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nHA composite scaffolds for bone tissue engineering
Author’s Accepted Manuscript Microscale electrohydrodynamic printing of biomimetic PCL/nHA composite scaffolds for bone tissue engineering Xiaoli Qu, Peng Xia, Jiankang He, Dichen Li www.elsevier.com
PII: DOI: Reference:
S0167-577X(16)31495-1 http://dx.doi.org/10.1016/j.matlet.2016.09.035 MLBLUE21473
To appear in: Materials Letters Received date: 29 June 2016 Revised date: 23 August 2016 Accepted date: 10 September 2016 Cite this article as: Xiaoli Qu, Peng Xia, Jiankang He and Dichen Li, Microscale electrohydrodynamic printing of biomimetic PCL/nHA composite scaffolds for bone tissue engineering, Materials Letters, http://dx.doi.org/10.1016/j.matlet.2016.09.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Microscale electrohydrodynamic printing of biomimetic PCL/nHA composite scaffolds for bone tissue engineering Xiaoli Qu, Peng Xia, Jiankang He*, Dichen Li State key laboratory for manufacturing system engineering, Xi’an Jiaotong University, Xi’an, Shaanxi 710049, China [email protected]
Abstract Electrohydrodynamic printing provides an innovative strategy to fabricate high-resolution tissue-engineering scaffolds with fiber orientation and scale similar to native extracellular matrix. However, few studies have been conducted to incorporate functional nanobiomaterials into microscale fibrous structures. In this study, we presented to fabricate microscale poly (ε-caprolactone) (PCL) and hydroxyapatite nanoparticles (nHA) composite scaffolds with the average fiber diameter of 8.85 ± 1.12 μm based on electrohydrodynamic 3D printing to better mimic collagen fibers and HA nanocrystals in natural bones. The composite scaffolds exhibited good biocompatibility and facilitated cell alignment and proliferation in vitro. This strategy might be useful to regulate cellular microenvironment in multiscale and multimaterial levels for improved tissue regeneration. Graphical abstract
Keywords: Electrohydrodynamic printing, bone tissue engineering, microscale scaffold, hydroxyapatite nanoparticles
1．Introduction
Natural bone is a typical hierarchical biocomposite containing collagen fibers and hydroxyapatite (HA) crystals (Fig. 1a), which determine its mechanical properties and biological functions
[1]
. Recapitulating such structural and compositional
organization in synthetic scaffolds can provide a biomimetic microenvironment for cellular attachment, proliferation and differentiation and finally facilitate bone regeneration
[2]
. To mimic the structural microenvironment of natural bone,
electrospinning has been widely used to fabricate nanoscale fibers from various biopolymers
[3]
, which were further
reinforced with nHA [4]. However, the electrospun scaffolds were mostly limited to two-dimensional membranes and the orientation of the micro/nanoscale fibers were poorly controlled. Electrohydrodynamic printing (EHDP), combining the principles of electrospinning and additive manufacturing, has emerged as a new subject to fabricate high-resolution three-dimensional (3D) fibrous structures in a controlled manner [5]. Ahmad et al. employed EHDP to generate 3D patterns of nHA topographies in a high resolution [6]. Thian et al. prepared various nHA patterns by EHDP to mimic the dimension of mineral components in natural bone that enhanced cellular differentiation
[7]
. Rasekh et al. coupled EHDP with solvent evaporation techniques to fabricate PCL/nHA composite
scaffolds with fine fiber arrangements
[8]
. However, few studies have been conducted to fabricate microscale composite
bone scaffolds based on melt EHDP. In this study we aim to use melt EHDP to fabricate microscale PCL/nHA scaffolds with precise microfiber orientations and arrangements to better mimic collagen fibers and HA crystals composites in natural bone. The biological properties of the resultant composite scaffolds were evaluated by cell culture experiments.
