Buccal Fat Pad–Derived Stem Cells in Three-Dimensional Rehabilitation of Large Alveolar Defects: A Report of Two Cases

Bone regenerative therapies, especially stem cell–based treatment, requires a consistent source of osteoprogenitor cells.¹  Although bone marrow aspirates reveal the most well-known origin of stem cells for bone tissue engineering,^2–5  the invasive procedure for harvesting the tissue and decreasing osteogenic capability of bone marrow mesenchymal stem cells (BMMSCs) after a certain age^1,6  challenge its beneficial application. Thus, a multitude of alternative sources, including dental tissues,^7–10  skeletal muscle,¹¹  and adipose tissue,^12–14  have been proposed. Among these recent sources, adipose-derived stem cells (AdSCs) were successfully used in several in vitro,^15–17  in vivo,^18–20  and clinical investigations^21–23  for bone repair. In comparison to BMMSCs, AdSCs revealed longer conservation in culture and maintained their proliferation and osteogenic differentiation capabilities.²⁴  Moreover, these multipotent cells in adipose tissues are easily accessible. Also, their harvesting procedure is less invasive, and they are abundant in the human body.^1,24  Buccal fat pad (BFP) is a specialized form of subcutaneous rich vascularized fatty tissue in the oral cavity,²⁵  which has been investigated as an attractive graft for bone and periodontal repair in oral surgery.^26–29  Furthermore, BFP has been shown to be a valuable source of AdSCs, capable of differentiating into osteoblast.³⁰ 

In order to reconstruct large bone defects in the craniofacial region, which are caused by congenital factors, trauma, and various pathologies,³¹  a cell-based approach can be beneficial in overcoming some drawbacks of conventional methods, such as obtaining iliac crest autograft, which causes a considerable morbidity in the donor site.^32–35  In this regard, application of AdSCs originated from BFP, that is, BFP-derived stem cells (BFPSCs) can simplify surgical procedures and diminish clinical risks compared with large autograft harvesting.³⁶  However, based on our investigation, no published articles have assessed stem cells derived from BFP for large craniofacial rehabilitations. This case report sought to evaluate and describe the potential clinical application of AdSCs originated from BFP in combination with conventional guided bone regeneration (GBR) as protected healing space for reconstruction of large alveolar defects after extraction of multiple teeth extractions.

In the current study, 2 patients presenting to the Department of Oral Maxillofacial Surgery, Shahid Beheshti University of Medical Sciences, were identified. The clinical procedures were confirmed by the Ethics Committee of Shahid Behesti University of Medical Sciences. The patients gave informed consent for BFP harvesting and surgical procedures.

Three weeks before graft surgery, patients' BFPs were exposed through a vestibular incision in the posterior part of the mandible as previously described.³⁷  Next, 3–5 mL of adipose tissue was excised and sent to the laboratory. The obtained adipose tissues were placed in a solution of 1% collagenase type I (Sigma-Aldrich, St Louis, Mo) for 1 hour at 37°C in an incubator shaker. Then, cells were resuspended in growth medium containing Dulbecco Modified Eagle Medium (Life Technologies, Carlsbad, Calif) and 10% autogenous human serum. Cell suspension was transferred to a T-25 flask and incubated at 37°C and 5% CO₂. Cells were cultured after 80% to 90% confluency. The medium was changed every 3 days. Cells were trypsinized using 0.25% trypsin-ethylenediaminetetraacetic acid (Life Technologies).³⁸ 

In order to characterize the human AdSCs, cell surface expression of CD90, CD44, CD73, CD105, CD34, and CD45 was measured by fluorescence absorbance cell sorting analysis using standard protocols. For the flow cytometry process, phycoerythrin (PE)–conjugated monoclonal antibodies and fluorescent isothiocyanate (FITC)–conjugated monoclonal antibodies were used according to the specific device (BD FACSCalibur; BD Biosciences, Franklin Lakes, NJ). Then, anti-CD44-FITC, anti-CD73-PE, anti-CD90-FITC, anti-CD45-FITC, anti-CD105-PE, and anti-CD34-PE (EXBIO Praha, Vestec, Czech Republic) were applied at 2 mg/mL. Stem cells dyed with FITC-labeled mouse immunoglobulin G were used as a negative control. After washing with phosphate-buffered saline, all specimens were fixed using 1% paraformaldehyde. Flow cytometry data were analyzed using FlowJo 7.6.1 software (FlowJo LLC, Ashland, Ore); samples with >90% fluorescent-labeled cells were considered positive.

