Functionalized collagen scaffold based on 3D printing and application thereof in achilles tendon repair and heterotopic ossification prevention

Abstract

The invention relates to the technical field of biomedical engineering and tissue regeneration, and particularly discloses a functionalized collagen scaffold based on 3D printing and application of the functionalized collagen scaffold in achilles tendon repair and heterotopic ossification prevention. And integrating the CD26 + tendon stem cell exosome which is pretreated by the BMP inhibitor and modified by the collagen binding peptide to form the 3D printing scaffold. According to the invention, the composite scaffold is prepared by integrating a bifunctional strategy of BMP signal inhibition and collagen targeted slow release and combining a 3D printing technology, so that the problems of insufficient regeneration effect and high incidence of heterotopic ossification (HO) in the existing tendon repair technology are obviously solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering and tissue regeneration technology, in particular to a functionalized collagen scaffold based on 3D printing and its application in Achilles tendon repair and prevention of heterotopic ossification. BACKGROUND

[0002] In recent years, tissue engineering scaffolds combined with exosome therapy have shown potential in the field of tendon repair, but there are still the following key problems.

[0003] Heterotopic ossification (HO) risk: existing technologies use TSPC-derived exosomes to promote tendon regeneration, but unmodified exosomes can activate the BMP signaling pathway, leading to osteogenic differentiation (such as increased Runx2 expression), exacerbating HO formation.

[0004] Targeted delivery defects: traditional PLGA / collagen scaffolds can load exosomes, but lack specific binding mechanisms, leading to rapid release of exosomes and difficulty in maintaining local effective concentrations.

[0005] Single function: existing technologies focus on a single target (regeneration or anti-osteogenesis), and BMP-12-loaded scaffolds promote collagen synthesis but do not inhibit HO. SUMMARY

[0006] The purpose of the present application is to provide a functionalized collagen scaffold based on 3D printing and its application in Achilles tendon repair and prevention of heterotopic ossification, which breaks through the above limitations by pre-treating exosomes with BMP inhibitors and modifying collagen-binding peptides (CBD), achieving the synergistic effect of regeneration and anti-osteogenesis, and optimizing the mechanical strength of the scaffold through 3D printing process, significantly improving clinical applicability.

[0007] The present application adopts the following technical solutions:

[0008] A functionalized collagen scaffold based on 3D printing, the scaffold is a composite substrate combining polylactic acid-glycolic acid copolymer (PLGA) and type I collagen, integrating CD26 + tendon stem cell exosomes, forming a 3D printed scaffold.

[0009] Preferably, the composite substrate is composed of polylactic acid-glycolic acid copolymer (PLGA) and type I collagen, forming a porous network structure with mechanical strength and biocompatibility.

[0010] Preferably, the BMP inhibitor is LDN193189.

[0011] Preferably, the preparation process of the scaffold includes the following steps:

[0012] Step 1: CD26 + TSPCs isolation and culture;

[0013] Step 2: Exosome preparation and modification:

[0014] Pre-treatment: After TSPCs progenitor cells were seeded to 80% confluence, BMP inhibitor LDN193189 (1 mM) was used to treat for 24-48 hours to induce osteogenic inhibitory phenotype of exosomes;

[0015] Exosome collection: After 48 hours of culture with exosome-free complete medium, exosomes were purified by ultracentrifugation (100,000g, 70 min);

[0016] CBD modification: gcGFP-CBD-Lamp2b lentivirus vector was constructed and transduced into TSPCs to make exosome membrane express CBD peptide segment, and Western blot was used to verify the expression of Lamp2b-CBD fusion protein;

