Clinically relevant doses of vitamin A decrease cortical bone mass in mice

Excess vitamin A has been associated with decreased cortical bone thickness and increased fracture risk. While most studies in rodents have employed high dosages of vitamin A for short periods of time, we investigated the bone phenotype in mice after longer exposure to more clinically relevant doses. For 1, 4 and 10 weeks, mice were fed a control diet (4.5 µg retinyl acetate/g chow), a diet modeled from the human upper tolerable limit (UTL; 20 µg retinyl acetate/g chow) and a diet three times UTL (supplemented; 60 µg retinyl acetate/g chow). Time-dependent decreases in periosteal circumference and bone mineral content were noted with the supplemented dose. These reductions in cortical bone resulted in a significant time-dependent decrease of predicted strength and a non-significant trend toward reduced bone strength as analyzed by three-point bending. Trabecular bone in tibiae and vertebrae remained unaffected when vitamin A was increased in the diet. Dynamic histomorphometry demonstrated that bone formation was substantially decreased after 1 week of treatment at the periosteal site with the supplemental dose. Increasing amount of vitamin A decreased endocortical circumference, resulting in decreased marrow area, a response associated with enhanced endocortical bone formation. In the presence of bisphosphonate, vitamin A had no effect on cortical bone, suggesting that osteoclasts are important, even if effects on bone resorption were not detected by osteoclast counting, genes in cortical bone or analysis of serum TRAP5b and CTX. In conclusion, our results indicate that even clinically relevant doses of vitamin A have a negative impact on the amount of cortical bone.

Porcupine inhibitors impair trabecular and cortical bone mass and strength in mice.

WNT signaling is involved in the tumorigenesis of various cancers and regulates bone homeostasis. Palmitoleoylation of WNTs by Porcupine is required for WNT activity. Porcupine inhibitors are under development for cancer therapy. As the possible side effects of Porcupine inhibitors on bone health are unknown, we determined their effects on bone mass and strength. Twelve-week-old C57BL/6N female mice were treated by the Porcupine inhibitors LGK974 (low dose = 3 mg/kg/day; high dose = 6 mg/kg/day) or Wnt-C59 (10 mg/kg/day) or vehicle for 3 weeks. Bone parameters were assessed by serum biomarkers, dual-energy X-ray absorptiometry, µCT and histomorphometry. Bone strength was measured by the 3-point bending test. The Porcupine inhibitors were well tolerated demonstrated by normal body weight. Both doses of LGK974 and Wnt-C59 reduced total body bone mineral density compared with vehicle treatment (P < 0.001). Cortical thickness of the femur shaft (P < 0.001) and trabecular bone volume fraction in the vertebral body (P < 0.001) were reduced by treatment with LGK974 or Wnt-C59. Porcupine inhibition reduced bone strength in the tibia (P < 0.05). The cortical bone loss was the result of impaired periosteal bone formation and increased endocortical bone resorption and the trabecular bone loss was caused by reduced trabecular bone formation and increased bone resorption. Porcupine inhibitors exert deleterious effects on bone mass and strength caused by a combination of reduced bone formation and increased bone resorption. We suggest that cancer targeted therapies using Porcupine inhibitors may increase the risk of fractures.

Affecting osteoblastic responses with in vivo engineered potato pectin fragments.

Pectins, complex plant-derived polysaccharides, are novel candidates for biomaterial nanocoatings. Pectic rhamnogalacturonan-I regions (RG-I) can be enzymatically treated to so-called modified hairy regions (MHR). We surveyed the growth and differentiation of murine preosteoblastic MC3T3-E1 cells on Petri dishes coated with RG-Is from native or genetically engineered potato tubers. Uncoated tissue culture polystyrene (TCPS) and aminated (AMI) dishes served as controls. MHRPTR_GAL sample was depleted of galactose (9 mol % galactose; 23 mol % arabinose) and MHRPTR_ARA of arabinose (61 mol % galactose; 6 mol % arabinose). Wild-type (modified hairy region from potato pectin (MHRP)_WT) fragment contained default amounts (58 mol % galactose; 13 mol % arabinose) of both sugars. Focal adhesions (FAs) indicating cellular attachment were quantified. Reverse transcriptase polymerase chain reaction (RT-PCR) of alkaline phosphatase and osteocalcin genes indicating osteoblastic differentiation was performed along with staining the produced calcium with tetracycline as an indicator of osteoblastic differentiation. Osteoblasts proliferated on all the samples to some extent. The control surfaces performed better than any of the pectin samples, of which the MHRP_WT seemed to function best. FA length was greater on MHRPTR_GAL than on other pectin samples, otherwise the mutants did not significantly deviate. RT-PCR results indicate that differences between the samples at the gene expression level might be even subtler. However, tetracycline-stained calcium-containing mineral was detected merely only on uncoated TCPS. These results indicate the possibility to affect bone cell growth with in vivo-modified pectin fragments, consecutively providing information on the significance of certain monosaccharides on the biocompatibility of these polysaccharides.

Perinatal exposure to environmental contaminants detected in Canadian Arctic human populations changes bone geometry and biomechanical properties in rat offspring.

