Hair testing for drugs of abuse

Hair testing for drugs of abuse is a developing technology, which offers the possibility of longer detection times than is commonly obtained with urine analysis. It is the main method for evaluation of an individual's drugs of abuse history. In many countries hair analysis is routinely used to detect drug abuse in forensic cases, occupational and traffic medicine and clinical toxicology. Hair analysis in pregnant women, neonates and infants is a useful tool for the detection of drug exposure in utero. In Croatia hair testing for drugs of abuse is performed at the Institute for Medical Research and Occupational Health. Three-year experience in drugs of abuse analysis in hair is described. In 331 hair samples (270 from adolescents and 61 from adults) opiates and metabolites, cocaine, methadone, and amphetamines were analyzed by gas chromatography/mass spectrometry. Most prevalent drugs of abuse in adolescents were amphetamines, and in adults heroin. From the examples cited and samples analyzed it is evident that hair testing is emerging as a reliable biological marker for cumulative account of individual exposure to drugs of abuse.     

External contamination of bovine hair with beta2-agonist compounds: evaluation of decontamination strategies

Hair analysis has shown great potential in the control of illegal use of veterinary drugs such as beta2-agonists. However, it has been shown that hair can be externally contaminated with drugs which can lead to false positive results. Exposure of bovine hair to aqueous solutions of beta2-agonist compounds results in incorporation of these drugs into the hair. Standard hair washing procedures found in the literature: detergent (Tween-20), phosphate buffer or organic solvents (dichloromethane or methanol) cannot eliminate this external contamination. Beta2-agonists can be extracted from hair very efficiently with 0.1 M HCl, the extraction kinetics of externally and endogenously accumulated clenbuterol at room temperature are different which makes it feasible to discriminate between them. Treatment of hair samples with a 0.1 M HCI solution for 2 h at room temperature results in a ratio of clenbuterol content in the wash solution to clenbuterol content in the washed hair equal to or less than 0.25 for samples from treated cattle; whereas this ratio is equal to or higher than 0.70 for externally contaminated samples. The design of the study was intended to resemble the plausible scenario of hair being sampled a short time after external contamination. A similar study to detect external contamination for hair sampled a long time after exposure is in progress.     

Hair analysis for abused and therapeutic drugs

This review focuses on basic aspects and recent studies of hair analysis for abused and therapeutic drugs and is discussed with 164 references. Firstly, biology of hair and sampling of hair specimens have been commented for the sake of correct interpretation of the results from hair analysis. Then the usual washing methods of hair samples and the extraction methods for drugs in hair have been shown and commented on. Analytical methods for each drug have been discussed by the grouping of three analytical methods, namely immunoassay, HPLC–CE and GC–MS. The outcomes of hair analysis studies have been reviewed by dividing into six groups; morphine and related, cocaine and related, amphetamines, cannabinoids, the other abused drugs and therapeutic drugs. In addition, reports on stability of drugs in the living hair and studies on drug incorporation into hair and dose–hair concentration relationships have been reviewed. Applications of hair analysis to the estimation of drug history, discrimination between OTC drug use and illegal drug use, drug testing for acute poisoning, gestational drug exposure and drug compliance have also been reviewed. Finally, the promising prospects of hair analysis have been described.     

Reorganization of actin during repair of hair bundle mechanoreceptors

Hair bundle mechanoreceptors can be damaged by over-stimulation or by exposure to calcium-free buffers. Provided the trauma is slight, hair bundles recover, although the subcellular mechanisms for such recovery are poorly understood. Hair bundle mechanoreceptors on tentacles of sea anemones are especially resilient, recovering from severe trauma within several hours. During the recovery period, large protein complexes are secreted called “repair proteins” containing replacement linkages for those lost during trauma. In the present study, we find that recovery requires reorganization of the actin-based cytoskeleton in hair bundles. F-actin is first partially depolymerized and then repolymerized in hair bundles based on confocal microscopy. Furthermore, stereocilia show considerable motility during repair based on field emission scanning electron microscopy of hair bundles fixed at 1 min intervals after exposure to exogenously supplied repair protein complexes. Recovery of vibration sensitivity occurs at the organismal level within 8 min. Paradoxically, a full recovery of morphology of hair bundles requires approximately 45 min and a recovery of F-actin levels requires approximately 40 min. Similarly, a full recovery of mechanoelectric responses of hair cells requires approximately 45 min. Thus, it appears that the recovery of responsiveness at the organismal level precedes a full recovery of hair bundles.     

