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1

Mesenchymal stem cells proliferation and osteogenic differentiation on polymeric scaffolds and microspheres for bone tissue engineering

Walczak Kamila, Krok-Borkowicz Małgorzata, Pamuła Elżbieta

Engineering of Biomaterials

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2023

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vol. 26, no. 170

14--19

EN

In this study, we aimed to compare how the microstructure and architecture of polymer supports influence adhesion, growth and differentiation of human mesenchymal stem cells (hMSC) in the context of bone tissue engineering. We manufactured poly(L-lactide-co-glycolide) (PLGA) three-dimensional supports in the form of microspheres by emulsification and porous scaffolds by solvent casting/ porogen leaching. HMSC were seeded on both materials and on control tissue culture polystyrene (TCPS, bottom of the wells) and cultured in basal or osteogenic medium for 1, 3, 7 and 14 days. HMSC proliferation and osteogenic differentiation were studied using lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) assays, respectively. Furthermore, cell morphology and viability were analyzed after live/dead fluorescence staining. The results show that the optimized emulsification conditions allowed the production of PLGA microspheres with a median size of 95 µm. The PLGA scaffolds had a porosity of 82.1% ± 4.2% and a pore size of 360 µm ± 74 µm. HMSC cultured on control TCPS in osteogenic medium were more spread and polygonal than those in basal medium. They were characterized with a lower proliferation rate, as shown by the LDH results, but higher ALP activity. This suggests that hMSC osteogenic differentiation was achieved. The same tendency was observed for cells cultured on microspheres and scaffolds. Cell proliferation was more efficient on both materials and control in growth medium as compared to differentiation medium. The amount of ALP, i.e. a marker of osteogenic differentiation, was elevated, as expected, in differentiation medium. However, on day 14 cells cultured on the scaffolds in basal medium exhibited the same osteogenic potential as those cultured in differentiation medium. In general, both microspheres and scaffolds promoted hMSC adhesion, proliferation, and osteogenic differentiation and may be used for bone tissue engineering.

2

Fabrication and properties of βTCP/Zeolite/Gelatin scaffold as developed scaffold in bone regeneration: in vitro and in vivo studies

Yazdanian Mohsen, Tabesh Hadi, Houshmand Behzad, Tebyanian Hamid, Soufdoost Reza Sayyad, Tahmasebi Elahe, Karami Ali, Ghullame Sayede

Biocybernetics and Biomedical Engineering

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2020

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Vol. 40, no. 4

1626--1637

EN

Synthetic scaffolds, as an alternative to allograft and xenograft scaffolds, are suitable for bone regeneration. This study aimed to synthesize a composite biomaterial of zeolite and beta-tricalcium phosphate (bTCP) to obtain a biocompatible material with physical and mechanical properties in bone regeneration. One scaffold without zeolite (bZG 0) and two scaffolds with different amounts of zeolite (bZG 1 and bZG 2) were synthesized. The scaffolds were evaluated by FTIR, XRD, compressive strength test, MTT assay, and radiographic and histological analyses. The XRD results confirmed the presence of bTCP and ZSM-5 phases in the composite scaffolds and also, indicated that the addition of gelatin decrease the crystallinity of composite scaffolds. FTIR revealed the gelatin, b-TCP and ZSM-5 functional groups in the composite structure. bZG 2 group had the maximum porosity among the scaffolds (74%) ranging in size from 61-600 mm. Compressive strength test showed that the Young's modulus changed from 23 MPa to 59 MPa, and the zeolite nanostructure was the most influential factor responsible for this change. The MTT assay showed the superiority of bZG 2, and the macroscopic and microscopic results at 4, 8, and 12 weeks revealed the maximum bone regeneration and formation of bone trabeculae in the bZG 2 and bZG 1 groups, respectively. The zeolite scaffold showed the superior mechanical, radiographic and histological properties compared with the control and non-zeolite scaffold. bTCP/ Zeolite/ Gelatin scaffold can be an appropriate candidate for medical application in bone regeneration.

