Abstract
Current wound healing models generally employ full-thickness or irregular split wounds. Consequently, assessing the type of healing at varying wound depths and determining the deepest level at which wounds can regenerate has been a challenge. We describe a wound model that allows assessment of the healing process over a continuous gradient of wound depth, from epidermal to full-thickness dermal loss. Further, we investigate whether green fluorescent protein–labeled bone marrow mesenchymal stem cells (BM-MSCs/GFP) transplantation could regenerate deeper wounds that might otherwise lead to scar formation. A wound gradient was created on the back of 120 Sprague Dawley rats, which were randomized into the BM-MSCs/GFP and control group. These were further subdivided into 6 groups where terminal biopsies of the healing wounds were taken at days 1, 3, 5, 7, 14, and 21 post-operatively. At each observed time point, the experimental animals were anesthetized and photographed, and depending on the group, the animals euthanized and skin taken for rapid freezing, haemotoxylin and eosin staining, and vascular endothelial growth factor (VEGF) immunohistochemistry. We found the deepest layer to regenerate in the control group was at the level of the infundibulum apex, while in the BM-MSCs/GFP group this was deeper, at the opening site of sebaceous duct at hair follicle in which had the appearance of normal skin and less wound contraction than the control group (P value less than .05). The expression of VEGF in BM-MSCs/GFP group was higher than that in control group (P value less than .05). The number of vessels increased from 2.5 ± 0.2/phf of control group to 5.0 ± 0.3/phf of BM-MSCs/GFP (P value less than .05). The progressively deepening wound model we described can identify the type of wound repair at increasing depths. Further, topical transplantation of BM-MSCs/GFP significantly improved regeneration of deeper wounds from infundibulum apex (maximum depth of control group regeneration) to the opening site of sebaceous duct at hair follicle level.
Keywords: skin wound, MSCs, regeneration
Abstract
Les modèles actuels de cicatrisation des plaies font généralement appel à des plaies pleine épaisseur ou de forme irrégulière. Il est donc difficile d’évaluer le type de cicatrisation à diverses profondeurs et de déterminer la profondeur maximale à laquelle les plaies se régénèrent. Les auteurs décrivent un modèle de plaie qui permet d’évaluer le processus de cicatrisation d’après un gradient continu de la profondeur de plaie, entre la perte épidermique et la perte dermique pleine épaisseur. Ils ont également examiné si la transplantation de cellules souches mésenchymateuses marquées de protéines fluorescentes vertes (BM-MSCs/GFP) peut régénérer des plaies plus profondes qui formeraient autrement des cicatrices. Les chercheurs ont créé un gradient de plaie sur le dos de 120 rats Sprague-Dawley divisés de manière aléatoire entre le groupe BM-MSCs/GFP et le groupe témoin. Ils ont ensuite subdivisé ces deux groupes en six groupes, dans lesquels ils ont prélevé des biopsies terminales des plaies en voie de cicatrisation les 1er, 3e, 5e, 7e, 14e et 21e journées après l’opération. À chaque journée d’observation, ils ont anesthésié et photographié les animaux expérimentaux et, selon le groupe, les ont euthanasiés et en ont prélevé la peau en vue de leur congélation rapide, de leur coloration HE et de l’immunohistochimie VEGF. Les chercheurs ont découvert que l’apex infundibulum était la couche la plus profonde à se régénérer dans le groupe témoin, mais que dans le groupe BM-MSCs/GFP, la régénération était plus profonde, à l’entrée du follicule pileux dans la glande sébacée, a repris l’apparence de la peau normale et se contractait moins que dans le groupe témoin (p<0,05). L’expression du VEGF dans le groupe BM-MSCs/GFP était plus élevée que dans le groupe témoin (valeur p inférieure à 0,05). Le nombre de vaisseaux observé était de 2,5±0,2/phf dans le groupe témoin et de 5,0±0,3/phf dans le groupe BM-MSCs/GFP (p<0,05). Le modèle de plaie de plus en plus profonde peut déterminer le type de réparation selon la profondeur. De plus, la transplantation topique de BM-MSCs/GFP améliore considérablement la régénération des plaies plus profondes, qui passe de l’apex de l’infundibulum (profondeur de régénération maximale dans le groupe témoin) à l’entrée du follicule pileux dans la glande sébacée.
