Defective Mineralization in X-Linked Hypophosphatemia Dental Pulp Cell Cultures.

X-linked hypophosphatemia (XLH) is a skeletal disease caused by inactivating mutations in the PHEX gene. Mutated or absent PHEX protein/enzyme leads to a decreased serum phosphate level, which cause mineralization defects in the skeleton and teeth (osteomalacia/odontomalacia). It is not yet altogether clear whether these manifestations are caused solely by insufficient circulating phosphate availability for mineralization or also by a direct, local intrinsic effect caused by impaired PHEX activity. Here, we evaluated the local role of PHEX in a 3-dimensional model of extracellular matrix (ECM) mineralization. Dense collagen hydrogels were seeded either with human dental pulp cells from patients with characterized PHEX mutations or with sex- and age-matched healthy controls and cultured up to 24 d using osteogenic medium with standard phosphate concentration. Calcium quantification, micro-computed tomography, and histology with von Kossa staining for mineral showed significantly lower mineralization in XLH cell-seeded scaffolds, using nonparametric statistical tests. While apatitic mineralization was observed along collagen fibrils by electron microscopy in both groups, Raman microspectrometry indicated that XLH cells harboring the PHEX mutation produced less mineralized scaffolds having impaired mineral quality with less carbonate substitution and lower crystallinity. In the XLH cultures, immunoblotting revealed more abundant osteopontin (OPN), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) than controls, as well as the presence of fragments of these proteins not found in controls, suggesting a role for PHEX in SIBLING protein degradation. Immunohistochemistry revealed altered OPN and DMP1 associated with an increased alkaline phosphatase staining in the XLH cultures. These results are consistent with impaired PHEX activity having local ECM effects in XLH. Future treatments for XLH should target both systemic and local manifestations.

Molecular alterations of bone quality in sequesters of bisphosphonates-related osteonecrosis of the jaws.

UNLABELLED: Compared to healthy bone, the intrinsic bone materials properties in the pre-existing lamellar bone are altered in jaw bone sequesters of bisphosphonates (BP)-related osteonecrosis. INTRODUCTION: The aim of this study was to evaluate the human jaw bone quality, especially intrinsic bone material properties among sequesters of osteonecrosis of the jaw (ONJ) induced by BP. METHODS: Bone sequesters were obtained from 24 patients suffering from ONJ following a BP treatment. Within BP-exposed bone samples, benign-BP and malignant-BP groups were distinguished in relation to the underlying disease: osteoporosis and bone metastases or multiple myeloma, respectively. Healthy cadaveric cortical jaw bone samples were used as controls. The physicochemical parameters of bone samples - mineral/organic ratio, relative proteoglycan content, crystallinity, monohydrogen phosphate content, and type-B carbonate substitution - were evaluated by Raman microspectroscopy. Representative Raman spectral features of bones control and BP-exposed bone sequesters were identified with the Partial-Least-Square Discriminant Analysis (PLS-DA). RESULTS: BP-exposed bone sequesters are characterized by a significant increase of mineral to organic ratio (+12 %) and a significant decrease of relative proteoglycan content (-35 %), thus regulating initial collagen matrix mineral deposition. Structural changes on mineral components are revealed by a significant decrease of both crystallinity (-2 %) and mineral maturation (-41 %) in the BP-exposed bone sequesters compared to healthy bones. These modifications were also observed distinctly in both benign-BP and malignant-BP groups. In addition, a shift of the phosphate ν1 band was highlighted by PLS-DA between bones control and BP-exposed bone sequesters, revealing a disruption of the apatitic phosphate environment in the jaw bone sequesters. CONCLUSIONS: The present data show that jaw bone quality can be altered with an overmineralization and ultrastructural modifications of apatitic mineral in bone sequesters of BP-related ONJ.

No anti-angiogenic effect of clinical dosing regimens of a single zoledronic acid injection in an experimental bone healing site.

