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.

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.

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.

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.

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.

Organization of apatite crystals in human woven bone.

The organization of collagen fibrils differs in woven bone and lamellar bone, and it reflects certain aspects of the nature of the mineral crystals associated with them. In order to investigate the morphology and distribution of apatite crystals in woven bone, mineralized collagen fibrils and isolated crystals from the mid-diaphyses of human fetal femurs were observed with scanning and transmission electron microscopy and high-resolution electron microscopy. A number of features of woven bone were observed for the first time by these means. Similar to mature crystals from lamellar bone, the apatite crystals in woven bone are also platelet-shaped. However, most likely because of a high rate of old bone resorption and new bone formation in woven material, the average crystal dimensions are considerably smaller than those of mature crystals in lamellar bone. Apatite crystals were noted on the surface of collagen fibrils in woven bone. In densely packed woven bone, the periodicity of mineral deposited on individual fibrils is in registration over many fibrils. In addition to their association with collagen surfaces, crystals also appear distributed in both extrafibrillar and intrafibrillar collagen regions. In both cases, the minerals are crystalline and defect-free. These characteristics provide insight into the spatial and temporal relation between collagen and mineral that is the basis for the structure and organization of the mineral comprising human woven bone.

Mechanical properties of skeletal bone in gene-mutated stöpsel(dtl28d) and wild-type zebrafish (Danio rerio) measured by atomic force microscopy-based nanoindentation.

An atomic force microscopy (AFM)-based nanoindenter was used to evaluate the mechanical properties of skeletal bones in wild-type and gene-mutated zebrafish (Danio rerio), stöpsel(dtl28d). Both skeletons were isolated from adult zebrafish and tested under a load of 5 mN. It was found that stp/stp bone has a similar nanohardness but significantly greater elastic modulus compared with that of wild-type bone. The residual indenter impressions using AFM and the fracture surfaces of both bones using scanning electron microscopy were examined and showed that the bone of zebrafish becomes more brittle after the stp mutation. This first observation of the alteration of bone mechanical behavior by gene mutation in zebrafish system is of scientific and clinical relevance to many areas of study, such as bone fracture and fragility mechanisms in human heritable disorders and bone-materials fabrication via gene engineering.

F+ ion implantation induced cell attachment on intraocular lens.

Cell attachment on the polymethylmethacrylate (PMMA) intraocular lens (IOL) was studied by ion implantation. F+ ion implantation was performed at an energy of 80 keV with fluences ranging from 5 x 10(12) to 5 x 10(15) ions/cm2 at room temperature. The cell attachment tests gave interesting results in that the number of the platelets, the neutral granulocytes, and the macrophages adhering on the surface of the IOLs was reduced significantly after F+ ion implantation. The optimal fluence was about 3 x 10(14) to 4 x 10(14) ions/cm2. The hydrophobicity imparted to the surface was also monitored. At the same time, no appreciable change in the tensile strength and the optical transmittance of the implanted samples was observed. X-ray photoelectron spectroscopy (XPS) and fourier transfer infrared (FTIR) analysis showed that F+ ion implantation caused the cleavage of some pendants, the oxidation of the surface, and the formation of some new F-containing groups. These results were responsible for the cell attachment changes.

Morphological behaviour of osteoblasts on diamond-like carbon coating and amorphous C-N film in organ culture.

Similar to diamond-like carbon (DLC) coating, amorphous carbon nitride (C-N) films can be extremely hard and wear-resistant. They may serve as candidates for the solution to the problem of aseptic loosening of total hip replacements. Morphological behaviour of osteoblasts on silicon, DLC-coated silicon and amorphous C-N film-deposited silicon in organ culture was investigated by scanning electron microscopy. Cells on the silicon wafers were able to attach, but were unable to follow this attachment with spreading. In contrast, the cells attached, spread and proliferated on the DLC coatings and amorphous C-N films without apparent impairment of cell physiology. The morphological development of cells on the coatings and films was similar to that of cells in the control. The preliminary results support the biocompatibility of DLC coating and are encouraging for the potential biomedical applications of amorphous C-N film.

Preparation of bioactive Ti6Al4V surfaces by a simple method.

Boiling diluted alkali incubation was found to be an effective way to prepare bioactive Ti6Al4V surfaces, whether polished or not, as indicated in vitro after immersion in two different supersaturated calcification solutions (SCSs). The induction of calcium phosphate (Ca-P) precipitation from the SCSs is most probably made possible by the formation of a new TiO2 surface layer and a large number of submicron-scaled etched pits therein. The morphologies and composition of the Ca-P deposited from different SCSs are entirely different from each other. The processes on Ti6Al4V surfaces during treatment and immersion were investigated in detail by means of scanning electron microscopy combined with energy dispersive X-ray analysis, X-ray photoelectron spectroscopy and X-ray diffraction.

Variation of nanomechanical properties of bone by gene mutation in the zebrafish.

