Method of Removing and Using Tissue for Allografts

Abstract

The present invention corresponds to a new alternative coverage for skin defects by harvesting and subsequent cryopreservation of a total skin allograft. The present invention seeks to provide a solution to the problem of temporary and definitive coverage of complex wounds. The proposed solution corresponds to a method of harvesting, processing and subsequent use of a skin allograft for coverage of complex wounds.

Description

Technical Field

[0001] The present invention is part of the technical field of tissue extraction and processing for grafts.

Background Art

[0002] Extensive skin damage occurs not only mainly in burn patients, but also in other situations such as trauma and skin diseases that cause destruction and de-epithelialization of very large parts of the body, which requires the search for covering and skin substitutes. However, despite all recent technological developments, autologous skin grafting remains the standard treatment as it has no risk of rejection and thus achieves final coverage and resolution. It must be emphasized, however, that when the affected area is a particularly large skin surface, there is a drawback in that the donor area is affected and insufficient.

[0003] Skin allografts (CA) obtained from allogeneic individuals are excellent substitutes as they perform the same functions as autologous skin, but are rejected due to immune action and are used only as temporary coverings and may ultimately become vectors for infectocontagious diseases. Currently, the number of donors is small, the management of skin extraction, processing, and storage for clinical use is complex, and furthermore, according to the latest general technical standards for tissue extraction, preservation, and transplantation of the Chilean Ministry of Health (MINSAL, February 2018), only cadaveric donors are reserved, so only limited partial-thickness CAs can be obtained, and there are large-scale clinical experiments in the literature on this.

[0004] While there are many clinical benefits and there is a shortage of CAs, particularly in living donors where a large amount of skin must be drying out for clinical improvement due to health status, as in people who have undergone body contouring surgery, such as abdominoplasty, for aesthetic and / or reconstructive reasons, the possibility of exploring potential skin donors is opened, and thus, whole-skin CAs (CAWS) can be obtained.

[0005] The clinical trials indicated have reconfirmed that body contouring surgery can be a source of CA. In the specific case of cryopreserved allograft with skin (CAWSC), its viability and many elements of the donor dermis, especially the fibroblasts adhering to the recipient, are maintained, serving as an intermediate covering for some patients and the final covering for others.

[0006] In regions with low overall and specific skin donation rates, and other regions where its use has not yet been legalized mainly for cultural and religious reasons, the use of CA from living donors in patients undergoing body contouring procedures, especially in the case of abdominoplasty, becomes an option. From 2017 to 2019, the number of effective skin donors in Chile was only 4, with 3,051 cm 2 , 1,069 cm 2 , and 6,839 cm 2 of skin obtained (all from cadaveric donors). In our experiments, approximately 302 cm 2 of clinically useful processed skin is obtained for each abdominoplasty, reflecting the potential of this procedure as an important source of CA and CAWS.

[0007] The present invention is a new alternative covering for skin defects that acts as a skin regeneration matrix, temporary for some patients and final for others. Some patients require autologous transplantation to complete the method, while others may be managed by advanced treatments to conclude the treatment method. It has three unique features, namely, a) obtained from living donors, b) including the entire layer of skin, and c) preserved by cryopreservation, so that viable tissue can be obtained.

[0008] The prior art documents similar to the present invention are described below.

[0009] US Patent Application Publication No. 2020 / 246508 describes a scaffold material prepared from decellularized natural tissue. This product has been shown to be suitable for use as a tissue filler. The difference from the evaluated invention is the fact that the tissue of the present invention is not decellularized and no growth factors are added. Furthermore, its use does not seem to be suitable for the treatment of large burns.

[0010] US Patent Application Publication No. 2014 / 316262 describes a method for identifying perforating veins in order to identify ideal sites for skin flap harvest. This method consists of injecting a contrast agent and then identifying the veins in the area by illumination. This document corresponds to the general state of the art and does not affect the evaluated invention.

[0011] US Patent Application Publication No. 2013 / 108683 discloses methods and compositions for the treatment of wounds of various severities and locations. This composition corresponds to a biologically compatible matrix supplemented with growth factors. The claims show that this product can help promote or improve grafts such as skin flaps. The difference from the evaluated invention is clear since this product can be used to supplement the evaluated invention.

[0012] US Patent Application Publication No. 2006 / 073592 describes a method for tissue matrix storage. Specifically, it mentions the dermis, but this method attempts to preserve decellularized tissue. Therefore, this document is considered to be part of the general state of the art and will not affect the evaluated invention.

