Dermal filler composition containing hyaluronic acid and hydroxyapatite and method for producing same

Abstract

HA and HAp-based compositions and methods for producing them in the field of soft tissue fillers. Optionally, the transdermal filler is useful for enhancing facial tissue augmentation by adding volume to facial tissue, correcting wrinkles and wrinkle folds, and restoring a smooth facial appearance. Optionally, the transdermal filler comprises uncrosslinked or crosslinked HA chemically bonded to HAp. [Selected Figure] Figure 2

Description

[Technical Field]

[0001] The present invention relates generally to soft tissue fillers, and specifically to dermal and subcutaneous implantable fillers based on hyaluronic acid and hydroxyapatite, both separately and in combination. [Background technology]

[0002] Skin consists of three layers, each performing a specific task. The thin, tough outer layer is the epidermis. The epidermis varies in thickness from 0.05 mm on the eyelids to 0.8–1.5 mm on the soles and palms of the hands. Most of the cells in the epidermis are keratinocytes. New keratinocytes develop from cells in the deepest layer of the epidermis, called the stratum basale, and migrate toward the surface of the epidermis to replace older cells. The stratum corneum, the outermost part of the epidermis, is a waterproof barrier that prevents most bacteria, viruses, and chemicals from entering the body. Melanocyte cells are scattered throughout the stratum basale and produce the pigment melanin, which contributes to skin color and filters ultraviolet light. The epidermis also contains Langerhans cells, part of the skin's immune system that protects the body against infection. Beneath the epidermis is the dermis. The dermis varies in thickness from 0.6 mm on the eyelids to 3–5 mm on the soles and palms of the hands.

[0003] The dermis is a thick layer of fibrous elastic tissue made primarily of collagen, elastin, and fibrillin, which gives skin flexibility and strength. Collagen fibers make up 70% of the dermis and provide strength and toughness, while elastin maintains normal elasticity and flexibility. The dermis contains nerve endings that sense pain, touch, pressure, and temperature. It also contains sweat glands that produce sweat in response to heat and stress, helping to cool the body. It also contains oil (sebaceous) glands that store oil (sebum) in hair follicles, thereby keeping the skin hydrated and soft. The dermis also contains hair follicles that help regulate body temperature, protect against injury, and enhance sensation. The dermis also contains blood vessels that nourish the skin and help regulate body temperature. Beneath the dermis is the hypodermis, also known as the subcutaneous fat layer or hypodermis. This layer contains approximately 50% of body fat. Fat is contained in living cells called adipocytes and held together by fibrous tissue. The fat layer attaches the skin to bones and muscles, helps insulate the body, provides protective padding, and acts as an energy storage area.

[0004] Facial aging is the manifestation of a dynamic process that occurs over the course of a lifetime, resulting in surface wrinkling, topography, and loss of facial volume due to cumulative effects on the skin, soft tissue, and craniofacial skeleton. These effects of aging include gravity, progressive bone resorption, loss of tissue elasticity, and redistribution of subcutaneous fat, resulting in loss of fullness. Hormonal imbalances, sun exposure, mental stress, diet, work habits, disease, substance abuse, and smoking can also affect facial attractiveness.

[0005] Bone atrophy and the dynamics of bone expansion and loss contribute to the aging of the craniofacial skeleton. Bone resorption can result in a loss of biometric volume, and the overlying soft tissues and skin, lacking structural support from bone, can undergo significant changes. For example, resorption of the maxilla can result in a loss of support for the upper lip and displacement of the cheek fat pads, which can contribute to the formation of perioral and nasolabial folds, respectively. A youthful face is characterized by a rounded facial shape, with a balanced distribution of superficial and deep fat imparting fullness to the soft tissues. Fat redistribution and loss gradually result in a loss of soft tissue support and fullness, resulting in soft tissue depression, such as fat loss in the forehead and cheekbones, and fat gain in the chin and nasolabial folds.

[0006] When considering facial appearance and structure, it is convenient to divide the face into three regions: the upper third includes the forehead and brows, the middle third includes the midface and nose, and the lower third includes the chin, jawline, and neck. The midface includes the eyes, nose, lips, and cheekbones (the central facial triangle) and primarily contributes to the perception of facial aesthetics and attractiveness.

[0007] The upper surfaces, including the forehead, brow, temporal, and upper eyelid regions, are affected by a loss of subcutaneous fullness, resulting in forehead lines. Muscle action in this area is relatively hidden in young people by the subcutaneous fullness of the forehead. With aging, this fullness is lost, resulting in wrinkles and overlapping wrinkles. The impression of brow ptosis is the result of a loss of lateral brow support and loss of upper eyelid fullness.

[0008] 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 the result of a loss of subcutaneous fullness around the orbicularis oculi muscle. The border of the orbicularis oculi muscle also becomes more pronounced, contributing to the development of overlapping malar crescents and nasochin wrinkles over the zygomatic eminence. Depression of the periorbital tear trough occurs with age as fat deposits in the lower eyelid are depleted, creating a thin appearance. Loss of fullness between the orbicularis oculi muscle and the lower eyelid brings the tissues closer together, resulting in a darker skin tone and a tired, worn-out appearance of the eyes, even after a good night's sleep. Dark pigmentation can also be attributed to dermal melanin deposition. Overlapping nasolabial wrinkles occur due to the descent of ptotic cheek fat. The nose is also subject to aging effects such as loss of fullness between the eyebrows, nasal and upper back (cephalic nose), and nasal tip ptosis, which contribute to the illusion of increased nasal length. A drooping chin can also contribute to the appearance of increased nasal length. The lower facial features, including the chin, jawline, and neck, exhibit fat accumulation with age, resulting in a relative excess of skin and loss of jawline definition. Fat descent relative to the mandibular border results in the formation of a facial jowl. The "sagging neck" deformity is the result of unsupported skin ptosis, a chin pad, and downward pulling of the platysma muscle, resembling turkey wattle. Additionally, with age, contraction of the platysma muscle creates a vertical fibrous band in the neck.

