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Age-dependent changes were associated with increases in cortical porosity, non-enzymatic collagen cross-links, and absolute collagen content, as will be discussed below. The cause of the initial damage is hypothesized Mohsin et al. Disruption of the structure of the bone cells that are embedded in mineral, the osteocytes, also can contribute Verborgt et al. For example, in an animal model of early premature aging, the klotho mouse, osteocytes die and their interaction with the matrix is altered Suzuki et al. The extent of this micro-damage increases exponentially with age in humans Schaffler et al.

It is likely that both the inability to repair the cracks Hirano et al. Morphology describes the shapes geometry of bones, in terms of whether they are long bones such as the femur and tibia , short bones such as the bones of the feet and hands , or flat bones such as the calvaria or breast bones. The morphological traits that determine strength are the sizes and the shapes of the bones Jepsen, There are compact areas cortices and spongy areas trabecular found in the ends of all long bones and in the central region of other bones see Fig.

Bones change in shape to facilitate their mechanical functions—being strong enough to withstand large forces and streamlined enough to minimize energy demands Seeman, ; Wang and Seeman, ; Seeman, , Jepsen, Fig.

Long bone geometrical changes with age. Cortical drift in the tibias of human bones with age. Changes in periosteal and endosteal remodeling around the bone are non-uniform throughout life. Note that the AP drift is larger from 2 to 9 yrs, and that the ML drift is greater from 9 to 14 yrs. Right panel adapted from Goldman et al. This can be seen in animal models, as well as in humans. In mice, for example, cortical bone size, trabecular bone volume, and bone strength decline with age after 3 mos corresponding to achievement of peak bone mass in this species Ramanadham et al.

In mature rats from mos of age , the only change reported in bone structure is an increase in the cross-sectional moment of inertia distribution of the bone around the central axis , due to the expansion of the outer diameter periosteal deposition of their bones, with a thinning of the cortical walls endosteal resorption LaMothe et al.

However, in a more recent study, when the trabecular bones in proximal tibias of month-old and 5-month-old rats were compared, mineral density, bone volume fraction, and trabecular number were significantly reduced in the aged rats compared with the younger rats Pietschmann et al. Serum markers of bone formation were also reduced in the older rats.

In bovine samples Nagaraja et al. Human samples, although more difficult to assess in such numbers, show similar patterns of morphological changes with age. In necropsied tibias from males ranging in age from 17 to 46 yrs, significant changes in mechanical properties were correlated with the size slenderness of the tibia in an age-independent fashion, with increased mechanical weakening, and more brittle behavior observed with increasing age Tommasini et al.

The shape of long bones changes during development Fig. Cortical drift occurs when formation is decreased and resorption increased on the endosteal surface, while bone is deposited on the periosteal surface. This leads to an increase in bone diameter and, in the case where endosteal resorption is greater than formation, cortical thinning. Cortical drift occurs rapidly during pre-pubertal growth, levels off after closure of the epiphyseal plate, and increases again in the elderly, often resulting in weaker bone with a wider diameter and significantly thinner cortices.

Cortical drift does not occur uniformly around the bone diameter. Like many of the transformations we will discuss, it is the response to mechanical demands and biological signaling that results in the alteration in bone geometry throughout life Goldman et al. In humans, there is some debate Seeman, as to whether the patterns of resorption and formation are similar in men and women, but this is beyond the scope of this review.

It must be noted, however, that within trabecular bone, the age-related loss in men is predominantly due to thinning of the individual struts, while in women the loss is due to a decrease in connectivity Aaron et al. Thus, in most animals studied, as well as in humans, bone gets stiffer with age, and the cross-sectional area trabecular surface over which mechanical load is distributed decreases. In the healthy individual, bone formation and resorption are in a state of balance. The variations in bone morphology are related to the changes in this balance between bone formation and bone remodeling.

While these changes do not affect all bones equally, the general trends are similar. The reason for these morphological changes is related to genetics, the loading of the bones, and the activity of the cells. There are several types of cells in bone Fig. The majority of the bone cells are of mesenchymal cell origin: Several factors can initiate bone remodeling, which, in a balanced system, begins with resorption by osteoclasts and ends with formation by osteoblasts.

Signals from external sources Wnts and from within the bone osteocyte apoptosis contribute to osteoblast and osteoclast differentiation and activity, a selection of which is shown in this figure. Osteoblastogenesis is regulated by numerous pathways, some of which are illustrated in Fig.

Some of the osteoblasts become small, quiescent bone-lining cells along inactive surfaces. The remaining osteoblasts are surrounded by mineral and extend long processes dendrites , which allow signaling and nutrition to pass from cell to cell through channels in the bone called canaliculi.


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They have a lifespan of 1 to 50 yrs Manolagas and Parfitt, , and they are the cells that are most responsive to mechanical signals Matsuo, ; Rochefort et al. These cells maintain contact with other osteocytes and the surface through gap junctions.

