89-91 - Polskie Stowarzyszenie Biomateriałów

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Introduction Nowadays, various methods of xenogeneic tissues stabilization are proposed for the purpose of preparing many tissue-derived biomaterials [1-3]. One of commercial methods of tissues stabilization is the crosslinking of extracellular matrix by glutaraldehyde (GA) action [4,5]. However, GA is responsible for cytotoxic effect [6] and induction of calcification [7]. Moreover, a key role in the premature biodegradation of tissues stabilized in various ways is played by the cellular debris [7] and phospholipids [8]. Decellularization of xenogeneic tissues may contribute to reduction of these failures [9]. The tissue treatment with trypsin in EDTA solution belongs to the most often studied methods of the tissues decellularization [10-12]. However, some risk of connective fibers damage is possible. The aim of this work was to determine nanostructure of bovine pericardium after trypsin treatment, using atomic force microscopy (AFM). materials and methods Bovine pericardium from hearts of 5–6 month old domestic cattle (Bos taurus) was obtained from the local abattoir and subsequently transported to the laboratory in buffered physiological saline solution (PBS; pH 6.5) at 4°C according to Simionescu and co-workers [13] procedure for the pericardium selection for bioprosthetic heart valves. Fatty tissue and sections with heavy vasculature were gently removed from prepared samples. Tissue samples were modified by incubation under continuous shaking in the solution containing trypsin and EDTA (0.5% trypsin and 0.2% EDTA; Sigma), and PBS in the ratio 1:10, at 37°C for 48 hours. During this period, the digestion solution was changed twice [14]. The nanostructures of native and modified tissue samples were evaluated by AFM. AFM imaging was performed using MultiMode 3 (di-Veeco, CA) working in the tapping mode under atmospheric conditions. Two standard AFM signals were registered: the signal corresponding to the topography of the sample (Height) and the differential signal (Deflection), which is useful for direct observation. Before measurements, tissue samples were gently air-dried, at room temperature in the laminar flow box, until the excess of water had evaporated from the samples’ surfaces [2]. All AFM images were processed using the software package WSxM (Nanotec Electronica, Spain) [15]. Results and discussions fIg.1. afm deflection (a) and height (B) images (1.0×1.0mm) of the native bovine pericardium; (C) represents an axial profile of the collagen fibril taken along the marked line in the height image (B). fIg.2. afm deflection (a) and height (B) images (1.0×1.0mm) of the trypsintreated bovine pericardium; (C) represents an axial profile of the collagen fibril taken along the marked line in the height image (B). FIGURE 1 shows the AFM Deflection (FIG.1A) and Height (FIG.1B) images of native structure of bovine pericardium. Generally, parallel arrangement of collagen fibers was revealed. However, some fibril groups formed by 2-3 fibers and more run in various directions (FIG.1A). Topography analysis of single fiber is presented in FIGURE 1C, where an axial profile taken along the marked line on the surface of collagen fiber in the Height image (FIG.1B) was shown. Collagen fibers in native bovine pericardium showed the regular D-banding pattern characteristic for collagen fibrils type I with the distance of 68-78nm. The soaking of bovine pericardium in PBS solution with trypsin and EDTA resulted in non significant changes in tissues’ morphology. FIGURE 2 shows the AFM Deflection (FIG.2A) and Height (FIG.2B) images of bovine pericardium treated in that manner. Generally, surfaces of trypsin-treated connective tissue samples were free of the extracellular matrix debris. It is important that the collagen fibers structure remains intact as it is clearly visible in FIG.2A. Topography of collagen fiber representing tissue treated with trypsin in EDTA-PBS solution, presented as an axial profile (FIG.2C) taken along the marked line on the fibril surface in the Height image (FIG.2B), reveals regular periodicity. The boundaries between bands are more visible (FIG.2C), which probably results from removal of low molecular proteins from the tissue structure. These data correspond to results of our earlier studies. We have shown that electrophoretic profile of proteins released from porcine pericardium treated in the same way revealed the lack of polypeptides with molecular weight below 24 kDa [12]. Demonstrated results of AFM studies of bovine pericardium treated with trypsin in EDTA-PBS solution do not reveal any failures on the fibers’ surface in the nanoscale. Conclusions Although the enzymatic decellularization belongs to the most promising methods of allogeneic and xenogeneic tissues stabilization, the biodegradation processes in modified tissues are complex and still not recognized. It has been found upon presented results that enzymatic “purification” of bovine pericardium surface may influence the reduction of biodegradation processes by elimination of cellular debris and immunogenic agents. The most important aspect of this finding is the lack of deterioration of collagen fibers in the tissue surface layer. acknowledgements This work was financially supported by Silesian Medical University.
