Expression of bone morphogenetic protein-2 after using chitosan gel with different molecular weight on wound healing process of dental extraction

Background: Bone morphogenetic protein-2 (BMP-2) is bone stimulator which capable of inducing differentiation of mesenchymal cells into osteoblast, stimulating bone formation in wound healing process of dental extraction. Chitosan is polymer composed N-acetylD-glucosamine unit that has been used in various applications in wound healing process and bone tissue engineering. Purpose: The objective of this research was to analyzed expressions of BMP-2 for 7,14 and 21 days after using chitosan gel with different molecular weight on wound healing process of dental extraction. Method: The research was an experimental laboratory study. Rattus nornegicus strain wistar male, aged 8-16 weeks, divided into 3 treatment groups namely group 1 and II which given chitosan gel 1 % with high and low molecular weight and group III as control which were not given chitosan gel. Chitosan gel were applied into the socket of dental extraction. Rat was decaputated 7,14 and 21 days after chitosan gel application and the jaw in the treated regions and control group were cut for immunohistochemical examination to observe BMP-2. Data were analyzed using ANOVA test. Result: The result of this research showed significant differences on BMP-2 for 7,14 and 21 days observation (p<0,05). The increasing of BMP-2 were found in the group which given chitosan gel with high molecular weight. Conclusion: It may be concluded that chitosan gel with high molecular weight can enhance the expresion of BMP-2 on wound healing process of dental extraction.


introduction
Bone morphogenetic protein (BMP)-2 has been widely used as an effective growth factor in bone tissue engineering.It is a member of the transforming growth factor-β (TGFβ) superfamily of multifunctional cytokines which has remarkable ability to induce bone formation and bone tissue reconstruction.It plays critical roles in osteogenesis and bone metabolism. 1 The bone matrix is rich in growth factors, among which bone morphogenetic proteins (BMP s ), that are synthesized and secreted by osteoblast and incorporated into the matrix during bone formation.The BMP s , released during osteoclastic bone resorption, are capable of inducing differentiation of mesenchymal cells into osteoblasts (osteinduction), stimulating bone formation in both remodeling and repairing process. 1,2hitosan is carbohydrate copolymer composed of glucosamine and N-acetyl-D-glucosamine units.Chitosan has a number of properties including biocompatibility, biodegradability, mucoadhesiveness and wound healing capabilities that make it useful as a biomaterial. 3Chitosan is very versatile and can be prepared as films, gels, sponges and other forms and has been used in various applications including wound healing and bone tissue engineering. 2,3iological activity of chitosan depend on their molecular weight and deacetylation degree.The deacetylation degree and molecular weight are important parameter, which could influences the performance of chitosan in many its application.The deacetylation degree is related to the ability of chitosan to form isoelectric interaction with other molecules.The deacetylation degree more than 75% will make more amide group formed, so chitosan will become have high chemical reactivity, and be able to interact with proteins and other organic matrices, such as anionic glycosaminoglycans and proteoglycans, and extracellular matrix macromolecule.The molecular weight is the sum of the atomic masses of molecule. 4,5It is one factor that determine the mucoadhesive properties.Chitosan with high molecular weight powder become chitosan gel with good physical characteristic, higher viscosity, neutral pH and have good mucoadhesive. 6Chitosan is able to increase transforming growth factor beta 1 (TGF β1), platelets release transforming growth factor (PDGF), fibroblasts growth factor-2 (FGF2), BMP expression of mRNA on the seventh day. 6,7In previous study, injectable chitosan gel has been found significantly enhance the osteoblastic differentiation of mouse osteoblast precursor cells.Chitosan gel formulation can release BMP-2 that enhance osteoblastic activity of bone cells by measuring alkaline phosphatase (ALP) specific activity of preosteoblast mouse bone marrow stromal cells. 8MP-2 has been widely investigated for bone healing due to its properties.BMP-2 recruits stem cells to the bone healing, promotes angiogenesis and causes differentiation of stem cells into osteoblast. 3,6In the wound healing process of dental extraction, it have different phases of alveolar healing were recognized by histological examination in socket.At the end of 1 st week, the socket were filled granulation tissue and there was osteoid matrix deposition.At the end of 2 nd week, there was progressive bone formation, and the end of 3 rd week, most socket filled with thicker bone trabecula surrounding interconnecting space. 2 The increasing expression of BMP-2 on wound healing process of dental extraction could be achieved by using chitosan gel.The aim of this study was to analyzed expressions of BMP-2 for 7, 14 and 21 days after using chitosan gel with different molecular weight on wound healing process of dental extraction.
Chitosan gel 1% (w/v) was made with diluted one gram of chitosan powder in 100 ml acetic acid 2%.It added with NaOH 1,25% solution to get neutral pH.The mixture was stirred until the gel was completely formed.After homogenization, the gels were stored in closed containers at ambient temperature until use.The characteristic of chitosan gel was evaluated includes solubility, pH, viscosity, physical characteristic, homogeneity, consistency, and duration of storage time.The homogeneity test of gel carried out using glass plates after the powder diluted in acetic acid 2%.It should be observed on optimized homogeneous.Consistency test could be done by using a penetrometer or mechanically sentrifugator.Gel without precipitation will produce a good consistency.Physical characteristic test or organoleptic analysis during the storage time includes change of colour, form of formulation gel and odorless.Measurement of viscosity using viscometer Ostwald (capillary method). 8,9he research was an experimental laboratory study.Rattus nornegicus strain wistar male, aged 8-16 weeks, divided into 3 treatment groups namely group 1 and II which given chitosan gel 1% with high and low molecular weight and group III as control which were not given chitosan gel.0.1 ml chitosan gel were applied into the socket of dental extraction.Rat was decaputated 7, 14 and 21 days after chitosan gel application and the jaw in the treated regions and control group were cut for immunohistochemical examination to analyze expression of BMP-2.Fixation was performed using 10% buffer formalin and decalcification applying eDTA.Further process was dehydration and continued by clearance.The tissue could be cut using microtome in 4-6 µm thickness.Deparafin and rehydration were subsequently performed.BMP-2 monoclonal antibody was diluted by antibody diluents.It was washes by PBS.Streptavidin-biotin was dropped and incubated for 30 minutes, washed by PBS.Counterstained using haematoxyline and washed by flowing water and dried.It was given entelan and covered by cover glass.Light microscope was applied and the evaluation was done.The measuring result were analyzed using Anova test.It analyzed the comparison between chitosan treated with high molecular weight group, lower molecular weight group and the control groups (p<0.05).

