Agata Przekora

Biomedical potential of chitosan/HA and chitosan/β-1,3-glucan/HA biomaterials as scaffolds for bone regeneration — A comparative study

The aim of this work was to compare biomedical potential of chitosan/hydroxyapatite (chit/HA) and novel chitosan/β-1,3-glucan/hydroxyapatite (chit/glu/HA) materials as scaffolds for bone regeneration via characterization of their biocompatibility, porosity, mechanical properties, and water uptake behaviour. Biocompatibility of the scaffolds was assessed in direct-contact with the materials using normal human foetal osteoblast cell line. Cytotoxicity and osteoblast proliferation rate were evaluated. Porosity was assessed using computed microtomography analysis and mechanical properties were determined by compression testing. Obtained results demonstrated that chit/HA scaffold possessed significantly better mechanical properties (compressive strength: 1.23 MPa, Youngs modulus: 0.46 MPa) than chit/glu/HA material (compressive strength: 0.26 MPa, Youngs modulus: 0.25 MPa). However, addition of bacterial β-1,3-glucan to the chit/HA scaffold improved its flexibility and porosity. Moreover, chit/glu/HA scaffold revealed significantly higher water uptake capability (52.6% after 24h of soaking) compared to the chit/HA (30.7%) and thus can serve as a very good drug delivery carrier. Chit/glu/HA scaffold was also more favourable to osteoblast survival (near 100% viability after 24-h culture), proliferation, and spreading compared to the chit/HA (63% viability). The chit/glu/HA possesses better biomedical potential than chit/HA scaffold. Nevertheless, poor mechanical properties of the chit/glu/HA limit its application to non-load bearing implantation area.

Chitosan/β-1,3-glucan/calcium phosphate ceramics composites—Novel cell scaffolds for bone tissue engineering application

Bone tissue engineering put emphasis on fabrication three-dimensional biodegradable porous scaffolds that possess ability to enhance adhesion, proliferation and differentiation of osteoblast cells, therefore supporting bone regeneration and functional bone tissue formation. The aim of this work was to fabricate novel tri-component scaffolds composed of chitosan, β-1,3-glucan, and bioceramics and to evaluate their basic structural, mechanical, and biological properties. It should be noted that we are the first who describe fabrication and characterization of tri-component composites containing β-1,3-glucan. Microstructure of novel composites was visualized by computed tomography scanning and SEM. Compressive strength and Youngs modulus of the composites were evaluated by compression testing. The biocompatibility was assessed in vitro by cytotoxicity, cell attachment and cell proliferation tests using human foetal osteoblast cell line. Our results demonstrated that novel composites possess good compressive strength as the effect of polysaccharide components of scaffolds, are very elastic, are non-toxic, favourable to cell adhesion and promote cell proliferation. However, novel biomaterials revealed relatively low Youngs modulus values. Thus, we infer that fabricated novel composites are promising materials for bone tissue engineering application as cell scaffolds to fill small bone losses rather than as massive bone fillers exposed to mechanical load.

In vitro evaluation of the risk of inflammatory response after chitosan/HA and chitosan/β-1,3-glucan/HA bone scaffold implantation.

The aim of the study was to evaluate in vitro the risk of inflammatory response induced by chitosan/hydroxyapatite (chit/HA) and novel chitosan/β-1,3-glucan/hydroxyapatite (chit/glu/HA) bone scaffolds. The inflammatory response was assessed via measurement of proinflammatory cytokine and ROI production by human monocytes, macrophages, and osteoblasts stimulated with investigated scaffolds. Moreover, adsorption of human serum/plasma proteins to the tested materials was determined. Both biomaterials did not induce intracellular ROI generation by monocytes, macrophages, and osteoblasts and did not stimulate proinflammatory cytokine (IL-6 and TNF-α) production by inflammatory cells. Moreover, the chit/glu/HA material induced increased TNF-α production by osteoblasts that is believed to enhance osteogenic differentiation. Thus, it was demonstrated that chit/HA and chit/glu/HA scaffolds carry a low risk of biomaterial-induced inflammatory response and are promising materials as bone scaffolds for bone tissue engineering and regenerative medicine applications.

Do novel cement-type biomaterials reveal ion reactivity that affects cell viability in vitro?

