RGD-conjugated copolymer incorporated into composite of poly(lactide-co-glycotide) and poly(L-lactide)-grafted nanohydroxyapatite for bone tissue engineering.

Various surface modification methods of RGD (Arg-Gly-Asp) peptides on biomaterials have been developed to improve cell adhesion. This study aimed to examine a RGD-conjugated copolymer RGD/MPEG-PLA-PBLG (RGD-copolymer) for its ability to promote bone regeneration by mixing it with the composite of poly(lactide-co-glycotide) (PLGA) and hydroxyapatite nanoparticles surface-grafted with poly(L-lactide) (g-HAP). The porous scaffolds were prepared using solvent casting/particulate leaching method and grafted to repair the rabbit radius defects after seeding with autologous bone marrow mesenchymal cells (MSCs) of rabbits. After incorporation of RGD-copolymer, there were no significant influences on scaffold's porosity and pore size. Nitrogen of RGD peptide, and calcium and phosphor of g-HAP could be exposed on the surface of the scaffold simultaneously. Although the cell viability of its leaching liquid was 92% that was lower than g-HAP/PLGA, its cell adhesion and growth of 3T3 and osteoblasts were promoted significantly. The greatest increment in cell adhesion ratios (131.2-157.1% higher than g-HAP/PLGA) was observed when its contents were 0.1-1 wt % but only at 0.5 h after cell seeding. All the defects repaired with the implants were bridged after 24 weeks postsurgery, but the RGD-copolymer contained composite had larger new bone formation and better fusion interface. The composites containing RGD-copolymer enhanced bone ingrowth but presented more woven bones than others. The combined application of RGD-copolymer and bone morphological protein 2 (BMP-2) exhibited the best bone healing quality and was recommended as an optimal strategy for the use of RGD peptides.

An Elastic Mineralized 3D Electrospun PCL Nanofibrous Scaffold for Drug Release and Bone Tissue Engineering.

Complex shaped and critical-sized bone defects have been a clinical challenge for many years. Scaffold-based strategies such as hydrogels provide localized drug release while filling complex defect shapes, but ultimately possess weaknesses in low mechanical strength alongside a lack of macroporous and collagen-mimicking nanofibrous structures. Thus, there is a demand for mechanically strong, extracellular matrix (ECM) mimicking scaffolds that can robustly fit complex shaped critical sized defects and simultaneously provide localized, sustained, multiple growth factor release. We therefore developed a composite, bi-phasic PCL/hydroxyapatite (HA) 3D nanofibrous (NF) scaffold for bone tissue regeneration by using our innovative electrospun-based thermally induced self-agglomeration (TISA) technique. One intriguing feature of our ECM-mimicking TISA scaffolds is that they are highly elastic and porous even after evenly coated with minerals and can easily be pressed to fit different defect shapes. Furthermore, the bio-mimetic mineral deposition technique allowed us to simultaneously encapsulate different type of drugs, e.g., proteins and small molecules, on TISA scaffolds under physiologically mild conditions. Compared to scaffolds with physically surface-adsorbed phenamil, a BMP2 signaling agonist, incorporated phenamil composite scaffolds indicated less burst release and longer lasting sustained release of phenamil with subsequently improved osteogenic differentiation of cells in vitro. Overall, our study indicated that the innovative press-fit 3D NF composite scaffold may be a robust tool for multiple-drug delivery and bone tissue engineering.

Gelatin-crosslinked pectin nanofiber mats allowing cell infiltration.

Pectin nanofiber mats are promising tissue engineering scaffolds but suffer from poor cell infiltration. In this study, gelatin, a collagen derived cell adhesive protein, was used to crosslink the electrospun nanofibers of periodate oxidized pectin. Cell culture experiment results demonstrated that cells were able to grow into the gelatin-crosslinked pectin nanofiber mats rather than only spread on mat surface. The nanofiber mats showed moderate mechanical strength, with a maximum tensile strength of up to 2.3 MPa, an ultimate tensile strain of up to 15%, and were capable of degrading gradually over 4 weeks or even longer periods in simulated body fluids. Thus, gelatin-crosslinked pectin nanofiber mats hold a great potential for soft tissue regeneration.

Preparation and in vitro characterization of scaffolds of poly(L-lactic acid) containing bioactive glass ceramic nanoparticles.

