ULOGA αSMA PERIVASKULARNIH STANICA TIJEKOM STVARANJA TERCIJARNOG DENTINA

OBJECTIVES: The aim of this study was to examine the contribution of perivascular cells to odontoblasts during the development, growth, and repair of dentin and examine the effects of early and limited exposure of perivascular cells to fibroblast growth factor 2 (FGF2) in vivo using mouse molars as a model. MATERIAL AND METHODS: We used an inducible, Cre-loxP in vivo fate-mapping approach to examine the contributions of the descendants of cells expressing the αSMA-CreERT2 transgene to the odontoblast lineage. RESULTS: In vivo lineage-tracing experiments showed the contribution of αSMA-tdTomato+ cells to a small number of newly formed odontoblasts during primary dentinogenesis. Experiments revealed that mild injury to dentin first led to activation of aSMA-tdTomato+ cells in the entire pulp chamber. After their activation aSMAtdTomato+ cells migrated towards the site of injury, gave rise to pulp cells and a few odontoblasts that became integrated into the existing odontoblast layer expressing Col2.3- GFP and Dspp. Using an experimental pulp exposure model in molars to induce reparative dentinogenesis, we demonstrate the contribution of αSMA-tdTomato+ cells to cells secreting reparative dentin. Our results demonstrate that αSMA-tdTomato+ cells differentiated into Col2.3-GFP+ cells composed of both Dspp+ odontoblasts and Bsp+ osteoblasts. Early delivery of exogenous FGF2 to exposed pulp led to proliferative expansion of αSMA-tdTomato+ cells and their accelerated differentiation into odontoblasts. Results showed that the calcified bridge/reparative dentin in FGF2-treated pulps were lined with an increased number of Dspp+ odontoblasts and devoid of BSP+ osteoblasts. CONCLUSION: Our findings identify a population of mesenchymal progenitor cells capable of giving rise to a second generation of odontoblasts during reparative dentinogenesis. The increased number of odontoblasts derived from αSMAtdTomato+ cells and the formation of reparative dentin devoid of osteoblasts provide in vivo evidence for the stimulatory effects of FGF signaling on odontoblast differentiation from early progenitors in dental pulp.CILJ ISTRAŽIVANJA: Reparativna dentinogeneza je regenerativni proces koji dovodi do stvaranja dentinskog mineraliziranog mosta koji pridonosi održavanju vitaliteta zubne pulpe. Reparativna dentinogeneza nastaje nakon intenzivne ozljede dentina koja dovodi do smrti odontoblasta i uključuje aktivaciju, proliferaciju i migraciju mezenhimnih matičnih stanica do mjesta ozljede i njihovu diferencijaciju u drugu generaciju odontoblasta i odontoblastima – sličnim stanica. Identitet stanica kćeri stanica i signalni putovi uključenih u proces reparativne dentinogeneze su još uvijek nedovoljno istraženi i