Calcium phosphate bioceramics have been studied as bone filler materials for years and have become a component of many commercial products. It is widely known that surface-reactive biomaterials may cause changes in the concentration of crucial ions in the surrounding environment, thereby affecting cell metabolism and viability. The aim of this study was to produce five cement-type biomaterials and characterize their phase composition using X-ray diffraction method, and porosity and pore size distribution using mercury intrusion porosimeter. We then evaluated ion interactions of the novel biomaterials with the surrounding environment (culture medium). A commercially available bone substitute, HydroSet™ (Stryker®), was used as a reference. MTT and NRU cytotoxicity tests were performed to assess the effect of changes in the concentration of crucial ions (calcium, magnesium, phosphate) on osteoblast metabolism and viability in vitro. Our study clearly indicated that various biomaterials demonstrated different ion reactivity and consequently may cause changes in ion concentration in the local environment. Critically low or high values of calcium, magnesium, and phosphate concentrations in the medium exerted cytotoxic effects on the cultured cells. Moreover, we discovered that the chemical composition of the culture medium had a substantial influence on ion interactions with biomaterials.

Biological properties of novel chitosan-based composites for medical application as bone substitute

Hydroxyapatite is the main inorganic component of bones and teeth. In order to improve mechanical properties and surgical handiness of bioceramics, a plasticizing agent e.g. polysaccharide can be added. Chitosan is a polysaccharide with biological properties that make it an ideal component of bioceramics-based composites for medical application as bone substitute. In this study, biocompatibility of two types of novel krill chitosan-based composites was evaluated. In vitro experiments were carried out using human foetal osteoblast cell line. Cytotoxicity, cell adhesion, and bone ALP activity tests were performed to assess biocompatibility of the composites. Osteoblast growth on composites was observed using confocal microscope. Our results demonstrated that fabricated novel composites are non-toxic, are favorable to cell adhesion and growth, and provoke increase in b-ALP activity with time, thus inducing osteoblast differentiation. Based on this data composites have promising clinical potential as a bone defect filler in regenerative medicine. It is worth emphasizing that our work resulted in fabrication of flexible and surgical handy, bone substitutes that possess absolute biocompatibility with structural and mechanical properties similar to trabecular bone.

Addition of 1,3-β-d-glucan to chitosan-based composites enhances osteoblast adhesion, growth, and proliferation

The aim of this work was to prove using two osteoblastic cell lines that addition of bacterial 1,3-β-D-glucan to chitosan-based biocomposites significantly enhances adhesion, growth, and proliferation of osteoblast cells. Cytotoxicity of materials was evaluated indirectly using fluid extracts and by direct-contact method using live/dead double fluorescent staining. Cell adhesion was determined quantitatively by LDH total test and cell proliferation was assessed by confocal microscope observation. Obtained data clearly prove that addition of 1,3-β-D-glucan to the bi-component chitosan/bioceramic materials significantly enhances adhesion, growth, and proliferation of osteoblast cells. The results demonstrated that all investigated biomaterials were non-toxic and allowed for cell attachment. However, significantly better osteoblast growth was observed on scaffolds containing 1,3-β-D-glucan. Thus, it may be inferred that scaffolds modified with glucan are more promising materials for bone tissue engineering application than bi-component chitosan/bioceramic composites.

The cytotoxicity assessment of the novel latex urinary catheter with prolonged antimicrobial activity

The purpose of this study was to evaluate the in vitro biocompatibility of the novel Sparfoxacin (SPA)-treated latex catheter with previously performed prolonged antimicrobial activity. Rectangular-shaped test samples of silicone latex catheter were fabricated according to patented procedure permitting the immobilization of SPA on heparin (HP)-coated catheter by means of mixed, covalent and non-covalent bonds. Samples subjected to cytotoxicity assay were divided into four groups: (1) the untreated catheter, (2) HP-coated catheter, (3) HP-coated catheter with SPA immobilized in low SPA concentration solution (SPA-L treated sample), and (4) high SPA concentration solution (SPA-H treated sample). Then the samples were placed directly into green monkey kidney (GMK) cell monolayer for 24 h. After the incubation period, cytotoxicity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The degree of cytotoxicity of each sample was evaluated according to the reference value represented by the control cells cultured without catheter sample. Statistical significance was determined by repeated t-test (P < 0.05). The cytotoxic effect of treated and untreated catheters was also estimated by microscopic observations of GMK cells morphological changes. SPA-treated catheters demonstrated high survival rates in MTT assay (>93%) on the contrary to the untreated catheters (6.13%) and HP-coated catheters (80.90%). Moreover, microscopic observation of GMK cells exposed to SPA-treated samples revealed no morphological changes and no cell growth reduction. We suggest that SPA-treated latex catheters are biofilm formation resistant (as we revealed in our previous work), considerably less toxic than untreated ones, and can be undoubtedly used in urological practice.

Titanium coated with functionalized carbon nanotubes--a promising novel material for biomedical application as an implantable orthopaedic electronic device.

