Limitations of human occipital scalp hair follicle organ culture for studying the effects of minoxidil as a hair growth enhancer

Minoxidil induces new hair growth in approximately one-third of patients with androgenetic alopecia after 1 year of treatment. With several conflicting reports in the literature based on small-scale studies, the current study aimed to clarify whether organ culture of human scalp anagen VI hair follicles is a suitable in vitro test system for reproducing, and experimentally dissecting, the recognized in vivo hair-growth-promoting capacity of minoxidil. Hair shaft elongation was studied in terminal anagen VI hair follicles microdissected from the occipital scalp of 36 healthy adults. A total of 2300 hair follicles, approximately 65 per individual, were tested using modifications of a basic organ culture protocol. It is shown here that minoxidil does not significantly increase hair shaft elongation or the duration of anagen VI in ex vivo culture despite several enhancements on the conventional methodology. This disparity to what is seen clinically in minoxidil responders may be explained by the following: (i) use of occipital (rather than frontotemporal or vertex) hair follicles; (ii) use of, already maximally growing, anagen VI hair follicles; (iii) a predominance of hair follicles from minoxidil unresponsive-donors; (iv) use of minoxidil rather than its sulfate metabolite; and/or (v) use of a suboptimal minoxidil dosage. This disparity questions the usefulness of standard human hair follicle organ culture in minoxidil research. Unexpectedly, minoxidil even inhibited hair shaft elongation in the absence of insulin, which may indicate that the actual hair-growth-modulatory effects of minoxidil depend on the concomitant local presence/absence of other growth modulators.     

In vivo induction of hair growth by dermal cells isolated from hair follicles after extended organ culture

Successful hair follicle organ culture has been established for some time, but hair growth in vitro is limited and generally terminates prematurely in comparison with in vivo. The reasons why growth stops in culture are as yet unknown. In this investigation, adult rat vibrissa follicles for which growth in culture is limited to about 10 d, were maintained in vitro for a minimum of 20 d after the hair shaft stopped growing. The pattern of fiber growth and long-term follicle pathology reflected the initial hair cycle stage at the time of isolation. Furthermore, there was evidence that a group of follicles put into culture when in late anagen were attempting to cycle in vitro. Microscopy showed that, in spite of widespread pathologic changes to the follicle epithelium, dermal cells in the follicle showed remarkable resilience. Their viability was confirmed when primary cell cultures were established from isolated dermal tissue. These cells labeled positively for alpha-smooth muscle actin, an established marker of hair follicle dermal cell phenotype in vitro. Moreover, isolated dermal tissue induced hair growth when implanted into inactivated hair follicles in vivo. These data confirm that the cessation in hair growth is not due to a loss of the inductive capacity in the dermal component. Long-term organ culture may provide opportunities to investigate factors that are expressed or lost during hair growth cessation. In addition it may be possible to develop this method further to obtain a reliable and predictable model of hair follicle cycling in vitro.     

T-oligos as differential modulators of human scalp hair growth and pigmentation: a new “time lapse system” for studying human skin and hair follicle biology in vitro?

Small DNA oligonucleotides homologous to the 3′ overhang of human telomeres, called T-oligos, stimulate pigmentation in human epidermal melanocytes in vitro and in vivo. They induce UV-mimetic effects in the absence of DNA-damage, however, it is unknown how T-oligos affect human hair follicle keratinocyte and melanocyte functions in situ. Here, we present the first evidence that these oligonucleotides are powerful modulators of pigmentation and growth of microdissected, organ-cultured human scalp hair follicles. Hair follicles were incubated with T-oligo or vehicle control and were then assessed for changes in hair shaft length, follicle morphology, pigmentation, proliferation and apoptosis. After only 48 h, T-oligos induced a fourfold increase in pigmentation of human anagen VI hair bulbs, while hair matrix keratinocyte proliferation was reduced by 65%, without apparent changes in hair bulb cell apoptosis. This corresponded well with a significant inhibition of hair shaft elongation, which was not accompanied by premature catagen induction in anagen VI hair follicles. These diametrically opposed effects of T-oligos on human hair follicle melanocytes (stimulation of melanogenesis) versus human hair bulb keratinocytes (inhibition of proliferation) in situ illustrate that human hair follicle organ culture offers an excellent tool for T-oligo research. They suggest that T-oligos deserve to be further explored for the management of clinical hair growth and pigmentation disorders, and raise the possibility that this model may offer a unique “time lapse system” for studying skin and hair follicle biology and DNA repair strategies under physiologically relevant conditions.     

Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture

Please cite this paper as: Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Experimental Dermatology 2010; 19: 305–312. Abstract: The organ culture of human scalp hair follicles (HFs) is the best currently available assay for hair research in the human system. In order to determine the hair growth‐modulatory effects of agents in this assay, one critical read‐out parameter is the assessment of whether the test agent has prolonged anagen duration or induced catagen in vitro. However, objective criteria to distinguish between anagen VI HFs and early catagen in human HF organ culture, two hair cycle stages with a deceptively similar morphology, remain to be established. Here, we develop, document and test an objective classification system that allows to distinguish between anagen VI and early catagen in organ‐cultured human HFs, using both qualitative and quantitative parameters that can be generated by light microscopy or immunofluorescence. Seven qualitative classification criteria are defined that are based on assessing the morphology of the hair matrix, the dermal papilla and the distribution of pigmentary markers (melanin, gp100). These are complemented by ten quantitative parameters. We have tested this classification system by employing the clinically used topical hair growth inhibitor, eflornithine, and show that eflornithine indeed produces the expected premature catagen induction, as identified by the novel classification criteria reported here. Therefore, this classification system offers a standardized, objective and reproducible new experimental method to reliably distinguish between human anagen VI and early catagen HFs in organ culture.     

培养毛乳头细胞生物学特性和毛囊重建的研究

目的 观察毛乳头细胞在体内外诱导毛囊再生和支持毛囊生长情况。方法 采用免疫组化、原位杂交、毛囊器官型培养和裸鼠移植技术，观察不同传代培养的毛乳头细胞碱性成纤维细胞生长因子，内皮素和干细胞因子的表达变化情况。结果 低传代培养的毛乳头细胞的内皮素和干细胞因子表达较强，传代6代后减弱。用低传代培养的毛乳头细胞与毛囊上皮细胞在毛囊器官型培养模型中可见毛囊样结构形成，移植到裸鼠后可见较为完整的毛囊形成。用低传代培养的毛乳头细胞与毛囊上皮细胞按一定比例混合后直接注射到裸鼠皮下，也可见毛囊样结构形成。发现毛乳头细胞诱导毛囊再生的能力与其表达内皮素和干细胞因子的强弱相关。结论 低传代培养的毛乳头细胞在体内外均具有诱导毛囊再生的能力，并且与其表达内皮素和干细胞因子的强弱相关。     

何首乌、女贞子等中药煎剂对体外培养的猪毛囊毛发生长的影响

目的 探讨何首乌、女贞子等中药混合煎剂对体外培养的猪毛囊毛发生长的影响。方法 将离体培养的猪毛囊分为对照组(Williams E培养基)和中药组(Williams E培养基+中药煎剂),显微镜下观察各组毛囊毛发生长和毛球部形态变化,并用末端脱氧核苷酸转移酶介导dUTP缺口末端标记法(TUNEL)检测各组毛囊中的凋亡细胞。结果 培养第7天,对照组毛囊毛发生长长度低于中药组(P<0.001);对照组毛囊TUNEL阳性细胞数明显高于中药组(P<0.001);毛球形态观察,对照组毛囊出现退行性变化,而中药组毛囊仍维持生长期毛囊形态。结论 何首乌、女贞子等中药煎剂能在一定程度上抑制猪毛囊细胞内凋亡,延缓生长期毛囊进入退行期。     

