In Vitro Osteogenic Induction Of Human Umbilical Cord Mesenchyme Cells Induced By Substance P

A large number of patients need medical treatments for various alveolar bone defects due to trauma,infection,or tumor resection each year.It is difficult to accomplish early functional restoration,like immediate placement of dental implant in post-extraction sites,in such poor conditions.Autogenousbone grafts have been used to repair bone defects.But there are problems in harvesting bone tissues,such as donor site trauma and limitation of the quantity of the bone grafts.Biomaterials with regenerative potential are investigated to meet the complex morphological structures and functional requirement of the reconstruction of maxillofacial skeleton.Composite biomaterials that architecturally and biologically compatible to natural bone were envisioned with several key considerations highlighted: the capacity to fill various bone defect,load bearing property,osteoconductivity,osteoinductivity,biocompatibility,and individual adaptability.Bone engineering techniques need large amount of stem cells to construct bioactive scaffold.At present,the main problems are the lack of seed cells,difficulties in stem cell harvesting,unstable efficiency and safety in osteogenic induction of stem cells.It is important to find a kind of practicable stem cell for clinical application that is readilyaccessible,safe and functional.This study uses human umbilical cord mesenchymal stromal(multipotential mesenchymal stromal cells)as a seed cell.Neuropeptides substance P(SP)is used as an osteogenic inducing bioactive factor.The osteogenic induction of the pluripotent stem cells in vitro is explored.It is hoped that this can provide a new solution to the above problems.Human umbilical cord mesenchymal cells(hUCMSCs)are obtained from Wharton's jelly in the umbilical cord that has been discarded from newborns.The cells can be isolated through collagenase digestion or inoculation of tissue blocks directly in culture dishes,and have the multi-differentiation potential of mesenchymal cells.The sources of h UCMSCs are extensive,accessible,and non-invasive.No medical ethics barriers exist because the cells are derived from waste tissue.Related experiments have confirmed that the cell is capable to differentiate into functional tissue cells such as bone,cartilage,fat,muscle,etc.Theyhaveuniqueadvantage to be used as bone tissue engineering seed cells.In previous studies,induction of human umbilical cord mesenchymal cell for osteogenic differentiation using artificially prepared induction medium containing dexamethasone,sodium glycerophosphate,and ascorbic acid.The solution has complex formulation,inefficient induction capacity,and low cytotoxicity,In vitro cultured cells frequently died.In this study,neurotransmitter polypeptide substance P(SP)was used to induce osteogenic differentiation of human umbilical cord mesenchymal cells.SP is a small molecule with highly conserved amino acid sequence(RPKPQQFFGLM).It exists widely in different species and in various types of tissues.Studies have shown that SP has the same amino acid sequence composition in humans,rabbits,and rats.When SP was first discovered,its main function was as a neurotransmitter.Recent studies have shown that SP is also a messenger molecule in a wound induced repair system and is an important bioactive factor that mobilizes stem cells and promotes wound healing.Compared with the commonly used macromolecule protein such as BMP and otherosteogenic factors,the SP has a simple amino acid sequence.Synthesis of the polypeptides is convenient and low cost.SP has better potential for clinical application due to its advantages in biosafety,bionic mechanism in osteogenic differentiation.In vitro experiments showed that SP canstimulate the proliferation and differentiation of bone marrow stromal cells(BMSCs)in a concentration-dependent manner.But the effect of SP on human umbilical cord mesenchymal cells has not been investigated.On the basis of previous studies of our research group,the purpose of this experimental study is to clarify the effect of SP on the proliferation and differentiation of human umbilical cord mesenchymal cells,and to explore the optimal conditions for SP-induced osteogenic differentiation of human umbilical cord mesenchymal cells.Further,a three-dimensional culture environment was constructed by using self-assembled peptide RAD16-I material to achieve in vitro three-dimensional culture of human umbilical cord mesenchymal cells.Self-assembling peptides are composed of amino acids with special sequence,with special structures such as polar and non-polar faces,and amino acids complementarily charged,so that hydrogen bonds,coordination bonds,and van der Waals forces are generated between short peptides,making them spontaneously and regularly assembled to form a polypeptide nanofiber network structure.The self-assembling peptide hydrogel have several advantages: 1)It is similar to the extracellular