An In vitro Study Comparing the Survival of Hair Follicles in Various Storage Media at Different Temperatures for Hair Transplant Procedure

ABSTRACT

Aim:

The purpose of this in vitro hair graft study was to better understand how different storage media affect the survival of hair follicles at different temperatures during a hair transplant procedure.

Materials and Methods:

In this study, hair follicles (n = 60) were harvested from 3 healthy male volunteers. Follicles were randomly assigned to the following groups: Group A: Storage media phosphate-buffered saline (PBS), platelet-rich plasma (PRP), platelet-rich fibrin (I-PRF), and Dulbecco’s Modified Eagle Medium (DMEM). Group B: Storage media were placed at the following temperatures: 4°C, 26°C (room temperature), and 37°C (body temperature). The viability of hair follicles was checked using the explant culture method. Cell outgrowth was observed after incubation at 37°C in DMEM containing 10% FBS.

Results:

Explant cultures of hair follicles stored at 4°C and 26°C did not show outgrowth of the cells after 7 days of culture. Explant cultures of hair follicles stored at 37°C did show outgrowth of the cells after 7 days of culture. Furthermore, these study results indicated that 10% DMEM preserves hair follicles more effectively than PBS, PRP, and I-PRF.

Conclusion:

According to the study’s findings, freezing graft storage options might not be the best option. Instead of 4°C or room temperature 26°C, 37°C has been shown to improve hair graft survival. Furthermore, these study results indicated that 10% DMEM preserves hair follicles more effectively than PBS, PRP, and I-PRF. The study concluded that maintaining hair follicles in 10% DMEM at 37°C would prolong graft life and improve therapeutic outcomes.

INTRODUCTION

Hair loss is a distressing issue that affects a significant percentage of the population, including up to 85% of males and 40% of females, and its prevalence grows with age in both sexes.[1] In cases with androgenic alopecia, hair transplantation is the most efficient way to restore hair. In recent years, breakthroughs in hair transplant techniques have been made as the procedure’s popularity has grown significantly.[2] Male-pattern baldness can now be treated with follicular unit extraction (FUE), which uses smaller grafts (1–3 hairs per graft) and produces significantly better results than previous follicular unit transplantation (FUT) methods.[3] However, this has increased the labor intensiveness of the hair transplantation process.

The drawback of this method is that it takes hours to prepare thousands of micrografts from donor scalp skin with the help of several assistants, and the patient must wait during that period. The ability to employ thousands of hair grafts at once would be made possible by the preservation of hair grafts for delayed transplantation.[4] The preservation of grafts while they are ready to be implanted outside the body is essential in hair restoration surgery in order to achieve maximum growth of transplanted follicles. The most popular holding solutions historically have been cooled saline or Ringer’s lactate.[5] Longer out of the body times are a result of the increasing number of grafts used in hair transplant surgeries. The need for oxygen in most tissues is relatively significant. Unfortunately, the tissue has very poor oxygen solubility. Aerobic energy is inhibited by sudden blood flow interruptions. Ischemia, or insufficient blood flow, is caused when grafts are removed for transplantation. When circulation is lost, cells are also deprived of essential metabolites and cannot remove waste. Such occurrences contribute to cell death.[6]

The tolerances for maintaining optimal health vary among different cell types. For instance, the brain cell has a limited ischemia tolerance, which lasts only 6 min at 37°C. However, when the body temperature drops below 17°, this increases to almost 60 min.[7] Kidneys can withstand substantially longer periods of nonhypothermic ischemia, after 1 h, a majority of tubules are completely necrotic.[8] According to Kim and Hwang, hair follicles at normal temperature may withstand even longer periods of ischemia.[9]

Ischemia during hair transplant surgery can linger for a long time, frequently more than 4–6 h. Therefore, it is crucial to preserve hair follicles during surgery in solutions that also maintain the cellular components including the hair matrix, inner and outer root sheath.[10] There are numerous holding solutions for both the intracellular and extracellular components. Intracellular storage solutions, which resemble the intracellular space that cannot pass through the cell membrane, are hypertonic solutions with increased potassium and decreased sodium concentrations.[11] This contributes to osmotic stability. The osmotic gradient is crucial because it maintains cell viability despite the drop in temperature and the deactivation of cell pumps that regulate water. These storage solutions with elevated osmotic pressure reduce the likelihood of cell swelling by preventing passive water entry. Extracellular storage solutions are isotonic solutions with an ion complement resembling that of plasma, simulating the usual extracellular environment of cells, which include tissue culture media, normal saline, Ringer’s lactate, and BSS. When temperatures are low, these solutions provide mediocre preservation. In addition, they cause cell enlargement at lower temperatures.[12]

