3D Printed Fe Scaffolds with HA Nanocoating for Bone Regeneration

Subscriber access provided by RYERSON UNIVERSITY

Article

3D printed Fe scaffolds with HA nano-coating for bone regeneration Chen Yang, Zhiguang Huan, Xiaoya Wang, Chengtie Wu, and Jiang Chang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00885 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 28, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

3D printed Fe scaffolds with HA nano-coating for bone regeneration

Chen Yang †§, Zhiguang Huan †, Xiaoya Wang †, Chengtie Wu †, and Jiang Chang*†

†State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, China. §University of Chinese Academy of Sciences, Beijing 100049, P.R.China *Corresponding Author: E-mail: [email protected] (J.C.), Tel./Fax: 86-21-52412804.

Keyword: 3D printing, HA nano-coating, bone scaffold, tissue engineering

1

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 28

Abstract Pure iron (Fe) has been investigated as a cardiovascular stent in recent years for its biodegradation property and blood compatibility. It also has great potential for bone healing, especially for load-bearing areas based on its inherent mechanical property which is high enough for bone regeneration. However, the conventional manufacturing methods restrict its application as bone scaffold for uncontrollable architecture and stiffness. Also, the cytotoxicity makes it impossible for in vitro culture as bone tissue engineering scaffold. To solve these deficiencies, in the present study, we applied 3D printing technique with a modified coating strategy together to precisely control the macropore structure and surface nanostructure of final scaffolds. Our results showed that the compressive mechanical properties of the 3D printed Fe scaffolds were in the natural bone range and the hydroxyapatite (HA) coating was highly bonded to the substrate, significantly improved the viability and alkaline phosphatase (ALP) activity and osteogenic differentiation of the rabbit bone marrow mesenchymal stem cells (rBMSCs) on the scaffold. This study indicates that the 3D printed Fe scaffolds with controllable nanostructured HA coating may be a promising candidate for bone tissue engineering applications.

2


Page 3 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. Introduction One challenging issue in bone tissue regeneration is to assist the restoration of large bone defects which cannot be healed by the host itself. Since autografts and allografts show various disadvantages including a second surgery or the high risk of disease transmission, porous scaffolds made of synthetic biomaterials have been paid a lot of attention 1-2. The 3D porous structures of biomaterials can accelerate the bone ingrowth through interconnected pores. However, well designed 3D porous scaffolds are still difficult to be achieved due to the limitation of traditional methods such as foaming and casting technique, which make porous structure inaccurate and uncontrollable

3-4

. Instead of the restrictions of the conventional

methods, additive manufacturing techniques including 3D printing are fast developed in recent years. Customized porous scaffolds can be produced by 3D printing with pre-designed shape and inner-architecture

5-7

. For non-load bearing applications, various efforts has been

put into both polymers and ceramics, especially biodegradable substitutes such as polycaprolactone (PCL) and tricalcium phosphate (TCP)

8-10

. Porous scaffolds made of these

polymers and ceramics possess significate capacity in bone repairing of critical defects in non-load bearing areas. However, the inner insufficient mechanical properties make them impossible for restoring of load bearing bones. Metallic scaffolds are obviously the best choice to provide support for healing bones in load-bearing areas, which can cope with the initially mechanical requirements. In spite of the fact that metals and alloys have been used as bone implants for a long time, such as titanium (Ti) and its alloys which are nowadays the main orthopaedic implants in clinical applications due to their superior biocompatibility and stability

11-12

. However, the superior corrosion

resistance makes Ti non-biodegradable in body and the high stiffness may cause bone failure due to the stress shielding, which lead researchers to put more efforts on biodegradable and stress-compatible metals. Magnesium (Mg)- and iron (Fe)-based metals are the two mainly 3



Page 4 of 28

biodegradable metals which also possess mechanical properties more aligned to nature bone as compared to Ti-based metals

13-15

. Mg-based metals have great potential in orthopaedic

applications for their osteoconductive and fully bioresorbable properties

16-17

. However, the

degradation rate of Mg is too rapid which may cause high pH and no match time for bone healing 18. On the contrary, the degradation rate of Fe is quite slow as compared to Mg, which offers sufficient time and mechanical support for new bone ingrowth. Still, many researchers believe that the degradation rate of Fe is too slow and they try to promote the degradation process by developing different Fe-based alloys or refining the microstructures of the materials

