Anisotropic scaffold of polycaprolactone, polyvinyl alcohol and cellulose nanocrystal for immune regulation and bone regeneration and its application

Abstract

The present invention manufactures a polycaprolactone (PCL) scaffold by 3D printing, and The present invention relates to a method for manufacturing a nanofiber-laminated PCL scaffold comprising adding polyvinyl alcohol to cellulose nanocrystals and then producing cellulose nanocrystals and polyvinyl alcohol nanofibers through electrospinning, and rolling the nanofibers onto the side walls of the three-dimensional printed PCL scaffold, and to applications related to immunomodulation and bone regeneration of the scaffold manufactured by the method. The scaffold of the present invention can serve as an immunomodulatory bone graft, particularly under conditions such as postoperative immunodeficiency.

Description

Technology Field

[0001] The present invention relates to an anisotropic scaffold of polycaprolactone, polyvinyl alcohol, and cellulose nanocrystals for immune modulation and bone regeneration, and to applications thereof. Background Technology

[0002] Postoperative immunosuppression, particularly the loss of cell-mediated immunity, is frequently observed due to increased release of immunosuppressive hormones such as catecholamines, prostaglandins, and cortisol, depending on the degree of surgical stress and tissue damage.

[0003] Factors such as blood transfusion, hypothermia, dehydration, and anesthesia can further impair immunity. A weakened immune system after surgery is associated with a worse prognosis, including delayed wound healing and infection. During bone injury, the initial response of the innate immune system is known to activate the recruitment of M1 macrophages. M1 macrophages, which are predominantly present at the wound site, release pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), and interleukin-1β (IL-1β). These pro-inflammatory cytokines promote the removal of damaged tissue and recruit stem cells. However, the timely transition of M1 macrophages to the M2 phenotype is critical for effective regeneration. Therefore, the dynamics of immune regulation are an important aspect that can significantly dictate bone tissue regeneration.

[0004] Various plasma proteins, including albumin, fibronectin, and vitronectin, are deposited on the implant surface immediately after transplantation. These anabolic proteins significantly influence the recruitment, adhesion, and activity of immune cells at the implant site. Platelet-derived TGF-, CXCL2, and CXCL8, released from blood clots and deposited on the implant, can attract neutrophils from the circulation, and integrins expressed by neutrophils help bind to the implant surface coated with proteins. This type of binding stimulates neutrophil activation and triggers an inflammatory response, thereby stimulating immune cell recruitment. Prolonged neutrophil activation causes chronic inflammation and delays tissue healing.

[0005] Meanwhile, neutrophils can also contribute to tissue regeneration through the secretion of growth factors, thus aiding in the remodeling of mesenchymal stem cells (MSCs) and the resolution of inflammation as previously discussed. Similar to neutrophils, the release of chemoattractants from platelets and blood clots, such as TGF-, CXCL4, and leukotrienes, can induce monocytes / macrophages to the transplant site.

[0006] Proteins absorbed by the implant with the help of integrins promote macrophage adhesion. As macrophages accumulate at the implantation site, chemoattractants are secreted to recruit additional macrophages, and activated macrophages tend to phagocytose biomaterials. In the early stages of inflammation, inflammatory macrophages produce various harmful substances detrimental to tissue healing, such as reactive oxygen species, degrading enzymes, and acids. In the later stages of inflammation, macrophages polarize into an M2-type phenotype that expresses anti-inflammatory cytokines to promote tissue repair. Dendritic cells can detect biomaterials via Toll-like receptors when activated by the ligand moiety of the protein layer adsorbed to the implant. Dendritic cells can attach to fibronectin via integrins. Albumin can stimulate dendritic cells to produce the anti-inflammatory cytokine IL-10, thereby promoting the resolution of inflammation and tissue repair.

[0007] In contrast, vivonectin caused dendritic cells to express IL-12p40, which is associated with CD4+ T cell proliferation that can interfere with tissue regeneration. Overall, proteins adsorbed onto the implant play a crucial role in mediating interactions between the implant and immune cells. Consequently, altering the surface properties of biomaterials can alter protein deposition and regulate immune cell responses.

[0008] Immunomodulatory micro / nano platforms developed in bone tissue engineering (BTE) have focused primarily on utilizing stem cell or biomaterial-based immunomodulation. Mesenchymal stem cells (MSCs) possess immunomodulatory functions and can secrete cytokines in response to inflammatory signals.

[0009] Therefore, MSCs have been used to treat numerous bone disorders, including graft-versus-host disease and immune disorders.

[0010] Liu et al. fabricated a multifunctional piezoelectric device by developing a biomimetic periosteum for immunomodulatory-induced osteogenesis using biodegradable poly(3-hydroxybutyric acid-co-3-hydrovaleric acid) (PHBV), polydopamine-modified hydroxyapatite (PHA), and barium titanate (PBT). Periosteum formed by spin coating method. The developed biomimetic periosteum exhibited improved surface hydrophilicity, mechanical properties, degradation behavior, and electrical stimulation directing M2 polarization of macrophages and osteogenic differentiation of mesenchymal stem cells (MSCs). The developed piezoelectric periosteum can serve as an effective future implant without the need for loading additional cytokines, growth factors, or cells [Liu H, Shi Y, Zhu Y, Wu P, Deng Z, Dong Q, et al. Bioinspired Piezoelectric Periosteum to Augment Bone Regeneration via Synergistic Immunomodulation and Osteogenesis. ACS Applied Materials & Interfaces. 2023;15:12273-93].

[0011] [Prior Art]

[0012] Korean Patent Publication No. 10-2023-0004525 The problem to be solved

[0013] The present invention was conceived in response to the above necessity, and the objective of the present invention is to provide a novel scaffold for immune regulation and bone regeneration. means of solving the problem

[0014] To achieve the above objective, the present invention manufactures a polycaprolactone (PCL) scaffold by 3D printing, and

[0015] Cellulose nanocrystals and polyvinyl alcohol nanofibers are prepared by adding polyvinyl alcohol to cellulose nanocrystals and then electrospinning.

[0016] A method for manufacturing a nanofiber laminated PCL scaffold is provided, comprising rolling the nanofibers onto the side walls of the three-dimensional printed PCL scaffold.

[0017] In one embodiment of the present invention, the PCL scaffold is preferably coated with arginine, but is not limited thereto.

[0018] In another embodiment of the present invention, the cellulose nanocrystals are preferably cationic nanocrystals, but are not limited thereto.

