Bone is an exquisitely sophisticated organ/tissue in mammals. Bone is generally viewed as the main component of the skeleton, providing mechanical and structural support to the rest of the organs and systems. This function is indispensable for life, both during the growth and development period as well as during adult life. However, bone also provides the unique architecture and microenvironment that preserves the niches that maintain immature stem cells. This inadequately recognized function is also essential, because these stem cells are required for tissue repair and regeneration during adult life. In this respect, besides providing mechanical support, bone holds and supports the main reservoir of cells needed to sustain tissue integrity and function throughout our lives. Thus, understanding how bone is made and maintained during life is central to developing adequate strategies to preserve a healthy skeleton as we age; so that proper mechanical support, structural integrity, and tissue repair capacity is maintained.
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References
Olsen BR, Reginato AM, Wang W. Bone development. Annu Rev Cell Dev Biol 2000;16:191–220.
Pechak DG, Kujawa MJ, Caplan AI. Morphological and histochemical events during first bone formation in embryonic chick limbs. Bone 1986;7:441–458.
Foster JW, Dominguez-Steglich MA, Guioli S, et al. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature 1994;372:525–530.
Wagner T, Wirth J, Meyer J, et al. Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 1994;79:1111–1120.
Bi W, Deng JM, Zhang Z, et al. Sox9 is required for cartilage formation. Nat Genet 1999;22:85–89.
Iyama K, Ninomiya Y, Olsen BR, et al. Spatiotemporal pattern of type X collagen gene expression and collagen deposition in embryonic chick vertebrae undergoing endochondral ossification. Anat Rec 1991;229:462–472.
Gerber HP, Vu TH, Ryan AM, et al. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 1999;5:623–628.
Park SR, Oreffo RO, Triffitt JT. Interconversion potential of cloned human marrow adipocytes in vitro. Bone 1999;24:549–554.
Doherty MJ, Ashton BA, Walsh S, et al. Vascular pericytes express osteogenic potential in vitro and in vivo. J Bone Miner Res 1998;13:828–838.
Schiller PC, D'Ippolito G, Brambilla R, et al. Inhibition of gap-junctional communication induces the trans-differentiation of osteoblasts to an adi-pocytic phenotype in vitro. J Biol Chem 2001; 276:14133–14138.
Holtrop ME. The ultrastructure of bone. Ann Clin Lab Sci 1975;5:264–271.
Parfitt AM. The actions of parathyroid hormone on bone: relation to bone remodeling and turnover, calcium homeostasis, and metabolic bone disease. Part I of IV parts: mechanisms of calcium transfer between blood and bone and their cellular basis: morphological and kinetic approaches to bone turnover. Metabolism 1976;25:809–844.
Zhang D, Weinbaum S, Cowin SC. Electrical signal transmission in a bone cell network: the influence of a discrete gap junction. Ann Biomed Eng 1998; 26:644–659.
Zhang D, Cowin SC, Weinbaum S. Electrical signal transmission and gap junction regulation in a bone cell network: a cable model for an osteon. Ann Biomed Eng 1997;25:357–374.
Schiller PC, Mehta PP, Roos BA, et al. Hormonal regulation of intercellular communication: parathyroid hormone increases connexin 43 gene expression and gap-junctional communication in osteoblastic cells. Mol Endocrinol 1992;6:1433– 1440.
Schiller PC, D'Ippolito G, Roos BA, et al. Anabolic or catabolic responses of MC3T3-E1 osteoblastic cells to parathyroid hormone depend on time and duration of treatment. J Bone Miner Res 1999;14:1504–1512.
Schiller PC, D'Ippolito G, Balkan W, et al. Gap-junctional communication mediates parathyroid hormone stimulation of mineralization in osteo-blastic cultures. Bone 2001;28:38–44.
Schiller PC, D'Ippolito G, Balkan W, et al. Gap-junctional communication is required for the maturation process of osteoblastic cells in culture. Bone 2001;28:362–369.
Civitelli R, Beyer EC, Warlow PM, et al. Con-nexin43 mediates direct intercellular communication in human osteoblastic cell networks. J Clin Invest 1993;91:1888–1896.
Lecanda F, Warlow PM, Sheikh S, et al. Con-nexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J Cell Biol 2000;151:931–944.
Furlan F, Lecanda F, Screen J, et al. Proliferation, differentiation and apoptosis in connexin43-null osteoblasts. Cell Commun Adhes 2001;8:367–371.
