Immobilizing osteogenic growth peptide with and without fibronectin on a titanium surface: effects of loading methods on mesenchymal stem cell differentiation

Abstract

In this study, to improve the osseointegration of implants, osteogenic growth peptide (OGP) and fibronectin (FN) were loaded within mineral, which was formed on titanium, through adsorption and coprecipitation methods. The release profiles of OGP loaded by either adsorption or coprecipitation and the effects of the loading methods to immobilize OGP with and without FN on rat mesenchymal stem cell (rMSC) osteogenic differentiation were studied. The coprecipitation approach slightly reduced the initial burst release, while the adsorption approach provided a more sustained release. Dual loading of OGP and FN further improved cell attachments compared with either OGP or FN alone. Dually loaded OGP and FN also had a positive impact on rMSC proliferation and osteogenic differentiation. The difference in methods of loading OGP with and without FN also had some effects on osteogenic differentiation. Compared with coprecipitated OGP alone, adsorbed OGP enhanced later differentiation, such as osteocalcin secretion and matrix mineralization. Simultaneously adsorbed OGP and FN led to higher proliferation and higher osteogenic differentiation in both early and late stages compared with sequentially loaded OGP and FN. rMSC culture clearly indicated that simultaneously adsorbed OGP and FN could improve osseointegration, and this treatment represents a potential method for effective surface modification of dental and orthopedic implants.

Keywords: adsorption; coprecipitation; osseointegration.

Figure 1

Sketch map of the loading of bioactive molecules on alkali-treated Ti. Abbreviations: Alkali-treated Ti, alkali-treated titanium substrate; DPBS, Dulbecco’s phosphate-buffered saline.

Figure 2

Scanning electron microscope images of representative samples. Notes: Samples from each of the following groups were examined: alkali-treated titanium substrate (A), 24-hour mineralization (B); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP (C); 24-hour mineralization, OGP adsorption (D). Abbreviation: OGP, osteogenic growth peptide.

Figure 3

XRD patterns of representative samples. Notes: Samples from each of the following groups were examined: alkali-treated titanium substrate (Alkali-treated Ti); 24-hour mineralization (Control); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP (cO); 24-hour mineralization, OGP adsorption (aO). Abbreviations: XRD, X-ray diffraction; OGP, osteogenic growth peptide; A, apatite; O, octacalcium phosphate; T, titanium.

Figure 4

In vitro cumulative release of FITC-OGP in PBS over the 14-day time period. Notes: Samples were prepared via 24-hour mineralization, 24-hour coprecipitation of mineral and FITC-OGP (cO) and 24-hour mineralization, FITC-OGP adsorption (aO). The values represent the mean ± standard deviation (n=3). Abbreviations: FITC-OGP, fluorescein isothiocyanate-labeled OGP; OGP, osteogenic growth peptide; PBS, phosphate-buffered saline.

Figure 5

Total DNA quantified of rMSCs adhered to the representative samples in serum-free growth medium for 3 hours. Notes: The samples were prepared as follows: 24-hour mineralization (Control); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP (cO); 24-hour mineralization, OGP adsorption (aO); 24-hour mineralization, FN adsorption (aF); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP, FN adsorption (cO/aF); 24-hour mineralization, OGP and FN adsorption (aOF). The values represent the mean ± standard deviation (n=4 or 5). In the table, pairwise comparisons of each of the groups were performed using the Student–Newman–Keuls post hoc comparison test, and significance is P<0.05. Abbreviations: OGP, osteogenic growth peptide; FN, fibronectin; rMSCs, rat mesenchymal stem cells.

Figure 6

Confocal microscope images of rMSCs cultured on the representative samples in serum-free growth medium for 3 hours. Notes: The samples were prepared as follows: 24-hour mineralization (Control); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP (cO); 24-hour mineralization, OGP adsorption (aO); 24-hour mineralization, FN adsorption (aF); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP, FN adsorption (cO/aF); 24-hour mineralization, OGP and FN adsorption (aOF). DAPI stained the nuclei (blue), and rhodamine phalloidin stained the cytoskeleton (red). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; OGP, osteogenic growth peptide; FN, fibronectin; rMSCs, rat mesenchymal stem cells.

Figure 7

Cell proliferation of rMSCs cultured on the representative samples in growth medium for 1, 3, 5, and 7 days. Notes: The samples were prepared as follows: 24-hour mineralization (Control); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP (cO); 24-hour mineralization, OGP adsorption (aO); 24-hour mineralization, FN adsorption (aF); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP, FN adsorption (cO/aF); 24-hour mineralization, OGP and FN adsorption (aOF). The values represent the mean ± standard deviation (n=4 or 5). In the table, pairwise comparisons of each of the groups were performed using the Student–Newman–Keuls post hoc comparison test, and significance is P<0.05. Abbreviations: CCK-8, Cell Counting Kit-8; OGP, osteogenic growth peptide; FN, fibronectin; rMSCs, rat mesenchymal stem cells.

Figure 8

Normalized ALP activities with respect to total protein of rMSCs cultured on the representative samples for 3, 7, and 14 days. Notes: rMSCs were cultured on samples in mineralizing medium as follows: 24-hour mineralization (Control); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP (cO); 24-hour mineralization, OGP adsorption (aO); 24-hour mineralization, FN adsorption (aF); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP, FN adsorption (cO/aF); 24-hour mineralization, OGP and FN adsorption (aOF); or rMSCs were cultured on 24-hour mineralization samples in osteogenic differentiation medium (Positive control). The values represent the mean ± standard deviation (n=4 or 5). In the table, pairwise comparisons of each of the groups were performed using the Student–Newman–Keuls post hoc comparison test, and significance is P<0.05. Abbreviations: ALP, alkaline phosphatase; OGP, osteogenic growth peptide; FN, fibronectin; rMSCs, rat mesenchymal stem cells.

Figure 9

OC production of rMSCs cultured on the representative samples for 7, 14, and 21 days. Notes: rMSCs were cultured on samples in mineralizing medium as follows: 24-hour mineralization (Control); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP (cO); 24-hour mineralization, OGP adsorption (aO); 24-hour mineralization, FN adsorption (aF); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP, FN adsorption (cO/aF); 24-hour mineralization, OGP and FN adsorption (aOF); or rMSCs were cultured on 24-hour mineralization samples in osteogenic differentiation medium (Positive control). The values represent the mean ± standard deviation (n=4 or 5). In the table, pairwise comparisons of each of the groups were performed using the Student–Newman–Keuls post hoc comparison test, and significance is P<0.05. Abbreviations: OC, osteocalcin; OGP, osteogenic growth peptide; FN, fibronectin; rMSCs, rat mesenchymal stem cells.

Figure 10

The calcium deposition content of rMSCs cultured on the representative samples for 28 days. Notes: rMSCs were cultured on samples in mineralizing medium as follows: 24-hour mineralization (Control); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP (cO); 24-hour mineralization, OGP adsorption (aO); 24-hour mineralization, FN adsorption (aF); 24-hour mineralization, 24-hour coprecipitation of mineral and OGP, FN adsorption (cO/aF); 24-hour mineralization, OGP and FN adsorption (aOF); or rMSCs were cultured on 24-hour mineralization samples in osteogenic differentiation medium (Positive control). The values represent the mean ± standard deviation (n=4 or 5). In the table, pairwise comparisons of each of the groups were performed using the Student–Newman–Keuls post hoc comparison test, and significance is P<0.05. Abbreviations: OGP, osteogenic growth peptide; FN, fibronectin; rMSCs, rat mesenchymal stem cells.