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This study investigates the morphology, microstructure, compressive behavior, biocorrosion properties, and cytocompatibility of magnesium (Mg)-aluminum (Al) alloy (AE42) scaffolds for their potential use in biodegradable biomedical applications. Mg alloy scaffolds were successfully synthesized via a camphene-based freeze-casting process with precisely controlled heat treatment. The average porosity was approximately 52% and the median pore diameter was ∼13 μm. Salient deformation mechanisms were identified using acoustic emission (AE) signals and adaptive sequential k-means (ASK) analysis. Twinning, dislocation slip, strut bending, and collapse were dominant during compressive deformation. Nonetheless, the overall compressive behavior and deformation mechanisms were similar to those of bulk Mg based on ASK analysis. The corrosion potential of the Mg alloy scaffold (-1.44 V) was slightly higher than that of bulk AE42 (-1.60 V), but the corrosion rate of the Mg alloy scaffold was faster than that of bulk AE42 due to the enhanced surface area of the Mg alloy scaffold. As a result of cytocompatibility evaluation following ISO10993-5, the concentration of the Mg alloy scaffold extract reducing cell growth rate to 50% (IC50) was 10.7%, which is higher (less toxic) than 5%, suggesting no severe inflammation by implantation into muscle.

Hydrostatic Extrusion (HE) was successfully applied to achieve grain refinement in titanium Grade 2. Cellular response (WST-1 assay) and protein adsorption tests (colloidal gold method) were performed to investigate the effects of severe plastic deformation on the biocompatibility of titanium. STEM observations were performed in order to characterize the microstructure of the deformed metal followed by correlation with it properties. This also made it possible to better understand the phenomena occurring at the surface. The surface characteristics, including wettability, surface energy and X-ray photoelectron spectroscopy (XPS), were used to determine those factors responsible for the increase in the biocompatibility of the extruded samples. The nanostructure had beneficial effect on proliferation and attachment of SaOS-2 cells. Grain refinement affected protein adsorption behavior. Non-specific adsorption represented by albumin was favored as a result of the high density of non-equilibrium defects. After the HE process, the biocompatibility of the titanium increased due to microstructural changes affecting the properties of the native passive oxide layer. Grain refinement promoted the formation of charged sites on the surface which improved protein adsorption and increased the amount of hydroxyl (OH−) groups. Surface free energy measurements revealed that this was attributed to a high acid-base component, γAB. Indirect XPS studies demonstrated that, after the HE process, the oxide layer on the titanium surface was thinner, which contributed to greater homogeneity and better corrosion resistance. The most possible explanations for the improved biocompatibility of the nanostructured materials are also discussed in this paper.

The present study was designed to investigate the biodegradation behavior of Mg alloy plates in the maxillofacial region. For in vitro analysis, the plates were immersed in saline solution and simulated body fluid. For in vivo, the plates were implanted into the tibia, head, back, abdominal cavity, and femur and assessed at 1, 2, and 4 weeks after implantation. After implantation, the plate volumes and the formed insoluble salt were measured via micro-computed tomography. SEM/EDX analysis of the insoluble salt and histological analysis of the surrounding tissues were performed. The volume loss of plates in the in vitro groups was higher than that in the in vivo groups. The volume loss was fastest in the abdomen, followed by the head, back, tibia, and femur. There were no statistically significant differences in the insoluble salt volume of the all implanted sites. The corrosion of the Mg alloy will be affected to the surrounding tissue responses. The material for the plate should be selected based on the characteristic that Mg alloys are decomposed relatively easily in the maxillofacial region.

This study was designed to examine osteoconductive effects of Mg in rats tibia. The animals were sacrificed after 1, 2, and 8 weeks. The elemental analysis was performed using SEM/EDX at week 1. Following X-ray micrography at weeks 2 and 8, samples were embedded in paraffin. The expression of osteocalcin was observed by immunohistochemical staining. The element concentrations of fibrous capsules around the specimens were also measured by ICP-MS. The concentrations of Ca and P on the surface of the Mg specimen increased in SEM/EDX. The tissue specimen showed new bone formation on the bone surface near the implanted area. The concentrations of Mg, Ca, and P were high in the fibrous capsules surrounding Mg. Implantation induced differentiation of osteoblasts, and this process was considered to be associated with new bone formation. Induction of cell differentiation may be influenced by corrosion products in addition to corroding magnesium.

In this study, the surface of magnesium metal was electrochemically engineered for enhanced biocompatibility and controlled degradation in body fluid. Firstly, a plasma electrolytic oxidation (PEO) coating was formed on magnesium, followed by electrochemical deposition of calcium phosphate (CaP) using an unconventional electrolyte. Cytocompatibility tests using L929 cells revealed that the PEO-CaP coating significantly improved the biocompatibility of magnesium. In vitro electrochemical degradation experiments in simulated body fluid (SBF) showed that the PEO-CaP coating improved the degradation resistance of magnesium significantly. The corrosion current density (icorr) of the PEO-CaP coated magnesium was ∼99% and ∼97% lower than that of bare magnesium and the PEO-only coated magnesium, respectively. Similarly, electrochemical impedance spectroscopy (EIS) results showed that the polarisation resistance (RP) of the PEO-CaP coated magnesium was one-order of magnitude higher as compared to the PEO-only coated magnesium and two-orders of magnitude higher than the bare magnesium, after 72 h immersion in SBF. Scanning electron microscopy (SEM) analysis revealed no localized degradation in the PEO-CaP coated magnesium. The study demonstrated that the PEO-CaP coating is a promising combination for enhancing the biocompatibility and reducing the degradation of magnesium for potential biodegradable implant applications.

In this study, ultrafine grain (UFG) SS316L was produced using an equal channel angular pressing (ECAP) process at two different die angles namely 120° and 126°. The effect of different die angles on mechanical, corrosion, and surface properties were thoroughly investigated. Furthermore, the subsequent effect on the cytotoxicity of SS316L was investigated. The microstructure observation shows ECAP processing has produced an elongated, finer grain size at 120° than 126°. The ECAP processing also increases the hardness of SS316L. There is no change in wettability and surface roughness observed. However, the ectrochemical measurement reveals that ECAP processing improves the corrosion resistance of SS316L. The cytocompatibility of ECAPed SS316L was evaluated by both a direct and an extract methods, finding the contribution of grain refinement by ECAP processing.

