Therapeutic effects of rosmarinic acid in alloplastic bone graft applied rat calvarial bone defect model

Öz

Aim: Many reasons such as trauma, neoplasms, infections, and congenital anomalies can cause calvarial bone defects. Various graft types have been tried and also treatment methods have been investigated in order to eliminate bone defects in the cranial region and to stimulate bone regeneration. In our study, we aimed to investigate the effects of Rosmarinic acid (RA) on the rat calvarial bone defect model with graft material. Method: Wistar male rats (n=32) were divided into 4 in the study; defect group as control (n=8), defect + RA treated (n=8), defect + graft (n=8), defect+graft+RA group (n=8). Under anesthesia, the frontal bone was opened and the periosteal flap was removed using a periosteal lever, and a circular full-thickness bone defect (5 mm) was created in the midline. In groups 2 and 3, allograft material was placed in the defect area, the tissue was sutured and closed. RA (100mg/kg) was administered orally for 7 days. After 28 days, all animals were sacrificed and calvarial bones were removed, fixed with 10% formalin, decalcified with 5% Ethylenediamine tetraacetic acid (EDTA), cut sections 4-6 μm thick after routine histological preparation and stained with Hematoxylin and eosin (HE). Bone Morphogenetic Protein-2 (BMP-2) was immunohistochemically labeled and examined under a light microscope. Alkaline phosphatase (ALP), creatine kinase (KK), total antioxidant level (TAS), total oxidant level (TOS) and oxidative stress index (OSI) parameters were studied from blood samples. Results: TAS value was found to be significantly higher in the defect + graft + RA group compared to the defect group (p<0.05). The OSI value was significantly lower in the defect + graft+RA group compared to the defect group. It was found that inflammatory cells were scattered in a small number of solitary forms within the graft area, and dilatation and congestion in blood vessels were decreased. It was observed that the bone tarbecules near the main bone area were enlarged, the matrix structure in which osteoblastic activity became evident and the osteocytes has settled in the lacunae. Conclusion: As a result, it was observed that with RA+Graft application, induced osteoblastic activity in the calvarial defect model, resulted in osteocyte formation and new bone development.

Anahtar Kelimeler

• Calvarial defect
• , rosmarinic acid
• immunohistochemistry
• BMP
• rat

Alloplastik kemik grefti uygulanmış sıçan kalvarial kemik defekt modelinde rosmarinik asidin terapötik etkileri

Öz

Amaç: Travma, neoplazmalar, enfeksiyonlar ve doğuştan anomaliler gibi pek çok neden, kalvaryal kemik kusurlarına neden olabilir. Kraniyal bölgedeki kemik kusurlarını gidermek ve kemik yenilenmesini uyarmak için çeşitli greft tipleri denenmiş ve tedavi yöntemleri araştırılmıştır. Çalışmamızda Rosmarinik asidin (RA) greft materyali uygulanmış sıçan kalvaryal kemik defekt modeli üzerindeki etkilerinin araştırılması amaçlanmıştır. Yöntem: Çalışmada Wistar erkek sıçanlar (n=32) dörde bölündü; kontrol olarak defekt grubu (n=8), defekt+RA uygulamalı grup (n=8), defekt+greft (n=8), defekt+greft+RA uygulamalı grup (n=8). Anestezi altında frontal kemik açılarak ve periosteal elevatör ile periosteal flep çıkarıldı ve orta hatta dairesel tam kalınlıkta kemik defekti (5 mm) oluşturuldu. Grup iki ve üçte defekt bölgesine allogreft materyal yerleştirildi, doku dikilerek kapatıldı. RA (100mg/kg) oral yolla 7 gün süre ile uygulandı. 28 gün sonra tüm hayvanlar sakrifiye edildi ve kalvaryal kemikler çıkarıldı, %10’luk formalin ile tespit edildikten sonra, %5’lik EDTA (Etilendiamin tetraasetik asit) ile dekalsifiye edildi, rutin histolojik preparasyondan sonra 4-6 μm kalınlığında kesitler kesildi ve Hematoksilen Eosin ile boyandı. BMP-2 (Bone morphogenetic proteins) kemik morfogenetik proteinleri immunohistokimyasal olarak işaretlendi ve ışık mikroskobu altında incelendi. Kan örneklerinden alkalen fosfataz (ALP), kreatin kinaz (KK), total antioksidan seviyesi (TAS), total oksidan seviyesi (TOS) ve oksidatif stres indeksi (OSI) parametreleri çalışıldı. Bulgular: TAS değeri defekt+greft+RA grubunda defekt grubuna göre anlamlı olarak yüksek bulunmuştur (p<0.05). OSI değeri defekt+greft+RA grubunda defekt grubuna göre anlamlı olarak düşüktür. Greft alanı içinde inflamatuvar hücrelerin az sayıda soliter olarak dağıldığı, kan damarlarında dilatasyon ve konjesyonun azaldığı tespit edildi. Ana kemik bölgesine yakın kemik tarbeküllerin genişlediği osteoblastik aktivitenin belirginleştiği matriks yapısının olgunlaştığı ve osteositlerin laküna içinde yerleştiği görüldü. Sonuç: Sonuç olarak, RA+Greft uygulaması ile kalvariyal defekt modelinde osteoblastik aktiviteyi indükleyerek osteosit oluşumuna ve yeni kemik gelişimine neden olduğu gözlenmiştir.

Anahtar Kelimeler

• kalvaryal defekt
• rosmarinik asit
• immunohistokimya
• sıçan
• BMP

Kaynakça

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