The expression of proinflammatory cytokine and proinsulin by bone marrow-derived mesenchymal cells for fracture healing in long term diabetic mice

doi:10.21203/rs.3.rs-2659650/v1

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Research Article

The expression of proinflammatory cytokine and proinsulin by bone marrow-derived mesenchymal cells for fracture healing in long term diabetic mice

https://doi.org/10.21203/rs.3.rs-2659650/v1

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published 18 Jul, 2023

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Background

Diabetes mellitus (DM) causes bone dysfunction due to poor bone quality and leads to severe deterioration of quality of life. The mechanisms of bone metabolism in DM remain unclear, although chemical and/or mechanical factors are known to disrupt the homeostasis of osteoblasts and osteoclasts. The purpose of this study was to identify the biochemical characteristics of osteoblasts and osteoclasts, using a mouse fracture model of long-term hyperglycemia (LT-HG).

Methods

C57BL/6J mice and green fluorescent protein (GFP)-positive bone marrow transplanted C57BL/6J mice with LT-HG in which hyperglycemia was maintained for 2 months were used in this study. After the experimental fracture, we examined the immunohistochemical expression of proinsulin and tumor necrosis factor (TNF) -α at the fracture site. C57BL/6J fracture model mice without hyperglycemia were used as the control sample.

Results

In the LT-HG mice, osteoblasts showed an irregular arrangement at the fracture site. The osteoclasts were scattered with a decrement in the number of nuclei. The positive expression of proinsulin was seen in mesenchymal stem cells (MSCs) with neovascularization 2 and 3 weeks after fracture. Immunopositivity for TNF-α was seen in immature chondrocytes or MSCs with neovascularization at 2 weeks, and the number of positive cells was not decreased at 3 weeks. Examination of GFP-grafted hyperglycemic mice showed that the majority of cells at the fracture site were GFP-positive. Immunohistochemistry showed that the rate of double positives was 15% for GFP and proinsulin and 47% for GFP and TNF-α.

Conclusion

LT-HG induced an increase in the number of proinsulin and TNF-α positive cells derived from systemic bone marrow cells. The proinsulin and TNF-α positive cells cause both bone formation and bone resorption, and they suppress inflammatory cytokines and impair glucose metabolism.

long-term hyperglycemia

fracture healing

TNF-α

proinsulin

bone marrow transplantation

The prevalence of diabetes mellitus (DM) is increasing globally due to lifestyle changes, urbanization, population growth, and aging demographics, and the number of diabetic patients is expected to exceed 700 million by 2025 [1, 2]. However, the treatment strategies for DM and DM-related complications, such as peripheral neuropathy, renal disorder, and increased risk of fractures, remain unclear. Bone strength consists of two factors, bone mass and bone quality, with poor bone quality considered more important than bone loss in diabetic bone dysfunction [3]. According to a meta-analysis by Vestergaard et al., patients with DM type 1 and 2 have an increased risk of hip fractures compared to non-diabetics [4]. In addition, the risk of delayed union or nonunion after fracture increases 2.2 to 3.4 times [5, 6]. Therefore, diabetic bone dysfunction is one of the important factors that can decrease activities of daily living and quality of life.

The balance between bone formation and bone resorption is important for maintaining bone homeostasis. The causes of poor bone formation in DM may be due to increased bone marrow (BM) adiposity of mesenchymal stem cells (MSCs) and impaired differentiation and mineralization of osteoblasts [7–10]. In previous studies by Kojima et al. [11] and Terashima et al. [12], bone marrow cells (BMCs) have been found to produce proinsulin and TNF-α and fuse with each organ cell to cause cytopathy in diabetic hepatic, renal, and peripheral neuropathy models. Furthermore, in a short-term hyperglycemia mouse fracture model in which hyperglycemia was maintained for 2 weeks, the sizes of the osteoclasts and the resorption pits formed were significantly smaller. Moreover, the expression of DC-STAMP, a putative pivotal gene for osteoclast fusion, decreased in osteoclasts in DM mice. Together, these results highlight the importance of impaired bone resorption due to osteoclast dysfunction [13]. DM patients are exposed to hyperglycemia for long periods of time, ranging from years to decades. It remains to be elucidated how proinsulin- and TNF-α-producing cells appear in the bone tissue of long-term diabetic patients and how they are able to alter the balance between bone formation and bone resorption.

