| Research background Tissue engineering:an effective solution to bone defects.Various degrees of bone defects caused by injury, bone tumors and osteonecrosis are very common in clinic. However, due to the limitation of bone graft sources, especially for large bone defects, the treatment for bone defects remains a big problem. The development of tissue engineering has provided new breakthroughs for bone defect treatment. Research hotspots in the field of orthopedics have continuously search for stem cells with high proliferation and differentiation ability, and an abundant source. This field of study has also ventured in searching for stem cells with osteogenesis factors that could extensively promote proliferation and differentiation, and the best new scaffold materials that could exhibit an optimal repair effect for bone defects. Osteogenesis results from the combined action of osteogenesis stem cells, surrounding scaffolds and regulatory osteogenic factors. It is of vital importance to obtain stable osteogenic precursor cells (seed cells) that can induce osteogenic differentiation. The establishment of embryonic stem cells (ESCs) has opened a door for exploring the regulation mechanism of stem cell biology and evaluating its potential in the treatment of many kinds of diseases. However, ESC research has encountered many social and ethical problems. Bone marrow mesenchymal stem cells (BMSCs) have been once considered as the best source of cells in bone tissue engineering. However, during the process of clinical application, autologous bone marrow stem cell therapy requires the extraction of a large number of cells from patients; which is an invasive process. Moreover, limited extraction volume (maximum of 40 ml, approximately 1.2×109 nucleated cells) has severely restrained its applications.Adipose-derived stem cells (ADSCs)Recent studies have confirmed that ADSCs could be isolated and cultured. Similar to mesenchymal stem cells (MSCs), ADSCs are derived from mesoderm and ectoderm at their early development stage. They can differentiate into cells of various germ sources including cartilage cells, osteoblasts and fat cells. They have the capacity for self-renewal and multi-directional differentiation potential, and are featured by bone marrow MSCs. Two-hundred ml of adipose tissue (1×106 ADSCs) could be obtained from adults through vacuum liposuction under local anesthesia. This is approximately 40 times the volume of BMSCs in 40 ml of bone marrow. ADSCs have advantages such as easily available, small trauma, high content, proliferative capability, and no immune rejection. ADSCs can be obtained from autologous cells without ethical controversy, making them a type of ideal seed cells in tissue engineering. Studies have shown that ADSCs can easily import foreign genes in vitro, which make them alternative target gene carriers in gene therapy.Bone morphogenetic protein-9 (BMP-9)BMP-9, a member of the transforming growth factor-β (TGF-β) superfamily, is similar in structure and highly conservative. BMP-9 can effectively induce osteogenic differentiation in vivo and in vitro. The study of Kang et al. has demonstrated that BMP-9 is one of the strongest members of the TGF-β superfamily, which induce osteogenic differentiation in MSCs. BMP-9, a kind of important regulatory factor of osteogenic differentiation, is more effective in inducing osteogenic differentiation in traditional applications, compared to BMP-2,-6 and-7. These results reveal a significant breakthrough in the promotion of osteogenic differentiation in MSCs and in bone tissue engineering with gene strengthening osteogenic activity.Icariin (ICA)In recent years, Chinese authors found that ICA, a traditional Chinese medicine composition, displays an excellent protective effect on osteoporosis when spayed in rats. They can also promote proliferation and osteogenic differentiation in BMSCs cultured in vitro. ICA has a number of advantages, including simple extraction technique, wide sources, low cost, and is a kind of traditional Chinese medicine monomer with great application prospects. Recent study results have confirmed that ICA can induce osteogenic differentiation in MSCs, and can be used to promote repair of bone defects through tissue engineering.Scaffold materials:Nano-hydroxyapatite/collagen (nHAC)Present research hotspots in the area of scaffold materials have utilized the imitation of the compositional and structural characteristics of native bones, as well as the mineralization process and development of bionic bone repair materials, for bone repair. nHAC, a kind of nanocomposite bone obtained by the Department of Materials of Tsinghua University, was constructed according to bionic principles. Approved in April 2005, this was the first non-medical product sold and applied in the domestic market. nHAC resembles native bones in both microstructure and composition. Furthermore, nHAC have the same pore structure as cancellous bone, and the same hierarchy as native bones. nHAC can be utilized as a good cell carrier, which provides a good microenvironment for cell growth, proliferation and differentiation. Moreover, nHAC-included bone repair materials have demonstrated good performance in the clinical repair of bone defects in animal experiments.Based on the aforementioned information, this project has carried out the following study:(1) the transfection of adipose tissue stem cells with BMP-9 to sustain the local expression of BMP-9; (2) the composition of ICA and nHAC to assemble into a three-dimensional controlled-release scaffold, inducing the repaired area to continuously release inducers and nutrients of cells (epimedium glycoside); (3) the use of a controlled-release scaffold as a carrier of ADSCs, in which the cell scaffold complex (BMP-9 transfected ADSCs+nHAC scaffold complex with ICA) was