Diabetes reduces mesenchymal stem cells in fracture healing through a TNFα-mediated mechanism
- 2k Downloads
Diabetes interferes with bone formation and impairs fracture healing, an important complication in humans and animal models. The aim of this study was to examine the impact of diabetes on mesenchymal stem cells (MSCs) during fracture repair.
Fracture of the long bones was induced in a streptozotocin-induced type 1 diabetic mouse model with or without insulin or a specific TNFα inhibitor, pegsunercept. MSCs were detected with cluster designation-271 (also known as p75 neurotrophin receptor) or stem cell antigen-1 (Sca-1) antibodies in areas of new endochondral bone formation in the calluses. MSC apoptosis was measured by TUNEL assay and proliferation was measured by Ki67 antibody. In vitro apoptosis and proliferation were examined in C3H10T1/2 and human-bone-marrow-derived MSCs following transfection with FOXO1 small interfering (si)RNA.
Diabetes significantly increased TNFα levels and reduced MSC numbers in new bone area. MSC numbers were restored to normal levels with insulin or pegsunercept treatment. Inhibition of TNFα significantly reduced MSC loss by increasing MSC proliferation and decreasing MSC apoptosis in diabetic animals, but had no effect on MSCs in normoglycaemic animals. In vitro experiments established that TNFα alone was sufficient to induce apoptosis and inhibit proliferation of MSCs. Furthermore, silencing forkhead box protein O1 (FOXO1) prevented TNFα-induced MSC apoptosis and reduced proliferation by regulating apoptotic and cell cycle genes.
Diabetes-enhanced TNFα significantly reduced MSC numbers in new bone areas during fracture healing. Mechanistically, diabetes-enhanced TNFα reduced MSC proliferation and increased MSC apoptosis. Reducing the activity of TNFα in vivo may help to preserve endogenous MSCs and maximise regenerative potential in diabetic patients.
KeywordsAnti-TNF Cytokine Diabetes Forkhead Fracture healing Hyperglycaemia Inflammation Mesenchymal stem cell Tumour necrosis factor
Cluster designation-271 (also known as p75 neurotrophin receptor)
Forkhead box protein O1
Human bone-marrow-derived mesenchymal stem cell
Mouse mesenchymal stem cell
Mesenchymal stem cell
Stem cell antigen-1
Diabetes mellitus is one of the most common metabolic diseases, with 439 million people estimated to be affected worldwide by 2030 . Diabetic patients have a greater risk of fracture and an increased risk of delayed union and non-union fracture healing [2, 3, 4]. Though the aetiology of diabetic complications is multifactorial, chronic inflammation is thought to play a critical role in several diabetic complications [5, 6]. It has been implicated in diabetic osteopaenia and reduced bone formation associated with impaired fracture repair in humans and in animal models of diabetes [7, 8, 9].
Fracture healing is a regenerative process that involves the coordinated activity of inflammatory cells, endothelial cells, stem cells, chondrocytes, osteoblasts and other cell types . In an experimental animal model of fracture healing, mice with streptozotocin (STZ)-induced diabetes exhibited increased inflammation, enhanced osteoclastogenesis, loss of cartilage in the callus and reduced bone formation [7, 11, 12].
An inflammatory response is indispensable for proper fracture healing. Mice deficient in TNFα receptor or IL-6 show delayed fracture healing [13, 14]. However, animals with diabetes or rheumatoid arthritis exhibit elevated TNFα levels and display impaired fracture healing [7, 15]. TNFα inhibition increases fracture callus size in mouse models of diabetes .
