Johannes P.T.M. van Leeuwen

Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: an in vitro study.

BackgroundAlthough pulsed electromagnetic field (PEMF) stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical effects of PEMF. Here, we performed in vitro experiments to investigate the effect of PEMF stimulation on human bone marrow-derived stromal cell (BMSC) metabolism and, specifically, whether PEMF can stimulate their osteogenic differentiation.MethodsBMSCs derived from four different donors were cultured in osteogenic medium, with the PEMF treated group being continuously exposed to a 15 Hz, 1 Gauss EM field, consisting of 5-millisecond bursts with 5-microsecond pulses. On culture day 1, 5, 9, and 14, cells were collected for biochemical analysis (DNA amount, alkaline phosphatase activity, calcium deposition), expression of various osteoblast-relevant genes and activation of extracellular signal-regulated kinase (ERK) signaling. Differences between treated and control groups were analyzed using the Wilcoxon signed rank test, and considered significant when p < 0.05.ResultsBiochemical analysis revealed significant, differentiation stage-dependent, PEMF-induced differences: PEMF increased mineralization at day 9 and 14, without altering alkaline phosphatase activity. Cell proliferation, as measured by DNA amounts, was not affected by PEMF until day 14. Here, DNA content stagnated in PEMF treated group, resulting in less DNA compared to control.Quantitative RT-PCR revealed that during early culture, up to day 9, PEMF treatment increased mRNA levels of bone morphogenetic protein 2, transforming growth factor-beta 1, osteoprotegerin, matrix metalloproteinase-1 and -3, osteocalcin, and bone sialoprotein. In contrast, receptor activator of NF-κB ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation.ConclusionsPEMF exposure of differentiating human BMSCs enhanced mineralization and seemed to induce differentiation at the expense of proliferation. The osteogenic stimulus of PEMF was confirmed by the up-regulation of several osteogenic marker genes in the PEMF treated group, which preceded the deposition of mineral itself. These findings indicate that PEMF can directly stimulate osteoprogenitor cells towards osteogenic differentiation. This supports the theory that PEMF treatment may recruit these cells to facilitate an osteogenic response in vivo.

Iodothyronine deiodinase enzyme activities in bone.

Euthyroid status is essential for normal skeletal development and maintenance of the adult skeleton, but the mechanisms which control supply of thyroid hormone to bone cells are poorly understood. Thyroid hormones enter target cells via monocarboxylate transporter-8 (MCT8), which provides a functional link between thyroid hormone uptake and metabolism in the regulation of T3-action but has not been investigated in bone. Most circulating active thyroid hormone (T3) is derived from outer ring deiodination of thyroxine (T4) mediated by the type 1 deiodinase enzyme (D1). The D2 isozyme regulates intra-cellular T3 supply and determines saturation of the nuclear T3-receptor (TR), whereas a third enzyme (D3) inactivates T4 and T3 to prevent hormone availability and reduce TR-saturation. The aim of this study was to determine whether MCT8 is expressed in the skeleton and whether chondrocytes, osteoblasts and osteoclasts express functional deiodinases. Gene expression was analyzed by RT-PCR and D1, D2 and D3 function by sensitive and highly specific determination of enzyme activities. MCT8 mRNA was expressed in chondrocytes, osteoblasts and osteoclasts at all stages of cell differentiation. D1 activity was undetectable in all cell types, D2 activity was only present in mature osteoblasts whereas D3 activity was evident throughout chondrocyte, osteoblast and osteoclast differentiation in primary cell cultures. These data suggest that T3 availability especially during skeletal development may be limited by D3-mediated catabolism rather than by MCT8 mediated cellular uptake or D2-dependent T3 production.

Mechanism and function of high vitamin D receptor levels in genetic hypercalciuric stone-forming rats

The functional status and mechanism of increased VDR in GHS rats were investigated. Basal VDR and calbindins were increased in GHS rats. 1,25(OH)2D3 increased VDR and calbindins in controls but not GHS rats. VDR half‐life was prolonged in GHS rats. This study supports the mechanism and functional status of elevated VDR in GHS rats.

Ageing and vitamin D deficiency: effects on calcium homeostasis and considerations for vitamin D supplementation.

Vitamin D is a fat-soluble, seco-steroid hormone. In man, the vitamin D receptor is expressed in almost all tissues, enabling effects in multiple systems of the human body. These effects can be endocrine, paracrine and autocrine. The present review summarises the effects of ageing on the vitamin D endocrine system and on Ca homeostasis. Furthermore, consequences for vitamin D supplementation are discussed.

An age-dependent interaction with leptin unmasks Ghrelin's bone-protective effects

The mutual interplay between energy homeostasis and bone metabolism is an important emerging concept. Ghrelin and leptin antagonize each other in regulating energy balance, but the role of this interaction in bone metabolism is unknown. Using ghrelin receptor and leptin-deficient mice, we show that ghrelin has dual effects on osteoclastogenesis, inhibiting osteoclast progenitors directly and stimulating osteoclastogenesis via a more potent systemic/central pathway. Using mice with combined ghrelin receptor and leptin deficiency, we find that this systemic osteoclastogenic activity is suppressed by leptin, thus balancing the two counterregulatory ghrelin pathways and leading to an unchanged bone structure. With aging, this osteoclastogenic ghrelin pathway is lost, unmasking the direct protective effect of ghrelin on bone structure. In conclusion, we identify a novel regulatory network linking orexigenic and anorectic metabolic factors with bone metabolism that is age dependent.