placed into the body to treat large segmental radius defects in rabbits. These studies included both in vitro and in vivo experiments, and were conducted in the cellular and animal level, respectively. The efficiency of the osteogenic development of ADSCs induced by BMP-9 and ICA was observed. The above study was intended for providing theoretical and experimental evidences for the treatment of bone defects.Objective(1) To establish a simple and efficient separation, passage and culture of rabbit ADSCs in vitro, and confirm its multidirectional and osteogenic differentiation potential. (2) To transfect ADSCs with adenovirus-carrying hBMP-9 (Ad-hBMP-9). This would provide a basis for sustainable ADSCs, the efficient and stable expression of BMP-9, and its subsequent osteogenesis in vivo. (3) To further explore the efficiency of the osteogenesis of allogeneic ADSCs in vivo:using scaffolds composed of allogeneic ADSC-and ICA-modified BMP-9 to repair large segmental radius defects in rabbits.Methods1. The isolation, culture and identification of ADSCs:inguinal hypodermic adipose tissues of New Zealand white rabbits were isolated under aseptic conditions after anesthetic management, cut into fat particles, and was later placed under shock digestion using 0.1% type I collagen enzymes at a constant temperature of 37℃. Basal medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% double resistance (penicillin, streptomycin) were used to adherently cultivate, amplify, purify and passage rabbit ADSCs. Cell morphology and proliferation was observed and evaluated to draw the cell growth curves. Osteogenesis, adipogenesis and chondrogenesis capacity, as well as the expression of surface marker antigens, were analyzed and identified after induction.2. Using adenovirus as a carrier to transfect adipose stem cells with Ad-hBMP-9: adenovirus carried by BMP-9 directly infected P3 ADSCs. Phase contrast microscope and fluorescence microscope were utilized to observe transfection effect and growth morphology. MTT method was used to detect the proliferation of ADSCs transfected by Ad-hBMP-9, in order to draw the growth curve of ADSCs. The expression of BMP-9 mRNA was detected by RT-PCR. The osteogenesis of ADSCs was determined by alkaline phosphatase staining.3. A 15-mm defect model of rabbit radial was established. The experimental animals were divided into seven groups (eight animals in each group, the total number was 56). According to groupings:group A, ADSCs+nHAC+BMP-9+ICA; group B, A ADSCs+nHAC+BMP-9; group C, ADSCs+nHAC+ICA; group D, ADSCs+nHAC; group E, nHAC+ICA; group F, nHAC; group G, blank control (note:BMP-9 expression plays a role via transfection into ADSCs). X-ray detection was used to observe experimental rabbit radius under anesthesia at postoperative day 30,60,90 and 109. These measurements were scored according to the Lane-Sandhu X-ray method. The radius was taken out to implement the overall, biomechanical and histological observation of the defect after the execution of two batches of experimental rabbits at postoperative day 60 and 109. A computer image analysis system was used for quantitative analysis, in order to determine the osteogenesis area ratio of the bone defect regions, and to explore the efficiency of osteogenesis in vivo.Results1. Isolation, culture, identification and differentiation of ADSCsA large number of fibroblasts that appear like rabbit ADSCs were successfully isolated and cultivated by collagenase digestion and the adherent culture method. These cells had good morphology (similar to fibroblasts, appear as a fusiform or polygonal, and grow in clusters) and extensive proliferation ability. ADSCs have the capacity of osteogenesis, adipogenesis and chondrogenesis though culturing in vitro, and induce differentiation. Surface markers of third generation rabbit ADSCs exhibited positive CD34 and CD 105 expression, and a prominent expression of Sca-1; but there was no CD45 and SSEA-1 expression. Furthermore, they have the characteristics of stem cell surface molecules.2. BMP-9 transfection of ADSCs(1) ADSCs can be successfully transfected by gene adenovirus-carrying BMP-9. (2) ADSCs infected by the virus were observed by fluorescence microscopy. The nucleus and cytoplasm of cells revealed green fluorescence with different fluorescence intensity. A large quantity of green fluorescence appeared after 24 hours of transfection. The transfection of ADSCs with Ad-hBMP9 exhibited an efficiency of 30.6 ± 4.2%, according to expression rate statistics after 24 hours of transfection. Fluorescence intensity was increased and enhanced after 48-72 hours. Transfection rate was as high as>80% after seven days. Meanwhile, cells appeared as clusters, cell volume increased, and part of the cells had an oval morphology. ADSCs not infected by the virus revealed green fluorescence when observed under a fluorescence microscope. (3) ADSCs transfected by Ad-hBMP9 demonstrated an identical growth curve with that of normally cultured ADSCs. (4) RT-PCR test results revealed that cell transfection groups display a positive hBMP-9 mRNA expression, while positive stripes were not observed in the non-transfection groups. (5) ALP staining was positive after ADSC transfection. ALP staining revealed a large number of black cells with black particles in the cell cytoplasm on the 7th day after transfection with Ad-hBMP9. These particles appeared bigger with deeper dyeing. Non-transfection groups:a small amount of dark particles are occasionally seen inside the cell with lighter dyeing.3. Animal experiment in vivo(1) X-ray inspection:Group A:Callus was formed in the recipient area at postoperative day 30. At postoperative day 60, the boundary between the callus and end host bone become blurred. Callus density was uneven, and the outline of the cortical bone appeared. Bone defects