Calcium carbonate particles: synthesis, temperature and time influence on the size, shape, phase, and their impact on cell hydroxyapatite formation.

To develop materials for drug delivery and tissue engineering and to study their efficiency with respect to ossification, it is necessary to apply physicochemical and biological analyses. The major challenge is labor-intensive data mining during synthesis and the reproducibility of the obtained data. In this work, we investigated the influence of time and temperature on the reaction yield, the reaction rate, and the size, shape, and phase of the obtained product in the completely controllable synthesis of calcium carbonate. We show that calcium carbonate particles can be synthesized in large quantities, i.e., in gram quantities, which is a substantial advantage over previously reported synthesis methods. We demonstrated that the presence of vaterite particles can dramatically stimulate hydroxyapatite (HA) production by providing the continued release of the main HA component - calcium ions - depending on the following particle parameters: size, shape, and phase. To understand the key parameters influencing the efficiency of HA production by cells, we created a predictive model by means of principal component analysis. We found that smaller particles in the vaterite state are best suited for HA growth (HA growth was 8 times greater than that in the control). We also found that the reported dependence of cell adhesion on colloidal particles can be extended to other types of particles that contain calcium ions.

Forming Anisotropic Crystal Composites: Assessing the Mechanical Translation of Gel Network Anisotropy to Calcite Crystal Form.

The promise of crystal composites with direction-specific properties is an attractive prospect for diverse applications; however, synthetic strategies for realizing such composites remain elusive. Here, we demonstrate that anisotropic agarose gel networks can mechanically "mold" calcite crystal growth, yielding anisotropically structured, single-crystal composites. Drying and rehydration of agarose gel films result in the affine deformation of their fibrous networks to yield fiber alignment parallel to the drying plane. Precipitation of calcium carbonate within these anisotropic networks results in the formation of calcite crystal composite disks oriented parallel to the fibers. The morphology of the disks, revealed by nanocomputed tomography imaging, evolves with time and can be described by linear-elastic fracture mechanics theory, which depends on the ratio between the length of the crystal and the elastoadhesive length of the gel. Precipitation of calcite in uniaxially deformed agarose gel cylinders results in the formation of rice-grain-shaped crystals, suggesting the broad applicability of the approach. These results demonstrate how the anisotropy of compliant networks can translate into the desired crystal composite morphologies. This work highlights the important role organic matrices can play in mechanically "molding" biominerals and provides an exciting platform for fabricating crystal composites with direction-specific and emergent functional properties.

Recombinant transgelin-like protein 1 from Mytilus shell induces formation of CaCO3 polymorphic crystals in vitro.

Transgelin is an actin cross-linking/gelling protein of the calponin family, which is associated with actin stress fibres, cell motility, adhesion and the maintenance of cell morphology. Transgelin-like proteins (TLPs) have also been identified as shell matrix proteins (SMPs) in several mollusc species; however, the functions of TLPs in biomineralization remain unknown. Transgelin-like protein 1 (TLP-1) was previously identified from the shell of Mytilus coruscus as a novel 19 kDa SMP with a calponin homology (CH) domain. To understand the role of TLP-1 in shell formation, the expression level and localization of the TLP-1 gene in biomineralization-related tissues were determined in this study. Furthermore, recombinant TLP-1 was expressed in a prokaryotic expression system with codon optimization, and an anti-rTLP-1 antibody was prepared based on the expressed recombinant TLP-1 (rTLP-1) protein. In vitro, rTLP-1 induced the formation of CaCO3 polymorphic crystals with distinct morphologies and inhibited crystallization rate and crystal interactions. Immunohistochemical, immunofluorescence, and pull-down analyses using the anti-rTLP-1 antibody revealed the specific locations of TLP-1 in biomineralization-related tissues and shell myostracum layer, and suggested the existence of a possible TLP-1 interaction network in the shell matrix. Our results are beneficial for understanding the functions of TLP-1, particularly through its CH domain, during shell mineralization.

Functionalised calcium carbonate as a coformer to stabilize amorphous drugs by mechanochemical activation.

