In vivo biocompatibility and degradation behavior of Mg alloy coated by calcium phosphate in a rabbit model.

This study is conducted to investigate the biocompatibility and biodegradation behavior of calcium phosphate-coated Mg alloy in vivo. Calcium phosphate (Ca-P) was coated on the Mg alloy (AZ31) by a chemical process. Samples of Ca-P coated rods, the naked alloy rods, and degradable polymer as controls were implanted into the thighbone of rabbits to investigate the bone response at the early stage. The reduction in implant volume was determined by micro-computed tomography and three-dimensional reconstruction of the remaining Mg alloy segmented from the bone matrix. It was observed that the biodegradation rate of naked Mg implant is faster than that of the coated ones. The bone-implant interface was characterized in sections by scanning electron microscopy with energy-dispersive spectroscopy. Biodegradation or reaction layer was formed on the surface of Mg alloy implants and direct contact with the surrounding bone. The layer was mainly composed of Ca, P, O, and Mg. After 8 weeks of post-operation, paraffin sections were generated for histomorphologic analysis; 100% implants were fixed and no inflammation was observed. Histological analysis showed that new bone tissue is formed around the Mg implants, and no fibrous capsule was found. Blood examination showed that the biodegradation of the Mg implant caused little change to blood composition. Ca-P coating on Mg alloy substrate might be an effective method to reduce the biodegradation rate of Mg alloy in vivo and improve the surface bioactivity of Mg alloy implants.

Application of nerve growth factor by gel increases formation of bone in mandibular distraction osteogenesis in rabbits.

The long period of bony consolidation is a concern in mandibular distraction osteogenesis (DO). We have previously shown that repeated local injections of human nerve growth factor beta (NGFß) can appreciably improve bony consolidation in a rabbit model of DO. The present study was designed to test the effect of a single injection of human NGFß delivered by collagen/nano-hydroxyapatite/kappa-carrageenan gels to sites of new bony formation in DO. Rabbits underwent mandibular DO at a rate of 0.75 mm/12h for 6 days. At the end of the distraction period, the following injections were given percutaneously into the callus (n=6 in each of the four groups): human NGFß in the gel; human NGFß in saline; the gels alone; and saline alone. Fourteen days after the end of distraction, mechanical testing, histological and histomorphometric variables of the new bone were evaluated. Histologically, the NGFß group had more advanced consolidation than the other three groups. Both maximal load and bone volume/total volume in this group were significantly higher than in the other three (P<0.05). In conclusion, the delivery of human NGFß in the gels results in better acceleration of new bone formation than when it is given in saline, and may be a possible way to shorten the duration of craniofacial DO.

Electrospun collagen-chitosan nanofiber: a biomimetic extracellular matrix for endothelial cell and smooth muscle cell.

Electrospinning of collagen and chitosan blend solutions in a 1,1,1,3,3,3-hexafluoroisopropanol/trifluoroacetic acid (v/v, 90/10) mixture was investigated for the fabrication of a biocompatible and biomimetic nanostructure scaffold in tissue engineering. The morphology of the electrospun collagen-chitosan nanofibers was observed by scanning electron microscopy (SEM) and stabilized by glutaraldehyde (GTA) vapor via crosslinking. Fourier transform infrared spectra analysis showed that the collagen-chitosan nanofibers do not change significantly, except for enhanced stability after crosslinking by GTA vapor. X-ray diffraction analysis implied that both collagen and chitosan molecular chains could not be crystallized in the course of electrospinning and crosslinking, and gave an amorphous structure in the nanofibers. The thermal behavior and mechanical properties of electrospun collagen-chitosan fibers were also studied by differential scanning calorimetry and tensile testing, respectively. To assay the biocompatibility of electrospun fibers, cellular behavior on the nanofibrous scaffolds was also investigated by SEM and methylthiazol tetrazolium testing. The results show that both endothelial cells and smooth muscle cells proliferate well on or within the nanofiber. The results indicate that a collagen-chitosan nanofiber matrix may be a better candidate for tissue engineering in biomedical applications such as scaffolds.

Modification of bone graft by blending with lecithin to improve hydrophilicity and biocompatibility.

