Hydroxyapatite nano bioceramics optimized 3D printed poly lactic acid scaffold for bone tissue engineering application

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Hydroxyapatite nano bioceramics optimized 3D printed poly lactic acid scaffold for bone tissue engineering application Sudip Mondala, Thanh Phuoc Nguyenb, Van Hiep Phamb, Giang Hoanga, Panchanathan Manivasagana, Myoung Hwan Kimc, Seung Yun Nama,b,c, Junghwan Oha,b,c,∗ a

Marine-Integrated Bionics Research Center, Pukyong National University, Busan, 48513, Republic of Korea Department of Biomedical Engineering and Center for Marine-Integrated Biotechnology (BK21 Plus), Pukyong National University, Busan, 48513, Republic of Korea c Interdisciplinary Program of Marine-Bio, Electrical & Mechanical Engineering, Pukyong National University, Busan, 48513, Republic of Korea b

ARTICLE INFO

ABSTRACT

Keywords: Hydroxyapatite Polylactic acid (PLA) Scaffold Bone tissue engineering Composites

To achieve optimum functionality and mechanical properties of advanced manufacturing-based scaffolds for biomedical application, it is important to study their mechanical strength by 3D-printing at different orientations. This study examined the effects of printing at different orientations on the mechanical properties of synthesized 3D-polylactic acid (PLA) and hydroxyapatite-modified PLA (PLA-HAp) scaffolds. A total number of 30 samples were printed in three orientations on the XY plane: 0°, 45°, and 90°. Finite element modeling and simulation was employed to identify the strongest scaffold in terms of compression strength, which is the primary criterion for load bearing bone tissue scaffolds. These findings indicate that 3D-printing at an orientation of 90° on the XY plane resulted in a scaffold with the highest compression strength. Moreover, the fabricated PLA scaffolds showed very poor cell attachment and proliferation on their surface, which is not suitable for their biomedical application. This study additionally showed the optimization of a very simple post-fabrication modification technique with nano HAp for better cell attachment and proliferation with enhanced mechanical properties. The post-fabrication modification of PLA scaffolds by nano-HAp results in excellent cell attachment property with enhanced mechanical strength and stability of up to 47.16% for 90° 3D-printed PLA-HAp scaffolds.

1. Introduction After blood transfusion bone is recognized as one of the most widely transplanted tissues in terms of medical treatment [1]. In tissue engineering, the fabrication of a suitable scaffold has gained considerable attention due to its challenging role in cell attachment and proliferation on its surface without affecting its mechanical strength [2]. Bone fracture is the most common type of injury occurred by many reasons such as accidental trauma, disease, metabolic failures, and aging. Presently, the metallic materials are the most commonly used internal fracture fixation agent, due to their high mechanical strength [3]. Metallic materials did not provide the optimum therapy for trauma fixation due to their certain limitations, such as, mismatch mechanical strength between the metallic materials and the host tissues, high risk of inflammation caused by the immunogenic reactions of released metallic ions, and non-biodegradable properties [4]. Therefore, research on developing biodegradable materials for human body fracture/trauma managements are till now in the first priority level. In near future, it will likely to be replaced the metallic implants completely with more

∗

suitable bio materials such as polymers, ceramics, or their composites [5]. Poly lactic acid (PLA) is a well-studied biodegradable polymer reported to have multifunctional applications including medical implant device fabrication, and as a scaffold material in tissue engineering. The reasons behind the popularity of PLA include its incomparable biocompatibility, significant production of nontoxic byproducts during biodegradation and approved clinical trials conducted by the US Food and Drug Administration (FDA) [6]. PLA has gained much consideration in the recent era due to its excellent bioresorbability, enhanced biocompatibility, and biodegradablity with nontoxic byproduct formation. The non-exhaustive application of PLA can be employed in different fields including medical and food, industries such as in antimicrobial product development, bone tissue engineering, 3D –printed scaffold fabrication, and surgical suturing in addition to as drug carrier agents [7–10]. Recently, a huge number of nanomaterials, such as silica, apatite, HAp, titanium dioxide, metal nanoparticles (silver, gold, platinum, copper, etc.), cellulose, and carbon nanomaterials (nanofiber, nanotubes, and graphene nanosheet), have been incorporated with PLA to construct customized

Corresponding author. Marine-Integrated Bionics Research Center, Pukyong National University, Busan, 48513, Republic of Korea. E-mail address: [email protected] (J. Oh).

https://doi.org/10.1016/j.ceramint.2019.10.057 Received 29 July 2019; Received in revised form 23 September 2019; Accepted 7 October 2019 0272-8842/ © 2019 Published by Elsevier Ltd.