MATERIALS AND METHODS

In fact, the most abundant gap junction protein in bone, connexin 43, is required for bone modeling and remodeling Matsuo, ; Rochefort et al. Theoretically, mechanical loading results in fluid flow within the canalicular-lacunar network, resulting in cell-level shear forces, membrane deformation, and tension in elements connecting osteocytes with the canalicular walls, which results in the release of anti-apoptotic factors.

Osteocyte apoptosis due to disruption of the canalicular network or lack of mechanical stimulus is hypothesized to regulate osteoclast and osteoblast function to induce the remodeling of the local bone tissue Rochefort et al. The other major bone cells, the osteoclasts, are of hematopoietic origin, and they are multinucleated giant cells responsible for removing bone resorption following signals from osteoblasts and osteocytes.

In male rats and in all mice studied to date, osteoclast differentiation is impaired in old as compared with younger animals Cao et al. Another interesting finding is that osteoclasts develop to a greater extent when they encounter older bone in vitro , so that older bone may be resorbed preferentially Henriksen et al. Some of the factors involved in the differentiation of these cells are shown in Fig. These factors and the way they control the development of the skeleton have been reviewed by Provot et al.

All normal cells, including osteoblasts, osteoclasts, and osteocytes, have a limited lifespan, which is controlled by the number of replication cycles and external factors Fig. The Hayflick limit of cell division indicates that cells can undergo only a limited number of divisions, and that the number of such divisions declines with age Martin et al. In the cell nucleus, the lengths of the telomeres at the ends of genes are presumably the determinant of that number.

A telomere is a repetitive length of DNA and associated proteins that provide stability to the ends of chromosomes. With each cell division, telomeres decrease in length due to the inability of the cell to fully replicate this region, and once the telomere length reaches some critical level, cell senescence and apoptosis are initiated Calado, Self-renewing hematopoietic and mesenchymal stem cells have low levels of an enzyme, telomerase, which extends their life cycle significantly compared with that of mature cells.

However, the activity of this enzyme is not indefinite and decreases with age Calado, Damage to telomeres by UV light and oxidative stress may also accelerate telomere shortening, thereby curtailing the lifespan of cells Muller, Factors affecting cell senescence. The inset shows the activity of an enzyme, telomerase, that allows stem cells to extend the length of the telomeres and therefore maintain an almost indefinite capacity for self-renewal.

There are many factors that also contribute to telomere shortening, which may lead to abnormal cell behavior. The accumulation of damaged cells and the inability of a depleting stem cell population to replace these cells with aging may affect tissue function. Adapted from Muller It has been noted in mice null for telomerase-specific genes that the first generation of the knock-out animals had no advanced aging phenotype, presumably due to telomere reserves that retained their chromosome stabilizing functions Blasco et al.

However, mice from the third generation and beyond exhibited systemic impaired organ function, tissue atrophy, and classic age-related disorders such as osteoporosis Lee et al. Werner syndrome in humans results in the premature onset of age-associated diseases with accelerated telomere attrition after puberty Martin, However, Wrn -null mice do not demonstrate any of the classic symptoms of the disease until the knock-out is developed in mice with telomerase-deficient backgrounds, further highlighting the role telomere maintenance in the onset of the premature aging phenotype Chang et al.

Studies in humans have found that telomere length was associated with decreased development of age-associated diseases; the connection between telomere length and longevity is still in question Sahin and DePinho, Cells often show changes in gene expression and activity with age, perhaps due to altered gene mRNA translation. A study by Kennedy and Kaeberlein found that treatments that decrease mRNA translation in animals increase their lifespan. As recently reviewed Wu et al. With age, the amount of bone deposited with each cycle of remodeling decreases Szulc and Seeman, , possibly due to a reduction in the number of cell precursors of osteoblasts, a reduction in the number of stem cells from which these precursors are derived, or a reduction in the lifespan of osteoblasts.

The signals that lead to differentiation of osteoblast precursors decrease with age Lee et al. The number of hematopoietic cells, which are osteoclast precursors, declines with age in non-human primates Lee et al. The net result is a decrease in the amount of bone with age, starting fairly early in life Szulc and Seeman, Few studies have examined whether human bone cell differentiation is age-dependent.

In one study of 80 patients aged yrs, bone marrow stromal cell gene expression was characterized for several markers of differentiation: There was also a slight not significant decrease with increasing age in the osteoblast marker, Runx2. In another study Zhou et al. There was also an age-dependent decrease in marrow stromal cell proliferation and osteoblast differentiation. All of these indicate that cell differentiation and proliferation in human bone is age-dependent.

