Osteopontin stabilization and collagen containment slows amorphous calcium phosphate transformation during human aortic valve leaflet calcification.

Calcification of aortic valve leaflets is a growing mortality threat for the 18 million human lives claimed globally each year by heart disease. Extensive research has focused on the cellular and molecular pathophysiology associated with calcification, yet the detailed composition, structure, distribution and etiological history of mineral deposition remains unknown. Here transdisciplinary geology, biology and medicine (GeoBioMed) approaches prove that leaflet calcification is driven by amorphous calcium phosphate (ACP), ACP at the threshold of transformation toward hydroxyapatite (HAP) and cholesterol biomineralization. A paragenetic sequence of events is observed that includes: (1) original formation of unaltered leaflet tissues: (2) individual and coalescing 100's nm- to 1 µm-scale ACP spherules and cholesterol crystals biomineralizing collagen fibers and smooth muscle cell myofilaments; (3) osteopontin coatings that stabilize ACP and collagen containment of nodules preventing exposure to the solution chemistry and water content of pumping blood, which combine to slow transformation to HAP; (4) mm-scale nodule growth via ACP spherule coalescence, diagenetic incorporation of altered collagen and aggregation with other ACP nodules; and (5) leaflet diastole and systole flexure causing nodules to twist, fold their encasing collagen fibers and increase stiffness. These in vivo mechanisms combine to slow leaflet calcification and establish previously unexplored hypotheses for testing novel drug therapies and clinical interventions as viable alternatives to current reliance on surgical/percutaneous valve implants.

Mgp High-Expressing MSCs Orchestrate the Osteoimmune Microenvironment of Collagen/Nanohydroxyapatite-Mediated Bone Regeneration.

Activating autologous stem cells after the implantation of biomaterials is an important process to initiate bone regeneration. Although several studies have demonstrated the mechanism of biomaterial-mediated bone regeneration, a comprehensive single-cell level transcriptomic map revealing the influence of biomaterials on regulating the temporal and spatial expression patterns of mesenchymal stem cells (MSCs) is still lacking. Herein, the osteoimmune microenvironment is depicted around the classical collagen/nanohydroxyapatite-based bone repair materials via combining analysis of single-cell RNA sequencing and spatial transcriptomics. A group of functional MSCs with high expression of matrix Gla protein (Mgp) is identified, which may serve as a pioneer subpopulation involved in bone repair. Remarkably, these Mgp high-expressing MSCs (MgphiMSCs) exhibit efficient osteogenic differentiation potential and orchestrate the osteoimmune microenvironment around implanted biomaterials, rewiring the polarization and osteoclastic differentiation of macrophages through the Mdk/Lrp1 ligand-receptor pair. The inhibition of Mdk/Lrp1 activates the pro-inflammatory programs of macrophages and osteoclastogenesis. Meanwhile, multiple immune-cell subsets also exhibit close crosstalk between MgphiMSCs via the secreted phosphoprotein 1 (SPP1) signaling pathway. These cellular profiles and interactions characterized in this study can broaden the understanding of the functional MSC subpopulations at the early stage of biomaterial-mediated bone regeneration and provide the basis for materials-designed strategies that target osteoimmune modulation.

3D-printed PCL framework assembling ECM-inspired multi-layer mineralized GO-Col-HAp microscaffold for in situ mandibular bone regeneration.

