Determination of bone regeneration potential of murine embryonic stem cells and bone marrow mesenchymal stem cells

U ovom istraživanju analiziran je koštani regeneracijski potencijal mezenhimnih matičnih stanica (MMS) koštane srži i embrionalnih matičnih stanica (EMS). U tu je svrhu upotrijebljen transgenični mišji model αSMARFP/Col2.3GFP. MMS su identificirane kroz izražaj crvene fluorescentne bjelančevine - RFP (od engl. red fluorescent protein) koja je pod kontrolom promotora za α-aktin glatkih mišića (αSMA). Kako bi se pratila diferencijacija MMS-a i EMS-a u zrele osteoblaste, upotrijebljena je zelena fluorescentna bjelačevina - GFP (od eng. green fluorescent protein), čiji je izražaj pod kontrolom 2,3 kb fragmenta štakorskog promotora za kolagen tip I (Col 2.3). Za obje vrste stanica napravljena je analiza osteogenog potencijala in vitro i analiza in vivo za EMS. Nakon poticanja koštane diferencijacije u uvjetima in vitro, MMS koštane srži koje izražavaju biljeg αSMARFP diferenciraju se u Col2.3GFP+ stanice, tj. u zrele osteoblaste, pokazuju sposobnost mineralizacije i u znatnoj mjeri izražavaju tipične biljege zrelih osteoblasta (osteokalcin i koštanu sijalobjelančevinu). EMS pokazuju slabu aktivnost biljega Col2.3GFP nakon poticanja koštane diferencijacije u uvjetima in vitro. Također pokazuju slab porast koštanih diferencijacijskih biljega, koji bi potvrdili diferencijaciju EMS-a u zrele osteoblaste. Primijećena je znatna aktivnost alkalne fosfataze i prisutnost mineraliziranog matriksa. Rezultati pokazuju da je uspješnost koštane diferencijacije EMS-a in vitro znatno manja u odnosu na koštanu diferencijaciju MMS-a koštane srži, koristeći se standardnim protokolima za koštanu diferencijaciju matičnih stanica. S druge strane, u uvjetima in vivo koristeći se modelom stvaranja teratoma, EMS pokazuju sposobnost diferencijacije u zrele osteoblaste koji izražavaju biljeg Col2.3GFP. Genski konstrukt Col2.3GFP može se upotrebljavati kako bi se potvrdila diferencijacija EMS-a u zrele osteoblaste.Introduction Bone marrow mesenchymal stem cells (MSCs) have been used in regenerative bone biology for more than a decade, as they can be easily recovered from patients. More recently some of the focus in this field has shifted towards the use of embryonic stem cells (ESCs). Previous studies reported that ESCs can be induced to differentiate into cells showing a mature osteoblastic phenotype by culturing them under osteoinductive conditions. This study proposes determination of the bone regeneration potential of murine bone marrow MSCs and ESCs. Materials and methods For this purpose, a murine model (dual transgenic mice αSMARFP/Col2.3GFP) has been utilized. After isolation of cells from the bone marrow of long bones, MSCs have been identified through the expression of alpha-smooth muscle actin (αSMA) promoter directed RFP. To track the transition of MSCs into mature osteoblasts, a bone-specific fragment of rat type I collagen promoter driving green fluorescent protein (Col2.3GFP) has been utilized. In addition, ESC lines have been derived from the same αSMARFP/Col2.3GFP transgenic mice, allowing identification of cells at the mesenchymal stage (αSMARFP) and at mature osteoblast stage (Col2.3GFP). In vitro analysis of osteogenic potential has been made for both types of cells and in vivo analysis (teratoma assay) of osteogenic potential for ESCs. Results Cultures of bone marrow MSCs were established, and after one week a population of αSMARFP expressing cells was noticed. Following cell sorting, replated αSMARFP+ cells (20-30%) were induced to osteogenesis, and strong expression of osteoblast specific transgene Col2.3GFP was noticed, confirming osteogenic ability of αSMARFP+ cells. Osteogenic differentiation was confirmed by detection of mineralization with von Kossa method and by up-regulation of markers of mature osteoblast lineage cells; osteocalcin and bone sialoprotein. Following osteogenic induction in ESCs, expression of alkaline phosphatase and subsequent mineralization (detected by von Kossa staining) was observed. After one week of osteogenic induction, ESCs began to express αSMARFP. This expression was localized to the peripheral area encircling a typical ESC colony. Nevertheless, these αSMARFP+ cells did not show activation of the Col2.3GFP, even after 6 weeks of osteogenic differentiation in vitro. Also, small increases in expression in some of the bone markers analyzed (Colagen type I, bone sialoprotein) were noticed, but most of them appeared minimal compared to the levels expressed by the MSCs, even after six weeks of osteogenic induction. In contrast, in vivo teratoma assay by ECSs showed bone formation and strong Col2.3GFP signal in the areas that stain positive for mineralization by von Kossa. Conclusion These results show that MSCs compare to ESCs, have much more capability to differentiate into mature osteoblast, express specific bone markers and Col2.3GFP transgene after osteogenic induction in vitro. On the other hand, in the functional in vivo assay ESCs show capability to form bone and activate Col2.3GFP marker. The results obtained indicate that detection of alkaline phosphatase activity and mineralization of ESCs cultured under osteogenic conditions is not sufficient to demonstrate osteogenic maturation. This study indicates the utility of the promoter-visual transgene approach to assess the commitment and differentiation of stem cells into the osteoblast lineage. To current knowledge, this study is the first to utilize the expression of Col2.3GFP transgene in the ESCs derived from transgenic mice