[0019] In another embodiment of the present invention, the method preferably adds, but is not limited to, the step of rolling the nanofibers onto the side walls of the three-dimensional printed PCL scaffold and then placing them in a chamber.

[0020] In addition, the present invention provides a method for inducing dynamic polarization of M1 macrophages and M2 macrophages by treating a composite scaffold prepared by the method of the present invention with M1 macrophages and M2 macrophages.

[0021] In addition, the present invention provides a composition for inducing dynamic polarization of M1 macrophages and M2 macrophages comprising a composite scaffold prepared by the method of the present invention as an active ingredient.

[0022] In addition, the present invention provides an immunomodulatory bone graft composition comprising a composite scaffold prepared by the method of the present invention as an active ingredient.

[0023] In addition, the present invention provides an antibiotic composition for viruses, Gram-positive bacteria, and Gram-negative bacteria comprising a composite scaffold prepared by the method of the present invention as an active ingredient.

[0024] In one embodiment of the present invention, the composition preferably incorporates a fluid shear stress (FFS) treatment process, but is not limited thereto.

[0025] In another embodiment of the present invention, the composition preferably increases the expression of Tlr4 and IL 15 genes, but is not limited thereto.

[0026] In addition, the present invention provides a method for increasing the expression of CCL3 (cytokine ligand 3) and TNFR2 (Tumor necrosis factor receptor type II) by applying fluid shear stress (FFS) to a composite scaffold prepared by the method of the present invention during in vitro culture, compared to when such FSS is not applied.

[0027] Furthermore, the present invention provides a method for increasing the expression of ALP and OCN among bone formation markers by applying fluid shear stress (FFS) during the process of culturing stem cells in vitro on a composite scaffold prepared by the method of the present invention, compared to a case where such FFS is not applied.

[0029] The present invention will be described below.

[0030] In this invention, anisotropic scaffolds of polycaprolactone (PCL), polyvinyl alcohol (PVA), and cellulose nanocrystals (CNC) were developed for immunomodulation and bone regeneration. Immunomodulation tests revealed that the kinetics of the fabricated scaffolds of this invention can influence macrophage polarization and bone regeneration. The results of this invention suggest that the fabricated scaffolds can induce M1 and M2 macrophage polarization over time through the topological and physicochemical properties of PVA and PVA@cCNC, reduce surgery-induced oxidative stress, and promote immunomodulation-induced bone regeneration.

[0031] The inventors also investigated the function of mechanically activated ion channels in macrophage polarization through RNA sequencing analysis and elucidated the effect of fluid shear stress (FFS) physical stimulation on transcriptome changes in macrophage activity. Under the influence of FFS, RNA-seq data showed differential expression of cation channels in cells cultured on PCL_PVA@cCNC scaffolds. In particular, the major participation of K+ ion channels in the dynamic macrophage polarization process was confirmed.

[0032] The inventors also observed the overexpression of two important genes (Tlr 4 and Il 15) involved in preventing bacterial invasion. Applying FFS stimulation to the complex PCL_PVA@cCNC scaffold is suitable for tissue regeneration through dynamic macrophage polarization, primarily through the differential expression of K+ ion channel activity.

[0033] The modulation of M1 and M2 polarizations was synergistically controlled by nanotopological cues as well as changes in chemical composition between PVA and PVA@cCNC nanofibers due to the presence of cCNC. Coated arginine induces macrophage polarization toward the M2 phenotype, while cCNC promotes M1 polarization.

[0034] Based on the results of the present invention, the inventors expect that the prepared composite can be used as a potential immunomodulatory bone graft for clinical application. RNA-seq data showed differential expression of cation channels in cells cultured on a PCL_PVA@cCNC scaffold in response to physical stimuli inducing fluid stress.

[0035] Specifically, the inventors observed the dominance of K+ ion channel involvement in the dynamic macrophage polarization process resulting from the cationic CNC present in the fabricated structures. Additionally, we observed stimulus-induced upregulation of two key genes (Tlr 4 and Il 15) associated with preventing bacterial invasion in PCL_PVA@cCNC cultured cells.

[0036] Furthermore, the expression of the M2 marker increased in cells cultured on PCL regardless of the presence or absence of stimulation. Since the fabricated scaffold is a composite structure of an electrospun mat covering the outer wall of a 3D-printed PCL scaffold, its unique fabrication allows Raw 264.7 cells to undergo dynamic macrophage polarization for 24 hours. Therefore, FFS stimulation of the composite PCL_PVA@cCNC scaffold is suitable for tissue regeneration through dynamic macrophage polarization, primarily via the differential expression of K+ ion channel activity.

[0038] The present invention will be described in detail below.

[0039] Composite Scaffold Fabrication and Characterization

[0040] Figure 2A shows the sequential steps in the 3D printing of a PCL scaffold. A PCL scaffold with an interconnected channel network with highly controlled porosity was printed using a 15x10mm CAD model. A total of 40 layers were printed. The interior of the printed scaffold forms a cross-stranded reticular structure, creating ≥50 square channels, each 10mm in size. The thickness of each strand was measured to be 0.5mm. However, although PCL is suitable for bone regeneration, it has been reported to be highly hydrophobic, which often limits cell matrix attachment.

[0041] Therefore, the fabricated PCL scaffold was coated with L-arginine. The loaded arginine was identified using FT-IR spectroscopy. The FT-IR spectrum of L-arginine corresponds to the NH, C=N, C=O vibration at 1559.46 cm⁻¹. -1 , 1610.79cm -1 , 1670.94cm -1 It was observed to have a distinct peak at 1724 cm⁻¹ for the PCL scaffold corresponding to the C=O stretching vibration. -1 A distinct peak was observed at . Other characteristics are 2863.54 (symmetric CH2 stretching) and 2946.95 cm⁻¹. -1 (Asymmetric CH2 Stretching) and 1293cm -1 The peak is at (CO and CC stretching). The FT-IR spectrum of arginine-coated PCL is at 1633 cm⁻¹. -1 (Amide II) and 1549cm -1 The appearance of a peak at (amide II) was shown, indicating that arginine was successfully loaded onto the PCL scaffold. The two amide bands generated by the nucleophilic attack of arginine on the carbonyl groups of PCL can be interpreted as evidence of arginine-PCL surface interactions. 1510–1580 cm⁻¹ -1 The first band of the range is associated with the NH bending vibration and corresponds to amide II. 1600–1700 cm -1The second band in between corresponds to amide I and is assumed to be associated with C=O (carbonyl) stretching vibrations and CN group vibrations (Fig. 2B, left panel). Additionally, the FE-SEM image of the PCL scaffold revealed an etched surface, resulting in the proliferation of surface wrinkles on the PCL scaffold (Fig. 2B, right panel).

