| BACKGROUNDChronic wounds are intractable clinically. Much work has been done to improve chronic wound healing. But development of novel therapies on chronic wounds is hindered due to lacking of preclinical animal models which closely mimic human pathophysiology. In order to observe the potentially effects of experimental treatment, many models of impaired healing have been developed [1,2,3]. Due to the similarities of anatomy and physiology [4] between swine and human, and even the response on radiation in a similar time-and dose-dependent manner[5], swine is the most available animal as chronic wound model. The delayed wound-healing model with Yorkshire pigs or Yucatan pigs were made[3,6,7]. A major shortcoming is that if the growth speed of pigs is too fast, it will influence the result of experimental therapy because wound healing rate and growth factor profiles are different in full-thickness wounds in swine of various ages [7]. Further, when is the optimal time for swine to be chosen as a chronic wound model after radiation?It was reported that low-level laser irradiation can positively affect human ADSCs in vitro[8].The characterization of the responses of human stem cells, e.g. neural stem cells, mesenchymal stem cells, hematopoietic stem cells, to ionizing radiation also has been investigated. Human bone marrow-derived mesenchymal stem cells were able to retain their defining stem cell characteristics[9,10,11]. However, these responses were overwhelmingly performed in vitro with a low-dose radiation (<10Gy), and in vivo with a high-dose ionizing radiation were seldom reported. Accordingly, our second hypothesis was that, with the different postradiation time, the characterization of radiation-injured adipose-derived stem cells (r-ADSCs) is comparable to normal adipose-derived stem cells (n-ADSCs) in vivo.Adipose-derived stem cells (ADSCs) secrete many potentially beneficial growth factors and cytokines, which are helpful to wound healing [12,13]. In our study, we created chronic wound model in micro pig by radiation according to different postradiation time and implanted autologous adipose-derived stem cells (ADSCs) on chronic wound. Then we examined the effects of ADSCs on wound healing compared with vehicle control medium or normal wounds.OBJECTIVE1、To create a standard chronic wound model in micro pig by radiation according to different postradiation time.2、To compare the difference between radiation-injured adipose-derived stem cells (r-ADSCs) which harvested at different postradiation time and normal adipose-derived stem cells (n-ADSCs) in vivo.3、To investigate the effect of autologous adipose-derived stem cells (ADSCs) on chronic wound model.METHODS 1. Delivery of irradiationTwo female Micro pigs (Micropig(?), Medikinetics Co, Ltd.Korea)weighing30to32kg were used. Animal care was performed according to the Care and Use of Laboratory Animals. After anesthetized via intramuscular and intravenous injection of tiletamine-zolazepam (Zoletil(?); Virbac Laboratoires, Carros, France) and xylazine hydrochloride (Rompun(?); Bayer, Lerverkusen, Germany),pigs were transported to the Department of Radiation Oncology, Dongsan hospital of Keimyung University. Each pig received a single18-Gy dose per an18×8-cm area and there were three this area on back each pig(Figure1A), exposed to the linear accelerator(Clinac iX, Varian Medical system, France).Then, pigs were transported to animal lab and housed in standard condition for two weeks.2. Preparation of normal adipose-derived stem cells (n-ADSCs) and r-ADSCs2.1. Isolation and culture of normal adipose-derived stem cells (n-ADSCs)Adipose tissue excised in the nonirradiated dorsum though a6-cm-long×4-cm-wide paraspinous flap. After harvesting adipose tissue, the flap was closed with1-0nylon suture. Adipose tissue was trimmed and transferred to sterile50-ml conical tubes containing25ml phosphate-buffered saline (PBS).Then fat tissue was minced with No.10blades after washed with PBS twice. The total volume of fat tissue minced is nearly40ml for each pig, which was subsequently digested with0.075%Collagenase type Ⅰ(Worthington Biochemical Corporation) in PBS at37℃for1h under moderate constant agitation. After that, culture medium containing high-glucose DMEM and10%FBS was added to halt the enzymatic activity. After centrifuge, the supernatant was discarded, and the pellet was re-suspended and filtered through100-um cell strainer to remove tissue debris. The suspension was centrifuged again at1500rpm for5min, and re-suspended in DMEM with FBS, seeded into1000culture dishes, and incubated at37℃with5%CO2.The medium was changed. Cells at the first passage were frozen. According to schedule, cells were thawed and cultured. Cells at the third passage were used for cell therapy on wounds. Similarly, normal adipose-derived stem cells (n-ADSCs) used for cell assay were harvested from adipose tissue excised from normal wound creation as negative control group.2.2. Isolation and culture of radiation-injured adipose-derived stem cells (r-ADSCs)There was three times