Fabrication and characterization of photosensitive non-isocyanate polyurethane acrylate resin for 3D printing of customized biocompatible orthopedic surgical guides

Abstract

Three-dimensional (3D)-printed orthopedic surgical guides have the potential to provide personalized precision treatment. Non-isocyanate polyurethane (NIPU) is commonly used in the 3D printing of biomedical materials but its application in the orthopedic surgical guide is limited by poor mechanical properties and biocompatibility. In this study, we fabricated non-isocyanate polyurethane acrylate (NIPUA) photosensitive resin with superior biocompatibility and mechanical properties required for 3D-printed orthopedic surgical guides. NIPU prepolymer was synthesized by a ring-opening reaction and a ring acrylation reaction. NIPUA was further synthesized using polyethylene glycol diacrylate (PEGDA) as a modified material based on sustainable synthesis with reduced synthesis time. NIPUA showed the best tensile and flexural strengths when the PEGDA content reached 12 wt.%. NIPUA exhibited higher thermal stability, hemocompatibility, superior biocompatibility to ME3T3-E1 bone cells and C1C12 muscle cells, and non-immunogenic effect toward macrophages compared with commercial photosensitive resins. Commercial resins triggered a severe inflammatory response during in vivo implantation, but this effect was not observed during NIPUA implantation. Transcriptome analysis showed downregulation of cell death and cell cycle disruption-related genes, such as CDK2, CDKN1a, and GADD45a, and upregulation of autophagy and anti-tumor activity-related genes, such as MYC, PLK1, and BUB1b, in NIPUA-treated MC3T3-E1 cells compared with commercial resin-treated MC3T3-E1 cells. In conclusion, NIPUA resin showed excellent mechanical and thermal properties as well as good biocompatibility toward bone cells, muscle cells, and macrophages, suggesting its possible application in the 3D printing of customized orthopedic surgical guides.

1. Introduction

8081The orthopedic surgical guide has been widely used in the clinic to increase surgical accuracy and reduce surgery time, trauma, pain, and swelling^[1,2]. Traditional polymers such as acrylonitrile butadiene styrene (ABS), polylactide (PLA), and polyethylene glycol (PEG) are widely used in the field of three-dimensional (3D) printing of biomedical materials^[3-6]. However, these materials-based orthopedic surgical guides have various disadvantages, such as poor accuracy, easy deformation, cytotoxicity, and high cost^[2,7]. Therefore, novel, cost-effective polymers with the required mechanical properties and excellent biocompatibility are in high demand for the 3D printing of orthopedic surgical guides.

Photosensitive resins initiated by polyurethane are commonly used in the 3D printing of surgical guides^[8,9]. The excellent mechanical strength, biocompatibility, flexibility, and hydrophobicity of polyurethane are suitable for medical device applications^[10,11]. The reaction of polyols with isocyanates produces polyurethanes. The reaction of amines with highly toxic phosgene compounds produces isocyanates^[12]. These toxic residual compounds continue to leach out during surgery, which seriously threatens the patient’s health^[13]. Despite recent advances in reducing such toxicity, toxic phosgenation of amine-terminated lysine esters-produced polyisocyanates are still the precursors of polyurethanes. The unreacted polyisocyanates remain in the final polymer as a toxic substance. To avoid these challenges, nontoxic methods to produce isocyanate-free polyurethanes should be developed.

Non-isocyanate polyurethane (NIPU) is a new type of polyurethane with a similar structure to traditional polyurethane. There are four main synthetic pathways: condensation polymerization, rearrangement reaction, ring-opening polymerization, and polyaddition polymerization. The first three methods mostly use toxic raw materials, produce by-products, and have harsh reaction conditions. Cyclic carbonates (CCs) and polyfunctional amine polyaddition is the common way to obtain NIPU. During this process, the primary and secondary –OH groups are formed alongside urethane linkages. This method completely avoids the usage of isocyanates in the synthesis process and eliminates the use of hazardous chemical compounds during synthesis^[14,15]. However, the curing process makes hybrid resins hard and brittle, resulting in poor mechanical properties, which limits the application of NIPU in the 3D printing of orthopedic surgical guides^[16,17]. Acrylation modulates the mechanical properties of NIPU. Polyethylene glycol (PEG), a commonly used biomedical material, is often chemically modified with acrylate groups to form polyethylene glycol diacrylate (PEGDA) with photopolymerization properties^[18]. PEGDA has good flexibility and suitable polarity due to its molecular chain and is well-compatible with acrylic resins^[19,20]. However, the use of PEDGA for the acrylation of NIPU to improve mechanical properties for biomedical applications has not been reported yet.

