow for an arthroscopic delivery system to assist the healing of cartilage defects.     

Tissue engineering of human cartilage and osteochondral composites using recirculation bioreactors

Chondrocytes isolated from human foetal epiphyseal cartilage were seeded dynamically into polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to produce tissue-engineered cartilage. Several culture techniques with the potential to provide endogenous growth factors and other conditions beneficial for de novo cartilage synthesis were investigated. Osteochondral composite constructs were generated by seeding separate PGA scaffolds with either foetal chondrocytes or foetal osteoblasts then suturing the scaffolds together before bioreactor cultivation. This type of co-culture system provided direct contact between the tissue-engineered cartilage and developing tissue-engineered bone and yielded significant improvements in cartilage quality. In the cartilage section of the composites, the concentrations of glycosaminoglycan (GAG) and total collagen were increased by 55% and 2.5-fold, respectively, compared with control cartilage cultures, while levels of collagen type II were similar to those in the controls. The osteochondral composites were harvested from the bioreactors as single units with good integration between the cartilage and bone tissues. Only the cartilage layer contained GAG while only the bone layer was mineralised. In other experiments, co-culture of tissue-engineered cartilage with pieces of ex-vivo cartilage or ex-vivo bone did not improve the quality of the cartilage relative to control cultures. Addition of 10(-6) M diacerein to the culture medium also had no effect on the properties of engineered cartilage. This work demonstrates the beneficial effects of generating cartilage tissues in contact with developing bone. It also demonstrates the feasibility of producing composite osteochondral constructs for clinical application using recirculation column bioreactors.     

Human mesenchymal stem cells induced by growth differentiation factor 5: an improved self-assembly tissue engineering method for cartilage repair

Previous studies have shown that novel scaffold-free self-assembled constructs can be an ideal alternative for cartilage tissue engineered based on scaffolds, which has many limitations. However, many questions remain, including the choice of seeding cells and the role of growth differentiation factor 5 (GDF-5) in constructing self-assembled engineered cartilages. Moreover, whether the optimum construct is effective in human chondral defect repair is still unknown. In this study, we generated self-assembled constructs of human mesenchymal stem cells (hMSCs) using four different approaches: direct self-assembly of hMSCs with or without GDF-5, and predifferentiated hMSCs self-assembly with or without GDF-5. Histological, immunohistochemical, and biochemistry analyses indicated that the constructs generated from predifferentiated hMSCs induced by GDF-5 (Group D2) exhibited up-regulated glycosaminoglycan (GAG) and type II collagen expression and contained higher amounts of GAG and total collagen than any other group. After 3-weeks of in vitro culturing of the constructs in a chondral defects explant culture system, the contructs from Group D2 were stably adhered to the surface of the cartilage matrix. Immunohistochemically, the repair tissue was positive for type II collagen, toluidine blue, and safranin O. These data demonstrated that the generation of self-assembled tissue-engineered cartilage from chondrogenically differentiated hMSCs induced by GDF-5 is a promising therapeutic strategy for cartilage repair.     

Bioartificial cartilage

Cartilage is a highly differentiated tissue. Its three-dimensional composition of cells and matrix is able to resist intensive mechanical loads. The capacity of cartilage tissue for regeneration is limited. Chondrocytes are responsible for matrix production of cartilage tissue. Enzymatic isolation and expansion of chondrocytes with cell culture techniques has been improved in the last years. These cells can be cultured on different three-dimensional culture systems suitable for transplantation to repair localized cartilage defects. Two types of bioresorbable polymer fleece matrices (PLLA and a composite fleece of polydioxanone and polyglactin) and lyophilized dura as a biological carrier are tested. Phenotypic and morphological appearance of the cultured articular rabbit chondrocytes is preserved on all three types of transport media. Production of glycosaminoglycans has been shown by Alcian blue staining, production of collagen by azan staining. Chondroitin 4- and 6-sulfate are detected immunohistochemically in the created constructs. The different carriers have specific characteristics regarding their suitability for the creation of bioartificial cartilage. This tissue is transplantable into articular cartilage defects and could, therefore, improve the minor intrinsic healing capacity of cartilage tissue.