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 Table of Contents  
Year : 2013  |  Volume : 3  |  Issue : 2  |  Page : 114-125

GMP-level adipose stem cells combined with computer-aided manufacturing to reconstruct mandibular ameloblastoma resection defects: Experience with three cases

1 Institute of Biomedical Technology, University of Tampere; Department of Eye, Ear and Oral Diseases, Tampere University Hospital, Tampere, Finland
2 Institute of Biomedical Technology, University of Tampere, Tampere; Department of Oral and Maxillofacial Surgery, University of Oulu, Oulu, Finland
3 Department of Eye, Ear and Oral Diseases, Tampere University Hospital, Tampere, Finland
4 Department of Oral and Maxillofacial Surgery, Central Hospital of Central Finland Health Care District, Jyväskylä, Finland
5 Institute of Biomedical Technology, University of Tampere, Tampere, Finland

Date of Web Publication3-Oct-2013

Correspondence Address:
George K Sándor
Institute of Biomedical Technology, University of Tampere, Biokatu 12 Krs 6, 33520 Tampere
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Source of Support: The Finnish Funding Agency for Technology and Innovation (TEKES) and the Competitive Research Funding of Tampere University Hospital

DOI: 10.4103/2231-0746.119216

PMID: 24205470

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Background: The current management of large mandibular resection defects involves harvesting of autogenous bone grafts and repeated bending of generic reconstruction plates. However, the major disadvantage of harvesting large autogenous bone grafts is donor site morbidity and the major drawback of repeated reconstruction plate bending is plate fracture and difficulty in reproducing complex facial contours. The aim of this study was to describe reconstruction of three mandibular ameloblastoma resection defects using tissue engineered constructs of beta-tricalcium phosphate (β-TCP) granules, recombinant human bone morphogenetic protein-2 (rhBMP-2), and Good Manufacturing Practice (GMP) level autologous adipose stem cells (ASCs) with progressively increasing usage of computer-aided manufacturing (CAM) technology. Materials and Methods: Patients' three-dimensional (3D) images were used in three consecutive patients to plan and reverse-engineer patient-specific saw guides and reconstruction plates using computer-aided additive manufacturing. Adipose tissue was harvested from the anterior abdominal walls of three patients before resection. ASCs were expanded ex vivo over 3 weeks and seeded onto a β-TCP scaffold with rhBMP-2. Constructs were implanted into patient resection defects together with rapid prototyped reconstruction plates. Results: All three cases used one step in situ bone formation without the need for an ectopic bone formation step or vascularized flaps. In two of the three patients, dental implants were placed 10 and 14 months following reconstruction, allowing harvesting of bone cores from the regenerated mandibular defects. Histological examination and in vitro analysis of cell viability and cell surface markers were performed and prosthodontic rehabilitation was completed. Discussion: Constructs with ASCs, β-TCP scaffolds, and rhBMP-2 can be used to reconstruct a variety of large mandibular defects, together with rapid prototyped reconstruction hardware which supports placement of dental implants.

Keywords: Adipose-derived stem cells, beta-tricalcium phosphate, bone morphogenetic protein, computer-aided design

How to cite this article:
Wolff J, Sándor GK, Miettinen A, Tuovinen VJ, Mannerström B, Patrikoski M, Miettinen S. GMP-level adipose stem cells combined with computer-aided manufacturing to reconstruct mandibular ameloblastoma resection defects: Experience with three cases. Ann Maxillofac Surg 2013;3:114-25

How to cite this URL:
Wolff J, Sándor GK, Miettinen A, Tuovinen VJ, Mannerström B, Patrikoski M, Miettinen S. GMP-level adipose stem cells combined with computer-aided manufacturing to reconstruct mandibular ameloblastoma resection defects: Experience with three cases. Ann Maxillofac Surg [serial online] 2013 [cited 2021 Aug 4];3:114-25. Available from: https://www.amsjournal.com/text.asp?2013/3/2/114/119216

  Introduction Top

While many strategies have been proposed for the management of mandibular bony defects following avulsive trauma or ablative surgery, [1] autogenous bone grafts are considered the "gold standard" for reconstructive bone surgery [2] to the present day.

