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. Author manuscript; available in PMC: 2023 Feb 16.
Published in final edited form as: J Biomed Mater Res A. 2018 Sep;106(9):2507–2517. doi: 10.1002/jbm.a.36446

Three-dimensional Porous PPF-co-PLGA Scaffolds for Tissue Engineering

Wei Wu 1,2,3, Xifeng Liu 1,2, Zifei Zhou 1,2,4, A Lee Miller II 2, Lichun Lu 1,2,*
PMCID: PMC9933994  NIHMSID: NIHMS1785067  PMID: 29707898

Abstract

Three-dimensional structural scaffolds have played an important role in tissue engineering, especially broad applications in areas such as regenerative medicine. We have developed novel biodegradable porous polypropylene fumarate-co-poly (lactic-co-glycolic acid) (PPF-co-PLGA) scaffolds using thermally induced phase separation (TIPS), and determined the effects of critical parameters such as copolymer concentration (6, 8, and 10 wt%) and the binary solvent ratio of dioxane:water (78/22, 80/20, 82/18 wt/wt%) on the fabrication process. The cloud-point temperatures of PPF-co-PLGA changed in parallel with increasing copolymer concentration, but inversely with increasing dioxane content. The compressive moduli of the scaffolds increased with greater weight composition and dioxane:water ratio. Scaffolds formed using high copolymer concentrations and solvent ratios exhibited preferable biomineralization. All samples showed biodegradation capability in both accelerated solution and phosphate-buffered saline (PBS). Cell toxicity testing indicated that the scaffolds had good biocompatibility with bone and nerve cells, which adhered well to the scaffolds. Variations in the copolymer concentration and solvent ratio exercised a remarkable influence on morphology, mechanical properties, biomineralization and biodegradation, but not on the cell viability and adhesion of the cross-linked scaffolds. An 8 to 10 wt% solute concentration and 80/20 to 82/18 wt/wt dioxane:water ratio were the optimum parameters for scaffold fabrication. PPF-co-PLGA scaffolds thus possess several promising prospects for tissue engineering applications.

Keywords: Three-dimensional scaffolds, interconnected porosity, phase separation, tissue engineering, biomineralization

1. Introduction

Tissue engineering is an important, ongoing area of regenerative medicine, and the intent of this developing field is to reconstruct and even restore tissue defects.1–2 In the past several decades, various biomimetic materials and scaffolds have been designed to regenerate injured tissue. To achieve structural support and eventually induce regeneration, natural or artificial substitutes and scaffolds must possess special characteristics, including considerable and reasonable interconnected porous architectures with sufficient cellular affinity and mechanical properties, acceptable biodegradability, favorable biocompatibility, and sometimes bone-binding and delivery functions.3–7

Poly (lactic-co-glycolic acid) (PLGA) polymer, which has excellent biodegradability, has aroused widespread use in research. As a copolymer, the hydrolyzates of PLGA are lactic acid and glycolic acid, which are common products of physiologic metabolic processes. Therefore, PLGA is frequently used as a controlled-release drug carrier, in gene delivery, and as a component of artificial composite materials.8,9 At the same time, some disadvantages of PLGA, such as low mechanical properties and acidic degradation products, were reported.10,11 To overcome its inherent weaknesses and fulfill the demands of tissue engineering applications, one or more other materials and PLGA are usually combined into composite substances by physical blending or the use of covalently bound copolymers.12–14

Poly (propylene fumarate) (PPF) is a linear polyester with unsaturated double bonds, which have the ability to crosslink via chemical or photo-initiation, and exhibits satisfactory biomechanical properties. As a result, PPF has been used as a supporting material in load-bearing and bone conduction grafts.5, 15, 16 Furthermore, PPF can be printed into three-dimensional (3D) scaffolds with desirable microstructure and suitable properties for regenerative medicine applications.17–19

