For years hair transplant physicians have experimented with holding solutions for grafts, Very few studies, however compare holding solutions for follicular transplants. In fact, there are many questions researchers ask when investigating holding solutions and temperature:
- Should we chill follicular grafts?
- If so, what is the ideal temperature?
- What happens when we chill tissue in specific storage solutions?
- Could some holding solutions actually be dangerous to our grafts?
More recently, some have considered the addition of antioxidants, ATP, and oxygen to their holding solutions. In fact, there is a new product under research called Vitasol, which has liposomes containing ATP. What are the benefits of such a product, if any?
Research on organ transplant preservation has looked at larger organs such as the liver, kidney, pancreas, and myocardium. This paper, in turn, will examine the current state and science of preserving hair follicle transplants via holding solution and temperature.
The following is a review of literature pertaining to follicular survival rates and health. The hope is that it will stimulate further study in this important topic. A few studies look at storage mediums and temperature. Unfortunately, they have yet to show any reproducible evidence. While gaining greater knowledge, no specific protocols exist to improve the survival of hair. Each hair restoration specialist picks their holding solution and temperature according to training and industry updates. Further, the design of many of these studies do not withstand statistical scrutiny.
- A study by Kim found that storage at 4 °C in normal saline does not improve graft survival unless the grafts are out-of-body for more than 6 hours. After 6 hours preserving grafts at 4 oc appears beneficial. Nonetheless, after 6 hours follicular survival decreased whether or not the follicles were preserved at 4 °C. (1)
- Perez-Meza found no difference in the survival of grafts at ambient temperature vs. 4°C between 4-6 hours in normal saline. (26)
- A study by Beehner found that cold storage in normal saline improves the survival rate of grafts exposed to crush injury.(2)
- Uebel observed that the survival of hair increased by 15% when stored in platelet rich plasma (PRP), though this has not been reproduced or confirmed.(3)
- Cooley has suggested that free radical scavengers may protect against reperfusion injury but no statistically significant studies show they improve hair graft survival.(4) He also noted that oxygen might benefit the survival of hair follicle grafts, and later suggested we add ATP. (5)
- Following a study using mice, Qian proposed that an oil on cuttings(OCC) storage solution is superior to the more commonly used 4°C normal saline for hair grafts' long term preservation. (6)
- Raposio found no statistically significant difference between survival and growth rates of grafts stored at room temperature and those preserved at 1 °C.(7)
- Krugluger demonstrated that transient effluvium was prevented in 6 patients whose grafts were stored in PBS-buffered tissue culture medium (TCM) with nitric oxide added as an inhibitor of apoptosis.(8)
- In another study, Krugluger found that grafts stored in PBS containing B12 revealed increased elongation of the hair follicle grown in tissue culture compared with those stored in PBS alone.(9) However, it remains unclear whether elongation is a good indicator of graft vitality.
- Parsley presented a photograph of a single patient in which he compared graft growth following storage in Hypothermosol on one side
- Beehner found that grafts stored in chilled
- Forhair's Dr.Cole showed that the pH declined in normal saline regardless of temperature as a function of time but that pH remained constant in PBS, which contains buffers.(11)
The Beehner study compares chilled saline survival rates to survival rates from Hypothermosol containing ATP. Empirical observation shows that the latter has better yield. (10) We note the survival rates in Table 1.
The first study documents that Hypothermosol and similar intracellular solutions are superior to a chilled extracellular (normal saline) storage solution. We detail the difference between these two fluid types below.
Intracellular and Extracellular Solutions
"Intra-" and "extracellular" holding solutions have several superficial similarities. All are liquids that, through various chemical effects, can also have constant oxygen levels. Beyond tangible appearances, the two holding solution types are vitally different.
- Intracellular -These solutions incorporate cellular structures into the liquid, allowing a solution's nutrients or vitamins to permeate the transplant
- Extracellular -These solutions isolate cellular structures, isolating the transplant and not allowing nutrients in or out
In this study, Beehner planted 10 single-hair grafts and 20 two-hair grafts into boxes at 2, 4, 6, 8, 24, 48, 72, and 96 hours after strip harvesting. Also demonstrable is that follicular diameter is superior with grafts stored in Hypothermosol and ATP. The only exception is follicles planted at the 6-hour mark. Limmer previously examined the effect of chilled saline solution on the survival rate of grafts stored up to 48 hours prior to transplantation.(12)
Further statistical analysis might be necessary in both studies. The 4-hour survival being no better than 48-hour survival, for instance, seems counterintuitive.Follicle survival rate in Beehner's chilled saline study was far less than in Limmer's study -with the exception of 6 and 8 hours out of body. The comparison suggests that chilled saline may produce a lower survival rate (Table 2).
