3.1. Construction and identification of recombinant human-like collagen
Using genetic engineering technology, we constructed RHC according to the construction schematic shown for recombinant plasmid of pET3c-hlcollagen in Fig. 1A. The results from nucleic acid electrophoresis in Fig. 1B showed that the RHC gene was successfully ligated into the expression vector. After induction of E.coliBL21 containing recombinant plasmid RHC with IPTG at either 30 °C or 37 °C for 4h, or 20 °C overnight, soluble RHC was expressed at the highest level at 37 °C (Fig. 1C). Subsequently, RHC protein was purified using affinity chromatography on a NI Sepharose 6 Fast Flow column combined with gel filtration Sephadex G-25. Purity of RHC were detected by SDS-PAGE gel electrophoresis (Fig. 1D) and western blot (Fig. 1E), and in both of them a single band was obtained. Identity were confirmed by MALDI-TOF MS and HPLC-based peptide mapping (the data were not shown). We conducted at least three consecutive batches of 50L fermentation process tests, and the mean of RHC protein expression accounted for 48% of the total protein in the bacterial cell supernatant (the data were not shown).
3.2.RHC/EGF promotes proliferation and migration
Fibroblast proliferation is critical for rapid closure of wounds during wound healing. We performed cell proliferation experiments on NIH/3T3 cells to detect the proliferative activity of RHC, EGF and RHC/EGF. Cell proliferation experiments found that RHC did not promote the proliferation of NIH/3T3 cells. But EGF and RHC/EGF (1: 1) can significantly promote cell proliferation (Fig. 2A). On the other hand, Keratinocytes (HaCaT cell) are essential for effective re-epithelialization and ECM construct during wound healing(Piperigkou, G?tte, Theocharis, & Karamanos). HaCaT cell are regarded as a good in vitro model of the skin epidermal layer, and can be used to assess the therapeutic effects of compounds on tissue regeneration.(Burlando et al.) In vitro wound healing assay using a created wound gap by HaCaT cells showed that, in RHC and RHC/EGF group, wound closures by cell migration in HaCaT cell were significantly promoted compared to those of non-treatments or EGF group (Fig. 2B). After 12 h and 24 h incubation in HaCaT cells, markedly higher wound closure percentages were monitored. The migration characteristics of HaCaT cells on RHC, EGF, and RHC/EGF were examined by scratch wound assay (Fig. 2B). After 24 h incubation in HaCaT cells, a significantly higher wound closure percentages were monitored in RHC (78 ± 3.12%) and RHC/EGF (91.6 ± 1.62%) versus those in Control (46.3 ± 1.82%) and EGF (62.3 ± 2.12%), showing a significant difference (P <0.01) (Fig. 2C). The proliferation and migration of fibroblasts and keratinocytes play a vital role in the rapid healing of wounds. Experiments show that RHC has no obvious effect on the proliferation of NIH/3T3 cells, but can significantly promote HaCaT cell migration. In contrast, EGF can significantly promote NIH/3T3 cells, but has no obvious effect on HaCaT cell migration. When we combined RHC and EGF, we found that they have a synergistic effect. RHC/EGF can both promote the proliferation of NIH/3T3 cells and migrate HaCaT cells. Among them, RHC/EGF and EGF have similar activities to promote the proliferation of NIH/3T3 cells. Surprisingly, compared with RHC and EGF, RHC/EGF has a significant effect on promoting HaCaT cell migration. This is beneficial for accelerating wound healing. These data also clearly show that RHC/EGF have the potential for use in cell therapies to treat cutaneous wounds.
3.3. RHC- EGF promote NIH/3T3celladhesion and extension
It has long been known that cell adhesion importantly influences the cell proliferation, migration, differentiation, and even the assembly of individual cells into the three-dimensional tissues of animals.(Gumbiner, 1996) To investigate the effect of RHC, EGF or RHC/EGF on cell adhesion, NIH/3T3 cells were cultured in serum free medium and plated on 96-well plates coated with RHC, EGF or RHC/EGF for 2 h. The cell adhesive activity of the NIH/3T3 cells was then evaluated by crystal violet assay. As shown in Fig. 2D, RHC and RHC/EGF groups found to significantly promoted cell adhesion compared with the control and EGF groups. Among them, the RHC/EGF group had the largest number of cells attach and were well-spread and exhibited the typical fibroblast cell morphology. We also determined the average number of cells that attached to monolayers presenting control, RHC, EGF and RHC/EGF (Fig. 2E). The largest number of cells attached to RHC/EGF, with approximately 130 cells attached per field of view. This significant decrease in adhesion at control and EGF reveals, attachment dropped to 20-30 cells per field.
To further analyze the adhesion activity of RHC/EGF to NIH/3T3 cells. In a separate experiment, we allowed cells to adhere for four hours to each of the monolayer substrates and then fixed and stained the samples to compare the development of the cytoskeleton (Fig. 2F). We observed that cells spread well on the RHC/EGF surfaces, and these cells also displayed organized actin cytoskeletons spanning the entire cells, indicating that the biological adhesion between them and substrates experienced a progression from substrate attachment, spreading, and cytoskeleton development. In contrast, we found a lack of organized actin structures in cells attached to control and EGF. In order to quantify the spread of attached cells, we calculated the cell area by image J (Fig. 2G). The results show that the spread area of NIH/3T3 cells in RHC/EGF is significantly larger than that of control and EGF. These results indicated that RHC/EGF has considerable adherent activity on NIH/3T3 cells. Previous studies revealed that collagen, the major structural protein found in the ECM of many tissues, is rich in arginine-glycine-aspartic acid (RGD), a cell adhesion motif that is capable of promoting cell adhesion.(Shekhter, Fayzullin, Vukolova, Rudenko, & Litvitsky, 2019) RHC we construct through genetic engineering has RGD. We speculate that exposure to RGD is the main factor that accounts for the enhancement of cell adhesion by RHC.