Synthesis and 3D Printing of PEG-Poly(propylene fumarate) Diblock and Triblock Copolymer Hydrogels

Abstract

PEG-based hydrogels are used widely in exploratory tissue engineering applications but in general lack chemical and structural diversity. Additive manufacturing offers pathways to otherwise unattainable scaffold morphologies but has been applied sparingly to cross-linked hydrogels. Herein, mono methyl ether poly(ethylene glycol) (PEG) and PEG-diol were used to initiate the ring-opening copolymerization (ROCOP) of maleic anhydride and propylene oxide to yield well defined diblock and triblock copolymers of PEG-poly(propylene maleate) (PPM) and ultimately poly(propylene fumarate) (PPF) with different molecular mass PEG macroinitiators and block length ratios. Using continuous digital light processing (cDLP) hydrogels were photochemically printed from an aqueous solution which resulted in a 10-fold increase in elongation at break compared to traditional diethyl fumarate (DEF) based printing. Furthermore, PPF-PEG-PPF triblock hydrogels were also found to be biocompatible in vitro across a number of engineered MC3T3, NIH3T3, and primary Schwann cells.

Graphical Abstract

*Note for reviewers: The far right hydrogel image was captured in brightfield mode on a Keyence BZ-X700 at 2X magnification.

Synthetic hydrogels, while used widely for a number of tissue engineering applications, are limited in the number and type of chemical groups used for network formation and structural elements that can be used to manipulate network structure.^1–2 Furthermore, they generally require several criteria to be clinically relevant. In addition to maintaining cell viability, a combination of suitable mechanical properties with sufficient processing flexibility to yield morphologically and structurally complex scaffolds is desirable.³ A number of reports have demonstrated that both mechanical properties as well as scaffold topology significantly influence cell proliferation, migration and differentiation; therefore, a functional material should concomitantly mimic the mechanical properties of the target tissue while providing a processing handle to produce complex, high resolution structures.^4–20

Hydrogels are networks of hydrophilic polymers that can be tailored to match a broad range of design parameters as a consequence of their tunable mechanical properties and intrinsic network morphology.²¹ For example, the modulus of poly(ethylene glycol) (PEG) oxime hydrogels can be tuned using a kinetically-controlled crosslinking reaction independent of chemistry, concentration, and stoichiometry.²² The moduli of other hydrogel systems can be adjusted by changing the weight percent of the hydrogel.²³ Furthermore, functionalization of hydrogels has afforded investigations into the role of exogenous bioactive ligands on cell behavior.^20,24,25 Microfabrication techniques such as microcontact printing, templated photolithography, and microfluidic molding have each afforded access to micrometer and nanometer-sized structures.²⁶ However, many hydrogel systems are limited to casting fabrication techniques, greatly limiting the variety as well as resolution control of scaffold architectures that can be produced.

Additive manufacturing has provided avenues to morphologically complex structures unachievable by other processing methods.²⁷ This technique provides a number of advantages in the development of tissue engineering scaffolds as features such as porosity and increased surface area promote host infiltration within a synthetic implant.²⁸ Additive manufacturing modalities such as fused deposition modeling (FDM) have been applied to hyaluronic acid and sodium alginate-based gels.²⁹ Furthermore, a recent report demonstrated the use of continuous digital light processing (cDLP) to print PEG-diacrylate (PEG-DA) hydrogels into high resolution structures.³⁰ cDLP methods using the stepwise production of UV cured layers to build a structure from a photo-reactive liquid resin.³¹ This technique can produce extremely fine (e.g. <120 μm) features and through the use of computer aided modeling (CAM) can be tailored to create scaffolds for patient-specific defects.³² However, the viscosity of the polymer resin must be sufficiently low that it can flow readily (e.g.. η* ≤ 1–3 Pa.s.), requiring the use of a solvent or diluent for many polymers.

