The Influence of Citrus Pectin and Polyacrylamide Modified with Plant-Derived Additives on the Properties of α-TCP-Based Bone Cements.

Materials based on highly reactive α-tricalcium phosphate (α-TCP) powder were developed and evaluated. Furthermore, the impact of different polymeric additives, such as citrus pectin or polyacrylamide (PAAM) modified with sago starch, neem flower, or rambutan peel, on the physiochemical and biological properties of the developed materials was assessed. The addition of modified PAAM shortened the setting process of bone cements and decreased their compressive strength. On the other hand, the addition of citrus pectin significantly enhanced the mechanical strength of the material from 4.46 to 7.15 MPa. The improved mechanical properties of the bone cement containing citrus pectin were attributed to the better homogenization of cementitious pastes and pectin cross-linking by Ca2+ ions. In vitro tests performed on L929 cells showed that 10% extracts from α-TCP cements modified with pectin are more cytocompatible than control cements without any additives. Cements containing PAAM with plant-derived modifiers show some degree of cytotoxicity for the highly concentrated 10% extracts, but for diluted extracts, cytotoxicity was reduced, as shown by a resazurin reduction test and live/dead staining. All the developed bone substitutes exhibited in vitro bioactivity, making them promising candidates for further biological studies. This research underscores the advantageous properties of the obtained biomaterials and paves the way for subsequent more advanced in vitro and in vivo investigations.

Physicochemical Properties of Inorganic and Hybrid Hydroxyapatite-Based Granules Modified with Citric Acid or Polyethylene Glycol.

This study delves into the physicochemical properties of inorganic hydroxyapatite (HAp) and hybrid hydroxyapatite-chitosan (HAp-CTS) granules, also gold-enriched, which can be used as aggregates in biomicroconcrete-type materials. The impact of granules' surface modifications with citric acid (CA) or polyethylene glycol (PEG) was assessed. Citric acid modification induced increased specific surface area and porosity in inorganic granules, contrasting with reduced parameters in hybrid granules. PEG modification resulted in a slight increase in specific surface area for inorganic granules and a substantial rise for hybrid granules with gold nanoparticles. Varied effects on open porosity were observed based on granule type. Microstructural analysis revealed increased roughness for inorganic granules post CA modification, while hybrid granules exhibited smoother surfaces. Novel biomicroconcretes, based on α-tricalcium phosphate (α-TCP) calcium phosphate cement and developed granules as aggregates within, were evaluated for compressive strength. Compressive strength assessments showcased significant enhancement with PEG modification, emphasizing its positive impact. Citric acid modification demonstrated variable effects, depending on granule composition. The incorporation of gold nanoparticles further enriched the multifaceted approach to enhancing calcium phosphate-based biomaterials for potential biomedical applications. This study demonstrates the pivotal role of surface modifications in tailoring the physicochemical properties of granules, paving the way for advanced biomicroconcretes with improved compressive strength for diverse biomedical applications.

The influence of silane coupling agents on the properties of α-TCP-based ceramic bone substitutes for orthopaedic applications.

Biomaterials based on α-TCP are highly recommended for medical applications due to their ability to bond chemically with bone tissue. However, in order to improve their physicochemical properties, modifications are needed. In this work, novel, hybrid α-TCP-based bone cements were developed and examinated. The influence of two different silane coupling agents (SCAs) - tetraethoxysilane (TEOS) and 3-glycidoxypropyl trimethoxysilane (GPTMS) on the properties of the final materials was investigated. Application of modifiers allowed us to obtain hybrid materials due to the presence of different bonds in their structure, for example between calcium phosphates and SCA molecules. The use of SCAs increased the compressive strength of the bone cements from 7.24 ± 0.35 MPa to 12.17 ± 0.48 MPa. Moreover, modification impacted the final setting time of the cements, reducing it from 11.0 to 6.5 minutes. The developed materials displayed bioactive potential in simulated body fluid. Presented findings demonstrate the beneficial influence of silane coupling agents on the properties of calcium phosphate-based bone substitutes and pave the way for their further in vitro and in vivo studies.

Novel Double Hybrid-Type Bone Cements Based on Calcium Phosphates, Chitosan and Citrus Pectin.