INTRODUCTION: An anti-angiogenic effect of bisphosphonates has been reported in different experimental models. Zoledronic acid is currently administered in osteoporotic patients as a single 5 mg injection once a year and its vascular effect in bone has not been yet evaluated. MATERIALS AND METHODS: The vascular dose effect of a single injection of zoledronic acid was evaluated on healing vascularization developed under a bone chamber implanted on the calvaria of 30 rats. After 3 weeks of healing, the rats were randomized into 3 groups receiving an injection of either physiologic saline solution (PSS) or zoledronic acid tested at 120 microg/kg, the equivalent of a 5 mg dose of zoledronic acid in humans (Z120), and 400 microg/kg, a supra-pharmacologic dose (Z400). A longitudinal follow-up of the healing vascular network was carried out at days (D) 1, 3, 6, 9, 12, 15 and 28 after injection by intravital imaging. Variations in vascular density, total length of the vascular network and mean diameter of vascular network branches were determined by image analysis (Aphelion software). RESULTS: A decrease was observed in both vascular density and total length of the network in control and treated groups (time effect). No difference in variation in vascular density was observed between the PSS group and the Z120 group at any time point (p=NS). A trend to a higher decrease in vascular density was noted between D12 and D15 in the Z400 group. A significant decrease in total length was noted at D15 in the Z400 group (p=0.03) compared to the PSS group, whereas no change was noted in rats treated with 120 microg/kg compared to PSS rats on any of the follow-up days (p=0.2). No variation in mean diameter of vascular network branches was noted in any of the three groups at any of the follow-up days (p=0.53). CONCLUSION: A single injection of clinically relevant dosing regimens of zoledronic acid may not have a notable impact on vascularization in bone sites. The anti-angiogenic effect of bisphosphonates seems to express itself, in our model, at higher doses than those used in patients treated for osteoporosis.

Effects of high dose of zoledronic acid on superficial vascular network of membranous bone sites: an intravital study on rat calvarium.

UNLABELLED: We evaluated the impact of high dose of zoledronic acid on the superficial vascular network parameters of a membranous bone site. During a 5-day follow-up, significant reduction of the vascular density is observed only in the treated group. INTRODUCTION: The superficial vascularization is of great importance in membranous bone-healing process. A new rat calvarium intravital model was developed to study the short-term effect of a single high dose of zoledronic acid infusion on the superficial vascularization. METHODS: Optical bone chambers were implanted in the bone tissue surface of Sprague-Dawley rats' calvarium. Nine rats were injected i.v. with 400 µg/kg of zoledronic acid (Z group), and nine rats were injected with vehicle (PSS group). A 5-day follow-up of the vascular network was made by the use of pictures analysis method. RESULTS: The vascular density significantly decreases only in Z group but there was no significant difference between groups at individual time points. The total length of the vascular network decreases significantly in Z group only (p=0.003) with a significant higher decrease at D3 (p=0.04) and D5 (p=0.02) compared with control. The vascular density related to the smaller vessels (width, 5-10 µm) decreases significantly between T0 and D5 in Z group only (p=0.002). CONCLUSIONS: With the help of an original intravital animal model a significant modifications on the total length of the vascular network and the vascular density of small vessels are highlighted on a membranous bone site.

Composition of bone and apatitic biomaterials as revealed by intravital Raman microspectroscopy.

Microcharacterization of biominerals allows a better understanding of the pathophysiological events that occur in calcified tissues and synthetic biomaterials. Different methods have been extensively used to conduct such investigations. A new model for the intravital study of the composition and structure of membranous bone by Raman microspectroscopy is described. Titanium bone chambers equipped with a fused-silica optical window were implanted transcutaneously in the calvaria of New Zealand rabbits. The implanted optical windows were well tolerated, and spectral acquisitions were performed without any additional invasive procedure. Bone and implanted apatitic biomaterials were analyzed at different times after surgery. All Raman bands were unambiguously identified in the bone and biomaterial spectra. The main PO4 and CO3 Raman bands in bone spectra were consistent with those found in the carbonated apatite spectrum. The major collagen bands were always observed around 1200-1300 (amide III) and 1600-1700 (amide I) delta cm(-1) and, 1400-1470 and 2800-3100 delta cm(-1) (bending and stretching modes of CH groups, respectively). The phenylalanine (Phe) band was identified in all spectra at 1003 delta cm(-1) and overlapped that of the weak HPO4(2-) ion. The CH bands frequently overlapped the lipid bands. However a distinct protein and lipid bands were detected at 2950 and 2852 delta cm(-1), respectively. In bone areas close to blood vessels, the Raman signature of hemoglobin was detected with a characteristic band at 754 delta cm(-1). The changes observed in bone varied as a function of time and location. The composition and structure of all of the biomaterials studied--including those that were resorbable--seemed to remain stable over time and location. We report for the first time the complete intravital study of Raman spectra of bone and calcium phosphate biomaterials over a period of 8 months. This new approach does not require specimen preparation and allows simultaneous observation of mineral and organic bone constituents over time, which therefore should provide insightful information about their relationship.

