Intra-Individual Variability of Human Dental Pulp Stem Cell Features Isolated from the Same Donor.

It is primarily important to define the standard features and factors that affect dental pulp stem cells (DPSCs) for their broader use in tissue engineering. This study aimed to verify whether DPSCs isolated from various teeth extracted from the same donor exhibit intra-individual variability and what the consequences are for their differentiation potential. The heterogeneity determination was based on studying the proliferative capacity, viability, expression of phenotypic markers, and relative length of telomere chromosomes. The study included 14 teeth (6 molars and 8 premolars) from six different individuals ages 12 to 16. We did not observe any significant intra-individual variability in DPSC size, proliferation rate, viability, or relative telomere length change within lineages isolated from different teeth but the same donor. The minor non-significant variances in phenotype were probably mainly because DPSC cell lines comprised heterogeneous groups of undifferentiated cells independent of the donor. The other variances were seen in DPSC lineages isolated from the same donor, but the teeth were in different stages of root development. We also did not observe any changes in the ability of cells to differentiate into mature cell lines-chondrocytes, osteocytes, and adipocytes. This study is the first to analyze the heterogeneity of DPSC dependent on a donor.

The Effects of Cryogenic Storage on Human Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) are a type of easily accessible adult mesenchymal stem cell. Due to their ease of access, DPSCs show great promise in regenerative medicine. However, the tooth extractions from which DPSCs can be obtained are usually performed at a period of life when donors would have no therapeutic need of them. For this reason, it is imperative that successful stem cell storage techniques are employed so that these cells remain viable for future use. Any such techniques must result in high post-thaw stem cell recovery without compromising stemness, proliferation, or multipotency. Uncontrolled-rate freezing is not a technically or financially demanding technique compared to expensive and laborious controlled-rate freezing techniques. This study was aimed at observing the effect of uncontrolled-rate freezing on DPSCs stored for 6 and 12 months. Dimethyl sulfoxide at a concentration of 10% was used as a cryoprotective agent. Various features such as shape, proliferation capacity, phenotype, and multipotency were studied after DPSC thawing. The DPSCs did not compromise their stemness, viability, proliferation, or differentiating capabilities, even after one year of cryopreservation at -80 °C. After thawing, they retained their stemness markers and low-level expression of hematopoietic markers. We observed a size reduction in recovery DPSCs after one year of storage. This observation indicates that DPSCs can be successfully used in potential clinical applications, even after a year of uncontrolled cryopreservation.

Cryopreservation of Dental Stem Cells.

Nowadays, regenerative and reparative medicine has grown in popularity. Dental stem cells are easily accessible source of adult stem cells. They can be harvested by a tooth extraction or spontaneous deciduous tooth exfoliation. They have to be isolated, expanded and stored until time they would be needed for individual stem cell therapy. Cryopreservation is both a short-term and long-term storage of tissues or cells at sub-zero temperatures. There are several methods of cryopreservation requiring different technologies. The objective of this review is to compare them and highlight their advantages and disadvantages.

Elution kinetics of vancomycin and gentamicin from carriers and their effects on mesenchymal stem cell proliferation: an in vitro study.

BACKGROUND: Musculoskeletal infections remain a major complication in orthopedic surgery. The local delivery of antibiotics provides the high levels required to treat an infection without systemic toxicity. However, the local toxicity of antibiotic carriers to the mesenchymal stem cells, as a result of both the peak concentrations and the type of carrier, may be significant. METHODS: To address this concern, the elution kinetics of vancomycin and gentamicin from several commercially available antibiotic carriers and several carriers impregnated by a surgeon (10 ml of each sterile carrier were manually mixed with a 500 mg vancomycin and an 80 mg gentamicin solution, and the duration of impregnation was 30 min) were assessed. Moreover, the effects of these antibiotic carriers on stem cell proliferation were investigated. The following two types of stem cells were used: bone marrow and dental pulp stem cells. RESULTS: The high eluted initial concentrations from antibiotic impregnated cancellous allogeneic bone grafts (which may be increased with the addition of fibrin glue) did not adversely affect stem cell proliferation. Moreover, an increased dental pulp stem cell proliferation rate in the presence of antibiotics was identified. In contrast to allogeneic bone grafts, a significant amount of antibiotics remained in the cement. Despite the favorable elution kinetics, the calcium carriers, bovine collagen carrier and freeze-dried bone exhibited decreased stem cell proliferation activity even in lower antibiotic concentrations compared with an allogeneic graft. CONCLUSIONS: This study demonstrated the benefits of antibiotic impregnated cancellous allogeneic bone grafts versus other carriers.

