This study evaluated human gingival fibroblast and mesenchymal stem cell migration, proliferation, and membrane stiffness using perforated collagen membranes. Cells were cultured from gingival tissue and tested for surface markers. Migration was assessed using transwell assays with membranes perforated at 0.2, 0.4, and 0.7 mm. Proliferation was measured using MTT assays. Membrane stiffness was analyzed by tensile testing. Cell migration increased with larger perforations while proliferation was similar. Membranes remained stiff with perforations up to 0.7 mm. The results suggest perforated membranes could enhance regeneration by allowing cell migration but smaller pores may better prevent tissue ingrowth.

1. IN VITRO EVALUATION OF THE HUMAN GINGIVAL
FIBROBLAST/ GINGIVAL MESENCHYMAL STEM CELL
DYNAMICS THROUGH PERFORATED GUIDED TISSUE
MEMBRANES: CELL MIGRATION, PROLIFERATION AND
MEMBRANE STIFFNESS ASSAY
PRESENTED BY
BIBINA GEORGE
GUIDED BY
DR. JAYASHREE A. MUDDA
Gamal et al.
J PERIODONT RES 2017

2. STEM CELLS
PRIMITIVE CELLS WITHVERY HIGH POTENTIAL AND INFINITE ABILITY OF SELF
RENEWAL AND DIFFERENTIATION INTO OTHER CELL TYPES.

3. GINGIVAL HEALING
Growth Factors
ECM Proteins
Gingival
Fibroblasts
Invitro
mRNA
variables
BMP-2/4ALP
PDL Fibroblasts
•< BMP-4
•Same level of ALP &
BMP-2

4. BMP & ALP
 BMPs
o TGF – β superfamily of proteins
o Growth & Differentiation factors
o Chemotactic agents
o (+) Angiogenesis & migration, prolif and
differentiation of MSC into cartilage &
bone forming cells
Rao SM et al, 2013.
 ALP
o Physiological mineralization
o Locally ↑ inorganic phosphate levels
Anderson et al,1995.
o PDL & ECM cells
Groenvald et al, 1996.
o Acellular cementum formation
Beertsen & Van den Bos,1991

5. STUDIES TESTING REGENERATING POTENTIAL OF GINGIVAL
FIBROBLASTS
1. Buurma & Coworkers :Genes------------- Ging. Fibroblasts-------------BMP----------Hard Tissue
Formation
2. Tomar et al : GMSCs more advantageous than bone marrow derived MSCs ------ Cell therapy
& Regenerative medicine
3. Fawzy El-Sayed et al : Exp.Animals ---------- Autologous GMSCs-----------Pdl Defects---------
Bone, Cementum & Pdl formation.
Gingival tissue occlusion Concept tested by Wilkesjo et al

6. AIM
 To test gingival fibroblasts and their associated GMSC locomotion and proliferation
through resorbable collagen membrane.
 Perforated membrane stiffness was also evaluated to figure out the membrane
perforation diameter that could negatively affect membrane stiffness, which could
adversely affect the space keeping effect of the membranes.
Hypothesis

7. MATERIAL AND METHODS
Human gingival tissue sample collection --------3 adult donors(21,24 and 31 years)
GMSC culturing (2-3 days)
Dulbecco’s MEM
Subculturing (every other week)
Α- modified
Eagle’medium
Epithelial layer
removed
Culture medium replaced
every 2-3 d over 21 d
period
PHASE- CONTRAST MICROSCOPY
1.1. Surface markers testingSurface markers testing
2.2. GMSC Migration AssayGMSC Migration Assay
3.3. MTT Cell Proliferation AssayMTT Cell Proliferation Assay
4.4. SEM of Perforated MembraneSEM of Perforated Membrane

8. 1.1. Surface markers testingSurface markers testing
1st
adherent cell appeared at day 6
GMSC –plastic adherence to tissue
culture flasks
Flow cytometric analysis
2.2. GMSC Migration AssayGMSC Migration Assay
5. Tensile Strength & Modulus of
Elasticity : Mech testing of
membranes
12 mm diameter
Collagen coated PTFE Transwells
Cultured GMSC
Blood Clot
Grp I: 0.2 mm
Grp II:0.4 mm
Grp III:0.7 mm
48h
Crystal violet staining
Neubauer hemocytometer
Inverted microscope
3.3. MTT Cell Proliferation AssayMTT Cell Proliferation Assay
Based on reduction of terazolium
salts.
Lower cell chamber + MTT
Reagent -----purple ppt
+detergent----
Spectrophotometer-----Absorbance

9. STATISTICAL ANALYSIS
 ANOVA
 Kruskal Wallis Test

10. RESULTS

16. DISCUSSION
 Critical sized periodontal and alveolar bone defects usually suffer limited
regenerative capacity
o limited cellularity and
o lack of adequate space provision.
 Occlusive membranes

17. Gamal and Iacono suggested the use of perforated barrier membranes for the
treatment of intrabony defects.
They claimed that, membrane perforations may allow for the participation of cells
derived from the gingiva (GMSC) and periosteum in the regenerative process
• The present study was conducted to prove feasibility of selective gingival fibroblasts and GMSC
migration through selected perforation diameters of collagen membranes.
• To achieve optimal outcomes of perforated GTR and bone regeneration membranes, it was important
to test the optimal pore diameter that allows for maximum cell migration through membrane
perforations without allowing for the passage of the rapidly growing gingival connective tissue.
• preventing pressure atrophy over the newly formed tissue in the defect area.

18. BLOOD CLOT FIBRIN
 Matrix for cellular migration
 Chemotactic potential through its content of growth factors
 Stimulate the proliferation of wound fibroblasts and
 provide signals for gene expression,
 induce expression of appropriate integrin receptors on the fibroblasts, which are
essential for cellular migration

19. Irwin et al. who studied the morphologic characteristics of fibroblasts obtained
from papillary and reticular layers of human gingival tissue. They reported a much
smaller size of gingival fibroblasts than the selected pore diameters.
• SEM analysis suggests that more predictable outcomes could be achieved than
of the same group’s previous clinical findings by more adjustment of membrane
perforations to be totally occlusive for gingival connective tissue elements.

20. Gamal et al. reported higher gingival crevicular fluid levels of BMP-2, vascular
endothelial growth factor and platelet- derived growth factor-BB following
the use of perforated membranes as compared to an occlusive one .
As stated by Scantlebury, space provision and maintenance are essential design criteria
for membranes used in GTR. He reported that the contour and volume of new bone
formed in the defects is determined by the space created and protected by the barrier
membrane. Membrane stiffness prevents its collapse into the defect.

21. CONCLUSION
 Macromembrane perforations of 0.2, 0.4 and 0.7 mm are suitable pore diameters that could maintain
membrane stiffness and allow for cellular migration.
 However, these membrane perforation diameters do not allow for total gingival connective tissue
isolation.This could affect negatively the optimal outcomes of perforated membrane use.
 More investigations are required to test smaller membrane diameters to reach the ideal perforation
diameter that could be totally occlusive for gingival connective tissue elements and permeable for
gingival fibroblasts with their associated mediators and GMSC populations.
 A membrane perforation diameter that allows for selective GMSC migration needs also to be
investigated.