WO2019067942A1 - Bioactive implants and methods of making - Google Patents

Abstract

Provided herein are bioactive implants, methods of making, and methods of delivery. In certain aspects, bioactive implants are described that can be used to help repair subcutaneous soft-tissue deficits in a subject and/or treat pain in a subject. Also provided herein are methods of making, use, and administration thereof. The bioactive implants can be prepared by harvesting cells or tissue from a donor and selectively lysing the cells or tissue to obtain the intracellular content. Also provided herein are delivery devices for delivering the bioactive implants described herein and kits that include the bioactive implants described herein.

Description

BIOACTIVE IMPLANTS AND METHODS OF MAKING

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application serial number 62/565,967 filed on September 29, 2017, having the title "BIOACTIVE IMPLANTS AND METHODS OF MAKING," the disclosure of which is incorporated herein in its entirety.

BACKGROUND Correction of subcutaneous soft tissue defects in humans is a significant problem that faces the field of medicine, especially that of plastic surgery. Currently available implants, which aim to correct such defects, have limited capacity to interact with the environment in which they are implanted. This limited capacity to interact with the host tissue precludes such implants from reaching their full potential, and in many cases, can prevent successful outcomes for such implants. Furthermore, the implantation of existing implants can be invasive, causing undue trauma at the site of implantation, and the implantation of implants that can maintain a desired shape after implantation, while staying functional, is lacking.

Additionally, bioactive implants suffer from poor flowability through traditional applicators or injectors, impeding successful implantation of such. As such, there exists a need for improved bioactive implants, as well as methods of making bioactive implants in addition to methods for delivery of medical implants.

SUMMARY

Described herein are bioactive implants, methods of making, and methods of using. Bioactive implants (referred to herein as medical implants) as described herein can comprise collagen, hyaluronan, protein, amino acid, peptide, or glycosaminoglycan, individually or in combination. Medical implants can further comprise biocompatible material and/or growth factors. Medical implants can include a plurality of cells from one or more sources. Cells or growth factors of medical implants as described herein can be each independently derived from adipose, bone marrow, blood, amniotic fluid, or from another matrix such as intestine, bladder, placental, or other soft tissue. Medical implants can be acellular and/or delipidized. Medical implants as described herein can comprise decontaminated adipose, dermis, or fascia, individually or in combination. Medical implants can be derived from allograft, autograft, xenograft, or synthetic sources. The source of cells or growth factors of medical implants as described herein can be a different source than the other components of the medical implant.

Described herein are methods of treatment. In certain aspects, described herein are a method of treating pain in a subject. Methods of treating pain can comprise delivering medical implants as described herein to one or more vertebral discs in the spine of a subject in need thereof in a therapeutic amount. Methods of treatment and/or treating pain can comprise delivering medical implants as described herein to a joint of a subject in need thereof in a therapeutic amount. Methods of treatment as described herein can comprise delivering medical implants as described herein to a subject in need thereof in a therapeutic amount.

In certain aspects, methods of treatment can be methods of treating soft-tissue defects.

Methods of treating soft-tissue defects can comprise delivering medical implants as described herein to a subject in need thereof in a therapeutic amount.

Described herein are methods of providing support or cushioning to treat soft-tissue defects in a subject. Such methods can comprise delivering medical implants as described herein to a subject in need thereof in a therapeutic amount.

Also described herein are methods to increase growth factor binding. Methods of increasing growth factor binding as described herein can comprise providing a bone composition in a solution; adding an acid to the solution, thereby exposing the bone composition to an acid to demineralize the bone composition; and adding a buffer or base to raise the pH of the solution from a first level to a second level, wherein the second level is more basic than the first level, after at least partial demineralization of the bone. Methods to increase growth factor binding can also comprise isolating the bone composition, mineral that has precipitated of solution, or both. In certain aspects, the extracellular matrix of the bone and/or bone composition can be removed prior to adding an acid to the solution.

Described herein are kits containing medical implants as described herein. Kits as described herein can comprise of medical implants as described herein and a delivery device (such as an applicator; also referred to as an applicator). In certain aspects, the medical implant is loaded in the delivery device in the kit and comes ready for a user's use.

Medical implants as described herein can be medical implants prepared according to any of the methods as described herein, individually or in combination.

Described herein are methods of enriching bound growth factors in a medical implant. Such methods comprise providing a scaffold in solution; combining the scaffold in solution with growth factors or agents; and raising the pH of the solution from a first level to a second level, wherein the second level is more basic than the first, by adding a base or buffer to the solution. Such methods can further comprise isolating the scaffold. The scaffold can be derived from allograft or xenograft tissue sources or created synthetically. The scaffold can be comprised of comprised of collagen, hyaluronan, or other protein, amino acid, GAG, and/or biocompatible material. Growth factors or agents can comprise VEGF, bFGF, aFGF,TGFB-1 , PDGF, IGF, or any other growth factor or tissue growth stimulative agent or differentiation agent, individually or in combination. Growth factors or agents comprise VEGF, bFGF, aFGF.TGFB- 1 , PDGF, or IGF, individually or in combination.

Also described herein are methods of providing shape memory for a medical implants.

Methods for providing shape memory for a medical implant can comprise providing the medical implant; and providing energy to the medical implant. The medical implant can be frozen before providing the energy. The medical implant can be hydrated before providing the energy. One or more additives can be added to the medical implant, before providing the energy or after providing the energy. Additives can be one or more of a vasodilator, a growth factor, or an amino acid. Medical implants can be placed in a mold of a desired shape before providing the energy. The mold can be constructed of a polymer or other material that can be confirmed to a shape and then becomes rigid after conforming to the shape. The mold can be configured to receive the medical implant in a desired configuration according to the shape of the destination to which the implant will be implanted. Medical implants can be freeze dried before or after providing the energy. Medical implants can be sterilizing after providing the energy. Medical implants can be terminally sterilized after the energy is provided.

Medical implants as described herein can be three-dimensional medical implants. Medical implants as described herein can be derived from an allograft. Medical implants as described herein can be primarily comprised of collagen. Medical implants as described herein can be micronized soft tissue. Medical implants as described herein can be a particulated medical implant.

BRIEF DESCRIPTION OF THE DRAWINGS Further aspects of the present disclosure will be readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

FIG. 1 is an embodiment of an applicator 100 according to the present disclosure. FIG. 2 shows a portion of an embodiment of a needle 101 of an applicator 100 according to the present disclosure, from a side of the applicator 100 different than what is shown in FIG. 1.

FIG. 3 is another embodiment of an applicator according to the present disclosure. FIG. 4 is an embodiment 200 according to the present disclosure.

FIG. 5 is an embodiment 300 according to the present disclosure.

FIG. 6 is a flow chart illustrating an embodiment of the method 1000.

FIG. 7 is a flow chart illustrating an embodiment of the method 2000. FIG. 8 is a flow chart illustrating an embodiment of the method 3000.

FIG. 9 is a flow chart illustrating an embodiment of the method 4000.

FIG. 10 is a flow chart illustrating an embodiment of the method 5000.

FIG. 1 1 is a graph depicting the modulation of flow characteristics in terms of mix energy.

FIG. 12 is a graph of volume retention versus non-protein/GAG mass according to an embodiment of the present disclosure.

FIGs. 13A and 13B are before (FIG. 13A) and after (FIG. 13B) thermal images taken to visualize temperature differences created by subcutaneous fat insulation. FIG. 13A was taken of the subject before application and FIG. 13B was taken of the subject about a week later. Notice the change on one side of the body.

FIG. 14 is an example of a crosslinked shape-formed medical implant according to the present disclosure.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology, physiology, modern surgical techniques, microbiology, organic chemistry, biochemistry, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

Definitions

In describing the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.

As used herein, "about," "approximately," and the like, when used in connection with a numerical variable, generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval for the mean) or within .+-.10% of the indicated value, whichever is greater.

As used herein, ""effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages.

As used herein, "therapeutic" refers to treating or curing a disease or condition.

As used herein, "preventative" refers to hindering or stopping a disease or condition before it occurs or while the disease or condition is still in the sub-clinical phase.

As used herein, "concentrated" used in reference to an amount of a molecule, compound, or composition, including, but not limited to, a chemical compound, polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that indicates that the sample is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than that of its naturally occurring counterpart.