One such polymer that has shown great utility for cDLP is poly(propylene fumarate) (PPF), a degradable, unsaturated polyester. First reported by Mikos and coworkers and developed specifically for bone tissue regeneration, this UV-crosslinkable polymer provides robust mechanical strength while also degrading into a Krebs-cycle constituent (fumaric acid) and a ubiquitous food additive (propylene glycol).³³ While the first described syntheses utilized step-growth polymerizations, Coates and coworkers demonstrated the ring-opening copolymerization (ROCOP) of propylene oxide (PO) and maleic anhydride (MA) in the presence of a cobalt catalyst to produce well-controlled PPM that could be converted to PPF upon isomerization.³⁴ Producing narrow molecular mass distribution polymers can be useful in regenerative medicine applications as chain length and distribution can affect degradation, mechanical properties, and the fidelity of the scaffold structure.^35,36,37 Magnesium ethoxide has since been utilized as a ROCOP catalyst to produce PPF albeit with less control but avoiding the toxicity of cobalt. With the use of diethyl fumarate (DEF) as a viscosity modifier and reactive diluent, PPF was formed into cell viable scaffolds using cDLP.²⁷ Finally, a recent report further demonstrated the well-controlled ROCOP of PPF using Mg(BHT)₂(THF)₂ as a catalyst, resulting in molecular mass distributions (Ɖ_m) similar to those reported by Coates and coworkers.³⁸ Furthermore, chain end-functionalization was achieved in this system using a functional alcohol initiator, simultaneously introducing a reactive handle for post-polymerization and post-printing functionalization.

The facile ROCOP initiation of PPF using a number of primary alcohols led to the notion of polymerizing PPF from the end of a PEG-diol chain to produce robust, 3D-printable hydrogel precursors. Multiblock poly(propylene fumarate-co-ethylene glycol) with broad molecular mass distributions were reported previously by Suggs et al., employing a step-growth polymerization followed by a transesterification mechanism to incorporate ethylene glycol subunits into PPF.³⁹ In contrast, this paper seeks to present the formation of well-ordered block copolymers of PEG_nPPF_m and PPF_mPEG_nPPF_m utilizing the magnesium-based catalyst. Formation of hydrogels by photo-crosslinking will be explored, both in the context of casting and 3D-printing by cDLP, as well as the resultant swelling and tensile mechanical properties.

Poly(propylene maleate) (PPM) was formed from the alternating ROCOP of MA and PO in the presence of Mg(BHT)₂(THF)₂ catalyst, initiated from the alcohol chain end of both methyl ether PEG and PEG-diol to form diblock (PEG_nPPM_m) and triblock (PPM_mPEG_nPPM_m) copolymers (Figure 1A and B) (Table 1), respectively. Using a molar ratio of 1:5 (catalyst:initiator), polymerizations exceeded 90% conversion in all cases except for entries 2 and 6 as determined by integration of the residual maleic anhydride peak in ¹H NMR spectroscopy. The lower conversions can be attributed to larger head space in the reaction vessel: PO boils at 34 °C and must be condensed back into solution. It was experimentally determined that by minimizing this head space, the reactions are driven to higher conversion. The polymers were then isomerized to the PEG_nPPF_m and PPM_mPEG_nPPM_m species by treatment with diethylamine (Figure 1C and D). The final polymer products were characterized by MALDI-TOF mass spectrometry to show the PPF repeat unit of 156 Da and the PEG repeat unit of 44 Da (Figure 1E). The final molar mass distributions of the diblock copolymers (Table 1, Entries 1–9) were calculated via end group analysis from ¹H NMR and size exclusion chromatography (SEC), while the triblock copolymers were analyzed by SEC as a consequence of potentially identical end groups (i.e. no terminal methyl group for analysis, end groups could be MA on either chain end, PO group on either chain end, or a combination of the aforementioned). In this case, the end group analysis via ¹H NMR demonstrated a consistent increase in number-average molecular weight (M_n) with targeted degree of polymerization, indicating the successful synthesis of blocks of varying lengths. Comparatively, the SEC data did not display such a linear increase of molecular mass with increasing block length. However, this can be attributed to the use of poly(styrene) standards, whose hydrodynamic radii in tetrahydrofuran (THF) solvent is expected to much different than that of the block copolymers and the relatively low molecular mass.

