KR102510662B1 - Three-dimensional scaffold with microchannels and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a three-dimensional scaffold having microchannels and a method for manufacturing the same, a porous scaffold for tissue regeneration having a hierarchical microchannel structure, and a solution in which polycaprolactone and camphene are dissolved in a solvent are mixed with ethanol and distilled water. It relates to a method for manufacturing a porous scaffold for tissue regeneration that is ejected and molded into the body.
When implanted in vivo, the scaffold of the present invention induces unique biological responses such as regulating immune or inflammatory responses, promoting angiogenesis, and promoting stem cell recruitment without the help of exogenous biological molecules, cells, or drugs, thereby inducing damage or disease. can effectively repair and regenerate tissues that receive it. In addition, according to the manufacturing method of the present invention, scaffolds having such excellent effects can be efficiently manufactured, and in particular, 3D printing technology can be applied, enabling easy and mass production.

Description

Three-dimensional scaffold with microchannels and manufacturing method thereof

The present invention relates to a three-dimensional scaffold having microchannels and a method for manufacturing the same, a porous scaffold for tissue regeneration having a hierarchical microchannel structure, and a solution in which polycaprolactone and camphene are dissolved in a solvent are mixed with ethanol and distilled water. It relates to a method for manufacturing a porous scaffold for tissue regeneration, which is ejected and molded into the body.

The in vivo microenvironment of damaged and diseased tissue can ultimately be controlled through extrinsic treatment to accelerate the process of repair and regeneration. While the use of biological components such as bioactive molecules and cells holds great potential for restoring regenerative function, there are still many hurdles to overcome. For example, obtaining cells requires an additional surgical biopsy of donor tissue and multiple steps to expand purified cells in vitro. The regenerative effect of stem cells (eg, mesenchymal stem cells (MSC)) in clinical trials has not yet been clearly demonstrated. Moreover, bioactive signaling molecules, such as growth factors and engineered peptides, are susceptible to proteolysis in the body, requiring high doses otherwise their biological potency is compromised.

On the other hand, if the fabricated scaffold can interact with endogenous biological entities (molecules and cells) to correctly reproduce the developmental process of the in vivo microenvironment, many practical advantages including safety and high clinical utility are considered. Given that cells communicate in vivo with the extracellular matrix (ECM) for feedback and adopt surrounding physiological conditions, the provided (with exogenous ECM) scaffold material reproduces the developmental process of the microenvironment and tissue repair and remodeling. can regulate a series of biological events related to

Making scaffolds and matrices with appropriate physiochemical properties (e.g., ligand type and density, nano/microtopology, strength, and pore structure) is important for immune-response, angiogenesis, cell homing, implantation, and It has been shown to influence several important events in tissue healing and repair, including differentiation. For example, surface chemistry altered by functional groups or bound ligands has had a profound effect on the adhesion and growth of various cell types. Nano-/micro-patterned surfaces can guide the orientation and migration of cells, and the strength (or stiffness) of gels is often tuned to direct MSCs to a bone- or adipogenic lineage. Moreover, the pore structure of a scaffold is an important three-dimensional feature that can significantly affect angiogenesis and tissue invasion.

In addition to these properties, the present invention addresses, interestingly, new features of scaffolds - 'microchannels' - responsible for many biological events in a unique way that aid in tissue healing and repair. In particular, this is in stark contrast to the phenomenon observed in conventional non-microchannelized scaffolds. Since the microchannels (tens of micrometers) of the present invention are open-channel microchannels and constitute the entire stem of a three-dimensional (3D)-printing scaffold, macroporous 3D geometries (hundreds of micrometers) generally made by 3D printing programs meter) can be regarded as an additional third dimension. The present inventors have studied in vivo biological (biomolecules and cells) microchannelized scaffolds (referred to as 'μCh') in terms of immune/inflammatory responses, angiogenesis, and stem cell homing and transplantation from hours to days to weeks. attention was paid to the reaction. This series of tissue reactions is commonly used to evaluate the biocompatibility of a material and is therefore considered a milestone for tissue healing and regenerative materials. In particular, the recruitment of early immune and inflammatory cells (eg, neutrophils, monocytes, macrophages) and the types of chemokines and cytokines secreted have been found to be important in determining several subsequent biological steps involved in tissue regeneration. Moreover, facilitating the migration and transplantation of resident stem cells to damaged sites is an attractive strategy to turn a highly damaged state into a repair and regenerative phase. In addition, a microenvironment capable of promoting neovascularization is very useful for nutrient supply, cell survival and invasion, and ultimately promotion of tissue healing cell function. Therefore, designing and discovering 3D scaffolds that can advantageously modulate these biological events are important for regenerative therapies using materials without extrinsic help from cells and biological molecules.

S. Pacelli, S. Basu, J. Whitlow, A. Chakravarti, F. Acosta, A. Varshney, S. Modaresi, C. Berkland, A. Paul, Strategies to develop endogenous stem cell-recruiting bioactive materials for tissue repair and regeneration, Adv. Drug Deliv. Rev. 120 (2017) 50-70.

Accordingly, the main object of the present invention is to provide a scaffold capable of effectively repairing and regenerating damaged or disease-affected tissues without the help of exogenous biological molecules or biological components such as cells.

Another object of the present invention is to provide a method for efficiently producing the above scaffold.

According to one aspect of the present invention, the present invention provides a porous scaffold for tissue regeneration having a hierarchical microchannel structure.

In the scaffold of the present invention, it is preferable that the porosity of the microchannel of the scaffold is 5% or more.

In the scaffold of the present invention, the scaffold is preferably made of polycaprolactone.

In the scaffold of the present invention, the scaffold is preferably manufactured by a molding method in which a solution in which polycaprolactone and camphene are dissolved in a solvent is discharged into a mixed solution of ethanol and distilled water.

In the scaffold of the present invention, the molding method is preferably a molding method in which a plurality of struts cross each other to form air gaps.

In the scaffold of the present invention, it is preferable that the temperature of the mixed solution of ethanol and distilled water is 30 to 50 ° C.

In the scaffold of the present invention, the solution in which polycaprolactone and camphene are dissolved in a solvent is preferably a solution in which polycaprolactone and camphene are dissolved in a weight ratio of 1:2 to 2:1.

In the scaffold of the present invention, the solvent is preferably acetone.

According to another aspect of the present invention, the present invention provides a method for manufacturing a porous scaffold for tissue regeneration by discharging a solution in which polycaprolactone and camphene are dissolved in a solvent into a mixed solution of ethanol and distilled water.

In the manufacturing method of the present invention, it is preferable to form a plurality of struts while crossing each other to form air gaps.

In the production method of the present invention, it is preferable that the temperature of the mixed solution of ethanol and distilled water is 30 to 50 ° C.

In the production method of the present invention, the solution in which polycaprolactone and camphene are dissolved in a solvent is preferably a solution in which polycaprolactone and camphene are dissolved in a weight ratio of 1:2 to 2:1.

In the production method of the present invention, the solvent is preferably acetone.

When implanted in vivo, the scaffold of the present invention induces unique biological responses such as regulating immune or inflammatory responses, promoting angiogenesis, and promoting stem cell recruitment without the help of exogenous biological molecules, cells, or drugs, thereby inducing damage or disease. can effectively repair and regenerate tissues that receive it. In addition, according to the manufacturing method of the present invention, scaffolds having such excellent effects can be efficiently manufactured, and in particular, 3D printing technology can be applied, enabling easy and mass production.

