Fibrinogen Modified Three-layer Demineralized Bone Scaffold With Different Stiffness Improves Osteochondral Defect Repair

Osteochondral injury is a common orthopedic disease that can cause joint pain and seriously affect people’s daily life.Due to the complex structure of the osteochondral interface and the special structure of the articular cartilage,limiting self-repair and regeneration,there are still major challenges in clinical treatment.In the early resersch,decalcified bone matrix(DBM)with different matrix stiffness was prepared by controlling different decalcification times.In this paper,from the perspective of biomechanics,a decalcified bone scaffold with three different matrix stiffness structures was constructed.The osteogenesis and cartilage properties of fibrinogen(Fg)was combined to modify DBM.The effect of Fg-modified different matrix stiffness scaffolds(Fg+DBM)on the repair of osteochondral injury.MSCs were inoculated on scaffolds to study the effects of MSCs viability,osteogenic and chondrogenic differentiation.SD rats were subcutaneously examined for the effects of scaffolds on endogenous cell infiltration,angiogenesis and endogenous osteogenesis and chondrogenic differentiation.The scaffolds were implanted into the osteochondral lesions of New Zealand white rabbits to investigate the repair effect of osteochondral damage.The following main conclusions were obtained in the study:(1)Successfully prepared three layers of Fg modified DBM scaffolds with different stiffnessThe compression test was carried out using E1000.Different matrix stiffness(High: 174.208±44.330 MPa,Medium: 21.214±6.922 MPa,Low: 0.67833±0.269 MPa)can provide a suitable biomechanical microenvironment for cell survival and growth.The experimental results show that the modification of Fg does not affect the stiffness of the scaffold.(2)The scaffold can promote the proliferation of MSCs and promote the differentiation of MSCs into osteoblasts and chondrocytes in different layers.Fg-modified scaffolds did not affect MSCs cell viability.It was found that Fg did not affect the secretion of extracellular matrix and deposition of collagen fibers.Alkaline phosphatasr(ALP)staining,typeⅠ collagen(Col Ⅰ),osteopontin(OPN)and osteocalcin(OC)immunohistochemistry results: High group of Fg+DBM scaffold can effectively promote the osteogenic differentiation of MSCs,which indicates that the High group of Fg+DBM scaffold has better osteogenic ability.Safranin O-fixed green staining,immunohistochemical staining of type Ⅱ collagen(Col Ⅱ)and type Ⅹ collagen(Col Ⅹ)revealed that the Medium and Low group of Fg+DBM scaffold could promote the cartilage differentiation of MSCs,indicating that the Medium and Low group of Fg+DBM scaffold have better cartilage ability.(3)scaffold promotes angiogenesis in vivo and endogenous cells differentiate into osteogenesis and chondrogenesis in different layersThe study found that the Medium and Low of DBM scaffolds were significantly degraded in the High grade group at 8 weeks after subcutaneous transplantation,indicating that the DBM stents in the Medium and Low groups have higher degradation ability.The Fg+DBM scaffold was significantly degraded in the Medium and Low groups compared to the DBM scaffolds.The High group of Fg+DBM scaffolds has a better ability to promote microvascular production.Immunohistochemical staining of Col Ⅰ,Col Ⅱ and Col Ⅹ revealed that the High group of Fg+DBM scaffold promoted osteogenic differentiation of endogenous cells after subcutaneous transplantation for 4 and 8 weeks.And the Medium and Low group of Fg+DBM scaffolds can promote endogenous cell cartilage differentiation.This indicates that endogenous cells have a certain ability to promote bone regeneration in the High group of Fg+DBM scaffold,and in the Medium and Low groups of the Fg+DBM scaffolds have certain cartilage regeneration ability.Rabbit osteochondral defects were sampled 4 weeks after surgery.Gross observation showed that Fg+DBM scaffold had better osteochondral repair ability than DBM scaffold.