Clinical Regenerative Potential of Cultured Gingival Fibroblast- Mesenchymal Stem Cells in Treatment of Periodontal Intrabony Defects (Randomized Clinical and Biochemical Trial)
Phase: NA
Conditions: Periodontal Intrabony Defect
Sponsor: Ain Shams University

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Regenerative Potential of Cultured Gingival fibroblast- 
Mesenchymal Stem Cells in Treatment of Periodontal 
Intrabony Defects (Randomized Clinical and 
Biochemical Trial) 
Protocol of thesis 
22- 1- 2016 
Submitted by : Mahetab Mohamed 
Abdal- wahab
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
22- 1- 2016 

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Introduction: 
     Fibroblasts are biologically dynamic and morphologically 
heterogeneous and are the most plentiful connective tissue cells, with 
different structures depending on their location and activity. The main 
function of fibroblasts is to keep the structural integrity of connective 
tissues by continuously secreting precursors of the extracellular 
matrix. Fibroblasts may act as a novel clue for in situ tissue repair and 
contribute to cellular mechanisms of mesenchymal stem cell-like 
features under normal or pathological conditions (Haniffa et al 2007). 
. 
    Nyman et al.,1989. were claimed that GTR procedure aims at the 
reconstruction of a periodontal ligament (PDL) with proper oriented 
and organized collagen fibers inserted in newly formed cementum and 
newly regenerated alveolar bone. Guided tissue membranes are 
employed in the hope of excluding epithelium and gingival connective 
tissue from the root surface due to that they interfere with periodontal 
regeneration and provide mechanical support for clot formation. 
 
  Melcher 
hypothesis 
were 
experimentally 
established 
and 
histologically verified by Karen et al. They have shown that 
periodontal regeneration occurs when gingival epithelial cells or 
fibroblasts are excluded from the wound space and periodontal 
ligament cells are allowed to migrate and populate the wound space. 
On the other side of research, other studies were reported that gingival 
connective tissue cells may contribute to the regenerative process. 
(Lallier et al 2005,(Bartold PM and Narayanan AS.2006). 
  
    A later study by Mitrano et al.2010 showed that gingival tissue cells 
fulfill the minimal criteria proposed by the International Society for 
Cellular Therapy to be defined as mesenchymal stem cells. Human 
gingival tissue-derived mesenchymal stem cells GMSCs are easy to 
isolate and proliferate faster than BMSCs without any use of growth 
factors. They have a constant morphology , they maintain a normal 
karyotype and telomerase activity in long-term cultures and are not 
tumorigenic(Tomar et al.,2010,Tang et al., 2011) . GMSCs exhibit 

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strong suppressive effects on the proliferation and activation of human 
peripheral blood mononuclear cells (PBMC)(Zhang et al., 2009). 
 
      MSC are currently defined as plastic adherent, multipotential 
fibroblast-like cells expressing CD73, CD105 and negative for the 
hematopoietic markers CD14, CD34 and CD45(Dominici M et al., 
2006) , but that properties and markers are also shared by fibroblasts. 
Osteoblastic, 
chondrogenic, 
adipogenic 
differentiation 
from 
fibroblasts has also been described (Haniffa MA eta l., 200). 
Fibroblasts have also the immunosuppressive properties. In fact, it had 
been comprehensively demonstrated that fibroblasts from various 
tissue sites inhibit mitogen and allo-antigen stimulated T-cell 
proliferation (Sarkhosh et al., 2003) and IFNγ production in exactly 
the same vein as more recent reports using MSC (Klyushnenkova EN 
et al., 1998). 
    A novel perforated collagen membrane as a sensory system that 
could enable participation of gingival fibroblasts and gingival stem 
cells in GTR procedures (Gamal et al., 2013). That approved in a 
clinical study that the utilization of a perforated membrane (PM) 
improved clinical outcomes significantly more than those observed 
with the use of occlusive membranes.  It has likewise been suggested 
that growth and differentiation factors from cells in the gingival 
connective tissue could pass through the membrane perforations and 
augment regeneration. Gamal et al. reported that PM use was 
associated with significantly higher GCF levels of BMP-2, PDGF-BB 
and VEGF compared to occlusive membrane (OM) treated sites. They 
hypothesized that; blood clot occluded membrane perforations could 
allow for more physiologic growth factor release compared to 
occlusive membranes (Gamal et al., 2014,2015) 
    Since cellular responses mainly dependent on specific interactions 
with extracellular matrix components(ECM), growth factors, or cell 
surface receptors, ECM act as a constitutive part of stem cell  niche, 
ECM components are key players of the niche instructive power(R. 
Peerani, P.W. Zandstra.2010) These extracellular macromolecules, by 
their assembly and three-dimensional arrangment, supply a 
microenvironment in which the signals deriving from cell–ECM 
interaction, as well as soluble and ECM-bound factors, are integrated 

