JP6803067B2 - Skull joint member - Google Patents

Description

The present invention relates to a skull joining member made of β-tricalcium phosphate and a method for producing the same.

Bone plays a very important role in supporting and protecting various parts of the living body. In particular, the brain, which is important for living organisms, is protected by being covered with a skull. When the skull is lost due to trauma or tumor resection, it is required to be reconstructed at an early stage to protect the brain. In addition, since appearance deformation such as skin depression occurs, early reconstruction is desired for aesthetic reasons.

Examples of the method for reconstructing the skull include a method of filling and reconstructing a defect with autologous bone (own bone), resin, metal, bioceramics and the like. In the reconstruction method using autologous bone, a part of the skull, ilium, ribs, etc. is collected, filled in the defect, and the skull is reconstructed. Since it uses the patient's own tissue, it has excellent stability after replacement compared to artificial bone. On the other hand, it is said that the burden on the patient is heavy because deformation and scars due to bone defects remain at the collection site and are painful.

Metals such as titanium have biocompatibility and are said to be excellent in strength when used for reconstruction of the skull. However, it is said that it is non-absorbable and remains in the living body, and a foreign body reaction may be observed after transplantation into the body.

In the reconstruction method using bioceramics such as hydroxyapatite and tricalcium phosphate (TCP), the defect is filled in the form of a molded body, fibrous, reticular, powder or the like, and the skull is reconstructed. It has been reported that these have very high biocompatibility, are highly resorbable when transplanted into the body, and are replaced by bone. In addition, there are many reports that it is preferable that these artificial bones are porous.

In Patent Document 1, a calcium phosphate porous body or porous granules having a porosity of 50 to 90% and communicating pore diameters of 50 to 1000 μm and 5 μm or less is filled in the bone defect portion, and the upper surface is coated with an organic material that can be absorbed in vivo. The skull is being reconstructed by covering it. However, since the reconstructed member of Patent Document 1 is a scaffold material in the form of particles or granules, there is a problem that the scaffold material easily flows out in the living body.

Patent Document 2 describes a plug implant made of an expandable material, and shows a porous structure in which a filament layer made of an expandable material such as bioabsorbable polycaprolactone is laminated. According to Patent Document 2, the plug implant can be easily used and does not require any means for attaching to the bone. However, since the holes of the plug implant communicate three-dimensionally and the surface area of the implant is large, it may take time to regenerate the bone tissue, and the regeneration of the bone tissue in the skull is not always sufficient. ..

Patent Document 3 describes a three-dimensional matrix of monetite having a structured porosity, which has a vertical cylindrical macropore having a diameter of 350 to 650 μm in the structure of the three-dimensional matrix. A three-dimensional matrix is described characterized in that the holes penetrate the matrix longitudinally from one end to the other and the macro holes are separated by a distance of 0.4 to 0.6 mm. According to Patent Document 3, these matrices have advantageous properties such as biocompatibility, resorption, bone induction and revascularization, and are therefore best for cell colonization and proliferation. Since it can be used as a scaffold, it is said that these matrices can be used for tissue engineering and bone regeneration. However, since the three-dimensional matrix is made of monetite, its strength is not sufficient, and the pore diameter and pitch of macropores are limited. In addition, the mechanical strength was not sufficient to apply the three-dimensional matrix to skull defects, and the orientation of macropores was not investigated.

Japanese Unexamined Patent Publication No. 2003-10310 Japanese Patent Application Laid-Open No. 2007-512803 Japanese Patent Publication No. 2011-529429

The present invention has been made to solve the above problems, and an object of the present invention is to provide a skull joining member having good biocompatibility and excellent formation of bone tissue. Also provided are suitable methods of manufacturing such skull joint members.

The above-mentioned problem is a skull joint member made of β-tricalcium phosphate (hereinafter referred to as β-TCP), and the member is provided with a plurality of communication holes that communicate vertically from one surface to the other. The skull is reconstructed by arranging members in the bone defect so that the hole diameter of the communication hole is 100 to 1500 μm and the virtual axis connecting the dura mater and the periosteum at the shortest distance and the direction of the communication hole are substantially parallel. It is solved by a skull joint member, which is characterized by being used to.

At this time, the thickness of the member is preferably 1 to 20 mm. Further, the porosity of the member is preferably 20 to 60%.

