Histological and Histomorphometric Evaluation of

Hydroxyapatite Based Biomaterials in Surgically Created

Defects Around Implants In Dogs.
Journal: Journal of Periodontology
Manuscript ID JOP-17-0469.R3
Manuscript Type: Original Article
Date Submitted by the
Author: 06-Jul-2018
Complete List of Authors:
Attia, Mai; Al-Azhar University Faculty of Dental Medicine for Girls, Oral
Medicine, Periodontology, Diagnosis and Radiology, Department
Shoreibah, Eatimad; Faculty of Dental Medicine, Al-Azhar University
(Girls Branch), Oral Medicine, Periodontology, Diagnosis and Radiology
Mohammed, Hend; Faculty of Dental Medicine, Al-Azhar University (Girls
Branch), Oral Medicine, Periodontology, Diagnosis and Radiology
Attia-Zouair, Mohamed; Faculty of dental medecine, oral pathology;
Ayad, Mohamed; Faculty of Veterinary Medicine,Cairo University ,
Veterinary Medicine
Key Words: Experimental design, Graft(s), Histology
Journal of Periodontology

Histological and Histomorphometric Evaluation of Hydroxyapatite Based

Biomaterials in Surgically Created Defects Around Implants in Dogs.

Mai S. Attia*, Hend M. Mohammed†, Mohammed G. Attia‡,Mohamed A. Abd El Hamid§,

Eatemad A. Shoeriabah¶

*Associate Professor, Department of Oral Medicine, Periodontology, Diagnosis and

Radiology.Faculty of Dental Medicine, Al-Azhar University (Girls Branch), Cairo, Egypt.
†Candidate for master’s degree, Department of Oral Medicine, Periodontology, Diagnosis
and Radiology.Faculty of Dental Medicine, Al-Azhar University (Girls Branch), Cairo, Egypt
‡Professor, Department of Oral Pathology.Faculty of Dental Medicine, Al-Azhar
University(Boys Branch), Cairo, Egypt.
§Professor, Department of Surgery, Anesthesia and Radiology, Faculty of
VeterinaryMedicine,Cairo University

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/JPER.17-0469.

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¶ Professor, Department of Oral Medicine, Periodontology, Diagnosis and Radiology.Faculty
of Dental Medicine, Al-Azhar University (Girls Branch), Cairo, Egypt.

Correspondence address:
Mai Shafik Attia
125 Omr Abn Elkhatab street, Al-zahraa buildings, Nasr city, Egypt
Mobile Number; +02 01093361936
E-mail address: mai_shafik@yahoo.com
Running title: Various hydroxyapatite in surgically created bone defects.
Keywords: Expiremental design, Graft(s); Histologic evaluation.
Summary Sentence: Various types of hydroxyapatitebone graft are effective in treatment of
surgically created defects around dental implants in dogs.
Figures-4; Tables-2; References-38; Words-2870

Abstract
Aim: The present study evaluated histologically and histometrically the efficacy of micro-,
nano or mixed composite of hydroxyapatite graft in treatment of surgically created defects
around dental implants in mongrel dogs.

Materials and Methods: Immediate implant was used after extraction of lower third
premolar in mongreal male dogs. Critical size defects were created in intact proximal alveolar
bone to each implant. The defects were divided randomly into four groups of two animals
based on biomaterials used for treatment: (1) received no treatment (negative control); (2)
defects treated with nanohydroxyapatite bone graft; (3) defects treated with micro
hydroxyapatite bone graft; and (4)defects treated with a mixed composite of micro and nano
hydroxyapatite. Animals were sacrificed at 2 months and histologic and histometric
evaluation was carried out.
Results: The amount of new bone formed with nano hydroxyapatite bone graft was highly
significant than that obtained by micro or mixed composite of hydroxyapatite. Defects treated
by mixed hydroxyapatite showed the greatest value in mean area percent of collagen fibers
using Masson trichrome stain.
Conclusion: The present study demonstrated that nano hydroxyapatite bone graft was better
than micro or mixed hydroxyapatite bone graft in new bone formation in standardized
surgically created defects around dental implants. However, longer period is necessary to

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determine the time taken for complete resorption of bone graft materials and their
replacement with new bone.

