Method for preparing dermal filler composition comprising hyaluronic acid and hydroxyapatite, and dermal filler composition comprising hyaluronic acid and hydroxyapatite

Abstract

The invention provides a composition based on HA and HAp in the field of soft tissue filling and a preparation method thereof. Optionally, a dermal filler is used to increase facial tissue uplift by adding a volume to the facial tissue, correcting wrinkles and laws, and restoring the smooth appearance of the face. Optionally, the dermal filler includes an uncrosslinked or crosslinked HA that is chemically bonded to the HAp.

Description

[0001] This invention is a divisional application of the invention patent with application number 2018800147144, application date October 9, 2018, entitled "Method for preparing a dermal filler composition containing hyaluronic acid and hydroxyapatite and a dermal filler composition containing hyaluronic acid and hydroxyapatite". Technical Field

[0002] The present invention generally relates to soft tissue fillers, and more specifically, to dermal and subdermal graft fillers based on hyaluronic acid and hydroxyapatite, respectively and in combination. Background Technology

[0003] The skin consists of three layers, each performing a specific function. The outermost, thin, and hard layer is the epidermis. The epidermis is 0.05 mm thick (eyelids) to 0.8-1.5 mm thick (sole of the feet and palms). Most epidermal cells are keratinocytes. New keratinocytes originate from cells in the deepest layer of the epidermis, called the basal layer, and migrate to the surface of the epidermis, replacing older cells. The stratum corneum (the outermost layer of the epidermis) acts as a waterproof barrier, preventing most bacteria, fungi, and chemicals from entering the body. Melanocytes are distributed throughout the basal layer and produce melanin, which contributes to skin color and filters ultraviolet radiation. The epidermis also contains Langerhans cells, which are part of the skin's immune system, helping the body fight infection. Below the epidermis is the dermis. The epidermis is 0.6 mm thick (eyelids) to 3-5 mm thick (sole of the feet and palms).

[0004] The dermis is a thick layer of fibrous and elastic tissue, mostly composed of collagen, elastin, and fibrils (which give the skin its flexibility and strength). Collagen fibers make up 70% of the dermis, giving it strength and resilience, while elastin maintains normal elasticity and flexibility. It contains nerve endings that sense pain, touch, pressure, and temperature. It contains sweat glands that produce sweat in response to heat and stress, thus helping to cool the body. It contains sebaceous glands that collect oil (sebum) into hair follicles, keeping the skin moist and supple. It contains hair follicles that help regulate body temperature, providing protection from damage and enhancing sensation. It contains blood vessels that supply nutrients to the skin and help regulate body temperature. Below the dermis is the subcutaneous tissue, also known as the subcutaneous fat layer or subcutaneous tissue. It is a layer of fat, accounting for approximately 50% of the body's fat. Fat is contained within living cells called adipocytes, held together by fibrous tissue. The fat layer attaches the skin to the bones and muscles and helps to insulate the body, thus providing a protective cushion and acting as an energy storage area.

[0005] Facial aging is a sign of a dynamic process, resulting from the cumulative effects on the skin, soft tissues, and craniofacial bones, leading to wrinkles, loss of facial contours, and the formation of facial lines. This dynamic process occurs throughout our lives. These aging effects include gravity, progressive bone resorption, decreased tissue elasticity, redistribution of subcutaneous fat, and loss of fullness. Hormonal imbalances, sun exposure, stress, diet, work habits, illness, drug abuse, and smoking can also affect facial attractiveness.

[0006] Bone atrophy, along with the dynamics of bone expansion and osteoporosis, are the causes of craniofacial skeletal aging. Bone resorption can lead to loss of biomarker volume and covering soft tissues, and the skin undergoes significant changes without the structural support of bone. For example, maxillary resorption can lead to loss of upper lip support and displacement of the zygomatic fat pad, thereby contributing to perioral wrinkles and nasolabial folds, respectively. A youthful face is characterized by a round facial shape due to the balanced distribution of superficial and deep fat, which gives the soft tissues fullness. The redistribution and loss of fat leads to a gradual loss of soft tissue support and fullness, resulting in soft tissue decline, such as fat loss in the forehead and cheekbones, and fat gain in the jawline and nasolabial folds.

[0007] When considering facial appearance and structure, it is convenient to divide the face into three regions: the upper third includes the forehead and brow bone, the middle third includes the midface and nose, and the lower third includes the jawline, chin contour, and neck. The midface, including the eyes, nose, lips, and cheekbones (central facial triangle), is most helpful in perceiving facial beauty and attractiveness.

[0008] The upper face (including the forehead, brow, temples, and upper eyelid area) is affected by the loss of subcutaneous fullness, resulting in forehead lines. Muscle activity in this area is relatively concealed in youth due to the subcutaneous fullness of the forehead. As we age, this loss of fullness produces wrinkles and nasolabial folds. Drooping eyebrows are a result of the loss of brow support and upper eyelid fullness.

[0009] The midface (including the eyes, nose, lips, and cheekbones (the central facial triangle)) is affected by a loss of subcutaneous fullness. "Crow's feet" are a result of this loss of subcutaneous fullness around the orbicularis oculi muscle. Furthermore, the orbicularis oculi muscle's boundaries are prominent and contribute to the formation of a malar crescent at the cheekbone prominence and tear trough line. Periocular tear trough hollowing is formed as fat accumulates in the atrophied lower eyelid with age, creating a tired appearance. Between the orbicularis oculi muscle and the lower eyelid, loss of fullness causes the tissues to converge, resulting in a darker skin tone and a tired, fatigued look even after a good night's sleep. This dullness also contributes to melanin deposition in the skin. Nasolabial folds are caused by the drooping of cheek fat. The nose also undergoes aging processes, such as loss of fullness between the eyebrows, at the root of the nose, and on the upper bridge of the nose (tip), and a drooping tip, which contributes to the illusion of a longer nose. A drooping chin can also contribute to the appearance of a longer nose. The lower face (including the lower jaw, jawline, and neck) exhibits fat accumulation with age, resulting in relatively excess skin and a loss of jawline definition. Fat descending to the jawline causes sagging skin on the chin. The "turkey neck" deformity is characterized by unsupported skin, drooping chin pads, and downward pull of the platysma muscle, resembling the red, fleshy folds of a turkey's throat. In addition, with aging, the platysma muscle contracts, leading to vertical fibrous bands in the neck.

