Image1_Effects of single and repeated shock wave application on the osteogenic differentiation potential of human primary mesenchymal stromal cells and the osteoblastic cell line MG63 in vitro.jpg

Introduction: Extracorporeal shock wave therapy is a non-invasive and effective option for treating various musculoskeletal disorders. Recent literature indicates that the parameters for extracorporeal shock wave therapy, such as the optimal intensity, treatment frequency, and localization, are yet to be determined. Studies reporting on the effects of shock wave application on primary mesenchymal stromal cells (MSCs) as well as osteoblastic cell lines in vitro are barely available and not standardized.Methods: In this study, we designed a special setup to precisely expose primary MSCs and the osteoblastic cell line MG63 to shock waves and subsequently analyzed the resulting cellular responses using standardized protocols to investigate their viability, proliferation behavior, cytokine secretion, and osteogenic differentiation potential in vitro. The shock wave transducer was coupled to a specifically designed water bath containing a 5 mL tube holder. Primary human MSCs and MG63 cells were trypsinated and centrifuged in a 5 mL tube and exposed to single and repeated shock wave application using different intensities and numbers of pulses.Results: Single treatment of MSCs using intensities 5, 10, 15, and 20 and pulse numbers 100, 250, 500, 750, and 1,000 at a constant pulse repetition frequency of 1 Hz resulted in a decreased viability and proliferation of both cell types with an increase in the intensity and number of pulses compared to controls. No significant difference in the osteogenic differentiation was observed at different time intervals in both cell types when a single shock wave application was performed. However, repeated shock wave sessions over three consecutive days of primary MSCs using low intensity levels 0.1 and 1 showed significant osteogenic differentiation 4-fold higher than that of the extracted Alizarin Red S at day 14, whereas MG63 cells showed no significant osteogenic differentiation compared to their corresponding controls. More specifically, repeated shock wave application triggered a significant downregulation of COL1A1, upregulation of RUNX2, and sustained increase of OCN in primary MSCs but not in the cell line MG63 when induced toward the osteogenic differentiation.Discussion: The effects of shock wave application on MSCs make it an effective therapy in regenerative medicine. We established a protocol to analyze a standardized shock wave application on MSCs and were able to determine conditions that enhance the osteogenic differentiation of MSCs in vitro.</p

Calcium incorporation in alginate beads with MSCs.

<p>For MSC beads, we found an increase in the uptake of AR from day -21 (A) before differentiation and day 0 (B). The Dex-treated beads then showed a much more intense staining, and at higher magnification, the Dex-treated cells (D) showed a pericellular matrix and dark red calcium depositions around the cells, which was not seen at this intensity in the other, T3-treated samples (C).</p

Markers of hypertrophy in chondrocyte and MSC culture.

<p>Shown is the relative change in Col type X mRNA in chondrocytes (A) and MSCs (B) and the total level of ALP activity (C and D, respectively). Both markers demonstrate that for articular chondrocytes, T3 can induce terminal differentiation in the cells, while chondrogenically differentiated MSCs show a similar response but to dexamethasone instead.</p

Calcium incorporation in alginate beads with chondrocytes.

<p>Between day -14 (A) and day 0 (B) no major increase was seen in AR uptake. Chondrocyte seeded beads treated with T3 (C) showed then a more heterogeneous stain of AR after induction, as compared to the dex-treated group (D).</p

Proteoglycan visualization in alcian blue staining of alginate beads.

<p>Freshly prepared beads (A) only have slight background staining of alcian blue, almost all proteoglycans are removed from the cells during the digestion process. In the overview after serum preculture and five weeks of culture (B), the cells impose as round, singular cells with surrounding deep-blue pericellular matrix and further removed matrix in the gel. No differences can be visualized in the GAG content between T3-treated (C) and Dex-treated (D) chondrocytes.</p

Experimental layout and timepoints.

<p>MSCs were harvested from the tuber ischiadicum and chondrocytes from fetlock joints, and both cells were encapsuled in alginate beads. While the MSCs were differentiated chondrogenically for three weeks (day -21 to day 0), the chondrocytes were kept in serum medium for two weeks (day -14 to day 0). The beads were then submitted to hormonal treatment for five weeks (day 0 to day 35) with weekly samples for analysis.</p

Production of DNA, GAG and HYP and collagen mRNA expression during chondrogenic MSC differentiation.

<div><p>The upper graph A shows the changes in total DNA content per bead (or per pellet, respectively) during the differentiation and the increase of proteoglycans and hydroxyproline as a sign of extra cellular matrix production. Both pellet cultures and alginate bead cultures show an increase in GAG, but only in alginate beads, also collagen is produced and retained. Shown is the mean of three samples per animal (n=6) +/- standard error of the mean (SEM).</p> <p>The lower graph B shows the fold-increase (mean +/- SD) of collagen mRNA, compared to undifferentiated MSCs kept in monolayer in expansion medium for 3 weeks. The bars compare alginate beads with and without TGF-β1 vs. pellet cultures with and without TGF-β1. Alginate beads with TGF-β1 show the highest increase in Col II mRNA expression, indicating that the culture system is well suited for chondrogenic differentiation of the MSCs.</p></div

Stained cryosections of a pellet culture and an alginate bead.

<p>Alcian blue stain of 8 µm Cryosections of an alginate bead culture (A) and an MSC pellet culture (B) after 21 days of chondrogenic differentiation. Proteoglycans show in blue. The gaps in the alginate bead section are artifacts from the cutting. The scale bar represents 500 µm.</p

GAG content over time in the treatment groups.

<p>Shown is the relative GAG per DNA content of chondrocytes (A) and MSCs (B), with day 0 being the reference and indicating the beginning of the hormonal treatment. Absolute amounts of GAG reached more than 60 µg GAG/µg DNA or more than 80 µg of GAG per bead in the Dex group. After changing to serum-free hormonal treatment medium, Dex enhanced the GAG production significantly more than T3 or control medium (seen from day 21 on, with P < 0.001) and showed a general increase over time. A further increase with T3 after preculture serum showed only at day 28 (P < 0.01 vs. day 0), the control group had no significant increase in GAG/DNA after change to serum free medium. The MSCs behaved similar in their response to Dex, with significant increase in GAG production after two weeks (P < 0.01 at day 14 and 21 vs. day 0, and P < 0.0001 afterwards), but T3 seemed to rather decrease GAG production.</p

Hydroxyproline production in chondrocytes.

<p>Total amount of HYP was quantified and calibrated to the total amount of DNA, to equalize for production merely due to increased or decreased cell number. Shown are the results of the quantification in the articular chondrocytes (A) and the MSCs (B) before and during hormonal treatment, as mean +/- SD.</p