Transplantation of autologous mesenchymal stem cells during repair of detached rotator cuff muscle in sheep

Published: 28-01-2019| Version 1 | DOI: 10.17632/hwdxv5dvhk.1
Contributor:
Martin Flück

Description

The data describes the molecular, cellular and anatomical effect of the implantation of autologous mesenchymal stem cells (MSCs) in degenerated rotator cuff muscle of sheep. The aim of the onvestigation was that MSC injection would halt muscle-to-fat conversion subsequent to tendon repair by promoting growth and regeneration of muscle fibers. Degeneration was induced for infraspinatus muscles of the right shoulder of Swiss Alpine sheep (n=14) by tendon release via osteotomy of the greater tuberosity. MSCs were prepared from bone marrow in a group of animals (T-MSC, n=8). They were characterised for their differentiation potential, fluorescently-labelled and grown as microtissues, before 0.9 Mio of them were injected in a specific area in the released muscle during tendon reattachment (repair). Two days (n=1), 2 weeks (n=1) and six weeks after repair (n=6) the effects of MSC implantation was characterised and compared against a group of sheep (n=6) where infraspinatus muscle was repaired after osteotomy but not being treated with MSCs. Anatomical effects on muscle volume and composition were documented with radiological imaging of the entire muscle at baseline, during repair and six weeks after repair, and in MSC-injected, media-injected and non-injected areas six weeks after repair. Animal mass was determined with a balance. Cellular and molecular effects were characterised in biopsies being collected six weeks after repair from MSC-injected, media-injected and non-injected areas. Cellular effects were characterised by histological analysis of muscle sections for fiber type, fat and extracellular matrix composition. Molecular measures comprised biochchemical measures of the expression of myogenic (myogenin), regeneration (tenascin-C) and adipogenic markers (PPARG). Measurements were subjected to a (repeated) analysis of variance (ANOVA) with post hoc test of Fisher to identify statistically significant effects.

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Degeneration and repair of infraspinatus muscle - Osteotomy of the greater tuberosity was carried out using an oscillating saw. 16 weeks after osteotomy, rotator cuff repair was performed by reattaching the bonechip to its original site, or as near as possible with attachment of the remaining sutures. Radiological imaging - Transverse sections of the entire infraspinatus muscle were recorded with Magnet Resonance Imaging (Philips Ingenia 3T with dStream body coil Solution, Philips AG; Zurich, Switzerland) and Computed Tomography (Somatom ART; Siemens Medical Solutions, Erlangen, Germany) from animals under general anesthesia immediately after tendon release, after 6 weeks, before repair and before sacrifice. Data were evaluated using the DICOM viewer OsiriX v.5.6 32-bit (Pixmeo). MSC preparation - MSCs were extracted from bone marrow aspirate, characterized, fluorescently-labelled with Q-Dots (QTracker Cell Labeling Kit, Invitrogen, Life Technologies), and seeded to be grown as microtissues in Dulbecco's modified eagle medium (DMEM) according to established procedures (Emmert et al. Biomaterials 2013 Mar;34(10):2428-41; Emmert et al. Biomaterials 2013 Jun; 34 (27): 6339-54; Mirsaidi et al. Curr Protoc Stem Cell Biol. 2013 Nov 13;27:Unit 2B 5). MSC implantation - Four-times 180 microtissues (0.9 Mio cells) in 0.4 ml DMEM were separately injected through a 27G needle during repair surgery in a 2 cm x 2 cm-sized area in the lateral portion of infraspinatus muscle being demarcated with radiodense surgical suture. Another area was injected with DMEM alone (media-injected), or only demarcated (non-injected). Sample collection – At the end of the animals were euthanized and the repaired and contralateral infraspinatus muscles were rapidly excised. A tissue piece was cut out from the MSC-injected, media-injected or not-injected, of group T-MSC and anatomically corresponding muscle areas from the untreated group T, and the contralateral muscles. Histological analysis - Formalin-fixed samples were processed to quantify the area percentages covered by muscle fiber types, fat and extracellular matrix, and MSCs based on the microscopic evaluation of immunochemically stained structures and Q-Dot fluorescence essentially as shown (Gerber et al. J Shoulder Elbow Surgery 2009 Mar-Apr;18(2):163-71; Benn MC. Vergleichende Darstellung des Nandroloneffekts auf die strukturelle Muskelveränderung des M. infraspinatus im Schafmodell für Rotatorenmanschettenruptur zum Zeitpunkt der Ruptur und chirurgischen Reparation. Universität Zürich; 2015). Biochemical analysis – Soluble proteins were extracted from paraffin embedded blocks with a modification of a published protocol (Addis et al. Proteomics. 2009 Aug;9(15):3815-23). 10 µg protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted. Subsequently, the abundance of myogenin, tenascin-C, and PPARG protein was estimated respective to actin with enhanced chemiluminescence.