Harutsugi Abukawa

Reconstructing Mandibular Defects Using Autologous Tissue-Engineered Tooth and Bone Constructs

PURPOSE Current strategies for jaw reconstruction require multiple operations to replace bone and teeth. To improve on these methods, we investigated simultaneous mandibular and tooth reconstruction, using a Yucatan minipig model. MATERIALS AND METHODS Tooth and bone constructs were prepared from third molar tooth tissue and iliac-crest bone marrow-derived osteoblasts isolated from, and implanted back into, the same pig as an autologous reconstruction. Implants were harvested after 12 and 20 weeks and evaluated by x-ray, ultrahigh-resolution volume computed tomographic (VCT), histological, and immunohistochemical analyses. RESULTS Small tooth structures were identified, and consisted of organized dentin, enamel, pulp, and periodontal ligament tissues, surrounded by new bone. No dental tissues formed in implants without tooth-bud cells, and bone regeneration was observed to a limited extent. Immunohistochemical analyses using tooth-specific and bone-specific antibodies confirmed the identity of regenerated tissues. CONCLUSIONS This pilot study supports the feasibility of tissue-engineering approaches for coordinated autologous tooth and mandible reconstruction, and provides a basis for future improvement of this technique for eventual clinical use in humans.

Tissue engineered hybrid tooth–bone constructs

Proper rehabilitation of craniofacial defects is challenging because of the complexity of the anatomy and the component tissue types. The ability to simultaneously coordinate the regeneration of multiple tissues would make reconstruction more efficient and might reduce morbidity and improve outcomes. The craniofacial complex is unique because of the presence of teeth, in addition to skin, bone, cartilage, muscle, vascular, and neural tissues since teeth naturally grow in coordination with the craniofacial skeleton, our group developed an autologous, tooth-bone hybrid model to facilitate repair of mandibular defects in the Yucatan minipig. The hybrid tooth-bone construct was prepared by combining tooth bud cell-seeded scaffolds with autologous iliac crest bone marrow derived stem cell-seeded scaffolds, which were transplanted back into surgically created mandibular defects in the same minipig. The constructs were harvested after 12 and 20 weeks of growth. The resulting bone/tooth constructs were evaluated by X-ray, ultra high-resolution volume computed tomography (VCT), histological, immunohistochemical analyses, and transmission electron microscopy (TEM). The observed formation of small tooth-like structures consisting of organized dentin, enamel, pulp, cementum, periodontal ligament, and surrounded by regenerated alveolar bone, suggests the feasibility for regeneration of teeth and associated alveolar bone, in a single procedure. This model provides an accessible method for future clinical applications in humans.

Osteoclastogenesis on tissue-engineered bone.

Bone remodeling plays an important role in bone function. To date, bone tissue-engineering research has focused primarily on bone formation from osteoblasts. This study demonstrates that osteoclastogenesis can occur on a mineralized polymer scaffold. Porcine bone marrow-derived mesenchymal stem cells (pMSCs) and hematopoietic cells were isolated from the bone marrow of Yucatan minipigs (n = 3) and cultured separately. pMSCs were differentiated into osteoblasts, seeded on porous poly(D,L-lactic-co-glycolic acid) foams, and cultured in a rotating oxygen-permeable bioreactor system. Once the cell-polymer constructs had started to mineralize, the hematopoietic cells were added and cocultured to include osteoclastogenesis. The cultured constructs were evaluated by histochemical and microscopic examination. Our results show that osteoblasts and osteoclasts were successfully differentiated from bone marrow on the scaffolds. This is the first demonstration of osteoclast formation on mineralized polymer surfaces.

Clinical application of a custom-made bioresorbable raw particulate hydroxyapatite/poly-l-lactide mesh tray for mandibular reconstruction

Mandibular reconstruction using particulate cancellous bone and marrow (PCBM) allows functional oral reconstruction. Although ready-made titanium trays are the most common material used in this method, they have some disadvantages such as difficulty in making them form a suitable contour for the defect, and the need for removal. A forged composite of raw particulate hydroxyapatite (HA)/poly-l-lactide (PLLA) is a bioresorbable material that is stronger than pure PLLA and induces bone formation more rapidly. We present two cases successfully treated with custom-made bioresorbable HA/PLLA mesh trays for mandibular reconstruction. A 29-year-old woman with recurrent ameloblastoma and a 66-year-old man with a recurrent keratinized odontogenic tumor of the mandible gave informed consent for this reconstruction technique. Mesh sheets of HA/PLLA were customized by a rapid prototyping method based on computed tomography (CT) data. Marginal resection of the tumor was carried out, and PCBM was harvested from the bilateral posterior iliac crests. PCBM and platelet-rich plasma were transferred to the tray, and the tray was fixed rigidly with HA/PLLA screws. In the second case, dental implants were inserted. There has been no bone resorption for over 2 years since reconstruction in these two cases, and the inserted dental implants have been free from any complications 1 year after loading. The average CT value in Hounsfield units (HU) of the implant sites of two cases was 790. In conclusion, the customized HA/PLLA tray was easily adapted to the mandible, and fine bone quality was obtained. These cases show that this tray system contributed to functional oral rehabilitation with dental implants.

