All PhD Theses

S. Grefte

Improving the regeneration of injured muscle


A scientific essay in Medical Sciences

DOCTORAL THESIS defended in public on 2nd of October 2011


In chapter 1, the background and rationale of the study is explained. The field of skeletal muscle engineering together with strategies to improve muscle regeneration is introduced and the outline of the study is presented. The aim of the present study was to improve muscle regeneration and to inhibit fibrosis in full-thickness muscle defects using scaffold-based approaches.

Chapters 2 and 3 present an overview of the biological aspects of skeletal muscle development and regeneration with the main focus on satellite cells. Satellite cells regenerate the muscle tissue by migrating to the site of injury where they proliferate, differentiate, and form myofibers. The specific micro-environment of the satellite cells, the niche, controls satellite cell behavior. In addition, a large diversity of growth factors regulates satellite cell activity after injury. Since the formation of scar tissue can prevent the recovery of full muscle function, three different approaches to improve muscle regeneration and to inhibit fibrosis are discussed: growth factor-, cell-, and scaffold-based therapies. For large muscle defects mainly the scaffolds-based approach is suitable, which is the focus in the next studies.

In chapter 4 an in vivo model for muscle regeneration in recurrent strain injury is established. The results showed that satellite cell activation around the defect, revealed by Pax7 and MyoD expression, was not affected by the implantation of a cross-linked collagen scaffold in the lacerated M. soleus. However, these cells were absent inside the scaffold and muscle regeneration inside the defect was impaired. It was concluded that the implantation of a cross-linked collagen scaffold into the lacerated M. soleus mimics a muscle discontinuity caused by a fibrotic wedge and can be used to evaluate new treatment modalities for recurrent strain injuries.

A new wound model that mimics full-thickness muscle defects and induces spontaneous fibrosis is described in chapter 5. By loading non cross-linked collagen scaffolds with SDF-1α, an attempt was made to improve muscle regeneration. The results showed that in this model a significant amount of fibrotic tissue was formed. The implantation of SDF-1α-loaded collagen scaffolds induced migration of Pax7+ satellite cells towards the regenerative zone around the wounds within the first ten days post-surgery. However, these cells did not enter the scaffold and the numbers of myofibroblasts and collagen deposition were not affected after 56 days. In conclusion, this spontaneous muscle fibrosis model can be used to test scaffold-based therapies. Loading scaffolds with SDF-1α induced satellite cell migration but did not reduce fibrosis.

In chapter 6 the putative inhibition of fibrosis using decorin-loaded collagen scaffolds with or without SDF-1α in the spontaneous muscle fibrosis model is described. In vitro studies showed that the decorin-loaded collagen scaffolds induced a short-term release of decorin within the first 3 days. In vivo, the SDF-1α and/or decorin-loaded collagen scaffolds did not affect the numbers of myofibroblasts, activated fibroblasts, satellite cells, and fused myoblasts at 56 days post-surgery. Moreover, fibrosis was not reduced. It is concluded that the release window of decorin was probably too short to prevent fibrosis.

In chapter 7, the myogenic potential of muscle stem cells is studied in 2D- and 3D-cultures with collagen type I and Matrigel. The latter contains satellite cell niche factors. In the 2D-cultures, higher numbers of proliferating Pax7+ and MyoD+ cells were found on Matrigel than on collagen. In addition, differentiating muscle stem cells formed more and larger MyoD+ and Myogenin+ myotubes on Matrigel. In the 3D-cultures, myofibers were also longer in Matrigel, but short and rounded in collagen. MyoD and Myogenin mRNA levels were also higher in muscle stem cells cultured in Matrigel. It was concluded that muscle stem cells, both in 2D and 3D, lose their differentiation capacity in collagen but not in Matrigel, which might be caused by the presence of niche factors.

Because differences were described for head and limb muscles, the myogenic potential of these muscle progenitor cells is compared in chapter 8. The muscle progenitor cells derived from head and limb muscles showed equal proliferation capabilities in vitro. During differentiation, head and limb muscle progenitor cells formed equal numbers of fused myotubes and showed comparable mRNA expression levels of several Myh-isoforms. The number of Pax7+, MyoD+, and Myogenin+ cells in head and limb muscle progenitor cells also did not differ during proliferation and differentiation. Thus, head and limb muscle progenitor cells show similar myogenic capacities in vitro. The reported differences must therefore be due to the different microenvironments of the muscles.

In chapter 9, the results of the previous chapters are discussed in the wider perspective of skeletal muscle engineering. Suggestions for future research are; the further development of smart scaffolds that induce the migration and attachment of satellite cells. The alignment of regenerating myofibers should also be stimulated in the scaffolds. Furthermore, in vitro culture conditions need to be optimized to maintain the stem cell status of satellite cells, and to generate aligned functional muscle tissue.