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Vol 13, No 1 (2025) 
ISSN 2167-8677 (online) 

DOI 10.5195/d3000/2025.861 

 

	

	

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Topical Impact of Nanosimvastatin on Bone Fracture 
 
Luay H. Jalil  
 
College of Den*stry, Al-Farahidi University, Baghdad, Iraq 

Abstract 

Bone healing is a vert complex process. Fracture healing is a major challenge in orthopaedic 
prac<ce and research. Simvasta<n is an encouraging novel bone regenera<on inducer due 
to its safety, low cost, and clinical convenience. However, the specific mechanism of 
simvasta<n's effect on bone growth, as well as the best dose, are unknown. Simvasta<n is 
a biodegradable polymer that promotes bone growth. A total number of 16 adult albino 
rabbits were divided into two groups (8 rabbits /each). A defect was done in bone and filled 
with gel only for controls and treatment specimens were filled with nanoliposomal simvas-
ta<n. The results showed that the treatment group with nanosimvasta<n gel had a signifi-
cant improvement in bone healing compared to the control group. 

Open Access 
Cita%on: Jalil LH, et al. (2025) Topical Impact of Na-
nosimvasta%n on Bone Fracture. Den%stry 3000. 1:a001 
doi:10.5195/d3000.2025.861 
Received: February 24, 2025  
Accepted: March 16, 2025 
Published: June 26, 2025 
Copyright: ©2025 Jalil LH. This is an open access ar%cle 
licensed under a Crea%ve Commons AVribu%on Work 
4.0 United States License. 
Email: Luay.h.Jalil@@uofarahidi.edu.iq

Introduc)on 
Bone	healing	is	a	very	complex	process	that	
relies	on	the	coordinated	action	of	numerous	
cell	 lineages	 on	 a	 series	 of	 biological	 pro-
cesses.	Fracture	healing	assessment	is	a	ma-
jor	challenge	in	orthopedic	practice	and	re-
search	[1].	Bone	healing	requires	the	regen-
eration	 of	 a	 functional	 bone	 structure	 [2].	
Traditional	 therapies,	 such	 as	 bone	 grafts,	
and	different	types	of	synthetic	bone	substi-
tutes	 are	 still	 challenging	either	because	of	
their	plain	side	effects	or	because	they	are	Ci-
nancially.	 Tissue-engineering	 technique,	 in	
which	 osteogenic	 stem	 cells	 and/or	 bone	
growth	 factors	 are	 multiplied	 or	 loaded	 in	
extracellular	matrices	 in	 vitro	before	being	
transplanted	in	vivo	to	repair	bone	defects,	is	
the	most	 recent	 approach.	 The	 growth	 fac-
tors	stimulate	bone	formation,	while	the	ma-
trices	 operate	 as	 a	 scaffold	 that	 incremen-
tally	dissolves	as	bone	ingrowth	progresses.	

This	 should	 give	 the	 new	 bone	 sufCicient	
structure	and	functionality.	However,	due	to	
the	absence	of	a	solid	technique	for	amplify-
ing	 and	 guided	 differentiation,	 the	 use	 of	
stem	 cells	 is	 still	 in	 the	 experimental	 stage	
[3].	Simvastatin	 is	a	white,	crystalline	pow-
der	with	a	low	water	solubility.	It	possesses	
antihypercholesterolemia	 properties.	 It	
works	 by	 blocking	 the	 reductase	 of	 3-hy-
droxy-3-methylglutaryl	 coenzyme	 A	 (HMG	
Co-A).	This	enzyme	catalysis	cholesterol	pro-
duction.	Pravastatin	was	originally	reported	
to	 prevent	 heart	 transplant	 rejection	 in	
1990,	indicating	that	statins	had	an	immuno-
suppressive	 impact	 [4].	 Simvastatin,	 a	 cho-
lesterol-lowering	medicine,	was	found	to	im-
prove	bone	production	by	boosting	 the	 ex-
pression	of	the	bone	morphogenetic	protein-
2	(BMP-2)	gene	in	bone	cells	in	a	previous	in-
vestigation	of	osteoporosis.	Simvastatin	has	
also	 been	 linked	 to	 enhanced	 bone	 density	
and	a	 lower	risk	of	osteoporotic	fracture	in	

more	 recent	 clinical	 investigations.	 Simvas-
tatin	has	been	gaining	traction	as	a	promis-
ing	novel	bone	regeneration	inducer	due	to	
its	 safety,	 low	cost,	 and	clinical	availability.	
However,	the	speciCic	mechanism	of	simvas-
tatin's	effect	on	bone	growth,	as	well	as	the	
best	dose,	are	unknown.	The	success	of	bone	
repair	is	aided	by	a	variety	of	cytokines	and	
growth	factors	[5].	The	purpose	of	this	study	
was	to	determine	a	better	bone	regeneration	
inducer.	

