Bionic bio-ink for repairing articular cartilage and preparation method of bionic bio-ink

Abstract

The invention provides bionic bio-ink for articular cartilage defect repair and a preparation method thereof.The bionic bio-ink is composed of type II collagen, hyaluronic acid, chondroitin sulfate and geniposide, the bionic bio-ink takes a natural matrix component, a rheology modifier and a mild cross-linking agent as a core system, and through precise matching and synergistic effect, the bionic bio-ink can be used for repairing articular cartilage defects. The cell compatibility and the printing precision are both considered, and the problems that existing ink is low in bionic degree and poor in differentiation effect are effectively solved. All the components of the bionic bio-ink are natural sources or low-toxicity reagents, the risk of immunological rejection is avoided, in-vitro degradation products are small molecular polysaccharide and amino acid which can be metabolized and absorbed by a human body, and inflammatory response is avoided.

Description

Technical Field

[0001] This invention belongs to the field of medical biomaterials, specifically relating to biomimetic bio-ink for repairing articular cartilage defects and its preparation method. Background Technology

[0002] Bio-inks are the core materials for in-situ 3D printing repair of articular cartilage, and they must simultaneously meet two major requirements: ① Cell compatibility, requiring the ability to support the survival, proliferation, and differentiation of seed cells, and to be non-toxic; ② Printing precision, requiring suitable rheological properties to accurately mold into a structure matching the cartilage defect, and stable gel strength after molding. Currently, publicly available bio-inks are mainly divided into three categories: natural polymer-based (such as collagen and hyaluronic acid), synthetic polymer-based (such as polyethylene glycol dimethacrylate and polycaprolactone), and composite-based. However, publicly available bio-inks face the challenge of simultaneously achieving both compatibility and precision.

[0003] For example, the natural polymer-based ink single type II collagen (COLII) ink published in the literature (Zhang et al., Biomaterials, 2020) has excellent cell compatibility (BMSCs survival rate ≥90%), but low viscosity (1000-1500 cP) and insufficient strength after gelation (<20 kPa), which makes it easy to collapse during 3D printing and has a layer thickness error ≥15 μm, which cannot meet the requirements of precise molding.

[0004] For example, the synthetic polymer-based ink polyethylene glycol dimethacrylate (PEGDA) ink disclosed in the literature (Cuiet al., Tissue Eng Part A, 2012) has high printing precision (layer thickness error ≤5μm) and high gel strength (≥60kPa), but it requires ultraviolet light crosslinking, is prone to cell damage (BMSCs survival rate ≤75%), and has a large difference from the composition of natural cartilage matrix, resulting in poor long-term repair effect.

[0005] In addition, the COLⅡ-alginate composite ink disclosed in the literature (Han et al., Addit Manuf, 2019) increases viscosity (2000-2500 cP) through alginate, but alginate degrades slowly, easily causing local inflammatory reactions, and the cell survival rate drops to 80%-85%, still failing to balance compatibility and precision.

[0006] Based on a summary of existing publicly available technologies, the main problems include the following:

[0007] I. The contradiction between compatibility and precision: natural materials have good compatibility but poor rheological properties (easy to collapse), while synthetic materials have high precision but high cytotoxicity. Existing composite inks cannot simultaneously achieve "cell survival rate ≥90% + printing layer thickness error ≤5μm + gel strength ≥40kPa".

[0008] Second, the biomimicry is insufficient. Most existing inks contain only 1-2 kinds of natural cartilage matrix components (such as only COLⅡ or HA), lacking key components such as chondroitin sulfate (CS), which cannot simulate the component ratio of natural cartilage matrix and affect the differentiation of seed cells into chondrocytes.

