WO2012118305A2 - Procédé de production d'une greffe de tissu mou allogène contenant une cellule souche autologue - Google Patents

Procédé de production d'une greffe de tissu mou allogène contenant une cellule souche autologue Download PDF

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WO2012118305A2
WO2012118305A2 PCT/KR2012/001443 KR2012001443W WO2012118305A2 WO 2012118305 A2 WO2012118305 A2 WO 2012118305A2 KR 2012001443 W KR2012001443 W KR 2012001443W WO 2012118305 A2 WO2012118305 A2 WO 2012118305A2
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soft tissue
allogeneic
tissue support
allogeneic soft
support
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PCT/KR2012/001443
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English (en)
Korean (ko)
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WO2012118305A3 (fr
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심영복
이광일
장주웅
이정수
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주식회사 코리아본뱅크
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Priority claimed from KR1020120018650A external-priority patent/KR101352378B1/ko
Application filed by 주식회사 코리아본뱅크 filed Critical 주식회사 코리아본뱅크
Priority to US14/002,104 priority Critical patent/US9511172B2/en
Publication of WO2012118305A2 publication Critical patent/WO2012118305A2/fr
Publication of WO2012118305A3 publication Critical patent/WO2012118305A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells

Definitions

  • the present invention relates to a method for producing an allogeneic soft tissue implant implanted into a human body using a soft tissue derived from an allogeneic organism as a support.
  • the transplantation of the autologous tissue also requires continuous rehabilitation treatment, and unlike the original inherent ligaments, the transplanted autologous tissues are markedly inferior in physical properties and cannot be viewed as a practical treatment. Therefore, there is a need for allogeneic soft tissue implants that can maintain their physical properties, significantly reduce immune rejection, and replace them as closely as possible with their own soft tissues in order to treat damaged ligaments, ie, damaged soft tissues. to be.
  • the present invention is to produce an allogeneic soft tissue implant in which autologous stem cells are injected, based on the allogeneic soft tissue support derived from allogeneic organisms to efficiently replace damaged soft tissues of the human body.
  • the present invention includes the steps of obtaining an allogeneic soft tissue support obtained from a human or animal body; Washing and sterilizing the obtained soft tissue support; Treating the washed and sterilized homologous soft tissue support with an enzyme solution to remove immune rejection cells; Forming one or more pores on the allogeneic soft tissue support from which the immune rejection cells have been removed; Injecting autologous stem cells of a user into the formed pores; And culturing allogeneic soft tissue scaffolds into which the autologous stem cells have been injected.
  • the allogeneic soft tissue support may be ligaments or bone fragment connecting ligaments.
  • the washing and sterilizing step may include treating hydrogen peroxide on the obtained homogeneous soft tissue support and applying ultrasonic waves.
  • the treated hydrogen peroxide has a concentration selected from the concentration range of 0.1 to 5.0 percent (%)
  • the applied ultrasound may have an output selected from the range of 120 to 400 watts (W). have.
  • the washing and sterilizing step may include the step of treating the radiation protective material to the obtained homogeneous soft tissue support, and irradiating the radiation.
  • the radiation protection material to be treated is cobalt 60 (Co 60 ), and the radiation to be irradiated may be gamma rays selected from the range of 12 to 50 kilograms (kGy).
  • the step of removing the immune rejection cells may be treating an enzyme solution comprising trypsin, collagenase and protease.
  • the step of removing the immune rejection cells may further comprise the step of washing by washing with physiological saline after treating the enzyme solution to the washed and sterile homologous soft tissue support. .
  • the step of removing the immune rejection cells is treated with an enzyme solution to the washed and sterilized homologous soft tissue scaffold, followed by washing with physiological saline, followed by osmotic treatment with distilled water. It may further comprise a step.
  • the pore in the step of forming the one or more pores, may have a size selected from the range of 1 to 1000 micrometers ( ⁇ m).
  • the step of injecting the autologous stem cells may further comprise the step of injecting the autologous stem cell supporting promoter prior to or simultaneously with the injection of the autologous stem cells.
  • culturing the allogeneic soft tissue support may further comprise providing a physical stimulus to the allogeneic soft tissue support.
  • the physical stimulus may be provided to the allogeneic soft tissue support at a frequency of up to once per second.
  • the provision of the physical stimulus provides a tensile stimulus at both ends of the allogeneic soft tissue support, or provides a twist in both transverse directions of the allogeneic soft tissue support based on the longitudinal axis of the allogeneic soft tissue support.
  • the provision of the physical stimulus provides a tensile stimulus at both ends of the allogeneic soft tissue support, or provides a twist in both transverse directions of the allogeneic soft tissue support based on the longitudinal axis of the allogeneic soft tissue support.
  • providing tensile stimulation at both ends of the allogeneic soft tissue support extends both ends of the allogeneic soft tissue support in the longitudinal direction, and one end is 10% of the total length of the allogeneic soft tissue support. At the same time as extending in the following range may be to extend the other end in the range of less than 10% of the total length of the allogeneic soft tissue support.
