Sequence of ossification in the skeleton of growing and metamorphosing tadpoles of Rana pipiens

The order of ossification of bones in the skeleton of Rana pipiens during larval growth and metamorphosis has been determined from observations on specimens fixed in 70% alcohol and stained with alizarin red S. The axial skeleton ossifies in a generally cephalo-caudal sequence, beginning with the parasphenoid bone at Taylor-Kollros stages IV-IX, followed by vertebrae (V-IX) and then the urostyle (IX-XIV). Exoccipitals (VII-IX), frontoparietals (XI-XII) and prootics (XIII-XVII) are additional cranial bones which successively ossify before metamorphosis. With the onset of metamorphosis at stage XVIII jawbones and rostral bones of the skull ossify in the following succession: premaxilla, maxilla, septomaxilla, nasal, dentary, angular, squamosal, pterygoid, prevomer, mentomeckelian, quadratojugal, palatine, columella, posteromedial process of “hyoid.” The sphenethmoid does not ossify until after metamorphosis. Ossification of limbbones begins with the femur or humerus at stages X-XII and progresses proximo-distally to the phalanges by stages XIII-XV. Carpals, however, do not ossify until stage XXV or after metamorphosis. The ilium of the pelvic girdle begins to ossify at stages X-XII, but the ischium is delayed until stages XX-XXIII. Scapula and coracoid of the pectoral girdle undergo initial ossification at stages XII-XIV, suprascapula and clavicle at stages XIII-XV. The sternum does not begin to ossify until stage XXIV. The possible role of thyroid hormones in stimulating osteogenesis is discussed.     

Collagen heterogeneity within different growth regions of long bones of rachitic and nonrachitic chicks

The different anatomical regions involved in osteogenesis in the chick long bone have been examined for heterogeneities in collagen structure that might relate to the mechanism of ossification. Experimentally induced lathyrism was employed to enhance collagen solubility, and vitamin D deficiency to allow accumulation of osteoid, the precursor of bone matrix. The extractable lathyritic collagens of the cartilaginous and osseous regions of growing long bones from rachitic and non-rachitic chicks were examined for alpha-chain type and amino acid composition. In both groups of animals the growth plate and cartilaginous regions of the epiphysis gave collagen molecules of the constitution [alpha1(II)](3), whereas the ossifying regions contained [alpha1(I)](2) alpha2. The degree of hydroxylation of the lysine moieties was increased by approximately 50% in the alpha1(I)-chain and alpha2-chain of rachitic bone collagen. Since uncalcified osteoid is greatly enriched in rachitic bone, it is concluded that the collagen of osteoid has the configuration [alpha1(I)](2) alpha2, similar to that of bone matrix, but has an elevated hydroxylysine content. The possible relationship of this difference to the mechanism of calcification is discussed.     

Occurrence of osteoblast necroses during ossification of long bone cortices in mouse fetuses

Previous investigations concerned with in vitro osteogenesis and mineralization have revealed some indication of a participation of cell necroses in the course of calcification. These observations were confirmed by in vivo investigations on desmoid ossification in fetal mouse calvariae, where abundant necrotic osteoblasts were found at the mineralization border and in the osteoid. In the present study, ossification of long bone cortices from fetal mice was investigated by use of electron microscopy. Specimens obtained from the collection of the Institute of Anatomy, Free University of Berlin (mouse fetuses, forearm; rat fetuses, forearm) were reinvestigated for control purposes. In all cases, mineralization of osteoid was accompanied by cell necroses. Cell degeneration was characterized by swelling of the endoplasmic reticulum and loss of the plasma membrane resulting in freely distributed vesicular structures. Cell debris was incorporated within the mineral. Initially, cell necroses in the perichondrium occurred in the region surrounding the hypertrophic cartilage and the matrix of which showed spots of endochondral mineralization. Necrotic osteoblasts occurred simultaneously with mineralization of the osteoid. During further ossification of the long bone cortices, the number of necrotic cells increased markedly. In addition to necrotic cells, healthy osteoblasts, osteocytes and perichondral tissue were present, indicating that an artifact can be excluded. The importance of cell necroses in the process of mineralization is as yet unclear. Possibly, the cells act as calcium and/or phosphate stores, which are liberated by cell death to increase the amount of mineral constituents at sites of mineralization.     

