Lipogems Product Treatment Increases the Proliferation Rate of Human Tendon Stem Cells without Affecting Their Stemness and Differentiation Capability

Abstract

Increasing the success rate of rotator cuff healing remains tremendous challenge. Among many approaches, the possibility of activating resident stem cells in situ, without the need to isolate them from biopsies, could represent valuable therapeutic strategy. Along this line, it has been recently demonstrated that lipoaspirate product, Lipogems, contains and produces growth-factors that may activate resident stem cells. In this study, human tendon stem cells (hTSCs) from the rotator cuff were cocultured in a transwell system with the Lipogems lipoaspirate product and compared to control untreated cells in terms of cell proliferation, morphology, stem cell marker and VEGF expression, and differentiation and migration capabilities. Results showed that the Lipogems product significantly increases the proliferation rate of hTSCs without altering their stemness and differentiation capability. Moreover, treated cells increase the expression of VEGF, which is crucial for the neovascularization of the tissue during the healing process. Overall, this study supports that directly activating hTSCs with the Lipogems lipoaspirate could represent a new practical therapeutic approach. In fact, obtaining a lipoaspirate is easier, safer, and more cost-effective than harvesting cells from tendon or bone marrow biopsies, expanding them in GMP facility and then reinjecting them in the patient.

Figures

Figure 1

Schematic illustration of the Lipogems device and the transwell coculture system of hTSCs with the Lipogems product. The lipoaspirate was processed using the Lipogems device to obtain the final Lipogems product. A transwell coculture system was used to study the effects of the Lipogems product on primary cultures of hTSCs. Cells were seeded on the bottom of a 6-well plate in normal growth medium. Twenty-four hours after plating, the Lipogems product was transferred in the upper well of a transwell cell culture system (250 μL/transwell insert), separated from hTSCs in the lower well by a 0.4 μm microporous polycarbonate membrane. hTSCs and Lipogems product were maintained in coculture for successive analyses.

Figure 2

Effects of the Lipogems product on hTSCs morphology and proliferation. (a) Phase-contrast microphotographs (original magnification ×10) and (b) cell growth curves of hTSCs during 96 h treatment with the Lipogems product in normal growth medium and compared to control cells. All experiments were performed in triplicate. Error bars show the mean ± SD of three different experiments. p values were calculated using Student's t-test. Only p values < 0.05 are indicated: ∗p < 0.05; ∗∗p < 0.01, as compared to control cells.

Figure 3

Effects of the Lipogems product on hTSCs apoptosis. Flow cytometric analysis of hTSCs survival rate at 0, 48, and 96 h after the treatment with the Lipogems product (right panel) as compared to control cells (left panel), through double staining with Annexin V-FITC and PI. Early apoptotic cells (Annexin V-positive/PI-negative) are localized in the lower right region, late apoptotic and necrotic cells (Annexin V-positive/PI-positive) are localized in the upper regions, and vital cells (double negative) are localized in the lower left region.

Figure 4

Effects of the Lipogems product on tendon marker expression. (a) Gene expression of TNMD, Tenascin C, and COL1A1 by Real-Time PCR at 96 h of treatment with the Lipogems product. Values are expressed as fold-changes relative to control cells. Effects of the Lipogems product on the in vitro differentiation of hTSCs toward the adipogenic and the osteogenic phenotypes. Adipogenic and osteogenic differentiation ability of hTSCs cultured with the Lipogems product in the appropriate differentiation medium was evaluated by Oil Red O (b) and Alizarin Red-S (c) staining, respectively. (b) Lipid intracellular droplets (red) in the adipocytes were stained with Oil Red O solution. (c) Alizarin Red-S staining revealed the presence of calcium deposits (yellowish-brown). Typical results are shown. Original magnification ×10. Effect of Lipogems treatment on stem cells marker (Nanog, Oct4, and KLF4) (d) and VEGF (e) expression by Real-Time PCR at 96 h of treatment with the Lipogems product. Values are expressed as fold-changes relative to control cells. Effect of the Lipogems product on hTSCs migration. (f) Representative time-lapse migration images of control and Lipogems-treated cells. Images were acquired at 0 and 25 h in in vitro wound-healing assay. Original magnification ×5. The migration rate was measured by quantifying the total area of the wounded region lacking cells. The average percentages of recovered area obtained from three different experiments at 17, 20, 25, 30, and 42 h of treatment with the Lipogems product, as compared to control cells. All quantitative data are expressed as the means ± SD of three different experiments. p values were calculated using Student's t-test. Only p values < 0.05 are indicated: ∗∗p < 0.01, compared to control cells.