The effect of adsorbed serum proteins, RGD and proteoglycan-binding peptides on the adhesion of mesenchymal stem cells to hydroxyapatite

ARTICLE IN PRESS

Biomaterials 28 (2007) 383–392 www.elsevier.com/locate/biomaterials

The effect of adsorbed serum proteins, RGD and proteoglycan-binding peptides on the adhesion of mesenchymal stem cells to hydroxyapatite Amber A. Sawyera, Kristin M. Hennessyb, Susan L. Bellisa,b, a

Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA Department of Physiology and Biophysics, University of Alabama at Birmingham, Rm. 982A MCLM, 1918 University Blvd., Birmingham, AL 35294, USA

b

Received 8 May 2006; accepted 16 August 2006 Available online 6 September 2006

Abstract Prior studies from our laboratory have shown that RGD peptides increase the attachment of mesenchymal stem cells (MSCs) to hydroxyapatite (HA), however, RGD does not induce cell spreading when coupled to this type of biomaterial. In an effort to improve MSC spreading, and possibly cell attachment, proteoglycan-binding peptides (KRSR or FHRRIKA) were combined with RGD in the current study. It was found that the peptide combinations did not enhance MSC attachment relative to RGD alone, although a slight amount of spreading was elicited by both KRSR and FHRRIKA. Similar results were obtained with proteoglycan-binding peptides modified with a heptaglutamate domain, a motif that improves peptide tethering to HA. To determine whether differentiation status affected cell responses, MSCs were in vitro differentiated into osteoblasts, and evaluated as before. These experiments revealed that, like MSCs, osteoblasts did not adhere in greater numbers to the peptide combinations. Finally, none of the peptides or peptide combinations were able to stimulate the robust amount of cell adhesion and spreading elicited by serum-coated HA surfaces (of note, five different species of serum were tested). Given the propensity of HA to adsorb proadhesive proteins from blood/serum, we question the utility of functionalizing HA with RGD and/or proteoglycan-binding peptides. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cell adhesion; Hydroxyapatite; Mesenchymal stem cell; Protein adsorption; RGD peptide; Bone

1. Introduction The rationale behind designing biomimetic biomaterials is to create substrates that mimic the in vivo environment, thereby stimulating specific cellular or tissue responses such as bone regeneration. Many biomimetic approaches utilize the triamino acid sequence, RGD, a cell-binding domain present within several extracellular matrix proteins, to improve cell interactions with biomaterials [1]. The RGD motif is a well-established mediator of integrin-directed cell attachment, however, this short peptide sequence displays significantly less adhesion-promoting activity relative to the corresponding intact protein (e.g., fibronectin) [2,3]. ConCorresponding author. Department of Physiology and Biophysics, University of Alabama at Birmingham, Rm. 982A MCLM, 1918 University Blvd., Birmingham, AL 35294, USA. Tel.: +1 205 934 3441; fax: +1 205 975 9028. E-mail address: [email protected] (S.L. Bellis).

0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.08.031

sequently, some studies suggest that additional domains within RGD-containing matrix proteins may cooperate with RGD in regulating cell adhesion processes [4]. It is becoming apparent that interactions between integrins and cell surface proteoglycans may be particularly important for regulating cell adhesive behavior [5]. Proteoglycan-binding domains within selected matrix proteins, such as the heparin-binding domain of fibronectin (FN), appear to cooperate with integrin-binding domains in inducing cytoskeletal reorganization (i.e., cell spreading) [6,7]. Accordingly, several investigators have tested whether cell/biomaterial interactions could be enhanced by functionalizing material surfaces with combinations of RGD and proteoglycan-binding peptides. Peptides consisting of the sequence XBBXBX and XBBBXXBX, where B is a basic amino acid and X is a hydropathic amino acid, were reported by Cardin and Weintraub [8] to be potential heparin-binding domains that would likely bind cell surface proteoglycans. Several versions of this peptide sequence

