Effects of freeze-drying bovine pericardium using a combination of polyethylene glycol and trehalose_Journal of Biomedical Engineering

Authors：

HUANG Wei ^1,2,3 ,  LI Weijie ^1,2,3 , LIU Baolin ^1,2,3

1. Institute of Biothermal Science and Technology, University of Shanghai for Science and Technology, Shanghai 200093, P. R. China;
2. Shanghai Co-innovation Center for Energy Therapy of Tumors, Shanghai 200093, P. R. China;
3. Shanghai Technical Service Platform for Cryopreservation of Biological Resources, Shanghai 200093, P. R. China;

Corresponding author：

LI Weijie, Email: liweijie10@139.com

Keywords：

Freeze drying; Mechanical property; Thermal shrinkage temperature; Polyethylene glycol; Trehalose

DOI：

10.7507/1001-5515.202311035

Video：

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The freeze-drying is a technology that preserves biological samples in a dry state, which is beneficial for storage, transportation, and cost saving. In this study, the bovine pericardium was treated with a freeze-drying protectant composed of polyethylene glycol (PEG) and trehalose (Tre), and then freeze-dried. The results demonstrated that the mechanical properties of the pericardium treated with PEG + 10% w/v Tre were superior to those of the pericardium fixed with glutaraldehyde (GA). The wet state water content of the rehydrated pericardium, determined using the Karl Fischer method, was (74.81 ± 1.44)%, which was comparable to that of the GA-fixed pericardium. The dry state water content was significantly reduced to (8.64 ± 1.52)%, indicating effective dehydration during the freeze-drying process. Differential scanning calorimetry (DSC) testing revealed that the thermal shrinkage temperature of the pericardium was (84.96 ± 0.49) ℃, higher than that of the GA-fixed pericardium (83.14 ± 0.11) ℃, indicating greater thermal stability. Fourier transform infrared spectroscopy (FTIR) results showed no damage to the protein structure during freeze-drying. Hematoxylin and eosin (HE) staining demonstrated that the freeze-drying process reduced pore formation, prevented ice crystal growth, and resulted in a tighter arrangement of tissue fibers. The frozen-dried bovine pericardium was subjected to tests for cell viability and hemolysis rate. The results revealed a cell proliferation rate of (77.87 ± 0.49)%, corresponding to a toxicity grade of 1. Additionally, the hemolysis rate was (0.17 ± 0.02)%, which is below the standard of 5%. These findings indicated that the frozen-dried bovine pericardium exhibited satisfactory performance in terms of cytotoxicity and hemolysis, thus meeting the relevant standards. In summary, the performance of the bovine pericardium treated with PEG + 10% w/v Tre and subjected to freeze-drying could meet the required standards.

Citation： HUANG Wei, LI Weijie, LIU Baolin. Effects of freeze-drying bovine pericardium using a combination of polyethylene glycol and trehalose. Journal of Biomedical Engineering, 2024, 41(2): 368-375. doi: 10.7507/1001-5515.202311035 Copy

