Abstract
Objective
Vascularization is a critical process during bone regeneration/repair and the lack of tissue vascularization is recognized as a major challenge in applying bone tissue engineering methods for cranial and maxillofacial surgeries. The aim of our study is to fabricate a vascular endothelial growth factor (VEGF)-loaded gelatin/alginate/β-TCP composite scaffold by 3D printing method using a computer-assisted design (CAD) model.
Methods
The paste, composed of (VEGF-loaded PLGA)-containing gelatin/alginate/β-TCP in water, was loaded into standard Nordson cartridges and promptly employed for printing the scaffolds. Rheological characterization of various gelatin/alginate/β-TCP formulations led to an optimized paste as a printable bioink at room temperature.
Results
The in vitro release kinetics of the loaded VEGF revealed that the designed scaffolds fulfill the bioavailability of VEGF required for vascularization in the early stages of tissue regeneration. The results were confirmed by two times increment of proliferation of human umbilical vein endothelial cells (HUVECs) seeded on the scaffolds after 10 days. The compressive modulus of the scaffolds, 98 ± 11 MPa, was found to be in the range of cancellous bone suggesting their potential application for craniofacial tissue engineering. Osteoblast culture on the scaffolds showed that the construct supports cell viability, adhesion and proliferation. It was found that the ALP activity increased over 50% using VEGF-loaded scaffolds after 2 weeks of culture.
Significance
The 3D printed gelatin/alginate/β-TCP scaffold with slow releasing of VEGF can be considered as a potential candidate for regeneration of craniofacial defects.
1
Introduction
Critically-sized bone defects created due to infection, tumor resection, or traumatic fractures cannot be healed spontaneously, and external interventions are needed in most cases to regenerate new bone to restore, maintain or improve its function. Although autografts are considered as the gold standard treatment , it remains challenging for the clinicians to select between autografts, allografts, xenografts or engineered tissues . The limited access to bone grafts has led to attempts to develop tissue engineering techniques using the three factors of scaffold, growth factors and/or cells for achieving favorable outcomes in bone regeneration.
Composition and physical properties of the porous scaffold can directly influence the regeneration process; biomimetic scaffolds can yield a more favorable outcome . Additive manufacturing (AM) approaches can be used to fabricate scaffolds with tailored pore size and porosity for complex-shaped defects, the fabrication of which is problematic or impossible using other manufacturing methods reported in the literature . 3D printing/AM allows fabrication of 3D objects of virtually any shape from a computer aided design (CAD) . Two key factors for successful 3D printing fabrication are ink/binder selection and process parameter optimization. The advantage of 3D-printing is the fine control over various features of the scaffold . Also, one of the widely used techniques of low temperature fused deposition modeling provides mild condition of processing, which allows plotting of drug and biomolecules such as proteins and living cells . However, there is a significant challenge regarding development of suitable structural materials containing adequate amounts of bioactive components .
Calcium phosphate-based formulations have presented excellent osteoconductivity and biocompatibility in reconstructive surgeries for more than 30 years . Tricalcium phosphate (TCP), as one of the most widely used calcium phosphates in bone tissue engineering, has demonstrated osteogenic properties, phase stability and strong bond formation with the host bone tissue in different studies . 3D-printed TCP-based scaffolds could be considered as a proper choice for bone tissue engineering applications, since both the fine control over the structure and shape through 3D printing and osteoconductivity of the composition can be exploited. However, when encapsulation and release of sensitive biomolecules such as growth factors is required, high temperature post processes like sintering should be avoided. In this case, biopolymers such as gelatin and alginate might be used both as the binder to facilitate the printing process and as the matrix to incorporate growth factor carriers. Gelatin, a natural polymer obtained from partial hydrolysis of collagen, provides Arg-Gly-Asp (RGD) motifs that can mediate cell attachment via interaction with integrin . The sol–gel transition of gelatin can be exploited in 3D printing procedures. On the other hand, alginate, a natural polysaccharide, has been widely used as a biomaterial for bone tissue engineering because of its biocompatibility, non-immunogenicity and biodegradability . Alginate has been widely used as a thickener in the food industry and can be used to adjust the viscosity and rheological properties of the ink.