Plotkin LI, Manolagas SC, Bellido T. Transduction of cell survival signals by connexin-43 hemichan-nels. J Biol Chem 2002;277:8648–8657.
Noble BS, Stevens H, Loveridge N, et al. Identification of apoptotic changes in osteocytes in normal and pathological human bone. Bone 1997;20:273–282.
Noble BS, Peet N, Stevens HY, et al. Mechanical loading: biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone. Am J Physiol Cell Physiol 2003;284:C934–C943.
Plotkin LI, Aguirre JI, Kousteni S, et al. Bisphos-phonates and estrogens inhibit osteocyte apopto-sis via distinct molecular mechanisms downstream of extracellular signal-regulated kinase activation. J Biol Chem 2005;280:7317–7325.
Plotkin LI, Mathov I, Aguirre JI, et al. Mechanical stimulation prevents osteocyte apoptosis: requirement of integrins, Src kinases, and ERKs. Am J Physiol Cell Physiol 2005;289:C633–C643.
Ducy P, Zhang R, Geoffroy V, et al. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 1997;89:747–754.
Otto F, Thornell AP, Crompton T, et al. Cbfa1, a candidate gene for cleidocranial dysplasia syn- drome, is essential for osteoblast differentiation and bone development. Cell 1997;89:765–771.
Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteo-blasts. Cell 1997;89:755–764.
Mundlos S, Otto F, Mundlos C, et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 1997;89:773–779.
Ducy P, Starbuck M, Priemel M, et al. A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev 1999;13:1025–1036.
Kim S, Koga T, Isobe M, et al. Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcrip-tional program of osteoblast differentiation. Genes Dev 2003;17:1979–1991.
Lee KS, Hong SH, Bae SC. Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene 2002;21:7156–7163.
Zhang YW, Yasui N, Ito K, et al. A RUNX2/PEB-P2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleido-cranial dysplasia. Proc Natl Acad Sci U S A 2000;97:10549–10554.
Alliston T, Choy L, Ducy P, et al. TGF-beta-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation. Embo J 2001;20:2254– 2272.
Zamurovic N, Cappellen D, Rohner D, et al. Coordinated activation of notch, Wnt, and transforming growth factor-beta signaling pathways in bone morphogenic protein 2-induced osteogenesis. Notch target gene Hey1 inhibits mineralization and Runx2 transcriptional activity. J Biol Chem 2004;279:37704–37715.
Sowa H, Kaji H, Hendy GN, et al. Menin is required for bone morphogenetic protein 2- and transforming growth factor beta-regulated osteoblastic differentiation through interaction with Smads and Runx2. J Biol Chem 2004;279:40267–40275.
Sierra J, Villagra A, Paredes R, et al. Regulation of the bone-specific osteocalcin gene by p300 requires Runx2/Cbfa1 and the vitamin D3 receptor but not p300 intrinsic histone acetyltransferase activity. Mol Cell Biol 2003;23:3339–3351.
Wang W, Wang YG, Reginato AM, et al. Groucho homologue Grg5 interacts with the transcription factor Runx2-Cbfa1 and modulates its activity during postnatal growth in mice. Dev Biol 2004; 270:364–381.
Bialek P, Kern B, Yang X, et al. A twist code determines the onset of osteoblast differentiation. Dev Cell 2004;6:423–435.
Nakashima K, Zhou X, Kunkel G, et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002;108:17–29.
Akiyama H, Kim JE, Nakashima K, et al. Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors. Proc Natl Acad Sci U S A 2005;102:14665–14670.
Huang LF, Fukai N, Selby PB, et al. Mouse clavicular development: analysis of wild-type and cleido-cranial dysplasia mutant mice. Dev Dyn 1997; 210:33–40.
Inada M, Yasui T, Nomura S, et al. Maturational disturbance of chondrocytes in Cbfa1-deficient mice. Dev Dyn 1999;214:279–290.
Kim IS, Otto F, Zabel B, et al. Regulation of chon-drocyte differentiation by Cbfa1. Mech Dev 1999;80:159–170.
Jimenez MJ, Balbin M, Lopez JM, et al. Collagenase 3 is a target of Cbfa1, a transcription factor of the runt gene family involved in bone formation. Mol Cell Biol 1999;19:4431–4442.
