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Development of macroporous tricalcium phosphate with hyaluronic acid as the cell carrier as the subcutaneous filler
Ceramics International xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Ceramics International journal homepage: www.elsevier.com/locate/ceramint
Development of macroporous tricalcium phosphate with hyaluronic acid as the cell carrier as the subcutaneous filler ⁎
Yung-Chin Yanga, , Chien-Chung Chenb, Wan-Chien Wua, Wei-Ling Hsua, Shu-Chen Tsengc,d a
Institute of Materials Science and Engineering, National Taipei University of Technology, Taipei, Taiwan Graduate Institute of Biomedical Materials & Tissue Engineering, Taipei Medical University, Taipei, Taiwan c Department of Plastic Surgery, Tri-Service General Hospital, Taipei, Taiwan d Sun Space Clinic, Taipei, Taiwan b
A R T I C L E I N F O
A BS T RAC T
Keywords: A. Sintering B. Porosity D. Apatite E. Biomedical applications
The purpose of this study is to develop macroporous TCP (tricalcium phosphate) with irregular shape and the use of HA (hyaluronic acid) gel as the lubricating agent of a composite hydrogel for cell carrier. The porous TCP particle with meteorite shape was made by the solid-state reaction by adding different ratios of the PMMA (Poly(methyl methacrylate)). The subcutaneous filler was made by mixing hyaluronic acid gel with TCP particles and live cells. For the in vitro testing, the mouse fibroblasts cell line L929 was employed for analysis of the toxicity and biocompatibility of the material. The cells were cultured on the material for one to three days followed by the WST-1 (Water soluble tetrazolium) and LDH (Lactate dehydrogenase) analysis. The results of LDH and WST-1 showed that the composite HA/β-TCP was not cytotoxicity. The advantage of this product is the meteorite shaped macroporous TCP particle in the hyaluronic acid gel that could form a linkage channel in the gel. When the fat cell mixed with the composite HA/TCP gel, the fat cells will not be covered with the hyaluronic acid for a long time, and hence the cell will not die of hypoxia or the lack of growth factors provided by the subject’s body fluid. As the proliferation of fat cells progressed, it became a good filler of the injected area.
1. Introduction Since the aging of the global population, aesthetic medicine mainly in anti-aging has become an important issue in human medical research. In recent years, soft tissue fillers have been used as the material to repair the contours especially in cosmetic surgeries. Hyaluronic acid, hereinafter referred to as HA, is a high molecular weight biopolysaccharide, with the structure closely related to GAG (glycosaminoglycan). HA is widely distributed in the intercellular matrix and extracellular matrix of the connective tissue of animals and human bodies. The concentration and molecular weight of hyaluronic acid distributed in different tissues differs significantly. The content in the umbilical cord, joint synovial fluids, skin, eye vitreous body is higher than the levels found in the blood serum [1]. HA is widely used as a soft tissue filler but because of its rapid degradation and absorption by the host’s body, frequent refilling is required. Current existing solution involves the injection of the host’s own fat tissue and HA into the target area with hopes of achieving a more permanent repair [2,3]. Although this method can effectively extend the effect of filling, the effect of self-filling and repairing due to the growth of the fat cells is not reliable because the fat cells become wrapped by the HA creating a barrier for the diffusion of nutrients, signaling factors and waste; all which are ⁎
critical for the long term survival of the fat tissues. Therefore, the purpose of this study is to develop a porous structure of tricalcium phosphate ceramic particles with irregular giant porous structure on the surface. The particles will be mixed with the gel-like filler of HA and the fat cells to produce micro-channels in the filler allowing the body fluid to circulate so the fat cells can survive. While the degradable ceramic particles are gradually absorbed, the growing fat cells replace their positions to achieve the effect of long-term self-filling effect. Calcium and phosphorus ratio of tricalcium phosphate (TCP, Ca3(PO4)2) is 1.5, which is between 1 and 1.67. At different sintering temperatures, there are two kinds of calcium phosphate crystalline phases, α phase and β phase. Compared with the slow degradation rate of hydroxylapatite and the rapid degradation rate of α-TCP, β-TCP has a relatively moderate degradation rate. Therefore, β-TCP was chosen as the composite filling materials [4–6]. 2. Material and methods 2.1. Sintering processes In this study, nano-sized β-TCP (Ca3(PO)4)2, Sigma) powder adding
Corresponding author. E-mail address: [email protected] (Y.-C. Yang).
http://dx.doi.org/10.1016/j.ceramint.2017.05.317
0272-8842/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Yang, Y.-C., Ceramics International (2017), http://dx.doi.org/10.1016/j.ceramint.2017.05.317
Ceramics International xxx (xxxx) xxx–xxx
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directly with the same parameters, crushed, sieved with pore size of 300 µm to obtain porous β-TCP particles. The surface and microstructure of the specimen were observed with SEM (S-4700, Hitachi, Japan), the composition of the material and the crystalline phase were analyzed with XRD (D2 Phaser 2nd Gen, Bruker, USA), the porosity and density of the material were measured by Archimedes method, and the specific surface area of β-TCP by BET method (Barrett-JoynerHalenda, ASAP 2000, Micrommeritcs, USA).
