New Stemcell Treatment Photos... wow?

[One]

Doctor can be more clear? You can get 5000 grafts from 1000 grafts or 5000 hair to 1000 grafts?

As usual, so far, only words. There is not even your own case complete with FUE or FUT, I'm sorry we do not know what you're worth as a surgeon.

Dr nigam do you still alive?

[One]

Doctor can be more clear? You can get 5000 grafts from 1000 grafts or 5000 hair to 1000 grafts?

As usual, so far, only words. There is not even your own case complete with FUE or FUT, I'm sorry we do not know what you're worth as a surgeon.

Dr nigam do you still alive?

[drnigams]

Dear ONE,I hope i do not die before i convert NW7 to nw2/3 with hm and doubling.
iF i am correct are you the same as just one of the other forum.
Regarding your question i can double 2OOO(5000 hair) follicular units to 4000(10000) follicular units by in vitro bisection at the level of DP. With activated stemcells and DP uncultured and cultured cells.
As per the recommendation of few members ,both at my clinic and website the traditional fue work is being reduced and replaced by doubling and hm except when the patient is not able to afford doubling which is 4 times the cost of fue.
Case study of 2000FU hairdoubling to 4000FU starts mid next week.

Dr nigam do you still alive?

[534623]

As per the recommendation of few members ,both at my clinic and website the traditional fue work is being reduced and replaced by doubling and hm except when the patient is not able to afford doubling which is 4 times the cost of fue.

Case study of 2000FU hairdoubling to 4000FU starts mid next week.

So let me get this straight:

1) On one hand you reduced and replaced traditional hair transplant procedures with follicle doubling procedures (including some sort of HM whatever - or as soon as you find it out yourself what you actually do).

2) On the other hand, you try to find out with case studies, whether or not your follicle doubling procedures, which you now offer to your patients in the 1st place (see 1), will work on a consistent basis at all in your hands =THE reason for your case studies - right?

Dr. Nigam,
do you know at all, how much is 1+1= ?

[Boldy]

So let me get this straight:

1) On one hand you reduced and replaced traditional hair transplant procedures with follicle doubling procedures (including some sort of HM whatever - or as soon as you find it out yourself what you actually do).

2) On the other hand, you try to find out with case studies, whether or not your follicle doubling procedures, which you now offer to your patients in the 1st place (see 1), will work on a consistent basis at all in your hands =THE reason for your case studies - right?

Dr. Nigam,
do you know at all, how much is 1+1= ?

maybe he means that the HM and some cultured cells are responsible for 2000 extra hairs..?

[One]

Doctor we are still waiting for his scientific research with which to compare, I see that changes its web site but does not put the most important things!



Hair Regeneration from Transected Follicles in Duplicative
Surgery: Rate of Success and Cell Populations Involved


MARCO TOSCANI, MD, SABRINA ROTOLO, PHD, SIMONA CECCARELLI, PHD, STEFANIA MORRONE, PHD,
GIOVANNI MICALI, MD, NICOLO ` SCUDERI, MD, LUIGI FRATI, MD,y ANTONIO ANGELONI, MD, AND
CINZIA MARCHESE, PHD


BACKGROUND: The use of bisected hair follicles in hair transplantation has been previously reported,
but the capacity of each half to regenerate the entire hair has not been clarified.

OBJECTIVE: To evaluate duplicative surgery rate of success and to analyze the cell populations involved
in hair regeneration.

METHODS: We screened 28 patients undergoing duplicative surgery. Approximately 100 hair follicles
from each patient were horizontally bisected and implanted. Upper and lower portions were stained for
the known epithelial stem cell markers CD200, p63, b1-integrin, CD34, and K19.

RESULTS: Similar percentages of hair regrowth after 12 months were observed when implanting the
upper (72.770.4%) and lower (69.271.1%) portions. Expression of CD200, p63, and b1-integrin was
detected in both portions, whereas K19 and CD34 stained different cell populations in the upper and
lower fragment, respectively.

CONCLUSION: Duplicative surgery might represent a successful alternative for hair transplantation,
because both portions are capable of regenerating a healthy hair. Moreover, our results suggest the
possible presence of stem cells in both halves of the follicle.

The authors have indicated no significant interest with commercial supporters.


