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Effects of material and surface functional group on collagen self-assembly and subsequent cell adhesion behaviors
Colloids and Surfaces B: Biointerfaces 116 (2014) 303–308
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effects of material and surface functional group on collagen self-assembly and subsequent cell adhesion behaviors Jing He, Yao Su, Tao Huang, Bo Jiang, Fang Wu ∗ , Zhongwei Gu National Engineering Research Centre for Biomaterials, Sichuan University, Chengdu 610064, PR China
a r t i c l e
i n f o
Article history: Received 4 September 2013 Received in revised form 7 January 2014 Accepted 8 January 2014 Available online 19 January 2014 Keywords: Hydroxyl group Collagen self-assembly Cell adhesion Collagen fibrous network Cytoskeleton
a b s t r a c t Collagen fibrous network not only provides structural support for cells but also serves as critical environment modulating various cell functions. Various factors would influence the collagen self-assembly but the effect of substrate surface on such process has been rarely studied. Here we examined the effects of materials (Ti and hydroxyapatite) and their surface characteristics (with and without the enrichment of hydroxyl group) on collagen self-reconstitution and fibrous network formation, and on subsequent cell adhesion and cytoskeleton organization of mesenchymal stem cells (MSCs). For both Ti and hydroxyapatite (HA) substrates, the enrichment of hydroxyl group ( OH) on substrate surfaces promoted the collagen self-reconstitution and facilitated the formation of the fibrous network after 4 h immersion in phosphate buffer solution (PBS), while all samples showed clear fibrous network formation after 2 day soaking in PBS. Compared with the Ti surfaces, the HA surfaces facilitated the self-reconstitution of collagen, leading to a more mature fibrous network with a twisted structure and enhanced lateral aggregation of fibrils. The fibrous network difference resulted in different behaviors of the subsequent MSC adhesion and spreading. The MSCs had the best adhesion and cytoskeleton organization on the OH enriched HA surface with collagen modification. Our results suggested that both the material selection and the hydroxyl group significantly influenced the collagen self-assembly and fibrous network formation and, as a result, the subsequent cell adhesion behaviors. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The extracellular matrix (ECM) is a complex organization composed of a wide variety of structural proteins and proteoglycans secreted by the cells [1,2]. The ECM proteins are able to bind growth factors and cytokines and can interact with bone cells via integrins or other specific cell surface receptors, thus controlling cell shape, motility, growth, survival and differentiation [3–6]. Type I collagen, the primary structural element in ECM and the major structural protein of bone, forms a fibrous network that not only provides a structural support for cells but also acts as an important regulator of cell behavior [7,8]. The fibrous architecture can provide a favorable microenvironment for cell attachment, binding of growth factors, and orchestrating signal and cellular events [9,10]. Recent studies have further suggested that the collagen fibers play a critical role for stem cells to sense the mechanical feedback and may ultimately affect the cell-fate decisions [11]. A proper mechanical feedback would be critical for MSC migration [12] and other MSC functions.
∗ Corresponding author. Tel.: +86 13438050329. E-mail addresses: [email protected], [email protected] (F. Wu). 0927-7765/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2014.01.009
It has been well known that solubilized collagen can selfassemble into fibrous structures in vitro with the characteristic axial periodic structure. Various factors would influence the kinetics of the self-reconstitution process, such as collagen concentration, pH value, temperature and ionic strength [9,13–15]. Collagen has often been used with other inorganic materials to form composites for bone regeneration applications [16]. The intrinsic property and surface characteristic of the inorganic material would likely exert a great influence on collagen fibril formation, thus affecting subsequent cellular response to the materials. However, the effects of material and its surface characteristic on the selfassembly of collagen molecules into the fibrils and fibrous network have been rarely studied. The most commonly used inorganic materials in orthopedic applications are Ti and hydroxyapatite (HA) [17,18]. Widely used in joint prosthesis and dentistry applications, the titanium is bio-inert despite of its excellent mechanical property. Alkali-heat treatment has been commonly used to make titanium surfaces bioactive by forming a bioactive sodium titanate layer on the titanium surface, in association with the increase of OH groups on the titanium surface [19,20]. HA is the major inorganic component of the natural bone and has been used as bioactive coating on Ti implants in artificial joints. HA coatings with different surface function groups could be synthesized using two different plasma spraying processes: the
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conventional air plasma spray (APS) process and the more recently developed liquid precursor plasma spraying (LPPS) process. During the APS process which uses powder as the feedstock material, serious dehydroxylation would occur due to the high process temperature, leading to the large loss of the OH group in the APS HA coatings. In the LPPS process which uses liquid precursor as the feedstock, the dehydroxylation effect is suppressed and as a result, the OH group is largely retained in the resultant LPPS HA coatings [18]. Therefore, Ti and HA substrates were prepared with diminished and enriched OH surface group respectively, as a result of different treatments applied to the substrates. The aim of this study was to examine the effects of material selection (Ti, HA) and the surface functional group on collagen self-assembly and the formation of the fibrous network, as well as its implication on subsequent cell adhesion behaviors.