The lack of functional vascularization is a major challenge in the successful clinical approach of bone tissue engineering in the practice of reconstructive orthopedic and craniofacial surgery . As a result, the aim of bone tissue engineering is not only the culture of osteogenic cells on osteoconductive scaffold, but also the induction of angiogenesis to support the metabolic needs of bone. Although the main VEGF receptors expressed on endothelial cells, there are many VEGF receptors expressed on chondrocytes and osteoblasts . Consequently VEGF not only promotes angiogenesis but also play an important role in bone growth and repair . Therefore, an osteoconductive scaffold releasing VEGF could be an appropriate candidate for bone regeneration. However, encapsulation and sustained release of fragile biomolecules, such as growth factors, is very challenging. Researchers have increasingly become interested in using biodegradable polymers as host materials for drug delivery systems in the past few decades. These systems not only protect the encapsulated molecule against aggressive environments but also release the molecule in a sustainable manner. Poly (lactic-co-glycolic acid) (PLGA) has been widely used for growth factor sustained delivery in different forms including coating layers and microspheres . Growth factor loaded PLGA microspheres can be easily incorporated into the ink to be 3D printed.
The aim of current study was to develop a 3D printed TCP-based scaffold with sustained release of VEGF for bone tissue engineering applications. Gelatin and alginate were employed as the binders to facilitate 3D printing. In fact, we hypothesized that gelatin/alginate/TCP scaffold containing vascular endothelial growth factor (VEGF)-loaded PLGA microspheres, fabricated by 3D plotting according to a predesigned CAD-model, could be a potential candidate for treatment of bone defects.
2
Experimental procedure
2.1
Materials
β-TCP, phosphate buffer saline (PBS), Alginate powder and gelatin (Type A, Bloom 300 g) were purchased from Sigma-Aldrich, USA. Dichloromethane, CaCl 2 and N-hydroxysuccinimide (NHS) were obtained from Alfa Aesar, USA. PLGA was purchased from Corbion Purac, PURASORB ® PDLG 5010. HUVECs, Osteoblasts and VEGF were purchased from Cell Applications Inc., USA and finally, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) was obtained from TCI America, USA. DMEM and FBS were purchased from Sigma. MTT and Alamar blue assays were obtained from Sigma and life technologies, respectively. All materials were utilized as received, without any further purification.
2.2
Formulating the ink for printing
In the formulation, β-TCP and alginate were used as the main component and thickener, respectively. Gelatin was employed as the hardening agent because of its sol–gel transition. Since sol–gel transition temperature of the ink is influenced by gelatin concentration, it plays a key role in printability of the ink. The concentration of β-TCP and alginate were set at 30% (w/v) and 5% (w/v), respectively and the gelatin concentration was altered (1, 3 and 10% (w/v)). The effect of gelatin concentration on the sol–gel transition of the ink was then investigated using rheometry. The rheological measurements were performed using a shear rheometer (Kinexus, Malvern, UK) with a stainless steel parallel-plate geometry of 20 mm in diameter and a Peltier temperature control. The warm paste to be tested was loaded onto the pre-heated Peltier plate (33 °C) of the rheometer and the upper plate was lowered until it gently touched the surface of the sample at a gap distance of 0.3 mm, and excess material was removed.
Frequency sweeps for various formulations were performed at temperatures in decreasing 3 °C increments ranging from 33 °C to 9 °C in order to determine the sol–gel transitions. A constant shear strain of 1% (angular displacement of 4 × 10 −4 rad) was applied during frequency sweep (1–15 Hz, logarithmic). All the measurements were carried out within the linear viscoelastic regions.
The formulation with the sol–gel transition temperature closest to room temperature was selected for printing. The viscometry of the selected formulation was performed using the same geometry and gap distance. The shear stress and viscosity were measured at temperatures in decreasing 5 °C increments ranging from 35 °C to 15 °C. At each temperature, the rheological evaluation consisted of two consecutive shear cycles with no rotational pre-shear step. The shear rate varied linearly in ramp mode from 0 to 100 s −1 with 10 s −1 intervals in 2 min and then back to 0 s −1 . The total testing time was 12 min.
2.3
Microsphere loaded ink preparation
VEGF loaded PLGA microspheres were fabricated through a modified double emulsion-solvent evaporation technique. VEGF was dissolved in sterile PBS (3 μg in 0.2 ml) mixed with 1 mL of a 10% w/v PLGA solution in dichloromethane. The first emulsion was obtained through vortexing the mixture for 30 s. The resulting emulsion (W/O) was added dropwise into 20 mL of 3% aqueous solution of gelatin and stirred at 800 rpm at 30 °C for 5 min to produce a double W/O/W emulsion. After 5 min, the stirring speed was reduced to 500 rpm, and the solvent was allowed to evaporate at 30 °C for 3 h. VEGF free PLGA microspheres were produced using the same protocol except blank PBS was used to prepare the first emulsion. In the next step, alginate powder was added to the suspension of microspheres in gelatin aqueous solution and allowed to dissolve to yield the final concentration of 5% w/v. Finally, β-TCP powder was added and all the components were mixed and defoamed using a centrifugal mixer (Thinky, USA). The final concentration of β-TCP was adjusted to be 30% w/v. The uniform paste obtained was taken for printing.
