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A new type of biomaterial for artificial skin: dehydrothermally cross-linked composites of fibrillar and denatured collagens
Koide, M., K. Osaki, et al. (1993), J Biomed Mater Res 27(1): 79-87.
Abstract: A new type of biomaterial for artificial skin was developed as a form of sponge by combining fibrillar collagen (F-collagen) with gelatin. The sponge was physically and metabolically stabilized by introducing dehydrothermal cross links. To get the final product, various conditions in the preparation of sponges were evaluated by in vitro cellular responses and in vivo tissue reactions. Fibroblasts placed on a sponge of gelatin attached themselves to it, migrated well into the sponge, and remained inside it for at least 7 days. However, sponges of gelatin showed structural instability for hydrolytic degradation by the cells. Most fibroblasts appeared not to penetrate into the interior of a sponge of F-collagen but to remain on its surface when fibroblasts were placed on the sponge, suggesting poor attraction of F-collagen toward cells. Implantation experiments of sponges of F-collagen revealed an intense infiltration of neutrophils into the sponge, indicating F-collagen as an inducer of the inflammatory reaction. These aggravating characters of F-collagen sponges were greatly improved by blending gelatin with F-collagen. The new type of collagen-based biomaterials developed in the present study is expected to become a useful matrix substance for artificial skin.

A new way of incorporating silicon in hydroxyapatite (Si-HA) as thin films
Thian, E. S., J. Huang, et al. (2005), J Mater Sci Mater Med 16(5): 411-5.
Abstract: Bioactive silicon-containing hydroxyapatite (Si-HA) thin films that can be used as coatings for bone tissue replacement have been developed. A magnetron co-sputtering technique was used to deposit Si-HA films up to 700 nm thick on titanium substrates, with a silicon level up to 1.2 wt%. X-ray diffraction demonstrated that annealing transformed the as-deposited Si-HA films which were amorphous, into a crystalline HA structure. A human osteoblast-like (HOB) cell model was used to determine the biocompatibility of these films. HOB cells were seen to attach and grow well on the Si-HA films, and the metabolic activity of HOB cells on these films was observed to increase with culture time. Furthermore, mineralisation of the cell layers was observed after 8 weeks of culture. Based on the present findings, Si-HA of different film compositions demonstrate bioactive properties in-vitro, and indicate the potential as biocoatings for a wide variety of medical implants including load-bearing applications such as the femoral stem of hip replacement implants.

A novel antibacterial titania coating: metal ion toxicity and in vitro surface colonization
Heidenau, F., W. Mittelmeier, et al. (2005), J Mater Sci Mater Med 16(10): 883-8.
Abstract: Postoperative implant-associated infection is still an unresolved and serious complication in modern surgery. Antibacterial and biocompatible surfaces could both reduce infection rates and promote tissue integration. In this respect, a comparative study of the antibacterial as well as the biocompatible potential of different metal ions in vitro is presented. The assays used were growth inhibition tests with different metal salts carried out with tissue cells and bacteria under corresponding culture conditions. Additionally, in vitro tests in direct surface contact with tissue cells and bacteria onto a novel copper containing sol-gel derived titanium dioxide coating (Cu-TiO2) and a fourfold Cu-TiO2 coating were performed. The values were compared to a non-filled titanium dioxide coating and standard Ti6Al4V alloy. SEM-investigations were performed to approve the results of the in vitro tests.Among Ag+, Zn2+, Co2+, Al3+ and Hg2+, the growth inhibition tests revealed an outstanding position of copper ions as antibacterial but nevertheless bio-tolerant additive. These results were affirmed by the cell tests in direct surface contact and SEM-investigations, where best cell growth was found on the Cu-TiO2 coatings. Highest antibacterial properties with a tolerable cytocompatibility could be observed on the fourfold Cu-TiO2 coatings. Consequently, surfaces with custom-tailored antibacterial properties may be established and could be of particular interest in revision and tumor arthroplasty.