This, however, is not certain, as not all the follicles were preserved at 4°C.(1) Perez-Meza found no difference in the survival of grafts at ambient temperature vs. 4°C
Specific solutions seem to offer advantages to the individual transplant organ.(13) A variety of standard solutions work to preserve tissue. Individualized additives (custom solutions), in turn, can also be customized to improve the viability for different tissue types.(14) With regard to hair, no optimal protocol, solution, or temperature has yet been defined. With this in mind, we will look at the basic science behind biopreservation.
Basic Tenets and Theories
The oxygen requirement for many tissues is quite high, but the solubility of oxygen in tissue is quite low. Interruption in circulation rapidly leads to inhibition of aerobic energy production. Loss of circulation also deprives cells of necessary metabolites and eliminates removal of waste products.
Excision of grafts for transplantation results in ischemia, or inadequate blood supply. Such events contribute to cell death. Ischemia in hair transplant surgery can last many hours and often exceeds 4-6 hours. Advances in holding solutions and temperature reduction may offer ways to improve hair transplant survival.
For holding solution temperature, a 1 ooc decrease in temperature further decreases oxygen consumption by approximately 50% and is called the Q (10). Oxygen consumption at 5°C is about 6% of that which occurs at 37°C. Cooling provides short-term survival through decreasing metabolic functions, and, in turn, oxygen and nutrient demand. Hypothermia is a primary risk and protector. It is also far more than the single variable of reduced temperature, however. Hypothermia will protect only to a point. Without manipulative intervention, Hypothermia may lead to progressive cell injury during each of its three in-vitro phases (cooling, maintenance in the cold, and re-warming).
Fluctuation in temperature (chilling, warming, and re-chilling) may greater stress the cells. Keeping them at a specific temperature is probably best for integrity and health. At warmer temperatures, however, the cells' new energy consumption would possibly present a new set of problems. Hair follicles' rapid change in temperature from 8°C to 37°C can induce apoptosis. At the very least, such rapid temparture change is a "shock" to the cells of the hair follicle.
Gradual warming could be more optimal for cell survival.(18) With hypothermia, the biopreservative solution is reduced from 37°C to 20°-25°C or, more commonly, 0-10°C. The target of 4 oc has no rationale other than water reaching its maximum density at this temperature. Nevertheless, 4°C is most often quoted by hair transplant surgeons with regard to chilling their grafts. Cooling, however, has its own characteristics that results in a depletion of energy stores and the associated adenylates (esters of AMP). This is due to the failure of aerobic production of ATP and a subsequent failure of the energy-dependent ion pumps in the cell membrane. There is also a change in cell and organelle membranes, which become "leaky." In addition to the production of free radicals and depleted natural defense mechanisms to the free radicals, cooling provides multiple pathways for the initiation of apoptosis. (17) The lipid bilayer membrane may change phase to a gel state at a lower temperature from a more fluid state at a higher temperature. Ultimately, cooling by itself can be harmful to cells.
Different cell types have different tolerances for maintaining optimal health. (19) For example, the ischemic tolerance of the brain is only 6 minutes at 37 degrees. This, however, extends to nearly 60 minutes when the body temperature reduces to 17 degrees. (16) Kidneys are able to tolerate much longer periods of "warm ischemia" (i.e., nonhypothermic ischemia). Total necrosis of the majority of tubules occurs after 60 minutes. As indicated by Kim, hair follicles at room temperature tolerate even longer periods of ischemia. (1)
Therefore, it makes sense to look for a preservation solution and temperature that is ideal for the hair follicle. We must approach storage solutions with a molecular-based logic to design the ideal holding solution for specific cell types of the hair follicle. Solutions should support the cellular proteome, genome, and fragmentome in addition to cellular structures such as the mitochondria, cell membrane, and nucleus.
