Reconstruction of vocal fold scarring with mesenchymal stromal cell therapy

Tissue injury/scarring of the vocal folds (VFs) can be caused by various factors including external trauma, cancer treatment or inflammation, leading to damage within the vibrating layers and a decrease in viscoelastic functions. These effects ultimately result in severe voice problems. This condition leads to significant morbidity for patients. Approximately 5-20% of the population in Western countries suffer from chronic hoarseness and/or a voice disorder. Currently, there are no effective treatments available. Embryonic stem cells (ESCs) are pluripotent cells that can different into various cell types or tissues in the body. They are powerful candidates for cell therapy for many diseases. Mesenchymal stromal cells (MSCs) have been considered as a valuable source in regenerative medicine due to their immunomodulatory properties, ability to self-renew and multipotency. The overall aim of this PhD project was to identify whether stem cells, and in particular local injection of MSCs can modulate the early inflammatory response within scarred VFs, and promote the wound healing process. We have used a combination of in vitro and in vivo models to address the fundamental questions, before translating our findings to a clinical phase I/II trial evaluating the safety and efficacy of autologous MSC injection on vocal function in patients suffering with severe voice problems.

In study I, in a xenograft model, we showed that local injection of human embryonic stem cells (hESCs) induces sustained enhanced healing properties in injured VFs. Measurement of tissue viscoelasticity demonstrated improved function of VFs. Histological findings show a reduction in type I collagen and lamina propria (LP) thickness in hESC treated VFs compared to untreated VFs. Moreover, no hESCs derivates (teratoma and malignances) were observed in the transplanted VFs. In study II, we examined how MSCs modulate the early inflammatory response within injured rabbit VFs. Histological examination demonstrated that injected MSCs were able to reduce tissue inflammation, further corroborated by findings confirming a significant reduction in mRNA expression of the pro-inflammatory markers, interleukin (IL)-1β and IL-8. These findings were attributed to the ability of MSCs to reduce the level of cell death at the injury site and promote an anti-inflammatory milieu, as evidenced by increased levels of CD163+ cells. Despite low level persistence of the MSCs within the tissue, long-term effects on tissue repair were evident, with improved organization of the collagen matrix. Limitations, in terms of cell suspension leakage and retention of MSCs at the site of injury have been reported with the use of liquid delivery vehicles. Study III therefore addressed these issues, evaluating the effectiveness of MSC delivery to injured VFs in a hyaluronan (HA) hydrogel carrier. Our findings demonstrated that HA was non-immunogenic, with no adverse events reported, and degraded within the VF tissue within 1 month. Complementary in vitro studies demonstrated that while the HA hydrogel delayed the migration of MSCs, these cells remained responsive to their extracellular environment, including proinflammatory cues. The presence of a collagen matrix, as seen within the VF tissue, enhanced movement out of the gel potentially explaining why hydrogel delivery did not enhance persistence of the MSCs in the tissue. The study concluded that HA hydrogels may offer a safe and practical means to deliver MSCs to the VF, with minimal leakage, but no additional therapeutic effect could be seen in comparison to MSCs in liquid suspension. Based on the findings from studies II and III, we performed a phase I/II clinical trial in 16 patients with severe VF scarring suffering from severe voice problems (study IV). Patients were treated with a single injection of autologous MSCs, after surgical resection of scar tissue. The study reached its primary endpoint of safety, with no adverse side effects reported. Clinical evaluation using high speed laryngoscopy and phonation pressure threshold demonstrated improvement in VF viscoelastic function in 62-75% of patients, dependent on the parameter. Self-reporting by the patients, using the voice handicap index confirmed significant improvements in phonation in 50% of the patients, with no significant change in the remaining cohort.

In summary, stem cells and stromal cells were found to improve healing in scarred VFs. Local administration of MSCs into injured/scarred VFs dampens inflammatory responses by decreasing cell death due to injury and promoting an anti-inflammatory milieu. Delivery of the MSCs in a HA hydrogel reduces cell leakage, but does not offer therapeutic advantages compared to MSCs alone. Despite low-level persistence and no engraftment within the VF tissue, MSCs exerted long-term effects resulting in functional tissue repair. Our in vivo animal studies demonstrating the mechanisms by which MSCs support tissue repair were translated to the clinical setting, with local administration of autologous MSCs improving VF function and reducing morbidities associated with poor phonation in the treated patients. We conclude that local injection of MSCs may offer a safe and novel option for the treatment of VF scarring.

List of scientific papers

I. Svenssion B, Nagubothu SR, Nord C, Cedervall J, Hultman I, Ährlund-Richter L, Tolf A, Hertegård S. Stem cell therapy in injured vocal folds: A three-month xenograft analysis of human embryonic stem cells. Biomed Research International. 2015:754876.
https://doi.org/10.1155/2015/754876

II. Nagubothu SR, Sugars RV, Tudzarovski N, Andrén AT, Bottai M, Davies LC, Hertegård S, Le Blanc K. Mesenchymal stromal cells modulate tissue repair responses within the injured vocal fold. Laryngoscope. 2019 Mar 5.
https://doi.org/10.1002/lary.27885

III. Hertegård S, Nagubothu SR, Malmström E, Ström CE, Tolf A, Davies LC*, Le Blanc K*. Hyaluronan hydrogels for the local delivery of mesenchymal stromal cells to the injured vocal fold. Stem Cells and Development. 2019. Sep 1;28(17):1177-1190. *Joint senior author.
https://doi.org/10.1089/scd.2019.0102

IV. Hertegård S, Nagubothu SR, Malmström E, Le Blanc K. Treatment of vocal fold scarring with autologous bone marrow derived human mesenchymal stromal cells- first phase I/II human clinical study. [Manuscript]