WO2016208675A1

WO2016208675A1 - Freeze-dried platelet-rich plasma and use thereof

Info

Publication number: WO2016208675A1
Application number: PCT/JP2016/068682
Authority: WO
Inventors: 泉朝比奈; 佑哉中谷; 秀樹縣; 吉慶住田; 喬充古賀
Original assignee: 株式会社Ｔｅｓホールディングス; 国立大学法人長崎大学
Priority date: 2015-06-23
Filing date: 2016-06-23
Publication date: 2016-12-29

Abstract

Provided are freeze-dried platelet-rich plasma that is substantially free of exogenous low-molecular-weight sugars, and a method for producing a freeze-dried platelet-rich plasma comprising a step for freezing platelet-rich plasma at -20°C or less and a step for freeze drying the frozen platelet-rich plasma.

Description

Lyophilized platelet rich plasma and uses thereof

The present invention relates to freeze-dried platelet-rich plasma and use thereof. This application claims priority based on Japanese Patent Application No. 2015-125916 filed in Japan on June 23, 2015, the contents of which are incorporated herein by reference.

It is known that platelet-rich plasma is effective for regenerative treatment of various tissues such as skin, mucous membrane, bone and periodontal tissue. However, platelet-rich plasma basically needs to be prepared at the time of use, which has hindered its clinical spread. Therefore, a technique for preserving platelet-rich plasma is required.

For example, Patent Document 1 includes platelet-rich plasma that has been coated on a solid support and then freeze-dried, and is used after the platelet-rich plasma has been refrigerated for at least one day after being freeze-dried. The material for regenerative treatment is described. Non-Patent Document 1 describes platelet-rich plasma freeze-dried after absorption of trehalose.

JP 2011-120663 A

However, since the platelet-rich plasma described in Patent Document 1 has a solid support as an essential component, it may be difficult to gel the platelet-rich plasma, for example. In addition, the platelet-rich plasma described in Non-Patent Document 1 has a complicated manufacturing process, and a complicated procedure may be required at the time of reconstitution.

Therefore, an object of the present invention is to provide platelet-rich plasma that can be stored and can be produced and used more easily and a method for producing the same.

The present invention is as follows.
(1) Platelet-rich plasma that is lyophilized and substantially free of exogenous low molecular sugars.
(2) The platelet-rich plasma according to (1), which is not coated on a solid support.
(3) A platelet-rich plasma composition containing the platelet-rich plasma according to (1) or (2) and a solvent.
(4) The platelet-rich plasma composition according to (3), wherein the platelet concentration is 200 to 1000 (× 10 ⁴ cells / μL).
(5) A coagulum of the platelet-rich plasma composition according to (3) or (4).
(6) A method for producing freeze-dried platelet-rich plasma, comprising a step of freezing platelet-rich plasma at −20 ° C. or lower and a step of freeze-drying the frozen platelet-rich plasma.

According to the present invention, it is possible to provide platelet-rich plasma that can be stored and can be produced and used more easily and a method for producing the same.

10 is a graph showing the results of Experimental Example 4. 10 is a graph showing the results of Experimental Example 4. 10 is a graph showing the results of Experimental Example 4. It is a figure which shows the photograph which shows the result of Experimental example 5, and an evaluation result. (A) is a photograph showing an incision line of the head of a nude mouse in Experimental Example 6, and FIG. 5 (b) is a photograph showing a state in which a sample is transplanted into the nude mouse in Experimental Example 6, FIG. (C) is a photograph of a nude mouse immediately after transplanting a sample in Experimental Example 6. (A) is a photomicrograph showing the results of hematoxylin and eosin staining of a sample taken from a nude mouse in Experimental Example 6. (B) is an enlarged view of a region surrounded by a square in (a). The scale bar indicates 200 μm. (A) is a photomicrograph showing the results of hematoxylin and eosin staining of a sample taken from a nude mouse in Experimental Example 6. (B) is an enlarged view of a region surrounded by a square in (a). The scale bar indicates 200 μm. (A) is a photomicrograph showing the results of hematoxylin and eosin staining of a sample taken from a nude mouse in Experimental Example 6. (B) is an enlarged view of a region surrounded by a square in (a). The scale bar indicates 200 μm. (A) is a photomicrograph showing the results of hematoxylin and eosin staining of a sample taken from a nude mouse in Experimental Example 6. (B) is an enlarged view of a region surrounded by a square in (a). The scale bar indicates 200 μm. It is a graph which shows the calculation result of the bone formation rate in Experimental example 6.

