Changes in bone volume after irradiation with carbon ions

The effects of carbon ions on bone volume were studied by histological and morphometrical methods. Anesthetized rats irradiated with three different single doses (15, 22.5, or 30 Gy) of either carbon ions or gamma rays were sacrificed (5 rats/group) at 10 time points together with non-irradiated controls. Carbon ion-induced bone responses were qualitatively and quantitatively different from those induced by gamma irradiation at the same dose level. Irradiation with carbon ions resulted in a dose-dependent increase in bone volume, while gamma irradiation induced loss of bone volume, which was found to be independent of dose in the range studied over the same time course. After irradiation with carbon ions, bones showed thickening of the trabeculae, and the bone marrow became fibrous with fewer vessels. Carbon ion irradiation showed a greater effect on the bone-absorbing capability of osteoclasts than gamma irradiation. These observations suggest that irradiation with carbon ions may produce differential modulation of radiation-induced growth factor expression.     

Effects of 17 β-estradiol on the expression of interstitial collagenases-8 and −13 (MMP-8 and MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in ovariectomized rat osteoblastic cells

Estrogen plays an important role in maintaining normal bone metabolism via the direct or indirect regulation of bone cells. Osteoblastic cells, as the target cells of estrogen, can secrete multiple matrix metalloproteinases (MMPs) that participate in bone remodeling. It has been demonstrated that bone loss induced by estrogen deficiency is closely related to the abnormal expression of multiple MMPs in osteoblastic cells. However, the regulating action of estrogen on the expression of interstitial collagenases MMP-8 and MMP-13 in osteoblastic cells in vivo remains unclear. We used an ovariectomized osteoporotic rat model to analyze the changes in the histomorphometric parameters of bone after and without treatment with 17-estradiol (E₂); We also used immunohistochemistry and in situ hybridization to observe changes in the expression of mRNA and the proteins MMP-8, MMP-13 and TIMP-1 in osteoblastic cells in rat proximal tibia. In this study, we found that in the ovariectomized rat the expression of MMP-13 mRNA and protein increased markedly, whereas the expression of MMP-8 and TIMP-1 mRNA and protein did not change significantly. Our analysis showed that the expression of MMP-13 protein was correlated positively to bone trabecular separation, osteoid surface area, and negatively to trabecular numbers and the percentage of trabecula bone volume/total tissue volume. Our results suggest that MMP-13 plays an important role in estrogen deficiency-induced bone loss, while estrogen can inhibit bone resorption and reduce bone turnover rate by down-regulating the expression of MMP-13 in osteoblastic cells.     

Impaired bone fracture healing in matrix metalloproteinase-13 deficient mice

Vascular and cellular invasion into the cartilage is a critical step in the fracture healing. Matrix metalloproteinase-13 (MMP-13) is a member of the zinc-dependent endopeptidase family and plays an important role in remodeling of extracellular matrix. Therefore we investigated the possible involvement of MMP-13 in a murine model of stabilized bone fracture healing. Repair of the fracture in MMP-13 deficient (MMP-13(-/-)) mice was significantly delayed and characterized by a retarded cartilage resorption in the fracture callus. Immunohistochemistry indicated severe defects in vascular penetration and chondroclast recruitment to the fracture callus in MMP-13(-/-) mice. Consistent with the observations, the chondrocyte pellets cultured from the MMP13(-/-) mice exhibited diminished angiogenic activities when the pellets were co-cultured with endothelial cells. These results suggest that MMP-13 is crucial to the process of angiogenesis during healing of fracture, especially in the cartilage resorption process.     

Contributions of matrix metalloproteinases toward Meckel’s cartilage resorption in mice: immunohistochemical studies,including comparisons with developing endochondral bones

