The present invention relates to manufacture of a calcified tissue substitute.
Defects caused by infection, trauma, inflammation and tumorous and congenital aetiologies are the most common orthopaedic and dental conditions that require extensive surgical treatment. A number of bone substitutes have been developed as medical devices used in the injectable and implantable forms, but the majority of them lack sufficient osteoconductivity and osteoinductivity. In spite of this, bone substitutes have an extensive clinical application in the orthopaedic and dental fields with a market value of over $8 billion per year.
Nacre has been shown to induce bone healing, which can be traced back to the era of the Maya. It has been documented that nacre and pearls contain trace elements and bioactive proteins that can induce bone formation. However, the rapid degradation rate of the calcium carbonate in nacre has limited its application as an implantable or injectable bone substitute.
Accordingly, there is a need for an improved composition for repairing a calcified tissue.
Generation of porous materials for repairing a calcified tissue is an essential design criterion for improvement of material composition. Currently there are two main porous materials—prefabricated porous materials and a porous material whose porous structure is generated in situ after implantation. In general, prefabricated porous materials display weaker mechanical strength than the corresponding solid material, whereas materials rendered porous in situ may suffer uncontrolled mechanical failure after degradation of the component.
It is to be understood that if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art in Australia or any other country.
The invention utilises a multi-calcium salt resorption gradient that induces gradual pore formation in the calcified tissue substitute, thereby inducing new bone formation in vivo without compromising mechanical properties of the calcified tissue substitute.
Accordingly, a first aspect provides a method for producing a calcified tissue substitute, the method comprising reacting ground nacre, monocalcium phosphate, and water to produce brushite; grinding the brushite; reacting ground nacre with the ground brushite in disodium hydrogen phosphate solution.
A second aspect provides a calcified tissue substitute when produced by the method of the first aspect.
A third aspect provides use of ground nacre, monocalcium phosphate, and water in the manufacture of a calcified tissue substitute for repairing a calcified tissue.
A fourth aspect provides a method for repairing a calcified tissue, the method comprising contacting the calcified tissue with the substitute of the second aspect.
An alternative form of the fourth aspect provides the substitute of the second aspect for use in a method for repairing a calcified tissue, the method comprising contacting the calcified tissue with the substitute.
In an embodiment of any one of the aspects, the calcified tissue is tooth or bone.
A fifth aspect provides a delivery device comprising a first chamber and a second chamber, the first chamber comprising ground nacre and the second chamber comprising ground brushite, wherein the ground brushite is produced by reacting ground nacre, monocalcium phosphate, and water and grinding the resulting brushite, wherein the device is adapted to deliver the ground nacre and the ground brushite simultaneously in reacting proximity thereby causing the ground nacre and the ground brushite to react in the presence of disodium hydrogen phosphate solution.
The inventors have developed an improved calcified tissue substitute for repair of calcified tissue and a method for manufacturing the calcified tissue substitute. The invention utilises the different degradation time of the main components to convert the solid material into a porous structure in vivo. Namely, in the components, the remaining nacre particles have the fastest degradation rate, which begins to biodegrade through contact with body fluid. Once the dissolution of nacre occurs, the bioactive matrix will release, enhancing cell integration and inducing bone formation. Simultaneously, pores are generated allowing cell ingrowth. Brushite (or monetite), carbonated-apatite, and strontium substituted hydroxyapatite, which are other components of the calcified tissue substitute of the disclosure, have different solubilities compared to nacre, and these components will be biodegraded in 2 to 8 months. These different calcium orthophosphates will be gradually absorbed by the cellular activity of macrophages, osteoclasts and other cell types via phagocytosis and the body fluid-solid reaction, thereby increasing porosity and cell ingrowth. Finally, hydroxyapatite in the calcified tissue substitute can be kept in body for up to 2 years as an osteoconductive scaffold and entirely replaced by new bone by means of the cell-mediated resorption afterwards. If used, the bioinert drug carrier laponite will remain in the body.
Table 1 provides biodegradation rates of nacre-orthophosphate components that enable generation of a porous structure in the material surface in situ and thus allow cell and tissue ingrowth for calcified tissue repair.
