Keywords angiogenesis - biodegradation - chitosan - collagen - guided tissue regeneration -
inflammatory response
Introduction
A barrier membrane is commonly used in periodontal regenerative therapy, such as guided
tissue regeneration (GTR). The placement of membranes aims to prevent epithelial growth
toward the apical direction so that the space during cementum formation, periodontal
tissue adhesion, and new bone formation in the teeth suffering from defects can be
maintained.[1 ]
[2 ] The membrane selection by considering its biological and mechanical characteristics
is important to obtain the optimal treatment results. The characteristic membranes
must be biocompatible, nontoxic to the patient's body, have adequate mechanical and
physical properties, have appropriate degradation time for tissue formation, have
cell occlusivity, be able to facilitate the proliferation of regenerative cells, promote
angiogenesis, and vascularize tissue regeneration.[3 ]
GTR membranes are classified according to their degradation properties, specifically
as resorbable and nonresorbable. Resorbable membranes are preferred since they eliminate
the need for a secondary surgery for removal. Collagen is a natural material often
used in the making of resorbable membranes since it has good biological activities,
such as hemostasis, biocompatibility, and biodegradability, but it degrades quickly
before the periodontal tissue completely regenerates.[4 ] Various collagen membranes found in the market include pericardium membrane and
cross-linked pericardium membrane.
The pericardial membrane (PM) is a resorbable membrane derived from cow and pig pericardium
containing collagen and has been widely used in periodontal tissue regenerative therapy.
The PM shows a longer and more stable resorption pattern because it comes from a compact
area so its physical and mechanical properties are better, but it shows slow vascular
penetration that will hinder angiogenesis.[5 ] The degradation time of the collagen membranes can be prolonged through cross-linking
methods using physical or chemical procedures, such as ultraviolet light, hexamethylene diisocyanate , glutaraldehyde plus irradiation, and diphenylphosphoryl azide , but the process can reduce the tissue integrity, decrease vascularization and biocompatibility,
and increase the inflammatory response.[5 ]
[6 ] Based on these considerations, in this study the collagen membrane is integrated
with polymer, which is chitosan and polyvinyl alcohol (PVA) expected to be able to
meet the membrane characteristics required in regenerative therapy.
Chitosan in dental medicine is used as a bone regeneration material and to improve
the properties of other dental materials. Chitosan is a natural polymer of the polysaccharide
type deriving from crustacean exoskeletons, such as shrimps, crabs, or shellfish.
As a cross-linked agent, chitosan shows good biocompatibility, biodegradability, and
antimicrobial activities.[7 ]
[8 ] Besides that, chitosan is proven to have a pore structure that can facilitate differentiation
and cell proliferation as well as neovascularization.[9 ] However, the weakness of chitosan is its vulnerability, so it is necessary to choose
an additive that is compatible with a high mechanic characteristic, such as PVA.[10 ]
[11 ]
[12 ] PVA is a water-soluble synthetic polymer with very good biocompatibility, can be
easily processed, and is biodegradable with superior mechanical properties, so it
can be used as a blend to enhance the mechanical characteristics.[13 ]
[14 ]
The collagen mixture with polymer has the potential to be developed as the alternative
membrane for GTR. In a previous study, physical and mechanical properties were evaluated
in the collagen-chitosan-PVA membrane (CM), which had been irradiated with gamma rays.
The results indicated that gamma-ray irradiation had a positive effect on enhancing
the mechanical properties of the membrane. Additionally, scanning electron microscopy
morphological analysis revealed the presence of microstructural pores, which could
facilitate vascularization.[10 ]
No studies have been conducted on CMs associated with GTR, prompting an in vivo investigation on Wistar rats to investigate their impact on periodontal tissue regeneration.
This study aims to examine biodegradation, angiogenesis, and the inflammatory response
in CMs.
Materials and Methods
This study is an experimental study with a randomized control trial design using animals
as the subjects, which are 51 Wistar rats calculated with the Federer formula previously.
The rats selected were Wistar male white rats, with a weight of 150 to 250 g, aged
6 to 8 weeks, and in good health condition marked with active movement, responding
well when stimulated, no anatomy disorder, and no hair loss. Rats experiencing weight
loss, illness, or death during acclimatization were not involved in this study.
This study has obtained ethical approval from the Health Research Ethical Committee
of the Faculty of Medicine of Universitas Jenderal Achmad Yani (Unjani) Cimahi with
the number 001/UH4.08/2022. The use of animals as the subjects of the research considers
three main principles regarding animal use, which are to substitute humans with rats
as the subjects of the research because they have almost similar physiological conditions
with humans (replacement), to determine the limited quantity of animals used (reduction),
and to treat the animals used properly or ethically to meet the concept of animal
testing, which is to avoid pain in them (refinement).