2.4
Scaffold preparation
The paste, composed of gelatin/alginate/β-TCP in water, was loaded into standard Nordson cartridges and immediately used for printing the scaffolds.
The scaffolds were fabricated by 3D-printing (3D-Bioplatter EnvisionTEC, Germany). For printing, pressure of 2 bars was applied while the plotting speed was adjusted at 10 mm/s. Cartridge temperature, platform temperature and needle diameter were 28 °C, 10 °C and 450 μm, respectively.
The pore geometry of the scaffolds was designed as square. After printing, the scaffolds were transferred into a 50 mM CaCl 2 solution and incubated for 1 h at room temperature to allow crosslinking of alginate, followed by washing with deionized water three times. Afterwards, the scaffolds were transferred into a solution of 9.6% w/v EDC and 0.92% w/v NHS in 70%v/v ethanol and incubated for 2 h at room temperature for crosslinking of gelatin, followed by rinsing in deionized water and drying at room temperature.
2.5
Morphology, pore size and porosity
Morphology of the scaffolds’ pores was studied using 3D laser scanning digital microscopy (Olympus LEXT OLS 4000, Japan). The porosity of the scaffolds was measured through a liquid displacement method . Ethanol was selected as the displacement liquid, as it permeates through the scaffolds without swelling or shrinking the matrix. The porosity was determined using the following equation:
where W 1 is the weight of the sample in air, W 2 the weight of sample with liquid in pores, and W 3 the weight of sample suspended in ethanol.
2.6
Mechanical properties of the scaffold
The compressive strength and Young’s modulus of 3D printed scaffolds (3.5 × 2 mm D × H) were tested employing a mechanical testing machine (AGS-X, Shimadzu, Japan) equipped with a 5 kN load cell. The test was performed by compressing the scaffolds in z-direction at a constant rate of 1 mm/min.
2.7
Entrapment efficiency
The amount of VEGF actually loaded within the microspheres was measured after complete degradation of 1 mg of unloaded VEGF-loaded particles in 1 mL of 0.05 N NaOH under stirring. After 24 h, the solutions were centrifuged at 5000 rpm at room temperature and the supernatant was analyzed for VEGF content by ELISA kit (Cell Applications Inc., USA). Results are expressed as actual loading (ng encapsulated per mg of microspheres) and encapsulation efficiency (ratio of actual to theoretical loading × 100) ± standard deviation of values collected from three different batches.
2.8
Kinetic of VEGF release
In vitro release of VEGF from VEGF-loaded scaffolds was evaluated in buffer solution. Briefly, samples (10 mg) were suspended in 1 mL of phosphate buffered saline (120 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer salts) (PBS) at pH 7.4 previously filtered on 0.22 μm sterile filters (Millex1, Millipore, USA) and placed in a shaking incubator at 37 °C. At scheduled time intervals, 1, 2, 3, 5, 7 and 10 days after incubation, 0.1 ml of the release medium was withdrawn for analysis and replaced with the same volume of fresh filtered medium. Samples were centrifuged at 5000 rpm at room temperature and the supernatant was analyzed for VEGF content via ELISA kit. The concentration of VEGF was calculated as Nano gram per milliliter of the solution.
2.9
Cell culture and VEGF release effect on bioactivity
The scaffolds (5 × 2 mm D × H) were disinfected by three times immersion in 70% ethanol and rinsing with PBS for 15 min cycles.
