|Year : 2015 | Volume
| Issue : 1 | Page : 16-23
Hair follicle plasticity with complemented immune-modulation following follicular unit extraction
Reza P Azar1, Alexander H Thomas2, Gerd Lindner2
1 Zentrum für Moderne Haartransplantation/Centre for Modern Hair Transplantation, Berlin, Germany
2 University for Technologies Berlin, Institute of Biotechnology, Berlin, Germany
|Date of Web Publication||18-Mar-2015|
Technische Universitšt Berlin, Institute of Biotechnology, Berlin
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: During hair transplantation as an effective therapy for androgenetic alopecia, hair follicles were typically trans-located from the nonaffected occipital to the balding frontal or vertex region of the scalp. Although this is an autologous intervention, the donor and recipient hair follicle tissue differ in composition and local environment. Settings and Design: In two case studies, we investigated the changes in hair follicle morphology and the immune status of scalp and body hair follicles from different origins transplanted to the eyebrows and the frontal scalp using follicular unit extraction. Results: Quantitative histomorphometry and immunohistochemistry revealed a transformation in hair follicle length and dermal papilla size of the scalp, chest and beard hair follicles, which had been re-extracted after a 6-month period posttransplantation. Furthermore, a significant infiltration of B and T lymphocytes as well as macrophages could be observed most prominently in the infundibulum of transplanted hair follicles. Conclusion: The presented results demonstrate that hair follicle units from different body sites are capable to replace miniaturized or degraded hair follicles in different recipient areas like scalp or eyebrows as they keep their intrinsic capability or acquire the potential to readjust plastically within the beneficiary skin region. The essential secretory crosstalk underlying the observed tissue remodeling is possibly mediated by the infiltrating immune cells.
Keywords: Dermal papilla, hair follicle plasticity, hair transplantation, intermittent follicular unit extraction
|How to cite this article:|
Azar RP, Thomas AH, Lindner G. Hair follicle plasticity with complemented immune-modulation following follicular unit extraction
. Int J Trichol 2015;7:16-23
|How to cite this URL:|
Azar RP, Thomas AH, Lindner G. Hair follicle plasticity with complemented immune-modulation following follicular unit extraction
. Int J Trichol [serial online] 2015 [cited 2019 Jan 22];7:16-23. Available from: http://www.ijtrichology.com/text.asp?2015/7/1/16/153451
| Introduction|| |
Follicular unit extraction (FUE) is evolving as a common practice in hair transplantation surgery. ,, It allows for trans-locating single hair follicle units from one body site to another employing a micro-sized hollow needle. This includes for example hair transplantations from the occipital to the frontal region of the scalp but also from other body areas to the scalp or facial skin.  Although the basic morphology of hair follicles constitute similar cell and matrix components, the size, shape and distribution of hair follicles differ by origin.  Besides different phases of growth and the influence of hormones, beard hair follicles, for example, comprise a broader follicular bulb harboring a larger dermal papilla (DP) compared with scalp hair follicles. , In contrast, body hair from chest is shorter and has a less voluminous DP. This divergence in cellular quantities causes the different thickness of hair emerging from the skin surface. ,, Common practice in hair transplantation surgery, therefore, is to preferably transplant hair follicles of similar morphology at donor and recipient site to achieve cosmetically acceptable results. On the contrary, we here report in a study on two different cases of FUE surgeries, showing the ability of the donor hair follicle to reshape its morphology and iteratively converge to the recipient tissue environment within a period of 6 months after transplantation. One patient underwent chest, beard and scalp to scalp hair transplantation, and the other patient received a beard to eyebrow transplantation. Half a year later, the selected transplanted hair follicles were extracted again, characterized and compared with originating hair follicles. Quantitative histomorphometry and immunohistochemistry were utilized to investigate changes in hair follicle morphology and the immune status.
