Human hair can be roughly divided into three layers. The cuticle on the outermost side, the cortex, and the medulla, which is the innermost region forming the core of the hair. The medulla is generally considered to have a porous structure and may exist in a discontinuous state, fragmented in the axial direction of hair. However, there are often cases in which the medulla does not exist, and it is thus a very uncertain region [1]. Regarding its function, although some researchers theorize that it contributes to heat retention by insulation because it becomes hollow due to keratinization [2], there are many reports indicating that it does not contribute at all to the chemical and mechanical properties of hair [3],[4]. As can be seen, there are many unclear points regarding the role and significance of the medulla, and to date, it is rarely talked about in the cosmetics field.

We have previously discovered that the structure of the medulla can change by chemical treatment, and by applying a formulation of specific ingredients to hair, we succeeded in reducing the voids of the porous medulla, thereby transforming the hair into a transparent and more beautiful state. We subsequently termed our method of allowing specific components to act on the medulla, “Hair Medulla Care” [5]. Regarding the relationship between the medulla and appearance, it has been reported that the porous structure of the medulla scatters light in light-colored hair. Therefore, the reduced transmission of light reduces the transparency and the reduced reflection of light reduces the luster [6]. In our survey on grey hair in Japanese women, the optical characteristics of the medulla were found have a substantial effect, with the scattering of light due to its porous structure making grey hair more noticeable. We further identified that a porous medulla was observed in more than 90% of conspicuous grey hair, regardless of age. Therefore, we proposed that by improving the porous structure inside the medulla, we could improve the luster and increase the transparency of grey hair, thus making it less noticeable.

Therefore, in this study, we first focused on the penetrating power of hair medulla care for evaluating the extent to which it acts on the medulla, and evaluated the penetrability of several components into the medulla. As a result, we found that Hair Medulla Care dramatically improved the penetration of various components, especially cationic components. By utilizing this property, basic hair dye could penetrate deeper into the hair, and the hair could be dyed in a much deeper and darker color than in the control case. In addition, we determined that melanin, which previously could not penetrate into hair, showed penetration following our treatment. Thus, we can conclude that Hair Medulla Care can improve traditional hair care and hair color standards and has the potential to become a completely new hair treatment.

Penetration Effect of Hair Medulla Care

Hair Medulla Care Procedure: A total of 1 g Asian grey hair bundle (Beaulax) was washed with 2% sodium dodecyl sulfate for 2 min before use. Hair bundles or the hair removed from them were treated with a pH 6.5 Hair Medulla Care solution, containing a specific polypeptide, penetrant, reducing agent, and pH regulator, then washed with water and dried.

Penetration Evaluation Of Various Fluorescent Dyes: Fluorescein isothiocyanate (FITC; 343-03664, DOJINDO), an anionic fluorescent dye, rhodamine isothiocyanate (RITC; R1755, SIGMA-ALDRICH), a cationic fluorescent dye, and Nile Red (144-0811, FUJIFILM Wako), a lipophilic fluorescent dye, were used to evaluate the promotion of Fluorescein isothiocyanate (FITC; 343-03664, DOJINDO), an anionic fluorescent dye, rhodamine isothiocyanate (RITC; R1755, SIGMA-ALDRICH), a cationic fluorescent dye, and Nile Red (144-0811, FUJIFILM Wako), a lipophilic fluorescent dye, were used to evaluate the promotion of penetration after Hair Medulla Care. After performing Hair Medulla Care, 60 μg/ml FITC, 60 μg/ml RITC, or 4.8 μg/ml Nile Red were added to the Hair Medulla Care solution, 16 µm sections were prepared using a microtome (SM2000R, LEICA), and observations and evaluations were made with a fluorescence microscope (BZ-X810, KEYENCE). In addition, in order to compare the penetrating power of Hair Medulla Care with general penetrants and reducing agents, 20% urea aqueous solution, 20% ethanol aqueous solution, 6.5% ammonium thioglycolate, and 6.5% sodium sulfite were compared using 60 μg/ml RITC as the dye.

