Early adhesion of human mesenchymal stem cells on TiO(2) surfaces studied by single-cell force spectroscopy measurements.

Understanding the interactions involved in the adhesion of living cells on surfaces is essential in the field of tissue engineering and biomaterials. In this study, we investigate the early adhesion of living human mesenchymal stem cells (hMSCs) on flat titanium dioxide (TiO(2) ) and on nanoporous crystallized TiO(2) surfaces with the use of atomic force microscopy-based single-cell force spectroscopy measurements. The choice of the substrate surfaces was motivated by the fact that implants widely used in orthopaedic and dental surgery are made in Ti and its alloys. Nanoporous TiO(2) surfaces were produced by anodization of Ti surfaces. In a typical force spectroscopy experiment, one living hMSC, immobilized onto a fibronectine-functionalized tipless lever is brought in contact with the surface of interest for 30 s before being detached while recording force-distance curves. Adhesion of hMSCs on nanoporous TiO(2) substrates having inner pore diameter of 45 nm was lower by approximately 25% than on TiO(2) flat surfaces. Force-distance curves exhibited also force steps that can be related to the pulling of membrane tethers from the cell membrane. The mean force step was equal to 35 pN for a given speed independently of the substrate surface probed. The number of tethers observed was substrate dependent. Our results suggest that the strength of the initial adhesion between hMSCs and flat or nanoporous TiO(2) surfaces is driven by the adsorption of proteins deposited from serum in the culture media.

A Comparative In Vitro and In Vivo Study of Osteogenicity by Using Two Biomaterials and Two Human Mesenchymal Stem Cell Subtypes.

Background: Bone repair induced by stem cells and biomaterials may represent an alternative to autologous bone grafting. Mesenchymal stromal/stem cells (MSCs), easily accessible in every human, are prototypical cells that can be tested, alone or with a biomaterial, for creating new osteoblasts. The aim of this study was to compare the efficiency of two biomaterials-biphasic calcium phosphate (BCP) and bioactive glass (BG)-when loaded with either adult bone marrow mesenchymal stem cells (BMMSCs) or newborn nasal ecto-mesenchymal stem cells (NE-MSCs), the latter being collected for further repair of lip cleft-associated bone loss. Materials and Methods: BMMSCs were collected from two adults and NE-MSCs from two newborn infants. An in vitro study was performed in order to determine the best experimental conditions for adhesion, viability, proliferation and osteoblastic differentiation on BCP or BG granules. Bone-associated morphological changes and gene expression modifications were quantified using histological and molecular techniques. The in vivo study was based on the subcutaneous implantation in nude mice of the biomaterials, loaded or not with one of the two cell types. Eight weeks after, bone formation was assessed using histological and electron microscopy techniques. Results: Both cell types-BMMSC and NE-MSC-display the typical stem cell surface markers-CD73+, CD90+, CD105+, nestin - and exhibit the MSC-associated osteogenic, chondrogenic and adipogenic multipotency. NE-MSCs produce less collagen and alkaline phosphatase than BMMSCs. At the transcript level, NE-MSCs express more abundantly three genes coding for bone sialoprotein, osteocalcin and osteopontin while BMMSCs produce extra copies of RunX2. BMMSCs and NE-MSCs adhere and survive on BCP and BG. In vivo experiments reveal that bone formation is only observed with BMMSCs transplanted on BCP biomaterial. Conclusion: Although belonging to the same superfamily of mesenchymal stem cells, BMMSCs and NE-MSCs exhibit striking differences, in vitro and in vivo. For future clinical applications, the association of BMMSCs with BCP biomaterial seems to be the most promising.

3D environment on human mesenchymal stem cells differentiation for bone tissue engineering.

In this work a novel method was developed to create a three dimensional environment at a cellular level for bone tissue engineering. Biphasic calcium phosphate (BCP) particles of 140-200 microm were used in association with human mesenchymal stem cells (hMSCs). The cells seeded on these particles adhered and proliferated more rapidly in the first day of culture compared to culture on plastic. Analyses of hMSCs cultured without osteogenic factors on BCP particles revealed an abundant extracellular matrix production forming 3-dimensional (3D) hMSCs/BCP particles constructs after few days. Bone morphogenetic 2 (BMP-2), bone sialoprotein (BSP) and ALP gene expression using real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed that expression profiles were modified by the culture substrate while the addition of osteogenic medium enhanced bone markers expression. These results indicate that BCP particles alone are able to induce an osteoblastic differentiation of hMSCs that might be of interest for bone tissue engineering.