1 shows a manufacturing process of a scaffold (μCh) according to an embodiment of the present invention and characteristics of the prepared scaffold. (A) Manufacturing process of the scaffold. (B) Differences in the microstructure of scaffolds under various fabrication conditions. (C) SEM cross-sectional image of μCh. (D) Digital image of highly open channelized pores (red) and struts (green) in μCh. (E) Pore size distribution of μCh. (F) Comparison of physical properties related to microchannel formation of μCh and non-scaffold (control).
2 shows the in vivo immune/inflammatory response of the scaffold. (A) SEM images of cells adhered on each scaffold (Non and µCh) retrieved 24 hours after transplantation, with Image J showing adhered cells in yellow. (B) Fluorescence images of extracellular DNA stained with SYTOX (green) and nuclei stained with DAPI (blue), co-stained at 24 h, and (C) blue/green signal ratio, data values are shown as Mean±SD derived from 6 microscopic regions of 3 samples in group, statistical analysis performed using two-tailed t-test (***P < 0.001 vs. Non). (D, E) Immunostaining of cells at day 7; (D) macrophages co-stained with F4/80 (red) and CD11b (green), and (E) NOS2 (M1-specific marker, red) and arginase (ARG, M2-specific marker, green). Specific phenotypes visualized. (F) Image-based quantification of macrophage positive populations for NOS, ARG, or NOS/ARG. Data values are derived from three microscope images (x 200) for each specimen. S: scaffold area.
Figure 3 shows stem cell recruitment by μCh. (A) Scheme of an in vivo experiment to investigate endogenous cell recruitment. (B, C) Immunohistochemical staining of cells on day 14, showing the distribution of MSCs infiltrating the scaffold. (B) CD90-positive MSCs (green) co-stained with the fibroblast marker (red), low magnification, and (C) positive markers CD90 and CD54, high magnification of MSCs (green). White arrows: endogenous MSCs, S: transplanted scaffolds. (D) Ex vivo experimental design to analyze the effects on stem cell migration and chemokine secretion. (E) Optical images of migrating MSCs stained with crystal violet after transplanted scaffolds for 3 and 7 days were cultured in transwells for 24 h. (F) Quantification of migrated MSCs. (G) Detection of SDF-1 released from the recovered scaffold. Data values are means ± SD from 5 microscopic regions and 6 samples for each specimen. Statistical analysis was performed using one-way ANOVA with Tukey's post-hoc test (***P < 0.001 vs. Non).
Figure 4 shows the stimulation of angiogenesis by μCh. (A) In vivo experimental scheme to investigate neo-angiogenesis in scaffolds implanted for 14 days. (B) Immunohistochemical staining of cells with CD31 on day 14 after transplantation, showing neo-angiogenesis. Red arrows: newly formed blood vessels, S: transplanted scaffold area. (C) Quantification of number and area of blood vessels generated. Data values are mean ± SD derived from three microscopic regions of three specimens in each group. Statistical analysis was performed using a two-tailed t-test (***P < 0.001 vs. Non). (D) Extracellular experimental design to analyze the effect on endothelial function and angiogenic factor secretion. (E) Fluorescence image of tubes formed by HUVECs cultured for 4 hours with scaffolds recovered on day 3. Control medium supplemented with growth factors was used as a positive control. (F) Quantification of tube number and area. Data values are mean ± SD from 5 microscopic regions in each group. Statistical analysis was performed using one-way ANOVA (*P < 0.05 and **P < 0.01 vs. Non). (G) Quantification of VEGF released from scaffolds recovered on days 3 and 7. Statistical analysis was performed using a two-tailed t-test (*P < 0.05 and **P < 0.01 vs. Non).
Figure 5 shows reduced fibrosis by the μCh scaffold. (A) MT staining results of tissue samples on days 7, 14, and 21 show that fibrosis (bluish areas) is induced at the scaffold/tissue interface. S: implanted scaffold area. (B) Quantification of fibrous tissue thickness. Data values are means±SD from 5 microscopic regions of each specimen. Statistical analysis was performed by one-way ANOVA with Tukey's post-hoc test (***P < 0.001 vs. Non).
Figure 6 shows the molecules sequestered on the scaffold. (A) Total protein quantification from scaffolds recovered at 24 hours. Data values are mean±SD (n=3). Statistical analysis was performed using a two-tailed t-test (***P < 0.001 vs. Non). (B) Expression of putative ECM proteins (fibronectin, vitronectin, and fibrinogen) detected by Western blot and Ponceau S staining. (C) Immunostaining results of adsorbed fibronectin and vitronectin. (D) Protein analysis results of small molecules adsorbed to the scaffold recovered at 24 hours. (E) Representative proteins expressed at more than two-fold difference compared between scaffold groups.
Figure 7 shows the sequence of events occurring in the implanted μCh scaffold. Summarize these events over time frames of hours, days, weeks, months, as inferred from experimental results. These events, including neutrophil response, macrophage polarization, MSC recruitment and angiogenesis, reduce fibrotic capsule formation, which can favor accelerated tissue regeneration, such as bone regeneration. The ability of μCh scaffolds to sequester potential molecules (e.g., adhesive proteins, chemokines, and growth factors) that are collected in a timely manner or secreted from infiltrated cells may have an impact.
8 shows the ability of μCh in a bone regeneration model. (A) X-ray images of samples implanted in a rat cranial canal defect model for 6 weeks. (B) μCT images of samples implanted in a rat cranial canal defect model for 6 weeks. (C) Results of quantifying the volume, surface area, and surface density of newly formed bone in the defect area. Data values are mean±SD (n=3). Statistical analysis was performed using a two-tailed t-test (*P < 0.05 vs. Non). (D) H&E staining result of the sample. S: implanted scaffold area, OB: existing bone, NB: new bone. (E) Results of staining with Sirius red S and fast green to visualize deposited collagen and non-collagen proteins, respectively. S: implanted scaffold area. (F) Quantification results of proteins. Data values are means±SD from the four slide regions of each sample. Statistical analysis was performed using a two-tailed t-test (**P < 0.01 vs. Non).

Since the present invention can apply various transformations and have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, it should be understood that this is not intended to limit the present invention to specific embodiments, and includes all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

Terms used in this application are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, the terms "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Terms such as first and second may be used to describe various components, but the components should not be limited by the terms. These terms are only used for the purpose of distinguishing one component from another.

The scaffold of the present invention is a porous scaffold for tissue regeneration having a hierarchical microchannel structure, and the present invention was devised through new experimental results proving that the scaffold of the present invention has excellent effects related to tissue regeneration. .

According to the experimental results, the scaffold of the present invention induces unique biological responses such as regulating immune or inflammatory responses, promoting angiogenesis, and promoting stem cell recruitment when implanted in vivo, ultimately exhibiting excellent effects on tissue regeneration. . In particular, there is a great advantage in that such a reaction can be performed without the help of foreign chemokines or cytokines, separate drugs or cells. Among the possible reasons for this, it is believed that the altered profile of sequestering molecules involved in early cell adhesion and inflammation by the scaffold of the present invention plays an important role in the spatiotemporal regulation of the microenvironment, accompanying the subsequent beneficial cellular response. A series of interactions of these cells and molecules can orchestrate the repair and remodeling of tissues, including missing bone.

