Hydroxyapatite Injection and its Preparation Method

Abstract

This invention relates to hydroxyapatite injections and their preparation methods, belonging to the field of biomimetic biological repair materials. By modifying hydroxyapatite and compounding it, an injectable hydroxyapatite is obtained. Specifically, it relates to a self-assembled polypeptide and nano-sized hydroxyapatite composite injection and its preparation method. It has advantages such as injectability, strong biomimetic mineralization ability, and cell adhesion promotion, overcoming the shortcomings of existing microsphere fillers, and has significant bone repair promotion effects and good clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomimetic biological repair materials. By modifying hydroxyapatite, a compound hydroxyapatite that can be injected is obtained. Specifically, it relates to a self-assembled polypeptide, a nano-sized hydroxyapatite composite injection and its preparation method. Background Technology

[0002] The hydroxyapatite described in the published literature is mostly in the form of microspheres, with a size in the micrometer range. It is generally used for cosmetic injections and contour support, and is not entirely suitable for small, irregular bone loss cases.

[0003] For small, irregular, bone defects, commonly used bone repair materials such as bone particles and bone cement are difficult to fill small or irregularly shaped bone defects. They are also difficult to inject into the lesion site through fine needles. Existing common supplementary materials are like a plaster covering the missing area, but they do not fit completely and have poor integration with the bone structure itself. Such bone defects are difficult to repair effectively through traditional surgical methods.

[0004] Therefore, injectable bone substitutes have become a research hotspot in recent years. Existing technologies, such as patent CN118557795B, employ a biphasic composite of hydroxyapatite microspheres and cross-linked hyaluronic acid (HA) to address the hydroxyapatite sedimentation problem; CN118718088B modifies microspheres with amino / carboxyl groups to improve dispersion stability through electrostatic interactions. CN119236167B uses porous microspheres derived from animal bone, combined with collagen / guar gum / PEG, to form a filler. All of these solutions use microspheres as the main carrier, which presents problems such as large particle size, easy precipitation and sedimentation, and needle blockage during injection. For example, literature reports that in injectable gels containing a mixture of cross-linked hyaluronic acid and HA particles, HA microspheres are difficult to distribute uniformly, easily displacing and accumulating during injection, ultimately causing needle blockage. Therefore, existing injectable materials based on HA microspheres are still insufficient to meet the needs of minimally invasive injections and filling of irregular defects.

[0005] The nano-sized hydroxyapatite injection of the present invention can be precisely injected into the defect site through a minimally invasive method. It utilizes the guiding effect of self-assembled peptides to promote the directional deposition of nano-hydroxyapatite. It has strong nano-level penetration, stimulates growth, harmonizes the body, can be phagocytosed by cells, and naturally has a strong binding to simulate the mineralization process of natural bone tissue, so as to achieve the synchronous reconstruction of structure and function. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention utilizes nano-hydroxyapatite (D50 50–80 nm) and self-assembled collagen peptides to construct a biomimetic nanofiber / mineral phase composite system, which is then compounded with biodegradable polymers (PVA, PLGA / PLLA) to create an injectable agent with shear-thinning properties. This system avoids large-sized microspheres and combines permeability, injection feasibility, self-assembly-induced mineralization, and cell adhesion promotion functions, making it suitable for minimally invasive injection treatments in bone repair and dental fillings.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A hydroxyapatite injection, said injection comprising the following components by weight percentage:

[0009] Nano-sized hydroxyapatite 5–20 wt%

[0010] Self-assembled collagen peptides 0.5–4 wt%

[0011] The remainder consists of physiological buffer solutions and auxiliaries;

[0012] The nano-sized hydroxyapatite has a particle size D50 of 50–80 nm, the self-assembled collagen peptide contains a Gly-XY repeat sequence with an amino acid length of 9–30 residues, and the self-assembled collagen peptide contains 0–2 RGD fragments and has the ability to self-assemble into nanofibers at near physiological pH.

[0013] Preferably, the self-assembled collagen peptide consists of three functional domains: the first part is 1-3 Gly-XY repeating units to provide a tendency for triple helix or short fragment folding; the second part is 0-2 RGD or equivalent short peptides for cell adhesion to provide cell adhesion sites; and the third part is charged residues.

