Osteoclasts are thought to be solely responsible for the removal of bone matrix. However, we show here that osteocytes can also remove bone matrix by reversibly remodeling their perilacunar/canalicular matrix during the reproductive cycle. In contrast, no osteocytic remodeling was observed with experimental unloading despite similar degrees of bone loss. Gene array analysis of osteocytes from lactating animals revealed an elevation of genes known to be utilized by osteoclasts to remove bone including Tartrate Resistant Acid Phosphatase, TRAP, and cathepsin K that returned to virgin levels upon weaning. Infusion of Parathyroid Hormone Related Peptide, PTHrP, known to be elevated during lactation, induced TRAP activity and cathepsin K expression in osteocytes concurrent with osteocytic remodeling. Conversely, animals lacking the Parathyroid Hormone Type 1 receptor, PTHR1, in osteocytes failed to express TRAP or cathepsin K or to remodel their osteocyte perilacunar matrix during lactation. These studies show that osteocytes remove mineralized matrix through molecular mechanisms similar to those utilized by osteoclasts.

We cultured MSCs on an ECM made by bone marrow cells to attempt to reconstitute the MSC niche. This ECM promoted replication of mesenchymal progenitors and retention of their multipotentiality. We conclude that the marrow ECM facilitates expansion of mesenchymal progenitors and hypothesize that it plays an important role in the maintenance of MSC stemness.Introduction: Mesenchymal colony-forming cells of the bone marrow comprise mesenchymal stem cells (MSCs) and their transit amplifying progeny, which we term mesenchymal colony-forming units (MCFUs). These progenitors undergo self-renewal and can differentiate into many different cell types including osteoblasts. However, they lose their unique properties when cultured on tissue culture plastic. This indicates that a critical feature of the marrow microenvironment that facilitates retention of stem cell properties is missing in such culture systems. In other tissues, the extracellular matrix (ECM) forms part of the specialized niche that controls stem cell behavior. Therefore, we examined whether a marrow cell-derived ECM promotes retention of the stem cell characteristics of MCFUs in vitro. Materials and Methods: A cell-free ECM was prepared from cultured murine marrow adherent cells. The replication and multipotentiality of murine MCFUs maintained on this marrow cell-derived ECM were examined in vitro and in vivo and compared with the behavior of MCFUs maintained on plastic. Results: The marrow cell-derived ECM was made up of collagen types I, III, and V, syndecan-1, perlecan, fibronectin, laminin, biglycan, and decorin, similar to the composition of the marrow ECM. This ECM preparation promoted MCFU replication, restrained their "spontaneous" differentiation toward the osteoblast lineage, and preserved their ability to differentiate into osteoblasts or adipocytes. Moreover, transplantation of MCFUs expanded on the marrow cell-derived ECM into immunocompromised mice generated five times more bone and eight times more hematopoietic marrow compared with MCFUs expanded on plastic. Conclusions:The marrow ECM facilitates expansion of MCFUs in vitro while preserving their stem cell properties. We hypothesize that the ECM made by bone marrow cells plays an important role in the maintenance of MSC function.

Odontoblasts in dentin and osteocytes in bone contain dendritic processes. To test if their dendrites share a common feature, we compared their cellular morphology as visualized using scanning electron microscopy. Analysis of our data showed that both cells share an identical dendritic canalicular system and express extensive processes forming a complex network within the mineralized matrix. Because dentin matrix protein 1 (DMP1), an extracellular matrix protein, is highly expressed in both types of cells, we next tested, using a transgenic approach, whether a 9.6-kb Dmp1 promoter-4-kb 1st intron would be able to target Cre cDNA in these cells for expression/deletion of other genes in odontoblasts and osteocytes. We determined the specificity and efficiency of Cre activity by crossing Dmp1-Cre mice with ROSA26 reporter mice. Results showed that odontoblasts and osteocytes were specifically targeted, suggesting that this animal model will be useful for the preferential study of gene functions in both types of cells.

Osteocytes are the most abundant cells in bone yet are the most challenging to study as they are embedded in a mineralized matrix. We generated a clonal cell line called IDG-SW3 (for Immortomouse/Dmp1-GFP-SW3) from long bone chips from mice carrying a Dmp1 promoter driving GFP crossed with the Immortomouse, which expresses a thermolabile SV40 large T-antigen regulated by IFN-γ. Cells from these mice can be expanded at 33°C in the presence of IFN-γ and then allowed to resume their original phenotype at 37°C in the absence of IFN-γ. IDG-SW3 cells are Dmp1-GFP-negative and T-antigen-positive under immortalizing conditions but Dmp1-GFP-positive and T-antigen-negative under osteogenic conditions. Like osteoblasts, they express alkaline phosphatase and produce and mineralize a type I collagen matrix containing calcospherulites. Like early osteocytes, they express E11/gp38, Dmp1, MEPE, and Phex. Like late osteocytes, they develop a dendritic morphology and express SOST/sclerostin and FGF23, regulated by PTH and 1,25-dihydroxyvitamin-D3. When cultured on 3D matrices, they express Dmp1-GFP and sclerostin. When the 3D cultures are implanted in calvarial defects in vivo, they accelerate bone healing. This cell line should prove useful for studying osteoblast-to-osteocyte transition, mechanisms for biomineralization, osteocyte function, and regulation of SOST/sclerostin and FGF23.