A porcine acellular dermal matrix for hip labral reconstruction and its preparation method and application

Abstract

The application provides a pig acellular dermal matrix for hip joint labrum reconstruction and a preparation method and application thereof, and belongs to the technical field of biomedical materials. Natural pig dermal tissue is washed and incubated, then is treated with a solution containing EDTA-trypsin and a solution containing a surfactant in sequence, and finally is washed to obtain the pig acellular dermal matrix. The pig acellular dermal matrix is treated by a specific process, is used as a xenogenic hip joint labrum reconstruction material, and exhibits good biocompatibility and tissue guiding function. Meanwhile, the preparation method is simple, raw materials are widely sourced, and the limitation of a graft source in a current hip joint labrum reconstruction surgery is solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a porcine decellularized dermal matrix for hip labral reconstruction, its preparation method, and its application. Background Technology

[0002] Hip impingement syndrome is a common cause of hip pain, and acetabular labral tears are the most common type of hip joint disease. The acetabular labrum is a fibrocartilaginous structure attached to the rim of the acetabulum, playing a crucial role in the stability and sealing of the hip joint. Injuries to the acetabular labrum can lead to decreased hip joint stability, accelerating wear and degeneration, and potentially developing into osteoarthritis. This results in long-term hip pain and limited mobility, severely impacting patients' quality of life and athletic ability.

[0003] With the rapid development of minimally invasive hip arthroscopy, traditional treatment strategies for labral tears include arthroscopic debridement, suturing and repair, and labral reconstruction. Compared to simple debridement, labral reconstruction surgery preserves the structural integrity of the labrum and can significantly improve long-term function, especially in cases of severe labral degeneration or defects, where labral reconstruction has become the preferred treatment option.

[0004] Autologous iliotibial bands are the primary graft source for labral reconstruction. However, using autologous tendons for labral reconstruction has significant limitations, including limited donor tissue availability, a high risk of complications such as decreased sensation at the donor site, risks of bleeding and infection, and healing issues with the reconstructed graft. Therefore, developing novel labral reconstruction graft materials has become a new research trend in this field.

[0005] In view of the above, this application is hereby submitted. Summary of the Invention

[0006] The purpose of this invention is to provide a porcine decellularized dermal matrix for hip labral reconstruction, its preparation method, and its application. By processing the porcine decellularized dermal matrix using a specific process, it is used as a xenogeneic hip labral reconstruction material, exhibiting good biocompatibility and tissue guiding function. Furthermore, its preparation method is simple, and the raw materials are widely available, overcoming the limitations of existing graft sources in hip labral reconstruction surgery.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing a decellularized porcine dermal matrix for hip labral reconstruction, comprising the following steps:

[0009] S1: Clean and incubate natural pig dermal tissue to remove fatty components.

[0010] Preferably, before step S1, the method further includes slicing natural pig dermal tissue into thin slices.

[0011] Furthermore, the washing and incubation process includes, in sequence: washing with phosphate-buffered saline (PBS), washing with acetone, washing and incubating with ethanol, and washing again with phosphate-buffered saline (PBS).

[0012] Furthermore, the washing time with phosphate-buffered saline (PBS) is 1-3 hours; the washing with acetone is performed 1-5 times, each time for 20-40 minutes; the washing time with ethanol is 20-40 minutes; and the incubation with ethanol is 6-10 hours or overnight; when washing again with phosphate-buffered saline, the pH of the phosphate-buffered saline solution is 7.2-7.6; further, the pH of the phosphate-buffered saline solution is 7.4. The acetone is a 100% acetone solution.

[0013] Preferably, the ethanol used for washing has a mass concentration of 100%, and anhydrous ethanol is used to remove residual acetone and water. Ethanol has excellent miscibility with acetone and water; anhydrous ethanol is completely miscible with both residual acetone and water. By soaking and washing with anhydrous ethanol, residual acetone and water inside the tissue can be effectively "displaced," thereby ensuring that the tissue is completely infiltrated by anhydrous ethanol.

[0014] Preferably, the ethanol used for incubation has a mass concentration of 60-80%. This concentration of ethanol solution can gently reintroduce water, prevent structural damage, complete rehydration, and create an environment conducive to enzyme digestion.

[0015] S2: The porcine dermal tissue treated in step S1 is then treated sequentially with a solution containing EDTA-trypsin and a solution containing surfactant to remove cellular components.