Introduction
Several etiologic factors may be contributed to bone loss and the bone defects may be too
large for spontaneous and physiologic repair.1) There are many cases in which bone grafts are
needed for reconstruction of bone defects caused by trauma, tumors, infections, and
congenital defects.(2,3)Moreover, surgically created bone defects around implants have been
widely used in order to resemble extraction sockets and/or perimplantitis, for histologic or
histomorphometric analysis.(4,5)
Several experimental bone graft materials (autograft,allograft,xenografts and alloplasts) have
been utilized to elicit bone formation in bone defects and to accelerate their healing. (6) Bone
tissue engineering is a promising approach which is based on the substitution of engineered
tissue with restoration of functions during regeneration and subsequent integration with the
host tissue.(7)Hydroxyapatite (HA) ceramic has been widely used as bone substitute material in
bone defects for a long time and showed excellent biocompatibility, bioactivity, and
osteoconductive properties.(8) In addition, it has been shown to support osteoblast adhesion and
(9)
proliferation in vitro and to establish a strong bonding with the newly deposited bone
mineral phase in vivo.(10)

Synthetic Nano-crystalline hydroxyapatite (ncHA) material is different from microcrystalline

bone substitution materials regarding solubility, as its chemical composition corresponds to
that of bone mineral. The particle size was reported to be 18 nm in average, which allows for
an accelerated substitution by vital bone. (11) In fact, human bone tissue is a kind of composite
composed of nano-apatite crystals and collagen matrix. (12, 13)
In 2005, Thorwarth et al., examined the de novo bone formation in bony defects following
the insertion of autogenous bone alone versus an injectable nanoparticle hydroxyapatite alone
and in combination with 25% autogenous bone. Micro radiographic analysis indicated
mineralization rates in ncHA groups were not significantly lower than those found in the
autogenous bone group. Histologically, there was suitable osseointegration and
osteoconduction of the used material. Complete resorption of the nanoparticle hydroxyapatite
had taken place after 12 weeks. Thus, it was concluded that the evaluated nano
hydroxyapatite met the clinical requirements as a bone substitute material within the limits of
this experimental setting. (14)

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Regarding the comparison of hydroxyapatite bone graft based on different particle size, an
attempt had been made through this study to compare and evaluate histologically and
histometrically, the efficacy of micro-, nano- or mixed composite of hydroxyapatite bone
graft in treatment of surgically created alveolar bone defects around dental implants in dogs.
Materials and Methods

Study Design: Eight healthy, pathogen-free adult mongrel male dogs were used for the
study. The ethical committee clearance for the study was obtained. Each dog had two defects
sites: one on the right side (distal aspect of right 3rd premolar) and the second on the left side
(distal aspect of the left 3rd premolar) in the lower jaw.The defects were divided randomly
into four groups of two animals based on biomaterials used for treatment: Group1(Gp 1)
defects received no treatment (negative control); Group2(Gp 2)
defects treated with nano hydroxyapatite bone graft§with size 10-50 nm; Group3(Gp 3)
defects treated with micro hydroxyapatite bone graft¶ with size of 300-400 microns and
Group4(Gp 4) defects treated with a mixed composite of micro and nano hydroxyapatite bone
graft.

Animal Care and Maintenance:

The experimental animals were housed in standard cages, under quarantine for a period of 21
days after procurement. During this period they were vaccinated against diseases. All
experiments were conducted in the experimental Surgical Centre of the Faculty of Veterinary
Medicine, CairoUniversity. All animals were kept under the same nutrition and
environmental conditions. The study protocol was approved by the Research Ethics
Committee, Faculty of Dental Medicine For Girls, Al-Azhar University (Approval Number
REC15-081). The procedures were conducted inaccordance with the committee for the
purpose of control and supervision on experiments on animals (CPCSEA Guidelines) .(15)

Steps of Operative Procedure:(figure1)