In this study, we induced fractures in LT-HG mice in which hyperglycemia was maintained for 2 months and observed histological changes in osteoblasts and osteoclasts that appeared in the callus as well as changes in the expression of proinsulin and TNF-α. In order to investigate the origin of these cells, bone marrow transplantation (BMT) of GFP-positive cells was performed, and their cell distribution was observed. The purpose of this research was to analyze the mechanisms of bone formation and bone resorption in a diabetic bone dysfunction model and to obtain new knowledge for the development of future treatment methods.

Mouse husbandry and grouping

This research was approved by the Institutional Review Boards of the authors’ affiliated institutions. Eight-week-old male C57BL/6J mice (wild type, Japan SLC, Inc., Shizuoka, Japan) and C57BL/6-Tg (UBC-GFP) 30Scha/J mice (GFP-Tg mice, Jackson Laboratory, Bar Harbor, ME, USA) were used for this study. Mice were housed in our university double-barrier facility on a 12-h light cycle and given access to commercial chow and water ad libitum.

Figure 1 presents the mouse groupings in this study. For investigation of osteoblasts, osteoclasts, and proinsulin and TNF-α-producing cells at the callus formation site, mice were allocated randomly to two groups: with (LT-HG positive; n = 10) or without (LT-HG negative; n = 10) exposure to LT-HG with streptozotocin (STZ). For the GFP-positive BMT models, mice were selected with LT-HG (GFP and LT-HG positive; n = 5) and without LT-HG (GFP positive and LT-HG negative; n = 5). Each mouse was subjected to histological and immuno-histochemical examination at 2 or 3 weeks after fracture, after euthanization by perfused fixation under deep anesthesia.

Long term hyperglycemia model

We induced hyperglycemia using STZ (180 mg/kg body weight) (Nacalai Tesuque, Kyoto, Japan) injected into C57BL/6 J mice intraperitoneally. The control group mice without LT-HG were injected with citrate buffer (pH 4.5). Blood obtained from the tail vein was measured by Glutest-Ace (Sanwa kagaku, Nagoya, Japan). Hyperglycemia was diagnosed when blood glucose levels (BGL) reached over 300 mg/dl (Supplemental Data. a). Based on a study by Terashima et al., we defined 2 months of hyperglycemia exposure as the LT-HG group because mice are known to show diabetic neuropathy during 2 months of hyperglycemia exposure [12].

Fracture model

Mice were anesthetized by intraperitoneal administration of a mixed anesthetic (medetomidine 0.3 mg/kg, midazolam 4.0 mg/kg, butorphanol 5.0 mg/kg), then positioned laterally to make a transverse, open fracture of the right femur. A skin incision was made on the lateral side of the right femur, the muscle was incised in the direction of the fibers, the femur was exposed, and the central femur was cut with scissors without periosteal dissection. A 23-gauge needle was inserted into the femur for internal fixation, and the wound was sutured (Fig. 2a, b). The mice were permitted full weight bearing activity.

Bone marrow transplantation model

Recipient C57BL/6J mice were irradiated (9 G× for 5 min) using an X irradiation device (MBR-1520R, Hitachi Medical Corporation, Tokyo, Japan). The GFP-Tg mouse donors were euthanized by cervical dislocation, then the humerus, femur, and tibia were collected, and BMCs were washed out. The suspension of BMCs was centrifuged at 1500 rpm for 5 min, and the suspension was diluted with 0.1 M phosphate buffer saline (PBS) HBSS. Whole BMCs (4×10⁶ cells) were injected through the tail artery of a recipient mouse to make a GFP-positive BMT model (Fig. 2c, d).

