Quiescent adipose tissue-derived stromal cells in mechanically compliant hydrogel accelerate diabetic wound healing through enhanced functions and extended dwell time

doi:10.21203/rs.3.rs-4702328/v1

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Quiescent adipose tissue-derived stromal cells in mechanically compliant hydrogel accelerate diabetic wound healing through enhanced functions and extended dwell time

https://doi.org/10.21203/rs.3.rs-4702328/v1

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Despite the therapeutic potential of mesenchymal stem cells (MSC), there are few clinical applications. One of the reasons is that MSC administered by conventional methods quickly disappear from the recipient, which leads to insufficient efficacy. We previously reported that MSC become quiescent in hydrogel with stiffness mimicking that of bone marrow or adipose tissue. In this study, we investigated whether inducing quiescence in adipose tissue-derived stromal cells (ADSC), one type of MSC, may overcome such a weakness. ADSC became quiescent in biocompatible gel whose stiffness matched that of adipose tissue, even in the presence of tumor necrosis factor α and interferon γ to mimic an inflammatory environment in vitro. Quiescent ADSC exhibited enhanced anti-inflammatory functions and fibroblast migration in vitro, suggesting enhanced therapeutic functions of ADSC by quiescence. Subcutaneous transplantation of quiescent ADSC accelerated wound healing in diabetic mice, which can also be attributed to their extended dwell time at the transplanted site, without any pathological changes. Inducing quiescence in ADSC also led to elimination of accumulated reactive oxygen species. These results suggest that quiescent ADSC may enhance the efficacy of ADSC-based cell therapies to treat diabetic wound healing.

Mesenchymal stem cells (MSC) have drawn much attention in tissue regeneration and cell therapies due to their ability to self-renew and differentiate into multiple cell types, as well as their ability to produce multiple factors responsible for their immunomodulatory and anti-inflammatory functions ¹. In fact, the number of clinical trials applying MSC to treat various types of autoimmune diseases and inflammatory diseases is rapidly growing. However, few have been approved by the regulatory authorities and most have failed due to either insufficient efficacy or safety concerns ². One of the challenges stems from premature senescence of MSC, which is often observed in disease conditions, such as inflammation and metabolic stress, after transplantation. Once MSC become premature senescent in these environments, they exhibit pro-inflammatory functions instead of anti-inflammatory functions, which is called senescence-associated secretory phenotype (SASP) ². Therefore, it is crucial to establish a method to make MSC resistant to premature senescence and maintain their anti-inflammatory functions in disease conditions in order to improve the efficacy of MSC-based cell therapies.

The extremely low engulfment rate of MSC after transplantation is also one of the major challenges facing MSC-based cell therapies ^3–5. For instance, intravenous infusion of MSC has been most commonly used to deliver MSC, although it is known that the majority of infused MSC are trapped in the lung capillaries and thereafter lost before being able to exhibit a therapeutic effect ⁶. Furthermore, administration of MSC by alternative methods to intravenous infusion, such as intramuscular, intraperitoneal or intrabronchial administration, still suffers loss of MSC almost immediately after transplantation ³. Thus, extending the dwell time of MSC in vivo is also indispensable to promote MSC-based cell therapies.

Previous studies have shown that MSC become quiescent on a mechanically compliant substrate, such as a hydrogel whose stiffness mimics that of MSCs’ native habitat and whose surface is engineered to support adhesion for MSC ⁷. In addition, quiescent MSC exhibited enhanced differentiation capability over non-quiescent MSC ⁷, suggesting the possibility that inducing quiescence in MSC may potentiate their therapeutic effects.

In this study, we investigated whether or not inducing quiescence in adipose tissue-derived stromal cells (ADSC), one type of MSC most frequently studied for developing MSC-based cell therapies because of their strong immunomodulatory and anti-inflammatory functions, as well as easy access to the source, will bring beneficial effects on their cellular functions. Here, we provide evidence that quiescent ADSC cultured in 3-dimensional biocompatible gel showed potentiated anti-inflammatory functions in vitro and exhibited an extended dwell time in vivo as well, which led to accelerated diabetic wound healing. Furthermore, we present data that quiescence was associated with elimination of accumulated reactive oxygen species (ROS) from ADSC.