Porous nanocomposite scaffolds of poly(l-lactic acid) (PLLA) containing different quantities of bioactive glass ceramic (BGC) nanoparticles (SiO(2):CaO:P(2)O(5) approximately 55:40:5 (mol)) were prepared by a thermally induced phase-separation method. Dioxane was used as the solvent for PLLA. Introduction of less than 20wt.% of BGC nanoparticles did not remarkably affect the porosity of PLLA foam. However, as the BGC content increased to 30wt.%, the porosity of the composite was observed to decrease rapidly. The compressive modulus of the scaffolds increased from 5.5 to 8.0MPa, while the compressive strength increased from 0.28 to 0.35MPa as the BGC content increased from 0 to 30wt.%. The in vitro bioactivity and biodegradability of nanocomposites were investigated by incubation in simulated body fluid (SBF) and phosphate-buffered saline, respectively. Scanning electron microscopy, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy and X-ray diffraction were employed to monitor the surface variation of neat PLLA and PLLA/BGC porous scaffolds during incubation. PLLA/(20wt.%)BGC composite exhibited the best mineralization property in SBF, while the PLLA/(10wt.%)BGC composite showed the highest water absorption ability.

Surface modification of bioactive glass nanoparticles and the mechanical and biological properties of poly(L-lactide) composites.

Novel bioactive glass (BG) nanoparticles/poly(L-lactide) (PLLA) composites were prepared as promising bone-repairing materials. The BG nanoparticles (Si:P:Ca=29:13:58 weight ratio) of about 40nm diameter were prepared via the sol-gel method. In order to improve the phase compatibility between the polymer and the inorganic phase, PLLA (M(n)=9700Da) was linked to the surface of the BG particles by diisocyanate. The grafting ratio of PLLA was in the vicinity of 20 wt.%. The grafting modification could improve the tensile strength, tensile modulus and impact energy of the composites by increasing the phase compatibility. When the filler loading reached around 4 wt.%, the tensile strength of the composite increased from 56.7 to 69.2MPa for the pure PLLA, and the impact strength energy increased from 15.8 to 18.0 kJ m(-2). The morphology of the tensile fracture surface of the composite showed surface-grafted bioactive glass particles (g-BG) to be dispersed homogeneously in the PLLA matrix. An in vitro bioactivity test showed that, compared to pure PLLA scaffold, the BG/PLLA nanocomposite demonstrated a greater capability to induce the formation of an apatite layer on the scaffold surface. The results of marrow stromal cell culture revealed that the composites containing either BG or g-BG particles have much better biocompatibility compared to pure PLLA material.

Tailoring weight ratio of PCL/PLA in electrospun three-dimensional nanofibrous scaffolds and the effect on osteogenic differentiation of stem cells.

Three-dimensional (3D) scaffolds as artificial ECMs have been extensively studied to mimic the critical features of natural ECMs. To develop more clinically relevant 3D scaffolds, electrospun nanofibrous scaffolds with different weight ratios of PCL/PLA (i.e., 100/0, 60/40, and 20/80) were fabricated via the thermally induced (nanofiber) self-agglomeration (TISA) method. The hypothesis was that, with the weight ratio increase of stiffer and more bioactive PLA in the 3D PCL/PLA blend scaffolds, the osteogenic differentiation of human mesenchymal stem cells (hMSCs) would be enhanced. The results indicated that, all of the 3D scaffolds were elastic/resilient and possessed interconnected and hierarchical pores with sizes from sub-microns to ∼300 µm; therefore, the morphological structures of these scaffolds were similar to those of natural ECMs. The PLA80 scaffolds exhibited the best overall properties in terms of density, porosity, water absorption capacity, mechanical properties, bioactivity, and cell viability. Furthermore, with increasing the PLA weight ratio, the alkaline phosphatase (ALP) activity, calcium content, and gene expression level were also increased, probably due to the improved stiffness/bioactivity of scaffold. Hence, the novel 3D electrospun PLA80 nanofibrous scaffold might be desired/favorable for the osteogenic differentiation of hMSCs.

Composites of poly(lactide-co-glycolide) and the surface modified carbonated hydroxyapatite nanoparticles.

To improve the mechanical properties of the composites of poly(lactide-co-glycolide) (PLGA, LA/GA = 80/20) and the carbonate hydroxyapatite (CHAP) particles, the rice-form or claviform CHAP particles with 30-40 nm in diameter and 100-200 nm in length were prepared by precipitation method. The uncalcined CHAP particles have a coarse surface with a lot of global protuberances, which could be in favor of the interaction of the matrix polymer to the CHAP particles. The nanocomposites of PLGA and surface grafted CHAP particles (g-CHAP) were prepared by solution mixing method. The structure and properties of the composites were subsequently investigated by the emission scanning electron microscopy, the tensile strength testing, and the cell culture. When the contents of g-CHAP were in the range of 2-15 wt %, the PLGA/g-CHAP nanocomposites exhibited an improved elongation at break and tensile strength. At the 2 wt % content of g-CHAP, the fracture strain was increased to 20% from 4-5% for neat PLGA samples. Especially at g-CHAP content of 15 wt %, the tensile strength of PLGA/g-CHAP composite was about 20% higher than that of neat PLGA materials. The tensile moduli of composites were increased with the increasing of filler contents, so that the g-CHAP particles had both reinforcing and toughening effects on the PLGA composites. The results of biocompatibility test showed that the higher g-CHAP contents in PLGA composite facilitated the adhesion and proliferation properties of osteoblasts on the PLGA/g-CHAP composite film.