Arctic inhabitants consume large proportions of fish and marine mammals, and are therefore continuously exposed to levels of environmental toxicants, which may produce adverse health effects. Fetuses and newborns are the most vulnerable groups. The aim of this study was to evaluate changes in bone geometry, mineral density, and biomechanical properties during development following perinatal exposure to a mixture of environmental contaminants corresponding to maternal blood levels in Canadian Arctic human populations. Sprague-Dawley rat dams were dosed with a Northern Contaminant Mixture (NCM) from gestational day 1 to postnatal day (PND) 23. NCM contains 27 contaminants comprising polychlorinated biphenyls, organochlorine pesticides, and methylmercury. Femurs were collected on PND 35, 77 and 350, and diaphysis was analyzed by peripheral quantitative computed tomography and three-point bending test, while femoral neck was assessed in an axial loading experiment. Dose-response modeling was performed to establish the benchmark dose (BMD) for the analyzed bone parameters. Exposure to the high dose of NMC resulted in short and thin femur with reduced mechanical strength in offspring at PND35. BMD of femur length, cortical area, and stiffness were 3.2, 1.6, and 0.8 mg/kg bw/d, respectively. At PND77 femur was still thin, but at PND350 no treatment-related bone differences were detected. This study provides new insights on environmental contaminants present in the maternal blood of Canadian Arctic populations, showing that perinatal exposure induces bone alterations in the young offspring. These findings could be significant from a health risk assessment point of view.

Pectin-coated titanium implants are well-tolerated in vivo.

Multiform coated titanium implants are widely used in orthopedic and dental surgery. In this study, we have investigated the reactivity of pectin-coated titanium samples implanted under the latissimus dorsi-muscle fascia of rats. Samples were coated with two enzyme treated apple pectins; modified hairy regions (MHR-A and MHR-B) that differed in chemical structure. Aminated (AMI) and uncoated titanium (Ti) served as controls. The thicknesses of the peri-implant fibrous tissue capsules formed 1 or 3 weeks after implantation were measured as indicative of possible inflammatory reactions toward the biomaterials. After 1 week, the MHR-B implant was surrounded by a thicker fibrous capsule (42.9 microm) than any of the other sample types: MHR-A (33.2 microm), AMI (32.5 microm), and Ti (32.3 microm), the last one being the only statistically significant difference. After 3 weeks, however, this difference disappeared; the capsule thicknesses around MHR-B and Ti implants had decreased to the values found for AMI and MHR-A. Additionally, the capsule formation represents merely a stromal rather than an inflammatory reaction, as indicated by the absence of activated macrophages or foreign body giant cells in the capsules. These results indicate for the first time the in vivo tolerability of covalently linked pectins, and suggest the feasibility of pectin-coated bone and dental implants for clinical use.

Effect of modified pectin molecules on the growth of bone cells.

The aim of this study was to investigate molecular candidates for bone implant nanocoatings, which could improve biocompatibility of implant materials. Primary rat bone cells and murine preosteoblastic MC3T3-E1 cells were cultured on enzymatically modified hairy regions (MHR-A and MHR-B) of apple pectins. MHRs were covalently attached to tissue culture polystyrene (TCPS) or glass. Uncoated substrata or bone slices were used as controls. Cell attachment, proliferation, and differentiation were investigated with fluorescence and confocal microscopy. Bone cells seem to prefer MHR-B coating to MHR-A coating. On MHR-A samples, the overall numbers as well as proportions of active osteoclasts were diminished compared to those on MHR-B, TCPS, or bone. Focal adhesions indicating attachment of the osteoblastic cells were detected on MHR-B and uncoated controls but not on MHR-A. These results demonstrate the possibility to modify surfaces with pectin nanocoatings.

Microstructural properties of bone in rat vertebra after long-term clodronate treatment.

Bisphosphonates (BPs) are known to increase bone mineral density, but it is not known how this increase manifests at low hierarchic levels of the bone structure. The present study aimed to clarify the effects of the long-term use of clodronate on the microstructure and chemical composition of bone. The second lumbar vertebral body (L2) in growing rats, subjected to 32 weeks' treatment with clodronate at either a therapeutic dose of 2 mg/kg, or a high dose of 10 mg/kg, or physiological saline (control group), was studied by scanning electron microscopy for morphology, by backscattered electron image (BSE) for density, and by energy dispersive spectrometry for material analysis. BSE images showed that the degree of mineralization in the different areas of trabecular bone of the vertebral body varied in both the control and the study groups, but this variation seemed to be different in the control and study groups. BSE analysis showed that there was more high-density bone (white area) in the low-dose clodronate group than in the controls, but the difference between the high-dose clodronate group and the control group was not significant. The density of the white area (high-density bone) was slightly increased in the low-dose clodronate group. There were no differences in the density of the gray area (low-density bone) between the groups. Neither the distribution of Ca, P, or Mg, nor the total mineral content, was affected by the clodronate treatment. Our results indicate that long-term clodronate treatment at the therapeutic level increases the proportion of high-density bone in the vertebral body in non-osteoporotic rats.