Abstract: Background: Mesenchymal stem cell-conditioned medium (MSC-CM) is the substance extracted from mesenchymal stem cell culture, contains a lot of potential cytokines and growth factors as regenerative agent. Objective: The aim of this study was to investigate the role of MSC-CM on the burn wound regeneration pattern of white rat (Rattus norvegicus ). Methodology: Rat was anesthetized using combination of 10% ketamine and 2% xylazine at the dose rate of 75 and 5 mg kg–1 b.wt., respectively. The burn wound condition was created on the dorsal area of each rat. The burn wound area of control group was treated with Bioplacenton®, while MSC-CM group was topically treated with MSC-CM cream twice a day. The diameter of burn wound was measured every 5 days from wounded. Skin wound tissues were collected at 4 h, followed by 2, 5, 10, 15, 20, 25 and 30 days after wounded and then were processed by paraffin method. Tissue samples were visualized by using hematoxylin-eosin and Masson’s trichrome stain. The polymer-based immunohistochemistry method was employed to detect the presence of basic fibroblast growth factor (bFGF) as important growth factor in wound healing process. Result: The results showed that MSC-CM promote the recovery of skin burn wound in white rat, as indicated by: (1) Acceleration of wound closure, (2) Greater numbers of fibroblasts, (3) High density of collagen fibers and (4) Greater numbers of blood vessels in MSC-CM group compare with control group. Conclusion: Since the number of bFGF immunoreactive cells increased significantly during recovery proccess in MSC-CM group compare with control group, it was suggested that bFGF plays important role on the tissues regeneration of skin burn treated by MSC-CM.
INTRODUCTION
Mesenchymal Stem Cells (MSCs) are multipotent cells that have multiliniage differentiation capacity, regulate the inflammatory response and release the active biomolecules of paracrine signal that affect cell migration and proliferation1. In vitro examination showed that in the MSCs culture medium, there are growth factors secreted by MSCs that potential as reparative agents through paracrine signal mechanism1,2.
The MSC-CM is factors also known as secretome, microvesicle or exosome found in the MSC culture medium. Stem cell conditioned medium contains growth factors that have a role as tissue reparative agent. Some studies reported that stem cell conditioned medium can be derived from several types of cells, collected by various method can be applied to several types of degenerative diseases and it has been examined to various types of experimental animals3. The MSC-CM derived from fetal umbilical cord has multipotent differentiation and tissue regeneration capability1,2.
Burn wound is one of the most common case occurs and can lead to illness and death4. Skin burn wound healing can impact the quality of life due to long period healing, infection risk and hypertropic scar forming5. Burn scar can cause discomfort effect, functionally and aestheticaly6. Wound healing process is an important case to clinicians thus burn wound can be solved. There are 4 stages in wound healing process, hemostatic, inflammatory, proliferation and remodeling. Wound healing is a complex and dynamic process involved cellular interaction, vascular and biomolecular response, such as growth factor7,8. Basic fibroblast growth factor (bFGF) is one of growth factor that regulates fibroblast proliferation, extracellular matrix synthesis and angiogenesis9. In the early stage of wound healing, bFGF was secreted by macrophage and still can be identified until remodelling stage, several weeks after injury10. Moreover, bFGF also stimulate collagen synthesis and reduce scar forming found in wound healing process. Clinically, bFGF can be used to heal burn wound rapidly11. The objective of this study was to investigate the role of MSC-CM on the burn wound regeneration pattern of white rat (Rattus norvegicus).
MATERIALS AND METHODS
Animals: Forty-eight of 2 months old Wistar rats (Rattus norvegicus) were divided into 2 groups, they are control group as Bioplacenton® treated group and MSC-CM group which topically treated by using MSC-CM cream. Rats were adapted in individual cage for 7 days before wounded. Rats were feed with basal food and water ad libitum and were maintenance to 12 h light-dark cycle. This study was approved by Ethical Clearance Committee from Gadjah Mada University.
Wound creation, sample collection and tissue preparation: Rats were anasthetized using combination of 10% ketamine and 2% xylazine at the dose rate of 75 and 5 mg kg–1 b.wt., respectively12. One centimeter diameter of nail head was burned and put down on dorsal area of each rat for 5 sec until full-thickness burn wound was created. Bioplacenton® treated group was treated with Bioplacenton® (Bovine placenta extract 10%, neomycin sulphate 0.5%, gel base) while, MSC-CM cream treated group was treated with MSC-CM cream (1 mL MSC-CM, 10 g base cream, mixed until homogen) twice a day, respectively. Burn wound size was measured every 5 days after wounded. Skin wound tissues were collected at 4 h, followed by 2, 5, 10, 15, 20, 25 and 30 days after wounded. Skin wound tissues were fixed in bouin solution and processed with paraffin-embedded method. Five micrometers thickness of tissue samples were visualized by using Masson’s trichrome stain, hematoxylene-eosin stain and polymer-based immunohistochemistry method.
