Patent Application
20180177920
The present invention relates to doped biodegradable glass compositions and methods of treating nerve damage using doped biodegradable glass to deliver nanoparticles.
Ideal nerve repair biomaterials or scaffolds should be biocompatible and noninflammatory, yet flexible with adequate tensile strength to prevent nerve compression. The materials should be biodegradable with a porosity and permeability to supply adequate oxygen and nutrients. Nerve autografts remain the gold standard in nerve repair and regeneration because of their performance. However, autografts require additional surgery, donor site morbidity, and the loss of nerve function; all reasons that alternative materials in nerve repair and regeneration are needed.
Bioresorbable synthetic natural polymers such as type I collagen (for example the NeuraGen™ collagen tube available from Integra LifeSciences of Plainsboro, N.J.) have received attention over the last decade due to ease of production and controlled degradation. Type I collagen supports cell attachment, unidirectional proliferation, and growth in vivo. The disadvantages to synthetic natural polymers include irreproducibility of collagen type I and the need for immunosuppressant drugs to reduce host rejection. Resorbable synthetic polymers have been used extensively to repair peripheral nerves due to lower cost, simple fabrication, and proven efficacy. Compared to an autograft, these synthetic materials eliminate the shortcomings of the autograft.
Over the past 50 years many methods have been developed to produce different types of inorganic materials with different sizes, shapes and properties. Most of these methods use a soluble salt such as nitrate or carbonate to provide the desired ions in a solution and then use other chemicals or processes to precipitate or reduce from an ionized to neutral form. However, small materials such as nanomaterials can be formed through other routes like Sol-Gel processing, Microemulsion, Hydrothermal/Solvothermal processing, Templated synthesis, and Biomimetic Synthesis.
Therefore, a need exists for methods for creating and delivering nanoparticles of different chemical composition, size, and shape formed within a biodegradable, biocompatible inorganic glass, to improve nerve regeneration and improve the quality of life following injury to the spinal cord or peripheral nerves.
Provided herein is a biocompatible, biodegradable composite material for soft tissue repair, such as repair of nerve or nerve cells, and related methods of use. A biocompatible, biodegradable composite material as described herein includes a matrix material and a parent glass suspended within the matrix material, the parent glass including a glass and a dopant. The parent glass may release a plurality of dopant-based nanoparticles upon contact and reaction with a body fluid or a simulated body fluid. The glass may comprise, for example, a borate glass such as sodium tetraborate, or a borosilicate glass. The dopant may be selected from a metal ion, a transition metal ion, an oxide, a rare earth oxide, a halide, carbonate, a compound containing a cation, and any combination thereof. The dopant may be selected from CeO2, Ce2O3, Y2O3, and ZrO2 and mixtures of any two or more thereof. The dopant may be selected from Co, Ni, Cu, Ag, Au, Pt, Fe, Ru, Si, V, Cr, Mn, Fe, Ni, Zn, Sn, Sb, Zn, Ti, Y, Zr, W, La, Ce, Pr, Nd, Sm, Eu, Lu, Yb, Er, Ba, Ga, I, N, S, Si, and any combination thereof. The dopant may be selected from the combinations I/Ce, I/Y, I/Ce/P, and Cu/Zn/Sr/Fe. The parent glass may include Na2O.2B2O3.xCeO2 and x ranges from about 0.001 to about 0.30 moles. The biocompatible, biodegradable composite material may be degradable in vivo. The biocompatible, biodegradable composite material may further include a therapeutic agent. The matrix material of the biocompatible, biodegradable composite material may comprise a material selected from a polymer, a ceramic, and any combination thereof. The matrix material may for example be selected from collagen, laminin, fibrin, PCL, PLA, PLLA, PEG, PGA, PLGA and any combination thereof. The parent glass may have a conformation selected from irregular particles, microspheres, fibers, rods, ribbons and any combination thereof. Any of the foregoing conformations of a biocompatible, biodegradable composite material may be combined with a second matrix material, which may comprise any scaffold material such as collagen, laminin, fibrin and any combination thereof. A combination of any conformation of a biocompatible, biodegradable composite material and a second matrix material can be formed in any shape, such as for example a generally cylindrical conduit form.
