Bone Remodeling System Research Articles

Brown fat-specific mitoribosomal function is crucial for preventing cold exposure-induced bone loss

This study examines the interplay between ambient temperature, brown adipose tissue (BAT) function, and bone metabolism, emphasizing the effects of cold exposure and BAT mitochondrial activity on bone health. Utilizing ovariectomized (OVX) mice to model primary osteoporosis and BAT-specific mitochondrial dysfunction (BKO) mice, we evaluated the impact of housing temperature on bone density, immune modulation in bone marrow, and the protective role of BAT against bone loss. Cold exposure was found to universally reduce bone mass, enhance osteoclastogenesis, and alter bone marrow T-cell populations, implicating the immune system in bone remodeling under cold stress. The thermogenic function of BAT, driven by mitochondrial oxidative phosphorylation, was crucial in protecting against bone loss. Impaired BAT function, through surgical removal or mitochondrial dysfunction, exacerbated bone loss in cold environments, highlighting BAT’s metabolic role in maintaining bone health. Furthermore, cold-induced changes in BAT function led to systemic metabolic shifts, including elevated long-chain fatty acids, which influenced osteoclast differentiation and activity. These findings suggest a systemic mechanism connecting environmental temperature and BAT metabolism with bone physiology, providing new insights into the metabolic and environmental determinants of bone health. Future research could lead to novel bone disease therapies targeting these pathways.

Adverse jaw outcomes from immune checkpoint inhibitors for head-and-neck cancer? Case reports.

Radiation treatment plays a mainstream role in the management of head and neck cancers (HNSCC). Adverse effects from radiation therapy include osteoradionecrosis of the jaw, and rarely, pathological fracture. Immune checkpoint inhibitors (ICI) such as pembrolizumab are of growing relevance to the management of metastatic and recurrent HNSCC. Adverse impact on bone secondary to medications such as pembrolizumab and nivolumab have been sporadically documented in the literature. The objective of this manuscript is to raise awareness of possible increase in risk for adverse jaw outcomes in patients with HNSCC exposed to both radiation treatment to the jaws and ICI therapy. This manuscript documents adverse jaw outcomes including osteonecrosis and pathological fracture of the mandible in two patients receiving pembrolizumab for management of HNSCC and had received prior radiation treatment. A potential link between immunotherapy and adverse jaw outcomes is consistent with our growing understanding of osteoimmunology, investigating the closely interrelated processes in bone remodeling and immune system function, in health and disease. It is important to ascertain if pembrolizumab poses an incremental risk for such outcomes, beyond the risk from prior radiation, for patients managed with radiation treatment and ICI therapy for HNSCC. The general dentist may encounter such patients either in the context of facilitating dental clearance prior to initiation of chemotherapy, or rarely, with poorly explained jaw symptoms and must be alert to the possibility of occurrence of such adverse jaw events to facilitate timely diagnosis and optimal patient management.

Research progress in immunomodulation in orthodontic tooth movement

In the process of orthodontic tooth movement, the secretion of cytokines by immune cells or cell-cell interaction affects the regulation of osteoclast and osteoblast differentiation. Increasingly, studies have focused on the role in the immune system in orthodontic bone remodeling. Based on the biological role of different immune cells or cytokines, this article briefly presents the research progress of immunomodulation in orthodontic tooth movement and future perspective, hopefully providing a deeper and more comprehensive understanding of the biological mechanism in orthodontic tooth movement.

Comparative transcriptome analysis reveals molecular adaptations underlying distinct immunity and inverted resting posture in bats.