BACKGROUND: In recent years, natural bone extracellular matrix (ECM)-inspired materials have found widespread application as scaffolds for bone tissue engineering. However, the challenge of creating scaffolds that mimic natural bone ECM's mechanical strength and hierarchical nano-micro-macro structures remains. The purposes of this study were to introduce an innovative bone ECM-inspired scaffold that integrates a 3D-printed framework with hydroxyapatite (HAp) mineralized graphene oxide-collagen (GO-Col) microscaffolds and find its application in the repair of mandibular bone defects. METHODS: Initially, a 3D-printed polycaprolactone (PCL) scaffold was designed with cubic disks and square pores to mimic the macrostructure of bone ECM. Subsequently, we developed multi-layer mineralized GO-Col-HAp microscaffolds (MLM GCH) to simulate natural bone ECM's nano- and microstructural features. Systematic in vitro and in vivo experiments were introduced to evaluate the ECM-inspired structure of the scaffold and to explore its effect on cell proliferation and its ability to repair rat bone defects. RESULTS: The resultant MLM GCH/PCL composite scaffolds exhibited robust mechanical strength and ample assembly space. Moreover, the ECM-inspired MLM GCH microscaffolds displayed favorable attributes such as water absorption and retention and demonstrated promising cell adsorption, proliferation, and osteogenic differentiation in vitro. The MLM GCH/PCL composite scaffolds exhibited successful bone regeneration within mandibular bone defects in vivo. CONCLUSIONS: This study presents a well-conceived strategy for fabricating ECM-inspired scaffolds by integrating 3D-printed PCL frameworks with multilayer mineralized porous microscaffolds, enhancing cell proliferation, osteogenic differentiation, and bone regeneration. This construction approach holds the potential for extension to various other biomaterial types.

ECM-inspired calcium/zinc laden cellulose scaffold for enhanced bone regeneration.

Cellulose-based polymer scaffolds are highly diverse for designing and fabricating artificial bone substitutes. However, realizing the multi-biological functions of cellulose-based scaffolds has long been challenging. In this work, inspired by the structure and function of the extracellular matrix (ECM) of bone, we developed a novel yet feasible strategy to prepare ECM-like scaffolds with hybrid calcium/zinc mineralization. The 3D porous structure was formed via selective oxidation and freeze drying of bacterial cellulose. Following the principle of electrostatic interaction, calcium/zinc hybrid hydroxyapatite nucleated, crystallized, and precipitated on the 3D scaffold in simulated physiological conditions, which was well confirmed by morphology and composition analysis. Compared with alternative scaffold cohorts, this hybrid ion-loaded cellulose scaffold exhibited a pronounced elevation in alkaline phosphatase (ALP) activity, osteogenic gene expression, and cranial defect regeneration. Notably, the hybrid ion-loaded cellulose scaffold effectively fostered an M2 macrophage milieu and had a strong immune effect in vivo. In summary, this study developed a hybrid multifunctional cellulose-based scaffold that appropriately simulates the ECM to regulate immunomodulatory and osteogenic differentiation, setting a measure for artificial bone substitutes.

Nano-/micro-scaled hydroxyapatite ceramic construction and the regulation of immune-associated osteogenic differentiation.

Hydroxyapatite (HA) bioceramic is a promising substitute for bone defects, and the surface properties are major factors that influence bioactivity and osteoinductivity. In this study, two kinds of HA bioceramics with nanoscale (n-HA) and microscale (m-HA) surface topography were designed to mimic the natural bone, thus enhancing the stimulation of osteogenic differentiation and revealing the potential mechanism. Compared to m-HA, n-HA owned a larger surface roughness, a stronger wettability, and reduced hardness and indentation modulus. Based on these properties, n-HA could maintain the conformation of vitronectin better than m-HA, which may contribute to higher cellular activities and a stronger promotion of osteogenic differentiation of mesenchymal stem cells (MSCs). Further RNA sequencing analysis compared the molecular expression between n-HA and m-HA. Six hundred twenty-seven differentially expressed genes were identified in MSCs, and 17 upregulated genes and 610 downregulated genes were included when n-HA compared to m-HA. The GO cluster analysis and enriched Kyoto encyclopedia of genes and genome signaling pathways revealed a close correlation with the immune process in both upregulated (chemokine signaling pathway and cytokine-cytokine receptor interaction) and downregulated pathways (osteoclasts differentiation). It suggested that the nanoscale surface topography of HA enhanced the osteoinductivity of MSCs and could not be separated from its regulation of immune function and the retention of adsorbed protein conformation.

Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral.

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.