nepoznati. Cilj ove studije je ispitati ulogu perivaskularnih stanica u pulpi koja eksprimira alfa aktin glatkog mišića-GFP (αSMA-GFP) u reparativnoj dentinogenezi i ispitati učinke FGF signalne molekule na ovu populaciju. Koristili smo inducirani CreloxP rekombinacijski sustav (αSMA-CreERT2) za eksperimente praćenja staničnih loza in vivo i in vitro. Cilj je ispitati doprinos perivaskularnih stanica odontoblastima tijekom razvoja, rasta i regeneracije dentina i ispitati učinke rane i ograničene izloženosti perivaskularnih stanica fibroblastičnom faktoru rasta 2 (FGF2) in vivo na uzorku mišjih kutnjaka. MATERIJALI I METODE: Koristili smo inducirani, Cre-loxP in vivo pristup praćenja sudbine stanica da bismo ispitali doprinos potomaka stanica koji izražavaju αSMA-CreERT2 transgen u odontoblastastičnoj liniji. REZULTATI: Pokusi in vivo praćenja loze na kutnjacima pokazali su doprinos αSMA-tdTomato+ stanica malom broju novoformiranih odontoblasta tijekom primarne dentinogeneze. Eksperimenti su otkrili da je blaga trauma dentina najprije dovela do aktivacije SMA-tdTomato+ stanica u cijeloj pulpnoj komori. Postotak područja koja zauzimaju SMA-tdTomato+ stanice u zubima sa traumom dentina bio je značajno veći nego kod zuba bez ozljeda. Nakon njihove aktivacije SMA-tdTomato+ stanice migrirale su prema mjestu ozljede, diferencirale u pulpne stanice, te su nekoliko SMA-tdTomato+ odontoblasta koji su se integrirali u postojeći odontoblastni sloj izražavali Col2.3-GFP i Dspp. Koristeći eksperimentalni model direktnog prekrivanja pulpe u kutnjacima kako bi potakli reparativnu dentinogenezu, prikazali smo doprinos αSMA-tdTomato+ stanica stanicama koje izlučuju reparativni dentin. Naši rezultati pokazuju da su se αSMA-tdTomato+ stanice diferencirale u Col2.3-GFP+ stanice, te su sastavljene od Dspp+ odontoblasta i Bsp+ osteoblasta. Rana primjena egzogenog FGF2 nakon direktnog prekrivanja pulpe dovela je do jače proliferacije αSMA-tdTomato+ stanica i njihove ubrzane diferencijacija u odontoblaste. Rezultati su pokazali da je mineralizirani most reparativng dentina u kutnjacima tretiranim FGF2-om obložen povećanim brojem Dspp+ odontoblasta uz odsudstvo BSP+ osteoblasta. ZAKLJUČAK: Blaga ozljeda dovela je do aktivacije perivaskularnih SMA-tdTomato+ stanica koje su diferencirale u stanice pulpe, kao i nekoliko odontoblasta koji su integrirani u prethodno postojeći sloj odontoblasta. Naši nalazi identificiraju populaciju mezenhimalnih potomskih stanica koje mogu stvoriti drugu generaciju odontoblasta (odontoblastima – slične stanice) tijekom reparativne dentinogeneze. Povećani broj odontoblasta dobivenih iz αSMA-tdTomato+ stanica i stvaranje reparativnog dentina lišenog osteoblasta pružaju in vivo dokaze za stimulacijske učinke signalizacije FGF2-a na diferencijaciju odontoblasta u zubnoj pulpi

