Cryogenic 3D Printing Of Silicate Nanoclay Incorporated PLGA/β-TCP Scaffolds For Improved Bone Regeneration With Enhanced Vascularization

Background and ObjectiveBone defect is usually resulted from congenital disease,tumor resection or trauma.It remains challenging to manage critical sized bone defects due to the loss of bone mass and insufficient vascularization.Although autologous bone grafting is the gold standard for bone defect,its clinical application is constrained due to the limited donor sites and additional surgical trauma.Therefore,there is a dramatically increased need for biodegradable bone grafts.Tissue engineering scaffold with favorable mechanical strength,biomimetic structure and excellent bone osteogenic/angiogenic property is recognized as a promising platform for bone defect.Various osteoinductive and angiogenic growth factors or peptides have been incorporated into scaffolds to enhance bone formation,however,the instability and ease of inactivation of growth factors/peptides under certain physiological conditions limit their effectiveness.Two-dimensional nanoclay is a layered mineral.Due to the good biocompatibility,biodegradation and osteogenic effect,nanoclay has a good application prospect in bone regeneration.In the present study,a bifunctional silicate nanoclay(LAP)with potent to induce both osteogenesis and angiogenesis was incorporated into β-tricalcium phosphate/poly(lactide-coglycolide)(β-TCP/PLGA)composite to form a porous bone tissue engineering scaffold.The purposes are:1.to explore the printability of the present composite system;2.to explore the physical structure characteristic,biocompatibility,in vitro degradation performance and ion release of printed scaffold;3.to study the in vitro osteogenic and angiogenic effect of printed scaffold;4.to study the scaffold in vivo osteogenesis and the effect on H-type vessels.Methods1.PLGA/β-TCP/LAP(PTL)porous scaffold weas fabricated via cryogenic three-dimensional printing;The scaffolds were characterized by scanning electron microscopy(SEM),energy dispersive spectroscopy(EDX),X-ray diffraction(XRD),fourier transform infrared spectroscopy(FTIR);The compressive strength of the scaffolds was measured by using universal mechanical testing machine;The in vitro degradation performance and ion release of scafolds were evaluated after 8 weeks of immersion in PBS.2.The biocompatibility of the scaffold was evaluated by in vitro hemolysis,live/dead staining,and the adhesion performance of rat bone marrow mesenchymal stem cells(rBMSCs)and rat bone marrow derived endothelial cells(rBMECs)on scaffold.3.The in vitro angiogenic effect of scaffold was evaluated by rBMECs proliferation,scratches,tube formation test.The angiogenic gene expression level was assessed by real time quantitative PCR(qPCR).4.The in vitro osteogenesis of scaffold was evaluated by rBMSCs proliferation,ALP staining,and ARS staining.qPCR and immunofluorescence were used to detect the level of osteogenic genes and protein,respectively.5.The cranial critical bone defect model of rat was established.The in vivo osteogenic ability was evaluated by micro-CT and histological staining.Immunofluorescence was used to evaluate the formation of H-type vessels.Results1.The scaffolds with an average pore size of 450-500μm were successfully fabricated by cryogenic 3D printing.The scaffold exhibited a coarse surface and a large amount of micropores in 5-60μm was found in 20%and 40%PTL through SEM;The EDX results indicated that element magnesium,calcium,phosphorus,and silicon of LAP were uniformly distributed in scaffolds.2.The XRD and FTIR results showed that the characteristic peak of each component did not change before and after printing.The compressive strength of scaffold was 4-14 MPa on average which was comparable to that of human cancellous bone.3.The hemolysis rate of each group was less than 5%;No obvious dead cell was observed in live/dead staining;Compared with PT group,the proliferation of rBMSCs in 10%and 20%PTL was significantly promoted at day 1 and 3(P<0.05),while 40%PTL group presented a dramatically inhibited effect(P<0.05);All scaffolds did not show a promotion effect on the proliferation of rBMECs.rBMSCs and rBMECs co-cultured with PTL scaffolds showed better spreading and higher expression of adhesive plaques,while 40%PTL scaffolds showed an inhibition effect.4.Compared with PT group,PTL group had higher ALP activity and more mineralized nodules and the gene ALP,Runx2,Ocn and related protein were up-regulated.Besides,the PTL scaffold exerted osteogenic effect in a LAP concentration-dependent way.PTL scaffolds promoted the migration and tubulogenesis of rBMECs and up-regulated the expression of angiogenic genes(PDGFR,CD31,Tie2 and ItgaV)in a LAP concentration-dependent manner.5.There was only little bone formed at the edge of defect in blank group,while a large amount of bones were formed at the central of defect in PT and 20%PTL group.The bone volume of new bone in 20%PTL group was significantly higher than that in PT and blank group(P<0.05),and the trabecular separation degree in PT and 20%PTL group was significantly lower than that in blank group(P<0.05).6.Immunofluorescence result showed that the number and density of H-type vessels in 20%PTL group were higher than those in the blank and PT group.Conclusion1.PLGA/β-TCP/LAP scaffold with a coarse surface and porous structure was successfully fabricated by via cryogenic 3D printing.The printed scaffold exhibited a moderate pore size and suitable mechanical strength.2.PLGA/β-TCP/LAP was biodegradable and the active agents were continuously released from scaffold.3.The scaffolds showed an excellent biocompatibility and promoted the adhesion of rBMSCs and rBMECs within a certain range of LAP concentration.4.PLGA/β-TCP/LAP exerted in vitro angiogenesis by promoting the migration of rBMECs,improving tubulogenesis ability and up-regulating the expression of angiogenic genes in a LAP concentration-dependent way.5.PLGA/β-TCP/LAP exerted in vitro osteogenesis by promoting the proliferation,mineralization of rBMSCs and up-regulating the expression of osteogenic genes and proteins in a LAP concentration-dependent manner.6.PLGA/β-TCP/LAP scaffold showed a favorable in vivo osteogenesis and promoted the formation of H-type vessels.