2．Materials and methods 2.1. Materials PCL（Mw=80000 g/mol）with medical grade was bought from Jinan Daigang Biomaterial Co., Ltd (China). nHA was bought from Aladdin Reagent Co., LTD (China). Live/dead viability/cytotoxicity kit and Alexa Fluor 594 phalloidin were purchased from Invitrogen (USA) for cell viability and cytoskeleton staining. 4', 6-diamidino-2-phenylindole (DAPI) and AlamarBlue were purchased from Life Science International (USA) for cell nuclei staining and cellular
activities testing. All other reagents were analytical grade. To prepare PCL/nHA composite materials, 5 g PCL raw material was melted at the temperature of 80 o C. 0.4 g nHA particles were added into PCL melts and thoroughly stirred at the temperature of 80 o C for 2 h. The composite materials were allowed to solidify at the temperature of 4 o C for 1 h. The resultant PCL/nHA composites were cut into small pieces for future use.
2.2 Melt electrohydrodynamic printing of microscale PCL/nHA composite scaffolds The schematic process to fabricate PCL/nHA composite scaffolds is illustrated in Fig. 1b. Briefly, PCL/nHA composite pieces were loaded into a stainless syringe that was immersed in a temperature-controlled water circulating system. The temperature was increased to 70 o C to melt PCL/nHA composites. The glass slides were coated with a thin layer of transparent indium tin oxide (ITO) film as the conductive collecting substrate. To initialize the electrohydrodynamic printing process, the printing nozzle with the inner diameter of 340 μm was grounded and a conductive glass collector mounted onto a XY moving stage was connected with the positive terminal of a high voltage generator. The electric force between the nozzle and the collector would induce the electrohydrodynamic jetting of tiny filaments without needle blockage. The filament diameter was much smaller than that of the nozzle. By controlling the movement of the XY stage, a user-specific pattern could be obtained. A 3D microscale fibrous scaffold could be printed by stacking the tiny filaments in a layer-by-layer manner. In this study, the voltage, moving speed, nozzle-to-collector distance and feeding rate were fixed at 3 KV, 30 mm/s, 3 mm and 10 μL/h respectively. For cell experiment, a thin layer of agarose film was precoated onto the glass slide to avoid the attachment of cells on the glass surface.
2.3 Characterization of the microscale PCL/nHA composite scaffolds Scanning electron microscope (SEM, SU8010, Hitachi, Japan) was used to characterize the morphologies of the printed composite scaffolds after sputtering a thin layer of gold layer. To verify the existence of nHA in the fibrous structures, X-ray diffraction (XRD) was carried out using X-ray Diffractometer (XRD-7000, Shimadu, Japan) in the 2 θ range of
10°- 65° and a scan speed of 8°/min. Pure PCL and nHA were used as the control.
2.4 Cell culture Microscale PCL/nHA composite scaffolds were sterilized in 75% alcohol aqueous solution for 24 hours and then washed three times in PBS before cell seeding. MC 3T3-E1 cells were cultured in a basal culture medium of α-MEM (Hyclone) supplemented with 10% fetal bovine serum (Gibco) at 37 o C. The fibrous scaffolds were placed in a 6-well plate. The cells were trypsinized and seeded onto the scaffold surface at a density of 2.5×104 cells/cm2. After cell attachment for 30 minutes, 2 mL fresh culture medium was added to each well. The culture medium was changed every 2 days. To visualize cellular morphology and distribution on the microscale composite scaffolds, the cell-scaffold constructs cultured for 1 day were stained with Live/dead. After cultured for 3, 5 and 7 days, the constructs were fixed in 3.7% formaldehyde, treated with 0.1% Triton X-100 and stained with Alexa Fluor 594 phalloidin for cytoskeleton visualization. Cell nuclei were counterstained with DAPI. The fluorescent images were visualized with a confocal laser scanning microscope (A1, Nikon, Japan). In addition, the cell-scaffold constructs cultured for 3 and 5 days were further observed with SEM. The proliferation of MC-3T3 cells on microscale PCL/nHA composite scaffolds cultured for 1, 3, 5 and 7 days was evaluated via AlamarBlue assay.
