The Experimental Study Of Large Scale Tissue Engineering Bone Construction

When patients experience with large jaw bone defect caused by various reasons, it is very important to reconstruct bone tissue defect and restore facial appearance and mastication function for the improvement of the clinical survival quality. Bone defects of more than 6 to 8 mm can generally referred to as large scale bone defects. Clinical appropriate treatment should balance the defect area, the disease etiology, and the severity. Current clinical therapies for alveolar bone loss are limited to the use of artificial bone substitutes and auto genic and allogenic bone grafts. However, a number of limitations of the use of these conventional methods have led to the search for alternative approaches such as stem cell therapy and tissue engineering to tackle this clinical challenge. Compared to other methods, the use of tissue engineering bone has the advantages of possible large bone defect repair, no secondary injury, less immune problems, and no spread of disease, which opens a promising way to the objective of no damage bone defect repair.It has been suggested that tissue engineering requires four basic elements:stem cells, extracellular scaffolds, growth factors, and mechanical stimulation. Bone defect reparation by traditional tissue engineering method was as follows. Stem cells were initially seeded on scaffolds. After a certain period in vitro culture, the composites were transplanted in vivo for bone reconstruction. But tissue engineering bone have no blood supply system, which leads to the supply of nutrients and the discharge of wastes mainly rely on the limited diffusion and permeation. The disadvantages hinder large scale tissue engineering bone construction. So at present stage, bone tissue engineering methods are limited to the reparation of small size, thin bone defect in animal experiments.We targeted on the obstacles to construction of large scale tissue engineering bone through the following aspects of research:1) the in-house made bioreactor simulating oral mechanical environment, to promote the organization nutrition supply, waste removal, and to stimulate cell osteogenic differentiation; 2) the latest stem cell techniques, namely human induced pluripotent stem cells (hiPSCs), were used in the oral and maxillofacial fields for bone formation; 3) the synthesis of new composites hydroxyapatite/chitosan/gelatin (HCG) scaffolds. The tissue engineering bone osteogenic induction activity was improved by changing the nano-hydroxyapatite (nHA) crystal structure, and by increasing the nHA content in the HCG composites; 4) vascularization and construction of large scale tissue engineering bone, which was divided into 3 parts:scaffolds production, in vitro experiments, and in vivo test. Scaffolds production included individualized bone defect computer reconstruction and the internal pipes printed by polylactic acid (PLA); in the in vitro experiments, stem cells were seeded and nutrition was perfumed through internal pipes; and the composites were then subcutaneously transplanted into immunocompromised mice in vivo. They are completed through the following four experiments.Experiment 1:Tooth loss often results in alveolar bone resorption because of lack of mechanical stimulation. Thus, the mechanism of mechanical loading on stem cell osteogenesis is crucial for alveolar bone regeneration. We have investigated the effect of mechanical loading on osteogenesis in human dental pulp stromal cells (hDPSCs) in a novel in vitro model. Briefly,1×10/ hDPSCs were seeded into 1 ml 3% agarose gel in a 48-well plate.A loading tube was then placed in the middle of the gel to mimic tooth-chewing movement (1 Hz,3×30 min per day, n=3). A non-loading group was used as a control. At various time points, the distribution of live/dead cells within the gel was confirmed by fluorescence markers and confocal microscopy. The correlation and interaction between the factors (e.g. force, time, depth and distance) were statistically analysed. The samples were processed for histology and immunohistochemistry. After 1-3 weeks of culture in the in-house-designed in vitro bioreactor, fluorescence imaging confirmed that additional mechanical loading increased the viable cell numbers over time as compared with the control. Cells of various phenotypes formed different patterns away from the reaction tube. The cells in the middle part of the gel showed enhanced alkaline phosphatase staining at week 1 but reduced staining at weeks 2 and 3. Additional loading enhanced Sirius Red and type I collagen staining compared with the control. We have thus successfully developed a novel in-house designed in vitro bioreactor mimicking the biting force to enhance hDPSC osteogenesis in an agarose scaffold and to promote bone formation and/or prevent bone resorption.Experiment 2:Human embryonic stem cells and adult stem cells have always been the cell source for bone tissue