[0013] In the case of International Publication No. 0052149, protection is required for methods of applying biological materials to patients. Initially, it refers to cells, but there is also the option of using tissues, and an example of a tissue is a skin flap.

[0014] The difference from the evaluated invention is that, in this case, the state of the flap to be transplanted is not defined.

Prior Art Documents

Patent Documents

[0015]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Summary of the Invention

[0016] The present invention addresses the excision of all-skin allografts and subsequent cryopreservation for a new alternative covering of skin defects.

Means for Solving the Problems

[0017] The present invention aims to provide a solution to the problems of temporary and final covering of complex wounds.

[0018] The proposed solution corresponds to a method for excising, processing, and subsequent use of skin allografts for covering complex wounds.

[0019] This method includes three main stages, namely, (i) obtaining all-skin tissue from a living donor, (ii) obtaining a skin allograft by treating and preserving the tissue, and finally (iii) transplanting the CAWS in the final patient.

[0020] In a more specific embodiment, step i) of this method includes step a) surgical intervention, step b) tissue extraction, step c) packaging and identification, step d) storage, and step e) transportation to a processing facility.

[0021] On the other hand, the tissue treatment and storage steps include step f) processing, step g) cryopreservation and isolation, step h) verification, and recording.

[0022] Finally, the steps corresponding to the final process include step i) transportation and step j) clinical use.

[0023] In a more specific embodiment, step a) is carried out in the ward where the tissue is removed from the donor. In a specific process, a mark is made on the area where the tissue is to be incised. When the mark is made, an incision of the skin adipose tissue is carried out. When the skin adipose tissue is obtained, autologous extraction is performed.

[0024] In step b), the tissue obtained in step a) is transferred to an appropriate surface where total skin extraction including the dermis is carried out, and the fat is peeled off from the deeper dermis using appropriate tools. As quality control, in this step, tissue samples are also taken for aerobic and anaerobic microorganism cultures, and fungal cultures. Finally, the tissue is placed in a sterile container with physiological serum. Optionally, the physiological serum may contain antibiotics. The container is sealed while ensuring that the tissue is completely immersed in the serum.

[0025] In step (c), the container obtained in step (b) is stored in a sterile state, and the container is labeled with at least one code assigned to the tissue and data on the extraction date and time.

[0026] In d), the tissue is stored in an appropriate container to maintain the temperature between 2 and 8 °C. Then, in e), the tissue is transported to the processing facility as quickly as possible within a period of less than 36 hours while maintaining the temperature in the range of 2 to 8 °C.

[0027] When the tissue arrives at the processing facility, the tissue performs the following steps: 1. Measurement of CAWS, cutting of parts if necessary, re-examination of thin slices, washing cycle to reduce microbial load, sample collection number 2, and immersion in a cryopreservation solution for 30 minutes to 3 hours, 2. Preparation, trimming, measurement, packaging, and labeling of each obtained film, together with sample collection numbers 3 and 4 (cultivation) during the packaging stage and sample collection number 5 (cultivation) for backing.

[0028] In step g), a cryopreservation and antifreeze solution is used for cryopreservation and isolation. While waiting for the results of a series of cultures until irradiation, CAWS is frozen and maintained at -80 °C. C) Irradiation: Sterilize the tissue lot at an irradiation dose of 25 to 28 kGy in dry ice to maintain the cold chain.

[0029] In a specific embodiment, 10% glycerol is used as the cryopreservation and antifreeze agent.

[0030] Next, in step h), verification, transfer, and recording are performed: provision, re-examination of tissue extraction and processing, recording in a public tissue and organ transplantation database.

[0031] Finally, in the final step, in the transportation step i), the processed tissue is transported to the manufacturing facility in an optimal state for maintaining the cold chain (-80 °C), and in j), the risks and benefits of clinical use are notified to the recipient by obtaining the consent of the tissue recipient.

[0032] The present invention also includes the use of the CAWS of the present invention in the preparation or generation of a biological dressing useful for improving the wound bed for later autotransplantation, chronic and complicated wounds, coverage defects without autotransplantation or local skin flap conditions, contained laparostomy without the possibility of closure, and treatment after resection of melanoma.

