Table 5.
Preclinical in vivo studies of Tissue Engineering for non-scarring alopecia.
Cell Types and Scaffold | Type of Study | Experimental Design | Results | Conclusions | Reference |
---|---|---|---|---|---|
Mouse epidermal and dermal cells in a gel-like endogenous matrix or in Integra™ Bilayer Wound Matrix | In vivo study Athymic nude, hairy SCID, or normal mice of the same inbred strain |
Dissociated new-born mouse epidermal and dermal stem cell were mixed in different ratios. For gel-like endogenous matrix: 150–200 μL of cell suspension, containing 2–20 million cells, was pipetted onto a tissue cell culture insert. For each 1.5 cm2 piece of Integra, 12 million epidermal cells and 60 million dermal cells in 200 μL of serum-free medium. The intended area of skin to be grafted for hair bearing was excised in full thickness. |
1-At day 8, hair germs started to appear, which progress to the hair peg stage at about day 9. 2-Hairs can be seen on the surface of the wound as early as 11–15 days postgraft. 3-Histological sections of the skin at day 11 postgraft showed that normal layers of the skin were regenerated. 4-after hairs were clipped or plucked, they grew and reached normal length in about 2 months. |
1-Ratio of 1:5–10 for epidermal:dermal cells is optimal. 2-There are no differences of skin quality or hair growth when tissue culture inserts or IntegraTM are used. 3-After 18 months, hair growth and cycling are active. |
[76] |
Mouse embryonic epithelial and dermal cells using the organ germ method. Collagen gel |
In vivo study -C57BL/6 mice -C57BL/6-TgN (act-EGFP) mice -C57BL/6-TgN (act-EGFP) OsbC14-Y01-FM131 mice -Balb/c nu/nu mice |
Bioengineered hair follicle germs: 7.5 × 104 epithelial cells and 7.5 × 104 mesenchymal cells which were derived from skin from C57BL/6-TgN (act-EGFP) mouse embryos. After 2 days in culture, to develop and mature bioengineered hair follicles, they were transplanted into the sub-renal capsules of 8-week-old C57BL/6 mice At 14 days after engraftment, mature bioengineered hair follicles were harvested and dissected into a single or a couple of follicular units via stereomicroscopic observation. Mature bioengineered hair follicles were intracutaneously grafted into Balb/c nu/nu mice. |
1-After 7 to 10 days after the orthotopic transplantation, the wound was completely healed. 2-The eruption of the bioengineered pelage shaft was observed at 14 ± 1.8 (n = 30) days at a frequency of 90% (n = 33) 3-The bioengineered hairs repeatedly exhibited growth and regression. 4-The periods of growth and regression of bioengineered hair follicles lasted 11.0 (±2.6) days and 9.4 (±2.4) days, respectively. |
1-Bioengineered hair follicles generated by ectopic transplantation can functionally replace orthotopic FUT therapy. 2-Ectopic bioengineered pelage follicle connected to the epidermal layer of the skin, reproduced the stem cell niche and the hair cycle equivalent to the natural pelage, and repeatedly produced the same hair types during the hair cycles. |
[78] |
Mouse embryonic skin epithelial and mesenchymal cells. Epithelial cells from adult vibrissa-derived bulge region and primary cultured dermal papilla (DP) cells. Collagen gel |
In vivo study -C57BL/6 -C57BL/6-TgN (act-EGFP) OsbC14-Y01-FM131 mice -Balb/c nu/nu mice |
The bioengineered pelage follicle germs: mouse embryonic skin epithelial and mesenchymal cells (7.5 × 103 of each cell type) The bioengineered vibrissae follicle germ: epithelial cells (1 × 104 cells) isolated from the adult vibrissae-derived bulge region and primary cultured DP cells (3 × 103 cells) Bioengineered hair germs were ectopically engrafted into the sub-renal capsules of C57BL/6 mice or intracutaneously transplanted onto the back of Balb/c mice. |
1-Eruption and growth hair shafts were observed at a frequency of 94% (n = 78) and 74% (n = 62) for bioengineered pelage and vibrissae follicles, respectively. 2-Bioengineered pelage follicle and the vibrissae follicle formed correct structures comprising an infundibulum and sebaceous gland in the proximal region. 3-Bioengineered pelage follicle germs were found to produce all types of pelage hairs. 4-The bioengineered pelage and vibrissae follicles repeated the hair cycle at least 3 times during the 80-day period. |
1-Both bioengineered hair follicles produce follicles that can repeat the hair cycle, connect properly with surrounding skin tissues and achieve piloerection. | [79] |
Human Dermal Papilla (DP) cells and Keratinocytes (KT) in collagen-glycosaminoglycan scaffolds | In vivo study | Human or murine DP cells, combined with foreskin-derived keratinocytes (KT) or transduced KT with pBABE-puro encoding N-terminally truncated β-catenin (KT’) (expression induced by hydroxytamoxifen (4OHT) administration) A positive control fabricated with murine hair from newborn cells was also evaluated. After 10 days incubation at air–liquid interface, Engineering Skin Substitutes (ESS) were grafted to athymic mice and were evaluated for 6 weeks. |
