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Universal method for protein bioconjugation with nanocellulose scaffolds for increased cell adhesion
Materials Science and Engineering C 33 (2013) 4599–4607
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Universal method for protein bioconjugation with nanocellulose scaffolds for increased cell adhesion Volodymyr Kuzmenko a,b,1, Sanna Sämfors c,1, Daniel Hägg c, Paul Gatenholm b,c,⁎ a b c
Department of Microtechnology and Nanoscience, Chalmers University of Technology, Kemivägen 9, SE-412 96 Gothenburg, Sweden Wallenberg Wood Science Center, Chalmers University of Technology, Kemivägen 4, SE-412 96 Gothenburg, Sweden Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 4, SE-412 96 Gothenburg, Sweden
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Article history: Received 23 May 2013 Received in revised form 26 June 2013 Accepted 17 July 2013 Available online 30 July 2013 Keywords: Nanocellulose Protein bioconjugation Cell adhesion Cell morphology
a b s t r a c t Bacterial nanocellulose (BNC) is an emerging biomaterial since it is biocompatible, integrates well with host tissue and can be biosynthesized in desired architecture. However, being a hydrogel, it exhibits low affinity for cell attachment, which is crucial for the cellular fate process. To increase cell attachment, the surface of BNC scaffolds was modified with two proteins, fibronectin and collagen type I, using an effective bioconjugation method applying 1-cyano-4-dimethylaminopyridinium (CDAP) tetrafluoroborate as the intermediate catalytic agent. The effect of CDAP treatment on cell adhesion to the BNC surface is shown for human umbilical vein endothelial cells and the mouse mesenchymal stem cell line C3H10T1/2. In both cases, the surface modification increased the number of cells attached to the surfaces. In addition, the morphology of the cells indicated more healthy and viable cells. CDAP activation of bacterial nanocellulose is shown to be a convenient method to conjugate extracellular proteins to the scaffold surfaces. CDAP treatment can be performed in a short period of time in an aqueous environment under heterogeneous and mild conditions preserving the nanofibrillar network of cellulose. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The need for new functional biomaterials for applications such as drug delivery, biosensors, bioseparation, diagnostics and engineering new tissues is growing rapidly. In tissue engineering, biomaterials are used as scaffolds to provide support for the formation of new tissue and a substrate for infiltrating host cells to attach to, as well as a vehicle for implantation of donor cells. A development of biocompatible materials that exhibit good mechanical properties and at the same time facilitate cell adhesion is needed to create functional tissues in vitro and regenerate organs in vivo. Bacterial nanocellulose (BNC) has previously been demonstrated to be a good biomaterial for different tissue engineering applications due to its biocompatibility [1] and good mechanical properties [2]. It is chemically pure and can be produced in various shapes and sizes [3,4].
Abbreviations: BNC, Bacterial nanocellulose; CDAP, 1-Cyano-4-dimethylaminopyridinium tetrafluoroborate; HUVECs, Human umbilical vein endothelial cells; MSCs, Mesenchymal stem cells; ECM, Extracellular matrix; TEA, Triethylamine; DAPI, 4′,6-diamino-2-phenylindole. ⁎ Corresponding author at: Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 4, SE-412 96 Gothenburg, Sweden. Tel.: + 46 31 772 34 07; fax: + 46 31 772 34 18. E-mail addresses: [email protected] (V. Kuzmenko), [email protected] (S. Sämfors), [email protected] (D. Hägg), [email protected] (P. Gatenholm). 1 These authors contributed equally to this manuscript. 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.07.031
It has been investigated for use as a scaffold material in engineering various types of tissues such as skin [5], blood vessels [6], bone [7] and cartilage [8]. The ability of the BNC nanofibrillar network to interact with water might contribute to the good biocompatibility of BNC [9]. This might also hinder proteins and cells from interacting with the BNC surface. Poor cell interaction was observed in a study by Fink et al., where endothelial cells showed a lack of adhesion to BNC biomaterial [10]. In tissue engineering, control of scaffold–cell interaction is crucial. For many cell types, such as osteoblasts [11], neuronal cells [12], endothelial cells [13] and adipose-derived cells [14], cell attachment to the surface is important for the viability and subsequent tissue development. Recent studies have shown the effect of focal adhesion points on shape and consequently on the stem cell differentiation process [15,16]. An effective approach to enhance cell adhesion on biomaterials has been to mimic the extracellular matrix (ECM) [17,18]. Among the most commonly used proteins for this purpose are fibronectin and collagen since they are the most abundant proteins in the ECM involved in cell interactions [17]. In general, cell attachment to solid surfaces occurs by interaction between cell adhesion receptors, such as integrins, and the surface material [19]. Both fibronectin and collagen can connect to the cell integrins via a specific amino acid sequence (RGD sequence) called cell binding domains. This specific interaction between proteins and cells can be mimicked by binding these proteins (or amino acid sequences) to the surface of the chosen scaffold material [20]. In a study by Bodin et al., RGD sequences were introduced to the BNC
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surface by adsorption of xyloglucan-RGD bioconjugates, which showed improvement of endothelial cell adhesion [21]. In other studies, carbohydrate binding modules tied with RGD were used to modify BNC scaffolds to enhance cell adhesion [22,23]. The accessibility of cellulose for bioconjugation is determined by the hydroxylic content of its sugar backbone [24]. Crosslinking compounds can turn the hydroxyl residues into intermediate reactive derivatives having suitable leaving groups for nucleophilic substitution. As a result of reaction between activated hydroxyls and strong nucleophiles, such as amines, stable covalent bonds that connect carbohydrate and the amine-containing molecule are obtained [25]. Among the most common activating agents for cellulose are carbonyldiimidazole [26], N-hydroxysuccinimide esters [27], sodium periodate [28], epoxide compounds [29], tresyl- and tosyl-chloride [30], cyanogen bromide [31], cyanuric chloride [32] and several chloroformate derivatives [33]. The activation process usually requires nonaqueous solutions (i.e., dry dioxane, THF, acetone, DMF or DMSO) to prevent hydrolysis of the reactive intermediate products in aqueous solution [34]. Hydroxyl groups can be modified in an aqueous environment with anhydrides, chloroacetic acid [35] or radical-mediated oxidation with (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl [36] to produce carboxylate functionality for further conjugation purposes with the use of carbodiimides as crosslinkers. In all cases, the reaction takes several hours. Another approach has been to introduce electrostatic interaction by providing an electrical charge to the cellulose surface [37]. However, since all the proteins are amine-containing molecules, an aminecoupling process can be preferably used for covalent conjugation [38]. Swelling cellulose in water improves the availability of OH groups in heterogeneous reactions, which makes an aqueous environment more suitable for activation [39]. Unlike most of the activating agents mentioned above, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) is stable to hydrolysis at a wide pH range due to the resonative delocalization of the positive charge over the whole aromatic ring and thus makes it a very promising activating agent [40]. The exploitation of CDAP as an activating agent was mentioned first by Kohn to replace toxic cyanogen bromide [41]. It was later used for activating soluble polysaccharides to get protein–polysaccharide conjugate vaccines and immunological reagents [42,43] and for activating cellulose membranes for the development of immunosensors [44]. In this study, a unique scaffold made of bacterial nanocellulose is being developed to mimic the properties of a natural extracellular matrix. It is evaluated for its support of attachment and growth of different human cell types at 2D level. Emphasis is put on the use of CDAP in the surface activation of BNC and its subsequent bioconjugation with different proteins to increase cell adhesion on the scaffold material.
