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Quality of root filling by three different techniques in teeth after minimal root canal preparation and… Zaghwan, Ala M. H. 2016

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QUALITY	OF	ROOT	FILLING	BY	THREE	DIFFERENT	TECHNIQUES	IN	TEETH	AFTER	MINIMAL	ROOT	CANAL	PREPARATION	AND	GENTLEWAVE	CLEANING		by	 	Ala	M	H	Zaghwan			D.M.D.,	University	of	Tripoli,	2007		A	THESIS	SUBMITTED	IN	PARTIAL	FULFILLMENT	OF	THE	REQUIREMENTS	FOR	THE	DEGREE	OF			MASTER	OF	SCIENCE		in	 	THE	FACULTY	OF	GRADUATE	AND	POSTDOCTORAL	STUDIES		(Craniofacial	Science)			THE	UNIVERSITY	OF	BRITISH	COLUMBIA		(Vancouver)			July	2016			©	Ala	M	H	Zaghwan,	2016		 ii	Abstract		Objective:	 This	 study	 aimed	 to	 examine	 the	 quality	 of	 root	 filling	 in	 canals	obturated	 using	 three	 different	 filling	 techniques	 after	 minimal	 root	 canal	preparation	and	GentleWave	(GW)	cleaning.	Methods:	Root	 canals	of	30	mandibular	molars	were	 instrumented	 to	 size	20/.04	taper	 using	 hand	 K-files,	 and	 a	 Vortex	 Blue	 NiTi	 rotary	 file.	 Sodium	 hypochlorite	(NaOCl)	 6%	was	 used	 in-between	 each	 instrument	 insertion	 and	 the	 canals	were	finally	cleaned	using	a	7.5	min	irrigation	by	the	GW.	The	samples	were	scanned	with	micro-CT	using	a	voxel	size	of	30	µm	after	instrumentation	(pre-GW),	post-GW,	and	after	obturation.	The	samples	were	randomly	allocated	 into	 three	groups	(n	=	10)	with	regard	to	the	root	filling	method:	(1)	single-cone	with	AH	Plus	sealer	(SC/AH+),	(2)	 single-cone	 with	 GuttaFlow	 sealer	 (SC/GF),	 and	 (3)	 GuttaCore	 with	 AH	 Plus	sealer	 (GC/AH+).	 The	 reconstructed	 3D	 images	were	 analyzed	 for	 the	 volumetric	percentages	 of	 debris,	 filling	materials,	 gaps,	 and	 voids	 at	 three	 canal	 levels.	 Data	was	analyzed	statistically	using	Kruskal-Wallis	and	Mann-Whitney	U	tests.	P	values	<	0.05	were	considered	to	be	significant.		Results:	 The	 proportion	 (volume	%)	 gaps	 and	 voids	 proportions	 in	multi-rooted	canals	obturated	by	using	GC/AH+	and	SC/GF	was	significantly	lower	than	in	canals	filled	 with	 SC/AH+	 (P	 =	 0.000).	 Different	 values	 indicated	 a	 statistical	 difference	between	coronal,	middle	and	apical	gaps	and	voids	volumes	within	each	group.	The	mean	 volumes	 of	 coronal	 and	 middle	 gaps	 and	 voids	 were	 significantly	 different	from	 the	mean	 volume	 of	 apical	 gaps	 and	 voids	 in	 canals	 filled	 with	 SC/GF	 (P	 =	0.000),	(P	=	0.001).	The	quality	of	mesial	fillings	did	not	differ	statistically	from	the		 iii	distal	 fillings	 amongst	 the	 three	 groups.	 After	 conventional	 irrigation,	 4.58	 %	 of	canal	volumes	was	filled	with	debris	while	no	debris	was	found	after	GW	cleaning.		Conclusion:	 	A	high	quality	of	filling	was	achieved	by	GC/AH+	and	SC/GF	indicating	that	both	these	methods	are	suitable	to	fill	the	minimally	instrumented	root	canals	after	GentleWave	cleaning.																							 iv	Preface		The	research	question	and	the	study	design	were	identified	by	Dr.	Ala	Zaghwan	under	the	guidance	of	Dr.	Markus	Haapasalo	and	Dr.	Ya	Shen.	Selection	and	preparation	of	the	samples	were	accomplished	by	Dr.	Ala	Zaghwan.	Micro-CT	scans	were	performed	by	John	Schipilow	at	the	UBC	Faculty	of	Dentistry	Centre	for	High	Throughput	Phenogenomics	(CHTP).	The	collected	micro-CT	data	was	analyzed	by	Dr.	Ala	Zaghwan	under	the	supervision	of	Dr.	Markus	Haapasalo	and	Dr.	Ya	Shen.	Dr.	Ala	Zaghwan	prepared	the	Manuscript	with	editing	by	Dr.	Markus	Haapasalo,	Dr.	Ya	Shen.	The	study	was	approved	by	the	University	of	British	Columbia	Office	of	Research	Services,	Clinical	Research	Ethics	Board	(Certificate	Number:	H15-02793).												 v	Table	of	Contents			Abstract	.......................................................................................................................................	ii	Preface	........................................................................................................................................	iv	Table	of	Contents	.....................................................................................................................	v	List	of	Figures	...........................................................................................................................	ix	List	of	Abbreviations	............................................................................................................	xii	Acknowledgements	..............................................................................................................	xiv	Dedication	................................................................................................................................	xv	Chapter	1	:	Introduction	........................................................................................................	1	1.1	 History	of	Endodontic	Treatment	........................................................................	2	1.2			Cleaning	of	the	Root	Canal	System	.......................................................................	5	1.2.1			Mechanical	Cleaning	..........................................................................................................	5	1.2.2			Chemomechanical	Cleaning	............................................................................................	8	1.2.2.1			Irrigation	Solutions	....................................................................................................	8	1.2.2.2			Methods	of	Irrigation	.............................................................................................	10	1.2.2.2.1			Conventional	Syringe	Irrigation	...............................................................	10	1.2.2.2.2			EndoVac	Negative	Pressure	System	.......................................................	11	1.2.2.2.3			Sonic	and	Ultrasonic	Irrigation	.................................................................	11	1.2.2.2.4			Laser	Activated	Irrigation	(LAI)	...............................................................	12	1.2.2.2.5			GentleWave	System	(GW)	...........................................................................	14	1.3			Root	Canal	Obturation	...........................................................................................	16		 vi	1.3.1			Filling	Materials	................................................................................................................	17	1.3.1.1			Gutta-Percha	..............................................................................................................	17	1.3.1.2			Root	Canal	Sealer	.....................................................................................................	18	1.3.2			Obturation	Techniques	..................................................................................................	22	1.3.2.1			Cold	Lateral	Compaction	Obturation	..............................................................	22	1.3.2.2			Warm	Vertical	Compaction	Obturation	.........................................................	23	1.3.2.3			Single-Cone	Obturation	.........................................................................................	25	1.3.2.4			Carrier-Based	Obturation	....................................................................................	26	1.4			Micro-Computed	Tomography	............................................................................	28	Chapter	2	:	Rationale	and	Hypothesis	...........................................................................	30	Chapter	3	:	Materials	and	Methods	.................................................................................	32	3.1			Tooth	Selection	and	Preparation	.......................................................................	32	3.2			Root	Canal	Instrumentation	................................................................................	34	3.3			First	Micro-CT	Scan	of	the	Instrumented	Root	Canals	................................	36	3.4			Root	Canal	Irrigation	..............................................................................................	37	3.5			Second	Micro-CT	Scan	of	the	Cleaned	Root	Canals	.......................................	37	3.6			Assignment	of	Teeth	...............................................................................................	40	3.7			Root	Canal	Obturation	...........................................................................................	40	3.7.1			Group	I:	Single	GP	Cone	with	GuttaFlow	Sealer	..................................................	40	3.7.2			Group	II:	Single	GP	Cone	with	AH	Plus	Sealer	.....................................................	43	3.7.3			Group	III:	GuttaCore	with	AH	Plus	Sealer	..............................................................	44	3.8			Third	Micro-CT	Scan	of	the	Teeth	After	Root	Filling	...................................	45	3.8.1			Reconstruction	and	Analysis	of	the	Images	..........................................................	46		 vii	Chapter	4	:	Results	................................................................................................................	48	4.1	Gaps	and	Voids	Analysis	.........................................................................................	48	4.1.1	Comparison	of	Gaps	and	Voids	within	Each	Group	.............................................	52	4.1.1.1	Comparison	of	Gaps	and	Voids	in	the	Coronal,	Middle	and	Apical	Thirds	of	the	Root	Canals	within	Each	Group	..............................................................	52	4.1.1.2	Comparison	of	Mesial	and	Distal	Gaps	and	Voids	within	Each	Group	55	4.1.2	Comparison	of	Gaps	and	Voids	between	Groups	.................................................	57	4.1.2.1	Comparison	of	Coronal,	Middle	and	Apical	Gaps	and	Voids	between	the	Groups	...................................................................................................................................	57	4.1.2.2	Comparison	the	Proportion	of	Gaps	and	Voids	in	the	Mesial	and	Distal	Canals	between	the	Three	Groups	....................................................................................	60	4.2	Debris	Removal	by	the	GentleWave	System	....................................................	65	Chapter	5	:	Discussion	.........................................................................................................	66	Chapter	6	:	Conclusion	........................................................................................................	77	References	..............................................................................................................................	79	Appendices:	..........................................................................................................................	101	Appendix	A	–	Research	Plan	.......................................................................................	101	Appendix	B	–	Micro-CT	Settings	................................................................................	102	Appendix	C	–	Normal	Distribution	Tests	................................................................	103	Appendix	D	–	Total	Gaps	and	Voids	Data	Analysis	Using	Kruskal-Wallis	Test	..............................................................................................................................................	104	Appendix	E	–	Statistical	Analysis	of	Gaps	and	Voids	within	and	between	the	Three	Tested	Groups	at	Three	Canal	Levels	.........................................................	105		 viii	List	of	Tables			Table	3.1	Stratified	sampling	method.	...........................................................................................	32	Table	4.1	The	proportion	(%	mean	±	SD)	of	the	root	canal	space	filled	with	the	three	root	filling	materials.	.............................................................................................................................	50	Table	4.2	The	proportion	(%	mean	±	SD)	of	gaps	&	voids	of	the	total	root	canal	volume.	........................................................................................................................................................	50	Table	4.3	The	proportions	(%	mean	±	SD)	of	the	filling	materials	in	the	mesial	and	distal	root	canals	obturated	using	the	three	techniques	(SC/AH+,	SC/GF,	and	GC/AH+),	at	three	canal	levels	(coronal,	middle,	and	apical	thirds).	...............................	63	Table	4.4		The	proportion	(%	mean)	of	the	gaps	and	voids	in	mesial	and	distal	root	canals	obturated	using	the	three	techniques	(SC/AH+,	SC/GF,	and	GC/AH+),	at	three	canal	levels	(coronal,	middle,	and	apical	thirds).	......................................................................	64	Table	4.5	The	proportion	(%mean	volume)	of	debris	removed	by	the	GentleWave	system	at	the	three	canal	levels.	.......................................................................................................	65	Table	C.1	Kolmogorov-Smirnov,	Shapiro-Wilk	tests	of	data	distribution	normality………..……………………………………………………………………………………………….103	Table	C.2	Levene	statistics	of	data	distribution	normality…………………………………..103	Table	D.1	Total	mean	rank	of	gaps	and	voids	volumes	in	the	three	different	groups	(SC/AH+,	SC/GF,	and	GC/AH+)………………………………………………………………...............104	Table	D.2	Non-parametric	statistical	test	(Kruskal	Wallis	Test)	compares	SC/AH+,	SC/GF,	and	GC/AH+	groups……………..………………….…………………………………………….104	Table	E.1	Statistical	analysis	of	gaps	and	voids	within	and	between	the	three	different	groups	at	three	canal	levels……..………………………………………………………….105		 ix	List	of	Figures			Figure	1.1	(A)	The	GentleWave™	System	and	(B)	the	tip	of	the	cleaning	instrument	in	the	pulp	chamber	of	a	molar.		Image	courtesy	Sonendo	Inc.	..........................................	15	Figure	1.2	The	recommended	protocol	for	the	GentleWave	cleaning.	............................	16	Figure	1.3	AH	Plus	Jet	sealer	in	a	dual-barrel	syringe	and	a	mixing	tip.	.........................	20	Figure	1.4	GuttaFlow	2	sealer	in	a	dual-barrel	syringe	and	a	delivery	tip.	....................	21	Figure	1.5	Micro-CT	Specimen	Scanner-Center	for	High-Throughput	Phenogenomics.	Courtesy	Phenogenomics.dentistry.ubc.ca.	..............................................	29	Figure	3.1	Dental	surgical	microscope.	.........................................................................................	35	Figure	3.2	A	size	verifier	for	the	corresponding	GuttaCore	obturator.	...........................	36	Figure	3.3	Matching	process	from	three	different	projections	using	the	MeVisLab	software.	.....................................................................................................................................................	38	Figure	3.4	(A)	Visualization	of	reconstructed	images	show	the	overlapping	of	post-GW	(red	color	2D	slice)	and	pre-GW	(grey	color	slice)	from	the	axial	section	of	the	same	sample,	(B)	matched	pre/post-GW	scans	of	the	same	sample.	...............................	39	Figure	3.5	Lentulo	spiral.	.....................................................................................................................	41	Figure	3.6	Steps	of	root	canals	obturation	using	single-cone	technique	with	GuttaFlow	sealer......................................................................................................................................	43	Figure	3.7	Color-coded	Schilder	plugger	for	posterior	teeth.	..............................................	44	Figure	3.8	Segmentation	of	root	filling	material	using	MeVisLab	software.	The	highlighted	area	represents	the	filling	materials	(GP	+	sealer).	.........................................	47	Figure	4.1	Reconstructed	2-D	(left)	and	3-D	(right)	images	for	a	sample	in	the	(SC/AH+)	group,	showing	the	distribution	of	gutta-percha,	sealer,	and	voids	inside		 x	the	filled	root	canal	space.	(1	&	2)	axial	aspect,	(3	&	4)	coronal	aspect,	and	(5	&	6)	sagittal	aspect.	..........................................................................................................................................	49	Figure	4.2	Mean	volumes	percentages	of	the	filling	materials	within	three	groups	(1)	Single-cone	technique	with	AH	Plus	sealer	(SC/AH+),	Single-cone	technique	with	GuttaFlow	sealer	(SC/GF),	and	GuttaCore	technique	with	AH	Plus	sealer	(GC/AH+).	