Nano-composite of poly(L-lactide) and surface grafted hydroxyapatite: mechanical properties and biocompatibility.

In order to improve the bonding between hydroxyapatite (HAP) particles and poly(L-lactide) (PLLA), and hence to increase mechanical properties of the PLLA/HAP composite as potential bone substitute material, the HAP nano-particles were surface-grafted with PLLA and further blended with PLLA. The structure and properties of the composites were subsequently investigated by the mechanical property testing, the differential scanning calorimeter measurements (DSC), the scanning electron microscopy (SEM), the polarized optical microscopy (POM), and the cell culture. The PLLA molecules grafted on the HAP surfaces, as inter-tying molecules, played an important role in improving the adhesive strength between the particles and the polymer matrix. At a low content (approximately 4 wt%) of surface grafted-HAP (g-HAP), the PLLA/g-HAP nano-composites exhibited higher bending strength and impact energy than the pristine PLLA, and at a higher g-HAP content (e.g., 20 wt%), the modulus was remarkably increased. It implied that PLLA could be strengthened as well as toughened by g-HAP nano-particles. The results of biocompatibility test showed that the g-HAP existing in the PLLA composite facilitated both adhesion and proliferation of chondrocytes on the PLLA/g-HAP composite film.

Influence of membrane cholesterol and substrate elasticity on endothelial cell spreading behavior.

Interactions between implanted materials and the surrounding host cells critically affect the fate of bioengineered materials. In this study, the biomechanical response of bovine aortic endothelial cells (BAECs) with different membrane cholesterol levels to polyacrylamide (PA) gels was investigated by measuring cell adhesion and spreading behaviors at varying PA elasticity. The elasticity of gel substrates was manipulated by cross-linker content. Type I collagen (COL1) was coated on PA gel to provide a biologically functional environment for cell spreading. Precise quantitative characterization of changes in cell area and perimeter of cells across two treatments and three bioengineered substrates were determined using a customized software developed for computational image analysis. We found that the initial response of endothelial cells to changes in substrate elasticity was determined by membrane cholesterol levels, and that the extent of endothelial cell spreading increases with membrane cholesterol content. All of the BAECs with different cholesterol levels showed little growth on substrates with elasticity below 20 kPa, but increased spreading at higher substrate elasticity. Cholesterol-depleted cells were consistently smaller than control and cholesterol-enriched cells regardless of substrate elasticity. These observations indicate that membrane cholesterol plays an important role in cell spreading on soft biomimetic materials constructed with appropriate elasticity.

Preparation and in vitro characterization of novel bioactive glass ceramic nanoparticles.

SiO(2)-CaO-P(2)O(5) ternary bioactive glass ceramic (BGC) nanoparticles with different compositions were prepared via a three-step sol-gel method. Polyethylene glycol was selected to be used as the surfactant to improve the dispersion of the nanoparticles. The morphology and composition of these BGC nanoparticles were observed by ESEM and EDX. All the BGC particles obtained in this method were about 20 nm in diameter. XRD analysis demonstrated that the different compositions can result in very different crystallinities for the BGC nanoparticles. Bioactivity tests in simulated body fluid solution (SBF), and degradability in phosphate buffer solution (PBS), were performed in vitro. SEM, EDX, and XRD were employed to monitor the surface variation of neat poly(L-lactic acid), PLLA, foam and PLLA/BGC porous scaffolds during incubation. The BGC nanoparticles with lower phosphorous and relative higher silicon content exhibited enhanced mineralization capability in SBF and a higher solubility in PBS medium. Such novel nanoparticles may have potential to be used in different biomedical applications, including tissue engineering or the orthopedic field.

Development of bioactive and biodegradable chitosan-based injectable systems containing bioactive glass nanoparticles.

