The histopathological evaluation revealed that the administered therapy resulted in a better organization of collagen fibers and a decreased inflammatory infiltrate. The ultrasound evaluation showed a lack of lesion progression (lesion area). An increased number and intensity of the signals detected by the Power Doppler examination was observed; this result evidenced the presence of increased blood flow in the treated area of the TG. The therapy used did not result in any differences in the expression levels of any of the genes tested.
Tendon lesions can be induced by different methods. These techniques are basically divided into physical and enzymatic methods, such as the use of collagenase [4, 5, 8, 13], collagenase gel [24, 26], and surgical induction . Although several studies have been conducted on the subject, there is still no consensus on which is the best induction technique . The tendon lesion resulting from the use of collagenase for the induction of tendonitis is not identical to the lesion resulting from the etiopathogenesis of a naturally occurring injury. However, the collagenase method exhibits several advantages over the other methods, such as the development of hypercellularity, the loss of organization of the extracellular matrix (ECM), the increased vascularization, and the absence of inflammation mediated by inflammatory cells in the injured tendon . The results obtained from the induction of the tendon lesion using collagenase gel are consistent with those reported previously : it was possible to create consistent lesions, and there was minor extravasation of the enzyme to the epitendon.
The collection of adipose tissue, although considered an invasive procedure, proved to be a relatively simple technique [4, 5, 23], and no postoperative complications were observed in the animals. The isolation of the stromal vascular fraction and the subsequent culture of adMSCs until a sufficient amount was obtained (approximately 10 × 106 MSCs) for the implementation of the stem cell therapy were successfully achieved during the three week period used in this experiment. Although the quantification of the cell duplication of adMSCs was not performed in the present study, the data obtained are consistent with the results previously described in studies that reported the rapid growth of these cells in culture, which enables the generation of a higher concentration of progenitor cells in a shorter period of time [17, 33].
Some studies that use MSCs derived from bone marrow for the therapy of equine tendonitis use a marrow puncture to isolate the bone marrow supernatant to suspend the stem cells at the time of the therapy. There are reports of beneficial effects from this type of therapeutic combination due to the improvement in the therapeutic effect of the progenitor cells that is induced by the stimulant effects of the bone marrow supernatant . In addition to the bone marrow supernatant [15, 35], progenitor cells have been previously suspended in PBS [3, 4, 13], cell culture medium , autologous serum [5, 10], plasma , and platelet-rich plasma . In the present study, we chose to prepare the stem cell suspension in PC to combine the therapeutic effects of MSCs derived from the adipose tissue with the growth factors that are released by platelets after their activation. Our use of double centrifugation for the collection of the PC proved effective and enabled the collection of a PC with an average platelet number that was greater than that reported by previous studies [14, 25].
Although the PC was not activated in this study, there is clinical evidence that demonstrates an improvement in tendon repair after therapy using a non-activated PC . The positive results obtained with the use of non-activated PC can be explained by the in situ activation of platelets and the subsequent release of growth factors due to its stimulation by the components present in the local environment (for example, collagen) . The activation of platelets can be induced by several methods. A recent study in horses showed that CaCl2 induced the highest concentration of growth factors compared with the other various types of platelet activators tested. However, we chose to not exogenously activate the platelet concentrate prior to the tendon therapy due to our uncertainty of the safety of its use in vivo.
The therapy adopted in this study, which was followed by rest with gradual increases in physical activity, was proven feasible. There were no episodes of lameness or sensitivity in the tendon after the administration of the therapy. Some researchers believe that physical activity, when correctly and gradually applied, can result in decreased tendon recovery time .
The results of the clinical assessment are consistent with the results obtained in previous studies: there was no difference in the sensitivity and the average circumference of the central portion of the metacarpal region between the groups . There was also no difference in the degree of lameness between the groups . The ultrasound evaluation revealed improvement of the cross-sectional lesion area and its percentage in the TG compared with the CG. This result is consistent with previous studies that used MSCs for the therapy of equine tendonitis [8, 10, 37]. The lack of significant differences between the cross-sectional lesion area and its percentage at different time points after the therapy can be justified by the correction of the P-values resulting from Tukey’s test and by the accidental reduction of the size of the CG.
