In Vitro Reconstitution of Fibrillar Collagen Type I Assemblies at

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In Vitro Reconstitution of Fibrillar Collagen Type I Assemblies at Reactive Polymer Surfaces Katrin Salchert,* Uwe Streller, Tilo Pompe, Nicole Herold, Milauscha Grimmer, and Carsten Werner* Institute of Polymer Research Dresden & The Max Bergmann Center of Biomaterials Dresden, Hohe Strasse 6, 01069 Dresden, Germany Received February 16, 2004; Revised Manuscript Received April 27, 2004

The reconstitution of fibrillar collagen and its assemblies with heparin and hyaluronic acid was studied in vitro. Fibril formation kinetics were analyzed by turbidity and depletion measurements in solutions containing varied concentrations of collagen and glycosaminoglycans. Fibril-forming collagen solutions were further applied for the coating of planar substrates which had been modified with alternating maleic anhydride copolymer films before. The immobilized collagen assemblies were characterized with respect to the deposited amount of protein using ellipsometry and acidic hydrolysis/HPLC-based amino acid analysis, respectively. AFM, SEM, and cLSM were utilized to gain information on structural features and patterns formed by surface-attached fibrils depending on the initial solution concentrations of collagen. The results revealed that the addition of heparin and hyaluronic acid affected both the fibril dimensions and the meshwork characteristics of the surface-bound fibrils. Introduction Biomolecular engineering of cell scaffolds can be advantageously attained by the in vitro reconstitution of supramolecular structures of extracellular matrix components such as proteins, proteoglycans, and glycosaminoglycans on top of artificial biomaterials. Among the biomacromolecules utilized for that purpose collagen is distinguished by its abundance in the mammalian organism, its importance for the connective properties of tissues, and a wide variety of specific interactions with nearly 50 different molecules1 including glycosaminoglycans such as heparin, proteins such as fibronectin, and numerous growth factors. Collagen directly mediates cell adhesion and is therefore frequently used for the coating of cell culture flasks and dishes. Furthermore, collagenous materials are applied in tissue engineering products which were recently developed for applications in dermatology, orthopedics, or oral surgery.2,3 Using the multiple binding sites of collagen, artificial collagen matrixes have been loaded with angiogenic factors such as bFGF to promote neovascularization and tissue regeneration in vivo.4,5 Established protocols for the coating of cell culture carriers often make use of the solubilized and monomeric collagen I. However, the monomeric tropocollagen forms thin layers which do not resemble the naturally occurring functional meshwork of fibrillar collagen. Therefore, native-type fibrils were reconstituted from tropocollagen solutions in vitro6 resulting in a three-dimensional supramolecular assembly of * To whom correspondence should be addressed. Tel.: +49 351 4658408. Fax: +49 351 4658533. E-mail: [email protected] (K.S.). Tel.: +49 351 4658531. Fax: +49 351 4658533. E-mail: [email protected] (C.W.).

collagen. Importantly, these structures can implement other components of extracellular matrix and establish threedimensional meshworks providing tissue-mimetic environments for adherent cells.7 The latter fact has been recently utilized by collagen coatings of implant materials such as titanium alloys to enhance the cell adhesion to the metal oxide surface.8 The spontaneous self-assembly of monomeric collagen was attributed to the entropy gain upon binding of collagen molecules implying that hydrophobic interactions between collagen monomers are the major driving force for fibril formation.9 In addition to hydrophobic interactions, the contribution of water clusters bridging recognition sites on opposing helices was discussed as a further driving force for the fibril assembly.10 Furthermore, recognition sites for the nonhelical telopeptides of a docking collagen monomer are crucial for fibril formation and the lack of telopeptides increases the assembly time.11 Mechanistically, fibrillogenesis was described as a multistep process initiated by the formation of collagen dimers and trimers followed by a rapid lateral aggregation involving five trimers.12 Consecutive linear and lateral addition of further monomers or multimers gives rise to the formation of microfibrils which can further assemble into large fibrillar structures depending on the conditions of the fibrillogenesis.13 The stability of collagen-based coatings and threedimensional scaffolds can be enhanced by chemical crosslinking14 or freeze-drying.15 Methods recently suggested to enhance the stability of collagen bound to polymer surfaces comprise the activation of surface carboxy groups of by water-soluble carbodiimide and subsequent collagen immobilization,16 cross-linking of collagen to chemically generated amino groups on polycaprolactone membranes using

10.1021/bm0499031 CCC: $27.50 © 2004 American Chemical Society Published on Web 05/29/2004

