Influence of Extracellular Vesicles Isolated From Osteoblasts of Patients With Cox-Arthrosis and/or Osteoporosis on Metabolism and Osteogenic Differentiation of BMSCs

Abstract

Background: Studies with extracellular vesicles (EVs), including exosomes, isolated from mesenchymal stem cells (MSC) indicate benefits for the treatment of musculoskeletal pathologies as osteoarthritis (OA) and osteoporosis (OP). However, little is known about intercellular effects of EVs derived from pathologically altered cells that might influence the outcome by counteracting effects from "healthy" MSC derived EVs. We hypothesize, that EVs isolated from osteoblasts of patients with hip OA (coxarthrosis/CA), osteoporosis (OP), or a combination of both (CA/OP) might negatively affect metabolism and osteogenic differentiation of bone-marrow derived (B)MSCs. Methods: Osteoblasts, isolated from bone explants of CA, OP, and CA/OP patients, were compared regarding growth, viability, and osteogenic differentiation capacity. Structural features of bone explants were analyzed via μCT. EVs were isolated from supernatant of naïve BMSCs and CA, OP, and CA/OP osteoblasts (osteogenic culture for 35 days). BMSC cultures were stimulated with EVs and subsequently, cell metabolism, osteogenic marker gene expression, and osteogenic differentiation were analyzed. Results: Trabecular bone structure was different between the three groups with lowest number and highest separation in the CA/OP group. Viability and Alizarin red staining increased over culture time in CA/OP osteoblasts whereas growth of osteoblasts was comparable. Alizarin red staining was by trend higher in CA compared to OP osteoblasts after 35 days and ALP activity was higher after 28 and 35 days. Stimulation of BMSC cultures with CA, OP, and CA/OP EVs did not affect proliferation but increased caspase 3/7-activity compared to unstimulated BMSCs. BMSC viability was reduced after stimulation with CA and CA/OP EVs compared to unstimulated BMSCs or stimulation with OP EVs. ALP gene expression and activity were reduced in BMSCs after stimulation with CA, OP, and CA/OP EVs. Stimulation of BMSCs with CA EVs reduced Alizarin Red staining by trend. Conclusion: Stimulation of BMSCs with EVs isolated from CA, OP, and CA/OP osteoblasts had mostly catabolic effects on cell metabolism and osteogenic differentiation irrespective of donor pathology and reflect the impact of tissue microenvironment on cell metabolism. These catabolic effects are important for understanding differences in effects of EVs on target tissues/cells when harnessing them as therapeutic drugs.

Keywords: EVs; extracellular vesicles; mesenchymal stem cells; osteoarthritis; osteoblasts; osteogenic differentiation; osteoporosis.

FIGURE 1

Patient characteristics and μCT analysis of bone biopsies obtained from CA, CA/OP, and OP patients after hip replacement surgery. (A) Age of patients with CA, CA/OP, and OP at time point of operation. N = 9 (CA), 10 (CA/OP), 9 (OP); Y = years. (B) μCT analysis of bone explants from trabecular bone—trabecular thickness (Tb.Th.). N = 7 (CA), 8 (CA/OP), 9 (OP). (C) μCT analysis of bone explants from trabecular bone—trabecular number (Tb.N.). N = 7 (CA), 8 (CA/OP), 9 (OP). (D) μCT analysis of bone explants from trabecular bone—trabecular separation (Tb.Sp.). N = 7 (CA), 8 (CA/OP), 9 (OP). (E) Representative 3D-images of bone explants of CA, CA/OP, and OP patients after μCT analysis. Scanning was performed with a voxel size of 12 μm.

FIGURE 2

Isolation of osteoblast-like cells from bone explants of patients with CA, CA/OP, and OP. (A) Isolation process demonstrating cell outgrowth of osteoblast-like cells expressing osteoblastic characteristics from bone explants in two consecutive phases of explant (E)1, E2, and E3 cultures. Cells were either frozen immediately in passage 0 (P0) or frozen after additional passaging of the cells until P3 (modified from Dillon et al., 2012). (B) Time span of outgrowth of cells from E1 bone explants until confluency. N = 6–10. (C) Time span of outgrowth of cells from E2 bone explants until confluency. N = 5–9. (D) Growth time of cells in cell culture from time point of seeding the cells after thawing until confluency. N = 6–9.