Presurgical Preparation: Pre- anaesthetic medication was carried out using atropine‡‡(0.05
mg/kg intramuscular) and tiletamine-zolazepam§§ (5–10 mg/kgintramuscular).Subsequently,
anesthesia of 10 to 15 mg/kg thiopental sodium*** was given intravenous and theanimal was
placed on a 1 to 4% O2–N2O isoflurane mixture.
Incisions and Elevation of Flap: A crevicular incision was placed around the 2nd, 3rd and
4thpremolars. Then a mucoperiosteal flap was elevated to disclose the marginal aspect of the
ridge in order to facilitate tooth extraction. The third mandibular premolar (P3) was

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hemisected with the use of a diamond cylindrical bur under copious irrigation and then both
roots were removed with the use of forceps.
Surgical Procedure: Three walled intrabonysurgical defects were created distal to each third
premolar by No.801 straight fissure bur. The defects were 5 mm in dimension buccolingually
and mesiodistally and 7 mm cervicoapically, measured with a periodontal probe. Based on
the measurement of the extracted root by periodontal probe, the implant osteotomies were
drilled. Dental Titanium implants§§(11mm length and 3.4mm in diameter with a sandblasted
acid-etched surface) were inserted and primary stability was attained . Each dog received two
implants bilaterally in the distal extraction sockets of the third mandibular premolar.
Placement of the Bone Graft Materials: Surgically created defects distal to each implant
were filled with selected hydroxyapatite particles mixed with 1 to 2 drops of saline to obtain a
putty consistency. In Gp 4, equal proportion of nano- and micro-hydroxyapatite were mixed
under aspetic conditions and used. However, in Gp1, no bone graft was added in the defects.
Finally,mucoperiosteal flaps were replaced and secured using 4-0 braided silk sutures.
(Figure 1)

§Nano Bone, ArtossGmbh, Germany

‡‡Atropine®, Aguettant, Lyon, France
§§Zoletil® 100, Virbac, Carros, France
***Nesdonal®, Merial,Lyon, France
¶OseteoGen R (HA Resorb), Impladent LTD, USA
§§TUT-dental Company,Cairo, A.R.E

Post-Surgical Care: The animals were put under antibiotic coverage with ampicillin
cloxacillin 250mg given twice daily by intramuscular injection for five days. For alleviating
inflammation and pain, diclofenac sodium 25mg was given twice daily. The sutures were
removed on the seventh postoperative day under sedation and the surgical area was irrigated
with 0.2% chlorhexidine
gluconate solution.

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Post-Surgical Procedures: The animals were sacrificed at 2 months of healing period by
euthanasia using a lethal dose injection of Dolethal***
Fixation: the mandible was dissected with soft tissues. The implant and the surrounded
tissues were immersed in 10% buffered formalin and allowed to be fixed for 48 h. The jaw
was sectioned using a diamond cutter into blocks corresponding to the surgical sites.
Demineralization and Tissue Processing: Prior to decalcification, dental implants were
removed by performing counter clockwise rotation with speed 35 revolution per minute
(RPM). The blocks were placed in separate beakers containing decalcifying solution
(diamine-tetra acetic acid (EDTA)125 gm in one liter of distilled water and sodium hydroxide
as a buffer). This was
continuously agitated using a magnetic stirrer. The completion of decalcification was
determined by adding a chemical ammonium oxalate, to the decalcifying solution. The
decalcified block was subjected to ascending grades of alcohol (70%, 80%, 90% and 100%)
each for 4 h with 2 changes.
The dehydrated tissue was immersed in a clearing agent methyl benzoate. The cleared tissue
was immersed in paraffin wax at 56°c for 2 h with 2 changes to make a block. Multiple serial
sections of 5μm thickness were cut in the mesio-distal plane using a leica microtome. These
sections were placed on microscopic slides. The sections were then stained with:
1- Hematoxylin and Eosin stain (H&E) for general histological examination and assessment
of new bone formation. (16)
2- Masson Trichrome stain (MT) for detection of collagen fibers. (17)

***pentobarbitalsodique, Vetoquinol, Paris, France.

Image Analysis of the Data

The stained tissue section was examined using the light microscope to assess the prevalence
of positive cases and the localization of the stain within the tissues. In addition, an image
analysis computer system was used to assess positive area percentage of stain.The image
analysis was performed using the Leica EZ4 HD image analyzer computer system
(Switzerland) and the Leica
Qwin V3 image analyzer computer system (Switzerland).