血管生成素在人毛囊中的表达及其对毛发生长的影响

目的 探讨血管生成素在人毛囊中的表达及其意义。方法 分离完整的人生长期毛囊,提取总RNA,RT-PCR法检测血管生成素mRNA的表达,同时应用免疫组化法检测血管生成素蛋白在人毛囊中的表达。在此基础上,在体外培养的人毛囊中加入不同浓度的重组人血管生成素（0 ～ 200 ng/mL）,培养6 d后测量毛囊的生长长度。利用两步酶法分离培养人毛乳头细胞,加入不同浓度的重组人血管生成素（0 ～ 200 ng/mL）,48 h后,MTT法检测细胞增殖,流式细胞仪检测细胞周期。结果 RT-PCR显示人毛囊表达血管生成素mRNA,免疫组化法发现人毛囊的毛乳头和真皮鞘表达血管生成素蛋白。25 ～ 200 ng/mL重组人血管生成素呈浓度依赖性促进体外培养的人毛囊生长（P < 0.05）,而12.5 ～ 200 ng/mL重组人血管生成素能够明显促进体外培养的人毛乳头细胞增殖（P < 0.05）。流式细胞仪检测12.5 ～ 200 ng/mL重组人血管生成素能够显著增加S期细胞比率和细胞增殖指数（P < 0.05）。结论 血管生成素可能是一种促进毛发生长的因子。     

Leptin of dermal adipose tissue is differentially expressed during the hair cycle and contributes to adipocyte‐mediated growth inhibition of anagen‐phase vibrissa hair

Adipose tissue encircles the lower portion of anagen hair follicles and may regulate hair cycle progression. As leptin is a major adipokine, its level of expression from the dermal white adipose tissue during hair cycle progression was studied. The result shows that leptin level is differentially expressed during hair cycle, the lowest in early anagen phase, upregulated in late anagen phase and the highest in the telogen phase. On the other hand, leptin receptor is detected in keratin 15‐positive hair bulge epithelium of both anagen‐ and telogen‐phase hair follicles of mice pelage and vibrissa hair, and hair from human scalp. Leptin contributes to adipocyte‐mediated growth inhibition of anagen‐phase vibrissa hair as demonstrated in organ culture and coculture system. Our data suggest that leptin of dermal white adipose tissue might regulate hair growth and, therefore, hair cycle progression via leptin receptor on the hair follicle epithelium.     

The epidermal pentapeptide pyroGlu-Glu-Asp-Ser-GlyOH inhibits murine hair growth in vivo and in vitro.

Tissue growth may be controlled by negative feedback mechanisms. Recently, a pentapeptide, pyroGlu-Glu-Asp-Ser-GlyOH ('epidermal pentapeptide', EPP), which slows the growth of mouse epidermis in vivo and of mouse keratinocytes in vitro, was isolated from mouse epidermis. Since inhibitory molecules like EPP might be part of the feedback systems underlying hair growth control, we assessed the effect of synthesized EPP on the growth of hair follicles, using rodent in vivo and in vitro assays. We report for the first time that intraperitoneally injected EPP (30 nmol/animal/day over 6 days) significantly slows the growth of hair follicles in plucking-induced anagen skin of C57 B1-6 mice (as assessed by microscopic morphometry). Using an in vitro organ culture assay, EPP inhibits the incorporation of 3H-thymidine into mouse pelage anagen follicles. That this epidermal-derived peptide affects hair growth raises the possibility that hair growth may be regulated by an inhibition/disinhibition mechanism under participation of EPP-like molecules and that the epidermis may play a role in the control of hair growth.     

