Design Of Photocrosslinking Hyaluronic Acid-based Hydrogels And Their Application In Cartilage Tissue Engineering

Articular cartilage has limited self-repair due to lack of blood vessels,nerves,lymphoid tissues and progenitor cells.At present,intra-articular injection,cartilage planer,micro-fracture,cartilage transplantation are mainly used in clinical treatment,but still can not achieve satisfactory long-term repair effect.With the development of tissue engineering technology,it has been widely used in cartilage tissue repair because of its excellent treatment prospects.In the construction of tissue engineering scaffolds,compared with the traditional method of constructing bioscaffold materials and then planting seed cells,the more desirable way is to directly perform three-dimensional encapsulation of seed cells for injectable in situ or personalized repair 3D cell printing.As a three-dimensional seed cell material,the good cell compatibility of the hydrogel matrix material and the mildness of the gelation reaction are the key to ensure the survival of seed cells.Therefore,by selecting the matrix material and the gelation method,the designed hydrogel has a microenvironment suitable for the survival of the seed cells,and has a controlled injection in situ gelation or 3D printing personalized molding,which is significance for cartilage repaire by tissue engineering technique.The subject of this paper is to design and construct photocrosslinking hyaluronic acid-based hydrogels.In view of the problems in the treatment of articular cartilage defect with controllable injection and individualized repair,such as the three-dimensional packing of seed cells and the construction of cell survival microenvironment,the hydrogel systems with controlled injection performance,good mechanical properties,fast photocrosslinking properties,network structure patterning and 3D bioprinting were designed by photocrosslinking reaction which were used for three-dimensional encapsulated cells and showed good cell activity and cell proliferation.Firstly,the modification of hyaluronic acid with furanmethylamine was carried out,and the furan group-modified hyaluronic acid(HA-Furan)with a degree of substitution of 65%was successfully prepared.In the presence of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate(LAP),the HA-Furan aqueous solution was gelled under ultraviolet light(365nm,30mW/cm~2)for 30s.In order to further improve the mechanical properties of the hydrogel,Diels-Alder(DA)click chemical reaction between the furan group and the maleimide group was introduced into the controlled injection HA hydrogel system to construct a double crosslinked hyaluronic acid/polyethylene glycol(HA/PEG)hydrogel.The compressive modulus of the hydrogel was increased from 5.9 kPa to21 kPa.By reducing the illumination time(within 30 s)of ultraviolet light,the damage of the three-dimensionally encapsulated ATDC-5 cells was significantly reduced.After 7 days of culture,the encapsulated cells still show good cell viability.Secondly,through DA clicking chemistry between furan group and maleimide group,hyaluronic acid with cyclohexene structure(HA-Furan-Mal)was successfully constructed in this paper.Hydrogel was formed under ultraviolet light(365nm,13mW/cm~2)2s,with rapid gelation characteristics.With the increase of ultraviolet light time from 10s to 60s,the mechanical compressive stress of HA/PEG hydrogels with rapid optical crosslinking was increased from 18kPa to 72kPa when the strain was 55%.The three-dimensionally loaded ATDC-5 cells still showed good cell activity after 7 days of culture.Furthermore,by combining the Thiol-ene photocrosslinking reaction and the patterned mask,a concentric pattern hydrogel and a honeycomb pattern hydrogel were successfully obtained and the ATDC-5 cell encapsulation was achieved.The internal network structure of the concentric pattern hydrogel exhibits a loose network and a close network of alternating concentric circles.The internal network structure of the honeycomb pattern hydrogel exhibits a tight network surrounding the loose network.This provides an advantage for the maintenance of the good mechanical properties of the hydrogel and the supply of nutrients to the encapsulated cells.The compressive stress of the concentric pattern hydrogel and the honeycomb hydrogel at a compressive stress of 45%can reach 15.6 kPa and 16.4 kPa,respectively.ATDC-5 cells encapsulated in patterned hydrogels(concentric circles and honeycomb patterns)with network structure remained viable after 7 days of culture.It is worth mentioning that ATDC-5 cells have more obvious proliferation in patterned hydrogels with network structure.Finally,a photocrosslinked 3D bioprinted HA/PEG hydrogel containing mouse mesenchymal stem cells(mBMSCs)was prepared by fast photocrosslinking HA/PEG hydrogel system combined with 3D bioprinting technology.Photocrosslinking 3D bioprinted HA/PEG hydrogel at the print needle by UV light(365 nm,13 mW/cm~2)by utilizing the Thiol-ene fast photocrosslinking reaction between the norbornene-based group and the thiol group.Using a 27G(diameter 0.21 mm)stainless steel needle with an extrusion pressure of0.15 mPa and a line spacing of 1.0 mm,the print speed can be adjusted to 70 mm/s to obtain a regular 3D printed hydrogel.Through computer model design,a variety of personalized hydrogels,such as ring model,outer inner circle model and so on,were obtained.After multi-layer deposition,the height of the hydrogels could reach 3 cm.The three-dimensional encapsulation printing of mouse bone marrow mesenchymal cells(mBMSCs)was realized.After 7 days of culture,the three-dimensional encapsulated cells still had good cell activity and significant proliferation.