Buccal Fat Pad- Stromal Vascular Fraction as a New Regenerative Source in Treatment of Maxillofacial Bone Defects: A Case Series Study

The study aimed to evaluate the biological properties of the buccal fat pad (BPF) derived tissue stromal vascular fractions (tSVF) using a mechanical device (Minceolip®), compared to BFP- cellular SVF (cSVF) in vitro. Also, the eciency, safety, and feasibility of the BFP-tSVF were assessed in patients with various maxillofacial defects. Isolation of cSVF and-tSVF from BFP was performed through enzymatic and Mechanical devices. They were evaluated in terms of cell counting and viability, expression surface markers, kinetic growth, and tri-lineage differentiation. Ten patients with jaw bone defects received the BFP- tSVF along with an absorbable gelatin sponge in the clinical stage. Effectiveness and safety were measured by computed tomography. The BFP- tSVF showed high cell viability and cell surface markers, specically CD45, CD31, and CD34. There was no difference in their kinetic growth and multi-lineage differentiation potential between enzymatic and mechanical approaches. Bone regeneration was signicantly enhanced in the patients who received BFP- tSVF than control groups in both segmental (33.50%±4.95 vs. 5.00%±7.07, P-value: 0.042) and partial resection (89.33%±2.08 vs. 10.33%±10.02, P-value: 0.000). There were no intervention-related adverse events reported in the patients. These results conrmed that the BFP- tSVF is effective, safe, and feasible for maxillofacial bone defects patients. It will be necessary to evaluate of clinical eciency of BFP- tSVF for improving bone regeneration in future studies.


Introduction
Stromal vascular fraction (SVF) is used in a broad spectrum of various medical conditions, from threatening life (i.e., cardiomyopathy, pulmonary brosis) to less-lethal states (i.e., chronic wound, osteoarthritis) 1-5 . This fraction consists of heterogeneous cellular populations, such as mesenchymal progenitors, endothelial progenitors, supra-adventitial adipose stromal cells, and pericytes, which have essential roles in angiogenesis, anti-in ammatory, and anti-brosis 6 . SVF is classi ed into cellular-derived SVF (cSVF) and tissue-derived SVF (tSVF) 7 according to preparation procedure, including enzymatic and mechanical, respectively.
In the enzymatic harvesting procedure, cells' phenotypical and functional properties alter via the degradation of cell surface and extracellular matrix (ECM) proteins. Therefore, their cells' number and quality depend on isolation methodology, equipment, chemical reagents, and protease activity of each batch of enzymes 7 . The translation of the enzymatic digestion procedure into clinical needs to get the manufacturing grade (GMP) standards for operating protocols from food and drug administration (FDA) agencies 8 .
Recently, tSVF has attracted many scientists' attention by preserving tissue native microenvironment and original relevant characteristics. The novel devices were patented to improve the e ciency and feasibility of tSVF isolation according to three physical methods, including centrifugation, ltration, and gravity sedimentation [9][10][11][12][13] . Notably, the mechanical procedure is considered a "minimal manipulated approach" in the US and does not need to be approved by the FDA agencies 14 .
Buccal Fat Pad (BFP) is a primary adipose tissue source between the buccinator muscle and other super cial muscles, containing abundant neural-crest-derived stem cells and vascular supply [15][16][17][18][19][20] . This tissue has two main advantages: easy harvesting with minimal donor site morbidity and complication rate and also having the same size in all people, independent of fat distribution, body weight, and age 16,21 . Our previous experimental study indicated that BFP derived stem cells (BFPdSCs) had a higher proliferation rate, osteogenesis, and the expression of angiogenic surface cell markers compared to isolated stem cells from other adipose tissues, i.e., abdomen and hip 21 .
On the other hand, one of the main challenges in regenerative medicine is a reconstruction of critical-sized jaw bone defects, and either large span defect originated from an odontogenic tumor resection or vertical agerelated jaw atrophy. As a traditional standard treatment technique, autologous bone grafting is associated with several disadvantages, such as infection, donor site morbidity, high cost, and prolonged hospitalization 19 . With the current advances in cell research, cell-based regenerative medicine approaches move from the bench to the patients' bedside to restore these defects to functional capability and appropriate esthetic form. In recent years, the use of intact BFP or BFPdSCs has been suggested to improve the bone regeneration in maxillomandibular atrophy, post-excision defects, alveolar cleft defect augmentation of the maxillary sinus oor 17-20 . However, the application of BFP-tSVF to improve jaw bone regeneration is a main challenge in regenerative medicine.
In the current study, a novel mechanical device, called Minceolip®, was fabricated to isolating tSVF from intact BFP during surgery. The biological properties of these cellular properties, such as cell viability, cell surface markers, kinetic growth, and multi-differentiation potential, evaluate and compare with BFP-cSVF.
Moreover, the effectiveness of mechanical tSVF harvesting from a BFP local niche for promotion jaw bone regeneration assesses.

