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

[EN] 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 degrees 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.This research was funded by the Croatian Science Foundation (project IP-2014-09-3752) and the European Structural and Investments Funds (grant KK.01.1.1.07.0014.). The authors thank Inga Urli, Faculty of Science, University of Zagreb for providing Hek293 and hMSC cells. Compression experiments were carried out at the Centre for Biomaterials and Tissue Engineering (CBIT), Universitat Politecnica de Valencia, Valencia, Spain under the PID2019-106000RB-C21/AEI/10.13039/501100011033 project. The authors would like to thank Jorge Mas-Estelles for his generous assistance.Bauer, L.; Antunovic, M.; Gallego-Ferrer, G.; Ivankovic, M.; Ivankovic, H. (2021). PCL-Coated Multi-Substituted Calcium Phosphate Bone Scaffolds with Enhanced Properties. Materials. 14(16):1-19. https://doi.org/10.3390/ma14164403S119141

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

[EN] 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.This work has been supported in part by the Croatian Science Foundation under the project IP-2014-09-3752 by the Spanish Ministry through the MAT2016-76039-C4-1-R project (including the FEDER financial support). Joaquin Rodenas-Rochina acknowledges funding by the Conselleria de Educacion, Investigacion, Cultura y Deporte (Generalitat Valenciana) and co-funding by Fondo Social Europeo (FSE) through APOSTD grant (APOSTD/2016/006).Ressler, A.; Ródenas Rochina, J.; Ivankovic, M.; Ivankovic, H.; Rogina, A.; Ferrer, G. (2018). Injectable chitosan-hydroxyapatite hydrogels promote the osteogenic differentiation of mesenchymal stem cells. Carbohydrate Polymers. 197:469-477. https://doi.org/10.1016/j.carbpol.2018.06.029S46947719

Cellular hydrogels based on pH-responsive chitosan-hydroxyapatite system

[EN] 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 hydro gel'. 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 chitosanhydroxyapatite/NaHCO3 as a cell carrier, whose impact on the cell differentiation need to be confirmed by encapsulation of other cell phenotypes.This work has been supported in part by the Croatian Science Foundation under the project IP-2014-09-3752. The authors want to thank Carlos Garcia Fernandez from TA Instruments for helping with rheological measurements. G. Gallego Ferrer is grateful for the financial support of the Spanish Ministry of Economy and Competitiveness through the MAT2016-76039-C4-1-R project (including Feder funds)Rogina, A.; Ressler, A.; Matie, I.; Ferrer, G.; Marijanovic, I.; Ivankovic, M.; Ivankovic, H. (2017). Cellular hydrogels based on pH-responsive chitosan-hydroxyapatite system. Carbohydrate Polymers. 166:173-182. https://doi.org/10.1016/j.carbpol.2017.02.105S17318216

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

[EN] 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 SeO3 2¿ ions, and chitosan have been prepared by freezegelation 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.The financial supports of the European Regional Development Fund (grant KK.01.1.1.07.0014) , the PID2019-106000RB-C21/AEI/10.13039/501100011033 project from the Spanish Research Agency, and the L'Oreal-UNESCO "For Women in Science" Foundation are gratefully acknowledged.Ressler, A.; Antunovic, M.; Teruel Biosca, L.; Ferrer GG; Babic, S.; Urlic, I.; Ivankovic, M.... (2022). Osteogenic differentiation of human mesenchymal stem cells on substituted calcium phosphate/chitosan composite scaffold. Carbohydrate Polymers. 277:1-16. https://doi.org/10.1016/j.carbpol.2021.11888311627

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

[EN] In the present study, poly(epsilon-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(c-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 (0.88 MPa) and the elastic modulus (15.5 MPa) 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.The financial support of the Ministry of Science, Education and Sports of the Republic of Croatia (project 125-1252970-3005: "Bioceramic, Polymer and Composite Nanostructured Materials") and from the Spanish Ministry project DPI2010-20399-C04-03 is gratefully acknowledged. Rocio Ochoa-Fernandez is also acknowledged for her help with the mechanical tests.Milovac, D.; Gallego Ferrer, G.; Ivankovic, M.; Ivankovic, H. (2014). PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: Morphology, mechanical properties and bioactivity. Materials Science and Engineering C. 34:437-445. https://doi.org/10.1016/j.msec.2013.09.036S4374453