| Subjects and methods|| |
An informed, volunteering patient (male, 36 years old, Norwood-Hamilton Scale Va) , received FUE surgery  with short intermittent extraction and implantation of hair follicle units (IFUE) after giving his written consent. The donor area was shaved and disinfected with Kodan® tincture forte colorless (Schülke and Mayr, Germany). After local anesthesia with 0.25% bupivacaine containing 1:100,000 Adrenalin (both Jenapharm, Germany), hair follicles from chest, beard and occipital scalp were extracted using a 0.95 mm punch. These follicular units were transplanted into the frontal region of the scalp [Figure 1]b]. Six months postsurgery, five to six transplanted, as well as native chest, beard and occipital scalp follicular units were sampled and fixed in 4% formalin (Roti® -Histofix, Roth, Germany). Native beard hair follicles from the chin along with beard hair follicles that had been transplanted into the eyebrows 6 months before [Figure 1]c] were obtained by IFUE from a second patient (male, 24 year old) after receiving his written consent. Six to 13 native and transplanted follicles each were extracted by a 0.9 mm punch and were transferred to a 4% formalin solution for fixation.
|Figure 1: Recipient area of transplanted body, beard and scalp hair follicles. Beard, chest and scalp hair follicles were transplanted into defined areas of the frontal region of the scalp (a and b). (a) Is showing the occipital view of the scalp before and (b) after follicular unit extraction transplantation of differentially originated hair follicle units. Recipient sites for autologous beard (red), chest (white) and scalp (yellow) hair follicle units are highlighted, respectively. (c) Photograph of patient two who received beard hair follicle transplantation. After 6 months, selected hair follicles were extracted from defined sites of transplantation area for further investigations|
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The formalin-fixed samples from both patients were processed according to a standard paraffin-embedding protocol described elsewhere  and subsequently sectioned by a microtome to yield 3 μm thick tissue slices. The follicular units were sectioned longitudinally, and medial cuts of each sample were mounted on multiple glass slides to apply different staining techniques with 3-5 consecutive sections per glass slide.
To compare native and transplanted hair follicles from both patients, different staining protocols were applied to the sections. After deparaffinization, Hematoxylin and Eosin (H and E) (Thermo Fisher Scientific Inc., USA and Avantor Performance Materials Inc., USA, respectively) as well as immunohistological staining [Table 1] was performed. Primary antibody-bound antigens were visualized enzymatically with horseradish peroxidase-conjugated antibodies. 3-amino-9-ethylcarbazole was used as substrate and the incubation time uniformly was 20 min. H and E stained sections were embedded in Roti® -Histokitt (Roth, Germany) and IHC sections in Aquatex® (Merck Millipore, USA).
A digital microscope (BZ-9000, Keyence, Japan) and the associated software (BZ Analyzer, Keyence, Japan) were used to photographically document and examine all sections. Micro-photographs were taken from every intact section whereof preferably most central cuts were used for analysis. Multiple high magnification (×20) images were shot from each section and merged by the BZ Analyzer software to obtain a detailed overview on every hair follicular unit.
The evaluation of native and transplanted hair follicles was identical for samples from both patients and included the measurement of morphological parameters as well as the quantification of the immunological staining. The DP size, expressed as the absolute area of its most central cross-section, and the follicle length, measured from the bottom of the dermal cup to the emergence of the hair shaft from the epidermis, were the morphological parameters of interest. Intact sections of every staining regimen were measured for their DP size and follicle length to yield a maximal number of technical replicates.
On the contrary, the number of replicates for all immunological staining was limited to the maximum number of follicle sections per glass slide and staining. To quantify the occurrence of immune cells in the sections by anti-CD3, anti-CD20 and anti-CD68 staining, the absolute area of positive regions was measured.
