AIP Conference Proceedings 2344, 020012 (2021); https://doi.org/10.1063/5.0049156 2344, 020012
© 2021 Author(s).
Optimization of hybrid PVA/hFDM scaffold preparation
Cite as: AIP Conference Proceedings 2344, 020012 (2021); https://doi.org/10.1063/5.0049156 Published Online: 23 March 2021
Rizqa Inayati, Muhammad Suhaeri, Nuzli Fahdia, Melinda Remelia, and Radiana Dhewayani Antarianto
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Optimization of Hybrid PVA/hFDM Scaffold Preparation
Rizqa Inayati
1, Muhammad Suhaeri
2, Nuzli Fahdia
3, Melinda Remelia
4,5, Radiana Dhewayani Antarianto
3,6,a)1Master Program in Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta, DKI Jakarta 10430 Indonesia
2Rumah Sakit Universitas Indonesia, Kampus Universitas Indonesia, Depok, West Java, 16424 Indonesia
3Stem cell and Tissue Engineering Research Cluster, Indonesian Medical Education and Research Institute (IMERI), Universitas Indonesia, Salemba Raya, Jakarta, West Java, 10430, Indonesia
4Doctoral Program in Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta, DKI Jakarta 10430 Indonesia
5Department of Basic Biomedicine, Faculty of Medicine Universitas Kristen Indonesia, Jl Mayjen Sutoyo No 2, DKI Jakarta 13630 Indonesia
6Department of Histology, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta, DKI Jakarta 10430 Indonesia
a)Corresponding author: radiana.dhewayani@ui.ac.id
Abstract. Osteogenic differentiation from Mesenchymal Stem Cell (MSC) to osteoblast has a clinical significance which is very important for treating bone injuries, in the form of femoral fractures with the most cases in Indonesia. Various studies have been conducted to find the best scaffold that can improve osteogenic differentiation, one of which is the development of a hybrid scaffold made from natural biomaterials in the form of the extracellular matrix, and from synthetic biomaterials. The discovery of the best scaffold is not only focused on the source of the scaffold but also requires optimization of the method in making the scaffold. Therefore, the aim of this study is to find out the optimum method for making hybrid scaffolds that support osteogenic differentiation from MSC. Materials and methods: human Fibroblast- derived Matrix (hFDM) as a hybrid scaffold material collected from decellularized fibroblasts cultures from post-cleft- surgery reconstruction palatal skin. Fibroblast cell cultures were divided into two groups of cultures, cultures without Platelet Rich Plasma (PRP), and cultures with the addition of PRP. For decellularization, we performed optimization at the preparation stage of the decellularization solution, and the time of culture for decellularization. In the preparation of the decellularization solution, we divided it into two groups, NH4OH as material from the decellularization solution was diluted with PBS before mixing with 0.25% Triton X-100, and NH4OH was diluted directly in 0.25% Triton X-100. In optimizing the culture time for decellularization, we divided it into three groups, decellularization on the day when cell growth reached 100% confluent, decellularization on the 3rd day after 100% confluent (H + 3) cells, and decellularization on the 4th day after 100% confluent (H + 4) cells. Next, the hFDM matrix is collected and added Polyvinyl Alcohol (PVA) solutions to form a hybrid PVA / hFDM scaffold in the form of a hydrogel. Observations on hybrid PVA / hFDM scaffolds were made using an inversion microscope. Results and discussion: Optimization of methods for culture techniques found that the addition of PRP to fibroblast culture medium increased the rate of fibroblast proliferation. For the decellularization technique, it is known that the preparation of the decellular solution by diluting NH4OH directly in 0.25% Triton X-100 to obtain a final concentration of 50mM NH4OH is known to be effectively used in decellularizing fibroblasts. The optimum culture time is also known that a thicker hFDM matrix can be obtained on day 4 after 100% confluent (H + 4) cells.
Conclusion: Making a good hFDM hybrid PVA scaffold requires method optimization, ranging from fibroblast culture techniques and decellularization techniques. Proper optimization can produce a hybrid PVA / hFDM scaffold which is suitable for bone tissue engineering applications.