Osteogenic PTHs and vascular ossification—Is there a danger for osteoporotics?

Inflammation in vascular (mostly arterial) walls and heart valves triggered by the trans-endothelial influx of LDL particles and the action of subsequently modified (e.g., by oxidation) LDL particles can trigger true bone formation by valvar fibroblasts, by a subpopulation of re-differentiation-competent VSMCs (vascular smooth muscle cells) or by vascular pericytes. Vascular ossification can lead to heart failure and death. Elderly osteoporotic women who need osteogenic drugs to restore their lost skeletal bone are paradoxically prone to vascular ossification-the "calcification paradox." The recent introduction into the clinic of a potently osteogenic parathyroid hormone peptide, Lilly's rhPTH-(1-34)OH (Forteotrade mark), to reverse skeletal bone loss raises the question of whether this and other potently osteogenic PTHs still in clinical trial might also stimulate vascular ossification in such osteoporotic women. Indeed the VSMCs in human and rat atherosclerotic lesions hyperexpress PTHrP and the PTHR1 (or PTH1R) receptor as do maturing osteoblasts. And the evidence indicates that endogenous PTHrP with its NLS (nuclear/nucleolar localization sequence) does stimulate VSMC proliferation (a prime prerequisite for atheroma formation and ossification) via intranuclear targets that inactivate pRb, the inhibitory G1/S checkpoint regulator, by stimulating its hyperphosphorylation. But neither externally added full-length PTHrP nor the NLS-lacking PTHrP-(1-34)OH gets into the VSMC nucleus and instead they inhibit proliferation and calcification by only activating the cell's PTHR1 receptors. No PTH has an NLS and, as expected from the observations on the externally added PTHrPs, hPTH-(1-34)OH inhibits calcification by VSMCs and cannot stimulate vascular ossification in a diabetic mouse model. Encouraging though this may be for osteoporotics with their "calcification paradox," more work is needed to be sure that the skeletally osteogenic PTHs do not promote vascular ossification with its cardiovascular consequences.     

ELECTRON MICROSCOPY OF CARTILAGE AND BONE MATRIX AT THE DISTAL EPIPHYSEAL LINE OF THE FEMUR IN THE NEWBORN INFANT

An examination of the fine structure of cartilage and bone matrix at the distal epiphyseal line of the femur of a newborn infant has revealed the following information. Cartilage matrix is composed of a network of widely spaced fibers without obvious periodic banding. Calcification is first seen about the level of the third chondrocyte capsule distal to the furthest penetration of the capillaries. It starts as a haphazard deposition of crystals which have no obvious relationship to the location of the fibers. The process of calcification is completed before ossification commences but the central zone of matrix remains only partly mineralized. Bone matrix is formed over a bar of calcified cartilage. Fibers, recognizable as collagen, are deposited in a loose network in a narrow zone between the osteoblasts and cartilage. These fibers are 2 to 5 times as wide as the fibers in epiphyseal cartilage. Calcification then begins in the osteoid, crystals being first laid down irregularly on or close to the fibers. As they increase in number, the crystals tend to line up along the fibers and eventually are arranged so that the periodicity of the underlying collagen is emphasized. In such an area the fibers are more tightly packed than when uncalcified. There is no change observed in the calcified cartilage at this level. The extracellular matrices of this epiphyseal cartilage and bone can be distinguished from one another in the electron microscope.     

Growth cartilage calcification and formation of bone trabeculae are late and dissociated events in the endochondral ossification of <Emphasis Type="Italic">Rana catesbeiana</Emphasis>

Endochondral ossification in the growth cartilage of long bones from the bullfrog Rana catesbeiana was examined. In stage-46 tadpoles and 1-year-old animals, the hypertrophic cartilage had a smooth contact with the bone marrow and the matrix showed no calcification or endochondral bone formation. In spite of showing no aspects of calcification, the chondrocytes exhibited alkaline phosphatase activity and some of them died by apoptosis. However, matrix calcification and endochondral ossification were observed in 2-year-old bullfrogs. Calcium deposits appeared as isolated or coalesced spherical structures in the extracellular matrix of hypertrophic cartilage. Bone trabeculae were restricted to the central area at the sites where the hypertrophic cartilage surface was exposed to the bone marrow. Cartilage matrix calcification and the formation of bone trabeculae were not dependent on each other. Osteoclasts were involved in calcified matrix resorption. These results demonstrate that the calcification of hypertrophic cartilage and the deposition of bone trabeculae are late events in R. catesbeiana and do not contribute to the development and growth of long bones in adults. These processes may play a role in reinforcing bony structures as the bullfrog gains weight in adulthood. In addition, the deposition of bone trabeculae is not dependent on cartilage matrix calcification.     

