Huichang Gao

In Situ Synthesis of Robust Conductive Cellulose/Polypyrrole Composite Aerogels and Their Potential Application in Nerve Regeneration

Nanostructured conductive polymers can offer analogous environments for extracellular matrix and induce cellular responses by electric stimulation, however, such materials often lack mechanical strength and tend to collapse under small stresses. We prepared electrically conductive nanoporous materials by coating nanoporous cellulose gels (NCG) with polypyrrole (PPy) nanoparticles, which were synthesized in situ from pyrrole monomers supplied as vapor. The resulting NCG/PPy composite hydrogels were converted to aerogels by drying with supercritical CO2, giving a density of 0.41-0.53 g cm(-3), nitrogen adsorption surface areas of 264-303 m(2) g(-1), and high mechanical strength. The NCG/PPy composite hydrogels exhibited an electrical conductivity of up to 0.08 S cm(-1). In vitro studies showed that the incorporation of PPy into an NCG enhances the adhesion and proliferation of PC12 cells. Electrical stimulation demonstrated that PC12 cells attached and extended longer neurites when cultured on NCG/PPy composite gels with DBSA dopant. These materials are promising candidates for applications in nerve regeneration, carbon capture, catalyst supports, and many others.

Pickering high internal phase emulsion-based hydroxyapatite–poly(ε-caprolactone) nanocomposite scaffolds

Biocompatible, biodegradable and bioactive nanocomposite (NC) scaffolds with well-defined interconnected porous structures have attracted increasing attention in bone tissue engineering. In this work, we develop a facile method to fabricate poly(l-lactic acid)-modified hydroxyapatite (g-HAp)-poly(ε-caprolactone) (PCL) NC porous scaffolds by solvent evaporation based on water-in-dichloromethane (W/O) Pickering high internal phase emulsion (HIPE) templates, which are stabilized using g-HAp nanoparticles. The resultant porous scaffolds demonstrate interconnected and rough pore structures, which can be adjusted readily by varying g-HAp nanoparticle concentration, PCL concentration and the internal phase volume fraction. Moreover, the investigation of mechanical properties and in vitro biomineralization activity shows that the Youngs modulus, compressive stress and bioactivity of the fabricated porous scaffolds are significantly enhanced upon increasing the g-HAp nanoparticle concentration. In addition, in vitro drug release studies of the porous scaffolds using ibuprofen (IBU) as a model drug show that the loaded IBU displays a sustained release profile. In vitro cell culture assays confirm that mouse bone mesenchymal stem cells can adhere, spread, and proliferate on the porous scaffolds, indicating that the porous scaffolds are biocompatible. All these results suggest that the fabricated g-HAp-PCL NC scaffolds have a promising potential for bone tissue engineering application.

Tough and Cell-Compatible Chitosan Physical Hydrogels for Mouse Bone Mesenchymal Stem Cells in Vitro

Most hydrogels involve synthetic polymers and organic cross-linkers that cannot simultaneously fulfill the mechanical and cell-compatibility requirements of biomedical applications. We prepared a new type of chitosan physical hydrogel with various degrees of deacetylation (DDs) via the heterogeneous deacetylation of nanoporous chitin hydrogels under mild conditions. The DD of the chitosan physical hydrogels ranged from 56 to 99%, and the hydrogels were transparent and mechanically strong because of the extra intra- and intermolecular hydrogen bonding interactions between the amino and hydroxyl groups on the nearby chitosan nanofibrils. The tensile strength and Youngs modulus of the chitosan physical hydrogels were 3.6 and 7.9 MPa, respectively, for a DD of 56% and increased to 12.1 and 92.0 MPa for a DD of 99% in a swelling equilibrium state. In vitro studies demonstrated that mouse bone mesenchymal stem cells (mBMSCs) cultured on chitosan physical hydrogels had better adhesion and proliferation than those cultured on chitin hydrogels. In particular, the chitosan physical hydrogels promoted the differentiation of the mBMSCs into epidermal cells in vitro. These materials are promising candidates for applications such as stem cell research, cell therapy, and tissue engineering.

miR-29b-Loaded Gold Nanoparticles Targeting to the Endoplasmic Reticulum for Synergistic Promotion of Osteogenic Differentiation.

