Bioinspired poly (γ-glutamic acid) hydrogels for enhanced chondrogenesis of bone marrow-derived mesenchymal stem cells
Graphical abstract
Introduction
Cartilage tissue has limited capacity for spontaneous healing and regeneration due to its avascular structure and sparse distribution of progenitor cells [1], [2]. Over the past decades, tissue engineering strategies have emerged as an effective approach with the potential to stimulate cartilage regeneration in clinical settings. In addition, 3D scaffolds combined with stem cells are most often utilized in tissue engineering technology. Nevertheless, their practicalities are often limited due to uncontrolled biodegradability, insufficient bioactivity, and dependence on intricate instruments or procedures. These scaffolds are not qualified to serve as the temporary ECM for the stem cells deliveries. Thus, the need for novel functional scaffolds for cartilage regeneration has been the ongoing demand in cartilage tissue engineering.
Among the candidates for artificial 3D scaffolds, hydrogel scaffolds are one of the best, due to their characteristics of high hydrophilic, favorable biocompatibility, and structural resemblance to naturally occurring extracellular matrix (ECM) [3]. Recently, hydrogels based on polysaccharide and peptides have been the focused materials of choice in designing stem cell scaffold. These hydrogels demonstrate excellent biocompatibility, appropriate biodegradability, and facile synthetic pathway [4], [5]. In comparison, synthetic polymer hydrogels, such as poly(ethylene glycol) [6], poly(vinyl alcohol) [7], and polyacrylamide [8], exhibit a unclear safety profile for long-term use. Hence, polysaccharide and peptides based hydrogels are more suitable for clinical use, owing to their close mimicking of native ECM [9], [10]. This property can provide numerous biochemical and biophysical cues for stem cell viability and differentiation. As the 3D microenvironment provided by the ECM plays a significant role in influencing cell behaviors and directing stem cell fates, a variety of artificial ECM mimicking hydrogels based on polysaccharide or peptide have been designed as stem cells carriers in tissue engineering [11], [12]. However, to our knowledge, few reports have been found with regard to using polypeptides as biomimetic hydrogel of 3D fibrous protein in ECM for stem cell-engineered constructions.
γ-poly (glutamic acid) (γ-PGA), a naturally occurring polypeptide, possess excellent biocompatibility, biodegradability [13], as well as unique similarity to natural protein-based ECM [14], [15]. These superior properties inspired researchers to investigate ECM mimicking γ-PGA-based hydrogel for tissue engineering application [16], [17], [18]. For example, rabbit chondrocytes were encapsulated within the injectable γ-PGA hydrogels, which could deposit abundant ECM to induce ectopic cartilage formation in vivo [19]. In addition, there is sufficient evidence to suggest γ-PGA hydrogel scaffold with tunable microenvironment may act as a promising 3D artificial ECM for guiding 3D spreading and chondrogenic differentiation of MSCs [18].
Recently, mesenchymal stem cells (MSCs) have attracted increasing attention as the seed cell for cartilage tissue engineering, owing to their ease of isolation, the ability to attenuate inflammation, and pluripotent potential [20], [21], [22]. The outstanding advantages of MSCs in stem cell biology has made it crucial to develop desired culture systems to induce chondrogenic differentiation for cartilage repair.
In the present study, inspired by natural protein-based ECM, a type of polypeptide-based hydrogels comprising functionalized γ-poly (glutamic acid) was developed through Michael-addition reactions, without the need to add any harmful reagents (e.g., initiator, some unidentified enzymes in the natural polymers) or catalysts. The advantage effectively expanded applications of the γ-PGA hydrogel in the field of regenerative biology. The properties of the hydrogel like gelation times, micromorphology, swelling ratio, and rheological properties were controlled by varying the concentrations of the copolymer. Meanwhile, the hydrogel also demonstrated good mechanical property in compression tests. Rabbit bone-derived mesenchymal stem cells were incorporated with copolymers and hydrogels to investigate the cell viability and proliferation of BMSCs through live-dead assay and MTT assay in vitro. Additionally, in vitro BMSCs differentiation behaviors were investigated, and in vivo cartilage repair effect in a rabbit auricular cartilage defect model was evaluated systematically (Fig. 1).
Section snippets
Materials
γ-PGA (700 kDa) was obtained from Shikening Biotechonongy co., Ltd (Nanjing China). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxy-succini-mide (NHS), Cysteamine hydrochloride (CSA·HCl), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Glycidyl methacrylate (GMA) were obtained from Aladdin Industrial Corporation (Shanghai, China). Acridine orange (AO), ethidium bromide (EB) and Papain were purchased from Solarbio Life Sciences (Beijing, China). RGDC were
Synthesis and characterization of polymers
γ-PGA-GMA was synthesized by the epoxy ring opening reaction of γ-PGA and GMA(Fig. 2a). The synthesis of γ-PGA-GMA was confirmed by the 1H NMR spectrum. As shown in Fig. 2c, the spectrum of γ-PGA-GMA showed two new peaks at 6.27 ppm, 5.64 ppm, and both of the two peaks (a) were corresponded to the protons of GMA. The 6.27 ppm and 5.64 ppm were the chemical shifts of protons of vinyl group (CC) in GMA, and 4.13–4.52 ppm (d) was the chemical shift of methine protons in γ-PGA [23]. This indicated
Conclusion
In summary, a polypeptide-based hydrogel consisting of functionalized γ-poly (L-glutamic acid) was fabricated via thiol-Michael addition reactions, which can gel quickly and gently under physiological conditions. The physicochemical properties of the hydrogel, including gelation time, mechanical strength, swelling ratio, and degradation behaviors could be tailored by adjusting the copolymer concentrations. Specifically, the γ-PGA hydrogels exhibited outstanding capacity of anti-compression and
Declaration of Competing Interest
There are no conflicts of interest to declare.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31771049), Jiangsu Provincial Key Research and Development Program (BE2018731), State Key Laboratory of Materials-Oriented Chemical Engineering (ZK201806, KL18-06 and ZK201606) and Six Talent Peaks Project in Jiangsu Province (SWYY-046).
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