Introduction
The regenerative capacity of the skin depends on the depth of the injury. More superficial injuries have the capacity to regenerate fully, whereas deeper ones are more likely to heal by scar formation. The formation of scar tissue, especially in cosmetically sensitive areas like the face, can negatively impact the psychological well-being and quality of life of the patient. 1 Numerous interventions have been described, including the use of growth factors, in an attempt to induce regenerative healing without scar formation, with various degrees of success. 2 There is good evidence that bone marrow mesenchymal stem cells (BM-MSCs) can aid in the regeneration of full-thickness wounds. 3
Current animal models of skin wound healing employ full-thickness or irregular segmental defects. 3,4 The limitation to this model is that it only allows observation of the healing process at 1 fixed depth (eg, full thickness), and as such, it is not possible to simultaneously study the healing process along a spectrum of varying depths. Therefore, determining the exact depth where a wound can regenerate fully is still somewhat uncertain. 5
In this paper, we describe a model that examines wound healing on a continuum of progressively increasing depth, from epidermis to full-thickness dermis, on the back of Sprague Dawley (SD) rats, and observed the repair types at increasing depths in order to determine the deepest level where wounds can regenerate under the natural healing state. In order to further investigate the effects of BM-MSCs in regeneration of deep skin wounds, we transplanted green fluorescent protein (GFP)–labeled BM-MSC (BM-MSCs/GFP) in the wound, traced the transplanted cells, and observed whether BM-MSCs/GFP can regenerate deeper wounds that might otherwise heal by scar formation.
Materials and Methods
Culture and Passage of BM-MSCs/GFP
Sprague Dawley rat-derived BM-MSCs/GFP cells (Cyagen Bioscience Inc) were thawed and cultured according to the manufacturer’s instruction. When the confluence of BM-MSCs/GFP reached 80% to 90%, the cells were passaged at a ratio of 1:3. Cells were stained with trypan blue to calculate the ratio of live cells.
Increasing Wound Depth Model
A total of 120 healthy adult SD rats, both male and female, with a mean weight of 150 g, and kept in accordance with the Guide for the Care and Use of Laboratory Animals, were used. Rats were randomly assigned to BM-MSCs/GFPs and control groups. Each group was then randomly subdivided into 6 subgroups (n = 10), assigned for terminal biopsies to be performed on the first, third, fifth, seventh, 14th, and 21st post-operative day, respectively.
One day prior to surgery, the back of the experimental animal in each group was depilated with 8% sodium sulfide. After intraperitoneal injection of 10% chloral hydrate (7.5 mL/100 g), the rat was fixed on the operating table in a prone position. A rectangular wound (10 mm × 15 mm) was drawn, centered on the midpoint of the back, and disinfected with iodophor. A progressively deepening wound gradient was created, from epidermis to full-thickness dermal loss in a caudocranial direction, using a specialized tool (Equipment for Establishment of Continuously and progressively Deepening Wound, Patent No. ZL201520247576.8) with an adjustable blade (Figure 1). Hemostasis was achieved by applying pressure to the area with gauze.
Figure 1.
A, Equipment for establishment of continuously and gradually deepening wound. The required gradient of wound model was controlled by adjusting “height adjustment button.” After the bottom of “skin fixator” attached to skin, the blade was pushed forward along the “blade orbit” to create wound model. B, Wound model. The wound is superficial to deep in a cephalad-cranial direction. Right: skin excised. Left: magnified wound model. C, Cross section of wound under light microscope (H&E, ×40). Note gradient of skin loss from epidermis to full-thickness dermal loss. H&E indicates haemotoxylin and eosin.
Bone Marrow-MSCs/GFP and Control Group
For the BM-MSCs/GFP group, a solution of 1.0 × 106 MSCs/300 µL in PBS was injected into the wound according to Kim’s description. 6 For the control group, the same volume of PBS was used. A total of 300 µL was used; 100 µL for direct injection into superficial layer of the wound bed and 200 µL spaced in a radial pattern around the wound edge. After injection, a single layer of gauze was applied on the wound, and a further layer was applied and secured with tape to fix the dressing. Groups were kept in separate cages, with free access to food and water.
