The Mandibular Ridge Oral Mucosa Model of Stromal Influences on the Endothelial Tip Cells: An Immunohistochemical and TEM Study

Authors

  • Mugurel Constantin Rusu,

    Corresponding author
    1. Division of Anatomy, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
    2. MEDCENTER, Center of Excellence in Laboratory Medicine and Pathology
    • Discipline of Anatomy, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
    Search for more papers by this author
  • Andreea Cristiana Didilescu,

    1. Department of Morphological Science, Division of Anatomy, Faculty of Medicine and Pharmacy, “Dunărea de Jos” University, Galaţi, Romania
    Search for more papers by this author
  • Ruxandra Stănescu,

    1. Division of Oral Implantology, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
    Search for more papers by this author
  • Florinel Pop,

    1. Division of Pathologic Anatomy, Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
    Search for more papers by this author
  • Valentina Mariana Mănoiu,

    1. Department of Meteorology-Hidrology, Faculty of Geography, University of Bucharest, Romania
    Search for more papers by this author
  • Adelina Maria Jianu,

    1. Department of Anatomy, Faculty of Medicine, “Victor Babeş” University of Medicine and Pharmacy, Timişoara, Romania
    Search for more papers by this author
  • Marek Vâlcu

    1. Division of Plastic and Reconstructive Surgery, Clinic of Plastic Surgery and Reconstructive Microsurgery, Bucharest Emergency Hospital, Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
    Search for more papers by this author

Abstract

This study aimed to evaluate by immunohistochemistry and transmission electron microscopy (TEM) the morphological features of the oral mucosa endothelial tip cells (ETCs) and to determine the immune and ultrastructural patterns of the stromal nonimmune cells which could influence healing processes. Immune labeling was performed on bioptic samples obtained from six edentulous patients undergoing surgery for dental implants placement; three normal samples were collected from patients prior to the extraction of the third mandibular molar. The antibodies were tested for CD34, CD117(c-kit), platelet derived growth factor receptor-alpha (PDGFR-α), Mast Cell Tryptase, CD44, vimentin, CD45, CD105, alpha-smooth muscle actin, FGF2, Ki67. In light microscopy, while stromal cells (StrCs) of the reparatory and normal oral mucosa, with a fibroblastic appearance, were found positive for a CD34/CD44/CD45/CD105/PDGFR-α/vimentin immune phenotype, the CD117/c-kit labeling led to a positive stromal reaction only in the reparatory mucosa. In TEM, non-immune StrCs presenting particular ultrastructural features were identified as circulating fibrocytes (CFCs). Within the lamina propria CFCs were in close contact with ETCs. Long processes of the ETCs were moniliform, and hook-like collaterals were arising from the dilated segments, suggestive for a different stage migration. Maintenance and healing of oral mucosa are so supported by extensive processes of angiogenesis, guided by ETCs that, in turn, are influenced by the CFCs that populate the stromal compartment both in normal and reparatory states. Therefore, CFCs could be targeted by specific therapies, with pro- or anti-angiogenic purposes. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.

INTRODUCTION

The mitogenic signaling in mammalian cells is performed mainly by growth factors that interact with receptors localized at the plasma membrane level. Most of these receptors have a cytoplasmic tyrosine kinase activity domain. The interaction of the growth factors with the receptors, besides inducing the kinase activity of the receptor, activates signaling pathways that alter gene expression patterns and induce mitogenesis, or if deregulated, are related to carcinogenesis (Perona,2006).

Stem cell factor and its receptor tyrosine kinase CD117 (c-kit) were hypothesized to have a role in processes of tissue survival and repair (Da Silva et al.,2006; Stokman et al.,2010). The receptor c-kit belongs to the same subclass as platelet derived growth factor receptor (PDGFR) (Stokman et al.,2010). The ligand for PDGFR is platelet derived growth factor (PDGF), which is also involved in growth of connective tissue and wound healing (Perona,2006). Autocrine and paracrine PDGF signaling plays an important role in fibroblast and endothelial cell proliferation (Li et al.,2006). PDGFR and c-kit are also efficiently inhibited by Imatinib, a tyrosine kinase inhibitor (von Mehren,2006).