[0017] Step 3: Preparation of low-temperature rapid 3D printing Exos / PLGA / ColI scaffold: PLGA was dissolved in 1,4-dioxane at room temperature, and the homogeneous polymer solution suitable for low-temperature phase separation was obtained by magnetic stirring for 30 minutes. Lyophilized exosomes and type I collagen were added gently and stirred for 10 minutes to ensure uniform dispersion of the vesicles without affecting their integrity. The resulting bio-ink was printed at a speed of 5-10 mm / s through a 300 pm nozzle on an ultra-low temperature platform (-20 to -30°C) to print a scaffold with a designed filament diameter (about 300 pm) and interconnected pore size (about 500 pm) using LDM technology. These materials include CD26 + Exosomes of TSPCs (3D-Exos), LDN-treated exosomes (3D-LDN / Exos), and CBD-modified LDN-treated exosomes (3D-CBD@LDN / Exos). The prepared 3D-printed scaffolds were freeze-dried using a vacuum freeze dryer (FD8-5T, SIM, New Jersey, USA).

[0018] Step 4: Post-treatment and sterilization.

[0019] Preferably, Step 1, CD26 + The step of TSPCs isolation and culture includes:

[0020] Material collection: The Achilles tendon tissue of 6-week-old SD rats was taken and mechanically cut after removing the sheath;

[0021] Digestion: 1.5 mg / mL collagenase I + 2 mg / mL Dispase was used for digestion for 30 minutes, and the cell suspension was obtained by centrifugation (500g, 5 min);

[0022] Sorting: CD26 by flow cytometry + / CD90 + / CD73 + Cells, excluding CD45- / Ter119- / CD31-, were cultured in DMEM low glucose medium containing 10% FBS and 1% double antibody by volume concentration.

[0023] Preferably, step 4, the post-processing and sterilization step includes:

[0024] Freeze-drying: pre-freezing at -80℃ for 24 hours, vacuum drying for 48 hours, maintaining the scaffold porous structure;

[0025] Sterilization: ethylene oxide gas sterilization (temperature 40℃, humidity 60%, 4 hours).

[0026] Preferably, in step 3, the amount of exosomes added is 1×10 11 particles / mL.

[0027] Another object of the present application is to provide an application of the 3D printing-based functionalized collagen scaffold as described above in the repair of Achilles tendon and the prevention of heterotopic ossification, when the morphology of the scaffold is a rectangular net structure, the size: width 3-25mm, height 1-25mm, length 4-50mm (customizable), suitable for full-layer defect repair of Achilles tendon: pore characteristics: porosity 70%-85%, pore size 300-500μm, promoting cell infiltration and vascularization.

[0028] Preferably, when the morphology of the scaffold is a customized biomimetic structure, a net scaffold matching the patient's Achilles tendon anatomy is 3D printed based on CT / MRI data, which is used for complex defect or bone-tendon interface repair.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] The composite scaffold prepared by integrating the BMP signal inhibition and collagen-targeted slow-release dual-function strategies combined with 3D printing technology of the present application significantly solves the problems of insufficient regeneration and high incidence of heterotopic ossification (HO) in existing tendon repair technologies, and the specific advantages include:

[0031] Synergistic effect of regeneration and anti-ossification: exosomes pretreated with LDN193189 effectively inhibit the BMP pathway, and at the same time, through CBD modification, achieve the targeted slow release of exosomes in the scaffold, promote the ordered deposition of collagen I / III, and restore the mechanical properties to the level of natural tendons;

[0032] Strong clinical adaptability: The stent adopts a PLGA / collagen composite substrate, is compatible with 3D printing customized design, meets different defect forms and mechanical requirements, and the material meets the FDA standard, and supports minimally invasive implantation;

[0033] Long-term stability and safety: The CBD modification of the exosome through lentiviral transduction ensures targeted binding and avoids systemic toxicity, the degradation rate of the stent (6-8 weeks) matches the tendon healing period, and reduces the risk of secondary surgery. This technology provides an efficient and safe solution for sports injuries, senile degenerative tendon disease and postoperative ossification complications, and has clear industrialization prospects. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 CD26 + Characterization analysis chart of tendon stem cell derived exosome and LDN pretreated exosome;

