10.5 Tissue engineering approach with acellular matrix supports affected diaphragm of an atrophic mouse model with activation of resident cells

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Abstract

Myopathies are frequently caused by mutations in genes encoding for extracellular matrix (ECM) proteins, which gets progressively substituted by fibrotic tissue. Spinal muscular atrophy is a disorder caused by mutations in SMN gene. The same mutation in HSA-Cre, SmnF7/F7 mice generates a specific impairment of skeletal muscle with diaphragm displaying fibrosis and myofiber loss. Using a decellularized matrix obtained from healthy-mice diaphragm we aimed to ameliorate diaphragm condition of HSA-Cre, SmnF7/F7 mice.

We characterised the decellularised matrix after detergent enzymatic treatment (DET) establishing that 3 DET cycles are a good compromise between DNA content reduction (p < 0.001 vs fresh tissue) and ECM preservation. Collagen and elastin content was not statistically different from fresh tissue. Importantly, decellularised matrix possessed the same thickness and stiffness of a fresh diaphragm, and key cytokines such as VEGF and SDF1α were maintained.

Decellularised patches were surgically applied to affected diaphragm of HSA-Cre, SmnF7/F7mice and changes in terms of thickness, morphological and cytological aspects were evaluated. New collagen deposition was noticeable 15 days post implantation with evident features that a remodelling process began. The acellular patch was gradually re-populated, with an increasing presence of proliferating cells. After 30 days the patch was partially absorbed while, on the other hand, the weak native diaphragm underwent strong remodelling and increased in thickness.

In conclusion, we successfully developed an acellular diaphragm scaffold that exerted local cellular activation, turnover and matrix composition in a myopathic diaphragm. Minimally invasive applications of decellularised matrices to diaphragms of dystrophic patients may ameliorate respiratory outcome.

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