2. Materials and methods 2.1. Materials preparation 2.1.1. Preparation of Ti with and without the alkali-heat treatment The titanium disks (14 mm × 2 mm) with and without alkaliheat treatment were used as the Ti substrates. For the alkali-heat treatment, the titanium disks were first cleaned by 10 min ultrasound treatment in pure acetone, ethanol and distilled water, respectively. The alkali-heat treatment was preformed according to the protocol described by Kim [20], using 10 M NaOH aqueous solution for 24 h at 60 ◦ C. After the alkali treatment, the titanium disks were rinsed with distilled water, dried in an oven at 60 ◦ C, and heat treated at 600 ◦ C for 1 h in air with a heating rate of 5 ◦ C min−1 . The functional groups of the pure and alkali treated Ti (Ti and TiAH, respectively) substrates were analyzed by X-ray photoelectron spectrometer (XPS, XSAM800, UK).
2.1.2. Preparation of HA coatings through the APS and LPPS processes HA coatings, with thickness around 100 m, have been deposited on the Ti–6Al–4V (14 mm × 2 mm) alloy substrate, using the APS and LPPS processes respectively. The liquid precursor was selected as the feedstock during the LPPS process, which was atomized into mists and injected into the plasma jet, instead of the powder feedstock used in the APS process. More detailed information of the APS and LPPS processes can be found elsewhere [18,21]. The functional groups of the APS and LPPS coatings were analyzed by Fourier Transform Infrared Spectroscopy (FTIR, Nicolet, 170SX, Wisconsin, USA), by scratching the powders from the HA coating samples.
2.1.3. Preparation of type I collagen solution and surface modification with collagen Type I collagen derived from bovine skin (provided by National Engineering Research Centre for Biomaterial, Sichuan University) was dissolved in acetic acid (pH 4.0) at 4 ◦ C with a concentration of 7.0 mg/ml. The surfaces of the four kinds of samples (referred as Ti, Ti-AH, HA-APS, HA-LPPS hereafter) were modified with the above collagen solution. Onto each sample we added 400 l collagen solution. First, half of the solution (200 l) was dropped on each sample, followed by air drying in a laminar flow cabinet at room temperature. Afterwards, the same process was repeated and the remaining half of the collagen solution was applied to the substrate.
2.2. Fibril network formation The four groups of samples were immersed into Dulbecco’s phosphate buffer solution (PBS, pH 7.4, Sigma, USA) to initiate the self-assembly and fibril network formation at 37 ◦ C. After being cultured for 4 h and 2 days respectively, the samples were fixed with 2.5% glutaraldehyde overnight, dehydrated in graded ethanol and isoamyl acetate, and subjected to critical point drying. After gold coating, the morphologies of the collagen fibrils were observed using the scanning electron microscope (SEM, S4800, Tecnai F20, Tokyo, Japan). 2.3. Cell culture Mesenchymal stem cells (MSCs) were isolated from rabbit bone marrow (1-week-old, New Zealand rabbits), as previously described [22]. MSCs were expanded in 20 ml of a-MEM containing 20% fetal bovine serum (FBS) and 1% antibiotics and cells from passage 3 were used for all the experiments. MSCs were seeded onto the surfaces of the samples that were placed in 24-well plates, with an initial density of 2.0 × 104 cells/well. Each well consisted of 1 ml of PBS supplemented with 20% FBS. 2.4. Cell morphology 2.4.1. Scanning electron microscopy After the MSCs were cultured for 1 day, the samples were washed twice with PBS. The MSCs were fixed with 2.5% glutaraldehyde buffer, dehydrated by a graded series of ethanol, subjected to critical point drying and gold coating. The cell morphologies were observed using the SEM (S4800, Tecnai F20, Tokyo, Japan). 2.4.2. Confocal microscopy Following one-day incubation, the MSCs were visualized using a confocal microscope (Leica SP5, Germany). Prior to observation, the cells were fixed in 1 l/ml fluorescein diacetate (FDA)/PBS (FDA, PBS, Sigma, USA) solution for live cells. To visualize the actin cytoskeleton, MSCs were fixed in 4% paraformaldehyde solution, treated with 0.1% Triton X-100 and stained with phalloidin Alexa 594 (Sigma) and DAPI (Sigma). 3. Results and discussions 3.1. Material characterizations 3.1.1. XPS analysis for Ti group samples The XPS spectra of the Ti and Ti-AH surfaces are shown in Fig. 1. Deconvolution of the O 1s peak revealed that the oxygen existed as TiO2 form in both Ti and Ti-AH surfaces. The oxygen peak (532.6 eV) of chemically adsorbed basic hydroxyl was clearly visible in the Ti-AH spectrum but was barely detectable in the spectrum of the untreated Ti surface, suggesting enrichment of hydroxyl group at Ti-AH surface. 3.1.2. FTIR analysis for HA group samples Fig. 2 shows the FTIR spectra of the as-deposited HA-APS and HA-LPPS coatings. Strong adsorption bands of H2 O (3434 and 1634 cm−1 ) and PO4 3− groups (1091, 1044, 960, 601, and 569 cm−1 ) were observed in both coatings. However, the HA-LPPS coatings exhibited quite sharp and strong adsorption bands of OH− at 3570 and 630 cm−1 , compared with the APS sample. The enrichment of the hydroxyl group is typical to the HA-LPPS coating where dehydroxylation is significantly suppressed likely due to the presence of the water vapor during the plasma spraying process [18].
