Histological and immunohistochemical evaluation of two cell therapy protocols in equine suspensory ligament repair

L.C Garbin,A.C Vasconcelos,R.R Faleiros,H.M.F Mendes,A.S Soares,G.D Cassali

doi:10.1590/1678-4162-10683

Abstract

ABSTRACT This study aimed to histologically evaluate the quality of tissue repair in equine suspensory ligament treated with two cell therapy protocols. All four limbs of six animals were operated simultaneously to remove a fragment in each ligament using a skin biopsy punch. Two days later, intralesional injections were performed using bone marrow mononuclear fraction (BM group), cultivated cells derived from adipose tissue (AT group), saline (positive control group), or no treatment (negative control group), in such way that each horse received all treatments. After sixty days biopsies were performed for histological analysis (H & E, Masson’s trichrome and picrosirius red) and immunohistochemistry analysis (collagen type III). Histological findings (H & E and Masson’s trichrome), birefringence intensity (through picrosirius) and collagen type III expression (through immunohistochemistry) were analyzed. Samples from treated groups had better birefringence intensity (P=0.007) and fiber alignment scores were superior compared to controls, though not statistically significant (P=0.08). Presence of inflammatory cells and intense staining for collagen type III occurred in all groups demonstrating an active healing process. In conclusion, both protocols resulted in improvement of tissue repair indicating their potential to be used as an adjuvant treatment of equine suspensory ligament disorders.

Highlights

Suspensory ligament desmitis affects horses from different breeds, ages, and equestrian activities, causing prolonged illness and reducing the performance of athlete horses (Dyson, 1994; Dowling et al, 2000; McClure et al, 2004)
Similar results were observed in studies using the tendonitis model, where the studies failed to find any beneficial effect of cell therapy in reducing the number of inflammatory cells in the scar tissue (Schnabel et al, 2009, Carvalho et al, 2011)
Increase in tissue cellularity and angiogenesis was observed in tendons treated with cell therapy in the collagenase-induced tendonitis model but the lower level of neovascularization was reported in the groups treated with adipose tissue-derived mesenchymal cells (Carvalho et al, 2011) and with bone-marrow aspirates (Oliveira et al, 2011) compared to untreated groups

Summary

Introduction

Suspensory ligament desmitis affects horses from different breeds, ages, and equestrian activities, causing prolonged illness and reducing the performance of athlete horses (Dyson, 1994; Dowling et al, 2000; McClure et al, 2004). Despite the new techniques for early diagnosis and treatment, the risk of recurrence of ligament lesion can reach up to 93% (Dahlgren, 2007). Such high risk of recurrence leads to an increase in interest on ligament physiology and disease etiology (Dahlgren, 2007) leading to the development of new disease-modifying treatments, which would result in a more physiological repair tissue (Nixon et al, 2008). Cell therapy aims to repair, replace, and increase the biological functions of injured tissue through the use of autologous or allogeneic stem cells. Stem cells can potentially differentiate in tissuespecific cells and produce a functional extracellular matrix (Sutter, 2007) and modulate inflammation and cell apoptosis (Nixon et al, 2008)

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Conclusion