Summary of The Invention
The present invention proposes a repair composition for promoting tissue repair and/or replacement in a patient in need thereof. The repair composition includes an effective amount of a cell growth enhancing composition in combination with one or more inflammation inducing agents.
In the context of cell growth enhancing compositions, the use of autologous and/or non-autologous cell growth enhancing materials is also contemplated. The cell growth enhancing composition may comprise autologous components incorporating one or more non-autologous factors. Typical autologous cell growth enhancing compositions include platelet rich fibrin solutions, such as 5% to 40% platelet lysate solutions and/or platelet gels. Typical non-autologous growth compositions include recombinant growth factors such as insulin-like growth factors.
For inflammation inducing agents, agents that induce localized, microdispersion lesions at a site in need of repair are included, as well as osmotic agents, inflammatory cytokines, and/or sclerosing agents. In some cases, combinations of these agents may be used, such as a combination of a permeation agent and a hardening agent.
The repair composition of the present invention may further comprise essential nutrients useful for the site in need of repair (e.g., collagen, the corresponding site of repair being a knee joint in need of cartilage repair). In addition, the repair composition may include anabolic hormones (e.g., human growth hormone) for further tissue growth signaling at the repair site.
Finally, the repair composition of the invention may comprise stem cells, in particular isolated stem cells, e.g. isolated autologous or non-autologous mesenchymal stem cells. The stem cells can be delivered to the site in the form of a repair composition or in a form that is independent of the repair composition (i.e., completely and temporarily independent).
The present invention also provides several methods to facilitate tissue repair in a patient in need thereof. The methods include obtaining and preparing a repair composition (e.g., 5% to 40% platelet lysate from a patient presenting a repair site in need of treatment, and an inflammation inducing agent); applying the repair composition to the site of repair according to the embodiments of the present invention (in an amount necessary to tolerate and maintain repair); stem cells or other similar repair cells are optionally administered to the repair site to facilitate tissue repair at the site in need thereof. For certain aspects of the above methods, the repair composition is a first inflammation inducing agent composition, and a second cell growth enhancing composition, wherein the first composition is administered to the site of repair to induce local microtissue damage, followed by administration of the second composition to enhance cell growth at the site. In some cases, stem cells or other repair-like cells are administered to the site to enhance the repair process, usually with or slightly after administration of the cell growth enhancing composition. The methods described herein may include multiple applications of the restorative composition over a course of 1 week to 6 months.
Finally, the invention provides pharmaceutical compositions for therapeutic use. In some cases, the pharmaceutical composition is used to treat patients in need of repair of the damaged site, and in some cases, such patients also suffer from osteoarthritis, osteoporosis, or other similar degenerative diseases.
The above and various other features and advantages of the present invention will become apparent upon reading the following detailed description and review of the appended claims.
Detailed description of the invention
Embodiments of the present invention provide a repair composition for promoting tissue repair and/or tissue replacement in a patient in need thereof. For the purposes described herein, patient refers to any mammal, preferably a human, in need of the compositions and/or methods of the invention. In one embodiment, the repair composition comprises a therapeutically effective amount of a cell growth enhancing composition in combination with one or more inflammation inducing agents.
Embodiments of the invention include repair compositions wherein the cell growth enhancing composition is an autologous or non-autologous growth factor, including, for example, a recombinant growth factor. In other embodiments, the cell growth enhancing composition refers to a mixture of autologous and non-autologous growth factors.
In typical embodiments, the cell growth enhancing composition refers to one or more autologous growth factors from a patient in need thereof (i.e., a patient in whom there is a site of injury in need of repair). The cell growth enhancing growth factor may comprise autologous platelets and/or platelet lysate compositions.
Embodiments of the invention also include such repair compositions wherein the inflammation inducing agent is an agent capable of inducing microtissue or local damage at a site in need of tissue repair. Inflammation-inducing agents, as used herein, include osmotic agents, inflammatory cytokines, sclerosing agents, and the like. Thus, the repair composition may comprise autologous platelet lysate in combination with one or more inflammation inducing agents.
The prosthetic compositions of the present invention may be administered endoscopically and/or percutaneously through a surgical incision. The repair site of a patient for the purpose of the present invention refers to any site where tissue repair or regrowth is desired, for example, a knee requiring cartilage, a liver requiring hepatocytes, a bone requiring osteocytes, and the like.
Embodiments of the present invention also provide methods for promoting tissue repair in a patient in need thereof. The methods comprise administering a repair composition comprising a cell growth enhancing composition and one or more inflammation inducing agents; or administering one or more inflammation-inducing agents and the cell growth-enhancing composition sequentially.