In this study, we synthesized NIPU by the ringopening reaction of propylene carbonate and isophorone diamine and modified it using methacryloyl chloride. This synthetic route is environmentally friendly and shortens the synthesis time of NIPU. The main advantage over the traditional NIPU synthesis route is the avoidance of the toxic isocyanate monomer. As for the synthetic route without isocyanate monomers, the advantage is the use of a six-membered ring of amines for the reaction, which adds mechanical strength and intermolecular forces to the NIPU molecular chain. Meanwhile, non-isocyanate polyurethane acrylate (NIPUA) was obtained by adding PEGDA to improve the mechanical properties. The biocompatibility of NIPUA and commercial resins to bone cells, muscle cells, and macrophages were compared to investigate the possible application of NIPUA in the 3D printing of orthopedic surgical guides.

2. Materials and methods

2.1. Materials

Propylene carbonate (PC, 99.7%), isophorone diamine (IPDA, 99%), n-Hexane (97%), dichloromethane (99.5%), anhydrous magnesium sulfate, triethylene glycol dimethacrylate (TEGDMA, 99%), polyethylene glycol diacrylate (PEGDA), phenothiazine (PTZ, 98%), and 2-(Dimethylamino) ethyl-methacrylate (DMAEMA, 99%) were obtained from Shanghai Aladdin Bio- Chem Technology Company (China). Camphorquinon (CQ, 98%) was purchased from Shanghai Yuanye BioTechnology Company (China). Triethylamine (TEA, 99%) and methacryloyl chloride (MAC, 95%) were obtained from Shanghai Macklin Bio-Chem Technology Company (China). Sodium chloride (99.5%) and sodium bicarbonate were purchased from Tianjin chemical reagent factory (China). Commercial resin solution with epoxybased resin as the main component (Trans, White) was purchased from Stratasys company (USA). Triethylamine and dichloromethane were dried over 4A molecular sieves before use.

2.2. Synthesis of non-isocyanate polyurethane methacrylate

82NIPU was synthesized from PC and IPDA by a ring-opening reaction. Propylene carbonate (30.00 g, 0.29 mol) was added into a round bottom four-neck flask (250 mL) equipped with a mechanical stirring and reflux-condenser. The mixture was heated to 120°C, followed by dropwise addition of isophorone diamine (27.52 g, 0.16 mol) and stirring for 8–10 h until the 1791 cm^-1 carbonyl group peak disappeared. The product was then cooled down, and dissolved in 100 mL of dichloromethane. After vigorous stirring for 1.5 h, 300 mL of n-Hexane was added to extract the product. The white precipitate was washed with n-Hexane to completely remove the byproduct of ammonium and unreacted triethylamine. The resulting mixture was further dried under vacuum at 60°C to remove n-Hexane.

The schematic diagram of the preparation of 3D printing photosensitive resin is shown in Figure 1. NIPUMA was synthesized from NIPU and MAC by a ring acylation reaction. NIPU prepolymer (60.00 g, 0.16 mol) and triethylamine (36.65 g, 0.36 mol) were dissolved in 200 mL of anhydrous dichloromethane and cooled to 0°C in an ice bath. A solution of acryloyl chloride (38.71 g, 0.37 mol) in 100 mL of anhydrous dichloromethane was added dropwise with stirring. This chemical reaction was allowed to warm until its temperature was equivalent to room temperature, and the triethylamine hydrochloride salts were filtered off after 12 h. Then saturated sodium bicarbonate solution was added to get two phase-separated mixture. The product at the bottom layer was collected and washed with 400 mL brine and 400 mL distilled water. The PTZ (0.05 wt.%) was added as a free radical inhibitor, then rotary evaporation removed the organic solvent to obtain NIPUMA (yield: 93%).

Figure 1.

Schematic diagram of the preparation of NIPUA resin and the 3D printing procedure.