Please cite this article as: Sudip Mondal, et al., Ceramics International, https://doi.org/10.1016/j.ceramint.2019.10.057

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composite materials [7,11,12]. The only disadvantage of using PLA is the lack of the property to facilitate cell attachment on its surface and their proliferation due to its poor cellular attachment ability [13]. Initial cell attachment and proliferation are the most basic and critical criteria for any implantable scaffold material. From the perspective of cell biology, primary cells are mostly anchorage dependent which means they need a rigid surface for cell attachment and proliferation. Most cases of material rejection are a result of poor interaction between cells and implant surface. To overcome such harsh situations, it is essentially important to understand the surface property of newly fabricated materials before being applied to affected tissues [6]. A few strategies have been proposed such as enhanced cell proliferation by surface roughening [14], modification of surface topology using collagen, hyaluronic acid, peptides, and mussel adhesive proteins [15,16] etc. According to studies on HAp scaffolds, calcium phosphates (such as DCP, and TCP, etc.), promote cell attachment and proliferation on scaffold surface. Compared to normal scaffolds, HAp fabricated scaffolds allow faster cell attachment and proliferation [13,17,18]. HAp is a well-known bioceramics with excellent biological behaviors including osteoconductivity, osteoinductivity, and extreme biocompatibility [19,20]. HAp shows superior biocompatibility due to its chemical composition, which allows protein attachment on its surface by strong electrostatic interaction. With the evidence of its superior bioactivity, HAp rapidly interacts with organic molecules, proteins, and essential amino acids and efficiently heals affected hard tissues such as bones and teeth [21–23]. HAp is simultaneously used as hard tissue regeneration material due to its better mechanical strength when applied as a composite material [24]. The only limitation of pure HAp is its poor mechanical strength primarily concerned with low toughness and limited patient-specific application [25]. The ever demanding bioceramics have always proved to be promising in hard tissue replacement, whereas their clinical application is restricted due to poor mechanical properties [26]. To improve its feasibility, researchers globally are performing extensive studies to enhance the mechanical feasibility of this novel bioceramics [27,28]. The strategy to utilize composite biomaterials including ceramics and polymers with different combinations might play a key role in its future application in bone tissue engineering. Synergistic composites from PLA and HAp hold great potential for load bearing applications [29]. Researchers have developed several strategies to improve the bioactivity of printed scaffolds by incorporating CaP as a filler in polymer composites [30,31]. However, mostly the polymer matrix entrapped filler materials are not directly come in contact with cells due to the slow degradation of polymer matrix. To counter the problem an alternate strategy is aimed to uniformly distribute HAp materials on the PLA scaffold surface by the simple following technique. In this study, a facile post-fabrication modification technique is introduced to make 3D scaffolds more suitable for biomedical application (Fig. 1). The important aspects of this study, is to optimize the scaffold designing parameters by finite element modeling, and post-fabrication modification which helps in selecting the optimal printing parameters with enhanced mechanical stability and bioactivity. The scaffolds were fabricated using 3D-printer (MakerBot Z18 3D printer, USA) with different printing orientations (0°, 45°, and 90° printing angle). These fabricated scaffolds were further studied for compression tests (ASTM standard D695), and biological evaluation. Scanning electron microscopy (SEM) revealed the well distributed HAp nanoparticles incorporation on PLA surface. HAp loading on PLA scaffold promotes protein adsorption, which allows enhanced cell proliferation. The possible mechanism underlying increased cell adhesion is the formation of actin fibers, which significantly improved the expression of other proteins on MG-63 cells. Based on these results, the potential application of PLA-HAp composite can be recommended as promising biomaterials for bone substitute in tissue engineering.