In this work, the influence of the liquid phase composition on the physicochemical properties of double hybrid-type bone substitutes was investigated. The solid phase of obtained biomicroconcretes was composed of highly reactive α-tricalcium phosphate powder (α-TCP) and hybrid hydroxyapatite/chitosan granules (HA/CTS). Various combinations of disodium phosphate (Na2HPO4) solution and citrus pectin gel were used as liquid phases. The novelty of this study is the development of double-hybrid materials with a dual setting system. The double hybrid phenomenon is due to the interactions between polycationic polymer (chitosan in hybrid granules) and polyanionic polymer (citrus pectin). The chemical and phase composition (FTIR, XRD), setting times (Gillmore needles), injectability, mechanical strength, microstructure (SEM) and chemical stability in vitro were studied. The setting times of obtained materials ranged from 4.5 to 30.5 min for initial and from 7.5 to 55.5 min for final setting times. The compressive strength varied from 5.75 to 13.24 MPa. By incorporating citrus pectin into the liquid phase of the materials, not only did it enhance their physicochemical properties, but it also resulted in the development of fully injectable materials featuring a dual setting system. It has been shown that the properties of materials can be controlled by using the appropriate ratio of citrus pectin in the liquid phase.

Study on ßTCP/P(3HB) Scaffolds-Physicochemical Properties and Biological Performance in Low Oxygen Concentration.

The search for new materials for bone regenerative purposes is still ongoing. Therefore, we present a series of newly constructed composites based on ß tricalcium phosphate (ßTCP) and poly(3-hydroxybutyrate) bacteria-derived biopolymer (P(3HB)) in the form of 3D scaffolds with different pore sizes. To improve the polymer attachment to the ßTCP surface, the etching of ceramic sinters, using citric acid, was applied. As expected, pre-treatment led to the increase in surface roughness and the creation of micropores facilitating polymer adhesion. In this way, the durability and compressive strength of the ceramic-polymer scaffolds were enhanced. It was confirmed that P(3HB) degrades to 3-hydroxybutyric acid, which broadens applications of developed materials in bone tissue engineering as this compound can potentially nourish surrounding tissues and reduce osteoporosis. Moreover, to the best of our knowledge, it is one of the first studies where the impact of ßTCP/P(3HB) scaffolds on mesenchymal stem cells (MSCs), cultured in lowered (5%) oxygen concentration, was assessed. It was decided to use a 5% oxygen concentration in the culture to mimic the conditions that would be found in damaged bone in a living organism during regeneration. Scaffolds enabled cell migration and sufficient flow of the culture medium, ensuring high cell viability. Furthermore, in composites with etched ßTCP, the MSCs adhesion was facilitated by hydrophilic ceramic protrusions which reduced hydrophobicity. The developed materials are potential candidates for bone tissue regeneration. Nevertheless, to confirm this hypothesis, in vivo studies should be performed.

Influence of Natural Polysaccharides on Properties of the Biomicroconcrete-Type Bioceramics.

In this paper, novel hybrid biomicroconcrete-type composites were developed and investigated. The solid phase of materials consisted of a highly reactive α -tricalcium phosphate (α-TCP) powder, hybrid hydroxyapatite-chitosan (HAp-CTS) material in the form of powder and granules (as aggregates), and the polysaccharides sodium alginate (SA) or hydroxypropyl methylcellulose (HPMC). The liquid/gel phase in the studied materials constituted a citrus pectin gel. The influence of SA or HPMC on the setting reaction, microstructure, mechanical as well as biological properties of biomicroconcretes was investigated. Studies revealed that manufactured cement pastes were characterized by high plasticity and cohesion. The dual setting system of developed biomicroconcretes, achieved through α-TCP setting reaction and polymer crosslinking, resulted in a higher compressive strength. Material with the highest content of sodium alginate possessed the highest mechanical strength (~17 MPa), whereas the addition of hydroxypropyl methylcellulose led to a subtle compressive strength decrease. The obtained biomicroconcretes were chemically stable and characterized by a high bioactive potential. The novel biomaterials with favorable physicochemical and biological properties can be prosperous materials for filling bone tissue defects of any shape and size.

Silver Decorated ßTCP-Poly(3hydroxybutyrate) Scaffolds for Bone Tissue Engineering.