The aim of this study was to investigate the amorphization, physical stability and drug release of a model drug, carvedilol (CAR), when loaded onto functionalised calcium carbonate (FCC) using mechanochemical activation (vibrational ball milling). The solid-state characteristics and physical stability of CAR-FCC samples, prepared at different weight ratios and for different milling times, were determined using differential scanning calorimetry and X-ray powder diffraction. Upon milling CAR-FCC samples containing 50% CAR, amorphization of CAR was observed after 10 min. For CAR-FCC samples milled for either 30 or 90 min, it was found that CAR was amorphised at all ratios (10-90% CAR), but FCC remained crystalline. The glass transition temperature (Tgα) of the various CAR-FCC samples milled for 90 min was found to be similar (38 °C) for all ratios containing 20% CAR and above. The similar Tgαs for the different drug ratios indicate deposition of amorphous CAR onto the surface of FCC. For CAR-FCC samples containing 10% CAR, a Tgα of 49 °C was found, which is 11 °C higher compared with other CAR-FCC samples. This may indicate restricted molecular mobility resulting from CAR molecules that are in close contact with the FCC surface. The physical stability, under both stress (100 °C) and non-stress conditions (25 °C at dry conditions), showed that drug concentrations up to 30% CAR can be stabilized in the amorphous form for at least 19 weeks under non-stress conditions when deposited onto FCC, compared to less than a week physical stability of neat amorphous CAR. In vitro drug release showed that CAR-FCC samples containing 60% CAR and below can improve the drug release and generate supersaturated systems compared to neat amorphous and crystalline CAR. Samples with lower drug concentrations (40% CAR and below) can maintain supersaturation during 360 min of dissolution testing. This study indicates that the crystalline inorganic material, FCC, can facilitate amorphization of drugs, provide stabilization against drug crystallization, and improve dissolution properties of amorphous drugs upon mechanochemical activation.

Toxicological profile of calcium carbonate nanoparticles for industrial applications.

Calcium carbonate nanoparticles (CaCO3NPs) derived from CO2 are promising materials for different industrial applications. It is imperative to understand their toxicological profile in biological systems as the human and environmental exposures to CaCO3NPs increases with growing production. Here, we analyse the cytotoxicity of CaCO3NPs synthesized from a CaO slurry on two cell lines, and in vivo on zebrafish (Danio Rerio). Our results demonstrate the CaCO3NPs in vitro safety as they do not cause cell death or genotoxicity. Moreover, zebrafish treated with CaCO3NPs develop without any abnormalities, confirming the safety and biocompatibility of this nanomaterial.

Development and characterization of novel ambroxol sustained-release oral suspensions based on drug-polymeric complexation and polymeric raft formation.

The aim was to develop sustained-release aqueous suspensions of ambroxol utilizing drug-polymer complexation and raft-forming formulations. Ambroxol-carrageenan (ABX-CRG) complexation was studied for the optimum binding capacity, which was used to prepare the complex by kneading and coprecipitation. The prepared complex was characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry and powder X-ray diffractometry. The complex was formulated as suspensions in aqueous raft-forming vehicle of sodium alginate (NA) and calcium carbonate (CC). The suspensions differed in the molecular weight and concentration of NA, in addition to CC level and inclusion of CRG in excess of drug-polymer complexation. In 0.1 M HCl as simulated gastric fluid, the suspensions were observed for their ability to form rafts and studied for drug-release. The optimum sustained-release, raft forming and pourable formulation using high molecular weight NA, NA concentration of 18 mg/ml and CC concentration of 9 mg/ml was reached. Another optimum suspension was obtained by replacement of CC with excess CRG. However, pH dissolution profiles of the optimum suspensions revealed less pH sensitivity of the release consequent to this replacement as well as more stable ABX release upon aging. Relative to Gaviscon liquid, the optimum suspensions formed rafts of similar strength and higher resilience.

CaCO3 nanoparticles pH-sensitively induce blood coagulation as a potential strategy for starving tumor therapy.

Based on the concept of starving tumor therapy, in this study we put forward a new idea that the pH-sensitive Ca2+ delivery of calcium carbonate nanoparticles (CaCO3 NPs) induced blood coagulation of tumor vessels, and first explored the effect of CaCO3 NPs on the in vitro and in vivo blood coagulation by acid stimulus. CaCO3 NPs with a size of about 100 nm and a porous structure of several nanometers were synthesized in an emulsion system, which showed a high loading capacity (49%) of doxorubicin hydrochloride (DOX) with an encapsulation efficiency of 98% and a pH-sensitive drug delivery. The hemolysis test showed that CaCO3 NPs were blood compatible. The in vitro Ca2+ delivery and blood clotting tests indicated that CaCO3 NPs pH-sensitively released Ca2+, and caused rapid blood coagulation at pH 5.0 but no thrombus at pH 7.4. Confocal laser scanning microscopy showed that after uptake by MCF-7 or MDA-MB-231 breast cancer cells, CaCO3 NPs mainly distributed in endosomes/lysosomes within the initial 2 h and then decomposed by acid stimulus, leading to the intracellular delivery of Ca2+ that subsequently migrated outside the cells. CaCO3 NPs were nontoxic to NIH3T3 mouse fibroblasts, but highly toxic to both MCF-7 and MDA-MB-231 cells after loading DOX. After topical administration into the breast tumors of mice, CaCO3 NPs evoked significant thrombosis and hemorrhage of tumor vasculature by hematoxylin-eosin and Masson's trichrome staining. These results indicated that CaCO3 NPs could induce blood coagulation via acid stimulus, showing potential applications in blocking tumor vessels for starving tumor therapy.