Precise control of stem cells, such as human bone marrow-derived mesenchymal stem cells (hMSCs), is critical for the development of effective cellular therapies for tissue engineering and regeneration medicine. Emerging evidence suggests that several miRNAs act as key regulators of diverse biological processes, including differentiation of various stem cells. In this study, we have described a delivery system for miR-29b using PEI-capped gold nanoparticles (AuNPs) to synergistically promote osteoblastic differentiation. The cell proliferation assay revealed that AuNPs and AuNPs/miR-29b exert negligible cytotoxicity to hMSCs and MC3T3-E1 cells. With the assistance of AuNPs as a delivery vector, miR-29b could efficiently enter the cytoplasm and regulate osteogenesis. AuNPs/miR-29b more effectively promoted osteoblast differentiation and mineralization through induced the expression of osteogenesis genes (RUNX2, OPN, OCN, ALP) for the long-term, compared to the widely used commercial transfection reagent, Lipofectamine. With no obvious cytotoxicity, PEI-capped AuNPs showed great potential as an adequate miRNA vector for osteogenesis differentiation. Interestingly, we observed loading of AuNPs as well as AuNPs/miR-29b into the lumen of the endoplasmic reticulum (ER). Our findings collectively suggest that AuNPs, together with miR-29b, exert a synergistic promotory effect on osteogenic differentiation of hMSCs and MC3T3-E1 cells.

Engineering poly(lactic-co-glycolic acid)/calcium carbonate microspheres with controllable topography and their cell response

Polymeric porous microspheres can be used as functional vehicles in drug delivery and cell culture. However, the conventional porous microspheres are somewhat complicated with respect to preparation, and limited in composition and controllability. Here we report the preparation of poly(lactic-co-glycolic acid)/calcium carbonate (PLGA/CC) composite microspheres with uniform superficial macropores through a facile single-step method, where CC acts simultaneously as in situ pore-forming agent and reinforcing phase. The SEM images and quantified results from mercury intrusion porosimetry indicated that the size and density of the superficial macropores were highly controllable via changing the starting parameters such as size and content of CC particles and concentration of PLGA. Mouse bone mesenchymal stem cells were cultured on microspheres of different topographies to investigate the cell-substrate interaction. The results showed that cells adhered and grew well on all microspheres, while the topography with smaller and more discrete macropores exhibited the highest proliferation, indicating that cells responded to the topography of the microsphere. This work provides a novel approach to obtain diverse porous composite microspheres designed for tissue repair and study of cell-substrate interaction.

High internal phase emulsions stabilised by supramolecular cellulose nanocrystals and their application as cell-adhesive macroporous hydrogel monoliths

Nanosized celluloses are attractive building blocks to generate hierarchically advanced materials and have been gradually explored in emulsion applications. Here we report a high internal phase emulsion (HIPE) prepared by using supramolecular cellulose nanocrystals (CNCs) as Pickering stabilisers via one-step emulsification, and interconnected macroporous hybrid hydrogels were produced by utilizing this HIPE as a template. A quadruple hydrogen bonding moiety 2-ureido-4[1H]-pyrimidone (UPy) was firstly grafted onto the surface of cellulose nanocrystals through simple free radical polymerization. The polymer grafting was confirmed by elemental analysis and thermogravimetry. The UPy modified CNCs (CNC-UPy) exhibited superior emulsion stabilising ability compared to the pristine CNCs, and the oil-in-water emulsions with an internal phase volume ratio of 80% showed good long-term stability. The properties of resulting macroporous polyHIPE hydrogels, such as swelling behaviours, porous structures and mechanical strength, were investigated on the dependence of CNC-UPy concentrations. In addition, the macroporous hybrid hydrogel exhibits excellent cytocompatibility and cell adhesion as demonstrated by mouse bone mesenchymal stem cell (mBMSC) culture. With these promising properties, the developed hydrogels demonstrate great potential as active biological scaffolds for tissue engineering.