The proteome is all the proteins expressed by a genome and the fragmentome refers to the peptide fragments.
Cost of various products extant today:
- Normal Saline: $44.20 USD case of 12 1 OOOcc bottles
- BSS: $132.18 USD case of8 500cc bottles
- Ringer's Lactate: $146.53 USD case of 16 lOOOcc bottles
- Hypothermosol: $1740.00 USD case of30 lOOcc bottles
- Vitasol: (liposomal ATP) $1354.00 USD per liter
As you can see, the cost varies greatly between the different storage solutions, so it is important to examine the differences in the various storage solutions.
As mentioned, many storage solutions are available. The following two sections further detail intracellular and extracellular options.
Intracellular storage solution
These are hypertonic solutions with elevated K+ levels and reduced Na levels more similar to the intracellular space that is unable to cross the cell membrane. This provides osmotic support. Important, as reduction in temperature and water-controlling cell pumps that regulate the osmotic gradient are inactivated. The higher osmotic pressure found in these storage solutions inhibit passive influx of water and thereby reduce the risk of cell swelling. Examples of intracellular storage solutions include Hypothermosol, Collins, Euro Collins, Viaspan (University of Wisconsin Solution), CryoStor, Celsior, HTK-Custodial, Unisol, and KPS I.
Tissue stored in intracellular storage solutions should be chilled, but at what temperature? According to Dr. Abey Matthew, grafts stored in Hypothermosol should be kept below l2°C and above freezing with a recognized usage range of 2°-8°C.(20) The composition of Hypothermosol is noted below: (19)
Extracellular Storage Solutions
These are isotonic solutions with a plasma-like complement of ions that mimics the normal extracellular environment of cells. Examples include normal saline, Ringer's Lactate, BSS, and tissue culture media. These solutions offer lackluster preservation at reduced temperatures. They also lead to cell swelling at lower temperatures due to reasons explained above. For this theoretical reason, specialists should never chill extracellular storage solutions. Nevertheless, specialists commonly use these solutions in combination with hypothermic temperatures. To summarize, there are strong hypothetical reasons based upon decades of large organ transplant research that conclude the following:
- 1. Synthetic preservation solutions that specifically inhibit detrimental cellular changes like ischemia and hypoxia may be better than culture medium or saline, which specialists commonly use.
- 2. Chilling of grafts should only be done when the grafts are stored in an intracellular medium.
- 3. If using an extracellular medium to store grafts then they should avoid chilling tissues.
- 4. However, not chilling the tissue presents its own problems.The metabolic rate and associated ATP demands do not decrease while the supply of ATP dramatically diminishes with the loss of normal perfusion of the tissue.
The minimum essential characteristics for the ideal storage solutions address the following:
- I. Minimize cell and tissue swelling
- 2. Maintain appropriate ionic balance
- 3. Prevent a state of acidosis
- 4. Remove or prevent formation of free radicals
- 5. Provide substrates for the regeneration of high energy compounds
- 6. Stimulate recovery upon re-warming and reperfusion.
New cryoprotective strategies are also emerging that primarily focus on combating oxidative stress and cold or hypoxia-induced apoptosis. Lactobinate found in Viaspan, Hypothermosol, Celsior, Cardiosol, Churchill's solution, and others contribute to minimizing cell injury due to calcium influx and free radical formation. Calcium is a major factor in apoptosis. Other anions included in an ideal solution include gluconate, citrate, glycerophosphate, and anionic forms of aminosulphonic acids such as HEPES. Osmotic agents such as sucrose and mannitol are also usually common. Mannitol possesses properties such as hydroxyl radical scavenger and prostaglandin mediated vasodilatation. Macromolecular oncotic agents include human serum albumin, hetastarch, or hydroxyethyl starch (HES).