3．Results and discussion Fig. 1c shows the microscale PCL/nHA composite scaffolds fabricated by EHDP. The contour of the scaffold was reconstructed from the CT images of porcine knee joint in Mimics (Materialise, Belgium) and the scaffold model was designed in Pro/Engineer (PTC, USA) according to previous developed methods [9]. The internal fibrous structures were printed with a total layer number of 20 and a fiber spacing of 100 μm. The size of the printed microfibers (Fig. 1d) was relatively uniform with the average fiber diameter of 8.85 ± 1.12 μm, close to the size of living cells. However, the line spacing was not accurately achieved due to the coulomb repulsion force between the printed fibers and the limited resolution of the moving stage. Fig. 1e shows the XRD profiles of PCL/nHA composites, PCL and nHA. Typical 2 θ
peaks of PCL were mainly observed at 21.28° and 23.7° while the peaks of nHA were found at 25.74°, 32.86° and 39.68°, which are in agreement with previous finding
[10]
. Both peaks were clearly observed in the XRD pattern of PCL/nHA
composites, which confirmed the existence of nHA in the microscale fibers. Fig. 2a and 2b shows the cell viability on the microscale fibrous scaffolds after seeding for 1 day. It is clearly observed that almost all the cells were uniformly attached on the printed microfibers with few cells on the glass slides, which was further validated by the fluorescent images of cellular nuclei distribution as shown in Fig. 2c. To characterize the spreading behavior of living cells on the composite scaffolds at different culture time point, cytoskeletons were stained and visualized as shown in Fig. 2d-f. Most cells began to spread and elongate along the direction of the microfibers on day 3. When the cell-scaffold constructs were cultured from day 5 to day 7, the proliferated cells not only covered the whole surface of the printed microfibers, but also infiltrated to the porous region of the fiber spacing.
Fig. 1 Fabrication and characterization of microscale PCL/nHA composite scaffolds based on EHDP. (a) Schematic organizations of natural bone, (b) schematic of electrohydrodynamic 3D printing to fabricate biomimetic PCL/nHA scaffolds, (c) microscale composite scaffolds with predefined geometries, (d) SEM images of the microfibrous structures, (e) XRD patterns of PCL, nHA and composite scaffold.
Fig. 2 Characterization of cellular attachment, viability and spreading on the microscale composite scaffolds. (a-b) cell viability on day 1, (c) cell distribution on the microfibrous scaffolds on day 1. Fluorescent images of cellular skeletons after cultured for (d) 3 days, (e) 5 days and (f) 7 days.
Fig. 3a-b show the SEM images of the cell-scaffold constructs cultured for 3 and 5 days. These results are in agreement with fluorescent characterization on cytoskeletons. Fig. 3c shows the quantified results on cell proliferation in the composite scaffolds cultured from day 1 to day 7. Significant cell proliferation was observed and cell number on day 7 was almost two-fold in comparison with that on day 1. Together these results indicated that the biomimetic PCL/nHA composite scaffolds possessed good biocompatibility and facilitated cellular alignment and proliferation in vitro. These biological results are in agreement with previous investigations
[7, 11]
, which found that both electrohydrodynamically
printed microscale PCL and nHA patterns were favorable for cellular behaviors. In addition, compared with electrospinning and other printing techniques, the present EHDP based on PCL melt and nHA composites avoids the use of organic solvents and enables to fabricate biomimetic bone scaffolds in a well controlled manner. It might be useful to regulate cellular microenvironment in multiscale and multimaterial levels for enhanced bone regeneration.
Fig. 3 SEM characterization and proliferation quantification of the cell-scaffold constructs cultured for different time. (a) SEM images of cell-scaffold constructs cultured for 3 days, (b) SEM images of cell-scaffold constructs cultured for 5 days, (c) quantification of cell proliferation in the composite scaffolds.
4．Conclusion Microscale PCL/nHA composite scaffolds were successfully fabricated by melt electrohydrodynamic printing to mimic collagen fibers and HA crystals in natural bone. The average diameter of the PCL/nHA fibers was 8.85 ± 1.12 μm, close to the size of living cells. The microscale composite scaffolds exhibited good biocompatibility and facilitated cellular proliferation and alignment in vitro. We believe that this strategy can potentially be used to regulate cellular microenvironment in multiscale and multimaterial levels for improved tissue regeneration.
