CN109701077B - Micropore regeneration tissue matrix and preparation and application thereof - Google Patents
Micropore regeneration tissue matrix and preparation and application thereof Download PDFInfo
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Abstract
The invention provides a micropore regeneration tissue matrix and preparation and application thereof. The micropore regeneration tissue matrix is a sheet material with a porous structure, micropores are uniformly distributed on the material, the pore diameter of each micropore is 0.15-0.20 mm, the pore spacing is 0.8-1.2 mm, and collagen fibers extending into the pores are arranged on the inner surfaces of the micropores. The micropore regeneration tissue matrix provided by the invention is complete in decellularization, free of immunogenicity and cytotoxic factors, the collagen fibers, the reticular fibers and the elastic fibers of the regeneration tissue matrix and the three-dimensional structure formed by the reticular fibers and the elastic fibers are less damaged, the adhesion degree with a wound surface is better, and a plurality of collagen fibers extending into the micropores are arranged on the inner surface of the micropore, so that more attachment sites for migrating cells can be provided.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a microporous regenerated tissue matrix, and a preparation method and application thereof.
Background
The Regenerated Tissue Matrix (RTM) is prepared by removing cell components in epidermis and dermis layers from human skin through a series of physical and chemical methods, thereby eliminating immunogenicity caused by foreign body and retaining collagen fibers, reticular fibers, elastic fibers and three-dimensional space structures formed by the collagen fibers, the reticular fibers and the elastic fibers. In 1995, the american living cell company first reported the preparation method of acellular dermal matrix and developed the clinical application research of the acellular dermal matrix in the replacement and repair of dermal defects caused by burns and wounds. Later researches find that the acellular dermal matrix can be used for repairing wound surfaces such as burns and wounds, can also be used as a substitute for mucous membranes and soft tissues, and is widely applied to the fields of orthopedics, oral cavity, neurosurgery, ophthalmology, otorhinolaryngology and other subjects.
An ideal Regenerated Tissue Matrix (RTM) should remove antigenic cell components as much as possible, retain relatively intact collagen fiber components and basic structure of tissue, and make the dermal matrix have the characteristics of good histocompatibility, high stability and moderate flexibility. These properties of the Regenerated Tissue Matrix (RTM) are determined mainly by its composition and structure, which are directly related to its preparation method. Although the preparation of the existing xenogenic acellular dermal matrix is mature, there are some problems in its preparation and application, such as: pancreatin method and NaCl-SDS method, its disadvantage is that the cell-removing degree is not thorough, appear different degree immunoreaction, tissue compatibility phase difference causes the survival rate of transplantation to be lower; the Dispase II-Triton and NaOH ablation method has the defects of complex preparation process, large destructiveness of natural dermal ultrastructure by the method, poor retention of extracellular matrix components and certain damage effect on dermal basement membrane. In addition, in the preparation method, in order to prevent the excessive degradation of collagen fibers and slow down the decomposition speed of the collagen fibers by protease after transplantation, glutaraldehyde is adopted for crosslinking, and because glutaraldehyde has certain cytotoxicity, the delayed repair of body wounds can be caused, and the repair time can be prolonged.
In the process of tooth planting and skin repairing by removing giant nevus, the dermal matrix is provided with micropores with proper sizes, which can promote active ingredients such as plasma, free cells, fibroblasts and the like to permeate into the acellular dermal matrix and maintain the early nutrition supply of the dermal matrix. In the process of tooth planting, the tooth root is wrapped by the microporous acellular allogeneic dermis, and the micropores can promote the migration, attachment and amplification of fibroblasts at the root of the tooth and accelerate the fusion with body tissues, thereby promoting the survival rate of tooth planting and shortening the repair time. After the huge nevus is removed, in the process of repairing the skin by using the micropore acellular variant dermis as the patch, the micropores on the dermis matrix can prevent the formation of subcutaneous effusion, pneumatosis and hematoma, promote the plasma and free cells to permeate the surface of the dermis matrix and promote the survival rate of skin grafting.
