Elsevier

Biomaterials

Volume 32, Issue 19, July 2011, Pages 4433-4442
Biomaterials

Development of bioengineered human larynx

https://doi.org/10.1016/j.biomaterials.2011.02.055Get rights and content

Abstract

To date, only two human laryngeal allotransplants have been reported and, although they were successful, both patients required life-long immunosuppression. A bioengineered human larynx could represent a possible alternative to allotransplantation. Human larynxes were decellularized enzymatically to obtain acellular matrices. Histological and molecular analysis demonstrated that all cellular components and nuclear material were removed. SEM showed that decellularized matrices retained the hierarchical structures of the native larynx, and mechanical tests demonstrated that the decellularization did not significantly impaired the biomechanically properties of the obtained matrices. Immunohistochemical staining found residual angiogenic factors after decellularization, and CAM analysis demonstrated that acellular laryngeal scaffolds induce a strong in vivo angiogenic response. Using a decellularization method, we are now able to obtain, in a short and clinically useful time, natural bioengineered laryngeal scaffolds which could be use for partial or total implantation in humans.

Introduction

The larynx is a finely tuned sphincter that orchestrates swallowing, breathing, coughing and voice. In 2006, there were 11,826 new cases of laryngeal cancer in EU [1], and a smaller, but severely impaired, group of patients with non-functioning larynxes due to benign disease and trauma [2]. Since conventional reconstruction and prosthetics have failed to replace the complex functions of the human larynx, laryngectomy is, at the moment, the only possible therapeutical option for patients with laryngeal advanced trauma.

An attractive therapeutical alternative to laryngectomy, in patients with irreversible laryngeal disease, would be either total or partial laryngeal transplantation. To date, only two documented laryngeal transplants have been performed on humans [3], [4]. Both required life-long immunosuppression, and this may explain why this procedure has not gained worldwide acceptance [5]. More recently, it has been demonstrated that most of the larynx can be removed with the preservation of one muscle-nerve-joint unit and, as a consequence, without the need of neuromuscular activity regeneration [6], [7]. However, besides the good breathing outcome, voice and swallowing remain sub-optimal, due to the lack of a truly laryngeal architecture. It is not unrealistic to postulate, therefore, that the availability of natural or synthetic substitutes displaying equivalent anatomical, physiological and biomechanical properties of normal human larynxes would provide the right, complex architecture and dynamics for normal voice production and sphincter action.

In recent years, there has been considerable progress in the translation of tissue engineered organs into the clinic [8], [9], and decellularized tissues and organs have been successfully used as scaffolds for engineering a variety of tissues, including heart, liver and trachea [10], [11], [12], [13], [14], [15]. Unlike scaffolds made of synthetic materials, natural matrices, consisting mainly of extracellular matrix (ECM), are degraded by cellular enzymatic activity, releasing growth factors and peptides that could stimulate constructive tissue remodeling. Decellularized matrices have been recently considered for laryngeal regeneration [16], [17], [18]. Partial hemilaryngectomies in a canine model were reconstructed using porcine decellularized urinary bladder matrix [16], obtaining the regeneration of thyroid cartilage, epithelium, connective tissue, glandular structures and some skeletal muscles. The ECM-based repair resulted superior to that observed using control standard procedure [17], suggesting that ECM scaffolds could be promising templates also for constructive remodeling of laryngeal tissue [16], [17].

Although the ECM scaffold tends to facilitate tissue repair, the method of its preparation (decellularization approach) can dramatically alter the biomechanical properties of the resulting scaffold, compromising its ability to provide mechanical support during the remodeling process as well as altering the host remodeling response [10]. Using a detergent-enzymatic approach, we have successfully obtained a bioengineered human tracheal matrix, structurally and mechanically similar to native trachea and containing angiogenic factors which exert chemotactive and pro-angiogenic properties [19]. This human tracheal bioactive support provided a natural environment for cellular growth and differentiation [20], [21] and allowed us to perform the first-in-man transplantation of fully tissue-engineered organ (windpipe) [14].

The success of our study led us to hypothesize that the best regenerative substitute would be the natural laryngeal ECM, which contain all the cytoskeleton components necessary to provide structural, biochemical and biophysical requirements for suitable laryngeal remodeling. We have therefore applied a decellularization approach to human larynges to obtain bioengineered scaffold for in vivo partial or sub-optimal larynx regeneration. Herein we describe the complete biological, mechanical and pro-angiogenic characterization of decellularized human laryngeal matrices with the ultimate goal to explore the functional solution for larynx regeneration without the risk of rejection.

Section snippets

Matrix obtainment and characterization

Cadaveric larynxes, upon signed informed consent by the relatives and protocol approval from the Ethic Commission of University Hospital Careggi (Italy) and Italian National Transplant Service, were retrieved from 5 donors (Table 1). The eligibility criteria for harvesting were: donor age <40 years, absence of structural abnormalities, neck trauma, previous neck surgery (tracheotomy) and no presence of infection.

Human matrix characterization

All larynges were decellularized with a total of 25 consecutive DEM cycles. All the bioengineering process lasted 17 days. Fig. 1A shows the histological structure of a native larynx: the ciliated columnar epithelium lying on a basal membrane, the lamina propria with several seromucinous glands and the cartilage; by contrast, the decellularized samples showed an almost complete removal of cells and nuclear material from the ECM (Fig. 1B–F). Epithelial, glandular and muscular cells were

Discussion

The larynx is a vitally important mucosal organ which plays a primarily role in the phonation and protection of the lower airways. Currently, there is no good surgical, medical or prosthetic solution for patients with irreversible laryngeal disease (including trauma and advanced cancer), and laryngectomy (subtotal or total removal of the larynx) remains the main surgical treatment. This approach is, however, associated with several devastating personal and physiological outcomes (voice loss,

Conclusions

This work strengthen our results recently obtained with tracheal grafts. 25 cycles of the DEM generates a bioengineered human laryngeal matrix that is structurally and mechanically similar to native larynx (in all its different structures) and that contains angiogenic factors which exert pro-angiogenic properties. This is the first study which completely describes and characterizes the obtainment of human acellular laryngeal graft which is ready for in vivo implantation in humans and may

Acknowledgments

This investigation was completely supported by a grant (pd 239-28/04/2009, delibera GRT 1210/08) issued on the 28 December 2008 by the region Tuscany (Italy) entitled “Clinical laboratory for complex thoracic respiratory and vascular diseases and alternatives to pulmonary transplantation”.

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