Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate.

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Available online xxxKeywords: Articular cartilage Sodium alginate Chondrogenesis Extra-cellular matrix (ECM) Differentiation a b s t r a c t Aim: Alternate strategies to regenerate the damaged tissue require exogenous supply of several chondrocyte implants. There are inherent challenges to optimize an appropriate tissue culture methodology that can aid in enrichment of chondrocytes. The aim of the study was to explore the differentiation potential, expansion and growth kinetics of the human adipose derived stem cells (hADSCs) in alginate microspheres in comparison to chondrogenesis from the cartilage tissue.Methods: Isolated hADSCs and cartilage derived chondrocytes were cultured and characterized. The distribution of stem cells within alginate bead was imaged by scanning electron microscopy (SEM). Encapsulated hADSCs were monitored for their cell viability, cell proliferation and apoptosis via JC-1 staining, MTT assay and Annexin V assays respectively.The alginate cell constructs were analyzed for chondrogenic gene expression by RT-PCR.Results: The chondrocyte pellet culture from cartilage demonstrated lower growth potential as compared to alginate encapsulation. hADSCs were successfully encapsulated within alginate matrix with >80% cell viability. Apoptotic assays provided safety profile for the alginate during cell growth. The up-regulation of cartilage specific genes like TGF-b, collagen type-X, COMP was observed during the entire period of culture. Conclusion:The chondrogenesis in pellet culture from cartilage tissue conserved the chondrocyte phenotype better with rich GAG polysaccharides. However, owing to an enriched chondrocyte requirement, alginate as a scaffold design would aid in the treatment of large focal defects.

Fluorescent magnetic iron oxide nanoparticles have been used to label cells for imaging as well as for therapeutic purposes. The purpose of this study was to modify the approach to develop a nanoprobe for cell selection and imaging with a direct therapeutic translational focus. The approach involves physical coincubation and adsorption of superparamagnetic iron oxide nanoparticle-polyethylene glycol (SPION-PEG) complexes with a monoclonal antibody (mAb) or a set of antibodies. Flow cytometry, confocal laser scanning microscopy, transmission electron microscopy, iron staining, and magnetic resonance imaging were used to assess cell viability, function, and labeling efficiency. This process has been validated by selecting adipose tissue-derived cardiac progenitor cells from the stromal vascular fraction using signal regulatory protein alpha (SIRPA)/kinase domain receptor (KDR) mAbs. These markers were chosen because of their sustained expression during cardiomyocyte differentiation. Sorting of cells positive for SIRPA and KDR allowed the enrichment of cardiac progenitors with 90% troponin-I positivity in differentiation cultures. SPION labeled cardiac progenitor cells (1×10 5 cells) was mixed with gel and used for 3T magnetic resonance imaging at a concentration, as low as 12.5 μg of iron. The toxicity assays, at cellular and molecular levels, did not show any detrimental effects of SPION. Our study has the potential to achieve moderate to high specific cell selection for the dual purpose of imaging and therapy.

There is an increasing evidence suggesting the role of fork head boxP3 (FoxP3) in the development and the regulation of CD4+CD25+ Treg cells. T-cell regulatory mechanisms in rheumatoid arthritis patients were evaluated by the contributing factors such as pro-inflammatory cytokines, circulating immune complexes, HLA DR expression, ligand binding biomarkers, FoxP3 expression in paired samples of peripheral blood (PB) and synovial fluid (SF). These cellular responses were further correlated with the humoral immune responses such as anti-cyclic citrullinated peptides IgG (CCP), circulating immune complex-c1q IgG (CIC), immunoglobulin G (IgG) and immunoglobulin M (IgM) of the rheumatoid arthritis factor (RAF). The results suggest a definitive role of Tregs in the homeostatic control because there is an increase in FoxP3 (37%) and HLA-DR (45%) expression in the synovial fluid as compared to PB. Furthermore, humoral responses as a downstream effector mechanism are positively correlated with the pathogenesis of rheumatoid arthritis (RA). A positive relationship exists between quantitative anti-CCP production and the expression of HLA-DR. The study relates an increased and pivotal role of B cell activation in the synovial fluid thereby permitting the need to ablate the targeted B cell immune responses.