Autologous keratinocyte tissue fragment-activated plasma matrix composite membrane, and preparation method and application thereof

Abstract

This invention relates to the fields of biomedical engineering and regenerative medicine, and particularly to a plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments, its preparation method, and its applications. The composite membrane is obtained by mixing autologous keratinized epithelial tissue fragments with a liquid autologous plasma matrix in a specific ratio, utilizing tissue factors on the surface of the tissue fragments to trigger platelet activation, spontaneously forming a gel, and then pressing it into a membrane. This invention is the first to utilize the biointerface of autologous keratinized epithelial tissue fragments to activate platelets, eliminating the need for exogenous thrombin and achieving completely autologous in-situ gelation. The composite membrane formed after pressing possesses excellent mechanical properties and barrier function, and can be prepared in a single operation in the operating room without the need for in vitro cell culture. The composite membrane of this invention is used for oral soft tissue augmentation, such as widening of keratinized gingiva and alveolar ridge soft tissue augmentation, and has advantages such as no donor site damage, high biosafety, and ease of operation.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and regenerative medicine, and in particular to a composite membrane material that uses fragments of discarded gingival keratinized epithelial tissue removed during oral surgery as a bioactivator to trigger in-situ gelation of a liquid autologous plasma matrix, which is then pressed into a membrane, and its application in oral soft tissue regeneration. Background Technology

[0002] In the fields of oral and maxillofacial surgery, periodontology, and implantology, sufficient width and thickness of keratinized gingiva are key factors in maintaining periodontal tissue health and ensuring the long-term stability of implants. Insufficient or absent keratinized gingiva can lead to difficulties in cleaning, increased susceptibility to inflammation, pain when brushing, and even affect the health and aesthetics of the soft tissues surrounding the implant. Therefore, augmenting and restoring keratinized gingiva recession or defects has always been an important topic in clinical dentistry.

[0003] Currently, the "gold standard" procedure for keratotic gingival augmentation is free gingival grafting. This technique, used since the 1960s, involves harvesting a whole graft containing epithelium and underlying connective tissue from the patient's own palate and transplanting it to the recipient area with insufficient keratotic gingiva. Free gingival grafting has advantages such as definite effectiveness and significant keratotic gingival augmentation, and has been widely recognized as the most reliable treatment method for decades.

[0004] However, free gingival grafting has significant clinical drawbacks. First, this procedure requires creating a second surgical area in the patient's palate to harvest healthy tissue, leading to postoperative pain, bleeding, and delayed healing at the donor site, severely impacting the patient's postoperative comfort and quality of life. Second, the amount of tissue available in the palate donor site is limited, making it difficult to meet the needs of large soft tissue defects. Furthermore, the color matching between the free gingival graft and the recipient site often differs, affecting aesthetic results. These drawbacks have prompted clinicians and researchers to continuously explore alternative solutions to reduce or avoid donor site damage.

[0005] In recent years, autologous plasma matrix has been widely used in the field of oral soft tissue regeneration due to its advantages such as simple preparation, lack of immunogenicity, and richness in growth factors. Among them, platelet-rich plasma and platelet-rich fibrin are the most representative. Platelet-rich plasma is usually prepared by a two-stage centrifugation method, which requires the addition of anticoagulants and extrinsic thrombin for activation; platelet-rich fibrin, on the other hand, is prepared by a single-stage centrifugation method, which does not require the addition of any exogenous substances. It utilizes the glass tube wall to activate platelets, forming a dense membrane with a three-dimensional fibrin network structure, which can slowly release a variety of bioactive factors.

[0006] In clinical applications, platelet-rich fibrin membranes have been widely used in areas such as tooth extraction site preservation, peri-implant soft tissue healing, oral ulcer treatment, and periodontal tissue regeneration. Studies have shown that the application of platelet-rich fibrin can promote early soft tissue healing and reduce postoperative pain.

[0007] However, recent studies have revealed the limitations of plasma matrix alone in soft tissue regeneration. A systematic review study found that in a three-dimensional tissue-engineered oral mucosa model, liquid platelet-rich fibrin alone failed to significantly improve epithelial thickness, morphology, or proliferative capacity. The study clearly concluded that growth factors secreted by platelet-rich fibrin alone may be insufficient to effectively stimulate three-dimensional repair and regeneration of oral soft tissues. This finding reveals a core deficiency of plasma matrix alone: ​​its mechanism of action mainly relies on the release of "signals" and the migration of cells surrounding the recipient area (such as basal cells at the wound edge) to complete epithelial coverage. When the number of cells surrounding the recipient area is insufficient or migration is restricted, the repair effect of plasma matrix alone will be significantly reduced.

[0008] To compensate for the lack of "seed cells" in plasma matrix alone, researchers have attempted to combine plasma matrix with autologous tissue or cells to enhance tissue regeneration. However, in the field of oral soft tissue regeneration, existing technologies mainly follow the approach of free gingival transplantation, which involves transplanting a "whole tissue" containing connective tissue and epithelium. Its core relies on the induction of epithelial keratinization phenotype by connective tissue.

