Nguyen Thuy Ba Linh

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.

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.

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.

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.

Hybrid hydroxyapatite nanoparticles-loaded PCL/GE blend fibers for bone tissue engineering

In order to augment bone formation, a new biodegradable scaffold system was fabricated using different ratios of hydroxyapatite (HAp) blended with synthetic polymer polycaprolactone (PCL) and natural polymer gelatin (GE) followed by electrospinning method. Three different concentrations of HAp were used in PCL/GE to obtain a blend of 10, 30, and 50% (w/v) HAp–PCL/GE. These HAp-loaded PCL/GE blends were then compared with PCL/GE blends by different mechanical and biological in vitro and in vivo studies to understand the applicability of the system. Scanning electron microscopy, X-ray diffraction analysis, and tensile strength measurement were done to obtain physical properties. Fifty Percent HAp–PCL/GE blends possessed the highest mechanical strength. In vitro cytotoxicity and proliferation of osteoblast cells on the PCL/GE and HAp–PCL/GE scaffolds were examined and shown that addition of HAp in PCL/GE was beneficial by increasing cell viability (>85%) proliferation and cell-surface attachment. Expression of collagen and osteopontin was also found higher in 50% HAp–PCL/GE blends than the others. On the other hand, in vivo bone formation was examined using rat models and increased bone formation was observed in 50% HAp–PCL/GE blends within 6 weeks. Based on the combined results of this study, HAp–PCL/GE membranes were found to hold great promise for use in tissue engineering applications, especially in bone tissue engineering.

Evaluation of the cytocompatibility hemocompatibility in vivo bone tissue regenerating capability of different PCL blends

In this study, the optimized formulations of polycaprolactone (PCL) combined with poly(lactic-co-glycolic acid) (PLGA), gelatin (GEL), and biphasic calcium phosphate (BCP) were analyzed in terms of cytocompatibility with bone-related cells, hemocompatibility, and in vivo bone-regenerating capacity to determine their potentials for bone tissue regeneration. Fiber morphology of PCL/GEL and PCL/BCP electrospun mats considerably differs from that of the PCL membrane. Based on the contact angle analyses, the addition of GEL and PLGA was shown to reduce the hydrophobicity of these membranes. The assessment of in vitro cytocompatibility using MC3T3-E1 cells indicated that all of the membranes were suitable for pre-osteoblast proliferation and adhesion, with PCL/BCP having a significantly higher reading after seven days of incubation. The results of the in vitro hemocompatibility of the different fibrous scaffolds suggest that coagulation and platelet adhesion were higher for hydrophobic membranes (PCL and PCL/PLGA), while hemolysis can be associated with fiber morphology. The potential of the membranes for bone regeneration was determined by analyzing the microCT data and tissue sections of samples implanted in 5 mm sized defects (one and two months). Although all of the membranes were suitable for pre-osteoblast proliferation, in vivo bone regeneration after two months was found to be significantly higher in PCL/BCP (p < 0.001).

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).

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.

Fabrication of recombinant human bone morphogenetic protein-2 coated porous biphasic calcium phosphate-sodium carboxymethylcellulose-gelatin scaffold and its In vitro evaluation

Fabrication of bone substitutes, which are a combination of bio-ceramics and bio-polymer, is performed to meet the demand for bone regeneration after fracture or disease. In this study, sodium carboxymethylcellulosegelatin (NaCMC-GEL) hydrogel scaffold where the ratio of NaCMC and GEL was 1:2 and biphasic calcium phosphate (BCP) loaded NaCMC-GEL where the ratio of BCP, NaCMC and GEL was 1:1:2 hydrogel scaffold were fabricated successfully by the freeze-drying method. These hydrogel scaffolds were crosslinked by 0.75wt% genipin solution to check the swelling and ensure optimum degradation. Then BCP-NaCMC-GEL hydrogel was coated with recombinant human bone morphogenetic protein-2 (rhBMP-2). Detailed morphological and material characterization, such as porosity, micro-structural analysis, and chemical constituents of NaCMC-GEL and BCP-NaCMC-GEL hydrogel scaffolds was carried out. The study demonstrates that these hydrogel scaffolds have a porous structure and the pore size is optimum for bone tissue regeneration. PBS uptake and degradation behavior of the NaCMC-GEL, BCP-NaCMC-GEL and BMP-2-BCP-NaCMC-GEL hydrogel scaffolds were also observed. BMP-2-BCP-NaCMCGEL hydrogel scaffold showed higher cell viability, cell attachment, and proliferation of pre-osteoblast MC3T3-E1 cells than the NaCMC-GEL, BCP-NaCMC-GEL scaffolds, which was confirmed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, live-dead assay, and immunofluorescence assay. The BMP-2 release profile from BMP-2-BCP-NaCMC-GEL scaffold was also shown. The observations show that BMP-2-coated BCPNaCMC-GEL hydrogel scaffold is a promising material for bone tissue regeneration.

Effect of rat bone marrow derived–stem cell delivery from serum-loaded oxidized alginate–gelatin–biphasic calcium phosphate hydrogel for bone tissue regeneration using a nude mouse critical-sized calvarial defect model:

Blood serum contains various kinds of proteins which are necessary for tissue repair and regeneration process. Defect healing of fractured bone is initiated by the influx of blood and then clot formation. Thus, proteins in serum may have the ability to stimulate the bone regeneration process. In this work, we investigated the fabrication of serum-loaded oxidized alginate–gelatin–biphasic calcium phosphate hydrogels with various contents of blood serum (0%, 5%, 10%, and 15% in % v/v) to evaluate the stimulatory effect of serum proteins on bone regeneration. This system was also evaluated for rat bone marrow–derived stem cell delivery to get faster bone healing. The serum-loaded oxidized alginate–gelatin–biphasic calcium phosphate hydrogel samples were characterized by scanning electron microscopy, porosity meter, X-ray diffraction, and Fourier transform infrared for morphology and phase characterization together with their mechanical behavior. Protein release behavior, degradation, and swelling of the samples were studied. In vitro study was performed using bone marrow–derived stem cells to study cell attachment, viability, and proliferation. These studies revealed the best cell attachment and highest proliferation for 5% serum-loaded oxidized alginate–gelatin–biphasic calcium phosphate hydrogel scaffold. This composition also showed the ability to deliver stem cell in the defect zone which significantly improved the bone regeneration extent found in the in vivo animal model. In vivo study revealed that for the critical 5-mm calvarial defect into nude mouse skull, the 5% serum-loaded sample with bone marrow–derived stem cells shows the best bone regeneration potential.