In one embodiment, the methods comprise obtaining an autologous growth-enhancing composition from a patient in need of tissue repair; administering to the patient an inflammation inducing agent in a dose sufficient to induce local inflammation at a site in need of tissue repair; an autologous growth-enhancing composition is administered to a patient at a site in need of tissue repair in an amount effective to promote cell growth/expansion at the site. In certain embodiments, the inflammation inducing agent and the growth-enhancing composition are administered simultaneously in separate compositions; in other embodiments, the two agents are administered separately over a period of 24 to 96 hours (more preferably 72 to 96 hours). Multiple administrations can be carried out over a course of 1 to 6 months (or longer as determined by a health professional). In other embodiments, the inflammation-inducing agent and the growth-enhancing composition can be administered as a single composition in combination.
Defining:
the following definitions are provided to facilitate understanding of several terms used frequently herein and are not intended to limit the scope of the present disclosure.
"cell growth enhancing composition" refers to growth factors (e.g., recombinant FGF, recombinant TGF- β), autologous compositions (e.g., platelets, platelet rich fibrin, platelet rich plasma, platelet lysate, platelet gel), and other similar substances, and may include growth factors, cytokines, hormones, essential nutrients or other proteins, fatty acids, or carbohydrates.
"inflammation-inducing agent" refers to any agent that induces the development of astroglial damage at a site, including osmotic agents such as hypertonic glucose, inflammatory cytokines, e.g., MIP-1 α, MIP-1 β, and MIP-2, sodium morrhuate, pumice, and phenol.
"mesenchymal stem cells" or "MSCs" refer to pluripotent stem cells capable of differentiating into osteoblasts, chondrocytes, myocytes, adipocytes, neuronal cells, islet cells and other similar cells (see below). The source of the MSCs of the invention is typically taken from the iliac crest of a patient (or suitable donor, (non-autologous)) to be treated for repair, such patient being referred to herein as a "patient in need thereof" (note that other sources of adipose tissue, synovial tissue, connective tissue, etc. have recently been identified and are also considered to be sources of MSCs within the scope of the invention). In one embodiment, approximately 10-20cc of bone marrow has been harvested and "isolated" using the method described in Centeno U.S. patent application 60/761,441, or by plastic cement attachment as described in Caplan et al, U.S. Pat. No. 5,486,359. Each of the above references is incorporated herein in its entirety for all purposes.
"platelet lysate" refers to a mixture of natural growth factors contained in platelets, released by platelet lysis. This can be done by chemical means (i.e. CaCl)2) Osmotic process (using distilled H2O) or by a freeze/thaw procedure. The platelet lysate of the present invention may also be obtained from whole blood and may be prepared using the methods described in U.S. patent No. 5,198,357, which is incorporated herein by reference. Alternatively, the platelet lysate used herein may also be prepared from harvested bone marrow by a method employing Doucet (Doucet, Erneu et al, 2005, Journal of Cellular Physiology, 205 (2): 228-. A typical lysate contains about tens of millions to billions of platelets. As described by Martineau et al in Biomaterials, 200425 (18) p4489-503, which is incorporated herein by reference in its entirety, the platelet lysate itself contains the growth factors required to promote stable MSC growth. In typical embodiments, the platelet lysate is autologous and the amount is an amount that can be used for effective and stable use in the embodiments herein. It is important to note that when the amount of growth factors (e.g., TGF- β) in the platelet lysate is much lower than the typical amount of MSCs expanded in vitro, it is believed that using all of the low amounts of growth factors contained in the platelet lysate together produces a tremendous synergistic effect.
"protein", "peptide" and "polypeptide" are used interchangeably and refer to a polymer of amino acids, or a group of two or more polymers of amino acids that interact or bind.
"Stem cell" refers to any cell that has the characteristics of being undesignated, and capable of long-term renewal through cell division, and can be induced to become a cell with specialized function.
Tissue repair compositions of the present invention
The compositions of the present invention comprise tissue repair compositions having the ability to enhance tissue repair and/or replacement in a patient in need thereof. Such compositions generally have two distinct aspects, the first of which is directed to inducing a local inflammatory response (in some cases, by causing cell lysis by the inflammatory agent) at the site where tissue repair is desired; and a second aspect is directed to promoting cell growth (autologous or non-autologous cell growth enhancing substances) at the site. The combination of inflammation and induction of cell growth is more pronounced and unexpected than traditional tissue repair therapies. In certain embodiments, also included is a third aspect, i.e., autologous or non-autologous stem cells, for use in promoting the ability of a repair composition to repair or replace tissue at a site in need thereof.
