Lead intoxication alters basal and parathyroid hormone-regulated cellular calcium homeostasis in rat osteosarcoma (ROS 17/2.8) cells

Summary The skeleton is the major reservoir of lead and calcium in humans, and plays an important role in systemic calcium regulation. Lead perturbs normal calcium transport and second messenger function, directly or indirectly, in virtually all cells studies so far. Therefore, we and others have postulated that an early and discrete toxic effect of lead is perturbation of one or more loci within the calcium messenger system. To understand further the role of lead on calcium homeostasis in bone, we undertook this study to characterize calcium homeostasis and the effect of lead on calcium homeostasis in rat osteosarcoma (ROS 17/2.8) cells, which exhibit the osteoblast phenotype. ROS cells were incubated in medium containing ⁴⁵Ca for 20 hours. Monitoring the efflux of ⁴⁵Ca from the cultures for 210 minutes allowed for the determination of kinetic parameters defining steady state calcium homeostasis. Three distinct intracellular kinetic calcium pools characterized ⁴⁵Ca homeostasis. Treatment with either 400 ng parathyroid hormone (PTH)/ml culture medium for 1 hour or 25 M lead for 20 hours increased total cell calcium. Treatment with PTH caused a larger increase of cell calcium in lead-intoxicated cells than either lead intoxication or PTH treatment alone. This increase suggests that lead may perturb normal calcium-mediated PTH responsiveness of the osteoblast. These experiments further establish a kinetic model for the study of calcium homeostasis in osteoblastic bone cells. The studies also advance the hypothesis that lead-induced perturbations of calcium-mediated processes represent and early effect of lead toxicity at the cellular level.     

Response of three murine macrophage populations to particulate debris: Bone resorption in organ cultures

Particulate wear debris from bone cement or prosthetic components can stimulate macrophages to cause bone resorption. We compared the effect of particle composition (titanium and polymethylmethacrylate as inherent components of prosthetic materials or bone cement and polystyrene as a reference material) on the secretion of interleukin-1 and prostaglandin E₂ by periotoneal macrophages and monocyte/macrophage cell lines (P388D₁ and IC-21) and on the bone-resorbing activity of conditioned medium harvested from these particle-challenged macrophages. Titanium particles (1–3 μm) in peritoneal macrophage cultures exhibited significantly enhanced bone-resorbing activity measured as ⁴⁵Ca release, whereas polymethylmethacrylate and polystyrene exhibited this effect to a greater extent in the P388D₁ and IC-21 monocyte/macrophage cultures. Although exogenous prostaglandin E₂ and recombinant human interleukin-1 could significantly increase the ⁴⁵Ca release and indomethacin significantly reduced both the spontaneous calcium efflux and active ⁴⁵Ca release from calvarial bones labeled in vivo, the levels of interleukin-1 and prostaglandin E₂, alone or together, did not always correlate with the bone-resorbing activity of conditioned media. Thus, the actual levels of potent bone-resorbing agents (prostaglandin E₂ and interleukin-1) measured in conditioned tissue culture media did not necessarily reflect the bone-resorbing capability. An important result of this study is that different macrophage populations may respond differently to the same microenvironmental signal, which in our investigation was particulate wear debris of differing composition and size.     

Influence of estrogen and progesterone on matrix-induced endochondral bone formation

Summary The influence of estradiol and progesterone, alone or in combination, on the discrete phases of matrix-induced endochondral bone formation was investigated. Administration of estradiol and progesterone in combination increased mesenchymal cell proliferation, as indicated by [³H] thymidine incorporaton into acid precipitable material. However, ornithine decarboxylase (ODC) activity was significantly suppressed by the combination of estradiol and progesterone. Also, this treatment did not influence the³⁵SO₄ incorporation into proteoglycans on day 7. Mineralization of newly induced bone was quantitated by alkaline phosphatase,⁴⁵Ca incorporation into bone mineral and calcium content, and was found to be significantly increased by progesterone alone and in combination with estradiol in both matrix-induced plaques and tibial metaphysis. These results demonstrated the stimulatory role of progesterone in combination with estradiol in bone formation and mineralization.     