Every tissue has an optimal storage temperature depending on nature of the cell, chemical composition and its interaction to changes in temperature of external environment. The ideal temperature for heart muscle is between 10°C and 20°C. According to certain research, the kidney responds better to temperatures of 10°C than those of 5–0.5.[13] According to Parsley,[4] the ideal temperature range for storing hair follicle grafts is between 8° and 14°. Specific temperatures have a better effect on some tissues. Some tissues respond better to a specific holding solution at a particular temperature. Therefore, a study was conducted to evaluate standardize storage media for hair graft preservation for 24 h, To evaluate the temperature 4°C, 26°C (room temperature), 37°C (body temperature-incubator) for hair follicle viability in different storage media. Viability of hair micrograft was analyzed using explants culture.

MATERIALS AND METHODS

Collection of hair follicle

Three healthy male volunteers (age ~25–30 years) were included in this in vitro study. This study was approved by the Institutional Stem Cell Research Committee, Research and Recognition Committee, and Institutional Ethics Committee and Review Board.

Patients suffering from systemic diseases such as uncontrolled diabetes, uncontrolled hypertension, those on steroid therapy, and those with a history of chemotherapy/radiotherapy and bleeding disorders were excluded from the study.

Valid written informed consent was obtained from the volunteers. A total of 60 hair follicular units per volunteer were harvested by a hair transplant surgeon following institutional guidelines by FUE technique. Postoperative instructions for care of the wound were explained to the volunteers. Micrograft preparation of hair follicle was done. Each follicle was isolated as a whole, consistently maintaining a significant amount of tissue (epidermis, dermis, and subcutaneous fat) around the entire length of the follicle, keeping in mind that the maintenance of the structural integrity of each follicle and related structures was an essential requirement for their growth in vitro.

Storage of hair follicle

Follicles were randomly assigned to the following groups:

• Group A: Storage media [Figure 1]

  1. Phosphate-buffered saline (PBS) [Figure 1a]

  2. Platelet-rich plasma (PRP) [Figure 1b]

  3. Injectable form of platelet-rich fibrin (I-PRF) [Figure 1c]

  4. Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) [Figure 1d].

• Group B: Storage temperatures – Storage media were placed at the following temperature [Figure 2] 4°C, 26°C (Room temperature), 37°C (Body temperature-placed in incubator). Viability of hair follicles was checked by explant culture (10% DMEM) after 24 h [Figure 2].

Figure 1.

Storage medium for hair follicle: (a) phosphate-buffered saline, (b) platelet-rich plasma, (c) platelet-rich fibrin, (d) Dulbecco’s Modified Eagle’s Medium

Figure 2.

Hair follicle stored in phosphate-buffered saline, platelet-rich plasma, platelet-rich fibrin, 10% Dulbecco’s Modified Eagle Medium Medium at (a) room temperature (26°C), (b) 4°C, (c) 37°C

Explant culture of hair follicle

Hair follicles stored in different medium at various temperatures were transferred to a culture dish containing DMEM as the culture medium supplemented with 10% FBS and antibiotic-antimycotic solution to prevent bacterial contamination. Culture dish was placed in a humidified incubator at 37°C with 5% CO₂. This culture was changed every 2–3 days and the cell outgrowth was monitored with an inverted phase-contrast microscope (Olympus CKX53, Japan). Carefully monitoring of the culture conditions was done to analyze optimal growth and survival of the hair follicles.

RESULTS

Storage condition at 4°C

Hair follicles were stored in four different medium PBS, PRF, PRP, DMEM for 24 h at 4°C [Figure 3]. Hair follicle explant culture did not show outgrowth of the cells after 7 days culture with DMEM containing 10% FBS [Figure 4] in PBS, PRF, and PRP.

Figure 3.

Hair follicles stored in medium for 24 h at 4°C. (a) Phosphate-buffered saline, (b) platelet-rich fibrin, (c) platelet-rich plasma, (d) Dulbecco’s Modified Eagle Medium with 10% FBS

Figure 4.

Hair follicles stored at 4°C after 7 days explant culture. (a) Phosphate-buffered saline, (b) platelet-rich fibrin, (c) platelet-rich plasma, (d) Dulbecco’s Modified Eagle Medium with 10% FBS

Storage condition at room temperature (26°C)

Hair follicles were stored in four different medium PBS, PRF, PRP, and DMEM at 26°C for 24 h [Figure 5]. Hair follicle explant culture did not show outgrowth of the cells after 7 days culture with DMEM containing 10% FBS in PBS, PRP, and PRF [Figure 6].

