The Effect of Non Freeze-dried Hydrogel-CHA on Fibroblast Proliferation

Bone damage can be caused by variety of surgical procedures. Bone reconstruction has been developed lately is tissue engineering techniques. One of materials that proved to be effective as a scaffold in tissue engineering is a hydrogel. The addition of carbonate apatite (CHA) will produce a hydrogel-CHA material which is believed to improve the mechanical properties and biological similarities with the original bone. Scaffold is considered an important aspect in the field of tissue engineering, because it’s ability to mimic extracellular matrix of the damaged tissue. Fibroblasts are mesenchymal cells that can be readily cultured in the laboratory and play a significant role in epithelial-mesenchymal interactions, secreting various growth factors and cytokines. On certain condition, Fibroblast will differentiate into bone-forming cells, osteoblasts. Objective: to determine the effect of non freezedried hydrogels CHA on the number of fibroblasts. Methods: In the treatment groups (hydrogel and hydrogelCHA group), the static seeding, where cells and scaffolds were simply brought into contact, was performed. The other group contained only cells and growth media. Cells were seeded at a density of 2x104 cells/ml in a 96-well plate. Number of fibroblasts cell in each group was observed by light microscopy and quantitified by MTT assay on days 1, 2 and 3 post-application. Results: Proliferation of fibroblasts increased significantly on day 3rd after application of non freeze-dried hydrogel CHA (p< 0.05). Conclusion: Application of non freeze-dried hydrogel CHA may induce fibroblasts proliferation.


INTRODUCTION
Bone tissue has a variety of functions; advocates posture, protective vital organs such as those found in the skull and the chest cavity, bone marrow reservoir as a maker of blood cells, including as a means of passive motion. In addition, bone is also a backup storage of calcium, phosphate, and other ions. This specialized connective tissue composed of calcareous material between cells, ie bone matrix and 3 types of cells: osteocytes, osteoblasts, and osteoclasts. 1 Defects in bone tissue are the most common problem encountered in health fields, including in dentistry. Loss of alveolar bone as tissue structures of supporting teeth can lead to tooth loss, impaired chewing, complicates the installation of prosthesis, and worsen the appearance Large bone defects (critical defect) as a result of the variety of surgical procedures, trauma or variety of congenital and degenerative diseases are not able to regenerate, so that It is necessary to do scientific and clinical reconstruction. And also, it provides the physical and psychological effect on patients' lives. 2,3 Therapy often used to treat bone defects are the application of bone graft and barrier membrane, which are to induce new bone growth. 4 A Bone graft can be classified based on the origin of the donor, ie autograft, allograft, xenograft, and alloplastic. Autograft bone is a patient's bone and is the gold standard of bone graft substitutes. The weakness of the autograft is limited in getting the appropriate graft and causing injury in the donor area due to the operation to be performed. Allograft derived from other person, xenografts derived from animals at risk of disease transmission from donor to recipient. Alloplastic have drawbacks related to biocompatibility because the body's reaction to a foreign body may occur. Alloplastic has drawbacks related to biocompatibility because the body's reaction to a foreign body may occur. 5 Application of Barrier membrane is direct cells replenish the defect space with Guided Tissue Regeneration known (GTR) and Guided Bone Regeneration (GBR). This technique facilitates bone formation due to the barrier membrane will prevent fibrous connective tissue to grow into the defect space and allow only the cells of the bone tissue to fill the defect space. 6 However, GTR has the disadvantage associated with inadequate flap closure so that GTR treatment indication is very limited. 7 Advanced bone reconstruction is performed with tissue engineering techniques. One of the factors necessary for the cells can proliferate and differentiate the scaffold with cells that can be planted well. The ideal scaffold must be effectively transport osteoprogenitor cells, capable of providing mechanical support biocompatible and biodegradable. 8,9 One type of materials that is effectively used as scaffolds in tissue engineering is hydrogels.
Hydrogels can serve as a scaffold that provides structural integrity for bone formation, and have a high degree of tolerance when in contact with body tissues and fluids. 10,11 Hydrogels have a similar structure and composition of the extracellular matrix. The composition and structure of the hydrogel as a scaffold is expected to function as an artificial extracellular matrix. Extracellular matrix is a hydrophilic matrix 3D micro with two main structures: collagen fibers and filaments proteoglycans. 11 In the present study, the hydrogel that we used non-freeze dried hydrogels. The method of freeze-dried produce porous hydrogel scaffold. The drawback of the technique of freeze dried is a high degree of difficulty in controlling the porous. So it will be difficult to get a large porous size. 12 Although, the method of non-freeze dried, just dried hydrogel membrane with a temperature of 40 C in the refrigerator for four days. Using this method, the scaffold will have a lower porous. This can improve the performance of the scaffold as a barrier membrane better because smaller porous can prevent the infiltration of the networks that inhibit bone reconstruction, such as connective tissue. 7 Carbonate Apatite (CHA) with the chemical formula CA 10 (PO 4 )6 (CO 3 ) is an effective component of bone mineral, is used as a bone substitute material because of its chemical composition resembles bone tissue and inorganic materials resorption rate is quite high. 13,14 The incorporation of hydrogels and CHA aims to form a 3D scaffold with an interconnected porous structure that facilitates cell migration so that not only stick and spur the proliferation of fibroblasts. 15 Fibroblasts are the most common cells of connective tissue that play a role in synthesize collagen, elastin, glycosaminoglycans, proteoglycans, multiadhesive glycoproteins. Fibroblasts used in this study because it is non-immunogenic, easily bred, available and quickly grow. Fibroblasts also have non-tumorigenic fibroblastic cell line properties, namely the potential to differentiate into osteoblastic cells. It is suitable for application in bone tissue engineering. 1,16 Non-freeze dried hydrogels-CHA was expected to be applied to the bone defect. These applications will result in a hydrogel-CHA direct contact with other cells that are on the oral tissues, such as fibroblasts. The aim of this study was to determine the effect of non freezedried hydrogels -CHA on the number of fibroblasts.