The BFPSCs were also assessed for osteogenic differentiation. Cells at passage 3 were placed in Stem PRO (Life Technologies) for 14 days and then fixed using paraformaldehyde solution. Next, they were stained with Alizarin red for 5 minutes and imaged using light microscopy.

In this study, natural bovine bone mineral (NBBM) (1521, Cerabone granules, Botiss, Berlin, Germany) with a crystalline size of 1.0–2.0 mm, which mimics the same characteristic of the human normal bone, was used.³⁹  Five mg of NBBM was placed into 96-well plates, and BFPSCs at density of 10⁵ cells per well were seeded on them for 48 hours at 37°C and 5% CO₂.

The attachment of BFPSCs on NBBM was examined using scanning electron microscopy (SEM), and the cells on the scaffolds were fixed by 2.5% glutaraldehyde for 1 hour at room temperature. Dehydration cell scaffold was done in a series of increasing concentrations of ethanol in distilled water 30%, 70%, 80%, 90%, and 100% for 10 minutes per each concentration. The samples were air-dried for 24 hours. Finally, the scaffolds were sputter-coated with gold before imaging (SU3500, Hitachi, Tokyo, Japan).

In September 2015, a 19-year-old woman was referred to the clinic complaining of several impacted teeth in the maxillary and mandibular regions, which could not be forced to erupt and were recommended for surgical extraction by the orthodontist. The patient has cleidocranial dysplasia. She was a nonsmoker with no systemic health problem (aortic stenosis and aortic insufficiency [ASAI]). Preoperative radiographic assessment revealed 11 impacted teeth in the upper jaw and 13 in the lower jaw between the first molars (Figure 1). All teeth were removed after being exposed through the buccal bone with minimum bone removal. In deeply impacted teeth, thin labial bone was cut with a Piezosurgery saw blade (Mectron, Carasco, Italy) and out fractured to expose the teeth. A protected healing space was created by fixing the out-fractured bone or by using the adjacent lateral ramus cortical bone stabilized with a microplate (1.2 mm, Jeil, Seoul, South Korea). Then, NBBM (1521, Cerabone granules, Botiss) coated with BFPSCs was delivered and filled the space. After 6 months post-GBR, she underwent 6 and 7 dental implant (Alfit, SSO, IDHE Dental Implant Company, Bern, Switzerland) placements, respectively, in the maxilla and mandible. At 10 months postoperatively, panoramic radiographic evaluations revealed thorough survival of implants.

In December 2012, a 22-year-old man was referred to the clinic, complaining of several impacted teeth in the maxillary and mandibular regions, which had not achieved erupted, and the orthodontist suggested surgical extraction. This patient was non-smoker with no systemic condition (ASAI). Preoperative radiographic assessment showed 12 impacted teeth in the upper jaw and 10 in the lower jaw between the first molars (Figure 2). At 12 months post-GBR, he received 4 dental implants (Alfit, SSO, IDHE Dental Implant Company) in his maxilla and 7 dental implants (Alfit, SSO, IDHE Dental Implant Company) in the mandible. At 48 months postoperatively, panoramic radiographic evaluations revealed thorough survival of implants.

