Elsevier

Biomaterials

Volume 33, Issue 11, April 2012, Pages 3127-3142
Biomaterials

Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers

https://doi.org/10.1016/j.biomaterials.2012.01.015Get rights and content

Abstract

Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for clinical repair of skeletal defects. However, the exact mechanism of action and the stem cell–host-material interactions are still poorly understood. We studied if pre-conditioning of human periosteum-derived cells (hPDCs) in vitro could enhance, in combination with a CaP-based biomaterial carrier, ectopic bone formation in vivo. By culturing hPDCs in a biomimetic calcium (Ca2+) and phosphate (Pi) enriched culture conditions, we observed an enhanced cell proliferation, decreased expression of mesenchymal stem cell (MSC) markers and upregulation of osteogenic genes including osterix, Runx2, osteocalcin, osteopontin, and BMP-2. However, the in vitro pre-conditioning protocols were non-predictive for in vivo ectopic bone formation. Surprisingly, culturing in the presence of Ca2+ and Pi supplements resulted in partial or complete abrogation of in vivo ectopic bone formation. Through histological, immunohistochemical and microfocus X-ray computed tomography (μCT) analysis of the explants, we found that in situ proliferation, collagen matrix deposition and the mediation of osteoclastic activity by hPDCs are associated to their ectopic bone forming capacity. These data were validated by the multivariate analysis and partial least square regression modelling confirming the non-predictability of in vitro parameters on in vivo ectopic bone formation. Our series of experiments provided further insights on the stem cell–host-material interactions that govern in vivo ectopic bone induction driven by hPDCs on CaP-based biomaterials.

Introduction

Bone formation is mediated via an endochondral or intramembranous pathway. These events are body site-specific and pre-determined by the presence of a cartilaginous template or involving a direct mineralisation process of the collagen matrix [1]. In the case of ectopic bone induction through a stem cell-based approach using a calcium phosphate (CaP)-based biomaterial carrier, the process is often intramembranous [2]. This may resemble a bone remodelling process whereby the CaP granules are resorbed by the infiltrated osteoclasts and degraded chemically upon implantation [3]. In turn, the released calcium (Ca2+) and phosphate (Pi) ions are believed to stimulate osteogenic differentiation of the osteoprogenitors present in the vicinity [4] and these ions are converted to hydroxyapatite by the differentiated osteoblastic cells to form new bone matrix [5]. In this context, blood vessel in-growth or angiogenesis is essential to promote cell survival within the hypoxic mineralising tissue [6], and to serve as a gateway for delivering important cell types (such as osteoclast progenitors) and endogenous growth factors that are necessary for bone formation [7]. Therefore, the absence of any of these events within the implant and in the surrounding host environment will interfere with the bone induction process.

Hence, in designing an effective bone induction approach using CaP-based biomaterials, fine-tuning the physicochemical properties of a biomaterial to closely match the physiological events of bone formation is essential for successful osteogenesis. This is highlighted by the recent success on producing osteoinductive ceramic as autograft substitute for the healing of skeletal defects [8]. In cell-based approaches, it is believed that bringing the desired progenitor cells into a specific stage of differentiation prior to implantation is another strategy for predictive and robust bone formation [9]. These cells essentially need to have a high proliferative capacity in situ (to generate critical cell mass) and the capability to differentiate into bone matrix-producing cells, while establishing beneficial communications with the surrounding host environment to support bone growth and remodelling. However, the exact stage of differentiation required (through in vitro manipulation of culture parameters) for successful in vivo bone formation is currently not well defined. In this study, we incorporate Ca2+ and Pi ions as in vitro osteogenic differentiation agents to pre-condition multipotent mesenchymal stem cell (MSC)-like osteoprogenitors, i.e. human periosteum-derived cells (hPDCs), in a Ca2+ and/or Pi enriched culture environment that mimics the conditions observed at sites of bone resorption. This rationale is supported by our recent study showing that in vitro exogenous Ca2+ and Pi supplementation promote osteogenic differentiation of hPDCs in vitro [4] in conjunction with the osteoinductive effect of CaP reported by other studies [10], [11].