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have now been used in combination with RGD to improve cell attachment and spreading on a variety of biomaterials, although, to our knowledge, such a strategy has not yet been applied to synthetic calcium phosphates such as hydroxyapatite (HA) [9–13]. Previously, we [14–16] and others [17–21] reported that human mesenchymal stem cells (MSCs) and other osteoblast-related cells adhered better to RGD-coated HA coated than to uncoated HA substrates. However, we also found that RGD was not able to induce cell spreading [14], an event that is necessary for cell survival and osteoblastic differentiation. This was true for two types of RGD peptides; one selective for FN receptors, the other for vitronectin (VN) receptors. In the present investigation, we evaluated whether combinations of RGD and proteoglycan-binding peptides, specifically FHRRIKA [13] or KRSR, could enhance cell adhesion and/or spreading on HA, relative to RGD alone. Results from these studies indicated that the peptide combinations did not provide any significant benefit beyond that elicited by single peptide coatings. Most importantly, none of the peptide treatments were able to induce the same degree of cell adhesion and spreading as that observed on HA surfaces pre-coated with serum proteins. Given the capacity of HA to adsorb proadhesive proteins such as FN and VN from blood/ serum, in conformations that support the binding of both purified integrin receptors and MSCs [22], we question whether peptide coatings will improve MSC/HA interactions in the in vivo environment, where HA implants become very rapidly coated with blood.

2. Materials and methods 2.1. Preparation of peptide and serum-coated HA disks HA powder (Fisher Scientific, Pittsburgh, PA) was pressed into disks using a 5/8 in hardened steel pressing die under 3000 1b pressure. Pressed disks were sintered at 1000 1C for 3 h and allowed to cool in the furnace at decreasing intervals. All disks were washed extensively with Tris-buffered saline (TBS) and warmed to 37 1C prior to experimental use. The peptide GGGGGGGFHRRIKA (FHRRIKA, 1326 g/mol) and the peptide GGGGGGGKRSR (KRSR, 945 g/mol), which are reported to mimic the proteoglycan-binding regions of various bone-binding proteins [9,13], were synthesized by American Peptide Co., Inc. (Sunnyvale, CA). Synthesized modifications of the FHRRIKA peptide included EEEEEEEFHRRIKA (E7FHRRIKA, 1831 g/mol), EEEEEEEFHRRIKAK-FITC (E7FHRRIKA-FITC, 2348 g/mol), and GGGGGGGFHRRIKAK-FITC (FHRRIKA-FITC, 1326 g/mol). The cyclic peptide GPenGRGDSPCA (RGD, 948.1 g/mol) was also synthesized by American Peptide Co, Inc. The lyophilized peptides were reconstituted in deionized water and diluted in TBS to final peptide concentrations. HA disks were placed in the bottom of low-attachment cell culture dishes (to prevent the non-specific binding of cells to the wells of the tissue culture plate). The disks were then coated overnight at 4 1C with varying concentrations of RGD, FHRRIKA, KRSR, E7
FHRRIKA, E7FHRRIKA-FITC, FHRRIKA-FITC, or 100% fetal bovine serum (FBS, Hyclone, lot APA20464), human serum (HuS, Cellgro, lot G00830), goat serum (GS, Chemicon, lot 429822), rat serum (RS, Chemicon, lot 25030684), or donkey serum (DS, Chemicon, lot 25080164).

2.2. Cell culture MSCs were isolated from human bone marrow (male samples ranging from 21 to 45 years old), as previously described [14]. Briefly, cells were pelleted by low-speed centrifugation, resuspended in Dulbecco’s modification of Eagle’s medium (DMEM), and then applied to a Histopaque-1077 (Sigma, St. Louis, MO) column. A density gradient was established by centrifugation at 455g for 30 min, and MSCs were extracted from the media/Histopaque interface. MSCs were grown in standard growth media containing DMEM, 10% FBS, 1% amphotericin B, and 1% penicillinstreptomycin; cells from passages 2 to 13 were used for our experiments. Cells isolated in this manner had a homogenous and fibroblast-like appearance, and no osteoclasts or adipocytes were present (as measured by TRAP and Oil-O-red staining, respectively). For selected experiments, MSCs were differentiated into osteoblasts according to established protocols. Specifically, upon confluency, cells were fed osteogenic (OS+) media, a cocktail containing standard growth media supplemented with 0.05 mM aspartic acid, 10 mM b-glycerophosphate, 100 nM dexamethasone, and 100 ng/ml BMP-2 for 21 days. Mineralized nodules were imaged using a Nikon SMZ-U stereomicroscope on day 21. Human bone marrow samples were obtained with prior approval from the University of Alabama Institutional Review Board (Appendix A).