2. Materials and methods 2.1. Chemical reagents Calcium nitrate tetrahydrate [Ca(NO3)2·4H2O] diammonium hydrogen phosphate [(NH4)2HPO4], ammonium hydroxide (NH4OH) (28%), and 3-(4, 5-dimethythiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Aldrich (USA). 2.2. HAp -synthesis Chemical precipitation techniques were adopted to synthesize HAp nanomaterials (nHAp), using Ca(NO3)2·4H2O, and (NH4)2HPO4 as synthetic precursor chemicals [28]. First, 0.24 M Ca(NO3)2·4H2O suspension (23.61 g in 350 mL deionized water) was prepared and stirred continuously at room temperature. The pH of the freshly prepared solution was maintained at 11.0 by adding ammonia solution. Thereafter, 0.29 M (NH4)2HPO4 solution (7.92 g in 250 mL deionized water) was prepared. The freshly prepared 0.29 mL (NH4)2HPO4 solution was added dropwise to the calcium nitrate solution following which the transparent calcium nitrate solution turned milky white as HAp nanoparticles formed in the solution. The chemical reaction is represented as follows: 10 Ca(NO3)2·4H2O + 6 (NH4)2HPO4 + 8NH4OH → Ca10(PO4)6(OH)2 + 20NH4NO3 + 20H2O Finally, the white precipitate was separated by centrifugation at 5000 rpm for 5 min and dried at 85 °C. The obtained nHAp powder was calcined in air at 600 °C for 1 h and stored for further use. 2.3. Fabrication of 3D scaffold 3D-printing is the most promising, accurate, and reliable scaffold fabrication technique, which allows complex structure formation [32,33]. Polymer-based building materials are generally used as printing materials [34]. However, ceramics, metals, and composite materials are also used for 3D-printing under different conditions [35–40]. The design for the orientations scaffolds was drawn using Solidworks 2016 (Dassault Systèmes, France). Next, these designs were used to print scaffolds using a 3D-printer with a commercial PLA filament [41]. A hot extrusion nozzle with an initial diameter of 0.4 mm was used to extrude the melted PLA filament. The melted PLA filament was extruded onto a metallic platform by driving the printing nozzle in a pre-calculated pattern to fabricate the customized shape (Fig. 2). After completing a layer on the same plane, the printing nozzle tucked back to its original position and started printing the next layer. All printing parameters such as layer thickness, printer head velocity, feeding rate of the PLA filament, and spacing between adjacent filaments were predefined and set prior to printing. Printing is associated with the direction of PLA filament deposition toward “axial” (x); in the plane of the printing layers, the direction perpendicular direction to the axial is referred to as “transverse” (y). Finally, the z-direction is denoted as the out of plane. The present study illustrates the mechanical response of uniaxial 3D-printing, where the deposition of printing material is similar for all layers. 2.4. Post-fabrication scaffold modification with HAp The fabricated PLA scaffolds were further modified with synthesized HAp nanoparticles (Fig. 3). A very simple step of mild heating followed by sonication was employed for the uniform distribution of HAp nanoparticles over the PLA scaffold surface. To make uniform deposition

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Fig. 1. Schematic representation of 3D printed PLA scaffold surface modification by hydroxyapatite nanoparticles for enhanced bone tissue engineering application.

we have taken several precautions. After several trial and errors, we optimized the parameters to modify the PLA scaffold surface with homogenized nano HAp. In first step, the synthesized HAp was ball milled in a planetary milling machine (Retsch PM100, Germany) for 6 h (at 300 rpm) in dry condition using 2 mm zirconia balls to prepare homogenized nano HAp. Next, 1 g of the finely distributed homogenized HAp nanoparticles was mixed in 50 mL deionized water. The fabricated scaffolds were immersed in the HAp sterile water mixture and sonicated at 70 °C for 10 min. After sonication, the scaffolds were dried and heated at 72 °C for 15 min. After cooling at room temperature, the modified PLA-HAp scaffolds were further sonicated for 15 min immersed in DI water (at room temperature). The excess amount of unattached HAp nanoparticles was washed away. Finally, the scaffolds were dried and used for further studies. The only PLA scaffold filament

shows smooth surface during SEM study (Fig. 3a and b), whereas after treatment with nano HAp the smooth PLA surface becomes rough due to incorporation of HAp (Fig. 3c and d). The incorporation of HAp nanoparticles over PLA scaffold surface was optimized by several trial and error study. Excess amount of HAp nanoparticle incorporation may cause the blockage of scaffold pores which is not desirable for biomedical application (Fig. 3 e and f). 2.5. Finite element modeling of scaffold Finite element modeling is a mathematical calculation to determine the optimal scaffold model for different problems. In the present study, the theoretical power of mathematical calculations helped to determine the optimal scaffold model application in bone tissue engineering 3

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Fig. 2. 3D printed scaffold fabrication for tissue engineering application. Top view shows the schematic drawing from Solidworks 2016 (Dassault Systèmes, France). Bottom view shows the original SEM image of 3D-printed scaffold (MakerBot Z18 3D printer, USA).

