| The occurrence of total dislocation usually refers to tooth injury,which means the tooth is completely propped out of the alveolar socket,resulting in pulp and periodontal membrane damage at the same time,and the alveolar socket becomes very empty.Total dislocation of teeth is a common and frequently occurring disease in oral cavity.Among all dental dislocations,permanent anterior complete dislocation is particularly common,and it often occurs in adolescence.This kind of tooth injury is caused by sudden external forces,including falls,violence,traffic accidents and other factors,which make the tooth completely protrusion from the tooth socket,thus causing the pulp and periodontal ligament rupture.Tooth trauma is the most serious injury,and the prognosis is often poor,if the patient’s teeth are not properly handled,it may lead to the loss of chewing function and other untreatable dental trauma.Clinically,if the patient’s dental conditions permit,the lost teeth can be implanted again.Part of the problem with the implants is that they can no longer recreate the natural pulp and periodontal membrane.Most of the surviving teeth are also dead pulp teeth,which are gray and lack life.At the same time,due to the lack of periodontal membrane,the dental body and alveolar bone are connected by osseous integration.With the loss of the natural pulp and periodontal membrane,the tooth implant can disintegrate as the enamel disintegrates,and the root is absorbed,causing the patient to completely lose normal function.Therefore,the restoration and regeneration of the pulp and periodontal membrane of the tooth with total dislocation has become a major problem in the field of oral science and has not been solved yet.Tissue engineering refers to extracting stem cells with self-renewal and multi-directional differentiation potential from the body to cling to and stick on the specific biological scaffold material after multiplication culture,and then constructing composite tissue engineering applied to the body after composition in vitro culture and provide growth factors according to the needs of the body so as to promote tissue repair and tissue regeneration.The development of tissue engineering has provided new ideas and methods for periodontal pulp repair and regeneration of delayed replantation teeth.Seed cells,the general term for various types of stem cells used for regenerate tissues and organs in tissue engineering can be obtained from the body and isolated and expanded in vitro.After being combined with the scaffold structure,seed cells can be implanted into the damaged location of the tissue to become the origin cells for regeneration and repair.Currently,researchers believe stem cells can be divided into embryonic stem cells and adult stem cells according to the source.Embryonic stems are totipotent cells.However,due to the strict control embryonic stem cell in ethics and the control difficulty of its growth and differentiation,the focus of research in recent years is still focused on the direction of adult stem cells.Human umbilical cord mesenchymal stem cells(h UCMSCs),as a member of the mesenchymal stem cell family,are derived from umbilical cord tissue and have better multi-directional differentiation potential,self-renewal ability and low immunogenicity than other adult stem cells.As a result,many scholars have used h UCMSCs as seed cells to study tissue repair and regeneration.However,there are rare reports on implanting h UCMSCs as seed cells into the periodontal space and root canal to promote periodontal pulp regeneration.Concentrate growth factors(CGF)fibrin,as a new generation of platelet concentrate,is prepared by differential centrifugation.The large amount of fibrin collagen contained in CGF forms a three-dimensional network structure which is more stable than the previous two generations.This stable fibrin grid-like structure provides a stable physical platform for cell climbing and adhesion.Furthermore,the fibers are connected in a three-dimensional direction,which is beneficial for migration of cells,transportation of nutrients and oxygen and the discharge of metabolites.In addition,CGF contains most of the growth factors in the centrifuged blood.These growth factors are slowly released during the application process,which in turn makes the soft tissue healing of the patient be more rapid and natural.TAZ,also known as WWTR1 gene,is located on human chromosomes3q23-q24.Initially,it is discovered that TAZ,a binding protein,can enter the nucleus,bind to transcription factors,activate transcription factor activity and promote gene expression.Previous studies have shown that TAZ can inhibit the PI3K/Akt pathway of mesenchymal stem cells,thereby promoting osteogenic differentiation of cells.Down-regulation of the Smad4/TAZ axis can cause