Investigation of the influence of processing parameters on the properties of bioceramic materials based on calcium hydroxyapatite and calcium phosphate obtained by different sintering technique

Kalcijum-fosfatni biokeramički materiali na bazi kalcijum-hidroksiapatita (HAP) i trikalcijum-fosfata (TCP), zahvaljujući hemijskom sastavu, biokompatibilnosti, bioaktivnosti i osteokonduktivnosti imaju značajnu ulogu pri izradi koštanih implanta. Guste nanostrukturne i kontrolisano porozne forme biokeramičkih materijala na bazi hidroksiapatita i kalcijum-fosfata imaju veliku primenu u maksiofacialnoj, dentalnoj i ortopedskoj praksi. Pažljiva kontrola faznog sastava, veličine zrna, oblika i dimenzija pora, kao i mehaničkih svojstava sinterovanih biokeramičkih materijala jesu ključni faktori za dobijanje implanata optimalnih svojstava. Danas je ogroman broj istraživanja baziran upravo na definisanju optimalnih uslova procesiranja sa ciljem da se dobiju gusti i kontrolisano porozni biokeramički monofazni kalcijum-hidroksiapatitni i bifazni materijali na bazi HAP i TCP, pogodni za ugradnju u ljudski organizam. Cilj ove doktorske disertacije bio je proučavanje procesa formiranja gustih nanostrukturnih biokeramičkih materijala na bazi kalcijum-hidroksiapatita i kalcijum-fosfata mikrotalasnim sinterovanjem, spark plazma sinterovanjem i toplim presovanjem, polazeći od stehiometrijskih i kalcijum deficitarnih nanočestičnih prahova kalcijum-hidroksiapatita dobijenih modifikovanim precipitacionim sintezama. Ispitan je uticaj svojstava polaznih prahova HAP i parametara procesiranja na mikrostukturne i fazne karakteristike, kao i na mehanička svojstva dobijenih gustih sinterovanih biokeramičkih materijala. Definisan je uticaj mikrostrukturnih parametara na žilavost i tvrdoću dobijenih monofaznih i bifaznih biokeramičkih materijala. Nanoindentacijom je utvrđen uticaj veličine zrna i faznog sastava na nanomehaničke karakteristike gustih mikro i nanostrukturnih materijala na bazi HAP i HAP/β-TCP. Svojstva materijala dobijenih mikrotalasnim sinterovanjem, spark plazma sinterovanjem i toplim presovanjem upoređeni su sa svojstvima materijala dobijenih konvencionalnim sinterovanjem.Calcium phosphate bioceramics materials based on hydroxyapatite (HAP) and tricalcium phosphate (TCP), due to their chemical composition, excellent biocompatibility, bioactivity and osteoconduction have received considerable attention as suitable bioceramics for the manufacture of osseous implants. Dense nanostructured and controlled porous forms of hydroxyapatite and calcium phosphate are often used as reparation material in maxillofacial, dental and orthopedic surgery. Careful control of phase composition, grain size and shape and size of pores of sintered bioceramic materials in accordance with good mechanical properties can be key issue for a successful implant. A large number of studies were focused on determining the optimal conditions required to obtain dense or controlled porous HAP and HAP/TCP form suitable for incorporation in living bone. The aim of this PhD thesis was to investigate the processing of dense nanostructured pure hydroxyapatite and biphasic HAP/TCP bioceramics by microwave sintering, spark plasma sintering and hot pressing, starting from stoichiometric and calcium deficient nanosized HAP powders, obtained by two modified chemical precipitation syntheses. The effects of the processing conditions on the microstructure, phase composition and mechanical properties of the obtained materials based on HAP and TCP were investigated. The influence of microstructure and phase composition on the fracture toughness and hardness of sintered HAP and HAP/TCP bioceramics was also defined. The influence of the grain size and phase composition on the nanomechanical behaviour of dense nano and microstructured HAP and biphasic HAP/β-TCP bioceramic materials by nanoindentation was also investigated. The results obtained by microwave sintering, spark plasma sintering and hot pressing were compared with those obtained by conventional sintering methods..