3

Osteoblasts response to novel chitosan/agarose/hydroxyapatite bone scaffold – studies on MC3T3-E1 and hFOB 1.19 cellular models

Kazimierczak Paulina, Vivcharenko Vladyslav, Truszkiewicz Wiesław, Wójcik Michał, Przekora Agata

Engineering of Biomaterials

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2019

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Vol. 22, no. 151

24--29

EN

Since it is known that various cell lines may ex-press different behaviours on the scaffolds surface, a comprehensive analysis using various cellular mo-dels is needed to evaluate the biomedical potential of developed biomaterials under in vitro conditions. Thus, the aim of this work was to fabricate bone scaffolds composed of a chitosan-agarose matrix reinforced with nanohydroxyapatite and compare the biological response of two cell lines, i.e. mouse calvarial preosteoblasts (MC3T3-E1 Subclone 4) and human foetal osteoblasts (hFOB 1.19). Within this study, the osteoblasts number on the scaffold surface and the osteogenic markers level produced by MC3T3-E1 and hFOB 1.19 cells were determined. Furthermore, changes in calcium and phosphorous ions concentrations in the culture media dedicated for MC3T3-E1 and hFOB 1.19 were estimated after the biomaterial incubation. The obtained results proved that the fabricated biomaterial is characterized by biocompatibility and osteoconductivity since it favours osteoblasts attachment and growth. It also supports the production of osteogenic markers (collagen, bALP, osteocalcin) by MC3T3-E1 and hFOB 1.19 cells. Interestingly, the developed biomaterial exhibits different ion reactivity values in the two culture media dedicated for the mentioned cell lines. It was also revealed that mouse and human osteoblasts differ in the cellular response to the fabricated scaffold. Thus, the use of at least two various cellular models is recommended to carry out a reliable biological characterization of the novel biomaterial. These results demonstrate that the tested bone scaffold is a promising biomaterial for bone regeneration applications, however further biological and physicochemical experiments are essential to fully assess its biomedical potential.

4

Surface functionalization of poly(L-lactide-co-glycolide) membranes with amphiphilic poly(2-oxazoline) for guided tissue regeneration and treatment of bone tissue defects

Tryba A. M., Krok-Borkowicz M., Paluszkiewicz C., Pamuła E.

Engineering of Biomaterials

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2018

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Vol. 21, no. 147

16--20

EN

The main challenge of this research was to functionalize the surface of poly(L-lactide-co-glycolide) (PLGA) membranes with amphiphilic poly(2-oxazoline) (POx) in order to change PLGA chemical state and properties. Poly(2-oxazolines) are very powerful polymers, which thanks to active pendant groups can be easily functionalized with biologically active molecules or peptides. The membranes were prepared by dissolving PLGA, POx, and poly(ethylene glycol) (PEG, 1000 Da) in methylene chloride (DCM), followed by PEG leaching. POx molecules were preferentially adsorbed at the interface PLGA-POx-PEG thanks to affinity to both hydrophilic (PEG) and hydrophobic (PLGA) chains. The properties of the membranes were characterized with Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and wettability tests. Cytocompatibility of the materials in contact with osteoblast-like MG-63 cells was studied by evaluation of cell viability (Alamar-Blue test), live/dead and phalloidin/DAPI staining. The results show that the presence of POx influenced topography of the PLGA membranes, but did not have an impact on their wettability. All membranes were fo-und cytocompatible with model osteoblasts. Presence of POx resulted in better cell adhesion as shown by microscopic studies after fluorescence staining for nuclei and cytoskeleton actin filaments. In summary, one-step phase separation process between PLGA, PEG, and POx, dissolved in DCM followed by drying and PEG leaching resulted in cytocompatible PLGA membranes with immobilised POx, which might be considered for guided tissue regeneration technique in periodontology and in bone tissue engineering.

5

Structural variability of lyophilized collagen-based scaffolds: micro-CT 3D analysis

Bartos M., Suchy T., Spanko M., Foltan R.

Engineering of Biomaterials

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2018

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Vol. 21, no. 148

24

6

Innovative macroporous chitosan/agarose matrix-based biomaterial for bone tissue engineering applications

Kazimierczak P., Pałka K., Ginalska G., Przekora A.

