Novel functional profiling approach combining reverse phase protein microarrays and human 3-D ex vivo tissue cultures: expression of apoptosis-related proteins in human colon cancer

Cancer is caused by a complex pattern of molecular perturbations. To understand the biology of cancer, it is thus important to look at the activation state of key proteins and signaling networks. The limited amount of available sample material from patients and the complexity of protein expression patterns make the use of traditional protein analysis methods particularly difficult. In addition, the only approach that is currently available for performing functional studies is the use of serial biopsies, which is limited by ethical constraints and patient acceptance. The goal of this work was to establish a 3-D ex vivo culture technique in combination with reverse-phase protein microarrays (RPPM) as a novel experimental tool for use in cancer research. The RPPM platform allows the parallel profiling of large numbers of protein analytes to determine their relative abundance and activation level. Cancer tissue and the respective corresponding normal tissue controls from patients with colorectal cancer were cultured ex vivo. At various time points, the cultured samples were processed into lysates and analyzed on RPPM to assess the expression of carcinoembryonic antigen (CEA) and 24 proteins involved in the regulation of apoptosis. The methodology displayed good robustness and low system noise. As a proof of concept, CEA expression was significantly higher in tumor compared with normal tissue (p<0.0001). The caspase 9 expression signal was lower in tumor tissue than in normal tissue (p<0.001). Cleaved Caspase 8 (p=0.014), Bad (p=0.007), Bim (p=0.007), p73 (p=0.005), PARP (p<0.001), and cleaved PARP (p=0.007) were differentially expressed in normal liver and normal colon tissue. We demonstrate here the feasibility of using RPPM technology with 3-D ex vivo cultured samples. This approach is useful for investigating complex patterns of protein expression and modification over time. It should allow functional proteomics in patient samples with various applications such as pharmacodynamic analyses in drug development.

Bovine primary chondrocyte culture in synthetic matrix metalloproteinase-sensitive poly(ethylene glycol)-based hydrogels as a scaffold for cartilage repair

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

Assessment of the Matrix Degenerative Effects of MMP-3, ADAMTS-4 and HTRA1 injected into a bovine Intervertebral Disc Organ Culture Model

Study Design. In vitro study to develop an intervertebral disc degeneration (IDD) organ culture model, using coccygeal bovine intervertebral discs (IVDs) and injection of proteolytic enzymes MMP-3, ADAMTS-4 and HTRA1.Objective. This study aimed to develop an in-vitro model of enzyme-mediated IDD to mimic the clinical outcome in humans for investigation of therapeutic treatment options.Summary of Background Data. Bovine IVDs are comparable to human IVDs in terms of cell composition and biomechanical behavior. Researchers injected papain and trypsin into them to create an IDD model with a degenerated nucleus pulposus (NP) area. They achieved macroscopic cavities as well as a loss of glycosaminoglycans (GAGs). However, none of these enzymes are clinically relevant.Methods. Bovine IVDs were harvested maintaining the endplates. Active forms of MMP-3, ADAMTS-4 and HTRA1 were injected at a dose of 10μg/ml each. Phosphate buffered saline (PBS) was injected as a control. Discs were cultured for 8 days and loaded diurnally (day 1 to day 4 with 0.4 MPa for 16 h) and left under free swelling condition from day 4 to day 8 to avoid expected artifacts due to dehydration of the NP. Outcome parameters included disc height, metabolic cell activity, DNA content, glycosaminoglycan (GAG) content, total collagen content, relative gene expression and histological investigation.Results. The mean metabolic cell activity was significantly lower in the NP area of discs injected with ADAMTS-4 compared to the day 0 control discs. Disc height was decreased following injection with HTRA1, and was significantly correlated with changes in GAG/DNA of the NP tissue. Total collagen content tended to be lower in groups injected with ADAMTS4 and MMP-3.Conclusion. MMP-3, ADAMTS-4 and HTRA1 neither provoked visible matrix degradation nor major shifts in gene expression. However, cell activity was significantly reduced and HTRA1 induced loss of disc height which positively correlated with changes in GAG/DNA content. The use of higher doses of these enzymes or a combination thereof may therefore be necessary to induce disc degeneration

In situ fiber-optical monitoring of cytosolic calcium in tissue explant cultures

We present a fluorescence-lifetime based method for monitoring cell and tissue activity in situ, during cell culturing and in the presence of a strong autofluorescence background. The miniature fiber-optic probes are easily incorporated in the tight space of a cell culture chamber or in an endoscope. As a first application we monitored the cytosolic calcium levels in porcine tracheal explant cultures using the Calcium Green-5N (CG5N) indicator. Despite the simplicity of the optical setup we are able to detect changes of calcium concentration as small as 2.5 nM, with a monitoring time resolution of less than 1 s.

