Applied Materials Today
Tanshinone IIA-loaded aligned microfibers facilitate stem cell recruitment and capillary formation by inducing M2 macrophage polarization
Graphical abstract
Introduction
The body's own repair capabilities can be enhanced by utilizing inducible biomaterials loaded with bioactive substance [1], [2], [3]. Implantation of a cell-free scaffold to guide in situ tissue regeneration is a proven strategy for recruiting and then regulating the behavior of endogenous stem cells [4,5]. Compared with cell transplantation, this option possesses numerous advantages, including averting ethical issues associated with embryonic stem cells and avoiding related complications, such as immune rejection, uncontrollable cell fate, time-consuming and costly cell culture steps, and the possibility of tumor formation [6,7]. In addition, scaffolds-assisted tissue repair must achieve a rich and efficient vascular network capable of transporting sufficient oxygen and nutrients as well as metabolites, after implantation in vivo [8]. Indeed, previous efforts to produce pre-vascularized scaffolds in vitro have encountered difficulty in achieving integration with host microcirculatory vessels [9]. Thus, the construction of scaffold-enabling stem cell recruitment and vascularization would be highly beneficial to endogenous tissue regeneration.
Upon implantation of biomaterials into the body, macrophages are one of the first immune cells to respond, invading the scaffold and regulating inflammation by initiation, propagation, and resolution [10], [11], [12], [13], [14]. Macrophages typically remain at the tissue-scaffold interface throughout the entire tissue repair process, serving important roles in regulating inflammation and tissue regeneration [15,16]. Macrophages can switch between proinflammatory (M1) and pro-healing (M2) phenotypes, depending on different stimuli and responses to microenvironmental factors [17,18]. Appropriate and timely regulation of the macrophage phenotype switch is a key factor in the process of endogenous stem cell recruitment and vascularization following scaffold implantation [14,16,19]. Therefore, to ensure the full success of implanted scaffolds in restoring injured tissue, scaffolds should first establish an immunoregulatory milieu, and then promote stem cell recruitment and enhance vascularization [20], [21], [22].
The physical structure and biological activity of the biomaterials can be controlled to regulate macrophage phenotypic transition and modulate the immune microenvironment[14]. Zhang et al. provided evidence that, compared with solid membranes, random nanofibrous scaffolds enhanced MSC recruitment by inducing M1-to-M2 macrophage transition [23]. Compared with random nanofibers, oriented nanofibers promoted the phenotypic transformation of M2 macrophages more efficiently [24,25]. However, these aligned or randomly distributed nanofiber-based scaffolds limited cell migration into scaffolds, resulting in limited tissue regeneration in later stages [26]. Recent studies confirmed that, compared to nanometer-sized fibers, micrometer-sized fibers significantly promote cell migration and tissue integration [10,24,27]. Although these polymeric microfibrous scaffolds can provide physical and structural support, they lack intrinsic biological activity [3,28].
Tanshinone IIA (Tan IIA) is one of the main active pharmaceutical ingredients (APIs) extracted from Salvia miltiorrhiza Bge (Danshen in Chinese). It has been used for the treatment of coronary heart disease, stroke, and angina pectoris [29,30]. More importantly, Tan IIA has been shown to prevent articular cartilage degeneration, ameliorate symptoms of rheumatoid arthritis (RA), and enhance cartilage and nerve regeneration [31], [32], [33]. Also, our previous studies proved that Tan IIA exhibited strong anti-inflammatory, antioxidative, and anti-apoptotic properties, and also encouraged macrophage phenotype switching [34], [35], [36]. Here, we sought to incorporate the bioactivity of Tan IIA into polymer scaffolds. We hypothesized that aligned microfiber scaffolds loaded with Tan IIA could increase macrophage recruitment and drive them toward the M2 phenotype. In turn, this would promote endogenous regenerative capacity, involving the recruitment of stem cells and the enhancement of vascularization.
