Our experiments have demonstrated that the Bacterial Cellulose(BC) has good biocompatibility; cells infected with corresponding Adeno-associated virus (type 6, AAV6) can significantly increase the expression level of growth factor (EGF) and antimicrobial peptide (LL-37), which verifies that BCAID has the basic function of the auxiliary healing module; the blue light system can successfully initiate cellulase expression and achieve the degradation of BC membranes; the red light system eventually enables the apoptosis of engineered exogenous cells. As a whole, our results preliminary proved that our products is safe and efficient to help our targeted users to recover from severe damage of dermis. And we successfully manufacture our hardware, which can be used with our BC scaffold for adjuvant therapy.

BC as the tissue engineering scaffold for large areas and deep trauma makes it easier for cells to cling and proliferate. And finally the wound can heal more quickly and even restore to its original state with the help of BC. We therefore designed experiments on BC co-culture with human cells to demonstrate that BC has good biocompatibility, allowing the climbing and proliferation of our exogenous cells as well as the cells of the wound itself. We seeded HEK293T cells, BGC823 cells, and fibroblast ATCC CRL-2522 (BJ) on treated 1 cm×1 cm square BC membranes, and observed the growth status of the cells on the membrane at 1-2 days after inoculation. We observed under the microscope and found that all three types of human cell lines were well attached to and proliferated on the BC membranes (Fig.1). According to this result, we demonstrated that the BC has good biocompatibility.

Fig.1 Co-culture of BC membrane and cells for 2 days. a. BC membrane of uninoculated cells under 10× objective lens. b,c. HEK 293T cells under 4× and 20× objective lens. d. HEK293T cells adherent to the wall under 10× objective lens. e,f. BGC-823 cells under 10× and 20× objective lenses, respectively. g. BGC823 cells adherent to the wall under 20× objective lens. h,i. BJ cells grown under 10× and 20× objective lenses. j. BJ cells adherent to the wall under 10× objective lens.

Although our BC tissue engineering scaffold itself can promote wound healing, we added an AAV auxiliary module to accelerate the growth and proliferation of endogenous cells, and improved the antimicrobial ability. We constructed a recombinant AAV plasmid containing the target genes EGF and LL-37, and stably transfected the recombinant AAV6 into HEK293T cells for ELISA experiments to verify the ability to infect the cells and the expression level of the target genes.

Fig.2 shows the results of ELISA measurement of EGF content in the cell culture supernatant. According to the results, the level of EGF in the supernatant of the experimental group was 107.25% higher than that of the control group after 24 hours of incubation; the level of EGF in the supernatant of the experimental group was 53.10% higher than that of the control group after 72 hours of infection (Fig. 2b, 2c).

Fig.2 Determination of EGF content in cell culture supernatant by ELISA. a. The standard curve of EGF. b. Table of EGF content in culture supernatant of experimental group and control group at different time. c. Comparison of EGF content in culture supernatant between experimental group and control group at 24h or 72h.

Fig.3 shows the results of the ELISA measurement of the LL-37 content in the cell culture supernatant. As can be seen from the results, the level of LL-37 in the supernatant of the experimental group was 1746.71% higher than that of the control group after 24 hours of infection (Fig. 3b, 3c); the level of LL-37 in the supernatant of the experimental group was 75.28% higher than that of the control group after 72 hours of infection (Fig. 3b, 3c).

Fig.3 Determination of LL-37 content in cell culture supernatant by ELISA. a. The standard curve of LL-37. b. Table of LL-37 content in culture supernatant of experimental group and control group at different time. c. Comparison of LL-37 content in culture supernatant between experimental group and control group at different time.

The above results proved that the expression of EGF and LL-37 could be effectively increased by infecting cells with corresponding adeno-associated viruses, and the increase rate is high.

When our wound is completely healed, the BC tissue engineering scaffold can be also retired. Because the human body itself does not have the enzymes to degrade the BC, we hope to infect the BJ cells attached to the BC membranes through lentivirus, so that they can express the cellulase under blue light control, and the cells have the ability to degrade the BC.

To verify that the system we constructed could express and secrete the cellulase following activation by blue light , we harvested the lysates of the transiently transfected HEK293T cells and performed the SDS-PAGE and Western blot analysis.

Western blot results (Fig.4) showed that the cells transfected with empty plasmids and plasmids inserted with cellulase could express GAVPO protein (an important element of LightOn system on the plasmid scaffold), suggesting that our plasmids were successfully transferred into cells.

Fig.4 Western blot analysis of GAVPO. a. GAVPO is a constitutive expression protein (56 kDa) on the plasmid scaffold, which is dimerized under blue light (470 nm); b. GAPGH is a housekeeping protein (36 kDa) as internal reference control to normalize protein expression.