A histochemical comparison of methylene-blue/acid fuchsin with hematoxylin and eosin for differentiating calcification of stromal tissue

Benign and malignant connective tissue tumors consist of a fibrous component that contains varying amounts of one or more types of bone or other calcified tissue. Diagnosis of these connective tissue tumors often poses challenges for pathologists, because it is difficult to differentiate the organic matrix of osteoid from hyalinized stroma. To establish a definitive diagnosis, it sometimes is advantageous to demonstrate histologically by special staining either the type of calcification or the presence or absence of calcification. We compared the efficacy of methylene blue-acid fuchsin (MB-AF) to hematoxylin and eosin (H-E) for connective tissue tumors suspected to contain calcifications and to devise an optimal staining technique for calcification that would be specific, simple, and cost- and time-effective. We examined 50 benign and 45 malignant connective tissue tumors that were suspected to contain calcifications. Sections were stained with H-E and MB-AF and evaluated. MB-AF stained bone pink, which contrasted with blue soft tissue. After MB-AF staining, osteoid was faint pink in a blue stromal background. Osteoid was not visualized in H-E stained sections; it was stained the same shade of pink as stromal tissue. Dystrophic calcification and cementum could be identified equally well using either staining technique, but contrast was better after H-E staining. MB-AF staining of bone was comparable to H-E staining and could be used effectively to stain bone and osteoid. MB-AF is a simple, single step procedure. It also stains cementum blue with faint blue rimming and dystrophic calcification bluish-pink, but it cannot be used as a specific stain for types of calcification other than bone and osteoid.     

Further studies on the effect of tetracycline on the developing skeleton of the chick embryo

At intervals from 2 to 13 days of incubation, 2.5 mg of tetracycline hydrochloride was injected into the yolk sac of chick embryos. The femurs and mandibles were examined histologically at intervals between 10 and 17 days of embryonic age. The abnormalities which result include inhibition of mineralization of the developing osteoid trabeculae, retardation of erosion of the long bone cartilage model, and abnormal calcification of the cartilage matrix in the long bones. The major effects on cartilage maturation appear to occur after cellular hypertrophy has taken place and thus are found only in cartilage models which are being replaced by bone. While tetracycline does cause some retardation in the rate of osteoid deposition, the drug appears to affect intramembraneous bone formation in the mandible and femur primarily by retarding or temporarily inhibiting the rate of mineralization of the osteoid matrix. The results of this study indicate that the effects produced by tetracycline on developing bones are dependent upon the concentration of the drug and not upon the time of administration.     

Expression and localization of plasma transglutaminase factor XIIIA in bone.

Transglutaminases (TGs) are protein crosslinking enzymes involved in cell adhesion and signaling and matrix stabilization and maturation, in many cell types and tissues. We previously described that in addition to transglutaminase 2 (TG2), cultured MC3T3-E1 osteoblasts also express the plasma TG Factor XIIIA (FXIIIA). Here we report on the expression and localization of FXIIIA in bone in vivo and provide confirmatory in vitro data. Immunohistochemistry and in situ hybridization demonstrated that FXIIIA is expressed by osteoblasts and osteocytes in long bones formed by endochondral ossification (femur) and flat bones formed primarily by intramembranous ossification (calvaria and mandible). FXIIIA immunoreactivity was localized to osteoblasts, osteocytes, and the osteoid. RT-PCR analysis revealed FXIIIA expression by both primary osteoblasts and by the MC3T3-E1 osteoblast cell line. Western blot analysis of bone and MC3T3-E1 culture extracts demonstrated that FXIIIA is produced mainly as a small, 37-kDa form. Sequential RT-PCR analysis using overlapping PCR primers spanning the full FXIIIA gene showed that the entire FXIIIA gene is expressed, thus indicating that the 37-kDa FXIIIA is not a splice variant but a product of posttranslational proteolytic processing. Forskolin inhibition of osteoblast differentiation revealed that FXIIIA processing is regulated by the protein kinase A pathway.     