UV exposure inhibits intestinal tumor growth and progression to malignancy in intestine-specific Apc mutant mice kept on low vitamin D diet

Mortality from colorectal cancer increases with latitude and decreases with ambient UV radiation. We investigated whether moderate UV dosages could inhibit intestinal tumor development and whether this corresponded with UV‐induced vitamin D. FabplCre;Apc15lox/+ mice, which develop intestinal tumors, and their parents were put on a vitamin D‐deficient diet. Next to a control group, one group was vitamin D supplemented and another one group was daily UV irradiated from 6 weeks of age. Vitamin D statuses after 6 weeks of treatment were markedly increased: meanu2009±u2009SD from 7.7u2009±u20091.9 in controls to 75u2009±u200915 nmol/l with vitamin D supplementation (no gender difference), and to 31u2009±u200913 nmol/l in males and 85u2009±u200917 nmol/l in females upon UV irradiation. The tumor load (area covered by tumors) at 7.5 months of age was significantly reduced in both the vitamin D‐supplemented group (130u2009±u200925 mm2, pu2009=u20090.018) and the UV‐exposed group (88u2009±u20099 mm2, pu2009<u20090.0005; no gender differences) compared to the control group (202u2009±u200923 mm2). No reductions in tumor numbers were found. Only UV exposure appeared to reduce progression to malignancy (pu2009=u20090.014). Our experiments clearly demonstrate for the first time an inhibitory effect of moderate UV exposure on outgrowth and malignant progression of primary intestinal tumors, which at least in part can be attributed to vitamin D.

Extracellular vesicles: specialized bone messengers.

Mammalian cells actively secrete factors that contribute to shape their microenvironment. These factors either travel freely or they are enclosed within the lipid bilayer of extracellular vesicles (EVs), and regulate the function of neighboring and distant cells. EVs are secreted by a wide spectrum of cell types and are found in various biological fluids. They convey their message by mediating the horizontal transfer of bioactive molecules, such as proteins, mRNAs and miRNAs, between cells. Recent studies showed the vital roles of EVs in a wide range of physiological and pathophysiological processes. In this review, we highlight the recent developments in the newly emerging EV field, including their biogenesis, molecular content and function. Moreover, we discuss the role of EVs in bone biology and their promising applications in diagnosis, drug development and regenerative therapy.

Paracrine Signaling by Extracellular Vesicles via Osteoblasts

Extracellular vesicles (EVs), spherical bilayered proteolipids, behave as paracrine effectors since they are released from cells to deliver signals to other cells. They control a diverse range of biological processes by transferring proteins, lipids, and nucleic acids between cells and are secreted by a wide spectrum of cell types and are found in various biological fluids. EVs are formed at the plasma membrane or in endosomes and are heterogeneous in size and composition. Increasing understanding of the working mechanisms is promising for therapeutic and diagnostic opportunities. In this review, we will focus on the recent developments in this emerging field with special emphasis on the role of EVs in the bone microenvironment, with a central role for the osteoblasts in the communication with a diversity of cells, including bone metastases.

Comparative proteomic profiling of human osteoblast-derived extracellular matrices identifies proteins involved in mesenchymal stromal cell osteogenic differentiation and mineralization

The extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cell‐secreted ECMs have become of interest as they reproduce tissue‐architecture and modulate the promising properties of mesenchymal stem cells (MSCs). We have previously created an in vitro model of human osteoblast‐derived devitalized ECM that was osteopromotive for MSCs. The aim of this study was to identify ECM regulatory proteins able to modulate MSC differentiation to broaden the spectrum of MSC clinical applications. To this end, we created two additional models of devitalized ECMs with different mineralization phenotypes. Our results showed that the ECM derived from osteoblast‐differentiated MSCs had increased osteogenic potential compared to ECM derived from undifferentiated MSCs and non‐ECM cultures. Proteomic analysis revealed that structural ECM proteins and ribosomal proteins were upregulated in the ECM from undifferentiated MSCs. A similar response profile was obtained by treating osteoblast‐differentiating MSCs with Activin‐A. Extracellular proteins were upregulated in Activin‐A ECM, whereas mitochondrial and membrane proteins were downregulated. In summary, this study illustrates that the composition of different MSC‐secreted ECMs is important to regulate the osteogenic differentiation of MSCs. These models of devitalized ECMs could be used to modulate MSC properties to regulate bone quality.

Chapter 9 – Osteoblast Biology

Bone formation takes place throughout life in order to support growth, mechanical forces, bone turnover to meet metabolic needs, and the reparative process. The requirement for continuous renewal of bone through the remodeling process necessitates recruitment, proliferation, and differentiation of osteoblast-lineage cells. A contributing factor to bone loss in the aging skeleton is the decreased ability of bone-forming osteoblasts to replace bone removed by the activity of the bone-resorbing osteoclasts. It is now appreciated how numerous developmental, growth factor, cytokine, and hormone-responsive regulatory signals mediate competency for expression of genes associated with bone matrix synthesis and metabolic responses as a function of the stages of osteoblast growth and differentiation.