A pre-vascularized composite stent and a preparation method and application thereof

Abstract

The application provides a pre-vascularized composite scaffold and a preparation method and application thereof, and relates to the technical field of biomedical materials. The composite scaffold is made of a three-dimensional porous scaffold body and microvessel fragments (MVFs) loaded on the surface and / or internal pores of the scaffold body. The composite scaffold is made of a three-dimensional interconnected porous scaffold formed by a GelMA / PEGDA composite hydrogel, the scaffold body, MVFs as pre-vascularized functional units are introduced into the surface and / or internal pores of the scaffold, and the MVFs are secondarily fixed through a fibrin connection layer to obtain the composite scaffold. The pre-vascularized composite scaffold provided by the application can improve wound closure, tissue integration, collagen deposition and CD31 positive blood vessel formation. The application can be further developed and applied as a tissue engineering material for wound covering, promoting tissue integration or constructing a tissue engineering skin.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a prevascularized composite stent, its preparation method, and its application. Background Technology

[0002] Full-thickness skin defects are commonly seen in trauma, burns, infected debridement, and tumor resection, and are usually accompanied by the loss of the epidermis, dermis, and part of the subcutaneous tissue. Repairing this type of wound not only requires wound closure, but also requires implanted materials that can restore local blood supply as early as possible, support tissue integration, and reduce cell death and inflammatory response caused by hypoxia.

[0003] Current wound repair scaffolds typically employ natural collagen scaffolds, decellularized matrix, hydrogel dressings, or artificial porous scaffolds. While these materials provide some coverage and filling, insufficient vascularization in the early stages of implantation remains a significant factor limiting repair quality in full-thickness defects or thicker tissue engineering. The lack of available blood supply to the deeper layers of the scaffold in the early stages hinders the timely diffusion of oxygen and nutrients, easily leading to decreased cell viability, delayed scaffold integration, and unstable collagen remodeling.

[0004] Existing technologies have attempted to promote blood vessel ingrowth by delivering angiogenic factors such as VEGF, seeding endothelial cells or mesenchymal stem cells, or by adjusting pore size and porosity. However, growth factors suffer from short release cycles and difficulty in controlling dosage, single-cell seeding requires expansion culture and has unstable survival and network formation capabilities after implantation, and simple pore structure optimization is also insufficient to form a microvascular network with a natural vascular wall structure in a short period of time.

[0005] Microvascular fragments (MVFs) are typically obtained from adipose tissue through enzymatic digestion, sieving, and centrifugation. They are natural microvascular units composed of segments of arterioles, capillaries, and venules, retaining endothelial cells, pericytes, and some vascular wall-related structures. Compared to single endothelial cells, MVFs have a conformation closer to that of natural microvessels and possess the potential for sprouting, extension, and vascular network remodeling. Publicly available reports have shown that seeding adipose-derived MVFs onto collagen-glycosaminoglycan-based dermal substitutes, or combining them with bio-inks such as GelMA / HAMA / fibrinogen, can improve the vascularization of tissue-engineered materials.

[0006] However, existing MVF-based prevascularized scaffolds still have shortcomings: First, the pore structure of natural porous scaffolds is random, making it difficult to control the distribution and penetration depth of MVFs in the thickness direction; second, some hydrogel materials lack sufficient molding stability, making it difficult to simultaneously achieve cell compatibility, printability, and wet mechanical support; third, MVFs are prone to detachment from the scaffold surface or pore entrances during medium changes, transfers, or short-term culture; and fourth, the "prevascularized" state is often described conceptually in existing terminology, lacking clear and observable criteria for determination. Therefore, there is still a need for a composite hydrogel scaffold with controllable structure, stable MVF loading, and the ability to define the prevascularized state through objective morphological characteristics, along with its preparation method. Summary of the Invention

[0007] (a) Technical problems to be solved

[0008] To address the shortcomings of existing technologies, the main objective of this invention is to provide a prevascularized hydrogel composite scaffold loaded with microvascular fragments and its preparation method, thereby solving the problems of insufficient early vascularization of existing wound coverage and tissue engineering scaffolds, difficulty in standardizing scaffold pore structure, insufficient load stability of MVFs, and unclear boundaries of prevascularization state.

[0009] (II) Technical Solution

[0010] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0011] In a first aspect, the present invention provides a prevascularized composite stent, the composite stent comprising a three-dimensional porous stent body and microvascular fragments (MVFs) loaded in the surface and / or internal pores of the stent body.

