JP5763880B2 - Regenerative treatment materials - Google Patents

Description

  The present invention relates to a regenerative therapeutic material, and more particularly to a regenerative therapeutic material comprising platelet-rich plasma that has been coated on a solid support and then lyophilized.

  Platelet rich plasma (hereinafter referred to as “PRP”) fractionated from human blood has been frequently used in surgery as a fibrin glue for more than 50 years. In the 21st century, it has been noticed that cell growth factors are concentrated and contained in PRP in the field of oral surgery, and is now being applied in the field of cosmetic surgery as well as regenerative medicine. . However, since it cannot be stored for a long time, preparation in use is a principle in any case (Patent Document 1). Since the 1990s, the development of PRP storage technology by freeze-drying has progressed in order to expand the versatility for emergency response and first aid in field hospitals. In addition, a lyophilization method is being studied as one of the methods for preserving a large amount of PRP for the purpose of liver regeneration. However, it remains a problem that these freeze-dried PRPs require attention to be dissolved in an aqueous solution (Non-patent Document 1).

Pamphlet of International Publication No. WO2007 / 027178

Walkers, W.M. F. Et al., Cryobiology, 42: 79-87 (2001).

  Therefore, it is necessary to develop a regenerative treatment material that uses platelet-rich plasma and that does not require preparation or frozen storage at the time of use.

  The present invention provides a regenerative therapeutic material comprising 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 and stored for at least one day. To do.

  The regenerative treatment material of the present invention may be used after the platelet-rich plasma has been refrigerated for at least 30 days after lyophilization.

  In the regenerative treatment material of the present invention, the solid support may be selected from the group consisting of a textile product, a porous substrate, a granular material, and a foam.

  In the regenerative treatment material of the present invention, the solid support may be made of a biodegradable material.

  In the regenerative therapeutic material of the present invention, the solid support may be coated with a ligand of a cell-substrate adhesion receptor before the platelet-rich plasma is coated.

  In the regenerative treatment material of the present invention, the ligand may be atelocollagen.

  In the present invention, the solid support may be of any material and shape as long as it can maintain a substantially constant shape during the period from coating of platelet-rich plasma to lyophilization. Preferred materials for the solid support in the present invention are short-term absorbable biodegradable polymers or bioabsorbable polymers such as polyglycolic acid, polylactic acid and copolymers thereof, propanediol, poly (L-lactide- Co-epsilon-caprolactone), polycaprolactone and their copolymers, and biodegradable or bioabsorbable polymers that are long-term degradable and polybutylene succinate, polyvinyl alcohol, chitosan and other biodegradable polymers or bio Including, but not limited to, absorbent polymers and non-biodegradable polymer silk. Preferred shapes of the solid support in the present invention include textile products such as woven fabrics, knitted fabrics, nonwoven fabrics, stitches, cotton, cloth, paper, filters, and porous substrates including granular materials and foams. It is not limited. Preferred textiles include those commonly used as wound dressings such as knitted fabrics, non-woven fabrics, stitches and cotton, as well as woven fabrics such as meshes and gauze fabrics.

  The term “coating” used in the present invention means that a solid support is covered with a solution containing a ligand such as PRP or atelocollagen. That is, the coating means that the PRP is solidified on the surface of the solid support, the solution penetrates through the fiber gaps and pores of the solid support by capillarity, and the presence of the solution. The solid support has a broad meaning including that the solution is absorbed by the solid support by forming a layer or membrane of the solution on the surface of the solid support in order to dissolve or decompose the solid support. .