The effect of diet calcium on fluoride toxicity in growing rats

The effect of dietary Ca in response to fluoride (F) treatment was investigated in rats. Rats were maintained on either adequate (0.5%) or high (2.0%) dietary Ca and given for 5 weeks, NaF in drinking water. The minimum NaF levels that inhibited body growth and reduced survival were 300 mg/L with 0.5% diet Ca and 550 mg/L with 2.0% diet Ca. With these toxic F doses, bone histology showed increased formation surfaces and thickened osteoid seams (osteoid index 6-7%). Fluoride doses 30% below toxic levels (200 and 350 mg/L for 0.5 and 2.0% diet Ca, respectively) had no demonstrable effect on bone. Additional diet Ca reduced F absorption from 76 +/- 3 to 47 +/- 3% for 0.5 and 2.0% diet Ca, respectively. Comparable absorbed doses of F produced comparable effects on bone and body growth but, with additional dietary Ca, these effects were observed with 50% lower serum and bone F levels. Variable response to NaF therapy can be produced in rats by alterations in dietary Ca alone. Results indicate that for clinical treatment the NaF dose needs to be adjusted on an individual basis but neither serum nor bone F levels can be used reliably to establish optimal doses.     

The differential response of cortical and trabecular bone to aluminum administration in the rat

Osteomalacia has been noted following in vivo aluminum (Al) loading in the rat by some investigators but not by others. To determine whether the response of bone to Al differs as a function of the skeletal site examined, quantitative histology of cortical and trabecular bone was done in the tibiae from control (C, n = 10), Al-treated (AL, n = 9), nephrectomized control (NX-C, n = 7), and nephrectomized Al-treated (NX-AL, n = 8) rats given 2 mg/day of Al for 4 weeks. Bone Al content was determined by histochemical methods. In cortical bone, osteoid seam width, osteoid volume, and percent osteoid area were similar for all groups. In contrast, for trabecular bone, both forming surface (means +/- SD) (5.2 +/- 3.4 vs 1.8 +/- 1.1%, P less than 0.05) and osteoid volume (1.7 +/- 0.7 vs 1.0 +/- 0.4%, P less than 0.05) increased from control values in AL, although osteoid seam width did not differ. In NX-AL, trabecular forming surface (20.2 +/- 6.7 vs 6.2 +/- 2.4%, P less than 0.01), osteoid area (13.2 +/- 5.7 vs 3.5 +/- 0.8%, P less than 0.01), and osteoid width (18.7 +/- 5.7 vs 9.7 +/- 2.3 micron, P less than 0.01) all were greater than in NX-C. Deposits of Al were undetectable in C and NX-C, were minimal in cortical bone in AL and NX-AL, but were present at 40.5 +/- 11.5 and 71.1 +/ 6.5% of trabecular surfaces in AL and NX-AL, respectively. Osteoid area and osteoid surface each correlated with trabecular bone Al. Thus, (a) osteoid accumulates in trabecular, but not in cortical, bone after 4 weeks of Al loading; (b) the extent of osteoid accumulation correlates with the bone Al content; and (c) the histologic response to Al in cortical and trabecular bone is related to local differences in the uptake of Al into bone.     

A Magnesium Based Phosphate Binder Reduces Vascular Calcification without Affecting Bone in Chronic Renal Failure Rats