As used herein, "isolated" means separated from constituents, cellular and otherwise, with which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated in nature. A non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart.

As used herein, "diluted" used in reference to an amount of a molecule, compound, or composition including but not limited to, a chemical compound, polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that indicates that the sample is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is less than that of its naturally occurring counterpart.

As used interchangeably herein, "subject," "individual," or "patient," refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. The term "pet" includes a dog, cat, guinea pig, mouse, rat, rabbit, ferret, and the like. The term farm animal includes a horse, sheep, goat, chicken, pig, cow, donkey, llama, alpaca, turkey, and the like.

As used herein, "biocompatible" or "biocompatibility" refers to the ability of a material to be used by a patient without eliciting an adverse or otherwise inappropriate host response in the patient to the material or a derivative thereof, such as a metabolite, as compared to the host response in a normal or control patient.

As used herein, "cell," "cell line," and "cell culture" include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included.

As used herein, "specific binding" refers to binding which occurs between such paired species as enzyme/substrate, receptor/agonist, antibody/antigen, and lectin/carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of the two species produces a non- covalently bound complex, the binding which occurs is typically electrostatic, hydrogen- bonding, or the result of lipophilic interactions. Accordingly, "specific binding" occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or enzyme/substrate interaction. In particular, the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs. Thus, for example, an antibody preferably binds to a single epitope and to no other epitope within the family of proteins.

As used herein, "control" is an alternative subject or sample used in an experiment for comparison purposes and included to minimize or distinguish the effect of variables other than an independent variable. As used herein, "positive control" refers to a "control" that is designed to produce the desired result, provided that all reagents are functioning properly and that the experiment is properly conducted.

As used herein, "negative control" refers to a "control" that is designed to produce no effect or result, provided that all reagents are functioning properly and that the experiment is properly conducted. Other terms that are interchangeable with "negative control" include "sham," "placebo," and "mock."

As used herein, "culturing" refers to maintaining cells under conditions in which they can proliferate and avoid senescence as a group of cells. "Culturing" can also include conditions in which the cells also or alternatively differentiate.

As used herein, "synergistic effect," "synergism," or "synergy" refers to an effect arising between two or more molecules, compounds, substances, factors, or compositions that is greater than or different from the sum of their individual effects.

As used herein, "additive effect" refers to an effect arising between two or more molecules, compounds, substances, factors, or compositions that is equal to or the same as the sum of their individual effects.

As used herein, "autologous" refers to being derived from the same subject that is the recipient.

As used herein, "allograft" refers to a graft that is derived from one member of a species and grafted in a genetically dissimilar member of the same species.

As used herein "xenograft" or "xenogeneic" refers to a substance or graft that is derived from one member of a species and grafted or used in a member of a different species.

As used herein, "autograft" refers to a graft that is derived from a subject and grafted into the same subject from which the graft was derived.

As used herein, "allogeneic" refers to involving, derived from, or being individuals of the same species that are sufficiently genetically different so as to interact with one another antigenicaly.

As used herein, "syngeneic" refers to subjects or donors that are genetically similar enough so as to be immunologically compatible to allow for transplantation, grafting, or implantation.

As used herein, "implant" or "graft," as used interchangeably herein, refers to cells, tissues, or other compounds, including metals and plastics, that are inserted into the body of a subject.

As used herein, "filler" refers to a substance used to fill a cavity or depression. The filler can fill the depression such that it is level with the surrounding area or that the cavity is filled, such that the depth of the depression or volume of the cavity is decreased, or such that the area that was the depression is now raised relative to the areas immediately surrounding the depression.

As use herein, "immunogenic" or "immunogenicity" refers to the ability of a substance, compound, molecule, and the like (referred to as an "antigen") to provoke an immune response in a subject.

As used herein, "exogenous" refers to a compound, substance, or molecule coming from outside a subject or donor, including their cells and tissues.

As used herein, "endogenous" refers to a compound, substance, or molecule originating from within a subject or donor, including their cells or tissues.

As used herein, "bioactive" refers to the ability or characteristic of a material, compound, molecule, or other particle that interacts with or causes an effect on any cell, tissue and/or other biological pathway in a subject.

As used herein, "bioactive factor" refers to a compound, molecule, or other particle that interacts with or causes an effect on any cell, tissue, and/or other biological pathway in a subject.

As used herein, "physiological solution" refers to a solution that is about isotonic with tissue fluids, blood, or cells.

As used herein, "donor" refers to a subject from which cells or tissues are derived.

As used herein, "slurry" refers to the resultant product from any of the methods described herein. Accordingly, the slurry can be in any form resulting from the processing described herein, including but not limited to, dehydrated slurry or tissue, paste, powder, solution, gel, putty, particulate and the like.

As used herein, "extra cellular matrix" refers to the non-cellular component surrounding cells that provides support functions to the cell including structural, biochemical, and biophysical support, including but not limited to, providing nutrients, scaffolding for structural support, and sending or responding to biological cues for cellular processes such as growth, differentiation, and homeostasis.

As used herein, "complete extracellular matrix" refers to extracellular matrix that has all components (proteins, peptides, proteoglycans, and the like) present and may or may not include other cells that are embedded in the extra cellular matrix.

As used herein, "decellularized extracellular matrix" refers to complete extracellular matrix that has been processed to remove any cells embedded within the extracellular matrix.

As used herein, "extracellular matrix component" refers to a particular component. By way of a non-limiting example, an extracellular matrix comportment can be a a specific class of comments (e.g. proteoglycans) or individual component (e.g. collagen I) that is separated or isolated from the other extracellular components. These components can be made synthetically. As used herein "hydrogel" refers to a network of hydrophilic polymer chains that are dispersed in water. "Hydrogel" also includes a network of hydrophilic polymer chains dispersed in water that are found as a colloidal gel.

As used herein "self-assembling peptides" refer to peptides which undergo spontaneous assembly into ordered nanostructures. "Self-assembling peptides" include di- peptides, lego peptides, surfactant peptides, molecular paint or carpet peptides, and cyclic peptides.

As used herein, "adipocyte" refers to a cell type also known as a lipocyte or fat cell. Adipocytes are the cells that primarily compose adipose tissue, specialized in storing energy as fat.

As used herein, "administering" refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.

As used herein, "effective amount" refers to an effective amount of medical implants as described herein to reduce the appearance of subcutaneous soft-tissue defects, to bolster the support of endogenous soft-tissue in the body which supports joints (for example, ankle joints, knee joints, vertebral discs, and the like, or to aid in the cushioning provided by soft- tissue at joints or on pads of the hands and/or feet.

As used herein, "primarily" means that a part of the whole is present in an amount greater than all other parts of the whole, greater than all other parts individually or in combination.

Discussion

Bioactive Implants and Methods of Making

Described herein are bioactive implants (also referred to herein as medical implants), methods of making, methods of delivery, and kits comprising bioactive implants.

As described herein, medical implants can comprise collagen, hyaluronan, protein, amino acid, peptide, and glycosaminoglycan. In embodiments of the present disclosure, the medical implants are primarily collagen-based and derived from allograft tissue. In embodiments according to the present disclosure, proteins and peptides are proteins and peptides derived from allograft tissue.

In certain aspects, medical implants can further comprise biocompatible material. In certain aspects, biocompatible material can be material that can product medical implants during processing and/or storage. In embodiments of the present disclosure, the biocompatible material is one or more of glycerol, propylene glycol, sugars, and preservatives, individually or in combination.

In certain aspects, medical implants can further comprise growth factors.

In certain aspects, medical implants can comprise a plurality of cells from one or more sources.

The cell or growth factors of the present disclosure can each independently be derived from adipose, bone marrow, blood, muscle, amniotic fluid, or from another matrix such as intestine, bladder, placental, or other soft tissue. Sources of such can be allograft, autograft, xenograft, or synthetic sources.

In certain embodiments, medical implants are acellular.

In certain aspects, medical implants can be delipidized. Lipids can be removed from medical implants according to well-known methods as known by skilled artisans.

In certain aspects, medical implants as described herein can comprise decontaminated adipose, dermis, or fascia, individually or in combination.

Medical implants as disclosed herein can be derived from allograft, autograft, xenograft, or synthetic sources.