The middle portion of Meckel’s cartilage (one of four portions that disappear with unique fate) degrades via hypertrophy and the cell death of chondrocytes and via the resorption of cartilage by chondroclasts. We have examined the immunolocalization of matrix metalloproteinase-2 (MMP-2), MMP-9, MMP-13, and MMP-14 (members of the MMP activation cascade) and galectin-3 (an endogenous substrate for MMP-9 and an anti-apoptotic factor) during resorption of Meckel’s cartilage in embryonic mice and have compared the results with those of developing endochondral bones in hind limbs. MMP immunoreactivity, except for MMP-2, is present in nearly all chondrocytes in the middle portion of Meckel’s cartilage. On embryonic day 15 (E15), faint MMP-2-immunoreactive and intense MMP-13-immunoreactive signals occur in the periosteal bone matrix deposited by periosteal osteoblasts on the lateral surface, whereas MMP-9 and MMP-14 are immunolocalized in the peripheral chondrocytes of Meckel’s cartilage. The activation cascade of MMPs by face-to-face cross-talk between cells may thus contribute to the initiation of Meckel’s cartilage degradation. On E16, immunopositive signaling for MMP-13 is detectable in the ruffled border of chondroclasts at the resorption front, whereas immunostaining for galectin-3 is present at all stages of chondrocyte differentiation, especially in hypertrophic chondrocytes adjacent to chondroclasts. Galectin-3-positive hypertrophic chondrocytes may therefore coordinate the resorption of calcified cartilage through cell-to-cell contact with chondroclasts. In metatarsal specimens from E16, MMPs are detected in osteoblasts, young osteocytes, and the bone matrix of the periosteal envelope, whereas galectin-3 immunoreactivity is intense in young periosteal osteocytes. In addition, intense MMP-9 and MMP-14 immunostaining has been preferentially found in pre-hypertrophic chondrocytes, although galectin-3 immunoreactivity markedly decreases in hypertrophic chondrocytes. These results indicate that the degradation of Meckel’s cartilage involves an activation cascade of MMPs that differs from that in endochondral bone formation.     

MMP-13 is induced during chondrocyte hypertrophy

During development, mRNA for matrix metalloproteinase-13 (MMP-13) is found associated with cartilage undergoing hypertrophy, suggesting that this collagenase plays a role in cell enlargement and/or cartilage calcification. Using chondrocytes from prehypertrophic cartilage of chick embryo sternae, we have examined the relationship between MMP-13 expression and the transition to hypertrophy. When hypertrophy was induced by serum-free culture with ascorbate and bone morphogenetic protein-2 (BMP-2), MMP-13 mRNA levels paralleled those for type X collagen. Chondrocytes from the caudal, nonhypertrophying portion of chick sternae expressed neither type X collagen nor MMP-13, confirming that MMP-13 mRNA is a marker for hypertrophy. Zymography with conditioned medium yielded a proteinase band at 59 kDa, which was absent in nonhypertrophic chondrocytes. A polyclonal antibody raised against chick MMP-13 reacted with the 59-kDa protein, confirming that it is MMP-13. Although mRNA for MMP-13 peaked at days 4-5 of culture, only low levels of MMP-13 activity were present, and the activity increased gradually in parallel with later increases in MMP-2. These results suggest that MMP-13 is activated by MMP-2 during chondrocyte maturation, and that the combination of both proteinases is required to prepare cartilage matrix for subsequent calcification, before endochondral ossification.     

Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.     

Microdistribution and local dosimetry of 226Ra in trabecular bone of the beagle

Sections of lumbar vertebral bodies of young adult beagle dogs have been analyzed autoradiographically to characterize and quantify the local distribution of 226Ra by means of a scanning microscope photometer. The animals received a single injection of 355 kBq/kg body weight and were serially sacrificed at 5 to 1381 days postinjection. Hotspot concentrations decreased from about 51 kBq/g bone at 5 days to 20 kBq/g at 1381 days postinjection. The diffuse concentration changed from 8.3 to 1.9 kBq/g. The mean 226Ra concentration in the trabecular areas scanned was initially higher and at the end of the observation period lower than the average calculated for the whole lumbar vertebral column. Density and area of, and fraction of bone activity in, hotspots virtually remained constant. With time hotspots tended to become translocated into bone volume. Mean dose rates to lining cells from both hotspots and diffuse labels decreased from about 210 mGy/d at early postinjection times to 105 mGy/d. This corresponds to 2.5 to 1.1 times the average skeletal dose rate. A discussion of the level of irradiation in terms of hit frequencies shows that osteoblasts in the initial phase of hotspot formation receive about 60 hits to their nucleus for the duration of bone formation. After about 6 months, however, the 226Ra concentration in new bone and the corresponding hit frequency appears to be low enough that interference with bone formation is unlikely. Morphometric measurements showed that abnormal bone accretion and thickening of trabeculae occurred. This was interpreted as an imbalance between bone formation and resorption. Both formation and resorption seem to be substantially lowered compared to control animals.     

Changes in osteoclasts after irradiation with carbon ion particles

We examined the effects of radiation on decreases in osteoclast numbers after regional irradiation of rats with carbon ions and gamma rays. Male Wistar rats were subjected to hind-leg irradiation with carbon ions (290 MeV/u) or gamma rays at doses of 15, 22.5, or 30 Gy. The effects of carbon ions and gamma rays on osteoclasts were studied using histologic and morphometric methods. At doses of 15 Gy and 22.5 Gy, osteoclast numbers increased transiently until day 5 after irradiation and then decreased rapidly in both the carbon ion and gamma ray irradiation groups. The carbon ion group showed reduced osteoclast size compared with the gamma ray group. Carbon ion irradiation had a more marked effect on osteoclast activity, and suppressed maturation to a greater extent than gamma irradiation. These observations suggest that carbon ion irradiation induces differential modulation of osteoclast growth factor expression.     