Alopecia areata: alterations in the hair growth cycle and correlation with the follicular pathology

A histopathological study was performed in 17 patients with alopecia areata to elucidate the changes in hair cycle dynamics. The findings confirm the view that the initial event in alopecia areata is the premature entry of anagen follicles into telogen, although some follicles survive for a time in a dystrophic anagen state. However, after re-entry into anagen takes place, growth appears to be halted in anagen III rather than anagen IV, as has previously been suggested. Follicles then return prematurely to telogen and these truncated cycles are repeated until the disease activity subsides. A new pathogenic hypothesis is presented which relates alterations in hair cycle dynamics to pathological changes within the anagen follicle and also provides an explanation for the formation of exclamation mark hairs and the non-destructive nature of the disease.     

小鼠触须毛囊体外培养的研究

目的 为了建立较理想的小鼠触须毛囊体外培养模型,对不鼠触须毛囊体外培养方法进行了研究。方法 通过小鼠触须毛囊体外无血清培养基的方法,对不同鼠龄及不同生长周期的毛囊进行了研究。结果 ①毛囊在Ｗｉｌｌｉａｍｓ－Ｅ加Ｌ－谷酰胺培养基上生长平均达８天;②通过对比不同阶段的生长期毛囊发现生长期－Ⅱ毛囊生长力明显优于其它各期毛囊;③对不同鼠龄毛囊体外增培养生长状况的研究发现出生３５天和６５天鼠优于出生７天乳鼠     

毛囊混合细胞在胶原/壳聚糖多孔支架上重建毛囊样结构

目的探讨利用毛囊混合细胞植入胶原／壳聚糖多孔支架内体外重建毛囊的可行性。方法用滴加法或注射法将体外培养的C57BL／6J近交系乳鼠背部皮肤毛囊混合细胞以不同传代数、不同细胞密度接种至胶原／壳聚糖多孔支架，倒置显微镜下观察支架表面或浅层的细胞生长或毛囊形成情况。将支架经10％甲醛固定后行组织学观察（H-E染色）。另用共聚焦激光扫描显微镜观察支架内活细胞的生长以及毛囊样结构的形成。结果在一定传代次数和细胞密度下，在支架内可形成具有毛干的毛囊样结构。激光共聚焦扫描显微镜发现团块内细胞排列呈同心圆状，整个三维结构似一长颈花瓶，且该结构仅见于注射法接种细胞的支架内。结论毛囊混合细胞植入胶原／壳聚糖多孔支架内体外可形成具有毛干的毛囊样结构。     

毛囊混合细胞植入胶原/壳聚糖多孔支架诱导裸鼠毛发生长及支架内形成血管样结构

目的 探讨利用毛囊混合细胞植入胶原,壳聚糖多孔支架内重建毛囊的可行性与支架内血管形成状态.方法 用注射法将体外培养的C57BL/6J近交系乳鼠背部皮肤毛囊混合细胞接种至胶原,壳聚糖多孔支架,培养2周后,将含毛囊混合细胞胶原/壳聚糖多孔支架植入裸鼠皮下,肉眼观察裸鼠背部毛发形成情况.6周后取移植区皮肤组织经10%甲醛固定后行组织学观察(HE染色).结果 移植至裸鼠皮下5周后,裸鼠背部在含毛囊混合细胞的胶原,壳聚糖多孔支架移植区皮肤出现毛发,丰长,6周后取皮肤组织作HE染色发现有分化成熟的毛囊形成,且支架内有血管样结构形成.而空白胶原/壳聚糖多孔支架移植区皮肤未发现毛生长,组织检杳也未发现有毛囊形成,且只有在支架表浅部位有少黑血管样结构形成.结论 毛囊混合细胞植入胶原/壳聚糖多孔支架内可诱导裸鼠毛发的形成,促进支架血管样结构的形成.     