2. Materials and methods 2.1. Materials Complex culture medium was prepared according to Matsuoka protocol [45]. Sodium hydroxide (NaOH), CDAP, acetonitrile, triethylamine (TEA), hydrochloric acid (HCl), glutaraldehyde, Triton X100, acetic acid and tert-butanol were obtained from Sigma-Aldrich (USA). Formaldehyde was purchased from Polysciences (USA). Lyophilized fibronectin bovine, collagen I bovine, rhodamine-phalloidin, 4′,6diamino-2-phenylindole and dilactate (DAPI) were obtained from Invitrogen (USA). Ethanol 99.7% was purchased from Solveco AB (Sweden). Endothelial basal media, endothelial growth supplements and human umbilical vein endothelial cells were purchased from Cell Application (USA). Mouse mesenchymal stem cells (MSCs, line C3H10T1/2) were purchased from ATCC (USA) and cultured in Basal Medium Eagle from HyClone (USA). Dulbecco's phosphate buffered saline solution (PBS 1×, without Ca & Mg) was purchased from PAA
Laboratories (Austria). MilliQ water used in all phases of the experiment was deionized and further purified with Millipore Synergy UV unit. 2.2. Methods 2.2.1. Scaffold production 24 well-plates were inoculated with Gluconacetobacter Xylinus (Acetobacter Xylinum susp. Sucrofermentas BPR2001, ATCC No. 700178). 0.5 ml of bacteria suspension and 1 ml of complex culture media were added to each well. The 24 well-plates were placed in an incubator for 48 hours at 30 °C. After 2 days of culture, pellicles of bacterial nanocellulose with a thickness of 2–3 mm and 1.6 cm in diameter had formed in each well. 2.2.2. Purification of BNC scaffolds The bacteria were removed from the BNC pellicles by placing them in NaOH (0.1 M) and heating in a shaking water bath (60 °C) for 1 hour. The pellicles were placed in a beaker with fresh NaOH (0.1 M) on a magnetic stirrer at room temperature overnight. The NaOH (0.1 M) was changed to fresh NaOH (0.1 M) two times a day for 2 days. To remove bacterial residues and neutralize the pH, the pellicles were placed in a beaker with deionized water on a magnetic stirrer. The water was changed to fresh deionized water two times a day for 4 days. Scaffolds were autoclaved and placed in pyrogen-free water in a shaking water bath (60 °C). The pyrogen-free water was changed two times a day for 4 days under sterile conditions in a LAF-hood. 2.2.3. CDAP modification of scaffolds To enhance cell adhesion to the bacterial nanocellulose, scaffolds were modified with CDAP as a crosslinker to bind necessary proteins to the surface of BNC. CDAP was dissolved in acetonitrile and mixed with 0.2 M TEA. To remove excess surface water from the scaffolds, they were wiped slightly by filter paper. The CDAP solution was added to the scaffolds by pipetting a few drops of solution on top of each scaffold. The solution was left to react for 1 minute, and 0.1 M HCl was added to stop the reaction. The scaffolds were rinsed with phosphate buffered saline solution (PBS) until neutral pH was reached. 2.2.4. Protein surface coating Lyophilized proteins were first dissolved and then diluted to the concentration of 100 μg/ml (fibronectin was dissolved in deionized water and diluted with PBS; collagen type I was diluted with 0.05 M acetic acid). 400 μl of protein solution was added to each scaffold (CDAP modified BNC and control samples of untreated BNC) and left to react overnight in 37 °C. The scaffolds were rinsed in PBS several times in order to remove free proteins. 2.2.5. Expansion of HUVECs Endothelial basal media was mixed with endothelial growth supplements to create endothelial cell growth media. A vial of HUVECs was thawed in a water bath at 37 °C. The cells were transferred to a 75-cm2 cell culture flask containing 15 ml of endothelial cell growth media. The cell culture flask was placed in an incubator (37 °C, 5% CO2, 95% relative humidity). The cells were split when they reached 80% confluency. 2.2.6. Expansion of MSCs One vial of MSCs was removed from the liquid N2 tank, thawed and transferred to basal medium eagle supplied with 10% fetal bovine serum and 1% antibiotics/antimyogenics. The cells were expanded in 75 cm2 flasks as described above. 2.2.7. Seeding of HUVECs and MSCs on BNC scaffolds Before cell seeding, scaffolds were rinsed two times in PBS and placed in 24 well-plates, one scaffold in each well. 200 μl of cell suspension was seeded on the scaffolds at a cell density of 3 × 105 cell/cm2 for the HUVECs and 2.53 × 105 cell/cm2 for the MSCs. The well-plates were
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Fig. 1. Cellulose bioconjugation using CDAP. SEM image of BNC on the top left. 1. The reaction of CDAP treatment. 2. The reaction of protein conjugation with CDAP-activated cellulose.
placed in an incubator (37 °C, 5% CO2, 95% relative humidity) for 4 hours to allow optimal cell attachment. 1.5 ml of cell media was then added to each scaffold and the well-plate was placed back into the incubator (37 °C, 5% CO2, 95% relative humidity); this was denoted day 0. Media was changed every 2 to 3 days and samples were taken at day 1, day 3 and day 8.
contact angle system at Attension Theta contact angle meter. A small drop of water was deposited on the surface, while 20 pictures were taken over a period of 10 seconds. To calculate the contact angle, Attension Theta Software was used. Measurements were done on untreated BNC, BNC + CDAP, BNC + fibronectin, BNC + collagen, BNC + CDAP + fibronectin and BNC + CDAP + collagen samples.
2.2.8. Contact angle measurements Samples were washed in sterilized deionized water two times and left to dry on aluminum foil in a LAF-hood. When the samples were completely dry, the contact angles were analyzed using a sessile drop
2.2.9. Electron Spectroscopy for Chemical Analysis (ESCA) Samples were washed in sterilized deionized water two times and left to dry on aluminum foil in a LAF-hood. When the samples were completely dry, the contact angles were analyzed with a Quantum
Fig. 2. High resolution carbon spectra and surface elemental composition (for C, O and N in %) from ESCA analysis on untreated BNC, CDAP, fibronectin, collagen, CDAP + fibronectin and CDAP + collagen treated BNC. Corresponding bonds: 1) C―C; 2') C―O or 2) C―O, C―N; 3') O―C―O or 3) O―C―O, O―C―N; 4') O―C―O or 4) O―C―O, O―C―N.