[42,43]. A detailed method for finite element modeling is described in the supplementary section. To identify the strongest model by finite element modeling that was considered simple for calculation, we fixed

the bottom surface and placed constant force on the top surface. In this model, because the load applies only axial force, this model was considered a bar model. We used finite element modeling to analyze the

Fig. 3. SEM analysis of (a) 3D printed PLA scaffold filaments (b) cross section of PLA filaments (c & d) optimized HAp modified PLA filament (e & f) non-optimized HAp loaded PLA filament. 4

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Fig. 4. The new model (a) 3D shape (b) 2D geometry dimensions and boundary.

model. Because the model has some identical layers, we analyzed the new model for simplicity, which reduces the number of layers (Fig. 4a). We used the 2D shape of the model for analysis using four elements and five nodes and under boundary conditions (Fig. 4b):

Then assembled matrix stiffness is:

[K ] = 3200

+ Fix the bottom face + Put the force on the top face P = 200 N

A1

A1 A1

he1

A2

he1

he1

; [K 2] = E

A1

he2

A2

he2

he1

3200

A1

A1

he1

A1

[K ] = E

he1

A1

he1

+

A2

he2

A2

0

he2

A1 + A2 A2 0 A2 A2 + A3 A3 3200 0 A3 A3 + A 4 0 0 A4

A2

he2 A2

he2

1280 = 0.4

Ai Ai

0 0 0 A4 A4

Q1 u1 Q2 u2 u3 = Q3 u4 Q4 u5 Q5

(2)

0 0 A4 A4

u2 0 u3 0 u4 = 0 u5 200

(3)

Apply in each model, we have: 0-degree model. Calculated area of cross section layers with dimension of each layer (Fig. 5 a).

0 A1 = 28.2743 mm2

A2

A3 = 7.0372 mm2

A2 = A4 = 15.9031 mm2

he2 A2

Then the expression (3) becomes:

he2

In this model, we have 4 elements and apply finite element method for linear element, we have Ai (i = 1, 2, 3, 4) are the constant and hie = 0.4 mm and E is the Young's modulus of PLA materials, E = 1280 MPa, then we have:

[K i]

A1 A1 0 0 A1 A1 + A2 A2 0 0 A2 A2 + A3 A3 0 0 A3 A3 + A 4 0 0 0 A4

with boundary conditions: u1 = 0; Q1 = Q2 = Q3 = Q4 = 0; Q5 = P0 Hence, the expression (2) becomes:

where, E is the Young's modulus of material, Ai is the cross section area at i element, hie is the element height. Hence, the assembled matrix stiffness with 2 elements is: he1

0 0 0 A4 A4

Then expression (1) becomes:

Determine the displacement in the model using finite element method, the model which has the smallest displacement is the strongest. In the finite element method, we use the equation Keue = Qe (1) to determine the displacement, where Ke is the assembled matrix stiffness; ue is the displacement; Qe is the load apply in the model. For the model has 2 elements, we have [Ke] = [K1] + [K2] with [K1], [K2] is the stiffness matrix at element 1, element 2, respectively. We can determine [K1], [K2] by the equation:

[K1] = E

A1 A1 0 0 A1 A1 + A2 A2 0 0 A2 A2 + A3 A3 0 0 A3 A3 + A 4 0 0 0 A4

3200

44.1774 15.9031 0 0

15.9031 22.9403 7.0372 0

0 7.0372 22.9403 15.9031

0 0 15.9031 15.9031

u2 0 u3 0 = u4 0 u5 200

we obtain: u2 = 0.0022 mm; u3 = 0.0061 mm; u4 = 0.0150 mm; u5 = 0.0190 mm. Compare value of u5 with result from software: Average u5 from software is 0.019258 mm, because software divides

