Effect of Platelet-Rich Plasma and other Key Factors on Hair Follicle Preservation

ABSTRACT

Background:

Hair graft preservation is an important factor that influences graft survival in hair transplantation.

Objective:

To investigate the benefits of adding platelet-rich plasma (PRP), and the effect of different storage solutions and temperatures on hair follicle preservation.

Materials and Methods:

This randomized-controlled study included 10 androgenetic alopecia patients who underwent hair transplantation. Forty-five hair grafts were collected from each patient and then randomized to 8 different culture conditions for 7 days. Hair grafts were cultured in Williams' Medium E or Ringer's lactate solution (RLS) at either 4°C or 37°C, and with or without 10% PRP supplementation.

Results:

In vitro hair growth in Williams' Medium E was significantly greater than in RLS. The 37°C temperature condition was found to be significantly better than the 4°C condition. The growth of hair grafts cultured with PRP was not significance difference from those without PRP. However, immunofluorescence staining for cytokeratin 15 showed greater expression in hair graft cultured with PRP.

Conclusion:

PRP may have a beneficial effect for preserving the viability of hair grafts. Williams' Medium E and 37°C temperature were found to be superior to RLS and 4°C relative to hair follicle growth in organ culture.

INTRODUCTION

Androgenetic alopecia is one of the most common types of nonscarring alopecia in both genders. In patients that fail to respond to medical treatment, hair transplantation surgery should be considered. The two currently used techniques for hair transplantation surgery are follicular unit transplantation and follicular unit excision. In follicular unit transplantation, a strip of scalp is removed under local anesthesia, after which the strip is separated into single follicular units under a microscope. Hair transplantation is a labor-intensive procedure that requires a lot of time and several assistants, especially in hair transplantation mega-sessions (3000–5000 grafts) that can take longer than 8 h to perform. Therefore, the preservation of hair follicles is an essential process and has a direct impact on the growth and survival of grafts.[1] Several different factors, including the type of storage solution, the temperature, and the supplementation added, can be modified to maximize the graft survival rate.[2] Qian et al. reported that both Ringer's lactate solution (RLS) and Dulbecco's Modified Eagle's Medium, which is a synthetic cell culture medium, could preserve hair follicles for up to 24 h for delayed hair transplantation.[3] Moreover, the addition of growth factor, such as epidermal growth factor or fibroblast growth factor, into the culture medium can help to promote hair growth.[4]

Platelet-rich plasma (PRP) is autologous plasma that has an above-average platelet concentration.[5] Alpha granules, which are a cellular component of platelets, contain numerous growth factors, including platelet-derived growth factor, transforming growth factor, vascular endothelial growth factor, insulin-like growth factor, and epidermal growth factor.[5] The effects of PRP have been studied in various dermatologic fields, including wound healing, anti-inflammation, melasma, and alopecia. Li et al. reported that PRP increased the proliferation of dermal papilla cells and stimulated hair growth in animal model.[6] The efficacy of PRP depends on many variables, such as the preparation protocol, the anticoagulants, and the platelet concentration. Several studies found that 10% PRP concentration had the maximum effect relative to the promotion of human fibroblasts and mesenchymal stem cell proliferation in vitro, whereas higher concentrations influenced a suppressive effect.[5,7] Another factor that influences the growth and viability of hair follicles in vitro is temperature.[2] Although 37°C is the most frequently used temperature for tissue culture, lower temperatures also confer benefit, such as reduced metabolic activity and potentially increased survival rate of transplanted tissues.[2]

Given the necessity of hair follicle preservation for delayed hair transplantation, the optimal storage solution and condition for maintaining hair follicle viability should be investigated and identified. In the present study, we evaluated the benefits of adding PRP, and the effect of different storage solutions and temperatures on hair follicle preservation.

MATERIALS AND METHODS

This randomized-controlled trial included 10 androgenetic alopecia patients who underwent strip harvesting or follicular unit transplantation at the Division of Hair Disorders and Hair Transplantation, Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, during the July 2015–December 2016 study period. This study was approved by the Siriraj Institutional Review Board (COA no Si 664/2013). Written informed consent was obtained from all study participants.