The image analyzer was first calibrated to convert the measurement units (pixels) produced
by the image analyzer program into actual micrometer units.The investigator selected a
specific field of examination at the surgically created defect distal to each dental. After
calculation of the corresponding dental implant length on the slide, the dimension of this
specific field was
determined according to the following equation; Dental implant length on slide /Actual
dental implant length(11mm) = surgical created defect on slide/ Actual surgical created defect

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length made (7 mm). The same equation was used for the width of defect. This specific field
for examination was 3.8mm in width and 5.3 mm in length to provide standardization and
reproducibility in each slide with each stain(Figure 2).

Statistical Analysis
Data were presented as mean and standard deviation (SD) values. One-way ANOVA was
used for comparisons between the different groups. Tukey’s post-hoc test was used for pair-
wise comparison between the groups when ANOVA test was significant. The significance
level was set at p ≤ 0.05. Statistical analysis was performed with SPSS 16.0 (Statistical
Package for Scientific Studies, SPSS, Inc., Chicago, IL, USA) for Windows.

Results

The postoperative healing was clinically uneventful in all eight dogs with no local or
systemic reactions. Descriptive histology and histomorphometric analysis, in the various
groups studied, utilizing the following stains: Hematoxin and eosin stain as well as Masson
trichrome stain were performed.
Histological analysis
The surgically created defects without any added bone graft showed a relatively wide space
surrounded by areas of fibrous as well as bony tissues especially at the base and periphery of
the defects. The central area was devoid of any newly formed tissues either fibrous or bony.
However, defects treated with nano-HA revealed multiple spaces that have been subdivided
by fibrillar and bony structures. The newly formed bone trabeculae are extensive and
intermingled with each other’s. In addition, a number of flattened, quiescent osteoblasts
lining the bone were seen. Less distribution of remnants of the graft material incorporated
inside the regenerated tissues was observed. (Figure 3)
Defects treated with micro-HA showed the persistence of extensively wide spaces in the
partially healed defect. These spaces were seen to be surrounded with fibrous tissue
formation and newly formed bone. The intercellular spaces in the mid-portion of the defect
did not show signs of bone formation. (Figure 4)
The histological defects treated with mixed composite of nano- and micro-HA revealed that
almost all defects had been filled with zones of new bone and condensing fibrillar connective
tissues which enveloped the periphery of defects. Some of the bony trabeculae showed
lamellar pattern and marrow tissue intermingled with each other.

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Histomorphometric Analysis
Table (1) Illustrates the mean value of area percent of newly formed bone in all groups. The
greatest mean value of area percent of newly formed bone was recorded in nano –HA group
(31.39±1.6), whereas the lowest mean value was recorded in group I , where no bone graft
was added (3.78±0.62). One way analysis of variance (ANOVA) test revealed a significant
difference between nano –HA group and all groups at 2 months (P<0.0001). Tukey’s post hoc
test revealed that the difference between Micro- HA and mixed composite of nano and micro-
HA group was not statistically significant.
Using Masson Trichrome Stain,Table (2) shows the mean value of area percent of collagen
fiber in the studied groups .The mean value of area percent of collagen fibers in Group 4
(Mixed composite of nano and micro HA) (23.5±6.08)was greater than in the other three
groups (P <0.001). Group 4 showed a statistically significant difference in mean value of area
percent of collagen fibers upon comparing with all groups at 2 months.However, the
difference in mean value of area percent of collagen fibers between the defects left untreated
and groups treated nano HA and /or micro HA groups was not statistically significant.