Angiogenesis is the process of developing vascular sprouts from existing blood vessels (Tung et al.,2012). This process, which occurs in response to specific signals, initially involves proliferation, sprouting, and migration of endothelial cells. The newly generated sprout is guided by migrating endothelial tip cells (ETCs). Endothelial cells are extremely sensitive to signal transduction from the extracellular microenvironment (Melo and Kalluri,2012). The tip cells are motile, invasive, and dynamically extend long filopodial protrusions (Adams and Eichmann,2010). The base of the endothelial sprout is formed by additional endothelial cells, termed stalk cells (Adams and Eichmann,2010). There is recently immunohistochemical evidence for a role of ETC driven angiogenesis in the oral mucosa (Stanescu et al.,2012).

Pericytes are polymorphic, elongated, multi-branched periendothelial cells covered by the same basement membrane as endothelial cells (Lu and Sood,2008). They can respond to angiogenic stimuli, guide sprouting tubes, provide endothelial survival signals, and have macrophage-like activities (Lu and Sood,2008).

Circulating fibrocytes (CFCs) were first described in 1994 (Bucala et al.,1994), and are bone marrow-derived mesenchymal/hematopoietic progenitor cells. These cells are able to migrate specifically to sites of tissue injury and exhibit mixed morphological and molecular characteristics of hematopoietic stem cells, monocytes and fibroblasts (Sumrall and Johnson,1973; Postlethwaite et al.,2004; Mori et al.,2005; Ebihara et al.,2006; Kisseleva et al.,2006; Lama and Phan,2006; Lang et al.,2006; Varcoe et al.,2006; Bellini and Mattoli,2007; Wu et al.,2007; Ishida et al.,2009; Mattoli et al.,2009; Strieter et al.,2009a,b; Curnow et al.,2010; Fan and Liang,2010; Mathai et al.,2010; Smith,2010; Uehara et al.,2010; Aldrich and Kielian,2011; Andersson-Sjoland et al.,2011; Grieb et al.,2011; Kao et al.,2011; Wada et al.,2011; Iqbal et al.,2012; Ohishi et al.,2012). Accumulating evidence supports the active role of fibrocytes in promoting angiogenesis (Hartlapp et al.,2001; Pilling et al.,2003). Fibrocytes express the stem-cell marker CD34, the panhematopoietic marker CD45, monocyte markers, and they produce components of the connective tissue matrix including collagen-1, collagen-III, and vimentin (Herzog and Bucala,2010). These cells are also positive for other markers, such as alpha-smooth muscle actin (α-SMA), CD44, CD105 (Bellini and Mattoli,2007).

Newly formed endothelial tubes become stable through the formation of a perivascular matrix and the recruitment of pericytes. Pericytes influence vessel stability by matrix deposition and/or by the release and activation of signals that promote endothelial cell differentiation and quiescence. The molecular mechanisms by which pericytes mediate vessel stability are not fully known (Lu and Sood,2008). The role of pericytes in angiogenesis is of great current interest as they represent a possible target for antiangiogenic therapies in various diseases which are dependent on angiogenesis (Morikawa and Ezaki,2011).

The oral cavity is the site of various infectious and inflammatory diseases with a high occurrence of angiogenesis due to the local anatomical conditions. For example, wound healing in the oral mucosa is faster and with less scar formation than that of normal skin (Okazaki et al.,2002; Szpaderska et al.,2005; Larjava et al.,2011). The mechanisms by which angiogenesis is regulated in oral tissues remains poorly understood (Szpaderska et al.,2005).

The hypothesis of this study was that the immune and ultrastructural phenotype of the stromal fibroblasts is suited to interfere with the processes of angiogenesis, but not with scar formation, in the oral mucosa. The aims of this study were: (1) to evaluate the morphological features of the oral mucosa ETCs by transmission electron microscopy (TEM), (2) to determine the immune and ultrastructural patterns of the stromal nonimmune cells, and (3) to evaluate which patterns are suitable for rapid and scar less healing of oral mucosa.

