Digital light 3D printing of artificial bone with star-shaped polycaprolactone-based polyurethane acrylate

Advanced medical materials and manufacturing technologies are highly in demand in artificial bones. Herein, a four-arm star-shaped polycaprolactone polyurethane acrylate (FPCLA) was designed and synthesized. The photosensitive character of FPCLA contributed to the rapid prototyping and personalized customization under digital light processing (DLP) 3D printing technology. The FPCLA was prepared by introducing unsaturated double bonds into polycaprolactone tetraethyl alcohol (PCLT). We characterized the physico-chemical properties of the material through FTIR, H-NMR, GPC, DSC and SEM. Cell behaviors on material were observed in vitro. In addition, we employed a DLP 3D printer to evaluate the feasibility of FPCLA to fabricate artificial bone model. The photocuring star polycaprolactone was confirmed in detail by detection method. SEM analyses demonstrated that FPCLA has good tenacity. The material can be used to fabricated artificial bone with a diameter of 3.02 mm at its narrowest by DLP 3D printing technology. The cell survival rates of CCK-8 and Live/Dead fluorescence staining experiments were both above 90%, which indicated safety and feasibility of such new-generation artificial bone made of synthetic polymers.


Introduction
Bone is critical organ of the human, which acts as a supportive structure for the human body, protects the interior tissues and gives the body its basic shape.There are many conditions that can cause bone defects, such as trauma, bone tumors, infections, and congenital defects [1,2].After injury, bone tissue can generally repair itself, when bone damage is severe, bone tissue is unlikely to heal itself after injury [3].Bone scaffold is a temporary mechanical structure designed to simulate the extracellular matrix of bone tissue [4].Bone repair material is one of the key factors for successful repair of segmental bone defects and scaffold.Currently, bone repair materials can be divided into autologous bones, and bone graft substitute materials [5][6][7].Autologous bone grafting needs to cause additional surgical trauma to patients, as well as bleeding, infection and other complications.In addition, the quantity of autologous bone is limited [8].The research on artificial bone replacement materials is conducted with a focus on metallic materials such as titanium alloys [9], cobalt alloys [10], and stainless steel [11], non-degradable polymer materials represented by ultra-high molecular weight polyethylene (UHMWPE), and degradable polymer materials represented by poly-lactic acid (PLA) [12,13].Moreover, degradable polymer materials exhibit excellent biocompatibility and osteogenesis, they increasingly became the most promising bone repair materials today [14].Thus, an increasing number of researchers have invested in the field of degradable polymer materials.And these works mainly revolve around the improvement of repair materials and the fabrication process of artificial bones.
Polycaprolactone (PCL) is a biodegradable aliphatic thermoplastic polyester with a high crystal degree, high flexibility, and high biodegradability [15,16].The US Food and Drug Administration (FDA) has approved PCL for use in biomedicine [17].Compared with most degradable materials such as poly-lactic acid (PLA) and cellulose, PCL has better toughness and degradability [18].Amsden [19], Sharma [20] and others that have worked on star shaped caprolactones and mixtures with other monomers.The study results verified that by adjusting the initial molecular weight and the proportion of monomer of the prepolymer, the physical properties of the material can be changed.Among the polymer materials, star-shaped polymers with the same molecular mass have higher segment density, lower fluid viscosity, and better characteristics as compared to linear polymers [21].
In recent years, the research and application of 3D printing technology in bone tissue engineering have greatly promoted bone defect regeneration and repair.Chakraborty J et al [22] used direct write technique to prepare the rat tibial defect scaffold model using hydroxyapatite (HAP); Zheng XX et al [23] fabricated porous titanium scaffolds for repairing mandible combined with selective laser melting (SLM) technology using titanium metal as raw material.With the development of materials and manufacturing technology, artificial bone scaffolds have made significant progress in biocompatibility and personalized customization.However, there are still deficiencies in mechanical performance and forming accuracy.Ultraviolet (UV) [24] curing facilitates the instant polymerization of unsaturated double bonds in oligomers or monomers under UV irradiation by inducing crosslinking.With the progress of imaging techniques, digital light procession has emerged.Compared to other 3D printing technologies, DLP 3D printing has the advantages of high resolution and high surface quality [25].So far, no relevant literature on the use of star shaped polycaprolactone combined with DLP 3D printing technology in bone defect repair has been reported.
In this paper, as indicated in figure 1, UV curable four-arm star shaped polycaprolactone was prepared by introducing unsaturated double bonds into its tetraol, and the chemical structure was characterized in detail.Further, DLP 3D printing was employed to probe the feasibility of preparing artificial bone model for repairing bone defects.On this basis, the biocompatibility evaluation of the material was explored.

