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

Luke G. Gutwein and Thomas J. Webster ^,

Department of Biomedical Engineering, Purdue University, 1296 Potter Building, West Lafayette, IN 47907-1296, USA

Received 27 August 2003;  accepted 21 October 2003.  Available online 9 December 2003.
Biomaterials
Volume 25, Issue 18 , August 2004, Pages 4175-4183

1. Abstract

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 
g/ml of cell culture media) for up to 6 h. 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.

Author Keywords: Author Keywords: Alumina; Titania; Apoptosis; Nanoparticle; Osteoblast; Wear debris; Orthopedic

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Particles and particle characterization

2.2. Substrates

2.3. Cell culture

2.4. Cell viability and density

2.5. Statistical analysis

3. Results

3.1. Particle characterization

3.2. Viability and osteoblast cell density in the presence of alumina particles

3.3. Viability and osteoblast cell density in the presence of titania particles

3.4. Viability and osteoblast cell density in the presence of alumina compared to titania particles

3.5. Osteoblast morphology

4. Discussion

5. Conclusions

Acknowledgements

References

3. 1. Introduction

In 1990, the total medical device sales for orthopedic implants totaled approximately 2.2 trillion dollars [1]. Since 1990, this number has been steadily growing. In fact, the 152,000 total hip replacements in 2000 is a 33% increase from the number performed in 1990 and a little over half of the projected number of total hip replacements (272,000) by the year 2030 in the United States alone [1]. Unfortunately, however, in 1997, 12.8% of the total hip arthroplasties were revisions of previously failed hip replacements [1]. The fact that such a high percentage of hip replacements performed every year are revision surgeries is not surprising when considering the life expectancy of the implant versus that of the patient receiving the implant. Consistently, over 30% of those requiring total hip replacements have been below the age of 65, yet, the longevity of orthopedic implants ranges from only about 12 to 15 years [1]. For this reason, the majority of those that receive an implant at age 65 or below will require at least one revision surgery. This suggests that the life expectancy of orthopedic prostheses is a recurring problem that has to be dealt with since clearly current approaches fail.

Motivated by this continuous need for orthopedic implant formulations with improved osseointegrative (that is, ability to bone to juxtaposed bone in situ) properties, research groups have previously designed, synthesized, and evaluated nanophase ceramic compacts with grain sizes less than 100 nm in diameter [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13]. Nanophase ceramics can be designed to possess the chemical composition, surface properties (specifically, topography), mechanical properties (namely, ductility), and grain size distribution similar to those of physiological bone (which contains about 70 wt% hydroxyapatite with grain sizes less than 100 nm [12]). In contrast, conventional materials (such as titanium and titanium alloys) do not simulate the chemical, surface, mechanical, or grain size distribution found in physiological bone [12].

In these studies, in vitro cellular models were utilized to determine the efficacy of nanophase alumina, titania, and hydroxyapatite to serve as bone prostheses [2, 3, 4, 5, 6, 7, 8, 9, 10 and 11]. Compared to respective conventional ceramic formulations, such experiments provided evidence of enhance adhesion of osteoblasts (bone-forming cells), decreased adhesion of fibroblasts (cells that contribute to fibrous encapsulation and callus formation events that may lead to implant loosening and failure), and decreased adhesion of endothelial cells (cells that line the vasculature of the body) on nanophase alumina, titania, and hydroxyapatite [4]. In fact, osteoblast adhesion increased by approximately 50% as alumina grain size decreased from 167 to 24 nm; in contrast, during the same 4-h time period, fibroblast adhesion decreased by 235% [4].

Moreover, studies provided evidence of enhanced proliferation, alkaline phosphatase activity, and concentration of calcium in the extracellular matrix when osteoblasts were cultured on nanophase compared to respective conventional (i.e., larger grain-size) alumina, titania, and hydroxyapatite formulations [10]. Specifically, three times more calcium was deposited on nanophase compared to conventional alumina after 28 days of culture [6]. Other research groups have recently confirmed these initial findings of increased functions of osteoblasts on nanophase compared to conventional ceramics [13]. Lastly, compared to respective conventional ceramic formulations, these studies also provided evidence of enhanced osteoclast (bone-resorbing cells) functions (specifically, synthesis of tartrate-resistance acid phosphatase (TRAP) and formation of bone-resorption pits) on nanophase ceramics [11]. In this manner, in vitro investigations have demonstrated enhanced functions of osteoblasts and osteoclasts which may result in the active formation and maintenance of healthy juxtaposed bone in vivo to improve orthopedic implant efficacy.