[0035] Figure 2 Preparation and characterization chart of 3D-CBD@LDN / Exos;

[0036] Figure 3 In vivo exosome release chart of 3D-CBD@LDN / exosome stent;

[0037] Figure 4 Image evaluation chart of ectopic ossification;

[0038] Figure 5 Histological analysis chart of regenerated Achilles tendon;

[0039] Figure 6 Immunohistochemical analysis and extracellular matrix quantification chart of regenerated Achilles tendon at 4 and 12 weeks after operation;

[0040] Figure 7 Biomechanical test chart of regenerated Achilles tendon at 4 and 12 weeks after operation. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0042] Please refer to Figures 1-7 The embodiment of the present application provides a functionalized collagen stent based on 3D printing. The stent is a composite substrate combining polylactic acid-glycolic acid copolymer (PLGA) and type I collagen, and integrates CD26 modified by BMP inhibitor (LDN193189) pretreatment and collagen binding peptide (CBD).+ Tendon stem cell exosomes, 3D printed scaffold formed.

[0043] In this embodiment, the composite substrate is formed by combining polylactic acid-glycolic acid copolymer (PLGA) and type I collagen, forming a porous network structure with mechanical strength and biocompatibility.

[0044] In this embodiment, the BMP inhibitor is LDN193189.

[0045] In Figure 1 , the BMP inhibitor is LDN193189.

[0046] (A) Exosome (Exos) and LDN pretreated exosome (LDN / Exos) extraction process schematic diagram.

[0047] (B) Flow cytometry sorting CD26 + Representative map of tendon stem cells, positive markers CD26, CD90 and CD73, negative markers Ter119, CD31 and CD45.

[0048] (C) Transmission electron microscopy images and nanoparticle tracking analysis show the morphological characteristics of Exos and LDN / Exos. Scale bar = 100 nm.

[0049] (D) Western blot detection results of exosome marker proteins CD9, CD63 and TSG101.

[0050] (E) Immunofluorescence images of Scx and Runx2 expression in bone marrow mesenchymal stem cells treated with Exos or LDN / Exos for 7 days. Scale bar = 200 microns. Data is expressed as mean ± standard deviation ("ns" means P>0.05, "***" means P<0.001).

[0051] In Figure 2 , the BMP inhibitor is LDN193189.

[0052] (A) Schematic diagram of 3D-CBD@LDN / Exos preparation process.

[0053] (B) Western blot analysis of Lamp2b protein expression in LDN / Exos and CBD@LDN / Exos.

[0054] (C) Scanning electron microscopy images of type I collagen, freeze-dried exosome powder and final scaffold, scale bar = 200 microns.

[0055] (D) Schematic diagram of scaffold stretching and corresponding tensile stress measurement results.

[0056] (E) Stent porosity statistics.

[0057] (F) Live / Dead cell staining of BMSCs cultured on TCPs, 3D-LDN / Exos and 3D-CBD@LDN / Exos for 3 days: green shows live cells, red shows dead cells, and cell viability quantification analysis is attached.

[0058] (G) Immunofluorescence images of Scx and Runx2 expression after co-culturing BMSCs on TCPs, 3D-LDN / Exos and 3D-CBD@LDN / Exos for 7 days, scale bar = 200 pm.

[0059] (H) Exosome cumulative release curve of 3D-LDN / Exos and 3D-CBD@LDN / Exos within 21 days. Data are presented as mean ± standard deviation ("ns" means P > 0.05, "**" means P < 0.01, "***" means P < 0.001).

[0060] In Figure 3 the

[0061] (A) In vivo imaging of DiR-labeled exosomes delivered through the scaffold implanted at the site of tendon defect, showing sustained release property.

[0062] (B) Mean radiant efficiency semi-quantitative analysis over time. Data are presented as mean ± standard deviation ("ns" means P > 0.05, "*" means P < 0.05, "***" means P < 0.001).

