tracing cd34+ stromal fibroblasts in palatal mucosa and

Microsc. Microanal. 21, 837–848, 2015doi:10.1017/S1431927615000598

© MICROSCOPY SOCIETYOF AMERICA 2015

Tracing CD34+ Stromal Fibroblasts in Palatal Mucosaand Periodontal Granulation Tissue as a Possible CellReservoir for Periodontal RegenerationAlexandra Roman,1,a Emőke Páll,1,2,a CarmenM. Mihu,3 Adrian S. Petruţiu,1 Lucian Barbu-Tudoran,4

Radu S. Câmpian,5 Adrian Florea,6,* and Carmen Georgiu7

1Department of Periodontology, Faculty of Dental Medicine, Iuliu Haţieganu University of Medicine and Pharmacy, 15 V.Babeş Street, 400012 Cluj-Napoca, Romania2Department of Veterinary Reproduction, Obstetrics and Gynecology, Faculty of Veterinary Medicine, University of AgriculturalSciences and Veterinary Medicine, 3-5 Mănăştur Street, 400372 Cluj-Napoca, Romania3Department of Histology, Faculty of Medicine, Iuliu Haţieganu University of Medicine and Pharmacy, 6 L. Pasteur Street,400349 Cluj-Napoca, Romania4Department of Molecular Biology and Biotechnologies, Faculty of Biology and Geology, Babeş-Bolyai University, 5-7 ClinicilorStreet, 400006 Cluj-Napoca, Romania5Department of Oral Rehabilitation, Faculty of Dental Medicine, Iuliu Haţieganu University of Medicine and Pharmacy, 15 V.Babeş Street, 400012 Cluj-Napoca, Romania6Department of Cell and Molecular Biology, Faculty of Medicine, Iuliu Haţieganu University of Medicine and Pharmacy, 6 L.Pasteur Street, 400349 Cluj-Napoca, Romania7Department of Pathology, Faculty of Medicine, Iuliu Haţieganu University of Medicine and Pharmacy, 8 V. Babeş Street,400012 Cluj-Napoca, Romania

Abstract: The aim of the present research was to trace CD34+ stromal fibroblastic cells (CD34+ SFCs) in thepalatal connective tissue harvested for muco-gingival surgical procedures and in granulation tissues fromperiodontal pockets using immunohistochemical and transmission electron microscopy. Immunohistochemicalanalysis targeted the presence of three antigens: CD31, α-smooth muscle actin (α-SMA), and CD34. In the palate,CD31 staining revealed a colored inner ring of the vessels representing the endothelium, α-SMA+ was located inthe medial layer of the vasculature, and CD34 was intensely expressed by endothelial cells and artery adventitialcells (considered to be CD34+ SFCs). Granulation tissue showed the same pattern for CD31+ and α-SMA, but adifferent staining pattern for CD34. Ultrastructural examination of the palatal tissue highlighted perivascular cellswith fibroblast-like characteristics and pericytes in close spatial relationship to endothelial cells. Theultrastructural evaluation of granulation tissue sections confirmed the presence of neovasculature andthe inflammatory nature of this tissue. The present study traced the presence of CD34+ SFCs and of pericytesin the palatal connective tissue thus highlighting once more its intrinsic regenerative capabilities. The clinical andsystemic factors triggering mobilization and influencing the fate of local CD34+ SCFs and other progenitors areissues to be further investigated.

Key words: tissue graft, immunohistochemistry, ultrastructure, CD34 antigen, pericytes

INTRODUCTION

The complex nature of tooth supporting tissues has ham-pered the development of effective and sustainable treat-ments to regenerate affected periodontal structures inperiodontitis lesions and in developmental defects such asgingival recessions. Classical surgical procedures treatinginfrabony defects or gingival recessions have been associatedwith good clinical outcomes (Cortellini et al., 2001; Trom-belli et al., 2002; Tonetti et al., 2004; Cairo et al., 2008;Chambrone et al., 2008), but the histological pictures have

been reported to be highly variable as only occasional trueperiodontal regeneration was demonstrated (Camelo et al.,1998; Mellonig, 2000; Paolantonio et al., 2001; Roman et al.,2010). Periodontal regeneration is still an unpredictableprocess (Diss et al., 2003) due to very complex mechanismsthat must develop in a very strict temporal and spatialsequence in order to synthesize all the components of theperiodontium (Karring et al., 1980; Nyman et al., 1980).