At this time, it is also preferable that the thickness of the wall surface between the communication holes is less than 400 μm. It is also preferable that the inside of the communication hole is filled with BMP.

The above problem is also solved by providing a method for manufacturing a skull joining member having a step of firing at 1000 to 1400 ° C. after providing a communication hole. At this time, it is a preferable embodiment to impregnate the member with a medium containing 0.1 to 1000 μg / mL of BMP after the step of firing.

The skull joint member of the present invention has good biocompatibility and is excellent in bone tissue formation. This is effective in protecting the brain of a patient having a skull defect, and enables early bone regeneration even in a skull that is difficult to reconstruct. According to the manufacturing method of the present invention, such a skull joint member can be easily manufactured.

Photographs of skull regeneration results using skull joint members in Examples 1 and 2. Photographs of skull regeneration results using skull joint members in Comparative Examples 1 and 2. Cross-sectional view of the TCP molded body in the reference example and the surface photograph after firing Photograph of bone regeneration result of molded body after firing treatment in reference example X-ray diffraction results of the molded product after firing in the reference example

Hereinafter, embodiments of the present invention will be described.

The skull joint member of the present invention is made of β-TCP, and the member is provided with a plurality of communication holes that communicate vertically from one surface to the other, and the hole diameter of the communication holes is 100 to 1500 μm and is hard. It is characterized in that it is used to reconstruct the skull by arranging the member in the bone defect portion so that the virtual axis connecting the membrane and the periosteum at the shortest distance and the direction of the communication hole are substantially parallel to each other. ..

In the present invention, it is important to arrange the member in the bone defect portion so that the virtual axis connecting the dura mater and the periosteum at the shortest distance and the direction of the communication hole are substantially parallel to each other. By arranging in this way, the direction of the communication hole becomes substantially perpendicular to the intercranial diploë, and the communication hole at this time may be referred to as a vertical communication hole.

As can be seen from the comparison between Examples and Comparative Examples described later, even in a skull joint member made of β-TCP and having a plurality of communication holes having a pore diameter of 100 to 1500 μm, the dura mater and the periosteum are connected at the shortest distance. Good bone formation was not confirmed in Comparative Examples 1 and 2 in which the members were arranged in the bone defect portion so that the virtual axis and the direction of the communication hole were substantially perpendicular to each other. By arranging in this way, the direction of the communication hole becomes substantially horizontal with respect to the intercranial diploë, and the communication hole at this time may be referred to as a horizontal communication hole. On the other hand, in Examples 1 and 2 in which the skull joint member is arranged so that the virtual axis connecting the dura mater and the periosteum at the shortest distance and the direction of the communication hole are substantially parallel in the bone defect portion, it is good. Bone formation was confirmed. As can be seen from the results of Examples 1 and 2, it was confirmed that bone formation progressed from the dural side to the periosteal side at the bone defect site in the skull. It can be seen that the dura mater and the periosteum are linearly connected through the vertical communication hole by the skull joint member of the present invention, osteoblasts are sufficiently supplied into the communication hole, and the formation of bone tissue is excellent.

The skull joining member of the present invention is made of β-TCP, but it may be composed of β-TCP as a main component and may contain α-TCP in a part thereof. The main component is usually 60% by mass or more. It is preferable that the skull junction member is substantially composed of β-TCP only because of its excellent bioabsorbability.

The hole diameter of the communication hole of the present invention is 100 to 1500 μm. If the diameter of the communication hole is less than 100 μm, cartilage may be easily formed during bone formation. The hole diameter of the communication hole is preferably 200 μm or more. Further, when the hole diameter of the communication hole is larger than 1500 μm, bone formation is difficult and bone reconstruction using the member as a scaffold may not be performed. The pore diameter of the communication hole is preferably 1200 μm or less, more preferably 600 μm or less, and further preferably 450 μm or less.

The shape of the communication hole formed in the skull joining member of the present invention is not particularly limited, and examples thereof include a circle, an ellipse, a triangle, a quadrangle, and a hexagon. If the shape of the communication hole is not a circle, the diameter equivalent to the circle is used as the hole diameter.

The thickness of the skull joining member of the present invention is appropriately adjusted according to the thickness of the skull, but is preferably 1 to 20 mm. If the thickness is less than 1 mm, the strength when applied to the skull defect may be insufficient. It is more preferably 1 to 10 mm.