2.3. Polymerization and 3D printing of NIPUA

NIPUA solution including NIPUMA monomer, TEGDA, PEGDA, 1 wt.% CQ photoinitiator was stirred at 50°C, 400 rpm for 15 min and then printed by LCD printing technology (LD-002H, CREALITY, China, 470 nm). The printed model structure was further drawn using a computer-assisted design (CAD) software. The NIPUA formulation is shown in Table 1. Commercial resin (trans and white) was also printed in the same way.

Table 1. NIPUA formula.

Sample	NIPUMA (wt.%)	PEGDA (wt.%)	TEGDMA (wt.%)	PI (wt.%)	Viscosity (mPa·s)
PEGDA-0	65	0	35	1	183.0
PEGDA-4	61	4	35	1	295.5
PEGDA-8	57	8	35	1	230.7
PEGDA-12	53	12	35	1	175.5
PEGDA-16	49	16	35	1	186.9
PEGDA-20	45	20	35	1	176.1
PEGDA-24	41	24	35	1	138.0

2.4. Characterization of physical property

2.4.1. FITR characterization

The transition of functional groups such as carbonyl, hydroxyl, and alkene in FTIR characterize the thermal ring-opening with polyamines, as well as the acylation. The transmission spectra in a region of 500–4000 cm^-1 were obtained in an infrared spectrometer (Thermo Scientific Nicolet iS50, USA).

2.4.2. NMR characterization

The ¹H nuclear magnetic resonance (NMR) spectra were determined by a Bruker AV400MHz NMR spectrometer (Bruker, Karlsruhe, Germany). NIPU and NIPUA were dissolved in dimethylsulfoxide (DMSO) and were injected into NMR sample tubes (5 mm diameter).

2.4.3. UV absorption analysis

TU-1901 visible light photometer was used to test the ultraviolet (UV) absorption of NIPUMA. NIPUMA 83was dissolved in dichloromethane and further diluted to different concentrations. The absorption beam of the product was measured with a range of 190–400 nm.

2.4.4. Light curing time test

The complete curing time of the resin was tested with UV light accessories (Anton Paar, Austria). The UV light power (465 nm) was 8.0 mW/cm² with a test thickness of 0.2 mm.

2.4.5. Characterization of mechanical properties

The tensile strength and flexural strength were conducted according to the standard of ASTM D638 and ASTM D790, respectively. Instron 5967 universal testing machine (USA) was used for testing the mechanical properties. The test temperature was 25°C.

2.4.6. Heat deflection temperature characterization

The heat deflection temperature of the resins was analyzed on an LJ-300B thermal deformation machine (USA) according to ASTM D648 standard. The load was 0.45 MPa, and the heating rate was 120°C.

2.4.7. Thermogravimetric analysis (TGA) characterization

The thermostability of samples was tested in a TGA instrument (TGA550, TA instrument, USA) in a temperature range of 30°C–600°C and 10°C/min heating rate in an N₂ atmosphere.

2.4.8. DMA characterization

Dynamic mechanical analysis (DMA) instrument (DMTA, Q800 TA Instrument, USA) was sued to test DMA. NIPUA was printed into a 4 × 10 × 40 mm³ rectangular block and heated from -80°C to 200°C at 3°C/min heating rate under N₂ with a three-point bending model.

2.4.9. Cell culture

Murine fibroblast L929, myoblast C2C12, preosteoblast MC3T3-E1, and macrophage RAW264.7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO, USA) supplemented with 10% v/v fetal bovine serum (FBS, GIBCO, USA) at 37°C and 5% CO₂.

2.4.10. In vitro test for biocompatibility

All the resins were sterilized through immersion in 75% ethanol for 2 h and three times washing with phosphate- buffered saline (PBS). CCK-8 assay (Cell Counting Kit-8, Dojindo, Japan) was used to analyze cell viability. NIPUA, Trans, and White resins were soaked with complete medium (5% FBS+95% H-DEMEM) for 24 h. MC3T3 and C2C12 cells (1 × 10⁴ cells/well) were seeded on 96- well plates. The cells were incubated with the extracted supernatant for 4, 12, and 24 h. At each time point, the cells were incubated with CCK-8 reagent for 1.5 h. A microplate reader (Sunrise, TECAN, Austria) was used to measure the absorbance at 450 nm.

