JP2018145169A - Gelling method for gelling platelet-rich plasma, and kit used for the gelling method - Google Patents

Abstract

[PROBLEMS] To gel platelet-rich plasma using human-derived human tissue kallikrein to enhance safety in wound healing and regenerative therapy for bone regeneration. A method for gelling platelet-rich plasma includes adding human tissue kallikrein to platelet-rich plasma and then placing the mixture in a plastic container for gelation. Human tissue kallikrein is salivary gland-derived glandular kallikrein derived from saliva, and is sterilized. Sterilization of saliva may be accomplished by filtration through a filter having a bore diameter of 0.45 μm or less. The liquid volume ratio of salivary gland-derived glandular kallikrein added to PRP is 0.03 or more. [Selection diagram] Fig. 1

Description

  The present embodiment relates to a method for gelling platelet-rich plasma and a kit used for gelling platelet-rich plasma used in the method.

  In recent years, regenerative treatment using platelet rich plasma (hereinafter referred to as “PRP”) as a treatment method for accelerating wound healing and bone regeneration using its own blood components in the dental field and orthopedic field. Is spreading.

  PRP is plasma with a higher platelet concentration than whole blood and is used to promote wound healing. In addition, a block material mainly composed of hydroxyapatite or β-tricalcium phosphate (hereinafter referred to as “β-TCP”) as a material for repairing a bone defect caused by trauma such as fracture or removal of a bone tumor. Alternatively, granular bone filling materials are widely used, but PRP is also used as a composite with bone filling materials by being mixed with such bone filling materials.

  The above-mentioned PRP is usually obtained by centrifuging collected blood, and is often used after gelation into a desired shape. When gelling PRP, a gelling material is usually used. Patent Document 1 describes calcium chloride because thrombin and an anticoagulant are used as a gelling material used when gelling a mixture of PRP and β-TCP.

International Publication No. 2002/040071

  However, thrombin is generally derived from bovine or human, and the use of calcium chloride or the like raises safety doubts.

  As a result of repeated studies to solve the above problems, it was found that PRP can be gelled using human-derived human tissue kallikrein.

In the PRP gelation method of this embodiment, PRP and human tissue kallikrein are mixed in a plastic container to gel the PRP.
Human tissue kallikrein is a glandular kallikrein derived from the salivary gland and is sterilized. A method for sterilizing saliva includes filtering with a filter having a bore diameter of 0.45 μm or less.

The liquid volume ratio of the glandular kallikrein derived from salivary glands to be added to PRP is 0.03 or more.
Add glandular kallikrein after adding bone prosthetic material to PRP. The bone grafting material contains hydroxyapatite and β-tricalcium phosphate.

  The kit used in the PRP gelation method of the present embodiment includes a plastic container for containing platelet-rich plasma and a filter having a bore diameter of 0.45 μm or less. Further, a bone prosthetic material may be provided.

  According to this embodiment, PRP can be gelled with salivary gland-derived glandular kallikrein collected from a subject.

It is a flowchart which shows the process of the gelatinization method of the platelet rich plasma which concerns on an Example. (A) A photograph in which blood is separated into a red blood cell layer, a buffy coat and a PRP in a test tube. (B) It is the photograph in which the cotton roll in a syringe which attached the filter was put. (C) It is the photograph of the state which is going to put sterilized saliva in the state in which the bone grafting material and PRP were put in the plastic petri dish. (A) It is the graph which showed time for PRP to gelatinize. (B) A photograph showing a drop of sterilized saliva. (A) is a photograph of a test tube showing PRP in a liquid state without adding PRP gelled with glandular kallikrein derived from salivary glands. (B) is a photograph showing a PRP gelled in the plastic test tube 16 (indicated by (+) in the figure) and a PRP that remains liquid in the plastic test tube 16 (indicated by (-) in the figure). .

As a result of intensive studies on whether or not PRP can be gelled with a substance derived from human self, the present inventor has found that PRP can be gelled in a short time using tissue kallikrein derived from human self. Kallikrein is divided into plasma kallikrein and tissue kallikrein.
Although plasma kallikrein is known to be involved in blood clotting, the involvement of tissue kallikrein is not clear. Tissue kallikrein includes salivary gland kallikrein or urinary kallikrein. In this example, salivary glandular kallikrein was used on the assumption that it was applied to regenerative medicine for oral tissues. Saliva can be sterilized using a sterile filtration filter attached to the tip of the syringe barrel. The sterilized saliva contains salivary glandular kallikrein.