Porte D, Tuckermann J, Becker M, et al. Both AP-1 and Cbfa1-like factors are required for the induction of interstitial collagenase by parathyroid hormone. Oncogene 1999;18:667–678.
Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 2004;20:781–810.
Church VL, Francis-West P. Wnt signalling during limb development. Int J Dev Biol 2002;46:927– 936.
Boyden LM, Mao J, Belsky J, et al. High bone den-sity due to a mutation in LDL-receptor-related protein 5. N Engl J Med 2002;346:1513–1521.
Little RD, Recker RR, Johnson ML. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med 2002;347:943–944; author reply 944.
Kato M, Patel MS, Levasseur R, et al. Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascu-larization in mice deficient in Lrp5, a Wnt core-ceptor. J Cell Biol 2002;157:303–314.
Holmen SL, Zylstra CR, Mukherjee A, et al. Essential role of beta-catenin in postnatal bone acquisition. J Biol Chem 2005;280:21162–21168.
Day TF, Guo X, Garrett-Beal L, et al. Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentia- tion during vertebrate skeletogenesis. Dev Cell 2005;8:739–750.
Hill TP, Spater D, Taketo MM, et al. Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell 2005;8:727–738.
Hu H, Hilton MJ, Tu X, et al. Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development 2005;132:49–60.
Mannstadt M, Juppner H, Gardella TJ. Receptors for PTH and PTHrP: their biological importance and functional properties. Am J Physiol 1999;277: F665–F675.
Swarthout JT, D'Alonzo RC, Selvamurugan N, et al. Parathyroid hormone-dependent signaling pathways regulating genes in bone cells. Gene 2002;282:1–17.
Qin L, Qiu P, Wang L, et al. Gene expression profiles and transcription factors involved in parathyroid hormone signaling in osteoblasts revealed by microarray and bioinformatics. J Biol Chem 2003;278:19723–19731.
Wang BL, Dai CL, Quan JX, et al. Parathyroid hormone regulates osterix and Runx2 mRNA expression predominantly through protein kinase A signaling in osteoblast-like cells. J Endocrinol Invest 2006;29:101–108.
Locklin RM, Khosla S, Turner RT, et al. Mediators of the biphasic responses of bone to intermittent and continuously administered parathyroid hormone. J Cell Biochem 2003;89:180–190.
Mikuni-Takagaki Y, Naruse K, Azuma Y, et al. The role of calcium channels in osteocyte function. J Musculoskelet Neuronal Interact 2002;2:252–255.
Sekiya H, Mikuni-Takagaki Y, Kondoh T, et al. Synergistic effect of PTH on the mechanical responses of human alveolar osteocytes. Biochem Biophys Res Commun 1999;264:719–723.
Chen X, Macica CM, Ng KW, et al. Stretch-induced PTH-related protein gene expression in osteo-blasts. J Bone Miner Res 2005;20:1454–1461.
D'Ippolito G, Schiller PC, Perez-stable C, et al. Cooperative actions of hepatocyte growth factor and 1,25-dihydroxyvitamin D3 in osteoblastic differentiation of human vertebral bone marrow stromal cells. Bone 2002;31:269–275.
D'Ippolito G, Diabira S, Howard GA, et al. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells. Bone 2006;39:513–522.
Erben RG, Soegiarto DW, Weber K, et al. Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and non- genomic functions of vitamin D. Mol Endocrinol 2002;16:1524–1537.
Paredes R, Arriagada G, Cruzat F, et al. Bone-specific transcription factor Runx2 interacts with the 1alpha,25-dihydroxyvitamin D3 receptor to up-regulate rat osteocalcin gene expression in osteo-blastic cells. Mol Cell Biol 2004;24:8847–8861.
Pan W, Quarles LD, Song LH, et al. Genistein stimulates the osteoblastic differentiation via NO/ cGMP in bone marrow culture. J Cell Biochem 2005;94:307–316.
Zallone A. Direct and indirect estrogen actions on osteoblasts and osteoclasts. Ann N Y Acad Sci 2006;1068:173–179.
Seeman E. Estrogen, androgen, and the pathogen-esis of bone fragility in women and men. Curr Osteoporos Rep 2004;2:90–96.
Jessop HL, Sjoberg M, Cheng MZ, et al. Mechanical strain and estrogen activate estrogen receptor alpha in bone cells. J Bone Miner Res 2001;16:1045–1055.