Fifty-one Wistar rats were randomly divided into three test groups and two control
groups. In the test groups, Wistar rats were treated by placing CM, PM, and cross-linked
pericardial membrane (CLPM). The negative control (NC) group of Wistar rats only went
through surgery without placing the membranes, while the normal control group did
not receive any treatment. Subsequently, these Wistar rats were terminated on days
0, 7, 14, and 30 to assess the level of biodegradation, the quantity of newly formed
blood vessels, and the inflammatory response.
Surgery Procedure
The experimental animals involved in this study had gone through the adaptation time
for 1 week at the Laboratory of Animal Experiments of the Faculty of Medicine of Unjani.
Those animals then went through rescreening 1 day before and 1 day after the treatment.
Prior to the procedure, the rats were cleaned, and they were anesthetized using ketamine
hydrochloride with a dose of 45 mg/kg of body weight and xylazine as much as 0.35 mg/kg
of body weight intramuscularly at the abdominal muscles. The incision was made at
the dorsum as long as 10 mm up to the subcutaneous layer under the dermis, and then
for the test group the CM membrane, PM, and CLPM were placed, followed by simple interrupted
suturing of the incision area.
Termination was performed by putting the rats into a chamber with CO2 inhalation, and then the membranes and tissue surrounding them were taken. The CM
group, PM group, CLPM group, and NC group were terminated on days 0, 7, 14, and 30,
while the normal control group (N) was terminated on day 0.
Membrane Biodegradation Analysis
Membrane biodegradation is the process of lacking the membrane thickness due to the
enzymatic activity. The application area on the rat's dorsum was cut transversally
with the size of 12×12×5 mm and taken, and then the remaining of membrane thickness
was measured using the Image J computer program on days 0, 7, 14, and 30.
Histological Analysis
The membranes and surrounding tissue were fixed in 10% neutral-buffered formalin solution
for 24 hours, and then we used the solution consisting of alcohol 70%, alcohol 80%,
alcohol 90%, alcohol anhydrides, toluene, and paraffin wax for dehydration and classification,
gradually over 1 day. The organ sample was sealed with an embedded device filled in
liquid paraffin and refrigerated. A microtome with a thickness of 5 μ m was used to slice the cold block. These sections were stained with hematoxylin and
eosin for histopathological analysis..
The number of new blood vessels formed was calculated manually under a binocular microscope
at 400× magnification from 5 fields of view of slide preparation. The inflammatory
response was observed through the calculation of the spread of the inflammatory cells,
which are eosinophil, monocyte, neutrophil, lymphocyte, and mast cells under the binocular
microscope at 100× magnification from 5 fields of view of slide preparation.
Statistical Analysis
In this study, the data obtained consisted of quantitative measurements. The normality
of the data was tested using the Kolmogorov–Smirnov test, which indicated that the
data were not normally distributed. Therefore, the analysis was performed using a
nonparametric analysis of variance, specifically the Kruskal–Wallis test. Subsequently,
post hoc analysis using the Mann–Whitney test was conducted to determine which groups
differed significantly at each observation time. The significance of the results was
assessed based on a p -value of < 0.05.
Results
The membrane biodegradation rate was calculated by measuring the remaining membrane
thickness. The measurement results in the CM, PM, and CLPM on days 0, 7, 14, and 30
are presented in [Fig. 1 ]. On day 7, the PM and CLPM groups were degraded completely, while the CM group remained
intact until day 30.
Fig. 1 The mean value of membrane thickness for Collagen-chitosan-polyvinyl alcohol membrane
(CM), Pericardial membrane (PM), and Crosslinked pericardial membrane (CLPM) on days
0, 7, 14, and 30.
Angiogenesis began on the 7th day in both the experimental and control groups. The
average number of the quantity of new blood vessels formed in the CM group was higher
compared with the PM group, CLPM group, and NC group in all the time of the observation,
where it shows that there is a significant difference statistically, with p -value < 0.05, as presented in [Fig. 2 ].
Fig. 2 The mean value of new blood vessels in the negative control (NC), collagen-chitosan-PVA
(CM), pericardial (PM), and cross-linked pericardial (CLPM) membrane groups (***:p < 0.001; **:p < 0.01; *:p < 0.05; ns: insignificant difference).
