Construction Of GelMA/PAMAM-MA Hydrogel And Its Role In Bone And Cartilage Defect Repair

The incidence rate of bone and cartilage defects caused by trauma,inflammation,tumor and other diseases is high,and is increasing with the aggravation of aging.Extracellular matrix(ECM)remodeling is an important part of defect repair,which is regulated by cells and growth factors.However,due to the limited supply of endogenous cells and growth factors in bone and cartilage defect areas,it is necessary to use bone and cartilage substitute materials to deliver exogenous cells and growth factors to help defect repair.At present,the commonly used graft materials,whether autologous grafts regarded as the "gold standard",allografts or metal,ceramic and polymer scaffolds,are difficult to meet the needs of ECM remodeling.Therefore,it is necessary to develop a kind of alternative materials for bone and cartilage matrix repair and regeneration.Hydrogels are three-dimensional network scaffolds crosslinked by hydrophilic polymers,which have good biocompatibilities and can not only deliver cells,provide a growth environment similar to natural ECM,but also transport and control the release of growth factors.Hydrogels are commonly used to construct biomimetic ECM scaffolds.Gelatin,as a common component in natural ECM,has good biocompatibility and biodegradability.Gelatin and its side chain modification products are commonly used in the synthesis of hydrogels.Among them,gelatin methacrylate(Gel MA)can be covalently crosslinked to form hydrogel scaffolds.Gel MA hydrogel is commonly used in cell culture and delivery because of their excellent biological properties.However,Gel MA hydrogel,as linear macromolecules,have relatively weak stability and mechanical properties.Due to the rapid liquid exchange rate and other reasons,the controlled release of growth factors cannot be realized.In this regard,Gel MA is difficult to meet the needs of bone and cartilage repair materials,and its physical properties need to be further optimized.Poly(amide amine)dendrimer(PAMAM)is a kind of spherical polymer with a large number of modifiable outer functional groups and internal cavities.It can not only realize the loading and delivery of bioactive macromolecules through surface modifiable functional groups and internal cavities,but also help maintain the stability of network structure and improve the mechanical strength of crosslinking network.Therefore,PAMAM has great potential to optimize the physical properties of Gel MA hydrogels to meet the needs of graft materials for bone and cartilage matrix repair.Based on the above research background,this dissertation proposes the research purpose: to construct substitute materials that meet the needs of ECM remodeling,and adjust the properties of the materials to meet the needs of different tissue defect repair,and finally used for bone and cartilage defect repair in vivo.In this dissertation,the fourth generation amino terminal PAMAM molecules were modified by methacrylic anhydride and introduced into Gel MA crosslinking network to construct line-sphere double factor hydrogel scaffold materials.Firstly,the physical properties of the hydrogels were adjusted and optimized by controlling the concentration of PAMAM molecules,and the growth factors were loaded and controlled by chemical crosslinking using the modifiable amino groups on the surface of PAMAM.Subsequently,the loaded mesenchymal stem cells were three-dimensional cultured and delivered,and the differentiation direction of stem cells was controlled by adjusting the physical properties of the hydrogels.A group of hydrogels with the best osteogenic differentiation induction ability was selected for chemically crosslinking the growth factors,delivering stem cells and growth factors to the bone defect areas and assisting bone repair and regeneration.Finally,the appropriate proportion of hydrogel was selected to deliver adipose stem cells to the cartilage defect area of knee joint for cartilage repair and regeneration.This dissertation provides a new design idea reference and reliable data support for the development of bone and cartilage graft materials.The main research contents and results of this dissertation are as follows.(1)Gel MA and methacrylic anhydride modified poly(amidoamine)(PAMAM-MA)were synthesized by modifying gelatin and PAMAM with methacrylic anhydride(MA).Four groups of Gel MA/PAMAM-MA hydrogels were synthesized by adjusting the concentration of PAMAM-MA.The pore sizes of the crosslinking networks of the four hydrogels decreased with the increase of PAMAM-MA concentration,and the liquid permeation rate also decreased.The stability and mechanical properties of the four groups of hydrogels gradually increased with the increase of PAMAM-MA concentration.Gel MA/PAMAM-MA hydrogels loaded with growth factor bone morphogenetic protein 2(BMP2)through chemical crosslinking have effectively reduced the release rate of BMP2.(2)Rat bone marrow mesenchymal stem cells(r BMSCs)meeting the criteria for stem cell identification were extracted and encapsulated in four groups of hydrogels for three-dimensional culture,all of which had high cell viabilities.The ability of inducing the osteogenic differentiation of stem cells in vitro of the four groups of hydrogels gradually increased with the increase of PAMAM-MA concentration,and the 5/5 group have the strongest performance.The hydrogel group of chemically-crosslinked BMP2 has the stronger ability of inducing osteogenic differentiation of stem cells in vitro than that of hydrogel group of physically-encapsulated BMP2.After subcutaneous implantation in rats,the hydrogel group of chemicallycrosslinked BMP2 has effectively reduced the inflammatory response by slowing down the release rate of BMP2.The hydrogels encapsulated with r BMSCs and loaded with growth factors in different ways were used to repair the cranial bone defects in rats.The hydrogel group of chemicallycrosslinked BMP2 had the best repair effect.(3)Rat adipose derived stem cells(r ASCs)meeting the criteria for stem cell identification were extracted.Gel MA/PAMAM-MA hydrogels(5/2group,experimental group)and Gel MA hydrogels(5/0 group,control group)were selected for three-dimensional culture of r ASCs.The cell viabilities of r ASCs in two groups of the hydrogels were more than 80% at each time point.The addition of PAMAM-MA limited the cytoskeleton spreading of r ASCs.With the addition of PAMAM-MA,the ability of inducing the chondrogenic differentiation of stem cells of the two groups of hydrogels was increased.Compared with Gel MA hydrogel,Gel MA/PAMAM-MA hydrogel encapsulated with r ASCs has a better effect on the repair of articular cartilage defects in rats.