Customized bioresin formulation for stereolithography in tissue engineering

Abstract

Recently, advancements in fabrication technology have brought a new aspect to the field of tissue engineering. By utilizing advanced techniques in 3D manufacturing and biomaterials, scientists have successfully created tissue engineering scaffolds with complex three-dimensional structures and customized chemical compositions that closely mimic the natural environment of living tissues. These methodologies show potential not only for developing therapies that restore lost tissue function but also for creating in vitro models that replicate living tissue. The current investigation involved the synthesis of methacrylated polycaprolactone (PCLMA) by incorporating methacryloyl chloride (Meth-Cl) into polycaprolactone (PCL) with a molecular weight of 80,000 Da. Afterwards, PCLMA was subjected to crosslinking with glycerol acrylate (GA) and, by utilizing Diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide (TPO) as a photoinitiator, achieved the three-dimensional (3D) printing of tissue materials using Stereolithography (SLA). Analytical techniques included nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Cell viability was investigated using Human Osteoblast (HOB) cells. The biocompatibility of glycerol acrylate (GA) crosslinked polymethacrylated polycaprolactone (PCLMA) was confirmed using cell viability experiments. Overall, the GA-crosslinked PCLMA bioresin, particularly PCLMA-8, shows promise for further use in tissue engineering applications.

Keywords

3-D printing Methacrylated Poly Caprolactone (PCLMA)tissue engineering photopolymerization stereolithograpy (SLA)

1 Introduction

Tissue engineering typically entails the integration of cells and biomaterials to fabricate tissue structures, aiming to substitute for or reinstate the normal functionalities of organs that have been impacted by disease or injury. The biomaterial scaffolds are engineered to offer mechanical reinforcement to the cells, facilitating their execution of essential tissue tasks [1 –3].

Lately, manufacturing technologies that utilize computer-aided design (CAD) have been used to create three-dimensional scaffolds with customizable properties at both the micro- and macro-scale levels. The utilization of additive 3D construction techniques presents potential benefits for tissue engineering. Significantly, the ability to separately control micro- and macro-scale characteristics enables the creation of multicellular formations that are crucial for the proper functioning of complex tissues. Additionally, the creation of vascular networks could enhance the growth of bigger tissue structures by bypassing the diffusion constraints found in traditional scaffolds. Moreover, the combination of clinical imaging data with CAD-based freeform production techniques shows potential for tailoring constructions to precisely fit the geometries of specific defects or injuries. This approach also has the potential to enable the mass manufacture of identical tissue constructions, which would be advantageous for applications in drug discovery and fundamental scientific research [4].

An important challenge in obtaining materials for tissue engineering is the choice of appropriate ingredients. The chosen material must accurately replicate the structural and functional properties of native tissue, while also meeting important requirements such as biocompatibility and non-toxicity [5 –7].

Significantly, polymers derived from polycaprolactone (PCL) and materials based on PCL have exhibited noteworthy effectiveness in replicating tissue properties. PCL, owing to its heightened mechanical strength, biocompatible characteristics, prolonged degradation kinetics, and facile manipulability, emerges as a preeminent selection among polymers for the mimicking of tissue characteristics [1, 8].

To regenerate intricate tissues that have specific biological and physicochemical requirements, it is crucial to choose the most suitable tissue engineering procedure. Gas foaming, solvent casting, electrospinning, phase separation, freeze casting, and freeze drying have been used for tissue fabrication; however, these methods do not adequately address the structural integrity, size, porosity, and interconnectivity of the void spaces. The use of additive manufacturing, also known as 3D printing, enables the fabrication of intricate architectures with adjustable layers and microstructures and reduces manufacturing expenses. 3D-printed structures not only facilitate cellular proliferation, transportation of nutrients, elimination of waste, and dispersion of growth factors with tissue mimicking abilities but also they assist in the formation of the internal vascular network [9 –11].