MATERIALS AND METHODS

Human adult bioptic material (reparatory mucosa of the edentulous alveolar crest of the mandible) was collected from six adult patients (four females and two males; mean age 33.5 years, SD 3.21), prior to healing abutment placement (second surgery). Three additional samples of mandibular ridge mucosa were collected from patients prior to the extractions of the third molar, and were used as control, non-reparatory samples. All samples were prepared for immunohistochemistry. Four samples of reparatory mucosa were prepared for TEM. Informed consent for use of the bioptic material with research purposes was obtained from the patients. The Bioethics Committee of the host institution approved the study.

The collected samples were fixed for 24 hr in buffered formalin (8%) and were processed with an automatic histoprocessor (Diapath, Martinengo, BG, Italy) with paraffin embedding. Sections were cut manually at 3 μm, and were mounted on SuperFrost® electrostatic slides for immunohistochemistry (Thermo Scientific, Menzel-Gläser, Braunschweig, Germany). Histological evaluations used 3-μm thick sections stained with Hematoxylin and Eosin.

The primary antibodies were used as follows: (a) anti-CD34 (clone QBEnd 10, Dako, Glostrup, Denmark, 1:50); (b) anti-CD117(c-kit) (clone T595, Leica Biosystems Newcastle, Newcastle Upon Tyne, UK, 1:20); (c) anti-PDGFR-α (clone C-20, Santa Cruz Biotechnology, Santa Cruz, CA, 1:500); (d) anti-Mast Cell Tryptase Ab-2 (clone AA1, Lab Vision, Fremont, CA, 1:1000); (e) anti-vimentin (clone V9, Dako, 1:50); (f) anti-CD44 (clone DF1485, Leica Biosystems Newcastle, 1:50); (g) anti-CD45 (clone RP2/18, RP2/22, Leica Biosystems Newcastle, 1:100); (h) anti-CD105 (polyclonal, Thermo Scientific, Pierce Biotechnology, Rockford, IL); (i) anti-α-SMA (Smooth Muscle Actin) (clone 1A4, Biocare Medical PM 001 AA, Biocare Medical, Concord, CA, 1:150); (j) anti-FGF2 (basic Fibroblast Growth Factor) (clone sc79, Santa Cruz Biotechnology, 1:50); (k) anti-Ki67 (clone MIB-1, Dako, 1:50).

Sections were deparaffinized, rehydrated and rinsed in phosphate-buffered solution (PBS) at pH 7.4. Retrieval by incubation in specific buffer was completed as follows: (a) for CD34 and vimentin: EDTA, pH 9; (b) for the other antibodies: 0.01 M citrate retrieval solution, pH 6. The standard ABC technique used a DAB protocol. Appropriate endogenous blocking peroxidase was completed before immunolabeling (0.1% BSA in PBS). Sections incubated with nonimmune serum served as negative controls. Sections were counterstained with Hematoxylin.

The microscopic slides were analyzed and micrographs were taken and scaled using a Zeiss working station: AxioImager M1 microscope with an AxioCam HRc camera and AxioVision digital image processing software (Carl Zeiss, Oberkochen, Germany).

Small tissue fragments were processed for TEM as previously described (Rusu et al.,2012a, c), and the grids were examined in a Philips electron microscope EM 208S (acceleration voltage of 80 kV). Micrographs were taken using a video camera Veleta (Olympus, Münster, Germany) using the iTEM Olympus Soft Imaging System.

