Regulation of Implant Surface Cell Adhesion: Characterization and Quantification of S-phase Primary Osteoblast Adhesions on Biomimetic Nanoscale Substrates Manus J.P. Biggs,1 R.G. Richards,2 N. Gadegaard,1 C.D.W. Wilkinson,1 M.J. Dalby1 1
Centre for Cell Engineering, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow, G12 8QQ, United Kingdom 2
AO Research Institute, AO Foundation, Clavadelerstrasse 8, Davos Platz, Switzerland
Received 8 December 2005; accepted 9 August 2006 Published online 14 November 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20319 ABSTRACT: Integration of an orthopedic prosthesis for bone repair must be associated with osseointegration and implant fixation, an ideal that can be approached via topographical modification of the implant/bone interface. It is thought that osteoblasts use cellular extensions to gather spatial information of the topographical surroundings prior to adhesion formation and cellular flattening. Focal adhesions (FAs) are dynamic structures associated with the actin cytoskeleton that form adhesion plaques of clustered integrin receptors that function in coupling the cell cytoskeleton to the extracellular matrix (ECM). FAs contain structural and signalling molecules crucial to cell adhesion and survival. To investigate the effects of ordered nanotopographies on osteoblast adhesion formation, primary human osteoblasts (HOBs) were cultured on experimental substrates possessing a defined array of nanoscale pits. Nickel shims of controlled nanopit dimension and configuration were fabricated by electron beam lithography and transferred to polycarbonate (PC) discs via injection molding. Nanopits measuring 120 nm diameter and 100 nm in depth with 300 nm center–center spacing were fabricated in three unique geometric conformations: square, hexagonal, and near-square (300 nm spaced pits in square pattern, but with 50 nm disorder). Immunofluorescent labeling of vinculin allowed HOB adhesion complexes to be visualized and quantified by image software. Perhipheral adhesions as well as those within the perinuclear region were observed, and adhesion length and number were seen to vary on nanopit substrates relative to smooth PC. S-phase cells on experimental substrates were identified with bromodeoxyuridine (BrdU) immunofluorescent detection, allowing adhesion quantification to be conducted on a uniform flattened population of cells within the S-phase of the cell cycle. Findings of this study demonstrate the disruptive effects of ordered nanopits on adhesion formation and the role the conformation of nanofeatures plays in modulating these effects. Highly ordered arrays of nanopits resulted in decreased adhesion formation and a reduction in adhesion length, while introducing a degree of controlled disorder present in near-square arrays, was shown to increase focal adhesion formation and size. HOBs were also shown to be affected morphologicaly by the presence and conformation of nanopits. Ordered arrays affected cellular spreading, and induced an elongated cellular phenotype, indicative of increased motility, while near-square nanopit symmetries induced HOB spreading. It is postulated that nanopits affect osteoblast–substrate adhesion by directly or indirectly affecting adhesion complex formation, a phenomenon dependent on nanopit dimension and conformation. ß 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:273–282, 2007
Keywords:
implant surface; cell adhesion; S-phage; nanoscale substrates
INTRODUCTION Biomaterials are increasingly becoming recognized as being crucial in tissue repair and regeneration. It is envisaged that future implantable devices will be capable of providing the vital biophysical Correspondence to: Manus J.P. Biggs (Telephone: þ44 (0) 141 339 8855 (ext. 0352); Fax: þ44 (0) 141 330 3730; E-mail:
[email protected]) ß 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
stimuli essential for normal cellular behavior and function. Research into fracture fixation and orthopedic biomaterials has led to the development of biomimetic materials to increase cellular adhesion and infiltration. Successful integration of an orthopedic prosthesis and bone must be associated with osseointegration and implant fixation, an ideal that can be approached via modification of the bone/ implant interface by either chemical or topographical means.1–3 In vitro studies have shown JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2007
