A self-waste-limb-source lymphatic vessel and lymph node transplantation kit and a preparation method thereof

Abstract

The application relates to the field of medical devices and discloses a self-abandoned-limb-source lymphatic vessel and lymph node transplantation kit and a preparation method, which comprises a collection module, an indocyanine green fluorescence navigation interface, ultramicro dissecting forceps and ultramicro scissors; a preservation module, a preservation cabin, a preservation liquid, an irrigation channel and an active rapid dyeing interface; the irrigation channel and the active rapid dyeing interface are communicated with the preservation cabin, and the preservation liquid is located in the preservation cabin; and an anastomosis sleeve is provided with a C-shaped buckle, an inner cavity is provided with a one-way valve, and an outer periphery is provided with a plurality of suture guide grooves. The kit recycles lymphatic vessels and lymph nodes in a non-infective amputation abandoned limb section into a self-transplant, thereby avoiding additional damage to a supply area.

Description

Technical Field

[0001] This invention relates to the field of medical devices, and in particular to an autologous waste limb lymphatic vessel and lymph node transplantation kit and its preparation method. Background Technology

[0002] Impaired lymphatic drainage at the stump after amputation can lead to stump lymphedema, causing swelling, difficulty in prosthesis fitting, skin breakdown, and pain. Traditional vascularized lymph node transplantation typically requires harvesting from healthy donor sites such as the groin, supraclavicular, submental, or greater omentum, posing an additional risk of donor site damage. Current ultramicroscopic lymphovenous anastomosis techniques lack specialized instrument combinations for amputation stump scenarios that can be integrated with ex vivo discarded limb lymphatic tissue. Furthermore, existing biodegradable anastomosis cannulas are often not specifically designed to address the low pressure, low flow rate, and reverse leakage suppression requirements unique to lymphatic vessels.

[0003] On the other hand, in non-infectious amputations, relatively intact superficial lymphatic vessels and regional lymph nodes often remain in the amputated distal limb segments. These tissues are discarded as medical waste in routine disposal procedures and are not systematically recycled.

[0004] Existing cell-engineered lymphoid tissue technologies often require a long in vitro construction period, which cannot meet the time requirement of obtaining autologous lymphoid tissue in the same surgery; there is currently a lack of integrated equipment solutions for the collection, preservation and anastomosis of ex vivo lymphoid tissue for amputation stumps. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an autologous waste limb-derived lymphatic vessel and lymph node transplantation kit, comprising: The acquisition module includes an indocyanine green fluorescence navigation interface, ultramicroscopic dissection forceps, and ultramicroscopic scissors; The preservation module includes a double-walled insulated preservation chamber, a preservation solution, a rinsing channel, and a rapid active staining interface. The double-walled insulated preservation chamber has a cavity inside. The rinsing channel and the rapid active staining interface are both located on the double-walled insulated preservation chamber and communicate with the cavity. The preservation solution is contained within the cavity. The anastomosis cannula is composed of a PCL / PLGA blend. A C-shaped buckle is provided at the first end of the anastomosis cannula. A one-way flap composed of the PCL / PLGA blend is provided in the middle section of the inner cavity of the anastomosis cannula. Multiple ultramicroscopic suture guide grooves are formed on the outer peripheral surface of the anastomosis cannula. The surface of the anastomosis cannula is coated with a PEG-PVP hydrophilic coating.

[0006] The anastomotic sleeve has an inner diameter of 0.20~0.80 mm, an outer diameter of 0.50~1.20 mm, a wall thickness of 0.08~0.20 mm, and a length of 3.0~8.0 mm; The opening angle of the C-type buckle is 60~110°; The number of the ultramicroscopic suture guide grooves is 4 to 8, and the width of each ultramicroscopic suture guide groove is 30 to 80 μm.

[0007] The opening pressure difference of the one-way lobe is 1~4 mmHg, and the closing response time is less than 50 ms; The thickness of the PEG-PVP hydrophilic coating is 2~8 μm.

[0008] The preservation solution contains heparin at a concentration of 5-25 IU / mL, adenosine at a concentration of 50-200 μmol / L, glucose at a concentration of 3-8 mmol / L, and HEPES at a concentration of 10-25 mmol / L. The preservation solution has an osmotic pressure of 280~320 mOsm / kg and a pH value of 7.20~7.45.

