Dual crosslinked pullulan-gelatin cryogel scaffold for chondrocyte-mediated cartilage repair: synthesis, characterization andin vitroevaluation.

Cryogels, a subset of hydrogels, have recently drawn attention for cartilage tissue engineering due to its inherent microporous architecture and good mechanical properties. In this study a dual crosslinked pullulan-gelatin cryogel (PDAG) scaffold was synthesized by crosslinking gelatin with oxidized pullulan by Schiff's base reaction followed by cryogelation. Chondrocytes seeded within the PDAG scaffolds and cultured for 21 din vitrodemonstrated enhanced cell proliferation, enhanced production of cartilage-specific extracellular matrix and up-regulated sulfated glycosaminoglycan without altering the articular chondrocyte phenotype. Quantitative reverse transcription-polymerase chain reaction-based gene expression studies, immunofluorescence, and histological studies demonstrated that the PDAG scaffold significantly enhanced the expression of chondrogenic marker genes such as type II collagen, aggrecan, and SOX9. Taken together, these results demonstrated that PDAG scaffold prepared by sequential Schiff's base reaction and cryogelation would be a promising cell-responsive scaffold for cartilage tissue engineering applications.

Glutamic acid-based dendritic peptides for scaffold-free cartilage tissue engineering.

Current treatment modalities for cartilage regeneration often result in the production of fibrous-type cartilage tissue at the defect site, which has inferior mechanical properties as compared to native hyaline cartilage. Further, effective treatments are not available at present, for preventing age-related as well as disease-related hypertrophic development of chondrocytes. In the present study, we designed and synthesized three sets of glutamic acid-based dendritic peptides, differing in degree of lipidation as well as branching. Each set constitutes of N-terminal protected as well as corresponding N-deprotected peptides. Altogether, six peptides [BE12, E12, BE3(12)4, E3(12)4, BE3OMe, E3OMe] were tested for their chondrogenesis enhancing potential in vitro, using rabbit adipose derived mesenchymal stem cells (ADMSCs). Immunohistochemical and gene expression studies as well as biochemical analyses revealed that the lipopeptides [E12 and BE3(12)4] are able to enhance chondrogenic differentiation of ADMSCs significantly (p < 0.001) as compared to control group (chondrogenic medium alone). Glycosaminoglycan content, and the chondrogenic marker genes like Aggrecan (Acan), Type II collagen (Col2a1), Hyaluronan synthase 2 (Has2), and SRY-box 9 (Sox9) expressions were found to be significantly increased in E12 and BE3(12)4 treated groups. Most importantly, the BE3(12)4 treated group showed significantly lower Type I collagen (Col1a2) and Type X collagen (Col10a1) transcript levels (p < 0.001), indicating its potential for hyaline cartilage formation and also to prevent hypertrophic development. Thus, the lipopeptides E12 and BE3(12)4 may be useful for preventing chondrocyte hypertrophy and realizing the hyaline nature of regenerated cartilage tissue in tissue engineering. STATEMENT OF SIGNIFICANCE: The current treatment modalities for degenerative cartilage diseases are unsatisfactory as the resultant regenerated cartilage is often fibrous in nature with inferior mechanical properties. Further, there is no proper treatment available for age-related development of chondrocyte hypertrophy at present. In this study we synthesized glutamic acid-based lipopeptides, which differ in the degree of lipidation as well as branching. We used a combinatorial approach of scaffold-free tissue engineering and dendritic lipopeptides to achieve hyaline-like cartilage tissue from adipose derived mesenchymal stem cells in vitro. Gene expression analysis revealed the down regulation of fibrous cartilage marker Col1a2 and hypertrophic marker Col10a1, suggesting that these lipopeptides may be useful for achieving mechanically superior hyaline cartilage regeneration in future.

Prognostic implication of the loss of TGFBR2 expression in oral carcinoma.