Small mandibular defects (<4-6 cm) are often treated with nonvascularized corticocancellous grafts harvested from the anterior or posterior iliac crest. [2],[3] Larger defects are commonly treated with vascularized fibula grafts [2],[4],[5] which offer a long pedicle length, wide vessel diameter, and the possibility to incorporate skin, muscle and bicortical bone components when such tissues are required. [6] However, the use of vascularized autologous tissue grafts requires healthy donor tissues and vessels. Morbidity of large bone graft harvesting is a deterrent to fibular bone flap use, [7],[8],[9],[10] therefore less morbid treatment strategies would be advantageous. [11]

Bone tissue engineering regenerates osseous tissue by combining biomaterials, bioactive molecules, and stem cells. [11],[12],[13],[14] One reliable source of stem cells for bone regeneration is autologous fat tissue which provides an abundant amount of adipose stem cells (ASCs). [15],[16] ASCs are capable of multiple lineage differentiation to chondrocyte, adipocyte, and osteoblast pathways. [15] The harvesting of fat tissue is simple and causes less morbidity when compared to standard bone harvesting techniques. [15],[16],[17],[18],[19] One other advantage of ASCs over human bone marrow stromal/stem cells (BMSCs) is that BMSCs are present in low frequency in the bone marrow, whereas ASCs can be retrieved in high numbers from adipose tissue and can easily be expanded in vitro.[14],[20] Using the tissue engineering model, [12] it has been possible to harvest autogenous ASCs from patients and use the cells to seed a resorbable scaffold. [21],[22]

Biomaterials of various types and forms can serve as scaffolds in bone tissue engineering, including granular forms and solid blocks. Although they have no load bearing ability, compared to blocks, granular forms present more surface area for cellularization. Granules are more quickly incorporated into host tissues than block forms which remodel more slowly. [23] Granules have a tendency to migrate within a wound and must be retained at the site of intended implantation by a containment mesh made of either titanium or a resorbable biomaterial. Granular calcium phosphate ceramics and related biomaterials such as hydroxyapatite possess macro- and microstructural properties that make them useful biomaterials in tissue engineering. Such properties affect cell attachment, survival, signaling, growth, and propagation. [23],[24] While some inorganic ceramics may also bind directly to bone, [25] the surfaces of other biomaterials may affect differentiation of stem cells to osteoblasts. [26]

Growth factors such as bone morphogenetic proteins (BMP) have recently been added to constructs in order to promote osteoinductive properties. [27],[28],[29]

Although major segments of human mandibular defects have been reconstructed with constructs containing growth factors such as recombinant human BMP-7 (rhBMP-7), [27],[30] reports regarding cell-seeded constructs are still few in number. [22],[24],[31],[32],[33],[34],[35],[36] While there has been much laboratory study of possible techniques, [37],[38],[39] most clinical reports describe the occasional single case success. [22],[31],[32],[33],[34],[35],[36]

One deficiency in planning complex reconstructive surgery using either autologous bone or tissue engineered constructs is the inability to predictably produce complex facial contours using commercially available reconstruction plates and meshes. Commercially available reconstruction plates are supplied as generic sizes and shapes being designed on the basis of the 'average' patient. These reconstruction plates are often made from titanium and are supplied as straight or slightly contoured metal plates with predrilled holes for retention by screws. The surgeon may spend considerable time during surgery bending and shaping the plate to fit the contour of the patient's bone. Titanium reconstruction plates currently used for mandibular defects are subject to both over-bending and a lack of passive fitting which may eventually lead to fatigue fractures. [40]

One solution to this problem is to use computer-guided surgical planning and additive manufacturing technology to produce a passive fitting implant designed for patient specific anatomical needs. [41] Progress in medical imaging and continued incremental increases in computer processing power available for both three-dimensional patient data acquisition and subsequent image processing make it possible for clinicians to diagnose, more accurately plan, simulate, and treat their patients. [42]

To date the most common use of additive manufacturing has been the fabrication of patient-specific skull models for preoperative planning. These skull models are fabricated using patient specific imaging data in the form of Digital Imaging and Communications in Medicine (DICOM) files, which are then converted into stereolithography (STL) files, and used to print the patient specific skull models. The use of such three-dimensional (3D) medical models helps surgeons to plan, simulate the planned operation, and manually pre-bend commercially available reconstruction plates. [43] Recent developments in the area of additive manufacturing allow the prefabrication of patient specific, customized, reconstruction plates using the patient's DICOM data. The advantages of rapid prototyped reconstruction plates are that they do not require intraoperative bending and offer improved passive fitting. In light of possible material failure, better structural designs may be developed according to individual anatomy at the design stage.

