Substrate-dependent gene regulation of self-assembled human MSC spheroids on chitosan membranes.

BACKGROUND: Three-dimensional (3D) multicellular spheroids of mesenchymal stem cells (MSCs) are generally regarded to have beneficial properties over MSCs in monolayer. Recent literatures have documented that MSCs can self-assemble into 3D spheroids with a greater capacity for differentiation into various cell types when grown on chitosan (CS), a biopolymer. The genomic modulation occurring in these MSC spheroids is thus of essential importance for understanding their uniqueness and therapeutic potentials. In this study, 3D spheroids self-assembled from human umbilical cord MSCs grown on CS membranes were analyzed by mRNA as well as microRNA microarrays, which helped identify the critical signaling events that may alter the cellular functions during the spheroid forming process. RESULTS: Genes screened from mRNA and microRNA cross-correlation analyses were further confirmed with the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. Results revealed the regulation of a significant number of calcium-associated genes, which suggested the crucial role of calcium signaling in CS-derived MSC spheroids. In addition, many genes associated with the multilineage differentiation capacities and those associated with the antiinflammatory and antitumor properties of MSCs were upregulated. The genetic modulation was significantly more remarkable and endured longer for MSC spheroids derived on CS substrates compared to those derived on a non-adherent (polyvinyl alcohol) substrate. CONCLUSIONS: Based on the study, the culture substrates used to prepare 3D MSC spheroids may predefine their properties through cell-substrate interaction.

Neocartilage formation from mesenchymal stem cells grown in type II collagen-hyaluronan composite scaffolds.

Three-dimensional (3D) collagen type II-hyaluronan (HA) composite scaffolds (CII-HA) which mimics the extracellular environment of natural cartilage were fabricated in this study. Rheological measurements demonstrated that the incorporation of HA increased the compression modulus of the scaffolds. An initial in vitro evaluation showed that scaffolds seeded with porcine chondrocytes formed cartilaginous-like tissue after 8 weeks, and HA functioned to promote the growth of chondrocytes into scaffolds. Placenta-derived multipotent cells (PDMC) and gingival fibroblasts (GF) were seeded on tissue culture polystyrene (TCPS), CII-HA films, and small intestinal submucosa (SIS) sheets for comparing their chondrogenesis differentiation potentials with those of adipose-derived adult stem cells (ADAS) and bone marrow-derived mesenchymal stem cells (BMSC). Among different cells, PDMC showed the greatest chondrogenic differentiation potential on both CII-HA films and SIS sheets upon TGF-ß3 induction, followed by GF. This was evidenced by the up-regulation of chondrogenic genes (Sox9, aggrecan, and collagen type II), which was not observed for cells grown on TCPS. This finding suggested the essential role of substrate materials in the chondrogenic differentiation of PDMC and GF. Neocartilage formation was more obvious in both PDMC and GF cells plated on CII-HA composite scaffolds vs. 8-layer SIS at 28 days in vitro. Finally, implantation of PDMC/CII-HA constructs into NOD-SCID mice confirmed the formation of tissue-engineered cartilage in vivo.

Self-patterning of adipose-derived mesenchymal stem cells and chondrocytes cocultured on hyaluronan-grafted chitosan surface.

The articular cartilage, once injured, has a limited capacity for intrinsic repair. Preparation of functionally biocartilage substitutes in vitro for cartilage repair is an attractive concept with the recent advances in tissue engineering. In this study, adipose-derived adult stem cells (ADAS) and chondrocytes (Ch) were cocultured in different population ratios on the surface of hyaluronan-grafted chitosan (CS-HA) membranes. The two types of cells could self-assemble into cospheroids with different morphologies. In particular, when ADAS and Ch were cocultured at an initial ratio of 7:3 on CS-HA surface, the expression of chondrogenic markers was upregulated, leading to preferred chondrogenesis of the cospheroids. Therefore, using the ADAS/Ch 7:3 cospheroids derived on CS-HA surface instead of using only a single type of cells may be favorable for future therapeutic applications.

Water-based synthesis and processing of novel biodegradable elastomers for medical applications.

Biodegradable elastomers in the form of polyurethane nanoparticles (NPs) were successfully synthesized based on the combinations of two hydrolysis-prone polyester diols by a green water-based process. The anionic nature of the polymers successfully rendered polyurethane NPs (30-50 nm) consisting of approximately 200-300 polymer chains. The mechanical properties and degradation rate could be adjusted by the types and ratios of the component oligodiols in the soft segment. We demonstrated the feasibility using these biodegradable NPs as building blocks to generate self-assembled morphologies in nanometric, micrometric, or bulk scale, bearing excellent elasticity and biocompatibility. The elastic NPs and their various assembled forms represent a series of smart biodegradable elastomers with potential medical applications.

