Bio-ink for DLP printing of bionic skin and its preparation and application

Abstract

The present invention discloses a bio-ink for DLP printing of bionic skin and its preparation and application, relating to the fields of biomedical materials and bioprinting. The bio-ink is composed of two skin ECM components that are chemically modified and proportioned. The modified photocurable collagen CoLMA and photocurable chondroitin sulfate CSMA both have the characteristics of rapid photocuring gel formation and good biocompatibility, which can meet the growth requirements of skin cells. Compared with single-component bio-ink, the composite CSMA-CoLMA bio-ink has better skin cell proliferation and adhesion promotion, and the gel storage modulus and elastic modulus values are close to those of natural skin. In addition, the two-component bio-ink is suitable for a variety of DLP printing equipment and printing methods on the market. The obtained cell-laden gel matrix can be adhered to the four sides of the Transwell chamber, meeting the construction requirements of bionic skin for cosmetic testing, and has clinical and commercial application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials and bioprinting technology, and specifically to a bio-ink for DLP printing of bionic skin, and its preparation and application, and more particularly to a bio-ink based on DLP light-curing bioprinting technology that can be printed on a Transwell chamber and can meet the growth requirements of skin fibroblasts and keratinocytes. Background Art

[0002] Although animal skin is physiologically similar to human skin, data suggest that in vitro / in vivo data obtained in animals and humans are poorly correlated. Since the 3R principle (Reduction, Replacement, Refine) was first proposed in 1957, many countries have been working to reduce and replace animal use, enacting legislation prohibiting animal testing and marketing bans in the cosmetics sector. To ensure the continued orderly development of the pharmaceutical and cosmetics industries, there is an urgent need to develop a more reliable and productive biomimetic skin model.

[0003] Cosmetics are usually applied to the human body surface by smearing or spraying, and then exert their specific functions. Currently, in vitro bionic skins that can be used for cosmetic toxicology and efficacy testing are all constructed using tissue engineering technology. Specifically, serum-free culture medium is used to reconstruct normal human skin cells isolated in vitro into active tissue similar to the structure of human skin through air-liquid interface differentiation technology in Transwell chambers. The main criteria for judging the success of bionic skin construction are: (1) having a layered structure similar to the epidermis; (2) the skin tissue surface is dry and closely adheres to the periphery of the chamber, and the culture medium does not leak from the periphery of the chamber to the surface; (3) the true epidermis does not shrink; and (4) there are no bubbles introduced by artificial manipulation in the dermal collagen layer.

[0004] Digital Light Processing (DLP) printing is a technology that uses a projection light source probe to solidify photopolymerizable bio-ink layer by layer "from top to bottom" or "from bottom to top", thereby achieving in situ rapid prototyping of 3D printed objects. The emergence of DLP bio-printing technology is a major advancement in the construction of in vitro bionic skin models. It is capable of creating multi-scale complex structures of human skin and other three-dimensional structures that are difficult to replicate in vitro through simple artificial processing methods. Unlike other methods of artificially preparing bionic skin in vitro, DLP printing requires the printing probe to be inserted into the Transwell chamber to perform in situ, layer-by-layer, photo-cross-linking molding of the bio-ink. Therefore, in order for the skin tissue after DLP in situ printing to fit tightly around the chamber, the printed tissue should have a certain swelling effect. Tissue swelling and adhesion to the chamber are the key to determining whether the air-liquid interface of skin cells can successfully differentiate into the multi-layer structure of the epidermis in the later stage. In summary, bio-inks for DLP printing of biomimetic skin should meet the following five main requirements: (I) be photocurable; (II) support skin cell adhesion and proliferation; (III) have mechanical properties similar to those of skin; (IV) have appropriate swelling capacity; and (V) be composed of a matrix material similar to that of skin.

[0005] Currently, most research on skin bioprinting is based on extrusion printing technology, using type I collagen (the main component of the dermal extracellular matrix (ECM)) as the only biomaterial. Type I collagen has high bioactivity, and the structural dimensions can remain unchanged after extrusion bioprinting. However, for this technology, collagen gel is formed by non-covalent cross-linking through hydrogen bond self-assembly, and hydrogels based on weak cross-linking have the problem of easy shrinkage under the traction of actin. However, photocurable printing bioink can greatly improve this problem, because collagen molecules can form covalently cross-linked collagen hydrogels after modification with methacrylamide, thereby improving shrinkage while retaining the excellent bioactivity of collagen molecules. However, at present, there are no reports on the preparation of collagen into photocurable bioink as a three-dimensional biomimetic skin tissue matrix. This may be related to the fact that the use of type I collagen alone as a biomimetic skin material cannot imitate the specific ECM environment of skin tissue.

[0006] It is worth noting that, in addition to collagen, skin tissue contains various glycosaminoglycans (GAGs), which play an important role in maintaining the volume and elasticity of the skin. Chondroitin sulfate (CS), a type of GAG, is a linear polysaccharide that is a component of the glycosaminoglycans in the extracellular matrix of the dermis. As a component of FDA-approved skin substitutes, it can be used to treat burns. Studies have shown that CS can accelerate the epidermal-dermal regeneration process and can be used to construct tissue-engineered skin. In addition, photocurable chondroitin sulfate (CSMA) has excellent photoinitiated gelation ability and swelling properties and has been widely studied in biomedical applications. As disclosed in Chinese patent CN 113087819 A, a double-modified compound, its preparation method, and application are described. Using hyaluronic acid or chondroitin sulfate molecules as the basic skeleton, after an aldehyde reaction, methacrylic acid molecules are grafted onto it to form a photocurable hydrogel compound. After adding a photoinitiator to the aqueous solution of the compound, the aldehyde groups react with amino groups in biological tissue to form chemical bonds, thereby adhering to the tissue; while the methacrylic acid groups undergo a cross-linking reaction under irradiation with ultraviolet or blue-violet light to form a hydrogel structure. This application increases the adhesion of the biological glue through a two-stage cross-linking reaction, thereby achieving the purpose of bonding tissues, stopping bleeding, and protecting wounds. However, it is important to note that although the single CSMA hydrogel has the biological activity of promoting cell adhesion and proliferation, it has low mechanical strength, making it difficult to directly apply it to the construction of biomimetic skin models.

