Specific proteins mediate enhanced osteoblast adhesion on nanophase ceramics

Osteoblast, fibroblast, and endothelial cell adhesion on nanophase (that is, materials with grain sizes less than 100 nm) alumina, titania, and hydroxyapatite (HA) was investigated using in vitro cellular models. Osteoblast adhesion was significantly (p < 0.01) greater after 4 h on nanophase alumina, titania, and HA than it was on conventional formulations of the same ceramics. In contrast, compared to conventional alumina, titania, and HA, after 4 h fibroblast adhesion was significantly (p < 0.01) less on nanophase ceramics. Examination of the underlying mechanism(s) of cell adhesion on nanophase ceramics revealed that these ceramics adsorbed significantly (p < 0.01) greater quantities of vitronectin, which, subsequently, may have contributed to the observed select enhanced adhesion of osteoblasts. Select enhanced osteoblast adhesion was independent of surface chemistry and material phase but was dependent on the surface topography (specifically on grain and pore size) of nanophase ceramics. The capability of synthesizing and processing nanomaterials with tailored (through, for example, specific grain and pore size) structures and topographies to control select subsequent cell functions provides the possibility of designing the novel proactive biomaterials (that is, materials that elicit specific, timely, and desirable responses from surrounding cells and tissues) necessary for improved implant efficacy.     

Enhanced osteoclast-like cell functions on nanophase ceramics

Synthesis of tartrate-resistant acid phosphatase (TRAP) and formation of resorption pits by osteoclast-like cells, the bone-resorbing cells, on nanophase (that is, material formulations with grain sizes less than 100nm) alumina and hydroxyapatite (HA) were investigated in the present in vitro study. Compared to conventional (that is, grain sizes larger than 100 nm) ceramics, synthesis of TRAP was significantly greater in osteoclast-like cells cultured on nanophase alumina and on nanophase HA after 10 and 13 days, respectively. In addition, compared to conventional ceramics, formation of resorption pits was significantly greater by osteoclast-like cells cultured on nanophase alumina and on nanophase HA after 7, 10, and 13 days, respectively. The present study, therefore, demonstrated, for the first time, enhanced osteoclast-like cell function on ceramic surfaces with nanometer-size surface topography.     

纳米羟基磷灰石对成骨细胞功能代谢影响的研究

比较纳米羟基磷灰石(nHA)和常规羟基磷灰石(cHA)对成骨细胞功能代谢影响方面的差异。采用化学沉淀法制备nHA粉体,采用压制成型和无压烧结工艺制备nHA与cHA的块体材料。将Wistar乳鼠颅骨体外原代分离培养的成骨细胞接种于nHA与cHA的表面,分别在7、14、21、28d时检测细胞内总蛋白、ALP活性及细胞基质钙含量。结果表明,所制备的nHA与cHA的平均粒径分别为55nm和0.78μm;第21和28d,nHA表面附着的成骨细胞ALP活性和细胞基质钙含量均高于cHA。该研究提示:与相应的cHA比较,nHA更能增强成骨细胞的功能及代谢活动。     

Evaluation of cytocompatibility and bending modulus of nanoceramic/polymer composites

