Marina Komrakova

Whole body vibration during fracture healing intensifies the effects of estradiol and raloxifene in estrogen-deficient rats

Current osteoporosis therapies aim to delay bone destruction and have additional anabolic effects. While they have demonstrated some positive effects on bone healing, more progress is needed in this area. This study used the well-known osteoporotic agents estrogen (E) and raloxifene (R) in conjunction with biomechanical whole body vibration (WBV) at a frequency of 70 Hz twice daily for six weeks to stimulate bone healing. Eighty-four 3-month old female Sprague-Dawley rats (12 per group) were bilaterally ovariectomized to develop osteopenia within eight weeks. Osteotomy of the metaphyseal tibiae was performed and fracture healing was then studied using mechanical tests, histomorphometry, computed tomography (μCT), and gene analysis. We found that E and R improved the structure of osteopenic bones as did WBV alone, although significant levels for WBV were seldom reached. Combination treatments significantly enhanced stiffness (R+WBV; p<0.05), endosteal bone (R+WBV; p<0.01), and trabecular density (E+WBV; p<0.05, R+WBV; p<0.05). In addition, the expression of osteoclast-specific Trap was significantly reduced after treatment with E, R, or their combination with WBV (p<0.01). The effects were additive and not inhibitory, leading us to conclude that the combined applications of WBV with E or R may improve the healing of osteopenic bones. The therapies studied are all currently approved for human use, suggesting ready applicability to clinical practice. To better understand the effects of WBV on osteopenic bones, the ideal vibration regime will require further study.

Impact of 4-methylbenzylidene camphor, daidzein, and estrogen on intact and osteotomized bone in osteopenic rats

The study investigated the influence of 4-methylbenzylidene camphor (4-MBC), daidzein, and estradiol-17β-benzoate (E(2)) on either intact or osteotomized cancellous bone in ovariectomized (Ovx) rats. Three-month old Ovx rats were fed with soy-free (SF) diet over 8 weeks; thereafter, bilateral transverse metaphyseal osteotomy of tibia was performed and rats were divided into groups: rats fed with SF diet and SF diet supplemented with 4-MBC (200 mg), daidzein (50 mg), or E(2) (0.4 mg) per kilogram body weight. After 5 or 10 weeks, computed tomographical, biomechanical, histological, and ashing analyses were performed in lumbar spine and tibia of 12 rats from each group. 4-MBC and E(2) improved bone parameters in lumbar spine and tibia, were not favorable for osteotomy healing, and decreased serum osteocalcin level. However, daidzein improved bone parameters to a lesser extent and facilitated osteotomy healing. For lumbar spine, the bone mineral density was 338±9, 346±5, 361±6, and 360±5 mg/cm(3) in SF, daidzein, 4-MBC, and E(2), respectively, after 10 weeks. For tibia, the yield load was 98±5, 114±3, 90±2, and 52±4 N in SF, daidzein, 4-MBC, and E(2), respectively, after 10 weeks. Serum daidzein was 54±6 ng/ml in daidzein group and equol was not detected. Alp and Igf1 genes were down-regulated in callus after daidzein and E(2) compared with 4-MBC (week 5). The response of bone tissue and serum markers of bone metabolism could be ordered: daidzein<4-MBC<E(2). Treatments were more effective after 5 vs 10 weeks. In SF group, bone structure was impaired after 5 weeks and improved after 10 weeks probably due to adaptation mechanisms to osteoporosis. In conclusion, it is conceivable that 4-MBC may improve bone tissue in osteoporotic organisms; osteoporotic patients with fractures could benefit from daidzein treatment.

Influence of intermittent administration of parathyroid hormone on muscle tissue and bone healing in orchiectomized rats or controls.