The Osteogenic Potential of Human Nondifferentiated and Pre-differentiated Mesenchymal Stem Cells Combined with an Osteoconductive Scaffold - Early Stage Healing.

Despite the huge research into stem cells and their regenerative properties for bone healing, there are still unanswered questions including the recipient's respond to the presence of the stem cells, the fate of stem cells inside the bone defect and the possible advantage in utilizing pre-differentiated cells. To address these problems, we used human multipotent mesenchymal stromal/stem cells (MSCs), GMP Grade, in a rat model of bone formation. In a "bioreactor concept" approach seven Wistar rats were implanted with 0.2 g of synthetic bone scaffold seeded with 2 × 106 MSCs, seven Wistar rats were implanted with 0.2 g of synthetic bone scaffold seeded with 1 × 106 predifferentiated osteoblasts and 1 × 106 pre-differentiated endothelial cells and 14 Wistar rats were implanted with 0.2 g of synthetic bone scaffold without seeded cells into an intramuscular pocket on the left side of their back. The right side of each rat was used as a control, and 0.2 g of synthetic bone scaffold was implanted into the intramuscular pocket alone. To see the early stage healing the samples were harvested 14 days after the implantation, MSCs were detected by positive DAPI and MTCO2 staining in 43% of all the samples implanted with MSCs, and no inflammation signs were present in any implanted animal. New vessels could be found in both groups implanted with MSCs, but not in the control group of animals. However, hematoxylin-eosin staining could not detect newly created bone within the implant in any of the groups. These results were in line with COLL1 staining, where we could detect positive staining only in three cases, all of which were implanted with un-differentiated MSCs. According to our findings, there were no benefits of using the pre-differentiated of MSC.

A Comparison of Three Rubber Dam Systems In Vivo--A Preliminary Study.

The aim of this study was to compare the isolation systems OptraDam® Plus and OptiDam™ with the conventional rubber dam in terms of objective and subjective parameters. The isolation systems were applied during the dental treatment of the patients. The time of preparation, placement, presence and removal were measured and the quality of isolation was evaluated. The median time of rubber dam placement was 76 s (Q1=62 s; Q3=111.25 s). The application time of OptraDam® Plus was significantly longer compared to the other systems (P ® plus. The results presented in this study could guide clinicians for choosing the most appropriate isolation system.

Human plasma and human platelet-rich plasma as a substitute for fetal calf serum during long-term cultivation of mesenchymal dental pulp stem cells.

AIMS: Our aims were to isolate and cultivate mesenchymal dental pulp stem cells (DPSC) in various media enriched with human blood components, and subsequently to investigate their basic biological properties. METHODS: DPSC were cultivated in five different media based on α MEM containing different concentrations of human plasma (HP), platelet-rich plasma (PRP), or fetal calf serum (FCS). The DPSC biological properties were examined periodically. RESULTS: We cultivated DPSC in the various cultivation media over 15 population doublings except for the medium supplemented with 10% HP. Our results showed that DPSC cultivated in medium supplemented with 10% PRP showed the shortest average population doubling time (DT) (28.6 ± 4.6 hours), in contrast to DPSC cultivated in 10% HP which indicated the longest DT (156.2 ± 17.8 hours); hence this part of the experiment had been cancelled in the 6th passage. DPSC cultivated in media with 2% FCS+ITS (DT 47.3 ± 10.4 hours), 2% PRP (DT 40.1 ± 5.7 hours) and 2% HP (DT 49.0 ± 15.2 hours) showed almost the same proliferative activity. DPSC's viability in the 9th passage was over 90% except for the DPSC cultivated in the 10% HP media. CONCLUSIONS: We proved that human blood components are suitable substitution for FCS in cultivation media for long-term DPSC cultivation.

A comparison of different rubber dam systems on a dental simulator.

