Introduction

Rabbit pinna is an effective experimental model for regeneration and restoration processes (Ten Koppel et al. 2001). Despite the differences between human and animal tissues, animals are intensively being used as models in developmental biology. Rabbit is a widely used animal model in tissue regeneration studies (Gorensek et al. 2004; Fröhlich et al. 2007).

Regenerative medicine, by using allogeneic or autologous cells in combination with a scaffold, offers the possibility of healing and repairing for different types of tissues (Langer and Vacanti 1993; Fröhlich et al. 2007). Regeneration is the ability to recreate lost or damaged tissues, organs, and limbs. Abstract regenerative biology has now been recognized as a new field with certain aims and goals. One direction of this new field is to understand the basic mechanisms by which tissues can be repaired and restored. The other direction examines the possibility of using this basic knowledge in promotion of tissue repair efficiency. Regeneration of tissues can occur by differentiation of stem cells (local or non-local) or by transdifferentiation of the local terminally differentiated cells. Meanwhile, in the past few years, a large body of data has been accumulated regarding the existence of stem cells and their participation in tissue renewal (Tsonis 2002). Regeneration of a lost limb occurs in two major steps, first de-differentiation of adult cells into a stem cell state similar to the embryonic cells and second, development of these cells into new tissue, more or less the same way as it was first developed. Some animals instead keep clusters of non-differentiated cells within their bodies, which migrate to the parts that need healing. A central question is whether the generation of progenitor cells during limb regeneration and mammalian tissue repair occur via separate or overlapping mechanisms. Limb regeneration depends on the formation of a blastema, from which the new appendage develops (Fröhlich et al. 2007). Epimorphic limb regeneration proceeds by rapid wound closure and is critically dependent on the formation of a multipotent mesenchymal growth zone, the blastema, which gives rise to the newly formed limb (Wallace 1981; Fröhlich et al. 2007).

Blastema tissue is a mass of undifferentiated cells capable of growth and regeneration into organs or body parts. Blastema are typically found in the early stages of an organism’s development such as in embryos, and in the regenerating tissues, organs, and bone (Tsonis 2002). At the end of the period of blastema formation (6 to 7 d after amputation), the cells begin to proliferate rapidly. At the beginning of the stage of proliferation, the cells have relatively abundant, granular cytoplasm, but the amount of cytoplasm decreases as cell division proceeds.

Dividing cells tend to be closely aggregated in groups at the tip of the limb in the older blastema, whereas in the younger one (7 to 10 d) they are more uniformly distributed (Hay 1958).

Many experiments have been performed in search for reagents to influence and improve the whole process of tissue regeneration. One candidate in this regard is vitamin A. Vitamin A is a generic term for a large number of related compounds. Retinol (an alcohol) and retinal (an aldehyde) are often referred to as preformed vitamin A. Retinal can be converted by the body to retinoic acid, the form of vitamin A known to affect the expression of certain genes. Retinol, retinal, retinoic acid, and related compounds are known as retinoids (Groff and Gropper 1999). Vitamin A, as an irreversibly oxidized form of retinoic acid, plays an important role as a hormone-like growth factor for epithelial and other cells (Berdanier 1997). Another reagent to influence the cellular behavior is vitamin C. Ascorbate (an ion of ascorbic acid) is required for a range of essential metabolic reactions in all animals and plants. It is an important constituent of the daily human diet and has been proven to promote wound healing in various wound models (Lanman and Ingalls 1937; Cabbabe and Korock 1986). Ascorbic acid is a common cofactor of many hydroxylating enzymes. It is essential for the synthesis of the important skin component collagen by hydroxylation of pro-collagen (Combs, 1992; Tsao 1997; Wahli et al. 2003) and is also involved in formation of cartilage and endothelium of the blood vessels. Moreover, some researches show more proliferation in cell culture when vitamin C is added (Wei et al. 2014). Given this range of essential roles, it is not surprising that many tissues are damaged in case of insufficient ascorbic acid intake and that the presence of adequate supplies of vitamin C is essential for efficient wound healing.