Figure 1.

Synthesis of PEGPPF di- and triblock copolymers initiated by (A) methyl ether PEG to produce poly(ethylene glycol-b-propylene maleate) and (B) PEG-diol to produce poly(propylene maleate-b-ethylene glycol-b-propylene maleate). (C) Isomerization using diethylamine as a catalyst yielded the poly(ethylene glycol-b-propylene fumarate) and (D) poly(propylene fumarate-b-ethylene glycol-b-propylene fumarate) products. (E) MALDI-TOF spectrum of poly(propylene fumarate-b-ethylene glycol-b-propylene fumarate). (F) Diffusion-ordered NMR spectroscopy (DOSY-NMR) spectra of poly(ethylene glycol-b-propylene fumarate) block copolymer (red line) and the homopolymer mixture of methyl ether PEG and PPM (black line).

Table 1.

Synthesis of PEGPPF Diblock and Triblock Copolymers

Entry	PEG Initiatora	Target DP PPFb	Temperature (°C)	Time (days)	MA Conversion (%)	Mnc (kDa)	Mnd (kDa)	MWd (kDa)	ƉMd
1	m 1000	6	100	3	94	1.9	2.4	3.7	1.59
2	m 1000	15	100	4	75	2.6	2.2	3.4	1.55
3	m 1000	25	100	5	91	3.5	1.6	2.2	1.38
4	m 2000	6	100	3	94	3.0	1.6	2.0	1.25
5	m 2000	15	100	4	83	4.6	2.6	4.5	1.70
6	m 2000	25	100	5	70	5.1	2.6	3.1	1.31
7	m 4000	6	100	3	93	5.1	5.2	7.0	1.36
8	m 4000	15	100	4	93	6.6	2.1	4.3	1.49
9	m 4000	25	100	5	94	7.0	3.9	4.4	1.12
10	d 1000	6	100	3	88	-	4.4	7.2	1.65
11	d 1000	15	100	4	94	-	2.6	2.9	1.11
12	d 1000	25	100	5	95	-	3.9	6.2	1.57
13	d 2000	6	100	3	93	-	0.5	0.5	1.12
14	d 2000	15	100	4	95	-	3.6	4.9	1.49
15	d 2000	25	100	5	97	-	4.6	7.5	1.61
16	d 4000	6	100	3	91	-	4.3	7.2	1.65
17	d 4000	15	100	4	99	-	6.7	10.0	1.51
18	d 4000	25	100	5	94	-	4.3	8.3	1.92

^aWhere m = methyl ether PEG, d = PEG-diol and values indicates molecular mass (Da).

^bBased on an initiator-to-monomer ratio.

^cBased on end-group analysis using ¹H NMR spectroscopy.

^dBased on SEC against poly(styrene) standards

To demonstrate that well-defined block copolymers had been synthesized diffusion-ordered NMR spectroscopy (DOSY-NMR) and quantitative ¹³C NMR spectroscopy experiments were employed. An example DOSY-NMR spectrum displays separate diffusional bands in a mixture of the two homopolymers compared to the block copolymer itself (Figure 1F). Furthermore, in the ¹³C NMR spectra, the carbonyl diad resonances of the PPF repeat unit (δ = 164.4 and 164.0 ppm) are split by PEG carbons adjacent to them. As the integration values of the carbonyl PPF peaks adjacent to PEG are lower than those adjacent to PPF, it stands to reason that a sharp interface between the two blocks exist. This can also be observed from the PEG peak (δ = 70.55 ppm) which is split by the PPF block interface (Figure S1).

