Aneta Zima

Application of β-1,3-glucan in production of ceramics-based elastic composite for bone repair

BackgroundUnsatisfactory surgical handiness is a commonly known disadvantage of implantable granular bioceramics. To overcome this problem, β-1,3-glucan, biotechnologically derived polysaccharide, has been proposed as a joining agent to combine granular ceramics into novel compact and elastic composite. Hydroxyapatite/glucan elastic material was processed and evaluated as a potential bone void filler.MethodologyThe procedure of composite formation was based on gelling properties of glucan. Its properties were studied using X-ray microtomography, SEM-EDS, FTIR spectroscopy, compression test and ultrasonic method. Sorption index was determined in phosphate buffered saline; bioactivity in simulated body fluid; sterility in growth broth and human blood plasma; implantation procedure in dog model.ResultsHAp/glucan composite is sterilizable, flexible and self-adapting to defect shape. It exhibits bioactivity, good surgical handiness, high sorption index and profitable mechanical properties, resembling those of spongy bone. Results of pilot clinical experiment on animal (dog) patients of a local clinic of animal surgery suggested good healing properties of the composite and its transformation into new bone tissue within critical-size defect.ConclusionsThe results obtained in this study confirm that flexible HAp/glucan composite has potential as a bone-substituting material. Promising results of pilot clinical experiment suggest that further in vivo experiments should be performed.

Biphasic mode of antibacterial action of aminoglycoside antibiotics-loaded elastic hydroxyapatite–glucan composite

Following the quest for new composite materials for bone tissue engineering, a novel elastic hydroxyapatite-glucan composite loaded with two aminoglycoside antibiotics was prepared. The porosity of the composite and the drug release profiles in closed-loop and semi-open systems were tested. The antibacterial activity of the drug was estimated against two Gram-positive and two Gram-negative bacterial strains causing orthopedic infections. It was found that the loaded antibiotic acted in a biphasic mode. The majority of the drug was released within 48-119 h in a pore-dependent manner and inhibited the bacterial growth in the culture medium. However, a small residual amount of the drug was bound to the composite microstructure via ionic interactions and acted as a short-lived barrier against bacterial adhesion to the composite, although the surrounding medium was no longer protected against bacterial infection. Sub-inhibitory concentrations of the released drug were observed in the medium only during the last two days of the experiment (minimized risk of occurrence of drug-resistant strains). Thus the novel drug-loaded elastic hydroxyapatite-glucan composite, demonstrating a biphasic mode of antibacterial action, may be recommended for antibiotic prophylaxis in bone substitute implantation, with less emphasis on the treatment of bone infections.

Cytocompatibility of the selected calcium phosphate based bone cements: comparative study in human cell culture

Calcium phosphate cements (CPC) are valuable bone fillers. Recently they have been also considered as the basis for drug-, growth factors- or cells-delivery systems. Broad possibilities to manipulate CPC composition provide a unique opportunity to obtain materials with a wide range of physicochemical properties. In this study we show that CPC composition significantly influences cell response. Human bone derived cells were exposed to the several well-characterized different cements based on calcium phosphates, magnesium phosphates and calcium sulfate hemihydrate (CSH). Cell viability assays, live/dead staining and real-time observation of cells in contact with the materials (time-laps) were performed. Although all the investigated materials have successfully passed a standard cytocompatibility assay, cell behavior in a direct contact with the materials varied depending on the material and the experimental system. The most recommended were the α-TCP-based materials which proved suitable as a support for cells in a direct contact. The materials which caused a decrease of calcium ions concentration in culture induced the negative cell response, however this effect might be expected efficiently compensated in vivo. All the materials consisting of CSH had negative impact on the cells. The obtained results strongly support running series of cytocompatibility studies for preclinical evaluation of bone cements.

