Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/
Title
The proliferation and differentiation of stem cells and myoepithelial cells of injured rat
submandibular glands are accelerated by collagen gel with bFGF
Author(s) 小林, 史卓 Journal , (): ‑
URL http://hdl.handle.net/10130/3629 Right
The proliferation and differentiation of stem cells and myoepithelial cells of injured rat submandibular glands
are accelerated by collagen gel with bFGF
Fumitaka Kobayashi
Department of Clinical Pathophysiology, Tokyo Dental College
1
Abstract
The purpose of this study was to investigate cells that contribute to the wound healing of externally damaged salivary gland tissues and the effects of bFGF in a collagen gel on that process. Submandibular glands were partially removed from rats by mechanical means and were filled with or without bFGF in collagen gels covered by Gore-Tex membrane. For immunohistochemical staining, a pan-cytokeratin, α-smooth muscle actin, vimentin, CD49f, c-kit and aquaporin 5 was used as primary antibodies. At after operation day (AOD) 5, small round cells and spindle-shape cells had invaded the collagen gel control group or bFGF group and they had dramatically increased at AOD 7. At AOD 10 and 14, cell proliferation and reduction of the collagen gel volume were observed control group or bFGF group. The collagen gel had disappeared at AOD 14 in with the presence of bFGF and at AOD 21 in the control group. The invading cells in the collagen gel were positive for vimentin, pan-cytokeratin, CD49f, c-kit and aquaporin 5 at AOD 5 and 7. However, cells of the bFGF group had significantly higher mRNA expression levels of vimentin, αSMA, keratin 13, keratin 19 and aquaporin 5 than those control group. This study suggests that using collagen gels and bFGF improves the potential of salivary gland regeneration.
Keywords: salivary glands, collagens, animal models, immunohistochemistry, wound healing
2
Introduction
Glandular tissues including salivary glands, have weak regeneration capacity at the cause of tissue damage,, since they are composed of well-differentiated epithelial cells（1-3). When salivary glands are damaged, the resulting hyposalivation disturbs both the pronunciation and swallowing acts as a lubricant and being involved in enzymatic digestion (4-6). Moreover, saliva contains antibacterial agents and antibodies that not only prevent microbial proliferation in the oral cavity but also play a key role in maintaining homeostasis in the oral cavity, such as pH maintenance, due to its buffering action (4-8). When those functions are lost, it significantly affects the patient's QOL (1-3).
Therefore, to regenerate damaged salivary gland tissues is an important challenge. Wound healing occurs starting with a vascular response followed by cell migration into the wounded area. In the case of sharp cut damage, firstly, inflammatory cells infiltration and degenerative changes of acinar cells occur (9, 10). However, intercalated ducts, which are usually positive for stem cell markers and myoepithelial cells, start to proliferate and differentiate into acinar cells (9, 10).
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However, when defect damage occurs in salivary gland tissues, it usually heals with scar tissue without regeneration of the salivary gland tissue (15).
Similar healing processes have also been reported in the pancreas (14, 16, 17), liver (18,19), intestine (20), lacrimal gland (21,22) and mammary glands (23). To regenerate severely damaged salivary glands, tissue engineering techniques, including cells, scaffolds and growth factors should be necessary.
Type I collagen gel has been used as one type of biological scaffold (24).
It is known that collagen gels play an important role in cellular proliferation and differentiation both in vitro (25-29) and in vivo (30).
Collagen gels facilitate wound healing (31). And it facilitate to produce luminal structural formation of the salivary gland (32).
Basic fibroblast growth factor (bFGF) is well-known as a mitogen which influences chemotaxis, cell differentiation, proliferation and regeneration of all kinds of tissue. bFGF is often used clinically and plays a role in the wound healing (33) of cartilage (34), skin (35), cornea (36), eardrum (37) and salivary glands (38-40). Additionally, bFGF effect to regeneration in periodontal tissue (54) and bone tissue (55).
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The aim of this study was to investigate the tissue regeneration of severely damaged salivary glands using bFGF in a collagen gel in vivo.