on wound creation totally for each pig. Every time when creating wounds, the fat tissue from radiation-injured zone was harvested, trimmed and transferred to sterile50-ml conical tubes containing25ml phosphate-buffered saline (PBS) after removing skin tissue including dermis carefully. Then tissue was washed, minced, digested and cultured as described for n-ADSCs. When the number of r-ADSCs was enough, it was used for cell proliferation assay and cell differentiation assays compare to normal adipose-derived stem cells (n-ADSCs).The latter, normal adipose-derived stem cells (n-ADSCs), was harvested from the normal wound group. According to the wound creation time, there were three groups on r-ADSCs:(1) r-ADSCs at2weeks postradiation (2R group),(2) r-ADSCs at4weeks postradiation (4R group),(3) r-ADSCs at6weeks postradiation (6R group). Similarly, three groups on n-ADSCs:(1) n-ADSCs at2weeks postradiation (2N group),(2) n-ADSCs at4weeks postradiation (4N group),(3) n-ADSCs at6weeks postradiation (6N group).3. Evaluation of radiation-injured adipose-derived stem cell (r-ADSCs) and normal adipose-derived stem cell (n-ADSCs)3.1. Cell proliferation assayR-ADSCs and n-ADSCs from wound creation were seeded at1×104cells/well in DMEM with1%FBS and10%FBS,respectively. The growth rates of the cells were determined using the Cell Counting Kit-8(CCK-8assay; company name). Media was changed every three days. CCK-8working solution was added at three-day intervals up to day30and the cells were incubated for2h at37℃. Absorbance was measured at450nm using a microplate spectrophotometer. The numbers of cells were counted in Cell Count (Korea).Cell growth curve was obtained by plotting the results. Also, cell morphology of r-ADSCs and n-ADSCs were showed by Crystal violet.3.2. Senescence-associated β-galactosidaseR-ADSCs and n-ADSCs were plated in24-well culture plates (1×104cells/well).Twenty-four hours later, the cells were washed with phosphate buffered saline and fixed with fix solution for15min and then washed thrice with phosphate-buffered saline and stained with staining solution (Senescence Detection Kit, Abeam, UK).After incubation at37℃overnight, positive cells were counted under light microscopy at100magnification.3.3. Colony Forming Units-FsR-ADSCs and n-ADSCs were resuspended and plated at a density of1×102～103cells in triplicate. Non-adherent cells were removed by media change twice a week. At day21, cells were fixed with4%paraformaldehyde for10minutes and stained with0.5%crystal violet in10%methanol for20minutes. After four washes, colonies formed by more than30fibroblast-like cells were counted under light microscope at low magnification. Results were expressed as CFU/million of nucleated cells plated.3.4. ImmunophenotypingR-ADSCs and n-ADSCs of the third passage were harvested by treatment with0.05%trypsin/0.53mM ethylenediamine tetraacetic acid (EDTA) and then washed twice with phosphate buffered saline (PBS). Cell aliquots (1×106cells/lml) were incubated for30min on ice with fluorescein isothiocyanate (FITC)-or phycoerythrin (PE)-conjugated monoclonal antibodies: CD31-PE, CD45-FITC, CD29-PE and CD90-FITC. Isotype-matched normal mouse IgGs were used as controls. Flow cytometry was performed on a fluorescence-activated cell sorter and data analysis was performed with Cell Quest software.3.5. Differentiation assayR-ADSCs and n-ADSCs were incubated with standard adipogenic or osteogenic differentiation media for7,14and20or18days, respectively. To quantify the adipogenic potential, cultures were stained with Oil Red O to show lipid droplets and analyzed by flow cytometry. To quantify the osteogenic potential, cultures were fixed with10%formaldehyde and stained with Alkaline phosphatase and Alizarin Red S, respectively. Alkaline phosphatase activity was detected by p-nitrophenyl phosphate (pNPP). Alizarin Red stain was solubilized with10%acetic acid neutralized with10%ammonium hydroxide and quantified spectrophotometrically (absorbance,492nm) in order to show mineral deposition. The effects on formation of bone-like nodules were also evaluated.4. Wound model and treatment4.1. Wound creationThere was three times on wound creation totally for each pig:(1) postradiation2weeks,(2) postradiation4weeks,(3) postradiation6weeks.Each pig was anesthetized as previously described, and was moved to animal operation room. After the hair was shaved, marking the wound on the dorsum skin with oil ink pen within a border of4cm2square,2cm from the irradiated zone boundary and6cm away from each other. The normal wound was made on another side of spine on the non-irradiated zone as negative control group. Every time in wound creation, there was three wounds including two irradiated wounds and one normal wound each pig. The two irradiated wounds were randomly divided to positive control group without ADSCs treatment and experiment group with ADSCs treatment. After marking, the skin was sterilized with betadin, and wounds were made with11#blade accurately. The excision tissue included skin and superficial fat layer without deep fat