Expressional Changes In Growth And Inflammatory Mediators During Achilles Tendon Repair In Diabetic Rats.

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1 Expressional Changes In Growth And Inflammatory Mediators During Achilles Tendon Repair In Diabetic Rats. Paul W. Ackermann, MD, PhD 1, Aisha Ahmed, PhD 1, Nicos Schizas, MD 1, Jian Li, MD, PhD 1, Mahmood Ahmed, MD, PhD 1, Claes- Göran Östensson, MD, PhD 1, Carol Hewitt 2, Paul T. Salo, MD 2, David A. Hart, PhD 2. 1 Karolinska Institutet, Stockholm, Sweden, 2 University of Calgary, Calgary, AB, Canada. Disclosures: P.W. Ackermann: 5; DJO Global, OPED Gmbh. A. Ahmed: None. N. Schizas: None. J. Li: None. M. Ahmed: None. C. Östensson: None. C. Hewitt: None. P.T. Salo: None. D.A. Hart: None. Introduction: Growth and inflammatory mediators are essential for connective tissue homeostasis and healing. Dysregulation of inflammation and metabolism are commonly observed in diabetes mellitus type 2, and may contribute to defective tissue homeostasis and healing. The present study aims to assess expressional changes in growth and inflammatory mediators in the intact and healing Achilles tendon of non-insulin dependent diabetic rats. Methods: The study included 5 male GK (with type-2 diabetes) and 4 Wistar (control) 14 month old rats. All rats were anesthetized and subjected to a blunt, complete right Achilles and plantaris tendon transection at the mid-part of the tendon. The torn ends were left unsutured and the rats were allowed free cage activity. At 2-weeks post-rupture (day 14) all the rats were euthanatized and the right (ruptured) and left (intact control) Achilles and plantaris tendons were dissected along with the gastrocnemious muscle and the calcaneal bone and immediately frozen for analysis by RT-qPCR and immunohistochemistry. RT-qPCR. Frozen tissues were homogenized and dissolved in Trizol reagent. RNA was extracted and further purified using the RNeasy MiniKit (Qiagen, USA). First-strand cdna were synthesized from 1 μg of total RNA and the quantification assays were performed to detect the relative mrna expression for VEGF, TB-4, TGF-β, IGF-1, COX-2 and inos. 18S was used as a validated house keeping gene. Immunohistochemistry. Dissected right and left tendons from each rat were fixed, sectioned and mounted on SuperFrost/Plus slides. Sections were immunostained with antiserum to VEGF, TB-4, TGF-β1, IGF-1, COX-2 and inos before incubation with secondary biotinylated antibodies. Sections were further incubated with ABC reagent followed by the application of diaminobenzidine (DAB) chromogen and counterstained with Hematoxylin. Sections were then dehydrated with 70, 95, 99% ethanol. Results: At day 14 post rupture, RT-qPCR analysis showed that all genes of interest (VEGF, TB-4, TGF-β1, IGF-1, COX-2 and inos) were expressed in measurable levels in intact and ruptured tendons of normal and diabetic rats. In injured tendons of diabetic GK rats, VEGF and TB-4 mrna levels were significantly down-regulated compared to injured tendons of Wistar controls. At 2 weeks post-injury, diabetic GK tendons exhibited higher mrna levels for TGF-β1, IGF-1 and COX-2, while inos mrna levels were lower compared to intact GK tendons. Within the Wistar controls, healing at 2 weeks post-injury led to significantly downregulated VEGF and inos mrna levels, while TGF-β1 mrna levels increased significantly compared to intact tendons of Wistar controls (Figure 1). The immunohistochemical analysis of proteins detected specific localisations and corroborated the differences in gene expression observed between the groups with small variations. In intact tendons of the diabetic GK and Wistar rats, no apparent differences in immunoreactivity were detected. Injured diabetic GK tendons exhibited much weaker VEGF, TB-4, TGF-β1, IGF-1 immunoreactivity with fewer positively stained tenocytes compared to injured Wistar controls. In both Wistar and diabetic GK injured tendons, positively stained cells were observed, especially localized to the injured areas (Figure 2). Discussion: We have previously demonstrated that in diabetes, tendons regenerative capability is compromised with corresponding expressional changes in collagen I and III and matrix metalloproteinase-13 in the GK rat model of diabetes mellitus type-2 (1, 2). In the present study, we further explored the mechanisms behind impaired regeneration in diabetic condition by analyzing growth and inflammatory genes, and their respective proteins vital to the normal healing process. Our results indicate that the inflammatory molecules and growth factors analyzed are actively involved in the healing process at this proliferative phase, two weeks post tendon rupture. We further observed decreased VEGF and TB-4 mrna and protein levels in injured tendons in diabetic rats compared to Wistar controls indicating an impaired angiogenesis response, since both VEGF and TB-4 are essential regulators of new vessel formation. Significance: Our data suggest that therapeutic modulation of TB-4 and VEGF might be a strategy for new regenerative approaches in operated, injured or in degenerative tendon diseases in diabetes. Acknowledgments: This study was supported by the Swedish Research Council and the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet (project nr. SLL ) and the Swedish National Centre for Sports Research. References: 1. Schizas N et al, Diabetes Mellitus impairs proliferative tendon repair. The 56th Annual Meeting of the

2 Orthopaedic Research Society Ahmed et al, Type 2 Diabetes Impairs Tendon Repair after Injury in a Rat Model. J Appl Physiol, 2012; 113(11):1784-

3 91.

4 ORS 2014 Annual Meeting Poster No: 1384

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