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4–5 nm. The results were evaluated using MultiPAK 6.0 software. Measurements were performed on untreated BNC, BNC + CDAP, BNC + fibronectin, BNC + collagen, BNC + CDAP + fibronectin and BNC + CDAP + collagen samples. 2.2.10. Scanning Electron Microscopy (SEM) Samples were washed two times in PBS to get rid of media residues and fixated for 2 hours in 2.5% glutaraldehyde in PBS. The glutaraldehyde was removed by rinsing the samples two times in PBS. The scaffolds were dehydrated by placing them in baths of increasing ethanol concentrations (70%, 80%, 90%, 95% and 99%). The samples were placed in t-butanol and placed in a -80 °C freezer until they were completely frozen. Samples were freeze dried in a lyophilizer (Heto, PowerDry, PL3000). Samples were mounted on SEM stubs with adhesive tape (TAAB) and sputter coated (Sputter Coater s150B, Edwards) with a thin gold layer for 1 minute. SEM analysis was performed with a Leo Ultra 55 FEG-SEM.
Fig. 3. Advancing water contact angles of untreated BNC, CDAP, CDAP + fibronectin and CDAP + collagen treated BNC.
2000 scanning ESCA microprobe using an Al Kα X-ray source with a beam size of 100 μm, and the angle between the detector and analyzed sample was 45°. The analyzed area was 500 × 500 μm with a depth of
2.2.11. Fluorescence microscopy (FM) Samples taken for FM analysis were washed in PBS two times to get rid of media residues. For fixation the scaffolds were covered in 3.7% formaldehyde in PBS and left for 2 hours. Samples were rinsed two times in PBS and transferred to 60% ethanol, where they were kept until analysis. Before staining of the samples, they were rinsed in PBS two times. 0.1% Triton X100 in PBS was added to cover the samples and they were left for 45 minutes. The solution was aspirated and the samples were washed two times in PBS. Rhodamine-phalloidin in PBS
Fig. 4. SEM images of HUVECs on unmodified BNC scaffolds and fibronectin modified scaffolds (with and without CDAP activation) after 1, 3 and 8 days of culturing.
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(10 μg/ml) was added to cover the samples and they were left for 30 minutes. The solution was aspirated and DAPI in PBS (5 μg/ml) was added to cover the samples for 30 minutes. The samples were rinsed two times in PBS. For analysis, a phase contrast microscope (Leica Digital Microscopes Inverted) was used together with the software Leica LAS AF.
3. Results and discussion 3.1. BNC scaffolds The bacteria-produced cellulose chains self-assembled into a dense network of nanofibers with similar dimensions as collagen fibers in the extracellular matrix in tissues (Fig. 1 left), i.e. about 1.6 × 5.8 nm in cross section [46]. To remove the bacterial remains, the BNC pellicles were washed with NaOH several times. Since cellulose is nitrogen free, and nitrogen is a constituent of medium components, nucleic acids and peptidoglycans present in bacterial cell wall, the amount of nitrogen was used as an evidence of the presence of bacterial residues in the pellicles [47]. It was confirmed using ESCA that there were no traces of nitrogen left in purified BNC (Fig. 2).
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3.2. CDAP modification of nanocellulose scaffolds The stability of CDAP (it is not hygroscopic and can easily be handled at room temperature in open air) and selectivity towards reaction with cellulosic hydroxyl groups make it a suitable reagent for modifications of BNC. After a few minutes of reaction, CDAP-activated cellulose (cyanate ester derivative) was generated (Fig. 1). The success of the reaction is demonstrated by the appearance of nitrogen (2.5%) in BNC activated with CDAP (Fig. 2). Moreover, side products were removed from the modified material before further interaction with proteins by a simple washing step in diluted HCl avoiding complicated purification procedures [40].
3.3. Bioconjugation with proteins Proteins were attached directly to CDAP-activated cellulose via the amine groups of surface amino acids at optimal pH of 9–10, forming an isourea bond (Fig. 1) [43]. High resolution C1s spectra of carbon from ESCA are shown in Fig. 2. C1s spectra are asymmetric and broadened. They were resolved to several components which were assigned to C―C (284.2 eV, the binding energy scale was charge referenced to this value as starting position),
Fig. 5. FM images of HUVECs on unmodified BNC scaffolds and fibronectin modified scaffolds (with and without CDAP activation) after 1, 3 and 8 days of culturing. Cell nuclei stained with DAPI shown in blue, actin filaments stained with rhodamine-phalloidin shown in red.
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C―O and C―N (285.9 eV), O―C―O and O―C–N (287.2 eV), O―C O and N―C O (288.2 eV) [48,49]. It is clear that peaks 1 and 3 were higher for the CDAP + fibronectin treated cellulose samples in comparison with cellulose treated with fibronectin only. The same phenomenon was present for CDAP + collagen treated versus collagen treated cellulose for peak 1, although the difference was slight for peak 3. This effect can be explained by the increase in proteins bonded to the surface, meaning the appearance of more C―C and O―C―N bonds (for 1 and 3 peaks, respectively) [50]. The atomic composition of the different samples after protein treatment is also presented in Fig. 2. Obviously, an untreated BNC is a polysaccharide made of carbon, oxygen and hydrogen only, it has no nitrogen. Since all the proteins contain nitrogen as an essential part of the structural amino acids, the nitrogen was found in all protein treated samples, as expected [51]. Protein adsorption was observed on the BNC surfaces that were not preliminarily activated with CDAP; however it was not as high as for the preactivated surfaces. It is vividly shown that cellulose surface modified with CDAP promoted protein attachment: for fibronectin, the nitrogen content increased from 5.7% to 11.8%, and for collagen from 1.5% to 3.8%. The higher percentages of nitrogen for fibronectin in comparison to collagen may be due to the much higher molecular mass of fibronectin, which can reach up to 500 kDa, as compared to collagen type I, with 139 kDa [52]. Water contact angle measurements shown at Fig. 3 confirmed the highly hydrophilic nature of native cellulose surface (the advancing
water contact angle was only 16°). After the treatment with the hydrophobic CDAP the surface of BNC samples expectedly had become less polar which was reflected in the increase of contact angle to 38°. After bioconjugation of CDAP-activated cellulose with proteins, the advancing contact angle reached 62° and 36° for fibronectin- and collagentreated samples respectively. Such a big alteration between differently modified surfaces can be partly explained by the contribution of hydrophilic amino acids (proline and hydroxyproline with polar OH groups), which are present in high amounts in collagen [53]. These amino acids make collagen surface more hydrophilic in comparison to the surface of fibronectin. Another explanation we can give is the smaller size of collagen molecules which were unable to cover the BNC surface as tightly as much bigger molecules of fibronectin [52]. The analysis of the surface modification confirmed that fibronectin and collagen were successfully conjugated to the BNC surface and that CDAP activation increased the yield of the attached proteins. 3.4. Cell studies To determine the effect of surface modification on cell adhesion, three types of surfaces were used: untreated BNC and BNC with adsorbed proteins as controls, and BNC bioconjugated with proteins via CDAP activation as the main object of investigation. HUVECs and MSCs were seeded on the different BNC substrates and evaluated by SEM and FM after 1, 3 and 8 days of culturing. FM images better reflect the level of cell adhesion than SEM images due to the
Fig. 6. SEM images of MSCs on unmodified BNC scaffolds and collagen modified scaffolds (with and without CDAP activation) after 1, 3 and 8 days of culturing.
