Effect Of Modified HBSS Solution On Biological Characteristics Of Human Periodontal Ligament Cells In Vitro

BackgroundDental trauma is a common dental problem among school children and young people. The prevalence of dental trauma is estimated to be15～20%.According to the American Academy of Pediatric Dentistry, tooth avulsion is defined as the "complete displacement of a tooth from its alveolar socket". It is one of the most serious of all dental trauma and constitutes as much as16%of all dental trauma [3].When a tooth is avulsed, the periodontal ligament is completely torn and small localized cemental damage, attachment damage and pulp necrosis also occurs. If the treatment for avulsed teeth is not appropriate, it will be result in teeth loss. This could particularly affect the patient’s face appearance, masticatory function and digestive function. For the young patients, this will atfect their physical and mental development.The ideal treatment of it is immediate replantation, thus reestablishing the natural nutrient supply to the periodontal ligament, thereby maximally maintaining the vitality and proliferation of the PDLC, enhancing the possibility of forming functional healing (FH). This will maximally preserve the patien’s injuried teeth, maintain the integrity of dentition. Although immediate replantation has been shown to have the best prognosis, but this rarely occurs. If immediate replantation is not possible, then the tooth must be stored in an adequate medium to maximally preserve the vitality of the PDLC attached to the root surface. The number of viable cells on the root surface decreases with increases drying time and that after2hours of extraoral time it is not possible to demonstrate cell vibaility. Cvek’s study demonstrated that13%of teeth kept in a dry state for15min,40%of those kept in a dry state for20～40min, and100%of those stored dry for more than60min showed signs of replacement resorption(RR).RR and inflammatory resorption(IR) are the major causes of failure of replanted teeth. Andreasen JO had analyzed the factors which impacted on FH following replantation. The results revealed that the following4factors had the strongest impact upon FH, in descending order of significance:stage of root development; length of the dry extraoral storage period; immediate replantation and length of the wet period.Some experimental studies have indicated that using a storage medium to preserve the vitality of the PDLC is a more critical prognostic factor than shortening the extraoral time to prevent IR and RR.An ideal storage medium should have a physiologic osmolality and pH, can maintain the viability and proliferation of the PDLC for a long time. Also it must be readily available and has a long shelf life. Hank’s balanced salt solution (HBSS) is recommended by The American Association of Endodontists as the best storage medium for avulsed teeth. HBSS is composed of essential nutrients necessary for cell survival, has an osmolality of290～320mOsm/L and a pH of7.2which is suitable for cell growth. Its widespread use has not been realized because it is not widely available in drug stores. Milk has gained wide acceptance as a storage medium as a result of containing some nutrients.growth factors and low bacterial content. However, whether the effectiveness of milk is as effective as HBSS is still controversial. Some studies show that it can only preserve cell viability for up to3h. ViaSpan, a storage medium for organ transplantation is also used for preserve viability of the PDLC. A study showed that ViaSpan was very effective in preserving the viability of lip fibroblasts for up to168h. Another study showed that neither IR nor RR was seen among the replanted dogs teeth stored in ViaSpan for6h or12h. Althogh ViaSpan has the capacity to maintain cell viability for a longer period, it’s expensive and is not readily available. Buttke TM considered that ViaSpan contained reduced glutathione (GSH) which could function as an antioxidant agent and protected PDLC from oxidative damage. His experiment also confirmed this hypothesis.The replanted dogs teeth stored in the HBSS supplemented with catalase (100U/ml) resulted in a statistically significant lower level of surface resorption than that stored in HBSS[3].A kind of storage medium for avulsed tooth named tooth rescue box is produced by Dentosafe in Germany. The medium was shown to maintain vitality and proliferation of PDLC for up to48h at room temperature in vitro. A long tern follow-up about the effectiveness of the tooth rescue box was reported. All the six avulsed teeth rescued in the tooth rescue box, the storage duration varied between1h and53h, showed FH. It can be seen that choosing an appropriate storage medium to keep the avulsed teeh wet plays an important role in maintaining the viability of PDLC and in improving the prognosis of replantation.At present, there are few researches about storage medium for avulsed tooth in our country. There also isn’t any commercial storage medium for avulsed tooth on sale. The purpose of this study is to finish the preparation of modified HBSS bases on HBSS, and compare it with HBSS on the biologic characteristic of PDLC in vitro. This study provides the primary experimental evidence for the development of a commercial storage medium for avulsed tooth.Chapter One:Primary culture and identification of human PDLCObjectives:To isolate and culture the human PDLC in vitro, and then identify the source of the cells.Methods:1. Isolate and culture of human