The Future in Graft Storage

When performing hair transplantation procedures, it is of the utmost importance to try to obtain the maximum survival rate possible of transplanted micrografts. Although numerous factors (such as dehydration of grafts, trauma in handling with forceps, etc.) play a critical role in the graft survival, a "metabolic pre-conditioning" of hair grafts by means of dedicated storage mediums should have value in striving to enhance the survival rate of grafts when performing hair transplantation surgery.

Knowing the best way to preserve the grafts is particularly important with the advent of megasessions (involving the transplant of a large number of very small grafts), since a significant period of time may elapse between graft harvesting and their implantation in the recipient area. At present, it is generally recommended to preserve the grafts at a low temperature (1ºC - 4ºC), in order to enhance the survival rate of the grafted hairs1. Indeed, it is assumed that, as in other major transplant procedures, lowering the metabolism of the grafts by means of a reduction of their temperature may be of some utility for enhancing their survival rate. On the other hand, since the development of tissue preservation regimes has achieved great successes in the preservation of vital organs and grafts in the interval between harvest and re-plantation into the recipient area, other additional methods should be considered.

In this regard, for example, the role of oxygen free radicals in tissue ischemia has been well documented. Oxygen free radicals in ischemic tissue are generated from a number of different sources. The relative importance of these mechanisms is not known. It would follow, however, that the prevention of action of radicals produced by all of these pathways would be more effective in preventing tissue damage than by blocking the radicals produced by a solitary pathway2. Similarly, analysis of cellular metabolism in hypoxic conditions reveals not only an accumulation of cytotoxic metabolites, but also a gradual depletion of cellular energy stores3.

Increased intracellular use of high-energy adenosine triphosphate (ATP) and decreased mitochondrial regeneration of ATP occur under anaerobic conditions3-6. These changes lead to the deterioration of normal cellular processes and eventual cellular demise.

The above-mentioned considerations can be can applied to the field of hair transplantation surgery. Deferoxamine has been shown2 to be a potent, non-selective scavenger of oxygen free radicals, while exogenous ATP is thought to replenish depleted intracellular ATP, since several observations support the notion of intracellular uptake of extracellular ATP by ischemic cells. Indeed, addition of the exogenous high-energy cellular substrate adenosine triphosphate-magnesium chloride has been shown to substantially improve cellular preservation and tissue viability in ischemic conditions6.

In a recent study7, normal human occipital scalp samples were obtained, from ten healthy male patients, during routine excision of benign scalp lesions. Isolation of anagen hair follicles was achieved by stereo-microscopic dissection. A total of 200 anagen hair follicles (20 follicles per each sample) were obtained.

Follicles from each of the ten occipital scalp samples were thus randomly assigned to one of the following group: Group A (control; n=10 follicles per each scalp sample; total n=100 follicles), and Group B (experimental; n=10 follicles per each scalp sample; total n=100 follicles). While follicles from Group A were preserved, for five hours, in a Petri dish filled with isotonic saline, follicles from Group B were preserved, for five hours, in a Petri dish filled with saline containing adenosine triphosphate-magnesium chloride 0.1 µmol/ml and deferoxamine mesylate 15 mg/ml.

Immediately after the five-hour period, hair follicles from both Groups were stored in 500 ml of Williams E medium with supplements as follows: 1% fetal calf serum, 10 mg/ml transferrin, 10 mg/ml insulin, 10 ng/ml sodium selenite, 10 ng/ml hydrocortisone, 100 U/ml penicillin, 100 mg/ml streptomycin, 2.5 mg/ml fungizone, and cultured for ten days8. Follicles were maintained free floating in individual wells of 24-well multi-well plates in an atmosphere of 37ºC, 5% CO2 - 95% air, and 100% humidity. This has permitted detailed measurements to be made on both the length and survival rates of the tested follicles.

