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Mesenchymal Stem Cell Proliferation and Expression of Growth Factors in Response to Extracorporeal Shock Wave Therapy Treatment

Take Home Message

While extracorporeal shock wave therapy (ESWT) has been shown to enhance the therapeutic potential of certain cell types, its effect on equine mesenchymal stem cells (MSCs) is not known. We report that ESWT does not enhance MSC proliferation in vitro, and that gene expression of selected growth factors is not affected. For proliferation cultures, the first application of ESWT decreased growth relative to unshocked MSCs, although subsequent ESWT treatments did not affect MSC proliferation. These data suggest that it may be possible to administer multiple ESWT treatments to defects containing MSCs without harming the repair response of transplanted MSCs.

Introduction

Extracorporeal shock wave therapy has been widely used to treat tendonitis in human (1) and veterinary medicine (2, 3). For equine patients, the use of ESWT on proximal suspensory desmitis has been suggested to improve the prognosis in the hindlimb (4). Similarly, promising evidence of increased collagen fibril accumulation (5) and neovascularization (2) in experimental models of equine tendonitis has been demonstrated with ESWT. A second promising therapy for equine tendonitis is injections of bone marrow MSCs (6, 7). When injected into tendon and ligament core lesions, MSCs have led to a higher return to work and lower reinjury rate than would be expected without treatment (7-9). To date ESWT or MSCs have been separately to treat tendonitis, although it remains possible that the combination of ESWT following MSC injection into core lesions may further enhance repair over the individual treatments. As a first measure of the effect of ESWT on equine MSCs, Drs. Kisiday, Frisbie, and McIlwraith evaluated MSC proliferation following ESWT using cell culture techniques that are similar to those used to prepare MSCs for clinical applications (7). In addition, we explored the effect of ESWT on MSC gene expression of growth factors that may influence healing of diseased tendon.

Methods

Proliferation: The effect of ESWT on MSC proliferation in monolayer culture was evaluated over a ten day timecourse. Equine MSCs were plated at a concentration of 12,000 MSCs/cm2 and maintained in baseline media containing 10% FBS. Half of the cultures were fed baseline media, while a second group was fed baseline media plus 2 ng/ml fibroblast growth factor 2 (FGF). Control groups received no additional treatment. Experimental groups were subjected to 500 pulses using energy level E2 prior to the first plating and in between passages. ESWT was administered to MSCs in suspension within 2 ml of baseline medium held within 15 ml centrifuge tubes. For each passage, MSCs were allowed to grow for two days, a point where control cultures in FGF-2 medium reached near confluence. At this point the cells were lifted using trypsin, counted, and replated at a concentration of 12,000 MSCs/cm2. The cultures were expanded as such five times, resulting in five passages over 10 days. At each passage, the cell yield was divided by the number of cells plated at the start of the experiment to obtain a fold-change in cell number. The log2 of the fold-change was calculated to determine the number of population doublings. This experimental protocol was repeated for MSCs from three animals.
 
Gene expression of growth factors: ESWT was administered to MSCs in suspension as previously described, and then seeded at 20x103 cells/cm2 in monolayer culture in alphaMEM with or without 10% FBS. Control cells that did not receive ESWT were seeded in parallel. After four hours, RNA was harvested from monolayer cultures and isolated using the RNeasy Mini Kit according to the manufacturer's instructions. RNA was reverse transcribed to cDNA in the presence of random hexamers. Semiquantitative real time PCR was performed for vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), and transforming growth factor beta (TGFβ) using the Applied Biosystems 7000 system and TaqMan Universal PCR Master Mix. For each sample, expression was normalized to 18S threshold values.
 
Statistical Analysis: Proliferation assays were conducted using MSCs from three donor horses, while gene expression analysis was evaluated using MSCs from four donor horses. Log transformed data were analyzed using a mixed model analysis of variance, with the donor animal used as a random effect. Individual comparisons were made using least square means procedure. For MSC proliferation through the first five passages, ESWT, FGF, and passage were considered main effects as well as their interactions. For cumulative proliferation through the first five passages, ESWT and FGF were considered main effects as well as their interactions. For gene expression analysis, ESWT and FBS were considered main effects as well as their interactions. Individual comparisons were made when main effects or interactions resulted in an f-value less than 0.05. For individual comparisons, p-values less than 0.05 were considered significant. Data are reported as mean +/- standard error of the mean.

Results

Proliferation: Interactions among ESWT, FGF, and passage were not significant (p = 0.91). When considering the effects of ESWT and passage independent of FGF, at passage one, ESWT cultures (0.71 +/- 0.17 population doublings) experienced 0.95 fewer population doublings than control cultures (1.66 +/- 0.17) (p < 0.001, Fig. 1). For passages two through five, the number of population doublings in ESWT cultures were not significantly different from controls for each passage (p = 0.24-0.95). When considering the effect of FGF independent of ESWT and passage, FGF+ cultures (1.86 +/- 0.09) averaged 0.59 more population doublings compared to FGF- cultures (1.27 +/- 0.09) (p < 0.001, data not shown). For the cumulative population doublings over the first five passages, interactions between ESWT and FGF were not significant (p = 0.32). When considering the effect of ESWT independent of FGF, ESWT cultures (7.35 +/- 0.40) resulted in 0.90 fewer population doublings than control cultures (8.25 +/- 0.40) (p < 0.005, data not shown). For MSCs from FGF+ cultures that were expanded through a sixth passage, the number of population doublings by MSCs that received ESWT throughout the first five passages was not significantly different from that in control cultures that did not receive ESWT (p = 0.87) (data not shown). MSCs from control cultures that were subjected to ESWT for the first time prior to seeding into passage six experienced approximately 0.3 fewer population doubling than cultures treated with ESWT throughout the six passages and untreated controls (p < 0.05).
 
Gene expression of growth factors: For IGF-1, TGFβ, and VEGF, interactions between ESWT and FBS were not significant (p = 0.67-0.79) (data not shown). When considering the main effect of FBS, gene expression of IGF-1 in FBS-free conditions were 4.3-fold higher than FBS cultures, while gene expression of TGFβ and VEGF in serum-free cultures conditions was 2.1- and 9.3-fold lower than controls (p < 0.05). When considering the main effect of ESWT, gene expression of IGF-1, TGFβ, and VEGF were not significantly different (p = 0.45-0.92).

Conclusion

ESWT and MSCs are used separately to treat equine tendonitis, and it is not know how ESWT applied following the injection of MSCs may affect healing. This in vitro study did not demonstrate a benefit of ESWT on MSC proliferation or gene expression of growth factors; however, our data suggest that the only negative effect of ESWT is a short-term decrease in MSC proliferation following the initial treatment only.

Acknowledgements

Funded by Pulse Veterinary Technologies, Alpharetta, Ga.

References

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