Raman investigation of calcium carbonate bone substitutes and related biomaterials.

The interaction between biomaterials used in surgical procedures and the host bone is not yet perfectly understood. It appears that these problems may have been encountered because of insufficient characterisation of the basic component used in the synthesis of such biomaterials. Calcium carbonate (CaCO3) is interesting for bone filling or regeneration procedures because of its resorbability. The aim of this work is to compare different CaCO3 biomaterials and their basic source with the help of microRaman spectroscopy. Bionacre and Biocoral are analysed. The main bands of carbonate internal modes are observed around 1084-86 and 704-12 cm-1. In the lattice modes region, for both Biocoral and synthetic aragonite, two bands at 206 and 155 cm-1 are observed. The eggshell, oyster shell and synthetic calcite samples exhibit bands at 281 and 155 cm-1. Three bands are present at 280, 206 and 155 cm-1 on the Bionacre sample. The 206 and the 280 cm-1 bands are due to aragonite and calcite forms respectively. Therefore it appears to be a mixture of aragonite and calcite whereas Biocoral is pure aragonite. Additional Raman investigations should be of great interest in evaluating the structural modifications and their influence on the biological behaviour of these biomaterials. Lity index, the percentage of prematurity and of low birth weight are the indices of national health status.

[Cleaning implantation burs. Observations using scanning electron microscopy]. / A propos du nettoyage des forets implantaires. Observation en microscopie electronique a balayage.

Drastic aseptic conditions are necessary in implantological treatments. A good sterilizing procedure of the specific instrumentation, like drills, is based on an efficient cleaning. Because of their design, the cleaning of drills is a real challenge. The aim of this investigation is to evaluate two different cleaning procedures usually used by implantologists. One is based on a manual cleaning, the other on an ultra-sonic cleaning. The instrument observed by scan-electron-microscope, is a I.T.I. system drill. The results show the superiority of the ultra-sonic cleaning. The manual cleaning is especially ineffective on the inside and the cutting part of the drill. Even if ultra-sonic cleaning is definitely a better procedure, it has to be improved. A extensive study should be conducted to optimize the cleaning parameters, if not, single-use drill should be definitely preferred by implantologists.

[Raman spectrometry applied to calcified tissue and calcium-phosphorus biomaterials]. / Spectrometrie raman appliquee aux tissus calcifies et aux biomateriaux phospho-calciques.

The rigid part of the human body consists essentially of carbonated apatite (calcium phosphate). Biologists don't have any tools to study this "mineral" phase, though its origin is organic. A new approach of some compounds like enamel or bone is obtained with the Raman micro-characterisation by a very fine analysis of chemical bonds in a micrometric scale. This method allows the characterisation, the analysis and the dosage of ions, like carbonate, acid phosphates, proteins and fatty acids. The identification of other organic or mineral compounds (e.g. calcium carbonate, calcium oxide, substitutant ions...) is also possible. The Raman microspectrometry can also be used to study the chemical and physical properties of biomaterials and their evolution after implantation in a dental or bone site. On synthetical calcium phosphate, beta-TCP, brushite and hydroxyapatite can be distinguished and the impurities found in plasma spray deposits can be measured. The detection of alpha-, beta-, or gamma-pyrophosphates could be obtained in some commercial beta-TCP. The Raman microspectrometry is the only non-destructive method which allows the identification of the chemical bonds in a micrometric scale and gives the "fingerprint" of the studied component.

Raman microspectrometry studies of brushite cement: in vivo evolution in a sheep model.

