CED
Experimental dermatology • Original article
Clinical and Experimental Dermatology
A simple and rapid model for hair-follicle regeneration in the nude mouse Y.-S. Su,1 Y. Miao,1 J.-D. Jiang,1 H. Liu,2 J. Hu2 and Z.-Q. Hu1 1 Department of Plastic and Reconstructive Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China; and 2Department of Burn and Plastic Surgery, The Shenzhen Baoan Hospital Affiliated to Southern Medical University, Shenzhen, China
doi:10.1111/ced.12563
Summary
Background. Methods for hair-follicle regeneration are important tools for investigating signalling and cytokines during hair-follicle morphogenesis and cycling. Several animal models for hair reconstitution have been established; however, these models have several shortcomings. Aim. To develop a simple and rapid model for hair induction in nude mouse. Methods. We designed an improved flap model (IFM) for hair regeneration based on the existing flap assay. Histological sections and scanning electron microscopy were used to evaluate the regenerated hair. The fates of grafted cells were traced by fluorescence. The time required for hair induction was analysed and compared. Results. IFM produced a large number of normal hairs, and the time required for hair induction using IFM was 20.67 0.67 days, compared with 29.33 0.67 days for the traditional flap assay. Conclusions. The time required for hair regeneration is considerably shortened with IFM. We speculate that this is due to increased blood supply at the transplantation sites.
Introduction The morphogenesis and cycling of the hair-follicle is a complex process that depends on intensive and continuous interactions between epithelial and dermal cells during the embryonic period.1 A series of reciprocal signals between these two components results in the formation of a functional follicle.2,3 The current hairfollicle reconstruction models include in vitro and in vivo regeneration. Some studies have successfully produced follicle structures in vitro;4–6 however, these studies could not fully mimic in vivo conditions, such as all the cytokines and nutrients.7,8 Several in vivo animal models for hair reconstitution have been established, such as the chamber assay,9,10 patch assay11 Correspondence: Professor Zhi-qi Hu, Department of Plastic and Reconstructive Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou City, China E-mail: [email protected] Conflict of interest: the authors declare that they have no conflicts of interest. Accepted for publication 29 July 2014
ª 2015 British Association of Dermatologists
and flap assay.12 However, all of these have drawbacks. The patch assay is a convenient model to reconstitute hair follicles, but the direction of growth of the regenerated hairs is random. The chamber assay can produce normal hair follicles with normal hair density, but is the most labour-intensive model, requiring complex transplantation procedures and a large number of cells.13 The flap assay was first introduced by Qiao et al.12 in 2008. Compared with other methods, this model has several advantages. First, it produces hair shafts of normal quality and density; second, it is labour-saving, because it requires fewer cells than other methods, and only mouse dermal cells and an intact epidermal sheet are needed; and third, the wounds do not need to be continuously exposed as in the chamber model, which can often cause infection and death. However, the flap assay also has a number of disadvantages; for instance, it takes the longest time of the three assays to produce follicles, and it causes greater trauma.14 In the current study, we aimed to design a simple and rapid model for hair reconstitution by modifying these deficiencies.
Clinical and Experimental Dermatology (2015) 40, pp653–658
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Methods All animal experiments were carried out under the approval of the Institutional Animal Care and Use Committee. Animals
Newborn C57BL/6J mice, 4–6 week-old athymic nude mice (BALB/cAJcl-nu) and newborn transgenic mice homozygous for green fluorescent protein (GFP) (Experimental Animal Centre, Southern Medical University, Guangzhou, China) were used. Preparation of epidermal sheet and dermal cells
Mouse epidermal sheet and dermal cells were isolated from C57BL/6J mice at natal day 0. After sterilization with 75% ethanol, the full thickness of the dorsal skin was removed and incubated in phosphate-buffered saline (PBS) with 0.1% dispase (Invitrogen, Grand Island, NY, USA) at 4 °C overnight. Each skin sample was washed three times with PBS, then the epidermis and dermis were separated with forceps. The epidermis sheet was put in a sterile culture dish containing Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM/10; Invitrogen) temporarily. The dermis was minced and digested in 0.2% collagenase (Sigma, St. Louis, MO, USA) at 37 °C for 1 h. After digestion, an equal volume of DMEM/10 was added, and the cell suspension was filtered sequentially through 100 lm and 40 lm mesh cell strainers. The cell suspension was centrifuged at 200 g for 5 min, then the cell pellet was resuspended in 1 mL DMEM/10 and counted. Finally, the cells were resuspended in 20 lL of DMEM as a slurry. Procedures for improved flap model and traditional flap assay
A lid from a 1.5 mL microcentrifuge tube (Eppendorf, Hamburg, Cologne, Germany) was used (Fig. 1a), and trimmed into a circle the same size as a 1.5 cm2 silicone plate (Fig. 1b). Once the dermal cells were prepared, the epidermal sheet was spread onto the smooth side of the plate with its basal side facing up. The slurry of dermal cells was pipetted evenly onto the centre of the basal side of the epidermis using a micropipette (Fig. 1c). The plate containing the epidermal sheet and dermal cells was placed in an incubator at 37 °C, and left for 30–60 min to evaporate excess liquid before transplantation.
