Exploration of the importance of viable cells in skin micrograft for wound healing

doi:10.21203/rs.3.rs-3678387/v1

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Exploration of the importance of viable cells in skin micrograft for wound healing

https://doi.org/10.21203/rs.3.rs-3678387/v1

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Background:

Skin micrograft (MG) has been reported to promote tissue regeneration and is clinically used for intractable wounds. MG contains diverse cells and an extracellular matrix (ECM); however, the importance of the proliferative potential of MG cells for wound healing is not clear. Therefore, we investigated the wound healing-promoting effect of MG, in which the cells were inactivated using several methods.

Methods:

Skin MG was prepared from the skin of C57BL mice using Rigenera protocol, and then four types of MGs were prepared: Fresh-MG, HHP-MG (treated with high hydrostatic pressure of 200MPa for 10 min), LN-MG (treated with liquid nitrogen for 20 min), and Heat-MG (treated heat with 80℃ for 30 min). The outgrowth potential, growth factor content (basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF)), and in vitro cell proliferation-promoting potential were examined for each type of MG. For in vivo studies, MG was used to treat murine skin defects. The wound area, neoepithelial length, granulation tissue formation, and newly formed capillaries were evaluated on day 7.

Results:

All types of MGs, except for Heat-MG, contained bFGF, EGF and VEGF, promoted cell proliferation, and accelerated wound closure. In addition, the neoepithelial lengths in the HHP-MG and LN-MG groups were significantly longer than those in the Heat-MG and control groups. The area of granulation tissue, and the number and area of capillaries in the Fresh-MG, HHP-MG, and LN-MG groups were significantly larger than those in the control groups.

Conclusions:

The results confirmed that proliferative cells are not essential for the promotion of wound healing by MG. This effect was attenuated in the Heat-MG group, suggesting that soluble factors, such as growth factors, which are inactivated by heat, play an important role.

Autologous micrograft

Rigenera protocol

High hydrostatic pressure

Viable cells

Inactivation

Wound healing

The autologous micrograft (MG) technique was first described by Reverdin in 1869. Subsequently, the Rigenera protocol[1] was reported as an advanced method for producing MG using a specific disposable medical device named Rigeneracons^® (Human Brain Wave Srl., Via Pinerolo, Torino, Italy). Rigeneracons^® is able to disaggregate a small piece of tissue into microtissues approximately ≤50 µm in size[2,3]. Skin MG, obtained following the Rigenera protocol, is regarded as an emerging therapy for skin injuries[4–9]. Some studies have shown that skin MG improves wound healing[4–7] and reduces scar formation[2,7]. Growth factors, tissue cells, and extracellular matrix (ECM) are thought to be the drivers[5,10,11] by which MG promotes tissue regeneration. Among these, the presence of stem cells is considered a key factor in the effectiveness of MG¹[12]. However, the mechanism of action of MG is controversial, and the importance of the proliferative potential of MG cells in wound healing remains unclear.

Therefore, in the present study, the effect of proliferative MG cells on the promotion of cell proliferation and wound healing was investigated, using MGs inactivated by high hydrostatic pressurization (HHP), liquid nitrogen (LN), or heat.

Preparation of MGs

C57BL/6JJcl mice (male, 8–9 weeks old) (CLEA Japan, Inc., Osaka, Japan) were used to prepare skin MG. Enough skin biopsies (8 mm in diameter) were harvested using an 8 mm punch (Kai Industries Co., Ltd., Tokyo, Japan) in equal amounts from three mice after euthanasia by carbon dioxide gas inhalation. The skin biopsies were allocated to the following four groups: Fresh, HHP, LN and Heat groups (each group has 12 skin biopsies). For the HHP treatment, plastic bags with skin biopsies were immersed in transmission fluid in the chamber of a pressurization machine (HPV-50C20-S-1; Sugino Machine Limited, Toyama, Japan) and pressurized at 200 MPa for 10 min. The LN group was frozen in LN for 20 min, and the Heat group was heated at 80℃ in a hot water bath for 30 min to inactivate the cells contained in the skin samples.