The invention overcomes the lack of sufficient osteoconductivity and osteoinductivity and rapid resorption of nacre by providing a method for coating calcium orthophosphate on the surface of nacre particles and by providing nacre particles coated with calcium orthophosphate made by the method. The invention also provides controlled co-release from the calcium orthophosphate-coated nacre particles of positively charged antibodies or molecules loaded onto appropriate additive particles, e.g. laponite particles.
The term “agents” or “additives” as used herein includes antibiotics, antibodies, anti-inflammatory molecules, bone growth stimulants such as OP-1, bone morphogenetic proteins (BMP) e.g. BMP-2, EP1A and the like as well as cells such as stem cells.
Without wishing to be bound by theory, the unreacted nacre in the calcified tissue substitute comprises bioactive compounds, thereby enhancing osteoinductivity. Further, exposed nacre is resorbed more rapidly than other calcium orthophosphates. The resorption gradient of four main different components offers a gradually pore-forming process that is accompanied by new bone formation after implantation. Compared with prior porous bone substitutes, traditionally made of calcium salt material, pores of the calcified tissue substitute of the invention are generated in vivo, thereby providing a stronger mechanical support in early stages of calcified tissue repair and enhancing osteoconductivity.
In use, the calcified tissue substitute develops pores in vivo owing to faster resorption of nacre relative to calcium orthophosphate, which solves the problem of slow resorption of calcium phosphate-based commercial products that hinders osteogenesis.
In any of the aspects of the invention, the calcified tissue may be a bone or a tooth.
As used herein, “repair” includes mending, restoring, reconstructing or rehabilitating the calcified tissue.
Repair may be needed due to infection, trauma, inflammation, or tumorous or congenital aetiologies.
As used herein, “repairing a calcified tissue” encompasses practices that ultimately treat another tissue or organ, for example spinal fusion where the object is to alleviate spinal canal narrowing and spinal cord and/or spinal nerve squeezing.
As used herein, a calcified tissue “substitute” refers to a material contacted with a calcified tissue to promote repair of the calcified tissue through osteogenesis, osteoconduction and/or osteoinduction. A calcified tissue “substitute” may also be referred to as nacre-calcium orthophosphate or a calcified tissue graft. In one embodiment, the calcified tissue substitute is used to fill a void.
As used herein, the term “osteogenesis” refers to the development and formation of calcified tissue. The process of osteogenesis may involve osteoconduction and/or osteoinduction.
The term “osteoconduction” refers to the passive process by which bone forming cells near the site of implantation move across a scaffold, i.e. the calcified tissue substitute, and replace the scaffold with new calcified tissue over time. Osteoconductive materials allow osteoblasts and osteoclasts to attach, migrate, grow and/or divide.
The term “osteoinduction” refers to the active process by which a calcified tissue substitute or graft recruits bone-forming cells to the site of implantation and induces them to form new bone. Osteoinduction refers to attraction, proliferation, and differentiation of early-lineage cells (e.g., mesenchymal stem cells or osteoprogenitor cells) into bone-forming cells, resulting in the formation of new calcified tissue.
The method for manufacturing the calcified tissue substitute uses nacre as a starting material and a further reactant, and the manufactured calcified tissue substitute comprises nacre. Nacre is an organic/inorganic biocomposite of aragonite mineral comprising calcium carbonate (CaCO3) and bioactive agents.
The nacre is ground or milled to a specific particle size distribution and then reacted with monocalcium phosphate, optionally as the hydrate (Ca(H2PO4)2·H2O), to form brushite crystals. The brushite crystals are ground or milled to a specific particle size distribution. The brushite crystals are optionally dried prior to grinding or milling. In one embodiment, the particle size of the ground brushite is less than the particle size of the ground nacre.
Without wishing to be bound by theory, the nacre particle size appears to be important in manufacturing the calcified tissue substitute. The particle size affects setting time—the finer the ground nacre particles, the faster the setting time of the calcified tissue substitute. Accordingly, the particle size of the ground nacre, and therefore the setting time of the calcified tissue substitute, may be adapted according to the repair required for the calcified tissue, inasmuch as different repair applications require different setting times.
Nacre and brushite may be ground or milled by ball milling, optionally high energy ball milling or planetary ball milling.
