Early inhibitory effects of zoledronic acid in tooth extraction sockets in dogs are negated by recombinant human bone morphogenetic protein.

PURPOSE: This study was conducted with 2 purposes. The first was to determine the effect of a single dose of zoledronic acid (ZA) on the healing of a tooth extraction socket in dogs. The second was to determine if placement of recombinant human bone morphogenetic protein-2 (rhBMP-2)/absorbable collagen sponge (ACS) - INFUSE, (Medtronic, Memphis, TN) into these extraction sockets would inhibit the inhibition on bone healing and remodeling by ZA. MATERIALS AND METHODS: Nine adult female beagle dogs (2 to 3 yr old) were placed into 3 groups of 3 dogs each. Group I received 15 mL of sterile saline intravenously; group II received 2.5 mg of ZA intravenously; and group III received 5 mg of ZA intravenously. Forty-five days after treatment, all dogs underwent extraction of noncontiguous right and left mandibular first molars and second premolars. In group I, the right mandibular extraction sockets had nothing placed in them, whereas the left mandibular sockets had only ACS placed in them. In groups II and III, the right mandibular sockets had rhBMP-2/ACS placed in them, whereas the left mandibular sockets had only ACS placed. All extraction sockets were surgically closed. Tetracycline was given intravenously 5 and 12 days later, and all animals were euthanized 15 days after tooth extraction. The extraction sockets and rib and femur samples were harvested immediately after euthanasia, processed, and studied microscopically. RESULTS: A single dose of ZA significantly inhibited healing and bone remodeling in the area of the tooth extractions. The combination of rhBMP-2/ACS appeared to over-ride some of the bone remodeling inhibition of the ZA and increased bone fill in the extraction sites, and remodeling activity in the area was noted. The effects of rhBMP-2/ACS were confined to the area of the extraction sockets because bone activity at distant sites was not influenced. CONCLUSIONS: A single dose of ZA administered intravenously inhibits early healing of tooth extraction sockets and bone remodeling in this animal model. The combination of rhBMP-2/ACS significantly increased bone fill and bone remodeling in these areas, negating much of the effect of the ZA.

Compound heterozygous loss of Ext1 and Ext2 is sufficient for formation of multiple exostoses in mouse ribs and long bones.

Multiple Hereditary Exostoses (MHE) syndrome is caused by haploinsufficiency in Golgi-associated heparan sulfate polymerases EXT1 or EXT2 and is characterized by formation of exostoses next to growing long bones and other skeletal elements. Recent mouse studies have indicated that formation of stereotypic exostoses requires a complete loss of Ext expression, suggesting that a similar local loss of EXT function may underlie exostosis formation in patients. To further test this possibility and gain greater insights into pathogenic mechanisms, we created heterozygous Ext1(+/-) and compound Ext1(+/-)/Ext2(+/-) mice. Like Ext2(+/-) mice described previously (Stickens et al. Development 132:5055), Ext1(+/-) mice displayed rib-associated exostosis-like outgrowths only. However, compound heterozygous mice had nearly twice as many outgrowths and, more importantly, displayed stereotypic growth plate-like exostoses along their long bones. Ext1(+/-)Ext2(+/-) exostoses contained very low levels of immuno-detectable heparan sulfate, and Ext1(+/-)Ext2(+/-) chondrocytes, endothelial cells and fibroblasts in vitro produced shortened heparan sulfate chains compared to controls and responded less vigorously to exogenous factors such as FGF-18. We also found that rib outgrowths formed in Ext1(f/+)Col2Cre and Ext1(f/+)Dermo1Cre mice, suggesting that ectopic skeletal tissue can be induced by conditional Ext ablation in local chondrogenic and/or perichondrial cells. The study indicates that formation of stereotypic exostoses requires a significant, but not complete, loss of Ext expression and that exostosis incidence and phenotype are intimately sensitive to, and inversely related to, Ext expression. The data also indicate that the nature and organization of ectopic tissue may be influenced by site-specific anatomical cues and mechanisms.

Effects of short-term zoledronic acid treatment on bone remodeling and healing at surgical sites in the maxilla and mandible of aged dogs.