Materials and Methods

Punch-hole procedure.

Experiments were performed in strict compliance with the University of Mashhad Animal Ethics Guidelines in accordance with the Iran Animal Welfare Act.

In this study, five male New Zealand white rabbits were provided from Razi Institute of Mashhad, each one weighing 2500 ± 100 g and aged about 2–3 mo. The organ of focus in this study was the pinna. Special punching devices were used for punch. The hairs of the area were shaved and disinfected by ethanol (70%). The rabbits then, were anesthetized by intramuscular injection of anesthetics including a mixture of ketamine 10% (Alfasan, Woerden, Holland) (1 ml/kg body weight) and xylazine 2% (Alfasan, Woerden, Holland) (0.5 ml/kg body weight). After that, the punch of the pinna was performed in the medial region of the ear, between the central arteries and peripheral veins, each hole with a diameter of 4 mm. Two days after the primary punch, the tissue around the punched holes was biopsied by a special gadget with a diameter of 5 mm. After that, the rings on the rabbit’s ear were immediately washed twice with normal saline, PBS2, and DMEM3 media, respectively. After the final washing, the pieces were placed in a six-well plate.

Tissue culture and characterization.

The biopsy materials (rings) were resuspended in culture medium containing DMEM (Gibco, Waltham, MA) supplemented with 15% fetal bovine serum (Sigma, St. Louis, MO), penicillin/streptomycin (1 μg/ml) and fungizone (0.06 μl/ml). For macroscopic studies and observation of changes of holes diameter, the rings were cultured for 100 d without any treatment. The medium was refreshed twice a week and the rings were reseeded in the six-well plates. On the other hand, for histological studies some of rings were cultured just 30 d in tissue culture and then the rings were fixed followed by fixation in Bouinʼs solution. These tissues were then subjected to microphotography by inverted microscope (Olympus IX-71).

The hole diameters were calculated using DP2-BSW software on the micrographs and the data were analyzed using Excel software.

Vitamin A and C preparations and treatments.

There were three groups of biopsied rings in vitamin treatments. The first group consists of the rings that were biopsied 2 d after the initial punch. The second and third groups were biopsied 4 and 6 d after initial punch, respectively (Appendix). All of the biopsied samples then were divided to two treatment groups. One of them was treated with vitamin A and the other treated with vitamin C for 30 d. In all our experiments, vitamin A (Sigma) was dissolved in DMSO4 and added to the culture in the form of a suspension prepared in DMEM. The experimental cultures were treated with vitamin A (1 μg/ml) or medium of DMEM and DMSO mixture. The stock solution of alcoholic vitamin A was stored in the dark condition in refrigerator and fresh vitamin A solution was prepared from the stock solution every week for our experiments.

Stock solutions containing 0.5 M of l-ascorbic acid (Sigma) were prepared in sterile distilled water and filter sterilized. Different working medium included 5 and 50 μg/ml of ascorbic acid prepared from the stock solution.

Tissue preparation for transmission electron microscopy.

The samples were punched using a puncher with a bigger diameter than the one that punched the first holes and the specimens were quickly fixed in 2.5% glutaraldehyde solution for half an hour in room temperature, and kept for 24 h in the refrigerator. After that, the specimens were washed in phosphate buffer (pH = 7.2) for 18 min with buffer changed at least four times. Next, fixation in 2% osmium tetroxide was done for 1–2 h in room temperature. The tissues were again washed once in phosphate buffer and twice in deionized water quickly. Dehydration steps were done completely with ethanol (Merck, Darmstadt, Germany) solutions of different concentration as follows: ethanol 70% (5–7 min), 95% (7–10 min), and 100% three times (each one 5 min).

After complete passage of the samples and resin curing by placing the capsules in 60°C for 2 d, the blocks were first trimmed and then cut by ultra-microtome. Lastly, they were stained with 5% lead citrate and 3% uranyl acetate, to be prepared for electron microscopy (LEO 912 AB).

Tissue preparation for light microscopy.