In one embodiment, the ground nacre particle size is about 1000 mm or less, about 900 mm or less, about 800 mm or less, about 700 mm or less, about 600 mm or less, about 500 mm or less, about 400 mm or less, about 300 mm or less, about 200 mm or less, about 100 mm or less, about 50 mm or less, or about 10 mm or less.
In one embodiment, the ground nacre particle size is about 1 mm or more, about 5 mm or more, about 10 mm or more, about 15 mm or more, about 20 mm or more, about 30 mm or more, about 40 mm or more, about 50 mm μm or more, about 60 μm or more, about 70 μm or more, about 80 μm or more, about 90 μm or more, or about 100 μm or more.
In one embodiment, the ground nacre particle size is about 50 μm to about 100 μm, about 50 μm to about 200 μm, about 50 μm to about 300 μm, about 50 μm to about 400 μm, or about 50 μm to about 500 μm.
In one embodiment, the ground brushite particle size is about 500 μm or less, about 400 μm or less, about 300 μm or less, about 200 μm or less, about 100 μm or less, about 90 μm or less, about 80 μm or less, about 70 μm or less, about 60 μm or less, about 50 μm or Jess, about 40 μm or less, about 30 μm or less, about 20 μm or less, about 10 μm or less, about 9 μm or less, about 8 μm or less, about 7 μm or less, about 6 μm or less, about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less.
In one embodiment, the ground brushite particle size is about 1 μm or more, about 2 μm or more, about 3 μm or more, about 4 μm or more, about 5 μm or more, about 6 μm or more, about 7 μm or more, about 8 μm or more, about 9 μm or more, about 10 μm or more, about 15 μm or more, about 20 μm or more, about 30 μm or more, about 40 μm or more, about 50 μm or more, about 60 μm or more, about 70 μm or more, about 80 μm or more, about 90 μm or more, or about 100 μm or more.
In one embodiment, the ground brushite particle size is about 10 μm to about 20 μm, about 10 μm to about 30 μm, about 10 μm to about 40 μm, about 10 μm to about 50 μm, about 10 μm to about 60 μm, about 10 μm to about 70 μm, about 10 μm to about 80 μm, about 10 μm to about 90 μm, about 10 μm to about 100 μm, about 10 μm to about 150 μm, or about 10 μm to about 200 μm.
In one embodiment, the calcified tissue substitute has a particle size of about 5 mm. In another embodiment, the calcified tissue substitute has a particle size of about 1 μm, about 5 μm, about 10 μm, about 50 μm, about 100 μm, about 500 μm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm.
As used herein, “particle” and “particulate” refers to separate constituent units but does not preclude the particles being fused into a solid. Therefore, “particle” and “particulate” refers to ground nacre and ground brushite as well as the calcified tissue substitute comprising fused nacre particles coated or surrounded by calcium orthophosphate. The particles may be a powder.
As used herein, “particle size” refers to the pore size of a selective mesh through which the particle passes. It follows that ground nacre and ground brushite having defined particle sizes may be selected by sieving using one or more meshes having defined pore sizes. Sieving and meshes are known in the art.
In one embodiment, the ground nacre and monocalcium phosphate are reacted in a ratio of about 4:10. In another embodiment, the ground nacre and monocalcium phosphate are reacted in a ratio of about 4:5, about 4:6, about 4:7, about 4:8, about 4:9, about 4:11, about 4:12, about 4:13, about 4:14, about 4:15, about 0.5:10, about 1:10, about 2:10, about 3:10, about 5:10, about 6:10, about 7:10, about 8:10, or about 9:10.
In one embodiment, the ground nacre and monocalcium phosphate are reacted at room temperature. In another embodiment, the ground nacre and monocalcium phosphate are reacted at about 4° C., about 10° C., about 15° C., about 20° C., about 25° C., about 30° C., about 35° C., about 37° C., about 40° C., about 42° C., about 50° C., or about 60° C.
If dried before grinding, the brushite crystals may be air-dried or freeze-dried, for example.
In the second stage of the method, additional ground nacre is mixed with the ground brushite and reacted in disodium hydrogen phosphate (Na2HPO4) solution.
