Effects And Mechanism Of Cryopreserved Living Micronized Amnion In Accelerating Diabetic Wound Healing

BackgroundDiabetic foot ulcer is a serious complication of diabetes mellitus, and approximately 25% of diabetic patients suffer from diabetic lower-extremity ulcer throughout their lives. Even with the standard therapies, including moist dressing, debridement, infection control, and wound offloading, these wounds still slowly heal, and 7%–20% of patients will eventually need an amputation despite standard care treatment. The development of new therapies to treat diabetic wounds and prevent foot ulcers from leading to amputations is thus urgent.Human amnion is the innermost layer of the placenta and consists of a thin epithelial layer, a thick basement membrane, and an avascular stroma. The use of amniotic membranes in skin transplantation was first reported in the early 1900 s and showed great potential in promoting wound healing. For the last 100 years, the therapeutic potential of human amnion/chorion tissue grafts in wound healing has been well established by hundreds of published paper, mainly by promoting epithelialization, alleviating inflammation, inhibiting scar formation and anti-infection. In addition, human amniotic epithelial cells(HAECs) retain a high level of pluripotency and amnion-derived cellular cytokine solution greatly improves wound healing in acute and chronic wound models. Recently, it has also been reported that HAECs promote acute wound healing in mice. Despite the great potential of amnion in treating various wounds, some drawbacks should be overcome to maximize its clinical potential. First, the present preservation techniques, such as freeze-drying, gamma sterilization or glycerol preservation, lead to the complete loss of HAECs viability of amnion. Second, human amnion is very thin with low mechanical strength, thus amnion in the form of large sheets does not easily completely flatten and adhere to the wound, and gradually dries, only used clinically as a temporary biological dressing. In additon, the therapeutic characteristics and mechanisms of HAECs directly used to treat chronic wounds, such as diabetic ulcers, are currently unknown.In this study, we processed human fresh amnion into micronized(300–600μm) amnion and further cryopreserved it in a serum-free stem cell cryopreservation medium for 6 months. The intact structure, cell viability and biologically active components of micronized amnion after cryopreservation were evaluated. Post-thawing micronized amnion was then transplanted to the wounds of db/db mice to evaluate its effect on repairing full-thickness skin defects. We found that the intact structure, cell morphology, and cell viability of the amnion were well-preserved by this cryopreservation method. Topical administration of cryopreserved living micronized amnion(LMAM) significantly promoted diabetic wound healing in the db/db mouse model, and the HAECs of the cryopreserved LMAM possessed a high engraftment potential after transplantation. The mechanisms were further explored, mainly focusing on inflammation and neovascularization through in vivo and in vitro experiments, and the role of amniotic matrix as a long-term dermal scaffold. Materials and Methods1. Human placentas(20 fetuses: 10 males, 10 females) were directly obtained from parturients and cut by self-made microskin cutter to obtain 300–600μm microparticles to be used as LMAM. The LMAM were resuspended in STEM-CELLBANKERTM and then preserved in a liquid nitrogen tank for 6 months. The effect of cryopreservation on LMAM was evaluated by the following examinations and fresh LMAM was used as control.(1) H&E staining was performed to explore the structure of post-thaw LMAM(2) Live/dead assay was conducted to detect LMAM viability(3)Scanning electronic microscopy was performed to detect cell membrane structure of HAECs(4) Living micronized amnion conditioned medium(LMAM-CM) was collected and the expression of growth factors, chemokines, and inflammatory cytokines in LMAM-CM was detected by using human growth factor antibody array G1,human chemokine array G1,and human inflammation antibody array G1(Ray Biotech, Inc.)2. The db/db mice were randomly and equally divided into three groups: LMAM group, DMAM(decellularized micronized amnion) group, and blank group. Cryopreserved LMAM or DMAM was smeared on wound surface of the mice from the LMAM and DMAM groups, respectively. The following tests were performed to evaluate the effect of cryopreserved LMAM on diabetic wound healing after transplantation.(1) Photographs of gross appearance of the wounds were taken regularly and the wound healing rate was calculated.(2) H&E staining was performed to explore the healed wound structure and LMAM degradation.(3) H&E staining and immunostaining against human mitochondria antibody were used to trace the fate of transplanted HAECs, and the TUNEL assay was conducted to detect HAEC apoptosis.(4) Immunostaining against F4/80 was performed to count the number of macrophages and flow cytometry was used to identify M1 or M2 macrophages in the wounds. Expression levels of the pro-inflammatory cytokines and healing-associated cytokines in the wounds were measured using ELISA.(5) Flow cytometry were performed to count the number of EPCs in peripheral blood, and immunostaining against CD34 and CD31 was conducted to calculate the number of hematopoietic stem cells and blood vessels in the wounds, respectively.3. LMAM-CM was collected and the following tests were performed to evaluate the effect of LMAM-CM on the function of bone marrow-derived mouse macrophages, HUVECs,human fibroblasts and keratinocytes.(1) Cell proliferation of macrophages,HUVECs,fibroblasts and keratinocytes was assessed using a CCK-8 kit.(2) Cell migration of macrophages, HUVECs,fibroblasts and keratinocytes was performed using transwell chambers assay.(3) Angiogenic effect of LMAM-CM on HUVECs was measured by tube formation assay.