[0012] Furthermore, the three-dimensional porous scaffold body is prepared by three-dimensional printing and photocrosslinking reaction of methacrylamide gelatin (GelMA), polyethylene glycol diacrylate (PEGDA), photoinitiator, and aqueous solvent. The three-dimensional porous scaffold body has interconnected mesh-like pores.

[0013] Furthermore, the mass-volume concentration of the methacrylamide gelatin (GelMA) is 5% to 20% (w / v), preferably 8% to 15% (w / v), and more preferably 10% to 12% (w / v).

[0014] Furthermore, the mass-volume concentration of the polyethylene glycol diacrylate (PEGDA) can be 1% to 10% (w / v), preferably 2% to 6% (w / v), and more preferably 3% to 5% (w / v).

[0015] Furthermore, the mass ratio of GelMA to PEGDA is 10:1 to 2:1, preferably 8:1 to 4:1.

[0016] Further, the photoinitiator is LAP or Irgacure 2959; when the photoinitiator is LAP, the crosslinking wavelength is preferably 365 nm or 405 nm, more preferably 405 nm; when the photoinitiator is Irgacure 2959, the crosslinking wavelength is preferably 365 nm.

[0017] Furthermore, the aqueous solvent is one or more of PBS, serum-free DMEM culture medium, and sterile deionized water.

[0018] Secondly, the present invention provides a method for preparing a prevascularized composite stent, wherein the composite stent is prepared according to the following steps:

[0019] (1) Preparation of GelMA / PEGDA precursor solution: Add GelMA to PBS or culture medium and stir to dissolve at 37-60℃; after GelMA is basically dissolved, add PEGDA and continue stirring; then add photoinitiator and stir in the dark to obtain a homogeneous precursor solution; preferably stir at 45-55℃ for 10-40 min, and after mixing, perform vacuum degassing or stand degassing for 1-15 min;

[0020] (2) Forming of three-dimensional printing bracket: Add the precursor liquid to the printing cylinder or syringe, connect the printing nozzle and perform three-dimensional printing according to the preset CAD model; the nozzle specification is preferably 18G~30G, more preferably 22G~27G; the printing pressure is preferably 20~150 kPa, more preferably 40~90 kPa; the printing speed is preferably 2~20 mm / s, more preferably 5~12 mm / s; the printing path is preferably 0° / 90° alternating.

[0021] (3) Crosslinking and curing: The printed scaffold prototype is placed under light to crosslink and cure, forming a GelMA / PEGDA composite scaffold; when using LAP, it is preferred to use a 405 nm light source and a light intensity of 5-30 mW / cm² for 10-120 s; when using Irgacure 2959, it is preferred to use a 365 nm light source for crosslinking; after crosslinking, it can be thoroughly washed and equilibrated with sterile PBS or culture medium to remove unreacted components;

[0022] (4) MVFs separation: Adipose tissue was taken and cut into 0.5-3 mm pieces under aseptic conditions. 3Small pieces are added to a collagenase digestion solution for enzymatic digestion; the collagenase is preferably type I collagenase, type II collagenase, or a combination thereof; the collagenase concentration is preferably 0.05%–0.30% (w / v), more preferably 0.08%–0.15% (w / v); the digestion temperature is preferably 37℃, and the digestion time is preferably 10–40 min, more preferably 15–25 min; after the enzyme digestion is completed, serum-containing culture medium is added to terminate the reaction, and then the particles are sieved through 500 μm and 30 μm sieves in sequence to collect MVFs in the range of 30–500 μm, and excessively fine fragments and excess enzyme solution are removed by low-speed centrifugation to collect MVFs in the target particle size range;

[0023] (5) MVF loading: Resuspend MVFs in a small amount of culture medium and introduce MVFs into the pores of the GelMA / PEGDA composite scaffold. Specifically, first pre-wet the GelMA / PEGDA composite scaffold, then add the MVF suspension in 1 to 5 drops at multiple points on the grid intersections and pore channel areas of the scaffold, and let it stand for 10 to 60 min; negative pressure assistance or low-speed centrifugation at 50 to 300 g for 1 to 5 min can also be used to promote the entry of MVFs into the pores inside the scaffold.

[0024] (6) Secondary fixation of fibrin junction layer: After MVFs are added to the stent and allowed to stand for 10-60 min, 10-50 μL of 2-8 mg / mL fibrinogen solution is added to each 10 mm diameter stent, followed by 5-30 μL of 0.5-5 U / mL thrombin solution. The stent is allowed to stand at room temperature for 5-15 min to form a dispersed fibrin junction layer in the contact area between MVFs and the stent.