In one embodiment, the disodium hydrogen phosphate solution has a pH of about 8.2 to about 9.5. In one embodiment, the disodium hydrogen phosphate solution has a pH of about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10, about 10.2, about 10.4, about 10.6, about 10.8, or about 11. In one embodiment, the disodium hydrogen phosphate solution has a pH of about 9.25.
In one embodiment, the disodium hydrogen phosphate solution is a 2.5% to 4% solution. In another embodiment, the disodium hydrogen phosphate solution is about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10%.
In one embodiment, the ground nacre and ground brushite are reacted in a ratio of about 4:10. In another embodiment, the ground nacre and ground brushite are reacted in a ratio of about 4:5, about 4:6, about 4:7, about 4:8, about 4:9, about 4:11, about 4:12, about 4:13, about 4:14, about 4:15, about 0.5:10, about 1:10, about 2:10, about 3:10, about 5:10, about 6:10, about 7:10, about 8:10, or about 9:10.
In a further embodiment of the second stage of the method, the reaction of ground nacre with the ground brushite in disodium hydrogen phosphate solution further comprises tetracalcium phosphate (Ca4(PO4)2O). There are three advantages in using tetracalcium phosphate in this stage: it reacts quickly with brushite to form hydroxyapatite, which leads to the initial setting; it may catalyse the reaction between nacre and brushite; and it offers a different resorption rate than nacre, brushite and carbonate apatite. The resorption gradient aids gradual generation of pores in the calcified tissue substitute after implantation.
In one embodiment, tricalcium phosphate (a-TCP) is used in place of tetracalcium phosphate, as a-TCP converts to hydroxyapatite through a hydration reaction.
In one embodiment, the ground nacre, ground brushite, and tetracalcium phosphate are reacted in a ratio of 6:9:5, 7:8:5, 8:7:5, 9:6:5, or 10:5:5 in disodium hydrogen phosphate solution.
As shown in example 2, varying the ratio of the ground nacre, ground brushite, and tetracalcium phosphate varies the characteristics of the calcified tissue substitute. As shown by X-ray diffraction (XRD), varying the proportion of ground nacre will vary the proportion of nacre in the calcified tissue substitute, which provides significant advantages. For instance, calcified tissue substitutes with a higher proportion of nacre, e.g. calcified tissue substitute E, will also have the highest proportion of bioactive agents and therefore the highest level of osteoinductivity, compared with calcified tissue substitutes with relatively lower proportions of nacre. As a corollary, calcified tissue substitutes with a lower proportion of nacre, e.g. calcified tissue substitute A, will have the highest proportion of apatite and therefore the highest level of osteoconductivity, compared with calcified tissue substitutes with relatively higher proportions of nacre. That is, calcified tissue substitutes with a relatively low proportion of nacre and high proportion of apatite will have a slower rate of resorption, thereby providing scaffolding or mechanical support for a longer period of time. Accordingly, the composition of the calcified tissue substitute can be varied according to the specific repair needed by the calcified tissue.
In one embodiment, the disodium hydrogen phosphate solution is reacted with other reactants in a solution: reactant ratio of about 1:4, about 1:3, about 1:2, or about 1:1, or about 10%, about 20%, about 25%, about 30%, about 33%, about 40%, about 50%, or about 60%.
In one embodiment, the ground nacre and ground brushite, and optionally tetracalcium phosphate, are reacted at about 37° C. This embodiment is particularly suited to surgery where the calcified tissue substitute is reacted and allowed to set and harden in situ. If the calcified tissue substitute is used for repair in a non-human animal whose body temperature is not 37° C., the ground nacre and ground brushite, and optionally tetracalcium phosphate, may be reacted at body temperature.
In another embodiment, the ground nacre and ground brushite, and optionally tetracalcium phosphate, are reacted at room temperature, or at about 4° C., about 10° C., about 15° C., about 20° C., about 25° C., about 30° C., about 35° C., about 37° C., about 40° C., about 42° C., about 50° C., or about 60° C. Reaction temperatures above or below about 37° C. are suited to manufacture of calcified tissue substitutes that are intended to be implanted pre-set and pre-hardened.
In one embodiment, the method further comprises adding an agent, optionally hectorite, laponite or alpha-calcium sulphate, that modifies setting time of the composition.