Skin MG was generated using the Rigenera protocol according to our previous study[3]. Briefly, two sheets of skin biopsy (φ8mm) (Fresh, HHP, LN or Heat group) were cut into small pieces using scissors, placed into Rigeneracons® with 1 mL saline solution (Otsuka Pharmaceutical Factory, Inc., Tokyo, Japan), and then processed for 2 min. Finally, we prepared four types of skin MGs: Fresh-MG, HHP-MG, LN-MG, and Heat-MG. The above process was performed three times (n = 3), and the MGs were used for the following experiments.

Cell proliferation assay of MGs

Cell proliferation was tested to evaluate the viability of cells in each MG group. Six additional skin samples were cut into small pieces and used as the positive control. Three milliliters of each MG solution (Fresh-, HHP-, LN-, and Heat-MG) was centrifuged at 1,000 rpm for 10 min, and MG was collected as the pellet at the bottom of the tube. Three milliliters of collagenase type I solution (0.2% w/v) was added to the MG in each group and the control skin sample, and incubated for 30 min at 37℃ on a shaker to dissolve the ECM and isolate cells in MG and skin. High-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (FUJIFILM Wako) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% antibiotic-antimycotic (anti-anti) (Gibco) was prepared. DMEM was added to stop the enzyme reaction and the cell suspension was filtered through a 100 µm cell strainer (Falcon, USA). After centrifugation at 1,000 rpm for 10 minutes, the supernatant was removed and resuspended in 3 mL of DMEM. Finally, the cell suspension was seeded in a total of 3 wells of a 12-well plate at 1 mL each. Seven days later, the number of cultured cells was evaluated using the WST-8 assay (Cell Counting Kit-8; Dojindo, Kumamoto, Japan), according to the manufacturer's protocol. Briefly, 0.1 mL of WST-8 solution was added in each well (n = 5) and incubated at 37℃ for 2 h, then the absorbance at 450 nm was measured using a microplate reader (Varioskan Flash, Thermo Fisher Scientific Inc., Tokyo, Japan).

The amount of growth factors contained in MGs

The levels of bFGF, EGF, and VEGF in the MGs were evaluated using enzyme-linked immunosorbent assay (ELISA). The MG solutions (Fresh-, HHP-, LN-, and Heat-MG) (n = 3) were centrifuged at 1,000 rpm for 10 min. Then, the supernatants were collected and stored at − 80 ℃ for the following experiments.

Samples (Fresh-, HHP-, LN-, and Heat-MG) were thawed and the amounts of bFGF, EGF, and VEGF were evaluated using the ELISA bFGF kit (MFB00; R&D Systems), EGF kit (MEG00; R&D Systems), and VEGF kit (MMV00-1; R&D Systems), respectively, according to the manufacturer’s instructions. Each sample was analyzed in duplicate.

Promotion of cell proliferation by MGs

To evaluate the effect of MGs on the proliferation of cells, NIH3T3 cells (JCRB0615; provided by the Health Science Research Resources Bank, Osaka, Japan) were used. The NIH3T3 cells were cultured in DMEM at 37°C in a water-saturated atmosphere of 95% air and 5% CO₂.

One day before MG administration (day − 1), 3T3 cell suspension (2.0 × 10⁴ cells/mL) with DMEM (1%FBS་1% anti-anti) was prepared and seeded in 12-well plates at 1 mL each. After culturing overnight (on day 0), 0.3 mL of skin MG suspension (Fresh-, HHP-, LN-, or Heat-MG; n = 5) was added to a cell insert that was placed in a well of the 12-well plate. Three independent plates were prepared simultaneously for each group to access cell proliferation on days 1, 2 and 3, respectively. No cell inserts were placed in the control group. On day 1, the medium was changed with DMEM (1%FBS་1% anti-anti). After 1, 2, and 3 days, the cell inserts were removed and cell proliferation was evaluated using the WST-8 assay.

