Differentiation of Osteoblasts on Pectin-Coated Titanium

Biomacromolecules 2008, 9, 2369–2376

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Differentiation of Osteoblasts on Pectin-Coated Titanium H. Kokkonen,† C. Cassinelli,‡ R. Verhoef,§ M. Morra,‡ H. A. Schols,§ and J. Tuukkanen*,† Department of Anatomy and Cell Biology, University of Oulu, Post Office Box 5000, 90014 Oulu, Finland, Nobil Bio Ricerche, Str. S. Rocco 36, 14018 Villafranca d‘Asti, Italy, and Laboratory of Food Chemistry, Wageningen University, Bomenweg 2, 6703HD Wageningen, The Netherlands Received April 4, 2008; Revised Manuscript Received July 8, 2008

The gold standard for implant metals is titanium, and coatings such as collagen-I, RGD-peptide, chondroitin sulfate, and calcium phosphate have been used to modify its biocompatibility. We investigated how titanium coated with pectins, adaptable bioactive plant polysaccharides with anti-inflammatory effects, supports osteoblast differentiation. MC3T3-E1 cells, primary murine osteoblasts, and human mesenchymal cells (hMC) were cultured on titanium coated with rhamnogalacturonan-rich modified hairy regions (MHR-A and MHR-B) of apple pectin. Alkaline phosphatase (ALP) expression and activity, calcium deposition, and cell spreading were investigated. MHR-B, but not MHR-A, supports osteoblast differentiation. The MHR-A surface was not mineralized, but on MHR-B, the average mineralized area was 14.0% with MC3T3-E1 cells and 26.6% with primary osteoblasts. The ALP activity of hMCs on MHR-A was 58.3% at day 7 and 9.3% from that of MHR-B at day 10. These data indicate that modified pectin nanocoatings may enhance the biocompatibility of bone and dental implants.

Introduction Osteoblasts arise from mesenchymal stem cells via the sequential and reciprocal processes of proliferation and differentiation. Differentiation includes bone matrix deposition, maturation, and calcification. The expression of many osteoblastspecific genes related to osteoid production as well as mineralization can be detected and analyzed as an indicator of osteoblast maturity and functionality.1,2 The cessation of proliferation and the commitment of the osteoprogenitor cells to differentiation is characterized by an increased expression of, for example, alkaline phosphatase (ALP), indicating the transition stage from matrix maturation to mineralization.1-3 Bone and dental implants are widely used in several clinical applications. Implant failure is a severe condition caused by compromised surgical procedures, biomechanics, and negative host tissue reactions. The ability of an implanted biomaterial to induce appropriate host responses in a particular application defines its biocompatibility.4 Biocompatibility thus includes proper interactions between an implant and the surrounding cells. This is partly mediated by production and deposition of bonespecific extracellular matrix (ECM) proteins, such as fibronectin (FN), onto the biomaterial surface.5 A specific amino acid motif (arg-gly-asp; RGD) of ECM proteins functions as a link between the ECM and anchorage-dependent osteoblastic cells, whose transmembrane integrin receptors, important components of the so-called focal adhesions (FAs), attach to the RGD motifs.6,7 To obtain adequate osseointegration of implants, the surface must be compatible with both biological phases of osteoblastic development, that is, proliferation and differentiation. To achieve this, various biomaterials and coatings are being investigated. For example, human osteoblastic cells have been differentiated in vitro on an algae-derived hydroxyapatite ceramic bone * To whom correspondence should be addressed. Tel.: +358 8 5375163. Fax: +358 8 537 5172. E-mail: [email protected]. † University of Oulu. ‡ Nobil Bio Ricerche. § Wageningen University.

