Osteogenic differentiation of human adipose-derived stem cells in 3D conditions - comparison of spheroids and polystyrene scaffolds.

Expansion and differentiation of adipose-derived stem cells (ADSCs) in vitro are routinely performed in two-dimensional (2D) environments. The study hypothesis was that the utilisation of three-dimensional (3D) culture conditions, mimicking the natural stem cell niche, might increase the osteogenic commitment of ADSCs. Therefore, human ADSCs were seeded in 3D culture systems lacking bioactive material components: spheroids and polystyrene scaffolds. ALP activity, a marker of early osteogenesis, was higher in ADSC spheroids and ADSC seeded on polystyrene scaffolds as compared to 2D cultures. Furthermore, the expression of the osteoblast marker genes Runt-related transcription factor 2 (RUNX2), osterix and integrin binding sialoprotein (IBSP) was significantly up-regulated in spheroids as compared to polystyrene scaffolds and 2D culture. Elevated levels of RUNX2 and IBSP in spheroids were confirmed at the protein level by Western blot and immunofluorescence, respectively. Bone mineral production was lower in spheroids than in polystyrene scaffolds and 2D culture at day 14. Curiously, adipocyte differentiation was downregulated in spheroids as compared to 2D-culture. Finally, to induce late differentiation events, cells were dissociated from spheroids after a 7 d osteogenic pre-differentiation culture and replated in 2D culture in osteoblast maturation medium. After a subsequent 14 d of maturation, cells produced bone mineral and osteocalcin proteins, which are late osteoblast markers. The present work showed that the 3D environment may provide additional stimuli for the commitment of ADSCs to the osteogenic lineage. Furthermore, the presented data may be valuable when designing protocols to prepare ADSCs for use in bone regeneration clinical studies.

Moisture-cured silicone-urethanes-candidate materials for tissue engineering: a biocompatibility study in vitro.

This study was performed to verify the response of human bone-derived cells (HBDCs) to moisture-cured silicone-urethanes (mcSUUs) in vitro, as the first step toward using them as scaffolds for bone tissue engineering. Good surgical handling, tissue cavity filling, stable mechanical properties, and potentially improved oxygen supply to cells after implantation justify the investigation of these nondegradable elastomers. A set of various mcSUUs were obtained by moisture-curing NCO-terminated prepolymers, synthesized from oligomeric siloxane diols of two different oligosiloxane chain lengths, and two different diisocyanates (MDI and IPDI), using two different NCO/OH molar ratios. Dibutyltindilaurate (DBTL) or N-dimethylethanolamine (N-met) served as catalysts. After 7 days of culture, cell number, viability, and alkaline phosphatase (ALP) activity were determined, and after 21 days, cell viability and collagen production were determined. Material characteristics significantly influenced the cell response. The mcSUUs prepared with DBTL (widely used in the syntheses of biomaterials) were cytotoxic. The MDI-based mcSUUs were significantly more favored by HBDCs than the IPDI-based ones in all performed tests. MDI-based material with low 2/1 NCO/OH and short chain length was the best support for cells, comparable with tissue-culture polystyrene (with ALP activity even higher). HBDCs cultured on porous scaffolds from this mcSUU produced a tissue-like structure in culture. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Effect of the heating temperature on the corrosion resistance of alkali-treated titanium.

The paper presents the results of examinations of the corrosion resistance of titanium after its being subjected to the surface modification by the alkali- and heat-treatments. The material examined was commercially pure titanium (grade 2). The samples were soaked in an aqueous 10M NaOH solution at 60 degrees C for 24 h and subsequently heated at 500, 600, or 700 degrees C for 1 h. The chemical composition of the surface layers was determined by X-ray photoelectron spectroscopy and secondary ion mass spectroscopy. The phases present in the layers were identified by XRD. The corrosion resistance was evaluated by electrochemical methods (Stern's method, potentiodynamic method, and impedance spectroscopy) at a temperature of 37 degrees C after short- and long-time exposures. The 13 h exposure was aimed to allow the corrosion potential to stabilize. The aim of the long-term exposures was to examine how the corrosion resistance of the modified samples changes during the exposure. Under the conditions prevailing during the experiments, the highest corrosion resistance was achieved with the samples heated at a temperature of 700 degrees C.

The effect of sodium-ion implantation on the properties of titanium.