[0025] (7) Prevascularization culture: After the fibrin junction layer is formed, culture medium is added, and the scaffold loaded with MVFs and fixed by the fibrin junction layer is placed in the culture system and cultured for 3 days at 37°C and 5% CO2 to allow MVFs to attach, extend, sprout and connect at least some segments inside the scaffold, thus obtaining the prevascularized composite scaffold; after the culture is completed, it is gently rinsed once with culture medium to remove unattached or free MVFs.

[0026] Furthermore, in step (3), the GelMA / PEGDA composite scaffold is a layered, stacked mesh structure with interconnected pores. The structural features of the GelMA / PEGDA composite scaffold include the following:

[0027] ①The preferred filament diameter is 100–500 μm, more preferably 150–350 μm;

[0028] ②The pore size is preferably 200–1200 μm, more preferably 300–800 μm;

[0029] ③The preferred layer height is 100–400 μm, more preferably 150–250 μm;

[0030] ④ The porosity is preferably 50% to 90%, more preferably 60% to 80%;

[0031] ⑤ The thickness of the support is preferably 0.5 to 6 mm, more preferably 1 to 3 mm;

[0032] ⑥ The wet compressive modulus of the stent is preferably 5 to 100 kPa, more preferably 10 to 50 kPa.

[0033] Further, in step (4), the MVFs are derived from subcutaneous fat, inguinal fat or abdominal adipose tissue of humans, mice or rats. After separation, the MVFs are in the form of cords, segments or short tubular structures, retaining endothelial cells, pericytes and some vascular wall related structures. The length of the MVFs is preferably 30 to 500 μm, more preferably 50 to 300 μm.

[0034] Further, in step (5), the MVFs in the GelMA / PEGDA composite stent have a loading rate of 1×10⁻⁶ per cubic centimeter of stent. 3 ~1×10 6 Strips, more preferably 1×10 4 ~5×10 5 For example, taking a 10 mm diameter, 2 mm thickness support as an example, the preferred MVFs load of a single support is 1 × 102. 4 ~5×10 5 strip.

[0035] Furthermore, in step (5), MVFs are distributed on the surface of the support, in the near-surface region, inside the pores, in the channel intersection region, or in a combination of the above regions.

[0036] Thirdly, the present invention provides the application of the above-mentioned prevascularized composite scaffold in the preparation of tissue engineering materials for wound coverage, promoting tissue integration, or constructing tissue-engineered skin.

[0037] (III) Beneficial Effects

[0038] The prevascularized composite stent provided by this invention has the following beneficial effects:

[0039] 1. The GelMA / PEGDA 3D printing scaffold provided by this invention has regular interconnected pores and good forming stability, which is beneficial for MVFs to enter, attach and sprout;

[0040] 2. The fibrin bonding layer used in this invention can improve the load stability of MVFs in the scaffold and reduce the detachment during fluid changes and transfers;

[0041] 3. The MVFs in the prevascularized composite stent provided by the present invention retain endothelial cells and vascular wall supporting cells, which can promote early vascularization after stent implantation.

[0042] 4. In a full-thickness skin defect model, the prevascularized composite scaffold provided by this invention can improve wound closure, tissue integration, collagen deposition, and CD31-positive angiogenesis.

[0043] In summary, the pre-vascularized composite scaffold provided by this invention effectively solves the problems of insufficient early vascularization, difficulty in standardizing scaffold pore structure, insufficient load stability of MVFs, and unclear boundaries of the pre-vascularized state in existing wound covering and tissue engineering scaffolds. This invention can be further developed and applied as a tissue engineering material for wound covering, promoting tissue integration, or constructing tissue-engineered skin. Attached Figure Description

[0044] Figure 1 Schematic diagram of the prevascularized composite scaffold fabrication process. Adipose tissue is digested and sieved to obtain microvascular fragments (MVFs); GelMA / PEGDA precursor solution is mixed, 3D printed, and photocrosslinked to form a porous mesh scaffold with a diameter of 10 mm; then MVFs are loaded into the pores of the scaffold and locally fixed by a fibrin junction layer. After in vitro culture for 3 days, a prevascularized composite scaffold with MVF budding and networking characteristics is formed. Figure 2 Morphology, budding, and immunofluorescence characterization results of MVFs. A: Bright-field observation of isolated MVFs, showing short tubular structures; scale bar = 30 µm; B: Budding results of MVFs in vitro culture; by day 7, MVFs were observed extending outward, budding, and forming branching structures; scale bar = 100 µm; C: Immunofluorescence staining results of MVFs, showing positive signals for α-SMA, IB4, vWF, and CD44, and counterstaining the cell nuclei with DAPI, indicating that the isolated MVFs retained microvascular-related cellular components and certain vascular wall structural characteristics. Scale bar = 20 µm.