Reconstitution of Fibrillar Collagen Assemblies

glutaraldehyde,17 photochemical immobilization to polystyrene cell-culture plates,18 and cyanogen bromide activation of polymer films having hydoxyl groups as poly(vinyl alcohol) or cellulose and subsequent immobilization of collagen.19 However, irradiation, chemical activation, as well as the application of cross-linking reagents can harm the polymer materials as well as the proteins themselves. Therefore, immobilization methods are required that cause no alterations of the polymer surface characteristics and of the protein properties. Chemical cross-linking was further used in some procedures for the covalent immobilization of glycosaminoglycans as chondroitin-6-sulfate,20 heparin,4,21 and hyaluronic acid22 to collagenous scaffolds. Glycosaminoglycans (GAG) are important components of the extracellular matrix which are able to specifically bind several proteins as cytokines and growth factors in addition to collagen and have been described to modulate (along with collagen) adhesion, migration, as well as the proliferation of cells. The in vitro formation of collagen-GAG-complexes was attributed to electrostatic interactions while the stability of the complexes was found to be enhanced for GAGs of high molecular weights and high charge density.20 In that line, preparation of stable cell scaffolds by complexation of collagen with hyaluronic acid (HA) or heparin (HE) can be reasonably expected to be successful due to the high molecular weight of HA and the very high charge density of HE as well as due to specific binding sites of collagen for HE.1 We describe herein the deposition of fibrillar collagen to planar model substrates which had been coated before with thin films of poly(octadecene-alt-maleic anhydride) to mediate the covalent attachment of fibrillar collagen and thus to enhance the stability of the immobilized layers of collagen and collagen-GAG complexes, respectively. The method does not require chemical or irradiation activation and thus protects the biomaterial collagen against changes of structure and chemical composition. Solution concentration effects and the complexation of collagen with HE or HA were examined with respect to the dynamics of fibril formation and by the comparison of structural features of the individual fibrils and the immobilized fibrillar meshwork. In contrast to other approaches described for the complexation or immobilization of GAGs to collagen,4,21 HE or HA were added to solutions of monomeric collagen to induce electrostatically driven complexation, and subsequently, fibrillogenesis was initiated. Materials and Methods Reconstitution of Fibrillar Collagen and Deposition to Planar Substrates. Collagen fibrils were reconstituted from a sterile solution of purified, pepsin-solubilized bovine dermal collagen I in 0.012 N HCl (Vitrogen, Cohesion Technologies, Palo Alto, CA) according to a modified protocol of the manufacturer. Eight parts of the acidic collagen solution (3.0 mg/mL) were mixed with one part of 10-fold concentrated PBS (Sigma, Steinheim, Germany) and one part 0.1 M NaOH. All components had to be kept in an ice bath before as well as after mixing. The pH of the mixture was tested to be 7.4. At this stage of the procedure, the concentration of

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the collagen solution was adjusted by addition of appropriate volumes of chilled PBS. Formation of fibrils was initiated by a temperature shift to 37 °C. Coating of glass slides or wafers was performed by the contact of the substrates to the collagen solution followed by a subsequent raise in temperature. In detail, a drop of 700 µL chilled nonfibrillar collagen solution was placed on top of a polymer modified planar substrate to cover about 2 cm2 and kept in a CO2-free incubator thermostated to 37 °C. After indicated times, the resulting collagen gel was removed from the surface leaving a thin collagen layer on the substrate. The modified surface was rinsed with PBS at least three times and subsequently with Milli-Q water (Millipore Corporation, Molsheim, France). For analytical tests, the protein layer was air-dried and the substrates were stored in closed containers. The deposition of tropocollagen on polymer modified substrates was performed by placing a diluted acidic collagen solution (0.1 mg/mL) or a collagen/PBS solution on top of a planar reactive polymer-modified substrate overnight. After removing the residual collagen solution, the surface was washed three times with PBS and closing with water. For protein quantification by acidic hydrolysis and amino acid analysis, only well-defined areas of 2 cm2 were coated with collagen using home-built immobilization chambers. Preparation of Collagen-GAG Assemblies. GAGs used for the preparation of conjugates with collagen were heparin sodium salt from porcine intestinal mucosa (HE) and hyaluronic acid potassium salt from human umbilical cord (HA; all from Sigma, Steinheim, Germany). Preparation of GAG-containing fibrils was accomplished by mixing a cooled nonfibrillar collagen solution, prepared as described above, with appropriate volumes of the polysaccharides in PBS to yield the indicated concentrations. Solutions were kept at 4 °C for at least 15 min. Fibril formation and immobilization to reactive polymer coated substrates was performed as for pure collagen fibrils. To analyze the structural composition of GAG-containing fibrillar collagen assemblies, fibrillogenesis of a 1.2 mg/mL collagen solution was performed in polypropylene tubes in the presence of different portions of HE and HA, respectively. After 2 h fibril formation at 37 °C, 400 µL of the gel was placed on top of glass slides and dried under a clean bench overnight at room temperature. The resulting layers were carefully rinsed with MilliQ water, dried again, and analyzed by SEM. For quantification of collagen that was not included into collagen fibrils, fibrillogenesis in the presence of GAGs was performed in polypropylene tubes for 2 h at 37 °C. The volume of all examined collagen gels was 1 mL. The fibrils were subsequently collected by centrifugation at 5000 g for 30 min. Samples of 70 µL were withdrawn from the supernatant and subjected to protein quantification by acidic hydrolysis and amino acid based HPLC analysis. Turbidity Measurements. Formation of fibrillar collagen and its conjugates with HE or HA was followed at 313 nm using a Specord S10 (Carl Zeiss, Jena, Germany) equipped with a thermostated cell holder. Preparation of Thin Polymer Films. For the preparation of stable thin films of poly(octadecene-alt-maleic anhydride)