Described herein also are methods of treatment. In an embodiment, disclosed herein is a method of treating pain in a subject. A method of treating pain in a subject can comprise delivering the medical implant of any one of claims 1 to 9 to a vertebral disc in the spine of a subject in need thereof in a therapeutic amount. A subject in need thereof can be a human subject experiencing pain. In certain aspects, a subject in need thereof can be a human subject experiencing joint (knee, ankle, wrist, any joint of the human body). A therapeutic amount can be about 0.1 cc to about 5cc. A therapeutic amount can be about 0.5cc to about 4.5cc. A therapeutic amount can be about 1cc to about 4cc. A therapeutic amount can be about 1.5cc to about 3.5cc. A therapeutic amount can be about 2cc to about 3cc. A therapeutic amount can be about 2.5cc.

In an embodiment, a method of treating pain in a subject can comprise delivering a medical implant as described herein to a knee of a subject in need thereof in a therapeutic amount. A subject in need thereof can be a human subject experiencing knee pain. A therapeutic amount can be 0.1 cc to 5cc. A therapeutic amount can be about 0.5cc to about 4.5cc. A therapeutic amount can be about 1cc to about 4cc. A therapeutic amount can be about 1.5cc to about 3.5cc. A therapeutic amount can be about 2cc to about 3cc. A therapeutic amount can be about 2.5cc.

In an embodiment, a method of treating pain in a subject can comprise delivering a medical implant as described herein to one or more vertebral discs of a subject in need thereof in a therapeutic amount. A subject in need thereof can be a human subject experiencing spinal and/or back pain, or other peripheral nerve pain as a result of nerve compression by vertebrae of the spine. A therapeutic amount can be 0.1 cc to 5cc. A therapeutic amount can be about 0.5cc to about 4.5cc. A therapeutic amount can be about 1 cc to about 4cc. A therapeutic amount can be about 1.5cc to about 3.5cc. A therapeutic amount can be about 2cc to about 3cc. A therapeutic amount can be about 2.5cc.

Described herein are methods of treating soft-tissue defects in a subject. Methods as described herein can comprise delivering the medical implant to a subject in need thereof in a therapeutic amount.

Described herein are methods of providing support or cushioning to treat soft-tissue defects in a subject. Methods as described herein can comprise delivering the medical implant of any one of claims 1 to 9 to a subject in need thereof in a therapeutic amount.

Described herein are methods of increasing growth factor binding. Methods of increasing growth factor binding as described herein can comprise providing a bone composition in a solution; adding an acid to the solution, thereby exposing the bone composition to an acid to demineralize the bone composition; adding a buffer or base to raise the pH of the solution in the presence of one or more growth factors from a first level to a second level, wherein the second level is more basic than the first level, after at least partial demineralization of the bone. The bone composition can then be isolated, as can the mineral that has precipitated of solution (for example calcium), or both. In an embodiment, the bone extracellular matrix is removed prior to raising the pH in order to precipitate the calcium from the bone.

Buffers or bases as described herein can raise the pH of a composition or a solution from a first level to a second level, wherein the second level is more basic than the first level. In certain embodiments, buffers or bases as described herein can comprise sodium hydroxide, water, sodium bicarbonate, phosphate buffer, sodium chloride, or calcium salt. Bone compositions as described herein can comprise mineralized bone, cancellous bone, or both. In certain aspects, the first pH level is an acidic pH level.

In certain aspects, medical implants as described herein are medical implants according to or created by any of the methods as described herein, individually or in combination.

Described herein are medical implant kits. Kits as described herein can comprise medical implants are described according to any of the methods as described herein, individually or in combination, and an applicator. In certain aspects, the medical implant of the kit is pre-loaded into the applicator.

Described herein are applicators that can implant medical implants as described herein. Applicators as described herein can be flexible applicators that can allow for accurate delivery of a fluid, gel, or scaffold. The applicator may contain a flexible tip, plunger/wire, crimped tube, and the like. Non-implantable portions of the applicator are configured to be removed after placement of the implant in the desired location of the subject. Embodiments of applicators as described herein can be used to implant medical implants and can be used to fill defects in skin, adipose, muscle, vasculature, or other soft tissue void.

FIG. 1 shows an embodiment of an applicator 100 according to the present disclosure.

The applicator 100 comprises a needle 101 (which can be a trocar needle and can have an opening along a longitudinal axis A in addition to the end B, as shown in FIG. 2) attached to a thread 105 via a mechanical joint 103 (the mechanical joint 103 can be a mechanical crimp joint). The thead 105 can be dehydrated and can be different sizes or lengths, but can be about 0.1 to about 1.0 mm in diameter or greater. In certain embodiments, the needle 101 can be a trocar needle and can be about 1 gauge to about 33 gauge on the Stubbs scale. In an embodiment, the needle 101 is a 27 gauge trocar (7.5 cm).

In certain aspects, the applicator 100 can be inserted subcutaneously into a dermal layer of a subject that contains soft-tissue defects, which are to be corrected. The applicator 100 can then be loaded with the medical implant, or the applicator 100 can be preloaded with the medical implant to be implanted. After insertion, the medical implant can be implanted with the applicator 100 by the addition of pressure (by way of a mechanical syringe or stylet coupled to the applicator 100), and the applicator 100 can then be removed from the dermal layer of the subject, leaving behind the medical implant. In an aspect, the epidermal layer of a subject can have a defect, such as a scar or a ripple, and the applicator 100 can be inserted subcutaneously below the defect. The applicator 100 can then implant the medical implant subcutaneously below the defect, and the applicator can then be removed from the subject.

FIG. 3 is a photograph showing another embodiment of an applicator as described herein. As shown in the photograph, an applicator as described herein can comprise a syringe and a stylet for delivering the medical implant into the subject.

In another embodiment, an applicator as described herein can comprise a hollow tube that may be curved to match the precise anatomy of a subject in a region of interest where there is a soft-tissue defect. The hollow tube can have a funnel at an end that is not inserted into the subject, and an outer ring at the opposite end that is to be inserted into the subject. A stylet can then be used to precisely deliver medical implants as described herein into the subject with the applicator.

FIG. 4 shows an embodiment according to the present disclosure 200. A medical implant (allograft) is a medical implant that is cut and/or otherwise formed into a tube 201 (a three-dimensional structure with a longitudinal axis of greater dimension than the diameter and/or circumference that is substantially circular when viewed in a direction along the longitudinal axis). A needle or wire 203 (or applicator 100) can be inserted into the allograft medical implant tube 201 along the longitudinal axis C or in a hole of the allograft medical implant tube 201 along the longitudinal axis C to provide rigidity. The allograft tube 201 can then be implanted into the location of interest in a subject, and the needle/wire 203 can be removed, leaving the medical implant in place in the subject while the need/wire 203 or applicator 100 is removed from the subject. The needle or wire 203 or applicator 100 can be packaged in a kit with the allograft tube 203, or a kit can comprise the needle or wire 203 or applicator 100 pre-loaded in the allograft medical implant tube 201.

FIG. 5 shows another embodiment according to the present disclosure. In another embodiment according to the present disclosure 300, a medical implant comprises allograft particles/pieces 303 that are preloaded into the hollow shaft of a needle 301 , the needle 301 comprising an implantation end E and a non-implanted end F. The hollow shaft of the needle 301 is also configured to receive a stylet 305 on an end that is not implanted into the subject. The implantation end E of the needle is inserted into a subject and positioned to where the medical implant is intended to be implanted in the subject. A stylet 305 can then push particles out of the needle 301 in the direction D into the desired location using a means such as a plunger in mechanical communication (not pictured) with the stylet 305, which are well known in the art. The needle 301 can then be removed.