Understanding how natural selection shapes unique traits in mammals is a central topic in evolutionary biology. The mammalian order Chiroptera (bats) is attractive for biologists as well as the general public due to their specific traits of extraordinary immunity and inverted resting posture. However, genomic resources for bats that occupy key phylogenetic positions are not sufficient, which hinders comprehensive investigation of the molecular mechanisms underpinning the origin of specific traits in bats. Here, we sequenced the transcriptomes of 5 bats that are phylogenetically divergent and occupy key positions in the phylogenetic tree of bats. In combination with the available genomes of 19 bats and 21 other mammals, we built a database consisting of 10918 one-to-one ortholog genes and reconstructed phylogenetic relationships of these mammals. We found that genes related to immunity, bone remodeling, and cardiovascular system are targets of natural selection along the ancestral branch of bats. Further analyses revealed that the T cell receptor signaling pathway involved in immune adaptation is specifically enriched in bats. Moreover, molecular adaptations of bone remodeling, cardiovascular system, and balance sensing may help to explain the reverted resting posture in bats. Our study provides valuable transcriptome resources, enabling us to tentatively identify genetic changes associated with bat-specific traits. This work is among the first to advance our understanding of the molecular underpinnings of inverted resting posture in bats, which could provide insight into healthcare applications such as hypertension in humans.

What Do Prescribers of Bone Modifying Agents Know about Medication-Related Osteonecrosis of the Jaw? Is Current Prevention Enough?

Osteonecrosis of the jaw represents interference by external and internal factors in the natural bone remodeling system. Numerous bone remodeling agents (BMAs), such as bisphosphonates, denosumab, and tyrosine kinase inhibitors, can lead to medication-related osteonecrosis of the jaw (MRONJ). This is a serious condition that ocurs as a side-effect of treatment in patients with osteoporosis or malignancies. Antiresorptive drugs are medications that target osteoclasts with the aim of preventing bone resorption and are used to treat osteoporosis, osteopenia, and a variety of other conditions, such as Paget’s disease. They are also used in cancer patients with active bone metastases where antiresorptive treatment is used for the prevention of skeletal complications. Poor dental health, infections, and especially dental surgery are the primary causes of MRONJ, while other risk factors, such as smoking, alcohol abuse, and diabetes mellitus, can also influence its development. Prevention is the key component of management, and thus awareness of the risk factors among prescribers is very important. The aim of our study was to evaluate current knowledge about MRONJ among BMA prescribers in an academic hospital and their awareness about oral health and dental check-ups. By using a custom-designed questionnaire addressed to general and internal medical practitioners, endocrinologists, rheumatologists, and oncologists as an instrument for collecting data, we tried to identify trends in BMA prescription among different specialists and their recommended preventative measures, with the aim of creating new strategies to prevent the occurrence of MRONJ. The survey revealed a low awareness among physicians of the potential risk factors and underlined the need for a concerted effort to improve patient management. In this sense, a multidisciplinary team approach that includes the patient, the drug prescriber, the dentist, and the oral surgeon could significantly improve the quality of life of patients with MRONJ.

Oral Complications in Cancer Patients-Medication-Related Osteonecrosis of the Jaw (MRONJ).

Medication-Related Osteonecrosis of the Jaw (MRONJ) was first reported in 2003. Despite the progress in the understanding of this oral complication in cancer patients for the past 18 years, there is still discussion about the best way to define MRONJ, prevent the complication, how to diagnose, and the options of treatment available. The initial reports associated MRONJ to bisphosphonates and denosumab, medications that work as bone-modifying agents. Later, other agents such as the antiangiogenics, have also been reported to cause the oral complication, either alone or in combination with antiresorptives. Initially, these medications were prescribed to patients with osteoporosis and cancers patients with bone metastasis. Today, because of the effect of the medications in the bone remodeling system, patients with several other diseases such as giant cell tumors, rheumatoid arthritis, Paget's disease of bone, fibrous dysplasia, osteogenesis imperfecta, are managed with these medications, significantly increasing the population of individuals at risk for developing MRONJ. This mini review focused on the cancer patient. It updates the dental clinician on the recent scientific literature about MRONJ and provides information on how to diagnose and manage patients being treated with these medications, suggests protocols to prevent the development of MRONJ, and present ways to manage those patients who develop the oral complication.

Protocol of Co-Culture of Human Osteoblasts and Osteoclasts to Test Biomaterials for Bone Tissue Engineering.