Oral squamous cell carcinoma (OSCC) is a disease that strikes many worldwide, accounting for more than 145,000 deaths annually. This study examined the role of Transforming Growth Factor Beta (TGFß) signalling alterations in oral carcinogenesis and also its influence on the disease prognosis. In presented study, we evaluated the protein-level alterations of core TGFß signalling members in 20 potentially malignant oral disorders (PMDs) - leukoplakia and submucous fibrosis and 87 oral cancer samples by western blotting. Further, we analysed the association between these alterations and prognosis of oral carcinoma. For statistical analyses, univariate test like Student's 't'-test to compare expression level of various genes and logrank test has been used to compare the Kaplan-Meier survival curves. The multivariate model such as Cox's proportional hazard regression was used to verify the independent influence of each variable on the survival endpoints. A gradual decrease in the expression of TGFß signalling members like SMAD2, SMAD4, TGFBR1 and TGFBR2 have been noted from normal to PMD in oral cancers. The bio-activeforms, SMAD2/3 also showed a similar trend. SMAD3 protein was downregulated significantly in the PMD stage itself. Thus an inverse correlation was observed between expression of TGFß members and oral cancer progression.Furthermore, oral cancer patients showing TGFBR2 downregulation exhibited poor disease-free survival (p=0.005) and poor overall survival (p=0.012).Thus, assessing the TGFBR2 protein levels can serve as one of the prognostic marker for oral cancer.

Prognostic implication of the loss of TGFBR2 expression in oral carcinoma.

Oral squamous cell carcinoma (OSCC) is a disease that strikes many worldwide, accounting for more than 145,000 deaths annually. This study examined the role of Transforming Growth Factor Beta (TGFß) signalling alterations in oral carcinogenesis and also its influence on the disease prognosis. In presented study, we evaluated the protein-level alterations of core TGFß signalling members in 20 potentially malignant oral disorders (PMDs) - leukoplakia and submucous fibrosis and 87 oral cancer samples by western blotting. Further, we analysed the association between these alterations and prognosis of oral carcinoma. For statistical analyses, univariate test like Student's 't'-test to compare expression level of various genes and logrank test has been used to compare the Kaplan-Meier survival curves. The multivariate model such as Cox's proportional hazard regression was used to verify the independent influence of each variable on the survival endpoints. A gradual decrease in the expression of TGFß signalling members like SMAD2, SMAD4, TGFBR1 and TGFBR2 have been noted from normal to PMD in oral cancers. The bio-activeforms, SMAD2/3 also showed a similar trend. SMAD3 protein was downregulated significantly in the PMD stage itself. Thus an inverse correlation was observed between expression of TGFß members and oral cancer progression.Furthermore, oral cancer patients showing TGFBR2 downregulation exhibited poor disease-free survival (p=0.005) and poor overall survival (p=0.012).Thus, assessing the TGFBR2 protein levels can serve as one of the prognostic marker for oral cancer.

The early manifestation, tumor-specific occurrence and prognostic significance of TGFBR2 aberrant splicing in oral carcinoma.

Alternative splicing is an important mechanism that can disrupt cell cycle control resulting in tumorigenesis. Although many alterations of Transforming Growth Factor Beta (TGFß) signaling are reported in cancers, the role of splice aberrations in destabilizing this signaling is the least understood mechanism. In this study, we compared TGFBR2 alternative splicing events in potentially malignant oral disorders (PMDs) and oral squamous cell carcinoma (OSCC) samples with those in normal samples. Interestingly, there were five alternatively spliced forms of TGFBR2 with a deficient kinase domain in OSCCs. The TGFBR2 aberrant splicing was tumor-specific, suggesting that selective splicing out of TGFBR2 kinase domain could be a mechanism misused by cancer cells for evading TGFß signaling-mediated anti-tumor activities. Moreover, these aberrant transcripts were present in PMDs as well, suggesting an early occurrence of these events during oral carcinogenesis and offering the possibility of early diagnosis of malignancy. Furthermore, OSCC patients who harbored these aberrantly spliced transcripts exhibited poor disease free survival (p=0.028) and poor overall survival (p=0.013). Thus, assessing the presence of these TGFBR2 transcripts can serve as a prognostic marker for oral cancer.

The microRNA networks of TGFß signaling in cancer.

In metazoans, the transforming growth factor ß (TGFß) signaling regulates a host of activities ranging from embryonic development to tissue homeostasis. The normal as well as tumor cells respond to this cytokine signaling pathway in a highly context-dependent manner. It acts as a potent tumor suppressor initially by inducing cell cycle arrest and apoptosis. But advanced tumors often misuse TGFß signaling for tumor progression by selectively disabling the tumor suppressor arm and using other properties of TGFß signaling such as induction of angiogenesis, epithelial to mesenchymal transition, and metastases. This dual role of TGFß in cancer remained a mystery until recently. But recent advances in the field of microRNA provided a deeper understanding about this dual nature of TGFß signaling in cancers. In the present review, we present an account of the role of microRNAs in deregulating TGFß signaling and modulating cancer cell behavior during tumor initiation and cancer progression. This review also includes a discussion on the recent advances in the deregulation of TGFß signaling in carcinogenesis.