The	bars	represent	the	standard	deviations.	..............................................................................	51	Figure	4.3	Proportion	of	gaps	and	voids	(%	mean)	at	the	three	canal	levels	in	the	SC/AH+	group.	..........................................................................................................................................	52	Figure	4.4	Proportion	of	gaps	and	voids	(%	mean)	at	the	three	canal	levels	in	the	SC/GF	group.	.............................................................................................................................................	53	Figure	4.5	Proportion	of	gaps	and	voids	(%	mean)	at	the	three-canal	levels	in	the	GC/AH+	group.	.........................................................................................................................................	54	Figure	4.6	Proportion	of	gaps	and	voids	(%	mean)	in	mesial	and	distal	root	canals	filled	with	the	SC/AH+	technique.	...................................................................................................	55	Figure	4.7	Proportion	of	gaps	and	voids	(%	mean)	in	mesial	and	distal	root	canals	filled	with	the	SC/GF	technique.	.......................................................................................................	56	Figure	4.8	Proportion	of	gaps	and	voids	(%	mean)	in	mesial	and	distal	root	canals	filled	with	the	GC/AH+	technique.	...................................................................................................	56	Figure	4.9	Proportion	of	gaps	and	voids	(%	mean)	in	the	coronal	thirds	of	canals	obturated	with	either	SC/AH+,	SC/GF,	or	GC/AH+	technique.	...........................................	57	Figure	4.10	Proportion	of	gaps	and	voids	(%	mean)	found	in	the	middle	third	of	the	canals	obturated	with	either	SC/AH+,	SC/GF,	or	GC/AH+	technique.	.............................	58	Figure	4.11	Proportion	of	gaps	and	voids	(%	mean)	found	in	the	apical	third	of	the	canals	obturated	with	either	SC/AH+,	SC/GF,	or	GC/AH+	technique.	.............................	59		 xi	Figure	4.12	Proportion	of	gaps	and	voids	(%	mean)	in	the	mesial	root	canals	filled	by	the	SC/AH+,	SC/GF,	and	GC/AH+	technique.	........................................................................	60	Figure	4.13	Proportion	of	gaps	and	voids	(%	mean)	found	in	distal	canals	obturated	using	the	SC/AH+,	SC/GF,	and	GC/AH+	technique.	..................................................................	61	Figure	4.14	The	proportions	(%)	of	sealer	and	gutta-percha	in	root	canals	of	three	samples	obturated	using	three	different	techniques;	SC/AH+	(first	row),	SC/GF	(second	row),	and	GC/AH+	(third	row).	A,	D	and	G	views	show	segmented	(sealer	+	GP)	inside	the	root	canals;	B,	E	and	H	views	show	segmented	GP	inside	the	root	canals;	and	C,	F	and	I	views	show	segmented	sealer	inside	the	root	canals.	................	62	Figure	4.15	The	proportion	(Means	±	SD)	of	filling	materials	in	mesial	and	distal	root	canals	obturated	using	the	three	techniques	(SC/AH+,	SC/GF,	and	GC/AH+),	at	three	canal	levels	(coronal,	middle,	and	apical	thirds).	..........................................................	63	Figure	4.16	The	proportion	(%	mean)	of	the	gaps	and	voids	in	mesial	and	distal	canals	obturated	using	the	three	techniques	(SC/AH+,	SC/GF,	and	GC/AH+),	at	three	canal	levels	(coronal,	middle,	and	apical	thirds).	......................................................................	64	Figure	4.17	The	amount	(%	mean	volume)	of	debris	removed	by	using	the	GentleWave	system	at	the	three	canal	levels.	.............................................................................	65	Figure	A.1	Research	plan…......……..…………………………………………..………………………...101	Figure	B.1	Micro-CT	settings………………….……………………………………………………….…102				 xii	List	of	Abbreviations			ASP……………………………………………………………………………………..apical	size	preparation	h....………………..………………………………………………………………………………………………hours	min……………………………………………………………………………………………………………minutes	NaOCl……………………………………………………………………………………..sodium	hypochlorite	EDTA…………………………………………………………………..ethylenediamine	tetra-acetic	acid	µm……………………………………………………………………………………………………..micrometers	µA……………………………………………………………………………………………………..microampere	kVp…………………………………………………………………………………….…………peak	kilovoltage	ms………………………………………………………………………………………………………..millisecond	sec……...……………………………………………………………………………………………………..seconds	ml……………………………………………………………………………………………………….……milliliter	°C………………………………………………………………………………………………….….degree	celsius	RCs………………………………………………………………………………………………….……root	canals	GC……………………………………………………………………………………………………..……GuttaCore	GF………………………………………………………………………………………………………….GuttaFlow	AH+…………………………………………………………………………………………………………...AH	Plus	SC………………………………………………………………………………………………………...single-cone	GP.……………………………………………………………………………………………………..gutta-percha	CSI………………………………………………………………………….conventional	syringe	irrigation	G…………………………………………………………………………………………………………………..gaugeANP……………………………………….………………………….......................apical	negative	pressure	SNI…………………………………………………………………………………..syringe-needle	irrigation		 xiii	PUI…………………………………………………….………………………..passive	ultrasonic	irrigation	CUI………………………………………………………………………..continuous	ultrasonic	irrigation	LAI…………………………………………………………………………………..laser	activated	irrigation		Er,Cr:YSGG......................................................erbium	chromium-yttrium-scandium-garnett					PIPS……………………………………………………….photon-initiated	photoacoustic	streaming		GW...........................................................................................................................GentleWave™	system	%...................................................................................................................................................percentage	CLC......................................................................................................................cold	lateral	compaction	WVC..............................................................................................................warm	vertical	compaction	ISO......................................................................International	Organization	for	Standardization		MTA..............................................................................................................mineral	trioxide	aggregate	SEM.......................................................................................................scanning	electron	microscope		Micro-CT..............................................................................................micro-computed	tomography	SD..................................................................................................................................standard	deviation	PGFA..................................................................................................................gutta-percha	filled	area		PSFA..................................................................................................................................sealer	filled	area									 xiv	Acknowledgements			First	 and	 foremost,	 I	 would	 express	 my	 sincere	 gratitude	 and	 acknowledge	 my	supervisor,	Prof.	Markus	Haapasalo,	for	giving	me	the	chance	to	be	one	of	the	master	students	 at	 UBC.	 I	 would	 like	 also	 to	 thank	 him	 for	 his	 professional	 guidance,	support,	 and	 for	 helping	 me	 to	 solve	 the	 obstacles	 that	 I	 have	 faced	 during	 the	preparation	of	 this	 research	project,	and	over	 the	 last	 two	years.	 	 I	will	be	always	grateful	for	having	had	the	opportunity	to	learn	from	his	wealth	of	knowledge.	My	 thanks	 are	 also	 extended	 to	 Dr.	 Ya	 Shen,	 as	 a	 member	 of	 my	 supervisory	committee,	 for	 her	 kind	 support	 and	 direction	 during	 the	 various	 stages	 of	 my	project,	particularly	on	 the	micro-CT	data	analysis.	This	study	wouldn’t	have	been	achieved	well	without	her	assistance.	I	would	 also	 like	 to	 thank	Dr.	 Fernanda	Almeida,	 as	 a	member	 of	my	 supervisory	committee,	for	her	contribution	and	revision	of	my	research	project.	I	 am	 thankful	 to	 Mr.	 John	 Schipilow	 at	 the	 Centre	 for	 High	 Throughput	Phenogenomics	for	his	assistance	with	micro-CT	scans.	Special	 thanks	 to	Dr.	HsingChi	von	Bergmann,	and	Dr.	Zhejun	Wang	 for	 their	help	and	skill	in	the	statistical	analysis	of	my	data.	Sincere	 thanks	 to	 my	 friends	 at	 UBC	 especially	 the	 kind	 group	 who	 work	 in	 Dr.	Haapasalo’s	lab,	they	made	my	time	at	UBC	enjoyable	and	incredible.				 xv	Dedication			First,	my	limitless	thanks	to	Allah	for	his	blessings	and	help	throughout	these	years	that	have	passed	me	to	reach	this	stage	of	knowledge	and	education.	I	dedicate	this	humble	work	to	my:		Father	(Mustafa)	&	Mother	(Fawzya),	The	strong	and	gentle	souls,	who	taught	me	to	trust	in	Allah,	believe	in	hard	work,	and	whose	love,	encouragement,	and	pray	for	day	and	night	made	me	able	to	get	such	success	and	achievements.	Loving	husband	(Faisel)	&	children	(Mohamed	&	Moad),	For	their	sacrifices	and	support	during	my	studies,	and	whose	love	is	one	of	the	greatest	gifts	of	my	life.	Beloved	sister	&	brothers,	I	am	thankful	for	the	inspiration,	encouragement,	and	for	supporting	me	to	achieve	the	goal	that	I	always	dreamed	of.		Supervisors	&	teachers,	Who	assist	me	in	my	educational	career,	my	eternal	thankfulness,	and	my	appreciation.			Friends	and	family,	Thank	you	for	supporting	and	encouraging	me	with	positive	words.	My	country	(Libya),	Hoping	that	peace,	safety,	and	security	come	back	to	my	country	soon.	I	had	promised	to	make	everyone	in	Libya	proud	by	the	achievement	of	this	academic	goal	and	I	hope	that	I	have	fulfilled	that	promise.			 1	Chapter	1 :	Introduction		Kakehashi	et	al.	(1)	in	1965	studied	the	pathological	changes	in	the	exposed	pulps	of	germ	free	laboratory	animals	before	and	after	contaminating	their	oral	cavities	with	a	 complex	microflora.	 They	 established	 that	 the	 presence	 of	microorganisms	 and	their	by-products	caused	pulp	necrosis	and	formation	of	periapical	granuloma	and	even	abscess	 (1).	 Following	 caries	or	 trauma,	 the	microorganisms	 invade	 the	 root	canal	system	through	dentinal	tubules	and	reach	the	area	that	eventually	becomes	necrotic	 and	 thereby	 a	 good	 environment	 for	 the	 microorganisms	 to	 multiply,	colonize	 and	 release	 their	 by-products	 (2).	 Following	 this,	 the	 spread	 of	 bacterial	antigens	 through	 the	 apical	 foramen	 and	 the	 lateral	 canals	 to	 the	 adjacent	periradicular	region	causes	apical	periodontitis.	The	host	immune	system	begins	the	repair	process	by	starting	the	inflammatory	reaction	in	order	to	kill	or	suppress	the	microbes,	 and	 to	 eliminate	 the	 necrotic	 tissue	 (3).	 The	 inflammatory	 processes	cause	 periapical	 tissue	 destruction,	 which	 will	 continue	 as	 long	 as	 the	 microbial	cause	 is	 not	 completely	 eradicated.	 After	 the	 root	 canal	 becomes	 necrotic,	 host	defense	 system	no	 longer	 can	effectively	 attack	 the	microbes	 inside	 the	 canal	 and	the	periapical	inflammation	continues	(2).	Hence,	endodontic	treatment	is	necessary	in	order	 to	prevent	 the	spreading	of	 infectious	microorganisms	and	 their	antigens	and	to	start	the	healing	process	(4).					 2	1.1 History	of	Endodontic	Treatment	The	word	“endodontics”	originates	from	two	Greek	words	(endo	means	inside	and	odont	 means	 tooth).	 Pierre	 Fauchard	 was	 the	 first	 one	 to	 introduce	 it	 for	 the	treatment	 of	 the	 root	 canal	 with	 an	 abscess	 in	 1728	 (5).	 The	 treatment	 included	removal	of	pulp	tissue	using	a	pin,	subsequently,	filling	the	empty	space	using	a	lead	foil.	 “Intentional	 endodontic	 treatment”,	 introduced	 by	 Bourdet	 in	 1757,	 was	 an	alternative	 treatment	 for	 the	 infected	 root	 canals	 (5).	 In	 that	 technique,	 the	 tooth	was	dislodged	 from	its	place	 in	order	 to	cut	 the	nerve,	 then	 immediately	returned	back	 to	 its	 socket.	 Later,	 in	 1766,	 Robert	Woofendale	 used	 another	 procedure	 to	treat	the	root	canals.	He	cauterized	the	pulp	with	a	hot	tool	to	relieve	the	pain	(5).	In	1809,	 Edward	 Hudson	 introduced	 a	 gold	 foil	 as	 the	 first	 filling	 material	 used	 in	endodontics	 (5).	 Many	 techniques	 and	 materials	 including	 paraffin,	 metal	 and	amalgam	were	being	used	as	filling	materials	following	the	use	of	gold	foil	material,	and	they	produced	satisfying	results	at	that	period	of	time	(5).	Baker	was	the	first	one	who	published	a	paper	in	the	American	Journal	of	Dental	Science	that	described	the	pulpal	tissues	removal	and	root	canal	filling	in	1839	(5).				In	the	1850s,	gutta-percha	appeared	as	a	material	of	choice	for	root	fillings	(5).	The	gutta-percha	was	mixed	with	other	components	 to	produce	a	cement	called	“Hill’s	stopping”.	The	cement	was	used	in	conjunction	with	chloroform	and	a	wooden	plug	to	 fill	 the	 root	 canal	 space	 (5).	 Consequently,	 several	 attempts	 were	 made	 to	improve	 the	 adaptability	 of	 gutta-percha	 to	 the	 root	 canal	walls.	Dubuque	 carried	out	the	first	attempt	when	he	softened	the	gutta-percha	by	using	heat	in	1865	(5).	It	had	also	been	noticed	that	if	the	endodontically	treated	tooth	was	isolated	from	the		 3	adjacent	teeth,	this	would	add	more	safety	and	control	to	the	working	area	during	root	canal	treatment.	Therefore,	Barnum	introduced	the	rubber	dam	by	using	a	thin	sheet	of	rubber	to	insulate	the	tooth	from	the	adjacent	teeth	and	tissues	(5).		In	 1867,	Magitot	 introduced	 the	 first	 pulp	 vitality	 test	 using	 electricity	 (5).	 It	was	believed	for	several	years	that	the	etiological	factor	of	pulpal	complications	was	the	lack	of	vitality	 in	 the	 tooth,	which	was	called	 the	 “vitality	 theory”.	 In	1879,	Tomes	revised	the	old	vitality	theory	to	a	septic	theory	(6).		He	explained	the	septic	theory	as	contamination	from	the	infected	dentine	reaching	the	root	canal	of	the	non-vital	tooth,	 which	 becomes	 infected,	 and	 subsequently	 the	 infection	 spreads	 into	 the	periradicular	area	(6).		The	changes	in	the	theories	resulted	in	a	belief	that	whether	the	pulp	was	vital	made	no	difference	if	the	pulp	chamber	and	canals	were	cleaned	with	 antiseptic	 agents.	 This	 concept	 encouraged	 the	 clinicians	 to	 use	 germicidal	agents	 to	 clean	 the	 root	 canals	 and	 eliminate	 the	 bacteria	 (6).	 	 The	 basis	 of	 root	canal	obturation	was	also	developed	from	this	concept.	In	1883,	Mills	and	Brooklyn	(6)	designated	a	process	in	which	an	orangewood	stick	was	introduced	into	a	root	canal	 after	 dipping	 the	 stick	 in	 creosote	 or	 carbolic	 acid	 solutions.	 Many	practitioners	 were	 developing	 filling	 materials	 and	 techniques	 subsequently	 to	introducing	the	wood	points	in	market	in	1895	(6).	They	also	incorporated	the	use	of	 x-ray	 radiation,	 first	 introduced	 by	 Kells	 in	 1896,	 for	 the	 examination	 of	 the	quality	of	the	root	fillings	(6).						 4	In	order	to	use	the	appropriate	antibacterial	agent	to	sterilize	the	root	canals,	it	was	crucial	to	detect	the	different	kinds	of	the	bacteria	that	caused	the	pulpal	diseases.	A	study	carried	out	by	Mayrhofer	in	1909	showed	that	the	pulp	infection	in	96%	of	the	cases	was	caused	by	streptococci	(7).	The	use	of	antimicrobial	filling	was	therefore	introduced	 by	 Hermann	 in	 1920	 (7).	 He	 advocated	 the	 use	 of	 calcium	 hydroxide	(Calxyl™)	 in	 pulp	 capping,	 pulpotomy,	 pulpectomy,	 and	 the	 treatment	 of	 infected	canals	 in	1930.	He	also	showed	that	a	secondary	dentin	could	be	 formed	when	an	amputated,	 vital	pulp	was	 covered	with	Calxyl	 (7).	 In	1925,	Rickert	 suggested	 the	use	of	a	sealer	with	a	gutta-percha	cone.	The	gutta-percha	was	first	 fitted	to	reach	the	 apical	 dentinocemental	 junction,	 then	 removed	 and	 coated	 with	 the	 sealer,	consequently,	 inserted	 into	 the	 canal	 and	 pressed	 tightly	 to	 distribute	 the	 sealer	apically	and	into	the	lateral	canals	(7).	The	technique	was	enhanced	by	allowing	an	instrument	to	be	inserted	into	the	canal	to	condense	the	gutta-percha	and	provide	more	space	 for	additional	points	 to	be	placed	 inside	 the	canal,	which	 is	nowadays	called	 “cold	 lateral	 condensation	 obturation	 technique”.	 At	 the	 same	 year,	 a	 new	spiral	shaped	 file	was	presented	by	Lentulo	 to	 facilitate	 the	 insertion	of	 the	sealer	inside	the	root	canal	(7).		