Zaman G, Jessop HL, Muzylak M, et al. Osteocytes use estrogen receptor alpha to respond to strain but their ERalpha content is regulated by estrogen. J Bone Miner Res 2006;21:1297–1306.
Li X, Cao X. BMP signaling and skeletogenesis. Ann N Y Acad Sci 2006;1068:26–40.
Zaidi SK, Sullivan AJ, van Wijnen AJ, et al. Integration of Runx and Smad regulatory signals at transcriptionally active subnuclear sites. Proc Natl Acad Sci U S A 2002;99:8048–8053.
Afzal F, Pratap J, Ito K, et al. Smad function and intranuclear targeting share a Runx2 motif required for osteogenic lineage induction and BMP2 responsive transcription. J Cell Physiol 2005;204:63–72.
Lee MH, Kim YJ, Kim HJ, et al. BMP-2-induced Runx2 expression is mediated by Dlx5, and TGF-beta 1 opposes the BMP-2-induced osteoblast differentiation by suppression of Dlx5 expression. J Biol Chem 2003;278:34387–34394.
Ryoo HM, Lee MH, Kim YJ. Critical molecular switches involved in BMP-2-induced osteogenic differentiation of mesenchymal cells. Gene 2006; 366:51–57.
Li T, Surendran K, Zawaideh MA, et al. Bone mor-phogenetic protein 7: a novel treatment for chronic renal and bone disease. Curr Opin Nephrol Hyper-tens 2004;13:417–422.
Green ED, Maffei M, Braden VV, et al. The human obese (OB) gene: RNA expression pattern and mapping on the physical, cytogenetic, and genetic maps of chromosome 7. Genome Res 1995;5:5–12.
Maffei M, Halaas J, Ravussin E, et al. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1995;1:1155–1161.
Wolf G. Leptin: the weight-reducing plasma protein encoded by the obese gene. Nutr Rev 1996;54:91–93.
Steppan CM, Crawford DT, Chidsey-Frink KL, et al. Leptin is a potent stimulator of bone growth in ob/ob mice. Regul Pept 2000;92:73–78.
Ducy P, Amling M, Takeda S, et al. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 2000;100:197–207.
Hess R, Pino AM, Rios S, et al. High affinity leptin receptors are present in human mesenchymal stem cells (MSCs) derived from control and osteo-porotic donors. J Cell Biochem 2005;94:50–57.
Canalis E. Mechanisms of glucocorticoid action in bone. Curr Osteoporos Rep 2005;3:98–102.
Ohnaka K, Taniguchi H, Kawate H, et al. Gluco-corticoid enhances the expression of dickkopf-1 in human osteoblasts: novel mechanism of gluco-corticoid-induced osteoporosis. Biochem Biophys Res Commun 2004;318:259–264.
Ohnaka K, Tanabe M, Kawate H, et al. Glucocor-ticoid suppresses the canonical Wnt signal in cultured human osteoblasts. Biochem Biophys Res Commun 2005;329:177–181.
Bassett JH, Williams GR. The molecular actions of thyroid hormone in bone. Trends Endocrinol Metab 2003;14:356–364.
Stevens DA, Hasserjian RP, Robson H, et al. Thyroid hormones regulate hypertrophic chon-drocyte differentiation and expression of parathyroid hormone-related peptide and its receptor during endochondral bone formation. J Bone Miner Res 2000;15:2431–2442.
Robson H, Siebler T, Stevens DA, et al. Thyroid hormone acts directly on growth plate chondro-cytes to promote hypertrophic differentiation and inhibit clonal expansion and cell proliferation. Endocrinology 2000;141:3887–3897.
Gruber R, Czerwenka K, Wolf F, et al. Expression of the vitamin D receptor, of estrogen and thyroid hormone receptor alpha- and beta-isoforms, and of the androgen receptor in cultures of native mouse bone marrow and of stromal/osteoblastic cells. Bone 1999;24:465–473.
Salto C, Kindblom JM, Johansson C, et al. Ablation of TRalpha2 and a concomitant overexpression of alpha1 yields a mixed hypo- and hyperthyroid phenotype in mice. Mol Endocrinol 2001;15:2115–2128.
Pereira RC, Jorgetti V, Canalis E. Triiodothyro-nine induces collagenase-3 and gelatinase B expression in murine osteoblasts. Am J Physiol 1999;277:E496–E504.
Engsig MT, Chen QJ, Vu TH, et al. Matrix metal-loproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 2000;151:879–889.