Expression and Regulation of Human Cytochrome P450 4F Isoforms in Tissue Samples and Under TNF-Alpha Challenges

CYP4F (Cytochrome P4504F) enzymes metabolize endogenous molecules including leukotrienes, prostaglandins and arachidonic acid. The involvement of these endogenous compounds in inflammation has led to the hypothesis that changes in the inflamed tissue environment may affect the expression of CYP4Fs during the pro-inflammatory state, which in turn may modulate inflammatory conditions during the anti-inflammatory state. We demonstrated that inflamed tissues have different levels of CYP4F isoform expression profiles in a number of human samples when compared to the average population. The CYP4F isoform expression levels change with the degree of inflammation present in tissue. Further investigation in cell culture studies revealed that inflammatory cytokines, in particular TNF-α, play a role in regulating the expression of the CYP4F family. One of the isoforms, CYP4F11, had different characteristics than that of the other five CYP4F family members. CYP4F11 metabolizes xenobiotics while the other isoforms metabolize endogenous compounds with higher affinity. CYP4F11 also was expressed at high quantities in the brain, and was up-regulated by TNF-α, while the other isoforms were not expressed at high quantities in the brain and were down-regulated by TNF-α. We identified the AP-1 protein of the JNK pathway as the signaling protein that causes significant increase in CYP4F11 expression. Since TNF-α stimulation causes a simultaneous activation of both JNK pathway and NF-κB signaling, we investigated further the role that NF-κB plays on expression of the CYP4F11 gene. We concluded that although there is a significant increase in CYP4F11 expression in the presence of TNF-α, the activation of NF-κB signaling inhibits CYP4F11 expression in a time dependent manner. The expression of CYP4F11 is only significantly increased after 24 hours of treatment with TNF-α; at shorter time points NF-κB signaling overpowers the JNK pathway activation. We believe that these findings may in the future lead to improved drug design for modulating inflammation.

Osteopontin (OPN) and neoplastic disease: Circulating levels and tissue distribution

OPN is a secreted phosphate containing protein which is expressed by osteoblasts and a variety of other cells in vivo. Data from in vitro studies has accumulated which relates OPN to cellular transformation. We hypothesize that OPN expression is associated with neoplastic disease in humans as suggested by cell culture models. The overall objective of the current study was to determine the tissue distribution of OPN in human malignancy and to determine whether or not a correlation exists between OPN serum levels and malignancy. At the inception of this project, no study had been made demonstrating the relevance of OPN expression with naturally occurring neoplastic disease in humans. To date, few studies have reported OPN distribution in human neoplasia and are limited by either the number of specimens analyzed or the technique used in analysis. In this dissertation study, OPN was purified from human milk and $\alpha$-OPN antiserum developed and characterized. Following antibody development, the distribution and prevalence of OPN in human oral squamous cell carcinoma and human prostate carcinoma was evaluated using immunohistochemical localization. OPN immunolocalization was found in a high percentage of oral epithelial dysplasia and oral squamous cell carcinoma in humans. One oral squamous cell carcinoma cells line, UMSCC-1, was found to express OPN mRNA using Northern blotting. OPN localized to a high percentage of primary prostate adenocarcinomas. OPN localized to 52% of androgen dependent cases and 100% of androgen independent cases. Androgen dependent cell lines such as LNCap and NbE showed minimal OPN mRNA expression while the androgen independent lines C4-2 and PC3 produced ample OPN mRNA. An OPN sandwich assay was developed and used to determine the serum level of OPN in normal males, patients with BPH (benign prostate hypertrophy), and patients with prostate carcinoma. No statistically significant difference was found in OPN serum levels among the three groups. However, a trend of increasing OPN in the serum was noted in patients with BPH and prostate cancer. ^

Organotypic slice cultures from rat brain tissue: a new approach for Naegleria fowleri CNS infection in vitro