Implantations in orthopedics are associated with a high risk of bacterial infections in the surgery area. Therefore, biomaterials containing antibacterial agents, such as antibiotics, bactericidal ions or nanoparticles have been intensively investigated. In this work, silver decorated ß tricalcium phosphate (ßTCP)-based porous scaffolds were obtained and coated with a biopolymer-poly(3-hydroxybutyrate)-P(3HB). To the best of our knowledge, studies using silver-doped ßTCP and P(3HB), as a component in ceramic-polymer scaffolds for bone tissue regeneration, have not yet been reported. Obtained materials were investigated by high-temperature X-ray diffraction, X-ray fluorescence, scanning electron microscopy with energy dispersive spectroscopy, hydrostatic weighing, compression tests and ultrahigh-pressure liquid chromatography with mass spectrometry (UHPLC-MS) measurements. The influence of sintering temperature (1150, 1200 °C) on the scaffolds' physicochemical properties (phase and chemical composition, microstructure, porosity, compressive strength) was evaluated. Materials covered with P(3HB) possessed higher compressive strength (3.8 ± 0.6 MPa) and surgical maneuverability, sufficient to withstand the implantation procedures. Furthermore, during the hydrolytic degradation of the composite material not only pure (R)-3-hydroxybutyric acid but also its oligomers were released which may nourish surrounding tissues. Thus, obtained scaffolds were found to be promising bone substitutes for use in non-load bearing applications.

Effect of Gold Nanoparticles and Silicon on the Bioactivity and Antibacterial Properties of Hydroxyapatite/Chitosan/Tricalcium Phosphate-Based Biomicroconcretes.

Bioactive, chemically bonded bone substitutes with antibacterial properties are highly recommended for medical applications. In this study, biomicroconcretes, composed of silicon modified (Si-αTCP) or non-modified α-tricalcium phosphate (αTCP), as well as hybrid hydroxyapatite/chitosan granules non-modified and modified with gold nanoparticles (AuNPs), were designed. The developed biomicroconcretes were supposed to combine the dual functions of antibacterial activity and bone defect repair. The chemical and phase composition, microstructure, setting times, mechanical strength, and in vitro bioactive potential of the composites were examined. Furthermore, on the basis of the American Association of Textile Chemists and Colorists test (AATCC 100), adapted for chemically bonded materials, the antibacterial activity of the biomicroconcretes against S. epidermidis, E. coli, and S. aureus was evaluated. All biomicroconcretes were surgically handy and revealed good adhesion between the hybrid granules and calcium phosphate-based matrix. Furthermore, they possessed acceptable setting times and mechanical properties. It has been stated that materials containing AuNPs set faster and possess a slightly higher compressive strength (3.4 ± 0.7 MPa). The modification of αTCP with silicon led to a favorable decrease of the final setting time to 10 min. Furthermore, it has been shown that materials modified with AuNPs and silicon possessed an enhanced bioactivity. The antibacterial properties of all of the developed biomicroconcretes against the tested bacterial strains due to the presence of both chitosan and Au were confirmed. The material modified simultaneously with AuNPs and silicon seems to be the most promising candidate for further biological studies.

Vimentin Cytoskeleton Architecture Analysis on Polylactide and Polyhydroxyoctanoate Substrates for Cell Culturing.

Polylactide (PLA), widely used in bioengineering and medicine, gained popularity due to its biocompatibility and biodegradability. Natural origin and eco-friendly background encourage the search of novel materials with such features, such as polyhydroxyoctanoate (P(3HO)), a polyester of bacterial origin. Physicochemical features of both P(3HO) and PLA have an impact on cellular response 32, i.e., adhesion, migration, and cell morphology, based on the signaling and changes in the architecture of the three cytoskeletal networks: microfilaments (F-actin), microtubules, and intermediate filaments (IF). To investigate the role of IF in the cellular response to the substrate, we focused on vimentin intermediate filaments (VIFs), present in mouse embryonic fibroblast cells (MEF). VIFs maintain cell integrity and protect it from external mechanical stress, and also take part in the transmission of signals from the exterior of the cell to its inner organelles, which is under constant investigation. Physiochemical properties of a substrate have an impact on cells' morphology, and thus on cytoskeleton network signaling and assembly. In this work, we show how PLA and P(3HO) crystallinity and hydrophilicity influence VIFs, and we identify that two different types of vimentin cytoskeleton architecture: network "classic" and "nutshell-like" are expressed by MEFs in different numbers of cells depending on substrate features.

