Biomimetic Scaffolds Based on Mn2+-, Mg2+-, and Sr2+-Substituted Calcium Phosphates Derived from Natural Sources and Polycaprolactone.

The occurrence of bone disorders is steadily increasing worldwide. Bone tissue engineering (BTE) has emerged as a promising alternative to conventional treatments of bone defects, developing bone scaffolds capable of promoting bone regeneration. In this research, biomimetic scaffolds based on ion-substituted calcium phosphates, derived from cuttlefish bone, were prepared using a hydrothermal method. To synthesize Mn2+-substituted scaffolds, three different manganese concentrations (corresponding to 1, 2.5, and 5 mol% Mn substitutions for Ca into hydroxyapatite) were used. Also, syntheses with the simultaneous addition of an equimolar amount (1 mol%) of two (Mg2+ and Sr2+) or three ions (Mn2+, Mg2+, and Sr2+) were performed. A chemical, structural, and morphological characterization was carried out using X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy. The effects of the ion substitutions on the lattice parameters, crystallite sizes, and fractions of the detected phases were discussed. Multi-substituted (Mn2+, Mg2+, and Sr2+) scaffolds were coated with polycaprolactone (PCL) using simple vacuum impregnation. The differentiation of human mesenchymal stem cells (hMSCs), cultured on the PCL-coated scaffold, was evaluated using histology, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction analyses. The expression of collagen I, alkaline phosphatase, and dentin matrix protein 1 was detected. The influence of PCL coating on hMSCs behavior is discussed.

Medical-Grade Poly(Lactic Acid)/Hydroxyapatite Composite Films: Thermal and In Vitro Degradation Properties.

Production of biocompatible composite scaffolds shifts towards additive manufacturing where thermoplastic biodegradable polymers such as poly(lactic acid) (PLA) are used as matrices. Differences between industrial- and medical-grade polymers are often overlooked although they may affect properties and degradation behaviour as significantly as the filler addition. In the present research, composite films based on medical-grade PLA and biogenic hydroxyapatite (HAp) with 0, 10, and 20 wt.% of HAp were prepared by solvent casting technique. The degradation of composites incubated in phosphate-buffered saline solution (PBS) at 37 °C after 10 weeks showed that the higher HAp content slowed down the hydrolytic PLA degradation and improved its thermal stability. Morphological nonuniformity after degradation was indicated by the different glass transition temperatures (Tg) throughout the film. The Tg of the inner part of the sample decreased significantly faster compared with the outer part. The decrease was observed prior to the weight loss of composite samples.

PCL/Si-Doped Multi-Phase Calcium Phosphate Scaffolds Derived from Cuttlefish Bone.

Increasing attention is focused on developing biomaterials as temporary scaffolds that provide a specific environment and microstructure for bone tissue regeneration. The aim of the present work was to synthesize silicon-doped biomimetic multi-phase composite scaffolds based on bioactive inorganic phases and biocompatible polymers (poly(ε-caprolactone), PCL) using simple and inexpensive methods. Porous multi-phase composite scaffolds from cuttlefish bone were synthesized using a hydrothermal method and were further impregnated with (3-aminopropyl)triethoxysilane 1-4 times, heat-treated (1000 °C) and coated with PCL. The effect of silicon doping and the PCL coating on the microstructure and mechanical and biological properties of the scaffolds has been investigated. Multi-phase scaffolds based on calcium phosphate (hydroxyapatite, α-tricalcium phosphate, ß-tricalcium phosphate) and calcium silicate (wollastonite, larnite, dicalcium silicate) phases were obtained. Elemental mapping revealed homogeneously dispersed silicon throughout the scaffolds, whereas silicon doping increased bovine serum albumin protein adsorption. The highly porous structure of cuttlefish bone was preserved with a composite scaffold porosity of ~78%. A compressive strength of ~1.4 MPa makes the obtained composite scaffolds appropriate for non-load-bearing applications. Cytocompatibility assessment by an MTT assay of human mesenchymal stem cells revealed the non-cytotoxicity of the obtained scaffolds.

