Histologic effects of demineralized bone matrix on regeneration of alveolar socket in diabetic rats.

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by  Kadkhodazadeh, Mahdi

ORIGINAL ARTICLE
Histologic effects of demineralized bone matrix on
regeneration of alveolar socket in diabetic rats
Mahdi Kadkhodazadeh1,Fatemeh Mollaverdi2,Hamid RezaAbdolsamadi3,
Ramin Azar4,Madjid Ghasemian Pour5*,ShahriyarAhmadpour6
1.DepartmentofPeriodontics,DentalSchool,HamadanUniversityofMedicalSciences,Hamadan,Iran.
2.Dentist,Hamadan,Iran.
3.DepartmentofOralMedicine,DentalSchool,HamadanUniversityofMedicalSciences,Hamadan,Iran.
4.DepartmentofOrthodontic,DentalSchool,HamadanUniversityofMedicalSciences,Hamadan,Iran.
5.Orthodontist,DentalResearchCenter,IranianCenterforEndodonticResearch,ShahidBeheshtiUniversityofMedicalSciences,Tehran,Iran.
6.HistologyandEmbryologyDepartment,MashadUniversityofMedicalSciences,Mashad,Iran.
ABSTRACT
INTRODUCTION: The aim of this in vivo study was to determine the effect of demineralized
bonematrix(DBM)onalveolarbonerepairintypeIdiabeticrats.
MATERIALSANDMETHODS:Thisstudywascarriedouton40adult(8weeks-old)albinorats
withanaverage weightof200-250grams.Theanimalsweredividedintofourgroups(n=10)as
follows:group1nondiabeticrats,group2,3and4werediabeticrats,group4ratstookoneunit
of insulin daily. Diabetes was induced by Alloxan Monohydrate through the tail veins of the
rats in groups 2-4. Only group 4 received insulin NPH 1 unit daily. After 10 days, the upper
rightincisorsofallsampleswereextractedandthesocketwasfilledwithDBMingroups3and
4. The animals were sacrificed at the end of week 1 and 2. The specimens were prepared and
stainedwithH&E.
RESULTS: Histological results of group 4 displayed osteoblastic activity and bone formation
withcollagenfibersatthe end ofthe first weekand thick bone trabeculae formation in vicinity
ofDBM atthe end ofsecond week. Ingroup 3, DBM showed some osteoinductivityatthe end
of the first week, but in some regions DBM particles were degraded by osteoclastic activity.
Bone trabeculae formed with a dispersed and separate pattern at the end of second week. In
group2hematomaandinflammation weredominanthistologicalfeaturesattheendoffirstand
second weeks;poorboneformation wasdetectedinthesetwo groups(2and3).In group1,the
resultswereasexpected.
CONCLUSION: It seems demineralized bone matrix simulate osteoblastic activity. [Iranian
EndodonticJournal2009;4(1):20-4]
KEYWORDS:Diabetes,Demineralizedbonematrix,Microscopy,Toothsocket.
Received:15Jun2008;Revised:06Oct2008;Accepted:08Nov2008
*Corresponding author at: Madjid Ghasemian Pour, Iranian Center for Endodontic Research, Dental School, Shahid
Beheshti University of Medical Sciences, Evin, Tehran, Iran. Tel: [phone], Fax: [phone]. E-mail:
[email protected]
INTRODUCTION proteinmetabolism(1).Decreaseinbonerepair
and formation are one of the consequences and
The two major types of diabetes are type 1, signs of diabetes (2). Demineralized freeze
formerly known as "insulin-dependent diabetes driedboneallograft(DFDBA)ordemineralized
mellitus" (IDDM) and type 2 formerly called bone matrix (DBM) and autogenous grafts are
"non-insulin-dependent diabetes mellitus some of the used materials in guided bone
(NIDDM)". During the past decade, medical regeneration (GBR) (3-5). DBM is one of the
management of diabetes has changed allograft materials employed in periodontal
significantly to minimize the debilitating surgery, bone regeneration, infection and
complications associated with this disease. trauma(6),firstutilizedbyUrist(7).
Type 1 diabetes is one of the metabolic This material had great success in repair of
diseases that involve increase in blood sugar calvarial bone defects (8). There have been
and disturbance in carbohydrate, lipid and controversial reports regarding the induction
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DBMandalveolarsockethealing
Figure1.Tissuesectionsfromthealveolarsocket Figure2.Sectionfromthetrabecularboneofthe
of the forth group [first week]. 1) DBM, 2) forthgroup[secondweek].1)Formedtrabeculas,
osteoblasts,3)Formingbone,4)Collagen 2)Osteoblasts,3)Osteocytes
and guided bone regeneration ability of DBM from 7 mmol (normal) before injection to 13
materials; however the increased popularity of mmol after injection. During treatment, blood
synthetic materials has limited the use of DBM sugarwaskeptstableatapproximately7mmol.
in maxillofacial surgeries (3). Induction and Ratsingroup4(insulintreated,diabetic)took1
guided bone formation of DBM may be due to unitofNPHinsulindaily(11).