The bioactivity of the VEGF released from the microspheres was examined in vitro by measurement of the proliferative capacity of HUVECs after VEGF treatment. HUVECs were cultured in modified endothelial cell growth medium (Cell Applications, USA). To control the impact of VEGF in the cultures, the media used did not contain VEGF. Experiments were performed with cells from passage 4. In order to assess the endothelial cell proliferation capacity after introduction of VEGF by the scaffolds, the HUVECs were seeded into 24-well culture plates at a density of 1.25 × 10 3 cells/well, and scaffolds were placed in an upper chamber by employing transwells (0.4 μm pore size, tissue culture treated polycarbonate membrane Corning, USA). Cells were incubated for 7 or 14 days, with VEGF-free or VEGF-loaded scaffolds, or medium alone as control. Cell proliferation in each experimental group was calculated by utilizing [3-(4,5-dimethylthiazol-2-yl)-1,5-diphenyltetrazulium bromide] (MTT, Sigma, USA) mitochondrial reaction. This assay was based on the ability of live cells to decrease a tetrazulium-based compound, MTT, to a purplish formazan product.
The results of the experiment were recorded as percentage absorbance relative to control sample [tissue culture polystyrene (TCPS)]. Furthermore, the percentage of HUVEC cell proliferation for certain days (7 and 14) was calculated using the equation: (mean optical density (OD) of the prepared scaffolds at certain day/mean OD of control sample at 7th day) × 100 .
2.10
Osteoblast proliferation
The capability of the prepared scaffolds (n = 3) to induce cell proliferation was evaluated utilizing normal human osteoblasts (HOB, Cell Applications, USA) by the Alamar blue assay. The scaffolds were disinfected using 70% ethanol as described. The cells were seeded (1 × 10 5 cells/cm 2 ) on the scaffold and cultured for 7 and 14 days. After the incubation period, 10% Alamar blue was added to the scaffold-call complexes. The optical density of the solution was determined at 570 nm using a micro-plate reader (Synergy HTX, BioTek, USA) in order to record the difference between samples and control group. Triplicate samples were analyzed for this experiment.
The results of the experiment were recorded as percentage absorbance relative to control sample [tissue culture polystyrene (TCPS)]. Furthermore, the percentage of osteoblast cell proliferation for certain days (7 and 14) was calculated using the equation: (mean optical density (OD) of the prepared scaffolds at certain day/mean OD of control sample at 7th day) × 100 .
2.11
Osteoblast and HUVEC adhesion
Cell adhesion was evaluated using scanning electron microscopy (SEM, JEOL JSM6510). The osteoblasts or HUVECs were seeded onto the scaffolds and allowed to attach. After 24 h, samples were taken out, washed with phosphate buffered saline (PBS) and fixed using Karnovsky’s fixative (composed of Paraformaldehyde-Glutaraldehyde) for 2 h. Samples were rinsed with PBS and post-fixed utilizing 1% Osmium Tetroxide solution. Sample were rinsed with PBS again and dehydrated employing ascending ethanol series (50, 70, 80, 95 and 100 v/v%). Finally, the samples were chemically dried using 50 and 100 v/v% Hexamethyldisilazane (HMDS, each for 10 min) and left at room temperature for complete drying. The imaging was conducted after sputter-coating with gold utilizing secondary electron modes at different magnifications.
2.12
Alkaline phosphatase activity
Alkaline phosphatase (ALP) activity (n = 3) was assessed utilizing an ALP assay kit (G-Biosciences, St Louis, MO, USA). The media was supplemented with osteoblastic medium one day subsequent to seeding. The cells were digested, collected, and lysed after 7 and 14 days’ culture in osteoblastic media. The lysates were reacted with p -nitrophenyl phosphate (p-NPP), and the absorbance of p-nitrophenol was measured at 405 nm employing a microplate reader. Cells cultured with osteoblastic medium but not seeded on a scaffold were used as the control.
The results of the experiment were recorded as percentage absorbance relative to control sample [tissue culture polystyrene (TCPS)]. Furthermore, the percentage of ALP activity for certain days was calculated using the equation: (mean optical density (OD) of the prepared scaffolds at certain day/mean OD of control sample at 7th day) × 100 .
2.13
Statistical analysis
All the computable results were articulated as mean ± standard deviation (SD), and were analyzed by two-tailed t-test. P-value <0.05 was considered to be statistically significant.
2
Experimental procedure
2.1
Materials
β-TCP, phosphate buffer saline (PBS), Alginate powder and gelatin (Type A, Bloom 300 g) were purchased from Sigma-Aldrich, USA. Dichloromethane, CaCl 2 and N-hydroxysuccinimide (NHS) were obtained from Alfa Aesar, USA. PLGA was purchased from Corbion Purac, PURASORB ® PDLG 5010. HUVECs, Osteoblasts and VEGF were purchased from Cell Applications Inc., USA and finally, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) was obtained from TCI America, USA. DMEM and FBS were purchased from Sigma. MTT and Alamar blue assays were obtained from Sigma and life technologies, respectively. All materials were utilized as received, without any further purification.
