Developing an animal model of Dupuytren’s disease by orthotopic transplantation of human fibroblasts into athymic rat

Abstract

BackgroundDupuytren’s disease (DD) is a slow, progressive fibroproliferative disorder affecting the palms of the hands. The disease is characterized by the formation of collagen rich- cords which gradually shorten by the action of myofibroblasts resulting in finger contractures. It is a disease that is confined to humans, and a major limiting factor in investigating this disorder has been the lack of a faithful animal model that can recapitulate its distinct biology. The aim of this study was to develop such a model by determining if Dupuytren’s disease (DD)- and control carpal tunnel (CT)-derived fibroblasts could survive in the forepaw of the nude rats and continue to exhibit the distinct characteristics they display in in vitro cultures.Methods1x107 fluorescently labeled DD- and CT-derived fibroblasts were transplanted into the left and right forepaws of nude rats respectively. Cells were tracked at regular intervals for a period of two months by quantifying emitted fluorescent signal using an IVIS imaging system. After a period of 62 days rat forepaw connective tissues were harvested for histology and total RNA was isolated. Human-specific probes were used to perform real time RT-PCR assays to examine the expression patterns of gene products associated with fibrosis in DD. Rat forepaw skin was also harvested to serve as an internal control.ResultsBoth CT- and DD-derived fibroblasts survived for a period of 62 days, but DD-derived cells showed a significantly greater level of persistent fluorescent signal at the end of this time than did CT-derived cells. mRNA expression levels of α-smooth muscle actin (α-SMA), type I- and type III- collagens were all significantly elevated in the forepaw receiving DD cord-derived fibroblasts in comparison to CT-derived fibroblasts. Masson’s trichrome stain confirmed increased collagen deposition in the forepaw that was injected with DD cord-derived fibroblasts.ConclusionsFor the first time we describe an animal model for Dupuytren’s disease at the orthotopic anatomical location. We further show that gene expression differences between control (CT) and diseased (DD) derived fibroblasts persist when these cells are transplanted to the forepaw of the nude rat. These preliminary findings indicate that, with further refinements, this animal model holds promise as a baseline for investigating novel therapeutic regimens to determine an effective strategy in treating DD.

Anti-fibrotic action of pirfenidone in Dupuytren’s disease-derived fibroblasts

“…Since we are aiming for topical application of PFD for use in DD patients, we expect that we can achieve the effect of the drug locoregionally with much less systemic exposure than used for treating other fibrotic conditions such as idiopathic pulmonary fibrosis (IPF). Future studies will focus on optimizing strategies to effectively deliver PFD to the palmar fascia, and to verify its ability to inhibit DD disease phenotype using a recently described orthotopic animal model of DD [31]. …”

“…Primary cultures of fibroblasts were isolated from freshly resected DD cord tissue and normal palmar fascia (CT cells) as previously described [31]. DD cord and CT fascial tissue samples were surgically resected at the Division of Upper Extremity Surgery, Department of Orthopaedic Surgery, Allegheny General Hospital, Pittsburgh, PA. All subjects signed the written informed consent forms, and the protocol was approved by the Allegheny-Singer Research Institute’s Institutional Review Board (IRB protocol no.…”

“…Following day, cells were switched to alpha-MEM medium containing 0.1 % dialyzed FBS for 24 h followed by treatment with different concentrations of PFD (0, 200, 400 and 800 μg/mL) with or without 10 ng/ml TGF-β1 in low-serum medium for additional 24 h. After the incubation period, cells were photographed, and MTT assay was performed using the CellTiter 96® Non-Radioactive Cell Proliferation Assay kit obtained from Promega Corporation (Madison, WI) [31]. Cells were replaced with fresh growth medium and dye solution and incubated at 37 °C for 2 h. After adding solubilization/stop solution, cells were incubated for another 1.5 h at 37 °C.…”

Comparative Efficacy of Commonly Available Human Bone Graft Substitutes as Tested for Posterolateral Fusion in an Athymic Rat Model

“…The ability of fat and marrow derived stem cells to survive in athymic nude mice has been shown. 60,73 Also, other human-derived tissues function in this animal model 74,75 ; however, this rodent model may not be a reliable and valid test of this class of grafting materials. DBM containing viable cells (Trinity Evolution Orthofix) failed to induce rigid fusions in athymic rats in another study as well.…”

Prevention of Recurrences in Dupuytren’s Contracture: Are We in the Right Side?

“…But the mechanisms triggering the disease are unknown. A recent study [7] has succeeded in reproducing DC in animal models, but such progress is far from being ready for clinical application.…”