Described herein is method of enriching bound growth factors in a medical implant, comprising: providing a scaffold in solution; combining the scaffold in solution with growth factors or agents; raising the pH of the solution from a first level to a second level, wherein the second level is more basic than the first, by adding a base or buffer to the solution. After the pH is made more basic, the scaffold can be isolated. In certain aspects, the scaffold can be derived from allograft or xenograft tissue sources or created synthetically. In certain aspects, the scaffold can be comprised of comprised of collagen, hyaluronan, or other protein, amino acid, glycosaminoglycan (GAG), and/or biocompatible material (for example, calcium, salt, glycerol, or other storage agents or biological preservatives known in the art). FIG. 6 is a flow chart illustrating an embodiment of the method 1000. According to the method 1000, a bone composition is provided in a solution 1010. The bone composition can comprise cortical bone, cancellous bone, or both. An acid can then be added to the solution 1020, followed by the addition of a base or buffer to the solution 1030 to raise the pH (make it more basic). The buffer or base can comprise one or more of sodium hydroxide, water, sodium bicarbonate, phosphate buffer, sodium chloride, or calcium salt. The bone composition can then be isolated after the pH is raised. Acids used may be weak or strong. Acids may include, but not limited to hydrochloric or phosphoric, sulphuric, glycolic, acetic, paracetic, citric, ascorbic, and the like. The solution can further comprise one or more growth factors. The method can further comprise adding one or more growth factors at any stage of the method. Growth factors, for example, can comprise vasoendothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), transforming growth factor beta-1 (TGFB-1), platelet-derived growth factor (PDGF), insulin-like growth factor IGF, or any other growth factor or tissue growth stimulative agent or differentiation agent, individually or in combination, or other growth factors as described herein.

FIG. 7 is a flow chart illustrating an embodiment of the method 2000. According to the method 2000, a bone composition is provided in a solution 2010. The bone composition can comprise cortical bone, cancellous bone, or both. An acid can then be added to the solution 2015, and the extracellular matrix (ECM) is then removed from the bone composition 2020. Next is the addition of a base or buffer to the solution 2030 to raise the pH (make it more basic). The buffer or base can comprise one or more of sodium hydroxide, water, sodium bicarbonate, phosphate buffer, sodium chloride, or calcium salt. The bone composition can then be isolated after the pH is raised. Acids used may be weak or strong. Acids may include, but not limited to hydrochloric or phosphoric, sulphuric, glycolic, acetic, paracetic, citric, ascorbic, and the like. The solution can further comprise one or more growth factors. The method can further comprise adding one or more growth factors at any stage of the method. Growth factors, for example, can comprise VEGF, bFGF, aFGF,TGFB-1 , PDGF, IGF, or any other growth factor or tissue growth stimulative agent or differentiation agent, individually or in combination, or other growth factors as described herein.

FIG. 8 is a flow chart illustrating an embodiment of the method 3000. According to the method 3000, a scaffold is provided in a solution 3010. The scaffold can be comprised of comprised of collagen, hyaluronan, or other protein, amino acid, GAG, and/or biocompatible material, and can be an allograft. Growth factors can then be added to the solution 3020, followed by the addition of a base or buffer to the solution 3030 to raise the pH (make it more basic). The buffer or base can comprise one or more of sodium hydroxide, water, sodium bicarbonate, phosphate buffer, sodium chloride, or calcium salt. The scaffold can then be isolated after the pH is raised. Growth factors, for example, can comprise VEGF, bFGF, aFGF,TGFB-1 , PDGF, IGF, or any other growth factor or tissue growth stimulative agent or differentiation agent, individually or in combination, or other growth factors as described herein.

FIG. 9 is a flow chart illustrating an embodiment of the method 4000. According to the method 4000, a medical implant is provided 4010 and energy is then provided to the medical implant 4020 to crosslink the medical implant or components thereof. The medical implant can be a particulated medical implant. The medical implant can be an allograft. The medical implant can be an allograft that is primarily comprised of collagen. The medical implant can be micronized soft tissue. The energy can be heat or irradiation (gamma, ebeam, uv, and the like). In certain aspects, the energy can be irradiation of an energy of about 0.5 to about 50 kGy). After energy is added, an additional energy can be added and the crosslinked medical implant can be terminally irradiated for the purpose of sterilization. The energy added for the purpose of sterilization can be less than the initial energy added 4020. The medical implant may be frozen before the step 4020. Additionally, the medical implant can be freeze-dried before or after the step 4020. Additionally, additives can be added to the medical implant before or after the step 4020. Additives can be vasodilator, a growth factor, or an amino acid, individually or in combination.

FIG. 10 is a flow chart illustrating an embodiment of the method 5000. According to the method 5000, a mold is provided 5010, that is configured to receive a medical implant, and is configured in a desired shape (for example, which can be complementary to a soft- tissue defect of a subject). A medical implant is then introduced into the mold 5015 and energy is then provided to the medical implant 5020. After the energy is provided, the medical implant can then be separated from the mold 5030. The medical implant can be a particulated medical implant. The medical implant can be an allograft. The medical implant can be an allograft that is primarily comprised of collagen. The medical implant can be micronized soft tissue. The energy can be heat or irradiation (gamma, ebeam, uv, and the like). In certain aspects, the energy can be irradiation of an energy of about 0.5 to about 50 kGy). After energy is added, an additional energy can be added and the crosslinked medical implant can be terminally irradiated for the purpose of sterilization. The energy added for the purpose of sterilization can be less than the initial energy added 5020. The medical implant may be frozen before the step 5020. Additionally, the medical implant can be freeze-dried before or after the step 5020. Additionally, additives can be added to the medical implant before or after the step 5020. Additives can be vasodilator, a growth factor, or an amino acid, individually or in combination.

Growth factors or agents according to methods of the present disclosure can comprise VEGF, bFGF, aFGF,TGFB-1 , PDGF, IGF, or any other growth factor or tissue growth stimulative agent or differentiation agent, individually or in combination.

Also described herein are methods of crosslinking medical implants in order to create a three-dimensional (3D) implant and to maintain shape memory. As described herein, methods of providing shape memory for a medical implant, can comprise providing the medical implant; and providing energy to the medical implant. In certain aspects, the energy can be heat or irradiation (gamma, ebeam, uv, and the like). In certain aspects, the energy can be irradiation of an energy of about 0.5 to about 50 kGy. In certain aspects, the energy can be irradiation of an energy of about 1 to about 45 kGy. In certain aspects, the energy can be irradiation of an energy of about 1.5 to about 40 kGy. In certain aspects, the energy can be irradiation of an energy of about 2 to about 35 kGy. In certain aspects, the energy can be irradiation of an energy of about 5 to about 30 kGy. In certain aspects, the energy can be irradiation of an energy of about 10 to about 20 kGy.The medical implants can comprise collagen, which can be crosslinked by the input of energy. The medical implants can comprise primarily collagen, meaning that collagen comprises the majority of the composition, or is present in an amount greater than any of the other components. In certain aspects, methods can include freezing the medical implant before providing the energy. In certain aspects, methods as described herein can include hydrating the medical implant before providing the energy. The medical implant can be placed in a mold of a desired shape before providing the energy. The medical implant can additionally be freeze dried and/or terminally irradiator (or sterilized by other sterilization means known in the art) after the application of energy.

In certain embodiments, one or more additives can be added to the medical implant, before providing the energy or after providing the energy. The additive can be a vasodilator, for example, herparin, arginine, citrulline, or other compositions known in the art to induce nitric oxide (NO) production, a growth factor, or an amino acid.

Medical implants are described herein can be particulated. In an embodiment, the medical implant can be micronized soft tissue.

Medical implants as described herein can be made from autograft, allogeneic, or xenograft sources and may contain collagen, and growth factors/cytokines such as (but not limited to) PDGF, FGF, bFGF, aFGF, VEGF, hepatocyte growth factor (HGF), IGF, angiopoietin (ANG), ANG-2, fibronectin, TGFbl , etc. Components of implants as described herein can be mixed together or layered as an injectable or structured implant. Medical implants as described herein can be particulated implants, and in certain aspects can be crosslinked.

Medical implants described herein can be implanted surgically, injected, microneedled, and/or applied topically.

Medical implants described herein can be derived from follicular, dermis, fascia, amnion, amniotic fluid, placenta, umbilical cord, muscle, blood, bone marrow, or adipose tissue, their ECM, soluble proteins, or interacellular proteins.

In certain aspects, medical implants as described herein can be derived from tissue that is >1 % adipose; >5% adipose; >10% adipose; >20% adipose; >30% adipose; >40% adipose; >50% adipose; >60% adipose; >70% adipose; >80% adipose; or about >90% adipose.

Medical implants as described herein can be particulated, gelatinized, solubilized, tissue pieces, or portions extracted. The implants described herein can be combined with a delivery enhancer, flow enhancer, preservative, storage agent, protease inhibitor, stabilizer, amino acids, radioprotectant, lyoprotectant, cryoprotectant, and/or the like.