mesenchymal stem cells to delay osteogenic differentiation and increase adipogenesis.However,TAZ on the proliferation and migration of human umbilical cord mesenchymal stem cells and human periodontal ligament cells co-culture system has not been reported yet.In this experiment,human-umbilical cord mesenchymal stem cells were isolated and extracted and cultured in vitro with CGF membrane prepared by differential centrifugation of venous blood to construct a tissue engineering complex.And then,the complex was implanted into the periodontal of the dog’s permanent anterior teeth with delayed replantation.The recovery and regeneration of the periodontal and dental pulp tissues of the delayed replanted teeth were observed histologically,and the mechanism of human umbilical cord mesenchymal stem cells to promote periodontal regeneration was initially explored from the perspective of molecular biology,providing theoretical and experimental basis of the success rate of clinical avulsion after delayed replantation and opening up new ideas and methods.This research consists of three parts,and the content of each part is summarized as follows.Part One Culture and Identification of Human Umbilical Cord Mesen-chymal Stem Cells in vitroObjective:To isolate and extract human umbilical cord mesenchymal stem cells,culture them in vitro and identify the morphological and biological characteristics.Methods:Collect the umbilical cord of 30 cm from healthy term newborns delivered by caesarean section,peel off the gel-like Wharton’s glue,separate and extract human umbilical cord mesenchymal stem cells with tissue block method,perform proliferation and culture in vitro,and take the third generation of logarithmic growth phase cells to observe the cell morphology with inverted microscope and scanning electron microscope and cellular immunohistochemical Vimentin staining.Flow cytometry was used to identify the source of the cells,cell growth curve was drawn to detect cell proliferation,and Alizarin red staining was used to detect the osteogenic differentiation of cells.Results:According to the observed results with an inverted microscope for about 10 days,the cells gradually climbed out of the edge of the tissue block and grew in adherent way.The primary cells were heterogeneous,with polygonal,star-shaped,short spindle or long spindle shape.When the cells grew to the third generation,the cells have good homogeneity,long spindle-shaped and fibrous,rich in cytoplasm and with large nucleus,and arranged in a whirlpool after proliferation.It was observed after electron microscopy scanning that the cells were large in size,with clear outline,most with protruding fusiform or polygonal structure.The nuclei were regular oval with large and clear nucleoli.It was observed a light microscope through immunohistochemical staining that vimentin staining was positive,cell cytoplasm was brownish yellow,nucleus was performed with blue dyeing;CK staining was negative,which was consistent with the characteristics of mesenchymal-derived cells.The results of flow cytometry showed that the surface markers of stem cells including CD29,CD44,CD146 and CD105were positively expressed,and the hematopoietic markers including CD34 and CD45 were negatively expressed,which showed that the cultured h UCMSCs were derived from mesoderm mesenchymal tissue.The cell proliferation curve was"S"-shaped.In Alizarin red mineralization experiment,after 21 days of culturing for osteogenic differentiation inducer,a large number of red calcified nodules were seen,which showed that the cultured h UCMSCs was characterized with proliferation and differentiation of stem cells.Summary:The isolated and extracted human umbilical cord mesenchymal stem cells are derived from mesoderm mesenchyme and are characterized with proliferation and differentiation of stem cells.Part Two Construction of tissue-engineered periodontal and dental pulp complex with human umbilical cord mesenchymal ste-m cellsObjective:Primary cultured human umbilical cord mesenchymal stem cells as seed cells are co-cultured with the prepared concentrated growth factor(CGF)in vitro to construct a tissue engineering complex.Histological observation and identification are performed.Observe the effect of CGF membrane in vitro on the activity of h UCMSCs;Observe the expression of transcriptional co-activator TAZ in h UCMSCs and the effect of CGF membrane on its expression;Construct taz gene overexpression and silencing cell lines,and observe the role of TAZ gene in this mechanism.Methods:Collect the venous blood of healthy volunteers and obtain CGF membrane with gauze film pressing method after differential centrifu-gation,perform histological sections staining with HE and observation of the microstructure with scanning electron microscope.After the human umbilical