Thermally induced phase separation (TIPS) is widely used to fabricate 3D porous architectural scaffolds with two thermodynamic states: nucleation in the stable region and spinodal decomposition in the unstable region. Only the polymer solution is quenched in the second state, which can form an interconnected network structure.20–22 The fabrication parameters can be regulated to obtain the desired interior morphologic characteristics using this method.23, 24 As compared to other three-dimensional (3-D) scaffold fabrication methods, the TIPS method has multiple advantages: i) facile fabrication process with major steps of heat, cool, then freeze dry; ii) cost effective, inexpensive equipment, e.g., three-dimensional printers, were required in the fabrication process; iii) tunable shapes and sizes of scaffolds by using different mold containers; iv) capable to incorporate multiple fillers within the scaffolds, only requires addition and mixing before freezing.

The 3D scaffolds of PPF/PLGA blend polymers and PPF incorporating PLGA microspheres have been previously reported, but most studies have concentrated on the functional release of a substance (such as a drug or gene) or just certain aspects of the material characteristics.25–28 In the present research, we introduce a novel copolymer polypropylene fumarate-co-poly (lactic-co-glycolic acid) (PPF-co-PLGA) that has favorable characteristics for tissue engineering applications. The properties of the scaffolds, which were manufactured by TIPS from various copolymer concentrations and binary solvent ratios, were characterized by scanning electron microscopy (SEM) and mechanical, biomineralization, degradation, and cellular adhesion testing. We also demonstrated the prospects of using these scaffolds in nerve and low load-bearing bone tissue engineering applications.

2. Materials and Methods

2.1. Synthesis of PPF-co-PLGA copolymer

PPF polymer terminated with hydroxyl groups was synthesized using diethyl fumarate monomers and 1,2-propylene glycol with zinc chloride as catalyst, as described in our previous report.29 Dried PPF was further reacted with D,L-lactide and glycolide monomer at 140 °C for 24 h using stannous octoate (Sn(Oct)2) as catalyst, as previously reported [Fig. 1(A)].30 The obtained PPF-co-PLGA copolymer was purified by dissolving in methylene chloride followed by precipitation in diethyl ether. This purification process was repeated three times and the purified copolymer was fully dried under vacuum. The number average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity index (PDI) of the PPF-co-PLGA copolymer were determined using gel permeation chromatography (GPC) and were 19,700 g mol−1, 59,100 g mol−1 and 2.9, respectively. The purified polymer was stored at −20 °C for future use.

Figure 1.

Figure 1.

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(A) Synthesis of polypropylene fumarate-co-poly(lactic-co-glycolic acid) (PPF-co-PLGA) copolymer from PPF and PLGA using stannous octoate (Sn(Oct)2) as catalyst. (B) Schematic demonstration of crosslinked 3D PPF-co-PLGA scaffold fabrication through thermally induced phase separation (TIPS).

2.2. Cloud point determination

According to a previously described method,31 the cloud-point temperatures of PPF-co-PLGA were evaluated by ocular turbidity in a binary solvent composed of 1,4-dioxane and water.

We tested the cloud point temperatures of PPF-co-PLGA copolymers fabricated using 1) various copolymer weight compositions (4, 5, 6, 7, 8, 9, 10, and 11 wt%) in a specific dioxane:water solvent (81/19 wt/wt%) and 2) a specific copolymer weight (7 wt%) in various dioxane:water solvents (77/23, 78/22, 79/21, 80/20, 81/19, 82/18, 83/17, and 84/16 wt/wt%). The copolymer-dioxane-water ternary system was bathed in 85°C water until the mixture became clear, and the cloud points were estimated when clouding was observed during the cooling procedure.