ULOGA αSMA PERIVASKULARNIH STANICA TIJEKOM STVARANJA TERCIJARNOG DENTINA

OBJECTIVES: The aim of this study was to examine the contribution of perivascular cells to odontoblasts during the development, growth, and repair of dentin and examine the effects of early and limited exposure of perivascular cells to fibroblast growth factor 2 (FGF2) in vivo using mouse molars as a model. MATERIAL AND METHODS: We used an inducible, Cre-loxP in vivo fate-mapping approach to examine the contributions of the descendants of cells expressing the αSMA-CreERT2 transgene to the odontoblast lineage. RESULTS: In vivo lineage-tracing experiments showed the contribution of αSMA-tdTomato+ cells to a small number of newly formed odontoblasts during primary dentinogenesis. Experiments revealed that mild injury to dentin first led to activation of aSMA-tdTomato+ cells in the entire pulp chamber. After their activation aSMAtdTomato+ cells migrated towards the site of injury, gave rise to pulp cells and a few odontoblasts that became integrated into the existing odontoblast layer expressing Col2.3- GFP and Dspp. Using an experimental pulp exposure model in molars to induce reparative dentinogenesis, we demonstrate the contribution of αSMA-tdTomato+ cells to cells secreting reparative dentin. Our results demonstrate that αSMA-tdTomato+ cells differentiated into Col2.3-GFP+ cells composed of both Dspp+ odontoblasts and Bsp+ osteoblasts. Early delivery of exogenous FGF2 to exposed pulp led to proliferative expansion of αSMA-tdTomato+ cells and their accelerated differentiation into odontoblasts. Results showed that the calcified bridge/reparative dentin in FGF2-treated pulps were lined with an increased number of Dspp+ odontoblasts and devoid of BSP+ osteoblasts. CONCLUSION: Our findings identify a population of mesenchymal progenitor cells capable of giving rise to a second generation of odontoblasts during reparative dentinogenesis. The increased number of odontoblasts derived from αSMAtdTomato+ cells and the formation of reparative dentin devoid of osteoblasts provide in vivo evidence for the stimulatory effects of FGF signaling on odontoblast differentiation from early progenitors in dental pulp.CILJ ISTRAŽIVANJA: Reparativna dentinogeneza je regenerativni proces koji dovodi do stvaranja dentinskog mineraliziranog mosta koji pridonosi održavanju vitaliteta zubne pulpe. Reparativna dentinogeneza nastaje nakon intenzivne ozljede dentina koja dovodi do smrti odontoblasta i uključuje aktivaciju, proliferaciju i migraciju mezenhimnih matičnih stanica do mjesta ozljede i njihovu diferencijaciju u drugu generaciju odontoblasta i odontoblastima – sličnim stanica. Identitet stanica kćeri stanica i signalni putovi uključenih u proces reparativne dentinogeneze su još uvijek nedovoljno istraženi i nepoznati. Cilj ove studije je ispitati ulogu perivaskularnih stanica u pulpi koja eksprimira alfa aktin glatkog mišića-GFP (αSMA-GFP) u reparativnoj dentinogenezi i ispitati učinke FGF signalne molekule na ovu populaciju. Koristili smo inducirani CreloxP rekombinacijski sustav (αSMA-CreERT2) za eksperimente praćenja staničnih loza in vivo i in vitro. Cilj je ispitati doprinos perivaskularnih stanica odontoblastima tijekom razvoja, rasta i regeneracije dentina i ispitati učinke rane i ograničene izloženosti perivaskularnih stanica fibroblastičnom faktoru rasta 2 (FGF2) in vivo na uzorku mišjih kutnjaka. MATERIJALI I METODE: Koristili smo inducirani, Cre-loxP in vivo pristup praćenja sudbine stanica da bismo ispitali doprinos potomaka stanica koji izražavaju αSMA-CreERT2 transgen u odontoblastastičnoj liniji. REZULTATI: Pokusi in vivo praćenja loze na kutnjacima pokazali su doprinos αSMA-tdTomato+ stanica malom broju novoformiranih odontoblasta tijekom primarne dentinogeneze. Eksperimenti su otkrili da je blaga trauma dentina najprije dovela do aktivacije SMA-tdTomato+ stanica u cijeloj pulpnoj komori. Postotak područja koja zauzimaju SMA-tdTomato+ stanice u zubima sa traumom dentina bio je značajno veći nego kod zuba bez ozljeda. Nakon njihove aktivacije SMA-tdTomato+ stanice migrirale su prema mjestu ozljede, diferencirale u pulpne stanice, te su nekoliko SMA-tdTomato+ odontoblasta koji su se integrirali u postojeći odontoblastni sloj izražavali Col2.3-GFP i Dspp. Koristeći eksperimentalni model direktnog prekrivanja pulpe u kutnjacima kako bi potakli reparativnu dentinogenezu, prikazali smo doprinos αSMA-tdTomato+ stanica stanicama koje izlučuju reparativni dentin. Naši rezultati pokazuju da su se αSMA-tdTomato+ stanice diferencirale u Col2.3-GFP+ stanice, te su sastavljene od Dspp+ odontoblasta i Bsp+ osteoblasta. Rana primjena egzogenog FGF2 nakon direktnog prekrivanja pulpe dovela je do jače proliferacije αSMA-tdTomato+ stanica i njihove ubrzane diferencijacija u odontoblaste. Rezultati su pokazali da je mineralizirani most reparativng dentina u kutnjacima tretiranim FGF2-om obložen povećanim brojem Dspp+ odontoblasta uz odsudstvo BSP+ osteoblasta. ZAKLJUČAK: Blaga ozljeda dovela je do aktivacije perivaskularnih SMA-tdTomato+ stanica koje su diferencirale u stanice pulpe, kao i nekoliko odontoblasta koji su integrirani u prethodno postojeći sloj odontoblasta. Naši nalazi identificiraju populaciju mezenhimalnih potomskih stanica koje mogu stvoriti drugu generaciju odontoblasta (odontoblastima – slične stanice) tijekom reparativne dentinogeneze. Povećani broj odontoblasta dobivenih iz αSMA-tdTomato+ stanica i stvaranje reparativnog dentina lišenog osteoblasta pružaju in vivo dokaze za stimulacijske učinke signalizacije FGF2-a na diferencijaciju odontoblasta u zubnoj pulpi

