Harnessing endothelial cells and vascularization strategies for nerve regeneration

Peripheral nerves are essential components of the human body’s communication system, transmitting signals between the central nervous system and various body parts. Damage resulting from trauma or disease can result in debilitating sensory and motor deficits. Nerve injuries, particularly those resulting in significant gaps in the nerve tissue, pose a formidable challenge for clinicians and researchers. Despite their limitations, including limited availability and donor site morbidity, nerve autografts remain the clinical gold standard for treating nerve injuries. Tissue engineering seeks to provide an alternative to the autograft with the fabrication of nerve repair constructs. These constructs have the potential to overcome the limitations of the autograft while still harnessing autograft biology with aligned biomaterials and therapeutic cells. A crucial aspect of nerve tissue engineering is the establishment of vascularization within the regenerating nerve tissue. This process plays a pivotal role in providing oxygen and nutrients to implanted cells, ensuring their long-term survival. Over recent years, it has become ever more apparent that the role of blood vessels in nerve regeneration extends beyond vascularization. Blood vessels, and the endothelial cells that form the vessel inner lining, serve prominent structural, regulatory, and modulatory roles in nerve regeneration (Shen et al., 2004; Cattin et al., 2015; Grasman and Kaplan, 2017; Witjas et al., 2019; Fornasari et al., 2022; Huang et al., 2023). The exploitation of this knowledge has led to the development of various effective nerve injury treatments in animal models (Muangsanit et al., 2021; Thibodeau et al., 2022; Huang et al., 2023). Therefore, the intricate interplay between vascularization and nerve tissue engineering could be the key to improving engineered nerve repair constructs.

Vascularization is critical in regenerating many organs and tissues within the human body, including nerves, with blood vessel formation occurring throughout organ development and tissue repair. During angiogenesis, sprouting blood vessels form an organized structure within tissues serving as a transport network for oxygen, nutrients, metabolites, waste, and cells; all of which are essential for sustainable tissue regeneration. Furthermore, endothelial cells actively regulate tissue homeostasis and repair. Endothelial cells secrete several growth factors necessary for tissue development and regeneration (Figure 1). Vascular growth factors released from endothelial cells contribute to the development of various tissues such as the heart, kidney, lung, bone, and nerve (Ramasamy et al., 2015). Shen et al. (2004) showed that secreted factors from brain endothelial cells activate Notch and Hes1 signaling, thereby promoting the self-renewal of neural stem cells, implying the critical role of endothelial cells in the neural stem cell niche in the maintenance of the nervous system throughout life. Endothelial cell-secreted factors also benefit the peripheral nervous system. For example, human umbilical cord vein endothelial cells (HUVECs) release brain-derived neurotrophic factors that enhance the outgrowth of dorsal root ganglion neurons in vitro (Grasman and Kaplan, 2017).

Figure 1.

Potential mechanisms through which the blood vessels regulate peripheral nerve regeneration following an injury.

After a peripheral nerve injury, circulating macrophages and neutrophils are recruited to the site of injury to facilitate the clearance of nerve debris during Wallerian degeneration. Consequently, aligned macrophage-induced blood vessels develop across the nerve injury site. These vessels serve as tracks, guiding Schwann cell migration, thereby directing the regrowing axons. Endothelial cells, which constitute the inner surface of these blood vessels, secrete autocrine factors as well as exosomes to promote axonal growth and stimulate Schwann cell proliferation. Furthermore, endothelial cells can produce extracellular matrix components, such as thrombospondin-1 and glycosaminoglycans, which regulate the surrounding microenvironment and enhance axonal regeneration. Adapted from Cattin et al. (2015). Created with BioRender.com.

