In vitro recellularization and characterization of sheep pulmonary valves after decellularization for preclinical studies

Year 2025, Volume: 40 Issue: 1, 515 - 528, 16.08.2024

Müslüm Süleyman İnal , Cihan Darcan , Ali Akpek

https://doi.org/10.17341/gazimmfd.1259974

Abstract

Decellularized grafts have shown promising results in tissue engineering. The aim of decellularization is to create a scaffold that is devoid of immunogenic components, has natural tissue architecture, and can provide cellularity again. The aim of our study is to obtain a living/regenerative valve by coculture of HUVEC and human dermal fibroblast cells on the pulmonary heart valves of decellularized young sheep and subsequently perform their in vitro characterization to determine its suitability for clinical studies. Various characterization tests were applied to sheep pulmonary heart valves decellularized by the detergent-based method. HUVEC and dermal fibroblast cells were seeded on the scaffold, and their viability was determined by MTT analysis, adhesion by SEM, and cell infiltration by histological staining. Finally, to determine the regenerative ability of the resulting live cap, collagen type I (Col1), collagen type III (Col3), and elastin (Eln) gene expressions were analyzed by PCR. The results showed that cell proliferation increased day by day on the scaffold. In histological findings, it was observed that cellular regeneration was almost completely achieved, especially in arterial wall samples. According to PCR findings, a significant increase in Col1, Col3, and Eln gene expressions was observed in arterial wall samples. In this study, for the first time in the literature, the regeneration potential of differentiated human cells on decellularized sheep heart valves was observed, and markers related to new ECM production were analyzed. In conclusion, we suggest that the decellularized heart valves of young sheep can be used as a starting matrix in tissue engineering studies, and clinical studies are needed.

Keywords

Tissue engineering, pulmonary valve, decellularization, xenograft, regeneration, biomaterial

Project Number

FCD-2021-4687

Koyun pulmoner kalp kapakçıklarının hücresizleştirilmesi sonrası preklinik çalışmalar için in vitro olarak yeniden hücrelendirilmesi ve karakterizasyonu

Abstract

Doku mühendisliğinde hücresizleştirilmiş greftler umut verici sonuçlar göstermiştir. Hücresizleştirmenin amacı immünojenik bileşenlerin olmadığı, doğal doku mimarisine sahip ve yeniden hücreselliği sağlayabilecek bir iskele oluşturmaktır. Çalışmamızın amacı, hücresizleştirilmiş genç koyunların pulmoner kalp kapakçıkları üzerinde HUVEC ve insan dermal fibroblast hücrelerinin kokültürü ile birlikte canlı/rejeneratif bir kapak elde etmek ve ardından klinik çalışmalara uygunluğunu belirlemek için in vitro karakterizasyonlarını gerçekleştirmektir. Deterjan tabanlı metodla hücresizleştirilen koyun pulmoner kalp kapakçıklarına çeşitli karakterizasyon testleri uygulanmıştır. HUVEC ve dermal fibroblast hücreleri iskele üzerine ekildi ve canlılıkları MTT analiziyle, adezyonu SEM ile, hücre sızması histolojik boyama ile belirlendi. Son olarak elde edilen canlı kapağın rejeneratif kabiliyetini belirlemek için kollajen tipI (Col1), kollajen tipIII (Col3), elastin (Eln) gen ifadeleri PCR ile analiz edildi. Sonuçlar, hücre çoğalmasının iskele üzerinde gün geçtikçe arttığını göstermiştir. Histolojik bulgularda özellikle arter duvarı örneklerinde yeniden hücreselliğin neredeyse tamamen sağlandığı gözlenmiştir. PCR bulgularına göre arter duvarı örneklerinde Col1, Col3 ve Eln gen ifadelerinde önemli bir artış gözlenmiştir. Bu çalışma ile birlikte literatürde ilk defa hücresizleştirilmiş koyun kalp kapakçıkları üzerinde farklılaşmış insan hücrelerinin rejenerasyon potansiyeli olduğu gözlenmiş ve yeni ECM üretimi ile ilgili belirteçler analiz edilmiştir. Sonuç olarak genç koyunların hücresizleştirilmiş kalp kapakçıklarının, doku mühendisliği çalışmalarında başlangıç matrisi olarak kullanılabileceğini, klinik araştırmalara ihtiyaç olduğunu öneriyoruz.

Keywords

Doku Mühendisliği,, pulmoner kapakçık, hücresizleştirme, ksenograft, rejenerasyon, biyomateryal

Supporting Institution

Yıldız Teknik Üniversitesi

Project Number

FCD-2021-4687

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