Tondravi MM, McKercher SR, Anderson K, et al. Osteopetrosis in mice lacking haematopoietic transcription factor PU.1. Nature 1997;386:81–84.
DeKoter RP, Singh H. Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science 2000;288:1439–1441.
Luchin A, Suchting S, Merson T, et al. Genetic and physical interactions between Microphthalmia transcription factor and PU.1 are necessary for osteoclast gene expression and differentiation. J Biol Chem 2001;276:36703–36710.
Simonet WS, Lacey DL, Dunstan CR, et al. Osteo-protegerin: a novel secreted protein involved in the regulation of bone density. Cell 1997;89:309–319.
Dai XM, Ryan GR, Hapel AJ, et al. Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononu-clear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Blood 2002;99:111–120.
Blair HC, Zaidi M. Osteoclastic differentiation and function regulated by old and new pathways. Rev Endocr Metab Disord 2006;7:23–32.
Choi SJ, Han JH, Roodman GD. ADAM8: a novel osteoclast stimulating factor. J Bone Miner Res 2001;16:814–822.
Rao H, Lu G, Kajiya H, et al. Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function. J Bone Miner Res 2006;21:1657–1665.
Whyte MP. Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization. Endocr Rev 1994;15:439–461.
McKane WR, Khosla S, Egan KS, et al. Role of calcium intake in modulating age-related increases in parathyroid function and bone resorption. J Clin Endocrinol Metab 1996;81:1699–1703.
Khosla S, Melton LJ, 3rd, Atkinson EJ, et al. Relationship of serum sex steroid levels and bone turnover markers with bone mineral density in men and women: a key role for bioavailable estrogen. J Clin Endocrinol Metab 1998;83:2266–2274.
Eastell R, Simmons PS, Colwell A, et al. Nyctohe-meral changes in bone turnover assessed by serum bone Gla-protein concentration and urinary deoxypyridinoline excretion: effects of growth and ageing. Clin Sci (Lond) 1992;83:375–382.
Duda RJ, Jr., O'Brien JF, Katzmann JA, et al. Concurrent assays of circulating bone Gla-protein and bone alkaline phosphatase: effects of sex, age, and metabolic bone disease. J Clin Endocrinol Metab 1988;66:951–957.
Riggs BL, Wahner HW, Seeman E, et al. Changes in bone mineral density of the proximal femur and spine with aging. Differences between the postmenopausal and senile osteoporosis syndromes. J Clin Invest 1982;70:716–723.
Calvi LM, Adams GB, Weibrecht KW, et al. Osteo-blastic cells regulate the haematopoietic stem cell niche. Nature 2003;425:841–846.
Zhang J, Niu C, Ye L, et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003;425:836–841.
Adams GB, Martin RP, Alley IR, et al. Therapeutic targeting of a stem cell niche. Nat Biotechnol 2007. In Press.
Yin T, Li L. The stem cell niches in bone. J Clin Invest 2006;116:1195–1201.
Kiel MJ, Yilmaz OH, Iwashita T, et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005;121:1109–1121.
Cipolleschi MG, Dello Sbarba P, Olivotto M. The role of hypoxia in the maintenance of hematopoi-etic stem cells. Blood 1993;82:2031–2037.
Cipolleschi MG, D'Ippolito G, Bernabei PA, et al. Severe hypoxia enhances the formation of ery-throid bursts from human cord blood cells and the maintenance of BFU-E in vitro. Exp Hematol 1997;25:1187–1194.
Reyes M, Lund T, Lenvik T, et al. Purification and ex vivo expansion of postnatal human marrow meso-dermal progenitor cells. Blood 2001;98:2615–2625.
D'Ippolito G, Howard GA, Roos BA, et al. Isolation and characterization of marrow-isolated adult multilineage inducible (MIAMI) cells. Exp Hematol 2006;34:1608–1610.
D'Ippolito G, Diabira S, Howard GA, et al. Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci 2004;117:2971–2981.
Breyer A, Estharabadi N, Oki M, et al. Multipotent adult progenitor cell isolation and culture procedures. Exp Hematol 2006;34:1596–1601.
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Schiller, P.C., D'Ippolito, G., Howard, G.A. (2009). Biology of Bone. In: Duque, G., Kiel, D.P. (eds) Osteoporosis in Older Persons. Springer, London. https://doi.org/10.1007/978-1-84628-697-1_1
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