Hepatocyte growth factor (HGF) adsorption kinetics and enhancement of osteoblast differentiation on hydroxyapatite surfaces

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Biomaterials 26 (2005) 2595–2602 www.elsevier.com/locate/biomaterials

Hepatocyte growth factor (HGF) adsorption kinetics and enhancement of osteoblast differentiation on hydroxyapatite surfaces M. Hossaina,b, R. Irwina,b, M.J. Baumannc, L.R. McCabea,b, a

Departments of Physiology and Radiology, Michigan State University, 2201 Biomedical Physical Science Building, East Lansing, MI 48824, USA b Molecular Imaging Research Center, Michigan State University, MI 48824, USA c Department of Chemical Engineering and Material Science, Michigan State University, East Lansing, MI 48824, USA Received 26 April 2004; accepted 27 July 2004 Available online 11 September 2004

Abstract Hepatocyte growth factor (HGF) is a growth factor that promotes angiogenesis (tissue vascularization), cell motility, and cell differentiation, making it a potentially beneficial coating for bone implants. However, very little is known about maximizing HGF attachment to surfaces of tissue-engineered scaffolds. Here, we examine methods and kinetics of HGF adsorption onto a dense hydroxyapatite (HA) surface (used in bone implants) and determine the influence of HGF coating on osteoblast phenotype/ differentiation. We demonstrate that incubating HA with HGF in solution (and not allowing the solution to dry) resulted in maximal surface adsorption that was not enhanced by extending incubation time beyond 2 days. Daily shaking of the coated HA surface did not remove adsorbed HGF. To further examine the effect of HA on osteoblast phenotype, MC3T3-E1 preosteoblasts were seeded onto HA or HGF-HA surfaces. Gene expression analyses indicate that HGF coating enhanced osteoblast differentiation as demonstrated by increased runx2 (a transcription factor important for osteoblast lineage and differentiation), alkaline phosphatase (marker of mid stage differentiation) and osteocalcin (marker of late stage differentiation) mRNA levels. Taken together, our results demonstrate that HGF can serve as an excellent bone implant coating based on its ability to readily adsorb to HA surfaces, maintain integrity over time, and enhance osteoblast differentiation. r 2004 Elsevier Ltd. All rights reserved. Keywords: Bone; Osteoblast; Runx2; Implant; HGF

1. Introduction The natural mineral phase of bone contains a hard ceramic calcium phosphate mineral salt phase called hydroxyapatite. A similar synthetic form of calcium phosphate used as a material for bone implants contains calcium and phosphorus (molar ratio of 1.67; Ca10 (PO4)6(OH)2) and is also called hydroxyapatite. Synthetic hydroxyapatite exhibits superior biocompatibility compared to other implant materials because of its chemical similarity to bone [1–4]. Hydroxyapatite can support osteoblast attachment and differentiation in Corresponding author. Tel.: +1-517-355-6475 X 1156; fax: +1517-355-5125 E-mail address: [email protected] (L.R. McCabe).

0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.07.051

vivo [5–7] and we have previously shown that hydroxyapatite stimulates osteoblast differentiation in vitro, as marked by increased expression of markers of osteoblast differentiation, alkaline phosphatase and osteocalcin [8]. A bone implant needs to exhibit rapid bone in growth to stabilize it within bone in the shortest amount of time. To further enhance and expand the beneficial properties of HA as a bone implant, HA surfaces are being coated with growth factors, matrix proteins, cellular proteins and attachment peptides to increase bone integration of implants by stimulating osteoblast growth and/or differentiation and/or recruitment/attachment. For example, Itoh et al. [9] demonstrate that coating hydroxyapatite surface with a synthetic peptide (arginine–glycine–aspartic acid sequence) enhances