Results
2.1 BFP-tSVF are a heterogeneous vital cell population that have all stemness properties The nucleated cell yield was 581.00 ± 20.075 × 10 3 per milliliter of BFP-tSVF, isolating using the Minceolip® device. Annexin-PI staining evaluated the percentage of viable nucleated cells after isolation of BFP-tSVF.
To evaluate the stemness properties of BFP-tSVF, including expansion rate, PDT, and multi-lineage differentiation, were analyzed and compared with isolated cSVF by ENZ protocol. Seven-day after cell culture, the morphology of isolated cells by both protocols was revealed that these cells had a spinal broblast-like shape with spiral growth. Also, the growth trend showed that the number of isolated cells by the mechanical protocols was signi cantly lower than in another group. Other differences between the two groups were the presence of tSVF fragments in the plate culture, and broblast-like cells were migrating from them outward ( Fig. 2 (A-B)).
The expansion rate in mechanical and ENZ groups was associated with a steady increase from PN2 to PN9 (39.67 and 32.84 fold, respectively). Although the mechanical protocol in isolated cells was higher than the ENZ technique in all PN, this difference was no statistically signi cant (Fig. 2C).
PDT increased in both groups during the time so that a statistically signi cant seen on 30 and 35 days compared to 5 days in intragroup comparison (P-value < 0.0001). Also, this time was signi cantly lower (Pvalue < 0.0001) in the mechanical group compared to the ENZ group at 30 days (5.02 ± 1.08 and 8.16 ± 1.25, respectively) and 35 days (8.9 ± 0.48 and 15.05 ± 0.58, respectively) ( Table 1).

BFP-tSVF promote bone regeneration in jaw bone regeneration
The evaluation of the adverse events' occurrence demonstrated that nine patients had uneventful healing, without any unexpected pain, signs of local in ammation, infection, and wound dehiscence. All patients showed considerable chewing and speech enhancement after six months. Desirable outcomes in terms of the esthetic and functional were also observed in all patients in a year follow-up. There was no report from loosening in screws or a history of jaw dislocation. However, the oral in ammatory exudates from the surgical site and pain were observed eight months postoperatively in a patient that was received BFP-tSVF based therapy based on segmental resection. The removal of a titanium reconstruction plate was performed to decrease in ammation for the patient. Thus, her pain and in ammatory signs have been removed after the treatment, and favorable bone healing was observed after one year.
Although all patients need autologous bone grafting from iliac crest bone after one year. This amount of autologous bone will be much lower for patients treated with BFP-tSVF. The radiographic analysis demonstrated that bone regeneration was statistically enhanced in BFP-tSVF (89.33 ± 2.08) than the control group (10.33 ± 10.02) in the marginal resection techniques (P-value: 0.000) (Fig. 5). Also, these patients did not require autologous bone grafting.