Apoptosis is a regulatory mechanism in most tissues and plays a role in normal tissue maintenance Carrington, The dysregulation of apoptosis contributes to the imbalance between bone resorption and formation, as well as changes in local tissue mechanical properties Carrington, It has been noted that osteocyte apoptosis increases with tissue age, which contributes to bone weakening independent of BMD through at least two mechanisms: Cell senescence may alter the responses of cells to apoptotic signals see Muller, , for review , although whether this is the case in bone cells has yet to be investigated.

There are multiple pathways that control differentiation and metabolism by the bone cells. These were recently reviewed in detail elsewhere Provot et al. Signaling by a family of genes called wingless-type MMTV integration site Wnt Williams and Insogna, is believed to regulate cellular aging in all cells, although the mechanism through which this occurs has not been completely accepted Brack et al.

A study comparing the expression levels of all genes in the Wnt pathway in bones of different ages, and in cells isolated from young and old bones, found that all Wnt-associated genes were decreased in adult and old mice compared with younger mice, with many being significantly decreased Rauner et al. Of note, transgenic overexpression of one of the Wnt genes Wnt 10b prevented bone mass loss in aged mice, again demonstrating the importance of the Wnt pathway in aging osteoblasts Bennett et al. Studies in an animal model of early senescence, the senescence-accelerated mouse strain P6 SAMP6 , reported that a soluble frizzled related protein, sFRP-4, a protein that increases osteoblast differentiation, is negatively associated with peak bone mass Nakanishi et al.

In the canonical Wnt pathway, Wnt proteins bind to a Frizzled family member transmembrane receptor to initiate the signaling cascade by activating the Disheveled protein Dsh by excessive phosphorylation. Recently, oxidative stress has been suggested to be an additional factor contributing to bone cell aging. The collagen provides the flexibility toughness to the bone structure, which provides resistance to impact loading, and serves as a template for the oriented deposition of mineral crystals. Collagen is secreted from the cell as triple-helical fibrils which self-associate to form larger fibrils and then fibers.

Extensive post-translational modifications hydroxylation, glycation occur before the fibrils associate within the cell. Once extruded from the cell, globular domains that help keep the fibrils soluble in the cell are cleaved. These fibrils are then stabilized and modified extracellularly by the formation of cross-links, based both on reduction of Schiff-bases and aldol condensation products within and between the fibrils, and by the addition of sugars to the collagen fibrils advanced glycation end-products [for review, see Saito and Marumo It is the cross-linking of the collagen fibrils that has the greatest impact on the strength of collagen fibrils.

There are two different categories of collagen cross-links, and they vary differently with age Fig. The cross-links that are formed enzymatically by lysyl hydroxylase and lysyloxidase enzymatic cross-links connect the N- or C-terminus of one collagen molecule to the helical region of another. They then mature, with age, to trivalent pyridinoline PYD and pyrrole PYL cross-links, which connect two terminal regions and a helical region, thereby increasing the stiffness of the collagen Vashishth et al.

Those formed by glycation- or oxidation-induced non-enzymatic processes, advanced glycation end-products AGEs , such as glucosepane and pentosidine, increase in formation as the collagen persists for longer times in the tissue. Limited numbers of non-enzymatic cross-links were found to be structurally related to the morphology of the trabecular bone Banse et al.

In a study of autopsy samples from individuals yrs old, Viguet-Carrin et al. The study also reported an increase in the amount of pyridinoline but not the other cross-links, a finding noted earlier in iliac crest trabecular bone Bailey et al. Age-dependent increases in both pentosidine and lysyl pyridinoline cross-links, but not in hydroxylysyl-pyridinoline cross-links, were found in cortical bone osteons Nyman et al.

This finding led the authors to speculate that the rate of non-enzymatic cross-linking increases with age, while formation of mature enzymatic cross-links may decrease. Such changes would result in decreased strength and toughness of the bone and, ultimately, decreased resistance to crack propagation. Similar findings were also reported for bovine bone Tang et al.

Thus, cross-links based on non-enzymatic glycation occur to a greater extent with age than do enzymatic cross-links.

Chronological formation of collagen cross-links. As collagen matures, reducible cross-links become non-reducible. Advanced glycation end-products AGEs also accumulate between the helical parts of the molecules as the collagen persists in the tissue. Both contribute to the stiffening of the collagen matrix with age, which may contribute to bone tissue properties. Adapted from Leeming et al.

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Formation of cross-links affects both the way the collagen mineralizes and the way micro-damage is propagated. There is also evidence to suggest that the accumulation of AGEs within bone tissue can be removed only by bone resorption, and their presence increases osteoclast activity while decreasing formation by osteoblasts, thereby contributing to the fragility of bone with age Viguet-Carrin et al.

Important features of the bone collagen network include the orientation of the collagen fibrils and the co-alignment of mineral crystals with the fiber axis of the collagen. Collagen orientation increases with tissue age, as is seen in images of osteons obtained by second-harmonic generation microscopy Fig.

