A Critical Analysis on

“Human Hair Keratins Promote the Regeneration of Peripheral Nerves in a Rat

Sciatic Nerve Crush Model”

By:
Jianyi Gao
Lei Zhang
Yusheng Wei
Tianyan Chen
Xianyan Ji
Kai Ye
Jiahong Yu
Bin Tang
Xiaochun Sun
Jiabo Hu

Submitted By:
Manto, Allysza A.
12 - Edison

Submitted To:
Ms. Emmylou C. Redoble
Adviser

Date of Submission:
April 5, 2023
 Axon regeneration and functional recovery after peripheral nerve injury

remains a clinical challenge. Peripheral nerve injury has been rising every year and

caused a heavy economic burden on patients and society (Burnett & Zager, 2004;

Navarro et al., 2007). To date, there is no effective treatment for peripheral nerve

injury, and biomaterials are considered a promising option. In recent years, a variety

of nerve repair materials have been developed, such as medical regenerative

collagen membranes, polyglycolic acid and polylactic acid copolymers (Toba et al.,

2001). However, the application of these biological materials in the body still has

diverse problems, including immune rejection, uncertainty of degradation speed,

toxicity of degradation products, limited sources of materials and high costs. This is

the part of the study where human hair keratin is introduced, which makes it even

more fascinating and leads to its selection as a subject of the critical analysis.

Human hair keratin is considered to be an excellent biomaterial to promote

the repair of peripheral nerve injury due to its good biocompatibility, nonimmune

response, good intercellular interaction and biodegradability (Lee et al., 2014; Hill et

al., 2010). The adhesion sequences LDV and RGD of keratin are also found in

several extracellular matrix proteins capable of supporting cell attachment

(Tachibana et al., 2002; Reichl, 2009; Verma et al., 2008). Previous studies have

shown that 12 kinds of cells, such as neural and bone marrow stem cells, have

obvious proliferation and growth in cell culture membranes prepared with keratin

nanoparticles (Reichl, 2009). Keratin can also form a uniform pore size keratin

sponge under a certain condition and will maintain its shape for several hours at a

high temperature of 60°C. This property distinguishes the keratin from relatively low

denaturation temperature collagen (Chevallay et al., 2000; Tachibana et al., 2002).

 In this study, the researchers hypothesized that keratin can activate repair-

related cells in vitro and that a prepared keratin sponge, as a dressing, can

accelerate the repair of sciatic nerve injury. Keratin is naturally produced in our body

(in our hair, nails, skin, glands, and organs), and to know that it can possibly be used

as a biomaterial to promote the repair of peripheral nerve injury makes it very

interesting.

The methods performed in this study are the following: (1) Human hair

extraction and fabrication of keratin sponge; (2) Electron microscopic (EM) analysis;

(3) Keratin coating; (4) Cells structure; (5) Primary cells culture; (6) Cell adhesion

assay; (7) Cell proliferation assay; (8) Cell migration assay; (9) Wound-healing

assay; (10) mRNA expression analysis; (11) Immunofluorescence (IF) staining; (12)

Rat sciatic nerve crush injury model; and (13) Sciatic functional index.

This study uses quantitative experimental research design, which for me is

appropriate since this study utilizes the scientific approach and it establishes

procedures to test the relationship among variables. Male SD rats weighing 180–

200 g were placed in a sterile environment. Twelve SD rats were randomly divided

into two groups (n = 6 per group): (1) control group and (2) keratin group. Unpaired

student’s t-tests were used to compare the differences between two groups. One-

way ANOVA was used to compare the differences among all the groups and LSD-

t test was used for further comparison between the two groups. Each experiment

was independently performed at least three times.

Human hair keratin has been gradually applied to the field of nerve injury due

to its good biological properties and plasticity. A high molecular weight keratin was

obtained by opening disulfide linkages and destroying hydrogen bonds by reducing

method (Hill et al., 2010). This kind of keratin molecule has less negative charge in

vivo and is apt to form a more stable structure (Waters et al., 2018).

As “repair cells” after injury, SCs play important roles in the re-establishment

of neural structure. In vitro experiments showed that keratin had neuroinducible

activity, and SCs grown in the keratin group had a series of changes in migration

capacity, proliferation activity and morphology. These changes were similar to the

activation of Epithelial-Mesenchymal Transition (EMT) program in SCs, which is the

key physiological response of cells involved in repair (Jessen & Farraj, 2019). SCs

can also secrete neurotrophic factors to nourish axons, prevent axon apoptosis and

stimulate cell proliferation by several pathways (Agthong et al., 2006; Yamazaki et

al., 2009; Terenghi, 1999). This study demonstrated for the first time that keratin can

promote neuronal axon extension in vitro. The DRG neurons seeded on keratin

group were superior to those of the control group in both quantity and length as

demonstrated in Fig. 5b. This change may be related to the activation of intracellular

cAMP levels by keratin, thereby increasing the intrinsic growth capacity of neurons

(Soto, 2017).