Inhibition of the effect of bFGF, EGF, and VEGF in MGs

We investigated whether the inhibition of bFGF, EGF, and VEGF receptors cancelled the cell growth-promoting effects of the MGs. Three inhibitors were prepared: an FGF receptor inhibitor (FGFRI) (Infigratinib (Selleck)), an EGF receptor inhibitor (EGFRI) (Erlotinib (Selleck)), and a VEGF receptor inhibitor (VEGFRI) (Regorafenib (Selleck)). The concentration of the inhibitor was determined according to pre-experiments, which was the maximum dose that did not affect cell activity after two days of culture (Fig. 2B).

A 3T3 cell suspension (2.0 × 10⁴ cells /mL) with DMEM (1%FBS་1% anti-anti) was seeded in 12-well plates at 1 mL each. After culturing overnight, 0.1 µM FGFRI, 0.01 µM EGFRI, 0.01 µM VEGFRI, and the mixture of the three was added, respectively. After 1 h, a cell insert (Falcon) with 0.3 mL Fresh-MG was placed in a well of a 12-well plate (n = 5). After culturing for 2 days, the WST-8 assay was used to evaluate cell proliferation. FGFRI strongly inhibited the promotion of cell growth by Fresh-MG compared with EGFRI and VEGFRI. Therefore, the effects of FGF inhibition on Fresh-, HHP-, LN-, and Heat-MG in promoting cell proliferation were subsequently evaluated by adding FGFRI according to the above procedure.

Application of MGs on murine skin defects

Thirty C57BL/6JJcl mice (male, 8–9 weeks old) were individually housed and maintained on a 12-h light/dark cycle in a temperature-controlled animal facility. On the day before surgery, the hair on the back of each mouse was shaved and depilated using a depilation cream. Spontaneously breathing mice were anesthetized using isoflurane (Pfizer Inc., Tokyo, Japan) during all painful procedures. The isoflurane concentration was kept at 1.5–2% to provide an appropriate level of anesthesia.

MG was combined with a collagen sponge (Pelnac Gplus) to apply MG stably to the wound surface, according to our previous report[3]. Briefly, 0.1 mL of MG suspension (Fresh-, HHP-, LN-, or Heat-MG) was added to a sheet of Pelnac Gplus (8 mm in diameter) and allowed to settle for 60 minutes. The mice were allocated to five groups (Fresh-, HHP-, LN-, and Heat-MG, and control; n = 6), and a full-thickness skin defect (8 mm in diameter) was created on the back of each mouse. In the four MG groups, a sheet of Pelnac Gplus impregnated with MG was applied to the skin defects with the silicon layer facing upward. The sheet was then sutured with the surrounding skin and covered with a silicone-faced wound dressing (SI Aid-Mesh, ALCARE Co., Ltd., Tokyo, Japan). Finally, a bandage and surgical tape (Silkytex, ALCARE Co., Ltd.) were used to prevent contamination and mechanical stress.

Evaluation of the effect of MGs on wound healing

Wound healing was evaluated on day 7 after surgery. The mice were euthanized by carbon dioxide gas inhalation. The wounds were photographed using a digital camera and harvested along with the surrounding tissue. The specimens were fixed in a 10% formalin buffer solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), embedded in paraffin, and sectioned axially in the central area of each wound. The sections were subsequently subjected to hematoxylin and eosin (HE) staining, Azan staining, and immunohistochemical staining for the anti-CD31 antibody.

For anti-CD31 staining, sections were deparaffinized and rehydrated, and heat-induced antigen retrieval was performed using ethylenediaminetetraacetic acid (EDTA) (Nichirei Biosciences Inc., Tokyo, Japan) at 98°C for 20 min. A rabbit monoclonal antibody (code: ab182981, Abcam plc., Cambridge, UK) and polymer reagent (simple stain mouse MAX PO; Nichirei Biosciences Inc., Tokyo, Japan) were used as primary and secondary antibodies, respectively.