A crestal incision was made molar to molar in the mandible and maxilla, with 2 releasing incisions. Subperiosteal dissection of the buccal and lingual mucosal flaps was performed gently. The impacted teeth were exposed with the delicate removal of the buccal plate after use of the Piezosurgery microsaw. In some places, superficially located teeth lead to out-fracture of thin covering bone. After extraction of impacted teeth, an extra-large (>6 cm) bone defect was created in the maxilla and mandible. A cortical autogenous bone block was harvested from the l00ateral ramus of the mandible on both sides. The obtained bone tissue was cut into 3 or 4 pieces. The thin labia plate and 1.2-mm microscrews with lateral ramus cortical plates were fixed together to create a protected healing space (Figures 3a and 4a). The space was filled with NBBM loaded with BFPSCs (Figures 3b and 4b). The whole construct was covered with collagen membrane (Jason Membrane, Botiss) and sutured (5/0 polyglactin 910, Vicryl, Ethicon, Sint-Stevens-Woluwe, Belgium) (Figures 3c and 4c). Subsequently, the retracted flap was replaced to its position and closed by continuous horizontal mattress sutures. All procedures were done in a hospital setting by an experienced oral and maxillofacial surgeon and his team.

Patients were all given 500 mg amoxicillin 3 times a day for a week, analgesics (ibuprofen 400 mg with or without acetaminophen codeine 300/10 mg depending on the severity of the pain), and 1 intramuscular injection of 8 mg dexamethasone (Alborz Darou, Tehran, Iran) and chlorhexidine mouthwash 0.2% (Behsa Pharmaceutical, Tehran, Iran) for 7 days postoperatively. In the consolidation period before implant therapy (4–6 months after surgery), patients were visited twice a week for the first month and monthly prior to dental implant placement. Figure 5 shows the clinical view of the new bone formation in case 1 before and after implant installation.

In order to evaluate clinical healing, soft tissue and the healing process of grafted tissue were checked biweekly for the first month postoperatively and then monthly for 6 months.

Patients had 2 cone beam computed tomography scans taken on the NewTom VG 9000 device (Quantitative Radiology SRL Co, Verona, Italy) before and 6 months after graft surgery. Panoramic view radiographs were taken in the follow-up periods after dental implant placement. A calibrated oral and maxillofacial surgeon qualitatively evaluated the amount of bone.

Trephine samples were obtained during implant installation. All specimens were fixed in 10% buffered-formalin for 5 to 7 days, and decalcified in formic acid and sodium citrate for 24 hours. The specimens were washed with tap water, dehydrated with ascending concentrations of ethyl alcohol, cleared in xylene (Sigma-Aldrich), and infiltrated with paraffin. Serial sections (5 μm) parallel to the midsagittal suture were cut from the center of each specimen using a microtome and stained with hematoxylin and eosin.

The sections were viewed and qualitatively analyzed for new bone formation, residual bone substitute, residual clot elements, soft tissue elements, and inflammatory reactions by 1 calibrated examiner using polarized light microscopy (SZX 9, Olympus, Tokyo, Japan).

Flow cytometric analysis of harvested and cultured BFPSCs indicated that more than 95 of the cells were positive for mesenchymal markers (CD73 , CD105, CD 44, and CD 90). Hematopoietic markers (CD34 and CD45) were expressed at very low percentages of the cells (Figure 6).

Alizarin red staining findings represented deposition of mineralized nodule-like structures and confirmed the osteogenic capability of the cultured cells (Figure 7).

Figure 8 presents the SEM image of BFPSCs in 3-dimensional scaffold culture after 48 hours. Stem cells demonstrated a similar spindle-shaped morphology and attachment to the NBBM.

All through the study, the patients' healing processes were uneventful, and there were no serious complication. In case 1, as seen in Figure 9, after 7 months radiographic assessment revealed the proper amount of newly regenerated bone in the upper and lower jaws (Figure 5). Radiographic evaluations revealed acceptable integration of bone substitute to the recipient healthy bone and 3-dimensional bone formation. In addition, clinical observation indicated a bony dense and vascularized tissue after dislocation of surgical flap. Figure 10a shows osseointegrated implants after 10 months.

In case 2, radiographic assessment showed appropriate amount of bone both in height and width for implant therapy after 12 months (Figure 11). Overall, the regenerated bone in case 2 was similar to that of the previous patient and depicted a bony hard clinical appearance and high vascularity during the specimen harvesting and implant placement process. Figure 10b shows osseointegrated implants after 48 months.