Similar to the accumulation of non-enzymatic cross-links, orientation is an age-dependent feature. Collagen orientation of osteons obtained by second-harmonic generation microscopy, where the intensity is proportional to the orientation of the collagen. The orientation increases with tissue age, as is apparent in the outer, older rings of the osteon, which have a brighter intensity. Image from a 6-year-old baboon courtesy of Jayme Burket. There are also age-dependent changes in the expression and relative presence of the non-collagenous proteins Ikeda et al. Such proteins, reviewed in detail elsewhere Zhu et al.

Thus, it is important to note that not only do their distributions change with age, but also the extent of their post-translational modification decreases with increasing age Plantalech et al. Decreased protein production with age was reported by Grynpas et al. This was also shown in cultures of undifferentiated osteoblasts pre-osteoblasts obtained from human trabecular bone from the embryo to age 60 yrs Fedarko et al. The relative distribution of the proteins expressed in culture was not constant, however, indicating that synthesis rates do vary.

Of the proteins studied, small proteoglycan content followed total protein content, but osteonectin, a cell-binding protein, increased in the teen years until puberty, and then steadily decreased thereafter. Similarly, in the klotho knockout mouse, a model of early senescence discussed later in this review, comparison of the distribution of proteins in 6-month-old mutant and wild-type mice of the same sex, background, and age showed increased histochemical staining for dentin matrix protein 1 DMP1 and osteopontin in the mutant mice.

DMP1 is a protein relatively specific for osteocytes; osteopontin is made by a multitude of cells Suzuki et al. Analysis of the data stresses the alteration in protein expression with age in the klotho mice, although the effect of genetic manipulation on the expression of these proteins is not known. This is seen not only in ash weight determination but also in the areal BMD, which is more frequently measured.

Introduction

BMD measurements have been compared by a variety of techniques for a variety of species, demonstrating the increase in mineral content during growth and development, and the decline with later aging Fig. Change in BMD of various species with age. All of the plotted species show an increase in BMD with age.

Male increases faster and female decreases earlier, but both sexes decline with age. Adapted from the following: The mineral found in bone is an analogue of the natural occurring mineral, hydroxyapatite [Ca 10 PO 4 6 OH 2 ]. Bone mineral crystals are nano-crystals of approximate dimensions They also contain a variety of inclusions and substitutions that also vary with age. Prevalent among these substituents is carbonate, which substitutes for hydroxyl and phosphate within the apatite surface and the crystal lattice LeGeros, The most comprehensive report describing how normal human bone mineral changes in composition and crystal size as a function of age was based on x-ray diffraction analyses by Hanschin and Stern , who examined homogenized iliac crest biopsies from patients aged yrs.

They found that the bone mineral crystal size and perfection increased during the first yrs and then decreased thereafter, slightly increasing in the oldest individuals. Maturation of the mineral, as shown by the line-broadening of the x-ray diffraction data, was later also noted in human embryonic vertebrae as a function of age Meneghini et al. While in some of these cases cortical and trabecular bone tissues were examined separately, there was no attempt to distinguish locations in the bone surface vs.

The same homogenized biopsy samples were analyzed by wavelength-dispersive x-ray fluorescence to quantify the carbonate substitution in the hydroxyapatite mineral as a function of age. The increase in carbonate content with age has also been reported based on Raman Tarnowski et al. Analysis of the FTIR data showed that the labile ions decreased with age, to a greater extent in cortical in contrast to trabecular bone, but in general, similar patterns of maturation were seen for both bone types when old and young bones were compared Kuhn et al.

It is important to note that all the x-ray diffraction data and most of the spectroscopic data came from homogenized bone. In the homogenized bulk bone samples of all species examined, parallel to the age-dependent increase in carbonate content, there is a decrease in acid phosphate content Rey et al. Bone apatite is non-stoichiometric, and is both hydroxyl- and calcium-deficient Cho et al.

Whether this is because the crystals are actually perfecting with time which argues against their incorporating carbonate ions or whether, as suggested by in vitro studies showing that osteoclasts preferentially resorb older bone Henriksen et al. To measure actual crystal size, rather than estimating the sizes of particles causing the broadening of the x-ray diffraction pattern of powdered bone mineral, investigators have used transmission electron microscopy and atomic force microscopy.

These studies confirm the sizes suggested by x-ray diffraction, but, to date, perhaps because of the amount of work required for each sample, and the site-to-site variation that occurs within the bony tissues Weiner and Traub, , there are few reports of the age-dependent variation in actual mineral crystal size.