substitute.8 The effect of some glycomolecules, such as chitosan,9 on the behavior of osteoblastic cells has also been tested. Pectins are plant-specific polysaccharides that provide mechanical strength for the cell walls of higher plants in addition to playing an important role in various cellular processes, for example, water binding, morphological development, and fruit ripening.10,11 Pectins are widely used in the food industry due to their gelling, thickening, and emulsifying properties: properties that are affected by the chemical characteristics of the raw material as well as extraction conditions and could be modified using chemical and enzymatic treatments. Pectins consist of socalled smooth regions and hairy regions. The smooth region is composed of repeated and partly methyl-esterified D-galacturonic acid units (R-1,4-linked homogalacturonan), whereas alternating D-galacturonosyl (R-1,4-linked)- and L-rhamnosyl (R-1,2linked) residues forming a rhamnogalacturonan backbone are present within the hairy regions of the pectin. Arabinans and arabinogalactans may form neutral sugar side chains of the rhamnogalacturonan, while xylogalacturonans may also be present as a pectin structural element. In addition to methyl esters, acetyl groups may also be found in the O-2 or O-3 position of the galacturonic acid residues within the hairy regions.11-13 Structural variables give rise to different physicochemical properties, such as total charge, side-chain branching, and molecular weight, of different pectins. The possibility of modifying these properties with a set of enzymes makes pectins interesting candidates for biomaterial coatings. For example, the hairy region can be separated and treated with various enzymes, for example, rhamnogalacturonase, endopolygalacturonase, and pectin methylesterase, yielding so-called modified hairy regions (MHRs).14-17 The recent findings with fibroblasts on pectin-grafted polystyrene Petri dishes have demonstrated that MHRs control fibroblast behavior via serum adhesive proteins such as fibronectin.18 Such a mechanistic interaction may also exist between MHRs and osteoblasts. Fibroblasts and osteoblasts derive from the same mesenchymal stem cells. Thus, MHR as a bioactive 6-10 nm nanocoating that is not resorbed or

10.1021/bm800356b CCC: $40.75  2008 American Chemical Society Published on Web 08/05/2008

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delaminated even in implants under mechanical loads may help control cell adhesion and behavior. In comparison, calciumphosphates and ceramic coatings may suffer from mechanical restrictions and mammalian-derived biomolecules, such as RGD peptides, may be resorbed from the surfaces. Thus, the importance of pectin coatings is that they are not metabolized by mammalian cells and the nanocoating may be more permanent than the biomolecules derived from mammalian origin. Moreover, pectins are relatively inexpensive and easy to obtain in comparison to, for example, recombinant proteins. Additionally, the human body cannot degrade pectins in tissues. When considering pectins as implant nanocoatings, it is also noteworthy that pectins have been reported to have various immunological influences, some of which, that is, anti-inflammatory properties,19,20 may be beneficial in implantations. However, in some cases, pectin fragments have also been shown to induce immunological responses.21 Pectin coating can be tailored by enzymatically modifying the length of the hairy regions, which determine the wettability of the coated surfaces. In this study, we have assessed two applederived MHRs (MHR-A and MHR-B) as novel candidate coating molecules for the widely used bone and dental implant material, titanium. These two types of tailored MHRs differ in their chemical and biological properties. MHR-A and MHR-B differ in terms of, for example, sugar contents and side chain length. MHR-B has shorter arabinan side chains, a lower degree of acetylation and more galactose and xylose than MHR-A.18 These MHRs represent the opposite types of tailored MHRs, which is an important starting point for comparing the effects of different MHRs on bone cells.18 MHR-B has previously been shown to enhance cell spreading and growth, in contrast to the MHR- A and the other MHRs studied, which lead to cell aggregation and decreased proliferation.18 The proliferation and differentiation of osteoblastic cell line, primary osteoblasts, and human mesenchymal cells on the MHR-coated titanium discs was investigated using both biochemical and molecular as well as microscopic methods to obtain preliminary information about bone formation on pectin nanocoatings coupled with titanium.

Materials and Methods Cell Cultures. MC3T3-E1 cells (subclone IV) murine calvarial preosteoblasts (ATCC, LCG Promochem) were cultured in Dulbecco’s modified Eagle’s medium, R-modification (R-MEM, Sigma or Gibco) containing 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin + 100 µg/mL streptomycin (Sigma), and 1% L-glutamine (L-Glu, Gibco). The cells were grown in a +37 °C incubator (5% CO2, 95% air) and divided 1:6 every third day before culturing on the test materials. The mineralization culture medium contained 50 µg/mL -2 L-ascorbic acid 2-phosphate (AsAP, Sigma) and 10 M β-glycerol phosphate disodium salt pentahydrate (βGP, Fluka). Ascorbic acid is needed for enhanced expression of osteoblast marker genes, as well as for collagen deposition and the ALP activity of differentiated osteoblasts, and functions synergistically with β-glycerol phosphate.22 Trypsinized MC3T3-E1 cells were cultured in mineralization medium on the test materials (104 cells/cm2) for 14 days. Primary osteoblasts differentiate from the bone marrow cell pool of 12-week-old male C57/BL6 mice. Mice were sacrificed with CO2 suffocation and cervical dislocation, then the femurs and tibias were removed and dipped in 70% ethanol. The bone ends were removed and the bone marrow flushed out of the bones with osteoblast culture medium, R-MEM (Sigma or Gibco) containing 10% FBS (Gibco: SPS Certified Orig. U.S.A.), 100 U/mL penicillin + 100 µg/mL streptomycin (Sigma), 1% L-Glu (Gibco), 50 µg/mL AsAP (Sigma), 10-2 M β-GP (Fluka), and 10-8 M dexamethasone (Dex, Sigma), using a 10 mL syringe with a 27 G needle. Dexamethasone is a glucocorticoid, which

