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. 2019 Dec 23;13(2):155–163. doi: 10.1007/s12195-019-00608-w

Transplantation of IL-10-Overexpressing Bone Marrow-Derived Mesenchymal Stem Cells Ameliorates Diabetic-Induced Impaired Fracture Healing in Mice

Keze Cui 1, Yuanliang Chen 1, Haibo Zhong 1, Nan Wang 2, Lihui Zhou 3, Fusong Jiang 4,✉
PMCID: PMC7048895  PMID: 32175028

Abstract

Background

Diabetes mellitus is characterized by hyperglycemia which displays insufficiency or resistance to insulin. One of the complications of diabetes is the increased risk of fracture and the impairment of bone repair and regulation. There have been evidences from previous studies that mesenchymal stem cells (MSCs) from bone marrow promote cartilage and callous formation. In addition, IL-10, an anti-inflammatory cytokine, has been observed to relieve inflammation-related complications in diabetes.

Methods

In this study, the role of IL-10-overexpressing bone marrow-derived MSCs (BM-MSCs) was examined in the diabetic mice model with femur fracture. MSCs were isolated from the BALB/c mice and IL-10 over expression was conducted with lentivirus transduction. The streptozotocin (STZ)-induced diabetes model with femoral fracture was established. BM-MSCs with IL-10 over expression were transplanted into the fracture area. The expressions of inflammatory factors IL-6, TNF-α and INF-γ were examined by qPCR and immunoblot; the biomechanical strength of the fracture site of the mice was examined and evaluated.

Results

Data showed that IL-10 overexpressed BM-MSCs transplantation decreased inflammatory response, promoted bone formation, and increased the strength of the fracture site in STZ-induced diabetic mice with femoral fracture.

Conclusion

IL-10 overexpressed BM-MSCs transplantation accelerated fracture repair in STZ-induced diabetic mice, which in turn provides potential clinical application prospects.

Electronic supplementary material

The online version of this article (doi:10.1007/s12195-019-00608-w) contains supplementary material, which is available to authorized users.

Keywords: Diabetes, Bone fracture, BM-MSCs, IL-10, STZ-induced diabetic mice

Introduction

Diabetes is a chronic metabolic disease characterized by high blood glucose attributable to the deficiency of, or resistance to, insulin secreted by the pancreas.7,17,27 The mortality of diabetes is due to its serious complications other than the disease itself. The complications involve various organs including the kidneys, heart, and nerves.15,18,19 In recent years, more and more studies have shown that diabetes may have negative influences on the skeletal system, such as decreased bone mass and loss of bone strength.14,21,23 It is noteworthy that the risk of fracture in diabetic patients is higher compared to healthy individuals, and diabetic patients have been observed to suffer delayed healing or, in worse cases, non-union after fracture.4,6 The mechanism of the effects of diabetes on fracture healing has not been fully elucidated to date. The shift of osteoblasts to adipocytes, the alteration of cellular and molecular properties in diabetic microenvironment are some of the hypotheses.3,9 Clinically, the treatment of slow or non-healing fractures caused by hyperglycemia are more difficult. Apart from effective antibiotics, surgical aseptic manipulation and regulation of blood glucose, there are currently no specific methods to treat this lesion. Little is known about the molecular pathways of osteoblast dysfunction caused by high glucose, nor are there any effective medication for treatment. Therefore, the lack of effective treatment of diabetic fractures has become a pressing issue needing to be resolved.

Bone marrow mesenchymal stem cells (BM-MSCs) are able to differentiate into osteoblasts and chondrocytes under certain conditions.11 They are easily isolated and purified from the bone marrow, as well as having a strong expanding ability in the laboratory. Studies have demonstrated that BM-MSCs are highly efficient in assisting with cartilage formation and bone regeneration after fracture when being transplanted back to the fracture area.5,8 Because of the advantages, BM-MSCs have become good candidate cells for repairing bone fracture. However, the mechanism of how the BM-MSCs promote fracture repairing remains poorly understood.