Development of highly porous calcium phosphate bone cements applying nonionic surface active agents.

A novel way of obtaining highly porous cements is foaming them with the use of nonionic surface active agents (surfactants). In this study, foamed calcium phosphate cements (fCPCs) intended for in situ use were fabricated by a surfactant-assisted foaming process. Three different surface active agents, Tween 20, Tween 80 and Tetronic 90R4, were used. The amount of surfactant, based on its critical micelle concentration and cytotoxicity as well as foaming method, was determined. It has been established that in order to avoid cytotoxic effects the concentration of all applied surfactants in the cement liquid phases should not exceed 1.25 g L-1. It was found that Tetronic 90R4 had the lowest cytotoxicity whereas Tween 20 had the highest. The influence of the type of surfactant used in the fabrication process of bioactive macroporous cement on the physicochemical and biological properties of fCPCs was studied. The obtained materials reached higher than 50 vol% open porosity and possessed compressive strength which corresponds to the values for cancellous bone. The highest porosity and compressive strength was found for the material with the addition of Tween 80. In vitro investigations proved the chemical stability and high bioactive potential of the examined materials.

Physicochemical and Biological Characterisation of Diclofenac Oligomeric Poly(3-hydroxyoctanoate) Hybrids as ß-TCP Ceramics Modifiers for Bone Tissue Regeneration.

Nowadays, regenerative medicine faces a major challenge in providing new, functional materials that will meet the characteristics desired to replenish and grow new tissue. Therefore, this study presents new ceramic-polymer composites in which the matrix consists of tricalcium phosphates covered with blends containing a chemically bounded diclofenac with the biocompatible polymer-poly(3-hydroxyoctanoate), P(3HO). Modification of P(3HO) oligomers was confirmed by NMR, IR and XPS. Moreover, obtained oligomers and their blends were subjected to an in-depth characterisation using GPC, TGA, DSC and AFM. Furthermore, we demonstrate that the hydrophobicity and surface free energy values of blends decreased with the amount of diclofenac modified oligomers. Subsequently, the designed composites were used as a substrate for growth of the pre-osteoblast cell line (MC3T3-E1). An in vitro biocompatibility study showed that the composite with the lowest concentration of the proposed drug is within the range assumed to be non-toxic (viability above 70%). Cell proliferation was visualised using the SEM method, whereas the observation of cell penetration into the scaffold was carried out by confocal microscopy. Thus, it can be an ideal new functional bone tissue substitute, allowing not only the regeneration and restoration of the defect but also inhibiting the development of chronic inflammation.

Comparative study on physicochemical properties of alpha-TCP / calcium sulphate dihydrate biomicroconcretes containing chitosan, sodium alginate or methylcellulose.

PURPOSE: Recently, the attention has been drawn to complex systems - biomicroconcretes composed of a bone cement matrix and resorbable granules or microspheres. This paper presents novel bone substitutes composed of α-tricalcium phosphate (α-TCP; cement matrix), calcium sulphate dihydrate granules (GCSD; aggregates in biomicroconcrete) and various polymers (chitosan, sodium alginate, methylcellulose) used for the improvement of material properties. The aim of this work was to study α-TCP-GCSD-polymer interactions and to compare the impact of organic additives on the physicochemical properties of biomicroconcretes. METHODS: Scanning electron microscopy (SEM), mercury intrusion porosimetry (MIP), X-ray diffractometry (XRD) as well as universal testing machine (INSTRON), Gilmore apparatus and pH/ conduct-meter were used. RESULTS: The chemical bonding between α-TCP matrix and CSD granules resulted in a compressive strength appropriate for low-load bearing applications (7-12 MPa) and clinically relevant setting times (8-33 min). Biomicroconcretes consisting of sodium alginate possessed the highest mechanical strength (12 ± 2 MPa). It has also been found that the dissolution-precipitation reactions of the α-TCP were retarded with the addition of chitosan and acetic acid. This effect was not observed in the case of methylcellulose and sodium alginate. Chemical stability and bioactivity of materials were demonstrated during in vitro studies in simulated body fluid. CONCLUSIONS: Materials containing calcium sulphate-based granules were surgically handy, possessed promising physicochemical properties and are supposed to ensure desired macroporosity as well as gradual resorption in vivo. It has been demonstrated that the presence of CSD granules and polymers influenced the physicochemical properties of composites.