Rickert	and	Dixon	in	1931	had	a	hypothesis	of	the	“	hollow	tube	effect”	”,	i.e.	if	gap	or	void	is	present	in	the	filling	material,	fluid	may	entrap	into	the	spaces	and	undergo	enzymatic	breakdown	(7).	These	products	may	reach	the	periapical	area	and	cause	an	inflammatory	reaction	(7).	Based	on	this	concept,	optimal	root	canal	obturation	is	necessary	for	the	success	of	the	endodontic	treatment.		 5	1.2			Cleaning	of	the	Root	Canal	System	The	aim	of	endodontic	treatment	is	achieved	by	thorough	mechanical	and	chemical	cleaning	of	 the	 root	 canals	 to	eradicate	 the	microorganisms	and	 their	by-products	(8).	 Several	 studies	 have	 shown	 that	 apical	 periodontitis	 is	 a	 consequence	 of	penetration	 of	microbes	 and	 their	 products	 to	 the	 periapical	 area	 (9,	 10).	 Hence,	apical	periodontitis	can	be	prevented	or	treated	by	endodontic	treatment	(11,	12).	The	failure	of	endodontic	therapy	can	lead	to	a	persistent	periradicular	lesion	due	to	incomplete	or	improper	elimination	of	bacteria	inside	the	root	canal	system	(12).		1.2.1			Mechanical	Cleaning	Instrumentation	 is	 considered	 as	 one	 of	 the	 most	 essential	 steps	 of	 root	 canal	treatment	(13,	14,	15).	It	removes	many/most	of	the	bacteria	inside	the	canals	and	thereby	 contributes	 to	 a	 favorable	 environment	 for	 the	 healing	 process	 (16).	 By	shaping	and	enlargement	of	the	root	canals,	 instrumentation	creates	enough	space	for	 the	 irrigants	 to	 be	 delivered	 throughout	 the	 root	 canal	 system,	 and	 the	 filling	material	to	be	properly	placed	into	the	canal	(17).	The	preparation	of	the	root	canals	should	 be	 conservative	 with	 regard	 to	 the	 apex	 to	 avoid	 irritation	 of	 the	periradicular	tissue,	and	to	reduce	the	susceptibility	to	tooth	fracture	(18).				Apical	 size	preparation	 (ASP)	 is	an	 important	 factor	 in	achieving	 the	goals	of	 root	canal	 preparation,	 by	 which	 the	 microorganisms	 and	 pathologic	 debris	 are	eliminated	 	 (19,	 20,	 21,	 22).	 There	 is	 a	 considerable	 variation	 in	 the	 literature	regarding	 ASP,	 Tan	 and	Messer	 (23)	 compared	 the	 quality	 of	 apical	 enlargement		 6	achieved	 by	 both	 hand	 files	 (K	 files)	 and	 the	 nickel-titanium	 (Ni-Ti)	 rotary	instruments	 in	 mandibular	 molars.	 They	 concluded	 that	 the	 mechanical	instrumentation	 was	 relatively	 insufficient	 to	 reduce	 the	 debris	 in	 the	 root	 canal	system.	They	did	note,	 however,	 that	more	debris	was	 eliminated	 from	 the	 apical	third	by	enlarging	the	apical	size	preparation	using	the	rotary	 files	(23).	Similarly,	several	 other	 studies	 have	 proved	 that	 efficient	 irrigation	 and	 superior	 bacterial	removal	were	achieved	when	larger	apical	size	files	were	used	to	prepare	the	root	canals	(24,	25,	26,	27).	The	size	and	the	 taper	of	 the	apical	preparation	could	also	affect	 the	 delivery	 of	 the	 irrigating	 solution.	 Some	 literature	 suggested	 using	#40/.04	 as	 an	 ideal	 size	 and	 taper	 to	 allow	 the	 maximum	 irrigation	 volume	 to	penetrate	 the	 apical	 third	 of	 anterior	 and	 posterior	 root	 canals	 (28,	 29).	 Another	study	suggested	 that	 file	 size	#30	 is	 the	minimum	 instrumentation	size	needed	 to	delivering	 the	 irrigating	 solutions	 to	 the	 apical	 portion	 of	 the	 mandibular	 molar	canals	(30).	Brunson	et	al.	(28)	examined	the	effect	of	both	file	size	and	taper	on	the	volume	 of	 irrigants	 that	 reached	 the	 apical	 third.	 The	 authors	 used	 forty	 single	rooted	teeth	and	designed	the	study	to	include	two	aims.	The	first	aim	was	to	detect	the	 smaller	 apical	 size	 that	 permits	 the	 irrigating	 solution	 to	 flow	 to	 the	working	length,	while	the	second	aim	was	to	detect	the	ideal	taper	that	lets	more	irrigants	to	penetrate	 to	 the	 working	 length.	 In	 order	 to	 achieve	 the	 first	 aim,	 they	 used	 the	same	taper	of	.06	with	different	file	sizes	ranged	from	#30	to	#45,	while	they	used	the	same	file	size	of	#40	with	different	tapers	ranged	.02-.08	to	achieve	the	second	aim.	 It	was	 concluded	 from	 this	 study	 that	 the	 greater	 the	 apical	 preparation	 size	and	taper,	the	grater	the	irrigating	solution	delivered	to	the	working	length	(28).	On	the	other	hand,	it	has	been	reported	that	“root	canal	preparation	with	GT	rotary	files		 7	to	apical	preparation	size	30	and	final	taper	0.04,	0.06,	and	0.08	did	not	affect	canal	cleanliness”	(31).		Extra	 caution	 should	be	 taken	when	 shaping	 canals	 especially	 those	with	external	root	concavities,	as	overpreparing	these	canals	weakens	roots	and	predisposes	the	tooth	to	fracture.	It	has	been	established	that	the	thicker	the	remaining	dentin,	the	greater	the	resistance	to	tooth	fracture	(32).	Coldero	et	al.	(33)	used	nickel-titanium	rotary	 instruments	 with/without	 apical	 enlargement	 to	 compare	 the	 intracanal	bacterial	 reduction.	 They	 found	 that	 using	 of	 rotary	 instruments	 in	 combination	with	 irrigants	 and	 apical	 canal	 enlargement	 contributed	 to	 eliminating	 E.	 faecalis	from	the	root	canal	system	(33).	Similarly,	a	study	by	Card	et	al.	(26)	showed	that	the	 greater	 the	 apical	 preparation	 size,	 the	 greater	 the	 intracanal	 bacteria	elimination	 from	 the	 infected	 root	 canals.	Moreover,	Rollison	et	al.	 (27)	 suggested	that	 instrumentation	 to	 an	 apical	 size	 of	 #35	 was	 less	 effective	 in	 debriding	 and	cleaning	the	root	canals	than	when	the	apical	preparation	size	was	#50.	In	contrast,	few	studies	found	no	significant	difference	in	removing	debris	and	bacteria	from	the	root	 canals	 when	 using	 different	 apical	 preparation	 sizes.	 	 Albrecht	 et	 al.	 (34)	 in	their	 study	 showed	 that	 when	 standardizing	 the	 taper	 to	 0.10,	 there	 was	 no	difference	in	debriding	the	root	canal	system	by	using	ProFile	G	T	instruments	of	20	and	 40	 apical	 size	 preparation.	 Moreover,	 Yared	 and	 Dagher	 	 (35)	 found	 no	difference	between	canals	prepared	to	a	size	25	and	those	prepared	to	a	size	40	in	terms	of	apical	disinfection.			 8	Anatomical	complexity	of	the	root	canal	system,	lateral	canals,	isthmus	area,	apical	ramification,	 and	 transverse	 anastomoses	 provide	 significant	 challenges	 for	endodontic	treatment	(36).	They	are	also	reasons	that	30-50%	of	the	canal	wall	area	remains	untouched	(37).	Further,	it	has	been	shown	that	the	level	of	bacteria	in	the	root	 canal	 cannot	 be	 significantly	 reduced	 by	 only	 using	 mechanical	 preparation	(38).	 Moreover,	 the	 endodontic	 irrigating	 solutions	 cannot	 be	 delivered	 to	 all	complexities	 of	 the	 root	 canal	 system	 by	 conventional	 methods	 (39).	 Hence,	remaining	 bacteria	 may	 develop	 into	 a	 biofilm	 that	 is	 even	 more	 difficult	 to	 be	removed	 by	 the	 commonly	 used	 methods	 of	 instrumentation,	 irrigation	 and	disinfection.			1.2.2			Chemomechanical	Cleaning	1.2.2.1			Irrigation	Solutions		The	 irrigating	solutions	are	used	with	files	or	with	special	 tools	during	cleaning	of	the	root	canal	system	to	eliminate	bacteria,	dissolve	necrotic	tissue,	facilitate	dentin	removal,	 remove	 the	 smear	 layer,	 dissolve	 inorganic	 tissue,	 and	 reduce	 the	instrument	friction	during	preparation	(40,	41,	42).	Sodium	hypochlorite	(NaOCl)	is	the	most	popular	irrigant	because	of	its	highly	antimicrobial	reaction	and	capability	to	dissolve	organic	matter,	necrotic	tissue	in	particular	(42,	43).	The	concentration	of	 NaOCl	 commonly	 used	 in	 endodontics	 is	 between	 0.5%	 and	 6%	 (44).	 Tissue	dissolution	 by	 NaOCl	 can	 be	 improved	 up	 to	 50-fold	 by	 optimizing	 the	concentration,	temperature,	flow	(agitation)	and	surface	tension	(45).			 9	The	smear	 layer	 is	a	1-2	µm-thick	 layer	that	 is	composed	of	organic	and	 inorganic	components	 including	mineralized	 and	unmineralized	 dentin,	 bacteria,	 and	 pulpal	tissue	remnants.	It	is	amorphous	layer	with	a	flat	surface	and	an	inner	layer	that	can	infiltrate	 a	 few	micrometers	 into	 the	 dentinal	 tubules	 (46).	 It	 has	 been	 suggested	that	the	presence	of	inorganic	constituents	(dentin	remnants)	in	the	smear	layer	is	responsible	for	the	reduced	killing	effectiveness	of	NaOCl	(1%)	against	Enterococcus	faecalis	(47).	The	smear	layer	cannot	be	removed	by	using	NaOCl	only.	However,	the	organic	portion	in	the	smear	layer	is	affected	by	NaOCl,	which	makes	it	more	prone	to	 the	 ethylenediamine	 tetra-acetic	 acid	 (EDTA).	 EDTA	 as	 a	 demineralizing	 agent	reacts	with	 the	 inorganic	portion	of	 the	 smear	 layer,	 and	 removes	hydroxyapatite	(48).	 	 In	 a	 systematic	 review,	 Shahravan	 et	 al.	 (49)	 showed	 that	 removal	 of	 the	smear	 layer,	 improves	 the	 “fluid	 tight	 seal”	 regardless	 the	 filling	 technique	 or	 the	sealer	used.		However,	using	of	EDTA	longer	than	the	recommended	time	can	cause	dentinal	tubular	damage	(50).		Chemomechanical	 cleaning	 is	 the	 integration	 of	 both	 mechanical	 flushing	 and	chemical	 action	 of	 the	 irrigant	 to	 remove	 as	much	 irritating	material	 as	 possible.	Irrigating	solutions	have	been	“activated”	using	different	methods	such	as	sonic	and	ultrasonic	activation	and	laser.				 10	1.2.2.2			Methods	of	Irrigation	1.2.2.2.1			Conventional	Syringe	Irrigation	A	 syringe	 attached	 to	 a	 needle	 has	 been	 the	 most	 widely	 used	 way	 to	 deliver	irrigating	 solutions	 into	 the	 root	 canal;	 the	 irrigation	 needle	 placed	 optimally	 2-3	mm	short	of	the	working	length.	Plastic	syringes	with	different	capacities	(1-20	ml),	needles	in	different	sizes	(25,	27,	30,	31	gauge),	and	various	needle	tip	designs	are	used	in	this	method.	Large	syringe	size	saves	the	time	of	solution	refill,	however,	the	apical	pressure	is	more	difficult	to	control	than	with	small	syringes	sizes.	Therefore,	use	of	 small	 size	 syringes	of	1-5	mL	 is	 recommended	 (44).	The	use	of	needle	 size	30/31	gauge	is	preferred	to	bigger	needles	to	enable	the	delivery	of	irrigants	to	the	most	apical	canal.	However,	the	high	risk	of	the	irrigating	solution	escaping	beyond	the	 apical	 foramen	 requires	 attention	 and	 caution	during	 application	 (44).	Needle	tips	are	manufactured	in	different	designs	such	as	open	and	closed	ended	needles;	some	have	 the	hole	on	 the	side	 (side	vented	needle),	 and	 the	one	or	more	outlets		(51).	During	root	canal	irrigation	the	needle	should	be	moved	up	and	down	while	it	is	inside	the	canal.	Size	#30-35	is	the	minimum	apical	size	preparation	required	for	the	placement	of	the	needle	to	a	maximum	insertion	depth	to	deliver	the	irrigating	solutions	to	the	apical	root	canal	(51).	Side	vented	(“safety”)	needles	can	be	placed	at	 0-1	 mm	 short	 of	 the	 working	 length,	 whereas	 open-ended	 needles	 should	 be	placed	 at	 2-3	 mm	 short	 of	 the	 working	 length	 to	 avoid	 apical	 extrusion	 of	 the	irrigants	(51).		 11	1.2.2.2.2			EndoVac	Negative	Pressure	System	Apical	 air	 bubbles	 that	 often	 form	 during	 the	 use	 of	 traditional	 methods	 of	delivering	the	irrigating	solutions	to	the	apex	can	prevent	the	solutions	reaching	to	the	apical	 area	of	 the	 root	 canal.	The	evacuation	process	of	 liquids	 through	apical	negative	pressure	is	the	method	that	secures	a	safe	flow	of	irrigating	solutions	down	to	the	depth	of	the	EndoVac	tip	in	the	apical	canal	and	then	back	(suction)	through	the	needle.		EndoVac	negative	pressure	system	is	used	to	safely	deliver	the	selected	solution	to	the	working	length	without	further	penetration	to	the	periradicular	area	(52,	 53,	 54).	 It	 consists	 of	 three	parts:	 the	Master	Delivery	Tip,	 the	macrocannula	and	the	microcannula.		1.2.2.2.3			Sonic	and	Ultrasonic	Irrigation	Although	 traditional	 syringe-needle	 irrigation	 has	 been	 considered	 as	 an	 effective	cleaning	method	for	 the	cleaning	of	 the	root	canal	system	for	many	years,	optimal	cleanliness	 cannot	 be	 achieved	 in	 all	 parts	 of	 the	 root	 canal	 system	 by	 using	 this	technique	(55,	56).		Therefore,	various	ways	to	improve	the	effects	of	syringe-needle	irrigation	 have	 been	 developed.	 A	 cavitation	 phenomenon	 occurs	 when	 an	ultrasonic	wave	 is	 applied	 in	 a	 liquid,	which	 creates	 vacuum	 bubbles	 (51).	 These	bubbles	become	larger	and	unsteady	till	they	explode	and	release	high	power	waves	and	 high	 temperature.	 The	 high-power	 stream	 generated	 has	 a	 strong	 cleaning	effect	 on	 the	 rough	 surfaces	 and	 unreachable	 areas	within	 the	 root	 canal	 system	(51).	Several	studies	compared	the	cleaning	efficacy	of	conventional	and	ultrasonic	agitation	 methods	 (57,	 58,	 59).	 Recent	 studies	 have	 examined	 the	 importance	 of		 12	ultrasonic	energy	in	maximizing	the	efficiency	of	the	irrigants	(55,	60).	Greporio	et	al.	 (39)	 compared	 the	 penetration	 ability	 of	 NaOCl	 into	 the	 simulated	 working	length	 and	 lateral	 canals.	 They	 found	 that	 the	 passive	 ultrasonic	 irrigation	 (PUI)	method	enhanced	the	ability	of	NaOCl	to	penetrate	the	lateral	canals	more	than	the	apical	negative	pressure	irrigation	(ANP).	However,	the	study	also	showed	that	ANP	secured	 the	 delivery	 of	 NaOCl	 to	 working	 length	 better	 than	 passive	 ultrasonic	irrigation	 (PUI)	 (39).	 Both	 PUI	 and	 Continuous	 Ultrasonic	 Irrigation	 (CUI)	 are	examples	 of	 ultrasonic	 activation	 irrigation	 devices.	 PUI	 agitates	 the	 irrigants	 by	using	a	metal	file,	which	can	have	a	smooth	or	rough	surface	and	which	is	placed	in	the	 root	 canal	without	 touching	 the	 canal	wall	 during	 application.	 Therefore,	 this	method	may	be	 inapplicable	 in	curved	and	minimally	 instrumented	canals	(51).	 In	some	of	the	new	CUI	equipment	such	as	ProUltra	PiezoFlow	(Dentsply	Tulsa	Dental	Specialties,	 Tulsa,	 OK,	 USA),	 irrigant	 flows	 into	 the	 root	 canal	 through	 a	metallic,	hollow	 tip	while	 the	 tip	 is	 oscillating	 at	 ultrasonic	 frequency	 (61).	 Several	 studies	have	suggested	that	this	type	of	CUI	can	further	improve	canal	cleanliness	(62,	63).	EndoActivator	 system	 is	 a	 model	 of	 sonic	 devices	 that	 attached	 to	 polymer	 tip	(Dentsply	 Tulsa	 Dental	 Specialties).	 The	 sonic	 vibrations	 move	 NaOCl	 quickly	around	the	canal	to	dissolve	necrotic	pulp	tissues	(45).			1.2.2.2.4			Laser	Activated	Irrigation	(LAI)	Irrigation	methods	have	also	been	developed	using	energy	 from	different	 types	of	lasers.	 	 Use	 of	 an	 Er,Cr:YSGG	 (erbium	 chromium-yttrium-scandium-garnett)	 laser	was	first	introduced	by	Blanken	and	Verdaasdonk	(64).	Matsumoto	et	al.	(65)	found	that	 the	 liquid	beside	the	 laser	 tip	evaporated	and	created	bubbles.	Once	the	 laser		 13	was	 stopped,	 the	 bubbles	 began	 to	 shrink	 and	 eventually	 collapsed	 due	 to	 the	increase	 of	 the	 surrounding	 pressure.	 The	 explosion	 of	 the	 bubble	 develops	 a	powerful	liquid	stream	without	rising	the	temperature.			Photon-Induced	Photoacoustic	Streaming	(PIPS)	This	technique	is	similar	to	LAI	with	a	difference	that	the	later	system’s	tip	is	placed	inside	 the	 canals	while	 the	 PIPS’s	 tip	 is	 placed	 at	 the	 orifice	 (66).	 The	 PIPS	 tip	 is	placed	 in	 the	pulp	chamber	with	a	continuous	 flow	of	 the	 irrigants	 to	keep	the	tip	immersed	 in	 the	 solutions	during	 the	procedure.	 Six	30s	 cycles	of	 laser	 activation	are	employed	by	this	method	(three	30s	of	NaOCl,	two	30s	of	water,	and	one	30s	of	EDTA).	 	A	 recent	 study	showed	 that	 the	samples	 irrigated	using	PIPS	system	with	6%	NaOCl	revealed	the	greatest	eradication	of	bacterial	biofilm	in	comparison	to	the	samples	cleaned	by	using	either	6%	NaOCl	or	PIPS	with	water	 (67).	Some	studies	have	shown	the	superior	efficacy	of	PIPS	 in	removing	the	dentine	debris,	bacteria,	smear	 layer	 and	 biofilm	 compared	 with	 the	 other	 conventional	 syringe-needle	irrigation	(SNI),	and	sonic	and	ultrasonic	irrigation	(56,	67,	68,	69,	70).	However,	it	has	been	shown	by	other	studies	that	the	removal	of	E.	faecalis	and	smear	layer	by	using	PIPS	with	NaOCl	was	equal	 to	 that	achieved	by	using	conventional	SNI	with	NaOCl	 +	 EDTA	 (71).	 Additionally,	 apical	 extrusion	 similar	 to	 that	 generated	 by	conventional	syringe-needle	irrigation	can	occur	also	with	the	PIPS	system	(72).				 14	1.2.2.2.5			GentleWave	System	(GW)	A	 novel	 technology	 for	 cleaning	 and	 disinfecting	 root	 canals	 (the	 GentleWave™	system)	 has	 recently	 been	 developed	 by	 Sonendo.	 