Interleukin-10 (IL-10) is a form of cell-derived anti-inflammatory cytokine, which inhibits inflammatory factors and in turn relieves inflammation. Recent researches have suggested that serum IL-10 plays a regulatory role in diabetes. The balance between IL-10 and inflammation-related factors is broken in a diabetic environment, and IL-10 level is lower than that of healthy individuals.13,16,20,29 Thus, IL-10 contributes to the extenuation of the complications of diabetes such as proliferative diabetic retinopathy and diabetic osteoarthritis.

To further explore the effects of BM-MSCs and IL-10 on fracture repair, we established a type I diabetes mice model by intraperitoneal injection of streptozotocin (STZ), and constructed a mouse femur fracture model.20,29 We isolated the mesenchymal stem cells from the bone marrow of the BALB/c mice and transduced these cells to the BM-MSCs with high expression of IL-10 (BM-MSCs/IL-10). After injecting the IL-10 overexpressed-BM-MSCs in situ into the fracture, the expressions of inflammatory factors INF-γ and IL6 were examined. The histological analysis of bone formation and mineralization was also checked, and the biotension of the fracture site of the mice were examined in each of the control and experimental groups.

Methods

Isolation and Culture of Mesenchymal Stem Cells (MSC) from Bone Marrow in BLAB/c Mice

The animal experiment was approved by Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. The food and commendation of the experimental mice were compliant with the guideline issued by Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. BLAB/c mice at the age of 8–12 weeks were purchased from SLAC (Shanghai, China). Mesenchymal stem cells (MSCs) were prepared as described in previous studies.22 In brief, BALB/c mice were sacrificed with euthanasia, and the tibia and femur were picked up. Bone marrow was collected from cavities of those bones. After removing the bone slags by filter, cells were suspended in DMEM/F-12, 50/50, 1 × (Corning cellgo, 10-092-CV) medium. The bone marrow cells were washed three times with DMEM and spun down at 1300 rpm, and were sorted with flow cytometric sorter (BD™ LSR II system). Cells with surface marker of CD29/CD44/CD90 positive and CD34/CD45 negative were considered to meet the criteria of MSCs (Fig. S1). Sorted MSCs were cultured in Eagle’s Minimum Essential Medium (EMEM) (ATCC® 30-2003™) with 1% of penicillin/streptomycin (GIBCO, Grand Island, NY). 2.5 × 107 cells were seeded in a 75 cm2 flask. Unattached cells were washed out by media change at 3 and 8 h respectively. Cells were passaged with trypsin digestion when they achieved 60–80% confluence and cultured in a new flask, the split rate was 1:1. The media were changed every 3 days. Cells were considered purified after 3 weeks culturing (Fig. 1a), and were used freshly either for injection to BLAB/c mice or transduction by lentivirus encoding IL-10.

Figure 1.

Figure 1

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Schematic depicts the experiment protocol. (a) BM-MSCs were isolated and cultured, and then transfected with MIG vector encoding IL-10. (b) BALB/c mice were intraperitoneal (i.p.) injection with streptozotocin (STZ) (40 mg/kg), and the mid-diaphyseal femur fracture model was constructed. BM-MSCs or IL-10 overexpressed-BM-MSCs were locally injected at fracture site, and three weeks later the mice were subjected to examination.

Lentivirus Transduction

The MIG (MSCV-IRES-GFP) vector harboring IL-10 (MIG-IL-10) and MIG viral vector were used to produce IL-10 overexpression lentivirus.5 Firstly, the two types of vectors were co-transfected to 293T cells with viral particles (gag-pol, virus-G and virus envelope) for 48 to 72 h depending on the transduction efficiency (green fluorescent protein positive cells > 80% were checked under the fluorescent microscope). The supernatant carrying lentivirus and IL-10 was ultra-concentrated to get higher titer. 10 µL of virus were added to the 6 well-plate of cultured mouse mesenchymal stem cells. After 48 h of transduction, GFP positive cells were sorted and expanded in DMEM + 10% FCS + 1% penicillin/streptomycin media. After 7-1– days of culture, cells were considered stably secreted IL-10 (BM-MSCs-IL-10). The expression of IL-10 secreted by the transduced cells was examined by real-time RT-qPCR and immunoblot.