Three-Dimensionally Printed Polycaprolactone and β-Tricalcium Phosphate Scaffolds for Bone Tissue Engineering: An In Vitro Study

PURPOSE The purpose of this study was to evaluate porcine bone marrow-derived progenitor cell (pBMPC) proliferation and penetration into a novel 3-dimensionally printed scaffold. MATERIALS AND METHODS Four different tissue engineering scaffolds to evaluate pBMPC proliferation and penetration were examined. Scaffolds were fabricated from polycaprolactone (PCL) or the combination of β-tricalcium phosphate (β-TCP) and PCL (50:50), with 2 separate channel sizes (1 mm [small (S)] vs 2 mm [large (L)]). Scaffolds were fabricated into 20 × 20 × 7-mm blocks by use of a TheriForm machine (Integra Life Sciences, Akron, OH). Four groups of scaffolds were examined for pBMPC proliferation and penetration: group 1, β-TCP/PCL S; group 2, β-TCP/PCL L; group 3, PCL S; and group 4, PCL L. Nonparametric mean (Kruskal-Wallis) and multiple comparisons tests were used to compare the 4 groups. RESULTS No shrinkage or deformation was noted in any of the scaffold groups after 2 weeks of culture. Mean surface cell counts ranged from 13.4 to 87.8 cells/0.57 mm(2), with group 1 (β-TCP/PCL S) having statistically significantly higher counts than the other groups (P < .001). Mean interior cell counts ranged from 10.9 to 75.6 cells/0.57 mm(2), with group 1 having the greatest interior cell count (P < .001). Total collagen formation ranged from 0.2% to 86%, with group 1 having the highest collagen formation (P < .001). CONCLUSIONS The 3-dimensionally printed scaffold (β-TCP/PCL) with 1-mm channels showed greater cellular proliferation, penetration, and collagen formation after a 2-week in vitro culture than the other scaffolds evaluated. β-TCP/PCL S scaffolds warrant further evaluation for bone tissue engineering in vivo.

Effect of ibuprofen on osteoblast differentiation of porcine bone marrow-derived progenitor cells.

PURPOSE Nonsteroidal anti-inflammatory drugs are commonly prescribed to reduce inflammation and pain. However, little is known about the direct effect of these drugs on the differentiation of bone marrow-derived progenitor cells into osteoblasts. The purpose of this study was to determine the effect of ibuprofen on osteoblast differentiation and proliferation in a minipig model. MATERIALS AND METHODS Bone marrow was aspirated from the minipig ilium, and porcine bone marrow-derived progenitor cells (pBMPCs) were isolated and expanded in standard culture medium. The pBMPCs were replated and differentiated into osteoblasts by use of osteogenic supplements (OS). Five groups were studied: negative control--pBMPCs in standard medium only; positive control--pBMPCs, standard culture medium, and OS; and 3 experimental groups--pBMPCs, standard culture medium, OS, and ibuprofen added in doses of 0.1, 1.0, and 3.0 mmol/L. Cell cultures were evaluated quantitatively by alkaline phosphatase (ALP) stain, von Kossa stain, and deoxyribonucleic acid (DNA) content. RESULTS pBMPCs cultured with OS and low-dose ibuprofen (0.1 mmol/L) showed ALP stain, von Kossa stain, and DNA content similar to pBMPCs cultured in OS (positive control). pBMPCs cultured in higher doses of ibuprofen (1.0 and 3.0 mmol/L) produced significantly less positive staining of ALP and von Kossa and decreased DNA content. CONCLUSION The results indicate that high-dose ibuprofen has a deleterious effect on pBMPC differentiation into osteoblasts whereas low-dose ibuprofen does not. The low dose of 0.1 mmol/L is the typical serum level when prescribed for clinical use.

Cyclo-Oxygenase–2 Expression Is Associated With Vascular Endothelial Growth Factor C Expression and Lymph Node Metastasis in Oral Squamous Cell Carcinoma

PURPOSE Cervical lymph node metastasis in oral squamous cell carcinoma (OSCC) is recognized as a poor prognostic factor, although its mechanism remains unclear. Recently, cyclo-oxygenase-2 (COX-2) level has been found to correlate highly with vascular endothelial growth factor C (VEGF-C) and lymph node metastasis, as in other solid tumors. However, there has been no report of this correlation in OSCC. Therefore, the aim of this study was to investigate whether COX-2 immunohistochemical expression in OSCC was associated with VEGF-C expression, histopathologic parameters, and lymph node metastasis. MATERIALS AND METHODS Lymphatic vessel density, VEGF-C, and COX-2 immunohistochemical expression were examined pathologically in 60 specimens of invasive OSCC. Relations of histopathologic parameters to lymph node metastasis were analyzed. RESULTS Expression levels of VEGF-C and COX-2 and lymphatic vessel density in the lymph node metastatic group were significantly higher than in the nonmetastatic group (P < .01). A significant correlation was found between the expression levels of VEGF-C and COX-2 (r = 0.512; P < .001). COX-2 expression was significantly related to lymph node metastasis (P = .004) and VEGF-C expression (P = .005). Univariate analysis showed that survival time was impaired by higher COX-2 and VEGF-C expression levels. Multivariate survival analysis showed that COX-2 expression was an independent prognostic factor. CONCLUSION This study showed that VEGF-C expression was upregulated by COX-2 in OSCC. High VEGF-C expression appears to promote peritumoral lymphangiogenesis. These data indicated that lymph node metastasis is promoted by COX-2 and VEGF-C in OSCC.

