Bone formation enhancer

Abstract

To provide a material useful for improving the efficiency and reliability of bone augmentation performed when implants are placed. [Solution] A bone formation enhancer containing photocrosslinkable collagen and a photocrosslinking initiator as active ingredients, which is used by applying to a bone filler packed into the bone formation area and then irradiating with light during bone formation at the time of implanting a dental implant.

Description

[Technical Field]

[0001] The present invention relates to an agent for enhancing bone formation in bone augmentation during dental implant placement. [Background technology]

[0002] Prosthetic treatment using implants requires sufficient bone of sufficient quality and quantity at the treatment site to allow the implant to be placed. Therefore, in areas with severe periodontal disease or areas where the surrounding alveolar bone has been lost along with the teeth due to trauma, bone augmentation is necessary to ensure sufficient bone volume. For areas with large bone resorption, guided bone regeneration (GBR) is widely used to create space using autologous bone and bone substitute materials, covering the area with a strong, moldable membrane, and has shown a high clinical success rate.

[0003] Autologous bone grafting is considered the "gold standard" for bone augmentation, but in reality, supplies are limited, and various bone substitute materials are used. Bone substitute materials are made from various inorganic substances such as hydroxyapatite, carbonate apatite, and bovine freeze-dried bone, but most are in granular form for ease of handling and efficient packing.

[0004] In the GBR method, granular bone substitute material is placed in the bone defect area, a membrane is placed on top of it, and after it is fixed with screw pins as necessary, the mucosal flap is sutured closed. However, because the bone substitute material is granular, it can fall off the surgical site, or it can be unable to maintain its shape due to the pressure or movement of the membrane after placement, or it can spill over into areas where it is not needed, or it can even get lost in the mucosal flap.

[0005] Bone substitute materials are generally soaked in saline to improve their operability, but attempts have also been made to mix them with collected blood, or to apply hyaluronic acid in the hope of increasing their cohesive power.The larger the bone augmentation, the more likely it is that the bone substitute will overflow from the recipient site, and the greater the pressure from the membrane and mucosal flap, making it necessary to maintain the bone substitute, but currently there are no materials specialized for this function.

[0006] Meanwhile, in recent years, various bioinks have been devised that enable 3D printing of structures containing living cells, and photocrosslinkable collagen, which crosslinks upon light irradiation, such as methacrylated collagen, is a useful bioink with high industrial value (e.g., Patent Document 1). However, there have been no reports of photocrosslinkable collagen being used for bone augmentation during implant placement, and it is completely unknown that it is useful for enhancing bone formation during bone augmentation. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2023-7562 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to providing a material that is useful for improving the efficiency and reliability of bone augmentation performed when placing a dental implant. [Means for solving the problem]

[0009] The inventors have conducted research in light of the above problems and have found that by filling the bone formation area with bone filler, applying photocrosslinkable collagen and a photocrosslinking initiator to the upper part of the bone formation area, and then irradiating it with light to crosslink the collagen, it is possible to suppress the dispersion and disintegration of bone filler granules from the surface of the bone formation area, promote the proliferation of osteoblasts, and enable reliable and rapid bone formation.

[0010] That is, the present invention relates to the following 1) to 6). 1) A bone formation enhancer containing photocrosslinkable collagen and a photocrosslinking initiator as active ingredients, which is used in bone formation at the time of implanting a dental implant by applying it to a bone filler packed into the bone formation area and then irradiating it with light. 2) The bone formation enhancer according to 1), wherein the photocrosslinkable collagen is collagen having a (meth)acryloyl group introduced therein. 3) The bone formation enhancer according to 1), wherein the photocrosslinkable collagen is methacrylated collagen. 4) The bone formation enhancer according to 3), wherein the collagen is type I collagen. 5) The bone formation enhancer according to any one of 1) to 4), wherein the light irradiation is with visible light having a wavelength of 405 nm. 6) The bone formation enhancer according to any one of 1) to 4), wherein the photocrosslinking initiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate. [Effects of the Invention]

