**11. Hair regrowth studies** *in vitro*

(application of stem cells or plasma-rich platelets and low-level laser therapy), even if clinical studies in this respect are lacking. A large variety of over-the-counter products claim to treat hair loss pathology: hair tonics, hair balms, hair masks, shampoos, leave in conditioners, topi-

Alternatives to traditional treatment are laser (low-level laser therapy) and platelet-rich

Gene therapy aims to deliver genetic material (DNA) into the patients' cells with either a prevention or therapeutic purpose. The therapeutic effect could theoretically be obtained by replacing the mutant gene that causes the disease with a healthy gene, inactivating a mutated gene that causes an imbalance in the organism or introducing a new gene that could fight a particular disease. For the introduction of the gene, a carrier called vector is used, and it usually consists of a modified virus (retrovirus) that will not produce a disease in the organism, but will deliver the gene by integrating the genetic material into the chromosome of a particular cell. The delivery pathway may consist of a direct injection into the tissue or it can be given

As new evidence shows that 80% of the baldness is genetic, gene therapy could be the solution, although it encounters technical problems that have not been solved up to the present [69]. Most of the hair loss complains in both female and male patients are due to the presence of androgenetic alopecia, caused by hyperandrogenism and sensitivity to dihydrotestosterone (DHT). It has been noticed that people naturally lacking from birth the 5-alpha reductase enzyme (which converts the testosterone to DHT) never develop androgenetic alopecia [50].

Human scalp has DHT-resistant follicles in the occipital area, this location being used to extract the hair follicles for transplant into the vertex or to the fronto-parietal area [53]. Gene therapy may be a solution in this case, if it can trigger the hair follicles with DHT-sensitive cells and change them into DHT-resistant follicles that could regrow hair without being affected by androgen hormones [70]. Another option would consist of the ribonucleic acid (RNA) interference to block the genes responsible for hair loss. Messenger ribonucleic acid (mRNA) represents the carrier of genetic information from the DNA out of the cell nucleus into the cytoplasm, where it is translated into specific proteins, such as receptors, enzymes. Small fragments of nucleic acids, such as small interfering RNAs (siRNAs), can target a specific gene and block the production of any type of protein in a cell. In hair loss, this technology could be used

in order to inhibit the androgen receptor (AR) and the 5-alpha reductase enzymes.

Up to the present, an attempt to effectively control delivery of small interfering RNA using biodegradable cationized gelatin microspheres in an animal model of disease was first performed in 2008. Researchers administered local injections of interleukin-4 and neutralizing anti-interferon-γ antibody in C3H/HeJ mice. They concluded that alopecia areata was effectively treated as the treatment suppressed CD8 T cell infiltrates around the hair follicles and

cal solutions, or foams function as potential anti-hair loss agents [43, 44, 63–68].

plasma (PRP) injections [47, 69].

**10. Hair follicle regrowth using gene therapy**

240 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

intravenously, to reach the blood flow [36].

Human hair follicles as research material for hair loss and regeneration involve ethical problems, an invasive collection method and a limited quantity of follicles available for extraction and testing [60, 61].

The first methods of isolation and maintenance of hair follicles in cell cultures go back to 1990, when several researchers used this method in order to study the biology of the hair cycle [74, 75]. Follicles were usually taken during face lifting surgery, but only a third were suitable for the isolation phase of the hair transplant, due to improper collection procedures. The follicles needed to be isolated from human scalp in a few hours, maintained at 2–6°C, in an Earl medium, combined with phosphate buffered saline solution, with calcium and magnesium added. Only the follicles that seemed intact were used.

*In vitro* hair research was supported by the identification of growth factor function in the process of hair regrowth and differentiation [76–79]. Philpott et al. have reported that in the absence of insulin, follicles prematurely enter the catagen stage [80]. Subsequent *in vitro* and *in vivo* studies, in murine and human models of hair follicles, have demonstrated that IGF-1 level is a regulation factor of hair growth and together with IGF-1 receptor influence hair growth cycle.