[0009] The primary goal of facial rejuvenation is the restoration of facial shape. Facial rejuvenation can be achieved through both surgical and non-surgical procedures. Surgical procedures include a wide range of procedures, from lifts to liposuction and fat transfer. Resurfacing techniques are used to modify the skin's surface and correct the effects of photoaging, such as fine lines, irregular pigmentation, and blemishes. Resurfacing is achieved through chemical peels, microdermabrasion, and laser resurfacing. Patients currently prefer and seek non-surgical procedures that can restore facial volume, correct facial asymmetries, or enhance existing facial features with immediate cosmetic results and short recovery times. This is achieved through a wide range of substances administered via injection. A primary use is for the treatment of lines, wrinkles, and creases to compensate for loss of facial volume. This includes injections of neurotoxins (using botulinum toxin to weaken muscles and minimize movement lines), volume fillers, and collagen stimulators.

[0010] Hyaluronic acid (HA) is a natural linear polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine linked by alternating β-1,3 and β-1,4 glycosidic bonds with molecular weights (MW) of up to a few million daltons (Da). Under physiological conditions, HA is found in its ionized form as hyaluronate, with counterions such as sodium and calcium, e.g., sodium hyaluronate (NaHA). In the human body, high concentrations are found in skin, umbilical cord, and vitreous humor. HA is a relatively rigid molecule due to restricted rotation of the glycosidic bond by the bulky N-acetyl groups adjacent to the glycosidic bond. Its naturally occurring conformation is helical, which is further strengthened by hydrogen bonds formed between different functional groups (e.g., amine, carboxyl, hydroxyl, etc.). The main sources of industrial-scale HA are derived from animal tissues such as chicken comb, which requires extensive purification, and from microbial streptococcal fermentation, such as HA fermentation from the bacterium Streptococcus zooepidemicus.

[0011] HA has important structural, rheological, physiological, and biological functions. Its ability to absorb large amounts of water and retain moisture in aqueous solutions, forming a highly viscoelastic substance, is coupled with its lack of immunogenicity and toxicity, allowing it to meet, coat, lubricate, and maintain mechanical stress within tissues. Therefore, HA has found diverse applications in the cosmetic, biomedical, pharmaceutical, and food industries. While biocompatible, HA is also biodegradable and readily degraded by enzymes such as hyaluronidase (enzymolysis), resulting in a relatively short residence time within tissues. Its half-life is approximately less than one week. Furthermore, HA cannot withstand high temperatures for any conceivable period and undergoes thermal hydrolytic degradation, which means it is degraded by free radicals. For example, autoclaving HA in aqueous solution at 121°C for several minutes significantly reduces its MW. This reduction exponentially accelerates above 60°C. HA is subject to enzymatic, thermal and radical degradation as well as mechanical degradation, e.g., shear force degradation. All of the above types of degradation have a relatively short residence time in tissues.

[0012] Chemical modification, functionalization, or derivatization of HA with organic groups allows for controlled alteration of its chemical and physical properties, resulting in novel biomaterials with new, desirable, and improved properties. Depending on the type and extent of modification, modified HA is a different chemical entity from native HA and may therefore be less natural, potentially altering its biocompatibility and even its biological behavior. Nevertheless, the benefits and consequences of such modifications must be determined for their potential medical applications. For example, HA has low solubility in organic solvents. Reaction with alcohols can change its lipophobicity to more lipophilic properties, thereby increasing its solubility in organic solvents. HA derivatives, due to their low solubility in water, may have a longer in vivo residence time. Furthermore, they may have improved interactions with molecules such as drugs. Another example is crosslinked HA hydrogels used as cosmetic implants. Transdermal cosmetic fillers, introduced into the skin to augment tissue, add volume, and reduce wrinkles, aim to extend the in vivo residence time of HA, which is essential for their clinical success.

[0013] The molecular weight of HA molecules for cosmetic and augmentation applications typically ranges from several hundred thousand to several million Da. Non-crosslinked HA, which can be used as a transdermal filler, has a high MW to maintain in vivo stability and is too viscous for injection through fine-gauge needles (generally in the 27G range or larger, and in practice, up to 30G). Low MW HA is easily injectable but lacks stability. One way to overcome this drawback is through chemical modification. HA chemical crosslinking is achieved by reacting non-crosslinked HA with a crosslinker to form an infinite three-dimensional (3D) network of HA that is no longer soluble in aqueous media, including HA hydrogels with improved in vivo stability. Nevertheless, this chemical reaction or modification step adds additional steps of handling chemical reagents and purifying the reaction products. Typically, the chemical reagents are water-soluble, and the chemical reaction and purification are carried out in aqueous media. As mentioned above, the benefits and results of the modification must be measured to determine its functionality for medical applications. As described therein, cross-linked HA gel overcomes the shortcomings of non-cross-linked HA and can be used as a dermal filler for cosmetic tissue augmentation, for example facial tissue augmentation by adding volume to the tissue, correcting wrinkles and wrinkle overlaps, and restoring a smooth appearance to the face.

[0014] Hydroxyapatite (HAp) has the following chemical formula: Ca 10 HAp has a Ca / P ratio of 1.67 (PO4)6(OH)2. HAp is a particulate calcium phosphate ceramic. It occurs naturally in geological deposits and in normal biological tissues, such as vertebrate bone, cartilage, enamel, dentin, and cementum, as well as in many pathological calcification sites, such as blood vessels and skin. Approximately 75% of human bone (all percentages are weight per unit mass unless otherwise specified) is composed of bioapatite, which provides bone with its rigidity and resistance to mechanical loads. Bioapatite is morphologically composed of flat, elongated particles with a hexagonal structure.

[0015] HAp is an implantable material that is thermodynamically stable under physiological conditions, has excellent biocompatibility (non-toxic, non-immunogenic), and bioactive properties, potentially stimulating cell formation and tissue repair. HAp can stimulate cellular responses and collagen synthesis, and is used as a cell transformation vehicle, gene delivery vehicle, and other applications. HAp has been found to be useful in tissue engineering applications, such as bone and tooth replacement materials, for repair and replacement. Many methods have been reported for the synthesis of HAp, including plasma spraying, hydrothermal synthesis, freeze-drying, sol-gel synthesis, phase transformation, mechanochemical synthesis, and chemical precipitation. The reported morphology of synthetic HAp is either monoclinic or hexagonal crystallites, and for medical purposes, the primary factors are the Ca / P ratio and powder particle size. HAp powders typically have particle sizes ranging from 10 μm to 100 μm. HAp in nanopowder form typically has particle sizes ranging from 1 nm to 100 nm. Typically, the HAp particles in the dermal filler are round, uniform, smooth, and have a small surface area.However, the HAp particles may also have an irregular shape, and may be porous, hollow, flower-like porous hollow microspheres, or any other suitable shape with a large surface area.