Influence of human parathyroid hormone (hPTH 1-34) on muscle and bone healing was studied in either orchiectomized (Orx at 8 months of age) or sham-operated male rats. Eleven-month-old Sprague-Dawley rats underwent bilateral transverse metaphyseal osteotomy of tibia and were divided into four groups (n=12): 1) sham-vehicle, 2) sham group-PTH everyday, 3) Orx-vehicle, 4) Orx-PTH everyday, and 5) Orx-PTH every other day. PTH dosage was 40  μg/kg body weight. After 5 weeks, fiber cross-sectional area, capillary density, and enzyme activity (lactate dehydrogenase, citrate synthase, and complex I) were measured in soleus (MS), gastrocnemius (MG), and longissimus (ML) muscles; tibiae were analyzed by computed tomographical, histological, and gene expression analyses. The effect of PTH in all rats was increased serum osteocalcin, cortical and callus densities and callus area. In sham rats capillary density was increased in limb muscles (MS: 1.3-1.7, MG: 1.2-1.4 capillaries/fiber), and rate of osseous bridging of osteotomy was enhanced (67-100%). In Orx rats serum creatine kinase was decreased (6670-2847 U/l), and bone genes (Igf-1, osteoprotegerin, and receptor activator of nuclear factor kB ligand) were up-regulated. Cross-sectional area, enzyme activity, food intake, weight of body, visceral organs, adipose tissue, MG, and MS were not affected by PTH. PTH had a favorable effect on muscle capillary density and improved bone healing being more effective in sham rats and having no adverse systemic effect. The effect was less if PTH was applied every other day. The findings may show up trends for therapeutic treatment of male patients.

Effect of daidzein, 4-methylbenzylidene camphor or estrogen on gastrocnemius muscle of osteoporotic rats undergoing tibia healing period

The effect of daidzein (D), 4-methylbenzylidene camphor (4-MBC) or estradiol-17beta-benzoate (E(2)) on muscle of osteoporotic rats during fracture healing was studied. After performing a metaphyseal tibia osteotomy in 96 osteoporotic 5-month-old female Sprague-Dawley rats, they received daily 50 mg D, 200 mg 4-MBC or 0.4 mg E(2) per kg body weight, or soy free (SF) diet up to 36 and 72 days. Mitochondrial activity, fiber area, and capillary density were analyzed in M. gastrocnemius. Osseous callus bridging of fracture was observed in half of the rats after 36 days. By day 72, fracture was healed in most of the animals. State 3 mitochondrial respiration significantly enhanced in E(2), 4-MBC and D groups versus SF after 36 days (30, 32 and 32 vs 23 pmol O(2)/s per mg). It declined after 72 days, however, in E(2) group it was still at a higher level versus SF (25, 23 and 21 vs 20 pmol O(2)/s per mg). Size of fast oxidative glycolytic (FOG) and fast glycolytic (FG) fibers, capillary density did not differ significantly between the groups, however, at day 36 an increase in D and 4-MBC groups was detectable. FOG diameter was 64, 66, 68, and 58 microm and FG diameter was 88, 98, 95, and 89 microm in SF, D, 4-MBC, and E(2) groups. The ratio of capillaries to muscle fiber was 1.1, 1.4, 1.3, and 1.1 in SF, D, 4-MBC and E(2) groups by day 36. D and 4-MBC react similar to estrogen thereby improving oxidative cell metabolism in severe osteoporotic rats. The level of mitochondrial activity was higher, though no significant morphological differences could be shown.

Evaluation of twelve vibration regimes applied to improve spine properties in ovariectomized rats

While whole-body vibration (WBV) has recently been introduced as a non-pharmacological therapy for osteoporosis, studies have shown that it has no significant effect on the lumbar spine in older women. However, the vibration protocols differed among studies, and the major factor influencing the outcomes is unclear. The intention of the present study was to evaluate the effect of WBV—vertical (v) or horizontal (h) and of different frequencies and application regimes (1 × or 2 ×/d)—on lumbar spine properties in ovariectomized rats (Ovx). Three experiments were conducted. Thirteen-week old female Sprague–Dawley rats were Ovx or left intact (Non-Ovx). After eight weeks, all of the rats underwent metaphyseal osteotomy of the tibiae. Five days later, the rats were divided into six groups (n = 15): 1) intact, 2) Ovx, and 3–6) Ovx exposed to WBV. In Experiment 1, groups 3–6 underwent 35 Hz-v, 50 Hz-v, 70 Hz-v, and 90 Hz-v, respectively. In Experiment 2, groups 3–6 underwent 30 Hz-h, 50 Hz-h, 70 Hz-h, and 90 Hz-h, respectively. In Experiment 3, groups 3–6 underwent 35 Hz-v, 70 Hz-v, 35 Hz-h, and 70 Hz-h, respectively. Vibration exposure was 15 min 1 ×/d in Experiment 1 and 2 and 2 ×/d in Experiment 3 for up to 30 days. Vertebral bodies were used in micro-computed tomography, biomechanical, ashing, and gene expression analyses. Vertical vibrations applied once a day favorably affected bone volume fraction (BV/TV) and Ca2 +/PO43 − and decreased Rankl gene expression. When applied twice a day, v-vibrations diminished mineral content. Horizontal vibrations (1 ×/d) reduced Ca2 +/PO43 − ratio and Opg mRNA level, whereas h-vibration (2 ×/d) normalized OC serum levels. Many of the other measured parameters did not reveal any significant differences between the vibrated groups and the untreated Ovx group. The effect of ovariectomy was confirmed by atrophied uterus, impaired biomechanical properties, and bone mineral density and BV/TV of the vertebral body. The findings of the present study indicate that application frequency rate and direction of vibration might influence spine response differently. However, we were unable to find any clearly beneficial or harmful effect of vibration regimes on the osteopenic lumbar spine in rats.