[0007] To address these challenges, this application proposes chemically modifying two components derived from the skin's ECM into a photopolymerizable bioink. Compared to single-component hydrogels, two-component hydrogels can achieve synergistic effects in overall performance or biological function, significantly improving the hydrogel's mechanical strength and better meeting the requirements of DLP-printed biomimetic skin bioinks. This approach holds promise for addressing pressing challenges in this field. Summary of the Invention

[0008] The purpose of the present invention is to solve the problems existing in the prior art and provide a bio-ink for DLP printing of bionic skin and its preparation and application. The bio-ink is composed of two main skin ECM components that are chemically modified and proportioned. Due to the toughening effect of the two-component printing hydrogel, the problem of insufficient mechanical strength of the single-component hydrogel is solved; and the obtained cell-laden gel matrix can be adhered to the four sides of the Transwell chamber, has good biocompatibility, can be used for DLP printing of bionic skin, and has great clinical and commercial application value.

[0009] In order to achieve the above technical objectives, the present invention is implemented by the following technical solutions: a method for preparing bio-ink for DLP printing of bionic skin, comprising the following steps:

[0010] 1) Preparation of photocurable chondroitin sulfate (CSMA): Chondroitin sulfate was added to phosphate buffer, the pH was adjusted to 3.5, glycidyl methacrylate was added dropwise, the reaction system was heated, stirred, cooled, precipitated, filtered, and lyophilized to obtain CSMA;

[0011] 2) Extraction of type I collagen CoL from rat tail;

[0012] 3) Preparation of photocurable collagen CoLMA: CoL was added to acetic acid, magnetically stirred, and the pH of the solution was adjusted to 7.4. Methacrylic anhydride was added dropwise to the solution, magnetically stirred, dialyzed, and freeze-dried to obtain CoLMA.

[0013] 4) Mixing CSMA, CoLMA solution and photoinitiator, and curing by irradiation with a DLP light source to obtain CSMA-CoLMA hydrogel.

[0014] Furthermore, in step 4), during mixing, CoLMA is first dissolved in 0.02N acetic acid solution at 4°C, the pH of the solution is adjusted to 7.4, and then CSMA, a photoinitiator and the CoLMA solution are mixed. The final mass concentrations of CSMA and the photoinitiator are 8-10% and 0.25%, respectively, and the final concentration of CoLMA is 3-9 mg / mL.

[0015] Furthermore, in step 4), the photoinitiator used is lithium phenyl-2,4,6-trimethylbenzoyl phosphite (LAP) or 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (I2959).

[0016] Furthermore, after the CSMA and CoLMA solutions and the photoinitiator were mixed, the final mass concentration of CSMA was 8%, and the final concentration of CoLMA was 6 mg / mL.

[0017] Furthermore, in step 4), the light curing time is 20 to 60 seconds.

[0018] Furthermore, in step 1), the mass volume ratio of chondroitin sulfate to glycidyl methacrylate is 1 g:3-12 mL; the reaction system is heated to 50-70° C. and stirred for 24-100 h.

[0019] Furthermore, in step 3), the mass volume ratio of CoL to methacrylic anhydride is 1 g:0.5-2.0 mL, the magnetic stirring process is carried out at 4° C., and the stirring reaction time is 24-72 h; the solution is dialyzed in 0.02 N acetic acid solution for 7 days, during which the dialysate is replaced every 8 h.

[0020] The bio-ink for DLP printing of bionic skin prepared by the above method is constructed by compounding modified photocurable chondroitin sulfate CSMA, photocurable collagen CoLMA and a photoinitiator.

[0021] The above-mentioned bio-ink for DLP printing of bionic skin is used in DLP printing of bionic skin. The bio-ink has a swelling function and can meet the growth requirements of skin fibroblasts and keratinocytes. It can be printed on the Transwell chamber and can swell to fit tightly around the Transwell chamber during the cell culture process.

[0022] The beneficial effects of the present invention are:

[0023] 1. The bio-ink for DLP-printed biomimetic skin prepared in this application is derived from the skin's ECM components and is composed of modified photocurable collagen CoLMA, photocurable chondroitin sulfate CSMA, and a photoinitiator. Because both CoLMA and CSMA have the characteristics of rapid photocuring to form a gel and good biocompatibility, they can meet the needs of skin cell growth;

[0024] 2. The bio-ink prepared in this application has an appropriate swelling capacity and can swell to a state of close adhesion around the perimeter of the Transwell chamber during cell culture. It can better meet the requirements of constructing bionic skin for cosmetic testing and has the characteristics of multi-adaptability, high throughput, rapidity, and stability.

[0025] 3. Compared with single-component bio-ink printing, the CSMA-CoLMA two-component bio-ink constructed by compounding CSMA and CoLMA in this application has better skin cell proliferation and adhesion promotion effects. The gel storage modulus and elastic modulus values are close to those of natural skin, making it more suitable for DLP printing of bionic skin.

[0026] 4. The two-component hydrogel constructed in this application has good toughening effect, which solves the problem of insufficient mechanical strength of single-component hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1A It is the synthetic route of CSMA;

[0028] Figure 1Bis the molecular weight distribution of CS, including the response to dw / dLogM / LogM, and the cumulative molecular weight percentage of CS components determined by gel chromatography (Cum W, %);

[0029] Figure 1C For CS, GMA and CSMA (500MHz, D2O) 1 H NMR spectrum;

[0030] Figure 1D FTIR spectra of original CS, GMA, and CSMA;

[0031] Figure 2 Synthesis and characterization data of CoLMA; Panel A shows the synthesis route of CoLMA; Panel B shows the SDS-PAGE analysis of protein marker, CoL, CoLMA, and CoL-C; Panel C shows the FTIR spectra of original CoL, MA, and CoLMA; Panel D shows the free amino group concentrations of CoL and CoLMA;