Dextran-40 is a colloid for oncotic support that improves efficient removal of erythrocytes by inhibiting red cell clumping. High magnesium concentrations and very low calcium levels show value in cardioplegia and myocardial preservation. We include some glucose is often included as a substrate. However, at a low concentration it prevents exogenous overload during hypothermia, a probably cause of lactate production and intracellular acidosis by anaerobic glycolysis. HEPES is an excellent buffer at low temperatures. Adenosine is an essential substrate for the regeneration of A~ during re-warming and also acts as a vasoactive component through vasodilatation. For this reason, some suggest the inclusion of ATP in graft storage solutions. Glutathione is an important cellular antioxidant and hydroxyl radical scavenger, as well as a cofactor for glutathione peroxidase. This enables metabolism of lipid peroxides and hydrogen peroxides, both potent free radicals. Finally, one might include molecules for oxygen delivery, calcium channel blockers, apoptosis inhibitors, and trophic factors.
Vitasol, liposomal ATP, contains 2.5mg/ml iipid with 5mM ATP. Cells and tissues (of the hair follicle size range) are best for extended periods of time at concentrations of 0.01-1 mg/ mllipid and thus ATP concentrations of O.l-2mM ATP to minimize purinergic effects. The purinergic effects will still occur, but at 0.1mM-lmM concentration range the P2Y receptors activate and actually inhibit apoptosis. For this reason, Vitasol should be diluted 1:10 in normal saline or Hypothermosol.(8)
Transitioning cells from a chilled state to room temperature in normal saline with Vitasol prior to warming to body temperature reduces "shock" to the cells. It may have other benefits. At room temperature cell metabolism is still reduced, which decreases ATP consumption. In addition, tissue stored at l4°-20°C is optimal for the uptake of the ATP by cells.(18)
Hypoxia does not always cause tissue damage. Reperfusion of tissue, however, shows marked and occasionally severe tissue damage. The reperfusion stage is when the ischemic organ is re-implanted and exposed to the body's circulation. There are several hypotheses as to the cause of this injury. These include free radicals, inflammatory cells, intracellular calcium accumulation, and loss of membrane phospholipids. Reperfusion injury is one theory behind poor growth that is occasionally seen in hair transplant surgery.
A free radical is a molecule with an unpaired electron denoted by a dot (R"). The electron renders the molecule highly unstable and reactive. The high reactivity initiates chain reactions that produce toxic free radicals.(21) Under normal conditions, cells produce small quantities of free radicals. This isp primarily since the electron's energy transfers to intermediate molecules such as NAD, FAD and ADP with the energy/electron/photon ultimately being stored as ATP.
O2-> O2*->H2O2->H2O + OH*
The hydroxyl free radical is the most reactive of all free radicals and will oxidize any organic molecule almost instantaneously. Superoxide dismutase detoxifies most of the free oxygen radicals made during a cell's normal metabolism. With reperfusion injury, the normal protective enzymes may be overwhelmed by the electron energy that couldn't be stored as ATP due to, among other things, the absence of O2. In addition, in ischemia, ADP becomes hypoxanthine, which is further converted to xanthine. This loss of substrate for ATP formation would appear to be a major impediment for the resumption of normal cellular respiration. Furthermore, during reperfusion, xanthine converts into uric acid and superoxide anion (202).
Other endogenous antioxidants such as glutathione, glutathione peroxidase, catalase, ascorbate (vitamin C), alpha-tocopherol (from vitamin E), and NADH act as free radical scavengers, but may be depleted during reperfusion.(22) Other drugs that may help include Allopurinol and compounds that chelate iron. Poor growth from hair transplant surgery may be minimizable by use of free radical scavengers in graft holding solutions.
Every organ has an optimal temperature for storage based on the interaction of hypothermia, the nature of the cell, and its chemical composition. For cardiac muscle, the optimal temperature is 10°-20° Celsius.(23) For the kidney, some studies show that 10° Celsius is superior to 5° and 5° is superior to 0.5° (23,25). We are yet to study the optimal temperature for hair grafts. Parsley, for example, feels the optimal storage temperature for hair follicle grafts is between 8°-14° Celsius, but he bases this on optimal temperatures for other organs rather than for hair follicles. (26)
For years my storage ice seemed to melt after a few hours, causing the temperature to rise abruptly in my storage medium. To overcome this shortfall, I began to fill my PVC containers with water and freeze them in a larger freezer. Upon taking them out of the freezer, I insulated them and placed an aluminum plate over the ice. This more effectively transfers heat from grafts. The additional insulation, the aluminum plate, and the fuJI container of ice help keep the temperature below 10°C for over 6 hours.
