Yingjun Wang

Genipin-cross-linked collagen/chitosan biomimetic scaffolds for articular cartilage tissue engineering applications

In this study, genipin-cross-linked collagen/chitosan biodegradable porous scaffolds were prepared for articular cartilage regeneration. The influence of chitosan amount and genipin concentration on the scaffolds physicochemical properties was evaluated. The morphologies of the scaffolds were characterized by scanning electron microscope (SEM) and cross-linking degree was investigated by ninhydrin assay. Additionally, the mechanical properties of the scaffolds were assessed under dynamic compression. To study the swelling ratio and the biostability of the collagen/chitosan scaffold, in vitro tests were also carried out by immersion of the scaffolds in PBS solution or digestion in collagenase, respectively. The results showed that the morphologies of the scaffolds underwent a fiber-like to a sheet-like structural transition by increasing chitosan amount. Genipin cross-linking remarkably changed the morphologies and pore sizes of the scaffolds when chitosan amount was less than 25%. Either by increasing the chitosan ratio or performing cross-linking treatment, the swelling ratio of the scaffolds can be tailored. The ninhydrin assay demonstrated that the addition of chitosan could obviously increase the cross-linking efficiency. The degradation studies indicated that genipin cross-linking can effectively enhance the biostability of the scaffolds. The biocompatibility of the scaffolds was evaluated by culturing rabbit chondrocytes in vitro. This study demonstrated that a good viability of the chondrocytes seeded on the scaffold was achieved. The SEM analysis has revealed that the chondrocytes adhered well to the surface of the scaffolds and contacted each other. These results suggest that the genipin-cross-linked collagen/chitosan matrix may be a promising formulation for articular cartilage scaffolding.

Enhancing Alendronate Release from a Novel PLGA/Hydroxyapatite Microspheric System for Bone Repairing Applications

PurposeThe goal of this study was to exploit the multifunction of PLGA based microsphere as efficient alendronate delivery and also as potential injectable cell carrier for bone-repairing therapeutics.Materials and MethodsNovel poly (lactic-co-glycolic acid) (PLGA)-hybridizing -hydroxyapatite (HA) microspheres loaded with bisphosphonate-based osteoporosis preventing drugs, alendronate (AL), are prepared with solid/oil/water (s/o/w) or water/oil/water (w/o/w) technique. Macrophage resistance was evaluated by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, DNA assay and Live/dead staining, and osteoblast proliferation and maturation was assessed by MTT assay, Alkaline phosphatase (ALP) activity assay and Real time-PCR.ResultsIn such fabricated AL laden PLGA/HA microspheric composites (abbreviated “PLGA/HA-AL”), the introduction of HA component has been proven capable of largely enhancing drug encapsulation efficiency especially when the single emulsion protocol is adopted. The in-vitro drug (AL) releasing profile of PLGA/HA-AL system was plotted basing over 30 days’ data collection. It indicates a sustained releasing tendency despite a minimal burst at the very beginning. The in-vitro bone-repairing efficacy of PLGA/HA-AL system was first tested with macrophages that are identified as precursors of osteoclasts and potentially responsible for osteoporosis. The results indicated that the AL release significantly inhibited the growth of macrophages. Additionally, as a central executor for osteogenesis, osteoblasts were also treated with PLGA/HA-AL system in vitro. The outcomes confirmed that this controlled release system functions to improve osteoblast proliferation and also enables upregulation of a key osteogenic enzyme ALP.ConclusionsBy pre-resisting osteoclastic commitment and promoting osteoblastic development in vitro, this newly designed PLGA/HA-AL controlled release system is promoting for bone-repairing therapeutics.

Novel mesoporous silica-based antibiotic releasing scaffold for bone repair.

Tissue engineering scaffolds with a micro- or nanoporous structure and able to deliver special drugs have already been confirmed to be effective in bone repair. In this paper, we first evaluated the biomineralization properties and drug release properties of a novel mesoporous silica-hydroxyapatite composite material (HMS-HA) which was used as drug vehicle and filler for polymer matrices. Biomineralization can offer a credible prediction of bioactivity for the synthetic bone regeneration materials. We found HMS-HA exhibited good apatite deposition properties after being soaked in simulated body fluid (SBF) for 7 days. Drug delivery from HMS-HA particle was in line with Ficks law, and the release process lasted 12 h after an initial burst release with 60% drug release. A novel tissue engineering scaffold with the function of controlled drug delivery was developed, which was based on HMS-HA particles, poly(lactide-co-glycolide) (PLGA) and microspheres sintering techniques. Mechanical testing on compression, degradation behavior, pH-compensation effect and drug delivery behavior of PLGA/HMS-HA microspheres sintered scaffolds were analyzed. Cell toxicity and cell proliferation on the scaffolds was also evaluated. The results indicated that the PLGA/HMS-HA scaffolds could effectively compensate the increased pH values caused by the acidic degradation product of PLGA. The compressive strength and modulus of PLGA/HMS-HA scaffolds were remarkably high compared to pure PLGA scaffold. Drug delivery testing of the PLGA/HMS-HA scaffolds indicated that PLGA slowed gentamycin sulfate (GS) release from HMS-HA particles, and the release lasted for nearly one month. Adding HMS-HA to PLGA scaffolds improved cytocompatibility. The scaffolds demonstrated low cytotoxicity, and supported mesenchymal stem cells growth more effectively than pure PLGA scaffolds. To summarize, the data supports the development of PLGA/HMS-HA scaffolds as potential degradable and drug delivery materials for bone replacement.