J. He et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 303–308
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Fig. 1. XPS spectra of Ti and Ti with alkali-heat treatment: (a) Ti; (b) Ti-AH.
Fig. 2. FTIR spectra of the as-deposited APS and LPPS HA coatings.
3.2. Fibrous network formation PBS solution was selected as the medium to study the selfreconstitution of the collagen fibrils, to minimize the masking of other influential factors and to better identify the influence from the inorganic substrate and hydroxyl group. Fig. 3 shows the SEM surface micrographs of the collagen modified Ti, Ti-AH, HA-APS, HA-LPPS sample surfaces, after 4 h immersion in the PBS solution. Significant difference in collagen fibril formation has been observed
on the two different Ti surfaces. A large number of collagen fibrils were found on the OH enriched Ti-AH surface (Fig. 3b), while almost no fibrils were found on the untreated Ti surface (Fig. 3a). Similarly, there were a large number of collagen fibrils on the OH enriched HA-LPPS surface (Fig. 3d), which were rarely found on the HA-APS surface (Fig. 3c). Lateral fibril aggregation was clearly observed at the HA-LPPS surface, with a twisted structure. The SEM micrographs in Fig. 4 show the collagen self-assembly on sample surfaces after immersion in PBS solution for 2 days. Significant fibril self-reconstitution was observed and collagen fibrous networks were formed for all the samples (Fig. 4a–d). This was not surprising since the collagen self-reconstitution was a thermodynamically spontaneous process. The lateral aggregations of the fibrils were more prominent in the OH enriched samples (marked by arrows in Fig. 4f) for both Ti and HA group samples. In addition, similar to samples after 4 h immersion, the HA-LPPS samples showed a twisted structure (Fig. 4h), which was absent in the Ti-AH samples. The twisted structure is likely more thermodynamically stable and desirable in bone regeneration materials due to its superior mechanical properties. Overall, our results clearly suggest that OH groups enhance the collagen fibril formation at both Ti and HA surfaces, especially at early stage (4 h). The OH group might provide suitable electrostatic interaction with the collagen molecules and fibrils, thus facilitating the self-reconstitution of the collagen fibrils. Yang et al. have reported that the interaction would occur between the OH groups from the surfaces and the carboxyl groups ( COOH) from the collagen. They found that the higher the amount of collagen absorbed on the surface, the more OH positions have been occupied [21].
Fig. 3. SEM images showing the collagen fibril formation on the different material surfaces after 4 h immersion in PBS: (1) Ti: a and e; (2) Ti-AH: b and f; (3) HA-APS: c and g; (4) HA-LPPS: d and h (a–d: 2000×; e–h: 20,000×).
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Fig. 4. SEM images showing collagen fibrous network formation on the different material surfaces after 2 days immersion in PBS: (1) Ti: a and e; (2) Ti-AH: b and f; (3) HA-APS: c and g; (4) HA-LPPS: d and h (a–d: 2000×; e–h: 20,000×).
It also appeared that the HA surface exhibited accelerated collagen fibril reconstitution compared with the Ti surface. The HA lattices possess two different binding sites (the C and P sites), which would have significant interactions with the different charged groups such as the carboxyl and amine groups in protein [23]. The existence of HA, the major inorganic component of the bone, likely played an important role in the self-reconstitution of collagen fibrils and collagen conformation, thus affecting the cell–matrix interactions [24]. It has been reported that proper conformation of collagen might expose the cryptic sites critical for binding additional cell surface receptors or growth factors [24,25]. 3.3. Cell adhesion The SEM images (Fig. 5) revealed the morphologies of MSCs adhered on different sample surfaces. Most of the MSCs were well flattened and favorably spread across all sample surfaces, as a result of collagen modification. Indeed collagen is a superior substrate for enhanced cell adhesion and cell–matrix interactions [10,22]. It also appeared that the collagen fibers served as the anchoring sites for MSC adhesion. As seen in Fig. 5b and d, a lot of filopodia and
lamelipodia extended from the MSCs were found to firmly adhere to the collagen fibers, showing better spreading of MSCs on collagen modified surfaces enriched with OH groups. As shown in Fig. 6, FDA staining revealed the shapes of the live MSCs after 1 day culture, under the examination of the confocal laser scanning microscopy. The MSCs exhibited better attachment and spreading on HA substrates than those on the Ti substrates. In addition, whether the substrate surface was enriched with OH group or not also led to drastic difference. A large number of cells were found on the OH enriched Ti-AH surface (Fig. 6b), while few cells were found on the Ti surface (Fig. 6a). Similar trend was found for the HA samples, and there were more MSCs attached to the OH enriched HA-LPPS surface (Fig. 6d) than those attached to the HAAPS surface without the OH enrichment (Fig. 6c). Overall, the cell number increased significantly and the cell attachment were also improved on surfaces enriched with hydroxyl functional groups, with the best adhesion and spreading of MSCs on the HA-LPPS coating surface. To further evaluate the cytoskeletal organization of MSCs on different sample surfaces, MSCs were stained with phalloidin and DAPI to visualize the actin cytoskeleton structure. As shown in
Fig. 5. SEM micrographs of MSCs cultured on the different material surfaces after 1 day: (a) Ti; (b) Ti-AH; (c) HA-APS; (d) HA-LPPS.