Surface treatments of titanium dental implants for rapid osseointegration.

The osseointegration rate of titanium dental implants is related to their composition and surface roughness. Rough-surfaced implants favor both bone anchoring and biomechanical stability. Osteoconductive calcium phosphate coatings promote bone healing and apposition, leading to the rapid biological fixation of implants. The different methods used for increasing surface roughness or applying osteoconductive coatings to titanium dental implants are reviewed. Surface treatments, such as titanium plasma-spraying, grit-blasting, acid-etching, anodization or calcium phosphate coatings, and their corresponding surface morphologies and properties are described. Most of these surfaces are commercially available and have proven clinical efficacy (>95% over 5 years). The precise role of surface chemistry and topography on the early events in dental implant osseointegration remain poorly understood. In addition, comparative clinical studies with different implant surfaces are rarely performed. The future of dental implantology should aim to develop surfaces with controlled and standardized topography or chemistry. This approach will be the only way to understand the interactions between proteins, cells and tissues, and implant surfaces. The local release of bone stimulating or resorptive drugs in the peri-implant region may also respond to difficult clinical situations with poor bone quality and quantity. These therapeutic strategies should ultimately enhance the osseointegration process of dental implants for their immediate loading and long-term success.

Study of osteoblastic cells in a microfluidic environment.

Bone tissue engineering consists of culturing osteoblastic cells onto synthetic three-dimensional (3D) porous scaffolds. The organization of bone cells into 3D scaffolds is crucial for ex vivo tissue formation. Diffusional rates of nutrients could be greatly improved by perfusing media through the 3D microporous scaffolds. However, bone cells cultured in vitro are responsive to a variety of different mechanical signals including fluid flow and shear stresses. In this work, we attempt to study osteoblastic cells behaviour cultured within microdevices allowing continuous and homogenous feeding of cells. We have fabricated polydimethylsiloxane PDMS microdevices with a 3D microstructured channel network. Mouse calvarial osteoblastic cells MC3T3-E1 were seeded at 2x10(6)cells/ml and cultured into the microdevices under flow rates of 0, 5, 35 microl/min. Cells attached and proliferated well in the designed microdevices. Cell viability was found around 85% up to 1 to 2 weeks for shear stress value under 5 mPa. The alkaline phosphatase (ALP) activity was enhanced 3- and 7.5-fold inside the microdevices under static and dynamic flow of 5 microl/min as compared to flat static cultures in PDMS coated Petri dishes. Therefore, osteoblastic cells could be successfully cultured inside the microdevices under dynamic conditions and their ALP activity was enhanced. These results are promising for bone cell growth and differentiation as well as future tissue regeneration using larger 3D microfluidic microdevices.

Cartilage and bone tissue engineering using hydrogels.

Tissue engineering is an emerging field of regenerative medicine which holds promise for the restoration of tissues and organs affected by chronic diseases, age-linked degeneration, congenital deformity and trauma. During the past decade, tissue engineering has evolved from the use of naked biomaterials, which may just replace small area of damaged tissue, to the use of controlled three-dimensional scaffolds in which cells can be seeded before implantation. These cellularized constructs aims at being functionally equal to the unaffected tissue and could make possible the regeneration of large tissue defects. Among the recently developed scaffolds for tissue engineering, polymeric hydrogels have proven satisfactory in cartilage and bone repair. Major technological progress and advances in basic knowledge (physiology and developmental biology) are today necessary to bring this proof of concept to clinical reality. The present review focuses on the recent advances in hydrogel-based tissue engineered constructs potentially utilizable in bone and cartilage regenerative medicine.

Bone regeneration strategies with bone marrow stromal cells in orthopaedic surgery.

Bone is the most transplanted tissue human with 1 million procedures every year in Europe. Surgical interventions for bone repair are required for varied reasons such as trauma resulting non-union fractures, or diseases including osteoporosis or osteonecrosis. Autologous bone grafting is the gold standard in bone regeneration but it requires a second surgery with associated pain and complications, and is also limited by harvested bone quantity. Synthetic bone substitutes lack the osteoinductive properties to heal large bone defects. Cell therapies based on bone marrow or ex vivo expanded mesenchymal stromal stem cells (MSCs) in association with synthetic calcium phosphate (CaP) bone substitutes may be alternatives to autologous bone grafting. This manuscript reviews the different conventional biological and synthetic bone grafting procedures as well as the more recently introduced cell therapy approaches used in orthopaedic surgery for bone regeneration. Some clinical studies have demonstrated safety and efficacy of these approaches but regeneration of large bone defects remain challenging due to the absence of rapid and adequate vascularisation. Future directions in the field of bone regeneration are presented, such as testing alternative cell sources or in situ fabrication of vascularized bone grafts in patients.