The alternative phosphate binder calcium acetate/magnesium carbonate (CaMg) effectively reduces hyperphosphatemia, the most important inducer of vascular calcification, in chronic renal failure (CRF). In this study, the effect of low dose CaMg on vascular calcification and possible effects of CaMg on bone turnover, a persistent clinical controversy, were evaluated in chronic renal failure rats. Adenine-induced CRF rats were treated daily with 185 mg/kg CaMg or vehicle for 5 weeks. The aortic calcium content and area% calcification were measured to evaluate the effect of CaMg. To study the effect of CaMg on bone remodeling, rats underwent 5/6th nephrectomy combined with either a normal phosphorus diet or a high phosphorus diet to differentiate between possible bone effects resulting from either CaMg-induced phosphate deficiency or a direct effect of Mg. Vehicle or CaMg was administered at doses of 185 and 375 mg/kg/day for 8 weeks. Bone histomorphometry was performed. Aortic calcium content was significantly reduced by 185 mg/kg/day CaMg. CaMg ameliorated features of hyperparathyroid bone disease. In CRF rats on a normal phosphorus diet, the highest CaMg dose caused an increase in osteoid area due to phosphate depletion. The high phosphorus diet combined with the highest CaMg dose prevented the phosphate depletion and thus the rise in osteoid area. CaMg had no effect on osteoblast/osteoclast or dynamic bone parameters, and did not alter bone Mg levels. CaMg at doses that reduce vascular calcification did not show any harmful effect on bone turnover.     

Experimental evaluation of ceramic calcium phosphate as a substitute for bone grafts.

Tricalcium phosphate (Synthos) is a bioceramic material which can be carved with a scalpel and wired into place as a bone graft would be. The process of bone replacement of the prosthesis begins with an ingrowth of cellular loose connective tissue, which is replaced later by dense connective tissue. Around the periphery of this dense fibrous connective tissue, osteoid tissue becomes evident and on later specimens this mixture seems to be converted to bone--which at first is in the form of spicules but later takes on the characteristics of lamellar bone (with tricalcium phosphate particles seen within its lacunae). The progressive replacement occurs in a circumferential pattern, but most heavily at the bone-prosthesis interface. Although the periosteum is beneficial, we do not feel that the major source of bone formation is as the soft tissue or subperiosteal area. The replacement of the tricalcium phosphate prosthesis is slower than we originally thought, or than reported by others. We have noted pockets of tricalcium phosphate, incompletely replaced, in dogs up to 18 months after implantation. We believe this may be related to the larger sized prostheses we used (2 x 2 cm blocks) with, therefore, longer distances that the ingrowth and calcification had to traverse.     

Expression of Na(+)/Ca(2+) exchanger isoforms (NCX1 and NCX3) and plasma membrane Ca(2+) ATPase during osteoblast differentiation

The ability to deliver calcium to the osteoid is critical to osteoblast function as a regulator of bone calcification. There are two known transmembrane proteins capable of translocating calcium out of the osteoblast, the Na(+)/Ca(2+) exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). In this study, we reveal the presence of the NCX3 isoform in primary osteoblasts and examine the expression of NCX1, NCX3, and PMCA1 during osteoblast differentiation. The predominant NCX isoform expressed by osteoblasts is NCX3. NCX1 also is expressed, but at low levels. Both NCX isoforms are expressed at nearly static levels throughout differentiation. In contrast, PMCA expression peaks at 8 days of culture, early in osteoblast differentiation, but declines thereafter. Immunocytochemical co-detection of NCX and PMCA reveal that NCX is positioned along surfaces of the osteoblast adjacent to osteoid, while PMCA is localized to plasma membrane sites distal to the osteoid. The expression pattern and spatial distribution of NCX support a role as a regulator of calcium efflux from osteoblasts required for calcification. The expression pattern and spatial distribution of PMCA makes its role in the mineralization process unlikely and suggests a role in calcium homeostasis following signaling events.     

The fate of mechanically induced cartilage in an unloaded environment.

According to mechanobiologic theories, persistent intermittent mechanical stimulation is required to maintain differentiated cartilage. In a rat model for bone repair, we studied the fate of mechanically induced cartilage after unloading. In three groups of rats, regenerating mesenchymal tissue was submitted to different loading conditions in bone chambers. Two groups were immediately killed after loading periods of 3 or 6 weeks (the 3-group and the 6-group). The third group was loaded for 3 weeks and then kept unloaded for another 3 weeks (the (3 + 3)-group). Cartilage was found in all loaded groups. Without loading, cartilage does not appear in this model. In the 3-group there was no clear ongoing endochondral ossification, the 6-group showed ossification in 2 out of 5 cartilage containing specimens, and in the (3 + 3)-group all cartilage was undergoing ossification. These results suggest a tendency of the cartilage to be maintained also under unloaded conditions until it is reached by bone that can replace it through endochondral ossification.Additional measurements showed less amount of new bone in the loaded specimens. In most of the loaded specimens in the 3-group, necrotic bone fragments were seen embedded in the fibrous tissue layer close to the loading piston, indicating that bone tissue had been resorbed due to the hydrostatic compressive load. In some specimens, a continuous cartilage layer covered the end of the specimen and seemed to protect the underlying bone from pressure-induced resorption. We suggest that one of the functions of the cartilage forming in the compressive loaded parts of a bone callus is to protect the surrounding bone callus from pressure-induced fluid flow leading to resorption.     