New biomaterials and scaffolds for bone tissue engineering (BTE) applications require to be tested in a bone microenvironment reliable model. On this assumption, the in vitro laboratory protocols with bone cells represent worthy experimental systems improving our knowledge about bone homeostasis, reducing the costs of experimentation. To this day, several models of the bone microenvironment are reported in the literature, but few delineate a protocol for testing new biomaterials using bone cells. Herein we propose a clear protocol to set up an indirect co-culture system of human-derived osteoblasts and osteoclast precursors, providing well-defined criteria such as the cell seeding density, cell:cell ratio, the culture medium, and the proofs of differentiation. The material to be tested may be easily introduced in the system and the cell response analyzed. The physical separation of osteoblasts and osteoclasts allows distinguishing the effects of the material onto the two cell types and to evaluate the correlation between material and cell behavior, cell morphology, and adhesion. The whole protocol requires about 4 to 6 weeks with an intermediate level of expertise. The system is an in vitro model of the bone remodeling system useful in testing innovative materials for bone regeneration, and potentially exploitable in different application fields. The use of human primary cells represents a close replica of the bone cell cooperation in vivo and may be employed as a feasible system to test materials and scaffolds for bone substitution and regeneration.

Role of Melatonin in Bone Remodeling around Titanium Dental Implants: Meta-Analysis

The theory, known as the “brain-bone axis” theory, involves the central nervous system in bone remodeling. The alteration of the nervous system could lead to abnormal bone remodeling. Melatonin produced by the pineal gland is a hormone that is characterized by its antioxidant properties. The aim of this meta-analysis was to examine the role of melatonin in the growth of new bone around titanium dental implants in vivo. A manual search of the PubMed and Web of Science databases was conducted to identify scientific studies published until November 2020. We included randomized clinical trials (RCTs) and animal studies where melatonin was used with titanium implants. Fourteen studies met the inclusion criteria. Quality was assessed using the Jadad scale and SYRCLE’s risk of bias tool. Our meta-analysis revealed that the use of melatonin during implant placement improves bone-to-implant contact percentages in animals (difference of means, random effects: 9.59 [95% CI: 5.53–13.65]), reducing crestal bone loss in humans (difference in means, random effects: −0.55 [95% CI: 1.10–0.00]). In animals, titanium implants using melatonin increase bone-to-implant contact surface 2–6 weeks after their placement and reduce crestal bone loss in humans following six months. The results of this meta-analysis should be taken with caution, due to the small samples and the large heterogeneity among studies.

Dendritic cells development into osteoclast-type APCs by 4T1 breast tumor T cells milieu boost bone consumption.

Bone metastases occur in 70% of patients with advanced breast cancer, causing severe morbidity and increased mortality due to osteolytic lesions driven by osteoclasts (OCs) inside the bone marrow (BM) microenvironment. A reciprocal vicious cycle between bone remodeling system and the tumor itself is established by the release of growth factors stored in the mineralized matrix, which in turn feed the tumor, changing tumor behavior and growth. However, BM is not a passive host microenvironment for circulating tumor cells, but instead can be actively modified by the primary tumor before metastatic spread occurs. Indeed, we have shown that T cells specific for the 4T1 mammary carcinoma cell line, are characteristically RANKL+ IL-17F+ CD4+ T cells. Those cells arrive in the BM before metastatic cells and set the pre-metastatic niche. In the absence of T cell derived RANKL, there is no pre-metastatic osteolytic disease and bone metastases do not take place. Recently, dendritic cells (DCs), the main T cell partner at the beginning of the immune response, came into the spotlight as a potential source of OCs progenitors under inflammatory conditions. Regarding bone metastasis, nothing is currently known about DCs plasticity or even its partnership with tumor induced T cells for BM pre-metastatic niche formation. Here, we show that splenic CD11c+ DCs stimulated with 4T1 conditioned media (CM) efficiently differentiated into mature and activated multinucleated giant cells (DC-OC) expressing TRAP and IL-23 cytokine. More important, 4T1 CM derived DC-OCs build a positive loop which amplifies the osteolytic phenomena by maintaining the RANKL+ Th17 T cells and by its own osteoclastic activity. In conclusion, our results indicate that differentiation of OCs from DCs may be achievable in the bone pre osteolytic disease context representing an alternative OC differentiation pathway. Besides being induced by high levels of T cells pro osteoclastogenic cytokines, especially by RANKL, DC-OC keep a positive feedback loop towards osteolysis, maintaining the pro-osteoclastogenic T cell phenotype in the BM.

