Zhongkui Hong

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.

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.

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.

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.

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.

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 scaffolds 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.

Increased vascular smooth muscle cell stiffness: a novel mechanism for aortic stiffness in hypertension.

Increased vascular stiffness is fundamental to hypertension, and its complications, including atherosclerosis, suggest that therapy should also be directed at vascular stiffness, rather than just the regulation of peripheral vascular resistance. It is currently held that the underlying mechanisms of vascular stiffness in hypertension only involve the extracellular matrix and endothelium. We hypothesized that increased large-artery stiffness in hypertension is partly due to intrinsic mechanical properties of vascular smooth muscle cells. After confirming increased arterial pressure and aortic stiffness in spontaneously hypertensive rats, we found increased elastic stiffness of aortic smooth muscle cells of spontaneously hypertensive rats compared with Wistar-Kyoto normotensive controls using both an engineered aortic tissue model and atomic force microscopy nanoindentation. Additionally, we observed different temporal oscillations in the stiffness of vascular smooth muscle cells derived from hypertensive and control rats, suggesting that a dynamic component to cellular elastic stiffness is altered in hypertension. Treatment with inhibitors of vascular smooth muscle cell cytoskeletal proteins reduced vascular smooth muscle cell stiffness from hypertensive and control rats, suggesting their participation in the mechanism. This is the first study demonstrating that stiffness of individual vascular smooth muscle cells mediates vascular stiffness in hypertension, a novel concept, which may elucidate new therapies for hypertension and for vascular stiffness.

Augmented Vascular Smooth Muscle Cell Stiffness and Adhesion When Hypertension Is Superimposed on Aging

Hypertension and aging are both recognized to increase aortic stiffness, but their interactions are not completely understood. Most previous studies have attributed increased aortic stiffness to changes in extracellular matrix proteins that alter the mechanical properties of the vascular wall. Alternatively, we hypothesized that a significant component of increased vascular stiffness in hypertension is due to changes in the mechanical and adhesive properties of vascular smooth muscle cells, and that aging would augment the contribution from vascular smooth muscle cells when compared with the extracellular matrix. Accordingly, we studied aortic stiffness in young (16-week-old) and old (64-week-old) spontaneously hypertensive rats and Wistar–Kyoto wild-type controls. Systolic and pulse pressures were significantly increased in young spontaneously hypertensive rats when compared with young Wistar–Kyoto rats, and these continued to rise in old spontaneously hypertensive rats when compared with age-matched controls. Excised aortic ring segments exhibited significantly greater elastic moduli in both young and old spontaneously hypertensive rats versus Wistar–Kyoto rats. were isolated from the thoracic aorta, and stiffness and adhesion to fibronectin were measured by atomic force microscopy. Hypertension increased both vascular smooth muscle cell stiffness and vascular smooth muscle cell adhesion, and these increases were both augmented with aging. By contrast, hypertension did not affect histological measures of aortic collagen and elastin, which were predominantly changed by aging. These findings support the concept that stiffness and adhesive properties of vascular smooth muscle cells are novel mechanisms contributing to the increased aortic stiffness occurring with hypertension superimposed on aging.

Temporal analysis of vascular smooth muscle cell elasticity and adhesion reveals oscillation waveforms that differ with aging

A spectral analysis approach was developed for detailed study of time‐resolved, dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion to identify differences in VSMC from young and aged monkeys. Atomic force microscopy (AFM) was used to measure Young’s modulus of elasticity and adhesion as assessed by fibronectin (FN) or anti‐beta 1 integrin interaction with the VSMC surface. Measurements demonstrated that VSMC cells from old vs. young monkeys had increased elasticity (21.6 kPa vs. 3.5 kPa or a 612% increase in elastic modulus) and adhesion (86 pN vs. 43 pN or a 200% increase in unbinding force). Spectral analysis identified three major frequency components in the temporal oscillation patterns for elasticity (ranging from 1.7 × 10−3 to 1.9 × 10−2 Hz in old and 8.4 × 10−4 to 1.5 × 10−2 Hz in young) and showed that the amplitude of oscillation was larger (P < 0.05) in old than in young at all frequencies. It was also observed that patterns of oscillation in the adhesion data were similar to the elasticity waveforms. Cell stiffness was reduced and the oscillations were inhibited by treatment with cytochalasin D, ML7 or blebbistatin indicating the involvement of actin–myosin‐driven processes. In conclusion, these data demonstrate the efficacy of time‐resolved analysis of AFM cell elasticity and adhesion measurements and that it provides a uniquely sensitive method to detect real‐time functional differences in biomechanical and adhesive properties of cells. The oscillatory behavior suggests that mechanisms governing elasticity and adhesion are coupled and affected differentially during aging, which may link these events to changes in vascular stiffness.

Coordination of Fibronectin Adhesion with Contraction and Relaxation in Microvascular Smooth Muscle

AIMS The regulation of vascular diameter by vasoconstrictors and vasodilators requires that vascular smooth muscle cells (VSMCs) be physically coupled to extracellular matrix (ECM) and neighbouring cells in order for a vessel to mechanically function and transfer force. The hypothesis was tested that integrin-mediated adhesion to the ECM is dynamically up-regulated in VSMCs during contractile activation in response to a vasoconstrictor and likewise down-regulated during relaxation in response to a vasodilator. METHODS AND RESULTS VSMCs were isolated from the Sprague-Dawley rat cremaster muscles. Atomic force microscopy (AFM) with fibronectin (FN)-functionalized probes was employed to investigate the biomechanical responses and adhesion of VSMCs. Responses to angiotensin II (Ang II; 10(-6) M) and adenosine (Ado; 10(-4) M) were recorded by measurements of cell cortical elasticity and cell adhesion. The results showed that Ang II caused an immediate increase in adhesion (+27%) between the probe and cell. Cell stiffness increased (+70%) in parallel with the adhesion change. Ado decreased adhesion (-15%) to FN and reduced (-30%) stiffness. CONCLUSION Changes in the receptor-mediated activation of the contractile apparatus cause parallel alterations in cell adhesion and cell cortical elasticity. These studies support the hypothesis that the regulation of cell adhesion is coordinated with contraction and demonstrate the dynamic nature of cell adhesion to the ECM. It is proposed that coordination of adhesion and VSMC contraction is an important mechanism that allows for an efficient transfer of force between the contractile apparatus of the cell and the extracellular environment.