In the present invention, a 'microchannel' or 'micropore' refers to a channel or void having a diameter of several to several tens of μm, and may be distinguished from a channel having a diameter of hundreds of μm. To be more specific, in the present invention, 'microchannel' or 'micropore' refers to a channel or pore with a diameter greater than 1 μm and less than 100 μm. The scaffold of the present invention has a plurality of such microchannels.

Preferably, the scaffold of the present invention has an average microchannel diameter of 2 to 90 μm, more preferably 2 to 70 μm, more preferably 2 to 60 μm, more preferably 2 to 50 μm, more preferably is 5 to 40 μm, more preferably 10 to 30 μm.

The scaffold of the present invention preferably has a microchannel porosity of 5% or more, more preferably 10% or more, more preferably 20% or more based on the porosity calculated by the Archimedes method. With respect to the lower limit of each microchannel presented above, the porosity of the microchannel may be 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less.

In the present invention, 'hierarchical' in relation to channels or pores means that the channels or pores are layered according to the size of their diameter. For example, it may be layered with channels or pores between 1 and 90 μm in diameter and channels or pores with a diameter of 100 μm or more. It may preferably be layered with channels or pores between 1 and 50 μm in diameter and channels or pores between 100 and 500 μm in diameter.

The scaffold of the present invention is preferably a polymer material, more preferably a biodegradable polymer material, and more preferably a polycaprolactone material.

In addition, the scaffold of the present invention is preferably prepared by the manufacturing method of the present invention.

The present invention provides a method for manufacturing a porous scaffold for tissue regeneration in which a solution in which polycaprolactone and camphene are dissolved in a solvent is discharged into a mixed solution of ethanol and distilled water to form a scaffold as described above. . In this method, a polymer molded article is manufactured by discharging a polymer solution into a quenching medium. Here, a solution in which polycaprolactone and camphene are dissolved in a solvent corresponds to the polymer solution, and a mixed solution of ethanol and distilled water is used for quenching. It can be considered as medium. According to this manufacturing method, it is possible to manufacture a scaffold made of polycaprolactone and having a plurality of microchannels formed by sublimation of camphene. In addition, this method has the advantage that scaffolds of various shapes can be manufactured according to the molding method, and 3D printing technology can be applied, enabling easy production and mass production.

In the manufacturing method of the present invention, for the hierarchical porous structure of the scaffold, preferably, a plurality of struts are molded to form air gaps while crossing each other. For example, as shown in FIG. 1A, the polymer solution is discharged into the quenching medium so that a plurality of layers consisting of a plurality of parallel struts are alternately formed. According to this, channels or voids can be formed at the intersection of the struts, and the size of the channels or voids can be adjusted by the same method as adjusting the spacing between the struts, so that microchannels and hierarchical It is possible to easily form channels or voids distinguished by. At this time, it is preferable that the size of the channel or void created by the intersection of the struts has a diameter of 100 to 500 μm.

In the production method of the present invention, it is preferable to set the temperature of the mixed solution of ethanol and distilled water to 30 to 40 °C. According to the experimental results of the present invention, the structure (particularly, the size) of the channel formed on the scaffold may vary depending on the difference in temperature. When the above temperature conditions are used, the structure is suitable for exhibiting excellent tissue regeneration effect. of microchannels can be formed.

In the production method of the present invention, the solution in which polycaprolactone and camphene are dissolved in a solvent is preferably a solution in which polycaprolactone and camphene are dissolved in a weight ratio of 1:2 to 2:1. More preferably, the weight ratio of polycaprolactone and camphene is 1: 1.5 to 1.5: 1, more preferably 1: 1.3 to 1.3: 1, more preferably 1: 1.2 to 1.2: 1, more preferably Preferably, it is a solution dissolved in a weight ratio of 1:1.1 to 1.1:1.

In the production method of the present invention, the solvent is preferably acetone. In this case, polycaprolactone and camphene are preferably dissolved at 1 to 50% (w/w), more preferably at 10 to 50% (w/w), more preferably at 20 to 40% (w/w) , more preferably dissolved at 25 to 35% (w/w).

Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

1. Materials and Methods

1.1. 3D Printing and Characterization of Scaffolds with Hierarchical Microchannels

1.1.1. Raw material solutions and 3D printing

Polycaprolactone (PCL) (MW 80000 Da, Sigma-Aldrich, St. Louis, MO, USA) pellets were dissolved in acetone at 30% (w/w). To create microchannel pores in struts, camphene (C ₁₀ H ₁₁ , Sigma-Aldrich, Missouri, USA) was dissolved together with PCL in acetone (Daejeong, Korea). The ratio of camphene/PLC was 1:1 by weight. This composite solution was homogenized overnight by hot ball milling at 50°C.

The scaffold was designed with a CAD (computer aided design) program, and the solution ejection was performed using a 3D printing system (Ez-ROBO3, Iwashita, Japan). The scaffold was made in a grid shape (size of 12 mm × 12 mm × 1 mm) with square pores of 300 μm × 300 μm. The PCL and camphene-PCL solutions were discharged through a nozzle into a temperature-controlled ethanol/distilled water (1:1 volume ratio) bath using continuous air pressure. The resulting struts were stacked to create a three-dimensional scaffold. The temperature of the water bath was maintained at 0 or 37°C. After the scaffold solidified, it was dried overnight at room temperature.

1.1.2. Characterization of 3D scaffolds

The surface morphology and microstructure of the scaffold were observed with a scanning electron microscope (SEM; JSM-6510, JEOL, Japan). The size of micropores was measured from SEM images. The interconnected pore structure was characterized by 3D X-ray microtomography (ZEISS) with a resolution of 500 nm, and torsion properties were calculated (n=40). To confirm that camphene was completely removed from the scaffold, ATR-FT-IR (Varian 640-IR, Australia) was used. Porosity was measured using the Archimedes method. Briefly, the weight of the scaffold (Ws), the weight of pure ethanol (W1), the weight of scaffold + ethanol (W2), and the weight of ethanol after removing the scaffold (W3) were measured using the following formula and the porosity was calculated.

Porosity (%) =

Surface area and density were measured using a mercury porosimetry (PM33, Quantachrome, Florida, USA). Total water uptake was calculated using the following formula using the starting weight (W0) and the weight of the scaffold after water uptake (W1).

Total water absorption (%) =

For cross-sectional images, samples were cut with a cryotome and stained with FITC dye. Images obtained with a fluorescence microscope (Life Technology, New York, USA) were analyzed with Image J software. The mechanical behavior of the scaffold was examined by Instron. A sample prepared in a size of 5 mm × 5 mm × 5 mm was pressed with a 1 kN load cell and a stress-strain curve was recorded.