[0010] The primary goal of facial rejuvenation is the restoration of facial contours. Facial rejuvenation can be achieved through surgical and non-surgical procedures. Surgical procedures range from lifting to liposuction to fat transfer. Facial reshaping techniques are used to reshape the skin's surface and correct the effects of photoaging, including fine lines, irregular pigmentation, and blemishes. Facial reshaping is accomplished through chemical peels, microdermabrasion, and laser facial reshaping. Today, patients prefer and seek non-surgical procedures that can restore facial contours, correct facial asymmetry, or even enhance existing facial features (with immediate cosmetic results and short recovery times). This is achieved through a variety of substances (administered via injection). The main uses are to treat lines, wrinkles, and nasolabial folds, thereby compensating for loss of facial contours. This includes injectable neurotoxins (botox is used to weaken muscles and reduce dynamic lines), contour fillers, and collagen stimulants.

[0011] Hyaluronic acid (HA) is a naturally occurring linear polysaccharide composed of repeating disaccharide units formed by alternating β-1,3 and β-1,4 glycosidic bonds of glucuronic acid and N-acetylglucosamine, with a molecular weight (MW) reaching millions of Daltons (Da). Under physiological conditions, HA is found in its ionized form, hyaluronic acid salt, with sodium, calcium, etc., as balancing ions, such as sodium hyaluronate (NaHA). High concentrations are found in the skin, umbilical cord, and vitreous humor in the human body. HA is a relatively rigid molecule due to the restriction of glycosidic bond rotation by the large N-acetyl group adjacent to the glycosidic bond. Its naturally occurring structure is helical, further enhanced by hydrogen bonds formed between different functional groups (e.g., amine, carboxyl, and hydroxyl groups). The main sources of industrial-grade HA are animal tissues, such as rooster combs, which require extensive purification; and microbial streptococcal fermentation, such as HA fermentation from *Streptococcus zooepidemicus*.

[0012] Hyaluronic acid (HA) possesses significant structural, rheological, physiological, and biological functions. HA has the ability to adsorb large amounts of water and retain moisture, thus forming a highly viscoelastic substance in aqueous solutions. Due to its adsorption capacity and viscoelasticity, coupled with a lack of immunogenicity and toxicity, it can fill, lubricate, and retain mechanical stress in tissues. Therefore, HA has various applications in the cosmetic, biomedical, pharmaceutical, and food industries. Although HA is biocompatible, it is biodegradable and readily degraded by enzymes such as hyaluronidase (enzymatic degradation), resulting in a relatively short residence time in tissues. Its half-life is on the order of less than a week. Furthermore, HA cannot tolerate elevated temperatures for any considerable period. It undergoes thermal degradation, meaning it degrades via free radicals and hydrolysis. For example, the half-life (MW) of HA decreases dramatically when autoclaved at 121°C for several minutes in aqueous solution. The MW decrease accelerates exponentially above 60°C. In addition to enzymatic, thermal, and free radical degradation, HA undergoes mechanical degradation, such as degradation due to shear forces. All of the types of degradation mentioned above have a relatively short residence time in tissues.

[0013] Chemical modification, functionalization, or derivatization of HA using organic groups can controllably alter its chemical and physical properties, resulting in new biomaterials with novel and improved properties. Depending on the type and extent of modification, modified HA is a different chemical from natural HA and can therefore be less natural, with alterations to biocompatibility and even its biological behavior. While benefits and outcomes should be measured, modification leads to its use in medical applications. For example, HA has low solubility in organic solvents.

[0014] Reaction with alcohols can transform its lipophilic properties into a more lipophilic one, thus increasing its solubility in organic solvents. When HA derivatives are less soluble in water, they can exhibit an increased in vivo residence time. Furthermore, they can have improved interactions with molecules such as pharmaceuticals. Another example is cross-linked HA hydrogels used as cosmetic grafts. Cosmetic dermal fillers introduced into the skin to fill tissue, enhance contours, and reduce wrinkles are expected to have a prolonged in vivo residence time for HA, which is important for their clinical success.

[0015] The molecular weight (MW) of HA for cosmetic and dermal filler purposes is typically on the order of hundreds of thousands to millions of Da. Uncrosslinked HA, which can be used as a dermal filler, has a high MW, thus maintaining its solubility in vivo, and is too viscous to be injected through fine-gauge needles (often referred to as 27G and above, practically up to 30G needles). Low MW needles are easier to inject but lack solubility. One way to overcome this drawback is through chemical modification methods similar to crosslinking. Chemical crosslinking of HA is achieved by reacting uncrosslinked HA with a crosslinking agent, thereby forming an infinite 3D network of HA that is no longer water-soluble in the aqueous medium that constitutes the HA hydrogel (which has improved in vivo solubility). However, this chemical reaction or modification is accompanied by additional steps of chemical reagent treatment and purification of the reaction products. Typically, the chemical reagents are water-soluble, and the chemical reaction and purification are carried out in an aqueous medium. As mentioned above, one should measure the benefits and results leading to its use in medical applications. Following this statement, cross-linked HA gel overcomes the disadvantages of uncross-linked HA and can be used as a dermal filler, tissue filler for cosmetic purposes, such as lifting facial tissue by adding contours to facial tissue, correcting wrinkles and nasolabial folds, and restoring a smooth appearance to the face.

[0016] Hydroxyapatite (HAp) has the following chemical formula: Ca 10 (PO4)6(OH)2 (Ca / P ratio = 1.67). HAp is a granular calcium phosphate ceramic. HAp is naturally formed in geological deposits, as well as in normal biological tissues (such as the bones, cartilage, enamel, dentin, and cementum of vertebrates) and in many sites of pathological calcification (such as blood vessels and skin). 75% w (all percentages are given by weight / weight unless otherwise stated) of human bone constitutes bioapatite, thus imparting hardness and resistance to mechanical stress to the bone. The morphology of bioapatite is that of flat, elongated grains with a hexagonal structure.