Preparation of FPCLA
Before the reaction, the PCLT was kept in a vacuum drying oven at 105 °C for 2 h to remove the small amount of water contained in the reagent.Firstly, 15.56 g IPDI (0.07 mol) and 0.148 g stannous iso caprylate (0.6%) were added into a straight three-necked flask equipped with a mechanical stirrer, thermometers, and a constant pressure burette.The mixture was heated to 30 °C and added dropwise with 9.11 g HEMA (0.07 mol).Next, 40.00 g PCLT (0.005 mol) was added into a straight three-necked flask when the content of NCO in the reaction system reached the theoretical value as monitored by the din-butylamine method.The solution was stirred at 30 °C, and the product of the first step was added to the flask.After the dropwise addition of the product was completed, the above mixture was reacted at 80 °C until the absorption peak of NCO disappeared, and the transparent viscous liquid was collected.During the second stage of the reaction, the change in NCO content was monitored by infrared spectroscopy.

Characterization and mechanical property 2.3.1. Fourier-transform infrared spectroscopy (FTIR)
Blank tablets were coated with 2 mg of the reaction products.Thereafter, infrared spectroscopy was performed using a Nicolet iS20 Fourier transform infrared spectrometer (Thermo Fisher Scientific).The scanning times were 32, the resolution was 4 cm −1 , and the scanning range was 400-4000 cm −1 .

Proton nuclear magnetic resonance spectroscopy (H-NMR)
The sample composition and structure were analyzed by the AVANCE NEO 400 M NMR spectrometer (Bruker, Germany).The sample was dissolved in deuterodimethylsulfoxide, and tetramethylsilane (TMS) was used as the internal standard.

Gel permeation chromatography (GPC)
A PL-GPC220 gel permeation chromatograph (Agilent, United States) was used to measure the relative molecular mass and distribution of the sample.The mobile phase was tetrahydrofuran, the test temperature was 40 °C, the sample concentration was 5 mg ml −1 , the injection volume was 1 ml, the flow rate was 1.0 ml min −1 , and the reference substance was polystyrene.

Differential scanning calorimetry (DSC)
Using a DSC2500 differential scanning calorimeter (TA Company, United States), 5-10 mg vacuum-dried polyurethane acrylate samples were heated in a nitrogen atmosphere from 40 °C to 200 °C at the rate of 10 °C min −1 for 5 min to delete thermal history.The samples were then cooled from 200 °C to −50 °C at the rate of 10 °C min −1 , which was held for 5 min.The samples were again heated from −50 °C to 200 °C at 10 °C min −1 to obtain a heating curve.The glass transition temperature (Tg) of FPCLA was then analyzed.
2.4.DLP 3D printing and testing 2.4.1.Model printing TPO photoinitiator with a mass ratio of 1% was added to the prepared polyurethane acrylate prepolymer.Next, the above mixture was heated and stirred at 60 °C for 0.5 h to obtain photosensitive resin materials for photocuring 3D printing.The model of human femurs was designed by SolidWorks software (Dassault Systems Solidworks Corp., USA) and the construct models were then sliced into images using Photon Workshop software (Anycubic Technology Co., Ltd., China).The commercial DLP 3D printer (Anycubic Photon Ultra, China) comprises a 405 nm light-emitting diode and a customized resin tray with heating functions.The printing temperature was set at 60 °C, the exposure time was 5 s, and initial exposure time was 20 s.After printing, the sample was cleaned in ethanol and then cured in a post curing machine (Anycubic Wash and Cure 2.0) for 10 min.