Engineering of Biomaterials

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2018

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Vol. 21, no. 148

13

7

Microstructure of chitosan-based, ZnO-doped antibacterial composite

Ciołek L., Biernat M., Jaegermann Z., Zaczyńska E., Czarny A., Jastrzębska A., Olszyna A.

Engineering of Biomaterials

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2017

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Vol. 20, no. 143 spec. iss.

18

8

Co-polymeric biomaterials for bone tissue engineering

Chatzinikolaidou M., Kaliva M., Papadimitriou L., Georgopoulou A., Mygdali ., Vamvakaki M.

Engineering of Biomaterials

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2017

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Vol. 20, no. 143 spec. iss.

6

9

The effect of zinc oxide doping on mechanical and biological properties of 3D printed calcium sulfate based scaffolds

Aldemir Dikici B., Dikici S., Karaman O., Oflaz H.

Biocybernetics and Biomedical Engineering

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2017

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Vol. 37, no. 4

733--741

EN

Fabrication of defect-matching scaffolds is the most critical step in bone tissue engineering. Three-dimensional (3D) printing is a promising technique for custom design scaffold fabrication due to the high controllability and design independency. The objective of this study is to investigate the effect of zinc oxide (ZnO) doping on mechanical and biological characteristics of 3D printed (3DP) calcium sulfate hemihydrate (CSHH) scaffolds. Crystalline phases, wettability, compressive strength and Young's modulus, human bone marrow derived mesenchymal stem cells (hMSCs) attachment, proliferation and morphology were investigated. XRD results showed that CSHH powder transformed into gypsum after the printing process due to the water content of binder. Contact angle measurements indicated that ZnO doped CSHH scaffolds have hydrophilic character, which stimulates cell attachment. The mechanical and cell culture studies demonstrated that increasing the ZnO doping concentration both mechanical strength and cell proliferation on CSHH scaffolds were enhanced.

10

The promise of composite polymers for bone tissue engineering

Amedee J.

Engineering of Biomaterials

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2016

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Vol. 19, no. 138 spec. iss.

7

EN

The repair of bone defects is of particular interest for orthopaedic, oral, maxillofacial, and dental surgery. Bone loss is conventionally reconstructed by bone grafting. Depending on size and location of the defect, this method has limits and risks. In addition, in the context of reconstruction of the craniofacial skeleton after radiation therapy, we need to improve therapeutic options for patients suffering from such disastrous sequelae of radiation therapy. While the use of BMPs has been approved for bone regeneration applications, their use is contraindicated in a carcinological context, due to concerns that these anabolic growth factors may contribute to tumor cell proliferation. Moreover, the main limitations are to regenerate a functional vasculature [1] and to restore bone innervation that also played a major role for bone tissue regeneration [2,3]. In such context, biomaterials such as calcium phosphate matrices, free of reparative cells, cannot offer sufficient potential for supporting especially vascularization of newly formed bone. Polymers and mainly composite based-polysaccharides, because of their versatility, their possible supplementation with a mineral phase (i.e hydroxyapatite particles), have immense potential for mimicking bone tissue, by trapping osteogenic and angiogenic factors and then promoting both osteogenesis and angiogenesis [4,5]. The other challenge in the field of bone tissue engineering is to favour anchorage of sensory neurons within 3D matrices that could produce neurotrophic factors [6], activate the coupling of osteogenesis and angiogenesis. Here, we will describe a cell-free approach for bone tissue engineering [7] using injectable composite polymers, their in vitro and in vivo validation in preclinical models from small to large animals. We will also show how composite polymer chemistry can also favour cell interactions between mesenchymal stem cells, endothelial cells and stimulate bone tissue regeneration.

11

Influence of chemical compositions on the structure and the bioactive properties of SiO2-CaO and SiO2-CaO-P2O5 bioactive glasses

Zagrajczuk B., Dziadek M., Cholewa-Kowalska K., Łączka M.

Engineering of Biomaterials

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2016

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Vol. 19, no. 138 spec. iss.

69

12

Evaluation of cellulose/hydroxyapatite scaffolds for bone tissue engineering: studies in vitro and in vivo

Liesiene J., Baniukaitiene O., Daugela P., Pranskunas M., Juodzbalys G., Babenko N.