19-20

. Nevertheless, the biocompatibility of Fe is still the crucial concern which

hinders its application in bone tissue regeneration. Although, pure iron has also been studied as blood vessel stents in recent years and no local toxicity is observed

21-22

, the

cytocompatibility is critical as cytotoxicity is obviously observed when cells are directly seeded on pure Fe substitutes

20, 23-26

, which makes it impossible for bone tissue engineering

applications. To solve this problem, surface modification such as bioceramic coating is one of the approaches which can ameliorate the interfacial biocompatibility and increase the osseointegration. Hydroxyapatite (HA), the main mineral component of bone, shows excellent bone-integration after coating on metallic implants

27-29

. Nowadays, many coating

techniques like plasma spraying and electrophoretic deposition have been used to generate the homogeneity of HA coating

30

. However, the morphology of the coating and the high

bonding strength between coating and substrates are hard to guarantee, which are important for the cytocompatibility and in vivo osteointegration 31. Herein, we firstly applied 3D-printing method to fabricate pure Fe scaffolds with tailored mechanical property. Subsequently, we developed a modified method to “grow” HA on the surface of 3D printed Fe scaffolds with controllable nano-morphology and high bonding

4


Page 5 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


strength, which allows rabbit bone marrow mesenchymal stem cells (rBMSCs) to survive directly on the scaffolds, further proliferate and osteogenically differentiate.

2. Materials and methods 2.1 Fabrication and characterization of the 3D printed Fe scaffolds The printable Fe paste was prepared by mixing 10 g of pure (99.5%) Fe powders (Haotian Nano Technology, China) with 2 g of an aqueous Hydroxypropylmethylcellulose (HPMC) solution (1.5 wt.%) as binder. Fe scaffolds were printed based on a precise 3D plotted system (Nano-Plotter NP 2.1, GeSiM, Grosserkmannsdorf, Germany) and the printing device which was developed by the Fraunhofer Institute for Materials Research and Beam Technology (Dresden, Germany). The geometric structure, pore size and porosity of the printed scaffolds were designed by a computer program (GeSiM Scaffold Generator). The paste was pushed out from the syringe by the pressure of 200–400 kPa and the printing speed was 10 mm s-1. In this study, a lay-dawn pattern of 0°-45°-90°-135° was designed to keep more cells on scaffolds. The filament gap of the scaffolds was 1.0 mm and layer thickness was 0.30 mm. The obtained Fe scaffolds were dried at room temperature overnight and cured at 300 ℃ in air for 2 h to remove HPMC binder. The cured Fe scaffolds were further sintered at 1120 ℃ for 3h under argon protection. The overall morphologies of the 3D printed Fe scaffolds were observed using an optical Nikon D90 camera. The macropore structure of the 3D printed Fe scaffolds were visualized by an optical microscope (S6D, Leica, Germany). The phase of the sintered Fe scaffolds was characterized by X-ray diffraction (XRD) (D8ADVANCE, Bruker, Germany). 2.2 Fabrication and characterization of HA coating on the 3D printed Fe scaffolds Nano-HA was coated on Fe scaffolds via a modified hydrothermal procedure

32

.

Typically, 7.49 g of ethylenediaminetetraacetic acid calcium disodium salt (EDTA-Ca-Na2) 5