[0032] Figure 3 Preparation and characterization data of CSMA-CoLMA hydrogel; among them, panel A is a schematic diagram of the composition of CSMA-CoLMA bio-ink and gel network formation; panel B is a photograph of the photocured CSMA-CoLMA hydrogel after compounding with different ratios of CSMA and CoLMA; panel C is the swelling curve of CSMA-CoLMA hydrogel; panel D is the degradation curve of CSMA-CoLMA hydrogel; panel E is the SEM image of the internal cross-section of CSMA-CoLMA hydrogel; panel F is the pore size data obtained by quantifying the pore size and distribution range of the SEM image of the internal cross-section of the hydrogel using Image J software;

[0033] Figure 4 The rheological and mechanical properties test data of CSMA-CoLMA hydrogel; among them, panel A is the oscillatory strain sweep result at a frequency of 1 Hz, from 0.01 strain to 10% strain; panel B is the frequency sweep curve of the hydrogel in the frequency range of 0.1 to 10 Hz; panel C is the average storage modulus (G′) and loss modulus (G″) data calculated from panel B; panel D is the stress-strain curve of CSMA-CoLMA hydrogel before 90% strain; panel E is a picture of CSMA-CoLMA-8-6 hydrogel compressed using a texture analyzer; panel F is the elastic modulus of CSMA-CoLMA hydrogel, data are expressed as mean ± standard deviation, three independent experiments in each group, P value was calculated using one-way analysis of variance, *P<0.05, **P<0.01, ***P<0.001 vs. CSMA-CoLMA-8-0;

[0034] Figure 5 Figure 3 shows the culture and identification results of primary rat skin cells. Panel A shows the morphology of KCs cultured in culture flasks, the morphology of second-generation KCs under light microscopy (cultured in culture flasks), and the expression of CK 10 (keratin-10, green), CK 14 (keratin-14, green), and CK 19 (keratin-19, green) in KCs. Panel B shows the morphology of DFs cultured in culture flasks, the morphology of second-generation DFs under light microscopy (cultured in culture flasks), and the expression of Vim (vimentin, green) in KCs.

[0035] Figure 6 Figure 3 is the cytotoxicity, cell proliferation and cell adhesion test data of CSMA-CoLMA hydrogel; among them, panel A shows the cell survival rate of KCs after culture in CSMA-CoLMA hydrogel extract for 1, 3 and 5 days; panel B shows the cell survival rate of DFs after culture in CSMA-CoLMA hydrogel extract for 1, 3 and 5 days; panel C is the comparison of cell proliferation ability of CSMA-CoLMA hydrogel loaded with KCs after culture for 1, 3 and 5 days; panel D is the comparison of cell proliferation ability of CSMA-CoLMA hydrogel loaded with DFs Comparison of cell proliferation ability after 1, 3, and 5 days of culture on CoLMA hydrogels; Panel E is the SEM image of KCs adhesion in CSMA-CoLMA hydrogels; Panel F is the SEM image of DFs adhesion in CSMA-CoLMA hydrogels; Data are expressed as mean ± SD and are from at least three independent experiments. P values were calculated using one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group and CSMA-CoLMA-8-0;

[0036] Figure 7 CLSM shows three-dimensional images of KCs-loaded CSMA-CoLMA gel tissue culture after live / dead cell staining on days 1, 3, and 5. Red indicates dead cells and green indicates live cells.

[0037] Figure 8 CLSM shows three-dimensional images of DFs-loaded CSMA-CoLMA gels after live / dead cell staining on days 1, 3, and 5 of tissue culture. Red indicates dead cells and green indicates live cells. The multicolor labeled layer view shows the distribution of active DFs in the CSMA-CoLMA gel (blue on the surface and red on the bottom).

[0038] Figure 9 This is the basic structure of the "bottom-up" DLP printer on the market, where 1-blue light, 2-light source, 3-material tank, 4-printing material, 5-printing interface;

[0039] Figure 10 is the high performance gel permeation chromatogram of CS;

[0040] Figure 11 The standard curve for glycine determination by ninhydrin method;

[0041] Figure 12 The CSMA-CoLMA hydrogel is tightly attached to the surrounding areas of the Transwell chamber after swelling; Panel A shows a top view of the Transwell chamber; Panel B shows a side view of the Transwell chamber. DETAILED DESCRIPTION

[0042] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0043] Example 1

[0044] 1. Preparation of photocurable chondroitin sulfate (CSMA) bioink

[0045] 1 g of chondroitin sulfate (CS) was added to 50 mL of phosphate buffer solution (PBS, pH 7.4) and fully dissolved at room temperature for 2 h. After adding 0.1 M HCl to adjust the pH to 3.5, 7.5 mL of glycidyl methacrylate (GMA) was added dropwise under constant stirring. The reaction system was heated to 60°C and stirred for 72 h. The resulting solution was cooled to 0°C in an ice bath, precipitated twice in ethanol, filtered, and lyophilized to obtain CSMA.

[0046] 2. Extraction of Type I Collagen (CoL) from Rat Tail

[0047] Use sterile surgical forceps to pull white collagen fibers from the tendon sheath of the rat tail tendon. After incubating with acetone for 5 minutes, transfer the collagen fibers to 70% isopropanol and incubate for another 5 minutes. The collagen fibers are then placed in 0.02N acetic acid solution and magnetically stirred at 4°C for at least 48 hours. Centrifuge at 12,000 rpm for 45 minutes and freeze-dry to obtain rat tail type I collagen.

[0048] 3. Preparation of Photocurable Collagen (CoLMA) Bioink

[0049] 1 g of CoL was added to 250 mL of 0.02 N acetic acid and magnetically stirred at 4°C until completely dissolved. 1 M NaOH was added to adjust the pH of the solution to 7.4, and then 1.035 mL of methacrylic anhydride (MA) was added dropwise to the solution, and the reaction was magnetically stirred for 72 hours. After the reaction, the reaction solution was transferred to a 3500 Da dialysis bag and dialyzed against 0.02 N acetic acid solution for 7 days, during which the dialysate was replaced every 8 hours. CoLMA was then freeze-dried to obtain.

[0050] 4. Characterization of CSMA and CoLMA Bioinks

[0051] CSMA was recorded on a Bruker Avance 600-MHz NMR using deuterated water as the solvent. 1 H-NMR spectrum.