Osteogenic differentiation of human mesenchymal stem cells on substituted calcium phosphate/chitosan composite scaffold.

Ionic substitutions are a promising strategy to enhance the biological performance of calcium phosphates (CaP) and composite materials for bone tissue engineering applications. However, systematic studies have not been performed on multi-substituted organic/inorganic scaffolds. In this work, highly porous composite scaffolds based on CaPs substituted with Sr2+, Mg2+, Zn2+ and SeO32- ions, and chitosan have been prepared by freeze-gelation technique. The scaffolds have shown highly porous structure, with very well interconnected pores and homogeneously dispersed CaPs, and high stability during 28 days in the degradation medium. Osteogenic potential of human mesenchymal stem cells seeded on scaffolds has been determined by histological, immunohistochemical and RT-qPCR analysis of cultured cells in static and dynamic conditions. Results indicated that ionic substitutions have a beneficial effect on cells and tissues. The scaffolds with multi-substituted CaPs have shown increased expression of osteogenesis related markers and increased phosphate deposits, compared to the scaffolds with non-substituted CaPs.

Compressive Strength of Conventional Glass Ionomer Cement Modified with TiO2 Nano-Powder and Marine-Derived HAp Micro-Powder.

The aim of this research was to investigate the compressive strength (CS), breaking strength (BS), and compressive modulus (CM) of conventional glass ionomer cement (GIC) modified with TiO2 nano particles, marine-derived hydroxyapatite (md-HAp) microparticles (<45 µm), and a combination of TiO2 NP and md-HAp particles. The materials used in the study were conventional GIC Fuji IX GP Extra (GC Corporation, Tokyo, Japan), TiO2 powder P25 (Degussa, Essen, Germany), and HAp synthesized from cuttlefish bone and ground in a mortar to obtain md-HAp powder. md-HAp was characterized using FTIR and SEM analysis. There were four groups of GIC samples: (i) Fuji IX control group, (ii) powder modified with 3 wt% TiO2, (iii) powder modified with 3 wt% HAp, and (iv) powder modified with 1.5 wt% TiO2 + 1.5 wt% HAp. Measurements were performed in a universal testing machine, and CS, BS, and CM were calculated. Statistical analysis was performed using ANOVA and Tukey's tests. CS, BS, and CM differed significantly between the Fuji IX control group and all experimental groups while differences between the experimental groups were not statistically significant. The addition of TiO2 NP, md-HAp micro-sized particles, and a combination of TiO2 and md-HAp reduced the CS, BS, and CM of conventional GICs when mixed at the powder/liquid (p/l) ratio recommended by the manufacturer.

PCL-Coated Multi-Substituted Calcium Phosphate Bone Scaffolds with Enhanced Properties.

Ionic substitutions within the hydroxyapatite lattice are a widely used approach to mimic the chemical composition of the bone mineral. In this work, Sr-substituted and Mg- and Sr-co-substituted calcium phosphate (CaP) scaffolds, with various levels of strontium and magnesium substitution, were prepared using the hydrothermal method at 200 °C. Calcium carbonate skeletons of cuttlefish bone, ammonium dihydrogenphosphate (NH4H2PO4), strontium nitrate (Sr(NO3)2), and magnesium perchlorate (Mg(ClO4)2) were used as reagents. Materials were characterized by means of X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Whole powder pattern decomposition refinements of XRD data indicated that increased magnesium content in the Mg- and Sr-co-substituted scaffolds was related to an increased proportion of the whitlockite (WH) phase in the biphasic hydroxyapatite (HAp)/WH scaffolds. In addition, refinements indicate that Sr2+ ions have replaced Ca2+ sites in the WH phase. Furthermore, PCL-coated Mg-substituted and Sr- and Mg-co-substituted scaffolds, with the HAp:WH wt. ratio of 90:10 were prepared by vacuum impregnation. Results of compression tests showed a positive impact of the WH phase and PCL coating on the mechanical properties of scaffolds. Human mesenchymal stem cells (hMSCs) were cultured on composite scaffolds in an osteogenic medium for 21 days. Immunohistochemical staining showed that Mg-Sr-CaP/PCL scaffold exhibited higher expression of collagen type I than the Mg-CaP/PCL scaffold, indicating the positive effect of Sr2+ ions on the differentiation of hMSCs, in concordance with histology results. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis confirmed an early stage of osteogenic differentiation.

