Electrospun fibers for tendon and ligament repairs

SEM image of electrospun yarn showing high degree of fiber alignment.

Electrospun fibers have been used or tested for use as implantable scaffolds for numerous organ and tissue repair due to its generally good cell adhesion and proliferation properties. While conventional electrospinning only produces flat two dimensional membrane, development in the process have lead to the ability to produce fiber bundles. This has opened the possibility of using electrospun fibers in tendon and ligament applications.

The extracellular matrix (ECM) of ligament and tendon is made out of longitudinally aligned nanofibers. Electrospun nanofibrous yarn with longitudinal alignment closely resembles them and this provides a physical stimulus for the cells. Ligament cells cultured on aligned electrospun nanofibrous scaffold has been shown to align along the length of the fibers. To improve the mechanical properties of electrospun scaffold, braiding may be used [Benjamin et al 2017]. Alternatively, electrospun fibers may be coated on microfibers and twisted or braided to give the final construct [Naghashzargar et al 2015]. Periodontal ligament cells cultured on aligned fibers showed higher metabolic activity and better proliferation on aligned electrospun fiber scaffold compared to randomly oriented fibers and cast films [Shang et al 2010]. Study by Bosworth et al (2014) has shown that human mesenchymal stem cells (hMSC) seeded on electrospun yarn demonstrated differentiation towards tendon lineage when cultured under mechanical loading.

Several strategies have been explored to take advantage of electrospun structures for ligament and tendon repairs. These are broadly divided into cellular or acellular methods. In acellular methods, electrospun fibers are implanted directly into the injury site without any pre-seeded cells. Scaffolds employing this method may comprise of random or aligned fibers. With cellular strategy, cells were seeded into the scaffold prior to implantation. Seeding of the cells may be carried out either just before implanting or cultured for a fixed duration either for cell adhesion or expansion. In vivo tests of both acellular and cellular electrospun scaffold has shown promising results with significant remodelling [Petrigliano et al 2015; Vaquette et al 2017]. An advantage of electrospinning is the relative ease of incorporating active ingredients into the scaffold. Nitric oxide (NO) is known to be beneficial for angiogenesis and Chen et al (2021) used a NO-loaded metal-organic frameworks (MOFs) for encapsulating within a polycaprolactone (PCL)/gelatin (Gel) aligned coaxial scaffolds (NMPGA) for tendon repair. A Cu-based MOFs nanoparticles were used to load NO. A solution consisting of this NO/MOFs and PCL would form the core and gelatin, the shell of the electrospun core-shell fibers using a co-axial nozzle. In vitro release of NO was found to be stable over 15 days without any burst release at the first 48 h. An in vivo rat patellar tendon model showed increased blood perfusion near the injured tendon. Collagen maturity and recovery of biomechanical strength of the injured tendon was also reported to be faster compared to the control 4 weeks post surgery. Therefore, NMPGA has the potential to be used as a tendon regenerative scaffold.

In vivo studies have also supported the use of electrospun fibers in ligament repairs. Petrigliano et al (2015) tested the performance of unoriented electrospun polycaprolactone (PCL) coated with collagen in an anterior cruciate ligament (ACL) rat model. After 12 weeks, secreted collagen showed evidence of orientation which means that remodeling is actively taking place and it has the potential to transform to native ACL form given sufficient time. The mechanical strength and stiffness of the scaffold also increased with implantation duration up to 12 weeks which was the end point of the experiment. Vaquette et al (2017) pre-seeded low fiber density polycaprolactone (PCL) electrospun membrane with sheep mesenchymal stem cells (sMSCs) and cultured for 4 weeks in vitro before braiding with two other bundles for the final implantable scaffold. Mechanical tests showed a stress/displacement J-curve that is similar to native ligament for the resultant scaffold which was not exhibited by non-cellularized scaffold. In vivo tests using immunocompromized rat also showed significant remodelling and well colonization by host cells on the implanted scaffold.