The most widely used technique for hair transplantation is the implantation of individual hair follicle units (FUs), although the availability of donor hairs limits it. Attempts to create hairlines using bisected hair follicles have been previously reported. This method is based on the transection of one FU into two growing follicles to regenerate new hairs. The general consensus shows 50% to 70% of success using this procedure, but the biological basis of the capacity of each half to regenerate the entire hair has not been elucidated. Hair follicles consist of a shaft surrounded by concentric layers of epithelial cells, the inner (IRS) and outer (ORS) root sheaths, a mesenchymal layer surrounding the epithelial core, and a sebaceous gland, which is an outgrowth at the side of the hair germ (Figure 1A). Hair follicles have their own stem cell ‘‘niche’’ in a region of the ORS known as bulge,6 in which cells with slow cycling potentiality were identified as keratinocyte stem cells (KSCs).7 Cross-talk between mesenchymal dermal papilla cells (DPCs) and KSCs initiate hair growth. Such cross-talk is crucial for normal development of the hair follicle, as well as for hair cycling, because the multipotent KSCs are stimulated to proliferate and differentiate through interactions with the underlying mesenchymal DPCs. Bulge stem cells are able to generate new follicles at each hair cycle and exhibit a specific repertoire of cell-surface molecules. Recently, many studies have been performed to characterize hair follicle stem cells, searching for the expression of various markers previously shown to be involved in hair follicle cycling, such as CD34, p63, cytokeratin (K19), b1-integrin, and CD200. The CD34 transmembrane protein has been found to be expressed in the ORS of mouse and human hair follicles in cells whose proliferation contribute to form the lower part of the follicle. Transcription factor p63 belongs to a family of structurally related proteins that includes the tumor suppressor proteins p53 and p73; it has been suggested that it is involved in the signalling pathways of the hair follicle cycle. Some studies also indicate that K19, which has been found to be expressed in cells of the basal layer of epidermis as well as in the bulge area, is a suitable marker for


epithelial stem cells of human hair follicles. We based the choice of b1-integrin marker on evidence that b1-integrin-positive cells show a high clonogenic potential and that b1-integrin mutant mice exhibit severe skin blistering and hair defects. Moreover, cultures of isolated bulge cells have been shown to strongly express K19 and b1-integrin.20 More recently, the transmembrane protein CD200, a modulator of the immune response, has been detected in the outermost layer of the ORS throughout the length of the mouse hair follicle or localized in human bulge cells, suggesting a role of this molecule in affording immune privilege to KSCs. Therefore, we believe that, in the present study, the use of these markers might represent an appropriate approach to assay the presence of cellular elements capable of regenerating the hair follicle.

In this report, we screened 28 patients undergoing hair duplicative surgery to evaluate the rate of success of this procedure. We analyzed the expression of the above-mentioned markers in both hair fragments. Subsequently, we cultured cells from upper and lower portions of the microdissected hairs to establish their in vitro behavior.

Materials and Methods

The Ethical Committee of the University Sapienza of Rome approved the protocol, and written consent was obtained from each donor. Twenty-eight patients (24 men and 4 women) were enrolled. Approximately 100 hair follicles from each patient were horizontally sectioned under light microscope below the origin of the arrector pili muscle. The procedure was standardized by cutting all follicles at one-third of their length from the papilla. The two portions were implanted in androgenetic alopecia bald sites, choosing standardized areas to the right (upper) or left (lower) of selected markers, such as angiomatous lesions (8 patients), melanocytic nevi (14 patients), or little scars (6 patients). To selectively follow up the transplanted hairs, all the grafted areas were photographed before and after transplantation. The percentage of hair regrowth (%HR) was evaluated as follows: %HR= (y1-y0/z)X100, where y1 = number of total hairs in a selected and oriented circular area of 1 cm diameter at the followup time (6 or 12 months), y0 = number of preexisting hairs in the same area at the time of transplantation, z = number of total FU (entire or transected hairs) transplanted in the same area. At the 12-month follow-up, 20 hair specimens, cut close to the skin surface, were collected from each group, and their diameter was measured using a microscope equipped with an ocular micrometer. The percentage of hair diameter (%HD) was evaluated as follows: %HD= (xr/xd)X100, where xr = diameter in mm of regrowth hairs and xd = diameter in mm of donor hairs. Hair fragments were assayed for the expression of specific markers using immunohistochemistry (IHC) or immunofluorescence (IFA).