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Fig. 7. SEM images of MSCs: (A) cell with a rounded shape on the untreated BNC; (B) well-stretched cell on the CDAP + collagen treated BNC.
treatment of the samples before SEM analysis (cells with weak adhesion might de-attach). SEM analysis of HUVECs cultivated on different surfaces is shown in Fig. 4. After day 1, no big differences in cell amount could be detected; however, cells on the BNC + CDAP + fibronectin treated surfaces exhibited a more stretched-out morphology than the cells on the other surfaces. After day 3, dissimilarities could be seen in the amount of cells on the three different surfaces. Many cells were found spread out on the surface on the CDAP preactivated samples while, for the untreated BNC and BNC coated with fibronectin,
not many cells could be detected. After 8 days, the difference was even larger. The few cells found on the untreated BNC and the BNC + fibronectin samples had a rounded morphology, indicating that they were de-attaching, while the cells on the CDAP + fibronectin treated surfaces formed almost a confluent layer after 8 days. FM images of HUVECs on the fibronectin treated surfaces and untreated surfaces also confirmed the effect of CDAP treatment (Fig. 5). A difference in morphology of the cells seeded on the untreated surface and the fibronectin treated surface was already observed at day
Fig. 8. FM images of MSCs on unmodified BNC scaffolds and collagen modified scaffolds (with and without CDAP activation) after 1, 3 and 8 days of culturing.
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1. The cells on the CDAP activated surfaces had more defined actin filaments while the filaments on the untreated BNC and BNC + fibronectin were not exactly aligned. After day 3 and day 8, not many cells could be found on the untreated surfaces. In contrast, on the CDAP treated surfaces, many cells were found after day 3 and day 8 that adhered to the surface and whose actin filaments were well defined and aligned. The cells acquired the cobblestone morphology associated with endothelial cells, as opposed to the more stretched out morphology of cells grown on untreated BNC or BNC + fibronectin. This indicates that conjugation of the protein to the BNC surface is preferable compared to the simple adsorption of the protein. To prove that the method using CDAP for the bioconjugation of proteins to cellulose is universal, MSCs were seeded on BNC conjugated with collagen type I. SEM analysis of cells seeded on the different surfaces showed that the bioconjugation in fact increased the amount of cells growing on the BNC surface (Fig. 6). After day 1, cell morphology was already markedly different when as compared with untreated surfaces, surfaces treated with only collagen and CDAP + collagen treated surfaces. Very few cells were found on the first two types of surfaces. The cells detected had a rounded shape, demonstrating poor cell adhesion to the surface (Fig. 7A). On the CDAP + collagen modified surfaces, many cells were spread out on the surface, creating an almost confluent layer, with a stretched-out morphology indicating the healthy status of the cells (Fig. 7B). After day 3 and day 8, the amount of cells, as well as their morphology, on the three different surfaces remained about the same. MSCs on the collagen treated surfaces and untreated surfaces were analyzed with FM as well (Fig. 8). Differences in morphology and the total number of the attached cells seeded on the untreated surface, the collagen treated and CDAP + collagen treated surface were already observed at day 1. First of all, the cells on the CDAP activated surfaces had more defined actin filaments; in contrast, the filaments on the other two types of surfaces were not really aligned. Second, CDAP treated pellicles were almost completely covered with the attached cells, forming a confluent layer, whereas not many cells could be found on the untreated BNC and treated with collagen only. The situation did not change significantly after 3 and 8 days of cell culturing. The positive influence of CDAP treatment on cell adhesion is shown once again.
4. Conclusions This study demonstrated that cell adhesion can be increased by bioconjugation of extracellular matrix proteins with the scaffold surface via a convenient two-step reaction using CDAP as an effective activation agent. Biocompatible nanofibrillar cellulose was used as a scaffold material suitable for cell study and potential tissue engineering. Using BNC as substrate with covalently bonded proteins was shown to enhance attachment of two different types of cells, forming confluent layers of healthy cells. The cells on the CDAP + protein treated surfaces were found in large quantity and had an appropriate morphology with defined actin filaments throughout all the days of cell culturing. In contrast, much fewer cells were attached to the substrate of the untreated BNC scaffolds and the BNC scaffolds with extracellular proteins simply adsorbed on the surface even after the first day of culturing, and the shape of those cells was not at all satisfactory.
Acknowledgements The Wallenberg Wood Science Center funded by Knut and Alice Wallenberg Foundation is greatly acknowledged for their financial support. We also thank Dr. Ergang Wang, Anders Mårtensson, Anne Wendel, and Johan Sundberg for their experimental assistance, as well as Janet Vesterlund for proofreading the English version of the manuscript.
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Volodymyr Kuzmenko obtained his Master of Science degree in 2012 at Chalmers University of Technology, where he continued his work as a PhD student at the Department of Microtechnology and Nanoscience collaborating with Wallenberg Wood Science Center, WWSC. The main focus of his research is on synthesis, modification and application of nanomaterials from renewable resources.
Sanna Sämfors got her master degree in Biotechnology from Chalmers University of Technology in 2013. Her master thesis focused on development of vascularized scaffolds for tissue engineering applications. She is currently continuing work on further development of these scaffolds.
Dr Daniel Hägg got his doctorate on genetics and genomics in the cardiovascular field from Göteborg University, Sweden, in 2008. Since then, he has shifted his research focus towards tissue engineering, primarily adipose tissue, first during his post-doctoral visit at Columbia University, New York, and at his current position at Chalmers University, Sweden. His main interests are 3D cell culture and cell differentiation.
