Oxytocin and bone quality in the femoral neck of rats in periestropause.

The objective of this study is to identify whether oxytocin (OT) contributes to the reduction of osteopenia in the femoral neck of rats in periestropause. Animals in irregular estrous cycles received two NaCl injections (0.15 mol/L) or OT (134 µg/kg) over a 12-h interval, and after thirty-five days without treatments, the biological sample collection was performed. The oxytocin group (Ot) demonstrated the highest enzymatic activity of alkaline phosphatase (p = 0.0138), lowest enzymatic activity of tartrate-resistant acid phosphatase (p = 0.0045), higher percentage of compact bone (p = 0.0359), cortical expression of runt-related transcription factor 2 (p = 0.0101), osterix (p = 0.0101), bone morphogenetic protein-2/4 (p = 0.0101) and periostin (p = 0.0455). Furthermore, the mineral-to-matrix ratio (ν1PO4/Proline) was higher and type-B carbonate substitution (CO3/ν1PO4) was lower (p = 0.0008 and 0.0303) in Ot group. The Ot showed higher areal bone mineral density (p = 0.0050), cortical bone area (p = 0.0416), polar moment of inertia, maximum, minimum (p = 0.0480, 0.0480, 0.0035), bone volume fraction (p = 0.0166), connectivity density (p < 0.0001), maximal load (p = 0.0003) and bone stiffness (p = 0.0145). In Ot percentage of cortical pores (p = 0.0102) and trabecular number (p = 0.0088) was lower. The results evidence action of OT in the reduction of osteopenia, suggesting that it is a promising anabolic strategy for the prevention of primary osteoporosis during the periestropause period.

Effect of pre-treatment of strength training and raloxifene in periestropause on bone healing.

BACKGROUND: There is evidence that strength training (ST) and raloxifene (Ral) treatment during periestropause promotes better bone quality. We wanted to determine whether the skeletal benefits of ST or Ral treatment, performed during periestropause, would persist after fracture. Therefore, the present study aimed to analyze the influence of pre-treatment with ST and administration of Ral during periestropause on bone healing after total unilateral osteotomy. METHODS: Senescent female Wistar rats between 18 and 21 months of age, performed ST on a ladder three times per week, were administered Ral by gavage (2.3 mg/kg/day), or an association of both. After 120 days, the treatments were interrupted, and a total osteotomy was performed on the left tibia in all animals. They were euthanized 1 and 8 weeks post-osteotomy. RESULTS: The administration of Ral during periestropause worsened the biochemical and oxidative profile, decreased gene expression of markers related to bone resorption and remodeling, which negatively affected the physicochemical properties; this lead to changes in the bone callus microarchitecture and mass, as well as a decrease in callus resistance to torsional deformation, resulting in lower tissue quality during bone healing. In contrast, ST performed prior to the osteotomy resulted in better bone healing, improvement of the biochemical and oxidative profile, alteration of the genetic profile in favor of bone formation and resorption, as well as the physic-ochemical properties of the callus. These changes led to better microarchitecture and bone mass and increased callus resistance to torsional deformation, confirming its beneficial effect on the quality of bone tissue, providing acceleration of bone consolidation. The combination of therapies at this exercise intensity and drug dosage showed a negative interaction, where the negative effect of Ral overcame the positive effect of ST, leading to decreased tissue quality in the bone healing process. CONCLUSIONS: This study indicates that in addition to excellent non-pharmacological therapy and action in the prevention of osteoporosis, ST performed during the aging period may increase bone quality at the onset of healing and provide improved bone consolidation. Furthermore, the anti-osteoclastogenic effect of Ral shown in this model delayed the bone repair process, resulting in considerable clinical concern.

Oxytocin effects on osteoblastic differentiation of Bone Marrow Mesenchymal Stem Cells from adult and aging female Wistar rats.