  • providing a twist in both transverse directions of the allogeneic soft tissue support rotates both ends of the allogeneic soft tissue support, while at the same time rotating one end in a range of 45 degrees or less in a clockwise direction. It may be to rotate the end in a range of 45 degrees or less counterclockwise.
  • the culturing step may be carried out in a range of 7 days or less.
  • allogeneic soft tissue implants capable of efficiently replacing damaged soft tissues of the human body can be prepared by injecting and culturing autologous stem cells into allogeneic soft tissue supports derived from allogeneic organisms.
  • Figure 1 illustrates the individual steps for implementing a method for producing allogeneic soft tissue implants according to one embodiment of the present invention.
  • Figure 2 shows the individual steps for implementing the support cleaning and sterilization step included in the method for producing allogeneic soft tissue implants according to an embodiment of the present invention.
  • Figure 3 shows the individual steps for implementing the step of removing the immune rejection response cells included in the method for producing allogeneic soft tissue implants according to an embodiment of the present invention.
  • Figure 4 illustrates a method for implementing a pore forming step included in the method for producing allogeneic soft tissue implants according to an embodiment of the present invention.
  • Figure 5 shows the individual steps for implementing the autologous stem cell injection step included in the method for producing allogeneic soft tissue implants according to an embodiment of the present invention.
  • Figure 6 shows the step of providing a physical stimulus in the culture of allogeneic soft tissue support included in the method for producing allogeneic soft tissue implants according to an embodiment of the present invention.
  • Figure 7 shows the tensile strength measurement results before and after the clearant process treatment of allogeneic tibial tendon according to an embodiment of the present invention.
  • Figure 8 shows the results of the measurement of tensile strength following irradiation after the normal irradiation and clearant process treatment of the tibia tendon of humans and pigs according to an embodiment of the present invention.
  • Figure 9 shows a scanning electron microscope (SEM) photograph of the surface and cross-section of the allogeneic soft tissue support before and after the step of removing the immune rejection cells included in the method for producing allogeneic soft tissue implants according to an embodiment of the present invention.
  • Figure 10 shows a hematoxylin & eosin staining picture of the cross section of the allogeneic soft tissue support before and after the step of removing the immune rejection cells included in the method for producing allogeneic soft tissue implants according to an embodiment of the present invention.
  • Figure 11 shows the results of measuring the DNA residual amount of pig tibia tendon before and after decellularization according to an embodiment of the present invention.
  • stem cells carrying collagen gel 0.5%) and (B) stem cells carrying collagen gel (1.0) according to the concentration of stem cells 1x10 5 , 2x10 5 and 5x10 5 cells / cm 2 according to an embodiment of the present invention. %) Results of measurement of cell proliferation rates on the first, third and seventh days of culture are shown.
  • FIG. 13 shows the results of real time PCR analysis of stem cell-supported gel-grafted tibial tendon before and after physical stimulation in a bioincubator according to one embodiment of the present invention.
  • Figure 14 shows the results of GAG content analysis of the stem cell-supported gel transplant group subjected to physical stimulation in the incubator according to an embodiment of the present invention.
  • Figure 15 shows the results of collagen content analysis of the stem cell-supported gel transplant group subjected to physical stimulation in the incubator according to an embodiment of the present invention.
  • FIG. 1 illustrates the individual steps for implementing a method for producing a homologous soft tissue scaffold in accordance with one embodiment of the present invention.
  • Method for producing allogeneic soft tissue implant comprises the steps of obtaining (100) an allogeneic soft tissue support obtained from a human or animal body; Washing and sterilizing the obtained allogeneic soft tissue support (200); Treating the washed and sterilized allogeneic soft tissue support with an enzyme solution to remove immune rejection cells (300); Forming one or more pores in the allogeneic soft tissue support from which the immune rejection cells have been removed (400); Injecting an autologous stem cell of the user into the formed pore (500); And culturing the allogeneic soft tissue support infused with the autologous stem cells (600).
  • the method for preparing allogeneic soft tissue implants includes a step 100 of obtaining an allogeneic soft tissue support obtained from a human or animal body.
  • the soft tissue generally refers to tissues that connect, support, or surround organs or other structures of the body as tissues in the body, not bone, and include, for example, ligaments, tendons, Achilles tendons, tibiaris tendons, and the like. There is this.
  • the soft tissue is mainly distributed in the site supporting or maintaining the weight of the body, and serves to support and maintain repetitive movement, for example bending, twisting, rotation, and the like.
  • the soft tissue homograft refers to an implant including soft tissue that can be mutually transplanted between the same individual or between medically recognized homologous individuals.
  • the allogeneic soft tissue support refers to a support that is directly removed from its body or is a structure of the body obtained separately from a medically recognized homogenous individual and replaced in the treatment site. In addition, the allogeneic soft tissue support may be legally collected from a donated body.
  • the allogeneic soft tissue support may be a structure itself separated from the body, but may be combined with other body structures for proper implantation.
  • the allogeneic soft tissue support may be the ligament itself, but may also be a structure associated with one or more bone fragments, such as a bone fragment-ligament complex structure or a bone fragment-ligament-bone fragment complex structure.