RESULTS

The Immunohistochemical Study

Stromal cells (StrCs) of the non-reparatory oral mucosa were found to be positive for a CD34/CD44/CD45/CD105/PDGFR-α/vimentin immune phenotype (Figs. 1 and 2). They had a fibroblastic appearance, were spindle-shaped and bipolar, sending off immune positive prolongations at the opposed poles. However, they were CD117/c-kit negative (Fig. 2B). Such StrCs were frequently located in periendothelial positions. Mast cells (MCs) were found within the lamina propria, both in the papillary and reticular layers. MCs were tryptase- and CD117/c-kit -positive (Fig. 1A,B). CD34 labeling identified extensive processes of sprouting angiogenesis (Fig. 1A) within the lamina propria. The endothelial stalk cells of resident microvessels were positively stained, similar to the ETCs involved in an extensive process of angiogenesis and configuring a rich stromal network, both in the papillary and the reticular layers. The ETCs were projecting short filopodial processes with a brush-like disposition at the periphery of cells, and were also giving off long moniliform prolongations sending in turn collaterals from their dilated segments in a “hook anchoring” fashion.

Figure 1.

Oral resident mucosa of the mandibular ridge, non-reparatory. A. CD34 positive sprouting microvessels (arrows). B. CD45 positive labeling of endothelial and StrCs. C. CD44 positive stromal labeling. D. Endothelial (arrows) and stromal (arrowheads, inset) CD105 positive labeling. E. Vimentin-positive endothelia and StrCs. F. α-SMA positive periendothelial but negative stromal labeling.

Figure 2.

Oral resident mucosa of the mandibular ridge, non-reparatory. A. Tryptase-positive MCs (arrows). B. CD117/c-kit only labels MCs (arrows). C. PDGFR-α positive StrCs. D. FGF 2 positive stromal reaction. E. FGF 2 positive epithelial suprabasal cells (arrow, inset). F. Epithelial basal (arrow) and suprabasal (arrowhead) cells positively labeled with Ki67 antibodies; few StrCs are also positively labeled (*). G. Detail of (F): Ki67 positive StrCs (arrows). H. Epithelial suprabasal (arrowhead) and stromal (arrows) Ki67 positive cells; immune negative reaction of the basal epithelial layer.

The stromal immune phenotype of the reparatory oral mucosa was similar to that in non-reparatory samples: CD34/CD44/CD45/CD105/PDGFR-α/vimentin positive (Figs. 3 and 4). The CD117/c-kit labeling led to a consistent, positive stromal reaction (Fig. 4B). The presence of MCs, tryptase- and CD117/positive (Fig. 4) was also recorded within the reparatory mucosa of the mandibular ridge. PDGFR-α positive labeled StrCs of fibroblastic appearance had long, immune positive prolongations (Fig. 4D). The microvascular bed was identified with CD34 antibodies. Processes of sprouting angiogenesis were suggested by the presence of stromal-embedded ETCs. CD34-positive cells of fibroblastic appearance were identified in the microvessel region of the papillary layer. The cellular extensions of the tip cells appeared to be in contact with adjacent StrCs.

Figure 3.

Reparatory mucosa of the mandibular ridge. Stromal networks (arrows) are built up by cells (arrowheads) positive for a large set of immune markers: CD34 (A), CD45 (B), CD44 (C), CD105 (D), and vimentin (E). Antibodies for α-SMA labeled vascular mural cells, but not StrCs or networks.

Figure 4.

Reparatory mucosa of the mandibular ridge. Tryptase-positive MCs (A). StrCs and networks are positive for the CD117/c-kit antibody (B), as also are for the PDGFRα antibodies (C). PDGFRα positive StrCs are spindle shaped and bipolar (D, arrow). Negative immune reaction for FGFs antibodies (E). Ki67 positive labeling of the suprabasal epithelial layer (F).

Positive α-SMA labeling of vascular smooth muscle cells and pericytes was found in both reparatory and control samples (Figs. 1 and 3). StrCs and networks were α-SMA negative in control samples (Figs. 1 and 3). In reparatory samples the α-SMA stromal labeling was also negative, except the perifibrotic stromal layer in which a positive α-SMA/CD117/CD45 phenotype was identified (Fig. 5). The perifibrotic epithelial proliferation appeared to be minimal, as demonstrated by the Ki67 labeling (Fig. 5A).Ki67 antibodies labeled epithelial (basal and suprabasal) cells in reparatory and control samples (Figs. 2 and 4). Ki67 positive endothelial and StrCs (Fig. 2) were scarcely found within the lamina propria.