[0045] Figure 3Structural characterization results of GelMA / PEGDA composite hydrogel and 3D printed scaffold. AB: Photocrosslinking of GelMA / PEGDA composite hydrogel and morphological retention after demolding; C: Infrared spectra of GelMA, PEGDA, and GelMA / PEGDA composite hydrogel; DE: Fiber placement path and mesh model of the 3D printed scaffold; F: Macroscopic morphology of the printed GelMA / PEGDA scaffold; Scale bar = 5 mm. G: Pore structure of the scaffold under scanning electron microscopy. The results show that the GelMA / PEGDA system can form a stable porous mesh scaffold with a connected pore structure suitable for MVF loading and budding extension.

[0046] Figure 4 Cell compatibility of GelMA / PEGDA scaffolds and the attachment and growth of MVFs on the scaffolds. A: Live / Dead staining results in the blank culture medium group; B: Live / Dead staining results in the GelMA / PEGDA scaffold extract group; Scale bar = 50 µm; C: Scanning electron microscopy observation of the attachment, growth, and extension of MVFs on the GelMA / PEGDA 3D printed scaffolds. The results showed that the GelMA / PEGDA scaffold extract did not show significant cytotoxicity, and the scaffold surface and pore structure could support the attachment and extension of MVFs.

[0047] Figure 5 Statistical results of full-thickness skin defect wound repair and wound closure rate in each group. A: Representative wound photographs of the Control group, Scaffold group, and MVFs-Scaffold group on postoperative days 0, 3, and 7; B: Statistical results of wound closure rate on postoperative days 3 and 7 in each group. The results showed that the wound closure rate of the MVFs-Scaffold group was higher than that of the Control group and the Scaffold group, suggesting that the prevascularized composite scaffold loaded with MVFs helps promote wound closure. *P<0.05 vs MVFs-Scaffold.

[0048] Figure 6 Representative images of H&E staining and Masson staining on postoperative day 7 for each group. H&E staining was used to observe re-epithelialization, inflammatory cell infiltration, and tissue integration; Masson staining was used to evaluate collagen deposition and arrangement. Scale bar = 200µm.

[0049] Figure 7 Representative images of CD31 immunofluorescence staining on postoperative day 7 for each group. Green signals indicate CD31-positive areas, and blue signals indicate DAPI-counted cell nuclei, used to evaluate angiogenesis in the wound area. Scale bar = 10 µm. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] Example 1

[0052] The prevascularized composite stent provided by this invention is prepared according to the following steps, as illustrated in the schematic diagram below. Figure 1 As shown:

[0053] (1) Weigh GelMA and add it to PBS, stir at 50°C for 20 min to dissolve it completely; add PEGDA and continue stirring for 10 min; then add LAP and stir in the dark to obtain a precursor solution containing 10% (w / v) GelMA, 3% (w / v) PEGDA and 0.15% (w / v) LAP; optionally, commercially available GelMA with a degree of methacrylation of about 70% to 90% is used, and PEGDA with a molecular weight of about 700 Da is used.

[0054] (2) After the precursor solution was allowed to stand and degas, it was added to the printing cylinder. A porous mesh scaffold with a diameter of 10 mm and a thickness of 2 mm was printed using a 25G nozzle, a printing pressure of 60 kPa, a printing speed of 10 mm / s and a layer height of 200 μm, following an alternating filament laying path of 0° / 90°. After printing, it was cross-linked and cured by irradiation with a 405 nm light source and a light intensity of 15 mW / cm² for 30 s. Then it was washed with sterile PBS and pre-equilibrated in a culture medium to obtain a GelMA / PEGDA composite hydrogel scaffold. The pore size of the representative sample was about 500±80 μm and the wet compressive modulus was about 18.6±3.4 kPa.

[0055] (3) Take rat inguinal adipose tissue, cut the adipose tissue into small pieces of about 1 mm³ under sterile conditions, add 5 mL of 0.10% (w / v) type I collagenase digestion solution for every 1 g of tissue, and digest at 37℃ with gentle shaking for 20 min; after digestion, add culture medium containing 10% serum to stop the reaction, sieve through 500 μm and 30 μm sieves, and collect MVFs by centrifugation at 100 g for 5 min to obtain MVFs with a length of about 50 to 300 μm.