Discussion
Complete regeneration of lost tissues should be the ultimate endpoint for the field of
regenerative medicine and engineering. Thus, significant progress has been made in recent
years with the introduction of various metallic and polymeric materials structured at the
nanoscales. (18,19)
To our knowledge, the present study was the first reported to experimentally assess
histologically and histometrically the efficacy of micro-, nano- or mixed composite of
hydroxyapatite bone graft in the treatment of surgically created defects around dental
implants in dogs.
It is highly recommended to use critical-sized surgically created defects(CSD) in large animal
models to clearly evaluate the capacity of the tissue-engineered bone substitute for its final
clinicalapplication. (20) Moreover, models for bone reconstruction in dogs are suitable for the
creation of defects, implantation of grafts and analysis of reconstruction.(21)
In this study, CSDs of size 5mm in width and 7 mm in length were created in order to prevent
spontaneous healing. Botticelli et al. (22) suggested that the width of the circumferential
defect should be more than 1.25 mm in order to properly evaluate the effectiveness of
grafting materials on bone regeneration. This suggestion was supported by Polyzois et al.
(23), who used a similar surgical procedure by creating a circumferential defect 2.37 mm in
width.
In the current study, there was new bone formation at the base and periphery of the defects in
the four studied groups at eight weeks. Areas with no bone or fibrous tissue formation were
observed in the central zone in Group 1 defects with no bone graft placement. In defects
treated with nano-HA, micro-HA and mixed composite, a fusion and coalescence of new

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bony trabeculae was noticed. Subsequently, closely condensing fibrils were shown to be
intermingled with bony tissues.However, Irregular pattern of bony trabeculae as well as
connective tissue fibers with varying degree of density were seen among these groups. This
may be attributed to a fundamental factor governing optimal integration of HA with bone
which is the dimension of the crystals. Nano HA particles with dimensions closer to the size
of natural crystals found in vertebrate hard tissues (from 1 to 10 nm), have been reported to
mimic the extracellular matrix of bone in size and structure. (24)

Based on histomorphometric analysis of the amount of new bone formation, defects treated
bynano-HA showed a statistical significant difference in mean value of new bone formation
upon comparing with defects treated by micro-HA or mixed composite of HA. This was in
accordance with the study which demonstrated that nano-HA had osteoconductive properties
and behaved adequately as grafting material for bone regeneration when compared to Bio Oss
in experimental defects prepared in rat calvaria.(25)

To initiate bone formation, cells need to adhere and proliferate. Surface roughness is an
important consideration when selecting a bone-inducing substrate. A substrate with
superficial roughness shows a higher capacity for adhesion when compared with a smooth
surface.(26-28)Nano-sized material have a stimulatory effect on mesenchymal stem cells (29)
with an increase in protein absorption and osteoblast adhesion as compared with traditional
micro-sized HA.(30,31)Moreover, it was demonstrated that nano-HA stimulates the local
alveolar osteoblasts to produce relevant bone specific BMPs, which are known to initiate and
regulate bone formation starting from the progenitor cells.(32)

In addition, the biodegradation of HA scaffolds may take place by dissolution or

fragmentation with subsequent phagocytosis by macrophages,(33) but also by the activity of
osteoclasts.(34,35)The latter mechanism of biodegradation was considered favorable, because
mimicry of the physiological bone processes should create optimal surfaces for colonization
with osteoblasts and vascular tissue. The degree of osteoclast activity on HA scaffolds
depends on material qualities such as crystal size and surface roughness.(34)Furthermore, the
nano-HA influenced a significant increase in the expression of several important markers of
osteogenesis such as alkaline
phosphatase, osteocalcin and osteonectin.(32)

Regarding the amount of newly formed collagen fibers which had been reported using
Masson trichrome stain, mixed composite of HA revealed the highest value of mean area
percent of newly formed collagen fibers(23.5). There was a non significant difference
between mean area of new collagen in the nano-HA group which was(13.039) , micro HA
group which was(12.558)and defects treated with no bone graft group which was (10.965).

These results could be explained by the difference in biodegration rate of micro and nano
hydroxyapatite particles, early vascularization and their action on the surrounding cells.

