- Email: [email protected]
VISCOELASTIC PROPERTIES OF AN ACELLULAR, XENOGENIC TISSUE ENGINEERING SCAFFOLD
Presentation 0031, Soft Tissue. 13:30, Room 105
S418
VISCOELASTIC PROPERTIES OF AN ACELLULAR, XENOGENIC TISSUE ENGINEERING SCAFFOLD R.W. Chan1, 2, C.C. Xu2 and M. Rodriguez2 1 Otolaryngology – Head & Neck Surgery, 2 Graduate Program in Biomedical Engineering, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9035, U.S.A. Email: [email protected] INTRODUCTION An implantable tissue replacement for the human vocal fold lamina propria has been developed in our laboratory. A novel proteolytic enzyme-free, detergent-free saline based protocol was employed to decellularize the bovine vocal fold lamina propria into a three-dimensional, biodegradable, acellular extracellular matrix (ECM) scaffold that can be repopulated with fibroblasts for the repair and replacement of pathological tissue. Viscoelastic properties of the scaffold under shear deformation were quantified at frequencies of up to 250Hz. Results showed that the elastic shear modulus and viscous shear modulus of the fibroblast-repopulated scaffold were comparable to those of the human vocal fold lamina propria, specifically the superficial layer (vocal fold cover) which is primarily responsible for vocal fold oscillation. These findings supported the biomechanical benefits of the scaffold as a xenograft for vocal fold reconstruction and regeneration.
Figure 1: Morphology of the bovine acellular scaffold (Masson’s trichrome staining). Intact basement membrane structure (solid arrow) and an ECM network of collagen (dashed arrow) can be observed (total magnification = 40 X) Figure 2 shows that the shear elasticity or stiffness (G’) of the acellular scaffold was higher than that of the native bovine vocal fold, but became much lower to a level comparable to that of the human vocal fold cover upon recellularization with human vocal fold fibroblasts.
METHODS A novel proteolytic enzyme-free, detergent-free saline based protocol was developed to decellularize the bovine vocal fold lamina propria into a biodegradable, acellular xenogenic ECM scaffold [1]. Briefly, the protocol involved the use of a highly hypertonic salt (3M NaCl) solution creating an extreme osmotic stress for the native cells, followed by cycles of osmotic stress with incubation of the specimens in isotonic PBS solution, 70% ethanol, and PBS solution again. Ribonuclease and deoxyribonuclease were also added to remove the nucleic materials. Viscoelastic shear properties of the scaffold were quantified by a controlled-strain, linear simple shear rheometer system that is capable of direct measurements of the complex shear modulus (G*) of low-modulus biomaterials and soft tissues at frequencies of up to 250Hz [2]. The elastic shear modulus (G’), viscous shear modulus (G’’), and dynamic viscosity (K’) of the specimens were quantified. RESULTS AND DISCUSSION Figure 1 shows the structure of the decellularized scaffold, demonstrating the complete removal of native bovine epithelial cells and fibroblasts, with the basement membrane and the fibrous proteins well preserved for repopulation by human cells. The three-dimensional structure of the ECM protein network was also intact. Infiltration of cells in the acellular scaffold was observed 7-10 days after seeding of primary culture human vocal fold fibroblasts onto the lateral, cut surface of the scaffold, corresponding to the deep layer of the vocal fold lamina propria.
Journal of Biomechanics 40(S2)
Figure 2: Elastic shear modulus (G’) of the bovine acellular scaffold, the fibroblast-repopulated scaffold and the human vocal fold lamina propria (vocal fold cover). These findings demonstrated the potential of the acellular scaffold as a substrate for the regeneration and reconstruction of the vocal fold lamina propria, particularly with regard to the biomechanical benefits of the scaffold for facilitating vocal fold oscillation and voice production. REFERENCES 1. Xu CC, et al. A biodegradable, acellular xenogeneic scaffold for regeneration of the vocal fold lamina propria. Tissue Eng, in press. 2. Chan RW, et al. 76th Annual Meeting of The Society of Rheology, Lubbock, TX, 2005. ACKNOWLEDGEMENTS This work was supported by NIH grant R01 DC006101.