Resins (φ 20 cm) were put into 12-well plates, and cells were seeded (5 × 10⁴ cells per well). After 12 h of culture and fixation, DAPI (Sigma-Aldrich, USA) was used to stain the nuclei, and TRITC-Phalloidin (Solarbio, China) was used to stain the cytoskeletons of the cells. A confocal laser scanning microscopy (CLSM; TCS SP8, Leica, Germany) was used to take the fluorescence images.

2.4.11. Genes expression of inflammatory factors

The expression of inflammatory markers was analyzed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Briefly, RAW264.7 cells were seeded in 6-well culture plates (2 × 10⁵ cells/well). After culturing for 24 h, extracts from NIPUA, Trans, and White groups were used to treat cells for 24 h. Escherichia coli lipopolysaccharide (LPS, 1 ng/mL) was added as a positive control for inflammation induction. Total RNA was extracted by Trizol reagent (Invitrogen, USA) and transcribed into cDNA using RT Reagent Kit (TAKARA, Japan) according to manufacturers’ instructions. The primers are listed in Table S1 ^{(2.1MB, pdf)} (in Supplementary File).

2.4.12. RNA sequencing

MC3T3-E1 cells were cultured in extracted solution for 4 h. Three replicates were employed in each group for bulk RNA sequencing. The library preparation, sequencing, and analysis were completed by the ShangHai Origin Gene Company (China)

2.4.13. Animal study

84The animal study was approved by the committee of the laboratory animal center at Guangdong Huawei Testing Co, LTD (No.202209004). NIPUA, Trans, and White resins were respectively printed into 0.5 × 0.2 × 0.2 cm³ sheets and implanted into the back of FVB mice (six per group). Muscle tissues were collected after implantation for 7 days. Tissues were fixed and paraffin-embedded. The tissue sections were stained with hematoxylin & eosin (H&E). Tissue sections were observed under a Nikon microscope (Nikon, Tokyo, Japan).

2.5. Statistical analysis

The data were analyzed by GraphPad Prism 8. Data are presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed to determine statistically significant differences (p < 0.05).

3. Results and discussion

3.1. Synthesis and characterization of NIPUMA

The transformation of functional groups monitored by FTIR indicates the reactions between propylene carbonate and isophorone diamine (Figure 2A). During ring-opening polymerization, the peak areas of carbonyl at 1791 cm^-1 for propylene carbonate were significantly decreased. A new peak that appeared at 1698 cm^-1 indicates that carbonyl has been consumed through the ring opening reaction^[21]. A new broad peak that appeared at 3000–3500 cm^-1 is attributed to the generation of a new hydroxyl group. A peak that appeared in NIPU at 3323 cm^-1 was resulted from the stretching vibration absorption peak of the secondary amine (-NH) connected to the ester bond. A single peak between 3300 cm^-1 and 3500 cm^-1 was found, indicating that there is no residual IPDA monomer in the product. The characteristic absorption peak of in-plane deformation and expansion vibration at 1636 cm^-1 is attributed to the terminal olefin C=C in NIPUMA. The 946 cm^-1 and 814 cm^-1 peaks are associated with the C-H bond of monosubstituted alkene in the methacrylate appeared with a new C=C bond formation generated by acylation.

Figure 2.

Synthesis and characterization of NIPUA. (A) FTIR spectrum, (B) ¹HNMR spectrum (400 MHZ, DMSO-d₆, 25°C), (C) mass spectrum, and (D) UV–visible absorption spectroscopy.

The chemical shift of different hydrogen atoms was further confirmed by NMR (Figure 2B). In particular, the hydroxyl absorption peak disappeared at 4.5–4.8 ppm and the C=C absorption peak appeared at 5.6–6.3 ppm, which indicated the synthesis of NIPUMA-prepolymer. ¹HNMR (DMSO-d₆, 25°C) was as follows: δ (ppm)=0.79 (s, 3H, CH₃), 0.86 (s, 6H, C(CH₃)₂), 0.92 (d, J = 11.8 Hz, 3H, CH₃), 0.97 (d, J = 11.2 Hz, 3H, CH₃), 1.07 (s, 2H, CH₂), 1.2085 (d, J = 6.5 Hz, 2H, CH₂), 1.43 (d, J = 12.3 Hz, 2H, CH₂), 1.87 (s, 3H, CH₃C=C), 1.94 (s, 3H, CH₃C=C), 2.72 (s, 2H, CH₂NH), 3.02 (m, 1H, CHNH), 3.57 (d, J = 7.7 Hz, 2H, CH₂O), 4.07 (m, 1H, CHO), 4.19 (m, 1H, CHO), 4.96 (d, J = 10.0 Hz, 2H, CH₂O), 5.67 and 6.02 (2s, CH2=C(CH₃), 2H), and 5.76 and 6.25 (2s, CH₂C=C(CH₃), 2H).