2.2. Biocompatibility test of the β-TCP The cells used in this experiment were L929 mouse connective tissue cells, growth conditions of the cells were observed daily with an optical microscopy [7]. The cells would secrete an acidic substance and turn the medium from red to yellow and when the medium became orange, the medium must be replaced. Downstream processing was carried out once the bottom of the dish was fully covered with cells. In the observation of cell adhesion, the cells were cultured on four kinds of porous β-TCP bulk specimen with different amounts of PMMA. The specimens with the cells at a concentration of 2×104 cells/well were placed in a 24-well microtiter plate, and placed in a CO2 incubator (37 ºC, 5% CO2) after adding 300 μl cell culture medium. After 24 h, the samples were removed and the cells that remained in the dish was counted (viable cells/dead cells). The specimens containing cells were fixed with glutaraldehyde, dried at the critical point and gold-plated. SEM was used to observe the cell morphology and adherence. Cytotoxicity tests were performed according to ISO 10993-5. The test was divided into two parts: the effect of the liquid extract on the cells, and the effect of the direct contact with the material on the cells. As the test specimen was solid, the test method used in this experiment was a direct contact with the material. The specimens were transferred to a 96-well microtiter plate and designed the control group (medium only) and the experimental group. After the cells were collected in trypsin-EDTA tubes, cells with a concentration of 3×104 cells/well were placed on the specimens and 200 μl of cell culture medium was added to culture the cells. After a day of culturing, the medium was aspirated, and the fresh medium and WST-1 (10 μl/well) were added, incubated
Fig. 1. XRD patterns of porous β-TCP assed with 90 wt% PMMA at different sintering temperatures.
polymethylmethacrylate (PMMA, Sigma) was used as a pore-forming agent, and β-TCP porous particles was prepared by low temperature molding and high temperature sintering. β-TCP were mixed with 30, 50, 70, and 90 wt% PMMA respectively by ball milling, and the mixture was dried at 60 °C until the mixture was fully dried. 3 wt% of PVA (Polyvinyl alcohol) was added to the mixture as the binder which was followed by grinding and crushing. The samples were then sieved to obtain the initial powder with particle size of between 100 and 300 µm. The bulk specimens for analysis were prepared by placing the initial powder in a mold and applying a pressure (2 ton/30 s) to form the pellets. The pellets were sintered in a high-temperature furnace. Since the tricalcium phosphate had a phase change of α-TCP at 1180 °C or more, and the phase structure required for this experiment was β-TCP, the sintering temperature parameters were set at 1000 °C, 1050 °C, 1100 °C and 1150 °C. The sintering process above was subjected to two-stage sintering with the heating rate of 2 °C/min. The temperature was held at 550 °C for 2 h to burn off the PVA binder and PMMA poreforming agent, and then raised to the sintering temperature of 3 h. The porous ceramic particles were prepared by sintering the initial powder
Fig. 2. Surface morphology of sintered β-TCP with 50 wt% PMMA at different temperature (a) 1000 °C, (b) 1050 °C, (c) 1100 °C, (d) 1150 °C.
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Fig. 4. The relationship between the sintering temperature and; (a) the porosity of the βTCP and(b) density of bulk TCP. Table 1 The specific surface area (m2/g) of different β-TCP porous particles sintered at different temperatures.
Fig. 3. Surface morphology of sintered β-TCP added with 90 wt% PMMA at different temperatures (a) 1050 °C, (b) 1100 °C, (c) 1150 °C.
in a CO2 incubator for 1 h, shaken gently for 1 min, and the absorbance (OD value, absorbance value is proportional to cell viability) of the cells was measured via ELISA reader (Wavelength = 450 nm) (Tecan, Trading AG, Switzerland). The cytotoxicity test was performed by transferring half of the cell culture medium (200 μl) to another 96well microtiter plate in the process of adding the agent and cells during the WST-1 test, measure the absorbance via ELISA reader (Wavelength = 490 nm) to determine the highest total toxicity standard value by Total lysis.
PMMA T (°C)
30 wt%
50 wt%
70 wt%
90 wt%
1000 °C 1050 °C 1100 °C 1150 °C
1.2315 1.4048 0.7734 0.6534
1.4486 1.5712 1.1002 0.8131
1.3445 1.3097 0.8329 0.7805
1.3759 0.9982 0.8423 0.6498
3. Results and discussion 3.1. Characterization of materials The XRD results (Fig. 1) shows that there were no phase change in the composition of the β-TCP with the addition of the pore-forming agent PMMA sintered at 1000 °C, 1050 °C, 1100 °C and 1150 °C. This indicated that there was no phase change during the process of sintering β-TCP powder into porous ceramic particles at different temperatures. The relationship between microstructure of the porous β-TCP ceramics and the sintering temperature observed with SEM was shown in Fig. 2, where the β-TCP was mixed with 50 wt% of PMMA. The powder agglomerated and as the sintering temperature increased, the powder coarsened with increasing obvious porous structures. The effects of adding different proportions of PMMA powder to the mixture
2.3. β-TCP/HA jelly filler Porous β-TCP particles with four different proportions of PMMA (30, 50, 70, and 90 wt%) were added into 3% HA in four different mixing ratios (0, 1, 5, 10 wt%) and stirred at a heating temperature of 60 °C for 2 h or more in the beaker until the two materials were thoroughly mixed. The homogeneous gelatinous composite material (βTCP particles + hyaluronic acid) was aspirated into a centrifuge tube with an injection syringe, then the viscosity of the mixture was measured and compared with TA laminar rheometer (AR-1000, NEW CASTLE, DE, USA).