Development of macroporous bioceramic materials based on multi-ion doped calcium-hydroxyapatite coated with chitosan

In recent years, scientists have been working on developing biocompatible materials that closely mimic the structure and properties of natural biological tissues for their application in hard tissue regeneration and controlled drug release. Human bones and teeth primarily consist of calcium-phosphate crystals with small amounts of various ions incorporated into their crystal structure. The aim of this study was to examine the possibility of processing macroporous bioceramic scaffolds based on calcium-hydroxyapatite (HAp) doped with magnesium (Mg), strontium (Sr) and fluorine (F) ions, subsequently coated with polymer chitosan. In this study, the doped HAp powder was synthesized by a hydrothermal process, and scaffolds were made using the sponge replica method, sintered and then coated with the chitosan. The influence of dopant ions and chitosan on the scaffold's microstructure, mechanical properties, bioactivity, cytotoxicity and drug release properties was examined. Energy dispersive spectroscopy confirmed that Mg and Sr are incorporated in all powder samples, while the presence of F was confirmed in samples synthesized with 1 and 2 mol.% F in the precursor solution. The phase composition of powders and scaffolds determined by X-ray analysis showed the presence of HAp and β-tricalcium phosphate phase (β-TCP) in scaffolds. In the compressive strength (CS) test, coated scaffolds showed significantly higher CS compared to uncoated scaffolds. Scanning electron microscopy was used to examine the morphology of nanostructured powders, microstructure, and the bioactivity of the scaffolds. The uncoated scaffolds showed satisfactory bioactivity after being immersed in simulated body fluid for 28 days, while lower bioactivity was observed in the coated scaffolds due to the slow degradation of chitosan. The synthesized scaffolds also demonstrated to have a positive impact on cell viability, even slightly stimulating the cell proliferation. Additionally, scaffolds were shown to successfully release drug. In concusion, the addition of ions and chitosan polymer significantly improved the properties of the obtained scaffolds, which indicates their potential application in tissue engineering and controlled drug release.Twenty-First Young Researchers’ Conference - Materials Science and Engineering: Program and the Book of Abstracts; November 29 – December 1, 2023, Belgrade, Serbi

The effect of hydrothermal synthesis parameters on cation-doped calciumhydroxyapatite

Calcium hydroxyapatite (HAP) presents the main mineral component of human bones and teeth, and thus is widely used bioceramic material for thehard tissue repair and regeneration. The biological HAP is never found pure in nature butdoped with multiple therapeutic ions, such as Cu, Mg, Sr, Zn, etc., which are found to play important roles in bone metabolism and growth. Hence, foreign cations have been introduced into the synthetic calcium hydroxyapatite, in order to induce a specific biological response after implementation, such as osteogenesis, angiogenesis, improved cell attachment and proliferation. However, the presence of the cations leads to the lattice distortion of the calcium-hydroxyapatite, resulting in different physico-chemical and mechanical properties. The hydrothermal synthesis of calcium hydroxyapatite leads to nanosized rod-like particles, which were found to possess properties close to those of the biological HAP. The aim of this study was to investigate the effect of hydrothermal synthesis parameters on physico-chemical and mechanical properties of mono- and binary cation-doped calcium hydroxyapatite by employing XRD, SEM and Hardness by Vickers tests. The temperature applied during the hydrothermal synthesis (150- 180 °C) was found to influence the hardness of the HAP based compacts sintered at 1200 °C.IX Serbian Ceramic Society Conference - Advanced Ceramics and Application : new frontiers in multifunctional material science and processing : program and the book of abstracts; September 20-21, 2021; Belgrad

The influence of the voltage on formation and morphology of hydroxyapatite/titanium composite coatings

This study focused on investigating the effects of voltage and deposition time on the formation and morphology of hydroxyapatite/titanium oxide coatings, which were obtained using an in-situ synthesis process. The coatings were prepared under various voltage conditions and deposition times to analyze their characteristics. To evaluate the morphology of the coatings, optical microscopy and field emission scanning electron microscopy (FE-SEM) techniques were employed. Additionally, Fourier transform infrared spectroscopy (FTIR) was utilized to confirm the presence of hydroxyapatite coatings on the substrate. The results revealed that an increase in voltage led to a more uniform coating. The voltage parameter had a significant impact on the morphology of the initial hydroxyapatite powder, causing the agglomerates to disperse and resulting in smaller particle sizes within the coating. Furthermore, the FTIR analysis provided solid evidence of the presence of hydroxyapatite within the obtained coatings. These findings emphasize the importance of voltage control and deposition time in the in-situ synthesis process for achieving desirable hydroxyapatite/titanium oxide coatings. Understanding the influence of these parameters on coating formation and morphology can contribute to the development of improved techniques for biomedical applications, such as enhanced osseointegration in orthopedic and dental implants