The aim of this study was to reconstruct segmental mandibular defects resulting from ameloblastoma resection in a small series of non-radiated patients using a tissue engineered construct consisting of beta-tricalcium phosphate (β-TCP) granules, rhBMP-2 in conjunction with ASCs. Additionally, the authors sought to gradually incorporate a 3D computer-aided surgical workflow, from diagnosis to surgical treatment, in order to provide patients with complimentary hardware to help restore mandibular ameloblastoma resection defects.

  Materials and Methods Top

A total of three patients with recurrent ameloblastomas requiring segmental mandibular resection were included in this study. There were two males and one female with an average age of 49.3 years ranging from 43 to 55 years [Table 1]. The planned mandibular segmental resection defects ranged from 6.0 to 10.0 cm in length with an average defect size of 8.2 cm [Figure 1], [Figure 2] and [Figure 3]. There was no history of radiation therapy in any of the three cases. Two patients were in good health. Case 3 was congenitally blind. Case 1 had been previously presented in a single case report [36] and was now included with further follow-up as part of this series of three patients.
Figure 1: Preoperative panoramic radiograph of case 1 with recurrent ameloblastoma at symphyseal region of anterior mandible

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Figure 2: Preoperative panoramic radiograph of case 2 with recurrent ameloblastoma at left body and ramus of mandible. Note the proximity of the lesion to the most distal mandibular dental implant

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Figure 3: Preoperative panoramic radiograph of case 3 with recurrent ameloblastoma at the left body of the mandible

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Table 1: Patient details of cases with mandibular defects treated with good manufacturing practice level adipose stem cells

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The combination of ASCs and biomaterials are considered to be an advanced therapy combined medicinal product according to the regulations of the European Medicines Agency. The treatment was carried out with the approval of FIMEA, the Finnish Medicines Agency under hospital exemption of patient specific treatment (Dnro 615/11.01.06./2010). The approval to use ASCs for research purposes was granted by the Ethics Committee of the Pirkanmaa Hospital District, Tampere, Finland (R03058) within the ethical principles of the Declaration of Helsinki. Prior to surgery, the patients were thoroughly informed about the procedures, which they approved and to which they gave their written consent.

Planning and reverse engineering

Prior to planning the operation 3D stereolithographic medical skull models (Planmeca, Helsinki, Finland) were fabricated using the patients' preoperative computed tomography (CT) noncompressed DICOM voxel-based data set. The medical skull models allowed visualization and assessment of the planned resection defects and were also used to pre-bend commercially available reconstruction plates and titanium meshes for later use in the reconstructions.

The acquired patient DICOM data sets were also imported into Pro Model Romexis virtual surgical planning software program (Planmeca, Helsinki, Finland) after which patient specific 3D preoperative planning was performed [Figure 4], [Figure 5] and [Figure 6]. The novel Pro Model Romexis software program was capable of performing virtual mandibular resections, manipulate bony segments, and mirror the anatomy of the unaffected side [Figure 7]. The calculated virtual reconstructions were then converted into STL software file format to generate the information needed to fabricate custom-made medical skull models, saw guides, and reconstruction plates. The custom-made saw guides were produced using an additive manufactured process with a Z printer 450 (Z Corporation Rock Hill, SC, USA), a commercial printer which creates objects by curing composite powder and ink jetting. The reconstruction plates were manufactured using titanium powder and selective laser sintering (SLS) technology. This method of additive manufacturing uses a laser beam to fuse and solidify titanium powder. Successive layers of powder are deposited and raster scanned creating a highly accurate medical-grade titanium (Ti6A14V ELI) custom reconstruction plate.
Figure 4: Virtual preoperative planning using Romexis software in case 2. The picture illustrates saw guides precisely over the resection position

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Figure 5: The computer generated image of case 2 shows the reconstruction plate over the area planned for resection

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Figure 6: This computer generated virtual planning image of case 2 shows the resection and shape of reconstruction plate based on the mirrored healthy side of mandible

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Figure 7: This computer generated virtual planning image of case 3 shows the resection jig in place with the shape of reconstruction plate based on the mirrored healthy side of mandible

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ASCs isolation and cell culturing

Three to four weeks prior to mandibular resection, approximately 50-100 ml of subcutaneous adipose tissue was harvested from the anterior abdominal wall [Figure 8] to be used for ex vivo tissue culturing and future autologous reimplantation. Additionally, 50-60 ml of autologous serum was obtained for the expansion of autologous stem cells.
Figure 8: Three weeks prior to mandibular resection, 50-60 ml of autologous serum and approximately 50-100 ml of subcutaneous adipose tissue was harvested from the anterior abdominal wall for ex vivo tissue culturing and autologous reimplantation

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Patient specific autologous ASCs from cases 1, 2, and 3 were isolated and expanded in vitro in Good Manufacturing Practice (GMP) clean room according to standard operating procedures at Regea Cell and Tissue Center, Institute of Biomedical Technology, University of Tampere and BioMediTech. Isolation and expansion procedures were performed as reported previously with minor modifications. [14],[21] The adipose tissue was minced and digested with recombinant collagenase NB 6 (Invitrogen, Paisley, Scotland, United Kingdom; GMP grade; SERVA Electrophoresis GmbH, Heidelberg, Germany).