Small-animal models for testing macroporous ceramic bone substitutes.

The aim of this study was to compare the bone colonization of a macroporous biphasic calcium phosphate (MBCP) ceramic in different sites (femur, tibia, and calvaria) in two animal species (rats and rabbits). A critical size defect model was used in all cases with implantation for 21 days. Bone colonization in the empty and MBCP-filled defects was measured with the use of backscattered electron microscopy (BSEM). In the empty cavities, bone healing remained on the edges, and did not bridge the critical size defects. Bone growth was observed in all the implantation sites in rats (approximately 13.6-36.6% of the total defect area, with ceramic ranging from 46.1 to 51.9%). The bone colonization appeared statistically higher in the femur of rabbits (48.5%) than in the tibia (12.6%) and calvaria (22.9%) sites. This slightly higher degree of bone healing was related to differences in the bone architecture of the implantation sites. Concerning the comparison between animal species, bone colonization appeared greater in rabbits than in rats for the femoral site (48.5% vs. 29.6%). For the other two sites (the tibia and calvaria), there was no statistically significant difference. The increased bone ingrowth observed in rabbit femurs might be due to the large bone surface area in contact with the MBCP ceramics. The femoral epiphysis of rabbits is therefore a favorable model for testing the bone-bonding capacity of materials, but a comparison with other implantation sites is subject to bias. This study shows that well-conducted and fully validated models with the use of small animals are essential in the development of new bone substitutes.

Inhibition of osteolysis and increase of bone formation after local administration of siRNA-targeting RANK in a polyethylene particle-induced osteolysis model.

Receptor activator of nuclear factor kappa-B (RANK) and RANK-ligand are relevant targets for the treatment of polyethylene particle-induced osteolysis. This study assessed the local administration of siRNA, targeting both human RANK and mouse Rank transcripts in a mouse model. Four groups of mice were implanted with polyethylene (PE) particles in the calvaria and treated locally with 2.5, 5 and 10 µg of RANK siRNA or a control siRNA delivered by the cationic liposome DMAPAP/DOPE. The tissues were harvested at day 9 after surgery and evaluated by micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) immunohistochemistry for macrophages and osteoblasts, and gene relative expression of inflammatory and osteolytic markers. 10 µg of RANK siRNA exerted a protective effect against PE particle-induced osteolysis, decreasing the bone loss and the osteoclastogenesis, demonstrated by the significant increase in the bone volume (P<0.001) and by the reduction in both the number of TRAP(+) cells and osteoclast activity (P<0.01). A bone anabolic effect demonstrated by the formation of new trabecular bone was confirmed by the increased immunopositive staining for osteoblast-specific proteins. In addition, 5 and 10 µg of RANK siRNA downregulated the expression of pro-inflammatory cytokines (P<0.01) without depletion of macrophages. Our findings show that RANK siRNA delivered locally by a synthetic vector may be an effective approach for reducing osteolysis and may even stimulate bone formation in aseptic loosening of prosthetic implants.

Biomimetic calcium phosphate coatings on Ti6AI4V: a crystal growth study of octacalcium phosphate and inhibition by Mg2+ and HCO3-.

The biomimetic approach for coating metal implants allows the deposition of new calcium phosphate (Ca-P) phases. Films elaborated at physiological conditions might have structures closer to bone mineral than hydroxylapatite (HA) plasma-sprayed coatings. In this study, different Ca-P coatings have been deposited through a two-step procedure. After cleaning and etching, Ti6Al4V plates were pretreated by soaking in a simulated body fluid (SBF), i.e., a solution containing inorganic components in concentration more or less similar to body fluids: a thin amorphous carbonated Ca-P layer precipitated on the metal substrate. Second, by soaking these thinly coated metal substrates in another SBF, with different concentrations, the thin amorphous carbonated Ca-P layer led to the fast precipitation of a second and thick Ca-P layer. Different SBF solutions were used in order to investigate the influence of magnesium and carbonate ions. From SBF containing only Ca2+ and HPO4(2-) ions, an octacalcium phosphate layer grew epitaxially on the substrate. When Mg2+ was added into this SBF, the coating was composed of Ca-deficient apatite crystals, while the addition of HCO3- in SBF led to the formation of a B-carbonated apatite layer. Magnesium and carbonate acted as inhibitors of crystal growth. The three phases obtained by our biomimetic process are closer to bone mineral structure than plasma-sprayed HA. Therefore, the obtained results may be particularly relevant for the development of biomimetic Ca-P coatings with optimal bioactivity.