This paper deals with the surface modification of titanium by sodium-ion implantation and with the effect of this modification on structure, corrosion resistance, bioactivity and cytocompatibility. The Na ions were implanted with doses of 1 x 10(17) and 4 x 10(17) ions/cm(2) at an energy of 25 keV. The chemical composition of the surface layers formed during the implantation was examined by secondary-ion mass spectrometry (SIMS) and X-ray photoelectron spectroscopy (XPS), and their microstructure--by transmission electron microscopy (TEM). The corrosion resistance was determined by electrochemical methods in a simulated body fluid (SBF) at a temperature of 37 degrees C, after exposure in SBF for various times. The surfaces of the samples were examined by optical microscopy, by scanning electron microscopy (SEM-EDS), and by atomic force microscopy (AFM). Biocompatibility of the modified surface was evaluated in vitro in a culture of the MG-63 cell line and human osteoblast cells. The TEM results indicate that the surface layers formed during the implantation of Na-ions are amorphous. The results of the electrochemical examinations obtained for the Na-implanted titanium samples indicate that the implantation increases corrosion resistance. Sodium-ion implantation improves bioactivity and does not reduce biocompatibility.

Osteoblast response to the elastic strain of metallic support.

It is known that metallic elements of joint endoprostheses undergo elastic strain due to their mechanical function. This is one of the factors which may be responsible for the loosening of endoprostheses. Since mechanisms involved in it remain unclear, it seems valuable to verify if cells responsible for bone regeneration are affected by a strain of the implant. Our experiment examines the influence of elastic strain applied to Ti6Al4V samples on osteoblasts cultured on their surface in vitro. Human bone-derived cells are observed in contact with metallic plates. Titanium alloy was chosen as a support since it is one of the most commonly used materials for stems in joint endoprostheses. Cyclic elastic deformation of 0.1% was applied to the support once daily for 7 days. Two thousand cycles were applied each time. Samples which were not subject to strain served as control. After the observation period XTT assay was performed, alkaline phosphatase activity as well as osteocalcin concentration and nitric oxide secretion were determined and compared with the results obtained in the control group. It was found that the number of viable cells in the mechanically stimulated population was significantly higher than in control, while both alkaline phosphatase activity and osteocalcin concentration were significantly lower in the experimental group. Nitric oxide secretion was found in the culture which was subject to elastic strain, but not in the control. The possible clinical implication is that elastic strain of the metallic endoprostheses may influence osteoblasts which are in contact with the implant in vivo.

Effect of dual ion implantation of calcium and phosphorus on the properties of titanium.

This study is concerned with the effect of dual implantation of calcium and phosphorus upon the structure, corrosion resistance and biocompatibility of titanium. The ions were implanted in sequence, first Ca and then P, both at a dose of 10(17) ions/cm2 at a beam energy of 25 keV. Transmission electron microscopy was used to investigate the microstructure of the implanted layer. The chemical composition of the implanted layer was examined by XPS and SIMS. The corrosion resistance was determined by electrochemical methods in a simulated body fluid (SBF) at a temperature of 37 degrees C. The biocompatibility tests were performed in vitro in a culture of human-derived bone cells (HDBC) in contact with the tested materials. The viability of the cells was determined by an XTT assay and their activity by the measurements of the alkaline phosphatase activity in contact with implanted and non-implanted titanium samples. The in vitro examinations confirmed that, under the conditions prevailing during the experiments, the biocompatibility of Ca + P ion-implanted titanium was satisfactory. TEM results show that the surface layer formed by the Ca + P implantation is amorphous. The corrosion resistance of titanium, examined by the electrochemical methods, appeared to be increased after the Ca + P ion implantation.

Effect of calcium and phosphorus ion implantation on the corrosion resistance and biocompatibility of titanium.

This paper is concerned with the corrosion resistance and biocompatibility of titanium after surface modification by the ion implantation of calcium or phosphorus or calcium + phosphorus. Calcium and phosphorus ions were implanted in a dose of 10(17) ions/cm(2). The ion beam energy was 25 keV. The microstructure of the implanted layers was examined by TEM. The chemical composition of the surface layers was determined by XPS and SIMS. The corrosion resistance was examined by electrochemical methods in a simulated body fluid (SBF) at a temperature of 37 degrees C. The biocompatibility was evaluated in vitro. As shown by TEM results, the surface layers formed during calcium, phosphorus and calcium + phosphorus implantation were amorphous. The results of the electrochemical examinations (Stern's method) indicate that the calcium, phosphorus and calcium + phosphorus implantation into the surface of titanium increases its corrosion resistance in stationary conditions after short- and long-term exposures in SBF. Potentiodynamic tests show that the calcium-implanted samples undergo pitting corrosion during anodic polarisation. The breakdown potentials measured are high (2.5 to 3 V). The good biocompatibility of all the investigated materials was confirmed under the specific conditions of the applied examination, although, in the case of calcium implanted titanium it was not as good as that of non-implanted titanium.