The purpose of this study was to test the hypothesis that more recently developed rubber dam systems (OptraDam ® Plus and OptiDam™) are faster and easier to handle, and that the quality of isolation is not decreased. The rubber dam systems were applied in standard conditions on a dental simulator in several model clinical situations. The time of preparation, application and removal were measured and the quality of isolation was evaluated. The median time of rubber dam placement was 51 s (Q1 = 38 s; Q3 = 79 s). The shortest median time of application was with OptiDam™ (42 s), followed by a conventional rubber dam (53 s), and finally the longest was with OptraDam® Plus (58 s). The median volume of fluid remaining in the isolated space after 5 minutes was 9.5 mL (Q1 = 8 mL; Q3 = 10 mL). The largest median volume of remaining water was with OptiDam™ (10 mL), followed by a conventional rubber dam (9.5 mL) and the least with OptraDam® Plus (8.5 mL). The afore-stated hypothesis about the advantages of modern rubber dam isolation systems was accepted for OptiDam™, but rejected for OptraDam® Plus. The results could contribute to decision-making concerning the choice of rubber dam system.

The effect of fetal calf serum on human dental pulp stem cells.

AIMS: Authors studied potential side effects of fetal calf serum (FCS) in cultivation media on human dental pulp stem cells (DPSC) during long-term cultivation. METHODS: Two lines of DPSC obtained healthy donors (male 22 years, female 23 years) were used. Both lines were cultivated under standard cultivation conditions in four different media containing 10% or 2% FCS and substituted with growth factors. During long-term cultivation proliferation ability, karyotype and phenotype of DPSC were measured. RESULTS: Both lines of DPSC cultivated in a media containing 2% FCS and ITS supplement showed the highest number of population doublings. On the other hand the proliferation rate of DPSC cultivated in a media with 2% FCS without ITS supplement was slowest. Proliferation rate of DPSC cultivated in 10% FCS media with or without FGF-2 was comparable. DPSC cultivated in a media with 10% FCS showed a significantly higher amount of chromosomal aberrations. These chromosomal aberrations do not seem to be clonal but surprisingly we found large amounts of tetraploid cells in the 9th passage in both media containing 10% FCS. CONCLUSIONS: Our study proved that cultivation of DPSC in media containing higher concentration of FCS has critical side effects on cell chromosomal stability.

Comparison of COVID-19 epidemic among Czech dentists and the Czech general population.

Dentists are one of the professional groups most at risk for COVID-19 infection. Enhanced protective measures in dentistry have been adopted worldwide; however, it is unclear to what extent they were sufficient. To assess whether the protective measures outweighed the high infection risk, we compared COVID-19-related data between Czech dentists and the general Czech population. The data was obtained through a survey study attended by 15.8% of Czech Dental Chamber members. Data of the general population were acquired from the Czech Ministry of Health database. By the end of May 2022, COVID-19 full vaccination and 1st booster dose rates among study participants were 85.8% and 70.1%, respectively, which is significantly higher (p < 0.0001) compared to the Czech general population aged over 24 years (74.9% and 49.4%, respectively). To the same date, PCR/Antigen test verified COVID-19 prevalence among participants was 41.7%, and 49.9% among the general population (p < 0.0001). Prevalence and reinfection rates among individuals who received the 1st booster were significantly lower than among individuals without the booster or full vaccination (p < 0.0001). Persons who received the booster showed a faster return to work, shorter and different types of complications. Willingness to future vaccination was positive among 79.7% of respondents. Mandatory vaccination for healthcare workers and the general population was supported by 62.0% and 49.0%, respectively. The results showed that the high risk of COVID-19 infection associated with dentistry did not lead to higher COVID-19 prevalence among respondents compared to the general population.

Low, medium, and high molecular weight hyaluronic acid effects on human dental pulp stem cells in vitro.