largely disappeared, while typical bone healing images are observed. Callus had been shaping on postoperative day 90, while the new bone cortex structure become clear and displayed a natural connection with the broken bone. The medullary cavity was basically recanalized, and was finally and completely recanalized at postoperative day 109. The scaffold material was basically degradated. Group B:A small amount of callus was formed at postoperative day 60. Cloud-like high density shadows were observed in bone defects, and these defects remained. Callus continued to shap and revealed a natural connection with the broken bone at postoperative day 109. The medullary cavity was basically recanalized, and the scaffold materials were basically degraded. Group C:Images of callus formation were observed in the bone defect areas at postoperative day 109, and the broken ends were connected. However, defects were still present. The medullary cavity did not pass, and scaffold materials were basically degraded. Group D and Group E:A small amount of callus was formed at postoperative day 90. Cloud-like high density shadows were observed in the bone defects, and these defects remained. Connections between the broken ends was not yet been established at postoperative day 109. Group F:A small amount of callus was formed in the area of the bone defect at postoperative day 109. Small amounts of cloud-like high density shadows appeared, and scaffold materials were basically degraded. Group G:Broken ends of the bone completely hardened without any connection between the radial-end at postoperative day 109, appearing as the nonunion of bone. Moreover, Lane-Sandhu X-ray scoring of bone defect healing revealed that the score in group A was significantly higher compared with the other groups, and groups B and C were significantly better compared with groups D, E, F and G (.P<0.05).(2) Histologic examination:a large quantity of trabecular bone in the hole of the scaffold materials was visible in the bone defects in group A at postoperative day 60, and the new trabecular bone demonstrated a scattered distribution in the bone matrix. In addition, new bone tissues form around the trabecular bone. The bone pit was scattered among these, and crumb-like scaffold materials that endured the degradation were observed. Trabecular bone was formed in the micropore of most scaffold materials in the bone defects in group B, and osteocytes were scattered within the bone matrix of the new trabecular bone. A large quantity of trabecular bone was formed in the bone defects in group A, and bone marrow-like samples appeared between the trabecular bone at postoperative day 109; forming a pulp cavity and leaving the medullary cavity unobstructed. A large quantity of trabecular bones were observed in group B. A large number of new bone tissues in its surroundings were formed, and the medullary cavity was clear. The new bone formation was observed in the bone defects in group C at postoperative day 60 and 109. Groups D, E and F had a small amount of new bone hyperplasia, with scattered osteoid formation. No osteogenesis phenomenon and broken end closing were observed in group G during the whole observation period.In addition, for the use of a semi-quantitative histology slice test to analyze the proportion of the osteogenesis area in various bone defects:the bone area ratio in group A was found to be significantly higher than that in other groups at postoperative day 60 and 109; and the ratio in groups B and C were better than in groups D, E and F (P<0.05). No obvious new bones were formed in group G, and the area ratio was not calculated.(3) Biomechanical measurements:these were obtained from the experimental radius (n=4) at postoperative day 109, and biomechanical measurements were implemented: fracture resistance in group A was significantly higher than in other groups, while fracture resistance in groups B and C were significantly higher than in groups D, E, F and G (P<0.05).Conclusion(1) A large number of fibroblasts that appeared like rabbit ADSCs were successfully isolated and cultivated from adipose tissues in vitro via collagenase digestion and the adherent culture method. The obtained rabbit ADSCs displayed high purity, uniform morphology, good stability and an excellent capacity of proliferation amplification.(2) Rabbit ADSCs isolated and cultured in the experiment exhibited the surface molecular characteristics of stem cells.(3) The experiment confirmed that recombinant adenoviruses mediated the transfection of the hBMP-9 gene into ADSCs in vitro, with characteristics of high safety and transfection efficiency. Ad-hBMP-9 transfected ADSCs exerted an effective BMP-9 mRNA expression. The recombinant adenoviruses are a type of ideal carrier.(4) ADSCs could successfully maintain the sustained expression of BMP-9 after transfection by Ad-hBMP-9.(5) Cells maintained relatively strong proliferation ability, and these cells were transformed into cells characterized by osteogenic capacity after Ad-hBMP-9 transfection. The hBMP-9 genes successfully induced the differentiation of ADSCs into osteoblasts after transfection.(6) BMP-9 modified ADSCs in combination with ICA/nHAC scaffold materials were implanted into 15mm segmental bone defects of radius defects in rabbits. Imaging examination at postoperative day 30,60,90 and 109, and histologic and biomechanical examination at postoperative day 60 and 109 revealed that the repair of bone defects had been fulfilled. This study confirms that bones obtained from tissue engineering are effective in promoting bone regeneration, improving the repair effect of bone defects. Furthermore, this study provides a novel idea for the selection of seed cells in bone tissue engineering, as well as the design and establishment of bone tissue engineering complexes. |
PDF Full Text Request