PURPOSE: It is unknown whether zoledronic acid (ZA) interferes with initial bone healing at extraction and implant sites. The goal of this study was to examine the effect of short-duration ZA on bone remodeling and healing after surgical insult in an aged dog model. MATERIALS AND METHODS: Four 2- to 3-year-old male dogs were administered ZA (0.1 mg/kg per month for 4 months), and 3 age-matched untreated dogs received no drug. In both groups, after the ZA-treated group had completed receiving the drug, the third premolar was extracted unilaterally and 2 orthodontic mini-implants per jaw per dog were placed on the ipsilateral side. After a 6-week healing period, a pair of calcein bone labels were administered. Bone sections from the mandible, maxilla, rib, and femur were obtained. The percent necrosis in the alveolar and basal regions of tooth-supporting bone was assayed by lactate dehydrogenase, and dynamic histomorphometric parameters were quantified and analyzed by use of mixed models. RESULTS: All extraction sites healed uneventfully, and no lesions resembling osteonecrosis were detected. The total percent necrosis was limited to less than 1% for all the bone sites examined. The ZA reduced bone remodeling at both surgical sites (extraction sites and mini-implant site) and nonsurgical sites. Although there was a significant (P < .05) increase in bone formation rate at the surgical sites in the untreated group, this increase was not significant (P = .3) in the ZA-treated group. CONCLUSIONS: Bone remodeling occurs in ZA-treated animals at surgical sites. ZA dramatically reduced bone turnover, but no exposed lesions resembling osteonecrosis developed at extraction and mini-implant sites after the 4-month drug duration.

Cancer treatment dosing regimens of zoledronic acid result in near-complete suppression of mandible intracortical bone remodeling in beagle dogs.

Bisphosphonate doses used in cancer treatment are substantially higher than those used for osteoporosis. Little is known about the effects of these high doses on tissue-level remodeling suppression. The aim of this study was to assess the effects of cancer dosing regimens of zoledronic acid on tissue-level bone remodeling at different skeletal sites. Skeletally mature female beagle dogs were treated with monthly intravenous infusions of vehicle (VEH, saline) or zoledronic acid (ZOL, 0.067 mg/kg); an additional group of animals was treated daily with oral alendronate (ALN, 0.2 mg/kg/day). Doses of ZOL and ALN were, on a milligram per kilogram basis, consistent with those used for cancer and osteoporosis, respectively. Following either 3 or 6 months of treatment, animals were euthanized, and mandible, rib, and tibia were processed for dynamic bone histology. There was no evidence of oral lesions or bone matrix necrosis in the mandibles of any animals. After 3 months, the rate of intracortical bone remodeling in the mandible was significantly suppressed with ZOL (-95%) compared with VEH; by 6 months, ZOL had produced nearly complete suppression (-99%) compared with VEH. ZOL also significantly suppressed remodeling in the rib cortex at both 3 (-83%) and 6 (-85%) months compared with VEH; tibia cortex bone formation rate was nonsignificantly lower with ZOL treatment (-68% to -75%). Remodeling suppression in ZOL-treated animals was significantly greater than in ALN-treated animals at both the mandible and the rib; ALN and VEH were not different for any of the assessed parameters at any of the sites. Compared across skeletal sites, the absolute level of remodeling suppression with ZOL treatment was significantly greater at sites with higher remodeling, whereas the percent reduction was similar among the sites. These results document nearly complete intracortical remodeling suppression resulting from monthly intravenous zoledronic acid dosing, with changes being most dramatic at the mandible.

Relationship between embryonic histonic hyperacetylation and axial skeletal defects in mouse exposed to the three HDAC inhibitors apicidin, MS-275, and sodium butyrate.

