JP2013063924A - Method for accelerating gelling of platelet-rich plasma and/or platelet-poor plasma, kit and coagulation accelerator used therefor, and bone prosthetic material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method which can easily gel platelet-rich plasma and/or platelet-poor plasma to a desired shape in a short time, a kit and a coagulation accelerator used therefor, and a bone prosthetic material.SOLUTION: In the method for accelerating the gelling of platelet-rich plasma and/or platelet-poor plasma, a gelling agent is added to platelet-rich plasma and/or platelet-poor plasma, and the same is temporarily in contact with a coagulation accelerator and is thereafter stored in a plastic container to be gelled. The kit and the coagulation accelerator are used for the method. The same can be also used as a bone prosthetic material by adding a bone restoration material to the platelet-rich plasma and/or platelet-poor plasma after the temporary contact with the coagulation accelerator.

Description

  The present invention relates to a method for promoting gelation of platelet-rich plasma and / or platelet-poor plasma, and a kit, a coagulation promoting material, and a bone filling material used therefor.

  In recent years, platelet treatment (Platelet Rich Plasma: hereinafter sometimes referred to as “PRP”) is a treatment method for accelerating wound healing and bone regeneration using its own blood components in the dental field and orthopedic field. In addition, a treatment method using platelet-free plasma (hereinafter sometimes referred to as “PPP”) is becoming widespread.

  Of PRP and PPP, PRP is plasma with a higher platelet concentration than whole blood and is used to promote wound healing. Moreover, hydroxyapatite or β-tricalcium phosphate (hereinafter sometimes referred to as “β-TCP”) is a main component as a material for filling a bone defect caused by trauma such as a fracture or removal of a bone tumor. Block-shaped or granulated bone repair materials are widely used (see, for example, Patent Document 1), but PRP is mixed with such bone repair materials, so that bone defects and subchondral bone defects can be obtained. It is also used as a bone filling material.

  On the other hand, PPP is plasma having a lower platelet concentration than whole blood and is used in fields such as regenerative medicine. The above-described PRP and PPP are usually obtained by centrifuging collected blood, and are often used after gelation into a desired shape.

When gelling PRP or the like, a gelling agent is usually used. Patent Document 2 describes thrombin and calcium chloride as gelling agents used when gelling a mixture of PRP and β-TCP.
However, thrombin is generally derived from bovine or human and therefore has a safety problem against infection.

In order to solve this problem, Non-Patent Document 1 describes that PRP is gelled only with calcium chloride.
However, when gelation of PRP or the like is performed using only calcium chloride, there is a problem that the time required for gelation is long and the operability is poor.

Patent Document 3 describes a method for promoting the gelation of blood by adding glass to blood.
However, the gelation promotion method described in Patent Document 3 is a method for whole blood. Further, when glass is added to gel the blood, there is a problem that it is difficult to separate the gelled product from the glass.

JP 2001-137328 A International Publication No. 2002/040071 Pamphlet Japanese Examined Patent Publication No. 58-027933

Takashi Hatakeyama, 7 others, “Evaluation of Periodontal Tissue Regeneration Therapy Using Platelet-Rich Plasma (PRP) and Autologous Bone Grafting”, Japanese Periodontology, 2007, 49 (1), p. 71-76

  An object of the present invention is to provide a method capable of gelling platelet-rich plasma and / or platelet-poor plasma into a desired shape in a short time and a kit, and a kit, a coagulation promoting material, and a bone filling material used therefor It is to be.

  As a result of intensive studies to solve the above problems, the present inventors have found the following findings. That is, when gelation of PRP or PPP is performed in a glass container, gelation can be completed in a short time.

  However, when gelation of PRP or the like is completed in a glass container, there are the following problems. That is, PRP or the like needs to be gelled into a desired shape depending on the application to be used. For example, when PRP is used as the bone grafting material described above, it is necessary to gel PRP into a shape corresponding to the bone defect. In order to gel PRP into such a shape, it is necessary to form a glass container into a shape corresponding to a bone defect.

  However, the bone defect shape is often complicated, and it is difficult to form a glass container into such a shape. Therefore, it is difficult to gel PRP or the like into a desired shape with a glass container. Moreover, although it is necessary to prepare a container for every individual shape according to a case, a glass container is inferior in mass-productivity and it is difficult to respond to an individual shape.

