Abstract
Introduction and Objective
The use of microfragmented adipose tissue (mFAT) for the treatment of musculoskeletal disorders, especially osteoarthritis, is gaining popularity following the positive results reported in recent case series and clinical trials. The purpose of this study is to characterize mFAT in terms of structure, cell content and secretome (i.e. protein and microvescicles released as paracrine mediators), and to compare it with unprocessed lipoaspirate tissue, in order to understand the possible mechanisms of action and the benefit derived from tissue processing.
Materials and Methods
Unprocessed lipoaspirate (LA) and mFAT were obtained from 7 donors. Each tissue sample was divided in four aliquots: A) fixed in formalin for histological evaluation; B) enzymatically digested to harvest cells with the exclusion of adipocytes; C) cultured for 24 hours in serum-free DMEM to harvest secretome; D) freshly frozen for proteomic evaluation. Hematoxylin and eosin staning, as well as immunohistochemistry for CD31, CD90, CD146 were performed on aliquot A. Cell count, viability, senescence and immunophenotype were assessed on aliquot B. Culture medium from aliquot C was collected and used for proteomic analysis and micro-RNA extraction and quantitation from extracellular vesicles. Aliquot D was lysed, protein were extracted and analyzed using a high-throughput proteomic approach.
Results
Histological investigations showed a lower red blood cell content in mFAT with respect to LA, while the presence of blood vessels (CD31+), stromal cells (CD90) and pericytes (CD146) was similar in all samples. These results were confirmed by flow cytometry, with reduction of erythrocytes (CD235a+) by 76% and reduction of lymphocytes (CD45+) by 79% in mFAT compared to LA. Otherwise, the proportions of stromal cells, pericytes and endothelial cells in LA and mFAT remained comparable. The percentage of senescent cells resulted similar before and after tissue processing, with very low values (< 5%). The analysis of the miRNAs contained in the extracellular vesicles in culture media identified 376 miRNAs in LA secretome and 381 in mFAT secretome. A high correlation in the expression of these miRNAs within subjects (LA and mFAT of each donor) was observed (R2> 0.8), indicating that processing in mFAT does not significantly alter the portfolio of miRNAs associated with extracellular vesicles. Proteomic analysis of secretome revealed that 217 proteins significantly differ between LA and mFAT. In particular, protein associated with acute phase were less represented in mFAT secretome, while intracellular proteins were more frequent. Proteomic analysis of tissues demonstrated a reduction of protein related to extracellular matrix and of proteins closely related to peripheral blood contamination in mFAT with respect to LA.
Conclusions
Taken together, these results suggest that processing of LA into mFAT allow for removal of blood elements, in terms of red blood cells, lymphocytes, acute phase and complement system proteins, and for the reduction of extracellular matrix components. Otherwise, tissue structure, cell populations, cell viability and senescence are not influenced by tissue processing. Then, microfragmentation process represents a safe and efficient method for the application of adipose tissue properties to musculoskeletal disorders, allowing for the maintenance of all the effector elements for tissue regeneration while removing possible detrimental agents such as inflammatory mediators.