Small Angle Oscillatory Shear Rheology.

cDLP additive manufacturing involves crosslinking a photo-reactive polymer resin into a solid using ultraviolet light. This technique requires sufficiently low resin viscosities (i.e. <3 Pa.s) to allow the polymer to flow into the void space as the printed product is raised out of the resin. The resin form of pure PPF₃PEG₂₃PPF₃ block copolymer has a complex viscosity of about 35–50 Pa.s measured by small angle oscillatory shear (SAOS) rheology. These values are far too viscous for this manufacturing method and therefore must be diluted. Dean and coworkers have shown DEF is well suited for this purpose as a solution with a 1:1 ratio of polymer:DEF easily produces a usable viscosity (<1 Pa.s) for homopolymer PPF.³¹ Similarly, 3:1 and 1:1 ratios of DEF:PPF_mPEG_nPPF_m yielded viscosities <3 Pa.s for all variants of PPF_mPEG₂₃PPF_m as well as PEG₂₃PPF_m (Figure 2A).

Figure 2.

Viscometry and cDLP Printing of PPF_mPEG₂₃PPF_m with DEF. (A) DEF was used as a viscosity modifier for the extremely viscous homopolymer resin, 50 wt.% being found optimal for 3D printing. (B) cDLP printing schematic. (C) A CAD model of a gyroidal scaffold (left) was successfully printed from a 50 wt.% solution of PPF₃PEG₂₃PPF₃ in DEF.

cDLP Printing with DEF-Resin.

PPF_mPEG₂₃PPF_m (i.e 1 kDa PEG) triblock copolymers were successfully printed using a 1:1 mass ratio of polymer:DEF. A printing formulation previously developed by Dean et al. was added: 3 wt.% BAPO, an acylphosphine photoinitiator, 0.4% Irgacure 784, a titanocene-based initiator/light scattering agent, and 0.7% oxybenzone, a radical scavenger.³¹ To demonstrate the print resolution of PPF₃PEG₂₃PPF₃ polymer, a gyroidal scaffold with strut sizes ~100 μm in diameter was printed from a computer-generated model (Figure 2C). An optical image of the printed structure demonstrates the high resolution of PPF₃PEG₂₃PPF₃ from cDLP printing.

cDLP Printing in Aqueous Solution.

For tested PPF_mPEG_nPPF_m species with PEG block >1000 Da the polymers are insoluble in DEF, limiting its use as a viscosity modifier. Fortuitously, above a PEG block mass of 2000 Da (i.e. PEG₄₅ and PEG₉₁) the polymers become water-soluble and when dissolved yield usable viscosities for cDLP printing, which requires sufficiently low viscosity that the resin can flow (e.g. typically η* ≤ 3 Pa.s) (Figure 3A). This provides a two-fold advantage over previously utilized PPF:DEF mixtures by simultaneously achieving usable viscosities as well as removing the need for DEF, which as a reactive diluent is incorporated into printed scaffolds and therefore changes the material properties.

Figure 3.

PPF_mPEG_nPPF_m Hydrogels. (A) The water solubility of PPF_mPEG_nPPF_m copolymers with PEG >2000 Da produces suitable viscosities for cDLP at ~25 wt.%. (B). Swelling ratios of PPF_mPEG_nPPF_m hydrogels. (C) Stress-strain curves for water-printed PPF₃PEG₄₅PPF₃ and DEF-printed PPF₃PEG₂₃PPF₃tensile bars.

One challenge with cDLP is selecting a photoinitiator for a printing formulation. To crosslink the water soluble polymers, the photoinitiator lithium acylphosphinate (LAP) was used.⁴⁰ In addition to water solubility, LAP also has a much higher molar absorptivity coefficient when compared to the widely used Irgacure 2959 (in the range of 340–420 nm). Furthermore, it is also cytocompatible making it an ideal photoinitiator for cross-linking water-soluble PEGPPF.⁴¹ As the printer emits light at 405 nm this combination of properties made LAP an ideal choice to attempt cDLP printing from an aqueous solution, which proved fruitful in the successful printing of PPF₃PEG₄₅PPF₃.

PEGPPF Hydrogels.