Page 6 of 28

and 1.44 g of NaH2PO4 were dissolved in 80 mL deionized water. NaOH powders (AR, Shanghai, China) were used to adjust pH value to 9. Then 15 mL obtained solution was transferred into 25 mL Teflon autoclaves with a Fe scaffold inside and hydrothermally treated at 120 ℃ for 12 h hours, followed by cooling to room temperature naturally. The coated samples were ultrasonically cleansed in deionized water bath for 30 sec, then rinsed with ethanol and dried at 60 ℃ for 12 hours. To investigate the morphology of HA coating, the reaction condition of pH, temperature and time were changed as described. For cross-section examination, the coated Fe scaffolds were embedded with polymethyl methacrylate and longitudinally cut partially. The section was scrupulously polished to expose the exquisite structure of the coating. The HA coating obtained under the reaction condition of 120 ℃, pH 13 for 12 hours was chosen for in vitro biological study. To coat more HA minerals on Fe substrates, Fe scaffolds were coated twice on both sides (4 times in total) by repeating the same procedure. The morphology of HA coatings was observed by scanning electron microscopy (SEM) (S-4800, Hitachi, Japan). The elemental analysis was conducted using SEM with energy dispersive Xray (EDS). The phase of the coating was characterized by X-ray diffraction (XRD) (D8ADVANCE, Bruker, Germany). 2.3 Mechanical properties of the 3D printed Fe scaffolds and bonding strength of HA coatings The compressive yield strength and modulus of 3D printed Fe scaffolds (8 mm ×8 mm ×8 mm, filament gap of 1.0 mm and layer thickness of 0.30mm) with HA coatings (coated for 4 times under the reaction condition of 120 ℃, pH 13 for 12 hours for each time) were evaluated using a mechanical testing machine (AG-I, Shimadzu, Japan). The crosshead speed is 0.5 mm min-1. The scaffolds without HA coating were used as control. The initial linear portion of the stress-strain graph was selected to determine the compressive Young’s 6


Page 7 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


modulus of scaffolds. Five samples were tested for each group. The porosity of 3D printed Fe scaffolds was evaluated using a Micro-CT 40 scanner (Scaco Medical, Brüttisellen, Switzerland). Flat Fe cylindrical disks (diameter: 25 mm, thickness: 3 mm) were manufactured for the measurement of the bonding strength between the HA coating and Fe substrates using the same condition as that for preparation of 3D printed Fe scaffolds. The HA coating was processed for 4 times by the same procedure as described in section 2.3. The bonding strength of HA coatings on Fe substrate was tested using a modified method referring to the American Society for Testing and Materials (ASTM) F1147-05. Briefly, both side of Fe disks were attached with cylindrical Ti-6Al-4 V rods (diameter: 25 mm) by High-performance E-7 glue (Shanghai Institute of Synthetic Resin, Shanghai, China). The combined rods and Fe substrate were placed at 100 °C for 2 h to harden the glue before testing. A mechanical testing machine (Instron-5592, SATEC, USA) was used for the measurement. The crosshead speed of the testing was 2 mm·min−1, and five replications were measured in this experiment. 2.4 The effect of HA coatings on Fe ion release profile The HA coating was obtained under the reaction condition of 120 ℃, pH 13 for 12 hours for each treatment. Non-coated Fe scaffolds, Fe scaffolds with 1 time HA coating and Fe scaffolds with 4 times HA coating were immersed in 75% ethanol for 10 min and exposed in UV for 30 min as sterilization. The sterilized samples were then immersed in Eagle’s medium alpha (αMEM) media for 1d, 3d, 7d, 14d, 21d, and 28d, and the ratio of the media volume to the sample weight was 100 mLg-1 in a shaking bath at 37 ℃ to mimic the in vivo situation. At each time point, 1 mL media was collected from each sample for measuring and 1 mL new media was added. The Fe ion concentration was measured using inductively coupled plasma atomic emission spectrometry (ICP-AES) (VarianCo, USA).