Figure 5.

Fibrous nodule of the reparatory mucosa of the mandibular ridge. The thin epithelial covering of the nodule presents rare Ki67 positive cells (A, arrow). The StrCs surrounding the fibrous lesion positively labeled with α-SMA (B), CD117/c-kit (C), and CD45 (D) antibodies (arrowheads).

In control samples FGF2 labeling appeared to be consistently diffuse in the StrCs and positive epithelial suprabasal foci were observed (Fig. 2). In reparatory samples the immune reaction was negative (Fig. 4).

Ultrastructure

The general structure of the mucosa was readily identified and consisted of an epithelial layer and lamina propria. Immune resident cells (MCs, macrophages) and nonresident inflammatory cells were found in the stromal compartment, together with cells belonging to the fibroblastic lineage.

Within the lamina propria, nonimmune StrCs (Fig. 6) presenting particular ultrastructural features were identified. The nucleus was oval in shape, euchromatic, with peripherally condensed chromatin, and nucleoli were occasionally present. The cytoplasm had little endoplasmic reticulum and few mitochondria, and a well-defined cytoskeleton consisting of intermediate filaments. Thick cell processes extended from the cell bodies. Plasmalemmal caveolae were observed on the plasma membrane and within the cytoplasm multivesicular bodies were identified in regions neighboring the nucleus. Vesicles were identified on the extracellular side of the cell membranes. Because of the presence of few organelles involved in secretion, these StrCs were not identified as fibroblasts. On the other hand, fibroblasts with characteristic endoplasmic reticulum and mitochondria were scarcely observed.

Figure 6.

Ultrathin section of human oral mucosa of the edentulous ridge. The lower left panel depicts a general view of the reticular layer of lamina propria. 1. basal epithelial layer; 2, 2′. endothelial cell (ETCs), with intracytoplasmic Weibel-Palade bodies (arrowheads); 3.lymphocyte. A non-immune StrC (4) is detailed at higher resolution (digitally reconstructed image, by bi-dimensional sequenced concatenation of 20 serial electron micrographs). That StrC is equipped with a well-represented signaling apparatus: plasmalemmal caveolae (detailed in A, indicated by arrowheads) and multivesicular bodies (detailed in B, indicated by arrows); close contacts of that StrC and an endothelial cell (ETC) are depicted at a higher magnification (C, arrows).

Within the stromal compartment, cells which were ultrastructurally different from the immune and fibroblastic StrCs were identified. Because these cells distinctively contained Weibel–Palade bodies in their cytoplasm, but were not building endothelial tubes and were not embedded in the basal laminae, they were considered as being ETCs. Close contacts between these ETCs and the StrCs were observed (Fig. 6).

ETCs sprouting out from the endothelial tubes were observed and characterized. The cell nucleus was heterochromatic, with indentations. Perinuclear elongated Golgi sacs were identified. Within the cytoplasm, mitochondria and endoplasmic reticulum were present. Plasmalemmal caveolae were observed. ETCs were connecting the endothelial stalk cells by peg-and-socket and adheren junctions. ETCs prolongations were observed. Two patterns of these prolongations were encountered: (a) filopodia, located beneath the endothelial basal lamina, embedded within it, or projected within the stromal compartment; (b) long prolongations emerging from the basal lamina of the endothelial tubes and freely penetrating the stroma. The morphology of these long prolongations was similar to that observed in light microscopy: moniliform processes, with collateral branches leaving the dilated segments in a “hook anchoring” manner. Microfilaments were noted in these prolongations (Fig. 7). Pericytes were embedded within the basal laminae of the sprouting endothelial tubes, and were occasionally contacted by the ETCs filopodia.

Figure 7.

