The hair follicle is a dynamic structure in the body that includes different niches of stem cells. The hair follicle stem cells (HFSCs), are valuable and rich sources of stem cells because of their easy access, multipotency, non-oncogenic and abundance. Tissue engineering is based on scaffolds, stem cells and biomolecules. In the present study, we investigated the differentiation capacity of HFSCs into bone cells on the natural collagen scaffolds. Stem cells were extracted from the hair follicle bulge area of rats’ whisker with range of weight 150-200gr and 4-6 week old and expanded to the 3rd passage in stem cell growth medium, then treated and cultured in the osteogenic medium. After 21 days the result of morphological changes with invert microscope, the level of mineralization with Alizarin red and von Kossa staining and the expression of bone bone specific genes with reverse transcription-polymerase chain reaction(RT-PCR) were evaluated. Collagen hydrogel scaffold were prepared by dissolving in acetic acid and freeze-drying methods then studied by scanning electron microscope (SEM). The results of stem cell culturing in the scaffold and non-scaffold condition showed that both conditions affected by bone differentiation medium and changes to large and cubic morphology with star-shaped nucleus that are indicated with Osteopontin and Alkaline phosphatase gene expression and presence of red and black calcium mass in Alizarin red and von Kossa staining respectively. SEM results indicated the existence of collagen scaffold with porous surface of the nanofibers. Although more investigations are required, the results indicate bulge stem cells possess significant capacity for osteoblastic differentiation and Collagen scaffolds are found suitable place for the growth and differentiation of cells and may be useful for developing new bone regenerative medicine therapies.
Keywords: bulge, Collagen hydrogel, acetic acid , osteoblasts, morphology.
The skin contains a mini-organ, the hair follicle (HF), which is subjected to life-long remodeling via the hair cycle . Hair follicles originate of the interaction epithelium and mesenchyme during embryonic development, that is characteristic of mammals (1, 2). Stem cells are undifferentiated cells that divide during their life and can repair damaged tissues. These cells have two basic characteristics, self-renewal and the ability to become different types of cells (3). Hair follicle stem cells are located in an area called the bulge, these cells generate hair cells, epidermal glands, and hair rectus muscle (4, 5). Studies have shown that hair follicle stem cells can differentiate into other cells, such as adipocytes, bone, neurons, keratinocytes, glia, smooth muscle cells, and melanocyte. They have a special place in regenerative medicine. High proliferation and easy access to adult stem cells in the skin make them a valuable and rich source (6-9). Most bone damages are repaired in the body, but this process does not take place in some cases due to the depth of the lesion. Therefore, implants and organ transplant methods are used that are very painful and dangerous. To regenerate functional bone tissue using tissue engineering, 5 characteristics are required; osteoconductive and osteoinductive properties, osteogenic ability, immune rejection-free status, and mechanical load-bearing ability (10-12) . It is expected that in the near future, engineering, regenerative medicine, and cell therapy with tissue engineering will provide the foundation for repairing bone defects (13, 14). Bone tissue engineering is based on a combination of biomaterials, cells, and therapeutic factors (15) Osteoblasts have an important role in bone formation(16). Osteoblasts express different bone specific gene during the differentiation, in primery stage express alkaline phosphatase (ALP) and type 1 collagen, and in last stage osteoblasts express osteocalcin(17). Scaffoldes by creating structures that mimic the extracellular matrix involves very small spaces; it leads to the formation and differentiation of the target tissue (18-20). The most important features of scaffolds are bio-degradability and bio-compatibility (21). Natural polymers may provide a better biological environment for stem cells because they naturally contain parts that can send important signals during development (22). Biomaterial scaffolds for tissue engineering must be able to support the cellular structures and provide the necessary mechanical properties. Natural tendon and ligament tissue contain fibres and collagen fibrils, which create a fine environment for the participation of cells. The tissue has mechanical properties . So far, a lot of research has been done on collagen outside cells for potential use in tissue engineering. The collagen found in the extracellular mammalian body is responsible for tissue engineering, drug delivery systems, and wound healing (23, 24). In this study, collagen type 1 from rats’ tails provide a scaffold to grow the hair follicle stem cells and their capacity for osteoblast differentiation are evaluated. These properties would be useful for developing new methods for bone tissue engineering.