The length of each follicle was measured at magnification x20 immediately following the five-hour test period and at the end of the ten-day culture period, using a calibrated microscope. Total follicle length was computed as the distance from the base of the bulb to the end of the shaft. Follicles which lost normal follicular architecture due to degeneration late in the culture period were computed as not survived. Histology was accomplished, at the end of the ten-day culture period.9 Most of the dissected follicles grew and retained their morphology for the whole ten-day period of culture.

A statistically significant difference was found between the survival rate (considered as preservation of normal follicular architecture and absence of degenerative signs) of follicles from the Control Group (87%) and Experimental Group (98%). Photographs taken of freshly isolated and maintained hair follicles showed that the increase in length over ten days was not associated with any disruption of hair follicle architecture. The length increase always occurred by the production of keratinized hair shaft. All the survived follicles produced a measurable shaft elongation, and no statistically significant differences were found between the growth rate of follicles from the Control and the Experimental groups. Histologic analysis demonstrated that the survived follicles from both Groups always maintained a normal histologic appearance, even after 10 days in culture.

The reported study addressed the question of whether an "enhanced" preservation solution, containing adenosine triphosphate-magnesium chloride and deferoxamine mesylate, is suitable for preservation of hair grafts, and whether this pharmacological/metabolic treatment may further enhance viability of transplanted micrografts. The survival rate of follicles from the control Group was consistent with that reported by Limmer10, storing the grafts in chilled isotonic saline at 4ºC. Conversely, our experimental data were indicative of a significant increase in the survival rate of hair micrografts pre-treated with adenosine triphosphate-magnesium chloride and deferoxamine mesylate.

In conclusion, it should be taken into account that the study discussed was just an in vitro study, and that the obtained results should be validated by further in vivo studies. Similarly, further studies need also to be undertaken in order to evaluate and compare other storage mediums. As hair transplant procedures continue to increase in length, metabolic pre-conditioning of hair grafts should play an important role in ensuring maximum follicular survival.

REFERENCES
Fan J, Raposio E, Nordström REA: Minigraft preparation in surgical hair replacement. Scand J Plast Reconstr Hand Surg 1997; 31: 83-7.
Kohout M, Lepore A, Knight KR, et al.: Cool perfusion solutions for skin flaps: A new mixture of pharmacological agents which improves skin flap viability. Br J Plast Surg 1995; 48: 132-44.
Zimmerman TJ, Sasaki GH, Khattab S.: Improved ischemic island skin flap survival with continuous intraarterial infusion of adenosine triphosphate-magnesium chloride and superoxide dismutase: A rat model. Ann Plast Surg 1987; 18: 218-23.
Machiedo GW, Ghuman S, Rush BF, et al.: The effect of ATP-MgCl2 infusion on hepatic cell permeability and metabolism after hemorrhagic shock. Surgery 1981; 90: 328-32.
Maxild J.: Effect of externally added ATP and related compounds on active transport of p-aminohippurate and metabolism in cortical slices of rabbit kidney. Arch Int Physiol Biochim 1978; 86: 509-14.
Williams D, Reibel DK, Rovetto MJ.: ATP induced increase in ATP content in cultured myocardial cells. Fed Proc 1979; 38: 1389-93.
Raposio E, Cella A, Panarese P, Nordström REA, Santi PL.: Power-boosting the grafts in hair transplantation surgery: Evaluation of a new storage medium. Dermatol Surg (in press).
Raposio E, Filippi F, Levi G, Nordström REA, Santi PL.: Follicular bisection in hair transplantation surgery: An in vitro model. Plast Reconstr Surg 1998; 102 (in press).
Heidenhein M.: Uber die Maliorysche Bindegeweosfarbung mit Karmin und Azokarmin als Vorfarben. Ztsch F Wissensch Wikr 1915; 32: 361-4.
Limmer BL: Micrografts survival. In: Stough DB, ed. Hair Replacement: Surgical and Medical. St. Louis: Mosby Press 1996: 147-9.

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