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that osteoblast response is influenced by substrate morphology,4 and that extracellular matrix (ECM) synthesis and osteoblast differentiation are influenced by substrate microtopography,5 factors that have consistently been shown to affect osteoblast infiltration and adhesion in vivo when applied to an orthopedic device.6 Accordingly, nanogrooves, nanopits, and nanoislands have been shown to affect contact guidance in vitro and directly influence cell adhesion to a degree related to feature dimension, cell type, and cell density.7–10 Present studies indicate that fibroblasts react significantly to nanotopographical cues in vitro, perceiving the topography of an implanted surface via dynamic filopodial formation and extension to find sites topographicaly suitable for adhesion, growth, and maturation.11,12 The processes that mediate cellular reaction to nanoscale surface structures are not well understood, and may be direct (a result of the influence of the surface topography) or indirect (where the surface structure has affected the composition, orientation, or conformation of the adsorbed ECM components).13 Cells have been shown to react to different nanotopographical features in different ways; most pronounced, however, are changes in cellular adhesion, which appear to depend on the symmetry and spacing of the nanofeatures.14 Cells dependent on ECM anchorage require substrate adhesion before normal function is initialized. If adhesion is disrupted or prevented, the cell will die through a specialized apoptosis, anokis. It is the ability of cells, such as osteoblasts to adhere that allows signalling from the ECM to the nucleus via integrins, cytoskeleton, and molecular cascades (G-proteins, kinases, ions). These events ultimately control proliferation and new tissue formation.15,16 Cellular interactions with a biomaterial are mediated through transmembrane integrin receptors that are covalently coupled to the ECM. Focal adhesions (FAs) are peripherally located adhesion sites associated intimately with the actin cytoskeleton that form adhesion plaques of clustered transmembrane receptors at points of cellular adhesion.1 FAs contain structural and secondary signalling molecules crucial to cell adhesion and function.11 Presently, FAs are grouped according to their size, molecular composition, and spatial location, variables directly influenced by both the cell cycle and substrate cues indicating these adhesion complexes to be dynamic structural entities, associated with continual maturation and disassembly.17 Focal complexes (FXs) originate as dot-like structures of approximately 1 mm2, which mature JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2007
into ‘‘dash’’ FAs upon increased intracellular and/ or extracellular tension18 when integrin packing density can increase by a factor of three fold.19 Mature FAs are typically dash shaped, 2– 5 mm2, and contain vinculin, paxillin, and talin. Fibrillar adhesions (FBs) form as an elongation of dash type FAs in the direction of mature stress fiber assembly and specifically contain a5b1 integrin and tensin.12 As internal or external stresses are applied to membrane associated integrins, intracellular transduction responses can lead to FAs assembly and associated cytoskeletal strengthening,20 as well as activation of chemical signalling cascades and ultimately gene transcription.12 However, little is known regarding the effect substrate nanotopography has on the formation of FAs and cell–substrate integration. Fabricated model surfaces with defined nanoscale topographies are of great experimental value in obtaining data regarding the influence of inherent biomaterial nanotopography on osteoblast adhesion. Most synthetic surfaces encountered by cells following implantation of prosthetic devices have nanotopographic features if only because of their methods of manufacture, that is, roughness or tool marks.21 The use of lithographic and dry etching techniques derived from the microelectronics industry are facilitating investigation into the influence of nanotopography on cellular function and adhesion. Photolithography and more recently electron beam lithography (EBL), have been added to an advancing repertoire of potential tools for the generation of controlled substrate topography. This report is concerned with the influence of nanoscale pits with both highly ordered arrangement and a controlled degree of dissorder on primary human osteoblast (HOB) adhesion as indicated by adhesion subgroup quantification and cytoskeletal organization. Here, the HOBs have been cultured on a 120-nm diameter, 300-nm center–center spacing, 100-nm deep pit arrays originally produced by electron beam lithography in square, hexagonal (hex), and 50 nm displaced near-square (N-square) symmetries. Experimental substrates were injection moulded via a Nickel intermediary into polycarbonate (PC). To eliminate potential analytical errors and large standard deviations in adhesion quantification due to morphological alterations (i.e., cellular spreading, and hence, adhesion formation fluctuate with the cell cycle phase), adhesion analysis was carried out only in S-phase cells. 5-Bromodeoxyuridine (BrdU), a thymidine analogue, was used to label the HOBs cultured on the DOI 10.1002/jor
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nanopitted PC within S-phase. Cells were initially subjected to serum starvation followed by serum supplementation to induce S-phase in the cell population. Incorporation of BrdU into the nucleus of cells undergoing DNA synthesis was subsequently fluorescently labeled. FAs were also labeled in these cells via vinculin detection, a protein common to all integrin-mediated adhesions.
