| Skin injuries in burns, traumas as well as ulcers derived from various chronic diseases often destroy the structure of skin tissues, resulting in wound exposure and increased chances for bacterial invasion. Autologous skin grafting is a conventional therapeutic strategy for small skin damages. However, in cases of large skin loss, this method could be infeasible because of limited skin supply. Autologous skin grafting itself is with an additional injury to normal skin. Allogeneic and xenogeneic skin substitutes have been taken into consideration for clinical use, but immune rejection of the host to allografts and xenografts remains a serious challenge to overcome. Skin tissue engineering technology plays a significant role under these circumstances. Engineered skin-structural and functional tissue is formed by a creation of biomimetic scaffolds as extracellular matrix(ECM) to facilitate cell adhesion, proliferation and differentiation, which ultimately achieves the goal of skin reconstruction. Cell-compatible polymeric ultramembranes are widely used in tissue engineering. Here, we report a free-standing nanomembrane developed by a layer-by-layer(LBL) self-assembly technique and safe sacrificial substrate method. After ionizing, two oppositely charged polyelectrolytes alginate and chitosan were alternately deposited on a substrate of solidified gelatin block to fabricate an ultrathin nanomembrane. The space between two adjacent layers was about 200 nm. The thickness of the nanomembrane was proportional to the number of layers. The temperature-sensitive gelatin gel served as a sacrifice template. When gelatin was dissolved in medium at 37℃,the membrane automatically released from its disappearing substrate. In comparison to other fabrication methods, this new method shows its particular advantages. First of all, the free-standing nanomembrane separated from the gelatin base was obtained via a very ingenious and mild way, which reduced the mechanical damage for the membrane during the fabrication and then guaranteed its further study. Secondly, it largely shortened the fabrication time and reduced the materials for nanomembrane fabrication. At last, no organic materials were used during the fabrication, which was helpful to keep the biocompatibility of the nanomembrane. Culture of bone marrow mesenchymal stem cells(BMSCs) showed that the free-standing nanomembrane promoted cell adhesion and proliferation. BMSCs seeded on the membrane showed significantly higher survival and proliferation when transplanted to mouse dorsal skin with a full-thickness burn. This methodology therefore provides a fast and facile approach to construction of free-standing ultrathin scaffolds for tissue engineering. The biocompatibility and free-standing feature of the fabricated nanomembrane may be particularly useful for stem cell delivery and tissue reconstruction for burn wound healing.Method1. Fabrication of CHI/ALG free-standing nanomembrane by LBL self-assembly Positive charged chitosan solution and negative charged alginate solution were alternately deposited on a substrate of solidified gelatin solution(20% w/v) at room temperature(20℃) until achieving the expected layers(15 layers). Then the nanomembrane-gelatin complex was transferred into 37℃ distilled water. Finally a free-standing nanomembrane was obtained as the solid gelatin block gradually melted.2. SEM and TEM The nanomembrane was respectively observed under the SEM and TEM for its superficial and LBL structure after a series of appropriate processing.3. Water contact angle testing for each layer of CHI/ALG nanomembrane Different layers(from layer 1 to layer 10) of free-standing CHI/ALG nanomembranes were constructed by LBL. Then water contact angles of each layer were measured by the Contact Angle Measuring Machine.4. Cell culture in vitro BMSCs were firstly seeded on the nanomembrane in vitro, then LIVE/DEAD viability/cytotoxicity staining was used to test the vitality of the cells on the free-standing nanofilm after 3 days in culture. And cells cultured on the CHI/ALG nanomembrane and blank 96-well plates respectively were tested for their viability by the MTT assay.5. In vivo tests Make mice full-thickness skin defected models and set experiment groups as well as control groups. In the experiment groups, the wounds were covered with the CHI/ALG nanomenmbrane while without that in the control groups. GFP-BMSCs were respectively seeded on the wounds of each groups to messure the fluorescent intensity by the