[0009] More importantly, there has long been a prevalent technical bias in this field: the regenerative capacity of epithelial tissue is considered to depend primarily on basal stem cells, while cells above the basal layer (such as spinous cells and granular cells) are considered terminally differentiated cells, lacking the capacity for proliferation and regeneration. Therefore, in tissue engineering and clinical transplantation, epithelial tissue containing only basal cells is typically regarded as "non-regenerative" waste and discarded during surgery.

[0010] However, recent basic research has challenged this traditional understanding. An in vivo transplantation study demonstrated that basal cells lacking basal layer markers, like basal cells, can successfully regenerate stratified epithelium with a normal layered structure. This study clearly demonstrates that epithelial cells without a basal layer still possess a certain regenerative capacity in a suitable microenvironment. This discovery overturns traditional understanding and provides a solid scientific basis for using "non-basal" epithelial tissue as a cell source for regenerative medicine.

[0011] Furthermore, existing platelet activation methods also have limitations. Traditional platelet-rich fibrinogen relies on contact with the glass tube wall to activate platelets, while platelet-rich plasma requires the addition of exogenous activators such as bovine thrombin. The former has limited activation efficiency, while the latter carries the risk of immunogenicity from foreign proteins. Currently, no research reports a technical solution using autologous tissue fragments as a biological interface to activate platelets.

[0012] In summary, the existing technology has the following problems that urgently need to be solved: First, although free gingival transplantation is effective, it requires opening a second surgical area to harvest healthy tissue, which leads to donor site damage, significant postoperative pain, and low patient acceptance. Second, although autologous plasma matrix alone does not have donor site damage, its function depends on growth factor signals to recruit recipient cells. Its repair capacity is limited in a three-dimensional tissue defect environment, and it lacks a direct source of "seed cells". Third, while attempts have been made to combine plasma matrix with autologous tissue, current technologies mostly follow the "whole tissue transplantation" model, failing to effectively utilize epithelial tissue discarded during surgery that does not contain basal layer structures, and also failing to address the cognitive barrier regarding whether these "non-basal layer" cells possess regenerative capabilities. Fourth, in the existing technology, the activation of liquid plasma matrix depends on contact with the glass tube wall or exogenous activators, and there is no technical solution that uses autologous tissue fragments as biological activators to trigger in-situ gelation. Fifth, the existing technology lacks a composite regenerative material that can avoid donor site damage, provide a direct cell source for the plasma matrix, and requires no exogenous additives. In particular, there is a lack of a simple, efficient, and room-only preparation method for the specific tissue type of oral keratinized epithelium.

[0013] To address the shortcomings of existing technologies, this invention aims to provide a plasma-based composite membrane activated by autologous keratinized epithelial tissue fragments, its preparation method, and its applications. This invention mixes surgically removed autologous keratinized epithelial tissue fragments (excluding the basal layer) with a liquid autologous plasma matrix. Tissue factors on the surface of the tissue fragments trigger platelet activation, initiating a coagulation cascade reaction. This allows the liquid plasma matrix to spontaneously form a fibrin gel in situ. Simultaneously, spinous and granular cells from the tissue fragments serve as seed cells for regeneration, promoting their migration, proliferation, and participation in the formation of new epithelium within the growth factor-rich microenvironment of the plasma matrix. This composite membrane is entirely derived from the patient's own tissue, requiring no secondary surgical area or exogenous activators. Furthermore, it can be prepared in a single procedure within the operating room, potentially overcoming multiple deficiencies of existing technologies and providing a safe, effective, and patient-friendly novel regenerative strategy for oral soft tissue augmentation. Summary of the Invention

[0014] To address the aforementioned issues, this invention provides a plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments. This composite membrane utilizes autologous keratinized epithelial tissue fragments prepared from discarded gingival tissue routinely removed during oral surgery. These fragments do not contain intact basal layer structures, and the spinous and granular cells they contain are traditionally considered terminally differentiated cells with no regenerative value. However, based on recent findings, this invention uses them as effective regenerative seed cells, thus eliminating the need for a second surgical area in the palate as required by free gingival grafting, and eliminating the need for in vitro cell culture or additional tissue sampling sites as required by existing cell-plasma matrix composite technologies. This achieves true "turning waste into treasure" and zero donor site damage. Simultaneously, this invention, for the first time, utilizes tissue factor on the surface of the autologous keratinized epithelial tissue fragments as a bioactivator. After mixing with a liquid autologous plasma matrix, the tissue factor triggers platelet activation, initiating a coagulation cascade reaction that converts fibrinogen into fibrin, spontaneously forming a three-dimensional fibrin gel network that encapsulates the tissue fragments. This process requires no addition of any exogenous thrombin, calcium chloride, or other chemical activators, avoiding the risk of xenogeneic protein immunogenicity associated with traditional platelet-rich plasma relying on exogenous activators (such as bovine thrombin), and overcoming the limitation of limited activation efficiency of traditional platelet-rich fibrin relying solely on glass tube wall contact. In the resulting composite membrane, the plasma matrix provides a microenvironmental signal rich in growth factors, the tissue fragments provide direct seed cells, and the fibrin gel network formed by both provides a three-dimensional scaffold, achieving a synergistic regenerative effect of "signal-cell-scaffold" integration. When the composite membrane is implanted into the defect site, cells in the tissue fragments migrate and proliferate from the fragment edges under the stimulation of growth factor signals, and migrate along the gel network, eventually forming new stratified keratinized epithelium. This solves the problem of limited repair capacity due to the dependence of plasma matrix on recipient cell migration. Furthermore, the preparation method described in this invention can be completed in one operation in the operating room, without the need for in vitro cell culture, with a total time not exceeding 40 minutes. Through systematic optimization of various technical parameters, the optimal balance between gel forming quality, cell viability, and regeneration efficiency is ensured. Thus, while avoiding donor site damage, requiring no exogenous additives, and being easy to operate, this provides a safe, effective, and patient-friendly regeneration strategy for oral soft tissue augmentation.