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(PO-MA) (Polysciences Inc., Warrington, PA), a copolymer solution in THF (Fluka, Deisenhofen, Germany) was spincoated (RC5, Suess Microtec, Garching) onto cleaned and aminosilanized SiO2 surfaces (silicon wafers or glass coverslips) prepared according to the procedure described elsewhere.23 Ellipsometric Measurements. The thickness values of the deposited collagen layers were determined by ellipsometry using the single-wavelength device (632 nm) ELX-02 (DRE Dr. Riss Ellipsometer Bau GmbH, Ratzeburg, Germany). The refractive indices were determined to 1.5037 for the POMA film and 1.6035 for the dried collagen layer, respectively, and kept invariant for the comparison of samples series. Coatings were performed in duplicate and the average thicknesses of the collagen layers were calculated from at least three measuring points on each wafer. Quantitative Protein Analysis. Quantification of immobilized collagen was performed by acidic hydrolysis and subsequent HPLC analysis as described elsewhere.24 Briefly, substrates were subjected to vapor hydrolysis in vacuo using 6 M HCl at 110 °C for 24 h and subsequently neutralized. The extraction of the amino acids from the surface was accomplished by repeated rinsing with a definite volume of 50 mM sodium acetate buffer at pH 6.8. The released amino acids were chromatographically separated after precolumn derivatization with ortho-phthalaldehyde on a Zorbax SBC18 column (4.6 × 150 mm, 3.5 µm, Agilent Technologies, Bo¨blingen, Germany) using an Agilent 1100 LC system (Agilent Technologies, Bo¨blingen, Germany) with fluorescence detection. Amino acids were quantified using an external standard, and analysis was controlled by the Chemstation-software Rev. 08.01 (Agilent Technologies, Bo¨blingen, Germany). Surface Characterization. The surface topography of the deposited collagen layers was investigated by atomic force microscopy (AFM) as well as by scanning electron microscopy (SEM). AFM measurements (Bioscope, Digital Instruments, Darmstadt, Germany) were accomplished on air-dried samples. Furthermore, the air-dried samples were gold coated with a sputter coater (SCD 050, BAL-TEC, Schalksmu¨hle, Germany) and examined by means of a scanning electron microscope (XL 30 ESEM FEG, FEI-Philips, Eindhoven, Netherlands). Laser Scanning Microscopy. Collagen fibrils and complexes of GAGs and fibrillar collagen were visualized utilizing FITC-labeled collagen, FITC-labeled HE (both: Molecular Probes, Leiden, Netherlands), and FITC-labeled HA (Sigma, Steinheim, Germany). Fluorescent labeled and unlabeled polysaccharides were mixed at a ratio of 1:40 to yield an average concentration of 2.4 mg/mL in PBS and chilled to 4 °C. FITC-labeled collagen was added to yield a portion of 2.5% in the collagen solution. Preparation of conjugates with collagen and coating of polymer modified glass slides was accomplished as described above. After removal of the gel from the supernatant, the surfaces were washed with PBS at least six times and mounted to a microscope slide. The surfaces were imaged using a confocal laser scanning microscope (TCS SP, Leica, Bensheim, Germany) with a 40× oil immersion objective.

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Figure 1. Turbidity measurements of collagen fibril formation. Dependency on time and concentration of the starting solution.

Figure 2. Turbidity measurements of fibril formation in the presence of HE and HA 2 h after initiation of fibril formation. Initial concentration of the nonfibrillar collagen solution was 1.2 mg/mL. Concentrations of the GAGs were 0.4, 1.2, and 5.0 mg/mL, respectively.

Results Turbidity Measurements. Turbidity measurements were utilized to follow the self-assembly of monomeric tropocollagen into collagen fibrils and collagen-GAG fibrils, respectively. As shown in Figure 1, different initial concentrations of pure monomeric collagen between 0.02 and 1.2 mg/mL caused gradual differences in the resulting, equilibrium, optical densities as well as in the process dynamics obvious from the curve shapes. Higher initial concentrations caused a faster adjustment of the maximum optical density and higher absolute values of the latter. Furthermore, the lag phase, the time period before the turbidity steadily increased, was extended for decreasing collagen concentrations. Presence of HE or HA at 0.4, 1.2, and 5.0 mg/mL in the collagen solutions resulted in similar optical densities of the supramolecular assemblies of collagen and the GAGs as compared to pure collagen (Figure 2) after 2 h of fibrillogenesis. However, higher portions of HE or HA caused a slight decline in the optical densities indicating that fibril formation was affected by both GAGs. Quantification of Residual Monomeric Collagen in Collagen-GAG Gels. Fibril formation of pure collagen was



Table 1. Dependency of the Thickness of Collagen Layers and the Total Amount of Collagen Immobilized to Thin Films of Poly(octadecene-alt-maleic anhydride) on Collagen Concentration and Contact Timea initial concentration 0.15 mg/mL

0.6 mg/mL

1.2 mg/mL

time

thickness by ellipsometry [nm]

protein quantification by acidic hydrolysis/ HPLC [µg/cm2]





10 min 30 min 90 min 24 h

2.8 ( 0.1 3.8 ( 0.2 7.2 ( 0.5 10.1 ( 0.1

0.51 ( 0.09 0.63 ( 0.04 0.74 ( 0.16 2.90 ( 0.61

7.1 ( 1.0 19.2 ( 12.0 33.1 ( 7.6 34.2 ( 1.1

0.92 ( 0.15 1.34 ( 0.49 5.26 ( 0.54 6.77 ( 1.11

11.5 ( 3.9 47.6 ( 20.4 64.5 ( 14.0 65.5 ( 14.7

1.26 ( 0.34 6.94 ( 3.49 10.37 ( 0.44 12.01 ( 0.72

a

Refractive indices used were 1.5037 for the maleic anhydride copolymer and 1.6035 for the dried collagen layer.