matrix,providing cells with a nanoscale three-dimensional microenvironment for better cell adhesion and growth.2)It is easy to be designedor modifiedin its amino acid residues that make up a polypeptide molecule to obtain specific biological activity.3)The hydrogel formed by the self-assembling peptide material has a water content of more than 99%,and the surface area is very large,allowing nutrients,biologically active molecules and oxygen to reach the cells inside the gels.4)The composition of the gel is confirmed,and the biocompatibility is good.There is no animal-derived pollution or immunogenicity.It can be absorbed or metabolized by the organism without side effects because the degradation product of the peptides is amino acid.A novel composite scaffold is primarily constructed withself-assembled peptide hydrogel and self-set calcium phosphate materials to simulate the natural bone composition and structure.It is aimed to form sustained release of SP in the scaffold material to induce the stem celldifferentiation.The in vitro transformation of the composite material is observed.We hopeto clarifythe osteogenic activity of the composite material,and make it a primary material model for future clinical application.Experiment 1: Primary Culture and Identification of h UCMSCsOBJECTIVE: To culture human umbilical cord mesenchymal cells(h UCMSCs),observe their microscopic morphology,identify the cells by various characteristic markers,and preliminary compare human umbilical cord mesenchymal cells(h UCMSCs)withhuman bone marrow mesenchymal cells(h BMSCs)on their osteogenic differentiationability.METHODS: Human Wharton's inner cells were obtained by enzyme digestion and cell surface marker molecules were identified by flow cytometry.Osteogenic-related ALP activity was detected by using osteogenic inducer to induce h UCMSCs and h BMSCs.Cellular mineral was observed by Alizarin Red staining.Ability.REULTS: Human umbilical cord Wharton's gel-derived cells obtained from primary culture have elongated spindles or irregular polygons,closely arranged and well-growthinvitro.They were identified as human umbilical cord mesenchymal cells(h UCMSCs)by flow cytometry with stem cell surface markers CD29,CD44,CD90,CD105 positive expression,and hematopoietic lineage markers CD34,CD45,CD106 negative expression,identified.Human bone marrow mesenchymal cells(h BMSCs)were purchased as control,and were prepared into osteoinductive culture medium.h UCMSCs and h BMSCs were induced by osteogenic differentiation.After 4 weeks,alizarin red staining was performed.Both cells formed obvious mineralized nodules.There was no significant difference in staining results.During osteogenic induction culture of h UCMSCs and h BMSCs,materials were collected in batches.The activity of ALP was detected and it showed that the ALP activity continued to increase.There was a significant difference from the normal culture medium culture control group.The difference was not significant.CONCLUSION: h UCMSCs can be successfully obtained from human umbilical cord Wharton's gel using type I collagenase digestion method.The differentiation of h UCMSCs and h BMSCs was compared using conventional osteogenic inductors.The results showed that the human umbilical cord mesenchymal cells(h UCMSCs)and human bone marrowwere confirmed.Human bone marrow cells(h BMSCs)have similar osteogenic differentiation ability.Experiment 2: Osteogenic Differentiation of h UCMSCs Induced by Different Concentrations of SPOBJECTIVE: To determine the feasibility of osteoblast differentiation induced by substance P(SP)in human umbilical cord mesenchymal cells(h UCMSCs)and the relationship between their induction and concentration.METHODS: SP was synthesized by peptide synthesizer and dissolved in stem cell culture medium to prepare induction culture medium at different concentrations(10-6M,10-8M,10-10M).Induction culture of h UCMSCs was performed,and cell ALP activity was periodically detected.Cell proliferation,osteogenic differentiation-related gene expression,and alizarin red staining were performed to observe the osteogenic differentiation of h UCMSCs.The feasibility and optimal concentration of SP for osteogenic induction of human umbilical cord mesenchymal cells(h UCMSCs)were explored.RESULTS: h UCMSCs were cultured with different concentrations of SP for 4 weeks.After alizarin red staining,significant mineralization was observed.There were visible calcium nodules in the culture wells,and most mineralized nodules formed in the 10-8 M SP group.h UCMSCs were cultured with different concentrations of SP to observe the cell ALP activity.The activity of ALP increased obviously from 1d to 7d.The activity of ALP was highest in the 10-8M SP group at 7d(P<0.05).In the experiment of h UCMSCs cell proliferation induced by SP with different concentrations,the OD values of the cells in the experimental group were increased compared with the control group,which indicated that SP could promote cell