The arithmetic mean was calculated from the technical replicates acquired from the image analyses to yield one value per follicle. GraphPad Prism (GraphPad Software, Inc. La Jolla, USA) was used to carry out statistics and data visualization. Data are expressed as mean ± standard deviation. One-way ANOVA in combination with Tukey's multiple comparison test was performed on the data from patient one to compare the different groups of native and grafted hair follicles. Student's t-test was carried out with the data sets from patient two to compare native beard hair follicles to beard hair follicles grafted into the eyebrows. P < 0.05 were considered as significant.
| Results|| |
No adverse effects were seen during and after FUE transplantation procedures. Complete and viable hair follicle units were obtained and transplanted using IFUE technique to ensure a maximum survival rate of the follicular units [Figure 1]. Here, approximately 100 follicular units were extracted and stored in saline to prevent dehydration. A maximum period of 30 min in storage solution guaranteed highest follicular unit viability before transplantation into preformed recipient sites. Visual inspection of the transplanted hair follicles and the adjacent skin surface 6 months posttransplantation could not detect any signs of inflammation or differences in the appearance regarding size, shape and color of transplanted hair follicles within the recipient area compared to native hair follicles of the same growth phase.
The follicle length of native beard, chest and scalp hair follicles from both patients generally was ranging from 3900 μm to 4300 μm. However, histological analysis of sectioned follicles was pointing out differences in length between nontransplanted and transplanted hair follicles. In samples from patient one, beard and scalp hair follicles were significantly shorter than their nongrafted controls [Figure 2]a and b]. The gathered histological data from patient two was consistent as beard follicles that had been grafted into the eyebrows were also significantly reduced in length compared with native beard hair follicle controls [Figure 2]c]. In general, hair follicles grafted by FUE showed a significant reduction in length after their 6-month residence in the recipient site. Most surprisingly, hair follicles transplanted from the occipital to the frontal region showed the most prominent reduction in length [Figure 2]b].
|Figure 2: Native beard, chest and scalp hair follicles have different morphological features that are characteristic for the body site they are derived from. The morphology of grafted hair follicles is changing independently on the follicles' former site of origin. (a) Photomicrographs showing the comparison of the measured follicle lengths of native beard, chest and scalp hair follicles. Dashed arrows demarcates length|
determination. (b and c) Quantification of the measured follicle length of native and transplanted hair follicles from different body sites to the scalp (b) and eyebrows (c). (d) Representative microscopic images showing hair bulbs of native beard, chest and scalp hair follicles with analyzed DP cross‑section area highlighted by dashed lines. (e and f) DP sizes of native and transplanted hair follicles from different body sites to the scalp (e) and eyebrows (f) as determined by measuring the maximum area of longitudinal DP cross‑sections. Scale bars indicate 100 μm (a) or 200 μm (d), respectively. DP: Dermal papilla
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The assessment of the maximum DP cross-section area as the main determinant for its size was performed on most medial sections. Among the samples from patient one, the DP size of native beard follicles was found to be significantly bigger than that of grafted beard hair follicles or of native chest and scalp hair follicles [Figure 2]d and e]. Beard follicles generally appeared more massive with thicker but slightly shorter follicles and a thicker hair shaft emerging from an enlarged DP [Figure 2]a and d]. The DP was larger in native beard hair follicles from patient two as well when compared to the group of beard follicles that had been residing in the eyebrows for 6 months [Figure 2]f]. Hence, not just the length, but also the DP size of transplanted hair follicles was decreasing 6 months posttransplantation. This shrinkage of follicular units was independent on the follicle region of origin and donor site-specific morphological features like a comparatively bigger DP of beard follicles seemed not to be conserved.
The quantification of anti-CD20 staining showed that the number of B lymphocytes was found to be massively increased in grafted follicular units 6 months postoperative compared to their native counterparts [Figure 3] a, b and [Figure 4] a, d]. Hair follicles from patient one demonstrated a higher occurrence of B cells than those from patient two [see [Figure 3]a and b for comparison]. While CD20 + populations only sporadically were present in native follicular units and almost exclusively found in the infundibulum [Figure 3]a and 4a], transplanted follicular units showed an strong increase in CD20 + cell number in the perifollicular dermis at the level of the isthmus, bulge and central hair follicle [Figure 3]a and 4d, arrows]. Clusters of CD20 + cells were primarily located in proximity to the entrance of the sebaceous gland [Figure 4]d]. No CD20 immunoreactivity was detected in the proximal hair follicle.