Further provided herein are methods of delivering nanoparticles to a region of interest, including placing any biocompatible, biodegradable composite material as described herein, in or in contact with a body fluid or simulated body fluid; and allowing the parent glass to react and/or degrade to form and release the nanoparticles. It should be understood that the body fluid or simulated body fluid may be combined or associated with a region of interest in the body, such as a body tissue. The region of interest may be for example any suitable location relative to nerve cells, which may be in a tissue or in a body, such as but not limited to a peripheral nerve, or the spine and/or spinal cord.
The following figures illustrate various aspects of the disclosure.
Corresponding reference characters and labels indicate corresponding elements among the views of the drawings. The headings used in the figures should not be interpreted to limit the scope of the claims.
Provided herein are materials and methods of nerve repair in which a biocompatible, biodegradable composite material is used to produce and deliver a nanomaterial. In various aspects, the biocompatible, biodegradable composite material comprises a glass, which is biocompatible and biodegradable, suspended in a matrix. The glass contains inorganic dopants which are released as ions when the composite comes into contact with a body fluid or simulated body fluid. A biocompatible, biodegradable composite material as disclosed herein may be used to produce and deliver nanomaterials to cells or tissues in vivo or in vitro.
As used herein, the term “subject” refers to an animal, including but not limited to a mammal including a human and a non-human primate (for example, a monkey or great ape), a cow, a pig, a cat, a dog, a rat, a mouse, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig).
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms as used herein and in the claims shall include pluralities and plural terms shall include the singular.
The use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
The term “body fluid” as used herein refers to a liquid that originates from inside the organism of a subject, including for example blood, interstitial fluid, cerebrospinal fluid, or lymph. The term “simulated body fluid” as used herein refers to a liquid having an ionic composition comparable to that of a body fluid. In non-limiting example, a simulated body fluid can be a buffered saline solution.
Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, and chemistry described herein are well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Any chemical, enzymatic or staining reactions, or purification techniques are performed according to manufacturer's specifications and protocols, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are also well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, diagnosis and treatment of all subjects, human and animal.
In one aspect, a biocompatible, biodegradable composite material comprises a glass comprising a nanomaterial. The nanomaterial is for example a nanoparticle. The size, shape, and chemical composition of a given type of nanoparticle can be controlled by the synthesis process. For example, chemical dopants can be added to the parent glass composition to contain the desired percent composition of compounds. By way of non-limiting example, dopants can include an oxide, halide, carbonate or any other type of compound that may contain or produce a desired cation. The parent glass for making the nanoparticles may be formed by melting the doped glass under controlled conditions (melting atmosphere, temperature and time) and cooling the glass in a controlled manner. The parent glass may then be degraded, in vivo or in vitro, forming the nanoparticles in the process.
Two primary types of biomaterials are available for use in connection with the present disclosure: biodegradable materials that degrade with time, or bio-inert materials that do not degrade. The bio-inert materials may be unreactive to provide long-term structural support. These may be used predominantly as scaffolding in bone and hard tissues for regeneration. In contrast, biodegradable materials react and degrade with time in the body, or when subjected to simulated body fluids (SBF) or cell culture media. These biodegradable materials may be better suited to soft tissue regeneration as with time, as the tissues regrow and infiltrate the damaged regions, the biodegradable material will react with simulated body fluids and be absorbed by the body as the scaffold is no longer required, thus leaving only the regenerated tissue in its place. Provided herein are biodegradable materials optimized for reacting and degrading with appropriate time for soft tissue repair, and while doing so, provide the needed structural scaffold and delivery system for advantageous chemical dopants and molecules. It may also have the ability to chemically cross-link and deliver drugs and growth factors. Optimization of this system may provide a better-suited soft tissue regenerative system than what currently exists for soft tissue regeneration and repair.