Effects of 8-Prenylnaringenin and Whole-Body Vibration Therapy on a Rat Model of Osteopenia

Background. 8-Prenylnaringenin (8-PN) is the phytoestrogen with the highest affinity for estrogen receptor-α (ER-α), which is required to maintain BMD. The osteoprotective properties of 8-PN have been demonstrated previously in tibiae. We used a rat osteopenia model to perform the first investigation of 8-PN with whole-body vertical vibration (WBVV). Study Design. Ovariectomy was performed on 52 of 64 Sprague-Dawley rats. Five weeks after ovariectomy, one group received daily injections (sc) of 8-PN (1.77 mg/kg) for 10 weeks; a second group was treated with both 8-PN and WBVV (twice a day, 15 min, 35 Hz, amplitude 0.47 mm). Other groups received either only WBVV or no treatment. Methods. The rats were sacrificed 15 weeks after ovariectomy. Lumbar vertebrae and femora were removed for biomechanical and morphological assessment. Results. 8-PN at a cancer-safe dose did not cause fundamental improvements in osteoporotic bones. Treatment with 8-PN caused a slight increase in uterine wet weight. Combined therapy using WBVV and 8-PN showed no significant improvements in bone structure and biomechanical properties. Conclusion. We cannot confirm the osteoprotective effects of 8-PN at a cancer-safe dose in primary affected osteoporotic bones. Higher concentrations of 8-PN are not advisable for safety reasons. Adjunctive therapy with WBVV demonstrates no convincing effects on bones.

Effect of the lipoxygenase-inhibitors baicalein and zileuton on the vertebra in ovariectomized rats

Osteoporosis is one of the most common diseases worldwide. In osteoporosis, vertebral fractures represent a major burden. Lipoxygenase (LOX) inhibitors such as baicalein and zileuton may represent a promising therapeutic option owing to their antioxidative effects and suppression of various inflammatory processes in muscle and bone. The effect of these LOX inhibitors on the spine was studied in osteopenic rats. Female Sprague-Dawley rats were divided two times into five groups: four groups each were ovariectomized (OVX) and one control group was non-ovariectomized (NON-OVX). Eight weeks after ovariectomy, three concentrations of baicalein (1mg/kg body weight [BW], 10mg/kgBW, and 100mg/kgBW) were administered subcutaneously daily in three OVX groups for 4weeks. Similarly, zileuton was administered in three concentrations via food for 5weeks. In vivo computed tomography (pQCT) of the spine was performed before the treatments and at the end of the experiment. Lumbar vertebrae were subjected to a compression test, micro-CT, and ashing analyses. After baicalein treatment, cortical bone mineral density (BMD) was improved; trabecular connectivity and trabecular BMD were diminished at high dose. After zileuton treatment, the total BMD, anorganic weight, trabecular nodes, and trabecular area were improved. The in vivo stress-strain index was increased and alkaline phosphatase activity in serum was enhanced after both treatments. A dose-dependent effect was not clearly observed after both treatments. The treatments using baicalein for 4 and zileuton for 5weeks were not sufficient to change the biomechanical properties and bone volume fraction (BV/TV). Overall, baicalein improved the cortical bone parameters whereas zileuton had a favorable effect on the trabecular structure. Moreover, both treatments increased the bone formation rate. Longer trials, a combination of both LOX inhibitors, and their effect at the cellular and molecular levels should be investigated in further studies.