2.3. Cell attachment assays Cell attachment to pre-coated HA disks was evaluated using a fluorescence-based cell attachment assay [23]. Near-confluent cells were labeled with a 2 mM Cell Tracker Green CMFDA fluorescent probe according to the vendor protocol (Molecular Probes, Eugene, OR, absorption 492 nm, emission 517 nm). Labeled cells were then detached from tissue culture flasks with a 0.05% trypsin/0.1% EDTA solution, followed by immediate incubation with trypsin inhibitor. The cells were subsequently washed in thiol/-serum-free media, and re-seeded onto peptide-coated HA disks at a density of 1  105 cells per disk. Cells were allowed to attach to the disks for 1 h, and then unattached cells were removed by three washes with PBS. The number of bound cells was quantified by permeabilizing cells with 1% Triton X-100, and then monitoring the fluorescence of the solutions using a fluorometer (BioRad Laboratories, Chicago, IL). The first set of attachment assays utilized 1–100 mg/ml RGD, FHRRIKA, or KRSR peptides, or 100% FBS pre-coated on HA disks. The second set included five different species of serum (FBS, GS, DS, RS, HuS) pre-coated on HA. Several cell attachment assays were performed on HA disks pre-coated with peptide combinations (mg/ml:mg/ml) of RGD:KRSR (10:10), RGD:FHRRIKA (10:10, 100:100, 100:10, and 10:100), and RGD:E7FHRRIKA (10:10, 100:100, 100:10, and 10:100).

2.4. Fluorescent microscopic analysis of cell morphology Cells were detached from tissue culture flasks as described above, and then seeded onto pre-treated HA disks. Following 1 h incubation at 37 1C and several washes with PBS, the cells were fixed in 3.7% formaldehyde/ TBS solution for 15 min. After two washes with PBS, cells were permeabilized by incubation in TBS containing 0.2% Triton X-100 for 2 min, and then again washed with PBS. All disks were incubated in 2% denatured bovine serum albumin for 10 min to block non-specific labeling. To detect polymerized actin, disks were incubated with phalloidin (1:200 dilution in TBS), conjugated to Alexa 488 (a green fluorescent dye) (Molecular Probes). After 1 h, disks were again washed and mounted on microscope slides in silicone gaskets (Sigma) with an n-propyl gallate (Sigma)/glycerol mounting solution. Cells were visualized using a Leitz Orthoplan fluorescent microscope.

2.5. Western blot HA disks were coated overnight at 4 1C with serum from one of five different species (rat, goat, donkey, human, and bovine). The next day,

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2.50 cell attachment

unbound proteins were washed away with several changes of PBS. Proteins adsorbed to the HA surface were desorbed by incubating HA disks in boiling SDS-buffer (50 mm Tris buffer, 2% SDS, 5% b-mercaptoethanol) for 10 min. Subsequently, the desorbed proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were either incubated with an antibody against FN (Chemicon), or VN (Santa Cruz), according to the vendor’s specifications. An HRP-conjugated secondary antibody was then added (Amersham Biosciences), and proteins were detected using enhanced chemiluminescence (Amersham Biosciences).

**

2.00 1.50 1.00

*

0.50 0.00 UNC

1

2.6. Visualization of immobilized peptides

2.7. Statistical analysis At least three independent experiments were performed for the assays described above, with each experiment performed in triplicate. Data sets were assessed using parametric techniques (One-Way ANOVA). If significant differences were found, Fisher’s Protected Least Significant Difference post hoc test was used to determine the level of significance. A 95% confidence level was considered significant.

3. Results HA disks were pre-coated with either RGD or one of two different proteoglycan-binding peptides, FHRRIKA or KRSR. Human MSCs were seeded onto the disks in serum-free media, and allowed to adhere for 1 h. As shown in Fig. 1, MSCs exhibited equivalent levels of cell attachment on HA disks pre-coated with RGD, FHRRIKA, or KRSR (presented in a range of concentrations), and the binding observed on all of the peptide-coated surfaces was consistently greater than that elicited by uncoated HA. However, cell attachment to the peptidecoated surfaces was significantly less than that observed on HA disks that were pre-coated with serum (FBS), indicating that serum proteins deposited onto HA facilitate better cell attachment than the proteoglycan-binding peptides or RGD peptides alone. To test whether cell adhesion could be improved by combining RGD with proteoglycan-binding peptides, HA disks were pre-coated with various ratios of RGD/KRSR or RGD/FHRRIKA and evaluated for cell attachment as before. Contrary to our expectation, none of the peptide combinations were able to increase cell-binding over that induced by singlepeptide coatings (Fig. 2). Moreover, while the peptide combinations stimulated greater cell attachment than

10 RGD

100

1

10

100

1

FHRRIKA µg/ml peptide coating

10

100 FBS

KRSR

Fig. 1. MSC attachment to HA disks pre-coated with RGD, FHRRIKA, KRSR, serum (FBS), or left uncoated (UNC). Cell-binding was equivalent on HA surfaces pre-coated with 1, 10, and 100 mg/ml of RGD, FHRRIKA, or KRSR, and was significantly less than attachment on FBS-coated HA. Results are from three independent experiments performed in triplicate. Asterisks indicate a significant difference from all treatments (*po0:05, **po0:0001). Bars ¼ means7SEM.