Gypsum-Related Impact on Antibiotic-Loaded Composite Based on Highly Porous Hydroxyapatite-Advantages and Disadvantages.

Highly porous hydroxyapatite is sometimes considered toxic and useless as a biomaterial for bone tissue regeneration because of the high adsorption of calcium and phosphate ions from cell culture media. This negatively affects the osteoblast's growth in such ion-deprived media and suggests "false cytotoxicity" of tested hydroxyapatite. In our recent study, we showed that a small addition of calcium sulfate dihydrate (CSD) may compensate for this adsorption without a negative effect on other properties of hydroxyapatite-based biomaterials. This study was designed to verify whether such CSD-supplemented biomaterials may serve as antibiotic carriers. FTIR, roughness, mechanical strength analysis, drug release, hemocompatibility, cytotoxicity against human osteoblasts, and antibacterial activity were evaluated to characterize tested biomaterials. The results showed that the addition of 1.75% gypsum and gentamicin caused short-term calcium ion compensation in media incubated with the composite. The combination of both additives also increased antibacterial activity against bacteria representative of bone infections without affecting osteoblast proliferation, hemocompatibility, and mechanical parameters. Thus, gypsum and antibiotic supplementation may provide advanced functionality for bone-regeneration materials based on hydroxyapatite of a high surface area and increasingly high Ca2+ sorption capacity.

Study of nano-hydroxyapatite tagged alkaline protease isolated from Himalayan sub-alpine Forest soil bacteria and role in recalcitrant feather waste degradation.

Purified calcium serine metalloprotease from Stenotrophomonas maltophilia strain SMPB12 exhibits highest enzyme activity at pH 9 and temperature range between 15 °C-25 °C. Enzyme supplemented with 40 µM Ca-Hap-NP (NP-protease) showed maximum elevated activity of 17.29 µmole/min/ml (1.9-fold of original protease activity). The thermostability of the enzyme was maintained for 1 h at 60 °C over an alkaline pH range 7.5-10, as compared to the NP untreated enzyme whose activity was of 8.97 µmole/min/ml. A significant loss of activity with EDTA (1.05 µmole/min/ml, 11.75 %), PMSF (0.93 µmole/min/ml, 10.46 %) and Hg2+ (3.81 µmole/min/ml, 42.49 %) was also observed. Kinetics study of NP-protease showed maximum decreases in Km (28.11 %) from 0.28 mM (NP untreated enzyme) to 0.22 mM (NP-protease) along with maximum increase in Vmax (42.88 %) from 1.25 µmole/min/ml to 1.79 µmole/min/ml at varying temperatures. The enhanced activity of NP-protease was able to efficiently degrade recalcitrant solid wastes like feather to produce value-added products like amino acids and helps in declogging recalcitrant solid wastes. The nano-enabled protease may be utilized in a smaller amount for degrading in bulk recalcitrant solid proteinaceous waste at 15 °C temperature as declogging agents providing an eco-friendly efficient process.

Hydroxyapatite nanoparticles promote TLR4 agonist-mediated anti-tumor immunity through synergically enhanced macrophage polarization.