Recombinant bone morphogenetic protein (BMP)-2 regulates costochondral growth plate chondrocytes and induces expression of BMP-2 and BMP-4 in a cell maturation-dependent manner

This study examined the effect of recombinant human bone morphogenetic protein-2 on several parameters of growth, differentiation, and matrix synthesis and on the endogenous production of mRNA of bone morphogenetic proteins 2 and 4 by growth plate chondrocytes in culture. Chondrocytes from resting and growth zones were obtained from rat costochondral cartilage and cultured for 24 or 48 hours in medium containing 0.05-100 ng/ml recombinant human bone morphogenetic protein-2 and 10% fetal bovine serum. Incorporation of [³H]thymidine, cell number, alkaline phosphatase specific activity, incorporation of [³H]proline into collagenase-digestible protein and noncollagenase-digestible protein, and incorporation of [³⁵S]sulfate were assayed as indicators of cell proliferation, differentiation, and extracellular matrix synthesis. mRNA levels T for bone morphogenetic proteins 2 andv4 were determined by Northern blot analysis. Recombinant human bone morphogenetic protein-2 increased the incorporation of [³H]thymidine by quiescent resting-zone and growth-zone cells in a similar manner, whereas it had a differential effect on nonquiescent cultures. At 24 and 48 hours, 12.5-100 ng/ml recombinant human bone morphogenetic protein-2 caused a dose-dependent increase in cell number and DNA synthesis in resting-zone chondrocytes. No effect was seen in growth-zone cell Recombinant human bone morphogenetic protein-2 stimulated alkaline phosphatase specific activity in resting-zone chondrocytes in a bimodal manner, causing significant increases between 0.2 and 0.8 ng/ml and again between 25 and 100 ng/ml. In contrast, alkaline phosphatase specific activity in growth-zone chondrocytes was significantly increased only between 12.5 and 100 ng/ml. Recombinant human bone morphogenetic protein-2 increased the production of both collagenase-digestible protein and noncollagenase-digestible protein by resting-zone and growth-zone cells, but incorporation of [³⁵S]sulfate was unaffected. Administration of recombinant human bone morphogenetic protein-2 also increased incorporation of [³H]uridine in both resting-zone and growth-zone chondrocytes; these cells produced mRNA for bone morphogenetic proteins 2 and 4. Bone morphogenetic protein-2 mRNA levels in both resting-zone and growth-zone chondrocytes increased in the presence of recombinant human bone morphogenetic protein-2; however, bone morphogenetic protein-4 mRNA levels in growth-zone cells decreased under its influence, and those in resting-zone cells were upregulated only with a dose of 10 ng/ml. This indicates that recombinant human bone morphogenetic protein-2 regulates chondrocyte proliferation, differentiation, and matrix production, and the effects are dependent on the stage of cell maturation. Resting-zone chondrocytes were more sensitive, suggesting that they are targeted by bone morphogenetic protein-2 and that this growth factor may have autocrine effects on these cells.     

Sequential differentiation of mesenchymal stem cells in an agarose scaffold promotes a physis‐like zonal alignment of chondrocytes

Chondrocytes of the epiphyseal growth plate (physis) differentiate and mature in defined linear zones. The current study examines the differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) into zonal physeal cartilage. hBMSCs were embedded in an agarose scaffold with only the surface of the scaffold in direct contact with the culture medium. The cells were differentiated using a two‐step system involving the sequential addition of TGFβ followed by BMP2. The resultant samples displayed a heterogenic population of physis‐like collagen type 2 positive cells including proliferating chondrocytes and mature chondrocytes showing hypertrophy, expression of early bone markers and matrix mineralization. Histological analysis revealed a physis‐like linear zonal alignment of chondrocytes in varying stages of differentiation. The less mature chondrocytes were seen at the base of the construct while hypertrophic chondrocytes and matrix mineralization was observed closer to the surface of the construct. The described differentiation protocol using hBMSCs in an agarose scaffold can be used to study the factors and conditions that influence the differentiation, proliferation, maturation, and zonal alignment of physeal chondrocytes. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1753–1759, 2012     

