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Acta Biomaterialia 3 (2007) 523–530 www.elsevier.com/locate/actabiomat Bone cell–materials interaction on Si microchannels with bioinert coatings a,b Russell Condie , Susmita Bose a, Amit Bandyopadhyay a,* a W.M. Keck Biomedical Materials Research Laboratory, School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164-2920, USA b Department of Bioengineering, University of Utah, Salt Lake City, UT, USA Received 20 April 2006; received in revised form 30 October 2006; accepted 7 November 2006 Abstract Bone implant life is dependent upon integration of biomaterial surfaces with local osteoblasts. This investigation studied the eﬀects of various microchannel parameters and surface chemistry on immortalized osteoblast precursor cell (OPC1) adhesion. Cell–materials inter- actions were observed within channels of varying length, width, tortuosity, convergence, divergence and chemistry. Si wafers were used to create four distinct 1 cm2 designs of varying channel dimensions. After anisotropic chemical etching to a depth of 120 lm, wafers were sputter coated with gold and titanium; and on another surface SiO2 was grown to vary the surface chemistry of these microchannels. OPC1 cells were seeded in the central cavity of each chip before incubation in tissue culture plates. On days 5, 11 and 16, samples were taken out, ﬁxed and processed for microscopic analysis. Samples were visually characterized, qualitatively scored and analyzed. Channel walls did not contain OPC1 migration, but showed locally interrupted adhesion. Scores for channels of ﬂoor widths as narrow as 350 lm were signiﬁcantly reduced. No statistically signiﬁcant preference was detected for gold, titanium or SiO2 surfaces. Bands of OPC1 cells appeared to align with nearby channels, suggesting that cell morphology may be controlled by topography of the design to improve osseointegration. Ó 2006 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Osteoblast adhesion; Osseointegration; Microchannel; Bone tissue engineering 1. Introduction causing eventual wear, inﬂammation and failure . How- ever, engineered biomaterial surfaces may improve implant Musculoskeletal disorders and bone deﬁciencies have stability by promoting cell adhesion, proliferation and dif- been established as being among the most important ferentiation. Various methods of improving osseointegra- human health conditions that exist today, costing more tion have been attempted. Meyer et al. identiﬁed several than $16 billion in products in 2006, and aﬄicting one in material physico-chemical properties that promote osteo- seven Americans . The success and lifetime of an implant blast adhesion . Dense, inert materials, such as Au, Ti, is largely dependent on the degree of osseointegration at SiO2 and steels, have relatively low bioactivity and rely pri- the material–bone interface, especially in load-bearing marily on morphological ﬁxation or press ﬁtting to bone orthopedic or dental applications [2,3]. A layer of ﬁbrous tissue [6–8]. Biological ﬁxation in porous materials and or soft tissues can accumulate at the surfaces of implant chemical ﬁxation in bioactive materials reduce soft-tissue materials . This may result in a modulus mismatch and encapsulation . Resorbable ceramics may further allow micromotion to disrupt integration with local bone, improve osseointegration if the rate of dissolution can be matched to that of tissue formation . * Corresponding author. Tel.: +1 509 335 4862. Nano- and micro-scale topography has been shown to E-mail address: email@example.com (A. Bandyopadhyay). inﬂuence cell–materials interactions [2,4]. Titanium sur- 1742-7061/$ - see front matter Ó 2006 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2006.11.001 524 R. Condie et al. / Acta Biomaterialia 3 (2007) 523–530 faces textured with 100 lm cavities were shown to favor A B osteoblast attachment and growth, while submicron-scale etching promoted diﬀerentiation . Bone tissue engineer- 2.5 mm [--------] ing on patterned collagen ﬁlms coated with calcium phos- phates and ﬁbrinogen showed preferential osteoblast alignment and orientation along the axis of parallel grooves . Similar experiments have shown osteoblasts