Acknowledgment
This work was financially supported by the National Natural Science Foundation of China (51422508, 51675412) and the Fundamental Research Funds for the Central Universities of China.
Reference [1] Nair AK, Gautieri A, Chang SW, Buehler MJ. Nature communications. 2013;4:1724. [2] Ramin Khajavi, Mina Abbasipour, Bahador A. Applied Polymer. 2015;133:42883. [3] Zhang B, Seong B, Nguyen V, Byun D. Journal of Micromechanics and Microengineering. 2016;26:025015. [4] Qian Y, Li L, Jiang C, Xu W, Lv Y, Zhong L, et al. International journal of biological macromolecules. 2015;79:133-43. [5] Ahmad Z, Rasekh M, Edirisinghe M. Macromolecular Materials and Engineering. 2010;295:315-9. [6] Ahmad, Z, Huang, J, Edirisinghe, M. J, Jayasinghe, S. N, Best, S. M, Bonfield, W, Brooks, R. A, Rushton, N. Journal of Biomedical Nanotechnology. 2006:2: 201-207. [7] San Thian E, Ahmad Z, Huang J, Edirisinghe MJ, Jayasinghe SN, Ireland DC, et al. Biomaterials. 2008;29:1833-43. [8] Rasekh M, Ahmad Z, Day R, Wickam A, Edirisinghe M. Advanced Engineering Materials. 2011:13: 296-335. [9] He J, Li D, Lu B, Wang Z, Zhang T. Rapid Prototyping Journal. 2006;12:198-205. [10] Uma Maheshwari S, Samuel VK, Nagiah N. Ceramics International. 2014;40:8469-77. [11] Rasekh M, Ahmad Z, Frangos CC, Bozec L, Edirisinghe M, Day RM. Acta biomaterialia. 2013;9:5052-62.
Highlights
PCL-nHA microfibrous scaffolds were printed via EHDP to mimic natural bone.
The composite microfibers were well controlled with the size close to living cells.
The biomimetic scaffolds directed cellular alignment and proliferation in vitro.
PII: DOI: Reference:
S0167-577X(16)31495-1 http://dx.doi.org/10.1016/j.matlet.2016.09.035 MLBLUE21473
To appear in: Materials Letters Received date: 29 June 2016 Revised date: 23 August 2016 Accepted date: 10 September 2016 Cite this article as: Xiaoli Qu, Peng Xia, Jiankang He and Dichen Li, Microscale electrohydrodynamic printing of biomimetic PCL/nHA composite scaffolds for bone tissue engineering, Materials Letters, http://dx.doi.org/10.1016/j.matlet.2016.09.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Microscale electrohydrodynamic printing of biomimetic PCL/nHA composite scaffolds for bone tissue engineering Xiaoli Qu, Peng Xia, Jiankang He*, Dichen Li State key laboratory for manufacturing system engineering, Xi’an Jiaotong University, Xi’an, Shaanxi 710049, China [email protected]
Abstract Electrohydrodynamic printing provides an innovative strategy to fabricate high-resolution tissue-engineering scaffolds with fiber orientation and scale similar to native extracellular matrix. However, few studies have been conducted to incorporate functional nanobiomaterials into microscale fibrous structures. In this study, we presented to fabricate microscale poly (ε-caprolactone) (PCL) and hydroxyapatite nanoparticles (nHA) composite scaffolds with the average fiber diameter of 8.85 ± 1.12 μm based on electrohydrodynamic 3D printing to better mimic collagen fibers and HA nanocrystals in natural bones. The composite scaffolds exhibited good biocompatibility and facilitated cell alignment and proliferation in vitro. This strategy might be useful to regulate cellular microenvironment in multiscale and multimaterial levels for improved tissue regeneration. Graphical abstract
Keywords: Electrohydrodynamic printing, bone tissue engineering, microscale scaffold, hydroxyapatite nanoparticles
1．Introduction
Natural bone is a typical hierarchical biocomposite containing collagen fibers and hydroxyapatite (HA) crystals (Fig. 1a), which determine its mechanical properties and biological functions
[1]
. Recapitulating such structural and compositional
organization in synthetic scaffolds can provide a biomimetic microenvironment for cellular attachment, proliferation and differentiation and finally facilitate bone regeneration