Such additives may also serve to provide controlled co-release from the calcified tissue substitutes (nacre-orthophosphate particles) of positively charged antibodies or molecules loaded onto appropriate additive particles, e.g. laponite particles. Such controlled co-release is known in the art (e.g. Xiao et al. Mater Sci Eng C Mater Biol Appl (2016) 60: 348-56; Ordikhani et al. J Mater Sci Mater Med (2015) 26(12): 269; Li et al. ACS Appl Mater Interfaces (2014) 6(15): 12328-34; Wang et al. ACS Appl Mater Interfaces (2014) 6(19): 16687-95).
In one embodiment, the calcified tissue substitute when produced by the method of the invention is particulate and comprises calcium orthophosphate on the particle surface.
As used herein, “orthophosphate” refers to the anion PO43−, and “calcium orthophosphate” covers multiple compounds comprising calcium and orthophosphate with varying stoichiometry. In one embodiment, the calcium orthophosphate may be hydroxyapatite.
In one embodiment, the calcified tissue substitute is set at 100% humidity and 37° C., or 100% humidity and body temperature if the calcified tissue substitute is used for repair in a non-human animal whose body temperature is not 37° C. This embodiment is particularly suited to surgery where the calcified tissue substitute is reacted and allowed to set and harden in situ. In another embodiment, the calcified tissue substitute is set at about 4° C., about 10° C., about 15° C., about 20° C., about 25° C., about 30° C., about 35° C., about 37° C., about 40° C., about 42° C., about 50° C., or about 60° C. In another embodiment, the calcified tissue substitute is set at about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 80%, about 70%, about 60%, or about 50% humidity. In another embodiment, the calcified tissue substitute is set at any combination of temperature and humidity from those recited above.
In one embodiment, the calcified tissue substitute has a peak load of greater than about 45 N. In another embodiment, the calcified tissue substitute has a peak load of greater than about 50 N, greater than about 100 N, greater than about 150 N, greater than about 200 N, greater than about 250 N, greater than about 300 N, greater than about 350 N, greater than about 400 N, greater than about 450 N, greater than about 500 N, greater than about 550 N, greater than about 600 N, greater than about 650 N, greater than about 700 N, greater than about 750 N, greater than about 800 N, greater than about 850 N, greater than about 900 N, greater than about 950 N, or greater than about 1000 N.
In one embodiment, the calcified tissue substitute has a compressive strength of greater than about 2.3 MPa. In another embodiment, the calcified tissue substitute has a compressive strength of greater than about 2.5 MPa, greater than about 3 MPa, greater than about 3.5 MPa, greater than about 4 MPa, greater than about 4.5 MPa, greater than about 5 MPa, greater than about 5.5 MPa, greater than about 6 MPa, greater than about 6.5 MPa, greater than about 7 MPa, greater than about 7.5 MPa, greater than about 8 MPa, greater than about 8.5 MPa, greater than about 9 MPa, greater than about 9.5 MPa, greater than about 10 MPa, greater than about 10.5 MPa, greater than about 11 MPa, greater than about 11.5 MPa, or greater than about 12 MPa.
As noted above, increased mechanical strength, e.g. increased peak load, reflects in part improved and prolonged osteoconduction.
In one embodiment, the calcified tissue substitute is implantable or injectable.
In one embodiment, the calcified tissue substitute has an initial setting time (Ti) of 30 min to 90 min. In another embodiment, Ti is about 30 min, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, about 60 min, about 65 min, about 70 min, about 75 min, about 80 min, about 85 min, or about 90 min.
In one embodiment, the calcified tissue substitute has a final setting time (Tf) of 60 min to 120 min. In another embodiment, Tf is about 60 min, about 65 min, about 70 min, about 75 min, about 80 min, about 85 min, about 90 min, about 95 min, about 100 min, about 105 min, about 110 min, about 115 min, or about 120 min.
An implantable calcified tissue substitute may be set and hardened before or after implantation. In contrast, an injectable calcified tissue substitute may be set or hardened after injection.
Injection of the calcified tissue substitute is facilitated by the delivery device of the fifth aspect. The delivery device provides means to delay setting and hardening of the calcified tissue substitute until the time of repair, often the time of surgery. The delay in setting and hardening is achieved by providing the ground brushite and ground nacre in separate chambers of the device, wherein activation of the device causes the ground brushite and ground nacre to react.