[0042] Next, the physicochemical properties of the electrospun PVA and PVA@cCNC nanofibers were analyzed. Figure 3A shows digital photographic images of the fabricated PVA and PVA@cCNC electrospun mats, demonstrating that there is no noticeable difference in the appearance of the PVA mat upon cCNC integration. However, close observation via FE-SEM images reveals denser fiber deposition in the PVA@cCNC mat compared to the PVA counterpart (Figures 3C and 3D).

[0043] In addition, the tensile strength of the PVA electrospun mat was observed to have a higher elastic modulus than that of the PVA@cCNC mat (elastic modulus, E= 0.00125 Pa). Close observation through FE-SEM analysis of the deformed fibers indicates that the difference in elastic modulus may have occurred due to the random and parallel fiber orientation of the mats in PVA and PVA@cCNC, respectively, as shown in Figures 3C and D.

[0044] Next, a synthetic structure was fabricated by overlaying a nanofiber mat onto a PCL scaffold, and the structure was cultured in a humidified chamber (Figs. 4A, B). Humidity in the air induced the dissolution of the PVA mat, creating distinct micro / nano topologies observed in the PCL scaffold overlaid with PVA@cCNC nanofibers (nano-islands). The formation of nano-islands was triggered by the partial dissolution of PVA, leaving the cCNC intact, as indicated in Figs. 4(C, D). The hydrophilicity of the nanocomposite was confirmed by contact angle measurements. The inventors observed a distinct increase in the hydrophilicity of the nanocomposite in the order of PCL < PA < PAP < PAPC (Fig. 4E).

[0045] In the present invention, PA: Polycaprolactone-Arginine, PAP: Polycaprolactone-Arginine-Polyvinyl alcohol, and PAPC: Polycaprolactone-Arginine-Polyvinyl alcohol-cationic cellulose nanocrystals.

[0047] Perfusion Bioreactor Design

[0048] The fabricated structure was tested for immunomodulatory potential using a custom-designed bioreactor. The bioreactor system consists of a circular metal body with six peripheral cylindrical chambers connected to a central well that receives incoming medium from a reservoir, as shown in Fig. 5(af).

[0049] Each peripheral chamber is designed with diameters of 10, 20, and 35 mm, arranged chronologically from top to bottom of the wells, creating three scaffold supports per well. Each scaffold supports a height of 10 mm, capable of simultaneously supporting three independent scaffolds. The culture medium is stored in storage vials made of borosilicate glass. These vials are connected to the chambers and flow control pumps via a tube system. The bioreactor chambers and culture medium vials are placed inside an incubator (37°C, 5% CO2, high humidity).

[0050] During the experiment, the scaffold in the bioreactor chamber is continuously streamed from top to bottom with the culture medium at a flow rate of 0.5 mL / min. As shown in Fig. 5(hk), no cytotoxicity was observed in Raw 264.7 and hBMSC under single or co-culture conditions. Additionally, as can be seen in Fig. 5(lp), the M1 phenotype of macrophages seeded on the scaffold with cationic CNC is observed, demonstrating the effect of CNC's immunomodulatory efficiency.

[0051] Mineralization ability and bone formation ability

[0052] The inventors confirmed the mineralization potential of hBMSCs cultured on scaffolds fabricated under static and dynamic conditions. Higher mineral deposition was observed on scaffolds coated with PVA nanofibers (Fig. 6).

[0053] In addition, analysis of bone formation marker genes revealed increased expression in the PAPC group during FFS, particularly increased expression of ALP and OCN. As shown in Figure 7(a, c), a decrease in Runx2 gene expression in the PA group during FSS was observed, which may be a result of Arg loss from the scaffold during FSS.

[0054] Furthermore, the results of the present invention show that FSS induced an increase in Piezo 1 expression in the PAP and PAPC groups. However, Piezo 1 expression decreased in the PA group, similar to Runx2 expression. SPARC and SPP1 expression in all groups was observed to decrease with FSS, indicating that Piezo 1 may play an important role in FSS-induced mechanical transformation. Relative gene expression is shown in Figure 7(b, d).

[0055] The inventors also observed macrophage polarization toward the M2 phenotype in PA and PAP scaffolds under static culture conditions. However, under fluid shear stress, the PAPC scaffold induced a sharp increase in the expression of M2 markers, which is negligible under static culture conditions, indicating that the polarization ability of macrophages can be fine-tuned using fluid shear stress on CNC-containing nanocomposites (Fig. 8).

[0056] The macrophage polarization ability of cCNC and Arg was found to regulate macrophage polarization. As can be seen in Tables 1, 2, and 3; the slow degradation of the fabricated composite scaffold can induce a longer duration of macrophage interaction with cCNC while delaying exposure to coated Arg.

[0057] In FSS, the inventors observed a dramatic increase in IL-6 expression in cCNCs containing scaffolds. IL-6, one of the important signaling factors, increases under fluid shear stress. Therefore, the dramatic increase in IL-6 expression in cCNC scaffolds indicates the role of cCNCs in IL-6 expression activation compared to other counterparts.

[0058] Next, the inventors analyzed the expression of TGFβ1 in FSS and the control group. A sharp increase in expression was observed in the CNC-containing group and the PA scaffold compared to the control group and the PAP scaffold. This indicates that the deposition of PVA on the scaffold interferes with the expression of the corresponding gene. While the presence of high Arg content in the PA scaffold promoted gene expression, the presence of cCNC along with FFS promoted TGFβ1 expression much more highly. A similar trend was observed in the expression of VEGF, Arg-1, and CD 163. Interestingly, iNOS expression was also observed in the cCNC-containing group in FSS. The overall expression demonstrates that cCNC containing the scaffold can regulate M1 and M2 macrophage polarization upon PPS stimulation compared to all other fabricated scaffolds.

[0059] DEG related to fluid shear stimulation

[0060] Upon application of fluid shear stimulation, the inventors observed upregulation of 639 genes and downregulation of 467 genes in the PCL scaffold under fluid shear stress for cells cultured under static conditions in the PCL scaffold.