Mode for Carrying Out the Invention

Examples

[0033] Fourteen patients who desired abdominoplasty to improve the aesthetic appearance of the abdomen were evaluated through individual diagnosis. When the evaluation process, preoperative examinations for the surgery, and informed consent were completed, they were invited to participate in the donation of skin from the dermal flap of the excised abdominal fat. Once it was confirmed that there were no exclusion criteria for tissue donation, the patients underwent a health check, routine clinical tests for tissue donation were performed (during the surgery to avoid any blank periods), informed consent for tissue donation was obtained, and they were registered in the Integrated Organ Donation and Transplant System (SIDOT, the Spanish acronym) in Chile. After donor selection, the method was divided into the following ten steps: a) surgical / abdominoplasty step, b) extraction, c) packaging and identification, d) storage, e) transportation to the processing facility, f) processing, g) cryopreservation / isolation, h) verification, transfer, recording, i) transportation, j) clinical use. All methods complied with the technical specifications for the extraction, storage, and transplantation of tissues by the Chilean Ministry of Health.

[0034] a. Surgical / abdominoplasty step: The extraction of the skin was performed in the ward, simultaneously with the abdominoplasty surgery, under general anesthesia with all aseptic and disinfection measures by the same surgical team (an anesthesiologist, two plastic surgeons, an organ extraction surgeon, a surgical assistant, a ward assistant, an anesthesia assistant, a ward nurse, and an organ extraction nurse). After marking an infraumbilical transverse ellipse on the skin (Figure 1), an incision was made on the skin flap of the abdominal fat, the redundant skin flap was excised, and then the team was divided. One part continued with the abdominoplasty, and the extraction of the skin was performed on an independent operating table.

[0035] b. Extraction of the skin: The skin flap of the fat skin excised in an elliptical shape was placed on another operating table. The extraction of the entire skin (including the dermis) was performed, and the fat was dissected from the deeper dermis using scissors. Tissue samples (3) were also taken for current (aerobic), anaerobic, and fungal cultures. The treated skin was placed in a sterile container containing 500 cc of physiological serum containing 1 g of cefazolin and 80 mg of gentamicin, and sealed while ensuring that the skin was completely immersed.

[0036] c. Skin packaging and identification: The skin containers were stored in at least 90-micron sterilized double bags. Each bag was evacuated to remove most of the air, sealed, and labeled with the tissue code and the date and time of excision.

[0037] d. Skin storage: The skin was transferred from the ward to a temporary storage location in a cooler with a refrigerant unit to maintain the temperature between 2 and 8 °C.

[0038] e. Skin transportation: For processing, the skin was sent by plane from Iquique to the National Tissue Bank (BNT, acronym in Spanish) in Santiago, ensuring that the container maintained a temperature between 2 and 8 °C and was always excised and sent within 36 hours.

[0039] f. Processing: CA was prepared as a graft at BNT in the following two stages: a): The skin flap was measured, parts were cut if necessary, the slices were reexamined, a washing cycle was performed to reduce the microbial load, the sample collection number for the culture was 2 (number 1 was during surgery), and it was immersed in a cryopreservation solution for 1 hour. b) Prepared, trimmed, measured, and each resulting film was packaged and labeled. Sample collection numbers 3 and 4 (culture) during the packaging stage. For backing, sample collection number 5 (culture).

[0040] g. Cryopreservation and isolation: 10% glycerol was used as the cryopreservation and antifreeze solution. CAWS was frozen and maintained at -80 °C while waiting for the results of a series of cultures until irradiation. C) Irradiation: The tissue lot was sterilized with an irradiation dose of 25 to 28 kGy in dry ice to maintain the cold chain.

[0041] h. Verification, transfer, recording: Method of provision, reexamination of tissue excision and processing, recording in SIDOT.

[0042] i. Transportation: Return of the processed tissue to the production facility in dry ice to maintain the cold chain (-80 °C).

[0043] j. Clinical use: The risks and benefits of this procedure were explained to the tissue recipient, informed consent was obtained for the use of human-derived tissue, and the recipient's data and the amount of tissue used were securely traceable and biovigilanced by a tissue transplantation form that included a code to identify it and possible adverse effects. The clinical indications for its use were a) preparation of chronic and complicated wounds to improve the wound bed for subsequent autografting, b) coverage defects without the state of autografting or local skin flaps, c) contained laparotomy with no possibility of closure, and d) after resection of melanoma. During the period immediately before surgery, when the size of the covering defect was measured, the CAWS was washed three times with warmed physiological serum (40°C or less) to remove the cryoprotectant. The recipient bed was prepared by scar excision of necrotic and inactivated tissue and disordered granulation areas, and the CAWS was fixed with points and / or brackets related to negative pressure therapy.

[0044] Results Samples from skin donors consisted of 14 female patients aged 31 to 55 years with an average age of 40 years, two of whom had a previous history of bariatric surgery. The excised skin surface was estimated using the elliptical area formula, and then the area decreased by removal of the edges during processing to leave the primary contraction of the excised total skin and multiple clinically useful sized films. The average values of the processed skin and clinically useful films were 302 cm 2 and 8.3 films per patient, respectively.