Recently, it has been suggested that oxytocin (OT) might play a role in the control of bone remodeling and in bone health of young and adult females. The purpose of this study was to evaluate the effect of osteogenic medium (OM) plus OT (OM + OT; 100 nmol/L) on osteoblastic differentiation of bone marrow mesenchymal stem cells (BMMSCs) from cyclic adult (12 months old) and acyclic aging (24 months old) female Wistar rats. After 14 days, OM + OT increased the oxytocin and oxytocin receptor in the BMMSCs from animals of both age groups relative to OM controls. Alkaline phosphatase activity was higher in the OM + OT than OM group in BMMSCs from 24-month-old female rats. OM + OT improved osteogenic differentiation, observed by anticipated mineralization and increased gene expression of bone morphogenetic protein 2, bone sialoprotein, osteopontin and osteocalcin in both aged relative to OM controls. These findings suggest a role for OT as an adjuvant to induce osteoblastic differentiation of BMMSCs from aged female rat.

Mate tea (Ilex paraguariensis) improves bone formation in the alveolar socket healing after tooth extraction in rats.

OBJECTIVES: The objective of this study was to investigate the effects of mate tea (MT) [Ilex paraguariensis] on alveolar socket healing after tooth extraction. MATERIALS AND METHODS: Sixteen male rats were divided into MT and control groups. MT was administered by intragastric gavage at a dose of 20 mg/kg/day for 28 days before and 28 days after right maxillary incisor extraction. The control group received an equal volume of water. Histopathological and histometric analysis of the neoformed bone area and osteocyte density were performed, as well as immunohistochemical analysis of osteocalcin (OCN), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), tartrate-resistant acid phosphatase (TRAP), and manganese superoxide dismutase (MnSOD) in the alveolar socket. Calcium, phosphorus, alkaline phosphatase (ALP) activity, total antioxidant capacity (TAC), and malondialdehyde (MDA) were measured in plasma, whereas TRAP activity was determined in serum. RESULTS: Histometry evidenced an increase in bone area (P < 0.0001) and osteocyte density (P < 0.0001). MT increased immunolabeling of MnSOD (P < 0.001), OCN (P < 0.0001), RANKL (P < 0.001), OPG (P < 0.0001), and TRAP (P < 0.001). Calcium and phosphorus concentrations did not differ between the groups. In addition, MT enhanced ALP (P < 0.05) and TRAP (P < 0.0001) activities. MT increased the TAC (P < 0.001), whereas it reduced MDA concentrations (P < 0.0001). CONCLUSIONS: MT increases bone area and osteocyte density in the alveolar socket healing on day 28 after tooth extraction. CLINICAL RELEVANCE: Regular MT ingestion improves the antioxidant defenses and bone formation, which is beneficial for alveolar socket bone healing after tooth extraction.

Model of hindlimb unloading in adult female rats: Characterizing bone physicochemical, microstructural, and biomechanical properties.

Prolonged bedrest and microgravity induce alterations to bone, leading to bone fragility and compromising the quality of life. In this study, we characterized the physicochemical changes, microstructure, and biomechanics of the femurs of female adult rats in response to hindlimb unloading for 21 days. Twenty 6-month-old Wistar female rats were distributed into control (CON) and hindlimb unloading (HLU) groups. Analysis the in vivo bone mineral density (BMD) by dual energy x-ray absorptiometry (DXA) from the femurs was performed at the beginning and end of the experiment; plasma levels of calcium, phosphorus, and alkaline phosphatase, tartrate-resistant acid phosphatase activity, assessed by spectrophotometry, and estradiol, measured by enzyme-linked immunosorbent assay, was performed after the experiment. We evaluated changes in the trabecular and cortical structure of the femur, after disuse, by micro-computed tomography with high resolution, for analysis of cortical porosity, Raman spectroscopy to measure the amount of physicochemical properties, and the biomechanical test to estimate the changes in biomechanical properties. Our results demonstrated that, after 21 days, HLU animals had decreased femoral BMD, deteriorated bone microarchitecture, particularly in the cortical compartment, with changes in the physicochemical properties and porosity, and reduced deformation capacity of the bone and resistance to the bone stresses. Nevertheless, this study showed the critical role of mechanical stimulation in maintaining the structure of the skeleton in female adults and that disuse, even for a few days, leads to microscopic changes in the structure of the bone matrix, which increases the risk of fracture.

Effects of strength training and raloxifene on femoral neck metabolism and microarchitecture of aging female Wistar rats.