Examination of antimicrobial potential in natural isolates of lactobacillus casei/paracasei group

Cilj ove studije je izučavanje antimikrobnog potencijala 52 prirodna izolata vrste L. casei/paracasei. Učestalost gena koji kodiraju BacSJ (bacSJ2-8/bacSJ2-8i genski klaster), acidocin 8912 (acdT), ABC-transporter (abcT) i pomoćni protein (acc) su takođe izučavani. Genski klaster bacSJ2-8/bacSJ2-8i prisutan je kod 49 (94.23%), a acdT kod 41 (78.85%) od 52 testirana soja. Četrdeset sojeva (76.92%) poseduje oba analizirana gena. Interesantno je da samo 17 sojeva (32.69%) koji poseduju bacSJ2-8/bacSJ2-8i genski klaster i/ili acdT gen proizvode bakteriocine. Soj L. paracasei BGNK1-62 poseduje bacSJ2-8/bacSJ2-8i genski klaster, ali ne proizvodi bakteriocin BacSJ što je verovatno posledica nedostatka abcT i acc gena. Nakon transformacije soja BGNK1-62 konstruktom pA2A koji poseduje abcT i acc gene ostvarena je proizvodnja bakteriocina BacSJ. Osim toga, utvrđeno je da soj L. paracasei BGGR2-66 proizvodi nov bakteriocin označen kao BacGR, koji je biohemijski okarakterisan, a određena je i njegova N-terminalna sekvenca.The aim of this study was to investigate the antimicrobial potential of 52 natural isolates of Lactobacillus casei/paracasei. The incidence of relevant genes encoding BacSJ (bacSJ2-8/bacSJ2-8i gene cluster), acidocin 8912 (acdT), ABC-transporter (abcT) and accessory protein (acc) was also studied. These genes were found to be widespread amongst the analyzed L. casei/paracasei strains. The bacSJ2-8/bacSJ2-8i gene cluster was present in 49 (94.23%) and acdT in 41 (78.85%) of the 52 tested strains. Forty of these strains (76.92%) harbored both analyzed genes. Interestingly, only 17 strains (32.69%) with the bacSJ2-8/bacSJ2-8i gene cluster and/or the acdT gene showed bacteriocin production. Strain L. paracasei BGNK1-62 contained the bacSJ2-8/bacSJ2-8i gene cluster, but did not produce bacteriocin BacSJ possibly due to absence of the abcT and acc genes. Hence, these genes were introduced into BGNK1-62 by transformation with constructed plasmid pA2A, after which BacSJ was produced. In addition, it was found that L. paracasei BGGR2-66 produced new bacteriocin designated as BacGR that was biochemically characterized and its N- terminal sequence was determined

Synthesis, characterization and toxicity studies of gelatin modified zinc oxide nanoparticles

Nanostructured zinc oxides are promising materials for numerous biomedical applications where they can serve as therapeutic agents or tools for sensing and imaging. Despite their favorable properties, wider use of zinc oxide nanoparticles in biomedicine is limited by toxicity issues. Therefore, new synthesis approaches should be devised to obtain zinc oxide nanoparticles which are safe-by-design. We present an innovative low-cost wet precipitation synthesis of gelatin modified zinc oxide nanoparticles at the gel/liquid interface. The diffusion of ammonia through the gelatin hydrogels of different porosities induces precipitation of the product in contact with the surface of the aqueous solution of zinc ions. After thermal treatment of the precipitate, adsorbed organic residues of decomposed gelatin act as modifiers of zinc oxide nanoparticles. We characterized the physicochemical properties of obtained gelatin modified zinc oxide nanoparticles by XRD, FTIR, DTA/TG, and SEM. The synthesized nanoparticles show hexagonal wurtzite structure and form flakelike aggregates. FTIR and DTA/TG analyses indicate that the thermal decomposition of adsorbed gelatin depends on the gelatin content of the hydrogel used in the synthesis. We also examined the viability of HepG2 cells, generation of intracellular reactive oxygen species, and genotoxicity using the MTS, DCFH-DA, and alkaline comet assay, respectively. Fabricated gelatin modified zinc oxide nanoparticles show very low toxicity potential at doses relevant for human exposure