(4) M1 macrophages was induced by IFN-γ and TNF-α. The effect of LMAM-CM on macrophages polarization was evaluated by observing morphological changes of macrophages under microscope, and real-time PCR to measure M1 and M2 macrophages marker gene. Results1.The viability of the cryopreserved LMAM after thawing was determined using the live/dead assay and compared with the result of fresh LMAM, showing that 92.94% ±2.27% and 73.08% ±2.95% of HAECs were viable in the fresh and cryopreserved LMAM, respectively. H&E staining revealed that, similar to that in the fresh LMAM, a single layer of HAECs was attached to a thick basement membrane in the cryopreserved LMAM immediately post-thawing. Further scanning electronic microscopy analysis showed that the membrane structure of HAECs in the cryopreserved LMAM group remained intact immediately post-thawing and was similar to that in the fresh LMAM group. No pores were seen on HAECs membrane in the cryopreserved LMAM group post thawing. Of the 41 growth factors represented in the human growth factor antibody array, AR, EGFR, CSF2, CSF3, HB-EGF, HGF,IGFBP-2, IGFBP-3, CSF1 R, NT-3, PDGFR, and TGF-β1 in LMAM-CM showed the highest expression levels. Of the 38 chemokines represented in the human chemokine antibody array, GRO, IL-8, MCP-1, and MIP-1b in LMAM-CM revealed the highest expression levels. Of the 39 inflammatory cytokines represented in the human inflammation antibody array, IL-6, IL-8, MCP-1, MIP-1b, TGF-β1, TNF-β, and TIMP-2 demonstrated the highest expression levels. Moreover, nearly all types of cytokines in LMAM-CM showed much higher expression levels than those in AECs-CM.2.1 At day 7 after wounding, the cryopreserved LMAM survived well, and the wound was moist and ruddy. At day 21 after wounding, most of the wounds in the cryopreserved LMAM group completely healed; the average wound healing rate in this group was 94.64% ±2.67%, which was significantly greater than that in the cryopreserved DMAM group(80.52% ±3.85%, p < 0.001) and blank group(68.40% ±5.37%, p < 0.001). At day 35 after transplantation, H&E staining of the sections showed that the dermal matrix was filled with microparticles in the cryopreserved LMAM and cryopreserved DMAM groups. In addition, an epidermal papillae-like structure was observed in the cryopreserved LMAM group, whereas no such structure was observed in the cryopreserved DMAM and blank groups. Thicker dermal layers were also observed in the cryopreserved LMAM and cryopreserved DMAM groups compared with the blank group. The LMAM grossly degraded 2 months after grafting observed through H&E staining.2.2 At day 5 after transplantation, HE staining and immunostaining showed that the HAECs of the cryopreserved LMAM survived well in the diabetic wounds and displayed tight adherence to the amniotic matrix and a tendency for multilayer growth, with only very few amniotic cells migrated elsewhere. In addition, TUNEL assay demonstrated that apoptosis of few HAECs had begun 7 days after transplantation. Immunostaining against human mitochondria antibody demonstrated that the HAECs were not observed in diabetic wounds 28 days after transplantation.2.3 The cells that stained positively for F4/80 were more prevalent in the wounds of the cryopreserved LMAM group than in those of the cryopreserved DMAM and blank groups 5 days after wounding. Interestingly, the number of infiltrated macrophages significantly reduced in the cryopreserved LMAM group 10 days after wounding than in the cryopreserved DMAM and blank groups. The cryopreserved LMAM significantly reduced M1 macrophages and increased M2 macrophages in day 10 wounds as determined by FACS analysis, whereas the cryopreserved DMAM did not significantly affect M1 or M2 macrophages in the wounds. The expression levels of IL-1β, TNF-α, and IL-6 significantly reduced in the cryopreserved LMAM-treated wounds compared with those in the other two groups. By contrast, the levels of TGF-β1, IGF-1, and VEGF significantly increased in the cryopreserved LMAM-treated wounds.2.4 At day 5 after transplantation, the percentage of EPCs in the peripheral blood was significantly higher in the cryopreserved LMAM group than that in the cryopreserved DMAM group and blank group. Moreover, the cells that stained positively for the hematopoietic progenitor cell marker CD34 were more prevalent in the wounds of the cryopreserved LMAM group than those in the other two groups 5 days after transplantation. Gross observation revealed that neovascularization was more obvious in the cryopreserved LMAM group than that in the other two groups at days 14 and 21 after wounding. This finding was consistent with the immunohistochemical results, showing that capillary density was much higher in the cryopreserved LMAM group at day 14.3. The macrophages treated with control media adhered onto the plate as a small round structure that failed to spread. IFN-γ + TNF-α caused many macrophages to project numerous protrusions, resulting in dendritic morphology, whereas IFN-γ + TNF-α + LMAM-CM induced elongation of the cells, which did not display numerous projections as observed in the cells grown in the IFN-γ + TNF-α cell medium. Furthermore, real-time PCR was used to further investigate the markers of the M1 or M2 macrophage. LMAM-CM strongly upregulated the M2 macrophage marker CD206 and downregulated the M1 marker CCR7. Moreover, LMAM-CM demonstrated considerable effect on the migration of macrophages, but not on the proliferation of macrophages. We examined the tube formation of HUVECs on Matrigel and determined its ex vivo angiogenic capacity. The LMAM-CM-treated HUVECs formed more networks than negative control group. In addition, LMAM-CM significantly promoted the migration and proliferation of HUVECs,fibroblasts and keratinocytes. Conclusions1. The intact structure, cell morphology, cell viability and biologically active components of the amnion were well-preserved by this cryopreservation method.2. Topical administration of cryopreserved LMAM significantly promoted diabetic wound healing in the db/db mouse model, and the HAECs of the cryopreserved LMAM possessed a high engraftment potential after transplantation.3. Cryopreserved LMAM significantly accelerated diabetic wound healing mainly by regulating inflammation, recruiting stem cells, promoting neovascularization, and making the amniotic stroma act as a dermal substitute.