For IHC, frozen sections (3–5 mm), obtained using a cryomicrotome (Microm HM 505N, Thermo Fisher Scientific Inc., Waltham, MA), were fixed in cold absolute methanol for 4 minutes; endogenous peroxidase activities were blocked using 0.03% hydrogen peroxide for 5 minutes; and sections were incubated for 1 hour at room temperature with antihuman CD200 (BD Biosciences Pharmingen, Bedford, MA), CD34, K19, p63, and b1-integrin (Santa Cruz Biotechnology, Santa Cruz, CA) (diluted 1:100 in phosphate buffered saline; PBS). Sections were then processed using avidin-biotin-peroxidase complex (Dako, Carpinteria, CA), counterstained with hematoxylin and permanently mounted under a coverslip. For anti-CD34 detection, antigen retrieval was achieved by heating sections in 10mM citrate buffer, pH 6, in a microwave for 15 minutes before endogenous peroxidase blocking. Control sections were prepared by omitting the primary antibody from the immunohistochemical procedure.



For IFA, frozen sections were incubated with the same primary antibodies, followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:50 in PBS; Cappel Research Products, Durham, NC). Nuclei were visualized using nuclear isolation medium-4,6-diamidino-2-phenylindole dihydrochloride (blue) or TOTO3 (red), a dimeric cyanine nucleic acid dye that stains nucleic acid (1:10,000 in PBS, Molecular Probes, Invitrogen Corporation, Carlsbad, CA). For double IFA, hair fractions were incubated with anti-CD200 followed by anti-K14 antibody (Santa Cruz Biotechnology).

Upper and lower portions were treated with 0.2% collagenase D (Boehringer, Mannheim, Germany) in Eagle minimum essential medium (MEM; ICN Biomedicals, Aurora, OH) containing 10% fetal bovine serum at 371C for 30 minutes and separately incubated in human hair follicle stem cell expansion
media (Celprogen, San Pedro, CA) supplemented with 24.3 mg/mL of adenine, 5 mg/mL of insulin, 5 mg/mL of human transferrin (Sigma-Aldrich, St. Louis, MO), 0.4 mg/mL of hydrocortisone (Calbiochem, La Jolla, CA), 10 ng/mL of human recombinant epidermal growth factor (hrEGF; Chiron, Emeryville,
CA), 100 iu/mL of penicillin (Sigma), and 25 mg/mL of gentamicin sulphate (Scheering, Pointe-Claire, QC, Canada). For flow cytometric analysis, single keratinocyte suspensions (1106 cells/mL) from hair portions or neonatal foreskin were stained with 1 mg/mL of allophycocyanin-conjugated anti-CD34
(BD Biosciences) or 1 mg/mL of anti-CD200 antibodies for 30 minutes at 41C, followed by FITCconjugated goat anti-mouse immunoglobulin G (MP Biomedicals, Irvine, CA) for 30 minutes. The percentage of positively stained cells over 10,000 events was evaluated using a FACS-Calibur flow cytometer and Cell Quest software (BD Biosciences).

Results and Discussion

The diagram of hair follicle structure in Figure 1A, modified from Fuchs, shows the exact localization of the bulge area in the upper two-thirds of the hair follicle, below the sebaceous gland and in correspondence to the arrector pili muscle origin. The dermal papilla is instead localized in the lower portion of the follicle, corresponding to the hair bulb. Figure 1B shows the two generated FU: the upper portion, which encompasses the bulge region, and the lower portion, containing the dermal papilla.

TABLE 1. Hair Regeneration in 28 Patients Undergoing Duplicative Surgery
Mean Percentage +/- Standard Deviation
6 months 12 months
Upper portion 53.7+/-0.3 72.7+/-0.4
Lower portion 48.2+/-1.1 69.2+/-1.1
Recipient’s follicle 56.4+/-1.3 78.4+/-1.5


Bulge and papilla of the same follicle were implanted in marked areas (Figure 1C) and separately followed up. Six months after grafting, transplanted hairs were regenerated with an average efficiency of 53.770.3% for the upper (n = 91) and 48.271.1% for the lower portion (n = 89), similar to the efficiency obtained with the entire follicle (56.471.3%) (n = 92) (Table 1). Twelve months after grafting (Figure 1C), the rate of regrowth was 72.770.4% for the bulge and 69.271.1%for the papilla, similar to that of the entire follicle (78.471.5%) (Table 1). Some previous works1,2 have reported that regenerated hair shafts were finer in caliber than the original donor hairs. We found that the caliber of hairs regenerated from entire follicles was 96.170.2% with respect to original donor hairs (100%). The new hairs obtained from bisected follicles were slightly finer than the donor hairs, although we found no difference in caliber between regenerated hairs derived from the upper (75.378.2%) or lower portion (74.474.1%) (Table 2).