Engineering of Biomaterials

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2016

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Vol. 19, no. 138 spec. iss.

73

13

Bioactive photocross-linkable hybrid materials for tissue engineering applications

Lewandowska-Łańcucka J., Mystek K., Mignon A., Vlierberghe S. van, Łatkiewicz A., Nowakowska M.

Engineering of Biomaterials

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2016

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Vol. 19, no. 138 spec. iss.

30

14

The influence of mineralization conditions on the effectiveness of enzymatic mineralization of hydrogels

Pietryga K., Reczyńska K., Pamuła E.

Engineering of Biomaterials

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2015

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Vol. 18, no. 131

2--7

EN

Polysaccharide hydrogels are widely used in food industry and medicine. Gellan gum (GG) recently gained a lot of attention as a promising material for tissue regeneration proposes due to its excellent biocompatibility and similarity to natural extracellular matrix. However, in unmineralized form it is not suitable for bone tissue engineering because of weak mechanical properties. Enzymatic mineralization (e.g. using alkaline phosphatase – ALP) is one of the methods of calcifying of hydrogels and it resembles natural processes occurring during bone healing. The aim of this research was to investigate mineralization of hydrogels and to improve properties of gellan gum scaffolds by adjusting processing conditions. Since ALP does not form with GG covalent bonds, during incubation in mineralization medium (solution of calcium glycerophosphate - CaGP) it is diffusing from the samples. Therefore, mineralization effectiveness depends on the interplay between incoming CaGP and outgoing ALP molecules. We hypothesize that better CaGP availability, especially in the first hours of incubation, can result in more effective and homogenous precipitation of calcium phosphates (CaP) in GG samples. To this end, samples with different GG and ALP concentration were subjected to two different mineralization regimes (more and less frequent CaGP exchanges). We proved that better CaGP availability (more frequent CaGP exchange) resulted in better mechanical properties (Young’s modulus) and more effective mineral formation (higher dry mass percentage) of the samples compared to the same samples mineralized with lower accessibility of CaGP. This may be related to the fact, that in presence of fresh organic substrates, more CaP are formed in the outer parts of the samples at the beginning of the process, that limit ALP diffusion and allow more uniform mineralization.

15

Effect of polylactide modification with β-TCP and lecithin on the properties of the material as a substrate for osteoblasts

Olkowski R., Stefanek A., Kaszczewski P., Ciach T., Lewandowska-Szumieł M., Kalaszczyńska I.

Engineering of Biomaterials

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2015

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Vol. 18, no. 131

8--11

EN

Polylactide (PLLA) containing β-TCP is biodegradable composite and an attractive biomaterial for bone tissue engineering, however, hydrophobicity of PLLA based composites is major limitation for their use as scaffolds for cell culture. In our study lecithin was used to improve hydrophilicity and cytocompatibility of PLLA/ β-TCP composite. Thin films of PLLA, PLLA/ β-TCP and PLLA/β-TCP/lecithin were manufactured by solvent-casting technique. Comparative analysis of all types of films was performed. Addition of β-TCP did not change hydrophilicity of PLLA. The hydrophilicity of PLLA/β-TCP/lecithin increased in comparison to PLLA and PLLA/β-TCP. Degradation of PLLA/β-TCP composite surpassed the degradation of PLLA while addition of lecithin diminished the degradation of composite. The cytocompatibility of composites were studied in 7 day long in vitro assay. Human bone derived cells were seeded on all tested surfaces. Cell viability was estimated by Live/Dead fluorescent staining and Alamar Blue test. Surprisingly, although lecithin addition improved hydrophilicity of the PLLA-based composite, adhesion and proliferation of human bone derived cells were markedly hampered on PLLA/β-TCP/lecithin in comparison to PLLA and PLLA/β-TCP. Despite positive effect we found of lecithin addition on hydrophilicity and stability of PLLA-based composite, its effect on cell attachment and proliferation is negative. Hence, incorporation of lecithin did not improve properties of PLLA/β-TCP/lecithin composite intended for bone tissue regeneration.