[0014] Preferably, the self-assembly induction conditions for the self-assembled collagen peptide are a peptide concentration of 0.5-5 mg / mL, a solution pH of 6.5-7.4, and a temperature of 20-37 ℃ for 30-120 minutes, forming nanofibers or fiber bundles with a diameter of 5-30 nm.

[0015] Preferably, the additives include: pH buffers and viscosity modifiers.

[0016] Preferably, the pH buffer is either a Tris solution or a phosphate solution.

[0017] Preferably, the viscosity modifier is poloxamer F127.

[0018] A second aspect of this invention provides a method for preparing a hydroxyapatite injection, comprising the following steps:

[0019] 1) Animal-derived bone is calcined and crushed to obtain primary hydroxyapatite (nHA). Then, liquid medium is added to grind large particles using the collision and shear force of grinding balls. The ball-to-material ratio is 10-20:1; the rotation speed is 300-600 rpm; the total ball milling time is 12-36 h with intermittent cooling; the obtained product is centrifuged, washed and dried to obtain nano-sized hydroxyapatite; the liquid medium is selected as ethanol, isopropanol or deionized water to minimize agglomeration.

[0020] 2) Disperse the obtained nHA in the dehydrated phase, add a dispersant and ultrasonically disperse, adjust the pH to 7.0–8.0 to obtain a stable nHA dispersion;

[0021] 3) Dissolve the collagen peptides in a weak acid or buffer solution, adjust the pH to 6.5-7.4, and incubate at 20-37°C for 30-120 minutes to initiate the self-assembly of collagen peptides;

[0022] 4) Mix the nHA dispersion obtained in step 2) with the peptide solution obtained in step 3) under low shear conditions to induce the peptide to assemble on / around the nHA surface and induce mineralization and template formation.

[0023] 5) Homogenize and degas at low speed, adjust pH and osmotic pressure to physiological levels to obtain the final injectable formulation; and store under sterile conditions.

[0024] By adopting the above technical solutions and adding dispersants to prevent re-agglomeration, and by implementing cooling measures to avoid local overheating that could lead to a phase transformation.

[0025] Preferably, the dispersant is at least one of sodium citrate or polyacrylic acid, and the amount of dispersant used is 0.1-3 wt% of the mass of nano-hydroxyapatite. The surface of the nano-hydroxyapatite is modified by the dispersant to improve its stability.

[0026] A third aspect of the present invention provides the use of a hydroxyapatite injection for minimally invasive injection filling of bone or tooth defects, for filling and penetrating microcracks or irregular depressions and promoting local mineralization and bone regeneration through the guiding action of the self-assembled polypeptide complex.

[0027] Preferably, the injection force is 5–30G needle, and the peak value of the syringe advance is <30N per 1mL.

[0028] Preferably, the injection can be compounded with a polymer, wherein the polymer is one of poly(L-lactic acid) (PLLA), polyvinyl alcohol (PVA), polylactic acid-glycolic acid copolymer (PLGA), or polyglycolic acid (PGA).

[0029] Furthermore, the polymer, such as PLGA / PLLA, is dispersed in the system in the form of nanoparticles or short fibers with a particle size of 150–400 nm or a fiber length of 3–5 μm.

[0030] The beneficial effects of this invention are:

[0031] 1) Composed of nano-sized HA and low-viscosity polymer solution, with moderate overall viscosity, it can be injected minimally invasively through a 25–30G needle. After injection, it has good fluidity and can easily penetrate into small and irregular defect areas such as bone sutures and depressions, with a filling effect superior to microsphere fillers, balancing good injectability and permeability;

[0032] 2) Collagen peptides self-assemble into a nanofiber network structure in solution and induce hydroxyapatite deposition in the network to form a bone-like matrix. Studies have shown that self-assembled peptides can nucleate hydroxyapatite in situ in vivo and significantly promote bone defect repair. This scheme utilizes the biomineralization ability of peptides to achieve one-step, cell-free bone regeneration.

[0033] 3) The biomimetic collagen fiber structure mimics the natural extracellular matrix, which is beneficial for osteoblast adhesion, migration and proliferation; the material is biodegradable, and the degradation products can be phagocytosed and cleared by macrophages. In addition, the synthetic peptide and polymer complex system can provide more adhesion sites for cells and promote tissue remodeling.