2.3. 3D scaffolds fabrication

The 3D PPF-co-PLGA scaffolds were manufactured using thermally induced phase separation (TIPS), and the fabrication schematic is shown in Figure 1. Briefly, the dioxane-water binary solvent (81/19 wt/wt% ratio) was prepared to dissolve PPF-co-PLGA (6, 8, or 10 wt%) in glass vials. Separately, 7 wt% copolymer was dissolved in various dioxane-water solvents (78/22, 80/20, or 82/18 wt/wt%) in glass vials. The ternary mixture was heated to 80°C for 20 minutes to allow the solutes to dissolve, and then quenched at room temperature for 2 minutes. Afterwards, the samples were crosslinked under UV radiation for 30 minutes, frozen at −80°C, and lyophilized for 3 days to obtain the final 3D scaffolds. The samples were stored at room temperature with 40% relative humidity before morphology evaluation.

2.4. Scanning electron microscopy

The morphology and internal structure of the PPF-co-PLGA scaffolds were detected by scanning electron microscopy (SEM) (S-4700; Hitachi Instruments, Tokyo, Japan). After being frozen in liquid nitrogen, the samples were broken apart to expose the internal structure. The scaffolds were put in a vacuum oven to dry, and then their surfaces were coated with gold-palladium for 3 minutes using a sputter coater. The structures of the porous scaffolds were detected, and images were captured at 400x, 1,000x, and 5,000x magnifications with a 3.5-kV accelerating voltage.

2.5. Mechanical testing

The mechanical compression capacities of the copolymer scaffolds were investigated using a dynamic mechanical analyzer (DMA 2980; TA Instruments, New Castle, Delaware), and stress-strain curves were analyzed using a previously described method.32 Briefly, cylindrical samples of approximately 5 mm in diameter and 8 mm in length were compressed at a strain rate of 0.001 mm/min. The initial slope of the stress-strain curve in the linear regions was used to calculate the compressive modulus.

2.6. In Vitro biomineralization

A conventional simulated body fluid (SBF) (pH 7.30) was prepared to assess the ability of PPF-co-PLGA scaffolds to facilitate biomineralization, as described in previous research.33–35 Cylindrical samples (5 mm diameter and 3 mm height) were fabricated using various copolymer weight compositions (6, 8, and 10 wt/wt % in an 81/19 wt/wt% dioxane:water solvent) and different dioxane:water ratios (7 wt% copolymer concentration in 78/22, 80/20, and 82/18 wt/wt% dioxane:water solvents). The specimens were immersed in 5 mL of 10 × SBF solution, and placed in a 37 °C incubator for 2 hours. The scaffolds were washed twice with distilled water after removing the SBF solution, and then frozen in liquid nitrogen for SEM and Fourier transform infrared spectroscopy (FTIR).

2.7. Fourier transform infrared spectroscopy

The absorption at different wavelengths of synthesized and biomineralized PPF-co-PLGA substances were detected using a Nicolet ATR-FTIR Continuum Infrared Microscope (Thermo Scientific, Waltham, MA), which has a wavenumber measurement range of 650 to 2300 cm−1.

2.8. In vitro degradation

Phosphate-buffered saline (PBS) and sodium hydroxide (NaOH) solution were used as the media to evaluate in vitro physiological and accelerated degradation of the copolymer scaffolds, respectively. Each 5 × 5 × 5 mm cubic scaffold was immersed in 5 mL of 0.02 mM NaOH or PBS solution, and incubated at 37 °C. For the NaOH-submerged scaffolds, the weight of the degraded sample was determined daily until the sample disappeared completely. The remaining mass of each sample in PBS was measured weekly for 8 weeks.

2.9. In vitro cytotoxicity

The biologic compatibility of PPF-co-PLGA scaffolds was investigated through co-culturing with MC3T3-E1 pre-osteoblast cells. To eliminate the biohazard of residual bisacylphosphine oxide, each 5 × 5 × 5 mm cubic sample was placed in PBS for 3 weeks and then sterilized with 70% ethanol, dried under vacuum, and rinsed with PBS prior to use. Cells were thawed and cultured in Minimum Essential Medium Eagle Alpha Modification (Mediatech, Inc, Manassas, Virginia) containing 10% fetal bovine serum and 1% streptomycin-penicillin in a 37 °C incubator set to 95% relative humidity and 5% carbon dioxide. The cells of the third passage were used for this assay. The cell culture supernatant medium was observed after trypsin digestion to calculate the density, and then the diluted suspension was placed into 24-well plates at a concentration of 20,000 cells/cm2 and incubated for 24 hours to allow the cells to adhere.