Keywords: fibroblast culture, decellularization, hybrid PVA/hFDM scaffold
INTRODUCTION
Femoral fractures are one of the most common types of fractures, followed by metatarsal, ankle, and tibial fractures [1]. Globally, the incidence of fractures is relatively low, at around 10 / 100,000 people per year [2]. The case in Indonesia, the incidence of fracture is categorized as high if seen based on transportation accident figures from the last few years. According to data from the Ministry of Health in 2018, those injury cases have increased from 2007- 2018. Injury prevalence increased from 7.5% in 2007 to 8.2% in 2013, and 9.2% in 2018 with the proportion of injuries of 67.9% lower limbs and 32.7% upper limbs [3]. Based on the data, as many as 100,000 incidents of fracture continue to be non-union. Non-union fractures occur when the repair process does not occur completely, or there are no signs of healing for 3 months [4]. Treatment failures are found due to a mismatch between the biomechanical demands of the fracture and the stability provided by the treatment chosen [5]. Therefore, until now various studies related to appropriate treatment are still being developed. One of them is by developing a bone tissue engineering approach.
Bone tissue engineering is a multidisciplinary science that aims to restore and enhance bone tissue functionality [6]. The approach with bone tissue engineering requires three main components that are often referred to as triad tissue engineering, including multipotent, scaffold cells that act as osteoconductive, and osteoinductive growth factor (GF) [7].
The approach to bone tissue engineering gives positive results to regeneration, and bone formation is an indication of the success of bone healing. This opinion refers to scientific publications reported from bone defect cases at Cipto Mangunkusumo Hospital (RSCM). Reported scientific publications focus on the use of MSC in the form of allogenic Umbilical Cord Mesenchymal Stem Cell (UC-MSC) in cases of femoral fractures that experience non-union [6], and autologous Bone Marrow Mesenchymal Stem Cell (BM-MSC) combined with Bone Morphogenetic Protein (BMP- 2) and Hydroxyapatite (HA) in cases of osteofibrous dysplasia with critical defects have successfully overcome bone defects without causing significant complications [7]. HA scaffold in its use, is often combined with calcium sulfate because HA has a slow bone formation ability [8]. Thus, scaffolds used in tissue engineering techniques continue to require refinement and development. Improvements to find the optimum method for making scaffolds, and the development of various sources to get the best scaffold is still being done to this day, both scaffolds from synthetic materials or scaffolds from natural ingredients. Polyvinyl Alcohol (PVA) is a synthetic biomaterial in the form of a polymer that is water-soluble and biocompatible. PVA in the form of hydrogel has been widely used in biomedical applications, one of which is for the manufacture of cell scaffolding [9]. In addition, scaffold from natural materials also needs to be developed, considering that scaffold also acts as a micro-cell expression in the cell differentiation process. Cell differentiation requires the same components as the original environment to mediate cell interactions with various GFs as controlling cell differentiation [10]. Human Fibroblast-Derived Matrix (hFDM) has been widely chosen to recapitulate matrices that are appropriate to the microenvironment of the cell and has been widely used in tissue engineering [11].
Therefore, in this study, we combine synthetic biomaterials in the form of Polyvinyl Alcohol (PVA) with natural biomaterials in the form of human Fibroblast-Derived Matrix (hFDM) as a material for making hybrid scaffold by optimizing the method of making hybrid PVA / hFDM scaffold which aims to obtain methods optimum for producing hybrid scaffold that is suitable for bone tissue engineering applications.
MATERIAL AND METHODS Fibroblast culture
hFDM was obtained from cryopreserved palatal fibroblast cells taken from the excised palatal mucosa skin of a 7- years-old patient who underwent cleft reconstruction surgery. Preparation of hFDM starting from thawing, culture, and harvest fibroblasts was carried out following procedures in the Stem Cell and Tissue Engineering Research Cluster (SCTE) Indonesian Medical Education and Research Institute of the University of Indonesia (IMERI UI). In this study, based on the presence of PRP in the culture medium, we made two different compositions of the culture medium.