  Gelation starts when human-derived tissue kallikrein is added to PRP obtained by centrifuging venous blood collected without an anticoagulant in a plastic tube. Although gelation of PRP occurs even with the same kind of tissue kallikrein, it is preferable to use it by matching with PRP from the subject, that is, own tissue kallikrein because of the risk of infection.

  When human-derived tissue kallikrein is added to PRP, the gelation time varies depending on the amount of tissue kallikrein. Note that gelation of PRP with human tissue kallikrein is considered to be the use of an intrinsic blood coagulation reaction, so the time required for gelation is 10 minutes plus or minus 2 minutes at 37 degrees Celsius.

  Hereinafter, the method for gelling platelet-rich plasma according to the present example will be described with reference to FIG. As shown in FIG. 1, in step S11, the subject's blood is first collected in the gelation method of the present embodiment. Do not add anticoagulants such as sodium citrate to the collected blood.

  Next, in step S13, the collected venous blood is centrifuged in a plastic tube to prepare PRP. Specifically, blood collected without an anticoagulant is put into a plastic test tube with 10 ml of venous blood. The test tube 16 is separated into three layers of an upper layer 11, an interface layer 12, and a lower layer 13 by a centrifuge not shown. The separated state is shown in the photograph of FIG. The separated lower layer 13 is a red blood cell layer, the separated interface layer 12 is a buffy coat, and the separated upper layer is a PRP. Centrifugation conditions are 400 g and 10 minutes. Hematological properties such as platelets and white blood cell counts contained in PRP separated and prepared under these conditions have been clarified by the inventors. And PRP is extract | collected with the syringe barrel which attached the catalan needle (18G) not shown.

  In step S15 of FIG. 1, saliva is absorbed by a cotton roll (roll men) placed on the bottom of the subject, and total saliva is collected.

  In step S17, the collected whole saliva is sterilized by filtration through a filter. Specifically, a cotton roll containing whole saliva is inserted into the cylinder of the syringe barrel and pushed, and then a filter is attached to the barrel head of the syringe barrel. The whole saliva extruded from the cotton roll is sterilized by filtering through a filter. FIG. 2B shows a state in which the filter 40 is attached to the cylinder head of the syringe barrel, and the cotton roll 20 containing the whole saliva is contained in the cylinder 31.

  The filter 40 is a single-layer filter having a diameter of φ10 to 16 mm for use in a syringe barrel, and has a pore diameter of φ0.22 μm to φ0.45 μm. The filter 40 makes viable bacteria contained in the whole saliva zero.

The viable cell count test was performed when the saliva of five subjects was sterilized with a filter 40 having a bore diameter of φ0.45 μmA. The test results are as follows.
Viable count method: Mixed culture method Medium used: Tryptic soy agar medium (BBL)
Culture conditions: 37 degrees Celsius (2 days) + room temperature (5 days)

Thus, if the filter 40 having a bore diameter of φ0.45 μm is used, the viable cell count becomes zero.

  In step S21 of FIG. 1, 2 ml of PRP collected in step S13 is placed in a plastic container (plastic petri dish or plastic test tube). Then sterilized saliva is added to the PRP and mixed. And place at 37 degrees. Then, gelled PRP is obtained by the blood coagulation cascade reaction.

  The amount of mixing PRP and sterilized saliva will be described later.

  In step S23, a bone grafting material is added to the gelled PRP to produce a composite material. That is, before adding the sterilized saliva to the PRP in step S21, the bone grafting material 50 is added to produce a composite material. FIG. 2C is a photograph of a state in which sterilized saliva is about to be put in a state where the bone grafting material 50 is put in the plastic petri dish 18 and the PRP 11 is further put.

  Examples of the bone prosthetic material include hydroxyapatite, β-TCP and the like, and these may be used alone or in combination of two or more.

  The bone prosthetic material 50 is preferably in the form of granules having an average diameter of 3 mm or less, but an adaptive size depending on the place of use is known.

  In this embodiment, since gelation is completed in a plastic container (plastic petri dish or plastic test tube), the PRP is easily gelled into a desired shape by using a container corresponding to the shape of each bone defect. It can be made.

  As a constituent material of the plastic container, for example, a polypropylene resin, a polyethylene resin, a polycarbonate resin, a polystyrene resin, or the like can be used as long as it is approved for the practice of regenerative medicine.