In an aspect, borate based biodegradable glass with added chemical dopants may be fabricated in many different forms such as nanoparticles, irregular shaped particles (frit), rods, spheres, and microfibers. This biodegradable glass may be used for soft tissue repair and nerve regeneration. The addition of chemical dopants such as Ce or Y has been shown to be neuroprotective to neurons in vitro, as well as prevent overproliferation of the scar forming fibroblasts. In various aspects, the forms of glass may be used as composites or mixed materials in scaffolds and nerve guide conduits to promote regeneration of peripheral and central nerves that have been damaged through trauma or disease, as well as promote the regrowth of nearly any type of soft tissue.
A biocompatible, biodegradable glass composite material as disclosed herein may include a parent glass that contains or releases desired cations, and/or creates nanoparticles. The parent glass may be semi-crystalline or non-crystalline/amorphous, and the nanoparticles may be crystalline or non-crystalline/amorphous. In one aspect, the parent glass reacts or degrades when exposed to fluid(s), for example a body fluid or a simulated body fluid (SBF) in vivo or in vitro, and may generate non-crystalline/amorphous nanoparticles. The parent glass may be, for example, biocompatible and non-toxic.
The materials synthesized may be formulated by methods to deliver inorganic nanomaterials in situ using biocompatible, biodegradable glasses. In one aspect, the nanomaterials are nanoparticles. The nanomaterials may be produced in two different ways. Methods of making nanoparticles include a solid-state (glassy-state) method to produce nanoparticles using a degradable glass as disclosed herein. In this method, nanoparticles may be produced in two different ways. In a first method, the desired nanoparticle base material (metal ions, transition metal ions, rare earth oxide, etc.) may be dissolved in a glass melt to form the parent glass. In one aspect, the parent glass may be doped with an oxide, halide, or other compound containing a desired cation. Then the parent glass may be reacted or degraded in a desired fluid in a laboratory (e.g., in vitro) or inside the body of subject (in vivo). As the glass reacts or degrades the desired ions may be released to form nanoparticles of a desired composition/size. A second method may be to dissolve the desired material in the molten glass and then form the nanomaterials inside the glass using techniques such as controlled heat treatments, radiation, etc., as will be understood by those of skill in the art. The glass may then be reacted or degraded in vitro or in vivo to release the nanoparticles. In this second method, the glass network may react when in contact with a body fluid or simulated body fluid, while the insoluble nanoparticles remain in the fluid, at least temporarily. The nanoparticles may for example remain in solution in vivo until they are removed or sequestered by the body.
The glass and other desired compounds may be mixed as powdered raw materials. In one aspect, the glass raw material may be a borate glass such as sodium tetraborate, or a borosilicate glass. In another aspect, the nanoparticle base material may be a dopant. The size, shape, and chemical composition of a given type of nanoparticle can be controlled by doping the parent glass with the desired amount of a chosen oxide, halide (e.g., F, CI, I), carbonate, or other type of compound (e.g., sulfate, oxalate, or nitrate) that contains the desired cation, (e.g., Ce, Y, Zr, Gd) and melting the doped glass under (a) controlled conditions (melting atmosphere, temperature and time), and (b) cooling the glass in a controlled manner. In an aspect, powdered sodium tetraborate and CeO2 may be mixed together to form a parent glass mixture of sodium borate glass.
In one aspect, the parent glass may have the molar composition of Na2O.2B2O3.xCeO2, where x ranges from about 0.001 to about 0.30 moles. In another aspect, the parent glass may have the molar composition xNa2O.2xB2O3.(1-3x)Al2O3, where x ranges from about 0 to about 0.2 moles. In an aspect, the parent glass may have the molar composition xR2O.yR′O.(1-x-y)B2O3, where R may be alkali ions such as Li, Na, K, etc. and R′ may be alkaline earth ions such as Mg, Ca, Sr, etc. and where x ranges from about 0 to about 0.5 moles and y ranges from about 0 to about 0.5 moles. In yet another aspect, the parent glass may be xR2O.yR′O. zR2″O3. m R2′″O5.(1-x-y-z)B2O3, where R may be alkali ions such as Li, Na, K, etc., R′ may be alkaline earth ions such as Mg, Ca, Sr, etc., R″ may be modifiers that form a R″2O3 oxide such as Al, Fe, etc., and R′″ may be modifiers that form a R′″2O5 oxide such as phosphorus (P) and x and y range from about 0 to about 0.5 moles, and z and m range from about 0 to about 0.2 moles.
