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

Yıl 2022, Cilt: 15 Sayı: 1, 88 - 99, 30.04.2022

Busra Deveci Ahmet Dağ Ela Tules Kadiroğlu Fırat Aşır Ebru Gokalp-ozkorkmaz Engin Deveci

https://doi.org/10.26559/mersinsbd.898835

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

Destekleyen Kurum

Dicle Üniversitesi Bilimsel Araştırma Projeleri (DUBAP)

Proje Numarası

DİŞ.20.003

Teşekkür

Bu çalışma Dt. Büşra DEVECİ'nin doktora tezinin bir parçasıdır ve Dicle Üniversitesi Bilimsel Araştırma Projeleri (DUBAP) tarafından DİŞ.20.003 numaralı proje ile desteklenmiştir.

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

Proje Numarası

DİŞ.20.003

Kaynakça

• Szpalski C, Barr J, Wetterau M, Saadeh PB, Warren SM. Cranial bone defects: Current and future strategies. Neurosurg Focus 2010;29(6):E8.
• Bosch C, Melsen B, Gibbons R, Vargervik K. Human recombinant transforming growth factor-beta 1 in healing of calvarial bone defects. J Craniofac Surg 1996;7(4):300-310.
• Kazancioglu HO, Ezirganli S, Aydin MS. Effects of laser and ozone therapies on bone healing in the calvarial defects. J Craniofac Surg 2013;24(6):2141-2146.
• Gupta MC, Maitra S. Bone grafts and bone morphogenetic proteins in spine fusion. Cell Tissue Bank 2002;3:255-267.
• Laçin N, İzol BS, Gökalp Özkorkmaz E, Deveci B, Tuncer MC. The effect of graft application and simvastatin treatment on tibial bone defect in rats. A histological and immunohistochemical study. Acta Cir Bras 34(4): e201900408.
• Younger EM, Chapman MW. Morbidity at bone graft donor sites. J Orthop Trauma 1989;3:192-195.
• Alagawany M, Abd El-Hack ME, Farag MR, et al. Rosmarinic acid: Modes of action, medicinal values and health benefits. Anim Health Res Rev 2017;18(2):167-176. doi:10.1017/S1466252317000081
• Lee JW, Asai M, Jeon SK et al. Rosmarinic acid exerts an antiosteoporotic effect in the RANKL induced mouse model of bone loss by promotion of osteoblastic differentiation and inhibition of osteoclastic differentiation. Mol Nutr Food 2015;59(3):386-400.
• Elbahnasawy ER, Valeeva Eman M. El-Sayed, Rakhimov I. The Impact of thyme and rosemary on prevention of osteoporosis in Rats. J Nutr Metab. 2019; https://doi.org/10.1155/2019/1431384
• Kim BS, Choi MK, Yon JH, Lee J. Evaluation of bone regeneration with biphasic calcium phosphate substitute implanted with bone morphogenetic protein 2 and mesenchymal stem cells in a rabbit calvarial defect model. Oral Surg Oral Med Oral Pathol Oral Radiol 2015;120:2
• Reddi AH. Bone and cartilage differentiation. Curr Opin Genet Dev 1994;4(5):737-744. doi:10.1016/0959-437x(94)90141-o.
• Hogan BL. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev 1996;10(13):1580-1594. doi:10.1101/gad.10.13.1580.
• Kawabata M, Imamura T, Miyazono K. Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev 1998;9(1):49-61. doi:10.1016/s1359-6101(97)00036
• Shegarfi H, Reikeras O. Review article: Bone transplantation and immune response. J Orthop Surg (Hong Kong) 2009;17:206-211. doi: 10.1177/230949900901700218.
• Kose O, Arabaci T, Yemenoglu H ve ark.. Influences of Fucoxanthin on alveolar bone resorption in induced periodontitis in rat molars. Marine Drugs. 2016;14(4):70. doi:10.3390/md14040070.
• Agarwal A, Gupta ND, Jain A. Platelet rich fibrin combined with decalcified freeze-dried bone allograft for the treatment of human intrabony periodontal defects: A randomized split mouth clinical trial. Acta Odontol Scand 2016;74(1):36-43. doi: 10.3109/00016357.2015.1035672.