cord mesenchymal stem cells identified in the first part of the experiment as seed cells were selected and cultured to the third logarithmic growth phase,the cell suspension was inoculated on the CGF membrane by digestion and centrifugation and cultured for 7 days in incubator incubate under the condition of 37~oC、5%CO2.Inverted microscope,histological section staining with HE and electron microscope scanning were used to observe and identify the construction of tissue engineering complex.A co-culture system involving human umbilical cord mesenchymal stem cells and CGF was established in vitro.MTT was used to detect cell proliferation,alizarin red staining was used to detect mineralization,immunofluorescence staining was used to detect osteopontin expression,and alkaline phosphatase activity was detected,and the effect of CGF on the proliferation and differentiation of human umbilical cord mesenchymal stem cells was observed.Western Blot was used to detect the expression of TAZ in nucleus and cytoplasm of human umbilical cord mesenchymal stem cells;lentiviral plasmids for overexpression of Tafazzin(pc-TAZ)and knockout of TAZ(sh TAZ)was prepared,and alizarin red staining was used to observe the effect of overexpression and knockout of TAZ on the osteogenesis ability of human umbilical cord mesenchymal stem cells(h UCMSCs).Results:According to the results of the HE staining observation of CGF membrane,CGF membrane was divided into three layers from the bottom,including red blood cell layer,white blood cell layer and fibrin mesh structure.Among of them,the fibrin mesh structure was considered as the dominant.According to the electron microscopy scanning results,it showed a dense three-dimensional three-dimensional network structure with small inter-fiber pores.After tissue engineering complex construction,h UCMSCs was seen crawling out of the surface of the CGF membrane under an inverted microscope.The cells around the membrane were in the shape of a long spindle and are closely arranged.The long axis of the cells was basically perpendicular to the surface of the CGF,and the cells grew in a radioactive way with the membrane as the center.Observed by electron microscopy scanning,it was showed that h UCMSCs was fully stretched on CGF membrane,adhered closely,grew well and had a normal morphology,growing in an evenly distributed way around or across the pores.The results of MTT detection,Alizarin red staining and immunofluorescence staining detecting the expression of osteopontin and alkaline phosphatase detection showed that CGF promoted the proliferation and osteogenic differentiation of h UCMSCs;the results of Western Blot detection showed that CGF fibrin membrane not only affected the basic physiological functions of umbilical cord mesenchymal stem cells,but also regulated the up-regulation of intracellular osteogenic-related genes including RUNX2,OCN and ALP m RNA.With the intervention of CGF fibrin membrane,the expression of TAZ in cytoplasm and nucleus was significantly up-regulated,indicating that CGF fibrin membrane promoted the nucleus transfer of TAZ;the results of Alizarin red detection showed that CGF further promoted the enhancement of the mineralization ability of h UCMSCs in addition to overexpression of TAZ,and inhibited TAZ knockout leading to weakening of the mineralization ability.Summary:Human umbilical cord mesenchymal stem cells can grow well on the CGF membrane,which has good biocompatibility,so the tissue engineered pulp and periodontium complex can be successfully constructed.CGF membrane can promote the proliferation and osteogenic differentiation of h UCMSCs.CGF can promote nucleus transfer of TAZ and its expression in the cytoplasm and nucleus of h UCMSCs.Overexpression of TAZ can promote the mineralization ability of h UCMSCs.The mineralization ability of h UCMSCs is weakened after knocking out TAZ;CGF can reverse the osteoinhibition effect after knocking out TAZ and enhance the osteogenic effect after overexpression of TAZ.Part Three Animal experiment on pulp and periodontal regeneration of the permanent anterior teeth with complete dislocationObjective:Implant the tissue-engineered complex constructed in vitro into the periodontal space of the permanent anterior teeth with delayed replantation of canine avulsion injury and the root canal system with the pulp tissue removed and observe the restoration and regeneration of the periodontal pulp tissue.Methods:Six healthy adult beagles with complete teeth and no other periodontal disease or systemic disease were used in the study.For each beagle,upper and lower incisor teeth were selected,and the experimental teeth were spaced,so a total of 36 teeth were obtained for 6 dogs.All 36 teeth were randomly digitized and divided into three experimental groups,including normal