In one embodiment, the first chamber and/or the second chamber of the delivery device of the fifth aspect further comprises disodium hydrogen phosphate solution. In another embodiment, the delivery device comprises a third chamber comprising disodium hydrogen phosphate solution.
In one embodiment, delivery from the delivery device comprises mixing the ground nacre and the ground brushite.
In one embodiment, the delivery device is a syringe.
As used herein, “reacting proximity” refers to a spatial relationship sufficient to allow the ground nacre to react with the ground brushite, and may entail mixing of the ground nacre with the ground brushite to react.
The calcified tissue substitute is for use in repairing a calcified tissue of a subject. Such calcified tissue may be, for example, worn, cracked, fractured, broken, abnormally formed or grown (e.g., a congenitally malformed bone), a combination thereof, or otherwise in need of repair or strengthening.
A “subject” as used herein may be human or a non-human animal, for example a domestic, a zoo, or a companion animal. In one embodiment, the subject is a mammal. The mammal may be an ungulate and/or may be equine, bovine, cervine, ovine, canine, or feline, for example. In one embodiment, the subject is a primate. In one embodiment, the subject is human. Accordingly, the present invention has human medical application, and also veterinary and animal husbandry applications, including treatment of domestic animals such as horses, cattle and sheep, and companion animals such as dogs and cats.
It will be appreciated by the person skilled in the art that the exact manner of administering to a subject a therapeutically effective amount of the calcified tissue substitute will be at the discretion of the medical practitioner with reference to the repair needed. The mode of administration, including combination with other agents, timing of administration, and the like, may be affected by the subject's likely responsiveness to treatment, as well as the subject's condition and history.
The calcified tissue substitute will be manufactured and administered consistent with good medical practice. Factors for consideration in this context include the particular calcified tissue being repaired, the particular subject in which the calcified tissue is being repaired, the clinical status of the subject, the site of administration, the method of administration, the scheduling of administration, possible side-effects and other factors known to medical practitioners. The therapeutically effective amount of the calcified tissue substitute to be administered will be governed by such considerations.
In embodiments, methods of repairing calcified tissue are provided. In some embodiments, the method comprises administering an effective amount of the calcified tissue substitute described herein to the calcified tissue (e.g., tooth, bone) in need of repair and/or strengthening. The calcified tissue substitute can be implanted or injected into and/or around the calcified tissue in need of repair or strengthening. When implanted, the calcified tissue substitute can be set before implantation or after implantation, and when injected, the calcified tissue substitute is set after implantation.
The term “therapeutically effective amount” refers to an amount of calcified tissue substitute effective to repair a calcified tissue in a subject.
Once produced the calcified tissue substitute of the present invention may be used as a carrier to other agents or additives including antibiotics, antibodies, anti-inflammatory molecules, bone growth stimulants such as OP-1, bone morphogenetic proteins (BMP) e.g. BMP-2, EPIA and the like as well as cells such as stem cells.
Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises”, means “including but not limited to”, and is not intended to exclude other additives, components integers or steps.
The invention will now be described with reference to the following, non-limiting examples.
The invention relates to a number of chemical reactions with nacre particles.
1. Pearl nacre was selected and ground into powder form (ground nacre). The powder was then sieved with selective meshes with different pore size from 50 to 400 μm.
2. Selected nacre particles with particle size 50 to 100 μm were mixed with monocalcium phosphate in the ratio of 4:10 at room temperature for 30 minutes.
3. Nacre particles were reacted with monocalcium phosphate in distilled water to form brushite crystal and CO2 at room temperature. The reaction occurred in a few minutes. The crystals were air-dried at room temperature for 3 days or freeze dried for 2-3 hours.
4. The brushite crystals were ground into powder with particle sizes between 10 to 100 μm (ground brushite).
5. The crystal brushite powder was mixed with nacre particles with particle size 50 to 400 μm in disodium hydrogen phosphate solution (pH 8.2˜9.5) in a ratio of 4:10 at room temperature. This reaction made the mixture harden in 90 minutes at 100% humidity at 37° C.
6. Additives including synthetic hectorite (Laponite) or alpha-calcium sulfate can be added to control the setting time for different applications as injectable or implantable devices.