Kokkonen et al. enhances the expression of osteoblast marker genes in primary osteoblasts and promotes the activity of ALP.23-25 The cells were centrifuged (1200 rpm, 8 min), resuspended in the medium, counted, and plated at a density of 106 cells/cm2 on the test materials. The cells were cultured for 14 days, and half of the medium was changed every third day. Human mesenchymal cells (hMC) were purchased from Cambrex Inc. (Walkersville, Md) and grown as directed. Briefly, cells were plated at 5 × 103 cells/cm2 for continuous passaging in control medium (Dulbecco‘s modified Eagle‘s medium, DMEM, supplemented with 10% FBS, 100 U/mL penicillin G, and 100 µg/mL streptomycin sulfate, and 1% L-Glu). The experimental medium was the osteogenic basal medium (Cambrex, PT3924) supplemented with 0.05 mM AsAP, 10 mM βGP (Sigma Chemical Co, St Louis, Mo), 0.1 µM Dex, and Mesenchymal Cell Growth Supplement (MCGS). Trypsinized cells were resuspended in the experimental cell culture medium and diluted to 1 × 105 cells/mL, after which 1 mL of the cell suspension was seeded onto the coated samples. Coated Test Materials. Osteoblasts were cultured on differentially coated titanium discs (Ø 6 mm). The substrates were surfacefunctionalized by plasma deposition of amino groups (aminated surface) and coated using carbodiimide condensation with different apple-derived modified hairy regions. Details of the coupling process have been reported elsewhere.13 Shortly, machined grade 4 cpTi discs where extensively rinsed in isopropanol, dried in an oven, and finally plasma-cleaned by Ar plasma. Discs were then surface-functionalized by the introduction of amino groups via deposition from allylamine plasma. Briefly, allylamine vapor was introduced from a reservoir kept at room temperature (RT) into a stainless steel, capacitively-coupled, parallel-plate reactor. The reactor is equipped with a radio frequency generator (13.56 MHz) and internal electrodes. The volume of the reactor chamber is about 3 dm3, and the distance between the electrodes is 10 cm. The monomer flow rate, as evaluated by the increase of the pressure with the pump turned off, is about 50 sccm (standard cubic centimeters per minute). The pressure, before switching on the discharge, was 200 mTorr. The samples were located on the watercooled grounded electrode, and deposition, using a discharge power of 100 W, was performed for 30 s, using pulsed plasma with a duty cycle of 4 ms on and 4 ms off. After treatment and prolonged rinsing in doubly distilled water, samples were dried under a laminar flow hood. In the second step, MHRs were covalently coupled to the surface amino groups on Ti. In brief, 5 mL of an unbuffered 0.5% MHR solution was placed into a Petri dish containing the Ti discs and coupled by carbodiimide-mediated condensation between carboxyl groups present in the MHR and amino groups of the surface. In particular, 0.04 g of dimethylaminopropylethylcarbodiimide hydrochloride (Fluka) and 0.03 g of N-hydroxysuccinimide (Fluka) were dissolved in the 5 mL of aqueous MHR solution. The reaction was carried out at RT overnight, and then samples were extensively rinsed in deionized water. Samples were dried under a laminar flow hood before cell adhesion experiments and surface characterization. MHRs had been produced using the commercial enzyme preparations Rapidase C600 (DSM Food Specialties) and Rapidase liq+ (DSM Food Specialties). Both enzyme mixtures release the rhamnogalacturonan region from the homogalacturonan part of the pectins producing two different MHRs. In further sections these coatings are reported as MHR-A (Rapidase C600) and MHR-B (Rapidase Liq+). The molecular contents of these fragments are reported in Table I. The substrate materials were treated with 10× penicillin-streptomycin solution (Sigma) at +4 °C for at least 2 h and rinsed with 1× PBS before applying the cells. Cells are also cultured on aminated (AMI) and on uncoated titanium (Ti) as controls. A parallel set of hMC samples was obtained by performing the surface modification process directly on polystyrene.13,20,26 This set of samples was used to follow the changes in cell morphology as a function of culturing time, because opaque Ti discs do not allow direct visualization.