Assessing chronic liver toxicity based on relative gene expression data

The risk associated with exposure to hepatotoxic drugs is difficult to quantify. Animal experiments to assess their chronic toxicological impact are time consuming. New quantitative approaches to correlate gene expression changes caused by drug exposure to chronic toxicity are required. This article proposes a mathematical model entitled Toxicologic Prediction Network (TPN) to assess chronic hepatotoxicity based on subchronic hepatic gene expression data in rats. A directed graph accounts for the interactions between the drugs, differentially expressed genes and chronic hepatotoxicity. A knowledge-based mathematical model estimates phenotypical exposure risk such as toxic hepatopathy, diffuse fatty change and hepatocellular adenoma for rats. The network's edges encoding the interaction strength are determined by solving an inversion problem that minimizes the difference between the observed and the predicted relative gene expressions as well as the chronic toxicity data. A realistic case study demonstrates how chronic health risk of three halogenated aromatic hydrocarbons can be inferred from subchronic gene expression data. The advantages of the TPN are further demonstrated through two novel applications: Estimation of toxicological impact of new drugs and drug mixtures as well as rigorous determination of the optimal drug formulation to achieve maximum potency with minimum side-effects. Prediction of animal toxicity may be relevant for assessing risk for humans in the future.     

Mercury concentrations in hair exposed in vitro to mercury vapor

Hair is often used as an index of environmental and industrial exposure to different metals. The interpretation of metal levels in hair is difficult because of the risk of external contamination. The aim of this study was to define the degree of external contamination of hair exposed in vitro to mercury vapor. Specimens of hair were exposed to concentration: 0.026, 0.21, and 2.7 mg Hg/m³ for 2–28 d. Mercury levels in hair increased during 28 d of exposure 2, 3 and 13, times, respectively, when compared to initial values. Mercury levels in hair exposed to the first and second (but not third) concentration of mercury vapor attained a steady state on the 21st d of exposure. The contamination of hair with mercury could not be removed by washing with water, solvent, and detergent. Hair may be used as an index of internal uptake of mercury provided that it was not externally exposed to mercury vapor. In cases of occupational exposure to mercury vapor, hair could become a useful tool for monitoring exposures.     

Gas chromatographic analysis of nicotine and cotinine in hair

Non-invasive validation of cigarette- or cigar-smoking behaviour is necessary for large population studies. Urine or saliva samples can be used for confirmation of recent nicotine intake by analysis of cotinine, the major metabolite of nicotine. However, this test is not suitable for validation of survey data, since the quantification of cotinine in saliva only reflects nicotine exposure during the preceding week. To validate information on tobacco use, we investigated hair samples for quantifying nicotine and cotinine by gas chromatography—mass spectrometry. Hair (about 50–100 mg) was incubated in 1 M sodium hydroxide at 100°C for 10 min. After cooling, samples were extracted by diethyl ether, using ketamine as an internal standard. Drugs were separated on a 12-m BP-5 capillary column, and detected using selected-ion monitoring (m/z 84, 98 and 180 for nicotine, cotinine and ketamine, respectively). Hair from non-smokers and smokers contained nicotine and cotinine. Although it is difficult to determine an absolute cut-off concentration, more than 2 ng of nicotine per milligram of hair can be used to differentiate smokers from non-smokers. Some applications of this technique are developed to determine the status of passive smokers, the gestational exposure in babies and the pattern of an individual's nicotine use by cutting strands of hair into sections of one-month intervals.     

Assessment of hair mineral analysis commercially offered in Germany.