[0052] Bruker Tensor 27 FTIR was used to detect CSMA and CoLMA in the range of 4000-400 cm -1 Infrared spectrum in the range with a resolution of 4cm -1 .

[0053] The degree of cross-linking of CoLMA was determined using the ninhydrin colorimetric method. CSMA and CoLMA test samples were boiled with a ninhydrin solution at 100°C for 2 minutes. The absorbance at 570 nm was recorded using a spectrophotometer to determine the number of free amino groups in the test samples. Glycine at various known concentrations (1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL) was used as a standard. The number of free amino groups is proportional to the Abs570 value, and various known concentrations of glycine were used to create a standard curve showing the relationship between glycine concentration and absorbance. The degree of cross-linking of the samples was then calculated.

[0054]

[0055] Wherein, Amino0 is the free NH2 concentration in the non-cross-linked sample, and Aminoc is the free NH2 concentration in the cross-linked sample;

[0056] CoL, CoLMA, and commercially available rat tail type I collagen (CoL-C) were characterized by 1D SDS-PAGE. Each protein sample was separated on an 8% sodium dodecyl sulfate polyacrylamide gel at a constant current of 120V for 2 hours and then stained with Coomassie blue. Protein bands were visualized using an image scanner.

[0057] 5. Preparation of CSMA-CoLMA Bioink

[0058] The photocurable bioink was composed of CSMA, CoLMA solution, and photoinitiator (lithium phenyl-2,4,6-trimethylbenzoyl phosphite, LAP) mixed at 4°C. All materials were sterilized by ultraviolet irradiation or filtration through a 0.22 μm filter membrane before use.

[0059] CoLMA was dissolved in 0.02N acetic acid solution at 4°C (CoLMA mass concentration of 9 mg / mL); 10× PBS and 1M NaOH were added to adjust the solution pH to 7.4; CSMA and LAP were mixed with the CoLMA solution to a final mass concentration of 8% and 0.25% for CSMA and LAP, respectively, and the final concentration of CoLMA was adjusted to 0, 3, 6, and 9 mg / mL. After the solution was mixed, a DLP light source (BP8600, EFL, China, providing 405 nm, 10 mW / cm 2 The CSMA-CoLMA hydrogel was obtained by irradiating the CSMA-CoLMA hydrogel for different time periods.

[0060] Related performance tests

[0061] 1. Determination of swelling and degradation behavior of hydrogels

[0062] The swelling degree of the obtained hydrogel was analyzed using the specific gravity method. First, a cylindrical hydrogel (6 mm thickness, 6 mm diameter) was prepared according to the above-mentioned bio-ink preparation method. After freeze-drying, it was weighed (W0) and rehydrated in PBS at 37°C. During the weighing, the excess buffer on the surface of the hydrogel was absorbed with a thin paper, and the weight of the gel (Wt) was recorded at different time points. The swelling degree of the gel was calculated according to the following formula (2):

[0063]

[0064] Wherein, Wt is the sample weight at different time points, W0 is the initial weight of the hydrogel;

[0065] Type I collagenase was used to test the degradation behavior of the hydrogel. First, cylindrical (6 mm thickness, 6 mm diameter) hydrogels were prepared using CSMA-CoLMA bio-ink. After freeze-drying, the initial weight of the hydrogel (M0) was recorded. Subsequently, the hydrogel was incubated with type I collagenase (50 U / mL) at 37 ° C. The weight of the hydrogel after freeze-drying (Mt) after different degradation times was recorded. The degree of degradation of the hydrogel was calculated according to formula (3):

[0066]

[0067] Wherein, Mt is the sample weight at different time points, and M0 is the initial weight of the hydrogel.

[0068] 2. Microstructure observation of hydrogel

[0069] The internal pore size of the photocrosslinked CSMA-CoLMA hydrogel was observed using a scanning electron microscope (SEM; Hitachi S-4800, JP). The photocrosslinked CSMA-CoLMA hydrogel was equilibrated in PBS at room temperature for 24 hours and then freeze-dried to completely remove the water from the gel. The freeze-dried sample was gold-sprayed and observed using a scanning electron microscope. The microstructure and pore size of the hydrogel were quantified using Image-J software.

[0070] 3. Measurement of rheological properties of hydrogels

[0071] The rheological properties of CSMA-CoLMA hydrogel were characterized using a TA DHR-2 rheometer equipped with a 20mm Peltier plate. The working gap distance of the plate was set to 1mm, and silicone oil was placed around the plate to prevent water evaporation. Before the experiment, a disc-shaped hydrogel with a diameter of 20mm and a thickness of 1mm was balanced in PBS. An oscillatory strain scanning mode was used, the frequency was set to 1Hz, and the test was performed in the range of 0.01-10% to determine the linear viscoelastic region of the hydrogel. Subsequently, a frequency sweep test was performed in the range of 0.1-10Hz to obtain the storage modulus (G′) and loss modulus (G″) of the hydrogel. During the measurement, a circulating water bath was used to maintain a constant temperature of 37±0.1°C for the Peltier plate to simulate the physiological conditions in vivo.

[0072] 4. Hydrogel mechanical properties test

[0073] The hydrogel's mechanical properties, including elastic modulus and stress-strain curves, were analyzed using a TMS-Touch texture analyzer. Cylindrical hydrogels (10 mm × 10 mm, according to GB / T 528) were prepared and allowed to swell in PBS. Measurements were performed in compression mode using a 250N pressure transducer. The initial distance and probe speed were set to 20 mm and 10 mm / min, respectively, until the sample fractured. The stress-strain curves were recorded using a microcomputer.

[0074] 5. Cell culture and identification of primary rat keratinocytes and fibroblasts

[0075] The characteristic markers of primary extracted rat skin cells were identified by immunofluorescence. Primary skin fibroblasts (DFs) and keratinocytes (KCs) were cultured at 1×10 4Cells were added to 6-well culture slides at a density of 100 μg / mL and incubated overnight at 37°C in 5% CO2. After attaching to the slides, the cells were rinsed with PBS and blocked with 0.5% bovine serum albumin (BSA) containing 0.025% Triton X-100 for 1 hour at room temperature. The cells were then incubated with primary antibodies rabbit anti-keratin 10 (CK10, 1:500 dilution), rabbit anti-keratin 14 (CK14, 1:500 dilution), and rabbit anti-keratin 19 overnight at 4°C. After washing three times with PBS, the cells were incubated with fluorescently labeled secondary antibodies for 2 hours at room temperature. Cell nuclei were counterstained with DAPI for 5 minutes and observed under an inverted fluorescence microscope. Fluorescence images were analyzed using Image J software.