Incorporation of tobramycin into biomimetic hydroxyapatite coating on titanium.

Calcium phosphate coatings containing an antibiotic were produced on titanium alloy (Ti6Al4V) implants using a biomimetic approach. Thin, amorphous calcium phosphate (ACP) coatings were first deposited onto Ti6Al4V plates by immersion in 5 times concentrated simulated body fluid (SBF), for 24h at 37 degrees C. The ACP-coated implants were then immersed in a supersaturated calcium phosphate (SCP) solution containing 0, 100, 200, 400, 600 or 800 mg/l of tobramycin for 48 h at 37 degrees C. A carbonated hydroxyapatite (CHA) layer, approximately 40 microm thick, was formed. Approximately 3 microg/mg of tobramycin was co-precipitated with the CHA crystals onto titanium alloy plates, using 800mg/l tobramycin in the coating solution. For comparison, plasma-sprayed calcium phosphate coatings were also immersed in solutions containing 100, 200, 400 or 1,000 mg/l of tobramycin for 10, 40 min, or 48 h. A maximum of about 0.3 microg/mg could be adsorbed onto the plasma-sprayed calcium phosphate coating with the comparable concentration of 800 mg/l in solution. The dissolution of coating and release of tobramycin were also measured in vitro using saline solution buffered at pH 5.0 or 7.3 at 37 degrees C. The release rate of tobramycin was faster at pH 7.3 than at pH 5, with 50 and 4 microg/ml/min, respectively. Tobramycin released from the biomimetic-coated plates could inhibit growth of Staphylococcus aureus bacteria. The result of this study, therefore, indicates that the biomimetic CHA coatings containing antibiotics could be used to prevent post-surgical infections in orthopaedic or trauma.

Nucleation of biomimetic Ca-P coatings on ti6A14V from a SBF x 5 solution: influence of magnesium.

Biomimetic Calcium-Phosphate (Ca-P) coatings were applied by using 5 times concentrated Simulated Body Fluid (SBF x 5) using Carbon Dioxide gas. This process allows the deposition of a uniform Ca-P coating within 24 h. A previous study of our process emphasized the importance of hydrogenocarbonate ions (HCO-3), a crystal growth inhibitor. The aim of the present study was to investigate the role of the other crystal growth inhibitor present in SBF x 5, Magnesium (Mg2+), on the Ca-P coating formation. Several SBF x 5 solutions were prepared with various Mg2+ and HCO3 contents. No Ca-P deposits were detected on Ti6A14V substrate soaked for 24h in a Mg-free SBF x 5 solution, whereas by increasing HCO-3 content in a Mg-free SBF x 5 solution, a Ca-P coating developed on Ti6A14V substrate. Therefore, it appeared that Mg2+ has a stronger inhibitory effect on apatite crystal growth than HCO-3. Nevertheless, Mg2+ plays also another important role as suggested by depth profile X-ray Photoelectron Spectroscopy (XPS) of the Ca-P coating obtained from SBF x 5 solution. Ca2+ and Mg2+ contents increased significantly at the titanium/coating interface. Therefore, Ca2+ and Mg2+ initiated Ca-P coating from SBF x 5 solution. The relatively high interfacial concentration in Mg2+ favors heterogeneous nucleation of tiny Ca-P globules onto the substrate. So physical adhesion is enhanced at the early stage of the coating formation.

Influence of ionic strength and carbonate on the Ca-P coating formation from SBFx5 solution.