The isolated cells were expanded for 21 days in basal media (BM) containing Dulbecco's modified Eagle medium/F-12 (Gibco Invitrogen, Paisley, Scotland, United Kingdom) with 15% of autologous serum, without antibiotics, and were subsequently passaged on confluency, detached mechanically with a cell scraper, and prepared for cell transplantation. This yielded an average of 10,240,000 cells (range 4,720,000-16,000,000) of passages three to four, for each of the three patients [Table 1]. These cells were combined with 40 mL of β-TCP granules (Chronos1, porosity 60%, granule size 1.4-2.8 mm; Synthes, Oberdorf, Switzerland) 48 h before the operation to allow cell attachment to the biomaterial. Prior to combining the cells with β-TCP, the material was incubated for 48 h in basal medium (BM) containing 12 mg rhBMP-2 (InductOS, Wyeth Europa, Berkshire, UK). Following the incubation, the BM containing rhBMP-2 was discarded.

Cell sterility and endotoxins were tested by Biovian Ltd. (Turku, Finland) according to methods described in the European Pharmacopoeia (Council of Europe, Strasbourg, France). The cells tested negative for mycoplasma contamination as determined by a mycoplasma polymerase chain reaction (PCR) kit (VenorGem; Minerva Biolabs GmbH, Berlin, Germany). The genomic integrity of hASCs was verified using a standard chromosome G-banding method. Karyotypic evaluation was performed as an outsourcing service in Medix Laboratories Ltd. (Espoo, Finland).

Cell attachment to the material and the cell viability were studied using live/dead staining prior to the cell transplantation operation. Briefly, the cell-biomaterial combination was incubated with a mixture of CellTracker™ green (5-chloromethylfluorescein diacetate (CMFDA, Molecular Probes, Eugene, Oregon, USA) and ethidium homodimer (EH-1; Molecular Probes). The viable cells (green fluorescence) and dead cells (red fluorescence) were detected with a fluorescence microscope [Figure 9].
Figure 9: Intraoperative photograph of case 2 showing reconstruction plate applied to mandible with resection lines transferred from resection jig onto the mandible

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ASCs were further expanded in vitro and the immunophenotype of the cells was analyzed by flow cytometry (FACSAria®, BDBiosciences, Erembodegem, Belgium). Monoclonal antibodies (MAb) against CD11a-allophycocyanin (APC), CD80-phycoerythrin (PE), CD86-PE, CD105-PE (R&D Systems Inc., Minneapolis, MN, USA), CD3-PE, CD14-phycoerythrin-cyanine (PECy7), CD19-PECy7, CD45RO-APC, CD54-fluorescein isothiocyanate (FITC), CD73-PE, CD90-APC (BD Biosciences), and CD34-APC, HLA-DR-PE (Immunotools GmbH, Friesoythe, Germany) were used [Table 2]. Analysis was performed on 10,000 cells per sample and unstained cell samples were used to compensate for the background autofluorescence levels.
Table 2: Surface marker expression of adipose stem cells by flow cytometry (FACS) of 10,000 cells analyzed. Data are presented as average and standard deviation obtained from all three patient samples

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Due to the limited amount of autologous serum for the subsequent in vitro analyses, basic medium supplemented with 10% human serum type AB (Lonza Bioscience Rockland, Romeoville, IL, USA), and 1% antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin; Invitrogen) were used.

Multipotency of the ASCs was analyzed by adipogenic, osteogenic, and chondrogenic differentiation. The differentiation capacity was evaluated after 14 days of differentiation induction in adipogenic, osteogenic or chondrogenic medium versus cells cultured in control medium. Briefly, for the in vitro osteogenic differentiation analyses, ASCs were maintained on a 12-well plate at a density of 2.5 × 10 3 cells/cm 2 in osteogenic media (OM), containing BM supplemented with 250 mM L-ascorbic acid 2-phosphate (Sigma Aldrich, St Louis MO, USA), 10 mM β-glycerophosphate (Sigma Aldrich) and 5 nM dexamethasone (Sigma Aldrich). The cultures were subsequently analysed by alkaline phosphatase staining (ALP). The cell cultures were fixed with a 4% paraformaldehyde solution (PFA) and stained with alkaline phosphatase according to the Sigma Aldrich Procedure No. 86 (#86R-1KT) to detect early osteogenesis.