However, when subjected to physiological loading forces (such as friction), wear debris may form from articulating components of orthopedic implants composed of ceramics. Studies have shown that wear particles induce bone loss, lead to implant loosening, and sometimes result in clinical failure of bone prostheses (such as hip, elbow, knee, ankle, etc.) [14, 15, 16, 17, 18, 19 and 20]. To date, several in vitro studies have examined the response of micron-size wear particles on cell (such as osteoblasts) function [21, 22, 23, 24, 25, 26, 27 and 28]. However, few (if any) studies have been conducted on osteoblast response to nanometer-size ceramic wear particles. Therefore, the present in vitro study investigated osteoblast viability and cell density in the presence of nanophase alumina and titania particles. Information from the present study is needed to fully evaluate the efficacy of nanophase ceramics to serve as the next-generation of orthopedic implants since clearly osteoblasts would be exposed to nanometer size wear debris if these materials were ever utilized as bone prostheses.

4. 2. Materials and methods

2.1. Particles and particle characterization

Nanophase alumina (23 nm size) and titania (32 nm size) particles were obtained from Nanophase Technologies, Corp. Nanophase ceramic particles were used as supplied by the manufacturer. To obtain larger conventional size (i.e., control) ceramic particles, alumina and titania nanophase powders were separately heated (in air at 10°C/min.) to 1200°C and sintered at this temperature for 2 h. Resulting nanophase and conventional ceramic particles were sterilized by UV light exposure for 5 min prior to experiments with cells.

Nanophase and conventional ceramic particle crystallinity was examined using X-ray diffraction (Phillips type PW2273/20). Nickel filtered Cu K
radiation (
=1.5406 nm) produced at 40 kV and 35 mA was used to scan the diffraction angles (2
) between 30° and 35° at every 0.02° for 20 s/angle. Diffraction signal intensity throughout the scan was monitored and processed using Scintag (Sunnyvale, CA) DMS software.

Ceramic particle size was determined through multiple point BET measurements using an SA 3100 gas adsorption analyzer (Beckman Coulter, Inc.) according to manufacturer's instructions. Agglomeration of nanophase and conventional particles in Dulbecco's modified eagle medium (DMEM; Hyclone) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin/streptomycin (Gibco), and 0.6% geneticin (Sigma) was determined using a Beckman Coulter N4 Plus instrument (Beckman Coulter, Inc.) according to manufacturer's instructions. Particle characterization studies were done at particle concentrations of 10,000, 1000, and 100 
g/ml as well as 10,000, 5500, and 1000 
g/ml for alumina and titania, respectively. In addition, particle characterization studies were completed 5 min after adding particles to the cell culture media; particles were vortexed during this time period to ensure proper mixing.

2.2. Substrates

Borosilicate glass coverslips (Fisher Scientific) were cleaned according to standard protocols [2]. Briefly, coverslips were separately degreased and sonicated in acetone and 70% ethanol, etched in 1 
NaOH solution for 1 h, and autoclaved at 135°C for 35 min before experiments with cells.

2.3. Cell culture

Human osteoblasts (bone-forming cells; ATCC: CRL-11372) were cultured in DMEM (supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.6% geneticin). The cells were cultured under standard conditions (37°C and 5% CO₂/95% air humidification). These cells are normal (non-transformed), bone-derived, osteoblasts. Cells were used as purchased from the vendor at low population numbers 1-3. These cells have been previously characterized as determined by alkaline phosphatase activity and their ability to deposit calcium [29].