1.2. Implantation of scaffolds in the subcutaneous site and analysis of tissue samples

1.2.1. Surgical procedure and sampling

Animal experiments were approved by the Animal Care and Use Committee of Ajou University. Animal care and experimental procedures were performed in accordance with the guidelines of the National Institutes of Health. Before surgery, the 3D printed scaffold was sterilized with ethylene oxide gas. C57BL/6 mice (male, 6 weeks old) and Sprague-Dawley rats (male, 6 weeks old) were used for scaffold implantation. Animals were anesthetized with a solution of tiletamine-zolazepam (30 mg/kg) and xylazine (10 mg/kg). To implant the scaffold, a 13 mm incision was made on each dorsal side of the rat and mouse. Each sample was placed in a subcutaneous pouch. After suturing, animals were maintained for as long as needed for analysis. For protein adsorption and immune cell infiltration experiments, the scaffold was first inserted into a stainless steel cage and then implanted into the subcutaneous tissue.

1.2.2. Observation of neutrophils and extracellular trap formation

The morphology of neutrophils adhering to the scaffold was observed by SEM. Twenty-four hours after implantation, samples were collected. Samples were washed with cold PBS and fixed for 4 hours at 4° C. in a 2.5% glutaraldehyde-2% paraformaldehyde solution. At 5 min intervals, samples were dehydrated with a graded series of ethanol solutions. Finally, the sample was rinsed twice with hexamethyldisilazane (HMDS, Sigma-Aldrich, Mo., USA) and dried in a fume hood. SYTOX green (Thermo Fisher Scientific, Massachusetts, USA) dye was used to detect extracellular nucleic acids in early immune cells. After 24 hours of implantation, the recovered samples were washed with cold PBS and fixed in a 4% formaldehyde solution at 4°C for 4 hours. Samples were then incubated with SYTOX green solution for 15 minutes. Intracellular nuclei were stained with Hoechst 33342 solution.

1.2.3. Macrophage invasion and differentiation

Samples recovered on day 7 from transplantation were washed with cold PBS and fixed in 1% formaldehyde solution for 4 hours at 4°C. After washing three times with PBS, the samples were soaked in a 20% sucrose solution at 4°C until they settled to the bottom. For cryosectioning, OCT compound (Thermo Fisher Scientific, Massachusetts, USA) was added to the sample and frozen at -20°C. Using a cryostat, frozen samples were cut to a thickness of 4 μm and transferred to coated slides. Slides were blocked with 1% (w/v) bovine serum albumin (BSA) for 30 min and incubated in CD11b, F4/80, NOS2, and arginase (ARG) primary antibody solution (1 in PBS) for 2 h at room temperature. :100 dilution, Santa Cruz Biotechnology, CA, USA). The slides were then incubated for 1 hour with the secondary antibody along with the specific marker probe solution. Cell nuclei were stained with Hoechst 33342 solution for 3 minutes and samples were mounted.

1.2.4. Detection of endogenously colonized MSCs

Samples collected on days 3 and 7 were washed and fixed. After soaking in a 20% sucrose solution, the OCT compound was added to the washed sample, frozen at -20 ° C, cut and transferred to a slide. To observe endogenous MSCs, slides were blocked and treated with CD90 and CD54 antibody solutions (1:100 dilution in PBS, Santa Cruz Biotechnology, CA, USA) for 2 hours at room temperature. Fibroblasts were stained with a fibroblast marker antibody (1:100 dilution in PBS, Santa Cruz Biotechnology, CA, USA). After washing 3 times, slides were incubated with secondary antibody for 1 hour and counterstained with Hoechst 33342 solution.

1.2.5. In vivo observation of angiogenesis

Sample slides obtained from the aforementioned frozen sectioning procedure were dehydrated with serial ethanol. Samples were pretreated with H ₂ O ₂ and pepsin for antigen retrieval. Samples were blocked and treated sequentially with CD31 antibody solution (1:100 dilution in PBS, Santa Cruz Biotechnology, CA, USA), secondary antibody solution, and streptavidin-HRP. After DAB development, samples were counterstained with hematoxylin, dehydrated with ethanol and xylene, and mounted.

1.2.6. fibrosis assay

Scaffolds implanted for 7, 14, and 21 days were used for fibrosis analysis. At each period, samples were collected, frozen sectioned on slides, and stained with Mason's Trichrome (MT) staining kit (Polysciences, Inc., Pa., USA). Fibrosis thickness was measured from the collagen fibers (blue) formed around the scaffold based on the optical images.

1.3. In vitro study of stem cell establishment and angiogenesis

1.3.1. Migration test of human MSCs

For migration studies, human tonsillar-derived MSCs (hMSCs) were used after approval by the Bioethics Committee of Ajou University Hospital (AJIRB-BMR-SMP-17-494). The procedure was performed according to the relevant regulations. Human tonsil tissues were obtained from the Department of Otorhinolaryngology, Ajou University Hospital (Suwon, Korea). Tissue discarded during surgery was used for cell extraction, and informed consent was obtained from all donors or parents or legal representatives. Tonsil tissue was digested in collagenase solution (0.075%) (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37°C, and the extracted cells were incubated in 1% antibiotic-antimycotic (Invitrogen-Gibco, Carlsbad, CA). California, USA) and αMEM medium supplemented with 10% PBS were resuspended and maintained.

To identify hMSCs, the extracted cells were analyzed using FACS (fluorescence-activated cell sorting). Assays were performed with MSCs from passage 4. Fluorescent dye-conjugated antibodies (CD90, CD29, CD44, CD45, and HLA-DR) were bound to each specific antigen on the hMSC surface. Adipogenesis, osteogenesis, and chondrogenesis of hMSC were confirmed by culturing in each induction medium. For lipogenesis, hMSCs were maintained in medium containing 5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 1 mM dexamethasone (Sigma-Aldrich), and 10 mg/mL insulin (Sigma-Aldrich). After 14 days, the cultured cells were stained with Oil Red O staining solution. To the osteogenic medium, 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 50 μM L-ascorbic acid (Sigma-Aldrich), and 100 nM dexamethasone were added. Differentiation was confirmed by Alizarin Red S (ARS; Sigma-Aldrich) staining. Chondrogenesis was assessed with 1x insulin-transferrin-selenium-G, 100 nM dexamethasone, 100 μg/ml sodium pyruvate, 1.25 mg/ml BSA, 50 μg/ml ascorbic acid, 40 μg/ml L-proline, and 10 ng/ml TGF-beta. was induced in low-glucose Dulbecco's Modified Eagle's medium (DMEM; Gibco). Chondrogenic cells were stained with Safranin O.

The hMSC migration assay was performed using a transwell chamber (24-well format with 8 μm pore size; Costar, Cambridge, MA, USA). MSC (1×10 ⁴ ) was added on top of the insert along with low-FBS medium (α-MEM with 3% FBS; Welgene, Daegu, Korea). After 24 hours, samples harvested on day 3 or day 7 of implantation were placed at the bottom of the well. hMSCs were co-cultured for 24 hours, fixed with 5% glutaraldehyde, and stained with 1% crystal violet solution. Images were obtained with optical and fluorescence microscopy (EVOS FL Auto, Thermo Fisher Scientific, Massachusetts, USA).