Scanning electron microscopy (SEM)
The printed object was broken after becoming brittle in liquid nitrogen for 1 h, and its cross-sections was intercepted.A Quorum SC7620 sputtering coating instrument was then used to spray gold for 45 s, with a gold spray rate of 10 mA.Subsequently, the ZEISS Sigma 300 scanning electron microscope (ZEISS, Germany) was used to photograph the morphology of the sample, with an acceleration voltage of 3 kV during the morphology photography, and the detector SE2 was used as a secondary electron detector.
2.5.Evaluation of cytocompatibility 2.5.1.Cell culture HUVECs were cultured in DMEM high-glucose medium containing 1% penicillin/streptomycin and 10% fetal bovine serum at 37 °C with 5% CO 2 and saturated humidity.The cells were digested with 0.25% trypsin at a cell density of 80%-90% per culture dish at 37 °C.The isolated cells were collected after centrifugation, cleaned with PBS buffers, and centrifuged again to remove the supernatant.The collected cells were re-suspended in the DMEM medium at a density of 1 × 10 5 cells per dish, and the cells were then placed in a new culture dish.The cells were kept in an incubator until the growth density was 60%-70% for subsequent experiments.

Preparation of material extract
According to the GB/T-16886 or ISO-10993 regulations, the FPCLA diaphragm and polyethylene film were cut into 10 mm × 10 mm × 2 mm square chips, and the membrane was sterilized by ethylene oxide.According to the ratio of sample surface area to leaching medium (3 cm 2 /1 ml), the material was added to a DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin.After incubating for 72 h at 37 °C, polyurethane acrylic ester and polyethylene material extract were obtained.

CCK-8 Experiment
HUVECs in the logarithmic growth stage were collected and inoculated in a 96-well plate with 4 × 10 4 cells/ well.Material extracts diluted to 5%, 10%, 15%, and 20% were used as experimental groups, while polyethylene extracts were used as the negative control group.Cells were cultured at 37 °C and 5% CO 2 for 72 h.After incubation, 100 μl CCK-8 solution was added to each well and incubated in the dark at 37 °C for 2 h.The absorbance of the samples was measured at a wavelength of 450 nm using a SPARK 10 M enzyme marker (TECAN, Switzerland).The cytotoxicity of the material was calculated as follows: Cell Survival Rate % absorbance of the experimental group absorbance of the negative control group 100% Live/Dead fluorescence staining HUVECs in logarithmic growth phase were collected and inoculated in a 24-well plate with an inoculation density of 8 × 10 4 cells/well.The cells were divided into two groups, 1000 μl diluted material extracts with concentrations of 5 mg ml −1 , 10 mg ml −1 , 15 mg ml −1 , and 20 mg ml −1 were added into each well for the experimental group, respectively.And an equal amount of complete medium was added into each well for the control group.Cells were cultured at 37 °C and 5% CO 2 for 72 h in incubator (WIGGENS WCI-180, Germany).Then, 24-well plates were washed with PBS and added 500 μl staining solution (including 0.5 μl Calcein-AM, 1 μl PI).The mixture was placed for 15 min, protected from light.Then, the live (green fluorescence) or dead (red fluorescence) cells in each group were viewed under a fluorescence microscope (Olympus FV1200, Japan).

Results and discussion
3.1.Characterization of polycaprolactone polyurethane acrylate 3.1.1.Fourier-transform infrared spectroscopy Figure 2 displays the infrared spectra of polyurethane acrylate at different reaction periods.The characteristic absorption peak of the NCO group gradually decreased and diminished over time, indicating the reaction completion of the NCO group.The green line displays the infrared spectrum of the final synthesized polycaprolactone polyurethane acrylate.In figure 2, according to reference literature [26], the O-H stretching vibration absorption peak at 3500 cm −1 was not observed, while the peak at 3360 cm −1 corresponded to the stretching vibration absorption peak of the N-H bond in -NHCO.The NCO characteristic peak at 2240-2275 cm −1 was not observed, implying that the NCO group had completely reacted.The stretching vibration absorption peak of C=O at 1724 cm −1 indicated that the carbamate bond was formed.The absorption peak at 1635 cm −1 was a C=C stretching vibration peak, while the characteristic absorption peak of the C-H bond on the C=C double bond was located at 810 cm −1 .