J. He et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 303–308
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Fig. 6. CLSM photos of MSCs cultured on the different material surfaces with FDA dye for live cells after 1 day: (a) Ti; (b) Ti-AH; (c) HA-APS; (d) HA-LPPS.
Fig. 7d, the phalloidin staining clearly revealed the presence of stress fibers on OH enriched HA-LPPS surface. The cytoskeletal tension is reported to have an important influence on cell differentiation lineages through a RhoA-ROCK (Rho-associated protein
kinase) mechanism [26]. The contractile force generated internally through cytoskeleton organization is also a critical part for mechanotranductions [27] and may potentially play important role in activation of canonical Wnt and other signal pathways, which
Fig. 7. CLSM photos of MSCs cultured on the different material surfaces with phalloidin and DAPI staining after 1 day: (a) Ti; (b) Ti-AH; (c) HA-APS; (d) HA-LPPS.
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modulate osteogenic differentiation [28]. The absence of such stress fibers on OH deficient Ti (Fig. 7a) and APS-HA (Fig. 7c) surfaces further substantiated the positive influence of OH functional group on collagen network formation and the subsequent cytoskeletal organization of MSCs. Recent studies suggest that the collagen fibers play a significant role for stem cells to sense the mechanical feedback, and even affect the cell-fate decisions [11]. A proper mechanical feedback would also be important to modulate MSC migration [12] and other MSC functions. Our study suggests both the material itself and the hydroxyl group have critical influence on collagen self-assembly and the subsequent cell adhesion and cytoskeleton organization. The OH enriched HA surface (as in natural bone) is the best substrate for the formation of collagen fibrous network, and resulted in the best cell adhesion and stress fiber formation of MSCs. The combination of hydroxyapatite and collagen (the major inorganic and organic components of bone) might provide suitable substrate for cell attachment, spreading, and cytoskeletal organization. 4. Conclusions We have examined the effects of material selection and surface characteristic on collagen fibril reconstitution. The results indicated that the OH groups had a strong influence on the kinetics of collagen self-assembly and the fibrous network formation. In addition, the hydroxyapatite surface might also play an important role in promoting the collagen self-assembly and formation of a fibrous network compared with the Ti surface. Such influence was critical in cell adhesion, cytoskeletal organization and likely other biological functions of MSCs. The OH enriched HA surface was the best substrate for collagen fibrous network formation and subsequent MSC adhesion. The results might have important implications in biomaterial surface design for bone regeneration applications. Acknowledgements The study was supported by the National Basic Research program (No. 2012cb619103), Natural Science Foundation grants (Nos.
81271702 and 31170922), the Research Fund for the Doctoral Program of Higher Education (No. 20120181110058), and Program for New Century Excellent Talents in University (NCET-12-0387). References [1] Y. Kang, S. Kim, A. Khademhosseini, Y. Yang, Biomaterials 32 (2011) 6119–6130. [2] T. Rozario, D.W. Desimone, Dev. Biol. 341 (2010) 126–140. [3] G.C. Reilly, A.J. Engler, J. Biomech. 43 (2010) 55–62. [4] W.P. Daley, K.M. Yamada, Curr. Opin. Genet. Dev. 23 (2013) 408–414. [5] S.S. Wolter, A. Ngezahayo, B.N. Chichkov, Exp. Cell Res. 319 (2013) 1553–1561. [6] J.W. Lee, R. Juliano, Mol. Cells 17 (2004) 188–212. [7] B.A.C. Harley, L.J. Gibson, Chem. Eng. J. 137 (2008) 102–121. [8] C.Z. Liu, J. Bionic Eng. 5 (2008) 1–8. [9] K.E. Kadler, D.F. Holmes, J.A. Trotter, J.A. Chapman, Biochem. J. 316 (1996) 1–11. [10] J. He, B. Jiang, Y. Dai, J.Y. Hao, Z.K. Zhou, F. Wu, Z.W. Gu, Biomaterials 34 (2013) 6580–6588. [11] B. Trappmann, J.E. Gautrot, J.T. Connelly, D.G.T. Strange, Y. Li, M.L. Oyen, M.A.C. Stuart, H. Boehm, B. Li, V. Vogel, J.P. Spatz, F.M. Watt, W.T.S. Huck, Nat. Mater. 11 (2012) 642–649. [12] D.J. Montell, Science 322 (2008) 1502–1505. [13] G.C. Na, L.J. Butz, R.J. Carroll, J. Biol. Chem. 261 (1986) 12290–12299. [14] L. Sang, X.L. Wang, Z.H. Chen, J. Lu, Z.W. Gu, X.D. Li, Carbohydr. Polym. 82 (2010) 1264–1270. [15] D.I. Zeugolis, R.G. Paul, G. Attenburrow, Acta Biomater. 