Non-limiting examples of nanoparticle base material dopants for producing metallic ionic nanomaterials include Co, Ni, Cu, Ag, Au, Pt, Fe, and Ru. Non-limiting examples of nanoparticle base material dopants for producing oxides, phosphate, and borate nanoparticles may include Si, V, Cr, Mn, Fe, Ni, Zn, Sn, Sb, Zn, Ti, Y, Zr, W, La, Ce, Pr, Nd, Sm, Eu, Lu, Yb, and Er. Dopants that may be used for soft tissue regeneration include, but are not limited to Ag, Ba, Ce, Co, Cu, Fe, Ga, I, Mn, N, S, Si, Sr, Ti, Y, and Zn, and a mixture of elements such as: I/Ce (Ce2), I/Y(Y2), I/Ce/P, and Cu/Zn/Sr/Fe (CZSF). Two or more of these dopant elements may be combined within the nanoparticles. In an aspect, Y or La doped CeO2 nanoparticles may be produced (co-doped cerium oxide-yttrium nanoparticles). In another aspect, the parent glass may contain up to about 4 dopants.
The parent glass mixture may be heated from about 600° C. to about 1000° C., forming a parent glass melt. In an aspect, the parent glass mixture may be heated to about 1000° C. for about 1 hour. The parent glass melt may be cooled to from about 20° C. to about 25° C. in various aspects. In an aspect, the parent glass melt may be cooled to room temperature to form the parent glass. In this aspect, the nanoparticles may form during the dissolution of the glass in aqueous solution.
In one aspect, a solid amorphous (glass) or semi-crystalline (glassceramic) material may be used to make nanoparticles. For example, nanoparticles can be formed by giving the glass a prescribed heat treatment such that the desired nanoparticles are formed within the melted glass matrix, which is then cooled and solidified. The resulting solid glass matrix containing a controlled and specified amount of nanoparticles can then be delivered to a desired site in humans or animals in several ways.
The heat treatment of the glass may include cooling the glass melt to a certain temperature and holding it for a chosen time before cooling to room temperature. In another aspect, the heat treatment may include cooling the glass melt to room temperature and then reheating to a certain temperature and holding for a chosen time before re-cooling to room temperature. In yet another aspect, the glass melt may be bubbled with gas to form the nanoparticles within the parent glass. The bubbling gas may be reducing (forming gas, CO/CO2 mixture), neutral (N2, Ar, He), or oxidizing (pure oxygen). The gas flow rate may be from about 0.1 cm3/min to about 1000 cm3/min depending upon the size/volume of the melt. The bubbling time may range from about 5 minutes to about 72 hours depending up on the melt temperature and composition, dopants in the melt, and melt size/volume. The bubbling temperature may be any temperature where the viscosity of the melt is low enough to permit the gas to escape from the melt. In one aspect, the viscosity may be lower than 10 poise.
The nanoparticles may be made and/or released by reacting the glass in contact with a desired fluid under certain reaction conditions (e.g., of temperature, pressure, pH, etc.). Physiological conditions can be used. Alternatively the temperature for reacting the glass in vitro may range from about −20 to about 120° C. under ambient pressure or up to about 500° C. under higher pressures. The pH of the fluid for reacting the glass in vitro may range from about 2 to about 12. In one aspect, the glass composite materials can be designed to react or degrade over the span of minutes to years. Additionally, it will be understood that by changing various conditions such as temperature or solution compositions, it may be possible to form a variety of nanomaterials with special/different properties.
In another aspect, the body fluid or simulated body fluid may contain added organic or inorganic stabilizers or surfactants to stabilize the nanoparticles and prevent them from agglomerating. Non-limiting examples of such stabilizers include tetrahydrofuran (THF), ethylene glycol (EG), hexadecylamine (HDA), mercaptosuccinic acid (MSA), poly(vinylpyrrolidone) (PVP), CTAB, and Polyvinyl Alcohol (PVA).