[Freeze-dried platelet-rich plasma]
In one embodiment, the present invention provides platelet rich plasma that is lyophilized and substantially free of exogenous low molecular sugars.

The freeze-dried platelet-rich plasma according to the present embodiment can be stored and can be more easily produced and used. Here, being storable means that the activity can be maintained even when stored at 4 ° C. for one month or longer.

Examples of the activity of platelet-rich plasma include gel forming activity described later, the activity of growth factors contained in platelet-rich plasma, and the like.

In the lyophilized product of the present embodiment, examples of the low molecular sugar include sugars having an action of improving resistance to freezing of animal cells and the like, such as trehalose, sucrose, and maltose.

In addition, the phrase “substantially free of exogenous low-molecular sugars” means that sugars that improve freezing resistance are not added from the outside during freeze-drying of platelet-rich plasma. The presence of sugar is acceptable.

In addition, an anticoagulant may be added to the blood at the time of collecting blood used for the production of platelet-rich plasma, and glucose may be added to the anticoagulant. Is not included in exogenous low-molecular sugars.

As described in Non-Patent Document 1, conventionally, in order to preserve platelet-rich plasma by freeze-drying, it has been thought that low molecular sugars such as trehalose need to be absorbed in advance by platelets. On the other hand, the inventors have found that platelet-rich plasma can be lyophilized while maintaining the activity without performing treatment such as absorption of low-molecular sugars into platelets.

The lyophilized product of this embodiment can be produced more easily because it does not require treatment such as absorption of low molecular sugars by platelets. Moreover, the lyophilized body of platelet-rich plasma described in Non-Patent Document 1 needs to be prehydrated by allowing it to stand in a saturated water vapor environment at 37 ° C. before reconstitution. On the other hand, when reconstituting the freeze-dried platelet-rich plasma according to the present embodiment, prehydration is not essential and can be used more easily.

It is preferable that the freeze-dried body of this embodiment is not coated on the solid support.
The platelet-rich plasma of this embodiment can be lyophilized and stably stored even if it is not coated on a solid support.

In other words, the freeze-dried body of the present embodiment includes “platelet-rich plasma freeze-dried after being coated on a solid support, and used after the platelet-rich plasma has been refrigerated for at least one day after being freeze-dried. It may not be “a material for regenerative treatment”.

Here, the solid support means, for example, a short-absorbable biodegradable polymer or bioabsorbable polymer such as polyglycolic acid, polylactic acid, and copolymers thereof; propanediol, poly (L-lactide-co-) Long-degradable biodegradable polymers or bioabsorbable polymers such as epsilon-caprolactone), polycaprolactone and copolymers thereof; polybutylene succinate, polyvinyl alcohol, chitosan and other biodegradable polymers or bioabsorbable polymers; Examples include a support made of silk, which is a non-biodegradable polymer.

Examples of the shape of the solid support include fiber products such as woven fabrics, knitted fabrics, nonwoven fabrics, stitches, cotton, cloth, paper, and filters; and porous substrates including granular materials and foams. Examples of the textile products include woven fabrics such as meshes and gauze fabrics; those commonly used as wound dressings such as knitted fabrics, nonwoven fabrics, stitches, and cotton.

Also, being coated means that the solid support is covered with a solution containing platelet-rich plasma. That is, being coated means that the platelet-rich plasma is coagulated on the surface of the solid support, the above solution is permeated by capillary action through the fiber gaps and pores of the solid support, The solid support is dissolved or decomposed in the presence of the above solution, and the above solution is absorbed by the solid support by forming a layer or membrane of the above solution on the surface of the solid support. means.