[0015] In a first aspect, the present invention provides a plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments, the composite membrane being spontaneously formed by mixing the following components: (a) Autologous keratinized epithelial tissue fragments, said fragments being derived from discarded gingival tissue removed during oral surgery in a patient, and said fragments not containing intact basal layer structures, said fragments containing cells including spinous cells and granular cells. (b) Liquid autologous plasma matrix.

[0016] After the components (a) and (b) are mixed, the tissue factor on the surface of the autologous keratinized epithelial tissue fragments triggers platelet activation in the liquid autologous plasma matrix, initiating a coagulation cascade reaction that converts fibrinogen into fibrin, spontaneously forming a three-dimensional fibrin gel network that encapsulates the tissue fragments. The gel network supports cell migration, proliferation, and the formation of stratified epithelium within the tissue fragments. After compression, the gel forms a uniformly thick membrane material, and the exudate removed during the compression process contains serum components that have not participated in cross-linking.

[0017] As a preferred embodiment of the present invention, the liquid autologous plasma matrix is ​​a platelet-rich fibrin liquid precursor or platelet-rich plasma obtained by centrifugation of whole blood without anticoagulants.

[0018] As a preferred embodiment of the present invention, the size of the autologous keratinized epithelial tissue fragment is 0.5 mm³ to 3 mm³, for example, 0.5 mm³, 1 mm³, 1.5 mm³, 2 mm³, 2.5 mm³, 3 mm³, etc.

[0019] When the size of tissue fragments is less than 0.5 mm³, the shearing process can easily lead to excessive cell damage, and the fragments are too small to form a stable cell migration interface in the gel. When the size of tissue fragments is greater than 3 mm³, cells in the central region of the fragment are prone to necrosis due to limited diffusion of nutrients and oxygen, while the interface area for cell migration at the edge of the fragment is relatively insufficient, resulting in reduced cell migration efficiency. Furthermore, large fragments are inconvenient to handle during injection or implantation. Therefore, limiting the fragment size to 0.5 mm³ to 3 mm³ can balance cell viability, migration efficiency, and clinical operability.

[0020] As a preferred embodiment of the present invention, the volume ratio of the autologous keratinized epithelial tissue fragments to the liquid autologous plasma matrix is ​​1:5 to 1:20, for example, 1:5, 1:8, 1:10, 1:12, 1:15, 1:20, etc.

[0021] When the proportion of tissue fragments is too high (volume ratio greater than 1:5, i.e., insufficient plasma matrix), the total amount of fibrinogen in the mixture is limited, making it difficult to form a complete and continuous gel network. This results in insufficient mechanical strength of the composite membrane, failing to maintain the stability of the defect space. Simultaneously, the tissue fragments are too dense, lacking sufficient space for cell migration and proliferation after migration, affecting the uniform distribution of newly formed epithelial tissue. When the proportion of tissue fragments is too low (volume ratio less than 1:20, i.e., excessive plasma matrix), although gel formation is sufficient, the excessive spacing between fragments and insufficient cell density make it difficult for the migrated cells to coalesce into a continuous epithelial layer, leading to low regeneration efficiency. Limiting the mixing volume ratio to 1:5 to 1:20 ensures complete gel network formation, moderate fragment distribution, and effective coalescence of migrated cells, achieving optimal regeneration results.

[0022] As a preferred embodiment of the present invention, the composite membrane is formed without the addition of any exogenous thrombin, calcium chloride, or other chemical activators, relying entirely on the biological interface provided by the tissue fragments to complete platelet activation and gelation. The exposure of tissue factors on the surface of the tissue fragments is key to activation. This activation method avoids the immunogenicity risks that may be caused by foreign proteins (such as bovine thrombin) and does not require physical contact with the glass tube wall, achieving a completely autologous, mild, and controllable gelation process.

[0023] Secondly, the present invention provides a method for preparing a plasma matrix composite membrane activated by the above-mentioned autologous keratinized epithelial tissue fragments, the preparation method comprising the following steps: Step (1): Collect the patient's own venous blood and obtain liquid autologous plasma matrix by centrifugation. Step (2): Obtain the discarded gingival tissue removed during the patient's oral surgery, and cut it into fragments of 0.5 mm³-3 mm³ under aseptic conditions to obtain autologous keratotic epithelial tissue fragments. Step (3): Mix the liquid autologous plasma matrix obtained in step (1) with the autologous keratinized epithelial tissue fragments obtained in step (2) at a volume ratio of 1:5-1:20 to form a mixture. Step (4): Let the mixture obtained in step (3) stand at room temperature or 37°C for 5-30 minutes to activate platelets using tissue factors on the surface of the tissue fragments, spontaneously forming a gel complex. Step (5): Place the gel-like complex obtained in step (4) into a plasma matrix pressurizer and press it under a pressure of 0.5-10 N for 0.5-5 minutes to remove the exudate and form a membrane material with a thickness of 0.5-3 mm, thus obtaining the plasma matrix composite membrane activated by the autologous keratinized epithelial tissue fragments.

