Tissue engineering is a growing research area, with a few successful clinical results, aimed at developing reliable alternatives to conventional surgical strategies (e.g., auto- and allogenic tissue transplantation or artificial prosthesis implantation) for the treatment of human tissue and organ failure caused by defects, injuries or other types of damage [1
]. Tissue engineering relies on the combination of cells, biomaterials and bioactive molecules to generate replacement biological tissues and organs for a wide range of medical conditions. The most common approach involves the employment of a highly porous biodegradable support, commonly referred to as the scaffold, which acts as a temporary template providing a cell adhesion substrate and mechanical support, and guiding the regeneration processes [2
]. In the last two decades, a great variety of biodegradable materials and processing techniques have been investigated for the development of scaffolds with proper physico-chemical properties as well as macro-, micro- and nano-architecture features suitable for tissue growth in three dimensions [3
Polyhydroxyalkanoates (PHAs) are microbial aliphatic polyesters widely investigated for biomedical applications due to their biodegradability and biocompatibility, as well as the wide range of mechanical and processing properties of the numerous homopolymers and copolymers belonging to this class of renewable polymers [4
]. Different articles have reported on poly[(R)-3-hydroybutyrate] (PHB) and poly[(R)-3-hydroxybutyrate-co
-(R)-3-hydroxyvalerate] (PHBV) investigations, both in vitro and in vivo, for bone tissue regeneration approaches [5
]. Due to the relatively long alkyl side chain, poly[(R)-3-hydroxybutyrate-co
-(R)-3-hydroxyhexanoate) (PHBHHx) exhibits lower crystallinity, a broader processing window and higher elasticity compared with PHB and PHBV [8
]. Among the different investigated biomedical applications, PHBHHx has been proposed as scaffolding material for bone regeneration thanks to its piezoelectric behavior and cytocompatibility when cultured with osteoblasts and bone marrow cells [9
]. In addition, recent articles showed that PHBHHx in the form of microgrooved membrane [15
], aligned nanofibers [16
] or carbon nanotubes-loaded composite materials [17
] well supports the osteogenesis of human mesenchymal stem cells.
As defined by the American Society for Testing and Materials (ASTM), Additive Manufacturing (AM) refers to the process of joining materials to make objects from three-dimensional (3D) model data, usually layer upon layer, as opposed to subtractive manufacturing methodologies [18
]. The introduction of a number of AM techniques, such as stereolithography and fused deposition modeling, into the tissue engineering field has allowed the enhancement of control over scaffold structure at different size scales (from macro- to micrometric scale) in terms of external shape and porous structure [19
]. They involve a computer-controlled layered manufacturing process based on a sequential delivery of energy and/or materials starting from a 3D digital model to build up 3D polymeric scaffolds with a predefined geometry and internal porosity. Advanced computer-aided design and manufacturing approaches enable a high degree of automation, good accuracy and reproducibility for the fabrication of clinically-sized, anatomically-shaped scaffolds with a tailored porous structure characterized by a fully interconnected network of pores with customized size and shape. However, despite the promising results and widespread research on PHAs for tissue engineering applications, their narrow melt processing temperature window [20
] has hindered the application of AM techniques for their processing into 3D porous scaffolds. Computer-aided wet-spinning (CAWS), a hybrid AM technique based on the computer-controlled deposition of a solidifying polymeric fiber extruded directly into a coagulation bath, was recently applied to process PHBHHx into 3D scaffolds with tailored geometry and networks of macropores as well as a homogenous microporous matrix [22
The aim of this study was to investigate the suitability of the CAWS technique for the fabrication of scaffolds made of PHBHHx blended with poly(ε-caprolactone) (PCL). PCL is an aliphatic polyester that has been widely investigated for biomedical applications receiving FDA approval and CE Mark registration for a number of drug delivery and medical device applications [24
]. Thanks to its good processing properties, tunable mechanical properties and slow biodegradation, PCL is seen as one of the most versatile scaffolding materials for the development of long-term biodegradable bone implants. Recent studies have investigated the blending of PHAs with PCL and other synthetic polyesters as a cost-effective strategy for combining the advantages of the two polymers and achieving additional desirable properties [21
]. As an example, a research activity on PCL/PHBHHx blend membranes by solvent processing showed that by optimizing the weight ratio between the two components it was possible to enhance the resulting mechanical properties in comparison with PCL and PHBHHx alone [27