[0017] Hap is a transplantable material that is thermodynamically stable under physiological conditions. It possesses excellent biocompatibility (non-toxic and non-immunogenic) and bioactivity, stimulating cell formation and tissue repair. It can stimulate cellular responses, collagen synthesis, and serve as a transformation medium and gene delivery medium. Hap has applications in tissue engineering, such as as a bone and tooth replacement material for repair and replacement purposes. Various methods for HAp synthesis have been reported, including plasma spraying, hydrothermal synthesis, freeze-drying, sol-gel, phase transition, mechanochemical synthesis, and chemical precipitation. The reported morphology of synthesized HAp is monoclinic or hexagonal microcrystals. Important factors for medical applications are the Ca / P ratio and the particle size of the powder. Typically, the particle size of HAp powder ranges from 10 μm to 100 μm. Typically, the particle size of HAp nanopowder form ranges from 100 μm. Generally, HAp particles in dermal fillers are spherical, uniform, smooth, and have a low surface area. However, HAp particles can have irregular shapes; they can be porous, hollow, flower-shaped porous hollow microspheres, or any other suitable shape with a high surface area.

[0018] Typically, HA-based dermal and subcutaneous fillers contain hundreds of thousands to millions of Da of HA, optionally with bifunctional or polyfunctional crosslinking agents that form covalent bonds with HA under alkaline or acidic conditions. Examples include diepoxides such as 1,4-butanediol diglycidyl ether (BDDE) and 1,2-ethylenediol diglycidyl ether (EDDE), which react with hydroxyl groups under alkaline conditions to form ether bonds but react in an acidic manner to form esters. The most common dermal fillers on the market are crosslinked with crosslinking agents such as BDDE and divinyl sulfone (DVS). However, any other bifunctional or polyfunctional crosslinking agent can be used for crosslinking, including but not limited to epoxy compounds that react directly with HA, DVS, formaldehyde, aziridine, amino acids, esters, carbodiimides, etc. HA can be crosslinked by using bifunctional or polyfunctional molecules as side groups, where one side is bonded to HA, and the other side is bonded to other monofunctional, bifunctional, or polyfunctional crosslinking agents or molecules of multiple crosslinking agents in their original state to bond the two ends of the side group.

[0019] Furthermore, it can contain uncrosslinked HA in the range of hundreds of thousands to millions of Da, thus facilitating extrusion through fine-gauge needles. Additionally, it can contain one or more active supplements, each with its own unique properties, such as anesthetic agents, antioxidants, vitamins, etc. Some examples include, but are not limited to, lidocaine, mannitol, and vitamin C. The concentration of HA in HA dermal fillers can range from 1 mg / ml to 50 mg / ml, more specifically 15-30 mg / ml, with each concentration being most suitable for specific areas of tissue filling and elevation.

[0020] A typical method for preparing cross-linked HA dermal fillers involves several basic steps known to those skilled in the art. The first step is the hydration and drying of the HA. The next step is the introduction of the HA into a cross-linking agent, thereby cross-linking the HA under alkaline or acidic conditions. The next step involves removing residual cross-linking agent by dialysis and swelling of the cross-linked HA, typically using purified water or phosphate-buffered saline (PBS). For example, dialysis may be performed for several days in a dialysis bag with a critical value of 12,000 MW until some of the residual cross-linking agent is removed. The next step involves the addition of an active supplement, the addition of uncross-linked HA, and homogenization of the entire composition. The next step involves sterilization, thereby completing the HA dermal filler with suitable dermal filler properties (e.g., rheological properties, extrusion force, pH, osmotic pressure, etc.).

[0021] Typically, HAp dermal fillers contain up to 60% w / w of HAp microspheres, all suspended in a high-viscosity liquid or gel serving as a carrier material. The HAp particles in the dermal filler are typically spherical, uniform, smooth, with a diameter of approximately 10 to 100 μm, most preferably 25 to 50 μm, and have a low surface area. In one example, the HAp microspheres are suspended in a carrier composed of carboxymethyl cellulose (CMC), glycerol, and water. In another example, the HAp microspheres are suspended in a carrier composed of HA and water. In yet another example, the HAp microspheres are suspended in a carrier composed of cross-linked HA as described above, wherein the HAp microspheres are added during or after the HA cross-linking process. The concentrations of these compositions and related components are known to those skilled in the art; for example, a typical HAp concentration in such compositions is 55.7% or lower. In all examples, the HAp microspheres are suspended in a high-viscosity liquid or gel, and no true bonds exist between the HA and HAp. The carrier serves as a tool for transferring HAp microspheres to designated areas for tissue filling and elevation, and prevents microsphere aggregation within the syringe and body. It prevents HAp deposition and phase separation. Furthermore, it holds the microspheres in their desired positions, preventing them from dispersing within the injection area. Invention Overview

[0022] In at least some embodiments, the present invention is characterized by HA and HAp-based compositions in the field of soft tissue fillers, and methods for preparing such compositions. Optionally, dermal fillers can enhance facial tissue elevation by incorporating a volume of facial tissue, correcting wrinkles and nasolabial folds, and restoring a smooth facial appearance. Optionally, the dermal filler comprises uncrosslinked or crosslinked HA chemically bonded to HAp. The binding of chemically anchored HA to the HAp surface has a synergistic effect in prolonging tissue elevation because both components degrade slowly, without diminishing their inherent ability to function as skin renewal components. These and other methods of the present invention, as well as the benefits thereof, will be more readily understood and appreciated in the detailed description of the invention. Brief description of the attached diagram

[0023] This document describes the invention by way of example only, with reference to the accompanying drawings. Referring now to the drawings in detail, it is emphasized that the specific cases shown are examples, intended only to illustrate embodiments of the invention, and are presented to provide a description of the principles and concepts of the invention that are believed to be most useful and readily understood. In this regard, no attempt has been made to show more detailed structural details of the invention than are necessary for a basic understanding of the invention, as described in the specification and appendices. Figure 1 This makes it obvious to those skilled in the art that various forms of the invention can be embodied in practice. In the accompanying drawings:

[0024] Figure 1 The image shows untreated HAp microspheres under SEM.