ULOGA αSMA PERIVASKULARNIH STANICA TIJEKOM STVARANJA TERCIJARNOG DENTINA

OBJECTIVES: The aim of this study was to examine the contribution of perivascular cells to odontoblasts during the development, growth, and repair of dentin and examine the effects of early and limited exposure of perivascular cells to fibroblast growth factor 2 (FGF2) in vivo using mouse molars as a model. MATERIAL AND METHODS: We used an inducible, Cre-loxP in vivo fate-mapping approach to examine the contributions of the descendants of cells expressing the αSMA-CreERT2 transgene to the odontoblast lineage. RESULTS: In vivo lineage-tracing experiments showed the contribution of αSMA-tdTomato+ cells to a small number of newly formed odontoblasts during primary dentinogenesis. Experiments revealed that mild injury to dentin first led to activation of aSMA-tdTomato+ cells in the entire pulp chamber. After their activation aSMAtdTomato+ cells migrated towards the site of injury, gave rise to pulp cells and a few odontoblasts that became integrated into the existing odontoblast layer expressing Col2.3- GFP and Dspp. Using an experimental pulp exposure model in molars to induce reparative dentinogenesis, we demonstrate the contribution of αSMA-tdTomato+ cells to cells secreting reparative dentin. Our results demonstrate that αSMA-tdTomato+ cells differentiated into Col2.3-GFP+ cells composed of both Dspp+ odontoblasts and Bsp+ osteoblasts. Early delivery of exogenous FGF2 to exposed pulp led to proliferative expansion of αSMA-tdTomato+ cells and their accelerated differentiation into odontoblasts. Results showed that the calcified bridge/reparative dentin in FGF2-treated pulps were lined with an increased number of Dspp+ odontoblasts and devoid of BSP+ osteoblasts. CONCLUSION: Our findings identify a population of mesenchymal progenitor cells capable of giving rise to a second generation of odontoblasts during reparative dentinogenesis. The increased number of odontoblasts derived from αSMAtdTomato+ cells and the formation of reparative dentin devoid of osteoblasts provide in vivo evidence for the stimulatory effects of FGF signaling on odontoblast differentiation from early progenitors in dental pulp.CILJ ISTRAŽIVANJA: Reparativna dentinogeneza je regenerativni proces koji dovodi do stvaranja dentinskog mineraliziranog mosta koji pridonosi održavanju vitaliteta zubne pulpe. Reparativna dentinogeneza nastaje nakon intenzivne ozljede dentina koja dovodi do smrti odontoblasta i uključuje aktivaciju, proliferaciju i migraciju mezenhimnih matičnih stanica do mjesta ozljede i njihovu diferencijaciju u drugu generaciju odontoblasta i odontoblastima – sličnim stanica. Identitet stanica kćeri stanica i signalni putovi uključenih u proces reparativne dentinogeneze su još uvijek nedovoljno istraženi i nepoznati. Cilj ove studije je ispitati ulogu perivaskularnih stanica u pulpi koja eksprimira alfa aktin glatkog mišića-GFP (αSMA-GFP) u reparativnoj dentinogenezi i ispitati učinke FGF signalne molekule na ovu populaciju. Koristili smo inducirani CreloxP rekombinacijski sustav (αSMA-CreERT2) za eksperimente praćenja staničnih loza in vivo i in vitro. Cilj je ispitati doprinos perivaskularnih stanica odontoblastima tijekom razvoja, rasta i regeneracije dentina i ispitati učinke rane i ograničene izloženosti perivaskularnih stanica fibroblastičnom faktoru rasta 2 (FGF2) in vivo na uzorku mišjih kutnjaka. MATERIJALI I METODE: Koristili smo inducirani, Cre-loxP in vivo pristup praćenja sudbine stanica da bismo ispitali doprinos potomaka stanica koji izražavaju αSMA-CreERT2 transgen u odontoblastastičnoj liniji. REZULTATI: Pokusi in vivo praćenja loze na kutnjacima pokazali su doprinos αSMA-tdTomato+ stanica malom broju novoformiranih odontoblasta tijekom primarne dentinogeneze. Eksperimenti su otkrili da je blaga trauma dentina najprije dovela do aktivacije SMA-tdTomato+ stanica u cijeloj pulpnoj komori. Postotak područja koja zauzimaju SMA-tdTomato+ stanice u zubima sa traumom dentina bio je značajno veći nego kod zuba bez ozljeda. Nakon njihove aktivacije SMA-tdTomato+ stanice migrirale su prema mjestu ozljede, diferencirale u pulpne stanice, te su nekoliko SMA-tdTomato+ odontoblasta koji su se integrirali u postojeći odontoblastni sloj izražavali Col2.3-GFP i Dspp. Koristeći eksperimentalni model direktnog prekrivanja pulpe u kutnjacima kako bi potakli reparativnu dentinogenezu, prikazali smo doprinos αSMA-tdTomato+ stanica stanicama koje izlučuju reparativni dentin. Naši rezultati pokazuju da su se αSMA-tdTomato+ stanice diferencirale u Col2.3-GFP+ stanice, te su sastavljene od Dspp+ odontoblasta i Bsp+ osteoblasta. Rana primjena egzogenog FGF2 nakon direktnog prekrivanja pulpe dovela je do jače proliferacije αSMA-tdTomato+ stanica i njihove ubrzane diferencijacija u odontoblaste. Rezultati su pokazali da je mineralizirani most reparativng dentina u kutnjacima tretiranim FGF2-om obložen povećanim brojem Dspp+ odontoblasta uz odsudstvo BSP+ osteoblasta. ZAKLJUČAK: Blaga ozljeda dovela je do aktivacije perivaskularnih SMA-tdTomato+ stanica koje su diferencirale u stanice pulpe, kao i nekoliko odontoblasta koji su integrirani u prethodno postojeći sloj odontoblasta. Naši nalazi identificiraju populaciju mezenhimalnih potomskih stanica koje mogu stvoriti drugu generaciju odontoblasta (odontoblastima – slične stanice) tijekom reparativne dentinogeneze. Povećani broj odontoblasta dobivenih iz αSMA-tdTomato+ stanica i stvaranje reparativnog dentina lišenog osteoblasta pružaju in vivo dokaze za stimulacijske učinke signalizacije FGF2-a na diferencijaciju odontoblasta u zubnoj pulpi