Selenite Substituted Calcium Phosphates: Preparation, Characterization, and Cytotoxic Activity.

The aim of this study was to prepare a biomimetic selenium substituted calcium phosphate system for potential application in osteosarcoma therapy. Calcium phosphate (CaP) systems substituted with selenite ions were prepared by the wet precipitation method, using biogenic CaCO3 (derived from cuttlefish bone), CO(NH2)2-H3PO4, and Na2SeO3·5H2O as reagents. Starting reaction mixtures were prepared based on the formula for selenite-substituted hydroxyapatite, Ca10(PO4)6-x(SeO3)x(OH)2, with Ca/(P + Se) molar ratio of 1.67 and Se/(P + Se) molar ratio of: 0, 0.01, 0.05, and 0.10, respectively. The prepared CaP powders were characterized by Fourier transform infrared spectrometry, elemental analysis, scanning electron microscopy, X-ray powder diffraction analysis and Rietveld refinement studies. Phase transformation and ion release were analyzed during 7 days of incubation in simulated body fluid at 37 °C. The metabolic activity of healthy and osteosarcoma cell lines was assessed by cell cytotoxicity and viability test. The as-prepared powders were composed of calcium-deficient carbonated hydroxyapatite (HAp), octacalcium phosphate (OCP), and amorphous calcium phosphate (ACP). Along with the selenite substitution, the presence of Sr2+, Na+, and Mg2+ was detected as a result of using cuttlefish bone as a precursor for Ca2+ ions. Inductively coupled plasma mass spectrometry analysis showed that the Se/(P + Se) molar ratios of selenite substituted powders are lower than the nominal ratios. Heat treated powders were composed of HAp, α-tricalcium phosphate (α-TCP) and ß-tricalcium phosphate (ß-TCP). Doping CaP structure with selenite ions improves the thermal stability of HAp. The powder with the Se/(P + Se) molar ratio of 0.007 showed selective toxicity to cancer cells.

Characterization of Chitosan-Based Scaffolds Seeded with Sheep Nasal Chondrocytes for Cartilage Tissue Engineering.

The treatment of cartilage defect remains a challenging issue in clinical practice. Chitosan-based materials have been recognized as a suitable microenvironment for chondrocyte adhesion, proliferation and differentiation forming articular cartilage. The use of nasal chondrocytes to culture articular cartilage on an appropriate scaffold emerged as a promising novel strategy for cartilage regeneration. Beside excellent properties, chitosan lacks in biological activity, such as RGD-sequences. In this work, we have prepared pure and protein-modified chitosan scaffolds of different deacetylation degree and molecular weight as platforms for the culture of sheep nasal chondrocytes. Fibronectin (FN) was chosen as an adhesive protein for the improvement of chitosan bioactivity. Prepared scaffolds were characterised in terms of microstructure, physical and biodegradation properties, while FN interactions with different chitosans were investigated through adsorption-desorption studies. The results indicated faster enzymatic degradation of chitosan scaffolds with lower deacetylation degree, while better FN interactions with material were achieved on chitosan with higher number of amine groups. Histological and immunohistochemical analysis of in vitro engineered cartilage grafts showed presence of hyaline cartilage produced by nasal chondrocytes.

Strontium substituted biomimetic calcium phosphate system derived from cuttlefish bone.