Current research trends have been directed towarddeveloping tissue engineering cell-based techniques in orderto obtain the regeneration of periodontal or other oral tissues(Seo et al., 2004; Gronthos et al., 2006; Feng et al., 2010;Duan et al., 2011; Hynes et al., 2012), but there are still manyunanswered questions that prevent advancement of the fieldof ex vivo manipulated stem cells toward clinical utility

*Corresponding author. [email protected]; [email protected] A. and Páll E. equally contributed to the present study and can be regarded,therefore, as being main authors.

Received February 11, 2015; accepted April 6, 2015

mailto:[email protected]

mailto:[email protected]

(Chen et al., 2012; Hynes et al., 2012). From a clinicalpoint of view it seems reasonable to further investigatethe local intrinsic regenerative potential of the periodon-tium or other oral sources in order to exploit them fortreatments.

A huge amount of research has addressed the fieldof oral mesenchymal stem cells (MSCs) from many andcomplex perspectives, but little attention has been focused ona specific subset of fibroblasts ubiquitously distributedthroughout the body, harboring CD34 antigen, and recentlydenominated as CD34+ stromal fibroblastic cells (CD34+SFCs) (Díaz-Flores et al., 2014a, 2014b). Numerous nameshave been given to CD34+ SFCs leading to confusion, suchas: CD34+ stromal cells (Yamazaki & Eyden, 1995), CD34+fibrocytes (Barth & Westhoff, 2007), interstitial Cajal-likecells (Gherghiceanu & Popescu, 2005), adventitial proge-nitor cells (Sartore et al., 2001), CD34+ dendritic cells(Regezi et al., 1992), and telocytes (Popescu & Faussone-Pellegrini, 2010; Faussone-Pellegrini & Popescu, 2011).

CD34+ SFCs are the major fibroblastic cell componentsof the adventitial or external layer of the vasculatureconcentrating in the vascular adventitial layer of arteries andveins (Corselli et al., 2012), but they are also found in stromalpositions in the connective tissue of multiple anatomical sites(Barth & Westhoff, 2007). These quiescent-slow-cyclingresident cells have the ability to self-renew and to originatedaughter cells. Similar behavior occurs with other residentquiescent cells, such as endothelial cells, pericytes, and vas-cular smooth muscle cells (Díaz-Flores et al., 1994, 2009).CD34+ SFCs behave as MSC progenitors. CD34+ SFCs ofadipose origin give rise to MSCs (CD34− ) under cultureconditions (Braun et al., 2013) indicating that tissue-CD34+SFCs have mesenchymal potential. Their mesenchymalstem cell properties are currently the object of greatinterest; CD34+ SFCs have myofibroblastic, adipogenic,osteoblastic, and chondrogenic differentiation capacities(Díaz-Flores et al. 2014a, 2014b). Consequently, CD34+SFCs are important participants in wound healing, tissuerepair, fibrosis, and tumor stroma formation. Beyondthe above mentioned properties, the functional capacitiesof CD34+ SFCs also include immunomodulation,parenchymal regulation, and scaffolding support forother cells (Díaz-Flores et al. 2014a, 2014b), contributingto replenishment of stem cell niches (Haniffa et al., 2009).

Our team previously isolated MSCs from palatalconnective tissue grafts (Roman et al., 2012, 2013) and fromperiodontal granulation tissues (unpublished data) provid-ing arguments for the regenerative potential of these sources.In preparing the present study we presumed the presence ofCD34+ SFCs in palatal mucosa and periodontal granulationtissues. We also considered that further insight in themorphological and ultrastructural features of these tissuescould provide information on the characteristics anddistribution of CD34+ SFCs to be used in regenerativeperiodontology or for extrapolation for the regenerationof extra-oral sites. The aim of the present research wasto trace CD34+ SFCs in the palatal connective tissue

harvested for muco-gingival surgical procedures and ingranulation tissues from periodontal pockets using immuo-histochemical and transmission electron microscopy (TEM)evaluations.

MATERIALS AND METHODS

Collection of the SamplesThe oral samples were collected from patients undergoingperiodontal surgical treatment in the PeriodontologyDepartment of Iuliu Hatieganu University of Medicine andPharmacy, Cluj-Napoca. The study was approved by theEthical Board of the Iuliu Haţieganu University (Nos 505/19.12.2011 and 359/13.10.2014). The study protocol andprocedural details were explained to patients and writteninformed consent was obtained. In obtaining informedconsent and conducting the research, the study adhered tothe principles outlined in the Declaration of Helsinki onexperimentation involving human subjects.

