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.