Co-culture Of Chondrocytes Research Articles

Assessment of growth factor treatment on fibrochondrocyte and chondrocyte co-cultures for TMJ fibrocartilage engineering

Treatments for patients suffering from severe temporomandibular joint (TMJ) dysfunction are limited, motivating the development of strategies for tissue regeneration. In this study, co-cultures of fibrochondrocytes (FCs) and articular chondrocytes (ACs) were seeded in agarose wells, and supplemented with growth factors, to engineer tissue with biomechanical properties and extracellular matrix composition similar to native TMJ fibrocartilage. In the first phase, growth factors were applied alone and in combination, in the presence or absence of serum, while in the second phase, the best overall treatment was applied at intermittent dosing. Continuous treatment of AC/FC co-cultures with TGF-β1 in serum-free medium resulted in constructs with glycosaminoglycan/wet weight ratios (12.2%), instantaneous compressive moduli (790kPa), relaxed compressive moduli (120kPa) and Young’s moduli (1.87MPa) that overlap with native TMJ disc values. Among co-culture groups, TGF-β1 treatment increased collagen deposition ∼20%, compressive stiffness ∼130% and Young’s modulus ∼170% relative to controls without growth factor. Serum supplementation, though generally detrimental to functional properties, was identified as a powerful mediator of FC construct morphology. Finally, both intermittent and continuous TGF-β1 treatment showed positive effects, though continuous treatment resulted in greater enhancement of construct functional properties. This work proposes a strategy for regeneration of TMJ fibrocartilage and its future application will be realized through translation of these findings to clinically viable cell sources.

The construction of engineered cartilage by co-culture of chondrocytes and bone mesenchymal stem cells in vitro

Objective To explore the effects of co-culture of chondrocytes and bone mesenchymal stem cells (BMSCs) on constructing engineered cartilages, and confirm the most suitable ratio of chondrocytes to BMSCs. Methods Chondrocytes and BMSCs were isolated from articulars cartilages of rabbit (1 month old)cells (40 μl 4×107/ml) were seeded into a poly(lactic-co-glycolic acid) (PLGA) scaffold and cultured statically for 2 days. They were transferred into the cyclic pressures system, and cultured under cyclic pressures for 3 weeks. The engineered cartilages were harvested and examined by gross observation, histological staining, immunohistochemisty of collagen Ⅱ, the content of glyeosaminoglycans, GAGs, DNA and the percentage of collagen Ⅱ dyeing area. Results The engineered cartilages of the co-cultured groups grew bigger than those of the chondrocytes alone group, and their surfaces were smooth and glossy. The distributions of cartilaginous extracellular matrices in the co-cultured groups were more homogenous than those of the chondrocytes alone group.gen Ⅱ dyeing area of the co-cultured groups were higher than those of the chondrocytes alone group. The conConclusion Co-culture of chondrocytes and BMSCs could improve the quality of engineered cartilages. The Key words: Chondrocytes; Mesenchymal stem cells; Bone marrow cells; Tissue engineering

Bioactive stratified polymer ceramic-hydrogel scaffold for integrative osteochondral repair.

Due to the intrinsically poor repair potential of articular cartilage, injuries to this soft tissue do not heal and require clinical intervention. Tissue engineered osteochondral grafts offer a promising alternative for cartilage repair. The functionality and integration potential of these grafts can be further improved by the regeneration of a stable calcified cartilage interface. This study focuses on the design and optimization of a stratified osteochondral graft with biomimetic multi-tissue regions, including a pre-designed and pre-integrated interface region. Specifically, the scaffold based on agarose hydrogel and composite microspheres of polylactide-co-glycolide (PLGA) and 45S5 bioactive glass (BG) was fabricated and optimized for chondrocyte density and microsphere composition. It was observed that the stratified scaffold supported the region-specific co-culture of chondrocytes and osteoblasts which can lead to the production of three distinct yet continuous regions of cartilage, calcified cartilage and bone-like matrices. Moreover, higher cell density enhanced chondrogenesis and improved graft mechanical property over time. The PLGA-BG phase promoted chondrocyte mineralization potential and is required for the formation of a calcified interface and bone regions on the osteochondral graft. These results demonstrate the potential of the stratified scaffold for integrative cartilage repair and future studies will focus on scaffold optimization and in vivo evaluations.