The aim of the study was to fabricate titanium (Ti) material coated with functionalized carbon nanotubes (f-CNTs) that would have potential medical application in orthopaedics as an implantable electronic device. The novel biomedical material (Ti-CNTs-H2O) would possess specific set of properties, such as: electrical conductivity, non-toxicity, and ability to inhibit connective tissue cell growth and proliferation protecting the Ti-CNTs-H2O surface against covering by cells. The novel material was obtained via an electrophoretic deposition of CNTs-H2O on the Ti surface. Then, physicochemical, electrical, and biological properties were evaluated. Electrical property evaluation revealed that a Ti-CNTs-H2O material is highly conductive and X-ray photoelectron spectroscopy analysis demonstrated that there are mainly COOH groups on the Ti-CNTs-H2O surface that are found to inhibit cell growth. Biological properties were assessed using normal human foetal osteoblast cell line (hFOB 1.19). Conducted cytotoxicity tests and live/dead fluorescent staining demonstrated that Ti-CNTs-H2O does not exert toxic effect on hFOB cells. Moreover, fluorescence laser scanning microscope observation demonstrated that Ti-CNTs-H2O surface retards to a great extent cell proliferation. The study resulted in successful fabrication of highly conductive, non-toxic Ti-CNTs-H2O material that possesses ability to inhibit osteoblast proliferation and thus has a great potential as an orthopaedic implantable electronic device.

Fabrication and physicochemical characterization of porous composite microgranules with selenium oxyanions and risedronate sodium for potential applications in bone tumors

Nanocrystalline hydroxyapatite containing selenite ions (SeHA; 9.6 wt.% of selenium) was synthesized using wet method and subject to careful physicochemical analysis by powder X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, solid-state nuclear magnetic resonance, wavelength dispersive X-ray fluorescence, and inductively coupled plasma optical emission spectrometry. SeHA was then used to develop the selenium-containing hydroxyapatite/alginate (SeHA/ALG) composite granules. Risedronate sodium (RIS) was introduced to the obtained spherical microgranules of a size of about 1.1–1.5 mm in 2 ways: during the granules’ preparation (RIS solution added to a suspension of ALG and SeHA), and as a result of SeHA/ALG granules soaking in aqueous RIS solution. The analysis made using 13C and 31P cross-polarization magic angle spinning nuclear magnetic resonance confirmed the presence of RIS and its interaction with calcium ions. Then, the release of selenium (inductively coupled plasma optical emission spectrometry) and RIS (high-performance liquid chromatography) from microgranules was examined. Moreover, cytotoxicity of fabricated granules was assessed by MTT test. Selenium release was biphasic: the first stage was short and ascribed to a “burst release” probably from a hydrated surface layer of SeHA crystals, while the next stage was significantly longer and ascribed to a sustained release of selenium from the crystals’ interior. The study showed that the method of obtaining microgranules containing RIS significantly affects its release profile. Performed cytotoxicity test revealed that fabricated granules had high antitumor activity against osteosarcoma cells. However, because of the “burst release” of selenium during the first 10 h, the granules significantly reduced viability of normal osteoblasts as well.

Evaluation of the potential of chitosan/β-1,3-glucan/hydroxyapatite material as a scaffold for living bone graft production in vitro by comparison of ADSC and BMDSC behaviour on its surface

The opinion regarding the origin of adult stem cells that should be used for living bone construct generation is strongly divided in the scientific community. In this study, the potential of chitosan/β-1,3-glucan/hydroxyapatite (chit/glu/HA) material as a scaffold for bone regeneration applications was evaluated by behaviour comparison of adult stem cells derived from both origins-adipose derived mesenchymal stem cell (ADSC) tissue and bone marrow derived mesenchymal stem cells (BMDSCs). In the case of ADSC isolation, low and high negative pressures were applied during a liposuction procedure in order to determine if negative pressure settings may have an impact on subsequent cell behaviour in vitro. The obtained results demonstrated that the chit/glu/HA material is a promising candidate to be used for living bone graft production in vitro as both ADSCs and BMDSCs revealed a satisfactory proliferation and differentiation ability on its surface. Nevertheless, BMDSCs would be a better choice of adult stem cells since they were better spread, more strongly attached and showed a more superior proliferation and differentiation ability than ADSCs when cultured on the chit/glu/HA scaffold. However, if BMDSCs cannot be isolated, ADSCs may be used for bone construct production but lipoaspirate should be collected under low negative pressure (-200 mm Hg), as high negative pressure (-700 mmHg) applied during liposuction surgery may retard subsequent ADSC proliferation and type I collagen production.