7



Page 8 of 28

2.5 The effect of HA coatings on in vitro cytotoxicity of the 3D printed Fe scaffolds All Fe scaffolds (non-coated Fe scaffold, Fe scaffolds with 1 time HA coating and Fe scaffold with 4 times HA coating) were sterilized by immersing in 75% ethanol for 10 min and UV treating for 30 min. Modified Eagle’s medium alpha (αMEM) supplement with 10% fetal bovine serum (FBS), 100 µg/ml penicillin and 100µg/ml streptomycin were used as culture medium. Each scaffold (Ø10 mm X 3 mm) was initially seeded with an initial density of 1× 104 rabbit bone marrow mesenchymal stem cells (rBMSCs) (Allcells, Silicon Valley, CA, USA) in 48-well tissue culture plates and then incubated at 37 °C wiht 5% CO2. After 24 hours, the cell culture medium was changed for the first time, and then changed every two days. The viability of seeded cells was assessed using LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen Corporation, Karlsruhe, Germany) after 1day and 3days culturing. Briefly, the 3D printed Fe scaffolds with attached rBMSCs were rinsed with PBS and stained by two different dyes (2 µmol L-1 ethidium homoimer-1 and 4 µmol L-1 calcein AM dissolved in PBS) for 30 min at room temperature under the cover of aluminum foil preventing from light. The live and dead cells on scaffolds were visualized using confocal laser scanning microscopy (Leica, Wetzlar, Germany). 2.6 Cell proliferation and ALP activity of rBMSCs on the 3D printed Fe scaffold with HA coatings To measure the proliferation and ALP activity of rBMSCs on Fe scaffold with HA coatings (4 times), blank wells were used as control. Each scaffold (Ø10 mm X 3 mm) was initially seeded with an initial density of 1× 104 rabbit bone marrow mesenchymal stem cells (rBMSCs). To evaluate the cell proliferation, cells were cultured for 1, 3, 7, 14, and 21 days,

respectively. At each time point, the relative proliferation was assessed by CellTiter-LumiTM Plus Luminescent Cell Viability Assay kit (Beyotime, China) based on the measurement of ATP. Briefly, all samples were washed twice using PBS buffer before adding 200 µl of assay 8


Page 9 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reagent. After 10 mins incubation in room temperature with light preventing. The luminescence was read on a Microplate Reader (Cytation™ 5, BioTek, USA). ALP activities of rBMSCs seeded on Fe scaffolds with HA coating (4 times) was measured by transformating of p-nitrophenyl-phosphate (pNPP: Sigma, St. Louis, USA) into p-nitrophenol (pNP) after 7, 14 and 21 days culture. Briefly, samples were rinsed with PBS before adding 0.2% (v/v) Triton-X/EDTA solution and then incubated at -20℃ for 10 min. The collected cell lysates were centrifuged at 12000 rpm for 5 min. 200 µl pNPP working solution and 100 µl cell lysate supernatant were added in 96-well plate and incubated at 37 °C for 30 min. The enzyme activity was quantified by absorbance at 405 nm (BioTek, USA). ALP activity was normalized against the total protein content of the sample, which was assessed by BCA Protein Assay Kit (Pierce, Rockford, IL, USA). The absorbance OD value was read at 562 nm and the total protein content was calculated according to a series of BSA (Sigma) standards. 2.7 Real-time PCR analysis 3D printed Fe scaffolds (Ø30 mm ×3 mm) with 4 times HA coatings (the reaction condition of 120 ℃, pH 13 for 12 hours for each time) were seeded with a density of 2 × 105 cells/well in 6-well plates. At each timepoint, total RNA was isolated using Trizol reagent (Invitrogen, USA). The cDNA of each sample was reverse transcribed and quantitatively analysed on following genes: collagen 1 (COL1), osteocalcin (OCN), osteopontin (OPN), alkaline phosphatase (ALP), and bone morphogenetic protein-2 (BMP-2). All genes were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH, housekeeping gene). All experiments were performed in triplicate. The sequences of the specific primers used are as follows: GAPDH: 5′- TCACCATCTTCCAGGAGCGA-3′& 5′-CACAATGCCGAAGTGGTCGT-3′; COL I: 5′-CTTCTGGCCCTGCTGGAA AGGATG-3′& 5′-CCCGGATACAGGTTTCGCCAGTAG-3′; OCN: 5′-CCGGGAGCAGTGTGAGCTTA-3′& 5′-AGGCGGTCTTCAAGCCA TACT-3′; 9



Page 10 of 28

OPN: 5′-CACCATGAGAATCGCCGT-3′& 5′-CGTGACTTTGGGTTTCTACGC-3′; ALP: 5′-TGT GCGGGGTCAAGGCTAAC-3′ & 5′-GGCGTC CGAGTACCAGTTGC-3′; BMP-2: 5′-CGCCTC AAATCCAGCTGTAAG-3′& 5′-GGGCCACAATCCAGTCGTT-3′.

2.8 Statistical analysis All values were expressed as means ± standard deviations (SD) which were calculated by the one-way ANOVA analysis model. Statistically significant was considered when P value was below 0.5.