[0016] Furthermore, when using a solution containing EDTA-trypsin for treatment, the mass concentration of EDTA-trypsin is 0.20-0.30%, the treatment temperature is 0-5℃, and the treatment time is 18-28 hours.

[0017] Furthermore, when the treatment is performed using a solution containing a surfactant, the solution containing the surfactant includes sodium dodecyl sulfate solution and / or Triton X-100 solution.

[0018] Furthermore, when using a solution containing surfactant for treatment, the solution containing surfactant is replaced every 20-28 hours, and the total treatment time is 3-5 times.

[0019] Preferably, the surfactant-containing solution comprises a mixed solution of sodium dodecyl sulfate and Triton X-100. The combination of the two surfactants can more effectively remove cellular components (including cell membranes, cell nuclei, and intracellular substances) from tissues, while maximizing the preservation of the natural three-dimensional structure and bioactive components of the extracellular matrix.

[0020] Further, the mass concentration of sodium dodecyl sulfate in the mixed solution is 0.5-1.5%; the mass concentration of Triton X-100 in the mixed solution is 0.2-0.8%. Even further, the mass concentration of sodium dodecyl sulfate in the mixed solution is 1%; the mass concentration of Triton X-100 in the mixed solution is 0.5%.

[0021] S3: The tissue after decellularization in step S2 is rinsed to remove residual chemical reagents, and finally the porcine decellularized dermal matrix is ​​obtained.

[0022] Furthermore, the rinsing is performed using pure water; the rinsing time is 5-8 days.

[0023] Secondly, the present invention also provides a porcine decellularized dermal matrix for hip labral reconstruction, prepared using the aforementioned preparation method.

[0024] Thirdly, the present invention provides the use of the porcine decellularized dermal matrix of the second aspect in the preparation of a material for hip labral reconstruction.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) Using widely available pig dermis as raw material overcomes the bottleneck of limited autologous graft sources and provides ample material options for labial reconstruction.

[0027] (2) The proposed preparation method can completely remove cell nuclei and other immunogenic cell components from pig dermis, while perfectly preserving extracellular matrix structures such as collagen fibers, providing an excellent biological scaffold for tissue regeneration.

[0028] (3) Through in vivo rabbit animal model experiments, it was demonstrated that the porcine decellularized dermal matrix prepared in this invention can effectively promote the regeneration of labrum tissue after implantation, showing good biocompatibility and tissue guiding function.

[0029] (4) It fills the gap in the clinical application of xenogeneic alternatives for hip labral reconstruction, providing a potential and better treatment option for patients with acetabular labral tears, and has great clinical translational value and broad market prospects. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a photograph of natural pig dermal tissue.

[0032] Figure 2 This is a photograph of the decellularized dermal matrix of pigs after processing according to Example 1 of the present invention.

[0033] Figure 3 Comparison of hematoxylin-eosin (H&E) staining of natural porcine dermis (left) and porcine decellularized dermal matrix treated in Example 1 of this invention (right).

[0034] Figure 4 Comparison of DAPI cell nuclei staining in natural porcine dermis (left) and porcine decellularized dermal matrix treated in Example 1 of this invention (right).

[0035] Figure 5 Masson staining comparison of natural porcine dermis (left) and porcine decellularized dermal matrix treated in Example 1 of this invention (right).

[0036] Figure 6 This is a photograph of the decellularized porcine dermal matrix processed according to Example 1 of the present invention, used as a material for reconstructing the labrum of the rabbit hip joint.

[0037] Figure 7 This is a schematic diagram of the surgical procedure for rabbit hip labral reconstruction using porcine decellularized dermal matrix processed according to Example 1 of the present invention. From left to right, the steps are: incision, excision, punching, suture threading, and fixation.

[0038] Figure 8 This image shows a gross assessment comparison of the regenerated labrum at 8 weeks post-operation between the experimental group and the control group with labrum injury in the rabbit hip joint. The upper left image is a photograph of the acetabulum in the control group, the upper right image is a photograph of the acetabulum in the experimental group, the lower left image is a photograph of the femoral head in the control group, and the lower right image is a photograph of the femoral head in the experimental group. The yellow arrow points to the regenerated labrum.

[0039] Figure 9 The images show the hematoxylin-eosin (H&E) staining (left) and marson dye staining (right) of the porcine decellularized dermal matrix material from Example 2.