16

The influence of sintering conditions on microstructure and mechanical properties of titanium dioxide scaffolds for the treatment of bone tissue defects

Rumian Ł., Reczyńska K., Wrona M., Tiainen H., Haugen H. J., Pamuła E.

Acta of Bioengineering and Biomechanics

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2015

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Vol. 17, no. 1

3--9

EN

In this study the attempts to improve mechanical properties of highly-porous titanium dioxide scaffolds produced by polymer sponge replication method were investigated. Particularly the effect of two-step sintering at different temperatures on microstructure and mechanical properties (compression test) of the scaffolds were analysed. To this end microcomputed tomography and scanning electron microscopy were used as analytical methods. Our experiments showed that the most appropriate conditions of manufacturing were when the scaffolds were heat-treated at 1500 °C for 1 h followed by sintering at 1200 °C for 20 h. Such scaffolds exhibited the highest compressive strength which was correlated with the highest linear density and the lowest size of grains. Moreover, grain size distribution was narrower with predominating fraction of fine grains 10–20 μm in size. Smaller grains and higher linear density suggested that in this case densification process prevailed over undesirable process of grain coarsening, which finally resulted in improved mechanical properties of the scaffolds.

17

The influence of pre-coarsening on architectural and mechanical properties of highly porous titanium dioxide scaffolds for bone tissue engineering

Reczyńska K., Rumian Ł., Pamuła E., Haugen H. J., Tiainen H.

Engineering of Biomaterials

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2014

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Vol. 17, no. 128-129

96--98

18

Modyfikacja rusztowania kostnego chitosan/HA za pomocą β-1,3-glukanu znacząco poprawia jego biokompatybilność in vitro

Przekora A., Ginalska G.