Materials and Methods
In this experimental study, animals were used according to the guidelines of the Iranian Council for Use and Care of Animals. Wistar male rats (n=15, 4-6 week old, 150–200 gr) were procured from Medical Science, Ardabil, Iran. All rats were observed daily under controlled conditions 12-hour/12-hour light and dark cycle with cage cleaning and were given free access to water and food all the time.
Hair follicle stem cells isolation and cultivation
The animals were sacrificed using chloroform (Merck, Germany). The heads were rinsed with a mixture of betadine and hydrogen peroxide for five minutes, and after shaving were washed with 70% alcohol. The two sides of the upper lips, containing whisker follicles, were dissected and transferred under a sterile environment to a solution of PBS containing penicillin (100 U/ml), streptomycin (100 mg/ml), and amphotericin (0.5 µg/ml). The tissues were cut into small pieces, after which all the whisker follicles were lifted out. After rinsing, the follicles were removed into a 35-mm dish. The bulge region of all whisker follicles was separated with an insulin needle by transversal cutting under a dissecting microscope, after which the collagen capsule was incised. About 30 pieces of bulge samples containing stem cells were transferred into a flask containing Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (DMEM/F12, Gibco, Germany) with L-glutamine, 20% foetal bovine serum (FBS, Gibco, Germany), and 2% penstrept (Sigma, USA). Incubation took place at 37 °C, 95% humidity and 5% CO2. After approximately one week of initiation culture, bulge tissue sticking to the bottom of the flask and stem cells began to migrate. The cell growth at first was just in the colonies scattered around the tissues. After migration of stem cells, the tissues were removed from the flask and the stem cells were cultured uniformly over the flask with trypsin (0.125%, 0.02 EDTA, Gibco, Germany) treatment. According to the stem cell growth, culture medium was changed every three days (25-28) .
Prepare collagen scaffold
First, the animals were sacrificed with chloroform (Merck, Germany) and the collagen scaffold was produced, as described by Rajan et al. with a few changes (26). The tails and feet were briefly placed in 70% alcohol for disinfection. After the tails were dissected from the bodies, they were placed in a freezer at −80 °C and were transferred to 4 °C before the start of work to soften for at least for 24 hours. Tail skin, after disinfection of the cross sections, were drawn for isolating white tendons into the buffer container. Tendons were put in acetone and isopropanol for five minutes each for dehydration and finally dissolved in acetic acid 0.04 N (Merck, Germany) at 4 °C for 48 hours with a magnetic stirrer. The resulting gelatinous mass was kept at −20 °C for three days. After 24 hours of freeze-drying, a white powder was obtained which was used to prepare scaffolds. Thus, the powder was dissolved in acetic acid at 4 g/litre in 0.04 N, and was centrifuged at 12,000 rpm for 45 minutes. The supernatant containing collagen type 1 was isolated. Acetic acid solution pH was kept at about 3 to comply with the solution of 0/1 N NaoH (Merck, Germany) adjusted on 7 as a result of a hydrogel scaffold sheet that is cut out and transferred to plates to be used for cell culture and determining scaffolding characteristics(22, 29) .
Induction of osteoblastic differentiation
Bulge stem cells (1×
cm2) were detached from hair follicles and cultured in 6-well plate. Stem cells Differentiation into bone cells was induced by a differentiation medium containing Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Germany) and osteogenic supplements (50 mg ascorbic acid 3 phosphate, 10 nM dexamethasone, 10 mM β-glycerol phosphate), 10% (v/v) Fetal bovine serum (FBS), and 1% penicillin-streptomycin. The culture flask was incubated at 37 °C and 5% CO2 for 21 days by refreshing differentiation medium every three days (30).