METHODS
Non freeze-dried hydrogels-CHA preparation Solid hydrogels removed from the refrigerator 4°C and allowed to stand for 30 minutes at room temperature. Brief ly solid hydrogels weighing 0.5 grams and mixed with 5ml of distilled water, wait until all of the hydrogels to absorb water and swell (swelling). Then added gradually 0.2048ml phosphoric acid solution that has been diluted with 5ml distilled water. Once the hydrogel-CHA homogeneous neutralization process is carried out in order to obtain a neutral pH. Non freeze-dried hydrogel-CHA was made by disc blocks with a diameter of 1.6 cm and a weight of 150 mg. Furthermore, hydrogel-CHA stored in the freezer with -210C temperature for 24 hours and then freeze drying is done until the non freeze-dried hydrogel-CHA regardless of the block mold. 9 The process followed by cross-linking using the method hydrothermal (DHT) 72 hours 140 o C.

In vitro cell proliferation
Human fi broblasts that taken from male circumcision were a culture in DMEM medium supplemented with 10% FBS, 2ml Penicillin Streptomycin, and fungizone as much as 0.5ml, incubated under 5% CO 2 at 37 o C. Membrane hydrogel only and or hydrogel membrane-incorporated CHA sterilized in 96 wells in the microplate wells using sterile tweezers. After that, fi broblasts with a concentration of 2 x 104 cells/ml of microplate wells included in each using a micropipette.
Fibroblasts were grown on non-freeze dried hydrogels -CHA membranes with controlled and aseptic conditions. Observation of the development and proliferation of fi broblasts was monitored with a digital inverted microscope on day 1, 2, and day 3.

Cell proliferation test
MTT assay was carried out to determine the number of cells present on the samples. Absorbance was measured at 550nm using an Elisa Plate reader. Cells number were determined after incubation for day 1, 2 and 3 and all experiments were performed in triplicate.

