In vitro bioactivity of a biocomposite fabricated from HA and Ti powders by powder metallurgy method

Traditionally, hydroxyapatite was used as a coating material on titanium substrate by various techniques. In the present work, a biocomposite was successfully fabricated from hydroxyapatite and titanium powders by powder metallurgy method. Bioactivity of the composite in a simulated body fluid (SBF) was investigated. Main crystal phases of the as-fabricated composite are found to be Ti2O, CaTiO3, CaO, alpha-Ti and a TiP-like phase. When the composite is immersed in the simulated body fluid for a certain time, a poor-crystallized, calcium-deficient, carbonate-containing apatite film will form on the surface of the composite. The time required to induce apatite nucleation is within 2 h. In addition, the apatite is also incorporated with a little magnesium and chlorine element. It is found that Ti2O has the ability to induce the formation of bone-like apatite in the SBF. And a dissolve of the CaO phase could also provide favorable conditions for the apatite formation, by forming open pores on the surface of the composite and increasing the degree of supersaturation of the SBF with respect to the apatite.     

In vitro degradation and cytotoxicity of Mg/Ca composites produced by powder metallurgy

Mg/Ca (1 wt.%, 5 wt.%, 10 wt.% Ca) composites were prepared from pure magnesium and calcium powders using the powder metallurgy method, aiming to enlarge the addition of Ca content without the formation of Mg₂Ca. The microstructures, mechanical properties and cytotoxicities of Mg/Ca composite samples were investigated. The corrosion of Mg/Ca composites in Dulbecco’s modified Eagle’s medium (DMEM) for various immersion intervals was studied by electrochemical impedance spectroscopy measurements and environmental scanning electron microscope, with the concentrations of released Mg and Ca ions in DMEM for various immersion time intervals being measured. It was shown that the main constitutional phases were Mg and Ca, which were uniformly distributed in the Mg matrix. The ultimate tensile strength (UTS) and elongation of experimental composites decreased with increasing Ca content, and the UTS of Mg/1Ca composite was comparable with that of as-extruded Mg–1Ca alloy. The corrosion potential increased with increasing Ca content, whereas the current density and the impedance decreased. It was found that the protective surface film formed quickly at the initial immersion stage. With increasing immersion time, the surface film became compact, and the corrosion rate of Mg/Ca composites slowed down. The surface film consisted mainly of CaCO₃, MgCO₃·3H₂O, HA and Mg(OH)₂ after 72 h immersion in DMEM. Mg/1Ca and Mg/5Ca composite extracts had no significant toxicity (p > 0.05) to L-929 cells, whereas Mg/10Ca composite extract induced ～40% reduced cell viability.     

Porous titanium scaffolds fabricated using a rapid prototyping and powder metallurgy technique

One of the main issues in orthopaedic implant design is the fabrication of scaffolds that closely mimic the biomechanical properties of the surrounding bone. This research reports on a multi-stage rapid prototyping technique that was successfully developed to produce porous titanium scaffolds with fully interconnected pore networks and reproducible porosity and pore size. The scaffolds' porous characteristics were governed by a sacrificial wax template, fabricated using a commercial 3D-printer. Powder metallurgy processes were employed to generate the titanium scaffolds by filling around the wax template with titanium slurry. In the attempt to optimise the powder metallurgy technique, variations in slurry concentration, compaction pressure and sintering temperature were investigated. By altering the wax design template, pore sizes ranging from 200 to 400 microm were achieved. Scaffolds with porosities of 66.8 +/- 3.6% revealed compression strengths of 104.4+/-22.5 MPa in the axial direction and 23.5 +/- 9.6 MPa in the transverse direction demonstrating their anisotropic nature. Scaffold topography was characterised using scanning electron microscopy and microcomputed tomography. Three-dimensional reconstruction enabled the main architectural parameters such as pore size, interconnecting porosity, level of anisotropy and level of structural disorder to be determined. The titanium scaffolds were compared to their intended designs, as governed by their sacrificial wax templates. Although discrepancies in architectural parameters existed between the intended and the actual scaffolds, overall the results indicate that the porous titanium scaffolds have the properties to be potentially employed in orthopaedic applications.     