Effect of phosphorus-ion implantation on the corrosion resistance and biocompatibility of titanium.

This work presents data on the structure and corrosion resistance of titanium after phosphorus-ion implantation with a dose of 10(17)P/cm2. The ion energy was 25keV. Transmission electron microscopy was used to investigate the microstructure of the implanted layer. The chemical composition of the surface layer was examined by X-ray photoelectron spectroscopy and secondary ion mass spectrometry. The corrosion resistance was examined by electrochemical methods in a simulated body fluid at a temperature of 37 C. Biocompatibility tests in vitro were performed in a culture of human derived bone cells in direct contact with the materials tested. Both, the viability of the cells determined by an XTT assay and activity of the cells evaluated by alkaline phosphatase activity measurements in contact with implanted and non-implanted titanium samples were detected. The morphology of the cells spread on the surface of the materials examined was also observed. The results confirmed the biocompatibility of both phosphorus-ion-implanted and non-implanted titanium under the conditions of the experiment. As shown by transmission electron microscope results, the surface layer formed during phosphorus-ion implantation was amorphous. The results of electrochemical examinations indicate that phosphorus-ion implantation increases the corrosion resistance after short-term as well as long-term exposures.

Effect of calcium-ion implantation on the corrosion resistance and biocompatibility of titanium.

This work presents data on the structure and corrosion resistance of titanium after calcium-ion implantation with a dose of 10(17) Ca+/cm2. The ion energy was 25 keV. Transmission electron microscopy was used to investigate the microstructure of the implanted layer. The chemical composition of the surface layer was examined by XPS and SIMS. The corrosion resistance was examined by electrochemical methods in a simulated body fluid (SBF) at a temperature of 37 degrees C. Biocompatibility tests in vitro were performed in a culture of human derived bone cells (HDBC) in direct contact with the materials tested. Both, the viability of the cells determined by an XTT assay and activity of the cells evaluated by alkaline phosphatase activity measurements in contact with implanted and non-implanted titanium samples were detected. The morphology of the cells spread on the surface of the materials examined was also observed. The results confirmed the biocompatibility of both calcium-ion-implanted and non-implanted titanium under the conditions of the experiment. As shown by TEM results, the surface layer formed during calcium-ion implantation was amorphous. The results of electrochemical examinations indicate that calcium-ion implantation increases the corrosion resistance, but only under stationary conditions; during anodic polarization the calcium-ion-implanted samples undergo pitting corrosion. The breakdown potential is high (2.7-3 V).

Interaction between tissues and implantable materials.

Interaction between tissues and implantable materials is a factor of critical importance in biocompatibility studies. Bioactivity of implants is expected when the resorption or controlled integration of the implant with surrounding tissues is required. On the contrary, biomaterial inertness is suitable in the case of most load-bearing implants. Both desired and undesired consequences of partial implant biodegradation are discussed on the base of the authors' experimental work done on alumina and carbon-fiber-reinforced carbon composites (CFRC). Two examples of investigations of the interaction between bioceramics (CFRC, alumina, hydroxyapatite, and tricalcium phosphate) and cells in culture are shown as an alternative to the methods based on experimental implantation. The future of research on biomaterial-tissue interaction is discussed with respect to the developments in tissue engineering.

Interaction between carbon composites and bone after intrabone implantation.

Candidate carbon fiber reinforced carbon (CFRC) porous implant materials were evaluated for tissue compatibility in a rat model. Six different CFRCs of constant pore size (about 30 microm) were fabricated that had 9, 12, and 17% porosity with 2-nm3 matrix crystallites and 6, 12, and 20% at 25 nm3. They were implanted as femoral transverse diaphyseal pins that were 1.5 mm in diameter. At 5 and 45 weeks, implant and bone histologic specimens were evaluated histologically and by a scanning electron microscope and an electron microprobe. Also, regional lymph nodes and spleens were examined. By 45 weeks, direct implant-bone contact was observed over most of the interface in most specimens. At the implant surface, there was partial replacement of CFRC with host tissue. However, the microprobe showed that the implant-bone interface was chemically abrupt with no cross-diffusion of ionic species. Besides the surface effects, there was partial filling of the implant pores with tissue, including bone organized de novo deep within. This was observed histologically and confirmed by microprobe. Lymph nodes and spleens were histologically normal, and no carbon particles were found. None of the results were influenced by porosity or matrix crystallite size over the ranges studied. In summary, these porous CFRCs are partially degraded when in contact with bone and appear substantially tissue compatible. They may be useful as scaffolds for regrowth of bone.

Human osteoblast in contact with various biomaterials in vitro.

