Enzymatic in situ formed hydrogel from gelatin-tyramine and chitosan-4-hydroxylphenyl acetamide for the co-delivery of human adipose-derived stem cells and platelet-derived growth factor towards vascularization.

An injectable, in situ forming hydrogel system capable of co-delivering human adipose-derived stem cells (hADSC) and platelet-derived growth factor (PDGF) was investigated as a new system for tissue engineering, envisaged to support vascularization. The system consists of tyramine-conjugated gelatin and hydroxyphenyl acetamide chitosan derivative. Both are soluble and stable at physiologic conditions, which is a key factor for retaining viable cells and active growth factor. In situ gelation involved enzymatic crosslinking using horseradish peroxidase as a catalyst and hydrogen peroxide as an oxidant. Gel formation occurred within 30-90 s by controlling the concentration of polymers. PDGF release showed adequate release kinetics within the intended period of time and hADSC showed good compatibility with the hydrogel formulation based on the in vitro assay and subcutaneous implantation into BALB/c-nu/nu nude female mice. Immunohistochemical analysis confirmed viability of delivered hADSC. Histological analysis showed no immune reaction and confirmed blood vessel formation. The results implicate the hydrogel as a promising delivery vehicle or carrier of both cell and growth factor, which support vascularization for tissue engineering applications.

A Study of BMP-2-Loaded Bipotential Electrolytic Complex around a Biphasic Calcium Phosphate-Derived (BCP) Scaffold for Repair of Large Segmental Bone Defect.

A bipotential polyelectrolyte complex with biphasic calcium phosphate (BCP) powder dispersion provides an excellent option for protein adsorption and cell attachment and can facilitate enhanced bone regeneration. Application of the bipotential polyelectrolyte complex embedded in a spongy scaffold for faster healing of large segmental bone defects (LSBD) can be a promising endeavor in tissue engineering application. In the present study, a hollow scaffold suitable for segmental long bone replacement was fabricated by the sponge replica method applying the microwave sintering process. The fabricated scaffold was coated with calcium alginate at the shell surface, and genipin-crosslinked chitosan with biphasic calcium phosphate (BCP) dispersion was loaded at the central hollow core. The chitosan core was subsequently loaded with BMP-2. The electrolytic complex was characterized using SEM, porosity measurement, FTIR spectroscopy and BMP-2 release for 30 days. In vitro studies such as MTT, live/dead, cell proliferation and cell differentiation were performed. The scaffold was implanted into a 12 mm critical size defect of a rabbit radius. The efficacy of this complex is evaluated through an in vivo study, one and two month post implantation. BV/TV ratio for BMP-2 loaded sample was (42±1.76) higher compared with hollow BCP scaffold (32±0.225).

Bone formation of a porous Gelatin-Pectin-biphasic calcium phosphate composite in presence of BMP-2 and VEGF.

A composite scaffold of gelatin (Gel)-pectin (Pec)-biphasic calcium phosphate (BCP) was fabricated for the successful delivery of growth factors. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) were coated on the Gel-Pec-BCP surface to investigate of effect of them on bone healing. Surface morphology was investigated by scanning electron microscopy, and BCP dispersion in the hydrogel scaffolds was measured by energy dispersive X-ray spectroscopy. The results obtained from Fourier transform infrared spectroscopy showed that BMP-2 and VEGF were successfully coated on Gel-Pec-BCP hydrogel scaffolds. MC3T3-E1 preosteoblasts were cultivated on the scaffolds to investigate the effect of BMP-2 and VEGF on cell viability and proliferation. VEGF and BMP-2 loaded on Gel-Pec-BCP scaffold facilitated increased cell spreading and proliferation compared to Gel-Pec-BCP scaffolds. In vivo, bone formation was examined using rat models. Bone formation was observed in Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds within 4 weeks, and was greatest with Gel-Pec-BCP/BMP-2 scaffolds. In vitro and in vivo results suggest that Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds could enhance bone regeneration.

Functional nanofiber mat of polyvinyl alcohol/gelatin containing nanoparticles of biphasic calcium phosphate for bone regeneration in rat calvaria defects.