Establishment of the Mouse Model

Firstly, the streptozotocin (STZ)-induced diabetic models were constructed on the 12 weeks old of BALB/c mice. Streptozotocin selectively damages the β-cells in the pancreas and results in type-I diabetes. The low-dose (7.86 mL/g) of streptozotocin, which dissolved in saline and multiple (1 dose × 5 days) intraperitoneal injections (i.p) were given to the mice.24,26 After 1 week of the final injection, only the blood glucose concentrations of the mice higher than 270 mg/dL were selected to the experiment group.

Secondly, the femoral fractures were conducted using the STZ-induced diabetic mice model. The experiment for making femoral fractures was performed as per Reference 1. On the same day, 2 × 106 of IL-10 overexpressed-BM-MSCs cells in 50 µL PBS solution were injected to the mice to make IL-10 overexpressed-BM-MSCs group, 2 × 106 of BM-MSCs without carrying IL-10 and PBS alone were also injected to the mice to make the BM-MSCs group and control group separately. Luciferase gene was inserted into retroviral vector, and the bioluminescence imaging (BLI) was detected using the Xenogen In Vivo Imaging System on the 7th day post-transplantation (Fig. 3a). 21 days after injection, mice of each group were euthanized for further experiments (Fig. 1b).

Figure 3.

Figure 3

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Transplantation of BM-MSCs significantly inhibits inflammation in the fracture calluses of diabetic mice with femoral fracture. (a) In vivo optical bioluminescence imaging (BLI). Mid-diaphyseal femur fracture was constructed in STZ-induced diabetic mice model according to literatures. 50 μL PBS solution containing BM-MSCs or IL-10 overexpressed-BM-MSCs were locally injected at fracture site. Luciferase gene was inserted into retroviral vector, and BLI was detected using the Xenogen In Vivo Imaging System one-week post-transplantation by intravenously injecting luciferase substrate. (b) The IL-10 expressions in fracture calluses were detected using Western blotting one-week post-IL-10 overexpressed-BM-MSCs transplantation. β-Actin was used as a loading control. The fracture calluses were harvested after 3 weeks treatment, and the expressions of IL6 (c), TNF-α (d) and IFN-γ (e) were examined RT-PCR (upper) and Western Blot (lower). β-Actin was set as control protein. The mRNA levels were normalized to Control group. n = 8. *P < 0.05, **P < 0.01, ***P < 0.001.

Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction

Total RNA was extracted from the fracture callus tissues in different groups using AllPrep RNA/Protein kit (QIAGEN, Valencia, CA). cDNA synthesis was performed using iScript reverse transcription supermix (Bio-Rad; Hercules, CA). Primers were designed by Bio-Rad on 96 well plates. Quantitative RT-PCR for associated genes was performed using the SsoAdvanced Universal SYBR Green Supermix and run on 95 °C, 2 min; 95 °C, 5 s and 60 °C, 30 s, 40 cycles; and every 5 s increase 0.5 °C from 65 to 95 °C. All of these assays were conducted according to the manufacturer’s protocol. The primers were referenced according to literature.28

