**5. When are electrospun nanofibers not a good option?**

Electrospinning is an impressive technique however, the size of the fibres being nano, is a disadvantage when it comes to control. The limited control of the pore size (Electrospun scaffold) is a diameter dependent which reflects on the cellular infiltration (decrease due to smaller average pore size) [36].

Another reason being the degradation effect (introduced in the latter half of the introduction). The rapid degradation of nanofibrous constructs can adversely affect the ability of the scaffolds to support tissue growth. The structure of the nanofibers plays an important role, especially when it comes to nanoscale, the high surface area to volume ratio serves as the reason for its selection. However, in case of degradation effect, due to this property, the nanofibers are prone to hydrolytic degradation. Hence long-term processes should not employ such scaffolds as before the entire process (observation, selection or any other research studies) is completed, the culture will have no support to grow [36]. Crystallinity in polymers can treat this problem however the size of the fibres (diameter) are still a variable with a high probability of variation (purpose dependent and needs a lot of testing before it can be finally put into use). The poor infiltration of cells into scaffolds is still an issue to deal with, especially when we want to add various properties into nanofibers.

As mentioned in the introduction, electrospinning is an easy to setup and scalable technique. The cost parameter is in our favour whereas the volume imposes some difficulties in terms of production. It is quite difficult to produce a large volume scaffold and if the critical factors do not meet the threshold level the final structure might not be at its best form. This will drastically affect the application part. This will also affect special properties like antimicrobial/inflammatory/oxidant. The release of the drug will be questionable in such cases (**Table 2**).

The drug loading process when it includes a high amount of drug can result in a burst. When we submerge the fibres in aqueous solution (prone to hydrolytic degradation), the antimicrobial properties (e.g., antibiotics) are released in a short duration (might not last till it's required). This issue can be solved by using different set-ups. We have learned that electrospinning technique is an easy to set-up one, here if we want to use such nanofibers for a longer duration, a different set-up is required which introduces more complication [37].

When environmental factors are taken into consideration, most frequently relative humidity is studied. When this parameter is a settable, considering higher RH leads to thinner fibre diameter, an appropriate high RH level could be selected.


**Table 2.**

*Variations in polymer nanofibers size in ligament and tendon tissue engineering [36].*

Since high RH levels may lead to beads, too high RH levels cannot be selected. Whereas if an ambient RH cannot be controlled. Then RH is considered a disturbance of the electrospinning process and the required jet diameter, responding to the desired fibre diameter, should change with an ambient RH. The system's velocity should be adjusted along with the flow rate. This is done to obtain a required jet diameter (the application of electric field in the polymer solution will cause the droplets to take a conical shape) and control the fibre's diameter. This is where production will become an issue. Such adjustments are difficult and are highly variable. Hence, relative humidity should be studied to decide what type of parameter it is (settable or disturbance) to decide the production rate. The operating regime should be selected to achieve the desired fibre diameter while maximising production rate [38].

Along with relative humidity, temperature should also be taken into consideration. The average diameter of nanofibers produced by electrospinning changes significantly through variation of temperature and humidity. At a relatively higher temperature the solvent evaporation rate will increase and the viscosity of the precursor solution to be electrospun will decrease, and as a result thinner nanofiber would be obtained [39].