PDLC: Normal maxillary or mandibular premolars with no caries or periodontal disease for orthodontic treatment were collected. PDL tissues were scraped from the middle1/3of the root and then cultured by tissue explant combined enzyme digestion. The digestive juice was mixed by3mg/ml collagenase type I and4mg/ml dispase in a volume ratio of1’.1.2. Identification of human PDLC: The expression of vimentin and keratin were detected in the cultured human PDLC by immunohistochemical method.3. Drawing the growth curve of human PDLC: The growth curve of cultured human PDLC was detected by MTS assay.Results:1. Morphological characteristics of human PDLC: Cells swim out of the tissues at the fifth day of primary culture, and the cells aren’t significant difference in cell morphology, the majority are fibrous long shuttle, and a small number of the cells presented as polygonal or spindle-shaped, and hae round nucleus. At the twelfth day of primary culture, cells connected with cytoplasmic extensions and lamellipodia and reached80%convergence. Cells arranged with a certain degree of directionality, by spiral-type. After staining with HE, the nucleus of the cells appeared round and bule, the cytoplasm appeared red.2. Immunohistochemical characteristics of human PDLC: Immunohistochemistry showed that cells positive for vimentin staining, brown positive particles distributed uniformly in the cytoplasm; and negative for keratin staning, no brown positive particle was seen. It is confirmed that the cultured cells derived from the mesenchyme.3. Characteristics of the growth curve: In the first48h after inoculation, the cells grew slowly, and then gradually accelerated growing, and came into logarithm growth period on the third day and flat period on the seventh day.Chapter Two:Effect of modified HBSS on proliferation of human PDLC in vitroObjectives:To prepare the modified HBSS bases on HBSS, and to detect its effectiveness in maintaining the proliferation of human PDLC in vitro by MTS assay.Methods:1. Preparation of modified HBSS:100U/ml penicillin and streptomycin,8mg/L dexamethasone were added to HBSS. The above soultion was thoroughly mixed and divided into three equal parts, then reduced GSH was added, at the concentrations of1,3,5mmol/L. At last, the pH value of the solution is adjusted with NaOH (pH7.2-7.4), then the soultion was filtrated and disinfected for use. 2. The well-grown4th passage of human PDLC were seeded in culture plates of96wells. The proliferation of human PDLC stored in the above three modified HBSS, HBSS, HC-A kidney preservation solution and H2O at room temperature (25℃) for0.5～48h was detected by MTS assay.Results:1. The proliferation of human PDLC:HBSS and modified HBSS were more effective than HC-A and H2O from0.5-48h (P<0.05); there was no significant difference between HC-A and H2O (P>0.05); group3mmol/L and5mmol/L GSH were more effective than group HBSS from2-12h (P<0.05); group1mmol/L GSH was more effective than group HBSS from2-6h (P<0.05); and group5mmol/LGSH was more effective than other modified HBSS groups from2-12h (P<0.05). There was no significant difference between group1mmol/L GSH and group HBSS at1and12h (P<0.05). Thus, there was also no significant difference between the modified HBSS groups and group HBSS from24～48h (P>0.05).Chapter Three:Effect of modified HBSS on survival and clonogenic capacity of human PDLC in vitroObjectives:To evaluate the effectiveness of modified HBSS in maintaining the viability and clonogenic capacity of human PDLC in vitro by trypan blue exclusion assay and colony-formation assay.Methods:1. The well-grown4th passage of human PDLC were used. The viability of human PDLC stored in modified HBSS, HBSS and H2O at room temperature (25℃) for0.5～48h was determined by trypan blue exclusion assay. Then the percentage of living cells was calculated.2. The cells not used in trypan blue staining were used in the colnoy-formation assay. The cells were diluted, then were seeded in culture plates of6wells containing2.5ml37℃culture medium in a gradient concentration of50,100,200cells/well.Results:1. The percentage of suvival human PDLC:Group3mmol/L GSH and5mmol/L GSH were more effective than group HBSS from2-24h (P<0.05); group5mmol/L GSH was more effective than group1mmol/L GSH from2-24h (P<0.05);3mmol/L GSH was more effective than group1mmol/L GSH from6-24h (P<0.05); there was no significant difference between group5mmol/L GSH and3mmol/L GSH (P>0.05), so it was between group lmmol/L GSH and group HBSS. There was also no significant difference between the modified HBSS groups and group HBSS at0.5,1and48h (P>0.05). There had been no living cells remained in group H2O since0.5h.2. The cloning efficiency of human PDLC:Group5mmol/L GSH was more effective than group HBSS from1-12h (P<0.05), was more effective than group1mmol/L from2-12h and more effective than group3mmol/L from6-12h (P<0.05);3mmol/L GSH was more effective than group HBSS from1-6h (P<0.05) and was more effective than group1mmol/L only at2h (P<0.05);1mmol/L GSH was more effective than group HBSS only at6h (P<0.05). There was also no significant difference between the modified HBSS groups and group HBSS at0.5,24and48h (P>0.05).Conclusions:1. The proliferation and clonogenic capacity of human PDLC stored in modified HBSS were significantly higer than that stored in HBSS within12h. This effectiveness is increased with the increase of the concentration of GSH.2. The modified HBSS contains5mmol/L GSH maybe a more effective storge medium for avulsed tooth.