  • the allogeneic soft tissue support may be implemented in various sizes and / or shapes according to the body portion to be implanted.
  • the method for preparing allogeneic soft tissue implants includes a step 200 of washing and sterilizing the obtained allogeneic soft tissue support.
  • the washing and sterilizing step 200 according to the present invention may be implemented in various physical or chemical methods, respectively, but it is preferable to simultaneously apply the chemical method as well as the physical method.
  • the washing and sterilizing step 200 may be implemented in various methods for physically and / or chemically washing and sterilizing the obtained homogenous soft tissue support. Details thereof will be described below with reference to FIG. 2.
  • the method for producing allogeneic soft tissue implant according to an embodiment of the present invention as a third step, the step of removing the immune rejection cells by treating the washed and sterilized homogenous soft tissue support with an enzyme solution (300) It includes. Removing the immune rejection cells is implemented by treating an enzyme solution.
  • the enzyme solution is used to remove intrinsic cells present in the human or animal body prior to transplantation from which the allogeneic soft tissue support has been obtained. Intrinsic cells present in the body of a human or animal prior to the transplant may act as antigens to the immune system of another human or animal body. In this case, various immune reactions are induced in the transplanted body, and unexpected side effects may occur due to muscle pain or atrophy in the course of the immune response.
  • the method for preparing allogeneic soft tissue implants includes a step 400 as a fourth step, forming one or more pores on the allogeneic soft tissue support from which the immune rejection-reactive cells have been removed. .
  • the pore is a portion on which autologous stem cells to be described below are carried.
  • the pore is formed by applying a microneedle to the allogeneic soft tissue support.
  • the surface of the homogeneous soft tissue support 10 from which the immune rejection cells have been removed by the third step is stimulated by the microneedle 420 (eg, M100SWBL model, Korea). Is formed.
  • the size (diameter) of the pore 410 may be selected from the range of about 1 to about 1000 micrometers ( ⁇ m), it is preferably formed of a size (diameter) of about 10 micrometers ( ⁇ m).
  • the number of the pores 410 is not particularly limited, but it is preferable that as many pores as possible be formed in the surface area of the allogeneic soft tissue support 10.
  • a substance for appropriately anchoring the autologous stem cells may be further introduced before or after the autologous stem cells to be described below are introduced.
  • the additionally introduced material may include growth factors and the like.
  • the method for preparing allogeneic soft tissue implants includes a step 500 of injecting autologous stem cells of a user into the formed pores.
  • the autologous stem cells refer to stem cells present in the body to be transplanted.
  • the stem cell is generally defined as a cell having the possibility of being differentiated into various kinds of cells constituting the body, such as nerves, blood, cartilage, etc., if necessary to remain undifferentiated into specific cells.
  • the stem cells are currently used in a variety of regenerative medicine fields, and are sequentially replacing or supplementing classical drug treatments or surgical treatments.
  • adult stem cells are cells that constitute specific tissues in the body, and they provide a minimum amount of cells that exist in the body in small amounts and maintain a normal state when the body is externally affected. Do this.
  • the adult stem cells have an advantage of being capable of autologous transplantation because the immune rejection response is insignificant or nonexistent in the field of transplantation for organ regeneration.
  • the autologous stem cells according to the present invention may use various stem cells of the body to be transplanted, but it is preferable to use autologous stem cells obtained from bone marrow of the body to be transplanted.
  • the autologous stem cells are injected into the pores formed in the fourth step and incubated under appropriate conditions with the allogeneic soft tissue support in the culturing step to be described below to be an allogeneic soft tissue implant.
  • a method for preparing allogeneic soft tissue implants includes a step (600) of culturing allogeneic soft tissue support into which autologous stem cells are injected.
  • the culturing step 600 is implemented under appropriate conditions so that the allogeneic soft tissue scaffold into which the autologous stem cells are injected can be an allogeneic soft tissue implant with optimal transplant conditions.
  • the appropriate conditions may be considered appropriate temperature, proper pressure, suitable period.
  • the culturing step 600 may further include providing a physical stimulus to the allogeneic soft tissue support. The physical stimulus providing step will be described in detail later with reference to FIG. 6.
  • Figure 2 shows the individual steps for implementing the scaffold cleaning and sterilization step included in the method for producing a homogeneous soft tissue scaffold according to an embodiment of the present invention.
  • the washing and sterilizing the obtained allogeneic soft tissue support 200 may include treating hydrogen peroxide with the obtained allogeneic soft tissue support and applying ultrasonic wave 210. Treating the allogeneic soft tissue support with a radioprotectant and irradiating the radiation 220.
  • the hydrogen peroxide treatment and ultrasonic application step 200 may contribute to the strengthening of physical properties, such as tensile strength (tensile strength) of the homogeneous soft tissue support in a particular region.
  • tensile strength tensile strength
  • ligaments obtained from the human body are treated with hydrogen peroxide in the concentration range of about 0.1 to about 5.0 percent (%) and ultrasonic waves in the power range of about 120 to about 400 watts (W) result in increased tensile strength of the ligaments. Appeared.
