wound healing is a complex biological process involving close interactions between various cell types. Chronic non-healing wounds, such as diabetic wounds, exhibit dysregulated healing. They manifest as a prolonged inflammatory phase and fail to transition to the proliferative phase, inducing persistent inflammation. Furthermore, cells in the diabetic wound microenvironment exhibit dysregulation of pro- and anti-inflammatory cytokines, and angiogenesis is impaired. Specifically, fibroblasts within diabetic wounds exhibit unwanted phenotypes and behaviors, marked by reduced ECM synthesis and reduced proliferation and migration capabilities.
In order to solve this problem, Amir M Ghaemmaghami's team from the University of Nottingham, UK, developed active biological induction matrix cell induction materials to actively regulate the phenotype and behavior of fibroblasts , thereby promoting diabetic wound healing. Through screening a series of surface chemicals, the author discovered pTHFuA (pro-proliferation polymer), and for the first time successfully used active monomers to modify particles to deliver bio-instructive polymers in the body.
The relevant research results were published in "Adv. Mater." on November 28, 2022 under the title "Microparticles decorated with cell-instructive surface chemistries actively promote wound healing" .
- Fibroblast-guiding polymers discovered
For materials to promote wound healing, chemical cues must guide fibroblast attachment, spreading and proliferation while controlling differentiation into myofibroblasts. The rounded shape of fibroblasts indicates insufficient spreading, hampering cell function. The size of fibroblasts on the polymer also ranged from 5–175% (Fig. 1a). To understand the effect of the polymer on fibroblast proliferation and differentiation, the authors plotted the fold changes in proliferation index and α-SMA expression (relative to TCP), which allowed visualization of the phenotypic modulating ability of the polymer relative to the TCP control (Figure 1b). Both proliferation and differentiation are key phenotypic traits that determine wound healing outcome. α-SMA, a marker of fibroblast-to-myofibroblast differentiation, was used as a surrogate for fibroblast behavior in this study. myofibroblasts are key cell types involved in wound healing, and their persistence is associated with scar healing.
Furthermore, pTHFuA and pEGPEA were selected from the high-throughput screen because a) their coefficient of variation (COV) is low and thus reproducible, b) they can be polymerized using thermal radical polymerization, and c) they can be synthesized with surfactants to prepare microparticles (i.e., polymers that remove intrinsic cross-links). Therefore, polymers with higher fold changes in proliferation index or α-SMA expression indicate an inability to synthesize surfactant and therefore particles and/or a higher COV, reducing reproducibility. Therefore, the authors designated pTHFuA as the proliferative polymer and pEGPEA as the antiproliferative polymer .
Figure 1 Polymers discovered to modulate the fibroblast phenotype
The authors performed scratch tests on wound closure on anti-proliferative and proliferative polymer surfaces . After 48 hours, fibroblasts on the pro-proliferative surface had invaded 48% of the initial "wound" area, compared to 35% on the anti-proliferative surface (Figure 1d and e). At 96 hours, the wound closure rate was 82% for the proproliferative surface and 55% for the antiproliferative surface. This observation demonstrates the functional ability of pro-proliferative polymers to accelerate fibroblast proliferation and migration, leading to wound healing.
- Fabrication and characterization of microparticles
Polymeric microparticles with pro- and anti-proliferative surface chemistries are used to stimulate bioinstructive stromal cell niches in the wound environment. The microparticles are made using a droplet microfluidic process to achieve uniform particle size. The authors synthesized THFuA-co(ethylene glycol) methyl ether methacrylate (mPEGMA) and EGPEA-co-mPEGMA polymers as pro- and anti-proliferative surfactants, respectively. The core material used by to produce the microparticles is 1,6 hexanediol diacrylate, making the microparticles non-degradable.This simpler, non-absorbable system allows the bioguidance potential of surface chemistry to be studied without the complexity of biosorption in in vivo experiments.