To evaluate intra- and inter-laboratory agreement concerning hair mineral analysis and interpretation of results, hair samples from 2 volunteers were sent to seven laboratories, which commercially offer hair mineral analysis in Germany. 6 weeks later, another identical part from the hair sample of volunteer 1 was sent to all seven labs. Altogether, 50 elements were analyzed, 23 by all seven labs. For comparability, only the results for these 23 elements were assessed. The intra-laboratory reproducibility was evaluated by the 2 identical hair samples from volunteer 1. On the average, the reproducibility seems to be sufficient (median +/- 9.48% to +/- 20.59%), but for individual elements there were unacceptable out-rulers up to 100%. Only one lab classified all elements of the first and the second analysis of the identical hair sample in the same category (below, within, or above normal range). The others grouped 4 to 7 elements different. This is not tolerable. The inter-laboratory comparability was assessed by the results of the hair samples of both volunteers. For the sample of volunteer 1 at least the results of 6 (out of 23) elements were within an acceptable range of +/- 30% from the consensus value (= mean of all seven labs). For volunteer 2 this was only the case for 2 (!) elements. Differences of more than 100% were found for most other elements. Moreover, in the vast majority of the tested elements there was no comparability of the cLassification to the respective reference ranges of the different laboratories. For example, for volunteer 1 only 3 elements (our of 23!) were identically classified by all seven labs. As neither the analytical results nor the classification to the individual reference ranges by the laboratories correspond in tolerable borders, conclusions, drawn from these results, cannot be valid. Hair mineral analysis from these laboratories is unreliable. Therefore we must recommend to refrain from using such analysis to assess individual nutritional status or suspected environmental exposure.     

Validation of a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair using LC-MS/MS and its application to human and rat hair

Benzodiazepines and zolpidem are controlled in many countries due to their inherent adverse effects of a high degree of tolerance and dependence. Recently, as some of these drugs have become distributed illegally and available through media such as the Internet, their abuse is becoming a serious social problem. Hair is a useful specimen to prove chronic drug use. In the present study, a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using methanol, filtered and injected on the LC-MS/MS. The following validation parameters of the method were satisfactory: selectivity, linearity, matrix effect, recovery, process efficiency, intra- and inter-assay precision and accuracy and processed sample stability. The limit of detection (LOD) and the limit of quantification (LOQ) were the total drug detected from the sample. The LODs ranged from 0.005 ng (zolpidem) to 0.5 ng (bromazepam and chlordiazepoxide) and the LOQs were 0.25 ng in every analyte except for bromazepam and chlordiazepoxide, for which they were 0.5 ng. The developed method was successfully applied to five legal cases involving use of benzodiazepines and zolpidem and to an animal study on drug incorporation into hair. Diazepam and its three metabolites, as well as lorazepam, were detected in hair from both the multiple- and single-dose administration groups of lean Zucker rats. The concentration of diazepam was higher than those of its metabolites in both dark grey and white hair from the multiple-dose administration groups, with the mean concentration ranges from 0.16 to 0.51 ng/mg and from 0.10 to 0.24 ng/mg, respectively. The mean concentration ranges of lorazepam were from 0.05 to 0.37 ng/mg in dark grey hair and from 0.11 to 0.45 ng/mg in white hair from the multiple-dose administration groups. Hair pigmentation did not have any significant effect on the degree of the deposition of drugs and their metabolites in hair.     

基于毛干蛋白质组的族群推断技术的建立与验证

毛干是一种案件现场常见的生物物证,由于核DNA含量极少且高度降解,难以采用现有的短串联重复序列(short tandem repeat,STR)检验方法进行个人识别鉴定,目前仅使用线粒体DNA检验进行母系亲缘关系的判定,利用率非常低.毛干中蛋白质非常稳定,而且具有遗传多态性,表现为基因组中的非同义单核苷酸多态性(non-synonymous single nucleotide polymorphisms,ns SNPs),转录翻译后形成蛋白质序列中的单氨基酸多态性(single amino acid polymorphisms,SAPs).充分利用毛干蛋白质中蕴含的遗传信息,为案件提供线索和证据,是实际公安业务的迫切需求,具有重要的应用价值.本文选取了104份中国汉族的毛干样本进行蛋白质组的检测,共获得了703个SAP位点,位于460个蛋白质上,共推导出552个nsSNP位点.进一步筛选在所有样本中检出率超过15%的位点,获得了88个nsSNP位点,使用毛干样本对应的口腔拭子DNA对88个ns SNP位点进行一代测序验证.为评估发现的nsSNP位点对于人群的区分能力,以千人数据库(1 000 Genome Project)为参考数据库,采用聚类分析和群体匹配概率等方法对检测的19份毛干样本进行人群来源推断.结果显示,通过检测毛干蛋白质组中的ns SNP可以实现东亚、欧洲、非洲三大洲际人群的区分.     