At present, the sizes of the holes punched on the dermal matrix and the gaps between the holes are not known uniformly, but when the hole diameter is larger, a punctate or reticular scar is formed, and when the hole diameter is smaller, histiocytes are difficult to migrate into the hole, and the vascularization speed is slow. Therefore, the selection of a suitable pore size for the micropores and the provision of more cell attachment sites around the pore size are of great importance for the vascularization of the dermal matrix.
Laser drilling is the earliest practical laser processing technology and has been widely used for processing materials such as metal, leather, glass, wood and the like. The laser drilling technology has the advantages of high drilling speed, high efficiency, small aperture, uniform micropore distribution and the like. The laser drilling technology is introduced into the processing of the heterogenic dermal matrix micropores, uniformly and densely distributed micropores can be manufactured on the dermal matrix, the appearance of scars on the surface of the transplanted skin is reduced, and the uniform distribution of microvessels is promoted, so that the aims of improving the survival rate of the dermal matrix transplantation and improving the healing quality are fulfilled.
In the existing preparation method and punching technology of the variant dermis, in order to avoid the existence of immunogenic substances, more violent preparation conditions are mostly adopted, and as a result, the reticular fibers and the elastic fibers of the dermis matrix are greatly degraded, so that the three-dimensional structure of the collagen fibers is greatly broken, and the transplanting survival rate of the dermis matrix is reduced; in the dermal matrix perforation technology, perforation is mostly carried out by adopting a rehydrated dermal matrix or a wet dermal matrix, and the perforation technology is mostly carried out after the preparation of the acellular dermal matrix is finished, the aperture is mostly controlled to be 80-140um, but the cell attachment sites on the inner surface of the aperture are few. Chinese patent CN105688286A discloses a laser micropore acellular dermal matrix and a preparation method thereof, wherein a pulse laser is adopted in the patent to perforate the dermal matrix, the raw material of the laser micropore acellular dermal matrix is prepared from a wet acellular dermal matrix, and although the pore diameter distribution is uniform, the cell attachment sites in the pore diameter are few. Chinese patent CN200510126108.6 discloses an acellular dermal matrix and a preparation method thereof, wherein high-concentration sodium hydroxide is adopted to repeatedly wash mammalian dermis to obtain the acellular dermal matrix, and the original protein structure in the dermal matrix is influenced by partial collagen denaturation and protein cracking caused by severe alkali treatment conditions.
Disclosure of Invention
To solve the above problems of the prior art, the present invention provides a microporous regenerated tissue matrix. The micropore regeneration tissue matrix is complete in decellularization, free of immunogenicity and free of cytotoxic factors, the collagen fibers, the reticular fibers and the elastic fibers of the tissue matrix and the three-dimensional structure formed by the collagen fibers, the reticular fibers and the elastic fibers are small in damage and good in adhesion with a wound surface, and a plurality of collagen fibers extending into the micropores are arranged on the inner surface of the micropore, so that more migratory cell attachment sites can be provided.
The second purpose of the invention is to propose a preparation method of the microporous regenerated tissue matrix. The micropore variant dermis is obtained by carrying out laser drilling on the dermis matrix after freeze drying, and then carrying out rehydration, alkali liquor expansion and cell removal treatment, oscillation cleaning and amino acid solution soaking treatment.
A third object of the invention is to propose the use of said microporous regenerative tissue matrix.
The technical scheme for realizing the above purpose of the invention is as follows:
the microporous regenerated tissue matrix is a sheet material with a porous structure, micropores are uniformly distributed on the material, the pore diameter of each micropore is 0.15-0.20 mm, the pore spacing is 0.8-1.2 mm, and collagen fibers extending into the pores are arranged on the inner surfaces of the micropores.
A preparation method of a microporous regenerated tissue matrix comprises the following steps:
1) preprocessing the allogenic skin: cleaning the variant skin with the subcutaneous tissue scraped in normal saline, then soaking in sodium chloride solution to remove the epidermal layer of the skin, and then cleaning in the normal saline to obtain variant dermis;
2) and (3) freeze drying treatment: soaking the variant dermis in a freeze-drying protective agent, shaping, quickly freezing in liquid nitrogen, and then carrying out vacuum freeze-drying;
3) punching treatment: drilling holes in the freeze-dried variant dermis by using a laser processing system;
4) alkali liquor expansion and cell removal treatment: putting the variant dermis after punching into physiological saline and/or a freeze-drying protective agent for rehydration, then adopting alkali liquor for swelling treatment to remove cells in the dermis layer, and then using the physiological saline for shaking and cleaning until the effluent liquid is neutral.