Hyaluronic acid (HA), an extracellular biopolymer found throughout the human body, holds promise as a biocompatible and biodegradable scaffold material. High molecular weight (HMW) HA degrades, generating low molecular weight (LMW) fragments with distinct properties. These fragments can influence the behaviour of cells, including human dental pulp stem cells (hDPSCs) incorporated into HA-containing hydrogels or scaffolds. Therefore, a comprehensive examination of the impact of a range of HA molecular weights on hDPSCs is essential before designing HA-based scaffolds for these cells. hDPSC lines were cultured with LMW HA (800 Da, 1600 Da, 15 kDa), medium molecular weight HA (237 kDa), or HMW HA (1500 kDa) over six passages. The various molecular weights had negligible effects on hDPSCs viability, morphology, adhesion, or relative telomere length. Furthermore, the expression of key surface stemness markers (CD29, CD44, CD73, CD90) remained unaltered. HA did not induce osteogenic, chondrogenic, or adipogenic differentiation. Moreover, the potential for chondrogenic and osteogenic differentiation was not adversely affected by LMW or HMW HA. Various molecular weights of HA seem safe, biocompatible and therefore suitable components for hDPSCs-containing scaffolds. These findings affirm that the hDPCSs will not be negatively affected by HA fragments resulting from scaffold degradation.

Editorial of Special Issue "Hyaluronic Acid in Human Medicine".

Hyaluronic acid (HA) is an acidic, non-sulfated glycosaminoglycan that is intensively studied as a biodegradable and biocompatible material for scaffolding, regenerative medicine, and clinical applications [...].

Effect of Human Platelet Lysate as Cultivation Nutrient Supplement on Human Natal Dental Pulp Stem Cell In Vitro Expansion.

Despite several scientific or ethical issues, fetal bovine serum (FBS) remains the standard nutrient supplement in the mesenchymal stem cell cultivation medium. Cell amplification plays an important role in human stem cell therapies. Increasing interest in this field has supported attempts to find suitable human alternatives to FBS for in vitro cell propagation. Human platelet lysate (hPL) has recently been determined as one of them. Our study aimed to evaluate the influence of 2% hPL in the growth medium for in vitro expansion of human natal dental pulp stem cells (hNDP-SCs). The effect was determined on proliferation rate, viability, phenotype profile, expression of several markers, relative telomere length change, and differentiation potential of four lineages of hNDP-SCs. As a control, hNDP-SCs were simultaneously cultivated in 2% FBS. hNDP-SCs cultivated in hPL showed a statistically significantly higher proliferation rate in initial passages. We did not observe a statistically significant effect on mesenchymal stem cell marker (CD29, CD44, CD73, CD90) or stromal-associated marker (CD13, CD166) expression. The cell viability, relative telomere length, or multipotency remained unaffected in hNDP-SCs cultivated in hPL-medium. In conclusion, hPL produced under controlled and standardized conditions is an efficient serum supplement for in vitro expansion of hNDP-SCs.

Innovative Approach in the Cryogenic Freezing Medium for Mesenchymal Stem Cells.

The physical stresses during cryopreservation affect stem cell survival and further proliferation. To minimize or prevent cryoinjury, cryoprotective agents (CPAs) are indispensable. Despite the widespread use of 10% dimethyl sulfoxide (DMSO), there are concerns about its potential adverse effects. To bypass those effects, combinations of CPAs have been investigated. This study aimed to verify whether high-molecular-hyaluronic acid (HMW-HA) serves as a cryoprotectant when preserving human mesenchymal stem cells (hMSCs) to reduce the DMSO concentration in the cryopreservation medium. We studied how 0.1% or 0.2% HMW-HA combined with reduced DMSO concentrations (from 10% to 5%, and 3%) affected total cell count, viability, immunophenotype, and differentiation potential post-cryopreservation. Immediately after cell revival, the highest total cell count was observed in 10% DMSO-stored hMSC. However, two weeks after cell cultivation an increased cell count was seen in the HMW-HA-stored groups along with a continued increase in hMSCs stored using 3% DMSO and 0.1% HMW-HA. The increased total cell count corresponded to elevated expression of stemness marker CD49f. The HA-supplemented cryomedium did not affect the differential potential of hMSC. Our results will participate in producing a ready-to-use product for cryopreservation of mesenchymal stem cells.

COVID-19 Vaccination among Czech Dentists.