A novel application of multi-wavelength TIRF spectroscopy for real time monitoring of antithrombin interactions with immobilized heparin
Klinth, J. E., R. Larsson, et al. (2005), Biosens Bioelectron
Abstract: Real time interactions of antithrombin (AT) with Corlinetrade mark Heparin Surfaces (CHS) with one and two layers of heparin conjugate have been examined using a multi-wavelength TIRF spectroscopy technique with continuous flow. Fluorescently labeled AT, adsorbed from citrated human blood plasma, showed significantly higher signals on CHS compared to the cationic surface used to attach the heparin conjugate. The AT binding to CHS was very stable, also after exposure to soluble heparin at a concentration of 1.5IU/mL. Only a few percent of the bound AT were displaced from the surfaces by AT present in plasma after long-term exposure to plasma. In contrast, larger amounts of the freshly added AT had adsorbed to the surfaces, especially to the surface with two layers of heparin conjugate, indicating the presence of unsaturated AT binding sites. The amount of AT bound to the different surfaces was quantified after elution using an enzyme immunoassay (EIA). Characteristic emission spectra of proteins and fluorophores of labeled proteins, obtained at the surfaces after a long-term exposure to plasma, confirmed their presence at the surfaces. The multi-wavelength TIRF technique proved to be a useful tool when combined with other techniques to study the time course of interactions of fluorescently labeled proteins with biomaterials, even in a complex environment such as plasma.

A novel approach to AFM characterization of adhesive tooth-biomaterial interfaces
Yoshida, Y., B. Van Meerbeek, et al. (1999), J Biomed Mater Res 47(1): 85-90.
Abstract: A novel approach is proposed for studying tooth-biomaterial interactions with high resolution. Thus far, polished interfaces examined by AFM have not disclosed much detail, mainly due to the destruction of soft surface texture and the smearing of polishing debris across the interface that obscures the actual ultra-structure. Therefore the practical utility of diamond-knife microtomy as a sample preparation technique for imaging tooth-biomaterial interfaces by AFM with high resolution was tested in this study and compared to that of ultra-fine mechanical polishing techniques. The AFM images clearly demonstrated the enhanced potential of diamond-knife microtomy for nondestructively producing clean cross-sections through interfaces that allow the interfacial ultra-structure to be imaged by AFM with a resolution equaling that of TEM. This novel approach opens the field to the full range of scanning probe microscopy, including physical and chemical surface characterization of interfaces with a mix of soft and hard substrates.

A novel biomaterial: poly(dimethylsiloxane)-polyamide multiblock copolymer I. Synthesis and evaluation of blood compatibility
Furuzono, T., E. Yashima, et al. (1993), J Biomater Sci Polym Ed 5(1-2): 89-98.
Abstract: Aramid-silicone resins (PASs) consisting of aromatic polyamide (aramid) and poly(dimethyl-siloxane) (PDMS) segments were synthesized by low temperature solution polycondensation. For the evaluation of blood compatibility in vitro, two kinds of experiments were carried out. One was the thromboxane B2(TXB2) release test from platelets attaching to PAS and Biomer. The other was the observation of the platelet adhesion on the surfaces of PAS by scanning electron microscopy (SEM). The results indicated that PAS was bio-inert in vitro. The surface chemical composition of PAS films was investigated by means of electron probe micro analysis (EPMA), X-ray photoelectron spectroscopy (XPS), and dynamic contact angle measurements. The relationship between blood compatibility and surface composition of PAS is discussed.

A novel bioreactor for the dynamic flexural stimulation of tissue engineered heart valve biomaterials
Engelmayr, G. C., Jr., D. K. Hildebrand, et al. (2003), Biomaterials 24(14): 2523-32.
Abstract: Dynamic flexure is a major mode of deformation in the native heart valve cusp, and may effect the mechanical and biological development of tissue engineered heart valves (TEHV). To explore this hypothesis, a novel bioreactor was developed to study the effect of dynamic flexural stimulation on TEHV biomaterials. It was implemented in a study to compare the effect of uni-directional cyclic flexure on the effective stiffness of two candidate TEHV scaffolds: a non-woven mesh of polyglycolic acid (PGA) fibers, and a non-woven mesh of PGA and poly L-lactic acid (PLLA) fibers, both coated with poly 4-hydroxybutyrate (P4HB). The bioreactor has the capacity to dynamically flex 12 rectangular samples (25 x 7.5 x 2mm) under sterile conditions in a cell culture incubator. Sterility was maintained in the bioreactor for at least 5 weeks of incubation. Flexure tests to measure the effective stiffness in the "with-flexure" (WF) and opposing "against-flexure" (AF) directions indicated that dynamically flexed PGA/PLLA/P4HB scaffolds were approximately 72% (3 weeks) and 76% (5 weeks) less stiff than static controls (p<0.01), and that they developed directional anisotropy by 3 weeks of incubation (stiffer AF, p<0.01). In contrast, both dynamically flexed and static PGA/P4HB scaffolds exhibited a trend of decreased stiffness with incubation, with no development of directional anisotropy. Dynamically flexed PGA/P4HB scaffolds were significantly less stiff than static controls at 3 weeks (p<0.05). Scanning electron microscopy revealed signs of heterogeneous P4HB coating and fiber disruption, suggesting possible explanations for the observed mechanical properties. These results indicate that dynamic flexure can produce quantitative and qualitative changes in the mechanical properties of TEHV scaffolds, and suggest that these differences need to be accounted for when comparing the effects of mechanical stimulation on the development of cell-seeded TEHV constructs.