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Fig. 5. The optical density (O.D.) in 450 nm of the L929 cell in WST-1 test.
Fig. 6. The optical density (O.D.) in 490 nm of the L929 cell in LDH test. Fig. 8. The morphology of L929 cells cultured on β-TCP (70 wt% PMMA) mixed with hyaluronic acid after (a) 1 day, (b) 3 days, (c) 7 days.
to increase as the proportion of PMMA in the mixture reached 70 wt%. This was due to the agglomeration of the PMMA molecule, which combined into a large. According to previous studies [7,9], the pore size of the porous calcium phosphate was found to be better when larger 100 µm. Pores larger than 150 µm allowed the materials to have a cell living space, if the micropores existed simultaneously with the macropores, these pores formed a 3D matrix of channels which allowed body fluids to circulate, providing nutrients, metabolic waste and ion exchange [10]. The porosity and density of the prepared β-TCP porous ceramics were measured by Archimedes method. The results indicated that the higher the sintering temperature, the higher the density of the ceramic particles and this translated to a lower porosity. As shown in Fig. 4, the addition of 50 wt% PMMA had higher porosity compared with those with the addition of 70 and 90 wt% of PMMA. Potentially, at 50 wt% of PMMA was the critical threshold where at any higher proportion of PMMA led to severe agglomeration which fused into larger pores. Therefore, when the proportion of PMMA was 70 or 90 wt%, the porosity began to decreased; and this was confirmed in Fig. 3 that the pore size of PMMA became larger. From the measurement data of the specific surface area of Table 1, it can be found that the specific surface area was higher when the sintering temperature was lower. This was because initially the TCP particles were mainly small grains and when the sintering temperature increased, the grain size increased due to the agglomeration of the ceramic powder resulting in the decrease of specific surface area. Analyzing the amount of PMMA that was added, the specific surface area was higher when the PMMA proportion was
Fig. 7. Viscosity of the hydrogel composite materials with different amounts of porous ceramic particles.
was as shown in Fig. 3. In addition, unlike the micropores (~1 µm) the size of the macropores (50–100 µm) changed significantly as the proportions of the PMMA increased. Comparing Fig. 2 with Fig. 3, it can be seen that the pore size increased to 50–200 µm when the proportion of the PMMA reaches 90 wt%; which was also highly favorable for cell growth [7,8] as the irregular pore topography on the surface of the particles provided a porous channel for tissue fluid come into contact with the. In short, the pore size of the ceramic started 4
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Fig. 9. 3T3 cells cultured in β-TCP/HA colloid and the cell growth was observed after 1 day and 3 days, (a) Cells cultured in pure HA for one day, (b) cells cultured in pure HA for three days, (c) cells cultured in β-TCP/HA composite gel for one day and (d) cells cultured in β-TCP/HA composite gel for three days.
injected to function as a filler and hence the flow properties associated with this composite filler was highly critical for the overall success. Fig. 7 revealed the viscosity of HA gel with different amounts of β-TCP ceramic particles. The viscosity of all samples will similar when the shear rate up to 200. Therefore, the viscosity obtained in lower shear rate is noteworthy and it close to the situation of injection. With the increasing amount (0–5%) of β-TCP porous ceramics the viscosity of the gel increases as well. This was due to the friction between the powder and HA molecules caused by the addition of β-TCP, resulting in a denser HA gel with smaller pores. However, when the amount of TCP dopant reached 10 wt%, the friction between TCP particles dominated the viscosity, and the overall viscosity decreased and the overall fluidity increased. The viscosity of adding 1% TCP and 10% TCP are similar. Therefore, HA hydrogel compounded with 10 wt% porous β-TCP particles was expected to be a more suitable subcutaneous filling, molding material. L929 cells were cultured on β-TCP/HA composite. The sampling numbers of culturing days are 1 day, 3 days and 7 days. The material was fixed with glutaraldehyde and dried at the critical point after dehydration with ethanol; which was followed by growth morphology observation. Fig. 8 showed the photograph of L929 cells cultured on βTCP/HA material with 70 wt% of PMMA addition after 1 day, 3 days, and 7 days, and the morphology of the cell was clearly observed. The cells proliferated and attached on the β-TCP/HA hydrogel composite, and the derivative of the cells that attached on the surfaces and in the pores of the material were clearly observable. The results showed that β-TCP/HA had good cell-compatibility and cell-attachment ability to
50 wt% because β-TCP pores were smaller and more numerous (Fig. 2). This result was consistent with the data from Fig. 4. 3.2. Biocompatibility of the β-TCP Cells cultured on β-TCP and benchmarked against other materials that were helpful for cellular growth. The absorbance value will be proportional to the survival of the number of cells. The results of the WST-1 cell activity assay were analyzed with an ELISA reader. As shown in Fig. 5, the control group consisted of culture medium without any cells seeded. Specimen A consisted of 30 wt%, specimen B 50 wt% PMMA, specimen C 70 wt% PMMA and specimen D 90 wt% PMMA of PMMA pore-forming agent. Number of L929 cell growth within specimen C registered the highest value and seconded by specimen D; and this indicated that the large pore size found within specimen C was highly helpful for the proliferation of cells. Aside from that, LDH method was used to measure the number of the dead cells. In Fig. 6, it can be seen that group A -D compared with control group had almost no cytotoxicity; on the third day of culturing, there was no increase in the trend or any statistical differences. This indicated that β-TCP does not produce any substances that were detrimental to the survival of the cells. Therefore, β-TCP was not toxic to cells and had good biocompatibility. 3.3. β-TCP/hyaluronic acid composite material β-TCP/HA composite material was planned to be subcutaneously 5
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to proliferate and thrive; and with the gradual degradation of TCP and HA, the self-growing fat cells replaced the filler which maintained the shaping effect at the filling sites, achieving long-term filling effects while eliminating the need for repeated fillings.