[0016] Typically, HA-based percutaneous and subcutaneous fillers contain HA of approximately hundreds of thousands to millions of Da and, optionally, a bifunctional or multifunctional crosslinker that forms covalent bonds with HA under alkaline or acidic conditions. For example, under alkaline conditions, diepoxides such as 1,4-butanediol diglycidyl ether (BDDE) and 1,2-ethanediol diglycidyl ether (EDDE) react with hydroxyl groups to form ether linkages, whereas under acidic conditions, the resulting reaction instead forms esters. The most common percutaneous fillers on the market are crosslinked with BDDE and divinyl sulfone (DVS). However, any other bifunctional or multifunctional crosslinker, including, but not limited to, epoxy compounds, DVS, formaldehyde, polyaziridine, amino acids or esters, and carbodiimides, may also be used for crosslinking by direct reaction with HA. HA can be crosslinked by using bifunctional or polyfunctional molecules as side chains that bind to the HA on one hand and to an additional single bifunctional or polyfunctional crosslinker on the other hand, or several crosslinkers are used to join both ends of the side groups.

[0017] Additionally, HA may contain non-crosslinked HA of approximately hundreds of thousands to millions of Da to aid in extrusion through fine-gauge needles. Furthermore, HA may contain one or more active supplements, such as anesthetics, antioxidants, or vitamins, each with its own unique properties. Some examples include, but are not limited to, lidocaine, mannitol, and vitamin C. The concentration of HA in HA transdermal fillers can range from 1 mg / ml to 50 mg / ml, more specifically, from 15 to 30 mg / ml, with each concentration being optimal for a specific area for tissue filling and augmentation.

[0018] A typical methodology for preparing crosslinked HA dermal fillers involves several basic steps known to those skilled in the art. The first step is hydration of dried HA. The next step involves introducing HA into a crosslinker in either an alkaline or acidic environment to crosslink the HA. The next step typically involves dialysis to remove residual crosslinker and swelling the crosslinked HA using purified water or phosphate buffered saline (PBS). For example, dialysis is performed in a dialysis bag with a 12,000 MW cutoff for several days until most of the residual crosslinker is removed. The next step involves adding active supplements and non-crosslinked HA, and homogenizing the entire composition. The next step involves sterilization to produce an HA dermal filler with the appropriate properties for the dermal filler, such as rheological properties, extrusion force, pH, and osmotic pressure.

[0019] Generally, HAp transdermal fillers contain 60% w / w or less HAp microsphere particles uniformly suspended in a highly viscous liquid or gel that serves as a carrier material. Typically, the HAp particles in the transdermal filler are round, uniform, smooth, and have a small surface area, with a diameter of approximately 10-100 μm, most preferably 25-50 μm. In one example, the HAp microspheres are suspended in a carrier composed of carboxymethylcellulose (CMC), glycerin, and water. In another example, the HAp microspheres are suspended in a carrier composed of HA and water. In another example, the HAp microspheres are suspended in a carrier composed of crosslinked HA, as described above, to which the HAp microspheres are added during or after the HA crosslinking process. These compositions and relative component concentrations are known to those skilled in the art; for example, a typical HAp concentration in such compositions is 55.7% w / w or less. In all of the examples, the HAp microspheres are suspended in a highly viscous liquid or gel, and no actual bonding exists between the HA and the HAp. The carrier serves to transport the HAp microspheres to the area designated for tissue filling and augmentation and to prevent the microspheres from clumping both in the syringe and within the body. The carrier helps prevent HAp settling and phase separation. Furthermore, it prevents the microspheres from dispersing within the injection area, keeping them in the desired location. Summary of the Invention

[0020] The present invention, in at least some embodiments, features HA- and HAp-based compositions and methods for their manufacture in the field of soft tissue fillers. Optionally, the transdermal fillers are useful for enhancing facial tissue augmentation by adding volume to facial tissue, correcting wrinkles and wrinkle folds, and restoring a smooth facial appearance. Optionally, the transdermal fillers comprise uncrosslinked or crosslinked HA chemically bonded to HAp. The combination of HA chemically fixed to the surface of HAp has a synergistic effect of enhancing tissue over time without diminishing their inherent capabilities due to the slow degradation of both components, functioning as a skin rejuvenation component. These and other aspects and advantages of the present invention will be more readily understood and appreciated in light of the detailed description.

[0021] The present invention is described herein, by way of example only, with reference to the accompanying drawings. Referring now specifically to the drawings in detail, the details shown are by way of example and are for the sole purpose of illustrating embodiments of the invention, and are presented to provide what is believed to be the most useful and readily understood explanation of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, and the description taken together with the drawings will make apparent to those skilled in the art how several forms of the invention may be embodied in practice. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows untreated HAp microspheres in SEM. [Figure 2] FIG. 1 shows treated HAp microspheres in SEM after treatment with 3-glycidoxypropyltrimethoxysilane. DETAILED DESCRIPTION OF THE INVENTION

[0023] According to the background art, HAp microspheres are added to a carrier, which is a highly viscous liquid or gel. In one example, HAp microspheres are added to crosslinked HA, again functioning as a carrier. The combination of HA and HAp has an additive effect, not a synergistic one. The additive effect is as follows: HAp microspheres slowly degrade without causing a foreign body reaction, serving as a scaffold for natural tissue growth and HA hydration and extracellular matrix support. A synergistic effect between HA and HAp is highly questionable due to the fact that the crosslinked 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 such a simple mixture, each component maintains its own unique properties without any synergistic effect.

[0024] Furthermore, it was claimed that adding HAp to HA before crosslinking could form an encapsulation structure of the following structure of HAp encapsulated by HA: This encapsulation structure was claimed to prolong the degradation of HAp until it decomposed, with HA acting as a shield to prevent degradation of HAp. Again, this is questionable due to the fact that there is no actual bond between HAp and HA in these prior art compositions.