MATERIALS AND METHODS Nanopatterning and Die Fabrication Samples were made in a three-step process of electron beam lithography, nickel die fabrication, and injection moulding. Silicon substrates were coated with ZEP 520A (Zeon corporation) resist to a thickness of 100 nm. Samples were baked for 1–2 h at 1808C prior to exposure in a Leica:BPG 5-HR100 beamwriter at 50 kV. An 80-nm spot size was used, resulting in pits 100 nm deep with a diameter of 120 nm. The pitch between the pits was 300 nm. Both square and hex symmetries were fabricated, while N-square was characterized by is a displacement of 5p nm from the center of the dots (in both x and y) where p is a random integer between 10 and þ10. After exposure the samples were developed in xylene at 238C for 60 s and rinsed in copious amounts of 2-propanol before being blown dry with filtered nitrogen. For more information about the procedure see ref. 22. Nickel dies were made directly from the patterned resist samples. A thin (50-nm) layer of Ni-V was sputter coated on the samples. That layer acted as an electrode in the subsequent electroplating process. The dies were plated to a thickness of ca. 300 nm. For more information about the procedure see ref. 23. Polymeric replicas were made in DVD grade PC by injection molding. (Macrolon DP1-1265, Bayer). PC was used, as it is known to have very good replication capabilities.24 Subsequent injection molded replicas possessed a nanoimprinted area of 1 cm2. Cell Culture Primary human osteoblasts (HOBs) derived from human femoral head biopsies and obtained from PromoCell1 (Heidelberg, Germany) were cultured according to PromoCell1 guidelines. Cells were cultured in 75-cm2 flasks in an osteoblast growth medium (OGM) encompassing 10% foetal calf serum supplied by PromoCell1. Culture medium was free of antibiotics. Experimental substrates were sterilized by three sequential 5-s immersions in 70% ethanol followed by two sequential 5-s immersions in HEPES (N-(2-hydroxyethyl)-piperazine-N0 -2-ethanesulfonic acid) buffer. Cells were cultured for 10 days before trypsinization and seeding onto planar control, hex, square, and N-square substrates at a density of 1 104 cells per DOI 10.1002/jor
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sample in 2 mL of complete medium. HOBs were incubated at 378C with a 5% CO2 atmosphere and OGM replaced twice weekly. Following 7 days of culture HOBs were maintained for 4 days without supplementation with fresh medium, inducing a brief period of serum starvation. Serum rich media was subsequently introduced to the culture following this period and cells allowed to metabolise for 17 h, giving rise to a population of cells possessing a synchronized nuclear cycle. Following this period the cells were cultured in 10 mM BrdU/ OGM for 3 h. Immunofluorescent Labeling HOBs on test materials were fixed in 4% formaldehyde in PBS (phosphate buffered solution), with 1% sucrose at 378C for 15 min. Once fixed, the samples were washed with PBS. Samples were permeabilized with buffered 0.5% Triton X-100 (10.3 g sucrose, 0.292 g NaCl, 0.06 g MgCl2, 0.476 g HEPES buffer, 0.5 ml Triton X-100, in 100 mL water, pH 7.2) at 48C for 5 min. Nonspecific binding sites were blocked with 1% BSA (bovine serum albumin) in PBS at 378C for 5 min and subsequently incubated for 2 h with a 1:200 concentration antivinculin [monoclonal antihuman raised in mouse (clone hVin1; IgG1), Sigma, Poole, UK] followed by incubation in 1:100 concentration of anti-BrdU/DNase solution for 1 h (378C) (RPN2001, Amersham Biosciences cell proliferation kit, Uppsala, Sweden). Simultaneously, rhodamine conjugated phalloidin was added for the duration of this incubation (1:100 in 1% BSA/PBS, Molecular Probes, OR). Nonspecific charges (e.g., remaining formaldehyde) were neutralized with 0.5% Tween 20/PBS (5 min 3) to minimize background labeling. A secondary, biotin conjugated antibody (1:50 in 1% BSA/PBS, monoclonal horse antimouse (IgG; Vector Laboratories, UK) was added for 1 h (378C) followed by subsequent washing as above. A FITC conjugated streptavidin third layer was added (1:50 in 1% BSA/PBS, Vector Laboratories, UK) at 48C for 30 min, and given a final wash. Samples were mounted in Vectorshield mountant for fluorescence (Vector Laboratories, UK), then viewed with a Zeiss Axiovert 200M microscope with a A Zeiss Plan Neofluor 40 (0.75 NA) lens. Image manipulation in Adobe1 Photoshop was then used to transpose the color layers to show adhesion complexes (vinculin) in green, actin in red, and S-phase nuclei in blue. Image Analysis For adhesion quantification Image J was used (downloaded from the National Institute of Health, Bethesda, MD; free download available at http://rsb.info.nih.gov/ij/). This used automated detection of vinculin labeling of which approximately 20 S-phase cells were analyzed on each material from three material replicates. Adhesion number and length were quantified. Criteria for adhesion classification was according to size restrictions described in the current literature.1,12 Adhesion complexes measuring less than 2 mm were assigned as FXs, those from 2–5 mm were designated as FAs, while JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2007
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adhesion complexes over 5 mm in length were accordingly classified as FBs. Statistics Data was log2 normalized and analyzed using Tukey ANOVA. Results of p < 0.05 were considered significant (differences p < 0.005 denoted by a single filled star, p < 0.001 denoted by two filled stars).
