Extracellular matrix networks in bone remodeling

Bones are constantly remodeled throughout life to maintain robust structure and function. Dysfunctional remodeling can result in pathological conditions such as osteoporosis (bone loss) or osteosclerosis (bone gain). Bone contains 100s of extracellular matrix (ECM) proteins and the ECM of the various bone tissue compartments plays essential roles directing the remodeling of bone through the coupled activity of osteoclasts (which resorb bone) and osteoblasts (which produce new bone). One important role for the ECM is to serve as a scaffold upon which mineral is deposited. This scaffold is primarily type I collagen, but other ECM components are involved in binding of mineral components. In addition to providing a mineral scaffolding role, the ECM components provide structural flexibility for a tissue that would otherwise be overly rigid. Although primarily secreted by osteoblast-lineage cells, the ECM regulates cells of both the osteoblast-lineage (such as progenitors, mature osteoblasts, and osteocytes) and osteoclast-lineage (including precursors and mature osteoclasts), and it also influences the cross-talk that occurs between these two oppositional cells. ECM influences the differentiation process of mesenchymal stem cells to become osteoblasts by both direct cell-ECM interactions as well as by modulating growth factor activity. Similarly, the ECM can influence the development of osteoclasts from undifferentiated macrophage precursor cells, and influence osteoclast function through direct osteoclast cell binding to matrix components. This comprehensive review will focus on how networks of ECM proteins function to regulate osteoclast- and osteoblast-mediated bone remodeling. The clinical significance of these networks on normal bone and as they relate to pathologies of bone mass and geometry will be considered. A better understanding of the dynamic role of ECM networks in regulating tissue function and cell behavior is essential for the development of new treatment approaches for bone loss.     

Distribution of EGF and its receptor in growing red deer antler

Autografts of the osteogenic part of early antler buds placed elsewhere on the skull have been shown by others to give rise to an antler at the site of grafting. This antler becomes covered in velvet skin, is shed at the end of the growing season and will regrow the following year. Thus, it can be concluded that the nature of antler velvet skin is primarily determined by the underlying osteogenic antler tissue to which it is attached. We hypothesise that a paracrine mechanism operates here and is central to communication between the antler osseous compartment and the integument. A signalling system comprising epidermal growth factor (EGF) and its receptor (EGFR) is known to be expressed in osteogenic cells and to play an important role in skin development and growth. This system may therefore play a significant role in determining the nature and speed of growth of velvet skin via paracrine signalling from osteogenic tissue. We have used bright-field microscope immunohistochemistry to determine the distribution of EGF and its receptor in developing red deer antler osseous compartment and integument. EGF was localized throughout the epidermis and epidermal appendages, in cells of the mesenchyme, in chondrocytes, and in cells of the osteoblastic lineage, including osteoprogenitor cells, osteoblasts and osteocytes. There was strong evidence supporting nuclear and nucleolar staining in sebaceous glands and in keratinocytes. The EGFR was similarly expressed in mesenchyme, chondrocytes and osteoblasts. In skin, the distribution of the EGFR was more localized, being expressed strongly in the deeper cells of the epidermis but not in superficial layers, and was absent from nuclei of cells of the epidermis and its appendages. We conclude that this signalling system is widely distributed in growing antler in a manner which suggests it is predominantly autocrine. No clear-cut evidence for paracrine signalling pathways for this system in either integument or osseous compartments was found. The pattern of distribution of the EGFR in the integument was similar to that seen by others in adult human skin. By contrast, in developing antler osseocartilage, the patterns of distribution were similar to those seen in rodent fetal bone. We conclude that antler consists of rapidly growing fetal osseocartilage overlayed by mature velvet.     

Cellular and molecular phenotypes of osteogenic cells isolated from the medullary bone of the hen in vitro

In this study, cells isolated from hen medullary bone were cultured to examine their matrix formation. Furthermore, we compared medullary bone cells with rat bone marrow cells regarding the temporal changes in osteoblast developmental markers. Medullary bone cells were positive for alkaline phosphatase (ALP) activity and formed bone nodules, apparent with Alcian blue and von Kossa staining. The intensity of these stains became stronger with the maturation of those bone nodules. In this developmental process, the expression patterns of osteoblast phenotypes of medullary bone cells differed from those of rat bone marrow cells. ALP mRNA was expressed at the maximum level in the proliferation stage and gradually decreased in medullary bone cells, but that expression showed the opposite pattern in rat bone marrow cells. Medullary bone cells strongly expressed two non-collagenous protein mRNAs from the early stages, but the expression of these mRNAs in rat bone marrow cells increased only in the later stages. These results suggest that the features of medullary bone osteoblasts differ from those of mammalian osteoblasts and are reflected in the characteristics of medullary bone in vivo.     

