Tugas Mandiri Farmakologi II

Histopathological Analysis of Korticosteroidantibiotic

Preparation and Propolis Paste Formulation as Intracanal
Medication After Pulpectomy: an in vivo study

Oleh:

Sofia Dwi Nurmalitasari

021511133046

FARMAKOLOGI II - DEPARTEMEN BIOLOGI ORAL

FAKULTAS KEDOKTERAN GIGI-UNAIR

Semester Genap - 2017/2018
 ABSTRAK

Obat intracanal dalam terapi pulpectomy digunakan untuk mengurangi rasa sakit
dan proses inflamasi pada pulpa dan jaringan periapikal. Propolis telah dikenal
sebagai antibiotik alami dan telah menjadi subyek penelitian dalam bidang
kedokteran dan kedokteran gigi karena sifat terapeutik seperti antibiotik, analgesik
dan efek anti-inflamasi. Tujuan: untuk melaksanakan study in vivo mengenai
respon jaringan periapikal untuk pasta propolis bila digunakan sebagai obat
intrakanal pada gigi anjing setelah pulpectomy. Bahan dan Metode: gigi seri 72
anjing dipilih untuk percobaan. Setelah persiapan biomekanik saluran akar diisi
dengan obat kortikosteroid-antibiotik, pasta propolis, dibuat dua kontrol yaitu
non-obat (kontrol negatif) atau non-pulpectomy sama sekali (kontrol positif).
Obat-obat dibiarkan di dalam saluran akar selama 7, 14 atau 28 hari. Pada akhir
periode percobaan disiapkan bagian histologis dan dianalisis menggunakan
mikroskop optik. Bagian yang diamati secara histologis diklasifikasikan menurut
skor mewakili peristiwa inflamasi yang diamati: kehadiran neutrofil
polimorfonuklear, eosinofil polimorfonuklear, limfosit dan sel plasma, makrofag,
kondensasi berserat dan abses. Hasil: Ada perbedaan yang signifikan secara
statistik antara reaksi jaringan yang disebabkan oleh dua zat yang diuji, setelah
periode percobaan yang berbeda, dengan jaringan periapikal yang kontak dengan
pasta propolis menunjukkan reaksi peradangan lebih sedikit dibandingkan dengan
obat kortikosteroid antibiotik. Kesimpulan: respon jaringan rendah dari pasta
propolis menunjukkan bahwa bahan ini dapat dianggap sebagai pilihan untuk
pengobatan saluran akar setelah pulpectomy.

Kata kunci: Endodontik. Propolis. Pulpectomy. Rongga pulpa gigi.

 PENDAHULUAN

1.1 Latar Belakang

Pada perawatan endodontik diperlukan untuk menggunakan obat

intrakanal, dalam terapi pulpectomy obat intrakanal adalah obat yang memiliki
kemampuan untuk menyembuhkan proses peradangan dan infeksi serta memiliki
potensi induksi penyembuhan. Beberapa bahan kimia dan agen terapi telah
diusulkan sebagai obat intrakanal salah satunya adalah gabungan dari
kortikosteroid-antibiotik. Obat kortikosteroid-antibiotik yang komersial digunakan
saat ini dalam kedokteran gigi yaitu Otosporin ®, karena sebagai obat intrakanal
memiliki komponennya yaitu: polimiksin B sulfat, sulfat neomycin, hidrokortison
dan berbasis air. Persiapan ini digunakan untuk mengurangi vasodilatasi,
menurunkan eksudasi cair, dan juga vasokonstriksi langsung pada pembuluh darah
kecil. Meskipun kombinasi ini memiliki banyak sifat serta manfaat yang
diinginkan sebagai obat intrakanal pulpectomy namun, jika digunakan untuk
waktu yang lama dapat mengurangi aktivitas metabolisme seluler dan bahkan
menghambat atau menunda perbaikan jaringan. Sehubungan dengan tujuan untuk
perbaikan jaringan, propolis telah dikenal sebagai antibiotik alami dan telah
menjadi subyek penelitian dalam kedokteran dan kedokteran gigi.
Propolis adalah suatu campuran kompleks yang mengandung zat resin dan
balsamic yang dihasilkan oleh lebah yang dikumpulkan dari pucuk daun tanaman
Apis mellifera, yang tanaman tersebut digunakan untuk transportasi ke sarang
mereka serta dimodifikasi dengan penambahan sekresi mereka sendiri yaitu air
liur, serbuk sari dan lilin. Propolis mengandung flavonoid yang dianggap sebagai
primary utama komponen biologis aktif, diketahui dapat bertanggung jawab untuk
sebagian besar sifat terapeutik. Dalam kedokteran gigi, propolis telah digunakan
untuk pengobatan ulkus aphthous, Candida albicans, necrotizing akut ulseratif
gingivitis (UNUG), gingivitis dan periodontitis dan telah terbukti memiliki
aktivitas antimikroba terhadap Streptococcus mutans dan polymicrobial yang
ditemukan pada saluran akar yang nekrotik. Sifat-sifat propolis antara lain adalah
tindakan analgesik, anti virus, anti jamur dan tindakan anti protozoal,
imunostimulasi, efek antioksidan dan tindakan anti inflamasi. Semua sifat ini
menimbulkan sebuah keinginan untuk menggunakan propolis sebagai obat
intrakanal di pulpectomy di mana ada kebutuhan untuk kedua efek analgesik dan
anti-inflamasi.