Dr. Paul Gatenholm is currently professor of Biopolymer Technology at Chalmers University of Technology, Director of Biosynthetic Blood Vessels Laboratory, Coordinator of EAREG program and Director of Graduate School at Wallenberg Wood Science Center, WWSC. He is also Adjunct Professor at Virginia Tech, Wake Forest University and Wake Forest Institute for Regenerative Medicine in Winston-Salem, North Carolina, USA. Dr. Gatenholm is a material scientist with interest in biomimetic design of new biomaterials that can replace or regenerate tissue and organs. His research includes biological fabrication through the use of enzymes, cells, and the coordination of biological systems.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Universal method for protein bioconjugation with nanocellulose scaffolds for increased cell adhesion Volodymyr Kuzmenko a,b,1, Sanna Sämfors c,1, Daniel Hägg c, Paul Gatenholm b,c,⁎ a b c
Department of Microtechnology and Nanoscience, Chalmers University of Technology, Kemivägen 9, SE-412 96 Gothenburg, Sweden Wallenberg Wood Science Center, Chalmers University of Technology, Kemivägen 4, SE-412 96 Gothenburg, Sweden Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 4, SE-412 96 Gothenburg, Sweden
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Article history: Received 23 May 2013 Received in revised form 26 June 2013 Accepted 17 July 2013 Available online 30 July 2013 Keywords: Nanocellulose Protein bioconjugation Cell adhesion Cell morphology
a b s t r a c t Bacterial nanocellulose (BNC) is an emerging biomaterial since it is biocompatible, integrates well with host tissue and can be biosynthesized in desired architecture. However, being a hydrogel, it exhibits low affinity for cell attachment, which is crucial for the cellular fate process. To increase cell attachment, the surface of BNC scaffolds was modified with two proteins, fibronectin and collagen type I, using an effective bioconjugation method applying 1-cyano-4-dimethylaminopyridinium (CDAP) tetrafluoroborate as the intermediate catalytic agent. The effect of CDAP treatment on cell adhesion to the BNC surface is shown for human umbilical vein endothelial cells and the mouse mesenchymal stem cell line C3H10T1/2. In both cases, the surface modification increased the number of cells attached to the surfaces. In addition, the morphology of the cells indicated more healthy and viable cells. CDAP activation of bacterial nanocellulose is shown to be a convenient method to conjugate extracellular proteins to the scaffold surfaces. CDAP treatment can be performed in a short period of time in an aqueous environment under heterogeneous and mild conditions preserving the nanofibrillar network of cellulose. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The need for new functional biomaterials for applications such as drug delivery, biosensors, bioseparation, diagnostics and engineering new tissues is growing rapidly. In tissue engineering, biomaterials are used as scaffolds to provide support for the formation of new tissue and a substrate for infiltrating host cells to attach to, as well as a vehicle for implantation of donor cells. A development of biocompatible materials that exhibit good mechanical properties and at the same time facilitate cell adhesion is needed to create functional tissues in vitro and regenerate organs in vivo. Bacterial nanocellulose (BNC) has previously been demonstrated to be a good biomaterial for different tissue engineering applications due to its biocompatibility [1] and good mechanical properties [2]. It is chemically pure and can be produced in various shapes and sizes [3,4].
Abbreviations: BNC, Bacterial nanocellulose; CDAP, 1-Cyano-4-dimethylaminopyridinium tetrafluoroborate; HUVECs, Human umbilical vein endothelial cells; MSCs, Mesenchymal stem cells; ECM, Extracellular matrix; TEA, Triethylamine; DAPI, 4′,6-diamino-2-phenylindole. ⁎ Corresponding author at: Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 4, SE-412 96 Gothenburg, Sweden. Tel.: + 46 31 772 34 07; fax: + 46 31 772 34 18. E-mail addresses: [email protected] (V. Kuzmenko), [email protected] (S. Sämfors), [email protected] (D. Hägg), [email protected] (P. Gatenholm). 1 These authors contributed equally to this manuscript. 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.07.031
It has been investigated for use as a scaffold material in engineering various types of tissues such as skin [5], blood vessels [6], bone [7] and cartilage [8]. The ability of the BNC nanofibrillar network to interact with water might contribute to the good biocompatibility of BNC [9]. This might also hinder proteins and cells from interacting with the BNC surface. Poor cell interaction was observed in a study by Fink et al., where endothelial cells showed a lack of adhesion to BNC biomaterial [10]. In tissue engineering, control of scaffold–cell interaction is crucial. For many cell types, such as osteoblasts [11], neuronal cells [12], endothelial cells [13] and adipose-derived cells [14], cell attachment to the surface is important for the viability and subsequent tissue development. Recent studies have shown the effect of focal adhesion points on shape and consequently on the stem cell differentiation process [15,16]. An effective approach to enhance cell adhesion on biomaterials has been to mimic the extracellular matrix (ECM) [17,18]. Among the most commonly used proteins for this purpose are fibronectin and collagen since they are the most abundant proteins in the ECM involved in cell interactions [17]. In general, cell attachment to solid surfaces occurs by interaction between cell adhesion receptors, such as integrins, and the surface material [19]. Both fibronectin and collagen can connect to the cell integrins via a specific amino acid sequence (RGD sequence) called cell binding domains. This specific interaction between proteins and cells can be mimicked by binding these proteins (or amino acid sequences) to the surface of the chosen scaffold material [20]. In a study by Bodin et al., RGD sequences were introduced to the BNC
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surface by adsorption of xyloglucan-RGD bioconjugates, which showed improvement of endothelial cell adhesion [21]. In other studies, carbohydrate binding modules tied with RGD were used to modify BNC scaffolds to enhance cell adhesion [22,23]. The accessibility of cellulose for bioconjugation is determined by the hydroxylic content of its sugar backbone [24]. Crosslinking compounds can turn the hydroxyl residues into intermediate reactive derivatives having suitable leaving groups for nucleophilic substitution. As a result of reaction between activated hydroxyls and strong nucleophiles, such as amines, stable covalent bonds that connect carbohydrate and the amine-containing molecule are obtained [25]. Among the most common activating agents for cellulose are carbonyldiimidazole [26], N-hydroxysuccinimide esters [27], sodium periodate [28], epoxide compounds [29], tresyl- and tosyl-chloride [30], cyanogen bromide [31], cyanuric chloride [32] and several chloroformate derivatives [33]. The activation process usually requires nonaqueous solutions (i.e., dry dioxane, THF, acetone, DMF or DMSO) to prevent hydrolysis of the reactive intermediate products in aqueous solution [34]. Hydroxyl groups can be modified in an aqueous environment with anhydrides, chloroacetic acid [35] or radical-mediated oxidation with (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl [36] to produce carboxylate functionality for further conjugation purposes with the use of carbodiimides as crosslinkers. In all cases, the reaction takes several hours. Another approach has been to introduce electrostatic interaction by providing an electrical charge to the cellulose surface [37]. However, since all the proteins are amine-containing molecules, an aminecoupling process can be preferably used for covalent conjugation [38]. Swelling cellulose in water improves the availability of OH groups in heterogeneous reactions, which makes an aqueous environment more suitable for activation [39]. Unlike most of the activating agents mentioned above, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) is stable to hydrolysis at a wide pH range due to the resonative delocalization of the positive charge over the whole aromatic ring and thus makes it a very promising activating agent [40]. The exploitation of CDAP as an activating agent was mentioned first by Kohn to replace toxic cyanogen bromide [41]. It was later used for activating soluble polysaccharides to get protein–polysaccharide conjugate vaccines and immunological reagents [42,43] and for activating cellulose membranes for the development of immunosensors [44]. In this study, a unique scaffold made of bacterial nanocellulose is being developed to mimic the properties of a natural extracellular matrix. It is evaluated for its support of attachment and growth of different human cell types at 2D level. Emphasis is put on the use of CDAP in the surface activation of BNC and its subsequent bioconjugation with different proteins to increase cell adhesion on the scaffold material.
