A Preliminary Study On Novel Osteogenic Materials Composed Of Collagen Or Collagen-mimic Substance

Collagen is an important organic component of bone.The collagen content in normal bone tissue is about 20% in weight.Collagen is a stable fibrous protein with triple helix structure,insoluble in water,and participates in formation of extracellular matrix(ECM).The semi-crystalline fibers formed by collagen can provide the necessary anti-stress and elastic supporting force for attaching cells,and play important roles in cells’ migration,proliferation,and differation.Collagen type I(COL-I)is the major component of bone extracellular matrix and critical in the maintenance of bone cell functions.It has been a most commonly used bone tissue engineering scaffold.The natural porous structure and fantastic hydrophilicity of COL-I make it an ideal material for bone cells attachment and ingrowth.Scaffolds of COL-I have been proved effective for bone regeneration in the form of sponges,membranes or gels.However,the strength of collagen is very low and the degradation rate is high in vivo.Simple collagen scaffolds are not appropriate for repairing bone defects in a wide range,or requiring bone grafting with load capacity.It is necessary to make composed artificial biomaterials to meet the requirements of oral and maxillofacial skeleton reconstruction.And the morphological structure and functional components of the maxillofacial bones are useful guidance for the investments.In the previous research of this group,calcium phosphate cement(CPC)was used to simulate the natural bone inorganic components to construct tissue engineering bone substitute materials.The obvious advantage of the materal is that it has sufficient load strength and can be used as a supporting material.CPC is moldable before self-set and can be filled into irregular bone defects.At the same time,CPC also has osteoconductive ability,good biocompatibility and bio-safety.It can be used as carrier for bioactive agents due to its mild self-setting reaction.However,its shortcomings are also prominent.In terms of osteogenesis,CPC still lacks osteoinductive activity and cannot form bone by itself.The degradation rate of CPC in the body is unable to synchronize with the ingrowth of normal bone tissue.In order to solve these problems,we choose to construct collagen-CPC composite materials.The permeability and hydrophilicity of collagen increase the porosity and pore diameter of composite CPC scaffold.The biomechanical and other properties were tested in vitro.And the biological properties of the composites were observed in vivo.We hope identify an effective approach for the construction of CPC-based bone engineering scaffolds through incorporation of collagen to promote material transformation to bone in vivo.The collagen materials currently used in research are mostly made from heterogeneous animal sources.There are contradictions in preparation of the collagen to preserve its biological activity and eliminate antigenicity and pathogenicity.The osteogenic performance of the material was unstable,which limited its applications.To obtain human originate collagen or collagen-like materials with better bio-activity and safety became one focus of our research.Based on previous research of the group,we proposed to construct a novel composite hydrogel scaffold with self-assembled peptide RAD16-I and neurotransmitter peptide Substance P(SP).Human umbilical cord mesenchymal stromal cells(hUCMSCs)were used as seed cells in the hydrogel.The neural peptide substance P has osteo-inductivity.And hUCMSCs were induced through stereoculture in vitro for a long time.The biological characteristics,osteogenic differentiation phenotype and histological structure of RAD16-I/SP/ hUCMSCs complex were observed.And the feasibility of artificially constructing of human collagenalike stem cell complex was explored.Part I: A viable method for improving the biological activity of self-curing calcium phosphate materials by collagenOBJECTIVE: To explore a viable method for improving the biological activity of calcium phosphate scaffold by using collagen as an organic component.METHODS: Rat tail tendon collagen type I(COL-I)lyophilized sponge,neuropeptide polypeptide substance P(SP)solution and self-curing calcium phosphate(CPC)powder was used to construct pure CPC(group A),CPC/SP(group B),CPC/COL-I(group C)and CPC/SP/COL-I(group D).Detecting and analyzing the setting time,phase composition,ultrastructure and mechanical properties of each group by XRD,SEM,etc.The annular bone defect of rabbit femurs were prepared.Then CPC and CPC composites were implanted respectively.The rabbits were sacrificed after 8 weeks.The osteogenic transformation of CPC composite in vivo was observed.RESULTS: The initial setting time and terminal setting time of composites in group C and group D were prolonged compared with group A and group B.The initial setting time of group A and group B was statistically different from group C and group D,respectively(P<0.05).XRD analysis of CPC and its composites showed that the addition of COL-I and SP did not change the phase composition of CPC,nor introduce other inorganic components.Scanning electron microscopy of the composite materials showed that the pore size between the particles after setting was less than 10-15μm before the COL-I sponge was introduced.But the structure and bonding mode of the materials changed after the COL-I sponges were introduced,and the pore diameter of the composites up to about 70 μm.It is found that the compressive strength of Group C and Group D was slightly lower than that of Group A and Group B after the composites were fully cured and dried.But there was no statistical