4 (2008) 1646–1656. [16] A. Ficai, E. Andronescu, V. Trandafir, C. Ghitulica, G. Voicu, Mater. Lett. 64 (2010) 541–544. [17] X.Y. Liu, P.K. Chu, C.X. Ding, Mater. Sci. Eng. R 47 (2004) 49–121. [18] Y. Huang, L. Song, T. Huang, X.G. Liu, Y.F. Xiao, Y. Wu, F. Wu, Z.W. Gu, Biomed. Mater. 5 (2010) 054113. [19] S. Nishiguchi, H. Kato, H. Fujita, M. Oka, H.M. Kim, T. Kokubo, T. Nakamura, Biomaterials 22 (2001) 2525–2533. [20] H.M. Kim, F. Miyaji, T. Kokubo, T. Nakamura, J. Biomed. Mater. Res. A 32 (1996) 409–417. [21] Q.Q. Wang, W. Li, B.C. Yang, J. Biomed. Mater. Res. A 99 (2011) 125–134. [22] J. He, T. Huang, L. Gan, Z.K. Zhou, B. Jiang, Y. Wu, F. Wu, Z.W. Gu, J. Biomed. Mater. Res. A 100 (2012) 1706–1715. [23] K. Kandori, S. Toshima, M. Wakamura, M. Fukusumi, Y. Morisada, J. Phys. Chem. B 114 (2010) 2399–2404. [24] R.O. Hynes, Science 326 (2009) 1216–1219. [25] V. Vogel, Annu. Rev. Biophys. Biomol. Struct. 35 (2006) 456–488. [26] R. McBeath, D.M. Pirone, C.M. Nelson, K. Bhadriraju, C.S. Chen, Dev. Cell 6 (2004) 483–495. [27] M.A. Wozniak, C.S. Chen, Nat. Rev. Mol. Cell Biol. 10 (2009) 34–43. [28] A. Liedert, D. Kaspar, R. Blakytny, L. Claes, A. Ignatius, Biochem. Biophys. Res. Commun. 349 (2006) 1–5.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effects of material and surface functional group on collagen self-assembly and subsequent cell adhesion behaviors Jing He, Yao Su, Tao Huang, Bo Jiang, Fang Wu ∗ , Zhongwei Gu National Engineering Research Centre for Biomaterials, Sichuan University, Chengdu 610064, PR China
a r t i c l e
i n f o
Article history: Received 4 September 2013 Received in revised form 7 January 2014 Accepted 8 January 2014 Available online 19 January 2014 Keywords: Hydroxyl group Collagen self-assembly Cell adhesion Collagen fibrous network Cytoskeleton
a b s t r a c t Collagen fibrous network not only provides structural support for cells but also serves as critical environment modulating various cell functions. Various factors would influence the collagen self-assembly but the effect of substrate surface on such process has been rarely studied. Here we examined the effects of materials (Ti and hydroxyapatite) and their surface characteristics (with and without the enrichment of hydroxyl group) on collagen self-reconstitution and fibrous network formation, and on subsequent cell adhesion and cytoskeleton organization of mesenchymal stem cells (MSCs). For both Ti and hydroxyapatite (HA) substrates, the enrichment of hydroxyl group ( OH) on substrate surfaces promoted the collagen self-reconstitution and facilitated the formation of the fibrous network after 4 h immersion in phosphate buffer solution (PBS), while all samples showed clear fibrous network formation after 2 day soaking in PBS. Compared with the Ti surfaces, the HA surfaces facilitated the self-reconstitution of collagen, leading to a more mature fibrous network with a twisted structure and enhanced lateral aggregation of fibrils. The fibrous network difference resulted in different behaviors of the subsequent MSC adhesion and spreading. The MSCs had the best adhesion and cytoskeleton organization on the OH enriched HA surface with collagen modification. Our results suggested that both the material selection and the hydroxyl group significantly influenced the collagen self-assembly and fibrous network formation and, as a result, the subsequent cell adhesion behaviors. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The extracellular matrix (ECM) is a complex organization composed of a wide variety of structural proteins and proteoglycans secreted by the cells [1,2]. The ECM proteins are able to bind growth factors and cytokines and can interact with bone cells via integrins or other specific cell surface receptors, thus controlling cell shape, motility, growth, survival and differentiation [3–6]. Type I collagen, the primary structural element in ECM and the major structural protein of bone, forms a fibrous network that not only provides a structural support for cells but also acts as an important regulator of cell behavior [7,8]. The fibrous architecture can provide a favorable microenvironment for cell attachment, binding of growth factors, and orchestrating signal and cellular events [9,10]. Recent studies have further suggested that the collagen fibers play a critical role for stem cells to sense the mechanical feedback and may ultimately affect the cell-fate decisions [11]. A proper mechanical feedback would be critical for MSC migration [12] and other MSC functions.