Four kinds of biodegradable polymers were employed to prepare bioresorbable coatings on Mg-2.0Zn-0.98Mn (ZM21) alloy to understand the relationship between polymer characteristics, protective effects on substrate corrosion, cytocompatibility and cell functionality. Poly-l-lactide (PLLA), poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) or poly(lactic-co-glycolic) acid (PLGA) was spin-coated on ZM21, obtaining a smooth, non-porous coating less than 0.5μm in thickness. Polymer coating characterization, a degradation study, and biocompatibility evaluations were performed. After 4 w of immersion into cell culture medium, degradation of PLGA and PLLA coatings were confirmed by ATR-FTIR observation. The coatings of PLLA, PHB and PHBV, which have lower water permeability and slower degradation than PLGA, provide better suppression of initial ZM21 degradation and faster promotion of human osteosarcoma cell growth and differentiation.

This study aimed to investigate the potential of a skin care product composed of hyaluronic acid (HA) and collagen (Col) sponge containing epidermal growth factor (EGF), vitamin C derivative (VC), glucosylceramide (GC), poly(γ-glutamic acid) (PGA), and argentine (Arg). High-molecular weight HA aqueous solution, hydrolyzed low-molecular weight HA aqueous solution, and heat- denatured Col aqueous solution were mixed, into which each aqueous solution containing EGF, VC, GC, PGA, or Arg were added, followed by freeze-drying to obtain a spongy EGF-incorporating skin care product (EGF-skin care product). In order to evaluate the first efficacy of EGF, fibroblast proliferation was assessed after 6 days of cultivation in the conditioned medium prepared by dissolving EGF-skin care product in a conventional culture medium. The fibroblast densities increased more effectively in conditioned medium with EGF than in control medium without EGF. In order to evaluate the second efficacy of EGF, the amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) produced by fibroblasts were assessed in a wound surface model. A fibroblast-incorporating Col gel sheet (cultured dermal substitute: CDS) was elevated to the air- medium interface, onto which a spongy sheet of EGF-skin care product was placed and cultured for 7 days. The condition covered with or without EGF-skin care product is divided into (+) EGF or (-) EGF, respectively. Fibroblasts in the CDS released 3.7 times more VEGF and 25 times more HGF in (+) EGF compared with (-) EGF. In another experiment, an aqueous solution of EGF-skin care product was added onto CDS and cultured for 7 days. Aqueous solutions were prepared and stored at 4°C or 37°C for a different period of 1 day, 2 weeks, and 4 weeks, respectively. Fibroblasts in CDS under different condition released similar amount of VEGF and HGF. This result indicated that the efficacy of EGF was maintained even after preservation at 37°C for 4 weeks. These findings suggest that EGF-skin care product can be used on damaged skin surface by placing its spongy sheet or its solution.

Calcium phosphate (CaP) was electrochemically coated on a magnesium–calcium (Mg–Ca) alloy using an unconventional electrolyte and a pulse-potential method. The CaP particles of the coating were relatively large, flat, and irregularly oriented; however, they covered the entire alloy surface with a coating thickness of 5 μm. Cytocompatibility tests using L929 cells inoculated in Eagle minimum essential medium supplemented with 10% (v/v) fetal bovine serum (E-MEM+FBS) revealed that CaP coating improved the cytocompatibility of the alloy. It also showed effective suppression of Mg2+ ion release from the substrate of the coated alloy and consequently reduced the pH increase of the medium. In vitro degradation experiments using electrochemical techniques in simulated body fluid (SBF) also suggested significant enhancement of the alloy degradation resistance by CaP coating. Potentiodynamic polarization results showed that the corrosion current density of the coated alloy was ∼95% lower than that of the bare meta...

We synthesized Fe foams using water suspensions of micrometric Fe2O3 powder by reducing and sintering the sublimated Fe oxide green body to Fe under 5% H2/Ar gas. The resultant Fe foam showed aligned lamellar macropores replicating the ice dendrites. The compressive behavior and deformation mechanism of the synthesized Fe foam were studied using an acoustic emission (AE) method, with which we detected sudden localized structural changes in the Fe foam material. The evolution of the deformation mechanism was elucidated using the adaptive sequential k-means (ASK) algorithm; specifically, the plastic deformation of the cell struts was followed by localized cell collapse, which eventually led to fracturing of the cell walls. For potential biomedical applications, the corrosion and biocompatibility characteristics of the two synthesized Fe foams with different porosities (50% vs. 44%) were examined and compared. Despite its larger porosity, the superior corrosion behavior of the Fe foam with 50% porosity can be attributed to its larger pore size and smaller microscopic surface area. Based on the cytotoxicity tests for the extracts of the foams, the Fe foam with 44% porosity showed better cytocompatibility than that with 50% porosity.

The use of laser 3D printers is very perspective in the fabrication of solid and porous implants made of various polymers, metals, and its alloys. The Selective Laser Melting (SLM) process, in which consolidated powders are fully melted on each layer, gives the possibility of fabrication personalized implants based on the Computer Aid Design (CAD) model. During SLM fabrication on a 3D printer, depending on the system applied, there is a possibility for setting the amount of energy density (J/mm3) transferred to the consolidated powders, thus controlling its porosity, contact angle and roughness. In this study, we have controlled energy density in a range 8&ndash, 45 J/mm3 delivered to titanium powder by setting various levels of laser power (25&ndash, 45 W), exposure time (20&ndash, 80 &micro, s) and distance between exposure points (20&ndash, 60 &micro, m). The growing energy density within studied range increased from 63 to 90% and decreased from 31 to 13 &micro, m samples density and Ra parameter, respectively. The surface energy 55&ndash, 466 mN/m was achieved with contact angles in range 72&ndash, 128&deg, and 53&ndash, 105&deg, for water and formamide, respectively. The human mesenchymal stem cells (hMSCs) adhesion after 4 h decreased with increasing energy density delivered during processing within each parameter group. The differences in cells proliferation were clearly seen after a 7-day incubation. We have observed that proliferation was decreasing with increasing density of energy delivered to the samples. This phenomenon was explained by chemical composition of oxide layers affecting surface energy and internal stresses. We have noticed that TiO2, which is the main oxide of raw titanium powder, disintegrated during selective laser melting process and oxygen was transferred into metallic titanium. The typical for 3D printed parts post-processing methods such as chemical polishing in hydrofluoric (HF) or hydrofluoric/nitric (HF/HNO3) acid solutions and thermal treatments were used to restore surface chemistry of raw powders and improve surface.