Amphiphilic silver-delaminated clay nanohybrids and their composites with polyurethane: physico-chemical and biological evaluations.

Silver nanoparticles (AgNPs) exhibit size-dependent bactericidal activity. They are, however, subject to aggregation when added to most hydrophobic polymers. In this study, a surfactant-modified delaminated clay (surfactant-capped nanosilicate platelets, or "NSQ") was employed to be a nanocarrier for AgNPs. The nanohybrid of AgNPs and NSQ ("AgNP/NSQ") in aqueous solution showed remarkable bactericidal effect as well as negligible cytotoxicity and immune response at a concentration of 10 ppm. Poly(carbonate)urethane (PCU) with different hard/soft segment ratios was synthesized and used in the preparation of the nanocomposites, PCU-AgNP/NSQ. Based on TEM observation, AgNP/NSQ was well dispersed in the resulting nanocomposites. The reinforcing effects of AgNP/NSQ in nanocomposites were distinct for PCU with different hard/soft segment ratios. The phase-separated structure of PCU-AgNP/NSQ nanocomposites was investigated by small-angle X-ray scattering (SAXS). PCU-AgNP/NSQ containing 75 ppm of Ag demonstrated superior microbiostatic effect, as well as better biodurability and lower foreign body reaction than the commercial Pellethane 2363-80A.

Spheroid formation and enhanced cardiomyogenic potential of adipose-derived stem cells grown on chitosan.

Mesenchymal stem cells may differentiate into cardiomyocytes and participate in local tissue repair after heart injury. In the current study, rat adipose-derived adult stem cells (ASCs) grown on chitosan membranes were observed to form cell spheroids after 3 days. The cell seeding density and surface modification of chitosan with Arg-Gly-Asp-containing peptide had an influence on the sizes of ASC spheroids. In the absence of induction, these spheroids showed an increased level of cardiac marker gene expression (Gata4, Nkx2-5, Myh6, and Tnnt2) more than 20-fold versus cells on the tissue culture polystyrene (TCPS) dish. Induction by 5-azacytidine or p38 MAP kinase inhibitor (SB202190) did not further increase the cardiac marker gene expression of these spheroids. Moreover, the enhanced cardiomyogenic potential of the spheroids was highly associated with the chitosan substrates. When ASC spheroids were plated onto TCPS with either basal or cardiac induction medium for 9 days, the spheroids spread into a monolayer and the positive effect on cardiomyogenic marker gene expression disappeared. The possible role of calcium ion and the up-regulation of adhesion molecule P-selectin and chemokine receptor Cxcr4 were demonstrated in ASC spheroids. Applying these spheroids to the chronic myocardial infarction animal model showed better functional recovery versus single cells after 12 weeks. Taken together, this study suggested that the ASC spheroids on chitosan may form as a result of calcium ion signaling, and the transplantation of these spheroids may offer a simple method to enhance the efficiency of stem cell-based therapy in myocardial infarction.

Surface phosphorylation for polyelectrolyte complex of chitosan and its sulfonated derivative: surface analysis, blood compatibility and adipose derived stem cell contact properties.

Many studies have tried to look for the application of chitosan in tissue engineering since its structure is similar to glycoaminoglycans, the main components of the extracellular matrix. Previous studies had indicated that the incorporation of sulfonic or phosphonic functionalities would be beneficial to the growth of certain cells. However, no study has explored the effect of incorporation of both above-mentioned anionic functionalities onto the chitosan structure. In this study, we have surface-phosphorylated the polyelectrolyte film formed by chitosan and water-soluble sulfonated chitosan with the aim to incorporate phosphonic and sulfonic functionalities onto the film surface. Surface analyses by ESCA and ATR-FT-IR have shown that these two functional groups have been successfully grafted onto the surface, and that the ratio of P/S was dependent upon the weight ratio of phosphorylation agents added. Blood compatibility evaluation indicated that phosphorylated polyelectrolyte complexes extended the plasma recalcification time as compared to non-treated chitosan and direct-phosphorylated chitosan film. In addition, these phosphorylated polyelectrolyte complexes showed similar or slightly less platelet reactivity than the non-phosphorylated counterpart. In contrast, significant platelet activation and adhesion were noted on the direct-phosphorylated chitosan. This implicated the incorporation of sulfonic acid onto the phosphorylated surface can increase the platelet compatibility. An adipose-derived stem cell incubation study has demonstrated that the incorporation of both phosphonic and sulfonic acid functionalities onto the chitosan surface can enhance the stem cell growth. Therefore, the phosphorylated polyelectrolyte complexes were not only blood compatible but also stem cell compatible, and could be a novel biomaterial in tissue-engineering applications.