Superficially porous poly(lactic-co-glycolic acid)/calcium carbonate microsphere developed by spontaneous pore-forming method for bone repair

Biodegradable polymer microspheres have been increasingly attracting attention in bone repair. In this work, superficially porous poly(lactic-co-glycolic acid)/calcium carbonate (PLGA/CC) composite microsphere is prepared by spontaneous pore-forming emulsion method. In addition to being the component that neutralizes the acidic products from PLGA degradation, calcium carbonate (CC) also constructs macropores on the surface of microsphere and thus exempts the use of extraneous porogens, which simplifies the preparation process and avoids problems related to extraneous porogen. It is found that the size of CC plays the most essential role in forming the superficial macropores because it determines the water absorption capacity of CC. Release of dexamethasone from the superficially porous composite microspheres shows a mild burst and the subsequent tri-phase profile. Meanwhile, the cell culture test demonstrates that MG-63 cells grow well on the superficially porous composite microsphere and proliferate better than those on pure PLGA microsphere. Therefore, this versatile composite microsphere has a promising application for bone repair.

Effective Spatial Separation of PC12 and NIH3T3 Cells by the Microgrooved Surface of Biocompatible Polymer Substrates

Most organs and tissues are composed of more than one type of cell that is spatially separated and located in different regions. This study used a microgrooved poly(lactic-co-glycolic acid) (PLGA) substrate to guide two types of cocultured cells to two spatially separated regions. Specifically, PC12 pheochromocytoma cells are guided to the inside of microgrooves, whereas NIH3T3 fibroblasts are guided to the ridge area in between neighboring parallel microgrooves. In addition, the microgrooved structures can significantly promote the proliferation and neural differentiation of PC12 cells as well as the osteogenic differentiation of NIH3T3 cells. Therefore, the microgrooved PLGA surface with separated PC12 and NIH3T3 cells can serve as a potential model system for studying nerve reconstruction in bone-repairing scaffolds.

Microgrooved Polymer Substrates Promote Collective Cell Migration To Accelerate Fracture Healing in an in Vitro Model

Surface topography can affect cell adhesion, morphology, polarity, cytoskeleton organization, and osteogenesis. However, little is known about the effect of topography on the fracture healing in repairing nonunion and large bone defects. Microgrooved topography on the surface of bone implants may promote cell migration into the fracture gap to accelerate fracture healing. To prove this hypothesis, we used an in vitro fracture (wound) healing assay on the microgrooved polycaprolactone substrates to study the effect of microgroove widths and depths on the osteoblast-like cell (MG-63) migration and the subsequent healing. We found that the microgrooved substrates promoted MG-63 cells to migrate collectively into the wound gap, which serves as a fracture model, along the grooves and ridges as compared with the flat substrates. Moreover, the groove widths did not show obvious influence on the wound healing whereas the smaller groove depths tended to favor the collective cell migration and thus subsequent healing. The microgrooved substrates accelerated the wound healing by facilitating the collective cell migration into the wound gaps but not by promoting the cell proliferation. Furthermore, microgrooves were also found to promote the migration of human mesenchymal stem cells (hMSCs) to heal the fracture model. Though osteogenic differentiation of hMSCs was not improved on the microgrooved substrate, collagen I and minerals deposited by hMSCs were organized in a way similar to those in the extracellular matrix of natural bone. These findings suggest the necessity in using microgrooved implants in enhancing fracture healing in bone repair.

Engineering poly(lactic-co-glycolic acid)/hydroxyapatite microspheres with diverse macropores patterns and the cellular responses

Present studies on the topographic effects of substrates on cell functions are limited to planar substrates, which are usually not applicable in bone repair. Specific patterns are rarely constructed on 3D substrates. Here spherical substrates with macroporous topography were obtained to explore cellular responses. Macropores with tunable density were generated on the surfaces of poly(lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) microspheres by using HA particles as the pore-forming source. Different densities of macropores represented different topographies and were found to influence the morphology, proliferation and osteogenic differentiation of human fetal mesenchymal stem cells (fMSCs). The microspheres with a medium density of macropores most benefitted proliferation and differentiation of fMSCs compared with the low and high density ones. This study reveals the role of macroporous spherical surfaces in affecting cell function and may guide the design of functional substrates in bone repair.