1.2 Tujuan

Untuk melakukan eksperimental melalui studi in vivo pada respon jaringan

periapikal menggunakan pasta propolis sebagai obat intrakanal pada gigi anjing
setelah pulpectomy.
 BAHAN dan METODE

Sebanyak saluran akar gigi seri 72 dari 6 anjing berusia 2 sampai 4 tahun
dipilih untuk percobaan. Protokol yang digunakan telah disetujui oleh lokal etika
Komite (CEP ulbra: 2003-015A; Cep UPF: 276/2004). Hewan-hewan itu berada
di bawah anestesi umum dilengkapi dengan infiltrasi anestesi lokal ke daerah
prosedur seluruh operasi. jaringan dan gigi lembut disterilisasi menggunakan
solusi alcoholiodine (Vico-farma, Lages, SC, Brasil).
Saluran akar dari 66 gigi yang diakses pada permukaan bukal gigi
menggunakan diamond bur # 1012 (Metalurgica Fava Industria e Comercio Ltda.,
Franco da Rocha, SP, Brazil) dengan kecepatan tinggi dengan pendingin water-
air. Setelah melakukan radiografi pada setiap gigi individual, panjang kerja
didirikan pada 1 mm singkat dari akar apeks. Berikut membersihkan saluran akar,
persiapan biomekanik dilakukan dengan menggunakan teknik manual sequential.
Saluran akar diinstrumentasi menggunakan Flexofile ® file K (Dentsply, Maillefer
Instrumen SA, Ballaigues, Swiss), finishing dengan instrumen # 40. Kanal yang
diairi dengan 2 mL larutan pada setiap perubahan instrumen, bergantian antara 1%
sodium hypochlorite (Vico-farma) dan 17% trisodium asam
ethylenediaminetetraacetic (EDTA) (Vico-farma) dengan pH 7,3. Setelah
persiapan kemomekanis selesai, melewati puncak 2 mm di luar panjang bekerja
file # 20. Kanal kemudian dikeringkan dengan poin kertas penyerap # 40 (Tanari
Industria e Comercio Ltda., Paraíba do Sul, RJ, Brasil) yang telah diautoklaf.
Saluran akar kemudian dikemas dengan obat untuk dianalisis: di masing-masing 6
anjing, lima saluran akar yang dikemas dengan larutan 10 mg / mL hidrokortison /
3400 U / mL neomycin sulfat / 10.000 U / mL polimiksin B sulfat, persiapan
kortikosteroid antibiotik dan 5 lain yang dikemas dengan pasta propolis
eksperimental.
Salah satu saluran akar dari setiap anjing dikemas dengan gliserin, yang
dianggap sebagai kelompok pembawa (kontrol negatif), dan gigi yang lain tidak
menerima pengobatan apapun, dianggap sebagai kelompok non-obat (kontrol
positif). Pilihan obat intrakanal dan kontrol dilakukan secara acak. persiapan
kortikosteroid-antibiotik disimpan dalam tabung anestesi bersih dan disterilkan
dan solusi diperkenalkan ke kanal dengan jarum suntik Carpule dan jarum 27G set
1 mm. Pasta propolis eksperimental sebelumnya dimanipulasi berikut komposisi:
ekstrak propolis 1%, titanium dioksida, gliserin. Pasta disimpan dalam tabung
steril dan pada saat saluran akar mengisinya diperkenalkan menggunakan # 35
Lentulo spiral (Pró Dent, São Paulo, SP, Brazil), dikalibrasi dalam dua milimeter
dari saluran akar panjang dan berjalan bekerja pada kecepatan rendah searah
jarum jam. Dalam kasus kedua jenis obat, penyisipan dihentikan ketika refluks
adalah diamati di pintu masuk kanal. Rongga itu kemudian dibersihkan, diisi
dengan kapas steril dan disegel dengan lapisan 2 mm dari semen ionomer gelas
(Vidrion R ® - SS White Artigos Dentários Ltda., Rio de Janeiro, RJ, Brasil),
disusun sesuai dengan instruksi pabriknya.
Hewan-hewan coba harus beristirahat selama 28 hari dengan obat intrakanal,
selama waktu tersebut mereka dipantau oleh dokter hewan. Setelah 7, 14 atau 28
hari dengan obat intrakanal, anjing-anjing di eutanasia dan sampel dikumpulkan.
Jaringan dibersihkan menggunakan perfusi intravena dari 4% buffered
paraformaldehyde. Rahang atas dan rahang bawah telah dihilangkan dan di
postfixed 10% formalin netral. Sampel demineral di EDTA 4,13% pada pH 7,2,
mengikuti protokol proses parafin, lima mikrometer bagian histologis dan
hematoxylin dan eosin. Bagian dianalisis menggunakan mikroskop optik dengan
perbesaran × 100, × 200 dan × 400 oleh pemeriksa tunggal, yang merupakan
seorang histologist berpengalaman dan buta terhadap bahan yang diuji. Subjek
analisis adalah daerah periapikal dan spesimen dengan tingkat terbesar
peradangan dipilih dari masing-masing sampel. Pengamatan kualitatif berikut
dicatat: kehadiran neutrofil polimorfonuklear, eosinofil polimorfonuklear, limfosit
dan sel plasma, makrofag atau sel raksasa, kondensasi berserat dan abses. Setiap
elemen disebabkan nilai numerik sesuai dengan protokol yang diusulkan oleh
Figueiredo, et al. 12 ( 2001). Sekitar 30 hari setelah analisis asli dari bagian, 10%
dari mereka dianalisa untuk menguji kesepakatan intraexaminer antara peristiwa
yang diamati dan diberikan skor. Hasil diuji menggunakan faktorial ANOVA,
dilanjutkan dengan tes Tukey Mann Whitney dan, dengan tingkat signifikansi 5%
(SPSS 12.0). Perjanjian Intraexaminer dinilai menggunakan W Kendall, dengan
tingkat signifikansi 5% (SPSS 12,0).
 HASIL