Histological and immunohistochemical examination

Mice were deeply anesthetized with the same anesthetic mixture described above and perfused with PBS followed by a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer. After perfusion fixation, the intramedullary needle was removed, and the specimens were decalcified in ethylenediamine tetra acetic acid disodium for 1 week at 4°C. The specimens were then dehydrated through a graded ethanol series, embedded in paraffin, and the sections were cut at 4 µm. To evaluate the morphology of the callus, the callus was stained with hematoxylin-eosin (HE). Immunostaining was performed using the ABC method, with anti-OC antibody (1:100; ab93876, Abcam, Cambridge, UK), anti-RANK antibody (1:50; 64C1385, Abcam, Cambridge, UK), anti-proinsulin antibody (1:1000; Progen Biotechnik, Heidelberg, Germany), and anti-TNF-α antibody (1:100; ab6671, Abcam, Cambridge, UK). The specimens were examined under an optical microscope (FXA, Nikon, Tokyo, Japan), and sections were evaluated and measured using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).

Frozen sections of non-decalcified tissue were prepared using the Kawamoto film method [14]. Mice were anesthetized and perfusion-fixed using the same method described above. The collected right femur was placed in an embedding agent (SCEM, Section-Lab Co. Ltd., Hiroshima, Japan), submerged in hexane, and cooled with liquid nitrogen to create a freeze-embedded block. The blocks were trimmed in a cryostat using a tungsten carbide knife (TC-65, Leica Microsystems Co., Ltd., Hiroshima, Japan), and a film (Cryofilm Type 2C, Section-Lab Co., Ltd., Hiroshima, Japan) was applied to the thin section surface to prepare sections of 4 µm thickness. The cryosections were dried at room temperature for 60 s, and the sections were placed in 100% alcohol. Staining was performed using the floating method, and the primary antibodies were anti-proinsulin antibody (1:1,000; Progen Biotechnik, Heidelberg, Germany) and anti-TNF-α antibody (1:100; ab6671, Abcam, Cambridge, UK). Alexa 405 anti-rabbit IgG antibody (ab175651, Abcam, Cambridge, UK) and Alexa 647 anti-guinea pig IgG antibody (ab150187, Abcam, Cambridge, UK) were used as fluorescence-labeled secondary antibodies. After staining, the sections were polymerized and cured with cover glass using encapsulating materials (SCMM-R2, Section-Lab Co., Ltd., Hiroshima, Japan) and encapsulation equipment (UV Quickcryosection Mounter, Section-Lab Co., Ltd., Hiroshima, Japan). Sections were evaluated with a confocal laser scanning microscope (TCS SP8 X, Leica, Wetzlar, Germany), and images were merged for cell count and semiquantitative evaluation.

Statistical analysis

Correlation analyses of cell counts via immunohistochemical examination were performed using the Mann–Whitney U test. P < 0.05 was considered statistically significant. These analyses were performed using SPSS software version 25 (IBM Corp., Armonk, NY, USA).

At the beginning of the experiment, the average body weight (BW) was 21.66 g (± 0.843) for the non-hyperglycemia control group (HT-LG^–) and 20.98 g (± 0.72) for the long-term hyperglycemia group (LT-HG⁺). After 2 months, the BW was 26.32 g (± 0.865) for the HT-LG^– group and 20.5 g (± 1.78) for the LT-HG⁺ group (Supplemental Data. b). The mean BGL of the HT-LG^– group remained at 120–140 mg/dl (± 17.92–23.42) during the experimental period; that of the LT-HG⁺ group was 119.6 mg/dl (SD 13.45) before STZ injection and 360–390 mg/dl (± 50.95–66.16) afterward (Supplemental Data. a).