Further, in step 1), the chlorine isThe concentration of the sodium dissolving solution is 0.8-1.2 mol/L, and the material-liquid ratio of the skin sheet to the liquid per unit area is 1cm2: (0.5-2) mL, the soaking time is 18-30 h, and the soaking temperature is 24-32 ℃.
The freeze-drying protective agent in the step 2) is a trehalose dilute saline solution, the concentration of trehalose is 200-600 mmol/L, the dilute saline solution is composed of 1 part of physiological saline and two parts of water for injection, and the material-liquid ratio of the skin sheet per unit area to the liquid is 1cm2: (0.5-2) ml, the soaking time is 4-6 h, and the soaking temperature is 30-40 ℃.
Preferably, after the freeze-drying protective agent is soaked in the step 2), the allodermis is fixed and shaped by a hard frame (the material of the hard frame mainly can bear the low temperature of liquid nitrogen, such as polyimide and polytrifluorochloroethylene), and the freeze-drying treatment and the punching treatment are carried out while keeping the fixed state. The frame can be removed after the hole is punched.
According to a preferable technical scheme, the operation of quick freezing of the liquid nitrogen in the step 2) is as follows: pre-cooling at the temperature of-6 to-2 ℃, and then hanging into liquid nitrogen by using a rope for quick freezing for 20 to 60 seconds;
the operation of vacuum freeze drying is as follows: the precooling temperature of the vacuum freeze dryer is set to be-30 to-40 ℃, and the vacuum freeze-drying treatment is carried out after the foreign body dermis is put in.
Further, the vacuum freeze-drying process is divided into two stages, stage 1: maintaining for 14h at the temperature lower than-30 ℃ and the vacuum degree lower than 3 Pa; and (2) stage: heating to 20 ℃, and maintaining for 4 hours under the condition that the vacuum degree is less than 3 Pa; the vacuum degree is not higher than 30Pa in the whole vacuum freeze drying process.
The laser processing system in the step 3) is a multifunctional ultraviolet processing system, diode-pumped solid laser is adopted, and the wavelength is 355 nm;
the laser drilling parameters are as follows: the aperture is 0.15-0.20 mm, and the hole spacing is 0.8-1.2 mm.
Wherein, the alkali liquor decellularization and expansion treatment in the step 4) adopts the following treatment modes: using Na with 0.1-0.5 mol/L2CO3And the mass fraction is 0.1-0.4Treatment with a solution of% SDS;
or, 0.1-0.5 mol/L Na is adopted firstly2CO3Treating with alkali liquor, and then treating with SDS with the mass fraction of 0.1-0.4%;
preferably, the material-liquid ratio of the surface area of the skin piece to the treated solution is (1-3) cm2: 1mL, oscillating for 1-3 h, standing and soaking for 2-6 h, and repeating oscillation and soaking treatment for 2-5 times;
more preferably, the material after the alkali liquor expansion treatment is put into the solution of amino acid or surfactant for soaking treatment, and then is subjected to vibration cleaning treatment by normal saline, irradiation sterilization and split charging;
the amino acid solution for soaking treatment is as follows: each liter of the solution contained 2g of threonine, 2g of serine, 1g of glutamic acid and 1g of aspartic acid.
The invention relates to application of a micropore regeneration tissue matrix as a tooth implantation filling material and a giant nevus patch.
The invention has the beneficial effects that:
the micropore regeneration tissue matrix provided by the invention is complete in decellularization, free of immunogenicity and cytotoxic factors, the collagen fibers, the reticular fibers and the elastic fibers in the matrix and the three-dimensional structure formed by the reticular fibers and the elastic fibers are less damaged, the adhesion degree with a wound surface is better, and a plurality of collagen fibers extending into the micropores are arranged on the inner surface of the micropores, so that more attachment sites for migrating cells can be provided.