2. Materials and methods 2.1. Materials Complex culture medium was prepared according to Matsuoka protocol [45]. Sodium hydroxide (NaOH), CDAP, acetonitrile, triethylamine (TEA), hydrochloric acid (HCl), glutaraldehyde, Triton X100, acetic acid and tert-butanol were obtained from Sigma-Aldrich (USA). Formaldehyde was purchased from Polysciences (USA). Lyophilized fibronectin bovine, collagen I bovine, rhodamine-phalloidin, 4′,6diamino-2-phenylindole and dilactate (DAPI) were obtained from Invitrogen (USA). Ethanol 99.7% was purchased from Solveco AB (Sweden). Endothelial basal media, endothelial growth supplements and human umbilical vein endothelial cells were purchased from Cell Application (USA). Mouse mesenchymal stem cells (MSCs, line C3H10T1/2) were purchased from ATCC (USA) and cultured in Basal Medium Eagle from HyClone (USA). Dulbecco's phosphate buffered saline solution (PBS 1×, without Ca & Mg) was purchased from PAA
Laboratories (Austria). MilliQ water used in all phases of the experiment was deionized and further purified with Millipore Synergy UV unit. 2.2. Methods 2.2.1. Scaffold production 24 well-plates were inoculated with Gluconacetobacter Xylinus (Acetobacter Xylinum susp. Sucrofermentas BPR2001, ATCC No. 700178). 0.5 ml of bacteria suspension and 1 ml of complex culture media were added to each well. The 24 well-plates were placed in an incubator for 48 hours at 30 °C. After 2 days of culture, pellicles of bacterial nanocellulose with a thickness of 2–3 mm and 1.6 cm in diameter had formed in each well. 2.2.2. Purification of BNC scaffolds The bacteria were removed from the BNC pellicles by placing them in NaOH (0.1 M) and heating in a shaking water bath (60 °C) for 1 hour. The pellicles were placed in a beaker with fresh NaOH (0.1 M) on a magnetic stirrer at room temperature overnight. The NaOH (0.1 M) was changed to fresh NaOH (0.1 M) two times a day for 2 days. To remove bacterial residues and neutralize the pH, the pellicles were placed in a beaker with deionized water on a magnetic stirrer. The water was changed to fresh deionized water two times a day for 4 days. Scaffolds were autoclaved and placed in pyrogen-free water in a shaking water bath (60 °C). The pyrogen-free water was changed two times a day for 4 days under sterile conditions in a LAF-hood. 2.2.3. CDAP modification of scaffolds To enhance cell adhesion to the bacterial nanocellulose, scaffolds were modified with CDAP as a crosslinker to bind necessary proteins to the surface of BNC. CDAP was dissolved in acetonitrile and mixed with 0.2 M TEA. To remove excess surface water from the scaffolds, they were wiped slightly by filter paper. The CDAP solution was added to the scaffolds by pipetting a few drops of solution on top of each scaffold. The solution was left to react for 1 minute, and 0.1 M HCl was added to stop the reaction. The scaffolds were rinsed with phosphate buffered saline solution (PBS) until neutral pH was reached. 2.2.4. Protein surface coating Lyophilized proteins were first dissolved and then diluted to the concentration of 100 μg/ml (fibronectin was dissolved in deionized water and diluted with PBS; collagen type I was diluted with 0.05 M acetic acid). 400 μl of protein solution was added to each scaffold (CDAP modified BNC and control samples of untreated BNC) and left to react overnight in 37 °C. The scaffolds were rinsed in PBS several times in order to remove free proteins. 2.2.5. Expansion of HUVECs Endothelial basal media was mixed with endothelial growth supplements to create endothelial cell growth media. A vial of HUVECs was thawed in a water bath at 37 °C. The cells were transferred to a 75-cm2 cell culture flask containing 15 ml of endothelial cell growth media. The cell culture flask was placed in an incubator (37 °C, 5% CO2, 95% relative humidity). The cells were split when they reached 80% confluency. 2.2.6. Expansion of MSCs One vial of MSCs was removed from the liquid N2 tank, thawed and transferred to basal medium eagle supplied with 10% fetal bovine serum and 1% antibiotics/antimyogenics. The cells were expanded in 75 cm2 flasks as described above. 2.2.7. Seeding of HUVECs and MSCs on BNC scaffolds Before cell seeding, scaffolds were rinsed two times in PBS and placed in 24 well-plates, one scaffold in each well. 200 μl of cell suspension was seeded on the scaffolds at a cell density of 3 × 105 cell/cm2 for the HUVECs and 2.53 × 105 cell/cm2 for the MSCs. The well-plates were
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Fig. 1. Cellulose bioconjugation using CDAP. SEM image of BNC on the top left. 1. The reaction of CDAP treatment. 2. The reaction of protein conjugation with CDAP-activated cellulose.
placed in an incubator (37 °C, 5% CO2, 95% relative humidity) for 4 hours to allow optimal cell attachment. 1.5 ml of cell media was then added to each scaffold and the well-plate was placed back into the incubator (37 °C, 5% CO2, 95% relative humidity); this was denoted day 0. Media was changed every 2 to 3 days and samples were taken at day 1, day 3 and day 8.
contact angle system at Attension Theta contact angle meter. A small drop of water was deposited on the surface, while 20 pictures were taken over a period of 10 seconds. To calculate the contact angle, Attension Theta Software was used. Measurements were done on untreated BNC, BNC + CDAP, BNC + fibronectin, BNC + collagen, BNC + CDAP + fibronectin and BNC + CDAP + collagen samples.
2.2.8. Contact angle measurements Samples were washed in sterilized deionized water two times and left to dry on aluminum foil in a LAF-hood. When the samples were completely dry, the contact angles were analyzed using a sessile drop
2.2.9. Electron Spectroscopy for Chemical Analysis (ESCA) Samples were washed in sterilized deionized water two times and left to dry on aluminum foil in a LAF-hood. When the samples were completely dry, the contact angles were analyzed with a Quantum
Fig. 2. High resolution carbon spectra and surface elemental composition (for C, O and N in %) from ESCA analysis on untreated BNC, CDAP, fibronectin, collagen, CDAP + fibronectin and CDAP + collagen treated BNC. Corresponding bonds: 1) C―C; 2') C―O or 2) C―O, C―N; 3') O―C―O or 3) O―C―O, O―C―N; 4') O―C―O or 4) O―C―O, O―C―N.
