Reduced Bone Volume Research Articles

Effects of locally applied adipose tissue-derived microvascular fragments by thermoresponsive hydrogel on bone healing

Insufficient vascularization is a major cause for the development of non-unions. To overcome this problem, adipose tissue-derived microvascular fragments (MVF) may serve as vascularization units. However, their application into bone defects needs a carrier system. Herein, we analyzed whether this is achieved by a thermoresponsive hydrogel (TRH). MVF were isolated from CD-1 mice and cultivated after incorporation into TRH, while non-incorporated MVF served as controls. Viability of MVF was assessed immunohistochemically over a 7-day period. Moreover, osteotomies were induced in femurs of CD-1 mice. The osteotomy gaps were filled with MVF-loaded TRH (TRH + MVF), unloaded TRH (TRH) or no material (control). Bone healing was evaluated 14 and 35 days postoperatively. MVF incorporated into TRH exhibited less apoptotic cells and showed a stable vessel morphology compared to controls. Micro-computed tomography revealed a reduced bone volume in TRH + MVF femurs. Histomorphometry showed less bone and more fibrous tissue after 35 days in TRH + MVF femurs compared to controls. Accordingly, TRH + MVF femurs exhibited a lower osseous bridging score and a reduced bending stiffness. Histology and Western blot analysis revealed an increased vascularization and CD31 expression, whereas vascular endothelial growth factor (VEGF) expression was reduced in TRH + MVF femurs. Furthermore, the callus of TRH + MVF femurs showed increased receptor activator of NF-κB ligand expression and higher numbers of osteoclasts. These findings indicate that TRH is an appropriate carrier system for MVF. Application of TRH + MVF increases the vascularization of bone defects. However, this impairs bone healing, most likely due to lower VEGF expression during the early course of bone healing. Statement of significanceIn the present study we analyzed for the first time the in vivo performance of a thermoresponsive hydrogel (TRH) as a delivery system for bioactive microvascular fragments (MVF). We found that TRH represents an appropriate carrier for MVF as vascularization units and maintains their viability. Application of MVF-loaded TRH impaired bone formation in an established murine model of bone healing, although vascularization was improved. This unexpected outcome was most likely due to a reduced VEGF expression in the early phase bone healing.

Partial-width injuries of the rat rotator cuff heal with fibrosis

ABSTRACTPurpose: Identify the healing outcomes following a partial-width, full-thickness injury to the rotator cuff tendon-bone attachment and establish if the adult attachment can regenerate the morphology of the healthy attachment.Hypothesis: We hypothesized that a partial-width injury to the attachment would heal via fibrosis and bone remodeling, resulting in increased cellularity and extra-cellular matrix deposition, reduced bone volume (BV), osteoclast presence, and decreased collagen organization compared to shams.Materials and Methods: A partial-width injury was made using a biopsy punch at the center one-third of the rat infraspinatus attachment. Contralateral limbs underwent a sham operation. Rats were sacrificed at 3 and 8 weeks after injury for analyses. Analyses performed at each time point included cellularity (Hematoxylin & Eosin), ECM deposition (Masson’s Trichrome), BV (micro-computed tomography; microCT), osteoclast activity (Tartrate Resistant Acid Phosphatase; TRAP), and collagen fibril organization (Picrosirius Red). Injured and sham shoulders were compared at both 3 and 8 weeks using paired, two-way ANOVAs with repeated measures (Sidak’s correction for multiple comparisons).Results: Cellularity and ECM deposition increased at both 3 and 8 weeks compared to sham contralateral attachments. BV decreased and osteoclast presence increased at both 3 and 8 weeks compared to sham contralateral limbs. Collagen fibril organization was reduced at 3 weeks after injury compared to 3-week sham attachments.Conclusions: These findings suggest that a partial-width injury to the rotator cuff attachment does not fully regenerate the native structure of the healthy attachment. The injury model healed via scar-like fibrosis and did not propagate into a full-width tear after 8 weeks of healing.

PHOSPHO1 is essential for normal bone fracture healing: An Animal Study.