[Reconstituted platelet-rich plasma]
In one embodiment, the present invention provides a platelet-rich plasma composition containing the platelet-rich plasma and solvent described above. It can also be said that the platelet-rich plasma composition of the present embodiment is a reconstituted platelet-rich plasma obtained by adding a solvent to the above-mentioned freeze-dried platelet-rich plasma.

As shown in the examples described later, the reconstituted platelet-rich plasma of this embodiment is fresh platelet-rich plasma that has not been lyophilized even after the freeze-dried form of platelet-rich plasma has been stored for a long time. Can exhibit the same activity.

The platelets contained in the platelet-rich plasma obtained by reconstitution of the above-mentioned freeze-dried platelet-rich plasma have a history of freeze-drying once. Therefore, it is predicted that the surface structure of platelets is different from that of fresh platelet-rich plasma. However, it is difficult to specify how the surface structure of platelets changes, and whether or not the platelet-rich plasma is a reconstituted lyophilized body of the above-mentioned platelet-rich plasma based on the surface structure of platelets or the like. It is also difficult to specify.

Examples of the solvent include sterilized water, physiological saline, and phosphate buffer. The solvent may be added to the lyophilized platelet-rich plasma in an amount that is equivalent to the platelet-rich plasma before lyophilization, or may be added in an amount that is less than that before lyophilization. In the former case, platelet-rich plasma having a platelet concentration equivalent to that before lyophilization is obtained. In the latter case, platelet-rich plasma having a platelet concentration higher (concentrated) than platelet-rich plasma before lyophilization is obtained.

In one embodiment, the reconstituted platelet-rich plasma of this embodiment may have a platelet concentration of 200-1000 (× 10 ⁴ cells / μL). The platelet concentration of general platelet-rich plasma is 100 to 200 (× 10 ⁴ cells / μL). Therefore, platelet-rich plasma having a platelet concentration of 200 to 1000 (× 10 ⁴ cells / μL) is concentrated about 2 to 5 times compared with general platelet-rich plasma. The reconstituted platelet-rich plasma of this embodiment may have a platelet concentration of 200 to 800 (× 10 ⁴ cells / μL) or 200 to 600 (× 10 ⁴ cells / μL).

As shown in the examples described below, concentrated platelet-rich plasma can more strongly promote tissue regeneration in vivo compared to unconcentrated platelet-rich plasma.

[Platelet-rich plasma gel]
In one embodiment, the present invention provides a clot (gel) of the platelet-rich plasma composition described above. The gel according to the present embodiment is obtained by reconstituting a platelet-rich plasma with a solvent and further coagulating it. The gel of the present embodiment can be produced, for example, by adding calcium chloride to reconstituted platelet-rich plasma and activating and coagulating a blood coagulation factor contained in the plasma.

More specifically, prothrombin contained in plasma is changed to thrombin by the action of thromboplastin released from platelets and calcium ions, and fibrinogen in plasma becomes fibrin by the enzyme activity of thrombin. Fibrin binds to each other by the action of calcium ions and the like, forming a network structure and solidifying.

The gel of the present embodiment serves as a scaffold for cells, and can keep platelet-rich plasma locally for a long period of time, and can gradually release growth factors and the like contained in the platelet-rich plasma. It is useful for regenerative treatment of various tissues such as bone and periodontal tissue.

In the preparation of the gel of the present embodiment, for example, a bone filling material or the like may be added to the platelet-rich plasma. In addition, autologous serum may be added to platelet-rich plasma to improve clotting activity.

[Method for producing freeze-dried platelet-rich plasma]
In one embodiment, the present invention provides a method for producing freeze-dried platelet-rich plasma comprising the steps of freezing platelet-rich plasma at −20 ° C. or lower and freeze-drying the frozen platelet-rich plasma. provide. It can be said that the production method of this embodiment is a method for preserving platelet-rich plasma.