Typical repair compositions described herein include a combination of at least one or more inflammation-inducing agents and at least one or more cell growth-enhancing compositions. In one embodiment, a cell growth enhancing composition for use herein may comprise one or more self-factors. In another embodiment, the cell growth enhancing composition used herein may comprise one or more non-self factors. In other embodiments, the repair composition comprises a combination of autologous and non-autologous growth factors.
Typical autologous growth factors used herein include: platelets, platelet rich plasma, platelet rich fibrin, platelet lysate, or a mixture thereof.
As described herein, exemplary non-autologous factors include recombinant growth factors (e.g., epidermal growth factor, fibroblast growth factor-2, vascular endothelial growth factor, insulin-like growth factor, transforming growth factor-beta, and platelet derived growth factor). Recombinant growth factors can be purchased from a number of manufacturers (e.g., RDJ, inc., Bio Vision inc., Bio Clone inc., etc.) or obtained by well-known isolation and purification techniques.
In addition, the repair compositions of the present invention may comprise autologous growth factors enriched with recombinant growth factors (e.g., platelet lysates doped with recombinant transforming growth factor-beta, prepared from patients in need of tissue repair).
Embodiments herein may include a repair composition comprising one or more inflammation inducing agents. As defined herein, an inflammation inducing agent refers to a drug that induces local cellular damage, and in some cases, hypertonic glucose, sodium morrhuate, pumice, phenol, and/or one or more inflammation inducing cytokines. The inflammation inducing cytokines used herein include macrophage inflammatory protein-1 (MIP-1), MIP-1 alpha, MIP-1 beta and MIP-2.
In one embodiment, the patient is treated with a remedial composition comprising 5-50% hypertonic glucose. In a second embodiment, the patient is treated with a remedial composition comprising sodium morrhuate at a dosage of 1% to 10%. Finally, the remedial composition may comprise phenol and may be administered to a patient in need thereof at a dosage of between about 1% and 20%. The total amount of the present invention in this respect may vary, but the amount of reagent per administration may be between 1 and 5 ml.
In addition, the inflammation inducing agent of the present invention may contain a substance that exacerbates local damage, thereby enhancing the effectiveness of the repair composition of the present invention. Materials used herein include gels, hydrogels, and foams. In some cases, such gels, hydrogels, and/or foams are bioabsorbable. These high density mixtures can then be diluted by the body's own reparative response, or can be diluted back to the 0.9% physiological range by subsequent treatment with physiological saline. Thus, for example, the repair composition may contain agents (gels, hydrogels, foams) that exacerbate local injury, which may be combined with other inflammation inducing agents (including hypertonic glucose, sodium morrhuate, phenol, and the like).
In typical embodiments, the repair composition comprises a cell growth enhancing composition of autologous growth factors (e.g., platelet lysate). Preferably between about 5% and about 40%, more typically 5% and 20% of the platelet lysate, although other concentrations may be used. Platelet lysate solutions can be obtained and prepared according to the methods and compositions described in U.S. patent application 11/773,774 (which has been incorporated herein by reference for all purposes) (other methods have been discussed above). The total amount of platelet lysate preparation administered to a patient may be between 1 ml and 40 ml, and in some cases, between 1 ml and 20 ml.
One problem with the clinical use of platelet lysates in patients is the difference in bioavailability and concentration of growth factors in a particular platelet lysate. Thus, without specific biological analysis to determine the factor levels in the lysate, administration of the lysate becomes difficult. The studies discussed in U.S. patent application 11/773,774 clearly show that some patients achieve the maximum possible in vitro amplification with 5% lysate, while others achieve the maximum amplification with increasing PL concentration to 400%. Although growth factor assays are clinically available and widely used, the bioavailability of these growth factors remains elusive.
In this case, the culture expansion availability data of the patient's platelet lysate provides data on the activity of these growth factors (as discussed in U.S. patent application 11/773,774, and incorporated herein by reference). This data can be used to determine the optimal platelet lysate% (i.e., culturing autologous MSCs with different amounts of autologous platelet lysate) for treating the target patient.
The repair compositions of the present invention may also contain necessary nutrients (e.g., collagen, glycosaminoglycans, amino acids, peptides, proteins, sodium pyruvate, glucose, glutamine, ribonucleosides, deoxyribonucleosides, carbohydrates, essential oils, and the like) to further promote tissue repair. Thus, the platelet lysate solution may incorporate collagen and various amino acids to facilitate the patient's repair process.