layer. The depth of wounds was approximately between1.5to2.0cm.According to the wound creation time and whether receive ADSCs treatment, there were different wounds:(1) irradiated wounds created at postradiation2weeks;(2) irradiated wounds created at postradiation2weeks with ADSCs treatment;(3) normal wounds created at postradiation2weeks;(4) irradiated wounds created at postradiation4weeks;(5) irradiated wounds created at postradiation4weeks with ADSCs treatment;(6) normal wounds created at postradiation4weeks;(7) irradiated wounds created at postradiation6weeks;(8) irradiated wounds created at postradiation6weeks with ADSCs treatment;(9) normal wounds created at postradiation6weeks.4.2. ADSCs transplantation on woundsThere were twelve irradiated wounds in total. The ADSCs-grafted group was randomly chosen from irradiated wounds. ADSCs-transplanted wounds were injected with autologous ADSCs at subcutaneous plane between the dermis and superficial fat layer, which was approximately5mm away from wound border. All wounds in ADSCs-grafted group received ADSCs injection at post wounding4weeks and6weeks, respectively. Each ADSCs-grafted wound received approximately10×106cells in2ml DMEM each time (between1×106cells to2×106cells in every1cm2of wound).4.3. Wound healing rateTo examine the rate of wound healing in different wound group, photographs of the wound region were obtained with digital camera once a week after wound creation. Photographs were uploaded to a computer and were analyzed with Visitrak Wound Measurement Device (Smith&Nephew, Australia). All photographs were taken with a sterilized paper ruler which was used for calibration and standardization, allowing subsequent quantitative analysis.4.4. Histological analysisBefore ADSCs application, biopsy was done in both irradiated wounds and normal wounds in three different time wound creation at post wounding0week,2weeks,4weeks. For all irradiated wounds with ADSCs treatment, ADSCs were applied at post wounding4weeks and6weeks. Then biopsy was done at in irradiated wounds with ADSCs treatment, irradiated wounds without ADSCs treatment and normal wounds at post wounding9weeks. Samples were fixed in10%formaldehyde, embedded in paraffin and cut into5um sections. Sections were stained with hematoxylin and eosin (H&E) for general histology analysis.To evaluate the level of histological changes more objectively, the level of acute and chronic inflammation were scored as0-4points by two investigators who were not informed of the experiment procedures. For epithelialization, applying the histological score0-2points, H&E stained slides were scored. Regarding evaluation factors, as the index of the level of acute inflammation, the numbers of neutrophils and eosinophils were scored. As the index of the level of chronic inflammation, the numbers of lymphocytes and plasma cells were counted. In addition, since the presence or absence of giant cells is closely associated with chronic inflammation, giant cells were scored as0-4points by the identical method, and analyzed semi-quantitatively.RESULTS1. Comparison between r-ADSCs and n-ADSCs1.1Cell proliferation assayAfter stained by Crystal violet, cell morphology was inhomogeneous and smaller visibly in6R group and similar among2N,4N,6N,2R and4R groups.There was no statistical difference in cell number between2N,4N and6N groups.And2R group and4R group had similar proliferation ability whereas there was statistically significant decrease in6R group (p<0.01) comparing with normal groups,2R and4R groups from cell growth curve. With the medium containing10%FBS, the proliferation rate of ADSCs in normal groups was significantly higher than those in2R,4R,6R groups (p<0.05), measured by CCK-8assay. There was no statistical difference in2R,4R,6R groups. Meanwhile, with the medium containing1%FBS as cell stress test, the proliferation rate of ADSCs in6R group was significantly higher than those in normal group,2R,4R and6R groups(p<0.05). This may be because ADSCs in6R group began to recover.Positive immunostaining for senescence-associated β-galactosidase was rarely observed in2N,4N and6N groups, but was easily seen in6R group.Viable ADSCs colony formation were significantly more abundant in2N,4N,6N groups than in2R,4R,6R groups, and the rarest in6R group(p=0.013).1.2. ImmunophenotypingTo characterize the phenotypes of r-ADSCs and n-ADSCs, flow cytometry was performed. The results showed that both of r-ADSCs and n-ADSCs expressed surface antigens such as CD29,CD90. In contrast, no expression of the hematopoietic and endothelial lineage markers (CD31and CD45) was observed.1.3. Cell differentiation assayAfter cultured in adipogenic induction media, n-ADSCs were induced into adipogenic lineage, which was confirmed by Oil Red O staining at day14,20. This adipogenic differentiation was also observed in2R and4R groups but the ability decreased progressively. The lipid contents were rarely founded in6R group either at day14or day20. The lipid contents in6R group was significantly fewer than that in normal group,2R groups (p<0.05).Meanwhile, after osteogenic induction, ADSCs differentiated into osteoblasts as demonstrated by both of Alkaline phosphatase staining and Alizarin Red S staining at day7,14,18. As early marker of osteogenic differentiation, alkaline phosphatase (AP) activity was showed in all groups on day7. On day14, observation of AP activity was hindered in all groups whereas bone-like nodules formed in all groups. On day18, AP activity increased and bone-like nodules turned to be bigger and darker than before in all groups. Interestingly, there were no significant differences in the contents of AP activity by pNPP between normal group,2R group,4R group and6R group. Mineral deposition was detected in all groups on day7, got stronger gradually in all groups on day14and18, measured by Alizarin Red S staining. On day14, comparing to normal group, Alizarin Red S staining was stronger in2R group (p<0.05) and weaker in6R group (p<0.05).However, there were no significant differences in Alizarin Red S staining between normal group,2R group,4R group and6R group at day18. Still, it was observed that the patterns of colonies were different between normal groups and radiated groups. In normal groups, the distribution of colonies equalized.2. wound healing2.1Wound healing rateFor normal wounds, the granulation tissue was fresher and grew faster on wound bed, and wound contracture was visible even at post wounding1week. But for the irradiated wounds, the granulation tissue was close to gray color and grew slowly. Compare to irradiated wounds created at postradiation2weeks, hard crust were more visible in irradiated wounds created at postradiation4weeks. And irradiated wounds created at postradiation6weeks were worse irradiated wounds created at postradiation4weeks.Analysis of wound healing rates was defined as the gross epithelialization of wound bed. Photographs of wound were taken on each week after wound creation.Closure of wounds in normal group was significantly greater than that in irradiated group without ADSCs treatment and irradiated group with ADSCs treatment. There were no significant differences in the wound healing rates between irradiated group without ADSCs treatment and irradiated group with ADSCs treatment. During the early4weeks post wound creation, before ADSCs treatment, the wound healing rate in irradiated group created at postradiation2weeks increased markedly at post wounding1week. For irradiated group created at postradiation4weeks and6weeks, this situation was delayed and happened at post wounding2weeks and3weeks, respectively. At post wound creation4weeks, the wound healing in irradiated group created at postradiation6weeks was significantly delayed (p<0.05).2.2Histology analysis on woundsAt wounding day, no inflammation was observed in all normal groups and irradiated groups. At post wounding2weeks, epithelialization is completed in all normal groups. The severity of acute and chronic inflammation is more increased in irradiated group created at postradiation4weeks and6weeks comparing irradiated group created at postradiation2weeks. Epithelialization is not completed in irradiated groups.At post wounding4weeks, acute inflammation is not present in all normal groups. In all irradiated groups, acute inflammation is more decreased than that at post wounding2weeks. However, acute inflammation is persistent. In all irradiated groups, the severity of chronic inflammation is markedly increased. The most prominent group on chronic inflammation is irradiated group created at postradiation6weeks. Epithelialization is not completed in all irradiated groups.At post wounding9weeks, acute inflammation is absent and chronic inflammation is mildly noted in all normal groups. The comparison between irradiated groups without ADSCs treatment and irradiated groups with ADSCs treatment is relatively similar finding. Chronic inflammation is markedly decreased in all irradiated groups. In irradiated groups without ADSCs treatment, chronic inflammation is slightly more increased than that in irradiated groups with ADSCs treatment. No histological difference between irradiated groups with ADSCs treatment and irradiated groups without ADSCs treatment. Epithelialization is finished in all groups.CONCLUSION1. In this study, we evaluated a chronic wound model in Micropig(?) by radiation according to different postradiation time. Our findings showed that it may be most suitable to create skin defect at postradiation6weeks when chronic wound model induced by radiation is made. Comparing to normal wounds healed in5weeks, wound healing in all irradiated wounds was delayed obviously about4weeks (Fig6.B). In all irradiated groups, the severity of chronic inflammation is markedly increased comparing to normal wounds. This suggested that ionizing radiation with a single18-Gy dose had a negative effect on wound healing and delay this procedure. At post wound creation4weeks, before ADSCs treatment, the wound healing rate in irradiated group created at postradiation6weeks was ranked the slowest, followed by4weeks and2weeks. Histological