In an attempt to simulate the microstructure and mechanical properties of natural bone, novel nanoceramic/polymer composite formulations were fabricated and characterized with respect to their cytocompatibility and mechanical properties. The bending moduli of nanocomposite samples of either poly(L-lactic acid) (PLA) or poly(methyl methacrylate) (PMMA) with 30, 40, and 50 wt % of nanophase (<100 nm) alumina, hydroxyapatite, or titania loadings were significantly (p < 0.05) greater than those of pertinent composite formulations with conventional, coarser grained ceramics. The nanocomposite bending moduli were 1-2 orders of magnitude larger than those of the homogeneous, respective polymer. For example, compared with 0.06 GPa for the 100% PLA, the bending modulus of 50/50 nanophase alumina/PLA composites was 3.5 GPa. Osteoblast adhesion on the surfaces of the nanophase alumina/PLA composites increased as a function of the nanophase ceramic content. Most importantly, osteoblast adhesion on the 50/50 nanophase alumina/PLA substrates was similar to that observed on the 100% nanophase ceramic substrates. Similar trends of osteoblast adhesion were observed on the surfaces of the nanophase titania/polymer and nanophase hydroxyapaptite/polymer composites that were tested. In contrast, fibroblast adhesion on the nanophase composites was either similar or lower than that observed on the conventional composites with either PLA or PMMA and minimum on all tested neat nanophase substrates. The calcium content in the extracellular matrix of cultured osteoblasts was also enhanced on the nanoceramic/PLA composite substrates tested as a function of the nanophase ceramic loading and duration of cell culture. The results of the present in vitro study provide evidence that nanoceramic/polymer composite formulations are promising alternatives to conventional materials because they can potentially be designed to match the chemical, structural, and mechanical properties of bone tissue in order to overcome the limitations of the biomaterials currently used as bone prostheses.     

Increased viable osteoblast density in the presence of nanophase compared to conventional alumina and titania particles

In the present in vitro study, osteoblast (bone-forming cells) viability and cell density were investigated when cultured in the presence of nanophase compared to conventional (i.e. micron) alumina and titania particles at various concentrations (from 10,000 to 100 microg/ml of cell culture media) for up to 6h. Results confirmed previous studies of the detrimental influences of all ceramic particulates on osteoblast viability and cell densities. For the first time, however, results provided evidence of increased apoptotic cell death when cultured in the presence of conventional compared to nanophase alumina and titania particles. Moreover, since material characterization studies revealed that the only difference between respective ceramic particles was nanometer- and conventional-dimensions (specifically, phase and chemical properties were similar between respective nanophase and conventional alumina as well as titania particles), these results indicated that osteoblast viability and densities were influenced solely by particle size. Such nanometer particulate wear debris may result from friction between articulating components of orthopedic implants composed of novel nanophase ceramic materials. Results of a less detrimental effect of nanometer--as compared to conventional-dimensioned particles on the functions of osteoblasts provide additional evidence that nanophase ceramics may become the next generation of bone prosthetic materials with increased efficacy and, thus, deserve further testing.     

Osteoblast adhesion on nanophase ceramics.

Osteoblast adhesion on nanophase alumina (Al2O3) and titania (TiO2) was investigated in vitro. Osteoblast adhesion to nanophase alumina and titania in the absence of serum from Dulbecco's modified Eagle medium (DMEM) was significantly (P < 0.01) less than osteoblast adhesion to alumina and titania in the presence of serum. In the presence of 10% fetal bovine serum in DMEM osteoblast adhesion on nanophase alumina (23 nm grain size) and titania (32 nm grain size) was significantly (P < 0.05) greater than on conventional alumina (177 nm grain size) and titania (2.12 microm grain size), respectively, after 1, 2, and 4 h. Further investigation of the dependence of osteoblast adhesion on alumina and titania grain size indicated the presence of a critical grain size for osteoblast adhesion between 49 and 67 nm for alumina and 32 and 56 nm for titania. The present study provides evidence of the ability of nanophase alumina and titania to simulate material characteristics (such as surface grain size) of physiological bone that enhance protein interactions (such as adsorption, configuration, bioactivity, etc.) and subsequent osteoblast adhesion.     