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

[EN] In the present study, we examined the potential of using highly porous poly(epsilon-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. (C) 2014 Elsevier B.V. All rights reserved.The financial support of the Ministry of Science, Education and Sports of the Republic of Croatia (Project 125-1252970-3005: "Bioceramic, Polymer and Composite Nanostructured Materials"), the Spanish Ministry project DPI2010-20399-C04-03 and the L'Oreal ADRIA-UNESCO national fellowship program for Women in Science is gratefully acknowledged.Milovac, D.; Gamboa-Martinez, TC.; Ivankovic, M.; Ferrer, G.; Ivankovic, H. (2014). PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: in vitro cell culture studies. Materials Science and Engineering C. 42:264-272. https://doi.org/10.1016/j.msec.2014.05.034S2642724

Antibacterial activity of silver doped hydroxyapatite toward multidrug-resistant clinical isolates of Acinetobacter baumannii

Bacteria Acinetobacter baumannii is a persistent issue in hospital-acquired infections due to its fast and potent development of multi-drug resistance. To address this urgent challenge, a novel biomaterial using silver (Ag+) ions within the hydroxyapatite (HAp) lattice has been developed to prevent infections in orthopedic surgery and bone regeneration applications without relying on antibiotics. The aim of the study was to examine the antibacterial activity of mono-substituted HAp with Ag+ ions and a mixture of mono-substituted HAps with Sr2+, Zn2+, Mg2+, SeO32- and Ag+ ions against the A. baumannii. The samples were prepared in the form of powder and disc and analyzed by disc diffusion, broth microdilution method, and scanning electron microscopy. The results from the disc-diffusion method have shown a strong antibacterial efficacy of the Ag-substituted and mixture of mono-substituted HAps (Sr, Zn, Se, Mg, Ag) toward several clinical isolates. The Minimal Inhibitory Concentrations for the powdered HAp samples ranged from 32 to 42 mg/L (Ag+ substituted) and 83–167 mg/L (mixture of mono-substituted), while the Minimal Bactericidal Concentrations after 24 h of contact ranged from 62.5 (Ag+) to 187.5–292 mg/L (ion mixture). The lower substitution level of Ag+ ions in a mixture of mono-substituted HAps was the cause of lower antibacterial effects measured in suspension. However, the inhibition zones and bacterial adhesion on the biomaterial surface were comparable. Overall, the clinical isolates of A. baumannii were effectively inhibited by substituted HAp samples, probably in the same amount as by other commercially available silver-doped materials, and such materials may provide a promising alternative or supplementation to antibiotic treatment in the prevention of infections associated with bone regeneration. The antibacterial activity of prepared samples toward A. baumannii was time-dependent and should be considered in potential applications.Peer reviewe

