In situ inflammatory-regulated drug-loaded hydrogels for promoting pelvic floor repair

Highlights

• Proposing a new anti-inflammatory biomaterial of drug loaded hydrogel.

• Ensuring stable structure and multi-pore distribution of the hydrogel.

• Realizing high-capacity loading of the drug into the hydrogel and control release.

• Achieving good biocompatibility for cell attachment and tissue regeneration.

Abstract

Biomedical hydrogel has been widely used as regenerative biomaterials, however, an immune inflammatory response of hydrogel constantly crops up in body due to crosslinking agent, external stimulus or small molecule residues. Here we present a strategy to treat pelvic organ prolapse (POP) by combining both anti-inflammatory and promote tissue regeneration, using drug-loaded hydrogel to reconstruct the pelvic floor and minimize multiple inflammations. Photo-crosslinked gelatin hydrogel (GelMA) loaded with Puerarin (Pue) regulate inflammation by inhibiting the aggregation of neutrophils and eosinophils, simultaneously intervene the matrix regenerating/remodeling via TGF-β/MMPs pathway to repair the fascia of pelvic floor in rabbit models (POP model). The assessment of inflammatory cytokines expression (IL-3, IL-6, TNF-α, TGF-β1) in human uterus fibroblasts (HUVs), and extracellular matrix (ECM) related factors (COL-1, COL-3, MMP2, MMP9) was performed in rabbit. Immune microenvironment was analyzed by immunohistochemistry in rabbit samples. Pue-loaded GelMA (Pue@GelMA) down regulate inflammatory cytokines (IL-3 and IL-6) and matrix metalloproteinase 2/9 (MMP 2/9), and up regulate 1/3 type collagen (COL-1/3) in vitro. In this study, Pue@GelMA was able to regulate immune microenvironment through restricting the aggregation of neutrophils and eosinophils and remodel the distribution of ECM collagen in vivo. In the POP model, Pue@GelMA can effectively inhibits the inflammatory response caused by material implanted and promote fascia regenerate. This Hydrogel drug loading system was considered as an safe and effective method to treat POP without persistent complications, and it can also be applied to other prolapse diseases (e.g., intestinal hernia) or complex diseases treatment.

Introduction

Biomaterials are widespread in various medical applications due to their ability to interact with the body and modify physiological functions. Recently, biomaterials have been increasingly used to regulate complex body microenvironments, including providing mechanical support for damaged tissues and promoting the host tissues regeneration with loading drugs, repairing the damaged organs [[1], [2], [3], [4]]. Chemically synthesized polymer biomaterials, such as polypropylene (PP) and polyethylene, have been widely used in clinical practice due to their superior cure rate [5,6]. However, foreign body responses often occur around the materials, which related to the aggregate of inflammatory cells, such as lymphocytes, plasma cells, macrophages and foreign body giant cells [7]. For example, using PP mesh to treat pelvic organ prolapse (POP) may end up with 29%–50% mesh exposure, erosion and other serious complications [8]. That means most unmodified biomaterials lack good biocompatibility, which may cause severe body rejection and application failure [9]. Thus, improve the biological property of biomaterials, which refer to the biocompatibility and the inducibility of biological function, is the first step for their applications.

The repair process after implantation is similar to the wound healing process, whereby inflammatory response is the first to act [10]. Biomaterials must be able to play the crucial repair role in the inflammatory microenvironment, otherwise, the inflammatory response would inhibit cell proliferation and differentiation, leading to the cell alteration, exudation and proliferation in the local tissue, which is damaging for tissue regeneration and repair [11]. Furthermore, improving the biocompatibility of biomaterials and introducing specific bio-inducing functions are key steps in the development of biomaterials.