To test our hypothesis, we optimized the electrospinning process to prepare random and oriented microfiber scaffolds for controlled release of Tan IIA. We first compared the effect of microfibers scaffolds loaded with Tan IIA of different concentrations on the immunoregulation of macrophages. In vitro studies revealed that oriented microfibers loaded with 1 μM Tan IIA significantly promoted M2 macrophage polarization, as proven by changes in gene and secretory factor profiles. Conditioned medium collected from macrophages cultured on 1 μM Tan IIA loading scaffolds significantly improved BMSC proliferation and migration and regulated their multiple biological behaviors. Similarly, conditioned medium promoted endothelial cell proliferation and migration, as well as tube formation. Subcutaneous implantation of Tan IIA-loaded aligned microfiber scaffolds further confirmed our findings observed in vitro and also demonstrated the integration of implanted scaffolds with surrounding tissues. Overall, these results suggested that aligned microfiber scaffolds loaded with bioactive substances can recruit stem cells and promote vascularization by modulating immune responses.
Section snippets
Materials
Poly (caprolactone) (PCL) pellets (Mn,80 KDa) were purchased from Sigma (USA). Tan IIA is provided by Seebio Biotech (Shanghai) Co, Ltd (China). Acetone, methanol, chloroform, and many other analytical reagents were obtained from Tianjin Chemical Reagent Company (Tianjin, China). Sprague Dawley rats were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). The Animal Experiments Ethical Committee of Nankai University approved all animal
Morphology and release properties of the microfibrous scaffolds
PCL scaffolds with random or aligned microfibers loaded with Tan IIA of different concentrations were fabricated by a modified electrospinning technique [41]. Scaffold color was darkened with increasing Tan IIA concentration, as visualized by stereomicroscope (Supplementary Fig. 1a). SEM images showed the distinct arrangement of the random (RF) and aligned microfiber (AF) scaffolds, and the fiber morphology exhibited no obvious changes after loading of Tan IIA (Fig. 1a). The corresponding
Discussion
Using immunomodulatory biomaterials to fully exploit the endogenous regenerative ability of tissues provides a feasible and effective strategy for improving tissue regeneration [17,[43], [44], [45]]. In this study, we prepared aligned microfiber (AF) scaffolds loaded with the bioactive Tan IIA to promote macrophage M2 phenotype transition and subsequent MSC recruitment and vascularization, as shown in Schematic 1.
Multiple physical factors, such as fiber diameter [17], stiffness [45], and
Conclusion
We developed Tan IIA-loaded aligned microfiber scaffolds, which enabled endogenous stem cell recruitment and vascularization by modulating macrophage phenotypes and inhibiting inflammation responses. AF-1 significantly promoted RAW 264.7 macrophages polarization toward the M2 phenotype. Conditioned medium collected from macrophage cultured on AF-1 scaffolds improved BMSC proliferation and migration, regulated their multiple biological functions, and promoted HUVEC proliferation, migration, and
Data availability
The data in this work are available in the manuscript or Supplementary Information, or available from the corresponding author upon reasonable request.
CRediT authorship contribution statement
Shan Gao: Writing - original draft, Funding acquisition, Writing - review & editing. Lina Wang: Methodology, Validation, Writing - original draft. Yu Zhang: Formal analysis, Methodology. Lan Li: Resources, Visualization. Yunsha Zhang: Funding acquisition, Formal analysis. Xiumei Gao: Resources, Formal analysis. Jingyuan Mao: Resources, Formal analysis, Supervision. Lianyong Wang: Funding acquisition, Conceptualization. Lichen Wang: Formal analysis. Hongjun Wang: Investigation,
Declaration of Competing Interest
All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by grants from Tianjin Science Foundation for Distinguished Young Scholars (17JCJQJC46200), the National Key R&D Program of China (NO.2018YFC1704500, 2017YFC1103500), National Natural Science Foundation of China project (81774050, 81972063, 81904054, 81503505, 31670990), Natural Science Foundation of Tianjin (17JCYBJC29000), Training Program Foundation for Innovative Research Team of Higher Education in Tianjin during the 13th Five-Year Plan Period (NO.TD13-5050),
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These authors contributed equally to this work.