the presence of bone morphogenic protein Ten days after diabetes induction, general
(BMP). These proteins are considered anesthesia was performedby10mg/kgKetamin
important factors in the formation of limbs in HCl (Alfanso, the Netherland) and 1mg/kg
addition to their key role in bone regeneration chlorpromazine injection, and then upper right
(9). Investigations to introduce and recognize incisor of each rat was extracted. After
DBM as a grafts material are still in process hemostasis was achieved ingroups 3 and 4, the
(10).Hence theaimofthisinvivostudywas to dental sockets were filled with mixed saline
determine the effects of DBM on alveolar bone and DBM, the area was then sutured. In groups
repairofdiabeticrats(typeI). 1 and 2 dental sockets were not filled. Finally,
2 mL of pentabioticveternario (sigma type
MATERIALSANDMETHODS antibiotic)wasinjectedintramuscularly(11).
At the end of two weeks, five rats from each
This in vivo study was performed according to group was randomly separated and beheaded
other similar studies (8,11-13). All experiments under general anesthesia and placed in 10%
were conducted according to the guideline of formalin. After that, decalcification was
local animal use and care committees and performed by means of formic and chloridric
executedaccordingtonationalanimallaw. acids (Kiyankaveh, Iran) for one week and 5-
Forty 8-week old albino rats with an average µm tissue sections were prepared and stained
weight of 200-250 grams were selected and by hematoxylin and eosin (Padtan Teb Inc.,
randomlydividedinto4groupsof10each(11). Tehran,Iran).
Group 1 was non diabetic and the other 3 In this study, Kim’s method was used to
groups were diabetic. For creating diabetes in harvest DBM (13). After separating long
diabetic groups, we diluted a vial of femoral and tibia bones of 20 rats, the end of
monohydrate Alexon (St. Louis, MO, USA) these bones were placed in cold sterile distilled
with buffer saline, then injected 52 mg/kg of water, the bone marrow was extruded and the
this solution to the rats immediately after its attached tissues from bone surfaces were
preparation through tail veins by insulin separated. The following steps were performed
syringe(12). sequentially:
Five days after injection, blood samples were 1) Immersionof the specimens inabsolute pure
derived through retro-orbital venous sinus by ethanolfor1hour;
means of micropipette in samplesofgroups2, 2)Immersioninethyletherforhalfanhour;
3, and 4. Blood sugar level showed an increase 3)Placingitinanoven(temperatureof36ºC)
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Kadkhodazadehetal.
Figure 3. Tissue section from the alveolar socket Figure 4. Tissue section from trabecular bone of
of the third group [first week]. 1) DBM, 2) the third group [second week]. 1) Bone
Cellularinfiltration,3)Formingbonetrabeculas trabeculas,2)Vessels,3)Osteocytes
Figure 5. Tissue section from the alveolar socket Figure 6. Tissue section fromthe formedbone in
of the second group [First week]. 1) Bone, 2) the alveolar socket of the second group [second
Connectivetissue,3)Hematoma week]. 1) Connective tissue, 2) Osteoblasts, 3)
Osteocytes,4)Vessels
for1night;
end of the second week, intramembranous
4)Millingandpowderingthebone;
bone, vessel and connective tissue formation
5) Immersing in 0.5% normal chloridric acid
occurred(Figure1andFigure2).
solutionsfor3handthenirrigatingwithwater;
In the third group (diabetic and DBM),
6) Immersinginethanolsolutionfor 1hourand
inflammationandcellularinfiltrationoccurredat
inetherforhalfanhour; the end of the first week. Limited bone
7) Putting in oven in the temperature of 36ºC formation around DBM particles was detected;
for1night; activated and DBM fragmenting macrophages
8) Keeping the prepared material in the and osteoclasts were also observed (two rats,
temperatureof2-4ºC. 40%). However, no changes occurred in some
All mentioned procedures were performed in areas. In this group, new-formed trabecula,
thesterilecondition. connective tissue and lots of dilated blood
vessels were seen at the end of the second week
RESULTS (Figure 3 and Figure 4). In group 2 (diabetic),
hematoma was the dominant tissue profileatthe
At the end of the first week, osteoblastic end of the first week. At the end of the second
activity and osseous trabeculas formation week,limitedbone formation,connective tissue,
occurred around DBM and collagen fibers in dilated vessels and intramembranous bone
group 4 (insulin treated). DBM particles were formationoccurred(Figure5andFigure6).