In vivo behavior of biomicroconcretes based on α-tricalcium phosphate and hybrid hydroxyapatite/chitosan granules and sodium alginate.

The current studies provide insights into how predictions based on results of physicochemical and in vitro tests are consistent with the results of in vivo studies. The new biomicroconcrete type materials were obtained by mixing the solid phase, composed of hybrid hydroxyapatite/chitosan granules and highly reactive α-tricalcium phosphate powder, used as the setting agent. This approach guaranteed a good adhesion of the continuous cement phase to the surface of granules. It has been demonstrated that developed biomicroconcretes are surgically handy, possessed favorable physicochemical and biological properties and can be considered as effective bone implant material. The hierarchical porosity and compressive strength (2-6 MPa) similar to cancellous bone made them suitable for low-load bearing applications. Despite the fact that final setting times of biomicroconcretes were longer than recommended in the literature (i.e., exceeded 15 min), their short cohesion time allows for a successful implantation in a rabbit femoral defect model. Histological analysis and Raman studies revealed newly formed bone tissues around the sides of implanted materials. Furthermore, the process of neovascularization and reconstruction of the bone tissue, as well as a reverse scaffolding process, was visible. No signs of inflammation or adverse tissue reactions were observed during the experiment.

Influence of Selected Surfactants on Physicochemical Properties of Calcium Phosphate Bone Cements.

The influence of the three nonionic surface active agents such as Tween 20, Tween 80, and Tetronic 90R4 on hydrolysis, setting reaction, microstructure, and mechanical properties of alpha tricalcium phosphate (α-TCP) based materials was determined. The study revealed that the addition of any of the surfactants mentioned above slightly prolonged the setting time of the tested cements (up to 5 min). On the other hand, it was found that surfactants influence the long-term hydrolysis reaction. The addition of surfactants also affected the microstructure of the final materials, especially after incubation in a simulated body fluid. Surface active agents also had an impact on mechanical behavior of the obtained cements. Sorbitan esters, Tween 20 and Tween 80, decreased compressive strength in comparison to the reference material (6.56 ± 1.59 MPa) to 3.54 ± 1.18 and 3.68 ± 1.03 MPa, respectively. Interestingly, Tetronic 90R4, never used before as an additive to calcium phosphate bone cements (CPCs) caused a 2-fold increase of this value (up to 13.28 ± 1.59 MPa). All the developed materials exhibited bioactivity in vitro. The obtained results shed new light on surfactants as CPCs additives. They should not only be considered as foaming agent or binders, but also they deserve more attention as modifiers affecting the physicochemical properties of α-TCP based materials.

Novel multicomponent organic-inorganic WPI/gelatin/CaP hydrogel composites for bone tissue engineering.

The present work focuses on the development of novel multicomponent organic-inorganic hydrogel composites for bone tissue engineering. For the first time, combination of the organic components commonly used in food industry, namely whey protein isolate (WPI) and gelatin from bovine skin, as well as inorganic material commonly used as a major component of hydraulic bone cements, namely α-TCP in various concentrations (0-70 wt%) was proposed. The results showed that α-TCP underwent incomplete transformation to calcium-deficient hydroxyapatite (CDHA) during preparation process of the hydrogels. Microcomputer tomography showed inhomogeneous distribution of the calcium phosphate (CaP) phase in the resulting composites. Nevertheless, hydrogels containing 30-70 wt% α-TCP showed significantly improved mechanical properties. The values of Young's modulus and the stresses corresponding to compression of a sample by 50% increased almost linearly with increasing concentration of ceramic phase. Incomplete transformation of α-TCP to CDHA during preparation process of composites provides them high reactivity in simulated body fluid during 14-day incubation. Preliminary in vitro studies revealed that the WPI/gelatin/CaP composite hydrogels support the adhesion, spreading, and proliferation of human osteoblast-like MG-63 cells. The WPI/gelatin/CaP composite hydrogels obtained in this work showed great potential for the use in bone tissue engineering and regenerative medicine applications.

