Study Of Temperature-activated Cryogel-PRP In Promoting ADSCs Osteogenesis And Bone Repair

BackgroundBone defects often exist in patients with trauma,infection,and bone tumor.And the lack of repair materials has been one of the clinical problems.Traditional bone repair materials are limited in clinical application due to their various shortcomings.In recent years,with the development of cell biology and materials science,tissue-engineered bone with seed cells,growth factors,and biomaterials provides a new method for the repair of bone defects.Adipose-derived stem cells(ADSCs)are a kind of adult stem cells with multi differentiation potential,which can differentiate into various cells with different properties and functions,and have been widely used in the field of tissue engineering.Different from the traditional single growth factor,platelet-rich plasma PRP,a platelet-derived biological agent,has been widely used in clinic to improve bone healing in recent years due to its safety and enriched multiple growth factors.However,there are many problems with its traditional activation and application methods.Methacrylic acid acylated gelatin(GelMA)is a biologically synthesized porous network structure hydrogel.It is an ideal biomaterial for growth factor loading.It has been widely used in the repair of the myoardium and skeletal muscle tissue.In this paper,a new type of bio-hydrogel was constructed by the use of GelMA and platelet-rich plasma(PRP).Some innovative efforts were made in controlling the release of multiple growth factors to promote osteogenesis and bone tissue engineering in vitro.ObjectiveA compound PRP biotype frozen hydrogel was prepared.This material,as a biological scaffold,has a three-dimensional network structure with good mechanical properties.It can activate PRP through temperature induction and release its growth factors,to satisfy the needs in different growth stages of osteogenesis and promote the long-term osteogenesis of ADSCs,and the repair of bone defects.Method1.synthesized the precursor of hyperelastic frozen hydrogel,GelMA with grafting reaction.Prepared the platelet-rich plasma(PRP)with Landerberg’s method,and detected the platelet concentration.Different concentrations of PRP combined with GelMA,PEGDA,and dopamine to develop PRP-Cryogel;PRP-Cryogel biocompatibility and characterization were detected.The release curve of the PRP-Cryogel activated growth factor was measured and compared with that of traditional thrombin-activated PRP gel.2.SD rat adipose tissue-derived mesenchymal stem cells(ADSCs)were extracted and passaged and amplified,and their stem cell characteristics were identified.Three-dimensional culture of ADSC in bio-hydrogel scaffolds,ADSCs proliferation and osteogenic differentiation ability in various tissue engineering scaffolds,and biocompatibility of PRP-Cryogel in vivo and in vitro were detected.3.The animals were divided into five groups:blank control group(Ⅰ),cryogel group(Ⅱ),cryogel+ADSCs group(Ⅲ),PRP cryogel group(Ⅳ)and PrP cryogel+ADSCs group(V).The rat skull bone defect model was made and implanted with tissue-engineered bone materials.After 8 weeks,the specimens were taken out for bone imaging analysis and histological examination.Result1.the cryogel(PRP-Cryogel)prepared from gelatin PRP composite was confirmed by IR analysis.Scanning electron microscopy revealed that Cryogel,PRP-1-Cryogel,and PRP-2-Cryogel formed three-dimensional porous network structure.The swelling rate and mechanical strength of hydrogels were improved by compound PRP,and PRP-2-Cryogel was particularly evident in 5 different concentrations of cold gel.After 30 days of immersion in PBS,the mass remained above 20%2.ELISA method was used to determine the release of a large number of high growth factors on the first 3 days of the traditional PRP gel.After 8 days,the concentration decreased significantly and the release decreased.The TGF-beta and PDGF in the temperature-activated bio-hydrogel were released slowly and steadily in one month.The activated percentage of CD63 positive platelets in PRP,inactive PRP-2-cryogel and activated PRP-2-cryogel at 37℃ were 9.5%,33.0%,and 73.1%respectively by flow cytometry,which indicated that the production process had little influence and most platelets were activated by temperature.3.ADSCs were identified as stem cells,oil red O staining was positive after adipogenic induction,and alizarin red S staining was positive after osteogenic induction.The ADSCs cultured on a new hydrogel three-dimensional scaffold showed a good growth trend through scanning electron microscopy.The survival rate of PRP-2-cryogel cells was significantly higher than that of other groups by living/dead cell staining.CCK-8 assay and in vitro experiments showed that PRP-2-cryogel had the best biocompatibility for cells and tissues.The results showed that the ADSCs cell viability was significantly higher than that of other groups.4.When cultured in vitro under osteogenic induction,the expression level of alkaline phosphatase(ALP)in the early stage of osteogenic differentiation of ADSCs was the highest in the PRP-2-cryogel group;the expression level of osteopontin(OPN)in the late stage of osteogenic differentiation was increased by qPCR detection and immunofluorescence analysis,while the expression level in PRP-2-cryogel group was the highest.Alizarin red S staining also showed that PRP-2-cryogel could promote bone formation.Oil red O staining showed that ADSCs cultured on three kinds of biological scaffolds did not produce adipocytes,which were harmful to the process of bone repair.5.8 weeks after the establishment of a rat skull defect model and implantation of a new composite hydrogel,according to the results of histological analysis and histological analysis,the bone repair effect of PRP-2-cryogel was better than that of cryogel+ADSCs,while the PRP-2-cryogel+ADSCs group had the best repair effect.ConclusionThe cryogel that encapsulated PRP showed an excellent porous structure,and could long-term release growth factors in a way of body temperature activation without any the addition of exogenous activators.In vitro studies,the PRP-cryogel accelerated the osteogenesis of ADSCs.In vivo studies,With the proper amount of PRP and ADSCs seeding,PRP-2-Cryogel promoted the repair of skull defects in rats.Our work has demonstrated that cryogel was a perfect carrier for PRP.The PRP-cryogel is an ideal bone tissue engineered material,had a good prospect in bone tissue repair.