  • the treated hydrogen peroxide has a concentration selected from a concentration range of 0.1 to 5.0 percent (%) and the applied ultrasonic waves are 120 It is preferred to have an output selected from the range of from 400 watts (W), in particular the hydrogen peroxide treated has a concentration range of 0.5% percent (%) and more preferably the applied ultrasound has a 120 watt (W) output.
  • W watts
  • the sterilization effect may be maximized by treating the obtained allogeneic soft tissue support with a radiation protective material and irradiating the radiation 220.
  • the homogeneous soft tissue scaffold may be sterilized by irradiation with radiation, but by using the radiation protective material, adverse effects may be minimized by radiation.
  • the radiation protective material to be treated may vary, but is preferably cobalt 60 (Co 60 ), the radiation to be irradiated may be selected within a variety of ranges, preferably selected from the range of 12 to 50 klogy (kGy) It is a gamma ray.
  • treating the obtained homogenous soft tissue support with hydrogen peroxide, applying ultrasonic wave 210 and treating the radiation protective material, and radiation Examining step 220 may be applied individually as well as applied separately.
  • the hydrogen homogenate is treated to the obtained homologous soft tissue support, and after applying the ultrasound (210), thereafter, the radiation protective material is treated. It is preferable to apply the step 220 of irradiating radiation.
  • Figure 3 shows the individual steps for implementing the step of removing the immune rejection cells included in the method for producing a homologous soft tissue scaffold according to an embodiment of the present invention.
  • the step of removing the immune rejection cells 300 includes treating the enzyme solution 310 and / or washing with water saline 320.
  • the enzyme solution may include various enzymes for removing immune rejection cells, but preferably includes trypsin, collagenase and protease.
  • the enzyme solution may contain about 0.25% trypsin (0.02% EDTA, invitrogen Corp., USA), about 3 mg collagenase A (0.15 U / mg, Sigma aldrich, USA) and about 15 mg protease (4.8 U / mg, Sigma aldrich, USA), and the enzyme solution may be stirred onto the homogenous soft tissue support with a solution of about 40 ml at about 37 ° C.
  • the step of removing the immune rejection cells (300) further comprises the step of washing (320) an enzyme solution to the washed and sterilized homologous soft tissue scaffold and additionally washing (320) with physiological saline. can do.
  • the step (300) of removing the immune rejection cells is treated with an enzyme solution in the washed and sterilized homogenous soft tissue support (310), washed with physiological saline (320), followed by osmotic treatment with distilled water ( 330) may further comprise the step of washing.
  • the physiological saline since the physiological saline is to be used for washing, the content, salinity, etc. of the components of the physiological saline are not particularly limited.
  • the physiological saline may be treated with the solution of about 40 ml at about 4 ° C. and about 120 rpm on the allogeneic soft tissue scaffold, and may be treated continuously for about 12 hours after three repeated treatments of about 1 minute. .
  • osmotic treatment with distilled water may be performed, and for example, ultrasonication may be performed at an output of about 240 watts (W) for about 5 minutes (using ultra sonicator). , Biofree, South Korea).
  • Figure 9a is a scanning electron microscope (SEM) photograph of the surface and cross-section of the allogeneic soft tissue support before removing the immune rejection cells included in the method for producing allogeneic soft tissue implants according to an embodiment of the present invention
  • Figure 9b is SEM (scanning electron microscope) photograph of the surface and cross-section of the allogeneic soft tissue support after the step of removing the immune rejection cells included in the method for producing an allogeneic soft tissue implant according to an embodiment of the present invention.
  • SEM scanning electron microscope
  • Figure 10a is a hematoxylin & eosin stained photo of the cross-section of the allogeneic soft tissue support before the step of removing the immune rejection cells included in the method for producing allogeneic soft tissue implants according to an embodiment of the present invention
  • Figure 10b is It is a hematoxylin & eosin staining picture of the cross section of the allogeneic soft tissue support after the step of removing the immune rejection cells included in the method for producing a homogenous soft tissue implant according to an embodiment. 9B and 10B, it can be seen that the cells of the allogeneic soft tissue graft were removed by removing the immune rejection-responsive cells.
  • Figure 5 shows the individual steps for implementing the autologous stem cell injection step included in the method for producing allogeneic soft tissue support according to an embodiment of the present invention.
  • injecting autologous stem cells of the user into the formed pores 500 further includes injecting autologous stem cell support promoting material 510 before or simultaneously with the injection of autologous stem cells 520. It may include.
  • the autologous stem cell supporting promoter is concentrated and lyophilized to a high concentration of a liquid containing water-soluble proteins such as collagen, gelatin, BMP2, BMP4, BMP7, PDGF, TGF-beta at high concentration, and then rehydrated to give a constant viscosity.
  • a material reconstituted with a chewy liquid autologous stem cells injected into the pore can be induced to properly settle in the allogeneic soft tissue support.