Processing, characterization, and in vitro/in vivo evaluations of powder metallurgy processed Ti-13Nb-13Zr alloys

This article presents details of processing, characterization and in vitro as well as in vivo evaluations of powder metallurgy processed Ti-13Nb-13Zr samples with different levels of porosity. Sintered samples were characterized for density, crystalline phases (XRD), and microstructure (SEM and EDX). Samples sintered at 1000 degrees C showed the highest porosity level ( approximately 30%), featuring open and interconnected pores ranging from 50 to 100 mum in diameter but incomplete densification. In contrast, samples sintered at 1300 and 1500 degrees C demonstrated high densification with 10% porosity level distributed in a homogeneous microstructure. The different sintering conditions used in this study demonstrated a coherent trend that is increase in temperature lead to higher sample densification, even though densification represents a drawback for bone ingrowth. Cytotoxicity tests did not reveal any toxic effects of the starting and processed materials on surviving cell percentage. After an 8-week healing period in rabbit tibias, the implants were retrieved, processed for nondecalcified histological evaluation, and then assessed by backscattered electron images (BSEI-SEM) and EDX. Bone growth into the microstructure was observed only in samples sintered at 1000 degrees C. Overall, a close relation between newly formed bone and all processed samples was observed.     

In vitro and in vivo characteristics of PCL scaffolds with pore size gradient fabricated by a centrifugation method

Polycaprolactone (PCL) cylindrical scaffolds with gradually increasing pore size along the longitudinal direction were fabricated by a novel centrifugation method to investigate pore size effect on cell and tissue interactions. The scaffold was fabricated by the centrifugation of a cylindrical mold containing fibril-like PCL and the following fibril bonding by heat treatment. The scaffold showed gradually increasing pore size (from approximately 88 to approximately 405 microm) and porosity (from approximately 80% to approximately 94%) along the cylindrical axis by applying the centrifugal speed, 3000 rpm. The scaffold sections were examined for their in vitro cell interactions using different kinds of cells (chondrocytes, osteoblasts, and fibroblasts) and in vivo tissue interactions using a rabbit model (skull bone defects) in terms of scaffold pore sizes. It was observed that different kinds of cells and bone tissue were shown to have different pore size ranges in the scaffold for effective cell growth and tissue regeneration. The scaffold section with 380-405 microm pore size showed better cell growth for chondrocytes and osteoblasts, while the scaffold section with 186-200 microm pore size was better for fibroblasts growth. Also the scaffold section with 290-310 microm pore size showed faster new bone formation than those of other pore sizes. The pore size gradient scaffolds fabricated by the centrifugation method can be a good tool for the systematic studies of the interactions between cells or tissues and scaffolds with different pore size.     

In vitro and in vivo bioactivity of single-chain interleukin-12

Interleukin-12 is a heterodimeric cytokine with potent immunoregulatory properties, making it a potential vaccine adjuvant and an immune response modulator. The study of its function is confounded by its heterodimeric structure. In order to facilitate the study of interleukin-12 in both in vitro and in vivo models, we constructed a single-chain porcine interleukin-12 gene and expressed the recombinant protein in Pichia pastoris. Single-chain porcine interleukin-12 was bioactive in vitro on both human and porcine cells as measured by its ability to induce proliferation of lymphoblasts and interferon-gamma secretion by lymph node cells. In contrast, the p40 subunit of porcine interleukin-12 alone did not induce proliferation or inhibit the activity of the single-chain porcine interkeukin-12. The in vivo bioactivity of single-chain porcine interleukin-12 was demonstrated in an oral immunization model where it increased antigen-specific IgA and IgG in jejunal mucus. These results indicate that binding of interleukin-12 to its receptor and transduction of intracellular signals requires both p40 and p35 subunits. The bioactivity of interleukin-12 expressed as a single polypeptide will facilitate its in vivo delivery and study of its structure and function.     

In vitro and in vivo degradability and cytocompatibility of poly(l-lactic acid) scaffold fabricated by a gelatin particle leaching method

Porous poly(l-lactic acid) (PLLA) scaffolds fabricated by a gelatin particle-leaching technique have good mechanical property and cytocompatibility, as demonstrated by a previous in vitro study. Here we investigate further the in vitro degradation of the scaffolds in terms of weight loss, water uptake, weight-average molecular weight, thermal behavior and morphology during a 39 week period in phosphate-buffered saline. The water uptake decreased dramatically during the initial stage due to release of the remaining gelatin, and then increased slightly with degradation time. The weight-average molecular weight decreased linearly as a function of time, while the crystallinity steadily increased with slightly decreased melting temperature. After degradation, many defects and big holes were seen in the scaffolds by scanning electron microscopy. Cartilage regeneration and scaffold disappearance in vivo were compared by implanting the construct into nude mice for 30-120 days. While the scaffolds maintained their intact pore structure after 23 weeks of degradation in vitro, they almost disappeared in vivo at the same time, implying a faster degradation rate in vivo. By 120 days after implantation, the scaffolds were hardly seen in the newly formed cartilage-like tissue. The regenerated cartilages could not maintain their predesigned shape after a long period of in vivo culture due to the weakening of the mechanical strength of the constructs as a result of PLLA degradation. The regions occupied initially by PLLA scaffold were filled later by collagen type II secreted by the chondrocytes, but with no evident basophilic proteoglycan.     

Processing, properties, and in vitro bioactivity of polysulfone-bioactive glass composites

The mismatch between the mechanical properties of bioceramics and natural tissue has restricted in several cases a wider application of ceramics in medical and dental fields. To overcome this problem, polymer matrix composites can be designed to combine bioactive properties of some bioceramics with the superior mechanical properties of some engineering plastics. In this work, polymer particulate composites composed of a high mechanical-property polymer and bioactive glass particles were produced and both the in vitro bioactivity and properties of the system were investigated. Composites with different volume fraction and particle size were prepared. In vitro tests showed that hydroxy-carbonate-apatite can be deposited on the surface of a composite as early as 20 h in a simulated body fluid. Ionic evolution from a composite with 40% volume fraction of particles was demonstrated to be similar to bulk bioactive glasses. The mechanical properties of some of the obtained composites had values comparable with the ones reported for bone. Moreover, a physical model based on dynamical mechanical tests showed evidences that the interface of the composite was aiding in the stress transfer process.     

In vitro and in vivo characterization of synthetic polymer/biopolymer composites

Collagen, extracted from rat tail tendons using dilute acetic acid, was fabricated into films for subsequent characterization and biocompatibility testing. The reconstituted collagen was characterized with infrared spectroscopy, solution viscosity, contact angle, and tensile testing techniques and was found to be pure with molecular and physical properties consistent with findings of previous researchers. Composites composed of collagen coated on urethane and Silastic Rubber films were fabricated to give improved tear resistance. The biocompatibility of the composites and individual polymers was evaluated by discs implanted in the paravertebral muscle of rabbits. After four weeks none of the materials induced any gross changes in the muscle. Histopathological evaluation revealed a fibrous capsule around all of the materials. Collagen and collagen composites exhibited a stronger reaction as evidenced by a larger fibroblast layer and a variety of inflammatory cells, lymphocytes, eosinophils, and macrophages. The urethane was rated with a response index of 1.5 versus 3.25 for the urethane/collagen composite; Silastic Rubber rated a response index of 1.67 versus 3.12 for the Silastic Rubber/collagen composite; collagen rated a response index of 3.3. The polyester sutures also induced a reaction with a larger fibrous capsule but fewer inflammatory cells as compared to collagen and collagen composites.     

In vitro bioactivity and degradation of polycaprolactone composites containing silicate fillers