The rings were removed from the media after certain periods of time for the purpose of histological studies. After 24 h fixation in Bouin’s solution, the tissues were dehydrated and were cleared with xylene. Then, tissues were impregnated and embedded in paraffin (Merck). After that, 5 μm sections were prepared using RM2145 microtome (Leica, Wetzlar Germany) and finally mounted on microscopic slides. Sections were stained with H&E, PAS and toluidine blue stains for histological observation.

Results

Macroscopic observations of the tissue growth.

The growth rate of the tissue specimens from rabbit ear, a perfect model of wound repair, was tested in current experiments. The healing process was assessed by measuring the weekly decrease in the punch diameter and the date of its full closure. Weekly analysis of data shows that over time, diameter rings in tissue culture medium were significantly reduced. This study was used just in control group not in treatment with vitamins (Fig. 1).

Figure 1.
figure 1

Weekly hole diameter measurement. (a) Weekly analysis of data shows that over time, diameter rings in tissue culture medium decreased significantly. (b) Figures (a) to (e) show the reducing of hole diameter during the time in one of biopsied rings in tissue culture.

Transmission electron microscopy observations.

Electron microscope observations of punched rings in culture medium show formation of cells with passible blastema cell appearances (Fig. 2A ). Figure 2B shows differentiated cells characteristic of chondrocytes with vacuoles containing polysaccharides. Twenty-four days after regeneration, activity of cellular secretion was observed. Also, vacuoles were shown to contain polysaccharides (Fig. 2C ).

Figure 2.
figure 2

Transmission electron microscopic observation. (A) Probably a blastema cell 7 d after regeneration. Organelles are not completed yet (magnification ×3300, TEM). (B) Chondrogenesis (magnification ×2500, TEM). A differentiated cell for chondrocyte formation with vacuoles containing polysaccharides. (C) Strong activity of cellular secretion in 24 days after regeneration, with vacuoles containing polysaccharides (magnification ×20,000, TEM).

Light microscopy observation–Observation of early blastema cells, their division and older blastema cells.

When histological rings biopsied from rabbit pinna and then seeded in specific medium, after few days some cells appear in the inner region at the cut edge of the ring. These cells are blastema cells that presumably could be created from dedifferentiation of cartilage cells (Fig. 3). Gene expression studies of blastema cells, the cells that released from the inner surface of ring during tissue culture of biopsied rings, have shown expression of oct4 and sox2 at RNA and Protein level. As a result, these cells showed stem cell characteristics (Mahmoudi et al. 2011).

Figure 3.
figure 3

Creation of blastema cells at the cut edge of the ring. (A) Biopsied ring of rabbit pinna. (B) A section of the inner region of the ring. (C) and (D) Appearance of blastema cells at the cut edge with magnification ×100 and ×40, respectively. The arrows indicate the blastema cells (H&E staining).

In this experiment, it was observed that some cells were designated as blastema cell, different from other neighboring cells in shape. They show high cytoplasm content, small nucleus, and are packed together (Fig. 4A discontinuous arrows). Dividing cells tend to be closely aggregated in groups in the older blastema, whereas in the younger blastema (10–15 d) they are more uniformly distributed. Observation of histological sections with light microscope showed that the older blastema cell has considerably less cytoplasm than the early blastema cells and lies farther away from the neighboring cells (Fig. 4A continuous arrows).

Figure 4.
figure 4

Observation of early blastema cells, their division and older blastema cells. (A) Early blastema cells. The cells that accumulate in the early blastema, 10 to 15 d after culturing, are strikingly different from the cells in their tissues of origin. These cells show high cytoplasm, small nucleus and near together (×200) H&E staining (discontinuous arrow). In older blastema cells, these cells have considerably less cytoplasm than the early blastema cell and lies farther away from neighboring cells (×400) H&E staining (continuous arrow). (B) Division of blastema cells (×400) H&E staining.

The next phase of regeneration is rapid proliferation of the blastema cells, bringing about an increase in the length of the regenerate. At the end of the period of blastema formation, 10–15 days after culturing, histological sections showed that the cells begin to proliferate rapidly (Fig. 4B ).