Culture group I is a non-PRP culture group, with a medium composition of 10% FBS, 1% Glutamax, 1% Abam, high
glucose DMEM. Culture group II is a culture group with the addition of 1% PRP, with a medium composition of 10%
FBS, 1% PRP, 1% Glutamax, 1% Abam, DMEM high glucose. Medium replacement is done once in 2-3 days.
Fibroblast Decellularization and K)'0 Preparation
Decellularisation of fibroblasts is carried out after cell growth reaches 100% confluent, post-confluent day 3, and post-confluent day 4. Before the fibroblasts are decellularized, a decellularization solution was prepared in the form of; 0.25% Triton-X 100 and 50 mM NH4OH (221228, Sigma). Based on the preparation method, the decellularization solution that we used was grouped into 2, namely group I; NH4OH was diluted with PBS, and group II; NH4OH is diluted directly with 0.25% Triton X-100. The process of decellularization of fibroblasts begins with the removal of the culture medium into a beaker glass and flushing with PBS. 400 μL of decellularization solution was put into each well using 1000 μL micropipette. The decellularization solution is pipetted out slowly through the well wall.
Decellularisation of fibroblasts results in a matrix called hFDM which is a thin sheet that can be observed using an inversion microscope. hFDM was rinsed with PBS several times and observed under an inversion microscope.
Flushing with PBS is done using a 1000 μL micropipette through the tube wall, the tip tips of the micropipette must not touch the bottom of the well, the well position is tilted with a slope of 450. The hFDM obtained is then evaluated under an inversion microscope. After that, the matrix is stored at -4°C in a PBS bath.
Preparation of PVA
7% (w/v) PVA (MW 146,000-186,000; 363065, Sigma) powder is dissolved in distilled water to form PVA solutions. (11) PVA solutions are inserted into the autoclave. After that, stirring was carried out using a magnetic stirrer without heating, lasting for 30 minutes with the aim of homogenizing PVA with distilled water. PVA solutions can be stored at room temperature for at least 1 day before use.
Hybrid PVA/hFDM Scaffold Preparation
hFDM is removed from -40 °C. PVA solutions are poured onto hFDM. PVA / hFDM scaffold is stored at -20 °C for at least 24 hours to form a hydrogel. PVA / hFDM scaffold is thawed. After that, the hybrid PVA / hFDM scaffold is carefully peeled from the culture plate using forceps and transferred to a new well plate in an upside-down position;
hFDM is above the PVA. Scaffold conditions are evaluated under an inversion microscope.
Morphology Observation of H\brLd Scaffold with Inverted Microscope
When observing, the hybrid PVA / hFDM scaffold is transferred to the glass object with the hFDM position under the PVA to facilitate the observation and determination of the microscope's focal point. Scaffold hybrid PVA / hFDM observation was carried out at 40 times magnification and images were captured using the Images software in the inverted microscope.
RESULT AND DISCUSSION
The success of making a hybrid PVA / hFDM scaffold depends on the following factors; good culture techniques, decellularization techniques, and preparation techniques, as well as PVA pouring. A good hybrid PVA / hFDM scaffold can be obtained after optimizing the method for each factor that affects the success of making PVA.
In this study, we performed a method optimization in the manufacture of PVA / hFDM scaffold which aims to obtain a hybrid PVA / hFDM scaffold that is suitable for bone tissue engineering applications. The first optimization we did was optimization on a fibroblast culture medium. Optimization of the culture medium aims to find out the composition of the right medium for fibroblast growth, that it is feasible to use, and to process for the decellularization stage for the manufacture of hFDM. The choice of the composition of the culture medium is known to influence the growth rate and differentiation ability of a cell [12]. We did the optimization by making a difference in the composition of the culture medium which was divided into two groups, namely group I using a non-PRP culture medium (Fig. 1.a),
and group II used a culture medium with PRP added (Fig. 1.b). Replacement of the culture medium is done once in every 2-3 days.
