Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts☆
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.
Section snippets
MLO-Y4 and MC3T3-E1 cell cultures
MLO-Y4 osteocyte-like cell line was a kind gift from Professor Lynda Bonewald (School of Dentistry, University of Missouri, Kansas City, MO). MLO-Y4 cell cultures were maintained in α-modified minimal essential medium (α-MEM, GIBCO BRL, Paisley, Scotland) supplemented with 5% fetal bovine serum (FBS, GIBCO BRL), 5% iron-supplemented calf serum (CS, HyClone Laboratories Inc., Logan, UT, USA) and antibiotics (penicillin/streptomycin, GIBCO BRL). All cultures were performed on collagen type I
MLO-Y4 CM stimulates the differentiation of primary osteoblasts
When mouse bone marrow cells were cultured in the presence of MLO-Y4 CM, there was an increase in the ALP activity (Fig. 1). The increase was statistically significant with 20% MLO-Y4 CM. No such stimulus could be observed with 20% MC3T3-E1 CM. The result was confirmed by histochemical staining for ALP and image analysis. The proportional area of ALP positive cells increased in the presence of MLO-Y4 CM (Fig. 2). Another marker of osteoblast differentiation, osteocalcin, behaved similarly,
Discussion
Osteoblasts are the bone-forming cells that produce new bone matrix into the sites where bone has previously been resorbed. The balance between bone formation and bone resorption is tightly controlled by the complex and highly organized interactions between cells and extracellular matrix. There are situations where bone formation exceeds the rate of bone resorption, resulting in thicker and mechanically stronger bones, for example, in response to mechanical loading. These effects can occur
References (43)
- et al.
A three-dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy
Bone
(2001) - et al.
Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture
Bone
(1998) - et al.
The quantitative histochemistry of brain. Enzyme measurements
J. Biol. Chem.
(1954) An improved automated procedure for the determination of calcium in biological specimens
Anal. Biochem.
(1967)- et al.
Relationships between osteocyte density and bone formation rate in human cancellous bone
Bone
(2002) - et al.
Changes in osteoblast phenotype during differentiation of enzymatically isolated rat calvaria cells
Bone
(1998) - et al.
IGF-I does not affect the proliferation or early osteogenic differentiation of human marrow stromal cells
Bone
(2003) - et al.
FGF-2 increases colony formation, PTH receptor, and IGF-1 mRNA in mouse marrow stromal cells
Biochem. Biophys. Res. Commun.
(2002) - et al.
Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts–correlation with prostaglandin upregulation
Biochem. Biophys. Res. Commun.
(1995) - et al.
Mechanically regulated expression of a neural glutamate transporter in bone: a role for excitatory amino acids as osteotropic agents?
Bone
(1997)
Glutamate signalling in non-neuronal tissues
Trends Pharmacol. Sci.
Osteocyte differentiation in the tibia of newborn rabbit: an ultrastructural study of the formation of cytoplasmic processes
Acta Anat. (Basel)
The cellular basis of bone turnover and bone loss: a rebuttal of the osteocytic resorption—Bone flow theory
Clin. Orthop.
How osteoblasts become osteocytes: a decreasing matrix forming process
J. Biol. Buccale
Morphological evidence of gap junctions between bone cells
Calcif. Tissue Int.
Establishment of an osteocyte-like cell line MLO-Y4
J. Bone Miner. Res.
Mechanical stimulation of gap junctions in bone osteocytes is mediated by prostaglandin E2
Cell Commun. Adhes.
Osteocytes inhibit osteoclastic bone resorption through transforming growth factor-beta: enhancement by estrogen
J. Cell. Biochem.
MLO-Y4 osteocyte-like cells support osteoclast formation and activation
J. Bone Miner. Res.
Functional gap junctions between osteocytic and osteoblastic cells
J. Bone Miner. Res.
Immunocytochemical demonstration of extracellular matrix proteins in isolated osteocytes
Histochem. Cell Biol.
Cited by (151)
Sweroside promotes osteoblastic differentiation and mineralization via interaction of membrane estrogen receptor-α and GPR30 mediated p38 signalling pathway on MC3T3-E1 cells
2020, PhytomedicineCitation Excerpt :As shown in Fig. 2B, sweroside significantly stimulated the ALP activity of MC3T3-E1 cells at all tested concentrations, which was similar to the effects of 17β-estradiol. Bone mineralized nodule formation is one of the most important phenomenon at the end stage of osteoblastic differentiation and represents the maturation of osteoblasts (Heino et al., 2004). To investigate whether sweroside enhanced osteoblastic mineralization of MC3T3-E1 cells, the mineralized nodules were stained by 1% Alizarin red S.
Osteocytes
2020, Marcus and Feldman’s OsteoporosisThe osteocyte
2019, Principles of Bone BiologyERK-estrogen receptor α signaling plays a role in the process of bone marrow mesenchymal stem cell-derived exosomes protecting against ovariectomy-induced bone loss
2023, Journal of Orthopaedic Surgery and ResearchMicrofluidic Co-culture Platforms for Studying Osteocyte Regulation of Other Cell Types under Dynamic Mechanical Stimulation
2022, Current Osteoporosis Reports
- ☆
Presented in part as an abstract at the International Bone and Hormone Meeting, Hamilton Island, Australia, 4–7 November, 2000 and at 30th European Symposium on Calcified Tissues, Rome, Italy, 8–12 May 2003.