Acellular scaffolds often demonstrate the capability to bind exogenous growth factors with their inherent glycosaminoglycans (GAGs), and with a controlled timed-release, they contribute to direct the matrix remodeling process by the host cells.18
In the present study, as GAGs in the bovine vocal fold lamina propria are water soluble, they could have been partially removed during the cycles of osmotic stresses induced during the saline-based decellularization process. Hence, the relative content of GAGs remaining in the bovine acellular scaffold could determine the levels of available growth factors and eventually the release and actions of the growth factors in vivo
. Our results showed that GAGs were indeed found in the acellular scaffold despite having undergone decellularization (). The level of GAGs in the acellular scaffold (0.297% by weight) was similar to that of acellular matrices derived from the bovine pericardia (0.09 to 0.57% by weight).18
The GAGs inherent of the bovine acellular scaffold could facilitate the loading and the release of growth factors in a timed-release fashion. This was consistent with the observed in vitro
HGF release profile (), which indicated that HGF could be loaded into the acellular scaffold and gradually released for over at least 7 days. Despite these promising results, further studies are required to determine the quantitative relationship between the level of HGF released, the degradation of HGF, and the activity of the HGF protein as a function of the GAG content inherent of the scaffold. Optimal methods for more accurate loading and delivery of growth factors through the modulation of GAGs in the acellular scaffold should be explored.
The H&E histological sections showed that on day 3, infiltration of acute inflammatory cells ( and ), including a large amount of lymphocytes, seemed to be delayed in the right vocal fold with the HGF-loaded scaffolds implanted. This finding was consistent with the observations of HGF attenuating the attachment of monocytes in vitro
and the sequestration of macrophages in the rat kidney.19
However, by day 7, the intensity of the inflammatory reaction appeared to be similar in both vocal folds, with or without HGF loading. Indeed, in the present study, cells were only identified by their morphology and the photomicrographs were not evaluated at multiple levels of sectioning. Moreover, Badylak and Gilbert20
reported that xenografts developed from ECM scaffolds elicited mainly Th2 lymphocyte response and M2 macrophage response, which were believed to be associated with anti-inflammatory wound healing and graft acceptance. Hence, in order to further examine the immune response that would be triggered by bovine acellular scaffolds with or without HGF, and the effect of such immune response upon downstream remodeling events, quantitative assessment of the degree of inflammation at multiple levels of histological sectioning as well as accurate identification and quantification of inflammatory cells using specific phenotypic markers should be pursued in future studies.
Results of the quantitative histological analysis indicated that the expressions of fibrous proteins including collagen I, collagen III and elastin synthesized by the host cells were generally reduced in the presence of HGF (). This finding was consistent with the well-documented anti-scarring properties of HGF.9
According to the manufacturer, the anti-type I collagen antibody could cross-react slightly with type III collagen but not vice versa. However, since the areas of sections stained by the anti-type I collagen antibody and by the anti-type III collagen antibody did not match with each other, any cross reaction of the anti-collagen type I antibody with collagen III was deemed negligible in the present study.3
The expression of fibronectin was elevated in the presence of HGF in the acute stage of injury (in day 3), agreeing with previous results observed in cultures of canine vocal fold fibroblasts.10
The fibronectin levels in vocal folds implanted with HGF-loaded scaffolds started to decrease afterwards and were below those of the control vocal folds at the subsequent time points. Given that fibronectin has shown chemoattractant properties for fibroblasts and other cells such as monocytes during wound healing,21
the decrease in fibronectin could be related to the observed attenuation of inflammatory cells infiltrating the HGF-loaded scaffolds ().