[0009] The volume of the cavity is 1~30 mL, and the double-walled insulated storage chamber can maintain an internal temperature of 2~6°C; The jaw tip width of the ultramicroscopic dissection forceps is 0.20~0.80 mm; The blade length of the ultramicro shears is 3.0~8.0 mm.

[0010] This invention also relates to a method for preparing an ex vivo lymphoid tissue component, wherein the preparation method is applied only to non-infectious discarded limb segments that have been separated from the body, and does not include surgical steps applied to living organisms, and includes the following steps: In vitro localization: Indocyanine green is injected into the non-infectious discarded limb segment via the indocyanine green fluorescent navigation interface, and the course of the superficial lymphatic plexus is located under a near-infrared light source; Ex vivo collection: Using the aforementioned ultramicroscopic dissecting forceps and ultramicroscopic scissors, ex vivo lymphatic vessel segments and ex vivo lymph nodes are separated from the aforementioned non-infectious discarded limb segments; Ex vivo preservation and irrigation: The separated ex vivo lymphatic vessel segments and ex vivo lymph nodes are placed in the preservation solution of the double-walled insulated preservation chamber, and the preservation solution is introduced into the cavity through the irrigation channel to irrigate the ex vivo lymphatic vessel segments; In vitro activity testing: Dyeing agent is introduced into the cavity through the rapid staining interface to stain and test the patency of the isolated lymphatic vessel segment; Ex vivo assembly: The tested ex vivo lymphatic vessel segment is fitted into the anastomotic cannula to form the ex vivo lymphatic tissue assembly.

[0011] In the in vitro positioning step, the concentration of the injected indocyanine green is 0.10~0.50 mg / mL, the injection volume at each point is 0.05~0.30 mL, and the number of injection points is 3~8; the excitation wavelength of the near-infrared light source is 760~810 nm.

[0012] In the ex vivo collection step, the diameter of the separated ex vivo lymphatic vessel segment is 0.20~0.80 mm and the length is 15~60 mm; the diameter of the separated ex vivo lymph node is 2~6 mm.

[0013] The time from the start of the in vitro positioning step to the completion of the in vitro assembly step is less than or equal to 120 minutes.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This kit is the first to recover lymphatic vessels and lymph nodes from non-infectious amputated limb segments as autologous grafts, avoiding additional damage to the donor site; the preservation module can maintain tissue viability and complete functional screening within the ex vivo time window; the flow-limiting anastomosis cannula improves the anastomosis effect of low-pressure lymphatic vessels through the one-way valve and guide groove structure, reduces reverse leakage, and provides integrated instrument support for stump lymphatic reconstruction. Attached Figure Description

[0015] Figure 1 : Axial cross-section diagram of the storage module; Figure 2 : Schematic diagram of the axial section and end view of the matching sleeve; Figure label: 1—Outer shell; 2—Insulating layer; 3—Cavity; 4—Ex vivo lymphatic graft; 5—Irrigation inlet; 6—Irrigation outlet; 7—Staining port; 8—Temperature sensor; 9—pH sensor.

[0016] 11—Outer wall; 12—C-type buckle; 13—One-way flap; 15—Suture guide groove; 16—Hydrophilic coating. Detailed Implementation

[0017] The terms “first,” “second,” “third,” “fourth,” etc. (if present) in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms “comprising” or “having,” and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0018] like Figure 1 , 2 As shown, an autologous waste limb-derived lymphatic vessel and lymph node transplantation kit includes: The acquisition module includes an indocyanine green fluorescence navigation interface, ultramicroscopic dissection forceps, and ultramicroscopic scissors; The preservation module includes a double-walled insulated preservation chamber, a preservation solution, a rinsing channel, and a rapid active staining interface. The double-walled insulated preservation chamber has a cavity inside. The rinsing channel and the rapid active staining interface are both located on the double-walled insulated preservation chamber and communicate with the cavity. The preservation solution is contained within the cavity. The anastomosis cannula is composed of a PCL / PLGA blend. A C-shaped buckle is provided at the first end of the anastomosis cannula. A one-way flap composed of the PCL / PLGA blend is provided in the middle section of the inner cavity of the anastomosis cannula. Multiple ultramicroscopic suture guide grooves are formed on the outer peripheral surface of the anastomosis cannula. The surface of the anastomosis cannula is coated with a PEG-PVP hydrophilic coating.