[0025] First, in an ideal state or system of encapsulated HAp and HA, there is a mechanism to protect the HAp from degradation due to encapsulation of HA until the capsule is broken. This ideal state occurs depending on the thickness of the capsule. However, even in this ideal state, there is no mechanism to protect against HA degradation. Second, the system is not ideal, and as a result, the majority of the HAp microspheres are not encapsulated. The state of the microspheres ranges from partial HA coverage of the HAp microspheres to no HA encapsulation. While not wishing to be limited by a single hypothesis, this is likely due to the fact that HAp microspheres are several orders of magnitude larger (end-to-end distance) than HA chains.

[0026] In the prior art compositions, these short chains are agitated, forming a local micro-3D network that does not necessarily trap the HAp microspheres, resulting in some encapsulated HAp microspheres and some unencapsulated HAp microspheres. Most of the encapsulated HAp microspheres may not be encapsulated due to the relative sizes described above. This result may also be considered simple mixing, not actual encapsulation.

[0027] Therefore, different dermal fillers based on HA and HAp can be injected separately into the same area to obtain the same benefits from the usual blending. Furthermore, simply adding a high concentration of HAp microspheres to cross-linked HA can have even more detrimental effects due to viscosity effects and high extrusion forces compared to non-cross-linked HA carriers. A simple solution to overcome this problem is to reduce the concentration of HAp microspheres. Nevertheless, using different types of transdermal fillers in the same area can help overcome this problem.

[0028] To achieve the synergistic effects of both HA and HAp, it is desirable to form an HA-based composite containing HAp as an additive. A composite is a blend of multiple additives with distinct functionalities and a polymer matrix that binds and connects them to form a polymer-based material with improved properties far exceeding those of the polymer itself, or to impart new properties. The properties of HA composite materials depend not only on the fractional volume of the HAp microspheres but also on their chemical and physical interactions with the HA gel, such as the polymer network and interfacial parameters (collectively described as miscibility, wettability, bonding, and "adhesion"). Adhesion is important for improving composite properties. Furthermore, improved adhesion also supports better dispersion of additives within the network.

[0029] Classically, a coupling agent is defined as a compound that creates a chemical bond between two dissimilar materials, usually an inorganic material and an organic material (such as HA and HAp). Coupling agents can also create interactions other than chemical bonds between two dissimilar materials, ranging from ionic interactions, hydrogen bonds, and weak van der Waals (VDW) interactions. As an example, a coupling agent may be applied during the mixing of a polymer with an additive. In another example, a coupling agent may be applied as a surface modification of the additive or as a new functional group introduced into the polymer chain that subsequently interacts with other moieties, as well as combinations thereof. All of the above are considered pathways for increasing adhesion between a polymer and an additive. Increased adhesion between an additive and a polymer can range from strong chemical bonds or electrical attractions to weak VDW interactions.

[0030] While any of the mechanisms of coupling agents described above can be used, the most preferred is the use of surface modification due to the sensitivity of HA to thermal, shear, enzymatic degradation, etc. Additionally, in the case of composites containing cross-linked HA, coupling agents introduced during mixing or as functional groups can be used to interfere with the cross-linking step of HA to form a cross-linked gel.

[0031] HA and HAp in contact may have slight VDW interactions, hydrogen bonds, or even ionic interactions that are not considered permanent. On the other hand, creating a covalent bond between HA and HAp that can be considered permanent results in the formation of a true composite material that combines the benefits of both HA and HAp. The simplest way to achieve a chemical bond between the HAp surface and the HA molecule is through the use of a coupling agent, specifically a surface treatment with an organofunctional silane. The silane surface treatment, a type of silane coupling agent, is selected depending on the surface chemistry and polymer of the additive. Other types of coupling agents, such as zirconates and titanates, can also be used in the same manner. Other routes to achieving a covalent bond between HA and HAp include the use of crosslinkers, chelating agents, or even the use of multiple coupling agents (referring to the additive surface, which can have the same effect as a chemical bond).

[0032] HAp has a tendency to aggregate due to VDW and hydrogen bonding interactions. Another advantage of using silanes is that they help prevent aggregation by eliminating these interactions and contributing to some degree to steric hindrance.

[0033] The most common chemical structure of organofunctional silanes is R-Si-X3, where X is an alkoxy group that can be hydrolyzed to a reactive group on the surface, and R is an organofunctional group that can react with the polymer matrix. Other types of organofunctional silanes with fewer than three alkoxy groups are also suitable for reaction with surfaces. Alternatively, X is a chlorine atom. Nevertheless, the use of these materials is commercially limited due to the difficulty of handling the corrosive, flammable, or toxic by-products associated with hydrolysis.

[0034] There are two main approaches to inserting silanes. a. Pretreatment - Pure silane or its solution in an organic solvent diluted with water is applied directly to the additive, which accelerates the reaction. This technique is usually achieved by spraying or immersing the additive in the silane agent (surface finishing). b. The silane is added to the polymer and then to the raw additive. This technique is considered rare.

[0035] In the case of HA and HAp, the preferred method is a pretreatment in which the HAp particles are coated with a silane before being introduced into the HA.

[0036] The industrial pretreatment process results in the formation of thick, three-dimensionally cross-linked silane multilayers (polysiloxane networks). Currently, silane monolayers can only be obtained by gas-phase silylation techniques, which are generally outside the scope of industrial practice.

[0037] The organofunctional group of the silane is selected based on the type of interaction favored between the polymer and the additive. This interaction can range from slight, weak VDW forces, hydrophilic-hydrophobic interactions, to chemical covalent bonding. A variety of R groups are possible, with the specific species selected depending on its affinity for the polymer matrix. Note that multiple types of silanes can be effectively used with a given polymer. Therefore, for a particular polymer, the most suitable organofunctional silane coupling agent selected will provide both chemical bonding and interdiffusion through the matrix to form a network with the polymer.

[0038] For example, silane surface treatments such as 3-glycidoxypropyltrimethoxysilane or 3-glycidoxypropyltriethoxysilane, or other epoxy-functional silanes, which act as BDDE crosslinkers and can participate in the chemical reactions that occur during HA gel curing to form bonds between the surface-treated HAp and HA.

