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Biomaterials 21 (2000) 1803 } 1810

Enhanced functions of osteoblasts on nanophase ceramics


Thomas J. Webster , Celaletdin Ergun , Robert H. Doremus , Richard W. Siegel , Rena Bizios *
Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180-3590, USA Department of Materials Science and Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180-3590, USA Received 18 January 2000; accepted 1 March 2000

Abstract Select functions of osteoblasts (bone-forming cells) on nanophase (materials with grain sizes less than 100 nm) alumina, titania, and hydroxyapatite (HA) were investigated using in vitro cellular models. Compared to conventional ceramics, surface occupancy of osteoblast colonies was signi"cantly less on all nanophase ceramics tested in the present study after 4 and 6 days of culture. Osteoblast proliferation was signi"cantly greater on nanophase alumina, titania, and HA than on conventional formulations of the same ceramic after 3 and 5 days. More importantly, compared to conventional ceramics, synthesis of alkaline phosphatase and deposition of calcium-containing mineral was signi"cantly greater by osteoblasts cultured on nanophase than on conventional ceramics after 21 and 28 days. The results of the present study provided the "rst evidence of enhanced long-term (on the order of days to weeks) functions of osteoblasts cultured on nanophase ceramics; in this manner, nanophase ceramics clearly represent a unique and promising class of orthopaedic/dental implant formulations with improved osseointegrative properties. 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Nanoceramics; Orthopaedic/dental; Osteoblasts; Cell proliferation; Alkaline phosphatase; Mineralization

1. Introduction Clinical complications with prosthetic devices are often due to insu$cient bonding to juxtaposed bone (i.e., lack of osseointegration) [1,2]. Osseointegration is needed to stabilize orthopaedic/dental prostheses in situ, to minimize motion-induced damage to surrounding tissues, and to increase overall implant e$cacy. Insu$cient bonding of juxtaposed bone to an orthopaedic/dental implant could be caused by material surface properties that do not support new bone growth. For this reason, synthesis of biomaterial surface properties which support osseointegration should be one of the key objectives in the design of the next generation of orthopaedic/dental implants. Recent in vitro studies of osteoblast functions on glass and titanium surfaces modi"ed with immobilized speci"c peptide sequences (for example, arginine}glycine}aspartic acid}serine (RGDS), lysine}arginine}serine}arginine

* Corresponding author. Tel.: 1-518-276-6964; fax: 1-518-276-3035. E-mail address: bizios@rpi.edu (R. Bizios).

(KRSR), etc. [3}6]) have reported enhanced cell adhesion, proliferation and deposition of calcium-containing mineral. Upon implantation, bioactivity of peptide sequences immobilized on biomaterials, however, may be a!ected because of macromolecular interactions. To date, only few attempts to design a biomaterial surface that (without peptide immobilization) elicits speci"c, timely and desirable responses from osteoblasts have been reported. The objective of the present study was to explore an alternative approach to produce novel biomaterials that, without peptide immobilization, promotes osteoblast functions. Ceramics possess exceptional biocompatibility properties with bone cells and tissues [7}10]. The cytocompatibility of conventional ceramics with grain sizes greater than 100 nm is well established [7}10]. In comparison, the biocompatibility of nanophase ceramics with grain sizes less than 100 nm is only, at best, partially understood [11}14]. Compared to respective conventional ceramic formulations, select, enhanced adhesion of osteoblasts has been observed on nanophase alumina, titania, and hydroxyapatite [11}14]. The mechanism(s) of these responses involve proteins (such as vitronectin [14]) since, in the absence of serum, osteoblast adhesion

0142-9612/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 0 7 5 - 2

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was greatly reduced and independent of ceramic grain size [11}14]. Adhesion of osteoblasts to material surfaces alone, however, is not adequate to achieve long-term osseointegration of orthopaedic/dental implants; subsequent osteoblast functions are required. For this reason, the present study investigated long-term (in the order of days to weeks) functions of osteoblasts on nanophase ceramics and provided the "rst evidence of enhanced osteoblast proliferation, alkaline phosphatase synthesis, and concentration of extracellular matrix calcium on nanophase alumina, titania, and hydroxyapatite.