For the in vitro adipogenic differentiation analyses, surplus ASCs were maintained on a 12-well plate at a density of 2 × 10 4 cells/cm 2 in adipogenic media containing BM supplemented with 33 μM biotin (Sigma Aldrich), 1 μM dexamethasone (Sigma Aldrich), 100 nM insulin (Life Technologies), and 17 μM pantothenate (Fluka, Buchs, Switzerland). In addition, 250 μM isobutylmethylxanthine (IBMX; Sigma) was used for the first 24 h of adipogenic induction. Cell cultures were fixed with 4% PFA and stained with 0.5% Oil Red O solution (Sigma Aldrich) for detection of oil droplets. For the chondrogenic differentiation, 1 × 10 5 cells were seeded on 24-well plates in a 10 μl volume as micromasses, and were allowed to adhere for 3 h in + 37°C prior to the addition chondrogenic medium; BM supplemented with 1% ITS + 1 (Sigma Aldrich), 50 μM ascorbic acid (Sigma Aldrich), 55 μM sodium pyruvate (Life Technologies), 23 μM L-proline (Sigma Aldrich), and 10 ng/ml TGF-β (Sigma Aldrich). The micromass pellets were fixed in 4% PFA, embedded in paraffin and sectioned at 5 μm thickness. Alcian blue solution was used to detect cartilaginous matrix glycosaminoglycans, with nuclear Fast Red (Biocare Medical, Concord, MA, USA) as counterstain.

Ameloblastoma resection protocol

Before segmental mandibulectomy all three patients were placed into maxillomandibular fixation unilaterally using fixation screws and elastic bands to maintain condylar position. An extraoral submental incision in case 1 or submandibular approaches in cases 2 and 3 were used to expose the parasymphysis or the body and ramus of the mandible on the defect side. A commercially available 2.4 mm generic reconstruction plate was used in cases 1 and 2 (Synthes, Obersdorf, Switzerland) and additive manufactured 2.4 mm custom-made titanium reconstruction plates (Planmeca, Helsinki, Finland) were attached to the affected hemimandible and fixed with 2.4 mm titanium screws in cases 2 and 3. After fixating the reconstruction plate, virtually simulated osteotomy lines from the preoperative planning were easily located intraoperatively with help of prefabricated saw guides [Figure 9], [Figure 10], [Figure 11] and [Figure 12] that were fixed on top of the already screw retained rapid prototyped reconstruction plate.
Figure 10: Intraoperative photograph of case 2 with superior resection line transferred from computer generated plan being cut with a piezosurgical tip

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Figure 11: Intraoperative photograph of case 2 showing both resection lines completed and resected fragment being lifted from wound without the need to remove the reconstruction plate. The reconstruction plate maintains correct orientations of the mesial and distal resection stumps

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Figure 12: Intraoperative photograph of case 2 with ASC containing granules of β-TCP and rhBMP-2 being packed beneath the titanium containment mesh

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The entire tumor with a margin of healthy tissue was excised as planned in each case [Figure 13]. The healthy ends of the resected mandibles were maintained in their correct orientations with the attached hardware. The defects resulting from ameloblastoma resection were immediately reconstructed using a preformed custom-made titanium mesh fixated onto the ends of the mandibular defects using 1.5 mm titanium screws. The space beneath the mesh was filled with rhBMP-2 and ASCs seeded β-TCP granules [Figure 14], [Figure 15], [Figure 16]. To avoid exposure of the titanium mesh to the oral cavity, the final height of the titanium mesh was kept lower than that of the resected mandibular bone. Routine layered wound closure was performed.
Figure 13: Intraoperative photograph of case 3 with reconstruction plate applied and resected fragment being lifted from the wound without the need to remove the reconstruction plate which was situated at the lower border of mandible. The reconstruction plate maintains correct orientations of the mesial and distal resection stumps

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Figure 14: Intraoperative photograph of case 3 showing the titanium containment mesh in place and its underlying space fi lled with granular scaffold of β-TCP and rhBMP-2