Related Indicators for Overall Evaluation of the Wounds
Wound healing is based on granulation tissue formation and epithelialization. 7 Evaluation of wound healing was based on the surface area of the wound that had healed. The surface area of the wound was calculated using the grid method; transparent 1 × 1 mm grid paper was laid on top of the wound, the area marked, and the number of squares counted, which corresponded to the total surface area. This was repeated at each time point. The rate of wound healing, to avoid the interference of wound contraction on wound size count, was expressed as the percentage of healing area versus the wound area at each time point.
Three specially trained technicians independently completed all tests and the averages were taken for statistical analysis. The animals group (BM-MSC vs control) was masked from the technicians.
Histopathological Examination
Depending on the group they were assigned to, the animals were euthanized on days 1, 3, 5, 7, 14, and 21 post surgery. Photographs of the wounds were taken and wounds harvested by scalpel for histological examination. The basal membranous layer and the normal skin 2.0 mm around the wound were included to ensure complete specimen collection. Following rapid freezing, haemotoxylin and eosin (H&E) staining and immunohistochemistry were performed.
Wound BM-MSCs/GFP count
The frozen sections were protected from light, and the distribution of BM-MSCs/GFP in the wounds and skin appendages was observed under a fluorescence microscope. Images were taken and the number of fluorescent cells at each time point was calculated using the Imagepro-plus image analysis system. The average number of cells in each slice were counted for each group.
Determining level of regeneration
Specimens were harvested with adjacent normal skin as a reference. Thickness of the epidermis, density, and arrangement characteristics of dermal collagen as well as the presence and completeness of skin appendages were evaluated. The deepest regeneration level was determined, according to Stone II’s description, 8 by making a marking line parallel to the epidermis through the deepest regeneration site on the original incision to determine the corresponding level on adjacent normal skin. According to the above method, the regeneration level of the wound under natural healing conditions (control group) was determined, and this was compared with the BM-MSCs/GFP group.
Blood vessel count
The number of vessels in 5 random high-power fields was counted by 3 specially trained technicians, and the average value was taken for statistical analysis.
Vascular endothelial growth factor immunohistochemistry and analysis of the average optical density
Vascular endothelial growth factor (VEGF) immunohistochemistry was performed according to previous reports. 9 Digital photographs of the sections were taken and the average optical density (IOD) of the positive staining cells on the vessel wall was quantitatively analyzed with Imagepro-plus 6.0 image analysis software (H: 0-30, S: 0-255, I: 0-230). All measurements were performed by 2 investigators blinded to the treatment procedure.
Statistical Analysis
The experimental data was expressed as means ± SD. The wound healing rate, BM-MSCs cell count, and percentage of immunohistochemically positive stained cells at each time point were analyzed by 2-way analysis of variance using the SPSS 13.0 statistical analysis software. The capillary count of the wounds on the fifth day after operation was tested with Dunnett. Wound regeneration level was statistically analyzed by using χ2 test, and a P values less than .05 was considered statistically significant.
Results
Characteristics and Passaging Ability of BM-MSCs/GFP
The indicators of BM-MSCs/GFP were in accordance with the identification criteria of BM-MSC in SD rats. 10 The split rate was 1:3. The rate of live cells stained by trypan blue was >95%.
Wound Contraction Rate and Healing Rate
After BM-MSCs/GFP transplantation, rats were given a normal diet and housed in their respective groups. All wounds were free of infection which defined as the presence of red swollen and purulent secretion.
Wound contraction rate
In the early stage of cell transplantation, the wounds of both groups were covered by a scab and the boundaries were clear. On day 10, the wound area of the 2 groups began to contract, but there was no significant difference between the two groups. By day 14, the wound contraction rate of the control group had increased (31.5% ± 0.4) compared with BM-MSCs/GFP group (20.8% ± 0.3), P value less than .05. The boundary between wound margin and surrounding normal tissue was clearly demarcated. On day 21, the contraction rate of wound surface of the control group reached 68.0% (±1.2), which was significantly higher than the BM-MSCs/GFP group 35.5% (±2.4; P value less than .05, Figure 2).
Figure 2.