  • the autologous stem cell supporting promoter is purely manufactured from bones of the human body without the addition of external substances, and therefore, there is no immune rejection response to the body to be transplanted, and is bioactive. Therefore, the additional injection of the autologous stem cell support promoting material, the autologous stem cells show significant bone induction ability and bone conduction ability in the allogeneic soft tissue support, and easily adapts to the body by maintaining viscosity by collagen components.
  • Figure 6 shows the step of providing a physical stimulus in the culture of allogeneic soft tissue support included in the method for producing allogeneic soft tissue support according to an embodiment of the present invention.
  • culturing the allogeneic soft tissue support into which the autologous stem cells are injected further includes providing a physical stimulus to the allogeneic soft tissue support (610).
  • the soft tissues present in the body are distributed to areas that support or maintain the weight of the body and serve to support and maintain repetitive movements such as bending, twisting, and rotation. Enemies are stimulated.
  • the alloplastic soft tissue graft can be efficiently differentiated within the implanted body and easily integrated into the implanted body's native tissue.
  • the physical stimulus may be variously implemented to be similar to the stimulus applied to the soft tissue present in the body, but preferably in relation to the motor stimulus, it may be a torsional and / or tensile stimulus.
  • the provision of the physical stimulus 610 provides a tensile stimulus 611 at both ends of the allogeneic soft tissue support, or the amount of the allogeneic soft tissue support based on the longitudinal axis of the allogeneic soft tissue support.
  • the physical stimulus including the tensile stimulus 611 and / or the torsional stimulus 612 may be provided at various frequencies, but is preferably provided at a frequency of less than once per second.
  • the culturing step 600 including the physical stimulus providing step 610 may be performed for various periods, but may be preferably performed within a range of about 7 days or less.
  • the ligament tissue of the pig was obtained and tested to determine whether the allogeneic soft tissue support can have a strength similar to that of the normal tissue of the human body.
  • About 10% tensile stimulation (pulling both ends to about 5%) and about 90 degrees torsional stimulation (rotating one end about 45 degrees clockwise, the other end about 45 degrees counterclockwise) ) was given once per second for 1 day, 4 days and 7 days to determine the respective intensity.
  • the maximum load value (Newton (N)) was about 354.9 Newtons (N) for the control group that provided no stimulation, about 380.8 Newtons (N) for the experimental group that provided stimulation for 1 day. It was measured about 418.6 Newtons (N) for the experimental group that provided stimulation for one day and about 818.1 Newtons (N) for the experimental group that provided stimulation for 7 days.
  • the results showed that porcine ligament tissues that were continuously provided with tensile and torsional stimuli could reach values close to the strengths similar to normal tissues of the human body.
  • FIG. 13 shows the results of real time PCR analysis of stem cell-supported gel-grafted tibial tendon before and after physical stimulation in a bioincubator according to one embodiment of the present invention.
  • Figure 14 shows the results of GAG content analysis of the stem cell-supported gel transplant group subjected to physical stimulation in the incubator according to an embodiment of the present invention.
  • the analysis of the content of GAG secreted in the culture medium of the stem cell-supported gel transplant group (5x10 5 cell / cm 2 ) inoculated on the surface of the pig tibia tendon in the incubator and the group without physical stimulation Proceeded. After culturing the cultures for 1, 3, 7 days, the absorbance was measured at 656 nm using a GAG assay kit. The linear regression of the standard chondroitin-4-sulfate curve yielded 99.9%.
  • the group with physical stimulation showed higher GAG content than the group without physical stimulation, and the group without physical stimulation showed 14.187 ⁇ 0.080 ⁇ g / ⁇ l on day 1, 25.542 ⁇ 0.599 ⁇ g / ⁇ l on day 3, and 36.346 ⁇ 0.843 ⁇ g on day 7 / ⁇ l, physical stimulation was 14.986 ⁇ 0.765 ⁇ g / ⁇ l on day 1, 33.298 ⁇ 0.936 ⁇ g / ⁇ l on day 3, and 44.791 ⁇ 0.087 ⁇ g / ⁇ l on day 7.
  • the stem cell supported gel transplantation group cultured with physical stimulation for 7 days was increased by about 23% compared to the group without stimulation.
  • Figure 15 shows the results of collagen content analysis of the stem cell-supported gel transplant group subjected to physical stimulation in the incubator according to an embodiment of the present invention.
  • the content of collagen secreted in the culture medium of the stem cell supporting gel transplanted group (5x10 5 cell / cm 2 ) inoculated on the surface of swine tibial tendon in the incubator and the group without physical stimulation Proceeded. After culturing the cultures for 1, 3, 7 days, the absorbance was measured at 550 nm using a collagen assay kit. The linear regression value of the standard collagen curve was 99.9%.
  • the group with physical stimulation showed higher collagen content than the group without physical stimulation, and the group without physical stimulation showed 0.769 ⁇ 0.012 ⁇ g / ⁇ l on day 1, 0.949 ⁇ 0.052 ⁇ g / ⁇ l on day 3, and 1.382 ⁇ 0.028 ⁇ g on day 7 / ⁇ l, physical stimulation was 0.811 ⁇ 0.106 ⁇ g / ⁇ l on day 1, 1.218 ⁇ 0.072 ⁇ g / ⁇ l on day 3, 1.803 ⁇ 0.065 ⁇ g / ⁇ l on day 7.