Lecithin was blended to improve the hydrophilicity and biocompatibility of bone graft containing poly(l-lactic acid) (PLLA). Solution blending and freeze drying were used to fabricate symmetrical scaffolds containing different percentages of lecithin (lecithin: PLLA = 0, 5, 10 wt%). Scanning electron microscopy showed that the scaffolds maintained the three-dimensional porous structure. A water uptake experiment proved the significant improvement of hydrophilicity of the blend scaffold. With the addition of lecithin, the compressive strength and compressive modulus decreased. When the weight ratio of lecithin to PLLA was up to 10%, the compressive strength was still more than the lower limit of natural cancellous bone. To test the biocompatibility of the scaffolds, cell culture in vitro and subcutaneous implantation in vivo were performed. MC3T3-E1 preosteoblastic cells were cultured on the scaffolds for 7 days. Methylthiazol tetrazolium assay and laser scanning confocal microscopy were used to exhibit proliferation and morphology of the cells. The subcutaneous implantation in rats tested inflammatory response to the scaffolds. The results proved the better biocompatibility and milder inflammatory reactions of the blend scaffold (lecithin: PLLA = 5%) compared with the scaffold without lecithin. The modified scaffold containing lecithin is promising for bone tissue engineering.

Bone regeneration by using scaffold based on mineralized recombinant collagen.

Bone regeneration was achieved in the 15-mm segmental defect model in the radius of rabbit by using the scaffold based on mineralized recombinant collagen for the first time. The recombinant collagen was recombinant human-like type I collagen, which was produced by cloning a partial cDNA that was reversed by mRNA from human collagen alpha1(I) and transferred to E. coli. The scaffold material nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid) (nHA/RHLC/PLA) was developed by biomimetic synthesis. Thermo gravimetric analysis, X-ray diffraction and scanning electron microscopy were applied to exhibit that the scaffold showed some features of natural bone both in main component and hierarchical microstructure. The percentages of organic phase and inorganic phase of nHA/RHLC were similar to that of natural bone. The three-dimensional porous scaffold materials mimic the microstructure of cancellous bone. In the implantation experiment, the segmental defect was healed 24 weeks after surgery, and the implanted composite was completely substituted by new bone tissue. The results of the implantation experiment were very comparable with that of the scaffold based on mineralized animal-sourced collagen. It is concluded that the scaffold based on mineralized recombinant collagen maintains the advantages of mineralized animal-sourced collagen, while avoids potential virus-dangers. The scaffold is a promising material for bone tissue engineering.

Biomedical modification of poly(L-lactide) by blending with lecithin.

Lecithin was, for the first time, blended with PLLA to prepare scaffold material for tissue engineering applications in the present study. Solution blending was used to incorporate Lecithin (containing 0-10 wt %) with PLLA to enhance the blend films biocompatibility, hydrophilicity and toughness while maintaining mechanical strength of PLLA. The results of FTIR-ATR analysis indicated that the amino groups of lecitin existed in the films. DSC analysis indicated that T(g) decreased with the increase of lecithin content in the blend films. The percentage elongation markedly increased with increase of lecithin content. The proliferation and viability of the vascular smooth muscle cell cultures on PLLA/Lecithin (containing 3-7 wt %) films were significantly enhanced compared to pure PLLA on tissue culture plates.

Atomic force and confocal microscopy for the study of cortical cells cultured on silicon wafers.

The primary cortical cells were selected as a model to study the adherence and neural network development on chemically roughened silicon substrates without any coatings using confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM). The silicon substrates have a nano-range roughness (RMS) achieved by chemical etching using hydrofluoric (HF) acid. After 7 days of culturing, the neurons were observed to connect together and form dense neural networks. Furthermore, AFM results revealed that some porous structures at a few micrometer range existed between the neuron cells and the silicon substrates. It is suggested that the porous structures are made of extracellular matrix (ECM) components and play an important role in the neuronal adhesion and neurite outgrowth on the inert silicon wafers.

Two types of mineral-related matrix vesicles in the bone mineralization of zebrafish.