Evolution of skin wound in BM-MSCs/GFP-treated and control group. Pictures in first line shows the evolution of skin wound in BM-MSCs/GFP-treated group. The scab was closely attached to the wound before day 7. The wound healing rate was 55.4% (±0.3) on day 14, and with hair growth and almost normal appearance on day 21. (From A to F are first, third, fifth, seventh, 14th, and 21st post-operative day, respectively). The second line is the evolution of skin wound in control group. Wound healing rate was 3.4 (±0.2) on day 14. Although the entire wound had healed by day 21, there was significant wound contraction and the border of the wound is more visible. (From A1 to F1 are first, third, fifth, seventh, 14th, and 21st post-operative day, respectively). The graph below plots the change in wound healing rate over time for 2 group. BM-MSC indicates bone marrow mesenchymal stem cell; GFP, green fluorescent protein.
Wound healing rate
Compared with the control group, the wound healing time of the BM-MSCs/GFP group was significantly shortened, and the role of BM-MSCs/GFP in promoting wound healing was demonstrated on the 10th day. By day 10, the scab on superficial wounds in the BM-MSCs/GFP group had started to lift off, showing an underlying epithelialized surface. The wound healing rate was 5.4% (± 0.3), while in the control group, the scab was closely adherent to the underlying wound bed (P value less than .05). On day 14, in the BM-MSCs/GFP group, except for the firm attachment of a scab in the distal deep wounds, the epithelialized area of the superficial wounds was further increased and the wound healing rate reached 55.4% (± 0.3), while the superficial wound in the control group began to epithelialize and the wound healing rate was only 3.4% (±0.2; P value less than .05). By day 17, wound epithelialization in the BM-MSCs/GFP group was complete, while wound healing rate in the control group was 73.6% (± 1.5), with none of the wounds fully healed (P value less than .05). By day 21, the skin color of the BM-MSCs/GFP group was similar to that of the surrounding normal skin. Although the epithelialization of the wound in the control group was complete, there was evident contraction and scar formation, and there was no obvious hair growth (Figure 2).
Histopathological Examination
Tracing of transplanted BM-MSCs/GFP in wound tissues
No fluorescent cells were found in the tissue sections of the control group whereas green fluorescent cells were seen in the wounds of the BM-MSCs/GFP group at all time points after transplantation. Among them, the green fluorescent cells of the BM-MSCs/GFP were mainly located on the original injection site of the wound bed on the first and third days after transplantation. The injection site was clustered and distributed unevenly, which was caused by the uneven distribution of topical injection during transplantation. The green fluorescent cells migrated into the deep wound from the fifth day after transplantation. In addition, the number of BM-MSCs/GFP after topical transplantation decreased over time, and the cells were distributed sporadically over the wound on day 21. This was less than 1% than on day 1 of transplantation (Figure 3).
Figure 3.
A, Number of transplanted BM-MSCs/GFP at each time point. The number of transplanted BM-MSCs/GFP reduced over time, and by day 21 were <1% total number transplanted. B, Tracing of transplanted BM-MSCs/GFP in wound tissues. The BM-MSCs/GFP mostly remained at original transplanted sites prior to day 3. Migration into deeper tissue was evident by day 5, with only scanty cells scattered in the wound by day 21. BM-MSC indicates bone marrow mesenchymal stem cell; GFP, green fluorescent protein.
Haemotoxylin and eosin staining
The healthy epidermis adjacent to the wound was composed of 3 to 4 layers of cells. On day 21, the wounds of the control group were completely epithelialized, and the regenerated epidermis on the proximal and superficial wound was the same as the normal skin. Collagen deposition below infundibulum apex was dense, and sebaceous glands and other skin appendages were formed, which was the typical pathological structure of scar tissue. 11 In the BM-MSCs/GFP group, the reticular layer over the opening of sebaceous duct on hair follicle was thicker, the thick collagen fibers inter-weaved, and the papillary collagen was fine, dense, and in a parallel arrangement to the epidermis, which was the same as the normal skin tissue structure, 12 whereas the collagen below the opening of sebaceous duct was dense, with significant differences from normal tissues, and scattered skin appendages were found in the area (Figures 4–5).
Figure 4.
View of BM-MSCs/GFP-treated group under light microscope (H&E). Bottom slide: The boundary of regeneration and scar is outlined in red. The green line started from deepest regeneration site on the original incision and paralleled the epidermis, which was used to determine the corresponding location on adjacent normal appendage. The deepest plane of wound regeneration in BM-MSCs/GFP group was at the opening of sebaceous duct on hair follicle. Top 2 slides: Magnified view of scar (left) and regeneration tissue (right). BM-MSC indicates bone marrow mesenchymal stem cell; H&E, haemotoxylin and eosin; GFP, green fluorescent protein; P, papilla; R, reticular; SG, sebaceous gland.