RESULTS
The fibroblasts were divided into 3 groups, i.e., fi broblasts cells only group (A), fi broblasts plus non freeze-dried hydrogel (B), and fi broblasts plus non freeze-dried hydrogel-CHA (C). Each group was divided into 3 subgroups will be observed the fi broblast proliferation on days 1, 2, and day 3 by ELISA reader. Each group contains 2x10 4 cells /ml. Group A is a group given only the media and fi broblasts.
Based on Figure 1 in general showed the phenomenon of the most increasing number of fi broblasts in the group of the given non freeze-dried hydrogen-CHA (C). The increasing number of fi broblasts also showed in fi broblasts only group (A) and the group of fi broblasts plus Non-freeze dried hydrogel (B) but less compare to group C. One-way ANOVA test result showed there was not signifi cance different on day 1 ( p>0.05) but day 2 and day 3 were signifi cance different (p<0.05). This result showed that the application of non freeze-dried hydrogel with or addition-CHA affects an increasing number of fi broblasts. Furthermore, to determine which treatment group had a signifi cant mean difference, further testing is done with Post-Hoc test analysis of LSD (Least Signifi cant Difference).
On day 2, Comparisons between groups A, B and C showed quite different phenomena in the mean number of fi broblasts but statistically were not signifi cance different (p>0.05). On day 3, Fibroblasts cells only group (A) showed significance different ( p<0.05) compare to fi broblasts plus non freeze-dried hydrogel-CHA (C) but not signifi cance different compare to fi broblasts plus non freeze-dried hydrogel (B) (p>0.05). Group fi broblasts plus non freeze-dried hydrogel (B) and fi broblasts plus non freeze-dried hydrogel-CHA (C) showed not signifi cance different (p>0.05).

DISCUSSION
The data analysis has been performed quantitatively by counting the number of fi broblasts. In this study, observations were made on days 1, 2, and day 3. Observation of the length of time for the application according to this research, it has conducted by previous studied. 16 They said that the fi broblasts did not change the morphological characteristics after incubation days 1, 2 and day 3. Another studied determined that during the process osteoblastic cells grown on the scaffold and suffered proliferation, differentiation, and promotes bone mineralization. 17 In the overall result study showed the phenomenon of an increase in the number of fibroblasts either the 1st, 2nd and 3rd day after application of nonfreeze dried hydrogel only and or non freeze-dried hydrogel-CHA compared fibroblasts without treatment. This is consistent with previous studied which stated that an increase in cell proliferation on given hydrogel treatment compared with not given treatment hydrogel. 18 Hydrogel as a scaffold has the capability of swelling. The size of the pores in the scaffold increased to facilitate the cells to migrate and grow in the 3D structure so that not only stick. 15 Porosity will facilitate cell migration because it provides immunoisolation that allows oxygen, nutrients and metabolic products diffuse into the hydrogel. 11 Interconnected porous structures serve to replace the old bone pores with new bone. 8 This study showed that the non freeze-dried hydrogel-CHA group (C) had a mean number of fibroblasts is much more than Fibroblasts cells only group (A) and the group of fibroblasts plus non freeze-dried hydrogel (B). Inorganic and organic composition of the hydrogel-CHA can stimulate fibroblast proliferation more rapidly. This phenomenon is caused by the hydrogel scaffold that can better cell adhesion thereby increasing migration, proliferation, organization, growth, and regulation of cells in tissue regeneration. 11 Other studies mentioned components such as calcium-phosphate CHA likely to be a determining factor in the increase in the swelling ability of the hydrogel, thus increasing the surface area, and the number of cells attached more. In addition, the hydrogel as a scaffold must be able to provide an appropriate microenvironment as initial conditions before the onset of tissue damage or the extracellular matrix. 11 The addition of CHA will provide the appropriate microenvironment to repair damaged bone tissue. The result is in line with our previous study that used vero cell fibroblasts (data not yet published).
Hydrogel-CHA is a porous scaffold is made using the freeze-drying method with thermally induced phase separation by lowering the temperature to encourage the separation of homogeneous polymer solution phase. 19 Dehydrothermal (DHT) is a method of the physical crosslinking covalent inter-chain polypeptide of collagen fibers at high temperature and vacuum. 20 Methods dehydrothermal (DHT) to serve the resulting elimination of water molecules and allowing cross-reactions take place throughout the process. Vacuum conditions required to avoid denaturation of collagen caused by the presence of water. 21 Dehydrothermal crosslinking with a growth rate of cells is much greater than the crosslinking with glutaraldehyde. 21 The combination of freeze drying and dehydrotermal (DHT) aims to provide sufficient mechanical strength to prevent damage to the pores during drying, and produces a matrix with porosity (interconnected porous) more than 90%. 20 Freeze drying and crosslinking can alter osteoblastic response through modification of the scaffold. 17

CONCLUSION
Application of non-freeze dried hydrogel -CHA can increase the amount of fibroblasts proliferation.