Silver Staining of Bone Prior to Decalcification for Quantitative Determination of Osteoid in Sections

Slices, 1-2 mm thick, of alcohol-fixed bone are immersed in 2% aqueous AgNO₃ in the dark for 48 hr. After thorough washing in running tap water, the silver phosphate formed at the interface of osteoid and calcified bone is reduced to a black deposit by 5% aqueous sodium hypophosphite containing 0.1 N NaOH, 0.2 ml/100 ml. The blocks are then immersed in 5% aqueous Na₂S₂O₃ and after further washing pass through a routine formic acid decalcification and paraffin wax embedding schedule. Sections cut at 5 μ thickness and counterstained with Van Gieson's picrofuchsin show a clear differentiation between osteoid tissue and the outer limit of calcification in trabecular or cortical bone, thus making them suitable for quantitative studies. The main advantage of the method is the production of intact stained sections without specialised embedding or cutting techniques.     

Chondrocyte apoptosis is not essential for cartilage calcification: evidence from an in vitro avian model

The calcification of cartilage is an essential step in the process of normal bone growth through endochondral ossification. Chondrocyte apoptosis is generally observed prior to the transition of calcified cartilage to bone. There are, however, contradictory reports in the literature as to whether chondrocyte apoptosis is a precursor to cartilage calcification, a co-event, or occurs after calcification. The purpose of this study was to test the hypothesis that chondrocyte apoptosis is not a requirement for initial calcification using a cell culture system that mimics endochondral ossification. Mesenchymal stem cells harvested from Stages 21-23 chick limb buds were plated as micro-mass cultures in the presence of 4 mM inorganic phosphate (mineralizing conditions). The cultures were treated with either an apoptosis inhibitor or stimulator and compared to un-treated controls before the start of calcification on day 7. Inhibition of apoptosis with the caspase inhibitor Z-Val-Ala-Asp (O-Me)-fluoromethylketone (Z-VAD-fmk) caused no decreases in calcification as indicated by radioactive calcium uptake or Fourier transform infrared (FT-IR) analysis of mineral properties. When apoptosis was inhibited, the cultures showed more robust histological features (including more intense staining for proteoglycans, and more intact cells within the nodules as well as along the periphery of the cells as compared to untreated controls), more proliferation as noted by bromo-deoxyuridine (BrdU) labeling, decreases in terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick-end labeling (TUNEL) staining, and fewer apoptotic bodies in electron microscopy. Stimulation of apoptosis with 40-120 nM staurosporine prior to the onset of calcification resulted in inhibition of calcium accretion, with the extent of total calcium uptake significantly decreased, the amount of matrix deposition impaired, and the formation of abnormal mineral crystals. These results indicate that chondrocyte apoptosis is not a pre-requisite for calcification in this culture system.     

Etude Histologique et Microradiographique du Cartilage Hémal de la Vertebre de la Carpe, Cyprinus carpio L. (Pisces, Teleostei, Cyprinidae)

The abdominal vertebrae of the adult carp retain a bulk of cartilage at the basement of the haemapophyses. This cartilage has two opposite directions of differentiation. There is an enchondral ossification of the hypertrophic calcified cartilage in its distal area whereas its proximal area is calcifying without previous hypertrophy. The calcification of this proximal area (hyaline calcified cartilage) is permanent and shows typical rings and waves of Liesegang. The calcification of the cartilage of the hemapophyses is of a globular type. The hyaline calcified cartilage is not a metaplastic bone. Other studies, specially with electron microscope, will allow us to understand the innermost process of the different stages of calcification in the cartilage of the carp.