Bioactivity of surface tethered Osteogenic Growth Peptide motifs

Abstract

Osteogenic Growth Peptide (OGP) motifs were tethered to SULFHYDRYL-BIND™ polystyrene plates tuning spatial presentation of the different functional domains. The effects of the different surface-tethered OGP motifs have been evaluated in mesenchymal stem cells (MSCs) osteogenesis. A culture medium without any osteogenic supplements was used in order to evaluate the OGP effect, avoiding any interference by additional factors. The motif YGFGG resulted to be the most interesting, strongly enhancing the expression of BMP2 that plays a fundamental role in bone regeneration. With this study we present new evidence of the role of OGP in osteogenesis and new insights into different OGP motifs able to act as best ligands for the design of innovative bioactive materials for osteogenesis promotion and regenerative medicine applications.

---

Introduction

Bone tissue regeneration is a well-orchestrated physiological process, in continuous remodeling throughout adult life. Bone marrow, anatomically and functionally related to bone, plays a fundamental role giving rise to cells that serve as precursors for the osteoblasts and osteoclasts required for osteogenesis and bone remodeling.^1–3 However, there are complex clinical conditions in which bone homeostasis and regeneration are compromised such as in skeletal reconstruction of large bone defects created by trauma, infection, and tumor resection or in osteoporosis.⁴ Currently, there is a plethora of different strategies to enhance the impaired bone regeneration ranging from autologous or heterologous bone graft to pharmaceutical treatment, from gene/cell therapy to tissue engineering.^4–6 Nevertheless none of these approaches is able to completely fix the bone regeneration deficit.

In the last few years, significant advances have been made in the field of biomaterials, mostly focused on making materials biologically active.^7–10 Employing the principles of biological recognition, the “gold standard” is to obtain a complex system combining physico-chemical biomimetic material features and smart biological cues.

Several growth factors are involved in bone homeostasis and are essential in the bone regeneration process. The biological activity of growth factors, in general, depends not only on their identity, but also on how they are accessible to the cells in space and over time. Under physiological conditions, the extracellular matrix plays a role in storing, displaying and releasing biomolecules. In fact, many growth factors bind to this substrate that serves as a sequestration site from which growth factor stores can be concentrated for enhanced local action or released for a heightened overall effect.¹¹

Recently, Osteogenic Growth Peptide (OGP) has attracted considerable clinical interest due to its relevant role in increasing bone formation and trabecular bone density.¹² In fact, OGP directly regulates the mesenchymal stem cells (MSCs) differentiation into osteoblasts and the matrix mineralization both in vitro and in vivo, however the exact mechanism of this process remains elusive.^13–15 OGP is a 14-mer peptide (ALKRQGRTLYGFGG) present in micromolar concentrations in mammalian serum,¹⁶ and its carboxy-terminal fragment [OGP(10–14)] has been reported to be the active portion of the full-length peptide.^13,17 Given these premises, OGP and fragments derived thereof may be interesting biocues to be linked to materials for bone tissue regeneration. Moreover, their effect in an immobilized and spatially oriented form has been scarcely investigated. To date, only a few groups have examined the effectiveness of OGP covalently immobilized into biomaterial scaffolds. In one of these studies, the full length and the fragment ¹⁰⁻¹⁴OGP were covalently linked to the scaffolds by their C-terminus,¹⁸ while another report considers the co-polymerization of the fragment ¹⁰⁻¹⁴OGP in the biomaterial, thus engaging both termini with the covalent linkage to the material.¹⁹ Here we wish to report a preliminary and systematic study of biological activity of immobilized OGP full length and peptide fragments ^1–9OGP (¹ALKRQGRT⁹L) and ¹⁰⁻¹⁴OGP (¹⁰YGFG¹⁴G) in relationship to their spatial exposition to the cells. The coating of surfaces with biocues is often based on physical adsorption processes and therefore easily achieved, but it is sensitive to the surrounding medium and also to biochemical degradation. Covalent anchoring on the other hand may offer several advantages over adsorbing strategies: covalent immobilization can be used to extend the half-life of a biomolecule, prevent its metabolism, and prevent migration from the implant site, maintaining its therapeutic concentration in loco.²⁰ Moreover, covalent linkage allows strict control over spatial orientation of the biomolecules, provided that the bonds between the material surface and the biocues occur chemoselectively. The immobilization of specifically oriented biomolecules is of critical relevance in bioactive material design and it strictly depends on ligand availability to cell receptors. In order to get better insights into different surface-tethered OGP peptidic sequences able to act as best ligands for osteogenesis promotion for the design of innovative bioactive materials for regenerative medicine applications we propose the study of the effect of OGP-mediated responses at the cell interface.