Macrophages represent the most prevalent immune cells in the tumor micro-environment, making them an appealing target for tumor immunotherapy. One of our previous studies showed that hydroxyapatite nanoparticles (HANPs) enhanced Toll-like receptor 4 (TLR4) signal transduction in macrophages. This study was proposed to investigate how HANPs manipulated the phenotype and function of macrophage against 4T1 breast tumors in the presence or absence of MPLA, a low toxic Toll-like receptor 4 (TLR4) agonist. The results demonstrated that the addition of HANPs to MPLA significantly promoted cytokine secretion and macrophage polarization toward a tumoricidal M1 phenotype. Further, the resulting supernatant from HANPs/MPLA co-stimulated macrophages enhanced 4T1 tumor cells apoptosis compared to that from macrophages treated with a single component or PBS control. In particular, we found HANPs elicited immunogenic cell death (ICD) indicated by the increased expression of "danger signals", including HMGB1, CRT and ATP in 4T1 cells. Subsequently, the ICD derivatives-containing supernatant from HANPs-treated 4T1 cells activated macrophage and shifted the phenotype of the cells toward M1 type. Moreover, in a tumor-bearing mice model, HANPs and MPLA synergistically delayed tumor growth compared to PBS control, which was positively associated with the promoted macrophage polarization and ICD induction. Therefore, our findings demonstrated a potential platform to modulate the function of macrophages, and shed a new insight into the mechanism involving the immunomodulatory effect of HANPs for tumor therapy. STATEMENT OF SIGNIFICANCE: Polarizing macrophage toward tumoricidal phenotype by harnessing Toll-like receptor (TLR) agonists has been proven effective for tumor immunotherapy. However, the immunomodulatory potency of TLR agonists is limited due to immune suppression or tolerance associated with TLR activation in immune cells. Herein, we introduced hydroxyapatite nanoparticles (HANPs) to MPLA, a TLR4 agonist. The results demonstrated that the addition of HANPs to MPLA promoted macrophage shift toward tumoricidal M1 phenotype, supported a "hot" tumor transformation, and delayed 4T1 tumor growth. Moreover, we found that HANPs elicited immunogenic cell death that produced "danger" signals from cancer cells thereby further facilitated macrophage polarization. This work is significant to direct the rational design of HANPs coupled with or without TLR agonists for tumor immunotherapy.

Silk fibroin-hydroxyapatite scaffolds promote the proliferation of adipose-derived mesenchymal stem cells by activating the ERK signal.

Adipose-derived mesenchymal stem cell (Ad-MSC) with capacities of releasing trophic factors and chondrogenic differentiation was a promising candidate for tracheal reconstruction. Silk fibroin (SF)- hydroxyapatite (HA) scaffolds were fabricated by the freeze-drying method. And Ad-MSCs were co-cultured on the scaffolds for 14 days in vitro. The role of the SF-HA scaffold in regulating the adhesion, growth, and proliferation of Ad-MSCs, and its potential mechanisms were investigated. The identity of Ad-MSCs was confirmed by cell morphology, surface markers, and differentiation characteristics. Cell proliferation, viability, and morphology were observed via CCK-8, live/dead assay, and scanning electron microscopy (SEM). Gene mRNA and protein levels were examined using quantitative real-time polymerase chain reaction and western blotting, respectively. SF-HA scaffolds showed excellent properties of promoting Ad-MSCs adhesion, growth, and proliferation for at least 14 days. In the CCK-8 assay, the relative OD value of Ad-MSCs cultured on SF-HA scaffolds increased (p < 0.001). Furthermore, live/dead staining showed that the fluorescent coverage increased with time (p < 0.05). SEM also showed that 3 days after inoculation, the coverage of Ad-MSCs on the SF-HA scaffolds was 78.15%, increased to 92.91% on day 7, and reached a peak of 94.38% on day 14. Extracellular signal-regulated kinase (ERK) mRNA and phosphorylated ERK (pERK) protein expression increased at day 3 (p < 0.05), followed by a significant decline at day 7 (p < 0.05). And ERK mRNA expression was positively correlated with Ad-MSCs proliferation (p < 0.05). In summary, the SF-HA scaffold co-cultured with Ad-MSCs is a promising biomaterial for tracheal repair by activating the ERK signal pathway.

Ultrastructural and Immunohistochemical Detection of Hydroxyapatite Nucleating Role by rRNA and Nuclear Chromatin Derivatives in Aortic Valve Calcification: In Vitro and In Vivo Pro-Calcific Animal Models and Actual Calcific Disease in Humans.