Figure 5.

Hair follicles stored in media for 24 h at room temperature −26°C. (a) Phosphate-buffered saline, (b) platelet-rich fibrin, (c) platelet-rich plasma, (d) Dulbecco’s Modified Eagle Medium with 10% FBS

Figure 6.

Hair follicles stored at 26°C after 7 days explant culture. (a) Phosphate-buffered saline, (b) platelet-rich fibrin, (c) platelet-rich plasma, (d) Dulbecco’s Modified Eagle Medium with 10% FBS

Storage condition at 37°C

Hair follicles were stored in four different medium PBS, PRF, PRP, and DMEM at 37°C [Figure 7]. Hair follicle explant culture showed outgrowth of the cells after 7 days culture with DMEM containing 10% FBS and PRP as shown in Figure 8. Hair follicle explant culture did not show outgrowth of the cells in PBS and PRF storage medium.

Figure 7.

Hair follicles stored in media for 24 h at 37°C. (a) Phosphate-buffered saline, (b) platelet-rich fibrin, (c) platelet-rich plasma, (d) 10% Dulbecco’s Modified Eagle Medium

Figure 8.

Hair follicles stored at 37°C after 7 days explant culture. (a) Phosphate-buffered saline, (b) platelet-rich fibrin, (c) platelet-rich plasma, (d) 10% Dulbecco’s Modified Eagle Medium

The ability to cultivate outer sheath cells from intact hair follicles was seen using an inverted phase-contrast microscope (Olympus CKX53, Japan) as shown in Figures 3, 5 and 7. The results of maintaining intact hair follicles in cell culture at 4°C, 26°, and 37°C are depicted in Figures 4, 6 and 8.

It demonstrates that, regardless of changes in the storage medium’s temperature, the colony formation capacity of PBS and PRF declined with preservation time. In 10% DMEM and PRP, the outer root sheath cells were grown. The survival of hair follicles kept in incubators at 37°C was higher than that of those kept in refrigerators (4°C) and at room temperature (26°C). When compared to follicles stored in PBS, PRP, or PRF than those preserved for 24 h in 10% DMEM at 37°C exhibit a substantial amount of cell outgrowth.

DISCUSSION

The most prevalent cause of hair loss that affects both genders equally is androgenetic alopecia, an inherited disease. Major advancements in hair restoration date back a century, and the ideas underlying them are based on skin grafting, a surgical procedure that has been employed for reconstructive purposes since antiquity.[14] Dr. Menahem Hodara of Turkey performed one of the very first hair transplant procedures in 1897. Dr. Hodara successfully treated favus-related hair loss by applying scalp tissue and hair from sections of the patient’s scalp that were untouched to the favus scars.[15] In the first part of the 20^th century, Japan would produce more advancement in hair restoration. Dr. Shojui Okuda developed hair transplant methods for the eyebrows, pubic region, and eyelashes in the 1930s.[16] He developed a punch graft method. This required small punches to harvest hair follicles from the skin that was 1–4 mm in diameter. To restore hair, these punch grafts were transplanted to other areas of the body as necessary.

The first hair transplant procedure was ever performed to treat male pattern baldness in 1952 by New York dermatologist Dr. Norman Orentreich.[17] Two additional contemporary methods of hair restoration were created in the 1980s. One involved strip excision, which restored the bald area of the scalp using a strip of donor grafts removed from the back of the scalp. The second technique involved using micrografts, which allowed donor graft strips to be cut into smaller grafts using a microscope. The invention and improvement of contemporary FUT and FUE are the results of these developments (FUE).[3,18] In the past 20 years, additional improvements to FUT and FUE techniques have been achieved to enhance the hairlines’ natural appearance. This entails being mindful of the hair follicles’ angles as well as how to arrange, space, and stagger the follicles to mimic natural hair growth.