The healing process of the fracture site was observed histologically. In the HT-LG^– group after 2 weeks (2W), a callus was formed around the fracture site, immature MSCs aggregated, blood vessels formed, chondrocyte areas and trabecular bone formation occurred, including osteoblasts and osteoclasts. At 3 weeks (3W), almost all of the chondrocytes were resorbed, blood vessel formation was accelerated, and trabecular bone formation was also accelerated (Fig. 3a, b). In the LT-HG⁺ group, a similar callus was observed at 2W, but the callus was also seen at 3W, and the chondrocyte areas persisted (Fig. 3c, d). The ratio of chondroid areas to the callus area was 24% at 2W and 3% at 3W in the HT-LG^– group, while it was 27% at 2W and 8% at 3W in the LT-HG⁺ group. In the LT-HG⁺ group, the chondroid area persisted substantially after 3W, and the callus resorption process was delayed (P < 0.05).

In addition, the callus at 2W was locally observed. In the LT-HG⁺ group, the arrangement of osteoblasts became irregular, the intercellular spaces were larger, and the number of cell aggregates decreased (Fig. 4a, d). The average number of osteoclasts was 7.5 in the HT-LG^– group and 6.3 in the LT-HG⁺ group under a 400X optical microscope. There was no significant difference in numbers between the two groups (P = 0.22) (Fig. 4b, c, e, f). However, the number of osteoclast nuclei was significantly decreased to 2.9 in the LT-HG⁺ group as compared to 4.2 in the HT-LG^– group (P < 0.05).

Immunostaining showed that the expression of proinsulin was negative in the HT-LG^– group (Fig. 5a, b). In contrast, in the LT-HG⁺ group, the MSCs surrounding the neovascular nests were strongly positive at 2W and 3W (Fig. 5e, f). In addition, the hypertrophic chondrocytes adjacent to trabecular bone and bone tissue were negative even in the LT-HG⁺ group. TNF-α expression was weakly positive in the chondrocytes adjacent to bone tissue and strongly positive in MSCs around small vessels in both groups. In the HT-LG^– group, the number of positive cells tended to decrease at 3W as compared to 2W, while in the LT-HG⁺ group, the number of positive cells tended to increase at 2W and 3W (Fig. 5c, d, g, h).

After BMT of GFP-positive cells, many GFP-positive cells could be observed in calluses in both HT-LG^–and LT-HG⁺ groups. Overlaying the immunolocalization of GFP-positive cells and proinsulin, GFP-positive and proinsulin-positive cells were found in MSCs around neovascular nests, and the proportion of GFP-positive cells (GFP⁺) and proinsulin-positive cells was 3% in the GFP⁺ / HT-LG^– and 15% in the GFP⁺ / LT-HG⁺ groups (P < 0.05) (Fig. 6a, c). GFP⁺ / TNF-α⁺ were also found in BMCs around the neovascular nests, accounting for 35% of the total GFP⁺ / HT-LG^– and 47% GFP⁺ / LT-HG⁺ (P = 0.07) (Fig. 6b, d). These results suggest that the proinsulin-positive and TNF-α-positive MSCs that collect in the calluses of the LT-HG groups are of BM origin.

The main feature of this study was that a LT-HG model was created and used. As compared to the study by Kasahara et al. that used a short-term hyperglycemia model, the LT-HG model showed significant thinning of the femoral cortex (Supplemental Data. c) and increased BM adiposity and bone fragility against blunt external force [13]. These results better approximate real DM clinical conditions.

It has been widely reported that bone formation reduces under hyperglycemia. Recently, it has been reported that the proliferative capacity of human BMCs is reduced after 2 weeks of culture under hyperglycemia [15, 16] and that induction of differentiation of mouse BMCs under hyperglycemia promotes apoptosis of BMCs [10]. In addition, some studies have reported that the differentiation of BMCs into adipocytes is promoted by hyperglycemia and that differentiation into osteoblasts is impaired. Inhibition of osteoblast maturation in cultured mouse tissue [7, 8], impaired osteoblast differentiation, and reduced mineralization in mouse BMCs have also been reported [10]. However, these reports are from in vitro models in which tissue or cells themselves are cultured under hyperglycemia, and there are no reports of in vivo studies in which LT-HG fracture models were created and studied.

