Elsevier

Biomaterials

Volume 34, Issue 13, April 2013, Pages 3270-3278
Biomaterials

Diverse functions of secreted frizzled-related proteins in the osteoblastogenesis of human multipotent mesenchymal stromal cells

https://doi.org/10.1016/j.biomaterials.2013.01.066Get rights and content

Abstract

Osteoinductive pretreatment of human mesenchymal stromal cells (hMSCs) has been widely accepted in bone tissue engineering before the use of cell transplantation; however, the mechanisms by which osteoinductive medium (OIM) enhances osteoblastic differentiation are not well understood. Using periodontal ligament-derived hMSCs, we identified key signalling molecules for osteoblastogenesis. Alkaline phosphatase activity induced by OIM, which contains ascorbic acid, β-glycerophosphate, and dexamethasone, was decreased by XAV939, which is an inhibitor of canonical WNT signalling, in a dose-dependent manner. A quantitative RT-PCR array demonstrated the upregulation of secreted frizzled-related protein (SFRP) 3 and the downregulation of SFRP4 during osteoinduction. Functional studies showed that SFRP3 promoted and SFRP4 suppressed the osteoblastic differentiation of hMSCs. In addition, SFRP3 inhibited non-canonical WNT signalling by binding WNT5A, which is a representative non-canonical WNT protein. These results indicate the involvement of the WNT signalling pathway during the osteoblastic differentiation of hMSCs. SFRPs oppositely control osteoblastogenesis through canonical and non-canonical pathways and may be useful for directing the lineage of hMSCs in cytotherapeutic use.

Introduction

Bone possesses the ability to heal itself by bone remodelling, which is a continuous process of bone resorption and bone formation. However, spontaneous healing is limited to relatively small defects, and systemic conditions affect bone healing [1]. Spontaneous healing does not occur for large bone defects or defects caused by systemic bone disorders, such as long-term steroid administration, menopause, kidney disease, and bisphosphonate-related osteonecrosis of the jaw [2], [3]. Because of the clinical requirement for bone repair, effective approaches to regenerate bone are needed and have been developed.

Recently, regenerative medicine using cells, growth factors, and scaffolds has been actively investigated as a means of tissue engineering [4]. For bone tissue engineering, it is important to mimic the native bone environment, which can be achieved using cell delivery substrates [5]. Generally, a combination of cells and scaffolds has been used [6]. Previous studies have shown that osteoinductive medium (OIM) containing ascorbic acid, β-glycerophosphate, and dexamethasone induces the osteoblastic differentiation of multipotent mesenchymal stromal cells (MSCs) in vitro, and culturing MSCs with OIM prior to cell transplantation enhances bone regeneration in vivo [7], [8]. Thus, OIM is widely used for the treatment of MSCs prior to transplantation [9]. However, the mechanisms by which OIM enhances osteoblastic differentiation are not well understood.

Bioactive molecules that promote bone formation, such as bone morphogenetic proteins (BMPs), have proven useful for bone regeneration. Some BMPs are able to induce ectopic bone formation [10], and BMP2 enhances the osteoblastic differentiation of well-established murine cell lines, such as MC3T3-E1 and C2C12 [11], [12]. In contrast, the effects of BMPs on human MSCs (hMSCs) in vitro are controversial. Although several studies have indicated that BMPs induce osteoblastic differentiation in vitro [12], [13], [14], [15], [16], other studies have shown that osteogenesis is not enhanced by BMPs [17], [18]. In this study, we hypothesised that other signalling pathways may be involved in the osteoblastic differentiation of hMSCs. We focused on WNT signalling because the osteoblastic differentiation of hMSCs is suppressed by a canonical WNT signalling inhibitor.

The WNT family consists of 19 proteins that regulate various critical functions, especially in embryogenesis [19], [20]. The WNT pathway functions through canonical and non-canonical pathways by binding to receptors called frizzled (FZD) [21]. Several classes of extracellular antagonists of WNT proteins, including cerberus, Wnt inhibitory factor 1 (WIF1), the secreted frizzled-related protein (SFRP) family, and the Dickkopf (DKK) family, have been identified. In addition, it has been suggested that the WNT signalling pathway is involved in cell differentiation, tissue homeostasis, oncogenesis, and bone formation [22]. Mice with a functional mutation of low-density lipoprotein receptor-related protein 5 (LRP5), a WNT co-receptor that acts with FZD in mediating the canonical pathway, show osteoporosis [23], and the overexpression of LRP5 leads to high bone mass syndrome [24]. The canonical pathway has been reported to activate the differentiation of hMSCs into osteoblasts [25]. In contrast, other studies have reported that the canonical pathway inhibits the osteoblastic differentiation of hMSCs [26], [27]. The present study investigated the involvement of WNT signalling in the osteoblastic differentiation of hMSCs.

Section snippets

Materials and methods

All experimental protocols were approved by the ethics committee of Tokyo Women's Medical University, Tokyo, Japan, and written informed consent was obtained from all subjects.

Characterisation of hMSCs

The hMSCs used in this study were examined to determine if they corresponded to the minimal criteria of MSCs stated by Dominici et al. [30]. Their characteristics were confirmed by flow cytometry analysis. The hMSCs expressed cell surface antigens of MSC markers (CD29-, CD44-, CD90-, and CD105-positive and CD14-, CD34-, and CD45-negative) in the same manner as hGFs, hADSCs, and hBMMSCs (Fig. 1). These cells were positive in the colony-forming assay (58.2 ± 7.8%) (Fig. 2A). To examine the

Discussion

Many studies have indicated that the pretreatment of MSCs with OIM prior to cell transplantation enhances bone regeneration [7]. However, the mechanisms by which OIM enhances osteoblastic differentiation remain to be understood. Because it is known that several signalling pathways, including WNT signalling and BMP signalling, have important roles in the bone formation process [32], we hypothesised that WNT signalling is involved in the osteoblastic differentiation of hMSCs. After confirmation

Conclusions

This study showed that SFRP3 and SFRP4 play important roles in the osteoblastic differentiation of hMSCs induced by OIM through canonical and non-canonical WNT signalling pathways. As SFRP3 and SFRP4 have the potential to regulate the osteoblastic differentiation of hMSCs, the modulation of SFRPs may be useful as a new approach to bone regeneration.

Acknowledgements

This study was partially supported by the Creation of Innovation Centres for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation Systems “Cell Sheet Tissue Engineering Centre (CSTEC)” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, the Global Centre of Excellence (GCOE) Program, the International Research Centre for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, and a Grant-in-Aid for

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