Increased osteoblast adhesion on nanophase metals: Ti, Ti6Al4V, and CoCrMo

Previous studies have demonstrated increased functions of osteoblasts (bone-forming cells) on nanophase compared to conventional ceramics (specifically, alumina, titania, and hydroxyapatite), polymers (such as poly lactic-glycolic acid and polyurethane), carbon nanofibers/nanotubes, and composites thereof. Nanophase materials are unique materials that simulate dimensions of constituent components of bone since they possess particle or grain sizes less than 100 nm. However, to date, interactions of osteoblasts on nanophase compared to conventional metals remain to be elucidated. For this reason, the objective of the present in vitro study was to synthesize, characterize, and evaluate osteoblast adhesion on nanophase metals (specifically, Ti, Ti6Al4V, and CoCrMo alloys). Such metals in conventional form are widely used in orthopedic applications. Results of this study provided the first evidence of increased osteoblast adhesion on nanophase compared to conventional metals. Interestingly, osteoblast adhesion occurred preferentially at surface particle boundaries for both nanophase and conventional metals. Since more particle boundaries are present on the surface of nanophase compared to conventional metals, this may be an explanation for the measured increased osteoblast adhesion. Lastly, material characterization studies revealed that nanometal surfaces possessed similar chemistry and only altered in degree of nanometer surface roughness when compared to their respective conventional counterparts. Because osteoblast adhesion is a necessary prerequisite for subsequent functions (such as deposition of calcium-containing mineral), the present study suggests that nanophase metals should be further considered for orthopedic implant applications.     

Transforming growth factor beta1 immobilized adsorptively on Ti6Al4V and collagen type I coated Ti6Al4V maintains its biological activity

Titanium and titanium alloys are often used for orthopedic and dental implants. Osseointegration of Ti6Al4V may be improved not only by precoating of the surface with extracellular matrix proteins like collagen type I but also by additional immobilization of growth factors. In the present study, transforming growth factor beta1 (TGF-beta1) which is known as an inducer of collagen synthesis was immobilized adsorptively on uncoated and collagen type I coated Ti6Al4V surfaces. TGF-beta1 was found immobilized slightly faster to collagen type I coated than to uncoated Ti6Al4V and released slower from the collagen coated material. Immobilized TGF-beta1 is biologically active for at least 3 weeks storage at 4 degrees C. Sterilization by ethylene oxide inactivates immobilized TGF-beta1. In osteoblasts cultured on implants with adsorptively immobilized TGF-beta1, mRNA level and specific catalytic activity of alkaline phosphatase as well as accumulation of calcium and phosphate were found reduced, whereas procollagen alpha1(I) mRNA level and the rate of collagen synthesis were increased.     

A comparison of the surface characteristics and ion release of Ti6Al4V and heat-treated Ti6Al4V

This work seeks to investigate the nanosurface characteristics and ion release for a Ti6Al4V alloy prepared by various methods (as received and heat treated at 1300 degrees C for 2 h) with three different passivation treatments (34% nitric acid passivation, 400 degrees C heating in air, and aging in 100 degrees C deionized water). The surface and nanosurface composition are not related to the surface passivation treatments and experimental materials as evaluated by energy dispersive spectroscopy and X-ray photoelectron spectroscopy (XPS) analyses. After passivation and autoclaving treatments, the specimens were immersed in 8.0 mM ethylenediaminetetraacetic acid (EDTA) in Hank's solution and maintained at 37 degrees C for periods of time up to 16 days. The 400 degrees C treated specimens exhibit a substantial reduction in constituent release, which may be attributed to the thicker thickness and rutile structure of the surface oxides. After soaking in Hank's-EDTA solution, a significant time-related decrease in constituent release rate is observed for all kinds of specimens throughout the 0-16 day experimental period. The thicker oxides may be a factor in the improved dissolution resistance. Upon immersion, nonelemental Ca and P are both detected on the surfaces of all kinds of specimens by XPS analysis, and this could be explained by the existence of two types of hydroxyl groups (acidic and basic OH groups) on the oxide surface of the specimens.     