In-vitro osteogenesis of synovium stem cells induced by controlled release of bisphosphate additives from microspherical mesoporous silica composite.

In this study, in-vitro osteogenesis was successfully induced in the highly chondrogenic synovium mesenchymal stem cells (SMSCs) by controlled release of a nitrogenous bisphosphonate additive--alendronate (AL) from a mesoporous silica (MS)-hydroxyapatite (HA) composite that was mediated in poly(lactic-co-glycolic acid) (PLGA) microspheres. This microspherical based controlled release system is constructed with three levels of degradable structures: (1) the AL drug was first hybridized with HA nanoparticles; (2) the HA-AL complexes were filled into the mesopores of MS particles by self-assembly in situ; and (3) the HA-AL-laden MS constructs (MSH-AL) were built in the bulk of PLGA microspheres. In comparison with any mono-component construct, the superiority of this multi-component system comes from two aspects of functionalities: (1) significantly greater loading capacity of the extremely hydrophilic drug-AL; and (2) better controlled profile of AL release. Based on this newly developed PLGA/MSH-AL releasing system, as recipients the SMSCs, which usually exhibit exclusively high chondrogenesis, demonstrated a strong osteogenic commitment. The results were verified by alkaline phosphatase (ALP) activity assay, calcium secretion assay, real time PCR and immunohistochemistry analysis. Considering the renewable source and high proliferative profile of SMSCs, the achievement of engineered SMSC osteogenesis with this PLGA/MSH-AL controlled release system would open a new door to major bony reparation and regeneration.

In vitro generation of osteochondral differentiation of human marrow mesenchymal stem cells in novel collagen–hydroxyapatite layered scaffolds

Integrated, layered osteochondral (OC) composite materials and/or engineered OC grafts are considered as promising strategies for the treatment of OC damage. A novel biomimetic collagen-hydroxyapatite (COL-HA) OC scaffold with different integrated layers has been generated by freeze-drying. The capacity of the upper COL layer and the lower COL/HA layer to promote the growth and differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes and osteoblasts respectively was evaluated. Cell viability and proliferation on COL and COL/HA scaffolds were assessed by the MTT test. The chondrogenic differentiation of hMSCs on both scaffolds was evaluated by glucosaminoglycan (GAG) quantification, alcian blue staining, type II collagen immunocytochemistry assay and real-time polymerase chain reaction in chondrogenic medium for 21 days. Osteogenic differentiation was evaluated by alkaline phosphatase activity assay, type I collagen immunocytochemistry staining, alizarin S staining and mRNA expression of osteogenic gene for 14 days in osteogenic medium. The results indicated that hMSCs on both COL and COL/HA scaffolds were viable and able to proliferate over time. The COL layer was more efficient in inducing hMSC chondrogenic differentiation than the COL/HA layer, while the COL/HA layer possessed the superiority on promoting hMSC osteogenic induction over either COL layer or pure HA. In conclusion, the layered OC composite materials can effectively promote cartilage and bone tissue generation in vitro and are potentially usable for OC tissue engineering.

Improved injectability and in vitro degradation of a calcium phosphate cement containing poly(lactide-co-glycolide) microspheres

An injectable calcium phosphate cement (CPC) containing 30 wt.% poly(lactide-co-glycolide) (PLGA) microspheres was developed in the present study. Sodium citrate solution was used as the cement liquid phase. The effects of sodium citrate concentration on the injectability, rheological properties, mechanical strength and self-setting properties of CPC containing PLGA microspheres were systematically investigated. The in vitro degradation behavior of the composite during immersion in phosphate buffer solution was also studied. With an increase in sodium citrate concentration, the viscosity and yield stress of the paste were reduced, thereby improving the injectability. At a sodium citrate concentration of 15%, the injectability of the paste reached 95%. The compressive strength of the specimen was also enhanced by the addition of sodium citrate. The specimens had a compressive strength of 32.24+/-2.72 MPa at 15% sodium citrate concentration, compared to 22.15+/-3.60 MPa for the specimen without sodium citrate. The in vitro degradation results demonstrate that incorporated PLGA microspheres can provide the required high strength to CPC in the early stage, which would gradually degrade to create macropores for bone ingrowth. In conclusion, an in situ macropore-generable CPC exhibited excellent injectability and high early strength, and should be a promising material for bone repair and bone reconstruction.