  In the present invention, PRP may be of any origin, provided that it has the required cell growth promoting activity, wound healing promoting activity, etc., and safety as a blood product is ensured. However, in consideration of species specificity, it is preferably derived from an individual of the same species as the patient to be subjected to regenerative treatment. Moreover, in order to ensure the safety as a blood product, it is more preferable that the PRP is purified from blood collected from the patient who is the subject of regenerative treatment. The preparation of PRP is described, for example, in Okuda, K .; (J. Periodontol. 74: 849-857 (2003)) and JP-A-2004-201799. Briefly, fresh whole blood collected from a patient is dispensed into a 10 mL centrifuge tube to which an anticoagulant containing citric acid and glucose is added, and a swing bucket rotor with a shortest diameter of about 57 mm and a longest diameter of about 140 mm is used. Centrifuge at 400 rpm for 10 minutes. When centrifuged, red blood cells are precipitated and platelets are collected between the supernatant plasma. Next, the platelets and plasma are transferred to another 10 mL centrifuge tube, centrifuged at 3,600 rpm for 15 minutes to precipitate the platelets, and the fraction collected by suspending in a minimum amount of plasma is used as platelet-rich plasma. The platelet-rich plasma of the present invention may be freshly collected from a patient or may be cryopreserved. Moreover, it is preferable that the provider of PRP is the patient himself. However, on the condition that safety as a blood product is ensured, PRP prepared from blood collected from humans other than patients, such as blood donation, may be used.

  In the present invention, lyophilization refers to drying by cooling and / or reduced pressure, and may be accompanied by centrifugation. For example, a woven fabric coated with a ligand such as PRP or atelocollagen is pre-frozen in a deep freezer at −80 ° C. for about 1 hour, and then at −45 ° C. and 20 Pa for about 10 minutes in a freeze drying apparatus. May be processed. In order to completely freeze-dry, it may be treated for another 20 to 50 minutes.

  In the present invention, refrigerated storage means storage at any temperature lower than room temperature that does not require a freezer for storage at -80 ° C to -20 ° C. Storage at a temperature in the range of 4 ° C to 10 ° C is more preferable.

  In the present invention, cell-substrate adhesion refers to adhesion between cells and extracellular matrix, and adhesion between cells and the surface of a culture vessel. The ligand of the cell-matrix adhesion receptor in the present invention includes, but is not limited to, collagen and atelocollagen, fibronectin and laminin.

  The material for regenerative treatment of the present invention can be used as a wound dressing material or an implant material embedded in a defect portion of hard tissue and / or soft tissue. Moreover, it can utilize for the objectives, such as a supply source of a cell growth factor, as a substitute of the conventional freeze-dried PRP.

  Since the regenerative treatment material of the present invention is used after being refrigerated for at least one day, there is no need for preparation at the time of use. Therefore, it is particularly useful for regenerative treatment in a small medical institution that does not have equipment such as a centrifuge. In addition, since the regenerative treatment material of the present invention can be refrigerated, there is no need for dry ice, deep freezer, etc. for transportation and storage. Therefore, it is useful as a substitute for frozen PRP from the viewpoint of energy consumption.

(Bioethics)
When using cultured cells, PRP and rats derived from humans, an experimental plan based on the ethical rules of Niigata University Medical and Dental General Hospital was approved by the Niigata University School of Dentistry Ethics Committee. Furthermore, with informed consent, each time the donor was explained about the collection of periosteal cells and PRP and the donation to the experiment, and written consent was obtained.

The bar graph of the experimental result which investigates the influence on the proliferation of the human periosteum origin cultured cell of a PRP coating woven fabric. Hematoxylin between control rat wound tissue (A) treated without bioabsorbable fabric and rat wound tissue (B) treated with PRP-coated bioabsorbable fabric -Optical micrograph of tissue stained with eosin. Tissues that were subjected to hematoxylin and eosin staining (A) and von Kossa staining (B), respectively, on adjacent thin slices of periosteum primary culture cultured on bioabsorbable woven fabric coated with atelocollagen and PRP Optical micrograph of the specimen.

  The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the appended claims.

Growth promotion effect of human periosteum-derived cultured cells Preparation of PRP Fresh whole blood collected from a patient was dispensed into a 10 mL centrifuge tube to which an anticoagulant containing citric acid and glucose was added, and the shortest diameter was about 57 mm and the longest diameter was about 140 mm. Were centrifuged at 2,400 rpm for 10 minutes. Red blood cells were precipitated by centrifugation, and platelets gathered with the supernatant plasma. The platelets and plasma were collected in another 10 mL centrifuge tube and centrifuged at 3,600 rpm for 15 minutes. Precipitated platelets were collected by suspending in a minimal amount of plasma, which was used as platelet rich plasma.