3.00 R = RGD F = FHRRIKA 2.50 cell attachment

The fluorescein conjugate (FITC) allows direct visualization of the immobilized peptide using fluorescent microscopy. HA disks were either pre-coated with 1, 10, or 100 mg/ml of FHRRIKA-FITC or E7FHRRIKA-FITC, as described above, or disks were left uncoated as controls. One disk from each peptide treatment of the like concentration was mounted on a microscope slide in a silicone gasket (Sigma). Collectively, each set of three disks was imaged using a fluorescent Nikon SMZ-U stereomicroscope (1  ). The relative amounts of bound peptide were quantified by using the Nikon Elements software package to measure the number of fluorescent pixels per disk (all disks are the same size). The fluorescence detected on uncoated disks (background) was subtracted from the FHHRIKA and E7FHRRIKA values.

385

K = KRSR **

2.00 1.50 1.00

*

0.50 0.00

UNC 10R

10F 10K

10R 10R 100R 10R 100R FBS 10K 10F 100F 100F 10F µg/ml peptide coating

Fig. 2. MSC attachment on HA pre-coated with varying concentrations and combinations of RGD, KRSR, or FHRRIKA, or serum (FBS). Equivalent cell attachment was observed on all HA surfaces pre-coated with either single peptides or peptide combinations. Conversely, cellbinding was significantly increased on FBS-coated HA, relative to peptide coated surfaces. Uncoated (UNC) HA promoted significantly less MSC attachment than all other treatments. Results are from three independent experiments performed in triplicate. Asterisks indicate a significant difference from all treatments (*po0:05, **po0:0001). Bars ¼ means 7SEM.

uncoated surfaces, it was far less than that demonstrated on FBS-coated HA. Proteoglycans are thought to play a particular important role in modulating cell spreading, therefore, the morphology of cells adherent to the peptide-coated surfaces was evaluated. As shown in Fig. 3, cells were very rounded on RGD-coated and uncoated surfaces, whereas MSCs were actively spreading and organizing stress fibers on FBScoatings. Interestingly, cells were slightly spread on surfaces pre-coated with FHRRIKA or KRSR, suggesting that the cells were engaging with the proteoglycan-binding peptides. However, the morphology of cells on HA disks coated with one-to-one ratios of RGD/FHRRIKA or RGD/KRSR was indistinguishable from that observed on

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Fig. 4. Morphology of MSCs attached to HA pre-coated with combinations of RGD and FHRRIKA peptides, or serum (FBS). In contrast to FBS-coated HA, surfaces pre-coated with combinations of RGD and FHRRIKA (RGD:FHRRIKA), at multiple coating concentrations (mg/ml:mg/ml), elicited only minimal cell spreading.

Fig. 3. Morphology of MSCs attached to HA surfaces pre-coated with FBS, RGD, KRSR, and/or FHRRIKA peptides. MSCs were few in number and retained a rounded morphology on uncoated (UNC) HA, and were significantly spread on HA pre-coated with serum (FBS). However, cells retained a rounded morphology on HA pre-coated with 10 mg/ml RGD peptides, and were slightly spread on HA pre-coated with 10 mg/ml of KRSR or FHRRIKA. Interestingly, the morphology of cells adherent to surfaces coated with RGD/KRSR or RGD/FHRRIKA peptide combinations was essentially identical to that observed on the surfaces with proteoglycan-binding peptides alone.

FHRRIKA or KRSR alone, indicating that the combinations were not able to induce the extensive spreading elicited by serum-coated HA. We further tested a range of ratios of RGD/FHRRIKA coatings (Fig. 4); however, once again, there appeared to be no improvement in cell spreading beyond that elicited by FHRRIKA alone. In order to determine whether the enhanced cell adhesion and spreading on FBS-coated surfaces was fetal bovine specific, we examined cell attachment and spreading on HA surfaces that were pre-coated with five different species of serum. As shown in Fig. 5, cell-binding was more than two-fold greater than that exhibited on uncoated surfaces, regardless of the type of serum used. Supporting

cell attachment

3.00 2.50 2.00 1.50 1.00

*

0.50 0.00 UNC

FBS

HuS DS serum-coated HA

RS

GS

Fig. 5. MSC attachment on HA pre-coated with multiple species of serum. Cell-binding was equivalent on HA pre-coated with fetal bovine (FBS), human (HuS), donkey (DS), rat (RS), and goat serum (GS). All of the serum-coated substrates promoted significantly greater cell attachment than uncoated (UNC) HA. Results are from three independent experiments performed in triplicate. Asterisks indicate a significant difference from all treatments (*po0:0001). Bars ¼ means7SEM.

