4.1 Ethical approval of the clinical study
The clinical study was performed according to the World Medical Association Declaration of Helsinki’s declared ethical principle. All clinical study protocols were submitted and approved by the Ethical Committee of the Shahid Beheshti University of Medical Sciences (Approval number IR.SBMU.REC.1398.061). Also, this clinical study was registered with registration numbers 20181205041860N1 at 26 September 2019. All healthy donors and patients were informed about surgery and harvesting of BFP, preparation of BFP- tSVF, possible risks and complications, and then signed the informed consent.
4.2 Experimental assessments
4.2.1 Harvesting and preparing of BFP
The BFP tissues were obtained from three healthy male donors (age range of 25 -35 years) by a vestibular incision distal to the maxillary second molar. This tissue was exposed by blunt dissection while preserving the thin covering membrane (Figure 6A). Then approximately 4 ml of the tissue was excised and delivered to the laboratory in Phosphate Buffered Saline (PBS) (Sigma-Aldrich, St. Louis, Missouri, the United States) supplemented with 5X penicillin/streptomycin antibiotic (Pen/Strep) ( Sigma-Aldrich, St. Louis, Missouri, the United States) and 1X amphotericin B (Sigma-Aldrich, St. Louis, Missouri, the United States). Also, cSVF and tSVF’ isolation was performed according to the below protocols after dividing them to equal volume.
- Enzymatic (ENZ) protocol: The BFP tissue was washed with PBS (Sigma-Aldrich, St. Louis, Missouri, the United States) and incubated with an equal volume of type 1 collagenase enzyme (3mg/ml) (Sigma-Aldrich, St. Louis, Missouri, the United States) at 37°C for 30 min and shaker condition. After centrifugation, the resultant pellet was washed and cultured in the standard medium, containing Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific Waltham, MA, and the United States) supplemented with 15% fetal bovine serum (FBS) (Thermo Fisher Scientific Waltham, MA, and the United States), and 1X pen/strep antibiotic.
- Mechanical protocol: Minceolip® device, was patented in the Intellectual Property Office of Real Estate Registration Organization of Iran (Application Number 13985040003006638) to isolation BFP- tSVF during surgery. This device manually disaggregated the minced BFP tissue into tSVF by passing the tissue from the filter (pore size is 100 µm) via a piston force (Figure 6 (B-E)). All parts of this device were fabricated from autoclaved materials (AISI 361L stainless steel and Fluorocarbon rubber), making it possible to sterilize by autoclave and use it several times. To perform experimental assessments, obtained BFP-tSVF were centrifuged at 500 g for 10 minutes. The supernatant was then thrown away, and the pellet was suspended and cultures in the standard medium.
4.2.2 Cell counting, viability, and phenotypic analysis
The number of nucleated cells obtained from BPF by the Minceolip® was freshly counted using a hemocytometer. According to the manufacturer, the percentage of BFP-tSVF viability was detected through Annexin V-FITC Apoptosis detected kit (Sigma-Aldrich, St. Louis, Missouri, and the United States)’s guideline. Briefly, the tSVF were washed with cold PBS twice and centrifuged at 300g for 5 minutes. The pellet was mixed and incubated with 1X binding buffer, 2 µl Propidium Iodide (PI), and 1 µl Annexin V for 10 minutes in darkness and room temperature.
Furthermore, to evaluate phenotypes, these BFP-tSVF were incubated in the blocking buffer and the various fluorochrome-conjugated antibodies for 30 minutes at 4°C. These antibodies were CD90-FITC, CD44-FITC, CD31-PE, CD34-PE, CD45-PE, and CD14-FITC supplying from EXBio, Vestec, Czech Republic. The viability and phenotypic analysis were then carried out by flow cytometer (Applied Biosystems, Foster City, CA, USA) and FLOWJO software 7.6.1 (Tree Star Software, San Carlos, California, USA).\
4.2.3 Cell culture and expansion rate
The cSVF and tSVF were cultured in 25-T flasks under a humidified atmosphere containing 5% CO2 at 37°. The morphology of cells was microscopically seen to evaluate growth trends and possible infection. At confluence, 2×104 cells were detached using 0.25% trypsin/1mM EDTA (Thermo Fisher Scientific Waltham, MA, and the United States) and seeded in the cell culture plate in the standard medium. The number of cells was counted at a density of approximately 70% using a hemocytometer up to eight passage numbers (PN). Then the expansion rate was calculated using the following formula (Equation 1) 24;
4.2.4 Population doubling time
The proliferation capability of isolated SVF using ENZ and mechanical protocols were assessed by calculating Population Doubling Time (PDT) based on the Patterson formula (Equation 2). To perform, a density of 2×104 of both SVF was cultured in the wells of 24 well plates with the standard medium. Then once every five days, the cells were detached using 0.25% trypsin/1mM EDTA and counted using a hemocytometer. Then the cells were re-cultured with an initial density.
4.2.5 Multi-lineage Differentiation potential
Two types of cells with a density of 2×104 cells/well were seeded into 12 well plates and incubated in the standard medium at the one PN. After complete confluence, this medium was separately exchanged with Stem Pro (Life Technologies, California, United States), adipogenic medium (Life Technologies, California, Unite d States), and chondrogenic medium (Life Technologies, California, United States) to different into osteoblast, adipocyte and chondrocyte, respectively for 14 days. After the induction period, the cells were stained with Alizarin Red, Oil Red O, and Toluidine Blue to visualize calcium deposits, lipid droplets, and proteoglycans development, respectively, according to the manufacture’s guidelines. The percentage of positive cells for all stains was quantified Image J (free download available at http://rsbweb.nih.gov/ij/) based on five images of each group.
4.3 Clinical assessments
4.3.1 Design of the clinical study
In the present study, the primary and secondary outcomes were to investigate the effectiveness, safety, and feasibility of isolated BFP- tSVF using the Minceolip® and the new bone formation after BFP- tSVF based therapy, respectively, in patients with maxillofacial bone defects. All the patients had the American society of anesthesiologists (ASA) I physical status classification system. According to eligibility criteria, ten patients with benign odontogenic tumors of the mandible were enrolled in this study, defined in Table 2. Our center's standard treatment procedure was to do a segmental or partial resection of the affected site and preserve the jaw with the reconstruction plate for at least one year. After that, the sites were reconstructed with the free non-vascular iliac bone graft. In this study, in half of the patients (BFP-tSVF group), the defects were filled with mechanical device derived BFP-tSVF with absorbable collagen sponge (AGS) (Gelfoam, Ethicon, Inc, Somerville, New Jersey) in the defect sites. Four patients underwent segmentally, and six patient’s experienced partial resection of the tumor site (Table 3). The defect site was determined by a 3 D-printed surgical guide and cut without damage to blood vessels and inferior alveolar nerve.
4.3.2 Safety and feasibility measurement
Safety was considered an adverse event because there is not enough knowledge about the relationship between tSVF therapy and its complications in patients with bone defects. According to consolidated standards of reporting trials (CONSORT), the Adverse event is defined as any anticipated and unexpected harm after intervention according to consolidated standards of reporting trials (CONSORT) statement 45. Hence the patients were carefully monitored for physical, radiological, and laboratory assessment by one expert oral-maxillofacial surgeon for one year after surgery, i.e., one week, one month, three months, and one year. The physical evaluation consisted of weight, body temperature, blood pressure, lymph node, neurological status. The radiological evaluation was screened by cone-beam computed tomography radiography. Laboratory assessments consisted of hematology index, C - reactive protein, erythrocyte sedimentation rate.
The protocols’ feasibility was assessed according to five factors; surgical manipulation for BFP harvesting, sterilization of Minceolip device, convenience, time, and BFP-tSVF preparation cost.
4.3.3 Radiographic assessment of the new bone formation
Cone-beam computed tomography (CBCT) images were obtained to measure the bone density in the defect site twice, once before maxillofacial surgery (BD1) and once 12 months after it (BD2). The radiographic images were converted to 256 to 4096 grayscales and established the bone defect area using ImageJ software. Then, the percentage of bone density was calculated based on the grayscales ratio at BD2 to BD1 by the following formula (Equation 3);
This measure was performed thrice by one expert investigator, and the mean of the reported scales was used.
4.4 Statistical analysis
Statistical analyses, including unpaired two-tailed Student’s t-test and two-way ANOVATukey’s post-hoc, were carried out using GraphPad Prism Software (GraphPad Software, INC., La Jolla, Calif.). Quantitative data were obtained in triplicates and presented as mean ± standard deviation (SD). P-value < 0.05 was considered statistically significant.