Factors influencing graft survival
   By William M Parsley MD The progress made in the field of hair restoration surgery over the past 15 years has been remarkable. Results are very natural looking and our understanding of full and receding hairlines has vastly improved. While outcomes are generally very good, with past reports of over 100% growth from grafts, experienced surgeons are still nagged by the inconsistencies of graft survival. Occasionally, grafts in an apparently excellent candidate will grow far less than 100% and the surgeon usually has no sound explanation. Many feel the answer lies within the basic fundamentals of hair restoration, but others believe that there are as of yet undiscovered factors which need to be disclosed. X- and H-Factors In the early 1980s, Norwood and Shiell proposed the term X-factor to describe unexplained poor survival of grafts beyond the control of the physician. They felt there was a little influence of X-factor in every case, but in 1-3% of patients it was significant. Norwood speculated that an autoimmune reaction might be involved. In 1994, Greco proposed the term H-factor to describe human errors leading to poor growth. He divided these into direct factors (manipulation, trauma) and indirect factors (drying, heat, staff fatigue). What Affects Graft Survival? The best answer is: “nearly everything.” The following are some of the primary factors to consider in graft survival: Selection of patients whose donor hair is of sufficient quality and vigour to survive transplantation and future loss to baldness. Selection of patients with a recipient area of sufficient health to support the grafts. Avoidance of direct and indirect physical trauma to the grafts on the day of surgery. Graft size and method of preparation. Selection of the best storage solution (including additives) and the decision as to whether or not chill that solution. Creation of recipient sites so that instrument size, density of sites, and depth of sites do not damage the recipient bed to the point that they impede survival of the grafts. Finding the best plan of post-op care. While we are far from having the answers we seek, there are some very helpful studies and case reports to help guide us. The following is a list of categories believed to be important to survival along with pertinent reports from the literature. The holding solutions in these studies were chilled unbuffered normal saline (UNS) unless otherwise noted. Hydration If there is one universally accepted factor in graft survival, it is hydration. In 2000, Gandelman, et al. published an article in Dermatologic Surgery studying 12 patients whose grafts were subjected to dehydration and trauma. Grafts were left on a surgical glove for 3 minutes and then examined under the light microscope (LM) followed by scanning and transmission electron microscopic (EM) analysis, if indicated. Major dam- age was observed by all modalities after dehydration—and planted dried grafts were found not to grow. This report was followed by a study by Beehner (Forum, 2007) in which 60 1-hair grafts and 60 2- hair grafts were allowed to dry on a wet Telfa pad for 16 minutes before placing. The grafts were getting stiff but were not brittle. Survival for 1-hair FUs was 60% and for 2-hair FUs was 82%, suggesting larger grafts give some protection against dehydration. Wetting the dried grafts before placing did not help. In a busy transplant setting, it is easy to lose a few grafts in each case from drying. Drying at the cutting stations, drying on the gloves, and undetected “popped” grafts continue to create a slight attrition from dehydration. The cure is persistent vigilance throughout the procedure. Physical Trauma The second part of Gandelman’s 2000 study showed no visible damage to grafts on light microscopy following trauma (bending, crushing, and stretching with forceps) and therefore EM was not performed. They admitted that LM could not necessarily rule out biological effects. Beehner found that soft crushing of the bulbs with a needle driver (rubber sleeves over the jaws) resulted in 64% survival versus a hard crush (35%). Interestingly, hard crushing of the bulge area resulted in a 0% survival for room temperature grafts versus 36% for chilled grafts, indicating that chilling provides a slight protection against physical damage. Beehner and Frechet (2006 Annual Scientific Meeting of the ISHRS) performed a transection study on slit minigrafts (SMG´s) in which intact SMG´s were compared with SMG´s that were transected at some point along the follicle. In Beehner’s grafts, intact SMG´s had a survival of 86% at 6 months, but dropped to 65% at 12 months; while transected minigrafts had a survival of only 49% at 6 months, dropping to 45% at 12 months. Frechet’s transected SMG´s had a survival in the range of 35%. These studies give evidence that trauma, including transection, results in a seriously reduced survival rate. Time Out of Body In one of the earliest and most quoted studies on FUs, Limmer (1992) recorded the following survival rates at different times out of the body. Using at least 200 FU grafts for each time frame, the survival was: 2 hours, 95%; 4 hours, 90%; 6 hours, 86%; 8 hours, 88%; 24 hours, 79%; 48 hours, 54%. A 1% loss per hour is a rough guide according to Dr. Limmer.   