(Platelet rich plasma)
Platelet rich plasma can be prepared by a general method. Specifically, first, peripheral venous blood is collected from a patient. At this time, an anticoagulant is added to the collected peripheral blood. As the anticoagulant, a common one can be used, and examples thereof include citric acid solution, sodium fluoride, ethylenediaminetetraacetic acid (EDTA) solution, ACD (acid-citrate-dextrose) solution, heparin and the like. .

Subsequently, the peripheral blood to which the anticoagulant has been added is placed in a centrifuge tube and centrifuged, for example, at 4 ° C. and 1000 to 1200 × g for 10 to 30 minutes. After centrifugation, the peripheral blood is separated into a plasma layer, a white blood cell layer (buffy coat), and a red blood cell layer in order from the top. Since platelets are accumulated above the buffy coat, platelet-rich plasma can be obtained by collecting the vicinity thereof.
For example, on the basis of the position of the buffy coat, mark 1/10 volume above the blood in the centrifuge tube, remove the platelet blood plasma above the mark, and remove platelets located below the mark The layer and buffy coat may be collected and used as platelet rich plasma.

Alternatively, platelet-rich plasma may be collected from peripheral blood using a commercially available instrument (for example, “Gravational Platelet Separation System”, Biomet).

(Step of freezing platelet-rich plasma)
In this step, the platelet-rich plasma is frozen. The freezing temperature may be −50 ° C. or lower, −80 ° C. or lower, −150 ° C. or lower, or −190 ° C. or lower. For freezing at −50 ° C. to −150 ° C., for example, a commercially available deep freezer can be used. For freezing at −190 ° C. or lower, for example, liquid nitrogen can be used.

(Freeze drying process)
In this step, the frozen platelet-rich plasma is lyophilized. The lyophilization is preferably performed within a few days after the platelet-rich plasma is frozen. For example, the platelet-rich plasma may be freeze-dried immediately after freezing or may be freeze-dried 24 hours after freezing.

Freeze-drying can be performed by a general method. For example, lyophilization may be performed using a freeze dryer (model “EYELA FD-1000”, Tokyo Rika Kikai Co., Ltd.) at −50 ° C. and 15 Pa.

The platelet-rich plasma after lyophilization may be stored at room temperature, but is preferably stored at a lower temperature. For example, it may be stored at 4 ° C. or may be stored at −20 ° C., for example.

Next, the present invention will be described in more detail with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[Experimental Example 1]
(Preparation of platelet-rich plasma)
Human peripheral venous blood was collected from three donors A, B and C. At this time, a volume of 10 w / v citrate solution as an anticoagulant was added to the collected peripheral blood at 1 volume per 9 volumes of peripheral blood.

Subsequently, peripheral blood to which an anticoagulant was added was placed in a centrifuge tube and centrifuged with a centrifuge (trade name “Medifuge”, manufactured by Silfrent). After centrifugation, the peripheral blood was separated into a plasma layer, a white blood cell layer (buffy coat), and a red blood cell layer in order from the top. Subsequently, the position of the buffy coat was used as a reference to mark an upper portion of 1/10 volume of blood in the centrifuge tube. Above the above mark, it was removed because it was sputum platelet plasma. Subsequently, the platelet layer and the buffy coat located below the mark were collected and used as platelet-rich plasma. Hereinafter, platelet-rich plasma may be referred to as (Platelet Rich Plasma, PRP).

[Experiment 2]
(Measurement of the number of blood cells in peripheral blood and platelet-rich plasma)
The numbers of white blood cells, red blood cells, and platelets in peripheral blood and platelet-rich plasma from each donor in Experimental Example 1 were measured. In addition, the concentration ratio of platelets in platelet-rich plasma based on the number of platelets in peripheral blood was also calculated. Table 1 shows the measurement results of the number of blood cells in the peripheral blood and platelet-rich plasma derived from each donor, and the platelet concentration rate. As a result, in all donors, the platelet concentration rate in fresh platelet-rich plasma (non-lyophilized platelet-rich plasma, fresh PRP) was about 6 times.

