Research Papers

Microstructure and Mechanical Property of Glutaraldehyde-Treated Porcine Pulmonary Ligament

[+] Author and Article Information
Huan Chen, Xuefeng Zhao

California Medical Innovations Institute,
San Diego, CA 92121

Zachary C. Berwick

3DT Holdings, LLC,
San Diego, CA 92121

Joshua F. Krieger, Sean Chambers

Cook Medical, Inc.,
Bloomington, IN 47402

Ghassan S. Kassab

California Medical Innovations Institute,
San Diego, CA 92121
e-mail: gkassab@calmi2.org

1H. Chen and X. Zhao contributed equally to this work.

2Corresponding author.

Manuscript received June 22, 2015; final manuscript received March 21, 2016; published online April 27, 2016. Assoc. Editor: Hai-Chao Han.

J Biomech Eng 138(6), 061003 (Apr 27, 2016) (9 pages) Paper No: BIO-15-1308; doi: 10.1115/1.4033300 History: Received June 22, 2015; Revised March 21, 2016

There is a significant need for fixed biological tissues with desired structural and material constituents for tissue engineering applications. Here, we introduce the lung ligament as a fixed biological material that may have clinical utility for tissue engineering. To characterize the lung tissue for potential clinical applications, we studied glutaraldehyde-treated porcine pulmonary ligament (n = 11) with multiphoton microscopy (MPM) and conducted biaxial planar experiments to characterize the mechanical property of the tissue. The MPM imaging revealed that there are generally two families of collagen fibers distributed in two distinct layers: The first family largely aligns along the longitudinal direction with a mean angle of θ = 10.7 ± 9.3 deg, while the second one exhibits a random distribution with a mean θ = 36.6 ± 27.4. Elastin fibers appear in some intermediate sublayers with a random orientation distribution with a mean θ = 39.6 ± 23 deg. Based on the microstructural observation, a microstructure-based constitutive law was proposed to model the elastic property of the tissue. The material parameters were identified by fitting the model to the biaxial stress–strain data of specimens, and good fitting quality was achieved. The parameter e0 (which denotes the strain beyond which the collagen can withstand tension) of glutaraldehyde-treated tissues demonstrated low variability implying a relatively consistent collagen undulation in different samples, while the stiffness parameters for elastin and collagen fibers showed relatively greater variability. The fixed tissues presented a smaller e0 than that of fresh specimen, confirming that glutaraldehyde crosslinking increases the mechanical strength of collagen-based biomaterials. The present study sheds light on the biomechanics of glutaraldehyde-treated porcine pulmonary ligament that may be a candidate for tissue engineering.