Engineering of Biomaterials

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2014

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Vol. 17, no. 128-129

20--22

PL

Inżynieria tkankowa kości kładzie nacisk na produkcje trójwymiarowego, porowatego rusztowania, które posiadałoby zdolność stymulowania adhezji, proliferacji i różnicowania osteoblastów. Takie rusztowanie wspierałoby proces regeneracji i tworzenia funkcjonalnej tkanki kostnej [1-3]. Celem niniejszej pracy było udowodnienie za pomocą 2 linii osteoblastycznych, że dodatek β-1,3-glukanu do rusztowania na bazie chitosanu i hydroksyapatytu (chit/HA) skutkuje wytworzeniem nowego, trójskładnikowego kompozytu chitosan/β-1,3-glukan/hydroksyapatyt (chit/glu/HA), który posiada lepszą biokompatybilność w porównaniu do dwuskładnikowego materiału chit/HA. Trójskładnikowe rusztowanie wyprodukowano poprzez modyfikację kompozytu chit/HA za pomocą bakteryjnego β-1,3-glukanu jak to zostało opisane wcześniej [2,3]. Eksperymenty in vitro przeprowadzono z zastosowaniem linii komórkowej prawidłowych ludzkich płodowych osteoblastów (hFOB 1.19) oraz linii komórkowej mysich preosteoblastów (MC3T3-E1 Subclone 4). Cytotoksyczność materiałów oznaczono metodą kontaktu bezpośredniego za pomocą podwójnego barwienia fluorescencyjnego „żywe/martwe komórki”. Kalceina-AM barwi na zielono jedynie żywe komórki, natomiast jodek propidyny barwi kwasy nukleinowe martwych komórek emitując czerwoną fluorescencję jader komórkowych. Wybarwione komórki obserwowano w mikroskopie konfokalnym. Liczbę osteoblastów przyklejonych do powierzchni rusztowań kostnych określono ilościowo po lizie komórek za pomocą testu LDH total. Wzrost i proliferację komórek na powierzchni biokompozytów oceniono poprzez obserwację w mikroskopie konfokalnym stosując podwójne barwienie fluorescencyjne cytoszkieltu i jąder komórkowych. Komórki linii hFOB 1.19 i MC3T3-E1 hodowano bezpośrednio na powierzchni biomateriałów przez 9 dni. Co trzeci dzień komórki barwiono za pomocą barwników fluorescencyjnych AlexaFluor635phalloidin i Hoechst 33342 w celu oceny ich morfologii oraz wzrostu ich liczby w czasie. Barwnik AlexaFluor635phalloidin zapewnia czerwoną fluorescencję filamentów cytoszkieletu, natomiast Hoechst 33342 barwi jadra komórkowe na niebiesko. Barwienie „żywe/martwe komórki” wykazało zgrupowania żywych, emitujących zieloną fluorescencje komórek na powierzchni obydwu biokompozytów (chit/HA i chit/glu/HA). Jednakże, komórki hFOB 1.19 porastające powierzchnię rusztowania chit/HA były okrągłe i nie wykazywały typowego dla ich morfologii podłużnego kształtu, co sugeruje, że nie przykleiły się do powierzchni chit/HA (RYS.1). Ponadto, na powierzchni materiału chit/HA zaobserwowano dość dużą liczbę martwych, czerwonych komórek linii hFOB 1.19. Komórki hFOB 1.19 hodowane na powierzchni chit/glu/HA były rozpłaszczone i miały podłużny kształt, co świadczy o ich dobrej adhezji do powierzchni tego materiału. Komórki linii MC3T3-E1 porastające powierzchnię obydwu materiałów były rozpłaszczone i miały typowy dla nich gwiazdkowaty kształt. Jedynie pojedyncze martwe, czerwone komórki MC3T3-E1 zaobserwowano na powierzchni tych kompozytów. Jednakże w porównaniu do rusztowania chit/glu/HA, zdecydowanie mniej komórek MC3T3-E1 było na powierzchni kompozytu chit/HA. LDH total test wykazał znacząco lepszą adhezję komórek hFOB 1.19 i MC3T3-E1 do powierzchni materiału chit/glu/HA (RYS. 2). Trzy godziny od momentu inokulacji rusztowań, do powierzchni kompozytu chit/HA przykleiło się 30% (1.6 x 104) komórek linii hFOB 1.19, natomiast do materiału chit/glu/HA 50% (2.6x104) komórek. W przypadku komórek linii MC3T3-E1, do materiału chit/HA przykleiło się 20% (1.9x104) komórek, a do kompozytu chit/glu/HA aż 70% Obserwacja mikroskopowa wykazała dobry wzrost i proliferację osteoblastów linii hFOB 1.19 i MC3T3-E1 jedynie na rusztowaniu chit/glu/HA (RYS. 3). Liczba komórek porastających powierzchnię chit/glu/HA wzrastała wraz z wydłużającym się czasem hodowli in vitro. Osteoblasty miały typową dla danej linii komórkowej morfologię i dobrze rozbudowany cytoszkielet. Fluoryzujące na niebiesko jądra komórkowe były również bardzo dobrze widoczne. Po 9 dniach prowadzenia hodowli, powierzchnia rusztowania chit/glu/HA była pokryta wielowarstwą komórek linii Hiob 1.19 i MC3T3-E1, które posiadały dobrze rozwiniętą sieć filamentów cytoszkieletu i liczne wypustki cytoplazmatyczne. Osteoblasty hodowane na materiale chit/glu/HA były rozpłaszczone i posiadały dobrze rozbudowaną strukturę cytoszkieletu, co sugeruje, że ten materiał sprzyja adhezji i proliferacji komórek. Udowodniono, że materiał chit/HA całkowicie nie sprzyja adhezji, wzrostowi i proliferacji komórek hFOB 1.19. Przez cały czas trwania eksperymentu na powierzchni chit/HA zaobserwowano jedynie pojedyncze, okrągłe komórki hFOB 1.19. Co więcej, ich liczba nie wzrastała w czasie, a komórki były drobne i okrągłe, co może świadczyć o tym, że były martwe. W przypadku komórek linii MC3T3-E1, 3 dni po inokulacji materiału chit/HA zaobserwowano jedynie pojedyncze komórki na powierzchni próbki (RYS. 3). Ponadto, komórki MC3T3-E1 były okrągłe i nie miały typowego gwiazdkowego kształtu, co świadczy o ich słabej adhezji do powierzchni chit/HA. Jednakże, liczba komórek MC3T3-E1 wzrastała w czasie i po 9 dniach prowadzenia hodowli na powierzchni materiału chit/HA zaobserwowano obszary o małej gęstości komórek MC3T3-E1, które miały gwiazdkowaty kształt, widoczny cytoszkielet i wypustki cytoplazmatyczne. Przeprowadzone eksperymenty in vitro oraz uzyskane zdjęcia z mikroskopu konfokalnego wyraźnie udowadniają, że dodatek β-1,3-glukanu do rusztowania chit/HA stymuluje adhezję, wzrost i proliferację komórek linii hFOB 1.19 i MC3T3-E1. Oba testowane biomateriały były nietoksyczne i pozwalały na wstępną adhezję komórek. Jednakże na powierzchni rusztowania zawierającego β-1,3-glukan zaobserwowano znacząco lepsze rozpłaszczanie się komórek, ich szybszy wzrost i proliferację. Analizując uzyskane wyniki można wysnuć wniosek, że nowy trójskładnikowy kompozyt jest obiecującym materiałem do stosowania w inżynierii tkankowej kości jako rusztowanie komórek mające za zadanie przyspieszenie procesów regeneracyjnych oraz tworzenie nowej, funkcjonalnej tkanki kostnej. (7x104) komórek.