proliferation in different concentrations,and the 10-8M SP group had the strongest promotion effect,and the difference was statistically significant(P<0.05).Different concentrations of SP induced h UCMSCs 3W and detected the expression of osteogenic genes: The expression of ALP in experimental groups was statistically different from that in control group,and the expression was highest in 10-8MSP group.The expression of COL in experimental groups was higher than that in control group.Group,10-8M SP group was statistically different,there was no statistical difference between the other groups;OCN expression in each concentration of experimental group was higher than the control group,10-8M SP group expression was statistically different,the other groups were not statistically different The expression of RUNX-2 in each concentration of experimental group was higher than that of the control group,only 10-8M SP group was statistically significant,and there was no statistical difference between the other groups.CONCLUTIONS: SP can promote the proliferation of h UCMSCs and induce osteogenic differentiation.The differentiation of h UCMSCs induced by different concentrations of SP has different performance.The suitable SP concentration for the proliferation and osteogenic differentiation of h UCMSCs is 10-8M.Experiment 3: Self-assembled polypeptide gel RAD16 sterilized hUCMSCsOBJECTIVE: To explore the feasibility and suitable conditions for the three-dimensional cultivation of h UCMSCs in self-assembled peptide hydrogel RAD16METHODS: The RAD16 material was dissolved in stem cell culture medium,placed in the cell incubator,allowed to stand for gelling.After self-assembly to form a hydrogel structure,the structure of the material was observed under an inverted microscope,a stereomicroscope,and a scanning electron microscope.Inoculation of h UCMSCs was performed on an inverted microscope.The growth state of the cells was observed,and the growth,proliferation,and cell morphology of the cells in the self-assembled polypeptide gel were observed by live/dead cell fluorescent staining,and the cell count and the percentage of viable cells were calculated.RESULTS: Scanning electron microscopy showed that RAD16 material consists of a large number of nanofibers and has a three-dimensional network structure.The fiber diameter is about 8 to 10 nm and the length can be several hundred nanometers.Nano-scale fibers are connected to each other to form a three-dimensional network structure.The mesh pores are micrometers.level.The h UCMSCs cultured for 1,3,5,and7 days in RAD16 were observed under inverted microscope at 10×40 magnification.The cells were aggregated and aggregated in the early stage after inoculation,and then gradually expanded.The cells gradually increased and showed a long spindle type or a polygonal irregular type.Live/dead cell fluorescence staining showed that the cells were generally stained green,indicating that they were living cells,and the number of cells gradually increased.The cell morphology gradually expanded,showing a long shuttle or irregular multi-type,only a very small amount of cells stained red,the percentage of living cells was always In a higher state.CONCLUTIONS: The self-assembled polypeptide RAD16 hydrogel material has good cell compatibility and good pore structure.Human umbilical cord mesenchymal cells(h UCMSCs)can stably grow,proliferate and migrate in the self-assembled gel three-dimensional culture system.Experiment 4: Self-assembled peptide gel inoculated with h UCMSCs and SP-loaded self-set calcium phosphate cementOBJECTIVE: To explore a feasible method for constructing osteogenic active materials by self-assembled peptide gels inoculated with h UCMSCs and SP-loaded self-curing calcium phosphate(CPC).METHODS: hUCMSCs were inoculated into self-assembled polypeptide hydrogel RAD16 materials,and compound with SP-loaded CPC materials.Inverted microscopes were used to observe the material transformation and cell proliferation and differentiation.Scanning electron microscopy was used to observe cell adhesion and material binding,and live/dead cells.The cells were fluorescently stained.And the cell morphology,cell count,and percentage of viable cells were observed and recorded.The osteogenic related gene expression was examined at different stages to understand the osteogenic efficiency of the composite material.RESULTS: The composite material(CPC-SP-hUCMSCs-RAD16)showed cell adhesion and osteogenic differentiation.ALP expression peaked on the 7th day and subsequently remained at a higher level.Collagen I was not expressed in the early stage.Subsequently,it was expressed from the 14 th day,increased significantly,and peaked on the 21 st day.The expression of OCN and RUNX-2 gradually increased with time,and alizarin red staining showed mineralization.CONCLUSION: The composite material CPC-SP-h UCMSCs-RAD16 can promote cell differentiation and adhesion,and express osteoblast-related genes and form mineralized stroma.