Masson’s trichrome stain and hematoxylene-eosin stain: Masson’s trichrome stain method was used for collagen scoring. Slides of tissue samples were deparaffinized in xylene and rehydrated in ethanol with gradual concentration. Slides were immersed in bouin solution and incubated overnight at room temperature. Slides were rinsed for 10 min in running water and then immersed in Weigert’s iron hematoxyline for 10 min. Slides were rinsed in destilated water and then immersed in biebrich scarlet solution for 5 min. Slides were rinsed in destilated water and immersed in phosphomolybdic-phosphotungstic acid solution for 10 min. Then, slides were immersed in anniline blue solution for 5 min, rinsed in destilated water and immersed in glacial acetic acid solution for 3 min. The last step for Masson’s trichrome stain was dehydrated in ethanol with gradual concentration, cleared in xylene and then mounted.
Hematoxylene-eosin stain was used for fibroplasia and angiogenesis examination. First step for this staining method was deparaffinized and rehydrated the slide. Slides were immersed in Harris hematoxylene solution for 4 min and immersed in running water for 10 min. Then, slides were immersed in eosin solution for 10 min. The last step, slides were dehydrated, cleared and mounted.
Immunohistochemical staining: Polymer-based immunohistochemistry method was used to detect basic fibroblast growth factor (bFGF) in rat skin burn wound. Slides were deparaffinized and rehydrated. Then, slides were rinse in running water for 10 min. Destillated water was placed in microwave for 20 min for pre-heating antigen retrieval. Slides were immersed in pre-heating destillated water for 10 min and washed in Phosphate Buffer Saline (PBS) 0.01 M (pH 7.4) for 5 min. Endogenous peroxidase activity was blocked by incubating slides in 3% H2O2 in absolute methanol (1 mL H2O2 30%, 9 mL absolute methanol) for 15 min at room temperature. Slides were washed in PBS 3 times for 5 min, respectively. Tissue on the slide was circled by using DAKOPEN®. Rabbit anti-bFGF (1:100, Bioss, USA) were used as primary antibody and applied overnight at 4°C.
Slides were washed in PBS 3 times for 5 min, respectively. The immunoreactive site were visualized using the N-Histofine® simple stain rat MAX PO kit and applied for 30 min. The N-Histofine® DAB-2 chromogen (Nichirei Biosciences Inc., Japan) was treated as a chromogen. Slides were counter stained with Harris hematoxylene and rinsed in running water for 10 min. Then, slides dehydrated, cleared and mounted.
Data analyze: Stained slides were examined under light microscope and photomicrograph was taken by Optilab® camera. The density of collagen fiber were analyzed semi-quantitatively and graded subjectively into 4 classes as shown in Table 1. The number of fibroblasts, blood vessels, bFGF immnureactive cells and burn wound diameter were analyzed qualitative and quantitatively. Five photomicrographs of fibroblasts, blood vessels and bFGF immunoreactive cells were taken randomly with 520 times magnification, respectively. Fibroblasts, blood vessels and bFGF immunoreactive cells were counted by using manual cell counter of ImageJ Software 1.46r. The quantitative data were evaluated and expressed as Mean±Standard Deviation. Significant differences between groups were determined by independent t-test using version 23 SPSS software for windows. The p-value less than .05 was considered statistically significant.
RESULTS
Fibroblasts: The MSC-CM cream application on normal rat skin burn wound stimulated fibroblasts migration and proliferation, so the number of fibroblast in MSC-CM treated group was greater than Bioplacenton® treated group (Fig. 1). Table 1 showed the number of fibroblasts between Bioplacenton® treated group and MSC-CM cream treated group were different significantly on 5, 10, 15, 20, 25 and 30 days after wounded, quantitatively.
Collagen fibers density: Extensive collagen deposition was detected in MSC-CM cream treated group, descriptively (Fig. 2). Based on Table 1, high density of collagen in MSC-CM cream treated group were detected on day 10 and very high density of collagen were detected on day 30 after wounded while in Bioplacenton® treated group, high density of collagen were detected on day 20 after wounded.