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osteoblast attachment via integrins, growth and differentiation in vitro. Similarly, Zambonin et al. [10] coated HA with insulin-like growth factor, a known stimulator of osteoblast proliferation and differentiation, and demonstrated increased osteoblast proliferation and alkaline phosphatase activity, a marker of early differentiation. Coating of HA with bone morphogenic proteins also promotes osteogenesis and may enhance biomaterial degradation [11]. Other approaches include those used by Denissen et al. [12] where HA implants are coated with alkaline phosphatase and a bisphosphonate, to allow their slow release into the extracellular environment and further enhance bone formation. Given that mature bone cells (i.e., osteoblasts) are highly metabolic [13] and require an adequate oxygen and nutrient supply to maintain function [13,14] it is logical to also incorporate angiogenic factors in implant coatings. Hepatocyte growth factor (HGF), also known as scatter factor, is an excellent candidate for an implant coating. HGF was identified in the early 1980s [15–17] and was subsequently determined to be a heterodimeric molecule composed of an alpha and beta chain [18]. The importance of HGF in organ development is demonstrated by HGF null mutation mice, which exhibit embryonic lethality [19,20]. HGF exhibits strong angiogenic properties through its ability to induce expression of vascular endothelial growth factor, another angiogenic factor, but also has angiogenic properties of its own [21,22]. Osteoblasts (including MC3T3-E1 cells) and osteoclasts express c-met, the receptor for HGF and produce HGF [23–25]. HGF has been demonstrated to stimulate both osteoblast proliferation and osteoclast chemotactic migration [24]. In combination with vitamin D, HGF promotes osteoblast differentiation of vertebral bone marrow cells [26] as well as chondroblast differentiation [27]. During the process of forming mature bone-like nodules, osteoblasts undergo three stages of development marked by elevated expression of gene subsets: proliferation (histones, collagen I), extracellular matrix maturation (alkaline phosphatase), and extracellular matrix mineralization (osteocalcin) [28,29]. Regulation of gene expression during osteoblast differentiation occurs primarily at the level of transcription although it is clear that post-translational modifications of key transcription factors also play a critical role. Runx2 is one of the transcription factors critical for osteoblast differentiation and ultimate bone formation. This is demonstrated by the inhibition of bone formation through inactivation of runx2 by deletion or mutation [30–35]. Runx2 binds to and regulates gene expression through the osteoblast-specific cis-acting element 2 (OSE2) [36,37] which is found in the promoter region of many major osteoblast-specific genes including bone sialoprotein, osteocalcin and matrix metalloproteinase13 [32,33,38,39].

In sum, HGF holds promise as a surface coating for bone implants based on its angiogenic and differentiation inducing properties. Recently, Zambonin et al. [40] reported an increase in osteoblast proliferation when cells are grown on HGF-coated HA compared to uncoated HA. Here, we determined the kinetics of HGF adsorption onto a dense HA surface and identified the influence of HGF coating on osteoblast gene expression. Our results demonstrate that HGF-coated HA enhances MC3T3-E1 differentiation relative to uncoated hydroxyapatite surfaces.

2. Material and methods 2.1. Preparation of HA discs HA powder (Hitemco Medical Applications, Old Bethpage, NY) was uniaxially pressed at 13.8 MPa (2000 psi) for 1 min using a Carver press (Carver Inc., Wabash, IN). The powder was pressed into green compacts, either 13 or 32 mm in diameter. The green compacts were carefully removed from the die, placed on an alumina plate and sintered in air at 1360 1C for 4 h in a 1612FL CM Box Furnace (CM Furnaces, Bloomfield, NJ), with molydisilicide heating elements at a heating and cooling rate of 10 1C/min. Following sintering, the average sample dimension was 27 mm in diameter, giving an average linear shrinkage of approximately 16%. This shrinkage is entirely expected as the sintering process takes the green compact from about 50% density up to nominal full density. Discs were sterilized by autoclaving for 1 h at 121 1C. Surface area was calculated assuming a circular and flat surface (SA=pr2). 2.2. HA disc coating Each HA disc (5.7 cm2) was placed in wells in 6-well plate and surrounded by agarose (0.7% agarose prepared in 1X phosphate buffered saline (PBS): NaCl 1.54 M, Na2HPO4–7H2O 0.15 M, KH2PO4 0.053 M, pH 7.4) such that the surface of the disc is left exposed. HA discs are subsequently washed with PBS and incubated, unless otherwise indicated, for 24–48 h at 4 1C with 200 ng/100 ul recombinant human HGF (PeproTech Inc., Rocky Hills, NJ) in PBS or with an equivalent volume of PBS alone as the control. 2.3. Comparison of different methods of adsorption on HA surface After 48 h of initial incubation with HGF, discs were either dry at room temperature (all liquid evaporated within 6 h) or further incubated for another 24 h at 4 1C with HGF. The former method, drying/evaproating the