ADSC became quiescent when cultured in 3-dimensions (3-D) using biocompatible gel

Previous studies have shown that bone marrow-derived mesenchymal stem cells became quiescent when they were cultured on polyacrylamide gel whose stiffness matched that of bone marrow or adipose tissue (approximately 250 Pa) ⁷. To examine the possible clinical application of these quiescent mesenchymal stem cells induced by the stiffness of the substrate, it is prerequisite to culture mesenchymal stem cells in 3-D using biocompatible hydrogel with the stiffness of bone marrow or adipose tissue. To this end, we adopted 3D-NanoFibGrow-Ⅰ gel, a commercially available biocompatible hydrogel whose properties matched our specifications ⁸. The proliferative activities of adipose tissue-derived stromal cells (ADSC), one type of mesenchymal stem cells widely used in preclinical or clinical studies for cell therapies and regenerative medicine, were analyzed during and after 3-D culture in 3D-NanoFibGrow-Ⅰ gel. As shown in Figure 1, approximately 40% of ADSC incorporated 5-ethynyl 2´-deoxyuridine (EdU) when they were cultured for 48 hours on regular tissue culture plates made of plastic with EdU added to the medium for the last 24 hours. Thus, approximately 40% of ADSC went through a cell cycle during the culture period with EdU. Treating the cells on regular tissue culture plates with a mixture of tumor necrosis factor α (TNFα) and interferon γ (IFNγ) to mimic a pro-inflammatory environment in an ex vivo setting did not affect proliferation of ADSC. On the contrary, ADSC in 3D-NanoFibGrow-Ⅰ gel failed to incorporate EdU. However, once ADSC were retrieved from the gel after 24 hours of culture in gel and reseeded on regular tissue culture plates, cells resumed proliferation at a similar level to that of cells that remained on tissue culture plates for the entire period. These results suggest that ADSC can become quiescent in biocompatible hydrogel and are able to resume proliferation when they attach to stiff materials.

Quiescence enhanced anti-inflammatory functions of ADSC in vitro

ADSC are known to exert anti-inflammatory functions by secreting multiple factors ¹, which has formed the basis for attempting to develop ADSC-based cell therapies for treating severe autoimmune diseases and inflammatory diseases ⁹. Here, the effect of inducing quiescence in ADSC on their production of anti-inflammatory factors was investigated in vitro. To this end, secretion of indoleamine 2,3-dioxygenase (IDO) and interleukin-6 (IL-6), two molecules that have been reported to play a major role in anti-inflammatory functions of MSC ^10,11, was evaluated. ADSC were treated with a mixture of TNFα and INFγ, which mimics a pro-inflammatory environment in vitro and is known to stimulate IDO secretion by MSC ¹². As shown in Fig. 2A, non-quiescent ADSC seeded on tissue culture plates were stimulated to secrete IDO by TNFα/INFγ treatment. Surprisingly, quiescence markedly increased TNFα/INFγ-stimulated IDO secretion by ADSC. The effect of inducing quiescence in ADSC on their IL-6 secretion under oxidative stress was also investigated. To this end, cells were exposed to either hydrogen peroxide (H₂O₂) or high glucose (50 mM glucose). As shown in Fig. 2B, quiescence markedly increased IL-6 secretion by ASDC. Both hydrogen peroxide treatment and high glucose treatment significantly attenuated IL-6 secretion in non-quiescent ADSC on plastic tissue culture plates. Quiescent ADSC exhibited markedly higher IL-6 secretion than non-quiescent ADSC even under oxidative stress. Although hydrogen peroxide treatment slightly but significantly attenuated IL-6 secretion, high glucose treatment failed to affect IL-6 secretion in quiescent ADSC. These results suggest that quiescence may potentiate anti-inflammatory functions of ADSC in a pro-inflammatory environment, including diabetes.

