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

Öz

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.

Anahtar Kelimeler

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

Destekleyen Kurum

Yıldız Teknik Üniversitesi

Proje Numarası

FCD-2021-4687

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

Öz

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.

Anahtar Kelimeler

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

Proje Numarası

FCD-2021-4687

Kaynakça

1. 1. Kodigepalli, K.M., Thatcher, K., West, T., Howsmon, D.P., Schoen, F.J., Sacks, M.S., Breuer, C.K., Lincoln, J., Biology and Biomechanics of the Heart Valve Extracellular Matrix, J Cardiovasc Dev Dis, 7, 57, 2020.
2. 2. Copeland, K.M., Wang, B., Shi, X., Simionescu, D.T., Hong, Y., Bajona, P., Sacks, M.S., Liao, J. Decellularization in Heart Valve Tissue Engineering. In Advances in Heart Valve Biomechanics, Springer International Publishing: Cham, 289–317, 2018.
3. 3. VeDepo, M.C., Detamore, M.S., Hopkins, R.A., Converse, G.L. Recellularization of Decellularized Heart Valves: Progress toward the Tissue-Engineered Heart Valve. J Tissue Eng, 8, 2017.
4. 4. Mozaffarian, D., Benjamin, E.J., Go, A.S., Arnett, D.K., Blaha, M.J., Cushman, M., Das, S.R., de Ferranti, S., Després, J.-P., Fullerton, H.J., et al., Heart Disease and Stroke Statistics-2016 Update. Circulation, 133, 2016.
5. 5. Ibrahim, D.M., Kakarougkas, A., Allam, N.K., Recent Advances on Electrospun Scaffolds as Matrices for Tissue-Engineered Heart Valves. Mater Today Chem, 5, 11–23, 2017.
6. 6. Jahnavi, S., Kumary, T.V., Bhuvaneshwar, G.S., Natarajan, T.S., Verma, R.S., Engineering of a Polymer Layered Bio-Hybrid Heart Valve Scaffold. Materials Science and Engineering: C, 51, 263–273, 2015.
7. 7. Fioretta, E.S., Motta, S.E., Lintas, V., Loerakker, S., Parker, K.K., Baaijens, F.P.T., Falk, V., Hoerstrup, S.P., Emmert, M.Y., Next-Generation Tissue-Engineered Heart Valves with Repair, Remodelling and Regeneration Capacity. Nat Rev Cardiol, 18, 92–116, 2021.
8. 8. Ciolacu, D.E., Nicu, R., Ciolacu, F., Natural Polymers in Heart Valve Tissue Engineering: Strategies, Advances and Challenges. Biomedicines, 10, 1095, 2022.