alignment, elongation and migration along parallel grooves on other surfaces as well [11,12]. However, research on C D bone cell–materials interaction along complex channels 500 μm |-| [-------------- 1.0 cm -------------] does not appear to have been studied well. Our current work investigates the hypothesis that immortalized osteoblast precursor cells (OPC1) may prolif- erate along microchannels with varying parameters such as length, width, tortuosity, convergence, divergence and sur- face chemistry. As bone cell–materials interaction is quite complex, we have created a few simpliﬁed structures to (i) understand these interactions, which is relevant in dental OPC1 cells suspended in media and orthopedic applications. Secondly, our work examines seeded in 120 micron deep pit the null hypothesis of a lack of detectable preference for 150 nm bio inert with 54.74º sidewalls OPC1 adhesion or proliferation on gold-, titanium- and sil- coating 380 micron Si wafer ica-coated surfaces. Finally, it reviews evidence that OPC1 cells may align with nearby channels, and that inclined sidewalls inhibit OPC1 adhesion. To study these hypothe- ses, OPC1 cells were cultured in various Si microchannels and analyzed by scanning electron microscopy (SEM). A semi-qualitative analysis revealed no signiﬁcant preference (ii) towards materials chemistry. However, narrow channels Fig. 1. (i) Top surfaces of four chips that are designed to test cell– were shown to inhibit adhesion and banding patterns. materials interactions along diverse microchannels. Design A varies channel length, B varies channel width, C varies convergent and divergent 2. Materials and methods patterns, and D varies channel tortuosity. (ii) Cross-sectional view of the microchannels. 2.1. Design and microfabrication To investigate OPC1 behavior along various channels, for chip fabrication. Wafers were subjected to high temper- 1 cm2 silicon chips were etched with four designs consisting ature wet oxidation at 1050 °C in mixed oxygen and nitro- of several channels branching out from a central cavity. gen environment. A 45 min ramp to 1050 °C was followed Design A varied channel length, B varied channel width, by 80 min soak time to grow 500 nm oxide layer on silicon. C varied channel divergence and convergence, and D var- Oxide layer on polished side of the wafer was then stripped ied channel tortuosity. Chips were designed using Corel using a buﬀered oxide etchant (BOE) solution of HF, Draw and micro-machined using anisotropic Si etching, NH4F, and H2O (10:1) for 10 min, while the back-side of and then coated with gold, titanium, and SiO2. Each the wafer was protected using semiconductor tape. This 1 · 1 cm square chip has a central cavity of 2500 · was followed by boron diﬀusion on the bare silicon side 2500 lm branching out into channels of standard width at 1125 °C for 110 min to result in 2.3 lm boron depth. and separation 500 lm. Fig. 1 shows all four designs. The oxide layer on the back-side prevents boron to diﬀuse Design A varied channel length from 500 to 4500 lm. onto silicon on this side. Boron diﬀusion results in borosil- Design B varied channel width as follows: 25, 50, 75, icate glass formation on the surface, which was stripped oﬀ 100, 200, 300, 400, 500, 600, 800 and 1000 lm. Design C to reduce stresses in the wafer. Borosilicate glass was had interrupted and uninterrupted convergent and diver- removed with a 20 min etching in BOE, followed by grow- gent channels 2500–3000 lm in length. Design D had chan- ing a sacriﬁcial oxide layer by low temperature oxidation nels of standard length 6000 lm (except channel 2) and (LTO) at 850 °C for 2 h. The sacriﬁcial LTO layer is then varied the channel tortuosity, or number of 90° bends, removed in BOE for 10 min and the ﬁnal LTO layer is including 0, 1, 3, 4, 5 and 10 bends. Six copies of each of grown, which acts as silicon dioxide windows for aniso- the four designs were arranged on a 76.2 mm diameter Si tropic silicon etching to fabricate silicon microchannels. wafer. Positive photolithography using AZ400K as photoresist Single side polished, p-type (1 0 0) silicon wafers with was carried out to create oxide mask on back-side of the 76.2 mm diameter and 380 ± 20 lm thickness were used wafer. The