Medical implants as described herein can be derived from a physiological solution containing cells such as blood, bone marrow, interstitial fluid, stromal vascular fraction, synovial fluid, amniotic fluid, and the like. Medical implants as described herein can be further purified using centrifugation, fluorescence, selective lysis, chromatography, filtration, separation, and the like.

Medical implants as described herein can be cellular (such as cellular dermis or adipose tissue) or acellular (such as acellular dermis or adipose tissue).

Additionally, medical implants as described herein can be refrigerated, frozen, or stored at ambient temperature. Medical implants as described herein can be dehydrated via lyophilization or supplied hydrated. Medical implants as described herein can be supplied in a syringe or a jar/bottle/vial.

Medical implants as described herein can be sterile filtered, tested per USP71 , or terminally sterilized via irradiation (gamma, ebeam, uv, and the like). Medical implants as described herein may be cross linked using chemical crosslinkers, heat, or irradiation (gamma, UV, ebeam, etc) to decrease degradation rate and improve volume retention.

Medical implants as described herein can be cleaned and disinfected using detergents, peroxides, antibiotics, water, and saline.

Medical implants as described herein can be cut into strips, sheets, or pieces. Medical implants can be ground or blended into fine particulate. Temperature control on cutting/grinding/blending may be used to help preserve growth factor content and prevent damage or denature proteins or other components.

Medical implant material (source tissue, final medical implants, or anything related to thereof or in between) may be screened/seived/filtered using syringes, needles, screens, seives, or filters. Tissue implant density may be controlled by filtration, dehydration, or centrifugation speeds (100-32000 rpm/g's).

Medical implants as described herein may have additives such as stabilizers (radioprotectants, lyoprotectants, or cryoprotectants, such as propylene glycol, glycerol, trehlose, sucrose, amino Acids, l-arginine, l-lysine, polysorbate, ascorbic acid, etc). Additionally, medical implants can be mixed by shear stress prior to injection/implantation/application to improve flowability, decrease heterogenocity, and decrease particle size.

In certain embodiments, medical implants as described herein can comprise a backbone of one or more collagens.

Methods as described herein can utilize medical implants as described herein

Methods as described herein can deliver medical implants as described herein to a subject in need thereof. Medical implants can be delivered to the skin of a subject in need thereof.

Medical implants employed in methods as described herein can be compositions comprising growth factors. In certain aspects, growth factor compositions may also contain cells (such as stem cells, keratinocytes, adipocytes, adipose derived stem cells, bone marrow derived stem cells, perivascular cells, muscle cells, stromal vascular fraction, and the like). In addition, growth factor compositions as described herein can contain ascorbic acid, hemoglobin, oxygenation molecules, vasodilators, amino acids (such as arginine, lysine, methionine, cysteine, or the remaining 16 amino acids). In certain aspects, medical implants as described herein may contain adipose-derived stem cells and/or adipocytes. Medical implants as described herein can be delivered to soft tissue, which in certain embodiments can be any tissue except for bone or cancellous bone. In certain aspects, medical implants can comprise bone or cancellous bone. In certain embodiments, viable cells can be added to the medical implants after the medical implants are prepared.

In certain embodiments, methods as described herein can utilize a single injection of medical implants to an area of the in need thereof, which can be an area suffering from a soft- tissue defect, or an area where shrinkage of adipose tissue is desired.

The amount of medical implants which is administered to a subject can vary and can be determined by the practitioner on an individual basis according to the subject and desired outcome. Factors which can determine the amount of medical implants administered to a subject can include the degree of the soft tissue defect, and the degree to which the subject desires treatment.

As described herein, medical implants can comprise a bioactive intracellular component. A bioactive intracellular component can be a platelet-derived growth factor, a hepatocyte growth factor, an insulin growth factor, an angiopoietin, a fibronectin, a transforming growth factor, a nerve growth factor, a fibronectin, an integrin, a bone morphogenetic protein, an epidermal growth factor, an insulin-like growth factor, a fibroblast growth factor, vascular endothelial growth factor, osteoprotegerin, and osteopontin, and various combinations thereof.

As described, an effective amount of a tissue implant can be an amount of tissue implant that contains a bioactive intracellular component at a concentration of at least at least 1 pg/g. As described, an effective amount of a tissue implant can be an amount of tissue implant that contains a bioactive intracellular component at a concentration of about 0 pg/g to about 100 mg/g. An effective amount of a tissue implant can be an amount of tissue implant comprising a-fibroblast growth factor is present at a concentration of at least 1 pg/g. An effective amount of a tissue implant can be an amount of tissue implant comprising β-fibroblast growth factor is present at a concentration of at least 1 pg/g. An effective amount of a tissue implant can be an amount of tissue implant comprising vascular endothelial growth factor is present at a concentration of at least 1 pg/g. An effective amount of a tissue implant can be an amount of tissue implant comprising acidic fibroblast growth factor and is present at a concentration of at least 1 pg/g. An effective amount of medical implants as described herein administered to a subject in need thereof to treat soft-tissue defects can be an amount of tissue implant that contains a bioactive intracellular component at a concentration of at least at least 1 pg/mL. An effective amount of medical implants as described herein administered to a subject in need thereof to treat soft-tissue defects can be an amount of tissue implant that contains a bioactive intracellular component at a concentration of at least at least 10 pg/mL. An effective amount of medical implants as described herein administered to a subject in need thereof to treat soft- tissue defects can be an amount of tissue implant that contains a bioactive intracellular component at a concentration of at least at least 100 pg/mL. An effective amount of medical implants as described herein administered to a subject in need thereof to treat soft-tissue defects can be an amount of tissue implant that contains a bioactive intracellular component at a concentration of at least at least 1000 pg/mL. An effective amount of medical implants as described herein administered to a subject in need thereof to treat soft-tissue defects can be an amount of tissue implant that contains a bioactive intracellular component at a concentration of at least at least 10000 pg/mL. An effective amount of medical implants as described herein administered to a subject in need thereof to treat soft-tissue defects can be an amount of tissue implant that contains a bioactive intracellular component at a concentration of at least at least 100000 pg/mL.

An effective amount of medical implants as described herein administered to a subject in need thereof to treat soft-tissue defects can be an amount of tissue implant that comprises one or more of: aFGF in an amount of at least 100,000 pg/mL; FGF in an amount of at least 100,000 pg/mL; acidic fibroblast growth factor (aFGF) in an amount of at least 100,000 pg/mL; basic fibroblast growth factor (bFGF) in an amount of at least 100,000 pg/mL; epidermal growth factor (EGF) in an amount of at least 10,000 pg/mL; hepatocyte growth factor activator (HGFa) in an amount of at least 100,000 pg/mL; hepatocyte growth factor b (HGFb) in an amount of at least 100,000 pg/mL; insulin-like growth factor 1 (IGF-1 ) in an amount of at least 10,000 pg/mL; platelet derived growth factor BB in an amount of at least 10,000 pg/mL; transforming growth factor β1 (TGF-βΙ ) in an amount of at least 10,000 pg/mL; and vascular endothelial growth factor (VEGF) in an amount of at least 5,000 pg/mL. In an embodiment, an amount effective comprises VEGF in an amount of about 5,000 pg/mL to about 1 ,000,000 pg/mL. In an embodiment, an amount effective comprises VEGF in an amount of about 66,000 pg/mL. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 0.01 cc to about 100cc. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 0.01 cc to about 1 cc. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 1 cc to about 10cc. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 10cc to about 100cc. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 10cc. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 2cc to about 9cc. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 3cc to about 8cc. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 4cc to about 7cc. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 5cc to about 6cc. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 1cc to about 20cc. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 2cc to about 19cc. Effective amounts of medical implants as described herein can be delivered to a subject in need thereof in a volume of about 5cc to about 15cc.

Also described herein are kits for treating soft-tissue defects. Kits as described herein can comprise one or more dosages of medical implants as described herein, wherein each of the one or more dosages contains an effective amount of medical implants as described herein. In certain aspects, kits can also comprise delivery devices (such as applicators or needles) according to methods and devices of the present disclosure. In certain embodiments, the medical implant of the kit can be pre-loaded into the delivery device and be ready to delivered by a practitioner to a subject.