[0019] As a preferred technical solution, the anastomotic sleeve has an inner diameter of 0.20~0.80 mm, an outer diameter of 0.50~1.20 mm, a wall thickness of 0.08~0.20 mm, and a length of 3.0~8.0 mm; As a preferred technical solution, the opening angle of the C-type buckle is 60~110°; As a preferred technical solution, the number of the ultramicroscopic suture guide grooves is 4 to 8, and the width of each ultramicroscopic suture guide groove is 30 to 80 μm.

[0020] As a preferred technical solution, the opening pressure difference of the one-way lobe is 1~4 mmHg, and the closing response time is less than 50 ms; As a preferred technical solution, the thickness of the PEG-PVP hydrophilic coating is 2~8 μm.

[0021] As a preferred technical solution, the preservation solution contains heparin at a concentration of 5-25 IU / mL, adenosine at a concentration of 50-200 μmol / L, glucose at a concentration of 3-8 mmol / L, and HEPES at a concentration of 10-25 mmol / L. As a preferred technical solution, the osmotic pressure of the preservation solution is 280~320 mOsm / kg, and the pH value is 7.20~7.45.

[0022] As a preferred technical solution, the volume of the cavity is 1~30 mL, and the double-walled insulating storage chamber can maintain an internal temperature of 2~6 °C; As a preferred technical solution, the width of the jaw tip of the ultramicroscopic dissection forceps is 0.20~0.80 mm; As a preferred technical solution, the blade length of the ultramicro shears is 3.0~8.0 mm.

[0023] This invention also relates to a method for preparing an ex vivo lymphoid tissue component, wherein the preparation method is applied only to non-infectious discarded limb segments that have been separated from the body, and does not include surgical steps applied to living organisms, and includes the following steps: In vitro localization: Indocyanine green is injected into the non-infectious discarded limb segment via the indocyanine green fluorescent navigation interface, and the course of the superficial lymphatic plexus is located under a near-infrared light source; Ex vivo collection: Using the aforementioned ultramicroscopic dissecting forceps and ultramicroscopic scissors, ex vivo lymphatic vessel segments and ex vivo lymph nodes are separated from the aforementioned non-infectious discarded limb segments; Ex vivo preservation and irrigation: The separated ex vivo lymphatic vessel segments and ex vivo lymph nodes are placed in the preservation solution of the double-walled insulated preservation chamber, and the preservation solution is introduced into the cavity through the irrigation channel to irrigate the ex vivo lymphatic vessel segments; In vitro activity testing: Dyeing agent is introduced into the cavity through the rapid staining interface to stain and test the patency of the isolated lymphatic vessel segment; Ex vivo assembly: The tested ex vivo lymphatic vessel segment is fitted into the anastomotic cannula to form the ex vivo lymphatic tissue assembly.

[0024] As a preferred technical solution, in the in vitro positioning step, the concentration of the injected indocyanine green is 0.10~0.50 mg / mL, the injection volume at each point is 0.05~0.30 mL, and the number of injection points is 3~8; the excitation wavelength of the near-infrared light source is 760~810 nm.

[0025] As a preferred technical solution, in the ex vivo collection step, the diameter of the separated ex vivo lymphatic vessel segment is 0.20~0.80 mm and the length is 15~60 mm; the diameter of the separated ex vivo lymph node is 2~6 mm.

[0026] As a preferred technical solution, the time from the start of the in vitro positioning step to the completion of the in vitro assembly step is less than or equal to 120 minutes.

[0027] Example 1: Preparation of isolated lymphoid tissue samples from the hind limbs of SD rats Animals: Healthy adult male Sprague-Dawley (SD) rats, weighing 280–320 g, n=4 per group; all procedures in this embodiment were approved by the Laboratory Animal Ethics Committee and complied with the relevant regulations on the use and welfare of laboratory animals.