The free-living amoeba Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM), a disease leading to death in the vast majority of cases. In patients suffering from PAM, and in corresponding animal models, the brain undergoes a massive inflammatory response, followed by haemorrhage and severe tissue necrosis. Both, in vivo and in vitro models are currently being used to study PAM infection. However, animal models may pose ethical issues, are dependent upon availability of specific infrastructural facilities, and are time-consuming and costly. Conversely, cell cultures lack the complex organ-specific morphology found in vivo, and thus, findings obtained in vitro do not necessarily reflect the situation in vivo. The present study reports infection of organotypic slice cultures from rat brain with N. fowleri and compares the findings in this culture system with in vivo infection in a rat model of PAM, that proved complementary to that of mice. We found that brain morphology, as present in vivo, is well retained in organotypic slice cultures, and that infection time-course including tissue damage parallels the observations in vivo in the rat. Therefore, organotypic slice cultures from rat brain offer a new in vitro approach to study N. fowleri infection in the context of PAM.

Co-culture of Notochordal Cells with Nucleus pulposus and Annulus fibrosus cells under normoxia and hypoxia

Introduction Notochordal cells (NC) are shifted back into focus due to their apparent action of activating other disc cells via indirect release of yet unknown factors into the medium (conditioned medium = CM).1,2 Recent evidence confirms the results from the late 1990s.3,4 Here, we test porcine (p) NC cultured in 3D and the influence of adding serum or using serum-free medium onto the culture on NC cells and its stimulating effects for subsequent coculture with primary bovine (b) nucleus pulposus (bNPC) and annulus fibrous cells (bAFC). Materials and Methods Primary pNC, bNPC, and bAFC were isolated from porcine tails (< 6-12 months age) or bovine tails (∼1 year age), which were obtained from the food chain (N = 4 repeats) within 4 hours postmortem. All cells were seeded into 1.2% alginate, each with a density of 4 × 106/mL. NC were then either cultured for 7 days in serum free medium (SFM = Dulbecco modified eagle medium [DMEM] supplied with ITS+, 50 µg/mL vitamin C and nonessential amino acids) or DMEM + 10% fetal calf serum (FCS). CM was produced from NC collecting 4 mL SFM and keeping approximately 30 beads for 7 days. Then, a coculture was set up in SFM for 14 days using indirect cell-cell contact (culture insert, high density pore, 0.4 µm) using a 50:50% ratio5 of pNC:bNP or bAF, or by addition of CM, respectively. The cell activity, glycosaminoglycan per DNA (GAG/DNA) ratio, and real-time RT-PCR of IVD relevant genes were monitored. Mass spectrometry was performed on the SFM and the cocultured medium as well as the CM of the pNC to identify possible key cytokines to the stimulatory effects. Results The results for cell activity confirmed that pNC are highly responsive on the nutritional condition in the culture (K-W test, p = 0.048) after 7 days of coculture. bNPC and bAFC did not respond significantly different to coculture or addition of CM with respect to cell activity. However, GAG/DNA ratio of pNC was significantly upregulated if they were initially pre-exposed to FCS and in coculture with bNPC after 14 days, for both normoxia and hypoxia (K-W, p = 0.03). The bNPC were stimulated by both, 1:1 coculture with pNC but also by addition of CM only, which resulted in approximately 200% increased GAG/DNA values relative to the day 0 state. However, this doubling of the GAG/DNA ratio was nonsignificant after 14 days. The aggrecan/collagen type 2 ratio as quantified from real-time RT-PCR pointed to a beneficial state of the bNPC if cultured either in indirect coculture with pNC or by the addition of CM (Fig. 1). The mass spectrometric analysis of the CM revealed that there was connecting tissue growth factor present (CTGF) among the cytokine CLC11, a cytokine that has been found to be expressed in skeletal tissues including bone marrow and chondrocytes among other factors that might have immunoregulatory and cell proliferative functions.

CCL5/RANTES is a key chemoattractant released by degenerative intervertebral discs in organ culture.