The choice of the most suitable 3D printing technology for tissue creation depends on factors like as production speed, resolution capabilities, and the properties of the raw materials. The main cell-free 3D printing methods utilized in medical applications are Selective Laser Sintering (SLS), Stereolithography (SLA), and Fused Deposition Modeling (FDM) [6, 12]. These approaches have seen significant progress, allowing for accurate fabrication using computer-aided designs and medical imaging data. Stereolithography is particularly notable for its capacity to create structures of various sizes, spanning from centimeters to submicron dimensions. It demonstrates much higher accuracy and clarity in comparison to other Solid Freeform Fabrication (SFF) methods. Most traditional fabrication techniques usually produce features that are between 50–200μm in size. However, there are multiple stereolithography systems currently on the market that can manufacture structures of several cubic centimeters with a remarkable precision of 20μm. These advancements have created opportunities for the creation of models tailored to individual patients, complex surgical parts, and customized items such as hearing aids and implants supported by molds, all within the field of biomedicine [2 , 12–14].

Although a significant amount of work has been done on this subject [14 –16], there is a noticeable lack of information about efforts to modify PCL polymers to photopolymerize and subsequently use, which is an easier method than synthesizing photopolymerizable PCL-derived polymers from their monomers. This particular study involved the synthesis of a photocurable polymer identified as PCLMA, derived from an 80,000 molecular weight PCL polymer, with the incorporation of methacryloyl chloride. Glycerol diacrylate (GA), possessing reactive terminal groups, was synthesized through an acryloylation reaction involving a modest molecular precursor, specifically glycerol. This process involved the introduction of acryloyl groups onto the glycerol molecule, resulting in GA that served as crosslinking agents in the fabrication of novel tissue engineering materials. Subsequently, the synthesized PCLMA polymer, along with GA as a crosslinker and TPO as a photoinitiator, were blended to formulate a bioresin intended for the production of tissue engineering biomaterial.

The bioresin formulations underwent photopolymerization utilizing a Stereolithography (SLA) 3D printer in order to enhance the bioresin formulation. An extensive examination of the structure was performed, using techniques such as nuclear magnetic resonance (NMR) and Fourier-transform infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) was used to conduct morphological assessments. Furthermore, evaluations pertaining to the survival and attachment of cells were conducted on the tissue biomaterial utilizing the human osteoblast cell line (HOB).

2 Experimental section

2.1 Reagents

Poly (caprolactone) Mn 80000 (PCL) were purchased from Aldrich. Methacryloyl chloride (97%, contains ∼200 ppm monomethyl ether hydroquinone as stabilizer) (Meth-Cl) and Diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide (TPO) were purchased from Aldrich and used as received. Unless explicitly stated otherwise, all additional chemicals and solvents were acquired from Sigma-Aldrich.

2.2 Synthesis of methacrylated PCL (1)

Polycaprolactone (PCL) with a number-average molecular weight (Mn) of 80000 g/mol was dissolved in tetrahydrofuran (THF) at a mass/volume ratio of 1:10 in order to functionalize the hydroxyl (-OH) groups within the polymer chain. Methacryloyl chloride (Meth-Cl) was added to the solution to neutralize the resulting methacrylic acid in the presence of an equimolar amount of triethylamine (TEA). This produced a photocurable polycaprolactone-methacrylate (PCLMA) product (see Scheme 1). The goal of adding TEA to the reaction was to intentionally neutralize the methacrylic acid produced by the chemical change. The methacrylation process took place in natural surroundings. The macromer in dichloromethane (DCM) precipitated when the reaction was completed and THF, TEA, and any leftover Met-Cl and methacrylic acid were removed. The isolated macromer was then subjected to trilateral washing.

Scheme 1

Synthesis procedure of PCLMA.

2.3 Synthesis of glycerol acrylate (GA) (2)

The acryloylation process described by Mohammed et al.was used to manufacture glycerol acrylate. A three-necked round-bottom flask holding 7.3 mL of glycerol was filled with nitrogen gas. The flask was then gradually filled with freshly made acryloyl chloride (16.0 mL) and dried pyridine (16.1 mL), and it was agitated for 45 minutes at 50°C in a nitrogen environment. For glycerol, acryloyl chloride, and pyridine, the molar ratios used were 1:2:2. To get rid of the pyridinium salt that was also created along with the glycerol ester, the final product was dissolved in ether [15]. The experimental process was shown in Scheme 2.