Bone resorption by experimentally induced osteogenic tumor

Summary To search for the mechanisms related to bone resorption by the osteogenic tumor, we obtained a osteogenic tumor with osteolytic capacity in rats by giving³²P. The tumor (HT-3) possessed osteoblastic phenotypes (production of alkaline phosphatase, positive response to parathyroid hormone and calcification of extracellular matrix at the metastatic lesion) and malignant nature (high incidence of pulmonary metastasis). When the HT-3 tumor was transplanted into the rat tibia, numerous acid phosphatase positive multinucleated cells were induced around the transplanted tumor. Primary cell cultures were established by enzymatic digestion. The conditioned medium of the tumor cells caused⁴⁵Ca-release from the femur of the chick embryos. Indomethacin blocked the⁴⁵Ca-release, evoked by the conditioned medium of tumor cells. These data suggest that the humoral osteoclast activating factor released from HT-3 tumor was prostaglandin like character.     

Osteoblastic cells from rat long bone. I. Characterization of their differentiation in culture

We here report the characterization for osteogenic markers alkaline phosphate (AP), osteocalcin, and mineral deposition of osteoblast cultures derived from cells migrating out from seven-day-old rat tibia fragments. The cells outgrown from bone fragments responded to stimulation with PTH with cAMP increase. We show in these cultures a high level of biosynthesis of type III collagen and osteonectin, and report the stimulatory effect that conditioned serum-free media (CM) from these cultures exert on the migration of endothelial cells (EA by 926). The cultures were kept for the initial seven days in a Coon's modified F12 medium, then were switched to a medium containing ascorbic acid and β-glycerophosphate (BGP), and cultured for another 41 days. They showed constitutive and timely restricted osteoblast markers and mineralization. Maximal AP activity occurred in concomitance with cell doubling, then fell to low levels by the time cultures were stationary. ⁴⁵Ca incorporation in the monolayer increased after four weeks of culture, in concomitance with the appearance of unmineralized nodules, and remained high throughout the phase of mineral deposition. Biosynthesis of collagens type I, type III, and type V was detected at all times; secreted newly synthesized collagens decreased overall, relative to total secreted newly synthesized proteins, and on a per cell basis, with progression of the culture, while the ratio of collagen type III/collagen type I increased. Osteonectin was detected by immunohistochemistry and high amounts of osteonectin were synthesized constitutively. Osteocalcin was detected on virtually all cells tested at 21 and 28 days. A preliminary step in neoangiogenesis is the migration of endothelial cells. CMs collected from osteoblast cultures at different times were potent inducers of endothelial cell migration and acted as such in a dose dependent fashion.     

Age-related decline in chondrocyte response to insulin-like growth factor-I: The role of growth factor binding proteins

The synthetic activity of chondrocytes in articular cartilage declines with age, possibly as a result of decreased sensitivity to anabolic growth factors such as insulin-like growth factor-I. The sensitivity of these cells to insulin-like growth factor-I is regulated, in pan, by the synthesis of insulin-like growth factor-I binding proteins. We hypothesized that, as cartilage ages, an increase in the expression of these binding proteins suppresses the synthetic response of chondrocytes to insulin-like growth factor-I, To test this hypothesis, we measured proteoglycan synthesis (incorporation of [³⁵S]sulfate per cell) in alginate cultures of chondrocytes from the articular cartilage of 1,3, 12, and 24-month-old rats. A dose-response to insulin-like growth factor-I was determined for cells from each age group; incorporation of [³⁵S]sulfate per cell declined with age, regardless of the dose. The sharpest decline was found between cells from the 1 and 3-month-old groups. Using the Western ligand b ot technique, we then compared the expression of insulin-like growth factor-1 binding protein in chondrocytes from the 1 and 3-month-old rats and found that it was increased in the cells from the older animals. Recombinant insulin-like growth factor-3, when added to the cell cultures of the 1-month-old rats, inhibited incorporation of [³⁵S]sulfate and blocked responses to insulin-like growth factor-I. These findings suggest that the age-related decline in the synthetic response of chondrocytes to insulin-like growth factor-I results, at least in part, from increased expression of insulin-like growth factor binding protein.     