To evaluate the presence of epithelial stem cells in the two portions, we analyzed the expression of known specific markers using IHC (Figure 2). CD200, a recently described marker of bulge stem cells, was detected in the outer root sheath (ORS) of the bulge




TABLE 2. Caliber of Hairs Regenerated After Duplicative Surgery
Donor Hair Caliber, Mean Percentage +/- Standard Deviation
Entire follicle 96.1 +/- 0.2
Upper portion 75.3 +/- 8.2
Lower portion 74.4 +/- 4.1
region, as previously shown25 and, to a lesser extent, in the lower portion (arrows). CD34 was expressed in the ORS of the lower portion (arrows), in keeping with its known downregulation in human bulge cells.15,25 The cytokeratin K19 was previously found in the bulge and also in a second region of ORS that may constitute a reservoir of stem cells.19,28,29 In our sections, K19 was consistently expressed in the bulge (arrow) and throughout the upper ORS. A strong signal for the p63 molecule was detected in the ORS of the entire hair shaft and also around the dermal papilla, previously reported to contain only cells with limited growth capacity.24 b1-integrin is considered a putative stem cell marker, because cells with higher levels of b1-integrin showed a higher colony-forming efficiency.11 Stronger immunoreactivity was evident in the ORS, although the entire follicle was labelled with different staining intensities. We then performed IFA on microdissected hairs to confirm these findings (Figure 3A and B). The upper hair (Figure 3A) clearly expressed CD200 and K19 in the ORS of the bulge region (arrows) and p63 and b1-integrin throughout the ORS (arrows). The upper hair (Figure 3A) clearly expressed CD200 and K19 in the ORS of the bulge region (arrows) and p63 and b1-integrin throughout the ORS (arrows). The lower hair (Figure 3B) showed the marked expression of p63 and b1-integrin in the ORS, whereas the K19 signal was virtually negative. Some CD200- positive cells were also detected in the lower portion, although double staining with CD200 and K14 (Figure 3C and D) showed only a partial co-localization of the two molecules (Figure 3D, arrows), suggesting that the positive signal in the lower portion of the follicle might be partially due to cells of the endothelial sheath.

Our screening for putative markers of the epithelial stem cell compartment, performed using IHC and IFA, demonstrated the presence of CD200-, b1-integrin-, and p63-positive cells in both halves of the follicle, whereas a differential


distribution was observed for K19 and CD34 molecules. In particular, K19-positive cells were found localized mainly in the bulge area, although previous reports have provided evidence of the presence of K19-labelled cells in the upper and lower thirds of the hair follicle during the anagen phase. However, a real consensus on the spatial distribution of K19 during the hair cycle has not been achieved, thus providing a possible explanation for our findings. By contrast, CD34-positive cells have been found to be mainly localized in the lower half of the hair, according to previous literature. Our findings about the expression and distribution of epithelial stem cell markers might contribute to identify the role of subsets of cells expressing these molecules on their surface in generating a complete hair. Primary cultures of keratinocytes obtained from the upper and lower portion of the same FU were different in terms of morphological features and proliferation ratio. Bulge-derived cultures formed large colonies (Figure 4A), and mitotic figures were often observed, suggesting a strong proliferative potential. In cultures derived from the lower portion, cell colonies had a smaller diameter (Figure 4A), and elements in cytokinesis were occasionally found. Hair-derived keratinocytes showed greater motility.