Immunoblot

Antibodies of IL-10, IL-6, TNF-α and IFN-γ were purchased from Millipore (Billerica, MA). For immunoblot, the fracture calluses in different groups were collected and grinded, tissues were lysed on ice in 1 × lysis buffer (Cell Signaling Technology, Danvers, MA) in 20 mM Tris–HCl buffer, pH 7.5, containing 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4 and 1% Triton) with 1 × Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO) or 1 × phosphatase inhibitor cocktail set II (Calbiochem, La Jolla, CA). 20 µg of total protein from each sample were resolved on 8–12% SDS Bis–Tris–Cl polyacrylamide gel with running buffer and transferred to 0.2 µm PVDF membranes (Bio-Rad, Hercules, CA). The membranes were probed with various antibodies. Blots were incubated with anti-rabbit or anti-mouse secondary antibodies conjugated with HRP from GE Healthcare life sciences (Little Chalfont Buckinghamshire, UK). The signals were visualized using Supersignal West Pico Chemiluminescent Substrate or Pierce ECL Western Blotting Substrate.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA was performed to evaluate the concentrations of IL-10 in the mice samples according to the manufacturer’s instructions.5 A sandwich ELISA kits (R&D Systems, Minneapolis, MN), with detection range of 10–300 pg/mL was purchased. The values were read at 450 nm in an ELISA reader, and the IL-10 value was calculated from specific calibration curves prepared with known standard solutions.

Histology and Histomorphometric Analysis

Samples of fractured femur sections were collected on the 21st day after fracture in the control, BM-MSCs and IL-10 overexpressed-BM-MSCs groups. The samples were then fixed for 24 h in cold 4% paraformaldehyde and subsequently decalcified for 5 weeks by incubation before embedding in paraffin blocks. Histological analysis of fractured femur sections from different groups was performed using alcian blue (blue; glycosaminoglycans and proteoglycans) and orange G staining (pink; bone and surrounding soft tissue). The percentage area of cartilage, bone and fibrotic tissue were quantified using Visiopharm software.1,2,6

Biomechanical Strength Analysis

We examined the biomechanical strength of the STZ-induced diabetic mice on day 14 after injection. The maximum torsion and yield torque were selected as indicators of biomechanical strength of the bone.25

Data Analysis

All data were presented as mean ± SD, and were analyzed by Student’s t test or one-way ANOVA analysis with a Tukey’s post hoc test. Experiments were repeated independently in triplicate.

Results

Establishment of the Experimental Mice Model

To establish the diabetic model, we prepared male BALB/c mice at the age of 12 weeks. The mice were randomly selected to set up the control group without any treatment. These control mice were implemented femoral fracture following the selection. The experimental mice were intraperitoneally (i.p.) injected with streptozotocin (STZ) (40 mg/kg) dissolved in 50 µL saline for 5 consecutive days (Fig. 1b). Next, we prepared the femoral fracture with the diabetic mice on the 7th day of injection (see method). Only the completed and transverse femoral fracture mice were accepted for further experiment (Fig. 1b). On the same day, the diabetic mice with femoral fracture were randomly separated into two groups, the BM-MSCs group and the IL-10 overexpressed-BM-MSCs group. IL-10 overexpressed-BM-MSCs were prepared as illustrated in Fig. 1a. 2 × 106 BM-MSCs (BM-MSCs group) or 2 × 106 BM-MSCs/IL-10 (BM-MSCs/IL-10 group) suspended in 50 µL PBS were locally administered around the fracture site. The mice were put back to their dens and fed with adequate food and water for a further 21 days. The blood of mice in each group was collected from their tails, the mice were then sacrificed and the calluses of femoral bone were collected for further use. Our data showed that the experimental mice model was successfully established (Fig. 1). Additionally, by examining the surface markers, we confirmed that the BM-MSCs used meet the criteria for MSCs (Fig. S1).

Overexpression of IL-10 in Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs) Decreased the Blood Glucose in BALB/c Mice

The concentration of blood glucose was examined on the 6th day. The average concentration of blood glucose in our diabetic mice was 300 mg/dL (Fig. 2a). The serum IL-10 concentrations in the control group and the STZ-induced diabetic mice group were examined by ELISA on the 15th day post-STZ injection. Our result showed that the diabetic mice had a much lower level of IL-10 concentration compared to the control mice, which suggest that the internal secretion of IL-10 was decreased alongside the hyperglycemia in STZ-induced diabetic mice (Fig. 2b). For further elucidation of the relationship between IL-10 and diabetes, the IL-10 expression (Fig. 2c) and IL-10 production (Fig. 2d) in transfected BM-MSCs or IL-10 overexpressed-BM-MSCs mice were examined by real-time PCR, Western blot and/or ELISA. Our results clearly showed that the RNA expression of IL-10 overexpressed-BM-MSCs group mice achieved a remarkable increase in folds compared with the BM-MSCs group mice. Moreover, the concentration of IL-10 reached 220 pg/mL in BM-MSCs/IL-10 group compared with 5 pg/mL in BM-MSCs group, and the differences were statistically significant (P < 0.001). The results indicated that IL-10 overexpressed-BM-MSCs group mice possessed high expressions of IL-10 both in RNA and protein level.