2.2. Substrate surface characterization The grain sizes of the alumina, titania, and hydroxyapatite of interest to the present study were determined by atomic force microscopy (using an Autoprobe CO Atomic Force Microscope) with image analysis software (Pro-scan version 1.5b) [12]. The average surface grain size of the as-pressed alumina compacts was 24 (nanophase) and 167 (conventional) nm when sintered at 1000, and 12003C, respectively. The average surface grain size of the as-pressed titania compacts was 39 (nanophase) and 4520 (conventional) nm when sintered at 600 and 12003C, respectively. The average surface grain size of the nanophase and conventional HA compact formulations was 67 (nanophase) and 179 (conventional) nm, respectively. 2.3. Cell cultures Osteoblasts were isolated via sequential collagenase digestions of neonatal rat calvaria according to established protocols [16] and cultured in Dulbecco's modi"ed Eagle medium (DMEM; Gibco), supplemented with 10% fetal bovine serum (Gibco) in standard cell culture conditions (that is, a 373C, humidi"ed, 5% CO /95% air environment). The osteoblastic  phenotype of these cells was determined by alkaline phosphatase activity and formation of calcium-containing mineral deposits in the extracellular matrix. Osteoblasts at population numbers 2}4 were used in the experiments. 2.4. Cell proliferation Osteoblasts in DMEM (supplemented with 10% fetal bovine serum) were seeded (2500 cells/cm) onto the substrates of interest to the present study. The cells were then cultured in DMEM (supplemented with 10% fetal bovine serum) under standard cell conditions for 1, 3, and 5 days. At the end of the prescribed time periods, osteoblasts were "xed in situ with 4% formaldehyde for 10 min. Osteoblasts cultured on opaque conventional (control substrates) and nanophase ceramics were stained with Hoechst (No. 33258; Sigma) #uorescent dye; the cell nuclei were thus visualized and the number of cells in each of "ve random "elds per substrate were counted using a #uorescence (365 nm excitation; 400 nm emission) microscope with image analysis software (Image Pro). Osteoblasts cultured on glass (reference substrate) were stained in situ with Commassie Brilliant Blue staining solution; adherent cells (on each of "ve random "elds per substrate) were thus visualized and counted using a light microscope. The average cell count per substrate was expressed as &cell density' or cells/cm of substrate surface area.

2. Materials and methods 2.1. Ceramic substrate synthesis and preparation Alumina (Al O ) and titania (TiO ) samples (discs    10 mm in diameter and 2 mm thick) were prepared as previously described [11}14]. Brie#y, nanophase alumina ( -phase; Nanophase Technologies Corporation) and titania (40% rutile and 60% anatase; Nanophase Technologies Corporation) powder were separately compacted in a tool-steel die via a uniaxial pressing cycle (0.2}1 GPa over a 10 min period). Ceramic grain size was controlled by changing alumina and titania compact sintering temperatures; nanophase alumina and titania compacts were sintered at 1000 and 6003C, respectively, to obtain "nal grain sizes less than 100 nm. Alumina and titania compacts were sintered at 12003C to obtain conventional "nal grain sizes greater than 100 nm. Conventional grain size alumina and titania samples served as control substrates. Hydroxyapatite (HA;Ca (PO ) (OH)) compacts (10 mm   in diameter and 2 mm thick) were prepared via wet chemistry techniques adopted from previously published methods [15]. HA grain size was controlled by changing the time and temperature of HA precipitation; speci"cally, the HA containing solution was stirred either at room temperature for 24 h to obtain grain sizes less than 100 nm (i.e., nanophase grain size HA) or at 903C for 3 h to obtain grain sizes greater than 100 nm (i.e., conventional grain size HA). Each HA-containing solution was then centrifuged, "ltered, dried at 603C for 8 h, heated in air at 103C/min from room temperature to a "nal temperature of 11003C, and sintered at this temperature for 60 min. Borosilicate glass coverslips (reference material) were etched in 1 N NaOH and prepared for experiments with cells according to standard protocols [16]. All ceramic samples were degreased, ultrasonically cleaned and sterilized (in a steam autoclave at 1203C for 30 min) according to standard laboratory procedures [16].