this result, cells were actively spreading and organizing stress fibers on all serum-coated substrates, whereas the cells remained completely rounded on uncoated surfaces (Fig. 6). These results indicate that the adsorbed serum proteins facilitating optimal MSC binding and spreading on HA are present within the serum of all species tested

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FBS

Hus

387

DS

GS

RS FN

FBS

Hus

DS

GS

RS VN

Fig. 7. HA surfaces adsorb FN and VN from the serum of five different species. HA disks were coated with human (HuS), bovine (FBS), rat (RS), goat (GS), or donkey serum (DS). The disks were then washed and the proteins on the surface solubilized in boiling SDS buffer. Desorbed proteins were resolved by SDS-PAGE, and then immunoblotted for either FN or VN. As shown, measurable quantities of FN and VN were detected on HA surfaces coated with all five types of serum. Note that quantitative comparisons cannot be made due to potential species-specific differences in the affinity of the antibody used for Western blotting.

Fig. 6. MSC morphology on HA disks pre-coated with multiple species of serum. MSCs exhibited active spreading and organized stress fiber formation on HA surfaces pre-coated with five different species of serum; fetal bovine (FBS), human (HuS), donkey (DS), rat (RS), and goat (GS).

to date. The integrin ligands, FN and VN, are known to be abundant within blood/serum, therefore we assayed for FN and VN deposition onto the HA surface. Briefly, HA disks were coated with the various species of serum; the substrates were then washed extensively to remove loosely bound proteins, and proteins were desorbed by incubation in boiling SDS-PAGE buffer. Desorbed proteins were resolved by SDS-PAGE and evaluated for FN and VN by Western blot analysis. As shown in Fig. 7, all of the serum coatings promoted the deposition of FN and VN onto the HA surface (note that a quantitative comparison cannot be made due to potential species-specific differences in the affinity of the antibody used for Western blot). Several studies have shown that the density and orientation of mimetic peptides like RGD are important determinants regulating cell responses to mimetic peptides [1,24,25]. Unfortunately, there are few methods available for covalently linking peptides to calcium phosphate biomaterials, and therefore these factors cannot be readily controlled for materials such as HA. In a prior study [15], we demonstrated that peptide tethering to HA could be significantly improved by modifying peptides with a heptaglutamate domain, a module that mimics the HAbinding domain of known bone-associated proteins. Given our data showing little apparent benefit in combining proteoglycan-binding peptides with RGD (Figs. 1–4), we

hypothesized that cell responses might be improved if the proteoglycan-binding peptides were tethered more tightly to the HA surface. To this end, we synthesized FHRRIKA with an E7 domain, as well as with a FITC tag, in order to allow direct visualization of the peptides. HA disks were coated with these peptides, and then examined under a fluorescent microscope. As shown in Fig. 8, a greater density of E7FHRRIKA, as compared with standard FHRRIKA was observed at all coating concentrations tested. Moreover, peptide density increased in correspondence with increasing initial coating concentrations, suggesting that the disks were not saturated at the lower concentrations. Although tethering of E7FHRRIKA to the surface of HA was improved via the E7 domain, this enhancement in coupling did not translate into increased cell attachment (Fig. 9). RGD/E7FHRRIKA and RGD/FHRRIKA coatings supported equivalent levels of cell attachment; however, as before, attachment on these surfaces was significantly less than that observed on serum-coated surfaces (of note, the peptides used for cell interaction studies did not have FITC tags). Likewise, cell morphology on RGD/E7FHRRIKA surfaces was similar to that exhibited on FHRRIKA or RGD/FHRRIKA surfaces; cells were slightly spread but were significantly less spread compared to the morphology of cells on serum-coated HA (Fig. 10). Some of the prior studies reporting improved cell attachment on combinations of RGD and proteoglycanbinding peptides utilized osteoblasts [9,13] rather than MSCs. Thus, we repeated some of our experiments using MSCs that were in vitro differentiated into osteoblasts. As shown in Fig. 11A, MSCs grown for 21 days in a standard osteogenic media acquire the ability to mineralize a matrix, one of the hallmarks of an osteoblastic phenotype. The in vitro-differentiated cells were seeded onto HA disks coated

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FHRRIKAFITC

E7FHRRIKAFITC

1 µg/ml

Uncoated

100 µg /ml

10 µg /ml

pixels per disk

(A) 125

FHRRIKA-FITC

100

E7FHRRIKA-FITC

75 50 25 0

(B)

