Diabetes Reduces Mesenchymal Stem Cells in Fracture Healing Through a Tnfα-Mediated Mechanism
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Diabetologia DOI 10.1007/s00125-014-3470-y ARTICLE Diabetes reduces mesenchymal stem cells in fracture healing through a TNFα-mediated mechanism Kang I. Ko & Leila S. Coimbra & Chen Tian & Jazia Alblowi & Rayyan A. Kayal & Thomas A. Einhorn & LouisC.Gerstenfeld& Robert J. Pignolo & Dana T. Graves Received: 31 July 2014 /Accepted: 19 November 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract (Sca-1) antibodies in areas of new endochondral bone forma- Aims/hypothesis Diabetes interferes with bone formation and tion in the calluses. MSC apoptosis was measured by TUNEL impairs fracture healing, an important complication in humans assay and proliferation was measured by Ki67 antibody. In and animal models. The aim of this study was to examine the vitro apoptosis and proliferation were examined in C3H10T1/ impact of diabetes on mesenchymal stem cells (MSCs) during 2 and human-bone-marrow-derived MSCs following transfec- fracture repair. tion with FOXO1 small interfering (si)RNA. Methods Fracture of the long bones was induced in a Results Diabetes significantly increased TNFα levels and streptozotocin-induced type 1 diabetic mouse model with or reduced MSC numbers in new bone area. MSC numbers were without insulin or a specific TNFα inhibitor, pegsunercept. restored to normal levels with insulin or pegsunercept treat- MSCs were detected with cluster designation-271 (also ment. Inhibition of TNFα significantly reduced MSC loss by known as p75 neurotrophin receptor) or stem cell antigen-1 increasing MSC proliferation and decreasing MSC apoptosis in diabetic animals, but had no effect on MSCs in normoglycaemic animals. In vitro experiments established Kang I. Ko and Leila S. Coimbra contributed equally to this work that TNFα alone was sufficient to induce apoptosis and inhibit Electronic supplementary material The online version of this article proliferation of MSCs. Furthermore, silencing forkhead box (doi:10.1007/s00125-014-3470-y) contains peer-reviewed but unedited protein O1 (FOXO1) prevented TNFα-induced MSC apopto- supplementary material, which is available to authorised users. sis and reduced proliferation by regulating apoptotic and cell : : * K. I. Ko C. Tian D. T. Graves ( ) cycle genes. Department of Periodontics, University of Pennsylvania, 240 S 40th α St, Levy 122, Philadelphia, PA 19104, USA Conclusions/interpretation Diabetes-enhanced TNF signif- e-mail: [email protected] icantly reduced MSC numbers in new bone areas during fracture healing. Mechanistically, diabetes-enhanced TNFα L. S. Coimbra reduced MSC proliferation and increased MSC apoptosis. Department of Physiology and Pathology, Araraquara Dental School, α State University of São Paulo, Araraquara, São Paulo, Brazil Reducing the activity of TNF in vivo may help to preserve endogenous MSCs and maximise regenerative potential in : J. Alblowi R. A. Kayal diabetic patients. Department of Oral Basic and Clinical Sciences, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia Keywords Anti-TNF . Cytokine . Diabetes . Forkhead . T. A. Einhorn : L. C. Gerstenfeld Fracture healing . Hyperglycaemia . Inflammation . Department of Orthopaedic Surgery, School of Medicine, Boston Mesenchymal stem cell . Tumour necrosis factor University, Boston, MA, USA R. J. Pignolo Abbreviations Department of Medicine, Perelman School of Medicine, University CD271 Cluster designation-271 (also known as p75 of Pennsylvania, Philadelphia, PA, USA neurotrophin receptor) FOXO1 Forkhead box protein O1 R. J. Pignolo Department of Orthopaedic Surgery, Perelman School of Medicine, hBMSC Human bone-marrow-derived mesenchymal stem University of Pennsylvania, Philadelphia, PA, USA cell Diabetologia mMSC Mouse mesenchymal stem cell that MSCs are thought to play in fracture healing, this deficit MSC Mesenchymal stem cell may significantly contribute to the negative effect of diabetes Sca-1 Stem cell antigen-1 on fracture repair. si Small interfering STZ Streptozotocin Methods Animals The research was carried out using 8-week-old male Introduction CD-1 mice purchased from Charles River Laboratories (Wilmington, MA, USA), conforming to a protocol approved Diabetes mellitus is one of the most common metabolic dis- by the American Association For Laboratory Animal Science eases, with 439 million people estimated to be affected world- (IACUC). The mouse model of type 1 diabetes induced by wide by 2030 [1]. Diabetic patients have a greater risk of STZ has been previously described [11, 16]. Briefly, multiple fracture and an increased risk of delayed union and non- injections of low-dose STZ (40 mg/kg, Sigma-Aldrich, St. union fracture healing [2–4]. Though the aetiology of diabetic Louis, MO, USA) were