[2]
. To mimic the structural microenvironment of natural bone,
electrospinning has been widely used to fabricate nanoscale fibers from various biopolymers
[3]
, which were further
reinforced with nHA [4]. However, the electrospun scaffolds were mostly limited to two-dimensional membranes and the orientation of the micro/nanoscale fibers were poorly controlled. Electrohydrodynamic printing (EHDP), combining the principles of electrospinning and additive manufacturing, has emerged as a new subject to fabricate high-resolution three-dimensional (3D) fibrous structures in a controlled manner [5]. Ahmad et al. employed EHDP to generate 3D patterns of nHA topographies in a high resolution [6]. Thian et al. prepared various nHA patterns by EHDP to mimic the dimension of mineral components in natural bone that enhanced cellular differentiation
[7]
. Rasekh et al. coupled EHDP with solvent evaporation techniques to fabricate PCL/nHA composite
scaffolds with fine fiber arrangements
[8]
. However, few studies have been conducted to fabricate microscale composite
bone scaffolds based on melt EHDP. In this study we aim to use melt EHDP to fabricate microscale PCL/nHA scaffolds with precise microfiber orientations and arrangements to better mimic collagen fibers and HA crystals composites in natural bone. The biological properties of the resultant composite scaffolds were evaluated by cell culture experiments.
2．Materials and methods 2.1. Materials PCL（Mw=80000 g/mol）with medical grade was bought from Jinan Daigang Biomaterial Co., Ltd (China). nHA was bought from Aladdin Reagent Co., LTD (China). Live/dead viability/cytotoxicity kit and Alexa Fluor 594 phalloidin were purchased from Invitrogen (USA) for cell viability and cytoskeleton staining. 4', 6-diamidino-2-phenylindole (DAPI) and AlamarBlue were purchased from Life Science International (USA) for cell nuclei staining and cellular
activities testing. All other reagents were analytical grade. To prepare PCL/nHA composite materials, 5 g PCL raw material was melted at the temperature of 80 o C. 0.4 g nHA particles were added into PCL melts and thoroughly stirred at the temperature of 80 o C for 2 h. The composite materials were allowed to solidify at the temperature of 4 o C for 1 h. The resultant PCL/nHA composites were cut into small pieces for future use.
2.2 Melt electrohydrodynamic printing of microscale PCL/nHA composite scaffolds The schematic process to fabricate PCL/nHA composite scaffolds is illustrated in Fig. 1b. Briefly, PCL/nHA composite pieces were loaded into a stainless syringe that was immersed in a temperature-controlled water circulating system. The temperature was increased to 70 o C to melt PCL/nHA composites. The glass slides were coated with a thin layer of transparent indium tin oxide (ITO) film as the conductive collecting substrate. To initialize the electrohydrodynamic printing process, the printing nozzle with the inner diameter of 340 μm was grounded and a conductive glass collector mounted onto a XY moving stage was connected with the positive terminal of a high voltage generator. The electric force between the nozzle and the collector would induce the electrohydrodynamic jetting of tiny filaments without needle blockage. The filament diameter was much smaller than that of the nozzle. By controlling the movement of the XY stage, a user-specific pattern could be obtained. A 3D microscale fibrous scaffold could be printed by stacking the tiny filaments in a layer-by-layer manner. In this study, the voltage, moving speed, nozzle-to-collector distance and feeding rate were fixed at 3 KV, 30 mm/s, 3 mm and 10 μL/h respectively. For cell experiment, a thin layer of agarose film was precoated onto the glass slide to avoid the attachment of cells on the glass surface.
