Anamarija Rogina

Preparation and characterization of nano-hydroxyapatite within chitosan matrix

Nano-composites that show some features of natural bone both in composition and in microstructure have been prepared by in situ precipitation method. Apatite phase has been prepared from cost-effective precursors (calcite and urea phosphate) within chitosan (CS) matrix dissolved in aqueous acetic acid solution. The compositional and morphological properties of composites were studied by means of Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) thermogravimetric analysis (TGA) and transmission electron microscopy (TEM). Depending on the reaction conditions (temperature, reaction time, glucose addition and pH control) in addition to hydroxyapatite (HA) as a major phase, octacalcium hydrogen phosphate pentahydrate (OCP) and dicalcium phosphate anhydrate (DCPD) were formed as shown by XRD and FTIR. Crystallite lengths of precipitated HA estimated by Scherrers equation were between 20 and 30 nm. A fibrous morphology (~400 nm) of HA observed by TEM indicates that HA nucleates on chitosan chains.

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.

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.

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.

Biomimetic design of bone substitutes based on cuttlefish bone-derived hydroxyapatite and biodegradable polymers: Biomimetic design of bone

Being a major component of bone tissue, hydroxyapatite is the most investigated calcium phosphate in the design and development of bone implants. The high brittleness and poor load-bearing properties have led researchers to manipulate hydroxyapatite performance by applying polymer or metal materials. The present study focuses on biomimetic approach of the hydroxyapatite synthesis from the cuttlefish bone in order to preserve highly porous structure. The low stiffness of hydroxyapatite scaffold was altered by thin polycaprolactone/poly(lactic acid) coating, resulting in remarkably 18-fold increase of Youngs modulus. The mechanical test revealed that poly(lactic acid) increases the stiffness of composite scaffolds which depends on the polycaprolactone/poly(lactic acid) volume ratio. The composite scaffolds are bioactive supporting the deposition of new calcium phosphates when incubated in simulated physiological medium for 21 days. Moreover, the culture of human embryonic kidney cells indicated non-cytotoxicity of the composite scaffolds with emphasis on the cell proliferation during three days of culture.