Morphological analysis of in vitro human hair growth

The histological and ultrastructural aspect of normal human hair follicles maintained ex vivo for 12 days was evaluated. Anagen hair follicles, dissected free of contaminating connective tissue, were maintained for up to 12 days in a serum-free medium. Macroscopic observations revealed continued viability for 12 days, at which time some follicles involuted in a manner morphologically similar to catagen. Increased growth of maintained follicles was measured from the abrupt ending of the connective tissue sheath (CTS), as no increase in this component was observed from initiation of culture. In general follicles maintained up to 8 days exhibited little divergence from normal in vivo morphologies including the persistence of functional hair bulb melanocytes — a marker of anagen. After this time melanin granules were present in dermal papilla cells, as occurs during impending involution in vivo. Heterotypic cell contact occurred in the middle to upper follicle between outer root sheath (ORS) keratinocytes and disorganized CTS. Herniation of some ORS cells away from the follicle and the occurrence of loose desmosomal junctions between ORS keratinocytes reflected loss of normal follicular cell interactions in upper follicles maintained after 8 days. Continued follicle growth correlated with the presence of mitotic matrix keratinocytes even at 12 days. After 12 days in culture most follicles involuted displaying apoptotic-like keratinocytes and hair bulb melanocytes and the presence of highly keratinized hair club structures. While most follicles exhibited this orderly sequence of events, a few follicles involuted after 24 h with synchronous degeneration of all cells. Two follicles exhibited upregulated cortical cell differentiation at the level of the dermal papilla (DP). Normal cell cytoplasmic constituents were replaced with ribosomes and keratin bundles. This study reveals for the first time the histology and ultrastructure of normal hair follicles in culture for up to 12 days in serum-free medium. Although involuted hair follicles may exhibit some features of catagen, it is possible that the mechanisms involved are entirely different.This study was supported in part by a grant to D. J. Tobin from Upjohn (UK)     

不同年龄成人头发的变化

目的 观察不同年龄成人头发的变化,探讨反映人老化的参照指标.方法 根据入选标准和排除标准征集志愿者,并依据年龄分为4组.头顶指定区域拍照后利用图像分析软件检测头发密度以及黑发率.该区域内随机拔取10根头发,利用目镜测微尺测量发干直径和毛球直径.同时,根据毛囊形态计算不同生长时期毛囊率.结果 共有96名志愿者加入本研究,男44例,女52例,年龄30~78岁.头顶头发密度、毛干直径、毛球直径、生长期毛囊率和黑发率随着年龄的增加逐渐减小,休止期毛囊率及白发率增多,并且60岁以上组明显减小,与其他3个年龄组差异有统计学意义,各组内部分指标存在性别差异.相关分析发现,毛干直径与毛球直径呈显著正相关.结论 头发的一些变化可能作为评价人老化程度的参照指标.     

Cyclical changes in rat vibrissa follicles maintained In vitro

In mammals hair growth is cyclical; however, the factors that regulate the hair growth cycle are still poorly understood. The recent development of methods for culturing hair follicles in vitro has proved an important tool to investigate many aspects of the regulation of hair follicle growth. At present, however, these models are based on the culture of anagen hair follicles and have only partially been used to address the cyclical nature of hair growth. In this study we have made use of the fact that in rodents the hair growth cycle is synchronized, well characterized, and relatively short. We have isolated vibrissa follicles from 12 d old rats and confirmed by histology that these follicles are in the anagen stage of their first hair growth cycle. We have then maintained these follicles in vitro, on Gelfoam supports, for up to 23 d (35 d of age) and compared their histology with in vivo follicles from equivalent age littermates. We observed that 12 d old follicles maintained in vitro for up to 23 d show changes in morphology that suggest that cultured rat vibrissa follicles retain cyclical activity in vitro. Cyclical changes in hair follicle morphology were only seen in follicles maintained on gelfoam supports and moreover, hair follicle size appears to be a key feature in determining the ability of the follicle to cycle in vitro. All follicles that showed cyclical changes in vitro, however, appeared to remain blocked in pro-anagen. These data suggest that the vibrissa follicle is a in vitro good model system with which to investigate hair cycle control. J Invest Dermatol 115:1152-1155 2000     