The remaining wound area was evaluated in photographs using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Neoepithelial length was measured on HE-stained sections, which was defined as the length from the nearest hair follicle at the wound edge to the end of the epithelium. The area of the newly formed granulation tissue in the wound was measured using Azan-stained sections.

Newly formed capillaries were measured in the anti-CD31-stained sections[13]. Capillaries were detected in the area of newly formed granulation, including the remaining material. A threshold was set for the brown stained tint, and then the regions above this threshold were counted and measured as the number and area of capillaries using BZ-X800 Analyzer software (Keyence Corp., Osaka, Japan).

Statistical analysis

All data are presented as mean ± standard deviation. We used the one-way analysis of variance with the least significant difference analysis between multiple groups. All statistical analyses were performed using IBM® SPSS® Statistics version 29. Statistical significance was set at p < 0.05.

Cell proliferation from the skin MGs

No cell proliferation was observed in the MG of any group (Fresh-, HHP-, LN-, and Heat-MG), whereas stable cell proliferation was observed in normal skin as a positive control after 7 days of culturing the collagenase-treated MG and normal skin (Fig. 1A). The results of the WST-8 assay were also consistent with this observation (Fig. 1B).

Promotion of 3T3 cell proliferation by MGs

The absorbance in the Fresh-, HHP-, and LN-MG groups was significantly higher than that in the Heat-MG and control groups on day 3, indicating that the three types of MGs (Fresh-, HHP- and LN-MG) promoted the proliferation of 3T3 cells, whereas Heat-MG did not promote proliferation at all (Fig. 2A).

The maximum concentration of the inhibitor, which did not affect cell activity after two days of culture, was 0.1 µM FGFRI, 0.01 µM EGFRI, and 0.01 µM VEGFRI, respectively. (Fig. 2B).

The promotional effect of Fresh-MG on cell proliferation was partly inhibited by the use of FGFRI, EGFRI, VEGFRI, or a mixture of all three inhibitors (Fig. 2C). In addition, the stimulatory effects of Fresh-, HHP-, and LN-MG on cell proliferation were inhibited by the FGFRI (Fig. 2D).

The amount of growth factors contained in MGs

The supernatants of Fresh-, HHP- and LN-MG contained bFGF, EGF and VEGF, whereas none were detected in Heat-MG. (Fig. 3)

The remaining wound area

The remaining wound areas in the control and Heat-MG groups were similar, indicating that Heat-MG does not accelerate wound closure. In addition, the wound areas in the Fresh-, HHP-, and LN-MG groups were significantly smaller than those in the Heat-MG group, indicating that except for Heat-MG, the other three MGs promoted wound area reduction (Fig. 4).

The neoepithelial length and granulation tissue formation

The neoepithelial lengths in the HHP- and LN-MG groups were significantly longer than those in the Heat-MG and control groups (Fig. 5A, B). The areas of newly formed granulation tissue in the Fresh-, HHP-, and LN-MG groups were significantly larger than that in the control group (Fig. 6A, B).

Histological assessment of newly formed capillaries

The number and areas of newly formed capillaries in the Fresh-, HHP-, and LN-MG groups were significantly larger than those in the control group (Fig. 7B, C).

Autologous skin MG following the Rigenera protocol is widely used in clinical practice and provides positive therapeutic outcomes, such as improving dehisced wounds[4,6], burn wounds[8,9], and full-thickness skin defects[7] and reducing scar formation[2,7]. The Rigenera protocol allows the extraction of MG smaller than 50 µm from dermal tissue without any enzymatic manipulation[14]. The generated MG contains all the skin tissue components, including tissue cells, various tissue factors, and ECM, which are beneficial for wound healing[15–17].