Preparation of PRP coated woven fabric 5, mm × 5 mm and 5 mm × 10 mm bioabsorbable woven fabric (Bikerill mesh, catalog number: VICRYL WOVEN MESH (Polyglactin 910) VWMM, Johnson & Johnson Co., Ltd.) Coated with 30 μL and 60 μL of PRP at room temperature for 3-5 minutes. The woven fabric was pre-frozen in a deep freezer at −80 ° C. for about 1 hour, and then treated in a freeze drying apparatus at −45 ° C. and 20 Pa for 30 to 60 minutes. (Hereinafter referred to as “PRP × 1” and “PRP × 2”, respectively). The PRP coated woven fabric was stored and stored in a refrigerator until use.

Cells Cultured cells derived from periosteum pieces collected from patients (hereinafter referred to as “human periosteum-derived cultured cells”) were used. The 6th passage of human periosteum-derived cultured cells in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum was a 6-well plate (Becton Dickinson, # 35390) with a cell insert (Becton Dickinson, # 353090). 353502 or # 55467) at 1 × 10 ⁵ per well. The culture was performed by adding 2 mL of Dulbecco's modified Eagle's medium (hereinafter referred to as “differentiation medium”) supplemented with 1% fetal calf serum to the wells and 1 mL to the cell insert, 37 ° C., 5% CO ₂ and It was carried out for 2 days under a saturated steam atmosphere. One PRP × 1 or PRP × 2 derived from the same patient from which the periosteum pieces were collected was immersed in the cell insert. Control experiments were a culture using a woven fabric not coated with PRP, and a culture using only differentiation medium without using PRP × 1 and PRP × 2.

Quantification of cell proliferation After culturing for 2 days, the cells were dissociated, and the number of cells was measured with a particle count analyzer (CDA-100X, Sysmex).

Results FIG. 1 is a bar graph of experimental results examining the effect of PRP-coated woven fabric on the proliferation of cultured cells from human periosteum. As shown in FIG. 1, the number of cells per well was 1.0 × 10 ⁵ in the control experiment, 1.2 × 10 ⁵ when using PRP × 1, and when using PRP × 2. It was 1.4 × 10 ⁵ pieces. This result suggests that the greater the size of the PRP-coated bicyclyl mesh, the higher the activity of promoting cell proliferation. The bioabsorbable woven fabric not coated with PRP did not show cell growth promoting activity (not shown). In this example, a PRP-coated woven fabric with a storage period of 1 day in the refrigerator was used, but the same cell growth promoting activity was observed with a PRP-coated woven cloth with a storage period of 30 days.

Effect of promoting healing of rat skin wound Wound and treatment The epidermis on the back of a locally anesthetized rat (F334, male, 6 weeks old) was incised and removed by 10 mm × 10 mm. A 10 mm × 10 mm bioabsorbable woven fabric coated with PRP (Bikerill mesh, catalog number: VICRYL WOVEN MESH (Polyglactin 910) VWMM, Johnson & Johnson Co., Ltd.) is placed at the wound site, and a surgical polyurethane is placed thereon Film (Surget, Nipro) was attached. The control experiment was a rat in which only the surgical polyurethane film was applied to the wound site without placing the bioabsorbable woven fabric.

Histological observation Wound tissue was removed 5 days after wound treatment, and a tissue specimen for light microscope observation was prepared. Thin section preparation and hematoxylin and eosin staining were performed according to standard methods well known to those skilled in the art.

Results FIG. 2 shows the rat wound tissue (A) from a control experiment treated without a bioabsorbable woven fabric and the rat wound tissue treated with a bioabsorbable woven fabric coated with PRP (A). It is an optical microscope photograph of the tissue stained with hematoxylin and eosin with B). The portion indicated by the asterisk (*) in FIG. 2 (A) is a granulation tissue formed subcutaneously. In the wound tissue (A) of the control experiment, granulation tissue was formed only in the subcutaneous part of the wound part, whereas in the wound tissue (B) treated with the bioresorbable woven fabric coated with PRP. A granulation tissue was formed throughout the subcutaneous part of the wound part. This result shows that the bioresorbable woven fabric coated with PRP has an effect of promoting wound healing. In this example, a PRP-coated woven fabric with a storage period of 1 day in the refrigerator was used, but a similar wound healing promoting effect was observed with a PRP-coated woven cloth with a storage period of 30 days.

