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The effect of tendon-derived progenitor cells to promote tendon healing in an equine tendinitis model

Flexor tendon injuries are among the most common musculoskeletal injuries which contribute to the loss of athletic use of horses. The healing response is prolonged in tendon injury and the resultant repair tissue is usually of inferior mechanical strength which is likely due to the low vascularity, low overall cellularity of the tissue, a low number of progenitor cells within the tissue, and healing under weight-bearing conditions. In this study collagenase-induced tendinitis lesions were created in the fore limbs of eight clinically normal horses. Autogenous tendon-derived progenitor cells were isolated and cultured and injected into one of the forelimbs and the opposite control forelimb received saline injection. A similar injury was created in one of the hind limbs and DiI stained tendon-derived progenitor cells were injected at 1, 2, 4 and 6 weeks prior to euthaniasia to determine the fate and progression of the cells injected at the above mentioned time points. Healing was assessed for 90 days using ultrasonography every 2 weeks and Magnetic Resonance Imaging (MRI) was performed prior to euthanasia. Superficial digital flexor tendons were harvested and subjected to histological analysis by light microscopy and fluorescent microscopy, mRNA and biochemical analysis and biomechanical testing.

Histology: Representative samples from tendons were fixed in 4% paraformaldehyde, mounted vertically on hinged multi cassette, dehydrated, infiltrated with paraffin (Leica ASP 300 Tissue Processor) and embedded blocks were sectioned at 6 micron intervals using a Leica Microtome (Leica RM2255  rotary microtome). Deparaffinized sections were stained for Hematoxilin and Eosin(H&E) to analyze the histological architecture and grade the collagen fiber pattern, cell numbers and neovascularization(Figure 1). Sections were also stained for Picro-sirius red (Figure 2) and toluidine blue to analyze the fiber pattern under polarization and proteoglycan content respectively.              
F:\273083 RF-PM H&E 40X.jpg     F:\273083 LF-PM H&E Pol.jpg
Figure 1: H&E staining of deparaffinized Section     Figure 2: Picro-sirius red staining under polarization

                
Cryosectioning, Fluorescent Microscopy and Image analysis: Samples of tendon injected with DiI stained progenitor cells were embedded in OCT after sucrose infiltration and sectioned using the cryostat (Leica CM3050 S cryostat) and images were acquired using fluorescence microscope (Zeiss Axiovert 200M with the Apotome) for the different time points mentioned. Rhodamine filter set was used to image the fluorescent cells injected with DiI and autofluorescence using a FITC filter set. DAPI counterstaining was performed on each section to image the nuclei of all cells present in the sample (figure 3B). Quantification of the DiI and DAPI stained cells were performed in the program Axiovision Automated Measurement Wizard using intensity based thresholding after brightness and contrast adjustments. Independent macro files were created and stored for both DiI and DAPI channels with contrast adjustments and threshold information and applied to all images equally before making measurements (Figure 3A and 3C). The area information in each channel is then exported as csv file format and opened in Microsoft Excel for further statistical analysis.
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Figure 3.  (A) Fluorescent quantification of progenitor cells staining with DiI corresponding to the red fluorescent labeling seen in (B).
(C) Fluorescent quantification of all cells (DAPI labeling)  which correspond to the blue fluorescent labeling seen in (B).

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Horse on the table      
    
Horse in MRI
    
Horse in MRI

Image courtesy and copyright - Dr.Allison Stewart, Department of Veterinary Clinical Medicine