Scheme 2

Formation of Glycerol acrylate.

2.4 Fabrication process of scaffolds using SLA

2.4.1 Formulation of Bioresin

Different weight ratios of PCLMA, glycerol acrylate (GA) as crosslinker, and the photoinitiator Diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide (TPO) were combined at ambient temperature in a light-absent environment in order to improve the formulation of PCLMA-GA bioresin. The solvent toluene was used to create a resin with a viscosity appropriate for the stereolithography technique, like the viscosity range of commercial resins falling between 200–300 MPa·s.

2.4.2 CAD Model of Scaffolds

A cylindrical bone scaffold model was conceptualized using computer-aided design (CAD) software, Autodesk Fusion 360 by Autodesk Inc. (San Rafael, CA, USA), as shown in Fig. 1. The model was designed to resemble a cylinder with a radius of 10 mm and an estimated height of ∼1 mm.

Fig. 1

3D CAD design of pattern.

2.4.3 3D Printing of Bioresin

The Sonic Mini 4K Resin 3D Printer (Phrozen3DP, Taipei City, Taiwan) was utilized to create bioresin compositions. A few of the Stereolithography (SLA) 3D printer’s parameters, such as the lifting speed (80 mm/min), bottom exposure duration, slice thickness (0.05 mm), and exposure time, were set up before the printing process started. After printing, the resultant cylinders were thoroughly cleaned three times with a 1:1 v/v ethanol/distilled water mixture in order to remove any remaining bioresin.

2.5 Characterization of PCLMA macromer and bioresins

2.5.1 Nuclear magnetic resonance (NMR) spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is a vital research technique that is mostly employed to clarify molecular structures through the utilization of the unique magnetic characteristics that are present in atomic nuclei. This method allows for the accurate separation of distinct functional groups that are present in the molecular structure that is being studied. Moreover, it provides unique signals that clarify the relationships among nearby groupings. PCLMA was characterized by proton nuclear magnetic resonance spectroscopy using a Varian UNITY INOVA 500 MHz device from Varian Medical Systems, Inc. After dissolving 20 mg of polymer samples in 0.5 mL of deuterated chloroform, the samples were analyzed at 500 Hz in an ambient environment. The peaks of the collected spectra were compared to 7.26 ppm of CDCl₃.

2.5.2 Fourier-transform infrared (FTIR) analysis PCLMA, GA and crosslinked bioresin

Using Fourier-transform infrared (FTIR) spectroscopy, functional groups within PCL, PCLMA, GA and crosslinked bioresin were identified. A Thermo Fisher Scientific NICOLET 6700 FTIR spectrometer (Waltham, MA, USA) with attenuated total reflectance (ATR) capability operating in the 400–4000 cm^-1 wavenumber range was utilized for the FTIR investigations.

2.5.3 Surface topography

The specimens’ surface topography was examined using scanning electron microscopy (SEM). The samples were coated with gold and palladium before being subjected to SEM examination. The samples were then analyzed with a Thermo Fisher Quattro S device, which was operated at an accelerating voltage between 10 and 15 kV.

2.6 Cell culture

Human osteoblast (HOB) cell line was kindly gifted by Prof. Ebru TOKSOY ÖNER, which was cultured in an aseptic environment. The cells were grown in a medium enriched with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The culture was kept at 37°C in an incubator with 5% CO₂.