The	 GentleWave™	 system	(Sonendo,	Laguna	Hills,	Orange	County,	CA,	USA)	consists	of	console	and	treatment	instrument,	 which	 resembles	 a	 handpiece	 (Figure	 1.1).	 The	 console	 consists	 of	numerous	electrical	circuits,	containers	for	the	different	 irrigating	solutions/waste	material,	 pressure	 producer,	 and	 degassing	 system.	 GentleWave	 treatment	instrument	 delivers	 the	 irrigants	 at	 a	 high	 flow	 rate	 of	 45	 ml/min	 to	 the	 pulp	chamber	and	consequently	to	the	entire	root	canal	system	(73).	When	the	 irrigant	flow	 hits	 the	 end	 plate	 of	 the	 tip	 of	 the	 nozzle	 in	 the	 pulp	 chamber,	 a	 stream	 of	irrigating	solution	interacts	with	the	static	fluid	in	the	pulp	chamber	and	creates	a	shear	 force,	 which	 according	 to	 the	 manufacturer	 produces	 a	 hydrodynamic	cavitation.	 High	 power	 waves	 of	 sounds	 are	 generated	 due	 to	 the	 strong	hydrodynamic	 cavitation	 that	propagates	 throughout	 the	whole	 root	 canal	 system	through	 the	 degassed	 fluids.	 The	 GentleWave™	 system	 utilizes	 three	 irrigating	solutions,	NaOCl,	EDTA	and	water	(Figure	1.2),	which	are	degassed	inside	the	main	console	before	being	delivered	 to	 the	pulp	 chamber.	The	degassing	process	of	 the	irrigating	 solutions	 aims	 to	 remove	 microscopic	 air	 bubbles	 from	 the	 fluids	 and	thereby	 eliminate	 the	 “vapor	 lock	 phenomenon”	 that	 often	 hinders	 the	 irrigants	from	 reaching	 the	 apical	 third	 (74,	 75).	 The	 tip	 of	 the	 handpiece	 is	 placed	 1	mm	above	 the	pulp	chamber;	no	or	minimal	 instrumentation	of	 size	#15/0.04	 taper	 is	recommended	 by	 the	manufacturer.	While	 the	 GentleWave™	 system	 is	 operating;	the	 pulp	 chamber	 is	 completely	 sealed	 to	 prevent	 the	 escape	 of	 NaOCl	 to	 the	adjacent	working	area.	The	excess	of	solutions	and	debris	are	evacuated	by	a	 five-	 15	points	 vented	 suction	 on	 the	 inner	 surface	 of	 GentleWave	 treatment	 instrument	creating	a	negative	pressure	within	the	root	canal	system.			Figure	1.1	(A)	The	GentleWave™	System	and	(B)	the	tip	of	the	cleaning	instrument	in	the	pulp	chamber	of	a	molar.		Image	courtesy	Sonendo	Inc.			The	 efficacy	 of	 the	 GentleWave	 system	 in	 tissue	 dissolution	 and	 cleaning	 of	 the	canals	 has	 been	 compared	 to	 a	 positive-	 pressure	 conventional	 syringe-needle	irrigation,	passive	ultrasonic	activation,	and	EndoVac.	The	studies	showed	that	GW	dissolved	 organic	matter	 eight	 times	 faster	 than	 the	 next	 best	 system	 tested	 (73,	76).		Haapasalo	et	al.	(77)	reported	that	negative	apical	pressure	was	created	by	the	GentleWave™	system,	which	improves	the	safe	use	of	this	system	in	comparison	to	positive	 pressure	 syringe-needle	 irrigation.	 Another	 study	 compared	 the	effectiveness	 of	 the	 GentleWave™	 system	 in	 removing	 calcium	hydroxide	 paste	 to	Syringe-needle	irrigation	and	PUI.	It	was	concluded	that	62.75%,	75.26%,	99.39%	of	Ca(OH)2	were	removed	 from	the	root	canals	by	using	conventional	 irrigation,	PUI,	and	GentleWave,	 respectively.	Furthermore,	GentleWave™	system	was	reported	 to		 16	have	a	high	success	rate	in	removing	fractured	files	from	root	canals	of	molar	teeth	in	vitro	(78).				Figure	1.2	The	recommended	protocol	for	the	GentleWave	cleaning.		1.3			Root	Canal	Obturation	The	purpose	of	root	canal	obturation	is	to	fill	the	canals	with	appropriate	materials	to	 fully	 occupy	 the	 space	 so	 that	 microorganisms	 cannot	 re-enter	 the	 root	 canal	system.	 Additionally,	 the	 microbes	 that	 remain	 within	 the	 canals	 following	 the	chemomechanical	cleaning	should	 	be	 isolated	from	nutrients	 in	the	pulp	chamber	and	in	the	tissue	fluids	by	achieving	a	good	seal	with	the	filling	materials	(79,	80).	Water		15	sec	8%	EDTA	2	min	Water	15	sec	3%	NaOCl		5	min	Total	cycle		7.5	min		 17	The	obturation	materials	consist	of	a	core	material	that	fills	most	of	the	canal	space,	and	 a	 sealer	 that	 fills	 the	 interface	 between	 the	 core	material	 and	 the	 canal	wall	(81).	 In	 addition,	 sealers	 often	 fill,	 at	 least	 partly,	 lateral	 canals.	 According	 to	Grossman	(82),	the	following	properties	are	desirable	for	root	filling	materials:		• Provide	a	maximum	sealing	ability	to	the	canal	ramifications	and	the	lateral	canals,	and	allow	for	a	fluid-tight	seal	• Biocompatible	with	the	periapical	tissues,	and	doesn’t	cause	any	irritation	or	discoloration	to	the	tooth	structure	or	the	surrounding	tissues	• Easy	to	be	applied,	and	not	difficult	to	be	removed	from	the	root	canals	when	the	endodontic	retreatment	is	required	• Resist	moisture	(nonresorbable),	and	not	shrink	after	being	inserted	into	the	canals	• Appear	as	a	radiopaque	material	on	the	radiograph	to	be	easily	distinguished	from	the	tooth	structure	• Prevent	 the	 bacterial	 growth	 within	 the	 root	 canal	 through	 its	bactericidal/bacteriostatic	effect		1.3.1			Filling	Materials	1.3.1.1			Gutta-Percha			The	 gutta-percha	 is	 the	 most	 widely	 used	 root	 filling	 material	 due	 to	 its	thermomechanical,	 physical,	 and	 sealing	 properties	 (83,	 84,	 85,	 86).	 It	 is	 derived	from	 a	 tree	 of	 the	 Sapotaceae	 family	 (87).	 Alpha	 and	 Beta	 forms	 are	 the	 two	crystalline	 phases	 of	 the	 gutta-percha;	 alpha	 is	 the	 natural	 form	 of	 gutta-percha		 18	while	beta	is	the	processed	form	of	gutta-percha.	Beta	form	is	a	result	of	cooling	the	warm	alpha	 form	at	 room	 temperature.	 Zinc	 oxide,	which	has	 some	 antimicrobial	effect,	 is	 the	major	 component	 of	 gutta-percha	 used	 in	 endodontics,	 it	 constitutes	about	 65%	 of	 gutta-percha.	 Gutta-percha	 constitutes	 20%,	 and	 the	 rest	 15%	 are	natural	waxes,	 (heavy)	metals	salts	and	plasticizers	(88).	Most	of	 the	gutta-percha	used	in	endodontics	is	in	beta	form	according	to	Goodman	(89).		Gutta-percha	 is	 a	 non-toxic	material,	 and	 no	 systemic	 reactions	were	 reported	 by	the	clinical	studies	when	using	the	gutta-percha	as	a	root	filling	material	(90).	The	gutta-percha	is	applied	together	with	a	sealer	because	of	its	poor	adaptability	to	the	canal	 walls	 if	 used	 alone	 (91).	 The	 gutta-percha-sealer	 as	 a	 filling	 material	 has	superior	 properties	 in	 comparison	 to	 many	 other	 filling	 materials	 and	 produces	good	 seal	 to	 the	 root	 canal	 system	with	 few	 voids	 and	 gaps	 inside	 the	 filling	 and	between	the	filling	and	the	canal	wall	(92).		1.3.1.2			Root	Canal	Sealer	Root	canal	sealers	are	responsible	for	producing	a	fluid-tight	seal	when	used	with	a	core	material	or	when	used	alone.	Additionally,	 they	can	 fill	 the	 interface	between	the	core	materials	such	as	gutta-percha,	and	the	canal	walls.	Moreover,	they	play	an	important	 role	 to	 fill	 the	 fins,	 lateral	 canals	and	 the	canal	 ramifications	within	 the	root	canal	systems.	Several	studies	have	shown	that	many	sealers	have	a	tendency	to	 be	 dissolved	 over	 time	 leaving	 a	 gap	 that	 is	 a	 potential	 target	 for	 bacterial	accumulation	 (93,	 94,	 95).	 Therefore,	 optimally	 the	 sealers	 should	 possess	 the		 19	following	properties	to	be	used	in	endodontic	therapy.	Grossman	(82),	showed	that	the	ideal	root	canal	sealer	should:	• Provide	good	seal	when	set	• Be	an	excellent	adhesive	to	the	root	canal	wall	and	the	core	material	• Be	radiopaque	• Be	dimensionally	stable	upon	setting	• Be	easily	mixed	and	manipulated	• Not	cause	tooth	structure	discoloration	• Be	bactericidal	or	at	least	bacteriostatic	to	depress	the	bacterial	growth	• Set	slowly	to	increase	the	working	time	• Not	be	dissolved	in	tissue	fluids	• Not	irritate	the	periradicular	tissues	• Be	easily	solved	by	a	solvent	if	necessary			Sealers	 should	 be	 applied	 properly	 to	 not	 extrude	 to	 the	 periradicular	 tissues	because	 most	 sealers	 have	 exhibited	 different	 degrees	 of	 toxicity,	 although	 the	toxicity	will	be	reduced	or	disappear	over	time	(96).	Sealers	are	classified	according	to	 their	 compositions	 into	 several	 categories;	 Zinc	 oxide-eugenol-based	 sealers,	epoxy	resin-based	sealers,	calcium	hydroxide-based	sealers,	silicone-based	sealers,	and	 glass-ionomer	 cement	 sealers.	 AH	 Plus	 sealer	 (Dentsply	 Tulsa	 Dental	Specialties)	 (Figure	1.3)	 is	one	of	 the	most	 common	epoxy	 resins	 cement	 that	has	been	used	in	endodontic	therapy	for	many	years.	It	has	been	modified	from	AH-26	to	 reduce	 its	 cytotoxicity	 by	 preventing	 the	 release	 of	 formaldehyde	 during	 the		 20	setting	process	(97).	Although	AH	Plus	sealer	is	less	cytotoxic	than	AH-26,	it	still	has	a	 cytotoxic	 effect	 	 (98,	 99,	 100,	 101).	 However,	 a	 recent	 study	 showed	 that	 after	setting	of	AH+,	fibroblast	cells	were	able	to	grow	on	the	surface	of	the	sealer,	similar	to	 bioceramic	 sealers	 (102).	 AH	 Plus	 has	 a	 considerably	 low	 flowability,	 low	 film	thickness,	 long	working	and	setting	time,	 low	solubility,	and	slight	shrinkage	upon	setting	in	comparison	to	other	sealers	(103).			Figure	1.3	AH	Plus	Jet	sealer	in	a	dual-barrel	syringe	and	a	mixing	tip.		GuttaFlow	Sealer	(GuttaFlow	2;	Coltène/Whaledent,	Langenau,	Germany)	 is	one	of	the	Silicon-Based	Sealers;	it	contains	small	gutta-percha	particles,	less	than	30	µm	in	diameter	in	a	silicone	sealer.	It	is	provided	in	a	dual-barrel	syringe	with	a	mixing	or	delivery	 tip	 (Figure	 1.4).	 It	 has	 been	 reported	 that	 the	 syringe-mixed	 endodontic	sealers	display	significantly	 lower	surface	porosity	than	hand-mixed	sealers	(104).		GuttaFlow	sealer	expands	upon	setting	(103)	and	is	characterized	by	a	thixotropic	property.	This	means	 that	 the	 sealer	 viscosity	 reduces	under	pressure.	Hence,	 the		 21	sealer	 flows	 into	 the	 small	 lateral	 canals	 and	 exhibits	 a	 good	 sealing	 ability	 in	comparison	 to	 other	 sealers	 including	 AH	 Plus	 sealer	 (105,	 106,	 107,	 108).	Accordingly,	a	study	by	Brackett	et	al.	(109)	showed	that	“The	use	of	GuttaFlow	with	a	 single	 gutta-percha	master	 cone	 creates	 an	 apical	 seal	 that	 is	 equivalent	 to	 that	produced	 with	 gutta-percha/AH	 Plus	 sealer	 using	 warm	 vertical	 compaction”.	Although	AH	Plus	sealer	penetrates	deeper	 in	the	dentinal	 tubules	than	GuttaFlow	sealer	 when	 the	 smear	 layer	 is	 removed	 (110),	 the	 penetration	 in	 the	 dentinal	tubule	 is	 affected	 by	 final	 irrigation	 method,	 root	 canal	 third	 and	 the	 root	 canal	sealer	used	(111).	AH	Plus	sealer	is	antibacterial	and	can	kill	bacteria	in	the	infected	dentin	 more	 efficiently	 than	 GuttaFlow	 sealer	 (112,	 113,	 114).	 However,	 the	GuttaFlow	sealer	is	biocompatible	with	the	periradicular	tissues	(99,	115).			Figure	1.4	GuttaFlow	2	sealer	in	a	dual-barrel	syringe	and	a	delivery	tip.			 22	1.3.2			Obturation	Techniques		A	 high	 quality	 root	 canal	 filling	 may	 be	 accomplished	 using	 one	 of	 the	 various	techniques.	 The	 selection	 of	 the	 technique	 depends	 on	 the	 final	 preparation	 size.	However,	 the	 operator	 preference	 and	 the	 modern	 technologies	 may	 be	 factors	 in	selecting	 the	proper	obturation	 technique	(116).	Cold	 lateral	compaction	(CLC),	warm	vertical	 compaction	 (WVC),	 single-cone	 obturation	 (SC),	 and	 carrier-based	 obturation	are	the	most	common	methods	to	fill	the	root	canal	space	(80).		1.3.2.1			Cold	Lateral	Compaction	Obturation		Cold	lateral	compaction	(CLC)	is	a	widely	used	root	filling	technique	(117).	It	 is	an	uncomplicated	 method,	 inexpensive,	 can	 be	 used	 in	 different	 clinical	 situations,	allows	 for	 working	 length	 control	 during	 compaction,	 and	 both	 retreatment	procedures	and	post	space	preparation	can	be	done	easily	 in	 the	root	canals	 filled	with	this	method	(118).	 	After	final	preparation	and	cleaning	of	the	root	canal,	 the	previously	selected	master	gutta-percha	cone	immersed	in	a	sealer	is	inserted	into	the	canal	 to	 the	working	 length.	A	proper	size	 finger	spreader	with	a	suitable	size	and	 taper	 is	 selected	 and	 inserted	 into	 the	 canal,	maximally	 1-2	mm	 short	 of	 the	working	 length,	 to	 compact	 the	 gutta-percha	 apically	 and	 laterally.	 Immediately	after	removing	the	spreader,	an	accessory	cone	coated	with	sealer	 is	placed	 in	the	space	 created	 by	 the	 spreader.	 Adding	 of	 accessory	 cones	 is	 continued	 until	 the	canal	 is	 filled	with	 gutta-percha	 cones,	 and	 the	 spreader	 insertion	 into	 the	 canals	only	extends	3	mm	or	 less	 from	the	canal	orifice.	The	gutta-percha	cones	are	then	severed	at	the	level	of	the	canal	orifice	by	a	heated	plugger	and	condensed	vertically		 23	to	about	1	mm	below	the	orifice	(119).	The	size	of	the	plugger	used	for	cold	vertical	compaction	 should	 be	 slightly	 smaller	 than	 the	 coronal	 canal	 diameter	 to	 avoid	potentially	harmful	forces	against	the	canal	wall	dentin.		Cold	 lateral	 compaction	 technic	may	 be	 difficult	 to	 use	 in	 severely	 curved	 canals,	abnormal	 root	 canal	 anatomy	or	when	 there	 is	 internal	 resorption	 (80).	The	poor	adaptation	of	the	filling	materials	to	the	canal	wall	and	formation	of	voids	within	the	fillings	are	also	drawbacks	of	using	this	technique	(120).	Additionally,	in	a	study	CLC	exhibited	lower	filling	mass	in	isthmus	areas	and	in	apical	regions	than	continuous	wave	 of	 condensation	 and	Ortho	MTA	 techniques	 (121).	 However,	 a	 recent	 study	has	shown	that	the	sealing	ability	achieved	by	CLC	is	comparable	to	that	achieved	by	injected	gutta-percha,	and	single	gutta-percha	point	techniques	(122).			1.3.2.2			Warm	Vertical	Compaction	Obturation		Warm	 vertical	 compaction	 technique	 (WVC)	 was	 first	 introduced	 by	 Schilder	 in	1967	(15).	‘Down-pack’	and	‘back-fill’	are	the	two	steps	of	this	technique.		The	root	canals	 filled	 by	 WVC	 are	 first	 prepared	 to	 a	 continuously	 tapered	 shape	 while	maintaining	 the	 original	 anatomy	 of	 the	 canal.	 Additionally,	 the	 apical	 foramen	should	be	kept	as	small	as	feasible	and	maintained	in	its	position.	Prior	to	starting	the	obturation	procedures,	proper	size	pluggers	are	selected.	Also,	the	master	cone	gutta-percha	 is	 trimmed	 to	 fit	 tightly	 0.5–1	mm	 short	 of	 the	 working	 length	 and	should	show	“tug	back”.	The	master	cone	is	coated	with	a	sealer	and	placed	in	the	canal	 until	 it	 fits	 the	 working	 length	 “tug-back”,	 and	 shows	 some	 resistance	 to	withdrawal.	A	heated	instrument	is	used	to	cut	the	cone	at	the	canal	orifice	and	the		 24	gutta-percha	cone	is	then	compacted	with	a	proper	size	plugger.	The	tip	of	the	heat	carrier	is	introduced	inside	3	mm	of	the	compacted	core	material	and,	subsequently,	removed	 to	 pull	 out	 the	 coronal	 part	 of	 the	 gutta-percha	 from	 the	 canal.	 Smaller	plugger	sizes	are	used	to	compact	the	remaining	gutta-percha,	followed	by	removal	of	additional	increments	of	the	gutta-percha	until	the	gutta-percha	fills	the	apical	4-5	mm	of	the	canal.	The	diameter	of	the	used	plugger	should	be	always	smaller	than	the	root	canal	at	the	different	canal	levels	to	avoid	interference	with	the	canal	walls.	The	 coronal	 space	 is	 then	 filled	 with	 3-4	 mm	 increments	 of	 heat-softened	 gutta-percha	 using	 injection	 system	 (backfill).	 Next,	 the	 gutta-percha	 increments	 are	compacted	using	a	plugger	of	proper	size	sequentially	till	 the	whole	canal	space	 is	filled	with	 the	 filling	material	 at	 the	 level	 of	 the	 canal	 orifice.	 A	 continuous	wave	condensation	technique	is	a	modified	technique	of	WVC	(123).	The	two	techniques	differ	 in	 the	source	of	carrier	heating	system,	where	 the	electricity	 is	employed	 in	heating	 of	 the	 carrier	 tip	 of	 the	 continuous	 wave	 of	 condensation.	 The	 electric	carrier	 tip’s	 temperature	can	be	rapidly	decreased	and	 increased;	 therefore,	 it	can	be	used	as	both	heat	carrier	and	a	plugger.			It	 has	 been	 shown	 that	 the	 adaptability	 of	WVC	 is	 better	 than	 CLC	 (124,	 125).	 In	addition,	 in	CLC	significantly	gutta-percha	and	more	sealer	are	present	 in	the	final	filling	than	WVC	filling.	Also,	a	significantly	higher	percentage	of	voids	are	found	in	CLC	 fillings	 when	 compared	 to	 WVC	 (126).	 However,	 the	 long-term	 success	 rate	achieved	 by	 warm	 vertical	 compaction	 technique	 is	 comparable	 to	 that	accomplished	by	the	cold	lateral	compaction	method	(127,	128).	