A method for preparing skeletal muscle fiber basal laminae

Abstract

Previous attempts to prepare skeletal muscle basal laminae (BL) for ultrastructural analyses have been hampered by difficulties in successfully removing skeletal muscle proteins and cellular debris from BL tubes. In the present study we describe a two phase method which results in an acellular muscle preparation, the BL of which are examined by light, transmission electron, and scanning electron microscopy. In the first phase, excised rat extensor digitorum longus muscles are subjected to x-radiation and then soaked in Marcaine to inhibit muscle regeneration and to destroy peripheral muscle fibers. The muscles are then grafted back into their original sites and allowed to remain in place 7-14 days to allow for maximal removal of degenerating muscle tissue with minimal scar tissue formation. In the second phase, the muscle grafts are subjected sequentially to EDTA, triton X-100, DNAase, and sodium deoxycholate to remove phagocytizing cells and associated degenerating muscle tissue. These procedures result in translucent, acellular muscle grafts which show numerous empty tubes of BL backed by endomysial collagenous fibers. These preparations should be useful for morphological analyses of isolated muscle BL and for possible in vitro studies by which the biological activity of muscle BL can be examined.

Decellularized Human Skeletal Muscle as Biologic Scaffold for Reconstructive Surgery

“…In particular, in the present paper, we compared different decellularization protocols. In the literature, the first decellularization protocol was developed by Carlson and Carlson [ 33 ], who proposed a method involving several steps: skeletal muscle was first treated with chelants and detergents (EDTA and Triton X-100), to disrupt and remove the cell membrane, then it was exposed to enzymatic digestion (DNase) and it was finally treated with sodium deoxycholate to remove cellular debris. Following decellularization, protocols were implemented and refined through the introduction of freezing/thawing cycles and further chemical (e.g., SDS) and enzymatic (e.g., trypsin, latrunculin B) reactions [ 14 , 15 , 16 , 17 , 18 , 34 , 35 ].…”

The pro-myogenic environment provided by whole organ scale acellular scaffolds from skeletal muscle

“…The muscle retains the distal tendon attached to one extremity and conserves its morphology and stiffness, including intact arterial and venous basement membranes, in the absence of endogenous cells. The first method designed to obtain skeletal muscle decellularization dates back to the early 1990s [29]. That method, as well as more recent ones, are based on long, specific series of enzymatic and detergent treatments aimed at selectively eliminating any trace of cellular constituents, i.e.…”

Decellularized tissue and cell-derived extracellular matrices as scaffolds for orthopaedic tissue engineering

“…Afterward the tissues were explanted and subjected sequentially to EDTA, Triton X-100, DNase and sodium deoxycholate to remove cells. Using this method they were able to obtain acellular muscle grafts [194]. Later studies reported decellularization methods that did not require the use of radiation or reimplanting the graft back into the donor.…”