慢性睡眠限制状态下大鼠髁突软骨MMP-1,MMP-13表达的变化

目的:探讨MMP-1,MMP-13在慢性睡眠限制引起大鼠髁突软骨结构变化中的表达变化及作用。方法:180只雄性Wistar大鼠随机分为3组(n=60):慢性睡眠限制组(CSR)、大平台组(LC)、笼养组(CON)。每组根据试验时间不同分别分为3个亚组(n=20):7天(7D)、14天(14D)、21天(21D)组。参考改良多平台法(MMPM)建立大鼠的慢性睡眠限制模型。通过HE染色观察大鼠髁突软骨的结构变化。通过免疫组化和实时定量PCR分别检测MMP-1和MMP-13的蛋白水平及m RNA水平的表达变化。结果:HE染色和扫描电镜结果显示,CSR组的大鼠髁突软骨出现了病理性的改变。与CON和LC组比较,CSR组MMP-1和MMP-13的m RNA转录和蛋白表达水平明显升高(P0.05)。结论:慢性睡眠限制能够引起大鼠颞下颌关节髁突软骨的病理性变化。MMP-1和MMP-13的表达水平的变化可能在大鼠髁突软骨病理性改变中起关键作用。     

Evaluation of the Effect of a Gamma Irradiated DBM-Pluronic F127 Composite on Bone Regeneration in Wistar Rat

Demineralized bone matrix (DBM) is widely used for bone regeneration. Since DBM is prepared in powder form its handling properties are not optimal and limit the clinical use of this material. Various synthetic and biological carriers have been used to enhance the DBM handling. In this study we evaluated the effect of gamma irradiation on the physical-chemical properties of Pluronic and on bone morphogenetic proteins (BMPs) amount in DBM samples. In vivo studies were carried out to investigate the effect on bone regeneration of a gamma irradiated DBM-Pluronic F127 (DBM-PF127) composite implanted in the femur of rats. Gamma irradiation effects (25 kGy) on physical-chemical properties of Pluronic F127 were investigated by rheological and infrared analysis. The BMP-2/BMP-7 amount after DBM irradiation was evaluated by ELISA. Bone regeneration capacity of DBM-PF127 containing 40% (w/w) of DBM was investigated in transcortical holes created in the femoral diaphysis of Wistar rat. Bone porosity, repaired bone volume and tissue organization were evaluated at 15, 30 and 90 days by Micro-CT and histological analysis. The results showed that gamma irradiation did not induce significant modification on physical-chemical properties of Pluronic, while a decrease in BMP-2/BMP-7 amount was evidenced in sterilized DBM. Micro-CT and histological evaluation at day 15 post-implantation revealed an interconnected trabeculae network in medullar cavity and cellular infiltration and vascularization of DBM-PF127 residue. In contrast a large rate of not connected trabeculae was observed in Pluronic filled and unfilled defects. At 30 and 90 days the DBM-PF127 samples shown comparable results in term of density and thickness of the new formed tissue respect to unfilled defect. In conclusion a gamma irradiated DBM-PF127 composite, although it may have undergone a significant decrease in the concentration of BMPs, was able to maintains bone regeneration capability.     

Regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in osteoblastic cells

In addition to their stimulating function on osteoclastic bone resorption, bone resorptive factors may regulate proteinases and related factors in osteoblastic cells to degrade bone matrix proteins. This study investigated the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in the cultures of mouse osteoblastic MC3T3-E1 cells, mouse primary osteoblastic (POB) cells, and neonatal mouse calvariae. Expression of either MMP-2, -3, -9, -11, -13, and -14 or TIMP-1, -2, and -3 was detected in MC3T3-E1 cells and POB cells. When the bone resorptive factors parathyroid hormone, 1,25-dihydroxyvitamin D(3), prostaglandin E(2), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha) were added to the cell cultures, MMP-13 mRNA levels were found predominantly to increase by all resorptive factors in the three cultures. mRNA levels of either MMP-3 and -9 or TIMP-1 and -3 were found to increase mainly by the cytokines IL-1beta and TNF-alpha. BB94, a nonselective MMP inhibitor, neutralized the (45)Ca release stimulated by these resorptive factors to an extent similar to that of calcitonin, strongly suggesting that bone resorptive factors function at least partly through MMP formation. We propose that MMP-13 mRNA expression in osteoblastic cells may play an important role in stimulating matrix degradation by both systemic and local resorptive factors, whereas either MMP-3 and -9 or TIMP-1 and -3 might modulate matrix degradation by local cytokines only.     