9. 9. Akpek A., Analysis of Biocompatibility Characteristics of Stereolithography Applied Three Dimensional (3D) Bioprinted Artifical Heart Valves, Journal of the Faculty of Engineering and Architecture of Gazi University, 33 (3), 929–938, 2018.
10. 10. Mela, P., Hinderer, S., Kandail, H.S., Bouten, C.V.C., Smits, A.I.P.M., Tissue-Engineered Heart Valves. In Principles of Heart Valve Engineering, Elsevier, 123–176, 2019.
11. 11. Boroumand, S., Asadpour, S., Akbarzadeh, A., Faridi-Majidi, R., Ghanbari, H., Heart Valve Tissue Engineering: An Overview of Heart Valve Decellularization Processes. Regenerative Med, 13, 41–54, 2018.
12. 12. Simionescu, D., Harpa, M.M., Simionescu, A., Oprita, C., Movileanu, I., Tissue Engineering Heart Valves – a Review of More than Two Decades into Preclinical and Clinical Testing for Obtaining the Next Generation of Heart Valve Substitutes. Romanian Journal of Cardiology, 31, 501–510, 2021.
13. 13. Ramm, R., Niemann, H., Petersen, B., Haverich, A., Hilfiker, A., Decellularized GGTA1-KO Pig Heart Valves Do Not Bind Preformed Human Xenoantibodies. Basic Res Cardiol, 111, 39, 2016.
14. 14. Hopkins, R.A., Bert, A.A., Hilbert, S.L., Quinn, R.W., Brasky, K.M., Drake, W.B., Lofland, G.K., Bioengineered Human and Allogeneic Pulmonary Valve Conduits Chronically Implanted Orthotopically in Baboons: Hemodynamic Performance and Immunologic Consequences. J Thorac Cardiovasc Surg, 145, 1098-1107, 2013.
15. 15. Godehardt, A.W., Ramm, R., Gulich, B., Tönjes, R.R., Hilfiker, A., Decellularized Pig Pulmonary Heart Valves—Depletion of Nucleic Acids Measured by Proviral PERV Pol. Xenotransplantation, 27, 2020.
16. 16. Schoen, F.J., Levy, R.J., Calcification of Tissue Heart Valve Substitutes: Progress Toward Understanding and Prevention. Ann Thorac Surg, 79, 1072–1080, 2005.
17. 17. Christ, T., Paun, A.C., Grubitzsch, H., Holinski, S., Falk, V., Dushe, S., Long-Term Results after the Ross Procedure with the Decellularized AutoTissue Matrix P® Bioprosthesis Used for Pulmonary Valve Replacement. European Journal of Cardio-Thoracic Surgery, 55, 885–892, 2019.
18. 18. Cicha, I., Rüffer, A., Cesnjevar, R., Glöckler, M., Agaimy, A., Daniel, W.G., Garlichs, C.D., Dittrich, S., Early Obstruction of Decellularized Xenogenic Valves in Pediatric Patients: Involvement of Inflammatory and Fibroproliferative Processes. Cardiovascular Pathology, 20, 222–231, 2011.
19. 19. Rüffer, A., Purbojo, A., Cicha, I., Glöckler, M., Potapov, S., Dittrich, S., Cesnjevar, R.A., Early Failure of Xenogenous De-Cellularised Pulmonary Valve Conduits-a Word of Caution. European Journal of Cardio-Thoracic Surgery, 38, 78–85, 2010.
20. 20. Abdolghafoorian, H., Farnia, P., Sajadi Nia, R.S., Bahrami, A., Dorudinia, A., Ghanavi, J., Effect of Heart Valve Decellularization on Xenograft Rejection. Exp Clin Transplant, 15, 329–336, 2017.
21. 21. Ramm, R., Goecke, T., Köhler, P., Tudorache, I., Cebotari, S., Ciubotaru, A., Sarikouch, S., Höffler, K., Bothe, F., Petersen, B., et al., Immunological and Functional Features of Decellularized Xenogeneic Heart Valves after Transplantation into GGTA1-KO Pigs. Regen Biomater, 8, 2021.
22. 22. Baraki, H., Tudorache, I., Braun, M., Höffler, K., Görler, A., Lichtenberg, A., Bara, C., Calistru, A., Brandes, G., Hewicker-Trautwein, M., et al., Orthotopic Replacement of the Aortic Valve with Decellularized Allograft in a Sheep Model. Biomaterials, 30, 6240–6246, 2009.
23. 23. Quinn, R.W., Hilbert, S.L., Converse, G.L., Bert, A.A., Buse, E., Drake, W.B., Armstrong, M., Moriarty, S.J., Lofland, G.K., Hopkins, R.A., Enhanced Autologous Re-Endothelialization of Decellularized and Extracellular Matrix Conditioned Allografts Implanted Into the Right Ventricular Outflow Tracts of Juvenile Sheep. Cardiovasc Eng Technol, 3, 217–227, 2012.
24. 24. Qiao, W., Liu, P., Hu, D., Al Shirbini, M., Zhou, X., Dong, N., Sequential Hydrophile and Lipophile Solubilization as an Efficient Method for Decellularization of Porcine Aortic Valve Leaflets: Structure, Mechanical Property and Biocompatibility Study. J Tissue Eng Regen Med, 12, 2018.
25. 25. van Steenberghe, M., Schubert, T., Gerelli, S., Bouzin, C., Guiot, Y., Xhema, D., Bollen, X., Abdelhamid, K., Gianello, P., Porcine Pulmonary Valve Decellularization with NaOH-Based vs Detergent Process: Preliminary in Vitro and in Vivo Assessments. J Cardiothorac Surg, 13, 34, 2018.
26. 26. Chauvette, V., Bouhout, I., Tarabzoni, M., Pham, M., Wong, D., Whitlock, R., Chu, M.W.A., El-Hamamsy, I., Lefebvre, L., Poirier, N., et al., Pulmonary Homograft Dysfunction after the Ross Procedure Using Decellularized Homografts—a Multicenter Study. J Thorac Cardiovasc Surg, 163, 1296-1305.e3, 2022.
27. 27. Converse, G.L., Buse, E.E., Neill, K.R., McFall, C.R., Lewis, H.N., VeDepo, M.C., Quinn, R.W., Hopkins, R.A., Design and Efficacy of a Single-Use Bioreactor for Heart Valve Tissue Engineering. J Biomed Mater Res B Appl Biomater, 105, 249–259, 2017.
28. 28. Quinn, R.W., Bert, A.A., Converse, G.L., Buse, E.E., Hilbert, S.L., Drake, W.B., Hopkins, R.A., Performance of Allogeneic Bioengineered Replacement Pulmonary Valves in Rapidly Growing Young Lambs. J Thorac Cardiovasc Surg, 152, 1156-1165.e4, 2016.
29. 29. Theodoridis, K., Tudorache, I., Calistru, A., Cebotari, S., Meyer, T., Sarikouch, S., Bara, C., Brehm, R., Haverich, A., Hilfiker, A., Successful Matrix Guided Tissue Regeneration of Decellularized Pulmonary Heart Valve Allografts in Elderly Sheep. Biomaterials, 52, 221–228, 2015.
30. 30. Tudorache, I., Calistru, A., Baraki, H., Meyer, T., Höffler, K., Sarikouch, S., Bara, C., Görler, A., Hartung, D., Hilfiker, A., et al., Orthotopic Replacement of Aortic Heart Valves with Tissue-Engineered Grafts. Tissue Eng Part A, 19, 1686–1694, 2013.
31. 31. Inal, M.S., Darcan, C., Akpek, A., Characterization of a Decellularized Sheep Pulmonary Heart Valves and Analysis of Their Capability as a Xenograft Initial Matrix Material in Heart Valve Tissue Engineering. Bioengineering, 10, 949, 2023.
32. 32. Quinn, R.W., Hilbert, S.L., Bert, A.A., Drake, B.W., Bustamante, J.A., Fenton, J.E., Moriarty, S.J., Neighbors, S.L., Lofland, G.K., Hopkins, R.A., Performance and Morphology of Decellularized Pulmonary Valves Implanted in Juvenile Sheep. Ann Thorac Surg, 92, 131–137, 2011.
33. 33. Lichtenberg, A., Tudorache, I., Cebotari, S., Ringes-Lichtenberg, S., Sturz, G., Hoeffler, K., Hurscheler, C., Brandes, G., Hilfiker, A., Haverich, A., In Vitro Re-Endothelialization of Detergent Decellularized Heart Valves under Simulated Physiological Dynamic Conditions. Biomaterials, 27, 4221–4229, 2006.
34. 34. Butcher, J.T., Nerem, R.M., Valvular Endothelial Cells Regulate the Phenotype of Interstitial Cells in Co-Culture: Effects of Steady Shear Stress. Tissue Eng, 12, 905–915, 2006.
35. 35. Liu, A.C., Joag, V.R., Gotlieb, A.I., The Emerging Role of Valve Interstitial Cell Phenotypes in Regulating Heart Valve Pathobiology. Am J Pathol, 171, 1407–1418, 2007.
36. 36. Dohmen, P.M., Hauptmann, S., Terytze, A., Konertz, W.F., In-Vivo Repopularization of a Tissue-Engineered Heart Valve in a Human Subject. J Heart Valve Dis, 16, 447–449, 2007.
37. 37. Baraki, H., Tudorache, I., Braun, M., Höffler, K., Görler, A., Lichtenberg, A., Bara, C., Calistru, A., Brandes, G., Hewicker-Trautwein, M., et al., Orthotopic Replacement of the Aortic Valve with Decellularized Allograft in a Sheep Model. Biomaterials, 30, 6240–6246, 2009.
38. 38. da Costa, F.D.A., Costa, A.C.B.A., Prestes, R., Domanski, A.C., Balbi, E.M., Ferreira, A.D.A., Lopes, S.V., The Early and Midterm Function of Decellularized Aortic Valve Allografts. Ann Thorac Surg, 90, 1854–1860, 2010.
39. 39. Quinn, R.W., Hilbert, S.L., Converse, G.L., Bert, A.A., Buse, E., Drake, W.B., Armstrong, M., Moriarty, S.J., Lofland, G.K., Hopkins, R.A., Enhanced Autologous Re-Endothelialization of Decellularized and Extracellular Matrix Conditioned Allografts Implanted Into the Right Ventricular Outflow Tracts of Juvenile Sheep. Cardiovasc Eng Technol, 3, 217–227, 2012.
40. 40. Iop, L., Bonetti, A., Naso, F., Rizzo, S., Cagnin, S., Bianco, R., Lin, C.D., Martini, P., Poser, H., Franci, P., et al., Decellularized Allogeneic Heart Valves Demonstrate Self-Regeneration Potential after a Long-Term Preclinical Evaluation. PLoS One, 9, e99593, 2014.
41. 41. Dainese, L., Guarino, A., Burba, I., Esposito, G., Pompilio, G., Polvani, G., Rossini, A., Heart Valve Engineering: Decellularized Aortic Homograft Seeded with Human Cardiac Stromal Cells. J Heart Valve Dis, 21, 125–134, 2012.
42. 42. Schenke-Layland, K., Complete Dynamic Repopulation of Decellularized Heart Valves by Application of Defined Physical Signals—an in Vitro Study. Cardiovasc Res, 60, 497–509, 2003.
43. 43. Converse, G.L., Buse, E.E., Neill, K.R., McFall, C.R., Lewis, H.N., VeDepo, M.C., Quinn, R.W., Hopkins, R.A., Design and Efficacy of a Single‐use Bioreactor for Heart Valve Tissue Engineering. J Biomed Mater Res B Appl Biomater, 105, 249–259, 2017.
44. 44. Zhou, J., Nie, B., Zhu, Z., Ding, J., Yang, W., Shi, J., Dong, X., Xu, J., Dong, N., Promoting Endothelialization on Decellularized Porcine Aortic Valve by Immobilizing Branched Polyethylene Glycolmodified with Cyclic-RGD Peptide: An in Vitro Study. Biomedical Materials, 10, 065014, 2015.
45. 45. Zhou, J., Ye, X., Wang, Z., Liu, J., Zhang, B., Qiu, J., Sun, Y., Li, H., Zhao, Q., Development of Decellularized Aortic Valvular Conduit Coated by Heparin–SDF-1α Multilayer. Ann Thorac Surg, 99, 612–618, 2015.
46. 46. Ye, X., Wang, H., Zhou, J., Li, H., Liu, J., Wang, Z., Chen, A., Zhao, Q., The Effect of Heparin-VEGF Multilayer on the Biocompatibility of Decellularized Aortic Valve with Platelet and Endothelial Progenitor Cells. PLoS One, 8, e54622, 2013.
47. 47. Paniagua Gutierrez, J.R., Berry, H., Korossis, S., Mirsadraee, S., Lopes, S.V., da Costa, F., Kearney, J., Watterson, K., Fisher, J., Ingham, E., Regenerative Potential of Low-Concentration SDS-Decellularized Porcine Aortic Valved Conduits In Vivo. Tissue Eng Part A, 21, 332–342, 2015.
48. 48. Theodoridis, K., Tudorache, I., Calistru, A., Cebotari, S., Meyer, T., Sarikouch, S., Bara, C., Brehm, R., Haverich, A., Hilfiker, A., Successful Matrix Guided Tissue Regeneration of Decellularized Pulmonary Heart Valve Allografts in Elderly Sheep. Biomaterials, 52, 221–228, 2015.