  If a plastic container excellent in moldability and mass productivity is adopted instead of the glass container, it is considered that the above-mentioned problems caused by the glass container can be solved. However, when gelation of PRP or the like is performed in the plastic container, The time required for gelation is longer than that of a glass container, and the operability is inferior.

  On the other hand, the present inventors have repeatedly investigated the reason why gelation of PRP or the like is promoted in a glass container. As a result, it was found that bringing PRP or the like into contact with glass or the like triggered the gelation reaction, and it was not necessary to bring PRP or the like into contact with glass or the like until gelation was completed. Based on this finding, as a result of further earnest research, PRP and the like were temporarily brought into contact with glass and the like, and then housed in a highly versatile plastic container, and gelled in this plastic container. When completed, it was found that it can be gelled into a desired shape easily in a short time, and the present invention has been completed.

That is, the method for promoting gelation of platelet-rich plasma and / or platelet-poor plasma of the present invention has the following constitution.
(1) A platelet-rich plasma, characterized in that a gelling agent is added to platelet-rich plasma and / or platelet-poor plasma, temporarily brought into contact with a coagulation promoter, and then housed in a plastic container for gelation. A method for promoting gelation of plasma and / or platelet-poor plasma.
(2) The method for promoting gelation according to (1), wherein the gelling agent is calcium chloride.
(3) The gelation promotion method according to (1) or (2), wherein the solidification promoting material is glass.
(4) The above-mentioned (1) to (1), wherein a bone repair material is added to the platelet-rich plasma and / or platelet-poor plasma after being temporarily brought into contact with the coagulation promoting material, and the gel is stored in a plastic container. The gelation promotion method according to any one of 3).
(5) The method for promoting gelation according to (4), wherein the bone repair material is β-tricalcium phosphate.
(6) The gelation promoting method according to (4) or (5), wherein the volume ratio of (bone repair material) / (platelet-rich plasma and / or platelet-poor plasma) is 0.05 to 2.0.

The kit of the present invention has the following constitution.
(7) A kit comprising a coagulation promoting material and a plastic container, wherein the kit is used for the gelation promoting method according to any one of (1) to (6).
(8) The kit according to (7), wherein the shape of the coagulation promoting material is a container shape, a filter shape, a rod shape, a fiber shape, or a bead shape.

The solidification promoting material of the present invention has the following configuration.
(9) A coagulation promoting material, characterized by being used in the gelation promoting method according to any one of (1) to (6).
The bone filling material of the present invention has the following configuration.
(10) A bone grafting material produced using the gelation promoting method according to any one of (4) to (6).

  According to the present invention, there is an effect that operability can be improved by gelling PRP or PPP in a short time. In addition, since a highly versatile plastic container is adopted as the container for completing the gelation, it becomes easy to produce a container corresponding to an individual shape corresponding to a case such as a bone defect, and therefore, PRP and PPP can be simplified. Can be gelled into a desired shape. In addition, the plastic container is more economical than the glass container, which is economically advantageous. Furthermore, gelation of PRP or PPP can be completed in a short time even with calcium chloride alone. Therefore, if calcium chloride is employed as the gelling agent, it is possible to avoid safety problems against infections caused by thrombin and to improve safety.

It is a flowchart which shows the process of the gelatinization acceleration | stimulation method of the platelet-rich plasma which concerns on one Embodiment of this invention, and / or platelet-poor plasma.

  Hereinafter, a method for promoting the gelation of platelet-rich plasma and / or platelet-poor plasma according to an embodiment of the present invention, and a kit, a coagulation promoting material, and a bone prosthetic material used therein will be described in detail with reference to FIG. . As shown in the figure, in the gelation promotion method of this embodiment, blood is first collected. It is preferable to add an anticoagulant to the collected blood. Thereby, PRP and PPP can be anticoagulated. Examples of the anticoagulant include sodium citrate.

  Next, the collected blood is centrifuged to prepare PRP and PPP. Specifically, first, centrifugation is performed for the first time, and the collected blood is separated into two layers, an upper layer and a lower layer. Of the separated upper and lower layers, the lower layer is the red blood cell layer. Appropriate conditions for the first centrifugation are about 300 to 1,000 G and about 1 to 20 minutes.