[0024] As a preferred embodiment of the present invention, the method is completed in one operation in the operating room, requiring no in vitro cell culture step and no addition of exogenous activators. The entire preparation process, from blood collection and tissue mincing to mixing and molding, takes no more than 40 minutes, seamlessly integrating with clinical surgery.

[0025] As a preferred technical solution of the present invention, the centrifugation in step (1) is horizontal centrifugation, the centrifugation acceleration is 60-1000g, such as 60g, 100g, 200g, 400g, 600g, 800g, 1000g, etc., the centrifugation time is 2-10 minutes, such as 2min, 4min, 6min, 8min, 10min, etc., and no anticoagulant is added.

[0026] When the centrifugation acceleration is below 60g, blood cell separation is insufficient, resulting in an excessive amount of red and white blood cells mixed in the liquid plasma matrix, affecting the purity and bioactivity of the final gel. When the centrifugation acceleration is above 1000g, it may lead to excessive platelet sedimentation or premature activation, reducing the content of effective platelets in the liquid plasma matrix and thus affecting the quality of gel formation. When the centrifugation time is below 2 minutes, separation is insufficient; when the centrifugation time exceeds 10 minutes, platelets may be damaged due to prolonged centrifugation. Limiting the centrifugation acceleration and centrifugation time within the above ranges can yield a stable liquid plasma matrix with high bioactivity.

[0027] As a preferred technical solution of the present invention, the standing time in step (4) is 5-30 minutes, such as 5min, 10min, 15min, 20min, 25min, 30min, etc., and the standing temperature is room temperature (20-25℃) or 37℃.

[0028] When the settling time is less than 5 minutes, the coagulation cascade reaction is not fully completed, the gel network is not fully formed, the mechanical strength of the composite membrane is insufficient, and it is difficult to maintain its three-dimensional morphology. When the settling time exceeds 30 minutes, although it does not affect gel formation, it prolongs the surgical waiting time, which is detrimental to clinical operation efficiency. Limiting the settling time to 5-30 minutes ensures that the gel is fully formed while also taking into account the convenience of clinical operation. Both room temperature and 37°C can maintain cell activity and enzyme reaction efficiency during settling, with 37°C being closer to physiological conditions and allowing for a shorter gelation time.

[0029] As a preferred technical solution of the present invention, the pressure during pressing in step (5) is, for example, 0.5 N, 1 N, 2 N, 5 N, 8 N, 10 N, etc., the pressing time is, for example, 0.5 min, 1 min, 2 min, 3 min, 4 min, 5 min, etc., and the film thickness after pressing is, for example, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, etc.

[0030] When the compression pressure is below 0.5 N or the compression time is less than 0.5 minutes, the exudate in the gel is not sufficiently removed, leaving excessive liquid inside the membrane material. This results in a loose membrane structure, insufficient mechanical strength, and susceptibility to breakage during subsequent use. When the compression pressure is above 10 N or the compression time exceeds 5 minutes, the fibrin network structure within the gel may be damaged, compressing and damaging cells in tissue fragments. Limiting the compression pressure, time, and membrane thickness within the above ranges allows for effective removal of exudate while ensuring cell viability, improving the density of the fibrin network, enhancing the membrane's mechanical strength and barrier function, and maintaining appropriate flexibility for clinical operation.

[0031] As a preferred embodiment of the present invention, the discarded gingival tissue includes, but is not limited to, gingival tissue removed during crown lengthening surgery, gingival tissue removed during impacted tooth extraction, or gingival tissue removed during gingival restoration surgery. These tissues are usually treated as medical waste in routine oral surgery. The present invention "turns waste into treasure" by obtaining autologous cell sources without the need to create a second surgical area.

[0032] Thirdly, the present invention provides the application of the above-mentioned autologous keratinized epithelial tissue fragment activated plasma matrix composite membrane or the composite membrane prepared by the above method in the preparation of medical devices for oral soft tissue augmentation.

[0033] As a preferred embodiment of the present invention, the oral soft tissue augmentation includes widening of keratinized gingiva, augmentation of alveolar ridge soft tissue, augmentation of peri-implant soft tissue, or repair of oral mucosal defects. After the composite membrane is implanted, the spinous and granular cells in the tissue fragments migrate and proliferate in the growth factor microenvironment provided by the plasma matrix, migrate along the gel network, and ultimately form new stratified keratinized epithelium on the defect surface.

[0034] Compared with the prior art, the present invention has the following beneficial effects.

