The Osteogenetic Efficacy And Mechanism Of Self-assembling Peptide/demineralized Bone Matrix Based On SCR Technology

BackgroundAutologous bone graft is still the most effective method in clinical practice for bone restoration. However, the donor sites of autogenous bone graft are caused the "second trauma," and take a limited amount of bone. This manner does not apply to the elder and the young especially. Therefore, the study of alternative autologous bone graft is important for clinical practice and social needs.The osteogenetic roles of autologous bone primarily due to the following characteristics: 1) osteogenesis, preosteoblasts/osteogenitor cells that are capable of differentiating into mature osteoblasts; 2) osteoinductive, the capable of osteogenic growth factors stimulating the preosteoblasts/osteogenitor cells to differentiate into osteoblasts and osteoclasts; 3) osteoconductive, scaffolds provide the necessary supporting role for bone growth. In recent years, an alternative method for autologous bone has been a focus and difficult field in orthopedics, the research focused on the following points: 1) Tissue engineered bone(TEB): it is the fusion of the main characteristics of autologous bone. In composite scaffolds of growth factor, cells are seeded on and given the necessary osteogenesis for bone restoration, especially bring the new hope for reconstruction of large bone defects. However, the longer cycle of stem cells amplification in vitro, the strict technology, the expensive treatment and ethical issues limit its clinical application; 2) local injection of bone marrow: bone marrow from patients is directly injected into restoration area, which are treated in bone cyst, nonunion and other difficult cases, and achieves excellent effect, but the amount of bone marrow that more than 200-300ml/time, the injections of multiple(about 2-3 times), and the bottleneck problem faced the unknown risks of amount of marrow aspiration and clinical acceptance of patients is not higher; 3) local injection of stem cells: the stem cell from centrifugal bone marrow in patients is injected into local bone restoration area. This method achieves gratifying efficacy in the treatment of avascular necrosis of the femoral neck, nonunion, but faces many insurmountable problems: such as the large amount of marrow aspiration(about 200-300ml), exogenous organisms reagent has to added into centrifugal separation reagent; 4) local injection of platelet-rich plasma: high density plasma is injected into bone restoration area by density gradient centrifugation of peripheral blood(or bone marrow), which is also facing many technical problems and clinical risk: such as the potential risks of exogenous biologics for the plasma separation, and the residence concentration is low in local bone restoration area as the fluid plasma. Rooted in the construction concept of autologous bone and tissue-engineered bone, Selective Cell Retention(SCR) technology has found their way into our surround. SCR technology can quickly integrate stem cells, growth factors, platelet and other ingredients of autologous bone marrow into the graft, and immediately transplant them into bone defect area in vivo. More important, the entire operation can be carried out simultaneously with the surgical procedure, and all operations can be done on the operating table. SCR technology not only can avoid the amplification of stem cell in vitro and ethical problems, but also avoid the potential risks of exogenous biological agents in existing enrichment technology. SCR technology brings a new method for alternative autogenous bone because of its clinical application and easy to industrialization.The key of SCR technology is to increase the rapid adhesion of function stem cell and related growth factors in bone marrow for bone grafts. At present, it is difficult to achieve this requirement with the single bone graft in clinical practice. We envision to increase the adhesive effect of the effective ingredient in bone marrow by bionics modified for allogenic demineralized bone matrix(DBM), which is the most commonly used bone graft in clinical practice. The usage amount of DBM is second only the autogenous bone in clinical bone grafts. However, DBM are not conducive to adhesion of cells and factors of bone marrow because of its large pore and damage surface structure due to decalcification, deproteinization and other operations. RADA16-I peptides are used to integrate