Poly(l-lactic acid)/vaterite composite materials were coated onto metallic magnesium substrates to control rapid degradation and to improve biocompatibility. Two types of composites were prepared by adding 30 and 60 wt% of vaterite to poly(l-lactic acid) (PLLA). The composite coating layer that contained 30 wt% vaterite in the PLLA matrix had almost no pores on the surface and suppressed the initial rapid degradation of the Mg substrate. After immersion in a culture medium for 7 days, pores of 0.5–1.0 μm in diameter formed on the surface. The composite coating layer that contained 60 wt% vaterite with pores of 1.0–2.0 μm in diameter on the surface did not suppress the degradation of the Mg substrate. During immersion, the pH of the media near the composite coating surfaces was maintained at 7.4–7.5 because of the degradation of PLLA and because the vaterite particles dissolved in the solution. Proliferation of murine osteoblast-like cells (MC3T3-E1) on the substrates was improved using composite coatings. Cells on the coating that contained 60 wt% vaterite had significantly higher proliferation than those on a bare Mg substrate. Our coating provides the optimum combination to suppress the initial Mg degradation and to promote cell growth on the coating surface by adjusting the vaterite content in the composite.

In recent years there are growing interest in fabrication of bulk nanostructured metals and alloys by using severe plastic deformation (SPD) techniques as new alternative in producing bulk nanocrystalline materials. These techniques allows for processing of bulk, fully dense workpiece with ultrafine grains. Metal undergoes SPD processing in certain techniques such as high pressure torsion (HPT), equal-channel angular pressing (ECAP) or multi-directional forging (MDF) are subjected to extensive hydrostatic pressure that may be used to impart a very high strain to the bulk solid without the introduction of any significant change in overall dimension of the sample. The change in the structure (small grain size and high-volume fraction of grain boundaries) of the material may result in the corrosion behavior different from that of the coarse-grained material. Electrochemical measurements were done to understand the corrosion behavior of TiNi alloys before and after HPT deformation. The experiment was carried out using standard three electrode setup (a sample as working electrode; a platinum wire as a counter electrode and a saturated calomel electrode in saturated KCl as a reference electrode) with the surface area of 26.42 mm2 exposed to the EMEM+10% FBS cell culture medium. The measurements were performed in an incubator with controlled environment at 37 °C and 5% CO2, simulating the cell culture condition. The potential of the specimen was monitored over 1 hour, and the stabilized potential was used as the open-circuit potential (EOCP). Potentiodynamic curves were scanned in the potential range from −0.5 V to 1.5 V relative to the EOCP, at a rate of 0.5 mV/s. The result of OCP-time measurement done in the cell culture medium shows that the OCP of HPT-deformed samples shifts towards to the more positive rather than that of BHPT samples. The OCP of deformed samples were ennobled to more than +70 mV for Ti-50mol%. The shift of OCP towards the nobler direction indicates the passive nature of native oxides formed on the surface of the samples. The polarization curve, on the other hand, indicates that the HPT deformation was found to shift the passive current to nobler region. The passive region current density is found to be lower than that of the BHPT, suggesting the passive film formed on the surface of HPT-deformed samples is more protective than that of the BHPT sample. This study has shown that nanocrystallization and amorphization induced by severe plastic deformation change the corrosion behavior of TiNi alloys.

Effect of high-pressure torsion (HPT) deformation on biocompatibility and surface chemistry of TiNi was systematically investigated. Ti–50 mol% Ni was subjected to HPT straining for different numbers of turns, N = 0.25, 0.5, 1, 5 and 10 at a rotation speed of 1 rpm. X-ray photoelectron spectroscopy observations after 7 days of cell culture revealed the changes in the surface oxide composition, enrichment of Ti and detection of nitrogen derived from organic molecules in the culture medium. Plating efficiency of L929 cells was slightly increased by HPT deformation though no significant difference was observed. Albumin adsorption was higher in HPT-deformed samples, while vitronectin adsorption was peaked at N = 1. HPT deformation was also found to effectively suppress the Ni ion release from the TiNi samples into the cell culture medium even after the low degree of deformation at N = 0.25.

This study aimed to develop a novel wound dressing composed of hyaluronic acid (HA) spongy sheet containing bioactive components. The wound dressing prepared by the freeze-drying method has a two-layered structure: an upper layer composed of cross-linked high-molecular-weight HA (HMW-HA) and a lower layer composed of low-molecular-weight HA (LMW-HA) containing arginine (Arg), magnesium ascorbyl phosphate (vitamin C derivative: VC), and epidermal growth factor (EGF) (referred to as EGF-dressing). A wound dressing containing only Arg and VC was prepared in a similar manner (referred to as EGF-free-dressing). The potential of each wound dressing was evaluated in animal tests using Sprague Dawley (SD) rats and diabetic mice. In the first experiment, each wound dressing was applied to a full-thickness skin defect in the abdominal region of SD rats. Wound conditions after 1 week and 2 weeks of treatment were evaluated based on macroscopic and histological appearance. A commercially available non-woven alginate wound dressing (Alg-dressing) was used in a control group. Both EGF-free-dressing and EGF-dressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with Alg-dressing. In particular, EGF-dressing promoted re-epithelialization. In the second experiment, each wound dressing was applied to a full-thickness skin defect in the dorsal region of diabetic mice. Wound conditions after 1 week and 2 weeks of treatment were evaluated based on macroscopic and histological appearance. A commercially available Alg-dressing was used in a control group. Both EGF-free-dressing and EGF-dressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with Alg-dressing. These findings indicate that EGF-free-dressing and EGF-dressing have the potential for more effective wound healing when compared with Alg-dressing. In particular, EGF-dressing has a higher potential for wound healing when compared with EGF-free-dressing.

This study aimed to investigate the potential of cultured dermal substitute (CDS) to release angiogenic growth factors when laminated with a membrane containing epidermal growth factor (EGF) as a top dressing. Membranes were prepared by air-drying a solution of hyaluronic acid (HA) and collagen (Col) with or without EGF. Membranes were designed to contain EGF at concentrations of 0, 0.1, 0.2 or 0.5 μg/cm2. CDS was prepared by incorporating fibroblasts into a collagen gel combined with a cross-linked HA spongy matrix, followed by culturing for 5 days. CDS was designed to contain fibroblasts at a density of 2 × 105 (Group I) or 4 × 105 cells/cm2> (Group II). CDS was elevated at the interface between air and culture medium, on the top of which each membrane was placed. This culture system was employed as a wound surface model. Metabolic activity of the fibroblasts in the CDS cultured for 7 days on a wound surface model was measured by MTT assay. The amounts of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) after 7 days of cultivation were measured by using ELISA. Membranes containing EGF ranging from 0.1 to 0.5 μg/cm2> facilitated production of both VEGF and HGF, as compared with control membranes without EGF. However, a membrane containing EGF at a concentration of 0.5 μg/cm2> failed to facilitate fibroblast cytokine production in Group I. These results demonstrated that the EGF-incorporating membrane was able to stimulate fibroblasts in the CDS to synthesize an increased amount of VEGF and HGF in a dose-dependent manner.

