Engineering Vascularized Bone Tissue Based On Cell-Sheet Techniques

1. Isolation, culture and the mutilineage potential of bonemarrow mesenchymal stem cells in vitro[Abstract] Purpose: To isolate and culture bone marrow mesenchymalstem cells (BMSCs) and observe the mutilineage potential of them．Methods:BMSCs were isolated and purified by adherence screening culture. Thecharacteristics and morphology of BMSCs were observed invertedmicroscopically and histochemically．Then under certain inductive conditions,BMSCs were made to differentiate into osteoblasts, adipocytes andchondrocyte, and identified in terms of Alizarin red, Oil Red O and toluidineblue staining respectively. Results: The cells cultured with this technique possesa general bionomics of BMSCs, appeared morphologically as spindle andcolony-shape, and expressed weakly positive of Alkaline phosphatase staining.Moreover, BMSCs were able to differentiate into osteoblasts、adipocyte andchondrocyte, expressing the specific phenotype respectively. Conclusions: The adherence screening method is relatively simple and feasible,by which BMSCscan be effectively isolated and purified. Furthermore, BMSCs have proliferationand multipotential ability.2. Endothelial progenitor cells isolation and culture[Abstract] Purpose: To isolate and culture endothelial progenitor cells(EPCs) and identify them. Methods: For angiogenic induction, MNCs werecultured in dishes coated with1%gelatin with M199medium supplementedwith10%fetal bovine serum and50μg/mL endothelial cell growth supplements,at37°C in a humidified atmosphere of5%CO2. After3days of culture,non-adherent cells were discarded by washing with PBS. The medium waschanged every3days. Subcultured using0.05%trypsin–EDTA. Cells of passage2–4were used for subsequent experiments. Bone-marrow derived EPCs wereidentified by immunofluorescence staining for CD31expression, ability of lectinbinding and capillary tube formation. Further characterisation of EPCs wasperformed by analysing transmission electron microscopy. Results: EPCscolonies appeared between5and7days of culture and defined as a central coreof rounded cells. Two weeks later, cells presented the characteristic cobblestonemorphology. Further, tubular network was formed, indicating endothelialphenotype. The differentiation status of bone marrow-derived EPCs wasconfirmed by binding UEA-1plant lectin and expressing endothelial markerprotein CD31. The Weibel-Palade body was also noticed by TEM in these cells,a critical feature for endothelial cells. Conclusions: When induced in culture,EPCs can be obtained, exhibit long term proliferative potential and are able todifferentiate into mature endothelial cells (ECs).3. Fabrication and osteogenic differentiation of BMSCs Sheet in vitro[Abstract] Purpose: The convenient approach of constructing BMSCssheets were explored. The histological construction and the osteogenicdifferentiation of sheets were analyzed. Methods: Firstly, we harvested BMSCscell sheets using a continuous culture method. Secondly, They can be detachedfrom the culture plate with a cell scraper and to be handled easily by forceps.Finally, histological and immunohistochemical examination, quantitativeanalysis of alkaline phosphatase activity, scanning electron microscopy (SEM)analysis and transmission electron microscopy (TEM) analysis were performedto evaluate the harvested sheet. Results: After continuous culturing in vitro,BMSCs cell sheets formed. They are semi-transparent, elastic, and rich in whitenodules. HE staining confirmed BMSCs cell sheets was composed of multipalelayers of cells and their extracellular matrix. Furthermore,the cell sheets showedhigh alkaline phosphatase(ALP) activities after osteoblastic differentiation. Themaintenance of the osteoblast specific phenotype was confirmed by positivestaining for ALP, as well as calcium deposits detected by Von Kossa andAlizarin red staining. In addition, both SEM and TEM analysis showed that cellswere embedded in their own matrix, while extensive collagen fibers and distinctmineral-like nodules were observed on the surface of the osteo-induced sheets.Conclusions: Our study indicates a new technique of constructing BMSCssheets is established and the obtained sheets maintain the potential of osteogenicdifferentiation.4. Engineering vascularized bone tissue using osteogenicBMSCs sheets/EPCs[Abstract] Purpose: To circumvent limitations associated with the use of alien