1-EF1 and WNT10B were significantly higher in 4OHT-treated ESS compared with vehicle-treated ESS but no hairs were observed in ESS with KTs’ and hDP cells. 2-Only ESS with mDP cells formed follicular structures, as confirmed by trichohyalin and keratin 10 immunostaining. |
1-Chimeric hair follicles were successfully generated in ESS containing combinations of mDP cells and KTs or KTs’, although they were deficient anatomically. 2-DP cells play an important role in the induction of hair morphogenesis in ESS. |
[83] |
Mouse Dermal Papilla (DP) cells and human keratinocytes in grafting chambers | In vivo study -Versican-GFP transgenic mice -Nude mice (bulb/c, nu/nu) |
DP cells were isolated from skin of Versican-GPF transgenic mice. Keratinocytes were isolated from human scalps tissues and neonatal foreskins. Human epidermal cells and DP cell fractions (containing 1 × 106–107 cells of each) were transferred to grafting chambers implanted on the dorsal skins of nude mice (bulb/c, nu/nu). Chambers were removed 1 week after grafting, and hair follicle formation was assessed at 3–4 weeks. |
1-DP cells were able to induce hair follicles together with the epidermal component but only when both epithelial and mesenchymal components were present. Hair pegs were formed a week after grafting. 2-When the number of epidermal cells was reduced to 1 × 106 cells (10% of DP cells), the efficiency of hair follicle reconstitution was mostly unchanged. 3-When human keratinocytes were included, hair follicle-like structures were formed at the graft sites 4 weeks later and innermost regions of the structures were clearly keratinized. 4-Human adult cells also have the same ability to differentiate into follicular keratinocytes as neonatal foreskin-derived epidermal cells. |
1-Results show that hair follicle-like structures consisting of human keratinocytes and murine mesenchymal cells are generated. 2-Epithelial-mesenchymal interactions exists between human and mouse cells. |
[84] |
Mouse dermal and human epidermal cells. Patch assay |
In vivo study | Mouse dermal and epidermal cells were freshly isolated from C57BL/6 used for control experiments. Human dermal papilla (DP) spheres (104 cells) were prepared from two-dimensional (2D) cultured DP cells using either low cell-binding plate or hydrocell plate and combined with freshly isolated mouse epidermal cells for implantation A total of 200 DP spheres (2 × 106 cells) prepared from human DP cells were mixed with fresh mouse epidermal cells (2 × 106 cells) and implanted. 50 DP spheres (5 × 105 cells) prepared from human DP cells were mixed with fresh mouse epidermal cells (5 × 105 cells) and implanted. Mice were killed 2 weeks after cell implantation in order to verify hair follicle induction. |
1-Hair follicle was observed in positive control experiments with mouse dermal and epidermal cells. 2-Hair follicle formation was observed when human DP spheres from various passages of culture were mixed with new born mouse epidermal cells. 3-Hair follicles were never observed when 2D cultures from the same population were use 4-The morphology and size of hair follicles induced by human DP spheres resembled the ones induced by mouse dermal cells. |
1-Using a reconstitution assay, sphere formation increases the ability of cultured human DP cells to induce hair follicles from mouse epidermal cells | [85] |
Human Dermal Papilla (DP) cells embedded into rat tail collagen type 1 and neonatal foreskin keratinocytes (NFK) Dermal-epidermal composites (DEC) |
In vivo study -Nude mice |
Human DP cells were isolated from temporal scalp dermis. DECs were constructed by combining DP cells with rat tail collagen type 1, adding NFKs on top and bringing the constructs to the air-liquid interface for 2 days before grafting onto female nude mice. |
1-Alkaline phosphatase activity was variable between samples, with cells from 3 of the donors showing alkaline phosphatase activity in more than 50% of the cells. 2-8 weeks after grafting, hair follicles (HFs) were observed in mice grafted with the 3 human DP cells with higher alkaline phosphatase activity. 3-HFs had a bulb, dermal sheath, hair matrix and cortex 4-Cells in the region of the DP and displayed alkaline phosphatase activity, normal reactivity with specific antibodies to human nestin and versican. 5-Basal layer of the outer root sheath was immunoreactive for keratin 15. |
1-Cultured specialized human cells such as DP cells can induce complete pilosebaceous units in vivo in the grafted DEC model. | [86] |