Conditioned medium from quiescent ADSC, but not from non-quiescent ADSC, enhanced fibroblast migration during in vitro wound healing

Promotion of fibroblast migration by ADSC is known to play a role in wound healing ¹³, and impaired wound healing in diabetic patients is one of the unmet needs in tackling diabetes ¹⁴. We investigated whether or not conditioned medium from quiescent ADSC enhanced fibroblast migration under a diabetic condition in vitro. To this end, mouse embryonic fibroblasts (MEF) were subjected to a high glucose environment, and their ability to fill a scratch gap was analyzed. As shown in Fig. 3A-B, administration of conditioned medium derived from quiescent ADSC cultured under high glucose significantly stimulated gap closure after 48 hours. These results suggest that quiescent ADSC secrete factors that accelerate wound healing.

Transplantation of quiescent ADSC promoted diabetic wound healing

The effect of transplanting quiescent ADSC, which showed enhanced anti-inflammatory and fibroblast migration functions in vitro, on diabetic wounds in streptozotocin (STZ)-induced diabetic mice (STZ mice) was investigated. To this end, a 1 x 1 cm rectangular incised wound was made on the back of either control non-diabetic mice or STZ mice, and either 3D-NanoFibGrow-Ⅰ gel or ADSC with/without 3D-NanoFibGrow-Ⅰ gel was subcutaneously injected at least 1 cm away from the wound. The area of the wounds was measured for the next 10 days, as reported previously ¹⁵. As shown in Figure 4A, the quickest wound healing was observed in control mice without diabetes transplanted with gel. STZ mice transplanted with a mixture of 3D-NanoFibGrow-Ⅰ gel and ADSC, in which ADSC remain quiescent, exhibited significantly enhanced wound healing on Day 3 compared to STZ mice transplanted with either gel only or ADSC only, although no significant difference was observed in the area of the wounds between each group on Day 10. Thus, subcutaneous transplantation of quiescent ADSC may accelerate wound healing in diabetic mice.

Quiescent ADSC in gel remained at transplanted site for prolonged period

Rapid disappearance of transplanted MSC has been a challenge in establishing MSC-based cell therapies ^4,5. In fact, a previous study reported that less than 1% of subcutaneously injected MSC could be detected ³. To compare the fate of administered ADSC between non-quiescent ADSC without gel and quiescent ADSC embedded in gel in vivo, cells were labeled with Vybrant CM-DiI and traced after subcutaneous transplantation. As shown in Fig. 4B, 7 days after transplantation, DiI-labeled cells could not be detected in tissue isolated from the injected site for mice transplanted with ADSC without gel (ADSC only), which is a conventional method for subcutaneous transplantation. On the other hand, accumulation of DiI-labeled cells was apparent in mice transplanted with quiescent ADSC in gel (Gel + ADSC). These results suggest that prevention of loss of ADSC from the transplanted site by having quiescent ADSC in gel, as well as enhanced anti-inflammatory and fibroblast migration functions of quiescent ADSC, may have contributed to accelerated wound healing.

It is of note that immune cell infiltration, as well as fibrotic changes, was not apparent in both mice transplanted with non-quiescent ADSC without gel (ADSC only) and mice transplanted with quiescent ADSC in gel (Gel + ADSC) at least 7 days after subcutaneous injection. The lack of acute rejection despite transplanting human ADSC into C57BL/6 mice may be attributable to the immunomodulatory functions of ADSC.

Quiescence was associated with elimination of reactive oxygen species (ROS) in ADSC