Significant variations of nanomechanical properties and fracture morphology between gene-mutated liliput(dtc232) (lil/lil) zebrafish skeletal bone and wild-type bone have been observed. Nanoindentation measurement disclosed that lil/lil bone has 36% lower nanohardness and 32% lower elastic modulus. The standard deviations of hardness and elastic modulus of lil/lil bone were both much higher than those of wild-type bone. SEM morphology of fracture surfaces further revealed that in bones after gene mutation, formative microcracks make the performance reduction and the increasing of brittleness. What is more, the plywood-like structure of the normal bone does not exist in the lil/lil bone.

Crosslinked collagen/chitosan matrix for artificial livers.

Matrices composed of collagen and chitosan may create an appropriate environment for the regeneration of livers. In this study, we have prepared, characterized and evaluated a new collagen/chitosan matrix (CCM). The CCM was made by using crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) in N-hydroxysuccinimide (NHS) and a 2-morpholinoethane sulfonic acid (MES) buffer system. The chemical characteristics were evaluated by Fourier-transformed infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The mechanical strength was measured by tensile tests. The platelet deposition and hepatocyte culture experiments show that CCM has excellent blood and cell compatibility. The results suggest that the CCM is a promising candidate matrix for implantable bioartificial livers.

Fast precipitation of calcium phosphate layers on titanium induced by simple chemical treatments.

A simple two-step chemical treatment, i.e. etching with HCl and H2SO4 followed by immersion in boiling dilute NaOH solution, has been developed by our group to prepare bioactive microporous titanium surfaces allowing fast deposition of a calcium phosphate layer (CPL) from an in vitro supersaturated calcification solution (SCS). In this work, a precalcification (Pre-Ca) procedure was applied by soaking the two-step treated titanium in Na2HPO4 and then saturated Ca(OH)2 solution before immersion in SCS to accelerate further the CPL precipitation. The treated titanium surfaces with Pre-Ca were characterized after 1, 2, 4, 8 and 16 h of immersion in SCS by means of scanning electron microscopy together with energy dispersive X-ray analysis, X-ray diffraction and infrared absorption analysis. It was observed that the CPL precipitation rate with Pre-Ca averaged 1 microm h-1, twice as fast as without Pre-Ca. No precipitation was observed on untreated titanium with Pre-Ca up to day 14 of immersion in the SCS.

Bone growth in biomimetic apatite coated porous Polyactive 1000PEGT70PBT30 implants.

We recently, developed a simple one-day one-step incubation method to obtain bone-like apatite coating on flexible and biodegradable Polyactive 1000PEGT70PBT30. The present study reports a preliminary biological evaluation on the coated polymer after implantation in rabbit femurs. The porous cylindrical implants were produced from a block fabricated by injection molding and salt leaching. This technique provided the block necessary mechanical integrity to make small cylinders (diameter 3.5 x 5 mm2) that were suitable for implantation in rabbits. The coating continuously covered the surface of the polymer, preserving the porous architecture of outer contour of the cylinders. Two defects with a diameter of 3.5 or 4 mm were drilled in the proximal and distal part of femur diaphysis. The implants were inserted as press-fit or undersized into the cortex as well as in the marrow cavity. The polymer swelled after implantation due to hydration, leading to a tight contact with the surrounding bone in both defects. The adherence of the coating on the polymer proved to be sufficient to endure a steam sterilization process as well as the 15% swelling of the polymer in vivo. The coated Polyactive 1000PEGT70PBT30 has a good osteoconductive property, as manifested by abundant bone growth into marrow cavity along the implant surface during 4-week implantation. A favorable bioactive effect of the coating with an intimate bone contact and extensive bone bonding with this polymer was qualitatively confirmed. Concerning the bone ingrowth into the porous implant in the defect of 4 mm diameter, only marginal bone formation was observed up to 8 weeks with a maximal penetration depth of about 1 mm. The pore interconnectivity is important not only for producing a coating inside the porous structure but also for bone ingrowth into this biodegradable material. This preliminary study provided promising evidence for a further study using a bigger animal model.

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.

Culture of neural cells on silicon wafers with nano-scale surface topograph.

The adherence and viability of central neural cells (substantia nigra) on a thin layer of SiO(2) on Si wafers with different surface roughness were investigated. Variable roughness of the Si wafer surface was achieved by etching. The nano-scale surface topography was evaluated by atomic force microscopy. The adherence and subsequent viability of the cells on the wafer were examined by scanning electron microscopy (SEM) and fluorescence immunostaining of tyrosine hydroxylase (TH). It is found that the surface roughness significantly affected cell adhesion and viability. Cells survived for over 5 days with normal morphology and expressed neuronal TH when grown on surfaces with an average roughness (Ra) ranging from 20 to 50 nm. However, cell adherence was adversely affected when surfaces with Ra less than 10 nm and rough surfaces with Ra above 70 nm were used as the substrate. Such a simple preparation procedure may provide a suitable interface surface for silicon-based devices and neurones or other living tissues.

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.

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.