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Figure 15: Immediate postoperative panoramic radiograph of case 2 showing reconstruction plates, titanium containment mesh, and granular scaffold in place on the ramus, angle, and body regions of the left mandible

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Figure 16: Immediate postoperative panoramic radiograph of case 3 showing reconstruction plates, titanium containment mesh, and granular scaffold in place on the body and angle region of the left mandible

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Dental implant operation protocol

After uneventful healing of 10 months in case 1 and 14 months in case 2, both patients underwent dental implant placement in the reconstructed and healed defects [Figure 17], [Figure 18], [Figure 19], [Figure 20]. Case 3 chose not to have dental implant treatment. In both case 1 and 2 the intraoperative clinical evaluation of the augmented defect sites revealed healthy non-inflamed soft tissues and dense hard tissue. After exposing the grafted sites, 6 Straumann SLActive implants were placed into case 1 and one Straumann SLActive bone level implant was placed into case 2 (Straumann AG, Basel, Switzerland) [Table 1]. Bone cores were retrieved during the implant insertion procedure from the bone necessary for removal at the implant osteotomy sites. The retrieved bone cores were processed to obtain histological material for further tests on cell viability, proliferation, and osteogenic differentiation in cases 1 and 2.
Figure 17: Panoramic radiograph taken 3.0 years after insertion of dental implants into the reconstructed symphysis of Case 1

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Figure 18: Immediate post dental implant insertion panoramic radiograph from Case 2 showing dental implant placed into the reconstructed alveolus of the left posterior mandible

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Figure 19: Periapical radiograph showing osseointegrated dental implant in Case 2 loaded with prosthetic crown

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Figure 20: Intraoral view of dental implant in Case 2 and its restored crown on the mandibular left first molar tooth

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The cells from the bone core samples were allowed to sprout and proliferate in BM in vitro and were subsequently analyzed by flow cytometry. MAb against surface proteins ALP-APC, CD105-PE, BMPR1-FITC, FGFR2-APC, FGFR3-PE, TGFβ1-PE, VEGFR1-PE, VEGFR2 -Peridinin chlorophyll protein (PerCP), VEGFR3-APC (R&D Systems Inc., Minneapolis, MN, USA), CD45RO-APC, CD90-APC (BD Biosciences), and CD34-APC, HLA-DR-PE (Immunotools GmbH, Friesoythe, Germany) were used. Further, intracellular markers VEGF-PE, latent TGFβ-PE and TWIST-1-FITC (R&D Systems) were used according to the manufacturer's protocols. The analysis was performed as described for ASCs above.

  Results Top

The reconstruction of the three mandibular defects with GMP level ASCs was successful in bridging the large defects averaging 8.2 cm (range 6.0-10.0 cm) with uneventful healing [Table 1]. Two of the three patients opted for reconstruction with dental implants [Figure 17], [Figure 18], [Figure 19], [Figure 20]. There were seven fixtures placed in two patients and six (86%) successfully osseointegrated and were loaded prosthodontically. The three patients have been under follow-up for an average of 28 months following their cell based mandibular reconstructions (range: 19-43 months).

In two of the three patients, the preoperative virtual designs were transferred as planned to the mandibular resection sites. The reconstruction plates were capable of maintaining the anatomical spatial relation of the ends of the resected mandible and there was excellent passive fit of the plates at their respective resection sites.

Laboratory results

ASCs recovered from the adipose tissue proliferated in the autologous serum and cell morphology was spindle-shaped [Figure 21]. The live/dead staining showed that viable and adherent ASC were transplanted with the β-TCP granules into the patient [Figure 22]. Osteogenic, adipogenic, and chondrogenic differentiation was evident in differentiated samples showing the multipotent nature of ASCs [Figure 23], [Figure 24], [Figure 25].
Figure 21: Case 2 in vitro microscopic images of adipose stem cells (ASCs) in culture 7 days post isolation

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Figure 22: Fluorescence microscope image of live/dead staining of ASCs on biomaterial, with live cells staining green and dead cells staining red, showing cell adhesion to the biomaterial and cell viability

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Figure 23: Alkaline phosphatase staining (red) on osteogenic differentiation cultures of ASCs

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Figure 24: Oil red O staining confirming adipose differentiation of ASCs cultured in monolayer

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Figure 25: Alcian blue staining confirming chondrogenic differentiation of ASCs cultured in micromass culture format