• Wozney JM, Rosen V. Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clin Orthop Relat Res. 1998;346:26-37.
• Jovanovic SA, Hunt DR, Bernard GW, et al. Long-term functional loading of dental implants in rhBMP-2 induced bone. A histologic study in the canine ridge augmentation model. Clin Oral Implants Res. 2003;14(6):793-803. doi: 10.1046/j.0905-7161.2003.clr140617.x9.
• Sorensen RG, Wikesjö UME, Kinoshita A, Wozney JM. Periodontal repair in dogs: evaluation of a bioresorbable calcium phosphate cement (Ceredex) as a carrier for rhBMP-2. J Clin Periodont. 2004;31(9):796-804. doi: 10.1111/j.1600-051X.2004.00544.x.
• Kang W, Liang Q, Du L, Shang L, Wang T et al. Sequential application of bFGF and BMP-2 facilitates osteogenic differentiation of human periodontal ligament stem cells. J Periodont Res 2019;54(4):424-434. doi: 10.1111/jre.1264411.
• Lee AR, Choi H, Kim JH, Cho SW, Park YB. Effect of serial use of bone morphogenetic protein 2 and fibroblast growth factor 2 on periodontal tissue regeneration. Implant Dent 2017;26(5): 664-673. doi: 10.1097/ID.0000000000000624.
• Kizildag A, Taşdemir U, Arabaci T, et al. Evaluation of new bone formation using autogenous tooth bone graft combined with platelet-rich fibrin in calvarial defects. J Craniofac Surg. 2019;30(6):662-1666.
• Acar AH, Yolcu Ü, Gül M ve ark. Micro-computed tomography and histomorphometric analysis of the effects of platelet-rich fibrin on bone regeneration in the rabbit calvarium. Arch Oral Biol 2015; 60(4):606-614.
• Scalbert A, Manach C, Morand C, Rémésy C, Jiménez L. Dietary polyphenols and the prevention of diseases. Crit Rev Food Sci Nutr 2005;45(4):287-306. doi:10.1080/1040869059096
• Moon DO, Kim MO, Lee JD, et al. Rosmarinic acid sensitizes cell death through suppression of TNF-alpha-induced NF-kappaB activation and ROS generation in human leukemia U937 cells. Cancer Lett 2010;288(2):183-4191. doi: 10.1016/j.canlet.2009.06.033.
• Sotnikova R, Okruhlicova L, Vlkovicova J, et al. Rosmarinic acid administration attenuates diabetes-induced vascular dysfunction of the rat aorta. J Pharm Pharmacol 2013;65(5):713-23, doi: 10.1111/jphp.12037.
• Lee HJ, Cho HS, Park E, et al. Rosmarinic acid protects human dopaminergic neuronal cells against hydrogen peroxide-induced apoptosis. Toxicol 2008;250(2-3):109-115. doi: 10.1016/j.tox.2008.06.010.
• Sanbongi C, Takano H, Osakabe N et al., Rosmarinic acid inhibits lung injury induced by diesel exhaust particles. Free Radic Biol Med 2003;34(8):1060–1069.
• Hsu YC, Cheng CP, Chang DM. Plectranthus amboinicus attenuates inflammatory bone erosion in mice with collagen-induced ar thritis by downregu lation of RANK L-induced NFATc1 expression. J Rheumatol 2011;38:1844-1857.
• Santiago-Mora R, Casado-Dı´az A, De Castro MD, Quesada-Go´mez JM. Oleuropein enhances osteoblastogenesisand inhibits adipogenesis: The effect on differentiation instem cells derived from bone marrow. Osteoporos Int 2011;22:675–684. doi:10.1007/s00198-010-1270-x
• Omori A, Yoshimura Yoshitaka D, Yoshiaki S. Rosmarinic acid and arbutin suppress osteoclast differentiation by inhibiting superoxide and NFATc1 downregulation in RAW 264.7 cells. Biomed Rep 2015;3.10.3892/br.2015.452.
• Notodihardjo FZ, Kakudo N, Kushida S, Suzuki K, Kusumoto K. Bone regeneration with BMP-2 and hydroxyapatite in critical-size calvarial defects in rats. J Craniomaxillofac Surg 2012;40(3):287-291. doi: 10.1016/j.jcms.2011.04.008.