Thermal oxidation enhances early interactions between human osteoblasts and alumina blasted Ti6Al4V alloy

Oxidation of Ti6Al4V at 500 degrees C for 1 h in air results in the formation of an outer ceramic layer that improves osteoblast behavior and decreases Ti and Al ion release. In this work, alumina blasted Ti6Al4V alloy has been thermally treated and its in vitro biocompatibility has been assessed. Roughness of the blasted alloy was not found significantly altered after heat treatment while chemical surface analysis indicated an increase in stable TiO(2) and Al(2)O(3) oxides. Cell attachment, spreading, cytoskeleton organization as well as cell proliferation, viability, and procollagen I peptide secretion of human primary osteoblasts, impaired on alumina blasted Ti6Al4V, were found to be greatly enhanced on the thermally oxidized blasted alloy. Other informative markers of the osteoblastic phenotype such as alkaline phosphatase, osteocalcin, osteoprotegerin, and mineralized nodule formation were evaluated and indicated that osteoblasts responded at the same extent on untreated and thermally treated blasted alloys. Taken together, our in vitro results indicate that thermal oxidation of alumina blasted Ti6Al4V may favor successful osseointegration by promoting early interactions of osteoblastic cells and the modified surface alloy.     

Fretting corrosion of CoCrMo and Ti6Al4V interfaces

Mechanically assisted corrosion (fretting corrosion, tribocorrosion etc.,) of metallic biomaterials is a primary concern for numerous implant applications, particularly in the performance of highly-loaded medical devices. While the basic underlying concepts of fretting corrosion or tribocorrosion and fretting crevice corrosion are well known, there remains a need to develop an integrated systematic method for the analysis of fretting corrosion involving metal-on-metal contacts. Such a method can provide detailed and quantitative information on the processes present and explore variations in surfaces, alloys, voltages, loadings, motion and solution conditions. This study reports on development of a fretting corrosion test system and presents elements of an in-depth theoretical fretting corrosion model that incorporates both the mechanical and the electrochemical aspects of fretting corrosion. To demonstrate the capabilities of the new system and validate the proposed model, experiments were performed to understand the effect of applied normal load on fretting corrosion performance of Ti6Al4V/Ti6Al4V, CoCrMo/Ti6Al4V, and CoCrMo/CoCrMo material couples under potentiostatic conditions with a fixed starting surface roughness. The results of this study show that fretting corrosion is affected by material couples, normal load and the motion conditions at the interface. In particular, fretting currents and coefficient of friction (COF)?vary with load and are higher for Ti6Al4V/Ti6Al4V couple reaching 3?mA/cm(2) and 0.63 at about 73?MPa?nominal contact stress, respectively. Ti6Al4V coupled with CoCrMo displayed lower currents (0.6?mA/cm(2)) and COF (0.3), and the fretting corrosion behavior was comparable to CoCrMo/CoCrMo couple (1.2?mA/cm(2) and 0.3, respectively). Information on the mechanical energy dissipated at the interface, the sticking behavior, and the load dependence of the inter-asperity distance calculated using the model elucidated the influence of mechanical factors on the experimental results. It was observed that the lowest amount of work was required to generate some of the highest fretting corrosion currents in Ti6Al4V/Ti6Al4V couples compared to the other combinations. The elements of the model presented here provide an excellent basis to explain many of the observed behaviors of these interfaces.     

Concentration-dependent effects of titanium and aluminium ions released from thermally oxidized Ti6Al4V alloy on human osteoblasts

Thermal oxidation treatments of Ti6Al4V, at 500 and 700 degrees C, for 1 h result in the formation of an outer "ceramic" layer of rutile, which enhances osteoblast response. In the present study, we have measured in vitro Ti and Al ion release from Ti64 alloy in the as-received state and after thermal oxidation treatments at 500 or 700 degrees C, to culture medium under standard cell-culture conditions. Concentrations of both Ti and Al released from both thermal oxidation treatments were lower than from polished alloy. Al was released from the treated or untreated surfaces in substantially lower extent than Ti. Titanium and aluminium ions affected primary human osteoblast proliferation, metabolic activity, and differentiation in a dose-dependent manner. Treatments with individual Ti or Al metal ions in similar concentration ranges than released from the surfaces did not alter osteoblast response, which also remained unaffected after treatments with combinations of Ti plus Al applied in the proportional relations than detected in ion-release experiments. We then selected higher concentrations of Ti that impaired osteoblast proliferation and differentiation, while the proportional lower concentrations of Al did not alter osteoblast behavior. In spite of its inert character, it was found that Al significantly enhanced the deleterious effect of Ti on osteoblast differentiation. Therefore, thermal oxidation treatments of Ti6Al4V alloy may improve the biocompatibility of the alloy by reducing both Ti and Al release, and thus attenuating ion-mediated interference with osteoblast differentiation.     

