Biocompatibility of hydroxyapatite scaffolds processed by lithography-based additive manufacturing

Abstract

The fabrication of hydroxyapatite scaffolds for bone tissue engineering applications by using lithography-based additive manufacturing techniques has been introduced due to the abilities to control porous structures with suitable resolutions. In this research, the use of hydroxyapatite cellular structures, which are processed by lithography-based additive manufacturing machine, as a bone tissue engineering scaffold was investigated. The utilization of digital light processing system for additive manufacturing machine in laboratory scale was performed in order to fabricate the hydroxyapatite scaffold, of which biocompatibilities were eventually evaluated by direct contact and cell-culturing tests. In addition, the density and compressive strength of the scaffolds were also characterized. The results show that the hydroxyapatite scaffold at 77% of porosity with 91% of theoretical density and 0.36 MPa of the compressive strength are able to be processed. In comparison with a conventionally sintered hydroxyapatite, the scaffold did not present any cytotoxic signs while the viability of cells at 95.1% was reported. After 14 days of cell-culturing tests, the scaffold was able to be attached by pre-osteoblasts (MC3T3-E1) leading to cell proliferation and differentiation. The hydroxyapatite scaffold for bone tissue engineering was able to be processed by the lithography-based additive manufacturing machine while the biocompatibilities were also confirmed.

Keywords

Additive manufacturing biocompatibility tissue engineering hydroxyapatite scaffolds

1. Introduction

Recently, bone transplants have been in a greater demand due to the increasing of active lifestyles, accidents, obesity, and aging populations. Bone grafts and substitutes have been forecasted to reach 3.3 billion US dollars of revenues by 2013, with an annual growth rate of 13.8% from 2006 to 2013 in the United States [1]. Therefore, bone tissue engineering (BTE), which is a process that initiates and develops bone tissues in order to replace, repair, or restore bone defects, is currently investigated to support this requirement.

The standard approach in BTE is to seed and culture cells such as osteoblasts, chondrocytes, or mesenchymal stem cells, on scaffolds. The scaffolds for BTE applications are three-dimensional porous structures imitating the extracellular matrix of bone. The structure should be highly porous with an interconnected pore network for an adequate nutrient supplement and metabolic waste removal [2]. The scaffold materials should be biocompatible and available for cell attachment, proliferation and differentiation. Besides, the mechanical properties should match with the tissue at the implant site.

For scaffold materials, Hydroxyapatite (HA) is widely used because its composition is closest to that of bone minerals. HA is a calcium phosphate ceramic which is biocompatible and is considered to be osteoconductive but not osteoinductive due to its favorable Ca/P ratio of 1.67 [3]. With the theoretical density at 3.16 g/cm³ in approximately, HA has a greater mechanical property than the other bioceramics such as tricalcium phosphate (TCP), or bioactive glasses. Although, the bioresorbabilty of HA is very low, its surface can provide nucleating site for the precipitation of apatite crystals in culture medium [3], which is favorable to cell attachment and cell growth.

To process HA to be scaffolds, Additive Manufacturing (AM) technologies play an important role to allow the scaffold can be tailored. AM technologies are the fabrication process that constructs three-dimensional (3D) objects by using the data from computational 3D models. Therefore, this technique enables the cellular structure and the pore architecture of scaffolds to be designed and created. Ideally, pore sizes should be controlled in order to match with the cell types with sufficient porosity and pore connectivity, while the mechanical properties of the structure are also concerned [4–7]. In this context, different AM techniques for the fabrication of HA have been investigated, including 3D printing [8–10], selective laser sintering [11,12] and rapid prototyping based on laser cladding [13].

Lithography is one of the AM techniques that constructs parts by using light to solidify the photosensitive materials. This technique has been developed in order to process cellular ceramic structures such as alumina, TCP or bioactive glasses [14,15]. To process such ceramics, the ceramic powder is mixed into a photosensitive resin resulting in the polymerizable ceramic slurry, which is fabricated layer by layer fashion until the entire object is built. Lithography-based additive manufacturing machine in this research uses a digital light processing (DLP) system as a light source to expose the light with certain light intensity and high resolution in order to construct the part in lithography technique [16]. The DLP of the machine projects a visible blue light (wavelength 460 nm) to solidify photosensitive slurry. As a result, the fabrication of parts with arbitrary design and high resolution, 50 µm in layer thickness and 40 µm in x–y plane can be achieved.

In this research, the DLP-based additive manufacturing machine in laboratory scale was built in order to investigate the processing of HA scaffolds used for BTE. The cellular structure was fabricated and its biocompatibilities were evaluated by the cytotoxic and cell-culturing tests. In addition, the density and compressive strength of the scaffolds were also characterized.