[0045] Clinical samples consisted of 10 patients aged from 2 months to 75 years (two procedures were performed on two patients) diagnosed with diabetic foot lesions (4), contained laparotomies (2), complex lower limb wounds (2), recurrent scalp sarcomas (1), and melanomas (1). Initial CA engraftment was observed in all patients, and rejection began 21 days later, as evidenced by color changes in the CA and the formation of superficial necrotic eschar. When this was removed, the major tissue was adherent to the recipient site. One patient received an autograft, and the other patients were managed by secondary healing as definitive treatment. The histology of the CA showed necrotic areas when the CA was removed mainly by polymorphonuclear infiltration, and an interface rich in fibroblasts and new blood vessels in the recipient bed. This interface or neodermis could be confirmed by imaging. In NMR of patients diagnosed with diabetic foot lesions and with a history of transverse midfoot osteotomy, the CA was observed as a non-captant superficial component and a deep component enhanced with a contrast agent, similar to the vascular stalked dermis. A series of clinical results are summarized in the following table.

[0046]

Table 1

[0047] For CAWS, rejection is a natural progression. In healthy individuals, this occurs between 8 and 10 days, and in extensive burns, it is delayed between 15 and 30 days due to immunosuppression of the immune system. In our experiments using CAWSC, rejection was delayed and became clinically apparent from the third week, characterized by polymorphonuclear infiltration of the epidermis. The rejection of CAWSC should not be interpreted as treatment failure but rather as an intermediate covering stage because part of the CAWSC is lost, part of it adheres to the recipient bed, and an interface of "neodermis" is obtained. On the one hand, the final covering is completed by autograft, and on the other hand, healing is completed by secondary healing, and there is also secondary contraction of the wound, reducing the size of the defect. Thus, CAWSC can be used as an alternative to the use of skin substrates, and in addition to covering structures such as bone, cartilage, and tendon, a thicker, more elastic, and better-quality coating can be obtained.

[0048] The therapeutic uses of CAWSC are diverse and include the diabetic foot lesions that we have focused on in a series of our studies, extensive complex covering defects, contained laparotomies, and after resection of skin tumors where biopsies are awaited for possible peripheral expansion and reconstruction.

Industrial Applicability

[0049] The present invention is applied in the clinical field, mostly temporarily but in some cases finally for covering complex wounds, and serves as an alternative to the use of skin matrices.

Claims

1. A method for treating and preserving a biological tissue patch that enables the supply of tissue for a skin graft of whole skin including dermis, comprising the following steps: i Measuring the biological patch, cutting parts if necessary, reexamining the slices, a washing cycle to reduce the microbial load, collecting a second sample for immersion in a cryopreservation solution for 30 minutes to 3 hours, obtaining a film of a skin allograft (CAWS) of whole skin for each part, ii Preparation, trimming, measurement, packaging, and labeling of each obtained film, together with collecting a third and a fourth sample for microbial culture control and a fifth sample for backup during the packaging stage, wherein in step ii, a cryopreservation and / or antifreeze solution is used for cryopreserving and further isolating the CAWS, and while waiting for the results of a series of cultures until irradiation, the CAWS during isolation is frozen and maintained at -80 °C, iii Irradiation: Sterilization of the CAWS at an irradiation dose of 25 to 28 kGy in dry ice for maintaining the cold chain A method comprising the above steps.

2. The method according to claim 1, wherein in step i), the tissue is treated on a suitable surface where the excision of the CAWS including dermis is carried out, and fat is peeled off from the deeper dermis using suitable tools.

3. The method according to claim 2, wherein as quality control, CAWS samples are also taken to perform aerobic and anaerobic microbial cultures and fungal cultures.

4. The method according to claim 1 or 2, wherein the CAWS is placed in a sterile container with physiological serum, and the container is sealed while taking care that the CAWS is completely immersed in the serum.

5. The method according to claim 4, wherein the physiological serum contains antibiotics.

6. The method according to claim 1, wherein the CAWS is stored in a suitable container to maintain the temperature between 2 and 8 °C, and then the CAWS is transported to the production facility while maintaining the temperature in the range of 2 to 8 °C within a period of less than 36 hours after being obtained.

7. The method according to claim 1, wherein the cryopreservation and / or antifreeze solution is 10% glycerol.

8. The method according to claim 6, wherein the CAWS is transported to the production facility in an optimal state for maintaining the cold chain (-80 °C).

Images