P. Carvajal Monroy
Muscle regeneration in the soft palate of the rat
A scientific essay in Medical Sciences
DOCTORAL THESIS defended in public on 29th of November 2016
In chapter 1, the background and rationale of this study is explained. Children with a cleft in the soft palate have difficulties with speech, swallowing and sucking. These patients are unable to separate the nasal from the oral cavity leading to air loss during speech. Although surgical repair ameliorates soft palate function by joining the clefted muscles of the soft palate, optimal function is in some cases not achieved. Adjuvant therapies based on tissue engineering might be suitable to improve muscle function after surgery. Therefore, the aim of this research project was to gain knowledge in the understanding of the biology and regeneration of branchiomeric head muscles. The regeneration of muscles in the soft palate after surgery is hampered because of (1) their low intrinsic regenerative capacity, (2) the muscle properties related to clefting, and (3) the development of fibrosis. Adjuvant strategies based on tissue engineering may improve the outcome after surgery by approaching these specific issues.
In chapter 2 an overview of myogenesis in the non-cleft and cleft palate, the characteristics of soft palate muscles and the process of muscle regeneration is presented. In addition, novel therapeutic strategies based on tissue engineering to improve soft palate function after surgical repair are proposed.
In chapter 3, a new in vivo model for the study of muscle regeneration in the soft palate of rats is established. This model is suitable to study muscle regeneration in the soft palate after surgical injury, and allows the development of novel adjuvant strategies to promote muscle regeneration. This offers new perspectives for the treatment of patients with cleft lip and/or palate, and for various other conditions in which the regeneration of head muscles is compromised.
In chapter 4, we study the long–term regeneration of the soft palate muscles after excisional injury. Muscle regeneration begins with the activation, recruitment and proliferation of satellite cells from the wound margins early in wound healing. However, differentiated myoblasts within the wound fail to form new myofibers and large amounts of collagen are deposited in the wounded area. Fibrosis as well as defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. In order to determine whether the intrinsic properties of satellite cells from branchiomeric head muscles also contribute to impaired muscle regeneration in the soft palate, in vitro culture systems are required.
Therefore, in chapter 5 a new protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. After isolation, low numbers of satellite cells are cultured on Matrigel spots of millimeter size to study their differentiation. This approach avoids the expansion and passaging of cells. In conclusion, this protocol offers new possibilities to study satellite cells derived from branchiomeric head muscles or other small muscles or muscle samples.
In chapter 6, the fiber type distribution and the expression of satellite cell markers were studied in ex vivo muscle tissue. Next, the in vitro differentiation and myotube formation of satellite cells isolated from representative muscles originating from the 1st, 2nd and 4th branchial arches were investigated. This was done with satellite cells from neonatal (2-week-old) and young (9-week-old) rats to study the effects of age. In this study, we demonstrate that not only fibrosis but also the intrinsic properties of satellite cells may be responsible for the poor regeneration of the palate muscles after (surgical) injury. Moreover neonatal satellite cells appear to differentiate less than young satellite cells.
In chapter 7, the results of the subsequent studies are discussed. We developed a model for compromised muscle regeneration in the soft palate by introducing a full-thickness defect. This model offers promising possibilities for further research. However, in the future, the use of larger cleft palate models may be necessary as they will approach the clinical situation more closely. We also developed a rapid and economical method for the isolation and study of satellite cells derived from head muscles or small muscle samples. We confirmed earlier observations that differentiation of satellite cells from branchiomeric head muscles is delayed when compared with muscles from the limb. This delay in differentiation might explain the poor regeneration of branchiomeric head muscles in response to injury. We also demonstrated that age-depended differences in stem cell properties can determine the regeneration of palate muscles after injury. Further research on age-dependent differences in stem cell properties will provide valuable information for clinical application in growing patients. Also, additional studies on satellite cells from clefted muscles from transgenic animals with a cleft are warranted. In the future, potential new therapies will have to be tested in larger animal models. This research project is the first step into the development of novel therapies to improve cleft palate repair and the quality of life of patients with a cleft in the palate.