Release of chemotactic factors in response to tissue damage has been described for different musculoskeletal tissues, including the intervertebral disc (IVD). This study investigated the chemoattractants that are released by induced degenerative IVDs and may be involved in recruiting mesenchymal stem cells (MSCs). Bovine caudal discs were cultured within a bioreactor and loaded under conditions that mimicked physiological or degenerative settings. Between days 4-6, medium was replaced by PBS, which was subsequently used for proteomic, ELISA and immunoprecipitation analyses of secreted chemokines and cytokines. A Boyden chamber assay was used to observe human MSC migration towards native and chemokine depleted media. Gene expression levels of chemokine receptors in human MSCs were analysed, and CCL5 was localised in bovine and human IVD by immunohistochemistry. Proteomic analysis revealed the presence of CCL5 and CXCL6 within conditioned media. Higher concentrations of CCL5 were found in the degenerative media, and a relationship was found between interleukin-1β and CCL5 concentration. Chemokine immunoprecipitation showed that MSCs had a significantly reduced chemotactic migration towards CCL5-immunoprecipitated and CCL5/CXCL6 co-immunoprecipitated media, whilst CXCL6 depletion did not change MSC chemotaxis. MSCs showed a significant increase in mRNA expression of the CCL5 receptors, CCR1 and CCR4, upon culture in degenerative media. Furthermore, CCL5 was identified in bovine and human disc tissue by immunohistochemistry. Hence, CCL5 may be a key chemoattractant that is produced and released by the intervertebral disc cells. Therefore, these factors could be used to enhance stem/progenitor cell mobilisation in regenerative therapies for early stages of disc degeneration.

Covalent immobilisation of VEGF on plasma-coated electrospun scaffolds for tissue engineering applications.

Recent findings in the field of biomaterials and tissue engineering provide evidence that surface immobilised growth factors display enhanced stability and induce prolonged function. Cell response can be regulated by material properties and at the site of interest. To this end, we developed scaffolds with covalently bound vascular endothelial growth factor (VEGF) and evaluated their mitogenic effect on endothelial cells in vitro. Nano- (254±133 nm) or micro-fibrous (4.0±0.4 μm) poly(ɛ-caprolactone) (PCL) non-wovens were produced by electrospinning and coated in a radio frequency (RF) plasma process to induce an oxygen functional hydrocarbon layer. Implemented carboxylic acid groups were converted into amine-reactive esters and covalently coupled to VEGF by forming stable amide bonds (standard EDC/NHS chemistry). Substrates were analysed by X-ray photoelectron spectroscopy (XPS), enzyme-linked immuno-assays (ELISA) and immunohistochemistry (anti-VEGF antibody and VEGF-R2 binding). Depending on the reaction conditions, immobilised VEGF was present at 127±47 ng to 941±199 ng per substrate (6mm diameter; concentrations of 4.5 ng mm(-2) or 33.3 ng mm(-2), respectively). Immunohistochemistry provided evidence for biological integrity of immobilised VEGF. Endothelial cell number of primary endothelial cells or immortalised endothelial cells were significantly enhanced on VEGF-functionalised scaffolds compared to native PCL scaffolds. This indicates a sustained activity of immobilised VEGF over a culture period of nine days. We present a versatile method for the fabrication of growth factor-loaded scaffolds at specific concentrations.

Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.

For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re-introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to <0.1%, whereas transcript levels encoding COL1 increased 370-fold as compared to primary chondrocytes. Flow cytometry for intracellular proteins revealed that chondrocytes acquired a COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to <2% in primary chondrocytes to passage six cells, the fraction of COL1 positive cells increased from <1% to >95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue.

L-mimosine increases the production of vascular endothelial growth factor in human tooth slice organ culture model.

AIM To assess the pro-angiogenic and pro-inflammatory capacity of the dentine-pulp complex in response to the prolyl hydroxylase inhibitor L-mimosine in a tooth slice organ culture model. METHODOLOGY Human teeth were sectioned transversely into 600-μm-thick slices and cultured in medium supplemented with serum and antibiotics. Then, pulps were stimulated for 48 h with L-mimosine. Pulps were subjected to viability measurements based on formazan formation in MTT assays. In addition, histological evaluation of pulps was performed based on haematoxylin and eosin staining. Culture supernatants were subjected to immunoassays for vascular endothelial growth factor (VEGF) to determine the pro-angiogenic capacity and to immunoassays for interleukin (IL)-6 and IL-8 to assess the pro-inflammatory response. Interleukin-1 served as pro-inflammatory control. Echinomycin was used to inhibit hypoxia-inducible factor-1 (HIF-1) alpha activity. Data were analysed using Student's t-test and Mann-Whitney U test. RESULTS Pulps within tooth slices remained vital upon L-mimosine stimulation as indicated by formazan formation and histological evaluation. L-mimosine increased VEGF production when normalized to formazan formation in the pulp tissue of the tooth slices (P < 0.05). This effect on VEGF was reduced by echinomycin (P < 0.01). Changes in normalized IL-6 and IL-8 levels upon treatment with L-mimosine did not reach the level of significance (P > 0.05), whilst treatment with IL-1, which served as positive control, increased IL-6 (P < 0.05) and IL-8 levels (P < 0.05). CONCLUSIONS The prolyl hydroxylase inhibitor L-mimosine increased VEGF production via HIF-1 alpha in the tooth slice organ culture model whilst inducing no prominent increase in IL-6 and IL-8. Pre-clinical studies will reveal if these in vitro effects translate into dental pulp regeneration.