Table 2. Deposition of Collagen-GAG Conjugates on Polymer Coated Wafersa

Figure 3. Quantification of collagen in the supernatants of collagen and collagen-GAG fibrils collected by centrifugation 2h after initiation of fibrillogenesis. Initial concentration of collagen was 1.2 mg/mL. Concentrations of the GAGs were 0.4, 1.2, and 5.0 mg/mL, respectively.

further compared to the fibril formation in the presence of different GAG portions with regard to the depletion of the collagen from the solution, i.e., by quantification of the amount of collagen not incorporated into fibrils. Therefore, collagen fibrils were collected by centrifugation and the concentration of collagen in the supernatants was determined by acidic hydrolysis and HPLC-based amino acid analysis as recently described elsewhere.24 Although fibrillogenesis of all samples was performed with initial collagen concentrations of 1.2 mg/mL, different concentrations of collagen were found in the supernatants after centrifugation (Figure 3). Supernatants from fibril containing solutions which were formed in the presence of 0.4 and 1.2 mg/mL HE contained less than the half collagen in comparison to supernatants resulting from fibrils formed in the presence of 5.0 mg/mL HE or from pure collagen. However, quantification of collagen in supernatants of spun down collagen fibrils which were formed in the presence of different HA portions provided collagen concentrations comparable to concentrations found in the supernatants of pure collagen. The dependency of the residual collagen concentration on the HA concentration was less pronounced in contrast to samples prepared in the presence of HE. Ellipsometry and Amino Acid Quantification of the Immobilized Layers. The concentration and time dependency of the collagen immobilization were studied by immersing reactive polymer substrates with solutions containing different concentrations of monomeric collagen and subsequent initiation of fibrillogenesis. After contact times indicated in Table 1, the substrates were evaluated using

immobilized assemblies

thickness of layer [nm]

collagen amount [µg/cm2]

collagen collagen/HE 0.4 mg/mL collagen/HE 1.2 mg/mL collagen/HE 5.0 mg/mL collagen/HA 0.4 mg/mL collagen/HA 1.2 mg/mL collagen/HA 5.0 mg/mL

81.3 ( 5.8 n.d. 29.3 ( 8.5 32.3 ( 1.6 n.d. 14.8 ( 2.0 30.9 ( 8.7

10.4 ( 0.4 2.7 ( 1.2 2.3 ( 0.3 2.3 ( 0.9 3.4 ( 1.3 1.8 ( 0.5 3.0 ( 0.8

a Fibrillogenesis and deposition of fibrillar collagen (1.2 mg/mL) was performed for 2 h in the presence of HE and HA (0.4, 1.2, and 5.0 mg/ mL, respectively). Thickness of the layers was ellipsometrically determined using the refractive index of 1.6035 for the dried collagen layer. The collagen amount was determined after acidic hydrolysis and subsequent HPLC analysis.

ellipsometric measurements and the surface bound protein was quantified by acidic hydrolysis and subsequent HPLC analysis. The data obtained reveal differences of the deposited collagen layers in the thickness and the protein amount. Increasing initial concentrations and contact times resulted in higher protein amounts on the surfaces as expected. Significantly thinner protein layers were obtained when immobilizing nonfibrillar tropocollagen for 2 h. By deposition from acidic solution (0.1 mg/mL collagen in 0.012 N HCl) and from buffered solution (0.1 mg/mL at pH 7.4), layer thicknesses of 0.5 and 4.8 nm (refractive index kept fixed at 1.6035), respectively, were obtained. The quantification of immobilized tropocollagen yielded 0.31 µg/cm2 for the deposition from the acidic and 0.71 µg/cm2 for the immobilization from the buffered solution. The coating of polymer-modified substrates with collagen-GAG conjugates was accomplished by the exposure of the slides or wafers to a mixture of dissolved collagen and HE or HA at different concentrations and subsequent initiation of fibrillogenesis. After removing the nonimmobilized gel from the supernatant, the resulting layers were air-dried and subsequently analyzed ellipsometrically. The determined layer thicknesses are summarized in Table 2. Note that these layer thickness data were obtained assuming invariant refractive index values and do therefore not represent the layer extension in the sense of the fibril dimensions imaged by SEM or AFM. Due to the application of this approach, the ellipsometric thickness may instead serve here as a measure of the immobilized amount of collagen (i.e., for comparison of hypothetical optical homo-

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Figure 4. SEM and AFM images of fibrillar collagen immobilized on thin layers of poly(octadecen-alt-maleic anhydride) after 90 min fibril formation and simultaneous deposition. (a) 0.15 mg/mL, (b) 0.6 mg/mL, (c) 1.2 mg/mL. The dimensions of AFM images were 50 × 50 µm.