Replacing animal models of osteoarthritis with 3‐dimensional models of articular cartilage and synovium

Osteoarthritis (OA) is an increasingly common clinical problem in ageing humans and companion animals. It affects load‐bearing synovial joints causing pain, inflammation, loss of mobility and significant morbidity. A number of animal models of OA are currently used to study disease pathogenesis and evaluate the efficacy of candidate anti‐inflammatory and anti‐arthritic drugs for clinical development. These animal models may involve injection of inflammatory agents into the joint, surgical creation of joint instability or surgical replication of joint trauma. Such models not only cause a considerable amount of pain and suffering but also none of them have a proven track record of predictability in human disease. Consequently, there is an acute need for developing novel and alternative in vitro models that closely mimic the disease. We have used articular cartilage and synovial tissues from horses and dogs euthanased for unrelated clinical reasons to establish several in vitro models of the joint. We have developed an explant model of articular cartilage inflammation, co‐cultures of primary synoviocytes and chondrocytes as well as 3‐dimensional cultures of chondrocytes and mesenchymal stem cells (MSCs). These approaches will help reduce the number of laboratory animals used in OA research and may provide a realistic alternative to experimental animals.

Characterization of auricular chondrocytes and auricular/articular chondrocyte co-cultures in terms of an application in articular cartilage repair

Cartilage injury remains a challenge in orthopedic surgery as articular cartilage only has a limited capacity for intrinsic healing. Autologous chondrocyte transplantation (ACT) is a suitable technique for cartilage repair, but requires articular cartilage biopsies for autologous chondrocyte expansion. The use of heterotopic chondrocytes derived from non-articular cartilage sources such as auricular chondrocytes may be a novel approach for ACT. The aim of the study is to evaluate whether co-cultured articular/auricular chondrocytes exhibit characteristics comparable to articular chondrocytes. Analysis of the proliferation rate, extracellular cartilage matrix (ECM) gene and protein expression (type II and I collagen, elastin, lubricin), beta1-integrins and the chondrogenic transcription factor sox9 in articular/auricular chondrocytes was performed using RTD-PCR, flow cytometry, immunofluorescence microscopy and Western blot analysis. Additionally, three-dimensional (3D) chondrocyte mono- and co-cultures were established. The proliferative activity and elastin gene expression were lower and that of type II collagen and lubricin was higher in articular compared with auricular chondrocytes. The species generally did not influence the chondrocyte characteristics, with the exception of type I collagen and sox9 expression, which was higher in porcine but not in human articular chondrocytes compared with both types of auricular chondrocytes. beta1-integrin gene expression did not differ significantly between the chondrocyte types. The type II collagen gene and protein expression was higher in articular chondrocyte monocultures and was slightly higher in co-cultures compared with monocultured auricular chondrocytes. Both chondrocyte types survived in co-culture. Despite their differing expression profiles, co-cultures revealed some adjustment in the ECM expression of both chondrocyte types.

Immunological response to tissue-engineered cartilage derived from auricular chondrocytes and a PLLA scaffold in transgenic mice

The immune response against biomaterials in tissue-engineered constructs could potentially worsen the outcome of tissue regeneration, but immunological reactions between host and donor in tissue-engineered constructs remain to be clarified. In the present study, we syngenically transplanted tissue-engineered cartilage constructs consisting of C57BL/6 mice auricular chondrocytes and poly- l-lactic acid scaffolds (MW:200,000) into EGFP transgenic mice of C57BL/6 background, and evaluated the response by the localization of donor-derived and host-derived cells, the latter of which were distinguished by the presence of EGFP. While donor-derived cells constituted the areas of regenerated cartilage, host-derived cells were increased in number for the initial two weeks, and then decreased and excluded to non-cartilage areas thereafter. Furthermore, EGFP positivity was mostly co-localized with that of F4/80, suggesting most of the host-derived cells in the tissue-engineered constructs could be macrophages. Immunohistochemical staining of the tissue-engineered cartilage constructs revealed expression of factors related to immune privilege in chondrocytes, such as macrophage migration inhibitory factor (MIF), fas ligand (FasL) and others. Co-culture of chondrocytes and macrophages in vitro increased the expression of MIF and FasL in the chondrocytes, suggesting that chondrocytes in tissue-engineered cartilage constructs could regulate the actions of host-derived macrophages by expressing factors related to immune privilege.

