Controlled release of hepatocyte growth factor from a bovine acellular scaffold for vocal fold reconstruction

Abstract

A bovine acellular scaffold was found to facilitate tissue remodeling in a rat model of vocal fold injury, whereas hepatocyte growth factor (HGF) has been shown to have an antiscarring effect in the larynx. This study examined the loading and release kinetics of HGF in vitro, and the potential of the acellular scaffold as a timed-release system for the delivery of HGF in vivo. Bilateral wounds were created in the posterior vocal folds of 20 rats, with HGF-loaded acellular scaffolds implanted into the wounds unilaterally, and scaffolds without HGF implanted into the contralateral vocal folds as control. The rats were humanely sacrificed after 3, 7, 30, and 90 days and their larynges were examined histologically and immunohistochemically. Expressions of key matrix proteins in the vocal fold coronal sections were quantified by digital image analysis. Results demonstrated a gradual, sustained release of HGF for at least 7 days in vitro, consistent with the detection of glycosaminoglycans inherent of the scaffold. In rat vocal folds implanted with HGF-loaded scaffolds, apparently fewer inflammatory cells were observed 3 days after surgery when compared to the control. The mean relative densities of collagen III and hyaluronic acid were significantly lower than those of the control 7 days after surgery. Scaffold implants were apparently degraded by 3 months in all animals, with no evidence of fibrosis or calcification. These data suggested that the bovine acellular scaffold could be promising for the exogenous delivery of select growth factors in vivo.

Figure 1

Coronal section of a typical bovine acellular scaffold section stained with Alcian blue, demonstrating the widespread distribution of glycosaminoglycans (total magnification = 40 ×).

Figure 2

(A) Profiles of hepatocyte growth factor (HGF) released from bovine acellular scaffolds loaded with HGF (9.01 ng HGF per mg scaffold) and from control scaffolds without HGF as a function of time (n = 5). Normalized concentration of HGF released (ng/ml per mg scaffold) is shown on the y-axis. (B) Cumulative levels of HGF released from the HGF-loaded scaffolds as a function of time (n = 5). Percentage of the HGF released with reference to the total HGF loaded is shown on the y-axis.

Figure 3

Histological coronal sections of rat laryngeal specimens stained with H&E, showing the HGF-loaded acellular scaffolds implanted into the right vocal fold wounds and control scaffolds without HGF implanted into the left vocal fold wounds (A) 3 days, (B) 7 days, (C) 30 days, and (D) 90 days after surgery (total magnification = 40 ×). Arrows indicate the implants in the experimental (right) vocal folds and the control (left) vocal folds. Parts (1), (2) and (3) show the interfaces between the control scaffolds and the host tissue 3 days, 7 days and 30 days after surgery, respectively, at a higher magnification (400 ×). Parts (4), (5) and (6) show the interfaces between the HGF-loaded scaffolds and the host tissue 3 days, 7 days and 30 days after surgery, respectively, at a higher magnification (400 ×) (AC = arytenoid cartilage; TA = thyroarytenoid muscle; TC = thyroid cartilage; S = implanted acellular scaffold).

Figure 4

Histological coronal sections of rat larynges showing HGF-loaded acellular scaffolds implanted into the right vocal fold wounds 3 days after surgery, with control scaffolds without HGF implanted into the left vocal fold wounds. (A) H&E (arrows indicating the implants on both sides); (B) Collagen type I; (C) Collagen type III; (D) Elastin; (E) Fibronectin; (F) Hyaluronic acid; and (G) Glycosaminoglycans.

Figure 5

Histological coronal sections of rat larynges showing HGF-loaded acellular scaffolds implanted into the right vocal fold wounds 7 days after surgery, with control scaffolds without HGF implanted into the left vocal fold wounds. (A) H&E (arrows indicating the implants on both sides); (B) Collagen type I; (C) Collagen type III; (D) Elastin; (E) Fibronectin; (F) Hyaluronic acid; and (G) Glycosaminoglycans.

Figure 6

Histological coronal sections of rat larynges showing HGF-loaded acellular scaffolds implanted into the right vocal fold wounds 30 days after surgery, with control scaffolds without HGF implanted into the left vocal fold wounds. (A) H&E (arrows indicating the implants on both sides); (B) Collagen type I; (C) Collagen type III; (D) Elastin; (E) Fibronectin; (F) Hyaluronic acid; and (G) Glycosaminoglycans.

Figure 7

Histological coronal sections of rat larynges showing HGF-loaded acellular scaffolds implanted into the right vocal fold wounds 90 days after surgery, with control scaffolds without HGF implanted into the left vocal fold wounds. (A) H&E (arrows indicating the implants on both sides); (B) Collagen type I; (C) Collagen type III; (D) Elastin; (E) Fibronectin; (F) Hyaluronic acid; and (G) Glycosaminoglycans.

Figure 8

Expressions of ECM proteins in the rat vocal fold implanted with the HGF-loaded acellular scaffold versus those in the vocal fold implanted with the control scaffold without HGF (n = 5): (A) Collagen type I; (B) Collagen type III; (C) Elastin; (D) Fibronectin; (E) Hyaluronic acid; (F) Glycosaminoglycans. * p < 0.05 for differences between the two groups.