XXI ISB Congress, Podium Sessions, Wednesday 4 July 2007
S418
VISCOELASTIC PROPERTIES OF AN ACELLULAR, XENOGENIC TISSUE ENGINEERING SCAFFOLD R.W. Chan1, 2, C.C. Xu2 and M. Rodriguez2 1 Otolaryngology – Head & Neck Surgery, 2 Graduate Program in Biomedical Engineering, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9035, U.S.A. Email: [email protected] INTRODUCTION An implantable tissue replacement for the human vocal fold lamina propria has been developed in our laboratory. A novel proteolytic enzyme-free, detergent-free saline based protocol was employed to decellularize the bovine vocal fold lamina propria into a three-dimensional, biodegradable, acellular extracellular matrix (ECM) scaffold that can be repopulated with fibroblasts for the repair and replacement of pathological tissue. Viscoelastic properties of the scaffold under shear deformation were quantified at frequencies of up to 250Hz. Results showed that the elastic shear modulus and viscous shear modulus of the fibroblast-repopulated scaffold were comparable to those of the human vocal fold lamina propria, specifically the superficial layer (vocal fold cover) which is primarily responsible for vocal fold oscillation. These findings supported the biomechanical benefits of the scaffold as a xenograft for vocal fold reconstruction and regeneration.
Figure 1: Morphology of the bovine acellular scaffold (Masson’s trichrome staining). Intact basement membrane structure (solid arrow) and an ECM network of collagen (dashed arrow) can be observed (total magnification = 40 X) Figure 2 shows that the shear elasticity or stiffness (G’) of the acellular scaffold was higher than that of the native bovine vocal fold, but became much lower to a level comparable to that of the human vocal fold cover upon recellularization with human vocal fold fibroblasts.
METHODS A novel proteolytic enzyme-free, detergent-free saline based protocol was developed to decellularize the bovine vocal fold lamina propria into a biodegradable, acellular xenogenic ECM scaffold [1]. Briefly, the protocol involved the use of a highly hypertonic salt (3M NaCl) solution creating an extreme osmotic stress for the native cells, followed by cycles of osmotic stress with incubation of the specimens in isotonic PBS solution, 70% ethanol, and PBS solution again. Ribonuclease and deoxyribonuclease were also added to remove the nucleic materials. Viscoelastic shear properties of the scaffold were quantified by a controlled-strain, linear simple shear rheometer system that is capable of direct measurements of the complex shear modulus (G*) of low-modulus biomaterials and soft tissues at frequencies of up to 250Hz [2]. The elastic shear modulus (G’), viscous shear modulus (G’’), and dynamic viscosity (K’) of the specimens were quantified. RESULTS AND DISCUSSION Figure 1 shows the structure of the decellularized scaffold, demonstrating the complete removal of native bovine epithelial cells and fibroblasts, with the basement membrane and the fibrous proteins well preserved for repopulation by human cells. The three-dimensional structure of the ECM protein network was also intact. Infiltration of cells in the acellular scaffold was observed 7-10 days after seeding of primary culture human vocal fold fibroblasts onto the lateral, cut surface of the scaffold, corresponding to the deep layer of the vocal fold lamina propria.
Journal of Biomechanics 40(S2)
Figure 2: Elastic shear modulus (G’) of the bovine acellular scaffold, the fibroblast-repopulated scaffold and the human vocal fold lamina propria (vocal fold cover). These findings demonstrated the potential of the acellular scaffold as a substrate for the regeneration and reconstruction of the vocal fold lamina propria, particularly with regard to the biomechanical benefits of the scaffold for facilitating vocal fold oscillation and voice production. REFERENCES 1. Xu CC, et al. A biodegradable, acellular xenogeneic scaffold for regeneration of the vocal fold lamina propria. Tissue Eng, in press. 2. Chan RW, et al. 76th Annual Meeting of The Society of Rheology, Lubbock, TX, 2005. ACKNOWLEDGEMENTS This work was supported by NIH grant R01 DC006101.
XXI ISB Congress, Podium Sessions, Wednesday 4 July 2007