Figure 2.

Figure 2

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Overexpression of IL-10 in bone marrow-derived mesenchymal stem cells (BM-MSCs). (a) To establish diabetic model, 12-week-old male BALB/c mice were intraperitoneal (i.p.) injection with streptozotocin (STZ) (40 mg/kg) for 5 consecutive days. The concentration of blood glucose was examined on 15th day. (b) The serum IL-10 concentrations in control mice and STZ-induced diabetic mice were detected by ELISA on 15th day. (c) BM-MSCs were isolated, cultured and transfected with MIG vector encoding IL-10. The IL-10 expression (c) and IL-10 production (d) in transfected BM-MSCs were examined Western blotting and ELISA 48 h post-transfection. n = 8. Data represent mean ± SD. **P < 0.01 and ***P < 0.001.

Transplantation of IL-10 Overexpressed-BM-MSCs Significantly Inhibits Inflammation in the Fracture Calluses of Diabetic Mice with Femoral Fracture

To elucidate whether BM-MSCs/IL-10 influences inflammation in the fracture calluses of diabetic mice, we injected 50 µL of PBS (control group) or 2 × 106 BM-MSCs (BM-MSCs group) or 2 × 106 IL-10 overexpressed-BM-MSCs (IL-10 overexpressed-BM-MSCs group) to BALB/c diabetic mice with femoral fracture. BLI was detected using the Xenogen In Vivo Imaging System one week post-transplantation by intravenously injecting luciferase substrate (Fig. 3a). Fourteen days after, we examined the inflammatory factor genes IL-6, TNF-α and IFN-γ in those mice with western blot, and the data showed that the IL-10 overexpressed-BM-MSCs group presented the highest IL-10 expressions both in mRNA and protein levels (Fig. 3b). The fracture calluses were collected and the inflammatory factors were examined by both qPCR and western blot, and the results are shown in Figs. 3c–3e. BM-MSCs group has decreased the expressions of IL-6, TNF-α and IFN-γ from 100 (control group) to 58, 62 and 57% respectively. Furthermore, the IL-10 overexpressed-BM-MSCs group has decreased the expressions of those inflammation factors to 28, 30 and 27% respectively, and apparent differences were observed (Figs. 3c–3e). These results indicated that IL-10 played the role of effectively decreasing the inflammation in diabetic mice with femoral fracture.

Transplantation of IL-10 Overexpressed-BM-MSCs Results in Expedited Endochondral Bone Formation and Mineralization

To further investigate the IL-10 overexpressed-BM-MSCs function on bone formation and mineralization, femoral bone tissues were collected in the control, BM-MSCs and BM-MDCs/IL-10 groups on day 21 post-BM-MSC transplantation. The percentage area of cartilage, bone, and fibrotic tissue were quantified using Visiopharm software (Figs. 4a–4d). IL-10 overexpressed-BM-MSCs group presented the lowest percentage area of cartilage and fibrotic tissue compared to control group and BM-MSCs group. IL-10 overexpressed-BM-MSCs group also presented the highest percentage area of bone. Histological analysis of fractured femoral sections from different groups showed consistent results as of the quantified percentage area analysis (Fig. 4a). The increased percentages of osteoblasts area and adipocytes areas (Fig. S2) also confirmed that overexpression of IL-10 promote the survival and differentiation of BM-MSCs. The results clearly indicated IL-10 expedited the endochondral bone formation and mineralization.

Figure 4.