L929 cells. When the number of culturing day increased from 1 day to 3 days, the cells continued to proliferate well up to day 7. This proved that inter-connected pore structure of the β-TCP/HA material was not only a good scaffold for cell proliferation but also acted as an excellent cell carrier for subsequent applications. The last part of the study was to mix 3T3 cells into β-TCP/HA composites, and perform cell culturing in vitro with this gel to simulate the conditions of the composite filler once injected subcutaneously. From the experiment results in Fig. 9, it is found that the number of viable cells in the hydrogel composite added with porous TCP ceramic particles was higher than that in pure HA, and the number of surviving cells on the third day was also far higher than that without the addition of porous ceramic particles. This was because the surface of the ceramic particles with irregularly shaped macropores and combined with other surface tensions resulted in the formation of gaps that were not filled with HA. The channels allowed tissue fluids flow past and brought nutrition to the cells which allowed the cells to survive. This meant the fat cells seeded into the ceramic composite would survive as it is being administered subcutaneously into the patient. As the TCP and HA gradually degrade, the selfgrowing fat cells will replace the filler maintaining the shaping effect of the filling site to achieve long-term and repeat-free subcutaneous filling.
Conflict of interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgements The first and second authors contribute equally to this work. Authors thank the budget support of National Taipei University of Technology - Taipei Medical University Joint Research Program (NTUT-TMU Joint Research Program: NTUT-TMU-101-06). References [1] R.D. Price, M.G. Berry, H.A. Navsaria, Hyaluronic acid: the scientific and clinical evidence, J. Plas. Recons. Aes. Surg. 60 (10) (2007) 1110–1119. [2] Y. Cao, Adipose tissue angiogrnesis as a therapeutic target for obesity and metabolic diseases, Nat. Rev. Drug. Discov. 9 (2010) 107–115. [3] K. Mineda, S. Kuno, H. Kato, K. Kinoshita, K. Doi, I. Hashimoto, H. Nakanishi, K. Yoshimura, Chronic inflammation and progressive calcification as a result of fat necrosis: the worst outcome in fat grafting, Plast. Reconstr. Surg. 133 (2014) 1064–1072. [4] M. Bohner, G.H. van Lenthe, S. Grünenfelder, W. Hirsiger, R. Evison, R. Müller, Synthesis and characterization of porous beta-tricalcium phosphate blocks, Biomaterials 26 (2005) 6099–6105. [5] N. Koça, M. Timuçina, F. Korkusuzb, Fabrication and characterization of porous tricalcium phosphate ceramics, Ceram. Int. 30 (2004) 205–211. [6] M. Yoshikawaa, N. Tsujia, T. Todaa, H. Ohgushib, Osteogenic effect of hyaluronic acid sodium salt in the pores of a hydroxyapatite scaffold, Mat. Sci. Eng. C27 (2007) 220–226. [7] J.A. Buckwalter, H.J. Mankin, Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation, Instr. Course Lect. 47 (1998) 487–504. [8] H. Nakjima, M. Goto, H. Hashimoto, Properties of α-tricalcium phosphatepolycarboxylic acid mixture and apatite formation (special issue)J. Dent. Res. 65 (1986) (Abstract 213). [9] T. Furukawa, D. Eyre, S.S. Koid, M.J. Glimcher, Biochemical studies on repair cartilage resurfacing experimental defects in the rabbit knee, J. Bone Jt. Surg. Am. 62 (1980) 79–89. [10] H. Yamasaki, H. Sakai, Osteogenic response to porous hydroxyapatite ceramics under the skin of dogs, Biomaterials 13 (1992) 308–312.