  After removing the lower layer, which is an erythrocyte layer, the upper layer is centrifuged a second time, thereby collecting the fraction of the lower layer substantially containing platelets, that is, PRP. Also, an upper fraction, ie, PPP, with relatively few platelets is collected. As conditions for the second centrifugation, about 1000 to 3000 G and about 2 to 20 minutes are appropriate. The method for preparing PRP and PPP is not limited to the above-mentioned centrifugation conditions as long as PRP and PPP can be prepared.

  A gelling agent is added to the obtained PRP and / or PPP. Examples of the gelling agent include calcium chloride and calcium gluconate, and calcium chloride is particularly preferable. The gelling agent may be added alone, or may be added after a solution previously dissolved or diluted with an aqueous medium or the like. Examples of the aqueous medium include water. The addition amount of the gelling agent is not particularly limited. For example, when calcium chloride is added, (calcium chloride) / (PRP and / or PPP) = 0.2 to 2 mg / ml is appropriate.

  Next, PRP and / or PPP to which the gelling agent is added is temporarily brought into contact with the coagulation promoting material. Thereby, gelation of PRP and / or PPP can be promoted. In this specification, “temporarily contact with a coagulation promoter” means that the operation of bringing PRP and / or PPP into contact with the coagulation promoter is completed at the same time as or immediately after the addition of the gelling agent. This is to be done until before, i.e., only during the period when PRP and / or PPP is still fluid. The time for contacting PRP and / or PPP with the coagulation promoting material is usually about 1 to 10 minutes, although it depends on the amount of PRP and / or PPP.

  Examples of the solidification promoter include glass (silicate glass), kaolin, bentonite, diatomaceous earth, diatomaceous earth, and silica sand, and glass is particularly preferable. Examples of the shape of the coagulation promoting material include a container shape, a (porous) filter shape, a (thin) rod shape, a fiber shape, and a bead shape.

  Examples of the method of bringing PRP and / or PPP into contact with the coagulation promoting material include, for example, a method of containing PRP and / or PPP in a container-shaped coagulation promoting material, a method of passing a filter-like coagulation promoting material, a rod shape, and a fiber shape Alternatively, a method of adding a coagulation accelerating material such as a bead and then separating the material may be used. When bringing PRP and / or PPP into contact with the coagulation promoting material, it is preferable to bring them into contact with each other in a rocked state. Thereby, the contact area of PRP and / or PPP and a coagulation promoter can be improved.

  In this embodiment, a bone repair material is added to PRP and / or PPP after being brought into temporary contact with the coagulation promoting material. Examples of the bone repair material include β-TCP, α-tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, hydroxyapatite, carbonate apatite, and the like. A mixture of the above may be used, and β-TCP is particularly suitable.

  The bone repair material is preferably in the form of granules having an average diameter of 3 mm or less, and more preferably in the form of granules having an average diameter of 1.5 mm or less. The smaller the average diameter is, the smoother the surface of the gelled product is, so that the adhesion is improved, and the bone regeneration can be accelerated by increasing the filling amount of the bone repair material.

  The amount of bone repair material added is preferably (bone repair material) / (PRP and / or PPP) = 0.05 to 1.0 g / ml, preferably 0.2 to 0.8 g / ml. Is more preferable. When the filling amount of the bone repair material is small, there is a possibility that the bone regeneration may be delayed, which is not preferable. Further, if the filling amount of the bone repair material is too large, the strength of the gelled product is lowered, and the handleability of the gelled product may be lowered, which is not preferable.

  Finally, PRP and / or PPP to which the bone repair material is added is accommodated in a plastic container, and gelation is completed in the plastic container to obtain a bone prosthetic material. In the present embodiment, as described above, since PRP and / or PPP are temporarily brought into contact with the coagulation promoting material, PRP and / or PPP can be gelled in a short time. Further, in this embodiment, since gelation is completed in a highly versatile plastic container, it is easy to produce a container corresponding to an individual shape corresponding to a case such as a bone defect, and therefore, PRP and / or Alternatively, PPP can be easily gelled into a desired shape.