Duration-dependent influence of dynamic torsion on the intervertebral disc: an intact disc organ culture study.

PURPOSE Mechanical loading is an important parameter that alters the homeostasis of the intervertebral disc (IVD). Studies have demonstrated the role of compression in altering the cellular metabolism, anabolic and catabolic events of the disc, but little is known how complex loading such as torsion-compression affects the IVD cell metabolism and matrix homeostasis. Studying how the duration of torsion affects disc matrix turnover could provide guidelines to prevent overuse injury to the disc and suggest possible beneficial effect of torsion. The aim of the study was to evaluate the biological response of the IVD to different durations of torsional loading. METHODS Intact bovine caudal IVD were isolated for organ culture in a bioreactor. Different daily durations of torsion were applied over 7 days at a physiological magnitude (±2°) in combination with 0.2 MPa compression, at a frequency of 1 Hz. RESULTS Nucleus pulpous (NP) cell viability and total disc volume decreased with 8 h of torsion-compression per day. Gene expression analysis suggested a down-regulated MMP13 with increased time of torsion. 1 and 4 h per day torsion-compression tended to increase the glycosaminoglycans/hydroxyproline ratio in the NP tissue group. CONCLUSIONS Our result suggests that load duration thresholds exist in both torsion and compression with an optimal load duration capable of promoting matrix synthesis and overloading can be harmful to disc cells. Future research is required to evaluate the specific mechanisms for these observed effects.

Prevention of vascular dysfunction and arterial hypertension in mice generated by assisted reproductive technologies by addition of melatonin to culture media.

Assisted reproductive technologies (ART) induce vascular dysfunction in humans and mice. In mice, ART-induced vascular dysfunction is related to epigenetic alteration of the endothelial nitric oxide synthase (eNOS) gene, resulting in decreased vascular eNOS expression and nitrite/nitrate synthesis. Melatonin is involved in epigenetic regulation, and its administration to sterile women improves the success rate of ART. We hypothesized that addition of melatonin to culture media may prevent ART-induced epigenetic and cardiovascular alterations in mice. We, therefore, assessed mesenteric-artery responses to acetylcholine and arterial blood pressure, together with DNA methylation of the eNOS gene promoter in vascular tissue and nitric oxide plasma concentration in 12-wk-old ART mice generated with and without addition of melatonin to culture media and in control mice. As expected, acetylcholine-induced mesenteric-artery dilation was impaired (P = 0.008 vs. control) and mean arterial blood pressure increased (109.5 ± 3.8 vs. 104.0 ± 4.7 mmHg, P = 0.002, ART vs. control) in ART compared with control mice. These alterations were associated with altered DNA methylation of the eNOS gene promoter (P < 0.001 vs. control) and decreased plasma nitric oxide concentration (10.1 ± 11.1 vs. 29.5 ± 8.0 μM) (P < 0.001 ART vs. control). Addition of melatonin (10(-6) M) to culture media prevented eNOS dysmethylation (P = 0.005, vs. ART + vehicle), normalized nitric oxide plasma concentration (23.1 ± 14.6 μM, P = 0.002 vs. ART + vehicle) and mesentery-artery responsiveness to acetylcholine (P < 0.008 vs. ART + vehicle), and prevented arterial hypertension (104.6 ± 3.4 mmHg, P < 0.003 vs. ART + vehicle). These findings provide proof of principle that modification of culture media prevents ART-induced vascular dysfunction. We speculate that this approach will also allow preventing ART-induced premature atherosclerosis in humans.

Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-βs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-β is essential for the formation of cartilage-like tissue and that alterations within the TGF-β signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-β1 was required to induce the formation of cartilage-like tissue. When TGF-β signaling was blocked by inhibiting the TGF-β receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-β receptors 1 and 2 and TGF-β2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-β. We propose that a decrease in the expression of the chondrogenic growth factor TGF-β2 and of the TGF-β receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-β signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.