Several studies have explored the mechanism of action of autologous MG[2,10,11,18,19] on wound healing. MG may contain not only different cell types from different tissues (connective tissue, epidermis, hair root, vessels, glands, and so on), but also skin-originated stem cells, including epithelial stem/progenitor and mesenchymal stem cells. Grafting cells have been reported to be involved in wound healing, and thus the cells in MG are thought to have an important role in promoting regeneration[20]. Some authors insist that the cells contained in implanted MG, especially stem cells, secrete cytokines and growth factors, which act on surrounding cells in a paracrine fashion[21], interacting with the ECM and growth factors to promote tissue generation and regeneration[5,22]. In contrast, Jimi et al.²[20] showed that grafted cells could not directly participate in granulation tissue formation and Balli et al.³[19] reported that MG without cells significantly accelerated cell migration and scratch wound closure. Thus, the hypothesis that the viable cellular components in the MG play a crucial role is controversial. Therefore, we investigated whether the presence of viable cells was essential for the efficacy of MG.

When the MG was cultured after treatment with collagenase solution, no cell proliferation was observed, suggesting that the proliferative capacity of the cells in the MG produced by the Rigenera protocol may have been reduced. Additionally, to eliminate the viability of cells in the MG, we used three different inactivation methods: HHP, LN and Heat.

The HHP technique has been applied for biological tissue inactivation and decellularization[23,24]. HHP at 200 MPa for 10 min inactivates cells by disturbing the inter- and intramolecular interactions of the tertiary and quaternary structures of proteins[25,26]. We previously applied 200 MPa of HHP to inactivate the skin and found that all cells contained in the skin were inactivated with no damage to the dermal component[27–29]. All cells in the skin were inactivated, and the structure of the ECM, the intact basal membrane, and the activity of growth factors were preserved[23,29]. In LN treatment, freezing devitalizes cells by inducing ice crystal formation and cell dehydration. Only one cycle of 20 minutes freezing at -196°C with LN was sufficient to ensure cell death[30]. It has been reported that protein activity is well preserved following LN treatment.

We also heated the skin to inactivate the cells. Thermal processing is a well-established sterilization method known as pasteurization, which is used to destroy pathogenic microorganisms in certain foods and beverages. When the skin is heated to temperatures above 65°C, irreversible structural changes occur[31]. It inactivates not only the living cells but also protein components to spoil the effect of MG. Furthermore, the majority of ECM and growth factors were inactivated after heating at 80°C for 30 min[32].

Except for the Heat-MG, the other three MGs promoted 3T3-cell proliferation. We confirmed that Fresh-, HHP-, and LN-MG contained bFGF, EGF, and VEGF. Inhibition of cellular utilization of bFGF, EGF, and VEGF weakened the proliferation effect of Fresh-, HHP-, and LN-MG. MG, which consists of a pool of growth factors, carries bioactive molecules that promote the migration of fibroblasts and keratinocytes[19]. Our results indicate that bFGF, EGF, and VEGF play crucial roles in the pool of growth factors of MG, and Fresh-, HHP-, and LN-MG have similar effects on cell proliferation, suggesting that MGs without viable cells have an equivalent effect as the Fresh-MG in vitro. In in vivo experiments using mouse skin defect wounds, HHP- and LN-MG promoted epithelialization, granulation, and angiogenesis, and accelerated wound area reduction, whereas Heat-MG lost these effects. This indicates that MG can exert its wound-healing effect even without viable cells and its effect is dependent on substances that are inactivated by heat.

As a limitation, the mechanisms by which Fresh-, HHP-, and LN-MG promote cell proliferation and wound healing remain unclear. Besides, the effect of cellular remnants in inactivated MG on wound healing remains to be explored in the future.

Considering that the mechanism of MG efficacy does not require viable cells, this opens up the possibility of a wide variety of MG sources, including MG of allogeneic origin, cryopreserved MG, and lyophilized MG. In particular, among several cell inactivation methods, HHP has the advantage of inactivating all the cells uniformly inside the MG in a short time, preserving protein properties. Allogeneic MG inactivated by HHP treatment could be a new modality for promoting wound healing that is convenient and easy to use.