Specific gene expression
The bulge stem cell RNA and induced cell RNA were extracted using TRIzol reagent (Invitrogen, UK) according to the manufacturer’s instructions. The total cDNA was synthesized by Oligo (dT)18, RT enzyme (Fermentas), and specific primers for Nanog, Oct4, and Sox2 in stem cells. In order to detect differentiation, we screened for expressions of Osteopontin (Ost) and Alkaline phosphatase (Alp) genes in differentiated cells, and β-actin was used as a positive control (Table 1).
Alizarin red staining
The control and treated cells were cultured in six-well plates for 21 days. When the medium was depleted, cells were washed with PBS and fixed with 4% (v/v) formaldehyde for 15 minutes at room temperature. They were then washed twice with PBS again. For staining, 1 ml of 1% Alizarin Red solution (Merck, Germany) was added per well. The plates were then incubated for 30 minutes at room temperature with shaking. They were then rinsed with distilled water and dried at room temperature. The red sediment calcium deposition was checked under a light inverted microscope and was photographed(31).
Von Kossa staining
The control and treated cells were cultured in six-well plates for 21 days. When the medium was depleted, cells were washed with PBS and fixed with 4% (v/v) formaldehyde for 15 minutes at room temperature. They were then washed twice with PBS again. For the stain, 1% silver nitrate solution (Merck, Germany) was added to plates for 30 minutes. The plates were placed under ultraviolet light for 20 minutes. After removing the silver nitrate solution, the plates were washed several times with distilled water and treated for five minutes with 2/5% thiosulfate solution (Merck, Germany). They were then washed with distilled water and dried at room temperature. The black sediment calcium deposition was checked under a light inverted microscope and was photographed (32)
Table 1: The primers which were used in the genes expression analysis
Bulge hair follicle stem cells
After sticking bulge follicle tissues to the floor of the culture flask, the cells migrated to the floor within two to three days. After about a week, a lot of cell colonies were seen around the tissues (Fig.1A, B). Through the colonies trypsin treatment, cells were spread uniformly over the floor with flattened morphology (Fig.1C). The pattern of Hair follicle stem cell specific genes expression shows that Nanog and Sox2 genes, depending on the length of the designed primers, were observed to be 414bp and 359bp respectively (Fig.1D).
Fig.1: A piece of Bulge tissue sticks to the floor of culture flask and cells migrate A. Hair follicle Stem cells colonies on the floor of the flask B. Stem cells are dividing with two morphologies , flattened and elongated on the floor of the flask C. Amplification of the HFSCs marker transcripts by RT-PCR (left to right: Size marker, β– actin, Oct4, Sox2 and Nanog ) D.
Differentiation to bone cells
Induction of the differentiation of the cells was carried out by the medium containing inducing factors. The differentiated cells showed cubic morphology with star shaped nucleus (Fig.2A, B). Differentiated cells stained by Alizarin Red and Von Kossa was shown by calcium deposits in the extracellular environment in red and black colours respectively (Fig.2D, F). The pattern of expression of bone-specific genes, Alkaline phosphatase, and Osteopontin show that genes expressed in differentiated cells were, depending on the length of the designed primers, observed to be 437bp and 461bp respectively (Fig.2G, H).
Fig.2:Large and cubic bone cells with star-shaped nucleus and control A, B. The mass of calcium red sediment in alizarin red stain and control C, D. The mass of calcium black sediment in Von Kossa stain and control E, F. Gene differentiation Ost, Alp in bone cells and stem cells in the presence of witness β– actin H,G.
After changing the pH from acidic to neutral, collagen hydrogel scaffolds were created. SEM studies showed a porous scaffold with cross-linked collagen fibres woven together (Fig.3). Investigation of scaffold porosity showed the most frequents pores with small pore size (1/4-10 micrometres), but highest surface of the scaffold were occupied with larger pores (10-22 micrometre) (Fig4). Light microscopic studies observed stem cells attached to the scaffold (Fig.5A). Study of cell differentiation at the surface of the scaffold at the 21st day, showed mass differentiated cells with calcium deposition, as the Alizarin red and Von Kossa staining led to calcium in red and black masses respectively (Fig.5 C, F)
Fig.3: Hydrogel collagen sponge A. Collagen B. Collagen hydrogel scaffold C. Scanning electron microscope image of the collagen hydrogel scaffold’s surface D.