Regarding bone resorption capacity under hyperglycemia, it has been reported that there is no difference in osteoclast function between hyperglycemic and nonhyperglycemic mice because there are no significant differences in bone metabolism markers (serum TRAP5b, urinary DPD) or proteolytic enzymes (cathepsin K) [7, 8]. Conversely, in bone calluses of mice treated with STZ injection and hyperglycemic for 3 weeks, osteoclast function is reportedly enhanced due to an increase in TRACP cells, aggrecanases (ADAMTS), and collagenases (MMP-13,16) [17]. In a human control study, type 2 DM patients have been reported to have lower bone resorption capacity due to lower bone metabolism markers (serum TRAP5b, serum CTX) [18]. As described above, there is no consensus regarding osteoclast function and bone resorption capacity under hyperglycemic conditions, so findings from LT-HG models, like the one in the present study, are considered important. Based on research by Kasahara et al., we believe that osteoclast function is reduced [13].

The callus in the LT-HG model of this study was characterized by delayed chondrocyte resorption, decreased osteoblast aggregation, impaired multinucleation of osteoclasts, and narrowing of neovascular vessels. This study also found that the arrangement of osteoblasts became irregular, the gap between cells was large, and the number of aggregated cells tended to be small. These results, similar to previous experiments with cultured cells, suggest that hyperglycemia exposure impairs BMCs, resulting in impaired differentiation and maturation into osteoblasts and abnormal osteoblast morphology. This study compared results at 2–3 weeks after fracture, as Kasahara et al. found the greatest difference in callus characterization during this time [13]. As a result, we freshly observed that osteoclasts in the callus did not increase in cell number and had fewer nuclei when LT-HG was maintained. This clearly indicates that BMCs are impaired under LT-HG exposure and that bone resorption capacity is reduced due to impaired differentiation and function of osteoclasts and osteoclasts. The results of this study suggest that the bone metabolic capacity of the LT-HG model has decreased function in both bone formation and bone resorption.

Insulin producing cells normally occur only in the pancreas and thymus [19]. However, Kojima et al. reported that hyperglycemia in mice and rats induces the expression of proinsulin positive cells, an insulin transcript, in the liver, adipose tissue, and bone marrow, and that hyperglycemia induces the appearance of proinsulin-producing cells in the BM [20]. Terashima et al. reported that diabetic neuropathy in mice is caused by cell fusion of proinsulin-positive cells in the sciatic nerve and dorsal root ganglia, with the fused cells expressing TNF-α and undergoing a high rate of apoptosis [12]. Diabetic neuropathy in STZ-treated DM mice is also reported to require proinsulin-positive BMCs to produce TNF-α [21]. This result suggests that proinsulin-positive cells appear at various sites. In this study, the expression of proinsulin and TNF-α was also observed in the callus area in the LT-HG model, suggesting that the BMCs themselves may be damaged by the same mechanism as diabetic neuropathy, resulting in a prolonged fracture healing process. These results suggest that proinsulin may be a potential biomarker for the early diagnosis of diabetic tissue damage.

Regarding the role of TNF-α, there are reports from BMT experiments in DM mice that diabetic BMCs themselves may express TNF-α and cause glomerular damage [22] and that diabetic BMCs fuse with hepatocytes, resulting in TNF-α production and causing diabetic hepatocyte injury [23]. Diabetes-enhanced TNF-α also reduces MSC proliferation, increases MSC apoptosis, and inhibits osteoblast differentiation via Indian Hedgehog signaling [24, 25]. In this study, the increase in proinsulin-positive and TNF-α-positive cells in bone marrow derived cells (BMDCs) around small blood vessels in the callus are thought to impair the proliferation of MSCs and their differentiation into osteoblasts, thereby reducing their osteogenic potential.

