Eine neue Nanofaser-Matrix für den kornealen Gewebeersatz

 

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Zusammenfassung

Hintergrund: Weltweit leiden circa 10 Millionen Menschen unter einem Sehverlust aufgrund eines Hornhautschadens. Im schlimmsten Fall besteht die einzige Therapieoption in einer Transplantation von kornealem Spendergewebe. In vielen Ländern existiert jedoch ein bedeutender Mangel an qualitativ geeignetem Spendergewebe, der zu verschiedenen Versuchen geführt hat, einen künstlichen Gewebeersatz zu entwickeln. In der vorliegenden Studie beschreiben wir die Entwicklung einer bioabbaubaren Nanofaser-Matrix aus Poly(glycerolsebazinsäure)(PGS)/Poly(ε-Caprolakton)(PCL)-Gemisch zum kornealen Gewebeersatz. Material und Methoden: Die Nanofaser-Matrizen wurden mittels eines modifizierten Elektrospinnverfahrens hergestellt. Die Biokompatibilität des Materials wurde in vitro durch einen kolorimetrischen MTT-Test zum Nachweis der vitalen kornealen Endothelzellen (HCECs) überprüft. Um eine potentielle immunologische Reaktion gegen die Matrizen zu untersuchen, wurden Matrixproben mononukleären Zellen aus peripherem Blut (PBMCs) ausgesetzt. Nach einer Inkubationszeit von 3 Tagen wurden die Zellen mittels Annexin-V-FITC/Propidiumjodid durch FACS-Analyse auf Apoptose und immunologische Reaktionen untersucht. Ergebnisse: Wir konnten erfolgreich zeigen, dass die Kultivierung von HCECs auf PGS/PCL-Matrizen möglich war. Nach 7 Tagen Kultivierung konnte mittels Mikroplattenabsorption eine signifikant höhere Zelldichte gemessen werden als an Tag 3 (p < 0,0001). Gemäß den MTT-Daten war keine Toxizität der Proben nachweisbar. Mit Hinblick auf PBMCs zeigten unsere FACS-Analysen keine Aktivierung oder Inhibierung von Apoptose und Immunreaktionen. Alle Matrix-Zusammensetzungen waren inert gegenüber naiven und aktivierten T-/B-/NK-Zellen und Monozyten. Folglich wurden Leukozytenüberleben und -aktivität durch die Matrizen nicht beeinflusst. Schlussfolgerungen: Es ist uns gelungen, ein gewebeähnliches Konstrukt zu entwickeln, das dem (menschlichen) Hornhautstroma ähnelt. Die Ergebnisse zeigen, dass PGS/PCL-Matrizen eine ideale Alternative für den kornealen Gewebeersatz darstellen, da sie eine hohe Biokompatibilität mit Endothel- und Blutzellen aufweisen.

Abstract

Background: An estimated 10 million people suffer worldwide from vision loss caused by corneal damage. For the worst cases, the only available treatment is transplantation with human donor corneal tissue. However, in numerous countries there is a considerable shortage of corneal tissue of good quality, leading to various efforts to develop tissue substitutes. The present study aims to introduce a nanofibrous scaffold of poly(glycerol sebacate) PGS as a biodegradable implant, for the corneal tissue engineering. Materials and Methods: Nanofibrous scaffolds were produced from PGS and poly(ε-caprolactone) (PCL) by a modified electro-spinning process. The biocompatibility of the material was tested in vitro by colorimetric MTT assay on days 3, 5, and 7 to test the cell viability of human corneal endothelium cells (HCEC). To examine a potential immunological reaction of the scaffolds, samples were exposed to mononuclear cells derived from peripheral blood (PBMCs). After an incubation period of 3 days, supernatants were assayed for apoptotic assessment and immunogenic potentials by annexin V FITC//propidium iodide and flow-cytometric analysis. Results: We could successfully demonstrate that cultivation of HCECs on PGS/PCL scaffolds was possible. Compared to day 3, cell density determined by microplate absorbance was significantly higher after 7 days of cultivation (p < 0.0001). According to the MTT data, none of the samples showed toxicity. Apoptotic assessments by FACS analysis showed that no composition stimulated apoptosis or activated PBMCs occurred. All the compositions were inert for native as well as activated T/B/NK cells and monocytes. It can be concluded that leukocytes and their activity was not affected by the scaffolds. Conclusion: A tissue-like scaffold mimicking the human stroma could be developed. The results indicate that PGS/PCL scaffolds could be considered as ideal candidates for corneal tissue engineering as they are biocompatible in contact to corneal endothelial cells and blood cells.

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