Human Hair Follicle Regeneration Following
Amputation and Grafting into the Nude Mouse

Colin A.B. Jahoda,* Roy F. Oliver Amanda J. Reynolds,* James C. Forrester, and Kenneth A. Hornet
"Department of Biological Sciences. University of Durham, Durham. U.K. and Department of Biological Sciences. University of
Dundee. and Department of Surgery, Ninewells Hospital. Dundee, Scotland. U.K

In this study we investigated the capacity of the human hair follicle to regenerate a fiber-forming bulb after its amputation. We removed the bases from terminal follicles from a variety of sites and transplanted the follicles onto athymic mice, either still attached to a skin graft or as subcutaneous implants of individual follicles. External hair growth was observed on the skin grafts, and histology of the follicles revealed restoration of dermal papillae and follicle bulb structures. This result suggests that the capacity of hair follicles to regenerate their lower structures after removal, which was first demonstrated on whisker follicles, may be a general phenomenon. It emphasizes the importance of specific cellular subpopulations within the follicle and the role of dermal-epidermal interactions in adult follicle activities. Key words: hair growth restoration/dermal-epidermal interactions/dermal sheath/transplantation. J Invest Dermatol 107:804-807, 1996

The physiologic regeneration of hair follicles during the adult hair cycle is an extraordinary natural phenomenon. That vibrissa follicles can reform active follicle bulbs after experimental amputation of up to a third of their bases is even more remarkable (Oliver, 1966a, 1966b) given the limited regenerative powers of mammalian systems. This phenomenon has been produced repeatedly with rodent vibrissae, both in situ and when follicles, or parts of follicles, have been transplanted ectopically to the kidney capsule (Oliver, 1966a, 1966b, 1967; Ibrahim and Wright, 1982; Kobayashi and Nishimura, 1989; Jahoda et al, 1992). The key initial event in the process is the formation of a new dermal papilla from the cells of the lower dermal sheath-the mesenchymal cells surrounding the sides of the follicle (Jahoda et al, 1992; Oliver, 1966b).

The question of whether human follicles have the same potential to regenerate as rodent vibrissa follicles has been the subject of some debate. Many of the positive reports arc anecdotal. Some years ago, apparent human follicle regeneration was demonstrated following split thickness removal of axilla skin to remove sweat glands (Inaba et al, 1979). Some commentators were doubtful, however, because the results were largely based on observations of renewed external hair growth. The alternative explanation put forward was that post-operative fiber production could have been the result of short telogen follicles, within the remaining split thickness skin, reverting to anagen (Montagna, 1980, 1984). Indeed, it was suggested that vibrissa follicles are in some way privileged in their regenerative powers. One recent report describes regeneration of individual human scalp follicle bases after autologous grafting (Kim and Choi, 1995).

Athymic mice have been used increasingly as recipients for hair growth studies (Gilhar and Krueger, 1987; Van Neste et al, 1987, 1991; Lichti et al, 1993; Scandurro et al, 1995). In the current work, we directly investigated the capacity of human hair follicles to regenerate by transplanting follicles from different human body sites into athymic mice, either as skin grafts or as implanted single follicles. Following removal of follicle bases, dermal papillae and then bulb structures were reformed, and fiber growth was restored.


MATERIALS AND METHODS

Animals:

Male athymic mice between 3 and 8 mo of age were used as recipients in these experiments. Two strains of nude mice were used. CBA and MF1 (supplied by Harlall Olac. Bicester, U.K.). Animals were kept in a pathogen-free facility until operated upon, and all surgical procedure were performed ill a filtered air flow hood.

Skin and Follicle Grafts

Specimens Skin Specimens were obtained from excised non-neoplastic human skill obtained from routine biopsies. Subjects were Caucasian of variable age and sex. and samples were from head. face. groin, or scrotal sac regions. Skin samples were stored at 4 degree celsius and used between a few hours and 7 d after biopsy.

Two surgical procedures were employed. In the first, small pieces of human skin with follicles still attached were grafted onto nude mouse skin. In the second. follicles were implanted into the mouse subcutaneously.

Only skin from body areas with relatively low follicle density was suitable for skin graft operations, as the aim was to use a small number of well spaced follicles whose positions could be reliably located. Head skin. with a dense population of straight follicles, had to be ruled out for this protocol because of the difficulties in separating individual follicles. Groin or scrotal sac skin (the latter with large follicles well spaced and lying at a shallow angle to the skin) was the preferred donor specimen for graft work.