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Nude mice were anaesthetized by intraperitoneal injection of 1% pentobarbital sodium, and the graft area of each mouse was cleaned with betadine solution. We created a single incision in the dorsal skin. After subcutaneous dissection, the plate was inserted between the panniculus carnosus of the skin flap and the musculoaponeurotic layer (Fig. 1d), and the incision was then sutured (Fig. 1e). Slurries with different numbers of dermal cells were transplanted. The traditional flap assay was performed as described previously.12 Briefly, a full-thickness, threesided, rectangular skin flap (08 9 10 mm) was made in the dorsal skin of recipient mice. The epidermal sheet, attached to a 25 9 15 mm silicone sheet, was placed onto the wound area with the basement membrane side of the epithelium facing up. Opening of the flap to allow gross hair observation
When the surface of the skin flap became raised and completely dark in colour (Fig. 1f), the flap was considered ready for opening. The incision was opened and the flap inverted to expose the graft to the exterior environment, and the wound was closed by suturing the wound edge to the base of the flap (Fig. 1g,h). Mouse cell labelling
To assess the fate of epidermal cells in the grafts, epidermal sheets were obtained from green fluorescent protein (GFP) transgenic mice for some experiments. To track the fate of the dermal cells in the grafts, we labelled the dermal cells with dilinoleyltetramethylindocarbocyanine perchlorate (DiI), a fluorescent cell-tracking dye (Invitrogen) prior to grafting, following the manufacturer’s instructions. The GFP-expressing epidermal sheet was co-transplanted with unlabelled dermal cells, while DiI-labelled dermal cells and unlabelled epidermal sheets were transplanted using IFM. Evaluation of hair reconstitution
Hair reconstitution was evaluated by visual and histological observation. For histological observation, the skin on the transplantation site was excised and fixed in 10% formaldehyde at room temperature for 24 h. Tissue was embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin (H&E). The fate of the epidermal and dermal cells in the grafts was assessed by fixing the grafts as before, freezing at 20 °C and sectioning (6–8 lm) for examination under fluorescence microscopy. To evaluate the
ª 2015 British Association of Dermatologists
Model for hair regeneration Y.-S. Su et al.
(a)
(b)
(c)
Figure 1 Procedure for improved flap
model. (a) The lid of a 1.5 mL microcentrifuge tube was used, and (b) trimmed into a circle to produce a silicone plate. (c) The lid was placed with the smooth side facing up and the rough side facing down, and the epidermal sheet was spread onto the smooth side of the plate with its basal side facing up. The slurry of dermal cells was then pipetted onto the basal side of the epidermis. (d) The silicone plate was inserted between the flap and muscle layer through a single incision, and (e) the incision was sutured. (f) The flap was considered ready for opening when the surface of the skin became raised and completely dark in colour(g) A full-thickness and three-sided skin flap was designed prior to opening the flap. (h) The flap was opened and inverted to expose the graft to the exterior, and the wound was closed by suturing the wound edges to the base of the flap. (f–h) The number of grafted dermal cells was 3 9 106.
(d)
(f)
quality of the regenerated hair shafts, we compared reconstituted hair with normal C57BL/6J hair by scanning electron microscopy (SEM). Statistical analysis
GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA) was used for all analyses. The Student t-test was used for comparing the days needed for hair regeneration in IFM and traditional flap assay. All P values were twotailed, with P < 0.05 considered statistically significant.