Porous calcite CaCO3 microspheres: Preparation, characterization and release behavior as doxorubicin carrier.

Porous CaCO3 microspheres are nowadays extensively used as a drug delivery system due to their excellent biocompatibility and degradability. However, the stability of vaterite CaCO3 microspheres is a major problem as a drug carrier. In this work, porous calcite CaCO3 microspheres are fabricated by calcination of gelatin-CaCO3 composite microspheres at 550 °C in air. The size and morphology of CaCO3 microspheres could be well regulated by the addition of gelatin. The structure of the as-prepared porous calcite CaCO3 microspheres is characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Compared with vaterite CaCO3 microspheres, porous calcite CaCO3 microspheres are more stable and exhibit good drug-loading and drug release properties for doxorubicin (DOX), an effective anticancer agent. This study provides an easy and novel approach to prepare stable porous calcite CaCO3 microspheres, which show promising potential as a carrier for DOX.

Magneto-Fluorescent Microbeads for Bacteria Detection Constructed from Superparamagnetic Fe3O4 Nanoparticles and AIS/ZnS Quantum Dots.

The efficient and sensitive detection of pathogenic microorganisms in aqueous environments, such as water used in medical applications, drinking water, and cooling water of industrial plants, requires simple and fast methods suitable for multiplexed detection such as flow cytometry (FCM) with optically encoded carrier beads. For this purpose, we combine fluorescent Cd-free Ag-In-S ternary quantum dots (t-QDs) with fluorescence lifetimes (LTs) of several hundred nanoseconds and superparamagnetic Fe3O4 nanoparticles (SPIONs) with mesoporous CaCO3 microbeads to a magneto-fluorescent bead platform that can be surface-functionalized with bioligands, such as antibodies. This inorganic bead platform enables immuno-magnetic separation, target enrichment, and target quantification with optical readout. The beads can be detected with steady-state and time-resolved fluorescence microscopy and flow cytometry (FCM). Moreover, they are suited for readout by time gated emission. In the following, the preparation of these magneto-fluorescent CaCO3 beads, their spectroscopic and analytic characterization, and their conjugation with bacteria-specific antibodies are presented as well as proof-of-concept measurements with Legionella pneumophila including cell cultivation and plating experiments for bacteria quantification. Additionally, the possibility to discriminate between the long-lived emission of the LT-encoded capture and carrier CaCO3 beads and the short-lived emission of the dye-stained bacteria with time-resolved fluorescence techniques and single wavelength excitation is demonstrated.

Development of CaCO3 microsphere-based composite hydrogel for dual delivery of growth factor and Ca to enhance bone regeneration.

Injectable scaffolds have attracted much attention because of their minimum surgical invasiveness. However, limited osteogenic induction property and low mechanical properties hampered their application in bone tissue engineering. CaCO3 microspheres, which possess osteoinductivity, rough surfaces and specific binding sites for BMP-2, were first fabricated; after BMP-2 uploading, microspheres were further entrapped in fibrin-glue hydrogel. CaCO3 microspheres were co-functionalized with casein and heparin. To obtain a high encapsulation of heparin and thus BMP-2 uploading, along with controlled release and simultaneous maintenance of the presence of vaterite which had osteogenic induction property, fabrication parameters were optimized and microspheres were characterized using XRD, FITR and SEM. The formed CaCO3 had a microsphere morphology of ∼1 µm. Both vaterite and calcite phases were present and the relative amount of calcite phase increased with the amount of heparin. Sample 25 mM_4-1Hep with the highest loading amount of heparin was selected as carrier for BMP-2 and BMP-2 loaded CaCO3 microspheres were further entrapped in fibrin-glue hydrogel (FC-B). For the as-prepared composite hydrogel, mechanical properties were characterized and the presence of CaCO3 significantly elevated the tensile strength; controlled release of BMP-2 was sustained until day 21. Based on ALP activity, alizarin red staining and RT-PCR, in vitro osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was found to be significantly enhanced under induction of FC-B. Rabbit tibia bone defect model was applied to evaluate its in vivo performance. After implantation for 4 weeks, presence of composite hydrogel was observed in defects. After 8 weeks, bone defects of FC-B group were nearly completely healed. Using the fact that autologous scaffolds can be derived based on fibrin-glue hydrogel, the well-designed BMP-2 loaded fibrin-glue composite hydrogel demonstrated good potential in bone tissue engineering.