7. After setting, the nacre particles were seen to have hydroxyapatite on their surface.
The chemical reaction of steps (2) and (3) is:
The chemical reaction of step (5) is:
An overview of the method of example 1 is provided in
Nacre was selected and ground into powder form. The powder was then sieved with selective meshes with pore size under 200 μm. Nacre particles were mixed with monocalcium phosphate in a ratio of 4:10 and the mixture reacted with distilled water at room temperature to form brushite crystal and CO2. The brushite crystals were dried at 37° C. then ground. As for example 1, the chemical reaction is:
The nacre particles were mixed with brushite particles and tetracalcium phosphate with five different ratios (A 6:9:5, B 7:8:5, C 8:7:5, D 9:6:5, E 10:5:5). The five ratios equate to 30% nacre, 35% nacre, 40% nacre, 45% nacre and 50% nacre, respectively. The mixture was reacted in 4% disodium hydrogen phosphate solution (pH 9.25) at room temperature with a liquid/powder (mL/g) ratio of 1:3. The mixed paste set and hardened at 100% humidity, 37° C. The chemical reaction is:
i. X-Ray Diffraction (XRD)
The five calcified tissue substitutes A to E were analysed by X-ray diffractometer (XRD, PANanalytical X′ Pert PRO, Netherlands). Samples of each calcified tissue substitute were scanned using a step scanning method with the step size of 0.0070° in the range from 10° to 90° of the diffraction angle (2Θ), the diffractometer was operated at 40 kV and 40 mA.
The XRD results (table 2) indicate the inorganic chemical composition of the nacre-calcium orthophosphate particles, i.e. the calcified tissue substitute.
The XRD data show that calcified tissue substitute E had the highest percentage of nacre, and accordingly the highest percentage bioactive composition, and therefore the highest osteoinductivity. Calcified tissue substitute A had the lowest percentage of nacre and the highest percentage of apatite. Consequently, the resorption rate of calcified tissue substitute A is the slowest, and therefore provides mechanical support for a greater period of time and is beneficial for large calcified tissue defects.
ii. Compression Test
Mechanical characterisation (table 3,
The compressive strength is calculated according to the following formula:
The compressive strength of the nacre-orthophosphate particles, i.e. the calcified tissue substitutes, is 2-3 times that of the commercial product. Therefore, the calcified tissue substitutes provide stronger mechanical support in calcified tissue defects.
iii. Scanning Electron Microscopy (SEM)
Scanning electron microscopy (SEM) in back scattered electron (BSE) and secondary electron (SE) mode was used to analyse the microstructure and morphology of the five calcified tissue substitutes.
The SEM micrographs of
iv. Setting Time
Initial and final setting times (tables 4 and 5) were determined by the Vicat method, which is a standard test method for time of setting of hydraulic cement paste by Vicat needles. The initial setting time is calculated as the time elapsed between the start of the reaction and the time when the needle penetration is 25 mm. The final setting time is calculated as the time elapsed when the needle does not penetrate the test material. The measurements were collected in triplicate and expressed as means+SD (n=5).
Tables 4 and 5 reveal that the setting time of the calcified tissue substitutes manufactured using finer particles as raw material is quicker than using coarser particles. Therefore, the particle size is critical for the setting time of calcified tissue substitutes.
To investigate cytocompatibility of the calcified tissue substitutes, HeLa, MLO-Y4 and MC3T3 cell lines were cultured on the calcified tissue substitutes (
Cell proliferation was tested by the colorimetric MTS method using hydroxyapatite as control.
v. Laser Scanning Confocal Microscopy—Cell Adhesion and Cytocompatibility
a. HeLa Cell Line (24 h)
HeLa cell culture shown in
b. MLO-Y4 Cell Line (72 h)
MLO-Y4 is an osteocyte-like cell line. MLO-Y4 cells cultured for 72 hours on the calcified tissue substitute A prepared according to example 2 using nacre particles, brushite particles and tetracalcium phosphate in the ratio 6:9:5. F-actin in cell membranes was labelled with fluorescent phalloidin (red). Laser confocal scanning microscopy showed the 3D structure of the cells' adhesion to the calcified tissue substitute (turquoise) and ingress into the calcified tissue substitute.