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2.5. Surface occupancy by osteoblast colonies Sterile Te#on fences were placed on the substrates of interest to the present study and covered the entire substrate surface except for a central circular area (3/16 inch diameter). Osteoblasts (25 000 cells) were seeded in the central well of the fences in DMEM (supplemented with 10% fetal bovine serum) and were allowed to adhere under standard cell culture conditions for 4 h. At that time, the fences were removed carefully (to avoid disturbing the adhering cells) and the medium in the wells was aspirated. The con#uent cell colony was gently rinsed with phosphate-bu!ered saline (to remove non-adherent cells) and was then cultured in DMEM (supplemented with 10% fetal bovine serum) under standard cell culture conditions for 2, 4 and 6 days. At the end of the prescribed time periods, cells adherent on glass (reference substrate) were "xed in situ with 4% formaldehyde for 10 min, stained with Commassie Brillant Blue, and visualized using light microscopy. Cells adherent on conventional (control substrates) and nanophase opaque ceramic substrates were "xed in situ with 4% formaldehyde for 10 min, stained with Hoechst 33258, and visualized using #uorescence (365 nm excitation; 400 nm emission) microscopy. Image Pro was utilized to determine the total surface area (expressed as cm) occupied by each cell colony. Surface areas occupied by osteoblast colonies on conventional ceramics for the same time periods served as respective controls. 2.6. Total intracellular protein content Osteoblasts (40 000 cells/cm) were seeded onto the substrates of interest to the present study and cultured in DMEM (supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbate (Sigma) and 10 mM -glycerophosphate (Sigma)) under standard cell culture conditions for 7, 14, 21, and 28 days. At the end of the prescribed time periods, osteoblasts were lysed using distilled water and three freeze}thaw cycles. Total protein content in the cell lysates was determined spectrophotometrically using a commercially available kit (Pierce Chemical Co.) and following manufacturer's instructions. For this purpose, aliquots of each protein-containing, distilled-water supernatant were incubated with a solution of copper sulfate and bicinchoninic acid at 373C for 30 min. Light absorbance of these samples was measured at 570 nm on a MR600 Spectrophotomectric Microplate reader (Dynatech). Total intracellular protein (expressed as mg) synthesized by osteoblasts cultured on the substrates of interest to the present study was determined from a standard curve of absorbance versus known concentrations of albumin run in parallel with experimental samples. Total intracellular protein synthesized by osteoblasts cultured on conventional ceramics served as controls.