The calcium-dependent regulation of spheroid formation and cardiomyogenic differentiation for MSCs on chitosan membranes.

Mesenchymal stem cells (MSCs) were recently found to form three-dimensional (3D) multicellular spheroids on chitosan membranes. The exact mechanism of spheroid formation, however, remains unclear. In this study, the regulation of spheroid formation for adipose derived adult stem cells (ADAS) grown on chitosan membranes was examined. By varying the membrane thickness, calcium concentration in culture medium, and acetylation extent of chitosan, the physico-chemical characteristics of chitosan that modulated spheroid formation was elucidated. The capacity of cardiomyogenic differentiation was further evaluated. Results suggested that the calcium binding capacity of chitosan may affect the cell-substrate and cell-cell interactions and critically influence the dynamics of spheroid formation. The intracellular calcium level was elevated for ADAS spheroids on chitosan. Chitosan-bound calcium was observed to enter the cells. The expression of N-cadherin was upregulated for ADAS spheroids on chitosan, evidenced by quantitative RT-PCR and Western blot. After the induction by 5-aza, the expression levels of cardiac marker genes (Gata4, Nkx2.5, Tnnt2, and Myh6) were remarkably enhanced (about four-fold) for ADAS on chitosan vs. tissue culture polystyrene or polyvinyl alcohol. Immunofluorescence staining confirmed the expression of cardiac-associated tight junction protein ZO-1 for ADAS grown on chitosan membranes. The gene expression of Wnt11 was significantly upregulated for ADAS spheroids on chitosan at 3 days and 12 days. We suggested that Wnt11 may be involved in the spheroid formation and cardiomyogenic differentiation of MSCs on chitosan membranes. Spheroids formed on the acetylated chitosan or polyvinyl alcohol membranes failed to show such behavior. The properties of MSC spheroids were therefore determined by the culture substrate.

Bioactivity and platelet adhesion study of a human thrombomodulin-immobilized nitinol surface.

Nitinol is a newly developed biomaterial that is gaining popularity in many biomedical applications. It has been reported that nitinol would not induce an inflammatory response and repulsion by the immunization after implantation in the human body. Besides, nitinol is a kind of shape memory alloy, which can memorize shapes at different temperatures. This can improve the convenience in surgery. However, nitinol has poor blood compatibility, so that further modification was needed to improve the antithrombogenicity. Human thrombomodulin (hTM), an endothelial-cell-associated glycoprotein, can be considered as a natural potent anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. In this study, the surface of nitinol was pre-activated by utilizing silanization with amino-terminated silane. The incorporated amino groups were available for the subsequent covalent immobilization of hTM by 2,4,6-trichloro-1,3,5-triazine (TCT), the coupling reagent. The surface density of immobilized hTM was determined by the Bradford method. The bioactivity of immobilized hTM and blood compatibility of various nitinol substrates were evaluated by the protein C activation assay and platelet adhesion test. It was observed that the immobilized hTM still had the ability to enhance protein C activation, though its activity was lower than the free hTM in solution. Furthermore, the platelet adhesion test showed that only a few platelets were adhered on the hTM-immobilized nitinol substrate. Therefore, the immobilization of thrombomodulin onto nitinol substrate could improve the blood compatibility of nitinol and might have the potential of application in antithrombogenic medical applications.

Surface characterization and in vitro platelet compatibility study of surface sulfonated chitosan membrane with amino group protection-deprotection strategy.

Glycosaminoglycans (GAGs) are the main components of the extracellular matrix (ECM). Studies have indicated that scaffolds modified by GAGs could improve cell proliferation and differentiation. Chitosan, the second-most abundant nature polysaccharide, has a structure similar to that of GAGs. Due to its relatively lower cost as compared to GAGs, many researchers have tried to incorporate sulfonate or carboxyl groups into the chitosan structure with the aim to form a GAG-like structure. However, these modifications were carried out on the reactive amino groups that were thought as the major character, resulting in special biological properties associated with chitosan. Such a decrease of amino-functional group density would very likely alter the specific biological properties of chitosan. Therefore, an amino group protection-deprotection strategy was explored in this study for surface sulfonation of chitosan membrane with the aim to imitate GAG structures. Various surface chemical characterization results, as well as surface zeta potential measurements have indicated that both sulfonate/sulfonic and amino functionalities were coexistent on the deprotected sulfonated chitosan specimen. In vitro platelet adhesion testing has shown that such a deprotected sulfonated chitosan membrane can increase the amount of platelet adhesion while keep those adhered remained unactivated. At the same time the presence of deprotected sulfonated chitosan film extended the plasma recalcification time value. With this protection-deprotection strategy, a further chemical grafting of bioactive molecules, such as RGD peptide, using the recovered amino functionalities, can be pursued on these sulfonated chitosan specimens.