In Vivo siRNA Delivery Using JC Virus-like Particles Decreases the Expression of RANKL in Rats

Bone remodeling requires a precise balance between formation and resorption. This complex process involves numerous factors that orchestrate a multitude of biochemical events. Among these factors are hormones, growth factors, vitamins, cytokines, and, most notably, osteoprotegerin (OPG) and the receptor activator for nuclear factor-kappaB ligand (RANKL). Inflammatory cytokines play a major role in shifting the RANKL/OPG balance toward excessive RANKL, resulting in osteoclastogenesis, which in turn initiates bone resorption, which is frequently associated with osteoporosis. Rebalancing RANKL/OPG levels may be achieved through either upregulation of OPG or through transient silencing of RANKL by means of RNA interference. Here, we describe the utilization of a viral capsid-based delivery system for in vivo and in vitro RNAi using synthetic small interfering RNA (siRNA) molecules in rat osteoblasts. Polyoma JC virus-derived virus-like particles are capable of delivering siRNAs to target RANKL in osteoblast cells both in vitro and in a rat in vivo system. Expression levels were monitored using quantitative real-time polymerase reaction and enzyme-linked immunosorbent assay after single and repeated injections over a 14-day period. Our data indicate that this is an efficient and safe route for in vivo delivery of gene modulatory tools to study important molecular factors in a rat osteoporosis model.

Effect of the Lipoxygenase Inhibitor Baicalein on Muscles in Ovariectomized Rats

Sarcopenia, a loss of muscle mass accompanying osteoporosis, leads to falls and fall-related injuries. Baicalein, as a phytochemical agent, has an antioxidative and anti-inflammatory effect in muscle. In this study, sixty-one female Sprague Dawley rats were divided into five groups: four groups were ovariectomized (OVX) and one control group was nonovariectomized (NON-OVX). Eight weeks after ovariectomy, three disparate concentrations (1 mg/kg body weight (BW), 10 mg/kg BW, and 100 mg/kg BW) of baicalein were applied subcutaneously daily in three OVX groups. Mm. soleus, gastrocnemius, and longissimus were extracted; their diameter, area, relation to body, and muscle weights as well as number of capillaries per fibre were recorded. In Mm. soleus and gastrocnemius, the baicalein effect (increasing number of capillaries per fibre) was proportional to the dose applied. The fibre diameters and area under baicalein treatment were significantly greater compared to OVX and NON-OVX groups. In M. longissimus, we observed a shift to type IIa fibres. Serum creatine kinase levels were significantly lower in highest baicalein concentration group. We conclude that baicalein can stimulate angiogenesis, though not fibre type-specific, in skeletal muscle and reduce the estrogen-related loss of fibre diameter and area in the skeletal muscle in rats. Therefore, a protective effect of baicalein on muscle cells can be assumed.

Efficiency of 48h vs. 24h Injection of Parathyroid Hormone forAmelioration of Osteopenic Spine Properties in Male Rats

Daily application of parathyroid hormone (PTH 1-34) is used for treatment of osteoporosis. It was investigated whether orchiectomy-induced osteoporotic changes in spine can be ameliorated by every 48h administration of PTH in aged male rats. Eight-month-old male Sprague-Dawley rats were sham operated (n=24) or orchiectomized (Orx, n=36) and maintained untreated over 12 weeks. Thereafter, both tibia underwent transverse metaphyseal osteotomy (Komrakova et al. 2011, J Endocrinol; 209:9-19) and rats were divided into 5 groups treated s.c. as follows: 1) sham vehicle; 2) sham PTH every 24h (PTH/24h); 3) Orx vehicle; 4) Orx PTH/24h; 5) Orx PTH every 48h (PTH/48h). PTH dosage was 40 �g/kg BW per injection. After 5 weeks, lumbar vertebral bodies were used in computed tomographical, biomechanical, histomorphological, ashing and gene expression analyses. Cortical and trabecular densities, biomechanical properties, serum osteocalcin level increased significantly after PTH treatments in all groups (yield load, sham: 232+17N, sham PTH/24h: 376+12N, Orx: 239+16N, Orx PTH/24h: 324+31N, Orx PTH/48h: 297+17N). Bone inorganic weight enhanced after daily PTH application in Orx rats. Bone gene expression did not differ (P>0.05) among the groups. Both PTH administration regimes (24h and 48h) improved impaired bone structure in osteopenic rats. Every 48h application was less effective, however, it improved bone properties to the level observed in healthy (sham) rats. Considering limitation of daily treatments known in humans, these results may be useful for further clinical studies.