exposed silicon was then etched away using R. Condie et al. / Acta Biomaterialia 3 (2007) 523–530 525 gluteraldehyde + 0.1 M cacodylate [Na(CH3)2 AsO2Æ3H2O] Silicon Wafer buﬀer at pH 7.2. Following three 10 min rinses with caco- dylate buﬀer, samples were immersed in 2% OsO4. Sample chips were rinsed in deionized H2O, then in 30%, 50%, 70%, 95% and 100% ethanol, followed by 1:1 ethanol/ace- Boron Diffusion tone, acetone and hexamethyldisilazane (HMDS). Samples were dried overnight, then mounted with carbon tape onto an SEM sample holder and sputtered with Au for 6 min to $200 nm in preparation for SEM. The process was repeated Low temperature oxidation with the remaining samples on days 11 and 16. One sample from each duplicate (of matching design, coating and cul- ture time) was selected for SEM analysis. Approximately 500 SEM images were taken, and samples were character- Photolithography ized and scored based on adhesion and proliferation. Scores were compared to show the eﬀects of material coatings and channel parameters on OPC1 behavior. Anisotropic Si Etching 2.3. SEM analysis and statistical analysis Fig. 2. Schematic of microchannel fabrication on Si using anisotropic OPC1 cell–materials interactions were observed using a etching. ﬁeld emission scanning electron microscope (Serion FEI, type 8206/02). Of the 72 chips (2 (duplicates) · 3 coat- ethylenediamine pyrocatechol (EDP) at 110 °C for 2 h to ings · 4 designs · 3 time points), 36 were analyzed; the bet- create microchannels 120 lm deep. Fig. 2 shows a sche- ter of each duplicate was selected. Channel beginnings, matic of the microchannel fabrication steps. For sputter central sections and endpoints, as well as cavity and surface coating, we used a DC/RF sputtering machine to deposit areas, were examined. Overall OPC1 conditions on each Ti and Au on etched microchanneled Si wafers. For Au sample chip were described in order of increasing cell den- layers, a 20 nm Ti was used as an adhesive layer prior to sity, adhesion and proliferation, as depicted in Fig. 3. Au deposition. The thickness of these layers was estimated Scores were assigned as follows: bare (density score = 0), based on established calibration data for this machine sparse (1), semi-conﬂuent (2), conﬂuent (3), banded (4) using deposition time. and layered (5). Bad cell adhesion or peeling was assigned a score of À1. An average score was assigned for each chip 2.2. Cell–materials interactions in cases of variation. Scores were tabulated, and a one-way analysis of variance (ANOVA) with a 95% conﬁdence level For the cell–materials interaction study, human osteo- was conducted to determine whether a signiﬁcant diﬀerence blasts cells were used. Cells were derived from an immortal- was present among scores for each material coating. ized osteoblastic precursor cell line (OPC1) established from human fetal bone tissue . Samples were sterilized by auto- 3. Results claving (Amerex Instruments Ltd., CA) for 20 min at 121 °C before cell culture. Cells were plated at a density of $20,000 OPC1 cells proliferated over the entire ﬂoor and top sur- cells in the central cavity of each chip and were cultured in faces of chips, uninhibited by sidewalls. Adhesion was McCoy’s 5A medium (with L-glutamine, without phenol characterized and scored for each sample and each channel red and sodium bicarbonate). The 20,000 cells per specimen of Design B. No signiﬁcant diﬀerence was found among were seeded in the central cavity and allowed to ﬂow into scores of Au-, Ti- or SiO2-coated surfaces. Narrow chan- (during overﬂow in seeding) the channels. Then 5% fetal calf nels inhibited OPC1 adhesion. Bands of cells appeared to serum and 5% bovine calf serum, 2.2 g lÀ1 sodium carbonate, align with nearest channels. 100 mg lÀ1 streptomycin and 8 lg mlÀ1 Fungizone (GibcoTM Labortories, Grand Island, NY) were added in the media. 