Increased osteoblast function on PLGA composites containing nanophase titania

Nanotechnology creates materials that potentially outperform, at several boundaries, existing materials in terms of mechanical, electrical, catalytic, and optical properties. However, despite their promise to mimic the surface roughness cells experience in vivo, the use of nanophase materials in biological applications remains to date largely unexplored. The objective of the present in vitro study was, therefore, to determine whether when added to a polymer scaffold, nanophase compared to conventional ceramics enhance functions of osteoblasts (or bone-forming cells). Results from this study provided the first evidence that functions (specifically, adhesion, synthesis of alkaline phosphatase, and deposition of calcium-containing mineral) of osteoblasts increased on poly-lactic-co-glycolic acid (PLGA) scaffolds containing nanophase compared to conventional grain size titania with greater weight percentage (from 10-30 wt %). Because the chemistry, material phase, porosity (%), and pore size of the composites were similar, this study implies that the surface features created by adding nanophase compared to conventional titania was a key parameter that enhanced functions of osteoblasts. In this manner, the study adds another novel property of nanophase ceramics: their ability to promote osteoblast functions in vitro when added to a polymer scaffold. For this reason, nanophase ceramics (and nanomaterials in general) deserve further attention as orthopedic tissue engineering materials.     

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.     

Enhanced functions of osteoblasts on nanometer diameter carbon fibers

The present in vitro study investigated select functions (specifically, proliferation, synthesis of intracellular proteins, alkaline phosphatase activity, and deposition of calcium-containing mineral) of osteoblasts (the bone-forming cells) cultured on carbon fibers with nanometer dimensions. Carbon fiber compacts were synthesized to possess either nanophase (i.e., dimensions 100 nm or less) or conventional (i.e., dimensions larger than 100 nm) fiber diameters. Osteoblast proliferation increased with decreasing carbon fiber diameters after 3 and 7 days of culture. Moreover, compared to larger-diameter carbon fibers, osteoblasts synthesized more alkaline phosphatase and deposited more extracellular calcium on nanometer-diameter carbon fibers after 7, 14, and 21 days of culture. The results of the present study provided the first evidence of enhanced long-term (in the order of days to weeks) functions of osteoblasts cultured on nanometer-diameter carbon fibers; in this manner, carbon nanofibers clearly represent a unique and promising class of orthopedic/dental implant formulations with improved osseointegrative properties.     

Nanocrystalline hydroxyapatite/titania coatings on titanium improves osteoblast adhesion

Bulk hydroxyapatite (HA) and titania have been used to improve the osseointegration of orthopedic implants. For this reason, composites of HA and titania have been receiving increased attention in orthopedics as novel coating materials. The objective of this in vitro study was to produce nanophase (i.e., materials with grain size less than 100 nm) HA/titania coatings on titanium. The adhesion of bone forming cells (osteoblasts) on the composite coatings were also assessed and compared with single-phase nanotitania and nano-HA titanium coatings. Nanocrystalline HA powders were synthesized through wet chemistry and hydrothermal treatments at 200 degrees C. Nanocrystalline titania powders obtained commercially were mixed with the nanocrystalline HA powders at various weight ratios. The mixed powders were then deposited on titanium utilizing a room-temperature coating process called IonTite. The results of the present study showed that such coatings maintained the chemistry and crystallite size of the original HA and titania powders. Moreover, osteoblasts adherent on single-phase nanotitania coatings were well-spread whereas they became more round and extended distinct filopodia on the composite and single-phase HA coatings. Interestingly, the number of osteoblasts adherent on the nanotitania/HA composite coatings at weight ratios of 2/1 and 1/2 were significantly greater compared with single-phase nanotitania coatings, currently-used plasma-sprayed HA coatings, and uncoated titanium. These findings suggest that nanotitania/HA coatings on titanium should be further studied for improved orthopedic applications.     

Human mesenchymal stem cell adhesion and proliferation in response to ceramic chemistry and nanoscale topography

Modification of the chemistry and surface topography of nanophase ceramics was used to provide biomaterial formulations designed to direct the adhesion and proliferation of human mesenchymal stem cells (HMSCs). HMSC adhesion was dependent upon both the substrate chemistry and grain size, but not on surface roughness or crystal phase. Specifically, cell adhesion on alumina and hydroxyapatite was significantly reduced on the 50 and 24 nm surfaces, as compared with the 1500 and 200 nm surfaces, but adhesion on titania substrates was independent of grain size. HMSC proliferation was minimal on the 50 and 24 nm substrates of any chemistry tested, and thus significantly lower than the densities observed on either the 1500 or 200 nm surfaces after 3 or more consecutive days of culture. Furthermore, HMSC proliferation was enhanced on the 200 nm substrates, compared with results obtained on the 1500 nm substrates after 7 or more days of culture. HMSC proliferation was independent of both substrate surface roughness and crystal phase. Rat osteoblast and fibroblast adhesion and proliferation exhibited similar trends to that of HMSCs on all substrates tested. These results demonstrated the potential of nanophase ceramic surfaces to modulate functions of HMSCs, which are pertinent to biomedical applications such as implant materials and devices.     