analysis confirmed that the severity of chronic inflammation was the most prominent in irradiated group created at postradiation6weeks. Ivan Hadad et al. found that after an initial increase in densities of microvessels possessing distinct lumens in skin exposed to18Gy of irradiation, microvascular densities declined dramatically over the course of the study, reaching a nadir at7weeks [6]. This supports our finding that it might be the optimal time for swine to be chosen as a chronic wound model at postradiation6weeks.2. Unlike previous studies in which the responses of stem cells on radiation were performed in vitro or with low dose radiation[9,8], we harvested ADSCs in radiation-injured zone directly. This experiment studied the radiation response of ADSCs to18-Gy ionizing radiation damage after2weeks,4weeks and6weeks of in situ damage processing, respectively. B. Mvula revealed that low level laser radiation can positively affect human adipose stem cells by increasing cellular viability, proliferation[8]. Nils H. Nicolay et al. revealed that human bone morrow-derived mesenchymal stem cells were able to retain their defining stem cell characteristics both on a functional level and regarding stem cell marker expression after exposure to10-Gy ionizing radiation [9]. Cmielova J showed that radiation with the doses up to20Gy significantly reduces proliferation of human bone morrow-derived mesenchymal stem cells [14]. Our data showed that r-ADSCs expressed surface antigens such as CD29, CD90and did not do the hematopoietic and endothelial lineage markers (CD31and CD45), which is same as n-ADSCs. The ability of proliferation, adipogenic differentiation on r-ADSCs in6R group decreased significantly comparing to r-ADSCs both in2R and4R group. Unlike the data suggested by Gaissmaier C that irradiation inhibits the initial phase of in vivo osteogenesis due to the cytostatic effect [15], our data revealed there were no significant differences in the contents of AP activity by pNPP between normal group,2R group,4R group and6R group. On day14, comparing to normal group, Alizarin Red S staining was stronger in2R group (p<0.05) and weaker in6R group (p<0.05).However, there were no significant differences in Alizarin Red S staining between normal group,2R group,4R group and6R group at day18. It may implicate that osteogenic differentiation on ADSCs was increased transiently in the early period after ionizing radiation. Panagiotakos G demonstrated that long term radiation injury is associated with irreversible damage to the neural stem cell compartment in the rodent subventricular zone[16]. The next step envisioned for this chronic model is to explore recovery procedure of radiation-injured ADSCs or continuation of radiation injury on ADSCs.3. Swine was chosen as animal model of wound healing because of the similarity to human skin anatomy, physiology and biochemistry [4]. It also responds similarly to radiation exposure [5] However, Yorkshire pigs used previously [6] grow rapidly during experiment, which hinders assessment of wound healing rate [7]. In our study, Micropig(?) was chosen as animal model in order to investigate wound contraction. Micropig(?) scarcely grows with restriction of feed during experiment, which makes itself a more adequate model to observe wound healing with time. Then, autologous ADSCs were harvested and applied on irradiated wound bed at post wounding4weeks. Lots of studies showed that the beneficial effects of stem cell transplantation are due to paracrine mechanisms with local release of cytokines to demonstrate the angiogenic potential and enhance wound healing in injured tissues [1,17,18,19,20,21]. At post wounding creation4weeks, either autologous ADSCs or medium was used on these chronic wound models. In irradiated groups without ADSCs treatment, chronic inflammation is slightly more increased than that in irradiated groups with ADSCs treatment. At post wounding creation6weeks,2weeks after the first time ADSCs injection, wound healing rate in irradiated groups with ADSCs treatment was faster than that in irradiated groups without ADSCs treatment. However, there were no significant differences in the wound healing rates between irradiated group without ADSCs treatment and irradiated group with ADSCs treatment. No histological difference between irradiated groups with ADSCs treatment and irradiated groups without ADSCs treatment. Future study will explore the better application method of autologous adipose-derived stem cells on chronic wounds.In conclusion our findings suggest that it may be most suitable to create skin defect at postradiation6weeks when chronic wound model induced by radiation is made. The ability of proliferation, adipogenic differentiation on r-ADSCs in6R group decreased significantly comparing to r-ADSCs both in2R and4R group. Interestingly the ability of osteogenic differentiation on r-ADSCs was the strongest in2R group. Single injection of autologous ADSCs is useful to epithelialization rate whereas no significant difference was found. |
PDF Full Text Request