Application of 3D-printed Polycaprolactone Scaffolds for both Bone and Neural Tissue Regeneration

Abstract

Background: There are still not investigated two different tissue regenerations at the same time with different scaffolds. Mandibular bone and mandibular nerve branch, such as inferior alveolar nerve tissue regeneration, are important issues and still face challenges caused by injuries such as extraction of impacted third molars, dental implants, and also cancer. Recent reports demonstrated that FDA-approved polycaprolactone scaffolds are effective for tissue regeneration. We hypothesized that bone marrow stem cell-loaded and modified 3D-printed polycaprolactone scaffolds could regenerate both mandibular bone and inferior alveolar nerves at the same time. The purposes of this study were as follows: 1) to investigate the mesh-structured 3D-printed PCL scaffolds coated with beta-tricalcium phosphate for bone tissue engineering in vitro for osteoblast-like cell regeneration. 2) to investigate the 3D-printed and poly-D-lysine-coated PCL scaffolds with a half-tubular array for neuron-like cell regeneration in vitro. 3) to investigate the mesh-structured 3D-printed polycaprolactone scaffolds coated with beta-tricalcium phosphate and tube 3D-printed polycaprolactone scaffolds with inner diameter half tubular array coated with chitosan and loaded with rabbit bone marrow stem cells for both mandibular bone and mandibular nerve regeneration in rabbits at the same time. 4) to investigate the mesh-structured 3D-printed polycaprolactone scaffolds coated with beta-tricalcium phosphate and tube 3D-printed polycaprolactone scaffolds coated with chitosan and seeded with pig bone marrow stem cells for both mandibular bone and inferior alveolar nerve regeneration in pigs at the same time. Methods: In vitro assays of viability and differentiation were carried out on a 3D-printed polycaprolactone scaffold of PC12 and MG-63 cells. The MTT assay is used for assessing cell viability. ALP is a mineralization assay used for MG-63 cell differentiation. Neuronal differentiation induced by nerve growth factor and expression of PC12 cell neuronal differentiation markers such as β3-tubulin and glial fibrillary acidic protein were carried out by immunofluorescence. In addition, experiments were extended to continue with in vivo experiments. Bone marrow stem cells were isolated from both rabbits and pigs, and seeded in the 3D-printed polycaprolactone scaffold for transplantation into the bones of the left mandible ramus side and mandibular nerve of the rabbit, the bone of the left mandible body side, and the inferior alveolar nerve of the pig, respectively. Created defects only in the bone of the mandibular ramus side of rabbits (right mandible) and the right mandible of the pigs were transplanted with acellular both bone and neural scaffolds as the control group. Micro-CT was performed for observation of bone-critical defect reconstruction. Cone beam computerized tomography irradiation was used for dynamic assessment. Four and eight weeks post-operation, animals were euthanized, and the mandibles of animals were sectioned and fixed for histological observation with Stevenel‘s blue, alizarin red , hematoxylin, and eosin staining and clinical findings. A confocal microscope was used to evaluate bone mineralization. Immunofluorescence was used for neuronal detection in the cellular 3D-printed polycaprolactone scaffold transplantation area. Both bone and neuronal polycaprolactone scaffolds from rabbit and pig experiments were fabricated with a fused deposition modeling 3D-printer. Results: The result shows that 3D-printed mesh polycaprolactone scaffolds coated with beta-tricalcium phosphate for bone regeneration and poly-D-lysine coated polycaprolactone scaffold with a 200 μm inner diameter of a half tubular array for neural regeneration were effective for both osteoblast like MG-63 and neuron like PC12 cells adhesion, growth and differentiation in vitro. Rabbit and pig bone marrow stem cell- loaded mesh-structured 3D-printed polycaprolactone dip coated with beta-tricalcium phosphate and tube structured 3D-printed and chitosan coated polycaprolactone scaffold with a 200 μm inner diameter of half tubular array implantation and reconstruction of the bone and nerve defects, stage of the bone mineralization, remodeling, and clinical finding results in the experimental group with cellular scaffolds were significantly higher resulted than the control group with a cellular scaffold of pig and without scaffolds of rabbit. Neuronal regeneration finding is not clear, however, rabbit bone marrow stem cell loaded and chitosan coated polycaprolactone scaffold areas have found some axons and determined by neurofilament-medium staining. Besides, we could not find neuronal regeneration results in pig animal experiments. Conclusion: This study is the first report demonstrating that 3D-printed mesh polycaprolactone scaffold dip coated with beta-tricalcium phosphate, poly-D-lysine coated polycaprolactone scaffolds with different inner diameters of half tubular arrays, and chitosan coated, different-sized tube polycaprolactone scaffold with a 200 μm inner diameter of half tubular array was capable of both bone and neuronal regeneration in vitro and in vivo. Rabbit bone marrow stem cell-loaded mesh polycaprolactone scaffolds were dip-coated with beta-tricalcium phosphate, and chitosan coated tube polycaprolactone scaffolds with a 200 μm inner diameter of a half tubular array was regenerated with both rabbit mandibular bone and mandibular nerve, respectively. This study was also the first report for in vivo observation of both bone (mesh PCL coated with tricalcium phosphate) and nerve (tube PCL with an inner diameter of a half tubular array coated with chitosan) scaffolds assembled with each other, such as “LEGO TOY” to regenerate both mandibular bone and mandibular and inferior alveolar nerve tissue at the same time in small and large experimental animals.