The vellus hair follicle in acne: hair growth and sebum excretion

Summary In this study we investigated the activity of the vellus hair follicle in acne. Hair growth and sebum excretion in vellus hair follicles were measured on the forehead and back of men, and on the forehead, cheek, and back of women with acne. Hair growth was assessed by computerized image analysis (phototrichogram), and sebum excretion by computer analysis using Sebutape®.
In patients with acne, marked differences were revealed when results were compared with recent data from healthy persons. In particular, the mean growth rate of vellus hairs was higher, whereas the percentage of anagen hairs was lower, and the duration of the anagen phase shorter in patients with acne than in healthy individuals. Hair growth and sebum excretion depended significantly ( P < 0.01) on the anatomical site (forehead 414 hairs/cm², 0.053 mm/day, 34%; back 93 hairs/cm², 0.16 mm/day. 21%),
In addition, analysis of hair growth revealed significantly higher values in females than in males for (i) percentage of anagen hairs ( P >0.01), (ii) for vellus hair length ( P <0.05), and (iii) for the duration of the anagen phase ( P <0.01).
The present study demonstrates that the activity of the vellus hair follicle is influenced by acne, and vice versa, and therefore its role in the aetiopathogenesis of acne should be reconsidered.     

Telogen skin contains an inhibitor of hair growth

We have investigated whether C57B1-6 mouse skin with all its follicles in the telogen stage of the hair cycle contains a hair-growth inhibitory activity, as opposed to skin with anagen follicles. Crude aqueous extracts of whole telogen mouse skin (TE), anagen skin (AE) or vehicle alone (V) were injected intraperitoneally into mice in which anagen had previously been induced by plucking of telogen hair follicles. Injection of TE, but not AE or V, significantly retarded the development of anagen follicles, as measured by macroscopic and quantitative microscopic hair growth parameters (skin pigmentation and thickness, appearance of trichohyaline granules) and the incorporation of tritiated thymidine into mouse skin from animals previously treated with either TE or V (skin organ culture). This inhibitory activity seemed to be localized to the epidermis and was also present in rat epidermis. We suggest that this apparently non-species-specific inhibitor present in telogen skin may play a role in regulating the hair cycle in rodents.     

Identification of clustered cells in human hair follicle responsible for MMP-9 gelatinolytic activity: consequences for the regulation of hair growth

BACKGROUND: The control of human hair follicle growth and differentiation is dependent upon several well-identified factors, including androgens, cytokines, and growth factors. In humans, alopecia androgenetica is a common aging process thought to be regulated through complex genetic imbalances, which also involve several of these crucial identified factors (and probably others not yet characterized), alone or in combination. Among these factors, epidermal growth factor (EGF), as well as pro-inflammatory cytokines, play a pivotal role, as evidenced by their direct inhibitory effects on hair growth both in vitro and in vivo. Following such treatments, the in vitro growth of hair follicles was rapidly arrested and deleterious modifications of hair morphology were also observed. AIM: Because these cytokines act, at least partly, through the induction of matrix metalloproteinases (MMP), and because tissue remodeling occurs during the hair cycle, we attempted to identify and localize MMP in the human pilosebaceous unit. METHOD: We used zymography to observe human hair follicles in culture in vitro. RESULTS: We observed that human hair follicles in culture in vitro mainly and almost exclusively produce MMP-2 and MMP-9 gelatinolytic activities. Furthermore, after stimulation with EGF, tumor necrosis factor-alpha (TNF-alpha), or interleukin-1alpha (IL-1alpha), MMP-9 production was strongly increased. Using immunohistochemistry, we then precisely localized MMP-9 in the lower part of the inner root sheath (Henle's layer) of control human anagen hair follicles. CONCLUSIONS: Cytokine- and EGF-induced upregulation of MMP-9 in the lower epithelial compartment of the human hair bulb is a major mechanism through which hair follicle involution, observed in alopecia, may occur.