Improvement of adipose tissue–derived cells by low-energy extracorporeal shock wave therapy
Introduction
Cell-based therapies with autologous adipose tissue–derived cells have shown great potential in several clinical studies in the last decades. In the field of aesthetic and reconstructive medicine an abundance of knowledge was accumulated in the last century [1] and later was extended through clinical studies in regenerative medicine and tissue engineering [2], [3], [4]. The majority of studies have been using the stromal vascular fraction (SVF), a heterogeneous mixture of fibroblasts, lymphocytes, monocytes/macrophages, endothelial cells, endothelial progenitor cells, pericytes and adipose-derived stromal/stem cells (ASC) among others [5], [6], [7], [8], [9], [10]. In clinical case studies and trials treating soft tissue defects [4], [11], [12], [13], [14], bone and cartilage defects [15], [16], [17], [18], [19], gastrointestinal lesions [20], immune disorders [21], [22], neurological injuries [23] and cardiovascular diseases [24], SVF and ASC have already proven their regenerative potential. Although possible clinical applications of autologous adipose tissue–derived cells are manifold, they are limited by drawbacks concerning stem cell identity and potency of the isolated cell population. Different cell isolation protocols and methods but also closed automated isolation devices bring up cell populations with variable content of potentially therapeutic cells within the fat graft or the SVF [25]. In addition, donor variability results in a highly heterogeneous cell composition and functionality, which may reduce reproducibility and efficacy, and increase the risk for transplantation of low-potent cells into the patient. SVF cells and ASC can be characterized with a distinct surface marker profile and have the ability to differentiate at least into the mesodermal lineages. This is defined in a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) together with the International Society for Cellular Therapy (ISCT) to provide guidance for standardization between different research groups [26]. Cultivation, purification and differentiation of ASC are standard procedures for clinical trials, but it remains difficult to meet the requirements of regulatory agencies for stem cell translation into clinics. To increase therapeutic cell potency, numerous strategies have been evaluated for activation of cells or cell material such as physical stimulation using low level light therapy (LLLT) [27], [28], [29], photobiostimulation [30], [31] or radio electric asymmetric conveyer [32]. Moreover, the efficiency of ASC transplants was improved by the addition of activated platelet-rich plasma (PRP) [33], [34] or growth factors [35].
In this study we aimed to improve stem cell properties and reduce donor variability by mild mechanical stimulation using extracorporeal shock wave therapy (ESWT).
Extracorporeal shock waves are sonic pulses, characterized by an initial increase, reaching a positive peak of up to 100 MPa within 10 ns, followed by a negative amplitude of up to -10 MPa and a total life cycle of less than 10 µs [36]. Biological responses are thought to be triggered by the high initial pressure, followed by a tensile force and the resulting mechanical stimulation [36]. ESWT has been applied for several decades in the clinics and has demonstrated beneficial effects on tissue regeneration in non-union fractures [37], [38], [39], ischemia-induced tissue necrosis [40] or post-traumatic necrosis, disturbed healing wounds, ulcers and burn wounds [41], [42]. We have previously shown that low-energy ESWT enhances proliferation and differentiation of ASC lines in vitro [43], [44]. These in vitro studies corroborate the clinical success of ESWT in wound healing, nerve regeneration and vascularization [45], [46].
In our experimental set-up, low-energy ESWT was applied to freshly isolated SVF cells from human adipose tissue aiming to equalize and enhance cell properties and functionality. To limit the degree of manipulation of the cells during the SVF isolation process we applied in a second approach ESWT directly on the freshly obtained human adipose tissue and compared it with ESWT on isolated SVF cells. Based on this, we studied cellular adenosine triphosphate (ATP) content, immunophenotype, cell yield, viability, colony-forming unit fibroblast (CFU-F) assay and protein secretome of the SVF. Furthermore, we cultured ASC from these SVF and investigated proliferation and differentiation potential toward the adipo-, osteo- and chondrogenic lineage.
Section snippets
SVF/ASC isolation
The use of human adipose tissue was approved by the local ethical board with patient's consent. Subcutaneous adipose tissue was obtained during routine outpatient liposuction procedures under local tumescence anesthesia. SVF isolation was performed as modified from Wolbank et al. [47] as follows. Briefly, 100 mL liposuction material was transferred to a blood bag (400 mL Macopharma) and washed with an equal volume of phosphate-buffered saline (PBS) to remove blood and tumescence solution.
Comparison of ESWT on human adipose tissue to ESWT on SVF cells
To identify the most beneficial set-up, ESWT was applied either directly on adipose tissue isolated via liposuction from five different donors or on freshly processed SVF cells derived from the same donors. Figure 2 and Figure 3, Figure 4, Figure 5 show the data of each single donor before (control condition, identical for both groups) and after the respective ESWT.
ESWT on adipose tissue enhances cellular ATP
Cellular ATP levels were assessed 2 h after cell seeding, comparing the untreated control group with ESWT-treated SVF and
Discussion
Based on our previous studies, application of low-energy ESWT is promising for promoting the regenerative quality of stem cells. Recently we have shown that shock wave treatment at an energy level of 0.09 mJ/mm2 enhanced adipogenic, osteogenic and Schwann-like cell differentiation of human ASC [43]. We have shown that shock waves at 0.1 mJ/mm2 and 300 impulses improve skin flap survival through neovascularization and early upregulation of angiogenesis-related growth factors in a rodent ischemic
Acknowledgments
We thank Dr. Matthias Sandhofer for providing liposuction material. Financial support from the Austrian Research Promotion Agency (FFG) project Liporegeneration (846062) and the FFG project Disease Tissue (845443) is gratefully acknowledged.
Disclosure of interests: We exclude any conflict of interest and guarantee that all authors are in complete agreement with the contents and submission of this article. Furthermore, we declare that our work is original research, unpublished and not submitted
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These authors contributed equally.