Irisan kontrol negatif menunjukkan gambaran histologis jaringan periapikal

yang normal, tanpa ada selular inflamasi yang signifikan. Di sisi lain, kontrol
positif terdapat neutrofil, limfosit dan makrofag dengan skor yang lebih tinggi
dibandingkan kontrol negatif menunjukkan proses inflamasi yang terjadi setelah
pulpectomy persiapan kimia-mekanis.
Mengenai peristiwa selular yang terdaftar untuk analisis, kondensasi berserat
tidak termasuk, walaupun asalnya tidak jelas dalam analisis: apakah karena respon
alami periodontal yang ligamen untuk teknik pulpectomy atau reaksi jaringan
terhadap bahan uji. Tidak ada eosinofil yang diamati pada salah satu bagian dari
salah satu kelompok. W Kendall menunjukkan bahwa perjanjian intra-pemeriksa
adalah 98,91%, yang diwakili oleh W = 0,119.
Gambar 1 menggambarkan variasi dalam jumlah neutrofil jika dianalisis
dengan mengacu pada kelompok-kelompok dan periode eksperimental. Kelompok
non-diobati memiliki skor terendah rata-rata dan tidak ada variasi sepanjang
waktu. Menganalisis cara ini dengan kaitannya dengan persiapan kortikosteroid-
antibiotik, teramati bahwa ada peningkatan terus-menerus dalam nilai rata-rata
sebagai periode eksperimental, dengan perbedaan yang signifikan secara statistik
(p = 0,007) terdeteksi oleh tes Tukey antara 7 dan 28 dan antara 14 dan 28 hari,
yang cukup mirip dengan kelompok pembawa. Menganalisis nilai rata-rata untuk
sel-sel ini untuk pasta propolis, tidak ada perbedaan yang signifikan antara periode
dianalisis (p = 0,47). Mungkin untuk menerapkan uji nonparametrik Mann-
Whitney untuk membandingkan zat pada setiap kali percobaan dalam isolasi
untuk setiap peristiwa inflamasi yang berbeda. Hal itu tidak ditemukan perbedaan
yang signifikan secara statistik antara zat diuji selama 7 hari; statistik perbedaan
yang signifikan yang terdeteksi untuk neutrofil antara pasta propolis dan persiapan
kortikosteroid-antibiotik, dan antara pasta propolis dan kelompok pembawa pada
14 dan 28 hari, dengan p = 0,05 dan 0,02, berturut-turut, dengan lebih tinggi skor
untuk persiapan kortikosteroid-antibiotik dan kelompok pembawa; ada perbedaan
antara persiapan kortikosteroid-antibiotik dan kelompok pembawa; ada perbedaan
antara pasta propolis dan kelompok non-obat.
Gambar 1- Perkembangan skor media neutrofil dari kelompok atas waktu eksperimental. (A)
perbedaan yang signifikan dari pasta propolis pada 14 hari; (B) perbedaan yang signifikan dari
pasta propolis pada 28 hari; (*) Perbedaan yang signifikan dalam substansi dari waktu ke waktu

Gambar 2- Perkembangan limfosit skor media dari kelompok atas waktu eksperimental. (A)
perbedaan yang signifikan dari pasta propolis pada 14 hari; (B) perbedaan yang signifikan dari
pasta propolis pada 28 hari; (*) Perbedaan yang signifikan dalam substansi dari waktu ke waktu

Dengan kaitannya dengan limfosit dan sel plasma, diilustrasikan pada Gambar
2, kelompok non-obat memiliki skor terendah rata-rata dan tidak ada variasi
sepanjang waktu. Meskipun zat uji menunjukkan nilai yang sama dalam 7 hari,
pada akhir periode percobaan perbedaan besar adalah diamati antara kelompok
eksperimen dengan skor media yang lebih tinggi untuk persiapan kortikosteroid-
antibiotik dan kelompok pembawa. Ketika masing-masing zat uji dianalisis dalam
isolasi dengan kaitannya dengan variabel waktu, persiapan kortikosteroid-
antibiotik menunjukkan perbedaan yang signifikan (p = 0,012), terdeteksi oleh tes
Tukey, antara 7 dan 14 dan antara 7 dan 28 hari. Dalam analisis ini, pasta propolis
sekali lagi menunjukkan tidak ada perbedaan yang signifikan antara kali (p =
0,41). Mann-Whitney tes nonparametrik menunjukkan tidak ada perbedaan yang
signifikan antara statiscally zat diuji selama 7 hari. Perbedaan yang signifikan
antara persiapan dan pasta propolis kortikosteroid-antibiotik, dan antara pasta
propolis dan kelompok pembawa pada 14 dan 28 hari, dengan p = 0,01 dan 0,02,
berturut-turut, dengan skor yang lebih tinggi untuk persiapan kortikosteroid-
antibiotik dan kelompok pembawa; ada perbedaan antara persiapan kortikosteroid-
antibiotik dan kelompok pembawa; ada perbedaan antara pasta propolis dan
kelompok non-obat.

Gambar 3- Perkembangan makrofag skor media kelompok selama waktu eksperimental

Gambar 4- Perkembangan skor media abses dari kelompok atas waktu eksperimental. (B)
perbedaan yang signifikan dari propolis paste pada 28 hari; (*) Perbedaan yang signifikan dalam
substansi melalui waktu

Kelompok non-obat memiliki skor terendah rata-rata dan tidak memiliki

variasi sepanjang waktu untuk makrofag dan sel raksasa. Analisis nilai rata-rata
antara kecenderungan ekspresi seluler untuk kelompok pembawa, pasta propolis
dan persiapan kortikosteroid-antibiotik menunjukkan bahwa peristiwa ini terus-
menerus hadir dalam jaringan yang dianalisis, dengan tidak ada perbedaan yang
signifikan antara mereka. Tidak ada perbedaan yang signifikan ketika masing-
masing kelompok eksperimen dianalisis sendiri dengan hubungannya dengan
variabel waktu (p> 0,05) (Gambar 3).
Gambar 4 mengilustrasikan nilai rata-rata untuk abses untuk kelompok
yang diuji. Dalam 7 hari abses praktis absen untuk semua kelompok. Kelompok
non-perlakuan dan kelompok pasta propolis tidak menunjukkan perbedaan yang
signifikan dari waktu ke waktu (p> 0,05). Namun, seiring waktu berlalu, ada
peningkatan drastis dalam penyusunan kortikosteroid-antibiotik dan kelompok
pembawa dengan perbedaan signifikan secara statistik (p <0,05), terdeteksi oleh
tes Tukey, antara 7 dan 14 dan antara 7 dan 28 hari. Untuk acara ini, persiapan
kortikosteroid-antibiotik dan kelompok pembawa memiliki skor yang lebih tinggi
bila dibandingkan dengan propolis pasta dan non-diperlakukan kelompok di 28
hari, dengan perbedaan yang signifikan (p <0,05) yang dideteksi dengan uji
nonparametrik Mann-Whitney; tidak ada statiscally perbedaan yang signifikan
antara zat selama 7 dan 14 hari.