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.     

LED照射对培养SW1353细胞MPP-3与MMP-13的影响

在骨关节疾病中,基质金属蛋白酶(matrix metalloproteinase,MMP)对关节软骨缺损的机理扮演着重要的角色。为了进一步明确低强度激光在关节软骨缺损中的治疗作用,应用细胞因子IL-1β与TNF-α刺激培养SW 1353细胞复制炎症细胞模型,观察发光二极管(LED)照射对培养细胞的生存率和死亡率的影响、细胞活性的影响、以及对MMP-3和MMP-13的调节作用。实验结果显示,10μg/L IL-1β与20μg/L TNF-α对培养细胞生存率无明显影响,而细胞活性明显下降(P<0.01),培养液中MMP-3和MMP-13含量明显上升(P<0.01,P<0.05);LED照射后可见与相应的模型组比较细胞死亡率下降(P<0.01)、培养液中MMP-3和MMP-13含量显著性降低在(P<0.01,P<0.05)。研究表明,LED照射对炎症因子刺激的SW 1 353细胞的MMP过多表达具有抑制性作用,可能对于关节软骨缺损性疾病的LED照射治疗起一定的指导意义。     

Effect of alendronate on endochondral ossification in mandibular condyles of growing rats

The replacement of the calcified cartilage by bone tissue during the endochondral ossification of the mandibular condyle is dependent of the resorbing activity of osteoclats. After partial resorption, calcified cartilage septa are covered by a primary bone matrix secreted by osteoblasts. Osteoadherin (OSAD) is a small proteoglycan present in bone matrix but absent in cartilage during the endochondral ossification. The aim of this study was to analyze the effect of alendronate, a drug known to inhibit bone resorption by osteoclasts, on the endochondral ossification of the mandibular condyle of young rats, by evaluating the distribution of osteoclasts and the presence of OSAD in the bone matrix deposited. Wistar newborn rats (n=45) received daily injections of alendronate (n=27) or sterile saline solution as control (n=18) from the day of birth until the ages of 4, 14 and 30 days. At the days mentioned, the mandibular condyles were collected and processed for transmission electron microscopy analysis. Specimens were also submitted to tartrate resistant acid phosphatase (TRAP) histochemistry and ultrastructural immunodetection of OSAD. Alendronate treatment did not impede the recruitment and fusion of osteoclasts at the ossification zone during condyle growth, but they presented inactivated phenotype. The trabeculae at the ossification area consisted of cartilage matrix covered by a layer of primary bone matrix that was immunopositive to OSAD at all time points studied. Apparently, alendronate impeded the removal of calcified cartilage and maturation of bone trabeculae in the mandibular ramus, while in controls they occurred normally. These findings highlight for giving attention to the potential side-effects of bisphosphonates administered to young patients once it may represent a risk of disturbing maxillofacial development.     

An in situ hybridization study of MMP-2, -9, -13, -14, TIMP-1, and -2 mRNA in fetal mouse mandibular condylar cartilage as compared with limb bud cartilage

The main purpose of this in situ hybridization study was to investigate MMPs and TIMPs mRNA expression in developing mandibular condylar cartilage and limb bud cartilage. At E14.0, MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the periosteum of mandibular bone, and in the condylar anlage. At E15.0 MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the perichondrium of newly formed condylar cartilage and the periosteum of developing bone collar, whereas, expression of MMP-14 and TIMP-1 mRNAs were restricted to the inner layer of the periosteum/perichondrium. This expression patterns continued until E18.0. Further, from E13.0 to 14.0, in the developing tibial cartilage, MMP-2, -14, and TIMP-2 mRNAs were expressed in the periosteum/perichondrium, but weak MMP-14 and no TIMP-1 mRNA expression was recognized in the perichondrium. These results confirmed that the perichondrium of condylar cartilage has characteristics of periosteum, and suggested that MMPs and/or TIMPs are more actively involved in the development of condylar (secondary) cartilage than tibial (primary) cartilage. MMP-9-positive cells were observed in the bone collar of both types of cartilage, and they were consistent with osteoclasts/chondroclasts. MMP-13 mRNA expression was restricted to the chondrocytes of the lower hypertrophic cell zone in tibial cartilage at E14.0, indicating MMP-13 can be used as a marker for lower hypertrophic cell zone. It was also expressed in chondrocytes of newly formed condylar cartilage at E15.0, and continuously expressed in the lower hypertrophic cell zone until E18.0. These results confirmed that progenitor cells of condylar cartilage are rapidly differentiated into hypertrophic chondrocytes, which is a unique structural feature of secondary cartilage different from that of primary cartilage.     

