Üç farklı mineral trioksit agregat simanının proenflamatuvar sitokin düzeyleri üzerindeki etkisinin değerlendirilmesi
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Dosyalar
Tarih
2020
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Çalışmamızın amacı, ProRoot MTA, MTA Repair HP ve Bio MTA+ simanlarının proenflamatuvar sitokin düzeyleri üzerindeki etkisinin değerlendirilmesi ve materyallerin sitotoksisitelerinin karşılaştırılmasıdır.
Test materyalleri üretici firmaların önerileri doğrultusunda karıştırıldı ve hazırlanan simanlar standart silindirik kalıplara yerleştirildi. Örnekler 370 C’de 48 saat inkübe edildikten sonra gama sterilizasyonuna tabi tutuldu.
Analizler için THP-1 orijinli insan monosit hücre hattı kullanıldı. Hücreler phorbol 12-myristate 13-acetate (PMA) ile farklılaştırıldı. Bir grup hücre ek olarak enflamasyonun başlatılması için lipopolisakkarit (LPS) ile de muamele edildi. Bu işlemi takiben, MTA siman blokları ile insan makrofaj hücrelerinin etkileşimi sağlandı ve 24 saat inkübe edildi. Makrofaj hücrelerinin ürettiği enflamatuvar sitokin yoğunlukları ELISA kiti ile ölçüldü. Bu amaçla TNF-α, IL-1β, IL-6 mediyatörleri analiz edildi. Çalışmada ayrıca test edilen MTA tiplerinin MTT yöntemiyle sitotoksik etkileri belirlendi. Veriler iki yönlü varyans analizi, Tukey çoklu karşılaştırma analizi, Kruskal Wallis testi ve Dunn’s çoklu karşılaştırma testi ile istatistiksel olarak değerlendirildi.
Bu çalışmadan elde edilen veriler doğrultusunda 72. saatin sonunda makrofaj canlılık değerleri ProRoot MTA 95,782 μg/ml****, MTA Repair HP 65,274 μg/ml*, Bio MTA+ 68,012 μg/ml* ve Pozitif kontrol grubu 58,767 μg/ml**** şeklinde gözlendi. LPS ile muamele edilmiş hücrelerin yer aldığı sitotoksisite analizinde ProRoot MTA 77.101 μg/ml, MTA Repair HP 69,211 μg/ml, Bio MTA+ 39,134 μg/ml, Pozitif kontrol grubu 88,438 μg/ml salınımda bulundu.
72. saatte gözlenen IL-1β değerleri Kontrol Grubu 9,857 pg/ml, ProRoot 30,082 pg/ml**, MTA Repair HP 22,503 pg/ml ve Bio MTA+ 58,660 pg/ml gözlendi. IL-6 seviyesi tüm gruplarda benzer oranda düşük iken, 24. saatte MTA Repair HP sitokin salınımını istatistiksel olarak anlamlı oranda baskıladı (P<0,05:*, P<0,01:**, P<0,001:***, P<0,0001:****). 72. saatte salınan TNF-α değerleri Kontrol Grubu 6,214 gp/ml, ProRoot 23,025 pg/ml**, MTA Repair HP 7,260 pg/ml ve Bio MTA+ 8,738 pg/ml şeklinde oldu (P<0,05:*, P<0,01:**, P<0,001:***, P<0,0001:****). LPS mevcudiyeti sitokin salınım miktarını ortamda siman bulunmasından bağımsız olarak arttırdı.
Değerlendirilen süre sonunda makrofaj hücrelerinin sağ kalım oranları yüksekten düşüğe doğru sırasıyla ProRoot MTA, Bio MTA+, MTA Repair HP ve Pozitif kontrol grubu olarak, LPS ile muamele edilmiş hücrelerin sağ kalım oranları ise Pozitif kontrol grubu, ProRoot MTA, MTA Repair HP ve Bio MTA+ olarak bulundu. 72. saatte gözlenen en yüksek IL-1β değerleri sırasıyla Bio MTA+, ProRoot MTA, MTA Repair HP ve Kontrol grubu, IL-6 değerleri Bio MTA+, MTA Repair HP, ProRoot MTA ve Kontrol grubu, TNF-α seviyeleri ise ProRoot MTA, Bio MTA+, MTA Repair HP ve Kontrol grubu şeklinde gözlendi. LPS ile farklılaştırılmış hücrelerden elde edilen en yüksek IL-1β değerleri sırasıyla Bio MTA+, MTA Repair HP, ProRoot MTA ve Kontrol grubu, IL-6 değerleri Kontrol grubu, ProRoot MTA, MTA Repair HP ve Bio MTA+, TNF-α değerleri ise MTA Repair HP, ProRoot MTA, Kontrol grubu ve Bio MTA+ olarak belirlendi.