Comparative in vitro study of calcium phosphate ceramics for their potency as scaffolds for tissue engineering

BACKGROUND Calcium phosphate ceramics have been widely considered as scaffolds for bone tissue engineering. Selection of the best support for cultured cells, crucial for tissue engineered systems, is still required. OBJECTIVE We examined three types of calcium phosphate compounds: α-tricalcium phosphate - the most soluble one, carbonate hydroxyapatite - chemically the most similar to the bone mineral and biphasic calcium phosphate - with the best in vivo biocompatibility in order to select the best support for osteoblastic cells for tissue engineered systems. METHODS Human osteoblasts were tested in direct contact with both dense samples and 3D scaffolds in either static or dynamic culture. Cell viability, cell spreading, osteogenic cell capacity, and extracellular matrix production were examined. RESULTS The obtained data indicate that biphasic calcium phosphate is the optimal cell-supporting material. In addition, dynamic culture improved cell distribution in the scaffolds, enhanced production of the extracellular matrix and promoted cells osteogenic capacity. CONCLUSIONS Biphasic calcium phosphate should be recommended as the most suitable matrix for osteogenic cells expansion and differentiation in tissue engineered systems.

How calcite and modified hydroxyapatite influence physicochemical properties and cytocompatibility of alpha-TCP based bone cements

Nowadays successful regeneration of damaged bone tissue is a major problem of the reconstructive medicine and tissue engineering. Recently a great deal of attention has been focused on calcium phosphate cements (CPCs) as the effective bone fillers. Despite a number of studies regarding CPCs, only a few compare the physicochemical and biological properties of α-TCP based materials of various phase compositions. In our study we compared the effect of several components (calcite, hydroxyapatite doped with Mg2+, CO32− or Ag+ ions, alginate, chitosan and methylcellulose) on the physicochemical and biological properties of α-TCP-based bone cements. The influence of materials composition on their setting times, microstructure and biochemical stability in simulated body fluid was determined. A number of in vitro laboratory methods, including ICP-OES, metabolic activity test, time-lapse microscopic observation and SEM observations were performed in order to assess biocompatibility of the studied biomaterials. The positive outcome of XTT tests for ceramic extracts demonstrated that all investigated cement-type composites may be considered cytocompatible according to ISO 10993-5 standard. Results of our research indicate that multiphase cements containing MgCHA, AgHA and calcite combined with αTCP enhanced cell viability in comparison to material based only on αTCP. Furthermore materials containing chitosan and methylcellulose possessed higher cytocompatibility than those with alginate.Graphical abstract

Novel self-gelling injectable hydrogel/alpha-tricalcium phosphate composites for bone regeneration : physiochemical and microcomputer tomographical characterization

Mineralized hydrogels are increasingly gaining attention as biomaterials for bone regeneration. The most common mineralization strategy has been addition of preformed inorganic particles during hydrogel formation. This maintains injectability. One common form of bone cement is formed by mixing particles of the highly reactive calcium phosphate alpha-tricalcium phosphate (α-TCP) with water to form hydroxyapatite (HA). The calcium ions released during this reaction can be exploited to crosslink anionic, calcium-binding polymers such as the polysaccharide gellan gum (GG) to induce hydrogel formation. In this study, three different amounts of α-TCP particles were added to GG polymer solution to generate novel, injectable hydrogel-inorganic composites. Distribution of the inorganic phase in the hydrogel was studied by high resolution microcomputer tomography (µCT). Gelation occurred within 30 min. α-TCP converted to HA. µCT revealed inhomogeneous distribution of the inorganic phase in the composites. These results demonstrate the potential of the composites as alternatives to traditional α-TCP bone cement and pave the way for incorporation of biologically active substances and in vitro and in vivo testing.