detectable with sharp borders and acellular In group 1, bone formation was limited to
surface or empty lacuna. These changes were periodontal ligament tissue area around the
clearly evident in four animals (80%).Atthe extracted tooth at the end of thefirstweek;
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DBMandalveolarsockethealing
Figure 7. Tissue section from the alveolar socket Figure8.Tissuesectionfromthealveolarsockets
of the control group [First week]. 1) Connective osseous trabecula of the control group [second
tissue,2)Cellsduringconversiontoosteoblast,3) week]. 1) Osteoblast, 2) Formated bone, 3)
Vessels,4)Fineformingtrabeculas Vessels,4)Connectivetissue
in flammation and dilated vessels were also Some reports claim that bone formation around
observed. At the end of second week, bony these particles is due to cytokine release from
islands formed by means of intramembranous DBM and invasion of multipotential cells to the
bone formation, dilated vessels, new injured area (15). It is likely that DBM releases
osteoblasts; connective tissue could also be cytokines or DMPs and that the cavities present
seen(Figures7,8) in the DBM particles take part in
biomineralization. Note that this mechanism
DISCUSSION requires important factors such as collagen,
appropriate pH and hydroxyl groups that
In diabetic rats treated with insulin (group 4), become defective in diabetes (2,16). Even
DBM had favorable effects on GBR. This though DBM has good ability to form bone and
caused the DBM particles to be surrounded by is an appropriate graft material; more
osteoblast cells and osteoid. Group 3 showed investigationsarestillrequired(10).
more bone formationthangroup2; therefore we Attheendofthefirstweek,finetrabeculaewere
can conclude that DBM may induce effective seen around DBM particles in group 3; cellular
GBR. According to these results DBM had a infiltration and inflammation were also
little inductive effect on the untreated diabetic dominant.At the endof the secondweek, minor
group(group4);thismaybeduetootherfactors osteogenesis became visible in some areas. Due
such as pH changes and protein catabolism to the inflammatory activity, destruction and
whichwilldecreaseitseffects. fragmentation of DBM particles, limited
It also seems that the control of blood sugar ontogenesis also occurred. In this group at the
with insulin is an effective measure to decrease end of the second week, cellular infiltration was
the severity of inflammation and protein still present around bone, although osteogenesis
catabolism in group 4 and increase the bone activitywas detected in non-treated areas. DBM
tissueformation. Final glycolysisproducts will effectsonguidedosteogenesiswereseenin40%
have a negative effect on bone repair and it of our samples; even though in some reports,
decreases bone formation. Chay et al. showed unpredictable biologic behavior of DBM is
that mesenchymal cells, preosteoblasts and mentioned (16). However, DBM osteogenesis
osteocytes couldbe seenaroundDBM particles ability in alveolar socket has been focused by
on the 15th day when implanting DMB in the Callan (3), but DBM effectiveness has been
cranium (14). In the control group, some decreased to some extent because of metabolic
changes were observed in PDL cells though no changes and when inflammation superimposes.
osteoblast and osteoid material was detected. It Samples in this group had obviously delayed
seems that DBM particles may induce bone osteogenesis.
formation by attracting multipotential cells and In group 1, at the end of the first week,
stimulatingtheirdifferentiationtoosteoblasts. osteogenesis was observed with peripheral to
IEJ-Volume4,Number1,Winter2009 23
Kadkhodazadehetal.
centraldirectioninsomepartsofremainedPDL. 6. Lynch SE, Genco RJ, Marx RE: Tissue
Current up to date research has demonstrated Engineering: Applications in Maxillofacial Surgery
that rapid osteogenesis after tooth extraction in and Periodontics. Chicago, Quintessence, 1999: pp.
normal tissue is the result of PDL collagen 90-121.
destructionaswellasthefibronectineffect. 7. Urist Mr: Bone morphogenetic protein in
biology and medicine. In: Lindholm TS , editor.
Bone morphogenetic proteins: Biology,
CONCLUSION
biochemistry and reconstructive surgery, Austin,
USA,R.G.LandesCompany,1996:p.7.
This study showed that DBM can have
8. Wang J, Glimcher MJ. Characterization of
inductive effects in rats with controlled
matrix-induced osteogenesis in rat calvarial bone
diabetes; DBM seems to stimulate
defects: II. Origins of bone-forming cells. Calcif
osteoprogenitor cells to differentiate into TissueInt.1999;65:486-93.
osteoblasts. A good recommendation would be 9. Hu ZM, Peel SA, Sandor GK, Clokie CM. The
to carry our further studies with DBM in osteoinductive activity of bone morphogenetic
diseased conditions and control the qualitive protein (BMP) purified by repeated extracts of
andquantitivefactors. bovinebone.GrowthFactors.2004;22:29-33.
10. Zuzarte-Luís V, Montero JA, Rodriguez-León
J, Merino R, Rodríguez-Rey JC, Hurlé JM. A new
ACKNOWLEDGEMENT
role for BMP5 during limb development acting
through the synergic activation of Smad and
This study was supported by the Dental
MAPKpathways.DevBiol.2004;272:39-52.
Research Center of Hamadan University of
11. Grandini SA, Brentegani LG, Novaes AB,
MedicalSciences.
Migliorini RH. Protein synthesis in wound after
tooth extraction in pancreatectomized diabetic rats.
ConflictofInterest:‘Nonedeclared’.
BrazDentJ.1990;1:25-30.
12. Tang LQ, Wei W, Chen LM, Liu S. Effects of
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