geneous layers containing the immobilized amounts of collagen). In comparison to pure fibrillar collagen that provided layers of about 81 nm thickness, the deposition of mixtures of collagen and polysaccharides resulted in noticeable thinner layers in the range between 14 and 32 nm. In addition, surface bound collagen deposited in the presence of HE or HA was quantified by acidic hydrolysis and subsequent HPLC based amino acid analysis (Table 2). The quantification of surface bound collagen confirmed the results of the ellipsometric measurements. Significantly lower amounts of collagen were found on the polymer coated surfaces where collagen fibrils were formed in the presence of GAGs. Surface Topography Analysis. Immobilization of pure collagen to reactive polymer coated substrates resulted in distinct shapes and surface patterns if different initial collagen concentrations were applied (Figure 4). Fibril formation and deposition was terminated after 90 min, and the resulting layers were assessed by SEM and AFM visualizing the arrangement of surface-bound collagen fibrils. Increasing the collagen concentration caused a different surface pattern. The lowest concentration of 0.15 mg/mL provided only isolated surface-bound fibrils, whereas tight fibrillar meshworks covering the surface completely were deposited from 0.6 and 1.2 mg/mL solutions. Bright points or regions in the AFM images were caused by interbreeding fibrils in the case of higher collagen concentrations whereby this effect was more pronounced at 1.2 mg/ mL due to the higher number of fibrils as well as the thicker protein layer. AFM and SEM were further utilized for the structural analysis of surface bound collagen fibrils reconstituted with and without addition of HE or HA, respectively. The technique offered information on the ultrastructure of collagen and collagen-polysaccharide conjugates. Comparison of the AFM images of collagen in Figures 5 and 6 provided significant differences in morphology as well as in the dimensions of the fibrils depending on the GAGs.

Furthermore, the impact of the GAG amount on the surface pattern of immobilized collagen-GAG layers was studied (Figure 6). In the case of fibrils reconstituted in the presence of polymer coated substrates, pure collagen with an initial concentration of 1.2 mg/mL provided well-established networks of collagen fibrils without gaps between the separate fibers. The fibrils revealed their characteristic band pattern indicating that the native structure was retained after deposition. Image processing of the AFM images using fast Fourier transformation (FFT) analysis revealed the conservation of the periodicity of the fibril band pattern. The average distance between the stripes in pure fibrillar collagen was 62 nm, in conjugates with HE 64.5 nm and in conjugates with HA 6364 nm. Fibrillogenesis of collagen in the presence of HE gave rise to a modified morphology of the deposited layer. Density, shape, as well as length of the fibrils significantly differed from pure collagen. Extremely wide and straight fibers but with distinct band patterns represented the shape of the deposited cofibrils. No impact of the HE amount on the fibril pattern could be detected; however, with increasing HE concentrations, a reduction of the fibril diameters was observed. The broad HE-containing collagen fibrils appeared markedly straight with tapered ends. The wide fibrils covered the area not completely, the surface between the fibers appeared not smooth indicating that the gaps were obviously filled with significantly thinner and shorter fibrils. The band pattern was apparent on the large fibrils and the fibrils themselves showed no twist. Fibril deposition to reactive polymer coated substrates in the presence of HA provided no dependency of the surface patterns on the deposited HA concentration. A relatively rough surface obviously consisting of small fibers was covered by a few separated fibrils only that showed the characteristic band pattern. The large twisted collagen fibrils formed in the presence of HA appeared thinner, longer, and less straight compared to the complexes with HE.



Figure 5. AFM images of collagen and collagen-GAG assemblies immobilized to reactive polymer coated surfaces. The collagen gel resulting from fibrillogenesis was removed after 90 min. Images size is specified in brackets.

Figure 6. SEM images of collagen and collagen-GAG assemblies to reactive polymer coated surfaces. The collagen gel resulting from fibrillogenesis was removed after 90 min.

To verify whether the surface bound fibrillar structures represent the characteristics of the “bulk phase” of the collagen-GAG gels well-defined volumes of fibrillar solutions were deposited to glass slides and subjected to SEM analysis after drying and washing steps (Figure 7). The results revealed quite different patterns as compared to the structures of collagen gels bound to reactive polymer coated glass slides (Figures 5 and 6). The number of fibrils visible on the desiccated gel bulk samples was clearly enhanced, but the fibril shapes corresponded to the covalently immobilized fibrils. With rising HE concentration, a definite decrease in fibril size and an increase in fibril number was observed. Furthermore, the structure of the fibrillar meshwork changed with increasing HE concentrations. Compared to HA containing preparations, the fibrils formed in the presence of HE were broader and straight with clearly tapered ends. Whereas a pronounced dependency of the number and the

size of deposited collagen fibrils on the HE portion was observed, structures of dried collagen gels formed in the presence of different HA portions provided no dependency on the HA concentration. Fluorescence Imaging. Complexation of fluorescent labeled HE or HA and fibrillar collagen on polymer coated glass slides enabled the visualization of fibrillar structures using confocal laser scanning microscopy (Figure 8). Compared to the fluorescent labeled layer of pure collagen, the HA containing assemblies provided less sharp contours, whereas HE induced straight fibrillar structures. However, areas of weaker fluorescence were detected next to the clearly stained structures of the collagen-GAG assemblies. Fluorescence staining was further used to compare the amounts of HE and HA bound to collagen in fibrillar structures. Therefore, relative fluorescence intensities of layers reconstituted from collagen solutions with different

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Figure 7. SEM images of deposited collagen-GAG assemblies produced by desiccation of 400 µL fibrillar collagen solution.