The porosity of the skull joint member of the present invention is preferably 20 to 60%. When the porosity is less than 20%, bone formation is performed in the communication hole, but the ratio of bone to the entire skull forming member is low, and the time until absorption into the living body may be long. On the other hand, if the porosity is greater than 60%, it may be difficult to maintain the shape of the skull joint member, and when it is placed on the head, it may be easily deformed by an external force. There is.

The thickness of the wall surface between the communication holes in the skull joining member of the present invention is preferably less than 400 μm. When the thickness of the wall surface is 400 μm or more, the ratio of bone to the entire skull forming member is low, and the time until it is absorbed into the living body may be long. The thickness of the wall surface is more preferably 300 μm or less, and further preferably 150 μm or less. The thickness of the wall surface is preferably 50 μm or more from the viewpoint of ease of manufacturing the communication hole.

It is preferable that the inside of the communication hole of the skull joining member of the present invention is filled with a filler such as an extracellular matrix. The extracellular matrix is not particularly limited, and examples thereof include collagen I, fibronectin, laminin, and Matrigel (registered trademark). By immersing the member in the filler, the filler can be filled inside the communication hole, and it is preferable to further centrifuge the immersed material or perform a treatment such as degassing under reduced pressure. ..

It is preferable that the inside of the communication hole of the skull joining member of the present invention is further filled with a bone morphogenetic factor such as a bone morphogenetic protein (BMP). This has the advantage of better formation of bone tissue. The specific BMP that can be used is not particularly limited, but for example, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-9, BMP-10, BMP- 11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-3b and the like. Of these, BMP-2, BMP-4, and BMP-7 are more preferable. The larger the amount of BMP, the greater the effect of promoting bone formation. The BMP content is preferably 1 to 500 μg and more preferably 10 to 200 μg with respect to 1 cm ^{3 of} the skull joining member.

The skull joint member of the present invention can be used by carving out from a large porous member so as to fit the bone defect portion of the skull, or a small porous member can be appropriately combined and used. The size of the skull joint member of the present invention is adjusted according to the bone defect portion of the skull.

The method for producing the skull joint member of the present invention is not particularly limited. Generally, when the firing temperature becomes high, an α-type crystal structure appears. When firing as an osteosynthesis member, firing at the highest temperature capable of maintaining a β-type crystal structure is preferable. A method having a step of forming a communication hole in the β-TCP member and then firing at 1000 to 1400 ° C. is preferable. When calcined at a temperature lower than 1000 ° C., a strong inflammatory reaction may be caused in the living body when placed on the head, and bone formation may not occur. The firing temperature is preferably 1150 ° C. or higher. In addition, when calcined at a temperature exceeding 1400 ° C., a strong inflammatory reaction may be caused in the living body when placed on the head, and the bone may be decomposed before being used as a scaffold for bone formation. is there. The firing temperature is preferably 1300 ° C. or lower. The method for producing the communication holes is not particularly limited, and examples thereof include extrusion molding, press molding, injection molding, mechanical drilling, and a method of bundling rod-shaped bodies having at least one through hole.

In the method for producing a skull joining member of the present invention, it is preferable to impregnate the member with a medium containing 0.1 to 1000 μg / mL of BMP after the firing step. From the viewpoint of better formation of bone tissue, the concentration of BMP is more preferably 20 μg / mL or more, further preferably 50 μg / mL or more. On the other hand, from the viewpoint of high cost, the concentration of BMP is more preferably 100 μg / mL or less. As the medium containing BMP, the above-mentioned extracellular matrix or the like can be preferably used.

Hereinafter, the present invention will be described in more detail with reference to Examples.

[Skull reconstruction test]
Example 1
The CaHPO ₄ · 2H ₂ O powder and CaCO ₃ 1: Water was added to a mixture 2 ratio, to obtain a 24 hours grinding kneaded to a slurry in a ball mill. The slurry was dried to obtain a molded product made of a rectangular parallelepiped TCP precursor having a length of 3.95 mm, a width of 3.95 mm, and a thickness of 2 mm having 91 communication holes having a diameter of 330 μm that communicate in the vertical direction. The molded product was heated to 1200 ° C. at a rate of 50 ° C. h- ¹ and held at that temperature for 1 hour for firing treatment to obtain a skull joining member made of β-TCP. The thickness of the wall surface between the communication holes of the skull joining member was about 100 μm, and the porosity was 49.8%. After the skull joint member is sterilized by an autoclave, the skull joint member is immersed in Matrigel (registered trademark) (manufactured by BD Bioscience) and centrifuged to fill the communication hole of the skull joint member with Matrigel (registered trademark). It was.