By comparing the amounts of growth factors and their effects on cell proliferation and wound healing in four types of MGs (Fresh-, HHP-, LN- and Heat-MG), we investigated whether the presence of viable cells in the MGs is essential. The results showed that MG promoted the proliferation of 3T3 cells and accelerated wound healing even without viable cells; this effect was attenuated by FGFRI, EGFRI, and VEGFRI, and was canceled by heat treatment. This suggests that the proliferative potential of the contained cells is not essential for the effectiveness of MG and that factors that can be inactivated by heat treatment, such as each growth factor, play an important role. Thus, MG without viable cells may represent a new therapeutic modality.

MG: Micrograft

bFGF: Basic fibroblast growth factor

EGF: Epidermal growth factor

VEGF: Vascular endothelial growth factor

ECM: Extracellular matrix

HHP: High hydrostatic pressure

LN: Liquid nitrogen

FGFRI: FGF receptor inhibitor

EGFRI: EGF receptor inhibitor

VEGFRI: VEGF receptor inhibitor

HE: Hematoxylin and eosin

Ethics approval and consent to participate

All animal experiments were conducted at Kyoto University, and the protocol was approved by the Animal Research Committee of Kyoto University Graduate School of Medicine (permit number: Med Kyo 22122). Title of the approved project: Investigation of the wound healing promoting effects. Date of approval: March 16th, 2022.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Conflict of interest

All authors declare no conflicts of interest in association with the present study.

Funding

This research was supported by the Translational Research Network Program (Seeds A) of the Japan Agency for Medical Research and Development (AMED) under grant number JP21lm020300.

Authors' contributions

Y.L. performed most of the experiments, analyzed the data, and wrote the manuscript. M.S., Q.Z., H.D., T.N., E.S., H.Y., and I.T. contributed to data analysis and edited the manuscript. M.S. and N.M. contributed to experimental design and approved the final version of the manuscript.

Acknowledgements

This work was partially supported by JSPS KAKENHI (grant number 21H03099). Yuanjiaozi Li was supported by JST SPRING (grant number JPMJSP2110).