4. Conclusions In this study, porous ceramic materials excellent for cell growth was successfully prepared. The ceramic particles have a size of between 100 and 300 µm. The internal porosity and the appearance of the particles were mainly related to the addition of PMMA. The addition PMMA over 50 wt% resulted in a large amount of aggregation molecules and caused the small pores to combine into large pores. When the addition amount of PMMA was between 70 wt% and 90 wt%, the porosity began to decrease and the pores began to enlarged, making them suitable for the purpose of this study. The addition of 10 wt% of porous β-TCP particles in HA hydrogel resulted in better fluidity and shaping effect. At the same time, this material was biocompatible and the material with the pore size between 100 and 300 µm was conducive for cell growth. Porous biodegradable ceramic particles with HA hydrogel can be used as the carrier of the cell, and subcutaneous filling and shaping can be performed after mixing the composite material with the patient's own adipocytes. The existence of porous ceramic particles allowed the cells
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Contents lists available at ScienceDirect
Ceramics International journal homepage: www.elsevier.com/locate/ceramint
Development of macroporous tricalcium phosphate with hyaluronic acid as the cell carrier as the subcutaneous filler ⁎
Yung-Chin Yanga, , Chien-Chung Chenb, Wan-Chien Wua, Wei-Ling Hsua, Shu-Chen Tsengc,d a
Institute of Materials Science and Engineering, National Taipei University of Technology, Taipei, Taiwan Graduate Institute of Biomedical Materials & Tissue Engineering, Taipei Medical University, Taipei, Taiwan c Department of Plastic Surgery, Tri-Service General Hospital, Taipei, Taiwan d Sun Space Clinic, Taipei, Taiwan b
A R T I C L E I N F O
A BS T RAC T
Keywords: A. Sintering B. Porosity D. Apatite E. Biomedical applications
The purpose of this study is to develop macroporous TCP (tricalcium phosphate) with irregular shape and the use of HA (hyaluronic acid) gel as the lubricating agent of a composite hydrogel for cell carrier. The porous TCP particle with meteorite shape was made by the solid-state reaction by adding different ratios of the PMMA (Poly(methyl methacrylate)). The subcutaneous filler was made by mixing hyaluronic acid gel with TCP particles and live cells. For the in vitro testing, the mouse fibroblasts cell line L929 was employed for analysis of the toxicity and biocompatibility of the material. The cells were cultured on the material for one to three days followed by the WST-1 (Water soluble tetrazolium) and LDH (Lactate dehydrogenase) analysis. The results of LDH and WST-1 showed that the composite HA/β-TCP was not cytotoxicity. The advantage of this product is the meteorite shaped macroporous TCP particle in the hyaluronic acid gel that could form a linkage channel in the gel. When the fat cell mixed with the composite HA/TCP gel, the fat cells will not be covered with the hyaluronic acid for a long time, and hence the cell will not die of hypoxia or the lack of growth factors provided by the subject’s body fluid. As the proliferation of fat cells progressed, it became a good filler of the injected area.
1. Introduction Since the aging of the global population, aesthetic medicine mainly in anti-aging has become an important issue in human medical research. In recent years, soft tissue fillers have been used as the material to repair the contours especially in cosmetic surgeries. Hyaluronic acid, hereinafter referred to as HA, is a high molecular weight biopolysaccharide, with the structure closely related to GAG (glycosaminoglycan). HA is widely distributed in the intercellular matrix and extracellular matrix of the connective tissue of animals and human bodies. The concentration and molecular weight of hyaluronic acid distributed in different tissues differs significantly. The content in the umbilical cord, joint synovial fluids, skin, eye vitreous body is higher than the levels found in the blood serum [1]. HA is widely used as a soft tissue filler but because of its rapid degradation and absorption by the host’s body, frequent refilling is required. Current existing solution involves the injection of the host’s own fat tissue and HA into the target area with hopes of achieving a more permanent repair [2,3]. Although this method can effectively extend the effect of filling, the effect of self-filling and repairing due to the growth of the fat cells is not reliable because the fat cells become wrapped by the HA creating a barrier for the diffusion of nutrients, signaling factors and waste; all which are ⁎
critical for the long term survival of the fat tissues. Therefore, the purpose of this study is to develop a porous structure of tricalcium phosphate ceramic particles with irregular giant porous structure on the surface. The particles will be mixed with the gel-like filler of HA and the fat cells to produce micro-channels in the filler allowing the body fluid to circulate so the fat cells can survive. While the degradable ceramic particles are gradually absorbed, the growing fat cells replace their positions to achieve the effect of long-term self-filling effect. Calcium and phosphorus ratio of tricalcium phosphate (TCP, Ca3(PO4)2) is 1.5, which is between 1 and 1.67. At different sintering temperatures, there are two kinds of calcium phosphate crystalline phases, α phase and β phase. Compared with the slow degradation rate of hydroxylapatite and the rapid degradation rate of α-TCP, β-TCP has a relatively moderate degradation rate. Therefore, β-TCP was chosen as the composite filling materials [4–6]. 2. Material and methods 2.1. Sintering processes In this study, nano-sized β-TCP (Ca3(PO)4)2, Sigma) powder adding
Corresponding author. E-mail address: [email protected] (Y.-C. Yang).
http://dx.doi.org/10.1016/j.ceramint.2017.05.317
0272-8842/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Yang, Y.-C., Ceramics International (2017), http://dx.doi.org/10.1016/j.ceramint.2017.05.317
Ceramics International xxx (xxxx) xxx–xxx
Y.-C. Yang et al.
directly with the same parameters, crushed, sieved with pore size of 300 µm to obtain porous β-TCP particles. The surface and microstructure of the specimen were observed with SEM (S-4700, Hitachi, Japan), the composition of the material and the crystalline phase were analyzed with XRD (D2 Phaser 2nd Gen, Bruker, USA), the porosity and density of the material were measured by Archimedes method, and the specific surface area of β-TCP by BET method (Barrett-JoynerHalenda, ASAP 2000, Micrommeritcs, USA).