Ai Ai 5

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Fig. 5. 0-Degree model (a) dimensions of cross section layer (b) analysis result from FEM software.

the model into more elements than theory so the results from software near the exact result (Fig. 5 b). For 45-degree model: Calculated area of cross section layers with dimension of each layer (Fig. 6 a): A1 = 28.2743 mm2

A3 = 15.9031 mm2

Then the expression (3) becomes:

3200

3200

15.9031 31.8062 15.9031 0

0 15.9031 31.8062 15.9031

0 0 15.9031 15.9031

u2 0 u3 0 u4 = 0 u5 200

we obtain: u2 = 0.0022 mm; u3 = 0.0061 mm; u4 = 0.0101 mm; u5 = 0.0140 mm. Compare value of u5 with results from software: Average u5 from software is 0.01467 mm, because software divides the model into more elements than theory so the results from software near the exact result (Fig. 6 b). For 90-degree model: Calculated area of cross section layers with dimension of each layer (Fig. 7 a): A1 = 28.2743 mm2

A3 = 15.9031 mm2

15.9031 31.8062 15.9031 0

0 15.9031 31.8062 15.9031

0 0 15.9031 15.9031

u2 0 u3 0 u4 = 0 u5 200

we obtain: u2 = 0.0022 mm; u3 = 0.0061 mm; u4 = 0.0101 mm; u5 = 0.0140 mm. Compare value of u5 with result from software: Average u5 from software is 0.0145 mm, because software divides the model into more elements than theory so the results from software near the exact result (Fig. 7 b). From the result of theory, we use the software to analyze to find the model which has the best stiffness. We apply the boundary conditions:

A2 = A4 = 15.9031 mm2

Then the expression (3) becomes:

44.1774 15.9031 0 0

44.1774 15.9031 0 0

+ Fixed the bottom face + Put on the top face a force P = 200 N Determine the displacement of the model, which model has the smallest displacement, that is the best model about stiffness. Here the result from Siemens NX software (Siemens PLM Software, Texas USA): From the result, we see the 90-degree model that has the smallest deformation then this model has the best stiffness (Fig. 8). Hence, 90degree orientation printed scaffold is the strongest one with respect to mechanical properties point of view.

A2 = A4 = 15.9031 mm2

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Fig. 6. 45-degree model (a) dimensions of cross section layer (b) analysis result from FEM software.

1 × 10 4 cells/cm2. The MTT assay was performed with PLA-HAp and PLA filaments on cultured MG-63 cells incubated for 24 h. MG-63 cells without any materials were used as control. After incubation, cells were treated with 10 μL of 0.5 mg/mL MTT reagent for 3 h in a CO2 incubator. After incubation, the medium was carefully discarded and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve formazan. In the MTT assay, cell suspension was measured using a Tecan Infinite F50 plate reader at wavelength of 570 nm.

3. Synthesized nano-HAp and 3D- printed scaffolds characterization Scanning electron microscope (SEM) was used to characterize the surface topography of 3D-printed scaffolds. To study the surface morphology of the developed scaffold, SEM (JSM 6700, JEOL, Japan) was performed. The HAp depositions on PLA-HAp scaffold were estimated using a thermogravimetric analyzer (TG-DTA, PerkinElmer Pyris Diamond). The crystallinity of the synthesized HAp was characterized by X-ray diffraction (Bruker AXS X-ray diffractometer) with radiation source from Cu Kα target (λ = 1.54 Å, 40 kV, and 20 mA). The mechanical compressive tests characterization was performed using Universal Testing Machine (Lloyd LR5K Plus) at room temperature (24 °C ± 2 °C).

Viable cells (%) =

Absorbance (570 nm) of treated cells X100 Absorbance (570 nm) of controlcells

3.1.2. Acridine orange (AO)/propidium iodide (PI) dual fluorescence based cell viability assay Cell viability, attachment, and proliferation were determined with MG-63 cell seeded PLA-HAp scaffolds stained with AO/PI fluorescent nucleic acid staining [27]. Cell culture plate with a diameter of 35 mm was used to incubate PLA-HAp and only PLA scaffold with MG-63 cells for 48 h. After incubation, the treated scaffolds were washed gently with PBS and finally stained with 200 μL (1 μg/mL) AO and PI for 10 min. Finally, PBS was used to wash untreated cells and finally analyzed under a 10 × magnified fluorescence microscope using 450–490 nm filter (LEICA DMI 3000B, Germany).