[0063] In Figure 4 the

[0064] (A) Representative micro-CT images of ectopic ossification at 4 and 12 weeks post-surgery in rat Achilles tendons.

[0065] (B) Quantitative analysis of ectopic ossification volume and density among the four groups. Data are presented as mean ± standard deviation ("ns" means P > 0.05, "*" means P < 0.05, "**" means P < 0.01, "***" means P < 0.001).

[0066] In Figure 5 the

[0067] (A) Representative images of H&E, MT and PR staining of Achilles tendon sections at 4 and 12 weeks post-surgery, with healthy rat Achilles tendon as the natural control group. Scale bar = 100 pm.

[0068] (B) Comparison of histological scores of regenerated Achilles tendons in the control group, 3D-Exos group, 3D-LDN / Exos group, and 3D-CBD@LDN / Exos group. Data are expressed as mean ± standard deviation. ("ns" indicates P > 0.05, "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001).

[0069] In Figure 6 which:

[0070] (A) Representative images of immunostaining of collagen type I and collagen type III. Scale bar = 100 microns.

[0071] (B) Quantitative detection of collagen and sulfated glycosaminoglycan (sGAG) deposition in each group. Data are expressed as mean ± standard deviation. ("ns" indicates P > 0.05, "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001).

[0072] In Figure 7 which:

[0073] (A) Schematic diagram of clamp installation: the upper clamp (containing sandpaper) fixes the tendon, and the lower clamp holds the calcaneus.

[0074] (B) Fracture load and ultimate stress values of each experimental group; the black dotted line indicates the average value of the natural Achilles tendon. Data are expressed as mean ± standard deviation ("ns" indicates P > 0.05, "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" P < 0.001).

[0075] When the shape of the scaffold is a cuboid net structure, the dimensions are: width 3-25 mm, height 1-25 mm, length 4-50 mm (customizable), suitable for full-thickness Achilles tendon defect repair: pore characteristics: porosity 70%-85%, pore size 300-500 microns, promoting cell infiltration and vascularization.

[0076] When the shape of the scaffold is a customized bionic structure, patient-specific design: based on CT / MRI data, 3D printing of a net-shaped scaffold matching the patient's Achilles tendon anatomy for complex defect or bone-tendon interface repair.

[0077] Example 1.

[0078] A 3D-printed functional collagen scaffold, the preparation process of which comprises the following steps:

[0079] Step 1: CD26 + TSPCs isolation and culture;

[0080] Material: Take the Achilles tendon tissue of 6-week-old SD rats, mechanically cut after removing the sheath;

[0081] Digestion: Digestion with collagenase I 1.5 mg / mL + Dispase 2 mg / mL for 30 min, centrifugation (500 g, 5 min) to obtain cell suspension;

[0082] Sorting: CD26 + / CD90 + / CD73 + Cells, excluding CD45- / Ter119- / CD31-, were cultured in DMEM low glucose medium containing 10% FBS and 1% double antibody by volume concentration.

[0083] Step 2: Exosome preparation and modification:

[0084] Pre-treatment: After the progenitor cells TSPCs were inoculated to 80% confluence, the BMP inhibitor LDN193189 (1 μM) was used for 24 hours to induce the osteogenic inhibition phenotype of exosomes;

[0085] Exosome collection: Exosomes were purified by ultracentrifugation (100,000 g, 70 min) after 48 hours of culture with exosome-free complete medium (10% exosome-free serum and 1% double antibody in low glucose complete medium);

[0086] CBD modification: gcGFP-CBD-Lamp2b lentiviral vector was constructed, TSPCs were transduced, and CBD peptide segment was expressed on the exosome membrane, and Western blot was used to verify the expression of Lamp2b-CBD fusion protein;

