Improvement of Decellularization Efficiency of Porcine Aorta Using Dimethyl Sulfoxide as a Penetration Enhancer.

Decellularization of tissues and organs enables researchers to obtain extracellular matrix (ECM) with the natural conformation and chemical composition of specific tissues. However, drawbacks exist such as the structural alteration of ECM or loss of some important components in ECM due to overexposure to chemicals during the decellularization process. In this study, porcine aorta was decellularized by sodium dodecyl sulfate (SDS). Dimethyl sulfoxide (DMSO) was used as a penetration enhancer in the decellularization process to enhance the penetration of SDS, consequently reducing the exposure time of SDS to treated tissues. It is revealed that by addition of DMSO to the decellularization process 64.4% more DNA was removed when compared with just SDS exposure within a 3 h reaction. Cross-validation by DAPI staining showed that, in the presence of DMSO, the penetration of SDS was improved and almost all cells were removed from the aorta within the 3 h exposure time. Collagen staining revealed that just SDS treatment showed less polarized collagen fibers, while the DMSO addition groups revealed denser and organized collagen fibers. Moreover 77% glycosaminoglycan content was preserved by addition of DMSO in resultant tissues. Scanning electron microscopy analysis of decellularized aortic matrix showed that ECM components remained in the adventitia layer with the addition of DMSO treatment, while the layer was removed with just SDS treatment. Biocompatibility assays proved that after washing the decellularized samples with media supplemented with 3% antibiotic and antimycotic solution for 2 days there was no cytotoxic effect related to the SDS + DMSO decellularization protocol. This study demonstrates that the new decellularization protocol not only improves the removal efficiency of cellular components but also protects the crucial ECM components.

Hydrostatic pressure stimulates the osteogenesis and angiogenesis of MSCs/HUVECs co-culture on porous PLGA scaffolds.

In native bone tissue regeneration, blood vessels, providing oxygen and nutrition for tissues, can promote the regeneration of bone and accelerate the repair of a defected area. In this study, Poly(D, L-lactic-co-glycolic acid) (PLGA) inverse opal scaffolds with high pore interconnectivity were fabricated and further modified with vascular endothelial growth factor (VEGF). The rat bone marrow derived mesenchymal stem cells (rMSCs) and human umbilical vein endothelial cells (HUVECs) were co-cultured onto the scaffolds to enhance vascularization for bone tissue regeneration. Cell attachment, viability, proliferation, and morphology were detected by cell counting kit-8 (CCK-8) assay, live and dead staining and scanning electron microscopy (SEM). Hydrostatic pressure with 0-279 KPa and 1 Hz one hour per day for 7 days was applied to tissue engineered bone constructs to investigate whether the loading stimulation can promote osteogenesis and angiogenesis mutually evaluated in parallel by multiple in vitro assays and in an in vivo chicken chorioallantoic membrane (CAM) model. The results indicated that the immobilization of VEGF can improve biocompatibility of PLGA scaffolds and promote cell attachment and proliferation. The cell-scaffold constructs showed higher CD31 expression because of the angiogenic differentiation of rMSCs in hydrostatic loading culture condition in vitro. The in vivo CAM model experiment demonstrated that hydrostatic loading stimulated angiogenic differentiation of rMSCs can accelerate tubulogenesis. Furthermore, the new capillaries formed in cell-scaffold constructs were conducive to calcium deposition in vivo.