Osteoblast response to thermally oxidized Ti6Al4V alloy

We have recently reported that thermal oxidation treatments of Ti6Al4V at 500 degrees and 700 degrees C for 1 h result in the formation of an outer "ceramic" layer of rutile that do not decrease the high in vitro corrosion resistance of the alloy. In the present work, surface roughness was measured and found marginally increased as a consequence of oxidation of the alloy at 700 degrees C, but not at 500 degrees C. We have evaluated the biocompatibility of the oxidized surfaces, by assessing cell adhesion, proliferation, and differentiation of primary cultures of human osteoblastic cells. Compared with polished alloy, both thermal treatments increased osteoblast adhesion measured as cell attachment, beta1 integrin and FAK-Y397 expression, as well as cytoskeletal reorganization. Compared with treatment at 500 degrees C, thermal oxidation at 700 degrees C enhanced cell adhesion. Treatment at 700 degrees C transiently impaired cell proliferation and viability, which were not altered in alloys oxidized at 500 degrees C. Several markers of osteoblastic differentiation such as procollagen I peptide, alkaline phosphatase, osteocalcin, and mineralized nodule formation were found either unaffected or differentially increased by alloys treated either at 500 degrees or 700 degrees C. In addition, thermal oxidation at 700 degrees C also increased osteoprotegerin secretion. Taken together, our results indicate that thermal oxidation treatments at 500 degrees or 700 degrees C for 1 h improve the in vitro biocompatibility of Ti6Al4V.     

Biological performance of uncoated and octacalcium phosphate-coated Ti6Al4V

The in vivo behavior of a porous Ti6Al4V material that was produced by a positive replica technique, with and without an octacalcium phosphate (OCP) coating, has been studied both in the back muscle and femur of goats. Macro- and microporous biphasic calcium phosphate (BCP) ceramic, known to be both osteoconductive and able to induce ectopic bone formation, was used for comparison purpose. The three groups of materials (Ti6Al4V, OCP Ti6Al4V and BCP) were implanted transcortically and intramuscularly for 6 and 12 weeks in 10 adult Dutch milk goats in order to study their osteointegration and osteoinductive potential. In femoral defects, both OCP Ti6Al4V and BCP were performing better than the uncoated Ti6Al4V, at both time points. BCP showed a higher bone amount than OCP Ti6Al4V after 6 weeks of implantation, while after 12 weeks, this difference was no longer significant. Ectopic bone formation was found in both OCP Ti6Al4V and BCP implants after 6 and 12 weeks. The quantity of ectopically formed bone was limited as was the amount of animals in which the bone was observed. Ectopic bone formation was not found in uncoated titanium alloy implants, suggesting that the presence of calcium phosphate (CaP) is important for bone induction. This study showed that CaPs in the form of coating on metal implants or in the form of bulk ceramic have a significantly positive effect on the bone healing process.     

Attachment and proliferation of neonatal rat calvarial osteoblasts on Ti6Al4V: effect of surface chemistries of the alloy

This study examined the cell attachment and proliferation of neonatal rat calvarial osteoblasts on Ti6Al4V alloy as affected by the surface modifications. The modifications could alter simultaneously the surface chemistries of the alloy (elemental difference of Ti, Al, V, Cu and Ni about 300-600mum thick examined by EDS) as well as the XPS nano-surface characteristics of oxides on the metal surface (chemistries of oxides, amphoteric OH group adsorbed on oxides, and oxide thickness). Three materials including two from modifications and a control were examined. It is argued that a slight change of the nano-surface characteristics of oxides as a result of the modifications neither alters the in vitro capability of Ca and P ion adsorption nor affects the metal ion dissolution behavior of the alloy. This implies that any influence on the cytocompatibility of the materials should only be correlated to the effect of surface chemistries of the alloy and the associated metal ion dissolution behavior of the alloy. The experimental results suggest that the cell response of neonatal rat calvarial osteoblasts on the Ti6Al4V alloy should neither be affected by the variation of surface chemistries of the alloy in a range studied.     