engineering. However, their limitations are obvious, including ethical concerns and/or a short lifespan. The use of hiPSCs could avoid these problems. nHA is an important component of natural bone and bone tissue engineering scaffolds. However, its regulation on osteogenic differentiation with hiPSCs from human gingival fibroblasts (hGFs) is unknown. The purpose of the present study was to investigate the osteogenic differentiation of hiPSCs from patient-derived hGFs regulated by HCG scaffolds with different nHA ratios, such as HCG-111 (1 wt/vol% nHA) and HCG-311 (3 wt/vol% nHA). First, hGFs were reprogrammed into hiPSCs, which have enhanced osteogenic differentiation capability. Second, HCG-111 and HCG-311 scaffolds were successfully synthesized. Finally, hiPSC/HCG complexes were cultured in vitro or subcutaneously transplanted into immunocompromised mice in vivo. The osteogenic differentiation effects of two types of HCG scaffolds on hiPSCs were accessed for up to 12 weeks. The results showed that HCG-311 increased osteogenic-related gene expression of hiPSCs in vitro proved by quantitative real-time polymerase chain reaction, and hiPSC/HCG-311 complexes formed much bone-like tissue in vivo, indicated by cone-beam computed tomography imaging, H&E staining, Masson staining, and RUNX-2, OCN immunohistochemistry staining. In conclusion, our study has shown that osteogenic differentiation of hiPSCs from hGFs wasimproved by HCG-311. The mechanism might be that the nHA addition stimulates osteogenic marker expression of hiPSCs from hGFs. Our work has provided an innovative autologous cell-based bone tissue engineering approach with soft tissues such as clinically abundant gingiva.Experiment 3:nHA is an important component of human bone and bone tissue engineering scaffolds. A plethora of bone tissue engineering scaffolds have been synthesized so far, including nHA/chitosan/gelatin (nHA/CG) scaffolds; and for seeding cells, stem cells, especially induced pluripotent stem cells (iPSCs), have been a promising cell source for bone tissue engineering recently. However, the influence of different HA nano-particle morphologies on the osteogenic differentiation of hiPSCs from hGFs is unknown. The purpose of this study was to investigate the osteogenic differentiation of hiPSCs from hGFs seeded on nHA/CG scaffolds with 2 shapes (rod and sphere) of nHA particles. Firstly, hGFs isolated from discarded normal gingival tissues were reprogrammed into hiPSCs. Secondly, hiPSCs were seeded on rod-like nHA/CG (rod-nHA/CG) and sphere-shaped nHA/CG (sphere-nHA/CG) scaffolds respectively and then cell/scaffold complexes were cultured in vitro. Scanning electron microscope, hematoxyline and eosin (HE) staining, Masson’s staining, and quantitative real-time polymerase chain reaction techniques were used to examine hiPSC morphology, proliferation, and differentiation on rod-nHA/CG and sphere-nHA/CG scaffolds. Finally, hiPSCs composited with 2 kinds of nHA/CG were transplanted in vivo in a subcutaneous implantation model for 12 weeks; pure scaffolds were also transplanted as a blank control. HE, Masson’s, and immunohistochemistry staining were applied to detect new bone regeneration ability. The results showed that sphere-nHA/CG significantly increased hiPSCs from hGF proliferation and osteogenic differentiation in vitro. hiPSCs and sphere-nHA/CG composites generated large bone, whereas hiPSCs and rod-nHA/CG composites produced tiny bone in vivo. Moreover, pure scaffolds without cells almost produced no bone. In conclusion, our work provided a potential innovative bone tissue engineering approach using clinically discarded gingival tissues and sphere-nHA/CG scaffolds.Experiment 4:For the objectives related to large jaw defects reconstruction based on bone tissue engineering methods, we created the large scaffolds with internal pipes by personalized computer aided design and 3D printing controlled manufacturing, osteogenic differentiation was then enhanced by inside cell seeding, nutrition perfusion. Firstly, personalized large defect mold was printed according to patient’s cone-beam computed tomography (CBCT) data. The vascular branches were designed and printed by PLA. Secondly, patient’s defect based large HCG scaffolds with inside pipe branches were fabricated by lyophilization. Thirdly, through the internal pipes, hiPSCs were seeded inside scaffolds in vitro. Finally, the composites were then subcutaneously transplanted into immunocompromised mice in vivo. Afterwards, nutrition was perfumed in experiment group. The results showed personalized bone defect based large individual HCG scaffolds with internal pipes were created. Inside scaffold cell seeding and nutrition perfusion were better for hiPSCs proliferation and osteogenic differentiation, proved by HE staining and bone density measurement of CBCT data. Our findings provided an innovative finding for bone regeneration, into which more investigations should be done before clinical use.