Results Evaluation of hair reconstitution
Co-grafting fresh epidermal sheet with fresh dermal cells generated abundant short and black hairs on the graft site of mice treated with IFM. There was reconstruction of hair follicles, which developed a dense, natural appearance (Fig. 2a,b). Histological sections of the skin at the transplantation site showed normal skin layers, including epidermis, dermis, hair follicles and sebaceous glands (Fig. 2c). The reconstituted hair shafts were of good quality and dense, with normal appearance under SEM (Fig. 2d,e).
ª 2015 British Association of Dermatologists
(e)
(g)
(h)
Comparison and analysis of hair reconstitution in improved flap model and flap assays
For IFM, six transplantation experiments were carried out. All animals survived and all experiments produced black hairs successfully, with a success rate of 100% for hair reconstitution. However, because of the flap contraction, some hair follicles failed to grow out at the final stage, when the wound healed and the flap contraction ceased completely. Six transplantation experiments were also performed for the flap assay, and all produced hairs. The success rate for hair regeneration using this method was also 100%, but necrosis occurred in the distal part of flap in one case. Time-course evaluations were conducted to define the advantages and disadvantages of both assays. The time to opening of the flap and observing hair growth was 20.67 0.67 days for IFM and 29.33 0.67 days for the flap assay (Fig. 3). Thus, the time needed for hair induction was significantly shortened in IFM compared with flap assay. Hair regeneration caused by grafted cells
In the frozen sections examined under fluorescence micoscopy, GFP-positive epidermal cells were seen to
Clinical and Experimental Dermatology (2015) 40, pp653–658
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(a)
(b)
(c)
(d)
form the epithelial components of the neonatal follicles. All of these components were GFP-positive, while the dermal papilla and the dermis were GFPnegative (Fig. 4a,b). In addition, we observed a resident portion of GFP-expressing cells in the neonatal epithelium. When we examined the transplants containing DiIlabelled dermal cells co-grafted with negative epidermal sheet, all the dermal papillae in the neonatal follicles were DiI-positive, confirming that the grafted cells contributed to forming the neonatal follicles (Fig. 4c,d).
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Figure 2 Evaluation of hair reconstitu-
(e)
Clinical and Experimental Dermatology (2015) 40, pp653–658
tion. (a,b) Abundant normal hair grew on the graft site after grafting, with (c) reconstitution of the normal layers of the skin (haematoxylin and eosin, original magnification 9 200). Scale bar = 200 lm. (d) The reconstituted hair shaft was of good quality and appeared to be similar to (e) a normal hair shaft under scanning electron microscopy. Original magnification (d,e) 9 2000. Scale bar 20 lm.
Discussion The goal of this study was to develop a simple and rapid method for hair reconstitution. We evaluated the time needed for hair induction in both IFM and flap assays, and found that IFM took less time for hair reconstitution. A previous study had indicated that direct contact between epithelial and dermal components and better vascularization or oxygenation of the recipient site are critical for hair regeneration.15 In IFM, the flap was created with a single incision instead of a three-sided rectangular skin flap as in the flap
ª 2015 British Association of Dermatologists
Model for hair regeneration Y.-S. Su et al.
(a)
(c)
(b)
(d)
(e)
Figure 3 Comparison of the time required for hair induction in both assays. Gross observation in (a) improved flap model (IFM) and (b) tradi-
tional flap assay. (c) After 21 days of grafting, hair growth was visible (areas marked by black dotted line). (b) In IFM, a large number of short black hairs grew out when the flap was opened. (d) In the traditional flap assay, a layer of black mucosal tissue formed, but few hairs had grown out onto the surface of the flap. (e) Days required for hairs reconstitution per graft. The density of grafted dermal cells was 2 9 106 cells/cm2 in both assays. Means SEM are indicated; n = 6 per group; ****P < 0.001, unpaired t-test.