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protein coating, is a standard coating method used in the literature [40] and was chosen to allow comparison to other studies. Adsorbed HGF was removed from the surfaces by first rinsing with PBS for 20 min to remove any nonadsorbed HGF, and then denaturing and removing proteins with 20 ml of SDS lysis buffer (0.5 M Tris HCl pH 6.8, 30% glycerol, 10% SDS). To test if an additional PBS incubation would influence dry HGF adsorption, some experiments received an additional 1 ml of PBS (added to both liquid and dry discs) and samples were incubated for an additional 48 h. Adsorbed proteins were removed by incubating surfaces for 20 min with 20 ml of SDS lysis buffer. 2.4. Adsorption of HGF on HA surface with long-term incubation For long-term adsorption studies, following the coating procedure, 1 ml of 1X PBS was added to the wells containing discs and discs were further incubated for 2, 3, 4, 5, 6 or 9 days at room temperature. During the incubation period, samples were continually agitated on a horizontal orbital shaker at medium speed. At each time point PBS was removed and adsorbed HGF was removed using 20 mL of SDS lysis buffer. 2.5. Adsorption of HGF on HA surface with daily washes HA discs were coated, air-dried and 1 ml PBS added to wells. Subsequently, plates containing the PBS were continuously agitated at room temperature and PBS changed daily. HGF adsorbed to the HA surface was harvested using 20 ml of SDS lysis buffer at day 2, 3, 4, 5, 6 and 9.

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containing 10% fetal bovine serum. Once confluent, medium was supplemented with 2 mM b-glycerolphosphate (Sigma, St. Louis, MO) and 25 mg/ml ascorbic acid (Sigma). Osteoblasts were harvested 15 days after plating. 2.8. RNA analysis RNA was extracted, on day 15, using TRI Reagent RNA isolation reagent (Molecular Research Center, Inc., Cincinnati, OH). Integrity of the RNA was verified by formaldehyde–agarose gel electrophoresis. Two-step quantitative RT–PCR was performed to verify gene expression. First strand cDNA was synthesized by reverse transcription of 2 mg RNA, using the Superscript II Kit and oligo dT(12–18) primers (Invitrogen Life Technologies, Carlsbad, CA). One ml of cDNA was amplified by Real Time PCR, by using the SYBR Green Core Reagents (PE Biosystems, Warrington, UK) and Taq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA) with a final volume of 25 ml. Amplification was carried out with the following primers: alkaline phosphatase (CAG TAT GAA TTG AAT CGG AAC AAC C and CAG CAA GAA GAA GCC TTT GAG G), osteocalcin (ACG GATTCA CTA TTT AGG ACC TGT G and ACT TTA TTT TGG AGC TGC TGT GAC), and runx2 (GAC AGA AGC TTG ATG ACT CTA AAC C and TCT GTA ATC TGA CAT TGT CCT TGT G) [41]. Levels were expressed relative to cyclophilin (ATT CAT GTG CCA GGG TGG TGA C and CCG TTT GTG TTT TGG CCA GCA) an internal control. Real-time PCR was carried out and analyzed for 40 cycles, using iCycler software (Biorad, Hecules, CA). Gel electrophoresis, melting curves and sequencing were used to verify the integrity of a single PCR product (amplicon).

2.6. SDS–PAGE analysis of HGF 2.9. Statistical analysis The adsorbed HGF harvested from the HA discs was examined by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Gels were then Comassie stained (0.025% comassie Brilliant blue R250, 20% methanol, 7% acetic acid) for 1 h, destained with destaining solution (20% methanol, 7% acetic acid) for another hour, and lastly, silver stained using Bio-Rad Silver Stain Kit (Biorad, Hecules, CA). This method of double staining provided the strongest HGF signals. The intensity of stained HGF protein bands was analyzed by digital photography and subsequent optical densitometry, Scion Image (Scion Corporation). 2.7. Cell culture MC3T3-E1 (osteoblasts) were plated on HA discs at a concentration of 30,000 cells per cm2 for differentiation studies. Cells were fed every 2 days with alpha-MEM

The data was analyzed using Student’s t-test. P-values p0.05 were considered significantly different.

