Fibrin Based Microthread Scaffolds

Fibrin is a natural provisional matrix protein that is vital in the clotting of blood at a wound site. When tissue damage that results in bleeding occurs, thrombin, a clotting enzyme, polymerizes fibrinogen into fibrin at the wound site (O’Brien, 2016). Fibrin is made up of long fibrous chains that entangle platelets at the wound site to create a blood clot. Since fibrin is naturally found in the body and facilitates wound healing, it is a biocompatible material that supports cell adhesion and proliferation in the direction of the fibers. This makes fibrin a promising material to use as a biomimetic scaffold for wound healing. Fibrin scaffolds act as a delivery vehicle for growth factors to increase cell proliferation to the target site. As the adherent cells create new ECM, the fibrin scaffold degrades to allow the newly formed tissue to take over. Fibrin has been used to create multiple types of scaffolds including hydrogel scaffolds and microthread scaffolds (Litvinov, 2016).

Fibrin microthreads and fibrin bundles offer a number of benefits for skeletal muscle regeneration; they provide contact guidance for cell growth, act as a delivery vehicle for muscle- derived cells, promote functional skeletal muscle regeneration, and deliver necessary growth factors (O’Brien, 2016). Page et al. 2011, loaded fibrin microthread bundles with adult human cells harvested from an adult male’s muscle tissue. The cell-loaded fibrin bundles were placed into a large wound in the tibialis anterior (TA) of a mouse. After 2 weeks, the fibrin threads were no longer individually visible by gross inspection and there was evidence of physical contact between the native tissue and the fibrin threads at the wound site. Thirty days after implantation, the wounds sites with fibrin implants (about 8%) had significantly less collagen than untreated controls (about 55%) (Figure 5), implying that the fibrin scaffolds prevent the formation of scar tissue (Page, 2011).

Figure 5. Histological sections of wound healing in untreated mouse tibialis anterior defect (A) and fibrin microthread implanted mouse tibialis anterior defect (B). Masson’s Trichrome stain was used for collagen (blue) and muscle tissue (red). Quantification of collagen deposition at no implant (untreated) and implant wound site (C) (Page, 2011).

In addition, the cell-loaded fibrin microthread bundle implants improved the recovery of muscle strength 4 months after surgery. The tetanic intermittent and maximum force was measured on mice without a TA defect (baseline), on mice with an untreated TA defect (no implant), and mice with a TA defect treated with cell-loaded fibrin microthread implants (implant). After the injury was left to heal, the results of the maximum tetanic force indicated a positive linear trend of muscle function in implanted mice, reaching almost 100% recovery at 90 days post-surgery. No implant mice showed a negative linear trend of function and exhibited just over 50% of normal (baseline) muscle strength. These results reveal that cell-loaded fibrin microthreads promote functional skeletal muscle recovery in a large-scale muscle wound (Page, 2011).

Grasman et al. 2015, fabricated a biomimetic scaffold using fibrin microthreads and incorporated varying biochemical and biophysical cues to promote tissue regeneration. Researchers conducted a study using cell seeded microthreads as a scaffold in a rodent VML injury. Human muscle cells were seeded onto fibrin microthreads and then secured in bundles into a VML injury created in the tibialis anterior (TA) muscle of mice and examined after 90 days. Time-course examination of the maximum tetanic force revealed the fibrin microthread scaffolds helped restore strength to the injury site. The mean tetanic force of the uninjured muscle, non-innervated muscle, and treated muscle were 16 g, 10 g, and 14 g respectively. Histological analysis of the injury site indicated that the microthreads appeared to reduce scar formation at the wound site as well as promote myotube formation. Fibrin microthreads appear to be a promising therapy for VML injuries, but more in vitro studies need to be conducted in order to test alterations to structural, mechanical, biological, and chemical properties of the microthreads to improve results and better predict how the scaffold will perform in an in vivo wound healing environment (Grasman, 2015).

The Pins lab has continuously been working to enhance the biological, chemical, and physical properties of fibrin microthreads (Grasman, 2017). A study comparing fibrin hydrogel scaffolds and fibrin microthread scaffolds was conducted to determine which scaffolding material was able to best promote regeneration of functional tissue in a mouse VML model. A VML injury was created in the TA muscle of a mouse. After the formation of the injury one of 5 treatments was implanted into the wound site: no intervention (control), fibrin hydrogel,

uncrosslinked (UNX) microthreads, crosslinked (EDCn) microthreads, or crosslinked and hepatocyte growth factor loaded (EDCn-HGF) microthreads. HGF is key signaling molecule for cell adhesion and wound healing found in the basal lamina of the body. In large skeletal muscle

injuries, the basal lamina is destroyed and HGF is removed. The fibrin gel treatment was used as a control to study the effect of the microthread architecture on wound healing. Pins lab

performed force and histological experiments to determine which condition promoted functional wound healing.

Sixty days post injury, a tetanic force analysis and a histological and

immunohistochemical analysis were performed on the animal subjects. Researchers concluded that crosslinked fibrin microthreads loaded with HGF (EDCn-HGF) were the most promising scaffold technology to promote skeletal muscle growth in a uniaxial direction. HGF stimulates the activation and migration of satellite cells from the wound margin onto the scaffold and to the injury site (O’Brien, 2016). EDCn-HGF microthreads were able to produce 200% the force of the injured muscle. Fibrin gels were only able to produce about 125% the force of the injured muscle. As indicated in the histological analysis (Figure 6), the no intervention treatment shows excess collagen tissue represented by the blue arrows, while the fibrin gel treatment resulted in the formation of adipose tissue represented by the yellow arrows. The EDCn-HGF resulted in regenerated muscle tissue with myofibers in direct contact with the threads (green arrows). Both adipose tissue deposition and collagen deposition at the wound site do not lead to functional muscle regrowth because the fat cells and collagen fibers do not align themselves uniaxially along the scaffold to allow for maximum contraction of the muscle.

Figure 6: Histological analysis of no intervention (A), fibrin gel (B), and fibrin microthread (E) treatments. The bottom pictures are a cross section of the target site with blue arrows indicating fibrous tissue, yellow arrows

indicating adipose tissue, and green arrows indicating aligned myofibers (Grasman, 2015).

Microthreads appear to be a successful method to facilitate cellular migration and proliferation of native muscle cells with reduced collagen and adipose deposition and increased muscle strength (Grasman, 2015). However, tissue engineers continuously strive to improve the clinical outcome of biomaterial-based therapies. The biological properties, strength, immune response, recruitment of cells, and infiltration of cells, of fibrin microthreads could be optimized and tested in vitro to improve the clinical results of fibrin microthreads as a treatment for VML. Therefore, a 3D in vitro model is needed to mimic the biological environment while testing the effectiveness of several scaffolds for muscle cell growth.