N-square substrates had reduced order, by shifting the pits from perfectly square by 50 nm (Fig. 1C). The total area of imprinted nanotopography on each PC substrate measured 1 cm2. Material pit coverage was quantified as square, hex, and N-square possessing 12.6, 14.5, and 14.2% total pit coverage, respectively. The planar controls had an average Ra of 1.17 nm over mm2. Osteoblast Morphology and Cytoskeletal Organization
RESULTS Characterization of Fabricated Substrate Topography The injection molding process resulted in the fabrication of nanopit arrays measuring 120 nm in diameter, with an interpit pitch of 300 nm and a depth of 100 nm (Fig. 1A) in hex and square symmetries as verified by SEM (Fig. 1B and D). Substrates appeared morphologically unaffected by processing and free from polymer crazing.
HOB morphology varied, dependent upon which experimental substrate cells were cultured. On planar control, S-phase HOBs were well spread with cytoplasmic processes (Figs. 2A and 3A). On the highly ordered square and hex symmetries, the HOBs were less well spread with rounded, stellate morphologies (Figs. 2B and C and 3B and C). Conversely, on the N-square substrate, HOBs were well spread with large lamellae (Figs. 2D and 3D).
Figure 1. SEM image of ordered and near-ordered nanopitted substrates. Arrays of nanopits on poly(carbonate) substrates produced by electron beam lithography and injection moulding. (A) Nanopits and interpit dimentions in ordered nanopit arrays, (B) hexagonal, (C) near-square, (D) square. JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2007
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Figure 2. Fluorescent microscopy image of S-phase HOB morphology on nanopitted substrates. (A) Control substrate, cells were well spread. (B) Square substrate, cells were less well spread and adopted an elongated morphology. (C) Hex substrate, cells were not well spread and adopted a rounded or elongated morphology. (D) N-square substrate, cells were well spread. Green, vinculin; blue, S-phase nuclei.
HOBs on all materials formed contractile stress fibers (Fig. 3). However, these were significantly reduced in cells cultured on square and hex arrays (Fig. 3B and C) compared to those cultured on planar controls and N-square (Fig. 3A and D) arrays. Focal Contact Quantification Quantification of vinculin localized within adhesion complexes revealed differences in adhesion formation between S-phase HOBs cultured on square, hex, N-square, and control substrates (Fig. 4). Nanopatterned PC substrates induced quantifiable alterations in adhesion frequency DOI 10.1002/jor
(Fig. 4A and B), length (Fig. 5A), and number (Fig. 5B) in HOBs relative to those cultured on control substrates. Adhesion Frequency HOBs cultured on all experimental and control substrates demonstrated an increase in frequency of FXs in the 1.5–2 mm range. HOBs cultured on control substrates, however, possessed greater numbers of FXs measuring 1.5 mm relative to HOBs cultured on nanopit substrates. HOBs cultured on square arrays of nanopits formed increased numbers of FXs measuring 1 mm relative to cells cultured on all other experimental JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2007
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Figure 3. Fluorescent image of adhesion formation and stress fiber organization of S-phase HOBs on nanopitted substrates. (A) Control substrate. S-phase cells possessed large and numerous adhesions. Many stress fibers were observed. (B) Square substrate. Adhesion numbers were significantly reduced in relation to control substrates, FXs observed as the predominant adhesion type. Stress fiber formation was also reduced. (C) Hex substrate. Adhesion numbers were significantly reduced relative to controls. Stress fiber formation was also reduced. (D) N-square substrate. Adhesions were large and numerous. Many stress fibers were observed. Red, F-actin; green, vinculin; light blue, S-phase nuclei; dark blue, non-S-phase nuclei.