2.7. Alkaline phosphatase activity The method of Lowry (1954) was used to assay alkaline phosphatase activity in the cell lysates (prepared as described in the total intracellular protein content section) [17]. For this purpose, aliquots (100 l) of the distilled water supernatants were incubated with 500 l of reaction solution (containing 0.6 M 2-amino-2-methyll-propanol (pH"10.0), and 10 mM p-nitrophenylphosphate) solution (Diagnostic Kit C104; Sigma) at 373C for 30 min; the reaction of p-nitrophenol conversion to p-nitrophenylate was stopped by adding 1.5 ml of 0.25 N NaOH [18]. Light adsorbance of these samples was measured on a spectrophotometer (MR600 Spectrophotometric Microplate Reader; Dynatech) at 400 nm. The alkaline phosphatase activity (expressed as nano-moles of converted p-nitrophenol/min) was normalized by total intracellular protein synthesis (determined as described in the total intracellular protein content section) and expressed as nano-moles of converted p-nitrophenol/min/mg protein. Alkaline phosphatase activity of osteoblasts cultured on conventional ceramics served as controls. 2.8. Quantixcation of extracellular calcium The substrates of interest to the present study, seeded either with or without osteoblasts (40 000 cells/cm), were cultured in DMEM (supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbate (Sigma) and 10 mM -glycerophosphate (Sigma)) under standard cell culture conditions for 7, 14, 21, and 28 days. At that time, cells were lysed for the purpose described in the total intracellular protein content section. The extracellular matrix of each preparation of interest to the present study was treated with 0.6 N HCl at 373C overnight. After the prescribed time period, the amount of calcium present in the acidic supernatant was spectrophotometrically quanti"ed using a commerically available kit (Calcium Quanti"cation Kit C587-A; Sigma) and following manufacturer's instructions; light adsorbance of the samples was measured spectrophotometrically at 575 nm. Total calcium ( g/dl) was calculated from standard curves of adsorbance versus known concentrations of calcium measured in parallel with the experimental samples. Calcium concentration values were normalized by total protein synthesis (determined as described in the Total Intracellular Protein Content section); "nally, these results were reported as g of calcium/mg protein. In the case of calcium deposited on HA substrates, calcium concentration from HA samples maintained in acellular conditions was subtracted from all experimental values. 2.9. Statistical analysis Experiments were run in triplicate and repeated at three di!erent times per substrate. Numerical data were

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analyzed using standard analysis of variance (ANOVA) techniques; statistical signi"cance was considered at P(0.01.

greater on nanophase HA after 3 and 5 days of culture (Fig. 1c). 3.2. Osteoblast colony occupancy

3. Results 3.1. Osteoblast proliferation Compared to respective conventional formulations of the same ceramic and to borosilicate glass (reference substrate), osteoblast proliferation increased on all nanophase ceramics tested in the present study (Fig. 1a}c). Speci"cally, the proliferation of osteoblasts was signi"cantly (P(0.01) greater on nanophase, than on conventional, alumina (Fig. 1a) and titania (Fig. 1b) after 1, 3 and 5 days of culture. Osteoblast proliferation was similar on nanophase and on conventional HA after 1 day of culture (Fig. 1c), but was signi"cantly (P(0.01)

The present study provided evidence of decreased surface occupancy by osteoblasts cultured on nanophase ceramics than on respective conventional ceramic formulations and on borosilicate glass (reference substrate) (Fig. 2a}c). Speci"cally, compared to surface occupancy on borosilicate glass and on conventional titania, surface occupancy by osteoblast colonies cultured on nanophase titania was signi"cantly (P(0.01) less after all time periods tested (Fig. 2b); surface occupancy of osteoblasts cultured on nanophase alumina and HA was signi"cantly (P(0.01) less compared to respective conventional ceramic and borosilicate glass after 4 and 6 days of culture (Fig. 2a and c). Moreover, osteoblasts covered twice as large a surface area on conventional than on

Fig. 1. Osteoblast proliferation on ceramics. Rat calvarial osteoblasts in Dulbecco's modi"ed Eagle medium (supplemented with 10% fetal bovine serum) were seeded (2500 cells/cm) on the following substrates: (a) borosilicate glass (reference material), 167 nm grain size alumina (conventional), and 24 nm grain size alumina (nanophase); (b) borosilicate glass (reference material), 4520 nm grain size titania (conventional), and 39 nm grain size titania (nanophase); and (c) borosilicate glass (reference material), 179 nm grain size hydroxyapatite (conventional), and 67 nm grain size hydroxyapatite (nanophase). Osteoblast proliferation under standard cell culture conditions (373C, humidi"ed, 5% CO /95% air  environment) was determined after 1, 3, and 5 days. Values are mean$SEM; n"3; * P(0.01 (compared to respective conventional grain size ceramic); ? P(0.01 (compared to borosilicate glass).