2.4. Cell viability and density

Osteoblasts were lifted from tissue-culture polystyrene using a small amount of a solution containing 0.05% trypsin (Sigma) and 0.53 EDTA (Sigma) in Hank's balanced salt solution (free of CaCl₂, MgCl₂, and MgSO₄; Gibco). The cells were then quickly suspended in cell culture media and counted using a hemacytometer. Appropriate volumes of osteoblasts in cell culture media were seeded to obtain final concentrations of 2500 cells/cm² on borosilicate glass substrates.

Cells were cultured in DMEM (supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.6% geneticin) for 48 h under standard cell culture conditions. At that time, old medium was replaced with fresh culture media supplemented with either conventional or nanophase particles at concentrations of 10,000, 1000, and 100 
g/ml as well as 10,000, 5500, and 1000 
g/ml for alumina and titania, respectively. For references, old medium was replaced with fresh medium without particles. Cells were then cultured for an additional 2 or 6 h under standard cell culture conditions.

After the allotted time, substrates were rinsed with phosphate buffered saline to remove particles and non-adherent cells. Adherent cells were then fixed and stained using the Apoptosis Detection Kit, Annexin V-Cy3 (Sigma). For some of the substrates, representative images were obtained at 40× using a fluorescent microscope with image analysis software (Image Pro). Five random fields were counted in situ per substrate under a fluorescent microscope at 10× to determine cell viability (expressed as a percentage) and to determine the total number of viable and non-viable cells (expressed as cell density/cm² of substrate surface area). All experiments were run in triplicate and repeated at least three times.

2.5. Statistical analysis

Cell viability and density experiments were analyzed statistically using standard analysis of variance (ANOVA) techniques followed by Duncan's multiple range test; statistical significance was considered at p<0.05 [30].

5. 3. Results

3.1. Particle characterization

Mean particle diameters as quoted by the supplier (Nanophase Technologies, Corp.) were confirmed at 23 and 32 nm for the alumina and titania nanophase particles, respectively (Table 1). Conventional (that is, larger than 100 nm) mean particle diameters were 179 and 4120 nm for alumina and titania, respectively. Results of the present study also provided evidence that nanophase alumina and titania formed spherical agglomerations 620 and 430 nm in diameter, respectively, in the presence of the cell culture media (Table 1). Conventional alumina and titania particles formed spherical agglomerations 5831 and 6506 nm in diameter, respectively, in the cell culture media. It is interesting to note that although dry conventional alumina and titania particle dimensions were different, once added to the cell culture media, dimensions of conventional alumina and titania agglomerations were similar. Results also provided evidence of similar crystalline phases between nanophase and conventional alumina (
) and nanophase and conventional titania particles (40% rutile/60% anatase) (Table 1). Lastly, previous studies using electron spectroscopy for chemical analysis (ESCA) revealed similar chemistries between respective nanophase and conventional alumina/titania [11]. The different particle concentrations tested in the present study did not significantly alter the above mentioned particle characterizations (specifically, size, chemistry, and crystalline phase).

Table 1. Alumina and titania particle characterization

3.2. Viability and osteoblast cell density in the presence of alumina particles

Results of the present study provided evidence of increased (p<0.05 and p<0.01) osteoblast viability when cultured in the presence of nanophase compared to conventional alumina particles at 10,000 and 100 
g/ml concentrations after 2 h, respectively (Fig. 1). For example, while only 18% of osteoblasts were viable when cultured in the presence of conventional alumina particles, this number jumped to 56% when osteoblasts were cultured with nanophase alumina particles after 2 h. Moreover, compared to references (no particles), osteoblast viability was similar when cultured with 10,000 
g/ml of nanophase alumina. In contrast, osteoblast viability was statistically (p<0.05) lower when cultured with the same concentration of conventional alumina after 2 h. Cell viability in the presence of all other concentrations of nanophase and conventional alumina particles were similar compared to references after 2 h.

(7K)

Fig. 1. Increased osteoblast viability in the presence of nanophase compared to conventional alumina particles after 2 h. Data=mean±SD; N=3; ^*p<0.05, ^**p<0.01 (compared to respective concentration of conventional particles); ^‡p<0.05 (compared to references; no particles).