A novel coating biomaterial for intracranial aneurysms: effects and safety in extra- and intracranial carotid artery
Yasuda, H., S. Kuroda, et al. (2005), Neuropathology 25(1): 66-76.
Abstract: Methyl-2-cyanoacrylate, a widely used material for coating cerebral aneurysm, was recently withdrawn. The aim of the present study was to develop an alternative coating material for cerebral aneurysm, which is safe, effective and stable within the brain. In the first experiment, an aneurysm model of the common carotid artery was produced in a rabbit by the local application of elastase. The aneurysm produced was covered by no material (Group A), a cellulose cotton sheet and conventional methyl-2-cyanoacrylate (Group B), a newly produced polyglycolic acid felt and fibrin glue (Group C), or a cellulose cotton sheet and fibrin glue (Group D). Histological examination showed that the materials resulted in the formation of tight connective tissue around the artery, and that the material was completely replaced by the connective tissue after 12 weeks. This change was found exclusively in Group C, but not in Group A or the other materials, although a temporary thickening of the intima was also observed at the site of the elastase application in Group C. In Group D, a long-term, marked thickening of the intima was observed. In the second experiment, using an intracranial internal carotid artery from a beagle, the applied polyglycolic acid felt and fibrin glue to the intracranial artery induced the formation of connective tissue around the artery that was completely absorbed 16 weeks after surgery. There were no signs of intimal thickening or of adverse reactions in nervous tissue. The present results suggest that polyglycolic acid felt and fibrin glue is a possible candidate for a safe, effective biomaterial to wrap or coat cerebral aneurysm.

A novel composite scaffold for cardiac tissue engineering
Park, H., M. Radisic, et al. (2005), In Vitro Cell Dev Biol Anim 41(7): 188-96.
Abstract: One approach to the engineering of functional cardiac tissue for basic studies and potential clinical use involves bioreactor cultivation of dissociated cells on a biomaterial scaffold. Our objective was to develop a scaffold that is (1) highly porous with large interconnected pores (to facilitate mass transport), (2) hydrophilic (to enhance cell attachment), (3) structurally stable (to withstand the shearing forces during bioreactor cultivation), (4) degradable (to provide ultimate biocompatibility of the tissue graft), and (5) elastic (to enable transmission of contractile forces). The scaffold of choice was made as a composite of poly(dl-lactide-co-caprolactone), poly(dl-lactide-co-glycolide) (PLGA), and type I collagen, with open interconnected pores and the average void volume of 80 +/- 5%. Neonatal rat heart cells suspended in Matrigel were seeded into the scaffold at a physiologically high density (1.35 x 10(8) cells/cm(3)) and cultivated for 8 d in cartridges perfused with culture medium or in orbitally mixed dishes (25 rpm); collagen sponge (Ultrafoam) and PLGA sponge served as controls. Construct cellularity, presence of cardiac markers, and contractile properties were markedly improved in composite scaffolds as compared with both controls.