Determination of bone regeneration potential of murine embryonic stem cells and bone marrow mesenchymal stem cells

U ovom istraživanju analiziran je koštani regeneracijski potencijal mezenhimnih matičnih stanica (MMS) koštane srži i embrionalnih matičnih stanica (EMS). U tu je svrhu upotrijebljen transgenični mišji model αSMARFP/Col2.3GFP. MMS su identificirane kroz izražaj crvene fluorescentne bjelančevine - RFP (od engl. red fluorescent protein) koja je pod kontrolom promotora za α-aktin glatkih mišića (αSMA). Kako bi se pratila diferencijacija MMS-a i EMS-a u zrele osteoblaste, upotrijebljena je zelena fluorescentna bjelačevina - GFP (od eng. green fluorescent protein), čiji je izražaj pod kontrolom 2,3 kb fragmenta štakorskog promotora za kolagen tip I (Col 2.3). Za obje vrste stanica napravljena je analiza osteogenog potencijala in vitro i analiza in vivo za EMS. Nakon poticanja koštane diferencijacije u uvjetima in vitro, MMS koštane srži koje izražavaju biljeg αSMARFP diferenciraju se u Col2.3GFP+ stanice, tj. u zrele osteoblaste, pokazuju sposobnost mineralizacije i u znatnoj mjeri izražavaju tipične biljege zrelih osteoblasta (osteokalcin i koštanu sijalobjelančevinu). EMS pokazuju slabu aktivnost biljega Col2.3GFP nakon poticanja koštane diferencijacije u uvjetima in vitro. Također pokazuju slab porast koštanih diferencijacijskih biljega, koji bi potvrdili diferencijaciju EMS-a u zrele osteoblaste. Primijećena je znatna aktivnost alkalne fosfataze i prisutnost mineraliziranog matriksa. Rezultati pokazuju da je uspješnost koštane diferencijacije EMS-a in vitro znatno manja u odnosu na koštanu diferencijaciju MMS-a koštane srži, koristeći se standardnim protokolima za koštanu diferencijaciju matičnih stanica. S druge strane, u uvjetima in vivo koristeći se modelom stvaranja teratoma, EMS pokazuju sposobnost diferencijacije u zrele osteoblaste koji izražavaju biljeg Col2.3GFP. Genski konstrukt Col2.3GFP može se upotrebljavati kako bi se potvrdila diferencijacija EMS-a u zrele osteoblaste.Introduction Bone marrow mesenchymal stem cells (MSCs) have been used in regenerative bone biology for more than a decade, as they can be easily recovered from patients. More recently some of the focus in this field has shifted towards the use of embryonic stem cells (ESCs). Previous studies reported that ESCs can be induced to differentiate into cells showing a mature osteoblastic phenotype by culturing them under osteoinductive conditions. This study proposes determination of the bone regeneration potential of murine bone marrow MSCs and ESCs. Materials and methods For this purpose, a murine model (dual transgenic mice αSMARFP/Col2.3GFP) has been utilized. After isolation of cells from the bone marrow of long bones, MSCs have been identified through the expression of alpha-smooth muscle actin (αSMA) promoter directed RFP. To track the transition of MSCs into mature osteoblasts, a bone-specific fragment of rat type I collagen promoter driving green fluorescent protein (Col2.3GFP) has been utilized. In addition, ESC lines have been derived from the same αSMARFP/Col2.3GFP transgenic mice, allowing identification of cells at the mesenchymal stage (αSMARFP) and at mature osteoblast stage (Col2.3GFP). In vitro analysis of osteogenic potential has been made for both types of cells and in vivo analysis (teratoma assay) of osteogenic potential for ESCs. Results Cultures of bone marrow MSCs were established, and after one week a population of αSMARFP expressing cells was noticed. Following cell sorting, replated αSMARFP+ cells (20-30%) were induced to osteogenesis, and strong expression of osteoblast specific transgene Col2.3GFP was noticed, confirming osteogenic ability of αSMARFP+ cells. Osteogenic differentiation was confirmed by detection of mineralization with von Kossa method and by up-regulation of markers of mature osteoblast lineage cells; osteocalcin and bone sialoprotein. Following osteogenic induction in ESCs, expression of alkaline phosphatase and subsequent mineralization (detected by von Kossa staining) was observed. After one week of osteogenic induction, ESCs began to express αSMARFP. This expression was localized to the peripheral area encircling a typical ESC colony. Nevertheless, these αSMARFP+ cells did not show activation of the Col2.3GFP, even after 6 weeks of osteogenic differentiation in vitro. Also, small increases in expression in some of the bone markers analyzed (Colagen type I, bone sialoprotein) were noticed, but most of them appeared minimal compared to the levels expressed by the MSCs, even after six weeks of osteogenic induction. In contrast, in vivo teratoma assay by ECSs showed bone formation and strong Col2.3GFP signal in the areas that stain positive for mineralization by von Kossa. Conclusion These results show that MSCs compare to ESCs, have much more capability to differentiate into mature osteoblast, express specific bone markers and Col2.3GFP transgene after osteogenic induction in vitro. On the other hand, in the functional in vivo assay ESCs show capability to form bone and activate Col2.3GFP marker. The results obtained indicate that detection of alkaline phosphatase activity and mineralization of ESCs cultured under osteogenic conditions is not sufficient to demonstrate osteogenic maturation. This study indicates the utility of the promoter-visual transgene approach to assess the commitment and differentiation of stem cells into the osteoblast lineage. To current knowledge, this study is the first to utilize the expression of Col2.3GFP transgene in the ESCs derived from transgenic mice