(a)
(c)
(b)
(d)
Figure 4 Hairs on the grafts were
induced by grafted cells. The grafted epidermal sheet and dermal cells formed epidermal components and dermal papillae respectively. (a) Fluorescent image of green fluorescent protein (GFP)-expressing epidermal components from a frozen section of a newborn hair follicle. (b) Corresponding histological image of epidermal components. (c) Fluorescent image of DiI-positive dermal papilla from regenerated hair follicle after DiI fluorescent probe labelling. (d) Corresponding histological image of dermal papilla. Original magnification (a–d) 9 200. Scale bar 200 lm.
assay. Thus, the blood supply of the flap was better protected, which is essential for the survival of graft cells and regeneration of hair follicles in the early stages. The reason why we chose a microcentrifuge tube lid rather than a silicone sheet was that nude mice have loose skin, thus the space between the skin and muscle layer is relatively large. The grafted silicone sheet used in the traditional flap assay was soft and smooth, and thus slid easily over the surface of muscle
ª 2015 British Association of Dermatologists
layer, which affected transplantation. In fact, to prevent sliding, the area of the silicone sheet (25 9 15 mm) used in the traditional flap assay was much larger than the areas of its loaded epidermal sheet (8 9 1.0 mm) and of the flap, which increased trauma. By contrast, with IFM, the microcentrifuge tube lid, being a hard silicone plate (1.5 cm2 in size) with some whorls on the side that was placed in contact with the muscle layer, did not slide easily, so the
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epidermal sheet and dermal cells were kept in close contact with the panniculus carnosus of the flap and confined to a small area for interaction. Lee et al.16 used another method, seeding epithelial and dermal cells from neonatal mice into a scaffoldlike matrix (Integra Bilayer Wound Matrix; Integra LifeSciences, Plainsboro, NJ, USA), and then transplanting the matrix onto a wound bed caused by excision of a full-thickness skin section in the nude mouse. A large number of hair follicles were produced through this one-step procedure. In IFM, a single incision was created, the operation time was greatly shortened and bleeding was reduced during grafting. In addition, the silicone lid we used is cheap and easily available, although two operations are still needed.
Conclusion We have developed a simple and rapid model for hair regeneration by improving the previous flap assay. In this new technique, trauma is reduced and the time needed for hair induction is shortened considerably. We speculate the technique preserves local blood supply, which shortens the time for hair induction.
Acknowledgements This study was supported financially by the Natural Science Foundation of China (Grant No. 31170949). The study was conducted in the Central Laboratory of Southern Medical University, Guangzhou, China.
What’s already know about this topic? Several animal models for hair reconstitution
have been established; however, these models have a number of shortcomings.
What does this study add? We have developed a simple and rapid mouse
model for hair regeneration based on the traditional flap assay. The time for hair induction was considerably shortened. We speculate that this method may increase blood flow, thus shortening the time for hair induction in vivo.
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References 1 Millar SE. Molecular mechanisms regulating hair follicle development. J Invest Dermatol 2002; 118: 216–25. 2 Wang X, Tredget EE, Wu Y. Dynamic signals for hair follicle development and regeneration. Stem Cells Dev 2012; 21: 7–18. 3 Xiao S, Hu Z, Jiang J et al. Neonatal murine skin-derived cells transplanted using a mini-chamber model produce robust and normal hair. J Tissue Eng Regen Med 2013; doi:10.1002/term.1802. 4 Lindner G, Horland R, Wagner I et al. De novo formation and ultra-structural characterization of a fiber-producing human hair follicle equivalent in vitro. J Biotechnol 2011; 152: 108–12. 5 Lim TC, Leong MF, Lu H et al. Follicular dermal papilla structures by organization of epithelial and mesenchymal cells in interfacial polyelectrolyte complex fibers. Biomaterials 2013; 34: 7064–72. 6 Sriwiriyanont P, Lynch KA, Maier EA et al. Morphogenesis of chimeric hair follicles in engineered skin substitutes with human keratinocytes and murine dermal papilla cells. Exp Dermatol 2012; 21: 783–5. 7 Yoo B-Y, Shin Y-H, Yoon H-H et al. Hair follicular cell/ organ culture in tissue engineering and regenerative medicine. Biochem Eng J 2010; 48: 323–31. 8 Yang CC, Cotsarelis G. Review of hair follicle dermal cells. J Dermatol Sci 2010; 57: 2–11. 