The efficacy of a novel SWEEPS laser-activated irrigation compared to ultrasonic activation in the removal of pulp tissue from an isthmus area in the apical third of the root canal

This study aimed to evaluate the efficacy of Shock Wave Enhanced Emission Photoacoustic Streaming (SWEEPS) in the removal of remaining pulp tissue from the root canal isthmus area in lower molars and compare it with ultrasonically activated irrigation (UAI) and conventional needle irrigation (NI). Forty-one lower molars with isthmuses between mesial canals were included in the study. The teeth were randomly distributed into experimental groups (n = 12/each) based on the final irrigation protocol (SWEEPS, UAI, or NI) and a control group (C) (n = 5). The traditional access cavity of the mesial part of each tooth was made in all samples. The mesial root canals in the experimental groups were instrumented with a Wave One Gold Primary (25/.07) file using 3% sodium hypochlorite (NaOCl) while the distal canal served as a control for the presence of pulp tissue. No treatment was performed in the C group. Sections from the isthmus region were processed for histopathology to measure the remaining pulp tissue (RPT). The results were analyzed using analysis of variance and the Kruskal-Wallis test (& alpha; = 0.05). There were no significant differences in the relative surface area of root canals and isthmus among the groups (p > 0.05). Samples in the SWEEPS group had significantly less RPT than UAI, NI, and C (p = 0.003, 0.014, 0.003, respectively). There were no significant differences between the UAI and NI (p = 0.583). SWEEPS was the most efficient in debridement of the root canal isthmus area. UAI and NI showed similar but lower efficiency

FGF2 Enhances Odontoblast Differentiation by αSMA Progenitors In Vivo

The goal of this study was to examine the effects of early and limited exposure of perivascular cells expressing α (αSMA) to fibroblast growth factor 2 (FGF2) in vivo. We performed in vivo fate mapping by inducible Cre-loxP and experimental pulp injury in molars to induce reparative dentinogenesis. Our results demonstrate that early delivery of exogenous FGF2 to exposed pulp led to proliferative expansion of αSMA-tdTomato cells and their accelerated differentiation into odontoblasts. In vivo lineage-tracing experiments showed that the calcified bridge/reparative dentin in FGF2-treated pulps were lined with an increased number of Dspp odontoblasts and devoid of BSP osteoblasts. The increased number of odontoblasts derived from αSMA-tdTomato cells and the formation of reparative dentin devoid of osteoblasts provide in vivo evidence for the stimulatory effects of FGF signaling on odontoblast differentiation from early progenitors in dental pulp

Generation and characterization of DSPP- Cerulean/DMP1-Cherry reporter mice

To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3-GFP, pOBCol3.6-GFP, and DMP1-GFP), similar to the endogenous proteins, are also expressed by bone-forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP- Cerulean/DMP1-Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24-258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1-Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP-Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblast

Generation and characterization of DSPP‐Cerulean/DMP1‐Cherry reporter mice

To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3-GFP, pOBCol3.6-GFP, and DMP1-GFP), similar to the endogenous proteins, are also expressed by bone-forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP- Cerulean/DMP1-Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24-258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1-Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP-Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblast