| BACKGROUNDThe research of bone tissue engineering has a bright future in the development of clinical practice and it is prospective for a splendid market prospect in the near future. But, for a long time, the process of the tissue-engineered bone (TEB) construction and its mechanism are still lacking of objective and full understanding, which restricts the clinical application of TEB in repairing the bone defects especially the large segmental weight-loading bone defects.The basic concept of bone tissue engineering is that:the combination of seed cells, biomaterials (scaffold), cytokines and gene therapy necessary for bone formation and regeneration. Seed cell is one of the basic factors in construction the living TEB physical replacing the original bone tissues. It is also the most important precondition of the TEB research and clinical application in vitro or in vivo. At the same time, the cells, blood vessels, nerves and other tissues of host also play the very important role in the TEB construction. How to recognize the cells exactly in vitro or in vivo and trace their distribution, proliferation, differentiation and migration in the formation of TEB is of significant importance in the mechanism research of bone tissue engineering and in the optimizing the construction idea and model.Using the enhanced green fluorescent protein (eGFP) gene or the monomeric red fluorescent protein (mRFP) gene as the report gene to modify the target cells, so that the cells and even the subcellular structure can be effectively distinguished in vitro or in vivo. The technology is commonly used in tracer study in some soft tissues but seldom reported in the research of bone. For the physical properties of bone or biomaterials (scaffolds) are rigid, opaque and friable, regular methods could not observe and distinguish the fluorescent protein gene modified cells effectively and exactly. It still has few mature research procedure about this problem in China and at abroad. So the tracer study of TEB repairing the large segmental bone defects in situ is not only very important but also a challenge.OBJECTIVE1. To obtain the rabbit bone marrow-derived mesenchymal stem cell (rBMSC) long-term labeled by fluorescent protein stably and safely using gene modification technology.2. To investigate the mechanism of vascularization and neurotization TEB construction in situ repairing the femur defects by dual-fluoresent protein report gene modifying the seed cells and the vascular nerve tissues of host respectively.3. To tracer the TEB construction in situ repairing large segmental femur defects in mRFP transgenic mice by seed cells coming from eGFP transgenic mice.CONTENTSPart I Gene modification of rBMSC by lentivirus vector with eGFP or mRFP[Methods]①Phoenix A transient package cells and the 3rd LV vectors were used to produce LV-mRFP and LV-eGFP pseudotyping with vesicular stomatitis virus glycoprotein (VSV-G).②LV titers were detected by large-scacle-real-time titration method (LaSRT).③rBMSC-mRFP and rBMSC-eGFP were obtained after BMSC infected with LV-mRFP and LV-eGFP, then the proliferation and differentiation ability of rBMSC-mRFP were compared with wild type rBMSC by MTT and specific stain(ALP, Alizarin red S, toluidine blue, alcian blue and Oil Red O).[Results]①High-titer LV-mRFP and LV-eGFP were obtained. Mean titer of LV-mRFP is 5.2×10e6 TU/mL(n=5). the RFP% of rBMSC-mRFP is 81.1%(n=5), and it remains 35.4%(n=5) after culture ex vivo for more than 24w (25 passages).①Mean titer of LV-eGFP is 4.6×10e6 TU/mL (n=5), the GFP% of rBMSC-eGFP is 93.3%(n=5), and it remains 85.5%(n=5) after culture ex vivo for more than 24w (25 passages).③o difference of the proliferation curve was found between rBMSC-mRFP and wide type rBMSC. The rBMSC-mRFP remains the ability of osteogenic, chondrogenic and adipogenic differentiation.④ithout proper FCM antibodies to rabbit BMSC, we tried to use the FCM antibodies for human to detect the rabbit BMSC. However, flow cytometry results are negative.[Conclusions] We produced high titer LV-eGFP and LV-mRFP, after transfection with LV, rBMSC-eGFP and rBMSC-mRFP can stably express fluorescent proteins more than 6 months. The ability of proliferation and multilineage differentiation remain unchanged in rBMSC with the mRFP gene modification.Part II Tracer study of the the vascularization and neurotization TEB construction in vivo by dual-fluorescence protein report gene[Methods]①12 New Zealand rabbits randomly assigned to 4 groups,3 in each group, were used for the experiment. rBMSC-mRFP were implanted inβ-TCP scaffold (φ8 mm×15 mm, with microtubule pleural furrow, Bio-lu company France). Then the compound of seed cells and scaffolds were cultured under regular condition for 7 days before used in vivo. Two methods were used to implant seed cells in scaffold, including 1) negative pressure suction method and 2) MagIC method (invented by the author, patent applied). The effects of two methods were compared by MTT, cell count, semi-solid decalcification (patent applied) and frozen section, observation by fluorescence microscope, scanning electronic microscope (SEM) and laser scanning confocal microscope.②Animal operations:A 6-hole stainless steel mini-plate was placed in front of the rabbit femur. A reciprocating saw was then used to resect a 15mm length segment of central diaphysis under saline irrigation. Tissue-engineered bone was imbedded into the defect at this location. An enough length of femoral vascular and sensory nerve bundle were isolated and implanted into the microtubule pleural furrow ofβ-TCP scaffold and fixed with the mini-plate by thread. Before implantation, LV-eGFP pseudotyped with VSV-G was condensed by ultra-high-speed centrifugation, then used in situ to infect vascular and sensory nerve bundle by micro-injection.