9 Lichti U, Weinberg WC, Goodman L et al. In vivo regulation of murine hair growth: insights from grafting defined cell populations onto nude mice. J Invest Dermatol 1993; 101: 124S–9S. 10 Weinberg WC, Goodman LV, George C et al. Reconstitution of hair follicle development in vivo: determination of follicle formation, hair growth, and hair quality by dermal cells. J Invest Dermatol 1993; 100: 229–36. 11 Zheng Y, Du X, Wang W et al. Organogenesis from dissociated cells: generation of mature cycling hair follicles from skin-derived cells. J Invest Dermatol 2005; 124: 867–76. 12 Qiao J, Philips E, Teumer J. A graft model for hair development. Exp Dermatol 2008; 17: 512–18. 13 Jensen KB, Driskell RR, Watt FM. Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis. Nat protoc 2010; 5: 898–911. 14 Liang Y, Silva KA, Kennedy V, Sundberg JP. Comparisons of mouse models for hair follicle reconstitution. Exp Dermatol 2011; 20: 1011–15. 15 Aoi N, Inoue K, Kato H et al. Clinically applicable transplantation procedure of dermal papilla cells for hair follicle regeneration. J Tissue Eng Regen Med 2012; 6: 85–95. 16 Lee LF, Jiang TX, Garner W, Chuong CM. A simplified procedure to reconstitute hair-producing skin. Tissue Eng Part C Methods 2011; 17: 391–400.
ª 2015 British Association of Dermatologists
Experimental dermatology • Original article
Clinical and Experimental Dermatology
A simple and rapid model for hair-follicle regeneration in the nude mouse Y.-S. Su,1 Y. Miao,1 J.-D. Jiang,1 H. Liu,2 J. Hu2 and Z.-Q. Hu1 1 Department of Plastic and Reconstructive Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China; and 2Department of Burn and Plastic Surgery, The Shenzhen Baoan Hospital Affiliated to Southern Medical University, Shenzhen, China
doi:10.1111/ced.12563
Summary
Background. Methods for hair-follicle regeneration are important tools for investigating signalling and cytokines during hair-follicle morphogenesis and cycling. Several animal models for hair reconstitution have been established; however, these models have several shortcomings. Aim. To develop a simple and rapid model for hair induction in nude mouse. Methods. We designed an improved flap model (IFM) for hair regeneration based on the existing flap assay. Histological sections and scanning electron microscopy were used to evaluate the regenerated hair. The fates of grafted cells were traced by fluorescence. The time required for hair induction was analysed and compared. Results. IFM produced a large number of normal hairs, and the time required for hair induction using IFM was 20.67 0.67 days, compared with 29.33 0.67 days for the traditional flap assay. Conclusions. The time required for hair regeneration is considerably shortened with IFM. We speculate that this is due to increased blood supply at the transplantation sites.
Introduction The morphogenesis and cycling of the hair-follicle is a complex process that depends on intensive and continuous interactions between epithelial and dermal cells during the embryonic period.1 A series of reciprocal signals between these two components results in the formation of a functional follicle.2,3 The current hairfollicle reconstruction models include in vitro and in vivo regeneration. Some studies have successfully produced follicle structures in vitro;4–6 however, these studies could not fully mimic in vivo conditions, such as all the cytokines and nutrients.7,8 Several in vivo animal models for hair reconstitution have been established, such as the chamber assay,9,10 patch assay11 Correspondence: Professor Zhi-qi Hu, Department of Plastic and Reconstructive Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou City, China E-mail: [email protected] Conflict of interest: the authors declare that they have no conflicts of interest. Accepted for publication 29 July 2014
ª 2015 British Association of Dermatologists
and flap assay.12 However, all of these have drawbacks. The patch assay is a convenient model to reconstitute hair follicles, but the direction of growth of the regenerated hairs is random. The chamber assay can produce normal hair follicles with normal hair density, but is the most labour-intensive model, requiring complex transplantation procedures and a large number of cells.13 The flap assay was first introduced by Qiao et al.12 in 2008. Compared with other methods, this model has several advantages. First, it produces hair shafts of normal quality and density; second, it is labour-saving, because it requires fewer cells than other methods, and only mouse dermal cells and an intact epidermal sheet are needed; and third, the wounds do not need to be continuously exposed as in the chamber model, which can often cause infection and death. However, the flap assay also has a number of disadvantages; for instance, it takes the longest time of the three assays to produce follicles, and it causes greater trauma.14 In the current study, we aimed to design a simple and rapid model for hair reconstitution by modifying these deficiencies.