1 H nuclear magnetic resonance spectroscopy
Figure 3 displays the 1 H-NMR spectra of FPCLA in CDCl 3 .The acrylic double bond signal (=CH 2 ) was detected at 5.60 ppm, while 6.14 ppm was the H signal connected by N in the carbamate bond (OOC-NH-CH 2 ).Likewise, the H signal connected by N in the carbamate bond formed at the other end of IPDI was also at 5.60 ppm, indicating the presence of a carbamate bond.The signal of 4.06-4.31ppm was the carbamate and acrylate group connected to CH 2 , whereas 3.80 ppm was the methylene proton peak of the acyl group in the hydroxyethyl of the PCL tetraol backbone.Additionally, 2.32 ppm was the methylene proton peak of the ester group in the PCL backbone, while 2.92 ppm was the CH 2 connected to the amino end of carbamate.The H signal in the double-bonded C-connected methyl group (=C-CH 3 signal in methyl groups introduced by hydroxyethyl methacrylate) was 1.95 ppm, and there were two sets of multiple peaks at 1.65-1.38ppm, which included CH 2 in the non-ester group of the PCL framework and CH 2 in the IPDI backbone.The signal of H in the methyl group in the IPDI backbone was at 1.05-0.94ppm.The results were consistent with the predicted target structure.

Gel permeation chromatography
The relative molecular weight and distribution of the sample were analyzed using GPC, and the results are displayed in table 1.The molar ratio of the designed raw materials n1 (PCLT): n2 (IPDI): n3 (HEMA) was 1:4:4, which theoretically implied the complete reaction between IPDI and PCL tetraol to obtain a polyurethane prepolymer terminated with diisocyanate.The number-average molecular weight (Mn) and weight-average molecular weight (Mw) of the samples (table 1) had small deviations from the theoretical values and low dispersion coefficients.

Differential scanning calorimetry
Due to the regular molecular structure and low molecular branching of the polyurethane acrylate prepolymer, its repeating units have a strong order, leading to the crystallization of the polyurethane prepolymer into a white solid at room temperature that melted upon heating (figure 4).The glass transition temperature (Tg) of polyurethane acrylate was measured by DSC to provide a basis for its application.According to DSC analysis, the glass transition temperature (Tg) of polyurethane acrylate was 40 °C, which was lower than that of PCL (55 °C).This could be due to the reaction between HEMA and the isocyanate group to reduce the hydrogen bonds within or between molecular chains and disrupt the order between the original PCL segments [27].

DLP 3D printing 3.2.1. DLP 3D printing of femoral model
DLP 3D printing heavily depends on the viscosity of the resin [25].Compared with the linear polymer, branched polymers have lower viscosity, higher cross-linking density, and better mechanical properties [28].Thus, we choose branched PCLT and introduce unsaturated double bond to it for photocuring.In order to ensure that the photosensitive resin is still in the liquid state at room temperature, and reduce the printing viscosity of the resin, we have customized and modified the resin tray of the printer to achieve heating function (figure 5).Printing within the range of 55 to 60 °C can effectively reduce the viscosity of the resin without causing thermocuring.SolidWorks software was used to design a human femur model with the size reduction of 10-fold and printed it on DLP 3D printer (figure 6).The 3D printed artificial femur model is transparent white with a slightly rough surface.The printed femur model has a layer thickness of 0.05 mm, with a diameter of 3.02 mm at its narrowest, 9.39 mm at its distal end and a height of 50.63 mm.The overall structure of the femur model is largely consistent with the initial design structure.However, the femoral head of the model is slightly flattened, and the shaping  effect of the details needs to be further improved.The printed femur model is used for subsequent SEM experiments.At present, several scholars have used PCL as a matrix for research on bone scaffolds.However, their manufacturing processes are mostly focused on electrospun nanofibers and fused deposition modeling (FDM) [29][30][31].Yedekci et al [32] used PCL as the main raw material for bone tissue engineering, demonstrating its excellent biological performance.
We have drawn on the experience of previous research and further incorporated photocuring technology.This experiment uses DLP 3D printing technology to fabricate artificial bones, which endows the material with the advantages of DLP 3D printing for rapid customization of personalized implants and the excellent performance of star-shaped polycaprolactone.Polycaprolactone has good processability, the blending of polycaprolactone with other materials can manufacture composite materials with excellent biological activity and mechanical properties.DLP 3D printing can provide customized implants with higher printing accuracy and lower time costs and extend the applications into multidisciplinary areas.