Coculture of Human Embryonic Stem Cells and Human Articular Chondrocytes Results in Significantly Altered Phenotype and Improved Chondrogenic Differentiation

Human embryonic stem (hES) cells have been suggested as a cell source for the repair of cartilage lesions. Here we studied how coculture with human articular chondrocytes affects the expansion potential, morphology, expression of surface markers, and differentiation abilities of hES cells, with special regard to chondrogenic differentiation. Undifferentiated hES cells were cocultured with irradiated neonatal or adult articular chondrocytes in high-density pellet mass cultures for 14 days. Cocultured hES cells were then expanded on plastic and their differentiation potential toward the adipogenic, osteogenic, and chondrogenic lineages was compared with that of undifferentiated hES cells. The expression of different surface markers was investigated using flow cytometry and teratoma formation was studied using injection of the cells under the kidney capsule. Our results demonstrate that although hES cells have to be grown on Matrigel, the cocultured hES cells could be massively expanded on plastic with a morphology and expression of surface markers similar to mesenchymal stem cells. Coculture further resulted in a more homogenous pellet and significantly increased cartilage matrix production, both in high-density pellet mass cultures and hyaluronan-based scaffolds. Moreover, cocultured cells formed colonies in agarose suspension culture, also demonstrating differentiation toward chondroprogenitor cells, whereas no colonies were detected in the hES cell cultures. Coculture further resulted in a significantly decreased osteogenic potential. No teratoma formation was detected. Our results confirm the potential of the culture microenvironment to influence hES cell morphology, expansion potential, and differentiation abilities over several population doublings.

Osteogenic Differentiation of Bone Marrow Stromal Cells Induced by Coculture with Chondrocytes Encapsulated in Three-Dimensional Matrices

Endochondral ossification implicates chondrocyte signaling as an important factor in directing the osteogenic differentiation of mesenchymal stem cells in vivo. In this study, the osteoinductive capabilities of articular chondrocytes suspended in alginate hydrogels were analyzed via coculture with bone marrow stromal cells (BMSCs). In particular, the effect of chondrocyte coculture time on the mechanism underlying this osteogenic induction was examined. Chondrocytes were suspended in alginate beads and cultured above BMSCs in monolayer. Beads containing chondrocytes were removed after 1, 10, or 21 days of coculture. Quantitative reverse transcriptase polymerase chain reaction was used to assess the expression of alkaline phosphatase, bone morphogenetic protein-2, and osteocalcin by BMSCs after days 1, 8, 14, and 21. Calcium deposition was also assayed to characterize the extent of mineralization within cultures. Results indicate that osteogenic differentiation of BMSCs is initiated upon brief exposure to chondrocyte signaling, but requires continued exposure in order to progress fully and maintain an osteoblastic phenotype.

Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle‐derived stem cells expressing bone morphogenetic protein 4 and soluble flt‐1

The control of angiogenesis during chondrogenic differentiation is an important issue affecting the use of stem cells in cartilage repair, especially with regard to the persistence of regenerated cartilage. This study was undertaken to investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the blocking of VEGF with its antagonist, soluble Flt-1 (sFlt-1), on the chondrogenesis of skeletal muscle-derived stem cells (MDSCs) in a rat model of osteoarthritis (OA). We investigated the effect of VEGF on cartilage repair in an immunodeficiency rat model of OA after intraarticular injection of murine MDSCs expressing bone morphogenetic protein 4 (BMP-4) in combination with MDSCs expressing VEGF or sFlt-1. In vivo, a combination of sFlt-1- and BMP-4-transduced MDSCs demonstrated better repair without osteophyte formation macroscopically and histologically following OA induction, when compared with the other groups. Higher differentiation/proliferation and lower levels of chondrocyte apoptosis were also observed in sFlt-1- and BMP-4-transduced MDSCs compared with a combination of VEGF- and BMP-4-transduced MDSCs or with BMP-4-transduced MDSCs alone. In vitro experiments with mixed pellet coculture of MDSCs and OA chondrocytes revealed that BMP-4-transduced MDSCs produced the largest pellets, which had the highest gene expression of not only type II collagen and SOX9 but also type X collagen, suggesting formation of hypertrophic chondrocytes. Our results demonstrate that MDSC-based therapy involving sFlt-1 and BMP-4 repairs articular cartilage in OA mainly by having a beneficial effect on chondrogenesis by the donor and host cells as well as by preventing angiogenesis, which eventually prevents cartilage resorption, resulting in persistent cartilage regeneration and repair.