Calcification starts with hydroxyapatite (HA) crystallization on cell membranous components, as with aortic valve interstitial cells (AVICs), wherein a cell-membrane-derived substance containing acidic phospholipids (PPM/PPLs) acts as major crystal nucleator. Since nucleic acid removal is recommended to prevent calcification in valve biosubstitutes derived from decellularized valve scaffolds, the involvement of ribosomal RNA (rRNA) and nuclear chromatin (NC) was here explored in three distinct contexts: (i) bovine AVIC pro-calcific cultures; (ii) porcine aortic valve leaflets that had undergone accelerated calcification after xenogeneic subdermal implantation; and (iii) human aortic valve leaflets affected by calcific stenosis. Ultrastructurally, shared AVIC degenerative patterns included (i) the melting of ribosomes with PPM/PPLs, and the same for apparently well-featured NC; (ii) selective precipitation of silver particles on all three components after adapted von Kossa reactions; and (iii) labelling by anti-rRNA immunogold particles. Shared features were also provided by parallel light microscopy. In conclusion, the present results indicate that rRNA and NC contribute to AVIC mineralization in vitro and in vivo, with their anionic charges enhancing the HA nucleation capacity exerted by PPM/PPL substrates, supporting the concept that nucleic acid removal is needed for valve pre-implantation treatments, besides better elucidating the modality of pro-calcific cell death.

Human adipose mesenchymal stromal cells growing into PCL-nHA electrospun scaffolds undergo hypoxia adaptive ultrastructural changes.

Human Adipose-Derived Mesenchymal Stem/Stromal Cells (hAD-MSCs) have great potential for tissue regeneration. Since transplanted hAD-MSCs are likely to be placed in a hypoxic environment, culturing the cells under hypoxic conditions might improve their post-transplantation survival and regenerative performance. The combination of hAD-MSCs and PCL-nHA nanofibers synergically improves the contribution of both components for osteoblast differentiation. In this work, we hypothesized that this biomaterial constitutes a hypoxic environment for hAD-MSCs. We studied the cellular re-arrangement and the subcellular ultrastructure by Transmission Electron Microscopy (TEM) of hAD-MSCs grown into PCL-nHA nanofibers, and we compared them with the same cells grown in two-dimensional cultures, over tissue culture-treated plastic, or glass coverslips. Among the most evident changes, PCL-nHA grown cells showed enlarged mitochondria, and accumulation of glycogen granules, consistent with a hypoxic environment. We observed a 3.5 upregulation (p = 0.0379) of Hypoxia Inducible Factor (HIF)-1A gene expression in PCL-nHA grown cells. This work evidences for the first time intra-cellular changes in three-dimensional compared to two-dimensional cultures, which are adaptive responses of the cells to an environment more closely resembling that of the in vivo niche after transplantation, thus PCL-nHA nanofibers are adequate for hAD-MSCs pre-conditioning.

Embryotoxicity and visual-motor response of functionalized nanostructured hydroxyapatite-based biomaterials in zebrafish (Danio rerio).

Hydroxyapatite (HA) is a biomaterial widely used in biomedical applications. Many studies have shown that ionic substituents can be incorporated into HA to produce a mineral composition more similar to natural bone tissue with more favorable biological characteristics for application in bone regeneration. However, its potentially toxic effects need to be evaluated before full approval for human use. For this purpose, an embryotoxicity test was performed on zebrafish according to OECD guideline 236. Zebrafish embryos were exposed to 1 or 3 microspheres of alginate containing nanoparticles of HA and carbonate (CHA), strontium (SrHA), and zinc-substituted HA (ZnHA) from 4 to 120 h post-fertilization (hpf). Lethality and developmental endpoints were evaluated. In addition, larval behavior at 168 hpf was also analyzed to observe whether biomaterials adversely affect optomotor and avoidance responses (neurotoxicity), as well as the oxidative stress pattern through qPCR. After 120 h exposure to all microspheres with different patterns of crystallinity, porosity, nanoparticle size, surface area, and degradation behavior, there was no mortality rate greater than 20%, indicating the non-embryotoxic character of these biomaterials. All experimental groups showed positive optomotor and avoidance responses, which means that embryo exposure to the tested biomaterials had no neurotoxic effects. Furthermore, larvae exposed to one SrHA microsphere showed a better optomotor response than the control. Furthermore, the biomaterials did not change the pattern of mRNA levels of genes related to oxidative stress even after 120 hpf. The growing number of new HA-based biomaterials produced should be accompanied by increased studies to understand the biosafety of these compounds, especially in alternative models, such as zebrafish embryos. These results reinforce our hypothesis that ion-substituted HA biomaterials do not impose toxicological effects, cause development and neuromotor impairment, or increase oxidative stress in zebrafish embryos being useful for medical devices and in the process of bone regeneration.