Bone-marrow-derived MSCs differentiate to chondrocytes and osteoblasts, which play essential roles in endochondral ossification of fracture calluses. MSCs also function as trophic mediators that promote angiogenesis, have anti-apoptotic effects and reduce inflammation . Therapeutic use of MSCs improves outcomes in the healing response to stroke, myocardial infarction and long bone fracture in animal models [18, 19, 20], and in the treatment of inflammatory diseases in phase 1 and 2 human clinical trials [21, 22]. Although MSCs are important in the repair of many different tissues, relatively little is known about the impact of diabetes on MSCs in fracture repair. Here, we determined that diabetes affects the number of MSCs in areas of new endochondral ossification in fracture healing in mice. Diabetes interferes with MSC proliferation and promotes MSC apoptosis in vivo through a mechanism that involves diabetes-enhanced TNFα levels. Thus, in fracture healing, diabetes-induced inflammation creates a suboptimal environment for MSC survival and ability to proliferate. Given the critical role that MSCs are thought to play in fracture healing, this deficit may significantly contribute to the negative effect of diabetes on fracture repair.
The research was carried out using 8-week-old male CD-1 mice purchased from Charles River Laboratories (Wilmington, MA, USA), conforming to a protocol approved by the American Association For Laboratory Animal Science (IACUC). The mouse model of type 1 diabetes induced by STZ has been previously described [11, 16]. Briefly, multiple injections of low-dose STZ (40 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) were administered to mice daily for 5 days. Mice were considered to be diabetic when blood glucose levels exceeded 13.78 mmol/l (250 mg/dl). Mice were diabetic for at least 3 weeks prior to starting experiments (see electronic supplementary material [ESM] Table 1 for blood glucose levels). A closed–transverse fracture of the mouse tibia or femur was carried out as previously described [23, 24]. In one set of experiments, mice were treated with slow-release insulin as previously described . In another set of experiments, pegsunercept was administered by intraperitoneal injection (4 mg/kg, Amgen, Thousand Oaks, CA, USA) 10 days post-fracture and every 3 days thereafter. The animals were killed by cervical dislocation and cardiac puncture under deep sedation with ketamine/xylazine/acepromazine. The samples of long bone were collected by carefully trimming the surrounding muscles.
Paraffin sections and histomorphometric analysis
Specimens were fixed, decalcified and embedded in paraffin as previously described . Haematoxylin and eosin (H&E) staining was performed to determine the centre of the callus, where the callus was widest. All histology was performed in sections taken close to the centre of the callus. The amount of bone formation was determined by measuring the bone area within the callus in tissue sections. Data are presented as mm2 bone per callus and percentage of bone area per total callus area. Osteoblasts were identified as cuboidal cells in clusters of three or more cells in contact with the bone surface in Toluidine-Blue-stained sections. For each specimen, five to ten random fields throughout the areas of new endochondral ossification were examined under ×400 magnification.
Antigen retrieval was induced by pressure heating (>120°C) paraffin slides in 10 mmol/l sodium citrate (pH 6.0) using a 2100-Retriever (Aptum, Southampton, UK). A list of antibodies and reagents used is provided in ESM Table 2. For immunohistochemistry, the primary antibody was localised by incubation with a biotinylated secondary antibody and then avidin–horseradish peroxidase. For some analyses, a scale of immunopositive index was used as has been previously described : 0 (no immunostaining); 1 (<10% lightly immunostained cells); 2 (<10% moderate-to-darkly immunostained cells); 3 (10–25% moderate-to-darkly immunostained cells); 4 (25–40% moderate-to-darkly immunostained cells). For immunofluorescence, primary antibody was localised by a biotinylated secondary antibody. To enhance the signal, avidin–biotin peroxidase enzyme complex and tyramide signal amplification was used. Visualisation was achieved using Alexa 546-conjugated streptavidin or fluorescein-conjugated avidin. For double immunofluorescence, residual biotin sites and peroxidase activity associated with the first primary antibody were blocked with avidin/biotin-blocking reagent and 3% (vol./vol.) hydrogen peroxide. IgG control experiments were carried out for each primary antibody, and images were acquired using the same exposure time. All histological images were analysed by two independent examiners under blinded conditions.