It has been reported that elevated levels of ROS, such as those observed in aged or prematurely senescent mesenchymal stem cells, impair the therapeutic effect of these cells ¹⁶¹⁷. In fact, oxidative stress, which is expected to cause ROS accumulation ¹⁷, decreased IL-6 secretion by non-quiescent ADSC in vitro (Figure 2B), suggesting a potential role of elimination of ROS accumulation in the beneficial effects of quiescent ADSC. Thus, we investigated whether or not there is an association between quiescence and ROS levels. As shown in Figure 5, non-quiescent ADSC plated on plastic tissue culture plates exhibited ROS accumulation even under a normal glucose level (5.5 mM). Because of the technical difficulty of evaluating ROS levels in ADSC while they are in 3D-NanoFibGrow-I gel, quiescent ADSC were replated onto plastic tissue culture plates to visualize ROS. Interestingly, ADSC replated onto plastic tissue culture plates after maintaining quiescence in 3D-NanoFibGrow-Ⅰ gel lacked ROS accumulation. Thus, these results suggest that quiescence in ADSC is associated with elimination of accumulated ROS, which may be involved in the beneficial effects of quiescent ADSC in diabetic wound healing.

The immunomodulatory and anti-inflammatory functions of MSC, including ADSC, are strongly expected to exert therapeutic effects on a wide variety of age-related diseases ². However, it has been found that when they are exposed to an inflammatory environment for a prolonged period, MSC actually become pro-inflammatory instead ². SASP of MSC under an inflammatory environment is a serious challenge in clinical applications of MSC-based cell therapy, since affected tissues and organs are basically inflamed, which could be one of the reasons for insufficient efficacy of MSC-based cell therapies ¹⁸. In fact, non-quiescent ADSC cultured on plastic tissue culture plates exhibited stress-induced SASP (Figs. 2B and 3). However, inducing quiescence in those once non-quiescent cells successfully eliminated SASP even in the presence of stimuli that cause oxidative stress in ADSC. Therefore, regardless of preexisting SASP due to premature senescence, ADSC may be able to exert therapeutic effects for age-related diseases, including diabetes, once they become quiescent. Our results suggest that even ADSC isolated from patients, which are supposedly inflamed and showing SASP, could serve to treat inflammatory conditions, once they are converted to a quiescent state.

Conditioned medium from quiescent ADSC, which contains secretome and extracellular vesicles originated from these cells, effectively promoted fibroblast migration in vitro (Fig. 3), and transplanted quiescent ADSC in gel away from a wound accelerated diabetic wound healing (Fig. 4). It has been considered that the beneficial effects of MSC are attributable to their secretome and extracellular vesicles ⁹. Thus, therapeutic effects of quiescent ADSC can be expected, both by transplanting them in patients, and by administering the culture medium or its extracts to patients. It is also noteworthy that quiescent ADSC exhibited beneficial effects even under a diabetic condition (Figs. 2B, 3 and 4). Thus, quiescent ADSC-based cell therapies may provide solutions for diabetic complications even without the necessity of appropriate glycemic control, which may be sometimes difficult to achieve ¹⁹. Further in vivo and clinical research is necessary to explore these possibilities.

ADSC have been administered without a scaffold intravenously or locally (e.g. intramuscularly or intratracheally) in clinical studies to date. Our data suggest that quiescence may eliminate SASP from ADSC. Thus, if quiescent ADSC were to be used for disease treatment, ADSC might be administered with hydrogel as their scaffold in order to make ADSC quiescent. There are apparent advantages of using a scaffold; rapid disappearance of cells is one of the major challenges for present MSC-based cell therapies in which cells are administered without a scaffold ^3–5. To overcome this issue, a large amount of MSC has been used for each treatment, which necessitates vast ex vivo expansion of MSC. Furthermore, most intravenously injected MSC are trapped in lung capillaries, instead of targeting inflamed tissues/organs, which raises the concern of pulmonary embolism due to administration of a large amount of MSC. ADSC embedded in gel can be injected as shown in Fig. 4, which shows that ADSC persist in gel for a prolonged period. In fact, a substantial amount of ADSC in gel persisted locally for at least 7 days after transplantation when compared with ADSC without gel (Fig. 4B), which may explain accelerated wound healing in mice with quiescent ADSC in gel. Thus, loss of cells is not a concern, and administration of a large amount of ADSC may not be necessary, which should make ADSC-based cell therapies safer and easier to access for patients. One potential drawback of administering ADSC in hydrogel is that the inflamed tissues and organs in patients may not necessarily be in close proximity to the administered ADSC. Inflammation could even be systemic. Nevertheless, as shown in Fig. 4A, transplanting quiescent ADSC in hydrogel away from the wound site still exhibited beneficial effects, suggesting that ADSC embedded in hydrogel may be able to control inflammation in remote tissues and organs. However, further research is necessary to establish such a remote effect of quiescent ADSC.