In any of the foregoing aspects, the nanoparticles may be less than 10 nm. In any aspect, the nanoparticles created from the parent glass may range in size from about 2 nm to about 60 nm. In any aspect, the nanoparticles may range in diameter from about 2 nm to about 20 nm, from about 10 nm to about 30 nm, from about 20 nm to about 40 nm, from about 30 nm to about 50 nm, and from about 40 nm to about 60 nm. In any aspect, it will be understood that the size of the nanoparticles may be affected by the composition of the parent glass, temperature, and pH.
II. Biomaterials from the Nanoparticles and Biodegradable Glass
A biodegradable glass composite material as disclosed herein includes for example a parent glass containing or capable of forming nanoparticles, and a matrix material. Nanoparticles can be formed in various ways. Glass raw materials may be mixed with nanoparticle base materials to form a parent glass mixture. The parent glass mixture is heated to a first temperature, forming a parent glass melt. The parent glass melt is cooled to a second temperature to solidify the parent glass melt. In so doing, the parent glass may be formed into a desired shape using standard shaping techniques as known in the art, which may then in use be degraded or otherwise react upon contact with a body fluid or simulated body fluid, wherein the nanoparticles are created as the parent glass reacts or degrades. For example, a borate glass composite containing cerium may in use produce ceria nanoparticles. Methods of making nanoparticles include the solid-state (glassy-state) method and other methods as described in detail above in section I. Methods of using a biodegradable glass composite material as disclosed herein are described in further detail below in section III.
The parent glass or matrix material may further include one or more therapeutic agent. This may include chemotherapeutics, growth factors, angiogenic compounds, or any other substance that may be beneficial to the region of interest. The therapeutic agent may diffuse out of the parent glass faster than the nanoparticles are formed. In an aspect, the therapeutic agent may provide a synergistic effect with the nanoparticles. The therapeutic agent may, for example, be copper, iron, zinc or strontium.
The nanoparticles and parent glass may be incorporated into many forms such as, for example, small irregularly shaped glass particles (frit), microfibers, microspheres, thin flexible polymer sheets, and micron diameter rods.
Delivery of nanoparticles created from the biodegradable glass may include injection of the nanoparticles alone, or as a suspension within a matrix material of soluble gels that will degrade with time (hours to weeks), insertion by surgical intervention, or placement at desired locations such that the linked or unlinked nanoparticles can be released over time into the body fluids at the site as the glass degrades in the body fluids. In an aspect, the nanoparticles may be suspended in a matrix material. In another aspect, the parent glass may be suspended in the matrix material. In yet another aspect, the parent glass may be on the surface of the matrix material.
In one aspect, the matrix material may be, but is not limited to collagen, laminin, fibrin, polycaprolactone (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly-L-lactide (PLLA), polyethylene glycol (PEG), any other biodegradable polymer matrix material, and combinations thereof. The matrix material and parent glass may be present in a mixture ratio of matrix material to parent glass from about 20:80 to about 40:60, from about 30:70 to about 50:50, from about 40:60 to about 60:40, from about 50:50 to about 70:30, and from about 80:20 to about 60:40. The parent glass may be present within the matrix material as a gradient from high dopant concentration to low dopant concentration. In another aspect, the parent glass may be evenly distributed throughout the matrix material. Multiple dopants with varying release rates and/or solubility may be included within a matrix material to provide a delayed release or gradient.
In another aspect, a biocompatible, biodegradable composite material may formed initially as a flat sheet which is then rolled in the form of a generally cylindrical conduit. Such a conduit may be any size, and in one example can be usefully formed to be about 10 mm to about 20 mm long, about 3 mm to about 5 mm in outer diameter, and have a thickness of about 3 to about 4 rolls of the polymer sheet, wherein the thickness ranges from about 25 μm to 250 μm. The conduit may be porous in one aspect. In another aspect, as described in further detail below, a biocompatible, biodegradable composite material in various conformations may be combined with a second matrix material. For example, a matrix material sheet containing a biodegradable composite material may be rolled into a conduit shape which may be filled with another matrix material containing a biodegradable composite material with the same or a different dopant. In one of many possible examples, a biodegradable composite material comprising PCL as the matrix and formed as a sheet may then be formed into a conduit which contains, or surrounds a gel-like scaffold material such as collagen or fibrin, in which a second biodegradable composite material is suspended or contained.