[0061] On the other hand, it was observed that 897 genes were upregulated and 650 genes were downregulated in the cationic CNC-containing PCL (PCL_PVA@cCNC) scaffold during FSS.

[0062] Among the two groups, as shown in Figure 9 (a, b) and Table 4, 367 genes were upregulated and 258 genes were downregulated in both PCL and PCL_PVA@cCNC scaffolds during FFS.

[0063] The inventors first investigated gene expression related to the macrophage differentiation process. The inventors observed the highest expression of VEGF in all groups, but a sharp decrease in expression was observed in the group subjected to fluid shear stimulation. Furthermore, CNC containing PCL scaffolds exhibited minimal gene expression, indicating fluid shear-induced downregulation of genes. NOS2 expression, a key marker of M1 macrophages, was observed to be highest in the CNC-containing group under static culture conditions. However, expression decreased in the CNC-containing group under dynamic culture conditions. Arg1 expression also decreased under fluid shear stimulation.

[0064] In contrast, no changes in expression were observed for IL6 and CD163 genes. RNA-seq data indicated that most macrophage polarization genes, with the exception of TGF β1, were downregulated in macrophages exposed to mechanical stimulation, suggesting that mechanical stimulation may delay the overall polarization process of the macrophage population (Fig. 9c, Table 5).

[0065] DEG involved in macrophage polarization and inflammation

[0066] The inventors observed eight major genes involved in macrophage polarization and inflammatory processes, and the data are shown in Figure 10 (a, b) and Table 6.

[0067] The most significant observation in the data is related to the expression of Tlr4 in CNCs containing PCL scaffolds under fluid shear stimulation, as Tlr4 is involved in the recognition of pathogens, including viral components as well as Gram-negative and Gram-positive bacteria. This represents substantial evidence regarding the M1 polarization ability of CNCs under mechanical stimulation. At the same time, we observed a rapid increase in the expression of Il15 in PCL_PVA@cCNC constructs under FFS, which plays a crucial role in the prevention of bacterial infections. The upregulation of both Tlr4 and Il15 significantly supports cellular M1 activity in the fabricated constructs.

[0068] DEG involved in ion channel activity

[0069] One of the important aspects controlling the perception of external stimuli to the internal cellular environment is ion channels. As fluid shear stress causes dynamic macrophage polarization, the inventors investigated ion channel expression profiles at the transcriptomic level.

[0070] The inventors observed a total of 85 ion channels that were differentially regulated according to FSS in the control and treatment groups, as shown in Fig. 11 (a, b) and Table 7.

[0071] The inventors observed significant differences in the expression of cation channels, including potassium (K+) channels, calcium (Ca2+) channels, and sodium (Na2+) channels. A total of 16 K+ channels were observed to be differentially expressed, of which 8 were upregulated and 4 were downregulated. Ion channels for Ca2+ ions each exhibited 5 types of upregulation and downregulation. In contrast, in FFS, two Na2+ channels were upregulated and one was downregulated. Among the anion channels, chloride ion channels (Cl-) were observed to be affected by stimulation. Of the 5 Cl- channels, 3 were upregulated while 2 were downregulated.

[0072] The inventors hypothesize that the role of cation channels in the overall macrophage dynamic polarization process is derived from the PCL_PVA@cCNC configuration containing cation CNCs. The presence of cationic surface groups in CNCs promoted differential expression of cationic channels in macrophages, which ultimately resulted in the overall dynamic polarization behavior of Raw 264.7 cells.

[0073] Figure 11c shows the differential expression of proteins expressed in FFS. The inventors observed the involvement of CCL3 (cytokine ligand 3) as a key mediator in the M1 polarization of macrophages. In addition, they observed an increased expression of TNFR2 (tumor necrosis factor receptor type II), which is involved in the anti-inflammatory response. Effects of the invention

[0075] The inventors fabricated an arginine-coated PCL scaffold laminate using a mixed topology of aligned PVA and PVA@cCNC and random nanofibers to control dynamic macrophage polarization and bone regeneration. It is expected that the fabricated composite of the present invention can serve as an immunomodulatory bone graft for immunodeficient patients, particularly in postoperative immunosuppression scenarios.

[0076] In addition, the inventors further investigated the molecular mechanism of the FSS-induced dynamic macrophage polarization process through RNA-seq analysis. The results of the invention revealed the involvement of ion channels that are important in determining the dynamic macrophage polarization behavior due to the unique structure of the structure of the invention when subjected to shear stimulation. The inventors expect that elucidating the mechanism will be of great help in the fine-tuning of future smart scaffolds that operate through dynamic immune regulation. Brief explanation of the drawing