1 µg/ml

10 µg /ml

100 µg /ml

Fig. 8. E7FHRRIKA and FHRRIKA peptides bound to HA disks. Peptides were synthesized with a fluorescein-conjugate (FITC) to allow for direct visualization of the immobilized peptides using fluorescent microscopy. (A) The stronger fluorescent intensity on the E7FHRRIKA-FITC-coated surfaces at all coating concentrations tested indicates that the heptaglutamate-conjugated peptides bind more efficiently to HA surfaces, compared to FHRRIKAFITC peptides. The top left panel represents a schematic diagram of the samples visualized by fluorescent microscopy; peptide-coating concentrations were 1, 10, and 100 mg/ml. (B) The relative amounts of bound peptide were quantified by using the Nikon Elements software package to measure the number of fluorescent pixels per disk (all disks are of the same size). The fluorescence detected on uncoated disks (background) was subtracted from the FHHRIKA and E7FHRRIKA values.

with combinations of RGD/FHRRIKA and evaluated for cell attachment. Similar to results obtained with undifferentiated MSCs, osteoblast attachment was greater on RGD/FHRRIKA-coatings, as compared with uncoated HA, but significantly less than binding to serum-coatings (Fig. 11B). Likewise, as seen in Fig. 12, osteoblasts remained rounded on uncoated and RGD-coated HA, and were slightly spread on FHRRIKA-, and RGD/ FHRRIKA-coated HA. In contrast, osteoblasts were well-spread on serum-coated HA, although the shape of these cells was more cuboidal than that of MSCs, again consistent with an osteoblastic phenotype. Collectively these results suggest that, as with MSCs, combinations of RGD and proteoglycan-binding peptides did not improve the attachment or spreading of osteoblasts adherent to HA, relative to that facilitated by adsorbed serum proteins.

4. Discussion The RGD motif is an important mediator of integrindirected cell attachment, but this short peptide sequence exhibits significantly less adhesion-promoting activity than full-length integrin-binding proteins (e.g., FN) [2,3]. Furthermore, soluble RGD peptides only partially block bone cell adhesion to intact adhesive proteins, suggesting cells use alternative domains within RGD-containing ligands for adhesion processes [4,26]. Many studies have now shown that the proteoglycan-binding domains of several adhesive proteins can modulate cell spreading and stress fiber formation in a variety of cell types [6,7]. Hence, it is believed that cell surface proteoglycans cooperate with integrins in regulating maximal cell adhesion and spreading [5]. Indeed, several studies have demonstrated improved cell attachment and spreading by modifying biomaterials

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cell attachment

2.50

R = RGD

F = FHRRIKA

E 7F = E 7FHRRIKA

**

2.00 1.50 1.00

*

0.50 0.00

UNC 10 R

10F 10 E 7F 10:10 100:100 10:100 100:10 FBS RGD : E 7FHRRIKA µg/ml peptide coating

Fig. 9. MSC attachment to HA surfaces pre-coated with serum (FBS) or with RGD, FHRRIKA, or E7FHRRIKA peptides, either singly or in combination. Cell attachment was equivalent on all peptide-coated HA surfaces, including the RGD/E7FHRRIKA combinations (mg/ml:mg/ml), and was significantly greater on serum-coated HA. Results are from three independent experiments performed in triplicate. Asterisks indicate a significant difference from all treatments (*po0:05, **po0:0001). Bar ¼ means7SEM.

389

with both integrin and proteoglycan-binding domains [9–13]. In this study, we modified HA with two reported proteoglycan-binding peptides, FHRRIKA [13] or KRSR [9], in combination with RGD, and hypothesized that the peptide combinations would stimulate cell-binding and spreading above that of single-peptide coatings. Contrary to our hypothesis, combinations of RGD/ KRSR or RGD/FHRRIKA did not elicit any increase in MSC attachment compared to cell responses on HA surfaces pre-coated with RGD alone, and these responses were significantly less than those observed on serum-coated HA. However, we did see a slight improvement in cell spreading on proteoglycan-binding peptide surfaces compared to RGD-coated HA and uncoated HA, suggesting that cell surface receptors were engaging with the proteoglycan-binding peptides. Similar to studies performed by Dee et al. [9], our study showed cell-binding to be equivalent on RGD-coated surfaces compared to KRSRcoated surfaces; however, unlike these investigators, we did

Fig. 10. Morphology of cells on HA pre-coated with E7FHRRIKA, or a combination of RGD and E7FHRRIKA. MSCs were slightly spread on all surfaces, including HA pre-coated with E7FHRRIKA alone, or with a combination of RGD and E7FHRRIKA peptides (mg/ml:mg/ml) (without FITC tags). These results indicate that, even though E7FHRRIKA peptides are bound more efficiently to HA via an ionic linkage (E7), the combination of RGD and E7FHRRIKA peptides stimulates only minimal cell spreading on HA.