Although HGF has been reported to increase HA production by human vocal fold fibroblasts,11
the mean level of HA was only slightly increased in day 3 in vocal folds implanted with HGF-loaded scaffolds. By day 7, the level of HA in the experimental group was actually statistically lower than that of the control. A previous report on acute vocal fold scarring in a rat model showed a maximum increase in HA production 5 days post-surgery, followed by a substantial decrease by 7 days.22
No significant difference in HA level was observed between the scarred vocal folds and the control. Similar trends in HA changes were also reported in a rabbit vocal fold scarring model.23
Such transient changes in HA in early-stage wound healing were also observed in the present study in the vocal folds with HGF-loaded scaffold implants, but not in the control vocal folds. According to our previous study,3
the acellular scaffold alone could actually increase HA production, although not reaching statistical significance. Furthermore, even though HA is often considered as anti-fibrotic and it is a key to the maintenance of optimal viscoelasticity of the vocal folds,8, 24
HA has also been shown to be associated with xenograft rejection.25
Based on these somewhat conflicting findings in the literature, our findings highlighted the need for further studies to elucidate the effect of the acellular scaffold on HA production as a function of HGF loading and release. No difference in HA level was observed between the two groups after 30 days and 90 days.
Comparing to the results of our previous study in which acellular scaffolds were only implanted into one vocal fold in each rat,3
the relative levels of ECM proteins in the present study seemed to be higher by 90 days, except for GAGs. Furthermore, peak levels of collagen I, collagen III and elastin were reached in day 7 or day 30 in the present study, instead of at the earlier time point (day 3) in the previous study.3
This could be a consequence of the bilateral implantation, which might have increased the host response level, possibly affecting the overall level of protein synthesis as the wound healing progressed. Proteoglycans and GAGs including HA, decorin, biglycan, and fibromodulin contribute to regulate various important ECM functions and properties such as biocompatibility, fibrous protein formation, and viscoelastic properties.24
The observed lower levels of GAGs in vocal folds implanted with HGF-loaded scaffolds () may be related to the delayed infiltration of host fibroblasts surrounding the implant, but further studies are required to examine the exact effect of HGF on different proteoglycans and GAGs individually. In future studies, more quantitative methods such as homogenization of vocal folds en bloc followed by enzyme-linked immunosorbent assays (ELISAs) could be used to better quantify the levels of the various ECM proteins as compared to the present study. Also, real-time polymerase chain reaction (RT-PCR) could be adopted to examine the gene expressions of host cells following the implantation.
Despite the general differences found between the experimental and the control vocal folds, it seemed that the HGF-loaded scaffold showed a rather weak effect in the current study. It is postulated that the effect of HGF on the host cells may have been attenuated by the cytokine signaling associated with the wound healing as well as the host reaction to the acellular scaffolds. Also, the sustained release, degradation, and activity of HGF in vivo were not known, which would likely significantly affect the therapeutic outcome of the HGF being delivered. Optimization of growth factor delivery with a sustained activity as well as optimal graft implantation are necessary for the success of tissue engineering approaches involving acellular scaffolds loaded with growth factors.
In the present study, the bovine acellular scaffolds were implanted into the more posterior, cartilaginous portion of the vocal fold instead of into the membranous vocal fold due to difficulties in the surgical procedure in the small rat vocal tract.3
Implantation of the acellular scaffolds into the membranous vocal fold should be targeted in future studies, in order to explore the potential of the acellular scaffold loaded with HGF or other growth factors for reconstruction of the vocal fold lamina propria. Moreover, more intensive, FDA-approved sterilization procedures instead of the use of only 70% ethanol and UV light could be conducted in future studies. Typical sterilization procedures (such as oxidation and alkylation), however, could have adverse effects on the structure and integrity of acellular ECM scaffolds and should be investigated cautiously.26, 27
There is also a potential concern on the use of HGF for the clinical treatment of voice disorders involving laryngeal cancer. In addition to its well documented anti-scarring effect, HGF has been known to increase the motility and scattering of some epithelial cells, such as tumor cells found in hypopharyngeal carcinoma.28, 29
Thus, it could be potentially unsafe to use HGF in patients with head and neck cancer and laryngeal cancer, which make up a significant portion of the clinical population with vocal fold scarring. The clinical applications and concerns of HGF are beyond the scope of the present study, but they should be addressed in further studies. Future studies should also examine the potential of the bovine acellular scaffold for the in vivo
delivery of alternative growth factors and drugs.