Biologic Composition And Method Of Use

Abstract

A biologic composition responsive to inflammation has an allograft scaffold matrix for injection or implantation. The allograft scaffold matrix has donor quiescent and/or senescent cells. The donor quiescent and/or senescent cells react in response to signaling of inflammation from host cells or matrix. The reaction to signaling causes the donor quiescent and/or senescent cells to secrete anti-inflammatory cytokines and secrete exosomes to initiate regeneration of the area of the inflammation. The biologic composition further has a cryoprotectant. The cryoprotectant is a polyampholyte, preferably the polyampholyte is an -poly-L-lysine. The cryoprotectant is not DMSO or glycerol based. The cryoprotectant is suitable for direct implantation without washing from the allograft scaffold matrix in either a diluted or non-diluted state.

Claims

1. A method of treating and repairing a degenerative condition comprising implanting a viable allograft comprising donor quiescent and/or senescent cells into a degenerated area exhibiting inflammation, wherein the implanting causes the donor quiescent and/or senescent cells to receive signaling from host cells indicating inflammation, and wherein the signaling activates the donor quiescent and/or senescent cells and causes the donor quiescent and/or senescent cells to secrete anti-inflammatory cytokines and exosomes directed to the host cells, thereby reducing the inflammation and initiating regeneration of the degenerated area.

2. The method of claim 1 wherein the activation of the donor quiescent and/or senescent cells in response to the signaling causes secretion and exchange of microvesicles, membrane rafts, miRNA, proteins, growth factors, and/or cytokines, and ions appropriate for tissue function.

3. The method of claim 1, wherein the degenerated area is a bone, cartilage, neurological tissue, or ischemic heart tissue.

4. The method of claim 1, wherein the donor quiescent and senescent cells are derived from bone marrow.

5. The method of claim 1, wherein the donor senescent cells are derived from placental tissue.

6. The method of claim 1, wherein the allograft further comprises an allograft scaffold matrix in the form of chips, fibers, or particles or combinations thereof, wherein the allograft scaffold matrix is made of bone, cartilage, nucleus pulposus, or dermis material, and wherein the donor quiescent and senescent cells are seeded onto or loaded into the allograft scaffold matrix.

7. The method of claim 1, wherein the allograft scaffold matrix in the form of allograft chips, fibers, or particles or combinations thereof comprises mineralized, demineralized, partially demineralized, or combinations of mineralized and demineralized bone chips, bone fibers or bone particles or any combination thereof.

8. The method of claim 6, wherein the allograft scaffold matrix further comprises nucleus pulposus particles or fibers or combinations thereof.

9. The method of claim 6, wherein the allograft scaffold matrix further comprises proteins.

10. The method of claim 1, wherein the donor quiescent and/or senescent cells are pre-treated with a cryoprotectant.

11. The method of claim 10, wherein the cryoprotectant is a polyampholyte.

12. A method of treating a degenerative intervertebral disc comprising implanting a viable allograft scaffold matrix having donor quiescent and/or senescent cells into a degenerative intervertebral disc exhibiting inflammation, wherein the implanting causes the donor quiescent and/or senescent cells to receive signaling from host cells indicating inflammation, and wherein the signaling activates the donor quiescent and/or senescent cells and causes the donor quiescent and/or senescent cells to secrete anti-inflammatory cytokines and exosomes directed to the host cells, thereby reducing the inflammation and initiating regeneration of the nucleus polposus of the degenerative intervertebral disc.

13. The method of claim 12, wherein the height of the treated degenerative intervertebral disc is increased relative to an untreated degenerative intervertebral disc.

14. The method of claim 12, wherein the activation of the donor quiescent and/or senescent cells in response to the signaling causes secretion and exchange of microvesicles, membrane rafts, miRNA, proteins, growth factors, and/or cytokines, and ions appropriate for tissue function.

15. The method of claim 12, wherein the donor quiescent and senescent cells are derived from bone marrow.

16. The method of claim 12, wherein the donor senescent cells are derived from placental tissue.

17. The method of claim 12, wherein the allograft further comprises an allograft scaffold matrix in the form of chips, fibers, or particles or combinations thereof, wherein the allograft scaffold matrix is made of bone, cartilage, nucleus pulposus, or dermis material, and wherein the donor quiescent and senescent cells are seeded onto or loaded into the allograft scaffold matrix.