2.3 Characterization of the microscale PCL/nHA composite scaffolds Scanning electron microscope (SEM, SU8010, Hitachi, Japan) was used to characterize the morphologies of the printed composite scaffolds after sputtering a thin layer of gold layer. To verify the existence of nHA in the fibrous structures, X-ray diffraction (XRD) was carried out using X-ray Diffractometer (XRD-7000, Shimadu, Japan) in the 2 θ range of
10°- 65° and a scan speed of 8°/min. Pure PCL and nHA were used as the control.
2.4 Cell culture Microscale PCL/nHA composite scaffolds were sterilized in 75% alcohol aqueous solution for 24 hours and then washed three times in PBS before cell seeding. MC 3T3-E1 cells were cultured in a basal culture medium of α-MEM (Hyclone) supplemented with 10% fetal bovine serum (Gibco) at 37 o C. The fibrous scaffolds were placed in a 6-well plate. The cells were trypsinized and seeded onto the scaffold surface at a density of 2.5×104 cells/cm2. After cell attachment for 30 minutes, 2 mL fresh culture medium was added to each well. The culture medium was changed every 2 days. To visualize cellular morphology and distribution on the microscale composite scaffolds, the cell-scaffold constructs cultured for 1 day were stained with Live/dead. After cultured for 3, 5 and 7 days, the constructs were fixed in 3.7% formaldehyde, treated with 0.1% Triton X-100 and stained with Alexa Fluor 594 phalloidin for cytoskeleton visualization. Cell nuclei were counterstained with DAPI. The fluorescent images were visualized with a confocal laser scanning microscope (A1, Nikon, Japan). In addition, the cell-scaffold constructs cultured for 3 and 5 days were further observed with SEM. The proliferation of MC-3T3 cells on microscale PCL/nHA composite scaffolds cultured for 1, 3, 5 and 7 days was evaluated via AlamarBlue assay.
3．Results and discussion Fig. 1c shows the microscale PCL/nHA composite scaffolds fabricated by EHDP. The contour of the scaffold was reconstructed from the CT images of porcine knee joint in Mimics (Materialise, Belgium) and the scaffold model was designed in Pro/Engineer (PTC, USA) according to previous developed methods [9]. The internal fibrous structures were printed with a total layer number of 20 and a fiber spacing of 100 μm. The size of the printed microfibers (Fig. 1d) was relatively uniform with the average fiber diameter of 8.85 ± 1.12 μm, close to the size of living cells. However, the line spacing was not accurately achieved due to the coulomb repulsion force between the printed fibers and the limited resolution of the moving stage. Fig. 1e shows the XRD profiles of PCL/nHA composites, PCL and nHA. Typical 2 θ
peaks of PCL were mainly observed at 21.28° and 23.7° while the peaks of nHA were found at 25.74°, 32.86° and 39.68°, which are in agreement with previous finding
[10]
. Both peaks were clearly observed in the XRD pattern of PCL/nHA
composites, which confirmed the existence of nHA in the microscale fibers. Fig. 2a and 2b shows the cell viability on the microscale fibrous scaffolds after seeding for 1 day. It is clearly observed that almost all the cells were uniformly attached on the printed microfibers with few cells on the glass slides, which was further validated by the fluorescent images of cellular nuclei distribution as shown in Fig. 2c. To characterize the spreading behavior of living cells on the composite scaffolds at different culture time point, cytoskeletons were stained and visualized as shown in Fig. 2d-f. Most cells began to spread and elongate along the direction of the microfibers on day 3. When the cell-scaffold constructs were cultured from day 5 to day 7, the proliferated cells not only covered the whole surface of the printed microfibers, but also infiltrated to the porous region of the fiber spacing.
Fig. 1 Fabrication and characterization of microscale PCL/nHA composite scaffolds based on EHDP. (a) Schematic organizations of natural bone, (b) schematic of electrohydrodynamic 3D printing to fabricate biomimetic PCL/nHA scaffolds, (c) microscale composite scaffolds with predefined geometries, (d) SEM images of the microfibrous structures, (e) XRD patterns of PCL, nHA and composite scaffold.