Hair analysis for opiates: evaluation of washing and incubation procedures

Hair analysis of drugs of abuse has been a subject of interest from a clinical, social and forensic perspective for years because of the broad time detection window after intake in comparison to urine or blood. However, the correct and reliable interpretation of opiates findings in an authentic hair sample requires optimalisation and standardisation of decontamination and incubation procedures. Comparing various published methods, we have found some variability in them and no unequivocal recommended procedure for starting with a method directly. Therefore, various combinations of solvents, of various polarity, as washing solvents were tested for removing opiates from the external surface of real hair samples. The yields of opiates from these washings were compared with the yields from the interior of the hair matrix after digestion with various procedures. The opiates after digestion were cleaned up from resulting solution on extraction columns with mixed solid-phase and analysed by GC–MS in standard EI mode after silylation. The efficiencies of neutral (Söerensen buffer, pH 7.4), acid (0.1 M HCl) and basic (1 M NaOH) digestion of the hair matrix were evaluated and the relative recoveries for morphine, codeine, dihydrocodeine and hydrocodone were compared. As it is very problematic to imitate the reference hair sample with a specific amount of analytes incorporated inside, which can be used for calibration to get a close estimate of the quantities of analytes inside the solid authentic sample, the total digestion of a hair sample in basic medium was considered to be a very important reference basis for quantitative determinations. The ratios of hydrolysis of labile 6-acetylmorphine or acetylcodeine were tested and evaluated in practical routine conditions of acid or neutral digestion of hair. Comparing the three methods of incubation of authentic hair samples, the methods using 1 M NaOH or 0.1 M HCl yielded higher recoveries of total equivalents of morphine or codeine, whereas the incubation in Söerensen buffer allowed the reflection of real ratios of labile metabolites and/or parent compounds in an original sample. This method has been shown to be capable of detecting hydrocodone in hair with other opiates concomitantly and to indicate the drug abuse pattern of a person at various time intervals in the past.     

Detection of illegal clenbuterol use in calves using hair analysis: Application in meat quality control

This study describes a real-life situation involving nine calves, 106 days old, which received oral doses of clenbuterol administered through their milk. Powdered skim milk containing 6.7 mg of clenbuterol was given daily for fifteen days under supervision (i.e. 100 mg per calf for the whole study) to seven calves, and two calves did not receive the drug. Hair samples and urine were taken and subjected to analysis by gas chromatography–mass spectrometry. Hairs were pulverized in a ball mill and 100 mg were incubated in a mildly acidic medium. The sample clean-up procedure involved solid-phase extraction on C₁₈ cartridges. Metoprolol was used as the internal standard for quantitation, after formation of methylboronate derivatives. The calibration curve for clenbuterol in hair was linear in the range 20–5000 pg/mg. The limit of detection of clenbuterol was 16 pg/mg in hair and 0.14 ng/ml in urine. Hair testing was effective after 7–10 days of treatment, and concentrations were in the range of 20 to 4372 pg/mg. Urinalysis can detect clenbuterol for up to two weeks after discontinuation of the drug. Conveniently, this is around the time when the hair samples attain greatest sensitivity. Therefore, the combination of the two matrices appears to be the method of choice for testing for the illegal use of drugs in meat-producing animals.     

Determination of thyroxine in the hair of newborns by radioimmunoassay with high-performance liquid chromatographic confirmation

Hair analysis is often used in forensic toxicology to study, retrospectively, chronic exposure of individuals to drugs, and consequently newborn hair may become an ideal sample to study intrauterine exposure to xenobiotics as well as to endogenous compounds. As a tool to investigate a supposed maternal thyroxine (T₄) supply to the congenital hypothyroid fetus, we devised to use the analysis of T₄ extracted from newborn hair. In the present paper, the analytical method based on T₄ extraction from hair followed by a radioimmunoassay is described. To verify the nature of the T₄-like immunoreactive material present in newborn hair, it was further studied by HPLC fractionation with radioimmunoassay of the eluted fractions. On the basis of a clear correspondence between retention times of T₄ standard and T₄-immunoreactive compound extracted from hair, we assigned this immunoreactive material to T₄. Then, we determined T₄ hair concentrations in 19 control newborns at birth and 12 congenital hypothyroid infants at 22 days of life. Values obtained from hypothyroid infants (31.47±8.8 pg/mg_hair, mean±S.D.) were not significantly lower than those obtained from healthy newborns at birth (36.10±13.2 pg/mg_hair). Such results are in agreement with the hypothesis of a maternal supply of thyroxine to the fetus through placental crossing.     