Crosstalk between bone remodeling and blood coagulation-anticoagulation system

骨リモデリングは，骨芽細胞と破骨細胞が相互に密接に関わる制御システムである．骨では，骨芽細胞による骨形成と破骨細胞による骨吸収が恒常的に行われており，この機能的に相反する性質を持つ細胞のバランスによって骨のホメオスタシスが保たれている．骨の基質蛋白質の一つであるオステオカルシンの合成にはビタミンKが必要とされる．同様に，血液凝固因子や凝固制御因子の一部についても，それらの蛋白質の翻訳後の修飾にビタミンKが必要とされる．最近，ビタミンK依存性凝固制御因子の一つであるプロテインCの活性型である活性化プロテインC（activated protein C: APC）が骨芽細胞の増殖を促進し，また破骨細胞の分化を抑制することにより，骨リモデリングを制御・調節することが明らかになった．本稿では，骨リモデリングとその制御に密接に関わる血液凝固系のクロストークについて，筆者らの研究結果も含めて概説する．

Analysis of the effects of vitamin d insufficiency on osseointegration of dental implants

Background Vitamin D is a key factor in the union of innate and adaptive immunity, and its deficiency may influence on osseointegration process. Aim/Hypothesis the objective of this research was to investigate the effects of vitamin D insufficiency on osseointegration of dental implants. Our hypothesis is that the insufficiency of D vitamin can interfere on clinical osseointegration process. Materials and Methods Ten participants underwent 25-hydroxy-vitamin D (25- (OH) D) serum levels and were classified according to the Endocrine Society's Clinical Practice Guidelines- 5 participants with Vitamin D = 25- (OH)D-21 Insufficiency -29 ng ml (Group D-Insufficient) and 5 participants Sufficient in vitamin D = 25- (OH) D-30-100 ng ml (Control Group). Osseointegration was assessed through clinical, digital radiographic and Osstell ISQ imaging in the 3rd month after implant placement. Laboratory analyses included the evaluation of the gene expression of OPG RANKL in the two groups, at the time of implant implantation (Time 1) and at the time of reopening of the implant and clinical evaluation (Time 2). Results The clinical-radiographic results evidenced adequate clinical osseointegration in all participants of the research. The OPG RANKL levels did not present a statistically significant difference between the groups and or between the intervals of analysis. Conclusion and Clinical Implications This study concluded that despite altered serum levels of vitamin D, there is no clinical correlation with osseointegration deficiency and bone remodeling system. Considering the aspects evaluated in this research, D vitamin insufficiency is not a real contraindication for implant placement.

A structure preserving model order reduction method for calcium homeostatic system

Calcium Homeostasis is a complex physiological process. Its mathematical model results in high order differential equation. In this paper, a model order reduction technique, based on time scale separation is proposed for a 27th order Calcium Homeostasis and Bone Remodeling (CHBR) system. The original state-space model after linearization has been decoupled into three reduced order subsystems: ”Very-Slow”, ”Slow” and ”Fast”, at the same time preserving the structure of the system. The time and frequency response of individual reduced order model has been compared with the response of the original system. Furthermore, the effect of administering a therapeutic daily moderate dose of PTH has been studied with the help of reduced order models.

Effect of osteoporosis treatment agents on the cortical bone osteocyte microenvironment in adult estrogen-deficient, osteopenic rats

Though osteoporosis is a significant cause of disability worldwide, treatment with pharmacologic agents decreases risk of fragility fracture. Though these treatments act through the bone remodeling system to improve bone mass, it is unclear if they alter the response of bone to mechanical loading at the level of the osteocyte. This pre-clinical study determined the relationship between microstructural bone tissue properties and osteocyte lacunar size and density to strain around osteocytes with standard osteoporosis treatment or sequential therapies. Six-month-old female ovariectomized (OVX) Sprague-Dawley rats were cycled through various sequences of pharmacological treatments [alendronate (Aln), raloxifene (Ral) and human parathyroid hormone-1,34 (PTH)] for three month intervals, over nine months. Linear nanoindentation mapping was used to determine Young's modulus in perilacunar and bone matrix regions around cortical bone osteocyte lacunae. Measurements of lacunar diameter and density were completed. Treatment-related differences in Young's modulus in the perilacunar and bone matrix regions were not observed. We confirmed previous data that showed that the bone matrix region was stiffer than the perilacunar matrix region. Whole bone material properties were correlated to perilacunar matrix stiffness. Finite element models predicted a range of mechanical strain amplification factors estimated at the osteocyte across treatment groups. In summary, though the perilacunar matrix near cortical osteocyte lacuna is not as stiff as bone matrix further away, osteoporosis treatment agents do not affect the stiffness of bone tissue near osteocyte lacunae.