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Standard growth media

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21 days, OS+ media R = RGD

F = FHRRIKA

1.60 1.20

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0.80 0.40 0.00 UNC

(B)

FBS

10R

10F

10R 10F

µg/ml peptide coating

Fig. 11. Osteoblast attachment to HA surfaces pre-coated with serum (FBS), RGD, and/or FHRRIKA peptides. (A) MSCs were grown for 21 days in osteogenic (OS+) media and are shown forming mineralized nodules (arrow) on tissue culture plastic, an indication of an osteoblastic phenotype. (B) Osteoblasts differentiated from MSCs bound equivalently to HA pre-coated with 10 mg/ml RGD or FHRRIKA, or a combination of the peptides, and bound in significantly greater numbers to FBS-coated HA. Results are from three independent experiments performed in triplicate. Asterisks indicate a significant difference from all treatments (*po0:05, **po0:0001). Bars ¼ means7SEM.

not see additive cell-binding on surfaces coated with RGD/ KRSR combinations. Conversely, a more recent study by Dettin et al. [12] reported that a combination of RGD/ KRSR did not increase osteoblast adhesion on tissue culture plastic, in comparison to surfaces of KRSR or RGD alone. Finally, Rezania and Healy [13] reported equivalent binding of rat calvarial osteoblasts to FHRRIKA, RGD, and RGD/FHRRIKA quartz surfaces at 4 h, and they also observed slight cell spreading on quartz surfaces pre-coated with combinations of RGD/FHRRIKA at 30 min. However, cell spreading and matrix mineralization were enhanced on the combined RGD/ FHRRIKA quartz surfaces at time points beyond 4 h, suggesting that proteoglycan-binding peptides may influence osteoblast responses that occur after initial cell attachment. There are several factors that could account for the observed variability in cell responses to RGD/proteoglycan-binding peptide combinations. First, a number of different cell sources have been used for these types of studies, for example, human MSCs and rat calvarial osteoblasts. To address the possibility that cell differentiation status might regulate responses to the proteoglycanbinding peptides, we repeated our experiments using human MSCs that had been in vitro-differentiated into osteoblasts. These studies showed that, as with MSCs, osteoblasts did not adhere to the RGD/KRSR or RGD/

Fig. 12. Morphology of osteoblasts on HA pre-coated with serum (FBS), RGD, and/or FHRRIKA peptides. Osteoblasts in vitro-differentiated from MSCs were few in number and retained a rounded morphology on uncoated (UNC) HA surfaces, but were well spread and exhibited stress fiber formations on FBS-coated HA. Interestingly, spread cells exhibited a cuboidal-like shape, a morphology that is characteristic of osteoblasts. Cells were fairly rounded on 10 mg/ml RGD-coated HA and were only minimally spread on HA pre-coated with 10 mg/ml FHRRIKA or a combination of 10 mg/ml RGD and FHRRIKA peptides.

FHRRIKA combinations in any greater numbers than to RGD alone. A second factor that likely contributes to differences in cell responses to proteoglycan-binding peptides is the physicochemical properties of the specific biomaterial, for example, topography, wettability, chemistry, etc. These properties are known to influence both the amount and conformation of peptides that adsorb to the material surface. Prior investigations of RGD/proteoglycan-binding peptide combinations have been performed using glass coverslips [9], quartz [13], tissue culture plastic [10,12], and polymers [11], but not with any type of synthetic calcium phosphate. Thirdly, the method used to couple peptides to biomaterials may be a key factor in surface bioreactivity, because the coupling method regulates both peptide density and orientation. In the study by Dee’s group, peptides were covalently linked to glass coverslips, whereas Healy’s group covalently coupled peptides to quartz. Unfortunately, the chemistry of calcium phosphate biomaterials does not readily lend itself to covalent peptide linkage. Therefore, most studies of biomimetic peptides on HA have relied on non-specific adsorption.