18. The method of claim 17, wherein the allograft scaffold matrix in the form of allograft chips, fibers, or particles or combinations thereof comprises mineralized, demineralized, partially demineralized, or combinations of mineralized and demineralized bone chips, bone fibers or bone particles or any combination thereof.

19. The method of claim 17, wherein the allograft scaffold matrix further comprises nucleus pulposus particles or fibers or combinations thereof.

20. The method of claim 17, wherein the allograft scaffold matrix further comprises proteins.

21. The method of claim 12, wherein the donor quiescent and/or senescent cells are pre-treated with a cryoprotectant.

22. The method of claim 21, wherein the cryoprotectant is a polyampholyte.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The invention will be described by way of example and with reference to the

[0041] accompanying drawings in which:

[0042] FIG. 1 is a schematic diagram of a cross section depicting a model of a healthy intervertebral disc with the annulus fiber at the peripheral ends and the nucleus pulposus in the central area. The nucleus pulposus schematically showing disc cells dispersed and suspended in water (H.sub.2O).

[0043] FIG. 2 is a schematic evidencing early disc degeneration as evidenced by few disc cells and loss of water with an associated reduction in disc height in the occurrence of pro-inflammatory cytokines.

[0044] FIG. 3 is a schematic showing donor quiescent and/or senescent cells implanted into a degenerative disc with an allogeneic disc matrix scaffold of nucleus pulposus and normal saline.

[0045] FIG. 4 is an enlarged view of FIG. 3, showing the quiescent and/or senescent donor cells surrounded or suspended in the matrix scaffold on the left below the disc and the host cells exhibiting conditions of inflammation.

[0046] FIG. 5 is a depiction of how the host cells of the degenerative condition signal the implanted quiescent and/or senescent donor cells.

[0047] FIG. 6 is a depiction of how the quiescent and/or senescent donor cells respond to the signal of the host cells and activation of the quiescent and/or senescent donor cells to release anti-inflammatory cytokines, growth factor and exosomes to reduce host cell inflammation and rebuild the disc structure by regeneration.

[0048] FIG. 7 depicts a restored disc after using a composition made in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0049] With reference to FIGS. 1 through 7, various schematic views of the present invention are shown. The diagrammatic schematic views depict one embodiment directed to the regenerative repair of an intervertebral disc. This exemplary depiction is only a single representative example. In fact, and as claimed herein, the invention is directed to any degenerative or injured tissue repair wherein the host or patient's cells are exhibiting inflammatory responses. This condition can include bone defects or injury, cartilage damage, nerve damage, organ issues such as the heart, kidneys, pancreas etc.

[0050] One of the innovative breakthroughs of the present invention is the use of allograft scaffold matrix which has been seeded and loaded with donor cells that may be quiescent or senescent. These donor senescent and/or quiescent cells react to signals emitted by the host cells, key signals being a broad spectrum of cytokines, growth factors, or exomes that have been consistently paired with inflammation.

[0051] An allograft with donor senescent cells is unique in that these allogeneic cells, for all intents and purposes, appear lifeless, or at least unresponsive when procured from donor site. Senescent cells typically are incapable of cell division and thus cannot be readily expanded by culturing through cell division. These cells are not dead and are in fact viable, but are at rest and dormant, incapable of replicating themselves until triggered by the proper signal.

[0052] In molecular biology, the culturing and expanding of mesenchymal stem cells has been a key to achieving large quantities of these pluripotent primitive cells in sufficient quantities to be of a therapeutic value. Once collected in sufficient quantities, the cells can be exposed to differentiating factors that align a trajectory based on the appropriate cytokines to which the cells are exposed. In example, agents known for inducing osteoinductivity have been defined that direct cells down an osteogenic lineage towards bone formation. A method of inducing osteogenic differentiation of multi lineage-inducible cells has been shown by culturing the lineage agnostic, but multi-lineage capable cells in an osteogenic medium. One appropriate, and well-established osteogenic medium comprises ascorbic acid 2-phosphate, -glycerophosphate, and dexamethasone. Osteogenic differentiation often is accounted by demonstrating the expression of Runx2, osteocalcin, collagen I(Xl, or bone sialoprotein.

