Full length articleVascular Endothelial Growth Factor-Capturing Aligned Electrospun Polycaprolactone/Gelatin Nanofibers Promote Patellar Ligament Regeneration
Graphical abstract
Introduction
Ligament injury is one of the common sports injuries, which may result in joint instability and dysfunction [1,2]. In the United States, from 2002 to 2014, the overall rate of anterior cruciate ligament (ACL) reconstruction increased by 22 %, from 61.4 per 100,000 person-years (PYs) in 2002 to 74.6 per 100,000 PYs in 2014. The rates of ACL reconstruction in adolescents aged 13 to 17 years increased dramatically over the 13-year study period (isolated, +37 %; ACL + meniscal repair, +107 %; ACL + meniscectomy, +63 %) [3]. Patients whose symptoms cannot be relieved by the traditional treatment of ligament injury require surgical repair [4]. The gold-standard treatment for the surgical management of ligament injuries includes grafting of autologous tissues; which, is however complicated by the lack of the suitable numbers of transplantable autologous tissues and/or donor-site associated infection risks [5]. The use of the allogenic grafts is also limited by the immunogenicity risks [5,6]. Alternatively, tissue-engineered scaffolds have garnered considerable interest of the research community [7,8]. However, inherently less vascularity of ligament tissues impedes the regenerative response by limiting the availability of oxygen and nutrients [6], [7], [8], [9]. Consequently, developing angiogenic scaffolds for the repair of ligament may hold great promise.
Growth factors play a vital role in tissue repair by effectively promoting cellular processes, such as cell viability, proliferation, and differentiation [10]. Being an integral component of vascular morphogenesis, vascular endothelial growth factor (VEGF) plays a crucial role in inducing neovascularization by promoting the growth, migration, and viability of endothelial cells (ECs) [11,12]. Accordingly, VEGF has been widely exploited for the treatment of ligament injuries [13], [14], [15]. However, being a large molecular weight protein, VEGF exhibits short half-life and poor retention at the injury site, which may abrogate its therapeutic benefits while posing additional side effects [16]. The localized and sustained presentation of growth factors at the injury site may avoid risks associated with their high concentration [17]. Additionally, being large molecular weight proteins, it is difficult to synthesize or incorporate growth factors into scaffolds [18]. On the other hand, short peptide sequences with the biological activity similar to the growth factors (GFs) may be beneficial to mimic the therapeutic benefits of GFs [19]. The in vitro or in vivo sequestration of GFs by using functional biomaterials is also an enticing avenue, which has been realized by installing GFs binding domains or peptides sequences into biomaterials [20]. Recently, Adini et al. demonstrated that the use of the specific prominin-1 derived VEGF binding peptide (BP) consisting of 12-amino acids (DRVQRQTTTVVA) can enhance the biological activity of exogenous and endogenous VEGF in vitro and in vivo to promote angiogenesis [19], [20], [21], while avoiding the potential risks of exogenously delivered or covalently-tethered GFs on scaffold materials [22,23]. Therefore, the method of grafting or adsorbing peptides on the surface of scaffold materials to sequester GFs may be of considerable significance to endow the scaffolds with the biological functions [24,25].
Herein, we hypothesized that the incorporation of BP into aligned electrospun nanofibrous scaffolds may orchestrate VEGF at the injury site, thus leading to a functional tissue repair. Electrospinning, being a versatile and a cost-effective technology for fabricating tissue-engineered scaffolds has received enormous interest of the scientific community [26]. The aligned nanofiber scaffolds prepared by electrospinning can guide the directional growth of tissues and organization of cells [27]. We have chosen biocompatible and biodegradable polycaprolactone/gelatin (PCL/Gel) hybrids as scaffold materials and adsorbed BP onto the surface of the fibers via van der Waals forces and hydrophobic interaction [28,29]. The VEGF binding ability of BP-adsorbed PCL/Gel nanofibers was discerned in vitro and potential of the scaffolds for the ligament regeneration was discerned in vivo. The functional scaffolds may orchestrate the endogenous VEGF at the injury site to promote neo-vessel formation and ligament regeneration, which may have broad applicability.
Section snippets
Materials
PCL (Mw, 80 kDa) and gelatin from porcine skin (Type B, 48722-500G-F) were purchased from Sigma-Aldrich, Co., Ltd (Shanghai, China). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was acquired from Shanghai Darui Fine Chemical Co., Ltd. (Shanghai, China). Glutaraldehyde was obtained from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Rhodamine-B conjugated BP (DRVQRQTTTVVA) was custom-synthesized by China Peptides Co., Ltd (Shanghai, China). The carboxyl groups (‒COOH) of the rhodamine were
Characterization of PCL/Gel and BP@PCL/Gel Scaffolds
The morphology of PCL/Gel and BP@PCL/Gel scaffolds was discerned by SEM (Figs. 1A, 1D). Nanofibers were mainly aligned in the horizontal direction; few fibers were inclined (Fig. S2). Since for the immobilization of BP, scaffolds were immersed in the BP solution, we also assessed the morphology of PCL/Gel and BP@PCL/Gel scaffolds after incubation in PBS and BP solution, respectively. Both types of scaffolds displayed wavy fibers after immersion into the PBS or the BP solution, which is ascribed
Discussion
VEGF, which plays an important role in angiogenesis, was identified, isolated, and cloned 32 years ago [46]. It has been confirmed that VEGF plays an important role in tissue repair by promoting the survival, migration, and proliferation of endothelial cells (ECs), and formation of neo-vessels in vitro and in vivo [47]. However, to achieve the reasonable concentration of VEGF that can induce vascularization, its high concentration may be required, which may induce potential toxicity risks,
Conclusions
In this study, the PCL/Gel based aligned nanofiber scaffolds were fabricated by electrospinning, which were next modified with the BP to enhance VEGF binding. The BP@PCL/Gel scaffolds exhibited biocompatibility, biodegradability, and VEGF-binding ability. The scaffolds containing BP facilitated HUVECs tubular formation and wound healing in vitro. The implantation of the BP containing scaffolds into an injured patellar ligament model in rats in vivo led to the regeneration of ligament tissues
Declaration of Competing Interest
The 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 the National Key R&D Program of China (2016YFC1100300), National Natural Science Foundation of China (Nos. 32050410286, 81772339, 81972129, 31771023, 81911530136, 81811530750, 82072521, 8217091186 and 82111530200), The Fundamental Research Funds for the Central Universities (2232019A3-07), The Key Clinical Medicine Center of Shanghai (2017ZZ01006), Sanming Project of Medicine in Shenzhen (SZSM201612078) and The Introduction Project of Clinical Medicine Expert Team for
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These authors contributed equally to this work.