Biomimetic calcium-phosphate (Ca-P) coatings were applied on Ti6Al4V by using simulated body fluids concentrated by a factor 5 (SBFx5). The production of SBFx5 solution was possible by decreasing the pH of the solution to approximately 6 using CO2 gas. The subsequent release of this mildly acidic gas led to a pH rise and thus, increasing supersaturation. After immersion for 5(1/2) h a Ca-P coating on Ti6Al4V plates and a precipitate simultaneously formed at pH = 6.8. Sodium chloride (NaCl) and hydrogencarbonate (HCO3) contents were studied in relation to CO2 release and coating formation by changing their individual concentration in SBFx5 solution. On one hand, NaCl-free or low NaCl-content SBFx5 solution led to the earlier aspecific precipitation in the solution than for SBFx5 solution. In contrast, Ca-P coating was formed later and was thinner than the coating obtained in regular SBFx5 solution. High ionic strength delayed precipitation and favored Ca-P heterogeneous nucleation on Ti6Al4V. On the other hand, HCO3- content increased the pH of the solution due to its buffering capacity and influenced the release rate of dissolved CO2. Thus, HCO3- content strongly affected the supersaturation and Ca-P structure. Furthermore, HCO3- favored the attachment of Ca-P mineral on Ti6Al4V by decreasing Ca-P crystal size resulting in a better physical attachment of Ca-P coating on Ti6Al4V substrate.

Biomimetic and electrolytic calcium phosphate coatings on titanium alloy: physicochemical characteristics and cell attachment.

Biomimetically deposited octacalcium phosphate (OCP) and carbonate apatite (BCA) as well as electrolytically deposited carbonate apatite (ECA) were considered as promising alternatives to conventional plasma spraying hydroxyapatite. This study compared their physicochemical characteristics and cell attachment behavior. The physicochemical characteristics included scanning electron microscopy observation, X-ray diffraction analysis, Fourier transform infrared spectroscopy analysis, surface roughness, coating thickness, dissolution test and scratch test. Cell attachment tests included morphology observation with stereomicroscopy and scanning electron microscopy as well as cell number count with DNA content assay. The OCP coating had 100% crystallinity and was about 40 microm thick, composed of large plate-like crystals of 30 microm, with the lowest surface roughness (R(a)=2.33 microm). The BCA coating had 60% crystallinity and was approximately 30 microm in thickness, composed of small crystals of 1-2 microm in size, with the highest surface roughness (R(a)=4.83 microm). The ECA coating had intermediate characteristics, with 78% crystallinity, 45 microm thickness, crystals of 5-6 microm and an average roughness of 3.87 microm. All coatings could be seen by eyes dissolving quickly and completely into acidic simulated body fluid (simulated physiological solutions-SPS, pH 3.0) but slowly and incompletely into neutral SPS (pH 7.3). It was suggested that the main factor determining coating dissolution in acidic SPS was the solubility isotherm, while some other factors including crystallinity and crystal size joined to determine coating dissolution in neutral SPS. In regard to adhesive strength, results of scratch test showed the critical load at the first crack of coating (L(c1)) was tightly related to crystal size as well as their arrangement, while the critical load at the total delamination of coating (L(c2)) was also related to the coating thickness. The ECA coating had the highest values. Owing to higher dissolution rate and globular appearance, BCA coating demonstrated the best goat bone marrow stromal cells attachment at 1 day or 3 days, followed by OCP and ECA coating.

Proteins incorporated into biomimetically prepared calcium phosphate coatings modulate their mechanical strength and dissolution rate.

In a previous investigation, we demonstrated that when bovine serum albumin (BSA) is biomimetically co-precipitated with Ca(2+) and PO(4)(3-) ions upon titanium-alloy implants, it becomes incorporated into the crystal lattice and is not merely deposited on its surface. Moreover, the protein elicited a change in crystal structure from an octacalcium phosphate type to a carbonated apatite one, which bears a closer resemblance to natural bone mineral. In the present study, we investigated the dissolution rate and mechanical strength of such BSA-containing coatings as a function of protein concentration within the bathing medium (10 ng/ml to 1.0 mg/ml). BSA-containing coatings released Ca(2+) ions more slowly (5 ppm/min) than did non-BSA-containing ones (10 ppm/min), but this rate did not change as a function of protein concentration within the bathing medium. In contrast, the strength of coatings increased almost linearly as a function of protein concentration within the bathing medium, indicating that BSA incorporated into the crystal lattice enhances its mechanical strength in a concentration-dependent manner.

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

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

Bone formation by mesenchymal progenitor cells cultured on dense and microporous hydroxyapatite particles.