2.6.1 Cell viability assay

The cell viability tests were performed according to the steps outlined in the ISO 10993-5 guidelines. These guidelines include test methods for figuring out how harmful medical devices are to cells in the lab. The human osteoblast (HOB) cell line was used in this study as opposed to the L929 cell line that is typically used in the test protocol. Following ISO 10993-12, extracts from the crosslinked bioresins produced by the Stereolithography (SLA) 3D printer were prepared. The MTT assay, which uses a tetrazolium salt that is soluble in water, was used to assess the vitality of resin cells. When the MTT molecule gets into living cells, it is broken down by mitochondrial succinate dehydrogenase, which creates purple formazan crystals that cannot be dissolved. The number of live cells is directly correlated with the intensity of the color that results. The HOB cell lines were grown in DMEM, which had 10% fetal bovine serum (FBS) and 1.0% penicillin/streptomycin added. Bioresins underwent a 72-hour in-vitro cytotoxicity study at three distinct concentrations (25, 50, and 100μg/ml). In 96-well tissue plates, cells were planted at a density of 4×10³ cells/well and allowed to grow to 80% confluence over a period of three days. After sample preparation according to ISO 10993-12 cells were exposed to different concentrations of bioresin extracts in new media and were cultured for longer periods of time. After being exposed, the cells were cultured for four hours with a 10% MTT solution added to the medium. Cells were treated with a 0.1 mg/ml solution of sodium dodecyl sulphate (SDS) in 0.01 M HCl after incubation [16]. At 570 nm, absorbance values were obtained with a Multiscan Go spectrophotometer (Thermo Fisher, USA).

2.6.2 Fluorescent Microscopy

Following treatment with crosslinked bioresin extracts, the cells were examined under a fluorescence microscope after staining with 4^′,6-Diamidino-2-phenylindole dihydrochloride (DAPI). Subsequently, the images obtained were documented. To be more precise, we subjected 8×10³ cells to standard cell culture conditions. These cells were then placed on a chamber slide and incubated for a period of 48 hours. Following the incubation period, the culture medium was replaced with a treatment medium containing DAPI. After an additional fifteen minutes of incubation, the cells were subjected to two successive washes with phosphate-buffered saline (PBS). Subsequently, photos were captured using a fluorescence microscope with DAPI filter illumination.

2.7 Statistical analysis

The statistical investigations employed one-way analysis of variance (ANOVA), and post hoc analysis was conducted using Tukey’s multiple comparison test. The average values of the obtained research findings were presented together with their corresponding standard deviations (±SD). Each experimental procedure was conducted a minimum of three times. P values less than 0.05 show statistical significance, while P values less than 0.01 or 0.001 indicate a highly significant effect.

3 Results and discussions

3.1 Synthesis and properties of PCL macromer

3.1.1 PCL macromer characterization

Meth-Cl and TEA were used in a reaction to methacrylate polycaprolactone (PCL), resulting in PCLMA. The macromeric samples of PCLMA were analyzed using proton nuclear magnetic resonance (NMR) spectroscopy. Figure 2 shows the proton NMR spectra of PCLMA.

Fig. 2

¹H NMR spectrum of PCLMA.

The characteristic peaks of PCL appear at 1.7, 2.35, and 4.1 ppm. Hydrogens of α carbon to ester carbonyl appeared at 2.35 ppm. The hydrogens of -O-CH₂–group revealed at 4.1 ppm [20]. After methacrylation, methacrylate peaks were depicted at 5.6 and 6.1 ppm. The intensity of the peaks so weak. The results obtained are compatible with the literature and show that the synthesis has been carried out successfully [17].

3.1.2 FTIR

The FTIR spectrum in Fig. 3 reveals distinctive peaks associated with PCL, including asymmetric and symmetric -CH₂ bonds at 2945.32 and 2865.41 cm^-1, respectively, as well as carbonyl bonds (C = O) at 1720.78 cm^-1. Moreover, in the crystalline phase, the FTIR spectrum of PCL exhibits bands corresponding to C-O, C-C, asymmetric and symmetric C-O-C bonds. Specifically, C-O and C-C bands are discerned at 1293.38 cm^-1, while asymmetric and symmetric C-O-C bands manifest at 1238.73 and 1155.36 cm^-1, respectively. Furthermore, the IR band at 3440.47 cm^-1 signifies the stretching of the -OH group of carboxylic acid, and peaks at 1470.87 and 1364.91 cm^-1 are associated with the stretching of the -CH₂ and -OH groups, respectively [3].

Fig. 3

FTIR spectrum of PCL, PCLMA, GA, and PCLMA-8.

Within the synthesized PCLMA polymer, a minor shoulder around the 1500 cm^-1 wavenumber indicates the occurrence of the methacrylation reaction, signifying the successful attachment of the methacrylate moiety to the PCL polymer. Additionally, newly formed peaks at 1257 cm^-1 and 1162 cm^-1 are attributed to ester formation resulting from C = O stretching. Also, the results support the NMR analysis.