[0087] Wherein, when constructing the gcGFP-CBD-Lamp2b lentivirus vector, the CBD peptide coding sequence needs to be inserted into the C-terminal of the Lamp2b extracellular domain by molecular cloning, and the gcGFP fluorescent marker is connected by IRES or self-cleavage 2A peptide to ensure the correct expression of the fusion protein. When transducing TSPCs, log phase cells (density 60-70%) were taken, and virus particles with MOI 10-20 were co-incubated with 8 μg / mL polybrene for 12 hours to complete the infection under the condition of 37°C, 5% CO2, and then the complete culture medium was replaced after 72 hours. The transduction population was enriched by flow sorting of gcGFP positive cells, and expanded to the 5th generation to ensure stable expression. After collecting the cell supernatant, the dead cells and debris were removed by 300g, 2000g centrifugation, and finally the exosomes were enriched by 100,000g ultracentrifugation for 70 minutes (4°C). When verifying by Western blot, 20 μg of exosome lysate was subjected to 12% SDS-PAGE electrophoresis, and after being transferred to the PVDF membrane, it was blocked with 5% skim milk for 1 hour, and then rabbit anti-Lamp2b (1:1000) and CBD monoclonal antibody (1:500) were used for 4°C incubation overnight, HRP labeled sheep anti-rabbit secondary antibody (1:5000) was incubated at room temperature for 1 hour, and ECL was developed. Untransduced cell exosomes and TSG101 need to be set as negative controls and internal references, and the Lamp2b-CBD fusion protein band is expected to be visible at about 75 kDa.

[0088] Step 3: Preparation of low-temperature rapid 3D printing Exos / PLGA / ColI scaffold: At room temperature, PLGA was dissolved in 1,4-dioxane, and the homogeneous polymer solution suitable for low-temperature phase separation was obtained by magnetic stirring for 30 minutes. The freeze-dried exosomes (1×10 11 Collagen I were added gently and stirred for 10 minutes to ensure uniform dispersion of the vesicles without affecting their integrity. The obtained bio-ink was printed on an ultra-low temperature platform (-20 to -30°C) at a speed of 5-10 mm / s through a 300 μm nozzle, and a designed scaffold was printed by LDM technology. These materials include CD26 + Exosomes of TSPCs (3D-Exos), LDN-treated exosomes (3D-LDN / Exos), and CBD-modified LDN-treated exosomes (3D-CBD@LDN / Exos). The prepared 3D printed scaffold was freeze-dried by a vacuum freeze dryer (FD8-5T, SIM, New Jersey, USA);

[0089] Step 4: Post-processing and sterilization:

[0090] Freeze-drying: pre-freezing at -80°C for 24 hours, vacuum drying for 48 hours, and maintaining the porous structure of the scaffold;

[0091] Sterilization: Ethylene oxide gas sterilization (temperature 40℃, humidity 60%, 4 hours).

[0092] The present application can adjust the exosome loading concentration (0.5-2×10 11 The particle / mL) or the degradation rate of the scaffold (controlled by the molecular weight of PLGA) according to the clinical needs, further expanding the application range.

[0093] Verification example 1: cuboid 3D printed scaffold repair rat Achilles tendon defect

[0094] 1) Model: 20-month-old rat Achilles tendon 6mm full-thickness defect.

[0095] 2) Implantation: 3D-CBD@LND / Exos scaffold (width 3mm, length 6mm, height 1mm) was fixed by modified Kessler suture.

[0096] 3) Results:

[0097] ① In vivo sustained-release experiment: 3D-CBD@LDN / Exos group can maintain red fluorescence signal at the injury site for up to 21 days, which indicates that compared with 3D-LDN / Exos scaffold, the scaffold has more persistent exosome sustained-release characteristics.