Effect of grain refinements and amorphization by high-pressure torsion (HPT) on surface chemistry was investigated on TiNi. X-ray diffraction and micro-Vickers tests were used to check the phase changes and hardness before and after HPT. X-ray photoelectron spectroscopy was used to observe the changes in the natural passive film formation on the surface. Phase analysis reveals the change of crystalline TiNi to nanostructured one with increased hardness with straining by HPT. Grain refinement and amorphization caused by HPT reduce the amount of metallic Ni in the passive films and also increase the thickness of the film.

The biocompatibility of a recently developed Ni-free Ti–16Nb–3Mo–1Sn (at.%) superelastic alloy was investigated both in vitro and in vivo. In addition, static water contact angle (WCA) and electrochemical tests were carried out. Commercial purity Ti (cp-Ti), which is already being used as a clinical material, was used as the control material. The alloy showed a stable corrosion behavior similar to that of the cp-Ti. The WCA measurements showed that the alloy exhibited hydrophilic properties that contributed to cell attachment to implants, as evident by the cytocompatibility tests. According to the in vivo implantation tests conducted on 30 adult BALB/c rats for periods up to 12 weeks, the tissue reaction around the implants was similar for both the cp-Ti and the alloy, and no significant difference was found in almost all parameters analyzed. Due to its stable superelastic properties accompanied with excellent biocompatibility and high corrosion resistance, we believe that this alloy is considered as a promising substitute for the biomedical materials containing Ni or other toxic elements.

Novel 316 L stainless steel (SS) foam with 85% porosity and an open pore diameter of 70–440 μm was developed for hard tissue application. The foam sheet with a 200-μm diameter had superior cell proliferation and penetration as identified through in vitro experiments. Calcification of human osteosarcoma cells in the SS foam was observed. Multi-layered foam preparation is a potential alternative technique that satisfies multi-functional requirements such as cell penetration and binding strength to the solid metal. In tensile tests, Young's modulus and the strength of the SS foam were 4.0 GPa and 11.2 MPa respectively, which is comparable with human cancellous bone.

Biodegradable magnesium (Mg) alloys are the most promising candidates for osteosynthesis devices. However, their in vivo corrosion behaviour has not been fully elucidated. The aim of this study was to clarify the influence of the physiological environment surrounding Mg alloys on their corrosion behaviour. A Mg-1.0Al alloy with a fine-grained structure was formed into plates using titanium (Ti) as a control. These plates were implanted into the subperiosteum in the head, subcutaneous tissue of the back, and in the muscle of the femur of rats for 1, 2 and 4 weeks. The volumes of the remaining Mg alloy and of the insoluble salt deposition and gas cavities around the Mg alloy were determined by microtomography, and the volume losses were calculated. Then, the tissue response around the plates in each implantation site was examined histopathologically, and its relation to the respective volume loss was analyzed. These analyses determined that the Mg alloy was corroded fastest in the head, at an intermediate level in the back, and slowest in the femur. The insoluble salt deposition at the Mg alloy surface had no influence on the volume loss. Gas cavities formed around the Mg alloy at all implantation sites and decreased after 4 weeks. Histopathological examination revealed that the Mg alloy exhibited good biocompatibility, as was seen with Ti. In addition, vascularized fibrous capsules formed around the plates and became mature with time. Notably, the volume loss in the different anatomical locations correlated with capsule thickness. Together, our results suggest that, to facilitate the successful clinical application of Mg alloys, it will be necessary to further comprehend their interactions with specific in vivo environments.

Magnesium (Mg) coated with four kinds of polymers, poly ( l -lactic acid) (PLLA)-high molecular weight (HMW), PLLA-low molecular weight (LMW), poly (ɛ-caprolactone) (PCL)-HMW and PCL-LMW, and uncoated Mg were immersed under cell culture condition to study the degradation/corrosion behavior of the polymer-coated Mg. The releases of Mg2+ are measured during the immersion. Surface morphology and chemical composition are observed and identified by SEM and EDX. The tomography is obtained by X-ray CT observation and degradation rate is calculated by image analysis after 10-day immersion. All kinds of polymer-coated Mg showed significantly low release of Mg2+ (p

In recent years, magnesium and its alloys have been investigated as biodegradable metallic materials in cardiovascular stents and bone implants. However, rapid corrosion rate in the early stage of the degradation process greatly influences the cytocompatibility and hinters their application. In this research, biodegradable polymer films are prepared under same coating condition by spin coating in order to improve the early corrosion resistance and cytocompatibility of Mg. The results present that uniform, nonporous, amorphous PLLA and semi-crystalline PCL films are coated on Mg. PLLA film shows better adhesion strength to Mg substrate than that of PCL film. For both PLLA and PCL, low molecular weight (LMW) film is thinner and exhibits better adhesion strength than high molecular weight (HMW) one. SaOS-2 cells show significantly good attachment and high growth on the polymer-coated Mg, demonstrating that all the polymer films can significantly improve the cytocompatibility in the 7-day incubation. The pH measurement of the immersion medium and the quantification of released Mg(2+) during the cell culture clearly indicate that the corrosion resistance of Mg substrate is improved by the polymer films to different extents. It can be concluded that both PLLA and PCL films are promising protective coatings for improving the initial corrosion resistance and cytocompatibility.