3. Results To begin to address methods for optimizing adsorption of HGF to HA, we utilized a staining procedure (Coomassie and silver staining of acrylamide gels) that allowed us to determine the level of HGF adsorbed to the HA surface. Fig. 1 demonstrates that a linear relationship (r ¼ 0:97) exists between the amount of purified HGF loaded onto a gel (SDS-PAGE) and the intensity of protein staining. We focused on the amount/ intensity of the 69 kDa subunit of HGF, which was readily visible by our method of protein staining, in extracts of pure HGF and in HGF removed from HA

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surfaces. We applied this technique to determine relative levels of surface-adsorbed HGF in subsequent experiments. Two approaches can be taken to adsorb a protein onto a surface. The first is to incubate the surface with the protein in solution and the second is to incubate the protein in solution and allow the solution to evaporate. We examined the effect of both methods on HGF adsorption to HA. Fig. 2A indicates that HGF in solution (200 ng HGF in 100 ml of PBS) readily adsorbs to the HA surface and is not removed with PBS washing. The total amount of HGF adsorbed to the HA surface (based on the band intensity relative to known amounts of purified HGF) was approximately 50% (109 ng721, n ¼ 3) of the total amount contained in the incubation solution. Consistent with this finding, we found approximately 50% of the HGF in the supernatant, see Fig. 2C. In contrast, when the HGF-solution is allowed to dry, the adsorption of HGF to HA is modest, nearly 30% of the level adsorbed under liquid conditions (Fig. 2B). Correspondingly, we see more HGF in the supernatant (Fig. 2C). Addition of liquid to the dried HGF coating, and subsequent incubation for 48 h, regained maximal HGF adsorption onto HA surfaces (Fig. 2B). This demonstrates that drying HGF does not cause degradation or prevent HGF from readsorbing to HA surfaces (if put into solution). To determine if extended periods of incubation could further enhance adsorption (over a 2 day incubation),

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(C) Fig. 2. HGF effectively adsorbs to HA surfaces when HGF is in solution. HA discs were incubated with 200 ng HGF in 100 ml PBS. After 48 h the HGF solution was removed (liquid, L) or the HGF solution was allowed to evaporate (dry, D). Discs were then washed with PBS and the adsorbed HGF removed and separated by 10% SDS–PAGE. (A) A representative gel stained with Coomassie followed by silver stain is shown. The bar graph shows the level of adsorbed HGF in solution compared to dry conditions (set at 100%). Values are the average of 3 separate experiments7SE. (B) Following the incubation period in 2A, 1 ml of PBS was added to both conditions which were incubated for an additional 48 h. A representative gel stained with Comassie followed by silver stain is shown. The bar graph shows the level of adsorbed HGF in both conditions relative to levels remaining after drying which were set at 100%. Values are the average of 3 separate experiments7SE. (C) Following the incubation period in 2A, liquid and dried discs were washed with PBS and the adsorbed HGF as well as the HGF in the wash (supernatant) were collected, separated by SDS–PAGE and stained with Coomassie followed by silver stain.

we coated each HA disc with 200 ng HGF and harvested adsorbed HGF proteins after 2, 3, 4, 6, and 9 days of incubation. Fig. 3 demonstrates that extending the incubation time with HGF coating does not further enhance HGF adsorption to HA. To determine longterm affinity of absorbed HGF to the HA surface, coated discs were washed continually under stringent agitation throughout the time course and the remaining adsorbed HGF was harvested and levels quantitated. As shown in Fig. 4, HGF remained associated with the HA surface even after continual daily washes. Although there was a trend toward decreased HGF adsorption after 9 days, this was not statistically different from levels seen after a 2-day incubation. After determining methods for maximal HGF adsorption to HA surfaces, we next determined the influence of HGF coating on osteoblast differentiation. Based on our adsorption studies, we incubated HA discs with HGF (in solution) for 48 h and osteoblasts were

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Fig. 5. HGF coating induces alkaline phosphatase and osteocalcin mRNA levels in osteoblast. Osteoblasts cultured for 15 days on HA (control) or HGF coated HA (HGF) were harvested for RNA analyses. Alkaline phosphatase (AP) and osteocalcin (OC) mRNA levels, markers of osteoblast differentiation, were determined by real time RT–PCR. Levels are expressed relative to cyclophilin (cyclo.) mRNA levels (a non-modulated control gene). Values are expressed as an average fold increase7SE relative to control cultures (set at 1) and represent an n of 3 separate experiments;  po0:05: As shown in images, levels of PCR products (amplicons) were confirmed during the linear phase of amplification (25 cycles) by gel electrophoresis and ethidium bromide staining.