∗ Corresponding author. Tel.: +86 13438050329. E-mail addresses: [email protected], [email protected] (F. Wu). 0927-7765/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2014.01.009
It has been well known that solubilized collagen can selfassemble into fibrous structures in vitro with the characteristic axial periodic structure. Various factors would influence the kinetics of the self-reconstitution process, such as collagen concentration, pH value, temperature and ionic strength [9,13–15]. Collagen has often been used with other inorganic materials to form composites for bone regeneration applications [16]. The intrinsic property and surface characteristic of the inorganic material would likely exert a great influence on collagen fibril formation, thus affecting subsequent cellular response to the materials. However, the effects of material and its surface characteristic on the selfassembly of collagen molecules into the fibrils and fibrous network have been rarely studied. The most commonly used inorganic materials in orthopedic applications are Ti and hydroxyapatite (HA) [17,18]. Widely used in joint prosthesis and dentistry applications, the titanium is bio-inert despite of its excellent mechanical property. Alkali-heat treatment has been commonly used to make titanium surfaces bioactive by forming a bioactive sodium titanate layer on the titanium surface, in association with the increase of OH groups on the titanium surface [19,20]. HA is the major inorganic component of the natural bone and has been used as bioactive coating on Ti implants in artificial joints. HA coatings with different surface function groups could be synthesized using two different plasma spraying processes: the
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conventional air plasma spray (APS) process and the more recently developed liquid precursor plasma spraying (LPPS) process. During the APS process which uses powder as the feedstock material, serious dehydroxylation would occur due to the high process temperature, leading to the large loss of the OH group in the APS HA coatings. In the LPPS process which uses liquid precursor as the feedstock, the dehydroxylation effect is suppressed and as a result, the OH group is largely retained in the resultant LPPS HA coatings [18]. Therefore, Ti and HA substrates were prepared with diminished and enriched OH surface group respectively, as a result of different treatments applied to the substrates. The aim of this study was to examine the effects of material selection (Ti, HA) and the surface functional group on collagen self-assembly and the formation of the fibrous network, as well as its implication on subsequent cell adhesion behaviors.
2. Materials and methods 2.1. Materials preparation 2.1.1. Preparation of Ti with and without the alkali-heat treatment The titanium disks (14 mm × 2 mm) with and without alkaliheat treatment were used as the Ti substrates. For the alkali-heat treatment, the titanium disks were first cleaned by 10 min ultrasound treatment in pure acetone, ethanol and distilled water, respectively. The alkali-heat treatment was preformed according to the protocol described by Kim [20], using 10 M NaOH aqueous solution for 24 h at 60 ◦ C. After the alkali treatment, the titanium disks were rinsed with distilled water, dried in an oven at 60 ◦ C, and heat treated at 600 ◦ C for 1 h in air with a heating rate of 5 ◦ C min−1 . The functional groups of the pure and alkali treated Ti (Ti and TiAH, respectively) substrates were analyzed by X-ray photoelectron spectrometer (XPS, XSAM800, UK).
2.1.2. Preparation of HA coatings through the APS and LPPS processes HA coatings, with thickness around 100 m, have been deposited on the Ti–6Al–4V (14 mm × 2 mm) alloy substrate, using the APS and LPPS processes respectively. The liquid precursor was selected as the feedstock during the LPPS process, which was atomized into mists and injected into the plasma jet, instead of the powder feedstock used in the APS process. More detailed information of the APS and LPPS processes can be found elsewhere [18,21]. The functional groups of the APS and LPPS coatings were analyzed by Fourier Transform Infrared Spectroscopy (FTIR, Nicolet, 170SX, Wisconsin, USA), by scratching the powders from the HA coating samples.
2.1.3. Preparation of type I collagen solution and surface modification with collagen Type I collagen derived from bovine skin (provided by National Engineering Research Centre for Biomaterial, Sichuan University) was dissolved in acetic acid (pH 4.0) at 4 ◦ C with a concentration of 7.0 mg/ml. The surfaces of the four kinds of samples (referred as Ti, Ti-AH, HA-APS, HA-LPPS hereafter) were modified with the above collagen solution. Onto each sample we added 400 l collagen solution. First, half of the solution (200 l) was dropped on each sample, followed by air drying in a laminar flow cabinet at room temperature. Afterwards, the same process was repeated and the remaining half of the collagen solution was applied to the substrate.