[0035] Firstly, this invention utilizes tissue factor from the surface of autologous keratinized epithelial tissue fragments as a bioactivator. When mixed with a liquid autologous plasma matrix, it triggers platelet activation and initiates a coagulation cascade reaction, spontaneously converting fibrinogen into fibrin to form a three-dimensional gel network. The entire process requires no addition of exogenous thrombin or calcium chloride, completely eliminating the immunogenicity risks associated with traditional platelet-rich plasma relying on bovine thrombin. It also overcomes the low efficiency and instability of traditional platelet-rich fibrin membranes, which rely solely on the glass tube wall for activation. Furthermore, the gel is pressed into a membrane, effectively removing uncrosslinked exudate and resulting in a denser fibrin network. The resulting composite membrane has uniform thickness and moderate flexibility, exhibiting significantly superior tensile strength and barrier function compared to unpressed simple gels or conventional platelet-rich fibrin membranes. Secondly, this invention overcomes the long-standing technical prejudice in the field that "keratinized epithelial cells without a basal layer have no regenerative value." For the first time, these traditionally recognized terminally differentiated cells are used clinically as effective regenerative seed cells, transforming discarded gingival tissue routinely removed during oral surgery into a valuable resource. Unlike free gingival transplantation, this eliminates the need for a second surgical area in the palate and in vitro cell culture, completely eliminating complications such as donor site pain and bleeding, and significantly improving patient comfort. Based on this, this invention constructs a three-in-one synergistic regeneration system of "signal-cell-scaffold": plasma matrix releases growth factors to provide regeneration signals; spinous and granular cells in keratinized epithelial tissue fragments maintain high vitality under the protection of a fibrin network, enabling them to migrate from the fragment edges, proliferate, and form stratified keratinized epithelium; the dense fibrin network after compression into a membrane provides support for cell migration and acts as a physical barrier to prevent rapid soft tissue invasion. In vitro culture shows that the composite membrane of this invention can form typical stratified epithelial islands within 14 days, which cannot be achieved by simply using platelet-rich fibrin or simple tissue fragments. Furthermore, this invention has systematically optimized various technical parameters, enabling the entire preparation method to be completed in one operation in the operating room, with a total time not exceeding 40 minutes, greatly reducing the technical threshold and cost. In summary, the composite membrane provided by this invention is entirely derived from the patient's own tissue, exhibiting high biocompatibility, excellent mechanical strength, and definite regeneration effect. It can be used in various oral soft tissue regeneration scenarios, such as keratotic gingival widening, alveolar ridge soft tissue augmentation, and peri-implant soft tissue augmentation, providing a novel regeneration strategy for oral soft tissue augmentation that is donor-site-free, requires no exogenous addition, is simple to operate, safe, and effective. Detailed Implementation

[0036] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0037] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0038] Example 1

[0039] This embodiment provides a plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments. The preparation method of the composite membrane includes the following steps: (1) Collect 10 mL of the patient’s own venous blood, place it in a plastic blood collection tube without anticoagulant, and quickly put it into a horizontal centrifuge. Centrifuge at 700g for 8 minutes to obtain the upper liquid autologous plasma matrix (platelet-rich fibrin liquid precursor), about 5-6 mL. (2) Obtain the waste gingival tissue removed during the patient's crown lengthening surgery, and cut it into fragments of about 1 mm³ (range 0.5-3 mm³) under sterile conditions using ophthalmic scissors to obtain autologous keratotic epithelial tissue fragments, about 0.2 g. (3) The liquid autologous plasma matrix obtained in step (1) and the autologous keratinized epithelial tissue fragments obtained in step (2) are mixed at a volume ratio of 1:10 and gently stirred with sterile surgical instruments to form a mixture. (4) Transfer the mixture obtained in step (3) to a sterile culture dish and let it stand in a constant temperature incubator at 37°C for 15 minutes. The tissue factor on the surface of the tissue fragments will activate the platelets and spontaneously form a gel complex. (5) The gel-like complex obtained in step (4) is transferred to a dedicated plasma matrix pressurizer and pressed at a pressure of 2 N for 2 minutes to remove the exudate and form a membrane material with a thickness of about 1.5 mm, thus obtaining the plasma matrix composite membrane activated by the autologous keratinized epithelial tissue fragments.

[0040] In this embodiment, the obtained composite membrane is light yellow, has a smooth surface, and is flexible. Tissue fragments are evenly distributed in the membrane, exudate is effectively removed, and the membrane material is easy to cut and implant.

[0041] Example 2

[0042] This embodiment provides a plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments. The preparation method is basically the same as that of Example 1. The difference from Example 1 is that the mixing volume ratio in step (3) of this embodiment is 1:20, the standing time is 30 minutes, the standing temperature is room temperature, the pressing pressure in step (5) is 1 N, and the pressing time is 3 minutes.

[0043] In this embodiment, the obtained composite membrane gel network is more compact and the spacing between tissue fragments is larger, making it suitable for clinical scenarios that require a larger coverage area but have a smaller thickness of soft tissue defects.

[0044] Example 3

[0045] This embodiment provides a plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments. The preparation method is basically the same as that of Example 1. The difference between Example 1 and Example 1 is that the mixing volume ratio in step (3) of this embodiment is 1:5, the standing time is 10 minutes, the standing temperature is 37°C, the pressing pressure in step (5) is 3 N, and the pressing time is 1 minute.

[0046] In this embodiment, the obtained composite membrane tissue fragments have a high density and a fast gel formation rate, making it suitable for clinical scenarios that require thicker soft tissue augmentation or rapid prototyping.