]. Although the great versatility of the CAWS technique in customizing PHBHHx scaffold’s shape and internal architecture, in the case of inter-fiber deposition distances larger than 200 μm (i.e., 500 and 1000 μm), a well-defined porosity along the Z axis was not achieved due to the slow solidification of the coagulating fiber. On the other hand, the optimization of PCL processing by CAWS has enabled the employment of large inter-fiber deposition distances (i.e., 500 and 1000 μm) for the fabrication of 3D scaffolds with a homogeneous porosity in the cross-section characterized by a Z axis pore size in the range of hundreds of micrometers [28
]. Since a pore size larger than 100 μm is recommended to achieve enhanced bone tissue regeneration and vascularization [31
], blending PHBHHx with a polymer showing better processing properties was investigated during the present study as an effective strategy to develop scaffolds meeting the aforementioned structural parameters requirement. For this purpose, the CAWS conditions for the fabrication of PHBHHx/PCL scaffolds with different ratios between the two blend components were investigated. Optimized PHBHHx/PCL scaffold prototypes were characterized in comparison with PHBHHx scaffolds for their morphology by means of scanning electron microscopy (SEM) under backscattered electron imaging, thermal properties by means of thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), and mechanical properties under compression using a uniaxial testing machine. The scaffold’s biocompatibility was evaluated in vitro by employing the MC3T3-E1 murine preosteoblast cell line. Cell response, in terms of viability, proliferation and morphology was investigated by tetrazolium salts (WST-1) and confocal laser scanning microscopy (CLSM).
2. Materials and Methods
Poly(ε-caprolactone) (PCL, CAPA 6800, Mw = 80,000 g·mol−1) was supplied by Perstorp UK Ltd (Warrington, Cheshire, UK) and used as received. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx, 12% mol HHx, Mw = 300,000 g·mol−1) was kindly supplied by Tsinghua University (Beijing, China). PHBHHx was purified before use according to the following procedure: (i) the polymer was dissolved in 1,4 dioxane (5% w/v) under stirring at room temperature for 1 h; (ii) the solution was filtered under vacuum using filter paper; (iii) the filtrate was slowly dropped into 10-fold volume water to precipitate PHBHHx; (iv) after precipitation the polymer was collected by filtering; (v) the polymer was washed with distilled water and then ethanol, and vacuum dried and stored in a desiccator. All the solvents and chemical reagents were purchased from Sigma-Aldrich (Italy) and used as received without further purification.
2.2. Scaffolds Fabrication
PHBHHx solutions were prepared by dissolving the polymer in tetrahydrofuran (THF) at 32 °C under stirring for 2 h at a concentration of 25% w/v. For the preparation of PHBHHx/PCL solutions, PCL was dissolved in THF at 32 °C under stirring for 2 h and then the desired amount of PHBHHx was added to the polymer solution. The mixture was left under stirring for 2 h at 32 °C until a homogenous solution was obtained. Solutions with different PHBHHx/PCL weight ratios (3:1, 2:1 and 1:1) and a total concentration of the polymeric phase of 12% w/v were prepared.
Scaffolds were fabricated by means of a subtractive rapid prototyping system (MDX 40A, Roland MID EUROPE, Acquaviva Picena, Italy) modified in-house by replacing the milling head unit with a programmable syringe pump system (NE-1000; New Era Pump Systems Inc., Wantagh, NY, USA) to enable the deposition of polymeric solutions with a controlled 3D pattern (Figure 1
]. The 3D geometrical scaffold parameters were designed using an algorithm developed in Matlab software (The Mathworks, Inc., Natick, MA, USA). The desired polymeric solution was placed into a glass syringe fitted with a metallic needle (Gauge 23) and injected at a controlled feeding rate directly into an ethanol coagulation bath by using the syringe pump. Scaffold fabrication was carried out by employing a deposition trajectory aimed at the production of scaffolds with a 0–90° lay-down pattern, distance between fiber axis of 500 μm and layer thickness of 100 μm. The optimized initial distance between the tip of the needle and the bottom of the beaker (Z0
) was 1.5 mm. The effect of different processing parameters, such as the deposition velocity (Vdep
) and the solution feed rate (F), on fiber collection and morphology was evaluated to produce blend scaffolds with a different PHBHHx/PCL ratio (Table 1
). By employing the optimized fabrication parameters, cylindrical samples with a designed diameter of 15 mm and height of 5 mm were fabricated. The samples were removed from the coagulation bath, left under a fume hood for 24 h, placed in a vacuum chamber at about 0.5 mbar for 48 h and then stored in a desiccator for at least 72 h before characterization.