Poly-L-ornithine/fucoidan-coated calcium carbonate microparticles by layer-by-layer self-assembly technique for cancer theranostics.

Recently, the layer-by-layer (LbL) self-assembly technology has attracted the enormous interest of researchers in synthesizing various pharmaceutical dosage forms. Herewith, we designed a biocompatible drug delivery system containing the calcium carbonate microparticles (CaCO3 MPs) that coated with the alternatively charged polyelectrolytes, i.e., poly-L-ornithine (PLO)/fucoidan by LbL self-assembly process (LbL MPs). Upon coating with the polyelectrolytes, the mean particle size of MPs obtained from SEM observations increased from 1.91 to 2.03 µm, and the surface of LbL MPs was smoothened compared to naked CaCO3 MPs. In addition, the reversible zeta potential changes have confirmed the accomplishment of layer upon a layer assembly. To evaluate the efficiency of cancer therapeutics, we loaded doxorubicin (Dox) in the LbL MPs, which resulted in high (69.7%) drug encapsulation efficiency. The controlled release of Dox resulted in the significant antiproliferative efficiency in breast cancer cell line (MCF-7 cells), demonstrating the potential of applying this innovative drug delivery system in the biomedical field.

Hybrid PCL/CaCO3 scaffolds with capabilities of carrying biologically active molecules: Synthesis, loading and in vivo applications.

Designing advanced biomaterials for tissue regeneration with drug delivery and release functionalities remains a challenge in regenerative medicine. In this research, we have developed novel composite scaffolds based on polymeric polycaprolactone fibers coated with porous calcium carbonate structures (PCL/CaCO3) for tissue engineering and have shown their drug delivery and release in rats. In vivo biocompatibility tests of PCL/CaCO3 scaffolds were complemented with in vivo drug release study, where tannic acid (TA) was used as a model drug. Release of TA from the scaffolds was realized by recrystallization of the porous vaterite phase of calcium carbonate into the crystalline calcite. Cell colonization and tissue vascularization as well as transplantability of developed PCL/CaCO3+TA scaffolds were observed. Detailed study of scaffold transformations during 21-day implantation period was followed by scanning electron microscopy and X-ray diffraction studies before and after in vivo implantation. The presented results demonstrate that PCL/CaCO3 scaffolds are attractive candidates for implants in bone regeneration and tissue engineering with a possibility of loading biologically active molecules and controlled release.

Delivery systems for agriculture: Fe-EDDHSA/CaCO3 hybrid crystals as adjuvants for prevention of iron chlorosis.

Fe-EDDHSA/CaCO3 hybrid crystals are synthesized and tested in vitro to determine their effect in treating iron chlorosis in kiwifruit plants, used as a proof of concept. Under the alkaline conditions provided by the calcareous substrate, plants release protons that dissolve the hybrids and trigger Fe uptake. These CaCO3 hybrids represent a new system for active molecule delivery in agriculture.

Controlling nucleation and growth of nano-CaCO3 via CO2 sequestration by a calcium alkoxide solution to produce nanocomposites for drug delivery applications.