ObjectivesBone fracture healing is regulated by a series of complex physicochemical and biochemical processes. One of these processes is bone mineralization, which is vital for normal bone development. Phosphatase, orphan 1 (PHOSPHO1), a skeletal tissue-specific phosphatase, has been shown to be involved in the mineralization of the extracellular matrix and to maintain the structural integrity of bone. In this study, we examined how PHOSPHO1 deficiency might affect the healing and quality of fracture callus in mice.MethodsTibial fractures were created and then stabilized in control wild-type (WT) and Phospho1-/- mice (n = 16 for each group; mixed gender, each group carrying equal number of male and female mice) at eight weeks of age. Fractures were allowed to heal for four weeks and then the mice were euthanized and their tibias analyzed using radiographs, micro-CT (μCT), histology, histomorphometry and three-point bending tests.ResultsThe μCT and radiographic analyses revealed a mild reduction of bone volume in Phospho1-/- callus, although it was not statistically significant. An increase in trabecular number and a decrease in trabecular thickness and separation were observed in Phospho1-/- callus in comparison with the WT callus. Histomorphometric analyses showed that there was a marked increase of osteoid volume over bone volume in the Phospho1-/- callus. The three-point bending test showed that Phospho1-/- fractured bone had more of an elastic characteristic than the WT bone.ConclusionOur work suggests that PHOSPHO1 plays an integral role during bone fracture repair and may be a therapeutic target to improve the fracture healing process.Cite this article: M. W. Morcos, H. Al-Jallad, J. Li, C. Farquharson, J. L. Millán, R. C. Hamdy, M. Murshed. PHOSPHO1 is essential for normal bone fracture healing: An Animal Study. Bone Joint Res 2018;7:397–405. DOI: 10.1302/2046-3758.76.BJR-2017-0140.R2.

CD109 deficiency induces osteopenia with an osteoporosis-like phenotype in vivo.

Osteoporosis is a global public health problem that is increasing along with an aging population. A major determinant of osteoporosis is high bone turnover, which results from osteoclast activation. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, a deficiency that leads to a psoriasis-like skin inflammation in mice. Although the expression of CD109 has been reported in mouse pre-osteoclast cells, its function in osteoclasts in vivo remains largely unknown. To investigate the physiological role of CD109 in bone metabolism, we analyzed bones from wild-type and CD109-deficient adult mice. Micro-computed tomography analysis of the femur (thigh bone) showed that bone volume was lower in CD109-deficient mice than in wild-type mice. Bone histomorphometric analysis showed not only a reduction in bone volume but also an increase in bone turnover in CD109-deficient mice as compared with wild-type mice. Additionally, we measured serum levels of several markers of bone turnover and found a significant increase in the N-terminal telopeptide of type I collagen, a bone resorption marker, as well as alkaline phosphatase, a bone formation marker, in CD109-deficient mice. These results indicate that CD109 deficiency induces a high-turnover, osteoporosis-like phenotype, which suggests that CD109 plays a role in bone metabolism in vivo.

Green tea prevents vascular disturbs and attenuates periodontal breakdown in long-term hyperglycaemia in T1D rats.

The effects of green tea on the modulation of vascularization during the progression of spontaneous periodontitis in long-term hyperglycaemia in streptozotocin-induced type 1 diabetic (T1D) rats were evaluated. Wistar rats normoglycaemic (NG) and T1D were divided into two control groups, which received water (NG-W and T1D-W) and two experimental groups that received green tea (NG-GT and T1D-GT). Periodontal structures were evaluated by microtomographic and histological analyses. Number of immunostained cells for VEGF (NcVEGF+/mm2 ) and CD31 (NcCD31+/mm2 ), as well microvessel density (MVD) in the periodontal ligament (PDL) were evaluated. Long-term hyperglycaemia in T1D-W rats induced vascular alterations in PDL with a reduction of 36% in MVD, a decrease of 33% in NcCD31+/mm2 and an increase of 53% in NcVEGF+/mm2 . Concomitantly, a severe degree of periodontitis with higher reduction in bone volume and periodontal bone level was observed. In T1D-GT, green tea maintained the MVD, NcCD31+/mm2 and NcVEGF+/mm2 in the PDL similar to normoglycaemic groups. Clinically, in T1D-GT rats, green tea reduced dental plaque accumulation and the degree of periodontitis when compared to T1D-W. Daily green tea consumption has a therapeutic effect on the diabetic vascular disorder in PDL and the progression of periodontitis in long-term hyperglycaemia in T1D rats.

