Application Of Surface Functionalized PLGA Magnetic Microspheres In Isolation,expansion And Bone Regeneration Of Bone Marrow Mesenchymal Stem Cells

Mesenchymal stem cells（MSCs）have the characteristics of multi-directional differentiation potential,self-renewal,and secretion of a variety of bioactive substances.From animal experiments to clinical studies,MSCs provide hope for the treatment of many diseases.In this study,through the material composition regulation and surface functionalization of the microspheres,different Poly（lactic-co-glycolic acid）（PLGA）magnetic microspheres were prepared to optimize the separation,expansion and culture,harvest,induced differentiation and tissue repair of bone marrow mesenchymal stem cells（BMSCs）.MSCs need adequate numbers for cell therapy,and the microsphere-based MSCs culture method has attracted more and more attention.The microspheres provide a larger surface area to volume ratio and improve space utilization efficiency.Conventional microsphere culture usually needs centrifugation when harvesting cells,which inevitably causes some damage and loss to the cells.Therefore,this study attempted to develop magnetic microspheres to improve efficiency and convenience during medium replacement,cell harvest,and washing.The separation and purification of cells is a prerequisite for a variety of biomedical applications,while the traditional antibody-based sorting method is often expensive and highly dependent on instruments and equipment,making it difficult to apply on a large scale.In addition to purification,the maintenance of MSCs phenotype and differentiation potential is also an aspect that limits its application.This study performed anti-fouling treatment with Polyethylene glycol（PEG）on the surface of PLGA magnetic microspheres,and then grafted BMSCs-specific polypeptide E7,to construct an interface for separation and sorting based on selective adhesion of BMSCs.When three kinds of cells including fibroblasts,macrophages and BMSCs are present at the same time,the efficiency of the selective functionalized PLGA magnetic microspheres for adhering BMSCs to the surface reaches nearly 80%,and the phenotype and differentiation potential of BMSCs proliferated on the surface can be well maintained.As a tissue filling material,microspheres have the following advantages:microspheres can be shaped more easily than fixed-profile materials,and can fill irregular gaps and cavities.Pores can be formed between the microspheres to allow the exchange of nutrients and metabolic wastes with the surrounding physiological fluids,thereby promoting metabolism and local vascularization.In order to endow PLGA magnetic microspheres with more biological functions,this study further coupled OGP_10-14,an osteogenic growth peptide with good bone induction and differentiation effect,to the surface of the microspheres and investigated its ability to induce osteogenic differentiation of BMSCs.As cell delivery vector,microspheres have been widely used in the field of regenerative medicine.This way also preserves the bioactive substances secreted during cell proliferation,as trypsin digestion is not required.In recent years,Static magnetic field（SMF）has been applied to bone tissue engineering because of its controllable magnetic intensity and convenience for treatment.The combination of PLGA magnetic microspheres and SMF might be a promising strategy for bone defect repair:The magnetic microspheres could not only regulate the osteogenic differentiation of BMSCs under the magnetic field condition,but also solve the problem of short residence time of single magnetic nanoparticles at the bone defect.In order to verify this idea,this study evaluated the osteogenic effect of the bispeptide grafted PLGA magnetic microspheres,which was the most effective in osteogenesis in the previous experiment,on BMSCs under the action of SMF,and further used the microspheres as the delivery carrier of BMSCs,and investigated the tissue repair effect of the microspheres combined with SMF on the rat skull critical bone defect model.The experiments in this paper were divided into four main parts as follows:（1）Preparation and characterization of PLGA magnetic microspheres and study on cytotoxicity and cell proliferation in vitro:oleic acid modified superparamagnetic nanoparticles with saturation magnetization of 51.4 emu/g,regular shape and average particle size of 15.03±2.47 nm were synthesized by thermal decomposition method.The results of Fourier transform infrared spectroscopy（FTIR）and Thermogravimetric analysis（TGA）indicated that the oleic acid molecule was successfully grafted onto the surface of the nanoparticles.Furthermore,the effect of different preparation parameters on the size of PLGA magnetic microspheres was analyzed by the high-voltage electrostatic droplet technology,and PLGA magnetic microspheres with the average diameter of 273.07±19.09μm were successfully prepared.Among them,the oleic acid molecule was conducive to enhancing the combination of the magnetic nanoparticles and the PLGA matrix and reducing their dissociation from the composite microspheres,thereby reducing the toxicity of the composite microspheres.The cytotoxicity experiment of the extract verified the low toxicity of PLGA magnetic microspheres.The results of cell experiments showed that,compared with the two-dimensional plane,with the prolongation of culture time,the advantage of large surface area to volume ratio of PLGA magnetic microspheres gradually appeared,and the secretion efficiency of cytokines was higher,which was more conducive to maintaining the pluripotency of BMSCs and reducing cell senescence.In the culture medium,the PLGA magnetic microspheres gradually sink in the absence of a magnetic field,and the microspheres gather around the magnetic field in the presence of the magnetic field,so that the culture medium is convenient to replace,wash and other links,the steps are simplified,and the damage to cells caused by centrifugation is avoided.