Observation of spontaneous differentiation of blastema cells–Chondrogenesis. Signs of blastema cell differentiation toward making chondrocytes appeared after lining the cells up in the center of the newly formed tissue, 25 d after culture. Morphology of the cells inside the cartilage-like tissue was graded. Normal mature cartilage cells are round shaped and located in the lacunae of the cartilage matrix. Immature cartilage cells are smaller and often triangular. In addition to morphological characteristics, the staining of the cartilage matrix with toluidine blue was classified. Blue staining of the matrix suggests a high glycosaminoglycan content indicating the presence of the cartilage. Intensity of the stain distinguishes mature and young immature cartilage from each other; where the matrix of mature cartilage is darker than the immature one. Also in the histological sections, chondrogenesis was observed in both sides of length and width (Fig. 5A ).

Figure 5.
figure 5

Chondrogenesis. (A) (1) Degenerated old chondrocytes, (2) Length chondrogenesis, and (3) Width chondrogenesis. (B) Left: Collagen in scar tissue has been shown with pic-indigocarmine staining. Right: Regenerated condrogenic tissue in punched place. This tissue probably been created by blastema cells in this section (Pas staining). (C) Cell division of chondrocytes. No significant change observed in chondrogenesis with vitamin A and C compared to the control group.

After application of vitamins A and C as an inducer or differentiating agent on the culture, the sections were observed under the microscope. No significant change was obvious in chondrogenesis, unlike the control group (spontaneous chondrogenesis) (Fig. 5B ). Chondrocyte division is shown in Fig. 5C .

Reepithelialization. The cellular morphology of the epithelium tissue in the defect was graded. The formation of granulation tissue in an open wound allows the reepithelialization phase to take place, as epithelial cells migrate across the new tissue to form a barrier between the wound and the environment (Verlhac and Gabaudan 1994). Basal keratinocytes from the wound edges are the main cells responsible for the epithelialization phase of wound healing (Combs 1992). They advance in a sheet across the wound site and proliferate at its edges, ceasing movement when they meet in the middle.

In this experiment, our observations show that all the steps of reepithelialization occurred in the culture of blastema tissues spontaneously (control group) (Fig. 6A ). The data did not show any significant differences in epithelium formation between control group (without any treatment) and vitamin A group (Fig. 6A ), but in treatment with vitamin C, epithelialization occurred faster than control group and vitamin A group (data not shown).

Figure 6.
figure 6

Reepithelialization. (A) Spontaneous differentiation of blastema cells to epithelial cells. (1) Degenerated old epithelium, (2) newly formed epithelial layer from blastema cells, (3) basal layer. (B) Induced differentiation of blastema cells to epithelial cells by vitamin C. More epithelial layers were formed than spontaneous differentiation. Arrows show the columnar type of epithelium in primary layers. This result was obtained 30 d after culture of tissue rings.

At 2, 4, and 6 d after primary punch, the rings were biopsied and cultured. As mentioned in the “Materials and Methods” section, vitamin C treatment was performed with two different concentrations, including 5 and 50 μg/ml for 30 d. All three groups (biopsied ring after 2, 4, and 6 d after primary punch) that were treated by vitamin C showed significantly increasing epithelium thickness with increasing of vitamin concentration (P < 0.05). The maximum increasing of epithelium thickness was related to the rings that biopsied 4 days after primary punch, but not significant compared with the other two groups (Fig. 7).

Figure 7.
figure 7

Comparison of epithelium thickness with different concentration of vitamin C treatment; 5 μg/ml and 50 μg/ml of vitamin C. Significantly increasing epithelium thickness was shown with increase in dosage of vitamin (P < 0.05). The maximum increasing of epithelium thickness was related to the rings that were biopsied 4 d after initial punch, but not significant compared with other two groups.

Observation of differentiation in blastema tissue–Osteogenesis. In this study, we observed differentiation in the blastema tissues as a result of vitamin treatments. Our data showed significant epithelization in the vitamin C-treated samples, while in those treated with vitamin A; the presence of osteogenesis was more significant (Fig. 8).