Assessing the potential of a "color effect" for hair analysis of 11-nor-9-carboxy-delta(9)-tetrahydrocannabinol: analysis of a large sample of hair specimens

It has been hypothesized that hair color may play a role in the concentration of various drugs of abuse in hair. Several studies have shown that melanin in hair appears to play a binding role for at least some commonly abused drugs. However, these studies have been limited by a number of factors when assessing the clinical significance of a hypothesized melanin or color effect. This study evaluates the possible effect of hair color on the concentration of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (c-THC) in human hair. The analysis is based on 3886 positive c-THC hair specimens drawn from a universe of approximately 80000 specimens of scalp hair harvested from the posterior vertex of the head. Analysis of variance of color categorization by c-THC concentration shows that c-THC concentration does not have a significant association with hair color (Hair Color F = 1.148, p =.332) and therefore does not have a demonstrable "color effect".     

Hair pulling and eating in captive rhesus monkey troops

Hair pulling and eating has not yet received attention in the nonhuman primate literature. Hair pulling and eating was recorded 388 times in two heterogeneous troops of healthy rhesus monkeys that were kept according to modern management practices. The behavior in question consists of the following sequence: pulling with the fingers (1/3 of cases) or with the teeth (2/3 of cases) tufts of hair from one's own or from a partner's coat; chewing the hair and finally swallowing it; the undigested material is excreted in the feces. Hair pulling was almost exclusively (378/388) partner-directed. It was observed 364 times between animals whose dominance relationships were known; it was performed in 96% (349/364) of observations by a dominant but only in 4% (15/364) of observations by a subordinate monkey. The recipient of hair pulling showed typical fear and/or avoidance reactions. In both troops young animals (2-8 years of age) engaged in hair pulling and eating significantly more often than old animals (10-26 years of age). There was no evidence that nutritional, toxicological or climatic factors were responsible for the manifestation of this behavior. It was concluded that, similar to trichotillomania in man, wool pulling and eating in sheep and muskox, and feather picking in poultry, hair pulling and eating is an aggressive behavioral disorder in rhesus monkeys reflecting adjustment problems to a stressful environment.     

The significance of hair for face recognition

Hair is a feature of the head that frequently changes in different situations. For this reason much research in the area of face perception has employed stimuli without hair. To investigate the effect of the presence of hair we used faces with and without hair in a recognition task. Participants took part in trials in which the state of the hair either remained consistent (Same) or switched between learning and test (Switch). It was found that in the Same trials performance did not differ for stimuli presented with and without hair. This implies that there is sufficient information in the internal features of the face for optimal performance in this task. It was also found that performance in the Switch trials was substantially lower than in the Same trials. This drop in accuracy when the stimuli were switched suggests that faces are represented in a holistic manner and that manipulation of the hair causes disruption to this, with implications for the interpretation of some previous studies.     

Seasonal differences in hair growth rates of captive aye-aye,red ruffed,and black-and-white ruffed lemurs

Hair can be an important source of biological information, providing a record of such things as pollutant exposure, hormonal levels, and stable isotope ratios. Hair as a biological sample is easily accessible, easily stored, and resists degradation. Analysis of hair is particularly useful when studying rare and endangered species, such as lemurs, since it can be sampled noninvasively. However, to better interpret the results of stable isotope or other analyses, it is important to understand hair growth rates. We measured hair growth rates in captive aye-ayes (Daubentonia madagascariensis), red ruffed lemurs (Varecia rubra), and black-and-white ruffed lemurs (Varecia variegata), both in winter (December 23, 2013, to January 13, 2014) and summer (July 10–31, 2013) at the Duke Lemur Center. Hair growth per week in all three species of lemurs differed significantly between the boreal summer and boreal winter. The aye-aye, black-and-white ruffed lemur, red ruffed lemur had a mean weekly hair growth of 0.195, 0.209, and 0.232 cm, respectively, in the summer. While the aye-aye, black-and-white ruffed lemur, and red ruffed lemur had a mean weekly hair growth of 0.239, 0.464, and 0.479 cm, respectively, in winter. We found no effect of age on hair growth rates, neither in the boreal summer, nor in the boreal winter for black-and-white ruffed lemurs and red ruffed lemurs. A pregnant black-and-white ruffed female displayed negligible hair growth during the northern winter, suggesting that pregnancy may affect the partitioning of resources away from such things as hair growth.     