The repair compositions of the present invention may also include anabolic hormones (e.g., human growth hormone, testosterone, and the like). In addition, for example, a subject anabolic hormone may be incorporated into the platelet lysate prior to administration to a patient in need thereof.
Finally, the repair compositions described herein may comprise autologous or non-autologous stem cells to enhance repair and regrowth of the repair site. In one embodiment, Mesenchymal Stem Cells (MSCs) have been prepared and expanded according to us patent application 11/773,774 (previously incorporated herein by reference) and implanted into a repair site. It should be noted that other stem cells or cell types are also encompassed within the scope of the present invention, however, MSCs are identified herein as one potential embodiment.
Recently, Centeno et al (us patent application 11/773,774) described a method for expanding MSCs using a growth channel and autologous platelet lysate. In addition, methods of transplanting certain levels of growth factors (platelet lysate or platelets) along with expanded MSCs into a patient at a site in need of repair are described. The levels of these growth factors are determined by the percentage of platelet lysate required to achieve optimal ex vivo (ex-vivo) expansion of certain cells. These techniques can also be used to provide sufficient numbers of MSCs for administration to a patient in need thereof. A method of promoting tissue repair in a patient in need thereof:
embodiments described herein include methods of performing therapeutic repair of a site in a patient in need thereof. For example, therapeutic repair of degenerative intervertebral discs or articular cartilage in need thereof. Other examples include myocardial replacement within the heart.
The methods described herein include a preliminary determination of parameters for optimal treatment of a repair site in a patient. For example, determining what and how much of an inflammation inducing agent will produce the best effect on the site of injury, and determining what and how much of a cell growth enhancing composition (autologous, non-autologous, mixed, etc.) should be used. In this regard, the site should have enough micro-damage to guide cellular repair mechanisms while avoiding more extensive damage to sites that do not heal. In addition, it is determined whether to use the repair composition of the present invention, or whether to first use the inflammation inducer composition and then contact it with the cell growth enhancing composition. The repair composition is applied to the repair site of the patient prior to performing the injury site analysis.
Therapeutic applications
The repair composition of the present invention provides an optimal repair condition/environment for the repair of a site in a patient in need thereof. The repair composition not only induces micro-tissue damage, thereby signaling inflammatory factors in the patient, but also initiates and/or promotes cell growth at the site. In certain embodiments, ex vivo cultured stem cells are implanted into the relevant environment to further increase the likelihood of successful repair of the repair site.
The repair compositions described herein may be formulated as pharmaceutical compositions and administered to a patient, preferably a mammalian host (including a human patient), in need thereof. The remedial composition may be formulated in a variety of forms to suit the chosen route of administration.
Embodiments carried herein include prosthetic compositions comprising a pharmaceutically acceptable carrier and/or a specific delivery drug.
For administration of the compositions as injectable solutions or suspensions, the remedial compositions may be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents, for example, sterile oils (including synthetic mono-or diglycerides) and fatty acids (including oleic acid).
The repair composition solution or suspension may be prepared with water, isotonic saline (PBS), optionally mixed with a non-toxic surfactant. The dispersant may also be prepared from glycerol, liquid polyethylene, ethylene glycol, vegetable oils, glycerol acetate and mixtures of the foregoing. Depending on the customary use and storage conditions, the remedial composition carried herein may contain one or more preservatives to prevent the growth of microorganisms.
Therapeutic applications as used herein refers to the use of the compositions and methods of the present invention for treating a patient having a damaged site requiring tissue repair. Sites of injury requiring repair (i.e., repair sites) include joints requiring cartilage repair and/or regrowth, bones requiring bone repair or regrowth, tendons/ligaments requiring repair or regrowth, organ repair requiring functional cell repair and/or regrowth (e.g., myocardial growth within the heart), and the like.
In certain embodiments, the invention is directed to therapeutic applications for patients suffering from diseases that limit the intrinsic cell repair or regrowth capacity of their repair site. For example, patients suffering from osteoarthritis, osteoporosis, avascular necrosis, will benefit from the promoted repair compositions and methods of treatment of the present invention.
The present invention is directed to creating a simulated inflammatory condition to induce tissue repair. Most animal studies in this field are performed in acute injury models (i.e., injuries that were created experimentally and are still acute or subacute when MSCs are introduced to promote tissue repair). This is a poor alternative to a model of chronic osteoarthritis in the absence of acute injury. Studies by the inventors in this field have shown that creating acute osmotic mini-lesions contributes to the meniscal repair associated with MSCs (see example 1). In such cases, we induced injury using hypertonic glucose delivered transdermally, and then delivered cultured expanded MSCs transdermally (expansion was performed according to us patent application 11/773,774 (incorporated herein by reference for this purpose)).