  Examples of the constituent material of the plastic container include polypropylene resin, polyethylene resin, polycarbonate resin, polystyrene resin, photocurable resin, and the like, and rubber elasticity such as silicone rubber, thermoplastic elastomer, natural rubber, and synthetic rubber. It may be the plastic material shown. The plastic container may be, for example, a general-purpose disposable container, or may be an individual shape according to a case such as a bone defect produced by an optical modeling method, and the shape is simply a plate shape. However, it may be tube-shaped. Gelation may be usually performed by standing at room temperature (23 ° C.).

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited only to a following example.

First, blood was collected and the collected blood was stored in a blood bag manufactured by Terumo Corporation. This blood bag contains 28 ml of CPDA solution. The components (W / V%) of the CPDA liquid are as follows.
Sodium citrate hydrate 2.630
・ Citric acid hydrate 0.327
・ Glucose 2.900
・ Sodium dihydrogen phosphate 0.251
・ Adenine 0.0275

  Next, blood was taken out from the blood bag via a syringe made of polyethylene resin, and each syringe was centrifuged to collect PPP. Centrifugation was performed at 2,000 G for 10 minutes.

  A 2% by weight calcium chloride solution was added to and mixed with the obtained PPP to obtain a mixed solution. The amount of calcium chloride solution added was (calcium chloride solution) / (PPP) = 0.05 ml / ml. Next, the obtained mixed liquid was accommodated in a glass container which is a coagulation promoting material, and was manually rocked for 3 minutes.

  On the other hand, granular β-TCP having an average diameter of 0.1 mm is placed in a polyethylene resin container, which is a plastic container, at a ratio of (β-TCP) / (PPP) volume ratio of 0.8. In advance. And the above-mentioned liquid mixture was added and stirred in this polyethylene resin container, it left still at room temperature (23 degreeC), and the time which gelation completion, ie, gelation time, was measured. The results are shown in Table 1.

  In addition, the judgment whether it gelatinized was performed by visual observation. The gelation time means the time from the addition of the calcium chloride solution in a polyethylene resin container to the completion of gelation.

[Comparative example]
First, PPP was collected in the same manner as in the above-described example to obtain a mixed solution with a calcium chloride solution. Next, except that the obtained mixed solution was not housed in a glass container, it was added to the container made of polyethylene resin containing β-TCP and stirred in the same manner as in the above-described example, and the mixture was stirred at room temperature. The gelation time was measured by standing at (23 ° C.). The results are shown in Table 1.

  As is clear from Table 1, the example in which the coagulation promoting material was temporarily brought into contact and then contained in a plastic container and gelled was compared with the comparative example in which the coagulation promoting material was not brought into temporary contact. It can be seen that the gelation time is short and the operability is excellent.

Claims (10)

  A platelet-rich plasma and / or a platelet-containing plasma and / or a platelet-containing plasma and / or platelet-poor plasma, characterized by adding a gelling agent, temporarily contacting the coagulation promoting material, and then placing the gel in a plastic container. Alternatively, a method for promoting gelation of platelet-poor plasma.   The gelling promotion method according to claim 1, wherein the gelling agent is calcium chloride.   The method for promoting gelation according to claim 1, wherein the solidification promoting material is glass.   The bone repair material is added to the platelet-rich plasma and / or platelet-poor plasma after being temporarily brought into contact with the coagulation promoter, and the gel is stored in a plastic container and gelled. The gelation promotion method as described.   The method for promoting gelation according to claim 4, wherein the bone repair material is β-tricalcium phosphate.   The method for promoting gelation according to claim 4 or 5, wherein a volume ratio of (bone repair material) / (platelet-rich plasma and / or platelet-poor plasma) is 0.05 to 2.0.   A kit comprising: a coagulation accelerating material; and a plastic container, wherein the kit is used for the gelation accelerating method according to claim 1.   The kit according to claim 7, wherein a shape of the coagulation promoting material is a container shape, a filter shape, a rod shape, a fiber shape, or a bead shape.   A coagulation promoting material characterized by being used in the gelation promoting method according to any one of claims 1 to 6.   The bone grafting material produced using the gelation promotion method in any one of Claims 4-6.

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