Biofabricated macrophage and fibroblast membranes synergistically promote skin wound healing

Abstract Effective skin wound healing is a complex process involving anti‐inflammation, fibrosis, matrix reconstruction, and angiogenesis. This work aimed to integrate the macrophage‐mediated anti‐inflammation and fibroblast‐assisted matrix reconstruction for enhanced skin wound healing. Herein, we utilized the cytomembranes derived from repolarized M2 macrophages and fibroblasts to prepare the natural biologics. Results showed that the inflammatory M1 macrophages were repolarized to M2 phenotype by the M2 macrophage cytomembranes. As a consequence, the cytomembranes of M2 macrophage could facilitate the wound closure in mice. Furthermore, the addition of fibroblast membranes to the macrophage cytomembranes contributed to a better matrix reconstruction, neovascularization and angiogenesis. Next, we used a transforming growth factor‐β (TGF‐β) inhibitor to attenuate cutaneous scar formation. Therefore, our modality could promote skin wound healing and effectively suppress scar formation in the preclinical murine skin wounds. The cytomembrane biologics might provide a biocompatible and versatile tool for wound healing.

producing various cytokines that stimulate new capillary growth, collagen synthesis, and tissue fibrosis. [4][5][6] Once the wound is cleaned up by inflammation, the next phase defined as proliferation is demanded for new generated tissues to fill and cover the wound surface. 7 As the key reparative cell, fibroblast closely correlates with tissue regeneration due to its immigration along the fibrin network. [8][9][10] It initiates reepithelization from the wound edges by secreting fibronectin and collagen, which contributes to matrix reconstruction. 11 Meanwhile, neovascularization and angiogenesis get activated, serving for the wound closure between tissue gaps. 12 Eventually, the regeneration gradually ceases in the following remodeling phase, new generated tissues are reorganized. 13 To be noted, the M2 macrophage also play another important role in directing fibroblasts to express genes and secrete proteins, thus generating the M2 macrophage-fibroblast crosstalk to promote extracellular matrix (ECM) deposition. 14 Until now, numerous studies have been focused on the modulation of cellular behaviors of both macrophage and fibroblast, chasing positive resolution of inflammation and supportive cell proliferation. [15][16][17] Very recently, membrane has been developed as an efficient tool kit in biomaterial research. [18][19][20] The allogenic or autologous provenance of cytomembrane effectuates informative communication being conveyed directly to targeted cells or tissues with high efficiency and excellent biocompatibility. 21,22 In most cases, cytomembranes are used as decoys or carriers to deliver drugs or nanoparticles, preventing them from being captured and cleared away by host mononuclear phagocyte system (MPS). [23][24][25] Moreover, specific biological molecules (e.g., growth factors, and signal molecules) are expressed on the surface of cytomembranes or even their fragments, endowing them with peculiar biofunctions. 26,27 However, most of the cytomembrane strategies are involved with chemical materials or synthetic drugs, the biosecurity problems like chemical toxicity or longterm retention are of great concern in clinical trial. 28,29 Previously, we have manufactured a tumor vaccine based on fused cytomembrane (derived from macrophages) to induce robust immune responses and prevent tumor recurrence. 30 Thus, we intend to take further advantages of the bio-affinitive and co-stimulatory merits in cytomembrane, proposing a chemical materials-, exogenous antigens-and adoptive cells-free modality to provide a biosafe solution for skin wound repair.
Herein, allogenic macrophages and fibroblasts were first obtained from the bone marrow and the skin of mice, respectively (Scheme 1).
The macrophages were induced into M2-like phenotype in conditioned culture medium. The cytomembranes of M2 macrophages and fibroblasts were then extracted and mixed into the suspended biologics (named as MM). This cytomembrane biologics can efficiently reverse over-inflammatory M1 macrophages to M2 phenotype due to the inclusive anti-inflammatory macrophage membranes, accelerating the "inflammation" phase of the wound healing cascade. Next, significant angiogenesis and re-epithelization were both activated and restored by the fibroblast membranes in the MM biologics, expediting the "proliferation phase." These positive bioreactions quickly built the basis for the generation of new matrix, serving for the full-thickness wound closure. Finally, in order to reduce excessive collagen deposition and to avoid disfiguring scars, we intentionally intervened the scarring process by injecting TGF-β inhibitor in the late stage to S C H E M E 1 Schematic illustration of the mixed membranes (MM) biologics that promote skin wound healing. develop scarless healing. In all, this cytomembrane-based strategy modulated two dominating cells (i.e., macrophage and fibroblast) in the inflammation and proliferation phases of wound healing. This strategy may provide new insight for the safe and efficient skin wound repair and shed some light on the future of nature-derived biomaterials.