In our previous study we demonstrated that bone marrow-derived mesenchymal stem cells became quiescent on the surface of 250 Pa polyacrylamide gel ⁷. In this study we investigated whether quiescent MSC have clinical relevance. To this end, we adopted 3D biocompatible commercially available soft gel as a substrate for ADSC. ADSC in 3D-NanoFibGrow-I gel, whose stiffness is similar to that of 250 Pa polyacrylamide gel ⁸, also exhibited reversible cell cycle arrest (Fig. 1) and apparently eliminated SASP due to premature senescence, which cells encountered before being embedded in 3D-NanoFibGrow-I gel (Figs. 2B and 3). Thus, it is safe to say that quiescent MSC on 2D soft polyacrylamide gel were successfully reproduced in ADSC cultured in 3-D biocompatible gel, which will make possible future studies to investigate whether quiescent ADSC have clinical relevance.

High glucose treatment causes both oxidative stress and hyperosmotic stress, both of which have been reported to induce cellular responses in MSC ²⁰. Thus, cellular responses caused by high glucose treatment could be attributable to either oxidative stress or hyperosmotic stress. However, non-quiescent ADSC exhibited attenuated IL-6 secretion, whereas quiescent ADSC showed resistance to high glucose treatment, which was reproduced in response to hydrogen peroxide treatment (Fig. 2B). Therefore, it is reasonable to consider that oxidative stress inhibits IL-6 secretion by non-quiescent ADSC, but quiescence markedly upregulates IL-6 secretion and makes ADSC resistant to oxidative stress in terms of IL-6 secretion.

Immune cell infiltration, as well as fibrotic changes, was not apparent at least up to 7 days after transplanting human ADSC into mice with a normal immune system (Fig. 4B), which was also true for the mice approximately 2 months after transplantation (data not shown). Thus, it is safe to say that the immunomodulatory functions of ADSC are maintained under quiescence. Also, 3D-NanoFibGrow-Ⅰ gel might be able to serve as a scaffold for ADSC for cell therapies in clinical settings. However, it is apparent that further studies are required to address these safety issues.

Although accelerated wound healing in STZ mice by quiescent ADSC in gel was apparent 3 days after transplantation, all mice including non-diabetic mice exhibited a similar level of wound healing 10 days after transplantation (Fig. 4A). Previous studies by others have also reported that STZ mice show completion of wound healing at the same time as non-diabetic control mice ¹⁵. Therefore, completion of wound healing eventually should be considered a limitation of using STZ mice, and initial acceleration during the healing process should be the endpoint in this diabetic wound model.

Non-quiescent ADSC on plastic tissue culture plates exhibited ROS accumulation, even when they were cultured with low glucose DMEM (5.5 mM glucose). However, these cells lost accumulated ROS despite being in a non-quiescent state, if they were once subjected to quiescence (Fig. 5). Thus, quiescence may be associated with elimination of accumulated ROS, which might explain the beneficial effects of quiescent ADSC. However, a causal relationship between quiescence and enhanced anti-inflammatory functions or elimination of SASP is not clear at this time and further research is required.

In conclusion, inducing quiescence in ADSC by embedding them in 3-D biocompatible gel accelerated diabetic wound healing presumably by eliminating SASP due to oxidative stress and by extending the dwell time of ADSC in vivo. Thus, application of quiescent ADSC in hydrogel may contribute to increasing the efficacy without compromising the safety of ADSC-based cell therapies.