The effect of nanotopography on calcium and phosphorus deposition on metallic materials in vitro

To date, long-term functions of osteoblasts leading to calcium and phosphorus mineral deposition on nanometals have not been determined. Nanometals are metals with constituent metal particles and/or surface features less than 100 nm in at least one dimension. For this reason, the objective of this in vitro study was to determine the amount of calcium and phosphorus mineral formation on microphase compared to nanophase Ti, Ti6Al4V, and CoCrMo cultured with and without osteoblasts (bone-forming cells). The results of this study provided the first evidence of significantly greater calcium and phosphorus deposition by osteoblasts and precipitation from culture media without osteoblasts on nanophase compared to respective microphase Ti6Al4V and CoCrMo after 21 days; the greatest calcium and phosphorus mineral deposition occurred on nanophase CoCrMo while the greatest calcium and phosphorus mineral precipitation without osteoblasts occurred on nanophase Ti6Al4V. No differences were found for any type of Ti: wrought, microphase, or nanophase. Moreover, increased calcium and phosphorus mineral content correlated to greater amounts of underlying aluminum content on Ti6Al4V surfaces. Since, compared to microphase Ti6Al4V, nanophase Ti6Al4V contained a higher amount of aluminum at the surface (due to greater surface area), this may provide a reason for enhanced calcium and phosphorus mineral content on nanophase Ti6Al4V. Regardless of the mechanism, this study continues to support the further investigation of nanometals for improved orthopedic applications.     

Altered responses of chondrocytes to nanophase PLGA/nanophase titania composites

Chondrocyte (cartilage-synthesizing cells) cell density and synthesis of select intracellular proteins by chondrocytes were investigated on novel nanophase poly-lactic/glycolic acid (PLGA) and titania composites in the present in vitro study. Nanophase PLGA films were created by chemically treating conventional (or micron-structured) PLGA films with 10N NaOH for 1h. Titania particle dimensions in ceramic compacts were controlled by utilizing either conventional (i.e., micron) or nanometer grain size titania. Composites of either conventional or nanophase PLGA with either conventional or nanophase titania at 70/30wt% were also created. Compared to surfaces with a conventional or micron topography, results provided the first evidence of stagnant confluent cell densities on nanostructured surfaces at time points between 1 and 7 days. Moreover, compared to surfaces with a conventional topography, increased chondrocyte intracellular synthesis of alkaline phosphatase and chondrocyte expressed protein-68 (proteins that have been correlated with the functions of chondrocytes) were observed on nanophase PLGA/nanophase titania composites. The present study, thus, provided the first evidence of different chondrocyte responses to nanostructured PLGA/nanophase titania composites; in light of other reports demonstrating increased functions of bone cells on the same materials, such data indicates that further investigation of these materials at the bone-cartilage interface should be conducted.     

Practice of a testing bench to study the effects of cyclic stretching on osteoblast-orthopaedic ceramic interactions