| Extraction and characterization of allogenic macrophages and fibroblasts
The bone marrow-derived macrophages (CD11b + ) were successfully obtained from BALB/c mice and polarized into M2-phenotype (CD206 + ) in conditioned culture medium (Figure 1a). Likewise, fibroblasts were extracted from the skin of the identical BALB/c mice. And the extracted fibroblasts showed the same expression level as another fibroblast cell line, namely, NIH 3T3, in the marker proteins such as Ecadherin and Vimentin demonstrated by Western Blot (Figure 1b). In addition, these two fibroblast cell lines showed great resemblance in morphology, the marker protein Vimentin was fluorescently visualized ( Figure 1c). Under microscope, the extracted fibroblasts appeared with a more spindle shape than NIH 3T3 (Figure 1d), which was completely rational considering its progenitor source from the skin.
2.2 | The MM biologics regulate macrophages' polarity and prevent redundant inflammation Next, we obtained the cytomembranes of bone marrow-derived macrophages (BMDMs) induced M2 macrophages and skin-derived fibroblasts, these cytomembranes were mixed into membrane biologics (named MM) for further use (Scheme 1). Since the MM biologics contained the membrane fragments of M2 macrophages, we expected it to reprogram endogenous macrophages toward M2 phenotype for the resolution of inflammation. Thus, the polarity of macrophages in different subtypes was surveyed under membrane treatment. First, the extracted BMDMs or RAW264.7 cells were induced into M1 or

| The MM biologics promote migration, proliferation, and activation of fibroblasts
We hypothesized that the MM biologics would promote the proliferation phase of wound healing by facilitating migration, proliferation, and activation of fibroblasts. To test this hypothesis, we first performed the scratch assay on the NIH 3T3 cell, which was certified as the cell model fibroblast, 31 to determine the fibroblastic migration status. In Figure 3a,b, the NIH 3T3 cells showed the highest migration rate under the MM treatment, while the FbM and M2M were similarly less effective. Furthermore, the transwell chamber assay also validated that the MM biologics prompted more NIH 3T3 cells to migrate or invade (Figure 3c,d). In addition, the fluorescent living/dead cell images and CCK8 assay both confirmed that all the membranes accelerated the NIH 3T3 cells to proliferate (Figure 3e-g), in which the MM showed a significantly higher promotion. This phenomenon was also seen in two other cell lines of fibroblasts, that is, mouse embryonic fibroblast (MEF, Figure S1) and the skin-derived fibroblasts

| The MM biologics accelerate wound healing in vivo
To evaluate the in vivo healing effect of the MM biologics, the skin defect animal model was built in BALB/c mice. Figure 4a showed the treatment schedule of the skin wound. The mice were retrieved at specific time intervals and fluorescently imaged in Figure S5 and their phosphorylation levels in NIH 3T3 cells. "Ctr" represents untreated group. Data are presented as mean ± SD (n = 3). "#" is the intragroup comparison with "Ctr," and "*" is the intergroup comparison. * or #, p < 0.05; ** or ##, p < 0.01; *** or ###, p < 0.001 to suppress scarring at the wound edges in the late stage ( Figure 4b).
To show the general effect of the MM biologics, the animal study was also performed in the KM mice model. Similar results were seen in To illustrate the myofibroblast activation and neovascularization, immunohistochemical staining (CD31) and dual immunofluorescence staining (CD31/α-SMA) were presented in Figure 5. α-smooth muscle actin (α-SMA) has been proved to be involved with the conversion of fibroblasts to myofibroblasts, 34 which further led to migration, proliferation, and production of ECM components. While CD31 was a sensitive and specific marker for vascular differentiation. 35 Data showed that groups containing fibroblastic membrane fractions (FbM and MM) had stronger effects to promote myofibroblast activation. And also, both the density and number of blood vessels were significantly higher in the MM-treated group than those in the other groups, referring to a promoted revascularization and matrix reconstruction. In the inhibitor-treated groups, they only showed small difference from the untreated groups, which certified that the TGF-β inhibitor would not disturb the dominating effect of cytomembranes.
Next, to elaborate the in vivo regulatory mechanism of MM, the skin tissues of the wounds were collected and analyzed by flow cytometry assay. In Figure 6a, the M1 macrophages (CD86 + ) were reduced after the membrane treatment for 3 or 6 days, among which the MM showed the strongest effect. It was noted that the

