Expression of cell growth and bone specific genes at single cell resolution during development of bone tissue‐like organization in primary osteoblast cultures

Abstract

Primary cultures of calvarial derived normal diploid osteoblasts undergo a developmental expression of genes reflecting growth, extracellular matrix maturation, and mineralization during development of multilayered nodules having a bone tissue-like organization. Scanning electron microscopy of the developing cultures indicates the transition from the uniform distribution of cuboidal osteoblasts to multilayered nodules of smaller cells with a pronounced orientation of perinodular cells towards the apex of the nodule. Ultrastructural analysis of the nodule by transmission electron microscopy indicates that the deposition of mineral is confined to the extracellular matrix where cells appear more osteocytic. The cell body contains rough endoplasmic reticulum and golgi, while these intracellular organelles are not present in the developing cellular processes. To understand the regulation of temporally expressed genes requires an understanding of which genes are selectively expressed on a single cell basis as the bone tissue-like organization develops. In situ hybridization analysis using 35S labelled histone gene probes, together with 3H-thymidine labelling and autoradiography, indicate that greater than 98% of the pre-confluent osteoblasts are proliferating. By two weeks, both the foci of multilayered cells and internodular cell regions have down-regulated cell growth associated genes. Post-proliferatively, but not earlier, initial expression of both osteocalcin and osteopontin are restricted to the multilayered nodules where all cells exhibit expression. While total mRNA levels for osteopontin and osteocalcin are coordinately upregulated with an increase in mineral deposition, in situ hybridization has revealed that expression of osteocalcin and osteopontin occurs predominantly in cells associated with the developing nodules. In contrast, proliferating rat osteosarcoma cells (ROS 17/2.8) concomitantly express histone H4, along with osteopontin and osteocalcin. These in situ analyses of gene expression during osteoblast growth and differentiation at the single cell level establish that a population of proliferating calvarial-derived cells subsequently expresses osteopontin and osteocalcin in cells developing into multilayered nodules with a tissue-like organization.

Sandblasted-acid-etched titanium surface influences in vitro the biologicalbehavior of SaOS-2 human osteoblast-like cells

“…Osseointegration of titanium dental implants is a complex phenomenon depending on several factors and characterized by a long sequence of events, such as the homing of multipotent mesenchymal cells, a cell proliferation phase coupled with the expression of some ECM proteins, the induction of genes related to maturation and organization of bone ECM, and finally a matrix mineralization phase 31,[48][49][50][51][52][53][54][55][56][57] . Different treatments of titanium surface can modify the implant microstructural properties that in turn are able to affect bone formation processes 11,15,44,55,58,59) .…”

Identification of the GATA Factor TRPS1 as a Repressor of the Osteocalcin Promoter

“…blasts occurs at the onset of mineralization and increases with mineral deposition (52,53). To evaluate the role of TRPS1 in osteoblast mineralization, we infected bone marrow stromal cells with retrovirus expressing Trps1 or lentivirus expressing shRNA targeting Trps1 and allowed the cells to differentiate.…”

Engineering growing tissues

“…Rat-derived calvarial osteoblasts (RCO) were isolated from newborn (Ͻ3 days old) Lewis rat pups (pregnant Lewis rats were purchased from Harlan-Sprague-Dawley) by using a reported method (14). Two experimental groups were investigated: (i) primary RCO cells alone, and (ii) a 2:1 ratio of RCO cells to BAC cells, keeping the transplantation cell number constant in both conditions.…”