Some histone deacetylase inhibitors (HDACi) have recently been related to teratogenic effects in rodents. Skeletal defects have been directly associated with embryonic hyperacetylation of somitic nuclei after valproic acid or trichostatin A exposure in vivo. Albeit the antitumoral activity of HDACi has been classically related to chromatin condensation due to histonic lysine hyperacetylation, nonhistonic proteins have also been suggested as an HDACi target. The aim of this work was the study of the effects of three HDACi (apicidin, API; MS-275; sodium butyrate, BUT) on mouse development and their activity on embryonic histonic and nonhistonic proteins. Pregnant mice were ip treated with 10 mg/kg body weight API, 25 mg/kg MS-275, 2000 mg/kg BUT or with the vehicle alone on day 8 post coitum. Embryos were extracted 1, 2, or 3 h after treatment and Western blotting (using antibodies antihyperacetylated histone H4, antiacetylated lysine, or antitubulin) and immunohistochemistry (using the antibody antihyperacetylated histone H4) were performed. Fetuses, explanted at term of gestation, were double stained for bone and cartilage to detect skeletal abnormalities. The studied HDACi were teratogenic. The specific axial skeletal malformations were fusions or homeotic respecifications. These molecules induced hyperacetylation restricted to somitic histones. The hyperacetylation index of histone H4 as well as immunohistochemical and skeletal analyses indicated BUT as the less active molecule. These new data on effects of API, MS-275, and BUT on development suggest histonic hyperacetylation as the mechanism for the induction of the observed skeletal abnormalities.

Antiremodeling agents influence osteoblast activity differently in modeling and remodeling sites of canine rib.

Antiremodeling agents reduce bone loss in part through direct actions on osteoclasts. Their effects on osteoblasts and bone formation activity are less clear and may differ at sites undergoing modeling vs. remodeling. Skeletally mature intact beagles, 1-2 years old at the start of the study, were treated daily with clinically relevant doses of alendronate (0.10 or 0.20 mg/kg), risedronate (0.05 or 0.10 mg/kg), raloxifene (0.50 mg/kg), or vehicle (1 mL/kg). Dynamic bone formation parameters were histologically assessed on periosteal, endocortical/trabecular, and intracortical bone envelopes of the rib. Raloxifene significantly increased periosteal surface mineral apposition rate (MAR), a measure of osteoblast activity, compared to all other treatments (+108 to +175%, P < 0.02), while having no significant effect on MAR at either the endocortical/trabecular or intracortical envelope. Alendronate (both 0.10 and 0.20 doses) and risedronate (only the 0.10 dose) significantly (P < or = 0.05) suppressed MAR on the endocortical/trabecular envelope, while none of the bisphosphonate doses significantly altered MAR at either the periosteal or intracortical envelopes compared to vehicle. Based on these results, we conclude that (1) at clinically relevant doses the two classes of antiremodeling agents, bisphosphonates and selective estrogen receptor modulators, exert differential effects on osteoblast activity in the canine rib and (2) this effect depends on whether modeling or remodeling is the predominant mechanism of bone formation.

Cell yield, proliferation, and postexpansion differentiation capacity of human ear, nasal, and rib chondrocytes.

Human ear, nasal, and rib chondrocytes were compared with respect to their suitability to generate autologous cartilage grafts for nonarticular reconstructive surgery. Cells were expanded for two passages in medium containing 10% fetal bovine serum without (control) or with transforming growth factor beta(1) (TGF-beta(1)), fibroblast growth factor 2 (FGF-2), and platelet-derived growth factor bb (PDGF-bb) (TFP). Expanded cells were cultured as three-dimensional pellets in chondrogenic serum-free medium containing insulin, dexamethasone, and TGF-beta(1). Chondrocytes from all three sources were successfully isolated, increased their proliferation rate in response to TFP, and dedifferentiated during passaging. Redifferentiation by ear and nasal, but not rib, chondrocytes was enhanced after TFP expansion, as assessed by the significant increase in glycosaminoglycan (GAG)/DNA content and collagen type II mRNA expression in the resulting pellets. TFP-expanded ear and nasal chondrocytes generated pellets of better quality than rib chondrocytes, as assessed by the significantly higher GAG/DNA content and collagen type II mRNA expression, and by the relative stain intensities for GAG and collagen types I and II. In conclusion, postexpansion cell yields suggest that all three sources investigated could be used to generate autologous grafts of clinically relevant size. However, ear and nasal chondrocytes, if expanded with TFP, display superior postexpansion chondrogenic potential and may be a preferred cell source for cartilage tissue engineering.