The palatal tissue sample was collected from a 24-year-old man treated with coronally advanced flap and aconnective tissue graft in order to cover a Miller class Igingival recession (Miller, 1985). The patient had good oralhygiene and no relevant systemic diseases, and was anonsmoker. An ~12/6mm split-thickness connective tissuegraft (including adipose tissue) was harvested from the palatalpremolar region using the single incision technique (Hürzeler& Weng, 1999). The waste tissue parts resulting from adap-tation of the graft onto the recipient surface were used forhistological, immunohistochemical, and TEM evaluation.

The granulation tissue was harvested from a 39-year-oldchronic periodontitis female patient during a modified flapoperation (Kirkland, 1931; Wennstrom et al., 2008). Thegranulation tissue was removed from the apical inner surfaceof the flaps with surgical scissors (Micro Curved CastroviejoScissor; Hu-Friedy Mfg. Co., Chicago, IL, USA).

Other parts of the waste tissues were used for the isola-tion of MSCs (data not shown).

Histological AnalysisSpecimens were fixed in buffered neutral 4% formaldehydefor 24 h and processed using progressing concentrations ofethanol and xylene (Leica TP 1020, Leica MicrosystemsNussloch GMbH, Nussloch, Germany). Then, the specimenswere embedded in paraffin (Leica EG 1150H, Leica Micro-systems Nussloch GMbH) and cut into 4-µm-thick sections,which were stained with hematoxylin and eosin.

ImmunohistochemistryThree paraffin blocks corresponding to three surface anti-gens to be detected were prepared from each tissue source(palatal connective tissue and periodontal granulationtissue). For each block, eight slides (seven slides for stainingsand one slide as control) were prepared. The immunohisto-chemical analysis evaluated 48 sections; 4-µm-thick sections

838 Alexandra Roman et al.

were cut from the paraffin blocks and attached to silane-coated slides (DakoCytomation, Glostrup, Denmark) thatwere then deparaffinized, treated with xylene, andrehydrated with decreasing concentrations of ethanol. Thesections were unmasked with Novocastra Epitope RetrievalSolution (10×) pH = 9 (Leica BioSystems Newcastle Ltd.,Newcastle Upon Tyne, UK) for 20 min at 95°C in a pressurecooker and washed with distilled water. Endogenous perox-idase activity was neutralized using the Novocastra Perox-idase Block (Leica Microsystems Nussloch GMbH,Nussloch, Germany) for 5 min and then washed with BondWash Solution (BWS, 10×) (Leica) for 5 min three times.This was followed by application of Novocastra ProteinBlock (Leica) for 5 min to reduce nonspecific binding ofprimary antibodies and then washed with BWS. Sectionswere subsequently incubated with optimal diluted primaryantibodies for 30 min (DakoCytomation). Primary anti-bodies used in this study were monoclonal mouse antibodiesanti-Human CD34, CD31, and α-SMA (DakoCytomation)as follows: CD34 (dilution 1:25), code no. M7165; CD31(dilution 1:20), code no. M0823; α-SMA (dilution 1:50), codeno. M0851. Specimens were washed with BWS for 5 min twotimes. The Novocastra Post Primary Block (Leica) wasapplied for 30 min to enhance penetration of the subsequentpolymer reagent. The NovoLink Polymer (Leica) applied for30 min recognized mouse immunoglobulin and thus detec-ted any tissue-bound antibody. Sections were washed andfurther incubated with the substrate/chromogen DAB (3,3'-diaminobenzidine), prepared from Novocastra DAB Chro-mogen (Leica) (50 µL) and NovoLink DAB Substrate Buffer(polymer) (Leica) (1 mL) and then washed with runningwater as recommended (Novocastra brochure). Reactionwith peroxidase produced a visible brown precipitate at theantigen site. Sections were counterstained with NovocastraHematoxylin (Leica), washed with water, dehydrated withprogressive concentrations of ethanol, treated with xylene,and coverslipped with NeoMount® (Merk KGaA, Darmstadt,Germany). The positive internal control marked vascularendothelial cells of the arteries with antibodies anti-CD31

and anti-CD34 and of the smooth muscle fibers of the mediallayer with antibodies to anti-α-SMA. For preparing the negativecontrol primary antibodies were excluded from the protocol.

The sections were examined for histology (CMM) andimuunohistochemistry (CG) with a Leica DM 750 microscope(Leica, Leica Microsystems Heidelberg, Mannheim, Germany)and photographed with Leica ICC 50 HD (Leica, Leica Micro-systems Heidelberg) camera connected to the microscope.

TEMThe tissue samples were processed for TEM according tousual protocols (Hayat, 2000; Watt, 2003). Samples werefixed with 2.7% glutaraldehyde (Electron Microscopy Sci-ences, Hatfield, PA, USA) in 0.1 M phosphate buffered saline(PBS) for 2 h, washed four times with 0.1 M PBS, postfixedwith 1% osmium tetroxide (Fluka, Buchs, Switzerland) in0.15 M PBS for 1.5 h, and washed with 0.15M PBS. Next,they were dehydrated in an acetone series (30–100%) andembedded in Epon 812 (Fluka). Ultrathin sections cut with adiamond knife on a Leica UC6 (Leica Microsystems, Wet-zlar, Germany) were contrasted with uranyl acetate (Merck,Darmstadt, Germany) and lead citrate (Fluka). The sectionswere examined on a JEOL JEM 1010 TEM (JEOL Ltd.,Tokyo, Japan), and images were captured using a Mega ViewIII camera (Olympus, Soft Imaging System, Münster,Germany).

RESULTS

Histological FeaturesThe palatal connective tissue was fibrous, displaying somefibroblasts and a well-developed vasculature; a fully devel-oped histological structure of arteriolae was observed (inti-mal, medial, and adventitial layers). Collagen fibers withdifferent orientations and the presence of nervous fasciculeswere also observed. Connective tissue cells were highlightedby their nucleus having different shapes, sizes, and tinctorial

Figure 1. Histological features of the harvested tissue samples: (a) palatal connective tissue (arrow, fibroblast); (b) peri-odontal granulation tissue (long arrow, lymphocyte; short arrow, plasma cell; arrowhead, macrophage; black asterisk,oblique sections of peripheral nervous fibers; white asterisk, transverse sections of peripheral nervous fibers) (hemato-xylin and eosin stain).

Oral CD34+ Stromal Fibroblastic Cells 839

aspects; the nuclei of fibroblasts were noticeable. No sign ofinflammation was present (Fig. 1a).

The periodontal granulation tissue was highlyvascularized, with a high-grade inflammatory infiltrate.Among the heterogeneous cell populations one could iden-tify lymphocytes, macrophages and plasma cells, andsome fibrocites. Peripheral myelinic nerve fibers in differentsection incidences were present (Fig. 1b).

ImmunohistochemistryCD31 staining of the palatal samples revealed a colored innerring of vessels, thin, compact, and complete, representing theendothelium that stained positively for CD31 (Figs. 2a, 2b).In the vicinity of the vascular structures of the adipose palataltissue no CD31 antigens were detected.

In the palatal connective tissue, α-SMA+ cells werelocated in the medial layer of the vasculature, and in thestroma of the palatal adipose tissue. Peripheral α-SMA+ cellsof the vessels were more intensely stained than the inner ones(Figs. 2c, 2d).

In palatal connective tissue samples, CD34 was intenselyexpressed by cells of the intimal layer (endothelial cells), andby adventitial layer cells of arteries, which led to the forma-tion of two concentric circles “sandwiching” the unstainedmedial layer (Figs. 3a, 3b). Beside CD34+ cells delineatingthe vascular peripheral ring other CD34+ cells were present.Thus, CD34+ cells were present in the endoneurium andaround the perineurium of the transverse sections of nerves

contained in the palatal connective tissue. These CD34+ cellshad small oval nuclei (Fig. 3c). In the stroma of the adiposepart of the palatal tissue, CD34+ cells with a bipolar andstar-like appearance were observed closely associated withadipocytes; the cytoplasmic processes of CD34+ cells formeda delicate network surrounding adipocytes (Fig. 3d).

The same pattern of staining of the granulation tissuefor CD31+ and α-SMA was observed as for the palatal tissue(Figs. 4a–4d). In capillaries of the granulation tissue,α-SMA+ cells were disposed in a single layer and formed auniform-stained discontinuous ring. Elongated cells withprimary extensions were found on the long axis of the vesselsand secondary prolongations were also present (Fig. 4c).

The periodontal granulation tissue showed a differentCD34-staining pattern. The difference between the granu-lation tissue and the palatal tissue was the monocircle stainedaspect of the vasculature due to staining of the intimallayer and absence of the exterior CD34+ stained layer(Figs. 4e, 4f).

TEMIn a transverse section of a palatal blood vessel, endothelialcells showed a protruding nucleus into the vascular lumen(Fig. 5a). In a longitudinal section, the relatively simpleultrastructure of endothelial cell cytoplasm was noted, withfew organelles, mostly concentrated in the perinuclear zone(Fig. 5b). A characteristic feature was the concentration ofsmall vesicles (pinocytotic) adjacent to the endothelial cell

Figure 2. Immunohistochemical staining of palatal tissue sections: (a) CD31 staining of intimal layer of palatal vessels;(b) CD31 staining of intimal layer of palatal vessels; (c) α-SMA+ vascular cells; (d) α-SMA+ vascular cells. SMA,smooth muscle actin.


membranes (Fig. 5c). A fibroblast with a perivascular location(Fig. 5a) showed prominent nucleoli, characteristic of activecell protein synthesis. The nucleus was slightly indented andrelatively large in relation to the small cytoplasm content inthe cell body, showing patches of heterochromatin, particu-larly near the nuclear membrane (Fig. 5d). Scarce cisternae ofrough and smooth endoplasmic reticulum, few mitochondria,and a small Golgi apparatus were observed. Long cytoplasmicextensions of the fibroblasts had multiple surface folds andcontained densely packed rough endoplasmic reticulum. Thefibroblast processes surrounded collagen fibers leading to acompartmentalized organization of the tissue (Fig. 6a).Perivascular fibroblasts surrounded pericytes (Fig. 6b).

Pericyte-like cells identified in palatal samples incomple-tely enveloped the endothelial cells. A pericyte covered two orthree endothelial cells incompletely, showing an elongated cellbody from which arose an elaborate system of longitudinal andcircumferential branches (Figs. 5a, 5c, 6c). The nuclear regionof pericytes was found immediately adjacent to the endothe-lium (Fig. 5b). Nuclei showed dense patches of hetero-chromatin, in close proximity to the nuclear membrane(Fig. 6d). Their cytoplasm was electron dense and containedsmall mitochondria, a few profiles of rough endoplasmicreticulum, free- and membrane-bound ribosomes, andmultilamellar bodies. The pericyte processes showed somemembranes of endoplasmic reticulum, free ribosomes,mitochondria, and plasmalemal vesicles (Fig. 6d). Pericyteswere embedded by the basement membrane together withendothelial cells.

In periodontal granulation tissue fibroblasts demon-strated an intense secretory activity highlighted by the pre-sence of intracellular collagen and a rich rough endoplasmicreticulum (Figs. 7a, 7b). Newly formed vascular structuresand a rich inflammatory infiltrate harboring a wide celldiversity were also observed (Fig. 7c, 7d).

DISCUSSION

The present research presumed the presence of theprogenitors of CD34+ SFCs mesenchymal cells in palataltissue samples and in periodontal granulation tissues, inorder to highlight the intrinsic regenerative potential ofautogenous soft tissue grafts and inflamed periodontaltissues as a premise to regenerate the periodontium affectedby disease, and as a source for regenerative cells. In thisstudy, tissue samples from the two above mentioned oralsources were evaluated using histological, immunohisto-chemical, and ultrastructural analyses in order to trace thelocation of CD34+ SFCs.

The presence of CD34+ cells in the palatal connectivetissue was demonstrated in several locations: (a) at the levelof the inner layer of the vasculature; (b) in the adventitiallayer of vasculature, forming a positive exterior stained ring,thus seconding the inner CD34+ cell sheet; (c) in palatalnerves; and (d) in close association with adipocytes havingcytoplasmic processes that formed a delicate network aroundadipose tissue cells. The inner CD34+ ring of the vasculature

Figure 3. CD34+ cells in palatal sections: (a) CD34-stained vessels; (b) CD34-stained vessels; (c) CD34+ cells aroundperineurium and in endoneurium; (d) CD34+ cells around adipocytes.


was undoubtedly the endothelium because this layer isknown to harbor CD34 (and CD31) antigens; the endothelialcells were negative for α-SMA (CD34+, CD31+, α-SMA−).These findings are in agreement with the observations ofother studies (Pusztaszeri et al., 2006; Barth & Westhoff,2007; Díaz-Flores et al., 2014a).