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Duan, X. , and Sheardown, H. , 2006, “ Dendrimer Crosslinked Collagen as a Corneal Tissue Engineering Scaffold: Mechanical Properties and Corneal Epithelial Cell Interactions,” Biomaterials, 27(26), pp. 4608–4617. [CrossRef] [PubMed]
Geiger, M. , Li, R. H. , and Friess, W. , 2003, “ Collagen Sponges for Bone Regeneration With rhBMP-2,” Adv. Drug Delivery Rev., 55(12), pp. 1613–1629. [CrossRef]
Cooper, C. , Moss, A. A. , Buy, J. N. , and Stark, D. D. , 1983, “ CT Appearance of the Normal Inferior Pulmonary Ligament,” Am. J. Roentgenol., 141(2), pp. 237–240. [CrossRef]
Godwin, J. D. , Vock, P. , and Osborne, D. R. , 1983, “ CT of the Pulmonary Ligament,” Am. J. Roentgenol., 141(2), pp. 231–236. [CrossRef]
Rabinowitz, J. G. , and Wolf, B. S. , 1966, “ Roentgen Significance of the Pulmonary Ligament,” Radiology, 87(6), pp. 1013–1020. [CrossRef] [PubMed]
DeLaria, G. A. , Phifer, T. , Roy, J. , Tu, R. , Thyagarajan, K. , and Quijano, R. C. , 1993, “ Hemodynamic Evaluation of a Bioprosthetic Venous Prosthesis,” J. Vasc. Surg., 18(4), pp. 577–584; discussion 584–586. [CrossRef] [PubMed]
Vesely, I. , 2005, “ Heart Valve Tissue Engineering,” Circ. Res., 97(8), pp. 743–755. [CrossRef] [PubMed]
Haugh, M. G. , Murphy, C. M. , McKiernan, R. C. , Altenbuchner, C. , and O'Brien, F. J. , 2011, “ Crosslinking and Mechanical Properties Significantly Influence Cell Attachment, Proliferation, and Migration Within Collagen Glycosaminoglycan Scaffolds,” Tissue Eng. Part A, 17(9–10), pp. 1201–1208. [CrossRef] [PubMed]
Xu, B. , Chow, M.-J. , Zhang, Y. , Xu, B. , Chow, M.-J. , and Zhang, Y. , 2011, “ Experimental and Modeling Study of Collagen Scaffolds With the Effects of Crosslinking and Fiber Alignment,” Int. J. Biomater., 2011, p. 172389. [CrossRef] [PubMed]
Billiar, K. L. , and Sacks, M. S. , 2000, “ Biaxial Mechanical Properties of the Native and Glutaraldehyde-Treated Aortic Valve Cusp: Part II—A Structural Constitutive Model,” ASME J. Biomech. Eng., 122(4), pp. 327–335. [CrossRef]
Vesely, I. , and Lozon, A. , 1993, “ Natural Preload of Aortic Valve Leaflet Components During Glutaraldehyde Fixation: Effects on Tissue Mechanics,” J. Biomech., 26(2), pp. 121–131. [CrossRef] [PubMed]
Holzapfel, G. A. , Gasser, T. C. , and Ogden, R. W. , 2000, “ A New Constitutive Framework for Arterial Wall Mechanics and a Comparative Study of Material Models,” J. Elasticity Phys. Sci. Solids, 61(1–3), pp. 1–48. [CrossRef]
Kroon, M. , and Holzapfel, G. A. , 2008, “ A New Constitutive Model for Multi-Layered Collagenous Tissues,” J. Biomech., 41(12), pp. 2766–2771. [CrossRef] [PubMed]
Chen, H. , Luo, T. , Zhao, X. , Lu, X. , Huo, Y. , and Kassab, G. S. , 2013, “ Microstructural Constitutive Model of Active Coronary Media,” Biomaterials, 34(31), pp. 7575–7583. [CrossRef] [PubMed]
Hollander, Y. , Durban, D. , Lu, X. , Kassab, G. S. , and Lanir, Y. , 2011, “ Experimentally Validated Micro Structural 3D Constitutive Model of Coronary Arterial Media,” ASME J. Biomech. Eng., 133(3), p. 031007. [CrossRef]
Lanir, Y. , 1983, “ Constitutive Equations for Fibrous Connective Tissues,” J. Biomech., 16(1), pp. 1–12. [CrossRef] [PubMed]
Chen, H. , Liu, Y. , Zhao, X. , Lanir, Y. , and Kassab, G. S. , 2011, “ A Micromechanics Finite-Strain Constitutive Model of Fibrous Tissue,” J. Mech. Phys. Solids, 59(9), pp. 1823–1837. [CrossRef] [PubMed]
Zulliger, M. A. , Fridez, P. , Hayashi, K. , and Stergiopulos, N. , 2004, “ A Strain Energy Function for Arteries Accounting for Wall Composition and Structure,” J. Biomech., 37(7), pp. 989–1000. [CrossRef] [PubMed]
Lanir, Y. , 1979, “ A Structural Theory for the Homogeneous Biaxial Stress-Strain Relationships in Flat Collagenous Tissues,” J. Biomech., 12(6), pp. 423–436. [CrossRef] [PubMed]
Dahl, S. L. M. , Vaughn, M. E. , and Niklason, L. E. , 2007, “ An Ultrastructural Analysis of Collagen in Tissue Engineered Arteries,” Ann. Biomed. Eng., 35(10), pp. 1749–1755. [CrossRef] [PubMed]
Einat, R. , and Yoram, L. , 2009, “ Recruitment Viscoelasticity of the Tendon,” ASME J. Biomech. Eng., 131(11), p. 111008. [CrossRef]
Farquhar, T. , Dawson, P. R. , and Torzilli, P. A. , 1990, “ A Microstructural Model for the Anisotropic Drained Stiffness of Articular Cartilage,” ASME J. Biomech. Eng., 112(4), pp. 414–425. [CrossRef]
Lokshin, O. , and Lanir, Y. , 2009, “ Micro and Macro Rheology of Planar Tissues,” Biomaterials, 30(17), pp. 3118–3127. [CrossRef] [PubMed]
Sacks, M. S. , 2003, “ Incorporation of Experimentally-Derived Fiber Orientation Into a Structural Constitutive Model for Planar Collagenous Tissues,” ASME J. Biomech. Eng., 125(2), pp. 280–287. [CrossRef]
Campagnola, P. J. , Clark, H. A. , Mohler, W. A. , Lewis, A. , and Loew, L. M. , 2001, “ Second-Harmonic Imaging Microscopy of Living Cells,” J. Biomed. Opt., 6(3), pp. 277–286. [CrossRef] [PubMed]
Gauderon, R. , Lukins, P. B. , and Sheppard, C. J. , 2001, “ Optimization of Second-Harmonic Generation Microscopy,” Micron, 32(7), pp. 691–700. [CrossRef] [PubMed]
Chen, H. , Liu, Y. , Slipchenko, M. N. , Zhao, X. , Cheng, J.-X. , and Kassab, G. S. , 2011, “ The Layered Structure of Coronary Adventitia Under Mechanical Load,” Biophys. J., 101(11), pp. 2555–2562. [CrossRef] [PubMed]
Chen, H. , Slipchenko, M. N. , Liu, Y. , Zhao, X. , Cheng, J.-X. , Lanir, Y. , and Kassab, G. S. , 2013, “ Biaxial Deformation of Collagen and Elastin Fibers in Coronary Adventitia,” J. Appl. Physiol., 115(11), pp. 1683–1693. [CrossRef] [PubMed]
Mansfield, J. , Yu, J. , Attenburrow, D. , Moger, J. , Tirlapur, U. , Urban, J. , Cui, Z. , and Winlove, P. , 2009, “ The Elastin Network: Its Relationship With Collagen and Cells in Articular Cartilage as Visualized by Multiphoton Microscopy,” J. Anat., 215(6), pp. 682–691. [CrossRef] [PubMed]
Raub, C. B. , Unruh, J. , Suresh, V. , Krasieva, T. , Lindmo, T. , Gratton, E. , Tromberg, B. J. , and George, S. C. , 2008, “ Image Correlation Spectroscopy of Multiphoton Images Correlates With Collagen Mechanical Properties,” Biophys. J., 94(6), pp. 2361–2373. [CrossRef] [PubMed]
Schriefl, A. J. , Reinisch, A. J. , Sankaran, S. , Pierce, D. M. , and Holzapfel, G. A. , 2012, “ Quantitative Assessment of Collagen Fibre Orientations From Two-Dimensional Images of Soft Biological Tissues,” J. R. Soc., Interface, 9(76), pp. 3081–3093. [CrossRef]
Chow, M.-J. , Turcotte, R. , Lin, C. P. , and Zhang, Y. , 2014, “ Arterial Extracellular Matrix: A Mechanobiological Study of the Contributions and Interactions of Elastin and Collagen,” Biophys. J., 106(12), pp. 2684–2692. [CrossRef] [PubMed]
Fata, B. , Carruthers, C. A. , Gibson, G. , Watkins, S. C. , Gottlieb, D. , Mayer, J. E. , and Sacks, M. S. , 2013, “ Regional Structural and Biomechanical Alterations of the Ovine Main Pulmonary Artery During Postnatal Growth,” ASME J. Biomech. Eng., 135(2), p. 021022. [CrossRef]
Timmins, L. H. , Wu, Q. , Yeh, A. T. , Moore, J. E. , and Greenwald, S. E. , 2010, “ Structural Inhomogeneity and Fiber Orientation in the Inner Arterial Media,” Am. J. Physiol.: Heart Circ. Physiol., 298(5), pp. H1537–H1545. [CrossRef] [PubMed]
Zoumi, A. , Lu, X. , Kassab, G. S. , and Tromberg, B. J. , 2004, “ Imaging Coronary Artery Microstructure Using Second-Harmonic and Two-Photon Fluorescence Microscopy,” Biophys. J., 87(4), pp. 2778–2786. [CrossRef] [PubMed]
Kato, Y. P. , Christiansen, D. L. , Hahn, R. A. , Shieh, S. J. , Goldstein, J. D. , and Silver, F. H. , 1989, “ Mechanical Properties of Collagen Fibres: A Comparison of Reconstituted and Rat Tail Tendon Fibres,” Biomaterials, 10(1), pp. 38–42. [CrossRef] [PubMed]
Minns, R. J. , Soden, P. D. , and Jackson, D. S. , 1973, “ The Role of the Fibrous Components and Ground Substance in the Mechanical Properties of Biological Tissues: A Preliminary Investigation,” J. Biomech., 6(2), pp. 153–165. [CrossRef] [PubMed]
Wolinsky, H. , and Glagov, S. , 1964, “ Structural Basis for the Static Mechanical Properties of the Aortic Media,” Circ. Res., 14(5), pp. 400–413. [CrossRef] [PubMed]
Bailey, A. J. , Light, N. D. , and Atkins, E. D. T. , 1980, “ Chemical Cross-Linking Restrictions on Models for the Molecular Organization of the Collagen Fibre,” Nature, 288(5789), pp. 408–410. [CrossRef] [PubMed]
Khor, E. , 1997, “ Methods for the Treatment of Collagenous Tissues for Bioprostheses,” Biomaterials, 18(2), pp. 95–105. [CrossRef] [PubMed]
Weadock, K. , Olson, R. M. , and Silver, F. H. , 1983, “ Evaluation of Collagen Crosslinking Techniques,” Biomater., Med. Dev., Artif. Organs, 11(4), pp. 293–318. [CrossRef]