Figure 8.
figure 8

Osteogenesis. All results for this treatment were obtained 10 d after treatment. Differences between these data correspond to the timing of tissue punch (biopsy of rings from rabbit ear after primary punch). (A) Cartilage matrix mineralization (the ring was biopsied 2 d after primary punch) (B) mineralization and calcification (the ring was biopsied 4 days after primary punch). (C) Formation of bone lamellae (the ring was biopsied 6 days after primary punching). Arrows show compact bone lamellae (H&E staining, ×40). (D) Higher magnification (×100) from figure C with H&E staining and (E) van Gieson’s picro-fuchsin staining show bone formation (×100). Continuous arrow show bone formation and discontinuous arrow show cartilage tissue in (D) and (E). Osteogenesis was only observed when vitamin A was used as a differentiation factor.

The osteogenesis can be observed in three phases (1) changing the chondrocytes to osteocyte or in other words cartilage matrix mineralization (Fig. 8A ), (2) mineralization and calcification and (Fig. 8B ) and (3) formation of compact bone lamellae (osteon) (Fig. 8C ).

As a summary, comparison of spontaneous differentiation and induced differentiation (by vitamins A and C) has been shown in Table 1.

Table 1. Histomorphological grading of differentiation

Discussion

Wound healing is a dynamic, interactive process involving soluble mediators, blood cells, extracellular matrix, and parenchymal cells. Wound healing has three phases of inflammation, tissue formation, and tissue remodeling, which overlap in time (Heldin and Westermark 1999; Singer and Clark, 1999). The primary goals of the treatment of wounds are rapid wound closure and a functional and aesthetically satisfactory scar. Recent advances in cellular and molecular biology have greatly expanded our understanding of the biological processes involved in wound repair and tissue regeneration and have led to improvements in wound care (Fröhlich et al. 2007).

Rabbit ear cartilage has been used to study tissue repair processes of skin, perichondrium, and cartilage (Ten Koppel et al. 2001; Brockes and Kumar 2002). The rabbit’s ear is large and its cartilage is easily accessible. Hence, multiple punch-hole defects can be made in one ear. Implantation of cartilage grafts is technically not difficult and the whole procedure causes minimal morbidity to the rabbit. It is known that the growth of rabbit elastic cartilage in vivo (Moskalewski et al. 1979) and also during cultivation in vitro is accompanied by the enlargement of chondrocytes (Yang et al. 2001). This has also been observed during in vitro cultivation of chondrocytes from rabbit hyaline cartilage, fibroblasts (Bayreuther et al. 1991), and urothelial cells, and is connected with aging and the lower proliferation potential of cell culture (Groff and Gropper 1999). In our experiments, a similar phenomenon was observed.

Much research in cell and tissue culture remains to be conducted before the model of chondrogenesis is adequately described. Specifically, little is currently known regarding how various chemical and mechanical factors may be manipulated over time and with each other to control this process. It is reasonable to expect that the perichondrium may serve a similar function in regeneration. The cells of the perichondrium are capable of proliferation, migration, and differentiation, all the requirements of cells needed to replace the lost cartilage and perhaps serve as the precursors of blastema cells. Cartilage cells, both in vitro and in vivo, can lose their distinctive chondrocyte structure, become more fibroblastic, and even stop producing cartilage-type collagen in favor of the fibroblastic type (Hay 1959; Steen 1970; Uitto 1979; Wallace 1981). The perichondrium may serve as an active source of blastema cells. Cartilage regeneration in the mammalian ear may be both from a blastema and tissue proliferation of the perichondrium, as noted by other authors (Hay 1959; Liosner and Vorontsova 1960; Uitto 1979). Blastema cells generated in our study is probably due to the loss of specific structure and dedifferentiation of perichondrium or cartilage cells has occurred.