3.1. Materials interactions Cells were maintained at 37 °C under an atmosphere of 5% CO2 and 95% air. Culture medium was changed every 48 h SEM images showed numerous OPC1 cells adhered to for the duration of experiment. Since overﬂow still remains the material surface of 35 of the 36 chips examined. Cells an issue for cell seeding, future experiments will focus on were attached to channel ﬂoors and walls, the central ﬂoor developing an improved approach. and the top surface. Material coating made no visibly On day 5, 24 samples (two copies of each design and apparent diﬀerence in OPC1 adhesion or morphology, as coating combination) were removed, ﬁxed, processed and is evident in Fig. 4. Samples cultured for 5 days generally sputtered coated with gold in preparation for SEM. Cells appeared more globular and were attached to the material were ﬁxed for 1 h at 22 °C in 2% paraformaldehyde + 2% by thin extensions. Samples ﬁxed on days 11 and 16 526 R. Condie et al. / Acta Biomaterialia 3 (2007) 523–530 Fig. 3. Examples of qualitative characterization of OPC1 adhesion and proliferation on various samples and corresponding scores assigned for semi- quantitative analysis. generally appeared to be more elongated and conﬂuent, the anisotropic etching, the narrowest ﬁve channels have and were often banded together. no ﬂoor, but form a ‘‘V’’ at the bottom. As shown by Cell density varied from bare to layered, according to Fig. 6, OPC1 populations were visibly less dense within the descriptions listed in Section 2 and in Table 1. The table narrow channels and showed evidence of poor adhesion, also shows material adhesion scores corresponding to each including cracking and peeling. Narrow channels appeared sample. The average scores for gold, silica and titanium to break up banding patterns of diﬀerentiating OPC1 cells. were 2.58, 2.75 and 2.83, respectively. The average scores Edges and corners appeared to induce peeling and disrupt for day 5, 11 and 16 were 2.33, 2.25 and 2.58, respectively. banding. Fig. 5 compares adhesion scores for gold, titanium and sil- Adhesion scores for each channel of nine sample chips ica surfaces. A Kruskal–Wallis non-parametric one-way for days 5, 11 and 16 are displayed in Table 2. Average ANOVA test  showed no signiﬁcant diﬀerence among scores for the narrowest ﬁve channels varied from 1.17 to adhesion scores for gold, titanium or silica. 1.53. Average scores for the other channels generally increased from 1.22 to 2.94. Top surface and center ﬂoor 3.2. Narrow channel inhibition average scores were 3.28 and 3.44, respectively. Fig. 7 illus- trates these scores. Scores generally increased with channel Design B varied channel width from 25 to 1000 lm at ﬂoor widths of 50–350 lm, and increased slightly further the surface and from 0 to 750 lm at the ﬂoor. Because of with the nearly unlimited channel widths of the center ﬂoor R. Condie et al. / Acta Biomaterialia 3 (2007) 523–530 527 4.00 3.50 3.00 Average Score 2.50 2.00 1.50 1.00 0.50 0.00 Gold Titanium Silica Error = Standard Error Mean Fig. 5. Comparison of OPC1 adhesion scores on gold, titanium and silica coatings. An ANOVA test showed no signiﬁcant diﬀerence among mean scores for the three materials at the 95% conﬁdence level. and top surface. The slight variance within this pattern at 250 lm is well within one standard deviation. A trend line describes a mutually increasing relationship between Fig. 4. Comparison of OPC1 morphology on: (a) gold, (b) titanium and (c) silica after 16 days in culture from the central well. Table 1 Adhesion scores on Au, Ti, and silica surfaces Chip design Gold surface Titanium surface Silica surface Day 5 Design A 2 1 1 Design B 1 3 4 Design C 2 3 3 Design D 2 3 3 Day 11 Design A 4 2 2 Design B 3 3 3 Design C 3 3 1 Design D 2 0 1 Day 16 Design A 3 5 4 Design B 3 3 4 Design C 2 3 5 Design D 4 4 3 Fig. 6. SEM images of OPC1 cells on microchannels of Design B. (a) Channels of surface width 25, 50, 75 and 100 lm. Sidewalls meet at the Mean 2.58 2.75 2.83 bottom of each channel. Channels 200, 300 and 400 lm wide at the surface Standard deviation 0.90 1.29 1.34 are shown in (b). Banding patters are broken and cells are sparsely Qualitative descriptions of OPC1 adhesion are correlated to a score on the scattered in channels less than 400 lm wide. Peeling frequently occurs at left. Scores for 36 samples are tabulated on the right. edges. 