3. Results 3.1 Characterization of 3D printed Fe scaffolds and HA coating The prepared Fe scaffolds can be fabricated in different shapes and size (Fig. 1a-c). The line width and distance as well as the porosity can be controlled by designed program (Fig. 1d-f). The pores are highly interconnected which is shown in the top view images (Fig. 1d and 1e) and side-view image (Fig. 1f). A lay-dawn pattern of 0°-45°-90°-135° was designed to keep more cells on the scaffolds (Fig. 1b-c). It shows that the Fe scaffold became rougher and the colour of the scaffold became light after HA coating (Fig. 1c). XRD patterns (Fig. 2) show that 3D printed Fe scaffolds consist of γ-Fe phases (Fig. 2a) and the component of coating was confirmed as HA (Fig. 2b).

10


Page 11 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 1. Overview of 3D printed Fe scaffolds: (a) Fe scaffolds prepared by the 3D-printing method with different design; (b-c) Scaffolds designed for in vitro study without coating (b) and with 4 times HA coating (c); (d-e) Top view of 3D printed Fe scaffolds with different porosity; (f) Side view of 3D printed Fe scaffolds.

Fig. 2. XRD patterns of the surface of 3D printed Fe scaffold before coating (a) and after coating (b)

3.2 Effects of reaction parameters on the morphology of HA coatings The morphologies of HA coating under different experimental conditions are shown in Fig. 3 and Fig. 4. There is no special micro or nanostructures on the surface of pure Fe scaffold which is shown in Fig. 3a. After coating, Nanosheet structured HA or nanorod structured HA was obtained at 120 ℃ for 12h depending on the pH value of the initial solution indicating the significant effect of reaction pH on the morphology of HA coatings. Nanosheet structured HA was obtained at pH 6 (Fig. 3b) and nanorod structured HA was obtained at pH 9 (Fig. 3c) and pH 13 (Fig. 3d). These nanorods became compact and ordered when the pH value increased to 13.

11



Page 12 of 28

Fig. 3. SEM images of HA coating prepared in initial solution with different pH value at 120 ℃ for 12h: (a) Fe scaffold without HA coating; (b-d) HA coating generated at pH =6, pH =9, pH=13, respectively. The reaction temperature also showed clear effect on the coating morphology. None HA coating was formed on Fe scaffold at the temperature of 60 ℃ with the initial solution at pH 9 and reaction time of 12h (Fig. 4a). Nanorod structured HA can be obtained at 120 ℃ (Fig. 4b) and 180 ℃ (Fig. 4c), and these nanorods became smaller and dense when the temperature increased from 120 ℃ (Fig. 4b) to 180 ℃. The effect of reaction time on HA coating morphology with the initial solution at pH 9 and reaction temperature at 120 ℃ was investigated and the results are shown in Fig. 4d-f. It is clear to see that more and more HA 12


Page 13 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nanorods were formed with the increase of reaction time which reveals that HA coating was “growing” on the Fe scaffolds with time.

Fig. 4. SEM images of HA coating prepared at pH 9 with different reaction temperature or time: (a-c) HA coatings generated at 60 ℃, 120 ℃ and 180 ℃ for 12h, respectively; (d-f) HA coatings generated at 120 ℃ for 6h, 12h and 24h, respectively. The surface morphology and cross view of SEM-EDS mappings of HA coatings for 1 time and 4 times are shown in Fig.5. It reveals that the thickness of HA coating on 3D printed Fe scaffolds increased from 10 µm (Fig. 5c-d) to about 120 µm (Fig. 5e-f) after 4 times coating. However, the relatively ordered HA morphology (Fig. 5a) was compromised (Fig. 5b).

