2. ECM Digestion

The ECM in the musculoskeletal system’s fibrous connective tissue comprises of aligned collagen fiber bundles that function as microstructural reinforcement to impart mechanical strength to the whole system ^[20][21][22]. The ECM imparts tissue resistance against external stresses along the fiber length and provides the strength to withstand the external load. During an injury to such tissues, the ECM–collagen microstructure gets disrupted, and hence the mechanical strength and load-bearing capacity of the tissue is altered. During the course of self-healing at the injury site, the tissue develops a scar mass that consists of a disordered high-density collagen fiber structure ^[22]. The application of a tissue-engineered scaffold at that site is limited because of the already deposited dense ECM of the scar. This is because the naturally existing high-density ECM bears less porosity, which hinders cell proliferation, adhesion, and migration. Hence, it acts as the barrier for endogenous tissue repair, resulting in inadequate healing. In this regard, researchers have suggested the use of the collagen digestion approach as an effective strategy for managing the efficient working of a nanofibrous implant. In this course, several studies have shown effective wound healing while applying digestion of wound edges using matrix-digesting enzymes that are immobilized on nanofibers. Examples of such enzymes that many researchers use as biocatalysts for tissue repair are collagenase, trypsin, hyaluronidase, etc. ^[23][24][25].

An excellent illustration of the application of this strategy to tissue repair is the one demonstrated by Qu et al. for restoration after knee meniscal injury ^[26]. It is well known that tearing in joint menisci leads to the destruction of collagen microstructure and the development of dense collagen scar that often results in degenerative osteoarthritis ^[27][28]. The primary treatment for meniscal injuries is meniscectomy or resection of the affected meniscal area through the surgery. However, the recovery rate during such a procedure is relatively low, and the meniscectomy imparts only temporary pain relief. Several patients need a second surgery, resulting in increased morbidity rates. Furthermore, the surgical procedures may damage articular cartilage and meniscal fibrocartilage, requiring total arthroplasty in some cases ^[29][30].

The basic idea behind employing a biocatalytic approach for such tissue repair is to decrease the density of meniscal scar to reduce the impedance in the healing process and allow easy cell migration, proliferation, and matrix deposition. Qu et al. fabricated collagenase-incorporated nanofibers for the repair of knee meniscal injury, employing the biocatalysis of collagen degradation in the dense matrix of wound scar. The nanofiber scaffolds were fabricated by electrospinning using 8% w/v polyethylene solution (in 1:1 ethanol/water) or 40–50% w/v polyethylene oxide/poly(ε-caprolactone) solution containing 1.25% w/v collagenase for 10–15 min onto glass coverslips. The in vitro release studies indicated that the nanofibers released the collagenase enzyme in a controllable manner. The scaffolds were evaluated in vitro for meniscal repair in juvenile and adult bovine menisci, as indicated in Figure 1. The scaffolds were placed in the meniscus defects, which significantly reduced the matrix integrity, increased the porosity, and reduced the levels of proteoglycans and collagen at the wound edge. This resulted in an increase in the cell infiltration and integration of fibrils, leading to the closure of more than 90% of the wound within four weeks. The annulus-core boundary of the meniscus displayed relatively higher cell density.

Figure 1. Reduction in the adult bovine meniscal matrix density and cellularity increase through collagenase-immobilized nanofiber treatment. (A) Schematic showing meniscal annulus and core. (B) Histological analysis of repair constructs using hematoxylin and eosin staining (left) and 4′,6-diamidino-2-phenylindole (right) staining. Reproduced from ^[26]. Copyright (2013) Elsevier Ltd.

Feini et al. adopted the same approach for the fabrication of biocatalytic nanofibers that incorporated a chemotactic agent in addition to the immobilized collagenase ^[31]. These researchers hypothesized that in addition to reducing the stiffness and density of native ECM, it was essential to guide the adult repairing cells to migrate to the wound site. Therefore, a chemotactic agent (i.e., platelet-derived growth factor-AB) was used to encapsulate in the nanofibers. These nanofibers were fabricated by electrospinning using 15% hyaluronic acid, 35% poly(ethylene oxide), and 50% poly(ε-caprolactone). Various nanofibrous scaffolds of different compositions that were evaluated are shown in Figure 2.

Figure 2. Nanofibrous scaffolds used for in vivo evaluation of collagenase-immobilized nanofibers. (a) Animal model for tissue repair evaluation: scaffolds placed subcutaneously in rats; (b) composition of various scaffolds used by Feini et al. in their study. PCL: poly(ε-caprolactone); PEO: poly(ethylene oxide); HA: hyaluronic acid; PDGF-AB: platelet-derived growth factor-AB. Reproduced from ^[31]. Copyright (2017), Feini Qu et al.