References [1] Cwalina B., Turek A., Nożyński J., Jastrzębska M., Nawrat Z. Structural changes in pericardium tissue modified with tannic acid. Int J Artif Organs. 28 (2005) 648-53. [2] Jastrzebska M., Barwiński B., Mróz I., Turek A., Zalewska- Rejdak J., Cwalina B. Atomic force microscopy investigation of chemically stabilized pericardium tissue. Eur Phys J E Soft Matter. 16 (2005) 381-8. [3] Turek A., Cwalina B., Pawlus-Łachecka L., Dzierżewicz Z. Influence of tannic acid and penicillin on pericardium proteins stability. Engineering of Biomaterials 69-72 (2007) 84-86. [4] Carpentier A., Lemaigre G., Robert L., Carpentier S., Dubost C. Biological factors affecting long-term results of valvular heterografts. J Thorac Cardiovasc Surg. 58 (1969) 467-83. [5] Cwalina B., Turek A., Miśkowiec M., Nawrat Z., Domal-Kwiatkowska D. Biochemical stability of pericardial tissues modified using glutaraldehyde or formaldehyde. Engineering of Biomaterials 23-25 (2002) 64-67. [6] Gendler E., Gendler S., Nimni, M.E. Toxic reactions evoked by glutaraldehyde-fixed pericardium and cardiac valve tissue bioprosthesis. J Biomed Mater Res. 18 (1984) 727-36. [7] Levy R.J., Schoen F.J., Anderson H.C., Harasaki H., Koch T., Brown W., Lian J.B., Cumming R., Gavin J.B. Cardiovascular implant calcification: a survey and update. Biomaterials 12 (1991) 707-14. [8] Pathak C.P., Adams A.K., Simpson T., Phillips R.E. Jr, Moore M.A. Treatment of bioprosthetic heart valve tissue with long chain alcohol solution to lower calcification potential. J Biomed Mater Res A. 69 (2004) 140-4. [9] Schmidt C.E., Baier J.M. Acellular vascular tissues: natural biomaterials for tissue repair and tissue engineering. Biomaterials 21 (2000) 2215-31. [10] Cebotari S., Lichtenberg A., Tudorache I., Hilfiker A., Mertsching H., Leyh R., Breymann T., Kallenbach K., Maniuc L., Batrinac A., Repin O., Maliga O., Ciubotaru A., Haverich A. Clinical application of tissue engineered human heart valves using autologous progenitor cells. Circulation 114 (2006) I132-7. [11] Tudorache I., Cebotari S., Sturz G., Kirsch L., Hurschler C., Hilfiker A., Haverich A., Lichtenberg A. Tissue engineering of heart valves: biomechanical and morphological properties of decellularized heart valves. J Heart Valve Dis. 16 (2007) 567-73. [12] Turek A, Cwalina B, Nożyński J. Biochemical and morphological properties of pericardium tissues after decellularization. Engineering of Biomaterials 77-80 (2008) 110-113. [13] Simionescu D., Simionescu A., Deac R. Detection of remnant proteolytic activities in unimplanted glutaraldehyde-treated bovine pericardium and explanted cardiac bioprostheses. J Biomed Mater Res. 27 (1993) 821-9. [14] Cebotari S., Mertsching H., Kallenbach K., Kostin S., Repin O., Batrinac A., Kleczka C., Ciubotaru A., Haverich A. Construction of autologous human heart valves based on an acellular allograft matrix. Circulation 106 (12 Suppl 1) (2002): I63-I68. [15] Horcas I., Fernández R., Gómez-Rodríguez J.M., Colchero J., Gómez-Herrero J., Baro A.M. WSXM: a software for scanning probe microscopy and a tool for nanotechnology. Rev Sci Instrum. 78 (2007) 013705. moRPhologICal STudIES of TISSuES STaBIlIzEd By gluTaRaldEhydE and TannIC aCId ARTUR TUREK 1 *, ANDRZEJ MARCINKOWSKI 2 , BEATA CWALINA 3 , JERZy NOżyńSKI 4 , ZOFIA DZIERżEWICZ 1 1dePArtMent oF bioPhArMAcy, MedicAl uniVerSity oF SileSiA, SoSnowiec, PolAnd 2centre oF PolyMer And cArbon MAteriAlS, PoliSh AcAdeMy oF Science, zAbrze, PolAnd 3dePArtMent oF enVironMentAl biotechnoloGy, SileSiAn uniVerSity oF technoloGy, Gliwice, PolAnd 4FoundAtion For cArdiAc SurGery deVeloPMent, zAbrze, PolAnd *MAilto: AtureK@ViP.interiA.Pl abstract Despite the disadvantages of glutaraldehyde (GA)stabilization of tissues, it is the method most often used for xenogeneic tissues preparation. Nowadays, partial elimination of drawbacks of this method is achieved by using GA in the mixture with other crosslinking reagents, which completes the stabilization effects and acts synergistically. The aim of this work was to determine microstructure and nanostructure of porcine pericardium stabilized by GA and tannic acid (TA). The microstructure was examined by optical microscopy and the nanostructure by atomic force microscopy (AFM). Different results on the level of micro- and nanostructure were observed. No essential changes in the tissue morphology after crosslinking with GA and TA were observed under optical microscope, but significant morphological differences were revealed in AFM studies. Keywords: glutaraldehyde, tannic acid, porcine pericardium, microstructure, nanostructure [Engineering of Biomaterials, 89-91, (2009), 32-34] Introduction Glutaraldehyde (GA) is the crosslinking agent most often used in stabilization of xenogeneic tissues [1]. However, GA is cytotoxic [2] and, moreover, the GA-stabilized tissues are susceptible to premature calcification [3]. Procedures of the tissue stabilization with GA are modified by use of GA low concentrations. Partial elimination of drawbacks in the GAstabilized tissues may also be achieved by using GA in the mixture with other crosslinking reagents, which completes the stabilization effects and acts synergistically. Lately, the attention has been paid on tannic acid (TA) as stabilizing reagent [4-6]. According to these data, the tissue treatment with TA alone or with mixture of GA and TA is proposed as a new method of the connective tissues stabilization. TA-treatment leads to the decrease in the tissue calcification [7] and the increase of extracellular matrix integrity [4]. The aim of this work was to determine microstructure and nanostructure of porcine pericardium stabilized by GA and TA. materials and methods Porcine pericardium from hearts of 5–6 month old domestic pig (Sus scrofa domestica) was obtained from the local