Differential effects of serotonin reuptake inhibitors fluoxetine and escitalopram on bone markers and microarchitecture in Wistar rats

Evidence from several studies indicates that the long-term treatment of selective serotonin reuptake inhibitors (SSRIs) is associated with a decrease in bone mass and increase the risk of fractures. The present work evaluated and compared the effect of treatment with two SSRIs viz. fluoxetine and escitalopram on bone biomarkers (P1NP and βCTX) in male Wistar rats. In addition, the effect of these drugs on bone microarchitecture of lumbar and tibia bones was carried out. Fluoxetine (8.2 mg/kg) treatment for 40 days significantly reduced (P < 0.01) the levels of the P1NP while escitalopram (2.0 mg/kg) was without such effects. Both drugs were devoid of any effects on bone resorption marker βCTX. The pCREB levels were reduced by both the antidepressants but the reduction was significantly (P < 0.001) marked in case of fluoxetine. The micro-CT data revealed that fluoxetine, but not escitalopram, treatment resulted in reduced bone volume fraction, trabecular thickness and number while increased trabecular separation, trabecular pattern factor and connectivity density in the proximal tibial metaphysis. No significant changes were, however, discernible in lumbar bones. The study shows that fluoxetine reduces bone formation possibly through reduced pCREB mediated by the action of gut serotonin in osteoblasts and that escitalopram can be a better treatment option as far as adverse effects on bone are concerned.

Intermittent PTH 1-34 administration improves the marrow microenvironment and endothelium-dependent vasodilation in bone arteries of aged rats.

Inflammation coincides with diminished marrow function, vasodilation of blood vessels, and bone mass. Intermittent parathyroid hormone (PTH) administration independently improves marrow and vascular function, potentially impacting bone accrual. Currently, the influence of marrow and intermittent PTH administration on aged bone blood vessels has not been examined. Vasodilation of the femoral principal nutrient artery (PNA) was assessed in the presence and absence of marrow. Furthermore, we determined the influence of PTH 1-34 on 1) endothelium-dependent vasodilation and signaling pathways [i.e., nitric oxide (NO) and prostacyclin (PGI2)], 2) endothelium-independent vasodilation, 3) cytokine production by marrow cells, and 4) bone microarchitecture and bone static and dynamic properties. Young (4-6 mo) and old (22-24 mo) male Fischer-344 rats were treated with PTH 1-34 or a vehicle for 2 wk. In the absence and presence of marrow, femoral PNAs were given cumulative doses of acetylcholine, with and without the NO and PGI2 blockers, and diethylamine NONOate. Marrow-derived cytokines and bone parameters in the distal femur were assessed. Exposure to marrow diminished endothelium-dependent vasodilation in young rats. Reduced bone volume and NO-mediated vasodilation occurred with old age and were partially reversed with PTH. Additionally, PTH treatment in old rats restored endothelium-dependent vasodilation in the presence of marrow and augmented IL-10, an anti-inflammatory cytokine. Endothelium-independent vasodilation was unaltered, and PTH treatment reduced osteoid surfaces in old rats. In conclusion, the marrow microenvironment reduced vascular function in young rats, and PTH treatment improved the marrow microenvironment and vasodilation with age. NEW & NOTEWORTHY This study investigated the influence of the marrow microenvironment on bone vascular function in young and old rats. An inflamed marrow microenvironment may reduce vasodilator capacity of bone blood vessels, diminishing delivery of blood flow to the skeleton. In young rats, the presence of the marrow reduced vasodilation in the femoral principal nutrient artery (PNA). However, intermittent parathyroid hormone administration (i.e., a treatment for osteoporosis) improved the marrow microenvironment and vasodilator capacity in old PNAs.

Loading of the Condylar Cartilage Can Rescue the Effects of Botox on TMJ.