Cytokine profiles in conditioned media from cultured human intervertebral disc tissue

Background Cytokines released from intervertebral discs cultured in vitro have not been profiled, and the effect of these cytokines on human bone marrow stem cells is yet to be studied.

Materials and methods Intervertebral discs from 14 patients who had undergone spinal fusion surgery were cultured separately in vitro. Conditioned media were collected after 48 and 96 h of culture in serum-free Minimum Essential Medium (MEM). Profiling of the cytokines was conducted using pooled media. Conditioned medium from each patient was also tested in human bone marrow stem cell culture, and incorporation of alkaline phosphatase and ³H-thymidine incorporation was evaluated.

Results Of the 18 cytokines screened, 12 were found to be positive, but only eotaxin, IP-10, Rantes IL-6 and IL-8 seemed to be present at high levels. There was a close correlation between IL-6 and IL-8 levels in the medium (R=0.90, p < 0.001). When the conditioned media were added to human bone marrow stem cell cultures, cellular proliferation was stimulated (p=0.02), but alkaline phosphatase activity remained unchanged. Cellular proliferation correlated negatively with IL-6 levels (R=-0.44, p=0.04).

Interpretation Intervertebral discs secrete certain cytokines into the medium when cultured in vitro, and conditioned media from cultured intervertebral discs stimulate proliferation of bone marrow stem cells.     

Effect of 1,25(OH)2D3 and 24,25(OH)2D3 on calcium ion fluxes in costochondral chondrocyte cultures

Summary Vitamin D₃ metabolites have been shown to affect proliferation, differentiation, and maturation of cartilage cells. Previous studies have shown that growth zone chondrocytes respond primarily to 1,25(OH)₂D₃ whereas resting zone chondrocytes respond primarily to 24,25(OH)₂D₃. To examine the role of calcium in the mechanism of hormone action, this study examined the effects of the Ca ionophore A23187, 1,25(OH)₂D₃, and 24,25(OH)₂D₃ on Ca influx and efflux in growth zone chondrocytes and resting zone chondrocytes derived from the costochondral junction of 125 g rats. Influex was measured as incorporation of⁴⁵Ca. Efflux was measured as release of⁴⁵Ca from prelabeled cultures into fresh media. The pattern of⁴⁵Ca influx in unstimulated (control) cells over the incubation period was different in the two chondrocyte populations, whereas the pattern of efflux was comparable. A23187 induced a rapid influx of⁴⁵Ca in both types of chondrocytes which peaked by 3 minutes and was over by 6 minutes. Influx was greatest in the growth zone chondrocytes. Addition of 10⁻⁸–10⁻⁹ M 1,25(OH)₂D₃ to growth zone chondrocyte cultures results in a dose-dependent increase in⁴⁵Ca influx after 15 minutes. Efflux was stimulated by these concentrations of hormone throughout the incubation period. Addition of 10⁻⁶–10⁻⁷ M 24,25(OH)₂D₃ to resting zone chondrocytes resulted in an inhibition in ion efflux between 1 and 6 minutes, with no effect on influx during this period. Efflux returned to control values between 6 and 15 minutes.⁴⁵Ca influx was inhibited by these concentrations of hormone from 15 to 30 minutes. These studies demonstrate that changes in Ca influx and efflux are metabolite specific and may be a mechanism by which vitamin D metabolites directly regulated chondrocytes in culture.     

Regeneration after physeal distraction of the radius in sheep

In 18 sheep, gradual physeal distraction of the distal radius was performed by external fixation. Separation of the physis from the metaphysis occurred in all the sheep. Prior to bone formation, collagen fibers were organized according to the direction of the distraction, and lamellar bone was formed if the collagen bundles were lineally organized. In the area where the collagen was disorganized, and also near the periosteum, woven bone was first formed, which was then remodeled into lamellar bone. In some sheep separate groups of chondrocytes were isolated in the metaphyseal area after the distraction. In 2 animals a double physis was formed in the metaphyseal area. This was obviously due to the separation of proliferative chondrocytes in the metaphysis as a result of distraction. After consolidation of the distraction area, this zone of chondrocytes, located in the metaphysis, was still producing new bone 20 weeks postoperatively.     

