Bone regeneration by systemic transplantation of mesenchymal stem cells (MSCs) is problematic due to the inability to control the MSCs’ commitment, growth and differentiation into functional osteoblasts on the bone surface. Our research group has developed a method to direct the MSCs to the bone surface by conjugating a synthetic peptidomimetic ligand (LLP2A) that has high affinity for activated α4β1 integrin on the MSC surface, with a bisphosphonates (alendronate) that has high affinity for bone (LLP2A-Ale), to direct the transplanted MSCs to bone. Our in vitro experiments demonstrated that mobilization of LLP2A-Ale to hydroxyapatite accelerated MSC migration that was associated with an increase in the phosphorylation of Akt kinase and osteoblastogenesis. LLP2A-Ale increased the homing of the transplanted MSCs to bone as well as the osteoblast surface, significantly increased the rate of bone formation and restored both trabecular and cortical bone loss induced by estrogen deficiency or advanced age in mice. These results support LLP2A-Ale as a novel therapeutic option to direct the transplanted MSCs to bone for the treatment of established bone loss related to hormone deficiency and aging.

Anti-resorptive and anabolic agents are often prescribed for the treatment of osteoporosis continuously or sequentially for many years. However their impact on cortical bone quality and bone strength is not clear. Methods Six-month old female rats were either sham operated or ovariectomized (OVX). OVX rats were left untreated for two months and then were treated with vehicle (Veh), hPTH (1-34) (PTH), alendronate (Aln), or raloxifene (Ral) sequentially for three month intervals, for a total of three periods. Mid-tibial cortical bone architecture, mass, mineralization, and strength were measured on necropsy samples obtained after each period. Bone indentation properties were measured on proximal femur necropsy samples. Results Eight or more months of estrogen deficiency in rats resulted in decreased cortical bone area and thickness. Treatment with PTH for 3 months caused the deposition of endocortical lamellar bone that increased cortical bone area, thickness, and strength. These improvements were lost when PTH was withdrawn without followup treatment, but were maintained for the maximum times tested, six months with Ral and three months with Aln. Pre-treatment with anti-resorptives was also somewhat successful in ultimately preserving the additional endocortical lamellar bone formed under PTH treatment. These treatments did not affect bone indentation properties. Summary Sequential therapy that involved both PTH and anti-resorptive agents was required to achieve lasting improvements in cortical area, thickness, and strength in OVX rats. Anti-resorptive therapy, either prior to or following PTH, was required to preserve gains attributable to an anabolic agent.

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Introduction Individual agents used to treat human osteoporosis reduce fracture risk by ~50-60%. Since agents that act with complementary mechanisms are available, sequential therapies that mix anti-resorptive and anabolic agents could improve fracture risk reduction, when compared to monotherapies. Methods We evaluated bone mass, bone microarchitecture, and bone strength in adult ovariectomized (OVX), osteopenic rats, during different sequences of vehicle (Veh), parathyroid hormone (PTH), alendronate (Aln), or raloxifene (Ral) in three 90 day treatment periods, over nine months. Differences among groups were evaluated. The interrelationships of bone mass and microarchitecture endpoints, and their relationship to bone strength were studied. Results Estrogen deficiency caused bone loss. OVX rats treated with Aln monotherapy had significantly better bone mass, microarchitecture, and bone strength than untreated OVX rats. Rats treated with an Aln drug holiday had bone mass and microarchitecture similar to the Aln monotherapy group, but with significantly lower bone strength. PTH-treated rats had markedly higher bone endpoints, but all were lost after PTH withdrawal without follow-up treatment. Rats treated with PTH followed by Aln had better bone endpoints than those treated with Aln monotherapy, PTH monotherapy, or an Aln holiday. Rats treated initially with Aln or Ral, then switched to PTH, also had better bone endpoints, than monotherapy treatment. Rats treated with Aln, then PTH, and returned to Aln had the highest values for all endpoints. Conclusion Our data indicate that anti-resorptive therapy can be coupled with an anabolic agent, to produce and maintain better bone mass, microarchitecture, and strength than can be achieved with any monotherapy.

Unperturbed fetal development is essential for future health of an individual. Previous studies have linked diseases of aging to harmful alterations that happen during fetal development. Given the significant long-term impact that intrauterine environment has on an individual's life, it was hypothesized that maternal stress during pregnancy will have negative effects on the offspring's prenatal and postnatal growth. To test this, twenty-eight female and seven male Wistar rats (Rattus norvegicus) were purchased and bred to produce 176 offspring. During pregnancy, dams were randomly divided into four groups (n=7, per group) and immobilization stress induced as follows; Group 1 (GW1): immobilization stress on days 1-7 of pregnancy, Group 2 (GW2): on days 8-14, Group 3 (GW3): on days 15-21, Group 4 (Controls): left undisturbed. Maternal cortisol hormone, food intake, and weight gain were monitored during pregnancy. Pups were raised under normal laboratory conditions and sacrificed at ages: 4, 8, 12, and 16 weeks to determine the effect of prenatal stress. At necropsy, the tibia was removed and processed for histology. Differences among groups were determined by T-test or analysis of variance (ANOVA). Linear regression analysis was performed to establish the relationship between stress in utero and indicators of bone development in offspring. P values ≤ 0.05 were considered significant. Cortisol hormone levels in controls were lower than those of stressed animals. Stressed dams consumed 12.5% less food per day compared to controls. Animals in GW1 and GW2 gained less weight during pregnancy but had larger litters than did GW3 or the control group. Offspring born to GW3 were heavier compared to all other groups. GW3 offspring had a higher rate of bone formation. In conclusion, stress during pregnancy resulted in increased cortisol and reduced food intake in mothers, but faster growth and higher weight gain in offspring compared to controls.