[0039] A common BDDE curing mechanism for HA involves the reaction of BDDE with 1,4-butanediol diglycidyl ether (Chemical Formula). This has two functional epoxy groups that, under alkaline conditions, create a stable covalent ether bond between HA and the crosslinker, just as 3-glycidoxypropyltrimethoxysilane has a functional epoxy group that can crosslink HAp to HA. The epoxy group, the R-group of 3-glycidoxypropyltrimethoxysilane, can act as a crosslinker, fulfilling the role of BDDE. As mentioned above, the crosslinker is also a route to achieve covalent bonding between HA and HAp. In this case, BDDE can bond between HA and HAp, on the one hand by bonding to the HA and on the surface of the HAp, forming ether linkages or, in extreme cases, by quenching radicals on the HAp surface. The challenge with such a system is controlling the degree of crosslinking, both between the HA chains themselves and between HA and HAp, due to competition for crosslinking sites on both HA and HAp. Higher concentrations of BDDE are required to achieve comparable or any (if any) properties with surface treatment of HAp followed by cross-linking to form a HA and HAp composite.

[0040] Another possibility is vinyltrimethoxysilane or vinyltriethoxysilane or vinyltrichlorosilane as examples of chlorosilanes or other vinyl-functional silanes that can act as DVS crosslinkers and participate in the chemical reaction that occurs during HA gel curing to form bonds between the surface-treated HAp and HA.

[0041] A common DVS curing mechanism for HA involves reaction with DVS. DVS has two functional vinyl groups that form stable covalent ether bonds between HA and the crosslinker under alkaline conditions, similar to the vinyl group in vinyltrimethoxysilane, which can crosslink HAp to HA. The vinyl group in vinyltrimethoxysilane, which is the R group, can act as a crosslinker and function as a DVS.

[0042] The number of possibilities for crosslinking HA and HAp is enormous. Another example is adipic acid dihydrazide (ADH) as the crosslinker, and 3-aminopropyltriethoxysilane or 3-aminopropyltrimethoxysilane. Alternatively, other amino-functional silanes can act as crosslinkers in the role of ADH. However, as explained above, due to the sensitivity of HA, not all reactions are favorable. Examples of silanes, such as epoxy-functional silanes and vinyl-functional silanes, mimic the most common HA crosslinking procedures in the dermal filler market and can be easily implemented in HA and HAp composites, with or without crosslinked HA compositions. As previously mentioned, during silylation on the surface of HAp microspheres, a three-dimensional crosslinked silane multilayer is formed. Due to the three-dimensional structure, the surface is never completely covered with bound silanes. Therefore, there are silane-treated and silane-free domains on the surface, giving the surface the shape of isolated or interconnected treated patches, with larger untreated areas visualized as islands in a sea or interconnected (bridged) islands. The surface-bound silane domains are islands that extend into a sea of ​​free HAp surfaces, and the surface texture of the islands within the sea can be tailored depending on the concentration of silane used.

[0043] While not wishing to be limited to changing the concentration, other methods may be applied to adjust the surface shape and affinity, such as Janus particle technology. Janus particles are antisymmetric particles with two different physical properties, such as HAp particles with a silane-sized hemispherical surface and an untreated hemispherical surface. The surface shape and affinity differ between the two hemispheres. Various methods exist and can be applied to form Janus particles. For example, a masking method is used in which particles are trapped at the interface between two phases, the untreated hemisphere is masked, and the other hemisphere is sized, thus modifying the particle surface only on one side.

[0044] Another example of this method of adjusting the surface shape and affinity is the use of a mixture of silane reagents with different functionalities, such as a mixture of silanes that can react with HA chains and silanes that cannot. In extreme cases, Janus particles are formed, with one hemisphere sized with a reactive silane and the other with a non-reactive silane. In any of the examples mentioned, the silane-treated areas are isolated in larger untreated areas, or have the shape of interconnected treated patches. The surface treatment is on the nanometer scale, with microspheres sized on the micron scale. This type of surface shape slows the decomposition of HAp, allowing it to function as a scaffold for natural tissue growth. It is reasonable to assume that in such structures, decomposition may be slower than that of untreated HAp microspheres.

[0045] HA chains are adsorbed onto the surface of surface-treated HAp. The adsorbed HA chains form trains (the portion of the chain in contact with the surface), loops (the portion of the chain not in contact with the surface, connected by two trains), and two tails (the portion of the chain not in contact with the surface, connected by a train on one side and free on the other). Adsorption involves physical interactions and the formation of chemical bonds, known as physisorption and chemisorption, respectively. Therefore, it is reasonable to assume that the trains of HA chains covalently adhere to the surface, making adsorption irreversible and permanent. This structure implies that HAp microspheres also act as cross-linking sites for the formed gel. Given that the cross-linking agent concentration can be reduced in the presence of surface-treated microspheres with the same cross-linking effect, it is reasonable to assume that these microspheres can, to some extent, replace the cross-linking agent. Additionally, the effect of the microspheres as cross-linking sites is to increase local mechanical strength, which may support a more stable volume effect at anchor sites, especially in deep wrinkles. In addition, HA chains attached to surfaces are less susceptible to enzymatic degradation. It is reasonable to assume that these portions of the chains, defined as the trains, are less susceptible to enzymatic degradation because they are surface-bound. Degradation of portions of the chains, defined as the loops and tails, depends on their relative size and accessibility to enzymes. Therefore, the entire HA chain proximal to the HAp microspheres is less prone to enzymatic degradation, which may support a stronger volumizing effect and longer lifespan in the injected area. Additionally, the improved mechanical strength of HA composites containing HAp as crosslinks, with the microspheres acting as anchor points, may significantly aid in areas where dynamic wrinkles and wrinkle overlap require greater resistance to muscle action. From the above, it is clearly understood that HA containing chemically bonded HAp particles can be considered a composite, a reinforced gel, with improved properties, such as mechanical resistance and degradation resistance, or whose properties can be tailored depending on the required application.Other types of surface-treated microspheres (such as polylactic acid microspheres), which may be biodegradable or non-biodegradable, inert, or irritating, may be used to immobilize HA chains.

[0046] As mentioned above, HA and HAp in contact in a simple mixture, rather than a complex, may have slight VDW interactions, hydrogen bonds, or even ionic interactions that are not considered permanent. Upon injection, the mixture is exposed to body tissues and fluids that may have the same slight VDW, hydrogen bonds, or ionic interactions with either HA or HAp, which may result in the loss of the interaction between HA and HAp, as opposed to the permanent covalent chemical interaction between HA and HAp.