③one formation throughout the defects was assessed by radiograph at the 4w,8w,12w and 24w post-operation.3 animals in each group were sacrificed at 4,8,12 and 24 weeks after the implantation operations. Postmortem general and histology observation were also undertaken to examine bone formation. 4% paraformaldehyde PBS was used to perfuse the operation leg of rabbit to make the TEB and the related tissues fixed.10% EDTA were used for decalcification.④In order to study the mechanism, the specimens were processed for gross observation, real-time PCR and serial frozen sections after decalcification. The sections were used to observe under the fluorescence invert microscope or Laser scanning confocal microscope, or to make HE and Masson sections etc.[Results]①agIC can inoculate more cells intoβ-TCP scaffold compared with negative pressure suction method, the cell numbers in scaffold 12h after inoculation is (4.17±0.15)×10e6 vs (1.37±0.15)×10e6 (t=22.450, P=0.000) and the OD value detected by MTT is (2.96±0.06) vs (1.01±0.02) (r＝52.695, P=0.000). We also get the same results by fluorescence invert microscope, scanning electro-microscope, laser scanning confocal microscope, semi-solid decalcification method and frozen sections.②he titer after one cycle of freeze-thaw is 56.3%(n=5)than that before freeze. After ultra-high-speed centrifugation, the mean titer of LV condensed from 3.0×10e6 TU/mL(n=5) to 220x10e6 TU/mL (n=5).③X-ray detection shows that:8w post-operation, the scaffold begins to degradation and much bony callus begin cover the bone defections; 12w after operation, there are signs of healing; 24w post-operation, some pulp chamber begin to rebuilted.④Using the fluorescent microscope or laser scanning confocal microscope, frozen sections at different timepoints shows that more and more mRFP cells growing in the hole of scaffold,8w after the implantation, there are a plenty of collagen fibers growing through the holes of the scaffold; 12w post-operation, there are many mRFP cells in newly formed bone and cartilage. With the time prolonged, the hole mixed together and disappeared, the bony materials increased but the fluorescence attenuated. We can find some small vessels with red fluorescence in the holes of scaffold, bone matrix and the surrounding tissues. We also can observe some concomitant artery-vein in medullary canal and some middle vascular and nerve bundle at the connection of scaffold and tissues with green fluorescence. Some fat tissues can show both the red and green fluorescence.⑤RQ-PCR showes that, in the middle of TEB, in the connection of TEB with normal bone and in the tissues near the insert vascular and sensory nerve bundle, mRFP expression is 0.465±0.110,0.034±0.029 and 0.097±0.010 (F=43.641, P=0.000); while the eGFP expression is 0.003±0.001,0.003±0.002 and 0.026±0.009 (F=4.199, P=0.028); VEGF gene expression is 14.095±2.887,9.422±0.815 and 13.851±1.310 (F=8.728, P=0.003). Moreover, gene expressions of CGRP, CGRP-R, NK-R and VIP-R are also have some same tendency that from the most to the less are in the middle of TEB, in the connection of TEB with normal bone and in the tissues near the insert vascular and sensory nerve bundle (P≤0.014). However, there is no difference between NPY and NPY-R (P>0.05) among the three tissues.[Conclusions] A serial of techniques used in the research of this part formed a practical and reliable research system in the tracer study of bone tissue engineering named FATTEB (fluorescence aided tracer of TEB), using this system we can figure out the roles of eGFP and mRFP gene modified cells during the construction of TEB in details objectively and microscopically. We found that the seed cells (mRFP modified rBMSC) not only grow and proliferate inside the scaffold to form the endochondral ossification and the newly bone, but also proliferate and differentiated to vessels in bone matrix and around the insert vascular bundles. Some vessels and fat tissues in the rebuilt medullary canal and some nerve fibers in newly formed cartilages come from the tissues of host inserted artificially. The proliferation of GFP and RFP cells has close relationship with angiopoiesis and neurogenesis. The vascular net of TEB is built by seed cells and vessels of host nearby. Real time PCR results hint that during the construction of TEB, genes related with angiopoiesis and neurogenesis express very actively. The migration and development of the functional cells can be figured out by the eGFP or mRFP gene they were modified.Part III Initial tracer study of TEB construction in situ with eGFP and mRFP transgenic mice models[Methods]①GFP transgenic female FVB mice were used as donors, all the cells in the mice are eGFP positive. The bone marrow cells were separated from the bone marrow of femur, and cultured with murine BMSC special complete conditional culture medium (Saiye Company, Guangzhou, China). With regular adherent culture method, eGFP-mBMSCs were obtained 3 weeks later.②5×10e6 eGFP-mBMSC cells were inoculated in theβ-TCP scaffold (φ3mm×5mm, with microtubule pleural furrow, Bio-lu company, France) by MagIC innoculation method. After culture for 7 days, the compounds of seed cells and scaffolds were used in the operation in vivo. (③mRFP transgenic FVB mice, male, anesthetized with sterile 1.5% Pentobarbital Sodium, then a 5mm length segmental resect of femur central diaphysis were made by circular saw, a 23 G stainless steel needle was used as internal fixation. Observation of the bone healing and the stability of internal fixation post-operation of the mice model with large segmental bone and periosteum defects.④aking the mice models by the same method as before and implant the compounds of BMSC-eGFP seed cells and (3-TCP scaffold into the defect, internal fixation needle was insert in the pleural furrow of the scaffold and closely binded by-silk thread. Bone formation throughout the defects was assessed radiographically 1,2 and 3month after the implantation.⑤otal body perfusion fixation was performed with paraformaldehydum, then different samples were collected for frozen sections. The femur were also made frozen sections after decalcification.⑥eal-time PCR:fresh samples were collected immediately after sacrifice, mRNA was extracted and then reverse transcripted to cDNA, eGFP and ABL genes in different tissues were detected by real-time PCR.[Results]①GFP-mBMSC innculated in theβ-TCP grows well.②5mm bone and periosteum defects could not self-cure by the mRFP transgenic FVB mice 4 months post-operation. And the internal fixation is stable and reliable.③3 months after operation, the biomaterials were absorbed gradually and some high density matrix began to cover the defection.④3 months after operation, gross samples were separated from the operation femur, completed cortical bone has covered the bone defects. Observation by fluorescent microscope or laser scanning confocal microscope, the frozen sections shows that:there are many eGFP and mRFP cells growing in the hole of relic scaffold, new-forming collagen with red fluorescence insertting into the holes. New cartilage and bone formation have already can be seen in some holes and the scaffold near medullary canal. Most cells in the holes of scaffold are eGFP positive, some mRFP cells distribute around the holes. We can find that the red and green cells mixed with each other to form the new cartilage and bone tissues so that some yellow or brownish red fluorescence could be observed there. The rebuilt marrow cavity was full of many eGFP positive cells. Some red fluorescence collagens grow into the TEB and some red fluorescence small vessles growing in the middle of hole. We can find some middle or small vessels around the TEB constructed with eGFP positive cells and mRFP positive cells together. Green fluorescence could also be observed in some muscles near the operation area.⑤eal time PCR results:the eGFP/ABL expression in the middle of TEB, in the connection of TEB with normal bone and in the muscles of operation femur are 0.436±0.036, 0.140±0.038 and 0.098±0.024 (P=0.000). eGFP cells in the middle of TEB> in the connection of TEB with normal bone> in the muscles of operation femur, which is in accordance with the results by microscope observation.[Conclusions] With the help of eGFP and mRFP transgenic mice model and the resource of seed cells, we can exclude the interference of the virus transfection efficiency and specificity to the research results, so that the tracer study and mechanism investigation could become more overall and objective. Large segmental bone defects and stable internal fixation animal model of FVB-mRFP transgenic mice laid groundwork for the following tracer and TEB repairing study. We got direct and definite proofs of that seed cells in scaffold and tissues of host both involved in the formation of TEB. eGFP-mBMSC implanted in scaffold has the ability of multi-directional differentiation to chondrocyte, osteoblast, muscle cells and vessels in vivo.SUMMARY AND PROSPECTWe established the FATTEB system for the research of TEB in vivo and ex vivo. It makes the research of TEB construction more objective and microscopic. It should be a valuable platform for investigating the bone tissue engineering mechanisms and further optimizing the idea and model of construction for TEB. The MagIC method invented by author is beneficial to the inoculation of seed cells with scaffold and further be helpful to the TEB formation in vivo. Semi-solid decalcification method is very useful in the tracer study of seed cells compound with osteal biomaterial ex vivo and also suitable for tracer study of TEB construction at the early stage in vivo. Real time PCR provides a kind of sensitive, effective and precise test facility for genes detection in TEB. Large segmental bone defects and stable internal fixation animal model of mRFP transgenic FVB mice laid groundwork for the following tracer and TEB repairing study.In conclusion, our tracer study results suggested that:seed cells (BMSCs) combined with host’s cells and tissues all involved in the construction of TEB and its related tissues, but they mainly distribute in different tissues with some regularity. Our tracer study performed in the rabbit model and the dual-fluorescence protein transgenic mice model, which can be used to study the interaction among cell-to-cell, cell-to-matrix/tissue in details.A small step in basic research might lead to a great step in clinical works. With the development of our research and the improvement of larger animal model, it will hopfully improve the scientific research and clinical application of bone tissue engineering. |
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