As with ligament repairs, In vivo test has also been carried out for tendon repair using electrospun fibers. Rashid et al (2020) constructed a multi-layered scaffold made of mainly electrospun polydioxanone (PDO) aligned fibers, electrospun polycaprolactone (PCL) random fibers and woven PDO fibers. PCL with its lower melting point was used as a bonding agent to hold the scaffold together after heat treatment. A sheep, digital flexor tendon injury model was used to determine the efficacy of the scaffold. At 3 months, the electrospun component of the scaffold has integrated with the host tendon tissue with significant ingrowth and proliferation of endogenous cells in the electrospun fibre. Much of the electrospun PDO material has degraded and remnants were rarely seen. However, no cells were observed on the woven component indicating that the electrospun fibres were more effective in directing cell migration and proliferation. Neovascularization of collagen containing tissue formed within the electrospun layers were also observed which is crucial in supporting tendon healing. Rashid et al (2020b) have also done a comparative in vivo study between electrospun polydioxanone (PDO) fibers twisted yarn and PDO monofilament suture for treatment of deep digital flexor tendon (DDFT) longitudinal surface defect within the digital sheath of a sheep model. Three months after implantation, there were no residual scaffolds from either from the electrospun suture or the monofilament with mild or no adhesive to the sheath. With the electrospun PDO fibers yarn, the suture areas were populated with highly cellular and vascularized tissue. However, in the PDO monofilament, the defect area was devoid of cells and without any increase in vascularity. The difference in the cellular infiltration was attributed to the high porosity of electrospun fiber yarn compared to the nonporous mono-filament. High porosity facilitates fluid penetration and this increases the hydrolytic degradation of the material.

(A) Diagram representing different layers in the electrospun patch. Seven layers of aligned electrospun PDO fibers are sandwiched between 6 layers of thin PCL electrospun grids (acting as an adhesive) in an alternate manner forming the electrospun component of the patch. The electrospun component is bound to a woven PDO monofilament layer by a single layer of aligned electrospun PCL fibers. (B) Photograph of patch sample showing white electrospun PDO layer (facing tendon) and blue woven PDO layer. (C-E) Representative scanning electron microscopy (SEM) images showing electrospun layer. (F-H) Woven layer of the patch at various magnifications shown [Rashid et al 2020].

In tendon/ligament injuries, one challenge is the reintegration of the ruptured tendon or ligament to the bone. Electrospun membrane has been tested positively as guided bone regeneration graft [Dimitriou et al 2012] and the same concept may be applied for re-establishment of tendon/ligament to bone interface. Chen et al (2018) tested the effectiveness of electrospun random and dual-layer aligned-random silk fbroin poly(l-lactic acid-co-e-caprolactone) (P(LLA-CL)) nanofibrous scaffolds (ARS) in tendon-to-bone healing in a rabbit extra-articular model. Autologous Achilles tendon was wrapped either in ARS or electrospun random silk fibroin/P(LLA-CL) membrane and passed through the bone tunnel, while the control group was unwrapped Achilles tendon transplanted directly. Various parameters such as ultimate load-to-failure and stiffness, collagen maturity and new bone formation was better with electrospun membrane wrap and the ARS wrap demonstrating the best results in the 12 weeks study. Zhu et al (2019) investigated the effect of adding kartogenin (KGN) to electrospun aligned polycaprolactone (PCL) for treating rotator cuff tear (RCT). The challenge in treating RCT is the difficulty of regenerating the fibrocartilage zone. KGN is a small molecule drug that was found to promote chondrocyte differentiation and is blended into PCL solution before electrospinning. Animal studies were carried out on a rat model by severing its rotator cuff tendon and removal of its fibrocartilage. The tendon was repaired to its anatomic footprint by transosseous repair with the aid of KGN loaded electrospun aligned PCL membrane. Repairs with PCL/KGN membrane showed the highest level of collagen-fibrils formation from week 2 onwards compared to group without membrane and with PCL only membrane. The collagen fibrils were also better organised in the groups with aligned electrospun membranes. Pull out tests at week 8 showed significant better ultimate load to failure for the group treated with PCL/KGN membrane compared to PCL only and no membrane treatment. These studies showed the benefits of using electrospun aligned fiber membrane in RCT repair in particular with the addition of KGN.