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4–5 nm. The results were evaluated using MultiPAK 6.0 software. Measurements were performed on untreated BNC, BNC + CDAP, BNC + fibronectin, BNC + collagen, BNC + CDAP + fibronectin and BNC + CDAP + collagen samples. 2.2.10. Scanning Electron Microscopy (SEM) Samples were washed two times in PBS to get rid of media residues and fixated for 2 hours in 2.5% glutaraldehyde in PBS. The glutaraldehyde was removed by rinsing the samples two times in PBS. The scaffolds were dehydrated by placing them in baths of increasing ethanol concentrations (70%, 80%, 90%, 95% and 99%). The samples were placed in t-butanol and placed in a -80 °C freezer until they were completely frozen. Samples were freeze dried in a lyophilizer (Heto, PowerDry, PL3000). Samples were mounted on SEM stubs with adhesive tape (TAAB) and sputter coated (Sputter Coater s150B, Edwards) with a thin gold layer for 1 minute. SEM analysis was performed with a Leo Ultra 55 FEG-SEM.
Fig. 3. Advancing water contact angles of untreated BNC, CDAP, CDAP + fibronectin and CDAP + collagen treated BNC.
2000 scanning ESCA microprobe using an Al Kα X-ray source with a beam size of 100 μm, and the angle between the detector and analyzed sample was 45°. The analyzed area was 500 × 500 μm with a depth of
2.2.11. Fluorescence microscopy (FM) Samples taken for FM analysis were washed in PBS two times to get rid of media residues. For fixation the scaffolds were covered in 3.7% formaldehyde in PBS and left for 2 hours. Samples were rinsed two times in PBS and transferred to 60% ethanol, where they were kept until analysis. Before staining of the samples, they were rinsed in PBS two times. 0.1% Triton X100 in PBS was added to cover the samples and they were left for 45 minutes. The solution was aspirated and the samples were washed two times in PBS. Rhodamine-phalloidin in PBS
Fig. 4. SEM images of HUVECs on unmodified BNC scaffolds and fibronectin modified scaffolds (with and without CDAP activation) after 1, 3 and 8 days of culturing.
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(10 μg/ml) was added to cover the samples and they were left for 30 minutes. The solution was aspirated and DAPI in PBS (5 μg/ml) was added to cover the samples for 30 minutes. The samples were rinsed two times in PBS. For analysis, a phase contrast microscope (Leica Digital Microscopes Inverted) was used together with the software Leica LAS AF.
3. Results and discussion 3.1. BNC scaffolds The bacteria-produced cellulose chains self-assembled into a dense network of nanofibers with similar dimensions as collagen fibers in the extracellular matrix in tissues (Fig. 1 left), i.e. about 1.6 × 5.8 nm in cross section [46]. To remove the bacterial remains, the BNC pellicles were washed with NaOH several times. Since cellulose is nitrogen free, and nitrogen is a constituent of medium components, nucleic acids and peptidoglycans present in bacterial cell wall, the amount of nitrogen was used as an evidence of the presence of bacterial residues in the pellicles [47]. It was confirmed using ESCA that there were no traces of nitrogen left in purified BNC (Fig. 2).
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3.2. CDAP modification of nanocellulose scaffolds The stability of CDAP (it is not hygroscopic and can easily be handled at room temperature in open air) and selectivity towards reaction with cellulosic hydroxyl groups make it a suitable reagent for modifications of BNC. After a few minutes of reaction, CDAP-activated cellulose (cyanate ester derivative) was generated (Fig. 1). The success of the reaction is demonstrated by the appearance of nitrogen (2.5%) in BNC activated with CDAP (Fig. 2). Moreover, side products were removed from the modified material before further interaction with proteins by a simple washing step in diluted HCl avoiding complicated purification procedures [40].
3.3. Bioconjugation with proteins Proteins were attached directly to CDAP-activated cellulose via the amine groups of surface amino acids at optimal pH of 9–10, forming an isourea bond (Fig. 1) [43]. High resolution C1s spectra of carbon from ESCA are shown in Fig. 2. C1s spectra are asymmetric and broadened. They were resolved to several components which were assigned to C―C (284.2 eV, the binding energy scale was charge referenced to this value as starting position),
Fig. 5. FM images of HUVECs on unmodified BNC scaffolds and fibronectin modified scaffolds (with and without CDAP activation) after 1, 3 and 8 days of culturing. Cell nuclei stained with DAPI shown in blue, actin filaments stained with rhodamine-phalloidin shown in red.
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C―O and C―N (285.9 eV), O―C―O and O―C–N (287.2 eV), O―C O and N―C O (288.2 eV) [48,49]. It is clear that peaks 1 and 3 were higher for the CDAP + fibronectin treated cellulose samples in comparison with cellulose treated with fibronectin only. The same phenomenon was present for CDAP + collagen treated versus collagen treated cellulose for peak 1, although the difference was slight for peak 3. This effect can be explained by the increase in proteins bonded to the surface, meaning the appearance of more C―C and O―C―N bonds (for 1 and 3 peaks, respectively) [50]. The atomic composition of the different samples after protein treatment is also presented in Fig. 2. Obviously, an untreated BNC is a polysaccharide made of carbon, oxygen and hydrogen only, it has no nitrogen. Since all the proteins contain nitrogen as an essential part of the structural amino acids, the nitrogen was found in all protein treated samples, as expected [51]. Protein adsorption was observed on the BNC surfaces that were not preliminarily activated with CDAP; however it was not as high as for the preactivated surfaces. It is vividly shown that cellulose surface modified with CDAP promoted protein attachment: for fibronectin, the nitrogen content increased from 5.7% to 11.8%, and for collagen from 1.5% to 3.8%. The higher percentages of nitrogen for fibronectin in comparison to collagen may be due to the much higher molecular mass of fibronectin, which can reach up to 500 kDa, as compared to collagen type I, with 139 kDa [52]. Water contact angle measurements shown at Fig. 3 confirmed the highly hydrophilic nature of native cellulose surface (the advancing
water contact angle was only 16°). After the treatment with the hydrophobic CDAP the surface of BNC samples expectedly had become less polar which was reflected in the increase of contact angle to 38°. After bioconjugation of CDAP-activated cellulose with proteins, the advancing contact angle reached 62° and 36° for fibronectin- and collagentreated samples respectively. Such a big alteration between differently modified surfaces can be partly explained by the contribution of hydrophilic amino acids (proline and hydroxyproline with polar OH groups), which are present in high amounts in collagen [53]. These amino acids make collagen surface more hydrophilic in comparison to the surface of fibronectin. Another explanation we can give is the smaller size of collagen molecules which were unable to cover the BNC surface as tightly as much bigger molecules of fibronectin [52]. The analysis of the surface modification confirmed that fibronectin and collagen were successfully conjugated to the BNC surface and that CDAP activation increased the yield of the attached proteins. 3.4. Cell studies To determine the effect of surface modification on cell adhesion, three types of surfaces were used: untreated BNC and BNC with adsorbed proteins as controls, and BNC bioconjugated with proteins via CDAP activation as the main object of investigation. HUVECs and MSCs were seeded on the different BNC substrates and evaluated by SEM and FM after 1, 3 and 8 days of culturing. FM images better reflect the level of cell adhesion than SEM images due to the
Fig. 6. SEM images of MSCs on unmodified BNC scaffolds and collagen modified scaffolds (with and without CDAP activation) after 1, 3 and 8 days of culturing.