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A few investigators have used surface silanization to generate HA substrates with covalently linked RGD peptides [17,19]. These substrates elicited better cell adhesion than uncoated HA, suggesting that the silanization procedure was successful in generating surfaces with bioactive RGD molecules. However, silanization markedly alters the HA surface, and therefore there is a good likelihood that silanization may block the adsorption of proadhesive proteins from blood/serum. The key question, which remains to be tested, is whether the benefits of covalently linked RGD and/or proteoglycan-binding peptides will outweigh the potential disadvantages associated with loss in adsorption of native integrin-binding proteins such as FN and VN. Silanization could also interfere with favorable aspects of the dynamic calcium phosphate chemistry when HA implants are placed in the bone microenvironment. The fact that HA biomaterials mimic native bone, which makes them highly interactive with the in vivo environment, may be precisely why this material is so highly osseoconductive. As an alternative to covalent linkage, our laboratory has tested whether the addition of a heptaglutamate domain (E7) to peptides can be used to increase peptide coupling through ionic mechanisms, which would not be expected to significantly alter the HA surface. The E7 domain and other acidic amino acid motifs have been shown to bind directly to HA [18,20,21,27]. We previously reported that modifying RGD with an E7 domain significantly improved the tethering of RGD to HA, and moreover, this domain did not interfere with the attachment of MSCs to the RGD part of the E7-RGD molecule [15]. In the current study, the addition of an E7 domain to proteoglycan-binding peptides improved peptide tethering to HA, as expected, but this did not appear to influence cell behavior. As observed with the non-specifically adsorbed standard FHRRIKA peptide, combining E7-FHRRIKA with RGD did not enhance cell attachment relative to RGD alone. All of the peptides, both singly and in combination, stimulated better cell attachment than uncoated HA, which at first glance might suggest a benefit for modifying HA with biomimetic peptides. However, none of the peptide coatings elicited the same degree of cell attachment and spreading as that induced by adsorbed serum proteins; notably, this was found to be true for five different species of serum. We speculate that adsorbed FN and/or VN may be responsible, at least in part, for the organized and active cell spreading seen on serum-coated HA. It is known that FN and VN are found abundantly in blood/serum [28,29], and depletion of VN from serum diminishes the attachment and spreading of explanted human bone cells [30,31]. Furthermore, osteoblastic cells have been shown to depend primarily on adsorbed FN or VN for initial adhesion and spreading on a number of biomaterials [32,33]. Previously, we reported that HA adsorbs substantially more FN and VN from serum compared to stainless steel and titanium [22], and these proteins are adsorbed in conformations that support the binding of both purified integrins and MSCs.

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In an additional study, we observed that function-blocking antibodies against the av integrin subunit, which associates with either b3 or b5 integrin subunits to form FN- and VNbinding receptors, significantly disrupted MSC attachment to serum-coated HA [16]. In light of these collective results, we hypothesize that, in vivo, the adsorption of endogenous blood proteins, including FN and VN, to HA may provide a more favorable surface for MSC attachment than RGD or RGD/proteoglycan-binding peptide combinations. In sum, the potential utility of pro-adhesive mimetic peptides is likely to depend upon the nature of the interaction between the individual biomaterial and the in vivo microenvironment. Numerous studies have shown that RGD and other pro-adhesive factors improve the performance of relatively inert biomaterials, which do not readily adsorb endogenous proteins [1,34]. However, HA is a highly adsorptive and reactive material, and therefore, any mimetic strategy applied to HA must be considered in light of how the functionalized surface will interact with molecular and cellular events occurring at the bone/ implant interface. The finding that adsorbed serum proteins promote better cell attachment and spreading than RGD/proteoglycan-binding peptides, combined with the inherent challenges associated with covalently linking peptides to the HA surface, calls into question the utility of functionalizing HA surfaces with pro-adhesive peptides.

5. Conclusion Combinations of RGD and proteoglycan-binding peptides (KRSR or FHRRIKA) did not significantly enhance MSC attachment or spreading on HA surfaces beyond that elicited by single peptides alone. Moreover, none of the peptide-modified surfaces were able to stimulate the same degree of cell attachment and spreading as that observed on serum-coated HA. These results suggest the possibility that optimal cell-material interactions may be evoked by native HA exposed to blood in vivo, rather than by surface functionalization with RGD/proteoglycan-binding peptides, although additional studies are needed to test this hypothesis. Importantly, these studies underscore the concept that, when designing biomimetic strategies for tissue engineering, one must consider how the unique surface properties of any given biomaterial will integrate with endogenous processes such as protein adsorption.

Acknowledgments The research reported in this study was supported by the NIH (AR51539 to SLB), as well as a T32 pre-doctoral training grant from the University of Alabama at Birmingham Center for Metabolic Bone Disease (AAS). The authors gratefully acknowledge Mr. Albert Tousson for assistance with the stereomicroscopy, and Dr. Anne Woods for helpful suggestions.

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