Gambar 5- A- Non-diperlakukan sampel - citra daerah periapikal dari akar dengan penampilan
normal; B- Propolis setelah 28 hari - ringan reaksi inflamasi periapikal; C- Propolis setelah 28 hari
- makrofag hadir di wilayah periapikal (arrowed); D- Otosporin 28 hari - reaksi inflamasi yang
intens dengan abses (arrowed); E- Otosporin 28 hari - pembesaran lebih besar dari (D). F-
Otosporin 28 hari - zona pengungsi abses terlihat pada (E). M-makrofag; N-neutrofil; L-
lymphoplasmacytic menyusup. Bar: AE 200 m; FG 50 pM

Gambar 5 menunjukkan gambar perwakilan dari sampel yang diuji setelah

28 hari kontak dengan jaringan periapikal. Gambar 5A adalah gambar perwakilan
dari kelompok non-diobati: jaringan periapikal normal, tanpa sel-sel inflamasi.
Dalam penyusunan kortikosteroid-antibiotik (Gambar 5e-F), adalah mungkin
untuk mengamati adanya peradangan intens setelah 28 hari, sedangkan ada
peradangan periapikal hanya ringan setelah paparan pasta propolis (Gambar 5B-
D).
 PEMBAHASAN

Selama pulpectomy penghapusan pulp dan persiapan kimia-mekanis akan

menjadikan jaringan periapikal ke dalam proses inflamasi akut, yang menjelaskan
hasil dari kelompok pembawa dalam percobaan ini. Seperti diketahui oleh
literatur, peristiwa selular utama yang harus dipertimbangkan ketika mencoba
untuk mengamati respon inflamasi awal dalam jaringan adalah neutrofil dan
adanya abses. Ketika neutrofil yang hadir itu menandakan bahwa proses akut
terjadi, di mana sel-sel ini terus direkrut mengandung peradangan. Hal ini adalah
sel pertama yang bermigrasi dari pembuluh darah dan untuk mencapai lokasi
agresi. Namun demikian, jika agresi yang intens dan neutrofil tidak mampu
menampungnya cepat, abses bisa terbentuk dari penumpukan neutrofil mati,
cairan jaringan dan materi yang abnormal serta menciptakan nanah.
Obat-obatan berdasarkan kortikosteroid bekerja pada sintesis lipocortin
dan vasocortin, menghambat pembentukan enzim edema dan A2 phospholipasis,
sekali menghambat enzim ini, fosfolipid membran tidak dapat dikonversi menjadi
asam arakidonat. Oleh karena itu, siklooksigenase dan lypooxygenase diblokir.
Persiapan kortikosteroid juga bertindak pada histamin, heparin, dan bradicinin,
yang merupakan mediator kimia penting dalam tahap awal peradangan akut.
Propolis berbasis obat-obatan mengandung flavonoid yang dianggap sebagai
primary utama komponen biologis aktif, bertanggung jawab untuk sebagian besar
sifat terapeutik diketahui Flavonoid ikut campur dalam metabolisme asam
arakidonat dan campur tangan dalam siklooksigenase dan lypooxygenase sintesis.
Beberapa zat turunan dalam kaskade ini adalah prostaglandin, tromboksan dan
leukotrien yang penting untuk menjaga integritas dari reaksi inflamasi. Oleh
karena itu, diharapkan bahwa penggunaan persiapan kortikosteroid dan obat-
obatan berbasis propolis menyebabkan perubahan dalam respon inflamasi dalam
jaringan seperti itu diamati dalam penelitian ini dan dibahas. Bila hasil dianalisis,
persiapan kortikosteroid-antibiotik memiliki skor tinggi untuk kehadiran neutrofil
dan abses, terutama dari 14 hari dan seterusnya. Neutrofil menyusup intens, yang
terus menerus hadir dan tidak surut dari 28 hari, menunjukkan tingkat keparahan
respon inflamasi untuk persiapan kortikosteroid-antibiotik.
 Hal tersebut telah dibahas dalam literatur bahwa jika obat ini dibiarkan
kontak dengan sisa jaringan apikal untuk waktu yang lebih lama respon jaringan di
wilayah ini dapat menjadi benar-benar dikompromikan. Di sisi lain, bisa
dimungkinkan bahwa setelah 28 hari, tidak lebih kortikosteroid-antibiotik harus
dalam saluran akar dan fakta ini bisa menjelaskan mengapa persiapan
kortikosteroid-antibiotik memiliki reaksi inflamasi yang sama di 28 hari sebagai
kelompok pembawa (gliserin), yang dianggap zat inert. Sebaliknya, skor untuk
neutrofil dan abses pada kelompok pasta propolis menggambarkan reaksi jaringan
yang lebih menguntungkan untuk proses perbaikan, karena berarti mereka rendah
dan tetap konstan sebagai periode percobaan, cukup mirip dengan kelompok non-
obat. Ini menguatkan studi oleh Bretz, et al. 4 ( 1998), Al-Shaher, et al. 2 ( 2004)
dan Silva, et al. 32 ( 2004), yang telah menunjukkan bahwa pemberian zat
berbasis propolis mengarah ke jaringan menunjukkan reorganisasi normal tanpa
peningkatan vaskular atau peristiwa seluler, mempertahankan rendahnya tingkat
peradangan. Hal ini penting untuk menekankan bahwa, dalam penelitian ini,
setelah 7 hari percobaan, semua zat yang diuji memiliki perilaku yang sama:
rendahnya tingkat sel-sel inflamasi. Perbedaan antara dua zat yang terutama
diamati setelah 14 dan 28 hari periode eksperimental, dengan skor untuk neutrofil
dan abses yang mirip dalam 7 hari.
Hasil ini mendukung indikasi untuk menggunakan obat berbasis
kortikosteroid untuk waktu tidak lebih dari 7 hari. Dengan kaitannya dengan
kehadiran limfosit dan sel plasma, peristiwa ini menandai kehadiran proses kronis.
Nilai rata-rata untuk persiapan kortikosteroid antibiotik digunakan pada 14 dan 28
hari tampak lebih mengkhawatirkan karena mereka menunjukkan bahwa ada
intens yang mengarah lymphoplasmacytic, yang tidak surut sebagai periode
percobaan. Mengenai tidak adanya eosinofil, kehadiran mereka sangat erat terkait
dengan proses alergi dan dengan demikian, meskipun literatur yang menjelaskan
kasus alergi terhadap propolis, setidaknya dalam penelitian ini, efek ini tidak
diamati. Menganalisis peristiwa seluler secara keseluruhan, diamati bahwa pada
kelompok persiapan kortikosteroid-antibiotik, mereka meningkatkan sebagai
periode percobaan, menunjukkan kemunduran proses inflamasi. Ini tidak terjadi
dengan pasta propolis, karena tidak ada perbedaan yang signifikan antara
peristiwa dianalisis terjadi selama periode percobaan. Pengamatan ini telah
dikonfirmasi dalam sebuah penelitian oleh Silva, et al. 32 ( 2004), di mana ia
menunjukkan propolis yang lebih efektif dalam mengurangi proses inflamasi akut
dari persiapan kortikosteroid-antibiotik. Temuan ini mengkonfirmasi aktivitas
anti-inflamasi yang disebutkan dalam studi sebelumnya yang dipublikasikan.
Berdasarkan hasil yang diperoleh, penelitian ini menunjukkan bahwa pasta
propolis adalah alternatif untuk obat intrakanal dalam kasus pulp atau proses
inflamasi periapikal. Lamanya waktu kontak dengan jaringan ini merupakan
faktor penting dan jika perawatan endodontik tidak dapat diselesaikan dalam satu
sesi, menggunakan persiapan kortikosteroid-antibiotik untuk jangka waktu lebih
dari 7 hari dapat memperburuk proses inflamasi.
Pada eksperimen, pasta propolis berkontak langsung dengan jaringan
periapikal setelah pulpectomy tampaknya tidak menjadi masalah. Selanjutnya,
pasta propolis lebih efektif dalam mengontrol respon inflamasi selama periode
eksperimental 28 hari. Di sini ditunjukkan bahwa pasta propolis eksperimental
memiliki nilai terendah dari peristiwa sel-sel inflamasi, dengan nilai rata-rata
sama dengan kelompok kontrol negatif. Hal ini menunjukkan bahwa pasta
propolis dapat dianggap sebagai kemungkinan obat intrakanal. Namun, penting
untuk menekankan bahwa ini adalah bagian dari studi primer dengan pasta
propolis eksperimental. Ilmu dasar dan eksperimental klinis harus dilakukan untuk
lebih mengetahui sifat-sifatnya dan mungkin digunakan dalam perawatan
endodontik.
 KESIMPULAN
Pasta propolis dapat dianggap sebagai pilihan untuk pengobatan intrakanal setelah
pulpectomy, karena menimbulkan efek respon inflamasi perapikal yang rendah
dengan tidak adanya variasi perubahan sel sepanjang periode eksperimental bila
dibandingkan dengan persiapan kortikosteroid-antibiotik.
 www.scielo.br/jaos