Characterization of nano-hydroxyapatite incorporated carboxymethyl chitosan composite on human dental pulp stem cells.

AIM: To compare the odontogenic differentiation potential of a composite scaffold (CSHA) comprising of nano-hydroxyapatite (nHAp) and carboxymethyl chitosan (CMC) with Biodentine on human dental pulp stem cells (hDPSCs). METHODOLOGY: A CSHA scaffold was prepared through an ultrasonication route by adding nHAp and CMC (1:5 w/w) in water medium followed by freeze-drying. Physicochemical characterization was achieved using scanning electron microscopy with energy-dispersive X-ray spectroscopy, X-ray diffraction and Fourier transform infrared spectroscopy. In-vitro bioactivity and pH assessments were done by soaking in simulated body fluid (SBF) for 28 days. The angiogenic and odontogenic differentiation abilities were assessed by expression of vascular endothelial growth factor (VEGF) and Dentine sialophosphoprotein (DSPP) markers on cultured hDPSCs by flow cytometry and RT-qPCR at 7, 14 and 21 days. Cell viability/proliferation and biomineralization abilities of CSHA were compared with Biodentine by MTT assay, alkaline phosphatase (ALP) activity, Alizarin Red Staining (ARS) and osteopontin (OPN) expression on hDPSCs following 7 and 14 days. Data were statistically analysed with Kruskal Wallis and Friedman tests as well as one way anova followed by appropriate post hoc tests (p < .05). RESULTS: Characterization experiments revealed a porous microstructure of CSHA with pore diameter ranging between 60 and 200 µm and 1.67 Ca/P molar ratio along with the characteristic functional groups of both HAp and CMC. CSHA displayed bioactivity in SBF by forming apatite-like crystals and maintained a consistent pH value of 7.70 during 28 days' in vitro studies. CSHA significantly upregulated VEGF and DSPP levels on hDPSCs on day 21 compared with day 7 (p < .05). Further, CSHA supported cell viability/proliferation over 14 days like Biodentine with no statistical differences (p > .05). However, CSHA exhibited increased ALP and ARS activity with an intense OPN staining compared with Biodentine after 14 days (p < .05). CONCLUSION: The results highlighted the odontogenic differentiation and biomineralization abilities of CSHA on hDPSCs with significant VEGF and DSPP gene upregulations. Further, CSHA exhibited enhanced mineralization activity than Biodentine, as evidenced by increased ALP, ARS and OPN activity on day 14. The nHAp-CMC scaffold has the potential to act as an effective pulp capping agent; however, this needs to be further validated through in-vivo animal studies.

Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral / 国际口腔科学杂志·英文版

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.

[Study on promoting bone formation in osteoporotic zebrafish by lithium-doped hydroxyapatite nanowires].