Application to Cosmetics

Evaluation of Dyeing of Basic Hair Dyes: The grey hair bundle was treated with our Hair Medulla Care formulation, and subsequently dyed with a basic hair dye, and the dyeing was visually evaluated. In addition, after performing the same treatment on a hair sample cut out from a hair bundle, a section was prepared using a microtome, and dye penetration was evaluated by bright-field observations with a transmissive bright-field microscope (VHX-5000, KEYENCE).

Evaluation of Melanin Penetration: As an application of Hair Medulla Care technology, we examined whether this treatment could facilitate the infiltration of melanin into grey hair to restore its black color. A total of 1% melanin (M8631, Sigma-Aldrich) was added to the Hair Medulla Care solution, which was then applied to the 10% grey hair wig (Beaulax), allowed to stand for 12 hours or more, treated with shampoo, washed, and dried. This process was repeated 7 times and the results were visually evaluated. The same treatment was performed using eumelanin dissolved in purified water as a control.

Penetration Effect of Hair Medulla Care

Penetration of Various Fluorescent Dyes: Hair Medulla Care increased the penetration of all anionic, cationic, and lipophilic components into the hair medulla. With the anionic FITC, the fluorescence signal of the medulla was weaker than that of the cuticle and cortex, and with the cationic RITC, the medulla was uniformly and deeply stained. On using lipophilic Nile Red, the fluorescence signal of the medulla was more prominent than that of the cortex (Figure 1). It is known that there is an osmotic barrier between the cuticle and the cortex [7], but it was found that the use of Hair Medulla Care allowed compounds to pass through, regardless of their charge, hydrophilicity, or hydrophobicity, allowing the compounds to penetrate into the cortex and medulla. In addition, the difference in stain ability between FITC and RITC suggests that cationic components may pass through the permeation barrier between the cortex and medulla more easily than anionic components.

When the stained image of hair infiltrated with Nile Red by Hair Medulla Care was observed at a high magnification, it was found that the staining of the cell membrane complex (CMC) of the cortex was prominent (Figure 2). From this, we determined that the lipophilic component penetrates into the medulla by Hair Medulla Care, allowing the fibrous structure inside to be deeply dyed. Since it has been reported that medulla is a lipid-rich tissue [8], it is possible that the lipophilic component showed a higher affinity for the medulla than the cortex and became localized there.

Figure 1: Enhancing penetration of various substances by Hair Medulla Care.

Observation of the penetration of the anionic fluorescent dye FITC (A and A’), the cationic fluorescent dye RITC (B and B’), and the fat-soluble fluorescent dye Nile Red (C and C’) with a fluorescence microscope. Hair Medulla Care-free Control (A, B, and C) and Hair Medulla Care treatment (A’, B’, and C’) were compared and evaluated. Hair Medulla Care clearly increased the penetration of FITC and RITC into the cortex and medulla, but the penetration into the medulla was more pronounced for RITC than for FITC (A, A’, B, and B’). Nile Red had the same effect in that the penetration into the cortex and medulla clearly increased, but it localized to the medulla more prominently than to the cortex (C and C’).

Scale bar represents 50 μm.

Figure 2: Hair Medulla Care enhances penetration through cortex CMC.

A section of hair infiltrated with Nile Red, a fat-soluble fluorescent dye, by Hair Medulla Care is shown (A and A’). There is a higher localization to the medulla than to the part of the cortex close to the medulla, and in the cortex, stained images like CMC that border cortex cells can be seen (A’; arrows).

Scale bar represents 30 μm (A) and 10 μm (A’).

Comparison with Other Penetration Techniques: The penetrating power of Hair Medulla Care was compared with that of urea and ethanol, which are often used as penetrants for keratin fibers, as well as ATG and sodium sulfite, which are reducing agents. As a result, both ethanol and urea, which are penetrants, slightly increased the penetration of RITC into the cuticle, but did not change its penetration into the cortex or medulla. In addition, the reducing agents ATG and sodium sulfite made it possible to penetrate a part of the cortex, but the components did not reach the medulla, and the penetration was extremely poor in some regions (Figure 3).

From this, we surmised that conventional penetrants and reducing agents cannot pass through the permeation barrier between the cuticle and cortex to allow dye components to permeate into the hair. Conversely, Hair Medulla Care was shown to be useful because it allowed deep and uniform penetration into the hair, even as far as into the medulla.