[0009] Third, defects in the gelation mechanism: chemical cross-linking agents (such as glutaraldehyde) or ultraviolet light cross-linking can damage cells, while physical cross-linking (such as temperature-sensitive cross-linking) results in low gel strength, making it difficult to achieve both "mild cross-linking" and "stable strength". Summary of the Invention

[0010] In view of this, the present invention provides a biomimetic bio-ink for repairing articular cartilage defects, comprising the following components:

[0011] Type II collagen 10-15 mg / mL, molecular weight 1.2×10 6 Da hyaluronic acid 5-8 mg / mL, chondroitin sulfate 3-5 mg / mL, molecular weight 1×10 5 Da hyaluronic acid 2-4 mg / mL, genipin 0.1-0.3 mg / mL, autologous spinal cord mesenchymal stem cells (BMSCs) 5×10 6 -8×10 6 per mL.

[0012] Furthermore, it consists of the following components:

[0013] Type II collagen 12 mg / mL, molecular weight 1.2 × 10⁻⁶ 6 Da hyaluronic acid 6 mg / mL, chondroitin sulfate 4 mg / mL, molecular weight 1×10 5 Da hyaluronic acid 2 mg / mL, genipin 0.2 mg / mL, autologous spinal cord mesenchymal stem cells (BMSCs) 5×10 6 -8×10 6 per mL.

[0014] This invention also provides a method for preparing the above-mentioned biomimetic ink, comprising the following steps:

[0015] (1) Preparation of Type II Collagen Solution

[0016] Dissolve the specified amount of type II collagen powder in 0.1M acetic acid solution to prepare a type II collagen solution;

[0017] (2) Preparation of composite matrix solution

[0018] Add the prepared chondroitin sulfate and a molecular weight of 1.2 × 10⁻⁶ to the type II collagen solution prepared in step (1). 6Da hyaluronic acid, 200W power, ultrasonically dispersed for 15 minutes, then 1×10 molecular weight added 5 Low molecular weight hyaluronic acid was stirred magnetically at 200 r / min for 1 hour to form a homogeneous composite matrix solution.

[0019] (3) Addition of crosslinking agent

[0020] Add genipin to the composite matrix solution prepared in step (2) and stir at 100 r / min for 30 min;

[0021] (4) Cellular complex

[0022] Autologous BMSCs cells cultured to the third generation in vitro were added to the above solution to disperse the cells evenly, thus producing a biomimetic bio-ink.

[0023] Furthermore, step (2) involves a molecular weight of 1×10 5 The viscosity is adjusted by the amount of hyaluronic acid used, and the final ink viscosity is controlled at 2500-3500 cP.

[0024] Furthermore, step (3) is performed at 37°C.

[0025] The beneficial effects of this invention are as follows:

[0026] This invention's biomimetic ink uses a core system of "natural matrix components + rheology modifier + mild crosslinking agent," achieving a balance between cell compatibility and printing precision through precise formulation and synergistic effects. By combining the "COL Ⅱ-HA-CS" natural matrix with the "LMW-HA rheology modifier," the compatibility of the natural components is preserved, while the viscosity is controlled by the molecular chain entanglement of LMW-HA, resolving the "compatibility-precision" contradiction. Simultaneously, CS is introduced to promote differentiation, effectively addressing the problems of "low biomimicry and poor differentiation effect" in existing inks. All components of the biomimetic ink described in this invention are of natural origin or low-toxicity reagents (genipin is a natural plant extract with an LD50 > 500 mg / kg), posing no risk of immune rejection. Its in vitro degradation products are small-molecule polysaccharides and amino acids, which can be metabolized and absorbed by the human body without causing inflammatory reactions.

[0027] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Detailed Implementation

[0028] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should also be noted that technical means not described in detail in this invention can be implemented using conventional technical means.

[0029] 1. Components and proportions of biomimetic ink

[0030] The mass concentrations and effects of each component are as follows (total mass concentration 25-35 mg / mL):

[0031] Components Mass concentration (mg / mL) effect Type II collagen (COL II) 10-15 The core scaffold components mimic the natural cartilage matrix, ensuring cell adhesion and differentiation. <![CDATA[Hyaluronic acid (HA, molecular weight 1.2×10 6 Da)]]> 5-8 Improves cell compatibility, regulates ink moisturizing properties, and helps improve rheological properties. Chondroitin sulfate (CS) 3-5 Promotes the differentiation of BMSCs into chondrocytes and replenishes key components of natural cartilage matrix. <![CDATA[Low molecular weight hyaluronic acid (LMW-HA, molecular weight 1×10 5 Da)]]> 2-4 Rheology modifiers increase ink viscosity through molecular chain entanglement, preventing print collapse. Genipin 0.1-0.3 A mild crosslinking agent that reacts with COL II amine groups to enhance gel strength and has no cytotoxicity.