difference in compression intensity between the groups(P>0.05).The elastic modulus of the composites of group C and group D were lower than that of group A and group B by the combination of COL-I materials,and the results were statistically significant(P<0.05).The results of the hard tissue specimens in the animal experiment showed that the CPC around the implant was not completely degraded.There was no obvious new bone formating around the implants.In the CPC composite materials group,the composite materials were completely degraded,and new bone ingrowth around the implants were observed.CONCLUSION: The CPC/COL-I composites have good biological and mechanical properties.The addition of COL-I significantly increased the microscopic pore diameter of the composites.It also increased the ductility of the composites.The the phase composition,crystal structure and microstructure of CPC were uneffected by introducing COL-I and SP.The introduction of collagen can effectively improve the biological activity and degradation rate of CPC materials.It is a feasible method for constructing bioactive scaffold materials.Part II: Study on construction of collagen-like matrix by self-assembled peptide complex hUCMSCsOBJECTIVE: To construct a matrix environment using self-assembled collagen-like peptides RAD16-I and SP,inoculated hUCMSCs in vitro.Observing the transformation of the complex to collagen-like bone.METHODS: Mesenchymal stem cells in Wortton gel of human umbilical cord tissue were obtained by enzymatic digestion method.Immunofluorescence and flow cytometry were performed on cell surface markers.The hUCMSCs were induced by osteogenic and adipogenic differentiation.Cell differentiation was observed by alizarin red and oil red O staining.The self-assembled collagen-like peptide RAD16-I and SP were used to construct a collagen-like matrix environment,hUCMSCs were cultured in stereo mode for 1-4w.Cell proliferation,viability,osteogenic related gene expression,histological features,injectable features etc.were performed.in the study.The growth of hUCMSCs in collagen-like matrix environment of RAD16-I/CPC composites were observed.RESULTS: The human umbilical cord mesenchymal stem cells obtained by primary culture.They had long spindle shapes and grown well.The purity of the cells was more than 90% by immunofluorescence.The stem cell surface antigen marker CD29 was identified by flow cytometry.The expression of CD44,CD73,CD90 and CD105 was positive,and the expression of hematopoietic markers CD34,CD45 and CD106 was negative.It was identified as hUCMSCs.The hUCMSCs were cultured in adipogenic environment for 4 weeks.Lipid droplets were found in the study group.No obvious lipid droplets were formed in the control group.Osteogenic induced hUCMSCs for 4 weeks,we found mineralized nodules were formed in the study group.The control group had no mineralized knots.The hUCMSCs cultured in the collagen-like matrix were observed under an inverted microscope at 40×,we found that the cells in the stereogenic culture group were aggregated at the early stage after inoculation,and then gradually expanded.The cells increased gradually,showing a long fusiform or irregular polygon.Cell proliferation assay(CCK-8)calculated the OD values of the cells in each group at 1d,2d,3d,5d and 7d.The cell proliferation rate of the control group was the lowest at each time point.Each group were logarithmized increasing at 3-5d.On the 7th day,the proliferation of the other three groups was statistically different than that of the control group(P<0.05).There was no statistical difference between the study groups.Fluorescence staining of live/dead cells showed that the cells were generally greenstained,indicating that most of them were living cells.The number of cells gradually increased,and the cell morphology gradually extended with a long fusiform or irregular polygon.Only a very small amount of cells were red-stained.The percentage of viable cells stayed in a high state in the whole process.The results of real-time quantitaty PCR showed that the expression of bone-related genes ALP,OCN,RUNX-2 and COL-1 was significantly higher in SP group and SP/RAD16-I group than the other two groups.After 4 weeks of culture,the three-dimensional culture group and the SP-containing planar culture group showed mineralization.Histological observation showed that the cells in the threedimensional culture group were aggregated in a mass.There was a collagen-like matrix environment around the cells,which was larger than the two-dimensional planar group.The cell/collagen-like matrix composites in 3D culture group accounted for more than 90% of the viable cells after injection.The cells could adhere tightly to the materials and the state of cells after being combined with CPC composites was in form.CONCLUSION: hUCMSCs can be successfully obtained from human umbilical cord tissue by collagenase type I digestion.The multi-directional differentiation ability of hUCMSCs were observed by osteogenic induction and adipogenic induction.The hUCMSCs can grow,proliferate and migrate stably in a three-dimensional culture system of rib collagen matrix constructed from self-assembled collagen peptides.It was more active and efficient than two-dimensional planar culture.The osteogenesis gene expression level is also higher than that of the planar culture group.The self-assembling peptide RAD16-I can promote the growth,proliferation and differentiation of stem cells as a stereo culture/transplant system.Building a collagen-like matrix that can maintain an active state.The combination of RAD16-I/SP/ hUCMSCs can be used as a collagen-like tissue engineering material for preclinical studies in regenerative medicine.