[0028] Methods: Under isoflurane inhalation anesthesia (induction 4%, maintenance 2%), rats were euthanized according to predetermined endpoint criteria, and the right hind limb was removed whole as an isolated limb segment; all subsequent steps were performed on this isolated hind limb segment, not on live animals: S1. In vitro localization: In the superficial lymphatic plexus of the dorsum of the foot of the isolated hind limb segment, inject indocyanine green 0.25 mg / mL at 5 points, 0.10 mL at each point; locate the course of the superficial lymphatic plexus under a 785 nm near-infrared light source; S2. Ex vivo collection: Using ultramicroscopic dissecting forceps with a jaw width of 0.30 mm and ultramicroscopic scissors with a blade length of 5.0 mm, 3–5 lymphatic vessel segments with a diameter of 0.20–0.40 mm and a length of 5–15 mm, and 2–3 popliteal / groin lymph nodes with a diameter of 1–3 mm were separated from the ex vivo hind limb segment. S3. In vitro preservation and perfusion: The above in vitro samples were placed in a 2 mL SD rat scale-down preservation chamber at 4 °C; and perfused for 60 s with a preservation solution containing 10 IU / mL heparin, 100 μmol / L adenosine, 5 mmol / L glucose, 20 mmol / L HEPES, osmotic pressure 300 mOsm / kg, pH 7.35 at a pressure of 10 mmHg and a flow rate of 0.3 mL / min. S4. In vitro activity test: The samples were stained with calcein for 60 s and tested for patency under 12 mmHg pressure via the activity staining interface. The endothelial activity ratio and lumen patency rate were recorded. S5. Ex vivo assembly: The qualified ex vivo lymphatic vessel segment is fitted into an anastomosis cannula with an inner diameter of 0.30 mm, a length of 4.0 mm, and 6 50 μm suture guide grooves to complete the assembly of the ex vivo graft component.

[0029] Results: The preliminary experimental data of this embodiment are shown in Table 3.

[0030] Example 2: Verification of the M1 acquisition module using ex vivo instruments and tissue stands Steps S1–S2 of Example 1 were repeated on isolated hindlimb segments of SD rats. The success rate, collection time, and lymphatic segment integrity score of the ex vivo collection were compared between the M1 collection module of this invention (jaw tip width 0.30 mm, ultramicroscopic scissor blade length 5.0 mm, indocyanine green concentration 0.25 mg / mL, 5 points × 0.10 mL, 785 nm illumination) and a control instrument combination (ordinary microforceps, without indocyanine green navigation) without this kit. Each group had n=4, and results are expressed as mean ± SD. This example is an ex vivo instrument / tissue test and does not involve in vivo surgery.

[0031] Example 3: Ex vivo preservation performance of the M2 preservation module (based on ex vivo lymphoid tissue samples) The preservation solution formulation is shown in Table 2. Lymphatic vessel segments derived from isolated hind limb segments of SD rats were randomly assigned to either the control group (4 °C saline) or the preservation solution group (4 °C, formulation shown in Table 2), with n=4 in each group. After preservation in a 2 mL scale-down SD rat preservation chamber for 60 min, endothelial cell viability, lumen patency, LYVE-1 positive endothelial integrity score, TUNEL apoptosis index, and the proportion of cells still congruent after 60 min were measured. Preliminary experimental data are shown in Table 3.

[0032] Example 4: Ex vivo test bench performance of M3 anastomotic sleeve Using a silicone simulated lumen (0.30 mm inner diameter, simulating SD rat lymphatic vessels) and a PBS perfusion bench, the one-way valve opening differential pressure, closing response time, and reverse leakage rate of the cannula of this invention (0.30 mm inner diameter, PCL / PLGA 75:25, six 50 μm guide grooves, 2 μm PEG-PVP coating) and a non-degradable silicone control cannula were measured. Simultaneously, the mechanical consistency before and after shelf life was evaluated under storage conditions of 4–25 °C. Each group had n=4. This embodiment is a purely ex vivo instrument bench test and does not involve any animal or human studies.

[0033] Example 5: Overall evaluation of this kit in a hindlimb amputation-lymphedema model in SD rats. Animals and Ethics: Healthy adult male SD rats, weighing 280–320 g, were used. They were divided into a sham-operated group, a model group, and a kit group, with n=4 in each group. All procedures were approved by the Laboratory Animal Ethics Committee, and standards for anesthesia, analgesia, infection monitoring, and human endpoints were established. This example is only for verification purposes and does not constitute a step in the in vitro preparation method described in claim 3, nor does it involve human research.

[0034] Modeling and Treatment: Under isoflurane inhalation anesthesia (induction 4%, maintenance 2%), a hindlimb amputation-lymphedema animal model was established in SD rats at the knee joint plane. Ex vivo lymphatic tissue collection and assembly of the ex vivo lymphatic tissue components (S1–S5) as described in Example 1 were performed on the excised distal limb segment. The model group only underwent amputation modeling, while the kit group was evaluated in the animal model according to ethically approved protocols to assess the effectiveness of the ex vivo lymphatic tissue components in reconstructing lymphatic drainage at the stump. Four weeks post-operation, the arrival time of ICG lymphocyte imaging, stump subcutaneous edema coefficient, increase in stump circumference, lymphatic vessel density (HE / LYVE-1), and VEGFR-3 positive area ratio were evaluated in each group. Preliminary experimental data are shown in Table 4.