A half-century ago, backscattered electron imaging suggested that there was a gradient of mineral density around osteons Jowsey, ; Barer and Jowsey, , with the youngest bone, closest to the blood vessels Haversian canals , being the least dense. Within cortical bone, a series of almost concentric circles surrounds the blood vessel, cutting a cone through the bone see Fig. With the application of infrared microspectroscopy and microspectroscopic imaging to bone for review, see Carden and Morris, ; Boskey and Pleshko Camacho, , this pattern was extended to mineral crystallinity, carbonate content, and acid phosphate content, as well as to the maturity of the collagen in osteons Fig.

Other studies scanning across cortical bone, or going from the formative surface to the resorptive surface of cortical or trabecular bone in rodents Boskey et al. The wide range of values and large coefficients of variation in the whole bone samples noted above could thus be partly explained by the relative contributions of younger and older bone, or by the relative numbers of new and old osteons examined.

Similar spatial variations were noted by Raman microspectroscopy Akkus et al. Thus, as summarized in the Table , there are parallel tissue-age- and animal-age-dependent changes in bone mineral composition. Heterogeneity of FTIR parameters in baboon osteonal bone. C Crystallinity for each baboon sample as a function of tissue age distance from the osteon center 0. D Collagen cross-link ratio maturity for each baboon sample 0.

Modified from Gourion-Arsiquaud et al. Much of the information available on age-dependent changes in bone has come from animal studies and in particular from models where there is early senescence. Knockout of the klotho gene, which is predominantly expressed in the kidney, leads to premature aging starting at about 4 wks of age, while overexpression extends the mouse lifetime Masuda et al.

Overexpression can rescue the premature aging changes noted in bones and teeth; hence this is an excellent model for studying age-related changes over a short time frame. In this model, rats avoid serious systemic disorders while demonstrating low-turnover bone loss, which may allow investigators to study bone fragility independent of the effects of decreased estrogen levels of OVX rats. To investigate the role of BMPs during bone remodeling, we generated a postnatal osteoblast-specific disruption of Bmpr1a that encodes the type IA receptor for BMPs in mice. Mutant mice were smaller than controls up to 6 months after birth.

Irregular calcification and low bone mass were observed, but there were normal numbers of osteoblasts. The ability of the mutant osteoblasts to form mineralized nodules in culture was severely reduced. Interestingly, bone mass was increased in aged mutant mice due to reduced bone resorption evidenced by reduced bone turnover. The mutant mice lost more bone after ovariectomy likely resulting from decreased osteoblast function which could not overcome ovariectomy-induced bone resorption.

In organ culture of bones from aged mice, ablation of the Bmpr1a gene by adenoviral Cre recombinase abolished the stimulatory effects of BMP4 on the expression of lysosomal enzymes essential for osteoclastic bone resorption. Bone formation is a well characterized process; however, little is known about the molecular mechanisms that regulate bone remodeling, the physiological process through which bone mass is maintained constant.

Remodeling consists of two distinct phases: Differentiated osteoblasts are the only cells responsible for bone formation. Bone formation is thought to be regulated by hormones and by locally acting growth factors 2. They were discovered by their ectopic bone formation activity when implanted locally in soft tissues 5. Over the past decade, the phenotypes of mice with mutations in genes coding for this group of proteins and their receptors uncovered the essential roles for BMPs in wide variety of developmental processes, including skeletal development and patterning 6 - 9.

However, despite its powerful ability to induce ectopic osteogenesis, the essential role of BMPs in bone formation and bone metabolism in the adult skeleton has not been established 10 because of embryonic lethality resulting from mutations of genes encoding the most potent BMPs for bone formation, BMP2 and BMP4, and their receptors 11 - Mice homozygous for this null allele died by embryonic day 8.

Bmpr1a is expressed in most tissues throughout development and after birth 13 , Expression of a dominant-negative form of BMPRIA in a cultured cell line or chick limb buds suggests that signaling through this receptor regulates apoptosis and adipocyte differentiation 15 , Overexpression of a constitutive-active form of BMPRIA in chicken limb buds suggests that signaling through this receptor also can regulate chondrocyte differentiation Because Bmpr1a is expressed in osteoblasts 20 , we designed a postnatal, differentiated osteoblast-specific disruption of Bmpr1a to elucidate the requirement of BMP signaling for bone formation Mice —The generation of Bmpr1a conditional null mice was reported elsewhere Briefly, one loxP site was placed in intron 1, and two others were placed in intron 2 flanking a PGK-neo cassette fn allele.

After germ line transmission, mice heterozygous for the fn allele were mated with CMV-Cre transgenic mice to remove the neo cassette by Cre-dependent recombination in vivo fx allele. Both the fn allele and the fx allele behaved as wild type, indicating that the presence of the PGK-neo cassette or the loxP sites in the Bmpr1a locus did not reduce Bmpr1a activity For the studies reported here we used both the fn and fx alleles.

All experimental procedures were performed according to ethical guidelines approved by local authorities. Genotyping —Mouse genotypes were initially determined by Southern blot described elsewhere Subsequently, each allele was identified by PCR see Fig. Osteoblast-specific disruption of Bmpr1a causes reduced growth.