Examples of soft tissues which can experience defects can include bone marrow, blood, adipose, skin, muscle, vasculature, cartilage, ligament, tendon, fascia, pericardium, nerve, and hair. These soft tissues may also include organs such as the pancreas, heart, kidney, liver, intestine, and stomach. In certain aspects, as used herein soft tissue can be any tissue for example, mesodermal, endodermal, and ectodermal tissues. Examples of these tissues include bone marrow, blood, adipose, skin, muscle, vasculature, cartilage, ligament, tendon, fascia, pericardium, nerve, and hair.

As such, the medical implants can be further sterilized to reduce the microorganism contamination to less than about 10^"3 microorganisms. Typical sterilization methods include, but are not limited to, combinations of washing with or without pressurization, centrifugation with various chemicals such as alcohols and/or detergents, and combining antibiotics with low- dose radiation. While these processing methods reduce the amount of microorganism contamination, they also can damage the medical implant and result in the loss of many intracellular proteins and molecules.

In an embodiment, a soft tissue implant contains a bioactive intracellular component of an adipose cell and a carrier substrate, where the soft tissue implant is prepared by harvesting an adipose cell from a donor, selectively lysing the adipose cell to obtain the bioactive intracellular components and combining the bioactive intracellular component with a carrier substrate. In some embodiments, the soft tissue implant can be directly administered to a subject in need thereof.

Accordingly, also provided are soft medical implants, grafts, and fillers produced by the methods described herein. Also provided are devices for containing and/or delivering the soft medical implants, grafts, and fillers produced by the methods described herein and kits containing the soft medical implants, grafts, fillers and/or devices described herein. The methods, soft medical implants, grafts, fillers, devices, and kits described herein offer several advantages to current soft tissue grafts at least insofar as they incorporate endogenous intracellular components, while minimizing the immunogenicity of the soft tissue implant.

Other compositions, compounds, methods, devices, systems, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.

Soft tissues include, any tissue or organ that is not bone, including, but not limited to adipose tissue, muscle, cartilage, tendons, and ligaments. In one embodiment, the harvested cells are adipose cells. The soft tissues can be autologous, allogeneic, xenogeneic, or syngeneic in origin. In order to minimize immunogenicity, the use of autologous cells is most advantageous. In other words, it is preferred if the harvested cells were obtained directly or indirectly (i.e. from an in vitro culture containing cells from the subject to receive the implant) from the subject that is to receive the soft tissue implant. In an embodiment, autologous adipose cells are harvested. In other embodiments, the tissue or cells are allogeneic.

As previously mentioned, in some embodiments, the harvested soft tissue cells are cultured in vitro for an amount of time using suitable cell culture methods generally known in the art. One having ordinary skill will appreciate that the culture conditions will vary depending on the cell type. In some embodiments, cells from adipose tissue are cultured in vitro for about 1 day to about 6 months. In some embodiments, the cultured cells are harvested as previously described. In an embodiment, adipose cells are harvested from a donor and cultured in vitro, until harvested as previously described.

Medical implants as described herein can comprise growth factors, particularly vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor beta 1 (TGFbl), acidic fibroblast growth factor (aFGF), insulin-like growth factor (IGF). Any given soft tissue protein and/or other bioactive factor can be present in the soluble soft tissue protein composition at a concentration of 0 μg/g to about 100 mg/g of isolated protein in the final product, dehydrated or otherwise provided.

In some embodiments, soluble soft tissue protein composition can include a stabilizer composition or stabilizer compounds. Suitable stabilization compounds can include, but are not limited to preservatives, antibiotics, antivirals, antifungals, pH stabilizers, osmostablizers, anti-inflammants, anti-neoplastics, chemotherapeutics, immunomodulators, chemoattractants, growth factors, anticoagulants, or combinations thereof. The stabilization solution can increase shelf life of the soft tissue soluble protein composition and/or reduce denaturation of proteins during dehydration, sterilization, and/or storage. In addition, other materials, such as nitrogen, can be used to help reduce free radical formation and denaturation during sterilization. In some embodiments, the stabilization solution per cc of final product can be about 1 mg sucrose, about 5mg Glycine, about 3.7 mg l-Glutamic Acid, about 0.02 mg NaCI, and about 0.02 mg Polysorbate-80. In another embodiment, the stabilizer solution may contain glycerol, amino acids, polaxomers, carbomers, carbohydrates, polysaccharides, sugars, or salts.

In some embodiments, the final volume of a medical implant according to compositions and methods as described herein can be at least 1 cc, or 1 cc to about 100 cc, about 1 cc to about 50 cc, 1 cc to about 25 cc, about 1 cc to about 20 cc, about 1 cc to about 10 cc. The final soluble soft tissue protein product can be dehydrated or reconstituted to achieve a desired volume or particular protein concentration or composition.

Cells as described herein can be bone-marrow derived stem cells that are supported physiologically by a fibrous tissue called the stroma in the subject. There are two main types of stem cells in bone marrow: (1) hematopoietic stem cells and (2) bone marrow mesenchymal stem cells (bmMSCs). bmMSCs can differentiate into a variety of cells types including without limitation, fibroblasts, chondrocytes, osteocytes, myotubes, stromal cells, adipocytes, astrocytes, and dermal cells. In addition to bmMSCs, bone marrow stroma contains other types of cells including fibroblasts (reticular connective tissue) macrophages, adipocytes, osteoblasts, osteoclasts, red blood cells, white blood cells, leukocytes, granulocytes, platelets, and endothelial cells.

Proteins as described herein can be proteins derived from soluble bone marrow protein compositions that can contain proteins and/or other non-recombinant bioactive factors derived from bone marrow mesenchymal stem cells, fibroblasts, chondrocytes, osteocytes, red blood cells, white blood cells, leukocytes, granulocytes, platelets, and/or osteoclasts. The proteins can be intracellular proteins or membrane associated proteins. Such proteins include without limitation, bone morphogenetic proteins (BMPs) {e.g. BMP-1 , BMP-2, BMP-3, BMP-4, BMP- 5, BMP-7 and BMP-8a), transforming growth factors (TGF-βΙ , TGF^2), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs) (e.g. IGF-1), fibroblast growth factors (FGFs) (e.g. aFGF (acidic fibroblast growth factor) and bFGF (basic fibroblast growth factor)), vascular endothelial growth factor (VEGF), platelet derived growth factor - BB (PDGF-BB), osteoprotegerin (OPG), and osteopontin (OPN).

Additional examples of cells as described herein are adipose stem cells, apidocytes, mesenchymal stem cells, bone marrow stromal cells, progenitor cells, etc. , that can remain viable in medical implants or in the tissue of the subject once the medical implants are implanted. EXAMPLES

Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure. Furthermore, one of skill in the art would recognize that the examples of the present disclosure presented below may be combined with one another. Example 1 : Medical implants for providing cushioning or support for soft tissue defects

Described herein is a medical implant that is at least partially comprised of collagen, hyaluronan, protein, amino acid, peptide, glycosaminoglycan (GAG), and/or biocompatible material that may cushion or support soft tissue defects such as dermis, adipose, muscle, joint, spinal disc, nerve, hair, fascia, vasculature, stroma, or other defects either created surgically, naturally occurring, or due to loss due to aging or disease (such as HIV lipoatrophy). Medical implants as described herein may be derived from allograft, autograft, xenograft, or synthetic sources. The medical implants as described herein can be used for joint injections (such as knee, finger, toes, ankles, hips, elbows, shoulders, jaw, or other joints), spinal disc (ie vertebral disc in a spine) injections, breast reconstruction, nipple reconstruction, foot pad atrophy, arthritis, or used subcutaneously, subdermally, intradermally, sub glandular, interglandular, inter peritoneal, or other region of the body. Medical implants as described herein may be used for any application that would benefit from support and cushioning in a subject. Medical implants as described herein may be combined with cells or growth factors from other sources such as adipose, bone marrow, blood, amniotic fluid, or from another matrix such as intestine, bladder, placental, or other soft tissue. These other sources may be allograft, autograft, xenograft, or synthetic sources.

Example 2: Medical implants for the alleviation of pain

A medical implant as described herein (and in Example 1 above) partially comprised of allogeneic, acellular, delipidized, and decontaminated adipose, dermis, and/or fascia was injected into spinal disc and knee of patients to reduce pain and delay or eliminate the need for surgical intervention relating. After injection, patients had pain relief and did not require immediate surgery.

Example 3: A flowable medical implant

Described in the present example is a cut or particulated medical implant comprising collagen, hyaluronan, or other protein/glycosaminoglycan (GAG). The particulated medical implant can fill a soft tissue defect or void and is capable of flowing out of a cannula. The particulated medical implant can be least partially comprised of particles <2mm in size. The particles may have an aspect ratio of greater that about 110% so that one perpendicular dimension is larger than the smallest dimension.

In the present example, a particulated implant partially comprised of adipose, dermis, and/or fascia that has particles that are 1 mm in smallest dimension but 1.5mm or greater in a perpendicular dimension can be injected into adipose voids to correct soft-tissue defects in a subject in need thereof, defects such as rippling of breast implants, asymmetry of the breast, wrinkles in the face or neck, cellulite defects, liposuction defects, lumpectomy cavity, tumor resections, and the like. Example 4: Adjusting flow of medical implants

Described in the present example is a medical implant at least partially comprised of collagen, hyaluronan, protein, amino acid, peptide, glycosaminoglycan (GAG), or biocompatible material that is flowable out of a cannula. The medical implant may have different flow characteristics at different temperatures or pH levels. Ph adjustment (such as pH lower than 7) can be used to decrease crosslinkinking or selfassembly of collagen, proteins, or peptides that may be present in the medical implant. The implant may also be more flowable with altered pH or at temperatures above/below 37C, room temperature, or implantation temperature.

A medical implant comprising collagen and derived from adipose, dermis, and/or fascia was tested for flowability at different temperatures on the first pass of extrusion/mixing. FIG. 11 is a graph showing energy expended on first mix. Example 5: Flexible applicator for medical implant delivery

Described in the present example is a flexible applicator that can allow for accurate delivery of a fluid, gel, or scaffold. The applicator can comprise a flexible tip, plunger/wire, crimped tube, and the like. Non-implantable portions of the applicator would be removed after placement of the implant in the desired location. Examples of possible embodiments are attached. Flexible applicators as described herein can be used for the implantation of medical implants as described herein. Medical implants as described herein can be used for the treatment of soft-tissue defects in a subject, for example to fill defects in skin, adipose, muscle, vasculature, or other soft tissue void.

FIG. 3 is a photograph of an embodiment of a flexible applicator as described herein.

Example 6: Enrichment and increased binding of growth factors

Described herein are medical implants that can be implanted/injected into a subject, or applied onto a subject in a location where growth factors/agents can be exposed to biocompatible scaffolding material where the combination of scaffold/growth factor solution has an acidic pH.

The scaffold may be acidic and/or the growth factors can be in an acidic medium. The binding of those growth factors onto the scaffold can be increased by bringing the pH of the growth factors/scaffold combination to a higher pH. The pH change can approach about a neutral pH (>=4) or go into a basic pH. The pH can be elevated using a buffer or a base. Growth factors may consist of proteins, cytokines, peptides, amino acids, etc.

Growth factors/agents according to the present example may be VEGF, bFGF, aFGF, TGFB-1 , PDGF, IGF, or any other growth factor or tissue growth simulative agent or differentiation agent.

Scaffolds may be derived from allograft or xenograft tissue sources or created synthetically. The scaffolds may be at least partially comprised of collagen, hyaluronan, or other protein, amino acid, GAG, and/or biocompatible material

Acids used may be weak or strong. Acids may include, but not limited to hydrochloric or phosphoric, sulphuric, glycolic, acetic, paracetic, citric, ascorbic, and the like.

Bases or buffers can be sodim hydroxide (NaOH), water, sodium bicarbonate, phosphate buffer, sodium chloride (NaCI), Calcium salt, or any other material that will raise pH of an acidic solution.

In an embodiment of the present example, bone can be combined with growth factors/agents with an acidic pH. The pH of the bone/growth agents can then be raised by the addition of sodium bicarbonate. The resulting bone is growth factor enriched and growth factors/agents are bound to the bone at a level higher than before treatment with the sodium bicarbonate.

In an embodiment of the present example, adipose tissue can be combined with growth factors/agents with an acidic pH. The pH of the adipose/growth agents can then be raised by the addition of sodium bicarbonate. The resulting adipose is growth factor enriched and growth factors/agents are bound to the adipose at a higher level than before sodium bicarbonate. Neutralization of the pH to 5 resulted in lower growth factor content than neutralization to pH 6. Neutralization of the pH to 6 resulted in lower growth factor content than neutralization to pH 7. Neutralization of the pH to above about 7.0 resulted in higher growth factor content than neutralization to pH less than about 7.0.

In an embodiment of the present example, a scaffold containing collagen can be combined with growth factors/agents with an acidic pH. The collagen/growth agents are then raised in pH by the addition of sodium bicarbonate. The resulting collagen is growth factor enriched and growth factors/agents are bound to the collagen at a higher level.

In an embodiment of the present example, mineralized cortical or cancellous bone

(sourced from allograft or xenograft tissue sources or created synthetically) is exposed to up to 1 M HCL or other acid (0.5-0.6N for example), and then after at least partial demineralization, the solution pH is raised by adding a buffer or base to above pH 4. The growth factors and growth agents that were bound to the mineral (and loosely bound to collagen or lipid) are released into the solution and raising the pH causes those growth factors/agents to bind to the extracellular matrix of the bone. Also, mineral (such as calcium) may precipitate out of solution and have attached growth factors/agents.

Example 7: Medical implant with elevated VEGF concentration

Disclosed according to the present example is a medical implant derived from blood that has an elevated VEGF concentration by lysing and harvesting VEGF from the intracellular space of red blood cells. The implant may be used in combination with platelets from that same donor. Implant may be frozen, freeze-dried, allogeneic, autologous, or xenographic. Example 8: Removing desired cell populations from medical implants

Disclosed according to the present example is a medical implant derived from allogeneic or xenograft tissue where certain cell populations are removed and desired cell populations are retained. Implant may be cryopreserved and stored frozen until use and decontaminated using antimicrobials such as antibiotics or other non-toxic materials.

In an embodiment according to the present example, adipose tissue is ground very lightly to resize the particles and partially or completely kill adipocytes and treated with water to remove non-desired cell populations, for example red blood cells and white blood cells. The progenitor/stem cells can still remain. The implant can be treated with antibiotics such as gentamycin, vancomycin, and amphotericin B. The implant can be aliquoted into individual containers and cryoprotectant is added prior to freezing. Example 9: Induction of a higher volume retention of medical implants

Disclosed according to the present example is a medical implant that has greater amino acid/peptide/protein/GAG content (such as collagen or hyaluronan) to induce a higher volume retention for correction of soft tissue defects/voids. Particulated implants may be further concentrated by centrifugation or rehydration in smaller volumes to increase protein/GAG concentration per unit volume.

In an embodiment according to the present example, a collagen containing implant derived from human tissue (such as adipose, dermis, or fascia) was tested at different densities and resulted in higher volume retention with increased density. FIG. 12 is a graph of volume retention versus non-protein/GAG mass.

Example 10: Improved flowability of medical implants

Disclosed according to the present example is a medical implant that can be crosslinked and self-assembled by energy deposition (such as irradiation, light, or heat). The medical implant can then be sheared to improve flowability through smaller gage needles. Shear may be introduced by rapidly cycling the flowable implant through a small aperture >5mm.

Example 11 : Reduction of the size and appearance of adipose tissue

Disclosed according to the present example is a medical implant or topical that can reduce the size and appearance of adipose tissue. The implant can be comprised of a protein, amino acid, GAG such as chitosan. The material can be injected, microneedled, or topically applied to the skin. The material can contain additives to help transport into the skin, adipose, or subcutaneous layers to aid in function. The material can help in the release of triglycerol, reducing the size of adipocytes, killing adipocytes, tightening the skin, or any other way to reduce the appearance of adipose tissue. An enzyme can be added to the medical implant according to the present example (such as lysozyme) to help with the activity. Also, additives such as L-carnitine, ascorbic acid, caffeine, forms of glucosamine, or l-arginine may also be added to improve function.