2 hs ……… 95% 4 hs ……… 90% 6 hs ……… 86% 8 hs ……… 88% 24 hs .........79% 48 hs .........54% While it might seem that time out of the body is a predictable critical factor, Unger’s study on 4mm grafts planted within 2 minutes of removal had no increased survival over those planted after an hour and no improvement over the survival of Limmer’s FU grafts planted after 8 hours. Measuring survival at 4 months, 184 of 218 hairs (84%) reinserted at 2 minutes survived compared to 212 of 218 (97%) reinserted at 60 minutes (Walter Unger, presenting to AAD meeting, Dallas, Texas, 1977). Perhaps measuring at 8 months would have revealed a higher survival rate. In an attempt to find a method for delayed graft re-implantation, Kurata, et al. compared organ culture survival (as measured by hair shaft elongation) for follicles stored for various periods of time at 4°C in Hanks solution, Dulbecco’s modified Eagle’s medium (DMEM), RPMI, and saline before culture with DMEM in a CO2 chamber. The pH buffers were not identified. After 24, 36, and 48 hours storage, survival in saline was significantly lower than the other solutions; however, none of the grafts grew in organ culture after 48 hours of cold storage in any of the solutions. Ten grafts were preserved for 7days in DMEM at 4°C then planted under the panniculus carnosus in athymic mice. At 5 months, 6 grafts were still growing. It is clear that long-term storage of grafts would be a significant advancement but is still a work-in progress. Chilling versus Non-chilling Using unbuffered normal saline, Raposio, et al. reported an 87% survival of chilled (1°C) versus 88% room temperature (RT) (26°C) storage of grafts for 5 hours followed by organ culture for 10 days in Williams E media. No survival was defined as loss of normal follicular architecture. The hair shaft elongation rate between the two groups was also similar. Jiange, et al. (2005) compared chilled storage in Ringer’s solution for 1–7 days at 0°C versus 4°C followed by (1) outer root sheath culture and (2) implantation under the panniculus carnosus of athymic mice. Survival following storage at 0°C was modestly better than at 4°C for all time periods of storage for both ORS cultivation and implant survival, with both categories showing no significant growth after cold storage for 7 days. Qian, et al. reported on human hair follicles implanted into athymic mice after several periods of storage at 0°C in Ringer’s solution compared to 0°C in DMEM culture media. Growth after 24 hours of storage followed by implantation into athymic mice for 5 months was 84% for Ringer’s versus 72% for DMEM. Results were also better with Ringer’s at 48 and 72 hours, but with considerably reduced survival. No regrowth was seen after being held in either solution for 7 days. The ability to culture outer root sheath cells after 24 hours of graft storage was also better with Ringer’s (95%) versus DMEM (86%). The value of chilling is well established in general organ transplantation. Kidneys, for example, show up to a tenfold increase in survival time in chilled storage compared to room temperature storage. Hair follicles do not appear to be as sensitive to RT, but studies indicate that there is an increasing loss sometime after 6 hours. However, studies have not been continued long enough to know at what time period the break point occurs; therefore, more research is needed to determine the maximum room temperature storage time for hair follicles. Holding (Storage) Solutions Beginning in the late 1950s, hair grafts have predominantly been stored in unbuffered normal saline (UNS). Some of the best results reported in our field have been with the use of this solution. But is it the best solution or are grafts just pretty resilient? When compared to other storage solutions, saline has generally shown decreased survival. There have been quite a few articles written on the subject recently, but a few brief comments will be made here. PH: Being unbuffered, UNS has a variable pH, usually in the range of 5.0. Normal human serum has a pH of 7.4. Increasing acidity has a known negative effect on tissue survival. The effect of using UNS on follicular tissue pH is not known at this time. Researchers will generally buffer normal saline with phosphate (PBS) before conducting tissue studies. Plasma-Lyte A has a pH of 7.4, using an acetate buffer. DMEM most commonly contains a natural bicarbonate buffer and is designed to be used at 37 degrees in vitro in controlled chambers with 5-10% CO2. In open air, DMEM can become alkaline and may not be healthy for hair grafts. DMEM used in hair studies normally contains the more expensive HEPES buffer, which works well in open air situations. Advanced intracellular balanced solutions most commonly use HEPES, particularly in those meant to be chilled as it adapts to temperature changes. It should be noted that DMEM is not specifically approved as a transplant storage media (personal correspondence with Sigma-Aldrich Co.). Osmolality and electrolyte balance: Osmolality of normal serum ranges from 280–310 mOsmol/L. UNS has an acceptable osmolality of 308. Advanced solutions use osmotic buffers because there is a higher concentration of impermeable solutes intracellularly versus extracellularly. Membrane pumps are altered during cold storage. Adding impermeable solutes, such as lactobionate and dextran, as osmotic buffers helps to maintain the proper balance, particularly in chilled solutions. Additives to holding solutions: En 1998, Swineheart no encontró