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Materials and methods 1. Animals
This study was conducted in compliance with the Guidelines for the Treatment of Experimental Animals at the Tokyo Dental College (Approval Number 243208). Twenty-eight adult male Sprague-Dawley rats, each weighing about 200 g (Sankyo Lab Service, Tokyo, Japan), were used in this study. During the experimental periods, none of the animals were infected or died.
2. Preparation of collagen gels
The acid-soluble type 1 collagen solution (3 mg/ml, pH 3) derived from porcine tendon (Cellmatrix Type I-A; Nitta Gelatin Inc, Osaka, Japan) gives transparent gels with a consistently high gel strength after reconstitution and was used in this study.
3. Preparation of bFGF in the collagen gel
Fifty ng recombinant bFGF powder (Pepro Tech Inc, Rocky Hill, NJ, USA) was dissolved in 1 ml phosphate-buffered saline (PBS) to make the bFGF stock solution. This bFGF solution was diluted in the collagen gel described above to a final concentration of 500 ng/ml bFGF (51) in
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the collagen gel.
4. Wound model in submandibular glands
The rats were anesthetized by an intraperitoneal injection of thiopental (0.2ml/100g) (Ravonal; Mitsubishi Tanabe, Osaka, Japan). A skin incision, approximately 2 cm, was made along the center of the anterior neck using a surgical knife. Both sides of the submandibular glands (SMGs) were exposed and a defect which pass through the SMGs was made using a biopsy punch, 3 mm in diameter (Kai Industries, Gifu, Japan) without injuring the principal artery and main duct (Fig. 1. A, B).
After arrest of bleeding with gauze, collagen gels without bFGF (control group) or with bFGF (bFGF group) were inserted into the defects (Fig. 1. C, D). The defects with collagen gels were covered by a Gore-Tex membrane (GORE, Tokyo, Japan) to prevent the migration of surrounding fibroblasts into the collagen gel (Fig. 1. E). The skin incision was then sutured using a 4-0 non-absorbable suture.
5. Histological observations
Two rats in each group were euthanized at after the operation days (AOD) 5, 7, 10, 14 and 21 (n=20). Animals were anesthetized by an
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intraperitoneal injection of thiopental (Ravonal; Mitsubishi Tanabe, Osaka, Japan) and perfusion fixation was performed by transcardial injection of 10% neutral buffered formalin for 1 hr. After the perfusion fixation, SMGs were removed by mechanical means and were immersed in the same fixative solution for 24 hr at room temperature. The specimens were dehydrated in ethanol before being embedded in paraffin. Paraffin sections, 4 μm in thickness, were cut horizontally using a sliding microtome. For light microscopic observations, paraffin sections were stained with hematoxylin and eosin (HE).
6. Measurement of collagen gel volume
The area of collagen gel in the specimens was measured using an Axio microscope system in each group at each of the time periods (mm2).
7. Immunohistochemical observations
Paraffin sections of specimens at AOD 5 and 7 were used for immunohistochemical observation. After being deparaffinized with xylol, they were microwaved with a 0.01 M citric acid buffer solution (pH 6.0) for 15 min at 65°C for antigen retrieval. Sections were incubated in 3% hydrogen peroxide with methanol for 30 min at room
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temperature to block endogenous peroxidase activity. To block non-specific binding, the sections were treated with 3% bovine serum albumin for 10 min at room temperature. The monoclonal antibody supplied in the kit was used as the primary antibody. The sections were incubated at 4°C overnight and then were incubated with a biotinylated secondary antibody, NICHIREI-Histofine simple-stain MAX-PO® (Nichirei, Tokyo, Japan), for 30 min at room temperature. Thereafter, the sections were rinsed with PBS and were stained with NICHIREI-Histofine simple-stain Diaminobenzidine® (Nichirei) and counterstained with hematoxylin. Specimens were observed by light microscopy (Axio-photo 2; Carl Zeiss, Oberkochen, Germany).