Figure 4

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Transplantation of IL-10 overexpressed-BM-MSCs results in expedited endochondral bone formation and mineralization. (a) Histological analysis of fractured femur sections from different groups was performed 2 weeks after fracture using alcian blue (blue; glycosaminoglycans and proteoglycans) and orange G staining (pink; bone and surrounding soft tissue). The percentage area of (b) cartilage, (c) bone, and (d) fibrotic tissue were quantified using Visiopharm software. The red arrows denote cartilage area, the asterisks denote bone and the circles denote fibrotic tissue. Data were presented as mean ± SD. n = 8. *P < 0.05, **P < 0.01.

Femoral Fractures Transplanted with IL-10 Overexpressed-BM-MSCs Exhibits Increased Biomechanical Strength

To examine the IL-10 functions in diabetic mice with femoral fracture, the biomechanical strength of fracture was checked. The indicators of the maximum torsion and the yield torque were used (Fig. 5).25 As anticipated, the IL-10 overexpressed-BM-MSCs group exhibited the highest value of the maximum torsion compared to the BM-MSCs group and the control group. Similarly, the examination of yield torque showed consistent results as the maximum torsion did. Significant differences were observed (Figs. 5a and 5b). Our results indicated that the transplantation of IL-10 overexpressed-BM-MSCs to femoral fracture exhibited increased biomechanical strength in STZ-induced diabetic mice.

Figure 5.

Figure 5

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Fractures transplanted with BM-MSCs/IL10 exhibit increased biomechanical strength. (a) Maximum torsion and yield torque (b) treated fractures was significantly increased over untreated controls. Data were presented as mean ± SD. n = 8. **P < 0.01, ***P < 0.001.

Discussion

Several researches have described that patients with diabetes have increased risk of bone fracture.27 The mesenchymal stem cells possess the ability to differentiate to osteoblasts and chondrocytes and provide a potential treatment for fracture. In addition, IL-10, an anti-inflammation factor, plays a role to relieve inflammatory responses in diabetes complications such as proliferative diabetic retinopathy and diabetic osteoarthritis. Based on these evidences, we established a STZ-induced type I diabetes model, transplanted with MSCs carrying IL-10 in BALB/c mice. Our purpose in this study was to elucidate the relationships between the diabetic fracture, the mesenchymal stem cells and the IL-10.

We successfully established the diabetic mice model with femoral fracture, transplanted with mesenchymal stem cells carrying IL-10. Our diabetic mice showed significantly decreased intrinsic IL-10 compared with control mice (Fig. 2b), which indicated that the IL-10 secretion was impaired in diabetic mice. Meanwhile, when we transduced IL-10 to MSCs and transplanted the cells to the mice, the IL-10 significantly increased both in RNA and protein level (Figs. 2c and 2d). We then examined the inflammatory factor expressions in control, BM-MSCs and BM-MSCs/IL-10 group, as shown in Figs. 3c–3e. The inflammatory factors IL-6, TNF-α and IFN-γ were all down-regulated in the IL-10 overexpressed-BM-MSCs group and significant differences were observed (P < 0.001). Our data clearly provided the evidence that IL-10 down-regulated the inflammation in diabetic mice. We then calculated the percentage of cartilage, bone and fiber tissue in the experiment groups. The expected result showed that the IL-10 overexpressed-BM-MSCs group achieved the highest percentage of bone formation and the lowest percentage of fiber tissue. In addition, we measured the percentages of the osteoblasts and adipocytes areas in these groups and the IL-10 overexpressed-BM-MSCs group showed the highest percentages of those cells (88 and 9% respectively, Fig. S2). These results provide further evidence that IL-10 plays a positive role in promoting bone formation (Fig. 4). Finally, the strength of bone was checked and the IL-10 overexpressed-BM-MSCs group presented the highest bone Maximum torsion and Yield torque (Fig. 5). From our data exhibited above, we conclude that IL-10 overexpressed-BM-MSCs transplantation accelerates fracture repair in diabetic mice.

