Osteoblast migration in vertebrate bone

Bone formation, for example during bone remodelling or fracture repair, requires mature osteoblasts to deposit bone with remarkable spatial precision. As osteoblast precursors derive either from circulation or resident stem cell pools, they and their progeny are required to migrate within the three‐dimensional bone space and to navigate to their destination, i.e. to the site of bone formation. An understanding of this process is emerging based on in vitro and in vivo studies of several vertebrate species. Receptors on the osteoblast surface mediate cell adhesion and polarization, which induces osteoblast migration. Osteoblast migration is then facilitated along gradients of chemoattractants. The latter are secreted or released proteolytically by several cell types interacting with osteoblasts, including osteoclasts and vascular endothelial cells. The positions of these cellular sources of chemoattractants in relation to the position of the osteoblasts provide the migrating osteoblasts with tracks to their destination, and osteoblasts possess the means to follow a track marked by multiple chemoattractant gradients. In addition to chemotactic cues, osteoblasts sense other classes of signals and utilize them as landmarks for navigation. The composition of the osseous surface guides adhesion and hence migration efficiency and can also provide steering through haptotaxis. Further, it is likely that signals received from surface interactions modulate chemotaxis. Besides the nature of the surface, mechanical signals such as fluid flow may also serve as navigation signals for osteoblasts. Alterations in osteoblast migration and navigation might play a role in metabolic bone diseases such as osteoporosis.     

Chitosan sponges as tissue engineering scaffolds for bone formation

Rat calvarial osteoblasts were grown in porous chitosan sponges fabricated by freeze drying. The prepared chitosan sponges had a porous structure with a 100-200 microm pore diameter, which allowed cell proliferation. Cell density, alkaline phosphatase activity and calcium deposition were monitored for up to 56 d culture. Cell numbers were 4 x 10(6) (day 1), 11 x 10(6) (day 28) and 12 x 10(6) (day 56) per g sponge. Calcium depositions were 9 (day 1), 40 (day 28) and 48 (day 56) microg per sponge. Histological results corroborated that bone formation within the sponges had occurred. These results show that chitosan sponges can be used as effective scaffolding materials for tissue engineered bone formation in vitro.     

Osteogenic differentiation of purified,culture-expanded human mesenchymal stem cells in vitro

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM β-glycerophosphate (βGP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM βGP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number or APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes. J. Cell. Biochem. 64:295–312. © 1997 Wiley-Liss, Inc.     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

Growth kinetics,self-renewal,and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation

Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity, and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 ± 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime. J. Cell. Biochem. 64:278–294. © 1997 Wiley-Liss, Inc.     

miR-542-3p suppresses osteoblast cell proliferation and differentiation,targets BMP-7 signaling and inhibits bone formation

MicroRNAs (miRNAs) are short non-coding RNAs that interfere with translation of specific target mRNAs and thereby regulate diverse biological processes. Recent studies have suggested that miRNAs might have a role in osteoblast differentiation and bone formation. Here, we show that miR-542-3p, a well-characterized tumor suppressor whose downregulation is tightly associated with tumor progression via C-src-related oncogenic pathways, inhibits osteoblast proliferation and differentiation. miRNA array profiling in Medicarpin (a pterocarpan with proven bone-forming effects) induced mice calvarial osteoblast cells and further validation by quantitative real-time PCR revealed that miR-542-3p was downregulated during osteoblast differentiation. Over-expression of miR-542-3p inhibited osteoblast differentiation, whereas inhibition of miR-542-3p function by anti-miR-542-3p promoted expression of osteoblast-specific genes, alkaline phosphatase activity and matrix mineralization. Target prediction analysis tools and experimental validation by luciferase 3′ UTR reporter assay identified BMP-7 (bone morphogenetic protein 7) as a direct target of miR-542-3p. It was seen that over-expression of miR-542-3p leads to repression of BMP-7 and inhibition of BMP-7/PI3K- survivin signaling. This strongly suggests that miR-542-3p suppresses osteogenic differentiation and promotes osteoblast apoptosis by repressing BMP-7 and its downstream signaling. Furthermore, silencing of miR-542-3p led to increased bone formation, bone strength and improved trabecular microarchitecture in sham and ovariectomized (Ovx) mice. Although miR-542-3p is known to be a tumor repressor, we have identified second complementary function of miR-542-3p where it inhibits BMP-7-mediated osteogenesis. Our findings suggest that pharmacological inhibition of miR-542-3p by anti-miR-542-3p could represent a therapeutic strategy for enhancing bone formation in vivo.     