13



Page 14 of 28

Fig. 5. The surface morphology and cross view of SEM-EDS mappings of HA coatings prepared by 1 time and 4 times treatment at 120 ℃, pH 13 for 12 hours for each time: (a-b) surface morphology of HA coatings for 1 time (a) and 4 times (b); (c-d) SEM image (c) and merge images of EDS elemental mappings (d) over the cross-section of HA coatings for 1 time; (e-f) SEM image (e) and merge images of EDS elemental mappings (f) over the crosssection of HA coatings for 4 times. (Red: Fe, green: HA) 3.3 Mechanical property of 3D printed Fe scaffolds and the bonding strength of HA coatings. The compressive properties of 3D printed Fe scaffolds with and without HA (coated 4 times) coatings are presented in Table 1 as well as the porosity of each group. Due to the high porosity of 67.5%, the compressive properties of 3D printed Fe scaffolds dropped to a natural bone level with compressive yield strength of 141.25 MPa and compressive young’s modulus of 1.25 GPa. The porosity, compressive yield strength and compressive young’s modulus of HA coated scaffold were about 66.2%, 139.46 MPa and 1.22 GPa, respectively. There was no significant difference between 3D printed Fe scaffolds with and without coatings suggesting that the coating process didn’t affect the mechanical properties of the scaffolds. The bonding strength of the HA coating (coated 4 times) was about 38.12 Mpa.

14


Page 15 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. The porosity, mechanical property of 3D printed Fe scaffolds with and without coatings and the bonding strength of the HA coatings.

3.4 The effect of HA coatings on Fe ion release from the scaffolds The Fe ions released from 3D printed Fe scaffolds were measured and the results are shown in Fig. 6. Before HA coating, the Fe ions released rapidly from 3D printed Fe scaffold which was 19.7 mg/L in the first day and accumulated to 132.2 mg/L up to 28 days. After 1 time HA coating, Fe ion concentration dropped obviously, which was 6.0 mg/L in the first day and accumulated to 44.1 mg/L in 4 weeks. Furthermore, with 4 times HA coating, the Fe ion concentration was declined to 0.3~1.9 mg/L.

Fig. 6. Fe release profile of 3D printed Fe scaffolds without HA coating, with 1 time HA coating and with 4 times HA coatings in α-MEM.

15



Page 16 of 28

3.5 The effect of HA coatings on in vitro cytotoxicity of the 3D printed Fe scaffolds Fluorescence microscopy of live/dead assay shows that rBMSCs were all dead (red) on pure Fe scaffolds after 1 day and 3 days (Fig. 7a and d). About half cells survived (green) on 1 time coating scaffolds after 1 day culture (Fig. 7b). However, most of them were dead after 3 days (Fig. 7e). In contrast, very few cells were dead on 4 times HA coating scaffolds (Fig. 7c), and a higher density of live cell attachment was observed after 3 days culture (Fig. 7f).

Fig. 7. Fluorescent images from the live/dead assay of live (green) and dead (red) rBMSCs attached after 1 day (a-c) and 3 days (d-f) culture on 3D printed Fe scaffold without HA coating (a, d), with HA coating for 1 time (b, e) and with HA coatings for 4 times (c, f). The proliferation of rBMSCs cultured on 3D printed Fe scaffolds with 4 times HA coating after 1,3,7,14 and 21 days are shown in Fig. 8. Cells proliferated with time on both 3D printed Fe scaffolds with 4 times HA coating and blank wells, and no significant difference between the two groups was observed.

16


Page 17 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 8. The proliferation of rBMSCs culture on 3D printed Fe scaffold with HA coatings (4 times) as compared to blank culture wells after 1,3,7,14 and 21days.

3.6 ALP activity of rBMSCs on the 3D printed Fe scaffold with HA coatings The ALP activity of rBMSCs on 3D printed Fe scaffolds with 4 times HA coating was measured and the results are shown in Fig. 9. It revealed that the ALP activity of rBMSCs increased apparently with time throughout the test period (7, 14 and 21days.) on both 3D printed Fe scaffolds and blank wells. Also, cells on Fe scaffolds with 4 times HA coating had significantly higher ALP activities than that of the blank group at each time point, which indicates enhanced osteogenic properties of the scaffolds. 17



Page 18 of 28

Fig. 9. ALP activities of rBMSCs on 3D printed Fe scaffolds with HA coating (4 times) as compared to blank wells after being cultured for 7,14 and 21days. (**p < 0.01 and *p

3D Printed Fe Scaffolds with HA Nanocoating for Bone Regeneration

Recommend Documents