3.1. Biological study 3.1.1. Cytotoxicity study of PLA-HAp Human MG-63 osteoblast-like cell line was purchased from KCLB (South Korea) and further cultured with 10% fetal bovine serum and antibiotic supplemented Dulbecco's modified Eagle medium (DMEM) at 37 °C in a 5% CO2 incubator. With sufficient cell confluency, 96-well culture plates were seeded with 100 μL of DMEM at a concentration of 7

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Fig. 7. 90-degree model (a) dimensions of cross section layer (b) analysis result from FEM software.

Fig. 8. Computational modeling of three different printed scaffolds for compressive displacement analysis. 8

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3.2. Mechanical characterization (compressive stress)

4. Results and discussions

Compression is the most common form of accident which results in skeletal fractures. Compressive stress examined to determine the maximum compressive load carrying ability of scaffolds. The ultimate loadcarrying ability of scaffolds is crucial to express and improve their strength and quality. For application in bone tissue engineering, the measurement of the compressive strength of scaffold is crucial. However, tensile strength is important for soft tissues such as the cartilage, which functions with flexibility and under tension. The compressive study of the developed test sample was performed at room temperature (24 °C ± 2 °C), in relative humidity of 55 ± 5RH. The cross-directional displacement speed of the UTM zig was set at 1.0 mm/ min without any preloading. The standard ASTM D695 protocol was followed to study compressive stress. The compressive modulus was determined by calculating slope obtained from the stress-strain curve in the elastic region of scaffolds during compressive study.

4.1. XRD analysis PLA-HAp and PLA scaffolds were characterized by XRD analysis (Fig. 9a and b). The diffraction peaks revealed the crystalline property of HAp. Wide-angle X-ray diffraction scattering showed broad characteristic peaks of PLA. The PLA material scaffold contained a maximum amount of D-lactide isomer, which enhances its crystalline property from amorphous nature (Fig. 9 a). The X-ray diffractogram for PLA scaffold exhibits a significant peak at ~16° originating predominantly from the intermediate form of ordering polymer chains between the amorphous and crystalline forms. For PLA-HAp composites, a sharp crystalline peak was recorded at a 2θ value of ~31°, on being subjected to the (211) plane of pristine HAp [28]. The XRD peaks for PLA-HAp scaffold recognized at 25°, 27°, 33°, 38°, 46°, 49°, 52°, 62°, and 64° (2θ angle) were associated with the (002), (210), (112), (130), (222), (213), (004), (214), and (304) planes, respectively [44].

Fig. 9. XRD analysis of (a) PLA and (b) PLA-HAp. TG analysis of (a) HAp, PLA-HAp (b) TG-DT analysis of PLA.

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4.2. TG-DT analysis

exact alignment of defect free atomic orientation (Fig. 10 c). The selected area electron diffraction (SAED) pattern of synthesized HAp nanoparticles were identified and marked in agreement with already calculated XRD patterns (Fig. 10 d).

The heating effect and thermal stability are significant characteristics of composite materials. The results of TG analysis of synthesized HAp, PLA-HAp, and PLA scaffolds are shown in Fig. 9 c and d. The TG analysis was performed from 35 °C to 800 °C in inert atmosphere at a heating rate of 10 °C/min. The results revealed a maximum weight loss of approximately 2.17%, and 6.75% for HAp and PLA-HAp respectively. In PLA, the weight loss was ~97% indicating a very poor thermal stability. The higher weight loss is observed for HAp and PLA-HAp composites at ~300 °C temperature. The initial thermal degradation occurs (up to 300 °C) due to the evaporation of entrapped and adsorbed water molecule. Further decomposition occurred at higher temperatures due to the chemical and crystalline changes in nanoparticles [45]. TG-DT analysis was performed for only PLA material to understand its thermal stability and thermal behavior. After 306 °C temperature the PLA material showed rapid degradation due to thermal effect resulting in an endothermic sharp peak (361 °C). With further heating an exothermic peak was observed at ~390 °C corresponding to the cold crystallization of PLA material.