[0040] Figure 10 The images show the hematoxylin-eosin (H&E) staining (left) and marson dye staining (right) of the porcine decellularized dermal matrix material from Example 3. Detailed Implementation

[0041] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0042] As used in this article:

[0043] "Prepared from" is synonymous with "comprising". The terms "comprising", "including", "having", "containing", or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0044] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–5” is disclosed, the described range should be interpreted as including ranges “1–4”, “1–3”, “1–2”, “1–2 and 4–5”, “1–3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0045] In these embodiments, unless otherwise specified, the portions and percentages are all by weight.

[0046] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0047] Example 1

[0048] A method for preparing porcine decellularized dermal matrix for hip labral reconstruction:

[0049] (1) Take fresh natural pig dermal tissue (such as...) Figure 1As shown in the image, subcutaneous fat and residual tissue were removed using a scalpel, and the tissue was cut into 2 x 2 cm slices. First, the slices were washed with phosphate-buffered saline (PBS) at pH 7.4 for 2 hours. Next, they were washed three times with 100% acetone for 30 minutes each time to remove lipid components. Subsequently, they were washed with 100% ethanol for 30 minutes, followed by overnight incubation in 70% ethanol for further degreasing and sterilization. Finally, they were thoroughly washed four times with phosphate-buffered saline (PBS) at pH 7.4.

[0050] (2) The tissue obtained in step (1) was placed in 0.25% EDTA-trypsin solution and incubated at 4°C for 24 hours to digest the intercellular connections and loosen the cells. Then, it was carefully and thoroughly washed with phosphate-buffered saline (PBS) at pH 7.4 to remove trypsin. Subsequently, the tissue was placed in a mixed washing buffer containing 1% sodium dodecyl sulfate (SDS) solution and 0.5% Triton X-100 solution and swirled at room temperature for 96 hours, with the washing buffer being replaced with fresh solution every 24 hours, to lyse the cells and elute cell debris.

[0051] (3) Rinse the tissue obtained in step (2) continuously with double-distilled water (or ultrapure water) for at least one week until no bubbles are generated in the rinsing solution, to ensure that chemical detergents such as SDS or enzymes are completely removed. The product obtained after treatment is porcine decellularized dermal matrix, which is white and translucent in appearance, such as... Figure 2 As shown.

[0052] To evaluate the effectiveness of this invention, material characterization analysis was performed using histopathological staining, with the specific steps as follows:

[0053] The acellular porcine dermal matrix tissue prepared in Example 1 and the natural porcine dermal tissue used as a control were fixed in 4% paraformaldehyde solution for 24 hours. After routine dehydration, clearing, and paraffin embedding, they were embedded in paraffin and cut into 4-micrometer-thick sections. The paraffin sections were stained with hematoxylin-eosin (H&E), DAPI, and Masson staining, respectively.

[0054] The staining results are as follows: Figure 3 , Figure 4 and Figure 5 As shown. H&E staining ( Figure 3 ) and DAPI staining ( Figure 4 The results showed that, compared to the abundant and clearly visible cell nuclei in natural porcine dermis, no cell nuclei remained in the decellularized porcine dermal matrix, indicating that the decellularization process was very thorough. Masson staining ( Figure 5The results showed that, compared with natural pig dermis, the collagen and muscle fiber structures in the extracellular matrix of decellularized pig dermis were preserved intact, proving that the present invention effectively removes cells while causing less damage to the natural structure of the matrix.

[0055] Furthermore, by constructing a rabbit hip labral defect model, the effect of porcine decellularized dermal matrix on promoting labral regeneration was examined.

[0056] The specific operating steps are as follows: Six-month-old male New Zealand white rabbits were selected and anesthesia was administered via isoflurane inhalation. Under aseptic conditions, both hip joints of the rabbits were selected for surgery, with the left hip joint serving as the control group and the right hip joint serving as the experimental group.

[0057] Control group: A labral injury model was constructed. The skin, subcutaneous tissue, fascia and muscle layers of the left hip joint were incised layer by layer, the joint capsule was incised to expose the hip joint, the upper outer part of the labrum of the acetabulum was removed, and then the layers were sutured.

[0058] Experimental group: Constructing a labral reconstruction surgical model. After removing part of the labrum above and outside the acetabular rim, two holes were made at the 11 o'clock and 1 o'clock positions on the acetabular rim, and a suture was inserted into each hole. Porcine decellularized dermal matrix was trimmed to the size of the original labrum, as shown below. Figure 6 As shown, the labrum is reconstructed by tying sutures to the edge of the acetabulum. Figure 7 As shown.