（2）Selective functionalization of PLGA magnetic microspheres and application thereof in separation and expansion of BMSCs:firstly,performing anti-fouling treatment on the PLGA magnetic microspheres by using PEG,and then grafting E7 polypeptide capable of specifically recognizing and binding to BMSCs on the surface of the microspheres,thereby completing selective functionalization of the PLGA magnetic microspheres.The cell adhesion experiments showed that the surface of PEG-modified PLGA magnetic microspheres effectively prevented cell adhesion,but the magnetic microspheres could selectively adhere to BMSCs after grafting with E7 polypeptide.This study cultured fibroblasts,macrophages and BMSCs on the surface of the microspheres individually or jointly and found that the selectively functionalized PLGA magnetic microspheres showed strong selective adhesion efficiency of BMSCs（up to nearly 80%）under either static or dynamic culture conditions.In addition,the stemness retention of BMSCs expanded on the surface of such microspheres was examined using flow cytometry and tri-lineage induced differentiation assays for osteogenesis,lipogenesis,and chondrogenesis.The experimental results showed that the prepared selectively functionalized PLGA magnetic microspheres with strong selective adhesion to BMSCs and the ability to maintain the cellular phenotype and differentiation potential of BMSCs are promising for the application of BMSCs isolation and expansion.（3）Osteogenic-induced functionalization of PLGA magnetic microspheres and their osteogenic effects in vitro:Based on the previous work,OGP_10-14 was grafted onto the surface of magnetic microspheres to complete the osteogenic-induced functionalization of PLGA magnetic microspheres with double peptide grafting.Physicochemical characterization showed that both E7 and OGP_10-14 peptides were successfully coupled to PEG molecules on the surface of PLGA magnetic microspheres.The biosafety of PLGA magnetic microspheres was investigated by intradermal reaction assay and in vitro hemolysis assay.The biological activity and in vitro osteogenic ability of the bipeptide-grafted PLGA magnetic microspheres were evaluated using cell proliferation,cell selectivity analysis,alkaline phosphatase quantification,and expression of osteogenic-related genes and proteins.In terms of biological activity,the bipeptide-grafted PLGA magnetic microspheres promoted both selective adhesion of BMSCs and osteogenic induction of BMSCs.The results of osteogenic differentiation experiments showed that the osteogenic differentiation effect of bipeptide-grafted PLGA magnetic microspheres was better than that of microspheres grafted with single peptide and without grafting.（4）Osteogenesis-inducing functionalized PLGA magnetic microspheres,in vitro and in vivo osteogenesis effect under SMF:The influence of dipeptide grafted PLGA magnetic microspheres on BMSCs under medium intensity SMF was further studied.The experimental results show that BMSCs on the surface of PLGA magnetic microspheres grafted with dipeptide have higher proliferation efficiency,larger adhesion area and better osteogenic ability after being combined with SMF.In terms of mechanism,the activation of ERK/P38 and BMP-2/Smad may promote the differentiation of BMSCs into osteogenesis in this process.Subsequently,the bispeptide grafted PLGA magnetic microspheres loaded with BMSCs were implanted into the critical bone defect of the rat skull,and the rats lived in medium-intensity SMF.Micro-CT results showed that the skull defect area was basically covered 6 weeks after operation.Twelve weeks after operation,the defect had been repaired more completely.The bone tissue fractions at two time points were 39.86±4.05%and 60.30±1.96%respectively.Observing the bone tissue section,it was found that the new bone tissue was densely and evenly distributed in the defect area12 weeks after operation.Immunofluorescence pictures showed that osteogenesis-related proteins were expressed in large quantities in bone tissue sections.No pathological changes were observed in the visceral tissue sections of rats,and there were no abnormalities in peripheral blood and biochemical analysis,which indicated that the magnetic microspheres could be safely applied in vivo.The above results show that the bispeptide modified PLGA magnetic microspheres can be used as BMSCs delivery carrier and can be used for magnetic therapy bone tissue repair.The results of this study show that surface functionalized PLGA magnetic microspheres provide a new strategy for BMSCs separation and expansion,and provide basic experimental basis and new ideas for bone repair.