Histopathological analysis of corticosteroid-

antibiotic preparation and propolis paste
formulation as intracanal medication after
pulpectomy: an in vivo study
Isabela França de Almeida Santos RAMOS 1, Michelle Tillmann BIZ2, Niraldo PAULINO3, Amarilis SCREMIN4,
Álvaro DELLA BONA5, Fernando Branco BARLETTA6, José Antônio Poli de FIGUEIREDO7

1- MSc, Department of Biology and Health Science, University of Planalto Catarinense, SC, Brazil.
2- PhD, Adjunct Professor, Morphology Science Department, Federal University of Santa Catarina, Florianópolis, SC, Brazil.
3- PhD, Biomedication Research and Development Group, Professional Masters Program in Pharmacy, Bandeirante University of São Paulo, São Paulo,
SP, Brazil. 4- MSc, Department of Pharmacy, Barriga Verde University Center, Florianópolis, SC, Brazil.
5- PhD, Senior Professor and Postgraduate Program, Dental School, University of Passo Fundo, Passo Fundo, RS, Brazil.
6- PhD, Professor Postgraduate Program, Lutheran University of Brazil; Adjunct Professor, University of Santa Cruz do Sul, Santa Cruz do Sul, RS, Brazil.
7- PhD, Coordinator of the Postgraduate Program in Dentistry, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, RS, Brazil.

Corresponding address: Prof. Michelle Tillmann Biz - Campus Universitário Reitor João David Ferreira Lima - Centro de Ciências Biológicas - Departamento - de
Ciências Morfológicas - Trindade - Florianópolis - Santa Catarina - Brasil - 88040-970 - Phone: (+55) 48 3721 9229 - e-mail: micbiz@uol.com.br

Received: November 12, 2009 - Modification: May 13, 2010 - Accepted: May 30, 2010

abstract

Intracanal medication in pulpectomy therapy is used between appointments with the

of reducing pain and inflammatory processes in pulp and periapical tissues.
objective

Propolis has been known as a natural antibiotic and has been subject of medical and dental
research due to its therapeutic properties such as antibiotic, analgesic and anti-inflammatory
effects. Objective: The aim was to carry out an in vivo evaluation of the periapical tissue
response to propolis paste when used as an intracanal medication in the teeth of dogs after
pulpectomy. Material and Methods: 72 dog’s incisors were selected for the experiment. After
biomechanical preparation the root canal was filled with a corticosteroid-antibiotic
preparation, experimental propolis paste, non-medicament (negative control) or non-
pulpectomy at all (positive control). The medications were left inside the root canal for 7, 14
or 28 days. At the end of the experimental period histological sections were prepared and all
laboratories processes for Harris hematoxylin and eosin staining was proceeded followed by
the analysis using an optical microscope. Sections were classified according to a score
representing the inflammatory events observed: the presence of polymorphonuclear
neutrophils, polymorphonuclear eosinophils, lymphocytes and plasma cells, macrophages
and/or giant cells, fibrous condensation and abscesses. Results: There were statistically
significant differences between the tissue reactions caused by the two substances being
tested, after different experimental periods, with the periapical tissue that was in contact
with propolis paste exhibiting fewer inflammatory reactions in comparison to corticosteroid-
antibiotic preparation. Conclusions: The low tissue responses from propolis paste suggest
that this material could be considered as an option for root canal medication after
pulpectomy.

Key words: Endodontics. Propolis. Pulpectomy. Dental pulp cavity.