administered to mice daily for 5 days. complications is multifactorial, chronic inflammation is Mice were considered to be diabetic when blood glucose thought to play a critical role in several diabetic complications levels exceeded 13.78 mmol/l (250 mg/dl). Mice were diabet- [5, 6]. It has been implicated in diabetic osteopaenia and ic for at least 3 weeks prior to starting experiments (see reduced bone formation associated with impaired fracture electronic supplementary material [ESM] Table 1 for blood repair in humans and in animal models of diabetes [7–9]. glucose levels). A closed–transverse fracture of the mouse Fracture healing is a regenerative process that involves the tibia or femur was carried out as previously described [23, coordinated activity of inflammatory cells, endothelial cells, 24]. In one set of experiments, mice were treated with slow- stem cells, chondrocytes, osteoblasts and other cell types [10]. release insulin as previously described [11]. In another set of In an experimental animal model of fracture healing, mice experiments, pegsunercept was administered by intraperitone- with streptozotocin (STZ)-induced diabetes exhibited in- al injection (4 mg/kg, Amgen, Thousand Oaks, CA, USA) creased inflammation, enhanced osteoclastogenesis, loss of 10 days post-fracture and every 3 days thereafter. The animals cartilage in the callus and reduced bone formation [7, 11, 12]. were killed by cervical dislocation and cardiac puncture under An inflammatory response is indispensable for proper frac- deep sedation with ketamine/xylazine/acepromazine. The ture healing. Mice deficient in TNFα receptor or IL-6 show samples of long bone were collected by carefully trimming delayed fracture healing [13, 14]. However, animals with the surrounding muscles. diabetes or rheumatoid arthritis exhibit elevated TNFα levels and display impaired fracture healing [7, 15]. TNFα inhibition Paraffin sections and histomorphometric analysis Specimens increases fracture callus size in mouse models of diabetes were fixed, decalcified and embedded in paraffin as previous- [16]. ly described [11]. Haematoxylin and eosin (H&E) staining Bone-marrow-derived MSCs differentiate to chondrocytes was performed to determine the centre of the callus, where the and osteoblasts, which play essential roles in endochondral callus was widest. All histology was performed in sections ossification of fracture calluses. MSCs also function as trophic taken close to the centre of the callus. The amount of bone mediators that promote angiogenesis, have anti-apoptotic ef- formation was determined by measuring the bone area within fects and reduce inflammation [17]. Therapeutic use of MSCs the callus in tissue sections. Data are presented as mm2 bone improves outcomes in the healing response to stroke, myocar- per callus and percentage of bone area per total callus area. dial infarction and long bone fracture in animal models Osteoblasts were identified as cuboidal cells in clusters of [18–20], and in the treatment of inflammatory diseases in three or more cells in contact with the bone surface in phase 1 and 2 human clinical trials [21, 22]. Although Toluidine-Blue-stained sections. For each specimen, five to MSCs are important in the repair of many different tissues, ten random fields throughout the areas of new endochondral relatively little is known about the impact of diabetes on ossification were examined under ×400 magnification. MSCs in fracture repair. Here, we determined that diabetes affects the number of MSCs in areas of new endochondral Immunohistochemistry/fluorescence Antigen retrieval was in- ossification in fracture healing in mice. Diabetes interferes duced by pressure heating (>120°C) paraffin slides in with MSC proliferation and promotes MSC apoptosis 10 mmol/l sodium citrate (pH 6.0) using a 2100-Retriever in vivo through a mechanism that involves diabetes- (Aptum, Southampton, UK). A list of antibodies and reagents enhanced TNFα levels. Thus, in fracture healing, diabetes- used is provided in ESM Table 2. For immunohistochemistry, induced inflammation creates a suboptimal environment for the primary antibody was localised by incubation with a MSC survival and ability to proliferate. Given the critical role biotinylated secondary antibody and then avidin–horseradish Diabetologia peroxidase. For some analyses, a scale of immunopositive en-us/). For insulin treatment and in vitro experiments, one- index was used as has been previously described [12]: 0 (no way ANOVA was used to determine statistical significance immunostaining); 1 (<10% lightly immunostained cells); 2 (p<0.05). For pegsunercept-treatment experiments and ap- (<10% moderate-to-darkly immunostained cells); 3 (10–25% propriate in vitro experiments, two-way ANOVA followed moderate-to-darkly immunostained cells); 4 (25–40% by Tukey’s