We had developed novel titanium (Ti) foam sheet with original slurry foaming method. The products had an average pore size under 500µm in diameter, volumetric porosities over 80%, and chemical composition corresponding to grade 4 in ISO 5832-2; Implants for surgery ® Metallic materials. The Ti foam had the tensile strength of 9 to 20MPa similar to cancellous bone strength and anisotropic characteristics. Considering the application of our Ti foam for surface modification of hip or knee prostheses, we prepared Ti foam sheet joined with CP Ti, Ti­ 6Al­4V, or Zr­2.5Nb plate to evaluate the effect of joined solid metals on cell proliferation and corrosion resistance. Plate materials did not show any significant difference on cell proliferation test but affected the anode corrosion rates. Our novel Ti foam exhibited high applicability to the surface modification not only of Ti but also of Ti­6Al­4V and Zr­2.5Nb orthopedic implants. [doi:10.2320/matertrans.M2011325]

Collagen fibril/(calcium phosphate and carbonate) composite coatings on 316L stainless steel were developed with a cathodic deposition technique. The response of SaOS-2 osteoblast-like cells to the collagen/calcium salt-coated 316L steel was investigated. The collagen fibrils were self-assembled on the 316L steel surface and immobilized by their partial incorporation into a calcium salt layer electrodeposited cathodically in Hanks’ solution. The amount of calcium salt depended on the applied cathodic potential. The mineralization of collagen fibrils was observed. The collagen coverage localized and the composition of calcium salts varied on the same specimen. Such non-uniform surfaces affected the cell response. The observed outlines of cell bodies and nuclei on the thin collagen coating were clearer than those on the thick collagen coating in most cases. The collagen coating did not significantly influence the mean viability of cells on the whole specimen surface. Interestingly, the alkaline phosphatase activity per cell on the collagen/calcium salt-coated specimens was higher than that on the as-received specimen. It was revealed that cathodic deposition is an effective technique to immobilize collagen fibrils on a 316L steel surface.

The control of degradation rate of bioabsorbable magnesium devices is crucial for their biomedical applications. In this study, the influence of anodizing voltages and autoclaving on the degradation behavior of anodized pure magnesium was examined by immersion tests in a culture medium for 14 d. The anodization and autoclaving varied the morphology of surface film. Porous films were formed at 7 V and 100 V, and non-porous films were formed at 2 V and 20 V. The microscopic appearance of the anodized films did not change by autoclaving. The degradation rate on Day 1 was the highest and subsequently decreased to a quasi-steady state within the initial 3–5 d. The 7 V- and 100 V-anodized specimens showed the highest and the lowest quasi-steady degradation rate, respectively. The autoclaving significantly retarded the degradation of the anodized specimens. These facts revealed that anodization and autoclaving are useful for the control of the degradation rate of magnesium and its alloys. The porous anodized films showed local corrosion, whereas the non-porous anodized film formed at 20 V did not show apparent local corrosion. The local corrosion was prevented by autoclaving. These results suggest that the occurrence of local corrosion depends on the porous morphology of surface film.

Anticorrosion coatings are crucial for practical applications of magnesium alloys, which are used to reduce the weight of vehicles, aircraft, electronics enclosures etc. Hydroxyapatite (HAp) potentially offers high corrosion resistance and no environmental toxicity because its thermodynamic structural stability is high and it is a basic component of bone. However, direct synthesis of HAp on magnesium in aqueous solutions has been a scientific challenge because Mg ions prevent HAp crystallization. A new method of direct synthesis of HAp on magnesium was developed using a Ca chelate compound, which can maintain a sufficiently high concentration of Ca ions on the magnesium surface to overcome prevention of HAp crystallization with Mg ions. Highly crystallized HAp coatings were successfully formed on pure magnesium and AZ series alloys. Corrosion behavior of HAp-coated pure magnesium was examined by cyclic dry and wet tests with 1 g m −2 NaCl on the surface and polarization tests in a 3.5 wt% NaCl solution. A HAp-coated pure magnesium showed no noticeable corrosion pits after the dry and wet test. HAp-coated specimens showed 10 3 –10 4 times lower anodic current density than as-polished specimen in the polarization test. The results demonstrate the remarkable anticorrosion performance of HAp coatings on magnesium for the first time.

Electrochemically formed spinel-lithium manganese oxides were synthesized from manganese hydroxides prepared by a cathodic electrochemical precipitation from various concentrations of manganese nitrate solutions. Two types of manganese hydroxides were formed from diluted and concentrated Mn(NO3)2 aqueous solutions. Uniform and equi-sized disk shaped Mn(OH)2 crystals of 0.2–5 μm in diameter were obtained on a Pt substrate after the electrochemical precipitation from lower concentration of ranging from 2 mmol dm−3 to 2 mol dm−3 Mn(NO3)2 aq., while the grass blade-like precipitate which is ascribed to manganese hydroxide with 20–80 μm long and 1–5 μm wide were formed from concentrated Mn(NO3)2 aq. Both manganese hydroxides gave the electrochemically formed spinel-LiMn2O4 onto a Pt sheet, which is ready for electrochemical measurement, after calcination of the Li incorporated precipitate at 750 °C without any additives. While the shape and size of the secondary particle frameworks (aggregates) of the electrochemically formed spinel-LiMn2O4 can be controlled by the electrolysis conditions, the nanostructured primary crystals of 200 nm in diameter were obtained in all cases except that the fiber-like nanostructured spinel-LiMn2O4 crystals with 200 nm in diameter were obtained from concentrated Mn(NO3)2 aq. Though these two types of electrochemically formed spinel-LiMn2O4 showed well-shaped CVs even in higher scan rates, it would be suitable for high power density battery applications. These behaviors are assumed to be ascribed to the crystal size and shape of the processed spinel-LiMn2O4.

Calcium phosphate precipitated on pure magnesium from artificial plasma (modified Hanks’ solution) was varied by anodization and autoclaving, aiming the control of corrosion rate of bioabsorbable magnesium. Rough and smooth anodized film was formed depending on anodizing voltage in 1 N NaOH. The amount of calcium phosphate precipitated on the porous film was 2–3 times larger than that on the smooth film. The Ca/P ratio on the porous film was slightly higher than that on the smooth film. The autoclaving did not significantly influence the morphology of anodized film; however, the precipitation of calcium phosphate was restricted. No significant local corrosion occurred after the immersion in modified Hanks’ solution. It is demonstrated that the precipitation of calcium phosphate on magnesium can be controlled by anodization and autoclaving.

We present a new technique for the evaluation of polymer blood compatibility that makes use of a microchannel array flow analyzer and we describe and characterize the flow dynamics of this instrument. The blood compatibility of four polymers is quantitatively and qualitatively assessed and the results discussed. The blood is allowed to flow through the channels of a polymer coated micro-fluidic chip under adjustable pressure. The chip surface is investigated using optical microscopy during the blood flow and by scanning electron microscopy afterwards. Polymers known for having good blood compatibility exhibited higher flow rate values. Platelets were observed adhering, aggregating and obstructing the channels of the chips coated with polymers known for having poor blood compatibility. This technique has remarkable qualities such as a small blood volume requirement for material tests (100 μL), tuneable flow regimes and the use of human blood.