Having generally described this invention, a better understanding will be obtained by reference to the following examples, which are provided by way of illustration and are in no way intended to be limiting.
Examples
Example 1: therapeutic uses of embodiments of the invention
About 20 ml of whole bone marrow was extracted from two patients, CD (40 years old, white man) and JV (28 years old, white man). CD was diagnosed with severe knee osteoarthritis prior to surgery based on medial > lateral meniscal mucoid degeneration, while JV was diagnosed with chronic tubocele rupture of the posterior horn of the medial meniscus prior to surgery.
Each patient was placed prone on an OR table and the area to be harvested was anesthetized with 1% lidocaine, and then 10cc of bone marrow blood was drawn from the left and right PSIS areas, respectively, using sterile disposable trocar.
Whole bone marrow was centrifuged at 100g for 4-6 minutes to separate plasma from RBCs. The plasma was removed, placed in a separate tube, and centrifuged at 1000g for 10 minutes to form a spherical nucleated cell fraction. Nucleated cells were washed once with PBS, counted, and then suspended in DMEM + 10% Platelet Lysate (PL) at 1x106Cells per square centimeter were seeded into monolayer culture flasks. Cultures were placed at 37 ℃/5% CO2And (4) in a humid environment. After 3 days, the medium was changed and the majority of the non-adherent cell population was removed. The MSC population was grown 6-12 days after inoculation. When growing to near confluence, the colonies were trypsinized for 30-60 seconds to separate only the MSCs that formed the clusters. MSCs were then re-seeded at a density of 12,000 cells/square centimeter in DMEM + 5%, 10% or 20% PL. After 40-50% confluency was achieved, each culture was subcultured at 1: 3. After MSCs grew to 3-5 passages, they were suspended in Phosphate Buffered Saline (PBS). The patient is asked to return to the clinic and sign a consent form.
The following procedure was performed on the patient:
1. first, each patient was treated with 12.5% glucose (hypertonic agent), and then a local anesthetic was injected intra-articularly through the medial inferior extremity of the affected knee via the c-arm.
After 2.3-5 days, after the first injection produced acute inflammatory response subsided, cultured expanded autologous MSCs in PBS were injected with 10% platelet lysate.
The patient has been provided with, and has been given, a modified VAS questionnaire and a functional rating index questionnaire before, 1 month after, and 3 months after surgery, respectively. The physical therapist has performed knee mobility measurements on the patient before, 1 month after and 3 months after the operation, respectively. In addition, pre-operative MRI was performed on a GE 3.0T magnet with a proton density rapid spin sequence in the sagittal coronal plane. Images at 1 month and 3 months after surgery were obtained by matching the number of shots (NEX), the number of repetitions (TR) and the number of echoes (TE). The same examiner for each region of interest (ROI) used a triplicate pursuit (trace) for quantitative meniscus and articular cartilage volume (volume) analysis with commercially available image processing software (OSIIS-Digital Imaging Unit, Division of Medical information, University Hospital of Geneva). And the standard deviation between these three traces and the mean has been calculated. In addition, the medial weight bearing femoral defect area is also tracked and calculated in a similar manner.
Results (see table 1):
table 1: MRI volume changes of femoral cartilage and meniscus before, 1 month after, 3 months after and 6 months after surgery:
the results of the above examples demonstrate the surprising efficacy of embodiments of the invention, as well as the utility of using compositions and methods of treatment according to the invention.
Example 2: MSC amplification in a glucose environment
To ensure that the various growth factors (TGF- β, FGF, IGF, PDGF) common in platelet lysates can be exposed to a hypertonic environment and still function to support the growth of mesenchymal stem cells, the following experiments have been performed using culture-expanded human MSCs:
the method comprises the following steps:
to 0.8 ml of 10% platelet lysate, 0.2 ml of 50% glucose was added. Under separate conditions, 0.2 ml of phosphate buffered saline was added to 0.8 ml of 10% Platelet Lysate (PL). Both samples were incubated at 37C, 5% CO2 for 1 hour. Then, 1 ml of each suspension was taken out and added to 9 ml of basal alpha-mem medium to obtain a final ratio of 10% PL and 1% glucose. Thereafter, 100,000 cells in each suspension were seeded in individual wells of a 6-well plate. After 48 hours incubation, all cells exhibited normal morphology.
As a result:
PL + ═ glucose-containing
PL- ═ control
This example shows that MSCs can be efficiently expanded in a glucose (inflammation inducer) environment, which is a surprising unexpected result.