Two types of mineral-related matrix vesicle, multivesicular body (MVB) and monovesicle, were detected in the skeletal bone of zebrafish. Transmission electron microscopy and energy dispersive spectroscopy (EDS) analyses of the vesicular inclusions reveal that both types of vesicles contain calcium and phosphorus, suggesting that these vesicles may be involved in mineral ion delivery for the bone mineralization of zebrafish. However, their size and substructure are quite different. Monovesicles, whose diameter ranges from 100 nm to 550 nm, are similar to the previously reported normal matrix vesicles, while MVBs have a larger size of 700-1000 nm in nominal diameter and possess a substructure that is composed of smaller vesicles with their average size around 100 nm. The presence of mineral-related MVBs, which is first identified in zebrafish bone, indicates that the mineralization-associated transportation process of mineral ions is more complicated than is ordinarily imagined.

Hyaluronic acid hydrogels with IKVAV peptides for tissue repair and axonal regeneration in an injured rat brain.

A biocompatible hydrogel of hyaluronic acid with the neurite-promoting peptide sequence of IKVAV was synthesized. The characterization of the hydrogel shows an open porous structure and a large surface area available for cell interaction. Its ability to promote tissue repair and axonal regeneration in the lesioned rat cerebrum is also evaluated. After implantation, the polymer hydrogel repaired the tissue defect and formed a permissive interface with the host tissue. Axonal growth occurred within the microstructure of the network. Within 6 weeks the polymer implant was invaded by host-derived tissue, glial cells, blood vessels and axons. Such a hydrogel matrix showed the properties of neuron conduction. It has the potential to repair tissue defects in the central nervous system by promoting the formation of a tissue matrix and axonal growth by replacing the lost tissue.

Hyaluronic acid hydrogel immobilized with RGD peptides for brain tissue engineering.

In this paper, hyaluronic acid hydrogels with open porous structure have been developed for scaffold of brain tissue engineering. A short peptide sequence of arginine-glycine-aspartic acid (RGD) was immobilized on the backbone of the hydrogels. Both unmodified hydrogels and those modified with RGD were implanted into the defects of cortex in rats and evaluated for their ability to improve tissue reconstruction. After 6 and 12 weeks, sections of brains were processed for DAB and Glees staining. They were also labeled with GFAP and ED1 antibodies, and observed under the SEM for ultrastructral examination. After implanting into the lesion of cortex, the porous hydrogels functioned as a scaffold to support cells infiltration and angiogenesis, simultaneously inhibiting the formation of glial scar. In addition, HA hydrogels modified with RGD were able to promote neurites extension. Our experiments showed that the hyaluronic acid-RGD hydrogel provided a structural, three-dimensional continuity across the defect and favoured reorganization of local wound-repair cells, angiogenesis and axonal growth into the hydrogel scaffold, while there was little evidence of axons regeneration in unmodified hydrogel.

Regulation of charged groups and laminin patterns for selective neuronal adhesion.

Primary neuronal cultures on substrates patterned with extracellular matrix proteins such as laminin have yielded much information regarding the physiological characteristics of neuronal cells in vitro. Surface charge also influences neuronal adherence, and a positive charge can have stimulatory effects. The attraction between laminin patterns and polycation films are of interest in the study of neuronal adhesion. We cultured primary hippocampal neurons on poly(ethylenimine) (PEI) films with laminin grids and evaluated their viability and morphology by means of fluorescent microscopy after 5-7 days. The results showed that the neurons did not form networks on the laminin grids. It is inferred that the PEI films were more favourable for neuronal adhesion than the laminin grid.

AFM study of hippocampal cells cultured on silicon wafers with nano-scale surface topograph.

The rat hippocampal cells were selected as model to study the interaction between the neural cells and silicon substrates using atomic force microscopy (AFM). The hippocampal cells show tight adherence on silicon wafers with nano-scale surface topograph. The lateral friction force investigated by AFM shows significant increase on the boundary around the cellular body. It is considered to relate to the cytoskeleton and cellular secretions. After ultrasonic wash in ethanol and acetone step by step, the surface of silicon wafers was observed by AFM sequentially. We have found that the culture leftovers form tight porous networks and a monolayer on the silicon wafers. It is concluded that the leftovers overspreading on the silicon substrates are the base of cell adherence on such smooth inert surfaces.