To understand cellular response against applied potential on different electrode materials, cell morphology and cell coverage of murine fibroblast L929 cultured on Ti and Pt electrodes were examined. Anodic and cathodic potentials were applied on Ti and Pt using a potentiostat. At the anodic potentials of 800 and 950 mV vs. Ag/AgCl, no cells kept a normal shape on both Ti and Pt. At the cathodic potential of −350 mV, the cells on Ti were normally extended ; whereas some of the cells on Pt were aggregated or round. At −1000 mV, the cells had spherical shape on Ti, while the Pt surface was covered by white precipitates with no cell remained. The change in cell morphology and the decrease in coverage at anodic potentials were significantly larger than those at the cathodic potentials. The morphological change of L929 on Ti was rather smaller than that on Pt at the same potentials, which is probably because the electric filed around the cells on Ti is smaller than that on Pt owing to the surface oxide film on Ti. These facts indicate that the cell response to the applied potential depends on the azimuth of electric field and electrode materials.

Tensile, compression and fatigue tests were carried out on a commercially extruded Mg-Al-Zn alloy having an average grain size of about 15 mm. The tensile and compression tests at room temperature showed that the yield strength in tension was much higher than that in compression. The lower yield strength in compression resulted from its texture. The effects of the mechanical anisotropy on the fatigue behavior and its deformed microstructure were also investigated under a stress ratio of R ¼ 0:1 and � 1. The fatigue strength, stress amplitude, at N ¼ 10 7 cycles under R ¼� 1 and 0.1 was about 120 and 90 MPa, respectively. The mechanical properties and the deformed microstructure observations indicated that the formation of deformation twins was related to the between maximum stress and the yield strength in tension and compression: the deformation twins were formed in the sample (maximum stress is higher than the yield strength) and showed no deformation twins in the sample (maximum stress is lower than the yield strength). [doi:10.2320/matertrans.MRP2007292]

Magnesium and its alloys are potential candidates for bioabsorbable stents. The degradation rate of an indwelled magnesium stent is expected to be controlled under a blood flow. The influence of the controlled flow on the polarization and impedance behavior of pure Mg was thus investigated in a 0.6 mass% NaCl solution using a rotating electrode. The existence of a flow caused an increase in the anodic current density as well as a decrease in the impedance for a few hours of immersion, indicating the acceleration of Mg dissolution and the retardation of the growth of the surface hydroxide film. Interestingly, the existence of a flow retarded the breakdown of the surface film. After the surface film was broken down, the impedance did not depend on the rotation speed. To precisely evaluate the degradation rate of magnesium and its alloys for use in stents, the flow rate of the test solution should be controlled. [doi:10.2320/matertrans.MRA2008011]

Metallic glasses are highly attractive because of their superior strength, relatively low Young’s modulus and superb corrosion resistance[S1], etc. By combining a metallic glass with different materials, synergy effect can be expected in the mechanical properties and corrosion resistance, for example. Fabricating a thick metallic glass layer on a substrate material by thermal spray process is one way to bond these materials. In such practice, however, the temperature of the sprayed metallic glass and that of the substrate must be kept relatively low in order to avoid crystallization and/or oxidation of the amorphous deposits. Warm Spray is a modified version of HVOF spraying developed by National Institute for Materials Science (NIMS) suited for spraying of heat sensitive feedstock materials. In the process, high-velocity solid particles heated to the temperatures below its melting point are projected on to the substrate. A Zr-base metallic glass powder was warm sprayed onto cylindrical substrates of 316L stainless steel to various thicknesses. Negligible crystallization and oxidation were observed in the deposited layer of the glass alloy. The linear relationship between the thickness of the glass alloy layer and the Young’s modulus of the composite bar demonstrated that the mechanical property of such composite structures can be controlled. [doi:10.2320/matertrans.T-MRA2007882]

To avoid the toxic effect of released nickel ions and compounds from conventional stainless steels, nickel-free austenitic stainless steels have been developed. We previously established a new manufacturing process to produce nickel-free austenitic stainless steel that involves nitrogen adsorption treatment. Although the cytocompatibility of nickelfree austenitic stainless steel produced using this method has been evaluated using two viability assay, molecular level analysis, such as gene expression analysis, has not been previously performed. In the present study, the cytotoxicity of our nickel-free austenitic stainless steel, as well as of commercially available metal materials, was evaluated by analysis of heat shock protein 70B’ (HSP70B’) gene expression as a stress response marker. Furthermore, to investigate the effect of metal materials on cytotoxicity, HSP70B’ gene expression was quantified using human osteoblast-like SaOS-2 cells, human monocyte THP-1 cells and the mouse macrophage cell line J774A.1. We found no significant differences in HSP70B’ expression among the various metal materials, including the nickel-free austenitic stainless steel, indicating that the nickel-free austenitic stainless steel produced using our nitrogen adsorption method has the same cytocompatibility as commercially available metal materials.

New short-term evaluation of material blood compatibility was attempted using a microchannel array with human blood under a flow condition. The microchannel array chips were made of silicon, having 8,736 microchannels of 10 microm-wide, 30 microm-long, and 4.5 microm-deep on the average, as the models of capillary blood vessels. Titanium, chromium, albumin and collagen were coated onto the chips to examine the difference of material blood compatibility and the effect of protein adsorption on it. The time for the first 100 microl portion of whole blood to pass through the channels (blood pass-through time, BPT) was measured under a pressure difference of 20 cmH2O. Simultaneously, the flow behavior of blood cells was observed by an optical microscope. The BPT tends to correlate well with the level of platelet adhesion. The highest BPT as well as platelet adhesion was observed on collagen, followed by titanium, chromium, silicon, and albumin. These results indicate that the BPT can detect the different levels of platelet adhesion and thrombus formation on microchannel surface and that the protein adsorption onto chip surface can influence BPT. We concluded that this method could be applied to evaluate initial blood compatibility of materials within several minutes in vitro.