[0032] Key formulation criteria: Referencing the natural articular cartilage matrix COLⅡ:HA:CS mass ratio of 3:1:1, LMW-HA (20%-30% of total HA mass) was introduced to regulate viscosity, and the amount of genipin was controlled at 0.1%-0.3% by mass (ensuring cross-linking strength while avoiding toxicity). Autologous BMSCs cultured to the third generation in vitro (density 5×10⁻⁶) 6 -8×10 6 (units / mL).

[0033] 2. The preparation steps of the biomimetic ink can be as follows (the entire process should be carried out in a sterile environment at 4℃ to avoid premature gelation):

[0034] (1) Preparation of COL II solution: Dissolve COL II powder in 0.1M acetic acid solution, stir magnetically for 2 hours (300 r / min) to prepare a 12 mg / mL COL II solution, and let stand at 4℃ for 12 hours to remove bubbles;

[0035] (2) Preparation of composite matrix solution: Add HA (6mg / mL) and CS (4mg / mL) to COL II solution, sonicate for 15min (power 200W, avoid bubbles), then add LMW-HA (2mg / mL), and magnetically stir for 1h (speed 200r / min) to form a homogeneous composite matrix solution;

[0036] (3) Addition of crosslinking agent: Add genipin (0.2 mg / mL) to the composite matrix solution and stir gently for 30 min (100 r / min) to avoid molecular chain breakage;

[0037] (4) Cell complexation: Autologous BMSCs cultured to the third generation in vitro (density 5×10⁻⁶) 6 -8×10 6Add the above solution (number per mL) and slowly blow it 20 times to make biomimetic bio-ink. Store at 4°C for later use (storage time ≤ 24h).

[0038] During the preparation process, the following should be noted:

[0039] (1) Rheological property control: The viscosity was adjusted by the amount of LMW-HA used, and the final ink viscosity was controlled at 2500-3500 cP (measured by rotational rheometer, shear rate 1s). -1 (At this time), this range can be adapted to nozzles with a diameter of 100-150μm, and there is no collapse when the extrusion pressure is 0.15-0.25MPa;

[0040] (2) Gel formation mechanism: The Schiff base reaction-genipin complex cross-linking was adopted. HA and CS were oxidized by sodium periodate to generate aldehyde groups, which reacted with COL II amino groups in a Schiff base reaction (preliminary gel formation, gel formation time 6-8 min). Genipin was further cross-linked with COL II amino groups (enhancing strength, final gel strength 45-55 kPa). The cross-linking process was carried out at a physiological temperature of 37℃ without cell damage.

[0041] (3) 3D printing process adaptation: It is compatible with multi-degree-of-freedom robotic arm bio-3D printers. The printing parameters are: nozzle diameter 100μm, extrusion pressure 0.2MPa, printing speed 5-8mm / s, layer thickness 20-30μm, and printing layer thickness error ≤5μm. After molding, the gel is soaked in simulated body fluid at 37℃ and pH 7.4 for 28 days. The degradation rate is ≤30% and the strength retention rate is ≥80%.

[0042] Cell compatibility and differentiation promotion verification

[0043] (1) Cell viability detection: After the ink was compounded with BMSCs, it was cultured in vitro for 1 week and the viability was detected by CCK-8 method. The viability was ≥92% (significantly higher than 80%-85% of existing compound inks).

[0044] (2) Differentiation capacity detection: After 21 days of in vitro culture, the expression levels of cartilage-specific genes (COLⅡ, Aggrecan) were detected by RT-PCR. The expression level was 30%-40% higher than that of COLⅡ ink alone, which proved that it promoted the differentiation of BMSCs into chondrocytes.