Graft Preparation All procedures were performed with sterile instruments in areas cleaned with alcohol wipes. In sterile modified Eagle's medium under a dissecting microscope (Nikon. Kingston Upon Thames. U.K.), skin was gradually cleared of fat and dermal tissue using fine curved iridectomy scissors. Between one and three large anagen follicles were left in place, and the remainder were removed. The chosen follicles were as far apart as possible but away from the edge of the specimens. After interfollicular material had been cleared as near as possible to the epidermis, the perifollicular region was carefully trimmed further (Fig 1a). At this point it was often necessary to cut out much of the sebaceous gland to ensure



that no vellus or telogen follicles remained. Where this proved impossible, the whole pilosebaceous structure was removed. Where two anagen follicles were inseparable and shared a common follicular opening, they were both used. After cleaning, the bases of follicles were cut off with iridectomy scissors, just above the bulb, and then the fibers were plucked. Control skin bad all visible follicles removed, but the uppermost regions of a few pilosebaceous structures were left in place.

For subcutaneous implantation grafts, individual follicles from skin specimens of all body regions were isolated, and surrounding material was , dissected away in saline as described above. In many cases this involved removal of nearly all of the sebaceous gland. The bases of follicles were transected, and the fibers plucked out as described above (Fig 1b).


Skill Grafting Recipient mice were anesthetized with pentobarbitol [80 mg per kg body weight, Nembutal (Rhone merieux, Harlow, U.K.)] diluted in phosphate-buffered saline, administered intraperitoneally. A piece of skin corresponding to the shape of the graft, but slightly smaller (usually -1 cm2 ) was aseptically removed from the mid-dorsal region of each animal. The graft of skin with amputated follicles was then carefully placed in position. because the dermis had been trimmed, the grafts usually fit neatly at the same depth as the host skin. Occasionally they were sutured in place, but usually it only required strips of Op-Site Johnson & Johnson, Raritan, NJ) to hold the grafts in place. Each experimental animal received a single graft.

Follicle Implants For subcutaneous grafts, animals were anesthetized as described above, and a small incision was made in the skin. Up to ten individual follicles were then implanted beneath the skin, at the level of the panniculus Calrnosus. The incision was sutured, and covered with Op-Site spray dressing. A variation of this procedure involved the prior establishment of a bed of subcutaneous granulation tissue. For this, a disc of roughened glass (diameter ~1 cm) was inserted into the subcutaneous site 10 d or so prior to the operation. The disc was removed and the amputated follicles were put into the chamber that had been formed.

Grafts were observed at intervals and biopsied from 7 d to 5 mo postoperatively. Specimens were photographed, fixed in formol saline, and processed for routine wax histology. Sections of 8 pm were stained with a combination of Weigert's hematoxylin, Curtis's ponceau S, and Alcian I blue.


RESULTS


The outline of all grafts was easily visible on the host mouse skin, the human skin showing darker pigmentation over time. After 8 week, small external fibers were visible on a number of grafts, always emerging from pre-established positions of the amputated follicle openings. Where two bases were removed 6:0111 inseparable follicles, double fibers were observed externally (Fig 2/a.). Where single follicle bases were amputated, isolated fibers were observed (Fig 2/a). Both pigmented and nonpigmented fibers were observed, but there was no apparent correlation between pigmentation of the inter-adnexial epidermis and the production of pigmented or unpigmented hair fibers. Long-term grafts were biopsied at between 8 week and 5 mo postoperatively. At biopsy it was apparent that some follicles were in anagen, because active bulbs with functional melanocytes were detected beneath the grafts (Fig 2c). Histology confirmed the presence of follicles actively producing hair fibers from hair bulbs with apparently normal dermal papillae and matrices (Fig 3/a.b.c). In the later biopsies, however, the majority of follicles that had produced fiber externally had an appearance somewhere between catagen and telogen (Fig 3d,e). They had shortened somewhat, and the undifferentiated lower follicle epithelium partially surrounded dermal papillae whose cells were condensed and displayed little extracellular space (Fig 3e). In total, just over a third of the experimental follicles (12 of 35) demonstrated lower bulb regeneration and hair growth (Table I). Several other specimens revealed unusual cyst formation. None of the control grafts produced external fibers or showed signs of regeneration when examined histologically.

More information of the events following amputation came from earlier biopsies. At 7 d, changes comparable to those observed in regenerating vibrissa follicles were already visible. The glassy membrane had thickened, and dermal cells had accumulated at the cut base of the follicle in contact with the follicular epidermis (Fig 3.f). At 36 d, a new dermal papilla had formed (Fig 3g).