INTRODUCTION used between appointments with the objective of

reducing pain and inflammatory processes in pulp
During endodontic treatment it may desirable and periapical tissues. The choice of this agent
or necessary to use an intracanal medication. In depends on its biologic characteristics: be non-
pulpectomy therapy intracanal medication is irritant, control of the intensity and duration of
J Appl Oral Sci. 50 2012;20(1):50-6
Histopathological analysis of corticosteroid-antibiotic preparation and propolis paste formulation as intracanal medication after pulpectomy: an in vivo study
inflammatory processes and infection, and have
potential healing induction11. Several chemicals and
therapeutic agents have been suggested as an
intracanal medication: in this case, a combined
corticosteroid-antibiotic could be selected5,25
In spite of being originally designed as an
otology medication, a commercial corticosteroid-
antibiotic preparation, Otosporin® is currently used
in Dentistry as an intracanal medication due to its
components: polymyxin B sulphate, neomycin
sulphate, hydrocortisone and an aqueous base 11,16.
This preparation reduces vasodilation, decreases
liquid exudation, and also presents a direct
vasoconstrictive action on small blood vessels 10.
Nevertheless, though this combination has many
properties that are desirable as a pulpectomy
intracanal medication, if it is used for prolonged
periods it can reduce cellular metabolic activity and
even impede or delay tissue repair16.
In relation to tissue repair, propolis has been
known to be a natural antibiotic and has been the
subject of medical and dental research. Propolis is a
complex mixture of resinous and balsamic
substances collected from plants by Apis mellifera
bees, which transport them to their hives, modifying
them with the addition of their own secretions,
pollen and wax. Propolis contains flavonoids which
are considered as the main primary biologically
active component, responsible for a large proportion
of its known therapeutic properties17,22. In dentistry,
propolis has been used for the treatment of
aphthous ulcers, Candida albicans, acute necrotizing
ulcerative gingivitis
(UNUG), gingivitis and periodontitis 9,21, and it has
been proven to have antimicrobial activity
against Streptococcus mutans8 and polymicrobial
cultures collected from necrotic root canals28.
Among the properties of propolis are: analgesic
action6,27, antiviral, antifungal and antiprotozoal
actions1,3,7,22,30, immunostimulation24,31, antioxidant
effects30, antiinflammatory action24,27. All these
properties led to the question whether it would be
possible to use propolis as an intracanal medication
in pulpectomy where there is a need for both
analgesic and anti-inflammatory effects.
The objective of this study was therefore to
evaluate the periapical tissue response to an
experimental propolis paste when used as intracanal
medication in dog’s teeth after pulpectomy.
MATERIAL AND METHODS
A total of 72 incisors’ root canals of 6 dogs
aged 2 to 4 years were selected for the
experiment. The protocol used was approved by
the local Ethics Committee (CEP ULBRA: 2003-
015A; CEP UPF: 276/2004).
The animals were kept under general anesthetic
supplemented by infiltration of local anesthetic into
the procedure area throughout the operation. Soft
tissues and teeth were sterilized using alcohol-iodine
solution (Vico-farma, Lages, SC, Brazil).
The root canals of the 66 teeth were accessed
on the buccal surface of the tooth using a
diamond bur #1012 (Metalúrgica Fava Indústria e
Comércio Ltda., Franco da Rocha, SP, Brazil) at
high speed with water-air cooling. After carrying
out radiography on each tooth individually, the
working length was established at 1 mm short of
the radiographic root apex.
Following root canal cleaning, biomechanical
preparation was carried out using a sequential manual
technique. The root canal was instrumented using
Flexofile® K files (Dentsply, Maillefer Instruments
S.A., Ballaigues, Switzerland), finishing with an
instrument #40. Canals were irrigated with 2 mL of
solution at each instrument change, alternating
between 1% sodium hypochlorite (Vico-farma) and
17% trisodium ethylenediaminetetraacetic acid
(EDTA) (Vico-farma) with pH 7.3. Once
chemomechanical preparation was complete, the
apex was breached passing 2 mm beyond working
length a file #20. Canals were then dried with
absorbent paper points #40 (Tanari Indústria e
Comércio Ltda., Paraíba do Sul, RJ, Brazil) that had
been autoclaved.
The root canals were then packed with the
medications to be analyzed: in each of the 6
dogs, five root canals were packed with a solution
of 10 mg/mL hydrocortisone/3,400 U/ mL
neomycin sulfate/10,000 U/mL polymyxin B
sulfate, a corticosteroid-antibiotic preparation
(Zest Farmacêutica Ltda., São Paulo, SP, Brazil)
and another 5 were packed with experimental
propolis paste. One root canal of each dog was
packed with glycerin, which was considered as
vehicle group (negative control), and another
tooth did not receive any treatment, considered
as the non-treated group (positive control). The
choice of intracanal medication and the controls
was randomized.
Corticosteroid-antibiotic preparation was stored
in a clean and sterilized anesthetic tube and the
solution was introduced into the canal with a
Carpule syringe and a 27G needle set 1 mm short
of the working length.
The experimental propolis paste was previously
manipulated following the composition: 1% propolis
extract, titanium dioxide, glycerin. The paste was
stored in a sterilized tube and at the moment of the
root canal filling it was introduced using a #35
Lentulo spiral (Pró Dent, São Paulo, SP , Brazil),
calibrated in two millimeters from the root canal
working length and running at low speed in a
clockwise direction. In the cases of both types of
medication, insertion was stopped when reflux was