Formation of a regenerative blastema following limb amputation is believed to occur through a process of dedifferentiation (Corcoran and Ferretti 1999). After limb amputation, the tissues in the stump, such as muscle and cartilage, lose their tissue characteristics that distinguish them. This process leads to the formation of the blastema (Tsonis 2000; Brockes and Kumar 2002). Cells within the blastema are undifferentiated, but soon after a period of proliferation, these cells redifferentiate to build a faithful replica of the lost part of the limb (Tsonis 2004). Previous research has shown that blastema cells have an important role in the process of healing, but most of these studies were performed on amphibians are in vivo (Morrison et al. 2006). Blastema cell accumulation also occurs in rabbits. Holes punched in rabbit ears are repaired by regeneration of new tissues from blastemas found on the periphery of the wounds (Williams-Boyce and Daniel 1980; Williams-Boyce and Daniel 1986). In this study, for the first time, we managed to culture punched rings of the rabbit’s ear as an in vitro mammalian model and demonstrated the development of blastema tissues without any treatment in period of 100 d.

The second objective of this study is to gather some information about morphologic processes of blastema development in mammals. There have been no reports of morphologic studies of blastema tissue from rabbit pinna with electron microscope so far and this makes our study a novel one. In morphologic (cytological) assessments, ultra structural, and quantitative investigations (organelle surfaces comparisons), the development of blastema tissue is studied on the basis of time. A prospect of this research can be the usage of the rabbit pinna for injured tissue regeneration.

Retinoids have long been associated with wound healing, but objective data, until recently, have been scarce. Deficiency of retinoic acid (RA5), commonly known as vitamin A, retards repair. Secondly, retinoids restore steroid-retarded repair toward normal. Because vitamin A tends to suppress fibroblasts in cell culture and stimulate steroid-treated macrophages to initiate reparative behavior in tissue, we favor the hypothesis that retinoids are particularly important in macrophagic inflammation, which plays a central role in the control of wound healing (Hunt 1986).

Retinoic acid is an influential factor used in differentiation of stem cells to more committed fates, echoing retinoic acid’s importance in natural embryonic developmental pathways. It is thought to initiate differentiation into a number of different cell lineages by unsequestering certain sequences in the genome. RA via the RA receptor influences the process of cell differentiation, hence, the growth and development of embryos. During development, there is a concentration gradient of retinoic acid along the anterior-posterior (head-tail) axis. Cells in the embryo respond differently to retinoic acid depending on the amount present (Kligman et al. 1984). In addition of differentiation stimulation, vitamin A increase cell growth and population doubling time (Emura et al. 1988). In contrast with the studies that report the differentiating effect of vitamin A, our data introduced the bone formation of rabbit ear tissue (blastema cells) after use of vitamin A as a differentiation factor. This data showed that differentiation due to vitamin A could be either stage specific or source dependent. The finding of bone in one of the older regenerated rabbit ear tissue samples reflects the earlier report by Goss and Grimes (Williams-Boyce and Daniel 1980), who described it as being deposited in the ear by the process of endochondral ossification. Goss notes that ossification in regenerating rabbit ear tissue tends to occur circumferentially just within the original edges of the wound (Goss 1983). The other reagent that was used in our study was ascorbic acid. Ascorbic acid (AA1) is a water-soluble vitamin that is necessary for normal growth and development. Vitamin C is necessary to form collagen, an important protein used to make skin, scar tissue, tendons, ligaments, and blood vessels. Vitamin C is essential for the healing of wounds, and for the repair and maintenance of cartilage, bones, and teeth. Also, it is required for the growth and repair of tissues in all parts of the body (Mason 2007; Hamrick and Counts 2008; Rakel 2012).

Our study supports other previous research about vitamin C is required for growth of tissues. The epithelium treated with vitamin C in our study showed significant growth over time. Also, our results showed increasing growth with use of increasing in concentration of that vitamin.

In summary, the rabbit pinna’s punch-hole model is an efficient method for first use of an in vitro model in tissue engineering to study wound repair, and differentiation of blastema tissue. Regarding the potentials of this model, creating the tissue bank from histological rings for further tissue engineering studies is suggested. According to sensitivity and high potency of this model in interaction with different reagents, it seems that the use of this model as a bio indicator is a fantastic idea. Since this sample is the source of blastema cells and as these cells show stem cell characteristics, this model can be used for stem cell studies in the future.