Furthermore, the mass spectrum of NIPUMA m/z = 510.8 (C₂₆H₄₂N₂O₈, Calcd. 510.29) indicated the synthesis of the NIPUMA monomer (Figure 1C). In Figure 1D, the synthesized NIPUMA showed a strong and broad peak at 250 nm with a higher peak, which is the characteristic absorption peak of the acrylate group. These results demonstrated that the double bond is successfully grafted on the NIPU molecule, and the acrylic products were obtained.

3.2. Light curing time analysis

The curing time of photosensitive resin under different PEGDA content (0 wt.%–24 wt.%) is shown in Figure S1 ^{(2.1MB, pdf)} (in Supplementary File). It can be seen from the figure that the curing time of all photosensitive resins was between 160 and 200 s when they reached the maximum curing modulus.

3.3. Mechanical properties of NIPUA

The tensile strength of NIPUA was gradually enhanced from 27.74 MPa to 63.93 MPa with increasing PEGDAcontent (Figure 3A). The elongation at the breaking point of the resin was increased from 11.9% to 59.2% (Figure 3A). The tensile modulus of the resin was 2706 MPa when the resin did not contain PEGDA. The Young’s modulus of resins was changed, and the optimum value existed at the PEGDA content of 12 wt.% (Figure 3B). The maximum value of bending strength was 90.78 MPa at the PEGDA content of 12 wt.% (Figure 3C). PEGDA was the long-chain molecule in the resin, resulting in a stronger net structure and higher bending properties. When the PEGDA content was more than 12 wt.%, both the bending strength and tensile strength were reduced, which were caused by incomplete curing during the subsequent process. Furthermore, the bending modulus of the photosensitive resin was slowly decreased with increasing PEGDA content (Figure 3D). No significant change in bending modulus was observed at the PEGDA content of 12 wt.%.

Figure 3.

Mechanical properties of NIPUA. (A) Tensile strength, (B) strength modulus, (C) flexural strength, (D) flexural modulus, (E) TGA curves, and (F) DTG curves.

Generally, the highest tanδ indicates glass transition temperature (T_g), symmetrical and narrow tanδ peaks show the homogeneity of the material^[22]. A loss factor tanδ showed homogenous nature of NIPUA. The storage modulus E’ measured the rigidity. The storage moduli of PEGDA-0, PEGDA-4, PEGDA-12, and PEGDA-16 were higher than those of PEGDA-8, PEGDA-20, and PEGDA-24, which demonstrated the importance of crosslinking degree in storage modulus. (Figure 3E) The dynamic E’ curve demonstrated the trend of the material E’ with temperature, which was a visual indication of the stiffness of NIPUA. As shown in Figure 3F, the E’ of NIPUA was above 3000 MPa after light-curing, with a general trend toward a sequential decrease. According to Table S2 ^{(2.1MB, pdf)} (in Supplementary File), the PEGDA-16 group showed the highest crosslinking density, this result was per the TGA result. Suitable crosslinking can increase the number of effective chains, but when the crosslinking density is too86 large, the molecular weight between the joints is small, and the fragment heat transfer and stress transfer ability is reduced. The number of effective chains and chain support inhomogeneity become larger with the crosslinking density. Despite the PEGDA-16 having higher crosslinking density, PEGDA-12 still has the best mechanical properties.

3.4. Thermal properties of NIPUA

The heat deflection temperature is the limit for the application of photosensitive resins. As shown in Figure 4A, PEGDA content influenced the heat deflection temperature of the photosensitive resin, with a variation range from 66.9°C to 79.5°C. The maximum temperature was reached when the PEGDA content was 12 wt.%. These data indicated that the resin did not deform at room temperature.