The nanofibers were able to successfully release the collagenase with an initial 20% showing a burst release and the remaining 80% released in a controlled manner over a period of 5 h in a phosphate buffer saline. On the other hand, 67% of platelet-derived growth factor-AB was released over a period of 12 days. The nanofibers were evaluated for their tissue repairing application in meniscal defects. The results showed increased cellularity at the wound site and integration with the adjoining tissues. The nanofibers were assessed for their meniscal repair potential in vivo in athymic rats by inserting the nanofibrous scaffolds subcutaneously. This revealed the role of the dense extracellular matrix in hindering the healing process of the wound. The degradation of the wound matrix collagen increased significantly with the use of the novel collagenase scaffold, while allowing for the formation of its own cellular matrix. This was evident from the appearance of interconnected thin collagen fibers that were found to bridge the scaffold with the wound tissue within four weeks (Figure 3).

Figure 3. Effect of collagenase-immobilized, platelet-derived growth factor-AB nanofibrous scaffolds on tissue integration, matrix formation, and cellularity. (a) Histological images of hematoxylin and eosin-stained wound after scaffold fixation. (b) Histological images of the wound after immunostaining showing collagen I and II; (c–e). The cell infiltration, normalized collagen II signal within the scaffold and % defect integration (% integrated tissue at the wound site) for various scaffolds are also presented. # = p < 0.05 vs. Scaffold, ** = p < 0.05 vs. Scaffold + P. These figures are reproduced from ^[31]. Copyright (2017), Feini Qu et al.

In these results, the cell proliferation and new collagen formation were more evident in the case of collagenase-immobilized scaffolds. An important aspect of these novel scaffolds is that the collagen degradation takes place only up to 300 µm from the wound edge. The study further revealed endogenous cell-mediated wound closure by recruitment was possible through the chemotactic property of the scaffolds.

Another target for extracellular digestion as a fast tissue repair facilitation strategy is the chondroitin sulfate proteoglycan digestion. The formation of a glial scar is considered as one of the major factors restricting the nerve regeneration process during the healing of a spinal cord injury. Therefore, a glial scar remains a chief therapeutic target for the treatment of spinal cord injury and efforts are being put forward to devise methods for eradicating glial scar to facilitate adequate reconstruction. Because the significant component of such a scar is chondroitin sulfate proteoglycans, several researchers have used a biocatalytic approach to eradicate these glial scars associated with spinal cord injury by using the enzyme chondroitinase as the biocatalyst. There is not much literature available demonstrating this application of the biocatalytic nanofibers; however, Liu et al. have applied this approach to the treatment of spinal cord injury ^[32]. Presently, intrathecal injection of chondroitinase ABC (lasting for weeks) is the central treatment approach for spinal cord injury, which is quite invasive, apart from chondroitinase ABC being thermal sensitive and susceptible to degradation in the host. Additionally, the use of intrathecal chondroitinase ABC injection is restricted because it overflows beyond the injection site, reducing the amount reaching the actual injury site. Liu et al. fabricated collagen scaffolds containing neurotrophin-3 and chondroitinase ABC for the repair of spinal cord injury. These nanofibers were able to release both neurotrophin-3 and chondroitinase ABC in a sustained manner, thus avoiding the rapid clearance from the site of action, which generally happens with conventional drugs. A similar combination for spinal cord injury treatment has already been attempted by other researchers who demonstrated an enhanced locomotor function and sensory axon growth in rats ^[33]. However, hydrogels are generally isotropic and weak. Furthermore, hydrogels exhibit poor mechanical and morphological properties, which are pivotal for cell alignment, migration, and proliferation. On the other hand, nanofibers fulfill all these requirements and provide an excellent alternative as a scaffold for neurotrophin-3 and chondroitin ABC delivery for neuronal repair in the treatment of spinal cord injury. Nanofibers’ excellent topographical properties enable their use in spinal cord injury due to their ability to increase cells, as demonstrated by Chew et al. using Schwann cells ^[34]. Further, when incorporating neurotrophic factors in the topographically favorable nanofibers, an increase in nerve regeneration was observed ^[35]. As demonstrated by Liu et al., nanofibers have been used in the treatment of acute spinal cord injury in rats ^[36].

The nanofibers fabricated by Liu et al. provided sustained release of both neurotrophin-3 and chondroitin ABC, minimizing the chances of losing therapeutic agents. These researchers used rat tail extracted collagen scaffolds for immobilization of neurotrophin-3 and chondroitin ABC. The immobilization was achieved by crosslinking through microbial transglutaminase crosslinking. In addition to this, heparin was added to increase the protective effect on chondroitin ABC. This was evident because the chondroitin ABC showed 32% bioactivity after 32 days compared with the 1.9% action after 22 days.