Calcium carbonate is an extremely attractive material in a plethora of biomedical applications. Intensive efforts have recently been made to achieve the control over its nucleation and subsequent aggregation, growth and crystallization; focusing on bringing insight into the role of precursors, solvents and templates. Having analyzed the recently acquired knowledge, we addressed this challenge using CO2 sequestration synthesis, using an unusual reactant, a solution of calcium ethoxide, Ca(OC2H5)2, as precursor. By tailoring the reaction conditions, it was possible to produce extremely small and rather size-uniform single-phase calcite CaCO3 nanoparticles, forming sols and subsequently gels in the applied medium. According to DLS and nanoparticle tracking analysis the particles are only to a minor extent aggregated in the mother liquor and can form transparent gels on concentration in less polar media, but produce large aggregates 400-800nm in size when dried and subsequently transferred to aqueous media. Complete drying of solutions renders xerogel type materials with only moderate active surface area, as identified by nitrogen adsorption, due to aggregation with development of densified surface layers. Such behaviour is typical for the sol-gel synthesis of particles possessing enhanced surface reactivity. The aggregation on drying was used to produce hybrid nanocomposites, with the hydrophobic model component, ß-carotene, introduced in solution in a non-polar co-solvent and model medicine - ibuprofen. The obtained nanocomposite particles, characterized by SEM, TEM, XRD, AFM and FTIR studies, are hierarchically structured spheroidal aggregates about 200nm in size with uniform distribution of the organic components present in the amorphous state. The composite particles are stable in neutral aqueous environments but are readily dissolved in acidic medium or even in PBS at pH = 7.40, releasing the hydrophobic organic component in the form of a relatively stable colloid solution. Efficient release of ibuprofen as model drug was achieved in both acidic and PBS medium and could be slowed down by the addition of ß-carotene as hydrophobic component. STATEMENT OF SIGNIFICANCE: The proposed sol-gel synthesis of CaCO3 proved to create unprecedented size of CaCO3 nanoparticles with striking size uniformity. The obtained results clearly demonstrate their ability to incorporate hydrophobic components in a nanocomposite matrix converting them into amorphous nano sized particles, building stable colloids via release in acidic medium. Transfer of a sol produced in organic medium into water in the presence of albumen surfactant results in relatively uniform micro particles about 1µm size. The obtained materials show characteristics attractive for use in drug delivery and potentially also a variety of other industrial applications.

Two-way Valorization of Blast Furnace Slag: Synthesis of Precipitated Calcium Carbonate and Zeolitic Heavy Metal Adsorbent.

The aim of this work is to present a zero-waste process for storing CO2 in a stable and benign mineral form while producing zeolitic minerals with sufficient heavy metal adsorption capacity. To this end, blast furnace slag, a residue from iron-making, is utilized as the starting material. Calcium is selectively extracted from the slag by leaching with acetic acid (2 M CH3COOH) as the extraction agent. The filtered leachate is subsequently physico-chemically purified and then carbonated to form precipitated calcium carbonate (PCC) of high purity (<2 wt% non-calcium impurities, according to ICP-MS analysis). Sodium hydroxide is added to neutralize the regenerated acetate. The morphological properties of the resulting calcitic PCC are tuned for its potential application as a filler in papermaking. In parallel, the residual solids from the extraction stage are subjected to hydrothermal conversion in a caustic solution (2 M NaOH) that leads to the predominant formation of a particular zeolitic mineral phase (detected by XRD), namely analcime (NaAlSi2O6∙H2O). Based on its ability to adsorb Ni2+, as reported from batch adsorption experiments and ICP-OES analysis, this product can potentially be used in wastewater treatment or for environmental remediation applications.

Biochar provides a safe and value-added solution for hyperaccumulating plant disposal: A case study of Phytolacca acinosa Roxb. (Phytolaccaceae).

In this work, an innovative approach using biochar technology for hyperaccumulator disposal was developed and evaluated. The heavy metal enriched P. acinosa biomass (PBM) was pyrolyzed to produce biochar (PBC). Both PBM and PBC were characterized with X-ray diffraction (XRD) for crystal phases, scanning electron microscopy (SEM) for surface topography, and analyzed for elemental composition and mobility. The results revealed that whewellite, a dominant crystal form in biomass, was decomposed to calcite after pyrolysis. Elemental analysis indicated that 91-99% total non-volatile elements in the biomass were retained in the biochar. The toxicity characteristic leaching procedure (TCLP) results revealed that 94.6% and 0.15% of total Mn was extracted for biomass and biochar, respectively. This suggests that mobility and bioavailability of Mn in biochar was much lower relative to pristine biomass. Batch sorption experiment showed that excellent removal of aqueous silver, lead, cadmium, and copper ions can be achieved with PBC. Findings from this work indicated that biochar technology can provide a value-added solution for hyperaccumulator disposal.

Mesoporous phenylalanine ammonia lyase microspheres with improved stability through calcium carbonate templating.