Results and discussion

Surface tethering of OGP motifs

Suitably functionalized full-length OGP (Pep 1 and Pep 2, Fig. 1), ^1–9OGP (Pep 3 and Pep 4, Fig. 1) and ¹⁰⁻¹⁴OGP (Pep 5 and Pep 6, Fig. 1) were chemoselectively tethered to polystyrene plates in order to alternatively expose to cell receptors their C-terminal or N-terminal domain. Toward this aim, a thiol group was inserted by means of a terminal cysteine residue at the desired end of each peptide. Hence, the control over spatial presentation of the tethered peptides was achieved by a Michael addition between the thiol group of the terminal cysteine introduced ad hoc on OGP peptides and sulfhydryl binding polystyrene plates (SULFHYDRYL-BIND™, Corning NY), exposing maleimide units on their surface (Fig. 2). The effectiveness of the coupling was checked by FTIR spectroscopy (ESI): the carbonyl band at 1707 cm⁻¹ typical of the conjugated system of the maleimide unit disappears after coupling, due to the loss of the resonating π system.

Fig. 1 Peptide sequences used for the present study.

Fig. 2 Immobilization strategy.

Unbound native full-length OGP (Pep 7, Fig. 1), ^1–9OGP (¹ALKRQGRT⁹L, Pep 8, Fig. 1), and ¹⁰⁻¹⁴OGP (¹⁰YGFG¹⁴G, Pep 9, Fig. 1) were used as control peptides added to culture media.

Proliferation and osteogenic behavior of tethered/non-tethered OGP motifs

As an early stage evaluation, a screening was performed for comparison of all the immobilized OGP sequences (Pep 1–6) and the three non-tethered OGP sequences (Pep 7–9). Assessment of MSCs proliferation was performed by quantification of metabolically active cells by the use of the MTT assay. No statistically significant differences were seen among groups at 7 days, compared to the cells only group used as the control. MSCs proliferation seemed not to be negatively affected by the presence of the immobilized or non-immobilized OGP sequences. An exception was seen for Pep 5 that showed a pronounced decrease in cell proliferation (Fig. 3a). At the same time, the Alkaline Phosphatase activity (AP) was quantified as a measure of osteogenic induction of MSCs, cultured with media lacking any osteogenic supplement. Results have been normalized to total number of cells present in the well according to results obtained from the MTT assay, and expressed as percentage of AP activity with respect to the cells only group (Fig. 3b). Although no statistical differences were seen, it was well-rendered that Pep 5 strongly affects MSCs, confirming that the tethered OGP carboxy-terminal fragment is still active. If on one side Pep 5 reduced cell proliferation, on the other side it enhanced MSCs differentiation towards an osteogenic phenotype. Instead, the non-tethered Pep 9, although corresponding to the active portion of the full-length peptide, seems not to provide any osteogenic stimulus to MSCs in culture. These results demonstrate the importance of the spatial orientation of the C-terminal portion of OGP to its bioavailability to cell-surface receptors and therefore to its biological action on osteogenesis.

Fig. 3 Analysis of the early effect of OGP peptide sequences on MSCs proliferation, as quantified by the MTT assay (a) and osteogenic activity, measured by Alkaline Phosphatase quantification (b).

Detailed studies with tethered Pep 1, Pep 5, and Pep 9

Tethered Pep 5 was shown to be the most active in the variation of cell proliferation and AP activity. In order to better characterize its biological behaviour, we conducted further studies in comparison with Pep 1 (tethered full-length OGP that exposes the same active motif YGFGG) and the non-tethered motif YGFGG (Pep 9).

Proliferation effect of tethered Pep 1, Pep 5 and Pep 9

Looking again at the cell proliferation (Fig. 4), now in more detail up to day 14, we observed that for the first 3 days MSCs proliferated without any differences among groups and similarly to the cells cultured without OGP sequences. Starting from day 7, MSCs cultured with Pep 5 began to slow down their proliferation, confirming previous data, and this trend was emphasized at day 14, where significant differences resulted by two-way ANOVA. In detail, Pep 5 strongly decreased cell proliferation with respect to the cells only and the Pep 9 groups (p ≤ 0.05 and p ≤ 0.001, respectively). Pep 9, which corresponds to the active non-tethered YGFGG motif, presents the highest values of cell proliferation at day 14, with p ≤ 0.05 with respect to Pep 1, the immobilized full-length peptide.

Fig. 4 Cell proliferation for Pep 1, Pep 5, Pep 9 and cells only group, quantified by the MTT assay. *p ≤ 0.05, ***p ≤ 0.001.

It is also noticeable that Pep 5 showed the lowest cell proliferation effect, even if compared to Pep 1. This effect might correlate with the stimulation of osteogenic differentiation. In order to verify this hypothesis, detailed cell morphology and gene expression studies were performed with tethered Pep 1 and 5, and free Pep 9.