[0011] The bone formation enhancer of the present invention can prevent the dispersion and disintegration of bone filler granules from the surface of the bone formation area during bone formation at the time of implanting a dental implant, thereby reliably maintaining the bone filler, and can also promote the proliferation of osteoblasts, thereby enabling reliable and rapid bone formation. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a flow chart showing one mode of use of the bone formation enhancer of the present invention. [Figure 2] Vibration-resistant effect of hydroxyapatite granules with cross-linked collagen. [Figure 3] Cross-linked collagen promotes osteoblast proliferation. DETAILED DESCRIPTION OF THE INVENTION

[0013] In the present invention, the term "photocrosslinkable collagen" refers to collagen that is photocrosslinked into which photocrosslinkable functional groups have been introduced. The photocrosslinkable collagen used in the present invention is not limited to any particular type as long as it can adhere bone fillers and induce bone tissue regeneration, but preferred examples include photocurable modified collagens in which (meth)acryloyl groups or organic groups containing (meth)acryloyl groups have been introduced into collagen molecules as photocrosslinkable functional groups. Here, (meth)acryloyl refers collectively to acryloyl and methacryloyl. Such photocrosslinkable collagen can be produced by a known chemical synthesis method, for example, by reacting collagen with a (meth)acrylic acid derivative in a hydrophilic solvent.

[0014] In the present invention, the photocrosslinkable collagen is preferably collagen having methacryloyl groups (also referred to as "methacrylated collagen"), and is sold, for example, by Advanced BioMatrix as "PhotoCol (registered trademark) Methacrylated Collagen." Such methacrylated collagen is collagen methacrylamide (CMA), which is obtained by methacrylating the free amino acids of bovine type I collagen (the ε-amino groups of lysine residues and the α-amino group at the N-terminus) (Gaudet, ID, Characterization of Methacrylated Type-I Collagen as a Dynamic Photoactive Hydrogel, Biointerphases, 2012 December; 7(1): 25.).

[0015] As for the collagen into which (meth)acryloyl groups have been introduced, it is preferable that 20% or more, for example 20 to 40%, of the collagen molecules are (meth)acrylated, from the viewpoint of enhancing bone formation.

[0016] Collagen of multiple types, differing in amino acid sequence, peptide length, presence or absence of fibrogenicity, etc., has been isolated and identified from living organisms, and any type can be used in the present invention, but type I collagen is preferred from the viewpoint of enhancing bone formation. The origin of collagen is not limited, but collagen from mammals such as bovine, porcine, equine, chicken, and human is preferred, and human-derived collagen can be expressed from human placenta cDNA or produced by culturing human fibroblasts.

[0017] In the present invention, the photocrosslinkable collagen is used together with a photocrosslinking initiator. The photocrosslinking initiator is not particularly limited as long as it generates radical species upon irradiation with light (UV or visible light). From the viewpoint of low toxicity and low irritation, examples include lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), camphorquinone, 9-fluorenone, and Irgacure 2959 (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone), Irgacure 819 (phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide), and Irgacure 184 (1-hydroxycyclohexylphenyl ketone), all of which are manufactured by Ciba Specialty Chemicals. However, visible light-activated photocrosslinking initiators such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), camphorquinone, and 9-fluorenone are preferred, with LAP being more preferred due to its high water solubility and cytocompatibility.

[0018] In the present invention, the photocrosslinkable collagen is preferably used in a state of being dissolved in an acidic solvent, more preferably as a solution containing the above-mentioned photocrosslinking initiator, from the viewpoints of the solubility of the collagen, low irritation, and low toxicity. Here, the acidic solvent may be, for example, an aqueous solution of acetic acid, and a 10 to 50 mM aqueous solution of acetic acid is preferred. A suitable example of an acidic solution containing photocrosslinkable collagen is a solution prepared by dissolving photocrosslinkable collagen in a 20 mM aqueous acetic acid solution at a concentration of 2 to 10 mg / mL, preferably 3 to 8 mg / mL. A suitable buffer solution can be added to such a solution to adjust the pH to 6 to 7. A kit combining methacrylated collagen and a photocrosslinking initiator is sold by Advanced BioMatrix as "PhotoCol (registered trademark) Methacrylated Type I Collagen Crosslinkable Hydrogel Kit."

[0019] The solution containing such photocrosslinkable collagen may further contain disinfectants, local anesthetics, wound healing agents, epidermal growth factors, and other medicinal ingredients as appropriate.