[0056] (4) Resuspend the MVFs in a small amount of endothelial support medium, using approximately 5 × 10⁶ scaffolds per 10 mm diameter scaffold. 4The loading amount of MVFs is added in 3 to 5 drops to the surface of the scaffold and the area where the pores intersect. After standing for 20 minutes, the MVFs are initially introduced and attached to the scaffold pores. Then proceed to step (5) to fix the fibrin junction layer.

[0057] (5) After MVFs are added to the stent and allowed to stand for 20 min, 20 μL of 4 mg / mL fibrinogen solution is added to each 10 mm diameter stent, followed by 10 μL of 1 U / mL thrombin solution. The stent is then allowed to stand at room temperature for 8 min to allow a dispersed fibrin interface layer to form between the MVFs and the stent. After adding culture medium, the stent is incubated at 37°C and 5% CO2 for 3 days. After incubation, the stent is gently rinsed once with culture medium to remove any unattached or free MVFs. The amount of fibrin system used in this step should be controlled to avoid forming a monolithic gel that embeds the stent or blocks the pores of the stent.

[0058] Example 2

[0059] Characteristics of MVFs themselves, stent compatibility, and verification of MVF growth on stents

[0060] 1. Verification of MVFs' own features

[0061] The MVFs obtained in step (3) of Example 1 were subjected to bright-field observation, in vitro budding observation, and immunofluorescence staining. In bright-field observation, the MVFs obtained on the day of isolation were resuspended in culture medium, gently mixed, and a suitable amount of suspension was dropped onto a culture dish for observation and photography using an inverted microscope. In in vitro budding observation, the isolated MVFs were inoculated into culture plates pretreated with matrix gel, cell culture medium was added, and the plates were cultured at 37°C and 5% CO2. Bright-field photography was performed at the start of culture and 7 days after culture. In immunofluorescence staining, the MVFs obtained on the day of isolation were used as the main staining target. After collection, the MVFs were placed on a glass slide or suitable culture medium, fixed with 4% paraformaldehyde, washed with PBS, permeabilized with 0.1% Triton X-100, and then non-specific binding sites were blocked with 5% BSA. Subsequently, α-SMA antibody, vWF antibody, and IB4 lectin were added and incubated. After washing, the corresponding fluorescently labeled secondary antibodies were added, and the cell nuclei were counterstained with DAPI. Finally, the cells were observed under a fluorescence microscope.

[0062] Figure 2 These are representative images of MVFs, which exhibit short tubular structures with a length of approximately 230 μm. Figure 2 A); By day 7 of in vitro culture, MVFs were observed to bud outwards, extend, and form branching structures. Figure 2B). Immunofluorescence results showed that MVFs expressed vascular-related markers such as α-SMA, IB4, and vWF, and a positive CD44 signal was observed. α-SMA, IB4, and vWF reflect the vascular structural characteristics of MVFs from different perspectives. Among them, IB4 and vWF mainly indicate the presence of microvascular endothelial components in MVFs, suggesting that they retain the basis for forming vascular lumen-like structures; α-SMA indicates that MVFs still contain vascular smooth muscle cells, pericytes, and other vascular wall supporting cells, indicating that MVFs are not simply endothelial cell clusters, but retain a certain degree of microvascular wall structural integrity. Although CD44 is not a vascular-specific marker, it can serve as a reference indicator for cell adhesion, extracellular matrix binding, and interstitial / perivascular phenotypes. Its positive expression suggests the presence of cellular components in MVFs that are conducive to cell-matrix interaction, scaffold attachment, and subsequent migration and budding.

[0063] 2. Characterization of the scaffold body structure

[0064] Figure 3 Figures A and B show the macroscopic gelation and morphology retention results of the GelMA / PEGDA composite hydrogel. As can be seen from the figures, the GelMA / PEGDA composite hydrogel exhibits a certain degree of integrity and morphological stability after photogelation. It maintains its basic shape even after inversion and demolding, without significant collapse or rapid disintegration, indicating that this composite system possesses the stability required for subsequent printing, transfer, and cell loading operations.