Because of their biodegradability, poly ε-caprolactone (PCL) and its copolymers have been used for soft tissue regeneration applications including peripheral nerves. PCL slowly degrades in vivo and its degradation can take several years depending upon its molecular weight. Furthermore, the degradation rate of PCL can be altered by polymerization with other polymers such as poly-lactic acid (PLA). Other copolymers may be used in place of PCL. Addition of inorganic materials like biodegradable glass to a biodegradable polymer improves the mechanical strength and enhances wetting properties of certain polymers, which can improve cell adhesion. Together these properties of copolymers provide the structural scaffold to develop a mixed biomaterial consisting of biodegradable glasses to help heal and repair wounds, regenerate nerves and repair other soft tissues such as blood vessels, and soft tissues such as muscle, liver, and kidney.
In one aspect, biodegradable glass composites may be fabricated in PCL or another such polymer as shown in
The thickness of the dried films, measured with a micrometer at several locations, may be about 60±10 μm. In various aspects, the thickness of the dried films may range from about 45 μm to about 55 μm, from about 50 μm to about 60 μm, from about 55 μm to about 65 μm, from about 60 μm to about 70 μm, and about 65 μm to about 75 μm. In one aspect, the composition of the 13-93 B3 borate glass (in wt. %) may be: 53% B2O3, 20% CaO, 12% K2O, 6% Na2O, 5% MgO, and 4% P2O.
Bioglass composites may be fabricated consisting of varying percentages of PCL and 13-93 B3 glass chemically doped with varying types and quantities of dopants. Additionally, in place of PCL, other types of polymers that are inert and degrade with time may be substituted with many other polymer materials used commonly in conduits including, but not limited to poly-lactic acid (PLA), poly-L-lactic acid (PLLA), and poly-lactic-co-glycolic acid (PLGA).
In one aspect, the biocompatible, biodegradable composite material is placed in contact with a body fluid or simulated body fluid. This may be at or near or in association with a region of interest in body of a mammal such that the nanoparticles are released into the body fluid(s) at the region of interest as the glass degrades upon contact with a body fluid(s), such as blood, interstitial fluid(s), cerebrospinal fluid, or lymph. A region of interest may for example be a peripheral nerve, or the spine and/or spinal cord. Alternatively, a biocompatible, biodegradable composite material is placed in contact with a body fluid or simulated body fluid in vitro.
For example, chemical dopants such as cerium oxide (CeO2-x) nanoparticles have been shown to prevent the death of HT22 nerve cells, protect rat endothelial cells, increase the life span of brain cells, and provide benefits for the survival of damaged spinal cord cells in rats. Cerium oxide nanoparticles may also be used to treat, perhaps prevent, Alzheimer's, Parkinson and Huntington's diseases, and multiple sclerosis. Other elements such as Y, Zn, and Ag may also be neuroprotective, anti-oxidative, and overall provide improved health of cells. Therefore, a number of types of chemical dopants may be added to the glass composition at low weight %.
As described above and shown for example in
It should be understood that many and various comparable structural iterations are contemplated by the fact that the biodegradable glass composite material is amenable to formation into many and various shapes and sizes for building such structures The resulting structure can be used in the aforementioned method to support suturing tissue repair, for example, to support the nerve endings for peripheral repair and/or for embedding the mixed materials for nerve regrowth. Multiple different biodegradable glass composite materials, each with a different dopant could be used in combination. The nanoparticle dopants themselves may be released as the glass reacts with a body fluid or simulated body fluid, providing a timed release of dopants, growth factors, and/or therapeutic agents to promote tissue repair. Further, for example, structures embedded into an injectable matrix such as a collagen gel can be injected at the site of injury.
In addition, similar concepts may be utilized for central nervous tissue repair as in a spinal cord injury (
The results described below illustrate the individual aspects for the feasibility and optimization of this system. Together, they will help to determine how the biodegradable glass types and various forms could function, and with particular dopants/factors/drugs to include for the greatest benefit towards regeneration for the particular tissue. For instance, in the case of tissues that are highly susceptible to oxidative stress, like nerves, including anti-oxidant dopants would be beneficial.