Cell morphology studies with tethered Pep 1, Pep 5 and Pep 9

In order to analyse MSC morphology in the presence of OGP peptides, phalloidin was used as a specific marker for actin filaments, which allowed the analysis of MSCs attachment, morphology and spreading on the tissue culture coated surfaces. No differences were observed among cells seeded in the presence of immobilized Pep 1 and Pep 5 and non-tethered Pep 9 at day 3; MSCs morphology is comparable to the cells only group (Fig. 5A–D). These data were confirmed also by hematoxylin and eosin staining at day 7 (Fig. 5E–H). The anchoring of OGP sequences did not negatively influence the cell adhesion and subsequently the cell morphology that, as shown, resulted completely analogous to the cell only and the Pep 9 group. MSC morphology is predictive of osteogenic potential²¹ and it is important to underline that cells were grown without any osteogenic supplements in cell culture media, with the exception of OGP sequences, and for this reason no morphological changes, typical of the late step of the MSCs commitment, was detected.

Fig. 5 Morphological analysis of MSCs cultured in the presence of OGP peptides. (A–D) Immunofluorescence for phalloidin at 3 days (green: actin filaments, blue: cell nuclei marked by DAPI), (E–H) hematoxylin and eosin staining at 7 days. (A and E) Pep 1; (B and F) Pep 5; (C and G) Pep 9; (D and H) control. Scale bars: 50 μm.

Gene expression profile of osteogenic markers with tethered Pep 1, Pep 5 and Pep 9

In order to investigate in detail the effect of active motif YGFGG on MSCs osteogenesis, cells were incubated with tethered Pep 1, with tethered Pep 5, and non-tethered motif YGFGG (Pep 9) for 7 and 14 days. Cells were cultured without any additional osteogenic supplements, and the results were expressed as changes in the gene expression level of the relevant genes Runt-related transcription factor 2 (RUNX2), Bone Morphogenetic Protein 2 (BMP2) and osteonectin (SPARC), calculated by normalization to the reference GAPDH and by normalization to the cell only group, cultured under the same conditions (Fig. 6).

Fig. 6 Gene expression profile for RUNX2, BMP2, Col1A1 and SPARC, for MSCs cultured for 7 and 14 days in the presence of Pep 1, Pep 5 and Pep 9. Fold-change expression is presented as relative to control cells with no peptides and using normalization against a GAPDH reference. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Pep1 and Pep 5 showed a significant increase in the mRNA level of the early marker involved in commitment and differentiation of MSCs. In detail, RUNX2 expression seems to be up regulated by the presence of Pep 5 after 7 and 14 days of culture (2.6 and 1.7 fold-change with respect to the cells only group). Pep 1 showed a time dependent increase in RUNX2 expression, in fact only after 14 days of culture an increase of 2.7 fold in the mRNA level of RUNX2 has been observed compared to the cells only group. In contrast, no effect was found in regulation of RUNX2 expression by Pep 9 (Fig. 6).

BMP2 gene expression is strongly up regulated by the presence of Pep1 and Pep 5 but not by Pep 9. Pep 1 showed the same time dependent effect observed in RUNX2 expression with respect to the cells only group increasing the BMP2 expression of ∼42 and ∼110 fold-change at day 7 and 14 respectively. Similarly, Pep 5 increased BMP2 expression of about 49 and 60 fold-change at day 7 and 14. The increase of RUNX2 and BMP2 mRNA levels exerted by Pep 1 and Pep 5 was also found to be statistically different compared to the expression induced by Pep 9 at both time points (Fig. 6).

No difference was found among groups for SPARC expression after 7 days of culture among the groups. Instead, after 14 days of culture Pep 5 showed a small increase in mRNA expression of SPARC gene (∼1.6 fold change compared to the cells only group), theoretically the mRNA levels would be greater in later time points because of their expression is upstream regulated by RUNX2.

As we can see, both Pep 1 and Pep 5 presented significant increase in gene expression levels for the osteogenic markers RUNX2, SPARC and especially for BMP2. Pep 9 presents values identical to the control.

Conclusions

The motif YGFGG resulted to be the most interesting peptide of this study. In particular, when specifically exposing the C-terminus to cell receptors, as in the case of tethered Pep 1 and Pep 5, osteogenic markers are expressed. These results are extremely promising in particular for the BMP2 expression. In fact, we demonstrated that both tethered Pep 1 and Pep 5 strongly enhance the expression of BMP2 that plays a fundamental role in bone regeneration. Presently, the clinical treatments with BMP2, approved by U.S. Food and Drug Administration, consist of the incorporation of the full functional protein into bone implant and its release stimulates bone formation.²² However, the cost of BMP2 treatments is extremely high, hence the identification of short and low cost peptides that could modulate the expression of key protein in the bone regeneration process may have a strong impact on bone tissue therapies.