Materials and Methods           

We	 applied	 the	 prepared	 gel	 to	 a	 surgical	
damage	to	the	left	rabbit	femur	with	follow-
up	of	the	healing	process.	
We	used	simvastatin	in	a	vehicle	gel	that	in-
cluded	cholesterol	and	 lecithin	and	simvas-
tatin	phosphate	buffer	(pH	7.4).	
The	 chloroform	 Cilm	 approach	was	 used	 to	
standardize	a	variety	of	liposomal	solutions.	
Cholesterol	and	lecithin	were	combined	and	



  Topical Impact of Nanosimvasta1n on Bone Fracture 

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dissolved	 in	 a	 9	ml	 organic	 combination	of	
chloroform	and	methanol	(2:1),	respectively.	
A	thin	coating	of	lipids	was	deposited	inside	
the	round	bottom	Clask	of	the	device	after	the	
organic	solvent	was	evaporated	using	a	ro-
tary	evaporator.	With	vigorous	shaking,	sixty	
ml	of	0.2	M	phosphate	saline	buffer	pH	7.4	
containing	1mg	s	simvastatin	[6]	and	1	mM	
EDTA	was	added	to	the	deposited	Cilm,	and	
liposome	suspension	formed	instantly	[7].	
A	 total	of	 sixteen	adult	albino	rabbits	were	
used	 in	 current	 study	 (2.2-3	 Kg).	 Animals	
were	kept	in	individual	cages	and	were	kept	
in	 similar	 conditions	 (22-24	 C0)	with	 light	
and	 dark	 cycle,	 as	 well	 as	 unlimited	 pellet	
meal	and	water	ad	libitum.	Before	the	surgi-
cal	procedure,	a	veterinary	professional	per-
formed	 an	 assessment	 to	 examine	 the	 ani-
mal's	overall	health	and	condition	[8].	
Food	was	 stopped	9-10	hours	before	 anes-
thesia	 was	 administered.	 Animal	 hair	 was	
shaved	off	the	left	femur,	and	the	region	was	
disinfected	with	a	povidone	 iodide	solution	
[9].	Food	was	not	eaten	for	eight	to	ten	hours	
before	 anaesthesia	was	 administered.	 Each	
animal	was	given	0.2	mg	of	vagal	tonus	to	re-
duce	 intramuscular	 atropine	 sulphate	 dose	
of	mg/kg	 injection.	Anesthesia	was	used	 to	
put	 the	 animals	 to	 sleep.	 Ketamine	 (50	
mg/kg)	was	injected	intramuscularly.	
intramuscular	 injection	 of	 (weight	 in	 kilo-
grams)	 and	 intramuscular	 injection	 of	
(weight	 in	 kilograms)	 diazepam	 (5.0–10.0	
mg/kg	body	weight).	Antimicrobial	prophy-
laxes	 (preoperative	 antimicrobial	 prophy-
laxes)	consisting	of	ceftrioxone	was	injected	
at	a	rate	of	50	mg/kg.	The	defect	that	done	in	
bone	was	Cilled	with	nanoliposomal	simvas-
tatin	for	treatment	groups	and	Cilled	with	gel	
only	 for	 control	 groups;	 sectioned	 femurs	
were	 Cixed	 in	 10%	 formalin	 in	 phosphate	
buffer	saline	PBS	0.1	M	for	48	hrs.	Two	con-
centrations	 were	 excluded	 from	 the	 study	
(2%	and	0.5%).	sectioned	femurs	were	Cixed	
in	10%	formalin	 in	phosphate	buffer	saline	
PBS	0.1	M	for	48	hrs.:	At	the	end	of	the	exper-
imental	periods	after	6th	weeks.	The	areas	of	
connective	 tissue	and	bone	were	measured	
in	relation	to	the	overall	measurement	area	
in	histomorphometric	evaluations.	To	avoid	
any	potential	bias,	the	middle	section	of	each	
core	was	chosen.	I	used	the	image	J	analysis	
tool	to	investigate.	
𝐏𝐞𝐫𝐜𝐞𝐧𝐭𝐚𝐠𝐞	𝐨𝐟	𝐜𝐡𝐚𝐧𝐠𝐞	%