PURPOSE: To observe the regulatory effect of lithium-doped hydroxyapatite nanowires on bone metabolism in osteoporotic zebrafish induced by dexamethasone. METHODS: Pure hydroxyapatite nanowires(nHA) and hydroxyapatite nanowires doped with 10% lithium ions (Li-nHA) were prepared by using hydrothermal method, and then material characterization was performed. The juvenile zebrafish cultured for 3 days(3dpf) were selected and co-cultured with nHA and Li-nHA extracts up to 7dpf. A negative(0.1% DMSO) control group was set up and transgenic zebrafish Tg(ola.sp7:nlsGFP) was used to select the best concentration for promoting bone formation. The osteoporotic zebrafish were induced by dexamethasone and incubated with nHA and Li-nHA extracts. The wild-type zebrafish was stained with alizarin red and the osteogenic differentiation was observed in transgenic zebrafish. Real-time quantitative PCR was adopted to detect osteogenic maker genes, such as zinc finger transcription factor (SP7), alkaline phosphatase (ALP), osteoprotegerin (OPG), Runt related transcription factor 2(Runx2) and osteocalcin (OCN). Statistical analysis was performed with GraphPad Prism 9.3 software. RESULTS: nHA and Li-nHA promoted bone formation and up-regulated expression levels of ALP, OCN, Runx2, SP7 and OPG of osteoporotic zebrafish. Compared with nHA, Li-nHA significantly increased the mineralization specific staining area and cumulative optical density of zebrafish bone, and the expression of osteogenic maker genes was also significantly increased. CONCLUSIONS: Doping lithium ions in nano hydroxyapatite can enhance its osteoinductive properties, and Li-nHA can effectively improve bone formation of osteoporotic zebrafish.

A specialized metabolic pathway partitions citrate in hydroxyapatite to impact mineralization of bones and teeth.

Citrate is a critical metabolic substrate and key regulator of energy metabolism in mammalian cells. It has been known for decades that the skeleton contains most (>85%) of the body's citrate, but the question of why and how this metabolite should be partitioned in bone has received singularly little attention. Here, we show that osteoblasts use a specialized metabolic pathway to regulate uptake, endogenous production, and the deposition of citrate into bone. Osteoblasts express high levels of the membranous Na+-dependent citrate transporter solute carrier family 13 member 5 (Slc13a5) gene. Inhibition or genetic disruption of Slc13a5 reduced osteogenic citrate uptake and disrupted mineral nodule formation. Bones from mice lacking Slc13a5 globally, or selectively in osteoblasts, showed equivalent reductions in cortical thickness, with similarly compromised mechanical strength. Surprisingly, citrate content in mineral from Slc13a5-/- osteoblasts was increased fourfold relative to controls, suggesting the engagement of compensatory mechanisms to augment endogenous citrate production. Indeed, through the coordinated functioning of the apical membrane citrate transporter SLC13A5 and a mitochondrial zinc transporter protein (ZIP1; encoded by Slc39a1), a mediator of citrate efflux from the tricarboxylic acid cycle, SLC13A5 mediates citrate entry from blood and its activity exerts homeostatic control of cytoplasmic citrate. Intriguingly, Slc13a5-deficient mice also exhibited defective tooth enamel and dentin formation, a clinical feature, which we show is recapitulated in primary teeth from children with SLC13A5 mutations. Together, our results reveal the components of an osteoblast metabolic pathway, which affects bone strength by regulating citrate deposition into mineral hydroxyapatite.

Zinc- and magnesium-doped hydroxyapatite-urea nanohybrids enhance wheat growth and nitrogen uptake.

The ongoing and unrestrained application of nitrogen fertilizer to agricultural lands has been directly linked to climate change and reductions in biodiversity. The agricultural sector needs a technological upgrade to adopt sustainable methods for maintaining high yield. We report synthesis of zinc and magnesium doped and undoped hydroxyapatite nanoparticles, and their urea nanohybrids, to sustainably deliver nitrogen to wheat. The urea nanohybrids loaded with up to 42% nitrogen were used as a new source of nitrogen and compared with a conventional urea-based fertilizer for efficient and sufficient nitrogen delivery to pot-grown wheat. Doping with zinc and magnesium manipulated the hydroxyapatite crystallinity for smaller size and higher nitrogen loading capacity. Interestingly, 50% and 25% doses of urea nanohybrids significantly boosted the wheat growth and yield compared with 100% doses of urea fertilizer. In addition, the nutritional elements uptake and grain protein and phospholipid levels were significantly enhanced in wheat treated with nanohybrids. These results demonstrate the potential of the multi-nutrient complexes, the zinc and magnesium doped and undoped hydroxyapatite-urea nanoparticles, as nitrogen delivery agents that reduce nitrogen inputs by at least 50% while maintaining wheat plant growth and nitrogen uptake to the same level as full-dose urea treatments.