1.	Iung B, Vahanian A. Epidemiology of valvular heart disease in the adult. Nat Rev Cardiol, 2011, 8(3): 162-172.
2.	Roberts W C, Makhdumi M, Salam Y M. Body mass index in patients with operatively-excised congenitally bicuspid aortic valves comparing those with stenotic to those with purely regurgitant valves. Am J Cardiol, 2022, 181: 102-104.
3.	Snyder Y, Jana S. Strategies for development of decellularized heart valve scaffolds for tissue engineering. Biomaterials, 2022, 288: 121675.
4.	Yang Y, Wang Z, Chen Z, et al. Current status and etiology of valvular heart disease in China: a population-based survey. BMC Cardiovasc Disord, 2021, 21(1): 339.
5.	Tam H, Zhang W, Feaver K R, et al. A novel crosslinking method for improved tear resistance and biocompatibility of tissue based biomaterials. Biomaterials, 2015, 66: 83-91.
6.	Attia R Q, Raja S G. Surgical pericardial heart valves: 50 Years of evolution. Int J Surg, 2021, 94: 106121.
7.	Bezuidenhout D, Williams D F, Zilla P. Polymeric heart valves for surgical implantation, catheter-based technologies and heart assist devices. Biomaterials, 2015, 36: 6-25.
8.	Hofferberth S C, Saeed M Y, Tomholt L, et al. A geometrically adaptable heart valve replacement. Sci Transl Med, 2020, 12(531): eaay4006.
9.	Sundt T M, Jneid H. Guideline update on indications for transcatheter aortic valve implantation based on the 2020 American College of Cardiology/American Heart Association Guidelines for Management of Valvular Heart Disease. JAMA Cardiol, 2021, 6(9): 1088-1089.
10.	Tam D Y, Rocha R V, Wijeysundera H C, et al. Surgical valve selection in the era of transcatheter aortic valve replacement in the Society of Thoracic Surgeons Database. J Thorac Cardiovasc Surg, 2020, 159(2): 416-427.
11.	Head S J, Celik M, Kappetein A P. Mechanical versus bioprosthetic aortic valve replacement. Eur Heart J, 2017, 38(28): 2183-2191.
12.	Grebenik E A, Gafarova E R, Istranov L P, et al. Mammalian pericardium-based bioprosthetic materials in xenotransplantation and tissue engineering. Biotechnol J, 2020, 15(8): e1900334.
13.	Polak R, Rodas A C D, Chicoma D L, et al. Inhibition of calcification of bovine pericardium after treatment with biopolymers, E-beam irradiation and in vitro endothelization. Mater Sci Eng C, 2013, 33(1): 85-90.
14.	Vesely I. The evolution of bioprosthetic heart valve design and its impact on durability. Cardiovasc Pathol, 2003, 12(5): 277-286.
15.	Sanchez D M, Gaitan D M, Leon A F, et al. Fixation of vascular grafts with increased glutaraldehyde concentration enhances mechanical properties without increasing calcification. ASAIO J, 2007, 53(3): 257-262.
16.	Brockbank K G, Wright G J, Yao H, et al. Allogeneic heart valve storage above the glass transition at -80°C. Ann Thorac Surg, 2011, 91(6): 1829-1835.
17.	Salinas S D, Clark M M, Amini R. The effects of −80 °C short-term storage on the mechanical response of tricuspid valve leaflets. J Biomech, 2020, 98: 109462.
18.	Sui Y, Fan Q, Wang B, et al. Ice-free cryopreservation of heart valve tissue: The effect of adding MitoQ to a VS83 formulation and its influence on mitochondrial dynamics. Cryobiology, 2018, 81: 153-159.
19.	Zouhair S, Aguiari P, Iop L, et al. Preservation strategies for decellularized pericardial scaffolds for off-the-shelf availability. Acta Biomater, 2019, 84: 208-221.
20.	Brockbank K G, Lightfoot F G, Song Y C, et al. Interstitial ice formation in cryopreserved homografts: a possible cause of tissue deterioration and calcification in vivo. J Heart Valve Dis, 2000, 9(2): 200-206.
21.	Merivaara A, Zini J, Koivunotko E, et al. Preservation of biomaterials and cells by freeze-drying: Change of paradigm. J Control Release, 2021, 336: 480-498.
22.	Bjelosevic M, Seljak K B, Trstenjak U, et al. Aggressive conditions during primary drying as a contemporary approach to optimise freeze-drying cycles of biopharmaceuticals. Eur J Pharm Sci, 2018, 122: 292-302.
23.	Mehanna M M, Abla K K. Recent advances in freeze-drying: variables, cycle optimization, and innovative techniques. Pharm Dev Technol, 2022, 27(8): 904-923.
24.	Leirner A A, Tattini V, Pitombo R N M. Prospects in lyophilization of bovine pericardium. Artif Organs, 2009, 33(3): 221-229.
25.	Wang S, Oldenhof H, Goecke T, et al. Sucrose diffusion in decellularized heart valves for freeze-drying. Tissue Eng Part C Methods, 2015, 21(9): 922-931.
26.	Goecke T, Theodoridis K, Tudorache I, et al. In vivo performance of freeze-dried decellularized pulmonary heart valve allo- and xenografts orthotopically implanted into juvenile sheep. Acta Biomater, 2018, 68: 41-52.
27.	Bhatnagar B, Tchessalov S. Advances in freeze drying of biologics and future challenges and opportunities. Drying Technologies for Biotechnology and Pharmaceutical Applications, 2020: 137-177.
28.	Payne K J, Veis A. Fourier transform IR spectroscopy of collagen and gelatin solutions: deconvolution of the amide I band for conformational studies. Biopolymers, 1988, 27(11): 1749-1760.
29.	Vidal Bde C, Mello M L. Collagen type I amide I band infrared spectroscopy. Micron, 2011, 42(3): 283-289.
30.	Whelan A, Duffy J, Gaul R T, et al. Collagen fibre orientation and dispersion govern ultimate tensile strength, stiffness and the fatigue performance of bovine pericardium. J Mech Behav Biomed Mater, 2019, 90: 54-60.