Hydrogels are gels dispersed in water capable of holding large amounts of water in their three-dimensional network due to the hydrophilic functional groups attached to the polymer backbone [12]. This characteristic makes hydrogels ideal as drug delivery vehicles, playing their several physiological function, water-locking, applicability, permeability, irritating responsiveness (pH, temperature, electrical stimulation) [13]. The hydrogel Gelatin-Methacryloyl (GelMA), obtained by the derivatization of gelatin with methacrylic anhydride (MA), is a photosensitive biomaterial with various biomedical applications such as tissue repair, scaffolding, and as a drug delivery vehicle [[14], [15], [16], [17]]. However, an immune inflammatory response might occur after the implantation of GelMA for the following reasons. First, GelMA may have cytotoxicity, because the unreflect MA and 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (PI 2959) from photo-crosslinking are toxic. Simultaneously, the implantation stimulates the differentiation of macrophage and the migration of neutrophils, leading to the secretion of inflammatory factors and acute inflammatory responses. On the other hand, the slow degradation of high-concentration GelMA causes foreign body responses stimulating fibroblastic heteromorphic hyperplasia, and forms a fibrous wrap, which is isolated from defective tissue.

Macrophage secretes inflammatory factors to stimulate neutrophils cross the endothelium and migrates rapidly to the surround of implant, followed by the acute inflammation. Influenced by T lymphocyte factor, the activated macrophages differentiate into type I (M1) and type II macrophages (M2), and a classic non-specific foreign body response is triggered [18]. Furthermore, TNF-α increases significantly the expression of matrix metalloproteinase 1/3 (MMP1/3), promotes collagen degradation, and inhibits tissue repair process [[19], [20], [21]]. M2, on the contrary, has an anti-inflammatory effect, where the secretion of transforming growth factor beta 1 (TGF-β1) can inhibit the expression of both inflammatory factor IL-3/6, promoting the transformation of fibroblasts into myofibroblasts, and increasing the expression of 1/3 type collagen (COL-1/3) responsible to promote the wound healing and tissue repair. Therefore, the balance of M1/M2 in the tissue microenvironment is very important for the immunomodulation after GelMA implantation, where the assessment of inflammation level can be achieved by balancing TNF-α/TGF-β1.

Targeting the immune inflammation response of hydrogel implantation, M. Xu proposed an antibacterial hydrogel which take advantage of silver ion to crosslink polyethylene glycol [22,23]. Pue is a traditional Chinese herbal medicine extracted from plant flavonoids [[24], [25], [26]]. It is sparingly soluble in water and has been used clinically for the treatment of various diseases, such as POP, osteoporosis, diabetes and cardiovascular diseases [[27], [28], [29]]. Y. Zhang found Pue was useful to treat osteoarthritis as reduces the expression of inflammation factors, IL-1β and TNF-α, meanwhile, it reported TNF-α decreases the expression of MMP2, which regulates collagen deposition, and promotes cartilage formation and repair [28,30]. Y. Li has shown that Pue against degradation of the extracellular matrix in pelvic tissues with POP, which accelerate the regeneration and repair of fascia to treat pelvic organ prolapse [19]. Thus, it is possible to take the anti-inflammatory properties of Pue to regulate the tissue microenvironment, reduce the immune inflammation caused by hydrogel, upregulate the secretion of collagen by fibroblasts to promote tissue regeneration [31].

In this study, we propose an anti-inflammatory biomaterial, Pue@GelMA, which downregulate the expression of inflammatory cytokines, promote the fascia regenerating/remodeling by TGF-β/MMPs pathway, and accelerate the reconstruction of damaged tissue of the pelvic floor. We showed that the implantation of Pue@GelMA improved the early stage inflammation by reducing the expression of inflammatory factors TNF-α, IL-3 and IL-6, and promoted fibroblast adhesion and growth on hydrogel. Meanwhile, the sustained-release of Pue improves the proliferation of type I collagen, which accelerates the regeneration and repair of gynecological pelvic floor fascia tissue. Pue promotes long-term fibroblast proliferation and collagen deposition on ECM, achieving the high-strength reconstruction of the pelvic floor fascia. Systematic in vitro experiments were performed to investigate the physical and chemical properties of Pue@GelMA, as well as the proliferation and toxicity of fibroblasts after exposure of Pue@GelMA and the expression of inflammatory factors TNF-α, TGF-β1, IL-3 and IL-6, and the proliferation of COL-1 and COL-3. In vivo experiments were carried out to evaluate the anti-inflammation properties, as well as safety and biological effect using a rabbit abdominal wall model (Fig. 1).