WVC	is	not	an	easy	technique	 especially	 in	 curved	 canals	 where	 the	 rigid	 pluggers	 are	 difficult	 to	 be		 25	introduced	deep	into	the	canal.	 In	such	cases,	 the	curved	canals	may	require	more	preparation	 and	 enlargement,	 which	 may	 increase	 the	 chance	 of	 tooth	 fracture	because	of	the	extensive	tooth	structure	loss	(129).	The	filling	material	extrusion,	as	a	 result	 of	 applied	 forces	 used	 in	 WVC,	 is	 also	 one	 of	 the	 disadvantages	 of	 the	method	 (130).	 Despite	 the	 limited	 clinical	 signs	 of	 adverse	 periodontal	 effects	following	 thermoplastic	 obturation	 methods	 (79),	 the	 heat	 produced	 from	 the	heated	 carrier	 during	 the	 obturation	 can	 affect	 the	 periodontal	 ligament	 (131).	Another	disadvantage	of	using	this	technique	is	the	difficulty	of	length	control,	and	the	dimensional	changes	of	the	filling	upon	cooling	of	the	material.			1.3.2.3			Single-Cone	Obturation		The	single-cone	technique	was	established	and	became	popular	with	the	 initiation	of	standardization	of	filling	cones	and	endodontic	files	(ISO)	in	the	1960s	(132).	The	single-cone	technique	utilizes	the	use	of	a	single	GP	point	with	a	variable	amount	of	sealer	 at	 room	 temperature	 and	 without	 using	 of	 condensation.	 The	 sealer	 is	inserted	 into	 the	prepared	 and	 cleaned	 canals	by	 injecting	 it	 via	 special	 tips,	 or	 it	introduced	into	the	canals	by	using	Lentulo	or	bidirectional	spiral.	Consequently,	the	pre-measured	gutta-percha	is	inserted	to	fit	with	the	tug-back.	A	heated	instrument	is	used	to	cut	the	excess	of	the	gutta-percha	cone	at	the	level	of	the	canal	orifice.	The	coronal	portion	of	GP	cone	is	then	slightly	vertically	condensed	using	a	non-heated	plugger	 with	 a	 diameter	 smaller	 than	 the	 coronal	 canal	 diameter.	 	 Single-cone	technic	is	an	easy,	simple	and	fast	endodontic	obturation	technique	(133,	134,	135).	However,	 in	 terms	 of	 the	 obturation	 quality,	 bacterial	 penetration,	 marginal	infiltration,	 and	 sealing	ability,	previous	 research	has	 shown	 that	 the	 fillings	done		 26	with	 the	 single-	 cone	 technique	have	 comparable	 or	 lower	quality	 than	 fillings	by	other	obturation	techniques	(136,	137,	138,	139).			1.3.2.4			Carrier-Based	Obturation		In	 1978,	 Johnson	 introduced	 a	 technique	 of	 delivering	 thermoplasticized	 gutta-percha	into	the	root	canal	space	(140).	The	idea	of	a	carrier-based	obturator	started	by	using	stainless	steel	file	that	was	placed	into	the	root	canal	about	1-2	mm	from	the	working	length.	The	file	was	marked	at	2	mm	away	from	the	orifice	level	while	it	was	still	in	the	canal.	The	mark	or	the	sign	was	made	to	facilitate	the	removal	of	the	undesirable	 portion	 of	 the	 file	 after	 placing	 the	 filling	material.	 The	 file	was	 then	coated	with	warmed	gutta-percha	and	inserted	 in	the	canal	after	coating	the	canal	with	a	sealer.	Subsequently,	the	portion	of	the	file	above	the	mark	was	removed	and	the	 excess	 gutta-percha	 condensed.	 In	 recent	 years,	 (Thermafil®)	 was	 developed,	and	 the	 metal	 core	 material	 was	 replaced	 with	 a	 plastic	 material.	 The	 clinical	outcomes	of	this	obturation	technique	are	comparable	to	other	techniques,	and	the	sealing	ability	is	satisfactory	with	less	time	consumed	for	the	application	(141).	It	is	an	effective	 technique	even	 in	curved	canals,	and	the	amount	of	core	material	and	gutta-percha	 in	 the	 final	 filling	by	using	 this	 technique	 is	 significantly	higher	 than	that	produced	by	both	lateral	compaction	and	warm	vertical	compaction	(142,	143,	144,	 145).	Other	 studies	 have	 shown	 that	 the	 success	 rate	 of	 the	 treatment	 using	Thermafil	 technique	 is	 comparable	 to	 CLC	 technique	 (143,	 146,	 147).	 However,	complete	removal	of	filling	material	from	the	root	canal	system	filled	with	Thermafil	is	difficult,	and	sometimes	impossible	(148).	Moreover,	the	filling	material	extrusion		 27	and	 the	 difficulty	 of	 post	 space	 preparation	 are	 also	 drawbacks	 of	 using	 this	technique	(149,	150).			In	2010,	GuttaCore	obturator	 (Dentsply	Tulsa	Dental	 Specialties,	Tulsa,	Oklahoma,	USA)	 was	 introduced.	 The	 GuttaCore	 obturator	 is	 made	 from	 proprietary	 cross-linked	 gutta-percha,	 which	 maintains	 its	 shape	 when	 heated,	 allows	 for	 the	 post	space	preparation	easier	than	the	Thermafil	obturator,	and	can	be	removed	easily	if	the	root	canal	retreatment	is	indicated	(151,	152).			In	both	GuttaCore	and	Thermafil	techniques,	the	prepared	canal	is	verified	by	a	size	verifier,	which	corresponds	to	the	obturator	size	and	taper.	The	size	verifier	is	used	to	 facilitate	 the	 insertion	 of	 the	 obturator	 into	 the	 root	 canal	 till	 it	 reaches	 the	working	 length.	A	paper	point	 is	used	 to	deliver	a	small	amount	of	 sealer	 into	 the	coronal	portion	of	the	canal.	The	obturator	is	warmed	in	a	special	oven	to	a	specific	temperature	 as	 recommended	by	 the	manufacture.	 Subsequently,	 the	 obturator	 is	removed	and	 introduced	directly	 into	 the	canal	with	a	continuous	pushing	motion	over	5	seconds	 interval	until	 it	reaches	the	working	 length.	The	carrier	 is	 then	cut	off	at	 the	orifice	 level	by	using	a	 sharp	endodontic	 spoon	excavator	or	 long	shank	friction	 grip	 1/2	 round	 carbide	bur	mounted	on	 a	 high-speed	handpiece.	Next,	 the	gutta-percha	 is	 condensed	 vertically	 to	 reduce	 the	 dimensional	 changes	 resulting	from	 the	 filling	material	 cooling	 (153).	A	 study	by	Li	et	al.	 (154)	 showed	 that	 the	quality	of	obturation	attained	by	GuttaCore	was	not	differed	from	that	achieved	by	WVC	 in	oval-shaped	canals.	Moreover,	 the	bacterial	 leakage	 that	 resulted	by	using	GuttaCore	 technique	was	 similar	 to	 teeth	 filled	with	 the	 EndoSeal	MTA	 technique		 28	(155).	 Further,	 another	 study	 reported	 that	 GuttaCore	 technique	 produced	homogenous	 fillings	with	a	 low	 incidence	of	voids	and	a	high	proportion	of	gutta-percha	 in	 comparison	 to	 lateral	 compaction	 and	 single-cone	 techniques	 (156).	 It	was	also	concluded	that	the	“push-out	bond	strength”,	which	is	the	strength	of	the	bond	 between	 the	 carrier	 material	 and	 gutta-percha	 around	 it,	 is	 higher	 in	GuttaCore	obturator	than	 in	Thermafil	obturator	(157).	As	no	technique	covers	all	the	 requirements	 of	 an	 optimal	 root	 filling,	 the	 practitioner	 must	 recognize	 the	advantages	and	limitations	of	each	technique,	and	select	the	appropriate	obturation	method	for	each	clinical	situation.		1.4			Micro-Computed	Tomography	Nielsen	et	al.	(158)	introduced	the	micro-CT	into	endodontic	research	in	1995.	It	is	a	“non-destructive	method”	to	visualize	the	tooth	morphology	and	root	canal	system	without	destroying	 the	 samples.	Micro-CT	 (Figure	1.5)	 can	be	used	 to	 analyze	 the	entire	length	of	the	tooth	voxel	by	voxel	and	collect	the	data	e.g.	for	calculating	the	volumes	and	relative	proportions	of	different	filling	materials	(159).	It	has	also	been	used	to	measure	changes	in	root	canal	morphology	after	instrumentation	(160,	161,	162).	Moreover,	it	has	been	used	to	calculate	the	hard	tissue	debris	left	in	the	root	canals	space	after	instrumentation	(163,	164),	to	evaluate	the	quality	of	obturation	(162,	 165,	 166),	 and	 to	 assess	 the	 quality	 of	 retreatment	 (167,	 168).	 In	micro-CT	technology,	the	sample	is	exposed	to	x-ray	radiation	while	rotating	360°	around	its	vertical	axis	producing	a	large	number	of	2D	images	that	are	reconstructed	to	create	a	 3D	 image	 of	 the	 sample.	Micro-CT	 images	 are	 based	on	 threshold	 values,	which		 29	help	 to	 distinguish	 a	 specific	 structure	 from	 adjacent	 structure	 or	 material.	 The	tooth	 enamel,	 dentin,	 cementum,	 and	 the	 empty/filled	 root	 canal	 space	 can	 be	identified	 using	 correct	 threshold	 values.	Micro-CT	 cannot	 be	 used	 in	vivo	 studies	due	 to	 the	 high	 radiation	 doses	 generated	 by	 this	method	 and	 because	 of	 limited	sample	space.	Micro-CT	scanning	is	expensive	and	the	scanning	of	each	sample	takes	a	long	time	(159).	In	 spite	 of	 the	 limitations	 mentioned	 above,	 micro-CT	 is	 a	 valuable	 method	 in	endodontic	research	because	of	its	accuracy	and	non-destructive	features	(169).			Figure	1.5	Micro-CT	Specimen	Scanner-Center	for	High-Throughput	Phenogenomics.	Courtesy	Phenogenomics.dentistry.ubc.ca.	http://www.phenogenomics.dentistry.ubc.ca/equipment/MicroCTSpecimenScanner/			 30	Chapter	2 :	Rationale	and	Hypothesis		The	goal	of	endodontic	treatment	is	to	remove	microorganisms,	necrotic	tissue	and	microbial	 by-products	 from	 the	 root	 canal	 system	 (170).	 A	 successful	 endodontic	treatment	can	be	achieved	by	a	 thorough	canal	debridement,	effective	disinfection	and	 high	 quality	 obturation	 of	 the	 canal	 space	 (171).	 Instrumentation	 cleans	 the	canal	mechanically	and	creates	space	for	irrigation,	disinfection	and	the	root	filling.	Root	canal	irrigation	has	an	important	role	in	chemical	cleaning	and	disinfection	of	the	root	canal.		The	minimum	 instrumentation	 size	 required	 for	 the	optimal	 root	 canal	 therapy	 is	still	 debatable	 (172).	 An	 in	 vitro	 study	 concluded	 that	 a	 #30	 file	 is	 the	minimum	instrumentation	size	needed	for	the	irrigating	solutions	to	penetrate	the	apical	third	of	 the	root	canal	(30).	Another	study	showed	that	bacterial	counts	decreased	with	increasing	 size	 of	 the	 apical	 file	 (173).	 In	 contrast,	 contemporary	 studies	 support	minimal	 root	 canal	 preparation	 in	 order	 to	 preserve	 the	 tooth	 structure	 and	increase	the	fracture	resistance	of	the	tooth	(32,	174).			Recently,	a	new	multisonic	ultracleaning	(GentleWave™	System)	for	the	cleaning	of	infected	 root	 canals	 chemically	 and	 mechanically	 has	 been	 developed.	 Several	studies	have	established	the	greater	ability	of	GW	in	obtaining	complete	cleanliness	in	minimally	 instrumented	canals	 than	other	methods	(73,	76,	175).	Sigurdsson	et	al.	(176)	in	a	prospective	clinical	study	also	concluded	that	patients	treated	with	the		 31	GW	 had	 a	 high	 cumulative	 successful	 healing	 rate	 of	 97.3%	 12-months	 after	 the	treatment.		No	study	so	 far	has	examined	the	quality	of	root	 filling	 in	minimally	 instrumented	root	canals	cleaned	with	the	GW	system.	Thus,	the	specific	aim	of	this	study	was	to	examine	 the	 root	 filling	 quality	 in	 canals	 obturated	 using	 each	 of	 the	 following	methods:	(1)	single	GP	cone	with	AH	Plus	sealer	(SC/AH+),	(2)	single	GP	cone	with	GuttaFlow	 sealer	 (SC/GF),	 and	 (3)	 GuttaCore	 with	 AH	 Plus	 sealer	 (GC/AH+)	techniques	after	minimal	root	canal	preparation	and	GW	cleaning.		The	 null	 hypothesis	 (H0)	 is:	 There	 is	 no	 difference	 in	 the	 quality	 of	 root	 fillings	amongst	the	three-obturation	techniques	in	minimally	instrumented	root	canals.														 32	Chapter	3 :	Materials	and	Methods	3.1			Tooth	Selection	and	Preparation	Thirty	human	permanent	mandibular	first	and	second	molars	(90	root	canals)	were	used	 in	 this	study.	A	stratified	sampling	was	 followed	 in	 this	study	by	distributing	the	same	number	of	mesial	and	distal	root	canals,	mandibular	1st	and	2nd	molars	and	isthmuses	into	the	experimental	groups	(Table	3.1).	The	sample	size	calculation	for	the	 current	 study	 was	 based	 on	 a	 pilot	 study	 with	 alpha-type	 error	 of	 0.05	 and	power	 of	 0.80.	 The	 means	 and	 standard	 deviations	 were	 entered	 into	 statistical	software	(G*Power	3.	1.	9.	2	for	Mac	OS	X;	Heinrich	Heine,	University	at	Dusseldorf)	(126)	 yielded	 a	 sample	 size	 of	 10.	 Teeth	 extracted	 for	 reasons	 not	 related	 to	 this	study,	were	collected	from	the	faculty	of	Dentistry	clinics	at	the	University	of	British	Columbia,	and	from	different	dental	offices	in	Vancouver	and	Surrey,	BC.				 SC/AH+	 SC/GF	 GC/AH+	No.	of	1st	mandibular	molars		8	 8	 8	No.	of	2nd	mandibular	molars		2	 2	 2	Molars	with	2	root	canals		2	 2	 2	Molars	with	3	root	canals		6	 6	 6	Molars	with	4	root	canals		2	 2	 2	No.	of	mesial	root	canals		18	 18	 18	No.	of	distal	root	canals		12	 12	 12	Isthmuses	distribution	 8	(6	mesial	+	2	distal)	 8	(6	mesial	+	2	distal)	 8	(6	mesial	+		2	distal)		Table	3.1	Stratified	sampling	method.			 33	After	extraction,	the	teeth	were	placed	in	0.01	%	NaOCl	solution	and	stored	at	room	temperature.	Exclusion	criteria	for	teeth	in	the	present	study	were:	severely	curved	root	 canals	 (>30	 degrees)	 according	 to	 Schneider	 (177),	 previously	 filled	 root	canals,	deep	caries	or	restoration	extending	down	to	the	root	canal	wall,	internal	or	external	 resorption,	 incompletely	 formed	 roots	 or	 apical	 root	 fracture,	 extremely	obstructed	 or	 calcified	 root	 canals,	 and	 wide	 (open)	 apical	 foramen.	 Teeth	 were	inspected	 thoroughly	 and	 radiographed	 mesio-distally	 and	 bucco-lingually	 using	digital	 radiography	 (Planmeca	 Intra,	 Helsinki,	 Finland)	 with	 size	 #2	 Scan	 X	Phosphor	 storage	 imaging	 plates	 (Air	 Techniques,	 Melville,	 NY).	 The	 radiographs	were	 analyzed	 using	 digital	 radiography	 imaging	 software	 (Planmeca	 Romexis,	Helsinki,	Finland)	to	determine	if	the	tooth	met	the	inclusion/exclusion	criteria.			Once	the	teeth	were	selected,	calculus	and	soft	tissue	debris	were	removed	from	the	outer	 surface	 using	 hand-scaling	 instruments.	 After	 gaining	 an	 access	 cavity	with	high	 speed	 bur	 under	 water	 cooling,	 endodontic	 hand	 K-files	 #8	 and	 #10	 were	inserted	 in	each	root	canal	and	advanced	1	mm	through	the	apical	 foramen	of	 the	selected	 samples.	 Teeth	 with	 canals	 that	 could	 not	 be	 negotiated	 with	 these	 two	sizes	of	endodontic	files	were	excluded	from	the	study.	Selected	teeth	were	assigned	random	sample	numbers	that	were	carved	into	the	root	surface	using	a	long	shank	friction	 grip	 ¼	 round	 carbide	 bur	 (Tulsa	 Dentsply)	 mounted	 on	 a	 high-speed	handpiece.			 34	3.2			Root	Canal	Instrumentation	The	 clinical	 procedures	 were	 performed	 under	 magnification	 using	 the	 dental	operating	microscope,	and	were	all	done	by	the	same	dentist	(A.Z.)	(Figure	3.1).	A	standard	access	 cavity	preparation	was	made	 in	 the	 teeth	and	 the	working	 length	was	determined	by	 inserting	size	10	stainless	steel	K	 file	 (Dentsply	Maillefer)	 into	the	root	canal	until	the	file	was	just	visible	at	the	apical	foramen,	and	subtracting	1	mm	 from	 this	 file	 length	measurement.	 The	 root	 canals	were	 cleaned	 and	 shaped	using	 K-type	 endodontic	 hand	 files	 #8,	 #10,	 #15,	 #20	 (Dentsply	 Tulsa	 Dental	Specialties)	sequentially.	Before	proceeding	 to	 the	 larger	 file	size,	 it	was	crucial	 to	ensure	 that	 each	 file	 fit	 in	 the	 canal	 loosely	 at	 the	 working	 length.	 Final	 canal	preparation	was	performed	with	a	 size	20/.04	Profile	Vortex	Blue	nickel-titanium	rotary	endodontic	file	(Dentsply	Tulsa	Dental	Specialties).	One	ml	of	6%	NaOCl	was	used	between	each	instrument	by	syringe-needle	irrigation	using	a	31-gauge	needle	tip	(178).	Apical	patency	was	maintained	by	using	a	#10	K-type	file	after	each	larger	file.			 35		Figure	3.1	Dental	surgical	microscope.		After	the	instrumentation	was	finished,	size	20/.045	verifier	(Figure	3.2)	was	tried	in	 the	 canal.	 In	 cases	where	 the	 verifier	 could	 not	 reach	 the	 established	working	length,	minor	apical	shaping	was	carried	out	using	the	same	size	verifier.	This	apical	canal	 enlargement	was	 performed	 to	 ensure	 that	 the	 filling	material	matches	 the	exact	apical	diameter	and	the	determined	working	length.	After	the	correct	working	length	was	marked	on	the	size	verifier	using	a	silicone	stopper,	the	verifier	was	used	in	 rotary	 clockwise	motion	with	 slight	 apical	pressure	 at	working	 length	until	 the		 36	instrument	became	loose	in	the	canal	and	could	be	rotated	180°	passively	inside	the	root	canal.																																																		Figure	3.2	A	size	verifier	for	the	corresponding	GuttaCore	obturator.		3.3			First	Micro-CT	Scan	of	the	Instrumented	Root	Canals			Fifteen	 randomly	 selected	 samples	were	 scanned	 using	 a	Micro-CT	 100	 (SCANCO	Medical	 AG,	 Bruettisellen,	 Switzerland)	 to	 measure	 the	 amount	 of	 dentin	 debris	caused	by	the	instrumentation	with	the	following	settings:		Isotropic	voxel	size	of	30	µm,	tube	current	of	200	µA,	energy	of	90	kVp,	integration	time	of	500	ms	per	projection	with	2X	frame	averaging	(1	sec	total	integration	time)	to	 minimize	 noise,	 and	 0.1	 mm	 Copper	 filter	 to	 minimize	 any	 beam	 hardening	artifact.	