The purpose of this study is to evaluate whether the effects of botulinum neurotoxin (botox) injection into the masseter in the mandibular condylar cartilage (MCC) and subchondral bone could be rescued by compressive loading of the temporomandibular joint (TMJ). Twenty-four 6-week-old female mice (C57BL/6J) were used. Mice were divided in three groups: (1) Botox (n = 8); (2) Botox plus loading (n = 8); (3) Pure control (n = 8). Bone labels (3 and 1day before sacrifice) and the proliferation marker EdU (2 and 1day before sacrifice) were intraperitoneally injected into all groups of mice. Condyles were dissected and examined by micro-CT and histology. Sagittal sections of condyles were stained for TRAP, alkaline phosphatase, EdU, TUNEL, and toluidine blue. In addition, immunostaining for pSmad, VEGF, and Runx2 was performed. Bone volume fraction, tissue density, and trabecular thickness were significantly decreased on the subchondral bone of botox-injected side when compared to control side and control mice, 4 weeks after injection. Furthermore, histological analysis revealed decrease in mineralization, matrix deposition, TRAP activity, EdU, and TUNEL-positive cells in the MCC of the botox-injected side, 4 weeks after injection. However, compressive loading reversed the reduced bone volume and density and the cellular changes in the MCC caused by Botox injection. TMJ compressive loading rescues the negative effects of botox injection into the masseter in the MCC and subchondral bone.

Buccal-Lingual Bone Remodeling in Immediately Loaded Fresh Socket Implants: A Cone Beam Computed Tomography Study.

The aim of this study was to evaluate buccal-lingual bone remodeling after fresh socket implant placement and immediate loading at 3 years of follow-up by cone beam computed tomography (CBCT). A total of 96 implants were placed in fresh sockets in anterior maxillary regions and immediately loaded. The sockets were divided into two groups related to buccal bone thickness: group A, with a buccal bone thickness > 1 mm, and group B, with a thickness ≤ 1 mm. The CBCT scans were performed before tooth extractions and 3 years after implant placement, and measurements were assessed. At 3 years, all fresh sockets in both groups presented considerable buccal-palatal crestal reduction. In both groups, statistically significant (P < .05) bone loss was found between time points. There was no statistically significant difference in bone reduction between groups at 3 years of follow-up. Nevertheless, sockets with vertical axis presented more bone volume reduction than those with axis parallel to buccal bone after implant placement.

Paradoxical Effects of JZL184, an Inhibitor of Monoacylglycerol Lipase, on Bone Remodelling in Healthy and Cancer-Bearing Mice

Cancer associated bone disease is a serious complication in bone sarcomas and metastatic carcinomas of breast and prostate origin. Monoacylglycerol lipase (MAGL) is an enzyme of the endocannabinoid system, and is responsible for the degradation of the most abundant endocannabinoid in bone 2-arachidonoyl glycerol (2AG). Here, we report that the verified MAGL inhibitor JZL184 reduced osteolytic bone metastasis in mouse models of breast and prostate cancers, and inhibited skeletal tumour growth, metastasis and the formation of ectopic bone in models of osteosarcoma. Additionally, JZL184 suppressed cachexia and prolonged survival in mice injected with metastatic osteosarcoma and osteotropic cancer cells. Functional and histological analysis revealed that the osteoprotective action of JZL184 in cancer models is predominately due to inhibition of tumour growth and metastasis. In the absence of cancer, however, exposure to JZL184 exerts a paradoxical reduction of bone volume via an effect that is mediated by both Cnr1 and Cnr2 cannabinoid receptors. Thus, targeting MAGL is of potential therapeutic efficacy in primary bone cancer and bone metastasis, however, activation of the skeletal endocannabinoid system may limit the usefulness of MAGL inhibitors as osteoprotective agents. Funding Statement: The studies described were partly supported by funding from Cancer Research UK Development Fund (Edinburgh Cancer Research Centre, University of Edinburgh) and a programme grant from Arthritis Research UK. Declaration of Interests: A.I. Idris and S.H. Ralston are inventors on a patent concerning the use of cannabinoid receptor ligands for the treatments of bone diseases. Other authors declare no conflict of interest. Ethics Approval Statement: All procedures involving mice and their care were approved by and performed in compliance with the guidelines of Institutional Animal Care and Use Committee of Universities of Edinburgh (Scotland, UK), L’Aquila (L’Aquila, Italy) and Nantes (Nantes, France), and received institutional approval and conducted in conformity with laws and policies (UK Home Office and Italian and French authorities).