2.3. Morphological Characterization
The top-view and cross section (obtained by fracture in liquid nitrogen) of the scaffolds were analyzed by means of scanning electron microscopy (SEM, JEOL JSM 300, Tokyo, Japan) under backscattered electron imaging. The average fiber diameter and pore size, defined as inter-fiber distance, were measured by means of ImageJ 1.43u software on top-view micrographs with a 50X magnification. Data were calculated over 20 measurements per scaffold.
2.4. Thermal Analysis
Thermal properties of the scaffolds were evaluated by means of thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). TGA was performed using TGA Q500 instruments (TA Instruments, Milano, Italy) in the temperature range 30–600 °C, at a heating rate of 10 °C/min and under a nitrogen flow of 60 mL·min−1. The scaffold’s thermal decomposition were evaluated by analyzing weight and derivative weight profiles as functions of temperature. DSC analysis was performed using a Mettler DSC-822 instrument (Mettler Toledo, Novate Milanese (MI), Italy) in the range −100–200 °C, at a heating rate of 10 °C/min and a cooling rate of −20 °C/min, and under a nitrogen flow of 80 mL·min−1. By considering the first and second heating cycle in the thermograms, glass transition temperature (Tg) was evaluated by analyzing the inflection point, while melting temperature (Tm) and enthalpy (∆H) was evaluated by analyzing the endothermic peaks.
2.5. Mechanical Testing
The scaffold’s mechanical properties were analyzed under compression using an Instron 5564 uniaxial testing machine (Instron Corporation, Norwood, MA, USA) equipped with a 2 kN load cell. After the treatment to remove residual solvents, as previously described, the samples were preconditioned at 25 °C and 50% of humidity for 48 h and then characterized at room temperature. The test was carried out on cylindrical samples with actual diameters of around 15 mm and actual heights of around 4 mm (50 layers). Six samples of each kind of scaffold were tested at a constant crosshead displacement of 0.4 mm·min−1
between two parallel steel plates up to 85% strain [32
]. The stress was defined as the measured force divided by the total area of the apparent cross section of the scaffold, whilst the strain was evaluated as the ratio between the height variation and the initial height. Stress-strain curves were obtained from the software recording the data (Merlin, Series IX, Instron Corporation, Norwood, MA, USA). The compressive modulus was calculated as the slope of the initial linear region in the stress-strain curve, avoiding the toe region. Compressive yield strength and strain were considered at the yield point, and compressive strength was considered as the stress corresponding to 85% strain.
2.6. In Vitro Biological Evaluation
2.6.1. Cell Culture
Mouse calvaria-derived pre-osteoblast cell line MC3T3-E1 subclone 4 was obtained from the American Type Culture Collection (ATCC CRL-2593, Manassas, VA, USA) and cultured in Alpha Minimum Essential Medium (α—MEM, Sigma, Milan, Italy) supplemented with 2 mM·L-glutamine, 10% fetal bovine serum, 100 U/mL:100 μg/mL penicillin:streptomycin solution (GIBCO, Invitrogen Corporation, Milan, Italy) and antimycotic. Before experiments, cells were trypsinized with 0.25% trypsin-EDTA (GIBCO, Gaithersburg, MD, USA) solution and resuspended in complete α-MEM at a concentration of 3 × 104/mL. Scaffolds were seeded with 100 μL of cell suspension and the final volume was adjusted to 1 mL with complete medium. The specimens were then placed in an incubator with humidified atmosphere at 37 °C in 5% CO2. Osteogenic differentiation was induced 24 h after seeding by culturing cells in osteogenic medium prepared with α–MEM supplemented with ascorbic acid (0.3 mM) and β—glycerolphosphate (10 mM). The culture medium was replaced every 48 h and biological characterizations were carried out weekly at days 7, 14, 21 and 28. Cells grown onto tissue culture polystyrene plates were used as control.