In spite of numerous publications on the potential use of combinations of polycaprolactone (PCL)/bioactive fillers for bone regeneration, little information exists on the assessment of solid, nonporous composites prepared via solventless routes and consisting of unmodified, slowly degrading homopolymer with relatively low amounts of reactive fillers such as bioglass or calcium silicate (CS). Thus, composites of PCL with commercial CS and a bioactive glass (BG45S5) at 30wt.% were produced by melt mixing in a twin screw extruder. Neat fillers, PCL and their composites were immersed in simulated body fluid (SBF) and phosphate buffer saline and tested for in vitro bioactivity and degradation, respectively, over a 4 month period. Testing methods included scanning electron microscopy with energy dispersive X-ray analysis, X-ray diffraction (XRD), elemental analysis and weight and pH changes before and after immersion. Experiments with neat fillers indicated fast growth of calcium phosphate minerals having different textures; they included clusters and globules of mineral precipitates as well as needle-shaped nanosized crystallites and possibly other calcium phosphate structures with varying Ca/P ratio. The bioactive glass composite initially showed fast growth of the precipitated minerals and partial surface coverage after 1 week, whereas in the CS composite, growth and surface coverage increased as a function of immersion time (over a period of 4 weeks) in the SBF solution. XRD results showed early appearance (1 week) of hydroxyapatite for both types of composites with differences attributed to different dissolution rates and different surface reactions of the fillers. Both fillers appeared to enhance the hydrolytic degradation of the matrix. Overall, the limited observed bioactivity of both composites within the test period may be related to the hydrophobicity of the matrix, insufficient ionic activity since SBF was not replenished and the relatively low content of the low surface areas fillers. Optimization of filler properties, such as surface/volume ratio, surface chemistry and size range, appears as a most important factor that would provide, at the required high filler volume fractions, a balance of melt processability and bioactivity.     

In vitro behavior of sintered powder injection molded Ti-6Al-4V/HA

This article reports the morphology and mechanical properties of sintered powder injection molded Ti-6Al-4V/HA parts in a simulated physiological environment. Sintered Ti-6Al-4V/HA parts were immersed in a simulated body fluid (SBF) with ion concentrations that were comparable to those of human blood plasma for a total period of 12 weeks. At intervals of 2 weeks, the immersed Ti-6Al-4V/HA parts were analyzed with the use of scanning electron microscopy (SEM), X-ray diffractometry (XRD), and inductively coupled plasma-atomic emission spectroscopy (ICP-AES). Mechanical properties such as flexural strength, flexural modulus, compressive strength, and compressive modulus were also evaluated. Results showed that complete dissolution of the more soluble phases such as tricalcium phosphate (TCP), tetracalcium phosphate (TTCP), and calcium oxide (CaO) were found after 2 weeks of immersion in SBF. ICP analysis showed that high calcium concentration release of around 200 ppm was observed in the SBF solution after 2-4 weeks of immersion, indicating that dissolution has taken place. Next, a gradual decrease in calcium concentration release in the SBF solution was observed after immersion for 4-6 weeks, with increasing amounts of calcium phosphate precipitates being observed on the Ti-6Al-4V/HA surface. Mechanical properties such as strength and modulus were found to deteriorate during 2-4 weeks of immersion, followed by gradual increment as the immersion period increased. This study also showed that parts sintered at 1150 C exhibited faster dissolution and precipitation rates than parts sintered at 1050 C in a physiological environment.     

In vitro and in vivo evaluation of the surface bioactivity of a calcium phosphate coated magnesium alloy

Magnesium has shown potential application as a bio-absorbable biomaterial, such as for bone screws and plates. In order to improve the surface bioactivity, a calcium phosphate was coated on a magnesium alloy by a phosphating process (Ca–P coating). The surface characterization showed that a porous and netlike CaHPO₄·2H₂O layer with small amounts of Mg²⁺ and Zn²⁺ was formed on the surface of the Mg alloy. Cells L929 showed significantly good adherence and significantly high growth rate and proliferation characteristics on the Ca–P coated magnesium alloy (p < 0.05) in in-vitro cell experiments, demonstrating that the surface cytocompatibility of magnesium was significantly improved by the Ca–P coating. In vivo implantations of the Ca–P coated and the naked alloy rods were carried out to investigate the bone response at the early stage. Both routine pathological examination and immunohistochemical analysis demonstrated that the Ca–P coating provided magnesium with a significantly good surface bioactivity (p < 0.05) and promoted early bone growth at the implant/bone interface. It was suggested that the Ca–P coating might be an effective method to improve the surface bioactivity of magnesium alloy.     

Comparison of in vitro and in vivo bioactivity of SrO-CaO-ZnO-SiO2 glass grafts

A range of calcium-strontium-zinc-silicate glass grafts are developed. Following characterization, their ability to form an apatite layer in simulated body fluid (SBF) is evaluated. Concurrently, their in vivo biocompatibility is determined. These glasses are incapable of forming an apatite layer in SBF. However, in vivo, each glass is well tolerated with new bone formation apparent in close apposition to implanted particles and no evidence of an inflammatory response. Such results are contrary to much of the literature and indicate that forecasting a materials ability to bond to bone based on SBF experiments may provide a false negative result.     