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.The financial support of the Croatian Science Foundation (project: "Development of Biocompatible Hydroxyapatite Based Materials for Bone Tissue Engineering Applications") and L'Oreal-UNESCO Foundation 'For Women in Science' is gratefully acknowledged. The financial support from the Spanish Ministry of Economy and Competitiveness and the Feder funds through the MAT2013-46467-C4-1-R project is acknowledged by the Spanish co-authors. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. The authors want to acknowledge Pilar Gomez Tena and Sergio Mestre Beltran from Instituto de Tecnologia Ceramica, Castellon, Spain, for theirs assistance with porosity measurements.Rogina, A.; Rico Tortosa, PM.; Gallego Ferrer, G.; Ivankovic, M.; Ivankovic, H. (2016). In Situ Hydroxyapatite Content Affects the Cell Differentiation on Porous Chitosan/Hydroxyapatite Scaffolds. Annals of Biomedical Engineering. 44(4):1107-1119. https://doi.org/10.1007/s10439-015-1418-0S11071119444Azzaoui, K., A. Lamhamdi, E. M. Mejdoubi, M. Berrabah, B. Hammouti, A. Elidrissi, M. M. G. Fouda, and S. S. Al-Deyab. Synthesis and characterization of composite based on cellulose acetate and hydroxyapatite application to the absorption of harmful substances. Carbohydr. Polym. 111:41–46, 2014.Bacakova, L., E. Filova, M. Parizek, T. Ruml, and V. Svorcik. Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants. Biotech. Adv. 29:739–767, 2011.Bose, S., and S. Tarafder. Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: a review. Acta Biomater. 8:1401–1421, 2012.Chan, B. P., and K. W. Leong. Scaffolding in tissue engineering: general approaches and tissue-specific considerations. Eur. Spine J. 17:467–479, 2008.Dhandayuthapani, B., Y. Yoshida, T. Maekawa, and D. S. Kumar. Polymeric scaffolds in tissue engineering application: a review. Int. J. Polym. Sci. 1–19:2011, 2011.Dorozhkin, S. V. Calcium orthophosphate-based bioceramics. Materials 6:3840–3942, 2013.Frohbergh, M. E., A. Katsman, G. P. Botta, P. Lazarovici, C. L. Schauer, U. G. K. Wegst, and P. I. Lelkes. Electrospun hydroxyapatite-containing chitosan nanofibers crosslinked with genipin for bone tissue engineering. Biomaterials 33:9167–9178, 2012.Gerstenfeld, L. C., C. M. Edgar, S. Kakar, K. A. Jacobsen, and T. A. Einhorn. Osteogenic growth factors and cytokines and their role in bone repair. In: Engineering of Functional Skeletal Tissues, in Topics in Bone Biology, edited by M. C. Farach-Carson, A. G. Mikos, and F. Bronner. London: Springer, 2005, pp. 17–44.Harada, S.-I., and G. A. Rodan. Control of osteoblast function and regulation of bone mass. Nature 423:349–355, 2003.Ishihara, S., T. Matsumoto, T. Onoki, T. Sohmura, and A. Nakahira. New concept bioceramics composed of octacalcium phosphate (OCP) and dicarboxylic acid-intercalated OCP via hydrothermal hot-pressing. Mater. Sci. Eng. C 29:1885–1888, 2009.Karageorgiou, V., and D. Kaplan. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 26:5474–5491, 2005.Kirkham, G.R., Cartmell, S.H. Genes and proteins involved in the regulation of osteogenesis. In: Topics in Tissue Engineering, edited by N. Ashammakhi, R.L. Reis, and E. Chiellini, R.R.E.C., 2007. pp. 1–22.Lee, H., and G. H. Kim. Cryogenically fabricated three-dimensional chitosan scaffolds with pore size-controlled structures for biomedical applications. Carbohydr. Polym. 85:817–823, 2010.Lewandowska, K. Miscibility and interactions in chitosan acetate/poly(Nvinylpyrrolidone) blends. Thermochim. Acta 517:90–97, 2011.Li, J., D. Zhu, J. Yin, Y. Liu, F. Yao, and K. Yao. Formation of nano-hydroxyapatite cristal in situ in chitosan-pectin polyelectrolyte complex network. Mater. Sci. Eng. C 30:795–803, 2010.Martel-Estrada, S. A., C. A. Martínez-Pérez, J. G. Chacón-Nava, P. E. García-Casillas, and I. Olivas-Armendariz. Synthesis and thermo-physical properties of chitosan/poly(dl-lactide-co-glycolide) composites prepared by thermally induced phase separation. Carbohydr. Polym. 81:775–783, 2010.Martins, A. M., R. C. Pereira, I. B. Leonor, H. S. Azevedo, and R. L. Reis. Chitosan scaffolds incorporating lysozyme into CaP coatings produced by a biomimetic route: a novel concept for tissue engineering combining a self-regulated degradation system with in situ pore formation. Acta Biomater. 5:3328–3336, 2009.Martins, A. M., M. I. Santos, H. S. Azevedo, P. B. Malafaya, and R. L. Reis. Natural origin scaffolds with in situ pore forming capability for bone tissue engineering applications. Acta Biomater. 5:1637–1645, 2008.Mohamed, K. R., Z. M. El-Rashidy, and A. A. Salama. In vitro properties of nanohydroxyapatite/chitosan biocomposites. Ceram. Int. 37:3265–3271, 2011.O’Brien, F. J. Biomaterials & scaffolds for tissue engineering. Mater. Today 14:88–95, 2011.Osborn, J. F., and H. Newesely. The material science of calcium phosphate ceramics. Biomaterials 1:108–111, 1980.Rogina, A., M. Ivanković, and H. Ivanković. Preparation and characterization of nano-hydroxyapatite within chitosan matrix. Mater. Sci. Eng. C 33:4539–4544, 2013.Rogina, A., P. Rico, G. Gallego Ferrer, M. Ivanković, and H. Ivanković. Effect of in situ formed hydroxyapatite on microstructure of freeze-gelled chitosan-based biocomposite scaffolds. Eur. Polym. J. 68:278–287, 2015.Sarem, M., F. Moztarzadeh, and M. Mozafari. How can genipin assist gelatin/carbohydrate chitosan scaffolds to act as replacements of load-bearing soft tissues? Carbohydr. Polym. 93:635–643, 2013.Seibel, M. J. Biochemical markers of bone turnover part I: biochemistry and variability. Clin. Biochem. Rev 26:97–122, 2005.Shaltout, A. A., M. A. Allam, and M. A. Moharram. FTIR spectroscopic, thermal and XRD characterization of hydroxyapatite from new natural sources. Spectrochim. Acta A 83:56–60, 2011.Silva, S. S., S. M. Luna, M. E. Gomes, J. Benesch, I. Paskuleva, J. F. Mano, and R. L. Reis. Plasma surface modification of chitosan membranes: characterization and preliminary cell response studies. Macromol. Biosci. 8:568–576, 2007.Stein, G. S., J. B. Lian, A. J. van Wijnen, J. L. Stein, M. Montecino, A. Javed, A. K. Zaidi, D. W. Young, J.-Y. Choi, and S. M. Pockwinse. Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. Oncogene 23:4315–4329, 2004.Suvorova, E. I., F. Christensson, H. E. Lundager Madsen, and A. A. Chernov. Terrestrial and space-grown HAP and OCP crystals: effect of growth conditions on perfection and morphology. J. Cryst. Growth 186:262–274, 1998.Suzuki, O. Interface of synthetic inorganic biomaterials and bone regeneration. Int. Congr. Ser. 1284:274–283, 2005.Suzuki, O., S. Kamakura, T. Katagiri, M. Nakamura, B. Zhao, Y. Honda, and R. Kamijo. Bone formation enhanced by implanted octacalcium phosphate involving conversion into Ca-deficient hydroxyapatite. Biomaterials 27:2671–2681, 2006.Wagoner Johnson, A. J., and B. A. Herschler. A review of the mechanical behavior of CaP and CaP/polymer composites for applications in bone replacement and repair. Acta Biomater. 7:16–30, 2011.Wang, Y.-C., M.-C. Lin, D.-M. Wang, and H.-J. Hsieh. Fabrication of a novel porous PGA-chitosan hybrid matrix for tissue engineering. Biomaterials 24:1047–1057, 2003.Yuan, N. Y., Y. A. Lin, M. H. Ho, D. M. Wang, J. Y. Lai, and H. J. Hsieh. Effect of the cooling mode on the structure and strength of porous scaffolds made of chitosan, alginate and carboxymethyl cellulose by freeze-gelation method. Carbohydr. Polym. 78:349–356, 2009