Effect of maternal methionine pre-treatment on alcohol-induced exencephaly and axial skeletal dysmorphogenesis in mouse fetuses.

Alcohol is known to induce folate deficiency and impair methionine synthase activity. Exogenous folic acid (FA) administered periconceptionally has been shown to prevent the first occurrence and recurrence of neural tube defects (NTD) in humans. Since folate, vitamin B(12) and methionine are metabolically interrelated, it was decided to determine the effect of methionine pre-treatment on alcohol-induced NTD and axial skeletal defects in mouse embryos. Following administration of a single dose of 70 or 150 mg/kg of methionine, 0.03 ml/g body weight of ethanol solution (25% v/v of absolute alcohol in saline) was injected intraperitoneally into pregnant mice at critical stages of neural tube development. The controls were either non-treated or saline treated and pair-fed and pair-watered. Fetuses were collected on gestation day 18. Alcohol and methionine plus alcohol numerically enhanced embryonic resorption and induced a significant reduction in fetal body weight. Alcohol alone caused a 3-fold increase in the background frequency of exencephaly in gestation days 7 and 8 treatment groups. The low dose of methionine only numerically reduced the spontaneous exencephaly. Pre-treatment with methionine only produced a numerical but not statistically significant reduction in alcohol-induced exencephaly. The higher dose of methionine did not produce a particularly beneficial effect on embryonic survival, fetal body weight and occurrence of exencephaly. Alcohol-induced cleft palate and limb malformations were ameliorated by methionine pre-treatment. Craniofacial skeleton, vertebrae and ribs were extensively malformed both in the alcohol and methionine plus alcohol groups indicating a lack of rescue effects of methionine. Whereas supernumerary ribs and extra sternal ribs were augmented by methionine, occipitalization of the atlas vertebra was a malformation unique to the pre-treatment group. Plasma levels of several amino acids including that of methionine were significantly lowered by alcohol. Pre-treatment with methionine produced a dose dependent enhancement of only methionine concentration. These data suggest that pre-administration of methionine only rescues mouse embryos of certain non-neural malformations and that the lack of ameliorative effect on NTD and axial skeletal defects may be due to the fact that alcohol lowers the concentration of a number of amino acids and therefore, supplementation should comprise a mixture of these amino acids and possibly FA and vitamin B(12).

Vertebral and rib sarcoidosis: long-term clinical remission with methotrexate.

We describe a patient with bilateral hilar lymphadenopathy shown on a chest radiograph and supraclavicular lymphadenopathy. Biopsy of a supraclavicular lymph node showed non-caseating granulomas. A diagnosis of sarcoidosis was made and no treatment was given. One year later she complained of cervical and lumbar pain and decreasing strength of the right hand. Magnetic resonance imaging of the spine showed multiple lesions within the vertebral bodies of six vertebrae, and thoracic computed tomography showed partial destruction of the first right rib. A biopsy of the second lumbar vertebra demonstrated non-caseating granulomas. Corticosteroid treatment was unsuccessful and long-term remission of the symptoms was achieved with a weekly low dose of methotrexate.

Mineralization of cancellous bone after alendronate and sodium fluoride treatment: a quantitative backscattered electron imaging study on minipig ribs.

Fluoride stimulates bone formation, whereas bisphosphonates reduce bone resorption. In clinical trials, both treatments increase bone density, although sodium fluoride (NaF) increases and alendronate (bisphosphonate, ALN) decreases bone turnover. In a comparative study using minipigs an inverse correlation has been reported between bone turnover and elastic modulus. Small-angle X-ray scattering (SAXS) measurements of these bones revealed no structural deterioration of the collagen/mineral composite at the nanometer range for ALN-treated vertebra, whereas a slight increase of the average thickness of the mineral crystals as well as changes of the structure of the collagen/mineral composite were found in the bones of NaF-treated animals. In this study we used quantitative backscattered electron imaging (qBSE) to investigate the cancellous bones from ribs of minipigs treated with vehicle, NaF, or ALN. This method provides information on the local mineral concentration in the micrometer range. Mineralization spectra were obtained from each treatment group, and statistically significant differences between ALN and controls were found for the peak position, the peak height, the peak width, and the average calcium (Ca) concentration of the mineral distribution. The results reveal that the cancellous bone matrix was more uniformly mineralized after ALN treatment. The reduced bone turnover induced by ALN, documented histomorphometrically could be at the origin of this phenomenon. No significant differences were detected between NaF and control. Together with the earlier SAXS data these results may explain in part the increase in bone density and the improvement of biomechanical properties observed after ALN treatment in animals and in osteoporotic patients.

