Optimizing the decellularization process of human maxillofacial muscles for facial reconstruction using a detergent‐only approach

Abstract

Trauma, congenital diseases, and cancer resection cause muscle deformities of the human facial muscle. Muscle defects are either treated with local or distal flaps if direct closure is not possible. However, such surgical interventions are limited by donor morbidity and limited tissue availability. Decellularized scaffolds provide alternative strategies for replacing and restoring missing facial muscle by creating scaffolds that mimic the native tissue. This study aimed to develop a protocol to decellularize human zygomaticus major muscle (ZMM) and masseter muscle (MM). Three protocols were assessed including a detergent‐only treatment (DOT), detergent‐enzymatic treatment (DET) protocol, and a third nondetergent nonenzymatic treatment protocol. Scaffolds were then characterized via histological, immunofluorescent, and quantitative techniques to assess which protocol provided optimal decellularization and maintenance of the extracellular matrix (ECM). The results demonstrated three cycles of DOT protocol consisting of 2% sodium dodecyl sulfate for 4 hr was optimal for decellularization for both ZMM and MM. After three cycles, DNA content was significantly reduced compared with native ZMM and MM (p < .05) with preservation of collagen and glycosaminoglycan content and ECM on histological analysis. DET and nondetergent nonenzymatic treatment protocols were unsuccessful in decellularizing the ZMM and MM with residual DNA content after four cycles and caused ECM disruption on histological analysis. All protocols did not impair the mechanical properties and supported human fibroblast growth. In conclusion, the DOT protocol is effective in producing human decellularized muscle scaffolds that maintain the ECM. Further investigation of detergent only decellurization techniques should be explored as a first step to create effective scaffolds for muscle tissue engineering.

Optimizing the decellularization process of an upper limb skeletal muscle; implications for muscle tissue engineering

“…Overall, the DOT protocol has been successful in decellularizing both human FDS and maxillofacial muscles to produce a tissue scaffold . This study highlights the broad application of DOT protocols for decellularization of skeletal muscle.…”

“…All steps for decellularization of the muscle were carried out as per the previous article which included three protocols. The detergent only treatment (DOT) used 2% sodium dodecyl sulfate (SDS) for decellularization.…”

“…Following decellularization, the muscles were characterized to determine the efficacy of the decellularization protocols including DNA content and extracellular matrix (ECM) component quantification. After decellularization, the muscle samples produced were snap frozen in liquid nitrogen and stored at −70°C for DNA, collagen, and glycosaminoglycan (GAG) for ECM quantification studies, as per the previous study . Histological analysis was also used to quantify the ECM and DNA content within the scaffolds following fixation in paraformaldehyde for at least 48 hours.…”

“…However, due to its novel nature, limited literature exists, as the majority of research has utilized animal muscle and the described protocols have only been applied to relatively small tissue samples. This is an area that warrants further research as current literature also describes inconsistencies concerning which decellularization protocols are optimal . We have previously evaluated three techniques to evaluate human facial muscle decellularization and aimed to establish if the same decellularization technique using a detergent only approach could be used for upper limb muscle scaffold production …”

Rabbit thyroid extracellular matrix as a 3D bioscaffold for thyroid bioengineering

“…Studies have reported that Collagens are responsible for for maintaining the ECM structure. [26] Although it is widely considered decellularization with SDS is related to ECM ultrastructure disruption [27,28] but our SEM results demonstrated that the 3D ECM structures were preserved thus indicating that 1% SDS was safe to DTG scaffolds. Besides, LN and FN are involved in cell adhesion.…”