Ultrathin cut of human oral mucosa of the edentulous ridge. The lower left panel depicts a general view of a blood microvessel located beneath the basal epithelial layer (bec: basal epithelial cell); ETCs are identified sending prolongations (arrows), and one of these (*) is detailed at higher resolution (upper panel, digitally reconstructed image, by bi-dimensional sequenced concatenation of 19 serial electron micrographs). The prolongation (arrow) of the ETC (*) is moniliform and presents a series of dilated segments (arrowheads) which are branching points for hook-like collaterals. Ultrastructural details of the ETC prolongation are presented at higher resolution in (A) and (B). (C), the interendothelial stalk-to-tip cells “joint” is depicted, consisting of a peg-and-socket junction (arrow), and adherens junctions (arrowheads).

Newly formed endothelial tubes had a “signet ring” appearance on transverse or oblique cuts. The ETCs were bordering those tubes with a thin wall covered by the endothelial basal lamina, and were projecting filopodia into the lumen (Fig. 8). No pericytes were found embedded in the basal laminae of these newly formed tubes.

Figure 8.

Ultrathin cut of human oral mucosa of the edentulous ridge. A newly formed endothelial tube is presented, and it is limited by an ETC. A thin wall (arrows) of the tube can be observed beneath the endothelial basal lamina, opposite to an indented nucleus. Endoluminal filopodial projections of the tip cell are indicated by arrowheads.

DISCUSSION

The Microanatomy of ETCs

In sprouting angiogenesis, endothelial cells must orientate in the tissue environment to effectively invade tissues and form vascular patterns according to the local needs. ETCs respond to vascular endothelial growth factor-A (VEGF-A) by guided migration while the proliferative response to VEGF-A occurs in the sprout stalks (Gerhardt et al.,2003). ETCs prolongations, which are usually described as filopodia (Gerhardt and Betsholtz,2005; Cullen et al.,2011; Siemerink et al.,2012), act as environmental sensors (Cullen et al.,2011) and the ETCs undergo chemotaxis toward angiogenic factors (Tung et al.,2012). Sprouting ETCs are followed by proliferating endothelial stalk cells that are rapidly ensheathed by pericytes (Cullen et al.,2011).

The oral mucosa appears as a good model for studying the process of sprouting angiogenesis and the ETCs. The in situ evidence denotes intensive processes of angiogenesis, seemingly with no obvious differences between the normal and the reparatory mucosa. CD34 labeling and TEM evaluation of ETCs demonstrated that these cells have processes of two morphological types: (a) short protrusions, filopodial like, closely related to neighbor StrCs; (b) long protrusions which are moniliform, and not filopodial as usually considered (see the previous paragraph). The moniliform morphology could be determined by attractive and repulsive forces of the local environment, similar to those guiding axons during the formation of the insect tracheal system (Gerhardt et al.,2003; Gerhardt and Betsholtz,2005). Moreover, collaterals arising from the dilated segments of the ETCs long processes mechanically support the guided migration of those processes while the dilations of these processes could simply indicate their stage of elongation. Observed by TEM the presence of such moniliform processes in the stromal compartment could lead to misinterpretations. A novel class of interstitial cells was recently characterized: the telocytes, which are resident StrCs with prolongations termed telopodes (Kostin,2010; Popescu and Faussone-Pellegrini,2010; Popescu et al.,2011; Nicolescu et al.,2012; Rusu et al.,2012a,b). Telopodes are defined as cells presenting moniliform prolongations, with dilated segments termed podoms. Therefore, TEM distinction between telopodes of telocytes and the moniliform processes of ETCs should come only after identifying the cell body.

The Stromal Environment of ETCs in the Oral Mucosa

In both control and reparatory mucosa, StrCs were identified. These cells had a fibroblastic appearance: spindle shaped, bipolar and were immune positive for the same panel of markers: CD34/CD44/CD45/CD105/PDGFR-α/vimentin. As these cells had mixed morphological and molecular characteristics of hematopoietic stem cells, monocytes and fibroblasts, they were identified as CFCs (see also “Introduction” Section). CFCs were closely related to, or were contacting stromal ETCs. The presence of CFCs, which are believed to promote angiogenesis (Hartlapp et al.,2001; Pilling et al.,2003), appeared to support the extensive sprouting angiogenesis observed in the present study. It should be emphasized also that angiogenesis is not the only process that can occur in adult tissues. For example, endothelial progenitor cells may also participate in neovascularization during ischemic conditions (adult vasculogenesis), and these cells are bone marrow derived (Tepper et al.,2005). There are studies which suggest that bone marrow derived mesenchymal stem cells, endothelial progenitor cells and fibrocytes may be involved in wound healing processes (Wu et al.,2007).