Evidence that human bone cells in culture secrete insulin-like growth factor (IGF)-II and IGF binding protein-3 but not acid-labile subunit both under basal and regulated conditions.

Insulin-like growth factors (IGFs) are found in human circulation predominantly as part of a growth hormone (GH)-dependent complex of 125-150 kD, which is composed of three subunits: IGF-I or IGF-II, an acid stable IGF binding protein (IGFBP)-3, and an acid labile subunit (ALS). Although recent studies demonstrate that a number of cell types in culture secrete IGFs and IGFBP-3, very little is known with regard to the origin of circulating ALS. To test the hypothesis that human bone cells (HBCs), which produce abundant amounts of IGF-II and IGFBP-3, also produce ALS, we measured the IGF-I, IGF-II, IGFBP-3, and ALS levels using specific radioimmunoassays (RIAs) in the conditioned medium (CM) of untransformed normal HBCs and SaOS-2 osteosarcoma cells treated with various effectors (IGF-II, osteogenic protein-1 [OP-1, bone morphogenetic protein-7] and human GH) for 48 h. No detectable levels (< 3 ng/ml) of ALS were found in the CM of various HBC types under basal conditions. In contrast, CM collected from liver explants in culture contained significant amount of ALS prepared and assayed under identical conditions. The IGF-I level was also undetectable in the CM of various HBC types. In the IGF-II (3, 30 ng/ml)-treated HBC CM, the IGFBP-3 level was increased in a dose-dependent manner but neither IGF-I nor ALS could be detected. In the SaOS-2 cell culture, OP-1 (1, 100 ng/ml) increased both IGF-II and IGFBP-3 secretion but neither ALS nor IGF-I secretion. Treatment of HBCs with GH (1, 10, 100 ng/ml) had no significant effect on the secretion of either IGF-I, IGF-II, IGFBP-3, or ALS.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of methotrexate in the biology of free vascularized bone grafts. A comparative experimental study in the dog.

To evaluate the effects of methotrexate (MTX) on the biology of a vascularized bone graft (VBG), a study of adult dogs was performed. Different protocols of treatment using MTX, with or without the administration of citrovorum rescue factor (CRF), were studied. A controlled experiment involving 18 dogs was performed to evaluate the biology of a free vascularized rib graft used to reconstruct a 7-cm segmental defect in the femoral shaft. The grafts were examined at five, eight, and 14 weeks after surgery. Bone union, remodeling, and vasculature were found to be normal, despite a high rate of cortical necrosis in the early follow-up periods (30-40%). In a similar group of VBG, MTX was administered at a dose of 1500 mg/m2 body surface (intravenous) from the 14th postoperative day, every three weeks, until the animals were killed. Citrovorum rescue factor was administered to avoid the toxic side effects of the MTX. The results of this group did not differ from those of the control group as far as bone union, remodeling, vasculature, and viability of the graft were concerned. Methotrexate did not, for the most part, affect the biology of a free vascularized bone graft.

Toxic effects of Solanum malacoxylon on sheep bone.