Based on the post hoc test, on day 7, there was a significant difference between the
CM group and NC group (p = 0.001) and the PM group (p = 0.027), but there was no significant difference between the CM group and CLPM group
(p = 0.546). On day 14, there was a significant difference between the CM group and
NC group (p = 0.001) and the CLPM group (p = 0.024), but there was no significant difference between the CM group and PM group
(p = 0.609). On day 30, there was a significant difference between the CM group and
NC group (p = 0.004), but there was no significant difference between the CM group and PM group
(p = 0.168) or CLPM (p = 0.110).
In the histological image, it can be observed that the distribution of new blood vessels
in the CM group is greater compared with the NC, PM, and CLPM groups, as shown in
[Fig. 3 ].
Fig. 3 The histological picture in the NC, CM, PM, and CLPM groups on the 7th day with the
coloring of Hematoxylin Eosin with the enlargement of 400X; the blue arrow mark shows
new blood vessels.
The inflammation response indicates how the tissue reacts to the membrane, as observed
through eosinophils, neutrophils, monocytes, lymphocytes, and mast cells. Initially,
on day 0, the CM group had the highest average number of inflammatory cells compared
with the N, NC, PM, and CLPM groups, and it was significant statistically (p = 0.000). Along with the time progress of the observation, in the CM group the quantity
of inflammatory cells tended to decrease compared with the NC, PM, and CLPM groups,
which experienced an increase, and this can be seen in [Fig. 4 ]. This pattern was further evident in the average quantity on day 30, where the CM
group had fewer inflammatory cells compared with the NC, PM, and CLPM groups, and
it was significant statistically in the eosinophil cell (p = 0.005), neutrophil cell (p = 0.000), lymphocyte cell (p = 0.000), and monocyte cell (p = 0.000), but it was not significant statistically in the mast cell (p = 0.058).
Fig. 4 The mean value of inflammatory cells in the normal control group (N), negative control
(NC), collagen-chitosan-polyvinyl alcohol membrane (CM), pericardial membrane (PM),
and crosslinked pericardial membrane (CLPM) on days 0, 7, 14, and 30).
In the histological picture on the 30th day, the amount of the inflammatory cell spread
in the CM group was less than that in the NC, PM, and CLPM groups, and it can be seen
in [Fig. 5 ].
Fig. 5 The histological picture in the CM, PM, and CLPM groups on the 30 th day with the
coloring of Hematoxylin Eosin with the enlargement of 100X; the blue arrow mark shows
the inflammatory cells.
Discussion
This study compares the biodegradation rate, quantity of new blood vessels, and inflammatory
response through the observation of inflammatory cell spread between the CM, the PM,
and the CLPM inserted into the experimental Wistar rats. The ideal membrane characteristics
that are required to support the success of GTR, include the ability to maintain space
during the periodontal tissue regeneration process, facilitate the proliferation of
regenerative cells, enhance angiogenesis and vascularization for tissue regeneration,
ensure good biocompatibility, and possess adequate physical and mechanical properties.[3 ]
[4 ]
[15 ]
The findings regarding membrane biodegradation rates indicate that the CM degrades
more slowly than both the PM and the CLPM. The addition of PVA to the CM can enhance
its mechanical properties by improving tensile strength and elongation at break, which
influences the degradation process.[10 ] Our results are in line with a previous study from Zhou et al[16 ] that added PVA to the collagen-PVA membrane to slow down the degradation time. Zhuang
et al[17 ] also reported that there is an increase in tensile strength in a wet condition by
adding PVA to the chitosan membrane. Tensile strength and elongation at break indicate
that the strength and elasticity of a membrane become important physical parameters
in supporting its application.[18 ] The biodegradation rate of the GTR membrane has to be able to be controlled at least
4 to 6 weeks along with the healing rate to achieve the optimal periodontal tissue
regeneration.[15 ]
[19 ]
[20 ] This is consistent with research findings indicating that the CM remains intact
until week 4.
Ideal GTR membranes, besides having an appropriate degradation rate for periodontal
tissue growth, also stimulate the differentiation and proliferation of tissue regeneration
cells, including new blood vessels that supply oxygen and nutrients to the growing
tissue. In the wound-healing process, angiogenesis begins on day 7, where endothelial
cells migrate to the extracellular matrix area temporarily in the wound area, which
will then form new blood vessels.[21 ]
[22 ]
[23 ] The study results show that new blood vessels were already formed on the 7th day
in all groups with the average quantity of new blood vessels in the CM group being
much more than that in the PM and the CLPM groups. Rothamel et al,[24 ] in their study on the cow PM, reported that pericardial tissue has good physical
and mechanical properties, but it shows slow vascular penetration. Previously, Rothamel
and colleagues[24 ] reported that there is retardation of the angiogenesis process in the pericardial
collagen membrane because the PM structure is thicker without any pores, while the
collagen membrane structure has more pores. Based on that, the angiogenesis pattern
can be influenced by the membrane structure.