control group,untreated trauma group and tissue engineered repair group,with 12 teeth in each group.The experimental animals were fasted with water for 12 hours before operation,and Lu Mianning was injected into the hip muscle to induce anesthesia,and sodium pentobarbital was injected intramuscularly to deepen the anesthesia.The study mainly performed the dental position surgery according to the group number.Normal control group did not do any treatment;The untreated trauma group and the tissue engineering repair group all needed the intervention treatment of extraction of experimental teeth.The teeth in the latter two groups were extracted by tooth extraction forceps.Sterile gauze was used to hold the experimental teeth,and the high-speed turbo handpiece was cooled by cold water spray,and the 3mm dental tissue at the root tip of the tooth was cut with a high-speed sharp split drill.The pulp extraction needle was inserted into the root canal upward from the apical hole to remove the pulp tissue in the root canal.Nickel-titanium open files and K-shaped root canal files were used to increase the number of inverted preparations in the root canal,and the diameter of the apical hole at the cut was expanded to 1mm.Rinse the tooth root with normal saline,place it in a sterile stainless steel tray,and dry it for 1 hour at room temperature to ensure extensive necrosis of the periodontal ligament tissue.The root curette was used to gently remove the necrotic periodontal ligament.the teeth in the untreated group were not treated.The root of the removed pulp and periodontal tissue was reimplanted into the patient’s alveolar socket.In the tissue engineered restoration group,the prosthesis was poured into the prepared apical hole and placed into the root canal and pulp cavity,while the tissue engineered prosthesis wrapped in the apical area was re-implanted into the alveolar fossa.The light-curable flowing resin was used to bond the fixed fiber tape to the tooth surface as a periodontal splint to elastically fix the teeth in the operation area.The animals were put to death after 12 weeks,and the jaw bones were taken and fixed and cut and separated according to the tooth position into tissue blocks,with 5-8mm thick.In addition,the issue blocks containing experimental teeth and periodontal tissues were labeled,decalcified,and embedded and performed with section and HE staining.Tissue sectioning observation results were selected for comparing the restoration of periodontal and dental pulp in different groups and performed statistical analysis.Results:After 12 weeks of histological observation,it was found that the structure of dental pulp and periodontal ligament in the normal control group was clear.At the same time,the periodontal space was narrow,the collagen fibers were arranged in bundles,and the perforated fibers were not obvious.In the untreated group,there were pulp cavities,no pulp and anterior dentin formation,and broken residue(dentin disintegration).There is no periodontal ligament structure in periodontal tissue.At the same time,there are a large number of osteoclasts and resorption lacunae at the junction of proper alveolar bone and cementum.In the tissue engineering repair group,there was dental pulp regeneration in the dental pulp cavity,and it was found that the dental pulp structure was clear,there were dental pulp cells,a large number of collagen fibers and blood vessels,and dentin and calcium spheres could be seen in the dentin wall.Periodontal ligament formation.The collagen fibers were arranged diagonally,and the perforated fibers could be seen.Summary:Human umbilical cord mesenchymal stem cells and CGF membrane were used to construct tissue engineered dental pulp periodontal complex to repair the dental pulp and periodontal tissue after complete dislocation of permanent anterior teeth in dogs.Conclusions:1.It has been found that human umbilical cord mesenchymal stem cells can become ideal stem cells for dental pulp and periodontal regeneration.2.CGF membrane is an ideal scaffold material for dental pulp and periodontal regeneration.3.CGF membrane can promote the proliferation and osteogenic differen-tiation of h UCMSCs;CGF can promote the nucleus transfer of TAZ and pro-mote the expression of TAZ in the cytoplasm and nucleus of h UCMSCs;TAZ can regulate the mineralization of h UCMSCs;CGF can promote the viability of co-culture cells and reverse the inhibitory effect after knocking out TAZ and enhance the promoting effect after TAZ is over-expressed.4.To study the tissue engineering periodontal complex constructed by human umbilical cord mesenchymal stem cells and CGF membrane,so as to successfully repair regenerated periodontal tissue and obtain the most ideal tissue morphology. |
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