During wound healing of the oral mucosa, CFCs around fibrotic lesions acquired a myofibroblastic phenotype being α-SMA positive. Loss of progenitor markers and gain of a myofibroblastic phenotype correlates with the maturation of the CFCs (Chen et al.,2010). As CFCs populate the mucosa in the absence of mucosa wounds, a prompt phenotypic switch from CFCs to myofibroblasts occurred that likely ensured a quick wound contraction response. This would reduce scar formation during the healing process.

When observed in TEM most of the nonimmune StrCs had morphologies that suggested they were more involved with signaling than with collagen secretion, as are the canonical fibroblasts. The scarcity of secreting fibroblasts in the oral mucosa may provide clues to help understand reduced scar formation during oral mucosa healing, when compared to normal skin. This argument is supported by the results of an experiment which demonstrate that even though the population of fibroblasts increased immediately after surgery, at 4 weeks the fibroblasts density decreased (Berglundh et al.,2007).

The ultrastructural phenotype of CFCs resembled that described for interstitial Cajal cells (Faussone-Pellegrini and Thuneberg,1999; Thuneberg,1999; Davidson and McCloskey,2005) and for telocytes (Popescu and Faussone-Pellegrini,2010; Popescu et al.,2011). However, the interstitial Cajal cells populate the gastrointestinal tract, and the telocytes can be identified in TEM firstly based on the presence of telopodes (Rusu et al.,2012b), and secondly by different morphological features (Rusu et al.,2012a). Telopodes were not observed in any of the samples used in this study.

The CD117/c-kit phenotype of CFCs was negative in control samples, but it was positive in the reparatory samples, and this phenotypic switch could be related with the reparatory process. PDGFR-α positive and CD117/c-kit negative mesenchymal cells, juxtaposed to interstitial Cajal cells, were presumed as being involved in gastrointestinal functions (Iino et al.,2009; Iino and Nojyo,2009; Cobine et al.,2011), but their function is still controversial (Kurahashi et al.,2011). PDGFR positive cells were also associated with chronic tissue inflammation (Rubin et al.,1988). The PDGFR phenotype of the CFCs could be related to the extensive processes of angiogenesis that were observed in this study.

Structurally, PDGFs consist of dimeric isoforms of A and B chains in all three combinations possible with the α-receptor binding to all three forms of PDGF (Sundberg et al.,1993). PDGFR-α acts as an inductor of angiogenesis, while PDGFR-ß is essential for vascular stability (Zhang et al.,2009). Taking these into account, the CFCs and pericytes of the mandibular ridge mucosa appear to ensure an adequate microenvironment for processes of angiogenesis. Moreover, the induction of angiogenesis is also related to MCs. Specifically, MCs products stimulate migration and/or proliferation of endothelial cells and degrade the extracellular matrix to provide space for angiogenic sprouts to form, PDGF facilitates the recruitment of MCs to sites of angiogenesis, and the MC tryptase is an angiogenesis-inducing molecule (Meininger and Zetter,1992; Metcalfe et al.,1997; Norrby,2002; Ribatti et al.,2002). FGF acts also as a key factor in sprouting angiogenesis (Tepper et al.,2005; Woad et al.,2012). FGF2 was consistently expressed in non-reparatory, but not in reparatory samples; this suggests that FGF2 acts in physiological reparatory processes, but it may not intervene in wound healing.