528 R. Condie et al. / Acta Biomaterialia 3 (2007) 523–530 Table 2 Table of adhesion scores on Design B microchannels with varied width Path # Floor width (lm) Top width (lm) Design B adhesion scores Au 5 Au 11 Au 16 Ti 5 Ti 11 Ti 16 SiO2 5 SiO2 11 SiO2 16 Average 1 0 25 1 1 1.5 1 2 2 1 1 2 1.39 2 0 50 1 1 1.5 1 2 1 1.5 2 2 1.44 3 0 75 1 1 0 1 2 1.5 1 1.5 1.5 1.17 4 0 100 1 1 1 1 2 1 1 2 1.5 1.28 5 0 200 1 2 1 2 1 2 1.5 2 1.25 1.53 6 50 300 1 1 1.5 1 1 2 1 1 1.5 1.22 7 150 400 1 2 2 2 2 2 2 2 1.5 1.83 8 250 500 1 3 3 3 3 3 2 3 2.63 9 350 600 1 2 3 3 3 2 2.33 10 550 800 1 3 3.5 3 3 3 3 4 2.94 11 750 1000 1 3 3.5 3 3 3 3 4 2.94 Center ﬂoor 1.5 3.5 4 3 3.5 4 3.5 3.5 4.5 3.44 Surface 1 3 4 3 3 4 3 4 4.5 3.28 Average 1.00 1.82 1.96 1.91 2.1 1.94 1.91 1.72 2.23 1.88 Qualitative descriptions of OPC1 proliferation and adhesion are correlated to a score on the left. Mean scores for nine samples for days 5, 11 and 16 are tabulated on the right. 4.00 y = -3E-06x2 + 0.0048x + 1.297 R2 = 0.8843 3.50 error = Standard Error Mean 3.00 2.50 Average Score 2.00 1.50 1.00 0.50 0.00 Channel 25 50 75 100 200 300 400 500 600 800 1000 Top Floor Width (μm) Fig. 7. Plot comparing adhesion scores for channels of Design B. Channels vary from 25 to 1000 lm in width at the top surface. Channels narrower than 300 lm at the top surface had no ﬂoor or ﬂat surface at the bottom. The area is nearly unlimited for the top surface. adhesion score and path width. A Kruskal–Wallis non- of chips as well as within channels. Occasionally, bands parametric one-way ANOVA test showed a signiﬁcant dif- appeared to radiate out from exterior corners. Sidewalls, ference among scores of diﬀerent channel widths. Channel narrow channels and other obstacles often broke up band- sidewalls sloped at 54.74° did not visibly inhibit the prolif- ing and alignment patterns. Peeling of banded cells fre- eration of OPC1 cells. Fig. 8 is a characteristic SEM image quently occurred at channel edges as well. of a design B chip showing similar cell density on the chan- nel ﬂoors and walls, and on the surface. Because cells were 4. Discussion not conﬁned within channels under the conditions of this experiment, no substantive data were gathered on the In an eﬀort to better understand osseointegration, this eﬀects of channel tortuosity, divergence or convergence. experiment investigated immortalized OPC1 adhesion and In many images, especially those of samples with high proliferation on various micro-topographies and bioinert adhesion scores, bands of OPC1 cells appeared to be elon- coatings. The relative bioactivities of gold, titanium, and gated and uniformly oriented in local areas. Frequently, silica coatings were compared. Adhesion and proliferation they appeared to be aligned with the nearest channels, as behaviors based on observations of cell density along chan- seen in Fig. 9. This behavior occurred on the top surface nels of varied width and length were observed. Fabrication R. Condie et al. / Acta Biomaterialia 3 (2007) 523–530 529 channel ﬂoors and walls and the top surfaces of each sam- ple chip by day 5 and continued to extend and diﬀerentiate through day 16. Some cracking and peeling that reduced adhesion scores may have developed during ﬁxation and processing. It was hypothesized that OPC1 would exhibit no detect- able preference for gold, titanium or silica surfaces. These materials are described as bioinert in literature and are often used in bone implants or other biomedical devices. As illustrated in Fig. 4 and conﬁrmed by the Kruskal–Wal- lis non-parametric one-way ANOVA test, no signiﬁcant diﬀerence between adhesion scores for the three material coatings was observed. The migration of osteoblasts through various porous materials indicates their ability to conform to a variety of Fig. 8. SEM image of convergent channel of a Design B sample chip. topographies . As bone tissue growth along two-dimen- Sidewalls failed to contain OPC1 proliferation and migration. Cells sional paths does not appear to have been studied previ- adhered to the channel ﬂoor, side walls and top surface. ously, it was also hypothesized that OPC1 cells may proliferate along and adhere to channels of varying length, width, tortuosity, convergence and divergence. Design B varied channel width from 25 to 1000 lm at the surface and from 0 to 750 lm at the ﬂoor. As observed in Fig. 6, cultured cells were present in all channels, but sidewalls and narrow channels broke up banding patterns