There is increasing interest in the development of new tissue engineering strategies to deliver cells and bioactive agents encapsulated in a biodegradable matrix through minimally invasive procedures. The present work proposes to combine chitosan-beta-glycerophosphate salt formulations with bioactive glass nanoparticles in order to conceive novel injectable thermo-responsive hydrogels for orthopaedic reconstructive and regenerative medicine applications. The initial rheological properties and the gelation points of the developed organic-inorganic in situ thermosetting systems were revealed to be adequate for intracorporal injection. In vitro bioactivity tests, using incubation protocols in simulated body fluid (SBF), allowed the observation of bone-like apatite formation in the hydrogel formulations containing bioactive nanoparticles. The density of the apatite formed increased with increasing bioactive glass content and soaking time in SBF. These results indicate that the stimuli-responsive hydrogels could potentially be used as temporary injectable scaffolds in bone tissue engineering applications.

In vivo mineralization and osteogenesis of nanocomposite scaffold of poly(lactide-co-glycolide) and hydroxyapatite surface-grafted with poly(L-lactide).

Nanocomposite of hydroxyapatite (HAP) surface-grafted with poly(l-lactide) (PLLA) (g-HAP) shows a wide application for bone fixation materials due to its improved interface compatibility, mechanical property and biocompatibility in our previous study. In this paper, a 3-D porous scaffold of g-HAP/poly(lactide-co-glycolide) (PLGA) was fabricated using the solvent casting/particulate leaching method to investigate its applications in bone replacement and tissue engineering. The composite of un-grafted HAP/PLGA and neat PLGA were used as controls. Their in vivo mineralization and osteogenesis were investigated by intramuscular implantation and replacement for repairing radius defects of rabbits. After surface modification, more uniform distribution of g-HAP particles but a lower calcium exposure on the surface of g-HAP/PLGA was observed. Intramuscular implantation study showed that the scaffold of g-HAP/PLGA was more stable than that of PLGA, and exhibited similar mineralization and biodegradability to HAP/PLGA at the 12-20 weeks post-surgery. The implantation study for repairing critical radius defects showed that the scaffold of g-HAP/PLGA exhibited rapid and strong mineralization and osteoconductivity, and the incorporation of BMP-2 could enhance the osteogenic process of the composite implant. The new bone formation with the intact structure of a long bone was guided by the implant of g-HAP/PLGA.

Formation of a unique crystal morphology for the poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymer.

The crystallization behaviors of the poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymer with the PEG weight fraction of 0.50 (PEG(50)-PCL(50)) was studied by DSC, WAXD, SAXS, and FTIR. A superposed melting point at 58.5 degrees C and a superposed crystallization temperature at 35.4 degrees C were obtained from the DSC profiles running at 10 degrees C/min, whereas the temperature-dependent FTIR measurements during cooling from the melt at 0.2 degrees C/min showed that the PCL crystals formed starting at 48 degrees C while the PEG crystals started at 45 degrees C. The PEG and PCL blocks of the copolymer crystallized separately and formed alternating lamella regions according to the WAXD and SAXS results. The crystal growth of the diblock copolymer was observed by polarized optical microscope (POM). An interesting morphology of the concentric spherulites developed through a unique crystallization behavior. The concentric spherulites were analyzed by in situ microbeam FTIR, and it was determined that the morphologies of the inner and outer portions were mainly determined by the PCL and PEG spherulites, respectively. However, the compositions of the inner and outer portions were equal in the analysis by microbeam FTIR.

Hydroxyapatite surface modified by L-lactic acid and its subsequent grafting polymerization of L-lactide.

A new method of surface modification of hydroxyapatite nanoparticles (n-HA) by surface grafting reaction of l-lactic acid and ring-opening polymerization of l-lactide (LLA) was developed. Two modified HA nanoparticles were obtained: HA modified by l-lactic acid (l-HA) and HA grafting with poly(l-lactide) (PLLA; p-HA). The modified surface of n-HA was attested by Fourier transformation infrared, (31)P MAS NMR, and thermal gravimetric analysis. The results showed that l-lactic acid could be easily grafted onto the n-HA surface by forming a Ca carboxylate bond and initiated by the hydroxyl group of the grafted l-lactic acid and LLA could be graft-polymerized onto the n-HA surface in the presence of stannous octanoate. The highest grafting amounts of l-lactic acid and PLLA were about 33 and 22 wt %, respectively. The modified HA/PLLA composites showed good mechanical properties and uniform microstructure. The tensile strength and modulus of the p-HA/PLLA composite containing 15 wt % of p-HA were 67 MPa and 2.1 GPa, respectively, while those of the n-HA/PLLA composites were 45 MPa and 1.7 GPa, respectively. The elongation at the break of the l-HA/PLLA composite containing 15 wt % l-HA could reach 44%, in comparison with 6.5% of the n-HA/PLLA composites containing 15 wt % n-HA.