[0065] Figure 3 C shows the infrared spectra of GelMA, PEGDA, and the GelMA / PEGDA composite hydrogel. GelMA exhibits an OH / NH stretching vibration peak at approximately 3300 cm⁻¹, and amide I and amide II bands at approximately 1650 cm⁻¹ and 1540 cm⁻¹, respectively. PEGDA shows an ester group C=O stretching vibration peak at approximately 1720–1730 cm⁻¹ and a COC / CO stretching vibration peak at approximately 1100–1150 cm⁻¹. The presence of characteristic peaks related to both GelMA and PEGDA in the GelMA / PEGDA composite hydrogel suggests that PEGDA has been incorporated into the GelMA system. Combined with macroscopic gelation, morphology retention, and printing results, the formation of the composite hydrogel is supported. Figure 3 DE represents the 3D printing path and mesh model for the support frame. Figure 3 F represents the macroscopic morphology of the printed support. Figure 3G represents the pore structure under scanning electron microscopy. The results show that the GelMA / PEGDA precursor solution, after 3D printing, can form a regularly laid and stacked mesh-like scaffold with interconnected pores inside. These regularly connected pores facilitate the entry of culture medium into the scaffold, enabling the exchange of nutrients and metabolites. Furthermore, they provide a spatial basis for MVFs to enter the scaffold pores, attach to the scaffold wire surface, and germinate and extend within the pores. Therefore, Figure 3 The results collectively demonstrate that the stent body of the present invention has good forming stability, composite material characteristics, and a suitable MVFs load and pre-vascularized interconnected pore structure.

[0066] 3. Stent compatibility verification

[0067] GelMA / PEGDA scaffold extract was used to culture fibroblasts and perform Live / Dead staining. Scaffold compatibility was evaluated by culturing fibroblasts in the scaffold extract and performing Live / Dead staining. Specifically, the cross-linked and cured GelMA / PEGDA scaffolds were thoroughly washed with sterile PBS to remove unreacted components and residual photoinitiators; subsequently, the scaffolds were placed in complete culture medium for pre-equilibration. Using a scaffold with a diameter of 10 mm and a thickness of 2 mm as an example, 1 mL of culture medium was added to each scaffold, and the extract was collected at 37°C for 24 h. Fibroblasts were seeded in 24-well plates, and after cell adhesion and stable growth, either GelMA / PEGDA scaffold extract or blank complete culture medium was added for culture. The scaffold extract group was used to evaluate the effect of the scaffold extract on cell survival, while the blank culture medium group served as a control. Live / Dead staining was performed at 0 h and 24 h of culture. During staining, live cells were labeled with Calcein-AM and dead cells were labeled with PI. After incubation in the dark, the cells were gently washed with PBS and images were acquired under a fluorescence microscope.

[0068] The viable cell percentage was calculated as follows: At least three fields of view were randomly selected from each sample, and the number of green fluorescent positive viable cells and red fluorescent positive dead cells were counted respectively. The viable cell percentage was calculated using the following formula: Viable cell percentage (%) = (Number of green viable cells / (Number of green viable cells + Number of red dead cells)) × 100%. Representative results showed that the viable cell percentage in the scaffold extract group was 94.6% ± 3.1%. Figure 4 B), the blank culture medium group was 96.2% ± 2.4% ( Figure 4 (A, n=6, p>0.05) indicates that the GelMA / PEGDA scaffold extract did not show significant cytotoxicity to fibroblasts, and the scaffold itself has good cell compatibility.

[0069] 4. Validation of MVF growth on scaffold

[0070] After loading MVFs onto a GelMA / PEGDA 3D printed scaffold, their adhesion, growth, and extension on the scaffold surface and in the pore region were observed using scanning electron microscopy.

[0071] The results are as follows Figure 4 As shown in Figure C, MVFs can be attached to the interface between the scaffold wire and the pores and remain extended along the pore channels, indicating that the three-dimensional printed scaffold can provide a suitable spatial interface for MVF loading and prevascularization.

[0072] 5. Verification of the fixation effect of the fibrin junction layer

[0073] MVF-loaded scaffolds were divided into a non-connector layer group and a fibrin-connector layer group. After three consecutive media exchanges, the MVF retention rate within the scaffolds was calculated. Specifically, the same number of MVFs were added dropwise to the surface and pore areas of the GelMA / PEGDA scaffold, respectively. After standing for 20 min, the scaffolds were divided into the non-connector layer group and the fibrin-connector layer group. The non-connector layer group was directly replenished with culture medium; the fibrin-connector layer group was sequentially replenished with fibrinogen solution and thrombin solution to form a dispersed fibrin-connector layer in the contact area between the MVFs and the scaffold. Both groups underwent three consecutive media exchanges. At each exchange, the removed old culture medium and detached MVFs were completely collected, and an equal volume of fresh culture medium was added. After standing for 10 min, the next media exchange was performed. The number of MVFs in the collected media from each exchange was counted under a microscope and summed to represent the total number of MVFs lost during the media exchange process. The initial number of MVFs added to the stent is denoted as N0, and the total number of MVFs lost during the three fluid changes is denoted as Nwash. The actual number of MVFs loaded / retained in the stent is N0-Nwash, and the MVF retention rate is calculated as (N0-Nwash) / N0×100%.