Biodegradable and chemically doped glasses may provide multiple means to aid and promote neuronal regeneration. Many of these examples of biodegradable glass may be used in other tissues for tissue regeneration.
The following examples are included to demonstrate the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the disclosure. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes could be made in the disclosure and still obtain a like or similar result without departing from the spirit and scope of the disclosure, therefore all matter set forth is to be interpreted as illustrative and not in a limiting sense.
A sodium borate glass, with the molar composition of Na2O.2B2O3.xCeO2 (0.001<x<0.06), was prepared using sodium tetraborate (Alfa Aesar Ward Hill Mass., USA) and cerium oxide (CeO2) (Alfa Aesar Ward Hill, Mass., USA). The powdered raw materials were mixed, the homogeneous mixture was melted in a platinum crucible at 1000° C. for 1 hour, and stirred several times. The melt was poured on a cold steel plate and cooled to room temperature, forming a glass.
The glass was crushed to particles of less than 150 μm in diameter and 200 mg of the glass particles were reacted in 50 ml of DI water and simulated body fluid (SBF) at 37° C. for 12 hours. As the glass particles degraded the solution became cloudy due to formation of ceria nanoparticles. The solution was centrifuged to separate the nanoparticles from the solution, the residue was washed with fresh DI water, sonicated for 5 minutes, and then centrifuged for a second time. This procedure was repeated several times to insure that any boron or sodium that had dissolved from the glass was removed.
Finally, a drop of the solution containing the nanoparticles was placed on a 400 mesh copper TEM grid coated with a thin-holed carbon, dried at 50° C., and then placed in the TEM.
Nanoparticles formed in SBF were about 50±10 nm in diameter and had an amorphous (non-crystalline) structure with no lattice fringes/parallel lines or patterns. Nanoparticles of yttrium and zirconium oxide may be formed in DI water using the same procedure (
Table 2 provides examples of parent glass compositions, in mol % that have been melted.
Embryonic (E10-11) chick dorsal root ganglia (DRG) were acutely dissociated and incubated with doped glass in the form of small pieces of glass (frit). The DRG are comprised of 3 types of cells, neurons and two support cells, fibroblasts and glia cells. Control cells were grown in the absence of glass. B3 glass refers to the biodegradable borate glass. Additional chemical elements added to the B3 biodegradable borate glasses are referred to by the chemical abbreviation. For example, Ce is B3 glass doped with Cerium.
In the initial experiments, dissociated cells grew as well as, if not better than control cultures based on qualitative visualization only, for up to 10 days in culture with the following doped glasses: Ce1, Ce2, Fe, Ga, Zn, and B3. These glasses were used further for quantification with Live/Dead assay and counting cells with glass in the form of rods, microfibers and polymer/glass composite sheets. The doped glass that supported some neuronal or cellular growth by qualitative visualization includes: Y1, Y2, Ag, I2, Mn, N2, S, and Sr.