In vitro experiments
In vitro experiments were carried out with a mouse MSC line (C3T10H1/2) and primary human bone-marrow-derived MSCs (hBMSCs), which were isolated and characterised as previously described . Briefly, isolation of hBMSCs was achieved with plastic adherence, and hBMSCs were characterised as CD73+CD90+CD105+CD45− cells that were capable of differentiating into osteoblasts and adipocytes. MSCs were grown and maintained in α-MEM + 10% (vol./vol.) FBS and 1% (vol./vol.) antibiotic/antimycotic mix. For all in vitro experiments, early-passage cells were used as previously determined by population doubling [25, 26].
For the apoptosis assay, the cells were allowed to reach 70% confluence and were then incubated with TNFα (10 ng/ml) in α-MEM + 0.5% FBS and 1% antibiotic/antimycotic mix. Apoptotic cells were labelled with annexin V–biotin or cleaved caspase-3 antibody. Five to eight random images were taken per well for the analysis. For the proliferation assay, 10% FBS medium was used to stimulate proliferation for 12 h. The BrdU Assay Kit (Cell Signaling, Danvers, MA, USA) was used according to the manufacturer’s protocol. BrdU incorporation was quantified by using a 96 well plate reader at λ = 450 nm (Infinite M200 PRO, Tecan, Morrisville, NC, USA).
Transfection experiments were carried out using Lipofectamine 2000 reagent and on-target plus smartpool human forkhead box protein O1 small interfering (si)RNA (10 nmol/l) in reduced-serum Opti-MEM for 6 h. The extraction of mRNA from each treatment group was carried out using an RNAeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol.
All statistical analyses were carried out with a software program (Minitab ver.16, www.minitab.com/en-us/). For insulin treatment and in vitro experiments, one-way ANOVA was used to determine statistical significance (p < 0.05). For pegsunercept-treatment experiments and appropriate in vitro experiments, two-way ANOVA followed by Tukey’s post-hoc test was used to denote significance. All data represent means ± SEM.
Diabetes decreases CD271+ and Sca-1+ cells during endochondral ossification in healing fracture calluses
The diabetes-induced decrease in MSC number is due to enhanced TNFα levels
Diabetes increases apoptosis and decreases proliferation in MSCs
TNFα inhibition directly rescues diabetic effects in MSC apoptosis and proliferation
TNFα directly induces apoptosis and reduces proliferation in mouse MSCs and hBMSCs in vitro
Diabetes enhances forkhead box protein O1 nuclear localisation in MSCs in a TNFα-dependent manner
FOXO1 knockdown prevents TNFα-mediated MSC apoptosis and anti-proliferation
TNFα upregulates FOXO1-dependent apoptotic and cell cycle arrest genes in hBMSCs
A better understanding of how diabetes impairs fracture healing is important. Diabetes is an increasingly important healthcare issue and the disease has a significant impact on the skeleton and interferes with fracture repair [32, 33]. Here, we report the deleterious effects of diabetes on MSC viability during fracture healing. Diabetes reduced the number of endogenous MSCs in areas of endochondral bone formation, with the number restored to normal values by insulin treatment. To better understand mechanistically how diabetes affects MSCs, diabetic animals were treated with the TNFα inhibitor, pegsunercept.
Pegsunercept significantly increased the MSC number, demonstrating that diabetes-enhanced inflammation played a major role in reducing the MSC number. Furthermore, diabetes significantly increased MSC apoptosis and reduced proliferation in vivo. These negative effects were mediated by diabetes-enhanced inflammation. In vitro experiments demonstrated that TNFα directly increased MSC apoptosis and reduced DNA synthesis. The effect of TNFα was dependent on FOXO1 activity.