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in a functional manner to allow the maintenance of stem cell 
homeostasis (M.F. Pera, P.P.L. Tam, 2010). 
 
      We hyposethized that if the gingival fibroblasts translocated away 
from its extracellular matrix components into the periodontal defect to 
be subject to its same mechanical and biologic media, it could behave 
the same way as the MSCs. This experimental study was performed in 
order to evaluate clinically and radiographically regenerative 
potentials of cultured GMSCs and gingival fibroblasts into intrabony 
periodontal defects in human. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Aim of the study: 
Primary outcome: 
Clinical and radiographic evaluation of the regenerative potentials of 
cultured gingival fibroblasts and GMSCs carried by tri calcium 
phosphate  into intrabony periodontal defects in human. 
Secondary outcome: 
Biochemical analysis during the healing of intrabony periodontal 
defect . 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Subject and methods: 
   Twenty patients diagnosed as a moderate to severe chronic 
periodontitis will be  participate in this study, they will be selected 
from the outpatient clinic, Department of Oral Medicine and 
Periodontology, Ain Shams University. 
All patients will be  fully informed about the aim and purpose of this 
study. An informed consent will be signed by each patient. 
I-Patient Selection: 
A) Inclusion Criteria: 
Patients were selected according to these criteria. 
1- Age range from 25 to 50 years. 
2-Presence of at least two bilateral interproximal osseous defects 
estimated from radiographic evaluation and transgingival bone 
sounding ≥3mm of two or three osseous walls. 
3- Probing Depth ≥5mm after initial therapy. 
4- Attachment loss ≥5mm. 
5- Thick gingival biotype more than 1 mm with enough width of 
attached gingiva. 
B) Exclusion Criteria: 
1- Patients with systemic disease or compromised immune illness 
regarding Cornell medical index.  
2- Smoker's patients. 
3-   Pregnant and lactating females. 
4- Patients with regular use of medications that could compromise 
wound healing. 
5-Uncooperative patients (low compliance). 
 

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II-Patients grouping: 
Group 1:   
Ten periodontal defects will receive tri calcium phosphate alone.1  
Group 2: 
   The other ten periodontal defects will receive2 gingival MSCs - 
fibroblast culture in tri calcium phosphate as a scaffold.   
III- Clinical and Radiographic Assessments: 
For the selected sites, the following clinical parameters will be  
assessed preoperative (baseline), 3, and 6 months after the surgical 
procedure using the same William's graduated periodontal probe. 
1- Plaque index (PI) Löe 1967. 
2- Gingival sulcus bleeding index (SBI) Muhlemann, 1963. 
3- Probing depth (PD) Glavind and Loe, 1967. 
4- Clinical attachment level (CAL) Glavind and Loe, 1967. 
5- Gingival recession: considered the distance of the gingival margin 
from the cemento-enamel junction if there were recession 
preoperative, while if there is no recession the gingival margin above 
the cemento-enamel junction is considered preoperatively and 
postoperatively. 
Radiographic assessment: 
1- Assessed at baseline, 3, 6 and 9 months postoperatively using 
indirect digital radiography (Digora). 
2- Each radiograph was standardized using a paralleling device with 
an acrylic bite block that was fixed by the opposite arch by the 
indentations of the opposing teeth. 
                                                          
 
 