Osteoblast viability and differentiation with Me2SO as cryoprotectant compared to osteoblasts from fresh human iliac cancellous bone

The aim of this study was to compare the viability of human osteoblasts cryopreserved with Me2SO to that of fresh human iliac cancellous bone using cell culture techniques. Osteoblasts were obtained by spontaneous outgrowth of human iliac cancellous bone specimens in experiment I. In experiment II, human iliac cancellous bone was frozen with 10% Me2SO at -80 degrees C for 2 weeks and osteoblasts grew spontaneously after thawing at 37 degrees C by removing Me2SO with sucrose. The cells were grown in culture flasks containing DMEM as a culture medium, supplemented with 10% fetal calf serum. They were kept at 37 degrees C in a humidified atmosphere of 95% air and 5% CO2. Cells from the second passage were plated at a density of 5 times 10(3) cells/cm2 in 24-well plates. For detection of viability and differentiation, WST-1 assay, determination of alkaline phosphatase activity, concentration of procollagen I peptide, concentration of osteocalcin, and indirect immunofluorescence for osteopontin, collagen type I, integrin beta1, and fibronectin were applied. Experiments were conducted at four stages of confluence (days 4, 7, 14, and 21 after plating the cells). Based on the results of this study, we conclude that osteoblast-like cells survived cryopreservation and synthesized a range of markers that were consistent with this cell type.     

Inflammatory cytokines activate p38 MAPK to induce osteoprotegerin synthesis by MG-63 cells

Inflammatory bone diseases are characterized by the presence of pro-inflammatory cytokines that regulate bone turnover. Osteoprotegerin (OPG) is a soluble osteoblast-derived protein that influences bone resorption by inhibiting osteoclast differentiation and activation. In the present study, we demonstrate that interleukin-1beta and tumor necrosis factor alpha induce OPG mRNA production and OPG secretion by osteoblast-like MG-63 cells. Maximum induction of OPG secretion by either cytokine requires activation of the p38 mitogen activated protein kinase (MAPK) pathway but neither the p42/p44 (ERK) nor the c-Jun N-terminal MAPK pathways. Induction of OPG mRNA by either cytokine is also p38 MAPK dependent. Taken together, these data indicate that cytokine-induced OPG gene expression and protein secretion are differentially regulated by specific MAP kinase signal transduction pathways.     

Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hematopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen I, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis.     

Butyrate response factor 1 is regulated by parathyroid hormone and bone morphogenetic protein-2 in osteoblastic cells

Parathyroid hormone (PTH) exerts potent and diverse effects in bone and cartilage through activation of type 1 PTH receptors (PTH1R) capable of coupling to protein kinase A (PKA) and PKC. We have used macroarrays to identify zinc finger protein butyrate response factor-1 (BRF1) as a novel PTH regulated gene in clonal and normal osteoblasts of human and rodent origin. We further demonstrate that in human osteoblast-like OHS cells, biologically active hPTH(1-84) and hPTH(1-34) stimulate BRF1 mRNA expression in a dose- and time-dependent manner, while the amino-terminally truncated hPTH(3-84) which does not activate PTH1R has no effect. Moreover, using specific stimulators or inhibitors of PKA and PKC activity, the PTH-elicited BRF1 mRNA expression is mediated through the PKA signaling pathway. In mouse calvarial osteoblasts, BRF1 mRNA levels are upregulated by PTH(1-84) and reduced in response to bone morphogenetic protein 2 (BMP-2). Hence, our data showing that BRF1 is expressed in osteoblastic cells and regulated by PTH and BMP-2, suggest an important role for BRF1 in osteoblasts within the molecular network of PTH-dependent bone remodeling.     

Effects of Lactobacillus helveticus fermented milk on bone cells in vitro

Milk fermented with Lactobacillus helveticus (L. helveticus) contains small peptides such as isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), which inhibit the angiotensin converting enzyme (ACE). We investigated the effects of L. helveticus fermented milk whey (Lh-whey) and its components, sour milk whey, calcium and IPP and VPP peptides, on bone cells in vitro. An osteoblast assay was performed by determining the amount of deposited calcium as an index of bone formation in cultures of mouse osteoblasts formed from bone marrow-derived osteoblast precursor cells. An osteoclast assay was performed by determining the activity of tartrate-resistant acid phosphatase released into the culture medium in cultures of mouse osteoclasts formed from bone marrow-derived osteoclast precursor cells. The Lh-whey increased bone formation 1.3-1.4 times with the 1 × 10⁻⁵, 1 × 10⁻⁴ and 1 × 10⁻³ solutions. The IPP and VPP peptides also demonstrated a significant 5-fold activation of bone formation in in vitro osteoblast cultures, whereas the sour milk whey and calcium had no effect. No significant effects were observed on osteoclasts in vitro with any of the study products. L. helveticus fermented milk whey contains bioactive components that increase osteoblastic bone formation in vitro. The effect may be due to the ACE-inhibitory IPP and VPP peptides, which showed a similar effect to that of the L. helveticus fermented milk whey.