[0074] The results showed that the retention rate of MVFs in the group without the connecting layer was 67.3%±8.5%, while that in the group with the fibrin connecting layer was 88.6%±5.9% (n=6, p<0.05). The retention rate of MVFs was significantly higher than that in the group without the connecting layer, indicating that the fibrin connecting layer can improve the load stability of MVFs in the GelMA / PEGDA scaffold.

[0075] Example 3

[0076] Tissue integration validation of the prevascularized composite scaffold provided by this invention in a full-thickness skin defect model

[0077] A full-thickness skin defect model was established. The specific method was as follows: After anesthesia, the back of the experimental animals was shaved, disinfected, and draped. A circular full-thickness skin defect, reaching the subcutaneous fascia or dartos fascia layer, was prepared on the back using a skin biopsy punch or surgical scissors with a diameter of approximately 10 mm. Three groups were established: a Control group, a Scaffold group, and an MVFs-Scaffold group, with n=6 in each group. The Control group received only routine wound treatment and was covered with a sterile dressing; the Scaffold group received a GelMA / PEGDA scaffold without MVFs loaded in the defect area; and the MVFs-Scaffold group received a GelMA / PEGDA pre-vascularized composite scaffold loaded with MVFs and fixed via a fibrin junction layer in the defect area. All groups were covered with sterile dressings and fixed after treatment to reduce scaffold displacement and external contamination. The wound was photographed on postoperative days 3 and 7, and the area of ​​unhealed wound was measured using image analysis software. Wound closure rate was calculated as (initial wound area - non-healed wound area at the corresponding time point) / initial wound area × 100%. On postoperative day 7, wound and surrounding tissue were harvested, fixed, dehydrated, embedded, and sectioned for H&E staining, Masson staining, and CD31 immunofluorescence staining. H&E staining was used to observe re-epithelialization, inflammatory cell infiltration, and tissue integration; Masson staining was used to evaluate collagen deposition and arrangement; and CD31 immunofluorescence was used to evaluate angiogenesis in the wound area.

[0078] like Figure 5 As shown in Figure A, 3 days post-surgery, the wound area was significantly smaller in both the Scaffold and MVFs-Scaffold groups compared to the Control group. Figure 5 B represents the statistical analysis results: On the 3rd postoperative day, the wound closure rate of the MVFs-Scaffold group was 68.8±3.8%, which was significantly higher than that of the Control group and the Scaffold group (P<0.05); on the 7th postoperative day, the wound closure rate of the MVFs-Scaffold group reached 81.9%, which was significantly higher than that of the Control group and the Scaffold group (P<0.05).

[0079] Table 1. Wound closure rate results for each group (%)

[0080] Control <![CDATA[31.4±5.7 * ]]> <![CDATA[43.2±6.8 * ]]> Scaffold <![CDATA[49.1±5.0 * ]]> <![CDATA[61.4±7.1 * ]]> MVFs-Scaffold 68.8±3.8 81.9±5.9

[0081] Note: Compared to MVFs-Scaffold * P<0.05.

[0082] H&E staining results showed that in the Control group, the reepithelialization of the wound area was discontinuous, the new tissue filling was insufficient, and a large number of inflammatory cells were visible in the local area, indicating incomplete repair of the wound tissue structure. Figure 6In the Scaffold group, a certain degree of new tissue ingrowth was observed in the wound area, and cell infiltration and tissue integration were present in the scaffold area, but the continuity of re-epithelialization and tissue arrangement were still not ideal. In the MVFs-Scaffold group, the re-epithelialization of the wound surface was more continuous, the new tissue filling was more sufficient, and the scaffold integrated more tightly with the surrounding tissue, suggesting that the scaffold loaded with MVFs helps improve the quality of wound tissue repair. Masson staining results showed ( Figure 6 In the Control group, collagen deposition in the wound area was relatively low, and the collagen fibers were loosely and irregularly arranged. In the Scaffold group, some collagen deposition was observed, suggesting that the GelMA / PEGDA scaffold could provide support for new tissue ingrowth and collagen formation, but the local collagen arrangement was still not uniform. The MVFs-Scaffold group showed more complete collagen deposition, more uniform collagen fiber distribution, and more continuous arrangement, with the wound repair area exhibiting a better trend of tissue remodeling. These results suggest that pre-vascularized composite scaffolds loaded with MVFs not only facilitate wound closure but also promote re-epithelialization, tissue integration, and collagen deposition, thereby improving the quality of wound repair. Scale bar = 200 µm.