FIGURE 3

Characterization of viability and osteogenic capacity of osteoblasts isolated from patients with CA, CA/OP, and OP. (A) Comparison of osteoblast viability [optical density = OD]. P-values over bars represent differences between groups (CA; CA/OP; OP). ^§ (Red) = p = 0.0781, represents tendency compared to the CA/OP group at day 14. *(blue) = p ≤ 0.05, represents difference compared to the OP group at day 14. N = 6–9. (B) Matrix formation capacity was compared using Alizarin Red staining. Graph shows concentration of Alizarin Red in mM. P-values over bars or *(= p ≤ 0.05) represent differences between groups (CA; CA/OP; OP). *(red) = p ≤ 0.05, represents difference compared to the CA/OP group at day 14. N = 7–8. (C) Bone formation ability was compared by assaying intracellular ALP enzyme activity (U/L). P-values over bars or *(= p ≤ 0.05)/**(= p ≤ 0.01) represent differences between groups. ^§ (Green) = p = 0.0625, represents tendency compared to the CA/OP group at day 14. (CA; CA/OP; OP). N = 6–8.

FIGURE 4

Gene expression of BGLAP, COL1A1, Runx2, and ALP in CA, OP, and CA/OP osteoblasts after 28 (A) and 35 (B) days in osteogenic culture medium. These time points were chosen according to time points for EV isolation from the culture supernatants of osteogenic cultured CA, OP, and CA/OP osteoblasts. Results were calculated by the ΔCt method. N = 4.

FIGURE 5

EV characterization and validation. (A) Representative SEM pictures of BMSC EVs. White arrows label EVs with 97 nm (left image) and 115 nm (right image) diameter. Magnification: 60,000×. (B) Representative TEM pictures of BMSC EVs. Scale bar left image = 200 nm, magnification: 100,000×. Right image shows magnification of the EV in the green box. The size of objects was determined (red lines). Vertical line = 104 nm, horizontal line = 113 nm. (C) Western Blot analysis of EV specific surface markers CD9 (left image) and CD81 (right image) in BMSC, CA, CA/OP, and OP EVs and in the EV-depleted FCS^{depl– uc}. Lane 1 = MW ladder, Lane 2 = 5 μg; Lanes 3–6 = 8.2 μg; Lane 7 = 10 μl; Exposure time: left image = 3 min, right image = 10 min (Pierce femto Kit). For respective Ponceau Red images, see Supplementary Figure 3. (D) Test for EV uptake of BMSC-derived EVs into cultured BMSCs using PKH-26 (red) stained EVs (lower panel). PBS solution + PKH-26 stain was used as negative control (upper panel). Nuclei were counterstained with DAPI. Scale bar 100 μm.

FIGURE 6

Influence of EVs isolated from BMSCs and CA, CA/OP, and OP osteoblasts on BMSC metabolism. (A) Proliferation of BMSCs after cultivation for 2 days followed by stimulation for 24 h with EVs isolated from BMSCs and CA, CA/OP, and OP osteoblasts. N = 9. (B) Viability (WST-1 assay) of BMSCs after cultivation for 2 days followed by stimulation for 48 h with EVs isolated from BMSCs and CA, CA/OP, and OP osteoblasts. N = 9. (C,D) Apoptosis rate (Caspase 3/7 activity assay) of BMSCs after cultivation for 2 days followed by stimulation for 24 h with EVs isolated from BMSCs and CA, CA/OP, and OP osteoblasts. Fluorescence was measured 1 h and 24 h after addition of Caspase 3/7-assay buffer. N = 9. Results were calculated as percentage to the unstimulated control (BMSCs without EV stimulation/no EVs/Ø = 100%, shown by the dotted line). */**Significant differences to control Ø with p ≤ 0.05/0.01; §/§§Significant differences to BMSC EVs (EVs) with p ≤ 0.05/0.01. ^§ (red) = significant differences to CA/OP EVs with p ≤ 0.05. ^#(green) = tendency compared to CA EVs.

FIGURE 7

Influence of stimulation with EV from BMSCs and CA, CA/OP, and OP osteoblasts on the expression of osteogenic marker genes in BMSCs and their osteogenic differentiation ability. (A) Gene expression of BGLAP, COL1A1, RUNX2, and ALP was analyzed after cultivation for 13 days followed by a 24 h stimulation of BMSCs with EVs isolated from BMSCs and CA, CA/OP, and OP osteoblasts. Calibrator (= no EVs/Ø control) was set to 1 and results were calibrated as x-fold change to calibrator (= ΔΔCt method). N = 7. (B) ALP enzyme activity as a marker for bone formation activity in BMSCs after cultivation for 21 days followed by stimulation during the last 7 days (EV addition 3× with medium exchange) with EVs isolated from BMSCs and CA, CA/OP, and OP osteoblasts. N = 7. (C) Alizarin Red incorporation into the extracellular matrix of BMSCs after cultivation for 21 days followed by stimulation during the last 7 days (EV addition 3× with medium exchange) with EVs isolated from BMSCs and CA, CA/OP, and OP osteoblasts. N = 7. *Significant differences compared to no EVs control Ø with p ≤ 0.05. ^#Tendency to BMSC EVs with p = 0.0625. ^§ Significant differences compared to BMSC-EVs with p ≤ 0.05. ^§ (Red) = significant differences to CA/OP EVs with p ≤ 0.05.