Combining SDS-PAGE and Western blot, the cells could express the three cellulases, Cex, CenA and Bglx respectively under blue light illumination (Fig. 5a,b). To test whether the cellulase was secreted outside the cells, we collected the cell culture supernatant for Western blot, and the results showed that the endoglucanase (CenA) was successfully secreted into the medium following transfection (Fig. 5c).

Fig.5 Western blot analysis of cellulase. The molecular weights of CenA, Cex and Bglx are about 48 kDa, 51 kDa and 66 kDa, respectively. a. and b. Western blot analysis of cell lysates. c. Western blot analysis of culture supernatant.

Collectively, these results demonstrated that the blue light system we constructed can activate the expression and secretion of cellulase under blue light control.

We have verified that the blue light system could be initiated and trigger the expression of cellulase. Furthermore, we co-cultured the tranfected cell supernatant and the BC membrane to validate whether the cellulase secreted by the cells could degrade the BC tissue-engineered scaffold.

We observed the BC membranes under the microscope every 2 hours after adding the supernatant of the different experimental groups and the positive control group. According to the co-culture results (Fig. 6), the BC membrane was basically degraded in the positive control group one day later. Three days later, the surface of the BC membrane in well D (1:1 pCex, pCenA after ultrafiltration concentration) was irregular and cracked. Under the microscope, there were obvious cracks and flocculent floaters on the surface of the membrane, while the degradation of well C (1:1:1 pCex, pCenA, pBglx after ultrafiltration concentration) was unclear and only a small amount of flocculent substances appeared. These results showed that cellulase did exist in the culture medium, which could degrade BC membrane at a low speed.

Fig.6 Degradation of (BC) membrane by cellulase. The picture shows that the degradation of BC membrane in 6-well plate after coculture with cell supernatant for 3 days under the 4X microscope field of vision. a. Add PBS into well A as a negative control; b. Add 1ml 25mg/ml of commercial cellulase solution into well B as a positive control; c. Add 0.5ml of supernatant of cells tranfected with pCex, pCenA and pBglx followed by ultrafiltration concentration at 1:1:1 in well C. Add DMEM medium to 2ml; d. The supernatants of cells tranfected with pCex and pCenA followed by ultrafiltration concentration were added into well D at 1:1, respectively 750 uL. Add DMEM medium to 2ml.

After the degradation of BC membrane, the remaining BJ cells from the scaffold also have biosafety risks. Therefore, we designed a red light-controlled suicide system to induce BJ cell apoptosis under red light (660 nm) illumination.

To confirm whether the toxin protein MazF and VP64 red light elements could successfully expressed, we transiently introduced the REDMAP plasmid into HEK293T cells and analyzed the lysates by SDS-PAGE and Western blot. The cell lysates in SDS-PAGE (Fig. 7 a) of the control and experimental groups both exhibited protein bands around 14 kDa and 31 kDa. Combined with Western blot (Fig. 7 b and c), we found that the bands in the experimental group were MazF and VP64 red light element, while the control group were unspecific proteins of similar size.

Fig.7 a. REDMAP SDS-PAGE. VP64 stripes in the red box and MazF stripes in the green box. CG-C was the cell lysate of the control group and EG-C was the cell lysate of the experimental group. b. Western blot of VP64. CG-C is the cell lysate of the control group, EG-C is the cell lysate of the experimental group, and the theoretical size of VP64 is 30 kDa. c. Western blot of MazF. CG-C is the cell lysate of the control group, EG-C is the cell lysate of the experimental group, and the theoretical size of MazF is 13 kDa.

Together, these results demonstrated that HEK293T cells transiently infected with the REDMAP lentivirus can successfully express the VP64 red light element and the toxin protein MazF.

We observed the cells of the experimental group and the control group 46 hours after adding the transient transfection system. It was obvious that most of the cells in the experimental group were dead, while they became less adhesive, smaller, rounded and brighter, and began to detach in large areas; the cells in control group continued to grow with a normal morphology (Fig.8). This phenomenon suggests that the toxin protein MazF can cleave RNA and eventually induce apoptosis in response to red light.

Fig.8 Cell morphology of control group and experimental group under red light illumination. The cells in the experimental group were tranfected cells, while the cells in the control group were non-transfected cells.

In the future, we will design more and richer experiments to further verify the functions of each system.

We successfully manufacture the enclosure device by 3D printing and iteratively update LED functional circuit board to produce different colors of light. To collect the wound information, we installed a camera on the hardware. And we develop a mobile App to observe the state of fibroblasts growing on the BC membrane, the state of the BC membrane, and the process of wound recovery photoed by the camera. For more details: hardware.

Fig.9 The scene of 3D printing