Construction Of Silk Hydrogel System With Multiple Gradients And Osteochondral Differentiation Control In Vitro And In Vivo

Gradient biomaterials are considered as preferable matrices for tissue engineering due to better simulation of native tissues.The introduction of gradient cues usually needs special equipment and complex process but is only effective to limited biomaterials.Incorporation of multiple gradients in the hydrogels remains challenges.ObjectivesBeta-sheet rich silk nanofibers(BSNF)were used as building blocks to form gradient hydrogels under the joint action of crosslinking and electric field.Multiple gradient cues were introduced to different hydrogel systems and regulated finely through tuning different regulatory factors,confirming the universality of the strategy.Silk hydrogels with osteochondral differentiation gradient mechanical cues were developed to evaluate the feasibility of repairing complex interfacial tissues.Methods1.Based on the selective responsibility of beta-sheet rich silk nanofiber(BSNF)and amorphous silk nanofiber solutions(ASNF),GSNF hydrogel was developed from BSNF and ASNF blend solutions through HRP-crosslinking and electric field,where BSNF was used as reinforcement fibers to provide mechanical and oriented gradients while ASNF was crosslinked to form hydrogel matrices.The mechanical properties,secondary structure and micromorphology of the hydrogels were investigated to evaluate the distribution of multiple gradients.The ratios of ASNF and BSNF and the pre-crosslinking time before electrical field treatment were tuned to control the solution viscosity,which then determined the gradient density of the hydrogels.As a model drug,rhodamine was loaded on the BSNF nanofibers to prepare GSNF hydrogel,and the distribution characteristics of rhodamine as a small molecule drug was investigated through confocal microscopy images and rhodamine fluorescence intensity.Gelatin methacryloyl(Gel MA)and N-isopropylacrylamide(NIPAM)were used as alternative matrix of ASNF to form gradient hydrogels,confirming the universality of introducing gradient cues through BSNF.2.The osteochondral differentiation capacity of the hydrogels with gradient osteochondral mechanical cues were investigated in vitro to assess the feasibility of the hydrogels in interfacial tissue regeneration.Cell adhesion,proliferation and chondrogenicosteogenic differentiation behaviors of BMSCs on the hydrogels were evaluated to reveal the gradient osteochondral differentiation capacity of the hydrogels with gradient mechanical cues.3.Subcutaneous embedding model was used to assess the ectopic osteochondral induction of the gradient hydrogels in vivo.Hematoxylin & eosin(H&E)and immunochemical double-staining for Runx2/Sox9,OCN/COL II,and OPN/Acan were performed to reveal heterotopic ossification of hydrogels.Results1.With BSNF as building blocks,the introduction of multiple gradient signals in the GSNF hydrogels was achieved through the joint action of crosslinking and electric field,and the formation of gradient was related to the gradient distribution of BSNF.The blocks migrated to the anode along the electric field and gradually stagnated due to the solutionhydrogel transition of the systems,finally achieving gradient distribution of the blocks in the formed hydrogels.The gradient distribution of the blocks could be tuned easily through changing different factors such as solution viscosity,which resulted in highly tunable gradient of mechanical cues.The blocks were also aligned under the electric field,endowing orientation gradient simultaneously.Different cargos could be loaded on the blocks and form gradient cues through the same crosslinking-electric field strategy.The building blocks could be introduced to various hydrogels such as Gelatin and NIPAM,indicating the universality,which could provide new material support for construction of multiple gradients to silk hydrogel system.2.GSNF hydrogels had good cytocompatibility.The existence of multi gradients were favorable for the adhesion and spreading of BMSCs.The results of osteochondral differentiation in vitro indicated gradient osteogenic and chondrogenic differentiation behaviors of BMSCs when cultured on the different areas with gradient mechanical properties.3.The hydrogels were implanted subcutaneously and evaluate ectopic osteochondral tissue regeneration.The cells migrated into the hydrogels gradually following the degradation of the hydrogels.The hydrogels with highest stiffness were mostly occupied by the osteocytes while plenty of chondrocytes were found in the softest hydrogels.Both chondrocytes and osteocytes existed in the hydrogels with middle stiffness.The qualitative gene expression results confirmed the gradient chondrogenic-osteogenic transition on the hydrogels with mechanical gradients.All the results suggested similar distribution of osteocytes and chondrocytes to that native osteochondral tissues in vivo,indicating that the mechanical gradients provided suitable cues to tune osteogenic and chondrogenic capacity of the hydrogels.The oriented gradients provided additional benefits to tissue regeneration.The osteocytes aggregated to form aligned structures in the hydrogels with highest stiffness and best oriented structures,which were more similar to native bones.ConclusionIn summary,multifunctional building blocks,BSNF,were introduced to pattern hydrogels with tunable gradients using simple low voltage electric field.The gradients could be easily formed with a range of different material hydrogels.Several parameters such as solution viscosity could tune the gradient,endowing our systems with adjustability.Multiple gradients such as mechanical cues,oriented structures and different bioactive cargos were introduced to the hydrogel system simultaneously due to multifunction of the blocks,suggesting the advantages of designing complex niches.The hydrogels with mechanical gradients were fabricated to demonstrate the applications of the systems in tissue engineering.The hydrogels possessed suitable mechanical gradients to tune osteogenic-chondrogenic capacity,which was similar to that happened at the native osteochondral interface in vivo.Overall,the electric field-driven building blocks offer an easy and versatile strategy of developing hydrogels with multiple gradients for various complex tissues and interfacial tissue engineering.