Hyaluronic acid-poly-D-lysine-based three-dimensional hydrogel for traumatic brain injury.

Brain tissue engineering in the postinjury brain represents a promising option for cellular replacement and rescue, providing a cell scaffold for either transplanted or resident cells. In this article, a hyaluronic acid (HA)-poly-D-lysine (PDL) copolymer hydrogel with an open porous structure and viscoelastic properties similar to neural tissue has been developed for brain tissue engineering. The chemicophysical properties of the hydrogel with HA:PDL ratios of 10:1, 5:1, and 4:1 were investigated by scanning electron microscopy (SEM) and X-ray photoelectron spectrometry. Neural cells cultured in the hydrogel were studied by phase-contrast microscope and SEM. The incorporation of PDL peptides into the HA-PDL hydrogel allowed for the modulation of neuronal cell adhesion and neural network formation. Macrophages and multinucleated foreign body giant cells found at the site of implantation of the hydrogel in the rat brain within the first weeks postimplantation decreased in numbers after 6 weeks, consistent with the host response to inert implants in numerous tissues. Of importance was the infiltration of the hydrogel by glial fibrillary acidic protein-positive cells-reactive astrocytes-by immunohistochemistry and the contiguity between the hydrogel and the surrounding tissue demonstrated by SEM. These findings indicated the compatibility of this hydrogel with brain tissue. Collectively, the results demonstrate the promise of an HA-PDL hydrogel as a scaffold material for the repair of defects in the brain.

Hyaluronic acid hydrogel as Nogo-66 receptor antibody delivery system for the repairing of injured rat brain: in vitro.

Nogo-66 and NgR are important receptors inhibiting neuronal regeneration and therefore are targets for treating CNS injury. Antagonists of this receptor including blocking antibodies are potential therapeutic agents for CNS axonal injuries such as spinal cord and brain trauma. A new antibody (IgG) releasing system has been developed by covalently attaching IgG to the biodegradable hyaluronic acid (HA) hydrogel via the hydrolytically unstable hydrazone linkage, aiming to deliver the antibody of CNS regeneration inhibitors to the injured brain. In this paper we describe the synthesis, physico-chemical characteristics and test results of biological activity of antibody released from hyluronic acid hydrogel. To form the conjugates the antibody is attached to the polymer backbone using a condensation reaction between aldehyde group of the antibody and hydrazide group of the HA hydrogel. Furthermore, pH sensitive linkage-hydrozone has been formed between hydrogel and antibody. The amount of conjugated antibodies can reach 135 microg antibody/mg hydrogel in the dry state. At low pH, the antibodies released quite fast. However, the antibodies released much slower in neutral and alkaline environment. The bioactivity of antibody released from hydrogel was retained as demonstrated by indirect immunofluorescence technique.

Property variations in the prism and the organic sheath within enamel by nanoindentation.

Atomic force microscopy (AFM) combined with nanoindentation technique was used to definitely, site-specifically, test the nanomechanical properties, including nanohardness and elastic modulus, of the isolated domains within single enamel, the prisms and the surrounding sheaths, of mature human maxillary third molars. In this way, it is for the first time that evident differences of nanomechanical properties were revealed between these domains. The nanohardness and elastic modulus of the sheaths were about 73.6% and 52.7% lower than those of the prisms, respectively. Measuring the residual impressions with AFM supported the similar conclusion. The variations of mechanical properties in these domains are considered to be mainly relative to their different component and fibrils arrangement.

Measuring membrane potential and electric field of brainstem neurons in vitro by confocal microscopy.

A method of measuring transmembrane potential and electric field of neural cells cultured in vitro is described in this paper. The high resolution and scanning speed of the method make it considerable to be used to observe the viability of the neurons cultured on opaque substrate. Rhodamine 123 was used to stain the cells in order to display different intensity corresponding to transmembrane potential. The fluorescence data were collected by confocal laser scanning microscopy (CLSM). Then the data were processed to create the graphs of transmembrane potential and electric field. This is the first paper describes a reliable method for three-dimensional visualization of potential voltage of neurons at the best of our knowledge.

