*2.2. Gender and Age*

Although some behavioral di fferences are seen in neurons and Schwann cells associated with gender and age, their impact on clinical neurological recovery following nerve trauma is not clear. Thus, it has been observed that the density of epidermal nerve fibers decreases with age, and is lower in men compared with women [8]. This suggests age and gender influence innervation.

In hamsters, recovery of function following facial nerve injury is significantly faster in females than males [9]. While the administration of exogenous steroids accelerates axon regeneration in males, it has a lower impact on the rate of regeneration in females [9]. In rats, continuous exercise training increases the extent of axon regeneration in male, but not female, or castrated rats [10,11]. However, interval exercise training enhances axon regeneration in female, but not male rats [10,11].

In the male animal model, the influence on axon regeneration is associated with the expression of androgens [12]. They influence the extent of axon regeneration in peripheral nerves by regulating motor neurons' expression of brain-derived neurotrophic factor (BDNF) and its receptor, trkB [11,13,14]. These molecules, in turn, influence the extent of axon regeneration [10,12]. The e ffect of training on axon regeneration in females works through a di fferent mechanism than that of males [12]. Further studies are required to determine whether hormone treatment strategies may be e ffective in enhancing the extent of neurological recovery following nerve injury.

#### *2.3. Promoting Axon Regeneration Through Crushed Nerves*

Two to three days after a crush nerve injury, the severed axons begin to regenerate into the distal part of the nerve and continue to regenerate until they reach and reinnervate their original targets. Axon regeneration is promoted by the denervated Schwann cells in the distal portion of the nerve by their release of neurotrophic factors, and their extracellular matrix [15–21]. The greater the number of axons that regenerate through the distal nerve, the greater the extent of neurological recovery [19,22] Generally, the precision of target reinnervation is extremely high [19].

#### *2.4. Restoration of Function without Surgical Intervention*

Following a nerve transection, the nerve stumps normally retract, resulting in a gap of ≤3 mm. Despite this small gap, neurological recovery may develop without surgical intervention. This is due to a cascade of events involving the di ffusion of fibrinogen from leaky blood vessels into the nerve gap where it combines with thrombin. This leads to fibrinogen polymerization and the formation of a three-dimensional fibrin matrix within the gap [23]. This matrix provides passive support to axons, which allows them to regenerate to the distal nerve stump.

However, fibrin clots are converted into a matrix that actively promotes axon regeneration by the migration of Schwann cells into the fibrin clot from the central and distal nerve stumps. These Schwann cells release a cocktail of neurotrophic and wound healing factors that bind to the pure fibrin converting it from a passive three-dimensional matrix into one that actively promotes axon regeneration [24]. This results in a significant increase in the number of axons that regenerates across the gap [25].

The e fficacy of fibrin in promoting axon regeneration is increased by the platelets and mesenchymal stem cells that become entrapped in the fibrin clot in the nerve gap. They act by multiple mechanisms: (1) They release neurotrophic and other factors that act directly on the axons to promote regeneration [26]. (2) They release factors that promote Schwann cells of the distal nerve pathway to proliferate and release neurotrophic factors, which also enhance the extent of axon regeneration [27]. (3) The mesenchymal stem cells di fferentiate into Schwann cells, which release neurotrophic and other factors, thus enhancing the concentration of these factors and the extent of axon regeneration [28]. (4) Mesenchymal stem cells release factors that induce angiogenesis, which is essential for axon regeneration [29].

Although factors within the fibrin clot promote axon regeneration, factors are also required to direct axons across the nerve graft. Growth cones extend fine processes that sample the environment around them in search of factors to which they can adhere, and that both promote and direct their growth. When Schwann cell-released neurotrophic factors are distributed in a uniform concentration around neurons and their growth cones, neurite outgrowth is promoted, but the outgrowth is random. However, when neurons and their growth cones are exposed to a concentration gradient of those same factors in vitro and in vivo, the growth cones turn and increase the concentration gradient of the Schwann cell-released factors [19,30]. This is because, as the factors di ffuse away from the distal nerve stump, they create a concentration gradient of the factor, which is the highest at the distal nerve stump [30–33]. This directs the axons up the gradient and to the distal stump [19,30,33–35]. Once the axons reach the distal nerve stump, their regeneration continues to be promoted and directed through the distal nerve segmen<sup>t</sup> by the concentration gradient of Schwann cell-released factors ahead of them.

#### *2.5. Restoration of Function with Surgical Intervention—Anastomosis*

When a nerve has a clean transection, or when the nerve defect is small (only a few millimeters), the nerve stumps can be anastomosed, which leads to the restoration of function. However, to develop functional recovery, the repaired nerve must be tension-free [36]. When anastomosis is performed within 14 days of nerve trauma, functional recovery is good in about 80% of patients [37]. However, with increasing time between nerve trauma and anastomosis, the extent of recovery decreases [38–40]. The types of changes that occur over time that lead to this decrease in recovery are discussed below.

#### *2.6. Promoting Axon Regeneration Across Nerve Gaps*

When the gap between the nerve stumps is too long, anastomosis is not possible because the nerve cannot be stretched to extend across the gap. Therefore, to restore function to such nerves, the gaps must be bridged with a material that both supports and promotes axon regeneration entirely across the gap.