To study the surface, the microparticles were characterized using scanning electron microscopy (SEM) and time-of-flight-secondary ion mass spectrometry (ToF-SIMS) (Figure 2) . The particles without granules had the largest change of 4.8%, which is consistent with the absence of surfactant stable interface. The COV of microparticles with surfactant added was 3.4% (THFuA-co-mPEGMA) and 3.0% (EGPEA-co-mPEGMA), indicating that surfactant can effectively stabilize the surface of microparticles compared with microparticles without surfactant.
Figure 2 Fabrication and characterization of microparticles
- Biologically guided microparticles promote wound healing Fibroblast phenotype
Next, studied the adhesion and activity of fibroblasts on coated microparticles by measuring the total DNA content of cells. research shows that both pro- and anti-proliferative particles support higher cell viability. Fibroblast proliferation was studied by measuring the increase in cell number over 24-96 hours in culture (Fig. 3a). The number of attached fibroblasts increased approximately 3-fold with pro-proliferative particles compared to anti-proliferative particles (1.5-fold increase). To further characterize the functional response of fibroblasts, the gene expression of ECM markers type I collagen (α1) and type III collagen (α1) in cells cultured with pro- and anti-proliferation microparticles was studied using qPCR . This is due to the fact that fibroblasts are the major cell type involved in the expression of both markers. In fibroblasts cultured on anti-proliferative microparticles, both type I collagen and type III collagen were up-regulated, and the expression of gene and was increased by 7.1-fold and 13.5-fold respectively (Figure 3c).
Figure 3 Functional behavior and phenotype of fibroblasts on wounding particles
- Pro-proliferation and anti-proliferation particles affect diabetic wound healing in vivo
The healing of lateral full-thickness skin resection wounds in diabetic mice treated with pro-proliferation and anti-proliferation functionalized particles was compared with wounds that did not receive treatment (Figure 4a) . Although all wound areas decreased over time, the application of proliferating particles reduced the wound area at a faster rate (Fig. 4d). In contrast to the pro-proliferative particle distribution, H&E staining (Fig. 4b) revealed how anti-proliferative particles within the wound cavity dissipated to the sides of the wound, where granulation tissue was visible, and the central part of the wound bed was almost devoid of granulation tissue depth (GTD) and immune cell infiltration (Fig. 4d and e). Masson staining (Figure 4c) and subsequent analysis of collagen thickness (Figure 4e) showed that the addition of pro-proliferative microparticles to diabetic wounds resulted in increased collagen production, improved healing and regeneration compared to anti-proliferative particles and untreated wounds.
Figure 4 Histological results extracted from full-thickness wounds of diabetic mice exposed to anti-proliferative (pEGPEA) and pro-proliferative (pTHFuA) microparticles
In addition, cell proliferation immunostaining showed that the proliferative microparticle-treated wound had more BrdU-positive cells compared with the anti-proliferative wound (p0.0001) and the untreated wound (p0.0001) (Figure 4h). Type I collagen expression was higher (Fig. 5a) . The levels of collagen III in all wounds were similar (Fig. 5b), which indicated that the granulation tissue formed in the wounds treated with pro-proliferation particles was mainly composed of type I collagen and type III collagen, thus upregulating the ratio of type I/III collagen, which is related to the promotion of diabetic wound healing in mice.
Figure 5 Immunofluorescence staining of resected tissue from diabetic mice treated with anti-proliferative (pEGPEA) and pro-proliferative (pTHFuA) microparticles
In conclusion, The strategy of utilizing active bio-guidance microparticles to guide the functional phenotype of fibroblasts is a novel approach to exploit the remodeling potential of these cells, which have distinct pro- and anti-proliferative phenotypes and behaviors . Through screening a series of surface chemicals, the authors discovered pTHFuA (pro-proliferation polymer), and for the first time successfully used surface-active methods to modify microparticles to deliver bioinstructive polymers in the body.The pro-proliferative microparticles significantly promoted wound healing and tissue granulation compared to pEGPEA (anti-proliferative polymer)-coated microparticles and untreated wounds. The findings provide new insights into the suitability of an active non-eluting immune-inducing polymer (pTHFuA) to support diabetic wound healing and could potentially translate into clinical treatments for diabetic wounds, potential burns and other types of chronic or acute wounds .
Article source: https://doi.org/10.1002/adma.202208364
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