To identify the essential parameters for both the hPDCs and carriers, and to gain further understanding of the stem cell–host-material interactions that govern ectopic bone induction using a CaP-based biomaterial, we studied the in vivo ectopic bone forming capacity of distinct pre-conditioned hPDCs by seeding on a clinically used CaP biomaterial carrier, i.e. NuOSS (NuOSS-Collagen™, ACE Dental Supply Co.) and implanted subcutaneously into the nude mouse model [12] (Table 1). The amount of new bone formed for each treatment was evaluated and several biological markers [including cell proliferation, collagen matrix production, osteoclastic activity, blood vessel in-growth and the changes in physicochemical parameters of the CaP grains (e.g. grain size, grain surface area)] within the explants were assessed quantitatively. Non-CaP containing collagen sponges (deNuOSS, obtained by decalcifying NuOSS in EDTA) were used to study the critical role of CaP on ectopic bone formation by pre-differentiated hPDCs.

Section snippets

Chemical reagents

Analytical grade calcium chloride (CaCl2.6H2O, Applichem), disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O, Merck), sodium di-hydrogen phosphate dihydrate (NaH2PO4.2H2O, Merck), hepes solution (Sigma), dexamethasone (sigma), ascorbic acid 2-phosphate sesquimagnesium salt (AA-phos; sigma), glycerol-2-phosphate disodium salt hydrate (β-GP; sigma), dulbecco’s modified eagle medium (DMEM + GlutaMax™-1, Gibco), foetal bovine serum (FBS, Gibco) and bovine serum albumin (BSA, BD Biosciences) were

In vitro proliferation and osteogenic differentiation of hPDCs

Analysis of cellular metabolic activity showed that cells treated with any of the Ca2+ and/or Pi enriched media induced significantly higher cell growth than the standard culture condition (GM) at 21 days, where C8 resulted in the highest metabolic activity (1.4 fold higher), followed by P8C8, C8P8 and P8 (1.21–1.28 fold) and then GMC8 and GMP8 (1.07–1.14 fold) (Fig. 1a). This pro-mitogenic effect of C8 was comparable to the osteogenic medium (OM) treatment (1.47 fold higher than GM) albeit the

Discussion

In this study, we have provided data on the potential use of Ca2+ and Pi supplemented media to induce differentiation of hPDCs into the osteoblastic lineage in vitro. The possible mechanisms for the upregulation of the investigated genes by these ion treatments have been described previously [4], [24]. Surprisingly, the obtained in vitro osteogenic differentiation events through Ca2+ and Pi treatments were non-predictive for in vivo ectopic bone formation using CaP-based carriers, including

Conclusion

In this study, we have shown the potential use of Ca2+ and Pi supplemented media to pre-condition MSCs-like osteoprogenitors. Surprisingly, enhancement of in vitro differentiation resulted in a partial and complete loss of the ectopic in vivo bone forming capacity of hPDCs when seeded onto a CaP-based carrier. This has led to the identification of several in situ biological parameters that are of critical importance for hPDC driven in vivo bone formation. Our study has provided a more

Acknowledgements

We are grateful to Carla Geeroms for the excellent technical assistance on μCT scanning and image analysis for the quantification of bone formation. This work is funded by the K.U.Leuven IDO project 05/009 – QuEST (Y.C.C.), the Industrial Research Fund K.U.Leuven (E.D.), Stem Cell Institute of Leuven – K.U.Leuven (S.J.R.), the Agency for Innovation by Science and Technology in Flanders: IWT/OZM/090655 (G.K.), IWT/083128 (N.v.G.), the fund for Scientific Research Flanders: G.0500.08 & G.0982.11

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