After the strip harvesting procedure, 45 human hair follicles were collected from each patient and immediately transferred to the laboratory in a sterile tube containing RLS. This study had a 2 × 2 × 2 factorial design, with factors that consisted of culture medium (Williams' Medium E solution or RLS), temperature (4°C or 37°C), and PRP supplementation (with or without PRP). Therefore, this study had a total of 8 different culture conditions [Figure 1].

Figure 1.

Experimental protocol. Will E: Williams' Medium E, RLS: Ringer's lactate solution, H and E: Hematoxylin and eosin, CK-15: Cytokeratin-15, PRP: Platelet-rich plasma

In vitro culture of hair follicles

Hair follicles were cultured using a modification of a previously described method.[8,9] Forty hair follicles were randomly allocated to 8 culture conditions for a total of 5 hair follicles per condition [Figure 1]. Each individual hair follicle was placed in an individual well of a 24-well multiplate filled with culture medium (10 ml of Williams' Medium E or 10 ml of RLS). In addition, each culture medium was supplemented with 2 mmol/L L-glutamine (Glutamax^®), hydrocortisone 10 ng/ml, insulin 10 μg/ml, penicillin 100 U/ml, streptomycin 100 μg/ml, and amphotericin B 25 μg/ml. PRP 1 mL (10% of total volume of culture medium) was added to the culture medium in conditions assigned for PRP supplementation. Half of the complete set of the hair follicle conditions were incubated at 37°C in an atmosphere of 5% CO₂ and 95% air, and the rest were incubated at 4°C in the same atmospheric condition. The medium in each culture was changed on day 4 of culturing.

Platelet-rich plasma preparation

PRP was obtained from each patient's blood before the hair transplantation procedure. Twelve milliliter of venous blood was collected in ethylenediamine tetraacetic acid tubes and then immediately centrifuged for 5 min at 200 × g at room temperature (Clay Adams Dynac II Centrifuge; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The single-spin prepared PRP was collected and divided into two equal parts. The first part was added to the culture medium on day 0. The second part was preserved at 22°C with constant gentle agitation, and then added to the culture medium on day 4. To quantify the PRP, the platelets were counted on both day 0 and day 4.

Examination of hair follicles

Hair follicles were examined and photographed using a handheld digital microscope (Dino-Lite™ Polarized Microscope; AnMo Electronics Corporation, Hsinchu, Taiwan). Images of each follicle were taken on day 0, day 4, and day 7 of the experiment. The length of hair follicles was measured from digital images using DinoXcope software (AnMo Electronics Corporation). The total hair length was defined as the length measured from the top end of the hair shaft to the bottom end of the bulb.

Histologic changes in the in vitro hair follicle

Initially, five hair follicles were fixed with 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 4 μ as controls. Three of those hair follicles were stained with hematoxylin and eosin, and the other two hair follicles were subjected to labeling with cytokeratin 15 (CK15) by immunofluorescence staining (Sigma-Aldrich Corporation, St. Louis, MO, USA). After culturing for 7 days, the remaining follicular grafts (40 grafts) were fixed with 10% neutral-buffered formalin and then stained in the same fashion as previously mentioned. CK15 expression was evaluated by inverted fluorescence microscope (Ti-S Intensilight Ri1 NIS-D, Nikon Instruments, Inc., Tokyo, Japan). The intensity of CK15 immunostaining was determined by ImageJ image processing software program (https://imagej.nih.gov/ij/download.html).

Statistical analysis

All data analyses were performed using PASW Statistics version 18 (SPSS, Inc., Chicago, IL, USA). Data are shown as mean plus/minus standard deviation. Three-way factorial analysis of variance was used to identify significant difference among factors during in vitro culture. The difference in CK15 immunostaining intensity after culture for each factor was analyzed using Mann–Whitney U-test. A P < 0.05 was regarded as being statistically significant.

RESULTS

According to hair follicle growth after culturing in 8 different conditions, the growth was highest on both day 4 and day 7 in those cultured in Williams' Medium E at 37° [Table 1 and Supplementary Figure 1 ^{(855.3KB, tif)} ]. Hair growth at day 7 compared among different culture medium, temperature, and PRP conditions are shown in Table 2. We found that the growth of cultured hair follicles depended on both temperature and the type of culture media. Hair follicles cultured in Williams' Medium E continued to grow in vitro during culture, whereas only minimal growth was observed in the RLS group. We found the growth of hair follicles cultured in Williams' Medium E to be significantly greater than that of those cultured in RLS at both day 4 and day 7 (P < 0.0001) [Figure 2a]. Moreover, storage at 37°C significantly promoted hair follicle growth in vitro compared to storage at 4°C on both day 4 and day 7 (P < 0.0001) [Figure 2b].