DBM surface as biomimetic is a better choice, since it is safe and simμlated extracellular matrix(ECM). RADA16-I is a self-assembling peptide(SAP), the composition is monomer of 16 amino acids, and the repetitive sequence is arginine(R)-alanine(A)-aspartate(D). The final degradation product in vivo is an amino acid monomer, which has been confirmed its low risk and no significant immunogenicity in vivo experiments. SAP might to start the self-assembly process meeting Ca2+,Mg2+ and other cations under the physiological conditions of the human body, and to form a three-dimensional(3D) of nano-fiber network hydrogels, which contain water exceeds 90%, and has a certain mechanical strength, where is the suitable 3D microenvironment for the growth of stem cells, fibroblasts, endothelial cells and other cells. SAP can self-assemble into a hydrogel in the pores of DBM, and increase the interceptor role to marrow cells and growth factors in the role of SCR technology. Meanwhile, itself positive and negative charges of amino acid peptide monomer may increase cell and factor ingredients adhesion through the inter electrostatic adsorption.Thus, the first part, we should try to integrate DBM using self-assembling peptide RADA16-I, and analysis the characterization and stability of SAP/DBM composite.The osteogenic differentiation of MSCs in the SAP/DBM microenvironment were evaluated based on the SCR technology in the second part. The efficacy of osteogenesis of SAP/DBM composite for ectopic in nude mice and critical bone restoration in goat were evaluated in the third part. Finally, the mechanisms of osteogenesis of SAP/DBM based on SCR technology would be approached.Purpose1. We are tring to integrate DBM using self-assembling peptides RADA16-I, and analyze the characterization, stability and other characteristics of SAP/DBM composite, and are seeking an ideal scaffold for SCR technology.2. Evaluating the osteogenic differentiation of MSCs in the SAP/DBM in vitro, the ectopic bone restoration in vivo and the efficacy of osteogenesis of SAP/DBM composite in situ for goats based on SCR technology.3. Studying the probalble mechanisms of excellent osteogenesis of SAP/DBM based on SCR technology by protein chip technology and other methods.Method1. Self-assembly process of RADA16-I peptide was to start for modifying DBM by cation PBS, and to form a SAP/DBM composite. To observe the effect of self-assembling of peptides by atomic force microscopy(AFM), measure pore morphology by environmental scanning electron microscopy(ESEM), analysis its constituent elements by X-ray photoelectron spectroscopy(XPS), and further measure the stability of SAP/DBM composite by UV spectrophotometric spectrophotometer full wavelength spectroscopy rooting in the sensitivity between amino acids with UV.2. Bone marrow mesenchymal stem cells(BMSCs) of goats(person) were isolated and cultured by density gradient centrifugation, and the cell proliferation of BMSCs on SAP/DBM scaffolds was detected by MTT assay. The efficacy of osteogenic differentiation of BMSCs in SAP/DBM microenvironment was evaluated by laser confocal microscopy from morphology and fluorescence real-time quantitative polymerase chain reaction(RT-PCR) for quantitative determination.3. Model of ectopic bone formation in the gluteal muscle bag beside iliac of nude was prepared. The efficacy of osteogenesis of SAP/DBM based on SCR technology were calculated quantitatively by Micro-CT, and further evaluated by HE and Masson staining. More, large animal model of goat femur middle 2-cm critical bone defect in situ was used to validated the effect of bone formation by various methods of X-ray, CT(Micro-CT), histological staining at 8 and 16 weeks of after surgery.4. The enriched effect of bone marrow cells in SAP/DBM composite based on SCR technology was analyzed by flow cytometry, which including adhesion rate, the enrichment factor, etc. And specific cells were labeled by immune histochemistry. The types of enriched protein were analyzed by high-throughput protein microarray. The gene expression levels of osteogenesis were analyzed using PCR techniques by ectopic bone formation model of quadriceps muscle bag in nude, and further studied the the relationship between platelet-derived growth factor-BB(PDGF-BB) with other related proteins in vivo by protein microarray technology.Results1. Peptide RADA16-I could be successfully triggered by cation, and self-assembled to form a 3D shape of