Trovato L, Monti M, del Fante C, Cervio M, Lampinen M, Ambrosio L, et al. A new medical device rigeneracons allows to obtain viable micro-grafts from mechanical disaggregation of human tissues. J Cell Physiol. 2015;230:2299–303. https://doi.org/10.1002/jcp.24973.
Svolacchia F, De Francesco F, Trovato L, Graziano A, Ferraro GA. An innovative regenerative treatment of scars with dermal micrografts. J Cosmet Dermatol. 2016;15:245–53. https://doi.org/10.1111/jocd.12212.
Li Y, Sakamoto M, Sawaragi E, Nakano T, Katayama Y, Yamanaka H, et al. Comparison of wound healing effect of skin micrograft impregnated into two kinds of artificial dermis in a murine wound model. Plast Reconstr Surg - Glob Open. 2022;10:e4636. https://doi.org/10.1097/GOX.0000000000004636.
Baglioni E, Trovato L, Marcarelli M, Frenello A, Bocchiotti MA. Treatment of oncological post-surgical wound dehiscence with autologous skin micrografts. Anticancer Res. 2016;36(3):975–9.
De Francesco F, Graziano A, Trovato L, Ceccarelli G, Romano M, Marcarelli M, et al. A regenerative approach with dermal micrografts in the treatment of chronic ulcers. Stem Cell Rev Rep. 2017;13:139–48. https://doi.org/10.1007/s12015-016-9692-2.
Marcarelli M, Trovato L, Novarese E, Riccio M, Graziano A. Rigenera protocol in the treatment of surgical wound dehiscence. Int Wound J. 2017;14:277–81. https://doi.org/10.1111/iwj.12601.
Riccio M, Marchesini A, Zingaretti N, Carella S, Senesi L, Onesti MG, et al. A multicentre study: the use of micrografts in the reconstruction of full-thickness posttraumatic skin defects of the limbs—a whole innovative concept in regenerative surgery. Stem Cells Int. 2019;2019:1–10. https://doi.org/10.1155/2019/5043518.
Andreone A, De Hollander D. Case Report A case report on the effect of micrografting in the healing of chronic and complex burn wounds. Int J Burns Trauma. 2020;10(1):15–20.
Yamamoto Y, Fujihara H, Kirita M, Soejima K. Micronized dermal grafts (Rigenera™) and split-thickness skin grafts alone or in combination for deep dermal burn wounds. Burn Open. 2022;6:212–7. https://doi.org/10.1016/j.burnso.2022.11.001.
Jimi S, Kimura M, De Francesco F, Riccio M, Hara S, Ohjimi H. Acceleration mechanisms of skin wound healing by autologous micrograft in mice. Int J Mol Sci. 2017;18:1675. https://doi.org/10.3390/ijms18081675.
Trovato L, Naro F, D’Aiuto F, Moreno F. Promoting tissue repair by micrograft stem cells Delivery. Stem Cells Int. 2020;2020:1–2. https://doi.org/10.1155/2020/2195318.
Astarita C, Trovato L, Graziano A. Suitability of a progenitor cell-enriching device for in vitro applications. Coatings. 2021;11:636. https://doi.org/10.3390/coatings11060636.
Pusztaszeri MP, Seelentag W, Bosman FT. Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues. J Histochem Cytochem. 2006;54:385–95. https://doi.org/10.1369/jhc.4A6514.2005.
Tresoldi MM, Graziano A, Malovini A, Faga A, Nicoletti G. The role of autologous dermal micrografts in regenerative surgery: a clinical experimental study. Stem Cells Int. 2019;2019:1–8. https://doi.org/10.1155/2019/9843407.
Baghaban Eslaminejad M, Malakooty Poor E. Mesenchymal stem cells as a potent cell source for articular cartilage regeneration. World J Stem Cells. 2014;6:344–54. https://doi.org/10.4252/wjsc.v6.i3.344.
Jeremias TdaS, Machado RG, Visoni SBC, Pereima MJ, Leonardi DF, Trentin AG. Dermal substitutes support the growth of human skin-derived mesenchymal stromal cells: Potential tool for skin regeneration. PLoS ONE. 2014;9:e89542. https://doi.org/10.1371/journal.pone.0089542.
Huang H, Liu J, Hao H, Tong C, Ti D, Liu H, et al. G-CSF administration after the intraosseous infusion of hypertonic hydroxyethyl starches accelerating wound healing combined with hemorrhagic shock. Biomed Res Int. 2016;2016:5317630. https://doi.org/10.1155/2016/5317630.
Balli M, Chui JS-H, Athanasouli P, Abreu de Oliveira WA, El Laithy Y, Sampaolesi M, et al. Activator protein-1 transcriptional activity drives soluble micrograft-mediated cell migration and promotes the matrix remodeling machinery. Stem Cells Int. 2019;2019:1–19. https://doi.org/10.1155/2019/6461580.
Balli M, Vitali F, Janiszewski A, Caluwé E, Cortés-Calabuig A, Carpentier S, et al. Autologous micrograft accelerates endogenous wound healing response through ERK-induced cell migration. Cell Death Differ. 2020;27:1520–38. https://doi.org/10.1038/s41418-019-0433-3.
Jimi S, Takagi S, De Francesco F, Miyazaki M, Saparov A. Acceleration of skin wound-healing reactions by autologous micrograft tissue suspension. Med (Kaunas). 2020;56:321. https://doi.org/10.3390/medicina56070321.
Cheng AS, Yau TM. Paracrine effects of cell transplantation: strategies to augment the efficacy of cell therapies. Semin Thorac Cardiovasc Surg. 2008;20:94–101. https://doi.org/10.1053/j.semtcvs.2008.04.003.
Schultz GS, Wysocki A. Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen. 2009;17:153–62. https://doi.org/10.1111/j.1524-475X.2009.00466.x.
Boonyaratanakornkit BB, Park CB, Clark DS. Pressure effects on intra- and intermolecular interactions within proteins. Biochim Biophys Acta - Protein Struct Mol Enzymol. 2002;1595:235–49. https://doi.org/10.1016/S0167-4838(01)00347-8.
Li Y, Katayama Y, Nie I, Nakano T, Sawaragi E, Sakamoto M, et al. Development of a novel regenerative therapy for malignant bone tumors using an autograft containing tumor inactivated by high hydrostatic pressurization (HHP). Regen Ther. 2023;22:224–31. https://doi.org/10.1016/j.reth.2023.02.002.
Adkins I, Hradilova N, Palata O, Sadilkova L, Palova-Jelinkova L, Spisek R. High hydrostatic pressure in cancer immunotherapy and biomedicine. Biotechnol Adv. 2018;36:577–82. https://doi.org/10.1016/j.biotechadv.2018.01.015.
Le TM, Morimoto N, Ly NTM, Mitsui T, Notodihardjo SC, Munisso MC, et al. Hydrostatic pressure can induce apoptosis of the skin. Sci Rep. 2020;10:17594. https://doi.org/10.1038/s41598-020-74695-5.
Matsuura Y, Sakamoto M, Ogino S, Arata J, Morimoto N. Inactivated nevus tissue with high hydrostatic pressure treatment used as a dermal substitute after a 28-day cryopreservation period. Biomed Res Int. 2021;2021:1–9. https://doi.org/10.1155/2021/3485189.
Jinno C, Morimoto N, Mahara A, Liem PH, Sakamoto M, Ogino S, et al. Inactivation of human nevus tissue using high hydrostatic pressure for autologous skin reconstruction: a novel treatment for giant congenital melanocytic nevi. Tissue Eng Part C Methods. 2015;21:1178–87. https://doi.org/10.1089/ten.tec.2015.0054.
Morimoto N, Jinno C, Mahara A, Sakamoto M, Kakudo N, Inoie M, et al. The alteration of the epidermal basement membrane complex of human nevus tissue and keratinocyte attachment after high hydrostatic pressurization. Biomed Res Int. 2016;2016:1–9. https://doi.org/10.1155/2016/1320909.
Yamamoto N, Tsuchiya H, Tomita K. Effects of liquid nitrogen treatment on the proliferation of osteosarcoma and the biomechanical properties of normal bone. J Orthop Sci. 2003;8:374–80. https://doi.org/10.1007/s10776-002-0626-3.
Allenby AC, Fletcher J, Schock C, Tees TFS. The effect of heat, pH and organic solvents on the electrical impedance and permeability of excised human skin. Br J Dermatol. 1969;81:31–9. https://doi.org/10.1111/j.1365-2133.1969.tb16059.x.
Klamroth R, Gröner A, Simon TL. Pathogen inactivation and removal methods for plasma-derived clotting factor concentrates. Transfusion. 2014;54:1406–17. https://doi.org/10.1111/trf.12423.