[0053] Appropriate cytokines used to pilot cell lineage for cartilage differ from those guiding bone and methods and medium for inducing chondrogenesis might comprise exposure to dexamethasone, TGF-3, ascorbic acid 2-phosphate, sodium pyruvate, proline, insulin, transferrin, and selenous acid. These descriptions are clearly not intended to provide an exhaustive summary of all possibilities, but to offer methods that have been shown to be effective in phenotypic differentiation of multi-linage inducible cells that are initially lineage agnostic prior to exposure to the methods.

[0054] The donor senescent cells can be guided to differentiation by exposure to various allograft scaffold materials such as bone, cartilage, nucleus pulposus, dermis, but until activated remain dormant, at rest and unresponsive. The present invention has discovered that these donor senescent cells, when implanted into a patient, can be activated in response to signaling a condition of the host cells of inflammation. This causes an activation, but not automatically, in fact unless properly preconditioned, the donor senescent cells remain at rest and cannot activate.

[0055] The inventors of the present invention found that, if the donor senescent cells were pre-treated with a cryoprotectant of polyampholyte, upon later implantation or injection these donor senescent cells in the allograft will respond to signals from inflamed host cells causing a release of anti-inflammatory cytokines, growth factors and microvesicles including exosomes.

[0056] With reference to FIG. 1, a healthy intervertebral disc 10 is illustrated. Around the outer periphery is the annulus fibrosus 12. The annulus fibrosus 12 surrounds the nucleus pulposus 14. The nucleus pulposus 14, as shown, has a vast number of disc cells 20 embedded in the gel like substance in the middle of the spinal disc 10. The nucleus pulposus 14 consists of large vacuolated notochord cells, small chondrocyte-like cells, collagen fibrils, and proteoglycan aggrecans 15 that aggregate through the hydraulic chains. Attached to each aggrecan molecule 15 are glycosaminoglycan (GAG) chains of chondroitin sulfate and keratan sulfate of varying lengths and moiety. Aggrecan 15 is negatively charged, and although depicted in the illustration as floating negative charge in the matrix, the conformation of the molecule is richly structured and through a combination of branched morphology and electro-repulsive forces confers a structured charge which in turn, allows the nucleus pulposus 14 to attract H.sub.2O water molecules. Based on still incomplete understanding of causal and reaction dynamics, enzymes clip the molecules and core proteins, releasing fragments that reduces amount of structured water and subsequently glycosaminoglycans decrease over time with additional degeneration.

[0057] As shown in FIG. 1, a healthy intervertebral disc 10 has a height H.sub.N, whereas in FIG. 2 depicting an early disc degeneration, the number of disc cells 20 has been greatly reduced. The charge activity and water content in the nucleus pulposus 14 has been also reduced and the height H.sup.() is reduced as compared to the healthy disc.

[0058] As shown in FIGS. 3 and 4, the present invention is used in combination with a degenerative disc or a disc experiencing inflammation. A composition 30 of an allograft scaffold matrix 34 can be inserted into the disc 10. This allograft scaffold matrix 34 includes donor senescent and/or quiescent cells 32 and a cryoprotectant 50 and supplements the depleted volume of the degenerative disc's nucleus pulposus 14. When initially inserted into the disc 10, the composition 30 of allograft scaffold matrix 34 provides additional volume. Preferably, the allograft scaffold matrix 34, in the case of working on a degenerative disc 10, will include a scaffold that has nucleus pulposus particles either micronized or particalized into fibers or small particles. Preferably, the nucleus pulposus is processed from a donor with healthy discs and ground and micronized then freeze-dried. This freeze-dried nucleus pulposus can then be rehydrated with normal saline. Preferably, the mixture is 50 percent nucleus pulposus to 1 cc of normal saline. In addition, disc cells that have been acquired through placental tissue harvesting and manufacturing can be produced. The process is illustrated in U.S. Pat. No. 10,064,896 which is incorporated herein by reference in its entirety.

