Effect of Precursor Deficiency Induced Ca/P Ratio on Antibacterial and Osteoblast Adhesion Properties of Ag-Incorporated Hydroxyapatite: Reducing Ag Toxicity

Ag-containing hydroxyapatite (HA) can reduce risks associated with bacterial infections which may eventually require additional surgical operations to retrieve a failed implant. The biological properties of HA in such applications are strongly affected by its composition in terms of dopants as well as Ca/P stoichiometry, which can be easily controlled by altering processing parameters, such as precursor concentrations. The objective of this in vitro study was to understand the effect of variations in HA precursor solutions on antibacterial properties against Escherichia coli (E. coli) and for promoting osteoblast (bone-forming cell) adhesion on Ag incorporated HA (AgHA) which has not yet been investigated. For this, two groups of AgHAs were synthesized via a precipitation method by adjusting precursor reactants with a stoichiometric value of 1.67, being either (Ca + Ag)/P (Ca-deficient) or Ca/(P + Ag) (P-deficient), and were characterized by XRD, FTIR, and SEM-EDS. Results showed that Ag+ incorporated into the Ca2+ sites was associated with a corresponding OH− vacancy. Additional incorporation of CO32− into PO43− sites occurred specifically for the P-deficient AgHAs. While antibacterial properties increased, osteoblast adhesion decreased with increasing Ag content for the Ca-deficient AgHAs, as anticipated. In contrast, significant antibacterial properties with good osteoblast behavior were observed on the P-deficient AgHAs even with a lower Ag content, owing to carbonated HA. Thus, this showed that by synthesizing AgHA using P-deficient precursors with carbonate substitution, one can keep the antibacterial properties of Ag in HA while reducing its toxic effect on osteoblasts.

Recombinant IGF-1 Induces Sex-Specific Changes in Bone Composition and Remodeling in Adult Mice with Pappa2 Deficiency

Deficiency of pregnancy-associated plasma protein-A2 (PAPP-A2), an IGF-1 availability regulator, causes postnatal growth failure and dysregulation of bone size and density. The present study aimed to determine the effects of recombinant murine IGF-1 (rmIGF-1) on bone composition and remodeling in constitutive Pappa2 knock-out (ko/ko) mice. To address this challenge, X-ray diffraction (XRD), attenuated total reflection-fourier transform infra-red (ATR-FTIR) spectroscopy and gene expression analysis of members of the IGF-1 system and bone resorption/formation were performed. Pappa2ko/ko mice (both sexes) had reduced body and bone length. Male Pappa2ko/ko mice had specific alterations in bone composition (mineral-to-matrix ratio, carbonate substitution and mineral crystallinity), but not in bone remodeling. In contrast, decreases in collagen maturity and increases in Igfbp3, osteopontin (resorption) and osteocalcin (formation) characterized the bone of Pappa2ko/ko females. A single rmIGF-1 administration (0.3 mg/kg) induced short-term changes in bone composition in Pappa2ko/ko mice (both sexes). rmIGF-1 treatment in Pappa2ko/ko females also increased collagen maturity, and Igfbp3, Igfbp5, Col1a1 and osteopontin expression. In summary, acute IGF-1 treatment modifies bone composition and local IGF-1 response to bone remodeling in mice with Pappa2 deficiency. These effects depend on sex and provide important insights into potential IGF-1 therapy for growth failure and bone loss and repair.

Breast cancer–secreted factors perturb murine bone growth in regions prone to metastasis

Breast cancer frequently metastasizes to bone, causing osteolytic lesions. However, how factors secreted by primary tumors affect the bone microenvironment before the osteolytic phase of metastatic tumor growth remains unclear. Understanding these changes is critical as they may regulate metastatic dissemination and progression. To mimic premetastatic bone adaptation, immunocompromised mice were injected with MDA-MB-231–conditioned medium [tumor-conditioned media (TCM)]. Subsequently, the bones of these mice were subjected to multiscale, correlative analysis including RNA sequencing, histology, micro–computed tomography, x-ray scattering analysis, and Raman imaging. In contrast to overt metastasis causing osteolysis, TCM treatment induced new bone formation that was characterized by increased mineral apposition rate relative to control bones, altered bone quality with less matrix and more carbonate substitution, and the deposition of disoriented mineral near the growth plate. Our study suggests that breast cancer–secreted factors may promote perturbed bone growth before metastasis, which could affect initial seeding of tumor cells.