New biodegradable mats was successfully obtained by functional polyvinyl alcohol (PVA)/Gelatin (GE) blend fiber mats containing different BCP amounts (20, 40, and 50 w/v%) of biphasic calcium phosphate (BCP) nanoparticles for bone regeneration. BCP nanoparticles were loaded and dispersed successfully in the PVA/GE fibrous matrix. The addition of BCP was found to have increased fiber diameter, tensile strength, osteoblast cell adhesion, proliferation, and protein expression. Compared to the others, the 50% BCP-loaded electrospun PVA/GE fibers had the most favorable mechanical properties, cell attachment and growth, and protein expression. In vivo bone formation was examined using rat models, and increased bone formation was observed for the 50% BCP-loaded electrospun PVA/GE blends within 2 and 4 weeks. This result suggests that the 50% BCP-PVA/GE composite nanofiber mat has high potential for use in the field of bone regeneration and tissue engineering.

In vitro and in vivo evaluation of electrospun PCL/PMMA fibrous scaffolds for bone regeneration.

Scaffolds were fabricated by electrospinning using polycaprolactone (PCL) blended with poly(methyl methacrylate) (PMMA) in ratios of 10/0, 7/3, 5/5 and 3/7. The PCL/PMMA ratio affected the fiber diameter, contact angle, tensile strength and biological in vitro and in vivo properties of the scaffolds, and the 7/3 ratio resulted in a higher mechanical strength than 5/5 and 3/7. In vitro cytotoxicity and proliferation of MG-63 osteoblast cells on these blended scaffolds were examined by MTT assay, and it was found that PCL/PMMA blends are suitable for osteoblast cell proliferation. Confocal images and expression of proliferating cell nuclear antigen confirmed the good proliferation and expression of cells on the 7/3 PCL/PMMA fibrous scaffolds. In vivo bone formation was examined using rat models, and bone formation was observed on the 7/3 PCL/PMMA scaffold within 2 months. In vitro and in vivo results suggest that 7/3 PCL/PMMA scaffolds can be used for bone tissue regeneration.

Electrospinning of polyvinyl alcohol/gelatin nanofiber composites and cross-linking for bone tissue engineering application.

A three-dimensional polymer composite system consisting of polyvinyl alcohol/gelatin (PVA/GE) was fabricated via the electrospinning method and physically cross linked by methanol treatment. The effects of cross-linking between PVA/GE blend on physical, mechanical, and biological properties were investigated. After treating with methanol, PVA/GE mats become dense, hard, and aggregative with increased resistance to water dissolution. Osteoblasts like MG-63 cells were seeded on the surfaces of the cross linked PVA/GE mats and were found to attach firmly by expressing philopodial extensions. In addition, MTT assay and Western Blot analysis confirmed that the cells readily proliferated on the cross linked PVA/GE scaffolds. The osteoblast cell-matrix interaction demonstrated that the active biocompatibility of the mats was facilitated by using GE and cross-linking. In conclusion, our results suggest that cross-linked PVA/GE scaffolds hold promise for tissue engineering applications, especially in the field of artificial bone implant.

Fabrication of polyvinyl alcohol/gelatin nanofiber composites and evaluation of their material properties.

Electrospinning of polyvinyl alcohol (PVA), gelatin (GE), and a PVA/GE blend was conducted with the aim of fabricating biodegradable scaffolds for tissue engineering. The process parameters including the concentration of GE in PVA/GE blends, electrical field, and tip-to-collector distance (TCD) were investigated. Electrospinning processes were conducted at three different GE concentrations (PVA/GE = 2/8, 6/4, and 8/2), and the voltage and TCD were varied from 18 to 24 kV and 7 to 20 cm, respectively. The average diameter of the electrospun PVA, GE, and PVA/GE blend fibers ranged from 50 to 150 nm. The TCD had significant effects on the average diameter of the PVA/GE nanofiber, while changes in the voltage did not significantly affect the diameter of the PVA/GE nanofiber. The miscibility of the PVA/GE blend fibers was examined by differential scanning calorimetry, and X-ray diffraction was used to determine the crystallinity of the membrane. Tensile strength was measured to evaluate the physical properties of the membrane. Based on the combined results of this study, the PVA/GE membrane holds great promise for use in tissue engineering applications, especially in bone or drug delivery systems.