substrates. Adhesion frequency in HOBs on all substrates begins to decrease in the FA range (Fig. 4A). Frequency of FBs over approx. 10.5 mm in length decreased on all substrates except N-square. Adhesions complexes on N-square were observed to approach approx. 20 mm in length (Fig. 4B). Adhesion Length Mean FX length (adhesion complexes measuring less than 2 mm) in S-phase HOBs was approxiomately 1.3 mm on all experimental and control JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2007
substrates. Mean FA length (adhesion complexes measuring between 2 and 5 mm) was approximately 3 mm on all experimental substrates. Intersubstrate variation of FX and FA length was low (Fig. 5A). S-phase HOBs cultured on highly ordered nanopitted substrates formed less FBs in relation to control substrates, while FB formation in HOBs cultured on N-square substrates was increased relative to controls. Mean FB length in HOBs cultured on hex and square substrates was approximately 6.6 mm, while mean length of FBs in HOBs cultured on control substrates was 7.2 mm. HOBs cultured on N-square DOI 10.1002/jor
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Figure 4. Quantification of adhesion complex distribution in S-phase HOBs on nanopitted substrates. (A) FX distribution in HOBs was decreased on nanopitted substrates relative to controls. Square nanoarrays induced increased FX formation of adhesions measuring 1 mm in length. FA distribution decreased as adhesion length increased on all experimental substrates. (B) Frequency of FBs over approximately 10.5 mm in length decreased in all substrates but N-square. A significant number of adhesions complexes on N-square were observed to approach approximately 20 mm in length.
substrates however had a mean FB length of approximately 11 mm, a marked increase relative to controls. A proportion of adhesions on this substrate were observed to approach 20 mm in length. Adhesion Number Differences in S-phase HOB adhesion numbers were found to be more pronounced than topographically induced variation in adhesion length. Cells cultured on square arrays exhibited reduced FA and FB numbers relative to controls, but possessed increased numbers of adhesions measuring 1 mm in length, in agreement with measurements of length and distribution. Mean adhesion subgroup formation was approximately 25 FXs, 16 FAs, and 3 FBs per cell. HOBs cultured on hex substrates followed a similar trend of adhesion formation to HOBs cultured on controls, DOI 10.1002/jor
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Figure 5. Quantification of adhesion complex length and number in S-phase HOBs on nanopitted substrates. (A) Mean adhesion length for FXs and FBs was conserved among all materials. Cells cultured on N-square substrates showed a marked increase in FB length, being on average approximately 4.5 mm greater in length than the ordered nanopits symmetries. (B). Cells cultured on square arrays exibited reduced FA and FB numbers relative to controls, but possessed increased numbers of adhesions measuring 1 mm in length. HOBs cultured on hex substrates followed a similar trend of adhesion to HOBs cultured on controls, but with a significant reduction in adhesion subgroup number. HOBs cultured on N-square substrates formed decreased numbers of FXs and FAs relative to controls and formation of FBs was significantly increased. Results for (A,B) are mean SD.
but with a significant reduction in adhesion subgroup numbers of each type (by almost 50%). HOBs formed an average mean value of approximately 18 FXs, 20 FAs, and 3 FBs per cell (Fig. 5B). HOBs cultured on N-square substrates formed reduced numbers of FXs and FAs (25 and 27, respectivly) compared to controls, but formation of FBs was significantly increased, with a mean value of 17 per cell (Fig. 5B) (see Table 1 for relevant statistics). JOURNAL OF ORTHOPAEDIC RESEARCH FEBRUARY 2007
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Table 1. Statistically Significant Data Generated from Adhesion Complex Quantification Adhesion Number Comparison N-square FBs vs. Hex FBs N-square FBs vs. Square FBs Flat FBs vs. Hex FBs Flat FBs vs. Square FBs
Adhesion Length
Dif. of Means
p
Comparison
Dif. of Means
p
2.898 2.891 1.803 1.795