Induction of MSCs from a local site such as the periosteum or BM is important for fracture healing. In addition, one of the important roles of MSCs is their involvement in hematopoietic stem cell (HSC) proliferation and maintenance of HSC progenitors [26, 27]. Since the callus at 2W post-fracture in this study was composed of many GFP-positive BMDCs, the induction of BMDCs into the matrix with angiogenesis is considered important in the fracture healing process. In addition, there were more proinsulin-positive and TNF-α-positive BMDCs in the LT-HG⁺ group, suggesting that LT-HG exposure may cause abnormalities in the BMDCs themselves.

One limitation of this study is the biological response such as cytokine release during fracture creation. To minimize the effects of fracture as much as possible, an osteotomy model was used instead of a blunt fracture model, in addition to soft tissue protection. The present study was limited to the second and third weeks after the fracture. However, in the study by Kasahara et al. [13], the greatest difference in callus formation was observed 2–3 weeks after fracture. In addition, Kayal et al. reported markedly increased expression of RANKL, TRACP, and TNF-α at 12 and 16 days after fracture in DM mice [17]. Therefore, the evaluation period for this study was judged to be sufficient. Future studies, however, may wish to address different time points after fracture.

Proinsulin and TNF-α positive BMDCs were found to be induced in the LT-HG condition, and bone formation and bone resorption functions were impaired. From the perspective of suppressing inflammatory cytokines and impaired glucose metabolism, we think that the results of this study provide useful information for the development of new diagnostic methods, such as biomarkers for bone metabolism dysfunction caused by diabetes, and for the development of therapeutic methods, such as anti-cytokine therapy.

DM: Diabetes mellitus

LT-HG: Long-term hyperglycemia

GFP: Green fluorescent protein

TNF: Tumor necrosis factor

MSC: Mesenchymal stem cells

GDP: Gross domestic product

BM: Bone marrow

BMCs: Bone marrow cells

BMT: Bone marrow transplantation

IHC: immunohistochemistry

STZ: Streptozotocin

PBS: Phosphate buffer saline

HE: Hematoxylin-eosin

BGL: Blood glucose level

BW: Body weight

SD: Standard deviation

HT-GH (-): non-hyperglycemia control group

LT-HG (+): long-term hyperglycemia group

2W: 2 weeks

3W: 3 weeks

BMDCs: bone marrow derived cells

HSC: Hematopoietic stem cell

Ethics approval and consent to participate: All methods were carried out with the approval of the Animal Care Committee at the Shiga University of Medical Science (2017-6-12). All methods were carried or in accordance with guidelines set by the Animal Care Committee at the Shiga University of Medical Science. All methods were reported in accordance with the ARRIVE guidelines.

Consent for publication: Not applicable.

Availability of data and materials: All data generated or analyzed during this study are included in this published article or are available from the corresponding author on reasonable request.

Competing interests: The authors declare that they have no competing interests.

Funding: Department of Orthopaedic Surgery at Shiga University of Medical Science.

Authors’ contributions: H.F., H.K., T.T. and M.K. designed the experiments. H.F. performed the experiments and analyzed the data. All authors participated in data interpretation. H.F., H.K., T.Y., K.K., K.M., H.S. and S.I. wrote the manuscript. All authors revised the manuscript and approved the final manuscript. All experiments were performed at Department of Orthopaedic Surgery or Department of Stem Cell Biology and Regenerative Medicine, Shiga University of Medical Science, Japan.

Acknowledgements: The authors thank Y. Uratani, T. Yamamoto, and Y. Nakae for technical support as well as M. Kanaya and M. Kunimatsu for maintenance of the animal colony. Additionally, we would like to thank Editage (www.editage.com) for English language editing.

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The expression of proinflammatory cytokine and proinsulin by bone marrow-derived mesenchymal cells for fracture healing in long term diabetic mice

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Background

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Mouse husbandry and grouping

Long term hyperglycemia model

Fracture model

Bone marrow transplantation model

Histological and immunohistochemical examination

Statistical analysis

Results

Discussion

Conclusions

Abbreviations

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References

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