Role of S100A12 in the pathogenesis of osteoarthritis

S100A12 is a member of the S100 protein family, which are intracellular calcium-binding proteins. Although there are many reports on the involvement of S100A12 in inflammatory diseases, its presence in osteoarthritic cartilage has not been reported. The purpose of this study was to investigate the expression of S100A12 in human articular cartilage in osteoarthritis (OA) and to evaluate the role of S100A12 in human OA chondrocytes. We analyzed S100A12 expression by immunohistochemical staining of cartilage samples obtained from OA and non-OA patients. In addition, chondrocytes were isolated from knee cartilage of OA patients and treated with recombinant human S100A12. Real-time RT-PCR was performed to analyze mRNA expression. Protein production of matrix metalloproteinase 13 (MMP-13) and vascular endothelial growth factor (VEGF) in the culture medium were measured by ELISA. Immunohistochemical analyses revealed that S100A12 expression was markedly increased in OA cartilages. Protein production and mRNA expression of MMP-13 and VEGF in cultured OA chondrocytes were significantly increased by treatment with exogenous S100A12. These increases in mRNA expression and protein production were suppressed by administration of soluble receptor for advanced glycation end products (RAGE). Both p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) inhibitors also suppressed the increases in mRNA expression and protein production of MMP-13 and VEGF. We demonstrated marked up-regulation of S100A12 expression in human OA cartilages. Exogenous S100A12 increased the production of MMP-13 and VEGF in human OA chondrocytes. Our data indicate the possible involvement of S100A12 in the development of OA by up-regulating MMP-13 and VEGF via p38 MAPK and NF-κB pathways.     

Studies of the cellular cure for osteopetrosis by transplanted cells: specificity of the cell type in ia rats.

Spleen cells from normal rats are known to cure osteopetrosis in ia littermates within 3 weeks. In this study cell suspensions from liver, thymus, bone marrow, salivary gland, skeletal muscle and brain from normal rats were tested for their ability to cure osteopetrosis in ia littermates whose ability to reject these cells had been suppressed by whole-body irradiation. Cells from liver, thymus and bone marrow cured the disease as effectively as spleen cells from normal littermates. Mutants that received cells from salivary gland, muscle and brain remained osteopetrotic. These data suggest that some cell found in spleen, liver, thymus and bone marrow of 10-day-old normal rats, such as a lymphoid cell or stem cell, can restore hemopoiesis and bone resorption in osteopetrotic (ia) rats.     

Dietary zinc reduces osteoclast resorption activities and increases markers of osteoblast differentiation,matrix maturation,and mineralization in the long bones of growing rats

The nutritional influence of zinc on markers of bone extracellular matrix resorption and mineralization was investigated in growing rats. Thirty male weanling rats were randomly assigned to consume AIN-93G based diets containing 2.5, 5, 7.5, 15 or 30 μg Zn/g diet for 24 days. Femur zinc increased substantially as zinc increased from 5 to 15 μg/g diet and modestly between 15 and 30 μg/g (P<.05). By morphological assessment, trabecular bone increased steadily as dietary zinc increased to 30 μg/g. Increasing dietary zinc tended to decrease Zip2 expression nonsignificantly and elevated the relative expression of metallothionen-I at 15 but not 30 μg Zn/g diet. Femur osteoclastic resorption potential, indicated by matrix metalloproteinases (MMP-2 and MMP-9) and carbonic anhydrase-2 activities decreased with increasing dietary zinc. In contrast to indicators of extracellular matrix resorption, femur tartrate-resistant acid and alkaline phosphatase activities increased fourfold as dietary zinc increased from 2.5 to 30 μg Zn/g. Likewise, 15 or 30 μg Zn/g diet resulted in maximum relative expression of osteocalcin, without influencing expression of core-binding factor α-1, collagen Type 1 alpha-1, or nuclear factor of activated T cells c1. In conclusion, increased trabecular bone with additional zinc suggests that previous requirement estimates of 15 μg Zn/g diet may not meet nutritional needs for optimal bone development. Overall, the up-regulation of extracellular matrix modeling indexes and concomitant decrease in resorption activities as dietary zinc increased from 2.5 to 30 μg/g provide evidence of one or more physiological roles for zinc in modulating the balance between bone formation and resorption.