Sheu, M.-T. , Huang, J.-C. , Yeh, G.-C. , and Ho, H.-O. , 2001, “ Characterization of Collagen Gel Solutions and Collagen Matrices for Cell Culture,” Biomaterials, 22(13), pp. 1713–1719. [CrossRef] [PubMed]
Gauvin, R. , Marinov, G. , Mehri, Y. , Klein, J. , Li, B. , Larouche, D. , Guzman, R. , Zhang, Z. , Germain, L. , and Guidoin, R. , 2013, “ A Comparative Study of Bovine and Porcine Pericardium to Highlight Their Potential Advantages to Manufacture Percutaneous Cardiovascular Implants,” J. Biomater. Appl., 28(4), pp. 552–565. [CrossRef] [PubMed]
Lam, M. T. , and Wu, J. C. , 2012, “ Biomaterial Applications in Cardiovascular Tissue Repair and Regeneration,” Expert Rev. Cardiovasc. Ther., 10(8), pp. 1039–1049. [CrossRef] [PubMed]
Aguiari, P. , Fiorese, M. , Iop, L. , Gerosa, G. , and Bagno, A. , 2015, “ Mechanical Testing of Pericardium for Manufacturing Prosthetic Heart Valves,” Interact. Cardiovasc. Thorac. Surg., 22(1), pp. 72–84. [CrossRef] [PubMed]
Inoue, H. , Iguro, Y. , Matsumoto, H. , Ueno, M. , Higashi, A. , and Sakata, R. , 2009, “ Right Hemi-Reconstruction of the Left Atrium Using Two Equine Pericardial Patches for Recurrent Malignant Fibrous Histiocytoma: Report of a Case,” Surg. Today, 39(8), pp. 710–712. [CrossRef] [PubMed]
Shinn, S. H. , Sung, K. , Park, P. W. , Lee, Y. T. , Kim, W. S. , Yang, J.-H. , Jun, T.-G. , Lee, S.-C. , and Park, S. W. , 2009, “ Results of Annular Reconstruction With a Pericardial Patch in Active Infective Endocarditis,” J. Heart Valve Dis., 18(3), pp. 315–320. [PubMed]
Delille, J. P. , Hernigou, A. , Sene, V. , Chatellier, G. , Boudeville, J. C. , Challande, P. , and Plainfosse, M. C. , 1999, “ Maximal Thickness of the Normal Human Pericardium Assessed by Electron-Beam Computed Tomography,” Eur. Radiol., 9(6), pp. 1183–1189. [CrossRef] [PubMed]
Nam, J. , Choi, S.-Y. , Sung, S.-C. , Lim, H.-G. , Park, S. , Kim, S.-H. , and Kim, Y. J. , 2012, “ Changes of the Structural and Biomechanical Properties of the Bovine Pericardium After the Removal of α-Gal Epitopes by Decellularization and α-Galactosidase Treatment,” Korean J. Thorac. Cardiovasc. Surg., 45(6), pp. 380–389. [CrossRef] [PubMed]
Lu, S.-H. , Sacks, M. S. , Chung, S. Y. , Gloeckner, D. C. , Pruchnic, R. , Huard, J. , de Groat, W. C. , and Chancellor, M. B. , 2005, “ Biaxial Mechanical Properties of Muscle-Derived Cell Seeded Small Intestinal Submucosa for Bladder Wall Reconstitution,” Biomaterials, 26(4), pp. 443–449. [CrossRef] [PubMed]
Prevel, C. D. , Eppley, B. L. , Summerlin, D. J. , Sidner, R. , Jackson, J. R. , McCarty, M. , and Badylak, S. F. , 1995, “ Small Intestinal Submucosa: Utilization as a Wound Dressing in Full-Thickness Rodent Wounds,” Ann. Plast. Surg., 35(4), pp. 381–388. [CrossRef] [PubMed]
Schallberger, S. P. , Stanley, B. J. , Hauptman, J. G. , and Steficek, B. A. , 2008, “ Effect of Porcine Small Intestinal Submucosa on Acute Full-Thickness Wounds in Dogs,” Vet. Surg., 37(6), pp. 515–524. [CrossRef] [PubMed]
Boyd, W. D. , Johnson, W. E. , Sultan, P. K. , Deering, T. F. , and Matheny, R. G. , 2010, “ Pericardial Reconstruction Using an Extracellular Matrix Implant Correlates With Reduced Risk of Postoperative Atrial Fibrillation in Coronary Artery Bypass Surgery Patients,” Heart Surg. Forum, 13(5), pp. E311–E316. [CrossRef] [PubMed]
Fallon, A. , Goodchild, T. , Wang, R. , and Matheny, R. G. , 2012, “ Remodeling of Extracellular Matrix Patch Used for Carotid Artery Repair,” J. Surg. Res., 175(1), pp. e25–e34. [CrossRef] [PubMed]
Choy, J. S. , Mathieu-Costello, O. , and Kassab, G. S. , 2005, “ The Effect of Fixation and Histological Preparation on Coronary Artery Dimensions,” Ann. Biomed. Eng., 33(8), pp. 1027–1033. [CrossRef] [PubMed]
Kothari, H. , Kaur, G. , Sahoo, S. , Idell, S. , Rao, L. V. M. , and Pendurthi, U. , 2009, “ Plasmin Enhances Cell Surface Tissue Factor Activity in Mesothelial and Endothelial Cells,” J. Thromb. Haemostasis, 7(1), pp. 121–131. [CrossRef]
Louagie, Y. , Legrand-Monsieur, A. , Remacle, C. , Maldague, P. , Lambotte, L. , and Ponlot, R. , 1986, “ Morphology and Fibrinolytic Activity of Canine Autogenous Mesothelium Used as Venous Substitute,” Res. Exp. Med., 186(4), pp. 239–247. [CrossRef]