Refer to " Suitability of a progenitor cell-enriching device for in vitro applications. " by Antonio Celentano for a more detailed discussion of this concept, published in Coatings in 2021;11:636.
See " Acceleration of skin wound-healing reactions by autologous micrograft tissue suspension" by Shiro Jimi for a more in-depth discussion of this concept, published in Medicina (Kaunas) in 2020;56:321.
See " Autologous micrograft accelerates endogenous wound healing response through ERK-induced cell migration" by Martina Balli for a detailed exploration of this concept, published in Cell Death Differ in 2020;27:1520-38

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Exploration of the importance of viable cells in skin micrograft for wound healing

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Background

Materials and methods

Preparation of MGs

Cell proliferation assay of MGs

The amount of growth factors contained in MGs

Promotion of cell proliferation by MGs

Inhibition of the effect of bFGF, EGF, and VEGF in MGs

Application of MGs on murine skin defects

Evaluation of the effect of MGs on wound healing

Statistical analysis

Results

Cell proliferation from the skin MGs

Promotion of 3T3 cell proliferation by MGs

The amount of growth factors contained in MGs

The remaining wound area

The neoepithelial length and granulation tissue formation

Histological assessment of newly formed capillaries

Discussion

Conclusions

Abbreviations

Declarations

References

Footnotes

Status:

Version 1