  • the stem cell supported gel transplantation group cultured with physical stimulation for 7 days was increased by about 30% compared to the group without stimulation.
  • pig patella and tibia were obtained.
  • the obtained soft tissue support was sterilized using Clearant Process® (US2005 / 6,908,591B2), which was subjected to chemical agents to the tissue and then subjected to high dose gamma-ray sterilization.
  • Clearant Process® US2005 / 6,908,591B2
  • a radiation protection solution was prepared by mixing 16.2% propylene glycol, 22.1% DMSO, 2.7% mannitol and 3.8% tetrahalose and 60.0% purified water.
  • the homogenous gun to be sterilized was placed in a sterile pouch, a solution prepared in proportion to the weight of the tissue, and then subjected to thermal bonding packaging.
  • the tissue moisture is removed with a sterile towel (20-30% moisture residue), and the lyophilized tissue is freeze-dried and the cryopreserved tissue is packaged. Frozen storage.
  • the total time required for drug treatment of tissues was 25 hours, followed by an additional 24 hours for tissues for lyophilized storage.
  • sterilization was performed in gamma rays (0, 25, 50 KGy). After treatment with clearant, the bones of the irradiated bones were found to have an increased tensile strength compared to the case of normal irradiation.
  • Decellularization was performed to remove tissue antigens for cellular components from the washed and sterilized soft tissue scaffolds.
  • the decellularization process was performed using enzyme solution treatment, water treatment and osmotic treatment, and the specific method is as follows.
  • the frozen pork tendon tendon (12 cm long, 1 cm wide) was taken out of a -70 ° C. deep freezer, and then placed in sterile distilled water and thawed at 37 ° C. in a water bath (DongA, KOREA) for 30 minutes.
  • Enzyme cocktail solution after thawing [500 ml of 0.25% trypsin (T4049, SIGMA, USA), 37.5 ml of collagenase A (C0130, SIGMA, USA), protease (P4630, SIGMA, USA) 187.5] 500 Ten tibial tendons were placed in a 1,000 ml beaker containing ml, and stirred for 4 hours at 120 rpm and 37 ° C in a shaker incubator (NB-205V, N-BIOTEK, KOREA).
  • the enzyme cocktail solution was removed and 500 ml of saline was added. Thereafter, the mixture was washed by stirring in a shaker incubator at 120 rpm and 4 ° C. for 1 hour. This process was repeated three times, and the last washing process was carried out for 12 hours.
  • saline was removed and 500 ml of sterile distilled water was added. Then, it treated for 5 minutes on 240w conditions using the ultrasonic apparatus. Sterile distilled water was removed, and 500 ml of physiological saline was added and treated for 5 minutes under 240w conditions using an ultrasonic apparatus.
  • the dehydrated tissue was placed in a humidity controller (Dry keeper, Sanplatec, JAPAN) and dried at room temperature.
  • the dried tissue was fixed to the specimen holder using double-sided tape, and then coated with 200 ⁇ m thick platinum using a plasma sputter. Subsequently, the appearance morphology of the tissue was observed 30 and 300 times using a low magnification scanning electron microscope.
  • Tissue staining pretreatment was performed as follows. Tissue process (STP 120, Thermo scientific, USA) equipment was used under the following program conditions.
  • the program value (Programed value) One Formalin 12:00 60 One 2 Formalin 12:00 60 One 3 Alcohol 70% 01:30 60 One 4 Alcohol 80% 01:30 60 One 5 Alcohol 95% 01:30 60 One 6 Alcohol 100% 01:00 60 One 7 Alcohol 100% 01:00 60 One 8 Alcohol 100% 01:00 60 One 9 Xylene 01:30 60 One 10 Xylene 01:30 60 One 11 Paraffin 02:00 60 One 12 Paraffin 02:00 60 One
  • tissue block specimens In order to fabricate tissue block specimens, the tissues infiltrated with paraffin solution were placed on a hot base of a tissue embedding system (Histocentre 3, Thermo, USA), and paraffin was first applied. The tissue was put in slightly swollen state. After that, paraffin was filled up. After placing the paraffin block on a cold plate of the tissue embedding system and hardening for 10 minutes, the mold base was removed and stored at room temperature.
  • tissue embedding system Histocentre 3, Thermo, USA
  • tissue slicer Finess met, Thermo, USA
  • microtome blade were used to trim to 10 ⁇ m and tissue sections were made to 4 ⁇ m thick. Float in a container containing tap water, and then floated with a slide in a preheated water bath, the tissue sections were attached to each slide and dried at 60 °C.
  • the tissue slide was soaked three times for 10 minutes in a glass stain jar containing xylene. To perform the water procedure, the tissue slides were soaked for 5 minutes in the first glass staining vessel containing 99.9% alcohol. Tissue slides were transferred to a second glass staining vessel containing 99.9% alcohol for 5 minutes. Tissue slides were transferred to a second glass staining container containing 95% alcohol for 3 minutes. Tissue slides were transferred to a second glass staining vessel containing 80% alcohol for 3 minutes. Tissue slides were transferred to a second glass staining vessel containing 70% alcohol for 3 minutes. Transfer to running tap water for 1 minute.