scaffolds and isolated cells in bone tissue engineering, We herein reportthe engineering of a vascularized bone using bi-directional differentiationinduced bone marrow-derived cells,without alien scaffolds. Methods: Underosteogenic induction condition, the BMSCs were expanded and formed anosteogenic cell sheet using a continuous culture method and a scrapingtechnique. The other portion of the bone marrow cells was angiogenicallydirected to differentiate into endothelial progenitor cells (EPCs), which werethen combined with the BMSCs sheets. The BMSCs sheet-EPC complexes wereimplanted subcutaneously in immunocompromised mice. BMSCs sheets withoutEPCs were also implanted as a control. At4or8weeks, the mice weresacrificed and the samples were harvested for evaluations: micro-CT scanning,scanning electronic microscopy analysis, histologic and histomorphometricexamination. Results: The BMSCs sheet-EPCs complex turned out to be awell-vascularized bone, which grossly appeared a blood red hard block.Micro-CT scanning and histological examination confirmed new bone formationin vivo. Additionally, both its bone density and vascular density weresignificantly higher than the control group (p <0.05). Conclusions: OsteogenicBMSCs sheet coculture with EPCs can be used to engineer vascularized bonetissue without use of alien scaffold. The results demonstrated that introductionof EPCs could not only generate vascular network, but also increase boneformation for cell sheet bone engineering. This may be a promising strategy tofacilitate clinical application of tissue engineering bone (TEB).5．Engineering injectable vascularized bone using osteogenicBMSCs sheet/EPCs[Abstract] Purpose: To construct vascularized and injectable tissue-engineered bone (ITB) composed of EPCs, BMSCs and ECM, withoutuse of alien scaffolds. Methods: The BMSCs sheet-EPCs complexes wereinjected subcutaneously on the back of the nude mice. Gross examination,Micro-CT scanning, scanning electronic microscopy analysis, histologic andhistomorphometric examination were performed to evaluate theneovascularization and osteogenesis. Results: The results revealed that thisBMSCs sheet-EPCs complexes formed bone structure in heterotopic site of nudemice. Bone is formed by two distinct modes of ossification: Intramembranousossification was the main process of new bone formation at the periphery ofengineered constructs, while endochondral ossification was also observed by thepresence of calcified cartilage and hypertrophic chondrocytes within theconstructs. Conclusions: Our study indicates that BMSCs sheet-EPCscomplexes can be used to engineer vascularized and injectable bone tissuewithout use of alien carriers and the engineered bone might be considered as apromising strategy for bone repair. Our findings indicate that the injectablevascularized bone composed of EPCs, BMSCs and ECM, without use of alienscaffolds, may represent a novel strategy to bone defect. However, furtherstudies should be done to fully reveal the characteristics of this injectabletissue-engineered bone.6. Engineering large tubular bone constructs with naturalcoral combined with BMSCs sheets/EPCs[Abstract] Purpose: In this study, BMSCs sheets/EPCs were assembledwith cylindrical scaffolds made from nature coral (NA), for the engineering oflarge and functional bone grafts.Methods: Rabbits BMSCs were cultured toform a thin layer of cell sheets via osteogenic induction. On the other hand, EPCs were isolated and cultured. Then, tubular long bones were constructed bywrapping the BMSCs sheets/MSCs on to cylindrical scaffolds. In vitro,perfusion bioreactors were used to overcome issues of mass transport inculture. In vivo, constructs were implanted subcutaneously in rabbits. Thespecimens were harvested and evaluated by gross examination, micro-CTscanning, scanning electronic microscopy analysis and histological examinationafter implantation. Results: The results showed that the layered cell sheetsintegrated well with the scaffold, underwent classical osteogenic differentiation,with mineralized nodules visible both inside and outside the scaffold for up to8weeks culture in vitro. In vivo, Micro-CT scanning, SEM analysis andhistological examinations revealed neo trabecular bone were formed within theconstructs. Conclusions: These findings illustrate that large bone tissues similarto native bone can be regenerated utilizing BMSCs sheet techniques inconjunction with scaffolds.