2.2. Biocompatibility test of the β-TCP The cells used in this experiment were L929 mouse connective tissue cells, growth conditions of the cells were observed daily with an optical microscopy [7]. The cells would secrete an acidic substance and turn the medium from red to yellow and when the medium became orange, the medium must be replaced. Downstream processing was carried out once the bottom of the dish was fully covered with cells. In the observation of cell adhesion, the cells were cultured on four kinds of porous β-TCP bulk specimen with different amounts of PMMA. The specimens with the cells at a concentration of 2×104 cells/well were placed in a 24-well microtiter plate, and placed in a CO2 incubator (37 ºC, 5% CO2) after adding 300 μl cell culture medium. After 24 h, the samples were removed and the cells that remained in the dish was counted (viable cells/dead cells). The specimens containing cells were fixed with glutaraldehyde, dried at the critical point and gold-plated. SEM was used to observe the cell morphology and adherence. Cytotoxicity tests were performed according to ISO 10993-5. The test was divided into two parts: the effect of the liquid extract on the cells, and the effect of the direct contact with the material on the cells. As the test specimen was solid, the test method used in this experiment was a direct contact with the material. The specimens were transferred to a 96-well microtiter plate and designed the control group (medium only) and the experimental group. After the cells were collected in trypsin-EDTA tubes, cells with a concentration of 3×104 cells/well were placed on the specimens and 200 μl of cell culture medium was added to culture the cells. After a day of culturing, the medium was aspirated, and the fresh medium and WST-1 (10 μl/well) were added, incubated
Fig. 1. XRD patterns of porous β-TCP assed with 90 wt% PMMA at different sintering temperatures.
polymethylmethacrylate (PMMA, Sigma) was used as a pore-forming agent, and β-TCP porous particles was prepared by low temperature molding and high temperature sintering. β-TCP were mixed with 30, 50, 70, and 90 wt% PMMA respectively by ball milling, and the mixture was dried at 60 °C until the mixture was fully dried. 3 wt% of PVA (Polyvinyl alcohol) was added to the mixture as the binder which was followed by grinding and crushing. The samples were then sieved to obtain the initial powder with particle size of between 100 and 300 µm. The bulk specimens for analysis were prepared by placing the initial powder in a mold and applying a pressure (2 ton/30 s) to form the pellets. The pellets were sintered in a high-temperature furnace. Since the tricalcium phosphate had a phase change of α-TCP at 1180 °C or more, and the phase structure required for this experiment was β-TCP, the sintering temperature parameters were set at 1000 °C, 1050 °C, 1100 °C and 1150 °C. The sintering process above was subjected to two-stage sintering with the heating rate of 2 °C/min. The temperature was held at 550 °C for 2 h to burn off the PVA binder and PMMA poreforming agent, and then raised to the sintering temperature of 3 h. The porous ceramic particles were prepared by sintering the initial powder
Fig. 2. Surface morphology of sintered β-TCP with 50 wt% PMMA at different temperature (a) 1000 °C, (b) 1050 °C, (c) 1100 °C, (d) 1150 °C.
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Fig. 4. The relationship between the sintering temperature and; (a) the porosity of the βTCP and(b) density of bulk TCP. Table 1 The specific surface area (m2/g) of different β-TCP porous particles sintered at different temperatures.
Fig. 3. Surface morphology of sintered β-TCP added with 90 wt% PMMA at different temperatures (a) 1050 °C, (b) 1100 °C, (c) 1150 °C.
in a CO2 incubator for 1 h, shaken gently for 1 min, and the absorbance (OD value, absorbance value is proportional to cell viability) of the cells was measured via ELISA reader (Wavelength = 450 nm) (Tecan, Trading AG, Switzerland). The cytotoxicity test was performed by transferring half of the cell culture medium (200 μl) to another 96well microtiter plate in the process of adding the agent and cells during the WST-1 test, measure the absorbance via ELISA reader (Wavelength = 490 nm) to determine the highest total toxicity standard value by Total lysis.
PMMA T (°C)
30 wt%
50 wt%
70 wt%
90 wt%
1000 °C 1050 °C 1100 °C 1150 °C
1.2315 1.4048 0.7734 0.6534
1.4486 1.5712 1.1002 0.8131
1.3445 1.3097 0.8329 0.7805
1.3759 0.9982 0.8423 0.6498
3. Results and discussion 3.1. Characterization of materials The XRD results (Fig. 1) shows that there were no phase change in the composition of the β-TCP with the addition of the pore-forming agent PMMA sintered at 1000 °C, 1050 °C, 1100 °C and 1150 °C. This indicated that there was no phase change during the process of sintering β-TCP powder into porous ceramic particles at different temperatures. The relationship between microstructure of the porous β-TCP ceramics and the sintering temperature observed with SEM was shown in Fig. 2, where the β-TCP was mixed with 50 wt% of PMMA. The powder agglomerated and as the sintering temperature increased, the powder coarsened with increasing obvious porous structures. The effects of adding different proportions of PMMA powder to the mixture
2.3. β-TCP/HA jelly filler Porous β-TCP particles with four different proportions of PMMA (30, 50, 70, and 90 wt%) were added into 3% HA in four different mixing ratios (0, 1, 5, 10 wt%) and stirred at a heating temperature of 60 °C for 2 h or more in the beaker until the two materials were thoroughly mixed. The homogeneous gelatinous composite material (βTCP particles + hyaluronic acid) was aspirated into a centrifuge tube with an injection syringe, then the viscosity of the mixture was measured and compared with TA laminar rheometer (AR-1000, NEW CASTLE, DE, USA).