| DISCUSSION
Wound healing is a complex and orderly process including four overlapping phases. 34 Typical strategies to accelerate full-thickness wound repair have been focused on the promotion of one specific phase to cause a series of biological responses. 17,35,36 With the advancement of biomimetic material technology, cytomembranes have been developed to convey more complicated and systematic information due to the abundance of condensed co-stimulatory molecules. 29,37 However, few studies have explored whether cytomembrane can modulate the sequential bioactivities in the wound healing process. Previously, as the most important immune and reparative cells in wound repair, macrophages and fibroblasts have been individually manipulated by various biomaterials. [38][39][40][41] But the concurrent regulation over these two cells are usually failed due to the insufficient signaling molecules in a single material platform. 42 In this work, we take advantages of the To this end, we first successfully obtained BMDMs-derived M2 macrophages and skin-derived fibroblasts ( Figure 1) and then extracted their cytomembranes. The primal effects of these two membranes were tested in vitro. As in Figure 2, the re-education ability of M2 macrophage membrane to reverse pro-inflammatory M1 macrophages into M2 phenotype was characterized. It was not surprising to see that M2M and MM remodeled macrophages toward M2 phenotype because both of them contained M2 membrane. But it was noted that the fibroblast-derived FbM also enforced macrophages to demonstrate M2 phenotype. In fact, it had already been reported that the NIH 3T3 fibroblast-derived conditioned medium caused reduced proinflammatory cytokine production in macrophages, 14  Here, the encountering with homologous FbM might strengthen cellcell junction between fibroblasts, thus enhanced the matrix generation, and promoted the context-dependent fibroblastic behaviors. 16 Likewise, the M2M greatly influenced the biological behaviors of fibroblasts as well ( Figure 3). This phenomenon was implicated with the M2 macrophage-derived signals that directly affected the gene expression and protein secretion of fibroblasts, 43 which were closely correlated with the ECM deposition that changed fibroblasts' behaviors in feedback. Collectively, these results indicated to a well-inherited nature expressed in the MM from the cytomembranes of both M2 macrophage and fibroblast and was further amplified to exert pleiotropic effects on endogenous macrophages and fibroblasts.
In the preclinical in vivo study, accelerated wound healing was achieved in the MM-treated mice (Figure 4). To elucidate the roles of the two cytomembranes, we initiated investigation into the changes of macrophages at the wound site. In Figure 5, massive M1 macrophages (CD86 + ) were activated to cause inflammation in the first 3 days after surgery. And the population of M2 macrophages (CD206 + ) in untreated mice was kept low even at Day 6. In accordance with the in vitro results, M2M and MM were highly efficient to reprogram M1 to M2 macrophages. Interestingly, FbM also aroused the macrophages to repolarize in vivo at Day 3, which was consistent with the Western Blot data, we presented in vitro (Figure 2b,c). However, this repolarization was not long lasting under the disturbing in vivo microenvironment at Day 6, suggesting to a relatively poorer effect of FbM in the resolution of inflammation. This phenomenon certified that the particular biofunctions expressed in each cytomembrane were indispensable for the improved wound repair. And this conclusion was further supported by the CD31/α-SMA staining experiment ( Figure 5), in which FbM and MM both achieved preferable results, but M2M was less effective. Until now, by utilizing natural cytomembranes derived from M2 macrophages and fibroblasts, we successfully realized the dual regulation over both the inflammation and proliferation phases. But this expedited development in wound healing might cause another concern, that the overexpression of fibrosis would induce excessive collagen deposition and ECM misalignment, or even dysfunctional and disfiguring scars. 44,45 Therefore, we intervened the cellular proliferation and ECM production procedures by injecting TGF-β inhibitor in the late stage of wound healing. Results showed that ARG1 (M2 marker) was reduced, CD86 (M1 marker) was slightly re-upregulated ( Figure 6), and α-SMA (activated fibroblasts) was decreased ( Figure 5). With the interference of TGF-β inhibitor, the fibrotic overreactions were normalized, entering the advancement toward scarless wound healing.
This study was inspired by our previous work that the hybrid cell membranes from the tumor cells and polarized macrophages can be used as a tumor vaccine. 20 The regular membrane extraction method, which was presented in this study, mechanically broke the cells and obtained the mixed membranes by centrifugation. The membranous substances were highly efficient to convey desired functions and also came with pleiotropic effects. Here in the skin wound repair process, we further show that simple nature-derived membranes can modulate complex bioactivities.

| CONCLUSIONS
In this work, a series of injectable biologics based on cytomembranes extracted from M2 macrophages and fibroblasts were prepared to promote full-thickness wound healing. These cytomembrane biologics demonstrated unique biofunctions like remodeling pro-inflammatory M1 macrophages to anti-inflammatory M2 phenotype, and modulating cellular behaviors of fibroblasts. During the wound healing process, macrophages were initially recruited as one of the innate immune cells to activate the inflammatory responses. The cytomembrane biologics reprogrammed M1 macrophages to M2 phenotype, which significantly reduced the excessive inflammation process. In addition, the reeducated M2 macrophages were also intimately involved in fibroblastic responses and blood vessels regeneration. Meanwhile, the fibroblast membrane contained in the biologics promoted the migrative and proliferative behaviors of the endogenous fibroblasts, forcing them to transform into myofibroblasts. Thus, these aligning cascade led to a more preferable ECM production, as well as angiogenesis and tissue remodeling. By interfering the late stage of wound healing with the TGF-β inhibitor, the fibrotic overreactions were ceased and the regular collagen arrangement was promoted. Thus, the dual modulation over these two cell types synergistically accelerated the wound healing. This natural membrane-based biologics might be further developed as an efficient bridging material to enhance the material-bio interactions and inflict pleiotropic effect in regenerative studies.