Clinical and Experimental Dermatology (2015) 40, pp653–658
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Model for hair regeneration Y.-S. Su et al.
Methods All animal experiments were carried out under the approval of the Institutional Animal Care and Use Committee. Animals
Newborn C57BL/6J mice, 4–6 week-old athymic nude mice (BALB/cAJcl-nu) and newborn transgenic mice homozygous for green fluorescent protein (GFP) (Experimental Animal Centre, Southern Medical University, Guangzhou, China) were used. Preparation of epidermal sheet and dermal cells
Mouse epidermal sheet and dermal cells were isolated from C57BL/6J mice at natal day 0. After sterilization with 75% ethanol, the full thickness of the dorsal skin was removed and incubated in phosphate-buffered saline (PBS) with 0.1% dispase (Invitrogen, Grand Island, NY, USA) at 4 °C overnight. Each skin sample was washed three times with PBS, then the epidermis and dermis were separated with forceps. The epidermis sheet was put in a sterile culture dish containing Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM/10; Invitrogen) temporarily. The dermis was minced and digested in 0.2% collagenase (Sigma, St. Louis, MO, USA) at 37 °C for 1 h. After digestion, an equal volume of DMEM/10 was added, and the cell suspension was filtered sequentially through 100 lm and 40 lm mesh cell strainers. The cell suspension was centrifuged at 200 g for 5 min, then the cell pellet was resuspended in 1 mL DMEM/10 and counted. Finally, the cells were resuspended in 20 lL of DMEM as a slurry. Procedures for improved flap model and traditional flap assay
A lid from a 1.5 mL microcentrifuge tube (Eppendorf, Hamburg, Cologne, Germany) was used (Fig. 1a), and trimmed into a circle the same size as a 1.5 cm2 silicone plate (Fig. 1b). Once the dermal cells were prepared, the epidermal sheet was spread onto the smooth side of the plate with its basal side facing up. The slurry of dermal cells was pipetted evenly onto the centre of the basal side of the epidermis using a micropipette (Fig. 1c). The plate containing the epidermal sheet and dermal cells was placed in an incubator at 37 °C, and left for 30–60 min to evaporate excess liquid before transplantation.
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Clinical and Experimental Dermatology (2015) 40, pp653–658
Nude mice were anaesthetized by intraperitoneal injection of 1% pentobarbital sodium, and the graft area of each mouse was cleaned with betadine solution. We created a single incision in the dorsal skin. After subcutaneous dissection, the plate was inserted between the panniculus carnosus of the skin flap and the musculoaponeurotic layer (Fig. 1d), and the incision was then sutured (Fig. 1e). Slurries with different numbers of dermal cells were transplanted. The traditional flap assay was performed as described previously.12 Briefly, a full-thickness, threesided, rectangular skin flap (08 9 10 mm) was made in the dorsal skin of recipient mice. The epidermal sheet, attached to a 25 9 15 mm silicone sheet, was placed onto the wound area with the basement membrane side of the epithelium facing up. Opening of the flap to allow gross hair observation
When the surface of the skin flap became raised and completely dark in colour (Fig. 1f), the flap was considered ready for opening. The incision was opened and the flap inverted to expose the graft to the exterior environment, and the wound was closed by suturing the wound edge to the base of the flap (Fig. 1g,h). Mouse cell labelling
To assess the fate of epidermal cells in the grafts, epidermal sheets were obtained from green fluorescent protein (GFP) transgenic mice for some experiments. To track the fate of the dermal cells in the grafts, we labelled the dermal cells with dilinoleyltetramethylindocarbocyanine perchlorate (DiI), a fluorescent cell-tracking dye (Invitrogen) prior to grafting, following the manufacturer’s instructions. The GFP-expressing epidermal sheet was co-transplanted with unlabelled dermal cells, while DiI-labelled dermal cells and unlabelled epidermal sheets were transplanted using IFM. Evaluation of hair reconstitution
Hair reconstitution was evaluated by visual and histological observation. For histological observation, the skin on the transplantation site was excised and fixed in 10% formaldehyde at room temperature for 24 h. Tissue was embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin (H&E). The fate of the epidermal and dermal cells in the grafts was assessed by fixing the grafts as before, freezing at 20 °C and sectioning (6–8 lm) for examination under fluorescence microscopy. To evaluate the
ª 2015 British Association of Dermatologists
Model for hair regeneration Y.-S. Su et al.