The aim of this study was to prevent female osteoporosis using strength training (ST), raloxifene (Ral) or a combination of ST plus Ral during the natural female aging process, specifically in the periestropause period. For a total of 120 days, aging female Wistar rats at 18-21 months of age performed ST on a ladder three times per week, and Ral was administered daily by gavage (1 mg/kg/day). Bone microarchitecture, areal bone mineral density, bone strength of the femoral neck, immunohistochemistry, osteoclast and osteoblast surface were assessed. We found that the treatments modulate the bone remodeling cycle in different ways. Both ST and Ral treatment resulted in improved bone microarchitecture in the femoral neck of rats in late periestropause. However, only ST improved cortical microarchitecture and bone strength in the femoral neck. Thus, we suggest that performing ST during the late period of periestropause is a valid intervention to prevent age-associated osteoporosis in females.

llex paraguariensis decreases oxidative stress in bone and mitigates the damage in rats during perimenopause.

During perimenopause, oxidative stress increases, which may result in disruption of bone turnover, and consequently in osteoporosis. The use of antioxidants may be an effective nutritional approach to reducing osteoporosis in this period of life. Mate tea (MT) (Ilex paraguariensis), a typical and inexpensive beverage consumed in the Brazilian south-east, Argentina and Uruguay, increases antioxidant defense. Our hypothesis was that MT would decrease oxidative stress and mitigate bone deterioration. To test this, we analyzed oxidative stress markers of bone turnover, and local and systemic markers of bone metabolism of rats during natural perimenopause. Female Wistar rats (aged 16months) in proven perimenopause period received 20mg/kgBW/day of mate tea, by gavage (PM+MT Group, n=10) or water (PM Group, n=10). Female rats aged 4months (AD Group, n=10) received water. The treatment period was four weeks. MT minimized the deterioration of rat microarchitecture, characterized by increase in the bone trabecular area, number of osteocytes and areal bone mineral density. These results were accompanied by a lower level of malondialdehyde, an oxidative stress marker, in femoral tissue homogenate. Plasmatic tartrate-resistant acid phosphatase, a typical osteoclastic function marker, decreases after treatment, indicating a decrease in osteoclastic function. MT also modified the immunostaining pattern of bone metabolism markers, decreasing the receptor activator of nuclear factor kappa-B ligant (RANKL), superoxide dismutase isoform 2 (SOD2) and increasing osteoprotegerin (OPG), a decoy receptor for the RANKL, which positively modulates bone mass. These results suggested MT was capable of decreasing bone resorption by inhibiting the osteoclastogenesis in a RANKL-dependent signaling pathway activated by oxidative stress. Taken together, the results indicated that MT minimized bone loss in perimenopause and this effect is at least partly due to the decrease in oxidative stress, confirming our hypothesis.

Effects of strength training on osteogenic differentiation and bone strength in aging female Wistar rats.

The effects of strength training (ST) on the mechanical bone strength and osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) from adult, aged and exercised aged rats were determined. The exercised aged animals displayed higher values of areal bone mineral density, compression test, alkaline phosphatase activity (ALP) and biological mineralization, while oil red O staining for adipocytes was lower. ST increased gene expression of runt-related transcription factor 2 (Runx2), osterix (Osx) as well as bone matrix protein expression, and reduced expression of peroxisome proliferator-activated receptor gamma (Pparγ). The production of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) was lower in BMSCs of the aged exercised group. The ST practice was able to improve the bone mechanical properties in aged female rats, increasing the potential for osteogenic differentiation of BMSCs, reducing the adipogenic differentiation and pro-inflammatory cytokine level. In summary, the data achieved in this study showed that strength training triggers physiological responses that result in changes in the bone microenvironment and bring benefits to biomechanical parameters of bone tissue, which could reduce the risk of fractures during senescent.

Influência do treinamento de força associado ou não ao raloxifeno sobre o perfil transcricional e microestrutural ósseo de ratas Wistar naturalmente envelhecidas / Influence of strength training associated or not with raloxifene on transcriptional and microstructural bone profile of naturally aged female Wistar rats