Introducing RGD peptides on PHBV films through PEG-containing cross-linkers to improve the biocompatibility.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, has been a good candidate of biomaterial employed in tissue engineering. However, the PHBV film is hydrophobic and has no recognition sites for cell attachment. In this study, PHBV films are activated by ammonia plasma treatment to produce amino groups on the surface, followed by sequential reactions with a heterobifunctional cross-linker containing a segment of poly(ethylene glycol) (PEG) and further with RGD-containing peptides. XPS analyses of modified surfaces after each reaction step reveal that the RGD-containing peptides have been covalently grafted onto PHBV films. The result of cell viability assay indicates that the RGD-modified PHBV films exhibit a distinctly improved cellular compatibility. Moreover, according to the results of serum adsorption tests by optical waveguide lightmode spectroscopy (OWLS) and fibrinogen adsorption tests by enzyme-linked immunosorbent assay (ELISA) on unmodified and modified PHBV surfaces, the introduced PEG chains can significantly decrease the nonspecific adsorption of proteins from serum and fibrinogen from plasma, thus decreasing the risk of thrombus formation and improving the blood compatibility of implanted materials.

Effects of hydroxyapatite-containing composite nanofibers on osteogenesis of mesenchymal stem cells in vitro and bone regeneration in vivo.

Among a variety of polymers, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a microbial polyester, with biodegradable, nonantigenic, and biocompatible properties, is attracting more and more attention in tissue engineering. Hydroxyapatite (HA), similar to the mineral component of natural bone, is known to be osteoconductive, nontoxic, and noninflammatory. In this study, aligned and random-oriented PHBV nanofibrous scaffolds loaded with HA nanoparticles were fabricated through electrospinning technique. Mesenchymal stem cells (MSCs) derived from rat bone marrow were used to investigate the effects of HA and orientation of fibers on cell proliferation and differentiation in vitro. Cell proliferation tested with CCK-8 assay indicated that the MSCs attached and proliferated more favorably on random-oriented PHBV nanofibrous meshes without HA. After one, two and four weeks of cell seeding, osteogenic markers including alkaline phosphate (ALP), osteocalcin (OCN), and mineralized matrix deposits were detected, respectively. The results indicated that the introduction of HA could induce MSCs to differentiate into osteoblasts. Moreover, 3D PHBV/HA scaffolds made from aligned and random-oriented nanofibers were implanted into critical-sized rabbit radius defects and exhibited significant effects on the repair of critical bone defects, implying their promising applications in bone tissue engineering.

The effects of PHBV electrospun fibers with different diameters and orientations on growth behavior of bone-marrow-derived mesenchymal stem cells.

Microenvironments in which cells live play an important role in the attachment, growth and interactions of cells. To mimic the natural structure of extracellular matrices, electrospinning was applied to fabricate biomaterials into ultrafine fibers. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biocompatible and biodegradable polyester, has been shown to be an excellent biomaterial candidate for tissue engineering. In this study, five types of PHBV fibrous scaffolds with different diameters and orientations were obtained by changing solvents, concentration of electrospun solution and collector. Three kinds of scaffolds with good continuity and suitable mechanical properties, selected according to the morphology and mechanical properties of the scaffolds, were used for studying the influence of fiber diameter and orientation on growth behavior of bone-marrow-derived mesenchymal stem cells (MSCs). The results indicated that the random-oriented nanofibrous scaffold is most favorable for cell growth compared to other scaffolds, while the microfibrous scaffold resulted in the lowest viability of MSCs. The orientation of nanofibers showed a distinct effect on cell morphology by guiding cell skeleton extension. Both the random-oriented and aligned PHBV nanofibrous scaffolds showed to be good candidates for applications in tissue engineering.

Cellular compatibility of RGD-modified chitosan nanofibers with aligned or random orientation.

Aligned and randomly oriented chitosan nanofibers were prepared by electrospinning. The fibers were modified with the RGD cell-adhesive peptide through a heterobifunctional crosslinker containing a segment of poly(ethylene glycol) (PEG). PEG rendered the surface hydrophilic and provided flexible spacers, allowing the preservation of the bioactivity of further captured RGD peptides. NIH 3T3 cells were used to test the cellular compatibility of these chitosan nanofibrous scaffolds. Cell morphology and viability were investigated by SEM, fluorescent staining and cell counting. The results indicate that RGD-modified surfaces significantly improve the cellular compatibility of chitosan nanofibers and suggest a good candidate as a scaffold employed in tissue engineering.