The Use of a Dehydrated Cellularized Collagen Matrix to Replace Fibrotic Vocal Fold Mucosa.

OBJECTIVES: Fibrosis of the vocal fold lamina propria reduces vocal cord vibration resulting in a chronically hoarse voice. We describe a novel approach using umbilical cord-derived mesenchymal stem cells in a dehydrated collagen matrix (cellogen) to reconstruct the delicate balance of extracellular matrix within the vocal fold lamina propria whilst limiting the host inflammatory response to the implant. METHODS: Human umbilical cord-derived mesenchymal stem-cells were embedded in bovine type I collagen hydrogel and dehydrated using the RAFT™ 3D culture system. The extracellular matrix, cellular viability and composition, paracrine profile, and genomic profile were assessed and the scaffold engrafted onto the hind muscle of NUDE mice. RESULTS: The cells retained stem-cell markers following fabrication and secreted collagen III, fibronectin, and glycosaminoglycans within the scaffold. Electron microscopy showed the scaffold consisted of single strands of protein with interspersed bundles of a similar size to native vocal fold lamina propria. The use of the dehydration step improved cell viability and upregulated the expression of genes important in wound healing and matrix organization compared with unmodified collagen hydrogel carriers. The cells were shown to secrete exosomes and cytokines and, following engraftment within an immunocompromised mouse model, appeared to modulate the host inflammatory response compared with controls. CONCLUSION: This article provides a scalable cell-protein scaffold that with further modifications could provide a replacement for lost or damaged vocal fold mucosa. Further investigations are required to assess the mechanical properties of the scaffold and inhibit the differentiation of the umbilical cord-derived stem-cells following implantation. Laryngoscope, 134:882-893, 2024.

The use of basic fibroblast growth factor to improve vocal function: A systematic review and meta-analysis.

OBJECTIVES: This systematic review and meta-analysis examines if intralaryngeal injection of basic fibroblast growth factor 2 (FGF2) can improve voice outcomes in those with vocal disability. DESIGN: A Systematic review of original human studies reporting voice outcomes following intra-laryngeal injection of basic fibroblast growth factor 2 in those with vocal dysfunction. Databases searched were Medline (1946-July 2022), Embase (1947-July 2022), Cochrane database and Google Scholar. SETTING: Secondary or tertiary care centres that undertook the management of voice pathology Hospital. PARTICIPANTS: Inclusion criteria were original human studies reporting voice outcome measurements following intralaryngeal injection of FGF2 to treat vocal fold atrophy, vocal fold scarring, vocal fold sulcus or vocal fold palsy. Articles not written in English, studies that did not include human subjects and studies where voice outcome measures were not recorded before and after FGF2 injection were excluded from the review. MAIN OUTCOME MEASURES: The primary outcome measure was maximum phonation time. Secondary outcome measures included acoustic analysis, glottic closure, mucosal wave formation, voice handicap index and GRBAS scale. RESULTS: Fourteen articles were included out of a search of 1023 and one article was included from scanning reference lists. All studies had a single arm design without control groups. Conditions treated were vocal fold atrophy (n = 186), vocal cord paralysis (n = 74), vocal fold fibrosis (n = 74) and vocal fold sulcus (n = 56). A meta-analysis of six articles reporting on the use of FGF2 in patients with vocal fold atrophy showed a significant increase of mean maximum phonation time of 5.2 s (95% CI: 3.4-7.0) at 3-6 months following injection. A significant improvement in maximum phonation time, voice handicap index and glottic closure was found following injection in most studies assessed. No major adverse events were reported following injection. CONCLUSIONS: To date, intralaryngeal injection of basic FGF2 appears to be safe and it may be able to improve voice outcomes in those with vocal dysfunction, especially vocal fold atrophy. Randomised controlled trials are needed to further evaluate efficacy and support the wider use of this therapy.

The life-cycle and restoration of the human vocal fold.