Figure 8. Visualization of collagen fibrils by adding collagen-FITC (a), HA-FITC (b) and HE-FITC (c). Image size is 125 µm.

portions of fluorescence-labeled HE and HA, respectively, were compared. The preparation reconstituted in the presence of 0.4 mg/mL HE provided a relative fluorescence intensity of 6, whereas for an initial concentration of 1.2 mg/mL, an intensity of 31 was found. HA containing preparations provided intensities of 7 and 9 for 0.4 and 1.2 mg/mL of the GAG, respectively. A direct comparison of HE and HA containing preparations was not possible due to the different degrees of FITC-labeling of the different GAG molecules. Discussion The in vitro reconstitution of collagen type I fibrils was followed in situ starting from solutions containing different concentrations of monomeric collagen, and the obtained assemblies were thoroughly analyzed. Fibril formation was initiated by increasing the ion strength of an acidic collagen solution and subsequent neutralization before increasing the temperature. Turbidity analysis revealed the kinetics of the collagen fibrillogenesis induced by this procedure. The lag phase, an initial period characterized by the absence of any turbidity, and the maximum optical density were used as parameters. With increasing collagen concentrations, a shortening of the lag phase as well as an increase in the

opacity were found (Figure 1). In line with reports stating that the assembly process depends on the initiating procedure,6 a dependency of the fibrillar assembly on the starting concentration of the collagen solutions was confirmed. According to the model for fibrillar assembly established by Silver,12,25 the formation of linear dimers and trimers occurs during the lag phase. Thus, the shortening of the lag phase with increased collagen concentrations could be attributed to the enhanced rate of dimer and trimer formation. The impact of the monomer concentration on the generated amount of fibrils was reflected by the trend of the maximum optical density achieved in different collagen preparations. In the investigated concentration range, a linear dependency of the maximum optical density on the starting concentration was observed. Fibrillogenesis was also studied in the presence of HE and HA. For that aim, fibrillogenesis was initiated after the GAGs had been added to the solutions of the monomeric tropocollagen. Binding of GAGs to collagen may be caused by ionic interactions as observed by Yannas et al.20 for chondroitin6-sulfate and via specific ligand-binding sites as described for heparin.1 The dynamics of the fibril formation as well as the topography of deposited fibrils indicated that the presence of GAGs caused significant deviations from the


fibrillogenesis behavior of the plain collagen fibrils. Turbidity analysis of the fibrillogenesis at gradually varied portions of HE or HA and constant collagen concentrations of 1.2 mg/mL showed that the duration of the lag phase of the fibril formation was not significantly affected by the GAGs (data not shown). However, increasing portions of HE and HA caused a slight decline in the maximum optical densities (Figure 2) and the net fibril formation was obviously affected by the polysaccharides. As described by Gaskin et al.26 for microtubules, changes in the turbidity could not be attributed to changes in the fibril length. Thus, differences in turbidity values of fibrillar collagen had to be ascribed to either different amounts of fibrils or to varied fibril diameters or to both. Comparing the collagen concentrations in the supernatants of collagen gels after centrifugation (Figure 3) with turbidity data confirmed the impact of the GAG portion on the fibrillogenesis. The highest HE portion of 5.0 mg/ mL caused the lowest optical density in comparison to lower HE portions indicating that the amount or/and diameter of fibrils were different. In the supernatants of spun down fibrils, a higher collagen amount was found if the fibrillogenesis was performed in the presence of 5.0 mg/mL HE suggesting that less collagen was incorporated into fibrils. Comparing further turbidity and protein quantification data obtained for collagen in the presence of 0.4 and 1.2 mg/mL HE with those obtained for pure collagen, opposite results were found: A higher fraction of the total collagen was converted into fibrillar collagen in the presence of heparin indicating that these HE concentrations significantly support the complexation of collagen. The higher HE concentration of 5.0 mg/ mL obviously caused a certain degree of inhibition of fibril formation in comparison to pure collagen since a lower turbidity and a higher residual collagen amount were determined. The impact of HA on the fibril formation was less pronounced when compared to HE. Protein quantification provided in general lower residual collagen amounts compared to pure collagen suggesting the incorporation of a higher collagen portion into fibrils. No correlation of residual collagen in solution on the deployed HA amount could be established. Thus, the presence of HE and HA can be concluded to differently affect collagen fibrillogenesis. Fibril formation was strongly affected only in the presence of high GAG portions which possibly caused the disruption of water clusters bridging recognition sites of collagen monomers as recently described for the inhibition of the fibrillogenesis by sugars and polyols.10 A disruption of water clusters by inclusion of the macromolecules seemed to be improbable due to the large size of the GAGs applied in this study. Therefore, impurities consisting of low-molecular-weight GAGs which possibly occurred in low amounts in the preparations might have altered the fibrillogenesis in the presence of the highest GAG concentrations only. Although measurements of the turbidity allowed us to follow the fibril formation, they provided no data on the composition of fibrillar assemblies with regard to structural features of the fibrils. To obtain information on the fibril dimensions and further meshwork characteristics fibrils reconstituted from pure collagen solutions were deposited