Promjene u zdravlju usne šupljine tijekom rane faze ortodontske terapije

The aim of the study was to assess the influence of fixed orthodontic appliance on Streptococcus (S.) mutans and S. sobrinus counts in orthodontic patients with regard to their previous caries experience (Decayed, Missing and Filled Teeth (DMFT ) index) during the first 12 weeks of orthodontic treatment. Twenty-two patients that satisfied inclusion criteria (healthy systemic and periodontal condition, avoidance of antibiotic therapy and antiseptic mouthwashes in the past three months) were included. All clinical measurements took place prior to and 12 weeks after fixed orthodontic appliance placement, in the following order: 1) stimulated saliva flow (SS); 2) Simplified Oral Hygiene I ndex (OHI -S); and 3) DMFT . The method of polymerase chain reaction (PCR) was used to detect the presence of S. mutans and S. sobrinus at T1 and T2. T-test showed significant increase in DMFT index and SS between T1 and T2. Results also indicated significant improvement in OHI -S index. By use of the PCR method, S. mutans was detected in two patients at T1. At T2, two more patients had S. mutans, but the increase was not statistically significant. Using the same method, S. sobrinus was detected only in two patients at T2. In conclusion, fixed orthodontic appliances did not induce statistically significant changes in caries microflora even in the presence of enhanced oral hygiene habits.Svrha istraživanja bila je utvrditi utjecaj fiksnih ortodontskih naprava na broj kariogenih bakterija Streptococcus (S.) mutans i S. sobrinus u ortodontskih pacijenata s obzirom na njihov kariogeni status (indeks DMFT ) tijekom prvih dvanaest tjedana ortodontske terapije. U istraživanju je sudjelovalo 22 ispitanika koji su zadovoljili kriterije za uključenje: uredno opće i parodontno zdravlje, izbjegavanje antiseptika za ispiranje usne šupljine i antibiotika u prethodna tri mjeseca. Sva klinička mjerenja napravljena su prije (T1) i 12 tjedana nakon (T2) postavljanja fiksne ortodontske naprave sljedećim redoslijedom: 1) količina stimulirane sline (SS), 2) Simplified Oral Hygiene Index (OHI -S) i 3) DMFT . Reakcija lančane polimeraze (PCR) je korištena za otkrivanje prisutnosti S. mutans i S. sobrinus prilikom T1 i T2. T-test je pokazao značajan porast indeksa DMFT i SS između T1 i T2. Rezultati također pokazuju značajno poboljšanje indeksa OHI -S. Koristeći PCR metodu S. mutans je otkriven kod dvoje ispitanika na T1. Nakon 12 tjedana fiksne ortodontske terapije došlo je do porasta broja ispitanika kod kojih je otkriven S. mutans, ali porast nije bio statistički značajan. Istom metodom S. sobrinus je otkriven tek u dvoje ispitanika na T2. U zaključku, tijekom prvih 12 tjedana fiksne ortodontske terapije nije došlo do statistički zančajne promjene u kariogenoj mikroflori. Primjena individualnih edukativnih i preventivnih mjera oralne higijene može spriječiti nastanak štetnih nuspojava tijekom fiksne ortodontske terapije