Discussion
This study's main outcome was evaluating the biological properties and e ciency of mechanical derived BFP-tSVF. Moreover, we aimed to assess the safety and feasibility of BFP-tSVF, as a one-step surgical procedure, in a clinical condition for patients with maxillofacial defects.
The cell counting data indicated that BFP-tSVF isolation by Miniceolip® device extracted more cells than Given the promising results in the experimental phase about BFP-tSVF, e ciency, safety, and feasibility of the cellular product evaluated to promote bone regeneration in the patients with critical-sized jaw bone defects, including vertical age-related jaw atrophy and large span defect originated from odontogenic tumor resection.
Computed tomographic evaluation of the patients following surgery demonstrated new regenerate bone in the respected segmental patients who received BFP-tSVF (6.7 fold) and in marginal respected patients (8.6 fold) resection compared to control groups. In the respected segmental defect, this amount of bone formation during routine one year follow up reducing the amount of autologous iliac bone in a second surgery and can lead to a decrease in adverse events, such as pain, donor site morbidity, hospitalization, and cost, and increase patients' satisfaction. This advantage was more pronounced in marginal respected patients who received BFP-tSVF than the control group. Extraoral bone harvesting was eliminated in these patients. The patients may need bone augmentation from Intralock oral donor sites or using bone substitutes. These results were consistent with other clinical studies that indicated adipose-derived stem cells and fresh cSVF could enhance new bone regeneration in the different bone defects 1,19,41,42 .
In safety evaluation, a chronic in ammatory reaction in one patient received BFP-tSVF that was resolved by removing the titanium reconstruction plate related to the allergic reaction to Ti particles, activating macrophages, and increasing TNF-α 43, 44 . Hence, the safety parameters demonstrated that no related adverse events occurred during follow-up. This result agrees with our previous clinical studies that used nonmanipulated BFP as a membrane for covering the bone graft, had no adverse events in sinus augmentation 17 and the atrophied edentulous maxilla 18 .
The presented mechanical protocol in this clinical study by the Minceolip® device is feasible to isolate BFP-tSVF. This adipose tissue can harvest through local anesthesia with minimal adverse events for patients and cost in a short time. The preparation time for isolation SVF by the Minceolip device (10 min) was lower than the enzymatic method (80min). This mechanical procedure is a minimal manipulation procedure according to regulations of HCT/P (human cell, tissue, and cellular and tissue-based product) under Sect. 361 of the public health service ACT authorizes FDA 14 . It can be done at the operating theatre's surgical table, decreasing the cost and total time of regenerative technique compared to other enzymatic or lab process procedures. It is an enzymatic-free technique that does not alter the biological native of the tissue.

Conclusion
The experimental results suggested that the mechanical derived BFP-tSVF by delivering stromal and vascular cells may increase the surgical defects' regenerative capacity during the healing period. More studies with a larger sample size need to be more conclusive for the effectiveness of the minimal manipulation procedure in Jaw bone regeneration. 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 lter (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.

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.
Brie y, 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.

Cell culture and expansion rate
The cSVF and tSVF were cultured in 25-T asks under a humidi ed atmosphere containing 5% CO2 at 37°.
The morphology of cells was microscopically seen to evaluate growth trends and possible infection. At con uence, 2×10 4 cells were detached using 0.25% trypsin/1mM EDTA (Thermo Fisher Scienti c 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 ;

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 2×10 4 of both SVF was cultured in the wells of 24 well plates with the standard medium. Then once every ve 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.

Multi-lineage Differentiation potential
Two types of cells with a density of 2×10 4 cells/well were seeded into 12 well plates and incubated in the standard medium at the one PN. After complete con uence, 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 quanti ed Image J (free download available at http://rsbweb.nih.gov/ij/) based on ve images of each group.

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 classi cation system. According to eligibility criteria, ten patients with benign odontogenic tumors of the mandible were enrolled in this study, de ned 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 lled 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.

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 de ned 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 ve factors; surgical manipulation for BFP harvesting, sterilization of Minceolip device, convenience, time, and BFP-tSVF preparation cost.

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.

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 signi cant.    Experimental assessment of BFP-tSVF. A) Morphology of broblast-like cells was isolated using ENZ protocol after seven days from in vitro culture. B) Morphology of broblast-like cells and tSVF were isolated using Minceolip® protocol and tSVF fragment (red arrow). C) Evaluation of the expansion rate of isolated cells using ENZ and Minceolip device for nine passages. ENZ: Enzyme, BFP: Buccal Fat Pad, tSVF: Tissue Stromal Vascular Fraction. Scale bar=12 µm. The results are presented as mean ± SD from three healthy donors.

Figure 3
Multi-lineage differentiation of cells isolated using ENZ and mechanical protocols. A) Speci c staining, including Alizarin red, Oil Red O, and Toluidin Blue, for con rmation of osteogenesis, adipogenesis, and chondrogenesis. B) Assessment of The percentage of positive cells for these speci c staining. Error bars are SD (N=3). ENZ: Enzyme. The results are presented as mean ± SD from three healthy donors-the scale bar=12 µm.

Figure 4
Radiography image from a benign tumor in a patient who was treated using standard segmental resection surgical procedure. A) Tumor site before surgery, B) image after surgery to remove the tumor, and C) image of defect site after one year.

Figure 5
The radiographic evaluation in a patient who received BFP-tSVF using partial resection technique on the left side mandible. A and B) OPG and CBCT Before surgery, respectively. C and D) OPG and CBCT after one year, respectively.