Poly(ester‐urethane) scaffolds: effect of structure on properties and osteogenic activity of stem cells

The present study aimed to investigate the effect of structure (design and porosity) on the matrix stiffness and osteogenic activity of stem cells cultured on poly(ester‐urethane) (PEU) scaffolds. Different three‐dimensional (3D) forms of scaffold were prepared from lysine‐based PEU using traditional salt‐leaching and advanced bioplotting techniques. The resulting scaffolds were characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), mercury porosimetry and mechanical testing. The scaffolds had various pore sizes with different designs, and all were thermally stable up to 300 °C. In vitro tests, carried out using rat bone marrow stem cells (BMSCs) for bone tissue engineering, demonstrated better viability and higher cell proliferation on bioplotted scaffolds compared to salt‐leached ones, most probably due to their larger and interconnected pores and stiffer nature, as shown by higher compressive moduli, which were measured by compression testing. Similarly, SEM, von Kossa staining and EDX analyses indicated higher amounts of calcium deposition on bioplotted scaffolds during cell culture. It was concluded that the design with larger interconnected porosity and stiffness has an effect on the osteogenic activity of the stem cells. Copyright © 2013 John Wiley & Sons, Ltd.     

Joint mimicking mechanical load activates TGFβ1 in fibrin‐poly(ester‐urethane) scaffolds seeded with mesenchymal stem cells

Transforming growth factor‐β1 (TGF‐β1) is widely used in an active recombinant form to stimulate the chondrogenic differentiation of mesenchymal stem cells (MSCs). Recently, it has been shown that the application of multiaxial load, that mimics the loading within diarthrodial joints, to MSCs seeded in to fibrin‐poly(ester‐urethane) scaffolds leads to the endogenous production and secretion of TGF‐β1 by the mechanically stimulated cells, which in turn drives the chondrogenic differentiation of the cells within the scaffold. The work presented in this short communication provides further evidence that the application of joint mimicking multiaxial load induces the secretion of TGF‐β1 by mechanically stimulated MSCs. The results of this work also show that joint‐like multiaxial mechanical load activates latent TGF‐β1 in response to loading in the presence or absence of cells; this activation was not seen in non‐loaded control scaffolds. Despite the application of mechanical load to scaffolds with different distributions/numbers of cells no significant differences were seen in the percentage of active TGF‐β1 quantified in the culture medium of scaffolds from different groups. The similar level of activation in scaffolds containing different numbers of cells, cells at different stages of differentiation or with different distributions of cells suggests that this activation results from the mechanical forces applied to the culture system rather than differences in cellular behaviour. These results are relevant when considering rehabilitation protocols after cell therapy or microfracture, for articular cartilage repair, where increased TGF‐β1 activation in response to joint mobilization may improve the quality of developing cartilaginous repair material. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd     

Chondrogenic,hypertrophic, and osteochondral differentiation of human mesenchymal stem cells on three‐dimensionally woven scaffolds

The development of mechanically functional cartilage and bone tissue constructs of clinically relevant size, as well as their integration with native tissues, remains an important challenge for regenerative medicine. The objective of this study was to assess adult human mesenchymal stem cells (MSCs) in large, three‐dimensionally woven poly(ε‐caprolactone; PCL) scaffolds in proximity to viable bone, both in a nude rat subcutaneous pouch model and under simulated conditions in vitro. In Study I, various scaffold permutations—PCL alone, PCL‐bone, “point‐of‐care” seeded MSC‐PCL‐bone, and chondrogenically precultured Ch‐MSC‐PCL‐bone constructs—were implanted in a dorsal, ectopic pouch in a nude rat. After 8 weeks, only cells in the Ch‐MSC‐PCL constructs exhibited both chondrogenic and osteogenic gene expression profiles. Notably, although both tissue profiles were present, constructs that had been chondrogenically precultured prior to implantation showed a loss of glycosaminoglycan (GAG) as well as the presence of mineralization along with the formation of trabecula‐like structures. In Study II of the study, the GAG loss and mineralization observed in Study I in vivo were recapitulated in vitro by the presence of either nearby bone or osteogenic culture medium additives but were prevented by a continued presence of chondrogenic medium additives. These data suggest conditions under which adult human stem cells in combination with polymer scaffolds synthesize functional and phenotypically distinct tissues based on the environmental conditions and highlight the potential influence that paracrine factors from adjacent bone may have on MSC fate, once implanted in vivo for chondral or osteochondral repair.     

Tonsil‐derived mesenchymal stem cells (T‐MSCs) prevent Th17‐mediated autoimmune response via regulation of the programmed death‐1/programmed death ligand‐1 (PD‐1/PD‐L1) pathway

Our knowledge of the immunomodulatory role of mesenchymal stem cells (MSCs) in both the innate and adaptive immune systems has dramatically expanded, providing great promise for treating various autoimmune diseases. However, the contribution of MSCs to Th17‐dominant immune disease, such as psoriasis and its underlying mechanism remains elusive. In this study, we demonstrated that human palatine tonsil‐derived MSCs (T‐MSCs) constitutively express both the membrane‐bound and soluble forms of programmed death‐ligand 1 (PD‐L1), which enables T‐MSCs to be distinguished from MSCs originating from other organs (i.e. bone marrow or adipose tissue). We also found that T‐MSC‐derived PD‐L1 effectively represses Th17 differentiation via both cell‐to‐cell contact and a paracrine effect. Further, T‐MSCs increase programmed death‐1 (PD‐1) expression on T‐cells by secreting IFN‐β, which may enhance engagement with PD‐L1. Finally, transplantation of T‐MSCs into imiquimod‐induced psoriatic skin inflammation in mice significantly abrogated disease symptoms, mainly by blunting the Th17 response in a PD‐L1‐dependent manner. This study suggests that T‐MSCs might be a promising cell source to treat autoimmune diseases such as psoriasis, via its unique immunoregulatory features. Copyright © 2017 John Wiley & Sons, Ltd.     

