Temporal changes in matrix protein synthesis and mRNA expression during mineralized tissue formation by adult rat bone marrow cells in culture

Abstract

To characterize the bone-like tissue produced by rat bone marrow cells (RBMC) from young adult femurs, the synthesis of bone proteins and the expression of their mRNA were studied in vitro. RBMC plated at a density of 5 x 10(3) cells/cm2 and grown in the presence of 10(-8) M dexamethasone (Dex) and 10 mM beta-glycerophosphate (beta-GP) produced mineralized bone nodules, which were first evident at day 3 and increased markedly to day 13. However, in the absence of dexamethasone, few mineralized nodules were observed. The formation of mineralized nodules was reflected by the uptake of 45Ca, which also increased markedly to day 13. Analysis of bone protein expression by Northern and slot-blot hybridizations revealed an increase in mRNA levels of collagen type I (Col I), osteonectin/SPARC (ON), alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OC) during the formation of mineralized nodules. Whereas the Col I, ON, ALP, and OPN mRNAs were expressed before the formation of mineralized nodules was evident and were also expressed at various levels in the absence of Dex, the expression of BSP and OC mRNA was induced in the bone-forming cultures. The expression of BSP mRNA was correlated temporally with bone tissue formation, reaching maximal levels on day 16. In contrast, OC mRNA was expressed later and, following induction, increased over the 28 day culture period. Production of matrix proteins during the rapid formation of the bone tissue appeared to reflect the levels of the respective mRNAs. However, whereas some of the collagen and almost all of the SPARC were secreted into the culture medium, virtually all of the OPN and most of the BSP were extracted from the mineralized tissue matrix with EDTA. Some OPN and BSP were present in the medium, especially early in the culture, and a significant amount of BSP was also found associated with the collagenous tissue matrix. These studies point to the importance of Col I, ALP, OPN, and BSP, but not ON or OC, in the initial formation of bone tissue.

Differentiation of mesenchymal stem cells in heparin-containing hydrogels via coculture with osteoblasts

“…In order to unmask the signaling that can occur between MSCs and osteoblasts, dexamethasone was not used in our coculture system, but increases in ALP activity and mineralization were still observed in the hydrogels. The coculture medium did include β-glycerolphosphate, a source of phosphate, which has been shown to be necessary to initiate mineralization (Bellows et al 1992) yet does not greatly increase the expression of bone proteins such as bone sialoprotein and osteocalcin in a manner like dexamethosone (Yao et al 1994). Mineralization was not observed on the surface of 0% wt.…”

Osteogenic Potential and Histological Characteristics of Mesenchymal Stem Cell Sheet/Hydroxyapatite Constructs

“…We previously reported that a construct of porous biomaterial and cultured MSCs showed osteogenic potential [3,8,9]. The potential of the constructs was demonstrated by biochemical analyses of factors such as osteocalcin (OC) content and alkaline phosphatase (ALP) activity, as well as by the levels of gene expression of various bone matrix proteins [10].…”

Osteoanagenesis after transplantation of bone marrow-derived mesenchymal stem cells using polyvinylidene chloride film as a scaffold

“…The results revealed that cells with a short differentiation period showed better osteogenic potential than cells with a long differentiation period and produced good bone regeneration in vivo. It has been reported that the differentiation rate is influenced by the length of the differentiation period and that the properties of MSCs are lost as the differentiation progresses 12,14,33) . In the present study, the osteogenic potential differed between D-MSCs and MSCs.…”

“…The progenitor cells are referred to as mesenchymal stem cells (MSCs) and have the capability of differentiating into mesenchymal phenotypes, including osteogenic and chondrogenic cell types 8,9) . Bone marrow-derived MSCs are an attractive cell source for bone regeneration because of their ability for self-renewal with a high proliferative capacity and osteogenic differentiation potential [10][11][12][13][14] . Importantly, their in vitro differentiation can be achieved by culturing the cells on various materials including hydroxyapatite, β-TCP and polylactic acid (PLA) [15][16][17][18] .…”