SEM images of the 3D printed bone
The surface morphology of the printed FPCLA femur model fracture was observed by SEM.As depicted in figure 7, The surface of the fractured cross-section of the material is relatively rough with voids present, and the phenomenon of drawing into fibers was observed.There was no 'river-like crack' expansion on the fracture  surface, which indicated that the material had good malleability [33,34].No obvious pore structure was observed in the 2 μm and 20 μm cross section.The internal section of the material shows uniform distribution, indicating that the molecular has a high cross-linking density, and the molecular chains are tightly linked [35].At 200 μm and 20 μm images, there is no collapse inside it, and the fracture surface is stratified with no obvious  cracks and pits, which indicates the material is uniform and stable.The fracture showed traces of micro-cracks in the 2 μm cross section image, and 'fish scale' expansion was shown in the 20 μm image, irregular distribution of small bubbles appeared at 200 μm image, which may be caused by certain shrinkage pores after the cross-linking of the photosensitive resin.Compared with linear polycaprolactone with the same molecular weight, the fourarm star polycaprolactone had lower crystallinity, and this led to increased flexibility and kinematic ability of the chain segment for a decent strain capacity [36].

Cell counting kit-8 tests
For artificial bones, their biocompatibility are essential [37].The biological safety of PCL has already been verified.Wei PR et al [38] used polycaprolactone scaffold modified by insulin releasing PLGA nanoparticles to repair osteochondral defects.The in vivo experiments showed that after 8 and 12 weeks of implantation of insulin-PLGA/PCL scaffold into rabbit osteochondral defects, the repair effect of cartilage and subchondral bone was significant.However, the toxicity evaluation of photo-curable type PCL have rarely been reported.According to the GB/T-16886.5-2017Biological Evaluation Standards for Medical Devices, cell survival rates less than 70% indicate that the material has potential cytotoxicity.The results of the CCK-8 experiment are displayed in table 2 and figure 8.At 5%, 10%, 15%, and 20% dilution of the material extract, the cell survival rates after 72 h were all above 90%, indicating good cell compatibility of the material.
Currently, polycaprolactone is widely used in the preparation of biomaterials for drug release, bone tissue repair, and biodegradable vascular scaffolds due to its excellent biocompatibility [39][40][41][42].Polycaprolactone-based polyurethane acrylate has excellent mechanical properties and biocompatibility.The advancement of photocurable 3D printing technology highlights the great potential of the material in the field of tissue engineering [43].experimental group treated with extract was similar to that of the control group, and it was observed that the bottom of the confocal dish was covered with live cells emitting green fluorescence, indicating that HUVECs could proliferate and grow in the material extract.The light-cured four arm star shaped polycaprolactone material had no significant toxicity.

Conclusion
In this study, photosensitive polycaprolactone was used as the raw material to prepare artificial bone through DLP, giving the artificial bone the dual advantages of star shaped polycaprolactone and DLP technology.FPCLA not only has excellent mechanical properties and good biocompatibility, but also has the characteristics of high precision, high surface quality, rapid prototyping, and personalized customization of DLP 3D printing.We used FTIR, H-NMR, GPC, DSC, SEM and other methods to detect the synthesized materials, indicating that we have successfully prepared a UV curable star shaped polycaprolactone.To comply the low viscosity requirements of DLP 3D printing resin, we modified the printer to achieve heating function to reduce the viscosity of FPCLA.In addition, the cytotoxicity of the material was detected through CCK-8 and Live/Dead fluorescence staining experiments, and the results showed cell survival rate was above 90%.Altogether, the use of FPCLA can bring more choices to medicine with advanced DLP technology, showing a good prospect of organ regeneration.However, the experimental results presented in this paper may have some limitations, for example, material strength and long-term stability still need to be improved and verified.Further, biomechanical experiments and animal models are needed to validate the clinical application value of this material.

Figure 1 .
Figure 1.Schematic presentation of synthesis and fabrication of our FPCLA femur.

Figure 2 .
Figure 2. FTIR spectra of polyurethane acrylate at different reaction periods.

Figure 5 .
Figure 5. Modifications of the resin tray of the printer to achieve heating function and modified DLP 3D printer.

Figure 7 .
Figure 7. SEM images of the fracture surfaces of FPCLA.

3. 4 .
Live/Dead fluorescence staining Fluorescent images were obtained after Live/Dead staining (figure9).After the material extracts with different dilution concentrations were cultured with cells for 72 h, a small amount of red fluorescence emitted by dead cells was observed under the microscope in each group, while the full field of vision was green fluorescence emitted by living cells.The cell survival rate of all groups was above 90%, indicating that HUVECs had good cell activity in the extraction solution of the light-cured star-shaped polycaprolactone.The fluorescent intensity of

Figure 9 .
Figure 9. Live/Dead fluorescence staining of HUVEC cells at different culture in each group.

Table 1 .
Molecular weight and distribution of FPCLA.