Vascular Endothelial Growth Factor-Capturing Aligned Electrospun Polycaprolactone/Gelatin Nanofibers Promote Patellar Ligament Regeneration

Abstract

Ligament injuries are common in sports and other rigorous activities. It is a great challenge to achieve ligament regeneration after an injury due the avascular structure and low self-renewal capability. Herein, we developed vascular endothelial growth factor (VEGF)-binding aligned electrospun poly(caprolactone)/gelatin (PCL/Gel) scaffolds by incorporating prominin-1-binding peptide (BP) sequence and exploited them for patellar ligament regeneration. The adsorption of BP onto scaffolds was discerned by various techniques, such as Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, and confocal laser scanning microscope. The accumulation of VEGF onto scaffolds correlated with the concentration of the peptide in vitro. BP-anchored PCL/Gel scaffolds (BP@PCL/Gel) promoted the tubular formation of human umbilical vein endothelial cells (HUVECs) and wound healing in vitro. Besides, BP containing scaffolds exhibited higher content of CD31⁺ cells than that of the control scaffolds at 1 week after implantation in vivo. Moreover, BP containing scaffolds improved biomechanical properties and facilitated the regeneration of matured collagen in patellar ligament 4 weeks after implantation in mice. Overall, this strategy of peptide-mediated orchestration of VEGF provides an enticing platform for the ligament regeneration, which may also have broad implications for tissue repair applications.

Statement of significance

Ligament injuries are central to sports and other rigorous activities. Given to the avascular nature and poor self-healing capability of injured ligament tissues, it is a burgeoning challenge to fabricate tissue-engineered scaffolds for ligament reconstruction. Vascular endothelial growth factor (VEGF) is pivotal to the neo-vessel formation. However, the high molecular weight of VEGF as well as its short half-life in vitro and in vivo limits its therapeutic potential. To circumvent these limitations, herein, we functionalized aligned electrospun polycaprolactone/gelatin (PCL/Gel)-based scaffolds with VEGF-binding peptide (BP) and assessed their biocompatibility and performance in vitro and in vivo. BP-modified scaffolds accumulated VEGF, improved tube formation of HUVECs, and induced wound healing in vitro, which may have broad implications for regenerative medicine and tissue engineering.

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.