[0035] Example 6: Overall performance comparison between this kit and the internal control example. The overall performance of this kit was compared with that of Control Example A (without kit matching) and Control Example B (non-degradable silicone sleeve) under the conditions of SD rat hind limb segment ex vivo and ex vivo bench. Preliminary experimental data are shown in Table 5.

[0036] Example Data Table Table 1 Comparison of key parameters of the three modules of this kit with the scaled-down specifications of SD rats. Note: This table compares the general specifications of this kit with the scaled-down specifications for SD rats.

[0037] Table 24 °C Preservation Solution Formulation and Osmotic Parameters Table 3 Immediate preservation viability of isolated lymphoid tissue samples from the hind limbs of SD rats (mean±SD, n=4) Table 4. Lymphatic drainage function of the stump in the SD rat hindlimb amputation-lymphedema model 4 weeks after surgery (mean±SD, n=4) Table 5. Overall performance comparison between this kit and the internal control example (mean ± SD, n=4) 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 kit for autologous discarded limb lymphatic vessel and lymph node transplantation, characterized in that, include: The acquisition module includes an indocyanine green fluorescence navigation interface, ultramicroscopic dissection forceps, and ultramicroscopic scissors; The preservation module includes a preservation chamber, a preservation solution, a flushing channel, and a rapid active staining interface; the flushing channel and the rapid active staining interface are connected to the preservation chamber, and the preservation solution is located inside the preservation chamber; The anastomotic cannula is equipped with a C-shaped buckle, a one-way flap in the inner cavity, and multiple suture guide grooves on the outer periphery.

2. The transplantation kit according to claim 1, characterized in that, The anastomotic cannula and the one-way valve are made of a PCL / PLGA blend, and the surface of the anastomotic cannula is coated with a PEG-PVP hydrophilic coating with a thickness of 2~8 μm.

3. The transplant kit according to claim 1, characterized in that, The anastomotic sleeve has an inner diameter of 0.20~0.80 mm, an outer diameter of 0.50~1.20 mm, a wall thickness of 0.08~0.20 mm, and a length of 3.0~8.0 mm.

4. The transplant kit according to claim 1, characterized in that, The opening angle of the C-type buckle is 60~110°.

5. The transplant kit according to claim 1, characterized in that, The number of suture guide grooves is 4 to 8, and the width is 30 to 80 μm.

6. The transplant kit according to claim 1, characterized in that, The preservation solution contains 5-25 IU / mL heparin, 50-200 μmol / L adenosine, 3-8 mmol / L glucose and 10-25 mmol / L HEPES, with an osmotic pressure of 280-320 mOsm / kg and a pH of 7.20-7.

45.

7. A method for preparing an ex vivo lymphatic tissue assembly, using the kit as described in any one of claims 1 to 6, wherein the method is applied only to non-infectious, discarded limb segments that have been separated from the body, characterized in that... include: In vitro localization: Indocyanine green is injected via the indocyanine green fluorescent navigation interface to locate superficial lymphatic plexuses under near-infrared light source; Ex vivo collection: Lymphatic vessel segments and lymph nodes were extracted from discarded limb segments using the aforementioned ultramicroscopic dissecting forceps and ultramicroscopic scissors; In vitro preservation and irrigation: Lymphatic vessel segments and lymph nodes are placed in the preservation solution and irrigated through the irrigation channel; In vitro activity testing: Staining agent is input through the aforementioned rapid staining interface for staining and patency testing; Ex vivo assembly: The tested lymphatic vessel segment is fitted into the anastomotic cannula.

8. The preparation method according to claim 7, characterized in that, The concentration of the indocyanine green injected is 0.10~0.50 mg / mL, the injection volume at each point is 0.05~0.30 mL, and the number of injection points is 3~8; the excitation wavelength of the near-infrared light source is 760~810 nm.

9. The preparation method according to claim 7, characterized in that, The extracted lymphatic vessel segments had a diameter of 0.20~0.80 mm and a length of 15~60 mm; the extracted lymph nodes had a diameter of 2~6 mm.

10. The preparation method according to claim 7, characterized in that, The time from the injection of the indocyanine green to the completion of the in vitro assembly is less than or equal to 120 minutes.

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