[0047] Example 4

[0048] This embodiment provides a plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments. The preparation method is basically the same as that in Example 1. The difference from Example 1 is that in this embodiment, the tissue fragments in step (2) are cut into pieces with a size of 0.5 mm³ (minimum size), the mixing volume ratio in step (3) is 1:15, the standing time is 20 minutes, and the pressing pressure in step (5) is 0.5 N and the pressing time is 5 minutes.

[0049] Example 5

[0050] This embodiment provides a plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments. The preparation method is basically the same as that of Embodiment 1. The difference from Embodiment 1 is that in this embodiment, the tissue fragments in step (2) are cut to a size of 3 mm³ (maximum size), the mixing volume ratio in step (3) is 1:8, the standing time is 25 minutes, and the pressing pressure in step (5) is 5 N and the pressing time is 0.5 minutes.

[0051] Comparative Example 1 This comparative example provides a simple platelet-rich fibrin membrane, the preparation method of which includes the following steps: (1) Collect 10 mL of the patient’s own venous blood, place it in a glass blood collection tube without anticoagulant, and quickly put it into a horizontal centrifuge. Centrifuge at 700g for 8 minutes to obtain the upper solid platelet-rich fibrin clot. (2) Use sterile forceps to remove the platelet-rich fibrin clot, place it in a pressure vessel, and press it with 2 N for 2 minutes to obtain a simple platelet-rich fibrin membrane.

[0052] The difference between this comparative example and Example 1 is that no autologous keratinized epithelial tissue fragments were added, and the activation of platelet-rich fibrin depended on the contact between the glass tube wall and the biological interface of the tissue fragments.

[0053] Comparative Example 2 This comparative example provides a membrane of simple keratinized epithelial tissue fragments, the preparation method of which includes the following steps: (1) Obtain the discarded gingival tissue removed during the patient's crown lengthening surgery and cut it into 1 mm³ fragments under aseptic conditions. (2) Mix the fragments with physiological saline at a volume ratio of 1:10, let stand in a constant temperature chamber at 37°C for 15 minutes, and then press with a pressure of 2N for 2 minutes to obtain a simple tissue fragment membrane.

[0054] The difference between this comparative example and Example 1 is that no liquid autologous plasma matrix was added, no fibrin gel network was formed, and tissue fragments could not be encapsulated in the gel scaffold.

[0055] Comparative Example 3 This comparative example provides a composite membrane of extrinsically thrombin-activated platelet-rich plasma and tissue fragments, the preparation method of which includes the following steps: (1) Collect 10 mL of the patient’s own venous blood and place it in a blood collection tube containing an anticoagulant. Platelet-rich plasma is prepared by a two-stage centrifugation method. (2) Prepare autologous keratinized epithelial tissue fragments in the same way as in Example 1, step (2). (3) Mix platelet-rich plasma and tissue fragments at a volume ratio of 1:10, add bovine thrombin (final concentration 50 U / mL) and calcium chloride (final concentration 10 mM), mix and let stand at 37°C for 15 minutes to form a gel, then press with 2 N pressure for 2 minutes to obtain a composite membrane.

[0056] The difference between this comparative example and Example 1 is that exogenous bovine thrombin and calcium chloride were used to activate platelets, and an anticoagulant was added during the preparation of platelet-rich plasma.

[0057] Comparative Example 4 This comparative example provides a composite membrane with an excessively high proportion of tissue fragments (volume ratio 1:2, exceeding the scope of this invention 1:5-1:20). The preparation method is basically the same as that of Example 1, except that the mixing volume ratio in step (3) is 1:2, and the other steps are the same.

[0058] Comparative Example 5 This comparative example provides a composite membrane with an excessively low proportion of tissue fragments (volume ratio 1:30, exceeding the scope of this invention 1:5-1:20). The preparation method is basically the same as that of Example 1, except that the mixing volume ratio in step (3) is 1:30, and the other steps are the same.

[0059] Performance testing The composite membranes prepared in Examples 1-5 and Comparative Examples 1-5 were subjected to the following performance tests.

[0060] Test 1: Gel formation time and film formation effect Take 1 mL of each mixture prepared in the examples and comparative examples and inject it into a 2 mL centrifuge tube. Record the time required from the start of mixing to complete gel formation (the contents of the inverted centrifuge tube do not flow). After formation, prepare a film according to the respective pressing conditions and observe the integrity and uniformity of the film material. The results are shown in Table 1.

[0061] Table 1. Gel formation time and film formation effect sample Mixed volume ratio (tissue fragments: plasma matrix) Gel formation time (min) Membrane integrity Membrane uniformity Example 1 1:10 12 ± 2 whole uniform Example 2 1:20 25 ± 3 whole uniform Example 3 1:5 8 ± 1 whole uniform Example 4 1:15 18 ± 2 whole uniform Example 5 1:8 14 ± 2 whole uniform Comparative Example 1 / 35 ± 5 whole uniform Comparative Example 2 1:10 > 60 (ungelatinized) Unable to form a film / Comparative Example 3 1:10 3 ± 1 whole Relatively uniform Comparative Example 4 1:2 6 ± 1 Broken, easily cracked Uneven Comparative Example 5 1:30 32 ± 4 Complete but too thin uniform As shown in Table 1, the gel formation time of Examples 1-5 was 8-25 minutes, all significantly shorter than that of Comparative Example 1 (purely platelet-rich fibrin, 35 minutes), indicating that the biological interface provided by keratinized epithelial tissue fragments effectively accelerated the platelet activation and gelation process. Comparative Example 2 could not form a film due to the lack of plasma matrix; Comparative Example 3 formed the fastest but depended on exogenous bovine thrombin; Comparative Example 4 (fragment ratio too high, 1:2) produced a fast gel but the film was easily broken because too many fragments led to a discontinuous gel network; Comparative Example 5 (fragment ratio too low, 1:30) had a long gel formation time and an excessively thin film, resulting in poor practicality. The 1:5-1:20 range specified in this invention can balance gel formation speed and film integrity.