In an embodiment according to the present example, a solution of chitosan, lysozyme, ascorbic acid, carnitine, arginine, lysine, glucosamine, and other amino acids was applied using microneedling and topical lotion application to one half of a torso. FIGs. 13A and 13B are thermal images depicting before and after thermal images were taken to visualize temperature differences created by subcutaneous fat insulation. FIG. 13A is before application and FIG. 13B is after application of the topical about a week later. A change on one side of the body can be seen. Example 12: Crosslinking of medical implants

Disclosed according to the present example is a particulated medical implant containing collagen that can be crosslinked to form a contiguous scaffold by using energy such as irradiation. The medical implant may be hydrated and/or frozen during delivery of such energy. The medical implant may be dehydrated and compressed for less invasive delivery. The medical implant may gel or expand upon rehydration resulting in a porous shape memory implant. The self-assembly or crosslinking may also effect the water content, hydrophilicity, or hydrophobicity. Additives such as vasodilators, growth enhancers, or arginine may be added to help with vascularization and nutrient transport. The additives, such as amino acids (arginine, lysine, glucosamine, and the like, natural or synthetic) may also be used to help bulk up the implant giving it a higher viscosity, G', G", compression resistance, resiliency, or the like.

In an embodiment according to the present example (FIG. 14), soft tissue was micronized and poured into a mold. The material was frozen and irradiated at -10, 20, 30, and 40 kGy. After irradiation, the implants were contiguous in the shape of the mold. The higher the dose of irradiation, the more stiff and stronger the shapes became.

Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of separating, testing, and constructing materials, which are within the skill of the art. Such techniques are explained fully in the literature.

It should be emphasized that the above-described embodiments are merely examples of possible implementations. Many variations and modifications may be made to the above- described embodiments without departing from the principles of the present disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

Claims

I claim at least the following:

1. A medical implant, comprising:

collagen, hyaluronan, protein, amino acid, peptide, or glycosaminoglycan, individually or in combination.

2. The medical implant of claim 1 , further comprising biocompatible material.

3. The medical implant of any one of claims 1 or 2, furthering comprising growth factors.

4. The medical implant of any one of claims 1 to 3, furthering comprising a plurality of cells from one or more sources.

5. The medical implant of any one of claims 3 or 4, wherein the cell or growth factors are each independently derived from adipose, bone marrow, blood, amniotic fluid, or from another matrix such as intestine, bladder, placental, or other soft tissue.

6. The medical implant of any one of claims 1 to 5, wherein the medical implant is a cellular.

7. The medical implant of any one of claims 1 to 6, wherein the medical implant is delipidized.

8. The medical implant of any one of claims 1 to 7, wherein the medical implant comprises decontaminated adipose, dermis, or fascia, individually or in combination.

9. The medical implant of any of claims 1 to 8, wherein the medical implant is derived from allograft, autograft, xenograft, or synthetic sources.

10. The medical implant of any of claims 1 to 9, wherein the source of the cell or growth factors is a different source than the other components of the medical implant.

1 1 . A method of treating pain in a subject, comprising:

delivering the medical implant of any one of claims 1 to 9 to one or more vertebral discs in the spine of a subject in need thereof in a therapeutic amount.

12. A method of treating pain in a subject, comprising:

delivering the medical implant of any one of claims 1 to 9 to a joint of a subject in need thereof in a therapeutic amount.

13. A method of treatment, comprising:

delivering the medical implant of any one of claims 1 to 9 to a subject in need thereof in a therapeutic amount.

14. A method of treating soft-tissue defects in a subject, comprising:

delivering the medical implant of any one of claims 1 to 9 to a subject in need thereof in a therapeutic amount.

15. A method of providing support or cushioning to treat soft-tissue defects in a subject, comprising:

delivering the medical implant of any one of claims 1 to 9 to a subject in need thereof in a therapeutic amount.

16. A method of increasing growth factor binding, comprising:

providing a bone composition in a solution;

adding an acid to the solution, thereby exposing the bone composition to an acid to demineralize the bone composition;

adding a buffer or base to raise the pH of the solution from a first level to a second level, wherein the second level is more basic than the first level, after at least partial demineralization of the bone.

17. The method of claim 16, further comprising isolating the bone composition, mineral that has precipitated of solution, or both.

18. The method of any one of claims 16 or 17, wherein the buffer or base comprises one or more of sodium hydroxide, water, sodium bicarbonate, phosphate buffer, sodium chloride, calcium salt.

19. The method of claim 18, wherein the mineral that has precipitated out of solution comprises calcium.

20. The method of any of claims 16 to 19, wherein the bone composition comprises cortical bone, cancellous bone, or both.

21. A medical implant comprising a bone composition, mineral, or both prepared according to the methods of claim 16 to 20.

22. A medical implant kit, comprising:

a medical implant according to the methods of any of claims 16 to 20; and

an applicator.

23. The medical implant kit of claim 22, wherein the medical implant of the kit is preloaded into the applicator.

24. A method of enriching bound growth factors in a medical implant, comprising: providing a scaffold in solution;

combining the scaffold in solution with growth factors or agents;

raising the pH of the solution from a first level to a second level, wherein the second level is more basic than the first, by adding a base or buffer to the solution.

25. The method of claim 24, further comprising isolating the scaffold.

26. The method of claim 24 or 25, wherein the scaffold is derived from allograft or xenograft tissue sources or created synthetically.

27. The method of any one of claims 23 to 26, wherein the scaffold is comprised of comprised of collagen, hyaluronan, or other protein, amino acid, GAG, and/or biocompatible material.

28. The method of any one of claims 23 to 27, wherein the growth factors or agents comprise VEGF, bFGF, aFGF,TGFB-1 , PDGF, IGF, or any other growth factor or tissue growth stimulative agent or differentiation agent, individually or in combination.

29. The method of any one of claims 23 to 27, wherein the growth factors or agents comprise VEGF, bFGF, aFGF,TGFB-1 , PDGF, or IGF, individually or in combination.

30. The method of any one of claims 23 to 29, wherein the first level is an acidic pH.

31 . The method of any one of claims 23 to 30, wherein the buffer or base comprises one or more of sodium hydroxide, water, sodium bicarbonate, phosphate buffer, sodium chloride, calcium salt.

32. A medical implant kit, comprising:

a medical implant according to the methods of any of claims 24 to 31 ; and

an applicator.

33. The medical implant kit of claim 32, wherein the medical implant of the kit is preloaded into the applicator.

34. A method of providing shape memory for a medical implant, comprising:

providing the medical implant; and

providing energy to the medical implant.

35. The method of any one of claims 34 or 35, further comprising freezing the medical implant before providing the energy.

36. The method of any one of claims 34 to 35, further comprising hydrating the medical implant before providing the energy.

37. The method of any one of claims 34 to 36, further comprising adding an additive to the medical implant, before providing the energy or after providing the energy.

38. The method of claim 37, wherein the additive is a vasodilator, a growth factor, or an amino acid.

39. The method of any one of claims 34 to 38, wherein the medical implant is a particulated medical implant.

40. The method of any one of claims 34 to 39, wherein the medical implant is micronized soft tissue.

41 . The method of any one of claims 34 to 40, further comprising placing the medical implant in a mold of a desired shape before providing the energy.

42. The method of any one of claims 34 to 41 , further comprising freeze drying the medical implant before or after providing the energy.

43. The method of any one of claims 34 to 42, further comprising sterilizing the medical implant after providing the energy.

44. The method of any one of claims 34 to 43, wherein the energy is irradiation of an energy of about 0.5 to 50 kGy.

45. The method of any one of claims 42 or 44, wherein sterilizing the medical implant is terminally irradiating the medical implant.

46. A medical implant kit, comprising:

a medical implant according to the methods of any of claims 34 to 45; and

an applicator.

47. The medical implant kit of claim 46, wherein the medical implant of the kit is preloaded into the applicator.

48. The medical implant of any one of claims 1 to 46, wherein the medical implant is a three-dimensional medical implant.

49. The medical implant of any one of claims 1 to 48, wherein the medical implant is derived from an allograft.

50. The medical implant of any of claims 1 to 49, wherein the medical implant is primarily comprised of collagen.

51 . The method of any one of claims 16 to 21 , further comprising removing the extracellular matrix of the bone prior to raising the pH.

52. A medical implant kit, comprising:

a medical implant according to the method of claim 51 ; and

an applicator.

53. The medical implant kit of claim 52, wherein the medical implant of the kit is preloaded into the applicator.