[0025] Figure 2 The image shows HAp microspheres treated with 3-epoxypropoxypropyltrimethoxysilane under SEM. Invention Details

[0026] According to the background art, HAp microspheres are added to a carrier, which is a high-viscosity liquid or gel. In one example, HAp microspheres are added to cross-linked HA, where they again act as a carrier. HA and HAp have an additive effect, not a synergistic effect. The additive effect is as follows: HAp microspheres degrade slowly without foreign body reaction, thus acting as a scaffold for natural tissue growth, as well as HA hydration and support of the extracellular matrix. The synergistic effect between HA and HAp is problematic due to the fact that the cross-linked HA network and HAp microspheres form a simple mixture in which the HA gel is the continuous phase and the HAp microspheres are the dispersed phase. In this simple mixture, the various components retain their own properties without synergistic effects.

[0027] Furthermore, it is claimed that adding HAp to HA prior to crosslinking can form an encapsulated structure in which HAp is encapsulated by HA. This encapsulated structure is claimed to prolong the degradation of HAp along with HA until HA is degraded, thus acting as a barrier to prevent HAp degradation. However, this is also problematic because there is no actual bond between HAp and HA in these prior art compositions.

[0028] First, in an ideal state or system using HA encapsulation, there exists a mechanism that protects HA from degradation through HA encapsulation until the encapsulation gaps open. This ideal state depends on the thickness of the encapsulation. However, even in this ideal state, no mechanism exists to protect HA from degradation. Second, the system described is not ideal, so the result is that most HAp microspheres are not encapsulated. The state of the microspheres ranges from partially covered by HA to HAp microspheres without HA encapsulation. It is undesirable to be limited by a single assumption, which may be due to the fact that HAp microspheres are several orders of magnitude larger than HA chains (end-to-end distance).

[0029] In existing compositions, these short chains are agitated to form localized micro-3D networks, which do not necessarily trap HAp microspheres, resulting in some encapsulated HAp microspheres and some unencapsulated HAp microspheres. Most of the encapsulated HAp microspheres are likely unencapsulated due to their relative sizes, as explained above. Moreover, the result can be considered a simple mixing rather than true encapsulation.

[0030] Therefore, it is common practice to achieve the same gain by injecting different dermal fillers of the same area onto HA and HAp separately, thus mixing them. However, adding a high concentration of HAp microspheres only to cross-linked HA can have other detrimental effects on viscous action and result in high extrusion forces relative to uncross-linked HA carriers. A simple solution to overcome this problem is to reduce the concentration of HAp microspheres. However, using a different type of dermal filler again in the same area can help overcome the aforementioned problems.

[0031] To achieve the synergistic effect of HA and HAp, it is ideal to form an HA-based complex containing HAp as an additive. This complex is considered a blend of various additives and polymer matrices, wherein the additives possess different and distinct functionalities, and wherein the polymer matrices are bound and linked together to form the polymer-based material, which possesses improved properties superior to or imparts new properties to the inherent properties of the polymer. The properties of the HA complex material do not depend solely on the volume fraction of HAp microspheres, but also on the chemical and physical interactions with the HA gel, such as polymer network and interfacial parameters (compatibility, wettability, and adhesion, collectively referred to as "adhesion" in this invention). Adhesion is important for achieving improved complex properties. Furthermore, improved adhesion also facilitates the good dispersion of additives within the network.

[0032] Coupling agents are defined as compounds that, conventionally, provide chemical bonding between two dissimilar materials, typically inorganic and organic in the cases of HA and HAp. Coupling agents can also provide interactions between two dissimilar materials beyond chemical bonding, such as ionic interactions, hydrogen bonds, and even weak van der Waals (VDW) interactions. As an example, coupling agents can be added during the mixing of polymers and additives. In another example, coupling agents can be added as surface modifiers and / or new functional groups for additives (which are introduced onto the polymer chain and then interact with other parts). All of the above are considered pathways to increase the adhesion between the polymer and the additive. The increased adhesion between the additive and the polymer ranges from strong chemical bonds or electrostatic attraction to weak VDW interactions.

[0033] Any mechanism of use of the coupling agents described above is feasible, but surface modification is most advantageous due to the sensitivity of HA, such as the heat, shear, and enzymatic degradation mentioned above. Furthermore, the use of coupling agents in combination or as functional groups can interfere with the cross-linking phase of HA, thereby forming a cross-linked gel in the case of a complex containing cross-linked HA.

[0034] The associated HA and HAp can have interactions, hydrogen bonds, or even ionic interactions on the order of VDW, which are considered non-permanent. However, creating covalent bonds between HA and HAp (which can be considered permanent) will result in a true composite material that combines the benefits of both HA and HAp. The simplest way to form chemical bonds between the HAp surface and HA molecules is to use coupling agents, specifically, surface treatments with organofunctional silanes. The type of silane coupling agent and the silane surface treatment are chosen based on the surface chemistry of the additive and the polymer. Other types of coupling agents, such as zirconates, titanates, etc., can be used in the same manner. Other approaches to forming covalent bonds between HA and HAp can include using crosslinking agents, chelating agents, or even using multiple coupling agents in a row pointing towards the additive surface (which can achieve the same chemical bonding effect).

[0035] HAp tends to aggregate due to VDW and hydrogen bonding interactions. Another benefit of using silanes is that they help prevent aggregation, which is due to the elimination of these interactions and, in part, steric hindrance.

[0036] The most common chemical structure of organofunctional silanes is R-S1-X3, where X is an alkoxy group that can hydrolyze reactive groups on the surface, and R is an organic functional group that can react with the polymer matrix. Other types of organofunctional silanes with fewer than three alkoxy groups are also suitable for surface reactions. Alternatively, X is a chlorine atom. However, the commercial use of these materials is limited due to the difficulty in handling the corrosive, flammable, or toxic byproducts associated with hydrolysis.