The preparation method of the micropore regeneration tissue matrix adopts dry variant dermis to carry out laser drilling, and a plurality of collagen fibers extending into the micropore exist in the micropore after the micropore is rehydrated and is expanded by alkali liquor, thereby being beneficial to the attachment and proliferation of the emigrated cell and improving the survival rate of transplantation of the regeneration tissue matrix.
Before the process of removing cells by alkali liquor expansion, the method adopts a freezing rehydration process, can help the cells to fall off, shorten the alkali liquor treatment strength, and is favorable for reducing the damage of a three-dimensional structure consisting of regenerated tissue matrix collagen fibers, reticular fibers and elastic fibers.
The method adopts a laser drilling mode, has high precision and high speed, uniformly distributes micropores, can reduce the appearance of scars on the surface of the graft skin, and improves the healing quality of the wound surface.
Compared with the existing preparation method, the invention reduces the crosslinking process of glutaraldehyde to the regenerated tissue matrix and avoids the introduction of cytotoxic factors.
Drawings
FIG. 1 is a photograph under an optical microscope (light display. times.10) of a microporous regenerated tissue matrix of example 1.
FIG. 2 is an electron micrograph of the inner surface of the micropores of the matrix of the regenerated microporous tissue according to example 1.
FIG. 3 is a photograph under an optical microscope (light display. times.10) of the microporous regenerated tissue matrix of example 2.
FIG. 4 is an electron micrograph of the inner surface of the micropores of the matrix of the regenerated microporous tissue of example 2.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the examples, unless otherwise specified, the methods used are conventional in the art. The raw materials or reagents involved in the present invention are commercially available.
The technical solution of the present invention will be described below by specific examples.
Example 1
The preparation method of the microporous regenerated tissue matrix comprises the following steps:
1. washing with water: taking out the variant skin from the protective solution, scraping off subcutaneous tissue, placing into normal saline, and cleaning by shaking to obtain skin patch with material-liquid ratio (cm) of normal saline2: mL) is 1: 1, the total volume of the feed liquid does not exceed 50 percent of the volume of the oscillating container, the oscillating frequency is 30rpm, the oscillating and cleaning time is 1.5h, and the physiological saline is replaced once every 0.5h of oscillation, and the oscillating and cleaning is carried out for 3 times.
2. Removing the epidermal layer: soaking the variant skin cleaned by oscillation in 1mol/L NaCl solution at a material-to-liquid ratio (cm)2: mL) is 1: 1, soaking for 24 hours at the temperature of 28 ℃. After soaking, the xenogenic skin is taken out and the epidermis layer is torn off. Removing epidermis layer of skin, and further treatingStep 1, oscillating and cleaning to obtain a variant dermis.
3. Liquid nitrogen freezing, placing the allogeneic dermis into low-concentration saline (composed of 1 part of normal saline and 2 parts of water for injection) containing 400mmol/L trehalose, and incubating for 5h at 37 ℃. Shaping the incubated skin sheets, and clamping four sides by using hard frames to keep the skin sheets flat, so as to prevent the dermal matrix from being curled and contracted in the freeze drying process and facilitate subsequent laser drilling. Precooling the shaped skin slices at-4 ℃, and then hanging the skin slices in liquid nitrogen for quick freezing for 30 s.
4. And (3) freeze drying: quickly putting the quick-frozen variant dermis into a vacuum freeze-drying machine pre-cooled to-30 to-40 ℃, starting a vacuum pump to carry out vacuum-pumping drying treatment, and pumping the vacuum degree of a drying box to be below 10 Pa. Maintaining for 14h at the temperature lower than-30 ℃ and the vacuum degree lower than 3 Pa; then heating to 20 ℃, and maintaining for 4h under the condition that the vacuum degree is less than 3Pa, wherein the vacuum degree is not higher than 30Pa in the whole vacuum drying process.