The outer vascular CD34+ ring, which belonged to theadventitia, was irregular, loose, incomplete, and distinctivelyCD31 negative; this specific localization of the CD34+ cellsclearly delineates the medial layer from the adventitial layer.These adventitial CD34+ cells were also α-SMA negative.This antigen make-up of the adventitial cells could recom-mend them as CD34+ SFCs (Díaz-Flores et al., 2014a,2014b). Although CD34 is intensely expressed in endothelialcells, both endothelial cells and CD34+ SFCs could beeasily distinguished by their different response to CD31(anti-CD31 stains endothelial cells but not CD34+ SFCs)

(Díaz-Flores et al., 2014a). Since CD34 antigen isexpressed in fibroblasts in the adventitia and inendothelial cells of the tunica intima, the CD34 stainedvessels in the palate samples of the present study showeda double ring appearance as two concentric circlesseparated by the unstained medial layer (smooth muscle),as the literature describes (Lin et al., 2010; Díaz-Flores et al.,2014a). These adventitial CD34+ cells (CD34+ SFCs)reside in a mixed population of cells (macrophages,dendritic cells, and progenitor cells) (Majesky et al., 2011);the CD34+ cells in the adventitia are considered MSCprogenitors (Lin & Lue, 2013; Díaz-Flores et al., 2014a)and could be termed as “adventitial progenitor cells”(Lin & Lue, 2013). The CD34+ SFCs in the adventitiallayer have the potential of differentiating into maturehematopoietic cells, endothelial cells, and macrophages(Zengin et al., 2006).

Figure 4. Immunohistochemical staining of periodontal granulation sections: (a) CD31 staining of capillaries; (b) CD31staining of capillaries; (c) α-SMA+ cells (long arrow, primary prolongation; short arrow, secondary prolongation);(d) α-SMA+ cells; (e) CD34 staining of vasculature; (f) CD34 staining of vasculature. SMA, smooth muscle actin.


Figure 5. Ultrastructural aspects of palatal tissue: (a) transverse section of a capillary; (b) longitudinal section of acapillary; (c) endothelial cell surrounded by pericytic prolongations; (d) fibroblast with a perivascular location. FB,fibroblast; P, pericyte; Pp, pericyte processes; EC, endothelial cell; RBC, red blood cells; CF, collagen fibers; CL, capillarylumen; PV, pinocytotic vesicle; M, mitochondria.

Figure 6. Ultrastructural aspects of fibroblasts and pericytes in palatal sections: (a) fibroblast processes and the com-partmentalized organization of the tissue; (b) perivascular fibroblasts; (c) partial pericyte coverage of endothelial cells;(d) pericyte with a nucleus with dense patches of heterochromatin in close proximity to the nuclear membrane. FB,fibroblast; CF, collagen fibers; P, pericyte; Pp, pericyte processes; EC, endothelial cell; RBC, red blood cells; GC, granu-locyte; RER, rough endoplasmic reticulum; M, mitochondria.


CD34+ cells in the palatal nerves (in endoneurium andaround perineurium) may be considered as CD34+ SFCs(Díaz-Flores et al., 2014a); some authors consider thatthese cells may play a supportive role for Schwann cells(Weiss & Nickoloff, 1993).

The present study also identified in the palatalconnective tissue CD34+ SFCs alongside adipocytes andtheir microvasculature, having long, attenuated, bipolar, ormultipolar cytoplasmic processes. This finding is in agree-ment with the description provided (Díaz-Flores et al.,2014a, 2014b). Three major cell subsets were identified in theheterogeneous population of cells in the adipose tissue-derived vascular stromal fraction: adventitial stromalcell-like cells or adipose tissue-derived stromal (stem) cells(CD34+, CD146− , CD271±); pericyte-like cells (CD34−,CD146+, CD271±), and endothelial cells (CD34+ , CD31+ ,CD146+) (Braun et al., 2013). Therefore, cell precursors ofadipose tissue-derived stem cells were found in the CD34+stromal vascular fraction of adipose tissues (Maumus et al.,2011) and they can give rise to adipocytes, osteoblasts, andchondrocytes (Zuk et al., 2002).