In addition to autocrine signaling, exosomes have also been shown to play a role in nerve regeneration. Exosomes, extracellular nano-vesicles produced by cells containing lipids, nucleic acids, proteins, or metabolites, can modulate intercellular communication. Endothelial cell-derived exosomes have been shown to reduce ischemia-induced neuronal cell apoptosis using SH-SY5Y neural cells in vitro (Xiao et al., 2017). Of particular note in peripheral nerve injury, Huang et al. (2023) characterized the effects of endothelial cell exosomes on RSC96 Schwann cells in vitro, demonstrating the ability of endothelial cell exosomes to regulate Schwann cell phenotype by promoting the Schwann cell repair phenotype, increase Schwann proliferation and migration, and upregulate immune and neurotrophic factor secretion. This publication concluded with an in vivo study which assessed the benefit of treating rat sciatic nerve crush with an injection of endothelial cell exosomes under the epineurium. The exosome injection promoted functional recovery, axonal regeneration, angiogenesis, and remyelination after peripheral nerve crush. Upregulation of miR199-5p expression and PI3K/AKT/PTEN signaling pathway activation were deemed to be responsible for the beneficial effects of these exosomes (Huang et al., 2023). This work suggests exosomes, or drugs that act on the same pathways, should be explored as potential therapeutics for peripheral nerve injury. Further to exosomes, extracellular matrix produced by endothelial cells can also modulate tissue regeneration. Upon injury, endothelial cells can secrete matrix metalloproteinases which degrade various proteins in the extracellular matrix and produce extracellular matrix fragments, named matrikines, which trigger tissue regeneration, while also releasing matricellular proteins, including CCN proteins, thrombospondin-1, tenascin-C, and membrane-bound glycosaminoglycans, which can modulate endothelial basement membrane formation as well as the activation of regenerative signaling cascades (Figure 1; Witjas et al., 2019). It has been shown that tenascin-C effectively stimulates Schwann cell migration and axonal regrowth via a β1-integrin-dependent pathway (Zhang et al., 2016).

Along with the effects of endothelial cells, blood vessels also play a role in regeneration, beyond circulatory function. The significant role of blood vessels in regenerating peripheral nerves was observed by Cattin et al. (2015) who remarked on Schwann cells using the aligned blood vessels as tracks, thereby guiding the Schwann cell migration in peripheral nerve regeneration (Figure 1). These longitudinally oriented, macrophage-induced blood vessels which formed after peripheral nerve injury, also facilitated the formation of Bands of Büngner, which, in turn, promote axonal regeneration. Moreover, Fornasari et al. (2022) illustrated this natural biology holds true in nerve injuries treated by implantation of engineered nerve constructs, where the environment could differ from that observed in the absence of any intervention. This work saw polarized blood vessels, with the association of Schwann cells, form within conduits bridging an 8 mm rat median nerve gap: a result comparable to what was naturally observed by Cattin et al. (2015) (Fornasari et al., 2022). Taken together, blood vessels and endothelial cells present promising platforms for tissue regeneration, particularly in nerve tissues.

The development of vascularized nerve grafts spans several decades, with St. Clair Strange’s 1947 study demonstrating the successful transplantation of the ulnar nerve with its vascular supply to the median nerve, and Taylor and Ham’s 1976 advancement introducing a vascularized graft from the superficial radial nerve, utilizing the radial artery as its vascular source, for repairing damaged median nerves; the studies on vascularized nerve grafts were summarized in the review by Muangsanit et al. (2018). However, the major limitation involved with vascularized nerve grafts is the lack of available donor sites. Given the manifold benefits of blood vessels and endothelial cells, several tissue engineers have attempted to use these structures to accelerate nerve repair. An experimental study demonstrated that combining primary rabbit Schwann cells and vascular endothelial cells in 3D scaffolds creates vascularized nerve constructs. These constructs were effective in promoting nerve repair in rabbit models with a 20 mm gap in the sciatic nerve (Gao et al., 2013). Another study revealed that the co-culture of rat RSC96 Schwann cell line and rat vascular vein endothelial cell line in Chitosan/artemisia sphaerocephala scaffolds significantly enhanced the growth of dorsal root ganglion-derived neurons in vitro (Zheng et al., 2022). While there have been advances in creating various biomaterial constructs seeded with endothelial cells, there has been insufficient assessment regarding the alignment and the formation of tube-like structures, which are crucial in directing nerve regeneration (Cattin et al., 2015).