Pourbaix-Guided Mineralization and Site-Selective Photoluminescence Properties of Rare Earth Substituted B-Type Carbonated Hydroxyapatite Nanocrystals

Rare-earth labeling in biological apatite could provide critical information for the pathologic transition (osteoclastic) and physiologic regeneration (osteogenesis) of bone and teeth because of their characteristic site-sensitive fluorescence in different coordinative conditions of various tissues in many biological processes. However, the rare-earth labeling method for biological apatites, i.e., carbonated-hydroxyapatite, has been rarely found in the literature. In this paper, we report a Pourbaix-diagram guided mineralizing strategy to controllable carbonation and doping of rare-earth ions in the hydroxyapatite (HA) lattice. The carbonation process of hydroxyapatite was achieved by controllable mineralization in hydrothermal condition with K2CO3 as the carbonate source, which results into the pure B-type carbonated hydroxyapatite (CHA) with tunable carbonate substitution degree. All of the as-synthesized materials crystalized into P63/m (No. 176) space group with the lattice parameter of a decreases and c increases with the increasing of carbonate content in the reactants. Structural refinement results revealed that the substitution of planar CO32− is superimposed on one of the faces of PO43− tetrahedral sub-units with a rotation angle of 30° in reference to c-axis. All of the hydrothermally synthesized CHA nanocrystals show hexagonal rod-like morphology with the length of 70–110 nm and diameter of 21–35 nm, and the decreasing length/diameter ratio from 3.61 to 2.96 from low to high carbonated level of the samples. Five rare-earth cations, of Pr3+, Sm3+, Eu3+, Tb3+, and Ho3+, were used as possible probe ions that can be doped into either HA or CHA lattice. The site-preference of Tb3+ doping is the same in the crystallographic site of HA and CHA according to characteristic emission peaks of 5D4–7Fj (j = 3–6) transitions in their photoluminescent spectroscopy. Our work provides a controllable carbonation method for rare-earth labeling hydroxyapatite nanomaterials with potential biologically active implant powders for bone repair and tissue regeneration.

Nano-carbonated hydroxyapatite precipitation from abalone shell (Haliotis asinina) waste as the bioceramics candidate for bone tissue engineering

In this study, nano-carbonated hydroxyapatite (n-CHAp) was successfully synthesized with abalone shells ( Halioitis asinina) as the calcium source using precipitation methods with aging time variations, namely, 0 (without the aging process), 24, and 48 h. Based on an analysis of X-ray diffraction characterization, the spectrum of the n-CHAp is shown for all sample variations in aging time. The results of the calculation of lattice parameter values confirm that the phase formed is the B-type CHAp phase with the increasing crystallinity degree, crystallite size, particle size, and polydispersity which is confirmed by the presence of the CO32- functional group at 1438 cm−1 and 878 cm−1, that is, the B-type carbonate substitution characteristic. The presence of the carbonate ions identified as smaller during the extension of aging time causes the decreasing value of the Ca/P mole ratio but still has a value greater than the HAp Ca/P value (1.67), which is 1.80–1.72. Based on the transmission electron microscopy analysis, the nanometer-size of B-type CHAp particles was successfully obtained. According to the criteria for nanostructures, crystallographic properties, carbonate content, and chemical processes, B-type CHAp samples based on abalone shells ( Halioitis asinina) are one of the candidates in bioceramics for bone tissue engineering applications.