Dexamethasone-releasing biodegradable polymer scaffolds fabricated by a gas-foaming/salt-leaching method

Dexamethasone, a steroidal anti-inflammatory drug, was incorporated into porous biodegradable polymer scaffolds for sustained release. The slowly released dexamethasone from the degrading scaffolds was hypothesized to locally modulate the proliferation and differentiation of various cells. Dexamethasone containing porous poly(D,L-lactic-co-glycolic acid) (PLGA) scaffolds were fabricated by a gas-foaming/salt-leaching method. Dexamethasone was loaded within the polymer phase of the PLGA scaffold in a molecularly dissolved state. The loading efficiency of dexamethasone varied from 57% to 65% depending on the initial loading amount. Dexamethasone was slowly released out in a controlled manner for over 30 days without showing an initial burst release. Release amount and duration could be adjusted by controlling the initial loading amount within the scaffolds. Released dexamethasone from the scaffolds drastically suppressed the proliferations of lymphocytes and smooth muscle cells in vitro. This study suggests that dexamethasone-releasing PLGA scaffolds could be potentially used either as an anti-inflammatory porous prosthetic device or as a temporal biodegradable stent for reducing intimal hyperplasia in restenosis.     

Enhancing the bioactivity of zirconia and zirconia composites by surface modification

Among bioceramics, zirconia (ZrO(2)) and alumina (Al(2)O(3)) possess exceptional mechanical properties suitable for load-bearing and wear-resistant applications but the poor bioactivity of these materials is the major concern when bonding and integration to the living bone are desired. This article investigates two different approaches and their underlying mechanisms to improve the bioactivity of zirconia (3Y-TZP) and a zirconia composite with alumina (10Ce-TZP/Al(2)O(3)). Chemical treatment approach applied on 3Y-TZP where the substrates were soaked in 5M H(3)PO(4) to create chemically functional groups on the surface for inducing apatite nucleation. X-ray photoelectron spectroscopy (XPS) was used to detect chemical changes and X-ray diffraction (XRD) to monitor phase changes on the surface before and after acid treatment. Alternate soaking approach applied on 10Ce-TZP/Al(2)O(3) consisted of soaking the composite substrates in CaCl(2) and Na(2)HPO(4) solutions alternately to make a precursor for apatite formation. The bioactivity was evaluated by apatite-forming ability of surface-treated materials in simulated body fluid (SBF). Both methods resulted in the formation of hydroxyapatite on the surface of materials; however, alternate soaking approach showed to be a simpler, faster, and more effective method than the chemical treatment approach for enhancing the bioactivity of zirconia materials.     

电纺丝PLLA/HA复合纤维支架的制备及体外降解性能研究

随着组织工程和支架材料的技术发展，复合支架材料的制备成为当前研究的热点。本研究通过静电纺丝法制备了左旋聚乳酸／羟基磷灰石（PLLA／HA）复合纳米纤维膜，对纤维膜的结构形态进行分析，并研究其在与人体环境相近的磷酸盐缓冲溶液（pH7．4，37℃）中浸泡不同时间的体外降解过程。结果表明：HA纳米粒子与PLLA基体间存在化学键合，纳米粒子使纤维直径增大且表面粗糙程度增加，HA的引入抑制了PLLA降解过程中的自催化作用，减缓了PLLA的降解速度，复合纤维体系降解液的pH值在降解后期呈缓慢上升趋势。     

Microstructures and mechanical properties of powder injection molded Ti-6Al-4V/HA powder