 
  Digora Soredex USA www.sordex.com 

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The radiographic parameters were measured on 2.7 version of Digora 
software: 
1- Intrabony defect height.  
2- Defect surface area. 
3- Bone density 
IV- the gingival crevicular PDGF-BB levels at 1,3, 7, 14,and 21 day: 
The measurement of platelet derived growth factor level in the 
gingival crevicular fluid at different interval will detect the 
regenerative process in periodental defect.        
V-Surgical protocol: 
A) Surgical procedures: 
- gingival biopsy will be taken from retromolar area , the tissue will be 
kept in a-MEM medium in sterile container. And so it will be cultured 
for 14 days in biochemical department in Cairo University. 
- Biopsy medium is consists of Dulbecco’s modified Eagle’s 
medium (DMEM) plus penicillin and streptomycin.   
-  The samples will be cut into small pieces displaced on glass slides 
and placed in culture plate with basic medium (DMEM+ fetal 
bovine serum + penicillin and streptomycin)( Alireza M et al., 2014). 
- It will be maintained in an incubator at 37°C and humidified air 
(5% CO2, 95% air). From days 14 to 21, gingival fibroblast cell 
cultures will reach 80% confluence; the cells will be transferred to 
tissue culture flasks using a solution of trypsin and EDTA. 
-  After centrifugation, cells will be resuspended with  serum  
supplemented  medium and incubated in 50 cm²culture flask 
(Falcon). 
- After that the minimally invasive technique will be made at the site 
of the defect and the graft material will be applied.  

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-All patients will take antibiotics for 1 week (3 · 500mg 
amoxicillin*/day), (3.250mg metronidazole**/ day) and an anti-
inflammatory (3.alphintern***/ day). 
-Patients will instruct to rinse twice daily with a 0.12% chlorhexidine 
digluconate◘ mouth rinse and to avoid mechanical plaque removal at 
the site of surgery for 15 to 30 days.-Periodontal dressing was removed 
after 1 week. 
-Sutures will removed 10 day following surgery. 
-Patients will instruct not to brush for 2 weeks and not to floss the 
surgical area for 4 weeks. 
- After 15 days patient were instructed to use Bass technique for tooth 
brushing. 
VI-Postoperative surgical evaluation and assessment: 
- 
Weekly recall appointments will schedule during the first 6 weeks 
after surgery and once per month. 
-  The follow up period included general and oral examination, plaque 
and calculus removal when necessary, at the surgical site careful 
subgingival debridement will carry out with hand instruments. 
- 
Both clinical and radiographic parameters will be recorded at 
3and 6 months postoperative. 
*The following rule in the unit of Biochemistry and Molecular Biology 
will be applied to maintain cells culture: 
✓ Hood preparation 
✓ Sterile handling .All biological contaminated tissue culture 
plates, flask, and other non sharps will be placed in non – sharp 
biohazard waste container. However, an effort to minimize 
entry /exit from the hood will be made to minimize disturbances 
in laminar flow at the entrance, which may create the potential 
to waft in contaminants. Removal of all these biohazards will 
be under supervision of researcher.     
✓ Cleaning up  
                                                          
 
 * 
Amoxil MUP Egypt 
 ** 
Flagyl Sanofi Aventis Egypt 
 *** Alphintern Amoun Egypt 
 ◘Antiseptol Kahira Pharm Egypt 

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✓ Cell feeding , To enable cells to grow and divide in dish, they 
will be fed by the nutrients provided by the media. Old media 
will be replaced with fresh media every two or three days for 
most animal cell lines  
✓ Cell splitting ,Cell growing in dish begin to get crowded and 
then stop growing. This crowded state is called (confluence) and 
to maintain cells growth, confluent cultures will be split and 
reseeded into new culture dishes at a lower density. 
VII- Statistical analysis: 
• All data will be subjected to statistical analysis. 
• Description of quantitative variable will be in the form 
of mean and standard deviation (SD). 
• Qualitative variable will be described as number and 
percentage. 
• Comparison between quantitative variable will be 
carried out using student -t test when normally 
distributed, and Mannwhitney test for abnormal 
distribution. 
• Categorical data will be compared using Chi-square 
test. 
• Significant value will be defined as P values <0.05. 
This research will be reviewed by the research ethics 
commeitee faculty of dentistry ain shams university 
 
 
 
 
 
 
 

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