Antibodies to vimentin (Dako, Glostrup, Denmark; diluted 1 : 100) as a fibroblast marker, α-smooth muscle actin (αSMA) (Santa Cruz Biotechnology, Dallas, US: diluted 1:50) as a muscle marker, Pan-cytokeratin (Pan-CK) (Abcam, Cambridge, UK: diluted 1:40) as an epithelial cell marker, CD49f (Santa Cruz Biotechnology, diluted 1:50) as a stem cell marker, c-kit (Santa Cruz Biotechnology: diluted 1:50) as a stem cell marker, and aquaporin 5 (Abcam, diluted 1:500) as an acinar
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cell marker, were used as primary antibodies. Immunopositive cells for those antibodies were counted using an Axio microscope system in the collagen gel of each group.
8. Quantitative RT-PCR
For messenger RNA (mRNA) expression studies, total RNAs were extracted using the acid guanidium thiocyanate/phenol chloroform method as follows. Four rats of each group were sacrificed at AOD 7, and the SMGs were removed mechanically (n=8). The collagen gels were washed with PBS, and were then mechanically removed from the salivary glands using a stereoscopic microscope. The collagen gels were homogenized in 500 ml TRIsol Reagent (Invitrogen Corp, Carlsbad, CA, USA), according to the standard method, and total RNA was reverse-transcribed to complementary DNA (cDNA) using a QuantiTect Reverse Transcription Kit (Qiagen, Germantown, MD, USA).
Quantitative RT-PCR was carried out using TaqMan Gene Expression Assays (Applied Biosystems, Life Technologies Corp, Carlsbad, CA, USA) for the target genes: vimentin, αSMA, keratin 13 (expression in the differentiated epithelium such as ductal cells), keratin19 (expression
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in the undifferentiated epithelium such as basal cells and epithelial stem cells), aquaporin 5 and GAPDH (endogenous control) using the primers shown in Table 1. All PCR reactions were performed using a real time PCR 7500 fast system (Applied Biosystems). mRNA expression levels of genes of interest were normalized against the expression of GAPDH and are designated as an expression coefficient.
9. Statistical analysis
Quantitative data are presented as means ± standard deviation and were analyzed using one-way analysis of variance (ANOVA) and multiple-comparison Bonferroni’s test by the MS Excel 2008 add-in.
Differences where p value is < 0.05 are considered to be statistically significant.
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Results
1. Histological study
In the control group, many white blood cells mainly composed of leukocytes had infiltrated the collagen gel and oval-shaped cells and spindle-shaped cells were also observed near the edge of the wounded salivary glands in the collagen gel at AOD 5 (Fig. 2. A, E).
At AOD 7, the number of spindle-shaped cells had increased in the collagen gel, especially in the inner area of the collagen gel (Fig. 2. B, F).
The volume of the collagen gel decreased over time (Fig. 2. C, D, G, H) and was completely replaced by the host tissue with numerous cells at AOD 21 (Data not shown).
In the bFGF group, many cells were seen in the collagen gel compared with the control group at AOD 5 (Fig. 2. I, M). At AOD 7, cells had increased in the collagen gel uniformly (Fig. 2. J, N). The replacement of the collagen gel in the bFGF group was much faster than in the control group at AOD 10 (Fig. 2. K, O). At AOD 14, the collagen gel had almost completely disappeared and was replaced with numerous cells and collagen fibers (Fig. 2. L, P).
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2. Volume of collagen gel
The volume of the collagen gel in the defect of each of the groups was getting smaller day by day. In the bFGF group, the volume of the collagen gel was significantly smaller than the control group at each of the time periods (Fig. 3).
3. Immunohistochemical study
In the control group (Fig. 4. A-L), spindle-shaped cells located in the peripheral area of the collagen gel were positive for vimentin (Fig. 4. A) and αSMA (Fig. 4. B) at AOD 5. However, at AOD 5 none of the cells were positive for CD49f, c-kit, Pan-CK or aquaporin 5 (Fig. 4. C, D, E, F).