Biocompatibility of synthetic poly(ester urethane)/polyhedral oligomeric silsesquioxane matrices with embryonic stem cell proliferation and differentiation

Incorporation of polyhedral oligomeric silsesquioxanes (POSS) into poly(ester urethanes) (PEU) as a building block results in a PEU/POSS hybrid polymer with increased mechanical strength and thermostability. An attractive feature of the new polymer is that it forms a porous matrix when cast in the form of a thin film, making it potentially useful in tissue engineering. In this study, we present detailed microscopic analysis of the PEU/POSS matrix and demonstrate its biocompatibility with cell culture. The PEU/POSS polymer forms a continuous porous matrix with open pores and interconnected grooves. From SEM image analysis, it is calculated that there are about 950 pores/mm² of the matrix area with pore diameter size in the range 1–15 µm. The area occupied by the pores represents approximately 7.6% of the matrix area. Using mouse embryonic stem cells (ESCs), we demonstrate that the PEU/POSS matrix provides excellent support for cell proliferation and differentiation. Under the cell culture condition optimized to maintain self‐renewal, ESCs grown on a PEU/POSS matrix exhibit undifferentiated morphology, express pluripotency markers and have a similar growth rate to cells grown on gelatin. When induced for differentiation, ESCs underwent dramatic morphological change, characterized by the loss of clonogenecity and increased cell size, with well‐expanded cytoskeleton networks. Differentiated cells are able to form a continuous monolayer that is closely embedded in the matrix. The excellent compatibility between the PEU/POSS matrix and ESC proliferation/differentiation demonstrates the potential of using PEU/POSS polymers in future ESC‐based tissue engineering. Copyright © 2010 John Wiley & Sons, Ltd.     

Encapsulation of rat bone marrow stromal cells using a poly‐ion complex gel of chitosan and succinylated poly(Pro–Hyp–Gly)

Encapsulation of stem cells into a three‐dimensional (3D) scaffold is necessary to achieve tissue regeneration. Prefabricated 3D scaffolds, such as fibres or porous sponges, have limitations regarding homogeneous cell distribution. Hydrogels that can encapsulate cells such as animal‐derived collagen gels need adjustment of the pH and/or temperature upon cell mixing. In this report, we fabricated a poly‐ion complex (PIC) hydrogel of chitosan and succinylated poly(Pro–Hyp–Gly) and assessed its effect on cell viability after encapsulation of rat bone marrow stromal cells. PIC hydrogels were obtained successfully with a concentration of each precursor as low as 3.0–3.8 mg/ml. The maximum gelation and swelling ratios were achieved with an equal molar ratio (1:1) of anionic and cationic groups. Using chitosan acetate as a cationic precursor produced a PIC hydrogel with both a significantly greater gelation ratio and a better swelling ratio than chitosan chloride. Ammonium succinylated poly(Pro–Hyp–Gly) as an anionic precursor gave similar gelation and swelling ratios to those of sodium succinylated poly(Pro–Hyp–Gly). Cell encapsulation was also achieved successfully by mixing rat bone marrow stromal cells with the PIC hydrogel simultaneously during its formation. The PIC hydrogel was maintained in the culture medium for 7 days at 37°C and the encapsulated cells survived and proliferated in it. Although it is necessary to improve its functionality, this PIC hydrogel has the potential to act as a 3D scaffold for cell encapsulation and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of biomimetic zinc‐containing tricalcium phosphate (Zn–TCP) on the growth and osteogenic differentiation of mesenchymal stem cells

Several studies have shown the effectiveness of zinc‐tricalcium phosphate (Zn–TCP) for bone tissue engineering. In this study, marine calcareous foraminifera possessing uniform pore size distribution were hydrothermally converted to Zn–TCP. The ability of a scaffold to combine effectively with mesenchymal stem cells (MSCs) is a key tissue‐engineering aim. In order to demonstrate the osteogenic ability of MSCs with Zn–TCP, the scaffolds were cultured in an osteogenic induction medium to elicit an osteoblastic response. The physicochemical properties of Zn–TCP were characterized by XRD, FT–IR and ICP–MS. MSCs were aspirated from rat femurs and cultured for 3 days before indirectly placing four samples into each respective well. After culture for 7, 10 and 14 days, osteoblastic differentiation was evaluated using alizarin red S stain, measurement of alkaline phosphatase (ALP) levels, cell numbers and cell viability. XRD and FT–IR patterns both showed the replacement of CO₃^2– with PO₄^3–. Chemical analysis showed zinc incorporation of 5 mol%. Significant increases in cell numbers were observed at 10 and 14 days in the Zn–TCP group, while maintaining high levels of cell viability (> 90%). ALP activity in the Zn–TCP group was statistically higher at 10 days. Alizarin red S staining also showed significantly higher levels of calcium mineralization in Zn–TCP compared with the control groups. This study showed that MSCs in the presence of biomimetically derived Zn–TCP can accelerate their differentiation to osteoblasts and could potentially be useful as a scaffold for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.