diferencias significativas en la supervivencia del injerto comparando el almacenamiento en solución salina vs medio de cultivo orgánico (RPMI) ambos a 9ºC, con una supervivencia medida a 5 meses de 82% vs 84% respectivamente. Raposio, et al. (Derm Surg., 1998) reported that enhancing normal saline with ATP-MgCl and deferoxamine showed improved graft survival. Normal saline (control) was compared to the “enhanced” saline by storing grafts in these solutions at RT for 5 hours. Half of the grafts in the control and experimental groups were then placed in Williams E media and cultured in a controlled CO2 chamber for 10 days. The grafts in the enhanced solution had a 98% survival rate compared to 87% for the control. The other half of the grafts was studied by hair shaft elongation, which showed no significant difference in survival. Currently, work is ongoing with ATP, which normally has difficulty crossing the cell membrane. By using liposomes, ATP is able to easily enter the cells; but because the liposome incorporates into the cell membrane, the membrane can weaken with too high a concentration. In addition, the freeze-drying of the ATP needed for this process is very expensive. For these reasons, work is being conducted to determine the effectiveness of a safer, inexpensive preparation (lipo tripolyphosphate) topically for ATP supplementation during the post-operative period in hair transplantation. Ischemia Reperfusion Injury and HT Grafts During transplantation, tissues develop ischemia. In organs susceptible to IRI, upon reperfusion and exposure to oxygen, the conversion of hypoxanthine (a breakdown product of ATP) to xanthine releases free radicals and reactive oxygen species—and starts a cascade leading to cell death by apoptosis or sometimes necrosis. The free radicals released by apoptotic cell death (ACD) are particularly damaging to the double strands of DNA and the cell membrane, where they cause lipid peroxidation. This lipid peroxidation of the cell membrane releases malondialdehyde (MDA) and 4-hydroxy- alkenals (HAE), which are considered measurements of IRI. DNA breakdown during ACD can be measured by cytoplasmic histone-associated DNA fragments (HADF). Most transplanted organs are surgically reconnected to the body’s blood supply and are exposed to a sudden dramatic rise in oxygen tension. In contrast to common organ transplants, hair grafts are perfused passively for at least 3 days before being re- vascularised, thus not receiving a sudden “blast” of oxygen. For this reason, some question exists whether IRI occurs in hair transplantation. Cooley used the MDA assay to test 150 grafts in 7 patients. The test grafts were placed into the scalp and later removed to complete the ischemia/reperfusion cycle and then tested against control grafts that were never re-implanted. The MDA assay in test grafts revealed MDA levels elevated 200–600% over controls. Krugluger, et al. demonstrated a dramatic rise in HADF after 36 hours of culture in serum containing DMEM culture media. In addition, HADF was significantly reduced by storage in media containing antioxidants. In yet another study, Krugluger reported better growth and less shedding after adding various antioxidants to holding solutions. While more studies are needed, there certainly appears to be reasonable evidence for the existence of IRI and ACD in hair grafts.   Platelet Rich Plasma There is currently considerable interest in platelet rich plasma (PRP). PRP is rich in growth factors, among which are platelet derived growth factor (PDGF), transforming growth factor beta-1 (TGF ß-1), and vascular endothelial growth factor (VEGF). PRP has been used with benefit in both the donor strip and also grafts before placement. In 2005, Uebel presented a study in which grafts were dipped in the PRP created on the day of surgery from the patient’s blood. Grafts were dipped into the PRP for 15 minutes before implanting into the scalps of 23 patients. There was a 15% increase in graft survival in the PRP side compared to controls. PRP also looks promising in donor and recipient site healing. The negatives are that it is a little cumbersome and expensive to prepare. Freezing for Long-term Graft Storage In 2002, Adanali, et al. reported that grafts frozen for 2 weeks at –20°C (standard freezer) showed no damage under LM examination, suggesting that this might allow long-term graft preservation. In response, Jimenez performed a study of 150 grafts frozen for 1 hour, 5 days, and 7 days at –20°C before implantation. Survival after freezing for 1 hour was 20%; 5 days, 0%; and 7 days, 0%. This demonstrates the unreliability of LM to evaluate survival. At –20°C, ice crystals are constantly forming and reforming, killing the cells. Freezing tissue for storage requires much colder temperatures in order to create a “glass formation state” (no crystal movement), usually with liquid nitrogen. This is an involved process using cryoprotectants in which modifications for tissue type and timing of the freeze/thaw are critical. 10 UF/cm2 …....... 97% 20 UF/cm2 …....... 92% 30 UF/cm2 …....... 70% 40 UF/cm2 …....... 