Healing of periodontal tissues destroyed by periodontal disease does not usually result in the regeneration of the periodontium. One of the often repeated reasons is the lack of progenitor cell populations capable of restoring the different tissues of the periodontium1. The fi broblast is the predominant cell type in the soft connective tissue of the periodontium. These cells were described as the architect, builder and caretaker of the perodontum2. It is not clear whether

Immunolocalization of CD 34 positive progenitor cells in healthy human gingiva - a pilot study

Vamsi Lavu, Padmavathy R.* & Suresh R. Rao

Department of Periodontics, Sri Ramachandra Dental College, Sri Ramachandra University & *Department of *Department of *

Pathology, Royapettah Government Hospital, Chennai, India

Received November 21, 2007

Background & objectives: The gingiva is a tissue with a high turnover rate of both epithelial and connective tissue cells. In an attempt to identify the possible source of cells which maintain the tissue turnover, we used CD 34, a well established marker of peripheral blood stem cell in healthy human gingiva to determine the origin of progenitor cells in healthy gingiva.

Methods: Healthy human gingival samples (n=15) were collected from patients undergoing orthodontic extraction. Immunohistochemistry was done on 5 micron paraffi n fi xed section using the primary antibody CD34 and a universal secondary immunoperoxidase kit. The sections were examined for a golden brown stain indicative of a positive staining.

Results: Of the 15 samples 12 demonstrated a positive staining for the endothelial cells. Of these 12 samples, 11 demonstrated positive staining for stromal and paravascular cells and 10 a positive staining for the basal epithelium layers.

Interpretation & conclusions: The presence of CD 34 positive cells in gingiva in stromal, paravascular location, and basal layer of the gingival epithelium was demonstrated. We speculate that these could be fi broblastic progenitors originating from the peripheral blood stem cells and the positivity stained epithelial cells could be gingival epithelial stem cells.

Key words CD 34 expression - gingival - immunohistochemistry - progenitor cells

a single progenitor cell for fi broblasts, osteoblasts and cementoblasts exists in the adult periodontal tissue3. Several early studies have focused on the identifi cation of progenitor cell populations in the periodontal ligament of experimental animals4,5. The lack of specifi c markers for the fi broblast progenitors has hampered the precise identifi cation of the origin and location of these cells. It has been suggested3 that the cells of fi broblastic series may resemble, other well

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described self renewal systems in mammals, such as the skin, liver, peripheral blood, etc. CD 34 is a surface glycophosphoprotein expressed on haemopoietic stem and progenitor cells, small vessel endothelial cells and embryonic fi broblasts6. CD 34 positivity has been demonstrated for fi broblasts and stromal cells in tissues such as skin7, thyroid gland8, testicular stroma9

and also in the oral tissues like sub mandibular gland10, and oral mucosa11. The gingiva is a highly vascular tissue. The progenitors in gingiva may be arising from the peripheral blood haemopoietic stem cells which are CD 34 positive. To test the above hypothesis, CD 34 was used as a marker in the present study to identify putative progenitor cells in the healthy human gingiva and their location. The objective was to determine the possible origin of progenitor cells in healthy human gingiva and its implications in regeneration of the periodontium.

Material & Methods

The subjects who gave informed consent were selected, the others were not included. A total of twenty patients were chosen based on inclusion and exclusion criteria. The small sample size is because the study is a pilot study. No special procedure was followed for randomization. All the subjects had visited the Out Patient Department of Oral Surgery, Sri Ramachandra Dental College, Porur, Chennai, during April to August 2005 for the purpose of extraction of teeth for orthodontic purposes. Healthy, non smoker individuals, who had not undergone any periodontal treatment in the past six months were selected. Among these patients, those who had healthy gingiva as determined by clinical fi ndings such as colour, consistency, absence of bleeding on probing, absence of probing depth/loss of attachment were included in the study. Informed written consent was obtained from each patient prior to sample collection. The study has been approved by Institutional Ethics Committee of the Sri Ramachandra University. The healthy gingival samples were collected by surgical excision of a part of the papilla either distal or mesial to the tooth to be removed. The gingival samples were washed in sterile saline to remove the blood and fi xed with 4 per cent buffered formalin for 24 h12 following which these were embedded in paraffi n wax. The embedding in paraffi n wax and subsequent steps of tissue sectioning and staining were performed in the Pathology Division of Bharat Scans, Chennai. Two 5 µm thick sections were taken on 3-aminopropylt-riethoxysilane (APES) (Sigma, USA) coated slides,

one of which was used for hematoxylin and eosin (H&E) (Merck, Germany) staining, the other used for immunohistochemistry. The sections were de-parrafi nized and re-hydrated and antigen retrieval (by microwave processing) was performed. The primary antibody used was the antibody to CD 34 class III epitope (Chemicon International, USA Catalog No- CBL 555). The primary antibody was diluted with phosphate buffered saline, pH 7.4 to obtain an end dilution of 1: 2013. A Universal Secondary Immuno-peroxidase Kit was used (B-SAP Universal Staining Kit- Span Diagnostics). It contains the secondary antibody tagged with peroxidase and also the substrate for the enzyme. Following staining, the sections were examined under the microscope (Zeiss Microscope-Axiostar Plus with attached Samsung Photomicrography equipment, Romania) for a golden brown stain which is indicative of positive staining. A section of a pyogenic granuloma obtained from lip lesion was used as a positive control specimen for the antibody to CD 34. A drop of phosphate buffered saline (p(p( H 7.4) was used instead of the primary antibody in the negative control specimens. Counting of cells in the positively stained sections was done under 100 X oil immersion. The results were expressed as the number of positively stained cells per 10 high power fi elds (modifi cation of the method described by Mesa et al12). Basically, in the modifi ed method, the counting was done for 10 consecutive high power fi elds.