Hydroxyapatite (HA) microparticles, varying in size and microporosity, were evaluated in vitro and in vivo on their suitability to be used as a carrier in an injectable tissue engineered bone filler. Depending on their manufacturing method, either dense (HA-s) or microporous (HA-r) particles were produced in diameter ranges of 212-300 microm (HA-s and HA-r) and 500-706 microm (HA-s). After seeding and culturing goat mesenchymal progenitor cells on the various particles for 1 week, sheets were produced in which multilayers of cells and extracellular matrix held the particles together. Subcutaneous implantation of the constructs in nude mice for 4 weeks revealed abundant bone formation with the 212 to 300-microm diameter particle range. Up to 30% bone was formed in the available areas between the individual microparticles, while bone marrow was present in the samples containing microporous particles. Surprisingly, no bone or bone marrow formation was apparent with the 500 to 706-microm diameter range particles. These results show that size and microporosity of HA microparticles affect the osteogenic potential of cultured cells and indicate that particles in a diameter range of 212-300 microm may be used toward the development of injectable formulations of tissue-engineered bone.

A review of bioceramics and fibrin sealant.

This review focuses on bone substitute composites made by mixing ceramic biomaterials with fibrin sealants. Different biomaterials such as coral, bone-derived materials, bioactive glass ceramics, and synthetic calcium phosphate have been mixed with fibrin sealant, resulting in a combination of the biological properties of the two components. This type of association has not produced identical results in all studies. In the past for some, the addition of fibrin sealant to the biomaterial failed to produce any significant, positive effect on osteointegration, whereas others found a positive impact on bone colonization. Despite the negative biological effects reported previously, bioceramic-fibrin composites have been widely used in various types of bone surgery because they are easy to manipulate. In particular, the intra-operative preparation of these composites makes it possible to add bone growth factors or autologous osteoprogenitor cells prior to bone reconstruction. The bone growth factors and autologous osteoprogenitor cells associated with the bioceramic-fibrin composites should provide surgeons with tissue engineered grafts with enhanced osteointegrative properties. This review discusses both the advantages and disadvantages, as well as the future perspectives, of using bioceramic-fibrin composites in various clinical indications.

Incorporation of different antibiotics into carbonated hydroxyapatite coatings on titanium implants, release and antibiotic efficacy.

Carbonated hydroxyapatite (CHA) coatings were applied onto titanium implants by using a biomimetic precipitation method. Different antibiotics were incorporated into the CHA coatings and their release and efficacy against bacteria growth were studied in vitro. The following antibiotics were used within this study: cephalothin, carbenicillin, amoxicillin, cefamandol, tobramycin, gentamicin and vancomycin. Increased concentrations of antibiotics in the coating solution led to a higher quantity of antibiotic incorporated into the CHA coating. Some antibiotics were better incorporated than others depending on their chemical structure. Antibiotics, containing carboxylic groups such as cephalothin, carbenicillin and cefamandol, were better incorporated than antibiotics lacking these groups. A bacterial inhibition test on Staphylococcus aureus bacteria showed inhibition of growth for all antibiotics that were released from the CHA coating. A release test was conducted in phosphate buffer saline PBS at pH 7.4 and 37 degrees C and showed that antibiotics containing carboxylic groups like cephalothin were slower released from the CHA coating than others. These results suggest that certain antibiotics are able to bind/chelate with calcium, resulting in a better incorporation into the CHA coating and a slower release. Antibiotics incorporated in CHA coatings on titanium implants might be used to prevent post-surgical infections and to promote bone-bonding of orthopedic devices.

Remineralization of demineralized albumin-calcium phosphate coatings.

Calcium phosphate and bovine serum albumin were coprecipitated (under physiological conditions of temperature and pH) upon the surfaces of titanium-alloy samples, which thereby became coated with a dense, proteinaceous mineral layer 30-50 microm in thickness. Dissolution of the inorganic phase by treatment with acidic saline yielded a self-supporting protein scaffold, 7-10 microm in thickness. Energy-dispersive X-ray analysis and Fourier-transform infrared spectroscopy confirmed the absence of inorganic components from the demineralized albumin scaffolds. When titanium-alloy samples bearing these demineralized protein scaffolds were immersed in a supersaturated solution of calcium phosphate (again at physiological temperature and pH), they remineralized. These redux albumin-calcium phosphate layers corresponded in thickness to the original coatings. When titanium-alloy discs bearing the demineralized protein scaffolds were implanted ectopically (subcutaneously) in mice, they, too, remineralized. No uniform mineral layer was deposited upon the surfaces of naked titanium-alloy implants. To the best of our knowledge, this is the first demonstration of remineralization within the interstices of a noncollagenous protein scaffold, either in vitro or in vivo.