2.2. Fibril network formation The four groups of samples were immersed into Dulbecco’s phosphate buffer solution (PBS, pH 7.4, Sigma, USA) to initiate the self-assembly and fibril network formation at 37 ◦ C. After being cultured for 4 h and 2 days respectively, the samples were fixed with 2.5% glutaraldehyde overnight, dehydrated in graded ethanol and isoamyl acetate, and subjected to critical point drying. After gold coating, the morphologies of the collagen fibrils were observed using the scanning electron microscope (SEM, S4800, Tecnai F20, Tokyo, Japan). 2.3. Cell culture Mesenchymal stem cells (MSCs) were isolated from rabbit bone marrow (1-week-old, New Zealand rabbits), as previously described [22]. MSCs were expanded in 20 ml of a-MEM containing 20% fetal bovine serum (FBS) and 1% antibiotics and cells from passage 3 were used for all the experiments. MSCs were seeded onto the surfaces of the samples that were placed in 24-well plates, with an initial density of 2.0 × 104 cells/well. Each well consisted of 1 ml of PBS supplemented with 20% FBS. 2.4. Cell morphology 2.4.1. Scanning electron microscopy After the MSCs were cultured for 1 day, the samples were washed twice with PBS. The MSCs were fixed with 2.5% glutaraldehyde buffer, dehydrated by a graded series of ethanol, subjected to critical point drying and gold coating. The cell morphologies were observed using the SEM (S4800, Tecnai F20, Tokyo, Japan). 2.4.2. Confocal microscopy Following one-day incubation, the MSCs were visualized using a confocal microscope (Leica SP5, Germany). Prior to observation, the cells were fixed in 1 l/ml fluorescein diacetate (FDA)/PBS (FDA, PBS, Sigma, USA) solution for live cells. To visualize the actin cytoskeleton, MSCs were fixed in 4% paraformaldehyde solution, treated with 0.1% Triton X-100 and stained with phalloidin Alexa 594 (Sigma) and DAPI (Sigma). 3. Results and discussions 3.1. Material characterizations 3.1.1. XPS analysis for Ti group samples The XPS spectra of the Ti and Ti-AH surfaces are shown in Fig. 1. Deconvolution of the O 1s peak revealed that the oxygen existed as TiO2 form in both Ti and Ti-AH surfaces. The oxygen peak (532.6 eV) of chemically adsorbed basic hydroxyl was clearly visible in the Ti-AH spectrum but was barely detectable in the spectrum of the untreated Ti surface, suggesting enrichment of hydroxyl group at Ti-AH surface. 3.1.2. FTIR analysis for HA group samples Fig. 2 shows the FTIR spectra of the as-deposited HA-APS and HA-LPPS coatings. Strong adsorption bands of H2 O (3434 and 1634 cm−1 ) and PO4 3− groups (1091, 1044, 960, 601, and 569 cm−1 ) were observed in both coatings. However, the HA-LPPS coatings exhibited quite sharp and strong adsorption bands of OH− at 3570 and 630 cm−1 , compared with the APS sample. The enrichment of the hydroxyl group is typical to the HA-LPPS coating where dehydroxylation is significantly suppressed likely due to the presence of the water vapor during the plasma spraying process [18].
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Fig. 1. XPS spectra of Ti and Ti with alkali-heat treatment: (a) Ti; (b) Ti-AH.
Fig. 2. FTIR spectra of the as-deposited APS and LPPS HA coatings.
3.2. Fibrous network formation PBS solution was selected as the medium to study the selfreconstitution of the collagen fibrils, to minimize the masking of other influential factors and to better identify the influence from the inorganic substrate and hydroxyl group. Fig. 3 shows the SEM surface micrographs of the collagen modified Ti, Ti-AH, HA-APS, HA-LPPS sample surfaces, after 4 h immersion in the PBS solution. Significant difference in collagen fibril formation has been observed
on the two different Ti surfaces. A large number of collagen fibrils were found on the OH enriched Ti-AH surface (Fig. 3b), while almost no fibrils were found on the untreated Ti surface (Fig. 3a). Similarly, there were a large number of collagen fibrils on the OH enriched HA-LPPS surface (Fig. 3d), which were rarely found on the HA-APS surface (Fig. 3c). Lateral fibril aggregation was clearly observed at the HA-LPPS surface, with a twisted structure. The SEM micrographs in Fig. 4 show the collagen self-assembly on sample surfaces after immersion in PBS solution for 2 days. Significant fibril self-reconstitution was observed and collagen fibrous networks were formed for all the samples (Fig. 4a–d). This was not surprising since the collagen self-reconstitution was a thermodynamically spontaneous process. The lateral aggregations of the fibrils were more prominent in the OH enriched samples (marked by arrows in Fig. 4f) for both Ti and HA group samples. In addition, similar to samples after 4 h immersion, the HA-LPPS samples showed a twisted structure (Fig. 4h), which was absent in the Ti-AH samples. The twisted structure is likely more thermodynamically stable and desirable in bone regeneration materials due to its superior mechanical properties. Overall, our results clearly suggest that OH groups enhance the collagen fibril formation at both Ti and HA surfaces, especially at early stage (4 h). The OH group might provide suitable electrostatic interaction with the collagen molecules and fibrils, thus facilitating the self-reconstitution of the collagen fibrils. Yang et al. have reported that the interaction would occur between the OH groups from the surfaces and the carboxyl groups ( COOH) from the collagen. They found that the higher the amount of collagen absorbed on the surface, the more OH positions have been occupied [21].
Fig. 3. SEM images showing the collagen fibril formation on the different material surfaces after 4 h immersion in PBS: (1) Ti: a and e; (2) Ti-AH: b and f; (3) HA-APS: c and g; (4) HA-LPPS: d and h (a–d: 2000×; e–h: 20,000×).