Ethanolic Extract of Rubus coreanus Fruits Inhibits Bone Marrow-Derived Osteoclast Differentiation and Lipopolysaccharide-Induced Bone Loss

The inhibition of osteoclast differentiation/bone resorption is a well-known therapeutic strategy for controlling pathological and postmenopausal bone loss. Natural products that specifically inhibit osteoclastogenesis could therefore be developed as antiresorptive drugs for the treatment of metabolic bone disorders characterized by excessive osteoclastic bone resorption. We therefore examined the effects of Rubus coreanus extract (eeRc) on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced differentiation of bone marrow macrophages (BMMs) into osteoclasts and pit formation in vitro. Additionally, the in vivo effects of the eeRc were observed in mice with lipopolysaccharide (LPS)-induced bone erosion. In this study, we found that the ethanolic extract of Rubus coreanus fruits considerably suppressed the RANKL-induced differentiation of primary BMMs into osteoclasts and bone-resorbing activity of mature osteoclasts. Oral administration of eeRc attenuated LPS-induced bone loss in vivo, as demonstrated by the reversal of LPS-induced reduction in bone volume per tissue volume, bone mineral density, and trabecular number to some extent in eeRc-treated mice. In addition, eeRc slightly decreased the serum levels of C-terminal telopeptide fragments of type I collagen, the collagen-breakdown product generated by osteoclasts. Collectively, our results indicate that eeRc has the potential to inhibit bone loss by blocking osteoclast differentiation and could therefore be a promising natural product for the prevention and/or treatment of inflammatory bone loss.

Moderate hyperhomocysteinemia induced by short-term dietary methionine overload alters bone microarchitecture and collagen features during growth

AimsIn general, hyperhomocysteinemia is increasingly appreciated as a risk factor for various diseases, including osteoporosis. However, its effects in non-adults remain largely unknown. Our aim was to determine whether dietary-caused increased homocysteine levels have deleterious effects on bone structure during growth. Main methodsWe developed a model of moderate hyperhomocysteinemia caused by short-term methionine nutritional overload in growing rats. 30-days-old male Wistar albino rats were randomly assigned to either experimental group subject to a 30-days hypermethionine diet or control group. High-resolution 3D assessment of bone geometry and microarchitecture, as well as fluorescence spectroscopic analysis of bone matrix were performed. Key findingsShort-term moderate hyperhomocysteinemia (~30μmol/L) achieved in the study notably affected bone and cartilage characteristics. Parameters of the cortical bone geometry in the experimental group indicated peculiar reorganization of the bone cross-section. Trabecular bone microarchitecture was especially sensitive to hyperhomocysteinemia showing clearly negative bone balance in the experimental group (almost 30% reduced bone volume, mainly due to ~25% decrease in trabecular number as well as markedly reduced trabecular connections). Fluorescent spectroscopy of bone matrix revealed multiple alterations to collagen spectra due to homocysteine accumulation in bone, indicative of broken collagenous cross-links. SignificanceGiven that appropriate accrual of bone mass during growth has important effects on the risk of osteoporosis in adulthood, understanding the skeletal effects of dietary-induced hyperhomocysteinemia in non-adults is essential for interpreting its importance as a modifiable risk factor for osteoporosis and improving programs to preserve/re-establish bone health.

Radium-223 Inhibits Osseous Prostate Cancer Growth by Dual Targeting of Cancer Cells and Bone Microenvironment in Mouse Models.