A new experimental method has been used to study the behaviour of human osteoblasts cultured on bioceramics subjected to mechanical strains. The ceramics were alumina, hydroxyapatite (HA) and a duplex system composed of hydroxyapatite-covered alumina. The system applied 400 microdeformations for a 6-h period with a cycle frequency of 0.5 Hz to osteoblasts growing on ceramic-covered disks. The effects of strains on short-term cell viability, cell growth, alkaline phosphatase (ALP) activity, and collagen biosynthesis were assessed. When possible, the parameters (lactate dehydrogenase) were studied along the experiment in samples of the culture medium, in the other cases by comparison of stretched and unstretched cultures on the same ceramics with the same cell line. In relationship with the coating, mechanical strains resulted in a decrease in DNA corresponding to cell number, an LDH release during straining, an unchanged (alumina) or decreased (HA and duplex) ALP activity, a decrease (HA and duplex) of collagen and total protein synthesis or an increase of it (alumina). The stress-producing device and its associated protocol are shown to be suitable for investigating the behaviour of cells, cultured on biomaterials subjected to mechanical strain.     

Phenotypic expression of bone-related genes in osteoblasts grown on calcium phosphate ceramics with different phase compositions

Calcium phosphate ceramics with different hydroxyapatite (HA) and tricalcium phosphate (TCP) ratios have different chemical properties. Does the difference in phase composition affect osteoblast behavior? In this study, osteoblasts were cultured on 4 kinds of calcium phosphate ceramics, i.e. pure (HA), HT1 (HA/TCP, 70/30), HT2 (HA/TCP, 35/65), and pure TCP. Cell proliferation of SaOS-2 cells together with bone-related genes' mRNA expression and protein production in osteoblasts cultured on different calcium phosphate ceramics were detected at different time points. Data suggested that cell proliferation rate on TCP ceramics was lower than that on the other substrates tested. Generally, mRNA expressions for osteonectin and osteocalcin were similar among the four kinds of ceramics in most circumstances, whereas at six days, alkaline phosphatase mRNA expression was higher on HA and HT1 surfaces than on the other two materials. Collagen I mRNA expression was also affected by the phase composition of substrates. Osteocalcin and bone sialoprotein production in SaOS-2 cells was very similar no matter which ceramic surface the cells were grown upon. This study revealed that calcium phosphate ceramics substrate could support osteoblast growth and bone-related gene expression and its gene expression pattern explained the basis of the biocompatibility and bioactivity for calcium phosphate ceramics.     

Preparation of bioactive nanotitania ceramics with biomechanical compatibility

In this article, bioactive nanotitania ceramics with biomechanical compatibility was prepared by using an additive of hydroxyapatite or MgO as particle growth inhibitor. After sintering at 1000 degrees C, the particle size of nanotitania ceramics prepared by using HA as additive (HT) was much smaller than that prepared by using MgO as additive (MT). In simulated body fluid (SBF), HT could induce apatite formation in 4 days, while no apatite could be found on MT even after it was soaked in SBF for 14 days. After Ros17/28 osteoblasts were cultured on the materials for 1, 4, and 6 days, MTT results showed that the osteoblasts on the HT differentiated faster than that on the MT. Mechanical tests results showed that the bending and compressive strength of HT were 160 and 200 MPa, while those of MT were 70 and 88 MPa, respectively. These results demonstrated that it is suitable to prepare bioactive nanotitania ceramics, with biomechanical compatibility, by using HA as particle growth inhibitor.     

HA／TCP双相陶瓷与人成骨细胞生物相容性的体外实验研究

体外复合培养条件下,通过观察人成骨细胞与HA／TCP的复合生长及监测材料对细胞生理功能的影响来评价材料的生物相容性。研究发现,复合培养过程中成骨细胞首先贴附于材料的表面,进而攀附于材料边缘切刻处及材料表层微孔的边缘,以后逐渐长入孔中。最后,材料几乎为细胞所覆盖。复合培养的成骨细胞与正常培养的成骨细胞一样,具有分泌大量胶原纤维、表现强烈碱性磷酸酶活性和形成矿化胞外基质等成骨表型。细胞能够与材料很好地复合生长、材料对细胞生理功能又无明显的影响表明HA／TCP与人成骨细胞具有良好的生物相容性。     

Bone-implant interface mechanics of in vivo bio-inert ceramics

We have previously demonstrated that there was no significant difference between the affinity of bone to bio-inert ceramics and stainless steel in a histological study. In this study, the bone-implant interface shear strength of alumina ceramics (AI₂O₃), zirconia ceramics (ZrO₂), stainless steel (SUS316L) and sintered hydroxyapatite (HA) were compared in 19 dogs using a transcortical push-out model of the femur 4 and 12 wk after implantation. The interface shear strength of HA was significantly greater than that of alumina ceramics, zirconia ceramics and stainless steel (P < 0.001). There was no significant difference between bio-inert ceramics and stainless steel.     

