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1、Acellular tissue engineering scaffolds for vocal fold regeneration Roger W. Chan Otolaryngology Head & Neck Surgery Graduate Program in Biomedical Engineering University of Texas Southwestern Medical Center Dallas, Texas 75390-9035, U. S. A. roger.chanutsouthwestern.edu Abstract A variety of xenogen
2、eic and allogeneic extracellular matrix (ECM) scaffolds have been promising in various tissue engineering applications, such as the porcine small intestinal submucosa. Yet no particular scaffolds have been optimal for vocal fold regeneration. The decellularized human umbilical vein (HUV) is a novel
3、allogeneic scaffold that has shown some promise for cardiovascular tissue engineering. This study examines the potential of the HUV as an acellular scaffold for engineering the vocal fold lamina propria, in order to develop an implantable tissue substitute that can promote a natural ECM remodeling r
4、esponse. A novel saline-based decellularization protocol developed in our laboratory was used to fabricate a three-dimensional (3-D), biodegradable, acellular scaffold from native HUV tissue. Histological examination and scanning electron microscopy indicated that native cells in the HUV were remove
5、d with a fine 3-D structure of proteins and proteoglycans well preserved. Primary human vocal fold fibroblasts were cultivated on the abluminal surface of the acellular scaffold in vitro. Significant proliferation and infiltration of the fibroblasts in the scaffold were observed. These findings supp
6、orted the biocompatibility of the HUV scaffold, and its promise for vocal fold reconstruction and regeneration. Keywords-larynx, vocal fold, extracellular matrix scaffolds I. INTRODUCTION The human vocal fold is a highly differentiated layered structure optimally designed for vibration and sound pro
7、duction. In particular, the vocal fold lamina propria is primarily an extracellular matrix (ECM) that undergoes flow-induced self-oscillation under a unique micromechanical environment, including small- and large-amplitude oscillations (1-3 mm) at high magnitudes of acceleration (up to 300 g) and at
8、 high frequencies (100-300 Hz) 1. Vocal fold fibroblasts regulate the expressions of matrix proteins, such as collagen, elastin, decorin, fibromodulin, hyaluronan, and fibronectin, and disturbed patterns of regulation lead to various laryngeal pathologies such as vocal fold scarring, vocal polyps, c
9、ysts, and many other benign lesions. In order to engineer an implantable tissue replacement for the surgical reconstruction of such ECM disorders, this study examines the potential of a novel allogenic acellular scaffold, the human umbilical vein (HUV), for regeneration of the vocal fold lamina prop
10、ria ECM. II. METHODS Following the protocol reported by Daniel et al. 1, native HUV tissue was harvested and frozen onto a stainless steel mandrel with a 6mm diameter at -80C for two days before dissection. An automated dissection procedure using a high-speed, steel cutting tool was performed with t
11、he tissue/mandrel secured on a modified lathe, resulting in a uniform tissue thickness of 750m when frozen 1. After dissection, the HUV tissue was thawed by immersion in double distilled water at 5C for 1 hour. HUV tissue sections were treated with a novel proteolytic enzyme-free, detergent-free sal
12、ine-based decellularization protocol developed in our laboratory 2. Briefly, the protocol involved the use of a highly hypertonic salt (3M NaCl) solution creating an extreme osmotic stress for cells in the HUV, followed by cycles of osmotic stress with incubation in PBS solution and ethanol under me
13、chanical agitation. RNase and DNase were added to remove the nucleic acids exposed by rupture of the cell membrane 2. Antibiotics were used to maintain tissue sterility. Structure of the resulting acellular HUV scaffold was examined under light microscopy and scanning electron microscopy. Human voca
14、l fold fibroblasts from primary culture were seeded on the abluminal surface of the acellular scaffold, and were cultured in 5% CO2 at 37C for 21 days. III. RESULTS AND DISCUSSION Fig. 1 shows the structure of the decellularized HUV scaffold, demonstrating the complete removal of endothelial cells,
15、fibroblasts, and other cell types in the native HUV tissue. The 3-D structure of the matrix protein network is well preserved, as shown in scanning electron micrographs. 21 days after seeding onto the abluminal surface of the acellular scaffold, human primary vocal fold fibroblasts have proliferated
16、 and infiltrated in the acellular scaffold to a depth of around 200 microns, roughly the thickness of the connective tissue layer in the HUV scaffold (Fig. 2). 978-1-4244-4713-8/10/$25.00 2010 IEEE IV. CONCLUSION The results of this study showed that our saline-based decellularization protocol was c
17、apable of fabricating a 3-D, biodegradable, acellular scaffold from native HUV tissue. Preliminary findings on the histological structure of the scaffold, as well as proliferation and infiltration of human vocal fold fibroblasts in the scaffold demonstrated the in vitro biocompatibility of the acell
18、ular scaffold, supporting its potential for vocal fold tissue engineering applications. ACKNOWLEDGMENT This work was supported by NIH Grant R01 DC006101. We would like to thank Dr. Peter S. McFetridge for sharing his HUV tissue and dissection technology with us. REFERENCES 1 J. Daniel, K. Abe, P.S.
19、McFetridge, “Development of the human umbilical vein scaffold for cardiovascular tissue engineering applications,” ASAIO J., vol. 51, pp. 252261, 2005. 2 C.C. Xu, R.W. Chan, N. Tirunagari, “A biodegradable, acellular xenogeneic scaffold for regeneration of the vocal fold lamina propria.,” Tissue Eng
20、., vol. 13, pp. 551566, 2007. Figure 1: Morphology of an acellular HUV scaffold (H&E staining). Note the luminal surface to the right and abluminal surface to the left. Total magnification = 40. Figure 2: Proliferation and infiltration of human vocal fold fibroblasts on the abluminal surface of an acellular HUV scaffold (H&E staining). Total magnification = 100.