In vivo live imaging of bone cells.

There are as many as 200 cell types in the body, and highly sophisticated and varied life phenomena are carried out by cell migration to appropriate places at appropriate times following the appropriate interactions. Recent advances in optical imaging technology using multi-photon excitation microscopy have enabled visualization inside intact bone tissues in living animals without thin sectioning. Using such advanced techniques, the dynamic behaviors of living bone cells on intact bone tissue structures can be elucidated. Here, we focus on recent findings using intravital multi-photon imaging of dynamic biological systems, e.g., bone homeostasis. This novel approach has proven beneficial for understanding the mechanisms underlying the spatiotemporal nature of bone remodeling systems and for evaluating the specific modes of actions of novel drugs currently in development, which will contribute to a new chapter in bone and mineral research.

Activated protein C suppresses osteoclast differentiation via endothelial protein C receptor, protease-activated receptor-1, sphingosine 1-phosphate receptor, and apolipoprotein E receptor 2

IntroductionBone remodeling relies on a delicate balance between formation and resorption of bone tissues, processes in which bone-forming osteoblasts and bone-resorbing osteoclasts play central roles. Recently, we reported that anticoagulant activated protein C (APC) promotes osteoblast proliferation, but the role of the blood coagulation system in bone remodeling remains unclear. In this study, to further elucidate the relationship between bone remodeling and blood coagulation, we investigated the effect of APC on osteoclast differentiation. Materials and methodsNormal human osteoclast precursor cells were cultured in their growth medium including soluble RANKL, M-CSF, and FBS, and on days 4 and 7, the culture medium was replaced with the same medium containing various concentrations of APC, protein C (PC), sphingosine 1-phosphate (S1P) receptor agonist, FTY720, or APC+various substances without FBS. On day 8, TRAP-positive multinucleated cells (≥3 nuclei) were counted manually using a light microscope. The effects of APC on NF-κB and NFATc1 activation were evaluated using specific ELISA. ResultsAPC suppressed RANKL-induced osteoclast differentiation, and this APC-induced suppression of osteoclast differentiation was inhibited by zymogen protein C and aprotinin, a serine protease inhibitor. Immunohistochemistry and RT-PCR analyses suggested that endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1) were expressed in osteoclast precursor cells and osteoclasts. Both anti-PAR-1 antibody and anti-EPCR antibody (RCR-252), which blocks APC binding to EPCR, inhibited the APC-induced suppression of osteoclast differentiation. FTY720 had no effect on osteoclast differentiation. However, FTY 720 and S1P receptor antagonist, VP 23019, inhibited the APC-induced suppression of osteoclast differentiation. On the other hand, recombinant soluble human ApoER2 and anti-human ApoER2 inhibited the APC-induced suppression of osteoclast differentiation. Further, APC had no effect on NF-κB and NFATc1 activation. ConclusionsAPC suppresses human osteoclast differentiation mainly by inhibiting the formation of multinucleated cells via EPCR, PAR-1, S1P receptor, and ApoER2 in a manner that depends on APC protease activity.