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The flow cytometry results of ASCs [Table 2] and [Table 3] showed strong expression of mesenchymal markers CD73, CD90, and CD105; moderate expression of CD14, CD19, CD54, CD80, and CD86; and low or no expression of CD3, CD11a, CD34, CD45, and HLA-DR. However, the immunophenotype showed patient variability; especially in CD3, CD14, CD19, and HLA-DR; a tendency that has also been reported previously [14] [Table 2].
Table 3: Column diagram from flow cytometry data of adipose stem cells (ASCs). Columns illustrate mean surface marker expression levels with standard deviation error bars of all three patient samples

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The re-entry and bone drilling necessitated by the placement of dental implants or removal of parts of the edges of almost exposed titanium mesh after 10 and 14 months healing, allowed the opportunity to biopsy the healing construct and by obtaining cores at the implant osteotomy sites.

The flow cytometric analysis on cells derived from bone samples [Table 4] and [Table 5] showed strong expression of mesenchymal markers CD90, CD105, and TWIST-1; moderate expression of FGFR3 and intracellular VEGF; low expression of CD34, CD45, FGFR2, ALP, BMPR1, VEGFR1, VEGFR3, and latent TGFβ; and no expression of TGFβ1, HLA-DR, and VEGFR2. As seen in the ASCs, the surface marker expression showed patient variability, particularly in CD34, CD45, FGFR2, FGFR3, VEGFR1, and intracellular VEGF [Table 2].
Table 4: Surface marker expression of osteoblasts determined by flow cytometry (FACS) of 10,000 cells analyzed. Data are presented as average and standard deviation obtained from all three patient sampl

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Table 5: Column diagram from flow cytometry data of osteoblasts. Columns illustrate mean surface marker expression levels with standard deviation error bars of all three patient samples

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Histologic analysis of the recovered bone cores revealed signs of bone formation and remodeling. The β-TCP granules were interconnected by bridges of vital newly formed bone [Figure 26] and [Figure 27].
Figure 26: Hematoxylin and eosin stained section of bone from case 2 obtained from dental implant drilling site showing healthy bone (original magnification, ×200)

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Figure 27: Hematoxylin and eosin stained section of bone from case 3 obtained from titanium mesh removal site showing healthy woven bone and areas of angiogenesis (original magnification, ×400)

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  Discussion Top

The degree of involvement of computer-aided design (CAD) and additive manufacturing progressively increased over the time in which the three cases were treated. In the first case an additive manufactured medical skull model was used preoperatively to guide pre-bending of patient specific hardware including a generic reconstruction plate and titanium mesh. In the two cases that followed, custom-made reconstruction plates requiring no bending as well as resection jigs were made using CAD software, and the practitioners became more confident in using these tools. For example, in case 1 only a pre-bent generic reconstruction plate guided by the contours of the stereolithic skull was used. In case 2, the custom-made reconstruction plate made with CAD was used together with a pre-bent generic plate as the surgeons were not confident in the load bearing ability of solely the custom-made plate. The generic plate was omitted as redundant in case 3, once a finite element analysis model [44],[45],[46] specific for these custom-made plates became available to better predict the loading and stress distribution of the computer designed hardware. The accuracy of such CAD-computer aided manufacturing (CAD-CAM) based reconstructions using virtual surgical planning have recently been determined. [47]

This report describes successful mandibular reconstruction in a series of three patients using the approach of in situ ossification with GMP level ASCs [36] as one non-morbid source of autogenous mesenchymal stem cells (MSCs). In order to appreciate fully the cell source used, thorough in vitro analysis was implemented [Figure 21], [Figure 22], [Figure 23], [Figure 24], [Figure 25].

In this cohort, there was individual variability in cell numbers available for implantation from passages three to four as the average cell number was 10,240,000 cells with a range of 4,720,000-16,000,000 cells. The characterization data of the ASCs expanded in autologous serum correspond to previous results for ASCs, [36],[48] with positive expression of certain markers substantiating the mesenchymal origin of cells and negative expression of other markers suggesting the hematopoietic and angiogenic origin of cells. [48],[49] Nevertheless, very few reports have been published regarding the surface marker expression of ASC expanded in autologous serum. [14],[22],[36],[50] For the markers CD11a, CD54, CD80, and CD86 no results have previously been reported on ASCs cultured in human serum. The multipotency analyzes verified the stem cell capacities of the ASCs of all three patients. To our knowledge, the marker profile on culture expanded osteoblasts from core bone samples have not been reported previously. The surface markers show strong expression of mesenchymal markers as well as moderate to low expression of several angiogenic and osteogenic markers.