[0077] Figure 1 is a schematic diagram of a nanocomposite manufacturing process combining 3D printing and electrospinning. FIG. 2(A) shows the 3D printing of a PCL scaffold; (A1) CAD model of the design; (A2) 3D printing process; (A3) 10 X 15 mm printed structure; (A4) PCL scaffold after arginine coating. 2(B) FT-IR spectra of PCL, Arg, and PCL-Arg structures showing successful coating of PCL scaffolds with arginine (left panel); FE-SEM images of PCL scaffolds before and after arginine coating (right panel). Fig. 3(A) Optical image of electrospun PVA and PVA@cCNC mat, (B) FE-SEM image of PVA, PVA@cCNC nanofiber, (C and D) Tensile strength of PVA and PVA@cCNC nanofibers with corresponding FE - SEM images at the fiber breakpoint. Fig. 4(A) Optical image of the process of rolling electrospun PVA and PVA@cCNC mats onto a 3D-printed PCL scaffold, (B) Fiber coating on scaffold under high humidity conditions, (C) FE-SEM image of knit and fiber coating PCL scaffold, (D) FE-SEM image of PVA, PVA@cCNC nanofiber showing distinct nanofiber topology, (E) Measurement of the water contact angle of the fabricated scaffold. Fig. 5(AE) Fluid flow bioreactor setup showing cell culture conditions in a 3D environment, (hj) Analysis of cell viability of (hj) Raw 264.7, hBMSCs and co-culture under static and fluid shear dynamics (k) Analysis of cells co-cultured under a living-dead fluid flow environment, (lp) FE-SEM images of Raw 264.7 cells under static culture conditions show the effect of nanomaterials on macrophage polarization. Data are the mean ± SD of three experiments, with statistical significance at *p<0.05, **p<0.01, and ***p<0.001. Figure 6 shows the evaluation of mineralization potential of nanocomposite scaffolds prepared under static and dynamic conditions through ARS staining. Figure 7 shows a real-time q-PCR study on (a) bone formation and (b) expression of mechanical transformation marker genes after 7 days of culture under static and dynamic culture conditions, with data showing statistical significance at mean ± SD of 3 experiments, **p<0.01 and ***p<0.001. Figure 8 shows real-time qPCR analysis of macrophage M1 and M2 marker genes after 24 hours of culture under static and dynamic conditions; data represent the mean ± SD of three experiments, with statistical significance at **p<0.01 and ***p<0.001. P: PCL, PA: PCL-arginine, PAP: PCL-arginine-PVA, PAPCCNC: PCL-arginine-PVA cationic cellulose nanocrystals. Figure 9 is a figure showing differentially expressed genes (DEGs) related to the cell physiology of macrophages Raw 264.7; (a) Total number of DEGs associated with transcriptomic changes; (b) Heatmap showing DEGs involved in macrophage differentiation. Downregulated genes (blue) and upregulated genes (yellow) are indicated by color codes representing Z-scores from -0.5 to +0.5. (c) Graphic plot showing the differential expression pattern of macrophage polarization marker genes. Figure 10 is a figure showing differentially expressed genes involved in macrophage polarization and inflammation; (a) Diploid change of genes in all study groups; (b) DEG heatmap of the dynamic group compared to the static control group. FIG. 11 is a figure showing differentially expressed genes involved in ion channels; (a) DEG involved in ion channels of all research groups; (b) DEG heatmap of the dynamic group compared to the static control group; (c) Protein analysis of macrophage polarization under FFS. In the drawings of the present invention, PA: Polycaprolactone-Arginine, PAP: Polycaprolactone-Arginine-Polyvinyl alcohol, and PAPC: Polycaprolactone-Arginine-Polyvinyl alcohol-cationic cellulose nanocrystals. Specific details for implementing the invention

[0078] The present invention will be described in more detail below through non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, and the scope of the present invention should not be interpreted as being limited by the following examples.

[0080] Example 1: Materials

[0081] Polycaprolactone (PCL; Sigma-Aldrich, USA; CAS: 24980-41-4, Average Mol. Wt. 80K, ≥95%), Polyvinyl Alcohol (PVA; Daejung Chemicals, Korea; CAS: , Average Mol. Wt. 98-99%), Cationic Cellulose Nanocrystals (Cellulose Lab; Canada), 1-Ethyl-3-(3-Dimethylaminopropyl)Carbodiimide (EDC; Alfa Aeser, USA; CAS: ,98%), N-Hydroxysuccinimide (NHS); Alfa Aeser, USA; The antibiotics provided by CAS: 98%), sodium hydroxide (NaOH; Alfa Aeser, USA; CAS: 97%), Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS), Dulbecco's phosphate-buffered saline (DPBS), and trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) were purchased from Welgene Inc., South Korea. Bone induction medium, 4,6-diamino-2-phenylindole dihydrochloride (DAPI), and alizarin red (ARS) staining kits were purchased from Sigma-Aldrich, USA. WST-8 dye (EZ-Cytox Cell Viability Assay Kit®) and Alexa Fluor conjugated monoclonal antibodies were purchased from DoGenBio Co., Ltd., South Korea, and Santa Cruz Biotechnology, USA, respectively. TRIzol® reagents, Acridine orange, and Ethidium bromide stains were purchased from Invitrogen at Thermo Fisher Scientific, USA. The cDNA synthesis kit was purchased from Invitrogen in Gaithersburg. Bio-Rad Laboratories, USA supplied the SYBR Green Master Mix.The gene primers were supplied by BIONEER® Inc. in Daejeon, South Korea. The fully automated CELLINK® BIO-X 3D bioprinter was purchased from CELLINK Corporation in Sweden.

[0083] Example 2: Bioreactor Design

[0084] The bioreactor system consists of a circular metal body with six peripheral cylindrical chambers connected to a central well that receives incoming medium from the reservoir. Each peripheral chamber is designed with diameters of 10, 20, and 35 mm, arranged chronologically from top to bottom of the well, creating three scaffolds per well. Each scaffold support is 10 mm high and can simultaneously support three independent scaffolds. The culture medium is stored in a storage container made of borosilicate glass, coupled with the chambers and flow control pumps, via a tube system. The bioreactor chambers and the culture medium reservoir are placed inside an incubator (37 ºC, 5% CO2, high humidity). During the experiment, the scaffolds in the bioreactor chambers are continuously streamed from top to bottom along with the culture medium at flow rates of 0.1 mL / min, 0.25 mL / min, and 0.5 mL / min.

[0086] Example 3: PCL Ink Preparation and 3D Printing

[0087] Weigh 1.5g of PCL and pour it directly into a plastic printing cartridge (CELLINK Corporation, Sweden), and incubate with 2mL of dichloromethane at room temperature until completely dissolved to make a 75% PCL solution.

[0088] 3D structures were printed at room temperature using manufactured PCL ink. The 3D printed structures were designed using SolidWorks software (www.solidworks.com, Dassault Biosystems, France). Circular (15 × 10 mm) structures were carefully printed using a 22G (0.410 mm) plastic head needle at room temperature and a print head temperature of 40°C with a print pressure of 8 kPa. The print speed was maintained at 2 mm / s. The scaffolds were printed directly onto glass Petri plates containing sandpaper. After printing, the scaffolds were stored at room temperature until needed.

[0090] Example 4: Preparation of PVA and PVA@cCNC nanofibers by electrospinning

[0091] 14% PVA (wt. / vol) was prepared in deionized water by magnetic stirring at room temperature. The polymer solution was 10 cm 3 It was transferred with a plastic syringe (Fortuna Optima with Luerlock ti, Poulten & Graf, Wertheim, Germany) and a needle with a right-angled tip (G 27 1 1 / 2 inch, K51 Luerlock, Italy).

[0092] Similarly, the PVA@cCNC solution was prepared by dissolving PVA in a purchased cationic CNC solution (1% cCNC content) while maintaining a PVA concentration of 14%.

[0093] For electrospinning, 5 mL of each solution was delivered to a rotating drum covered with aluminum foil at a distance of 210 cm at a 40° angle at a constant flow rate of 2 mL / h. The drum's rotation speed was maintained at 3000 rpm. The operating voltage was 20.0 kV / cm at 40% relative humidity.