EN

Bone tissue engineering put emphasis on fabrication three-dimensional porous scaffolds that possess ability to enhance adhesion, proliferation and differentiation of osteoblast cells, therefore supporting bone regeneration and functional bone tissue formation [1-3]. The aim of this work was to prove using 2 osteoblastic cell lines that addition of β-1,3-glucan to chitosan/hydroxyapatite (chit/HA) scaffold results in fabrication of novel tri-component chitosan/β-1,3-glucan/hydroxyapatite (chit/glu/HA) composite that possesses better biocompatibility compared to bi-component chit/HA material. Tri-component scaffold was fabricated by modification of chit/HA composite with bacterial β-1,3-glucan as was described previously [2,3]. In vitro experiments were carried out using human foetal osteoblast cell line (hFOB 1.19) and mouse calvarial preosteoblast cell line (MC3T3-E1 Subclone 4). Cytotoxicity of the scaffolds was evaluated by direct-contact method using live/dead double fluorescent staining. The calcein-AM dye stains only viable cells giving green fluorescence and propidium iodide dye stains nucleic acids of only dead cells emitting red fluorescence. Stained cells were observed under confocal microscope. Cell adhesion to the scaffold surfaces was determined quantitatively after cell lysis by LDH total test. Cell growth and proliferation on the biocomposite surfaces were evaluated by confocal microscope observation using double fluorescent staining of osteoblast cytoskeleton and nuclei. HFOB 1.19 and MC3T3-E1 cells were cultured directly on the scaffold surfaces for 9 days and every third day cells were stained with AlexaFluor635phalloidin and Hoechst 33342 fluorescent dyes in order to assess cell morphology and increase in cell number. AlexaFluor635phalloidin dye provides red fluorescence of cytoskeletal filaments, while Hoechst 33342 gives blue fluorescence of nuclei. Live/dead double staining showed clusters of viable green fluorescent osteoblast cells on the surface of both biocomposite samples (chit/HA and chit/glu/HA). However, hFOB 1.19 cells growing on the chit/HA surface were spherical and did not reveal their typical lengthened shape what indicates that hFOB 1.19 cells were not attached to the chit/HA surface (FIG.1). Moreover, there were quite a lot of dead, red fluorescent hFOB 1.19 cells on the chit/HA material. HFOB 1.19 cells cultured on the chit/glu/HA sample were flattened and had lengthened shape what proves their good adhesion to the composite surface. MC3T3-E1 cells growing on both materials were flattened and revealed typical stellar shape. Only occasional dead red fluorescent cells were observed. However, there were meaningfully less MC3T3-E1 cells on the surface of chit/HA composite compared to chit/glu/HA sample. LDH total assay demonstrated significantly higher number of hFOB 1.19 and MC3T3-E1 cells attached to the chit/glu/HA compared to the chit/HA sample (FIG. 2). Three hours after cell inoculation there were 30% (1.6x104 cells) and 50% (2.6x10/4 cells) of hFOB 1.19 cells attached to the chit/HA and chit/glu/HA composites, respectively and 20% (1.9x104 cells) and 70% (7x104 cells) of MC3T3-E1 cells attached to the chit/HA and chit/glu/HA scaffolds, respectively. Microscopic observation showed good osteoblast growth and proliferation only on chit/glu/HA scaffold (FIG.3). The number of hFOB 1.19 and MC3T3-E1 cells growing on the chit/glu/HA increased with time during the in vitro culture. Osteoblasts revealed their typical morphology and had well extensive cytoskeleton. There were also well visible blue fluorescent nuclei. After 9-day culture, chit/glu/HA surface was covered by multilayer of hFOB 1.19 and MC3T3-E1 cells, which revealed extensive network of cytoskeletal filaments and numerous filopodia. Osteoblast cells cultured on the chit/glu/HA were well spread, flattened and generated large filamentous structure of the cytoskeleton what indicates that this scaffold is very favourable to cell adhesion and proliferation. The chit/HA biomaterial was proved to be completely unfavourable to adhesion, growth, and proliferation of hFOB 1.19 cells. Only single spherical hFOB 1.19 cells were observed on the chit/HA sample throughout the full length of the experiment. Moreover, the hFOB 1.19 cell number did not increase with time, cells were tiny and spherical what may indicate that were already dead. In the case of MC3T3-E1 cells, 3 days after cell seeding there were only individual MC3T3-E1 cells on the chit/HA surface (Fig. 3). Furthermore, visualized MC3T3-E1 cells were spherical and did not reveal typical stellar shape what indicates that cells were not well attached. However, the number of MC3T3-E1 cells increased with time and 9 days after cell inoculation there was low density culture of stellar shape MC3T3-E1 cells with visible cytoskeleton and filopodia on the chit/HA material. Conducted in vitro experiments and obtained confocal microscopy images clearly prove that addition of β-1,3-glucan to the chit/HA scaffold enhances adhesion, growth, and proliferation of hFOB 1.19 and MC3T3-E1 cells. Both investigated biomaterials were non-toxic and allowed for initial cell attachment. However, significantly better cell spreading, growth, and proliferation were observed on the scaffold containing β-1,3-glucan. Based on the obtained results, it may be inferred that novel tri-component composite is promising material for bone tissue engineering applications as cell scaffold to accelerate bone regeneration and new bone formation process.