J Appl Oral Sci. 51 2012;20(1):50-6
RAMOS IFAS, BIZ MT, PAULINO N, SCREMIN A, DELLA BONA A, BARLETTA FB, FIGUEIREDO JAP
observed at the canal entrance.
The cavity was then cleaned, filled with sterile
cotton and sealed with a 2 mm layer of glass
ionomer cement (Vidrion R® - SS White Artigos
Dentários Ltda., Rio de Janeiro, RJ, Brazil), prepared
in accordance with its manufacturer’s instructions.
The animals spent up to 28 days with intracanal
medication in place, during which time they were
monitored by a veterinarian. After 7, 14 or 28 days
with the intracanal medication in place, the dogs
were euthanized and the samples were collected.
Tissues were preserved by intravenous perfusion
of 4% buffered paraformaldehyde. The maxilla and
mandible were removed and postfixed in 10%
neutral formaldehyde. The samples were
demineralized in EDTA 4.13% at pH 7.2, following
the process protocol for paraffin, five micrometers
histological sections and hematoxylin and eosin
staining. The sections were analyzed using an
optical microscope (Olympus, Tokyo, Tokyo, Japan)
at ×100, ×200 and ×400 magnification by a single
examiner, who was an experienced histologist and
blind to the tested materials. The subject of analysis
was the periapical region and the specimen with the
greatest degree of inflammation was chosen from
each sample.
The following qualitative observations were
recorded: the presence of polymorphonuclear
neutrophils, polymorphonuclear eosinophils,
lymphocytes and plasma cells, macrophages and/ or
giant cells, fibrous condensation and abscesses.
Each element was attributed a numerical value
according to the protocol proposed by Figueiredo, et
al.12 (2001). Approximately 30 days after the
original analysis of the sections, 10% of them were
reanalyzed to test for intraexaminer agreement
between events observed and scores awarded.
The results were tested using factorial ANOVA,
followed by the Mann Whitney and Tukey’s tests, all
to a significance level of 5% (SPSS 12.0).
Intraexaminer agreement was assessed using
Kendall’s W, with a 5% significance level (SPSS
12.0).
RESULTS
The negative control slices showed a normal
histological periapical tissue, without a significant
inflammatory cellular event. On the other hand,
positive control presented neutrophils,
lymphocytes and macrophages with higher scores
than negative control showing the inflammatory
processes that occur after pulpectomy chemical-
mechanical preparation.
Regarding to the cellular events listed for
analysis, fibrous condensation was not included,
once its origin was not clear in the analysis: whether
due to a natural response of the periodontal
ligament to the pulpectomy technique or tissue
reaction to the test materials. No eosinophils were
observed on any of the sections from either of the
groups, being scored 1 for the analysis. Kendall’s W
showed that the intra-examiner agreement was
98.91%, represented by W=0.119.
Figure 1 illustrates variations in the number of
neutrophils when analyzed with reference to the
groups and experimental periods. The non-treated
group had the lowest mean scores and had no
variation throughout the time. Analyzing this event
with relation to corticosteroid-antibiotic preparation,
it is observed that there was a continuous increase
in the mean scores as the experimental period
elapsed, with statistically significant differences
(p=0.007) detected by the Tukey’s test between 7 and
28 and between 14 and 28 days, which was quite
similar to the vehicle group. Analyzing the mean scores
for these cells for the propolis paste, there was no
significant difference between the periods analyzed
(p=0.47). It was also possible to apply the Mann-
Whitney nonparametric test in order to compare the
substances at each experimental time in isolation for
each of the different inflammatory events. It was not
found any statistically significant differences between
the tested substances for
7 days; statistically significant differences were
detected for neutrophils between propolis paste and
corticosteroid-antibiotic preparation, and between
propolis paste and vehicle group at 14 and 28 days,
with p=0.05 and 0.02, consecutively, with higher
Figure 1- Progression of neutrophils medium scores of
the groups over the experimental time. (a) Significant
difference from propolis paste in 14 days; (b) Significant
difference from propolis paste in 28 days; (*) Significant
difference in the substance over time
Figure 2- Progression of lymphocytes medium scores of
the groups over the experimental time. (a) Significant
difference from propolis paste in 14 days; (b) Significant
difference from propolis paste in 28 days; (*) Significant
difference in the substance over time
J Appl Oral Sci. 52 2012;20(1):50-6
Histopathological analysis of corticosteroid-antibiotic preparation and propolis paste formulation as intracanal medication after pulpectomy: an in vivo study
scores for corticosteroid-antibiotic preparation
and vehicle group; no difference between
corticosteroid-antibiotic preparation and vehicle
group; no difference between propolis paste and
non-treated group.
With relation to lymphocytes and plasma cells,
illustrated in Figure 2, the non-treated group had
the lowest mean scores and had no variation
throughout the time. Although the test substances
exhibited similar values in 7 days, by the end of the
experimental period a large difference was
observed between the experimental groups with
higher medium score for corticosteroid-antibiotic
preparation and vehicle group. When each of the
test substances were analyzed in isolation with
relation to the variable time, corticosteroid-antibiotic
preparation exhibited significant differences
(p=0.012), detected by the Tukey test, between 7
and 14 and between 7 and 28 days. In this analysis,
the propolis paste once more exhibited no significant
differences between times (p=0.41).
The Mann-Whitney nonparametric test showed

