Compositions and methods of suspended hyaluronic acid microspheres for tissue and skin regeneration, augmentation, repair, and adjustment

Abstract

Disclosed herein is a novel, biocompatible, and bioresorbable hydrogel material composed of hyaluronic acid-derived small particles or microspheres, in a physiologically acceptable suspending agent. This material can be used for the regeneration, augmentation, repair, or adjustment of tissue and skin irregularities. This material, compositions, uses, and methods described herein provide a safe and effective alternative to existing methods for therapeutic, aesthetic and reconstructive procedures.

Description

PCT ApplicaƟon 134733-0001WO01 COMPOSITIONS AND METHODS OF SUSPENDED HYALURONIC ACID MICROSPHERES FOR TISSUE AND SKIN REGENERATION, AUGMENTATION, REPAIR, AND ADJUSTMENT CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This applicaƟon claims priority to U.S. Provisional Patent applicaƟon Number 63 / 552,444, filed February 12, 2024. All references, including publicaƟons, patent applicaƟons, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its enƟrety herein. BACKGROUND Field of the InvenƟon

[0002] The present disclosure relates to thefield of regeneraƟve medicine, including hydrogels, methods, uses, and composiƟons for at least one of Ɵssue and skin regeneraƟon, augmentaƟon, repair, and adjustment. SUMMARY

[0003] In an aspect of the disclosure, an injectable composiƟon is provided, comprising a physiologically acceptable suspending agent, and hyaluronic acid (HA) microspheres suspended in the physiologically acceptable suspending agent. In some embodiments, the HA microspheres are HA hydrogel microspheres. In some embodiments, the injectable composiƟon is a hydrogel for Ɵssue engineering or regeneraƟon. In some embodiments, the suspending agent is un-crosslinked hyaluronic acid. In some embodiments, the suspending agent is un-crosslinked collagen. In some embodiments, the suspending agent is bovine collagen. In some embodiments, the HA hydrogel microspheres have a diameter of 1-250 micrometers. In some embodiments, the HA hydrogel microspheres have a diameter of 20- 180 micrometers. In some embodiments, the microspheres swell to 10-100 Ɵmes their volume in pure water. In some embodiments, the material is ground to achieve a dry parƟcle diameter 0.01-25um.

[0004] In another aspect of the disclosure, a method of manufacturing an injectable composiƟon is provided. In some embodiments, the method comprises dissolving a hyaluronic acid powder comprising hyaluronic microspheres in deionized water to form aPCT ApplicaƟon 134733-0001WO01 hyaluronic acid soluƟon. In some embodiments, the method further comprises dissolving a surfactant into a mineral oil to form an oil phase. In some embodiments, the method further comprises adding the hyaluronic acid soluƟon to the oil phase. In some embodiments, the method further comprises mixing the hyaluronic acid soluƟon with the oil phase to form a stable water-in-oil emulsion.

[0005] In some embodiments, the method further comprises adding a crosslinking agent to the water-in-oil emulsion. In some embodiments, the method further comprises centrifuging the water-in-oil emulsion to separate hyaluronic acid microspheres from the oil phase. In some embodiments, the method further comprises washing the hyaluronic acid microspheres with Phosphate-buffered saline (PBS) or other washing agent. In some embodiments, the method further comprises immersing the hyaluronic acid microspheres in a soluƟon. In some embodiments, the method further comprises resuspending the hyaluronic acid microspheres in a buffer. In some embodiments, the method further comprises centrifuging the emulsion to separate the hyaluronic acid microspheres from the buffer. In some embodiments, the method further comprises placing the hyaluronic acid microspheres in at least one of a liquid, a gelaƟn, and a biocompaƟble hydrogel.

[0006] In some embodiments, a degree of degradaƟon is controlled by a degree of crosslinking with the crosslinking agent.

[0007] In some embodiments, the composiƟons described herein are for Ɵssue engineering and regeneraƟon.

[0008] In some embodiments, funcƟonal molecules, geneƟc material, compounds and / or formulaƟons are incorporated into the microspheres to deliver the molecules, geneƟc material, compounds, and / or formulaƟon into a target Ɵssue.

[0009] Other advantages and benefits of the disclosed assemblies, components and methods will be apparent to one of ordinary skill with a review of the following detailed descripƟon. DETAILED DESCRIPTION

[0010] Hydrogels, mimicking the body's matrix and boasƟng biocompaƟbility and tunability, are useful in Ɵssue repair. Their inherent bioresorbable property and minimally invasive delivery, coupled with controlled drug release and cell encapsulaƟon capabiliƟes, make them versaƟle tools for targeted Ɵssue regeneraƟon, wound healing, and beyond.PCT ApplicaƟon 134733-0001WO01 Hydrogels made from hyaluronic acid (HA) microspheres in a suspending agent, offer several advantages for therapeuƟc applicaƟons due to their biocompaƟble and bioresorbable nature. Unlike convenƟonal implant materials, which can be non-biocompaƟble and pose long-term risks associated with permanence or migraƟon, hydrogels derived from the appropriate size HA microspheres in suspending agent offer a significant advantage. Their biocompaƟble nature minimizes the potenƟal for adverse reacƟons and undesired immune responses, facilitaƟng safe and seamless integraƟon with the surrounding Ɵssue.

[0011] HA microspheres, suspended in compaƟble suspending agent, can be delivered via a syringe or other medical device. It should be appreciated that the HA microspheres referred to herein may or may not be hydrogel microspheres. For opƟmal applicaƟon in Ɵssue regeneraƟon and other therapeuƟc procedures, the ideal hydrogel parƟcle size, once swollen, lies within the range of 1-250 micrometers in diameter. In some embodiments, the ideal hydrogel parƟcle size, once swollen, measures between 20-180 micrometers in diameter. This precise size range, when suspended in a suspending agent, minimizes migraƟon, and ensures efficient Ɵssue integraƟon. Their small parƟcle size and pliability facilitate effortless passage through a needle or other suitable delivery device, minimizing discomfort and procedural Ɵme. The treatment process itself may be straighƞorward.

[0012] It is also contemplated that in some embodiments, the hydrogel parƟcle size is between 30-170 micrometers, between 40-160 micrometers, between 50-150 micrometers, between 70-130 micrometers, between 20-170 micrometers, between 20-150 micrometers, between 20-130 micrometers, between 40-180 micrometers, between 50-180 micrometers, between 70-180 micrometers, between 75-125 micrometers, or any other suitable parƟcle size in diameter.

[0013] In some embodiments, the HA microspheres may be suspended in collogen, uncross- linked bovine collagen, uncross linked hyaluronic acid, saline, a combinaƟon thereof, or other physiologically acceptable suspending agent(s). These carrierfluids can impact the performance of the HA microspheres and lead to beƩer treatment outcomes. Once HA microspheres are in suspension, the composiƟon, which in some preferred embodiments is a hydrogel, is placed into a syringe equipped with a needle or other delivery device suitable for the target Ɵssue. The injecƟon or treatment site is sterilized, and the composiƟon is carefully injected or placed into the desired locaƟon. The small parƟcle size may advantageouslyPCT ApplicaƟon 134733-0001WO01 ensure a smooth and controlled delivery, minimizing Ɵssue trauma. Once injected or placed, the hydrogel transiƟons into a stable and supporƟve matrix, effecƟvelyfilling in Ɵssue depressions and deficiencies. This immediate improvement is oŌen noƟceable and graƟfying for paƟents. This minimally invasive nature translates to several paƟent-centric benefits such as reduced discomfort, faster recovery, and outpaƟent treatment.

[0014] Mechanism of AcƟon:

[0015] HA hydrogel microspheres, when introduced to the body, elicit the immune system to respond by generaƟng connecƟve Ɵssue to encapsulate the microspheres, thus regenerate, augment, adjust and repair target site and Ɵssue. This immune response is the basis for the autologous Ɵssue engineering result of this disclosure. By controlling one, some, or all of the cross link density, surface texture, microsphere size, type of suspending agent, the raƟo of microspheres to suspending agent, the specificaƟon of the engineered Ɵssue volume, Ɵssue texture, target locaƟon, and result duraƟon can be modulated and achieved.

[0016] ApplicaƟons:

[0017] Some useful applicaƟons for the composiƟons described herein are:

[0018] Autologous Tissue Engineering: HA hydrogel, for example, a crosslinked HA hydrogel suspended in a hydrogel matrix, can be used to create scaffolds that mimic the natural extracellular matrix, providing a supporƟve environment for cell growth and differenƟaƟon. This allows scienƟsts to engineer Ɵssues to address deficiency, potenƟally revoluƟonizing regeneraƟve medicine.

[0019] AugmenƟng Tissue Structure in Lower Esophageal Sphincters (LES): HA hydrogel or HA microspheres-based hydrogels, for example, a crosslinked HA hydrogel suspended in a hydrogel matrix, can be injected into the lower esophageal sphincter (LES) of paƟents suffering from Gastro-Esophageal Reflux Disease (GERD) to reinforce the structure and funcƟons of the LES and prevents stomach acid fromflowing into the esophagus.

[0020] Reinforce Tissue Structure and FuncƟons in Urethral and Anal Sphincter: HA hydrogel or HA microspheres-based hydrogels can be used in the urethral walls and anal sphincter to elicit Ɵssue regeneraƟon. The reinforced structure can restore funcƟons of the urethral and anal sphincter and allow for improvement of symptoms of Urinary and Fecal InconƟnence.PCT ApplicaƟon 134733-0001WO01

[0021] Wrinkle and Fold Filling: The injectable nature of the hydrogel, for example, HA microspheres-based hydrogels, makes it ideal forfilling in wrinkles and folds on the face and neck, offering a non-surgical soluƟon for achieving a smoother and more youthful appearance.

[0022] Facial Contouring: The supporƟve nature of the hydrogel enables its use for augmenƟng lips, cheeks, chin, jawline, and nose, providing natural-looking volume and definiƟon for a more balanced facial profile.

[0023] Acne Scar CorrecƟon: Thefilling and supporƟve properƟes of the hydrogel, for example, HA microspheres-based hydrogels, may effecƟvely address acne scars and other skin depressions, promoƟng a smoother and more even skin texture.

[0024] SoŌ Tissue AugmentaƟon for ReconstrucƟve Surgery: In reconstrucƟve surgery, the hydrogel, for example, HA microspheres-based hydrogels, provides a valuable tool for soŌ Ɵssue augmentaƟon, aiding in the restoraƟon of lost Ɵssue volume and improving funcƟonal outcomes.

[0025] Drug Delivery: Due to its biocompaƟbility and ability to encapsulate various substances, HA is a promising material for drug delivery applicaƟons. By controlling the crosslinking process and other factors, scienƟsts can design HA microspheres-based hydrogels that release drugs in a controlled manner over Ɵme, targeƟng specific sites in the body and improving treatment efficacy.

[0026] Wound Healing: HA hydrogels, or HA microspheres-based hydrogels, can be applied to wounds to create a moist environment that promotes healing. They can also be used to deliver drugs or other therapeuƟc agents directly to the wound site for enhanced healing effects.

[0027] Gene Delivery: HA-based vectors can be used to deliver genes into cells, offering potenƟal for gene therapy to treat various diseases. These vectors can be designed to target specific cells and deliver the genes with high efficiency, opening new avenues for personalized medicine.

[0028] CosmeƟcs: HA can be used as a thickener and stabilizer in various cosmeƟc products, improving their texture, consistency, and shelf life. Its biocompaƟble nature makes it a safe and versaƟle ingredient for cosmeƟc formulaƟons.PCT ApplicaƟon 134733-0001WO01

[0029] The structural and physical properƟes of HA hydrogels are important for a variety of applicaƟons, including Ɵssue engineering, Ɵssue repair and drug delivery. In general, these properƟes can be controlled by controlling the degree of crosslinking on the material with more crosslinking leading to harder parƟcles. For example, the sƟffness of an HA hydrogel is determined by its crosslink density. Crosslinking is the process of joining two or more molecules together, and it can be achieved through a variety of chemical reacƟons. The higher the crosslink density, the sƟffer the hydrogel will be. The elongaƟon at break is a measure of the elasƟcity of an HA hydrogel. It is defined as the amount of stretch that the hydrogel can withstand before it breaks. ElasƟc hydrogels are able to stretch and return to their original shape, while inelasƟc hydrogels do not. The elongaƟon at break of a hyaluronic acid (HA) hydrogel is controlled by its crosslink density. The crosslink density is the number of bonds between the polymer chains in the hydrogel. A higher crosslink density results in a sƟffer hydrogel with a lower elongaƟon at break, while a lower crosslink density results in a more elasƟc hydrogel with a higher elongaƟon at break. The degradaƟon rate of an HA hydrogel is determined by its chemical composiƟon and crosslink density. HA hydrogels are biodegradable, which means that they break down over Ɵme. The degradaƟon rate of an HA hydrogel can be controlled by adjusƟng the crosslink density.

[0030] Examples:

[0031] The following non-limiƟng examples are provided as exemplary methods of preparing HA hydrogel microspheres and injectable composiƟons comprising HA hydrogel microspheres. It should be appreciated that other suitable crosslinking agents, surfactants, oils, buffers, washing agents, and other components are contemplated. It should also be appreciated that the amounts, Ɵmes, equipment, pressures, and temperatures described herein are provided as non-limiƟng examples, and that other suitable amounts, Ɵmes, equipment, pressures, and temperatures are also contemplated.

[0032] Example 1: Synthesis of HA hydrogel microsphere

[0033] Materials: Hyaluronic acid (HA) powder, Deionized (DI) water, 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), N-Hydroxysuccinimide (NHS), Oil phase (e.g., mineral oil), Tween 80 (surfactant), Phosphate-buffered saline (PBS), Calcium chloride (CaCl2) soluƟon (0.145 M), Phosphoric acid (H3PO4) soluƟon (0.087 M), Ammonium hydroxide (NH4OH) soluƟon (10% v / v).PCT ApplicaƟon 134733-0001WO01

[0034] Dissolve HA powder in DI water to achieve the desired concentraƟon. SƟr unƟl the HA is completely dissolved. Dissolve Tween 80 into mineral oil. Add the prepared HA soluƟon to the oil phase containing Tween 80 as the surfactant. Use a homogenizer or vortex mixer to vigorously mix the two phases unƟl a stable water-in-oil emulsion is formed. Add EDC and NHS to the emulsion under sƟrring to iniƟate crosslinking of the HA molecules. The amount of crosslinker can be adjusted to control the degree of crosslinking and the properƟes of the resulƟng microspheres. Allow the emulsion to stand for a period of Ɵme (~8 hours) to allow complete crosslinking of the HA. Centrifuge the emulsion at 1,000g to separate the HA microspheres from the oil phase. Wash the collected microspheres thoroughly with PBS to remove any residual oil and / or crosslinking agents. Immerse the HA microspheres in a soluƟon containing CaCl2and H3PO4, followed by immersion in NH4OH soluƟon. This step enhances the mechanical strength and biostability of the microspheres. Resuspend the microspheres in PBS or another appropriate buffer and allow them to swell fully. Centrifuge the emulsion (e.g., at 1,000g) to separate the HA microspheres from PBS or other buffer.

[0035] AŌer the parƟcles are isolated they can be sterilized prior to injecƟon. There are many various methods to sterilize such as UV irradiaƟon, ethanol sterilizaƟon, or plasma irradiaƟon, yet steam sterilizaƟon works well for the HA microparƟcles. To sterilize the microparƟcles, they are placed in a glass container and sealed. The autoclave is heated to 126°C and a pressure of 1.5 kgf / cm2 with a hold Ɵme of 5 minutes. The autoclave is cooled and the parƟcles are collected in a sterile environment. Cooling the autoclave quickly and limiƟng the exposure to high temperatures may be preferred as high temperatures for a long Ɵme could alter the structural integrated due to thermal degradaƟon. Although that is a concern, we experienced no degradaƟon aŌer this process. The sterilize HA parƟcles are then resuspended in a sterile buffer soluƟon using common asepƟc techniques. The sterilize suspension can be a buffer of uncross-linked hyaluronic acid, bovine collagen, collagen, saline or other suspending agent.

[0036] Example 2: InjecƟon of HA microspheres.

[0037] The prepared HA microspheres are placed in a matrix containing either bovine collagen, uncross-linked collagen, uncross-linked hyaluronic acid, saline, or other biocompaƟble gelaƟn or liquid to form a biocompaƟble carrier in an asepƟc environment. ThePCT ApplicaƟon 134733-0001WO01 soluƟon is placed in a sterile syringe and injected, for example, into the skin panniculus adiposus layer and panniculus carnosus layer.

[0038] Non-limiƟng Embodiments:

[0039] Embodiment 1. An injectable composiƟon, comprising: a physiologically acceptable suspending agent; and hyaluronic acid microspheres suspended in the physiologically acceptable suspending agent.

[0040] Embodiment 2. The injectable composiƟon of embodiment 1, wherein the hyaluronic acid microspheres are hydrogel microspheres.

[0041] Embodiment 3. The injectable composiƟon of any of embodiments 1-2, wherein the injectable composiƟon is a hydrogel for Ɵssue engineering or regeneraƟon.

[0042] Embodiment 4. The injectable composiƟon of any of embodiments 1-3, wherein the suspending agent comprises un-crosslinked hyaluronic acid.

[0043] Embodiment 5. The injectable composiƟon of any of embodiments 1-4, wherein the suspending agent is un-crosslinked collagen.

[0044] Embodiment 6. The injectable composiƟon of any of embodiments 1-5, wherein the suspending agent comprises bovine collagen.

[0045] Embodiment 7. The injectable composiƟon of any of embodiments 1-6, wherein the suspending agent comprises saline.

[0046] Embodiment 8. The injectable composiƟon of any of embodiments 1-7, wherein the hyaluronic acid microspheres have a diameter of 1-250 micrometers.

[0047] Embodiment 9. The injectable composiƟon of any of embodiments 1-8, wherein the hyaluronic acid microspheres have a diameter of 20-180 micrometers.

[0048] Embodiment 10. The injectable composiƟon of any of embodiments 1-9, wherein the microspheres swell to 10-100 Ɵmes their volume in pure water.

[0049] Embodiment 11. A method of manufacturing an injectable composiƟon, comprising: dissolving a hyaluronic acid powder comprising hyaluronic microspheres in deionized water to form a hyaluronic acid soluƟon.

[0050] Embodiment 12. A method of embodiment 11, further comprising dissolving a surfactant into a mineral oil to form an oil phase; adding the hyaluronic acid soluƟon to the oil phase; and mixing the hyaluronic acid soluƟon with the oil phase to form a stable water- in-oil emulsion.PCT ApplicaƟon 134733-0001WO01

[0051] Embodiment 13. A method of any of embodiments 11-12, adding a crosslinking agent to the water-in-oil emulsion.

[0052] Embodiment 14. A method of any of embodiments 11-13, centrifuging the water-in- oil emulsion to separate hyaluronic acid microspheres from the oil phase.

[0053] Embodiment 15. A method of any of embodiments 11-14, washing the hyaluronic acid microspheres with a washing agent.

[0054] Embodiment 136. A method of any of embodiments 11-15, wherein the washing agent comprises Phosphate-buffered saline.

[0055] Embodiment 17. A method of any of embodiments 11-16, immersing the hyaluronic acid microspheres in a soluƟon.

[0056] Embodiment 18. A method of any of embodiments 11-17, resuspending the hyaluronic acid microspheres in a buffer.

[0057] Embodiment 19. A method of any of embodiments 11-18, centrifuging the resuspended hyaluronic acid microspheres in the buffer to separate the hyaluronic acid microspheres from the buffer.

[0058] Embodiment 20. A method of any of embodiments 11-19, placing the hyaluronic acid microspheres in a biocompaƟble hydrogel.

[0059] Embodiment 21. A method of any of embodiments 11-20, wherein a degree of degradaƟon is controlled by a degree of crosslinking with the crosslinking agent.

[0060] Embodiment 22. A method of any of embodiments 11-21, wherein the composiƟon is for Ɵssue engineering and regeneraƟon, and wherein at least one of funcƟonal molecules, geneƟc material, compounds and formulaƟons are incorporated into the microspheres to deliver the at least one of the funcƟonal molecules, geneƟc material, compounds, and formulaƟon into a target Ɵssue.

[0061] Thus, specific materials, composiƟons, uses, and methods for suspended HA microspheres have been disclosed. It should be apparent, however, to those skilled in the art that many more modificaƟons besides those already described are possible without deparƟng from the invenƟve concepts herein. The invenƟve subject maƩer, therefore, is not to be restricted except in the spirit of the disclosure. Moreover, in interpreƟng the disclosure all terms should be interpreted in the broadest possible manner consistent with the context. In parƟcular the terms “comprises” and “comprising” should be interpreted as referring to thePCT ApplicaƟon 134733-0001WO01 elements, components, or steps in a non-exclusive manner, indicaƟng that the referenced elements, components, or steps can be present, or uƟlized, or combined with other elements, components, or steps that are not expressly referenced.

[0062] As used in the descripƟon herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the descripƟon herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0063] Reference throughout this specificaƟon to “one embodiment” or “an embodiment” means that a parƟcular feature, component, structure, or characterisƟc described in connecƟon with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specificaƟon are not necessarily all referring to the same embodiment. Furthermore, the parƟcular features, components, structures, or characterisƟcs may be combined in any suitable manner in one or more embodiments.

[0064] The word “exemplary” is used herein to mean “serving as an example, instance, or illustraƟon.” Any aspect described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects. Unless specifically stated otherwise, the term “some” refers to one or more.

[0065] The recitaƟon of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and including the endpoints. Unless otherwise indicated herein, each individual value is incorporated into the specificaƟon as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0066] Various modificaƟons to the embodiments described herein will be readily apparent to those skilled in the art, and the general principles described herein can be applied to other embodiments without deparƟng from the spirit or scope of the claims. Thus, it is understood that the scope of the claims fully encompasses other embodiments that may become obvious to those skilled in the art and that the scope of the claims are accordingly not limited.

[0067] CombinaƟons, described herein, such as “at least one of A, B, or C,” “one or more of A, B, or C,” “at least one of A, B, and C,” “one or more of A, B, and C,” and “A, B, C, or anyPCT ApplicaƟon 134733-0001WO01 combinaƟon thereof” include any combinaƟon of A, B, and / or C, and may include mulƟples of A, mulƟples of B, or mulƟples of C. Specifically, combinaƟons such as “at least one of A, B, or C,” “one or more of A, B, or C,” “at least one of A, B, and C,” “one or more of A, B, and C,” and “A, B, C, or any combinaƟon thereof” may be A only, B only, C only, A and B, A and C, B and C, or A and B and C, and any such combinaƟon may contain one or more members of its consƟtuents A, B, and / or C. For example, a combinaƟon of A and B may comprise one A and mulƟple B’s, mulƟple A’s and one B, or mulƟple A’s and mulƟple B’s.

Claims

PCT ApplicaƟon 134733-0001WO01 CLAIMS 1. An injectable composition, comprising: a physiologically acceptable suspending agent; and hyaluronic acid microspheres suspended in the physiologically acceptable suspending agent.

2. The composition of claim 1, wherein the hyaluronic acid microspheres are hydrogel microspheres.

3. The composition of claim 1, wherein the injectable composition is a hydrogel for tissue engineering or regeneration.

4. The composition of claim 1, wherein the suspending agent is un-crosslinked hyaluronic acid.

5. The composition of claim 1, wherein the suspending agent is un-crosslinked collagen.

6. The composition of claim 1, wherein the suspending agent is bovine collagen.

7. The composition of claim 1, wherein the suspending agent is saline.

8. The composition of claim 2, wherein the hyaluronic acid hydrogel microspheres have a diameter of 1-250 micrometers.

9. The composition of claim 2, wherein the hyaluronic acid hydrogel microspheres have a diameter of 20-180 micrometers.

10. The composition of claim 1, wherein the microspheres swell to 10-100 times their volume in pure water.

11. The composition of claim 1, wherein the material is ground to achieve a dry particle diameter of 0.01-25um.PCT ApplicaƟon 134733-0001WO01 12. A method of manufacturing an injectable composition, comprising: dissolving a hyaluronic acid powder comprising hyaluronic microspheres in deionized water to form a hyaluronic acid solution; dissolving a surfactant into a mineral oil to form an oil phase; adding the hyaluronic acid solution to the oil phase; and mixing the hyaluronic acid solution with the oil phase to form a stable water-in-oil emulsion.

13. The method of claim 13, further comprising: adding a crosslinking agent to the water-in-oil emulsion; centrifuging the water-in-oil emulsion to separate hyaluronic acid microspheres from the oil phase; washing the hyaluronic acid microspheres with Phosphate-buffered saline; after washing, immersing the hyaluronic acid microspheres in a solution; after immersing, resuspending the hyaluronic acid microspheres in a buffer; and after resuspending, centrifuging to separate the hyaluronic acid microspheres from the buffer; and placing the hyaluronic acid microspheres in a biocompatible hydrogel.

14. The method of claim 13, wherein a degree of degradation is controlled by a degree of crosslinking with the crosslinking agent.

15. The method of claim 13, wherein the composition is for tissue engineering and regeneration, and wherein at least one of functional molecules, genetic material, compounds and formulations are incorporated into the microspheres to deliver the at least one of the functional molecules, genetic material, compounds, and formulation into a target tissue.