As reported in the literature, the spectrum of glycerol exhibits absorption bands at 3282 cm-1 attributed to OH stretching vibration and 2932 cm-1 arising from the dissymmetry stretching vibration of C–H. Furthermore, the C–O stretching and C–O–H bending vibrations are discerned at 1029 and 1414 cm-1, respectively. Following acryloylation, the spectrum of acryloylated glycerol (GA) manifests new absorption peaks at 1730 cm-1, associated with the C = O stretching mode, and at 1635 cm-1, corresponding to the double bond vibration of C = C stretching in the acryloyl groups within the glycerol structure. These observed vibration peaks serve as confirmation of the successful acryloylation of glycerol [15]. The emergence of new peaks and the alteration in the positions of existing ones substantiate the effective chemical role of glycerol acrylate as a crosslinker in the bioresin.

3.2 Optimization of bioresin formulation

Optimization experiments were performed on crucial variables to facilitate the application of PCL-MA polymer on the stereolithography (SLA) equipment. The parameters considered the appropriate amounts of cross-linker and photo initiator in the bio-resin formulation. These amounts were determined based on the measurement of polymerization time under UV light exposure. The results of the optimization studies are displayed in Table 1.

Table 1
Optimization of Bioresin Formulation

Sample Name Quantity of Quantity of Proportion of Duration of

PCLMA (g) Crosslinker (g) Photoinitiator (%) Polymerization (min)

PCLMA-1 4,60 0,50 3 45

PCLMA-2 1,00 0,10 5 35

PCLMA-3 1,00 0,10 10 15

PCLMA-4 1,00 0,15 10 10

PCLMA-5 0,50 0,10 15 4

PCLMA-6 0,50 0,15 15 2.5

PCLMA-7 0,50 0,175 15 2

PCLMA-8 0,50 0,20 15 1

Sample Name	Quantity of	Quantity of	Proportion of	Duration of
PCLMA-1	4,60	0,50	3	45
PCLMA-2	1,00	0,10	5	35
PCLMA-3	1,00	0,10	10	15
PCLMA-4	1,00	0,15	10	10
PCLMA-5	0,50	0,10	15	4
PCLMA-6	0,50	0,15	15	2.5
PCLMA-7	0,50	0,175	15	2
PCLMA-8	0,50	0,20	15	1

The given data show that different concentrations of crosslinker and photoinitiator have noticeable influence on the time it takes for the material to cure. Increasing the amount of crosslinker in relation to the polymer content resulted in a decrease in the time required for polymerization [18]. Similarly, an increased concentration of photoinitiator showed a positive effect on reducing the time required for curing. The polymerization process shows variations depending on the content of the material being polymerized. The conversion rate is influenced by two factors: 1) the ratio of monofunctional monomers to bifunctional monomers in a mixture, and 2) the presence of high concentrations of initiator and co-initiator, which both contribute to faster polymerization kinetics [19].

Attaining a rapid and precise print finish is essential for maximizing efficiency in the 3D printing process. Therefore, a decision was made to further investigate the formulation that exhibited the shortest polymerization time for layers (PCLMA-8) in the stereolithography equipment (SLA) among the formulated compositions.

3.3 Surface characteristics

The characterization parameters of the PCLDMA/P-PLA composite, 3D printed scaffolds, such as morphology and surface roughness were observed via SEM (Fig. 4).

As observed in the SEM images, it is evident that the structure generated through computer-aided design (CAD) exhibits a high degree of accuracy. Furthermore, the presence of a porous structure, inherent to the utilized materials, is discernible. This porous architecture holds significance for facilitating cell migration, promoting cell growth, and facilitating nutrient and oxygen exchange among cells [13, 20].

Fig. 4

SEM images of PCLMA-8.