[0098] ② 3D-CBD@LDN / Exos group showed lower ectopic ossification volume and density than control group and 3D-Exos group at 4 weeks and 12 weeks. Although there was no significant difference between 3D-CBD@LDN / Exos group and 3D-LDN / Exos group at 4 weeks, but at 12 weeks, 3D-CBD@LDN / Exos group showed obvious reduction of ectopic ossification compared with 3D-LDN / Exos group. These results indicate that 3D scaffold loaded with LDN pretreated extracellular vesicles and modified by CBD can effectively inhibit tendon ectopic ossification.

[0099] ③ Modified Mankin score showed that the histological score of new tendon in 3D-CBD@LDN / Exos group was significantly lower than that in control group and 3D-Exos group. Although there was no significant difference between 3D-LDN / Exos group and 3D-CBD@LDN / Exos group at 4 weeks, but at 12 weeks, 3D-CBD@LDN / Exos group scored significantly lower than 3D-LDN / Exos group (P<0.05). These results indicate that compared with control group and 3D-Exos scaffold, scaffold loaded with LDN pretreated exosomes can significantly promote tendon regeneration, and CBD modification can further improve long-term regeneration effect. In addition, the porous and loose structure of 3D-CBD@LDN / Exos scaffold seems to be more conducive to tendon healing, while inhibiting ectopic ossification.

[0100] (4) At 12 weeks, the 3D-LDN / Exos group and the 3D-CBD@LDN / Exos group still maintained higher levels of collagen type I and III than the control group and the 3D-Exos group; notably, the 3D-CBD@LDN / Exos group exhibited particularly regular fiber arrangement. At 12 weeks, the 3D-CBD@LDN / Exos group exhibited significantly higher collagen and sulfated glycosaminoglycan deposition than the other groups. These results indicate that scaffolds loaded with LDN-pretreated exosomes can promote extracellular matrix synthesis over time, and that CBD modification can further improve fiber arrangement and enhance extracellular matrix (ECM) quality, thereby supporting superior tendon regeneration.

[0101] (5) At 12 weeks postoperatively, the mechanical properties of the control group, the 3D-Exos group, and the 3D-LDN / Exos group were still lower than those of the native tendon (failure load: 38.57 ± 3.32 N; ultimate stress: 22.47 ± 1.85 MPa), while the 3D-CBD@LDN / Exos group reached levels comparable to those of the native tissue. In addition, the failure load and ultimate stress of the 3D-Exos group and the 3D-LDN / Exos group were significantly higher than those of the control group, indicating that exosome-loaded scaffolds can enhance the mechanical recovery of Achilles tendon defects. Notably, the mechanical properties of the 3D-CBD@LDN / Exos group were significantly superior to those of the 3D-LDN / Exos group, indicating that CBD modification can further enhance the efficacy of LDN-pretreated exosomes in promoting tendon healing.

[0102] In summary, the core of the present application is:

[0103] 1. Dual-functional exosome design: Inhibit osteogenic differentiation tendency of exosomes by pretreating with BMP inhibitor LDN193189, combined with collagen-binding peptide (CBD) modification to achieve targeted and sustained release;

[0104] 2. 3D-printed composite scaffold: Use PLGA / collagen type I substrate combined with porous structure (porosity 70%-85%, pore size 300-500 pm) to provide mechanical support and cell infiltration environment;

[0105] 3. Synergistic mechanism: Exosome sustained release regulates tendon regeneration and inhibits HO.

[0106] It will be obvious to a person skilled in the art that the application is not limited to the details of the above-described exemplary embodiments, but can be implemented in other concrete forms without departing from the spirit or essential characteristics of the application. Therefore, the embodiments should be considered in all respects as illustrative and not restrictive, and the scope of the application is defined by the appended claims rather than the above description, and all changes falling within the meaning and range of equivalents of the elements of the claims are intended to be encompassed by the application.

