Positive impact of fibronectin in stimulation human adipose-derived mesenchymal stem cells attachment seeded into polytetrafluoroethylene patch for future surgical closure of atrial and ventricular septal defect

I Gde Rurus Suryawan1*, Anudya Kartika Ratri1, Andrianto Andrianto1, Meity Ardiana1, Ricardo Adrian Nugraha1 Department of Cardiology and Vascular Medicine, Faculty of Medicine Universitas Airlangga – Dr. Soetomo General Hospital, Surabaya – INDONESIA * Corresponding author: I Gde Rurus Suryawan, M.D., Ph.D. Corresponding address: Mayjend Prof. Dr. Moestopo 6-8, Surabaya INDONESIA Corresponding email: igde.rurus.s@fk.unair.ac.id


INTRODUCTION
Acyanotic congenital heart disease (CHD) such as Atrial Septal Defect (ASD) and Ventricular Septal Defect (VSD) are the most common CHD (50% of all CHD). They are often underdiagnosed because of lack of symptoms and only 20% of patients will require surgical intervention (1). Usually, a patch of fabric or pericardium is sewn over the ASD or VSD to close it completely.
Unfortunately, materials used for patch repair or reconstruction have limitations such as their inability to grow, repair, and remodel. Aneurysm formation, thrombosis, and the inability of patches to grow or remodel are important sources of morbidity and mortality after repair or reconstruction of cardiovascular structures, especially in children and young adults (2). Tissue engineering has become an evolving in vitro innovative approach for intended surgical replacement of congenital defects. By transplanting multi potential human adipose derived mesenchymal stem cells (hAMSCs) onto widely used PTFE patch, authors offer a new solution for its prosthetic limitations.
Fibronectin as an extracellular matrix protein was added to enhance cell-to-cell adherence, and cell-to-patch surface attachment.

Objective
To understanding positive impact of fibronectin to enhance hAMSCs cell-to-cell adherence and cell-to-patch surface attachment into PTFE patch for future ASD or VSD closure.

Scaffold materials
Cardiovascular synthetic patch made of polytetrafluoroethylene/PTFE (Gore-Tex®) was used. It is a durable, easy to handle, sterile synthetic patch designed for optimal tissue ingrowth while minimizing aneurysmal dilation in a wide variety of cardiovascular applications including cardiac, great vessel, and peripheral vascular reconstructions and surgeries. The patch was minced into 0.8 x 0.8 cm 2 pieces.

Mesenchymal stem cells culture and harvesting
Mesenchymal stem cells obtained by thawing of cryopreserved human adipose derived mesenchymal stem cells. They were thawed at 37°C and transferred in 5 mL of standard cell media and gently homogenized. 500 μL warm phosphate buffered saline (PBS) added into medium well for 2 minutes. Resuspension with PBS plus 500 μL Trypsin / EDTA solution (0,5%) and incubated for 5-10 minutes 37°C and 5% of CO2 until they reached 80% of confluence. The cell suspension was transferred for sterile centrifuge tube and centrifuged at 300 g for 5 minutes. After centrifugation, 200 μL warm PBS solution was added to each centrifuge tube. After another centrifugation, the supernatant was aspirated, and the cells were resuspended in approximately 500 µL of stromal medium. 1 mL aliquot cell dilution in trypsin blue were submitted to counting cells using hematocytometer. The cells then placed onto suitable culture plates.
For each sample, the culture was done at passage 5. Using light microscope, the cell culture showed spindle-like shaped morphology consistent with mesenchymal stem cell.

Identification of hAMSCs phenotype
All samples were analyzed for the expression of surface markers, using monoclonal antibodies against cluster of differentiation (CD) antigens, conjugated with fluorochromes, and analyzed after thawing by indirect immunofluorescence. Cultured cells were fixated with 15 mL methanol and CD90+, CD105+, CD45-antibodies were labeled FITC, rinsed with PBS and analyzed under fluorescence microscope for 15 minutes.

Fibronectin addition onto PTFE patch surface
Fibronectin solution 0.1% were used to soak patch scaffolds for approximately 2 hours duration, and then dried for 20 minutes for treatment group. As for control group, Fibronectin solution was not added on the culture.

Structure Examination with SEM
The samples were fixed with 2% Glutaraldehyde, followed by gradual dehydration using alcohol. Afterward, the specimens were critical point dried in CO2 using a Hitachi HCP-2 critical point dryer (Hitachi, Tokyo, Japan) and was subsequently gold sputtered using vacuum evaporator. The samples were examined with Jeol scanning electron microscope (Hitachi, Tokyo, Japan) and the images were captured by Nikon camera.

Statistical Analysis
Data results were expressed as mean + SD. Data and graphs were produced

CONCLUSION
Fibronectin has a positive impact on hAMSCs attachment seeded onto PTFE patch. These properties, in combination with their developmental plasticity, have generated tremendous interest because of the potential use of hAMSCs in regenerative medicine to replace damaged tissues. Longer duration of cell culture did not provide significant difference on cell-to-patch surface attachment.

AUTHOR CONTRIBUTIONS
A.K.R. conceived and planned the experiments, carried out the experiments, planned and carried out the simulations, contributed to sample preparation.