Histological and histochemical evaluation of human oral mucosa constructs developed by tissue engineering

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues is very common. In this context, tissue engineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we developed a new model of artificial oral mucosa generated by tissue engineering using a fibrin-agarose scaffold. For that purpose, we generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal oral mucosa using enzymatic treatments. Then we determined the viability of the cultured cells by electron probe quantitative X-ray microanalysis, and we demonstrated that most of the cells in the primary cultures were alive and had high K/Na ratios. Once cell viability was determined, we used the cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarose extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and the oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.

Analysis of TNFAIP3, a feedback inhibitor of nuclear factor-κB and the neighbor intergenic 6q23 region in rheumatoid arthritis susceptibility

INTRODUCTION Genome-wide association studies of rheumatoid arthritis (RA) have identified an association of the disease with a 6q23 region devoid of genes. TNFAIP3, an RA candidate gene, flanks this region, and polymorphisms in both the TNFAIP3 gene and the intergenic region are associated with systemic lupus erythematosus. We hypothesized that there is a similar association with RA, including polymorphisms in TNFAIP3 and the intergenic region. METHODS To test this hypothesis, we selected tag-single nucleotide polymorphisms (SNPs) in both loci. They were analyzed in 1,651 patients with RA and 1,619 control individuals of Spanish ancestry. RESULTS Weak evidence of association was found both in the 6q23 intergenic region and in the TNFAIP3 locus. The rs582757 SNP and a common haplotype in the TNFAIP3 locus exhibited association with RA. In the intergenic region, two SNPs were associated, namely rs609438 and rs13207033. The latter was only associated in patients with anti-citrullinated peptide antibodies. Overall, statistical association was best explained by the interdependent contribution of SNPs from the two loci TNFAIP3 and the 6q23 intergenic region. CONCLUSIONS Our data are consistent with the hypothesis that several RA genetic factors exist in the 6q23 region, including polymorphisms in the TNFAIP3 gene, like that previously described for systemic lupus erythematosus.

A combined approach for the assessment of cell viability and cell functionality of human fibrochondrocytes for use in tissue engineering.

Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5-P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5-P6 for cell therapy protocols.

Preoperative chemoradiotherapy in rectal cancer induces changes in the expression of nuclear β-catenin: prognostic significance.

BACKGROUND Preoperative chemoradiotherapy (CRT) is the cornerstone of treatment for locally advanced rectal cancer (LARC). Although high local control is achieved, overall rates of distant control remain suboptimal. Colorectal carcinogenesis is associated with critical alterations of the Wnt/β-catenin pathway involved in proliferation and survival. The aim of this study was to assess whether CRT induces changes in the expression of β-catenin/E-cadherin, and to determine whether these changes are associated with survival. METHODS The Immunohistochemical expression of nuclear β-catenin and membranous E-cadherin was prospectively analysed in tumour blocks from 98 stage II/III rectal cancer patients treated with preoperative CRT. Tumour samples were collected before and after CRT treatment. All patients were treated with pelvic RT (46-50 Gy in 2 Gy fractions) and 5-fluorouracil (5FU) intravenous infusion (225 mg/m2) or capecitabine (825 mg/m2) during RT treatment, followed by total mesorectal excision (TME). Disease-free survival (DFS) was analysed using the Kaplan-Meier method and a multivariate Cox regression model was employed for the Multivariate analysis. RESULTS CRT induced significant changes in the expression of nuclear β-catenin (49% of patients presented an increased expression after CRT, 17% a decreased expression and 34% no changes; p = 0.001). After a median follow-up of 25 months, patients that overexpressed nuclear β-catenin after CRT showed poor survival compared with patients that experienced a decrease in nuclear β-catenin expression (3-year DFS 92% vs. 43%, HR 0.17; 95% CI 0.03 to 0.8; p = 0.02). In the multivariate analysis for DFS, increased nuclear β-catenin expression after CRT almost reached the cut-off for significance (p = 0.06). CONCLUSIONS In our study, preoperative CRT for LARC induced significant changes in nuclear β-catenin expression, which had a major impact on survival. Finding a way to decrease CRT resistance would significantly improve LARC patient survival.