The	scanning	time	per	sample	was	3	h	and	30	min.		 37	3.4			Root	Canal	Irrigation	The	GentleWave™	system	(GW;	Sonendo	Inc.)	was	used	to	clean	all	30	samples.	The	GentleWave	 treatment	 instrument	 was	 applied	 onto	 the	 access	 opening	 of	 the	sample,	with	the	end	of	 the	nozzle	1	mm	above	the	pulp	chamber	 floor.	The	tip	of	the	 handpiece	 never	 touches	 the	 pulpal	 floor	 during	 the	 treatment	 procedures.	GentleWave	treatment	was	performed	with	3%	NaOCl	for	5	minutes,	distilled	water	for	 15	 seconds,	 8%	 EDTA	 for	 2	 minutes,	 and	 distilled	 water	 for	 15	 seconds	consequently	 for	 a	 total	 treatment	 time	 of	 7.5	 minutes	 as	 recommended	 by	 the	manufacturer	 (73)	 (Figure	 1.2).	 The	 irrigating	 solutions	 were	 released	 as	 a	 high	speed	flow	(45	ml/min)	onto	the	end	plate	of	the	nozzle	tip	and	further	into	the	pulp	chamber	and	root	canal	system	at	a	temperature	of	40°C.	During	operation	the	pulp	chamber	was	sealed	from	the	surrounding	working	area	to	prevent	the	dispersion	of	 the	 irrigating	 solutions.	 	 Excess	NaOCl,	water	 and	 EDTA	were	 sucked	 from	 the	pulp	chamber	through	small	holes	in	the	GentleWave	treatment	instruments.		3.5			Second	Micro-CT	Scan	of	the	Cleaned	Root	Canals	All	30	samples	were	scanned	using	a	Micro-CT	100	(SCANCO	Medical	AG)	with	the	same	settings	as	 in	 the	 first	 scan.	The	 settings	used	 in	 this	 study	were	 tested	 in	a	number	of	pilot	scans	to	optimize	the	quality	of	the	scanned	images.	The	data	was	analyzed	 using	 two	 software	 packages:	 Amira	 v.	 6.0	 (FEI	 Visualization	 Sciences	Group,	 SAS,	 Burlington,	 MA),	 and	 MeVisLab	 2.6	 OS	 X	 Edition	 (MeVis	 Medical	Solutions	AG,	Bremen,	Germany)	(179).			 38	Amira	 was	 used	 to	 create	 animations	 and	 images,	 while	 MeVisLab	 was	 used	 to	create	 images,	 and	 for	 matching	 and	 superimposing	 corresponding	 sample	 data	from	 the	 same	 tooth	 but	 from	 different	 scans,	 and	 to	 analyze	 data	 quantitatively	(Figure	 3.3).	 The	 image	 volumes	 of	 both	 scans	 for	 all	 samples	 were	 matched	 in	sagittal,	 coronal,	 and	 axial	 planes	 to	 facilitate	 visualization,	 identification,	 and	comparison	of	the	various	parameters	in	the	two	images	(Figure	3.4).					Figure	3.3	Matching	process	from	three	different	projections	using	the	MeVisLab	software.				 39		 																													A																																																																												B				Figure	 3.4	 (A)	 Visualization	 of	 reconstructed	 images	 show	 the	 overlapping	 of	 post-GW	 (red	 color	 2D	slice)	and	pre-GW	(grey	color	slice)	from	the	axial	section	of	the	same	sample,	(B)	matched	pre/post-GW	scans	of	the	same	sample.			The	data	of	 the	 first	and	second	scans	were	analyzed	 to	determine	 the	volumetric	percentage	of	debris	before	and	after	cleaning	with	 the	GW	system:	The	 following	equation	was	used	in	the	calculations	of	debris:	D%	=	!!!! 	X	100	D%	is	the	percentage	of	debris	volume	Z	is	the	volume	of	the	empty	canal	space	after	GW	cleaning.	Y	is	the	volume	of	the	empty	canal	space	before	GW	cleaning.	The	D%	was	measured	 for	 the	 entire	 canal	 volume	 and	 for	 the	 three	 canal	 levels	(coronal,	middle,	and	apical	thirds)	using	the	same	equation.		 40	3.6			Assignment	of	Teeth	The	 teeth	 used	 in	 the	 study	 (mandibular	 first	 and	 second	molars)	 had	 2	 –	 4	 root	canals.	 The	 number	 of	 root	 canals	 for	 each	 group	 was	 equalized	 (stratified	sampling)	to	avoid	the	effect	the	different	morphology	and	the	number	of	RCs	on	the	filling	quality.	The	teeth	were	then	randomly	divided	into	three	groups	according	to	the	filling	material	and	the	obturation	techniques.		3.7			Root	Canal	Obturation	After	instrumentation	the	canals	were	dried	using	matching	paper	points	(Dentsply	Tulsa	Dental	Specialties)	(Figure	3.6,	B).	Teeth	in	the	three	groups	were	filled	with	different	filling	materials	and	different	techniques.	Group	I	was	obturated	using	Single	GP	cone	with	GuttaFlow	sealer,	Group	II	was	filled	using	Single	GP	cone	with	AH	Plus	sealer,	and	Group	III	obturated	using	GuttaCore®	(GC)	(Dentsply	Tulsa	Dental	Specialties)	and	AH	Plus	sealer.		3.7.1			Group	I:	Single	GP	Cone	with	GuttaFlow	Sealer	A	Vortex	20/.04	gutta-percha	point	(Dentsply	Tulsa	Dental	Specialties)	was	used	in	canals	where	the	apical	size	was	20/.04	and	tug-back	was	observed	when	trying	the	cone.	Bigger.04	taper	GP	cones	were	used	when	canal	was	naturally	larger,	the	size	was	determined	by	inserting	GP	points	of	increasing	size	into	WL	until	the	correct	size	was	detected.		Larger	GP	points	were	used	in	the	distal	canals.	GuttaFlow®	2	-	Standard	Set	sealer	was	delivered	into	the	prepared	coronal	canal	from	the	5	ml		 41	Syringe	and	introduced	deeper	with	Size	#25	Lentulo	spiral	(Dentsply	Maillefer)	(Figure	3.5).					 				 		Figure	3.5	Lentulo	spiral.		The	Lentulo	 spiral	was	mounted	on	a	 contra-angle	 low	 speed	handpiece.	The	 two	thirds	of	the	Lentulo	was	coated	with	GuttaFlow	sealer	(Figure	3.6,	C),	and	inserted	into	the	canal	2	mm	short	of	the	working	length	(Figure	3.6,	D).	The	handpiece	was	operated	when	 the	Lentulo	was	 inside	 the	canal;	 the	Lentulo	was	 removed	slowly	out	of	the	canal	while	the	handpiece	was	still	working.	The	master	GP	was	inserted	and	pumped	twice	 inside	the	canal	 to	 insure	the	optimal	distribution	of	 the	sealer	inside	 the	 canal	 (Figure	 3.6,	 E).	 If	 necessary,	 additional	 amount	 of	 the	 sealer	was	added	 by	 using	 gutta-percha	 point	 (Figure	 3.6,	 F).	 Before	 the	 insertion	 of	master	cone,	 the	amount	of	 the	sealer	was	standardized	 for	all	 samples	by	 filling	 the	root	canal	with	a	sealer	up	to	1mm	short	the	canal	orifice	(Figure	3.6,	F).	The	apical	half	of	the	pre-fitted	master	cone	was	coated	with	sealer	and	introduced	into	the	canal	and	slowly	placed	to	working	length	(Figure	3.6,	G).	The	gutta-percha	point	was	cut		 42	using	a	hot	instrument	and	the	coronal	part	was	condensed	using	0.5,	0.6,	0.7,	and	0.8	 mm	 posterior	 Schilder	 plugger	 (Dentsply	 Maillefer,	 Ballaigues,	 Switzerland)	(Figure	3.6	H,	3.7).												 													 													 	A)	Pulp	chamber	after	GW	cleaning.	 B)	Close-up	image	of	pulp	chamber	and	canal	orifice	after	drying	of	the	canals.	C)	Lentulo	spiral	coated	with	GuttaFlow.	sealer.																			 D)	Sealer-coated	Lentulo	inside	the	distal	RC.			E)	Distribution	of	sealer	in	the	distal	RC.																						 F)	Filled	distal	canal	with	GF	sealer.			 43										 												 		Figure	3.6	Steps	of	root	canals	obturation	using	single-cone	technique	with	GuttaFlow	sealer.		3.7.2			Group	II:	Single	GP	Cone	with	AH	Plus	Sealer	A	Vortex	20/.04	master		gutta-percha	point	(Dentsply	Tulsa	Dental	Specialties)	was	used	 in	canals	where	 the	apical	size	was	20/.04	and	 tug-back	was	observed	when	trying	 the	 cone.	 Bigger	 .04	 taper	 GP	 cones	 were	 used	 when	 canal	 was	 naturally	larger,	 the	 size	was	 determined	 by	 inserting	GP	 points	 of	 increasing	 size	 into	WL	until	the	correct	size	was	detected.		Larger	GP	points	were	used	in	the	distal	canals.	AH	 Plus	 sealer	 (Dentsply	 Tulsa	Dental	 Specialties)	was	 used	 and	mixed	 following	manufacturer’s	 recommendation.	 The	 sealer	was	 inserted	 into	 the	 prepared	 canal	G)	Master	GP	inserted.	 H)	Condensation	with	a	Schilder	plugger.														I)	Mesial	root	canals	obturation.																																											 J)	Sealed	root	canals.			 44	by	 using	 size	 25	 Lentulo	 spiral.	 The	 Lentulo	 was	 used	 in	 a	 similar	 manner	 as	 in	group	I,	but	with	AH	Plus	sealer.	The	apical	half	of	 the	pre-fitted	master	cone	was	coated	 with	 the	 sealer	 and	 introduced	 into	 the	 canal	 and	 slowly	 introduced	 to	working	length.	The	gutta-percha	point	was	cut	at	the	coronal	end	of	the	root	canal	using	a	heated	instrument	and	compacted	using	0.5,	0.6,	0.7,	and	0.8	mm	posterior	Schilder	plugger	(Dentsply	Maillefer,	Ballaigues,	Switzerland)	(Figure	3.7).			Figure	3.7	Color-coded	Schilder	plugger	for	posterior	teeth.		3.7.3			Group	III:	GuttaCore	with	AH	Plus	Sealer	A	 size	 20	 GuttaCore	 Size	 Verifier	 (Dentsply	 Tulsa	 Dental	 Specialties)	was	 used	 to	verify	 passive	 fit	 at	 working	 length.	 A	 GuttaCore	 endodontic	 obturator	 that	corresponded	to	the	Size	Verifier	size	was	selected.	The	working	length	was	marked	on	 the	 obturator	 by	placing	 the	 silicon	 rubber	 stopper	 at	 the	working	 length	 that	coincides	 the	 size	 verifier’s	 calibration	 mark.	 A	 small	 amount	 of	 AH	 Plus	 sealer	(Dentsply	Tulsa	Dental	Specialties)	was	spread	into	the	coronal	third	of	the	canals	by	using	paper	point.	The	GuttaCore	obturator	was	heated	by	using	 the	GuttaCore		 45	oven	 (Dentsply	 Tulsa	 Dental	 Specialties)	 at	 the	 settings	 recommended	 by	 the	manufacturer.	 The	warm	obturator	was	 removed	 from	 the	 oven	 and	 immediately	placed	into	the	canal	pushing	smoothly	with	a	continuous	motion	over	a	period	of	5	seconds	until	the	desired	length	was	reached.	The	handle	was	bent	to	opposite	sides	to	 break	 it	 off	 (180),	 and	 the	 GP	 was	 condensed	 with	 0.5,	 0.6,	 0.7,	 and	 0.8	 mm	Schilder	plugger	slightly	below	the	pulp	chamber	floor.		The	samples	 from	all	 three	groups	were	stored	 in	an	 incubator	at	100%	humidity	and	37°C	 for	 five	 days	 to	 allow	 the	 sealers	 to	 completely	 set	 before	 the	micro-CT	scanning.		3.8			Third	Micro-CT	Scan	of	the	Teeth	After	Root	Filling		All	30	teeth	were	scanned	using	a	Micro-CT	100	(SCANCO	Medical	AG,	Bruettisellen,	Switzerland)	with	the	same	settings	of	the	first	and	second	scans.	A	resolution	of	30	µm	 (voxel	 size)	was	 chosen	 for	 the	 study	 as	 it	 has	 been	 used	 in	 several	 previous	studies	in	endodontics	and	is	sufficient	for	comparison	of	the	quality	of	root	fillings	with	different	materials	(175,	181,	182).							 46	3.8.1			Reconstruction	and	Analysis	of	the	Images		Voids	were	observed	both	in	the	2D	and	3D	images	within	the	region	of	interest.	The	apical	1	mm	from	the	WL	was	excluded	from	the	measurements	of	voids	as	in	many	teeth	 the	 root	 filling	 was	 slightly	 short	 and	 therefore	 this	 area	 was	 identified	 as	unfilled	 instead	of	 a	 large	 void.	 In	 other	words,	 voids	 in	 the	 root	 filling	 can	occur	only	in	that	area	of	the	root	canal	that	is	filled.	The	volume	of	filling	materials,	gaps,	and	 voids	was	 determined	 by	 analyzing	 the	 data	 from	 the	 second	 and	 third	 scan.	The	 difference	 between	 the	 volume	 of	 empty	 canal	 space	 and	 the	 volume	 of	 root	fillings	represented	the	volume	of	gaps	and	voids.	The	mean	volume	difference	was	calculated	for	each	group	and	these	values	were	compared	using	Kruskal-Wallis	and	Mann-Whitney	tests.	After	importing	the	data	to	MeVisLab	software	in	HDL	format,	the	root	canal	volume	was	divided	into	three	sections	of	equal	length	in	each	root	in	the	 apico-coronal	 direction	 using	 the	 Draw3DMacro	module.	 The	 filling	materials	and	 the	 empty	 canal	 space	 were	 segmented	 with	 RegionGrowingMacro	 module.	MeVisLab	is	a	software	program	in	which	a	lower	threshold	was	selected	to	include	the	 low	 grey	 scale	 values	 (empty	 canal	 space,	 gaps	 and	 voids),	 while	 an	 upper	threshold	 was	 adjusted	 to	 include	 the	 high	 grey	 scale	 value	material	 (root	 filling	materials).	 These	 threshold	 values	 were	 standardized	 when	 analyzing	 all	 the	samples	(Figure	3.8).		 47		Figure	 3.8	 Segmentation	 of	 root	 filling	 material	 using	 MeVisLab	 software.	 The	 highlighted	 area	represents	the	filling	materials	(GP	+	sealer).													 48	Chapter	4 :	Results		Two	and	three-dimensional	reconstructed	images	of	the	root	canal	filling	are	shown	from	 the	 coronal,	 axial	 and	 sagittal	 aspects	 in	 Figure	4.1.	Voids,	 sealer,	 and	 gutta-percha	were	 easily	 identified	with	 their	 different	 grey	 scale	 levels,	 except	 for	 the	GuttaFlow	group.	In	this	study,	the	volumes	of	the	filling	materials,	gaps,	and	voids	were	 measured,	 and	 the	 volumetric	 percentages	 were	 calculated.	 The	 calculated	percentages	were	compared	between	the	examined	groups	and	within	each	group	at	 three	 canal	 levels.	 Additionally,	 the	 volume	 of	 debris	 removed	 using	 the	GentleWave	system	was	measured	and	the	percentages	were	calculated.		4.1	Gaps	and	Voids	Analysis		The	data	 for	normal	distribution	and	 their	equality	of	variance	was	evaluated	and	proper	 statistical	 tests	 were	 chosen	 to	 analyze	 the	 volumetric	 percentages	difference	of	gaps	and	voids	between	groups	and	within	each	group	at	three	canal	levels.	The	data	did	not	display	normal	distribution	(Kolmogorov-Smirnov	test	and	Shapiro-Wilk	 test;	 p	 <	 0.05)	 or	 equality	 of	 variances	 (Levene’s-test;	 p	 <	 0.05).	Therefore,	 the	 data	 was	 analyzed	 with	 non-parametric	 tests.	 Kruskal-Wallis	 test	indicated	that	there	was	a	statistically	significant	difference	between	the	groups	(P	=	0.000).	Mann-Whitney	U-test	indicated	statistical	significances	between	SC/AH+	and	SC/GF	groups	 (P	=	0.000),	 and	between	SC/AH+	and	GC/AH+	groups	 (P	=	0.000).	However,	there	was	no	statistical	difference	between	SC/GF	and	GC/AH+	groups	(P	=	0.100).		 49				Figure	4.1	Reconstructed	2-D	(left)	and	3-D	(right)	images	for	a	sample	in	the	(SC/AH+)	group,	showing	the	distribution	of	gutta-percha,	sealer,	and	voids	inside	the	filled	root	canal	space.	(1	&	2)	axial	aspect,	(3	&	4)	coronal	aspect,	and	(5	&	6)	sagittal	aspect.					 50	The	means	and	 standard	deviations	 (SD)	of	 the	overall	proportion	 (%	of	 the	 total	canal	volume)	of	 filling	material,	gaps	&	voids	are	shown	in	Figure	4.2,	and	Tables	4.1,	 4.2.	 The	 GC/AH+	 (GuttaCore	 with	 AH	 Plus	 sealer)	 group	 showed	 the	 highest	percentage	of	 filling	materials	(98.79	%)	(Table	4.1),	and	the	 lowest	percentage	of	gaps	and	voids	(1.21	%)	(Table	4.2).	The	SC/AH+	(Single	cone	with	AH	Plus	sealer)	group	 had	 the	 lowest	 percentage	 of	 filling	 material	 (95.94	 %)	 (Table	 4.1),	 and	highest	percentage	of	gaps	&	voids	(4.06	%)	(Table	4.2).		 							 									 																																																Group	(n	=	10)	Overall	filling	volume	(%)	Means	and	SD		SC/AH+		95.94	±	3.32		SC/GF		98.66	±	0.93		GC/AH+		98.79	±	1.25			Table	4.1	The	proportion	(%	mean	±	SD)	of	the	root	canal	space	filled	with	the	three	root	filling	materials.		Group	(n	=	10)	Overall	gaps	and	voids	(%)	Means	and	SD		SC/AH+		4.06	±	3.32		SC/GF		1.34	±	0.93		GC/AH+		1.21	±	1.25		Table	4.2	The	proportion	(%	mean	±	SD)	of	gaps	&	voids	of	the	total	root	canal	volume.		 51		Figure	 4.2	 Mean	 volumes	 percentages	 of	 the	 filling	 materials	 within	 three	 groups	 (1)	 Single-cone	technique	 with	 AH	 Plus	 sealer	 (SC/AH+),	 Single-cone	 technique	 with	 GuttaFlow	 sealer	 (SC/GF),	 and	GuttaCore	technique	with	AH	Plus	sealer	(GC/AH+).	The	bars	represent	the	standard	deviations.																		 52	4.1.1	Comparison	of	Gaps	and	Voids	within	Each	Group	4.1.1.1	Comparison	of	Gaps	and	Voids	in	the	Coronal,	Middle	and	Apical	Thirds	of	the	Root	Canals	within	Each	Group		No	statistical	difference	was	found	between	the	coronal,	middle	and	apical	thirds	of	the	G&V	volumes	within	the	root	canals	obturated	with	the	SC/AH+	technique	(P	=	0.170)	(Figure	4.3).			Figure	4.3	Proportion	of	gaps	and	voids	(%	mean)	at	the	three	canal	levels	in	the	SC/AH+	group.									 53	The	 volumetric	 percentages	 of	 G&V	 that	 were	 detected	 in	 the	 apical	 third	 of	 the	multi-rooted	 canals	 after	 using	 the	 SC/GF	 technique	 were	 significantly	 different	from	that	detected	in	middle	and	coronal	thirds	within	the	same	group	(P	=	0.001,	P	=	0.000)	(Figure	4.4).				Figure	4.4	Proportion	of	gaps	and	voids	(%	mean)	at	the	three	canal	levels	in	the	SC/GF	group.											 54	The	statistical	analysis	of	the	proportions	of	gaps	and	voids	in	the	obturated	canals	using	GC/AH+	group	at	the	three	levels	showed	no	significant	difference	(P	=	0.753)	(Figure	4.5).				Figure	4.5	Proportion	of	gaps	and	voids	(%	mean)	at	the	three-canal	levels	in	the	GC/AH+	group.											 55	4.1.1.2	Comparison	of	Mesial	and	Distal	Gaps	and	Voids	within	Each	Group	The	overall	volume	of	gaps	and	voids	 in	mesial	and	distal	 canals	within	each	root	filling	group	were	compared	using	 the	Mann	Whitney	U-test.	