[0047] Thus, in a mixture, the HA chains and HAp microspheres degrade as if injected alone, but in a complex, both the HA chains and the HAp microspheres degrade much more slowly in a synergistic effect, which extends their in vivo retention time and thus tissue augmentation. As mentioned above, the use of a crosslinking agent can be considered chemical modification or crosslinking stabilization of HA, while the addition of chemically bound particles can be considered physical and chemical modification of HA by crosslinking to the particles. The latter, in addition to crosslinking stabilization, is also a method of stabilizing HA with particles in general, especially HAp.

[0048] Some additional benefits that may arise from the use of HA and HAp composites are the avoidance of the Tyndall effect due to the opacity of HAp or the reduction of whiteness in thin areas due to the translucency of HA. Additionally, HAp is radiopaque, allowing the entire composition to be easily detected by radiography during or after injection.

[0049] Overall, it can be considered as the chemical modification, functionalization, or derivatization of HA with inorganic groups, such as silane-surface-treated ceramic HAp, which allows the chemical and physical properties of HA to be altered in a controlled manner to obtain new biomaterials with new, desired, and improved properties.

[0050] As mentioned above, HA and bifunctional or polyfunctional crosslinkers can form covalent bonds under alkaline or acidic conditions. The addition of a polyfunctional crosslinker to a dendrimer can provide additional benefits, such as reduced viscosity and increased longevity within tissues. Dendrimers are macromolecules with precise molecular weights and multibranched structures that define an ellipsoidal or spherical shape. Dendrimers are assembled from a central core and a series of concentric, rooted layers in branches, called "generations," that define their shape. Typically, available functional groups capable of reacting with other moieties are located in the outermost generation (i.e., the outermost portion of the branches). For example, the reaction of HA with both BDDE (a biepoxy-functional crosslinker) and an epoxy-functional dendritic molecule can result in a softer gel, reducing the extrusion force from a fine-gauge needle, and the dendritic structure of the dendrimer crosslinks can result in a gel with longer longevity within tissues.

[0051] Alternatively, some or all of the uncrosslinked HA in the transdermal filler can be crosslinked using an epoxy-functional dendrimer, which maintains a low viscosity, aids in extrusion through a fine-gauge needle, and may provide a somewhat longer lifespan in tissue than uncrosslinked HA. Fine-gauge needles, for example, have gauges ranging from 27G to 30G. Optionally, the longer lifespan may be at least 20%, at least 50%, at least 100%, at least 200%, at least 500% longer than the lifespan of the HA transdermal filler composition, or any integer value therebetween. It is the crosslinked HA, which may be crosslinked with, for example, BDDE and a dendrimer, that provides the longer lifespan. The final gel product, such as a transdermal filler, may contain, for example, about 90% crosslinked gel and 10% uncrosslinked HA. The presence of uncrosslinked HA reduces the viscosity of the gel, making it smoother and easier to extrude from a syringe.

[0052] Non-limiting examples of such epoxy-functional dendrimers can be found in Multiply Functionalized Dendrimers: Protecting-Group-Free Synthesis through Sequential Thiol-Epoxy "Click" Chemistry and Esterification Reaction (RSC Advances, presented May 4, 2015, Khan et al.; see, e.g., molecule 8 as a non-limiting example of an epoxy dendrimer).

[0053] As mentioned above, HA and bifunctional or polyfunctional crosslinkers can form covalent bonds under alkaline or acidic conditions. For example, the addition of POSS® (polyhedral oligomeric silsesquioxane) cage molecules as polyfunctional crosslinkers, such as epoxy-functional POSS® (glycidyl POSS® (epoxy-functional silsesquioxane)), can provide several advantages, including reduced viscosity and longer lifespan in tissues. Different organic functional groups can be tailored to the base cage structure, such as the epoxy functional groups in glycidyl POSS®. For example, the reaction of HA with both BDDE and glycidyl POSS® molecules can result in a gel with a softer gel, lower extrusion force from a fine-gauge needle, and longer lifespan in tissues due to the cage structure of the glycidyl POSS® crosslinks. Alternatively, the non-crosslinked HA in the dermal filler can be partially or fully crosslinked using glycidyl POSS®, which maintains a low viscosity, aiding in extrusion through fine gauge needles and may exhibit some longer longevity in tissue than non-crosslinked HA.

[0054] Also, the above discussion of fine gauge and / or longer life may apply to this embodiment using POSS®.

[0055] Furthermore, POSS® modified with suitable organic functional groups can also function as a coupling agent, for example, when both silane and alkoxy functional groups, such as epoxy functional groups, are present in the base cage structure. Such POSS® structures can be linked to both ends of HA and HAp simply as a silane surface treatment. The procedure for applying POSS® as a coupling agent is similar to that described above.

[0056] While the above materials have featured polysaccharides, specifically HA chemically bonded to particulate calcium phosphate ceramics such as HAp, for example, by the use of coupling agents, this is by no means intended to be limiting. Other polysaccharides, such as heparosan, can follow the same procedure for bonding HA to HAp, and other types of surface-treated microspheres, such as biodegradable polylactic acid microspheres, can play the role of HAp in surface-immobilized HA chains.

[0057] The above description of percutaneous fillers, particularly facial percutaneous fillers, is by no means limiting. Other anatomical areas of the body, such as the neck, buttocks, chest, breasts, hands, and calves, can be filled or augmented. Percutaneous fillers can also be used as a non-surgical alternative or complement to surgery in nose reconstruction and tip grafting. Furthermore, percutaneous fillers can be used throughout the body to blur skin blemishes, such as scars, acne scars, and stretch marks. Additionally, HAp has been found to be beneficial in areas such as bone and dental reconstruction, where it is a primary component. Furthermore, HAp has been found to be beneficial in areas such as joint lubrication to improve joint mobility and shock absorption.

[0058] Example 1 - Silane Surface Treatment 1. Preparation of silanization solution (2% silane in total volume): 93.25% by volume. Methanol is mixed with 3.93% by volume of distilled water. 0.81% by volume of acetic acid is added to buffer the solution to pH 4.5-5.5. 2% by volume of functional silane is added to the solution. Lower silane concentrations can be used to adjust surface silane coverage.