In terms of cell numbers (both viable and not viable), compared to 10,000 
g/ml of conventional alumina particles, there were significantly (p<0.05) more osteoblasts in the presence of nanophase alumina at the same concentration after 2 h (Fig. 2). No other differences in cell numbers were observed between nanophase and conventional alumina after 2 h. Compared to references, there were significantly (p<0.05) less osteoblasts when cultured in the presence of any concentration of alumina (either nanophase or conventional) particles after 2 h.

(7K)

Fig. 2. Increased osteoblast numbers in the presence of nanophase compared to conventional alumina particles after 2 h. Cell numbers represent viable and non-viable cells. Data=mean±SD; N=3; ^*p<0.05 (compared to respective concentration of conventional particles); ^‡p<0.05 (compared to references; no particles).

Similar trends were observed after 6 h of culture (Fig. 3 and Fig. 4). That is, there was an increase (p<0.05) in osteoblast viability when cultured in the presence of nanophase compared to conventional alumina particles at a concentration of 10,000 
g/ml after 6 h (Fig. 3). In addition, the only statistical difference in viability when compared to references was when osteoblasts were cultured with conventional alumina at 10,000 
g/ml; for that data point, osteoblast viability was significantly (p<0.05) less. For both viable and non-viable cell numbers after 6 h, significantly (p<0.05) more osteoblasts were present when cultured with 10,000 
g/ml of nanophase compared to conventional alumina particles (Fig. 4). All cell numbers were statistically (p<0.05) less than references except for osteoblasts cultured with 100 
g/ml of nanophase alumina which was similar to references.

(7K)

Fig. 3. Increased osteoblast viability in the presence of nanophase compared to conventional alumina particles after 6 h. Data=mean±SD; N=3; ^*p<0.05 (compared to respective concentration of conventional particles); ^‡p<0.05 (compared to references; no particles).

(5K)

Fig. 4. Increased osteoblast numbers in the presence of nanophase compared to conventional alumina particles after 6 h. Cell numbers represent viable and non-viable cells. Data=mean±SD; N=3; ^*p<0.05 (compared to respective concentration of conventional particles); ^‡p<0.05 (compared to references; no particles).

When comparing 2-6 h osteoblast viability was similar for all experimental conditions except when cultured in the presence of 10,000 
g/ml of conventional alumina particles (Fig. 1 and Fig. 3). That is, osteoblast viability only significantly (p<0.05) decreased when cultured in the presence of 10,000 
g/ml of conventional alumina particles from 2 to 6 h. In contrast, osteoblast numbers (both viable and non-viable) were significantly (p<0.05) less from 2 to 6 h in the presence of both nanophase and conventional alumina at the 10,000 
g/ml particle concentration (Fig. 2 and Fig. 4). For all other concentrations, osteoblast numbers were similar from 2 to 6 h.

3.3. Viability and osteoblast cell density in the presence of titania particles

Similar to alumina, for titania, significantly (p<0.05) more viable osteoblasts were observed when cultured with 10,000 
g/ml of nanophase compared to conventional titania after 2 h (Fig. 5). Compared to conventional titania particles, osteoblast viability in the presence of all other respective concentrations of nanophase titania particles were similar after 2 h. The only experimental condition in which similar numbers of viable osteoblasts were determined compared to references was in the presence of 1000 
g/ml of either nanophase or conventional titania particles after 2 h; all other experimental conditions were significantly (p<0.05) less than references.

(6K)

Fig. 5. Increased osteoblast viability in the presence of nanophase compared to conventional titania particles after 2 h. Data=mean±SD; N=3; ^*p<0.05 (compared to respective concentration of conventional particles); ^‡p<0.05 (compared to references; no particles).

Also similar to alumina, significantly (p<0.05) more osteoblasts (both viable and non-viable) were counted when cultured with 10,000 
g/ml of nanophase compared to conventional titania particles after 2 h (Fig. 6). All cell numbers were significantly (p<0.05) less than references.

(5K)

Fig. 6. Increased osteoblast numbers in the presence of nanophase compared to conventional titania particles after 2 h. Cell numbers represent viable and non-viable cells. Data=mean±SD; N=3; ^*p<0.05 (compared to respective concentration of conventional particles); ^‡p<0.05 (compared to references; no particles).