2.6.2. Cell Viability and Proliferation
Cell viability and proliferation were measured by using the (4-[3-(4-iodophenyl)-2-(4 nitrophenyl)-2H-5-tetrazolium]-1,3-benzene disulfonate) (WST-1) assay (Roche Molecular Biochemicals, Monza, Italy), which is based on the mitochondrial conversion of the tetrazolium salt WST-1 into soluble formazan in viable cells. WST-1 reagent diluted 1:10 was added to the culture and incubated for 4 h at 37 °C. Measurements of formazan dye absorbance were carried out with a Biorad microplate reader at 450 nm, with the reference wavelength at 655 nm. The in vitro biological test was performed on triplicate samples for each material.
2.6.3. Morphologic Characterizations by Confocal Laser Scanning Microscopy (CLSM)
The morphology of the cells grown on the prepared meshes was investigated by means of CLSM. Cells were fixed with 3.8% paraformaldehyde in PBS 0.01 M pH 7.4 (PBS 1X), permeabilized with a PBS 1X/Triton X-100 solution (0.2%) for 15 min and incubated with a solution of 4′-6-diamidino-2-phenylindole (DAPI; Invitrogen) and phalloidin-AlexaFluor488 (Invitrogen) in PBS 1X for 60 min at room temperature in the dark. After dye incubation, samples were washed with PBS 1X before being mounted on a glass slide and sealed with resin for microscopic observation. A Nikon Eclipse TE2000 inverted microscope equipped with an EZC1 confocal laser and Differential Interference Contrast (DIC) apparatus was used to analyze the samples (Nikon, Tokyo, Japan). A 405 nm laser diode (405 nm emission) and an argon ion laser (488 nm emission) were used to excite DAPI and Alexa fluorophores, respectively. Images were captured with Nikon EZ-C1 software with identical settings for each sample. Images were further processed with GIMP (GNU Free Software Foundation) Image Manipulation Software and merged with Nikon ACT-2U software.
2.7. Statistical Analysis
The data are represented as mean ± standard deviation. Statistical differences were analyzed using one-way analysis of variance (ANOVA), and a Tukey test was used for post hoc analysis. A p-value < 0.05 was considered statistically significant.
The main result attained during the reported research activity is the development of an AM process based on the processing of polymeric solutions for the fabrication of PHBHHx-based blend scaffolds. This represents a novel approach to combining the advantages of PHA blending with other biocompatible polymers and the versatility of AM in supplying advanced fabrication tools for the development of scaffolds with customized macro- and microstructure. The developed manufacturing process meets both the product specification and good manufacturing practice requirements. In fact, it allows a good control of scaffold composition, external shape and internal porosity, it does not require thermal treatments that could cause material degradation, and involves the use of solvents allowed in medical device manufacturing that are completely removed from the scaffold during the fabrication and post-processing treatment.
The characterization analyses highlighted the versatility of the developed manufacturing process by demonstrating how PHBHHx/PCL blend scaffold composition, morphological features, thermal properties and mechanical parameters could be tuned in certain ranges by varying the ratio between the two blend components in the starting solution. In addition, the results obtained from the performed preliminary biological evaluations indicated that the developed scaffolds are able to sustain a good cell adhesion and proliferation, and after 28 days of culture, scaffolds were fully colonized by MC3T3-E1 preosteoblast cells.
As shown by recent studies, the CAWS technique is well suited for the development of PHBHHx scaffolds with a complex shape resembling that of an anatomical part and a tailored porous structure with advanced architectural features at different scale levels (e.g., longitudinal macrochannel, local micro/nanoporosity) designed to enhance tissue regeneration processes [22
]. The developed PHBHHx/PCL scaffolds can therefore represent advanced prototypes for the development of sophisticated PHAs-based blend constructs with tailored composition, anatomical shape, macroporosity and nanoporous morphology.