In vitro biocompatibility and bacterial adhesion of physico-chemically modified Ti6Al4V surface by means of UV irradiation

UV irradiation leads to a "spontaneous" wettability increase of the Ti6Al4V surface while preserving bulk properties of the alloy that are crucial for its performance as an orthopedic and dental implant. We hypothesized that UV treatment of Ti6Al4V may impair bacterial adhesion without compromising the good response of human bone-forming cells to this alloy. The in vitro biocompatibility of the Ti6Al4V surface, before and after UV irradiation, was analyzed by using human cells related to the osteoblastic phenotype. The adhesion processes of bacterial strains related to clinical orthopedic infections, i.e., Staphylococcus aureus and Staphylococcus epidermidis, were studied theoretically and in vitro, under dynamic and static conditions as well as in the presence or absence of shear forces. While human cell adhesion was not altered by UV irradiation of Ti6Al4V alloy, this treatment reduced not only initial bacterial adhesion rates but also the number of bacteria retained on the surface after the passage of two air-liquid interfaces on the previously adhered bacteria. This study proposes the use of UV treatment prior to implantation protocols as an easy, economic and effective way of reducing bacterial adhesion on the Ti6Al4V surface without compromising its excellent biocompatibility.     

钛合金Ti6Al4V表面钛酸锶生物薄膜的水热合成

采用氢氧化钠和硝酸锶混合溶液对Ti6Al4V合金进行水热处理,温度180℃,时间1、3和6 h。X射线衍射、扫描电镜、电子能谱和X射线光电子能谱分析表明,水热处理后钛合金表面形成了钛酸锶的纳米颗粒薄膜,颗粒尺寸80～230 nm,薄膜中含有极少量铝和钒元素。在无钙Hank’s平衡盐液中的动电位极化和电化学阻抗实验表明,与抛光试样相比,水热处理3h试样的耐蚀性大幅提高。显微硬度压入实验表明,钛酸锶薄膜具有良好的附着性和内聚力。水热处理制备钛酸锶薄膜的方法可用于钛合金Ti6Al4V骨科植入体的表面改性。     

Nanometer-scale surface modification of Ti6Al4V alloy for orthopedic applications

This communication presents a novel technology to enhance the biocompatibility of bioinert Ti6Al4V alloy as implant materials for orthopaedic application. The surface of Ti6Al4V alloy was electrochemically activated in NaOH solution to create a porous structure with nanometer topographic features and an alkaline environment, thus promoting the formation of bone-like hydroxyapatite coating and enhancing the bonding strength of the coating. This innovative activation process was proved to be effective and essential. The activated surface was confirmed to be pure TiO2 and the formed coating was characterized of pure hydroxyapatite with a nanometer-scaled grain size structure by means of XPS, FESEM/SEM/EDX, XRD, and TEM techniques.     

Biomimetic collagen/apatite coating formation on Ti6Al4V substrates

Hydroxyapatite and type I collagen are two major components of natural bone. They have been used extensively to engineer biomaterial surfaces for enhanced implant-bone integrations. In this study, a collagen/apatite composite coating was successfully deposited on treated Ti6Al4V surfaces by coprecipitation of type I collagen and apatite in a collagen-containing modified simulated body fluid (m-SBF). X-ray diffraction and Fourier-transform infrared spectroscopy results indicated that the coating achieved was a composite of bone-like carbonated apatite and collagen type I, which is similar to the composition of natural bone. The coating composition and morphology could be tailored by carefully adjusting the collagen concentration in the m-SBF. In addition, both the coating thickness and bonding strength were down regulated by the collagen concentration in the m-SBF. Collagen inhibited the apatite nucleation on the metallic substrate, and thereby reduced the bonding strength of the composite coating. In addition, an in vitro cell culture study was conducted on both the apatite and the collagen-apatite coating surfaces. Addition of collagen promoted osteoblast activities, which may lead to early bone formation.     