Fabrication and cellular biocompatibility of porous carbonated biphasic calcium phosphate ceramics with a nanostructure

Microwave heating was applied to fabricate interconnective porous structured bodies by foaming as-synthesized calcium-deficient hydroxyapatite (Ca-deficient HA) precipitate containing H(2)O(2). The porous bodies were sintered by a microwave process with activated carbon as the embedding material to prepare nano- and submicron-structured ceramics. By comparison, conventional sintering was used to produce microstructured ceramics. The precursor particles and bulk ceramics were characterized by transmission electron microscopy (TEM), dynamic light scattering, scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier-transformed infrared spectroscopy (FTIR) and mechanical testing. TEM micrographs and assessment of the size distribution showed that the needle-like precursor particles are on the nanoscale. SEM observation indicated that the ceramics formed by microwave sintering presented a structure of interconnective pores, with average grain sizes of approximately 86 and approximately 167nm. XRD patterns and FTIR spectra confirmed the presence of carbonated biphasic calcium phosphate (BCP), and the mechanical tests showed that the ceramics formed by microwave sintering had a compressive strength comparable to that obtained by conventional methods. Rat osteoblasts were cultured on the three kinds of BCP ceramics to evaluate their biocompatibility. Compared with the microscale group formed by conventional sintering, MTT assay and ALP assay showed that nanophase scaffolds promoted cell proliferation and differentiation respectively, and SEM observation showed that the nanoscale group clearly promoted cell adhesion. The results from this study suggest that porous carbonated biphasic calcium phosphate ceramics with a nanostructure promote osteoblast adhesion, proliferation and differentiation. In conclusion, porous carbonated BCP ceramics with a nanostructure are simple and quick to prepare using microwaves and compared with those produced by conventional sintering, may be better bone graft materials.     

Increased osteoblast and decreased Staphylococcus epidermidis functions on nanophase ZnO and TiO2

Many engineers and surgeons trace implant failure to poor osseointegration (or the bonding of an orthopedic implant to juxtaposed bone) and/or bacteria infection. By using novel nanotopographies, researchers have shown that nanostructured ceramics, carbon fibers, polymers, metals, and composites enhance osteoblast adhesion and calcium/phosphate mineral deposition. However, the function of bacteria on materials with nanostructured surfaces remains largely uninvestigated. This is despite the fact that during normal surgical insertion of an orthopedic implant, bacteria from the patient's own skin and/or mucosa enters the wound site. These bacteria (namely, Staphylococcus epidermidis) irreversibly adhere to an implant surface while various physiological stresses induce alterations in the bacterial growth rate leading to biofilm formation. Because of their integral role in determining the success of orthopedic implants, the objective of this in vitro study was to examine the functions of (i) S. epidermidis and (ii) osteoblasts (or bone-forming cells) on ZnO and titania (TiO(2)), which possess nanostructured compared to microstructured surface features. ZnO is a well-known antimicrobial agent and TiO(2) readily forms on titanium once implanted. Results of this study provided the first evidence of decreased S. epidermidis adhesion on ZnO and TiO(2) with nanostructured when compared with microstructured surface features. Moreover, compared with microphase formulations, results of this study showed increased osteoblast adhesion, alkaline phosphatase activity, and calcium mineral deposition on nanophase ZnO and TiO(2). In this manner, this study suggests that nanophase ZnO and TiO(2) may reduce S. epidermidis adhesion and increase osteoblast functions necessary to promote the efficacy of orthopedic implants.