Purpose: Radium-223 dichloride (radium-223, Xofigo), a targeted alpha therapy, is currently used for the treatment of patients with castration-resistant prostate cancer (CRPC) with bone metastases. This study examines the mode-of-action and antitumor efficacy of radium-223 in two prostate cancer xenograft models.Experimental Design: Mice bearing intratibial LNCaP or LuCaP 58 tumors were randomized into groups (n = 12-17) based on lesion grade and/or serum PSA level and administered radium-223 (300 kBq/kg) or vehicle, twice at 4-week intervals. X-rays and serum samples were obtained biweekly. Soft tissue tumors were observed macroscopically at sacrifice. Tibiae were analyzed by gamma counter, micro-CT, autoradiography and histology.Results: Radium-223 inhibited tumor-induced osteoblastic bone growth and protected normal bone architecture, leading to reduced bone volume in LNCaP and abiraterone-resistant LuCaP 58 models. Furthermore, radium-223 resulted in lower PSA values and reduced total tissue and tumor areas, indicating that treatment constrains prostate cancer growth in bone. In addition, radium-223 suppressed abnormal bone metabolic activity as evidenced by decreased number of osteoblasts and osteoclasts and reduced level of the bone formation marker PINP. Mode-of-action studies revealed that radium-223 was deposited in the intratumoral bone matrix. DNA double-strand breaks were induced in cancer cells within 24 hours after radium-223 treatment, and PSA levels were significantly lower 72 hours after treatment, providing further evidence of the antitumor effects.Conclusions: Taken together, radium-223 therapy exhibits a dual targeting mode-of-action that induces tumor cell death and suppresses tumor-induced pathologic bone formation in tumor microenvironment of osseous CRPC growth in mice. Clin Cancer Res; 23(15); 4335-46. ©2017 AACR.

Cross-sex testosterone therapy in ovariectomized mice: addition of low-dose estrogen preserves bone architecture.

Cross-sex hormone therapy (XHT) is widely used by transgender people to alter secondary sex characteristics to match their desired gender presentation. Here, we investigate the long-term effects of XHT on bone health using a murine model. Female mice underwent ovariectomy at either 6 or 10 wk and began weekly testosterone or vehicle injections. Dual-energy X-ray absorptiometry (DXA) was performed (20 wk) to measure bone mineral density (BMD), and microcomputed tomography was performed to compare femoral cortical and trabecular bone architecture. The 6-wk testosterone group had comparable BMD with controls by DXA but reduced bone volume fraction, trabecular number, and cortical area fraction and increased trabecular separation by microcomputed tomography. Ten-week ovariectomy/XHT maintained microarchitecture, suggesting that estrogen is critical for bone acquisition during adolescence and that late, but not early, estrogen loss can be sufficiently replaced by testosterone alone. Given these findings, we then compared effects of testosterone with effects of weekly estrogen or combined testosterone/low-dose estrogen treatment after a 6-wk ovariectomy. Estrogen treatment increased spine BMD and microarchitecture, including bone volume fraction, trabecular number, trabecular thickness, and connectivity density, and decreased trabecular separation. Combined testosterone-estrogen therapy caused similar increases in femur and spine BMD and improved architecture (increased bone volume fraction, trabecular number, trabecular thickness, and connectivity density) to estrogen therapy and were superior compared with mice treated with testosterone only. These results demonstrate estradiol is critical for bone acquisition and suggest a new cross-sex hormone therapy adding estrogens to testosterone treatments with potential future clinical implications for treating transgender youth or men with estrogen deficiency.

Abstract 253: Mammary tumorigenesis causes bone loss and dietary selenium supplementation does not affect such bone loss in male MMTV-PyMT mice

Abstract Cancer progression is accompanied by wasting that eventually results in cachexia characterized by significant weight loss and multi-organ functional failures. Limited clinical trials indicate that bone is adversely affected by cancer-associated wasting. To determine the effects of breast cancer on skeletal health, we performed micro-computed tomographic analysis of femurs and vertebrae collected from a recently completed study showing that dietary supplementation with selenium (methylseleninic acid, 2.5 mg Se/kg) reduced male mammary tumorigenesis in MMTV-PyMT mice. Compared to age-matched non-tumor-bearing mice (MMTV-PyMT negative), the presence of mammary tumors significantly reduced bone volume fraction, trabecular thickness and bone mineral density and increased the structure model index (an indicator of the plate- and rod-like geometry of trabecular structure) in femoral trabecular bone. Mammary tumor development did not affect vertebral trabeculae nor femoral and vertebral cortical bone, except it significantly reduced cortical bone thickness of vertebrae. There were no differences in aforementioned measurements between groups with or without selenium supplementation. In conclusion, mammary tumorigenesis causes bone loss and dietary selenium supplementation at 2.5 mg Se/kg, which is antitumorigenic, does not affect mammary tumor-associated bone loss in this male MMTV-PyMT breast cancer model. Citation Format: Lin Yan. Mammary tumorigenesis causes bone loss and dietary selenium supplementation does not affect such bone loss in male MMTV-PyMT mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 253. doi:10.1158/1538-7445.AM2017-253

The skeletal impact of the chemotherapeutic agent etoposide.