(a)
(b)
(c)
Figure 1 Procedure for improved flap
model. (a) The lid of a 1.5 mL microcentrifuge tube was used, and (b) trimmed into a circle to produce a silicone plate. (c) The lid was placed with the smooth side facing up and the rough side facing down, and the epidermal sheet was spread onto the smooth side of the plate with its basal side facing up. The slurry of dermal cells was then pipetted onto the basal side of the epidermis. (d) The silicone plate was inserted between the flap and muscle layer through a single incision, and (e) the incision was sutured. (f) The flap was considered ready for opening when the surface of the skin became raised and completely dark in colour(g) A full-thickness and three-sided skin flap was designed prior to opening the flap. (h) The flap was opened and inverted to expose the graft to the exterior, and the wound was closed by suturing the wound edges to the base of the flap. (f–h) The number of grafted dermal cells was 3 9 106.
(d)
(f)
quality of the regenerated hair shafts, we compared reconstituted hair with normal C57BL/6J hair by scanning electron microscopy (SEM). Statistical analysis
GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA) was used for all analyses. The Student t-test was used for comparing the days needed for hair regeneration in IFM and traditional flap assay. All P values were twotailed, with P < 0.05 considered statistically significant.
Results Evaluation of hair reconstitution
Co-grafting fresh epidermal sheet with fresh dermal cells generated abundant short and black hairs on the graft site of mice treated with IFM. There was reconstruction of hair follicles, which developed a dense, natural appearance (Fig. 2a,b). Histological sections of the skin at the transplantation site showed normal skin layers, including epidermis, dermis, hair follicles and sebaceous glands (Fig. 2c). The reconstituted hair shafts were of good quality and dense, with normal appearance under SEM (Fig. 2d,e).
ª 2015 British Association of Dermatologists
(e)
(g)
(h)
Comparison and analysis of hair reconstitution in improved flap model and flap assays
For IFM, six transplantation experiments were carried out. All animals survived and all experiments produced black hairs successfully, with a success rate of 100% for hair reconstitution. However, because of the flap contraction, some hair follicles failed to grow out at the final stage, when the wound healed and the flap contraction ceased completely. Six transplantation experiments were also performed for the flap assay, and all produced hairs. The success rate for hair regeneration using this method was also 100%, but necrosis occurred in the distal part of flap in one case. Time-course evaluations were conducted to define the advantages and disadvantages of both assays. The time to opening of the flap and observing hair growth was 20.67 0.67 days for IFM and 29.33 0.67 days for the flap assay (Fig. 3). Thus, the time needed for hair induction was significantly shortened in IFM compared with flap assay. Hair regeneration caused by grafted cells
In the frozen sections examined under fluorescence micoscopy, GFP-positive epidermal cells were seen to
Clinical and Experimental Dermatology (2015) 40, pp653–658
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Model for hair regeneration Y.-S. Su et al.
(a)
(b)
(c)
(d)
form the epithelial components of the neonatal follicles. All of these components were GFP-positive, while the dermal papilla and the dermis were GFPnegative (Fig. 4a,b). In addition, we observed a resident portion of GFP-expressing cells in the neonatal epithelium. When we examined the transplants containing DiIlabelled dermal cells co-grafted with negative epidermal sheet, all the dermal papillae in the neonatal follicles were DiI-positive, confirming that the grafted cells contributed to forming the neonatal follicles (Fig. 4c,d).
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Figure 2 Evaluation of hair reconstitu-
(e)
Clinical and Experimental Dermatology (2015) 40, pp653–658
tion. (a,b) Abundant normal hair grew on the graft site after grafting, with (c) reconstitution of the normal layers of the skin (haematoxylin and eosin, original magnification 9 200). Scale bar = 200 lm. (d) The reconstituted hair shaft was of good quality and appeared to be similar to (e) a normal hair shaft under scanning electron microscopy. Original magnification (d,e) 9 2000. Scale bar 20 lm.