[0037] There are two main techniques for inserting silanes:

[0038] a. Pretreatment—Pure silane or a solution thereof, in an organic solvent diluted with water (which accelerates the reaction), is directly added to the additive. Typically, this technique is accomplished by spraying or impregnating the additive with a silane reagent (surface polishing).

[0039] b. Adding silane to the polymer, followed by the addition of untreated additives. This technique is considered rare.

[0040] In the case of HA and HAp, the preferred method is pretreatment, wherein the HAp particles are silane coated before being introduced into the HA.

[0041] The result of industrial pretreatment processes is the formation of thick, three-dimensionally cross-linked silane multilayers (polysiloxane networks). Silane monolayers (which are currently only achievable through vapor-phase silane technology) are generally beyond the scope of industrial practice.

[0042] The organofunctional groups of silanes are selected based on the type of favorable interaction between the polymer and the additive. These interactions can range from weak VDW forces and hydrophilic-hydrophobic interactions to chemical covalent bonds. A large number of viable R groups exist, and specific types are selected based on their affinity for the polymer matrix. It should be noted that more than one type of silane can be used effectively with a given polymer. Therefore, for a particular polymer, the most suitable organofunctional silane coupling agent should achieve both chemical bonding and interdiffusion across the matrix, thereby forming a network with the polymer.

[0043] For example, silane surface treatments such as 3-epoxypropoxypropyltrimethoxysilane or 3-epoxypropoxypropyltriethoxysilane or other epoxy-functionally coupled silanes can act as BDDE crosslinking agents and participate in the chemical reactions that occur during HA gel curing, thereby forming bonds between the surface-treated HAp and HA.

[0044] The common BDDE curing mechanism of HA involves reaction with BDDE (chemical formula 1,4-butanediol dicondensate), which has two functional epoxy groups. Under basic conditions, these groups form stable covalent ether bonds between HA and the crosslinking agent, just as 3-epoxypropoxypropyltrimethoxysilane has functional epoxy groups that can crosslink HAp with HA. The R group and epoxy group of 3-epoxypropoxypropyltrimethoxysilane can act as BDDE and also as a crosslinking agent. As mentioned above, the crosslinking agent is also the pathway for forming covalent bonds between HA and HAp. In this case, BDDE can also bond between HA and HAp, either by bonding with HA on one hand and with the surface of HAp on the other, thus forming ether bonds, or under extreme conditions, undergoing radical quenching on the surface of HAp. The problem in this system is the need to control the degree of crosslinking in both aspects due to competition for crosslinking sites on HA and HAp: the crosslinking of the HA chains themselves and the crosslinking of HA and HAp. Higher concentrations of BDDE are required to achieve comparable results, or whether the HAp surface treatment method is followed by cross-linking to form HA and HAp complexes.

[0045] Another possibility is that vinyltrimethoxysilane, vinyltriethoxysilane, or vinyltrichlorosilane, as examples of chlorosilanes or other vinyl-functionalized silanes, can act as DVS crosslinking agents and participate in the chemical reactions that occur during the HA gel curing process, thereby forming bonds between the surface-treated HAp and HA.

[0046] The common DVS curing mechanism of HA involves reaction with DVS, which has two functional vinyl groups that, under alkaline conditions, form stable covalent ether bonds between HA and the crosslinking agent, just as vinyltrimethoxysilane has functional vinyl groups that can crosslink HAp with HA. The R group and vinyl group of vinyltrimethoxysilane can act as DVS and also serve as a crosslinking agent.

[0047] Several possibilities exist for successfully crosslinking HA with HAp. Another example is adipamide hydrazide (ADH) as a crosslinking agent, and 3-aminopropoxypropyltriethoxysilane, 3-aminopropoxypropyltrimethoxysilane, or other amino-functionalized silanes can act as ADH and serve as crosslinking agents. However, due to the sensitivity of HA as described above, not all crosslinking is advantageous. Examples of silanes with epoxy-functionalized and vinyl-functionalized groups are the most common processes for mimicking HA crosslinking found in the dermal filler market and can be readily implemented in HA and HAp complexes within crosslinked HA compositions or other substances. As mentioned above, three-dimensionally crosslinked silane multilayers are formed on the surface of HAp microspheres during silanization. Due to the three-dimensional structure, the bonded silanes do not completely cover the surface. Therefore, there are silane-treated regions and non-silane-treated regions on the surface, resulting in a surface morphology with separated or interconnected treated blocks over a larger untreated area, which can be viewed as islands in a sea or interconnected (bridged) islands. Surface-bonded silane regions are islands dispersed on the ocean surface that do not possess an HAp surface. The surface texture of these islands in the ocean can be created based on the concentration of silane used.

[0048] To avoid being limited by concentration variations, alternative methods can be used to create surface morphology and affinity, such as Janus particle technology. Janus particles are asymmetric particles with two distinct physical properties, such as HAp particles, which have a hemispherical surface sized by silane and an untreated hemisphere. Surface morphology and affinity differ between the two hemispheres. Different methods exist for forming Janus particles, and methods such as masking can be used, where particles are trapped at the interface between the two phases, allowing the untreated hemisphere to be masked while the other hemisphere is sized, thus modifying the particle surface only on one side.

[0049] Another example of such methods for creating surface morphology and affinity involves using silane reagents with mixtures of different functionalities, such as a silane that reacts with the HA chain and a silane that does not. In extreme cases, Janus particles are formed having a hemispherical surface sized using a reactive silane and another hemisphere sized using a non-reactive silane. In any of the examples mentioned, the silane-treated region has a morphology of separated or interconnected treated blocks over a larger untreated region. The surface treatment is on the nanoscale order of microspheres sized at the micrometer scale. This surface morphology allows for the slow degradation of HAp and acts as a scaffold for natural tissue growth. It is reasonable to assume that degradation can be slower in this structure than in untreated HAp microspheres.