Donma süresinin oldukça kısa olduğu bilinen MTA Repair HP materyalinin, optimum düzeydeki biyouyumluluğu ve antienflamatuvar özelliği göz önünde bulundurulduğunda, klinik uygulamalarda avantaj sağlayacağını öngörmekteyiz. Hücresel düzeyde elde edilen bu verilerin daha iyi anlaşılması için klinik çalışmalar ile desteklenmesi gerekmektedir.
The aim of this study was to compare the cytotoxicity of ProRoot MTA, MTA Repair HP and Bio MTA+ and to evaluate the effects of these materials on proinflammatory cytokine levels of human monocytes. MTA cements were prepared according to their manufacturers’ instructions, were placed in standard cylinder-shaped moulds and were incubated 24 hours at 370 C. THP-1 (human monocyte cells) cell lines were used for the assays. THP-1 cells were differentiated into macrophages by 24-hour incubation with phorbol 12-myristate 13-acetate (PMA). Then, a group of cells was activated by lipopolysaccharide (LPS). The cytotoxic effects of the samples were evaluated by colorimetric determination of mitochondrial reductase activity in viable cells at 24th, 48th and 72nd hours. In the second phase of the study, supernatants of cells used in cytotoxicity test were utilized to test the proinflammatory potential of the tested materials. IL-1β, IL-6 and TNF-α levels in supernatants were determined using enzyme linked immunosorbent assay (ELISA) kits. The data were examined with Two way ANOVA, Tukey’s Multiple Comparison Test, Kruskal Wallis test and Dunnett’s Multiple Comparison Test (P=0,05). Macrophage viability values at the end of 72 hours were observed as 95.782 μg/ml**** for ProRoot MTA, 65.274 μg/ml for MTA Repair HP *, 68.012 μg/ml* for Bio MTA+ and 58.767 μg/ml**** for positive control group (P<0,05:*, P<0,01:**, P<0,001:***, P<0,0001:****). In the cytotoxicity analysis involving LPS-treated cells, values observed as 77.101 μg/ml for ProRoot MTA, 69.211 μg/ml for MTA Repair HP, 39.134 μg/ml for Bio MTA+, and 88.438 μg/ml for positive control group. IL-1β values observed at 72 hours were 9.857 pg/ml in the control group, 30.082 pg/ml* in ProRoot, 22.503 pg/ml** in MTA Repair HP and 58.660 pg/ml* in Bio MTA+ (P <0,05: *, P <0,01: **, P <0,001: ***, P <0,0001: ****). While IL-6 levels were similarly low in all groups, MTA Repair HP statistically significantly suppressed cytokine secretion at 24th hour. The TNF-α values secreted at 72 hours were 6.214 pg/ml for the control group, 23.025 pg/ml** for ProRoot, 7.260 pg/ml for MTA Repair HP and 8.738 pg/ml for Bio MTA+ (P <0,05: *, P <0,01: **, P <0,001: ***, P <0,0001: ****). The presence of LPS increased the amount of the cytokine secretion regardless of cements. At the end of the evaluation period, the survival rates of macrophage cells were sorted in a descending order starting from high to low as ProRoot MTA, Bio MTA+, MTA Repair HP and positive control group, respectively. The survival rates of LPS-treated cells were ranked as positive control group, ProRoot MTA, MTA Repair HP and Bio MTA+. The highest IL-1β values observed at 72 hour were listed in order as Bio MTA+, ProRoot MTA, MTA Repair HP and the control group, whereas the IL-6 values were ranked from the highest as the control group, ProRoot MTA, MTA Repair HP and Bio MTA+. TNF-α levels were ranged as ProRoot MTA, Bio MTA+, MTA Repair HP, and the control group. The highest IL-1β values obtained from LPS differentiated cells were aligned as Bio MTA+, MTA Repair HP, ProRoot MTA and the control group. IL-6 values were ranked in a descending order as the control group, ProRoot MTA, MTA Repair HP and Bio MTA+, but TNF-α values were listed in order as Bio MTA+MTA Repair HP, ProRoot MTA and the control group. It is anticipated that MTA Repair HP, which is known to have a very short setting time, will provide an advantage in clinical applications, considering its optimum biocompatibility and anti-inflammatory properties. In order to provide a better understanding of the in vitro results of the present study, further clinical studies are required.