Figure 4.

Thermal properties of NIPUA. (A) Heat deflection temperature, (B) thermal stability, and (C) differential thermal gravity.

TGA characterized the internal structure and thermal properties of NIPUA (Figure 4B). The 5% mass decomposition temperature was significantly enhanced with the increase of PEGDA content. The highest temperature was 253.92°C. This may have resulted from more hydrogen bonds formed between the different N atoms^[23]. Furthermore, according to differential thermal gravity (DTG) curves (Figure 4C, Table S3 ^{(2.1MB, pdf)} in Supplementary File), there was only one maximum decomposition rate at 410°C, which illustrates one-step thermal degradation process^[21]. The maximum decomposition rate was slightly enhanced as PEGDA content increases, which may be resulted from the lower bond dissociation energy of C-O (330 kJ/mol) than that of C-N (337.7 kJ/mol)^[24].

3.5. Hardness and impact resistance of NIPUA

The hardness and notched impact strength properties of NIPUA was shown in Table 2. The hardness of the resin was decreased from 84 D to 81 D after PEGDA addition. There is a slight difference between hardness with different PEGDA contents. PEGDA is a flexible molecule and could increase the toughness of the resin and decrease its hardness. The notched impact strength of NIPUA was slightly reduced after PEGDA addition, which was resulted from incomplete curing of the resin and reduced impact strength by high PEGDA content.

Table 2. Resin hardness and impact strength performance.

Sample	Hardness (Shore D)	Notched impact strength (kJ/m2)
PEGDA-0	84 (±2)	3.56 (±0.33)
PEGDA-4	80 (±1)	2.97 (±0.35)
PEGDA-8	81 (±1)	3.13 (±0.49)
PEGDA-12	81 (±1)	3.01 (±0.39)
PEGDA-16	81 (±1)	3.27 (±0.55)
PEGDA-20	83 (±1)	3.74 (±0.09)
PEGDA-24	79 (±2)	2.62 (±0.55)

3.6. Corrosion resistance of NIPUA

The acid and alkaline resistance morphology of NIPUA is shown in Figure S2A ^{(2.1MB, pdf)} . The surface morphology of the resin was changed in 5 wt.% H₂SO₄ solution. The surface of PEGDA-4, PEGDA-8, and PEGDA-12 did not show significant morphology changes, indicating that the PEGDA addition enhanced the resistance against acid corrosion (Figure S2A ^{(2.1MB, pdf)} ). Alkaline erosion leads to black87 spots on the resin’s surface. NIPUA showed the best alkali corrosion resistance. The black spots that appeared on the surface of PEGDA-12 were smaller in size and less in number, which indicates that the NIPUA has better alkali corrosion resistance when the PEGDA content is 12 wt.%.

3.7. Dimensional accuracy test

The different sizes of microporous structures ranging from 500 μm to 2 mm were designed and printed to test the dimensional accuracy of 3D printing. The test results are shown in Figure S3 ^{(2.1MB, pdf)} . The experimental results show that a microporous structure below 500 μm can be formed. In addition, the smaller is the size of microporous structures in different shapes, the greater is the impact on the size error and shape integrity. This is related to material refractive index, equipment resolution, and other factors.

3.8. Hemocompatibility of NIPUA

The hemolysis test is used to evaluate the hemocompatibility of biomaterials. According to ISO 10993-4, biomaterials with a hemolysis rate <5% are considered safe to use as blood-contacting materials. In the positive control group, blood cell membrane was disrupted, resulting in hemolysis. The addition of PBS as negative control did not result in hemolysis. The hemolysis rate of the resins with different PEGDA contents was less than 0.5% (Figure 5A, 5B), indicating that the NIPUA has excellent hemocompatibility.

Figure 5.

Hemocompatibility test of NIPUA. (A) Hemolytic phenomenon and (B) hemolysis rate. (n = 4, ***p < 0.001, mean ± SD.)