New insights into the application of pair distribution function studies to biogenic and synthetic hydroxyapatites

Biogenic and synthetic hydroxyapatites are confounding materials whose properties remain uncertain, even after years of study. Pair distribution function (PDF) analysis was applied to hydroxyapatites in the 1970’s and 1980’s, but this area of research has not taken full advantage of the relatively recent advances in synchrotron facilities. Here, synchrotron X-ray PDF analysis is compared to techniques commonly used to characterise hydroxyapatite (such as wide angle X-ray scattering, Fourier-transform infrared spectroscopy and thermogravimetric analysis) for a range of biogenic and synthetic hydroxyapatites with a wide range of carbonate substitution. Contributions to the pair distribution function from collagen, carbonate and finite crystallite size were examined through principal component analysis and comparison of PDFs. Noticeable contributions from collagen were observed in biogenic PDFs when compared to synthetic PDFs (namely r < 15 Å), consistent with simulated PDFs of collagen structures. Additionally, changes in local structure were observed for PDFs of synthetic hydroxyapatites with differing carbonate content, notably in features near 4 Å, 8 Å and 19 Å. Regression models were generated to predict carbonate substitution from peak position within the PDFs.

Geochemistry and paleoenvironment of the phosphorites from the Ameki Formation, Niger delta, Nigeria

The phosphorites of the Ameki Formation occur as nodules, pellets as well as primary phosphatic shales and siltstones. Geochemical analysis of the phosphate samples was carried out to determine its chemical composition as well as its depositional environment. The methodology applied include XRF, INAA, XRD and thin section petrography using Polarizing Microscope. The XRF result identified CaO- P2O5- F as the major mineral group, SiO2, Al2O3, Fe2O3, MgO and TiO2, which show minor occurrences and Cr, U, Pb, V, Cu, Zn, Se and Cd that occur in trace amounts. The nodules and pellets are of medium to high grade (25- 34wt% P2O5) whereas the primary phosphatic shales and siltstones are of low to medium grade (4.5– 22wt% P2O5). The phosphorites comprise mostly of francolites. The mean index of refraction estimated by Becke-line method using Standard Polarizing Microscope gave 1.634 and1.636.XRD analysis yielded an average values of 9.243 (± 0.002) A° and 6.715 (± 0.002) A° for a and c crystallographic axes respectively with an axial ratio (c/a) of 0.726for unit cell parameter, suggestive of low degree of carbonate substitution. An increase in P2O5 content is found to be accompanied by increase in CaO, CO2 and F contents, but by a decrease in H2O, organic carbon, SiO2 and Fe contents indicative of amorphous solid phase of calcium phosphate. The phosphorite is interpreted to form under high biologic productive, shelf setting. High nutrient availability in the ancient sea is linked to upwelling along the West African Coastline during the Eocene. Keywords: Phosphorites; Geochemistry; Paleoenvironment; Ameki; Francolites; Nodules

A Comprehensive Microstructural and Compositional Characterization of Allogenic and Xenogenic Bone: Application to Bone Grafts and Nanostructured Biomimetic Coatings

Bone grafts and bone-based materials are widely used in orthopedic surgery. However, the selection of the bone type to be used is more focused on the biological properties of bone sources than physico-chemical ones. Moreover, although biogenic sources are increasingly used for deposition of biomimetic nanostructured coatings, the influence of specific precursors used on coating’s morphology and composition has not yet been explored. Therefore, in order to fill this gap, we provided a detailed characterization of the properties of the mineral phase of the most used bone sources for allografts, xenografts and coating deposition protocols, not currently available. To this aim, several bone apatite precursors are compared in terms of composition and morphology. Significant differences are assessed for the magnesium content between female and male human donors, and in terms of Ca/P ratio, magnesium content and carbonate substitution between human bone and different animal bone sources. Prospectively, based on these data, bone from different sources can be used to obtain bone grafts having slightly different properties, depending on the clinical need. Likewise, the suitability of coating-based biomimetic films for specific clinical musculoskeletal application may depend on the type of apatite precursor used, being differently able to tune surface morphology and nanostructuration, as shown in the proof of concepts of thin film manufacturing here presented.