Background: There are still not investigated two different tissue regenerations at the same time with different scaffolds. Mandibular bone and mandibular nerve branch, such as inferior alveolar nerve tissue regeneration, are important issues and still face challenges caused by injuries such as extraction of impacted third molars, dental implants, and also cancer. Recent reports demonstrated that FDA-approved polycaprolactone scaffolds are effective for tissue regeneration. We hypothesized that bone marrow stem cell-loaded and modified 3D-printed polycaprolactone scaffolds could regenerate both mandibular bone and inferior alveolar nerves at the same time. The purposes of this study were as follows: 1) to investigate the mesh-structured 3D-printed PCL scaffolds coated with beta-tricalcium phosphate for bone tissue engineering in vitro for osteoblast-like cell regeneration. 2) to investigate the 3D-printed and poly-D-lysine-coated PCL scaffolds with a half-tubular array for neuron-like cell regeneration in vitro. 3) to investigate the mesh-structured 3D-printed polycaprolactone scaffolds coated with beta-tricalcium phosphate and tube 3D-printed polycaprolactone scaffolds with inner diameter half tubular array coated with chitosan and loaded with rabbit bone marrow stem cells for both mandibular bone and mandibular nerve regeneration in rabbits at the same time. 4) to investigate the mesh-structured 3D-printed polycaprolactone scaffolds coated with beta-tricalcium phosphate and tube 3D-printed polycaprolactone scaffolds coated with chitosan and seeded with pig bone marrow stem cells for both mandibular bone and inferior alveolar nerve regeneration in pigs at the same time. Methods: In vitro assays of viability and differentiation were carried out on a 3D-printed polycaprolactone scaffold of PC12 and MG-63 cells. The MTT assay is used for assessing cell viability. ALP is a mineralization assay used for MG-63 cell differentiation. Neuronal differentiation induced by nerve growth factor and expression of PC12 cell neuronal differentiation markers such as β3-tubulin and glial fibrillary acidic protein were carried out by immunofluorescence. In addition, experiments were extended to continue with in vivo experiments. Bone marrow stem cells were isolated from both rabbits and pigs, and seeded in the 3D-printed polycaprolactone scaffold for transplantation into the bones of the left mandible ramus side and mandibular nerve of the rabbit, the bone of the left mandible body side, and the inferior alveolar nerve of the pig, respectively. Created defects only in the bone of the mandibular ramus side of rabbits (right mandible) and the right mandible of the pigs were transplanted with acellular both bone and neural scaffolds as the control group. Micro-CT was performed for observation of bone-critical defect reconstruction. Cone beam computerized tomography irradiation was used for dynamic assessment. Four and eight weeks post-operation, animals were euthanized, and the mandibles of animals were sectioned and fixed for histological observation with Stevenel‘s blue, alizarin red , hematoxylin, and eosin staining and clinical findings. A confocal microscope was used to evaluate bone mineralization. Immunofluorescence was used for neuronal detection in the cellular 3D-printed polycaprolactone scaffold transplantation area. Both bone and neuronal polycaprolactone scaffolds from rabbit and pig experiments were fabricated with a fused deposition modeling 3D-printer. Results: The result shows that 3D-printed mesh polycaprolactone scaffolds coated with beta-tricalcium phosphate for bone regeneration and poly-D-lysine coated polycaprolactone scaffold with a 200 μm inner diameter of a half tubular array for neural regeneration were effective for both osteoblast like MG-63 and neuron like PC12 cells adhesion, growth and differentiation in vitro. Rabbit and pig bone marrow stem cell- loaded mesh-structured 3D-printed polycaprolactone dip coated with beta-tricalcium phosphate and tube structured 3D-printed and chitosan coated polycaprolactone scaffold with a 200 μm inner diameter of half tubular array implantation and reconstruction of the bone and nerve defects, stage of the bone mineralization, remodeling, and clinical finding results in the experimental group with cellular scaffolds were significantly higher resulted than the control group with a cellular scaffold of pig and without scaffolds of rabbit. Neuronal regeneration finding is not clear, however, rabbit bone marrow stem cell loaded and chitosan coated polycaprolactone scaffold areas have found some axons and determined by neurofilament-medium staining. Besides, we could not find neuronal regeneration results in pig animal experiments. Conclusion: This study is the first report demonstrating that 3D-printed mesh polycaprolactone scaffold dip coated with beta-tricalcium phosphate, poly-D-lysine coated polycaprolactone scaffolds with different inner diameters of half tubular arrays, and chitosan coated, different-sized tube polycaprolactone scaffold with a 200 μm inner diameter of half tubular array was capable of both bone and neuronal regeneration in vitro and in vivo. Rabbit bone marrow stem cell-loaded mesh polycaprolactone scaffolds were dip-coated with beta-tricalcium phosphate, and chitosan coated tube polycaprolactone scaffolds with a 200 μm inner diameter of a half tubular array was regenerated with both rabbit mandibular bone and mandibular nerve, respectively. This study was also the first report for in vivo observation of both bone (mesh PCL coated with tricalcium phosphate) and nerve (tube PCL with an inner diameter of a half tubular array coated with chitosan) scaffolds assembled with each other, such as “LEGO TOY” to regenerate both mandibular bone and mandibular and inferior alveolar nerve tissue at the same time in small and large experimental animals.