2.2
Formulating the ink for printing
In the formulation, β-TCP and alginate were used as the main component and thickener, respectively. Gelatin was employed as the hardening agent because of its sol–gel transition. Since sol–gel transition temperature of the ink is influenced by gelatin concentration, it plays a key role in printability of the ink. The concentration of β-TCP and alginate were set at 30% (w/v) and 5% (w/v), respectively and the gelatin concentration was altered (1, 3 and 10% (w/v)). The effect of gelatin concentration on the sol–gel transition of the ink was then investigated using rheometry. The rheological measurements were performed using a shear rheometer (Kinexus, Malvern, UK) with a stainless steel parallel-plate geometry of 20 mm in diameter and a Peltier temperature control. The warm paste to be tested was loaded onto the pre-heated Peltier plate (33 °C) of the rheometer and the upper plate was lowered until it gently touched the surface of the sample at a gap distance of 0.3 mm, and excess material was removed.
Frequency sweeps for various formulations were performed at temperatures in decreasing 3 °C increments ranging from 33 °C to 9 °C in order to determine the sol–gel transitions. A constant shear strain of 1% (angular displacement of 4 × 10 −4 rad) was applied during frequency sweep (1–15 Hz, logarithmic). All the measurements were carried out within the linear viscoelastic regions.
The formulation with the sol–gel transition temperature closest to room temperature was selected for printing. The viscometry of the selected formulation was performed using the same geometry and gap distance. The shear stress and viscosity were measured at temperatures in decreasing 5 °C increments ranging from 35 °C to 15 °C. At each temperature, the rheological evaluation consisted of two consecutive shear cycles with no rotational pre-shear step. The shear rate varied linearly in ramp mode from 0 to 100 s −1 with 10 s −1 intervals in 2 min and then back to 0 s −1 . The total testing time was 12 min.
2.3
Microsphere loaded ink preparation
VEGF loaded PLGA microspheres were fabricated through a modified double emulsion-solvent evaporation technique. VEGF was dissolved in sterile PBS (3 μg in 0.2 ml) mixed with 1 mL of a 10% w/v PLGA solution in dichloromethane. The first emulsion was obtained through vortexing the mixture for 30 s. The resulting emulsion (W/O) was added dropwise into 20 mL of 3% aqueous solution of gelatin and stirred at 800 rpm at 30 °C for 5 min to produce a double W/O/W emulsion. After 5 min, the stirring speed was reduced to 500 rpm, and the solvent was allowed to evaporate at 30 °C for 3 h. VEGF free PLGA microspheres were produced using the same protocol except blank PBS was used to prepare the first emulsion. In the next step, alginate powder was added to the suspension of microspheres in gelatin aqueous solution and allowed to dissolve to yield the final concentration of 5% w/v. Finally, β-TCP powder was added and all the components were mixed and defoamed using a centrifugal mixer (Thinky, USA). The final concentration of β-TCP was adjusted to be 30% w/v. The uniform paste obtained was taken for printing.
2.4
Scaffold preparation
The paste, composed of gelatin/alginate/β-TCP in water, was loaded into standard Nordson cartridges and immediately used for printing the scaffolds.
The scaffolds were fabricated by 3D-printing (3D-Bioplatter EnvisionTEC, Germany). For printing, pressure of 2 bars was applied while the plotting speed was adjusted at 10 mm/s. Cartridge temperature, platform temperature and needle diameter were 28 °C, 10 °C and 450 μm, respectively.
The pore geometry of the scaffolds was designed as square. After printing, the scaffolds were transferred into a 50 mM CaCl 2 solution and incubated for 1 h at room temperature to allow crosslinking of alginate, followed by washing with deionized water three times. Afterwards, the scaffolds were transferred into a solution of 9.6% w/v EDC and 0.92% w/v NHS in 70%v/v ethanol and incubated for 2 h at room temperature for crosslinking of gelatin, followed by rinsing in deionized water and drying at room temperature.
2.5
Morphology, pore size and porosity
Morphology of the scaffolds’ pores was studied using 3D laser scanning digital microscopy (Olympus LEXT OLS 4000, Japan). The porosity of the scaffolds was measured through a liquid displacement method . Ethanol was selected as the displacement liquid, as it permeates through the scaffolds without swelling or shrinking the matrix. The porosity was determined using the following equation:
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