The study was approved by Institutional Ethics Committee Dr. D Y Patil Dental College and Hospital Pimpri Pune (DPU/484/2/20221). All the methods performed in the study are in accordance with the relevant guidelines and regulations. Informed consent was obtained from all subjects before tissue sample collection. This study was conducted at the Regenerative Medicine Laboratory (RML) at Dr. D.Y Patil Dental College and Hospital Pimpri Pune, Dr. D. Y Patil Vidyapeeth Pimpri, Pune, Maharashtra India. In this study commercially available type I collagen scaffold, previously cryopreserved GMSCs and different growth factors like FBS, i-PRF were used.
Collagen scaffold
Commercially available collagen membranes (COLOGIDE)25 were used as scaffolds in the present study based on the previously unpublished data. The COLOGIDE has a fibrous structure which creates porosity. It is obtained from bovine origin from a highly purified type I collagen (especially from controlled and certified animals to avoid any antigenicity).
Procurement of GMSCs
At RML the GMSCs were isolated from the excised gingival tissue obtained after the crown lengthening or gingivectomy procedures by the explant culturing method which were checked for their stemness. Confluent GMSCs were harvested and washed with phosphate-buffered saline (PBS) and characterized for stem cell surface markers such as CD34, CD45, HLADR, CD90, CD73, CD105 by using flow cytometry.
Procurement of the growth media/factors (FBS, i-PRF)
Fetal bovine serum (FBS (cat No. Himedia) was used for cell culture. FBS is the liquid fraction of clotted blood from foetal calves that is devoid of cells, fibrin, and clotting factors but includes a substantial amount of nutritional and macromolecular components necessary for cell proliferation. FBS’s main component is bovine serum albumin. FBS growth factors are necessary for the maintenance and development of cultured cells. FBS also includes a range of tiny molecules such as amino acids, carbohydrates, lipids, and hormones26.
The Injectable platelet rich fibrin (i-PRF)13 preparation: 20 ml blood withdrawn from a healthy adult volunteer by venepuncture of the antecubital vein. The blood was collected in two tubes (10 ml each) that did not have any anti-coagulant present in the tube. The test tubes were then centrifuged at 700 revolutions per minute (RPM) for 3 min at room temperature in a Choukroun’s Duo Quattro which resulted in the separation of three basic fractions because of differential densities: a yellow part, a buffy coat in the middle, and a red blood cell containing lower part. The i-PRF (1 ml from the top layer) was collected using an automated pipette. Because even little alterations in the fractionation process might alter the physical and biological features of the final products, special care was taken to collect just the superficial yellow coat (above the leukocyte-rich buffy coat). The procedure was completed as promptly as possible.
A total of 18 collagen scaffolds measuring about 1 cm x 1 cm were selected and included in the study and divided into 3 groups (6 in each group). Group I = Collagen Scaffolds preconditioned with FBS. Group II = Collagen Scaffolds preconditioned with i-PRF. Group III = Collagen Scaffolds preconditioned with FBS and i_PRF.
After preconditioning for 24 h with 3 different preconditioned medias all the collagen scaffolds were then seeded with cryopreserved 2 million GMSCs in 10% α MEM (Gibco) and incubated in a CO2 incubator (THERMO FISSURE) at 37 0 C for 7 days. The 10% α MEM was changed every 2–3 days.
For the evaluation of attachment and depth of penetration of the GMSCs all the 18 pieces of collagen scaffold of 1 cm x 1 cm each seeded with 2 million cryopreserved GMSCs in the 3 groups were further divided (cut) into 2 parts (total of 36). They were fixed in 4% formalin and carried in an Eppendorf tubes. 18 parts were sent for the histopathological analysis at the Department of Oral Pathology, Dr. D Y Patil Dental College and Hospital Pimpri Pune. Other 18 parts were sent for ESEM analysis at Kiran Icon Analytics, Sanpada, Navi Mumbai.
Histological analysis
18 samples were fixed in 4% formalin and carried in Eppendorf tubes. Pre-conditioned Collagen scaffold seeded with GMSCs were re-fixed with 10% formalin for 24 h. Dehydration was carried out using 60%, 70%, 80%, 90% alcohol (1/2 hour each) and with two changes of absolute alcohol (1 h each). Clearing with xylene (with two changes, 1 ½ hours each.). After clearing the scaffold were embedded in paraffin wax (with two changes of 1 ½ hours each.) Blocks were made with the embedding machine. Then thin section about 3–5 microns were made followed by H and E stain protocol. The slide was warmed for 3–4 min with a slide warmer to remove the excess of paraffin wax. After which it was kept in xylene for 3 min (with two changes each) followed by Alcohol for 3 min (with two changes each) and in distilled water for 10 min. After that haematoxylin staining was done for 4–5 min and was washed with running tap water for 1–2 min till bluing of the slide occurred. Eosin staining was done for 1 min followed by washing it under the running tap water. Lastly, it was kept in the alcohol dip and xylene dip followed by mounting with DPX. Microscopic analysis for the number of cells attached/ penetrated into the collagen scaffolds was performed using a microscope (40x ) in 5 different areas using the phase contrast microscope (OLYMPUS).
Environmental Scanning Electron Microscope (ESEM) analysis
Eighteen samples were fixed in 4% formalin and carried in Eppendorf tubes to the canter (Kiran Icon Analytics). The samples were analysed in Qaunta 200 model make FEI manufactured in the Netherlands at Kiran Icon Analytics Sanpada, Navi Mumbai. The samples were dried using a blower and lint-free paper. Once the samples were dried they were loaded and fixed on to the aluminium stubs using double-sided carbon tapes. The stubs were then placed into the chamber and were examined under low vacuum under 15kv and 20kv at room temperature using different magnifications (1000x, 2000x, 4000x, 8000x).
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- Source: https://www.nature.com/articles/s41598-024-77373-y