Bone Molecular Modifications Induced by Diagenesis Followed-Up for 12 Months.

After death, diagenesis takes place. Numerous processes occur concomitantly, which makes it difficult to identify the diagenetic processes. The diagenetic processes refer to all processes (chemical or physical) that modify the skeletal remains. These processes are highly variable depending on the environmental factors (weather, temperature, age, sex, etc.), especially in the early stages. Numerous studies have evaluated bone diagenetic processes over long timescales (~millions of years), but fewer have been done over short timescales (between days and thousands of years). The objective of the study is to assess the early stages of diagenetic processes by Raman microspectroscopy over 12 months. The mineral and organic matrix modifications are monitored through physicochemical parameters. Ribs from six humans were buried in soil. The modifications of bone composition were followed by Raman spectroscopy each month. The decrease in the mineral/organic ratio and carbonate type-B content and the increase in crystallinity reveal that minerals undergo dissolution-recrystallization. The decrease in collagen cross-linking indicates that collagen hydrolysis induces the fragmentation of collagen fibres over 12 months.

Short-term high-dose zoledronic acid enhances crystallinity in mandibular alveolar bone in rats.

Owing to its antiresorptive properties, zoledronic acid (ZOL) is commonly used in the management of benign as well as malignant bone diseases. This molecule targets sites where bone is actively remodeling, and high concentrations have been reported in the jaw. The purpose of this study was to investigate whether treatment of male rats with ZOL, at a dosage equivalent to that used for antitumor treatment, impacts the short-term qualitative properties of mandibular bone independent of bone remodeling. Thirty rats were randomly assigned to treatment either with ZOL or with serum-vehicle (control) (weekly injections: 100 µg kg-1 for 6 wk, n = 15 per group). Using the tetracycline double-labeling technique, remodeled bone areas, corresponding to the preferential site of bisphosphonate binding, were found in the alveolar bone along the alveolar bone proper. The composition of bone in these areas was characterized using Raman microspectroscopy and compared with adjacent, non-remodeled, older bone. The ZOL-treated group exhibited higher crystallinity in the remodeled bone areas (+2%), reflecting an early maturation of the apatite mineral after ZOL injection. Our findings highlight a direct and rapid effect of clinically relevant anti-tumoral ZOL doses on the qualitative properties of mandibular bone, especially on mineral crystallinity in the vicinity of the teeth, namely, the alveolar bone proper.

Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur.

Osteoporosis is characterized by a bone loss associated to an increased bone marrow adiposity; however, it is still unclear what kind of lipids are involved. Therefore, the main purpose of this study was to see if there is any local bone lipid changes related to osteoporosis, by using the ovariectomy-induced osteoporosis (OVX) rat model. Female SD rats (operated at 6 months of age for skeletal maturity) were divided in control SHAM and OVX groups (n = 6/group) and maintained for 9 month post-surgery. Lipids were analyzed in two compartments of femoral diaphyses: bone marrow (BM) and mineralized tissue (MT), by chromatographic methods. As expected, osteoporotic femurs had a larger BM mass associated with a two-fold increase of lipid content. The MT had a similar lipid enrichment, indicating that adiposity affected the mineral part as well. The main lipids concerned were triglycerides, sphingomyelin, phosphatidylcholine and phosphatidylserine in BM, and triglycerides and cholesterol esters in MT. The increase of both energy-storage and membrane-associated lipids in BM suggested that cell number and/or size was enhanced to allow more triglyceride storage. Interestingly, in MT of osteoporotic femurs, sphingomyelin was decreased, suggesting that its catabolism could be linked to osteoporosis. In both femoral compartments, fatty acid profiles were enriched in 14:0 and 16:1, lowered in 18:0 and 20:4 n-6, and two-fold higher stearoyl-CoA desaturase indexes (16:1/16:0 and 18:1/18:0 ratios), suggesting an increased de novo lipogenesis in osteoporotic femurs. Thus, the present study is first to report local changes of individual lipids in rat osteoporotic femurs and suggests that osteoporosis is a pathologic condition associated with an enhanced de novo lipogenesis. Further studies will be needed to better understand the consequences of these lipid changes in osteoporotic bones.

Mandibular bone is protected against microarchitectural alterations and bone marrow adipose conversion in ovariectomized rats.

Osteoporosis is a disease that leads to a loss of bone mass and to alterations in the bone microarchitecture that occur in a site-specific manner; however it remains controversial in the jaw. The involvement of bone marrow adipose tissue (BMAT) in the bone metabolism has been suggested in several physiopathological contexts, such as in aging and osteoporosis. To test whether the BMAT content is related to mandibular bone loss, this study aimed to investigate the potential correlations between the trabecular bone microarchitecture on one hand and BMAT content and its spatial distribution in relation to bone surface on the other hand during aging and ovariectomy (OVX) during a long-term follow-up in a mature rat model. No age-related microarchitectural or BMAT changes were observed in the mandible. The OVX-induced bone loss was three-fold lower in the mandible than in the tibia and was observed only in the alveolar bone (not in the condyle). We also report a delayed increase in the mandibular BMAT content that remained 4-6-fold lower compared to tibia. This low BMAT content in the mandible was located at a distance from the trabecular bone surface (only 5% in contact with the bone surface versus 87% in the tibia). These findings highlight a specific mandibular response to OVX, in particular fewer microarchitectural alterations compared to that in the tibia. For the latter, the trabecular bone thickness and surface were correlated with the BMAT content. Oral functions may have a protective effect on the mandibular BMAT conversion in an OVX context.

A Novel microCT Method for Bone and Marrow Adipose Tissue Alignment Identifies Key Differences Between Mandible and Tibia in Rats.