(a) (b)
FIGURE 1. Fibroblast culture in medium without PRP (a), in medium with PRP (b)
We made observations on the 4th day post culture. In group I, only a few cells grow. Whereas in group II, cell growth reached a confluence rate of 80%. Subsequent observations were made on the 6th-day post culture. In group I, the cells did not experience growth, the same as when observed on the 4th day. In group II, cells reach 100%
confidence and are ready to be harvested and subcultured. Observation in group I continued, until the 11th day of observation, there was no cell growth, the cell showed signs of apoptosis, in the form of granules on the cytoplasm of cells. So it was concluded that fibroblast culture in group I was not feasible because the fibroblast cells did not grow and were confluent, and even had apoptosis. The culture of fibroblasts in group II is feasible to be dissolved into the decellularization stage. In accordance with the requirements of fibroblast used in decellularization, it must use healthy fibroblasts with a confluency level reaching 100%. The absence of PRP in the culture medium causes the rate of cell proliferation to be very slow, even the cells do not experience a growth at all. This indicates that the presence of PRP in the culture medium is an important factor influencing the proliferation of fibroblasts. PRP is known to promote the proliferation of human dermal fibroblasts [13]. PRP contains various growth factors, including Platelet-derived Growth Factor (PDGF) [13, 14], basic Fibroblast Growth Factor (bFGF), Transforming Growth Factor-b (TGF-b) [14, 15], Insulin-Like Growth Factor 1 (IGF-1) [15]. IGF-1 has the ability to stimulate the proliferation and migration of fibroblast cells [16].
Next, we performed an optimization of the decellularization technique based on the different ways of preparing a decellularization solution. The decellularization solution we used consisted of NH4OH 50 mM, and 0.25% Triton X- 100. NH4OH plays a role in cell lysis. The Triton X-100 is a non-ionic surfactant that can affect membrane permeability and cause structural damage to cell membranes when used at certain concentrations, with a range of 0.19 to 0.20 mM [17]. In this treatment, the way to prepare the decellular solution is divided into two groups; group I, dilution of one of the decellularization (NH4OH) compositions was carried out using PBS (Fig. 2.a) before mixing with 0.25% Triton X-100, and group II, dilution of NH4OH was carried out directly in 0.25% Triton X-100 (Fig. 2.b).
(a) (b)
FIGURE 2. Optimization of decellularization solution preparation techniques on fibroblast decellularization. NH4OH in PBS prior to Triton-X mixture (a). NH4OH in Triton X (b)
Based on observations using a microscope, cells are still clearly visible in group I. This shows that the decellularization has not been successful. The failure of the decellularization process is thought to occur due to an improper concentration of decellularization solution. The treatment of dilution of NH4OH by using PBS before being mixed with Triton-X 100 can reduce the final concentration of NH4OH so that it is not in accordance with the previously determined concentration. Following the reference we used, the optimum concentration of decellularization solution was 50 mM NH4OH and 0.25% Triton X-100 (11). Proper decellularization produces a matrix of thin sheets with fibers that are visible when viewed under a microscope (Fig. 2.b). In this study, the images we obtained were not fully in focus at the time of observation. Finally, we found that the preparation of the decellularization solution was crucial to the success of decellularization. The optimum preparation is done by directly diluting NH4OH in 0.25%
Triton X-100 to obtain a final concentration of 50mM NH4OH. For the next technique, the decellularization solution is pipetted out slowly and gently through the well wall. After that, observations were made using a microscope.
Next, we optimize the decellularization technique based on the time of the culture of fibroblasts, which aims to obtain the optimum culture time in order to obtain a matrix that matches the criteria needed in making a hybrid scaffold. In the treatment of cultural time differences, divided into several groups, namely; decellularization is carried out when fibroblast culture reaches 100% confluent (Fig. 3.a), decellularization is done three days after fibroblast culture reaches 100% confluence (H + 3) level (Fig. 3.b), and decellularization is carried out four days after fibroblast culture reaches 100% confidence (H + 4) (Fig. 3.c).