Results

Of the 15 healthy gingival samples, 12 exhibited a positive staining for the CD 34 antigen. Eleven of these 12 samples demonstrated the presence of positively stained stromal/paravascular cells in the connective tissue (Fig. 1), and ten demonstrated positive staining for the basal and parabasal layers of the epithelium (Fig. 2). All the 12 samples demonstrated a positive staining for the endothelial cells. Cell membrane positivity for the CD 34 antigen was detected in the basal and parabasal layers of the epithelium. Of the ten positive samples, four exhibited a focal positive staining, whereas six exhibited a uniform staining of the entire basal layer. Of the eleven samples that stained positive for the connective tissue cells, six demonstrated more number of stromal cells than paravascular cells, and four demonstrated more number of para-vascular cells, with one section showing equal distribution of cells in the paravascular and stromal locations. Some cells had a fi broblastic morphology, whereas the others had a round or oval shape.

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LAVU et al: CD 34 EXPRESSION IN HUMAN GINGIVA 687

The positive control sample (pyogenic granuloma) demonstrated a positive staining of the endothelial cells (Fig. 3) and the negative control sample was characterized by complete absence of the golden brown stain (Fig. 4).

Discussion

Of the two prominent fi broblast populations in the periodontium- the gingival and periodontal ligament fi broblasts, the origin of gingival fi broblast is less clear. Tencate and co-workers14 suggested that gingival fi broblasts may be derived from the dental follicle cells or from the non-odontogenic gingival connective tissue. However, the origin of these cells in the adult periodontium is not known. The gingival epithelium

as well as connective tissue have a very high turnover rate and new cells are constantly produced to replace cells, which are lost through cell death or migration. Thus, existence of a lineage of cells, which constantly replenish the fi broblast population, cannot be doubted.

Krause and co-workers15 have shown that a single donor haemopoietic stem cell (HSC) could do more than just repopulate the marrow and haemopoietic system in the recipient irradiated mice. They found epithelial cells derived from the donor stem cells in the lungs, gut, and skin of the recipient mice. The most well characterized marker for the peripheral blood stem cells in humans as well as other mammals has been the sialomucin CD 34. Berardi et al16, found

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Fig. 1. Photomicrograph of a section of healthy human gingiva with positive staining of a para-vascular and stromal cell- Fig 2. Photomicrograph of a section of healthy human gingiva with a cell membrane positive staining of the basal and parabasal layer of gingival epithelium- Fig. 3. Photomicrograph of a section of pyogenic granuloma (Positive control) with a positive staining of the endothelial cells- Fig. 4. Photomicrograph of a section of healthy human gingiva in which no primary antibody was used. (Negative control) with no golden brown staining- Figs 1-4 - 40 X magnifi cation.

that CD 34 is expressed on a population of pluripotent progenitors that can be enriched from the human bone marrow. In another study Schmidt et al17 determined that CD 34 positive fi brocytes in the peripheral blood were precursors of bronchial fi broblasts.

A paravascular location for the fi broblast progenitors has been described by Nemeth et al18 in the gingiva of the cynomolgus monkey. An earlier work by Pender et alPender et alPender 19 did not describe any paravascular location but identifi ed two distinct progenitor connective tissue cell populations in the rat gingiva; one population was located in the mid papilla region and the other close to the epithelial attachment and cementum. Our study showed that CD 34 positive cells were present in the healthy human gingiva; similar to lower species, some of these cells had a paravascular location whereas some demonstrated a stromal location. Three of the 15 gingival samples were not stained. This could possibly be due to technical errors in processing or these sections might not have contained the target cells.

CD 34 positive cells have been identifi ed to be markers for stem cells in the epidermis of mouse skin20,21. Song et al22et al22et al studied the pathogenesis of Pterygium; a proliferative disease affecting the conjuctiva of humans. They hypothesized that adult stem cells from human bone marrow contributed to the development of Pterygium. They found that the basal layer epithelial cells were CD 34 positive in addition to being positive to a few other markers. In our study, we found cell membrane positivity for the CD 34 antigen in the basal and parabasal layers of the gingival epithelium.

The present study had certain limitations, such as the lack of use of negative markers (Thy-1, c-kit etc.) which would have more defi nitively established the presence of stem cells of haemopoietic origin.

In conclusion, in this pilot study, we demonstrated the presence of CD 34 positive cells in the healthy human gingiva. These cells in the paravascular location could be fi broblast progenitors and the possible precursors of these progenitors could be the peripheral blood stem cells. The results of this preliminary study warrant further investigation.

References

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Reprint requests: Dr Suresh R. Rao, Principal, Professor & Head, Department of Periodontics, Sri Ramachandra Dental CollegeSri Ramachandra University, No-1, Ramachandra Nagar, Porur, Chennai 600 116, Indiae-mail: chennai_dentist@yahoo.co.in

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