The interpretation of the evolution of the cross-sectional lesion area and its percentage in the TG and the CG between the time of therapy (Week 0) and the subsequent assessment times shows significant differences between the groups at Weeks 4 and 6. This result demonstrated that the therapy used in this study exhibited a preventive action on the progression of the lesion because the TG showed no increase in the cross-sectional lesion area or its percentage (Figure 3).
This absence of lesion progression can be largely explained by the anti-inflammatory and immunomodulatory action of the therapy [38, 39]. We hypothesize that the moment of therapy administration may have contributed to these data because the preventive action of the therapy on the progression of the tendon lesion in the TG can be more clearly demonstrated in the ultrasound evaluation (the tendon lesion is not yet fully developed at this point). If the therapy had been performed four weeks after the induction of the lesion, we would certainly not find this preventive action because the lesion would have already reached its maximum size. This statement is based on a recent study that used a technique similar to the induction of tendon injury with the collagenase gel that was used in the present study. The study demonstrated that the maximal cross-sectional area of the lesion and the maximal relative cross-sectional area of the lesion of the SDTF are found four weeks after lesion induction .
The use of Color Doppler ultrasound for the assessment of the vascularization in equine tendonitis  and after therapy for equine tendonitis with PC  has been described. Although Power Doppler does not provide information on the blood flow speed or direction, as can be obtained with the Color Doppler ultrasound, Power Doppler has a greater sensitivity to the presence of blood and to the blood volume, which enables the visualization of smaller vessels . The results of the assessment of the vascularization obtained in this study indicate that there was increased blood flow in the treated group six weeks after the start of the therapy compared with the control group (P = 0.05). This result suggests that a biopsy at Week 6 would more likely find a larger number of vessels in the treated group than in the control group; this would be true in both the histopathological analysis and the immunohistochemical evaluation using factor VIII. The vessels are poorly visualized with the Color Doppler evaluation of a healthy digital flexor tendon. Hypervascularity is expected during the tissue repair process, but there is decreased vascularity with the progress of tendon repair .
The semi-quantitative morphological evaluation demonstrated the histopathological improvement of the treated group compared to the control group; this improvement is based on the cumulative sum of histological scores (P = 0.049). This evaluation also demonstrated a reduction in the inflammatory infiltrate and an improved organization of the ECM. These data are in agreement with previously reported results [4, 5, 13]. An improved linearity and uniformity of the collagen fibers was also observed in the treated group compared with the control group.
The action of the anti-inflammatory therapy is evident after combining the analysis of the histopathological data with the ultrasound evaluation (Weeks 2, 4 and 6). The cross-sectional area and its percentage did not worsen with therapy in the TG (Weeks 2, 4 and 6), whereas there was an increase in the lesion in the same period in the CG. This result suggests a possible anti-inflammatory effect on the progression of the lesion that prevents it from increasing in size by either minimizing or stabilizing the degeneration of the tendon fibers. The decreased inflammation after the implantation of adMSCs is likely due to the immunosuppressive effect of these cells [39, 41, 42]. There are several mechanisms that compose the anti-inflammatory effects of MSCs that benefit tendon repair; these include an increase in chemokines, the suppression of cytokine secretion from dendritic cells, and a reduction in the effects of T lymphocytes and natural killer cells. These data highlight the potent anti-inflammatory and immunosuppressive effects of these cells. Given these potent immunomodulatory effects of MSCs, it is not surprising that these cells are being used in clinical studies of graft-versus-host disease, which is usually a fatal condition after organ transplantation .
The immunohistochemical evaluation found no difference in the expression of collagen type III and factor VIII between the different groups. Previous studies have described the use of mononuclear cells derived from adipose tissue and adMSCs for the therapy of equine tendonitis. These studies have demonstrated a decreased expression of type III collagen in the treated group [4, 5]. In addition, another study described the quantification of the blood vessels present in the SDFT treated with PC using factor VIII : a greater amount of vessels was found in the group that received therapy with a platelet concentrate. Factor VIII is a clotting pro-cofactor that is found only on intact and functional endothelial cells of blood vessels; thus, this factor is an important tool for the evaluation of tissue vascularization .