[0034] 4) Compared with traditional HA microsphere injections, this formulation does not contain large-sized particles, avoiding problems such as HA microparticle sedimentation, phase separation, and needle blockage during long-term storage or in vivo use. Nano HA remains stably suspended under the action of dispersants, and can fully combine with collagen peptides and polymers, giving the injection good uniformity and stability, maintaining long-term stability without precipitation, and synergistically with high molecular polymers, with adjustable mechanical and degradation rates, making it suitable for various clinical scenarios.

[0035] In summary, this invention innovatively combines nano-HA with self-assembled peptides to form an injectable fluid, which is significantly different from existing patented technologies. Compared with existing technologies based on HA microspheres, this solution abandons the microsphere structure and uses nanomaterials that can easily penetrate tissue fissures; it utilizes self-assembled peptides to guide the mineralization reaction, resulting in stronger biomineralization capabilities and better promotion of cell adhesion; and the overall system can be injected into minimally invasive areas, adapting to complex and irregular defect morphologies. This invention combines injection feasibility with biomimetic functionality, demonstrating significant bone repair promotion effects and broad application prospects. Attached Figure Description

[0036] Figure 1 Scanning electron microscope image of hydroxyapatite;

[0037] Figure 2 A schematic diagram of the targeted penetration of an injection agent to repair irregular small gaps in bone source. Detailed Implementation

[0038] The present invention will be further described below through specific embodiments. To make the inventive objectives, technical solutions, and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention.

[0039] Unless otherwise stated, all instruments and reagents used in the examples are commercially available or synthesized using conventional methods and can be used directly without further processing, and all instruments used in the examples are commercially available.

[0040] Materials and reagents

[0041] Sodium citrate (analytical grade), ≥99.0%, AR, purchased from Thermo Fisher Scientific;

[0042] Polyacrylic acid (PAA), molecular weight: 2,000–450,000, purchased from Aladdin;

[0043] Polyvinyl alcohol (PVA), degree of hydrolysis (87–99%), molecular weight (30,000–146,000), purchased from Aladdin;

[0044] PLGA, LA:GA ratio (50:50, 75:25, 85:15, etc.); molecular weight 10k–100k, purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0045] PLLA, molecular weight 50k–300k; L-configuration purity > 98%, purchased from Shenzhen Guanghua Weiye;

[0046] PBS, purchased from Sangon Biotech (Shanghai).

[0047] Example 1:

[0048] Formulation (total 100 wt% by weight): nano hydroxyapatite (nHA) 8.0 wt%; self-assembled collagen peptides 1.5 wt%; dispersant (sodium citrate) 0.3 wt%; Poloxamer F127 8.0 wt%; phosphate buffer and physiological excipients and water 82.2 wt%.

[0049] Summary of preparation key points: Following the steps of claim 7, calcined animal-derived hydroxyapatite powder was ball-milled (BPR 10:1, 400 rpm, 24 h total time, intermittent cooling) to D50 = 50–80 nm, centrifuged, washed, and dried; nHA was dispersed in deionized water, ultrasonically dispersed, and sodium citrate was added to adjust the pH to 7.0–7.4 to obtain a stable dispersion; self-assembled collagen peptides were dissolved in a weak acid and adjusted to pH 6.8, and incubated at 25 °C for 45 min to initiate self-assembly; the nHA dispersion and self-assembled peptides were mixed under low shear conditions, Poloxamer F127 was added, homogenized at low speed and degassed, and the final pH and osmotic pressure were adjusted to physiological levels; aseptically filled and stored at 2–8 °C.

[0050] Example 2:

[0051] Formulation: nHA 5.0 wt%; self-assembled collagen peptides 1.0 wt%; dispersant (PAA) 0.2 wt%; Poloxamer F127 2.0 wt%; buffer and excipients 91.8 wt%.

[0052] Summary of preparation key points: nHA is ball-milled using the same parameters but at a shorter time (12 h) to reduce surface activation (while still controlling D50 to 50–80 nm); after obtaining the dispersion, it is mixed with the peptide at 20–25 °C and incubated for 30 min. A small amount of Poloxamer F127 is added to adjust the viscosity to ensure shear-thinning behavior; the mixture is then homogenized at low speed, degassed, and aseptically filled. This formulation emphasizes low viscosity to improve post-injection fissure penetration and is suitable for filling very fine fissures or shallow depressions.