After 6 h, however, there were no statistical differences in osteoblast viability when cultured in the presence of nanophase compared to conventional titania at any concentration (Fig. 7). Compared to references, the number of viable osteoblasts was statistically (p<0.05) less when cultured with either 10,000 or 5500 
g/ml of nanophase and conventional titania (Fig. 7). For both nanophase and conventional titania, the number of viable osteoblasts at the 1000 
g/ml concentration was similar to references.

(5K)

Fig. 7. Similar osteoblast viability in the presence of nanophase and conventional titania particles after 6 h. Data=mean±SD; N=3; ^‡p<0.05 (compared to references; no particles).

The number of osteoblasts (both viable and non-viable) was greater (p<0.05) when cultured in the presence of nanophase compared to conventional titania at 10,000 and 5500 
g/ml particle concentrations after 6 h (Fig. 8). Osteoblast numbers were similar when comparing nanophase to conventional titania particles at the 1000 
g/ml concentration after 6 h. Compared to references, the only statistically similar number of osteoblasts was when cultured in the presence of 1000 
g/ml of nanophase titania; significantly (p<0.05) less osteoblasts were present for all other experimental conditions after 6 h.

(5K)

Fig. 8. Increased osteoblast numbers in the presence of nanophase compared to conventional titania particles after 6 h. Cell numbers represent viable and non-viable cells. Data=mean±SD; N=3; ^*p<0.05 (compared to respective concentration of conventional particles); ^‡p<0.05 (compared to references; no particles).

Comparing 2 and 6 h, osteoblast viability (Fig. 5 and Fig. 7) and the number of osteoblasts ( Fig. 6 and Fig. 8) were similar when cultured with any concentration of nanophase and conventional titania of interest to the present study.

3.4. Viability and osteoblast cell density in the presence of alumina compared to titania particles

Lastly, in general, osteoblast viability was much greater (p<0.05) when cultured in the presence of any concentration of either conventional or nanophase alumina compared to respective titania experimental conditions after 2 h (Fig. 1 and Fig. 5). The only exception to this rule was the similar osteoblast viability in the presence of 1000 
g/ml of nanophase and conventional alumina compared to the respective concentration of nanophase and conventional titania after 2 h (Fig. 1 and Fig. 5). After 6 h, osteoblast viability was similar between conventional alumina and conventional titania for all concentrations of interest to the present study (Fig. 3 and Fig. 7). In contrast, osteoblast viability was significantly (p<0.05) greater in the presence of nanophase alumina compared to nanophase titania for all concentrations after 6 h (Fig. 3 and Fig. 7).

Similar cell numbers (both viable and non-viable) were found for respective concentrations of nanophase and conventional alumina compared to nanophase and conventional titania after 2 h (Fig. 2 and Fig. 6) and 6 h (Fig. 4 and Fig. 8).

3.5. Osteoblast morphology

The present study not only provided evidence of differences in osteoblast viability and density, but also in osteoblast morphology depending on whether they were cultured in the presence of nanophase or conventional ceramic particles. For each concentration and time point in the present study, increased cell spreading was observed when osteoblasts were cultured in the presence of nanophase compared to conventional ceramics. As an example, osteoblasts morphology is illustrated in the presence of 100 and 1000 
g/ml of alumina and titania particles, respectively, in Fig. 9. Ceramic particles (which autofluoresce) are indicated by arrows in Fig. 9. As can be seen, osteoblast spreading increased when cultured in the presence of nanophase alumina ( Figs. 9c and d) compared to conventional alumina ( Figs. 9e and f) after 2 and 6 h. Similarly, osteoblast spreading increased when cultured in the presence of nanophase titania (Figs. 9g and h) compared to conventional titania after 2 and 6 h (Figs. 9i and j). Osteoblast morphology was more similar to that of references (no particles; Figs. 9a and b) when cultured in the presence of nanophase ceramics.

(40K)

Fig. 9. Representative fluorescent images of osteoblasts in the presence of nanophase and conventional ceramic particles. Magnification=40× original magnification. Arrows indicate particles.