A novel porous Ti6Al4V: characterization and cell attachment

For the first time, a highly porous strong Ti6Al4V was produced by using a "polymeric sponge replication" method. A polymeric sponge, impregnated with a Ti6Al4V slurry prepared from Ti6Al4V powders and binders, was subjected to drying and pyrolyzing to remove the polymeric sponge and binders. After sintering at a high temperature and under high vacuum, a porous Ti6Al4V was produced. Optical microscopical observation, environmental scanning electron microscopy observation (with energy-dispersive micro X-ray analysis), mechanical tests, and metallurgical analyses were performed on the obtained porous Ti6Al4V with regard to the porous structure (both macropores and micropores), mechanical properties, chemical composition, phase compositions, and cell attachment behavior. The porous Ti6Al4V made by this method had a three-dimensional trabecular porous structure with interconnected pores mainly ranging from 400 to 700 microm and a total porosity of about 90%. The compressive strength was 10.3 +/- 3.3 MPa and the elastic constant 0.8 +/- 0.3 GPa. MC3T3-E1 cells attached and spread well in the inner surface of pores. Being similar to cancellous bone with regard to both interconnected porous structure and mechanical properties, the resulting porous Ti6Al4V is expected to be a promising biomaterial for biomedical applications.     

Tailoring the surface properties of Ti6Al4V by controlled chemical oxidation

Many efforts have been made to promote cell activity at the surface of implants, mainly by modifying their topography and physicochemical properties. Here we demonstrate the feasibility of creating Ti6Al4V surfaces having both a microtexture and a nanotexture, and show that their properties can be tailored by controlling the length of exposure to a mixture of H2SO4 and H2O2. Scanning electron microscopy (SEM), combined with energy-dispersive X-ray spectroscopy (EDX), indicated that beta-phase grains, which surround larger alpha-phase grains, are etched more rapidly, resulting in a surface composed of microscale cavities with alpha-grain boundaries. Furthermore, high-resolution SEM and atomic force microscopy (AFM) revealed the presence on the surfaces of both alpha- and beta-phase grains of a network of nanopits with mean diameters ranging between 13 and 21 nm. The grain surface roughness increases from about 4 nm on untreated samples to about 12 nm after 4h of treatment. AFM analysis showed that the depth of microscale cavities can be varied in the 10-180 nm range by controlling the extent of chemical etching. Fourier transform infrared spectroscopy (FT-IR), combined with ellipsometry, established that the etching generated an oxide layer with a thickness in the range 15-45 nm. The resulting new surfaces selectively promote the growth of osteoblasts while inhibiting that of fibroblasts, making them promising tools for regulating the activities of cells in biological environments.     

Contact profilometry and correspondence analysis to correlate surface properties and cell adhesion in vitro of uncoated and coated Ti and Ti6Al4V disks

A fundamental goal in the field of implantology is the design of specific devices able to induce a controlled and rapid "osseointegration". This result has been achieved by means of surface modifications aimed at optimizing implant-to-bone contact; furthermore, bone cell adhesion on implant surface has been directly improved by the application of biomolecules that stimulate new tissue formation, thus controlling interactions between biological environment and implanted materials. Actually, methods for biochemical factor delivery at the interface between implant surface and biological tissues are under investigation; a reliable technique is represented by the inclusion of biologically active molecules into biocompatible and biodegradable materials used for coating implant surface. This paper focuses the application of three polymeric materials already acknowledged in the clinical practice, i.e. poly-L-lactic acid (PLLA), poly-DL-lactic acid (PDLA), and sodium alginate hydrogel. They have been used to coat Ti (Ti2) and Ti6Al4V (Ti5) disks; their characteristics have been determined and their performances compared, with specific regard to the ability in allowing osteoblast adhesion in vitro. Moreover, profilometry data analysis permitted to identify a specific roughness parameter (peak density) which mainly controls the amount of osteoblast adhesion.     

Strontium-substituted apatite coating grown on Ti6Al4V substrate through biomimetic synthesis

During the last few years Strontium has been shown to have beneficial effects when incorporated at certain doses in bone by stimulating bone formation. It is believed that its presence locally at the interface between an implant and bone will enhance osteointegration and therefore, ensure the longevity of a joint prosthesis. In this study we explore the possibility of incorporating Sr into nano-apatite coatings prepared by a solution-derived process according to an established biomimetic methodology for coating titanium based implants. The way this element is incorporated in the apatite structure and its effects on the stereochemistry and morphology of the resulting apatite layers was investigated, as well as its effect in the mineralization kinetics. By using the present methodology it was possible to incorporate increasing amounts of Sr in the apatite layers. Sr was found to incorporate in the apatite layer through a substitution mechanism by replacing Ca in the apatite lattice. The presence of Sr in solution induced an inhibitory effect on mineralization, leading to a decrease in the thickness of the mineral layers. The obtained Sr-substituted biomimetic coatings presented a bone-like structure similar to the one found in the human bone and therefore, are expected to enhance bone formation and osteointegration.     