Discussion The goal of this study was to develop a simple and rapid method for hair reconstitution. We evaluated the time needed for hair induction in both IFM and flap assays, and found that IFM took less time for hair reconstitution. A previous study had indicated that direct contact between epithelial and dermal components and better vascularization or oxygenation of the recipient site are critical for hair regeneration.15 In IFM, the flap was created with a single incision instead of a three-sided rectangular skin flap as in the flap
ª 2015 British Association of Dermatologists
Model for hair regeneration Y.-S. Su et al.
(a)
(c)
(b)
(d)
(e)
Figure 3 Comparison of the time required for hair induction in both assays. Gross observation in (a) improved flap model (IFM) and (b) tradi-
tional flap assay. (c) After 21 days of grafting, hair growth was visible (areas marked by black dotted line). (b) In IFM, a large number of short black hairs grew out when the flap was opened. (d) In the traditional flap assay, a layer of black mucosal tissue formed, but few hairs had grown out onto the surface of the flap. (e) Days required for hairs reconstitution per graft. The density of grafted dermal cells was 2 9 106 cells/cm2 in both assays. Means SEM are indicated; n = 6 per group; ****P < 0.001, unpaired t-test.
(a)
(c)
(b)
(d)
Figure 4 Hairs on the grafts were
induced by grafted cells. The grafted epidermal sheet and dermal cells formed epidermal components and dermal papillae respectively. (a) Fluorescent image of green fluorescent protein (GFP)-expressing epidermal components from a frozen section of a newborn hair follicle. (b) Corresponding histological image of epidermal components. (c) Fluorescent image of DiI-positive dermal papilla from regenerated hair follicle after DiI fluorescent probe labelling. (d) Corresponding histological image of dermal papilla. Original magnification (a–d) 9 200. Scale bar 200 lm.
assay. Thus, the blood supply of the flap was better protected, which is essential for the survival of graft cells and regeneration of hair follicles in the early stages. The reason why we chose a microcentrifuge tube lid rather than a silicone sheet was that nude mice have loose skin, thus the space between the skin and muscle layer is relatively large. The grafted silicone sheet used in the traditional flap assay was soft and smooth, and thus slid easily over the surface of muscle
ª 2015 British Association of Dermatologists
layer, which affected transplantation. In fact, to prevent sliding, the area of the silicone sheet (25 9 15 mm) used in the traditional flap assay was much larger than the areas of its loaded epidermal sheet (8 9 1.0 mm) and of the flap, which increased trauma. By contrast, with IFM, the microcentrifuge tube lid, being a hard silicone plate (1.5 cm2 in size) with some whorls on the side that was placed in contact with the muscle layer, did not slide easily, so the
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Model for hair regeneration Y.-S. Su et al.
epidermal sheet and dermal cells were kept in close contact with the panniculus carnosus of the flap and confined to a small area for interaction. Lee et al.16 used another method, seeding epithelial and dermal cells from neonatal mice into a scaffoldlike matrix (Integra Bilayer Wound Matrix; Integra LifeSciences, Plainsboro, NJ, USA), and then transplanting the matrix onto a wound bed caused by excision of a full-thickness skin section in the nude mouse. A large number of hair follicles were produced through this one-step procedure. In IFM, a single incision was created, the operation time was greatly shortened and bleeding was reduced during grafting. In addition, the silicone lid we used is cheap and easily available, although two operations are still needed.
Conclusion We have developed a simple and rapid model for hair regeneration by improving the previous flap assay. In this new technique, trauma is reduced and the time needed for hair induction is shortened considerably. We speculate the technique preserves local blood supply, which shortens the time for hair induction.
Acknowledgements This study was supported financially by the Natural Science Foundation of China (Grant No. 31170949). The study was conducted in the Central Laboratory of Southern Medical University, Guangzhou, China.
What’s already know about this topic? Several animal models for hair reconstitution
have been established; however, these models have a number of shortcomings.
What does this study add? We have developed a simple and rapid mouse
model for hair regeneration based on the traditional flap assay. The time for hair induction was considerably shortened. We speculate that this method may increase blood flow, thus shortening the time for hair induction in vivo.
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