[0062] Test 2: Cell viability and migration ability in tissue fragments The composite membranes prepared in Examples 1-5 and Comparative Examples 1-5 were placed in 6-well plates (membrane sample size approximately 10 × 10 mm). 2 mL of DMEM culture medium (containing 10% FBS) was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator. Membrane samples were removed on days 3, 7, and 14, respectively. Cell viability in tissue fragments was observed using Live / Dead staining, and cell migration from the membrane was observed using crystal violet staining. The results are shown in Table 2.

[0063] Table 2 Cell viability and migration ability sample Day 3 Cell viability (%) Day 7 ectopic cell density Epithelioid cell formation on day 14 Example 1 91 ± 3 +++ Visible multilayered epidermal islands Example 2 86 ± 4 ++ Scattered cell clusters Example 3 88 ± 4 +++ Visible multilayered epidermal islands Example 4 89 ± 3 +++ Visible multilayered epidermal islands Example 5 84 ± 5 ++ Scattered cell clusters Comparative Example 1 (No fragments) / Absent epithelial cells Comparative Example 2 42 ± 7 + Poor cell viability, no epithelium observed. Comparative Example 3 82 ± 5 ++ Scattered cells, no stratification observed. Comparative Example 4 65 ± 6 ++ A small number of cell clusters, unevenly distributed Comparative Example 5 90 ± 4 + Cell density too low, not confluent Note: ++ indicates high density (>500 cells / mm²), ++ indicates medium density (100-500 cells / mm²), + indicates low density (<100 cells / mm²), and / indicates not applicable.

[0064] As shown in Table 2, the cell viability of tissue fragments in Examples 1-5 was significantly higher than that in Comparative Example 2 (simple fragments without plasma matrix protection), indicating that the fibrin gel network formed by the plasma matrix provided nutritional support and protection for the cells in the fragments. Typical stratified epithelial islands were observed in Examples 1, 3, and 4 (volume ratios of 1:5, 1:10, and 1:15) on day 14 of culture, with 1:5 and 1:10 showing the best results. Examples 2 (1:20) and 5 (1:8 with larger fragment sizes) had lower cell migration densities and failed to form continuous epithelium. Comparative Example 4 (1:2) showed decreased cell viability due to excessive fragmentation leading to cell crowding and nutrient competition. Comparative Example 5 (1:30) had too low a cell density, making confluence difficult. The 1:5-1:20 range defined in this invention ensures appropriate fragment density, allowing cells to effectively confluence and form stratified epithelium after migrating from the fragment edges.

[0065] Test 3: Mechanical property testing of composite membranes The film-like materials obtained after pressing in Examples 1-5 and Comparative Examples 1-5 were cut into rectangular strips of 20 mm × 5 mm, and their tensile strength and elongation at break were tested using a universal testing machine (loading rate 10 mm / min). The results are shown in Table 3.

[0066] Table 3 Mechanical properties of composite membranes sample Tensile strength (kPa) Elongation at break (%) Membrane thickness (mm) Example 1 28.3 ± 3.7 35 ± 5 1.5 ± 0.1 Example 2 23.0 ± 2.8 40 ± 6 1.2 ± 0.1 Example 3 31.2 ± 4.6 30 ± 4 1.8 ± 0.2 Example 4 26.6 ± 3.7 38 ± 5 1.4 ± 0.1 Example 5 25.2 ± 3.1 33 ± 5 1.6 ± 0.2 Comparative Example 1 9.6 ± 1.3 55 ± 8 1.4 ± 0.2 Comparative Example 2 3.8 ± 1.2 12 ± 3 1.3 ± 0.2 Comparative Example 3 15.5 ± 2.3 25 ± 4 1.5 ± 0.1 Comparative Example 4 12.2 ± 2.1 18 ± 4 1.6 ± 0.2 Comparative Example 5 20.6 ± 2.5 45 ± 6 0.8 ± 0.1 As shown in Table 3, the tensile strength of the composite membranes in Examples 1-5 of this invention is significantly higher than that of Comparative Example 1 (simple platelet-rich fibrin membrane), Comparative Example 2 (simple fragment membrane), and Comparative Example 3 (exogenous thrombin composite membrane). This indicates that the composite of keratinized epithelial tissue fragments and plasma matrix, as well as the activation mechanism of the tissue fragment bio-interface, synergistically enhance the mechanical properties of the membrane. Comparative Example 4 (too many fragments) shows decreased tensile strength and increased brittleness; Comparative Example 5 (too few fragments) shows an excessively thin membrane with insufficient strength. The 1:5-1:20 range specified in this invention ensures that the membrane material possesses excellent mechanical properties.

