Abstract
PURPOSE: Orthopaedic musculoskeletal injuries affect millions of people each year. Conventional treatments are limited in scope and present potential side effects. The last decade has seen a tremendous interest in using biologics for regenerative medicine applications including stem cells. Despite therapeutic benefits, stem cells present several disadvantages. Recent evidence has attributed the beneficial effects of stem cells to their ability to secrete bioactive molecules such as growth factors (GFs), cytokines (CKs) and extracellular vesicles (EVs), including exosomes. The present study reports the development of a novel cell-free, stem cell-derived extract (CCM) formulation and characterized for the presence of GFs, CKs and EVs including exosomes. METHODS: The CCM was formulated from procured human progenitor endothelial stem cells (hPESCs). Randomly selected samples from different batches were analyzed for the presence of GFs and CKs using enzyme-linked immunosorbent assay, and EVs, including exosomes, using nanoparticle tracking analysis and single particle interferometric reflectance imaging sensing. The effect on human fibroblasts proliferation and bone marrow mesenchymal stem cells induced migration was analyzed using Alamar Blue cell proliferation assay and a Transwell migration assay, respectively. SUMMARY OF RESULTS: GFs including IGFBP 1, 2, 3 and 6, insulin, growth hormone, PDGF-AA, TGF-α, TGF-β1 and VEGF were detected. Anti-inflammatory cytokine, IL-1RA, was also detected. Membrane enclosed particles in the exosome size range and expressing exosome specific tetraspanins CD81 and CD9 were also detected, indicative of true exosomes. Cells treated with CCM showed significantly higher rate of cell proliferation and induction of stem cell migration compared to control. CONCLUSION: Analysis of the CCM formulation revealed the presence of GFs, CKs and EVs, including exosomes. This significantly enhanced the rate of cell proliferation and stem cell migration. The presence of multiple factors within one formulation and their ability to promote cell proliferation and stem cell migration may help reduce inflammation and pain and augment repair and regeneration of musculoskeletal tissues.
Highlights
During the past few decades, there has been a tremendous growth in the use of biologics for regenerative medicine applications [1,2]
Interleukin 1 receptor antagonist (IL-1RA), an anti-inflammatory cytokine was identified at significant levels in CCM relative to the control which had undetectable IL-1RA levels (Figure 1)
We describe the process of formulation of a novel cell-free stem cell-derived extract (CCM), and evaluated it for the presence of growth factors (GFs), CKs, and extracellular vesicles (EVs), including exosomes
Summary
During the past few decades, there has been a tremendous growth in the use of biologics for regenerative medicine applications [1,2]. Biologics currently available for clinical use include platelet rich plasma, bone marrow aspirate, lipoaspirate, amniotic allograft suspension, umbilical cord-derived Wharton’s Jelly, cord blood, and exosomes [3,4]. The efficacy of these biologics is attributed to the presence of stem cells, growth factors (GFs), cytokines (CKs), and extracellular vesicles (EVs), including exosomes [5,6]. MSCs present several disadvantages, including establishing a reliable source with stable phenotype, genetic instability and chromosomal aberrations, intravenous administration-related toxicity caused by physical trapping of the cells in the lung microvasculature, rejection by the host, formation of ectopic tissue, and tumorigenicity [11,12,13]
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