[0076] 6. Cytotoxicity Analysis

[0077] To test the cell compatibility of the hydrogel, the cytotoxicity of the CSMA-CoLMA hydrogel extract on mouse DFs and KCs was evaluated according to the ISO / TC194 guidelines. 1 g of hydrogel was immersed in 10 mL of complete culture medium and cultured at 37 ° C for 24 h to obtain the hydrogel extract. According to the ISO / TC194 guidelines, the relative growth rate (RGR) was used to evaluate the cytotoxicity stage of the CSMA-CoLMA hydrogel extract and was divided into 5 levels (Table 1). RGR was calculated according to formula (4):

[0078]

[0079] Among them, A1 is the absorbance of the experimental group at 570 nm, and A2 is the absorbance of the control group at 570 nm;

[0080] The comparison between the RGR and cytotoxic phases is as follows:

[0081]

[0082] 7. Live / dead (Calcin AM / PI) assay

[0083] In order to directly observe the proliferation of skin cells in the gel, the cell-loaded hydrogel samples were detected using a live / dead cell staining kit. 6 Cells were uniformly suspended in the prepared CSMA-CoLMA solution at a final concentration of 10 cells / mL to produce a cell-laden hydrogel ink. Curing conditions were set at a volume of 90 μL and a curing time of 40 seconds. After incubation for 1, 3, and 5 days, the cell-laden hydrogel samples were stained and observed using confocal laser scanning microscopy (CLSM).

[0084] result

[0085] 1. Synthesis and characterization of CSMA

[0086] Figure 1A The synthetic route of CS modified with GMA is shown. The molecular weight distribution of CS was determined by size exclusion chromatography and static laser light scattering. Figure 10 As shown, the molecular weight of CS is 17.58 ± 2.1 kDa, and the polydispersity index (PDI, Mw / Mn) is 1.55 ± 0.02, indicating that the particle size distribution of CS is relatively concentrated ( Figure 1B ).

[0087] By FTIR and 1 Its structure was characterized by H NMR. FTIR spectrum showed the characteristic band of CS at 3417 cm -1 (OH and NH); 2915 cm -1 (CH); 1719cm -1 (C=Oester); 1633cm -1 (C=O amide ,C=C);1574cm -1 (NH); 1421cm -1 (CH); 1315cm -1 (CN); 1131cm -1 (CO). In addition, at 1719cm -1 and 1633cm -1 The characteristic bands of CSMA and GMA were observed, indicating the ester carbonyl group (C=O) and the carbon-carbon double bond conjugated system (C=C) ( Figure 1D ).

[0088] pass 1 H-NMR further confirmed that CSMA was successfully synthesized. Figure 1C As shown, the characteristic CS signals are at 4.55 (d, O-CH2); 4.24 (m, H-1GlcUa and H-1GalNAc); 4.10-3.20 (m, H-2,3,4,5,6GlcUa and H-2,3,4,5,6GalNAc, O-CH, CH, 5×CH2, NH, and OH); and 2.07 (s, CH3). After the methacrylate compound is inserted into the CS backbone, three new signals are observed at 6.22, 5.80, and 1.99 ppm, labeled 12, 12′, and 13, respectively. The two protons on the vinyl double bond produce two unique peak signals at 6.22 and 5.80 ppm. The peak signal at 1.99 ppm is derived from the methyl group of the methacrylate compound, which is not present in the original CS. Comparing the intensity of the methacrylate protons with the intensity of the methyl protons in the acetamide of CS, the degree of methacrylation in CS is 46%.

[0089] 2. Synthesis and characterization of CoLMA

[0090] The free amino groups in the terminal peptide region of collagen can be replaced by methacrylate groups to obtain functionalized collagen molecules. CoLMA is synthesized based on the condensation reaction between free amino acids and methacrylic acid ( Figure 2 The molecular weight distribution of collagen fragments in CoL, CoLMA and CoL-C was characterized by SDS-PAGE gel electrophoresis. Figure 2 As shown in panel B, the protein fragments of CoLMA and CoL are identical to those of CoL-C, with four characteristic bands: a trimeric γ-component (~300 kDa) composed of three covalently cross-linked α chains ([α1(I)]2[α2(I)]), a dimeric β-component (~200 kDa) composed of two covalently cross-linked α chains ([α1(I)]2), and a mixture composed of a single monomeric α chain (~100 kDa). These results demonstrate that chemical modification of CoL maintains the biological activity of the protein without destroying its component fragments.

[0091] FTIR spectra such as Figure 2 As shown in the small figure C, the characteristic peaks of CoL and CoLMA are 3299 cm -1 (Amide A, NH); 2919 cm -1 (Amide B, CH2); 1628 cm -1 (Amide I, C=O); 1540 cm -1 (Amide II, NH, CN); 1234 cm -1 (Amide III, CONH2). In addition to retaining the characteristic peak of CoL, CoLMA also has a peak at 1633 cm -1 The characteristic bands of MA can be observed at 400 nm. In addition, compared with unreacted CoL, the characteristic bands of amide I and amide II of CoLMA showed higher intensities, indicating that new bonds were formed on the collagen chains and MA was successfully grafted onto the collagen molecular chains.

[0092] The percentage of free amino groups in collagen was determined by ninhydrin colorimetry and the grafting rate of CoLMA was calculated. Figure 11 As shown, there is a good linear relationship between the concentration of glycine standard in the range of 0.5-6 mg / L and the absorbance value. The standard curve equation of the ninhydrin colorimetric method is Y=0.12012x+0.01772(R 2 =0.99035). The free amino group concentrations of CoL and CoLMA were 2.57 mg / mL and 2.33 mg / mL, respectively ( Figure 2 (D in the middle panel). Compared with unmodified collagen, the grafting efficiency of CoLMA was 7%.

