Preparation And Osteogenic Properties Of Injectable SA-PRP Composite Porous Microcarriers

As the population ages and the incidence of orthopedic diseases increases,the number of patients with bone diseases worldwide has also increased dramatically.In recent years,the rapid development of bone tissue engineering has provided a new approach for the treatment of bone diseases.Microcarriers are a kind of tissue engineering scaffolds with spherical pores and particle size ranging from 50～450μm.Due to the excellent properties of various tissue engineering scaffold materials,microcarriers have become a research hotspot in tissue engineering scaffolds in recent years.Porous microcarriers have high specific surface area,which can provide more cell adhesion sites for tissue engineering seed cells.At the same time,they are injectable and can be directly injected into bone defects.It is expected to achieve minimally invasive repair of complex and irregular bone defects.,and improve the effect of bone repair and reconstruction,which has important clinical practice and scientific research significance.In addition,injectable microcarriers can avoid the pain and related inflammation caused by traditional surgical immersion surgery on patients,and can also reduce the economic burden and generate significant social and economic benefits.Tissue-engineered microcarriers provide a temporary attachment space for seed cell growth and new bone tissue formation,and are a core part of affecting cell seeding,proliferation,migration,and osteogenic differentiation.In addition,most of the cell carriers implanted into the body die due to the slow vascularization rate inside the material,which leads to the death of cells in a hypoxic or hypoxic environment for a long time,resulting in treatment failure.Therefore,the selection,design,construction and optimization of cell carriers and their materials have always been the key to bone tissue engineering research.In this paper,using sodium alginate（SA）,platelet-rich plasma（PRP）and nano-magnesium peroxide（MgO₂）as the main raw materials,two kinds of composite porous microcarriers,SA-PRP-Ca²⁺（SPC）and SA-PRP-MgO₂-Ca²⁺（SPOC）,were designed and constructed by emulsion method combined with freeze-drying technology.The effects of preparation process conditions:SA concentration,mixture ratio of SA and PRP,curing temperature and MgO₂concentration on the morphology and pore structure of the microcarriers were studied.The results show that the SA-PRP microcarriers prepared by mixing 1.75%（wt）SA and PRP with SA:PRP=2:1 and curing temperature of-15℃have the best morphology and pore structure,and the pore size is 108±14μm,the 150-350μm particle size ratio is 35%.In addition,based on the preparation of SA-PRP microcarriers,nano-MgO₂was embedded in the SA-PRP mixed system,and the effect of different MgO₂concentrations on the preparation of microcarriers was explored.The results show that the SA-PRP-MgO₂microcarriers prepared with a concentration of 2.0%（wt）MgO₂have the best spheroidization rate and pore size,with a pore size of 46±6.8μm and a particle size ratio of150-350μm of 47%.The physicochemical characteristics of the porous microcarriers,such as FT-IR,TG-DSC,porosity,in vitro degradation properties and oxygen release properties,were tested.Anhydrous ethanol was used for pre-crosslinking,and then Ca Cl₂was used for secondary cross-linking to reduce the solubility of the microcarriers in water.The FT-IR results indicated that Ca²⁺ions chemically cross-linked with the SA-PRP system,and MgO₂would oxidize the SA-PRP system to stabilize the aqueous phase of the microcarriers.TG-DSC results show that the microcarriers can withstand temperatures up to 200°C and have good biothermal stability.The porosity results show that both SA-PRP and SA-PRP-MgO₂microcarriers have more than70%porosity,which may increase again after crosslinking by Ca Cl₂solution.The in vitro degradation results showed that the microcarriers could provide a good living environment for seed cells within 4 weeks,indicating that both SPC and SPOC microcarriers had suitable degradation properties.The oxygen release results of the PBS system showed that the SPOC microcarriers supported controllable and continuous oxygen release for up to 25 days,and the oxygen release amount was up to 2.4 mg/L*g microcarriers.rBMSCs were seeded on SPC and SPOC porous microcarriers.The control group was SC,SOC,SPMC and SPOS.Then,the cell adhesion and proliferation performance and the osteogenic differentiation performance were determined qualitatively and quantitatively,respectively.The results of CCK-8 and DIO staining showed that SPC and SPOC microcarriers had good biocompatibility and could provide a good microenvironment for rBMSCs to adhere,grow and expand.In addition,SPOC microcarriers also have oxygen release properties,which can provide oxygen to cells and promote cell proliferation in a closed environment.SPC and SPOC microcarriers also have osteoinductive activity,which can induce the differentiation of rBMSCs into osteoblasts,and the specific marker ALP staining is positive.The results of SPMC and SPOS control groups indicated that Sr²⁺and Mg²⁺could synergize SA-PRP system to induce osteogenic differentiation of rBMSCs.