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Fig. 7. SEM images of MSCs: (A) cell with a rounded shape on the untreated BNC; (B) well-stretched cell on the CDAP + collagen treated BNC.
treatment of the samples before SEM analysis (cells with weak adhesion might de-attach). SEM analysis of HUVECs cultivated on different surfaces is shown in Fig. 4. After day 1, no big differences in cell amount could be detected; however, cells on the BNC + CDAP + fibronectin treated surfaces exhibited a more stretched-out morphology than the cells on the other surfaces. After day 3, dissimilarities could be seen in the amount of cells on the three different surfaces. Many cells were found spread out on the surface on the CDAP preactivated samples while, for the untreated BNC and BNC coated with fibronectin,
not many cells could be detected. After 8 days, the difference was even larger. The few cells found on the untreated BNC and the BNC + fibronectin samples had a rounded morphology, indicating that they were de-attaching, while the cells on the CDAP + fibronectin treated surfaces formed almost a confluent layer after 8 days. FM images of HUVECs on the fibronectin treated surfaces and untreated surfaces also confirmed the effect of CDAP treatment (Fig. 5). A difference in morphology of the cells seeded on the untreated surface and the fibronectin treated surface was already observed at day
Fig. 8. FM images of MSCs on unmodified BNC scaffolds and collagen modified scaffolds (with and without CDAP activation) after 1, 3 and 8 days of culturing.
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1. The cells on the CDAP activated surfaces had more defined actin filaments while the filaments on the untreated BNC and BNC + fibronectin were not exactly aligned. After day 3 and day 8, not many cells could be found on the untreated surfaces. In contrast, on the CDAP treated surfaces, many cells were found after day 3 and day 8 that adhered to the surface and whose actin filaments were well defined and aligned. The cells acquired the cobblestone morphology associated with endothelial cells, as opposed to the more stretched out morphology of cells grown on untreated BNC or BNC + fibronectin. This indicates that conjugation of the protein to the BNC surface is preferable compared to the simple adsorption of the protein. To prove that the method using CDAP for the bioconjugation of proteins to cellulose is universal, MSCs were seeded on BNC conjugated with collagen type I. SEM analysis of cells seeded on the different surfaces showed that the bioconjugation in fact increased the amount of cells growing on the BNC surface (Fig. 6). After day 1, cell morphology was already markedly different when as compared with untreated surfaces, surfaces treated with only collagen and CDAP + collagen treated surfaces. Very few cells were found on the first two types of surfaces. The cells detected had a rounded shape, demonstrating poor cell adhesion to the surface (Fig. 7A). On the CDAP + collagen modified surfaces, many cells were spread out on the surface, creating an almost confluent layer, with a stretched-out morphology indicating the healthy status of the cells (Fig. 7B). After day 3 and day 8, the amount of cells, as well as their morphology, on the three different surfaces remained about the same. MSCs on the collagen treated surfaces and untreated surfaces were analyzed with FM as well (Fig. 8). Differences in morphology and the total number of the attached cells seeded on the untreated surface, the collagen treated and CDAP + collagen treated surface were already observed at day 1. First of all, the cells on the CDAP activated surfaces had more defined actin filaments; in contrast, the filaments on the other two types of surfaces were not really aligned. Second, CDAP treated pellicles were almost completely covered with the attached cells, forming a confluent layer, whereas not many cells could be found on the untreated BNC and treated with collagen only. The situation did not change significantly after 3 and 8 days of cell culturing. The positive influence of CDAP treatment on cell adhesion is shown once again.
4. Conclusions This study demonstrated that cell adhesion can be increased by bioconjugation of extracellular matrix proteins with the scaffold surface via a convenient two-step reaction using CDAP as an effective activation agent. Biocompatible nanofibrillar cellulose was used as a scaffold material suitable for cell study and potential tissue engineering. Using BNC as substrate with covalently bonded proteins was shown to enhance attachment of two different types of cells, forming confluent layers of healthy cells. The cells on the CDAP + protein treated surfaces were found in large quantity and had an appropriate morphology with defined actin filaments throughout all the days of cell culturing. In contrast, much fewer cells were attached to the substrate of the untreated BNC scaffolds and the BNC scaffolds with extracellular proteins simply adsorbed on the surface even after the first day of culturing, and the shape of those cells was not at all satisfactory.
Acknowledgements The Wallenberg Wood Science Center funded by Knut and Alice Wallenberg Foundation is greatly acknowledged for their financial support. We also thank Dr. Ergang Wang, Anders Mårtensson, Anne Wendel, and Johan Sundberg for their experimental assistance, as well as Janet Vesterlund for proofreading the English version of the manuscript.
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Volodymyr Kuzmenko obtained his Master of Science degree in 2012 at Chalmers University of Technology, where he continued his work as a PhD student at the Department of Microtechnology and Nanoscience collaborating with Wallenberg Wood Science Center, WWSC. The main focus of his research is on synthesis, modification and application of nanomaterials from renewable resources.
Sanna Sämfors got her master degree in Biotechnology from Chalmers University of Technology in 2013. Her master thesis focused on development of vascularized scaffolds for tissue engineering applications. She is currently continuing work on further development of these scaffolds.
Dr Daniel Hägg got his doctorate on genetics and genomics in the cardiovascular field from Göteborg University, Sweden, in 2008. Since then, he has shifted his research focus towards tissue engineering, primarily adipose tissue, first during his post-doctoral visit at Columbia University, New York, and at his current position at Chalmers University, Sweden. His main interests are 3D cell culture and cell differentiation.
Dr. Paul Gatenholm is currently professor of Biopolymer Technology at Chalmers University of Technology, Director of Biosynthetic Blood Vessels Laboratory, Coordinator of EAREG program and Director of Graduate School at Wallenberg Wood Science Center, WWSC. He is also Adjunct Professor at Virginia Tech, Wake Forest University and Wake Forest Institute for Regenerative Medicine in Winston-Salem, North Carolina, USA. Dr. Gatenholm is a material scientist with interest in biomimetic design of new biomaterials that can replace or regenerate tissue and organs. His research includes biological fabrication through the use of enzymes, cells, and the coordination of biological systems.