The increased expression of the COL1A1, COL3A1, TNC, TNMD and SCX genes in the tendons of the TG and the CG compared with the HG are consistent with the results obtained in a previous study . Soon after tendon injury occurs, the expression levels of COL1A1 and COL3A1 increased . The expression level of TNC, which is an ECM protein that is synthesized during inflammation and tissue remodeling  and modulates the binding of cells to ECM components, also increased . TNMD regulates the proliferation of tenocytes and the maturation of collagen fibers . The increased expression of SCX in the injured tendons was consistent with the attempt to repair the tissue because this gene regulates the tendon progenitor cells  and is a marker of tendon and ligament development .
Although an increases expression of the tested genes was observed in the injured tendons (TG and CG) compared with the healthy tendons (HG), no difference was observed between the expression in the different injured groups (TG and CG). This result is consistent with the results obtained in previous studies [13, 26]. However, a trend for increases in the expression of COL3A1 was observed in the control group (P = 0.08). Soon after tendon injury, the gene expression levels of collagen type I and III increase. The decrease in the concentration of collagen type I compared with that of collagen type III is more closely related to the formation of scar tissue than to tendon regeneration . Thus, the lower expression of the COL3A1 gene in the treated group suggests that the tendons in the TG are better repaired than those in the CG. However, this result should be examined with caution because the difference in the expression levels of COL3A1 between the groups was not significant and because no increase in the collagen type III protein expression was observed in the immunohistochemical evaluation.
Only one study has reported differences in gene expression after the use of cell therapy for equine tendonitis. This study reported an increase in the expression of the gene cartilage oligomeric matrix protein (COMP) after tendonitis therapy with mononuclear cells derived from adipose tissue . A likely explanation for the lack of gene expression differences between the tendons of the TG, which exhibited improved histological and ultrasound characteristics, and the tendons of the CG is that the tested genes, although associated with tendon repair, are not specific. Thus, further research is necessary to select genes that are better markers of tendon repair [50, 51].
A limitation of the present study was the fact that the investigation did not determine the exact mechanism of action of the therapy that was used. This lack could have been due to trophic action (local modulation of cytokines), the replacement of cells from the injured tissue, local immunomodulation, or other mechanisms that favor the improvement of tissue repair evidenced by histopathological and ultrasound analyses.
There is great difficulty in the development of a well-designed large-scale study involving equines. The lack of basic knowledge of the behavior of adult stem cells and PC after their administration is also undeniable. Progenitor cells are believed to differentiate in the specific tissue site into which they were deployed (for example, tenocyte differentiation) and thus promote the production of the appropriate ECM. These cells can also synthesize bioactive proteins (for example, growth factors and cytokines) that promote the recruitment of endogenous stem cells and the anabolic stimulation of newly recruited cells and the mature cells of the tissue itself .
Similar to other experiments that used cell therapy with or without PC for the treatment of equine tendonitis [4–8, 11, 13, 26], the present study demonstrated a satisfactory result. However, it has limitations regarding the effects of the therapy used. The results demonstrate that there is no definition for the effectiveness of a therapy for the treatment of equine tendonitis. Based on the preventive result obtained with the adMSCs suspended in PC on the progression of the lesion in the ultrasound evaluation (cross-sectional lesion area and its percentage), further studies should be conducted to confirm this preventive action and the mechanism of action of the therapeutic association, as well as the action of progenitor cells and platelets alone as the lesion progresses. This conclusion is consistent with that described in a recent study on tendonitis, which states that prevention can have a significantly greater clinical impact than therapy due to the impossibility of tendon regeneration .
Future studies should be conducted to further elucidate the ideal time of administration of the equine tendonitis therapy, which is currently standardized between 7 and 45 days after the onset of the lesion. Additionally, further studies would contribute to the understanding of when the best therapeutic results are obtained and whether it really is possible to prevent further injury when the therapy is administered to a still-developing injury.
Basic studies that elucidate the real mechanism of action of the therapy used in tissue repair should also be conducted to confirm the differentiation of the stem cells used, analyze the release of trophic factors (for example, cytokines and growth factors), and verify the anti-inflammatory and immunomodulatory action of the therapy. Better knowledge of the etiopathogenesis of tendonitis associated with the knowledge of the mechanism of action of adult stem cells and activated platelets will enable improved clinical outcomes and result in the verification of the therapeutic efficacy of this new biotechnology.