A , 2-month-old females top and males bottom. Bar , 1 cm. B , body weight of female top and male bottom littermates. The number of animals is shown above each bar. C , PCR detection of Cre-dependent recombination. All mice analyzed here were heterozygous for the fn allele. All of the mice that carried the Og2-Cre transgene showed the deletion of exon2.

After 4 weeks, the cultures were scored for osteoblastic colonies by staining for alkaline phosphatase to assess osteoblast differentiation and von Kossa stain for mineral deposition Histomorphometric analyses were carried out according to standard protocols Mouse Calvarial and Tibia Organ Culture —The calvaria and tibia from aged mice 12 months old on average that were homozygous for fx were excised and cut in half along the sagittal suture calvaria or perpendicular to the long axis tibia. Each portion was placed in a well tissue culture dish containing 0.

Cre-dependent recombination was induced by infection with recombinant adenovirus that expresses Cre recombinase a gift from Dr. Tibia were flushed to remove bone marrow before RNA extraction. Primer sequences were as follows: To direct Cre recombinase in postnatal differentiated osteoblasts, we used transgenic mice that carry a 1. Cre recombinase activity in Og2 -Cre transgenic mouse line.

A , osteoblast-specific Cre recombinase activity was detected using CAT-lacZ reporter mice whole mount view, 1 week after birth. The failure of mineralization is interesting, implying that communication with vessels is necessary for matrix vesicle secretion by hypertrophic chondrocytes in growing bones. In some respects this is similar to rickets, in which vitamin D deficiency leads to elongation of hypomineralized growth plates.

Mineralization and normal growth can be restored if vitamin D levels and mineralization are normalized soon enough to prevent deformation of the bones This is in contrast to the mineralization of the hypertrophic chondrocytes in anlagen before formation of the bone collar, which occurs without vascular invasion Figure 1C.

Anatomy and Physiology Chapter 6 Part B: Bones and Skeletal Tissue Lecture

This difference suggests that there may be a critical size limit for diffusion of necessary factors into the hypertrophic zone. It may be, for example, that oxygen tension in small anlagen is still high enough to meet a mineralization threshold, and that as bones grow, hypoxia induces a circulation-dependent state for mineralization. Alternatively, specific developmental signals could permit mineralization of anlagen to proceed even without vessels in intimate contact with the mineralization front. The treatment blocked vascular invasion much as had the plastic sheets, and it led to a 3-tofold thickening of the growth plate, a halving of limb bone growth in length, and failure to recruit TRAP-positive multinucleated cells to the mineralized cartilage trabeculae of the lower hypertrophic zone.

Also strikingly similar to the study, cessation of treatment allowed recovery of vascular invasion and growth. VEGF production and availability in cartilage has also been found at very early stages. Sox9, the key chondrocyte transcription factor, was found to control the production of VEGF by chondrocytes at the onset of patterning during limb bud formation. In the process of forming primitive anlagen, VEGF secretion by the differentiating chondrocytes recruited the formation of a vascular meshwork surrounding the condensation Additionally, VEGF is secreted into the cartilage matrix by hypertrophic chondrocytes during bone growth and fracture repair, the latter being a process which locally recapitulates endochondral ossification.

Whether the osteoclast itself is pro-angiogenic remains unresolved They also showed that the angiogenic effects were dependent on MMP9. They suggested that those effects were caused by enhanced osteoclast migration due to their expression of MMP9, although it is possible that the collagenase activity of MMP9 was required to release VEGF from the matrix. Osteoclasts, first described in 24 ; see also 25 , have been investigated far more than either chondroclasts or septoclasts.

They are multinucleated cells formed by the fusion of hematopoietic cells of the monocyte lineage 26 — Many of the discoveries concerning their origin, differentiation, and mechanisms of bone removal have been decisively informed by studies of mutations that cause osteopetrosis in human patients and in animal models 29 , These and related investigations have revealed numerous steps required for osteoclast differentiation and bone resorptive activity.

Some mutations in genes needed for osteoclast differentiation lead to so-called osteoclast-poor osteopetrosis, and mutations in genes needed for function lead to osteoclast-rich osteopetrosis. Structurally, the mature, multinucleated osteoclast is a large, highly polarized cell with an extremely convoluted plasma membrane region called the ruffled border.