A ocorrência de doenças crônicas e degenerativas é significativamente maior nos organismos durante o envelhecimento, dentre elas, a osteoporose, que resulta em aumento no número de fraturas. As fraturas são as consequências mais dramáticas da osteoporose, sendo que do colo do fêmur é a mais severa, com maior incidência de morbidades e mortalidade. A menor concentração plasmática de estrogênio nas mulheres menopausadas, exerce ação primordial no desenvolvimento desta doença. Desta maneira, o objetivo deste estudo foi estudar a prevenção da osteoporose em decorrência do envelhecimento reprodutivo feminino, especificamente no período de periestropausa, utilizando treinamento de força (TF), raloxifeno (Ral) ou combinação de TF e Ral. Durante 120 dias, ratas Wistar no período do envelhecimento (18 a 21 meses) realizaram TF em escada três vezes por semana, receberam Ral (1mg/Kg/dia) por gavagem, ou realizaram TF associado ao tratamento com Ral. Microarquitetura óssea cortical e trabecular, densidade mineral óssea areal (DMOa), força óssea, imunoistoquímica (OCN, TRAP e SOST) e superfície de osteoclastos do colo do fêmur foram avaliadas, além de PCR (Runx2, Sp7, Alp, Bsp, Ocn, Rank, Rankl, Opg, Trap e Ctsk) e Western Blot (p-ERα e TRAP) do tecido ósseo de todo o fêmur. Os resultados demonstram que os tratamentos modularam o ciclo de remodelamento ósseo de maneiras diferentes: TF estimulou RNAm de marcadores osteoblásticos e osteoclásticos, enquanto Ral diminuiu marcadores osteoclásticos e TF associado a Ral aumentou marcadores osteoblásticos e diminuiu osteoclásticos. Ambos tratamentos resultaram em melhora da microarquitetura trabecular do colo do fêmur de ratas na periestropausa, todavia, apenas o TF foi capaz de melhorar além da microarquitetura trabecular, a cortical e força óssea. Desta maneira, sugerimos que a realização de TF, utilização de Ral ou a associação de TF e Ral durante a periestropausa são intervenções válidas na prevenção de osteoporose em decorrência do envelhecimento reprodutivo feminino, porém os efeitos do TF parecem ser superiores. Levando em consideração que a carga mecânica gerada pelo TF age também em tecidos não esqueléticos, concluímos que TF pode ser intervenção sistêmica para osteoporose. Esses resultados adicionam novas informações à literatura sobre terapêuticas preventivas para osteoporose e fornecem informações relevantes para estudos pré-clínicos(AU)

The association of aging with osteoporosis results in an increased number of fractures. In these fractures, the femoral neck is involved in 75% of affected women and is one of the most dramatic possible consequences. The aim of this study was to prevent female osteoporosis using strength training (ST), raloxifene (Ral) or a combination of ST plus Ral during the natural female aging process, specifically in the periestropause period. For 120 total days, aging female Wistar rats at 18-21 months of age performed ST on three times per week, and Ral was administered daily by gavage (1mg/kg/day). Bone microarchitecture, areal bone mineral density (aBMD), bone strength of the femoral neck, immunohistochemistry, western blotting (p-ERα and TRAP) and RT-PCR were assessed. We found that the treatments modulate the bone remodeling cycle in different ways. Both ST and Ral treatment resulted in improved bone microarchitecture in the femoral neck of rats in late periestropause. However, only ST improved cortical microarchitecture and bone strength in the femoral neck. In addition, ST stimulated mRNA levels of osteoclastic and osteoblastic markers, while Ral decreased mRNA levels of osteoclastic markers. The combined ST plus Ral therapy increased osteoblastic markers and decreased osteoclast markers. In this way, we suggest that SF, the use of Ral or the association of ST and Ral during periestropause are valid interventions in the prevention of osteoporosis due to female reproductive aging, but the effects of ST seem to be superior, taking into account that the mechanical load generated by ST also acts on nonskeletal tissues, we conclude that ST can be a systemic intervention for osteoporosis. These results add new information to the literature on preventive therapies for osteoporosis and provide relevant information for preclinical studies(AU)

Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis.