A skull defect model of an 8-week-old male Wistar rat was used for the study. After making an incision in the skin of the rat's head, the skull was cut with a diamond bar to preserve the dura and create one bone tissue defect of about 5 x 5 mm on each side. The skull joint member was sterilized by an autoclave, and the communication hole was placed at the bone defect site so that the direction of the communication hole was substantially parallel to the virtual axis connecting the dura mater and the periosteum at the shortest distance, and the head skin was sutured.

Rats were euthanized 3 weeks after implantation of the skull junction and the skull junction was removed along with the surrounding skull tissue. The excised tissue was fixed overnight with 4% paraformaldehyde, then decalcified with a 10% ethylenediaminetetraacetic acid (EDTA) solution, a paraffin block was prepared by a conventional method, and stained with hematoxylin and eosin (HE staining). Later, histological observation was performed. The results are shown in Figures 1-a and 1-b.

Example 2
A skull reconstruction test was carried out in the same manner as in Example 1 except that Matrigel (registered trademark) (manufactured by BD Bioscience) mixed with BMP-2 having a concentration of 80 μg / mL was filled in the communication holes of the skull joining member after the sterilization treatment. Was done. The BMP content of the entire skull joint member was about 1.24 μg. The results are shown in FIGS. 1-c and d.

Comparative Example 1
A molded product made of a rectangular parallelepiped TCP precursor was obtained in the same manner as in Example 1 except that the length was 3.95 mm, the width was 3.95 mm, and the thickness was 5 mm. The molded product baked at 1200 ° C. in the same manner as in Example 1 is cut at a width of 2 mm so that the cut surface is parallel to the communication hole, divided into two equal parts, and a skull joint member made of β-TCP. Got The same as in Example 1 except that the skull joint member sterilized by the autoclave was arranged at the bone defect site so that the direction of the communication hole was substantially perpendicular to the virtual axis connecting the dura mater and the periosteum at the shortest distance. Then, a skull reconstruction test was performed. At this time, the communication hole is in a state where the communication hole is in contact with the inter-skull diploë, that is, a horizontal communication hole. The results are shown in FIGS. 2-a and 2-a and b.

Comparative Example 2
Skull reconstruction test in the same manner as in Comparative Example 1 except that Matrigel (registered trademark) (manufactured by BD Bioscience) mixed with BMP-2 having a concentration of 80 μg / mL was filled in the communication hole of the skull joining member after the sterilization treatment. Was done. The results are shown in FIGS. 2-c and d.

[Results of tissue observation in skull reconstruction]
In the skull joint member arranged as in Example 1, the formation of bone tissue was observed even under the condition that BMP was not added (FIGS. 1-a and 1-b). The bone tissue was formed from the dural side, and the formation of bone tissue was observed up to the vicinity of the periosteum in the connected hole. Almost no inflammatory reaction was observed in the communicating hole, and good bone tissue was formed so as to be added to all the inner walls of the hole.

In the skull joint member (Example 2) arranged as in Example 1 and further added with BMP, more vigorous formation of bone tissue was observed than in Example 1 (Fig. 1-c, d). Bone tissue was formed so as to fill the entire hole with all communication holes, and the formation of bone tissue almost reached the outer periosteum. The formation of bone marrow tissue was also confirmed.

On the other hand, in the skull joint members arranged as in Comparative Example 1, bone tissue formation was observed even under the condition that BMP was not added, but only in the vicinity of the dura mater (Fig. 2-a, b). ). No formation of bone tissue was observed in the hole on the periosteal side.

Further, in the skull joint member (Comparative Example 2) arranged as in Comparative Example 1 and further added with BMP, the amount of bone tissue formed was increased as compared with Comparative Example 1, but bone was formed in all the holes. No formation was observed (Fig. 2-c, d).