4.4. Compressive stress analysis 4.4.1. PLA and PLA-HAp scaffolds The experimental results show three distinct regions in compressive stress-strain curve with linear elasticity, long plateau, and densification region. The compressive modulus and compressive yield strengths were calculated from initial linear region. The scaffolds were placed and compressed in the Z-direction of fabrication process. Compressive strength at yield, σ y was defined as the inter section of the stress–strain curve with the modulus slope at an offset of 1.0% strain. The ultimate compressive strength of fabricated 3D-printed PLA scaffold (at orientations of 90°, 45°, and 0°) was ~28 ± 0.2 MPa, ~5.1 ± 0.2 MPa, and ~2.5 ± 0.2 MPa, respectively (Fig. 11a). The 90° orientation 3Dprinted scaffold showed the maximum compressive strength. However, it was not sufficient for load-carrying application. Therefore, post-fabrication HAp modified PLA scaffold was also studied for mechanical characterization (Table 1). PLA-HAp scaffold exhibited a compressive strength of ~53 ± 0.2 MPa, ~21 ± 0.2 MPa, and ~16 ± 0.2 MPa, for 90°, 45°, and 0° orientations respectively, which were higher than that of PLA scaffold (Fig. 11 b). The pores on 3D-printed PLA scaffold possibly provide space for HAp nano particles during post-fabrication modification of scaffold. The HAp nanoparticles accumulation on PLA scaffold surface enhanced its surface roughness and controlled the porosity on modified PLA-HAp scaffold. This post-fabrication modification also enhances the

4.3. FE-SEM, TEM, HR-TEM, and SAED analysis of synthesized nano HAp The synthesized HAp nanoparticles are further characterized by FESEM analysis. The synthesized HAp nanoparticles are near elongated spherical in morphology and are agglomerated (Fig. 10 a). The TEM analysis revealed the ultra-nanostructure of HAp nanoparticles. The average particle size was calculated 36 ± 4 nm (Fig. 10 b). The high resolution TEM study confirms the interplanar spacing similar to pristine HAp lattice. The lattice fringes are perfectly parallel due to the

Fig. 10. FE-SEM analysis of (a) synthesized HAp nanoparticles and (b) PLA-HAp. TG analysis of (a) HAp, PLA-HAp (b) TG-DT analysis of PLA. 10

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Fig. 11. Compressive stress analysis of (a) PLA scaffold (b) PLA-HAp scaffold (c) a standard compression stress-strain curve for polymer materials. Table 1 Compressive strength analysis of different orientation angle printed PLA and HAp modified PLA (PLA-HAp) scaffolds. Sl. No.

1 2 3

Different orientation printed scaffold

0° 45° 90°

Table 2 Apparent porosity measurement of 3D-printed PLA and PLA-HAp scaffolds. Sl. No.

Compressive stress (MPa) PLA Scaffold

PLA-HAp scaffold

2.5 ± 0.2 5.1 ± 0.2 28 ± 0.2

16 ± 0.2 21 ± 0.2 53 ± 0.2

1 2 3

Porosity is the property of bearing pores or void space in a sample. In bone tissue engineering, the interconnected porous network is more appropriate for the transfusion of biological fluids, nutrients and allows space to regenerate new tissues and blood vessels. The porosity of 3Dprinted PLA scaffold was determined by liquid displacement method employing Archimedes’ Principle (ASTM B962) [46]. Fabricated scaffolds were weighed in dry state and next immersed in liquid under vacuum of 4 mm of mercury pressure for 2 h. Then, the soaked and suspended samples are weighed for finding out the % apparent porosity of the samples.

Wd Wa

PLA Scaffold

PLA-HAp scaffold

~74% ~61% ~56%

~69% ~54% ~47%

The noticeable porosity for 0°, 45°, and 90° orientation 3D-printed PLA scaffolds were obtained ~74%, ~61%, ~56% respectively. Whereas, the obtained apparent porosity for PLA-HAp scaffolds were ~69%, ~54%, ~47% respectively represented in Table 2. After modification with HAp nanoparticles PLA-HAp scaffold shows decreased porosity due to incorporation of nanoparticles. There is also a synergistic relation between porosity and compressive strength. With enhanced porosity the scaffold shows lower mechanical stability and vice versa. The maximum compressive stress of ~53 ± 0.2 MPa was obtained for 90° orientation printed PLA-HAp scaffold with a maximum porosity of ~47%.