Osteoblasts on Pectin-Coated Titanium


Table I. Chemical Compositions (mol %) of MHR-A and MHR-Ba sugar arabinose galactose galacturonic acid glucose mannose rhamnose xylose methyl groups/100 mol of galacturonic acid acetyl groups/100 mol of galacturonic acid rhamnose/galacturonic acid % (w/w) sugar a

MHR-A (mol %)

MHR-B (mol %)

50 10 26 1 0 5 8 40

11 20 37 3 0 11 18 34

55

11

0.19 66

0.3 78

Partly modified from ref 13.

Labeling and Microscopy. At culture day 10, 50 ng/µL tetracycline hydrochloride (Sigma-Aldrich) was added to the cultures. Tetracycline is an antibiotic that binds to calcium and can be visualized as a fluorescin stain. After 14 d in culture, the cells were fixed in a 3.0% paraformaldehyde (PFA) solution for 10 min at RT and rinsed three times with 1× PBS. The fixed cells were fluorescently stained with TRITC-phalloidin, Hoechst 33258 and paxillin antibody. TRITC-phalloidin stains Actin, and the Hoechst stain binds to the nuclei. The paxillin antibody marks paxillin molecules, protein components of FAs, indicating appropriate cellular attachment onto the underlying surface. A dilution of 1:200 of TRITC-phalloidin (Sigma Chemical Co.) was incubated for 20 min at +37 °C. The Hoechst 33258 stain (Sigma Chemical Co.) was used at a 1:800 dilution for 10 min at RT. For paxillin staining, 0.1% (v/v) Triton-X-100 in PBS is used for permeabilization of the cells (10 min on ice), after which the cells were treated with 0.2% BSA (30 min at RT). The staining reaction was performed on ice using 1:100 mouse monoclonal paxillin antibody (ZYMED Laboratories) for 45 min and with a secondary antibody (ALEXA Fluor 488 goat antimouse IgG, Molecular Probes) for 30 min. The stains were visualized with a confocal microscope (LSM 510, Zeiss). The cells were observed at 40× and 63× magnifications with water immersion objectives of high numerical aperture. Tetracyclinestained areas were quantified from a maximum of 10 confocal microscopic fields (40×) per sample type with a digital image analysis system (MCID-M5+, Imaging Research, Inc., Canada). These values represent the proportional areas, that is, tetracycline-labeled area/the total area of the microscopic field. Cell Counting and ALP Activity. At selected time intervals (3, 7, and 10 d) the number of the hMC on the samples was evaluated. Briefly, samples were removed from the wells, rinsed with PBS, and placed into a 24-multiwell plate. Cells were harvested from the sample surfaces by trypsin and counted in a hemocytometer. The ALP activity of the hMC was measured after 3, 7, and 10 d in culture. After rinsing the samples with PBS, 1 mL lysing solution (0.2% Triton X-100, Sigma) was added to each sample. After that, the lysates were incubated for 2 h at 37 °C with 1 mL of the enzyme substrate mixture solution containing 1 mM MgCl2 × 6H2O, 1.5 mM 2-amino2-methyl-1-propanol, and 20 mM p-nitrophenylphosphate. The reaction was stopped with 1 mL/well of 1 M NaOH. Enzyme activity was measured at a wavelength of 405 nm using a Tecan Genios microplate reader. The readings were compared with values measured on a standard curve obtained by the measurements of standard solutions of pnitrophenol. The specific ALP activity was determined as a ratio of absorbance to cell number as evaluated by a MTT assay. Briefly, at a given experimental time, the cells were washed with sterile PBS, after which PBS was replaced with 2 mL/well of MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide)-sodium succinate solution. The cells and MTT solution were incubated at 37 °C for 3 h. During this