Reference example 1
The CaHPO ₄ · 2H ₂ O powder and CaCO ₃ 1: Water was added to a mixture 2 ratio, to obtain a 24 hours grinding kneaded to a slurry in a ball mill. The slurry was dried to obtain a molded product made of a rectangular parallelepiped TCP precursor having 91 through holes having a diameter of 330 μm, a length of 3.95 mm, a width of 3.95 mm, and a thickness of 1 mm (porosity: 49.8). %). The obtained molded product was heated to 1100 ° C. at a rate of 50 ° C. h ^-1 and held at that temperature for 1 hour for firing treatment.

[Measurement of X-ray diffraction]
X-ray diffraction was measured using XRD (BRUKER D8 Advance) on the molded product (1100TCP) subjected to the firing treatment (X-ray: Cu-Kα ray, output: 40 KV, 40 mA). The scan angle range was 20 ° to 60 ° and the scan speed was 2 ° min- ¹ . Diffraction patterns were compared with the Joint Committee on Powder Diffraction Standards Powder Diffraction Files database.

[Observation of surface morphology]
The molded product after the firing treatment was coated with platinum, and the surface structure was observed using SEM (Philips XL30W10D). The results are shown in FIG.

[Bone regeneration test]
The test was performed on two healthy 4-week-old male Wistar rats. A bone defect was provided in the ear canal, and a molded body (1200 TCP) was implanted to compensate for the bone defect. After 4 weeks, the molding (1200TCP) was removed from the rat and fixed in neutral buffered formalin. The sample was immersed in 10% EDTA solution for 2 weeks to remove calcium. Subsequently, the sample was embedded in paraffin, the sections were HE-stained, and histological observation was performed. The results are shown in FIG.

Reference examples 2-6
The firing treatment was performed and evaluated in the same manner as in Reference Example 1 except that the temperature of the firing treatment was changed as shown in Table 1. The results are shown in Table 1 and FIGS. 3-5.

[X-ray diffraction result]
From the X-ray diffraction results of Reference Examples 1 to 6, it was found that the composition of the molded product after the firing treatment depends on the firing temperature (Table 1). In the samples having the firing temperatures of 1100, 1150 and 1200 ° C., only the peak of β-TCP was detected as the composition of the molded product after firing (Fig. 5-a). In the sample having a firing temperature of 1250 ° C., a β-TCP peak was mainly detected as the composition of the molded product after firing, and a slight α-TCP peak was also detected (FIG. 5-b). In the sample having a firing temperature of 1500 ° C., no β-TCP peak was confirmed, and only the α-TCP peak was detected (Fig. 5-c).

[Bone regeneration test results]
It can be seen that as the firing treatment temperature of the molded product increases, the number of particles decreases, the particle size increases, and the surface becomes smooth (FIG. 3-b). When a sample at each treatment temperature was implanted in the living body, a foreign body reaction such as infiltration of inflammatory cells was confirmed by 1100 TCP. Among the conditions in which only β-TCP was confirmed in the composition, 1200 TCP treated at the highest temperature reduced the inflammatory response and confirmed new bone cells (Fig. 4). In 1250 TCP, in which α-TCP was confirmed in the composition, an inflammatory reaction was confirmed again. In 1500 TCP, the embedded sample was dissolved during 4 weeks (Fig. 4). Therefore, it can be seen that the most preferable embodiment is to use a molded product obtained by firing at 1200 ° C.

Claims (7)

A skull junction member consisting of β-tricalcium phosphate.
The member is provided with a plurality of communication holes that vertically communicate from one surface to the other, and the hole diameter of the communication holes is 100 to 600 μm.
By arranging the member in the bone defect portion so that the virtual axis connecting the dura mater and the periosteum at the shortest distance and the direction of the communication hole are substantially parallel , bone tissue is formed in all of the communication holes. A skull joint member, characterized in that it is used to reconstruct a skull. The skull joining member according to claim 1, wherein the thickness of the member is 1 to 20 mm. The skull joining member according to claim 1 or 2, wherein the member has a porosity of 20 to 60%. The skull joining member according to any one of claims 1 to 3, wherein the thickness of the wall surface between the communication holes is less than 400 μm. The skull joining member according to any one of claims 1 to 4, wherein the inside of the communication hole is filled with BMP. The method for producing a skull joint member according to any one of claims 1 to 5, further comprising a step of firing at 1000 to 1400 ° C. after providing the communication holes. The method for producing a skull joining member according to claim 6, wherein after the firing step, a medium containing 0.1 to 1000 μg / mL of BMP is impregnated with the member.