to polymer-coated planar surfaces during fibrillogenesis and subsequently assessed by SEM and AFM images (Figure 4). Evaluation of the average fibril diameters by means of SEM provided no effect of initial collagen concentration on the fibril size indicating that differences in turbidity could only be attributed to the amount of fibrils in the gel. The differences in the fibril layer topography dependent on the initial concentration of collagen were similarly reflected by ellipsometric measurements and protein quantification by acidic hydrolysis and HPLC on modified planar substrates. Before protein immobilization, glass slides or wafers were coated with thin films (thickness about 4-5 nm) of a reactive polymer consisting of alternating units of maleic anhydride and alkyl chains.23 Alteration of the alkyl comonomers allows for adjustment of physicochemical surface characteristics which affect the behavior of biomolecules and cells as recently described by Renner et al.27 In our study, poly(octadecene-alt-maleic anhydride) films were invariably utilized. The alkyl chains of this copolymer are capable of mediating hydrophobic interactions between the surface and the protein while covalent immobilization can be accomplished by reaction of the anhydride moiety with lysine side chains of collagen. However, if fibrillogenesis was performed in the presence of cleaned glass slides or slides covered with poly(octadecene-alt-maleic acid), significantly lower collagen amounts (0.67 and 1.02 µg/cm2, respectively, starting from a 1.2 mg/mL collagen solution) were calculated on the surfaces after displacement of the resulting gel in contrast to collagen that was quantified on poly(octadecenealt-maleic anhydride) films (Table 1). The lack of a covalent anchorage of collagen results in an easier separation of collagen fibrils from hydrophilic as well as hydrophobic surfaces by the displacement of the bulk phase and/or by washing procedures. Therefore, contact of reactive polymer coated substrates to the fibril-forming collagen solutions can be considered to allow for the covalent immobilization of monomeric collagen and, through this, the anchorage of the fibrillar collagen assemblies. Deposition of tropocollagen from acidic solution provided no significant increase in layer thickness and consequently a low protein surface concentration was determined by amino acid analysis. The pH of the acidic collagen solution allowed no deprotonation of the lysine amino group. Therefore, no covalent attachment of the protein could be expected to occur and the immobilized collagen amount was considerably lower compared to tropocollagen deposited from neutral solutions. Ellipsometry and HPLC-based amino acid analysis provided consistent trends of the layer characteristics obtained from collagen solutions at different settings. A dependency of the immobilized amount on the initial concentration as well as on the contact time could be established. Both analytical methods demonstrated a linear growth of the protein layer during the first 90 min. At about 90 min contact time, saturation of collagen deposition occurred since only a slight increase of immobilized collagen after 24 h contact time was observed. The data were confirmed by the quantification of residual collagen (monomers) 2 h after the initiation of fibrillogenesis of 1.2 mg/mL collagen (Figure 3). About 0.22 mg/mL collagen were found in the supernatant

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of spun down fibrils at this time indicating that fibrillogenesis could have further continued from this solution. Altogether, variations of the solution concentrations and contact times were found to permit the adjustment of surface concentrations and structural characteristics of surface bound layers of collagen fibrils as imaged by AFM and SEM (Figure 4). As for pure collagen layers, measurements of the turbidity of fibril forming collagen solutions in the presence of GAGs cannot provide information on the structural composition of the gels. Therefore, fibrillar collagen-GAG assemblies were similarly further evaluated after immobilization from a fibril forming solution to reactive polymer surfaces. Deposited collagen-GAG assemblies exhibited significantly diminished layer thicknesses compared to the thickness of pure collagen fibril layers (Table 2). Furthermore, the topography of immobilized collagen-GAG assemblies (5.0 mg/mL GAG in the initial collagen solution) shows different surface patterns compared to pure collagen (Figure 5). By AFM, many small fibrils were detected in the immobilized collagen-GAG complexes and the deposited layers appeared smooth and thinner as compared to the pure collagen layers. This was obviously due to the reduced number of wide and elongated fibrils. As discussed above, turbidity measurements and the quantification of residual monomeric collagen in the supernatant demonstrated an impact of GAG concentration on the fibril formation. However, the patterns of the immobilized collagen-GAG assemblies (Figures 5 and 6) did not correspond to the data from turbidity measurements and quantification of residual monomeric collagen in collagenGAG gels regarding number and size of surface bound fibrils. Therefore, SEM images were taken from collagen gels which were desiccated on glass slides (Figure 7). The desiccated gels reflect the complete content of a collagen gel and thus represent its actual structural composition. In collagen gels formed in the presence of HE, the relation of broad collagen fibrils to smaller fibrillar structures was quite different compared to the covalently immobilized collagen-HE fibrils (Figure 5). Broad and elongated fibrils were also formed in the presence of HE, where low HE concentrations supported the formation of broad and clearly structured fibrils while in the presence of 5.0 mg/mL HE a meshwork of thinner fibrils was formed. These observations correlate well with the turbidity measurements and the results of the quantification of residual collagen monomers in collagen-HE gels. Thus, fibrillar structures formed in the presence of HE on polymer-coated substrates provided no image of the gel bulk phase. Fibrillogenesis in the presence of HA provided comparable results: The fibrils attached to the polymer coated surfaces (Figures 5 and 6) did not reflect the meshwork characteristics of the fibrils in the gel bulk phase (Figure 7). However, in contrast to the fibrillogenesis in the presence of HE, no significant impact of the HA concentration on the fibril size and number was observed. The cofibrils attached to reactive polymer coated surfaces were further characterized by the quantification of the surface bound amounts of collagen (Table 2). Confirming the surface topographic observations (Figures 5 and 6), significantly less collagen was determined in comparison to the layers of pure collagen. Obviously, the electrostatic interaction of the

Salchert et al.