Fig. 2 Characterization of cellular attachment, viability and spreading on the microscale composite scaffolds. (a-b) cell viability on day 1, (c) cell distribution on the microfibrous scaffolds on day 1. Fluorescent images of cellular skeletons after cultured for (d) 3 days, (e) 5 days and (f) 7 days.
Fig. 3a-b show the SEM images of the cell-scaffold constructs cultured for 3 and 5 days. These results are in agreement with fluorescent characterization on cytoskeletons. Fig. 3c shows the quantified results on cell proliferation in the composite scaffolds cultured from day 1 to day 7. Significant cell proliferation was observed and cell number on day 7 was almost two-fold in comparison with that on day 1. Together these results indicated that the biomimetic PCL/nHA composite scaffolds possessed good biocompatibility and facilitated cellular alignment and proliferation in vitro. These biological results are in agreement with previous investigations
[7, 11]
, which found that both electrohydrodynamically
printed microscale PCL and nHA patterns were favorable for cellular behaviors. In addition, compared with electrospinning and other printing techniques, the present EHDP based on PCL melt and nHA composites avoids the use of organic solvents and enables to fabricate biomimetic bone scaffolds in a well controlled manner. It might be useful to regulate cellular microenvironment in multiscale and multimaterial levels for enhanced bone regeneration.
Fig. 3 SEM characterization and proliferation quantification of the cell-scaffold constructs cultured for different time. (a) SEM images of cell-scaffold constructs cultured for 3 days, (b) SEM images of cell-scaffold constructs cultured for 5 days, (c) quantification of cell proliferation in the composite scaffolds.
4．Conclusion Microscale PCL/nHA composite scaffolds were successfully fabricated by melt electrohydrodynamic printing to mimic collagen fibers and HA crystals in natural bone. The average diameter of the PCL/nHA fibers was 8.85 ± 1.12 μm, close to the size of living cells. The microscale composite scaffolds exhibited good biocompatibility and facilitated cellular proliferation and alignment in vitro. We believe that this strategy can potentially be used to regulate cellular microenvironment in multiscale and multimaterial levels for improved tissue regeneration.
Acknowledgment
This work was financially supported by the National Natural Science Foundation of China (51422508, 51675412) and the Fundamental Research Funds for the Central Universities of China.
Reference [1] Nair AK, Gautieri A, Chang SW, Buehler MJ. Nature communications. 2013;4:1724. [2] Ramin Khajavi, Mina Abbasipour, Bahador A. Applied Polymer. 2015;133:42883. [3] Zhang B, Seong B, Nguyen V, Byun D. Journal of Micromechanics and Microengineering. 2016;26:025015. [4] Qian Y, Li L, Jiang C, Xu W, Lv Y, Zhong L, et al. International journal of biological macromolecules. 2015;79:133-43. [5] Ahmad Z, Rasekh M, Edirisinghe M. Macromolecular Materials and Engineering. 2010;295:315-9. [6] Ahmad, Z, Huang, J, Edirisinghe, M. J, Jayasinghe, S. N, Best, S. M, Bonfield, W, Brooks, R. A, Rushton, N. Journal of Biomedical Nanotechnology. 2006:2: 201-207. [7] San Thian E, Ahmad Z, Huang J, Edirisinghe MJ, Jayasinghe SN, Ireland DC, et al. Biomaterials. 2008;29:1833-43. [8] Rasekh M, Ahmad Z, Day R, Wickam A, Edirisinghe M. Advanced Engineering Materials. 2011:13: 296-335. [9] He J, Li D, Lu B, Wang Z, Zhang T. Rapid Prototyping Journal. 2006;12:198-205. [10] Uma Maheshwari S, Samuel VK, Nagiah N. Ceramics International. 2014;40:8469-77. [11] Rasekh M, Ahmad Z, Frangos CC, Bozec L, Edirisinghe M, Day RM. Acta biomaterialia. 2013;9:5052-62.
Highlights
PCL-nHA microfibrous scaffolds were printed via EHDP to mimic natural bone.
The composite microfibers were well controlled with the size close to living cells.
The biomimetic scaffolds directed cellular alignment and proliferation in vitro.