Objective: To better understand the challenges of designing therapies to treat damaged vocal fold lamina propria, it is essential to understand the biophysical and pathophysiological mechanisms involved in vocal fold development, maintenance, injury, and aging. This review critically analyses these points to try and direct future efforts and new strategies toward science-based solutions. Data Sources & Review Methods: MEDLINE, Ovid Embase, and Wed of Science databases were used to identify relevant literature. A scoping review was performed following the preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews checklist. Results: The layered arrangement of the vocal fold, develops during early childhood and is maintained during adulthood unless injury occurs. The stellate cells of the macular flava are likely to be important in this process. The capacity for vocal fold regeneration and growth is lost during adulthood and repair results in the deposition of fibrous tissue from resident fibroblasts. With advancing age, viscoelastic tissue declines, possibly due to cell senescence. Strategies aimed at replacing fibrous tissue within the vocal folds must either stimulate resident cells or implant new cells to secrete healthy extracellular protein. Injection of basic fibroblast growth factor is the most widely reported therapy that aims to achieve this. Conclusions: The pathways involved in vocal fold development, maintenance and aging are incompletely understood. Improved understanding has the potential to identify new treatment targets that could potentially overcome loss of vocal fold vibratory tissue.

GMP compliant isolation of mucosal epithelial cells and fibroblasts from biopsy samples for clinical tissue engineering.

Engineered epithelial cell sheets for clinical replacement of non-functional upper aerodigestive tract mucosa are regulated as medicinal products and should be manufactured to the standards of good manufacturing practice (GMP). The current gold standard for growth of epithelial cells for research utilises growth arrested murine 3T3 J2 feeder layers, which are not available for use as a GMP compliant raw material. Using porcine mucosal tissue, we demonstrate a new method for obtaining and growing non-keratinised squamous epithelial cells and fibroblast cells from a single biopsy, replacing the 3T3 J2 with a growth arrested primary fibroblast feeder layer and using pooled Human Platelet lysate (HPL) as the media serum supplement to replace foetal bovine serum (FBS). The initial isolation of the cells was semi-automated using an Octodissociator and the resultant cell suspension cryopreservation for future use. When compared to the gold standard of 3T3 J2 and FBS containing medium there was no reduction in growth, viability, stem cell population or ability to differentiate to mature epithelial cells. Furthermore, this method was replicated with Human buccal tissue, providing cells of sufficient quality and number to create a tissue engineered sheet.

Bioengineered airway epithelial grafts with mucociliary function based on collagen IV- and laminin-containing extracellular matrix scaffolds.

Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air-liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.

Using a Three-Dimensional Collagen Matrix to Deliver Respiratory Progenitor Cells to Decellularized Trachea In Vivo.

IMPACT STATEMENT: This article describes a method for engrafting epithelial progenitor cells to a revascularized scaffold in a protective and supportive collagen-rich environment. This method has the potential to overcome two key limitations of existing grafting techniques as epithelial cells are protected from mechanical shear and the relatively hypoxic phase that occurs while grafts revascularize, offering the opportunity to provide epithelial cells to decellularized allografts at the point of implantation. Advances in this area will improve the safety and efficacy of bioengineered organ transplantation.

Tissue-Engineered Larynx: Future Applications in Laryngeal Cancer.

PURPOSE OF REVIEW: This article reviews the latest developments in tissue engineering for the larynx with a specific focus on the treatment of laryngeal cancer. RECENT FINDINGS: Challenges in tissue engineering a total larynx can be divided into scaffold design, methods of re-mucosalization, and how to restore laryngeal function. The literature described a range of methods to deliver a laryngeal scaffold including examples of synthetic, biomimetic, and biological scaffolds. Methods to regenerate laryngeal mucosa can be divided into examples that use a biological dressing and those that engineer a new mucosal layer de novo. Studies aiming to restore laryngeal function have been reported, but to date, the optimum method for achieving this as part of a total laryngeal transplant is yet to be determined. SUMMARY: There is great potential for tissue engineering to improve the treatments available for laryngeal cancer within the next 10 years. A number of challenges exist however and advances in restoring function must keep pace with developments in scaffold design.