Figure 5.
View of control group under microscope (H&E). A, The boundary of regeneration and scar was outlined with red line, the green line started from deepest regeneration site on the original incision and paralleled the epidermis, which was used to determine the corresponding location on adjacent normal appendage. In the control group, the wound above the level of infundibulum apex regenerated without scar formation. A1&A2: Magnified view of scar (left) and regeneration tissue (right). B, Schematic drawing of hair follicle (from Panagiotis Mistriotis. Hair follicle: A novel source of multipotent stem cells for tissue engineering and regenerative medicine. Tissue Engineering: part B. 2013;19:265-279). The view of infundibulum is shown by the dotted line. H&E indicates haemotoxylin and eosin; HS hair shaft; SG, sebaceous gland.
The deepest wound regeneration level
Haemotoxylin and eosin staining was used to observe the thickness of epidermis at different depths, the formation of skin appendages, and the arrangement of collagen in dermis.
We found that topical transplantation of BM-MSCs/GFP could enhance regeneration level from the infundibulum apex in control group to the opening of sebaceous duct on hair follicles (Figures 4–5). There was a statistically significant difference between the 2 groups (P value less than .05).
Blood vessel count
On day 5, mature vascular wall structure was observed in the wound. The number of blood vessels in the BM-MSCs/GFP treatment group was 5.0 ± 0.3, which was higher than the 2.5 ± 0.2 in the control group (P value less than .05).
Expression of VEGF in vascular endothelial cells in the wound
Angiogenesis is a crucial step in wound healing. The increase of vessel can supply sufficient nutrition to granulation tissue and keratinocytes, thereby improving the healing quality and shortening healing time. The expression of VEGF in BM-MSCs/GFP group from the third day after cell transplantation was significantly higher than that in the control group (P value less than .05). There was no significant difference between the 2 groups after the 14th day (P value more than 0.05; Table 1, Figure 6).
Table 1.
The IOD of Vascular Endothelial Cell in the Wound.
| 1 day | 3 days | 5 days | 7 days | 14 days | |
|---|---|---|---|---|---|
| BM-MSC | 7840 (24) | 16 145 (31) | 18 058 (18) | 17 362 (19) | 8458 (12) |
| Control | 7515 (20) | 8064 (16)a | 8294 (12)a | 8307 (13)a | 8239 (13) |
Abbreviations: BM-MSC, bone marrow mesenchymal stem cell; GFP, green fluorescent protein; IOD, optical density.
a P value less than .05: compared with corresponding time point of observation in BM-BMCs/GFP group.
Figure 6.
A, Expression of VEGF in vascular epithelial cell in wound (×400). VEGF located on cytoplasm. Vascular epithelial cells stained brown are VEGF expression cells. The expression of VEGF in BM-MSCs/GFP group increased on third postoperative day. There was no significant difference between the 2 groups on day 14. B, Transplanted BM-MSCs/GFP in vascular walls. Green fluorescent could be seen on day 5 and reduced on day 7. GFP indicates green fluorescent protein; VEGF, vascular endothelial growth factor.
Distribution of BM-MSCs/GFP in vascular walls
On day 3, no obvious vascular-like structures were found on the wound. On day 5, blood vessel density in the BM-MSCs/GFP treatment group was significantly increased, and mature luminal structures were seen. Scattered green fluorescent cells were seen on the vascular wall and adjacent sites. No significant fluorescence-expressing cells were observed on the wall of the blood vessel and its surroundings after day 14 (Figure 6).
Discussion
The skin consists of epidermis and the dermis. The dermis is divided into a superficial papillary layer and a deep reticular layer. Removal of epidermis with mild loss of papillary layer could be completely regenerated. However, with the increased areas and/or the depth of the damaged reticular dermis, the risk of scar formation increases accordingly. 13 Our study found that topical transplantation of BM-MSCs/GFP could enhance regeneration from the infundibulum apex in the control group to the opening of sebaceous ducts on hair follicles. The transplanted cells migrated down into deeper layers of the wound over time, and blood vessel walls had a scattered distribution in the early process and almost disappeared after 21 days.
