USPIO-labeled SF/HA Scaffolds For Bone Repair Monitered By Non-invasive MR Imaging

Bone defects caused by trauma surgery,or tumor resection are very common and thus consume a large amount of medical resources annually.Recently,tissue engineering has attracted much attention as a promising alternative to nature bone grafts.Conventional tissue engineering involves the use of a combination of biomaterials,bioactive molecules and cells.Biomaterials comprise a key element in bone tissue engineering and provide a three-dimensional(3D)scaffold to support cell growth and formation of the extracellular matrix(ECM).Silk fibroin(SF),a protein harvested from domesticated silk worm cocoons,receives intensive attention as a material for use in fabrication of 3D porous scaffolds because of its unique mechanical properties,tunable biodegradation rate,good preservation of bioactive molecule activity.However,silk alone has high dissolubility and lower mechanical properties than native bone.Hydroxyapatite(HA)is chemically similar to the natural bone and exhibits excellent osteoconductivity.It has been demonstrated that the incorporation of nanoparticle Hydroxyapatite(nHAp)in SF scaffolds may enhances the mechanical property and also induces osetoinductivity.It is necessary for a qualified scaffold sustain cell adhesion,growth and differentiation.In previous studies,mesenchymal stem cells(MSCs)isolated from bone marrow were widely employed as a promising source of cells for skeletal regeneration,owing to their potential to differentiate into osteoblasts both in vitro and in vivo as well as their potent paracrine anti-inflammatory properties.However,it is difficult for researches to recognize and monitor the processes of biomaterial degradation in vivo.The non-invasive and successive observation can provide efficient information and minimize individual differences between animals,which might facilitate the clinical translation of tissue engineering.Noninvasive tracking of scaffolds degradation and bone regeneration in bone tissue engineering remains to be exploredMagnetic resonance imaging(MRI)is characterized by its safety,diverse functional sequences,excellent soft-tissue contrast,and penetration depth,it is widely used to monitor the molecular and functional changes that occur during biomaterial resorption and neo-tissue remodeling.Non-invasive monitoring approaches can help reduce the amount of animals used.The experiment data can be achieved repeatedly to prevent unnecessary sacrifice for histological analysis at different time points.Furthermore,ultrasmall superparamagnetic iron oxide(USPIO)is emerged out rapidly as a promising contrast agent in molecular MR imaging for tissue engineering.SPIO is a T2 weighted magnetic resonance imaging agent that can shorten the T2 time.Therefore,T2 weighted images become dimmed and exhibit negative enhancement effect.Due to their biocompatibility and superparamagnetic properties,USPIO has been intensively investigated as functional tools for cancer detection,drug delivery monitoring,stem cells tracking and implanted scaffolds labelingHere,we have developed for the first time a visualizable functional USPIO-labeled silk fibroin(SF)/nanoparticle Hydroxyapatite(nHA)composite system with which to semiquantitatively monitor the scaffold degradation process and clarify the bone regeneration using multiparametric MRI in situ.This system might provide meaningful insight into the noninvasive monitoring and longitudinal therapeutic efficacy of implanted material in bone tissue engineering.Objective:1.To synthesis SF/HA scaffolds labeled by different USPIO concentration,with assessment of physical-chemical properties.Then the optimum concentration had to be decided by MR imaging and its biocompatibility.2.To isolate BMSCs from SD rat,and seed them into USPIO labeled SF/HA scaffolds.Then to evaluate the ability of the scaffolds to enable the adhesion,proliferation,and osteogenic differentiation of BMSCs,and the features of MR imaging.3.To define the potential of BMSCs to form ectopic osteogenesis in a USPIO-SF/HA system with subcutaneous implantation in nude mice,and to monitor the scaffold degradation process and clarify the bone regeneration using multiparametric MRIMaterials and method:1.The HA/SF scaffold