[0050] HA chains can adsorb onto the surface of surface-treated HAp. The adsorbed HA chains on the surface form columns (a portion of the chain in contact with the surface), loops (a portion of the chain not in contact with the surface but bonded through two columns), and two tails (a portion of the chain not in contact with the surface but bonded on one side of the column and free on the other). Adsorption involves physical interactions and the formation of chemical bonds, referred to as physisorption and chemisorption, respectively. Therefore, it is reasonable to assume that some columns of HA chains are covalently attached to the surface, and that the adsorption is irreversible and permanent. This structure suggests that HAp microspheres also act as cross-linking sites in the formed gel. It is reasonable to assume that the concentration of the cross-linking agent can be lower in the presence of surface-treated microspheres while having the same cross-linking effect, thus these microspheres can, to some extent, replace the cross-linking agent. Furthermore, the role of the microspheres as cross-linking sites is to locally increase mechanical strength, act as anchoring points, and, particularly in deep wrinkles, contribute to a more stable volume effect. Additionally, the HA chains attached to the surface are not easily degraded by enzymes. It is reasonable to assume that this portion of the chain, defined as columns, is less prone to enzymatic degradation due to its surface bonding. The degradation of the portion of the chain defined as the loop and tail depends on their relative size and enzyme accessibility. Therefore, the entire HA chain near HAp microspheres is less prone to enzymatic degradation, which can contribute to more potent volume action and durable use in the injection site. Furthermore, HA complexes containing HAp exhibit improved mechanical strength upon cross-linking, and microspheres acting as anchoring points can greatly benefit areas where dynamic wrinkles and nasolabial folds require greater tolerance to muscle action. As mentioned above, it is clear that HA containing chemically bonded HAp particles is considered a complex, a reinforced gel with improved properties such as mechanical properties, degradation resistance, etc., or can be tailored to various properties depending on the desired application. Other types of surface-treated microspheres (biodegradable or non-degradable, inert or irritating) can be used to anchor HA chains, such as polylactic acid microspheres.

[0051] As mentioned above, in simple mixtures rather than complexes, the HA and HAp in contact can have interactions on the order of VDW, hydrogen bonds, or even ionic interactions, which are not considered permanent. Upon injection, the mixture is exposed to bodily tissues and fluids, where the same interactions as HA and HAp can exist on the order of VDW, hydrogen bonds, or even ionic interactions. This can lead to the loss of the interaction between HA and HAp, which is the opposite of the permanent chemical covalent interaction between HA and HAp.

[0052] Therefore, in mixtures, HA chains and HAp microspheres degrade as if injected individually, while in complexes, the synergistic effect lies in the slower degradation of HA chains and HAp microspheres, resulting in longer in vivo application, a longer residence time, and thus prolonged tissue elevation. As mentioned above, the use of cross-linking agents is considered a chemical modification or cross-linking stabilization of HA, while the addition of chemically bonded particles can be considered a physical and chemical modification of HA through cross-linking with the particles. Besides cross-linking stability, the latter is also a method of HA stabilization through both general and specific HAp particles.

[0053] Some additional benefits of using the HA and HAp complex include avoiding the Tyndall effect, which occurs because HAp is opaque or reduces whiteness in thin areas, while HA is translucent. Furthermore, HAp is opaque, making the entire composition easily detectable in radiographic imaging during or after injection.

[0054] In general, it can be viewed as the chemical modification, functionalization, or derivatization of HA using inorganic groups, such as silane-surfaced ceramic HAp, which allows for the controllable alteration of the chemical and physical properties of HA, thereby yielding new biomaterials with novel and improved properties.

[0055] As mentioned above, HA and bifunctional or polyfunctional crosslinkers can form covalent bonds under alkaline or acidic conditions. The addition of dendritic polyfunctional crosslinkers can provide another benefit: reduced viscosity and longer durability in tissues. Dendritic molecules are macromolecules with precise molecular weights and hyperbranched structures (defined as ellipsoidal or spherical shapes). Dendritic molecules form radially concentric layers (called “generations”) with a central core and a series of branches (defined by their shape). Typically, the most effective functional groups that can react with other molecules are located in the outermost generation (which is the outermost part of the branch). For example, the reaction of HA with BDDE (biepoxy functional crosslinker) and epoxy functional dendritic molecules can yield a softer gel, lower extrusion force through fine-gauge needles, and a gel with longer durability in tissues (due to the dendritic structure of the crosslinked dendritic molecules).

[0056] Alternatively, the uncrosslinked HA (partial or complete) in the dermal filler can be crosslinked using epoxy-functional dendritic molecules, thereby maintaining low viscosity, which can facilitate extrusion through fine-gauge needles and exhibit longer durability in tissue compared to uncrosslinked HA. Fine-gauge needles are, for example, needles with a gauge range of 27G to 30G. Optionally, the longer durability can be at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, or any integer value between these values, longer than the HA dermal filler composition. The longer durability is achieved using crosslinked HA, which can be, for example, crosslinked with BDDE and dendritic molecules. A gel product, such as the final dermal filler, can, for example, contain approximately 90% crosslinked gel and 10% uncrosslinked HA. The presence of uncrosslinked HA reduces the viscosity of the gel and lubricates it, thus facilitating extrusion from a syringe.

[0057] Non-limiting examples of such epoxy-functionalized dendritic molecules can be found in Multiply Functionalized Dendriticers: Protecting-Group-Free Synthesis through Sequential Thiol-Epoxy 'Click' Chemistry and Esterification Reaction (RSC Advances, submitted May 4, 2015, Khan et al; see, for example, molecule 8 as a non-limiting example of an epoxy dendritic molecule).