2. Materials and methods

2.1. DLP-based AM machine

The DLP-based additive manufacturing machine in laboratory scale was designed and fabricated in this research. The schematic setup of the machine is illustrated in Fig. 1(a).

Fig. 1.

Digital light processing system for processing hydroxyapatite photosensitive slurries: (a) schematic setup of the utilized machine, and (b) photograph of the device. (Colors are visible in the online version of the article; https://dx-doi-org.web.bisu.edu.cn/10.3233/BME-151549.)

The major components of the machine are a DLP-projector, a lifting control unit, a wiping control unit, a wiper and a building platform. This machine can fabricate parts by projecting visible blue light from the DLP-projector (Dell 4320, Dell Inc., USA) to the photosensitive material on the building platform. At the beginning, the wiper, which is controlled by the wiping control unit, is moved over the platform in order to spread out the material to ensure its thickness and surface smoothness. The unit consists of the stepping motor, 0.72°/step (Vexta PH544-N-A, Oriental motor Co., Ltd, Japan) and the linear- motion guide actuator (THK KR20, THK Co., Ltd, Japan). Then, the light which is the image of the cross-sectional area of the part selectively solidifies the material. After that, the material is full-filled manually and the building platform, which is controlled by the lifting control unit consist of the stepping motor, 0.72°/step (Vexta KP543-A, Oriental motor Co., Ltd, Japan) and the linear-motion guide actuator (Mecha industry Co., Ltd, Japan) is moved down with the distance of the part layer thickness. Finally, the whole process is repeated again until the entire part is built. The image of the machine is shown in Fig. 1(b).

2.2. Preparation of hydroxyapatite photosensitive slurry

The hydroxyapatite slurry was processed at 27% in approximately of solid loading by speed-mixing the hydroxyapatite powder (HAP-200, Lot No 10120201, Taihei Chemical Industrial Co., Ltd, Japan), and the photosensitive resin, which contains 98% of methacrylate-based monomers and 2% of photoinitiator. The density and the mean particle size of the HA powder were reported at 3.l6 g/cm³ and 9.21 µm respectively. The powder and the resin were mixed with the speed at 1,600 rpm for 3 min by the speed-mixing machine (Kurabo Mazerustar, KK-250S, Kurabo Industries Ltd, Japan).

2.3. Fabrication of hydroxyapatite scaffolds

At the beginning, computational 3D models of solid structure for density measurement and cellular structure for compressive test, cytotoxicity test and cell-culturing test were designed by CAD software (AutoFab RnD, Marcam Engineering GmbH, Germany). The scaffold models were created in a cylindrical shape with the dimension of 11.3 mm in diameter and 3 mm of thickness. In the next step, the DLP-based AM machine fabricated the samples by using the hydroxyapatite photosensitive slurry as a material. The slurry was solidified with 28.3 W/m² of light intensity for 15 s of the exposure time in each layer. The layer thickness was controlled at 50 µm. After the fabrication process, the heat treatment process was applied to the samples in order to remove the polymeric binders up to 500°C with the heat rate at 0.5°C/min. The parts were finally sintered at 1,300°C for 2 h to obtain a nearly dense hydroxyapatite product. The samples for biocompatibility tests were sterilized by a gamma radiation method.

2.4. Density and mechanical properties

The density of the solid samples was measured using Archimede’s method by a precision balance (AND GX-400, USA) and compared with a fully dense of hydroxyapatite. The samples were cylindrical in shape with the dimension of 8 mm in diameter and 3 mm in height. The compressive strength of the scaffolds was measured with 1 mm/min cross head speed using a universal testing machine (Instron 55R4502, USA). The cellular structure of the specimens was 500–700 µm of pore size and 77% of porosity with the dimension of 7 mm in diameter and 3 mm in height.

2.5. Biocompatibilities

2.5.1. Direct contact test

The samples were prepared by the DLP-based AM technique into circular discs with 7.8 mm in diameter and 2.25 mm in thickness. The sample discs were typically placed in the middle of a 35 mm dish and belt tightly with non-toxic dental wax. The MC3T3-E1 pre-osteoblasts were seeded onto the dish at a density of 8 × 10⁴ cells/dish and incubated for 48 h. Cell morphology and the toxic zone were evaluated by phase contrast light microscopy after exposure to the cells for 48 h. The cells were stained with 0.01% neutral red in phosphate buffer saline (PBS) for membrane integrity. The samples were tested in triplicate. Besides, the result of the samples was compared with hydroxyapatite samples which, are prepared by conventionally sintering method. The percentage of cell viability was determined by the average number of cells, which were counted in the area of 200 × 200 µm, in comparison with the conventionally sintered hydroxyapatite.