Blood vessels: The number of blood vessels in MSC-CM treated group was greater than Bioplacenton® treated group, descriptively (Fig. 3). Quantitative data on Table 1 indicate that the number of blood vessels wound were significantly different between Bioplacenton® treated group and MSC-CM cream treated group on 10, 15 and 20 days after wounded.
bFGF immunoreactive cells: Immunoreactivities of bFGF were detected in macrophages, fibroblasts and endothelial cells, which were distributed on dermis of wound area. The number of bFGF immunoreactive cells was greater in MSC-CM cream treated group than Bioplacenton® treated group, descriptively (Fig. 4). Based on Table 1, the bFGF immunoreactive cells were significantly different between Bioplacenton® treated group and MSC-CM cream treated group on 4 h and 2, 10, 15, 20, 25 and 30 days after wounded.
| Table 1: | No. of bFGF immunoreactive cells, fibroblasts, collagen fibers density, the number of blood vessels and wound size in Bioplacenton® treated group MSC-CM cream treated group |
| Mean±Standard Deviation, aSignificant at p<0.05, +: Less density, ++: Moderate, +++: High density, ++++: Very high density | |
| Fig. 1(a-f): | (a-c) No. of fibroblasts in Bioplacenton® treated group and (d-f) MSC-CM treated group. The number of fibroblasts (black arrow), (d) On day 5, (e) On day 15 and (f) On day 30 after wounded in MSC-CM treated group were detected greater than (a-c) Bioplacenton® treated group |
| Fig. 2(a-f): | (a-c) Collagen fibers density in Bioplacenton® treated group and (d-f) MSC-CM treated group. Collagen fibers in MSC-CM treated group were detected denser than Bioplacenton® treated group, (a) On day 10 after wounded less density of collagen fibers were detected in Bioplacenton® treated group, (d) While moderate density of collagen fibers were detected in MSC-CM treated group, (b) On day 20 after wounded moderate density of collagen fibers were detected in Bioplacenton® treated group, (e) While dense collagen fibers were detected in MSC-CM treated group, (c) On day 30 after wounded dense collage fibers were detected in Bioplacenton® treated group, (f) While high density of collagen fibers were detected in MSC-CM treated group |
Wound size: Taken together, the MSC-CM cream accelerated burn wound closure as shown on Fig. 5 as percentage of wound area on the day after wounded compared to wound area on day 0 in both treated group. On day 10 after wounded, wound crust of MSC-CM cream treated group was peeled and on 15 days after wounded, wound diameter of MSC-CM treated group were smaller than wound diameter of Bioplacenton® treated group, descriptively.
| Fig. 3(a-f): | (a-c) No. of blood vessels in Bioplacenton® treated group and (d-f) MSC-CM treated group. The number of blood vessels (black arrow), (d) On the day 10, (e) On day 15 and (f) On day 20 after wounded were detected greater than (a-c) Bioplacenton® treated group |
| Fig. 4(a-f): | (a-c) bFGF immunoreactive cells in Bioplacenton® treated group and (d-f) MSC-CM treated group. bFGF immunoreactive cells in MSC-CM treated group were showed the greater number compared to Bioplacenton® treated group, (a) On day 2 after wounded, bFGF immunoreactive cells (black arrow) in Bioplacenton® treated group were detected in fibroblasts, (d) While in MSC-CM treated group were detected in fibroblasts, macrophages and endothelial cells, (b) On day 15 after wounded, fibroblasts were the most detected as bFGF immunoreactive cells in Bioplacenton® treated group, (e) while in MSC-CM treated group fibroblasts and endothelial cells were detected as bFGF immunorecative cells, (c) On day 25 after wounded, fibroblasts and endothelial cells were detected as bFGF immunoractive cells in Bioplacenton® treated group and (f) MSC-CM treated group |
Wound was closed on 20 days after wounded in MSC-CM cream treated group. On 25 and 30 days after wounded wound burn were covered by hair (Fig. 6).
| Fig. 5: | Wound closure rate was expressed as percentage of wound area on the day after wounded compared to wound area on the day 0 in both treated group. The wound area percentage decreased on 5, 10, 15 and 30 days after wounded in both treated group. However, the wound area percentage of MSC-CM treated group shown that the wound closure area rate in the MSC-CM treated group were faster than the wound closure area rate in Bioplacenton® treated group |
| Fig. 6(a-b): | (a) Burn wound diameter in Bioplacenton® treated group and (b) MSC-CM treated group. On day 5 after wounded, burn wound in both treated group showed wound crust formation. On day 10 after wounded, wound crust in MSC-CM treated group were peeled. On day 15 after wounded wound size in MSC-CM treated group were smaller than Bioplacenton® treated group. Wound closure in MSC-CM treated group was showed on day 20 after wounded. Complete wound closure and hair growth in MSC-CM treated group were showed on day 30 after wounded, macroscopically |
Wound diameter of MSC-CM cream treated group was significantly different on 5 and 20 days after wounded compared to Bioplacenton® treated group, quantitatively (Table 1).