The prepared scaffolds were transferred to Transwell cell culture inserts and immersed in the medium. The co-cultivation period was 4 days, and the media were replaced every 2 days; afterwards, the media and scaffolds were removed and the adhesive cells were rinsed three times with Dulbecco’s PBS. Diluted MTS assay reagent (CellTiter 96, Promega, Madison, WI) was added to each cell well to quantify the cell population. The cell plate was incubated at 37 °C with 5% CO2 for 60 min. Then the reagent liquid was transferred to a 96-well plate for UV absorbance detection at 490 nm using a microplate reader (SpectraMax Plus 384, Molecular Devices, Sunnyvale, CA), and the absorbance values were converted to percent cell viability. The positive control groups were set as cells grew in the absence of copolymer scaffolds. The same test was conducted using PC-12 neuronal cells cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% FBS, 10% horse serum, and 1% streptomycin-penicillin.

2.10. In vitro cell attachment

MC3T3-E1 cells were used to evaluate the effects of scaffold on osteoblast responses and bone regeneration. The cell suspension and scaffold disks (approximately 5 mm in diameter and 8 mm in length) were prepared similar to that described in the cytotoxicity study. The sterile disks were transferred to 24-well cell culture plates, and the cells in 500 μL medium were carefully seeded in a dropwise manner onto the scaffold surface at a plating density of 30,000 cells/cm2. The cells on the scaffolds were incubated at 37 °C for 2 hours to allow them to adhere, and then an additional 500 μL of culture medium was placed into each well to ensure the samples were submerged. After 4 days of incubation, staining for fluorescent microscopy was conducted. Briefly, the cell-attached scaffolds were washed twice and fixed in 4% paraformaldehyde solution for 20 min, rinsed three times with PBS, then permeabilized with 0.5% Triton X-100. The cytoskeleton was stained with rhodamine-phalloidin (RP) for 60 min at 37 °C, and the cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 2 min at room temperature. The morphologies of attached cells were observed and imaged using an Axiovert 25 Zeiss light microscope (Carl Zeiss, Germany). The number of cells on each sample surface was counted using the Image J software, and the mean of 3 scaffolds was determined for each group. We also used PC-12 cells as representatives to assess the affinity of these scaffolds to neuronal cells.

2.13. Statistical Analysis

The experimental results were reported as mean ± standard deviation (SD). Tukey post-hoc test was used for one-way analysis of variance to detect statistical difference between groups. Difference was deemed significance at p < 0.05 and p < 0.01.

3. Results

3.1. Cloud point of copolymer

The cloud points of PPF-co-PLGA with different copolymer concentrations of 4, 5, 6, 7, 8, 9, 10, and 11 wt% in 81/19 wt/wt% dioxane-water ratio increased from 39.9 ± 0.35°C to 48.1 ± 0.42°C as the copolymer concentration increased. The values of 7 wt% copolymer composition in 77/23, 78/22, 79/21, 80/20, 81/19, 82/18, 83/17 and 84/16 wt/wt% dioxane-water solvent ratios reduced from 66.7 ± 1.06°C to 26.5 ± 0.85°C as the solvent ratios increased. The cloud points of the copolymer-dioxane-water mixture at different copolymer concentrations and solvent ratios are shown in Figure 2.

Figure 2.

Figure 2.

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Cloud points of PPF-co-PLGA fabricated using different concentrations (4, 5, 6, 7, 8, 9, 10, and 11 wt%) in a binary solvent comprised of 81/19 wt/wt% dioxane-water solvent. (B) Cloud points of PPF-co-PLGA fabricated using different dioxane-water ratios (77/23, 78/22, 79/21, 80/20, 81/19, 82/18, 83/17 and 84/16 wt/wt%) with 7 wt% copolymer concentration. Error bars represent SD.