6. 4. Discussion

Aseptic loosening is a frequent complication of orthopedic implants (such as hip, knee, etc.) [12, 16, 20 and 31]. Unsuitable fixation of an implant to juxtaposed bone could be caused by poor cementing technique during surgical implantation or insufficient osseointegration post-implantation [12]. Moreover, stress and strain imbalances around the biomaterial-bone interface during physiological loading conditions could lead to bone resorption, implant loosening, and clinical failure [32]. These events may contribute to malpositioning of the implant components and subsequent generation of debris through friction of articulating surfaces; such wear debris has been associated with osteolysis and, frequent, implant failure [16, 17, 18, 19, 20 and 31].

Wear debris concentrations, as determined by the average volume of wear from a metal/ultra high molecular weight polyethylene (UHMWPE) Charnely articulating hip joint, can range from 33.8 to 77.6 mm³/year [31]. For this purpose, by assuming a middle point of 50 mm³/year, a volume of an implant juxtaposed to tissue of approximately 100 mm³, and a 0.1 g mass of that implant, ceramic wear debris concentrations of 18,250 
g particles/ml for a period of 1 day can be obtained. These values are typically used in studies (including this one) elucidating effects of wear debris on functions of osteoblasts.

Moreover, since conventional implants possess grain sizes in the micron regime, wear particles in the range of 1-100 
m in diameter have also frequently been used in cell culture simulated wear debris studies. These reports highlight the influence of micron particle size, chemistry (i.e., polymer compared to ceramic), and shape on bone cell functions [23, 25, 26, 27, 28, 33 and 34]. For example, reports in the literature demonstrated a slight decrease in osteoblastic cell growth in the presence of 80 
m diameter
-alumina particles compared to 80 
m diameter polyethylene particles [33]. More importantly, studies have consistently correlated decreased osteoblast cell proliferation with smaller diameters of conventional (from 1 to 100 
m diameter) wear particles regardless of chemistry (for example, in alumina, polyethylene, titania, tantalum, etc.) [23, 25 and 28]; numerous reports indicate that wear debris between 1 and 5 
m in diameter initiate the most aggressive biological response [33, 35 and 36].

The present study confirmed literature reports of the detrimental effects of micron size particles on osteoblast viability (leading to apoptosis) and cell density [33 and 35]. However, the present study demonstrated, for the first time, that by decreasing alumina and titania particle diameters into the nanometer regime, the negative effects of particle size on osteoblast viability and cell density is decreased. That is to say, compared to respective conventional ceramic particles, this in vitro study provided the first evidence that viable osteoblast density was greater when cultured in the presence of either nanometer size alumina or titania particles. Furthermore, since a key objective in the present study was to utilize nanophase and conventional particles that varied only in size (that is, chemistry and material phase were similar), results of viable osteoblast cell densities when exposed to either alumina or titania particles were dependent only on the size of the ceramic particles. If formation of wear debris is unavoidable, such information of a less detrimental influence of nanophase ceramic particles on viable osteoblast densities implies that more attention should be given to the design of materials that generate nanometer—not micron-sized particulates; nanophase ceramics fulfill such a requirement.

7. 5. Conclusions

In summary, compared to conventional particle size alumina and titania compacts, reports in the literature show that nanophase (that is, those with particle sizes less than 100 nm in diameter) ceramic compacts possess a topography that selectively enhances osteoblast function leading to increased deposition of calcium-containing mineral [6]. The present study is the first of its kind to investigate osteoblast viability and density in the presence of alumina as well as titania nanometer-dimensioned particulates. Results of a more well-spread morphology and increased viable cell densities in the presence of nanophase alumina and titania particles, demonstrates that wear debris resulting from bone prostheses composed of nanophase ceramics may less adversely affect bone cell function compared to larger conventional ceramic wear particles. In this manner, the present study provided additional evidence that orthopedic implants composed of nanophase ceramics have the potential to become the next generation of bone prostheses with increased efficacy and thus deserve further investigation.

8. Acknowledgements

The authors of the present study would like to thank Ms. Rachel Price for technical assistance; Nanophase Technologies, Corp. for nanophase alumina and titania particles; as well as the Whitaker Foundation and the SURF program at Purdue University for financial support.

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