Role of endogenous zinc in the enhancement of bone protein synthesis associated with bone growth of newborn rats

The role of endogenous zinc in protein synthesis in the bone tissues of newborn rats was investigated in the present study. Femoral–diaphyseal and metaphyseal tissues were obtained at 1, 7, 14, 21, and 28 days after birth. Many protein molecules were found to be present in the diaphyseal and metaphyseal tissues using sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. Bone protein synthesis activity was enhanced by increasing age, and reached a plateau 21 days after birth. Protein synthesis in the diaphyseal and metaphyseal tissues obtained from 7- or 14-day-old rats was significantly decreased by the addition of dipicolinate (10⁻³ M), a chelator of zinc ion, into the reaction mixture, while it was significantly enhanced by zinc sulfate (10⁻⁴ M). When the diaphyseal and metaphyseal tissues obtained from 7- or 14-day-old rats were cultured for 48 h in a medium containing dipicolinate (10⁻³ M), bone protein synthesis was significantly reduced. This decrease was blocked completely by culture with the addition of zinc (10⁻⁴ M). Culture with zinc (10⁻⁵ and 10⁻⁴ M) alone had a stimulatory effect on the bone protein synthesis. Zinc (10⁻⁴ M)-induced increases in bone protein synthesis were completely blocked by culture with cycloheximide (10⁻⁶ M) or actinomycin D (10⁻⁷ M). The present study suggests that bone protein synthesis is enhanced with increasing age of newborn rats, and that endogenous zinc in bone tissues has a stimulatory role in the enhancement of protein synthesis with bone growth. Received: April 28, 2000 / Accepted: July 11, 2000     

Kinetics of β-glycerophosphate-induced endochondral mineralization In vitro. Calcium accumulation,alkaline phosphatase activity,and effects of levamisole

Summary Isolated mesenchymal limb bud cells from day-12 mouse embryos grown at high density in organoid culture at the medium/air interphase differentiate into chondrocytes and form cartilage nodules. Upon addition of -glycerophosphate (-GP), cartilage undergoes endochondral mineralization. This -GP-induced mineralization was investigated by measuring the calcium content in the cultures and the activity of alkaline phosphatase (AP) in the cell mass and the medium. Calcium incorporation depended on the amount of -GP added. After continuous treatment, mineralization began on day 8 of the culture period and increased linearly until day 15. In long-term cultures, periodical treatment for 6 days caused an increase in mineralization the older the cultures were, but the slope of increase was proportionately less steep. Treatment at the latest period on days 19–24 resulted in a markedly reduced mineralization. After short-term treatment (48 hours), mineralization increased also the older the cultures were and proceeded during further cultivation in -GP-free medium. This kinetic behavior indicates a dependency of mineralization on cartilage maturation in this in vitro system. AP activity increased enormously and nearly logarithmically in the cell mass in -GP-free medium, whereas -GP treatment inhibited this drastic increase. In the medium, considerable activities of AP were also measurable from day 10 onward. It increased in -GP-free medium up to day 14, but was diminished after mineralization had been induced. Levamisole inhibited AP activity dose dependently when added directly to the enzyme-containing medium (100% inhibition at 10^-3 M). Added to the cultures from day 7 to 14, it partially inhibited AP activity and mineralization at 5×10^-5 M; mineralization was totally inhibited at 10^-3 M, but AP activity was still present. This high concentration was cytotoxic, as revealed ultrastructurally and by GAG estimation. This in vitro system comprises cartilage development and maturation, -GP-inducible endochondral mineralization, and final degenerative changes; it may be an appropriate model for investigations on endochondral mineralization.     