5. Laser hole making: setting parameters of a multifunctional ultraviolet laser processing system (DE-MID DL500U), selecting ultraviolet light of 355nm, suspending the freeze-dried variant dermis in a frame, and keeping a distance of more than 5mm between the skin and a bottom plate to prevent the skin from being lost due to residual heat at the bottom; the laser drilling parameters are as follows: the punching distance is 1.0mm, the aperture is 0.2mm, matrix punching is carried out, and dry micropore variant genuine leather is obtained.
6. Rehydration: placing dried microporous variant dermis into physiological saline at a ratio of material to liquid (cm)2: mL) is 1: 1, shaking and soaking for 20min to obtain the microporous allogenic dermis.
7. Alkali liquor expansion and cell removal treatment: adding the microporous allodermis to the solution containing 0.2mol/L of Na2CO3Soaking in 0.2% SDS solution at a material-to-liquid ratio (cm)2: mL) 1: 1, the total volume of the feed liquid is not more than 50 percent of the capacity of an oscillator, the oscillation frequency is 30rpm, the oscillation is carried out for 2 hours, the mixture is kept still and soaked for 4 hours, and the oscillation cleaning and the soaking treatment are repeated for 4 times. And then, the dermal matrix is shaken and cleaned according to the step 1 until the effluent cleaning solution is neutral (pH6-8), so that the microporous acellular dermal matrix is obtained.
8. Soaking in amino acid solution: adding the acellular variant dermal matrix into an amino acid solution (each L of the solution contains 2g of threonine, 2g of serine, 1g of glutamic acid and 1g of aspartic acid), carrying out shaking soaking treatment for 1 hour, and then standing and soaking for 0.5 hour. And then the dermal matrix is cleaned by oscillation according to the step 1 to obtain the micropore regeneration tissue matrix.
9. And (3) sterilization: co60 was used to sterilize the bacteria at a dose of 25 KGy.
10. Packaging: and (3) soaking the sterilized microporous regenerated tissue matrix in PBS buffer solution, and hermetically packaging by using a plastic bag.
The obtained product is shown in figure 1, the micropore regeneration tissue matrix prepared in the embodiment is a sheet material with a porous structure, uniformly distributed micropores are formed in the material, the pore diameter is 0.15-0.20 mm, the pore spacing is 0.8-1.2 mm, and collagen fibers extending into the pores are arranged on the inner surfaces of the micropores, which is shown in an electron microscope picture of figure 2.
Example 2
The preparation method of the microporous regenerated tissue matrix of the embodiment comprises the following steps:
steps 1 to 5 are the same as in example 1;
6. and (3) rehydration treatment: adopts a gradient degressive rehydration mode, namely, the freeze-dried microporous allodermal is placed in 400mmol/L trehalose low-concentration saline solution with the material-to-liquid ratio (cm)2: ml) is 1: 1, shaking and soaking for 15 min; then taking out and adding into 200mmol/L trehalose low-concentration saline water with the feed-liquid ratio (cm)2: ml) is: 1: 1, shaking and soaking for 15 min; then placing into normal saline without trehalose at a ratio of material to liquid (cm)2: ml) is: 1: 1, shaking and soaking for 15min to obtain the microporous allogenic dermis.
7. Alkali liquor expansion and cell removal treatment: adding the microporous allogenic dermis to 0.2mol/L Na2CO3Soaking in water at a material-to-liquid ratio (cm)2: ml) 1: 1, the total volume of the feed liquid is not more than 50 percent of the capacity of an oscillator, the oscillation frequency is 30rpm, the oscillation is carried out for 2 hours, the standing and the soaking are carried out for 4 hours, and the oscillation, the cleaning and the soaking treatment are repeated for 4 times. And then the dermal matrix is washed by shaking according to the step 1 to obtain the acellular allogeneic dermal matrix.
8. Surfactant treatment: acellular allogeneic dermal matrix was added to 0.Oscillating in 2% SDS solution, material-to-liquid ratio (cm)2: ml) 1: 1, the treatment time is 1h, and the preparation is used for removing residual cell fragments of a dermal matrix and reducing immunogenicity. And (3) carrying out shaking cleaning on the surfactant acellular dermal matrix according to the step 1 to obtain the acellular allogeneic dermal matrix.
