| Tissue engineering and regenerative medicine provide a new way for the treatment of bone defects. Biodegradable polymer/HA scaffolds with controllable biodegradability, good processablity and bioactivity are extensively investigated in bone tissue engineering. In the conventional methods for fabricating the polymer/HA composite scaffolds such as particulate leaching, phase separation, electrospinning and solid freeform fabrication, the HA particles are premixed with the polymer solutions or polymer melts. Therefore, the bioactive HA particles are generally embedded in the polymer matrices and have little chance to expose on the pore surfaces, reducing significantly interactions between HA particles and cells. Therefore, the design and preparation of composite scaffold with HA coating is one of the supreme methods for obtaining ideal bone tissue engineering scaffolds.A porous poly(lactide-co-glycolide)(PLGA) scaffolds embedded spatially with HA particles on pore walls (PLGA/HA-S) were designed and fabricated by a porogen leaching method for bone tissue engineering. In this process, the key issue is to use the paraffin spheres coated with HA as the porogens, which were prepared via a "Pickering emulsion" method. Following a series procedures of heat treatment of the HA coated paraffin spheres, PLGA solution casting, freeze-drying and porogen leaching, the HA particles were successfully transferred to pore walls of the PLGA scaffolds. As the comparisons, PLGA/HA-M composite scaffolds with equal amount of HA nanoparticles (2%) in the PLGA matrix and pure PLGA scaffolds were prepared. With a same pore size of450～600μm and similar porosity (90～93%), the PLGA/HA-S and PLGA/HA-M scaffolds had significantly and insignificantly higher values of compression moduli than the PLGA scaffolds, respectively.Coating of nanosized hydroxyapatite facilitated the apatite growth in the simulated body fluid (SBF), showing enhanced bioactivity of PLGA/HA-S scaffold. The compressive modulus also increased substantially when a continuous apatite layer was formed on the pore walls of the scaffold. In vitro co-culture with preosteoblasts found that the cells distributed evenly on all the scaffolds and the preosteoblasts seeded on the PLGA/HA-S scaffolds showed the same proliferation as that on the PLGA/HA-M and pure PLGA scaffolds.In vitro culture of rat bone mesenchymal stem cells (BMSCs) found no difference of cell proliferation and morphology in all the scaffolds, but the alkaline phosphatase (ALP) activity in the PLGA/HA-S scaffolds were always highest, and were significantly improved in comparison with those in the PLGA scaffolds. In vivo implantation of the scaffolds/BMSCs constructs in rat calvarial critical-sized defects for4weeks confirmed that the PLGA/HA-S scaffolds could better improve the bone formation. The bone healing depends on viability of the transplanted cells. To investigate the cell viability, transplanted rat BMSCs were labeled with CM-Dil fluorochrome before they are implanted into the full-thickness calvarial defects (5mm in diameter). The red fluorescence in the defects area was observed by small animal in vivo fluorescence imaging system and and laser scanning confocal microscope after frozen section, respectively. The results showed that the transplanted BMSCs were still in the defect site and were alive even after4wk post-implantation in the defects.To investigate the influence of scaffolds on the Escherichia coli-derived recombinant human morphogenetic protein-2(ErhBMP-2) induced osteogenesis, BMSCs were seeded on PLGA/HA-S, PLGA/HA-M and PLGA scaffolds. And then the influence of scaffolds on osteogenic differentiation of BMSCs was examined in ErhBMP-2containing media. BMSCs cultured on PLGA/HA-S scaffolds exhibited higher alkaline phosphatase activity and enhanced expression of type I collagen (COL I) and osteocalcin (OCN) compared with PLGA/HA-M and PLGA scaffolds. The scaffold/ErhBMP-2composite constructs were implanted into the calvarial defects (5mm in diameter), while the constructs absent from ErhBMP-2served as the control. At4and8wk post-implantation, enhanced new bone formation was observed in PLGA/HA-S/ErhBMP-2implants, whereas nearly no new bone was observed in the group absent from ErhBMP-2.Growth factors are generally have a short-half life. To solve the problem, gene therapy was used in present study and N,N,N-trimethyl chitosan chloride (TMC) was employed as a vector for pDNA-BMP-2. In vitro culture of rat BMSCs demonstrated that rat BMSCs were transfected by the TMC/DNA complexes with an efficiency of13%and showed heterogeneous BMP-2expression for10days after transefected by TMC/pDNA-BMP-2. The PLGA/HA-S/(TMC/pDNA-BMP-2)/cell sheet constructs were implanted into the calvarial defects (5mm in diameter), while the scaffolds absent from BMSC sheet or gene complexes served as the control. In vivo expression of hBMP-2was detected by qRT-PCR at2weeks post-implantation. At4weeks post-implantation, new bone formation was evident in experimental group, whereas only a little bit of bone tissue was observed in the group absent from pDNA-BMP-2and BMSC sheet, At8weeks, new bone regenerated in the experimental group was significantly higher than constructs absence of pDNA-BMP-2or BMSC sheet. The qRT-PCR data also showed that bone specific genes expressed in the defect sites were much higher than those of the control groups.The composite scaffold incorporated with pDNA-BMP-2and BMSC sheet improved bone healing capability substantially. |
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