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Fig. 5. The optical density (O.D.) in 450 nm of the L929 cell in WST-1 test.
Fig. 6. The optical density (O.D.) in 490 nm of the L929 cell in LDH test. Fig. 8. The morphology of L929 cells cultured on β-TCP (70 wt% PMMA) mixed with hyaluronic acid after (a) 1 day, (b) 3 days, (c) 7 days.
to increase as the proportion of PMMA in the mixture reached 70 wt%. This was due to the agglomeration of the PMMA molecule, which combined into a large. According to previous studies [7,9], the pore size of the porous calcium phosphate was found to be better when larger 100 µm. Pores larger than 150 µm allowed the materials to have a cell living space, if the micropores existed simultaneously with the macropores, these pores formed a 3D matrix of channels which allowed body fluids to circulate, providing nutrients, metabolic waste and ion exchange [10]. The porosity and density of the prepared β-TCP porous ceramics were measured by Archimedes method. The results indicated that the higher the sintering temperature, the higher the density of the ceramic particles and this translated to a lower porosity. As shown in Fig. 4, the addition of 50 wt% PMMA had higher porosity compared with those with the addition of 70 and 90 wt% of PMMA. Potentially, at 50 wt% of PMMA was the critical threshold where at any higher proportion of PMMA led to severe agglomeration which fused into larger pores. Therefore, when the proportion of PMMA was 70 or 90 wt%, the porosity began to decreased; and this was confirmed in Fig. 3 that the pore size of PMMA became larger. From the measurement data of the specific surface area of Table 1, it can be found that the specific surface area was higher when the sintering temperature was lower. This was because initially the TCP particles were mainly small grains and when the sintering temperature increased, the grain size increased due to the agglomeration of the ceramic powder resulting in the decrease of specific surface area. Analyzing the amount of PMMA that was added, the specific surface area was higher when the PMMA proportion was
Fig. 7. Viscosity of the hydrogel composite materials with different amounts of porous ceramic particles.
was as shown in Fig. 3. In addition, unlike the micropores (~1 µm) the size of the macropores (50–100 µm) changed significantly as the proportions of the PMMA increased. Comparing Fig. 2 with Fig. 3, it can be seen that the pore size increased to 50–200 µm when the proportion of the PMMA reaches 90 wt%; which was also highly favorable for cell growth [7,8] as the irregular pore topography on the surface of the particles provided a porous channel for tissue fluid come into contact with the. In short, the pore size of the ceramic started 4
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Fig. 9. 3T3 cells cultured in β-TCP/HA colloid and the cell growth was observed after 1 day and 3 days, (a) Cells cultured in pure HA for one day, (b) cells cultured in pure HA for three days, (c) cells cultured in β-TCP/HA composite gel for one day and (d) cells cultured in β-TCP/HA composite gel for three days.
injected to function as a filler and hence the flow properties associated with this composite filler was highly critical for the overall success. Fig. 7 revealed the viscosity of HA gel with different amounts of β-TCP ceramic particles. The viscosity of all samples will similar when the shear rate up to 200. Therefore, the viscosity obtained in lower shear rate is noteworthy and it close to the situation of injection. With the increasing amount (0–5%) of β-TCP porous ceramics the viscosity of the gel increases as well. This was due to the friction between the powder and HA molecules caused by the addition of β-TCP, resulting in a denser HA gel with smaller pores. However, when the amount of TCP dopant reached 10 wt%, the friction between TCP particles dominated the viscosity, and the overall viscosity decreased and the overall fluidity increased. The viscosity of adding 1% TCP and 10% TCP are similar. Therefore, HA hydrogel compounded with 10 wt% porous β-TCP particles was expected to be a more suitable subcutaneous filling, molding material. L929 cells were cultured on β-TCP/HA composite. The sampling numbers of culturing days are 1 day, 3 days and 7 days. The material was fixed with glutaraldehyde and dried at the critical point after dehydration with ethanol; which was followed by growth morphology observation. Fig. 8 showed the photograph of L929 cells cultured on βTCP/HA material with 70 wt% of PMMA addition after 1 day, 3 days, and 7 days, and the morphology of the cell was clearly observed. The cells proliferated and attached on the β-TCP/HA hydrogel composite, and the derivative of the cells that attached on the surfaces and in the pores of the material were clearly observable. The results showed that β-TCP/HA had good cell-compatibility and cell-attachment ability to
50 wt% because β-TCP pores were smaller and more numerous (Fig. 2). This result was consistent with the data from Fig. 4. 3.2. Biocompatibility of the β-TCP Cells cultured on β-TCP and benchmarked against other materials that were helpful for cellular growth. The absorbance value will be proportional to the survival of the number of cells. The results of the WST-1 cell activity assay were analyzed with an ELISA reader. As shown in Fig. 5, the control group consisted of culture medium without any cells seeded. Specimen A consisted of 30 wt%, specimen B 50 wt% PMMA, specimen C 70 wt% PMMA and specimen D 90 wt% PMMA of PMMA pore-forming agent. Number of L929 cell growth within specimen C registered the highest value and seconded by specimen D; and this indicated that the large pore size found within specimen C was highly helpful for the proliferation of cells. Aside from that, LDH method was used to measure the number of the dead cells. In Fig. 6, it can be seen that group A -D compared with control group had almost no cytotoxicity; on the third day of culturing, there was no increase in the trend or any statistical differences. This indicated that β-TCP does not produce any substances that were detrimental to the survival of the cells. Therefore, β-TCP was not toxic to cells and had good biocompatibility. 3.3. β-TCP/hyaluronic acid composite material β-TCP/HA composite material was planned to be subcutaneously 5
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to proliferate and thrive; and with the gradual degradation of TCP and HA, the self-growing fat cells replaced the filler which maintained the shaping effect at the filling sites, achieving long-term filling effects while eliminating the need for repeated fillings.