results
Table 1 shown the evaluation of chitosan gel formulation.Chitosan gel considered as ideal formulation forms for wound healing dressing could have good characteristic and easy to applicate on dental socket.The expressions of BMP-2 for 7, 14 and 21 days observation after using chitosan gel with different molecular weight on wound healing process    11       of dental extraction shown in Figure 1, 2 and 3.The region which would be evaluated and observed in third apical socket.The expressions of BMP-2 in third apical socket was done 400 times magnification in all sub groups.In our study, the expressions of BMP-2 on wound healing process of dental extraction using chitosan was more increasing compared to the control group.The expressions of BMP-2 in 7, 14 and 21 days observation after using chitosan gel with high molecular weight was more increasing than the other groups.
Table 2 and Figure 4 shown the mean and standard deviation of each group from immunohystochemistry study done in 7, 14 and 21 days after treatment.The expressions of BMP-2 for 7, 14 and 21 days observation after using chitosan gel with different molecular weight on wound healing process of dental extraction.The data was analyzed using Kolmogorov-Smirnov statistical test.It showed normal distribution (p>0.05) in which fulfiiling the requirement of parametric test.The Anova test showed, expressions of BMP-2 in treatment group using chitosan gel with high molecular weight were significant difference compared to the treatment group using chitosan gel with low molecular weight and control group (p<0.05).