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time the yellow MTT solution was transformed by the cells’ mitochondrial dehydrogenase into insoluble blue formazan. By measuring the amount of formazan produced it is possible to measure mitochondrial activity, and, as a consequence, cell viability. At the end of the incubation period, the MTT solution was removed and replaced with 2 mL/well of 6.25% v/v 0.1 mol/L NaOH in dimethylsulfoxide in order to dissolve the formazan. The wells were swirled for 5 min until the purple color was uniform and the absorbance is evaluated at 560 nm. RT-PCR. Total RNA from cells growing on sample discs was isolated with a GenElute Mammalian Total RNA Kit (Sigma-Aldrich) according to the manufacturer‘s instructions at different time points depending on the cell type. RNA from MC3T3-E1 cells was isolated at days 3, 7, and 15 (denoted as 3 d, 7 d, and 15 d) and from primary osteoblasts at 10 and 15 d because of the presence of various cell types and slower settling of the cultures. The RNAs were concentrated with 5 M NaCl and ethanol precipitation, and the concentration and purity analyzed with a spectrophotometer (Eppendorf BioPhotometer). The RNAs were used in reverse transcriptase polymerase chain reactions (RT-PCR) in which RNA (9 ng) was reverse-transcribed to cDNA and subsequently amplified with PCR reactions using genespecific primer pairs (Sigma Proligo). cDNA syntheses were conducted with SuperScript III Reverse Transcriptase (Invitrogen), after which the cDNAs were amplified with DyNAzyme EXT DNA polymerase (Finnzymes) according to the manufacturers‘ instructions. Wild-type genomic mouse DNA and water were used as templates in negative control reactions. The expression of a housekeeping gene β-Actin was tested in order to control the amount and quality of the RNAs used in the RT-PCR reactions. The primer sequences for ALP were 5’GCCCTCTCCAAGACATATA-3’ (forward) and 5’-CCATGATCACGTCGATATCC-3’ (reverse) demarcating a product size of 372 bp,27,28 and for β-Actin 5’-TGGACTTCGAGCAAGAGATGG-3’ (forward) and 5’-ATCTCCTTCTGCATCCTGTCG-3’ (reverse) demarcating a product of 289 bp.28,29 The PCR reaction conditions for both genes started with 30 min at 48 °C followed by 10 min at 95 °C (ALP) or at 94 °C (β-Actin). ALP was produced in 30 cycles of 1 min 95 °C, 2 min 55 °C, and 1 min 72 °C. β-Actin synthesis consisted of 23 cycles of 1 min 94 °C, 45 s 62 °C, and 45 s 72 °C. The reactions were completed with an incubation for 7 min at 72 °C. DNA fragments were detected after 1% agarose (Sigma) gel electrophoresis by staining the fragments with 1 µg/mL SYBR Green I Nucleic Acid Stain (Fluka). DNA samples were loaded into the gel with gel loading solution (Sigma) and electrophoresed at 80 V for 45 min-1 h (Stratagene 10 × 10 Horizontal Electrophoresis Apparatus) in 1× Tris-acetate-EDTA buffer (Sigma). DNA bands were visualized with UV light (Alpha Innotech Corporation), and the sizes of the fragments were verified by comparison to GeneRuler 100 bp DNA ladder (Fermentas). Statistical Analyses. Data sets of the proportional tetracycline areas and the specific ALP activities were statistically analyzed with an ANOVA (Analysis of Variance) and a t test for two independent samples using SPSS 14.0 software. The normality of the data distributions is analyzed with a Kolmogorov-Smirnov test. A p-value less than or equal to 0.05 was considered statistically significant. Histograms are drawn with Origin6.0 software.

Results Cell Spreading and Attachment. Cells were stained with phalloidin and paxillin antibodies to visualize the spreading and attachment of the cells. Confocal images of MC3T3-E1 cells and primary osteoblasts growing on uncoated titanium discs (Ti), aminated discs (AMI), or discs coated with MHR-A and MHR-B are depicted in Figure 1. The confluency and spreading of the cells revealed by Actin stress fibers is clearly impaired on MHR-A compared to other sample types of which MHR-B and pure Ti seem to be the most favorable for cell spreading. On aminated surfaces cells have also spread, but the cell confluency

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Figure 1. Morphological appearance of MC3T3-E1 cells and primary murine osteoblasts are visualized on (A) MHR-A, (B) MHR-B, (C) AMI, and (D) Ti discs after 2 weeks of culture in mineralization medium (confocal images, 63× objective, picture framesize 146.23 µm × 146.23 µm for MC3T3-E1 cells and 206.78 µm × 206.78 µm for primary osteoblasts). Actin is seen as red and paxillin as green fluorescence.