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Simultaneously osteoclasts resorb and phagocytose calcium phosphate ceramics.(35,36) The
arrival of osteoclasts is triggered by bare mineralized bone surfaces created by the activity of
collagenases.(37)Activating signals are lacking on nano-HA embedded in connective tissue
where osteoclasts were rarely situated in resorption. (38) Thus, these findings could explain
themaintenance of the scaffold function of micro HA associated with the inactivity of
osteoclasts revealed more collagen formation without bone formation. Moreover, the space
maintaining and osteoconductive property of mixed composite may require an increased time
interval to show more favourable findings to allow complete resorption of micro HA and its
complete replacement with new bone.

Conclusion
The various types of hydroxyapatite particles used promoted comparable newly formed
tissues over the entire extension of the surgically created defects,independent of their
granular size,thusconfirming their biological osteoconductive property. Nanohydroxyapatite
might offer clues to the surrounding cells allowing them to recognize and attach to the
biomaterial and improve target function like proliferation, migration and tissue
differentiation. The final goal is to mimic the native tissue. However, this subject is still in
pre-clinical phase. For this reason, more studies on regeneration of bone defects using various
sizes of hydroxyapatie particles are needed.
Conflict of Interest:
The authors report no conflicts of interest related to this study.

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Figure legend
Figure (1): A surgically created defect was made and hydroxyapatite bone graft was inserted
in bone defects
Figure (2): A specific field of examination was demarcated corresponding to the defect
size(Masson trichrome stain)
Figure (3): Group 2 showed spaces surrounded with irregular bony trabeculae and connective
tissue fibers with varying degree of density. Flattened, quiescent osteoblasts lining the bone
were seen. Less distribution of remnants of the graft material was observed incorporated
inside the regenerated tissues (H&E stain x100; Masson trichrome stain x100).
Figure (4): Group 3 showed multiple spaces surrounded with fibrous tissue formation and
newly formed bone. Fragments filling the defect intermingled with fibrin meshwork were
noticed (H&E stain x100; Masson trichrome stain x100).

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Table 1: Area percent of newly formed bone and significance of the difference using
ANOVA test.

Table 1:Area percent of newly formed bone and significance of the difference using ANOVA

test.

Gp1 Gp2 Gp3 G4

Mean 3.784c 31.392a 21.958b 23.903b

Std Dev 0.621 1.602 8.272 7.082

Min 3.163 29.79 11.87 16.53

Max 4.405 32.994 31.23 31.95

Fvalue 132.61

P value <0.0001*

*significant at p<0.05;Tukey’s post hoc test: means with different superscript letters are

significantly different.

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Table 2: Mean percent of collagen fibers using Masson stain and significance of the
difference using ANOVA test

Table(2): mean percent of collagen fibers using Masson stain and significance of the

difference using ANOVA test

Gp1 Gp2 Gp3 Gp4

Mean 10.965b 13.039b 12.558b 23.5a

Std Dev 3.348 1.722 2.513 6.08

Min 7.617 11.317 8.93 18.45

Max 14.313 14.761 14.73 31.71

F value 29.67

P value <0.0001*

*significant at p<0.05 ;Tukey’s post hoc test: means with different superscript letters are

significantly different

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A surgically created defect was made and hydroxyapatite bone graft was inserted in bone
defects
127x95mm (300 x 300 DPI)

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A specific field of examination was demarcated corresponding to the defect size (Masson
trichrome stain)
254x190mm (96 x 96 DPI)

Group 2 showed spaces surrounded with irregular bony trabeculae and connective tissue
fibers with varying degree of density. Flattened, quiescent osteoblasts lining the bone were

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seen. Less distribution of remnants of the graft material was observed incorporated inside the
regenerated tissues (H&E stain x100; Massontrichrome stain x100).
127x95mm (300 x 300 DPI)

Group 3 showed multiple spaces surrounded with fibrous tissue formation and newly formed
bone.
Fragments filling the defect intermingled with fibrin meshwork were noticed (H&E stain
x100; Masson
trichrome stain x100).
127x95mm (300 x 300 DPI)

This article is protected by copyright. All rights reserved.