In both cases, new bone and osteoid matrix formation with incorporation into the NBBM were seen (Figure 12a and b). The trabecular pattern was mostly lamellar organization containing osteocytes within lacunae and rimmed via osteoblasts. There was no evidence of foreign body reaction, and there was considerable immune response in the obtained specimens from both patients.

Autogenous iliac crest bone graft is the gold standard for rehabilitation of large bone defects⁴⁰  due to its rapid incorporation to the host, the presence of a multitude of growth factors, and its osteogenic properties.⁴¹  However, there are some major drawbacks in its clinical application, such as the need for a second site of surgery, its limited tissue volume, morbidity of donor site, infection, and scar creation.^41–43  In addition, it is estimated that up to 80% of the osteocytes do not survive the transplantation process between the donor site and the surgical site, and they showed a resorption rate between 65% and 85%.^44–47  Moreover, from a biological perspective, the origin of iliac crest harvested bone is the mesoderm germinal layer, which generates bone by endochondral ossification process, whereas bone grafts that are obtained intraorally from the mandible turn into bone much faster because they generate bone by intraosseous bone formation.⁴⁸  Thus, the emergence of regenerative medicine to address these shortcomings of iliac crest harvested bone is pivotal. One of the most imperative purposes of bone tissue engineering is to seek a reliable source of stem cells that provide a proper number of stem cells for clinical administration with minimal invasiveness and morbidity. Nowadays, BMMSCs are the most common source of osteoprogenitor cells in bone tissue engineering^4,5  that are isolated from bone marrow aspirate. However, this invasive and painful process has only 0.001% to 0.1% of BMMSCs in the obtained cells compared with lipoaspirates, which contain 1% to 5% of ADCSs.⁴⁹  Moreover, the comparative investigations revealed similar osteogenic differentiation of AdSCs and BMMSCs.^50–52  In the current study, we used BFP as the adipose tissue source. In contrast to subcutaneous adipose tissue, BFP is a specialized mass of fat that is easy to harvest in oral surgery and causes less morbidity and complications in donor site.^36,53,54  Moreover, BFP size is approximately similar in various persons and does not depend on fat distribution and body weight.⁵⁵  Hence, it can be a primary source of stem cells for therapeutic purposes.

In spite of the intensifying amount of evidences on AdSCs and their excellent potential for bone regeneration, most in vitro investigations and only limited clinical trials have evaluated the safety and efficacy of AdSC use in humans.^{21,36,56–58}  In accordance with our results, AdSCs showed beneficial effects on bone healing in anterior mandibular,⁵⁶  maxillary,⁵⁷  and cranial⁵⁸  bony defects. This study evaluated the efficacy of BFP-derived mesenchymal stem cells for 3= dimensional bone regeneration in alveolar defects greater than 6 cm after extraction of multiple impacted teeth. The findings in these cases show that in large alveolar bone defects, application of BFPSCs with the GBR technique maintains appropriate bone regeneration for dental implant placement. One of our study's limitations is our evaluation methods. Although radiographic images cannot separate areas of new bone formation from bone substitutes, histologic assessments indicated a lamellar trabecular pattern containing osteocytes that represent a new bone formation area. Another limitation is that due to ethical considerations, we did not have any control to compare our findings.

With the limitation of our study, this efficacious clinical application of BFPSCs for regeneration of alveolar bone opens a window into this promising new field of research that requires further efforts, especially randomized controlled clinical trials and histomorphometric assessments to lead an evidence-based decision making.

Abbreviations

AdSC

adipose-derived stem cell

ASAI

aortic stenosis and aortic insufficiency

BMMSC

bone marrow mesenchymal stem cell

BFP

buccal fat pad

BFPSC

buccal fat pad stem cell

FITC

fluorescent isothiocyanate

GBR

guided bone regeneration

NBBM

natural bovine bone mineral

PE

phycoerythrin

SEM

scanning electron microscopy

The authors report no conflicts of interest.