Materials

Human ADSC and mouse embryonic fibroblasts (MEF) were purchased from Lonza (Basel, Switzerland). Nanofiber bacterial cellulose (3D-NanoFibGrow-Ⅰ) gels were purchased from Nano T-Sailing (Tokushima, Japan). Click-It EdU imaging kit and Vybrant CM-DiI Cell-Labeling Solution were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The ELISA kit for IL-6 was from R&D Systems (Minneapolis, MN, USA) and the ELISA kit for IDO was from Abcam (Cambridge, UK). C57BL/6 mice aged 8 weeks old were purchased from Charles River Laboratories (Wilmington, MA, USA). The ROS assay kit (Photo-oxidation Resistant DCFH-DA Dye) was from Dojindo (Kumamoto, Japan). All other chemicals were of analytical grade.

Cell culture

ADSC were maintained in low glucose Dulbecco’s modified medium (5.5 mM glucose) supplemented with 10% fetal bovine serum on tissue culture plastic tissue culture dishes. Mouse embryonic fibroblasts were maintained in high glucose Dulbecco’s modified medium (25 mM glucose) supplemented with 10% fetal bovine serum on tissue culture plastic tissue culture dishes. For high glucose treatment of ADSC, medium was switched to either high glucose Dulbecco’s modified medium (25 mM glucose) supplemented with 10% fetal bovine serum or glucose was added to the medium so that the final concentration reached 50 mM. 3D-NanoFibGrow-Ⅰ gel with/without ADSC were prepared according to the manufacturer’s instructions. Retrieval of ADSC from 3D-NanoFibGrow-Ⅰ gels was conducted by suspending a mixture of ADSC and gel with at least a 10 fold volume of phosphate buffered saline (PBS) and centrifuged at 300g for 5 minutes to sediment the cells but not the gel. Cells were resuspended in medium where approximately 70–80% of cells were recovered (data not shown).

Animal experiment

The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation at Tokushima University. All animal experiments were carried out in accordance with relevant guidelines and regulations and ARRIVE guidelines. At the end of each experiment mice were euthanized with overdose of isoflurane. For diabetic wound healing experiments, mice were intraperitoneally injected with either citrate buffer only (control) or citrate buffer containing STZ (200 mg/kg). Four weeks later, mice injected with STZ and with fasting blood glucose level higher than 200 mg/dl were considered to have developed diabetes. Control mice and STZ mice were subjected to a wound healing experiment followed by a subcutaneous injection of either 30 µl 3D-NanoFibGrow-Ⅰ plus 300 µl PBS or 3x10⁵ ADSC with/without 30 µl 3D-NanoFibGrow-Ⅰ plus 300 µl PBS in the back at least 1 cm away from the wound, as described in the text. For tracking ADSC after subcutaneous injection into mice, ADSC were labeled with Vybrant CM-DiI Cell-Labeling Solution (1 µg/ml) for 15 min at 37 ℃ according to the manufacturer’s instructions. Then 10⁶ cells alone or 10⁶ cells embedded in 30 µl 3D-NanoFibGrow-Ⅰ plus 300 µl PBS were subcutaneously injected into the back of mice. Five mice were used for each condition.

Proliferation Assay

Cells were incubated in the presence of EdU for 24 hours and EdU incorporation was visualized and compared with the images of Hoechst 33342 staining, which visualized nuclei, according to the manufacturer’s instructions.

Scratch Assay

To obtain conditioned medium, ADSC were first cultured with high glucose DMEM (25 mM glucose) supplemented with 10% FCS for 24 hours. Then 10⁴ ADSC with/without 3D-NanoFibGrow-Ⅰ gels were placed in each well of a 24-well tissue culture plate and their media were retrieved after an additional 24 hours of culture. Then, 5 x 10⁴ MEFs were seeded in each well of a 24-well tissue culture plate and incubated for 24 hours. A scratch was made in the monolayer of MEFs using a 1000 µl tip. To monitor the size of scratches as a function of time at the same locations, three marks were made at the bottom of each well. Cells were washed with medium to remove cellular debris and treated with either medium or conditioned medium derived from ADSC. Three samples were prepared for each condition. Thus, 9 images for each condition were taken at the marked areas at each time point. Wound area was measured using Image J.