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Fig. 4. SEM images showing collagen fibrous network formation on the different material surfaces after 2 days immersion in PBS: (1) Ti: a and e; (2) Ti-AH: b and f; (3) HA-APS: c and g; (4) HA-LPPS: d and h (a–d: 2000×; e–h: 20,000×).
It also appeared that the HA surface exhibited accelerated collagen fibril reconstitution compared with the Ti surface. The HA lattices possess two different binding sites (the C and P sites), which would have significant interactions with the different charged groups such as the carboxyl and amine groups in protein [23]. The existence of HA, the major inorganic component of the bone, likely played an important role in the self-reconstitution of collagen fibrils and collagen conformation, thus affecting the cell–matrix interactions [24]. It has been reported that proper conformation of collagen might expose the cryptic sites critical for binding additional cell surface receptors or growth factors [24,25]. 3.3. Cell adhesion The SEM images (Fig. 5) revealed the morphologies of MSCs adhered on different sample surfaces. Most of the MSCs were well flattened and favorably spread across all sample surfaces, as a result of collagen modification. Indeed collagen is a superior substrate for enhanced cell adhesion and cell–matrix interactions [10,22]. It also appeared that the collagen fibers served as the anchoring sites for MSC adhesion. As seen in Fig. 5b and d, a lot of filopodia and
lamelipodia extended from the MSCs were found to firmly adhere to the collagen fibers, showing better spreading of MSCs on collagen modified surfaces enriched with OH groups. As shown in Fig. 6, FDA staining revealed the shapes of the live MSCs after 1 day culture, under the examination of the confocal laser scanning microscopy. The MSCs exhibited better attachment and spreading on HA substrates than those on the Ti substrates. In addition, whether the substrate surface was enriched with OH group or not also led to drastic difference. A large number of cells were found on the OH enriched Ti-AH surface (Fig. 6b), while few cells were found on the Ti surface (Fig. 6a). Similar trend was found for the HA samples, and there were more MSCs attached to the OH enriched HA-LPPS surface (Fig. 6d) than those attached to the HAAPS surface without the OH enrichment (Fig. 6c). Overall, the cell number increased significantly and the cell attachment were also improved on surfaces enriched with hydroxyl functional groups, with the best adhesion and spreading of MSCs on the HA-LPPS coating surface. To further evaluate the cytoskeletal organization of MSCs on different sample surfaces, MSCs were stained with phalloidin and DAPI to visualize the actin cytoskeleton structure. As shown in
Fig. 5. SEM micrographs of MSCs cultured on the different material surfaces after 1 day: (a) Ti; (b) Ti-AH; (c) HA-APS; (d) HA-LPPS.
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Fig. 6. CLSM photos of MSCs cultured on the different material surfaces with FDA dye for live cells after 1 day: (a) Ti; (b) Ti-AH; (c) HA-APS; (d) HA-LPPS.
Fig. 7d, the phalloidin staining clearly revealed the presence of stress fibers on OH enriched HA-LPPS surface. The cytoskeletal tension is reported to have an important influence on cell differentiation lineages through a RhoA-ROCK (Rho-associated protein
kinase) mechanism [26]. The contractile force generated internally through cytoskeleton organization is also a critical part for mechanotranductions [27] and may potentially play important role in activation of canonical Wnt and other signal pathways, which
Fig. 7. CLSM photos of MSCs cultured on the different material surfaces with phalloidin and DAPI staining after 1 day: (a) Ti; (b) Ti-AH; (c) HA-APS; (d) HA-LPPS.
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modulate osteogenic differentiation [28]. The absence of such stress fibers on OH deficient Ti (Fig. 7a) and APS-HA (Fig. 7c) surfaces further substantiated the positive influence of OH functional group on collagen network formation and the subsequent cytoskeletal organization of MSCs. Recent studies suggest that the collagen fibers play a significant role for stem cells to sense the mechanical feedback, and even affect the cell-fate decisions [11]. A proper mechanical feedback would also be important to modulate MSC migration [12] and other MSC functions. Our study suggests both the material itself and the hydroxyl group have critical influence on collagen self-assembly and the subsequent cell adhesion and cytoskeleton organization. The OH enriched HA surface (as in natural bone) is the best substrate for the formation of collagen fibrous network, and resulted in the best cell adhesion and stress fiber formation of MSCs. The combination of hydroxyapatite and collagen (the major inorganic and organic components of bone) might provide suitable substrate for cell attachment, spreading, and cytoskeletal organization. 4. Conclusions We have examined the effects of material selection and surface characteristic on collagen fibril reconstitution. The results indicated that the OH groups had a strong influence on the kinetics of collagen self-assembly and the fibrous network formation. In addition, the hydroxyapatite surface might also play an important role in promoting the collagen self-assembly and formation of a fibrous network compared with the Ti surface. Such influence was critical in cell adhesion, cytoskeletal organization and likely other biological functions of MSCs. The OH enriched HA surface was the best substrate for collagen fibrous network formation and subsequent MSC adhesion. The results might have important implications in biomaterial surface design for bone regeneration applications. Acknowledgements The study was supported by the National Basic Research program (No. 2012cb619103), Natural Science Foundation grants (Nos.
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