1.3.2. Tube formation in HUVECs

HUVECs from passage 4 (PCS-100-010, ATCC) were used for tube formation assays. HUVECs were cultured in vascular cell basal medium (Gibco, Carlsbad, CA, USA) supplemented with endothelial cell growth kit-VEGF (Gibco). Cells were cultured at 37°C under 5% CO ₂ conditions. For the tube formation assay, a transwell chamber with 0.4 μm pore size was used. First, the wells were coated with Matrigel and incubated at 37° C. for 1 hour. 1×10 ⁵ HUVEC (ATCC, Manassas, VA, USA) was seeded on Matrigel. At the same time, the withdrawn sample was added to the top of the transwell. As a positive control, HUVECs were cultured in high-glucose DMEM containing low-serum growth supplement (Welgene, Daegu, Korea). High-glucose DMEM without supplements was used for the test group. After 4 hours, tube formation was observed under a microscope and stained with Calcein AM (Thermo Fisher Scientific, Massachusetts, USA).

1.3.3. Protein analysis using ELISA

After migration and tube formation tests, the presence of VEGF and SDF-1 in the recovered sample scaffolds was determined using VEGF and SDF-1 ELISA (enzyme-linked immunosorbent assay) kits (R&D system, Minnesota, USA), respectively. Quantification was performed according to the protocol.

1.4. Analysis of isolated molecules

1.4.1. Quantification of total protein

To measure the amount of total protein, samples collected 24 hours after transplantation were washed twice with cold PBS. Then, the samples were immersed on ice for 30 minutes in a lysis buffer containing proteinase and phosphatase inhibitors. The lysate was centrifuged at 13000 rpm for 20 minutes. The amount of total protein in the supernatant was measured using a protein assay kit (DC™, Bio-Rad Laboratories, Inc., CA, USA) based on the Bradford dye-binding method.

1.4.2. Detection of ECM molecules

After protein quantification, lysates were loaded on a 12% SDS/PAGE gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane. The position of the protein band was confirmed with Ponceau S solution. Membranes were blocked with 5% skim milk solution and incubated with fibronectin, vitronectin, or fibrinogen antibodies (1:1000, Santa Cruz Biotechnology, CA, USA). The HRP-linked secondary antibody was then combined with the primary antibody (1:2000). Bands were visualized using ECL reagent (Amersham, Piscataway, NJ, USA) and detected with LAS-4000 (Fuji, Japan). For immunohistochemistry, the collected samples were cryo-sectioned, blocked, and treated with fibronectin or vitronectin antibody solution (1:100 dilution in PBS, Santa Cruz Biotechnology, CA, USA) at room temperature for 2 hours. . Samples were then incubated for 1 hour with secondary antibodies along with specific marker probes. Samples were mounted without counterstaining and then visualized under a confocal laser scanning microscope.

1.4.3. proteomic analysis

Cytokines, chemokines, and growth factors sequestered in the scaffold were analyzed from samples collected 24 hours after transplantation. The lysate obtained from the sample was analyzed using a protein assay kit, and an equal amount of the protein lysate was loaded onto the membrane of a proteomic profiler array kit (R&D system, Minnesota, USA). The experimental procedure followed the manufacturer's instructions. The protein analysis results were visualized using a LAS device, and the intensity was quantified by gel analysis using ImageJ software.

1.5. Bone defect experiment in vivo

1.5.1. Cranial tube defect model and manipulation

This animal study was approved by the Animal Care and Use Committee of Ajou University. Animals (Sprague-Dawley rats, males, 10 weeks old) were maintained in a controlled environment with a humidity of 30-70% and a temperature of 20-24°C. Protocols for animal care followed guidelines from the National Institutes of Health. Anesthesia was performed as previously described. A longitudinal incision was made on the skull and two bone defects of 5 mm diameter were made in each rat using a trepine bur. Thereafter, the missing portion was filled with a scaffold prepared with a diameter of 5 mm and a thickness of 1 mm.

1.5.2. Evaluation of new bone formation

To visualize new bone growth in the calvarial defect model, a μCT scanner (Skyscan 1176; Skyscan, Artcellar, Belgium) was used. Six weeks after implantation, samples were collected and fixed with 4% formaldehyde. Based on the reconstructed 3D images using the computer analysis program CTAn (Skyscan), new bone growth was measured in the cylindrical region of interest, and the newly formed bone volume and surface area were obtained.

For histological analysis, calcium was removed from the samples with rapid Cal solution (BBC Chemical Co., Stanwood, WA, USA). Samples were dehydrated using ethanol and xylene and filled with paraffin solution. After cutting into 4 μm thick sections, slides were stained with Hematoxylin & Eosin (H&E) and Sirius Red S/Fast green kit (Chondrex, Inc., WA, USA). Histological images were visualized under an optical microscope (EVOS FL Auto, Thermo Fisher Scientific, MA, USA).

1.6. statistical analysis

All statistical analyzes were performed with GraphPad Prism software. Data are presented as mean ± standard deviation (SD). Differences between control groups were analyzed using one-way analysis of variance with two-tailed t-test or Tukey's post-hoc test.

2. Results

2.1. 3D printed scaffold with hierarchical microchannel structure

As shown in Figure 1A, a 3D printed polycaprolactone (PCL) scaffold having a hierarchical microchannel structure was prepared. In particular, camphene was used in the PCL solution to allow microchannels to be created during 3D printing in a quenching medium. The unique properties of camphene (eg, miscibility with PCL solvents, high volatility, and low sublimation temperature) make camphene-PCL solutions capable of 3D printing with various microchannel structures in one step. During 3D printing in the medium, the PCL-camphene struts precipitate together to form an interconnected network and further, the camphene sublimes and is removed from the microchannels. In the meantime, the microchannel structure can be determined by various process parameters (quenching medium type and temperature). The microchannels were created only in an ethanol/distilled water (DW) mixed-medium, and the temperature of this medium was decisive in determining the size of the microchannels. The microstructure of the 3D-printed scaffold under different conditions is shown in Figure 1B. When 3D printed in ethanol/DW mixed-medium, a highly microporous structure was created throughout the scaffold. Of note, these microvoids became smaller when printed at lower temperatures (less than 10 µm at 0 °C < tens of µm at 37 °C). Based on the theory of nucleation during solidification, a lower medium temperature modulates camphene nucleation during solidification, ultimately reducing the size of camphene-sublimated micropores. On the other hand, the pore structure printed in the DW-only medium was not uniform, and no pores were generated in the ethanol-only medium. As shown in the visualization of the cross-sectional area of the scaffold struts (Figure 1C and D), the resulting microvoids were highly open-channel throughout the scaffold. The micropore size distribution of the scaffold (printed at 37°C) was analyzed (Fig. 1E). According to the analysis, most of the pores in the bulk were several to several tens of micrometers with an average of 12.9 (±7.69 μm), while the pores on the surface were larger with an average of 21.1 (±16.6 μm). In addition, 3D X-ray microtomography analysis revealed a highly interconnected pore structure of the μCh scaffold with a tortuosity value of 2.18 (±0.75). Other physical properties of the scaffolds related to microchannel formation were also characterized (Fig. 1F). In particular, there were significant differences in porosity (scaffold density), surface area, and water absorption. The volume occupied by the microchannels is approximately 28% (based on the difference in porosity between microchannel and non-microchannel scaffolds, termed 'μCh' and 'Non', respectively, where 'μCh' is 37 °C, EtOH +DW, 'Non' is made only with PCL without using camphene). The microchannels increased the surface area and water absorption of the scaffold by more than 6 and 3 times, respectively. The microchannelized scaffold was found to be highly hydrophilic after treatment with 1N sodium hydroxide, allowing water to permeate rapidly; Accordingly, the substantially altered (increased) surface area and pore space will play an important role in interactions with biological entities such as proteins and cells in in vivo conditions. The mechanical behavior of the two scaffolds under compressive loading was found to be similar; densification at strains of 20 to 30% with very low slopes in the initial elastic and collapse regions; The calculated initial slope was slightly higher in 'Non' (0.36 kPa) than in 'μCh' (0.17 kPa), while the steepness in the densified region was almost the same (80 to 90 kPa). In the next part, the in vivo phenomena occurring in the microchannelized scaffold were carefully investigated in comparison with conventional scaffolds.