Mesenchymal stem cells in bone development,bone repair,and skeletal regenaration therapy

Bone formation in the embryo, and during adult fracture repair and remodeling, involves the progreny of a small number of cells called mesenchymal stem cells (MSCs). These cells continuously replicate themselves, while a portion become committed to mesenchymal cell lineages such as bone, cartilage, tendon, legament and muscle. The differentiation of these cells, within each lineage, is a complex multistep pathway involving discrete cellular trasitions much like that which occurs during hematopoiesys. Progression from one stage to the next depends on the presence of specific bioactive factors, nutrients, and other environmental cues whose exquisitely controlled contributions orchestrate the entire differentiation phgenomenon. As understanding of the cellular and molecular events of osteogenic differentiation of MSCs provides the foundation for the emergence of a new therapeutic technilogy for cell therapy. The isolation and in vitro mitotic expansion of autologous human MSCs will support the development of novel protocols for the treatment of many clinically challenging conditions. For example, local bone defects can be repaired through site-directed delivery of MSCs in an appropriate carrier vehicle. Generalized conditions, such as osteoporosis, may be treatable by systemic administration of culture-expanded autologous MSCs or through biopharmaceutical regimens based on the discovery of critical regulatory molecules in the differentiation process. With this in mind, we can begin to explore therapeutic options that have never before been available.     

Influence of electromagnetic fields on endochondral bone formation

Endochondral ossification is a basic physiological process in limb development and is central to bone repair and linear growth. Factors which regulate endochondral ossification include several biophysical and biochemical agents and are of interest from clinical and biological perspectives. One of these agents, electric stimulation, has been shown to result in enhanced synthesis of extracellular matrix, calcification, and bone formation in a number of experimental systems and is the subject of this review. The effects of electric stimulation have been studied in embryonic limb rudiments, growth plates, and experimental endochondral ossification induced with decalcified bone matrix and, in all these models, endochondral ossification has been enhanced. It is not known definitively whether electric fields stimulate cell differentiation or modulate an increased number of molecules synthesized by committed cell population and this is a fertile area of current study.     

Lutein,a carotenoid,suppresses osteoclastic bone resorption and stimulates bone formation in cultures

Lutein, a member of the xanthophyll family of carotenoids, suppressed IL-1-induced osteoclast differentiation and bone resorption. The survival of mature osteoclasts was also suppressed by lutein in cultures. When lutein was added to the cultures of osteoblasts, lutein enhanced the formation of mineralized bone nodules by elevating BMP2 expression and inhibiting sclerostin expression. Lutein may be beneficial for bone health.     

Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.     

CHIP regulates skeletal development and postnatal bone growth

C terminus of Hsc70-interacting protein (CHIP) is a chaperone-dependent and U-box containing E3 ubiquitin ligase. In previous studies, we found that CHIP regulates the stability of multiple tumor necrosis factor receptor-associated factor proteins in bone cells. In Chip global knockout (KO) mice, nuclear factor-κB signaling is activated, osteoclast formation is increased, osteoblast differentiation is inhibited, and bone mass is decreased in postnatal Chip KO mice. To determine the role of Chip in different cell types at different developmental stages, we created Chip^flox/flox mice. We then generated Chip conditional KO mice Chip^CMV and Chip^OsxER and demonstrated defects in skeletal development and postnatal bone growth in Chip conditional KO mice. Our findings indicate that Chip conditional KO mice could serve as a critical reagent for further investigations of functions of CHIP in bone cells and in other cell types.     

Effects of phospholipase D during cultured osteoblast mineralization and bone formation

Mammalian phospholipase D (PLD) mostly hydrolyzes phosphatidylcholine producing phosphatidic acid. PLD activity was previously detected in different osteoblastic cell models, and was increased by several growth factors involved in bone homeostasis. To confirm possible actions of PLD isoforms during mineralization process, we analyzed their effects in osteoblastic cell models and during bone formation. PLD1 expression, along with PLD activity, increased during differentiation of primary osteoblasts and Saos-2 cells, and peaked at the onset of mineralization. Subsequently, both PLD1 expression and PLD activity decreased, suggesting that PLD1 function is regulated during osteoblast maturation. In contrast, PLD2 expression was not significantly affected during differentiation of osteoblasts. Overexpression of PLD1 in Saos-2 cells improved their mineralization potential. PLD inhibitor Halopemide or PLD1-selective inhibitor, led to a decrease in mineralization in both cell types. On the contrary, the selective inhibitor of PLD2, did not affect the mineralization process. Moreover, primary osteoblasts isolated from PLD1 knockout (KO) mice were significantly less efficient in mineralization as compared with those isolated from wild type (WT) or PLD2 KO mice. In contrast, bone formation, as monitored by high-resolution microcomputed tomography analysis, was not impaired in PLD1 KO nor in PLD2 KO mice, indicating that the lack of PLD1 or that of PLD2 did not affect the bone structure in adult mice. Taken together, our findings indicate that PLD activity, especially which of PLD1 isoform, may enhance the mineralization process in osteoblastic cells. Nonetheless, the lack of PLD1 or PLD2 do not seem to significantly affect bone formation in adult mice.