[0059] 2.Silanization: The solution is stirred for 1 to 10 minutes, during which time silanol groups are formed in the solution. The HAp microspheres are then introduced into the solution for approximately 30 minutes. The HAp microspheres are then washed twice with pure methanol. The HAp microspheres are then placed in an oven at temperatures ranging from 70°C to 105°C for 1 to 24 hours to cure the silane layer, or left overnight at room temperature. The time and temperature depend on the type of silane; for example, 70°C for 24 hours is suitable for 3-glycidoxypropyltrimethoxysilane, preventing decomposition of the silane epoxy functional groups.

[0060] A high shear mixer, rotor-stator mixer, homogenizer, or probe-type sonicator can be used to prevent agglomeration of the microspheres.

[0061] Example 2 - Non-crosslinked HA transdermal filling formulation containing HAp microspheres Raw NaHA in any form, such as fiber or powder, with a molecular weight of 2 megaDa, is introduced into the alkaline solution, which is stirred, dissolved, and hydrated in the alkaline solution at room temperature for several hours until a uniform HA viscous liquid is formed.

[0062] 3-glycidoxypropyltrimethoxysilane-surface-treated HAp is introduced into the HA viscous liquid under stirring to form a dispersion. The dispersion is heated to 50°C for several hours to allow for bond formation between the HA and the surface-treated HAp. If the dispersion is highly viscous and the microspheres are immobilized in the dispersion due to high viscosity, this reaction may be performed without stirring. If the microspheres are unstable, a mixer or probe-type ultrasonicator can be used to prevent microsphere settling and aggregation. The dispersion is then cooled and returned to room temperature. The dispersion is then swelled and dialyzed against PBS in a dialysis bag with a 12,000 MW cutoff for several days to remove low-molecular-weight residues. The PBS is periodically replaced, and fresh PBS is introduced into the dispersion. For example, the degree of swelling is determined to form a dispersion containing 30% v / v or less of HAp. Due to the synergistic effect, the concentrations of both HA and HAp can be reduced, yet this concentration still has the same effect as common commercially available transdermal fillers. The dispersion is filled into a syringe, sterilized, and ready to use. Alternatively, the HA surface-bound HAp microspheres can be incorporated into other gel carriers composed of carboxymethylcellulose (CMC), glycerin, and water.

[0063] Example 3 - Crosslinked HA Dermal Filler Formulation Containing HAp Microspheres The basic steps of the crosslinking procedure are known to those skilled in the art. Raw NaHA, for example, 2 megaDa, in any form, such as fiber or powder, 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 uniform HA viscous liquid is formed.

[0064] BDDE and 3-glycidoxypropyltrimethoxysilane-surface-treated HAp are introduced into the HA viscous liquid under stirring to form a dispersion. The dispersion is heated to 50°C for several hours to allow for bond formation between the HA and the surface-treated HAp. If the dispersion is highly viscous and the microspheres are immobilized in the dispersion due to high viscosity, this reaction may be performed without stirring. If microsphere settling is unstable, a mixer or probe-type ultrasonicator can be used to prevent microsphere settling and aggregation. The composite gel is then cooled to room temperature. The composite gel is then swelled and dialyzed with PBS in a dialysis bag with a 12,000 MW cutoff for several days to remove low molecular weight residues, including excess BDDE. The PBS is periodically replaced, and fresh PBS is introduced into the composite gel. Non-crosslinked HA is added to the composite gel to help reduce extrusion force. For example, the composite gel may contain 30% v / v HAp. The composite gel is filled into a syringe, sterilized, and ready to use.

[0065] Example 4 - Testing of silane-treated particles HAp microspheres were prepared according to Example 1 above. Materials used: 1. HAp microspheres medical grade, spherical 15-60 microns, D50=35 microns. 2,3-Glycidoxypropyltrimethoxysilane >98% Sigma Aldrich 3. Methanol >99.9% HPLC Grade Sigma Aldrich 4. Acetic Acid >99.7% ACS Reagent Grade (Sigma Aldrich) SEM+EDS: Equipment: Phenom ProX desktop SEM

[0066] SEM+EDS analysis Scanning electron microscopy (SEM) combined with energy dispersive spectroscopy (EDS) techniques was used to obtain local chemical and elemental analyses of untreated and 3-glycidoxypropyltrimethoxysilane-treated HAp microspheres. SEM micrographs and EDS quantitative analyses of surface element concentrations were obtained.

[0067] SEM micrographs shown in Figures 1 and 2 show untreated and 3-glycidoxypropyltrimethoxysilane-treated HAp microspheres, respectively. Both sets of microspheres exhibited similar morphological structures, indicating that the application of the silane surface treatment to the HAp microspheres did not degrade the microspheres.

[0068] EDS analysis of untreated HAp revealed the presence of Ca, P, and O atoms as expected in HAp. As can be seen in Table 1, the atomic ratio of Ca / P is ∼1.67 as expected in HAp.

[0069] EDS analysis of 3-glycidoxypropyltrimethoxysilane-treated HAp revealed the presence of Si atoms in addition to Ca, P, and O atoms, indicating the presence of silane surface treatment. As mentioned above, as a result of the industrial pretreatment process, a thick three-dimensional cross-linked silane multilayer (polysiloxane network) was formed, which can lead to deviations in the Ca / P atomic ratio from the theoretical value of 1.67, as can be seen in Table 2, and also indicates the presence of silane surface treatment relative to the untreated surface. [Table 1] [Table 2]

[0070] Although the present invention has been described in conjunction with specific embodiments thereof, it is apparent that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, patent applications, and sequences identified by accession numbers mentioned herein are incorporated by reference in their entirety, just as if each individual publication, patent, patent application, or sequence identified by its accession number were specifically and individually indicated to be incorporated by reference herein. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention.

Claims

1. 1. A dermal filler composition comprising a polysaccharide chemically bonded to hydroxyapatite (HAp) microspheres, the polysaccharide is hyaluronic acid (HA) or heparosan; The HAp microspheres comprise an organofunctional silane-treated domain, an organofunctional silane-free domain, and a polysaccharide chemically bonded to the organofunctional silane-treated domain, and the organofunctional silane-free domain is not chemically bonded to the polysaccharide.

2. 2. The dermal filler composition of claim 1, wherein the organofunctional silane has at least one alkoxy group or at least one chlorine atom.

3. The organofunctional silane has the formula R—Si—X ３ 3. The dermal filler composition of claim 2, having the formula: wherein X is an alkoxy group that can be hydrolyzed to a reactive group on the surface, and R is an organic functional group that can react with the HA.