Full-thickness or irregular split wound models are widely used in studies looking at wound healing, 1,4 making it difficult to identify the type of healing taking place at varying wound depths in a single wound model. Therefore, the deepest level of wound that can regenerate with normal skin architecture remains uncertain. 5 The application of continuously and progressively deepening wound model has enabled accurate assessment of the type of healing at different wound depths.
We found that tissue defects above the infundibulum apex results in regeneration of normal skin, while injuries deeper to this level will inevitably result in scar formation. Therefore, hair follicle entrance can be used as a “STOP!” sign for dermabrasion procedure to avoid scar formation.
The BM-MSCs transplantation can improve the healing quality of full-thickness wounds and shorten the healing time, 14 with promising results in the field of wound regeneration research. 2,3 In this study, BM-MSCs/GFP almost disappeared after 21 post-operative days, findings similar to previous research. 15 The reason may be due to changes in the microenvironment signaling that affect survival of stem cells. The maximum depth of wound regeneration in the BM-MSCs/GFP group increased from the level of infundibulum apex (seen in control group) to the level of the opening of sebaceous duct. We observed scattered hair follicles, sebaceous glands, and other skin appendage structures in the healing tissue. Since the long-term survival of transplanted cells is low, the reason for increased regeneration is not only the proliferation and differentiation of transplanted stem cells, but also the effects of other factors such as native stem cells.
Stem cells in the dermis are mainly located at or below the opening of sebaceous ducts, while the reason for the regeneration level of the BM-MSCs/GFP group above the opening of sebaceous ducts may be that to a greater extent tissue damage above this layer left the niche of dermal stem cells undamaged. And after the resident stem cells in it migrated outward to participate in wound repair, the “vacant” niche provided ideal residence for transplanted BM-MSCs/GFP. 16 The increase in cell reserves within the niche made it possible for these cells to migrate to the wound surface and participate in wound repair. Despite a shorter survival time, BM-MSCs/GFP can also form a precursor of permanent skin structure, which plays an important role in the skin regeneration and serves as a basis for the resident stem cells to regenerate skin appendages. 17 Although scattered transplanted cells were found in the blood vessel wall at the level of regenerating wounds in our experiments, it was not possible to determine the cellular origin of the skin appendages in the scar tissue.
Angiogenesis is an important step in wound healing. 18 The formation of new blood vessels can provide adequate nutrition for wound repair. Our study found that the capillary density of BM-MSCs/GFP group increased significantly. Fluorescence microscopy showed that the transplanted cells were scattered in the vessel wall and its adjacent tissue. Meanwhile, VEGF expression in the wound vascular endothelial cells significantly increased compared with the control group, and VEGF can stimulate the proliferation and migration of endothelial cells and promote the formation of new blood vessels. 19 Therefore, we believe that BM-MSCs can promote the formation of new blood vessels in wounds. In addition to its own proliferation and differentiation, its secretion of VEGF also plays an important role in wound healing. 20
In summary, the progressively deepening wound model can accurately identify the type of wound repair on a continuum of increasing depth. Topical transplantation of BM-MSCs/GFP significantly improved the depth at which wounds can regenerate from the level of the infundibulum apex to the level of sebaceous duct opening at hair follicles.
Supplemental Material
Supplemental Material, change_in_authorship_form for Topical Transplantation of Bone Marrow Mesenchymal Stem Cells Made Deeper Skin Wounds Regeneration by Qin Yonghong, Li Aishu, Yazan Al-Ajam, Liao Yuting, Zhang Xuanfeng and Zhang Jin in Plastic Surgery
Footnotes
Authors’ Note: Qin Yonghong and Li Aishu contributed the same efforts to the manuscript, and we regarded 2 of them as the first authors. The animal experiments were performed in our laboratory with permission of the Ethics Committee of Lanzhou University (Lanzhou, China) on October 8, 1998.
Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.
ORCID iD: Qin Yonghong, MD, PhD https://orcid.org/0000-0003-0633-942X
Supplemental Material: Supplemental material for this article is available online.
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Supplementary Materials
Supplemental Material, change_in_authorship_form for Topical Transplantation of Bone Marrow Mesenchymal Stem Cells Made Deeper Skin Wounds Regeneration by Qin Yonghong, Li Aishu, Yazan Al-Ajam, Liao Yuting, Zhang Xuanfeng and Zhang Jin in Plastic Surgery