Figure 2. Confocal images (40× objective, picture framesize 230.32 µm × 230.32 µm) of tetracycline-stained calcium deposits of MC3T3-E1 cells and primary murine osteoblasts on (A) MHR-A, (B) MHR-B, (C) AMI, and (D) Ti.

is reduced compared to MHR-B and the titanium control. With MC3T3-E1 cells the major difference between MHR-A and the other sample types was the lower degree of confluency. However, the cell line attached on MHR-A with focal contacts, whereas primary osteoblasts were not able to make focal contacts on MHR-A. With both cell types, the greatest abundance of FAs was manifested on MHR-B and Ti samples. The inferiority of the MHR-A coating was further revealed by primary osteoblasts, in which the Actin cytoskeleton has not organized at all. The hMC grow and adhere well on MHR-B, AMI, and Ti surfaces, while they maintain a round morphology on MHR-A. Cell morphology switched to the expected cuboidal shape23 after 4-5 days of culture, except on MHR-A, where the cells are barely adherent. Calcium Quantification: Tetracycline Staining. The amount and appearance of the deposited calcium stained with tetracy-

cline varies between sample types with both MC3T3-E1 cells and primary osteoblasts (Figure 2). On MHR-A, no stained mineral can be observed in contrast to MHR-B, AMI, and Ti surfaces on which calcium formed relatively sizable clusters forming distinctive centers of mainly rounded depositions. The proportional areas of the bound tetracycline stain were evaluated from confocal microscopic fields of the samples. The tetracycline-labeled area on the MHR-A coating was statistically significantly lower (no stained calcium at all) than on any other sample type with both MC3T3-E1 cells as well as primary cells. On MHR-B the average mineralized area was 14.0% with MC3T3-E1 cells and 26.6% with primary osteoblasts. The mineralized area on AMI was 12.7 and 24.4% and on titanium 14.2 and 27.5%, respectively. The proportional tetracycline areas did not differ between MHR-B, AMI, and Ti with either of the cell types. These data are presented in Figure 3.



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Figure 3. Proportional tetracycline-stained areas (calculated from confocal microscopic fields; 40× objective) of MC3T3-E1 cells and primary murine osteoblasts. The differences were not statistically different. MHR-A is excluded from the graph since there was no visible mineralization.

Figure 4. Specific ALP activity of hMC grown on the four different samples tested in osteogenic medium. Differences between MHR-A and the other samples are significant (**) at 7 and 10 days. Differences between MHR-B and all the other samples are significant (**) at 10 days.

hMC-Specific ALP Activity. The results of the specific ALP measurements are reported in Figure 4: At day 3, the induction of osteoblastic differentiation was still occurring, and cells grown on all samples showed very low ALP activity; at day 7, cells grown on all samples that support cell adhesion (MHR-B, AMI, Ti) yield similar ALP values, while significantly lower activity is detected for MHR-A; and at day 10, when hMC morphology is fully osteoblastic, cells cultured on MHR-B show the highest ALP activity. RT-PCR. The expression of a housekeeping gene β-Actin indicated a uniform and adequate amount and functionality of all the RNAs (data not shown) except that of RNA isolated at day 3 from MHR-A samples, probably resulting from the low number of cells on this coating type. The ALP RT-PCR results are shown in Figure 5. At day 3, ALP expression of MC3T3E1 cells was not detected in any sample type except very faintly in Ti. However, at days 7 and 15, clear ALP bands were produced in all sample types. Similar results were obtained at time points 10 and 15 d with primary osteoblast RNAs. However, the band on primary culture MHR-A at 15 d is fainter than in the other sample types. As expected, neither of the negative controls showed ALP expression.

Discussion In this study we have assessed the effects of modified apple pectin fragments grafted on titanium on the proliferation and