DISCUSSION
The result of this study showed that skin tissue regeneration in burn wound of MSC-CM treated group faster compared with to Bioplacenton® treated group. MSC–CM derived from various of sources have been examinated in various kind of regenerative disease3,13 and factors secreted in conditioned medium might have more than one of regenerative action14. The MSC-CM was known containing cytokines and growth factors secreted by MSC that have important role in tissue regeneration process, such as Vascular Endothelial Growth Factor (VEGF), Platelet Derived Growth Factor (PDGF), bFGF, Epidermal Growth Factor (EGF), Keratinocyte Growth Factor (KGF) and Transforming Growth Factor-β (TGF-β). Some study reported, there were various type of cells that responded on MSC paracrine signal, regulated cells maintenance, proliferation, migration and gene expression, such as epithelial cells, endothelial cells, keratinocytes and fibroblasts9. In vitro study reported that MSC-CM was known as chemoattractan to macrophages, endothelial cells, keratinocytes and fibroblasts. Moreover, MSC-CM could stimulate fibroblasts and accelerate wound regeneration process15. Multipotent differentiation properties of MSC derived from fetal umbilical cord has immunoregulatory ability, so MSC derived from fetal umbilical cord were considered as a viable alternative source of stem cells for long-term clinical trials15. The MSC-CM derived from fetal umbilical cord tissue culture has been widely studied as an alternative therapy of to solve wound healing problem16. The bFGF is a potential growth factor for fibroplasia, angiogenesis, collagen synthesis and scar-forming reduction11,17. In normal skin, as growth factor, bFGF is detected in the cytoplasm of cells and in the extracellular matrix18 has angiogenic and mitogenic effect for tissue regeneration, wound healing and angiogenesis17.
Normal wound healing is complex and dynamic process. The process involves a series of coordinated occurrence start immediately after wound until new tissue formation19,20. In the normal process of wound regeneration, bFGF has been secreted by platelets in the early stage, hemostatic stage of wound healing21. Hemostatic plug is formed by platelets to hold the bleeding and stimulate the inflamatory stage22,23. Platelets also secrete TGF-β, EGF and PDGF. Cytokines secreted in the early stage of wound stimulate inflammatory stage, neutrophiles and macrophages migration to phagocyte wound debris21,24. The MSC-CM presumed contain of bFGF applicated on burn wound healing and hemostatic plug stimulated bFGF in hemostatic stage so, the number of bFGF immunoreactive cells were significant on 4 h after wounded. On the other hand, partial thickness burn wound (second-degree) treated with recombinant bFGF as early as arrival day of wounded, showed complete healing on 12 days25 and this finding was acceptable with bFGF immunoreactivity detected on rat skin during the 4-11 days after wounded10. In this study, bFGF immunorectivity was detected on full-thickness burn wound on 4 h, as early as wounded. Significant number of bFGF stimulated macrophages migration on 2 day after wounded. Macrophages as bFGF secreted cells have a role to stimulate fibroblasts and keratinocytes migration in the early stage of wound healing21. On day 5 after wounded, regeneration process were observed macroscopically. Proliferation proccess stimulate epidermal regeneration, characterized by keratinocyte presence on the edge o f wound to induce re-epitelialization, replacing the demage tissue26. In the early stage of wound, immunoreactivity of bFGF was immediately responded by fibroblasts and endothelial cells for their migration and proliferation18. When the number of neutrophiles and macrophages decreased, the proliferation stage was begun21. On day 5 after wounded, the number of bFGF immunoreactive cells were not significant. But, significant bFGF immunoreactivity on the previous day secreted by macrophages stimulated increased of fibroplasia activity. It cause the number of fibroblast was significant on 5 days after wounded. The MSC-CM derived from various of sources were potential to accelerate wound healing by their mitogenic effect. Injection of bone marrow-mesenchymal stem cell (BM-MSCs) conditioned medium subcutaneously accelerated acute and chronic excisonal wound by increased the number of myofibroblasts to promote wound closure. Excisional wound healing was complete on 6-8 days after bone marrow MSC-CM injection27.