Chemotherapeutics target rapidly dividing tumor cells yet also impact hematopoietic and immune cells in an off target manner. A wide array of therapies have negative side effects on the skeleton rendering patients osteopenic and prone to fracture. This study focused on the pro-apoptotic chemotherapeutic agent etoposide and its short- and long-term treatment effects in the bone marrow and skeleton. Six- to 16-week-old mice were treated with etoposide (20-25mg/kg) or vehicle control in short-term (daily for 5-9days) or long-term (3×/week for 17days or 6weeks) regimens. Bone marrow cell populations and their phagocytic/efferocytic functions were analyzed by flow cytometry. Blood cell populations were assessed by CBC analysis. Bone volume and area compartments and osteoclast numbers were measured by microCT, histomorphometry, and TRAP staining. Biomarkers of bone formation (P1NP) and resorption (TRAcP5b) were assayed from serum. Gene expression in bone marrow was assessed using qPCR. Flow cytometric analysis of the bone marrow revealed short-term etoposide reduced overall cell numbers and B220+ cells, with increased marrow apoptotic (AnnexinV+PI-) cells, mesenchymal stem-like cells, and CD68+, CD45+, and CD11b+ monocyte/myeloid cells (as a percent of the total marrow). After 6weeks, the CD68+, Gr1+, CD11b+, and CD45+ cell populations were still relatively increased in etoposide-treated bone marrow. Skeletal phenotyping revealed etoposide decreased bone volume, trabecular thickness, and cortical bone volume. Gene expression in the marrow for the leptin receptor and CXCL12 were reduced with short-term etoposide, and an increased ratio of RANKL/OPG mRNA was observed. In whole bone, Runx2 and osteocalcin gene expressions were reduced, and in serum, P1NP was significantly reduced with etoposide. Treatment with the antiresorptive agent zoledronic acid prior to etoposide increased bone volume and improved the etoposide-induced decrease in skeletal parameters. These data suggest that etoposide induces apoptosis in the bone marrow and significantly reduces parameters of bone formation with rapid reduction in bone volume. Pre-treatment with an antiresorptive agent results in a preservation of bone mass. Preventive approaches to preserving the skeleton should be considered in human clinical studies.

Sheep model for osteoporosis: The effects of peripheral hormone therapy on centrally induced systemic bone loss in an osteoporotic sheep model

Hypothalamic-pituitary disconnection (HPD) leads to low bone turnover followed by bone loss and reduced biomechanical properties in sheep. To investigate the role of peripheral hormones in this centrally induced systemic bone loss model, we planned a hormone replacement experiment. Therefore, estrogen (OHE), thyroxin (OHT) or a combination of both (OHTE) was substituted in ovariectomized HPD sheep, as both hormones are decreased in HPD sheep and are known to have a significant but yet not fully understood impact on bone metabolism. Bone turnover and structural parameters were analyzed in comparison to different control groups – untreated sheep (C), ovariectomized (O) and ovariectomized+HPD sheep (OH). We performed histomorphometric and HR-pQCT analyses nine months after the HPD procedure, as well as biomechanical testing of all ewes studied. In HPD sheep (OH) the low bone turnover led to a significant bone loss. Treatment with thyroxin alone (OHT) mainly increased bone resorption, leading to a further reduction in bone volume. In contrast, the treatment with estrogen alone (OHE) and the combined treatment with estrogen and thyroxin (OHTE) prevented HPD-induced bone loss completely. In conclusion, peripheral hormone substitution was able to prevent HPD-induced low-turnover osteoporosis in sheep. But only the treatment with estrogen alone or in combination with thyroxin was able to completely preserve bone mass and structure. These findings demonstrate the importance of peripheral hormones for a balanced bone remodeling and a physiological bone turnover.