Figure 3- Progression of macrophages medium scores

Figure 4- Progression of abscess medium scores of the
of the groups over the experimental time
groups over the experimental time. (b) Significant
difference from propolis paste in 28 days; (*) Significant
difference in the substance through the time

Figure 5- A- Non-treated sample – image of the periapical area of the root with a normal appearance; B- Propolis after
28 days – mild periapical inflammatory reaction; C- Propolis after 28 days – macrophages present in periapical region
(arrowed); D- Otosporin 28 days – intense inflammatory reaction with abscess (arrowed); E- Otosporin 28 days – greater
magnification than (D). F- Otosporin 28 days – displaced zone of abscess seen in (E). M-macrophages; N-neutrophils; L-
lymphoplasmacytic infiltrate. Bars: A-E 200 μm; F-G 50 μm
J Appl Oral Sci. 53 2012;20(1):50-6
RAMOS IFAS, BIZ MT, PAULINO N, SCREMIN A, DELLA BONA A, BARLETTA FB, FIGUEIREDO JAP
no statiscally significant differences between the
tested substances for 7 days. Significant
difference between corticosteroid-antibiotic
preparation and propolis paste, and between
propolis paste and vehicle group at 14 and 28
days, with p=0.01 and 0.02, consecutively, with
higher scores for corticosteroid-antibiotic
preparation and vehicle group; no difference
between corticosteroid-antibiotic preparation and
vehicle group; no difference between propolis
paste and non-treated group.
The non-treated group had the lowest mean
scores and had no variation throughout the time
for macrophages and giant cells. Analysis of the
mean scores between the tendencies of cellular
expression for vehicle group, propolis paste and
corticosteroid-antibiotic preparation showed that
these events were constantly present in the
tissues analyzed, with no significant differences
between them. There were no significant
differences when each of the experimental
groups was analyzed alone with relation to the
variable time (p>0.05) (Figure 3).
Figure 4 illustrates the mean scores for abscesses
for the tested groups. In 7 days abscesses were
practically absent for all groups. The non-treated group
and the propolis paste group did not exhibit any
significant difference over time (p>0.05). However, as
time passed, there was a drastic increase in the
corticosteroid-antibiotic preparation and vehicle group
with statistically significant differences (p<0.05),
detected by the Tukey test, between 7 and 14 and
between 7 and 28 days. For this event, corticosteroid-
antibiotic preparation and vehicle group had the higher
scores when compared with propolis paste and non-
treated group at 28 days, with a significant difference
(p<0.05) as detected by Mann-Whitney nonparametric
test; there was no statiscally significant differences
between the substances for 7 and 14 days.
Figure 5 shows representative images of samples
tested after 28 days in contact with periapical
tissues. Figure 5A is a representative image of the
non-treated group: normal periapical tissue, with no
inflammatory cells. In the corticosteroid-antibiotic
preparation (Figure 5E-F), it is possible to observe
the presence of intense inflammation after
28 days, whereas there is merely mild periapical
inflammation after exposure to the propolis paste
(Figure 5B-D).
DISCUSSION
During pulpectomy the pulp removal and
chemical-mechanical preparation will lead the
periapical tissues into an acute inflammatory
process5,25, which explains the result of the vehicle
group in this experiment. As it is known by the
literature, the main cellular events to be taken into
consideration when attempting to observe an initial
inflammatory response in tissues are neutrophils
and the presence of abscesses. When neutrophils
are present it signify that an acute process is
occurring, during which these cells are constantly
recruited to contain inflammation. These are the
first cells to migrate from blood vessels and to reach
the site of aggression. Nevertheless, if the
aggression is intense and neutrophils are unable to
contain it rapidly, abscesses may form from a
buildup of dead neutrophils, tissue liquids and
abnormal material, creating pus18.
Corticosteroid based medicaments acts on the
synthesis of lipocortin and vasocortin, inhibiting the
formation of edema and A2 phospholipasis enzymes,
once inhibiting this enzyme, membrane
phospholipids cannot be converted into arachidonic
acid. Therefore, the cyclooxygenase and
lypooxygenase are blocked20,33. Corticosteroid
preparation also acts on histamine, heparin, and
bradicinin, which are important chemical mediators
in the initial phases of acute inflammation 10,29.
Propolis-based medicaments contains flavonoids
which are considered as the main primary
biologically active component, responsible for a
large proportion of its known therapeutic
properties17,22. The flavonoids interfere in the
arachidonic acid metabolism and so interfere in the
cyclooxygenase and lypooxygenase synthesis. Some
of the substances derivate in this cascade are the
prostaglandins, thromboxane and leukotriene which
are important to keep the integrity of the
inflammatory reaction13,14. Therefore, it is expected
that the use of corticosteroid preparations and
propolis-based medicaments cause changes in the
inflammatory response in the tissue as it was
observed in this study and discussed in the following
sentences.
When the results were analyzed, corticosteroid-
antibiotic preparation had high scores for the
presence of neutrophils and abscesses, primarily
from 14 days onwards. The intense neutrophil
infiltrate, which was continuously present and had
not receded by 28 days, demonstrates the severity
of the inflammatory response to corticosteroid-
antibiotic preparation. It was already discussed in
the literature that if this medication is left in contact
with remaining apical tissues for longer periods the
tissue response in this region can become
completely compromised16. On the other hand, it
could be possible that after 28 days, no more
corticosteroid-antibiotic should be in the root canal
and this fact could explain why corticosteroid-
antibiotic preparation had the same inflammatory
reaction in 28 days as the vehicle group (glycerin),
which is considered an inert substance.
In contrast, the scores for neutrophils and
J Appl Oral Sci. 54 2012;20(1):50-6
Histopathological analysis of corticosteroid-antibiotic preparation and propolis paste formulation as intracanal medication after pulpectomy: an in vivo study
abscesses in the propolis paste group illustrate a
tissue reaction that is more favorable to the repair
process, since their means were low and remained
constant as the experimental period elapsed, quite
similar to the non-treated group. This corroborates
studies by Bretz, et al.4 (1998), Al-Shaher, et al.2
(2004) and Silva, et al. 32 (2004), which have
demonstrated that administering propolis-based
substances leads to tissues exhibiting normal
reorganization without increased vascular or cellular
events, maintaining low levels of inflammation.
It is important to emphasize that, in this
study, after 7 days of experiment, all tested
substance had the same behavior: low levels of
inflammatory cells. The differences between the
two substances were primarily observed after the
14 and 28-day experimental periods, with the
scores for neutrophils and abscesses being
similar in 7 days. This result supports the
indication for using corticosteroid-based
medications for periods no longer than 7 days16.
With relation to the presence of lymphocytes
and plasma cells, these events characterize the
presence of a chronic process. The mean values
for the corticosteroid-antibiotic preparation used
at 14 and 28 days appear to be more worrying
since they indicate that there was intense
lymphoplasmacytic infiltrate, which did not
recede as the experimental period elapsed.
Regarding the absence of eosinophils, their
presence is intimately related with allergic
processes and thus, despite the literature
describing cases of allergy to propolis 15,19,28, at
least in this study, this effect was not observed.
Analyzing cellular events as a whole, it was
observed that in the corticosteroid-antibiotic
preparation group, they increased as the
experimental period elapsed, indicating a
deterioration of the inflammatory process. This was
not the case with the propolis paste, since no
significant difference between the events analyzed
occurred during the experimental period.
These observations have been confirmed in a
study by Silva, et al.32 (2004), where it was
demonstrated that propolis is more effective at
reducing acute inflammatory processes than
corticosteroid-antibiotic preparation. These
findings confirm the anti-inflammatory activity
mentioned in earlier published studies23,27.
Based on the obtained results, this study shows
that propolis paste is a viable alternative for
intracanal medication in cases of pulp or periapical
inflammatory processes. Length of time in contact
with these tissues is a critical factor and, if an
endodontic treatment cannot be completed in a
single session, using corticosteroid-antibiotic
preparation for a period of more than 7 days may
worsen the inflammatory process. In this
experiment, propolis paste in contact with periapical
tissues after pulpectomy did not appear to be a
problem. Furthermore, the propolis paste was more
effective at controlling the inflammatory response
over the 28-day experimental period.
Here it was demonstrated that the experimental
propolis paste had the lowest value of inflammatory
cells events, with mean scores similar to the
negative control group. This indicates that the
propolis paste could be considered as an intracanal
medication possibility. However, it is important to
emphasize that this is part of a primary study with
the experimental propolis paste. Basic science and
clinical experimental should be performed to better
know its properties and possible use in Endodontic
treatments.
CONCLUSION
Within the context of the methodology proposed
and results observed, it is possible to conclude that
propolis paste caused a periapical mild inflammatory
response with no variation throughout the
experimental period when compared with
corticosteroid-antibiotic preparation; corticosteroid-
antibiotic preparation caused an increasing degree
of inflammatory response that was directly related
to the length of time, being most severe at 28 days.
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