Fibroblasts produce type III collagen during proliferation, facilitated wound closure. During proliferation stage, fibroblasts proliferation activity is higher due to the presence of TGF-β stimulate fibroblasts to secrete bFGF. Greater number of fibroblasts also induce higher collagen synthesis28. Collagen fiber is the major proteinsecreted by fibroblast, composed extracellular matrix to replace wound tissue strength and function29. Collagen fibers deposition was significant on 10 day after wounded. The number of fibroblasts increase significantly, may in correlation with the presence of abundance bFGF immunoreactivities on 10 days after wounded. Various kind of burn wound healing traditional therapy agent has been reported, one of them is emu oil. Similar to this study, burn wound was created by heating 1 cm diameter of nail head for 30 sec on the mice skin, treated with emu oil topically. Emu oil delayed inflammatory stages as indicated by the great number of inflammatory cells on 4 days and still detected on 14 days on the edge of wound, histologically. Although inflammatory stage was delayed, collagen fibers deposition in emu oil treated wound were siginificant on day 10 after wounded. Emu oil and MSC-CM stimulated early collagen fiber deposition30. The presence of significant bFGF on the day 2 after wounded were stimulated by umbilical cord MSC-CM. The bFGF promoted migration and proliferation of endothelial cells. Since endothelial cells have ability to produce bFGF, the increasing on the number of endothelial cells as a result of angiogenesis process, the numerous number of bFGF immunoreactive cells were detected in day 15 after wounded. High density of new blood vessels on exicional diabetic wound treated with Human Cord Blood (HCB) endothelial progenitor cells-conditioned medium (EPC-CM) were also detected on day 15 as indicated by the great number of von Willebrand factor (vWF) or hemostatic factor immunoreactivity31. Both, human umbilical cord MSC-CM32,33 and human cord blood EPC-CM promoted angiogenesis.
The end of normal regeneration process was indicated by cellular activity and angiogenesis reduction and granulation tissue formation, indicated by high tensile strength34. In remodelling stage, collagen fibers secreted by fibroblast cells were stopped when the wound gap was clossed35. Fibroblasts proliferation and collagen synthesis were reduced for a balance between collagen synthesis and degradation36. The reduction of acellular granulation tissue via fibroblasts apoptosis stimulation were stimulated by bFGF37. The bFGF immunoreactivity were detected significantly on 20 and 25 days after wounded. Burn wound regeneration in MSC-CM treated group were complete without acellular tissue forming. Human umbilical cord MSC-CM could use as wound healing alternative therapy32. The other studies reported that human umbilical cord MSC-CM accelerated exicional wound healing in diabetic mice as indicated by complete re-epithelization and lesser but thicker remodelling tissue on day 14 after wounded32. Similar to this study, human umbilical cord MSC-CM contained VEGF and bFGF promoted angiogenesis, fibroplasia, re-epithelization, hair follicles and remodelling tissue formation on rat incisional wound healing, histologically38.
In remodelling stage, matrix metalloproteinases (MMPs) secreted by macrophages were also stimulated bFGF to reduced cellular activity by suppressed type III collagen synthesis and degradated them into type I collagen. The MMPs also secreted by epithelial cells, endothelial cells and fibroblasts8. So, MMPs have a role in the balanced of fibroplasia and angiogenesis39. The MMPs stimulated bFGF for endothelial cells apoptosis through the anti-angiogenic factor. It caused optimal angiogenesis and avoided abnormal angiogenesis40. Burn wound treated with recombinant bFGF had the same finding that bFGF as an important growth factor in tissue repair process promoted angiogenesis, wound healing acceleration and improved scar quality25,41. The bFGF stimulation on 20 and 25 days after wounded could produce the vascular and cellular tissue balance. So, macroscopically, there were not scar tissue, complete wound closure and hair growth on 30 days after wounded. The MSC-CM act through autocrine and paracrine mechanism to increase the number of fibroblasts, collagen fibers density and the number of blood vessels.
CONCLUSION
The result of this study showed that skin tissue regeneration in burn wound of MSC-CM treated group faster compared with Bioplacenton® treated group as indicated by greater numbers of fibroblasts, high density of collagen fibers, greater numbers of blood vessels and finally accelerates the wound closure macroscopically. The presence of intense bFGF immunoreactivities and its extensive distribution in the skin tissues of the MSC-CM cream treated group may plays important roles on the regeneration process.
ACKNOWLEDGMENTS
This study was fully supported by the Grant for Scientific Research (PUPT UGM 2015) from the Directorate General of Higher Education (DIKTI), Ministry of Research, Technology and Higher Education of Indonesia, with contract number 112/LPPM UGM/2015.