3.2. Morphology of scaffolds

All scaffolds fabricated with different copolymer concentrations and solvent ratios had the characteristics of interconnected porous architectural structures with protuberances. The protuberances and pores of the 6 wt% scaffolds are unsmooth and rugged (Figure 3A), and as the copolymer concentration increased the pore walls became much smoother and the pores became larger, more symmetrical, and well ordered (Figure 3B and C). The 8 wt% scaffolds are especially well interconnected.

Figure 3.

Figure 3.

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Scanning electron microscopy (SEM) images of crosslinked 3D porous PPF-co-PLGA scaffolds fabricated using TIPS: (A) 6 wt%, (B) 8 wt%, and (C) 10 wt% copolymer in a 81/19 dioxane-water binary solvent, or (D) 78/22, (E) 80/20, and (F) 82/18 wt/wt% dioxane-water solutions at a 7% copolymer concentration.

As the dioxane content increased, the protuberances became much flatter, porosity decreased, pores became smaller, and strut thickness increased (Figure 3D–F). These changes indicate that PPF-co-PLGA can dissolve thoroughly in mixtures with higher organic solvent ratios, and this solubility affects microstructure formation during the phase separation process.

3.3. Mechanical properties

In the different concentration groups, the mean (SD) compressive moduli of the samples 6, 8, and 10 wt% were 2.55 (0.09), 3.30 (0.20), and 7.08 (0.22) MPa, respectively. The mean (SD) compressive moduli of the scaffolds 78:22, 80:20, and 82:18 wt/wt% were 2.25 (0.12), 3.12 (0.05), and 4.22 (0.05) MPa, respectively.

In the different copolymer concentration groups, the compressive moduli of samples fabricated using 6, 8, and 10 wt% copolymer compositions (in the 81/19 wt/wt% dioxane-water solvent) were 2.55 ± 0.09, 3.30 ± 0.20, and 7.08 ± 0.22 MPa, respectively, and significant differences exist between subgroups (P < 0.05) (Figure 4). In the different solvent ratio groups, the compressive moduli of scaffolds fabricated in 78/22, 80/20, and 82/18 wt/wt% dioxane-water solvents (with a 7 wt% copolymer concentration) were 2.25 ± 0.12, 3.12 ± 0.05, and 4.22 ± 0.05 MPa, respectively, and significant differences were also determined between subgroups (P < 0.05) (Figure 4).

Figure 4.

Figure 4.

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(A) Compressive moduli of PPF-co-PLGA scaffolds fabricated using 6, 8, and 10 wt% copolymer in a binary solvent of 81/19 dioxane-water ratio. (B) Compressive moduli of PPF-co-PLGA scaffolds fabricated using 78/22, 80/20, and 82/18 wt/wt dioxane-water solutions at a 7% copolymer concentration. Error bars represent SD. Significant differences at *P<0.05 and **P<0.01 are shown.

3.4. Ability of scaffolds to facilitate biomineralization

All samples exposed to the mineralizing fluid presented obvious mineral depositions, which led to the formation of rough interfaces covered with apatite-like substances. As the copolymer concentration and solvent ratio increased, biomimetic mineralized precipitations became more apparent. Almost all pores and protuberances formed on the surfaces of the scaffolds using a 10 wt% copolymer concentration (in an 81/19 wt/wt% dioxane-water solvent) or in 82/18 wt/wt dioxane-water solvent (with a 7 wt% copolymer concentration) were covered with mineralized crystallization layers in comparison with the scaffolds fabricated using lower copolymer concentrations or solvents with lower dioxane-water ratios, which had inadequate superficial coverage (Figure 5).

Figure 5.

Figure 5.