SCs are not the only important cells in the regenerative environment. After

nerve damage, macrophages are attracted to the site of injury, strictly regulate the

local inflammatory response to avoid excessive tissue damage and create a suitable

microenvironment for nerve regeneration (Fearing & Dyke, 2014; Waters et al., 2018;

Chen, 2015). LPS is a commonly used substance in the establishment of

inflammatory models. The increase of anti-inflammatory factor IL-10 after LPS

stimulation may reflect the protective immunity of cells. Keratin can downregulate the

expression of pro-inflammatory factors and upregulate anti-inflammatory factors, as

shown in Fig. 6. In addition, the researchers observed that a small number of SD rats
in control group showed inflammation reaction on the early postoperative,

characterized by the redness and fever of toes. This body’s excessive inflammatory

response to injury can recover spontaneously within a week. Due to the anti-

inflammatory effect of keratin sponge, this phenomenon did not occur in the keratin

group.

Ice crystals in keratin solution connected with each other at low temperature

to form a homogeneous porous structure, which transformed keratin from liquid to

solid. This spongy keratin has a porous structure and a certain mechanical strength,

high temperature resistance and insolubility in water. It is well known that the pore

structure plays an important role in the growth of new tissue and is beneficial to the

nutrient and waste exchange and the inhibition of scar formation by fibroblasts

(Tachibana et al., 2002; Wu et al., 2015; Ezra et al., 2014). The keratin sponge was

wrapped around the lesion to provide a suitable space for nerve repair, preventing

the adhesion to surrounding tissues and facilitating nerve regeneration. SD rats in

the keratin group had significant improvement in terms of footprint analysis and SFI

index compared with those in the control group from 14 days after surgery,

suggesting that wrapping the keratin sponge in the injured site accelerates axon

regeneration at an early stage, and lays a solid foundation for later functional

recovery. The recovery of myelin sheath at the distal of the regenerated axons was

further observed by TEM, the results of which showed that the axons in the keratin

group are arranged neatly and that the diameter and thickness of the myelin sheath

increase significantly, which is better than the control group and close to the normal

nerve group. Because the axon cannot regenerate to the target organ in time or the

regeneration effect is poor, the gastrocnemius wet weight and muscle fiber

morphology in the control group were lower than those in the keratin group at 21
days. Studies have shown that axons grow at a rate of 1–3 mm/d after peripheral

nerve injury, so the rate of degradation of keratin sponge should be adapted to the

rate of axonal regeneration appropriately. In this study, most of the kinetic function of

SD rats could be recovered in about 3–4 weeks after crush injury, so the degradation

cycle of keratin sponge prepared in our laboratory is sufficient for nerve regeneration

within 5 weeks.

Keratin has a wide range of applications and has been developed in other

areas such as the repair of heart, skin and bone tissue. Previous studies have noted

that an antibiotic-loaded keratin film shows high antibacterial effects in bacterial

sensitivity assays and can potentially prevent wound infection (Lv et al., 2016). In

addition, keratin dressing applied to the wound site of animal models can accelerate

the onset of epithelialization and faster wound healing compared with polyurethane

dressing (Paladini et al., 1996; Petchter et al., 2012). However, keratin still has

some limitations in its application to sciatic nerve transection injury, so it must be

combined with other materials to improve its mechanical strength. Nevertheless, the

researchers have reason to believe that keratin has great potential and prospect in

the repair of peripheral nerve injury. Other research opportunities that this study

presents include exploring and applying the said study to the central nervous

system, which is made up of the brain and spinal cord. It can also possibly be

applied to the optical nerve that fixes blurry sight or maybe blindness because of

keratin’s regenerating factor. Through further research, it can possibly be applied to

humans in the future which is of great help in healing the body.

In summary, the repair and regeneration after peripheral nerve injury is a

complex process. Keratin, as a natural biomaterial, can regulate the biological

activity of many kinds of cells, stabilize the microenvironment of the damaged site
and accelerate the regeneration of axons. Therefore, keratin has a good promoting

effect on both cell culture and tissue repair, but its potential regulatory mechanism

needs to be further explored.