The water-soluble species of PPF_mPEG_nPPF_m (i.e PEG₄₅ and PEG₉₁ blocks) were photo-crosslinked into hydrogels to examine their swelling properties and cell viability. Each gel was cast at 25 wt.% in water with a 5:1 molar ratio of reactive center (double bond):LAP to ensure uniform cross-linking. The swelling of PPF_mPEG₉₁PPF_m (i.e 4 kDa PEG) (Q, Eq S1) species is much higher than PPF_mPEG₄₅PPF_m for short PPF block lengths but quickly converges as the PPF block length increases (Figure 3B). This is a consequence of the increasing quantity of cross-linking sites which decrease the distance between cross-links and suppress swelling of the scaffold. Furthermore, the PPF block length increases concomitantly with hydrophobicity, which promotes the aggregation of the reactive centers compared to the shorter chains and potentially increases the number of cross-links formed. Finally, to assess the impact of the sterilization process for cell viability assays (i.e. incubation in ethanol followed by autoclaving) the swelling properties of cast hydrogels were measured following sterilization (Figure S12). The increased swelling ratios compared to unsterilized gels indicate that some degradation is occurring. This effect is most pronounced for the shortest PPF block lengths (m = 3) as the crosslink density is lowest in those materials, while the longer PPF blocks did not change as drastically. While the gels remained intact enough for cell viability assays, this indicates a softer sterilization method such as e-beam methods will be needed for clinical translation.

Tensile Properties of 3D printed PPF_mPEG_nPPF_m Hydrogels.

3D-printing from an aqueous solution yielded distinctly different mechanical properties compared to printing from a DEF-based resin. The tensile properties of water-printed PPF₃PEG₄₅PPF₃ material demonstrated a 10-fold increase in strain at break compared to DEF-printed PPF₃PEG₂₃PPF₃ (Figure 3C). This extension can be attributed to the lack of DEF thereby producing a network with greater distance between crosslinks. In the case of PPF₃PEG₂₃PPF_3, DEF is incorporated into the final network and produces a highly cross-linked and somewhat brittle material. In contrast, the lower crosslinking density in the water-printed hydrogel affords a notable increase in conformational relaxation modes of the PEG chains allowing them to extend farther before material failure. Surprisingly, the moduli of the two conditions were similar (PPF₃PEG₄₅PPF₃: 9.1 ± 0.1 kPa; PPF₃PEG₂₃PPF₃: 8.9 ± 0.1 kPa) indicating the extension at break can be tuned independently of modulus.

Cell Viability.

The potential of PPF_mPEG_nPPF_m (n = 45, 91) hydrogels for use in soft tissue applications, such as peripheral nerve regeneration, was evaluated using a viability assay with three different cell types. Mouse preosteoblast MC3T3-E1 cells have been frequently used for elucidating baseline cell responses for orthopaedic materials; Schwann cells are the principal glia that support neurons in the peripheral nervous system; and NIH 3T3 are a commonly used fibroblast cell line.^42–44 Not surprisingly, after 24 h of cell culture the dominant green fluorescence from live cells demonstrated that PPF_mPEG_nPPF_m hydrogels have high viability when each of the cell types were cultured on sterile disks of the cross-linked samples (Figure 4). These results indicate that the materials are nontoxic and exhibit compatibility with each of the three cell types. This was ideal as it promotes the potential for PPF_mPEG_nPPF_m as a biomaterial to become translationally relevant in a number of diverse applications.

Figure 4.

Viability of various engineered and primary cell lines on PPF_mPEG_nPPF_m hydrogels. (A) Representative fluorescent images of primary Schwann cells stained with Calcein AM (green, live cells) and ethidium bromide (red, dead cells). (B) The viability of NIH3T3, MC3T3, and Schwann cells normalized to glass slide controls was nearly quantitative.

Further exploration of cellular response can be achieved by tethering bioactive ligands. This could be achieved through the incorporation of functionalized epoxide monomers such as glycidyl propargyl ether for azide-alkyne cycloadditions, or by performing thiol-ene additions to unreacted double bonds in the backbone, the latter of which has been shown to exist postcrosslinking.^45,46 Through orthogonal chemical approaches, multiple peptide sequences could be tethered to the same scaffold.