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SEM images of the surfaces of PPF-co-PLGA scaffolds after in vitro biomineralization in simulated body fluid (SBF) at 37 °C for 2 hours. The samples were fabricated using (A) 6 wt%, (B) 8 wt%, and (C) 10 wt% copolymer concentrations in a binary solvent of 81/19 dioxane-water ratio, or using (D) 78/22, (E) 80/20, and (F) 82/18 wt/wt dioxane-water solutions at a 7% copolymer concentration.

The functional groups of the copolymer and its scaffold treated by the mineralizing solution were detected using FTIR spectroscopy. As shown in Figure 6, besides the intrinsic ions of the PPF-co-PLGA copolymer, the biomineralized specimen exhibited strong peaks at 1,038 and 688 cm−1 which correspond to phosphate groups (PO4).

Figure 6.

Figure 6.

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ATR-FTIR spectra of PPF-co-PLGA before and after biomineralization.

3.5. In vitro degradation

The scaffolds were fabricated using different concentrations and solvent ratios and submerged in PBS or 0.02 M NaOH for in vitro degradation over a certain period of time. All specimens exhibited biodegradable properties in both physiological PBS and accelerated NaOH solutions, and samples fabricated using lower polymer weight compositions and ratios degraded significantly faster than those using higher polymer content and solvent ratios. The scaffolds fabricated using a 6 wt% copolymer concentration (in an 81/19 dioxane-water solvent) and 78/22 wt/wt dioxane-water solvent (with a 7 wt% copolymer concentration) disappeared in NaoH and PBS at 6 days and 7 weeks, respectively. In contrast, scaffolds formed using 10 wt% and 82/18 wt/wt dioxane:water solvent degraded in 9 days in 0.02 M NaOH and still maintained some weight in PBS at 8 weeks (Figure 7).

Figure 7.

Figure 7.

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Percent weight loss of PPF-co-PLGA scaffolds fabricated using 6, 8, and 10 wt% copolymer concentrations in a 81/19 wt/wt% dioxane-water solution and using 78/22, 80/20, and 82/18 wt/wt% dioxane-water solution at 7 wt% copolymer concentration in (A) 0.02 mM NaOH solution for accelerated degradation and (B) PBS solution for simulated physiological degradation. Error bars represent standard deviation (SD).

3.6. Cytotoxicity

After culturing cells in the presence of PPF-co-PLGA scaffolds in the Transwell inserts for 4 days, we found no significant differences in cellular viability among the various copolymer concentration and solvent ratio groups and the positive control groups (cells cultured in the absence of polymer) for both MC3T3 and PC-12 cells (P > 0.05) (Figure 8).

Figure 8.

Figure 8.

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Viability of (A) MC3T3 cells and (B) PC12 cells detected by MTS assay after 4-day co-culturing with PPF-co-PLGA scaffolds. The scaffolds were fabricated using 6, 8, and 10 wt% copolymer concentration (in 81/19 wt/wt% dioxane-water solution) and using 78/22, 80/20, 82/18 wt/wt% of dioxane-water solution (at 7 wt% copolymer concentration). There were no significant differences between these groups and the positive control (cells cultured in the absence of polymer scaffolds). Error bars represent SD.

3.7. Cell adhesion

Both MC3T3-E1 and PC12 cells had excellent adherence to the PPF-co-PLGA scaffolds after 4 days of co-culturing, which were fabricated using different copolymer concentrations and solvent ratios. The cellular morphologic makeup of the cytoskeleton and nucleus were clearly shown after rhodamine-phalloidin and DAPI staining (Figure 9). The cells on the scaffold surface spread well, and some were confluent in rough areas. Cell counting was conducted at 4 days of proliferation. The MC3T3-E1 cell densities on the surfaces of scaffolds fabricated using 6, 8, and 10 wt% copolymer concentrations (in an 81/19 wt/wt dioxane-water solvent) were 57,151 ± 7,594, 52,364 ± 7,834, and 52,473 ± 5,866 cells/cm2, respectively, and 68,353 ± 8,808, 59,898 ± 8,073, and 56,835 ± 6,938 cells/cm2 on scaffolds fabricated in 78/22, 80/20, and 82/18 wt/wt% dioxane-water solvents (with a 7 wt% copolymer concentration), respectively (Figure 10).