=		
𝐝𝐞𝐟𝐞𝐜𝐭	𝐨𝐟	𝐛𝐨𝐧𝐞(𝐀) − 𝐜𝐨𝐧𝐭𝐫𝐚𝐬𝐭	𝐚𝐫𝐞𝐚(𝐁)	

𝐦𝐞𝐚𝐧	𝐨𝐟	𝐢𝐧𝐭𝐞𝐫𝐜𝐨𝐫𝐭𝐢𝐜𝐚𝐥𝐛𝐨𝐧𝐞	𝐚𝐫𝐞𝐚	(𝐂) − 𝐜𝐨𝐧𝐭𝐫𝐚𝐬𝐭	𝐚𝐫𝐞𝐚(𝐁)	 ∗ 100	

Volume	percentages	(percent)	of	new	bone	
were	 estimated	 using	 histomorphometric	
measurements	of	the	samples.	After	identify-
ing	the	external	and	internal	surfaces	of	the	
original	femur,	the	total	area	was	calculated,	
and	the	cavity	and	soft	tissue	(non-bone	re-
gion)	 were	 circled	 and	

deCined(10)determined	 the	 following	histo-
morphometric	parameter.	
Area	new	bone	%=	[(1	–	(non-bone	area	

/	total	area)]	×	100	(%)	

Results 
1. Figure	 1	 shows	 surgical	 procedure.	 At	 the	

end	of	the	study,	no	wound	infection	was	ob-
served.	(Figure	2).	Nano	simvastatin	showed	
mineralization	early	and	complete	bridging	
of	defect	was	seen	at	end	6th	week	after	treat-
ment	group,	in	contrast	to	control	group	that	
reported	less	evidence	of	healing	with	signif-
icant	differences	(p-value	0.03,	Table	1).	
In	Figure	3,	an	example	of	the	image	J	analy-
sis	taken	for	rabbit	femur.	The	area	of	the	de-
fect	was	replacement	with	low	density	bone	
of	granulation	tissue	in	the	controls	(Figure	
4).	

For	the	treated	animals,	there	was	signiCicant	
increase	of	cellular	component	of	the	newly	
formed	soft	callus	at	the	site	of	defect	com-
pared	 with	 6th	 week	 sections	 appeared	 as	
clusters	of	osteoblasts	surrounded	by	varia-
ble	amount	of	eosinophilic	pink	osteoid	par-
tially	mineralized	to	appear	slightly	grayish	
pink	at	some	foci,	generally	osteoid	precipi-
tation	 is	 more	 diffused	 compared	 with	 6th	
week		sections	(control)	(Figures	5	and	6).		

At	the	end	of	6th	week	of	treatment,	bone	for-

mation	 in	 the	 treatment	 group	was	 signiCi-

cantly	higher	than	control	group	(p=	0.000)	

(Table	2).	

Discussion 
Numerous	experimental	research	has	inves-
tigated	 the	use	of	 statins	 in	bone	 tissues	 in	
combination	 with	 various	 vehicle	 systems	
[10].	Simvastatin	works	by	increasing	the	ac-
tivity	of	osteoblasts	while	 inhibiting	the	ac-
tivity	of	osteoclasts.	This	is	accomplished	by	
bone	morphogenetic	 proteins	 (BMPs)	 driv-
ing	 osteoblastic	 cell	 development	 (through	
TNF-a-to-Ras/Rho/mitogen-activated	 pro-
tein	 kinase	 competition)	 [11].	 It	 was	 re-
ported	 that	 the	 outcome	 of	 radiological	 in-
vestigation	in	all	treatment	periods	revealed	
an	 increase	 in	 radiopacity	and	has	come	 to	
agreement	 with	 following	 gel	 production	
and	application	in	the	rabbit	femur,	after	tak-
ing	the	right	dose	[12].	Biological	complexity	
of	bone	healing	of	 fractures	 is	 still	an	open	
Cield	 for	research	 [13].	Tumors,	bone	 infec-
tion,	atrophy,	and	surgical	procedures	can	all	

