Scaffold-assisted cartilage tissue engineering using infant chondrocytes from human hip cartilage.

OBJECTIVE: Studies about cartilage repair in the hip and infant chondrocytes are rare. The aim of our study was to evaluate the use of infant articular hip chondrocytes for tissue engineering of scaffold-assisted cartilage grafts. METHOD: Hip cartilage was obtained from five human donors (age 1-10 years). Expanded chondrocytes were cultured in polyglycolic acid (PGA)-fibrin scaffolds. De- and re-differentiation of chondrocytes were assessed by histological staining and gene expression analysis of typical chondrocytic marker genes. In vivo, cartilage matrix formation was assessed by histology after subcutaneous transplantation of chondrocyte-seeded PGA-fibrin scaffolds in immunocompromised mice. RESULTS: The donor tissue was heterogenous showing differentiated articular cartilage and non-differentiated tissue and considerable expression of type I and II collagens. Gene expression analysis showed repression of typical chondrocyte and/or mesenchymal marker genes during cell expansion, while markers were re-induced when expanded cells were cultured in PGA-fibrin scaffolds. Cartilage formation after subcutaneous transplantation of chondrocyte loaded PGA-fibrin scaffolds in nude mice was variable, with grafts showing resorption and host cell infiltration or formation of hyaline cartilage rich in type II collagen. Addition of human platelet rich plasma (PRP) to cartilage grafts resulted robustly in formation of hyaline-like cartilage that showed type II collagen and regions with type X collagen. CONCLUSION: These results suggest that culture of expanded and/or de-differentiated infant hip cartilage cells in PGA-fibrin scaffolds initiates chondrocyte re-differentiation. The heterogenous donor tissue containing immature chondrocytes bears the risk of cartilage repair failure in vivo, which may be possibly overcome by the addition of PRP.

Analysis by cDNA microarrays of gene expression patterns of human adrenocortical tumors.

OBJECTIVES: Adrenocortical carcinoma (ACC) is a rare malignant neoplasm with extremely poor prognosis. The molecular mechanisms of adrenocortical tumorigenesis are still not well understood. The comparative analysis by cDNA microarrays of gene-expression patterns of benign and malignant adrenocortical tumors allows us to identify new tumor-suppressor genes and proto-oncogenes underlying adrenocortical tumorigenesis. DESIGN AND METHODS: Total RNA from fresh-frozen tissue of 10 ACC and 10 benign adrenocortical adenomas was isolated after histologic confirmation of neoplastic cellularity of at least 85%. The reference consisted of pooled RNA of 10 normal adrenal cortex samples. Amplified RNA of tumor and reference was used to synthesize Cy3- and Cy5-fluorescently labeled cDNA in a flip-color technique. D-chips containing 11 540 DNA spots were hybridized and scanned and the images were analyzed by ImaGene 3.0 software. RESULTS: The comparative analysis of gene expression revealed many genes with more than fourfold expression difference between ACC and normal tissue (42 genes), cortical adenoma and normal tissue (11 genes), and ACC and cortical adenoma (21 genes) respectively. As confirmed by real-time PCR, the IGF2 gene was significantly upregulated in ACCs versus cortical adenomas and normal cortical tissue. Genes that were downregulated in adrenocortical tumors included chromogranin B and early growth response factor 1. CONCLUSIONS: Comprehensive expression profiling of adrenocortical tumors by the cDNA microarray technique is a very powerful tool to elucidate the molecular steps associated with the tumorigenesis of these ill-defined neoplasms. To evaluate the role of identified genes, further detailed analyses, including correlation with clinical data, are required.

Expression profiling of Wilms tumors reveals new candidate genes for different clinical parameters.

Wilms tumor is the most frequent renal neoplasm in children, but our understanding of its genetic basis is still limited. We performed cDNA microarray experiments using 63 primary Wilms tumors with the aim of detecting new candidate genes associated with malignancy grade and tumor progression. All tumors had received preoperative chemotherapy as mandated by the SIOP protocol, which sets this study apart from related approaches in the Unites States that are based on untreated samples. The stratification of expression data according to clinical criteria allowed a rather clear distinction between different subsets of Wilms tumors. Clear-cut differences in expression patterns were discovered between relapse-free as opposed to relapsed tumors and tumors with intermediate risk as opposed to high risk histology. Several differentially expressed genes, e.g.TRIM22, CENPF, MYCN, CTGF, RARRES3 and EZH2, were associated with Wilms tumor progression. For a subset of differentially expressed genes, microarray data were confirmed by real-time RT-PCR on the original set of tumors. Interestingly, we found the retinoic acid pathway to be deregulated at different levels in advanced tumors suggesting that treatment of these tumors with retinoic acid may represent a promising novel therapeutic approach.

Programmed cell death protein 4 suppresses CDK1/cdc2 via induction of p21(Waf1/Cip1).

We show that the recently discovered tumor suppressor pdcd4 represses the transcription of the mitosis-promoting factor cyclin-dependent kinase (CDK)1/cdc2 via upregulation of p21(Waf1/Cip1). p21(Waf1/Cip1) inhibits CDK4/6 and CDK2. Decrease of CDK4/6 and CDK2 enhances the binding of pRb to E2F/DP, which in turn together bind to and repress the cdc2 promoter. Upregulation of CDK1/cdc2 accompanied by a malignant change was previously reported in colon cancer. We show that expression of pdcd4 as an indirect suppressor of CDK1/cdc2 is lost in progressed carcinomas of lung, breast, colon, and prostate. Furthermore, it seems that localization and expression of pdcd4 directly correlate with tumor progression. Finally, the CDK1/cdc2 inhibitor roscovitine reduces the proliferation of several tumor cell lines, suggesting that inhibition of CDK1/cdc2 may be a useful strategy against malignant transformation. Therefore, pdcd4 might serve as a novel target for antineoplastic therapies.