discussion
The proliferation and differentiation of sufficient progenitor cells are critical for bone healing.These processes are regulated by growth factors such as TGF-β and BMP S .BMP-2 can efficiency induce bone formation.Many materials, especially the natural polymers, have been researched for controlled release of BMP-2. 10,11Chitosan is naturally derived polysaccharide.It has gained much attention as biomaterial in tissue engineering application, due to its antimicrobial activity and biocompatibility. 10itosan is reported to be biocompatible, biodegradable and non toxic, having some biomedical properties such as healing accelerator, antifungal, hemostatic, antimicrobial and analgesic.Several study have shown the activity of chitosan in healing process, where it enhanced the infiltration of inflamamatory cells in the injured area and release growth factor. 6,12Chitosan is the partially deacetylated form poly-(1,4)-2-amino-2-deoxy-D-glucose) of chitin, and is a potential biomaterial for bone tissue engineering application. 1,10,12In the present study, the chitosan powder was mixed with blood of each person and filled in dental socket, and it found that bone tissue regeneration will be faster in chitosan-filled socket than untreated dental socket. 13n our study it was used chitosan gel material with different molecular weight to analyzed expressions of BMP-2 Rattus norvegicus on wound healing process of dental extraction.It showed the expressions of BMP-2 in 7 and 14 days observation after using chitosan gel was increasing and decreasing at 21 days observation.Chitosan

GROUP
is a polycationic complex carbohydrate with a structure similar to those of glycosaminoglycans specifically hyaluronic acid.Hyaluronic acid is thought to facilitate the migration and proliferation of progenitor cells.Previous studies have demonstared that chitosan might function as a substrate that enhances the migration and differentiation of osteoblast cells and conversely, it might inhibit the function of fibroblast and so indirectly facilitate osteogenesis. 14,15ts biological properties of chitosan, including chitosan biodegradation by lysozim that can change chitosan polymer form (N-acetyl-D-glucosamine) to dimer active form.N-acetyl-D-glucosamine dimer active form crosslinked with matrix macromolecules extracellular as well as stimulate increased TGF-ß and BMP expression of mRNA. 6,15Chitosan loading rhBMP-2 can facilitate bone regeneration of periodontal tissue.In the cavities filled with chitosan, not only the bone regeneration was faster, but also the density was similar to the density of the bone of the subject under study. 13ccording to the Figure 4, the expressions of BMP-2 in 7 and 14 days observation after using chitosan gel with high molecular weight was more increasing than the other groups.Chitosan was the material used to form gels due to its good gel forming property, biodegradability, biocompatibility and non toxic.Gels could be considered as ideal pharmaceutical forms to treat the dental socket.It have the ability to absorb exudates, which moisture on the wound surface.Besides, gels have high water vapor and oxygen permeability, as well as mechanical properties that resemble physiological soft tissue.Chitosan gel has a strong tissue-adhesive property. 12,17,18The characteristic of chitosan gel is related with its molecular weight.The absorbtion mechanism also depend upon the particle size of chitosan powder.The molecular weight related to particle size of powder.Molecular weight and particle size has been linked with viscosity, low molecular powder which small particle size will result formulation with a low viscosity.0][21] The viscosity of chitosan gel with high molecular weight more higher than formulation with low molecular weight.It have high viscosity and better mucoadhesive properties due to strong blood clot is formed in order to eliminate complications of dental extraction such as dry socket. 19,20,22Chitosan with high molecular weight have more biodegradable induce N-acetyl-Dglucosamine dimer active of chitosan cross-linked with glycosaminoglycan and glycoprotein that part of matrix macromolecules extracellular as well as stimulate increased BMP-2 that is growth factor of bone healing.However, it can promote the accleleration of wound healing process on dental socket. 15,16,22MP-2 can efficiently induce bone formation.It has been widely use as an effective growth factor in bone tissue engineering.Using C1C12 myoblast cells as in vitro models, the enhanced bioactivity of BMP-2 was attributed primarily to the stimulation of 6-O sulfated chitosan.A low dose of 6-O sulfated chitosan showed significant enhancement alkaline phosphatase activity and mineralization induced by BMP-2, as well as the expression of ALP and osteocalcin mRNA. 23ALP is an enzyme regarded to be important in the process of mineralization, since high levels of ALP would promote hydrolysis of phosphate, produce orthophosphate and increase the calcium deposition.ALP activity and calcium deposition are widely used as marker for early and late differentiation of osteoblast cells, respectively. 15ature osteoblast produce mineralized bone matrix, and the component of this matrix, such as calcium deposition and osteogenic markers (osteocalcin and osteopontin), are usually used as markers for late stage of osteoblast differentiation.Osteocalcin and osteopontin expression marks the late stage of osteoblast differentiation, and BMP-2 is an important factor for osteoblast maturation and osteoblast activity. 2,3,15In our study, the expressions of BMP-2 in 21 days observation after using chitosan gel with high molecular weight was decreasing.Probably, the activity of ALP has decreased.There was most socket filled with thicker bone trabecula surrounding interconnecting space in the end of the 3nd week.The major proportion of bone formation and the maximum mineral density has occurred in the end of the 2 nd week.In the end of the 2 nd week the activity of ALP has decreased 2 The using of chitosan gel with high molecular weight could increasing the expression of BMP-2.BMP-2 is major growth factor in bone healing.It may be concluded that chitosan gel with high molecular weight could accelerate bone healing process of dental extraction.