[0058] As mentioned above, HA and bifunctional or polyfunctional crosslinking agents can form covalent bonds under alkaline or acidic conditions. Adding POSS... ® (Polyhedral Oligomeric silsesquioxane) cage-like molecules as multifunctional crosslinking agents (e.g., epoxy-functional POSS) ® For example, glycidyl POSS ® (Epoxy-functionalized silsesquioxanes) can provide a variety of benefits in tissues, including reduced viscosity and increased durability. Different organic functionalities can be used to create basic cage-like structures, such as epoxypropyl POSS. ® The epoxy functional group in it. For example, HA with BDDE and epoxypropyl POSS. ® Molecular reactions can yield softer gels and lower extrusion forces through fine-gauge needles, as well as gels with higher persistence in tissues, due to glycidyl POSS. ® Cross-linked cage structure. Alternatively, uncross-linked HA (partial or complete) in dermal fillers can be made using glycidyl etheroplastin (GEP).® Cross-linking, which maintains lower viscosity, can facilitate extrusion through fine-gauge needles and exhibits a longer durability in tissue compared to uncross-linked HA.

[0059] The descriptions of fine specifications and / or high durability above also apply to the use of POSS. ® This implementation plan.

[0060] POSS made using suitable organic functionalities ® It can also be used as a crosslinking agent, for example, in cases where both alkoxy and epoxy functionality exist in the basic cage structure of silanes. This POSS ® The structure can couple the ends of HA and HAp, much like silane surface treatment. POSS ® The process of using it as a coupling reagent is similar to that described above.

[0061] Although the materials described above are characterized by polysaccharides, particularly HA chemically bonded to particulate calcium phosphate ceramics, such as HAp via coupling agents, this is not intended to limit them in any way. Other polysaccharides, such as heparin precursors, can be bonded via the same process as HA and HAp, as well as other types of surface-treated microspheres; for example, biodegradable polylactic acid microspheres can act as HAp in surface-anchored HA chains.

[0062] Although the above discussion pertains to dermal fillers, particularly facial dermal fillers, it is by no means intended to imply limitations. Other anatomical areas of the body can be filled or augmented, such as the neck, buttocks, chest, breasts, hands, and calves. Furthermore, it can be used as a non-surgical alternative or as a supplement in rhinoplasty and tip grafting. Additionally, it can be used throughout the body to blur skin markers such as scars, acne scars, and stretch marks. Moreover, it has beneficial uses in areas such as bone and teeth reconstruction (where HAp is a major component). Furthermore, it can have beneficial uses in areas such as joint lubrication (to improve joint flexibility and shock absorption). Example

[0063] Example 1 - Silane Surface Treatment

[0064] 1. Preparation of silane treatment solution (2% silane in total volume):

[0065] Mix 93.25% vol methanol with 3.93% vol distilled water. Add 0.81% vol acetic acid to buffer the solution to pH 4.5–5.5. Add 2% vol functionalized silane to the solution. Lower concentrations of silane can be used to create a silane surface coating.

[0066] 2. Silane treatment

[0067] The solution is stirred for 1–10 min, during which silanol groups are formed in the solution. HAp microspheres are introduced into the solution over approximately 30 min. The HAp microspheres are then rinsed twice in pure methanol. The HAp microspheres are then inserted into an oven at 70°C to 105°C for 1–24 hours to cure and complete the process, thereby forming a silane layer, or cured overnight at room temperature. The given duration and temperature are silane type dependent; for example, 70°C for 24 hours is suitable for 3-epoxypropoxypropyltrimethoxysilane, thus preventing degradation of the silane epoxy functional groups.

[0068] High-shear mixers, rotor-stator mixers, homogenizers, or probe-type ultrasonic generators can be used to prevent microsphere aggregation.

[0069] Example 2 - Uncrosslinked HA dermal filler formulation containing HAp microspheres

[0070] Raw material NaHA (in any form, such as fiber or powder, with an MW of 2 million Da) is introduced into an alkaline solution. The NaHA is stirred, dissolved, and hydrated in the alkaline solution at room temperature for several hours until a homogeneous viscous HA liquid is formed.

[0071] Under stirring, HAp surface-treated with 3-epoxypropoxypropyltrimethoxysilane is introduced into a viscous HA liquid to form a dispersion. The dispersion is heated to 50°C for several hours, thereby forming bonds between the HA and the surface-treated HAp. The reactants can be used without stirring in a highly viscous dispersion, and the microspheres are immobilized in the dispersion due to its high viscosity. In cases where the microspheres tend to sink, a mixer or probe-type ultrasonic generator can be used to prevent sinking and aggregation. The dispersion is then cooled to room temperature. The dispersion then swells and is dialyzed for several days using PBS in a dialysis bag with a critical value of 12,000 MW, thereby removing low molecular weight residues. The PBS is replaced regularly, introducing fresh PBS into the dispersion. The degree of swelling is measured, for example, resulting in a dispersion containing 30% v / v HAp or less. Due to synergistic effects, the concentrations of HA and HAp can be reduced while still achieving the same effect as commercially available common dermal fillers. The dispersion is filled into syringes, sterilized, and ready for use. Alternatively, HA surface-bonded HAp microspheres can be introduced into other gel carriers consisting of carboxymethyl cellulose (CMC), glycerol, and water.

[0072] Example 3 - Crosslinked HA dermal filler formulation containing HAp microspheres

[0073] The basic steps of the crosslinking process are known to those skilled in the art. Any form of raw material NaHA (e.g., 2 million Da) in fiber or powder form can be introduced into an alkaline solution. The NaHA is stirred, dissolved, and hydrated in the alkaline solution at room temperature for several hours until a homogeneous viscous HA liquid is formed.

[0074] Under stirring, BDDE and HAp surface-treated with 3-epoxypropoxypropyltrimethoxysilane are introduced into a viscous HA liquid to form a dispersion. The dispersion is heated to 50°C for several hours, thereby forming bonds between the HA and the surface-treated HAp. The reactants can be used without stirring in a highly viscous dispersion, which immobilizes the microspheres. In cases where the microspheres tend to sink, a mixer or probe-type ultrasonic generator can be used to prevent sinking and aggregation. The complex gel is then cooled to room temperature. The complex gel then swells and is dialyzed for several days using PBS in a dialysis bag with a critical value of 12,000 MW, thereby removing low molecular weight residues, including excess BDDE. The PBS is replaced regularly to introduce fresh PBS into the complex gel. The addition of uncrosslinked HA to the complex gel helps reduce extrusion forces. For example, the complex gel may contain 30% v / v HAp. The complex gel is then filled into syringes, sterilized, and ready for use.