OBJECTIVES: The aim of the study was to investigate changes in osteoblast number and viability in contact with biomaterials and to compare both tests. METHODS: Human primary culture osteoblasts were seeded on the polished surfaces of hydroxyapatite, alumina, titanium and surgical steel. After 4, 9, 14, 24 and 48h cultures were subjected to XTT viability assay. Subsequently Hoechst staining of nuclei was performed. Number of cells on each sample was counted. RESULTS: There were no differences in cell viability measured by means of XTT assay between osteoblasts cultured on hydroxyapatite and alumina during 48 hours of experiment. However on titanium as well as on surgical steel cell viability was significantly lower than on bioceramics. The lowest viability was noticed on surgical steel. Cell number on titanium was significantly higher than on steel. There were no differences between cell numbers on hydroxyapatite and alumina as well as between investigated bioceramics and metals. Nucleus number and the results of viability assay were compared. There was no correlation found between number and viability of cells. CONCLUSION: the results of a single test may not provide sufficient information on the interaction between cells and implant. Application of a battery of tests is necessary in material biocompatibility investigation in vitro.

Alternative methods for assessing biocompatibility and function of implant materials.

Biocompatibility testing is used to evaluate the host response to implantable materials and to assess their ability to perform in applications in which they are intended to interact with biological systems. In compliance with international and/or national standards, such assessment is based mainly on the results of experimental implantation into animal tissues. However, the development of in vitro experimental techniques creates new opportunities to observe and to understand the interaction of biomaterials with host tissue. The state-of-the-art application of alternative methods in biocompatibility testing is presented in this review article. It is discussed with respect to the Three Rs concept (reduction, refinement, replacement) of Russell & Burch. Perspectives on alternative methods in biocompatibility studies are discussed with regard to the possible role of biomaterials in tissue engineering.

Fixation of carbon fibre-reinforced carbon composite implanted into bone.

The push-out test of three types of biomaterials: carbon fibre-reinforced carbon (CFRC), hydroxyapatite (HA), and surgical steel (SS) implanted into rabbits' femurs was carried out. Hydroxyapatite was used as a positive control (good fixation expected in bone) and surgical steel was a negative one (potentially no fixation in bone). Regeneration of bone in contact with all implants was found three months after implantation. The shear strength between CFRC implants and bone was lower than with the HA implants and higher than the shear strength between the surgical steel and bone. Compressive strength of CFRC implants removed after the observation period was significantly lower than the compressive strength of non-implanted samples. It is concluded that the mechanical bonding between the CFRC implants and host tissues exists 3 months after intrabone implantation and is accompanied by a decrease of the strength of implants.

Bone-implant interface, objectivization of X-ray microanalysis.

Several techniques of the bone-implant interface uncovering in the proper way for X-ray microanalysis, were tested. The obtained results confirmed that the method of sample preparation has significantly influenced the results of investigation. The mechanical polishing, which is routinely used for preparation of surfaces to the X-ray microanalysis, introduces artifacts. Cutting by means of microtome, in the case of brittle implants, and sawing by utilizing diamond saw, in the case of metallic ones, were indicated as the optimal methods. Cleaning of the samples in the ultrasonic cleaner before covering them with conductive layer is required.

[Use of irradiation technique for obtaining and modifying biopolymers. Review]. / Zastosowanie techniki radiacyjnej do otrzymywania i modyfikacji biopolimerów. Praca pogladowa.

A review of papers concerning application of radiation techniques to the biopolymers production is presented. The nature of electron and gamma irradiation influence on polymers is outlined. Advantages of the method from the point of view of biocompatibility and bio-functionality of biopolymers are underlined. Among them the most important are the following: chemical purity of products, high efficiency of the method, expanded influence on polymers' structure, usefulness in the graft copolymerization, ability of avoiding enhanced temperature during polymerisation and sterility of products. Examples of biopolymers obtained or modified by means of irradiation techniques are gathered.

Aluminium release as a new factor in the estimation of alumina bioceramic implants.

Aluminium release from an alumina bioceramic was investigated parallel to standardized biocompatibility testing in an animal experiment. Alumina implants were introduced into both femurs of male rats and into the mandible of male guinea-pigs. After a period of 6 or 8 months animals were sacrificed. The bone surrounding the implant was examined by utilizing standard histological procedure. The material was well tolerated both in the rat and in the guinea-pig. The surface of the removed implants was examined in the SEM. No changes were observed. In both femurs of the 10 experimental and six control rats the aluminium content was determined by utilizing the atomic absorption spectroscopy method (AAS). It was found that the level of aluminium was essentially higher in the bones of the experimental animals. Therefore the safety of alumina introduction into human organisms for a long period becomes open to doubt, in spite of the favourable results of the short-term biocompatibility tests.