The in vitro and in vivo bone forming capacity of ASCs in combination with various scaffold materials, including β-TCP, has been reported by our group and others. [14],[22],[36],[51],[52] Furthermore, the use of osteogenic growth factor rhBMP-2 might have facilitated ossification. [51],[52] Nevertheless, contradictory reports have also been published, including reports of BMP-2 possibly not promoting osteogenic differentiation of human MSCs. [53],[54],[55] However, the transplanted ASCs produce cytokines and chemokines that act as homing signals for endogenous stem cells and progenitor cells to the site of injury. [56] The factors secreted by the ASCs and the osteoconductivity of β-TCP act synergistically towards producing a well-ossified construct. [56]

Titanium hardware was chosen over resorbable materials because at the time the cases were treated, the track record of resorbable hardware for major mandibular reconstruction was still lacking. Since the authors decided to use granules rather than a solid block, a containment mesh was required to house the scaffold. The authors were concerned that with the thin soft tissue coverage of the mandibular alveolus, the wound might not tolerate the additional pH drop resulting from degradation of a resorbable polymer mesh containing polylactic acid.

Titanium mesh was problematic in all three cases with thin soft tissue coverage and each of the three patients required resection of the superior borders of the mesh. For this reason the authors would prefer to use a thin custom-made passive fitting resorbable mesh once a suitable tested version becomes available. The authors considered many resorbable scaffolds such as bioactive glass but chose β-TCP granules because of its more rapid resorption profile. [23],[24],[25],[26] In the future, the authors could consider using scaffolds containing carriers which would release bioactive molecules such as growth factors and other drugs.

While both Warnke et al., 2004 [33],[34] and Mesimäki et al., 2009 [22] have reported single cases of mandibular and maxillary defect reconstruction using bone marrow or adipose derived stem cells, the current three cases are unique. Both groups of previous authors used an ectopic muscle pouch site for bone induction.

The current three cases used the same type of GMP level autologous stem cells as did Mesimäki et al., [22] but there was no need for an ectopic bone formation step. Instead the construct was put directly into its final recipient bed from the outset. This obviated the need for an ectopic bone formation site and this protocol has been called in situ bone formation. [32],[36]

Preparation of the recipient site by maximizing the contact of the construct with well vascularized muscle is most important with in situ bone formation. [36] There was no history of prior radiation therapy in these three cases. Unlike the present cases, the patient reported by Mesimäki et al., had a very complicated wound with pre-existing oronasal and oroantral fistulae, requiring soft tissue coverage on two sides. [22] In such complex cases requiring soft tissues in addition to bone, or in cases where there has been prior radiation therapy, these authors feel that it is best to use ectopic ossification as the preferred method over in situ bone formation.

The components of the constructs used in these reconstructions are thought to act synergistically. The ASCs serve as a reservoir of functional stem cells. The rhBMP-2 functions as a signaling molecule inducing osteogenic differentiation within its half-life. The β-TCP bone substitute acts as a scaffold to support osteogenesis and remodeling of bone. The sequence of these events is hopefully accelerated by the collective presence of these three entities.

One major drawbacks of this novel treatment was increased costs to the Finnish healthcare system. The GMP level ASC laboratory preparation costs were 10,000 Euros or approximately 12,000 US dollars for each patient. The cost of the use of planning software and production of additive manufactured saw guides and reconstruction plates was 1,000 euros or 1,200 US dollars more than using commercially available generic reconstruction hardware.

  Conclusion Top

Rapid prototyped saw guides and reconstruction plates combined with tissue engineered constructs containing osteogenic cells, such as GMP level ASCs, osteoinductive factors like rhBMP-2 in combination with a synthetic osteoconductive matrix β-TCP granules, can be used to reconstruct large ameloblastoma resection defects of the mandible. In situ bone formation is an attractive alternative for such large bony defects without a history of radiation therapy or without complicated wounds which require vascularized soft tissue transfers with an ectopic bone formation protocol.

  Acknowledgments Top

The authors wish to acknowledge Sari Kalliokoski, Anna-MaijaHonkala, MiiaJuntunen, TiiaTallinen, Hanna Kankkonen, and AnikkaHakamäki for their valuable contributions which made this work possible. This study has been financially supported by EVO (9L100), the Competitive Research Funding of Tampere University Hospital and the Finnish Funding Agency for Technology and Innovation (TEKES).

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21], [Figure 22], [Figure 23], [Figure 24], [Figure 25], [Figure 26], [Figure 27]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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