Other studies performed in 1990 have shown that transforming growth factor beta 2 (TGF-ß2) promotes anagen to catagen transition. Several inhibitors of hair follicle growth *in vitro* have been identified such as interleukins (IL-1 alpha and beta) and tumor necrosis factor (TNF-alpha). Researchers concluded that these cytokines play a significant part in the pathophysiology of hair inflammatory diseases. Although the factors that perform the transition *in vitro* from anagen to catagen have been discovered, inducing a full hair growth cycle has not been made possible yet. Murine models of hair follicles, isolated at different growth stages *in vitro* seem to maintain their cyclic activity and to illustrate their status *in vivo* [81].

On the other hand, healthy human dermal papilla cells, isolated from hair follicle, lose the ability to produce hair growth when being outside the body. Also, cycling hair follicles cannot be maintained in culture for any length of time [82].

The data that we now possess about the life and function of the hair follicle in health and disease rely on the successful research performed *in vivo* (experiments on natural animals and genetically manipulated models) and *in vitro* (cultures of a cell type—dermal papilla or organ culture of isolated cell follicles). The preference for one of the two experimental alternatives depends on several factors: the purpose of the research and the advantages and disadvantages involved.

harmful effect of research performed on animal models, scientists tend to reduce the number of animals used to obtain valid results, to refine the experimental technique, or replace it with

Evaluation of Animal Models Suitable for Hair Research and Regeneration

http://dx.doi.org/10.5772/intechopen.69698

243

A large variety of animals (mice, rats, hamsters, rabbits, sheep, and even stump-tailed macaque) provide useful models for the *in vivo* study of hair loss and regrowth, but 95% of

Mice represent an excellent model to study the hair cycle for several reasons: the first two cycles of the mouse hair follicle are synchronized; the mouse hair cycle is short, lasting for 3 weeks; hair follicles can be easily harvested and examined at specific time points in the cycle. Most importantly, the stages of the hair cycle have been well characterized in the mouse: anagen being morphologically subdivided into six stages and catagen into eight [22]. The periodic intervals of rodent hair cycles (especially the anagen-growing phase) seem to be less susceptible to iatrogenic influences [90]. The mouse hair cycle does not differ structurally from the human hair follicle cycle, except for the fact that during catagen the hair bulb is remodeled, but the vibrissae follicles do not retract. Scientists have recently discovered that a certain progenitor cell population in mice is analogous to the human cells, encouraging

Besides studying the normal hair cycle on mice, scientists also focussed on the growth waves and hormonal control [91]. Significant differences between species regarding the follicular function and limited androgen-sensitive models were noticed [92]. Spontaneous mutations have been discovered and studied on hairless, nude, and tabby mutants, waved and angora animals, leading to the identification of new genes involved in hair loss and opening the path

Transgenic mice, also known as "knockout mice," are mice with altered genome through the use of genetic engineering. This gene-targeting technique has revolutionized the biomedical research by offering researchers the ability to create a specific animal model for the most common human diseases. In order to select the most appropriate immunodeficient mouse models for research purpose, scientists also take into consideration: background strain, behavior, husbandry, disease susceptibility, life span, breeding performance, radiosensitivity, functionality of various endogenous immune system components, and leakiness (tendency to produce

Up to the present, immunodeficient mice (with T and B cells deficiencies) were used as models for autoimmune disease mechanisms and androgenetic alopecia studies. The androgen action upon the hair follicles has been studied on spontaneous and genetically engineered

The C57BL/6 mouse is the most popular laboratory rodent, widely used and studied, having its entire genome published. Research applications using this particular type of mice include immunology, cancer, neurodegerative disease, age-related hearing loss, bone density, diabetes,

nonanimal research methods.

**12.2. Animal models used in hair loss and regrowth**

research on this particular animal model.

for transgenic technology research [93, 94].

functional B and T cells as they age).

nude mutant mice [95].

the animals bred for research purposes are rats and mice [85–89].