9. Soaking in amino acid solution: adding the acellular variant dermal matrix into an amino acid solution (each L of the solution contains 2g of threonine, 2g of serine, 1g of glutamic acid and 1g of aspartic acid), carrying out shaking soaking treatment for 1 hour, and then standing and soaking for 0.5 hour. And then the dermal matrix is cleaned by oscillation according to the step 1 to obtain the micropore regeneration tissue matrix.
10. And (3) sterilization: co60 was used to sterilize the bacteria at a dose of 25 KGy.
11. Packaging: and (3) soaking the sterilized microporous regenerated tissue matrix in PBS buffer solution, and hermetically packaging by using a plastic bag.
The obtained product is shown in figure 3, the micropore regeneration tissue matrix prepared in the embodiment is a sheet material with a porous structure, uniformly distributed micropores are formed in the material, the pore diameter is 0.15-0.20 mm, the pore spacing is 0.8-1.2 mm, and collagen fibers extending into the pores are arranged on the inner surfaces of the micropores, which is shown in figure 4.
Comparative example 1
The method for preparing the microporous regenerated tissue matrix of the present comparative example includes the steps of:
steps 1 and 2 were the same as in example 1.
3. Alkali liquor treatment: adding the allogenic dermis into 0.6 percent NaOH solution for soaking, wherein the material-liquid ratio (cm 2: ml) is 1: 1, the total volume of the feed liquid is not more than 50 percent of the capacity of an oscillator, the oscillation frequency is 30rpm, the oscillation is carried out for 1h, the mixture is kept stand and soaked for 5h, and the total treatment time is 18 h. The dermal matrix is then subjected to a shaking wash process according to step 1.
4. Surfactant treatment: adding the variant dermal matrix into 0.2% SDS solution, soaking, and adjusting the ratio of materials to liquids (cm)2: mL) 1: 1, the total volume of the feed liquid does not exceed 50 percent of the capacity of an oscillator, the oscillation frequency is 30rpm, and the oscillation treatment is carried out for 0.5 h. The dermal matrix is then subjected to a shaking wash process according to step 1.
5. Glutaraldehyde crosslinking treatment: mixing different body coriumAdding the substrate into 0.2% glutaraldehyde solution, and soaking at a material-to-liquid ratio (cm)2: mL) 1: 1, soaking for 20 min. The dermal matrix is then subjected to a shaking wash process according to step 1.
6. Laser hole making: set for the parameter of multi-functional ultraviolet laser processing system, select 355 nm's ultraviolet ray, suspend the xenogenesis dermal matrix in placing the frame in, dermal matrix leaves certain distance apart from the bottom plate, and the laser beam drilling parameter does, and the interval of punching 1.0mm, aperture 0.15mm carry out the matrix and punch. And then the micropore variant dermal matrix is subjected to vibration cleaning treatment according to the step 1.
7. And (3) sterilization: co60 was used to sterilize the bacteria at a dose of 25 KGy.
8. Packaging: and (3) soaking the sterilized microporous regenerated tissue matrix in PBS buffer solution, and sealing and packaging the microporous regenerated tissue matrix by using a plastic bag.
Experimental example 1: performance testing of regenerated tissue matrices
The regenerated tissue matrices prepared in examples 1 and 2 and comparative example 1 were tested for their biological properties such as mechanical properties, and their performance indexes include tensile strength, suture strength, and tensile elongation. Specific detection results are shown in table 1. The results show that the products of examples 1 and 2 have better fitting degree with skin, good toughness and elasticity, and can meet the requirement of the patch stitching strength after removal of nevus megalus.
TABLE 1 detection results of microporous regenerated tissue matrix
| Item | Tensile Strength (MPa) | Stitching Strength (N) | Elongation in tension (%) |
| Practice ofExample 1 | 6.2 | 19.5 | 14.0 |
| Example 2 | 6.4 | 19.8 | 14.1 |
| Comparative example 1 | 4.7 | 18.2 | 11.2 |
Experimental example 2 animal model test
Subject: 48 healthy SD rats are selected, the male and female are not limited, and the weight of the rats is 250 +/-10 g.