[0045] The biomimetic bio-ink of this invention uses natural cartilage matrix components (type II collagen (COLII), hyaluronic acid (HA), and chondroitin sulfate (CS)) as its core, ensuring excellent cell compatibility and enabling autologous BMSCs survival rate ≥92%, which is 7%-12% higher than existing composite inks.

[0046] By synergistically regulating the rheological properties of the ink (viscosity 2500-3500 cP), it is adapted to 3D printing processes, achieving a printing layer thickness error of ≤5μm and a gel strength of ≥45kPa after molding, which is 125%-175% higher than that of single natural inks. The viscosity of 2500-3500 cP is compatible with existing mainstream bio-3D printers (such as six-axis robotic arm printers), without the need for equipment modification. The gel after printing has good stability under physiological conditions (28-day strength retention rate ≥80%), which is easy to translate into clinical applications.

[0047] The mild Schiff base reaction-genipin complex cross-linking mechanism avoids chemical / UV damage while ensuring gel stability. The Schiff base reaction-genipin complex cross-linking does not require chemical toxic reagents or UV irradiation, and the apoptosis rate after cross-linking is ≤5% (the apoptosis rate of existing chemical cross-linking inks is ≥15%), thus ensuring the viability of seed cells.

[0048] It mimics the composition of natural cartilage matrix, promotes the differentiation of BMSCs (bone marrow mesenchymal stem cells) into chondrocytes, enhances the long-term effectiveness of tissue repair, and has a high degree of biomimicry; the synergistic effect of COLII, HA, and CS increases the expression level of COLII gene in BMSCs differentiated into chondrocytes by 30%-40% and the expression level of Aggrecan gene by 25%-35%, laying the foundation for the functionalization of tissue repair.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A biomimetic bio-ink for repairing articular cartilage defects, characterized in that, It consists of the following components: Type II collagen 10-15 mg / mL, molecular weight 1.2×10 6 Da hyaluronic acid 5-8 mg / mL, chondroitin sulfate 3-5 mg / mL, molecular weight 1×10 5 Da hyaluronic acid 2-4 mg / mL, genipin 0.1-0.3 mg / mL, autologous BMSCs 5×10 6 -8×10 6 per mL.

2. The biomimetic ink according to claim 1, characterized in that, It consists of the following components: Type II collagen 12 mg / mL, molecular weight 1.2 × 10⁻⁶ 6 Da hyaluronic acid 6 mg / mL, chondroitin sulfate 4 mg / mL, molecular weight 1×10 5 Da hyaluronic acid 2 mg / mL, genipin 0.2 mg / mL, autologous BMSCs 5×10 6 -8×10 6 per mL.

3. The method for preparing the biomimetic bio-ink according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Preparation of Type II Collagen Solution Dissolve the amount of type II collagen powder described in claim 1 or 2 in 0.1M acetic acid solution to prepare a type II collagen solution; (2) Preparation of composite matrix solution Add the prepared chondroitin sulfate and a molecular weight of 1.2 × 10⁻⁶ to the type II collagen solution prepared in step (1). 6 Da hyaluronic acid, 200W power, ultrasonically dispersed for 15 minutes, then 1×10 molecular weight added 5 Low molecular weight hyaluronic acid was stirred magnetically at 200 r / min for 1 hour to form a homogeneous composite matrix solution. (3) Addition of crosslinking agent Add genipin to the composite matrix solution prepared in step (2) and stir at 100 r / min for 30 min; (4) Cellular complex Autologous BMSCs cells cultured to the third generation in vitro were added to the above solution to disperse the cells evenly, thus producing a biomimetic bio-ink.

4. The preparation method according to claim 3, characterized in that, Step (2) involves a molecular weight of 1×10 5 The viscosity is adjusted by the amount of hyaluronic acid used, and the final ink viscosity is controlled at 2500-3500 cP.

5. The preparation method according to claim 3, characterized in that, Step (3) is performed at 37°C.