[0020] As shown in the Examples below, when a photocrosslinkable collagen solution containing a photocrosslinking initiator is added to hydroxyapatite granules, which are a granular bone filler material placed on a glass plate, and the light-irradiated sample is then vibrated with a vibrator, the sample remains intact without any disintegration.Furthermore, when a photocrosslinkable collagen solution containing a photocrosslinking initiator is added to hydroxyapatite granules and osteoblast-like cells are cultured in a medium containing the light-irradiated sample, the proliferation of the cells is significantly promoted. Therefore, the photo-crosslinkable collagen and the photo-crosslinking initiator can be used as a bone formation enhancer for enhancing bone formation during bone augmentation when implanting a dental implant by applying them to a bone filler packed in the bone augmentation site and then irradiating them with light. Here, "enhancing bone formation" means the action of adhering and maintaining the bone filler in bone augmentation surgery, as well as promoting the proliferation of osteoblasts and increasing bone formation ability.

[0021] In the present invention, the bone augmentation technique used when placing a dental implant includes the Guided Bone Regeneration (GBR) method. The GBR method is a bone augmentation technique for ensuring sufficient bone mass in prosthetic treatment using dental implants. The GBR method involves filling a bone defect area where bone tissue is to be guided and regenerated with a bone filler, shaping it into a desired shape, and then covering the bone defect area with a specialized membrane to ensure the space necessary for new bone regeneration in order to prevent the intrusion of biological tissues other than bone tissue, such as gums, from the surrounding area and inhibiting bone regeneration. Known bone fillers include autologous bone, allogeneic bone (allogeneic bone), heterogeneous bone, and artificial materials (β-tricalcium phosphate (β-TCP), hydroxyapatite, carbonate apatite, etc.), and in the present invention, these can be appropriately selected and combined for use.

[0022] For light irradiation, a light source suitable for activating the photocrosslinking initiator used is used, and either ultraviolet or visible light may be used, but a visible light source (wavelength 380 to 800 nm) is preferably used. For example, it is preferable to use an LED irradiator capable of irradiating visible light with a wavelength of 405 nm or 420 nm. Light irradiation is carried out at an intensity and time that allows collagen crosslinking. For example, visible light with a wavelength of 405 nm is irradiated at an irradiation distance of 1 cm or less at 400 to 2000 mW / cm. 2 For example, irradiation is performed for 1 to 5 minutes under the above conditions.

[0023] When bone augmentation (GBR) is performed using the bone formation enhancer of the present invention, the bone augmentation site is filled with a bone filler and shaped into any desired form. Photocrosslinkable collagen is then applied to the top of the bone augmentation site together with a photocrosslinking initiator (specifically, a photocrosslinkable collagen solution containing the above-mentioned photocrosslinking initiator is dripped onto the surface), and the collagen is photocured by irradiation with light. The surface is then covered with a membrane, and the mucosal flap is closed. This procedure prevents the dispersion and disintegration of bone filler granules from the surface of the bone augmentation site, allowing the surface to withstand the pressure and frictional forces of the membrane coating, ensuring the bone augmentation site is maintained, and promoting the proliferation of osteoblasts, enabling reliable and rapid bone augmentation. Also, when using the bone formation enhancer of the present invention, as shown in FIG. 1, the membrane coating step in the GBR method can also be omitted. In this case, a photocrosslinkable collagen solution containing a photoinitiator is dropped (A) onto the upper part of the bone formation site, irradiated with light to photocure the collagen (B), and then the mucosal flap is sutured and closed (C).