Cell migration and cell differentiation promoting effect in human periosteum primary culture Preparation of PRP-coated woven fabric for human periosteum culture The bioabsorbable woven fabric (bicyclyl mesh) used in Example 1 is pre-coated with calcium stearate. In addition, since it is difficult for human cells to adhere only by coating with PRP, it is not suitable as a culture substrate for human periosteum fragments. Therefore, the bioabsorbable woven fabric for primary culture of the human periosteum piece of this example was coated with atelocollagen prior to the PRP coating. Specifically, a 7-10 mm × 7-10 mm bioabsorbable woven fabric (Bikerill mesh, catalog number: VICRYL WOVEN MESH (Polyglactin 910) VWMM, Johnson & Johnson Co., Ltd.) is a 3 mg / mL bovine dermis After coating with 50 μL of derived atelocollagen (Koken, IPC-30) solution for 15-30 minutes, lightly rinsed 3 times with sterile distilled water, dried on gauze and then room temperature with 120 μL of PRP prepared in Example 1 3-5 minutes.

Treatment of human periosteum pieces Periosteum pieces collected from a patient who provided PRP were put on a gauze moistened with a medium and aseptically transferred to a culture room, and were subjected to the following operations in a clean bench. The periosteum pieces were washed three times with PBS and then placed in the center on a woven fabric coated with atelocollagen and PRP. The periosteum pieces on the woven fabric were cultured in a plastic dish with a medium 199 medium containing 25 μg / mL vitamin C and 10% fetal calf serum for a total of 26 days.

Differentiation into osteoblasts The primary culture of the periosteum piece was carried out after 19 days in a medium 199 medium containing 10 nM dexamethasone, 10 mM β-glycerophosphate, 25 μg / mL vitamin C and 10% fetal calf serum. After switching, the cells were further cultured for 7 days.

Histological observation of primary culture of periosteum 26 days after the start of primary culture, a tissue specimen of periosteum culture on the woven fabric was prepared. Thin section preparation, hematoxylin-eosin staining and von Kossa staining were performed according to standard methods well known to those skilled in the art.

FIG. 3 shows hematoxylin-eosin staining (A) and von Kossa staining (B) on adjacent slices of primary periosteum cultures cultured on bioabsorbable woven fabric coated with atelocollagen and PRP, respectively. It is the optical microscope photograph of the tissue specimen which gave. In the hematoxylin-eosin stained specimen (A), active cell migration from the periosteum pieces was observed. In the von Kossa-stained specimen (B), a black stained portion was observed at the center of the periosteum piece, and calcified deposits were observed inside the periosteum piece. From this result, it was shown that the woven fabric coated with atelocollagen and PRP promotes cell migration from the periosteum piece and calcification in the periosteum piece. In this example, the woven fabric coated with atelocollagen and PRP was subjected to an experiment after being stored in a refrigerator for 1 day. However, the same cell migration and cell differentiation promoting effects were observed even in the coated woven fabric having a storage period of 30 days.

Claims (5)

Characterized in including that the platelet-rich plasma which has been lyophilized after being coated bioabsorbable fabric, cold storage material for regenerative therapy. Wherein the refrigerated material for the regeneration treatment is for long-term storage, refrigerated therapeutic materials for regenerative therapy of claim 1. The refrigerated storage material for regenerative treatment according to claim 1 or 2, wherein the bioabsorbable woven fabric is made of a polyester polymer of glycolic acid and lactic acid . The regenerative treatment according to any one of claims 1 to 3, wherein the solid support is coated with a ligand of a cell-substrate adhesion receptor before the platelet-rich plasma is coated. Material for refrigerated storage for. 5. The refrigerated storage material for regenerative treatment according to claim 4, wherein the ligand is atelocollagen.