[Experiment 3]
(Freeze-drying of platelet-rich plasma)
The platelet-rich plasma prepared in Experimental Example 1 was frozen in a deep freezer at −80 ° C. for 24 hours, and then freeze-dried (model “EYELA FD-1000”, Tokyo Rika Instruments) at −50 ° C. and 15 Pa. Lyophilized to obtain a lyophilized platelet-rich plasma.

[Experimental Example 4]
(Measurement of growth factor concentration in platelet-rich plasma)
A fresh platelet-rich plasma prepared in the same manner as in Experimental Example 1 was frozen at -20 degrees and thawed at 37 ° C. twice to extract growth factors, and a reference platelet-rich plasma (hereinafter referred to as “reference PRP”). ). In addition, the freeze-dried platelet-rich plasma prepared in Experimental Example 3 was stored at 4 ° C. for 1 month and then reconstituted at the same magnification by adding sterilized water (hereinafter referred to as “× 1FD-PRP”). ) And lyophilized platelet-rich plasma prepared in Experimental Example 3 at 4 ° C. for 1 month, and then reconstituted to 1/3 volume by adding sterile water (platelet concentration is 3 times concentrated) In the following, “× 3FD-PRP” was prepared.

For reference PRP, × 1FD-PRP and × 3FD-PRP, platelet-derived growth factor (PDGF) -BB, transforming growth factor (TGF) -β1, vascular endothelial growth factor ( The concentration of vascular (secondary growth factor, VEGF) was measured by an ELISA method using a commercially available kit. More specifically, a kit (trade name “Quantikine Human PDGF-BB ELISA”, catalog number “DBB00”, R & D Systems) was used for measurement of PDGF-BB. A kit (trade name “Quantikine Human TGF-β1 ELISA”, catalog number “DBB00”, R & D Systems) was used for the measurement of TGF-β1. In addition, a kit (trade name “Quantikine Human VEGF ELISA”, catalog number “DVE00”, R & D Systems) was used for VEGF measurement. The concentration of each growth factor was measured four times for each of the reference PRP, x1FD-PRP and x3FD-PRP derived from three donors.

FIG. 1 is a graph showing the measurement results of the PDGF-BB concentration in the standard PRP, × 1FD-PRP, and × 3FD-PRP. In the figure, the graph shows the average value, and the error bar shows the standard deviation. Further, “ns” indicates that there is no significant difference with a risk rate of less than 1%. The numbers in the figure indicate relative values of the PDGF-BB concentration with the PDGF-BB concentration in the reference PRP being 1.

As a result, it was shown that the concentration of PDGF-BB in x1FD-PRP was equivalent to the concentration of PDGF-BB in the reference PRP. In addition, the concentration of PDGF-BB in x3FD-PRP was shown to be about 2.8 times the concentration of PDGF-BB in reference PRP.

FIG. 2 is a graph showing the measurement results of the concentration of TGF-β1 in the standard PRP, × 1FD-PRP and × 3FD-PRP. In the figure, the graph shows the average value, and the error bar shows the standard deviation. Further, “ns” indicates that there is no significant difference with a risk rate of less than 1%. The numbers in the figure show the relative values of the TGF-β1 concentration, where the TGF-β1 concentration in the reference PRP is 1.

As a result, it was shown that the concentration of PDGF-BB in x1FD-PRP was equivalent to the concentration of TGF-β1 in the reference PRP. Further, it was shown that the concentration of TGF-β1 in × 3FD-PRP was about 2.5 times the concentration of TGF-β1 in reference PRP.

FIG. 3 is a graph showing the measurement results of the concentration of VEGF in the standard PRP, × 1FD-PRP, and × 3FD-PRP. In the figure, the graph shows the average value, and the error bar shows the standard deviation. Further, “**” indicates that there is a significant difference at a risk rate of less than 1%. The numbers in the figure indicate relative values of the concentration of VEGF, where the concentration of VEGF in the reference PRP is 1.