Grahic Jump Location
Fig. 1

A schematic figure of biaxial testing setup

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Fig. 2

Representative SHG and TPEF images demonstrating collagen and elastin structure at different depths of an unloaded pulmonary ligament specimen. (a, d) TPEF images for elastin, (b, e) SHG images for collagen, and (c, f) merged images. Row-wise: Z = 22.1 and 58.1 μm. columnwise: elastin, collagen, merged.

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Fig. 3

Representative SHG and TPEF images demonstrating fiber arrangement at different depths of a specimen under equibiaxial stretch (E11=E22=0.6). Row-wise: elastin, collagen, merged; columnwise: Z = 9.9, 21.9, 37.9, and 69.9 μm.

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Fig. 4

Quantitative measurement of fiber orientation angle based on SHG and TPEF images. (a) and (b) Fiber orientation angle measured based on images in Fig. 3. (c) The mean orientation angle at ten different depths of pulmonary ligament specimens. Error bar indicates standard deviation.

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Fig. 5

The stress–strain curves of fresh and glutaraldehyde-treated pulmonary ligaments, respectively, based on equibiaxial testing data. Symbols denote experimental mean stress components, and solid lines denote model predicted stresses. Error bar indicates standard error.

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Fig. 6

The stress–strain curves of a representative glutaraldehyde-treated sample under three biaxial protocols E11:E22  = 1.3:2, 2:1.3, 2:2. Symbols and solid lines denote experimental data and model predictions, respectively.

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Fig. 7

The stress–strain curves of a representative glutaraldehyde-treated sample under all biaxial protocols. Symbols and solid lines denote experimental data and model predictions, respectively.



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