Table 1.

Hair growth at day 4 and day 7 compared among different culture conditions

Culture medium	Temperature (°C)	Mean±SD
		Hair growth at day 4 (mm)		Hair growth at day 7 (mm)
		With PRP	Without PRP	With PRP	Without PRP
Williams’ Medium E	37	0.29±0.21	0.46±0.40	0.42±0.26	0.60±0.62
	4	0.10±0.09	0.09±0.06	0.16±0.11	0.13±0.07
Ringer’s lactate solution	37	0.04±0.05	0.03±0.06	0.05±0.07	0.05±0.09
	4	0.04±0.04	0.04±0.04	0.06±0.52	0.05±0.04

PRP – Platelet-rich plasma; SD – Standard deviation

Table 2.

Hair growth at day 7 compared among different culture medium, temperature, and platelet-rich plasma conditions

Condition	Hair growth at day 7 (mm) mean±SD	P	η2
Culture medium
Williams’ Medium E	0.33±0.39	<0.001*	0.24
Ringer’s lactate solution	0.05±0.06
Temperature (°C)
37	0.28±0.42	<0.001*	0.12
4	0.10±0.09
PRP
With	0.17±0.29	0.15	0.01
Without	0.19±0.31

*A P<0.05 indicates statistical significance by 2×2 × 2 factorial ANOVA. ANOVA – Analysis of variance; PRP – Platelet-rich plasma; SD – Standard deviation

Figure 2.

Mean hair follicle length at baseline, and after being cultured for 4 and 7 days in different (a) culture medium conditions, (b) temperature conditions, and (c) PRP supplementation conditions *Statistical significance (P < 0.001). PRP: Platelet-rich plasma, RLS: Ringer's lactate solution

After preparation of PRP, the final concentration of platelets was approximately 2.4 times greater than the baseline concentration. The mean platelet count in whole blood and PRP was 2.6 × 10⁵/μl and 6.4 × 10⁵/μl, respectively. In culture medium supplemented with PRP, the elongation of hair follicles did not reach statistical significance compared to those cultured in medium without PRP at day 7 (P = 0.15) [Figure 2c].

After 7 days of culture, the histology of hair follicles revealed similar morphological features compared to baseline in all conditions. CK15 expression, which is generally regarded as a stem cell marker at outer root sheath cells of the bulge and infrabulge regions of hair follicles [Figure 3a], were decreased compared to baseline in all conditions on day 7 [Figure 3b]. However, we found that hair follicle grafts preserved in storage media supplemented with PRP exhibited significantly higher levels of CK15 expression than those without PRP (P = 0.004). There was no significant difference in CK15 expression between the 4°C and 37°C groups and between the Williams' Medium E and RLS groups [Figure 3b].

Figure 3.

Immunohistochemistry for CK15. (a) CK15 immunoreactivity prominently marks outer root sheath cells at bulge and infrabulge areas (b) CK15 intensity before and after being cultured for 7 days in different PRP supplementation conditions, temperature conditions, and culture medium conditions. *Statistical significance (P = 0.004 [Mann–Whitney U-test]). PRP: Platelet-rich plasma, RLS: Ringer's lactate solution, CK15: Cytokeratin 15

DISCUSSION

PRP is an innovative therapy that has found application in a range of fields, including surgery, orthopedics, dentistry, dermatology, and esthetics. Various growth factors in PRP, such as platelet-derived growth factor, insulin-like growth factor-1, transforming growth factor-ß, epidermal growth factor, and fibroblast growth factor, can stimulate cell survival, proliferation, differentiation, vascularization, and angiogenesis.[6,10,11,12] A meta-analysis by Gupta and Carviel showed PRP to be a promising treatment for androgenetic alopecia patients due to its ability to promote hair restoration.[13] However, the benefit of adding or using PRP as a graft-holding solution was supported by only a few studies.[14,15] Moreover, other factors such as temperature and type of storage solution may influence the growth and survival of follicular grafts.