Arsenic removal from water using low-cost adsorbents: A comparative study

Inorganic arsenic removal from water using low-cost adsorbents is presented in this paper. Selective removal of As(III) and As(V) from water was performed with natural materials (zeolite, bentonite, sepiolite, pyrolusite and limonite) and industrial by-products (waste filter sand as a water treatment residual and blast furnace slag from steel production); all inexpensive and locally available. Kinetic and equilibrium studies were realized using batch system techniques under conditions that are likely to occur in real water treatment systems. The natural zeolite and the industrial by-products were found to be good and inexpensive sorbents for arsenic while bentonite and sepiolite clays showed little affinity towards arsenic. The highest maximum sorption capacities were obtained for natural zeolite, 4.07 mg As(V) g-1, and waste iron slag, 4.04 mg As(V) g-1

Mg/Cu co-substituted hydroxyapatite – Biocompatibility, mechanical properties and antimicrobial activity

The aim of this study was to improve the mechanical properties and to optimize antimicrobial activity of hydroxyapatite (HAP) by simultaneous doping with Mg and Cu ions in order to obtain material that would be able to assist in the bone/tooth healing process, prevent post-implementation infections and provide satisfying values of hardness and fracture toughness for biomedical application. Ion doping was done during the hydrothermal synthesis of HAP powders, whereby the content of Mg ions in the starting solution was varied between 1-20 mol. % with regard to Ca ions, while the amount of Cu ions was kept constant at 0.4 mol. %. The green compacts were sintered for 2 h at temperatures ranging 750–1200 °C depending on the Mg content, chosen in agreement with dilatometry results. Presence of Mg ions was found to favour transition from HAP to β−tricalcium phosphate phase (β−TCP), which enabled formation of biphasic HAP/β−TCP and pure β−TCP phase at 160 °C during hydrothermal synthesis. In vitro investigation of antimicrobial activity against Escherichia coli, Staphylococcus aureus and Enterococcus faecalis showed satisfactory antimicrobial activity. MTT assay performed on MRC-5 and L929 cell lines showed excellent cytocompatibility and cell proliferation. Maximum hardness by Vickers and fracture toughness values, 4.96 GPa and 1.75 MPa m1/2 respectively, were obtained upon addition of 5 mol. % Mg, as a consequence of the lowest grain size and porosity, as well as the highest densification rate. This is, to the best of our knowledge, the highest fracture toughness for HAP or β-TCP ceramics reported thus far.This is the peer-reviewed version ofthe article: Veljović, Đorđe, Matić, Tamara, Stamenić, Tanja, Kojić, Vesna, Dimitrijević Branković, Suzana, Lukić, Miodrag J., Jevtić, Sanja, Radovanović, Željko, Petrović, Rada, Janaćković, Đorđe, "Mg/Cu co-substituted hydroxyapatite – Biocompatibility, mechanical properties and antimicrobial activity" in Ceramics International, 45, no. 17, Part A (2019):22029, [https://doi.org/10.1016/j.ceramint.2019.07.219