Hierarchically biomimetic bone scaffold materials: nano-HA/collagen/PLA composite.

A bone scaffold material (nano-HA/ collagen/PLA composite) was developed by biomimetic synthesis. It shows some features of natural bone both in main composition and hierarchical microstructure. Nano-hydroxyapatite and collagen assembled into mineralized fibril. The three-dimensional porous scaffold materials mimic the microstructure of cancellous bone. Cell culture and animal model tests showed that the composite material is bioactive. The osteoblasts were separated from the neonatal rat calvaria. Osteoblasts adhered, spread, and proliferated throughout the pores of the scaffold material within a week. A 15-mm segmental defect model in the radius of the rabbit was used to evaluate the bone-remodeling ability of the composite. Combined with 0.5 mg rhBMP-2, the material block was implanted into the defect. The segmental defect was integrated 12 weeks after surgery, and the implanted composite was partially substituted by new bone tissue. This scaffold composite has promise for the clinical repair of large bony defects according to the principles of bone tissue engineering.

In vitro and in vivo degradation of mineralized collagen-based composite scaffold: nanohydroxyapatite/collagen/poly(L-lactide).

The objective of this article was to investigate the in vitro and in vivo biodegradation of a novel biomimetic bone scaffold composite, nanohydroxyapatite/collagen/poly(L-lactide), that could be used for bone tissue engineering. For evaluation of in vitro degradation specimens were immersed into 1% trypsin/phosphate-buffered saline solution at 37 degrees C. In vivo evaluation involved the implantation of samples into the posterolateral lumbar spine of rabbits, and the retrieved specimens were analyzed by Fourier transform-infrared spectroscopy. The results demonstrated that weight loss increased continuously in vitro with a reduction in mass of 19.6% after 4 weeks. During the experimental period in vitro, the relative rate of reduction of the three components in this material was shown to differ greatly: collagen decreased the fastest, from 40% by weight to 20% in the composite; hydroxyapatite content increased from 45 to 60%; and PLA changed little. The pore structure was maintained throughout the whole experimental period in vitro; however, the thickness of the walls of the pores decreased and the surface of the walls increased in roughness. In vivo, the ratio of collagen to hydroxyapatite appeared to be slightly higher near the transverse process than in the central part of the intertransverse process. This finding may have been due to new bone matrix formation extending from the transverse to the intertransverse process.

Hierarchical structural comparisons of bones from wild-type and liliput(dtc232) gene-mutated Zebrafish.

The alterations of hierarchical structures of bone by gene mutation in the zebrafish, which is associated with abnormal bone mineralization and bone disease, were reported for the first time in this paper. Bone samples from the liliput(dtc232) (lil) mutants as well as normal controls were studied by polarized light microscope, scanning electron microscope (SEM), transmission electron microscope (TEM), and atomic force microscope (AFM). Light microscopy examinations reveal that the lil bone has asymmetric mineralization and much thinner bone wall. The SEM studies show a lot of microcracks in lil bone wall. And the plywood-like structure of the normal bone does not exist in the lil bone, which is confirmed by the measurements of polarized light microscope. Furthermore, the TEM investigations display the collagen fibrils with two typical diameters. For the thinner collagen fibrils, the diameter of lil bone is about twice larger than that of the wild-type bone. And for the thicker one, there is a small increase in diameter after mutation and the band periodicity of the lil bone is similar with that of wild-type bone, which is consistent with the result of AFM. The morphologies of the minerals revealed that the mutated mineral was in bigger size and the shape was irregular but not plate-shaped.

Covalent immobilization of chitosan and heparin on PLGA surface.

Chitosan and heparin were covalently immobilized onto a poly(lactic acid-co-glycolic acid) (PLGA) surface using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) in a 2-morpholinoethane sulfonic acid (MES) buffer system. The properties of the modified PLGA surface and the control were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). The water contact angle of the modified film was greatly decreased and the element content on the surface of the films changed correspondingly. Platelet adhesion assay showed that blood compatibility of the chitosan/heparin modified film was improved while hepatocyte culture indicated that the cell compatibility of the modified film was enhanced.