  FIG. 3A is a graph showing the time for PRP of a woman whose subject is 33 years old to gel. The vertical axis of the graph is the time until gelation, and the horizontal axis is the amount of saliva. As shown in FIG. 3B, sterilized saliva 25 from the filter 40 was added drop by drop. Therefore, the horizontal axis indicates the case where 0 drops, 1 drop, 2 drops, 3 drops and 4 drops are added.

  FIG. 4A is a photograph showing a PRP gelled in the plastic test tube 16 (indicated by (+) in the figure) and a PRP that remains liquid in the plastic test tube 16 (indicated by (-) in the figure). It is. FIG. 4B is a photograph showing a PRP gelled in the plastic petri dish 18 (indicated by (+) in the figure) and a PRP remaining in a liquid state in the plastic petri dish 18 (indicated by (-) in the figure). is there. The amount of one drop of saliva 25 that came out of the filter 40 was 0.065 ml. Therefore, the sterilized saliva amount is 0.065 ml (1 drop), 0.130 ml (2 drops), 0.195 ml (3 drops), and 0.260 ml (4 drops).

  As described in step S21 of FIG. 1, 2 ml of PRP is put in the plastic tube 18, and sterilized saliva is put in and stirred. And the time until PRP gelled at 37 degrees Celsius was measured. PRP without saliva (0 drops) did not gel even after 20 minutes. If 1 drop of saliva is added, the PRP will gel in 17 minutes, 12.4 minutes if 2 drops, 10.6 minutes if 3 drops, 7.5 minutes if 4 drops are added. Turned into. That is, when the amount of saliva to be added was increased, the gelation time of PRP was shortened. As shown in FIG. 4 (a) or (b), the gelation was confirmed by tilting the PRP contained in the plastic test tube 16 or the plastic petri dish 18 and visually by the inventors.

  Since the gelation of PRP starts even at 0.065 ml / 2 ml, the sterilized saliva may be added to PRP at a liquid volume ratio of 0.03 ml / 2 ml. In general, the intrinsic blood coagulation time is 10 minutes plus or minus 2 minutes, so even if 0.260 ml (4 drops) or more is added, the gelation time is not significantly reduced. That is, it is considered that the gelation time is not shortened so much even if about 0.1 or more of sterilized saliva is added to the PRP liquid amount of 1. However, it is speculated that the amount of glandular kallikrein contained in the whole saliva to be collected may be influenced by physiological effects such as age, sex, and collection time period.

  The worker can easily gel RPR with glandular kallikrein collected from the subject using the above-described gelation method of gelling PRP. In this case, the plastic test tube 16 or the plastic petri dish 18 and the filter 40 having a pore diameter of φ0.45 μm or less are used. If these are prepared as a kit, PRP can be gelled. In addition, when using gelled PRP as a regenerative therapy, a bone prosthetic material can be further prepared in the kit.

DESCRIPTION OF SYMBOLS 11 ... Upper layer 12 ... Intermediate layer 13 ... Lower layer 16 ... Test tube 18 ... Plastic petri dish 31 ... Cylinder 40 ... Filter 50 ... Bone prosthetic material

Claims (8)

    A method for gelling platelet-rich plasma, wherein platelet-rich plasma is gelled by containing platelet-mixed plasma and human-derived human tissue kallikrein in a plastic container and mixing them.     The gelation method according to claim 1, wherein the human tissue kallikrein is a glandular kallikrein derived from salivary glands derived from sterilized saliva.     The gelation method according to claim 2, wherein the sterilization of the saliva includes filtering with a filter having a bore diameter of 0.45 μm or less.     The gelation method according to claim 2 or 3, wherein a liquid volume ratio of the sterilized saliva added to the platelet-rich plasma is 0.03 or more.     The glandular kallikrein is added after adding a bone filling material to the platelet-rich plasma, or the bone filling material is added after adding the glandular kallikrein to the platelet-rich plasma. The gelation method described.     The gelation method according to claim 4, wherein the bone grafting material contains β-tricalcium phosphate.   A kit for use in the gelation method according to any one of claims 2 to 5, comprising a plastic container for containing platelet-rich plasma and a filter having a bore diameter of 0.45 µm or less. The kit according to claim 7, further comprising a bone prosthetic material placed in the plastic container.