To quantify MSCs in vivo, specific markers were used, CD271 and Sca-1, which accurately identify MSC in areas with little haematopoietic tissue [28, 34]. Moreover, virtually all CD271+ and Sca-1+ cells were CD45−. CD271+ cells found in dermal and adipose tissue exhibit robust tri-lineage mesenchymal potential . In addition, CD271+ cells administered in vivo home to fracture sites in mice . Furthermore, Sca-1 is found on MSCs in murine synovial membrane and skeletal muscle that exhibits mesengenic properties [36, 37].
STZ-induced diabetes drastically reduced the number of MSCs in areas of new bone. Insulin treatment reversed serum glucose to normal levels in STZ-treated mice. It also restored the number of MSCs to normal levels in STZ-diabetic mice, together with the amount of new bone and osteoblast numbers. Thus, the reduction is not an untoward effect of STZ-induced diabetes in this model. Previous studies have demonstrated that bone marrow samples derived from diabetic mice have fewer MSCs compared with normoglycaemic animals [38, 39], and have significantly decreased MSC-colony-forming units ex vivo . Our studies demonstrate for the first time that diabetic animals exhibit lower numbers of local endogenous MSCs in fracture callus, a mesenchymal tissue, in accordance with decreased bone formation in diabetic fracture healing.
Here, we have identified TNFα-mediated MSC apoptosis and anti-proliferation as the two mechanisms that explain the reduced MSC numbers in diabetic fracture healing. MSC apoptosis was returned to normal values when TNFα was inhibited in vivo, and TNFα sufficiently induced caspase-3 activation and apoptosis in hBMSCs in vitro. Previous in vitro studies have demonstrated that high glucose levels stimulate apoptosis and induce senescence in adipose-derived MSCs . Consistent with these findings, a recent study showed that TNFα induced apoptosis in rat MSCs in vitro .
An essential component of an adequate healing process is the proliferative capacity of regenerative cells. MSC proliferation was reduced by 33% in diabetic fracture calluses, and TNFα inhibition significantly restored MSC proliferation. Moreover, TNFα induced upregulation of genes that interfere with progression through the cell cycle in a FOXO1-dependent manner. No previous in vivo studies have identified this mechanism for the effect of diabetes on MSC proliferation during fracture healing. Increased TNFα levels may delay the healing process in other mesenchymal tissues affected by diabetes.
FOXO1 is a transcription factor that regulates apoptosis and proliferation, and it is subject to activation by diabetic inflammatory stimuli in fibroblasts . Diabetes increased FOXO1 nuclear localisation in MSCs in diabetic fracture approximately sixfold, and TNF inhibition significantly reduced this increase. FOXO1 knockdown in hBMSCs prevented the negative impact of TNFα on MSC apoptosis and proliferation. This is consistent with previous reports that FOXO1 plays an adverse role in fibroblasts , microvascular cells  and chondrocytes  in diabetic conditions. Thus, a high level of FOXO1 activation may mediate some of the negative effects of diabetes and contribute to diabetic complications.
Treatment of diabetic fractures with MSCs can promote healing . Our studies suggest an alternative approach, which would be to enhance survival and support the proliferation of endogenous MSCs by reducing prolonged inflammation in diabetic fracture healing. This may be a simpler and more pragmatic approach than treatment of diabetic fractures with MSCs. Thus, targeting endogenous MSCs in order to maximise their regenerative potential using pharmacological intervention may ultimately be safer and more cost effective.
We would like to thank M. Foroozia (University of Pennsylvania, School of Dental Medicine) for technical assistance with this manuscript.
This work was supported by National Institute of Health/National Institute of Arthritis and Muscular and Skin Diseases (NIH/NIAMS) grant AR-060055-03 (DTG) and R01AG028873 (RJP).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
DTG, KIK and LSC contributed to the study conception and design. DTG is responsible for the integrity of the work as a whole. All authors contributed to the acquisition of data or analysis and interpretation of data, revised the manuscript and approved the final version of the manuscript to be published.
- 32.Clarke JL (2010) Building a coordinated care model for diabetes management. Popul Health Manag 13(Suppl 1):S3–S13Google Scholar