Cross-linked enzyme aggregates (CLEAs) have recently emerged as a promising method for enzyme immobilization due to its simplicity and low cost. However, a lack of good size and morphological control over the as-prepared CLEAs has limited their practical applications in some cases. Here, monodisperse spherical CLEAs of phenylalanine ammonia lyase (PAL microspheres) were prepared based on CaCO3 microtemplates. The preparation procedure involves filling porous CaCO3 microtemplates with the protein by salt precipitation, glutaraldehyde crosslinking, and dissolution of the microtemplates. The formulation of CaCO3 templates with controlled size was studied in detail. Characterization of the prepared PAL microspheres was investigated. The results showed that the PAL microspheres with high immobilization efficiency (79%) exhibited excellent stability, including increased tolerance to proteolysis, low pH, and denaturants, and excellent mechanical properties. For example, free PAL almost lost all activity after they were incubated in the presence of trypsin for 2min, whereas PAL microspheres still retained 95% of their initial activity. Moreover, scanning electron microscope, transmission electron microscope, and N2 adsorption-desorption isotherms revealed that the resultant PAL microspheres possessed good monodispersity and mesoporous structure instead of the amorphous clusters of conventional CLEAs with few pores. Compared with conventional CLEAs, the monodisperse PAL microspheres with mesoporous make them more potentially useful for biomedical and biotechnological applications.

Methotrexate intercalated calcium carbonate nanostructures: Synthesis, phase transformation and bioassay study.

The formation and stabilization of amorphous calcium carbonate (ACC) is an active area of research owing to the presence of stable ACC in various biogenic minerals. In this paper, the synthesis of calcium carbonate (CaCO3) under the participation of methotrexate (MTX) via a facile gas diffusion route was reported. The results indicated that the addition of MTX can result in the phase transformation of CaCO3, and then two kinds of hybrids, i.e., MTX-vaterite and stable MTX-ACC came into being. Interestingly, the functional agent MTX served as both the target anticancer drug loaded and effective complexation agents to modify and control the morphology of final samples. The examination of MTX-ACC biodegradation process revealed that the collapse of MTX-ACC nanoparticles was due to the synergistic effect of drug release and the phase transformation. Finally, our study also proved that MTX-ACC exhibited the most excellent suppressing function on the viability of cancer cells, especially after long-time duration.

Synthesis of calcium carbonate using extract components of croaker gill as morphology and polymorph adjust control agent.

Biomimetic synthesis of calcium carbonate with various polymorphs, sizes and morphologies by using organic substrates has become an interesting topic for the last years. Calcium carbonate has been synthesized by the reaction of Na2CO3 and CaCl2 in the presence of extract components of croaker gill. The products were characterized by powder X-ray diffraction (PXRD) and Fourier transform infrared (FT-IR) spectrum, and particle morphologies were observed by scanning electron microscope (SEM). The results show that at lower concentration yellow croaker gill extract has no effect on calcium carbonate crystal polymorph. Calcite was obtained only. But the morphologies of calcite particle change with the increase of the concentration. The corners of the particle change from angular to curved. However, with the further increase of the concentration of yellow croaker gill extract, the calcium carbonate obtained is a mixture of calcite and vaterite. The vaterite component in the mixture rises with increasing concentration of extract solution, indicating that the proteins from the yellow croaker gill during growth play a crucial role in stabilizing and directing the crystal growth.

Synthetic Calcite as a Scaffold for Osteoinductive Bone Substitutes.

Although a wide variety of biomaterials have been already proposed for use in bone tissue engineering, there is still need for man-made materials, which would combine support for osteogenesis with simplicity desirable for upscaling and costs reduction. In this study we have shown that synthetic calcite may serve as a scaffold for human osteoblasts transplantation. A simple dynamic system allows uniform and effective cell distribution. Cell viability and osteogenic phenotype were confirmed by XTT assay, alkaline phosphatase activity and selected osteoblast-specific genes expression. Extracellular matrix deposited by cells improved elasticity and made the whole system similar to the flexible composite material rather than to the brittle ceramic implants. It was revealed in the compression tests and also by the improved samples handling. Subcutaneous implantation of the cell-seeded calcite scaffolds to immunodeficient mice resulted in mineralized bone formation, which was confirmed histologically and by EPR analysis. The latter we propose as a method supplementary to histological analysis, for bone regeneration investigations. It specifically confirms the presence of bone mineral with a unique sensitivity and using bulk samples, which eliminates the risk of missing the material in the preparation. Our study resulted in development of a new osteogenic tissue engineered product based on man-made calcite.