Cytocompatibility of nickel-free Ti-Mo-Al shape memory alloys was evaluated and compared with that of a conventional Ti-Ni shape memory alloy. Two types of cytotoxicity tests were performed; in static and dynamic conditions. In the static condition, human normal diploid fibroblast HEL299 was cultured on the disks of the three materials and cell growth was examined. In the dynamic condition, the disks were rotated in extraction medium with zirconia balls at 37 � C for 14 days, and the collected extraction medium was added into HEL299 culture to examine its inhibitive effect on cell growth. Quantification of metallic elements in the collected extraction medium was also performed. The results showed that Ti-Mo-Al alloys had a rate of cell growth similar to that of Ti-Ni alloy in the static condition. The extraction medium of Ti-Ni alloy, however, tended to inhibit cell proliferation more than those of Ti-Mo-Al alloys. Nickel was detected in all of these extraction media, but that of Ti-Ni alloy was about three times higher than those of Ti-Mo-Al alloys. These facts indicate that developed Ti-Mo-Al alloys have higher cytocompatibility than the Ti-Ni alloy. [doi:10.2320/matertrans.48.361]

In the last few decades, there has been a remarkable progress in the field of minimally invasive surgery. Such progress has been supported by the invention and development of novel medical devices, such as stents, guide wires, and filters. Stents may be one of the most important devices used for various lesions including coronary, carotid, biliary, etc. Materials used for these devices are divers, ranging from metallic materials (e.g., stainless steels, cobalt-chromium alloys, nitinol, magnesium alloys, etc.) to biodegradable polymers. This chapter introduces the functions of stents and the currently used materials, and also gives some prospect for future materials and device development.

The melt polycondensation of the two kinds of the bis(hydroxydiphenylsilyl)arylene monomers, 1,4-bis(hydroxydiphenylsilyl)benzene and 4,4′-bis(hydroxydiphenylsilyl)biphenyl, was investigated. The thermal properties of the obtained polymers were studied by using a thermogravimetry analysis, a differential scanning calorimetry and a thermomechanical analysis. The IR spectra and the XRD patterns of the polymers indicated that the melt polycondensation occurred in the temperature range from 290 °C to 400 °C. The 5% weight loss temperatures of the polymers synthesized at above 350 °C was around or over 500 °C, so that these polymers exhibited high thermal stability. All the polymers which were obtained by the melt polycondensation of 1,4-bis(hydroxydiphenylsilyl)benzene exhibited the glass transition temperatures. On the contrary, the polymers obtained from 4,4′-bis(hydroxydiphenylsilyl)biphenyl at above 350 °C showed no phase transitions in the DSC measurements. From the analysis of the volatile compounds generated during the melt polycondensation of 4,4′-bis(hydroxydiphenylsilyl)biphenyl by using a gas chromatograph mass spectrometry and the comparison of the 13C CP-MAS NMR spectra of the polymers, it was shown that the cross-linking reaction resulting from the thermal decomposition of the monomers and the produced oligomers took place simultaneously. It was revealed that the melt polycondensation and simultaneous cross-linking reaction were effective to obtain the polysilarylenesiloxanes which have high thermal degradation temperature and high thermomechanical stability.

Polarisation test and impedance measurement were performed on powder consolidated Zr-Al-Ni-Cu amorphous alloy with and without fibroblast L929 cells incubated in medium for 7 d. L929 cells show well extended morphology, indicating that the surface of the alloy dose not show toxicity. L929 does not influence the values of cathodic and passive current densities and pitting potential of the alloy. Also, impedance behaviour is not changed with L929 cells. Those results indicate that the corrosion resistance of the powder consolidated Zr base amorphous alloy is not influenced with L929 cells. However, the pitting potential varies in the wide potential range regardless of the existence of L929 cells. Significant uniform corrosion sometimes occurs under impedance measurement at open circuit potential. It is supposed that the alloy becomes sensitive to electrochemical stimulation during the immersion in solution probably due to the defects such as grain boundary between original amorphous alloy powders.

A new type of fully aromatic polysilarylenesiloxanes showing high T g was synthesized via the solution polymerization from the bis(diphenylhydroxysilyl)arylene monomers. And the thermal properties of these monomers and polymers were investigated by using differential scanning calorimetry (DSC), and thermogravimetry analysis (TGA). The monomers containing the diphenylsilanol group showed higher melting temperatures and thermal degradation temperatures than those of the monomers containing the dimethylsilanol group. The obtained fully aromatic polysilarylenesiloxanes were insoluble powders, and showed no weight loss in air below 500 °C. No glass transition was detected according to the DSC measurements, whereas 3a and 3b showed melting points at 323 and 391 °C, respectively. However, 3b held solid-state at melting point. It would be caused by a cross-linking reaction occurring with melting simultaneously. It was found that these were the thermally stable polymer-solids consisted of the organosiloxane polymers.

β-type titanium alloys consisting of non-toxic elements, Ti–8Fe–8Ta, Ti–8Fe–8Ta–4Zr, and Ti–10Fe–10Ta–4Zr, were newly designed and developed for biomedical applications. Changes in the mechanical properties of the designed alloys with various heat treatments were discussed on the basis of the resultant microstructures. In addition, the corrosion resistance of the designed alloys was evaluated by polarization testing in Hank's solution. Conventional biomedical titanium (cp-Ti) and the titanium alloy Ti–6Al–4V ELI were also polarized for comparison. The structural phase of the designed alloys, after cold rolling and solution treatment, was only the β phase. Ultimate tensile strength and elongation to fracture of Ti–8Fe–8Ta, Ti–8Fe–8Ta–4Zr, and Ti–10Fe–10Ta–4Zr after solution treatment were 1066 MPa and 10%, 1051 MPa and 10%, and 1092 MPa and 6%, respectively. Ti–8Fe–8Ta and Ti–8Fe–8Ta–4Zr have higher strength than those of conventional biomedical titanium alloys such as Ti–6Al–4V ELI, Ti–6Al–7Nb, and Ti–13Nb–13Zr. In particular, the elongations at failure of Ti–8Fe–8Ta and Ti–8Fe–8Ta–4Zr were equal to those of Ti–6Al–4V ELI and Ti–6Al–7Nb. The designed alloys and conventional biomedical titanium alloys were spontaneously passivated in Hank's solution. The current density of cp-Ti and Ti–6Al–4V ELI was increased at a potential above 2.5 V. On the other hand, the current density of the designed alloys abruptly increased at a potential above 3.5 V. The designed alloys have the advantage over cp-Ti and Ti–6Al–4V ELI in their high resistance to pitting corrosion in biological environments. Therefore, new β-type titanium alloys designed in this study, Ti–8Fe–8Ta and Ti–8Fe–8Ta–4Zr, are expected to have good properties as biomaterials.

Friction-wear properties of a Co–29 mass%Cr–6 mass%Mo alloy (ASTM F799-95) but reducing the amount of nickel were evaluated with friction-wear test using a pin-on-flat type reciprocating friction tester in air and phosphate buffered saline, PBS(−), as a quasi-biological environment under applied stress of 1.0, 3.5 and 5.0 MPa to understand the performance of this alloy for metal-on-metal type artificial hip joints. In addition, metal ions dissolved in PBS(−) after the test was quantified. As a result, wear loss in PBS(−) is much smaller than that in air and increases with the increase of applied stress in both air and PBS(−), while the coefficient of friction in steady state during the test is larger in PBS(−) than in air, according to lubrication behavior of wear debris and PBS(−). Wear damage in PBS(−) is smaller than that in air, however more small scale of irregularity is observed in PBS(−) than that in air. Nickel and molybdenum are preferentially dissolved during the test.