Age-based differences in hair zinc of Vancouver preschoolers

Marginal zinc deficiency (MZD), the subclinical stage of zinc deficiency, is common in industrialized societies. Serum zinc, the most common biomarker of zinc status, lacks sensitivity and specificity to diagnose this deficiency. Hair zinc, however, is sensitive and specific enough to detect MZD in children. Differences in hair zinc associated with age and sex have been reported. These differences have not been investigated thoroughly; therefore, interpretation of the results of hair analyses is difficult. This cross-sectional study was designed to examine the hair zinc status of a group of Vancouver preschoolers (24-71 months) and assess the age- and sex-based differences in their hair zinc. Hair samples were obtained (n = 719) and analyzed for zinc using inductively coupled plasma mass spectrometry. Our results indicated a mean hair zinc of 115 +/- 43 microg/g with 17% below the low hair zinc cutoff (70 microg/g). Boys and girls had comparable mean hair zinc, while girls had a significantly higher occurrence of low hair zinc than boys (21% vs. 12%). Children <4 years of age had significantly lower mean hair zinc and higher rate of low hair zinc compared to children > or =4. Our study provides important reference values for the hair zinc of healthy North American preschoolers.     

Cross-Sectional Relationship Between Chronic Stress and Mineral Concentrations in Hair of Elementary School Girls

Chronic stress exposure is associated with diverse negative health outcomes. It has been hypothesised that stress may also negatively affect the body's mineral status. This study investigates the association between chronic stress and long-term mineral concentrations of calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), phosphorus (P) and zinc (Zn) in scalp hair among elementary school girls. Complete information on child-reported stress estimates (Coddington Life Events Scale (CLES)), hair cortisone and hair mineral concentrations, and predefined confounders in the stress–mineral relationship (i.e. age, body mass index, physical activity, diet, hair colour and parental education) was provided cross-sectionally for 140 girls (5–10 years old). The relationship between childhood stress measures (predictor) and hair minerals (outcome) was studied using linear regression analysis, adjusted for the abovementioned confounders. Hair cortisone concentrations were inversely associated with hair mineral concentrations of Ca, Mg, Zn and the Ca/P ratio. Children at risk by life events (CLES) presented an elevated Ca/Mg ratio. These findings were persistent after adjustment for confounders. This study demonstrated an independent association between chronic stress measures and hair mineral levels in young girls, indicating the importance of physiological stress–mineral pathways independently from individual or behavioural factors. Findings need to be confirmed in a more heterogeneous population and on longitudinal basis. The precise mechanisms by which stress alters hair mineral levels should be further elucidated.     

Hair follicular cell/organ culture in tissue engineering and regenerative medicine

Hair follicles are complex organs composed of the dermal papilla (DP), dermal sheath (DS), outer root sheath (ORS), inner root sheath (IRS) and hair shaft. Development of hair follicles begins towards the end of the first trimester of pregnancy and is controlled by epidermal–mesenchymal interaction (EMI), which is a signaling cascade between epidermal and mesenchymal cell populations. Hair grows in cycles of various phases. Specifically, anagen is the growth phase, catagen is the involuting or regressing phase and telogen is the resting or quiescent phase. Alopecia is not life threatening, but alopecia often causes severe mental stress. In addition, the number of individuals afflicted by alopecia patients has been increasing steadily. Currently there are two methods employed to treat alopecia, drug or natural substance therapy and human hair transplantation. Although drug or natural substance therapy may retard the progress of alopecia or prevent future hair loss, it may also accelerate hair loss when the medication is stopped after prolonged use. Conversely, the transplantation of human hair involves taking plugs of natural hair from areas in which occipital hair is growing and transplanting them to bald areas. However, the number of hairs that can be transplanted is limited in that only three such operations can generally be performed. To overcome such problems, many researchers have attempted to revive hair follicles by culturing hair follicle cells or mesenchymal cells in vitro and then implanting them in the treatment area.