This work describes and evaluates vaccination against COVID-19 among members of the Czech Dental Chamber during the pre-booster vaccination phase. A cross-sectional online survey was conducted between 23 June and 4 September 2021, among 2716 participants, representing 24.3% of all chamber members. A total of 89.5% of respondents stated that they were registered for vaccination against COVID-19, their vaccination had started or been completed, or had a medically relevant reason to avoid vaccination. A total of 79.6% of respondents stated that they were fully vaccinated, most of them with the Comirnaty (Pfizer−BioNTech) vaccine (88.3%). The vaccination rate among males was significantly higher than among females (p = 0.001, OR 1.48). The main reasons for vaccination were professional (91.5%). The share of fully vaccinated participants was significantly higher (p < 0.0001, OR = 8.17) compared to the Czech general population (30.8%). A COVID-19 vaccine breakthrough infection rate was 0.42%. The study shows that both the willingness to vaccinate and the proportion of fully vaccinated individuals among Czech dentists are high, and only about 10% of them refused vaccination based on reasons classified as not medically relevant.

COVID-19 among Czech Dentistry Students: Higher Vaccination and Lower Prevalence Compared to General Population Counterparts.

The restrictions on medical students' clinical education during the COVID-19 pandemic has affected their professional readiness and often lengthened their training. These negative impacts are often considered a necessary price as clinical education is hypothesized to be associated with a high risk of pandemic spread. This work assesses this hypothesis based on COVID-19 epidemiological data among Czech dentistry students and their comparison to data of the Czech general population of similar age. We addressed two of the five Czech medical faculties (Charles University, Faculty of Medicine in Hradec Kralove and in Pilsen) providing dentistry study program with a survey. A total of 240 students participated, representing a 66.9% response rate. Over 75% of respondents participated in clinical education during the pandemic. The school environment was identified as a place of infection by only 9.8% of respondents who were aware of where they were infected. Overall, 100% of students used FFP2 respirators, and 75.3% used face shields or protective glasses while working with patients. By the end of May 2022, COVID-19 full vaccination and 1st booster rates among students were 93.8% and 54.6%, respectively, which is significantly higher (p < 0.0001, OR 7.3, 95% CI 4.4−12; p < 0.0001, OR 3.7, 95% CI 2.9−4.8, respectively) compared to their peers from the general population (67.1% and 24.4%, respectively). A total of 75.4% of respondents supported mandatory COVID-19 vaccination for healthcare professionals. To the same date, PCR and/or antigen test verified COVID-19 prevalence among students was 37.1%, while among peers from the general population, it was 45.1% (p = 0.015, OR 1.2, 95% CI 1.0−1.5). The combination of extensive protective measures and high vaccination against COVID-19 led to significantly lower COVID-19 prevalence among the students compared to their general population counterparts.

Characterization of dental pulp stem cells from impacted third molars cultured in low serum-containing medium.

We isolated and expanded stem cells from dental pulp from extracted third molars using an innovative culture method consisting of low serum-containing medium supplemented with epidermal growth factor and platelet-derived growth factor BB. We evaluated the differentiation potential of these cells when they were growing either adherently or as micromass/spheroid cultures in various media. Undifferentiated and differentiated cells were analyzed by flow cytometry, immunocytochemistry and immunoblotting. The flow cytometry results showed that the dental pulp stem cells (DPSCs) were positive for mesenchymal stromal cell markers, but negative for hematopoietic markers. Immunocytochemical and/or immunoblotting analyses revealed the expression of numerous stem cell markers, including nanog, Sox2, nestin, Musashi-1 and nucleostemin, whereas they were negative for markers associated with differentiated neural, vascular and hepatic cells. Surprisingly, the cells were only slightly positive for α-smooth muscle actin, and a heterogeneous expression of CD146 was observed. When cultured in osteogenic media, they expressed osteonectin, osteopontin and procollagen I, and in micromass cultures, they produced collagen I. DPSCs cultured in TGF-ß1/3-supplemented media produced extracellular matrix typical of cartilaginous tissue. The addition of vascular endothelial growth factor to serum-free media resulted in the expression of endothelial markers. Interestingly, when cultured in neurogenic media, DPSCs exhibited de novo or upregulated markers of undifferentiated and differentiated neural cells. Collectively, our data show that DPSCs are self-renewing and able to express markers of bone, cartilage, vascular and neural tissues, suggesting their multipotential capacity. Their easy accessibility makes these cells a suitable source of somatic stem cells for tissue engineering.

Impact of COVID-19 on Czech Dentistry: A Nationwide Cross-Sectional Preliminary Study among Dentists in the Czech Republic.