The aim of this study was to compare the cytotoxicity of ProRoot MTA, MTA Repair HP and Bio MTA+ and to evaluate the effects of these materials on proinflammatory cytokine levels of human monocytes. MTA cements were prepared according to their manufacturers’ instructions, were placed in standard cylinder-shaped moulds and were incubated 24 hours at 370 C. THP-1 (human monocyte cells) cell lines were used for the assays. THP-1 cells were differentiated into macrophages by 24-hour incubation with phorbol 12-myristate 13-acetate (PMA). Then, a group of cells was activated by lipopolysaccharide (LPS). The cytotoxic effects of the samples were evaluated by colorimetric determination of mitochondrial reductase activity in viable cells at 24th, 48th and 72nd hours. In the second phase of the study, supernatants of cells used in cytotoxicity test were utilized to test the proinflammatory potential of the tested materials. IL-1β, IL-6 and TNF-α levels in supernatants were determined using enzyme linked immunosorbent assay (ELISA) kits. The data were examined with Two way ANOVA, Tukey’s Multiple Comparison Test, Kruskal Wallis test and Dunnett’s Multiple Comparison Test (P=0,05). Macrophage viability values at the end of 72 hours were observed as 95.782 μg/ml**** for ProRoot MTA, 65.274 μg/ml for MTA Repair HP *, 68.012 μg/ml* for Bio MTA+ and 58.767 μg/ml**** for positive control group (P<0,05:*, P<0,01:**, P<0,001:***, P<0,0001:****). In the cytotoxicity analysis involving LPS-treated cells, values observed as 77.101 μg/ml for ProRoot MTA, 69.211 μg/ml for MTA Repair HP, 39.134 μg/ml for Bio MTA+, and 88.438 μg/ml for positive control group. IL-1β values observed at 72 hours were 9.857 pg/ml in the control group, 30.082 pg/ml* in ProRoot, 22.503 pg/ml** in MTA Repair HP and 58.660 pg/ml* in Bio MTA+ (P <0,05: *, P <0,01: **, P <0,001: ***, P <0,0001: ****). While IL-6 levels were similarly low in all groups, MTA Repair HP statistically significantly suppressed cytokine secretion at 24th hour. The TNF-α values secreted at 72 hours were 6.214 pg/ml for the control group, 23.025 pg/ml** for ProRoot, 7.260 pg/ml for MTA Repair HP and 8.738 pg/ml for Bio MTA+ (P <0,05: *, P <0,01: **, P <0,001: ***, P <0,0001: ****). The presence of LPS increased the amount of the cytokine secretion regardless of cements. At the end of the evaluation period, the survival rates of macrophage cells were sorted in a descending order starting from high to low as ProRoot MTA, Bio MTA+, MTA Repair HP and positive control group, respectively. The survival rates of LPS-treated cells were ranked as positive control group, ProRoot MTA, MTA Repair HP and Bio MTA+. The highest IL-1β values observed at 72 hour were listed in order as Bio MTA+, ProRoot MTA, MTA Repair HP and the control group, whereas the IL-6 values were ranked from the highest as the control group, ProRoot MTA, MTA Repair HP and Bio MTA+. TNF-α levels were ranged as ProRoot MTA, Bio MTA+, MTA Repair HP, and the control group. The highest IL-1β values obtained from LPS differentiated cells were aligned as Bio MTA+, MTA Repair HP, ProRoot MTA and the control group. IL-6 values were ranked in a descending order as the control group, ProRoot MTA, MTA Repair HP and Bio MTA+, but TNF-α values were listed in order as Bio MTA+MTA Repair HP, ProRoot MTA and the control group. It is anticipated that MTA Repair HP, which is known to have a very short setting time, will provide an advantage in clinical applications, considering its optimum biocompatibility and anti-inflammatory properties. In order to provide a better understanding of the in vitro results of the present study, further clinical studies are required.
Açıklama
Anahtar Kelimeler
MTA, IL-1β, IL-6, TNF-α, Proenflamatuvar Sitokin, MTA, IL-1β, IL-6, TNF-α, Proinflammatory Cytokine