Effects of co‐cultures of meniscus cells and articular chondrocytes on PLLA scaffolds

The knee meniscus, a fibrocartilaginous tissue located in the knee joint, is characterized by heterogeneity in extracellular matrix and biomechanical properties. To recreate these properties using a tissue engineering approach, co-cultures of meniscus cells (MCs) and articular chondrocytes (ACs) were seeded in varying ratios (100:0, 75:25, 50:50, 25:75, and 0:100) on poly-L-lactic acid (PLLA) scaffolds and cultured in serum-free medium for 4 weeks. Histological, biochemical, and biomechanical tests were used to assess constructs at the end time point. Strong staining for collagen and glycosaminoglycan (GAG) was observed in all groups. Constructs with 100% MCs were positive for collagen I and constructs cultured with 100% ACs were positive for collagen II, while a mixture of collagen I and II was observed in other co-culture groups. Total collagen and GAG per construct increased as the percentage of ACs increased (27 +/- 8 microg, 0% AC to 45 +/- 8 microg, 100% ACs for collagen and 12 +/- 4 microg, 0% ACs to 40 +/- 5 microg, 100% ACs for GAG). Compressive modulus (instantaneous and relaxation modulus) of the constructs was significantly higher in the 100% ACs group (63 +/- 12 and 22 +/- 9 kPa, respectively) when compared to groups with higher percentage of MCs. No differences in tensile properties were noted among groups. Specific co-culture ratios were identified mimicking the GAG/DW of the inner (0:100, 25:75, and 50:50) and outer regions (100:0) of the meniscus. Overall, it was demonstrated that co-culturing MCs and ACs on PLLA scaffolds results in functional tissue engineered meniscus constructs with a spectrum of biochemical and biomechanical properties.

In vitro stage‐specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

Osteoarthritis is characterized by an imbalance in cartilage homeostasis, which could potentially be corrected by mesenchymal stem cell (MSC)-based therapies. However, in vivo implantation of undifferentiated MSCs has led to unexpected results. This study was undertaken to establish a model for preconditioning of MSCs toward chondrogenesis as a more effective clinical tool for cartilage regeneration. A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs and to study the detailed stages of chondrogenesis of MSCs, using a human MSC line, Kp-hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP]). In addition, committed MSCs were seeded into a collagen scaffold and analyzed for their neocartilage-forming ability. Coculture of hPi-GFP chondrocytes with Kp-hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules and stimulated cartilage developmental growth factors, including members of the transforming growth factor beta superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor. Furthermore, committed Kp-hMSCs acquired neocartilage-forming potential within the collagen scaffold. These findings help define the molecular markers of chondrogenesis and more accurately delineate the stages of chondrogenesis during chondrocytic differentiation of human MSCs. The results indicate that human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes. This model of in vitro chondrogenesis of human MSCs represents an advance in cell-based transplantation for future clinical use.

Induction of chondrogenic differentiation in cultured fibroblasts isolated from the inferior turbinate

To explore options of cell sources for regeneration of cartilage, turbinate fibroblasts were tested for their differentiation potential when cultured in three-dimensional scaffolds with growth factors and co-cultured with septal chondrocytes. Fibroblasts and chondrocytes were seeded on to an alginate sponge in different proportions and were stimulated with TGF-beta1 and IGF-I. Chondrogenic differentiation of fibroblasts was assessed by histology including immunohistochemical staining, which was examined by reverse transcriptase polymerase chain reaction (RT-PCR). Chondroid cells with extracellular matrices occupied the porous structures of alginate sponge and immunolocalizations for type II collagen became prominent at four weeks in culture. Expression of type II collagen and aggrecan mRNA were detected by RT-PCR. Co-culture of fibroblasts and chondrocytes showed comparable expansion of cells and extracellular matrix to culture of chondrocytes only. These findings suggest that human turbinate fibroblasts are apparently redirected toward chondrogenic phenotype by in vitro culture system under specific conditions. Co-culture of turbinate fibroblasts with septal chondrocytes would be an effective method to expand cartilage specific extracellular matrix.

Derivation of Chondrogenically-Committed Cells from Human Embryonic Cells for Cartilage Tissue Regeneration

BackgroundHeterogeneous and uncontrolled differentiation of human embryonic stem cells (hESCs) in embryoid bodies (EBs) limits the potential use of hESCs for cell-based therapies. More efficient strategies are needed for the commitment and differentiation of hESCs to produce a homogeneous population of specific cell types for tissue regeneration applications.Methodology/Principal FindingsWe report here that significant chondrocytic commitment of feeder-free cultured human embryonic stem cells (FF-hESCs), as determined by gene expression and immunostaining analysis, was induced by co-culture with primary chondrocytes. Furthermore, a dynamic expression profile of chondrocyte-specific genes was observed during monolayer expansion of the chondrogenically-committed cells. Chondrogenically-committed cells synergistically responded to transforming growth factor-β1 (TGF-β1) and β1-integrin activating antibody by increasing tissue mass in pellet culture. In addition, when encapsulated in hydrogels, these cells formed cartilage tissue both in vitro and in vivo. In contrast, the absence of chondrocyte co-culture did not result in an expandable cell population from FF-hESCs.Conclusions/SignificanceThe direct chondrocytic commitment of FF-hESCs can be induced by morphogenetic factors from chondrocytes without EB formation and homogenous cartilage tissue can be formed in vitro and in vivo.

Creating a spectrum of fibrocartilages through different cell sources and biochemical stimuli

In this study a scaffoldless approach was employed with two different cell sources and biochemical stimuli to engineer a spectrum of fibrocartilages representative of the different regions of the knee meniscus. Constructs composed of 100% fibrochondrocytes (FC) or a 50:50 co-culture of fibrochondrocytes and chondrocytes (CC) were cultured in 10% fetal bovine serum medium or serum-free "chondrogenic" medium, each +/-10 ng/mL TGF-beta1 (+T). Constructs from these two cell groups and four culture conditions were cultured for 6 weeks. By varying the cell type and presence of the growth factor, GAG per dry weight of the constructs spanned that of native tissue, ranging 16-45% and 1-7% in the CC and FC groups, respectively. Collagen density was most dependent on cell type and was significantly lower than tissue values. The collagen I/II ratio could be manipulated by cell type and serum presence to span the native range, from 3.5 in the serum-free CC group to over 1,000 in the FC groups treated with serum-containing medium. Using the CC cell group in the presence of serum-free medium dramatically increased the compressive stiffness to 128 +/- 34 kPa, similar to native tissue. Similarly, serum-free medium or TGF-beta1 treatment enhanced the tensile modulus by an order of magnitude, up to 3,000 kPa. Using two cell sources and manipulating biochemical stimuli, a range of fibrocartilaginous neotissues was engineered. Fibrocartilages such as the knee meniscus are characterized by heterogeneity in matrix and functional properties, and this work demonstrates a strategy for recreating these heterogeneous tissues.

Self-Assembly of Fibrochondrocytes and Chondrocytes for Tissue Engineering of the Knee Meniscus

Chondrocyte self-assembly in high-density scaffoldless culture has shown success in producing articular cartilage constructs, and a similar process could be applied to fibrocartilage tissue engineering. Three cell combinations were compared in self-assembly culture-100% chondrocytes, 100% meniscal fibrochondrocytes, and 50:50 co-cultures of fibrochondrocytes and chondrocytes with the goal of creating a proteoglycan, collagen I, and collagen II matrix similar to native meniscus. Two culture surfaces were also compared for self-assembly: agarose-coated wells and tissue culture plastic. After 4 weeks, the resulting self-assembled chondrocyte constructs were 10.24+/-0.63 mm in diameter and 0.96+/-0.14 mm thick, weighing 84.5+/-7.2 mg. Co-culture constructs were smaller and weighed 22.5+/-1.0 mg. In contrast, the fibrochondrocyte constructs contracted into spheres weighing 1.3+/-0.3 mg. Immunostaining showed collagen II in the chondrocyte constructs, both collagen I and collagen II in the co-cultures, and only collagen I in the fibrochondrocyte constructs. Collagen densities for chondrocyte, co-culture, and fibrochondrocyte constructs were 41+/-3, 38+/-3, and 20+/-2 microg/mg dry weight, and glycosaminoglycan densities were 230+/-2, 80+/-6, and 10+/-1 microg/mg dry weight, respectively. Self-assembled co-cultures, with their mixed collagen I and II matrix and robust gross characteristics, appear promising for tissue engineering of the knee meniscus.

Chondrogenic Differentiation of Human Embryonic Stem Cells: The Effect of the Micro-Environment

We have previously induced differentiation of embryonic stem cells (ESC) to specific phenotypes by manipulating the culture conditions, including the use of indirect co-culture. In this study, we hypothesized that co-culture with primary chondrocytes can induce human embryonic stem cells (hESC) to differentiate towards the chondrocyte lineage. Co-cultures of hESC and chondrocytes were established using well inserts, with control comprising hESC grown alone or with fibroblasts. After 28 days, after removal of the chondrocyte inserts, hESC differentiation was assessed, by morphology, immunocytochemistry, and reverse transcription polymerase chain reaction. hESC, co-cultured or grown alone, were also implanted into SCID mice on a poly-D, L-lactide scaffold, harvested 35 days later and assessed in the same way. hESC co-cultured with chondrocytes formed colonies and secreted extracellular matrix containing glycosaminoglycans (GAG). Quantitative assay showed increased synthesis of sulfated GAG in co-culture as compared with control hESC grown alone for the same period (p < 0.0001). In addition, co-cultured hESC expressed Sox 9 and collagen type II, unlike control hESC. Co-culture with fibroblasts did not induce chondrogenic differentiation. The implanted constructs with co-cultured hESC contained significantly more type II collagen (p < 0.01), type I collagen (p < 0.05), total collagen (p < 0.01), and GAG (p < 0.01) than those with hESC grown alone. Thus, we show for the first time differentiation of hESC to chondrocytes. Our results confirm the potential of the culture micro-environment to influence ESC differentiation and could provide the basis for future generation of chondrogenic cells for use in tissue repair and increase our understanding of the mechanisms that direct differentiation.

Co-culture of osteoblasts and chondrocytes modulates cellular differentiation in vitro

Biological integration of cartilage grafts with subchondral bone remains a significant clinical challenge. We hypothesize that interaction between osteoblasts and chondrocytes is important in regenerating the osteochondral interface on tissue-engineered osteochondral grafts. We describe here a sequential co-culturing model which permits cell–cell contact and paracrine interaction between osteoblast and chondrocytes in 3-D culture. This model was used to determine the effects of co-culture on the phenotypic maintenance of osteoblasts and chondrocytes. It was found that while chondrocytes synthesized a type II collagen and glycosaminoglycan (GAG) matrix, GAG deposition was significantly lower in co-culture. Alkaline phosphatase activity was maintained in osteoblasts, but cell-mediated mineralization in co-culture was markedly lower compared to osteoblast controls. These results collectively suggest that interactions between osteoblasts and chondrocytes modulate cell phenotypes, and the importance of these interactions on osteochondral interface regeneration will be explored in future studies.

Development of Controlled Heterogeneity on a Polymer-Ceramic Hydrogel Scaffold for Osteochondral Repair

Due to its intrinsically poor repair potential, injuries to articular cartilage do not heal and clinical intervention is required. Osteochondral grafts may improve healing while promoting integration with host tissue. We report here the development of an osteochondral graft based on a hybrid of a hyrogel and a polymer-bioactive glass composite (PLAGA-BG) microsphere scaffold. This novel osteochondral construct consists of three regions: gel-only, gel/composite interface, and a composite-only-region. The three phases differ in calcium phospate (Ca-P) or BG content. The objective of the current study is to investigate the effects of scaffold composition on chondrocyte response, and to evaluate the effects of co-culture on osteoblasts and chondrocyte growh and differentiation on the hybrid scaffold. The PLAGA-BG microsphere scaffold supported the growth of chondrocytes and initial results indicate that in the presence of BG, chondrocyte-mediated mineralization may be stimulated. Co-culture of osteoblasts and chondrocytes on the multi-phased scaffold with varied Ca-P content facilitated the formation of multiple matrix zones: a GAGrich chondrocyte region, an interfacial matrix rich in GAG+collagen, and a mineralized collagen matrix with osteoblasts. In summary, chondrocyte response has been optimized as a function of scaffold composition and the novel osteochondral graft has the potential to support the simultaneous formation of multiple types of tissue in vitro.

Clonal growth of human articular cartilage and the functional role of the periosteum in chondrogenesis

Clinical cartilage repair with transplantation of cultured chondrocytes, the first described technique introduced in 1994, includes a periosteal membrane but today cells are also implanted without the periosteal combination. The aim of this study was to see if the periosteum had more than a biomechanical function and if the periosteum had a biological effect on the seeded cells tested in an agarose system in which the clonal growth in agarose and the external growth stimulation could be analysed. Four different experiments were used to study the growth of human chondrocytes in agarose and the periosteal influence. Human chondrocytes were isolated and transferred to either primary or secondary agarose culture. After 4 weeks, the total number of clones >50 microm was counted. Cocultures of chondrocytes and periosteal tissue, cultures of chondrocytes with conditioned medium from chondrocytes, periosteal cells and fibroblast were used to study a potential stimulatory effect on growth and different cytokines and growth factors were analysed. It was found that the human chondrocytes had different growth properties in agarose with the formation of four different types of clones: a homogenous clone without matrix production, a homogenous clone with matrix production, a differentiated clone with matrix production and finally a differentiated clone without matrix production. The periosteum exerted a paracrine effect on cultured chondrocytes in agarose resulting in a higher degree of cloning. The chondrocytes produced significant amounts of interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-beta. The periosteum produced significant amounts of IL-6, IL-8 and TGF-beta. Cocultures of chondrocytes and periosteum demonstrated a potentiation of IL-6 and IL-8 release but not of TGF-beta and GM-CSF. Articular chondrocytes are able to form clones of different properties in agarose and the periosteum has a capacity of stimulating chondrocyte clonal growth and differentiation and secretes significant amounts of IL-6, IL-8, GM-CSF and TGF-beta. It may be that the repair of cartilage defects with seeded chondrocytes could benefit from the combination with a periosteal graft. The production of TGF-beta by implanted chondrocytes could influence the chondrogenic cells in the periosteum to start a periosteal chondrogenesis and together with the matrix from implanted chondrocyte production, a repair of cartilaginous appearance may develop; a dual chondrogenic response is possible.

Experimental study of in vitro chondrogenesis by co-culture of bone marrow stromal cells and chondrocytes

Chondrogenic microenvironments play a very important role in chondrogenesis of bone marrow stromal cells (BMSC). This study explored the feasibility of in vitro chondrogenesis by co-culture of BMSC and chondrocytes so as to confirm the hypothesis that chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of BMSC and thus promote in vitro chondrogenesis of BMSC. Porcine BMSC and auricular chondrocytes were in vitro expanded respectively and then were mixed at a ratio of 8:2 (BMSC:chondrocyte). 200 microl mixed cells(5.0 x 10(7)/ml) were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffold, 9 mm in diameter and 3 mm in thickness, as co-culture group. Chondrocytes and BMSC with the same cell number were seeded respectively onto the scaffolds as positive control (chondrocyte group) and negative control (BMSC group). 200 microl chondrocytes (1.0 x 10(7)/ml, equal to the chondrocyte number of co-culture group) alone were seeded as low concentration chondrocyte group. There were 6 specimens in each group. All specimens were harvested after in vitro culture for 8 weeks in DMEM plus 10% FBS. Gross observation, average wet weight measurement, glycosaminoglycan (GAG) quantification, histology and immunohistochemistry were used to evaluate the results. Cells in all groups had fine adhesion to the scaffolds and could secrete extracellular matrix. In both co-culture group and positive control group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture and formed homogenous mature cartilage after 8 weeks of in vitro culture. Furthermore, the neo-cartilages in both groups were similar to each other in gross appearance and histological features, and abundant type II collagen was also detected by immunohistochemistry in both groups. The average wet weight and GAG content of co-culture group were both more than 80% of those of positive control group. In negative control group, however, the constructs shrunk gradually during in vitro culture and cartilage-like tissue could only be observed at the edge area of the construct. In low concentration chondrocyte group, the constructs also shrunk gradually during in vitro culture and the average wet weight was below 40% of that of the positive control group although histology showed a small amount cartilage formation. Chondrocytes can provide a chondrogenic microenvironment to induce a chondrogenic differentiation of BMSC and thus promote the in vitro chondrogenesis of BMSC.