At biopsy, the specimens that were grafted subcutaneously for extended periods had an opaque bulbous appearance and had tended to merge or fuse in each other (Fig 4a). This time, however, histology revealed the presence of fibers inside these follicle capsules and of restored papillae and follicle bulbs (Fig 411) in just under a third of the cases (9 of 31; Table I).












DISCUSSION

We have demonstrated that the human hair follicle has a capacity to regenerate a dermal papilla and active bulb region following its amputation. The relatively low success rate may be attributed, in part, to the fact that many of the specimens were not used immediately after biopsy. Our observations support and extend a recent description of regeneration of the bases of human scalp.



Table I. Regeneration of Human Follicles Following
Amputation
Gkin Gragt Subcutancous Implants
No. of animals 25 6
No. of follicles grafted 35 31
No. of regenerating follicles 12 9



follicles grafted onto leg skin (Kim and Choi, 1995). A previous report of hair regeneration following surgery (Inaba et al, 1979) was criticized on the grounds that short telogen follicles may have produced the observed hair growth (Montagna, 1980, 1984). Our procedures Were designed to avoid this possibility. The dissection and tissue removal protocol in both skin grafts and isolated follicles obviated the possibility of the presence of vellus or telogen follicles. Furthermore, hairs were clearly observed to grow from the amputated terminal follicles. This was reinforced by the absence of hair growth in control skin grafts in which residual follicle structures and sebaceous glands were visible from follicles that had been amputated close to the skin surface. Histology revealed that some follicles were in all elongated telogen-like state, suggesting possible restrictions on their capacity to cycle. With the current protocol. the lack of movement Il'light be due to physical constraints of the relatively shallow graft site. In other work involving alopecic human skin grafted onto nude mice (Gilhar and Krueger, 1987; Van Neste et al, 1987, 1991) there was evidence that follicles might cycle, but thicker skin was initially grafted.

Short-term histologic evidence revealed the regeneration sequence. It showed that, as in whisker follicle end bulb regeneration (Oliver, 1966b; .Jahoda et al, 1992), regeneration occurred at the site of amputation without follicle shortening. Concerning the formation ofthe new dermal papilla, although we cannot absolutely rule out the possibility that mouse fibroblasts might have been recruited into dIe new structures, previous histologic evidence in vibrissa follicles shows that the new dermal papillae are formed from the cells of the lower dennal sheath (Oliver, 1966b;Jahoda et al, 1992). that the human follicle dermal sheath consists of two ultrastructurally distinct cell layers (Ito and Sato, 1990; KA Horne, unpublished) We have also noted that smooth muscle actin marking of the dermal sheath is confined to the inner layer Jahoda et al, 1991). The opaque bulbous structures produced by human follicles transplanted subcutaneously in the mouse were histologically similar to the collagen capsules that sorround vibrissa follicles. Therefore, one could surmise chat the outer layer of the human follide dennal sheath is analogous to the protective layer of the vibrissa follicle, whereas the inner layer consists of cells involved in maintenance of the glassy membrane. The inner sheath cells may also perhaps replenish or replace part of the papilla cell population durign the hair cycle.

In this experiment, no attempt was made to establish the level at which regeneration could take place. The question of follicle regeneration has become entangled with the debate about follicle stem cells, and the level at which they reside. Thus, one group has taken the regeneration of follicles removed at the level of the isthmus to imply that stem cells are high up (Inaba and Inaba, 1992), above the generally favored region of the bulge (Cotsarelis et al, 1990). The current observations do not shed any light on this issue, but the new epidermal bulb was clearly induced to form from outer root sheath cells at the level of amputation. The main thrust of our finding is to reinforce the concept that the cellular interactions underpinning the activities of the lower hajr follicle are Likely to involve universal mechanisms. They also highlight the understated role ofthc dermal sheath in follicle activities. Finally, the fact that some of the follicles that regenerated had produced pigmented fibers supports the idea that a reservoir melanocytes is retained in the outer root sheath above the bulb region the follicle throughout the hair cycle (Horikawa et al, 1996).