Osteogenic differentiation and proliferation potentials of human bone marrow and umbilical cord-derived mesenchymal stem cells on the 3D-printed hydroxyapatite scaffolds.

Mesenchymal stem cells (MSCs) are a promising candidate for bone repair. However, the maintenance of MSCs injected into the bone injury site remains inefficient. A potential approach is to develop a bone-liked platform that incorporates MSCs into a biocompatible 3D scaffold to facilitate bone grafting into the desired location. Bone tissue engineering is a multistep process that requires optimizing several variables, including the source of cells, osteogenic stimulation factors, and scaffold properties. This study aims to evaluate the proliferation and osteogenic differentiation potentials of MSCs cultured on 2 types of 3D-printed hydroxyapatite, including a 3D-printed HA and biomimetic calcium phosphate-coated 3D-printed HA. MSCs from bone marrow (BM-MSCs) and umbilical cord (UC-MSCs) were cultured on the 3D-printed HA and coated 3D-printed HA. Scanning electron microscopy and immunofluorescence staining were used to examine the characteristics and the attachment of MSCs to the scaffolds. Additionally, the cell proliferation was monitored, and the ability of cells to differentiate into osteoblast was assessed using alkaline phosphatase (ALP) activity and osteogenic gene expression. The BM-MSCs and UC-MSCs attached to a plastic culture plate with a spindle-shaped morphology exhibited an immunophenotype consistent with the characteristics of MSCs. Both MSC types could attach and survive on the 3D-printed HA and coated 3D-printed HA scaffolds. The MSCs cultured on these scaffolds displayed sufficient osteoblastic differentiation capacity, as evidenced by increased ALP activity and the expression of osteogenic genes and proteins compared to the control. Interestingly, MSCs grown on coated 3D-printed HA exhibited a higher ALP activity and osteogenic gene expression than those cultured on the 3D-printed HA. The finding indicated that BM-MSCs and UC-MSCs cultured on the 3D-printed HA and coated 3D-printed HA scaffolds could proliferate and differentiate into osteoblasts. Thus, the HA scaffolds could provide a suitable and favorable environment for the 3D culture of MSCs in bone tissue engineering. Additionally, biomimetic coating with octacalcium phosphate may improve the biocompatibility of the bone regeneration scaffold.

Synthesis of Antibacterial Hybrid Hydroxyapatite/Collagen/Polysaccharide Bioactive Membranes and Their Effect on Osteoblast Culture.

Inspired by the composition and confined environment provided by collagen fibrils during bone formation, this study aimed to compare two different strategies to synthesize bioactive hybrid membranes and to assess the role the organic matrix plays as physical confinement during mineral phase deposition. The hybrid membranes were prepared by (1) incorporating calcium phosphate in a biopolymeric membrane for in situ hydroxyapatite (HAp) precipitation in the interstices of the biopolymeric membrane as a confined environment (Methodology 1) or (2) adding synthetic HAp nanoparticles (SHAp) to the freshly prepared biopolymeric membrane (Methodology 2). The biopolymeric membranes were based on hydrolyzed collagen (HC) and chitosan (Cht) or κ-carrageenan (κ-carr). The hybrid membranes presented homogeneous and continuous dispersion of the mineral particles embedded in the biopolymeric membrane interstices and enhanced mechanical properties. The importance of the confined spaces in biomineralization was confirmed by controlled biomimetic HAp precipitation via Methodology 1. HAp precipitation after immersion in simulated body fluid attested that the hybrid membranes were bioactive. Hybrid membranes containing Cht were not toxic to the osteoblasts. Hybrid membranes added with silver nanoparticles (AgNPs) displayed antibacterial action against different clinically important pathogenic microorganisms. Overall, these results open simple and promising pathways to develop a new generation of bioactive hybrid membranes with controllable degradation rates and antimicrobial properties.