Calcium phosphate hydraulic cements are promising synthetic bone grafting materials. Brushite-based cements were implanted for 6 and 12 months in the distal condyle of sheep femur, and their in vivo evolution was investigated by Raman microspectrometry. This new technique can probe small volumes in the cubic micrometer range. Its resolution allows a very fine analysis of crystalline changes in calcium phosphate mixtures at the microscopic level. First, Raman spectra of pure brushite, monetite, and beta-tricalcium phosphate (beta-TCP) were recorded, in order to set a data base for the basic components of brushite cements. These spectra show significant differences in the vibration mode v1 for the phosphate ion (988 and 878 cm(-1) for brushite, 988 and 900 cm(-1) for monetite, 968 and 948 cm(-1) for beta-TCP). These differences are strong enough as to allow the qualitative and quantitative analysis of these crystalline phases in the cement. Implanted sheep femur samples were harvested after 24 and 52 weeks post-op, and prepared for Raman analysis in the form of 1-mm-thick sections. Implants at 24 weeks show a core of residual cement isolated from the surrounding bone by fibroconnective tissue. No trace of brushite was detected by micro-Raman analysis in this area, but instead, a mixture of beta-TCP and Type-B carbonated apatite, the latter being very close in composition and structure to the mineral fraction of normal bone in the vicinity of the implant. Implants recovered after 52 weeks show a decrease of the bone/residual cement perimeter, whereas new trabeculations are formed in the implanted zone; the small amounts of residual cement still present are substantially transformed into Type-B carbonated apatite containing small amounts of proteins. In the same area, some beta-TCP particles are also detected showing that, contrary to brushite, the excess beta-TCP originally present in the cement is not completely metabolized. In the implanted zone already converted into trabecular bone, Raman microspectrometry shows the characteristic spectrum of normal bone.

MicroRaman spectral study of the PO4 and CO3 vibrational modes in synthetic and biological apatites.

The carbonate and phosphate vibrational modes of different synthetic and biological carbonated apatites were investigated by Raman microspectroscopy, and compared with those of hydroxyapatite. The nu1 phosphate band at 960 cm-1 shifts slightly due to carbonate substitution in both A and B sites. The spectrum of type A carbonated apatite exhibits two nu1 PO43- bands at 947 and 957 cm-1. No significant change was observed in the nu2 and nu4 phosphate mode regions in any carbonated samples. The nu3 PO43- region seems to be more affected by carbonation: two main bands were observed, as in the hydroxyapatite spectrum, but at lower wave numbers. The phosphate spectra of all biominerals apatite were consistent with type AB carbonated apatite. In the enamel spectrum, bands were observed at 3513 and at 3573 cm-1 presumably due to two different hydroxyl environments. Two different bands due to the carbonate nu1 mode were identified depending on the carbonate substitution site A or B, at 1107 and 1070 cm-1, respectively. Our results, compared with the infrared data already reported, suggest that even low levels of carbonate substitution induce modifications of the hydroxyapatite spectrum. Increasing substitution ratios, however, do not bring about any further alteration. The spectra of dentine and bone showed a strong similarity at a micrometric level. This study demonstrates the existence of acidic phosphate, observable by Raman microspectrometry, in mature biominerals. The HPO42- and CO32- contents increase from enamel to dentine and bone, however, these two phenomena do not seem to be correlated.

Infrared and Raman microspectrometry study of fluor-fluor-hydroxy and hydroxy-apatite powders.

Visible Raman and infrared microspectrometry studies performed on fluorapatite and hydroxyapatite powders have shown similar results. Small modifications of the nu2 and nu4 PO(3-)4 tetrahedra bending modes are observed. A small frequency shift of the nu1 mode and modifications on the nu3 mode region accompanied with a simplification of the hydroxyapatite and fluorapatite respective spectra from seven to four bands were observed. A broad and weak band which could be attributed to the Ca-F bond is detected at 311 cm(-1) on the Raman fluorapatite spectra. The phosphate vibration modes are little disturbed by fluoride substitution. This could indicate that phosphate groups interact strongly between themselves and weakly with substituted atoms (i.e. hydroxyle and fluoride atoms). Whatever crystallographic model is considered, the number of bands observed is always lower than the number of calculated ones, even for hydroxyapatite, whose symmetry is lower than that of fluorapatite.