c. MC3T3 Cell Line (Up to 72 h)
MC3T3 is an osteoblast precursor cell line and was used for cell proliferation assays up to 72 h of culture. The result of the cell proliferation assay is shown in
To investigate biodegradation efficiency of the calcified tissue substitutes, cylindrical calcified tissue substitutes were implanted under the superficial fascia of the back of a rabbit. And, to investigate biocompatibility and osteoinductivity of the calcified tissue substitutes, the calcified tissue substitutes were implanted into an artificial bone defect site on the distal metaphysis region of rabbit femurs. Calcified tissue substitute C prepared according to example 2 was used in the animal studies, and hydroxyapatite was used as control.
Skeletally mature New Zealand white rabbits weighing 2.8+0.3 kg were used in the animal studies. Animals were allowed to acclimatize for 1 week before starting the experiment. They were kept separately in smooth-walled stainless-steel cages, one animal per cage, with food and water and handled in accordance with National Health and Medical Research Council (NHMRC) animal care guidelines. Morbidity, moribundity and general health observation (cage side) was performed once daily.
vi. Biodegradation Efficiency
New Zealand white rabbits were anesthetized by ear vein intravenous injection of Amylobarbitone (0.03 mg/kg of body weight). The cylindrical materials (10 mm in diameter, 5 mm in height, four nacre-calcium orthophosphate materials and four hydroxyapatite materials) were implanted under the animals' bilateral paravertebral superficial fascia through a dorsal midline approach (nacre-calcium orthophosphate materials in the left, hydroxyapatite materials in the right). One of each material was removed at four different time points (3 weeks, 6 weeks, 9 weeks or 12 weeks), weighed, and the percentage weight loss of each calculated. The test was performed in triplicate, and the mean±SD of weight loss percentages in each time course calculated.
vii. Biocompatibility and Osteoinductivety
New Zealand white rabbits were anesthetized by ear vein intravenous injection of Amylobarbitone (0.03 mg/kg of body weight). The animals were placed supine on the operating table and the experimental knee (right) shaved, prepared, and draped in a sterile fashion. The metaphysis region in right femur was entered by a longitudinal medial parapatellar incision and the patella was dislocated laterally to expose the femoral cortical bone. A circular defect, 5 mm in diameter, 5 mm in depth, 2.5 mm away from lineae epiphysialis of the femur, was created with a stainless steel dental drill. Then, the different calcified tissue substitutes (nacre-orthophosphate) or control material (hydroxyapatite) were implanted into different bone defect sites according to table 5. The incision was closed layer by layer. Each rabbit had only one femur operated on, and a total of 33 defects were created in 33 rabbits (
After 4 weeks and 8 weeks, all rabbits were sacrificed, the right femurs retrieved, and the soft tissues removed (
The retrieved femurs were scanned and reconstructed at high resolution using a micro-CT to observe the bone/implant interface (
From observation of the retrieved samples by naked eyes (
From the images of Micro-CT, after 4 weeks and 8 weeks, the surrounding bone tissue tightly connected with the nacre-calcium orthophosphate material, which indicated good osteointegration of the material (
Compared with the blank control group (
As shown in the results of Micro-CT data analysis (
In summary, as an important part of pre-clinical tests, the animal studies confirmed that, compared with conventional calcium phosphate materials, such as hydroxyapatite, the nacre-calcium orthophosphate material was a better bone substitute, due to its quick biodegradation rate, good biocompatibility and osteoinductivity.
Number | Date | Country | Kind |
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2017902108 | Jun 2017 | AU | national |
This application is a continuation-in-part of U.S. application Ser. No. 18/454,753, filed Aug. 23, 2023, which is a continuation of U.S. application Ser. No. 16/618,684, filed Dec. 2, 2019, which is a U.S. National Stage under 35 U.S.C. § 371 of International Application No. PCT/AU2018/050541, filed Jun. 1, 2018, which claims the benefit of Australian Application No. AU 2017902108, filed Jun. 2, 2017, all of which are incorporated by reference herein in their entirety.
Number | Date | Country | |
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Parent | 16618684 | Dec 2019 | US |
Child | 18454753 | US |
Number | Date | Country | |
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Parent | 18454753 | Aug 2023 | US |
Child | 18405569 | US |