Effect of in situ formed hydroxyapatite on microstructure of freeze-gelled chitosan-based biocomposite scaffolds

New in situ highly-porous chitosan/hydroxyapatite (CS/HA) biocomposite scaffolds have been prepared via freeze-gelation technique. Different content of in situ synthesized hydroxyapatite within chitosan solution was obtained by changing the amount of calcium and phosphate precursors. The composition of precipitated inorganic phase was characterized by X-ray diffraction analysis (XRD) and Fourier transformed infrared spectroscopy (FTIR), while morphology of scaffolds was imaged by scanning electron microscopy (SEM). SEM observations of cross section and surface area of prepared scaffolds have shown different microstructure and topography regarding to the HA content, which plays an important role in cell adhesion and proliferation, and nutrient transport. The MIT assay of scaffolds with different content of hydroxyapatite has shown no toxicity which is one of the main requirements for potential biomedical application. Likewise, the presented synthesis allows preparing the scaffolds with large and very well interconnected pores without obtaining toxic intermediate products.CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.Rogina, A.; Rico Tortosa, PM.; Gallego Ferrer, G.; Ivankovic, M.; Ivankovic, H. (2015). Effect of in situ formed hydroxyapatite on microstructure of freeze-gelled chitosan-based biocomposite scaffolds. European Polymer Journal. 68:278-287. https://doi.org/10.1016/j.eurpolymj.2015.05.004S2782876