Quantitative analysis was performed on B3, Ce1, Ce2, Y1, and Y2 doped glasses compared to control (without any glass added). The cells were dissociated, seeded onto wells with the glass added and incubated for 10 days. Wells were treated with Live/Dead Assay (Molecular Probes) as per the instructions, and fluorescent images were acquired for each of the wells (4 per condition) at both 7 and 10 days. Green fluorescence indicates the live cells (calcein) and red fluorescence indicates the dead cells (ethidium bromide). Images were acquired individually, merged with Image J, and live and dead cells were counted. Neurons were distinguished based on their physical aspects of the cells: larger cells bodies with long and thin processes growing from the cell body. Fibroblasts are wide and flat, and glia are small round bodies without any processes. The total number of cells from both live and dead conditions per merged images was calculated and the fraction of live neurons/total cells for percent live neurons was determined. The images in
To fabricate the PCL/biodegradable glass composites, or another such polymer, as shown in
Noticeable changes in the microstructure and morphology of the rods and composite polymer sheets occurred after immersion in SBF or cell culture media. The rods react and degrade with exposure time in the cell culture media (
Degradation of the 100% PCL and the 13-93 B3 composite in SBF was determined in two ways: weight loss measurements and analysis of the SBF solution with inductively coupled plasma (ICP) for elemental analysis of B, Ca, and P, as a function of time. The weight loss degradation profile for each composite is shown in
Uniaxial tensile testing was performed to assess the mechanical properties of the composites to confirm that the composites were strong enough for an initial repair, but degrade within an appropriate time. Unreacted composites were compared to composites reacted with media for 3 and 6 weeks. For this experiment, glass composites consisted of 50% 13-93 B3:50% PCL (B3), 50% 45S5 (Bioglass®): 50% PCL (45S5), and 25% 13-93 B3:25% 45S5:50% PCL (blend) were compared to 100% PCL. Peak stress, strain at break, and elastic modulus were calculated for all composite samples (
The biodegradable glass/PCL polymer sheets can be readily rolled and formed into a conduit. Conduits were formed by rolling the polymer sheet (single layer ˜0.06 mm) about 5×, gluing with collagen to form a nerve guidance conduit that was 15-16 mm long, 4.3 mm outer diameter and ˜0.3 mm inner thickness (although these dimensions can be easily modified). We sutured cadaver rat sciatic nerve with 9-0 sutures to one side of each end of the rolled 13-93 B3 conduits (
To test the effects of the chemically doped glasses on neurons, two main experiments were conducted to test whether the dopants were a) promoting or inhibiting survival, and b) promoting or inhibiting neuronal outgrowth as a model for nerve regeneration. To accomplish this, embryonic (E10-11) chick dorsal root ganglia (DRG) were dissociated. The “whole” DRGs are comprised of 3 types of cells: neurons and two support cell types called fibroblasts and glia. When dissociated, the types of cells can be distinguished from one another morphologically, neurons have brightly round and clustered cells bodies interconnected by neurite processes, fibroblasts are flattened broad cells, and glia are small, polarized cells. In addition, Live cells were stained with Calcein AM and for Dead cells with eithidium bromide (Live/Dead Assay; Molecular Probes). Cells were then counted as live neurons, live support cells, or dead cells. Percentage of live neurons compared to all live cells or total cells were calculated.
These types of experiments were repeated with biodegradable borate-based glass without dopants (B3, control) or with chemical dopants. The cells were dissociated, and cultured for 3 days prior to adding any biodegradable glasses. For this experiment, all of the cells were exposed to two, 1 cm pieces of glass that approximated ˜14 mg/mL of glass reacting in cell culture media. After 3, 7, or 10 days in culture with the biodegradable glass, cells were counted and analyzed for survival.
The response of DRG neurons to the biodegradable glass B3/PCL polymer sheets was determined by measuring the length of neurite outgrowth from whole DRGs after culture for 3 days on each doped polymer. Representative images of whole DRGs are shown, and demonstrate that the neurites survive and extend after culture on the doped polymer composites compared to the 13-93 B3 (
Further data was obtained by measuring the length of the single longest neurite to grow out from the whole dorsal root ganglia, when measured from the center of the ganglia after 3 days in culture. The whole ganglia were placed on top of a poly-L-caprolactone (PCL) polymer sheet of bioactive glass (50% polymer, 50% doped borate glass). As shown in
Interestingly, the chemical dopants that improve the neuronal survival of single dissociated cells as shown in
Whole chick dorsal root ganglia (DRG) were seeded onto aligned 13-93 B3 rods “glued” onto thin fibrin or collagen scaffolds to form a “raft” of biodegradable glass rods. The whole DRG were imaged growing on the aligned 13-93 B3 glass rods as shown in
All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
This application claims the benefit of U.S. Provisional Patent Application No. 62/048,146 filed Sep. 9, 2014 and U.S. Provisional Patent Application No. 62/048,148 filed Sep. 9, 2014, the entire disclosures of which are both incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US15/49184 | 9/9/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62048146 | Sep 2014 | US | |
| 62048148 | Sep 2014 | US |