Biomimetic triphasic strontium-substituted calcium phosphate (CaP) powders were prepared by wet precipitation method at 50°C, using CaCO3 , (NH2 )2 COH3 PO4 , and Sr(NO3 )2 as reagents. Calcite was prepared from biogenic source (cuttlefish bone). The synthesized powders have been characterized by elemental analysis, Fourier transform infrared spectrometry, X-ray diffraction, Rietveld refinement studies and cell viability test. Phase transformation and ion release were analyzed during 7 days of incubation in simulated body fluid at 37°C. The raw precipitated powders were composed of calcium deficient carbonated hydroxyapatite (HA), octacalcium phosphate (OCP), and amorphous calcium phosphate (ACP). After heat treatment at 1200°C ß-tricalcium phosphate (ß-TCP) was detected. Strontium substitution for calcium results in an increase of lattice parameters in HA, OCP, and ß-TCP. Sr2+ occupy the Ca(1) site in HA, Ca(3,4,7,8) sites in OCP and Ca(1,2,3,4) sites in ß-TCP. Along with Sr2+ substitution, presence of Mg2+ and Na+ ions was detected as a result of using biogenic calcium carbonate. The culture of human embryonic kidney cells indicated noncytotoxicity of the prepared CaP powders with emphasis on the cell proliferation during 3 days of culture.

Tuning physicochemical and biological properties of chitosan through complexation with transition metal ions.

The synthesis of biologically active scaffolds is focused on the design of cell-sensitive surface by applying cell-adhesive proteins or bioceramic micro (nano) particles. The emerging new strategy for manipulating the biological properties lies in the modification by trace metals found in the living organism. In this work, we have prepared biocompatible chitosan hydrogels modified by copper (II) and zinc (II) ions through complexation interactions. Due to the strong affinity of metal ions towards amino groups of chitosan, we obtained defined and ordered structures of metal ion-chitosan hydrogel without the formation of additional metal species. The physical and biological properties of complex hydrogels varied in metal ion concentration-dependent manner, from less stable cytocompatible to more stable cytotoxic structure for copper-chitosan system. Interestingly, zinc-chitosan complex hydrogels did show lower stability, but significantly higher biocompatibility with respect to the copper-containing hydrogels.

Injectable chitosan-hydroxyapatite hydrogels promote the osteogenic differentiation of mesenchymal stem cells.

Injectable hydrogels have emerged as promising biomaterials for tissue engineering applications. The goal of this study was to evaluate the potential of a pH-responsive chitosan-hydroxyapatite hydrogel to be used as a three-dimensional support for encapsulated mesenchymal stem cells (MSCs) osteogenic differentiation. In vitro enzymatic degradation of the hydrogel, during 28 days of incubation, in simulated physiological condiditons, was characterized by swelling measurements, molecular weight determination and SEM analysis of hydrogel microstructure. Osteogenic differentiation of encapsulated MSCs was confirmed by osteogenic Runx2, collagen type I and osteocalcin immunostaining and alkaline phosphatase quantification. The deposition of late osteogenic markers (calcium phosphates) detected by Alizarin red and von Kossa staining indicated an extracellular matrix mineralization.

Cellular hydrogels based on pH-responsive chitosan-hydroxyapatite system.

The development of bioactive injectable system as cell carrier with minimal impact on viability of encapsulated cells represents a great challenge. In the present work, we propose a new pH-responsive chitosan-hydroxyapatite-based hydrogel with sodium bicarbonate (NaHCO3) as the gelling agent. The in situ synthesis of hydroxyapatite phase has resulted in stable composite suspension and final homogeneous hydrogel. The application of sodium bicarbonate has allowed non-cytotoxic fast gelation of chitosan-hydroxyapatite within 4min, and without excess of sodium ions concentration. Rheological properties of crosslinked hydrogel have demonstrated possible behaviour as 'strong physical hydrogel'. The live dead staining has confirmed good viability and dispersion, as well as proliferation of encapsulated cells by the culture time. Presented preliminary results show good potential of chitosan-hydroxyapatite/NaHCO3 as a cell carrier, whose impact on the cell differentiation need to be confirmed by encapsulation of other cell phenotypes.