Corrosion behavior and biocompatibility of nanostructured TiO2 film on Ti6Al4V

The corrosion behavior and cell adhesion property of nanostructured TiO2 films deposited electrolytically on Ti6Al4V were examined in the present in vitro study. The nanostructured TiO2 film deposition on Ti6Al4V was achieved via peroxoprecursors. SEM micrographs exhibit the formation of amorphous and crystallite TiO2 nanoparticles on Ti6Al4V before and after being annealed at 500 degrees C. Corrosion behavior of TiO2-deposited and uncoated Ti6Al4V was evaluated in freely aerated Hank's solution at 37 degrees C by the measurement and analysis of open-circuit potential variation with time, Tafel plots, and electrochemical impedance spectroscopy. The electrochemical results indicated that nano-TiO2 coated Ti6Al4V showed a better corrosion resistance in simulated biofluid than uncoated Ti6Al4V. Rat bone cells and human aortic smooth muscle cells were grown on these substrates to study the cellular responses in vitro. The SEM images revealed enhanced cell adhesion, cell spreading, and proliferation on nano-TiO2 coated Ti6Al4V compared to those grown on uncoated substrates for both cell lines. These results suggested that nanotopography produced by deposition of nanostructured TiO2 onto Ti alloy surfaces might enhance corrosion resistance, biocompatibility, and cell integration for implants made of Ti alloys.     

Response of human endothelial cells to oxidative stress on Ti6Al4V alloy

Titanium and its alloys are amongst the most frequently used materials in bone and dental implantology. The good biocompatibility of titanium(-alloys) is attributed to the formation of a titanium oxide layer on the implant surface. However, implant failures do occur and this appears to be due to titanium corrosion. Thus, cells participating in the wound healing processes around an implanted material, among them endothelial cells, might be subjected to reactive oxygen species (ROS) formed by electrochemical processes during titanium corrosion. Therefore, we studied the response of endothelial cells grown on Ti6Al4V alloy to H(2)O(2) and compared this with the response of endothelial cells grown on cell culture polystyrene (PS). We could show that although the cell number was the same on both surfaces, metabolic activity of endothelial cells grown on Ti6Al4V alloy was reduced compared to the cells on PS and further decreased following prototypic oxidative stress (H(2)O(2)-treatment). The analysis of H(2)O(2)-induced oxidative stress showed a higher ROS formation in endothelial cells on Ti6Al4V than on PS. This correlated with the depletion of reduced glutathione (GSH) in endothelial cells grown on Ti6Al4V surfaces and indicated permanent oxidative stress. Thus, endothelial cells in direct contact with Ti6Al4V showed signs of oxidative stress and higher impairment of cell vitality after an additional oxidative stress. However, the exact nature of the agent of oxidative stress generated from Ti6Al4V remains unclear and requires further investigation.     

In vitro differentiation of human mesenchymal stem cells on Ti6Al4V surfaces

Long-term stability of arthroplasty prosthesis depends on the integration between the bone tissue and the implanted biomaterials, which requires the contribution of osteoblastic precursors and their continuous differentiation into the osteoblastic phenotype. Classically, these interactions are tested in vitro using mesenchymal stem cells (MSCs) isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue-derived stromal cells (AMSCs) may be a more convenient source of MSCs, according to their abundance and accessibility, but no data are available on their in vitro interactions with hard biomaterials. The aim of this work is to compare the osteogenic potential of human AMSCs and bone marrow-derived MSCs (BMMSCs) and to evaluate their response to Ti6Al4V alloy in terms of adhesion, proliferation and differentiation features, using the human osteosarcoma cell line SaOS-2 for comparison. The overall results showed that AMSCs have the same ability to produce bone matrix as BMMSCs and that Ti6Al4V surfaces exhibit an osteoinductive action on AMSCs, promoting their differentiation into functional osteoblasts and increasing bone formation. In conclusion, adipose tissue is a promising autologous source of osteoblastic cells with important clinical implications for bone tissue engineering.