[0095] Example 5: Fabrication of a composite scaffold

[0096] A nanofiber-layered PCL scaffold was fabricated by manually rolling fixed-dimensional PVA and PVA@cCNC nanofibers onto the side walls of a 3D-printed PCL scaffold. The composition was then placed in a humidified chamber for 30 minutes to promote moisture-induced dissolution of the PVA. The composite scaffold was placed in a custom-sealed chamber saturated with 50% glutaraldehyde vapor for 30 minutes to crosslink the PVA nanofibers. The scaffold was immersed in PBS and DMEM media to completely remove excess glutaraldehyde.

[0098] Example 6: Chemical Characterization

[0099] Morphological analysis

[0100] The morphology of the printed scaffold was analyzed using a scanning electron microscope (UR-SEM, Hitachi-S4800, California, USA) with an acceleration voltage of 15.0 kV / cm.

[0101] FT-IR analysis

[0102] 4cm using a Perkin Elmer FTIR analyzer (Frontier, Perkin Elmer, UK) -1 4000-1000cm with a resolution -1 Functional groups present in the sample were evaluated in transmission mode within the wavenumber range.

[0103] Tensile test

[0104] The mechanical properties of the fabricated scaffold were evaluated through tensile testing using a universal testing machine (UTM; MCT-1150, AND Inc., Japan).

[0105] Analysis was performed at a constant speed of 10 mm / min. Each sample was prepared in triple layers.

[0106] Contact angle measurement

[0107] To evaluate the surface hydrophilicity of the developed structures, the water dispersion of the PCL, PA, PAP, and PAPC surfaces was measured using a Contact Angle Analyzer (SEO-Phoenix MT (MAT), Malaysia). The hydrophilicity of the samples was evaluated via the adhesion drop method. A 5 μL drop was deposited using a syringe mounted vertically on the film surface. After applying water to the surface for 10 seconds, the contact angle (θ) and wetting energy at the interface were measured and recorded using a charge-coupled device (CCD) camera.

[0108] Decomposition study

[0109] The degradation behavior of the developed scaffold was investigated in PBS (0.2M) and protease solution at 25°C with some modifications, as previously reported elsewhere [Wan Y, Cao X, Wu Q, Zhang S, Wang S. Preparation and mechanical properties of poly (chitosan-g*?*DL*?*lactic acid) fibrous mesh scaffolds. Polymers for Advanced Technologies. 2008;19:114-23].

[0110] In this study, an enzyme concentration of 1 mg / mL was measured. To achieve this, the scaffold was immersed in a PBS and protease solution for a predetermined time. The scaffold was then removed from the PBS and protease medium and rinsed with distilled water. Excess water was extracted using tissue paper. The weight of the dried scaffold was measured. After 3 days, the protease solution was replaced. In vitro degradation was calculated using the following formula.

[0111]

[0113] Here, Wi and Wd are the initial and disassembled scaffold weights, respectively.

[0115] Example 7: In vitro study

[0116] cell culture

[0117] To induce bone formation, cells were cultured in a bone formation induction medium containing DMEM supplemented with 50 μg / mL L-ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone. hBMSCs were obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and cultured at 37°C in a humid atmosphere of 5% CO2 (Steri-Cycle 370 incubator, Thermo Fisher Scientific, USA) using DMEM supplemented with penicillin (10,000 units / mL), streptomycin (10,000 μg / mL), and amphotericin B (25 μg / mL).

[0118] hBMSCs were seeded onto printed GelMA and GelMA-nHAp scaffolds and cultured for 24 hours under static conditions. After cell attachment, one hBMSC-laden GelMA and one GelMA-nHAp scaffold were transferred to a bioreactor. Cells cultured under static culture conditions on both scaffolds were taken as a control set. Substance 5 cells were used in the present invention.

[0119] Cell viability analysis

[0120] hBMSC(1 x 10 4 Canine cells (100 μL medium) were seeded into 96-well plates and cultured with the developed scaffold at 37°C in 5% CO2 for selected periods (1, 3, and 5 days). hBMSCs cultured without the scaffold were considered as a control. Cell viability was analyzed using the WST-8 assay. After the desired culture period, 10 μL of WST-8 dye was added and cultured for 2 hours. The resulting formazan was quantified by measuring absorbance at 450 nm (625 nm as the reference value). All experiments were performed in triplicate, and data are expressed as mean OD ± standard deviation. Statistical significance was considered at p* < 0.05.

[0121] Mineralization Research

[0122] The effect of FSS on the mineralization of hBMSCs was evaluated by the ARS procedure 7 days after stimulation. Cultured cells on scaffolds were rinsed with PBS. Cells were fixed and permeated with 1 mL of 70% absolute ice-cold ethanol at RT for 15 minutes. Permeated cells were stained with 500 μL of 40 mM ARS (pH 4.2) stain for 10 minutes, followed by washing with deionized water to remove excess staining. Mineralization was documented using a fluorescence microscope.

[0123] RNA isolation and real-time PCR (qRT-PCR) analysis

[0124] The expression of bone formation marker genes, mechanotransduction genes, and macrophage polarization markers in FSS-treated cells and control cells was evaluated using qRT-PCR technology.

[0125] In summary, according to the manufacturer's instructions, cells (4 × 10 4 After culturing dog cells (100 μL medium) in bone formation induction medium under experimental conditions for 7 and 14 days, RNA was extracted using TRIzol® reagent (Thermo Fisher Scientific, USA).

[0126] The purity and concentration of the extracted RNA were evaluated using a spectrophotometer. cDNA was synthesized from 2 μg of RNA using reverse transcriptase and SYBR Green Master mix. mRNA expression was quantified by Bio-Rad Real-Time PCR (CFX96™ Maestro Real-Time System, Bio-Rad, USA). The reaction conditions consisted of 43 cycles of denaturation at 95°C for 15 seconds and amplification at 60°C for 1 minute. All experiments were performed in triplicate and normalized to the housekeeping gene GAPDH. Relative mRNA expression was compared using histograms. All samples were prepared in triplicate during the experiment. Specific gene primers used for qRT-PCR analysis have been previously reported [Ganguly K, Dutta SD, Jeong MS, Patel DK, Cho SJ, Lim KT. Naturally-derived protein extract from Gryllus bimaculatus improves antioxidant properties and promotes osteogenic differentiation of hBMSCs. PloS one. 2021;16:e0249291;Dutta SD, Hexiu J, Patel DK, Ganguly K, Lim KT. 3D-printed bioactive and biodegradable hydrogel scaffolds of alginate / gelatin / cellulose nanocrystals for tissue engineering. International Journal of Biological Macromolecules. 2021;167:644-58].