One of the serious complications in both type I and type II diabetes is the increased risk of osteoporosis and bone fracture, and worse is the healing delay and un-union which are common in the fractured parts of diabetic individuals.4 In patients of type I diabetes, bone fracture can be attributed to the absolute deficiency of insulin, which has an osteo-anabolic effect, resulting in a lower bone mineral density. On the other hand, type II diabetes patients often have a normal or higher bone density, which suggest impaired bone quality involved.15,17,19 The mechanism of diabetes affecting fracture healing has not been fully elucidated. The current research indicates that the cellular and molecular properties of bone tissue are altered, the function of osteoblasts is destroyed in the diabetic microenvironments, and these cells differentiate into adipocytes and subsequently impair fracture healing.9 Studies suggest that the up-regulation of PPARγ changes the mesenchymal stem cell fate with a shift from the osteoblast lineage to the adipocyte lineage, resulting in an osteoblast decrease.3

Mesenchymal stem cells (MSCs) are able to differentiate into chondrocytes and osteoblast.12 As such, MSCs have a therapeutic potential to reduce the time required for healing in patients with fractures. Presently, MSCs transplantation are not being used commercially for fracture treatment. However, the study of animal model described that MSCs transplantation could improve cartilage and bone formation.8,12 Clinical trials of MSCs transplantation have also been applied.10 Our experiments provide evidence consistent with findings in previous studies that MSCs transplantation helps callus formation and in turn strengthens the bone (Figs. 4 and 5).

Diabetes is known to display a strong inflammatory response. The secretion of certain inflammatory cytokines by peripheral blood monocytes can be stimulated by hyperglycemia.13 Therefore, it is associated with proliferative diabetic retinopathy and the diabetic osteoarthritis as well. IL-10 is known as an anti-inflammatory cytokine which plays a role in attenuating myocardial ischemia–reperfusion injury. Krishnamurthy et al. described that IL-10 could relieve the inflammatory response in myocardial infarction via suppressing HuR/MMP-9 and activating STAT3, thus enhancing capillary density. Mao et al. examined the IL-1β and IL-10 levels in proliferative diabetic retinopathy (PDR) and claimed that, although the absolute level of IL-10 had not decreased, the balance of IL-1β and IL-10 was broken, which resulted in the inaction of IL-10 in its anti-inflammatory function.16 Studies have also reported a significant reduction in IL-10 levels in type 2 diabetes.1,20,29 Our data presented a significant decrease of the inflammatory factors INF-γ, TNF-α and IL6 when we introduced the over-expression of IL-10. Diabetes with fracture is an outcome, while the factors contributing to this outcome, which also influence bone repair, are complicated. Our diabetes mice model fracture was the simple, although settled ones, it has limited relevance to the clinical scenarios of delayed and non-union bone fracture in people. Thus, the inherent limitations of rodent models should be acknowledged.

Taken together through our work, we explored the effects of BM-MSCs with high expression of IL-10 on STZ-induced mice with femoral fracture. We conclude that IL-10 overexpressed-BM-MSCs transplantation accelerates fracture repair in diabetic mice, which provides potential clinical application in future treatment processes.

Electronic supplementary material

Below is the link to the electronic supplementary material.

12195_2019_608_MOESM1_ESM.docx (111.5KB, docx)

Electronic supplementary material 1 (DOCX 113 kb)

Acknowledgments

This work was supported by the Natural Science Foundation of Hainan Province, China (819MS146) and the Seed Fund Program of Shanghai University of Medicine & Health Sciences (No. 2017N0612).

Conflict of interest

Keze Cui, Yuanliang Chen, Haibo Zhong, Nan Wang, Lihui Zhou and Fusong Jiang declare that they have no conflict of interest.

Ethical Approval

No human studies were carried out by the authors for this article. All animal studies were carried out in accordance with the guideline issued by Shanghai Jiao Tong University Affiliated Sixth People’s Hospital and approved by Shanghai Jiao Tong University Affiliated Sixth People’s Hospital.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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