2.2. Altered early immune and inflammatory responses to μCh scaffolds

Two types of scaffolds (μCh and Non, where 'μCh' was prepared in EtOH+DW at 37 °C, and 'Non' was prepared only with PCL without using camphene) were implanted subcutaneously in mice. First, the in vivo response of neutrophils and macrophages to the scaffold was investigated as an initial response (several hours to several days) of the body to recognize and judge foreign substances. In particular, neutrophils are first responders that can regulate the recruitment of the following macrophages through the release of bioactive molecules. When neutrophils adhere to the extracellular matrix, they are activated to expel deoxyribonucleic acid (DNA) and form neutrophil extracellular traps (NETs), and the formation of NETs is important for the promotion of further immune and inflammatory responses. Since most neutrophils migrate to the area around the foreign object within 24 hours after transplantation, samples were collected within 24 hours after transplantation for analysis.

SEM images of cells were obtained from scaffolds recovered at 24 hours (Fig. 2A). In the 'Non' group, some of the newly adherent cells were found within highly fibrotic NETs. In the 'μCh' group, cells escaped from the NET and were located inside the micropores created on the surface. Apparently, there was no difference in cell morphology between these groups. Cells were then co-stained with SYTOX green and Hoechst for fluorescence microscopic observation of extracellular DNA and cell nuclei (FIG. 2B). Since SYTOX green does not penetrate the plasma membrane, green and blue stained areas represent extracellular DNA and live cells, respectively. In the 'Non' group, the green signal appeared stronger and appeared overall, whereas in the 'μCh' group, it became weaker. The ratio of blue/green stained areas was quantified to identify significant differences; Most of the cells in the 'Non' group died and showed a green signal due to extracellular DNA, whereas most of the cells in the 'μCh' group survived and stained the nuclei (Fig. 2C). It is known that NET formation can act as a physical trap for foreign substances, including bacteria and biomaterials, to minimize damage around the tissue. Therefore, a low degree of NET formation indicates a reduction in adverse responders and allows for favorable progression of tissue repair. Fetz et al demonstrated in vitro that template-bound neutrophils express extracellular snares that induce different tissue integration outcomes depending on the components and diameter of the template. Early neutrophil action affects various biological responses such as inflammation, macrophage recruitment and differentiation, resolution of inflammation, fibrosis, and angiogenesis. Therefore, reduced NET formation in microchannelized scaffolds may hint at possible alterations in subsequent biological responses related to tissue repair and remodeling.

Next, the response of macrophages to the scaffold was investigated. In particular, macrophage populations and subpopulations following polarization are closely related to tissue regeneration. Since it is known that during the implantation period (usually between 2 and 4 days) macrophages migrate mainly into the implanted material, the samples collected on day 7 were evaluated for recruited macrophages and their polarization. First, adhesion of macrophages to the scaffold was visualized by co-staining of CD11b and F4/80 (Fig. 2D). The population of recruited macrophages appeared larger in the 'μCh' group (both on the surface and in the pores) compared to the 'Non' group. Next, polarization of macrophages was investigated. Indeed, macrophages are conventionally divided into activated (M1) or alternatively activated (M2) phenotypes, and the polarization of these macrophages determines subsequent cellular responses related to tissue remodeling and fibrosis. It has been reported that macrophage polarization is closely related to the physico-chemical properties of substances. The macrophage phenotype was monitored with fluorescent dyes of anti-NOS2 (M1-positive marker) and anti-arginase (M2-positive marker) (Fig. 2E), and the fraction of NOS2- or/and arginase-positive cells was quantified. (FIG. 2F). The number of cells positive only for NOS2 was almost twice as large in the 'Non' group (15%) as compared to 'μCh' (8%). On the other hand, the number of cells positive only for arginase increased significantly in 'μCh' (44%), and only a small number in 'Non' group (4%). Interestingly, cells positive for both NOS2 and arginase were generally high, especially in the 'Non' group (76% in 'Non' and 48% in 'μCh' group). While co-expression of the M1 and M2 markers indicates that some macrophages are beginning to resolve, significant expression of M2 in 'μCh' indicates that the cells are substantially shifting to the resolution phase.

From these results, we inferred that the microchannelized scaffold promotes the recruitment of macrophages and their subsequent M1 to M2 phase polarization. In many animal studies, macrophage depletion generally resulted in delayed and failed tissue repair, whereas replenishment of endogenous macrophages was able to induce successful regeneration, and highlighting macrophage populations is a necessary step in relaying the regeneration process. am. Altered neutrophil responses, such as reduced NET formation on microchannelized scaffolds and secretion potential of other bioactive molecules, may also be associated with macrophage recruitment and their M2 polarization. Indeed, it has been investigated that neutrophils secrete bioactive molecules differently depending on the structure and composition of the scaffold. Recently, increasing evidence supports that physical and chemical cues from matrices and scaffolds can modulate immune responses such as macrophage adhesion and polarization, ultimately altering the healing process. Interestingly, it has been reported that pore structures of 20 to 40 μm in size between different material parameters polarize macrophages from M1 to M2. The pore structure was also beneficial for penetration of recruited host cells, including MSCs. Indeed, the altered phenotype of macrophages and the molecules they secrete can recruit MSCs to mediate a damaged or diseased state into a repair phase. Similar to the recruitment of MSCs, other host cells involved in tissue repair and remodeling (eg, endothelial cells) may also be significantly affected by early immune/inflammatory processes; Here, the response of neutrophils and macrophages by microchannels of the scaffold. In the following, MSC homing and angiogenesis in vivo were investigated and correlated with early immune/inflammatory responses.

2.3. Transplantation into recruited stem cells and μCh scaffolds

After 14 days of transplantation, it was investigated whether endogenous MSCs were recruited to the scaffold (experimental design is shown in Figure 3A). CD90 and CD54 antibodies were used as MSC-specific markers. At low magnification, the CD90-positive signal appeared stronger in 'μCh' than in 'Non', indicating that more MSCs were recruited to the microchannelized scaffold (Fig. 3B). In addition, the expression of the co-stained fibrosis marker (red) was widespread in the 'Non' group, whereas it was limited near the 'μCh' scaffold (Fig. 3C). At high magnification, many endogenous MSCs (arrows) appeared to be located in and around the large pores (marked 'S') of the 'μCh' scaffold, which were not readily found in the 'Non' scaffold. When samples were H&E-stained, many endogenous cells were detected within the struts and throughout the interconnected microchannels. Interestingly, despite the lower weight and density of the original 'μCh' scaffold compared to 'Non', the recovered 'μCh' scaffold rapidly precipitated in the sucrose solution. This is probably due to an increase in density due to cell infiltration.

The recruitment of MSCs in an ex vivo model using tissue samples recovered through a transwell-migration assay (shown in Figure 3D) was further investigated. Scaffolds recovered on day 3 or 7 post-implantation were allowed to interact with MSCs to allow cells to migrate across the transwell to the scaffold, and the cells at the bottom of the transwell were stained. As a result, MSC migration to the recovered scaffold was significantly higher in 'μCh' than in 'Non' (Fig. 3E and F). The difference between the two scaffold groups recovered on day 7 was twice as high (44.6 ± 11.3 cells/m for 'Non' and 91.1 ± 21.9 cells/m for 'μCh'). Since MSC homing to the recovered scaffold was evident, we speculated that cytokines for stem cell homing might play a role in this phenomenon. The release of stromal cell-derived factor-1 (SDF-1) from the recovered scaffold was quantified by ELISA (Fig. 3G). Of note, the SDF-1 release from 'μCh' scaffolds recovered on day 7 was almost twice that of 'Non'. Interestingly, SDF-1 release showed a very similar pattern to MSC recruitment, suggesting that a role for SDF-1 in MSC homing to the scaffold may be crucial.

Recruitment of endogenous MSCs has been recognized as highly beneficial for the repair process, as MSCs release cytokines and growth factors that increase the ability of tissue repair cells to remodel the damaged microenvironment. In particular, the recruited MSCs have the ability to regulate immune responses, crosstalk with endothelial cells (or hepatocytes) for angiogenesis, and directly differentiate into the required cell lineage. Therefore, engineering scaffolds to recruit host stem cells and preserve their native regenerative potential is considered a promising alternative approach to exogenous stem cell transplantation. For this reason, most people have incorporated chemokines (eg, SDF-1, matrix P, etc.) into scaffolds to secrete chemokines appropriate for stem cell homing.

However, here we witnessed a stem cell homing phenomenon using a microchannelized scaffold without the use of exogenous chemokines. It is thought that inflammatory cells that respond early to this can manage this stem cell homing process. Stem cell homing chemokines (eg, SDF-1) are secreted primarily by inflammatory cells to initiate migration of cells involved in tissue repair and remodeling (eg, MSCs). Therefore, the inflammatory response shifted to the resolution phase (i.e., cell polarization to the M2 phase) within the μCh scaffold may have promoted the secretion of SDF-1, which accelerates tissue healing through directing MSCs to the site of injury.

2.4. Promoted Angiogenesis in μCh Scaffolds

Next, we investigated angiogenesis in vivo in microchannelized scaffolds (Fig. 4A). When harvested 14 days after implantation, samples were analyzed immunohistochemically (FIG. 4B). A CD31-positive signal in the scaffold was determined as tube formation. For reference, the number of newly formed blood vessels was significantly higher in 'μCh' than in the 'Non' group (Fig. 4C). Also, the size of blood vessels in the 'μCh' group was significantly larger than that in the 'Non' group. After confirming the ability of the microchannelized scaffold to induce angiogenesis in vivo, angiogenesis was further tested using an ex vivo tissue culture model involving recovered samples and transwell inserts (Fig. 4D). Human umbilical vein endothelial cells (HUVEC) were cultured with tissue samples harvested on days 3 and 7 and analyzed for tube network formation. Vascular-inducing medium supplemented with growth factors was used as a positive control. On the 'μCh' scaffold harvested on day 3, HUVECs showed stronger and expanded vascular networks (Fig. 4E). The number of tubes and the total tube area in the 'μCh' group were significantly higher than those in the 'Non' group (Fig. 4F). Interestingly, however, there was no significant difference in tube formation between 'μCh' and 'Non' recovered on day 7. As a result of confirming the release of VEGF from tissue samples recovered by ELISA quantitative method, 'μCh' was found to be significantly higher than 'Non' (Fig. 4G). Based on these results, it was demonstrated that microchannels significantly promote angiogenesis of 3D scaffolds with respect to the initial period and high VEGF release is thought to play some crucial role in angiogenesis induction.

Angiogenesis is essential for cell survival and tissue growth in the repair process, which precisely orchestrates the biological response promoted by polarized macrophages and bioactive molecules secreted from recruited MSCs. Here we observe that microchannelized scaffolds promote angiogenesis. In fact, the effect of many parameters of porous scaffolds, such as porosity, macro-pore size and interconnectivity, on angiogenesis has been extensively studied. For example, an increase in pore size and interconnectivity could enhance angiogenesis and consequently tissue repair. However, in most cases, the pore size considered was at least in the range of hundreds of micrometers, and in few cases, pores in the range of tens of micrometers were considered. There was one groundbreaking study demonstrating the effect of micropore size in channelized scaffolds on angiogenesis. These micropores were created in different sizes in parallel channeled scaffolds, and scaffolds with 30-40 μm micropores showed the highest angiogenesis, which is interestingly similar to what we found in our microchannelized scaffolds. do. Supporting the in vivo angiogenesis results, VEGF was released at significantly higher levels in the transplanted μCh samples, presumably due to growth factors and cytokines appropriate for tissue repair, such as the release of SDF-1 for MSC recruitment (here VEGF) seems to be due to the action of early recruited cells capable of secreting. On the other hand, homing MSCs can also promote the secretion of angiogenic factors, suggesting the possibility of mesenchymal-endothelial crosstalk.

2.5. Reduced fibrosis in μCh scaffolds

Fibrosis, the excessive and disordered deposition of collagen, has been a problem in implantable materials and is therefore often considered a negative sign in assessing the biocompatibility of many developed biomaterials. Excessive or long-lasting inflammation often leads to the formation of a thick fibrous capsule, predictably delayed subsequent healing and remodeling of the tissue. Indeed, the thick fibrous capsule can impede angiogenesis and invasion of host stem cells. Thus, fibrosis on the scaffold was investigated through MT staining for up to 3 weeks (FIG. 5A). A thick fibrous capsule was formed around the 'Non' scaffold, whereas this fibrosis was significantly reduced in the 'μCh' scaffold. The thickness of the fibrous capsule was quantified, and it was almost doubled for the 'μCh' scaffold in 'Non' (Fig. 5B). Looking again at the results of fibroblast/MSC co-staining at day 7, where fibroblasts were extensively present on the 'Non' scaffold but not on the 'μCh' scaffold (Fig. 3B), the deposition of collagenous fibers and the fibrotic response Fibroblasts infiltrated on a 'Non' scaffold are required. Taken together, the less fibrotic capsule formation around 'μCh' compared to the 'Non' scaffold is thought to be the result of a series of host events including inflammation, angiogenesis and stem cell recruitment.

2.6. Successive Cavitation of Isolated Molecules and Possible Roles

As demonstrated, the 'μCh' scaffold significantly promoted the process of angiogenesis and stem cell homing through an altered immune/inflammatory response, ultimately leading to reduced formation of the fibrotic capsule. Of note, the release of SDF-1 and VEGF from tissue samples was much higher in the 'μCh' scaffold, supporting their key role in angiogenesis and stem cell stimulation. Although SDF-1 and VEGF signaling molecules were initially speculated to be secreted from cells infiltrating the scaffold, they can also be sequestered in the scaffold. In this regard, it was envisioned that the 'μCh' scaffold would have the ability to sequester molecules. To test this hypothesis, protein uptake into scaffold samples retrieved 24 hours after implantation was analyzed.

First, the total amount of protein absorbed by the 'μCh' scaffold was quantified to be 9.84 mg, almost twice the level of the 'Non' scaffold (4.85 mg) (Fig. 6A). In addition, protein components attached to the scaffold were confirmed. We focused on ECM molecules known to be involved in early cellular events, including vitronectin, fibronectin, and fibrinogen. Interestingly, Western blot analysis (Fig. 6B) showed that all of these ECM molecules were absorbed at significantly higher levels by 'μCh' compared to 'Non'. Ponceau S staining results support that equal amounts of protein for both scaffold groups were loaded on the gel. In addition, immunostaining of samples for fibronectin and vitronectin showed stronger protein signals in 'μCh' compared to 'Non' scaffolds (Fig. 6C). Next, the isolation of a series of bioactive molecules including cytokines, chemokines and growth factors was analyzed using a protein assay kit (Fig. 6D).

The intensity of each point is summarized in Tables S1-3. Among the molecules sequestered on the scaffold within 24 hours, pro-inflammatory cytokines and chemokines were generally elevated. Interestingly, expression of inflammatory signals was lower in 'μCh' compared to 'Non' scaffold. In particular, the expression of CCL11, CCL12, CCL21, CXCL10, CXCL13, and MMP-9 differed more than twofold between the scaffolds (Fig. 6E and F). On the other hand, there was no significant difference in uptake of anti-inflammatory response-related molecules including IL-1ra, IL-4, IL-10, VEGF, CD105, and FGF21. It is therefore evident that the μCh scaffold changes the profile of the putative protein isolated. The expression profile of the chemokines identified here may reflect the time point of analysis (24 h) when an early inflammatory response predominates.

In particular, based on the analysis of the type and amount of proteins absorbed/sequestered into the scaffold at a very early time point, the 'μCh' scaffold firstly stimulates some ECM molecules that are critically involved in early cell adhesion processes such as stem cell establishment. Second, it is thought to have the ability to recruit much less molecules that upregulate inflammation. It is possible that engineered biomaterials modulate responses associated with chronic inflammation by modulating chemokine concentrations in the body. In a recent study, engineered hydrogels were shown to be very effective in inducing enhanced angiogenesis for tissue repair by sequestering molecules secreted from activated macrophages at an early stage, suggesting that biomaterials are potential candidates for creating a cellular microenvironment. Emphasize the importance of isolating molecules. The present finding that uptake of adherent proteins is increased while sequestration of proinflammatory cytokines is simultaneously decreased suggests recapitulation of the cellular microenvironment by the μCh scaffold, which can drive repair processes while inhibiting chronic inflammation. The fibronectin/vitronectin-rich surface serves to promote initial adhesion of cells (eg MSCs) and the stump matrix provides a temporary structure for host cell migration and invasion. Moreover, these adhesive molecules can promote neutrophils and macrophages to recognize and colonize surfaces. It has also been reported that fibrin-rich fibrous surfaces stimulate macrophages to polarize pro- and anti-inflammatory responses.

Collectively, a series of sequential events occurring in the 'μCh' scaffold in vivo can be summarized as shown in Fig. 7. Therefore, it is clear that the 'μCh' scaffold possesses unique physicochemical properties that accelerate the tissue repair process. Based on this, we further designed experiments to test the tissue regeneration ability of the 'μCh' scaffold with more tissue-specific models; Since bone regeneration also prefers such a series of sequential events, a bone defect model was introduced here. In particular, the in vivo effects on pro-angiogenesis and MSC recruitment by the 'μCh' scaffold were also confirmed in mouse-like mice.

2.7. Regenerative potential of μCh scaffolds presented in bone defect models

'μCh' scaffolds were implanted into calvarial defect mice for 6 weeks and tissue samples were analyzed if a series of initial in vivo events might be effective in new bone formation conditions. X-ray and micro-CT images of the samples showed a significant level of hard tissue formed within the defect area in the 'μCh' scaffold, but very limited in the 'Non' scaffold (Fig. 8A and B). As a result of quantification based on the reconstructed micro-CT images, the volume, surface area and density of new bone were higher in 'μCh' than in 'Non' scaffold (Fig. 8C). Histological (H&E) staining results showed that new bone was well formed within the defect area of the 'μCh' scaffold (Fig. 8D). Upon closer inspection, many cells were aggregated and embedded within the microchannels of the 'μCh' scaffold. Sirius Red S/Fast Green co-staining of the newly formed tissue revealed the deposition of collagenous (red) and non-collagenous (green) matrices within the scaffold. It was found that more matrix molecules were produced within the 'μCh' scaffold compared to the 'Non' scaffold (Fig. 8E). For reference, non-collagenous proteins were mainly deposited on the periphery of the collagen matrix, and collagen deposits were also found within the microchannels of the 'μCh' scaffold, indicating that the infiltrated cells also produced collagen molecules. In quantification, the amount of collagenous and non-collagenous proteins in the 'μCh' scaffold was significantly higher by almost 2-fold compared to the 'Non' scaffold; 4.83 (±0.44) vs. collagenous protein. 2.27 (±0.14) μg/section and 65.3 (±6.05) vs. non-collagenous proteins. 36.9 (±2.97) μg/section (FIG. 8F). While collagen is considered the initial bone matrix, non-collagenous proteins including osteocalcin, osteopontin, and osteonectin are primarily involved in the final stages of bone matrix formation. In particular, the immunohistochemical signal of osteopontin was more evident in tissue samples from the 'μCh' scaffold than from the 'Non' scaffold, indicating that bone matrix deposition was promoted by the microchannels.

Thus, in vivo results demonstrated that the 'μCh' scaffold could enhance the regenerative process of cells in bone defects, which, as discussed earlier, could be promoted by a series of favorable events in tissue healing. In vitro experiments also support that the 'μCh' scaffold is better than the 'Non' scaffold to promote attachment of MSCs and differentiation towards the osteogenic lineage. Although the process of regeneration in the same site does not exactly correspond to the process of tissue healing in other tissues (as shown in Fig. 7), key phenomena including inflammation, angiogenesis and stem cell recruitment can generally be shared in common, which ultimately MSCs will affect osteogenesis and osteogenesis. However, it is also possible that the microchannels of the scaffold, independently of other phenomena, directly modulate the osteogenic differentiation of MSCs (eg, due to topological effects).

In the above, the present invention has been shown and described in relation to specific preferred embodiments, but the present invention can be variously modified and changed without departing from the technical features or fields of the present invention provided by the claims below. It is clear to those skilled in the art that it can be.

Claims (13)

delete delete delete delete delete delete A method for manufacturing a porous scaffold for tissue regeneration having a hierarchical microchannel structure,
It is molded by discharging a solution in which polycaprolactone and camphene are dissolved in a solvent into a mixed solution of ethanol and distilled water,
The temperature of the mixed solution of ethanol and distilled water is 30 to 40 ° C,
The mixed solution of ethanol and distilled water is a mixed solution of ethanol and distilled water in a volume ratio of 1: 1,
A method for manufacturing a porous scaffold for tissue regeneration.
According to claim 7,
A method for manufacturing a porous scaffold for tissue regeneration, wherein the temperature of the mixed solution of ethanol and distilled water is 37 ° C.
delete delete delete delete delete

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