Surface nanoscale patterning of bioactive glass to support cellular growth and differentiation.

Bioactive glasses (BGs) have been widely used for bone tissue regeneration as they are able to bond directly with bone. Clinical applications of these materials are likely to be in particulate form. Nanoscale materials can mimic the surface properties of natural tissues, which have exhibited superior cytocompatible property and improved tissue regeneration. The objective of this study is to prepare bioactive glass particles with nanoscale or non-nanoscale surface features and investigate their microstructure, apatite-forming bioactivity and cellular response. The microstructure and micro-nanoscale surface morphology were controlled by adding a hydroxyl-carboxyl acid (citric acid) in the sol-gel process. Results shown that the addition of citric acid induced the formation of nanoscale surface structure and increased the specific surface area, pore volume and pore size of bioactive glass particles. The citric acid with low-concentration-derived sol-gel bioactive glasses (CBGs) resulted in an enhanced apatite-formation ability in simulated body fluids (SBF) compared to normal bioactive glasses. The attachment and proliferation of rat marrow mesenchymal stem cells (RMSCs) on CBGs (low concentration) were higher than those of normal BGs, demonstrating that the CBGs had the excellent cytocompatibility. RMSCs on CBGs (low concentration) expressed the higher alkaline phosphatase activity (ALP) than normal BGs and tissue culture plastic, revealing that CBGs can induced differentiation of RMSCs to the osteogenic lineage. Such improved physical and biological properties of CBGs (low concentration) should be useful in developing new bioactive glass materials for stem cell-based bone regeneration or biomimic tissue engineering scaffolds.

Hydroxyapatite coatings produced on commercially pure titanium by micro-arc oxidation

A porous hydroxyapatite (HA) coating on commercially pure titanium was prepared by micro-arc oxidation (MAO) in electrolytic solution containing calcium acetate and beta-glycerol phosphate disodium salt pentahydrate (beta-GP). The thickness, phase, composition morphology and biocompatibility of the oxide coating were characterized by x-ray diffraction (XRD), electron probe microanalysis (EPMA), scanning electron microscopy (SEM) with an energy dispersive x-ray spectrometer (EDS) and cell culture. The thickness of the MAO film was about 20 microm, and the coating was porous and uneven without any apparent interface to the titanium substrates. The result of XRD showed that the porous coating was made up of HA film. The favorable osteoblast cell affinity gives HA film good biocompatibility. HA coatings are expected to have significant uses for medical applications such as dental implants and artificial bone joints.

In Situ Fabrication of Nano-hydroxyapatite in a Macroporous Chitosan Scaffold for Tissue Engineering

A chitosan (CS)/hydroxyapatite (HAP) nanohybrid scaffold with high porosity and homogeneous nanostructure was fabricated through a bionic treatment combined with thermally-induced phase separation. The nano-HAP particles were formed in situ in the scaffold at room temperature instead of mechanically mixing the powders with the polymer component. The scaffold was macroporous with a pore size of about 100–136 μm. The nano-sized HAP particles with diameters of 90–200 nm were scattered homogeneously in the interactively connective pores. Both the improvement of the compressive modulus and yield strength of the scaffolds showed that the in situ nano-HAP particles reinforced the microstructure of the scaffold. The in vitro bioactivity study carried out in simulated body fluid (SBF) indicated good mineralization activity. The crystallization phenomenon suggested that the nano-HAP particles have positive impacts on directing apatite crystallization in the scaffold and led to the good bioactivity of the nanohybrid scaffold.

Carbodiimide crosslinked collagen from porcine dermal matrix for high-strength tissue engineering scaffold.

Naturally-derived collagens for tissue engineering are limited by low mechanical strength and rapid degradation. In this study, carbodiimide is used to chemically modify the collagen derived from porcine acellular dermal matrix (PADM). The results show that the strength and resistance of PADM to enzymatic digestion can be adjusted by the reconnection of free amino and carboxyl groups of the collagen fibers. The cytocompatibility of the crosslinked PADM was evaluated by cell adhesion and proliferation assays. The cell culture studies on crosslinked and uncrosslinked PADM showed that the modification does not affect the scaffolds biocompatibility. These results demonstrate that the PADM collagen materials can be strengthened through a low-cost, non-toxic crosslinking method for potential use in biomedical applications.