Nigel P. Hunt

Human adult craniofacial muscle‐derived cells: neural‐cell adhesion‐molecule (NCAM; CD56)‐expressing cells appear to contain multipotential stem cells

Skeletal muscle has been well characterized as a reservoir of myogenic precursors or satellite cells with the potential to participate in cellular repopulation therapies for muscle dysfunction. Recent evidence, however, suggests that the postnatal muscle compartment can be considered an alternative to bone marrow as a source of multipotent cells or muscle‐derived stem cells (MDSCs). MDSCs, when primed with appropriate environmental cues, can differentiate into a variety of non‐muscle cells. The present study describes the application of a new technique for the isolation of adult human myoblasts and putative MDSCs, based on microbead–immunomagnetic selection of CD56+ cells, derived from craniofacial skeletal muscle, and details changes in morphological/molecular phenotype of the purified cells when maintained in either a myogenic or a non‐myogenic milieu. Multiple immunofluorescence microscopy and two‐colour flow‐cytometric analysis of proliferating CD56+ cultures revealed positive staining for myogenic markers (CD56, desmin and M‐cadherin) as well as putative stem‐cell markers [the antigens CD34, CD90 and CD106, and Flk‐1 (fetal liver kinase‐1)/VEGFR‐2 (vascular‐endothelial‐growth‐factor receptor)]. Confluent cultures subjected to cycles of adipogenic or osteogenic induction contained either adipocytes or osteoblasts and myotubes. In conclusion, the CD56+ subpopulation within adult human skeletal muscle is heterogeneous and is composed of both lineage‐committed myogenic cells and multipotent cells (the candidate MDSCs), which are able to form non‐muscle tissue such as fat and bone.

Gelatinase-B (matrix metalloproteinase-9; MMP-9) secretion is involved in the migratory phase of human and murine muscle cell cultures

The remodelling of connective tissue components is a fundamental requirement for a number of pivotal processes in cell biology. These may include myoblast migration and fusion during development and regeneration. In other systems, similar biological processes are facilitated by secretion of the matrix metalloproteinases (MMPs), especially the gelatinases. This study investigated the activity of the gelatinases MMP-2 and 9 by zymography on cell conditioned media in cultures of cells derived from explants of the human masseter muscle and in the murine myoblast cell-line C2C12. Expression of MMP-9 by western blotting and TIMP-1, the major inhibitor of MMPs, by northern blotting, during all phases of myoblast proliferation, migration, alignment and fusion, was also measured. Irrespective of the origin of the cultures, MMP-9 activity was secreted only by single cell and pre-fusion cultures whilst MMP-2 activity was secreted at all stages as well as by myotubes. The loss of MMP-9 activity was due to the loss of MMP-9 protein expression. TIMP-1 mRNA was not detectable at the single cell stage but its expression increased as cells progressed through the pre-fusion and post-fusion stages to reach a maximal in myotube containing cultures. Migration of cells derived from human masseter muscle was inhibited, using a specific anti-MMP-9 blocking monoclonal antibody (6-6B). These data are consistent with the concept that regulation of matrix turnover via MMP-9 may be involved in the events leading to myotube formation, including migration. Loss of expression of this enzyme and expression of TIMP-1 mRNA is associated with myotube containing cultures. Consequently, the ratio between MMPs and TIMPs maybe important in determining myoblast migration and differentiation.

Identification of matrix metalloproteinases and their tissue inhibitors type 1 and 2 in human masseter muscle.

Changes in masticatory muscle structure and function are either developmental, as seen in anomalies of facial form, or adaptive, as seen during procedures such as orthognathic surgery and functional-appliance orthodontic therapy. Remodelling of muscle extracellular matrix is pivotal in these processes. This turnover is mediated via members of the family of enzymes known as matrix metalloproteinases (MMP) and inhibited by the tissue inhibitors of metalloproteinases (TIMP). The aim here was to investigate the in vivo pattern of expression and distribution of MMPs and TIMPs in masseter muscle of humans with both normal and abnormal facial forms. Masseter muscle biopsies were taken from 10 patients, four with long-face syndrome and six normal controls as confirmed by cephalometry. Immunohistochemical techniques were used to show the pattern and distribution of MMPs and TIMP proteins in the muscle. Zymography of tissue extracts was used to determine the presence of MMP activity. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the presence of MMP and TIMP-2 mRNA. MMP-1 was expressed around the individual muscle fibres, especially in those fibre surfaces in contact with the interstices of the connective tissue and around blood vessels. MMP-9 staining was less intense and was expressed in the interstices of the connective tissue and around blood vessels. Zymography of protein extracts confirmed that MMP-9 activity was present. MMP-2 and MMP-3 were not expressed in the samples, although MMP-2 mRNA could be detected by RT-PCR and its activity could be detected by zymography. Intense TIMP-1 staining was present around each muscle fibre, in the interstices of the connective tissue and surrounding blood vessels; TIMP-2 mRNA could be detected in all samples. These staining patterns were seen in all biopsies examined and were irrespective of the facial form of the donor. These findings provide evidence that the mechanisms required for matrix remodelling are present in the human masseter muscle.