[0127] Immunocytochemical staining

[0128] The expression of bone formation marker proteins was studied through immunocytochemical staining procedures. hBMSCs (4 × 10⁶ 4Canine cells (100 μL medium) were cultured for 7 days under static and FSS culture conditions. Medium without CPI was used as a control. Cell staining was performed by washing with PBS and fixing with 3.7% PFA for 15 minutes at room temperature. Next, cells were permeated by adding 0.1% Triton X-100 for 10 minutes at room temperature. Subsequently, cells were rinsed twice with PBS, blocked with 1% BSA, and incubated with 250 μL of mouse monoclonal antibodies against Runx2 and ALP. Nuclei were counterstained with 20 μL of 1 mg / mL DAPI solution for 2 minutes in the dark. Fluorescence images were captured using a fluorescence microscope at 40X magnification.

[0129] Transcriptome evaluation

[0130] Transcriptome analysis was performed to evaluate DEGs in various experimental groups. Seven days after osteogenic differentiation, total RNA was extracted using RNAzol (Sigma-Aldrich, USA) reagent and used to construct a standard RNA library. QuantiSeq 3' mRNA-Seq was performed using a next-generation RNA sequencer (Nova-Seq 6000, PE100 bp, CA, USA) in conjunction with the human reference genome hg19 and the UCSC genome database. Raw data were processed using ExDEGA graphical software (ebiogen, South Korea) and normalized by log2 (the mean of the normalized data for each group). Student's t-test was used for statistical analysis. Genes with a fold change greater than 2.0 and a p-value less than 0.05 were considered statistically significant. Potential interactions and co-expressed gene and protein networks were evaluated using STRING software (https: / / string-db.org / ).

[0132] Statistical analysis of all the aforementioned in vitro data was performed using OriginPro 9.0. One-way analysis of variance was used to determine statistical significance between the control and treatment groups. All data were expressed as mean and standard deviation. Significant differences were determined at *p<0.05, **p<0.01, and ***p<0.001. RNA sequencing data were normalized using TMM and CPM techniques with the edgeR utility. The obtained p-values ​​were adjusted for multiple comparisons with Benjamin and false discovery rates from DAVID software (https: / / david.ncifcrf.gov / tools.jsp).

[0134] Sample conditions PCL (g) PCL-Arg (g) PAP (g) PAPC (g) pH4 pH7 pH12 pH4 pH7 pH12 pH4 pH7 pH12 pH4 pH7 pH12 Buffer without enzyme 0.008 0.01 0.01 0.01 0.01 0.04 0.009 0.01 0.01 0.01 0.01 0.01 Buffer with enzyme 0.01 0.008 0.008 0.009 0.01 0.004 0.01 0.01 0.01 0.01 0.01 0.01

[0135] Table 1 shows the initial weight of the samples before incubating in various pH buffers.

[0136] Day(s) PCL (g) PCL-Arg (g) PAP (g) PAPC (g) pH4 pH7 pH12 pH4 pH7 pH12 pH4 pH7 pH12 pH4 pH7 pH12 0 0.008 0.01 0.01 0.01 0.01 0.004 0.009 0.01 0.01 0.01 0.01 0.01 1 0.008 0.01 0.01 0.01 0.01 0.004 0.009 0.01 0.01 0.01 0.01 0.01 2 0.008 0.01 0.01 0.01 0.01 0.004 0.009 0.01 0.01 0.01 0.01 0.01 3 0.008 0.01 0.01 0.01 0.01 0.004 0.009 0.01 0.01 0.01 0.01 0.01 4 0.008 0.01 0.01 0.01 0.01 0.003 0.009 0.01 0.01 0.01 0.01 0.01 5 0.008 0.01 0.01 0.01 0.01 0.004 0.009 0.01 0.01 0.01 0.01 0.01 6 0.008 0.01 0.01 0.01 0.01 0.004 0.009 0.01 0.01 0.01 0.01 0.01 7 0.008 0.01 0.01 0.01 0.01 0.004 0.009 0.01 0.01 0.01 0.01 0.01 14 0.008 0.01 0.01 0.01 0.01 0.004 0.009 0.01 0.01 0.01 0.01 0.01 21 0.008 0.01 0.01 0.01 0.01 0.004 0.009 0.01 0.01 0.01 0.01 0.01

[0137] Table 2 shows the remaining weight of the sample after incubating in various pH buffers without enzymes.

[0138] Day(s) PCL (g) PCL-Arg (g) PAP (g) PAPC (g) pH4 pH7 pH12 pH4 pH7 pH12 pH4 pH7 pH12 pH4 pH7 pH12 0 0.01 0.008 0.008 0.009 0.01 0.004 0.01 0.01 0.01 0.01 0.01 0.01 1 0.01 0.008 0.008 0.009 0.01 0.004 0.01 0.01 0.01 0.01 0.01 0.01 2 0.01 0.008 0.008 0.009 0.01 0.004 0.01 0.01 0.01 0.01 0.01 0.01 3 0.01 0.008 0.008 0.009 0.01 0.004 0.01 0.01 0.01 0.01 0.01 0.01 4 0.01 0.008 0.008 0.009 0.01 0.004 0.01 0.01 0.01 0.01 0.01 0.01 5 0.01 0.008 0.008 0.009 0.01 0.004 0.01 0.01 0.01 0.01 0.01 0.01 6 0.01 0.008 0.008 0.009 0.01 0.004 0.01 0.01 0.01 0.01 0.01 0.01 7 0.01 0.008 0.008 0.009 0.01 0.004 0.01 0.01 0.01 0.01 0.01 0.01 14 0.01 0.008 0.008 0.009 0.01 0.004 0.01 0.01 0.01 0.01 0.01 0.01 21 0.01 0.008 0.008 0.009 0.01 0.004 0.01 0.01 0.01 0.01 0.01 0.01

[0139] Table 3 shows the remaining weight of the sample after incubation with the enzyme.