[0067] Test 4: Barrier function test of composite membrane The composite membranes prepared in Examples 1, 3, 1, 3, and 4 were placed in the upper layer of a Transwell chamber, with culture medium added to the lower layer and a solution containing fluorescently labeled dextran (40 kDa) added to the upper layer. After 24 hours, the fluorescence intensity of the lower layer was measured, and the permeability was calculated. The results are shown in Table 4.

[0068] Table 4. Fluorescent label penetration (%) sample 24-hour fluorescence permeability (%) Example 1 5.2 ± 1.2 Example 3 4.8 ± 1.0 Comparative Example 1 28.5 ± 3.5 Comparative Example 3 18.2 ± 2.8 Comparative Example 4 22.6 ± 3.0 As shown in Table 4, the fluorescence permeability of the composite membranes in Examples 1 and 3 of the present invention is less than 6%, which is significantly better than that of Comparative Example 1 (simple platelet-rich fibrin membrane) and Comparative Example 3 (exogenous thrombin composite membrane). This indicates that the dense fibrin network formed by the present invention has a good barrier function, which can effectively block the penetration of macromolecules and cells, and provide a stable space for soft tissue regeneration.

[0069] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments, characterized in that, The composite membrane is a membrane material formed by spontaneously forming a gel after mixing the following components, followed by pressing to remove the exudate: (a) Autologous keratinized epithelial tissue fragments, said fragments being derived from discarded gingival tissue removed during oral surgery in a patient, and said fragments not containing intact basal layer structures, said fragments containing cells including spinous cells and granular cells. (b) Liquid autologous plasma matrix. After the components (a) and (b) are mixed, the tissue factor on the surface of the autologous keratinized epithelial tissue fragments triggers the activation of platelets in the liquid autologous plasma matrix, initiating a coagulation cascade reaction that converts fibrinogen into fibrin, spontaneously forming a three-dimensional fibrin gel network that encapsulates the tissue fragments. The gel network can support cell migration, proliferation and formation of stratified epithelium in the tissue fragments; the gel is pressed to form a uniformly thick membrane material, and the exudate removed during the pressing process contains serum components that have not participated in cross-linking.

2. The composite membrane according to claim 1, characterized in that, The liquid autologous plasma matrix is ​​platelet-rich fibrin liquid precursor or platelet-rich plasma obtained by centrifugation of whole blood without anticoagulants.

3. The composite membrane according to claim 1, characterized in that, The size of the autologous keratinized epithelial tissue fragments ranges from 0.5 mm³ to 3 mm³.

4. The composite membrane according to claim 1, characterized in that, The volume ratio of the autologous keratinized epithelial tissue fragments to the liquid autologous plasma matrix is ​​1:5 to 1:

20.

5. The composite membrane according to claim 1, characterized in that, The formation of the composite membrane does not require the addition of any exogenous thrombin, calcium chloride or other chemical activators, and relies entirely on the biological interface provided by the tissue fragments to complete platelet activation and gelation.

6. A method for preparing a plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments as described in any one of claims 1-5, characterized in that, Includes the following steps: Step (1): Collect the patient's own venous blood and obtain liquid autologous plasma matrix by centrifugation. Step (2): Obtain the discarded gingival tissue removed during the patient's oral surgery, and cut it into fragments of 0.5 mm³-3 mm³ under aseptic conditions to obtain autologous keratotic epithelial tissue fragments. Step (3): Mix the liquid autologous plasma matrix obtained in step (1) with the autologous keratinized epithelial tissue fragments obtained in step (2) at a volume ratio of 1:5-1:20 to form a mixture. Step (4): Let the mixture obtained in step (3) stand at room temperature or 37°C for 5-30 minutes to activate platelets using tissue factors on the surface of the tissue fragments, spontaneously forming a gel complex. Step (5): Place the gel-like complex obtained in step (4) into a plasma matrix pressurizer and press it under a pressure of 0.5-10 N for 0.5-5 minutes to remove the exudate and form a membrane material with a thickness of 0.5-3 mm, thus obtaining the plasma matrix composite membrane activated by the autologous keratinized epithelial tissue fragments.

7. The method according to claim 6, characterized in that, The centrifugation in step (1) is horizontal centrifugation with a centrifugation acceleration of 60-1000g and a centrifugation time of 2-10 minutes, and no anticoagulant is added.

8. The method according to claim 6, characterized in that, The pressure during pressing in step (5) is 1-5 N, the pressing time is 1-3 minutes, and the film thickness after pressing is 1-2 mm.

9. The use of the plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments as described in any one of claims 1-5 or the composite membrane prepared by the method described in any one of claims 6-8 in the preparation of a medical device for oral soft tissue augmentation.

10. The application according to claim 9, characterized in that, The increase in oral soft tissue includes widening of keratinized gingiva, increase in alveolar ridge soft tissue, increase in peri-implant soft tissue, or repair of oral mucosal defects; after the composite membrane is implanted, the spinous cells and granular cells in the tissue fragments migrate and proliferate in the growth factor microenvironment provided by the plasma matrix, forming new stratified keratinized epithelium.