Figure 3: Enhancing penetration of various dyes by Hair Medulla Care.

RITC permeation enhancement by common penetrants and reducing agents was evaluated by fluorescence microscopy of sections in comparison with penetrant-free control (A) and Hair Medulla Care (B). As a result, with 20% urea and 20% ethanol, the pigment remained in the cuticle as in the control (C and D), with 6.5% ATG and 6.5% sodium sulfite, penetration to a part of cortex was observed, but not to the medulla (E and F), and the degree of penetration was low depending on the part of the hair (E and F; arrows).

Scale bar represents 50 μm.

Application to Cosmetics

High Dyeing of Basic Hair Dyes: Since it was found that Hair Medulla Care particularly enhances the penetration of cationic components, we then evaluated the improvement in the function of basic hair dyes induced by Hair Medulla Care. As a result, we found that the dyeing ability increased remarkably after Hair Medulla Care, and the hair could be dyed much darker than usual (Figure 4, upper). When observing the sections of the same hair that had undergone the same treatment in order to evaluate the penetration of the dye, it was observed that the dye, which normally penetrates only to the cuticle, penetrated the entire hair including the medulla (Figure 4, lower). As an example of application of Hair Medulla Care, we used a dye to allow the easy visualization of penetration, but it may be possible to penetrate components other than the dye in the same way. For example, it may be possible to infiltrate hair care ingredients that could so far only act on cuticles but deep into the medulla, and to infiltrate wax-based styling ingredients into the hair. When discussing conventional styling agents, a common problem is that the smoothness of the hair surface and ability to pass fingers through the hair are lost in exchange for styling power. However, if it becomes possible to style hair using Hair Medulla Care, it may be possible to create hairstyles while preserving the texture of the surface. In this manner, Hair Medulla Care has the potential to dramatically improve the performance of any styling agent applied to hair.

Figure 4: Deeply dyeing of Basic hair dye.

After Hair Medulla Care, hair dyeing with basic hair dyes containing basic blue 75, basic red 213, basic red 76, and basic purple 2 resulted in a dramatically deep dyeing effect (A). When the hair was sectioned after dyeing and observed under a microscope, penetration was usually seen only until the cuticle (A’). On the other hand, when Hair Medulla Care was performed as a pretreatment, penetration into the medulla was observed (A’’).

Scale bar represents 50 μm (A’ and A’’).

Melanin-Based Black Hair Formation: We investigated the penetration of melanin into grey hair as an application of the penetration We investigated the penetration of melanin into grey hair as an application of the penetration promotion effect of Hair Medulla Care. When a test was conducted the grey hair was clearly colored with melanin, and the presence of grey hair was not prominent in the 10% grey hair wig. On the other hand, in the control in which melanin was dissolved in water and treated in the same manner, the results were the same as for the untreated case (Figure 5). Even after 7 days of continuous use, the melanin could be completely washed away with shampoo, indicating that melanin alone could not penetrate the hair at all. Until now, there is no product on the market that uses melanin itself as a dye because it was not possible to supplement melanin from outside the hair. For this reason, if people are concerned about grey hair, they have no choice but to use a conventional hair dye that may damage their hair and scalp. However, our study shows that the use of Hair Medulla Care makes it possible to supplement melanin in grey hair, something that was previously unattainable.

Figure 5: Restoring grey hair to black with melanin.

The results of melanin penetration with Hair Medulla Care applied to 10% grey hair wig are shown.?It can be seen that melanin alone does not penetrate the hair at all, but Hair Medulla Care leaves the brown color of melanin in the hair. As a result, the grey hair disappears and the hair attains a natural brown hair.

Here, we succeeded in delivering a basic pigment to the medulla, allowing the better dyeing of hair, using a technique called Hair Medulla Care. Our technique further allowed the infiltration of melanin into the hair, which was impossible until now.

Although many questions regarding the medulla remain, we believe that our research on medulla care has revealed new possibilities for hair care. We would like to continue research in the future with the expectation that the era of caring for the medulla will soon arrive for all kinds of hair treatment, such as hair care and hair coloring, in the same manner as for the cortex in the present day.

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