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The ruffled border is closely apposed to the bone surface and is the site of secretion of HCl and proteases — predominantly cathepsin K and MMP9 — to degrade the mineralized matrix, as well as for endocytosis of the dissolved bone. A sealing zone surrounds the ruffled border and consists largely of a thick ring of filamentous actin. Extremely active vesicle transport is essential for delivery of the acidification pumps and channels to the ruffled border, for exocytosis of proteases, and for endocytosis and subsequent transcytosis and secretion of solubilized bone matrix 34 , Osteoclast differentiation from precursor cells is stimulated by locally high concentrations of first, colony stimulating factor 1 CSF-1; M-CSF , signaling through its receptor c-fms 36 — Besides these growth factors and their signaling pathways, there are essential transcription, signaling, and effector proteins necessary for osteoclast differentiation and activity which have been reviewed in some detail elsewhere 29 , 30 , 46 , Among the transcription factors are c-fos, PU.

There are also many signaling proteins and effector proteins needed by osteoclasts for such tasks as vesicle trafficking, extracellular matrix breakdown, and acidification, including Snx10, c-Src, vATPase, TRAF6, ClC7, cathepsin K, carbonic anhydrase II, and plekhm1. It is generally used in reference to cells that look like osteoclasts but which seem to be digesting cartilage, not bone. A more recent histology text does give chondroclast a single mention One examined the loss of multinucleated cells resorbing mineralized cartilage in growth plates of ovariectomized rats and their restoration by estrogen treatment The other also looked at multinucleated cell formation in cartilaginous bone models, and it proposed that chondroitin sulfate in the extracellular matrix might have an inhibitory effect on their differentiation They showed light, transmission and scanning electron microscopic studies of calcified cartilage resorption.

Crumbling of matrix with mineral crystals penetrating between these foldings and fragmentation of collagen fibrils were also seen. Thus, from their earliest description, chondroclasts were seen as essentially identical to osteoclasts with the exception of their location on mineralized cartilage, as opposed to bone. Underscoring the similarity, if not identity, of osteoclasts and chondroclasts are the observations that: There have been reports of relatively minor differences between osteoclasts and chondroclasts. A comparative analysis of osteoclasts vs.

Alternatively, it is possible, perhaps even likely, that the chondroclasts at the COJ are in quite an early stage of differentiation and activity, which could render the ruffled border, the actin ring, and TRAP exocytosis all immature. Mutations that eliminate osteoclasts, thus causing osteoclast-poor osteopetrosis, also lead to loss of chondroclasts at the chondroosseous junction COJ and to growth plate dysplasias 8.

This provides some evidence that osteoclasts and chondroclasts may be identical, excepting only their substrate. Note that, unlike the longitudinal septa, the transverse septa are generally not mineralized in hypertrophic cartilage. One is the original report describing the septoclast. The other is from our laboratory, in which we examined septoclasts at the chondroosseous junction of the proximal tibia of wild type and osteoclast-deficient osteopetrotic rats with and without osteoclastogenic treatments They also looked at protein synthesis, and they carefully examined TEM images to characterize this unusual cell.

The septoclast is a perivascular cell adjacent to the tip of budding capillaries Figure 2. They are elongated, somewhat similar in appearance to fibroblasts, but are very rich in the lysosomal cysteine protease cathepsin B Figure 3. They are polarized, with a basal nucleus and cytoplasm that extends toward the terminal transverse septum of the growth plate. A ruffled membrane protrudes from the cytoplasm and closely approaches that septum. Cathepsin B is present in septoclasts in multivesicular bodies and dense bodies which are tightly packed in the cytoplasm Septoclasts were markedly more active in protein synthesis than their immediate neighbors.

The cartilage of the adjacent septum in the growth plate appeared partially degraded in the area of the septoclast ruffled membrane. The authors proposed that the septoclast is responsible for removal of the terminal septum, thereby permitting capillary invasion of the growth plate, contact with the hypertrophic cell, and release of contents of the chondrocyte lacuna. B Diagram of A with various cellular and structural constituents numbered: C Serial section to A at higher magnification in which the septoclast cell body and nucleus are visible, but the cytoplasmic extension is out of the plane.

Cathepsin B intensely labels the septoclasts, which are evident all along the chondroosseous junction COJ , between the hypertrophic chondrocyte zone HCZ and the metaphyseal bone MB. See 60 for methodological details. Perivascular cells, or pericytes, are known to include a subset which are mesenchymal stem cells capable of differentiating into osteoblasts, chondrocytes, myocytes, and adipocytes 61 , In addition, other stem cells inhabit the perivascular space, including hematopoietic stem cells and neuro-progenitors Overall, the septoclast, compared with other perivascular cells, remains understudied and poorly understood.

No cellular origin of the septoclast among the known subsets of pericytes has yet been identified. The toothless tl rat mutation is a CSF-1 loss-of-function that causes severe osteoclast-poor osteopetrosis in homozygous mutants 36 , 37 , Similar to other mutations in that class, growth is stunted, and growth plate cartilage is not removed, nor mineralized, nor invaded by vessels, and it accumulates over the post-natal period 8 , 65 — Indeed, deficiency and misdirection of septoclasts was observed, suggesting a relationship between septoclasts, capillary invasion of growth cartilage, and osteoclast differentiation factors Septoclasts remained fewer in number and disorganized despite CSF-1 injections Another rat model, osteopetrosis op — n.