Introduction While the anti-resorptive effects of the bisphosphonates (BPs) are well documented, many questions remain about their mechanisms of action, particularly following long-term use. This study evaluated the effects of alendronate (Ale) treatment on TGF-β1 signaling in mesenchymal stem cells (MSCs) and osteocytes, and the relationship between prolonged alendronate treatment on systemic TGF-β1 levels and bone strength. Methods TGF-β1 expression and signaling were evaluated in MSCs and osteocytic MLO-Y4 cells following Ale treatment. Serum total TGF-β1 levels, a bone resorption marker (DPD/Cr), three-dimensional microCT scans and biomechanical tests from both the trabecular and cortical bone were measured in ovariectomized rats that either received continuous Ale treatment for 360 days or Ale treatment for 120 days followed by 240 days of vehicle. Linear regression tests were performed to determine the association of serum total TGF-β1 levels and both the trabecular (vertebrae) and cortical (tibiae) bone strength. Results Ale increased TGF- β1 signaling in the MSCs but not in the MLO-Y4 cells. Ale treatment increased serum TGF-β1 levels and the numbers of TGF-β1-positive osteocytes and periosteal cells in cortical bone. Serum TGF-β1 levels were not associated with vertebral maximum load and strength but was negatively associated with cortical bone maximum load and ultimate strength. Conclusions The increase of serum TGF-β1 levels during acute phase of estrogen deficiency is likely due to increased osteoclast-mediated release of matrix-derived latent TGF- β1. Long-term estrogen-deficiency generally results in a decline in serum TGF-β1 levels that are maintained by Ale treatment. Measuring serum total TGF-β1 levels may help to determine cortical bone quality following alendronate treatment.

Anti-resorptive and anabolic treatments can be used sequentially to treat osteoporosis, but their effects on bone composition are incompletely understood. Osteocytes may influence bone tissue composition with sequential therapies because bisphosphonates diffuse into the canalicular network and anabolic treatments increase osteocyte lacunar size. Cortical bone composition of osteopenic, ovariectomized (OVX) rats was compared to that of Sham-operated rats and OVX rats given monotherapy or sequential regimens of single approved anti-osteoporosis medications. Adult female Sprague-Dawley rats were OVX (N = 37) or Sham-OVXd (N = 6). After 2 months, seven groups of OVX rats were given three consecutive 3-month periods of treatment with vehicle (V), h-PTH (1-34) (P), alendronate (A), or raloxifene (R), using the following orders: VVV, PVV, RRR, RPR, AAA, AVA, and APA. Compositional properties around osteocyte lacunae of the left tibial cortex were assessed from Raman spectra in perilacunar and non-perilacunar bone matrix regions. Sequential treatments involving parathyroid hormone (PTH) caused lower mean collagen maturity relative to monotherapies. Mean mineral:matrix ratio was 2.2% greater, mean collagen maturity was 1.4% greater, and mean carbonate:phosphate ratio was 2.2% lower in the perilacunar than in the non-perilacunar bone matrix region (all P < 0.05). These data demonstrate cortical bone tissue composition differences around osteocytes caused by sequential treatment with anti-osteoporosis medications. We speculate that the region-specific differences demonstrate the ability of osteocytes to alter bone tissue composition adjacent to lacunae.

Cigarette smoking is extremely common among pregnant women. The main active ingredient in cigarettes is nicotine. Nicotine is known to have negative effects on the cardiovascular system, respiratory system and birth weight among others. Maternal smoking during pregnancy has also been found to affect dental development in the offspring. The objective of this project was to assess the dose dependent effect of prenatal nicotine exposure on dental development in humans using rats (Rattus norvegicus) as an animal model. We hypothesized that maternal prenatal exposure to nicotine would affect dental development in the offspring. We also hypothesized that nicotine exposure in the early developmental stages would have long lasting negative effects on dental development. Rats are an ideal animal model for this study because of their short lifespan and continuously growing incisors.To test these hypotheses, 12 female and six male Long Evans rats were purchased and bred. During the 21 days of pregnancy, dams were randomly divided into four groups (n=2, per group) and injected subcutaneously with different concentrations of nicotine hydrogen tartrate (Sigma CAS Number 65‐31‐6) as follows; group 1 received a low dose of 1 mg/kg, group 2 received a medium dose of 2 mg/kg and group 3 received a high dose of 4 mg/kg. Maternal food intake and weight gain were monitored during pregnancy. At birth, pups were raised under normal laboratory conditions and weaned after three weeks.Teeth measurements are ongoing. Measurements are taken once a week and will continue for a period of 8 weeks. The rats are anaesthetized by ether. Grooves are then made on the labial surface of the right mandibular incisor teeth by means of a jeweller's file; this procedure is repeated whenever the grooves are abraded. The distance between the groove and the free gingival margin covering the interincisal septum is measured by vernier calipers, the latter being taken as the datum point. This is repeated at intervals of 2 and 7 days.Statistical analysisThe differences among groups on maternal food intake, weight gain and litter size were determined by analysis of variance. P values ≤ 0.05 were considered significant.ResultsThere was a significant difference between the experimental groups in terms of maternal weight gain (p≤0.05). However, there was no difference among the four groups when it came to food intake. Analysis of dental development is ongoing and results will be available and ready for presentation at the annual meeting.ConclusionFrom the current data, high dose nicotine treatment negatively impacted weight gain during pregnancy. Compared to the control group, the high dose treatment group gained the least amount of weight and had the smallest litter size. However nicotine treatment did not seem to affect food intake. More research is underway to determine the mechanisms underlying these observations.Support or Funding InformationNone Food Intake During PregnancyimageFood Intake During Pregnancy Weight Gain During PregnancyimageWeight Gain During PregnancyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.