A novel electrochemical synthesis of poly-3-methylthiophene-gamma-cyclodextrin film Application for the analysis of chlorpromazine and some neurotransmitters
Bouchta, D., N. Izaoumen, et al. (2005), Biosens Bioelectron 20(11): 2228-35.
Abstract: Gamma-cyclodextrin is an eight glucose unit conical structure, which possesses a hydrophobic internal cavity exhibiting supramolecular complexing properties and a hydrophilic exterior due to the presence of hydroxyl groups. In this work, we have synthesized for the first time a functionalized stable film of poly-3-methylthiophene combined with gamma-cyclodextrin (P3MT/gamma-CD) in tetrabutylammonium hexafluorophosphate/acetonitrile solution. A potentiostatic mode was employed for the film growth. Cyclic voltammetry and electrochemical impedance spectroscopy were used for the characterization of the new film. The resulting conducting polymer sensor offers interesting analytical performances such as: (a) fast and linear responses towards the neurotransmitters dopamine and l-dopa towards the neuroleptic chlorpromazine with low detection limits of 2 x 10(-7), 10(-6), and 10(-7)M, respectively, and (b) simultaneous detection and well-resolved signals between the compounds of interest and ascorbic acid. To the best of our knowledge, these LOD are among the lowest found in the literature. Optimization of parameters such as interference effect, perm-selectivity, and mechanical stability of the sensor are discussed.

A novel ELISA using PVDF microplates
Halim, N. D., A. W. Joseph, et al. (2005), J Neurosci Methods 143(2): 163-8.
Abstract: Here we describe the development of a novel specific, rapid ELISA system, which is performed on modified microplates where polyvinylidine fluoride (PVDF) forms the base of each well. The use of microplates with PVDF membranes as the solid phase allows for a greater binding capacity of protein in comparison to the solid phases of traditional ELISAs. The increased binding capacity of the solid phase provides for the direct binding of antigens, which can subsequently be assayed using a single, specific and well-characterized antibody. This direct assay system eliminates the need for two distinct antibodies that are often necessary in conventional two site ELISA systems. The system is able to specifically detect purified proteins as well as antigens in crude preparations of tissue homogenates. The PVDF-based ELISA performs with similar sensitivity and reproducibility as conventional two site ELISAs in tissue homogenates. The intra- and inter-assay coefficients of variation for the measurement of actin in crude rat brain homogenate were 2.36 and 5.15%, respectively.

A novel filler free silicone rubber biomaterial. II. Radiation chemical and physical evaluation
Chawla, A. S. (1978), J Bioeng 2(3-4): 231-40.
Abstract: Hexamethylcyclotrisiloxane was polymerized at 60 degrees C by gamma radiation to yield filler free silicone rubber (FFSR). The G (crosslinking) value of 23.5 was found for the FFSR polymerized for 4 hours, compared to about 2 for the pure polydimethylsiloxane (PDMS). The sol fractions for both FFSR and cross-linked PDMS varied between 5-7%. The FFSR and the cross-linked PDMS both had tensile strength of about 1 kg per cm2 but FFSR was not brittle and could be extended as much as 500% before breaking. It is believed that the marked improvement in physical properties of FFSR is due to the formation of domains of highly cross-linked material which act as "internal filler".

A novel high-resolution magnetic resonance imaging microscopy coil as an alternative to the multislice computed tomography in postoperative imaging of orbital fractures and computer-based volume measurement
Kolk, A., C. Pautke, et al. (2005), J Oral Maxillofac Surg 63(4): 492-8.
Abstract: BACKGROUND: Multislice computed tomography (MSCT) has been the modality of choice for postoperative detailed imaging of orbital trauma. Unfortunately, it involves extensive exposition of the lens to radiation, especially when taking multiple readings. Also, it holds beam hardening effects and limited imaging (delineation) of the reconstruction material. Alternative conventional magnetic resonance imaging (MRI) head coils (MRIhc) present reduced differentiation of anatomic structures caused by low signal and artifact appearance. A substantially improved depiction is made possible by a newly introduced MRI microscopy coil (MRImc), used for the first time in this field. PATIENTS AND METHODS: In this prospective study, 32 patients with extended orbital wall fractures (n = 36) were treated surgically using a polydioxanonsulfate (PDS) foil after reconstruction. Postoperatively, imaging was performed using MRImc, conventional MRI, and MSCT to evaluate the different imaging techniques. RESULTS: The position of the PDS foil could precisely be depicted in 29 of 36 fractures by MRImc, whereas by conventional MRI and MSCT the reconstruction material could only be detected in 25 and 24 of 36 fractures, respectively. In contrast to MRIhc, the new microscopy coil allows fast and high resolution imaging and therefore a clear differentiation of eventual postoperative complications (eg, dislocation of the PDS foil with secondary soft tissue entrapment resulting in limitation of ocular movements or enophthalmos). In 13 long lasting symptomatic cases, revision surgery could be avoided because of regular MRImc findings, apart from muscle swelling and hematoma. In 11 cases of inadequate PDS foil position resulting in revision, the mean volume of displaced tissue (VDT) of 0.62 cm3 correlated significantly to an enophthalmos of more than 2 mm. CONCLUSION: In this pilot study, MRImc proved to be highly superior to MRIhc and MSCT in postsurgical orbital imaging, especially for decision making regarding revision surgery.