Figure 1 .
Figure 1.The expressions of Bone morphogenetic protein-2 (BMP-2) at 7 days observation, magnification 400x.(A) Control group, without using chitosan; (B) Treatment group using chitosan gel with high molecular weight; (C) Treatment group using chitosan gel with low molecular weight.

Figure 2 .
Figure 2. The expressions of Bone morphogenetic protein-2 (BMP-2) at 14 days observation, magnification 400x.(A) Control group, without using chitosan; (B) Treatment group using chitosan gel with high molecular weight; (C) Treatment group using chitosan gel with low molecular weight.

Figure 1 .
Figure 1.The expressions of BMP-2 at 7 days observation, magnification 400x.(A) Control group, without using chitosan; (B) Treatment group using chitosan gel with high molecular weight; (C) Treatment group using chitosan gel with low molecular weight.

Figure 1 .
Figure 1.The expressions of Bone morphogenetic protein-2 (BMP-2) at 7 days observation, magnification 400x.(A) Control group, without using chitosan; (B) Treatment group using chitosan gel with high molecular weight; (C) Treatment group using chitosan gel with low molecular weight.

Figure 2 .
Figure 2. The expressions of Bone morphogenetic protein-2 (BMP-2) at 14 days observation, magnification 400x.(A) Control group, without using chitosan; (B) Treatment group using chitosan gel with high molecular weight; (C) Treatment group using chitosan gel with low molecular weight.

Figure 2 .
Figure 2. The expressions of BMP-2 at 14 days observation, magnification 400x.(A) Control group, without using chitosan; (B) Treatment group using chitosan gel with high molecular weight; (C) Treatment group using chitosan gel with low molecular weight.

Figure 3 .
Figure 3.The expressions of Bone morphogenetic protein-2 (BMP-2) at 21 days observation, magnification 400x.(A) Control group, without using chitosan; (B) Treatment group using chitosan gel with high molecular weight; (C) Treatment group using chitosan gel with low molecular weight.

Figure 3 .
Figure 3.The expressions of BMP-2 at 21 days observation, magnification 400x.(A) Control group, without using chitosan; (B) Treatment group using chitosan gel with high molecular weight; (C) Treatment group using chitosan gel with low molecular weight.

Figure 4 .
Figure 4.The graphic of expressions of BMP-2 on 7, 14 and 21 days using chitosan gel wi low molecular weight group and control group.

Figure 4 .
Figure 4.The graphic of expressions of BMP-2 on 7, 14 and 21 days using chitosan gel with high and low molecular weight group and control group.

Table 2 .
The mean and standard deviation BMP-2 of each group at 7, 14 and 21 days after treatment

Table 2 .
The mean and standard deviation BMP-2 of each group at 7, 14 and 21 days after treatment

Table 2 .
The mean and standard deviation BMP-2 of each group at 7, 14 and 21 days after