and signif- icantly inhibited adhesion. A plot of adhesion scores versus channel width, shown in Fig. 7, revealed an increasing trend with width. A signiﬁcant variation among the mean scores of various channel widths was conﬁrmed by the Kruskal– Wallis non-parametric one-way ANOVA test. These results support the key ﬁnding that channels of ﬂoor width nar- rower than 350 lm reduce OPC1 adhesion in these bioinert surfaces, suggesting a minimal width for ﬂat engineered bio- material surfaces for optimized integration with bone cells. This surface channel width requirement is somewhat larger than the 100 lm requirement of three-dimensional porous matrices quoted in the literature [16,17]. OPC1 cells have been shown to proliferate well in ceramic pores ranging from 300 to 500 lm in width . Similarly, foam pore sizes ranging from 150 to 710 lm did not signiﬁcantly aﬀect stro- mal osteoblast proliferation or function . Because 120 lm deep sidewalls sloped at 54.74° failed to contain OPC1 cell growth within channels under these experimental conditions, no substantive data were accumu- lated on the eﬀects of channel divergence or convergence, or tortuosity. However, the presence of healthy OPC1 cells in all areas of each chip shows their ability to adhere to such surfaces. Overﬂow during OPC1 seeding likely con- tributed to the broad proliferation patterns. Proliferating cells may have migrated over channel walls and across Fig. 9. OPC1 cells appear to be aligned with nearby sidewalls in SEM the top surfaces as well as along the ﬂoor, disrupting con- images (a) and (b). Banding patterns appear to follow channels around ﬁdence in deﬁnite cell migration pathways. In future exper- bends or radiate from corners. iments, channel surfaces may be covered with a glass cover slip or removable, microfacricated top to contain cell of eﬀective micro-patterned substrates for cell proliferation migration within the channel. The 2 ml suspension volume, was accomplished by oxidization of silicon wafers, photoli- corresponding to a 7.4 ml seeding volume, was selected as a thography, EDP etching and sputter coating with gold or minimum for statistically consistent seeding of 20,000 cell titanium or oxidation to form SiO2. Cultured cells covered cells. Seeding fewer cells in a lower-volume suspension 530 R. Condie et al. / Acta Biomaterialia 3 (2007) 523–530 may prevent overﬂow and enable channels to contain cell program under grant number 0453554 and the Oﬃce of proliferation. Analysis at earlier time points may provide Naval Research (Grant Number: N00014-1-04-0644). useful data as well. Moreover, other etching techniques such as plasma etching can be used to fabricate channels References with nearly 90° sidewalls that may better contain cell pro- liferation within the channels.  http://www.biomet.com/ci/investors/presentations/index.cfm. Abrupt angles at channel walls appeared to interrupt  Hedia HS, Mahmoud NA. Design optimization of functionally graded dental implant. Bio-Med Mater Eng 2005;14:133–43. banding and adhesion patterns, and frequently stimulated  Ratner BD, Hoﬀman AS, Schoen FJ, Lemons JE. Biomaterials peeling of banded cells away from the material surface. science: an introduction to materials in medicine. San Diego Therefore, straight surfaces may be recommended in engi- (CA): Elsevier Academic Press; 2004, p 154–5. neered biomaterial surfaces to improve osseointegration.  Dalby MJ, McCloy D, Robertson M, Wilkinson CDW, Oreﬀo ROC. We have also observed the apparent alignment of OPC1 Osteoprogenitor response to deﬁned topographies with nanoscale depths. Biomaterials 2006;27:1306–15. bands on ﬂoor or top surfaces with local channels. These  Meyer U, Joos U, Wiesmann HP. Biological and biophysical observations are in harmony with previous studies of principles in extracorporal bone tissue engineering. Int J Oral osteoblast alignment with parallel grooves fabricated in Maxillofac Surg 2004;33:325–32. various material surfaces, but expand the study to tortuous  Daculsi G, LeGeros RZ, Deudon C. Scanning and transmission and divergent channels [10,11,16]. Based only on visual electron microscopy, and electron probe analysis of the interface between implants and host bone. Osseo-coalescence versus osseo- observation, aligned bands appeared to follow channels integration. Scanning Microsc 1990;4:309–14. around corners and obstacles. An in-depth statistical anal-  Fredel MC, Boccaccini AR. Processing and mechanical properties of ysis of alignment is beyond the scope of this experiment, biocompatible Al2O3 platelet-reinforced TiO2. J Mater Sci but further studies recording the elongation and orienta- 1996;31:4375–80.  