3.9. Biocompatibility of NIPUA to bone and muscle cells

According to the physiochemical evaluation, PEGDA-12 in NIPUA was used for subsequent experiments. The orthopedic surgical guide has unavoidable contact with bone and muscle cells during surgery. The cytocompatibility of NIPUA resin toward MC3T3-E1 bone cells and C2C12 muscle cells was compared with commercial resin by cell proliferation assay and live-dead assay. Trans and White commercial resin were extremely cytotoxic to MC3T3-E1 and C2C12 cells. However, the NIPUA was cytocompatible with MC3T3-E1 and C2C12 cells as indicated by the absence of inhibitory effect on their proliferation at different time points of the culture (Figure 6C and D). In addition, the different formulations of NIPUA showed less cell toxicity to L929 cells (Figure S4A–C ^{(2.1MB, pdf)} ). These data demonstrated that NIPUA showed excellent biocompatibility.

Figure 6.

Biocompatibility of NIPUA. Cell viability of MC3T3-E1 (A) and C2C12 cells (B). Live-dead staining of MC3T3-E1 (C) and C2C12 cells (D). (n = 6, ***p < 0.001, mean ± SD.)

3.10. Evaluation of the inflammatory effect of NIPUA in vivo and in vitro

The biosafety of NIPUA was further evaluated in vivo. The commercial resin and NIPUA were implanted into the back muscles of mice. After 7 days, the resins attached with muscle tissues were collected and subjected to H&E staining. As shown in Figure 6A, the Trans and White groups caused severe muscular toxicity reactions. It demonstrated severe transverse muscle atrophy with varying degrees of hemorrhagic spots on the muscle edges, varying thickness of myocytes, thin muscle bundles, and partial cell necrosis^[25,26]. The nuclei were densely packed, with a large number of brown lipofuscin granules at the ends of the nuclei (black arrows). No significant toxicological reactions were observed in the NIPUA group. Inflammatory reactions were present in all three groups, which were caused by the rejection of the organism to the allogeneic graft contact. However, it is also obvious that the inflammatory cell infiltration was more severe in the Trans and White groups than in the NIPUA group. This indicated that NIPUA has good in vivo biocompatibility and is not prone to toxic side effects.

It was demonstrated that macrophage M1 and M2 polarization are important during the inflammatory responses. We further detected the inflammatory effect of NIPUA resins and commercial resins on Raw264.7 cells. LPS was administered as positive control. Pro-inflammatory factors including M1 markers IL-6, IL-1β, and TNF-α were significantly increased in the Trans group and White group compared to the NIPUA group (Figure 7B). While the anti-inflammatory factors including M2 markers IL-10 and TGF-β1 were significantly reduced in the commercial resins group, NIPUA did not significantly affect the gene expressions of inflammatory factors.

Figure 7.

(A) H&E staining of muscle tissue in contact with resins for 7 days. (B) mRNA expression of inflammatory markers in macrophages after contact with resins for 4 h. (n = 6, **p < 0.01, ***p < 0.001, mean ± SD.)

3.11. Preliminary investigation of the biosafety mechanism of NIPUA

RNA sequencing was used to further explore the regulatory mechanism of commercial resins in the cell cycle and apoptosis of MCET3-E1 cells. According to differentially expressed genes (DEGs) in Figure 8A, 1400 genes were upregulated while 1157 genes were downregulated in the NIPU group compared with the Trans group, whereas 901 genes were upregulated and 1234 genes were downregulated in the NIPU group compared to the White group. Furthermore, several DEGs were enriched in the cell according to the KEGG analysis (Figure 8B).

Figure 8.

Transcriptome analysis of MC3T3-E1 cells. (A) Volcano plot showing differentially expressed genes (DEGs). (B) The KEGG analysis of DEGs. (C) The quantitative analysis of DEGs related to KEGG pathways. (n = 3, ***p < 0.001, mean ± SD.)

88The genes related to the regulation of the cell cycle are presented in Figure 8C. In the NIPUA group, the CDK2 gene expression level was lower compared with the Trans or White group. CDK2, a negative regulator of the cell cycle, is activated by DNA damage or abnormal replication at cell cycle checkpoints, resulting in temporary cell cycle arrest^[27,28]. During apoptosis, CDK2 activity is upregulated and activated, resulting in the transformation of anti- apoptotic protein Bcl-xL to pro-apoptotic protein with a function similar to that of Bax/Bak^[29]. This demonstrated that compared to NIPUA, the commercial resins induce severe cell damage, which leads to cell cycle arrest.

The expression level of GADD45a in NIPUA group was downregulated compared with the Trans or White group. GADD45a induces apoptosis and DNA repair by controlling the cell cycle G2-M checkpoint^[30,31]. GADD45a plays a role in the process of DNA demethylation^[32] and8990plays different roles through p53, JNK, and p38 signaling pathways^[33,34]. When DNA is damaged, GADD45a upregulation promotes stem cell apoptosis^[35] Furthermore, CDKN1a was downregulated in NIPUA compared with the Trans or White group. CDKN1a is the most widely known cell cycle inhibitory protein with the broadest kinase activity^[36]. It is one of the most important downstream genes of the p53 gene and is a major component in the regulation of cell cycle arrest after DNA damage^[37]. Our results indicate that compared to NIPUA, the commercial resins are highly toxic, which affect the cell cycle and cause DNA damage.

In addition, MYC expression level was upregulated in NIPUA compared with the Trans or White group. There is positive feedback between MYC and Wnt signaling pathways^[38]. Several ligand-membrane receptor progrowth signaling pathways pass through MYC, such as Notch and EGFR^[39-41]. Low expression of MYC in commercial resins may activate cell cycle checkpoints, causing cell growth arrest and death or even carcinogenesis. The expression of PLK1 was upregulated in NIPUA compared with the Trans or White group. PLK1 plays an important role in mitosis, participating in centrosome migration and spindle assembly^[42,43]. There is a negative feedback regulation between a Fas-associated protein with the death domain (FADD) and PLK1. Inhibition of PLK1 activity impairs autophagy and weakens the interaction of FADD with downstream signaling proteins such as caspase-8^[44]. The upregulation of PLK1 levels in the NIPUA group reduced malignant cell proliferation and inhibited the possibility of carcinogenesis. BUB1b is involved in regulating the spindle assembly checkpoint (SAC)^[45]. The SAC maintains genomic stability by delaying cell division and ensuring proper chromosome segregation^[46,47]. Mutations or abnormal expressions of SAC proteins lead to the development of cancer^[48]. BUB1b directly interacts with CDC20 in the mitotic checkpoint complex, thereby inhibiting mitotic prophase^[49]. Thus, the high expression of BUB1b in the NIPUA group ensured the correct chromosome segregation and inhibited unlimited cell proliferation.

4. Conclusion

In this study, we first synthesized a low-toxic NIPU resin via the green synthesis method. We investigated the effect of different content of PEGDA on the physicochemical properties of the photosensitive resin. A non-isocyanate polyurethane NIPUA with strong mechanical properties, high thermal stability, and good biosafety was obtained. When the content of PEGDA was 12 wt.%, NIPUA reached its peak in tensile and flexural strength. In addition, the NIPUA photosensitive resin also showed good thermal stability and did not deform at 69°C. Most importantly, we evaluated the biocompatibility of NIPUA by comparing it with commercial resins. The commercial resins demonstrated more pronounced toxicity and may pose a safety hazard during implantation as a surgical guide, whereas the NIPUA resin has good biocompatibility in vitro and in vivo. This paper presents an ingenious method for the fabrication of medical-grade NIPUA from renewable materials, broadening the possibility of NIPUA photosensitive resin as a 3D bioprinting material and providing important direction for its clinical application.

Funding

This work was supported by Guangdong Basic and Applied Basic Research Foundation (2020B1515120075), Key Research and Development Program of Guangzhou (202007020002), and Dongguan Sci-tech Commissioner Program (20221800500032).

Conflict of interest

The authors declare no conflicts of interest.

Author contributions

Conceptualization: Zhichao Zheng, Janak L. Pathak Formal analysis: Yan Wang, Zhichao Zheng, Weiwei Feng Funding acquisition: Huade Zheng

Investigation: Yan Wang, Weicong Wu, Chuangang Yang Methodology: Lihong Wu

Resources: Weiwei Feng, Lihong Wu Project administration: Huade Zheng Supervision: Lihong Wu, Huade Zheng Visualization: Yan Wang Writing – original draft: Yan Wang

Writing – review & editing: Janak L. Pathak, Huade Zheng

Ethics approval and consent to participate

All animal experiments were approved by the committee of the laboratory animal center at Guangdong Huawei Testing Co, LTD (No.202209004).

Consent for publication

Not applicable.

Availability of data

The data will be provided upon reasonable request.