|Figure 3: Quantification of immune cells in native and transplanted hair follicles. B and T lymphocytes as well as macrophages are differently distributed in native and grafted hair follicles as determined by area measurement of CD3+, CD20+ and CD68+ regions. Quantification of B cells (a), T cells (c) and macrophages (e) in native and transplanted hair follicles from different body sites with the scalp as the recipient site for the grafts. Quantification of B cells (b) and T cells (d) in native and transplanted beard hair follicles with the eyebrows as the recipient site for the grafts|
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|Figure 4: Immunohistological staining reveals a massive infiltration of immune cells preferentially in the infundibulum of grafted hair follicles. Prominent staining for CD3+, CD20+ and CD68+ cells is more widespread in grafted hair follicles compared to controls. (a‑c) Representative photographs of native scalp hair follicles stained for CD20 (B cells), CD3 (T cells) and CD68 (macrophages). Positively stained regions are marked by arrows and scale bars indicate 200 μm in (a‑c) and 150 μm in (d‑f), respectively|
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The anti-CD3 antibody is a pan-T cell marker for detection of normal T cells. It is well-suited for detecting reactive T cells in tissues with lymphoid infiltrates and to distinguish between T and B lymphocytes. The number of CD3 + T lymphocytes in transplanted scalp hair follicles was dramatically increased compared to native scalp hair follicles [Figure 3] c, d and [Figure 4] b, e] while the increase in T lymphocytes was less prominent in beard and chest hair follicles transplanted to the scalp. Interestingly, most of the beard follicles from patient two that were grafted into the eyebrows showed a stronger increase in the number of T cells than the control group [Figure 3]d]. In general, considerably more T cells were encountered in native and grafted hair follicles from patient one than in those from patient two [Figure 3]c and d for comparison]. T cells had a similar distribution but a massively higher extent than B cells as they generally were clustered in the connective tissue around the sebaceous gland and distal outer root sheath (ORS) as well as in the surrounding perifollicular dermis [Figure 4]b and e]. In addition, CD3 + cells were also detected in epithelial compartments, namely in the basal epidermis and the proximal hair follicle connective tissue sheath. The majority of native follicular units showed no T cells in epithelial compartments while this was observed in grafted follicular units. As for B cells, no T cells were detected in the epithelium of the lower hair follicles neither in the grafted or nongrafted hair follicles.
Inhomogeneity in the distribution of CD68 + macrophages were not as distinct as seen for lymphocytes although increased numbers of these cells were found in the majority of occipital scalp hair follicles grafted to the frontal region [Figure 3]e and [Figure 4]c, f]. Beard hair follicles grafted into the eyebrows also appeared to more prominently show macrophages. Macrophage populations were considerably smaller compared with lymphocyte populations and mostly not clustered [Figure 4]c and f]. CD68 + cells appeared most frequently in the connective tissue near the distal ORS and sebaceous gland. However, they were also found to line the hair follicle epithelium in the connective tissue sheath. Some positively stained cells were even found in the DP and the hair matrix in some samples (data not shown).
| Discussion|| |
By quantitative histomorphometry and immunohistology, we have analyzed the morphological changes, cellular compositions and the immune status of autologous human anagen hair follicles which had been transplanted utilizing the IFUE technique and which were re-extracted 6 months after the translocation from different body sites to the scalp or eyebrows. Here we show that after this period the hair follicle displays a reduction in size of about 20-30% depending on the donor and recipient area chosen. Unexpectedly, the reduction lengthwise was highest in scalp to scalp transplantations. The most obvious reason for this finding seems to be that the anagen hair follicle still has not fully gone through the entire anagen phase reaching its final follicle length after 6 months. On the other hand, each follicle undergoes successive steps of fiber production (anagen), regression (catagen) and rest (telogen), which in humans last for an average of 3 years, 3 weeks and 3 months, respectively. ,, Given that after transplantation the vast majority of hair follicles first enters catagen and subsequently telogen, about two more month of reorganization should be more than sufficient to generate a new, fully regrown anagen hair follicle. Vogt et al. describe the average length of a terminal scalp hair follicle, tested in 21 individuals, to be 3864 ± 605 μm.  All measured native hair follicles in our case study are within the described range, while transplanted and extracted hair follicles 6 months later are slightly below it. The reduced follicle length, as seen following an altered cyclic behavior, might be a direct consequence of the recipient site-driven reduction of the DP size, which we additionally have observed in all transplanted hair follicles independent of origin and recipient site. The shrunken DP seemed to lose its full capacity to orchestrate the hair follicle growth by stimulating morphogen secretion.