This study will be extremely useful in developing novel gene-switcher scaffolds for novel tissue engineering platforms and devices.

Acknowledgements

This work has been supported by Fondazione Cariplo, Grant no. 2010-0378, MIUR under project PRIN 2010L9SH3K, and 5x1000 2010 Rizzoli Orthopaedic Institute fundings and CC wishes to acknowledge Fundação para a Ciência e a Tecnologia for the fellowship SFRH/BPD/87071/2012.

Notes and references

1. C. Szpalski, M. Barbaro, F. Sagebin and S. M. Warren, Tissue Eng., Part B, 2012, 18, 258.
2. A. Teti, Curr. Osteoporos Rep., 2011, 9, 264.
3. N. Udagawa, N. Takahashi, T. Akatsu, H. Tanaka, T. Sasaki, T. Nishihara, T. Koga, T. J. Martin and T. Suda, Proc. Natl. Acad. Sci. U. S. A., 1990, 87, 7260.
4. R. Dimitriou, E. Jones, D. McGonagle and P. V. Giannoudis, BMC Med., 2011, 9, 66.
5. K. E. Benders, P. R. van Weeren, S. F. Badylak, D. B. Saris, W. J. Dhert and J. Malda, Trends Biotechnol., 2013, 31, 169.
6. S. Bose, M. Roy and A. Bandyopadhyay, Trends Biotechnol., 2012, 30, 546.
7. J. R. Jones, Acta Biomater., 2013, 9, 4457.
8. K. E. Tanner, J. R. Soc., Interface, 2010, 7(suppl. 5), S541.
9. M. Navarro, A. Michiardi, O. Castano and J. A. Planell, J. R. Soc., Interface, 2008, 5, 1137.
10. K. Rezwan, Q. Z. Chen, J. J. Blaker and A. R. Boccaccini, Biomaterials, 2006, 27, 3413–3431.
11. I. D. Dinbergs, L. Brown and E. R. Edelman, J. Biol. Chem., 1996, 271, 29822.
12. I. Bab and M. Chorev, Biopolymers, 2002, 66, 33.
13. Z. Chen, X. Wang, Y. Shao, D. Shi, T. Chen, D. Cui and X. Jiang, Mol. Cell. Biochem., 2011, 358, 221.
14. L. Vanella, D. H. Kim, D. Asprinio, S. J. Peterson, I. Barbagallo, A. Vanella, D. Goldstein, S. Ikehara, A. Kappas and N. G. Abraham, Bone, 2010, 46, 236.
15. Z. X. Chen, M. Chang, Y. L. Peng, L. Zhao, Y. R. Zhan, L. J. Wang and R. Wang, Regul. Pept., 2007, 142, 16.
16. Z. Greenberg, M. Chorev, A. Muhlrad, A. Shteyer, M. Namdar-Attar, N. Casap, A. Tartakovsky, M. Vidson and I. Bab, J. Clin. Endocrinol. Metab., 1995, 80, 2330.
17. L. Matti, B. Battolla, S. Moscato, R. Fazzi, S. Galimberti, N. Bernardini, A. Dolfi and M. Petrini, Med. Sci. Monit., 2008, 14, BR103.
18. N. M. Moore, N. J. Lin, N. D. Gallant and M. L. Becker, Biomaterials, 2010, 31, 1604.
19. K. Sloan Stakleff, F. Lin, L. A. Smith Callahan, M. B. Wade, A. Esterle, J. Miller, M. Graham and M. L. Becker, Acta Biomater., 2013, 9, 5132.
20. L. Cipolla, L. Russo, N. Shaikh and F. Nicotra, Materials biofunctionalization for tissue regeneration, CRC Press, 2013.
21. F. Matsuoka, I. Takeuchi, H. Agata, H. Kagami, H. Shiono, Y. Kiyota, H. Honda and R. Kato, PLoS One, 2013, 8, e55082.
22. J. Even, M. Eskander and J. Kang, J. Am. Acad. Orthop. Surg., 2012, 20, 547.

---

Footnotes

† Electronic supplementary information (ESI) available: Experimental details in biology and chemistry. See DOI: 10.1039/c4md00112e

‡ These authors contributed equally to this work.

§ Present address: Department of Nanomedicine, The Methodist Hospital Research Institute, 6670 Bertner Ave, Houston, TX 77030 and Pain Therapy Service Fondazione IRCCS Policlinico San Matteo via Golgi 19, 27100 Pavia, Italy.

¶ Present address: INEB-Instituto de Engenharia Biomédica, Microenvironments for NEWTherapies Group, Porto 4150-180, Portugal.

---