Fig.4: Collagen hydrogel scaffold pore size distribution.
Fig.5: Stem cells attached to the collagen scaffold A. Bone cells with Alizarin red stain on the surface of scaffold with control cells B, C. Bone cells with Von Kossa stain on the scaffold with control cells D, E.
In this study, the isolation, growth, proliferation, and morphology of hair bulge stem cells and their differentiation into bone cells were performed in three-dimensional scaffold. Isolation of stem cells was performed in observed colonies around the area of the bulge tissue in the tissue culture flask. The colony formation is a special characteristic of the bulge area tissue culture. Approximately three to four days after cells migrated to the floor of the flask, sticking of tissue was carried out. Growth of cells was first slow and gradual, but after a few days was quicker and logarithmic. Two types of morphology were observed in bulge area tissue culture which were flattened and elongated stem cells. Nanog and Sox2 genes expression in the hair follicle derived cells in the presence of control β-actin proved stemness. Bone differentiation happened gradually by changing the morphology through seven days of treatment. Bone cells, in terms of morphology, are large and cubic cells with star-shaped nucleus that had differentiated compared to spindly stem cells. Osteopontin and Alkaline phosphatase genes expressed in the differentiated bone cells. Red and black mass of calcium was seen in Alizarin red and Von Kossa staining receptively. In this study, collagen hydrogel scaffolds were prepared using type 1 collagen from rat tail tendon. Scaffolds were prepared at several stages: collagen hydrogel, scaffold sponge, and collagen hydrogel scaffold. The SEM image shows abundent pores on the surface. Investigation of the scaffold pore size shows that the highest space occupy with big size of pores was while the number of small pores was higher. The scaffold pore size is important item in tissue engineering because very big pores cannot prepar stability and very small pores cannot provide essential material for cells and space for cells signaling. In addition to the cultured stem cells on the scaffold, differentiation into bone was done and could be seen through mass of the black and red colours in Von Kossa and alizarin red staining. The results show that hair follicle bulge stem cells have significant potential to differentiate into bone. Culture and differentiation of hair follicle stem cells into bone in three-dimensional hydrogel scaffold of collagen, because of collagen’s special properties, leads to differentiation into bone cells similar to natural bone morphology in the body. Recent studies have shown that hair follicle stem cells, because of easy access, autologous nature, high proliferation, and pluripotent properties, are one of the best sources of stem cells in regenerative medicine. Our knowledge about many of the properties is low and requires further study (33).In order to study these cells, cells located In rats’ whisker bulge areas are used a valuable niche in certain areas immediately below the upper part of the hair follicle glands, near the right muscle forefinger hair(34). Important characteristics of bulge stem cells are slow division and high colony formation, as shown in the experiment by Kobayashi and colleagues and other researchers (35) .Stem cells, for use in regenerative medicine, require a three-dimensional environment for the formation of organs; scaffolds provide such a three-dimensional environment. Cells cultured in the laboratory often done in two-dimensional culture dishes. The three-dimensional growth by mimicking the ECM makes a lot of changes in the function and formation of cells and has been highly regarded. A lot of scaffolds are produced by different methods to help repair the tissues and organs of our bodies (36) .Collagen scaffold is natural; it was originally designed to burn repair, and later used in tissue engineering (37).
These findings have enhanced our understanding of bulge area stem cells characters. We have shown that bulge stem cells can potentially differentiate into bone cells. Our experimental results suggest that the use of collagen scaffolds can be helpful in the differentiation into bone tissue. Since the bulge stem cells have high potential for differentiation into other cells, we recommend more research.
This research was financially supported by University of Mohagheg Ardabili, Ardabil, Iran. There is no conflict of interest in this study.