Mayr-Wohlfart U, Fiedler J, Gunther K-P, Puhl W, Kessler S. ¨ tion of each cell could validate the observation that cells Proliferation and diﬀerentiation rates of a human osteoblast-like cell align with the nearest obstacle. This alignment with topo- line (SaOS-2) in contact with diﬀerent bone substitute materials. J graphical features may allow tissue engineering of bone Biomed Mater Res 2001;57:132–9. cells on implant surfaces . In a recent publication sup-  Zinger O, Zhao G, Schwartz Z, Simpson J, Wieland M, Landolt D, porting this idea, bone tissue has been shown to preferen- et al. Diﬀerential regulation of osteoblasts by substrate microstruc- tially form within and along grooves 110 lm wide and tural features. Biomaterials 2005;26:1837–47.  Ber S, Torun Koseb G, Hasırcıa V. Bone tissue engineering on ¨ 70 lm deep in oxidized titanium dental implants, providing patterned collagen ﬁlms: an in vitro study. Biomaterials increased resistance to shear forces . Overall, our results 2005;26:1977–86. show that engineered surface topographies have potential  Dunn GA, Brown AF. Alignment of ﬁbroblasts on grooved surfaces to greatly improve osseointegration. described by a simple geometric transformation. J Cell Sci 1986;83:313–40.  Lenhert S, Meier MB, Meyer U, Chi Lifeng, Wiesmann HP. 5. Conclusions Osteoblast alignment, elongation and migration on grooved polysty- rene surfaces patterned by Langmuir–Blodgett lithography. Bioma- We have studied eﬀects of various microchannel param- terials 2005;26:563–70. eters on immortalized osteoblast precursor cell (OPC1)  Winn SR, Randolph G, Uludag H, Wong SC, Hair GA, Hollinger JO. Establishing an immortalized human osteoprecursor cell line: adhesion and proliferation. Microchannels were micoma- OPC1. J Bone Miner Res 1999;14:1721–33. chined on (1 0 0) Si wafers using anisotropic wet etching  William H Kruskal, Allen Wallis W. Use of ranks in one-criterion with varying designs to understand the inﬂuence of channel variance analysis. J Am Stat Assoc 1952;47(260):583–621. width, channel length, tortuosity, convergence and diver-  Kalita SJ, Bose S, Hosick HL, Bandyopadhyay A. Development of gence. We have also coated these channels with Au, Ti controlled porosity polymerceramic composite scaﬀolds via fused and SiO2 to vary the bio-inert surface chemistry. Among deposition modeling. Mater Sci Eng, C 2003;23:611–20.  Ishaug SL, Crane GM, Miller MJ, Yasko AW, Yaszemski MJ, Mikos all parameters, channel width is found to inﬂuence the AG. Bone formation by three-dimensional stromal osteoblast culture most in terms of cell attachment. A channel ﬂoor width in biodegradable polymer scaﬀolds. J Biomed Mater Res narrower than 350 lm has shown reduced OPC1 adhesion 1996;36:17–28. in these bioinert surfaces, suggesting a minimal width for  Klawitter JJ, Hulbert SF. Application of porous ceramics for the ﬂat engineered biomaterial surfaces for optimized integra- attachment of load bearing orthopaedic applications. Biomed Mater Symp 1971;2:161–7. tion with bone cells.  Bose S, Darsell J, Kinter M, Hosick H, Bandyopadhyay A. Pore size and pore volume eﬀects on alumina and TCP ceramic scaﬀolds. Mater Acknowledgements Sci Eng, C 2003;23:479–86.  Bandyopadhyay A, Bernard S, Xue W, Bose S. Calcium phosphate The authors thank Mr. Hongsoo Choi, Ms. Kakoli Das based resorbable ceramics: inﬂuence of MgO, ZnO and SiO2 dopants. J Am Ceram Soc 2006;89(9):2675–88. and Ms. Jessica Moore of WSU for experimental assis-  Hall J, Miranda-Burgos P, Sennerby L. Stimulation of directed bone tance. This work was supported through the National Sci- growth at oxidized titanium iimplants by macroscopic grooves: an ence Foundation: Division of Materials Research REU site in vivo study. Clin Implant Dent Relat Res 2005;7:76–82.
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