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Fig. 2. Osteoblast colony surface occupancy on ceramics. Surface occupancy by colonies of rat calvarial osteoblasts in Dulbecco's modi"ed Eagle medium (supplemented with 10% fetal bovine serum) was determined on the following substrates: (a) borosilicate glass (reference material), 167 nm grain size alumina (conventional), and 24 nm grain size alumina (nanophase); (b) borosilicate glass (reference material), 4520 nm grain size titania (conventional), and 39 nm grain size titania (nanophase); and (c) borosilicate glass (reference material), 179 nm grain size hydroxyapatite (conventional), and 67 nm grain size hydroxyapatite (nanophase). Osteoblast colony surface occupancy under standard cell culture conditions (373C, humidi"ed, 5% CO /95% air environment) was determined after 2, 4, and 6 days. Values are mean$SEM; n"3; * P(0.01  (compared to respective conventional grain size ceramic); ? P(0.01 (compared to borosilicate glass).

respective nanophase ceramic formulations after 6 days of culture. 3.3. Synthesis of alkaline phosphatase

lations, respectively. The highest (P(0.01) alkaline phosphatase activity was observed on nanophase titania (Fig. 3a}c). 3.4. Calcium content in the extracellular matrix

There were no detectable amounts of alkaline phosphatase activity by osteoblasts cultured on all substrates tested in the present study after 7 days and on titania, HA, and borosilicate glass after 14 days of culture. In contrast, alkaline phosphatase activity was signi"cantly (P(0.01) greater on nanophase alumina after 14 days of culture. Alkaline phosphatase activity was also signi"cantly (P(0.01) greater when osteoblasts were cultured on nanophase alumina, titania, and HA than on conventional formulations of the respective ceramics after 21 and 28 days of culture (Fig. 3a}c). Speci"cally, at 28 days of culture, synthesis of alkaline phosphatase by osteoblasts on nanophase alumina, titania, and HA was 36, 22, and 37% greater than on conventional ceramic formu-

For any time period tested, there were no detectable amounts of calcium deposited on the substrates of interest to the present study cultured in an acellular environment (data not shown). In addition, there were no detectable amounts of calcium in the extracellular matrix after 7 days of osteoblast culture on any substrate tested in the present study (Fig. 4a}c); after 14 days, calcium was only detected in the extracellular matrix of osteoblasts cultured on nanophase alumina. Calcium content in the extracellular matrix on all nanophase ceramics tested in the present study was signi"cantly (P(0.01) greater than on respective conventional ceramic formulations after 21 and 28 days of culture (Fig. 4a}c); speci"cally,

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Fig. 3. Alkaline phosphatase activity of osteoblasts cultured on ceramics. Rat calvarial osteoblasts in Dulbecco's modi"ed Eagle medium (supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbate and 10 mM -glycerophosphate) were cultured on the following substrates: (a) borosilicate glass (reference material), 167 nm grain size alumina (conventional), and 24 nm grain size alumina (nanophase); (b) borosilicate glass (reference material), 4520 nm grain size titania (conventional), and 39 nm grain size titania (nanophase); and (c) borosilicate glass (reference material), 179 nm grain size hydroxyapatite (conventional), and 67 nm grain size hydroxyapatite (nanophase). Alkaline phosphatase activity (nmol p-nitrophenol/min/mg protein) was determined after 7, 14, 21, and 28 days. Values are mean$SEM; n"3; * P(0.01 (compared to respective conventional grain size ceramic); ? P(0.01 (compared to borosilicate glass).

after 28 days, the content of calcium in the extracellular matrix of osteoblasts cultured on nanophase alumina, titania, and HA was 4, 6, and 2 times greater than on respective conventional ceramics. Moreover, compared to nanophase HA, the calcium content in the extracellular matrix was signi"cantly (P(0.01) greater after 28 days of cell culture on nanophase alumina and titania; calcium content was similar on nanophase alumina and titania during this time interval (Fig. 4a}c).