Total ROS assay

ADSC were cultured overnight with low glucose DMEM supplemented with 10% FCS either on plastic tissue culture plates or in 3D-NanoFibGrow-Ⅰ gels. Cells in gels were then retrieved and reseeded onto plastic tissue culture plates. Six hours later, when cells fully adhered to the plates, cells were fixed and ROS accumulated in them were visualized using a ROS assay kit according to the manufacturer’s instruction.

ELISA (IDO, IL-6)

First, 10⁴ ADSC were either directly seeded on the bottom of a plate and cultured in 200 µl medium or embedded in 20 µl 3D-NanoFibGrow-Ⅰ plus 200 µl medium in each well of a 96-well tissue culture plastic plate. Cells were treated as described in the Figure Legends. Then, 100 µl supernatant was collected from each sample, centrifuged at 1000g to remove cellular debris, and mixed with 100 µl fresh medium, which was then subjected to ELISA according to the manufacturer’s instructions.

Histological analysis

Mice were sacrificed, and tissue at the injected site of ADSC with/without 3D-NanoFibGrow-Ⅰ gel was isolated and fixed in 4% phosphate-buffered paraformaldehyde for 24 hours at room temperature. Then tissue sections were stained with hematoxylin and eosin (H&E) and subjected to either bright-field microscopy or fluorescence microscopy.

Imaging

Staining ADSC with Vybrant CM-DiI was carried out according to the manufacturer’s instructions. H&E staining was conducted at the Support Center for Advanced Medical Sciences, Tokushima University Graduate School of Biomedical Sciences. All images were obtained with a Keyence BZ-X800 microscope (Osaka, Japan). Images for each sample were obtained in at least 5 randomly selected fields.

Statistical analysis

P-values were calculated using one-way ANOVA with post-hoc Tukey test; values of p < 0.05 were considered significant.

Data availability statement

The data used to support the findings of this study are included within the article and are available from the corresponding author upon reasonable request.

MSC; mesenchymal stem cell, ADSC; adipose-derived stromal cell, MEF; mouse embryonic fibroblast, PBS; phosphate buffered saline, DMEM; Dulbecco’s Modified Eagles Medium, FCS; fetal calf serum, TNF; tumor necrosis factor, IFN; interferon, 2-D; two-dimensional, 3-D; three-dimensional, Pa; pascal, H&E; hematoxylin and eosin, SASP; senescence-associated secretory phenotype, IDO; indoleamine 2,3-dioxygenase, IL-6; interleukin-6, EdU; 5-ethynyl 2´-deoxyuridine, STZ; streptozotocin, ROS; reactive oxygen species.

Acknowledgements

This study was supported by the Support Center for Advanced Medical Sciences, Tokushima University Graduate School of Biomedical Sciences. This study was funded by a grant from Tokushima Prefecture and by Mechanogenic, Inc.

Author contributions

MF designed and supervised the study, carried out experiments and wrote the paper. AH, MY, NM and AT conducted animal experiments. TI, YU, MA and HY conducted histological testing, which was evaluated by HO and TO.

Competing interests

MF is the CEO of Mechanogenic, Inc, which funded this study.

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Competing interest reported. MF is the CEO of Mechanogenic, Inc, which funded this study. No other authors has competing interests.

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Quiescent adipose tissue-derived stromal cells in mechanically compliant hydrogel accelerate diabetic wound healing through enhanced functions and extended dwell time

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Abstract

Figures

Introduction

Results

ADSC became quiescent when cultured in 3-dimensions (3-D) using biocompatible gel

Transplantation of quiescent ADSC promoted diabetic wound healing

Quiescent ADSC in gel remained at transplanted site for prolonged period

Quiescence was associated with elimination of reactive oxygen species (ROS) in ADSC

Discussion

Materials and methods

Materials

Cell culture

Animal experiment

Proliferation Assay

Scratch Assay

Total ROS assay

ELISA (IDO, IL-6)

Histological analysis

Imaging

Statistical analysis

Data availability statement

Abbreviations

Declarations

References

Additional Declarations

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