The only way to reliably regrow hair in people who are experiencing pattern hair loss as well as hair loss from the majority of other reasons is through hair restoration surgery. The invention of FUE, subsequently known as follicular unit excision[19] or FUE, greatly enhanced hair transplantation. Consequently, the donor location can be used to harvest grafts without creating a scar on the patient. Grafts can also be taken from different body parts using FUE. The transection rate for FUE remains below 4% when correctly carried out with the most technologically equipped tools. Local anesthetics are used during the outpatient surgery. A skilled surgical team is required for an effective surgery to gain satisfying postoperative results.[20]

There is more than one factor that affects the success rate of hair transplant operation. The primary ones are; operator’s surgical skills, quality of donor area, tissue quality of the needy area and largeness of operation area. It is known that while hair transplanting, it takes much time to extract a hair follicle and transplant it. In the meantime, keeping hair follicles alive directly affects the success rate. Consequently, transplanting 1000 grafts or 5000 grafts will not be successful if the hair follicles are not alive.[21]

There are thousands of cells in each hair follicle. Keeping the cells alive is keeping the hair follicles alive. Goal of the hair restoration treatment is protecting the cellular structure to provide tissue integrity while extracting and transplanting hair follicles. Cells, which are fundamentals of living beings, need some certain conditions to stay alive.[22] Primarily, hair follicles that were taken from the donor field should be preserved in a proper liquid environment. Time that hair follicles stayed alive, changes according to liquid storage media properties. Our cells have a specific osmotic pressure. Cells take nourishment and store their foods owing to osmotic pressure. If we put a cell into a hypertonic environment it loses water, in a hypotonic environment deplasmolysis happens and if the process continues it results in cell death. In either case, lifetime of cell will become shorter. The best environment for a cell is an isotonic environment.[22,23]

Isotonic environments that have electrolyte balance are preferred to keep the hair follicles alive. Nowadays, 0.9% NaCl is widely used solution for preserving the hair follicles.[12] Generally a hair transplant operation takes 5–8 h according to graft number, tissue quality and donor area quality. If the necessary procedures are followed, hair follicles are going to stay alive for 12–14 h. The main advantage of a perfect graft-preserving solution would be an improvement in the quality of hair implanted grafts leading to an increase in survival rate of hair production from the transplanted grafts.[24] The harm brought on by ischemia phase-induced ionic imbalance, free radical generation, reperfusion injury, and changes in osmolality would be considerably reduced by the optimum holding solution. When creating solutions that retain hair follicles in place, consideration is given to the extracellular and intracellular body fluid environment’s makeup. An extracellular fluid called autologous plasma is isotonic with nutrients and growth factors produced from platelets.[24,25] It can be made by a surgeon and is affordable.

According to recent studies, there is no optimum temperature or holding solution for hair restoration surgery. In recent years, the temperature of graft storage has been examined, but the combined results have not proven definitive. In order to simulate the time spent out of the body during a typical transplant process, Raposio et al. dissected 240 hair follicles and placed half of them in saline at room temperature and the other half in cold saline (1°C). They did this for 5 h. Then, to simulate an in vivo setting, they cultivated follicles in an additional medium. They measured the length of these follicles after 10 days and divided them into those that grew and those that did not. They demonstrated that 88% of the follicles preserved in the cold and 87% of those cultivated at normal temperature both exhibited some microscopic proliferation. They came to the conclusion that there were no differences in the impacts of temperature storage given the almost comparable rates of growth.[26,27]

PBS[28,29] which is widely used in hair transplant surgery and is readily available, is used as the control group in this study for the storage medium of hair follicles. In comparison to the control group, PRP,[29,30] I-PRF (injectable platelet-rich fibrin),[31] and 10% DMEM were all used. PRP is a concentrate of platelets derived from the patient’s own blood. It is used in various medical and cosmetic procedures to promote healing and tissue regeneration. As blood-derived autogenous sources of many growth factors necessary for tissue repair and regeneration, PRP and I-PRF were included in the study.[32,33] DMEM, which is frequently used as a base medium to support the development of several distinct mammalian cells, was included in the study.

All hair follicle graft in the study was single follicular unit.[19] The harvested hair follicles were transported to storage medium and placed in an incubator where they were kept at body temperature 37°C in incubator, 4°C and room temperature 26°C. Using explant culture, hair follicles were cultured for outer root sheath cells.[34] Based on the outcome of explant culture, the vitality of hair follicles was evaluated. An inverted phase-contrast microscope (Olympus CKX53, Japan) was used to track the cell expansion while the culture was replaced every two to 3 days.

CONCLUSION

This electro microscopic research study of hair grafts was conducted in order to better understand how different storage media affect the survival of hair follicles at various temperatures during a hair transplant surgery. According to the study’s findings, freezing graft storage options might not be the best option. Instead of 4°C or room temperature 26°C, 37°C can result in better hair graft survival. The benefit of room temperature vanished when grafts were chemically or mechanically traumatized. Furthermore, this study results indicated that 10% DMEM preserves hair follicles more effectively than PBS, PRP, and I-PRF. The study concluded that maintaining hair follicles in 10% DMEM at 37° would prolong graft life and improve therapeutic outcomes.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.