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osteoblast differentiation, was increased nearly 3-fold (Fig. 6). These findings demonstrate that HGF surface coating can significantly influence osteoblast gene expression and enhance differentiation.

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Fig. 4. HGF adsorption to HA is stable and remains after daily washing. The graph indicates the percent of remaining HGF adsorbed onto HA surfaces, after daily PBS washes and agitation, relative to day 2 values (set at 100%). Values are averages obtained from four separate experiments7SE. A representative Coomassie and silverstained SDS–PAGE of adsorbed HGF extracted from HA surface prior to washing (day 2) and then following daily washes 3, 4, 5, 6, and 9 days after starting HGF coating.

directly seeded onto coated and uncoated HA surfaces. After 15 days osteoblasts were harvested, RNA extracted, and gene expression determined. Fig. 5 demonstrates that HGF coating significantly induces alkaline phosphatase mRNA levels, a marker of early osteoblast differentiation, by over 11-fold compared to uncoated HA surfaces. Osteocalcin expression, a marker of late stage differentiation, was also increased over two-fold in cells grown on HGF-coated HA, although the induction was not statistically significant (Fig. 5). Furthermore, expression of runx2, a key transcriptional activator of

To further enhance and expand the beneficial properties of HA as a bone implant, we examined the kinetics and osteogenic potential of HGF coating HA. Little is known about the actual adsorption kinetics of many implant surface coatings. For this reason, we tested several approaches to coat hydroxyapatite surfaces with a growth factor, HGF. While many adsorption protocols suggest that protein solutions should be dried, we found that drying the HGF-solution made the HGF attachment less stable; this was based on our finding that more than 60% of the dried HGF coating was removed with a single PBS rinse (Fig. 2B and C). In contrast, when the HGF solution is not allowed to dry, a significant amount of HGF (an average of 109 ng, or roughly 50% of the total amount of HGF in the coating mixture) remains on the HA surface even following rinsing. This level is nearly three-fold more than the dried condition. This finding supports the idea that growth factor coating protocols should include maintaining implant/scaffold surface and coating proteins in solution. The solution that we used, PBS, contains ions that most likely help to stabilize protein structure as well

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as interactions with surfaces [42]. Correspondingly, most surface coating protocols for collagen, an extracellular matrix protein which exhibits significant three-dimensional triple helical structure, do not incorporate drying [43]. Examination of HGF by staining of SDS–PAGE gels, allows quantitation based on the linearity of protein band staining intensity relative to increasing levels of purified HGF as indicated in Fig. 1. This approach also allows examination of protein degradation based on the presence of additional lower molecular weight bands. Degradation products may not be detected by other analyses (such as western or elisa assays) that are dependent upon the presence of a specific region of a protein, which could be degraded. Based on these measurements, three separate experiments demonstrated that the adsorption and integrity of HGF to HA was maximal after a 48 h incubation (in solution) and was maintained for at least 9 days after coating as demonstrated by Coomassie and silver staining. In addition to adsorption kinetics, our findings are consistent with other studies that suggest that HGF can

promote bone formation. HGF treatment and surface coating has been demonstrated to stimulate osteoblast proliferation [24,40] and thereby increase the number of bone forming cells. Here, we demonstrate that HGF surface coating can also enhance osteoblast differentiation, as marked by increased runx2, alkaline phosphatase and osteocalcin expression. Similarly, D’lippolito et al. [26] demonstrated that when HGF was coupled with vitamin D treatment, alkaline phosphatase expression was increased in vertebral bone marrow cells. However, they did not see an effect with HGF alone. Possible reasons for this discrepancy may stem from differences in stages of osteoblast development (pluripotent versus osteoblastic) as well as differences in serum factors that are present during the HGF treatment. Taken together, our results demonstrate that HGF readily adsorbs to HA surfaces, maintains integrity (does not degrade) over time, and enhances osteoblast differentiation. These characteristics make HGF a potential growth factor coating for bone implant/ scaffold surfaces. Future studies determining mechanisms of HGF-induced osteoblast differentiation and directly testing the influence of HGF coating on bone implant integration will contribute to our understanding of HGF-osteoblast interactions and the efficacy of HGF as a scaffold/implant coating in vivo.

Acknowledgements We thank Jianwei Xie, Sergiu Botolin and Ian Smith for their insightful discussions. This work was supported by grants from the National Science Foundation, NASA (NAG8-1575), NIH (DK061184) and the Michigan State University Foundation.

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