[0053] Example 3:

[0054] Formulation: nHA 15.0 wt%; self-assembled collagen peptides 2.0 wt%; dispersant (sodium citrate) 0.5 wt%; Poloxamer F127 6.0 wt%; buffer and excipients 76.5 wt%. Summary of preparation key points: nHA was ball-milled to the target particle size and subjected to intensive washing to remove free impurities; the self-assembled peptides were induced to assemble at 2.0 mg / mL for 60–90 min; the high-solids nHA dispersion was compounded with the peptides under low shear, and a moderate amount of Poloxamer was added to provide initial adhesion and shape retention; low-speed homogenization and degassing were followed by aseptic filling. Application scenarios: Auxiliary filling for dental restorations requiring a high initial mineralization rate or for large local defects.

[0055] Example 4:

[0056] Formulation: nHA 6.0 wt%; self-assembled collagen peptides 3.5 wt% (high peptide content to emphasize directed mineralization); dispersant PAA 0.2 wt%; Poloxamer F127 4.0 wt%; balance is buffer. Summary of preparation key points: The high peptide content enhances the density of the self-assembled network. During mixing, the peptides are first induced to self-assemble before being slowly added to the nHA dispersion, allowing nHA to be directionally deposited between the self-assembled fibers. The final system has moderate viscosity and excellent cell adhesion site density, suitable for applications where accelerated osteogenic initiation is desired.

[0057] Comparative Example 1: Microsphere HA gel, without self-assembled peptides

[0058] Formulation (representing existing technology logic): HA microspheres (particle size 30–60 μm) 12.0 wt%; Poloxamer F127 8.0 wt%; buffer and excipients 80.0 wt%; non-self-assembled collagen peptides.

[0059] Summary of preparation key points: Micron-sized HA microspheres were prepared by spray drying / spheroidization and dispersed in a Poloxamer matrix; the system has a high viscosity and the microspheres are the main solid phase.

[0060] Comparative Example 2: Microspheres HA + Low-Content Peptides

[0061] Formulation: HA microspheres (30–60 μm) 12.0 wt%; self-assembled collagen peptides 1.5 wt%; Poloxamer F12 78.0 wt%; buffer 78.5 wt%.

[0062] Summary of preparation key points: The same peptide was added to the microsphere matrix to test the effect of peptide introduction on the performance of the microsphere system.

[0063] The results of comparing Examples 1-4 with Comparative Examples 1-2 are shown in the table below:

[0064] Group Settlement height 7d (mm) Mineralization score (1–5) Cell compatibility 72h (%) Fracture penetration depth (mm) Example 1 1.0 3.2 92 6 Example 2 0.8 2.6 88 9 Example 3 0.5 4.5 90 4 Example 4 0.7 4.0 95 7 Comparative Example 1 6.0 2.0 75 1 Comparative Example 2 4.0 2.8 85 2

[0065] Results and key points of analysis:

[0066] Injectability and Permeability: Examples 1 and 2 demonstrate that the nanoscale HA and self-assembled collagen peptide composite system can be smoothly injected using a 25–30 G needle (injection force 10–20 N), far superior to the microsphere-based comparative (32–35 N), which exhibits a significant risk of needle clogging or high thrust. The low-Poloxamer content formulation in Example 2 demonstrates the best penetration depth (9 mm), suitable for extremely fine fractures, while sacrificing some initial mineralization rate.

[0067] Stability: After the addition of dispersant and self-assembly with peptides, the nano-system showed a significantly lower sedimentation height than the microsphere system, indicating that the nano-peptide complex has better suspension stability and anti-settling properties.

[0068] Induced mineralization and biocompatibility: Example 5 (high peptide content formulation) significantly improved mineralization score and cell compatibility, indicating that self-assembled collagen peptides can effectively serve as mineralization templates and provide more cell adhesion sites in nanosystems, thereby promoting osteoblast activity. Example 3 (high nHA) showed the highest mineralization score, making it suitable for dental or rapid mineralization scenarios, but it increased injection force and decreased permeability.

[0069] Compared with microspheres: Even with the addition of peptides (Comparative Example 2), the microsphere system is still inferior to the nano-peptide composite system of the present invention in terms of injection force, sedimentation and fissure permeability. This shows that the synergistic mechanism of morphology and size (nano rather than microspheres) + self-assembled peptides is the core of the superiority of the present invention.