cause	 bone	 defects	 in	 the	 maxillofacial	 re-
gion.	Rabbits	were	used	as	models	because	
they	acquire	skeletal	maturity	at	around	[6]	
months	of	 age.	 In	 comparison	 to	other	ani-
mals,	 the	 rabbit	 has	 a	 rapid	 bone	 turnover	
[14].	When	compared	 to	rats	and	mice,	 the	
rabbit	model	has	the	beneCit	of	being	easier	
to	manage	and	set	aside	for	greater	bone	ab-
normalities.	The	usage	of	males	was	limited	
to	 avoid	 possible	 hormonal	 changes	 in	 fe-
males,	which	 could	have	 inCluenced	 the	 re-
sults.	The	 cycle	of	 rabbit	bone	 repair	 takes	
approximately	42	days,	according	to	consen-
sus.	Allows	for	the	investigation	of	the	begin-
ning,	 intermediate,	and	 Cinal	stages	of	bone	
repair	evaluations	conducted	over	7,	21,	and	
42	days	.	Because	it	contains	red	bone	mar-
row	 and	 a	 considerable	 number	 of	 spongy	
bone	and	osteoprogenitor	cells,	the	femoral	
epiphysis	is	a	good	candidate	for	cavity	prep-
aration.	 The	 femoral	 epiphysis	 also	 allows	
for	the	creation	of	a	defect	[14].	The	goal	of	
this	 study	 is	 to	 see	how	simvastatin	affects	
different	pathways	of	bone	growth.	The	ef-
fect	of	 simvastatin	on	entochondrostosis	of	
long	 tubular	 bones	 and	 intra-membranous	
ossiCication	of	Clat	bones	was	investigated	us-
ing	a	rabbit	radial	defect	and	a	monkey	cal-
varia	defect	model,	respectively.	The	rabbit	
radial	 defect	 is	 the	 traditional	 model	 for	
studying	bone	formation,	with	a	20	mm	long	
defect	 regarded	 the	 upper	 limit	 for	 self-re-
pair	 [15].	 Simvastatin's	 ability	 to	 increase	
bone	formation	and	decrease	bone	loss	was	
Cirst	demonstrated	in	rats	after	stomach	ad-
ministration	and	subcutaneous	tissue	injec-
tion	[15].	

Conclusion 
The	current	study	showed	that	nano-simvas-
tatin	 gel	 as	 a	 treatment	 for	 fractures	 and	
bone	deformities	accelerated	their	recovery.	

References 
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Figure	3.	Rabbit	femur	for	test	(A)	and	control	animal	(B).	

	

Figure	1.	Surgical	procedure.	

Figure	2.	No	infection	was	observed.	
	

 

B A 



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Figure	4.	(A)	Photomicrograph	of	rabbit	left	femoral	bone	at	site	of	defect	at	control	group	after	2	weeks	of	defect	induction	showing	
the	original	mature	bone	(A)	and	the	soft	callus	tissue	formed	at	site	of	defect	composed	of	mesh	like	collagen	bundles	(B),	a	nidus	
or	foci	of	osteoid	precipitations	(C),	and	newly	formed	capillaries	(D).	MagniCication	165	X,	Staining	H&E.	

	

	

	

	

	

	

	

	

	
(B)	The	process	if	ossiCication	amount	of	maturation	and	mineralization	of	osteiod	is	much	more	progressed	at	peripherally	than	it	is	in	cen-

ter	of	newly	formed	callus.	

	

	

	

	

	

	

	

	

	
(C)	Few	remnants	of	Cibrin	casts	still	apparent,	no	sign	of	inClammatory	reaction,	few	newly	formed	capillaries	can	be	noted,	few	multinucle-

ated	osteoclasts	observed	at	noticed	sections.	

	

	



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Group	Statistics	

Group	 N	 Mean	 Standard	Deviation	 Standard.Error	Mean	 p-value	

Control	group	 8	 46.0000	 10.74709	 4.80625	 0.030*	

Treatment	group	 8	 64.4000	 11.37102	 5.08527	

Independent	T	Test	signiCicant	difference-p	value	≤0.05*	

	

	

Figure	5.	The	areolar	tissue	bridges	between	those	partially	ossiCied	segments	still	recognizable,	vasculari-
zation	and	capillaries	are	more	prominent.	Photomicrograph	of	rabbit,	femoral	bone	at	site	of	defect	at	
treated	group	after	6	weeks	of	defect	induction	showing	areolar	tissue	at	the	center	of	the	callus	demon-

strating	mature	capillaries	and	foci	of	proliferating	osteoblasts.	Staining	H&E.	

 
 

Figure	6.	Photomicrograph	of	rabbit	femoral	bone	at	site	of	defect	at	treated	group	after	6	weeks	of	defect	induc-
tion	showing	a	magniCied	view	of	previous	H&E	staining.		



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Table	2.	New	bone	after	created	defects	in	rabbit	femur	(n=8).	

Group	Statistics	

Group	 N	 Mean	 Standard	Deviation	 Standard	Error	Mean	 p-value	

Control	group	 8	 0.5720	 0.04970	 0.02223	 0.000	

Treatment	group	 8	 0.9020	 0.07190	 0.03216	

Independent	T	Test	signiCicant	difference-p	value	≤0.05