Role of tumor necrosis factor alpha in particulate-induced bone resorption

The purpose of this study was to determine the role of tumor necrosis factor alpha in bone resorption secondary to mediator release from macrophages exposed to cement particles. The J774 mouse macrophage cell line was exposed to polymethylmethacrylate particles for 24 hours and the resulting conditioned medium was analyzed for prostaglandin E₂, tumor necrosis factor alpha, interleukin-1 alpha and beta, and the ability to stimulate release of prostaglandin E₂ and ⁴⁵Ca from radiolabeled mouse calvaria. Macrophage exposure to polymethylmethacrylate particles led to a 9-fold increase in release of tumor necrosis factor alpha (p < 0.01), but did not lead to a significant increase in release of prostaglandin E₂, interleukin-1 alpha, or interleukin-1 beta when compared to unexposed cells. Exposure of the macrophages to polymethylmethacrylate particles over a time course from 30 minutes to 96 hours led to an increase in the release of tumor necrosis factor alpha that was initially detected at 30 minutes and was maximum at 48 hours. Incubation of the macrophage-polymethylmethacrylate conditioned medium with rat calvaria significantly increased the release of ⁴⁵Ca and prostaglandin E₂ from the bone. To study the role of release of tumor necrosis factor alpha in bone resorption, the macrophage-polymethylmethacrylate conditioned medium was then preincubated with anti-tumor necrosis factor alpha antibody prior to exposure of the conditioned medium to the calvaria. This preincubation was successful in significantly inhibiting ⁴⁵Ca release by calvaria (p < 0.01) to levels that were not significantly different from the levels of release by unexposed calvaria. Tumor necrosis factor alpha appears to play a critical role in initiating particulate-induced bone resorption. Exposure of macrophages to polymethylmethacrylate particles leads to a significant release of tumor necrosis factor alpha in a time-dependent fashion. This macrophage-polymethylmethacrylate conditioned medium stimulated release of prostaglandin E₂ and bone resorption in bone organ culture. The addition of anti-tumor necrosis factor alpha antibody to this in vitro system inhibited the bone resorption stimulated by the macrophage-polymethylmethacrylate conditioned medium and partially suppressed the production of prostaglandin E₂. The sequence of events in this model for particulate-induced bone resorption appears to be initiated by the production of tumor necrosis factor alpha by the macrophage, followed by production of prostaglandin E₂ by cells in bone, and then by bone resorption.     

Effects of polyethylene on macrophages

A system was developed to expose macrophages to polyethylene in vitro. Exposure of macrophages to these particles in isolation led to the release of tumor necrosis factor alpha and prostaglandin E₂. Exposure of macrophages in co-culture with osteoblasts to polyethylene particles increased the release of prostaglandin E₂ and also led to the release of interleukin-6. Incubation of radiolabelled calvariae with conditioned medium from macrophages exposed to polyethylene particles alone or to particles in co-culture with osteoblasts led to bone resorption reflected by release of ⁴⁵Ca. Incubation with pamidronate was effective in inhibiting resorption stimulated by conditioned medium from macrophages exposed to these particles alone or in co-culture with osteoblasts. This demonstrates that pamidronate, or other bisphosphonates, may be effective in inhibiting bone resorption at the implant/bone interface in association with the macrophage ressponse to particulate polyethylene. Further investigation into the possible use of pamidronate or other bisphosphonates in the treatment of aseptic loosening is warranted.     

Alternation in bone components with increasing age of newborn rats: role of zinc in bone growth