At AOD 7, vimentin, CD49f, c-kit, Pan-CK and aquaporin 5 positive cells were observed in the peripheral area of the collagen gel (Fig. 4. G, I, J, K, L). The αSMA-positive cells were observed in the entire area of the collagen gel (Fig. 4. H).
In the bFGF groups (Fig. 4. M-X), vimentin, αSMA, CD49f, c-kit and Pan-CK positive cells were observed in the collagen gel at AOD 5 (Fig. 4.
M, N, O, P, Q) but not positive for aquaporin 5 (Fig. 4. R).
At AOD 7, Cells located at the border of the remaining salivary tissue in
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the collagen gel were positive for CD49f (Fig. 4. U) and c-kit (Fig. 4. V). A Pan-CK and aquaporin 5 positive cells was observed diffusely in the peripheral area of the collagen gel (Fig. 4. W, X).
4. Positive cell ratio
At AOD 7, vimentin, αSMA, CD49f, c-kit, pan-CK and aquaporin 5 positive cells in the bFGF group showed significantly higher ratios compared to the control group (Fig. 5).
5. Quantitative RT-PCR
The mRNA expression levels of vimentin, αSMA, keratin 19, keratin 13 and aquaporin 5 were significantly higher in the bFGF group compared to the control group at AOD 7 (Fig. 6).
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Discussion
Intercalated ducts, striated ducts, excretory ducts, myoepithelial cells and stem cells except acinar cells should be engaged with the wound healing of salivary glands (41). However, the migration of non-specific fibroblasts which are located outside of the wound has to be avoided because those cells produce scar tissue (42). In this study, a Gore-Tex membrane was used to avoid the invasion of non-specific fibroblasts. Because proliferating myoepithelial cells which are positive for αSMA , ductal cells which was positive for pan-CK and stem cells which are positive for either CD49f or c-kit and those cells most probably originated from the remaining salivary gland. In the bFGF group, aquaporin 5 positive cells were observed in the collagen gel at AOD 7. It is thought that the experiment system used in this study was suitable for investigating regeneration of salivary glands.
It is known that the role of stem cells is related to cell differentiation, immunoregulation, the production of growth factors, re-epithelialization and vascularization (43, 44). In addition, stem cells are also assumed to be involved in signaling in the epithelial-mesenchymal interaction involved in tissue regeneration (43). Therefore, it is thought that the CD49f and c-kit
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positive stem cells contribute to both the proliferation of the myoepithelium and ductal cells and are also related to the regeneration of the glandular tissue itself.
It has been reported that in the case of the salivary gland regeneration model using duct ligation, firstly inflammatory reaction started and afterword atrophy and apoptosis of acinar cells occurred. In the meantime myoepithelial cells and stem cells proliferate (45). After the release of the ligation, regenerated intercalated duct cells originated from stem cells differentiated into acinar cells and myoepithelial cells are eventually located around the regenerated acinus. The myoepithelial cells are known that have an ability to produce laminin (46,47). Furthermore, the stem cell which is positive for CD49f and CD49f is a high affinity receptor for laminin (48). Taken together, it is suggests that at the beginning of the event, proliferated myoepithelial cells produce laminin which is bound by the stem cells and this stem cells probably differentiate into the ductal epithelium. Furthermore, aquaporin 5 positive cells were observed in the collagen gel at AOD 7 and this may suggest that regenerated intercalated duct cells might differentiate into acinar cells.
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Collagen gels are known as suitable scaffolds for cells to proliferate and differentiate (25-31). It has been reported that salivary gland tissue on collagen gels in primary cultures, the collagen gel inhibits the proliferation of fibroblasts, but promotes the three-dimensional proliferation of duct epithelial cells, and eventually the formation of the luminal structure by myoepithelial cells (49). It has been also reported that bFGF affects fibroblasts, vascular endothelial cells and epithelial cells that result in the promotion of tissue regeneration (50). bFGF also promotes the proliferation of myoepithelial and ductal cells of salivary glands (51). Furthermore, stem cells are known to protected by bFGF for their functional characterization (18, 40, 52). The results of the immunohistochemical study and RT-PCR analysis showed that cells positive for Pan-CK, α-SMA, CD49f and c-kit were much higher in the bFGF group than in the control group as was their mRNA expression levels. This suggests that bFGF in the collagen gel promotes the proliferation of ductal cells, myoepithelial cells, fibroblasts and stem cells and protects the characteristics of stem cells.