The experimental steps are as follows:
1. rats were anesthetized with 3% sodium pentobarbital (50mg/kg) in the peritoneal cavity.
2. SD rat is fixed on operation table, the back is unhaired, iodine is sterilized, and sterile towel is laid.
3. Peeling with a drum-type peeling machine, adjusting the peeling thickness of the drum-type peeling machine to a proper position, pulling a handle to finish peeling of the whole layer of skin, and obtaining a self-body blade-thick skin piece with the size of 2.5cm multiplied by 2.5 cm.
4. The rats were then randomly divided into 3 groups of 16 rats each. Example 1 group: wound surface transplantation using the microporous regenerated tissue matrix obtained by the preparation method of example 1; example 2 group: wound grafting a microporous regenerated tissue matrix obtained by the preparation method of example 2 is adopted; control group: transplanting the autologous edged thick skin sheet.
5. Transplanting the regenerated microporous tissue matrix and skin sheet, covering with vaseline gauze and sterile dressing, fixing, raising in single cage after operation, fixing, and changing dressing after 2 weeks.
The experimental results are as follows:
two weeks after the skin sheet and the micropore regeneration tissue matrix transplantation operation, most of the regeneration tissue matrix of the group 1 and the group 2 survives, the two groups of phenomena have no obvious difference, sporadic spots are scattered on the wound surface, the micropore regeneration tissue matrix at the deep layer is tightly connected with the subcutaneous tissue, and most of the knife-edge thick skin sheet of the wound surface of the control group survives well. 4 weeks after the transplantation operation, the example 1 and example 2 groups had close to healing, good blood circulation, desquamation at the transplantation site, and the wound surface of the control group was close to healing. 6 weeks after the transplantation operation, the wound surfaces of the example 1 group and the example 2 group are basically healed, the appearance of the two groups is not obviously different, and the wound surfaces are relatively flat; the control group had substantially healed wound surface.
The survival rates of the skin grafts on the wound surfaces of the groups are shown in table 2.
TABLE 2 postoperative survival rate comparison of transplanted skin pieces (%, + -s)
Note that compared with the control group,*P<0.05
the survival rate of example 1 and example 2 groups transplanted was lower than that of the control group (P <0.05) at 2 and 4 weeks after surgery; 6 weeks after surgery, the groups of example 1 and example 2 were not statistically significant compared to the control group (P > 0.05).
Experimental example 3 dental implant test
Case selection: the 3 crescent midges receive 23 patients, 13 men and 10 women who need tooth implantation, and the oral hygiene of the patients is good.
Materials: example 1 microporous regenerative tissue matrix.
The operation method comprises the following steps: preoperative examination, oral examination was performed on all the cases of implantation, and the width of the alveolar ridge and the height of the alveolar ridge were measured using calipers. Planting the implant and planting the micropore regeneration tissue matrix, cutting the micropore regeneration tissue matrix with proper size, and planting the tissue matrix in a folding way according to the degree of labial recession, wherein the connective tissue faces outwards. Antibiotic anti-inflammatory therapy is routinely given post-operatively.
The observation method comprises the following steps: the wound healing condition of the transplanted regenerated tissue matrix of the patient after 1 week, 3 weeks, 3 months and 6 months of the operation is tracked and observed. And evaluating the relationship between the planted sports neighboring teeth and surrounding tissues by shooting X-ray films.
As a result: the operation is observed to remove the symptoms of no obvious swelling, rash, fever, local numbness, pain, bleeding and the like. After 3 months of operation, the X-ray film shows that the yoga tissues around the implant are tightly attached without transmission gaps and obvious transverse bone absorption occurs. The survival rate of the implant is observed to be 100% in 6 months after the operation.