Figure 9.

Figure 9.

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Fluorescence images of MC3T3 cells after 4 days of culture on PPF-co-PLGA scaffolds fabricated using (A) 6 wt%, (B) 8 wt%, and (C) 10 wt% copolymer concentrations in a binary solvent of 81/19 dioxane-water ratio. Fluorescence images of MC3T3 cells after 4 days of culture on 7wt% PPF-co-PLGA scaffolds fabricated using (D) 78/22, (E) 80/20, and (F) 82/18 wt/wt% dioxane-water solutions. Fluorescence images of PC12 cells after 4 days of culture on (G) 6 wt%, (H) 8 wt%, and (I) 10 wt% PPF-co-PLGA scaffolds fabricated using 81/19 dioxane-water ratio. Fluorescence images of PC12 cells after 4 days of culture on 7 wt% PPF-co-PLGA scaffolds fabricated using (J) 78/22, (K) 80/20, and (L) 82/18 wt/wt dioxane-water solutions.

Figure 10.

Figure 10.

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Number of MC3T3-E1 cells after 4 days of culture on the surfaces of scaffolds fabricated using various copolymer concentrations (6, 8, and 10wt% in an 81/19 dioxane-water solvent) and (B) various dioxane-water ratios (78/22, 80/20, 82/18 at a 7% copolymer concentration). Number of PC12 cells after 4 days of culture on the surfaces of scaffolds fabricated using (C) various copolymer concentrations (6, 8, and 10wt% in an 81/19 dioxane-water solvent) and (D) various dioxane-water ratios (78/22, 80/20, 82/18 at a 7% copolymer concentration).

The PC12 cell densities on the surfaces of scaffolds fabricated using 6, 8, and 10 wt% copolymer concentrations were 83,062 ± 8,346, 70,961 ± 8,307, 73,154 ± 14,25 7 cells/cm2, respectively, and 80,459 ± 11,528, 89,459 ± 10,363, 68,176 ± 9,453 cells/cm2 on scaffolds fabricated in 78:22, 80:20, and 82:18 wt/wt% dioxane-water solvents, respectively (Figure 10). These differences were not significant among the various copolymer concentration and solvent ratio groups (P > 0.05).

4. Discussion

In the current study, cross-linked PPF-co-PLGA scaffolds were created using TIPS. The characteristics of the scaffolds with a porous microarchitecture were evaluated for their potential use in tissue engineering. TIPS has extensive applications in polymeric scaffold manufacturing.22 The technical principles of using TIPS to fabricate interconnected scaffolds were previously described,20 and the dioxane-water binary solvent was developed in an earlier study for inducing phase separation in a homogenized solvent.36 However, different polymers have their own properties, which influence solubility in different solvent systems and phase separation processes. In the present work, we explored the effects of various PPF-co-PLGA concentrations and dioxane-water ratios on the 3D structures and characteristics of scaffolds.

The solubility of PPF-co-PLGA in the dioxane-water solvent is reflected by the cloud point. The cloud points of the copolymers fabricated using different concentrations (4 to 11 wt %) gradually increased, and the cloud point ranges were wide between the various dioxane-water ratio groups (77:23 to 84:16 wt/wt%), but the step changes were basically homogeneous. Both copolymer weight composition and the binary solvent ratio could significantly affect solubility, but the solvent ratio has a greater role than concentration. This suggests that the water content in the binary solvent is the critical factor for liquid separation. Moreover, the cloud point is an indicator of phase separation, and therefore the cloud-point curves provide indications regarding the key solute concentrations (6, 8, and 10wt %) and binary solvent ratios (78:22, 80:20, and 82:18 wt/wt% dioxane-water) for scaffold fabrication.37