Effects of Mg Additives on Properties of Mg-Doped Hydroxyapatite Ceramics

In the studies undoped HA and HA modified with 0.3; 0.6; 0.9; 1.8 wt % of Mg were prepared by the wet method. Introduction of magnesium into HA structure influenced its thermal stability as well as phase composition, sinterability, microstructure, flexural strength and chemical stability of the obtained calcium-phosphate ceramics. The presence of magnesium promoted the decomposition of HA to βTCP above 800°C. Beyond a certain limit (0.9 wt %), Mg ions caused formation of MgO in Mg-HA ceramics. Chemical stability of Mg modified HA below 0.9 wt % Mg under in vitro conditions was similar to that of the undoped hydroxyapatite. Biological studies showed that the number of cells cultured on the surface of HA samples with 1.8 wt % Mg additive, probably due to the MgO content, was lower than on the pure HA ceramics.

Novel self-gelling injectable hydrogel/alpha-TCP composites for bone regeneration: physiochemical and micro-computer tomographical characterization

Mineralized hydrogels are increasingly gaining attention as biomaterials for bone regeneration. The most common mineralization strategy has been addition of preformed inorganic particles during hydrogel formation. This maintains injectability. One common form of bone cement is formed by mixing particles of the highly reactive calcium phosphate alpha-tricalcium phosphate (α-TCP) with water to form hydroxyapatite (HA). The calcium ions released during this reaction can be exploited to crosslink anionic, calcium-binding polymers such as the polysaccharide gellan gum (GG) to induce hydrogel formation. In this study, three different amounts of α-TCP particles were added to GG polymer solution to generate novel, injectable hydrogel-inorganic composites. Distribution of the inorganic phase in the hydrogel was studied by high resolution microcomputer tomography (µCT). Gelation occurred within 30 min. α-TCP converted to HA. µCT revealed inhomogeneous distribution of the inorganic phase in the composites. These results demonstrate the potential of the composites as alternatives to traditional α-TCP bone cement and pave the way for incorporation of biologically active substances and in vitro and in vivo testing.

Drug Release from Hydroxyapatite Implants with Different Microstructure and Phase Composition

The goal of our studies has been to determine under in vitro conditions the amount and rate of pentoxifylline release from the samples of modified hydroxyapatite [HAp-Ca10(PO4)6(OH)2] implants in the form of microporous blocks (heterogeneous system) as well as from hydroxyapatitegypsum pellets (homogeneous system). For the preparation of microporous hydroxapatite ceramics additives of calcium metaphosphate Ca(PO3)2 (5 and 10 wt. %) or hydrated magnesium orthophosphate Mg3(PO4)2·8H2O (10 wt. %) were used as modifiers. In the case of drug release from heterogeneous carriers, cylinders filled with 50 mg of PTX were used. In the homogeneous system the pellets made of HAp and CaSO4·1/2H2O powders with homogeneously incorporated of 50 mg PTX were applied. It has been shown that the process of drug release from multifunctional ceramic implants depends to a significant degree on the microstructure of the materials, and the type of carrier system (heterogeneous or homogeneous).

Novel self-gelling injectable hydrogel/alpha-tricalcium phosphate composites for bone regeneration: Physiochemical and microcomputer tomographical characterization [in press]

Mineralized hydrogels are increasingly gaining attention as biomaterials for bone regeneration. The most common mineralization strategy has been addition of preformed inorganic particles during hydrogel formation. This maintains injectability. One common form of bone cement is formed by mixing particles of the highly reactive calcium phosphate alpha-tricalcium phosphate (α-TCP) with water to form hydroxyapatite (HA). The calcium ions released during this reaction can be exploited to crosslink anionic, calcium-binding polymers such as the polysaccharide gellan gum (GG) to induce hydrogel formation. In this study, three different amounts of α-TCP particles were added to GG polymer solution to generate novel, injectable hydrogel-inorganic composites. Distribution of the inorganic phase in the hydrogel was studied by high resolution microcomputer tomography (µCT). Gelation occurred within 30 min. α-TCP converted to HA. µCT revealed inhomogeneous distribution of the inorganic phase in the composites. These results demonstrate the potential of the composites as alternatives to traditional α-TCP bone cement and pave the way for incorporation of biologically active substances and in vitro and in vivo testing.