CD34 is a 110 kDa transmembrane cell surface glyco-protein expressed in hematopoietic progenitor cells ofmyeloid and lymphoid lineage (Civin et al., 1984), endothe-lial cells (Fina et al., 1990), stromal cells in severalorgans (van de Rijn et al., 1994), mast cells, and neurons(Nielsen &McNagny, 2008). Potential functions of the CD34family include enhanced proliferation and blocking differ-entiation of stem or progenitor cells (Suga et al., 2009),

promoting or blocking cellular adhesion depending on thecellular environment (Lin et al., 2013), trafficking of hema-topoietic cells, and cell morphogenesis (Nielsen &McNagny,2008). Regarding the positivity for CD34 antigen of the cellsfrom the adipose tissue stromal vascular fraction it must bestated that many cell types that are CD34+ when freshlyisolated (e.g., endothelial or adipose tissue-derived stromalcells) lose CD34 expression in culture (Ning et al., 2006;Stolzing et al., 2012). Thus, MSC negativity for CD34 can beconsidered more likely a cell culture-induced phenomenon,and not necessarily an indication of their in vivo status.Standard antigen characterization of MSCs includes the lackof CD34 antigen expression (Dominici et al., 2006), but inthe light of recent knowledge whether CD34 is truly anegative marker for MSCs, this should be re-evaluated(Lin et al., 2013). CD34+ SFCs also express or can changeexpression of vimentin, CD10, CD117, CD99, S00 protein,desmin, or cytokeratin (Suster et al., 1998) depending on celllocation, temporal activity, and participation in pathologicalprocesses.

In our paper, ultrastructural examination of palatal tis-sue highlighted the presence of cells with fibroblast-likecharacteristics in the perivascular region. Ultrastructuralanalysis traced only capillaries in palatal tissue sections, butfibroblasts observed in paravascular regions could beequivalent to CD34+ SFCs identified by immunohisto-chemical analysis. The present ultrastructural examinationof the palatal samples also highlighted the presence ofpericytes in the capillaries in close spatial relationship to

Figure 7. Ultrastructural aspects of periodontal granulation tissue: (a) fibroblast synthesizing collagen fibers; (b) detailfrom extracellular matrix with collagen fibers; (c) longitudinal section of a blood vessel; (d) inflammatory infiltrate. FB,fibroblast; CF, collagen fibers; WBC, white blood cells.


endothelial cells with partial pericyte coverage or anumbrella-like structure around endothelial cells as describedby Allt & Lawrenson (2001).

Traditionally, pericytes are defined as extensively bran-ched cells located in nonmuscular microvessels, capillaries,and postcapillary venules (Majno, 1965), and in thesubendothelium in the outer layer of the media and in thevasa-vasora of large, medium, and small arteries and veins(Andreeva et al., 1998). A single pericyte in contact withseveral endothelial cells suggests that they may integrate andmediate some endothelial cell functions (von Tell et al.,2006). There are more and more data considering pericytesas the de facto MSCs due to demonstrations that pericytesdifferentiate into osteoblasts, chondrocytes, and adipocytes(Lin & Lue, 2013). No specific markers could be identifiedto highlight the perivascular localization of these cells(Díaz-Flores et al., 2009, 2014a; Dore-Duffy & Clearyn,2011) making future research difficult to carry on.

The presence in the palatal connective tissue of pro-genitor cells in different commitment stages (CD34+ SFCs,pericytes) highlighted the regenerative potential of the areaand could be a supplementary argument in the favor of theuse of autologous soft tissue grafts for increasing periodontalregeneration after root coverage surgeries.

As for the antigen make-up of the periodontal granula-tion tissue, a quite different pattern was recorded.Immunohistochemical CD34 staining revealed only thepresence of an inner vascular CD34+ ring as the granulationtissue contains only capillaries. As capillaries do not have awell-structured adventitial layer, a monocircle appearancedue to CD34+ endothelial cells (CD34+, CD31+, α-SMA−)characterized the granulation tissue samples. In capillariesthe presence of CD34+ SFC could not be excluded becausein the absence of the medial layer these cells could be locatedin close proximity of endothelial cells thus increasing thethickness of the stained ring. It was observed that incapillaries, the CD34+ layer was thicker in comparison withthe somehow flattened CD34+ inner layer of the palatalvasculature.

The presence of a CD34+, CD31−, α-SMA−, CD140b−population of cells was observed in capillaries (Lin et al.,2008), which is believed to be the equivalent of the CD34+,CD31−, α-SMA− adventitial progenitor cells (Lin & Lue,2013). Since capillaries lack the tunica adventitia, the term“vascular stem cells” was proposed to denote the CD34+,CD31−, α-SMA−, CD140b− population in both capillariesand larger vessels (Lin & Lue, 2013).