As a result, it was shown that the concentration of VEGF in x1FD-PRP was lower than the concentration of VEGF in the reference PRP. Further, it was shown that the concentration of VEGF in x3FD-PRP was about 1.5 times the concentration of VEGF in reference PRP.

[Experimental Example 5]
(Evaluation of gelation ability of platelet-rich plasma)
The lyophilized platelet-rich plasma prepared in Experimental Example 3 was immediately after preparation (immediately after FD), 1 day (after 1 day of FD), 3 days (after 3 days of FD), 1 week (after 1 week of FD), 2 weeks (after 2 weeks of FD) ) And 4 weeks (after 4 weeks of FD) Gel storage ability (fibrinization ability, coagulation ability) was evaluated for those reconstituted at an equal magnification by adding sterilized water after storage at 4 ° C. As a control, the same evaluation was performed on fresh platelet-rich plasma prepared in the same manner as in Experimental Example 1.

More specifically, β-tricalcium phosphate (trade name “Osferion (registered trademark) G1”, manufactured by Olympus) 25 mg, which is a bone filling material, is added to the above-mentioned reconstituted platelet-rich plasma or fresh platelet-rich plasma 100 μL. Then, 10 μL of autoserum and 10 μL of 2 w / v% calcium chloride aqueous solution were mixed, and the gelation state 10 minutes after mixing was evaluated according to the following criteria.
+: Completely solidified.
±: Partially solidified.
-: Not solidified.

FIG. 4 shows a photograph of the mixture after 10 minutes of mixing, a photograph of the platelet-rich plasma gel taken out with tweezers after 10 minutes of mixing, and an evaluation result of the gelation state of the platelet-rich plasma. As a result, it was clarified that the freeze-dried platelet-rich plasma exhibited the same gelation ability as fresh platelet-rich plasma even after 4 weeks of storage.

[Experimental Example 6]
(Evaluation of the ability of platelet-rich plasma to promote bone regeneration)
The ability of platelet rich plasma to promote bone regeneration was evaluated using a nude mouse subperiosteal model. Specifically, a sample described later was transplanted under the periosteum on the skull of a 6-week-old male nude mouse (BALB / c). Subsequently, the transplanted samples were taken out after 4 weeks and 8 weeks, decalcified specimens were prepared and stained with hematoxylin and eosin, and the ability to promote bone regeneration was evaluated by histological analysis.

FIG. 5 (a) is a photograph showing the incision line of the head of a nude mouse, FIG. 5 (b) is a photograph showing a state of transplanting the sample into the nude mouse, and FIG. 5 (c) is a photograph of the sample. It is the photograph of the nude mouse immediately after transplanting.

The following (i) to (iv) were used as samples.
(I) β-tricalcium phosphate (trade name “Osferion (registered trademark) G1,” manufactured by Olympus Co., Ltd.) (hereinafter referred to as “β-TCP sample” in this experimental example)
(Ii) β-tricalcium phosphate (trade name “Osferion (registered trademark) G1”, manufactured by Olympus Co., Ltd.), 25 mg, which is a bone filling material, 100 μL of fresh platelet-rich plasma, 10 μL of human serum (autologous serum) and 2 w / v 10 μL of 10% calcium chloride aqueous solution mixed and allowed to stand for 10 minutes to gel (hereinafter referred to as “fresh PRP sample” in this experimental example)
(Iii) In the preparation of the “fresh PRP sample”, the freeze-dried product of platelet-rich plasma prepared in Experimental Example 3 was stored at 4 ° C. for 1 month instead of fresh platelet-rich plasma, and then sterilized water was added. Samples that were reconstructed at the same magnification (hereinafter referred to as “× 1FD-PRP sample” in this experimental example)
(Iv) In preparation of the above “fresh PRP sample”, the lyophilized body of platelet-rich plasma prepared in Experimental Example 3 was stored at 4 ° C. for 1 month instead of fresh platelet-rich plasma, and then sterilized water was added. Samples reconstituted to 1/3 capacity (hereinafter referred to as “× 3FD-PRP sample” in this experimental example)