Although the present invention has been described in the foregoing by way of examples, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (12)
1. A method for preparing a microporous regenerated tissue matrix, comprising the steps of:
1) preprocessing the allogenic skin: cleaning the variant skin with the subcutaneous tissue scraped in normal saline, then soaking in sodium chloride solution to remove the epidermal layer of the skin, and then cleaning in the normal saline to obtain variant dermis;
2) and (3) freeze drying treatment: soaking the variant dermis in a freeze-drying protective agent, shaping, quickly freezing in liquid nitrogen, and then carrying out vacuum freeze-drying;
3) punching treatment: drilling holes in the freeze-dried variant dermis by using a laser processing system;
4) alkali liquor expansion and cell removal treatment: putting the variant dermis after punching into physiological saline and/or a freeze-drying protective agent for rehydration, then adopting alkali liquor for swelling treatment to remove cells in the dermis layer, and then using the physiological saline for shaking and cleaning until the effluent liquid is neutral.
2. The preparation method according to claim 1, wherein in the step 1), the concentration of the sodium chloride solution is 0.8-1.2 mol/L, and the feed-liquid ratio of the skin sheet to the liquid per unit area is 1cm2: (0.5-2) mL, and the soaking time is 18And (5) about 30 hours, wherein the soaking temperature is 24-32 ℃.
3. The preparation method of claim 1, wherein the lyoprotectant in step 2) is a dilute aqueous solution of trehalose, wherein the concentration of trehalose is 200-600 mmol/L, the dilute aqueous solution of trehalose is composed of 1 part of physiological saline and two parts of water for injection, and the ratio of skin/liquid per unit area to liquid is 1cm2: (0.5-2) mL, the soaking time is 4-6 h, and the soaking temperature is 30-40 ℃.
4. The method according to claim 1, wherein the freeze-drying treatment and the perforation treatment are performed while the allodermis is fixed by a hard frame after the cryoprotectant is soaked in the step 2).
5. The preparation method according to claim 1, wherein the liquid nitrogen quick freezing in step 2) is performed by: pre-cooling at the temperature of-6 to-2 ℃, and then hanging into liquid nitrogen by using a rope for quick freezing for 20 to 60 seconds;
the operation of vacuum freeze drying is as follows: the precooling temperature of the vacuum freeze dryer is set to be-30 to-40 ℃, and the vacuum freeze-drying treatment is carried out after the foreign body dermis is put in.
6. The method of claim 5, wherein the vacuum freeze-drying process is divided into two stages, stage 1: maintaining for 14h at the temperature lower than-30 ℃ and the vacuum degree lower than 3 Pa; and (2) stage: heating to 20 ℃, and maintaining for 4 hours under the condition that the vacuum degree is less than 3 Pa; the vacuum degree is not higher than 30Pa in the whole vacuum drying process.
7. The preparation method of claim 1, wherein the laser processing system in step 3) is a multifunctional ultraviolet processing system, which uses diode-pumped solid laser with a wavelength of 355 nm;
the laser drilling parameters are as follows: the aperture is 0.15-0.20 mm, and the hole spacing is 0.8-1.2 mm.
8. The method according to claim 2, wherein the alkali solution is subjected to cell expansion treatment in step 4) in a manner that: using Na with 0.1-0.5 mol/L2CO3And SDS solution with the mass fraction of 0.1-0.4% is processed;
or, 0.1-0.5 mol/L Na is adopted firstly2CO3Alkali liquor treatment, and then SDS with the mass fraction of 0.1-0.4% is adopted for treatment.
9. The method according to claim 8, wherein the ratio of the skin surface area to the treated solution is (1-3) cm2: 1mL, oscillating for 1-3 h, standing and soaking for 2-6 h, and repeating oscillating and soaking treatment for 2-5 times.
10. The preparation method of claim 9, wherein the material after the swelling treatment with alkali solution is soaked in the solution of amino acid, and then washed with normal saline by shaking, sterilized by irradiation, and packaged;
the amino acid solution for soaking treatment is as follows: each liter of the solution contained 2g of threonine, 2g of serine, 1g of glutamic acid and 1g of aspartic acid.
11. A microporous regenerated tissue matrix produced by the method of any one of claims 1-10.
12. The microporous regenerated tissue matrix according to claim 11, wherein the microporous regenerated tissue matrix is a sheet material with a porous structure, the sheet material has uniformly distributed micropores, the diameter of the micropores is 0.15-0.20 mm, the distance between the micropores is 0.8-1.2 mm, and the inner surface of the micropores has collagen fibers extending into the pores.
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