(a) (b) (c)
FIGURE 3. Optimization of decellularization time from fibroblast culture. 100% confluency (a), post-confluent day 3 (b) and post-confluent day 4 (c)
After optimization of decellularization, it was found that optimum decellularization can be carried out at H + 4 after fibroblast cells reach a 100% confidence level. The determination of optimum decellularization is done based on observations on the extracellular matrix formed. The existence of the matrix can be observed starting from the day when the culture of fibroblasts reaches 100% confluent, H + 3, and H + 4. The difference lies in visible matrix formation that is produced from the culture of the fibroblasts in low magnification microscopic observation. We observed that the longer the culture period, the matrix produced by fibroblasts appeared more clear and visible. So we decided the optimum decellularization time to be able to collect hFDM was at H + 4 after the cell reached the 100%
confidence level. The obtained hFDM is stored at -4 °C under submerged PBS conditions, hFDM must be kept wet.
Prior to PVA pouring, the PBS was pipetted out of the well, leaving an intact hFDM sheet.
The PVA solution is poured on top of hFDM sheet. PVA plays a role in maintaining the stability of hFDM. Viscous PVA solutions require certain techniques when poured on top of hFDM sheet, one of which is by cutting the tip from 1000 μl micropipette tips. After PVA solutions are poured onto hFDM, there are usually bubbles both on the surface and inside the scaffold. The existence of bubbles in the scaffold should be avoided, to remove the bubbles, we do vacuum suction using a syringe. Hybrid PVA / hFDM scaffold can be stored at -20 °C for at least 24 hours to produce hydrogel form. Prior to MSC seeding for further differentiation into osteocyte, the PVA/hFDM scaffold is reversed with the hFDM position above the surface of the scaffold, and the scaffold is moved to a new well plate (Fig. 4).
FIGURE 4. Preparation of hybrid PVA/hFDM scaffold
Finally, we want to show the morphology of hybrid PVA / hFDM scaffold based on macroscopic photos (Fig. 5.a), and microscopic photos (Fig. 5.b) taken using an inversion microscope with objective lens magnification of 40 times.
(a) (b)
FIGURE 5. Morphology of hybrid PVA/hFDM scaffold. Inside the well macroscopic appearance (a), observed under inverted microscope (b)
In the macroscopic picture, the hybrid scaffold looks clear white, with a rubbery structure resembling a gel. While on the microscopic picture, scaffold hybrid PVA / hFDM should look clear with interwoven fibrous threads of collagen fibers. However, in this observation, we have yet to obtain an image with the right focus. There is a streak of thread- like appearance but the image is still blurred.
TABLE 1. summarized the optimized method for preparation of hybrid PVA/hFDM scaffold from our experiments.
Fibroblast culture
medium Decellularization
solution Time for fibroblast
decellularization PVA pouring
Optimized preparation
protocol
+ PRP
NH4OH diluted directly with 0,25% Triton X-100
to obtain final NH4OH concentration of 50 mM.
Decellularization performed on post-confluent day 4
of fibroblast culture
Micropipette tips distal end were cutted to enable easy flow of thick, viscous PVA
solution PVA
solutions poured on
top of
hFDM Detachment
PVA/hFDM Scaffold were
removed to a new well plate
CONCLUSIONS
Making a good PVA / hFDM hybrid scaffold requires method optimization, ranging from fibroblast culture techniques and decellularization techniques. Proper optimization can produce a hybrid PVA / hFDM scaffold which is suitable for bone tissue engineering applications.
ACKNOWLEDGMENTS
This research was funded by the 2019 KIST School Project Grant. The research was supported by Nuzli Fahdia Mazfufah, S.Pd, M.Biomed, and Evah Luviah, S.Si, M.Biomed as research assistants in stem cells and tissue engineering cluster research. FKUI IMERI.
Authors' contributions: RDA is the principal research supervisor, responsible for study design, experimental analysis, revision, and editing of manuscripts. MS is the research co-supervisor, grant holder, responsible for research design, and experimental analysis. RI is the master's thesis research student, responsible for conducting research, research design, and experimental models, obtaining and analyzing results, as well as preparation of manuscripts. NF is the research assistant of SCTE IMERI who helped RI in performing and collecting the data. MR is the Doctoral student under the principal research supervisor who provides the cryopreserved fibroblast cells.
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