4.5. Scaffold porosity measurement

Ws Ws

0° 45° 90°

Apparent porosity (%)

and Suspended weight of the scaffold = Wa .

mechanical stability of the scaffolds for high load-carrying application with promising properties of osteoconduction and osteo-integration.

% Apparent Porosity =

Different orientation printed scaffold

4.6. Cytotoxicity of PLA and PLA-HAp 4.6.1. MTT assay and AO/PI dual fluorescence assay The cell scaffold response was evaluated using standard assays and imaging techniques represented as follows. Scaffolds were sanitized and seeded in 12 well plates with MG-63 cells at a concentration of 1 × 04 cells. Cells were allowed to fix on the scaffold surface and then

× 100

where.

Dry weight of the scaffold = Wd , Soaked weight of the scaffold = Ws , 11

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Fig. 12. MTT assay of PLA-HAp and PLA filaments (Left side). Figure (a & b) AO/PI staining of MG-63 cells attached on 90° printed PLA scaffold (c) schematic representation of PLA scaffold surface modification with HAp (d & e) AO/PI staining of 90° printed PLA-HAp scaffold with enhanced MG-63 cells attached and proliferated.

stained with PI and AO and observed under 488 nm wavelength to visualize under a fluorescence microscope. MTT assay was used to assess the proliferation of MG-63 cells on the scaffolds surface (Fig. 12). The MTT assay revealed the nontoxic properties of PLA and PLA-HAp material. Over 72 h MTT assay no significant cell death was observed. AO/ PI live and dead cell imaging showed that almost all live cells had attached to the scaffold surface. Remarkably, the attachment behavior of MG-63 cells was superior on PLA-HAp scaffold surface. Only PLA scaffold contained very few cells attached on its surface, whereas PLA-HAp scaffold was completely covered with healthy cells (Fig. 12 a-d). Moreover, the PLA-HAp scaffold surface allowed proliferation of cells. The present study revealed that HAp enhanced cell attachment and proliferation on the PLA-HAp scaffold surface.

63 osteoblast-like cells. Their firm attachment on scaffold surfaces showed cell proliferation network with connection between newly proliferated cells. 5. Conclusions A suitable scaffold must possess enhanced mechanical support with excellent cell attachment and proliferation capability on its surface. This study helps to determine the simplest scaffold fabrication technique (90° 3D-printing orientation) with most advanced mechanical stability and adequate porosity. Furthermore, a facile post fabrication modification on the 3D-printed PLA scaffold surface was attempted by incorporating HAp nanoparticles. The results revealed that compared with PLA scaffold, the post-fabricated HAp modified PLA scaffold influenced cell function, such as proliferation and differentiation. Cell attachment is highly influenced by the interaction of HAp nanoparticles on PLA scaffold surface, which adsorb proteins and facilitate cellular activity. The mechanical study also revealed the enhancement of compressive stress property (up to 47.16% for 90° 3D-printed PLA-HAp scaffold) due to the incorporation of HAp nanoparticles. The pores on 3D-printed PLA scaffold might provide space for HAp nano particles during post-fabrication modification of scaffolds. The maximum

4.7. SEM analysis of MG-63 osteoblast-like cell adhesion and proliferation on scaffold surface Human osteoblast-like MG-63 cells were used to study cell attachment and proliferation of synthesized and HAp-modified PLA scaffolds (Fig. 13). Cells were fixed with formaldehyde and observed by SEM to assess their morphology. SEM analysis revealed that PLA-HAp scaffolds served as excellent surface for the attachment and proliferation of MG-

Fig. 13. SEM analysis of (a & b) MG-63 cells attached and proliferated over PLA-HAp surface. 12

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compressive stress of ~53 ± 0.2 MPa was obtained for 90° orientation printed PLA-HAp scaffold with a maximum porosity of ~47%. As the main conclusion, PLA-HAp scaffolds have proved to be an excellent composite material with enhanced surface activity due to the coating of HAp nanoparticles, with a 90° 3D-printing approach for application in bone tissue engineering.

[20]

Declaration of competing interest

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[21]

The authors declare no conflicts of interest.

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Acknowledgement

[24]

This research is supported by a grant from Marine Biotechnology Program (20150220) funded by the Ministry of Oceans and Fisheries, Republic of Korea.

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ceramint.2019.10.057.

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