Hair transplantation surgery was pioneered by Orentreich in 1959 when he discovered the "donor dominance" of the donor skin graft over the recipient skin region. This led to the common practice to translocate unaffected follicular units from the occipital region to the frontal or vertex region in androgenetic alopecia-affected scalp skin. Reciprocally, some authors recently unfold an influence of the recipient tissue on the donor grafts. Hwang et al. for example showed macroscopically that chest hair follicles grow longer during a 1-year period when transplanted to the frontoparietal scalp region compared to nontransplanted chest hair follicles. The hair also becomes significantly thicker and the growth rate increases.  Woods and Campbell described in another 18 months study that chest hair follicles transplanted to the scalp grew 11 cm longer than nontransplanted native chest hairs. 
However, our data suggest an intensive crosstalk between the donor unit and the receiving skin environment leading to either dominating or recessive characteristics of the follicular units. In particular, the receiving skin tissue might change the morphogen gradient by sending certain signals to reduce the DP volume. Diametrically, the recipient skin is not capable of producing stimulating signals anymore as the donor tissue did before, which might also lead to a reduced DP size. As the size of the hair follicle is dictated by the volume of the DP, that is, by its secretory power which is dependent on the number and morphogen-secretion profile of DP fibroblasts, , this would implicate an altered growth behavior leading to a reduced hair follicle length. This secretory crosstalk is possibly influenced by a massive infiltrating population of immune cells, which we could also observe in all transplanted follicular units. It is well described, that immune cells are capable of secreting cytokines, growth factors and other signaling proteins that influence the hair follicle growth and cycling.  This is supported by the fact that powerful immunomodulatory agents like glucocorticoids and immunophilin ligands (e.g., cyclosporine) are highly effective in hair growth control. 
The hair follicle itself disrupt the continuous barrier function of the skin. Therefore, compared to the interfollicular epidermis, the distal portion of the hair follicle naturally harbors a higher density of antigen-presenting cells equipped to defend this port of entry for microbial invasion and allergens. In addition, it is quite evident that a certain increase in numbers of immune cells is primarily caused by the wound trauma set during the hair transplantation procedure, but this immune cell infiltration should decline within a few months. The observation of a massive immune cell infiltration might also been described by the Koebner phenomenon  or a partial collapse of the hair follicle immune privilege,  which is a product of multiple anatomical, physiological, and immunoregulatory processes.  Furthermore infiltration of immune cells in the upper hair follicle has been linked to the progression of androgenetic alopecia in striking contrast to alopecia areata, were the proximal hair follicle is infiltrated, too.  The implication of various activators of inflammation in the etiology of androgenetic alopecia has emerged from several independent studies. , However in contrast to a fibroplasia of the dermal sheath, which is suspected to be a common terminal process resulting androgenetic alopecia has not been detected in any of the samples investigated.
Nevertheless, while the functional role of the increased immune cell number in the infundibulum of transplanted hair follicles observed in this case study remains elusive, its potential involvement in the adaptation of size and shape of the donor hair follicle towards the recipient hair follicle population might be postulated.
Intermittent FUE is the method of choice when body hair is utilized to cover patchy or bald areas in facial or scalp hair transplantation surgeries. In contrast to the general opinion, hair follicle units from different body sites are highly suitable to replace miniaturized or degraded hair follicles in different recipient areas like scalp or eyebrows as they have the intrinsic potential to plastically readjust within the beneficiary skin region. Although the donor and sample size studied is very small and larger study data are desperately needed to confirm the observations made, these analyses elaborated here might pave the way for a broader usage of heterogeneously originated hair follicles in scalp and facial hair transplantations and to further explore the mechanism of adaptation of trans-located hair follicles.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]