BIOMIMETIC BIOREACTORS AS A TOOL FOR MORE RELEVANT BIOMATERIAL ASSESSMENT

Development of novel biomaterials for use in biomedical applications requires careful assessment due to the intended interactions with cells and tissues. Understanding biocompatibility, non-toxicity, and capability of promoting desired biological responses requires thorough characterization of biomaterial, including its chemical composition, surface properties, mechanical strength, degradation rate, etc. Traditional in vitro methods for evaluating biomaterials in cell monolayers are convenient but limited by the lack of specific biophysical signals found in vivo, which can lead to unreliable results. This in vitro-in vivo gap can result in the unnecessary sacrifice of a large number of animals for testing purposes. Therefore, there is a need for alternative approaches that beter mimic the in vivo environment and accurately predict the behavior of the biomaterial after implantation. Biomimetic bioreactors are primarily developed for tissue engineering to provide the key biochemical (e.g., nutrients, gases, growth factors) and biophysical signals (e.g., shear stress, hydrostatic pressure, mechanical strains) found in vivo and thus could be indispensable tools in physiologically relevant biomaterial assessment. Our group introduced the application of two biomimetic bioreactors for the physiologically relevant characterization of two types of composite biomaterials aimed for bone and osteochondral tissue engineering. In specific, macroporous composite scaffolds were produced using two natural polymers (gellan gum and alginate) as matrices imitating organic phase of bone tissue with incorporated particulate bioactive glass (BAG) and β-tricalcium phosphate (β-TCP) as hydroxyapatite (HAp) precursors. In addition, in osteochondral scaffolds, gellan gum hydrogel served as a cartilaginous layer on top of the porous composite base. Integrity and mechanical properties of all prepared scaffolds were monitored for 14 days under physiological levels of mechanical compression (up to 10% strain, compression rate 337.5 µm s-1) in a bioreactor with dynamic compression and medium perfusion. Bioactivity and HAp formation within the scaffolds were investigated in a perfusion bioreactor under the flow of simulated body fluid for up to 28 days. The scaffolds were assessed by SEM, EDS, and XRD analyses indicating a significant increase in HAp formation under bioreactor conditions as compared to static controls in all investigated samples. Moreover, the formed HAp crystals were more uniformly distributed throughout the scaffolds showing a more cauliflower-like morphology and thus, indicating potentials for bone/osteochondral tissue engineering applications. The obtained results confirm the high influence of experimental conditions on the outcomes of biomaterial characterization and importance of closely mimicking physiological conditions, thus puting forward biomimetic bioreactors as a means in this direction.Conference Abstracts / 33rd Annual Conference of the European Society for Biomaterials (ESB), 4-8 September, Davos, Switzerlan

Swelling behavior of synthesized poly(1-vinyl-2-pyrrolidone-co-vinyl acetate) hydrogels

Poly(1-vinyl-2-pyrrolidone-co-vinyl acetate) has great application in cosmetics, primarily in personal care products. It is used in drug delivery, and in a variety of biomedical applications. Also, significant is the application as an adsorbent of various pesticides. The aim of this paper is to examine structural characterization and the swelling behavior of synthesized poly(1-vinyl-2-pyrrolidone-co-vinyl acetate) copolymer hydrogels with 10 mol% of vinyl acetate and with 1.0; 1.5; 2.0; 2.5 and 3.0 mol% of cross-linker ethylene glycol dimethacrylate (EGDM). The characterization of the synthesized hydrogels was performed using FTIR spectroscopy. The swelling study was monitored gravimetrically until equilibrium was reached in solutions of different pH values (3.0, 6.0, 9.0) at a temperature of 25°C. The sensitivity of poly(1-vinyl-2-pyrrolidone-co-vinyl acetate) hydrogels to changes in external temperature was examined by monitoring the change in the equilibrium degree of swelling with increasing fluid temperature from 25ºC to 80ºC in a pH solution of 6.0. FTIR spectra of xerogels confirm the performed synthesis. Swelling of hydrogels poly(1-vinyl-2-pyrrolidone-co-vinyl acetate) was favored at a lower temperature (25°C), in a solution whose pH=6.0, when the sample with 1.0 mol% EGDM reaches the highest degree of swelling (αe=87.23) compared to the achieved capacity at a temperature of 80ºC (αe=20.74). The most intense phase transition was observed in the temperature range of 40-45°C. Based on the obtained results, the synthesized copolymers can be classified into negative thermosensitive hydrogels that have a lower critical dissolution temperature (LCST). pH sensitivity analysis led to the conclusion that the highest value of the equilibrium degree of swelling (αe=168.97) was observed in the hydrogel sample with 1.5 mol% EGDM in the solution whose pH=9 at 25°C. An increase in the molar content of cross-linkers in the hydrogels composition shows a decrease in swelling capacity at all pH values and temperaturesVIII International Conference on Radiation in Various Fields of Research : RAD 2020 : book of abstracts; Virtual Conferenc