  • the solution was transferred to Harris hematoxylin for 10 minutes and then soaked in running tap water for 3 minutes.
  • Porcine tibial tendon was frozen at ⁇ 20 ° C. for 24 hours before and after decellularization and water was removed using a lyophilizer. Thereafter, residual DNA was examined to confirm the amount of residual cells.
  • DNA quantification was measured using the DNEasy kit (69506-250, Qiagen, USA). The specific method is as follows. Before and after decellularization, 25 mg of lyophilized tissue was placed in a 1.5 ml microcentrifuge tube, followed by addition of 180 ⁇ l of ATL buffer. 20 ⁇ l of Proteinase K was added to the tube, and after stirring, the mixture was allowed to stand at 55 ° C. in a heating block until the tissue was completely decomposed (the stirring was performed every 2 hours). After confirming the decomposition, the mixture was mixed using a vortex mixer for 15 seconds. 200 ⁇ l of AL buffer was added to the tube, mixed using a vortex mixer, and allowed to stand for 10 minutes at 70 ° C. in a thermal block.
  • ⁇ l of 99.9% alcohol was added to the tube and mixed using a vortex mixer.
  • the mixed samples were placed in a DNeasy spin column coupled to a 2 ml collection tube and centrifuged (8,000 rpm) for 1 minute to remove the filtrate from the collection tube.
  • 500 ⁇ l of AW1 buffer was added to a DNeasy spin column coupled to a 2 ml collection tube, followed by centrifugation (8,000 rpm) for 1 minute to remove the filtrate from the collection tube.
  • 500 ⁇ l of AW2 buffer was added to a DNeasy spin column coupled to a 2 ml collection tube and centrifuged (17,000 rpm) for 3 minutes to allow the DNeasy membrane to dry completely.
  • cell supporting promoters were prepared in the following manner.
  • Collagen gel extracted from pig skin by treatment with pepsin in a sterile process system was purchased from Bioland Co., Ltd., and as a result of checking the test report, more than 99% of collagen was in type I form.
  • Reconstitution buffer for gelling collagen solution 2.2% NaHCO in 0.05 N NaOH aqueous solution 3 (90421-C, SAFC Bioscience, USA) and 200 mM HEPES (H4034, SIGMA, USA) were dissolved and the total volume was adjusted to 100 ml. Filter sterilization using a 0.2 ⁇ m syringe filter (16534, Satorius stedim, USA), and refrigerated 4 °C until use. PBS solution Filter sterilization using a 0.2 ⁇ m syringe filter, and refrigerated at 4 °C until use.
  • the final collagen solution was prepared by mixing an acid-soluble collagen gel solution: PBS (10 ⁇ concentrated solution): reconstitution buffer solution 8: 1: 1. When mixed, it was placed in a styrofoam box containing ice to prevent rapid gelation of the neutral collagen gel.
  • Human bone marrow-derived mesenchymal stem cells collected after centrifugation were divided into 1x10 5 , 2x10 5, and 5x10 5 cell / cm 2 groups, respectively, and slowly mixed with the neutral collagen gel prepared above to prevent bubbles from being generated.
  • 150 ⁇ l of the cell-supported collagen gel was dispensed into 48 well culture plate dishes and allowed to stand for 1 hour in an incubator (NB-203XLSP, N-Biotek, KOREA) at 37 ° C. and 5% CO 2 .
  • the medium used for culturing the stem cell loaded collagen gel was removed by vacuum suction method, and 200 ⁇ l of each medium mixed with EZ Cytox reagent was added to each well of a 48 well culture plate dish. Incubation was performed at 37 ° C. in a 5% CO 2 incubator for 1 hour. Absorbance was measured at 450 nm wavelength using an ELISA reader (1420, PerkinElmer, USA) (see FIG. 12).
  • the physical strength propensity of stem cell-supported gel grafts inoculated into tibial tendon was compared and analyzed according to physical stimulation conditions and time settings in the incubator. In other words, to determine the effect on the strength of the implant was carried out in the following way. Specimens were prepared in a size of 12 ⁇ 1 cm 2 (horizontal ⁇ vertical) and then mounted in the chamber. In the experimental group, 10% (2 mm each end) tension stimulation, 90 ° (torsion stimulation 45 ° in the clockwise and counterclockwise directions, respectively) and frequency of 1 Hz on the computer program. The experiment was conducted by giving a physical stimulus in the incubator for 1, 3, and 7 days under conditions (Table 3), and the control group was incubated for 7 days without physical stimulation in the incubator chamber.
  • RLT Plus buffer Into the collected tissue 30 mg 600 ⁇ l of RLT Plus buffer was homogenized. The supernatant was carefully removed by centrifugation and pipetting at maximum RPM for 3 minutes.
  • the gDNA removal spin column was placed in a 2 ml collection tube and homogenized to add dissolved tissue. The column was removed by centrifugation at 10,000 x g for 30 seconds, leaving the filtered solution. 600 ⁇ l of 70% ethanol was placed in a collection tube and pipetted to mix.