[0140] Gene Symbol DN1..SN1 DC1..SC1 Description Il1rl1 1.651881985 0.632014093 interleukin 1 receptor-like 1 Casp8 0.563516441 0.210842675 caspase 8 L3mbtl3 0.449832853 0.503509535 l(3)mbt-like 3 (Drosophila) Socs1 0.156321535 -0.201241566 suppressor of cytokine signaling 1 Nrros 1.667564275 1.533087355 negative regulator of reactive oxygen species Vegfa -1.314047655 -1.190185526 vascular endothelial growth factor A Cd4 0 0.204919462 CD4 antigen Il15 -0.587635548 0.725537878 interleukin 15

[0141] Table 4 is a list of major DEGs associated with the macrophage differentiation process.

[0143] Gene Symbol DN / SN DC / SC Il6 0 0 Tgfb1 0.09973 0.745032 Nos2 -2.79842 -2.97561 Vegfa -1.31405 -1.19019 Arg1 -0.66649 -0.57311 Cd163 0 0

[0144] Table 5 shows the expression profiles of polarization marker genes identified by RNA-seq analysis.

[0146] Gene symbol DN / SN DC / SC Description Nrros 3.177 2.894 negative regulator of reactive oxygen species Csf1r 3.372 1.424 colony stimulating factor 1 receptor Tlr2 2.204 2.760 toll-like receptor 2 Csf1 1.458 1.330 colony stimulating factor 1 (macrophage) Tlr4 2.516 3.060 toll-like receptor 4 Itgam 2.150 1.219 integrin alpha M Il15 0.665 1.654 interleukin 15 Myd88 2.163 1.778 myeloid differentiation primary response gene 88

[0147] Table 6 shows the expression profile of DEG involved in both macrophage differentiation and inflammation, as revealed by RNA-seq analysis.

[0149] Gene Symbol DN1..SN1 DC1..SC1 Cnga3 -0.197617166 0.166991748 Slc26a9 -0.371397532 -0.406160934 Tmco1 0.615711021 -0.402071862 Kcnk2 -1.242894804 -0.705245287 Cacnb1 -0.75503746 -0.613034365 Kcnh4 0 0.204919462 Ryr2 0 -0.217326742 Vdac2 0.406198133 -0.011987098 Kcnma1 0 0.384319527 Cacna1d -1.175393754 -0.158305869 Gsdmd 0.260550834 -0.903004435 Apol9a 2.252135559 1.501547168 Apol9b 1.98471803 0.818121669 Ano6 0.080178732 1.105481638 Cacnb3 -0.14132254 0.778810765 Scn8a 0.054410915 -0.406160934 P2rx6 0.297347566 0.384319527 Clcn2 -1.172840783 1.171409252 Atp5o 0.114924004 -0.618102021 Kcne2 0.652792096 0 Kcnj6 0.156321535 0 Clcn7 0.19360271 0.881131291 Rhag 0 -0.012407431 Kcnk7 0.652792096 0.384319527 Best1 0.080798174 1.047521637 Trpm6 0.156321535 0 Trpm3 0 0.204919462 Atp5c1 -0.387056672 -0.609205548 Cacnb2 0.156321535 -0.997102805 Cacna1b 1.043441801 1.460482567 Grin1 -2.452251693 -2.793044768 Cacnb4 1.100584805 -0.040167094 Scn1a 0.297347566 0 Chrna1 -0.667125282 0.464220753 P2rx3 0.543760147 0 Ryr3 0.543760147 0.204919462 Catsper2 -0.911641812 1.036597576 Trpm7 -0.004575831 0.514796917 Slc4a11 1.324636663 1.089934012 Slc12a5 1.563050229 0.644576203 Kcnb1 -0.127764118 1.004173445 Kcnab1 0.346142228 0.687497015 Gria2 0 0.204919462 Chrnb2 -0.390705904 1.136338405 Clca4a 0 0.384319527 Gabrr2 0.297347566 0.204919462 Grin3a -0.526477395 0.384319527 Rhd 0.25761413 -0.318073319 Clcn6 -0.6664936 -0.217326742 Kcnab2 -0.492914972 -1.681377036 Cacna2d1 0 -0.217326742 Kcnh2 -0.371397532 0 Asic3 -0.265625677 -1.433620861 P2rx7 0.588586803 0.893367648 P2rx4 0.752731467 1.073619407 Orai1 0.84056797 -0.35744758 Ttyh3 -0.308206977 -0.112731162 Clcn1 0 -0.982191802 Itpr1 0.30341254 0.412980915 Cacna1c 0 -0.217326742 Ttyh1 0.382763521 0.281336233 Kcnn4 0.132872918 -0.652813545 Ryr1 0.263780807 0.616751961 Kcnc3 -0.524122787 -1.242083754 Slc17a7 0.93774588 1.151193154 Trpm4 -0.037891089 0.95846038 Kcnj14 0 0.384319527 Trpm1 0.126222248 -0.569953287 Chrna10 0 0.204919462 Kcnq1 0 0.384319527 Ano1 0.297347566 0.204919462 Tpcn2 0.22768317 -0.438334061 Mcoln1 0.33487829 0.895633007 Clcn3 -0.182391968 0.167207612 Kcnn1 1.629067936 1.306450659 Ano8 0.872654641 0.01235127 Tmem38a 0.305073992 0.276756298 Cacna1a 1.013311461 1.215156709 Cngb1 0 -0.217326742 Trpc1 -0.197617166 0 Cacna2d2 0 -0.573114819 Scn11a 1.577592213 1.336979142 Kcnd1 -1.152766321 3.211324536 Gria3 0 -0.217326742 Gabre -0.197617166 0

[0150] Table 7 shows the expression profile of DEG involved in ion channel activity revealed by RNA-seq analysis.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 A composition comprising, as an active ingredient, a composite scaffold prepared by a method for preparing a nanofiber laminated PCL scaffold comprising preparing a polycaprolactone (PCL) scaffold by 3D printing, preparing cellulose nanocrystals and polyvinyl alcohol nanofibers through electrospinning after adding polyvinyl alcohol to cellulose nanocrystals, and rolling said nanofibers onto the side wall of said 3D printed PCL scaffold, wherein the composition is characterized by combining a fluid shear stress (FFS) treatment process. Claim 9 delete Claim 10 In claim 8, the above composition is an antibiotic composition for viruses, Gram-positive bacteria, and Gram-negative bacteria, characterized by increasing the expression of Tlr4 and IL 15 genes. Claim 11 delete Claim 12 delete

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