Cytocompatibility of nickel-free austenitic stainless steel manufactured by nitrogen adsorption treatment, Fe–Cr–Mo–N, was evaluated and compared with a conventional austenitic stainless steel, 316L, and nickel-free ferrite stainless steel, Fe–Cr–Mo, before nitrogen adsorption treatment. Two types of cytotoxicity tests were performed; static and dynamic conditions. In static conditions, human normal diploid fibroblast HEL299 was cultured on the disks of the three materials and cell growth was examined. In dynamic conditions, the disks were rotated in extracting medium with zirconia balls at 37 °C for 14 days, and the collected extracting medium was added into HEL299 culture to examine its inhibitive effect on cell growth. Quantification of metallic elements in collected extracting medium was also performed. As a result, Fe–Cr–Mo–N had higher cell growth than 316L in static and dynamic conditions. Nickel was detected in the extracting media of 316L and Fe–Cr–Mo, whereas no nickel was detected in that of Fe–Cr–Mo–N. These facts indicate that Fe–Cr–Mo–N has higher cytocompatibility than 316L and that the nitrogen adsorption treatment contributes to the higher corrosion resistance of Fe–Cr–Mo–N in the presence of wear.

The purpose of this study was to characterize the surface oxide films on 316L austenitic stainless steel located in various environments to estimate the reconstruction of the film in human body. Five kinds of specimens were prepared according to the following methods: polished in deionized water, autoclaved, immersed in Hanks’ solution, immersed in cell culture medium, and incubated with cultured cells. X-ray photoelectron spectroscopy (XPS) was performed to estimate the compositions of the surface oxide film and substrate and the thickness of the film. Surface oxide film on 316L steel after polished in water consists of iron and chromium oxides containing small amount of nickel, molybdenum, and manganese oxides. The surface oxide contained a large amount of OH − . Calcium phosphate was formed on/in the film after immersion in the Hanks’ solution and medium and incubated with the cells. Sulfate is adsorbed by the surface oxide film and reduced to sulfite and/or sulfate in cell culture medium and with culturing cells. The results in this study suggest that nickel and manganese are depleted in the oxide film and the surface oxide changes into iron and chromium oxides containing a small amount of molybdenum oxide in human body.

In this study, a new Ti-Zr-Nb-Sn alloy system was developed as Ni-free biomedical superelastic alloys with a large recovery strain and excellent biocompatibility. Ti-18Zr-(9-16)Nb-(0-4)Sn alloys were prepared by an Ar arc melting method and the effect of composition on the crystal structure and superelastic properties was investigated. A large superelastic recovery strain of 6.0% was observed in Ti-18Zr-12.5Nb-2Sn, Ti-18Zr-11Nb-3Sn, and Ti-18Zr-9.5Nb-4Sn alloys subjected to cold-rolling and solution treatment. XRD results showed that the large recovery strain of Sn-added alloys is due to a combination effect of a large transformation strain and a strong recrystallization texture. The Ti-18Zr-11Nb-3Sn alloy exhibited excellent cyclic stability with an extremely narrow stress hysteresis about 20MPa. Cytocompatibility was also examined using three types of cell lines, murine fibroblast L929, human osteosarcoma SaOS-2, and human umbilical vein endothelial cell HUVEC and the results showed that the Ti-18Zr-11Nb-3Sn alloy exhibited larger cell covering ratios when compared with those of the Ti-50.5Ni alloy for all kinds of cells.

Anti-adhesive products need to be designed while considering the concept of wound healing. Two main events must proceed simultaneously: facilitating wound healing in surgically excised tissue, as well as preventing injured tissue from adhering to the surrounding tissue. The present study aimed to develop an anti-adhesive spongy sheet composed of hyaluronic acid and collagen (Col) containing epidermal growth factor, and to investigate the potential of this spongy sheet using an in vitro wound surface model (placing a spongy sheet on a fibroblast-incorporating Col gel sheet) and an in vitro inter-tissue model (placing a spongy sheet between two fibroblast-incorporating Col gel sheets). These in vitro experiments demonstrated that this spongy sheet effectively stimulates fibroblasts to release an increased amount of vascular endothelial growth factor and hepatocyte growth factor, which are essential for wound healing to proceed succesfully. In addition, anti-adhesive performance of this spongy sheet was evaluated in animal experiments using Sprague Dawley rats. Under anesthesia, a 1 cm × 2 cm segment of peritoneum was superficially excised from walls, and the cecum was then abraded by scraping with a scalpel blade over a 1 cm × 2 cm area. A piece of spongy sheet was placed on the peritoneal defect. Both defects were placed in contact, and the incision was closed by suturing. Peritoneal condition was evaluated after one week. This spongy sheet was capable of facilitating the wound healing of surgically excised tissue and preventing surgically excised tissue from adhering to surrounding tissues.

The present study aimed to develop a two-layered cultured dermal substitute (CDS). The upper layer is a hyaluronic acid (HA) and collagen (Col) spongy sheet with or without epidermal growth factor (EGF). The lower layer is a HA spongy sheet and Col gel containing fibroblasts. The CDS is prepared in serum-free medium, followed by placing on the wound surface. Corresponding to clinical application, CDS was incubated in serum-free medium for a period of 1, 3 or 5 days, followed by placing onto the air and culture medium interface (wound surface model), and culture for 6 days using conventional culture medium supplemented with serum. Metabolic activity and cytokine production were considerably higher in EGF-incorporating CDS, as compared with EGF-free CDS. Metabolic activity of EGF-incorporating CDS was maintained for a period of 3 days, but decreased slightly after 5 days. EGF-incorporating CDS is able to effectively stimulate fibroblasts within CDS to release increased amounts of vascular endothelial growth factor and hepatocyte growth factor, which are essential for wound healing. CDS is promising for wound therapy, because there is no risk of cellular damage caused by cryopreservation, thawing and rinsing processes. The critical issue is how to reduce the cellular damage during a prolonged period of incubation in serum-free medium. EGF-incorporating CDS can be used after a period of 3-5 days incubation in serum-free medium. This period is sufficient for transport of CDS from manufacturing facilities to hospitals.