[0070] In summary, the injectable formulation of this invention exhibits excellent biomimetic bone healing capabilities and clinical applicability in bone defect repair. Through ball milling and dispersant modification, HA particles are prepared to the nanoscale and highly dispersed; combined with self-assembled collagen peptides, a highly hydrophilic bone-like matrix is ​​formed, significantly enhancing the biomineralization effect of the system. Compared with traditional HA microsphere injectable formulations, this approach eliminates the risks of large particle sedimentation and needle clogging, making it more stable and reliable during storage and use. Secondly, the material can be injected via a 25–30G fine needle for minimally invasive treatment, fully filling bone defect spaces of various shapes with precise filling. After injection, the self-assembled collagen network structure of the material continuously promotes osteoblast adhesion, secretion, and new bone formation, while the material degradation products are phagocytosed and absorbed by macrophages, promoting tissue remodeling. Finally, the synergistic effect of synthetic polymers (PLLA / PLGA / PVA), natural peptides, and nano-HA gives the material excellent mechanical properties, bioactivity, and biodegradability.

[0071] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A hydroxyapatite injection, characterized in that, The injection is composed of the following components by weight percentage: Nano-sized hydroxyapatite 5–20 wt%; Self-assembling collagen peptides 0.5–4 wt%; The remainder consists of physiological buffer solutions and auxiliaries; The nano-sized hydroxyapatite has a particle size D50 of 50–80 nm, the self-assembled collagen peptide contains a Gly-XY repeat sequence with an amino acid length of 9–30 residues, and the self-assembled collagen peptide contains 0–2 RGD fragments and has the ability to self-assemble into nanofibers at near physiological pH. The self-assembled collagen peptide consists of three functional domains: the first part is 1-3 Gly-XY repeating units to provide a tendency for triple helix or short fragment folding; the second part is 0-2 RGD or equivalent cell adhesion short peptides to provide cell adhesion sites; and the third part is charged residues; and forms nanofibers or fiber bundles with a diameter of 5-30 nm.

2. The hydroxyapatite injection according to claim 1, characterized in that, The self-assembly induction conditions for the self-assembled collagen peptides are: peptide concentration of 0.5-5 mg / mL, solution pH of 6.5-7.4, and temperature of 20-37℃ for 30-120 minutes.

3. The hydroxyapatite injection according to claim 1, characterized in that, The additives include: pH buffers and viscosity modifiers.

4. The hydroxyapatite injection according to claim 3, characterized in that, The pH buffer is either a Tris solution or a phosphate solution.

5. The hydroxyapatite injection according to claim 3, characterized in that, The viscosity modifier is poloxamer F127.

6. A method for preparing a hydroxyapatite injection according to any one of claims 1-5, characterized in that, Includes the following steps: 1) Animal bone was calcined and crushed to obtain primary hydroxyapatite (nHA). Then, liquid medium was added to grind large particles using the collision and shear force of grinding balls. The ball-to-material ratio was 10-20:

1. The rotation speed was 300-600 rpm, the total ball milling time was 12-36 h, and intermittent cooling was used. The obtained product was centrifuged, washed, and dried to obtain nano-sized hydroxyapatite. 2) Disperse the obtained nHA in the aqueous phase, add a dispersant and ultrasonically disperse, adjust the pH to 7.0–8.0 to obtain a stable nHA dispersion; 3) Dissolve the collagen peptides in a weak acid or buffer solution, adjust the pH to 6.5-7.4, and incubate at 20-37°C for 30-120 minutes to initiate the self-assembly of collagen peptides; 4) Mix the nHA dispersion obtained in step 2) with the peptide solution obtained in step 3) under low shear conditions to induce the peptide to assemble on / around the nHA surface and induce mineralization and template formation. 5) Homogenize and degas at low speed, adjust pH and osmotic pressure to physiological levels to obtain the final injectable formulation; and store under sterile conditions.

7. The method for preparing a hydroxyapatite injection according to claim 6, characterized in that, The dispersant is at least one of sodium citrate or polyacrylic acid, and the amount of the dispersant is 0.1-3 wt% of the mass of nano-hydroxyapatite. The surface of the nano-hydroxyapatite is modified by the dispersant to improve its stability.

8. A composition for repairing bone or tooth defects, characterized in that, The composition comprises a hydroxyapatite injection agent according to any one of claims 1-5, for minimally invasive injection filling of bone or tooth defects.

9. The composition according to claim 8, characterized in that, The composition further comprises a polymer selected from at least one of poly(L-lactic acid) PLLA, polyvinyl alcohol (PVA), polylactic acid-glycolic acid copolymer (PLGA), or polyglycolic acid (PGA).

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