Novel bioresorbable tricalcium phosphate/polyhydroxyoctanoate (TCP/PHO) composites as scaffolds for bone tissue engineering applications.

Development of new composite materials for bone tissue engineering is a constantly growing field of medicine. Therefore there is a continuous need in creating novel materials that can not only regenerate the defected tissue but also nourish it while the healing process progresses. Here we present a novel type of composite material that fulfils these requirements. The study describes creation of a composite with macroporous bioceramic core that is infiltrated with a thin biopolymer layer. The ceramic component, namely tricalcium phosphate (TCP), due to its mechanistic and bioactive properties may promote new bone creation as shown through the in vitro studies. To the best of our knowledge the coating layer was created for the first time from a representative of bacterially derived medium chain length polyhydroxyalkanoate polymers (mcl-PHA), namely polyhydroxyoctanoate (PHO). This polymer layer not only profoundly changed the stress-strain characteristics of the bioceramic foam but also released (R)-3-hydroxyacids and their dimers/trimers to the investigated environment. In the manuscript we have in depth characterised these materials employing a set of basic procedures, through 3D structure reconstruction and finishing with prolonged in vitro experiments.

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. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 822-828, 2018.

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.

Quantitative stereological analysis of the highly porous hydroxyapatite scaffolds using X-ray CM and SEM.

BACKGROUND: Material properties of the scaffolds as well as their microstructure are vital in determining in vivo cellular response. Three-dimensional (3D), highly porous scaffolds are used in tissue engineering to provide a suitable microenvironment and to support regeneration of bone. Both pore sizes and their architecture, in particular interconnection density, impact functionality of scaffold during its biomedical applications. OBJECTIVE: In this paper a comparative study of the microstructure of highly porous hydroxyapatite scaffolds produced via gelcasting of foamed slurries and replication of polyurethane sponge were carried out. METHODS: Quantitative stereological analysis of the microstructure was conducted using transmission X-ray computed microtomography (µCT) and scanning electron microscopy (SEM). Application of the X-ray microtomography allowed obtaining the 2D cross-sectional images of examined samples, and then the 3D reflection of individual samples. RESULTS: In our studies we proved that the distribution of pores in HAp bioceramics can be controlled by selection of the manufacturing method. In the case of material produced by the gelcasting method, the porosity of the samples was about ∼78 vol.%, while for the method of replication of the porous organic matrix it was higher ∼84 vol.%. Application of gelcasting method resulted in bioceramics with the macropores ranging from 95 µm to 158 µm (the modal value of 120 µm). Furthermore, micropores of size 34 µm-60 µm - so called "windows", were observed on spherical macropores surfaces. In the case of replication of polyurethane sponge only macropores from 295 µm to 337 µm (the modal value of 300 µm) were obtained. Application of µCT and SEM give more information than classical mercury intrusion porosimetry in studies of porous bioceramics. Developed materials met the criteria for porous bone substitutes. CONCLUSIONS: The results of quantitative description of microstructure allowed determining the differences between porous hydroxyapatite bioceramics obtained via replication of porous organic matrix and gelcasting of foamed slurry. The stereological analysis demonstrated, that bioceramics prepared via gelling of foamed slurry has a lower pore size and grains (1.1-1.9 µm) than the material obtained by the method of replication of polyurethane sponge (2.1-2.3 µm). Based on morphological analysis the porosity of tested materials was determined. In the case of material produce by the gelcasting, porosity of the samples was about ∼78 vol.%, while for method of replication of the porous organic matrix the porosity was higher and constituted ∼84 vol.%. Furthermore, evaluated materials varied in porosity and the pore size distribution. It was stated that the method of gelcasting resulted in hydroxyapatite bioceramics with the macropores diameter (95-158 µm), micropores so called "windows" (34-60 µm) - observed on spherical macropores walls and micropores of size 0.6 µm-1.3 µm, which were visible in sintered areas. When the method of replication of polyurethane sponge was applied only macropores from 295 µm to 337 µm were obtained. The comparable values of shape factors such as elongation, curvature of pours boundary and convexity, confirmed that macropores in both studied series had similar shape.