L929 cells. When the number of culturing day increased from 1 day to 3 days, the cells continued to proliferate well up to day 7. This proved that inter-connected pore structure of the β-TCP/HA material was not only a good scaffold for cell proliferation but also acted as an excellent cell carrier for subsequent applications. The last part of the study was to mix 3T3 cells into β-TCP/HA composites, and perform cell culturing in vitro with this gel to simulate the conditions of the composite filler once injected subcutaneously. From the experiment results in Fig. 9, it is found that the number of viable cells in the hydrogel composite added with porous TCP ceramic particles was higher than that in pure HA, and the number of surviving cells on the third day was also far higher than that without the addition of porous ceramic particles. This was because the surface of the ceramic particles with irregularly shaped macropores and combined with other surface tensions resulted in the formation of gaps that were not filled with HA. The channels allowed tissue fluids flow past and brought nutrition to the cells which allowed the cells to survive. This meant the fat cells seeded into the ceramic composite would survive as it is being administered subcutaneously into the patient. As the TCP and HA gradually degrade, the selfgrowing fat cells will replace the filler maintaining the shaping effect of the filling site to achieve long-term and repeat-free subcutaneous filling.
Conflict of interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgements The first and second authors contribute equally to this work. Authors thank the budget support of National Taipei University of Technology - Taipei Medical University Joint Research Program (NTUT-TMU Joint Research Program: NTUT-TMU-101-06). References [1] R.D. Price, M.G. Berry, H.A. Navsaria, Hyaluronic acid: the scientific and clinical evidence, J. Plas. Recons. Aes. Surg. 60 (10) (2007) 1110–1119. [2] Y. Cao, Adipose tissue angiogrnesis as a therapeutic target for obesity and metabolic diseases, Nat. Rev. Drug. Discov. 9 (2010) 107–115. [3] K. Mineda, S. Kuno, H. Kato, K. Kinoshita, K. Doi, I. Hashimoto, H. Nakanishi, K. Yoshimura, Chronic inflammation and progressive calcification as a result of fat necrosis: the worst outcome in fat grafting, Plast. Reconstr. Surg. 133 (2014) 1064–1072. [4] M. Bohner, G.H. van Lenthe, S. Grünenfelder, W. Hirsiger, R. Evison, R. Müller, Synthesis and characterization of porous beta-tricalcium phosphate blocks, Biomaterials 26 (2005) 6099–6105. [5] N. Koça, M. Timuçina, F. Korkusuzb, Fabrication and characterization of porous tricalcium phosphate ceramics, Ceram. Int. 30 (2004) 205–211. [6] M. Yoshikawaa, N. Tsujia, T. Todaa, H. Ohgushib, Osteogenic effect of hyaluronic acid sodium salt in the pores of a hydroxyapatite scaffold, Mat. Sci. Eng. C27 (2007) 220–226. [7] J.A. Buckwalter, H.J. Mankin, Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation, Instr. Course Lect. 47 (1998) 487–504. [8] H. Nakjima, M. Goto, H. Hashimoto, Properties of α-tricalcium phosphatepolycarboxylic acid mixture and apatite formation (special issue)J. Dent. Res. 65 (1986) (Abstract 213). [9] T. Furukawa, D. Eyre, S.S. Koid, M.J. Glimcher, Biochemical studies on repair cartilage resurfacing experimental defects in the rabbit knee, J. Bone Jt. Surg. Am. 62 (1980) 79–89. [10] H. Yamasaki, H. Sakai, Osteogenic response to porous hydroxyapatite ceramics under the skin of dogs, Biomaterials 13 (1992) 308–312.
4. Conclusions In this study, porous ceramic materials excellent for cell growth was successfully prepared. The ceramic particles have a size of between 100 and 300 µm. The internal porosity and the appearance of the particles were mainly related to the addition of PMMA. The addition PMMA over 50 wt% resulted in a large amount of aggregation molecules and caused the small pores to combine into large pores. When the addition amount of PMMA was between 70 wt% and 90 wt%, the porosity began to decrease and the pores began to enlarged, making them suitable for the purpose of this study. The addition of 10 wt% of porous β-TCP particles in HA hydrogel resulted in better fluidity and shaping effect. At the same time, this material was biocompatible and the material with the pore size between 100 and 300 µm was conducive for cell growth. Porous biodegradable ceramic particles with HA hydrogel can be used as the carrier of the cell, and subcutaneous filling and shaping can be performed after mixing the composite material with the patient's own adipocytes. The existence of porous ceramic particles allowed the cells
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