Efficacy of Preconditioned Adipose-Derived Stem Cells in Fat Grafting

Supplementation of Autologous Fat Grafts With Curcumin Preconditioned Adipose-Derived Stem Cells in the Treatment of Facial Contour Deformities

Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Higher Education Commission (Pakistan), Jinnah Burn and Reconstructive Surgery Centre, Lahore

The aim of this study is to evaluate the comparison of therapeutic potential of curcumin preconditioned adipose derived stem cells (ASCs) enrichment fat grafting, naïve ASCs enrichment fat grafting and conventional fat grafting to correct facial contour deformities that cause aesthetic complications in patients.

Human subcutaneous adipose tissue along informed consents will be collected in a lipoaspirate container or 20 cc B.D syringes under the sterilized conditions during the surgery. Fat aspirate collection will be based on certain selection criteria including pre-requisitioning non-diabetic, negative viral status patients. Adipose derived stem cells (ASCs) will be isolated from adipose tissue under sterile aseptic conditions. Firstly, the lipoaspirate container will be opened in the biosafety cabinet and drain off the blood carefully with the help of serological pipette. Then, fat will be washed thrice with 1X phosphate buffer saline (PBS) containing antibiotic and anti-mycotic solution, in order to get rid of blood vessels, hair and other type of connective tissue. After that, the tissue will be enzymatically digested with collagenase type I solution (1mg/ml prepared in Low Glucose Dulbecco's Modified Eagle Medium (LG-DMEM) and incubate for 45 minutes at 37°C on a shaker. After digestion, the enzyme will be inactivated by LG-DMEM augmented with 5% freshly isolated human serum and spin at 1200 rpm for 10 minutes. After centrifugation, fat will be discarded and the infranatant will be passed through 100 µm cell strainers to remove the debris. The filtrate will be centrifuged again for 1200rpm for 8 minutes. Pellet will be resuspended and plated in a sterile T-75 cm2 flask containing 10 ml LG-DMEM supplemented with 5% human serum. The flask will be placed in a humidified incubator at 37°C, 5% carbon dioxide (CO2). The media will be replenished after every third day until the cells got 80-90 % confluency. Cells of P2-P3 stage will be used in this study for immunocytochemistry, and intervention as well. For preconditioning, ASCs will be incubated with curcumin preconditioned medium for 24 h. Cell sterility and viability will be assessed prior to transplantation. For transplantation, 1,00,000 cells per ml of fat will be mixed, transferred to 10 cc syringes and injected at deformity site. Amount of fat will be predetermined by the clinician on the basis of facial asymmetry.

Patients will undergo conventional fat harvesting. Harvested fat will be mixed with cultured ASCs and grafted into recipient site with 1,00,000 cells/ml injected fat

Patients will undergo conventional fat harvesting. Harvested fat will be mixed with cultured ASCs preconditioned with curcumin and grafted into recipient site with 1,00,000 cells/ml injected fat

Volumetric evaluation of treated area will be done by measuring the thickness of subcutaneous tissue (in millimeters) by using baseline B mode ultrasound device. Volumetric evaluation of treated area will be done by measuring the thickness of subcutaneous tissue (in millimeters) before transplantation, 1 week after transplantation then after 3, 6 and 9 months by using baseline B mode ultrasound device.

Patients will be interviewed and pre-and post-operatively and digital images will be taken until 9 months of follow up.

Patients will be visually inspected for facial volume by two qualified plastic surgeons unaware of treatment after grafting to check whether curcumin pretreatment have effect on MSCs origin of ASCs.

Inclusion criteria Age 18-50 years (male or female) Body-mass index 20-30 kg/m² Suitable for liposuction as assessed by the expected cosmetic results Provided signed informed consent Exclusion criteria Systemic disorders include autoimmune diseases, hemorrhagic diseases, bone marrow aplasia, sepsis, cancer, and others Local disorders include acne, infections and others Risk factors include smoking, uncompensated diabetes, hypertension, and allergy to the drugs used for general anesthesia Microbiological contamination of either the cell culture or the transplanted tissue

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