This work evaluates the impact of the COVID-19 pandemic on Czech dentistry from March 2020 to March 2021. The assessment was based on questionnaires filled out by 3674 Czech dentists representing 42.6% of practicing dentists in the country. During March-May, 2020 (the first COVID-19 wave), 90.7% of dental practices remained open; however, only 22.8% of the practices continued to operate with no changes, 46.5% had fewer patients, 21.4% treated only acute cases, and 3.8% were closed. During September 2020-May 2021 (the second wave of COVID-19), 96.1% of dental practices remained open, 60.8% operated with no changes, 34.5% had fewer patients, 0.8% treated only acute cases, and 0.5% were closed. The reasons leading to the closure of Czech dental practices during the whole pandemic were a shortage of personal protective equipment (50.5%), a COVID-19 outbreak in the workplace (24.5%), fear of a possible self-infection (24.0%), and quarantine (20.5%). The time range of Czech dental practices closure during the whole pandemic was: 1-2 weeks (49.9%), 2-4 weeks (21.2%), and >1 month (0.8%). The greatest professional difficulties of Czech dentists during the pandemic were crisis operating management (55%), health safety and hygiene concerns (21%), shortage of personal protective equipment (21%), and difficulty working with the protective equipment (15%). In addition, 47.3% of dentists also observed a declining interest in preventive dental care, and 16.9% of them observed worse oral care of patients. These results show that despite the lack of protective equipment, dental care was maintained throughout the pandemic. Additionally, the pandemic negatively affected the patients' approach to dental care, indicating a deterioration in oral health as a possible delayed outcome of the COVID-19 pandemic.

The Effect of Cultivation Passaging on the Relative Telomere Length and Proliferation Capacity of Dental Pulp Stem Cells.

Telomeres are repetitive nucleoprotein DNA sequences that shorten with each cell division. The stem cells activate telomerase to compensate for the telomere loss. This study aimed to evaluate the effect of cultivation passaging on the relative telomere length and proliferation capacity of dental pulp stem cells. We used ten dental pulp stem cell (DPSC) lineages stored for 12 months using uncontrolled-rate freezing to reach the study's goal. We analyzed their proliferation rate, phenotype using flow cytometry, multipotency, and relative telomere length using a qPCR analysis. We determined the relative telomere length in the added study by performing analysis after one, two, and three weeks of cultivation with no passaging. We documented the telomere attrition with increasing passaging. The shorter the relative telomere length, the lower reached population doublings, and longer population doubling time were observed at the end of the cultivation. We observed the telomere prolongation in DPSCs cultivated for two weeks with no passaging in the added subsequent study. We concluded that excessive proliferation demands on DPSCs during in vitro cultivation result in telomere attrition. We opened the theory that the telomerase might be more efficient during cell cultivation with no passaging. This observation could help in preserving the telomere length during ex vivo DPSC expansion.

COVID-19 Prevalence among Czech Dentists.

This work evaluates the prevalence of coronavirus disease (COVID-19), a viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), among members of the Czech Dental Chamber. The assessment was based on an online questionnaire filled out by 2716 participants, representing 24.3% of all chamber members. Overall, 25.4% of the participants admitted they were diagnosed with COVID-19 by 30 June 2021, with no statistical differences between the sexes. While in the age groups under 50 the reported prevalence was around 30%, with increasing age, it gradually decreased to 15.2% in the group over 70 years. The work environment was identified as a place of contagion by 38.4% of the respondents. The total COVID-19 PCR-verified positivity was 13.9%, revealing a statistically lower prevalence (p = 0.0180) compared with the Czech general population, in which the COVID-19 PCR-verified positivity was ~15.6% (fourth highest rank in the world). The total infection-hospitalization ratio (IHR) was 2.8%, and the median age group of hospitalized individuals was 60-70 years. For respondents older than 60 years, the IHR was 8.7%, and for those under 40 years, it was 0%. Of the respondents, 37.7% admitted that another team member was diagnosed with COVID-19, of which the most frequently mentioned profession was a nurse/dental assistant (81.2%). The results indicate that although the dentist profession is associated with a high occupational risk of SARS-CoV-2 infection, well-chosen antiepidemic measures adopted by dental professionals may outweigh it.