Another possibility is that CD34+ SFCs were absent ingranulation tissue samples of the present study due to thechange of CD34+ expression that could take place bothin vivo as well as in vitro conditions. Thus, in repair throughgranulation tissue, stromal cell remodeling occurs with lossof CD34 expression (loss of CD34+ stromal cells) and sub-sequent gain of α-SMA expression due to myofibroblastdifferentiation (Barth et al., 2002; Barth & Westhoff, 2007).After 7 days of granulation tissue formation, stromal cellsexpressing CD34 were scarce (Díaz-Flores et al., 2014a).

The elongated α-SMA+ cells observed in our granula-tion tissue specimens had primary and secondaryprolongations and they partially encircled endothelial cells(observable unstained) from the abluminal side ofmicrovessels. The α-SMA+ cells correspond to the descrip-tion of perycites (Zimmermann, 1923; Ross et al., 2003; Diaz-Flores et al., 2009). In the present study the ultrastructuralanalysis of periodontal granulation tissue sections failed toobserve the presence of fibroblasts or pericytes in para-vascular locations. The ultrastructural observations con-firmed the highly inflammatory nature of the periodontalgranulation tissue.

Since in periodontitis lesions the periodontal maturetissue could not be regenerated in the presence of chronicinfection and possibly because of the lack of an appropriatescaffold to guide new tissue development, the immaturetissue persists up to treatment. The presence of infection inperiodontal granulation lesions removed during periodontalflap surgery was demonstrated (Ronay et al., 2013), which isnot surprising due to the high bacterial load in the sub-gingival area of the pockets in close relationship with thegranulation tissue. In the meantime, cells harboring MSCproperties were isolated from these highly infected tissues(Ronay et al., 2013, 2014). Other authors reported that MSCsisolated from inflamed periodontium had differentiation andregenerative capabilities (Park et al., 2011).

Cells with MSC properties were also isolated frominflamed dental pulp (Alongi et al., 2010; Yazid et al., 2014)and inflamed periapical tissues (granuloma) (Liao et al.,2011). There are conflicting data about whether the presenceof inflammation affects the proliferative and migratorypotentials of MSCs isolated from these inflamed oral tissuesin comparison with the cells isolated from healthy sites(Alongi et al., 2010; Park et al., 2011; Yazid et al., 2014;Yu et al., 2014; Pall et al., unpublished data) and furtherresearch should elucidate this issue.

As palatal connective tissues and periodontal granula-tion tissues harbor some types of progenitor cells (MSCs,pericytes, CD34+ SFC) some current clinical paradigmsshould be reconsidered. The elimination of adipose tissuefrom palatal connective tissue grafts during periodontalplastic surgeries (Borghetti & Monett-Corti, 2000) seems notto be mandatory as it translocates progenitor cells ontorecessed radicular surfaces, thus transmitting the progenitoror differentiating signal in the neighborhood. Moreover,surgical removal of granulation tissues from the periodontaldefects inevitably results in removal of progenitor cells thatmight potentially contribute to regeneration of the surgicalarea. On the other hand, granulation tissue formed aftertooth extraction differentiates into bone thus highlightingthe presence of some precursor cells in the healing area(Steiner et al., 2008). What differentiates the healing ofperiodontal infrabony defects from that of postextractionsockets may be the proximity of avascular tooth surface andthe highly infectious state of the former area.

The present study traced the presence of CD34+ SFCsand pericytes in the palatal connective tissue thus


highlighting once more its intrinsic regenerative capabilities.Pericytes were observed in periodontal granulation tissuesections. Despite the presence of cells in different commit-ment stages in palatal and periodontal granulation tissues,regeneration after surgeries in these areas is highly unpre-dictable. The clinical and systemic factors triggering themobilization of local CD34+ SCFs and other progenitors,influencing their fate and orchestrating periodontalregeneration, are issues to be clarified by further research.Until then the two investigated oral sources remain a valu-able cell reservoir for regenerative medicine.

ACKNOWLEDGMENT

Costs of the research were partially covered by the grant PNII-PT-PCCA-2013-4-1474 (No. Contract: 127/2014) andpartial by Iuliu Hatieganu University of Medicine andPharmacy (No. 1493/5/28.01.2014).

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tracing cd34+ stromal fibroblasts in palatal mucosa and

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