1. Iung B, Vahanian A. Epidemiology of valvular heart disease in the adult. Nat Rev Cardiol, 2011, 8(3): 162-172.
2. Roberts W C, Makhdumi M, Salam Y M. Body mass index in patients with operatively-excised congenitally bicuspid aortic valves comparing those with stenotic to those with purely regurgitant valves. Am J Cardiol, 2022, 181: 102-104.
3. Snyder Y, Jana S. Strategies for development of decellularized heart valve scaffolds for tissue engineering. Biomaterials, 2022, 288: 121675.
4. Yang Y, Wang Z, Chen Z, et al. Current status and etiology of valvular heart disease in China: a population-based survey. BMC Cardiovasc Disord, 2021, 21(1): 339.
5. Tam H, Zhang W, Feaver K R, et al. A novel crosslinking method for improved tear resistance and biocompatibility of tissue based biomaterials. Biomaterials, 2015, 66: 83-91.
6. Attia R Q, Raja S G. Surgical pericardial heart valves: 50 Years of evolution. Int J Surg, 2021, 94: 106121.
7. Bezuidenhout D, Williams D F, Zilla P. Polymeric heart valves for surgical implantation, catheter-based technologies and heart assist devices. Biomaterials, 2015, 36: 6-25.
8. Hofferberth S C, Saeed M Y, Tomholt L, et al. A geometrically adaptable heart valve replacement. Sci Transl Med, 2020, 12(531): eaay4006.
9. Sundt T M, Jneid H. Guideline update on indications for transcatheter aortic valve implantation based on the 2020 American College of Cardiology/American Heart Association Guidelines for Management of Valvular Heart Disease. JAMA Cardiol, 2021, 6(9): 1088-1089.
10. Tam D Y, Rocha R V, Wijeysundera H C, et al. Surgical valve selection in the era of transcatheter aortic valve replacement in the Society of Thoracic Surgeons Database. J Thorac Cardiovasc Surg, 2020, 159(2): 416-427.
11. Head S J, Celik M, Kappetein A P. Mechanical versus bioprosthetic aortic valve replacement. Eur Heart J, 2017, 38(28): 2183-2191.
12. Grebenik E A, Gafarova E R, Istranov L P, et al. Mammalian pericardium-based bioprosthetic materials in xenotransplantation and tissue engineering. Biotechnol J, 2020, 15(8): e1900334.
13. Polak R, Rodas A C D, Chicoma D L, et al. Inhibition of calcification of bovine pericardium after treatment with biopolymers, E-beam irradiation and in vitro endothelization. Mater Sci Eng C, 2013, 33(1): 85-90.
14. Vesely I. The evolution of bioprosthetic heart valve design and its impact on durability. Cardiovasc Pathol, 2003, 12(5): 277-286.
15. Sanchez D M, Gaitan D M, Leon A F, et al. Fixation of vascular grafts with increased glutaraldehyde concentration enhances mechanical properties without increasing calcification. ASAIO J, 2007, 53(3): 257-262.
16. Brockbank K G, Wright G J, Yao H, et al. Allogeneic heart valve storage above the glass transition at -80°C. Ann Thorac Surg, 2011, 91(6): 1829-1835.
17. Salinas S D, Clark M M, Amini R. The effects of −80 °C short-term storage on the mechanical response of tricuspid valve leaflets. J Biomech, 2020, 98: 109462.
18. Sui Y, Fan Q, Wang B, et al. Ice-free cryopreservation of heart valve tissue: The effect of adding MitoQ to a VS83 formulation and its influence on mitochondrial dynamics. Cryobiology, 2018, 81: 153-159.
19. Zouhair S, Aguiari P, Iop L, et al. Preservation strategies for decellularized pericardial scaffolds for off-the-shelf availability. Acta Biomater, 2019, 84: 208-221.
20. Brockbank K G, Lightfoot F G, Song Y C, et al. Interstitial ice formation in cryopreserved homografts: a possible cause of tissue deterioration and calcification in vivo. J Heart Valve Dis, 2000, 9(2): 200-206.
21. Merivaara A, Zini J, Koivunotko E, et al. Preservation of biomaterials and cells by freeze-drying: Change of paradigm. J Control Release, 2021, 336: 480-498.
22. Bjelosevic M, Seljak K B, Trstenjak U, et al. Aggressive conditions during primary drying as a contemporary approach to optimise freeze-drying cycles of biopharmaceuticals. Eur J Pharm Sci, 2018, 122: 292-302.
23. Mehanna M M, Abla K K. Recent advances in freeze-drying: variables, cycle optimization, and innovative techniques. Pharm Dev Technol, 2022, 27(8): 904-923.
24. Leirner A A, Tattini V, Pitombo R N M. Prospects in lyophilization of bovine pericardium. Artif Organs, 2009, 33(3): 221-229.
25. Wang S, Oldenhof H, Goecke T, et al. Sucrose diffusion in decellularized heart valves for freeze-drying. Tissue Eng Part C Methods, 2015, 21(9): 922-931.
26. Goecke T, Theodoridis K, Tudorache I, et al. In vivo performance of freeze-dried decellularized pulmonary heart valve allo- and xenografts orthotopically implanted into juvenile sheep. Acta Biomater, 2018, 68: 41-52.
27. Bhatnagar B, Tchessalov S. Advances in freeze drying of biologics and future challenges and opportunities. Drying Technologies for Biotechnology and Pharmaceutical Applications, 2020: 137-177.
28. Payne K J, Veis A. Fourier transform IR spectroscopy of collagen and gelatin solutions: deconvolution of the amide I band for conformational studies. Biopolymers, 1988, 27(11): 1749-1760.
29. Vidal Bde C, Mello M L. Collagen type I amide I band infrared spectroscopy. Micron, 2011, 42(3): 283-289.
30. Whelan A, Duffy J, Gaul R T, et al. Collagen fibre orientation and dispersion govern ultimate tensile strength, stiffness and the fatigue performance of bovine pericardium. J Mech Behav Biomed Mater, 2019, 90: 54-60.

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