4. 4. The dermal filler composition of claim 2, wherein the organofunctional silane is selected from the group consisting of epoxy-functional silanes, vinyl-functional silanes, or silanes characterized by amino-functional groups.

5. 5. The dermal filler composition of claim 4, wherein the epoxy-functional silane comprises one or more of 3-glycidoxypropyltrimethoxysilane or 3-glycidoxypropyltriethoxysilane.

6. 5. The dermal filler composition of claim 4, wherein the vinyl-functional silane comprises one or more of vinyltrimethoxysilane, vinyltriethoxysilane, or vinyltrichlorosilane.

7. 5. The dermal filler composition of claim 4, wherein the amino-functional silane comprises one or more of 3-aminopropyltriethoxysilane or 3-aminopropyltrimethoxysilane.

8. The dermal filler composition according to any one of claims 1 to 7, wherein the HAp microspheres have a particle size ranging from 10 μm to 100 μm.

9. The dermal filler composition according to any one of claims 1 to 7, wherein the HAp microspheres are in the form of nanopowder having a particle size ranging from 1 nm to 100 nm.

10. 10. The transdermal filler composition of any one of claims 1 to 9, wherein the HAp microspheres are microspheres with a small surface area, have a uniform or smooth shape, or alternatively are irregular, porous, hollow, flower-like porous hollow microspheres.

11. The transdermal filler composition according to any one of claims 1 to 10, further comprising a carrier suitable for insertion into a mammalian subject as a transdermal filler.

12. 12. The dermal filler composition of claim 11, wherein the mammalian subject is a human subject.

13. 13. The dermal filler composition of claim 12 adapted for enhancing facial tissue augmentation.

14. The dermal filler composition according to any one of claims 1 to 13, wherein the concentration of HA ranges from 1 mg / ml to 50 mg / ml.

15. 15. The dermal filler composition according to claim 14, wherein the concentration of the HA is in the range of 15 to 30 mg / ml.

16. The dermal filler composition according to any one of claims 1 to 15, further comprising one or more of an anesthetic, an antioxidant, or a vitamin.

17. The dermal filler composition according to any one of claims 1 to 16, further comprising a dendrimer used as a multifunctional crosslinking agent for crosslinking HA chains.

18. 18. The dermal filler composition of claim 17, wherein the dendrimer is present in an amount suitable to produce a composition characterized by having a softer gel with reduced viscosity and longer longevity in tissue.

19. 19. The transdermal filler composition according to claim 17 or 18, wherein portions of HA are non-crosslinked, and the dendrimer is used as a crosslinking agent to crosslink at least a portion of the non-crosslinked HA, thereby reducing the viscosity of the composition and assisting in extrusion of the composition through a fine gauge needle.

20. 20. The dermal filler composition of claim 19, wherein the fine gauge needle has a gauge ranging from 27G to 30G.

21. The dermal filler composition according to any one of claims 17 to 20, wherein the dendrimer comprises an epoxy-functional dendrimer.

22. 22. The dermal filler composition according to any one of claims 18 to 21, wherein the longer longevity ranges from at least 20% to at least 500% longer than that of HA dermal fillers.

23. The transdermal filler composition according to any one of claims 1 to 22, further comprising polyhedral oligomeric silsesquioxane cage molecules used as multifunctional crosslinking agents for crosslinking HA chains.

24. 24. The dermal filler composition of claim 23, wherein the polyhedral oligomeric silsesquioxane cage molecules are present in an amount providing the composition characterized by reduced viscosity and a softer gel with longer longevity in the tissue.

25. 25. The transdermal filler composition of claim 23 or 24, wherein a portion of the HA is uncrosslinked, and the polyhedral oligomeric silsesquioxane cage molecules are used as crosslinking agents to crosslink the portion of the uncrosslinked HA, thereby reducing the viscosity of the composition and assisting in extrusion of the composition through a fine gauge needle.

26. The transdermal filler composition according to any one of claims 23 to 25, wherein the polyhedral oligomeric silsesquioxane cage molecules are modified with suitable organic functional groups that function as coupling agents connecting the HA and the HAp microspheres.

27. 27. The dermal filler composition of claim 26, wherein the organic functional groups include alkoxy functional groups and epoxy functional groups.

28. 28. The dermal filler composition according to any one of claims 1 to 27, wherein the HAp microspheres are in the form of particles after forming Janus particles, and the particles comprise one hemispherical surface sized by silane and the other hemisphere untreated.

29. 28. The dermal filler composition according to any one of claims 1 to 27, wherein the HAp microspheres are in the form of particles after application of different silane reagents to form Janus particles, and different domains or halves of the particles are partially treated with different reagents.

30. 30. The dermal filler composition of claim 28, wherein the particles comprise chemically bonded HAp microspheres.

31. A method for producing the transdermal filler composition according to any one of claims 1 to 30, comprising: treating the surface of the HAp microspheres with an organofunctional silane to produce organofunctional silane-treated domains and organofunctional silane-free domains; chemically bonding a polysaccharide to said organofunctional silane-treated domain; The method of manufacturing includes the step of: the polysaccharide is hyaluronic acid (HA) or heparosan, and is not chemically bonded to the domain that does not contain the organofunctional silane.

32. 32. The method of claim 31, wherein the HAp microspheres are cured in an oven at a temperature ranging from 70°C to 105°C for 1 to 24 hours.

33. The method of claim 32, wherein the organofunctional silane comprises 3-glycidoxypropyltrimethoxysilane and the curing is carried out at 70° C. for 24 hours.

34. 32. The method of claim 31, further comprising applying the organofunctional silane to the HAp microspheres and allowing the HAp microspheres to cure overnight at room temperature.

35. 35. The method of any one of claims 31 to 34, comprising treating the HAp microspheres according to a masking method, trapping the HAp microspheres at the interface between two phases, thereby masking the untreated hemispheres and modifying the other hemispheres.

36. The method of claim 35, wherein the treatment is by a silylation process.

37. The method of any one of claims 31 to 36, comprising treating the HAp microspheres with different organofunctional silane reagents having different functional groups.

38. 38. The method of claim 37, wherein the treating comprises preparing Janus particles of the HAp microspheres using the different silane reagents.

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