Taken together, collagen gels as a scaffold induce the proliferation of ductal cells, myoepithelial cells and stem cells, but not their differentiation
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to acinar cells. However, in this study, aquaporin 5 positive cells appeared and this may suggest that the regeneration of acinar cells may be occurred.
Conclusion
This study suggests that using collagen gels with bFGF for the repair of salivary gland defects improves the potential of salivary gland regeneration.
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Table 1. Primers used for real-time reverse transcription-polymerase chain reaction
Primer Gene name Assay ID Cytokeratin 13 keratin 13 Rn01464229_m1 Cytokeratin 19 keratin 19 Rn01496867_m1 αSMA smooth muscle alpha-actin Rn01759928_g1
vimentin vimentin Rn00579738_m1
Aquaporin5 aquaporin5 Rn00562837_m1
GAPDH glyceraldehyde-3-phosphate Rn01775763_g1 (endogenous control) dehydrogenase
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Figure 1. A cylindrical defect, 3 mm in diameter and pass through the SMGs, was made in each salivary gland using a biopsy punch (A, B). After arrest of bleeding, collagen gels were placed in the wounds (C, D). Finally, salivary glands were over molded by a Gore-Tex membrane to prevent the migration of fibroblasts from the connective tissue and getting inside the collagen gels (E).
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Figure 2. Hematoxylin and eosin staining of submandibular glands after creation of the wound (original magnification ×25, ×100). Collagen gel (A-H), Collagen gel with bFGF (I-P). At after operation day (AOD) 5 (A, E, I, M), AOD 7 (B, F, J, N), AOD 10 (C, G, K, O) and AOD14 (D, H, L, P), the collagen gel was clearly reduced in size with time.
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Figure 3. The area of collagen gel applied to the wound site.
The area of collagen gel applied to the wound site decreased with time. The bFGF group showed a significantly faster disappearance of the collagen gel area compared to the control. Data represent means ± SD; *P < 0.05.
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Figure 4. Immunohistochemical staining (original magnification ×400).
Collagen gel (A-L) and Collagen gel with bFGF (M-X). Collagen gel stained with antibodies to vimentin (A, G, M, S), αSMA (B, H, N, T), Pan-CK (C, I, O, U), CD49f (D, J, P, V), c-kit (E, K, Q, W) and aquaporin 5 (F, L, R, X) at AOD 5 (A-F, M-R) and AOD 7 (G-L, S-X). In the control group, at AOD 5, vimentin and αSMA positive cells were observed into the peripheral area of the collagen gel. Pan-CK, CD49f, c-kit and aquaporin 5 positive cells were not observed. In the bFGF group, at AOD 5, CD49f and c-kit positive cells were observed but aquaporin 5 positive cells were not observed. In the both groups, at AOD 7, αSMA positive cells were observed in the inner area of the collagen gel. The vimentin, Pan-CK, CD49f, c-kit, aquaporin 5 positive cells were observed at peripheral areas of the collagen gel.
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Figure 5. Immunopositive cell ratio.
At AOD 7, vimentin, αSMA, CD49f, c-kit and aquaporin 5 showed a significantly higher ratio in the group with bFGF compared to the control group. Data represent means ± SD; *P < 0.05.
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Figure 6. mRNA expression levels
At AOD 7, mRNA analysis indicated that the expression of vimentin, αSMA, keratin 19, keratin 13 and aquaporin 5 was significantly higher in the group with bFGF compared to the control group. Data represent means
± SD; *P < 0.05.
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