Example

[0024] Example 1 Evaluation of the dispersibility of hydroxyapatite granules using photocrosslinkable collagen 1. Materials · Photocrosslinkable collagen: "PhotoCol (registered trademark) LAP kit" (Advanced BioMatrix) PhotoCol (registered trademark): Methacrylated type I collagen (lyophilized) · Photoinitiator: Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; Advanced BioMatrix) · Hydroxyapatite granules: "Apaseram (registered trademark)" (HOYA, Japan)

[0025] 2. Methods 0.5 ml of hydroxyapatite granules was placed on a glass plate, and 1 ml of a photocrosslinkable collagen solution (polymerization initiator added) prepared at a concentration of 6.0 mg / mL with 20 mM acetic acid <COL6> was dropped thereon. Visible light with a wavelength of 405 nm was irradiated at an irradiation distance of 1 cm with a 2000 mW / cm 2 condition for 1 minute using an LED irradiator (PenCure2000, MORITA), and cultured at 37°C for 5 minutes (COL6 group). A group with physiological saline dropped instead of COL6 was designated as the raw food group, and a group with nothing dropped was designated as the control group. The three groups were applied to a dental laboratory vibrator for 5 seconds, and the subsequent diffusion state was observed. The results are shown in FIG. 2.

[0026] 3. Results and Discussion As shown in Figure 2, in the control group, vibration caused the hydroxyapatite granules to disperse largely outside the glass plate. In the saline group, the hydroxyapatite granules did not disperse, but they did move significantly. In contrast, in the COL6 group, vibration did not cause any change in position, and no dispersion of the hydroxyapatite granules was observed. This suggests that cross-linked collagen increases resistance to vibration and contributes to morphological stability.

[0027] Example 2: Examination of the effect of photocrosslinkable collagen on osteoblasts 1.Material Photocrosslinkable collagen: "PhotoCol® LAP Kit" (Advanced BioMatrix) PhotoCol®: Methacrylated Type I Collagen (lyophilized) Photoinitiator: Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; Advanced BioMatrix) Hydroxyapatite granules: "Apaseram (registered trademark)" (HOYA, Japan) Mouse calvarial osteoblast-like cells (MC3T3-E1)

[0028] 2. Evaluation of osteoblast proliferation A 24-well plate was filled with 1 ml of hydroxyapatite granules, and photocrosslinkable collagen solutions (with crosslinking initiator added) prepared with 20 mM acetic acid at 3.0 mg / mL and 6.0 mg / mL concentrations (referred to as COL3 and COL6, respectively) were dropped onto the plate. The plates were then irradiated with visible light at a wavelength of 405 nm using an LED irradiator for 1 minute under the same conditions as in Example 1, and then cultured for 5 minutes (COL3 and COL6 groups, respectively). As a control group, saline was dropped onto the hydroxyapatite granules. 1000 mL of Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin was dropped into the wells. Next, MC3T3-E1 cells were added at 1 x 10 4Cells were seeded at 1000 cells / well and cultured for 1, 3, and 5 days. The medium was changed every 2 days. After the culture was completed, non-adherent cells were removed with phosphate buffered saline (PBS). The absorbance at 450 nm was measured as the luminescence intensity using the WST-8 method with a Cell Counting Kit-8 (Dojindo, Japan) on a microplate reader (Varioskan Flash 2.4, Thermo Fisher Scientific, USA). The results are shown in Figure 3.

[0029] 3.Results As shown in Figure 3, on day 1 of culture, cell numbers increased significantly in both the COL3 and COL6 groups compared to the control group. Furthermore, the COL6 group showed significantly higher initial cell adhesion ability than the COL3 group. The number gradually increased in all groups on days 3 and 5 of culture, and the difference between the groups widened further on day 5, with the COL6 group showing the highest proliferation ability. Cross-linked collagen has the effect of promoting osteoblast proliferation, and this effect was particularly pronounced when a 6.0 mg / ml photocross-linked collagen solution was used.

Claims

1. A bone formation enhancer containing photocrosslinkable collagen and a photocrosslinking initiator as active ingredients, which is used in bone formation at the time of implanting a dental implant by applying it to a bone filler packed into the bone formation area and then irradiating it with light.

2. The bone formation enhancer according to claim 1, wherein the photocrosslinkable collagen is collagen having a (meth)acryloyl group introduced therein.

3. The bone formation enhancer according to claim 1, wherein the photocrosslinkable collagen is methacrylated collagen.

4. The bone formation enhancer according to claim 3, wherein the collagen is type I collagen.

5. The bone formation enhancer according to any one of claims 1 to 4, wherein the light irradiation is irradiation with visible light having a wavelength of 405 nm.

6. The bone formation enhancer according to any one of claims 1 to 4, wherein the photocrosslinking initiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

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