[0107] Furthermore, it should be understood that although the specification is described in terms of embodiments, not every embodiment includes only one independent technical solution, and the specification is described in this way only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A functionalized collagen scaffold based on 3D printing, characterized in that, The scaffold is a composite substrate combining polylactic acid-glycolic acid copolymer and type I collagen, integrating CD26 cells pretreated with BMP inhibitors and modified with collagen-binding peptides. + 3D-printed scaffolds formed from tendon stem cell exosomes.

2. The functionalized collagen scaffold based on 3D printing according to claim 1, characterized in that, The BMP inhibitor is LDN193189.

3. The functionalized collagen scaffold based on 3D printing according to claim 1, characterized in that, The fabrication process of the stent includes the following steps: Step 1: CD26 + Isolation and culture of TSPCs; Step 2: Exosome preparation and modification: Pretreatment: After progenitor cells TSPCs were seeded to 80% confluence, they were treated with the BMP inhibitor LDN193189 for 24-48 hours to induce an exosome osteogenic inhibition phenotype. Exosome collection: The exosomes were cultured in a complete medium free of exosomes for 48 hours and then purified by ultracentrifugation. CBD modification: A gcGFP-CBD-Lamp2b lentiviral vector was constructed, and TSPCs were transduced to express CBD peptides on exosome membranes. Western blot was used to verify the expression of Lamp2b-CBD fusion protein. Step 3: Preparation of Exos / PLGA / Col I scaffold by low-temperature rapid 3D printing: At room temperature, PLGA was dissolved in 1,4-dioxane and magnetically stirred for 30 minutes to obtain a homogeneous polymer solution suitable for low-temperature phase separation; lyophilized exosomes and type I collagen were gently added and stirred for 10 minutes to ensure uniform dispersion of vesicles without affecting their integrity; the obtained bio-ink was printed through a 300μm nozzle at a speed of 5-10mm / s on an ultra-low temperature platform of -20 to -30℃, and a scaffold with designed filament diameter and interconnection aperture was printed using LDM technology; Step 4: Post-processing and sterilization.

4. A functionalized collagen scaffold based on 3D printing according to claim 3, characterized in that, Step 1, CD26 + The steps for isolating and culturing TSPCs include: Material collection: Achilles tendon tissue was collected from 6-week-old SD rats, and mechanically minced after removing the sheath. Digestion: The cells were digested with 1.5 mg / mL collagenase I + 2 mg / mL Dispase for 30 minutes, and then centrifuged to obtain a cell suspension. Sorting: CD26 screening by flow cytometry + / CD90 + / CD73 + Cells, excluding CD45- / Ter119- / CD31-, were cultured in DMEM low-glucose medium containing 10% FBS and 1% penicillin-dextrose antibody.

5. A functionalized collagen scaffold based on 3D printing according to claim 3, characterized in that, Step 4, the post-processing and sterilization steps include: Freeze-drying: Pre-freeze at -80℃ for 24 hours, vacuum dry for 48 hours, maintaining the porous structure of the support; Sterilization: Sterilization with ethylene oxide gas.

6. A functionalized collagen scaffold based on 3D printing according to claim 3, characterized in that, In step 3, the amount of exosomes added is 1×10⁻⁶. 11 Particles / mL.

7. The application of a 3D-printed functionalized collagen scaffold as described in any one of claims 1-6 in Achilles tendon repair and prevention of heterotopic ossification, characterized in that, When the scaffold is in the form of a rectangular mesh structure, its dimensions are: width 3-25mm, height 1-25mm, length 4-50mm, suitable for repairing full-thickness Achilles tendon defects. Pore characteristics: porosity 70%-85%, pore size 300-500μm, promoting cell infiltration and vascularization.

8. The application of a 3D-printed functionalized collagen scaffold in Achilles tendon repair and prevention of heterotopic ossification according to claim 7, characterized in that, When the scaffold is a customized biomimetic structure, a mesh scaffold that matches the patient's Achilles tendon anatomy is 3D printed based on CT / MRI data for the repair of complex defects or bone-tendon interfaces.

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