Construction Of Oriented Bone/Cartilage Integrated Scaffold Based On 3D Bioprinting And Its Performance Study

Articular osteochondral injury is an important risk factor for the development of osteoarthritis(OA)in later years,and the ability of self-repair after cartilage injury is limited,accompanied by subchondral bone degeneration and lesions,so osteochondral treatment and repair is needed.The traditional treatment methods of osteochondral defects have their own shortcomings,so it is difficult to reconstruct the multi-layer functional structure of articular osteochondral defects.Tissue engineering is the most promising method for the treatment of osteochondral defects in recent years.Ideal scaffolds for osteochondral tissue engineering need to have good mechanical properties,physical and chemical properties similar to extracellular matrix(ECM),and excellent biological activity to induce tissue regeneration.Hydrogel materials have great advantages in the above aspects.As a new technology for manufacturing scaffolds,3D bioprinting technology can build personalized scaffolds according to the requirements,with different holes and hierarchical structures,and can effectively simulate the tissue complexity and gradient structure of natural tissue.Therefore,by simulating the layered structure of natural articular cartilage,based on the highly similar properties of hydrogel and ECM and 3D bioprinting technology,a bilayered oriented bone-cartilage integrated scaffold was constructed in order to induce the integrated repair and regeneration of bone and cartilage.Articular cartilage is composed of chondrocytes distributed in extracellular matrix.Therefore,in order to treat cartilage injury,ECM should be made properly.A series of GMA/CS/SF cartilage layer hydrogel inks were prepared by using methacrylate gelatin(Gel MA),chitosan(CS)and silk fibroin(SF),and the best component 5GMA/5CS/7SF was optimized as the printing ink of cartilage layer scaffold.Using 3D bio-printing and directional freezing technique,combined with double-network cross-linking mechanism,the horizontal/vertical oriented scaffold of 5GMA/5CS/7SF cartilage layer was successfully constructed and compared with the non-oriented group.The experimental results showed that both the horizontal oriented group and the vertical oriented group of 5GMA/5CS/7SF cartilage scaffolds showed a linear pore structure with orientation,and the horizontal orientation structure and vertical orientation structure reduce the water content and swelling ratio,and increase the porosity up to more than 88%.The horizontally oriented structure had smaller contact angle and suitable degradation rate,and degraded to 50% on the 23 rd day,and the horizontally oriented structure improved the compressive capacity and structural stability of the cartilage layer scaffold,and the elastic modulus and maximum compressive strength reached 0.08 MPa and 0.07 MPa,respectively.Both horizontally oriented scaffolds and vertically oriented scaffolds had no cytotoxicity and are more conducive to cell proliferation,and had higher glycosaminoglycan sulfate content,which can provide an excellent cartilage tissue differentiation environment for ATDC5 cells.For the repair of subchondral bone layer,not only the strength of the material should be considered,but also excellent biological activity and osteoinductive activity should be considered.Therefore,a series of GMA/SA/HA subchondral bone layer hydrogel inks were prepared with Gel MA,sodium alginate(SA)and hydroxyapatite(HA),and the best component 5GMA/5SA/7.5HA was optimized as the printing ink of subchondral bone layer scaffolds.Using 3D bio-printing and directional freezing technique,combined with double-network cross-linking mechanism,the horizontal/vertical oriented scaffold of 5GMA/5SA/7.5HA cartilage layer was successfully constructed and compared with the non-oriented group.The experimental results showed that the horizontal and vertical orientation groups of the5GMA/5SA/7.5HA subchondral bone scaffolds showed linear pore structure with orientation.The horizontal orientation structure and the vertical orientation structure reduced the water content and swelling rate,and increased the porosity to more than80%.The vertical orientation structure had a hydrophilic and suitable degradation rate,which degraded to about 65% on the 44 th day.The vertical orientation structure improved the compressive capacity and structural stability of the subchondral bone layer scaffold,and the elastic modulus and the maximum compressive strength were up to 0.16 MPa and 0.13 MPa,respectively.The scaffolds in the horizontal orientation group and the vertical orientation group showed no cytotoxicity and were more conducive to cell proliferation.The scaffolds in the vertical orientation group had better mineralization ability and better ability to promote bone differentiation in the early,middle and late stages.According to the natural osteochondral structure,bone and cartilage are not two separate parts,but a whole which is closely connected by the transition layer.Therefore,5GMA/5CS/7SF and 5GMA/5SA/7.5HA simulated the cartilage layer and subchondral bone layer respectively,and selected Gel MA,CS and DA as the transition layer to increase the binding force(10KPa).The bilayered oriented osteochondral integrated scaffold(horizontal orientation of cartilage layer and vertical orientation of subchondral bone layer)was constructed by 3D printing and directional freezing technique,and compared with the bilayered non-oriented group.The experimental results showed that the structure of the upper layer with horizontal orientation and the lower layer with vertical orientation can be observed,and the transition layer was well combined.The double-layer oriented structure had higher water content and swelling rate,and the porosity decreased slightly,but still maintained above 70%.The double-layer oriented structure improved the compressive strength and structural stability of the bone/cartilage scaffolds.The horizontal orientation of the upper layer and vertical orientation of the lower layer of the double-layer oriented bone/cartilage scaffolds can promote the proliferation and differentiation of chondroblasts and osteoblasts,and had the potential to promote the repair and regeneration of osteochondral tissue.