| Extraction and culture of mouse skin fibroblasts
After removing the back hair as much as possible, the female BALB/c mice were sacrificed and the back skin tissues were harvested. The skin was soaked in PBS containing 6% penicillin-streptomycin for 5 min and washed with PBS for three times. After cutting the tissue into pieces, washed for three times with PBS and centrifuged at 800 g for 5 min to collect the precipitate. Treated by 0.25% trypsin and collagenase IV for 6 h. After filtering with 200 mesh filter, the filtrate was centrifuged at 800 g for 10 min to collect the cells and washed again with PBS for two times. The skin fibroblasts were cultured in 100 mm dishes with DMEM medium containing 20% FBS and 1% penicillin-streptomycin.

| Generation of bone marrow-derived macrophages
The female BALB/c mice were sacrificed, both femur and tibiae were collected, muscle attachment was removed. Intact bones were soaked in PBS containing 10% penicillin-streptomycin for 5 min and washed with PBS for three times. Both bone joint heads were cut off, and the bone cavities were flushed with DMEM medium using a 1 ml syringe.
After centrifugation at 400 g for 5 min, the red blood cells (RBCs) were lysed with lysis buffer. The cells were collected and cultured in 100 mm dishes containing DMEM medium added with 20% heatinactivated FBS, 1% penicillin-streptomycin; 24 h later, the culture medium was changed with 10% heat-inactivated FBS, 1% penicillinstreptomycin and GM-CSF (20 ng/ml). M1 macrophages can be harvested from the LPS (20 ng/ml) treatment, and M2 macrophages can be obtained after treatment with IL-4 (20 ng/ml). After 1 week of conditioned culturing, these macrophages were harvested for further use.

| Preparation of membrane biologics
The M2 macrophages and fibroblasts were mixed at the ratio of 1:1 (cell number) and collected with a cell scraper and washed with PBS for three times. After centrifugation at 400 g for 5 min, the cell pellets were resuspended in hypotonic solution containing 10 mM PMSF.
After incubating on ice for 10 min, the solution was ultrasonically broken in ice bath for 6 min and centrifuged at 700 g for 10 min. The supernatant was collected and centrifugated at 14,000 g for 40 min.
The precipitate was collected and freeze-dried, stored at À80 C for further use.

| CCK8 assay
The cells were seeded in a 96-well plate at a density of 5 Â 10 3 cells/ well. After being cultured in incubators for 4 h, the cells were incubated with FbM, M2M or MM, respectively. At the time interval of 0, 12, 24, and 48 h, the medium was discarded. The cells were collected and washed with PBS, followed by staining with freshly prepared CCK8 solutions for 2 h at 37 C. The absorbance used to calculate the cell viability was measured 450 nm.

| Scratch assay
The cells were planted in six-well plates. When the confluence reached 80%-90%, a uniform scratch was formed by a sterile pipette tip. The cells were cultured in serum-free medium as the control group (Ctr), or in the same medium added with FbM, M2M or MM. At 0 and 12 h after the scratch, the cell plates were observed under the bright field of electron microscope. The width of the scratch was measured to calculate the relative closure rate.

| Western blot
The cells were seeded in six-well plate at the density of 2 Â 10 5 cells/ well; 24 h later, FbM, M2M, or MM were added into the medium.
After another 24 h, the cells were washed with PBS. The 1% SDS lysis buffer was used to extract protein in samples and the BCA protein quantification kit was used to determine the concentration. A total of 10-20 μg protein was used for electrophoresis. The volume of protein and the corresponding volume of 5Â SDS loading buffer were calculated based on the measured concentration. After mixing, the samples were heated in a metal bath at 95 C for 10 min. Sodium dodecyl sulfate-polyacrylamide gels were prepared in advance, and the target proteins were separated at a constant pressure of 80 V after adding the protein sample. Subsequently, proteins were transferred to PVDF membrane under a constant current of 300 mA. The membranes were blocked in 5% bovine serum albumin at room temperature for 1 h and then incubated with the primary antibody including anti-β-Actin

| Animal imaging assay
The animal imaging assay was used to observe the degradation of membrane in the wound. The extracted cell membranes were prestained with DiO. The BALB/c mice were randomly divided into PBS group and M@DiO group. After injecting the membrane solution into the edge of wounds, the degradation pattern of cytomembrane was recorded by the animal imaging system (Lumina XR) at Days 2 and 4 after the surgery.