[0075] Example 4 - Testing of Silane-treated Particles

[0076] HAp microspheres were prepared according to Example 1 described above.

[0077] Materials used:

[0078] 1. HAp microspheres, medical grade, spherical, 15-60 micrometers, D50=35 micrometers.

[0079] 2. 3-Epoxypropoxypropyltrimethoxysilane >98% Sigma Aldrich

[0080] 3. Methanol >99.9% HPLC grade Sigma Aldrich

[0081] 4. Acetic acid >99.7% ACS reagent grade Sigma Aldrich

[0082] SEM+EDS:

[0083] Equipment: Phenom ProX Desktop SEM

[0084] SEM+EDS analysis

[0085] Scanning electron microscopy (SEM) coupled with energy-dispersive spectroscopy (EDS) was used to obtain local elemental chemical analyses of untreated HAp microspheres and HAp microspheres treated with 3-epoxypropoxypropyltrimethoxysilane. SEM micrographs of surface elemental concentrations were obtained, and EDS quantitative analysis was performed.

[0086] Figure 1 and 2 The SEM micrographs depicted show untreated and HAp microspheres treated with 3-epoxypropoxytrimethoxysilane, respectively. Both groups of microspheres exhibit morphological similarities, indicating that applying silane surface treatment to the HAp microspheres does not damage them.

[0087] EDS analysis of the untreated HAp showed the presence of Ca, P, and O atoms, as predicted by HAp. Table 1 shows that the Ca / P ratio was 1.67, as predicted by HAp.

[0088] EDS analysis of HAp treated with 3-epoxypropoxypropyltrimethoxysilane showed the presence of Si atoms in addition to Ca, P, and O atoms, indicating the presence of silane surface treatment. As mentioned above, the result of the industrial pretreatment process is the formation of a thick, three-dimensionally cross-linked silane multilayer (polysiloxane network), thus its Ca / P atomic ratio deviates from the theoretical value of 1.67 (as shown in Table 2), and indicates the presence of silane surface treatment relative to the untreated surface.

[0089] Element number Element symbols Element name Atomic concentration weight concentration 8 O oxygen 68.28 48.43 20 Ca calcium 19.82 35.22 15 P phosphorus 11.90 16.35

[0090] Table 1. Elemental analysis of untreated HAp microspheres

[0091] Element number Element symbols Element name Atomic concentration weight concentration 8 O oxygen 58.88 38.46 20 Ca calcium 25.75 42.13 15 P phosphorus 15.22 19.24 14 Si silicon 0.15 0.18

[0092] Table 2. Elemental analysis of HAp microspheres treated with 3-epoxypropoxypropyltrimethoxysilane

[0093] Although the invention has been described in connection with specific embodiments thereof, it will be apparent to those skilled in the art that many alternatives, modifications, and alterations will be readily apparent. Therefore, it should be noted that all such alternatives, modifications, and alterations falling within the spirit and broad scope of the appended claims are to be covered. All publications, patents, patent applications, and sequences identified by numbers referenced in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent, patent application, or sequence identified by number were expressly and individually indicated to be incorporated herein by reference. Furthermore, any reference or identification in this application should not be construed as an admission that such reference can be used as prior art of the invention.

Claims

1. A dermal filler composition comprising hyaluronic acid (HA) or heparin precursor chemically bonded to particulate hydroxyapatite (HAp), wherein the chemical bond is obtained through a coupling agent, and wherein the particulate hydroxyapatite (HAp) is pre-treated with a coupling agent.

2. The composition of claim 1, wherein the hyaluronic acid or heparin precursor is maintained in a non-crosslinked state prior to reaction with the particulate hydroxyapatite.

3. The composition of claim 1 or 2, further comprising a carrier suitable for injection into a mammalian subject as a dermal filler.

4. The composition of any one of claims 1 to 3, wherein the chains of the HA or heparin precursor are stabilized by crosslinking to the particles.

5. The composition of any one of claims 1 to 4, wherein the coupling agent is an organofunctional silane.

6. The composition of claim 5, wherein the organofunctional silane has at least one alkoxy group or at least one chlorine atom.

7. The composition of claim 5, wherein the organofunctional silane has the formula R-Si-X3, wherein X is an alkoxy group capable of hydrolyzing to a reactive group on the surface, and R is an organofunctional group capable of reacting with the HA or heparin precursor.

8. The composition of any one of claims 4 to 7, wherein the organofunctional silane is selected from an epoxy-functional silane, a vinyl-functional silane, or a silane having an amino-functional group.

9. The composition of claim 8, wherein the epoxy-functional silane comprises one or more of 3-glycidyloxypropyltrimethoxysilane or 3-glycidyloxypropyltriethoxysilane.

10. The composition of claim 8, wherein the amino-functional silane comprises one or more of 3-aminopropyltriethoxysilane or 3-aminopropyltrimethoxysilane.

11. A method of making the composition of claim 1, the method comprising: modifying the surface of particulate HAp with a coupling agent; and bonding HA or heparin precursor to the coupling agent that has modified the surface of the particulate HAp.

12. The method of claim 11, wherein the coupling agent is an organofunctional silane.

13. The method of claim 12, wherein the modifying the surface of particulate HAp with a coupling agent comprises applying the organofunctional silane to the particulate HAp followed by curing the particulate HAp at a temperature of 70 °C to 105 °C for 1 to 24 hours.

14. The method of claim 13, wherein the organofunctional silane comprises 3- glycidyloxypropyltrimethoxysilane and the curing is at 70 °C for 24 hours.

15. The method of claim 12, wherein the modifying the surface of the particulate HAp with a coupling agent comprises applying the organofunctional silane to the particulate HAp and curing the particulate HAp overnight at room temperature.

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