Monocyte chemotactic protein-1 deficiency attenuates and high-fat diet exacerbates bone loss in mice with Lewis lung carcinoma

Bone loss occurs in obesity and cancer-associated complications including wasting. This study determined whether a high-fat diet and a deficiency in monocyte chemotactic protein-1 (MCP-1) altered bone structural defects in male C57BL/6 mice with Lewis lung carcinoma (LLC) metastases in lungs. Compared to non-tumor-bearing mice, LLC reduced bone volume fraction, connectivity density, trabecular number, trabecular thickness and bone mineral density and increased trabecular separation in femurs. Similar changes occurred in vertebrae. The high-fat diet compared to the AIN93G diet exacerbated LLC-induced detrimental structural changes; the exacerbation was greater in femurs than in vertebrae. Mice deficient in MCP-1 compared to wild-type mice exhibited increases in bone volume fraction, connectivity density, trabecular number and decreases in trabecular separation in both femurs and vertebrae, and increases in trabecular thickness and bone mineral density and a decrease in structure model index in vertebrae. Lewis lung carcinoma significantly decreased osteocalcin but increased tartrate-resistant acid phosphatase 5b (TRAP 5b) in plasma. In LLC-bearing mice, the high-fat diet increased and MCP-1 deficiency decreased plasma TRAP 5b; neither the high-fat diet nor MCP-1 deficiency resulted in significant changes in plasma concentration of osteocalcin. In conclusion, pulmonary metastasis of LLC is accompanied by detrimental bone structural changes; MCP-1 deficiency attenuates and high-fat diet exacerbates the metastasis-associated bone wasting.

Benefits of Bifidobacterium animalis subsp. lactis Probiotic in Experimental Periodontitis.

This study evaluates effects of topical administration of probiotic bacteria of the genus Bifidobacterium on experimental periodontitis (EP) in rats. Thirty-two rats were divided into groups C (control; without EP), EP (EP only), C-HN019 (control+probiotic), and EP-HN019 (EP+probiotic). On day 0 of the experiment, animals of groups EP and EP-HN019 received cotton ligatures around mandibular first molars (MFMs). In groups C-HN019 and EP-HN019, 1 mL of suspensions containing Bifidobacterium animalis subsp. lactis (B. lactis) HN019 was topically administered in the subgingival region of MFMs on days 0, 3, and 7. In groups C and EP, topical administrations were performed using a sham suspension (without probiotic). All animals were euthanized at day 14. Gingival tissue, hemimandibles, and oral biofilm were collected. Data were statistically analyzed (P <0.05). Group EP presented greater bone porosity, trabecular separation, and connective tissue attachment loss (CTAL) as well as reduced bone volume than all other groups (P <0.05). In group EP-HN019, there were greater proportions of Actinomyces and Streptococcus-like species and lower proportions of Veillonella parvula, Capnocytophaga sputigena, Eikenella corrodens, and Prevotella intermedia-like species than group EP. Group EP-HN019 presented greater expressions of osteoprotegerin and β-defensins than group EP (P <0.05). Group EP presented greater levels of interleukin-1β and receptor activator of nuclear factor-kappa B ligand than group EP-HN019 (P <0.05). Topical use of B. lactis HN019 promotes a protective effect against alveolar bone loss and CTALs attributable to EP in rats, modifying immunoinflammatory and microbiologic parameters.

Molecular, Cellular and Pharmaceutical Aspects of Autologous Grafts for Peri-implant Hard and Soft Tissue Defects.

The lack of supporting hard and soft tissues always prevents the rehabilitation with dental implants. Among various hard and soft tissue augmentation procedures, autologous grafts have been considered to be the gold standard. Autologous mesenchymal stem cells (MSCs) from bone marrow, dental tissue and adipose tissue have been described as promising alternatives for bone regeneration in the field of dental implantation. Mucosal cells, gingival fibroblasts and dental progenitor cells (DPS) can enhance peri-implant soft tissue augmentation and regenerate periodontal tissues around dental implants. Obtained from patients, platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are enriched in autologous platelets, which contain a great deal of growth factors and cytokines that are conducive to the regeneration of both hand and soft tissues around dental implants. Pharmaceutical treatments for osteoporosis and diabetes should be locally applied with implant procedures to restrict the resorption of autologous bone grafts and reduction of bone volume. Although autografts hold great potentials for dental implants, new approaches should also be explored with minimally invasion donor sites methods such as tissue engineering combined with autologous three factors and bio-3D printing involving selfassembling cell aggregates.