3.4 Cell viability

The evaluation of cell viability was performed in accordance with the guidelines outlined in ISO 10993-5, which specifies the protocols for measuring the in vitro cytotoxicity of medical devices. This study used the human osteoblast (HOB) cell line instead of the commonly used L929 cell line mentioned in the usual testing technique. The extraction of chemicals from the PCLMA-8 followed the protocols outlined in ISO 10993-12. This section of the ISO 10993 directives sets out standards and offers information on how to prepare samples and select reference materials for evaluating medical devices in biological systems, following the ISO 10993 framework. Three separate concentrations, which corresponded to purity levels of 25%, 50%, and 100%, were given to the HOB cell line. The MTT method was used to assess cell viability. Figure 5 demonstrates the effect of extracts on the survival of cells. Following a 72-hour period, it was noted that the extracts did not reduce the viability of the HOB cell line. An inverse correlation between concentration and cell viability was noted, with increasing concentrations leading to a reduction in viability. Nevertheless, the recorded cell viability values consistently surpass 70%. Consequently, it is inferred that the samples do not induce toxic effects on HOB cells.

Fig. 5

The cell viability results of PCLMA-8 (n = 5). Cell viability assays were performed with HOB cells. As a control, DMEM complete media used only.

The data demonstrate consistency with the available literature [20 –22]. This congruence can be attributed to multiple sources. The PCL-based methacrylated polycaprolactone (PCLMA) polymer used in the formulations is known to be a biocompatible substance that has been approved by the Food and Drug Administration (FDA) [28]. Additionally, materials that have been exposed to ultraviolet (UV) radiation and produced by a Stereolithography (SLA)-type 3D printer are thoroughly cleansed using suitable solutions after the printing process. This painstaking washing guarantees the complete elimination of any potentially harmful substances. Furthermore, the cell viability test clearly demonstrates that these compounds do not degrade, leading to the production of harmful residues. Based on the results of the cell viability assays, it was shown that the PCLMA-8 sample is biocompatible with living human osteoblast (HOB) cells.

3.5 Fluorescent microscopy

Fluorescent microscope image (Fig. 6) and SEM image with HOB cells (Fig. 7) were captured to get more information about cell adhesion on the surface of the sample named PCLMA-8. The reason for choosing sample named PCLMA-8 is that, according to the results of physical, chemical, structural, and cytotoxicity tests, it is considered to have the optimum properties that a tissue material should possess. As it can be seen in Fig. 3.6 and 3.7 cells on surface of the samples showed carpet like covering with fully attached which considers it has good cellular attachment properties [23].

Fig. 6

Fluorescence microscope image of the PCLMA-8 with DAPI labeled HOB cells. The magnification factor of the microscope in use is set at a twenty-fold increase.

Fig. 7

SEM image of PCLMA-8 with HOB cells.

As evident from both fluorescence microscope and scanning electron microscope (SEM) images, human osteoblast (HOB) cells demonstrated adhesion to the PCLMA-8 sample.

4 Conclusions

The synthesis of PCLMA polymer was achieved by utilizing an 80000 molecular-weight PCL polymer as the precursor material in this research project. The effective formation of this PCLMA polymer was confirmed through thorough investigations utilizing FTIR and NMR methods. Afterwards, a bioresin was formulated utilizing the synthesized methacrylated polycaprolactone (PCLMA) polymer, glycerol acrylate (GA) as a cross-linker, and diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO) as a photoinitiator. The optimization of the bioresin formulation involved the systematic exploration of varying proportions of the cross-linker and photoinitiator. Specifically engineered for curing upon exposure to ultraviolet (UV) light, this bioresin formulation is designed to facilitate three-dimensional (3D) printing through a stereolithography device. Finally, cellular viability studies were performed on the human osteoblast (HOB) cell line at three different degrees of purity (25%, 50%, and 100%). The evaluation of cell viability was completed using the MTT method following a 72-hour incubation period. Consequently, the cell viability tests confirmed that the PCLMA-8 sample is biocompatible with living HOB cells. In conclusion, based on the knowledge gained from the products obtained in this study, it is advisable to conduct an in vivo investigation to determine if a specimen called PCLMA-8 can be used as a substitute for tissue engineering material in future research.

Footnotes

Acknowledgments

This work was supported by Türkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBİTAK, The Scientific and Technological Research Council of Türkiye) with project coded 1002-222M436 (2022–2023).

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used GPT-3.5/Open AI. In order to improve readability and language. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

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