Conclusions.

The availability and diversity of printable and resorbable materials will be critical to using additive manufacturing in regenerative medicine. Using a magnesium catalyst, PPF was successfully synthesized by ROCOP from the chain end of both methyl ether PEG and PEG-diol to produce a series of PEG_nPPF_m diblock and PPF_mPEG_nPPF_m triblock copolymers, respectively. Hydrogels printed in aqueous conditions demonstrated a noticeable increase in elongation at break compared to a DEF-printed variant. Additionally, the ability to manipulate strain at break independent of modulus was demonstrated. Finally, PPF_mPEG_nPPF_m (n = 45, 91) hydrogels were found to be compatible with MC3T3, NIH3T3, and Schwann cell lines demonstrating the potential of these materials in tissue-engineering applications. While this report constitutes only an initial demonstration, we are exploring many more specific applications of these materials across regenerative medicine and nanomedicine.

Materials and Methods:

Materials.

Methyl ether poly(ethylene glycol) was purchased from TCI (Portland, OR). All other commercial reagents and solvents were purchased from Sigma-Aldrich or Fisher Scientific and used as received unless noted otherwise. All reactions were performed under nitrogen unless noted otherwise.

Example synthesis of poly(propylene maleate-b-ethylene glycol-b-propylene maleate) (PPM_mPEG_nPPM_m):

PEG-diol (MW 1000) (0.911 g, 0.091 mmol), propylene oxide (0.668 mL, 9.54 mmol), maleic anhydride (0.935 g, 9.54 mmol), and Mg(BHT)₂(THF)₂ (110.6 mg, 0.182 mmol) were dissolved in 10 mL of toluene in an oven dried ampoule. The ampoule was sealed and heated at 100 °C for 72 hours. The resultant polymer was recovered by precipitation in excess hexanes at 40 °C and washed with phosphate buffer solution to remove excess starting material before drying on high vacuum. (97 % conversion) ¹H NMR (500 MHz, 303 K, CDCl₃): δ = 6.28–6.24 (t, OC(=O)CH=CHC(=O)O), 5.30–5.20 (m, CH₂CH(CH₃)O), 4.29–4.21 (m, CH₂CH(CH₃)O), 3.64 (m, OCH₂CH₂O), and 1.33–1.31 (m, CH₂CH(CH₃)O) ppm.

Isomerization of poly(propylene maleate-b-ethylene glycol-b-propylene maleate):

Poly(propylene maleate-b-ethylene glycol-b-propylene maleate) (1.00 g, 10 mol. eq. olefin) was dissolved into chloroform (50 mL). Diethylamine (0.151 mL, 0.15 mol. eq. olefin) was added to the solution and refluxed for 24 h under a nitrogen atmosphere. After cooling to room temperature, the organic solution was washed with phosphate buffer solution (150 mL, pH = 6) and the polymer was recovered through precipitation from hexanes. ¹H NMR (500 MHz, 303 K, CDCl₃): δ = 6.88–6.82 (m, OC(=O)CH=CH(=O)O), 5.30–5.20 (m, CH₂CH(CH₃)O), 4.29–4.21 (m, CH₂CH(CH₃)O), 3.64 (m, OCH₂CH₂O), and 1.33–1.31 (m, CH₂CH(CH₃)O) ppm.

3D Printing of PPF_mPEG_nPPF_m:

PPF_mPEG₂₃PPF_m was printed on an EnvisionTec Micro+ Advantage 3D printer using a 405 nm UV light projector. Resin formulations were prepared as follows: for printing with diethyl fumarate (DEF), the polymer was dissolved at 50 wt.% in DEF with an additional 3 wt.% BAPO, 0.7 wt.% Irg 784, and 0.4 wt.% oxybenzone.³¹ For printing from an aqueous solution, PPF_mPEG_nPPF_m (n = 45, 91) polymers were dissolved from 20–25 wt.% in water depending on their complex viscosities with 1:5 molar ratio of LAP photoinitiator to double bond in the polymer (determined by M_n ¹H NMR).