Role of Osteopontin in Systemic Lupus Erythematosus

Background: Systemic lupus erythematosus (SLE) is a multisystemic disease, caused by a variety of factors, which lead to immunological abnormalities. Osteopontin (OPN) is a pleiotropic protein, important in bone remodeling and immune system signaling. It produced by various cells, including immune cells, and played a key role in regulating T-helper 1/T-helper 2 balances, stimulating B lymphocytes to produce antibodies, regulating macrophages, neutrophils and inducing dendritic cells. Over expression of OPN has been associated with the pathogenesis of immune-mediated diseases. Objectives: To investigate the role of osteopontin in SLE and to assess its relation to disease activity and subclinical atherosclerosis. Subjects and Methods: This cross sectional case control study was conducted on 30 SLE patients fulfilling SLICCA /ACR criteria for SLE classification and 30 subjects sex- and age-matched apparently healthy as control group. All patients were assessed for SLE disease activity by the systemic lupus activity Index (SLEDAI). Serum OSP and Doppler ultrasound for carotid artery media thickness (CIMT) were measured in all patients and control. Results: Plasma OPN levels were significantly higher in SLE patients than in control group (P<0.001) and had significant positive correlation with disease activity (r 0,595, P<0.05) and CIMT (r = 0.400, P <0.001), but there was no significant correlation between CIMT and disease activity. Conclusion: serum level of OPN was higher in SLE patients and correlated with disease activity and CIMT, and can be used as a marker of subclinical atherosclerosis.

Analysis of the bone anabolic agent by intravital imaging technique.

Teriparatide, recombinant human PTH(1-34), is the only anabolic agent widely used for osteoporosis. This drug is thought to promote bone formation by modulating bone remodeling system, although the detailed mechanism remains unclear. Recently, we developed a novel intravital imaging technique to visualize mature osteoclasts and mature osteoblasts simultaneously. By means of this system, we revealed the effect of teriparatide on three-dimensional distribution and cell-to-cell interactions between these cells. Advances in these imaging studies may lead to further understanding of the intercellular network in bone metabolism.

Abstract P2-05-24: OPG+ bone marrow B cells induced by non-metastatic tumors inhibit the pre-metastatic bone niche induced by T cells

Abstract Using the 4T1 model of experimental breast cancer we had recently shown that cancer induced bone disease starts before metastatic colonization and is mediated by RANKL expressed by tumor specific T cells. The role of anti-tumor B cell immune response in the context of cancer induced bone disease has never been investigated. There is evidence in the literature that B cells are good prognostic markers for metastatic breast cancer. B cells have an intimate relationship with bone cells as they differentiate from HSC present on endosteal surfaces; cross-talk with skeletal system through the RANK-RANKL-OPG signaling axis; and produce OPG, a decoy receptor of RANKL. Here we used the BALB/c derived 4T1 (metastatic) and 67NR (non-metastatic) sibling cell lines of mammary mouse carcinomas. By day 7, 14 and 21 after tumor injection, B220+ BM B cells from 67NR+ animals produce high amounts of OPG in vitro in contrast to B220+ BM B cells of 4T1+ mice. In vitro, BM B cells from 67NR+ mice, but not from 4T1+, could inhibit the RANKL dependent, anti-4T1 T cell mediated-OC differentiation ascertained by TRAP enzymatic activity, morphology and osteolytic disk assay. Transference of BM B cells from 67NR+ mice together with 4T1 tumor cells to BALB/c mice led to inhibition of osteoclastogenesis, increased numbers of bone lining cells and mesenchyme stem cell. Besides acting directly on the bone remodeling system, these B cells also modulated T cell activity evidenced by diminished RANKL and IL-17F production. All the anti-osteolytic and pro-osteogenic activity of B cells modulate and inhibits the pre-metastatic niche formation. Indeed transference of such cells to 4T1 animals inhibited LN and BM metastatic colonization. We conclude that 67NR induced OPG+ B cells can inhibit pro-osteoclastic / pre-metastatic activity of tumor induced T cells, favoring a bone metastasis free-phenotype. These findings have implications, not only for the understanding of the direct contribution of B cells in the control of bone metastasis but also it might be a promising prognostic tool for predicting cancer-induced bone metastasis. Citation Format: Bonomo A, Monteiro AC, Leal AC, Fontão AP, Spinetti E, Balduino A. OPG+ bone marrow B cells induced by non-metastatic tumors inhibit the pre-metastatic bone niche induced by T cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-24.

Involvement of the Nonneuronal Cholinergic System in Bone Remodeling in Rat Midpalatal Suture after Rapid Maxillary Expansion.