The PM originates from a compact area, resulting in slow vascular penetration which
impedes angiogenesis.[5 ] Porosity in the membrane allows for the infiltration of nutrients into the defect
area, which can support tissue regeneration. Chitosan can be produced in a porous
structure enabling to do cell proliferation, cell migration, nutrient exchange, and
angiogenesis. Based on the previous study, the CM shows a microarchitecture of pore,
with pores sizes ranging from 1 to 50 µm, and pore interconnectivity, potentially
providing space for vascularization.[3 ]
Another GTR membrane characteristic that must be owned is good biocompatibility to
enable integration with host tissue without causing an inflammatory response or immunity
response to appear, which can disturb healing and cause harm to patients.[15 ]
[25 ] The inflammatory cells involved in wound healing are primarily polymorphonuclear
leukocytes (PMNs). Leukocytes can be found in the inflammation, proliferation, and
remodeling phases. During the inflammation phase, leukocytes migrate to the extravascular
area guided by chemotactic mediators released by damaged tissues. In this phase, PMNs
play a significant role in the phagocytosis process. Neutrophil is an inflammatory
cell appearing for the first time after injury, and it has the ability to produce
mediators that kill bacteria. Neutrophils are particularly prominent in the inflammatory
and early proliferation phases.[26 ]
[27 ]
[28 ] PMNs that are seen up to the end of the proliferation phase and the remodeling phase
can be the indicator of the persistent inflammation reaction, and if this is allowed
to continue, it will become chronic inflammation; meanwhile, the macrophage is discovered
in all three phases of wound healing and plays a greater role in the proliferation
and the remodeling phase. If leukocytes are still abundant at the end of the proliferation
and the remodeling phase, it is possible that the inflammatory process is still ongoing
and tissue damage is still occurring.[29 ]
[30 ]
Based on the data, in the CM group the quantity of cells decreases over time, while
in other groups, there are fluctuations of increase and decrease. Therefore, it can
be concluded that the wound-healing process in the CM group is relatively faster compared
with the PM and CLPM groups. This is in line with the study of Kusumastuti et al[31 ] who stated that the number of inflammatory cells will keep decreasing along with
the time because the wound-healing process is taking place. This wound-healing process
will be hindered if inflammatory cells keep increasing. Wahyuningsih[32 ] reported that the mast cell given in the castor bean plant extract experienced a
decrease in the number of cells faster. This shows an indication that the wound-healing
process is faster.
The chitosan content in the CM has a strong anti-inflammatory activity, which can
inhibit the growth of bacteria and fungi.[32 ] The study by Hartatiek et al[33 ] showed that there is an antibacterial activity decrease of Escherichia coli and Staphylococcus
aureus in the nanofiber composite of collagen-chitosan-PVA along with the chitosan concentration
decrease. Zheng et al[34 ] reported that the anti-inflammatory activity occurs in chitosan because of its polycationic
trait and the impermeable membrane is formed due to the interaction between the cationic
group in chitosan and the anionic group on the cell surface that can hinder nutrition
transport. Chitosan with low molecule weight can enter the bacterial cells, destroying
microbe cells and disturbing the metabolism.
In addition to its antibacterial effects, chitosan also has an antiallergic effect
by inhibiting histamine release. Eosinophil and mast cells play a role in the allergic
process. The granule in the mast cell contains histamine, when reacting with an antigen,
the mast cell releases its granule content indicating that the increase of the mast
and eosinophil cell quantity shows that there is an allergic reaction.[29 ] In this study, the quantity of eosinophil and mast cells in the CM group decreased
over time, while in the PM and CLPM groups, it increased. This indicates that there
was no allergic reaction in the collagen-chitosan-PVA.
The CM is expected to serve as an alternative membrane in the GTR process. However,
in this study, it was limited only to soft tissue without involving the hard tissue,
therefore, further study is necessary to investigate bone defects in experimental
animals to discover its influence toward the alveolar bone remodeling process.
Conclusion
Biodegradation, angiogenesis, and the inflammatory response in CMs showed better results
compared with other membranes. CMs exhibit potential for application in GTR treatment
for periodontal disease.