Unfortunately, ice-based systems can lead to the temperature abruptly falling below 4°C and then staying there for a prolonged period of time. We do not know if the temperature below 4°C has a negative impact on the survival of hair follicles. Furthermore, we do not know if the abrupt drop in temperature is as good as a gradual decline in temperature. Some theorize that a gradual decline in temperature is better for hair follicle grafts. Dr. Parsley first moves his hair follicle tissue to a room temperature holding reservoir and then transfers it to an ice-based system to achieve a more gradual decline in temperature from body temperature.(25)
We always traumatize tissue when we take it from the body. Then we further traumatize it upon chilling. Finally, it is traumatized once again when warmed. Based on this theory of traumatization, it may be harmful to our grafts if we allow our grafts to rise above the low temperature in our holding solutions to a temperature closer to room temperature and then suddenly shock them again by re-chilling them. Striving to maintain a constant temperature throughout the chilling process is ideal based on this rationalization.
Consequently, I have created a chilling pump that circulates a cool mixture of propylene glycol through my storage dishes. I've found that this system allows me to maintain a constant temperature and may benefit someone interested in doing studies into the optimal temperature for graft storage. More recently, I created the Graft Chilling Plate (GCP). The GCP is a compact chiller designed to hold one or two petri dishes and is capable of storing grafts at a stable temperature throughout the duration of the hair transplant procedure. It will hold multiple stainless steel cones (developed by Dr. Bill Parsley) that allow the transfer of multiple grafts at one time.
Current studies have not found an ideal temperature or holding solution for hair follicles. Selecting one, therefore, requires defining the optimal outcome. Our optimal outcome is both survival rates and the overall results our patients experience. Hair restoration specialist's selection of holding solutions and their temperature should always depend on that standard.
Holding Solutions and Anagen Effluvium
Some theorize that the ideal holding solution will prevent anagen effluvium. Dr. Krugluger created the Moser solution that apparently prevented anagen effluvium.(8) Unfortunate, then, that this solution never made it to the market and there is no long-term data to support his conclusions.
Furthermore, transferring FUE grafts to the scalp in less than 20 seconds also does not prevent anagen effluvium.(28) If such rapid transfer fails to prevent it then the chance of a holding solution making the difference is small. Stopping anagen effluvium requires preventing apoptosis of the more metabolically active progeny of stem cells. The more important cell to protect, meanwhile, is likely much less metabolically active stem cells such as mesodermal stem cells.
Holding Solutions, Temperature, and Hair Yield
Holding solutions' most important contribution is increased hair yield and quality. The optimal holding solution, therefore, best reduces tissue trauma. This includes reperfusion injury, free radical formation, and ion or osmolarity abnormalities from the ischemic phase.
As Beehner has shown, chilled grafts seem to tolerate crushing trauma better than grafts maintained at room temperature.(2) His follow-up study compared room temperature normal saline to Hypothermosol and ATP. He concludes chilling tissue in Hypothermosol and ATP results in a higher yield of hair. Another potential benefit from the optimal holding solution and the optimal temperature include the potential to prevent thin hair shafts. In fact, thin hairs probably fit within a spectrum where no growth is extreme. Theoretically, there is also a way to "supercharge" the hair fiber diameter, as I note about donor area adjacent to the strip scar. (29)
Temperature Depends on Holding Solution
We know that certain tissues respond better to specific temperatures. Furthermore, some tissues respond better to a specific or customized holding solution. It remains for us to define these in terms of hair follicles. We remain uncertain whether there is any benefit from chilling grafts below room temperature. We do know, however, that, for reasons discussed, there is strong evidence that only hypertonic solutions should be used when chilling grafts. Furthermore, that data also suggest to only use isotonic solutions when maintaining grafts at room temperature.
Despite this knowledge, hair transplant data at this time suggest that chilled grafts in normal saline or Ringer's Lactate seem to thrive just as well as when they are placed at room temperature in these solutions. It is uncertain whether follicle survival is better in hypertonic solutions at a cool temperature as is the case with other organs. There are a variety of different holding solutions of both the intracellular and extracellular composition. For reasons stated in this article, extracellular holding solutions should not be chilled. Conversely, as discussed, do not store grafts at room temperature in intracellular holding solutions.