Few studies sought to analyze the expression and function of the nonneuronal acetylcholine system in bone remodeling in vivo due to the lack of suitable models. We established a rat maxilla expansion model in which the midline palatine suture of the rat was rapidly expanded under mechanical force application, inducing tissue remodeling and new bone formation, which could be a suitable model to investigate the role of the nonneuronal acetylcholine system in bone remodeling in vivo. During the expansion, the expression pattern changes of the nonneuronal cholinergic system components and the mRNA levels of OPG/RANKL were detected by immunohistochemistry or real-time PCR. The value of the RANKL/OPG ratio significantly increased after 1 day of expansion, indicating dominant bone resorption induced by the mechanical stimulation; however after 3 days of expansion, the value of the RANKL/OPG ratio significantly decreased, suggesting a dominant role of the subsequent bone formation process. Increasing expression of Ach was detected after 3 days of expansion which indicated that ACh might play a role in bone formation. The mRNA expression levels of other components also showed observable changes during the expansion which confirmed the involvement of the nonneuronal cholinergic system in the process of bone remodeling in vivo. Further researches are still needed to figure out the detailed functions of the nonneuronal cholinergic system and its components.

Microfluidic co-culture system model to study the interplay between sensory neurons and mesenchymal stem cells: new clues towards osteogenesis

Event Abstract Back to Event Microfluidic co-culture system model to study the interplay between sensory neurons and mesenchymal stem cells: new clues towards osteogenesis Hugo Oliveira1*, Bruno P. Dos Santos1*, Diana I. Silva1*, Jacques Leng2* and Joelle Amedee1* 1 Inserm U1026, Université Bordeaux Segalen, BioIngénierie Tissulaire, France 2 UMR 5258 CNRS/SOLVAY/Univ. BORDEAUX, LaboratoireduFutur, France Introduction: The existence of nerve fibers in bone and the expression of neural factors in close vicinity to bone cells have been reported. Neuron-derived molecules have demonstrated to play a role on bone turnover [1] and accumulating evidence has revealed the importance of the sensory nervous system in the orchestration of bone repair [2]. The aim of this work is to establish a co-culture system between sensorial neurons and mesenchymal stem cells (MSC) and to study the cellular interplay, between these cells, in view of osteogenesis. Materials and Methods: In order to allow the establishment of co-culture between rat dorsal root ganglia (DRG) sensory neurons and rat bone marrow MSCs we fabricated PDMS microfluidic chambers, using standard photolithography techniques. This approach allows to physically separate cellular bodies from the distinct cell types, to use two different cell culture medias and to some extent to mimic the in vivo physiological environment. The primary cultures were co-cultured during 7 and 14 days in microfluidics system using DMEM (high glucose) with 2% B27 medium or DMEM with 10%FBS medium for DRGs and MSCs, respectively. At designated time points (i.e. 7 and 14 days), metabolic activity, immunostaining and gene expression profiles (qPCR) were performed. Results: DRGs were able to spread neurites through microchannels toward MSCs (Figure 1). Figure 1. Co-culture microfluidic system. DRG neurons and MSCs are cultured separately but cells can interact through channels. Immunocytochemistry of DRG neurons (anti-beta III tubulin, green) and MSCs (actin, red) co-cultures at 7 and 14 days of culture. MSCs metabolic activity was not influenced by the co-culture with DRGs. MSCs in co-culture conditions shown an upregulation of the osteogenic markers Runx2, Col1 and Sp7 (Osx). Additionally, in view of further evaluate the mechanism involved in this regulation we assessed the gene expression of SMAD1 and Ctnnb1 (beta-catenin) (Figure 2). Figure 2. Quantitative gene expression of Runx2, collagen type I (Col1), osterix (osx), beta cathenin (Bcath) and Smad 1 of MSCs in mono and co-culture with sensory neurons, at 7 and 14 days of culture (Aver±SD, n=3, * denotes p <0.05). Discussion: We show that that sensorial neurons can closely interact with MSCs and modulate their phenotype toward the osteogenic lineage. Further studies are underway in order to further understand the mechanism underlying this cellular communication. Conclusion: These co-culture systems will allow the broadening of current knowledge regarding the impact of the sensorial nervous system in bone remodeling and repair, and can open new avenues for the establishment of new strategies for bone tissue regeneration.