OBJECTIVE: We investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) from young and aged rats cultured on Ti. MATERIAL AND METHODS: Bone marrow MSCs derived from 1-month and 21-month rats were cultured on Ti discs under osteogenic conditions for periods of up to 21 days and osteoblast and adipocyte markers were evaluated. RESULTS: Cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of RUNX2, osterix, ALP, bone sialoprotein, osteopontin, and osteocalcin were reduced in cultures of 21-month rats compared with 1-month rats grown on Ti. Gene expression of PPAR-γ , adipocyte protein 2, and resistin and lipid accumulation were increased in cultures of 21-month rats compared with 1-month rats grown on the same conditions. CONCLUSIONS: These results indicate that the lower osteogenic potential of MSCs derived from aged rats compared with young rats goes along with the higher adipogenic potential in cultures grown on Ti surface. This unbalance between osteoblast and adipocyte differentiation should be considered in dental implant therapy to the elderly population.

Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis

ABSTRACT Aging negatively affects bone/titanium implant interactions. Our hypothesis is that the unbalance between osteogenesis and adipogenesis induced by aging may be involved in this phenomenon. Objective We investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) from young and aged rats cultured on Ti. Material and Methods Bone marrow MSCs derived from 1-month and 21-month rats were cultured on Ti discs under osteogenic conditions for periods of up to 21 days and osteoblast and adipocyte markers were evaluated. Results Cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of RUNX2, osterix, ALP, bone sialoprotein, osteopontin, and osteocalcin were reduced in cultures of 21-month rats compared with 1-month rats grown on Ti. Gene expression of PPAR-γ , adipocyte protein 2, and resistin and lipid accumulation were increased in cultures of 21-month rats compared with 1-month rats grown on the same conditions. Conclusions These results indicate that the lower osteogenic potential of MSCs derived from aged rats compared with young rats goes along with the higher adipogenic potential in cultures grown on Ti surface. This unbalance between osteoblast and adipocyte differentiation should be considered in dental implant therapy to the elderly population.

The effects of strength training and raloxifene on bone health in aging ovariectomized rats.

The aim of this study was to investigate the effects of strength training (ST) and raloxifene (Ral), alone or in combination, on the prevention of bone loss in an aging estrogen-deficient rat model. Aging Wistar female rats were ovariectomized at 14months and allocated to four groups: (1) non-trained and treated with vehicle, NT-Veh; (2) strength training and treated with vehicle, ST-Veh; (3) non-trained and treated with raloxifene, NT-Ral; and (4) strength training and treated with raloxifene, ST-Ral. ST was performed on a ladder three times per week and Ral was administered daily by gavage (1mg/kg/day), both for 120days. Areal bone mineral density (aBMD), strength, microarchitecture, and biomarkers (osteocalcin, OCN; osteoprotegerin, OPG; and tartrate-resistant acid phosphatase, TRAP) were assessed. Immunohistochemistry was performed for runt-related transcription factor 2 (RUNX2), osterix (OSX), OCN, OPG, TRAP, and receptor activator of nuclear factor kappa-B ligand (RANKL). The rats that performed ST (ST-Veh) or were treated with Ral (NT-Ral) showed significant improvements in aBMD (p=0.001 and 0.004), bone strength (p=0.001), and bone microarchitecture, such as BV/TV (%) (p=0.001), BS/TV (mm(2)/mm(3)) (p=0.023 and 0.002), Conn.Dn (1/mm(3)) (p=0.001), Tb.N (1/mm) (p=0.012 and 0.011), Tb.Th (1/mm) (p=0.001), SMI (p=0.001 and 0.002), Tb.Sp (p=0.001), and DA (p=0.002 and 0.007); there was also a significant decrease in plasma levels of OCN (p=0.001 and 0.002) and OPG (p=0.003 and 0.014), compared with animals in the NT-Veh group. Ral, with or without ST, promoted an increased immunolabeling pattern for RUNX2 (p=0.0105 and p=0.0006) and OSX (p=0.0105), but a reduced immunolabeling pattern for TRAP (p=0.0056) and RANKL (p=0.033 and 0.004). ST increased the immunolabeling pattern for RUNX2 (p=0.0105), and association with Ral resulted in an increased immunolabeling pattern for OPG (p=0.0034) and OCN (p=0.0024). In summary, ST and Ral administration in aged, estrogen-deficient Wistar female rats is associated with a decrease in bone turnover marker plasma levels, increased activity of cells that promote osteoblastogenesis, and decreased activity of cells that promote osteoclastogenesis; these are correlated with higher aBMD, bone strength, and bone microarchitecture at the femoral neck. The results indicate that strength training and Ral are potential tools to reduce the risk of fractures at clinically relevant sites.