4. Discussion Compared to metals and metal alloys (such as titanium and titanium alloys), conventional ceramics (such as alumina and hydroxyapatite of grain sizes greater than 100 nm) utilized for orthopeadic/dental implants have long been appreciated for their biocompatibility [7}10]; these material formulations, however, do not represent the ideal orthopaedic/dental implant due to insu$cient bonding of juxtaposed bone, which often leads to clinical failure [19}25]. Clearly, novel material formulations that promote and sustain osseointegration of surrounding

bone are needed to improve implant e$cacy. The results of the present in vitro study provided the "rst evidence of enhanced long-term functions (speci"cally, cell proliferation, synthesis of alkaline phosphatase, and concentration of calcium in the extracellular matrix) of osteoblasts on nanoceramics, that is ceramic formulations with grain sizes less than 100 nm. Initial events during cell-biomaterial interactions (such as cell adhesion and concomitant morphology) a!ect long-term functions, such as cell population motility, proliferation, and synthesis of extracellular matrix proteins of anchorage-dependent cells [26}29]. The dynamics of cell migration require continuous forming and breaking of focal contacts at the proximal and distal sides of the cell, respectively. Cell migration is inhibited on substrate surfaces that promote cell adhesion; in other words, contractile forces necessary for cell migration cannot overcome the strength of cell contact points formed on highly adhesive substrate surfaces [30,31]. Surface occupancy experiments have been utilized as an index of cell population motility [26]. The present results of decreased surface occupancy by osteoblasts cultured on nanophase ceramics are in agreement with our previous

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Fig. 4. Extracellular calcium deposited by osteoblasts cultured on ceramics. Rat calvarial osteoblasts in Dulbecco's modi"ed Eagle medium (supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbate and 10 mM -glycerophosphate) were cultured on the following substrates: (a) borosilicate glass (reference material), 167 nm grain size alumina (conventional), and 24 nm grain size alumina (nanophase); (b) borosilicate glass (reference material), 4520 nm grain size titania (conventional), and 39 nm grain size titania (nanophase); and (c) borosilicate glass (reference material), 179 nm grain size hydroxyapatite (conventional), and 67 nm grain size hydroxyapatite (nanophase). Extracellular calcium concentration ( g calcium/mg protein) was determined after 7, 14, 21, and 28 days. Values are mean$SEM; n"3;* P(0.01 (compared to respective conventional grain size ceramic); ? P(0.01 (compared to borosilicate glass).

studies demonstrating enhanced osteoblast adhesion on these novel ceramic surfaces [11}14]. Increased osteoblast adhesion coupled with decreased cell motility, as well as enhanced proliferation, synthesis of alkaline phosphatase and deposition of calcium-containing mineral has been observed on surfaces (for example, borosilicate glass and titanium) modi"ed with immobilized peptide sequences (such as arginine}glycine} aspartic acid}serine (RGDS) and lysine}arginine}serinearginine (KRSR) [3}5]) contained in extracellular matrix proteins such as vitronectin and collagen. Nanophase ceramics, as the present study proved for the "rst time, represent novel material formulations that without peptide immobilization enhance these functions of osteoblasts. For this reason, nanophase ceramics clearly represent a unique class of material formulations that promise enhanced bonding of orthopaedic/dental implants to juxtaposed bone and thus improved overall implant e$cacy.

Acknowledgements The authors would like to thank Dr. Abigail SnyderKeller (Wadsworth Center, New York State Department of Health) as well as Ms. Carol Charniga and Dr. Harry Kimmelberg (Division of Neurosurgery Research Laboratory, Albany Medical College) for neonatal rat calvaria, the source of the osteoblasts used in the present study. The contribution of nanophase alumina and titania powder by Nanophase Technologies Corporation is also acknowledged.

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