Human Mesenchymal Stem Cells Differentiation Regulated by Hydroxyapatite Content within Chitosan-Based Scaffolds under Perfusion Conditions.

The extensive need for hard tissue substituent greatly motivates development of suitable allogeneic grafts for therapeutic recreation. Different calcium phosphate phases have been accepted as scaffold's components with positive influence on osteoinduction and differentiation of human mesenchymal stem cells, in terms of their higher fraction within the graft. Nevertheless, the creation of unlimited nutrients diffusion through newly formed grafts is of great importance. The media flow accomplished by perfusion forces can provide physicochemical, and also, biomechanical stimuli for three-dimensional bone-construct growth. In the present study, the influence of a different scaffold's composition on the human mesenchymal stem cells (hMSCs) differentiation performed in a U-CUP bioreactor under perfusion conditioning was investigated. The histological and immunohistochemical analysis of cultured bony tissues, and the evaluation of osteogenic genes' expression indicate that the lower fraction of in situ formed hydroxyapatite in the range of 10⁻30% within chitosan scaffold could be preferable for bone-construct development.

In Situ Hydroxyapatite Content Affects the Cell Differentiation on Porous Chitosan/Hydroxyapatite Scaffolds.

Highly porous chitosan/hydroxyapatite composite structures with different weight ratios (100/0; 90/10; 80/20; 70/30; 60/40; 50/50; 40/60) have been prepared by precipitation method and freeze-gelation technique using calcite, urea phosphate and chitosan as starting materials. The composition of prepared composite scaffolds was characterized by X-ray diffraction analysis and Fourier transformed infrared spectroscopy, while morphology of scaffolds was imaged by scanning electron microscopy. Mercury intrusion porosimetry measurements of prepared scaffolds have shown different porosity and microstructure regarding to the HA content, along with SEM observations of scaffolds after being immersed in physiological medium. The results of swelling capacity and compressive strength measured in Dulbecco's phosphate buffer saline (DPBS) have shown higher values for composite scaffolds with lower in situ HA content. Viability, proliferation and differentiation of MC3T3-E1 cells seeded on different scaffolds have been evaluated by live dead assay and confocal scan microscopy. Our results suggest that the increase of HA content enhance osteoblast differentiation confirming osteogenic properties of highly porous CS/HA scaffolds for tissue engineering applications in bone repair.

PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: in vitro cell culture studies.

In the present study, we examined the potential of using highly porous poly(ε-caprolactone) (PCL)-coated hydroxyapatite (HAp) scaffold derived from cuttlefish bone for bone tissue engineering applications. The cell culture studies were performed in vitro with preosteoblastic MC3T3-E1 cells in static culture conditions. Comparisons were made with uncoated HAp scaffold. The attachment and spreading of preosteoblasts on scaffolds were observed by Live/Dead staining Kit. The cells grown on the HAp/PCL composite scaffold exhibited greater spreading than cells grown on the HAp scaffold. DNA quantification and scanning electron microscopy (SEM) confirmed a good proliferation of cells on the scaffolds. DNA content on the HAp/PCL scaffold was significantly higher compared to porous HAp scaffolds. The amount of collagen synthesis was determined using a hydroxyproline assay. The osteoblastic differentiation of the cells was evaluated by determining alkaline phosphatase (ALP) activity and collagen type I secretion. Furthermore, cell spreading and cell proliferation within scaffolds were observed using a fluorescence microscope.

Hydrothermal synthesis and thermal evolution of carbonate-fluorhydroxyapatite scaffold from cuttlefish bones.