Bone homeostasis is influenced by the bone marrow adipose tissue (BMAT). BMAT distribution varies from one anatomical location in the skeleton to another. We developed an advanced microfocus computed tomography imaging and analysis protocol that allows accurate alignment of both the BMAT distribution and bone micro-architecture as well as calculation of the distance of the BMAT adipocytes from the bone surface. Using this protocol, we detected a different spatial BMAT distribution between the rat tibia and mandible: in the proximal metaphysis of the tibia a large amount of BMAT (~ 20% of the total BMAT) was located close to the bone surface (< 20 µm), whereas in the alveolar ridge ~ 30% of the total BMAT was located between 40 and 60 µm from the bone surface. In the alveolar ridge of rats, the trabecular bone volume was 48.3% higher compared to the proximal metaphysis of the tibia (p < 0.0001) and the percentage of adiposity determined to the relative marrow volume was lower (1.5%) compared to the proximal metaphysis of the tibia (9%, p = 0.0002). Interestingly, in the tibia a negative correlation was found between the percentage of adiposity in the total volume and the trabecular thickness (r =- 0.74, p = 0.037). The present study highlights that in comparison to tibial proximal metaphysis, the mandibular bone exhibits a massive trabecular network and a low BMAT content with almost no contact with the bone surface. These findings are of great interest because of the importance of the fat-bone interaction and its potential relevance to several resorptive bone diseases.

Doses effects of zoledronic acid on mineral apatite and collagen quality of newly-formed bone in the rat's calvaria defect.

Due to their inhibitory effects on resorption, bisphosphonates are widely used in the treatment of diseases associated to an extensive bone loss. Yet, little is known about bisphosphonates effects on newly-formed bone quality. In the present study, adult male Sprague-Dawley rats (n=80) with a bone defect calvaria area were used and short-term effects of zoledronic acid (ZA) were studied on the healing bone area. Three ZA treatments were tested by using either: 1°) a low single dose (120µgZA/kg, n=10; equivalent to human osteoporosis treatment), 2°) a low fractionated doses (20µgZA/kg daily for 6days either a total of 120µg/kg, n=15), and 3°) a high fractionated doses, (100µgZA/kg weekly for 6weeks, n=15; equivalent to 6months of human bone metastasis treatment). For each treatment, a control "vehicle" treatment was performed (with an identical number of rats). After ZA administration, the intrinsic bone material properties were evaluated by quantitative backscattered electron imaging (qBEI) and Raman microspectroscopy. Neither single nor fractionated low ZA doses modify the intrinsic bone material properties of the newly-formed bone compared to their respective control animals. On the opposite, the high ZA treatment resulted in a significant decrease of the crystallinity (-25%, P< 0.05) and of the hydroxyproline-to-proline ratio (-30%, P<0.05) in newly-formed bones. Moreover, with the high ZA treatment, the crystallinity was positively correlated with the hydroxyproline-to-proline ratio (ρ=0.78, P<0.0001). The present data highlight new properties for ZA on bone formation in a craniofacial defect model. As such, ZA at high doses disrupted the apatite crystal organization. In addition, we report here for the first time that high ZA doses decreased the hydroxyproline-to-proline ratio suggesting that ZA may affect the early collagen organization during the bone healing.

Bone Samples Extracted from Embalmed Subjects Are Not Appropriate for the Assessment of Bone Quality at the Molecular Level Using Raman Spectroscopy.

Bone samples extracted from embalmed cadavers are commonly used as controls in the study of bone. The effects of embalmment on the molecular composition of bone are unknown. The objective of this study was to determine the effect of embalmment on the molecular composition and structure of bone, as evaluated by Raman spectroscopy. Bone samples of femoral heads from five embalmed donors and five fresh-frozen donors were compared using Raman microspectroscopy with DuoScan technology. Physicochemical parameters simultaneously describing the organic and mineral phases of bone were compared using the Mann-Whitney U test. Partial least squares discriminant analysis (PLS-DA) was used to determine specific Raman spectral features of each group. Study of the mineral phase showed a 15% reduction of the mineral-to-matrix ratio (p < 0.001), an 8% decrease of type B carbonate substitution (p < 0.001), and a 2% increase in crystallinity (p < 0.001) in the embalmed donors group compared to those of the fresh donors group. Regarding the organic phase of bone, the hydroxyproline-to-proline ratio was increased by 18% in the embalmed group (p < 0.001), with no variation in both the relative proteoglycan content (GAG/CH3) (p = 0.08) and collagen maturity (p = 0.57). PLS-DA showed that the embalmed group was characterized mainly by peaks assigned to hydroxyproline, lipids, and collagen. Embalmment induces significant modifications of the molecular composition of bone. Bone samples from embalmed subjects should be avoided as controls for Raman spectroscopy studies. Preservation procedures performed prior to bone sampling should be reported in studies using human cadaver samples.

Insulin-like growth factor 1 receptor and p38 mitogen-activated protein kinase signals inversely regulate signal transducer and activator of transcription 3 activity to control human dental pulp stem cell quiescence, propagation, and differentiation.

Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Y(low) stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration.

Human dental pulp stem cells differentiate into neural crest-derived melanocytes and have label-retaining and sphere-forming abilities.

Adult tissues contain highly proliferative, clonogenic cells that meet criteria of multipotent stem cells and are potential sources for autologous reparative and reconstructive medicine. We demonstrated that human dental pulp contains self renewing human dental pulp stem cells (hDPSCs) capable of differentiating into mesenchymal-derived odontoblasts, osteoblasts, adipocytes, and chondrocytes and striated muscle, and interestingly, also into non-mesenchymal melanocytes. Furthermore, we showed that hDPSC cultures include cells with the label-retaining and sphere-forming abilities, traits attributed to multipotent stem cells, and provide evidence that these may be multipotent neural crest stem cells.