regμlar nano web fiber structure,which pore diameter formed mostly in 5 ~ 500 nm interwoven fibers. The effect of the peptide self-assembling of the concentration of 0.5%, 1%(w/v) was ideal. The aperture of SAP/DBM was(20.22 ± 6.726μm) by ESEM, which provided the physical structure for cell retention. XPS showed RADA16-I integrated well with the DBM by analysis of element in different materials. UV spectrophotometer analysis showed that the stability of SAP/DBM composite was excellent.2. Typical characteristics of BMSCs were nuclear cytoplasm ratio, spindle-shaped or conical imitation. Cell proliferation of CCK-8 showed growth trend of gMSCs in the SAP/DBM scaffold was better than DBM. The growth states of cells were stained by rhodamine-phalloidin-DAPI through LCSM, and showed better in the SAP /DBM than that of DBM, which penetrate into the depth inner nesh of SAP, and covered the entire SAP/DBM scaffold, and cells grown in DBM showed more along the holes wall spreading regional limitations. Meanwhile, the cell grown of gMSCs in concentration between 0.5% and 1% of the SAP/DBM had no significant difference for SAP /DBM. Gene expression trend of MSCs were consistent between 0.5% and 1% of the SAP/DBM microenvironment by PCR. At 14 d, gene expression level of OCN and OPN is higher in SAP/DBM than that of DBM, the difference was statistically significant(P <0.05).3. The efficacy of osteogenesis of ectopic muscle bag in nude was evaluated at 8 weeks post-operatively, which new bone including bone volume to total bone volume(BV/TV), trabecular number(Tb/N), bone density(BMD) were higher in SAP/DBM groups than that of DBM group, the differences between them had significant statistical significance(P <0.05, n = 8). At 16 weeks post-operatively, bone restoration for critical femur defect in goats were very complete in SAP/DBM groups, where bone morphogenetic were to approach normal bone tissue. However, incomplete healing was found in DBM groups, where new bone showed to coexist of lamellar and woven bone structure. We had also found that had no significant difference in osteogenesis effect between 0.5% with 1% peptide construct.4. Cells flow sorting confirmed much more functional cells of bone marrow were adhesive to SAP/DBM than DBM. Further, more nucleated cells and less red blood were adhered uniformly to SAP/DBM than DBM by ESEM. Immuno histochemistry showed that adhesive proportion of monocytes(CD14 +), hematopoietic stem cells(CD34 +), MSCs(CD73 or CD90 +) were significantly higher in SAP/DBM than DBM. Protein microarray results showed enriching growth factor in SAP/DBM mainly including BMP-6, PDGF-BB, VEGF and EGF(concentration>10pg/ml). PCR results showed signal pathway Wnt/β-catenin/Runx-2 might play an important role in bone formation process for SAP/DBM transplanting into vivo. Further, NR4A1/SP1 complex might be activated by high concentration of PDGF-BB through Akt signal pathway, and induced MSCs of host-derived directionally migrated to bone restoration area for bone restoration by the protein chip technology.Conclusion1. RADA16-I self-assembling peptides is an excellent biomimetic modifications for DBM, can self-assemble into a nano-mesh fiber structure under physiological conditions, and form a stable SAP/DBM composite. These excellent characteristics will provide the basis for cell biology research in vitro and for bone restoration experiments in vivo.2. The proliferation of BMSCs in the SAP/DBM scaffold is better than that of DBM, and the osteogenic differentiation of MSCs in the marrow-enriched SAP/DBM microenvironment based on SCR technology is better than that of DBM also.3. The efficacy of osteogenesis of ectopic muscle bag beside iliac in nude for the SAP/DBM based on SCR technology is significant, and in the large animal bone defect restoration in situ, SAP/DBM can successfully promote 2-cm goat femur critical bone defects healing. Meanwhile, it is clarified that 0.5% and 1% peptide has no significant difference on the large animal bone defect restoration.4. The mechanisms of osteogenesis of SAP/DBM based on SCR technology is resulted from the more enriched stem cells and a higher concentration of growth factors, and signal pathway Wnt/β-catenin/Runx-2 may play an important role in bone restoration. Moreover, PDGF-BB may activate NR4A1/SP1 complex by raise Akt signal pathway, which may recruit host-derived MSCs directional migration to bone restoration area to further promote bone formation.