Solanum malacoxylon (Sm), a calcinogenic plant that contains 1,25-(OH)2D3 glycoside, was administered orally to sheep. Fifty milligrams of air-dried leaves three times a week caused an increased volume density of cancellous bone within lumbar vertebrae and an increased trabecular thickness. There was little remodeling activity at the end of a 180-day treatment period, and few trabecular bone surfaces had tetracycline double labels at this time. Bone biopsies taken at the end of a 1-month treatment demonstrated increased extent of bone-forming surfaces and osteoid volume. Sm caused a mineralization defect that was transitory but resulted in unmineralized lines and foci in osteones. These remaining foci of unmineralized bone were associated with the deposition of acid mucopolysaccharide, and acid mucopolysaccharide accumulation could be identified on all bone envelopes in 30-day biopsy specimens. A similar hyperostosis in the metaphyses of rats was produced by parenteral administration of 1,25-(OH)2D3 for 10 days.

Experimental aluminum intoxication and parathormone: effects on the mineralization process.

In order to study the effect of aluminum intoxication on bone and bone cells in normal animals and its relationship with hyperparathyroidism, and so to obtain further data on a pathogenetic role of this condition in inducing osteomalacia in uremic patients, 31 rats divided in four groups were injected intraperitoneally for 11 weeks with: Al (75.6 mg); Al-PTH (Al = 75.6 mg + PTH = 200 USP during the last week); C (saline solution), and C-PTH (saline solution + PTH = 200 USP during the last week). Al injection induced a consistent increase in the element in serum and tibia. PTH administration further enhanced Al content in tibia. The trabecular bone surfaces of Al-administered rats were stained by aluminon; the endosteal borders of their compact bone were always negative. Rib histomorphometry showed absence of osteomalacia in Al group and increase in osteoid in Al-PTH group, with development of mild osteomalacia. In these groups osteoclasts were less numerous than in controls. Dynamic bone parameters showed no separation of double tetracycline labels in trabecular bone of both Al-administered groups. Cortical bone was only slightly affected by treatment. All these data indicate that Al alone, in the quantity administered, does not induce osteomalacia in normal rats and that PTH, although given for a few days, enhances Al content in bone and induces osteoid increment. The reduction of tetracycline labels in all Al-treated animals is due to reduction of calcification and formation rate, which might be an index of osteoblast inhibition. The decrease in the number of osteoclasts suggests that Al might inhibit their formation.

The effects of continuous estradiol therapy on cortical bone remodeling activity in the spayed beagle.

Ribs from 6 oophorectomized and 4 17 beta-estradiol-supplemented spayed, 4-year-old Beagle dams were subjected to histomorphometric analyses to determine what effects continuous estradiol treatment of 9 months duration had on the Basic Multicellular Unit (BMU) of cortical bone remodeling. The findings of this study suggest that 17 beta-estradiol has a two-step mode of action. First, this estrogen directly suppresses the formation of new BMUs. Secondly, it causes uncoupling of the Resorption/Formation (R/F) mechanism within each BMU, together with the creation of an approximate 1:1 balance between bone resorption and bone formation. This balance is probably responsible for the preservation of cortical bone mass seen to occur with continuous estrogen replacement therapy.

Bone changes in mice after prolonged continuous exposure to a high concentration of carbon monoxide.

Quantitative and qualitative pathological changes in mouse bones are described after continuous exposure to 0.24% v/v of carbon monoxide in air for periods of up to 180 days. Planimetric measurements of five bones (parietal bone, sternum, lumbar vertebrae, ribs and shafts of femurs) showed a considerable increase in the amount of bone tissue in all bones except the femurs. The greatest amount of new bone formation was found, in decreasing order, in the skull, ribs, lumbar vertebrae and sternal segments. The cortex of the femur shafts showed some thinning, although there was more trabecular bone in the metaphyses. At the same time an expansion of marrow cavities was found in ribs, parietal bones and femurs. Possible pathomechanisms responsible for the excessive bone formation are explored. It is suggested that an increased rate of blood flow through the bone and marrow tissues played an important role.

Management of contaminated bone grafts.

Using an experimental animal model, the infection rate of contaminated bone grafts after irrigation with either normal saline, povidone-iodine, or a cefazolin solution was evaluated. Mechanical cleansing appears to be the important factor in preventing infection in these grafts, since all the solutions showed almost equal effectiveness. As the amount of bulk and dead space increases, particularly in Pseudomonas infections, povidone-iodine might be slightly superior, although this difference was not statistically significant.