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Tissue engineering of bone: the role of osteoblasts in osteogenesis and peri-implant bone healing

Wróbel E., Witkowska-Zimny M.

Engineering of Biomaterials

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2013

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Vol. 16, no. 119

2--7

EN

Osteoblasts are cells of mesenchymal origin, which rebuild resorbed bone by synthesizing bone matrix proteins and by inducing bone matrix mineralization. Osteoblasts play a crucial role in creating and maintenance of healthy bone architecture, bone repair, and peri-implant bone healing (osseointegration). These bone-forming cells are also involved in regulation of osteoclasts function, and hence bone resorption in osteoclastogenesis process. We have presented our own studies on the subsequent stages of differentiation of Human Bone-Derived Cells (HBDCs) that could be a good candidate as an autogenous source for reconstruction and rebuilding of own patient's bone using tissue engineering methods. In this review we discussed the biology of osteoblasts, compared with the HBDCs cultures, under the influence of growth factors (FGF-2, TGF-ß, IGF, PDGF) and hormones (PTH, 1,25-dihydroxyvitamin D3, leptin). Our review is also focused on the participation of intercellular adhesion proteins (cadherins, claudins, connexin, 'OsteoMacs'), transcription factors (Cbfal, Msx-2, Osx, ATF4), and others molecules (RANKL, OPG, BMP2, lactofferin, PPARY) in modulating osteoblasts functions on the basis of current reports, throwing new light on the involvement of osteoblasts during osteogenesis and peri-implant bone healing.

20

Synthesis and characterization of novel calcium phosphates/polyurethane composites for bone tissue engineering

Szczepańczyk P., Chłopek J., Pielichowska K.

Engineering of Biomaterials

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2013

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Vol. 16, no. 122-123 spec. iss.

71--72