The FGF2 positive epithelial phenotype that was found may be associated to a degree with epithelial dysplasia that may be supported, in turn, by the basal and suprabasal Ki67 positive labeling. For example, it was shown that in oral epithelial dysplasia, Ki67 is frequently expressed in the basal and suprabasal epithelial layers (always jointly) (Gonzalez-Moles et al.,2000).

Pericytes are a crucial target for anti-angiogenic therapies (Morikawa and Ezaki,2011). The presence of pericytes appears to protect endothelial cells against inhibition of VEGF signaling. The simultaneous inhibition of PDGF receptors on pericytes improves the effect of VEGF inhibitors on endothelial cells and thus enhances anti-angiogenic therapies (Hellberg et al.,2010). Pericytes are recruited from the periphery and are involved in blood vessel stabilization during ischemia-induced angiogenesis (Kokovay et al.,2006). Additionally, also bone marrow derived pericytes can contribute to vascularization (Cai et al.,2009).

A synergy between the endothelial cells and pericytes is essential to the stabilization and maturation of blood microvessels. Using an in vitro tissue-engineered model of angiogenesis, it was demonstrated that the newly formed endothelial tubes recruit pericytes from the fibroblast population in that model. PDGF appeared to be a major factor involved in the recruitment of pericytes (Berthod et al.,2012).

Some caution must be exercised when defining cells as pericytes to avoid confusion. A periendothelial cell embedded in the endothelial basal lamina is correctly termed as pericyte. Endothelial stalk cells are indeed closely related to pericytes, as also are the sprouting ETCs. However, ETCs emerge from the endothelial basal lamina and penetrate the connective stroma where various cells may influence them. This differs from pericytes. CFCs contact ETCs, and consequently, when a new endothelial tube is established, the CFCs could be recruited as pericytes. The density of MCs is also directly related to angiogenesis and microvessel density (Muramatsu et al.,2000; Takanami et al.,2000; Ribatti et al.,2007).

An experimental study performed in mice on a nerve scar model identified two types of pericytes: type B, desmin– and/or α smooth muscle actin–positive, and type A, expressing PDGFR–α and –ß, and CD13. It was observed that the lesion sites were populated with blood vessel sprouts with an increased density of associated type A pericytes, and many such pericytes “lost” contact with blood vessels at the lesion site and passed into the perivascular stroma (Goritz et al.,2011). Interestingly, in that study (Goritz et al.,2011), TEM revealed a telocyte morphology of the type A pericyte which, in our opinion, should be considered as a StrC after it breaks the basal endothelial lamina. Unfortunately, the respective study did not take into account and discuss these features, and neither did it consider the presence of ETCs within the scar tissue.

The scenario presented above is different from that described in studies of human skeletal muscle study that brought arguments to support the fact that “the cells identified so far as pericytes by their immunophenotype might also include other types of interstitial cells such as telocytes” (Suciu et al.,2012). This study did not distinguish the telocytes from ETCs, nor did it discuss the stromal influences on this peculiar cell type. Furthermore, it did not consider approaching a differential identification between telocytes and CFCs. It is, however, more supportive of our findings in the oral mucosa, in which the stromal compartment is already configured when ETCs invade it. The distinction between CFCs, lacking telopodes, and telocytes, defined mostly by their specific telopodes (Popescu and Faussone-Pellegrini,2010; Popescu et al.,2011; Nicolescu et al.,2012; Rusu et al.,2012a,b,c), is important since telocytes are resident StrCs, but CFCs are bone marrow derived cells.

Changes in wound bed vascularity are significantly less in oral wounds than in skin wounds (Szpaderska et al.,2005). The difference could be attributed to the CFCs which normally populate the oral mucosa. Maintenance and healing of the oral mucosa are supported by extensive processes of angiogenesis, likely guided by ETCs. The later are influenced by both CFCs and resident StrCs. There is some doubt, however, about the presence of telocytes in the oral mucosa stromal compartment.

Acknowledgements

The present research was funded (author #7) by the Sectoral Operational Programme Human Resources Development (SOP HRD), financed from the European Social Fund and by the Romanian Government; Grant number: POSDRU/89/1.5/S/60782.

Ancillary