[0093] 3. Preparation and characterization of CSMA-CoLMA hydrogel

[0094] CSMA and CoLMA were mixed in varying ratios, with the LAP concentration fixed at 0.25%, to create a two-component CSMA-CoLMA bioink. The photocuring time and ink concentration were then optimized to produce high-fidelity CSMA-CoLMA hydrogels.

[0095] The optimized photocuring parameters and results of the single-component CSMA hydrogel are as follows:

[0096]

[0097] Note: (a) √ and × represent successful and unsuccessful hydrogelation under the above light curing parameters, respectively; (b) A: Partial gelation, liquid; B: Complete gelation, elastic; C: Reduced elasticity, fragile;

[0098] The optimized photocuring parameters and results of the single-component CoLMA hydrogel are as follows:

[0099]

[0100]

[0101] Note: (a)√ and × represent successful and unsuccessful hydrogelation under the above light curing parameters, respectively. (b) A: Partial gelation with residual liquid; B: Complete gelation with elasticity; C: Reduced elasticity and brittleness;

[0102] The results show that: (1) The ink concentration directly affects the fidelity of the hydrogel structure. When the bio-ink concentration is too low, short-term photocuring cannot completely gelate. In contrast, as the ink concentration increases, the integrity of the hydrogel structure increases accordingly; (2) The most suitable photocuring parameters for CSMA hydrogel are: curing time 40s, LAP concentration 0.25%, CSMA concentration 8-10%; (3) The most suitable photocuring parameters for CoLMA hydrogel are: curing time 40s, LAP concentration 0.25%, CoLMA concentration 3-9mg / mL.

[0103] Figure 3 Panel A in the middle shows a schematic diagram of the preparation of CSMA-CoLMA hydrogels. A fixed CSMA concentration (8% by mass) was added to various concentrations of CoLMA (0, 3, 6, and 9 mg / mL) and then photocured to obtain CSMA-CoLMA hydrogels. The single-component hydrogel without CoLMA was designated CSMA-CoLMA-8-0, while the two-component hydrogels with varying concentrations of CoLMA were designated CSMA-CoLMA-8-3, CSMA-CoLMA-8-6, and CSMA-CoLMA-8-9, respectively.

[0104] The optimized photocuring parameters and results of CSMA-CoLMA hydrogel are as follows:

[0105]

[0106] Note: (a)√ and × represent successful and unsuccessful hydrogelation under the above light curing parameters, respectively. (b) A: Partial gelation, liquid; B: Complete gelation, elastic; C: Reduced elasticity, fragile;

[0107] The most suitable light-curing parameters for CSMA-CoLMA hydrogel are: curing time 40s and LAP concentration 0.25%. Figure 3 Panel B in the middle shows photos of CSMA-CoLMA hydrogels prepared with different compounding ratios.

[0108] The results of the hydrogel swelling test showed that the CSMA-CoLMA hydrogel swelled rapidly within the initial 120 minutes and reached equilibrium at 240 minutes. As the concentration of CoLMA increased, the hydrogel swelling decreased, which may be due to the increase in the degree of cross-linking between the chains, thereby improving the stability of the hydrogel matrix ( Figure 3 (Small image in center C).

[0109] The results of the hydrogel in vitro degradation test showed that the single-component CSMA hydrogel degraded very slowly, retaining 63% of its original mass after 25 days of degradation. The introduction of CoLMA greatly improved the degradation of the two-component CSMA-CoLMA hydrogel, and the degradation degree increased with the increase of CoLMA content ( Figure 3 (Small image in middle D).

[0110] SEM observation of the hydrogel microstructure and pore size showed that Figure 3 Small picture in E and Figure 3 As shown in panel F, the pore size of the CSMA-CoLMA-8-0 hydrogel is less than 100 μm and relatively uniform. The introduction of CoLMA significantly improves the pore size of the CSMA-CoLMA-8-0 hydrogel. As the CoLMA content increases from 0 mg / mL to 6 mg / mL, the pore size of the hydrogel increases and becomes more uniform, ranging from 100 to 200 μm, which is more suitable for tissue and cell growth. However, when the CoLMA content increases to 9 mg / mL, the pore size of the CSMA-CoLMA-8-9 hydrogel becomes less than 100 μm. This may be due to the increased degree of cross-linking between the chains, which promotes the formation of more covalent bonds between CoLMA and CSMA.

[0111] 4. Rheological and mechanical properties testing of CSMA-CoLMA hydrogel

[0112] To elucidate the differences in viscoelastic properties between the single-component CSMA hydrogel and the two-component CSMA-CoLMA hydrogel, the dynamic rheological behavior of the hydrogels was characterized. First, an oscillatory strain sweep was performed on the hydrogel to determine the linear viscoelastic region ( ) from 0.01 to 10% strain at a constant 1 Hz angular frequency. Figure 4 (Panel A). The storage modulus (G′) and loss modulus (G″) represent the elastic and viscous parts of the CSMA-CoLMA hydrogel, respectively. When the shear strain reaches 1%, the G′ and G″ of the CSMA-CoLMA hydrogel are almost constant, indicating that the 1% shear stress is in the linear viscoelastic region of the hydrogel. A frequency sweep of 0.1 to 10 Hz was performed with a 1% stress. Figure 4 As shown in panel B, in all groups, regardless of the CSMA-CoLMA ratio, G′ was always higher than G″ by about 6-8 times within the linear viscoelastic range, which means that the CSMA-CoLMA hydrogel is stable and behaves as an elastic hydrogel. Figure 4 As shown in panel C, the average G′ of the two-component CSMA-CoLMA hydrogels significantly increased with increasing CoLMA concentration compared to the single-component hydrogels (P < 0.01). The average storage moduli of the CSMA-CoLMA-8-0, CSMA-CoLMA-8-3, CSMA-CoLMA-8-6, and CSMA-CoLMA-8-9 hydrogels were 0.47, 1.4, 3.3, and 3.5 kPa, respectively. Notably, the average G′ of the CSMA-CoLMA-8-6 and CSMA-CoLMA-8-9 hydrogels approached that of native skin (~1900–5500 Pa).

[0113] The mechanical properties of the single-component CSMA hydrogel and the two-component CSMA-CoLMA hydrogel were compared by compression test. Figure 4 As shown in panel D, when the compressive strain reaches 45%, the stress of the two-component CSMA-CoLMA hydrogel continues to increase, while the single-component CSMA hydrogel breaks at 50% strain and reaches its maximum stress. Figure 4 Panel E in the middle shows the state of the hydrogel during the compression process. Compared with the broken block CSMA hydrogel after compression, the two-component CSMA-CoLMA hydrogel appears to be in a more complete state. Figure 4Panel F shows that the average elastic modulus of CSMA-CoLMA-8-0 is 13.01 kPa. In comparison, the corresponding values for CSMA-CoLMA-8-3 and CSMA-CoLMA-8-6 hydrogels reach 24.62 kPa and 30.34 kPa, respectively, close to that of natural skin (20 kPa). These results demonstrate that the toughening effect of the two-component CSMA-CoLMA hydrogel can significantly improve the poor mechanical properties of the single-component CSMA hydrogel. In summary, the CSMA-CoLMA-8-6 hydrogel exhibits superior mechanical properties, approaching those of natural skin.

[0114] 5. Culture and identification of primary rat skin cells

[0115] Primary keratinocytes (KCs) were extracted from the skin of newborn rats using the ability of keratinocytes to quickly adhere to type IV collagen matrices. Microscopic observation revealed that KCs initially grew slowly, but then began to grow rapidly after 3 to 5 days. The first fusion required 5 days of culture ( Figure 5 (Panel A, middle). Immunofluorescence analysis revealed high expression of the proliferation marker protein CK14 and low expression of the differentiation-related protein CK10 in these KCs. Furthermore, low levels of the stem cell marker protein CK19 were detected in the KCs. These experimental results demonstrate that the isolated KCs are in a state of high proliferation and low differentiation, with some cells retaining the characteristics of keratinocyte stem cells, making them suitable as seed cells for the next step in constructing artificial skin.

[0116] Primary fibroblasts (DFs) were obtained by trypsin digestion. Microscopic observation showed that DFs formed a tight monolayer after 4 to 7 days of culture ( Figure 5 (Panel B). Immunofluorescence staining results indicated high expression of vimentin, a characteristic fibroblast marker, in these DFs, confirming the activity and purity of KCs and DFs. To prevent senescence and differentiation of primary cells, subsequent experiments used secondary passage DFs and KCs.

[0117] 6. Cytotoxicity assay of CSMA-CoLMA hydrogel

[0118] Cytotoxicity is one of the important indicators for evaluating the biocompatibility of materials. This application uses the MTT method to evaluate the effect of CSMA-CoLMA hydrogel extract on the activity of primary skin cells. Figure 6As shown in panel A, there was no significant difference in RGR between KCs and the control group after 3 days of incubation with CSMA-CoLMA hydrogel extract. However, as the incubation time was extended to 5 days, the RGR of KCs was significantly higher than that of the control group (p<0.05). In addition, during the incubation with CSMA-CoLMA hydrogel extract, the RGR of DFs was significantly lower than that of the control group (p<0.05), but as the incubation time was extended, the RGR of DFs gradually increased ( Figure 6 In summary, CSMA-CoLMA hydrogel did not impair cell growth, and the biocompatibility of the hydrogel matrix with cells improved with prolonged culture time.

[0119] 7. Proliferation and adhesion of KCs and DFs in CSMA-CoLMA hydrogels

[0120] Photocurable bio-inks should support cell adhesion and proliferation, which are crucial for regulating various cellular physiological processes such as tissue reconstruction and regeneration. To evaluate the proliferation ability of KCs and DFs in hydrogels, KCs and DFs were cultured at a density of 1×10 6 The cells were encapsulated in CSMA-CoLMA bio-ink at a concentration of cells / mL, and the skin cell-loaded CSMA-CoLMA hydrogel matrix was obtained after photocuring. The cells were then cultured for 1, 3, and 5 days, and the cell proliferation ability was determined by MTT assay. Figure 6 Small picture in C and Figure 6 As shown in the middle panel D, cells proliferated rapidly with the extension of culture time. The cell viability of KCs on the 5th day increased by nearly 2 times compared with that on the 1st day, and the cell viability of DFs on the 5th day increased by nearly 3 times compared with that on the 1st day. SEM results showed that the cultured KCs and DFs adhered to the surface of the pore structure ( Figure 6 These results indicate that CSMA-CoLMA hydrogels can support the adhesion, growth, and proliferation of KCs and DFs.

[0121] To further observe the proliferation of KCs and DFs in the CSMA-CoLMA hydrogel matrix, three-dimensional imaging of skin cells was performed using CLSM on days 1, 3, and 5 using a live / dead cell staining kit. Figure 7 ) shows that KCs are evenly distributed and do not aggregate within the hydrogel. No significant red fluorescence-labeled dead KCs was observed during the culture process. Furthermore, the number of green-labeled live cells increased significantly with prolonged culture time and increased CoLMA ratio. This may be due to the high proportion of collagen added, which makes the pore size of the photo-crosslinked CSMA-CoLMA hydrogel matrix more uniform, thereby promoting the proliferation of skin cells.

[0122] From the top view and side view ( Figure 8) As can be seen, the proliferation trend of DFs in the hydrogel matrix is consistent with that of KCs. These results indicate that the CSMA-CoLMA-8-6 hydrogel matrix has good biocompatibility, can support the proliferation of skin cells, and has broad prospects in constructing three-dimensional biomimetic skin tissue.

[0123] 8. Printing artificial skin in Transwell chambers based on DLP printing technology

[0124] Printing biomimetic skin using a "bottom-up" DLP bioprinter

[0125] A common characteristic of bioprinters that perform "bottom-up" DLP printing is that the size of the printed material is always smaller than the size of the fluid reservoir. However, a key criterion for successful biomimetic skin construction is that the skin tissue adheres tightly to the perimeter of the chamber, and that the culture fluid does not leak from the perimeter of the chamber to the surface. This requires that the printed material possess a certain degree of swelling capacity, enabling it to swell to a tight fit around the perimeter of the Transwell chamber during the cell culture process after "bottom-up" printing. This differs from methods such as biomimetic skin printing using an integrated extrusion and light-curing bioprinter or biomimetic skin bioprinting using a solidified surface light source, which do not require the bio-ink to possess good swelling properties.

[0126] In addition, components from the skin's extracellular matrix (mainly composed of different types of collagen and glycosaminoglycans) are the best biomaterials for constructing bionic skin. Among them, glycosaminoglycan components (including chondroitin sulfate, dermatan sulfate, hyaluronic acid, keratan sulfate, heparin, etc.) have the swelling ability common to polysaccharide components.

[0127] Therefore, this application selects the bio-ink formed by compounding CSMA and CoLMA for DLP bioprinting bionic skin.

[0128] The basic structure of the common "bottom-up" DLP bioprinter on the market is as follows: Figure 9 As shown, Figure 9 The DLP printing light source of the bioprinter shown in the middle panel A is located below the liquid tank. The printing surface moves "from bottom to top" with the printed material, printing layer by layer. After printing is completed, the printed material will be separated from the liquid tank and adhere to the printing interface. Figure 9 The middle panel B shows that the structure of the bioprinter is that the printing surface carries the printed material under the liquid tank. The DLP printing light source moves "from bottom to top" and prints layer by layer. After printing is completed, the printed material will remain in the liquid tank and adhere to the printing interface.

[0129] The process of printing artificial skin in a Transwell chamber using the bio-ink formed by compounding CSMA and CoLMA is as follows:

[0130] I: Preparation of CSMA-CoLMA photocurable bio-ink loaded with skin cells:

[0131] The photocurable bio-ink is composed of CSMA, CoLMA solution, and photoinitiator LAP mixed at 4°C. All materials are sterilized by ultraviolet irradiation or filtration with a 0.22μm filter membrane before use. First, CoLMA is dissolved in 0.02N acetic acid solution at a concentration of 6mg / mL at 4°C, and then the pH of the solution is adjusted to 7.4 by adding 10×PBS and 1M NaOH. Subsequently, CSMA and LAP are added to the CoLMA solution at final concentrations of 8% and 0.25%, respectively. Finally, a skin cell suspension of a certain density is mixed with the bio-ink and added to the printer's liquid tank. The resulting CSMA-CoLMA hydrogel loaded with skin cells is printed using a "bottom-up" DLP printer.

[0132] II: Swelling and culture of CSMA-CoLMA hydrogels loaded with skin cells

[0133] The printed CSMA-CoLMA hydrogel loaded with skin cells was transferred to the Transwell chamber and immersed in the culture chamber until the gel was tightly attached to the periphery of the chamber. Figure 12 .

[0134] The above shows and describes the basic principles, main features, and advantages of the present invention. However, the above is only a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other implementation methods derived by any person skilled in the art without departing from the technical solution of the present invention should be included in the patent scope of the present invention.

Claims

1. A method for preparing bio-ink for DLP printing of bionic skin, characterized in that: The steps include: 1) Preparation of photocurable chondroitin sulfate (CSMA): Chondroitin sulfate was added to phosphate buffer, the pH was adjusted to 3.5, glycidyl methacrylate was added dropwise, the reaction system was heated, stirred, cooled, precipitated, filtered, and lyophilized to obtain CSMA. 2) Extraction of type I collagen CoL from rat tail; 3) Preparation of photocurable collagen CoLMA: CoL was added to acetic acid and magnetically stirred. The pH of the solution was adjusted to 7.

4. Methacrylic anhydride was added dropwise to the solution and magnetically stirred for reaction. The solution was dialyzed and freeze-dried to obtain CoLMA. 4) Compounding photocurable chondroitin sulfate (CSMA), photocurable collagen (CoLMA), and a photoinitiator to produce a bioink; the final mass concentrations of CSMA and the photoinitiator are 8-10% and 0.25%, respectively, and the final concentration of CoLMA is 3-9 mg / mL; In step 1), the mass volume ratio of chondroitin sulfate to glycidyl methacrylate is 1 g:3-12 mL; the reaction system is heated to 50-70° C. and stirred for 24-100 h.

2. The method for preparing the bio-ink for DLP printing of bionic skin according to claim 1, wherein: In step 4), during mixing, CoLMA is first dissolved in 0.02 N acetic acid solution at 4° C., the pH of the solution is adjusted to 7.4, and then CSMA, the photoinitiator and the CoLMA solution are mixed.

3. The method for preparing bio-ink for DLP printing of bionic skin according to claim 1, wherein: In step 4), the photoinitiator used is lithium phenyl-2,4,6-trimethylbenzoyl phosphite or 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone.

4. The method for preparing the bio-ink for DLP printing of bionic skin according to claim 2, wherein: After mixing the CSMA, CoLMA solution and photoinitiator, the final mass concentration of CSMA was 8% and the final concentration of CoLMA was 6 mg / mL.

5. The method for preparing the bio-ink for DLP printing of bionic skin according to claim 1, wherein: In step 3), the mass volume ratio of CoL to methacrylic anhydride is 1 g: 0.5-2.0 mL, the magnetic stirring process is carried out at 4°C, and the stirring reaction time is 24-72 h. The solution is dialyzed in 0.02 N acetic acid solution for 7 days, during which the dialysate is replaced every 8 hours.

6. Bio-ink for DLP printing of bionic skin, characterized in that: The bio-ink is prepared according to the method for preparing bio-ink for DLP printing of bionic skin according to any one of claims 1 to 5.

7. The bio-ink for DLP printing of bionic skin according to claim 6, characterized in that: The bio-ink is constructed by compounding modified photocurable chondroitin sulfate CSMA, photocurable collagen CoLMA and a photoinitiator.

8. The use of the bio-ink for DLP printing of bionic skin according to claim 7, wherein: This bio-ink has swelling function and can meet the growth requirements of skin fibroblasts and keratinocytes. It can be printed on the Transwell chamber and can swell to fit tightly around the Transwell chamber during the cell culture process.

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