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82 podsumowanie Ponad połowa chory
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84 materiały i metody syntezy Mono
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86 resonance lines Sequences Lactid
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88 powinny zmieniać swoich właśc
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90 ryS.4. naprężenie przy zerwani
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92 wyniki badań Wyniki badań in v
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96 o średnicy d 1=1,0mm i kącie 2
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98 pozauStrojowe badania nad tokSyC
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114 rys.1. a) Przykładowy obraz se
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116 analiza numeryczna i doświadcz
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120 mikroskopię świetlną, skanin
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122 dla połączeń stomatologiczny
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124 rys.1. krzywa tg i dtg degradac
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126 mikrostruktura i właściwości
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130 o normę ISO/TS 11405:2003: Den
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132 bezpośrednie oddziaływanie na
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134 Materiał Material Kompozyt C/S
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136 wPływ materiałów węglowych
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138 Materiał Material Nuclear carb
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140 Piśmiennictwo [1] Paluch D., S
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142 kompozytów znaleziono zależno
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144 określoną szybkością i w ok
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146 materiały i metody Włókna po
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148 zasTosoWanie spekTroskopii uV-V
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150 kompozyToWe Włókna polilakTyd
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152 zachoWanie elekTrochemiczne sTo
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154 piśmiennictwo [1]. M. C. Advin
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160 podziękowania Badania te były
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162 rys.4. Wpływ mocy mikrofalowej
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164 chorobach serca [4]. Wolne rodn
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166 WłaściWości paramagneTyczne
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168 piśmiennictwo [1] J. Marciniak
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170 rys.3. Wpływ mocy mikrofalowej
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172 dowym pochodzenia naturalnego.
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174 Wstęp Cladosporium herbarum to
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176 analiza WyTrzymałościoWa i od
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178 Wykres 2. prędkości przejści
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180 Ocena wybranych cech włóknino
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182 wnioski Parametr Oarameter Wytr
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184 sics SP 8800, stosując chlorof
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186 piśmiennictwo [1] Li S, Vert M
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192 mianowymi i ich rozpychaniem. N
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194 wstęp Zastosowanie nanokompozy
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198 wprowadzenie Częstą przyczyn
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200 Przeprowadzona ocena mikrostruk
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202 próbek zostały zaprezentowane
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216 rys.1.wykresy przedstawiające
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232 Farmakokinetyka - analiza zagad
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234 X 0-masa leku podana dożylnie
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236 włókno polimerowe [13]. W pon
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prądu w zakresie potencjałów wys
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inkubowano 15 minut w ciemności z
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Wartości średnie oraz parametry r
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skończonych. Dla potrzeb obliczeń
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ozwój polimerów w oparciu o natur
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poly(butylene succinate) modified b
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influence of surface (nano)roughnes
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combinatorial discovery approaches
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89-91 - Polskie Stowarzyszenie Biomateriałów

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