Mandibular reconstruction using a tray with particulate cancellous bone and marrow and platelet-rich plasma by an intraoral approach.

PURPOSE To evaluate the possibility of immediate mandibular reconstruction using particulate cancellous bone and marrow (PCBM), platelet-rich plasma (PRP), and a tray, we compared the postsurgical infection rate and bone formation in patients who received mandibular reconstruction with this method using either an intraoral or extraoral approach. PATIENTS AND METHODS We conducted a retrospective study of a series of 18 patients who underwent the mandibular reconstruction procedure using a mesh tray with PCBM and PRP, all performed by 1 surgeon. These cases were further divided into those treated by the intraoral approach and those treated by the extraoral approach. Clinical data, postoperative bone formation, and complications in the 2 groups were evaluated. The χ(2) examination and the Mann-Whitney U test were used for statistical analysis. RESULTS We could not detect any statistically significant differences in clinical data between the 2 groups, except for the timing of reconstruction. There were postoperative complications such as wound dehiscence and tray exposure, as well as infection of the reconstructed bone. The overall complication rate of the recipient sites in the intraoral group was 30% (3 of 10), whereas in the extraoral group, it was 0%. However, satisfactory bone formation was seen in all cases in the intraoral group (100% [10 of 10]) but only 87.5% (7 of 8) in the extraoral group. CONCLUSION We conclude that mandibular reconstruction using a tray with PCBM and PRP is a safe and reliable method for cases of benign tumor and trauma, even if immediate reconstruction is performed by an intraoral approach.

Growth factor directed chondrogenic differentiation of porcine bone marrow-derived progenitor cells.

BackgroundDespite advances in surgical technique, reconstruction of a mandibular condyle still causes significant donor-site morbidity. The purpose of this study was to compare the effect of 3 different growth factors and define optimal cell culture conditions for bone marrow-derived progenitor cells to differentiate into chondrocytes for mandibular condyle reconstruction. MethodsPorcine bone marrow-derived progenitor cells (pBMPCs) were cultured as a pellet for 2, 3, and 4 weeks under the following conditions: group 1, TGF-&bgr;3 + standard medium; group 2, TGF-&bgr;3 + BMP-2 + standard medium; group 3, TGF-&bgr;3 + IGF-1 + standard medium; and group 4, TGF-&bgr;3 + BMP-2 + IGF-1 + standard medium. Chondrogenic differentiation was evaluated using 3 lineage differentiation markers. ResultsThe mean type II collagen positive area increased over weeks 2, 3, and 4 in group 4 compared to all the other groups (ANOVA; P = 0.005). At week 4, there was significantly greater type II collagen production in group 4 compared to all the other groups (ANOVA; P = 0.003). The medium in group 4 produces the greatest amount of cartilage when compared to groups 1, 2, and 3, and that 4 weeks produces the greatest amount of type II collagen. ConclusionsThe results of this study indicate that the most efficacious medium for chondrogenic differentiation of pBMPCs was group 4 medium and the most type II collagen was produced at 4 weeks.

Application of custom-made bioresorbable raw particulate hydroxyapatite/poly-l-lactide mesh tray with particulate cellular bone and marrow and platelet-rich plasma for a mandibular defect: Evaluation of tray fit and bone quality in a dog model

The purpose of this study was to evaluate tray fit and bone quality of particulate cancellous bone and marrow (PCBM) mandibular reconstruction using custom-made bioresorbable forged composites of a raw particulate hydroxyapatite/poly-L-lactide (HA/PLLA) tray in a dog model. Mesh sheets of HA/PLLA were formed in a tray shape according to the mandible stereolithographs of 14 beagle dogs. Platelet-rich plasma (PRP) was obtained from venous blood, and PCBM was harvested from the iliac crest. Bone defects were made bilaterally on the lower borders of the mandible. The PCBM and PRP were mixed and compressed into the defects and a custom-made HA/PLLA or a manually adopted titanium tray was fixed by screws. Tray fit and bone qualities were evaluated using computed tomography, microfocus computed tomography and confocal laser scanning microscopy. In buccal side, there is no significant difference with tray fit between the HA/PLLA and the titanium type, but in lingual side, it was better in the HA/PLLA type than that of the Ti type. Bone volume fraction (BV/TV) had markedly increased on the HA/PLLA side at 12 months. In conclusion, the custom-made HA/PLLA tray was easily and accurately adapted to the mandible, and had achieved sufficient bone quality by 12 months.