2.5.2. Cell-culturing test

The cellular structures in cylindrical shape with 7.8 mm in diameter and 2.25 mm in thickness, around 500–700 µm of pore size and 90% of porosity were prepared for cell-culturing tests. The samples were sterilized by a gamma irradiation method. The MC3T3-E1 mouse pre-osteoblasts were used in this study. The samples were placed onto a 35 mm dish. Then, the pre-osteoblasts with the density of 5 × 10⁴ cells/specimen were directly seeded onto the surfaces of the samples. The cells were cultured in ∞-MEM supplement with 10% fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 µg/ml), and maintained at 37°C in a 5% CO₂ atmosphere. At 7 and 14 days of incubation periods, the samples with the attached cells were examined by scanning electron microscopy (SEM) (Jeol JSM-5410, Japan) to observe the cellular morphology of the pre-osteoblasts on the samples.

3. Results and discussion

The scaffold that was fabricated by the DLP-based additive manufacturing machine is shown in Fig. 2(a). The fabrication indicated that the photosensitive hydroxyapatite slurry using in this experiment are able to be processed by the machine. The light intensity of visible blue light from the DLP-projector at 28.3 W/m² for 15 s is sufficient to solidify the slurry in each layer. The appearance of the samples is comparable with the 3D model from CAD software.

Fig. 2.

The hydroxyapatite scaffold processed by DLP-based AM techniques: (a) the green part, and (b) the sintered part. (The colors are visible in the online version of the article; https://dx-doi-org.web.bisu.edu.cn/10.3233/BME-151549.)

Fig. 3.

Phase contrast light microscopy of cell morphology for cytotoxicity test after exposure to pre-osteoblasts for 48 h: (a) the hydroxyapatite sample processed by DLP-based AM techniques in comparison with (b) a conventionally sintered hydroxyapatite. (Colors are visible in the online version of the article; https://dx-doi-org.web.bisu.edu.cn/10.3233/BME-151549.)

The image of scaffolds after sintering is shown in Fig. 2(b). The shrinkage was reported at around 31% in the $X Y$ -plane and 25% in the Z direction. The properties of the sample are summarized in Table 1. The density of the solid sample was measured, and a result of 2.88 g/cm³ was obtained, which corresponds to 91% of the theoretical density of hydroxyapatite (3.16 g/cm³). The compressive strength of the cellular structure was reported to be 0.36 MPa with 77% of porosity and 500–700 µm of pore size.

The cytotoxicity of the sample was evaluated by the images of phase contrast light microscopy of pre-osteoblasts from the direct contact test that are shown in Fig. 3. The morphology of the cells that were contacted with the sample for 48 h (Fig. 3(a)) was comparable with the cell morphology of the conventionally sintered hydroxyapatite (Fig. 3(b)). The cells did not present any signs of cytotoxicity, which can lead to cell death. According to the images, the numbers of the cells in the area of 200 × 200 µm were counted and reported at 41 cells for the conventionally sintered hydroxyapatite and 39 cells for the samples, which is equal to 95.1% of cell viability.

Table 1

Summary of material properties of the hydroxyapatite scaffold processed by DLP-based AM techniques

Density of solid sample (g/cm³)	2.88
Compressive strength of cellular structure (MPa)	0.36
Pore size (µm)	500–700
Porosity of the cellular structure (%)	77

Fig. 4.

SEM images of pre-osteoblastic cells attached on the scaffolds for: (a) 7 days, 35 times, (b) 7 days, 350 times, (c) 14 days, 35 times, and (d) 14 days, 350 times of magnification.

SEM images of the scaffolds that were attached by pre-osteoblasts and cultured for 7 and 14 days in various magnifications are presented in Fig. 4. After 7 days of the incubation period, the cells were able to attach with and spread on the surface of the hydroxyapatite scaffold as shown in Fig. 4(a) and (b). After 14 days, the cells were proliferated and spread throughout to cover almost all of the surface area (Fig. 4(c) and (d)).

4. Conclusion

The DLP-based AM machine that was fabricated in this research is able to construct the scaffold from photosensitive HA slurries. The geometry of the scaffold is comparable with its original source, which is the CAD model. The building parameters such as the light intensity and the exposure time that were used in this experiment are sufficient to polymerize the slurry in order to fabricate the structure in AM techniques. The shrinkage of the sample after sintering at 31% in the $X Y$ -plane and 25% in the Z direction can be compensated in the designing step when the exact shape of the part is required. In addition, the density at 91% of the theoretical density of hydroxyapatite and the compressive strength at 0.36 MPa of the cellular structure with 77% of porosity are sufficient to be used as a scaffold for bone tissue engineering. The biocompatibility of the scaffold was confirmed by the results of direct contact and cell-culturing tests. As a result, the sample is free from the residual toxics, caused by this fabrication process.

Footnotes

Acknowledgement

This research was fully supported by grants from National Metal and Materials Technology Center (MTEC), a member of National Science and Technology Development Agency (NSTDA), Thailand.

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