79% An important and often-quoted study on 2 patients by Mayer, et al. in 2000 compared 2-hair FUs planted at various densities and measured at 8 months. Results showed the following survival: 10/cm2, 97%; 20/cm2, 92%; 30/cm2, 70%; 40/cm2, 79%. All sites were made with an 18g needle, which is quite large by today’s standards. In a 2006 study, Beehner studied 2 patients using densities of 20 and 30/cm2 into 19g needle sites and 40 and 50/cm2 into 20g needle sites. Results showed the following: 20/cm2 (95% patient 1, 87% patient 2); 30/cm2 (93%, 92%); 40/cm2 (70%, 100%); 50/cm2 (67%, 94%). While the results are inconsistent, this study seemed to indicate that recipient site size is important. A recent yet unpublished study tends to verify this, showing 98–100% survival at densities of over 60 and 70 FUs/cm2 while using small recipient sites. Survival at higher densities is influenced by a variety of factors, the most important of which are the site size, tissue handling, donor hair quality, and recipient site quality. Doctors new to the field would be well served to increase density slowly. . Skinny vs. Chubby In 1997, Seager performed a study on 88 “skinny” grafts in which trimming left the papillae with no surrounding tissue and compared them to 163 “chubby” grafts in which ample surrounding tissue was left. The survival rate was 89% and 113%, respectively. In 1999, Beehner compared survival in 60 “skinny” and 60 “chubby” grafts, but left an equal amount of tissue surrounding the dermal papillae. Result survival rates were 101% and 133%, respectively. More recent studies have not shown survivals much in excess of 100%, possibly due to better counting techniques. Regardless, it appears healthier for the grafts to leave a little tissue beyond the dermal sheath and papillae. Planting trauma and graft dehydration may be reduced with having just a little extra tissue. Intact versus Non-intact Grafts In 1999, Beehner performed a study comparing intact FU´s compared to grafts with the same number of hair follicles but containing follicles from two adjacent FUs that were subdivided. The grafts containing follicles from subdivided FUs actually had a slightly better survival rate, though not significant. From this study, it appears that it should be safe to divide FU´s, if needed. Lateral (Coronal) vs. Parallel (Sagittal) Grafts In 2006, Perez and Parsley performed a study using 2-hair grafts planted both laterally (l) and parallel (p) at densities of 30, 40, and 50 grafts/cm2. Results: 30/cm2, 70% (p) vs. 100% (l); 40/cm2, 86% (p) vs. 92% (l); 50/cm2, both 105%. All sites were made with a 19g needle. This small study, along with a general overview of results around the world, would tend to indicate that there may be no significant differences in survival using lateral versus parallel grafts. 30 UF/cm2…....... P: 70% L: 100% 40 UF/cm2 …....... P: 86% L: 92% 50 UF/cm2 …....... P y L: 105% Miscelaneas In the July/August 2007 issue of the Forum (Vol. 17, No. 4), Rinaldi, et al. used a twice daily topical post-op solution containing adenosine sulfate 0.1%, taurine 1.0%, and ornithine chloride 1.0% (called 1-3 atodine). Adenosine sulfate up-regulates vascular endothelial growth factor (VEGF) and follicular growth factor-7 (FGF 7), while taurine and ornithine stimulate outer root sheath growth. At 1 month, vessel diameter and hair shaft diameter were both larger than the placebo. Revascularization (using reflectance confocal microscopy) of the grafts was quicker by nearly threefold, and the follicle growth was improved. Could one of the keys to improved graft survival reside with VEGF? Yano, et al. demonstrated that perifollicular angiogenesis correlated with up-regulation of VEGF mRNA expression in murine outer root sheath keratinocytes, but not in dermal papillae cells. The role of the ORS being the primary site of VEGF up-regulation was also found in a study by Krugluger, et al. Transgenic over-expression of VEGF resulted in a strongly induced perifollicular angiogenesis; resulting in increased hair growth, follicle size, and shaft diameter. Systemic neutralizing anti-VEGF antibodies resulted in poor hair growth and reduced follicle size. Because the outer root sheath is more accessible to topical therapy than the dermal papillae, it is easy to speculate that the topical 1-3 atodine solution mentioned in the previous paragraph might be effective. General Impressions We have looked at graft survival from many viewpoints, but we have not yet satisfactorily uncovered some of the factors leading to inconsistencies in growth. In this author’s opinion, part of it may lie in the recipient bed and the speed of revascularization. Grafts placed immediately after harvesting don’t seem to grow significantly better than those placed several hours later. Rinaldi’s use of topical 1-3-atodine solution post-transplantation, the effects of PRP, the use of inhibitors of iNOS, and the work on up-regulating VEGF are all exciting. Grafts may take 3 or more days to re-vascularise. Anything to speed this process or support them in the interim logically might help. Preconditioning of grafts with growth factors and antioxidants while out of the body is also very promising. Additionally, isolated cases suggesting improved hair growth using hyperbaric oxygen (HBO) are encouraging, especially when one considers studies showing improved skin graft and flap survival with HBO. It should be pointed out that oxygen therapy is known to stimulate angiogenesis. In conclusion, there is much to be learned about hair graft survival. Fortunately, interest in research is growing rapidly. William M Parsley MD Translation: Dra. Ximena Vila
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