This scenario led us to propose the fabrication of aligned endothelial cells within collagen hydrogels for the treatment of nerve injuries. We hypothesized that the aligned tube-like structures from endothelial cells would recruit and guide Schwann cells as well as enhance vascularization, thus accelerating axonal regeneration. Collagen type I hydrogels were chosen as the scaffolds for the engineered constructs owing to their high biocompatibility, biodegradability, and resemblance to native nerve tissues. Aligned collagen gels, recognized for facilitating axon regrowth, are commonly used in nerve repair. The collagen gel 3D environment can be tailored to carry beneficial cells, growth factors, and/or drugs. In our work, the collagen gels were laden with HUVECs, widely used model endothelial cells, to promote vascularization and axonal regeneration in this proof-of-concept experiment. HUVECs were tested in mono- and co-culture constructs with Schwann cells to investigate potential synergistic effects. To achieve cellular self-alignment and endothelial tube formation, perforated tethering silicone conduits were used to house the cellular collagen gels, providing an ideal microenvironment for stable tube-like formation and cellular alignment within collagen hydrogels. Notably, constructs containing only HUVECs significantly promoted axonal regeneration and vascularization across the 10 mm rat sciatic nerve gap after 4 weeks post-operation. This outcome surpassed the performance of both conventional aligned Schwann cell constructs and co-cultures of HUVECs and Schwann cells (Muangsanit et al., 2021). The superior performance of HUVECs in promoting axonal regeneration and vascularization, compared with Schwann cell constructs and co-cultures, underscores the critical role of endothelial cells in nerve tissue engineering, which is more substantial than previously thought.

The growing understanding of the importance of angiogenesis in nerve regeneration, emphasizes the need for vascularization strategies in nerve repair protocols. Multiple approaches to vascularizing engineered nerve constructs have been and continue to be developed (Muangsanit et al., 2018). One recent such example is the work of Thibodeau et al. (2022) which tested a conduit made from a rolled multi-layered cellular sheet containing layers of fibroblasts, endothelial cells, and Schwann cells. Each layer was matured in culture before conduit formation and implantation into a 15 mm rat sciatic nerve gap. After 22 weeks, the motor function recovery observed from the nerve repair conduit was comparable to that of the autograft (Thibodeau et al., 2022).

To deepen our understanding of the interactions between blood vessels, endothelial cells and nerves, numerous in vitro models have been developed. These include 3D co-culture models of neuronal cells and endothelial cells from various sources, which have largely gained attention for their physiologically relevant microenvironment. A study by Osaki et al. (2018) employed this approach by establishing a co-culture model with human neural stem cell-derived motor neuron spheroids and induced pluripotent stem cell-derived endothelial cells. These cells were embedded in a microfluidic device filled with collagen, resulting in an enhancement in both neuronal functionality and microvascular network perfusion.

In summary, the multifaceted roles of blood vessels and endothelial cells in tissue repair, including structural and regulatory functions, highlight their significance in nerve tissue engineering. Evidently, focusing on incorporating endothelial cells or vessel-like structures holds promise as a therapeutic strategy for peripheral nerve injury. However, future research is required to enhance the preclinical efficacy and translational feasibility of the engineered constructs. These include the consideration of the source of endothelial cells, which could be clinical-grade pluripotent stem cells, and the selection of good manufacturing practice materials, which may be either synthetic hydrogels or bio-based hydrogels. Additionally, the manufacturing technology of anisotropic cellular structures could be further optimized, potentially through employing Gel Aspiration-Ejection techniques which utilize syringe-induced negative pressure to rapidly generate dense and robust aligned scaffolds (Muangsanit et al., 2020). While further studies and clinical trials are essential to validate these findings and facilitate their translation into practical applications, the potential impact and the perceived roles of endothelial cells in treating nerve injuries cannot be ignored.

This work was supported by the Royal Thai Government Scholarship (to PM).