labeled by USPIO was fabricated by mixing 1 mL of the 4%(w/v)SF solution,10 mg of HA and USPIO ranging from 0.25%-1.5%(w/w)and then pouring the mixture into a 48-well plate and freeze-drying for 24 h.The components,morphologies,chemical property and mechanical properties of the composite scaffolds were investigated by XRD,SEM and FTIR.The MR imaging features were measured for the scaffolds[0-1.5%(w/w)USPIO]in a clinical 3T MR scanner.Obtain Primary bone marrow stem cells from the SD rat,and the scaffolds were putted into a 48-well plate which was seeded with BMSCs.CCK-8 was used for elevating the biocompatibility.The optimal USPIO concentration was determined by the MR visualization and cytotoxicity assay of the composite scaffolds.2.We obtained Primary bone marrow stem cells from the SD rat.Observed the growth,proliferation and morphology of cells which inoculated on the materials by contrast microscope,electron microscopy,CCK-8 and Live/dead kit.Furthermore,we detected the mRNA levels of osteogenic-specific genes(ALP,Bmp,COLL I,Runx).The alkaline phosphatase activity of BMSCs cultured on the scaffolds after osteogenic induction was also measured.In addition,we analyzed the shape and signal changes of scaffolds under osteogenic induction by 3T MRI3.The ectopic bone formation experiment was performed in a USPIO-SF/HA system with subcutaneous implantation in nude mice.Animals were scanned with T2-weighted imaging(T2WI),T2 mapping,T2*mapping and QSM sequences to determine the image features and relaxation rates at 2,6,8 weeks after implantation Computer tomography(CT)scanning,Hematoxylin-Eosin(H&E)and masson's trichrome staining were also preformed.Finally,the quantitative measurement of SPIO in scaffolds was calculated by ICP-MSResults:1.The SF/HA composites blended with USPIO,at concentrations ranging from 0%to 1.5%(w/w),were successfully fabricated.The signal contrast in the prepared scaffolds gradually increased as the amount of USPIO increased on T2-weighted imaging,and composites with USPIO amounts from 0.75%to 1.5%(w/w)demonstrated more homogeneous imaging than others.Results showed the cell proliferation was affected in USPIO labeling concentration more than 1%(w/w).Therefore,0.75%(w/w)USPIO-labeled SF/HA scaffolds was used for the subsequent in vitro and in vivo assays because it displayed sufficient MR contrast and the best biocompatibility2.Either scaffolds labeled or non-labeled were enable for adhesion,proliferation,and differentiation of bone marrow mesenchymal stem cell.Most BMSCs attaching to the non-labeled or USPIO-labeled scaffolds remained viable with live/dead staining.The cells were spindle-shaped and flat,and aggregated into condensed clusters under SEM observation.The amount of cells in all of the scaffolds increased monotonously with increasing culture time.Because of the positive expression of ALP and bone genes,it was confirmed that BMSCs could differentiate into osteoblasts after osteogenesis induced in vitro.3.CT imaging of the nude mouse showed that the density of the scaffold enhanced gradually in both groups.While the signal of MRI varied,the signal of labeled scaffolds became more and more bright as time passed,with the value of R2 and R*2 declined.The T2 weigh imaging of unlabeled groups changed from hyperintensity to hypointensity.The H&E staining demonstrated that both group developed with scaffolds degradation and new bone formation,in keeping with CT and MR imaging.Conclusion:In summary,this work presents a novel functional system fabricated from USPIO-labeled SF/HA scaffolds,and confirms that the optimal USPIO concentration is 0.75%(w/w)by the MR visualization and cytotoxicity assay of the composite scaffolds.Either scaffolds labeled or non-labeled are proved not only to be enable for adhesion,proliferation,and differentiation of BMSCs,but also to be favorable for stable MR imaging in vitro.In vivo studies of nude mouse reveal that the SF/HA scaffolds can have a beneficial effect on ectopic osteogenesis and degradation of the composites was monitored longitudinally using MRI relaxation rate measurements.This USPIO-labeled SF/HA scaffolds with BMSCs co-cultured should provide a practical approach to monitoring materials degradation noninvasively and effectively promote bone regeneration.