[0083] Figure 7 This is a representative image of CD31 immunofluorescence staining on postoperative day 7. Quantitative results showed that the proportions of CD31-positive areas in the Control group, Scaffold group, and MVFs-Scaffold group were approximately 0.27±0.1%, 2.71±0.8%, and 4.14±1.5%, respectively. Compared with the Control group, the Scaffold group showed increased CD31-positive signal, while the proportion of CD31-positive area in the MVFs-Scaffold group was further increased (p<0.05), suggesting that the GelMA / PEGDA pre-vascularized composite scaffold loaded with MVFs can promote increased vascularization in the wound repair area. Scale bar = 10 µm.

[0084] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A prevascularized composite stent, characterized in that, The composite stent consists of a three-dimensional porous stent body and microvascular fragments (MVFs) loaded in the surface and / or internal pores of the stent body.

2. The prevascularized composite stent as described in claim 1, characterized in that, The three-dimensional porous scaffold body is prepared by three-dimensional printing and photocrosslinking reaction of methacrylamide gelatin (GelMA), polyethylene glycol diacrylate (PEGDA), photoinitiator, and aqueous solvent. The three-dimensional porous scaffold body has interconnected mesh-like pores.

3. The method for preparing a prevascularized composite stent as described in claim 1, characterized in that, The composite scaffold is prepared according to the following steps: (1) Preparation of GelMA / PEGDA precursor solution: Add GelMA to PBS or culture medium and stir to dissolve at 37-60℃; After the GelMA has mostly dissolved, add PEGDA and continue stirring. Then, a photoinitiator was added and stirred in the dark to obtain a homogeneous precursor solution; (2) 3D printing bracket forming: Add the precursor liquid to the printing cylinder or syringe, connect the printing nozzle and perform 3D printing according to the preset CAD model; (3) Crosslinking and curing: The printed scaffold prototype is placed under light to crosslink and cure, forming a GelMA / PEGDA composite scaffold; (4) MVFs separation: Adipose tissue was taken and cut into 0.5-3 mm pieces under aseptic conditions. 3 Small pieces are added to collagenase digestion solution for enzymatic digestion; after the enzyme digestion is completed, serum-containing culture medium is added to terminate the reaction, and then the MVFs in the range of 30-500 μm are collected by sieving through 500 μm and 30 μm sieves respectively. The excessively fine fragments and excess enzyme solution are removed by low-speed centrifugation, and the MVFs are collected. (5) MVFs loading: Resuspend MVFs in a small amount of culture medium and introduce MVFs into the pores of GelMA / PEGDA composite scaffold. Specifically, first pre-wet the GelMA / PEGDA composite scaffold, then add the MVFs suspension in 1 to 5 drops at multiple points to the intersection of the scaffold grid and the pore channel area, and let it stand for 10 to 60 min. (6) Secondary fixation of fibrin junction layer: After MVFs are added to the stent and allowed to stand for 10-60 min, 10-50 μL of 2-8 mg / mL fibrinogen solution is added to each 10 mm diameter stent, followed by 5-30 μL of 0.5-5 U / mL thrombin solution. The stent is allowed to stand at room temperature for 5-15 min to form a dispersed fibrin junction layer in the contact area between MVFs and the stent. (7) Prevascularization culture: After the fibrin junction layer is formed, add culture medium, place the scaffold loaded with MVFs and fixed by the fibrin junction layer in the culture system, and continue to culture for 1 to 7 days at 37°C and 5% CO2, preferably for 3 days; after the culture is completed, the scaffold can be gently rinsed with culture medium to remove unattached or free MVFs, and finally obtain the prevascularized composite scaffold fixed by the fibrin junction layer.

4. The use of the prevascularized composite scaffold as described in claim 1 in the preparation of tissue-engineered materials for wound coverage, promoting tissue integration, or constructing tissue-engineered skin.

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