The copolymer-lean phase has a primary role in the use of TIPS to fabricate scaffolds when the solute content in the solution is less than the critical content.22,38 When the newly formed, copolymer-rich, tiny drops are scattered over the copolymer-lean substance, an unsystematic scaffold architecture forms. In contrast, if the copolymer concentration exceeds the critical quantity, the copolymer-lean tiny drops form a substance that is distributed among the copolymer-rich phase. The organic content of the binary solvent is a key factor in the dissolution of the copolymer, and high solubility prevents the separation of the copolymeric molecular chains from the solution, which is the reason that the scaffolds formed in 80:20 and 82:18 wt/wt% dioxane-water solvents had smaller pores.

Biodegradable scaffolds with desired porosity could have broad applications for low load-bearing reconstruction, or regeneration, of soft tissues such as nerve and blood vessels.39,40 However, varied tissue types with vast intrinsic mechanics may have varied mechanical requirements for scaffold implants. Here, the porous PPF-co-PLGA scaffolds we fabricated in the current study, were successfully shown to have tunable compressive moduli by modulating polymer concentration, or altering the dioxane-water ratio. Further, the ability of synthetic biomaterials and scaffolds to facilitate biomineralization is crucial for tissue engineering, especially for the repair of bone and musculoskeletal defects. As a bionic electrolyte solution, SBF was used to test the bioactivity potential of an artificial scaffold. 33,41 By immersion in SFB solution, our PPF-co-PLGA porous scaffolds showed a substantial amount of mineral deposition on the surface, as evidenced by the SEM and FTIR results. These results indicate those cross-linked PPF-co-PLGA porous scaffolds may have satisfactory mineralization after implant for bone regeneration.

PPF and PLGA composite scaffolds showed considerable biodegradability in previous works.26–28, 42 The degradation rate of the novel PPF-co-PLGA substrates in the present study was shown to be dependent on the porous microstructure of the scaffold. The differences of biodegradation rate may be due to the variation in pore structures and porosity. Diversified pore structures or porosity may result in a heterogeneous transportation of solution into the interior of porous scaffolds, thus leading to varied bulk degradation rates.43,44 Due to the existence of multiple enzymes, the degradation in vivo is much more complicated, with typically faster degradation profiles.45 On the other hand, bone tissue regeneration normally happens in a few weeks, our PPF-co-PLGA porous scaffold with 7~8 weeks sustenance in vitro should be perfect to meet the supportive demands of a defective tissue growth before full degradation.46,47 After surface bone formation, these PPF-co-PLGA porous scaffolds were expected to gradually biodegrade, therefore giving more free space for a higher volume of bone to grow in, and thus facilitate tissue regeneration.

In brief, we have developed a novel PPF-co-PLGA copolymer and fabricated 3D cross-linked scaffolds using TIPS. The inner structures, mechanical properties, mineralization capacity, biodegradability, biologic toxicity, and cellular affinity of these scaffolds were evaluated to determine whether they have prospective applications in tissue engineering.

5. Conclusions

In the present research, a new synthetic copolymer was used to manufacture porous scaffolds using a dioxane-water binary solvent through TIPS. As shown by the characterization evaluations, the cloud point, morphologic structure, compressive modulus, in vitro biomineralization, and biodegradable properties were all affected by variations in the copolymer weight compositions and solvent ratios, which did not appreciably impact in vitro cellular viability and attachment. In our experiment, the scaffolds fabricated using all different copolymer concentrations and dioxane-water ratios exhibited a considerable, interconnected, porous internal structure, compressive mechanical properties, biomineralization ability, biodegradability, and cellular affinity. Among these, samples fabricated using 8 and 10 wt% copolymer concentrations and 80/20 and 82/18 wt/wt% dioxane-water ratios showed optimum properties. These novel biodegradable PPF-co-PLGA scaffolds are therefore promising candidates for various tissue engineering applications.

Acknowledgments

This work was supported by National Institutes of Health grant R01 AR56212.

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