The	 test	 revealed	no	statistical	difference	between	mesial	and	distal	canals	within	each	group	(SC/AH+,	SC/GF,	and	GC/AH+),	with	P	values	of	0.261,	0.433,	and	0.487,	respectively	(Figures	4.6,	4.7,	and	4.8).				Figure	4.6	Proportion	of	gaps	and	voids	(%	mean)	in	mesial	and	distal	root	canals	filled	with	the	SC/AH+	technique.		 56		Figure	4.7	Proportion	of	gaps	and	voids	(%	mean)	in	mesial	and	distal	root	canals	filled	with	the	SC/GF	technique.				Figure	4.8	Proportion	of	gaps	and	voids	(%	mean)	in	mesial	and	distal	root	canals	filled	with	the	GC/AH+	technique.		 57	4.1.2	Comparison	of	Gaps	and	Voids	between	Groups	4.1.2.1	Comparison	of	Coronal,	Middle	and	Apical	Gaps	and	Voids	between	the	Groups	Kruskal-Wallis	and	Mann-Whitney	U	tests	were	used	to	compare	the	total	volume	of	gaps	and	voids	in	the	coronal,	middle,	and	apical	thirds	of	the	canals	obturated	with	the	 three	 techniques.	 The	 coronal	 %G&V	 in	 canals	 filled	 by	 SC/AH+	 differed	statistically	 from	 the	 coronal	 %G&V	 found	 in	 canals	 obturated	 using	 SC/GF	 and	GC/AH+	 techniques	 (P	 =	 0.001,	 P	 =	 0.000).	 However,	 there	 was	 no	 statistical	difference	between	the	coronal	%G&V	between	the	SC/GF	and	GC/AH+	groups	(P	=	0.277)	(Figure	4.9).				Figure	4.9	Proportion	of	gaps	and	voids	(%	mean)	in	the	coronal	thirds	of	canals	obturated	with	either	SC/AH+,	SC/GF,	or	GC/AH+	technique.					 58	The	average	volume	of	gaps	and	voids	in	the	middle	thirds	of	the	SC/AH+	canals	was	five	 times	greater	 than	 in	 canals	 filled	with	 the	GC/AH+	 technique.	Kruskal-Wallis	test	showed	a	difference	between	the	three	groups	(P	=	0.000)	(Figure	4.10).			Figure	4.10	Proportion	of	gaps	and	voids	 (%	mean)	 found	 in	 the	middle	 third	of	 the	canals	obturated	with	either	SC/AH+,	SC/GF,	or	GC/AH+	technique.											 59	The	 canals	 filled	 using	 the	 SC/GF	 method	 presented	 the	 lowest	 total	 volume	 of	apical	 gaps	 and	 voids	 (0.68	 %),	 followed	 by	 GC/AH+	 (1.30	 %).	 On	 the	 contrary,	SC/AH+	group	accounted	for	the	highest	volume	of	apical	gaps	and	voids	(3.26	%)	(Figure	4.11).	There	was	a	statistically	significant	difference	between	the	apical	gaps	and	voids	between	the	SC/AH+	group	and	both	the	SC/GF	and	GC/AH+	groups	(P	=	0.000).	 No	 statistically	 significant	 difference	 was	 found	 between	 the	 SC/GF	 and	GC/AH+	groups	(P	=	0.429).				Figure	4.11	Proportion	of	gaps	and	voids	(%	mean)	found	in	the	apical	third	of	the	canals	obturated	with	either	SC/AH+,	SC/GF,	or	GC/AH+	technique.								 60	4.1.2.2	Comparison	the	Proportion	of	Gaps	and	Voids	in	the	Mesial	and	Distal	Canals	between	the	Three	Groups	The	overall	combined	volume	of	gaps	and	voids	in	the	mesial	canals	in	the	SC/AH+	group	 differed	 significantly	 from	 the	 mesial	 gaps	 and	 voids	 in	 the	 SC/GF	 and	GC/AH+	 groups	 (P	 =	 0.000).	 However,	 no	 statistically	 significant	 difference	 was	found	between	the	SC/GF	and	GC/AH+	groups		(P	=	0.734)	(Figure	4.12).				Figure	4.12	Proportion	of	gaps	and	voids	(%	mean)	in	the	mesial	root	canals	filled	by	the	SC/AH+,	SC/GF,	and	GC/AH+	technique.								 61	The	 total	 combined	 volume	 of	 gaps	 and	 voids	 in	 the	 distal	 canals	 of	 the	 SC/AH+	group	 differed	 significantly	 from	 the	 SC/GF	 and	 GC/AH+	 groups	 (P	 =	 0.000).	However,	 no	 statistically	 significant	 difference	 in	 distal	 canal	 gaps	 &	 voids	 was	found	between	the	SC/GF	and	GC/AH+	groups	(P	=	0.084)	(Figure	4.13).				Figure	4.13	Proportion	of	gaps	and	voids	(%	mean)	found	in	distal	canals	obturated	using	the	SC/AH+,	SC/GF,	and	GC/AH+	technique.					The	proportions	of	sealer	and	gutta-percha	in	the	root	canals	from	each	group	were	also	measured.	The	lowest	amount	of	sealer	and	the	highest	amount	of	gutta-percha	were	found	in	the	GC/AH+	group	(Figure	4.14).				 62		Figure	4.14	The	proportions	 (%)	of	sealer	and	gutta-percha	 in	root	canals	of	 three	samples	obturated	using	three	different	techniques;	SC/AH+	(first	row),	SC/GF	(second	row),	and	GC/AH+	(third	row).	A,	D	and	G	views	show	segmented	(sealer	+	GP)	inside	the	root	canals;	B,	E	and	H	views	show	segmented	GP	inside	the	root	canals;	and	C,	F	and	I	views	show	segmented	sealer	inside	the	root	canals.					 63	In	Summary:		Tables	4.3,	4.4,	and	Figures	4.15,	4.16	summarize	the	results	of	 the	proportions	of	the	root	filling	materials,	gaps	and	voids	in	the	different	groups.					 SC/AH+	 SC/GF	 GC/AH+		 Mesial	 Distal	 Mesial	 Distal	 Mesial	 Distal	Coronal	 96.07	±	3.44					94.53	±	5.02			98.47	±	0.86	 98.12	±	1.36		98.57	±	1.51	 	98.49	±	1.63	Middle	 96.09	±	2.76				95.51	±	2.97			98.69	±	1.34	 98.02	±	0.98	 99.03	±	0.71			99.28	±	0.47	Apical	 97.11	±	2.02					96.36	±	3.73			99.24	±	0.51	 99.41	±	0.51	 98.59	±	1.50	 	98.80	±	1.67		Table	4.3	The	proportions	(%	mean	±	SD)	of	the	filling	materials	in	the	mesial	and	distal	root	canals	obturated	using	the	three	techniques	(SC/AH+,	SC/GF,	and	GC/AH+),	at	three	canal	levels	(coronal,	middle,	and	apical	thirds).					Figure	4.15	The	proportion	(Means	±	SD)	of	filling	materials	in	mesial	and	distal	root	canals	obturated	using	 the	 three	 techniques	 (SC/AH+,	 SC/GF,	 and	 GC/AH+),	 at	 three	 canal	 levels	 (coronal,	middle,	 and	apical	thirds).											 64		 SC/AH+	 SC/GF	 GC/AH+		 Mesial	 Distal	 Mesial	 Distal	 Mesial	 Distal	Coronal	 3.93%	 5.47%	 1.53%	 1.88%		1.43%	 1.51%	Middle	 3.91%	 4.49%	 1.31%	 1.98%	 0.97%		0.72%	Apical	 2.89%	 3.64%	 0.76%	 0.59%	 1.41%	 1.20%		Table	4.4		The	proportion	(%	mean)	of	the	gaps	and	voids	in	mesial	and	distal	root	canals	obturated	using	the	three	techniques	(SC/AH+,	SC/GF,	and	GC/AH+),	at	three	canal	levels	(coronal,	middle,	and	apical	thirds).							Figure	4.16	The	proportion	(%	mean)	of	the	gaps	and	voids	in	mesial	and	distal	canals	obturated	using	the	 three	 techniques	 (SC/AH+,	 SC/GF,	 and	GC/AH+),	 at	 three	 canal	 levels	 (coronal,	middle,	 and	 apical	thirds).												-			2			4			6			8			10		Mesial	 Distal	 Mesial	 Distal	 Mesial	 Distal	SC/AH+													 SC/GF						 GC/AH+				%	volume	of	G&V	Mesial	and	distal	root	canals	filled	with	different	obturaJon	techniques	Coronal	Middle	Apical		 65	4.2	Debris	Removal	by	the	GentleWave	System	The	 debris	 volumes	 that	 were	 measured	 before	 the	 GW	 cleaning	 system	 are	presented	in	Table	4.5	and	Figure	4.17.	After	conventional	irrigation,	4.58	%	of	canal	volumes	were	filled	with	debris	while	no	debris	was	found	after	GW	cleaning.	The	apical	 third	 contained	 the	 highest	 %	 of	 debris	 volumes	 before	 the	 use	 of	 the	GentleWave™	system,	followed	by	middle,	and	coronal	thirds	of	the	root	canals.		 	Coronal		Middle		Apical		Total		3-D	Debris	%				3.83%	 		5.22%	 		5.83%	 	4.58%		Table	4.5	The	proportion	(%mean	volume)	of	debris	removed	by	the	GentleWave	system	at	the	three	canal	levels.		  	Figure	4.17	The	 amount	 (%	mean	volume)	of	 debris	 removed	by	using	 the	GentleWave	 system	at	 the	three	canal	levels.			-			1			2			3			4			5			6			7		Coronal	 Middle	 Apical	 Total	Debris	volume	(%)	Canal	level		 66	Chapter	5 :	Discussion		The	current	study	was	designed	to	compare	the	quality	of	root	filling	using	a	single-cone	 technique	 with	 either	 AH	 Plus	 or	 GuttaFlow	 sealers,	 and	 a	 thermoplastic	GuttaCore	 technique	with	AH	Plus	 sealer	 in	 teeth	 after	minimal	 canal	preparation	and	 the	 GentleWave	 cleaning.	 As	 the	 total	 volume	 of	 gaps,	 voids	 and	 the	 filling	materials	between	the	three	groups	was	statistically	significantly	different,	the	null	hypothesis	was	 rejected.	 	 Several	 studies	 have	 examined	 root	 canal	 debridement,	apical	pressure	created	during	irrigation,	and	tissue	dissolution	by	a	new	irrigation	method,	 the	GentleWave™	 system,	 in	minimally	 prepared	 canals	 (73,	 76,	 77).	 Our	study	was	 the	 first	 study	 to	 examine	 the	 quality	 of	 root	 filling	 in	 those	minimally	instrumented	 canals	 subjected	 to	 GW	 cleaning.	 The	 selection	 of	 the	 obturation	techniques	was	 a	 challenge	 since	 the	 final	 preparation	 size	 and	 taper	were	 small	(20/.04).	 	 Cold	 lateral	 compaction	 and	 warm	 vertical	 compaction	 were	 not	applicable	obturation	techniques	in	this	study	because	of	the	small	final	size	of	the	prepared	 canals.	 The	 single-cone	 technique	was	 selected	 to	 be	 used	 in	 this	 study	based	 on	 various	 studies	 that	 have	 shown	 comparable	 results	 in	 terms	 of	 sealing	ability	 between	 single-cone	 and	 both	 WVC	 and	 LC	 techniques	 (136,	 137).	 In	 the	current	study,	two	different	sealers	were	used,	AH	Plus	and	GuttaFlow	sealers.	The	sealers	 were	 selected	 based	 upon	 their	 potential	 properties	 for	 the	 filling	 of	minimally	 instrumented	 root	 canals	 (103,	 105,	 106,	 107,	 108).	 Both	 sealers	 are	widely	used	by	dentists	worldwide.	A	recent	study	showed	that	after	setting	of	AH+,	fibroblast	cells	were	able	to	grow	on	the	surface	of	the	sealer,	similar	to	bioceramic		 67	sealers	 (102).	 AH	Plus	 has	 a	 considerably	 low	 flowability,	 low	 film	 thickness,	 and	slight	shrinkage	upon	setting	in	comparison	to	other	sealers	(103).	GuttaFlow	sealer	expands	 upon	 setting	 (103)	 and	 is	 characterized	 by	 a	 thixotropic	 property.	 This	means	that	the	sealer	viscosity	reduces	under	pressure.	Hence,	the	sealer	flows	into	the	 small	 lateral	 canals	 and	exhibits	 a	 good	 sealing	 ability	 in	 comparison	 to	other	sealers	 (105,	 106,	 107,	 108).	 The	 GuttaCore	 technique	 was	 the	 other	 obturation	method	that	was	examined	in	this	study.	It	was	introduced	six	years	ago	as	a	further	development	of	the	Thermafil	system	and	has	been	used	since	by	many	as	a	method	of	root	canal	obturation.	However,	 it	has	not	been	used	in	minimally	instrumented	root	 canals.	 However,	 it	 has	 been	 proven	 that	 a	 filling	 with	 excellent	 ability	 in	sealing	 the	 prepared	 root	 canals	 and	 a	 homogenous	 filling	 could	 be	 achieved	 by	using	GuttaCore	technique	(156).	Based	on	the	above-mentioned	factors,	 the	three	filling	techniques/materials	were	selected	for	the	present	study.	It	would	have	been	possible	 to	 instrument	 the	 canals	 even	 smaller,	 to	 size	 15/.04,	 however,	 the	GuttaCore	obturator	is	not	available	in	size	15,	the	smallest	available	size		is	20/.04	(180).	Therefore,	the	apical	master	preparation	that	used	in	the	current	project	was	20/.04.		In	our	study,	the	confounding	factors	that	may	affect	the	incidence	of	gaps	and	voids	within	 the	 filling	 materials	 between	 the	 groups	 were	 minimized.	 The	 anatomical	configuration	 of	 the	 root	 canal	 system	 such	 as	 isthmus	 area	 might	 contribute	 to	creating	a	difference	in	the	distribution	of	gaps	and	voids.	Therefore,	the	stratified	sampling	was	followed	in	this	study	by	distributing	the	same	number	of	mesial	and	distal	root	canals	that	have	isthmuses	amongst	the	experimental	groups.	Regarding		 68	the	quality	of	the	canal	preparation	that	could	be	another	factor	affecting	the	fillings	quality,	 all	 samples	 were	 prepared	 by	 one	 operator	 (A.	 Z)	 following	 the	 same	standardized	procedures.	In	addition,	the	consistency	and	the	volume	of	the	sealer	are	important	issues	that	should	have	been	controlled	and	standardized	amongst	all	samples.	Hence,	many	pilot	 studies	were	done	 to	develop	a	method	 for	delivering	the	sealer	into	the	canals	in	a	way	that	allows	for	a	complete	fill	of	the	canal	space	with	 the	 sealer.	Therefore,	 a	 Lentulo	 spiral	was	used	 to	deliver	 the	 sealer	 in	both	SC/AH+	and	SC/GF	groups	into	the	root	canals	until	the	sealer	filled	the	canal	spaces	up	 to	 1	 mm	 below	 the	 canals	 orifices.	 This	 method	 was	 followed	 to	 ensure	 the	optimal	delivery	of	the	sealer	 into	the	root	canals	reaching	the	apical	area	and	the	lateral	canals	as	shown	in	Figure	3.6.	Although	the	amount	of	sealer	was	not	equal	within	all	root	canals	especially	those	that	differ	morphologically,	it	was	possible	to	optimally	fill	the	root	canals	and	their	ramifications	with	a	sealer	by	using	a	Lentulo	spiral.	The	obturation	procedures	were	also	carried	out	by	one	practitioner	(A.	Z).		In	 comparison	with	other	 studies	 that	 used	decoronated	 teeth	 (76,	 151,	 165),	 the	crowns	of	 the	samples	 in	 the	present	study	were	maintained	to	simulate	a	clinical	scenario	within	which	the	clinical	crown	of	the	tooth	is	usually	present.	Moreover,	the	access	cavity	was	prepared	conservatively	to	maintain	the	tooth	structure,	and	allow	 the	 placement	 of	 the	 GentleWave	 handpiece	 within	 the	 pulp	 chamber	 and	allow	its	proper	stabilization.		Multiple	 methods	 have	 been	 used	 in	 the	 literature	 to	 evaluate	 the	 quality	 of	 the	obturation	technique	and	the	root	fillings.	Radiographs	allow	a	 limited	assessment		 69	of	the	filled	canal	contents	by	giving	a	two-dimensional	view	of	a	three-dimensional	area	(183).	The	invasive	method	of	sectioning	the	roots	with	filled	root	canals	and	further	viewing	or	scanning	with	a	stereomicroscope	or	SEM	is	also	used	to	assess	the	fillings	quality.	A	loss	of	some	parts	of	the	filling	materials,	which	may	be	miss-interpreted	as	a	gap	or	a	void,	could	be	caused	by	such	invasive	methods.	Clearing	or	demineralization	 technique	 is	 time-consuming	 (184),	 and	 the	 long	 immersion	 in	alcohol	may	 affect	 the	 flowability	 of	 gutta-percha	 and	 its	 ability	 to	 be	 compacted.	Moreover,	 the	 tooth	 becomes	 weak	 against	 the	 forces	 of	 compaction,	 which	 may	lead	 to	 its	 fracture	 during	 obturation	 procedures	 (185).	 Recently,	 a	 non-invasive	(micro-CT)	method	has	gained	popularity	as	a	research	tool	in	various	applications	in	 dental	 research	 (158,	 159,	 160,	 161,	 162).	 In	 endodontics,	 micro-CT	 has	 been	used	 in	 studying	 the	 root	 canal	 anatomy	 and	 morphology	 before	 and	 after	instrumentation,	measuring	dentin	debris,	and	assessing	the	quality	of	root	fillings	done	using	different	filling	materials	and	techniques	(158,	159,	160,	161,	163).	It	is	a	relatively	rapid	method	that	provides	highly	accurate	3D	images	of	the	root	fillings	and	 voids.	 However,	 this	 technology	 is	 expensive,	 may	 not	 be	 available	 for	 all	institutions,	and	cannot	be	used	in-vivo	due	to	the	high	dose	of	radiation	utilized	by	this	technique.	In	the	present	study,	micro-CT	was	used	as	a	method	to	detect	gaps	and	voids	within	 the	 filled	 root	 canals,	 calculate	 their	 relative	proportions,	 and	 to	measure	 the	 volume	 of	 debris	 removed	 by	 cleaning	 the	 root	 canals	 with	 the	GentleWave™	system.		Despite	using	the	same	filling	method	for	the	two	single-cone	groups,	SC/AH+	and	SC/GF,	 in	 our	 study,	 the	 results	 revealed	 a	 statistically	 significant	 difference		 70	between	the	groups.	This	might	be	explained	by	the	findings	of	a	recent	study	(103)	that	showed	that	GuttaFlow	expands	upon	setting,	which	could	result	in	a	decrease	in	the	incidence	and	volume	of	gaps	and	voids	in	the	filled	canals.	In	contrast	to	AH	Plus	sealer	that	shrinks	upon	setting	possibly	increasing	the	possibility	of	formation	of	 gaps	 and	 voids.	 Another	 reason	 for	 the	 frequent	 presence	 of	 gaps	 and	 voids	 in	canals	 filled	 with	 SC/AH+	 may	 be	 the	 higher	 solubility	 of	 AH	 Plus	 sealer	 in	comparison	 to	 GuttaFlow	 sealer	 (103,	 186).	 A	 thixotropic	 property	 of	 GuttaFlow	facilitates	 sealer	 penetration	 in	 narrow	 canals	 and	 its	 ramifications,	 as	 the	 sealer	becomes	less	viscous	under	pressure.	In	support	of	our	results,	several	studies	have	shown	that	the	sealing	ability	of	GuttaFlow	sealer	was	significantly	higher	than	that	of	 AH	 Plus	 sealer	 with	 either	 single-cone	 or	 lateral	 compaction	 techniques	 (105,	106,	108).				Our	study	revealed	a	statistically	significant	difference	between	the	GC/AH+	group	and	the	SC/AH+	in	the	quality	of	filling	and	the	incidence	of	gaps	and	voids.	The	root	canals	 of	 these	 two	 groups	 were	 filled	 using	 two	 different	 obturation	 techniques	(GuttaCore	and	Single-cone)	with	the	same	sealer	(AH	Plus).	Similar	 findings	were	detected	 in	 a	 previous	 study	 when	 an	 invasive	 method	 together	 with	 a	 digital	stereomicroscope	 were	 used	 to	 compare	 gutta-percha	 filled	 area	 (PGFA),	 sealer	filled	area	(PSFA)	and	voids	in	root	canals	after	filling	using	GuttaCore,	single-cone	and	lateral	compaction	techniques	(156).	