The effect of zinc on bone growth in newborn rats supplied with lactation by maternal rats was investigated. Newborn rats were killed between 1 and 35 days after birth. Increasing age caused a significant increase in zinc content, calcium content, and alkaline phosphatase activity in the femoral-diaphyseal and metaphyseal tissues, while the bone deoxyribonucleic acid (DNA) content was significantly decreased because of elevation of mineral content. Oral administration of zinc sulfate (2.0 mg/100 g body weight; four times at 24-h intervals) to maternal rats from 1 day after birth induced a significant increase in zinc, alkaline phosphatase activity, DNA, and calcium content in the femoral-diaphyseal and metaphyseal tissues of newborn rats compared with those 7 or 14 days old. The results indicate that the increase in bone components results from lactation with zinc-containing milk of maternal rats. The femoral-metaphyseal tissues of newborn rats obtained at 7 days after birth were cultured for 24 h in a medium containing either vehicle or zinc sulfate (10⁻⁶ to 10⁻⁴ M) in vitro. Bone alkaline phosphatase activity and calcium and DNA content were significantly increased by zinc addition. These increases were completely prevented by the presence of dipicolinate (10⁻³ M), a chelator of zinc ion, or cycloheximide (10⁻⁶ M), an inhibitor of protein synthesis. The present study suggests that zinc plays a role in the development of bone growth in newborn rats. Received: December 1, 1999 / Accepted: February 8, 2000     

Bone growth in the neonatal rat

Long bones and mandibles from neonatal rats were studied to characterize the mechanisms regulating their growth and to describe the patterns of bone mineral acquisition. Mineral uptake was determined by measuring the rates of incorporation of radioactive calcium (⁴⁵Ca) into three metabolic pools within, or intimately surrounding, the bone. The incorporation into the pool of protein-bound calcium was low throughout the neonatal period, accounting for no more than 1% of the total calcium uptake during this period. The ionized calcium concentration, which was low throughout, increased at the time when formation of the mineral phase was found to be high. The largest fraction of the mineral, the complexed calcium, showed a gradually increasing rate of incorporation, lasting approximately until the age of weaning. The two bony tissues differed somewhat in the slopes at which the incorporation rates changed. From these studies of bone growth, it appears that the process of bone mineralization starts around the time of birth, gradually gains intensity and is maximal between approximately 10 and 18 days. It is speculated that the ionized calcium fraction may be the precursor of mineral formation.     

Regeneration after physeal distraction of the radius in sheep

In 18 sheep, gradual physeal distraction of the distal radius was performed by external fixation. Separation of the physis from the metaphysis occurred in all the sheep. Prior to bone formation, collagen fibers were organized according to the direction of the distraction, and lamellar bone was formed if the collagen bundles were lineally organized. In the area where the collagen was disorganized, and also near the periosteum, woven bone was first formed, which was then remodeled into lamellar bone. In some sheep separate groups of chondrocy tes were isolated in the metaphyseal area after the distraction. In 2 animals a double physis was formed in the metaphyseal area. This was obviously due to the separation of proliferative chondrocytes in the metaphysis as a result of distraction. After consolidation of the distraction area, this zone of chondrocytes, located in the metaphysis, was still producing new bone 20 weeks postoperatively.     

Influence of estrogens on bone resorption in organ culture

Estradiol benzoate, ethinyl estradiol, and human calcitonin were compared for their ability to inhibit the spontaneous or parathyroid extract-induced bone resorption in organ cultures of 19-day fetal rat fibulae. The criteria used for the assessment of bone resorption were: the release of⁴⁵Ca from paired prelabeled bone rudiments into the culture medium, the dry weight, and the number of osteoclasts per bone. Estradiol benzoate at concentrations of 1.3×10^–5 to 2.6×10^–4 M had no effect on the release of⁴⁵Ca from fetal fibulae or on the dry weight. Histologically, treated bones were well preserved and resembled the controls. Parathyroid extract (PTE) alone caused extensive bone resorption with numerous osteoclasts and enhanced⁴⁵Ca release without weight gain. The addition of estradiol benzoate to the culture medium did not prevent the resorptive action of PTE. Ethinyl estradiol alone provoked a dose-related inhibition of⁴⁵Ca release at concentrations of 3.4×10^–5 to 1.7×10^–4 M. However, this inhibition was accompanied by a decrease in bone dry weight and by various degrees of cellular damage. The same phenomenon was observed in PTE-treated fibulae. Human calcitonin, on the contrary, inhibited the release of⁴⁵Ca by decreasing the number of osteoclasts while the weight of the fibulae increased. The inhibitory action of ethinyl estradiol appears to be caused by a toxic effect on bone cells. It is concluded that estrogens have no direct physiological effect on bone resorption in vitro.