polyanions HE and HA with collagen may have inhibited the availability of the positively charged side chains of lysine for the covalent attachment of collagen to the polymer film. Furthermore, the presence of GAGs in collagen gels most probably resulted in a weaker cohesion of the fibril meshworks and therefore in a reduced amount of fibrils associated with the surface after removal of the gel volume phase. However, nearly no influence of the GAG portion on the covalently immobilized collagen amount was observed indicating that the relevant collagen-GAG interaction was already saturated at the lowest GAG concentrations applied in this study. The differences between the surface patterns of immobilized collagen-GAG assemblies compared to pure fibrillar collagen were attributed to strong interactions of collagen molecules with HE and HA, respectively. Collagen has a specific HE binding site,1 whereas no similar binding site is known for HA. The specific interactions of HE with collagen can be considered to enhance ordered fibril formation resulting in regular and straight fibrils. Furthermore, the HE concentration also affected fibril size since increasing heparin concentrations resulted in decreasing fibril diameters (Figure 7). We assume that two antidromic effects gave rise to this observation: The hydrophobic characteristics as well as the net charge of collagen were affected by the complexation with the polyanion heparin. Thus, fibril formation by hydrophobic interactions of collagen monomers could be suppressed by HE. On the other hand, the flexible HE chain could bridge two or more collagen monomers21 compensating their positive charges and thus, reducing their electrostatic repulsion. Obviously, both effects may have differently affected the fibril formation. This explanation could be similarly applied to fibrils reconstituted in the presence of HA. However, no dependency of fibril diameter on initial HA concentration was found (Figure 7). The large fibrils in preparations where the structures were covalently attached (Figures 5 and 6) appeared more twisted compared to collagen-heparin fibrils which could be attributed to the inhibitory effects of HA itself or its fragments on fibrillogenesis. Furthermore, HA is known to adopt a stiff helical conformation in solution.28 Therefore, the appearance of twisted collagen fibrils may be attributed to interactions of collagen to the coiled macromolecule giving the fibrils their characteristic shape. HA is also known to bind high amounts of water28 and might therefore act osmotically to compress the supramolecular structures. AFM and SEM provided no information on the localization of the GAGs in the fibrillar assemblies. However, the formation of collagen fibrils in the presence of fluorescent labeled GAGs could at least visualize the overall distribution of glycosaminoglycans in deposited collagen-GAG layers (Figure 8). This staining of fibrillar structures indicated the close contact of HE and HA to the fibrils. However, the images provided no detailed information on the GAG localization at the fibrils. Furthermore, labeled GAGs were utilized to compare surface bound collagen-GAG assemblies reconstituted in the presence of different GAG portions. Collagen-HA assemblies containing FITC-labeled HA provided no dependency of the collagen-bound HA on the


collagen-HA ratio before initiation of fibrillogenesis indicating a saturation of collagen with HA at the lowest concentration of 0.4 mg/mL or below. In contrast, a dependency of the collagen-bound amount of HE on the deployed amount was observed in similar experiments. The enhanced complexation of HE to collagen could be mediated by both nonspecific electrostatic interactions as well as by HE binding sites to collagen resulting in an enhanced attachment of HE to collagen in comparison to HA. The detection of fluorescence in close vicinity of the collagen-GAG fibrils but not along pure collagen fibrils similarly stained with FITC (Figure 6) further confirms the simultaneous generation of large and very small fibrils when fibrillogenesis was accomplished in the presence of HE or HA, respectively. Conclusions Fibrillar collagen and assemblies of collagen and glycosaminoglycans were reconstituted from solution and utilized for the surface modification of reactive polymer films. The dynamics of the fibrillogenesis of pure collagen were found to confirm an earlier proposed mechanism suggesting the initial formation of collagen dimers and trimers prior to the fibril assembly. Different initial concentrations of collagen gave rise to varied densities of the surface-bound meshwork. Reconstitution of fibrillar collagen in the presence of HE resulted in the formation of fibrils of larger and, at higher HE amounts, also significantly smaller diameters, whereas HA did not affect fibril formation in this manner. The probable cause for the coexistence of distinct fiber variants in the presence of GAGs was discussed in view of the physicochemical characteristics of the GAGs. The shape of the large fibrils was found to depend on the type of GAG. Lower amounts of collagen were immobilized in the presence of GAGs on reactive polymer surfaces which was attributed to the altered reactivity of GAG-associated collagen. The covalent immobilization of fibrillar collagen and collagen-GAG assemblies was accomplished without addition of chemical cross-linkers or the application of irradiation to perform photochemical reactions. Consequently, the exclusion of processes which could harm the biopolymer assemblies as well as the coated polymer materials enables the application of the method for the fast and simple modification of cell culture carriers and biomedical devices. Furthermore, surface bound fibrillar collagen combined with GAGs allows for the secondary loading of the coated materials with GAG-binding growth factors to control proliferation and differentiation of different cell types. Ongoing studies are dedicated to the further clarification of structure-property relations of collagen assemblies and coassemblies with GAG using complementary methods such as micro differential scanning calorimetry, polyelectrolyte titrations, and streaming potential measurements. Beyond that, surface-bound collagen and collagen-GAG assemblies are analyzed in cell culture experiments to verify the expected benefits of these carriers for the directed administration of specifically bound growth factors. Acknowledgment. The authors thank Katharina Go¨rner for assistance with protein hydrolysis and HPLC analysis.


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In Vitro Reconstitution of Fibrillar Collagen Type I Assemblies at

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