It	was	concluded	from	that	study	that	the	root	 filling	material	was	very	homogenous	 in	 the	GuttaCore	 filled	root	canals	with	high	 PGFA	 and	 low	 occurrence	 of	 voids.	 As	 to	 the	 dimensional	 stability	 of	 gutta-percha	 after	 setting,	 the	 high	 proportion	 of	 gutta-percha	 in	 the	 filling	 material		 71	maximizes	the	quality	of	fillings.	Many	sealers	shrink	upon	setting	and	may	become	more	soluble	in	tissue	fluids	over	time	(187,	188).	Therefore,	minimizing	the	sealer	amount	 has	 been	 thought	 to	 result	 in	 better	 filling	 quality.	 Somma	 et	 al.	 (189)	compared	 the	 carrier-based,	 system	B,	 and	 single-point	 techniques	 in	 conjunction	with	AH	Plus	sealer	using	micro-CT	and	found	similar	results	to	our	study.	Although	they	 did	 not	 find	 a	 statistically	 significant	 difference	 between	 the	 groups,	 the	carrier-based	 filling	 technique	had	higher	 percentage	 of	 fillings	 and	 less	 gaps	 and	voids	than	the	single-cone	technique.		In	 the	 present	 study,	 although	 the	 results	were	 not	 different	 between	 SC/GF	 and	GC/AH+	groups,	 the	GuttaCore	method	exhibited	 the	 lowest	 incidence	of	gaps	and	voids.	 A	 recent	 study	 that	 compared	 the	 bacterial	 leakage	 between	GuttaCore/AH	Plus	 sealer	 (GC),	 and	 single-cone/GuttaFlow	 sealer	 (GF)	 in	 teeth	with	 single	 root	canals	 using	 confocal	 laser	 scanning	 microscopy	 found	 similar	 findings	 to	 our	results	(155).	It	was	concluded	from	that	study	that	GC	group	showed	less	bacterial	leakage	than	the	GF	group.	Compared	to	SC/GF	method,	GC/AH	Plus	method:	1/	can	be	applied	easily		2/	produces	a	homogenous	filling	material	(156)	3/	utilizes	little	amount	of	sealer	(156)	4/	 can	 be	 easily	 removed,	 if	 necessary,	 in	 less	 time	 in	 comparison	 to	 other	obturation	techniques	(152,	154).	Therefore,	GuttaCore	technique	might	be	the	best	technique	 to	 fill	 the	 minimally	 instrumented	 root	 canals	 compared	 with	 other	obturation	techniques	that	were	examined	in	this	study.			 72	In	 a	 previously	 study	 (156),	 the	 evaluation	 of	 the	 PGFA	 and	 PSFA	 areas	 and	 the	incidence	of	voids	were	performed	at	2	mm,	4	mm,	6	mm,	and	8	mm	from	the	apex.	Similarly,	 in	 the	 present	work,	we	 divided	 the	 root	 canals	 into	 three	 equal	 thirds	(coronal,	middle,	apical),	and	measured	the	%G&V	and	filling	materials	volumes	in	each	third,	and	compared	the	volumes	amongst	 the	different	 thirds.	The	results	of	these	two	studies	are	similar	in	terms	of	canal	levels	where	the	coronal,	middle,	and	apical	voids	in	root	canals	filled	with	SC/AH+	were	higher	than	when	GC/AH+	was	used.	This	can	be	interpreted	by	the	increase	of	sealer	amount,	and	the	decrease	of	GP	 quantity	 at	 different	 levels	 of	 the	 canals	 obturated	 using	 the	 single-cone	technique.	The	 incidence	of	 coronal	gaps	and	voids	was	higher	 than	 in	 the	middle	and	 apical	 thirds	 in	 all	 groups	 in	 our	 study.	 In	 addition,	 the	 apical	 thirds	 were	associated	with	 lower	 percentages	 of	 gaps	 and	 voids	 in	 all	 groups	 except	 for	 the	GC/AH+	 group,	 where	 the	middle	 thirds	 had	 the	 lowest	 percentages	 of	 gaps	 and	voids.	Uneven	coating	of	the	GuttaCore	points	has	been	indicated	to	cause	pushing	of	gutta-percha	to	one	side,	which	can	lead	to	uncovering	of	the	core	from	the	gutta-percha	 and	make	 the	 carrier	 core	 “naked”	 especially	 in	 the	 apical	 third	 (93,	 190,	191).		In	the	present	study,	the	micro-CT	was	used	also	to	measure	the	debris	volume	that	was	 removed	 by	 the	 GentleWave	 system.	 The	 findings	 confirmed	 the	 high	effectiveness	of	the	device	in	cleaning	the	root	canal	system.	After	instrumentation	and	 conventional	 irrigation,	 an	 average	of	 4.58	%	of	 canal	 volume	was	 filled	with	debris.	The	most	debris	was	detected	in	the	apical	thirds	of	the	canals,	followed	by	middle	and	coronal	thirds,	while	no	debris	was	found	after	GW	cleaning.		 73	The	 cleaning	 of	 infected	 root	 canals	 by	 the	 elimination	 of	 bacteria,	 removal	 of	necrotic	 pulp	 tissue,	 killing	 of	 planktonic	 and	 biofilms	microorganisms,	 removing	the	smear	 layer,	and	dissolving	 the	organic	and	 inorganic	 tissues	are	goals	of	 root	canal	irrigation	(192,	193,	194).	The	anatomical	complexity	of	the	root	canal	system	and	 the	 inaccessibility	 to	 the	 entire	 endodontic	 space	 prevent	 the	 completely	cleaning	by	instruments	and	irrigants	alone	(44).	Therefore,	there	was	a	necessity	to	use	 methods	 to	 agitate	 the	 irrigating	 solutions.	 Several	 previous	 studies	 have	measured	the	effectiveness	of	different	irrigation	systems	and	irrigating	solutions	in	root	 canal	 debridement	 (195,	 196,	 197).	 All	 methods	 have	 their	 limitations	 in	different	clinical	situations.		Various	 assessment	methods	have	been	used	 to	 evaluate	 the	 effectiveness	 of	 root	canal	debridement.	A	microscope	at	 a	 specific	magnification	with	a	digital	 camera	has	been	used	in	some	studies	(198,	199).	The	debris	amount	was	scored	in	a	way	that	the	higher	the	score,	the	larger	the	debris	amount	(198,	199).	However,	the	low	sensitivity	 is	 one	of	 the	drawbacks	 of	 this	method	 in	 comparison	 to	 the	micro-CT	method.	Scanning	electron	microscopy	is	also	used	after	sectioning	the	root	canals	in	 order	 to	 establish	 the	 quality	 of	 cleaning	 and	 the	 quantity	 of	 remaining	 dentin	debris,	and	to	compare	the	effectiveness	of	different	irrigation	techniques	(198,	200,	201,	202).	These	are	 invasive	methods	 in	which	 the	 sample	 can	be	used	only	one	time	 to	 evaluate	 one	 irrigation	 technique.	 In	 contrast,	 when	 a	 non-invasive	technique	 such	 as	 micro-CT	 is	 used,	 the	 sample	 can	 be	 examined	 and	 analyzed	multiple	times	at	various	stages	of	the	treatment.			 74	In	a	 recent	 study	 that	aimed	 to	compare	 the	debridement	efficacy	of	 conventional	syringe-needle	 irrigation,	 and	 GW	 system	 after	 minimal	 canal	 preparation,	histological	 sections	 of	 the	 teeth	 were	 examined	 to	 compare	 the	 debridement	efficacy	 of	 the	 two	 irrigation	methods.	 After	 irrigation,	 each	 canal	was	 filled	with	buffered	formalin	in	order	to	fix	the	pulpal	tissue	and	processed	following	standard	procedures	for	tooth	histological	specimens	(76).	The	GW	system	had	significantly	greater	 capacity	 in	 the	 cleaning	 of	 the	 root	 canals	 than	 syringe-needle	 irrigation	method	 (76).	 In	 our	 study,	 the	 observation	 of	 complete	 removal	 of	 dentin	 debris	also	points	toward	the	effectiveness	of	the	GW	system	to	completely	remove	tissue	remnants	(such	as	dentin	debris),	which	may	be	important	in	facilitating	the	filling	of	hard-to-reach	areas	in	the	canal	system	with	either	sealer	of	warm	gutta-percha.	Micro-CT	scanning	has	also	been	used	by	others	to	compare	the	efficacy	of	various	irrigation	methods	in	removing	debris.	Paqué	et	al.	(163)	established	that	micro-CT	is	 an	 appropriate	 method	 for	 dentin	 debris	 assessment,	 and	 can	 be	 used	 for	calculating	 the	 dentin	 debris	 quantitatively.	 Additionally,	 the	 three-dimensional	reconstructed	 images	 of	 micro-CT	 scans	 enhance	 the	 qualitative	 observation	 of	dentin	 debris	 (163).	 The	 technique	 of	measuring	 the	 debris	 in	 our	 study	was	 the	same	 as	 the	 one	 used	 by	 Paqué,	 but	with	 different	 resolution	 (203).	 Both	 studies	used	mandibular	first	and	second	molars	because	it	has	been	shown	that	persistent	apical	 periodontitis	 occurs	most	 frequently	 in	molars	 (204,	 205).	 The	 anatomical	complexity	of	molar	root	canal	system	that	makes	complete	removal	of	biofilms	and	debris	 difficult	 is	 believed	 to	 be	 one	 of	 the	 most	 important	 factors	 of	 persistent	endodontic	 infections	 (206).	Paqué	et	al.	 (203)	 scanned	 the	 specimens	 four	 times,	before	 instrumentation	 and	 after	 applying	 different	 irrigating	 solutions.	 After		 75	registration	 of	 the	 outer	 contours	 of	 both	 scans,	 “Hard	 tissue	debris	was	 identified	and	calculated	as	follows:	voxels	that	were	identified	as	soft	tissue,	liquid,	or	air	(canal	volume)	in	the	preoperative	scan	but	then	were	filled	with	radiopaque	material	in	the	postoperative	scan	were	assumed	to	be	filled	with	hard	tissue	debris”	(203).	The	study	by	 Paqué	 et	 al.	 has	 some	 limitations;	 it	 was	 impossible	 to	 calculate	 the	 dentine	debris	that	accumulated	on	the	canal	walls.	However,	in	our	study,	we	were	able	to	calculate	 the	 dentin	 debris	 all	 through	 the	 canal	 because	we	were	 comparing	 the	canal	 spaces	 of	 pre-GW	 scans	 (after	 instrumentation)	 versus	 the	 post-GW	 scans.	Thus,	 no	 dimensional	 changes	 have	 occurred	 in	 the	 canal	 space	 except	 for	 the	missing	 of	 debris	 voxels	 after	 using	 the	 GW	 system.	 Therefore,	 the	 difference	between	 the	empty	canals	volumes	of	both	scans	was	considered	as	dentin	debris	volume	that	was	removed	by	using	the	GW	system.	In	the	present	study,	the	dentin	debris	volume	was	measured	at	three	different	canal	levels	in	order	to	identify	the	most	likely	areas	to	be	filled	with	dentin	debris.	We	found	that	although	the	apical	area	of	 the	root	canal	 is	 the	most	difficult	part	 to	reach	and	clean	by	conventional	needle	irrigation,	dentin	debris	was	completely	removed	also	in	the	apical	canal	by	the	GW	cleaning.		The	 limitation	 of	 this	 study	 is	 that	micro-CT	 can	 only	 be	 used	 to	 detect	 the	 hard	tissue	 debris	 while	 the	 remaining	 soft	 tissue	 is	 not	 detectable	 by	 this	 method	 of	assessment	(163).	In	addition,	the	resolution	used,	30	μm,	does	not	allow	detection	of	very	thin	layers	of	sealer	or	smallest	amounts	of	dentin	debris.	On	the	other	hand,	higher	resolution	takes	much	longer	time	to	scan	the	specimen,	is	expensive,	and	the	resulting	 amount	 of	 image	 data	 is	 challenging	 to	 handle	 without	 very	 strong		 76	processing	power.	It	is	possible	that	some	areas	identified	as	gaps	may	in	fact	have	been	voids,	but	the	sealer	at	the	area	of	the	void	next	to	canal	wall	dentin	may	have	been	too	thin	to	be	detected	in	the	micro-CT	scan.	However,	such	errors,	if	present	would	 not	 change	 the	 combined	 volume	 of	 gaps	 and	 voids.	 The	 use	 of	 30	 µm	resolution	may	be	not	an	optimal	voxel	 size	 to	detect	 the	dentin	debris.	However,	the	 results	 showed	 excellent	 filling	 of	 isthmus	 areas	 in	most	 cases,	 which	 clearly	indicates	 that	GW	effectively	 removed	 the	 debris	 and	 thereby	made	 it	 possible	 to	completely	filled	the	root	canals.	 	Despite	its	potential	shortcomings	the	resolution	of	 30	 µm	 seems	 good	 enough	 to	 compare	 the	 quality	 of	 the	 three	 different	 root	filling	methods	 that	were	used	 in	 this	study.	The	same	micro-CT	resolution	 is	also	used	in	many	instrumentation	studies	(175,	181,	182).	              	 77	Chapter	6 :	Conclusion		The	overall	proportion	(%)	of	gaps,	voids	and	the	filling	materials	in	the	canal	space	was	 statistically	 significantly	 different	 between	 the	 three	 groups.	 Thus,	 the	 null	hypothesis	that	there	would	be	no	difference	in	the	quality	of	root	fillings	amongst	single	 point	 technique	with	AH	Plus	 sealer	 (SC/AH+),	 single	 point	 technique	with	GuttaFlow	sealer	(SC/GF),	and	GuttaCore®	technique	with	AH	Plus	sealer	(GC/AH+)	was	rejected.			The	study	outcome	indicates	that	a	high-quality	obturation	can	be	done	in	minimally	prepared	root	canals	by	the	GC/AH+	and	SC/GF	methods.	However,	the	root	canals	obturated	with	GuttaCore	 technique	had	 the	 lowest	 incidence	and	 total	 volume	of	gaps	&	voids	and	the	highest	proportion	of	gutta-percha.	GuttaCore	exhibited	a	good	adaptability	 and	 suitability	 to	 fill	 minimally	 instrumented	 root	 canals	 after	GentleWave	 cleaning.	 Therefore,	 of	 the	 three	 techniques	 examined	 GuttaCore	technique	 might	 be	 the	 best	 technique	 to	 fill	 the	 minimally	 instrumented	 root	canals.		For	an	optimal	root	canal	filling,	the	GentleWave	system	makes	it	possible	to	fill	the	entire	root	canal	space	due	to	its	high	efficacy	to	remove	all	dentin	debris,	also	from	the	apical	third,	and	completely	clean	minimally	instrumented	root	canals.			This	study	was	the	first	to	examine	the	quality	of	fillings	in	minimally	instrumented	root	 canals	 after	 GW	 cleaning.	 It	 was	 carried	 out	 in	 conditions	 similar	 to	 clinical		 78	situation.	Therefore,	in	vivo,	the	root	fillings	in	minimally	instrumented,	GW-cleaned	and	obturated	 root	 canals	by	 two	of	 the	 three	 root	 filling	 techniques	 examined	 in	this	 study	can	be	expected	 to	have	comparable	quality	 to	 the	 fillings	 in	our	study.	Future	 studies	 should	 investigate	 the	 quality	 of	 root	 fillings	 in	 minimally	instrumented	 root	 canals	 using	 several	 different	 sealers,	 including	 bioceramic	sealers.	Further,	the	quality	of	root	filling	in	uninstrumented,	GW	cleaned	canals	of	different	size	should	also	be	studied.																					 79	References		 1. Kakehashi	S,	Stanley	HR,	Fitzgerald	RJ.	The	effects	of	surgical	exposures	of	dental	 pulps	 in	 germ-free	 and	 conventional	 laboratory	 rats.	 Oral	 Surg	Oral	Med	Oral	Pathol	1965;20:340-9.			 2. Nair	 PN.	 Pathogenesis	 of	 apical	 periodontitis	 and	 the	 causes	 of	endodontic	failures.	Crit	Rev	Oral	Biol	Med	2004;15:348-81.			 3. Baumgartner	 JC,	 Falkler	 WA	 Jr,	 Beckerman	 T.	 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Oral	Surg	Oral	Med	Oral	Pathol	Oral	Radiol	Endod	2005;99:231–52.										 101	Appendices:	Appendix	A	–	Research	Plan											Figure	A.1	Research	plan.	 		Root	canals	of	30	mandibular	1st	&	2nd	molars	were	shaped	and	cleaned	to	Size	#20/.04	Group	1	(n=10)	Single-cone	technique	+	AH	Plus	sealer	(SC/AH+)	 Group	2	(n=10)	Single-cone	technique	+	GuttaFlow	sealer	(SC/GF)	 Group	3	(n=10)	GuttaCore	technique	+	AH	Plus	sealer	(GC/AH+)	A	third	micro-CT	scan	of	the	teeth	was	carried	out,	and	the	reconstructed	images	were	analyzed	for	the	volumetric	percentage	of	gaps	and	voids	at	three	different	canal	levels	(coronal,	middle,	apical).	Root	canals	of	all	30	samples	were	cleaned	using	the	GentleWave	system	and	scanned	for	2nd	time		15	samples	scanned	using	micro-CT		 102	Appendix	B	–	Micro-CT	Settings						Figure	B.1	Micro-CT	settings.												 103	Appendix	C	–	Normal	Distribution	Tests	    							Table	C.2	Levene	statistics	of	data	distribution	normality.	     	Tests	of	Normality		 Groups	 Kolmogorov-Smirnova	 Shapiro-Wilk		 Statistic	 df	 Sig.	 Statistic	 df	 Sig.	Volumes_values	 Single	cone	+	AH	Plus	 .221	 60	 .000	 .796	 60	 .000	Single	cone	+	GuttaFlow	 .165	 60	 .000	 .876	 60	 .000	GuttaCore	+			AH	Plus	 .181	 60	 .000	 .768	 60	 .000	a. Lilliefors	Significance	Correction		Table	C.1	Kolmogorov-Smirnov,	Shapiro-Wilk	tests	of	data	distribution	normality.								 	Test	of	Homogeneity	of	Variances	Volumes_values			Levene	Statistic	 df1	 df2	 Sig.	14.517	 2	 177	 .000		 104	Appendix	D	–	Total	Gaps	and	Voids	Data	Analysis	Using	Kruskal-Wallis	Test	  Ranks		 Groups	 N	 Mean	Rank	Volumes_values	 	Single	cone	+	AH	Plus	 60	 130.87		Single	cone	+	GuttaFlow	 60	 76.08		GuttaCore	+	AH	Plus	 60	 64.55		Total	 180	 	Table	D.1	Total	mean	rank	of	the	gaps	and	voids	volumes	in	the	three	different	groups	(SC/AH+,	SC/GF,	and	GC/AH+).				            							Table	D.2	Non-parametric	statistical	test	(Kruskal	Wallis	Test)	compares	SC/AH+,	SC/GF,	and	GC/AH+	groups.									Test	Statisticsa,b		 Volumes_values	Chi-Square	 55.485	df	 2	Asymp.	Sig.	 .000	a.	Kruskal	Wallis	Test	b.	Grouping	Variable:	Groups		 105	Appendix	E	–	Statistical	Analysis	of	Gaps	and	Voids	within	and	between	the	Three	Tested	Groups	at	Three	Canal	Levels				 Table	E.1	Statistical	analysis	of	gaps	and	voids	within	and	between	the	three	different	groups	at	three	canal	levels:	SC/AH+		=	Single-cone	technique	with	AH	Plus	sealer	SC/GF					=	Single-cone	technique	with	GuttaFlow	sealer	GC/AH+	=	GuttaCore	with	AH	Plus	sealer	C															=	Coronal	third																																														*		=	No	statistical	difference	M														=	Middle	third																																															**	=	Statistical	difference	A															=	Apical	third			

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