Taguchi method with an L9 orthogonal array was employed to investigate the sintered properties of Ti-6Al-4V/HA tensile bars produced by powder injection molding. The effects of sintering factors at the 90% significance level: sintering temperature (1050 degrees C, 1100 degrees C and 1150 degrees C), heating rate (5 degrees C/min, 7.5 degrees C/min and 10 degrees C/min), holding time (30, 45 and 60 min) and cooling rate (5 degrees C/min, 20 degrees C/min and 40 degrees C/min) were investigated. Results showed that sintering temperature, heating rate and cooling rate have significant effects on sintered properties, whereas the influence of holding time was insignificant. It was found that a sintering temperature of 1100 degrees C, a heating rate of 7.5 degrees C/min and a cooling rate of 5 degrees C/min increased the relative density, Vicker's microhardness, flexural strength and flexural modulus. However, a further increment of sintering temperature to 1150 degrees C did not show any discernable improvement in the relative density and Vicker's microhardness, but there was a slight increase of 0.6% and 0.9% in the flexural strength and flexural modulus, respectively. Mechanically strong Ti-6Al-4V/HA parts with an open porosity of around 50% were developed.     

人重组粒细胞集落刺激因子突变体的构建、表达和其体内外生物学活性研究

目的为提高人重组粒细胞集落刺激因子（ｈｕｍａｎｒｅｃｏｍｂｉｎａｎｔｇｒａｎｕｌｏｃｙｔｅｃｏｌｏｎｙ－ｓｔｉｍｕｌａｔｉｎｇｆａｃｔｏｒ,ｒｈＧ－ＣＳＦ）的稳定性和活性,对ｒｈＧ－ＣＳＦ进行改造和体内、外活性研究。方法利用定向点突变技术,将人重组粒细胞集落刺激因子第１７位游离的半胱氨酸编码序列突变为丙氨酸序列,ＤＮＡ序列分析证明后,在大肠杆菌中表达突变蛋白。利用Ｇ－ＣＳＦ依赖细胞株ＮＦＳ－６０,对在不同温度及在人血浆中保存的Ｇ－ＣＳＦ蛋白（Ｇ－ＣＳＦ－Ａｌａ１７）和野生型的Ｇ－ＣＳＦ进行活性测定。腹腔注射后小鼠外周血白细胞计数检测其体内生物学活性。结果ＤＮＡ序列分析表明１７位半胱氨酸突变为丙氨酸,并在大肠杆菌中表达成功Ｇ－ＣＳＦ－Ａｌａ１７。纯化的突变蛋白对ＮＦＳ－６０细胞具有刺激活性;与野生型Ｇ－ＣＳＦ相比,在各种温度储存的突变蛋白保留更高的活性,与人血浆孵育５０小时后突变蛋白仍保持活性;小鼠一次性体内注射突变蛋白后外周血白细胞数明显高于野生型Ｇ－ＣＳＦ。结论Ｇ－ＣＳＦ突变体（Ｇ－ＣＳＦ－Ａｌａ１７）较野生型的Ｇ－ＣＳＦ具有更高的体外稳定性和体内造血刺激活性。     

In vitro and in vivo toxicity of rinsed and aged nanocellulose-polypyrrole composites

Novel composites of nanocellulose and the conducting polymer polypyrrole (PPy) are herein suggested as potential candidates for active ion-extraction membranes in electrochemically controlled hemodialysis. This study has defined processing parameters to obtain a biocompatible nanocellulose-PPy composite, and for the first time, the effect of the composite aging on cell viability has been studied. The influence of rinsing and extraction process steps, as well as aging under different conditions (i.e. in air, at -20°C and in argon), on the electroactivity and cytotoxicity of a PPy-nanocellulose composite has been investigated. The biocompatibility evaluation was based on indirect toxicity assays with fibroblasts and monocyte cell lines and an acute toxicity test in mice, while the electroactivity was evaluated by cyclic voltammetry experiments. The as-prepared composite did not induce any cytotoxic response in vitro or in vivo. Extensive rinsing and 48 h incubation in biological buffer previous to the preparation of the culture medium extracts were, however, necessary to obtain a noncytotoxic composite. The as-prepared composite was also found to exhibit acceptable electrochemical performance, which was retained upon 4 weeks storage in argon atmosphere. It was shown that aging of the composite had a negative effect on biocompatibility, regardless of the storage condition. Thus, to allow for longtime storage of electroactive nanocellulose-PPy hemodialysis membranes, the degradation of PPy upon storage must be controlled. The present results show that the biocompatibility of PPy composites depends on the rinsing and pretreatment of the composite material as well as the aging of the material.