[0059] With the present invention, the composition 30 of allograft scaffold matrix 34 and cryoprotectant 50 has a unique combination of donor senescent cells 32, saline, rehydrated previously freeze-dried nucleus pulposus particles and/or fibers that are preferably reduced to 400 microns or less, more preferably 300 microns or less suitable for direct injection through a 22 gauge needle or cannula. The nucleus pulposus, when injected with the composition 30, will receive the donor senescent cells 32. Importantly, it has been discovered that the senescent cells 32, by themselves without a coating or a treatment with a cryoprotectant 50, remain dormant when injected into a spinal disc. There is virtually no communication, the senescent cells 32 simply occupy space and do not respond to any cell signaling 21 from the host cells 20. However, by applying a cryoprotectant 50 of polyampholyte, preferably -poly-L-lysine, a non-glycerol, non DMSO cryoprotectant, which can be injected directly into the patient along with the allograft scaffold matrix 34, it has been determined that this coating 50 causes a field gradient that allows the donor quiescent and/or senescent cells 32 to be able to respond to signals 21 from the host cells 20. When this signaling occurs, the donor quiescent and/or senescent cells 32 are activated causing a release of anti-inflammatory cytokines and microvesicles 31. As shown, this release 31 helps reduce the inflammation of the host cells 20 and helps initiate a repair. In addition, exosomes 40 and other growth factors are released which helps in the regeneration of the degenerative disc 10, as shown in FIGS. 3 and 4.

[0060] With reference to FIG. 5, the signaling 21 is illustrated from the host cell 20 to the donor quiescent and/or senescent cell 32 plus the scaffold matrix 34 and cryoprotectant 50.

[0061] In FIG. 6, the signaling from the pro-inflammatory cytokines in the host cells 20 is dramatically reduced by an anti-inflammatory response 31 sending anti-inflammatory cytokines to the degenerative host cells 20.

[0062] With reference to FIG. 7, the restored intervertebral disc 10 is shown. The restored intervertebral disc 10 preferably over a period of 6 months time will have a height H.sub.R that closely approximates a healthy intervertebral disc.

[0063] This example of using polyampholyte coated 50 donor quiescent and/or senescent cells 32 in an allograft scaffold matrix 34 when used in the spine, is only one example of the application of the present invention. It is to be understood that these donor senescent cells can also be produced in a scaffold that would allow for bone regeneration. These donor quiescent and/or senescent cells could be used in a fashion similar to that which is described in U.S. Pat. No. 9,675,643 which is incorporated herein by reference in its entirety.

[0064] In the use of bone regeneration, the donor quiescent and/or senescent cells can come from bone marrow as described in U.S. Pat. No. 9,675,643. The donor senescent cells will be treated in a similar way to the previously mentioned example for the degenerative disc. They will be treated with a cryoprotectant with a polyampholyte. Once a bone allograft combination is produced this combination can be applied to any bone defect to repair either an injury and/or degenerative condition. Additionally, the scaffold can be mixed with neurological micronized material such as found in U.S. Pat. No. 9,402,869 is neural tissue composition which is incorporated herein by reference in its entirety. The scaffold then would have neural tissue that would activate repair and regeneration of damaged nerves. As can be seen, almost any degenerative condition where inflammation has occurred, is a receptive condition for the use of the present invention which is responsive to the inflammation of the host cells which will allow for a response of the donor quiescent and/or senescent cells in such a way that a regenerative repair of intervertebral disc, bone, cartilage, neurological tissue, ischemic heart and other organs can be accomplished with the use of donor senescent cells that otherwise would be dormant and incapable of providing any reparative response to the host cells.

[0065] Variations in the present invention are possible in light of the description of it provided herein. While certain representative embodiments and details have been shown for the purpose of illustrating the subject invention, it will be apparent to those skilled in this art that various changes and modifications can be made therein without departing from the scope of the subject invention. It is, therefore, to be understood that changes can be made in the particular embodiments described, which will be within the full intended scope of the invention as defined by the following appended claims.