The histological analysis was performed as follows. Based on the hematoxylin and eosin stained image, NIH Image (Image J), which is analysis software, was used to measure the area of the transplanted sample and the area of osteogenesis. Subsequently, the bone formation rate (%) was calculated by the following formula (1).
Osteogenesis rate (%) = Bone formation area (μm ² ) / Transplant sample area (μm ² ) × 100 (1)

FIG. 6 (a) is a photomicrograph showing the results of hematoxylin and eosin staining of a β-TCP sample taken from a nude mouse. FIG. 6B is an enlarged view of a region surrounded by a square in FIG. The scale bar indicates 200 μm.

FIG. 7 (a) is a photomicrograph showing the results of hematoxylin and eosin staining of a fresh PRP sample taken from a nude mouse. FIG. 7B is an enlarged view of a region surrounded by a square in FIG. The scale bar indicates 200 μm.

FIG. 8 (a) is a photomicrograph showing the results of hematoxylin and eosin staining of a x1FD-PRP sample taken from nude mice. FIG. 8B is an enlarged view of a region surrounded by a square in FIG. The scale bar indicates 200 μm.

FIG. 9 (a) is a micrograph showing the results of hematoxylin and eosin staining of a × 3FD-PRP sample taken from nude mice. FIG. 9B is an enlarged view of a region surrounded by a square in FIG. The scale bar indicates 200 μm.

As a result, in the group transplanted with β-TCP sample, bone formation was slightly observed on the skull. In addition, clear bone formation was observed in the group transplanted with the fresh PRP sample, × 1FD-PRP sample, and × 3FD-PRP sample, and bone replacement was promoted in the group transplanted with the × 3FD-PRP sample.

FIG. 10 is a graph showing the calculation results of the bone formation rate in nude mice transplanted with each sample. In the figure, the graph shows the average value, and the error bar shows the standard deviation. In addition, “**” indicates that there is a significant difference when the risk rate is less than 1%, and “*” indicates that there is a significant difference when the risk rate is less than 5%. Moreover, the number in a figure shows an average value.

The group transplanted with β-TCP sample had n = 3. In the group transplanted with fresh PRP sample, × 1FD-PRP sample, and × 3FD-PRP sample, n = 3 (total n = 9) for platelet-rich plasma derived from each of the three donors.

As a result, it was clarified that the group transplanted with the fresh PRP sample, × 1FD-PRP sample and × 3FD-PRP sample significantly promoted osteogenesis compared to the group transplanted with β-TCP sample. It was. In addition, the osteogenesis rate of the group transplanted with the × 1FD-PRP sample was not significantly different from the osteogenesis rate of the group transplanted with the fresh PRP sample. Further, it was revealed that the osteogenesis rate of the group transplanted with the × 3FD-PRP sample was significantly higher than the osteogenesis rate of the group transplanted with the × 1FD-PRP sample.

From the results of the above experimental examples, it was shown that the platelet-rich plasma can be stored for a long time without losing its function by freeze-drying. In addition, it was shown that platelets can be concentrated when reconstituted a freeze-dried form of platelet-rich plasma, thereby further promoting tissue regeneration.

Claims

Platelet-rich plasma that is lyophilized and substantially free of exogenous low-molecular sugars.
The platelet-rich plasma according to claim 1, which is not coated on a solid support.
A platelet-rich plasma composition comprising the platelet-rich plasma according to claim 1 or 2 and a solvent.
The platelet-rich plasma composition according to claim 3, wherein the platelet concentration is 200 to 1000 (× 10 4 cells / μL).
A coagulum of the platelet-rich plasma composition according to claim 3 or 4.
Freezing the platelet-rich plasma at −20 ° C. or lower;
Lyophilizing the frozen platelet-rich plasma;
A method for producing freeze-dried platelet-rich plasma comprising: