Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts

Abstract

Osteocytes are the most abundant cells in bone and there is increasing evidence that they control bone remodeling via direct cell-to-cell contacts and by soluble factors. In the present study, we have used the MLO-Y4 cell line to study the effect of osteocytes on the proliferation, differentiation and bone-forming capacity of bone marrow mesenchymal stem cells (MSC). Conditioned media (CM) from osteocytic MLO-Y4 and osteoblastic MC3T3-E1 cell lines were collected and added on mouse bone marrow cultures, in which MSC were induced to osteoblasts. There was a significant increase in alkaline phosphatase activity and osteocalcin expression in the presence of MLO-Y4 CM. No such stimulus could be observed with MC3T3-E1 CM. There was almost 4-fold increase in bone formation and up to 2-fold increase in the proliferation of MSC with MLO-Y4 CM. The highly proliferating bone marrow cells were negative for ALP and OCN, suggesting that they could represent early osteoblast precursors. MLO-Y4 CM did not enhance the viability of mature osteoblasts nor protected them of apoptosis. This is the first study to describe soluble signals between osteocytes and osteoblasts and there most likely are several still unidentified or unknown factors in osteocyte CM. We conclude that osteocytes have an active stimulatory role in controlling bone formation.

Introduction

Bone turnover is a complicated process, in which bone formation and bone resorption are tightly coupled both in time and in space. These processes are essential for the development, maturation, maintenance, and repair of bones. The proliferation and the differentiation of osteoblasts are important events during bone turnover and are controlled both by local growth factors as well as by systemic hormones. In their final phase of differentiation, osteoblasts become embedded deep within the mineralized bone matrix during bone formation and become osteocytes [1]. Osteocytes are the most abundant cells in bone, and in normal human bone, there are approximately 10 times more osteocytes than osteoblasts [2]. When osteoblasts turn into osteocytes, the cellular volume and protein synthesis level decrease [3] but long cell processes with gap junctions in the tip are being formed [4]. Osteocytes have a unique location in bone compared to osteoblasts and osteoclasts. They are regularly spaced throughout the matrix and still connected with each other via long processes. Osteocytes remain in contact also with bone surface and other cells [5]. This way they ensure the access of oxygen and nutrients and also the possibility of cell-to-cell signaling.

Due to the hard matrix around osteocytes, they have been difficult to study. Kato et al. [6] developed a unique osteocyte-like cell line, MLO-Y4, that was created using SV40 large T-antigen oncogene with osteocalcin promoter. The cell line was isolated from the long bones of transgenic mice and it appears to have the properties of primary osteocytes. MLO-Y4 cells have extensive dendritic processes, high osteocalcin production and low expression of collagen type I and alkaline phosphatase (ALP). These cells have functional gap junctions and their connexin-43 (Cx43) expression is regulated by fluid flow [7].

Since osteocytes can have a contact with the cells on the bone surface, either directly or by soluble factors via canaliculi, it is likely that they play a role in controlling bone turnover. Only recently, some reports about interactions between osteocytes and osteoclasts have been published. These studies suggest that osteocytes can control bone resorption both by soluble factors [8] and by direct cell-to-cell contact [9]. However, very little is known about the interactions between osteocytes and osteoblasts, even though functional gap junctions between osteoblasts and osteocytic MLO-Y4 cells have been demonstrated in vitro [10]. Aarden et al. [11] have studied the capacity of osteocytes to change their surrounding extracellular matrix by production of matrix proteins. This property may be important in the regulation of the calcification of the bone matrix immediately surrounding the cells, but possibly also by controlling the action of osteoblasts located further away. In the present study, we have used the MLO-Y4 cell line to study the effect of osteocytes on the proliferation, differentiation, and bone-forming capacity of bone marrow mesenchymal stem cells (MSC). Mouse bone marrow culture model was used to study the differentiation of osteogenic cells [12]. In this model, the process of bone formation is mimicked by the maturation of osteoprogenitors into active osteoblasts that finally calcify the extracellular matrix. Bone marrow MSC proliferation, osteoblast differentiation, and mineral deposition were significantly increased when osteocyte conditioned medium was added on the osteoblast cultures.