The Decelluarized Human Umbilical Vein (HUV) as an Allogeneic Scaffold for Vocal Fold Tissue Engineering

Date

2007-12-18

Authors

Rodriquez, Maritza Lizette

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Abstract

The human umbilical vein (HUV) has great potential for use as a biological tissue engineering scaffold. Thus far, it has been used as an extracellular matrix (ECM) scaffold for cardiovascular tissue engineering applications due to its many structural and biomechanical advantages. Since the HUV is vascular derived, the chemical and physical environment from which it came allows it to be more conducive to cell adhesion and ECM remodeling than currently available synthetic scaffold materials. The connective tissue component of the HUV is primarily the Wharton's Jelly (WJ), a gelatinous connective tissue surrounding the umbilical cord vessels. It is rich in peptide growth factors, glycosaminoglycans (GAGs), and proteoglycans, many of which are similar to those present in the vocal fold lamina propria. The vocal fold undergoes vibrations at relatively large amplitudes (1-2mm) and relatively high frequencies (100- 300Hz), thus requiring a scaffold that could endure such mechanical stimuli. The lamina propria consists of proteoglycans, glycosaminoglycans (especially hyaluronic acid), and fibrous proteins (collagen and elastin) that are optimally designed to withstand the unique mechanical stimuli. The natural biological constituents of the human umbilical vein, its structural stability, and the novel method for preparing uniform specimens make this tissue potentially useful as a vocal fold scaffold. Also, as a potential allograft, the possibility of interspecies viral infectivity is low. Native HUV tissue is obtained with an automated dissection method developed by Daniel et al. (2005), where HUVs are dissected from umbilical cords within two to three minutes, with uniform and repeatable dimensions using a custom built steelcutting machine. Preliminary experiments were conducted to determine the optimal decellularization protocol. Once this was determined, 15 cords were obtained to further examine the potential of the HUV as an acellular scaffold. Three cords from different donors (each sliced into 3 sections or scaffolds) were examined as follows: manually dissected, in the native state, decellularized, decellularized and cultured (controls), and recellularized. In these experiments, the HUV obtained was sliced into scaffolds of uniform dimensions (10-12mm x 17-20mm x 1-2mm), decellularized, recellularized with primary-culture human vocal fold fibroblasts, and cultured for 21 days. At the end of the 21 days, each scaffold was sliced into 3 sections, subjected to biomechanical testing, scanning electron microscopy (SEM) and histology. Using a custom-built linear simple shear rheometer, the viscoelastic properties of each scaffold section were determined. Preliminary experiments determined the optimal decellularization protocol based upon: the extent of decellularization, the depth of cellular infiltration, and their shear modulus (G') and dynamic viscosity (η') as compared to those of the human vocal fold. Recellularized scaffolds were determined to have G' and η' values similar to those of the human vocal fold cover obtained from a 79-year-old male and a 53-year-old female. Scanning electron microscopy (SEM) showed that viable fibroblast cells, as characterized by a lighter shade of gray and an elongated shape, attached to the abluminal surface of the scaffold. Native tissue, decellularized tissue, and control tissue all appeared to have an intact ECM protein network structure as shown by the scanning electron micrographs. Histological staining with hematoxylin and eosin, Alcian blue with and without hyaluronidase, Periodic acid-Schiff's reagent, Safranin-O, and Masson's Trichrome were used to examine cellular attachment, depth of cellular infiltration, the structure of the tissues and the scaffolds, and the expressions of various proteins and GAGs, including glycogen, hyaluronic acid, chondroitin sulfates A, B, and C, keratosulphate, mucins, sialomucins, and sulphated sialomucins. Results showed the presence of these proteins and GAGs in the native HUV tissue, varying extent of decreases in protein and GAG densities for the decellularized scaffold, and large decreases in protein and GAG densities for the control scaffold. However, the recellularized scaffold was able to regain some of the proteins and GAGs after 21 days of culture. Cell recovery results showed that the seeding of one million cells on each scaffold (with a seeding density of about 5,000 cells/mm2) led to an initial cell attachment of 1.7-1.8%, a faster proliferation than similar previous studies, and an average final cell count of around 480,000 cells per scaffold. These findings provided preliminary support to the potential of the acellular HUV scaffold as an allograft for the repair of vocal fold lamina propria lesions. The HUV consists of proteins conducive to cellular attachment and proliferation, has viscoelastic properties similar to those of the human vocal fold cover, demonstrates deep cellular infiltration, and can support the growth and recovery of a relatively large number of viable cells within a short period of time. Further studies involving a larger number of samples are needed to verify and extend the present findings.

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