in vivo imaging at different time point. Use the FACS to analyze the percentage of the GFP-positive mesenchymal stem cells harvested from the wounds and investigate the condition of wound healing at different time point in each group. Prepare frozen sections of every wound newly-formed tissue to observe the survival condition of BMSCs. Finally, observe the condition of epithelization by wound tissue HE staining.Results1. The CHI/ALG nanomembrane fabricated by LBL self-assembly is a biomimetic scaffold with 3D structure, and the space between two adjacent layers is about 200 nm, the whole thickness was in direct proportion to the number of total layers. It was demonstrated that LBL process was a success. The structure of the namomembrane varies as the material depositing. Water contact angles changed from layer to layer since the different hydrophilia of the two materials.2. BMSCs were cultured on the nanomembrane in vitro. More than 99% of the BMSCs showed a healthy appearance after 3 days in culture. A number of dividing BMSCs on the nanomembrane indicated its superb biocompatibility. Then, cells cultured on the CHI/ALG nanomembrane and blank 96-well plates respectively were tested for their viability by the MTT assay. After 72 h, the relevant concentration of the cells on the nanomembrane was prominently higher than that cultured in the blank 96-well plate.3. Total intensity of the GFP signal in the experimental group was significantly higher than that in the control group. The average signal intensity in the experimental group was 25.97±6.98(×104)while that in the control group was 6.00±2.84(×104), P<0.05.4. FACS results: The results showed that the ratio of living GFP-MSCs to all living cells The percentage of the GFP-MSCs on Day 5 was 33.58% in the control group and 66.78% in the experimental group, and on Day 7, 33.45% and 53.03% respectively. Statistical analysis of the data indicated that the CHI/ALG nanomembrane significantly enhanced survival of the transplanted cells. There was no significant difference between the two different time points in the same group.5. Mice wound healing after surgery: observe the condition of mice wound healing in all groups on day 1, 3, 5, 7, 9 after surgery respectively and measure the percentage of the remaining wounds. The results showed that all the wounds decreased as time went on. After one week, the wounds of the nanomembrane-MSC group showed significantly better recovery than the other groups at the same time point, the wounds of the blank control group showed the worst healing. On day 9, wounds of the nanomembrane-MSC group were nearly healed, and the percentage of the unhealed wound was statistically significant less than the other three groups, P <0.05.6. Frozen sections of the newly-formed tissue of the mice wounds showed that the fluorescent density and intensity in experimental group was obviously higher in comparison to that in control group. Images with same scale and size(800?m × 600?m) in each group were randomly selected to count the number of GFP-BMSCs. The average amount of green fluorescent cells in the nanomembrane group was 101.00 ± 15.51 while that in the control group was 25.25 ± 5.07, P<0.05.7. At 3d postsurgery, no significant difference was found for the length of the epithelial tongue of the wounds in the four groups. At 7d postsurgery, the length of the epithelial tongue in the nanomembrane+BMSCs group(1099.8μm) was notably longer than that in the nanomembrane group(764.4μm, P <0.05), BMSCs group(837.3μm, P <0.05) and control group(533.1μm, P <0.01).ConclusionA new fabrication for an ultrathin free-standing CHI/ALG nanomembrane was studied using a temperature-sensitive material as the base of LBL self-assembly, which reduced the mechanical damage for the membrane during the fabrication. It largely shortened the time for the manufacture and reduced the applied materials. At the same time, no organic materials were used during the fabrication, which was helpful to keep the biocompatibility of the nanomembrane. It also provided the condition for further study by seperating the nanomembrane from the gelatin base. The nanomembrane showed good biocompatibility without foreign material rejection and cytotoxicity. It had promoted the adhesion and proliferation of BMSCs when applied in vivo, which ultimately enhanced the survival rate of cells and promoted wound healing. The CHI/ALG nanomembrane is an ideal candidate of cell scaffolds. |
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