differentiation of various kinds of osteoblastic cell types; murine MC3T3-E1 preosteoblasts, murine primary osteoblasts, and human bone marrow mesenchymal cells. In general, osteoblasts prefer the MHR-B to the MHR-A coating. These results are strongly in accordance with our previous study concerning similarly coated tissue culture polystyrene (TCPS) substrata,26 in which we preliminary assessed the interactions between MHRs and bone cells. Cellular spreading indicated by Actin stress fibers is seen in all sample types with MC3T3-E1 cells (Figure 1). On MHR-B and Ti the cells are confluent, whereas on AMI and especially on MHR-A the cell layer is not confluent. hMCs behave in agreement with previous observations from different cell lines on MHR-coated TCPS.13 Primary cells are not capable of organizing an Actin cytoskeleton on MHR-A: only a diffuse Actin stain of nonviable cells is visualized (Figure 1). Another difference between cell types is revealed by the formation of FAs: Primary cells do not form FAs on MHR-A, whereas MC3T3-E1 cells do. Interestingly, this observation is partly contrary to our previous data26 in which MC3T3-E1 cells did not form focal adhesions on MHR-A-coated TCPS. This difference may be due to differences in surface topography. The Ti discs used in these experiments show the typical “machined” surface topography. While the roughness of machined Ti is comparatively low (Ra ≈ 0.300 nm),30 it is definitely higher than that of the TCPS samples we previously tested. The combination of the microrough topography and significant wettability gives rise to capillary effects that are not found on MHR-modified TCPS surfaces. We used antibiotic sterilization of the test samples with 10× concentration of penisillin-streptomycin, as done previously,13,26 and as it would be used in an industrial process of pectin-coated implants. No antibiotics were retained by the surface-linked sugars, as confirmed by extensive surface characterization work with XPS and ToFSIMS analysis (data not shown). An accurate and widely applicable method for quantitative evaluation of the mineralization efficacy of osteoblasts may be an intractable problem. We chose tetracycline staining to mark calcium in cell cultures.31,32 However, as demonstrated in Figure 2, tetracycline easily diffuses into the cells where it also stains the free intracellular calcium. This had to be taken into account when measuring the stained areas so that only the brightest signals clearly representing the extracellular mineral-bound calcium are screened. The calcification capacity of both MC3T3E1 and primary osteoblasts was analyzed by using tetracycline

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Figure 5. Products of ALP RT-PCR reactions at various time points (3, 7, and 15 days for MC3T3-E1 cells and 10 and 15 days for primary cells). Wells 21 and 22 represent negative controls (21, mouse genomic DNA; 22, pure water). DNA ladder is in well 23.

supplementation and by quantifying the proportional areas of the bound tetracycline. With both cell types, no tetracyclinestained mineral was detected on MHR-A, which reflects the inability of the cells to grow and differentiate properly on this MHR. However, on MHR-B and AMI, as well as on Ti, the cells have produced statistically uniform mineral amounts. The mineralization progression may also be dependent on the underlying material based on differences considerably more robust than nanocoatings, such as the MHRs in this study. For instance, the calcium deposition in the MC3T3-E1 cultures commenced later on titanium than on plastic.33 This should be kept in mind when investigating mineralization processes on a metallic surface, such as titanium. In addition to microscopic methods, we analyzed the differentiation of osteoblasts indicated by ALP expression and activity with spectrophotometric measurement of the specific ALP enzyme activity and RT-PCR. According to the hMC ALP activity results, osteoblasts produce only very low amounts of ALP at 3 d, which is consistent with the presumed proliferation phase preceding differentiation. This observation is supported by the MC3T3-E1 RT-PCR data, which showed that no detectable ALP was produced by day 3 in any sample type except faintly on Ti. Differences in ALP activity can be observed at day 7, when osteoblasts are supposed to begin differentiation: The ALP activity does not differ between cells cultured on MHR-B, AMI, and Ti, whereas on MHR-A the level of ALP activity is significantly lower than on the other sample surfaces further indicating the inferiority of the MHR-A coating to support osteoblastic differentiation. Interestingly, however, RTPCR reveals a visible ALP band also in the MHR-A sample. At day 10, the putative time point for differentiation, the ALP activity of cells grown on MHR-B significantly surpasses that of cells on all the other sample types, Ti included, whereas RTPCR again indicates constant ALP bands in every sample type with primary osteoblasts. These slight discrepancies between spectrophotometric and RT-PCR ALP data sets might be difficult to interpret because of the nonquantitative methodology of RT-PCR. Moreover, different subclones of MC3T3-E1 cells may fluctuate in terms of, for example, ALP expression.34 One possibility is that the deviations in the tendency of osteoblasts to differentiate as well as the timing on the different coatings may become more evident at the level of protein activity than at the level of gene expression. In addition, compared to the

