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Research

GENETICALLY ENGINEERED AND SYSTEMICALLY EXPRESSING KUSABIRA-ORANGE TRANSGENIC PIGS AS IN VIVO MODEL TO TRACE CELL RECRUITMENT AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION

8th Combined Meeting Of Orthopaedic Research Societies (CORS)



Abstract

Summary

A novel in vivo animal model to establish new surgical interventions for patients with ACL insufficiency.

Introduction

After ACL reconstruction, recruited cells from surrounding tissues play crucial roles in ligamentization to obtain adequate structural properties. To allow athletes to return sports activity sooner, these remodeling processes should be elucidated and be accelerated. However, in conventional animal models, it has been difficult to differentiate donor and recipient cells. Here we introduce the transgenic Kusabira-Orange pigs, in which cells produce fluorescence systemically, as in vivo model to trace cell recruitment after ACL reconstruction.

Methods

After the approval by the Institutional Animal Care and Used Committee, a transgenic pig that carries and produces red fluorescent Kusabira-Orange (KO) was established. Skeletally immature transgenic pigs (n=12) (20 wks old, 76.0 ± 17.5 kg) and wild type (WT) pigs were used as recipient and donor, respectively. For validation of the pigs as in vivo model, the ACL histological structure, cell shape, mitogenic activity, and migration activity were assessed and were compared to those of wild type pigs. The sensitivity and specificity of KO fluorescence under microscopy were analyzed. Histological analyses were conducted with HE, Masson trichrome (MT), and DAPI staining. The length change pattern in our ACL reconstruction was evaluated to validate the surgical procedure. After allograft ACL reconstruction with fresh-frozen flexor digital tendon of WT pigs, pigs were euthanised at 3, 6, 12, and 24 weeks postoperatively (3 pigs each) for the histological analyses.

Results

The histological analyses, and mitogenic/migration assays did not show any apparent differences between KO and WT pigs. The sensitivity and specificity of KO fluorescence revealed to be 98%. Maximal length change of the reconstructed ACL was less than 3.5 mm. Three weeks postoperatively, host cells producing KO fluorescence repopulated mainly at the peripheral part of the graft, while, interestingly, cells also located in inter-territorial space of collagen fascicles. More cells migrated towards the mid of the graft in 6–12 week. Cell distribution became homogeneous in parallel to matrix remodeling in 12–24 week.

Discussion

As far as we know, this is the first study to apply the genetically engineered pig producing a fluorescent protein as in vivo model to analyze biological remodeling processes after ACL reconstruction. ACL fibroblasts in KO pigs could be detected under fluorescence with high sensitivity and specificity. In addition, structural organization, mitogenic and migration activity were not different from those in WT. As for histological experiments, the recipient cells could be easily and effectively differentiated from donor cells especially 3 weeks postoperatively. Cells migrated in the inter-territorial region among collagen fascicles earlier than we expected. We are going to investigate angiogenesis, matrix remodeling, and structural properties in parallel to the cell migration.