A novel hyaluronan-based biomaterial (Hyaff-11) as a scaffold for endothelial cells in tissue engineered vascular grafts
Turner, N. J., C. M. Kielty, et al. (2004), Biomaterials 25(28): 5955-64.
Abstract: Current prosthetic small diameter vascular grafts show poor long-term patency rates, leading to the pursuit of a biological alternative. Hyaff-11 is a hyaluronan-based biodegradable polymer developed for tissue-engineering applications. This study aimed to determine whether human vascular endothelial cells attach to Hyaff-11 scaffolds and produce a subendothelial matrix. Two forms of fibrous, non-woven Hyaff-11 scaffolds: unpressed and pressed felts, were analysed. Attachment of human venous endothelial cells was investigated after 1, 5, 10 and 20 days in culture using SEM and confocal microscopy. The deposition of subendothelial matrix components was investigated by immunofluorescent staining. We demonstrate that endothelial cells adhere to the individual fibres of both unpressed and pressed scaffolds: with a seeding density of 1 x 10(6) cells/cm(2), 94% of the cells attached to Hyaff-11 fibres after 24 h. The pressed material provided the best environment for cell growth, allowing the formation of a complete endothelial monolayer after 20 days. Furthermore, endothelial cells on Hyaff-11 pressed felts deposited an organised subendothelial matrix containing laminin, fibronectin, type IV and type VIII collagen. This work indicates Hyaff-11 based biopolymers as suitable scaffolds to promote endothelialisation within the next generation of vascular grafts.

A novel immobilization method for single protein spFRET studies
Pal, P., J. F. Lesoine, et al. (2005), Biophys J 89(2): L11-3.
Abstract: We have developed a new method for immobilization of single proteins by utilizing streptavidin-biotin and protein L-antibody interactions on glass coverslips coated with polyethylene glycol. The method is particularly well suited for single-molecule fluorescence studies. A monomeric, detergent-solubilized bacterial transport protein, GlpT, and the dimeric cytoplasmic region of a mammalian transporter, cdAE1, were immobilized by our method with a high degree of specificity. The fluorescence from single molecules attached to the immobilized proteins was detected with a high signal/noise ratio. Single-pair fluorescence resonance energy transfer (spFRET) measurements on cdAE1 dimers indicate that the structure of the protein is not compromised and provide evidence that the cdAE1 protein can exist in at least two conformations under physiological conditions.

A novel method for biomaterial scaffold internal architecture design to match bone elastic properties with desired porosity
Lin, C. Y., N. Kikuchi, et al. (2004), J Biomech 37(5): 623-36.
Abstract: An often-proposed tissue engineering design hypothesis is that the scaffold should provide a biomimetic mechanical environment for initial function and appropriate remodeling of regenerating tissue while concurrently providing sufficient porosity for cell migration and cell/gene delivery. To provide a systematic study of this hypothesis, the ability to precisely design and manufacture biomaterial scaffolds is needed. Traditional methods for scaffold design and fabrication cannot provide the control over scaffold architecture design to achieve specified properties within fixed limits on porosity. The purpose of this paper was to develop a general design optimization scheme for 3D internal scaffold architecture to match desired elastic properties and porosity simultaneously, by introducing the homogenization-based topology optimization algorithm (also known as general layout optimization). With an initial target for bone tissue engineering, we demonstrate that the method can produce highly porous structures that match human trabecular bone anisotropic stiffness using accepted biomaterials. In addition, we show that anisotropic bone stiffness may be matched with scaffolds of widely different porosity. Finally, we also demonstrate that prototypes of the designed structures can be fabricated using solid free-form fabrication (SFF) techniques.