Similar pathology is seen in many mouse osteopetrotic mutations. Most osteoclast-rich osteopetrotic mice have normal-looking growth plates e. The thickened growth plates in those mutants are reminiscent of the growth plates isolated by polyethylene from capillaries 15 or by elimination of active VEGF from the area 18 , as described above. Further, they imply that the chondrocytes themselves are the source of those signals.

The most likely factor is of course VEGF. Indeed, RANKL expression has been detected in hypertrophic chondrocytes in normal and arthritic chondrocytes 77 , Although it has not been directly demonstrated that hypertrophic chondrocytes also supply CSF-1 at the COJ, it seems likely when one takes together the above-noted failure of systemic treatment or rescue, and the ability of hypertrophic cartilage implanted into renal capsules to support osteoclast differentiation Osteoclasts might also play an auxiliary signaling role in that process, perhaps analogous to their role in stimulating bone formation, as has been shown by the increased rates of bone synthesis in osteoclast-rich osteopetrosis compared with the osteoclast poor types reviewed in A mouse or rat with a chondrocyte-specific deletion of CSF-1, or rescue of tl rats or op mice also CSFdeficient with chondrocyte-specific expression are two means to test that idea.

In addition to septoclasts at the COJ, a further potential role for pericytes in cartilage is vascular invasion of non-mineralized articular cartilage. Secondary ossification centers form in epiphyses following the excavation of cartilage canals into the outer surface of epiphyseal cartilage. To accomplish this invasion, the physiological challenge is for capillaries to invade non-mineralized cartilage despite its active defenses against vascularization. In fact, due to its distinctive avascular nature, cartilage was correctly envisioned by Folkman and co-workers as a source of angiogenesis inhibitors to fight cancer progression.

They had succeeded in demonstrating anti-angiogenic properties of cartilage explants by 81 and in isolating the first anti-angiogenic substance from cartilage by It is synthesized by chondrocytes and promotes their proliferation and the synthesis of cartilage matrix components, including aggrecan and type II collagen. It has also been shown to stimulate production of protease inhibitors by chondrocytes 5 , 9 , Mechanisms by which the vascularization of epiphyseal cartilage is regulated are not well understood, although they have potential significance in pathological cartilage loss, for example in osteoarthritis In human development, capillary invasion of non-mineralized joint cartilage occurs in the femur in two phases during mid-gestation, from about week 8 through weeks 15— Interestingly, the canals appear to be cut by small clusters of fibroblastoid mesenchymal cells without any accompanying vessel formation The canals then remain devoid of cells for several weeks before vessels and perivascular tissues eventually form within them.

Thus, this process is fundamentally different from what occurs on the diaphyseal side of the growth plate. In rats, cartilage canal formation in the proximal tibia occurs through approximately the first 3 postnatal weeks, and it was studied in some detail by Lee and co-workers, in particular with respect to the degradative enzymes actively breaking down the cartilage 86 , Endothelial cells were observed in intimate contact with the degrading cartilage surface.

Osterix-driven excision of the IGF1 receptor resulted in mice with a slight delay and reduction in canal formation, suggesting a possible role for IGF1 from osteoblasts in the process The cells causing canal formation appeared to originate as perichondrial cells, but are otherwise not characterized. As in development and growth, these take place in close association with blood or marrow.

It occurs both on bone surfaces and within bone in close proximity either to the vascularized extracellular milieu, to the marrow, or adjacent to vessels in Haversian canals. There is no cartilage intermediary in normal bone turnover. In remodeling of the mature skeleton, there are by definition no chondroclasts because the basic multicellular unit removes bone that does not possess cartilage cores.

Due to the purely osseous matrix being degraded, the catabolic cells at these sites are unequivocally osteoclasts. In fracture repair the cell type name is not so clear-cut, given the comparative disorganization of the callus, with mineralized cartilage and bone matrix often intimately intertwined. Fractures can be repaired by both endochondral and intramembranous mechanisms.

At the site of injury, a hematoma forms and is soon followed by an inflammatory response. This in turn leads either to direct bone deposition in the form of intramembranous ossification, or to the recruitment of periosteal cells which migrate in, proliferate, and adopt a chondrocyte phenotype. The chondrocytes secrete a cartilaginous fracture callus that bridges the bone parts and also occupies a significant volume surrounding the injury site. Unlike in mature cartilage, the matrix fibers are disorganized.

The chondrocytes undergo hypertrophy, switching from type II to type X collagen expression, and mineralize the callus. At various sites throughout the callus, vessels invade and the cartilage is replaced by bone. Over time, the excess bone is removed to restore the normal bone contours. A key role for angiogenesis in fracture repair was also recently shown.