  • RNA extract Raise the RNeasy spin column in a new 2 ml collection tube and add 1,000 ⁇ l of mixture.
  • the lid was closed and centrifuged at 10,000 xg for 15 seconds and the filtrate was removed.
  • 700 ⁇ l of RW1 buffer was placed on an RNeasy spin column, centrifuged at 10,000 ⁇ g for 15 seconds, and the filtrate was removed.
  • 500 ⁇ l of RPE buffer was added to an RNeasy spin column, centrifuged at 10,000 ⁇ g for 15 seconds, and the filtrate was removed.
  • 500 ⁇ l of RPE buffer was added to an RNeasy spin column, centrifuged at 10,000 ⁇ g for 2 minutes, and the filtrate was removed.
  • the RNeasy spin column was placed in a new 1.5 mL collection tube, 50 ⁇ l of RNase-free water was added to the spin column membrane, and centrifuged at 10,000 ⁇ g for 1 minute to obtain RNA extract.
  • SYBR ® Premix Ex Taq (RR420Q, TaKaRa, JAPAN) for the Real time PCR analysis was performed in the following way. 12.5 ⁇ l of SYBR ® Premix Ex Taq (1x), 0.5 ⁇ l of 0.2 uM PCR Forward Primer, 0.5 ⁇ l of 0.2 uM PCR Reverse Primer, 2.0 ⁇ l of cDNA synthesis sample and 9.5 ⁇ l of DW water were mixed to adjust the total volume to 25 ⁇ l.
  • GAG Glycosaminoglycan
  • Sulfated Glycosaminoglycan (0-5 ⁇ g) was used as a standard reagent in a microcentrifuge tube, and the final volume was adjusted to 100 100 with purified water.
  • the medium of the stem cell supporting gel transplant group cultured for 1, 3 and 7 days was placed in a microcentrifuge tube, and the final volume was adjusted to 100 ⁇ l.
  • 1.0 ml of glycosaminoglycan Dye Reagent was added to all the prepared tubes, followed by mixing for 30 minutes using a vortex mixer. It was then centrifuged at 10,000 xg for 10 minutes. After centrifugation, the supernatant was removed and the tube was flipped over to remove the solution at the bottom or wall of the tube, and absorbent paper was used.
  • the collagen content was analyzed using the collagen assay kit. The detailed method is as follows. Collagen solution (0-50 ⁇ g) was used as a standard reagent in a microcentrifuge tube, and the final volume was adjusted to 100 ⁇ l with purified water.
  • the medium of the stem cell supporting gel transplant group cultured for 1, 3 and 7 days was placed in a microcentrifuge tube, and the final volume was adjusted to 100 ⁇ l.

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Abstract

Cette invention concerne un procédé de fabrication d'une greffe de tissu mou allogène dans laquelle une cellule souche autologue a été injectée en fonction d'une base de tissu mou allogène dérivée d'un organisme allogène, la présente invention permettant de fabriquer une greffe de tissu mou allogène pour remplacer efficacement un tissu mou endommagé du corps humain.
PCT/KR2012/001443 2011-02-28 2012-02-24 Procédé de production d'une greffe de tissu mou allogène contenant une cellule souche autologue WO2012118305A2 (fr)

Priority Applications (1)

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US14/002,104 US9511172B2 (en) 2011-02-28 2012-02-24 Method for manufacturing allogeneic soft-tissue transplant having autologous stem cell transplanted therein

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KR10-2011-0018216 2011-02-28
KR20110018216 2011-02-28
KR10-2012-0018650 2012-02-23
KR1020120018650A KR101352378B1 (ko) 2011-02-28 2012-02-23 자가 줄기 세포가 이식된 동종 연조직 이식체를 제조하는 방법

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100365573B1 (ko) * 1994-03-14 2004-08-25 크라이어라이프, 인크. 이식용처리조직및이의제조방법
KR100839875B1 (ko) * 2006-05-25 2008-06-19 코아스템(주) 관절연골 재생용 지지체
KR100920951B1 (ko) * 2007-08-01 2009-10-09 한양대학교 산학협력단 미분화 인간 지방조직 유래 줄기세포를 포함하는 골 재생용복합 지지체
KR20100005105A (ko) * 2007-05-06 2010-01-13 민병현 세포외 기질 지지체를 이용한 연골질환 치료용 조성물

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100365573B1 (ko) * 1994-03-14 2004-08-25 크라이어라이프, 인크. 이식용처리조직및이의제조방법
KR100839875B1 (ko) * 2006-05-25 2008-06-19 코아스템(주) 관절연골 재생용 지지체
KR20100005105A (ko) * 2007-05-06 2010-01-13 민병현 세포외 기질 지지체를 이용한 연골질환 치료용 조성물
KR100920951B1 (ko) * 2007-08-01 2009-10-09 한양대학교 산학협력단 미분화 인간 지방조직 유래 줄기세포를 포함하는 골 재생용복합 지지체

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