tetracycline area data collected at the final 2 week time point, the ALP activity may have different timing peaks on different sample surfaces, even though the actual mineral production subsequently balances out by the end of the culturing time between MHR-B, AMI, and Ti. On the other hand, the detection of the ALP gene product in the MHR-A sample at 15 d conflicts with the total absence of tetracycline-labeled calcium on the MHR-A samples. This might indicate that the observed calcification differences originate from some chemical or physical features - probably exhibited by the MHRs, affecting the mineral deposition downstream of ALP gene expression. However, cells even with very low levels of ALP expression can produce large amounts of mineralized matrix and many authors simply use ALP as an indicator of bone phenotype.35 Many physical and chemical properties of the material on which the cells are growing affect cellular processes. An important example is surface microtopography; for instance, as the roughness of the titanium surface increases, ALP activity, and nodule formation also increases, but proliferation of the fetal rat calvarial cells decreases.36 Similar results have been obtained with MG-63 osteoblasts, whose differentiation was enhanced on rough titanium microtopographies.37 Surface energy also plays a role; osteoblast proliferation is affected by the surface energy so that osteoconduction of an implant material can be enhanced by adding polar components onto the surface.38 The adsorption of proteins from the fluids infusing the biomaterial affects cellular attachment and proliferation, and these adsorption profiles may be at least partly defined by the coating molecules: For instance, the aforementioned chitosan coating on titanium promotes the attachment of HEPM osteoblast precursors via favoring the adsorption of fibronectin and albumin proteins despite a more hydrophobic chitosan surface.9 This exceptional relationship between cellular preference and surface wettability is also detected in our results, in which the more hydrophobic MHR-B is clearly more biocompatible than the more wettable MHR-A. FN adsorbed onto titanium promotes osteoblast attachment.39 The tendency of MHRs to contribute to or hinder protein, especially FN, adsorption could thus partly explain the observed differences in cellular growth in this study. Side chain length probably affects the protein adsorption profile so that a coating molecule carrying shorter side chains (MHRB) allows protein adsorption more effectively because the hindering steric repulsion is diminished.13


Notwithstanding the importance of glycomolecules in biology they have been underutilized in biomaterial research. Glycosylation is the most common form of protein and lipid modification. Recently the concept of the sugar code of biological information has been extensively studied. Monosaccharides represent an alphabet of biological information similar to amino acids and nucleic acids but with enormous coding capacity. Studies have shown that glycomolecules naturally exist in bone tissue, such as biglycan, a proteoglycan found in the bone ECM, and are needed for osteoblast differentiation.40-42 In general, collagen-binding small leucine-rich proteoglycans (sLRP), such as biglycan and decorin, are crucial for collagen fibrillogenesis and bone-implant contact formation.33,43 Thus, it could also be important to investigate the possible effects of MHRs on the function of bone ECM glycocomponents and study the sugar language of bone biology by the means of polymer science. The general knowledge of glycomolecules could pave the way also for applicative exploitation of certain macromolecules. For instance, a soluble chitooligosaccharide molecule has been shown to promote the proliferation and differentiation of human osteosarcoma-derived osteoblasts.44 Another recent example of the interactions of glycomolecules and osteoblasts are chondroitin sulfate-treated hMSC, which displayed the most ALP production during differentiation into osteoblasts on chondroitin sulfate-coated fabrics.45 These studies illustrate that glycomolecules affect osteoblast gene expression and morphology and are consistent with our conclusion that pectin polysaccharides can influence osteoblast maturation. Glycomolecule-binding lectins represent other interesting moieties worth considering in evaluating pectin coatings of bone implants: The resorbed bone tissue can be stained with wheat germ agglutinin (WGA) lectin.46 After osteoclastic activity in the resorbed bone area certain glycoepitopes are revealed that subsequently attract bone-forming osteoblasts to the site of resorption in order for bone remodeling to proceed properly. Identification of these sugar molecules could serve as guidance for enzymatic tailoring of pectin MHRs to enhance osteoblastcompatibility. On the cellular side, osteoclast inhibitory lectin (OCIL) is a type II cell membrane bound lectin, which binds sulfated glycosaminoglycans and a NK cell-associated receptor. OCIL is expressed in various cell types found in bone and inhibits osteoclast differentiation. It is required for normal bone remodeling in vivo and it inhibits osteoblast function in vitro.47 OCIL is an interesting example of sugar interaction essential for bone remodeling.

Conclusions Our results indicate that it is possible to affect cellular proliferation and differentiation with coatings of enzymatically modified pectin fragments applied to a material exploitable in bone and dental implantations, that is, titanium. MHR-B is a pectin fragment type that merits further study. In a broader subtext of biomaterial coatings it is important to note that MHRA-like coatings could have potential in nonadhesive clinical applications. The possible anti-inflammatory effects as well as the in vivo compatibility of these MHR types should also be investigated. Acknowledgment. This work was supported by EC-project PectiCoat NMP4-CT-2005-517036 and by the Finnish Graduate School for Musculo-Skeletal Disorders and Biomaterials. We thank Marja Nissinen and Marja Paloniemi for their valuable assistance with RT-PCR.


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BM800356B

Differentiation of Osteoblasts on Pectin-Coated Titanium

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