Phase composition, crystal structure and morphology of carbonated fluor/hydroxyapatite synthesized hydrothermally from aragonitic cuttlefish bones were studied by powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) combined with energy dispersive X-ray spectroscopy (EDS). The product of synthesis has been characterized as carbonated fluor/hydroxyapatite with carbonate incorporated inside channel (A-type) and substituted for the PO4(3-) group (B-type). The vibration band at 874 cm(-1) assigned to bending (ν2) mode undoubtedly confirmed carbonate substituted for PO4(3-) group, while the band at 880 cm(-1) was attributed to A-type carbonate substitution. The additional sharp and intense band at 865 cm(-1) considered as "non-apatitic" carbonate substitution is not assigned with certainty so far. Evolution of CO2 from tetrahedral (PO4(3-)) sites with the increase in heat-treatment temperature is evident by the changes in tetrahedral bond lengths and angles, as obtained by the Rietveld structure refinement. Also, changes in the isotropic temperature parameters for the 2a site point to A-type carbonate incorporation as well.

PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: morphology, mechanical properties and bioactivity.

In the present study, poly(ε-caprolactone)-coated hydroxyapatite scaffold derived from cuttlefish bone was prepared. Hydrothermal transformation of aragonitic cuttlefish bone into hydroxyapatite (HAp) was performed at 200°C retaining the cuttlebone architecture. The HAp scaffold was coated with a poly(ε-caprolactone) (PCL) using vacuum impregnation technique. The compositional and morphological properties of HAp and PCL-coated HAp scaffolds were studied by means of X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM) with energy dispersive X-ray (EDX) analysis. Bioactivity was tested by immersion in Hank's balanced salt solution (HBSS) and mechanical tests were performed at compression. The results showed that PCL-coated HAp (HAp/PCL) scaffold resulted in a material with improved mechanical properties that keep the original interconnected porous structure indispensable for tissue growth and vascularization. The compressive strength (0.88MPa) and the elastic modulus (15.5MPa) are within the lower range of properties reported for human trabecular bones. The in vitro mineralization of calcium phosphate (CP) that produces the bone-like apatite was observed on both the pure HAp scaffold and the HAp/PCL composite scaffold. The prepared bioactive scaffold with enhanced mechanical properties is a good candidate for bone tissue engineering applications.

Luminescence and Raman spectra of acetylacetone at low temperatures.

Raman spectra of acetylacetone were recorded for molecules isolated in an argon matrix at 10 K and for a polycrystalline sample. In the solid sample, broad bands appear superimposed on a much weaker Raman spectrum corresponding mainly to the stable enol form. The position of these bands depends on the excitation wavelength (514.5 and 488.8 nm argon ion laser lines were used), sample temperature, and cooling history. They are attributed to transitions from an excited electronic state to various isomer states in the ground electronic state. Laser photons have energies comparable to energies of a number of excited triplet states predicted for a free acetylacetone molecule (Chen, X.-B.; Fang, W.-H.; Phillips, D. L. J. Phys. Chem. A 2006, 110, 4434). Since singlet-to-triplet photon absorption transitions are forbidden, states existing in the solid have mixed singlet/triplet character. Their decay results in population of different isomer states, which except for the lowest isomers SYN enol, TS2 enol (described in Matanovic I.; Doslic, N. J. Phys. Chem. A 2005, 109, 4185), and the keto form, which can be detected in the Raman spectra of the solid, are not vibrationally resolved. Differential scanning calorimetry detected two signals upon cooling of acetylacetone, one at 229 K and one at 217 K, while upon heating, they appear at 254 and 225 K. The phase change at higher temperature is attributed to a freezing/melting transition, while the one at lower temperature seems to correspond to freezing/melting of keto domains, as suggested by Johnson et al. (Johnson, M. R.; Jones, N. H.; Geis, A; Horsewill. A. J.; Trommsdorff, H. P. J. Chem. Phys. 2002, 116, 5694). Using matrix isolation in argon, the vibrational spectrum of acetylacetone at 10 K was recorded. Strong bands at 1602 and 1629 cm(-1) are assigned as the SYN enol bands, while a weaker underlying band at 1687 cm(-1) and a medium shoulder at 1617 cm(-1) are assigned as TS2 enol bands.