REFERENCES


Cocsarclis G. Sun T-T. Lavkcr RM: Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle and skin carcinogenesis. Cell 61: 1329-1337,1990

Gilhar A. Krueger GG: Hair growth in scalp grafts from patients with alopecia areata and alopecia universalis grafted onto nude mice. arch Dermatol 123:-l4 -50.1987

Horikawa T. Norris DA. Johnson TW, Zekman T, Dunscomb N. Bennion SD. Jackson RL. Morelli JG: DOPA-negative mclanocytes in the outer root sheath of human hair follicles express premelanosomal antigens, but not melanosomal, antigen or the melanosome-assosiated glyecoproteins tyrosinase, TRP-l. and TRP-2.) Invest Dermatol 106:28-35. 1996

Ibrahim L. Wright EA: A quantitative study of hair growth using mouse .and rat vibrissal follcles. J Embryol exp Morphol 72:209-224.1982

Inaba M. AnthonY J. McKinstry C: Histologic study of the regeneration of axillary hair after removal with subcutaneous shaver J Invest dermatol 72:22-4 -231.1 979

Inaba M, Inaba Y: In: Inaba. M Inaba Y (eds) Human body odor Springer Verlag. Tokyo. 1992

Ito M. Sato Y: Dynamic ultrastructural changes of the connective tissue sheath of human hair follicles during the hair cycle Arch Dermatol Res 282:-434 441, 1990

Jahoda CAB. Horne KA. Mauger A. Bard S. Sengel P: Cellular and extracellular involvement in the regeneration of the rat lower vibrissa follicle. Development 114:887-897. 1992 Jahoda CAB. Horne KA. Mauger A. Bard S. Sengel P: Cellular and extracellular involvement in the regeneraltion of the rat lower vibrissa follicle. Development 114:887-897. 1992

Jahoda CAB. Reynolds AJ. Chaponnier C. Forester JC Gbbiani G: Smooth muscle a actin is a marker for hair follicle dermis in vivo and in vitro. J cell Sci 99:627-636. 1911

KimJC amd choi YC: Regrowth ofgarfted human sCalp hair after removal of the buib. Dermatol Surg 2-4:312-313.1995

Kobayashi K: Nishimura E: Ectopic growth of mouse whiskers from implanting lenghths of plucked vibrissa follicles. J invest Dermatol 92:278-282.1989

Lichti U. Weinberg WC. Goodman L. Ledbetter 5. Dooley TP. Morgan D. Yuspa SH: in vivo regulation of murine hair growth-insight's from grafting defined cell populations onto nude mice.J Invest Dermatol 101:s27-s32, 1993

Montagna wJ: Regeneration of axillary hair. J Invest Dermatol 75:202, 1980

Montanga WJ: Electrolysis and the problem of hair regrowth. Journal of Applied Cosmetology 2:6. 1984

Oliver R.F: Whisker growth after removal of the dermal papilla and lengths of the follicle in the hooded rat J Embryol Exp Morphol 15:331-347. 1966

Oliver RF: Histological studies of Whisker ,regeneration in the hooded rat by implantation Exp Marpho1 16:231-244,1966

Oliver RF: Ecropic regeneration or whisker growth in the hooded rat by implantation of dermal papillae. J Embryol Exp morphol 17:27-34.1917

Scandurro AB. Wang QZ. Goodman L. Ledbetter S. Dooley TP. Yuspa SH. Lichti U: Immortalized rat whisker dermal papilla cells co-operate with mouse immature hair follicle buds to activate type-IV procollagenase in collagen matrix coculture correlation with ability to promote hair follicle development in nude mouse graft's J Invest Dermatol 105: 177-183. 1995

Van Neste D, De Brouwer B. Dumortier M: Reduced linear hair growth rates of vellus and terminal hairs produced by human balding Scalp grafted onto nude mice. ann Ny Acad Sci 642:-180-482. 1991

Van Neste D. Warnier G. Thulliez. M, Van Hoof F: Human hair follicle grafts onto nude mice: morphological Study. In: Van Neste D. Lachapelle JM. Antoine JL (eds.) Trends in Human Hair Growth and Alopecia Research. Kluwer Academic. Dordrecht. Gormany. 1987. pp '117-131

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[justeone]

Just to clarify Dr Nigam . That is not me .
Great thread by the way , we appreciate your informative answer's and quick response time .

[Rogerrabbit]

Buuuuuuump!