[0059] Eight weeks after the modeling procedure, both acetabula were removed for gross evaluation. Results were as follows: Figure 8 As shown, in the control group (top left image), the labral defect was filled with only a small amount of scar tissue, without forming a structure resembling the labrum. In contrast, in the experimental group (top right image), newly formed, smooth white tissue resembling the natural labrum was visible at the defect site (indicated by the yellow arrow). This newly formed tissue fused well with the surrounding normal labral tissue, indicating that the implanted acellular porcine dermal matrix successfully guided the regeneration of the labrum and could protect the femoral head cartilage in the experimental group shown in the bottom right image.

[0060] It can be seen that the preparation of porcine decellularized dermal matrix material and the successful verification of the effect of this labrum reconstruction material in promoting labrum regeneration in in vivo rabbit animal experiments have demonstrated its feasibility and effectiveness as a xenograft for repairing acetabular labral tears, providing a solid experimental basis for clinical application.

[0061] Example 2

[0062] The difference from Example 1 is that in step (2), the surfactant contains only sodium dodecyl sulfate solution and does not contain Triton X-100 solution.

[0063] While using sodium dodecyl sulfate (SDS) solution alone can effectively lyse cells and remove cell nuclei, its effects are too drastic, disrupting the natural three-dimensional structure of the extracellular matrix and resulting in poor retention of key bioactive components such as glycosaminoglycans. The resulting decellularized matrix is ​​inferior to the ideal method using an SDS / Triton X-100 mixed solution in both structural integrity and bioactivity. Figure 9 As shown.

[0064] Example 3

[0065] The difference from Example 1 is that in step (1), the ethanol concentration of the ethanol incubated overnight is 100%.

[0066] While treatment with anhydrous ethanol alone can effectively replace and remove acetone and water from tissues, the lack of a gentle rehydration process with 60-80% ethanol directly leads to rapid shrinkage and deformation of the extracellular matrix in the subsequent hydration environment. This significantly disrupts its natural three-dimensional porous structure, making it unsuitable for subsequent applications as a tissue engineering scaffold. Figure 10 As shown.

[0067] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing porcine decellularized dermal matrix for hip labral reconstruction, characterized in that, Includes the following steps: S1: Clean and incubate natural pig dermal tissue; S2: The pig dermal tissue treated in step S1 is then treated sequentially with a solution containing EDTA-trypsin and a solution containing a surfactant; when using the solution containing EDTA-trypsin, the mass concentration of EDTA-trypsin is 0.20-0.30%, the treatment temperature is 0-5℃, and the treatment time is 18-28 hours. S3: The tissue after decellularization in step S2 is rinsed to obtain the porcine decellularized dermal matrix. In step S1, the cleaning and incubation sequentially include: cleaning with phosphate buffer solution, washing with acetone, washing and incubating with ethanol, and cleaning again with phosphate buffer solution; In step S1, the ethanol concentration used for washing is 100%; and / or, the ethanol concentration used for incubation is 60-80%. In step S2, the surfactant-containing solution includes a mixed solution of sodium dodecyl sulfate and Triton X-100; the mass concentration of sodium dodecyl sulfate in the mixed solution is 0.5-1.5%; and the mass concentration of Triton X-100 in the mixed solution is 0.2-0.8%.

2. The preparation method according to claim 1, characterized in that, In step S1, the washing time with phosphate buffer solution is 1-3 hours; And / or, the number of times acetone is used for washing is 1-5 times, each time for 20-40 minutes; And / or, the washing time with ethanol is 20-40 minutes, and the incubation with ethanol is 6-10 hours or overnight. And / or, when the phosphate buffer solution is used for washing again, the pH of the phosphate buffer solution is 7.2-7.

6.

3. The preparation method according to claim 1, characterized in that, In step S2, when the treatment is performed using a solution containing a surfactant, the solution containing a surfactant includes sodium dodecyl sulfate solution and / or Triton X-100 solution; And / or, when using a solution containing a surfactant for treatment, the solution containing the surfactant is replaced every 20-28 hours, for a total of 3-5 treatments.

4. The preparation method according to claim 1, characterized in that, In step S3, the rinsing is performed using pure water; And / or, the rinsing time is 5-8 days.

5. A porcine acellular dermal matrix for hip labral reconstruction, characterized in that, It is prepared using the preparation method according to any one of claims 1-4.

6. Use of the porcine decellularized dermal matrix of claim 5 in the preparation of a material for hip labral reconstruction.

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