In this study, we used single-spin PRP, which yielded a final mean platelet concentration of 6.4 × 10⁵/μl (2.4 times greater than the baseline concentration). According to a study by Anitua, et al., PRP with a platelet concentration above 3 × 10⁵/μl was sufficient to exert a therapeutic effect. The concentration of PRP is another factor that can affect the growth and viability of hair follicle grafts. Several studies found that PRP at 10% concentration had the largest effect on cell proliferation.[5,7] From our results, the growth of hair follicle grafts cultured with 10% PRP appeared to be less than in those without PRP; however, no significant difference between conditions was observed. A negative effect of PRP has been reported. A higher concentration of PRP results in the suppression of cell viability and proliferation of alveolar bone cells, human oral fibroblasts, and osteoblasts.[16,17] Nevertheless, we found that hair follicle grafts preserved in storage solution with 10% PRP supplementation had a higher expression of CK15, which is a stem cell marker of the hair follicle, compared to those without PRP after 7 days of culture. Therefore, adding PRP to hair storage solution might have a benefit for preserving hair follicle viability, but the optimal concentration of PRP still needs to be determined.

Several storage solutions, including normal saline, RLS, and Dulbecco's Modified Eagle's Medium, are commonly used for hair follicle graft preservation. Williams' Medium E was developed to enrich isolated rat hepatocyte cultures for polygonal epithelial cells, and for reducing the number of contaminating fibroblasts. Williams' Medium E contains a higher amount of trace elements, and it exhibits small variations in supplementation. A previous study reported that hair follicle grafts continued to grow after being cultured in Williams' Medium E for at least 7 days, while those preserved in normal saline showed no growth.[9] Our study evaluated the growth of hair follicle grafts compared between those cultured in Williams' Medium E and those cultured in RLS for 7 days. We found the growth rate of hair follicle grafts cultured in Williams' Medium E to be significantly greater than the growth observed in grafts cultured in RLS; however, immunohistochemistry of CK15 showed that Williams' Medium E was not better than RLS relative to the maintenance of cell viability after culture for 7 days. Moreover, it should be noted that sterile Williams' Medium E is not available, nor is it specifically approved as a transplant storage media.

Regarding temperature, Kurata et al. reported that hair follicles refrigerated at 4°C for more than 48 h seldom grew in organ culture. In contrast, a temperature of 4°C could maintain the viability of dermal papilla and outer root sheet cells for up to 7 days, and these follicles grew well after being transplanted into athymic mice.[18] Similar to the previous study, we found the growth rate of hair follicles preserved at 4°C to be inferior to the growth rate of those cultured at 37°C on both day 4 and day 7. However, the temperature seemed not to affect hair follicle viability as evidenced by CK15 staining. Hair follicle viability decreased in both the 4°C and 37°C groups after culturing for 7 days; however, the difference between groups was not statistically significant.

Limitations

This study has some mentionable limitations. First, the hair growth rates in all conditions in our study were low compared with previous studies. These differences may be due to differences in cultural protocol. In addition, the culture medium was changed less often than normal, and this may have adversely affected hair growth rates. Second, only one concentration of PRP was used in this study, which means that our results may not fully and/or accurately reflect the actual effect of PRP on hair preservation. Further studies with various concentrations of PRP should be conducted to identify the optimal concentration and precise effect of PRP in hair preservation.

CONCLUSION

PRP may have a beneficial effect on preserving the viability of hair follicle grafts. Williams' Medium E and 37°C temperature were found to be superior to RLS and 4°C temperature relative to hair follicle growth in organ culture; however, no significant difference was observed between those two conditions relative to the preservation of hair follicle viability. Additional controlled in vivo studies are needed to more clearly elucidate the advantage of adding or using PRP for preserving the viability of hair follicle grafts during hair transplantation.

Financial support and sponsorship

Siriraj Research Fund, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (grant no. R015732019).

Conflicts of interest

There are no conflicts of interest.

Supplementary Figure 1

Hair shaft culture (a) with PRP supplementation and (b) without PRP supplementation. PRP: Platelet-rich plasma