A novel method for the simultaneous, titrant-free control of pH and calcium phosphate mass yield
Lynn, A. K. and W. Bonfield (2005), Acc Chem Res 38(3): 202-7.
Abstract: Calcium phosphate (CaP) bioceramics have long been of interest for the unique properties that they exhibit as bone substitute materials. By harnessing the unique bone-bonding capacity of CaP's, biomaterials scientists have made great strides over the past 2 decades to produce novel materials to assist in the treatments of defects caused by trauma, disease, or both. In recent years, however, it has become apparent that the traditional set of techniques used to produce calcium phosphates does not satisfy all of the requirements necessary to meet the challenges of emerging applications. In particular, recent interest in (i) the synthesis of coprecipitated CaP/bioorganic composites and (ii) the investigation of the mechanisms of biomineralization has highlighted the need for new methods to control pH and CaP mass yield.

A novel method for the synthesis of the PEG-crosslinked chitosan with a pH-independent swelling behavior
Kulkarni, A. R., V. I. Hukkeri, et al. (2005), Macromol Biosci 5(10): 925-8.
Abstract: In this study, a simple method was developed to crosslink chitosan using poly(ethylene glycol) (PEG) with different molecular weights. Crosslinking of chitosan was confirmed by various spectral analyses. The differential scanning calorimetric (DSC) study indicated that the rigid crystalline structure of chitosan was decreased after crosslinking with PEG. The PEG-crosslinked chitosan (PEG-Ch) showed a pH-independent swelling behavior: swelled in both the simulated stomach (pH 1.1) and intestinal (pH 7.4) solutions. The swelling ratio of PEG-Ch increased significantly with a higher molecular weight of PEG used. In contrast, chitosan dissolved completely in a simulated stomach solution and showed a comparatively less swelling in a simulated intestinal solution. Thus, the prepared PEG-Ch could be a better biomaterial than chitosan in the development of orally sustained drug-delivery devices.

A novel method to enhance the stability of alginate-poly-L-lysine-alginate microcapsules
Wang, M. S., R. F. Childs, et al. (2005), J Biomater Sci Polym Ed 16(1): 91-113.
Abstract: Implantation of microencapsulated recombinant cells is an alternative approach to gene therapy. These genetically-engineered cells enclosed in microcapsules to deliver therapeutic recombinant products have been effective in treating several murine models of human diseases. However, the most commonly used microcapsules fabricated from alginate ionically cross-linked with calcium suffer from loss of long-term mechanical stability. We now report on a method to improve their stability by introducing additional polymers to provide covalent linkages via photopolymerization. Vinyl monomers and a photoinitiator were allowed to diffuse into the initially formed calcium-alginate microcapsules. In situ photopolymerization in the presence of sodium acrylate and N-vinylpyrrolidone substantially enhanced their mechanical strength. After four months of storage in saline, > 70% of these capsules remained intact in the osmotic pressure test, while the un-modified alginate microcapsules totally disintegrated. Tests of their permeability to polyethylene glycol of different molecular weight and their ability to support cell survival showed that these properties remained unaffected by the photopolymerization. Hence, these microcapsules modified by adding a network of vinyl polymers are promising candidates to use for long-term delivery of recombinant gene products in this cell-based method of gene therapy.

A novel non line-of-sight method for coating hydroxyapatite onto the surfaces of support materials by biomineralization
Macaskie, L. E., P. Yong, et al. (2005), J Biotechnol 118(2): 187-200.
Abstract: A novel method is described for the non line-of-sight coating of hydroxyapatite onto polyurethane reticulated foam and titanium discs. This utilises a biofilm of Serratia sp. NCIMB 40259 which, when challenged with a solution containing calcium chloride and phosphatase substrate, manufactures biofilm-bound material identified as hydroxyapatite by X-ray powder diffraction analysis. Non-invasive magnetic resonance imaging was used to visualize the biofilm coating throughout the foam labyrinth and to measure the thickness of the film within reticulated foam cubes in situ. The film developed within the cube matrices was similar to that measured on the surface of a glass slide. Using LaPO(4) deposition as a model system the metallised biofilm was visualised in two-dimensional slices throughout three-dimensional images acquired by magnetic resonance imaging. A similar encrustation of hydroxyapatite on the surface of biofilm grown on titanium discs was confirmed by scanning electron microscopy. Potential applications for bio-hydroxyapatite as possible bone implant precursors are discussed.

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