NATIVE SOFT TISSUE MATRIX FOR THERAPEUTIC APPLICATIONS

Abstract

A product for implantation within a soft tissue site of the human or animal body comprises a matrix of pulverized or morselized substantially non-mineralized native soft tissue (NSTM) of the human or animal body, provided in a therapeutic amount to induce growth of native tissue or organs and healing at the tissue site. The NSTM is composed of at least one soft tissue selected from the group consisting of cartilage, meniscus, intervertebral disc, ligament, tendon, muscle, fascia, periosteum, pericardium, perichondrium, skin, nerve, blood vessels, and heart valves or from organs such as bladder, lung, kidney, liver, pancreas, thyroid, or thymus. Preferably, the NSTM is composed of a soft tissue of the same type of tissue native to the repair site.

Claims

1. A product for introduction within a soft tissue site of the human body, comprising a suspension of a native soft tissue matrix (NSTM) consisting of a morselized or pulverized substantially non-mineralized native soft tissue of the human body, said NSTM suspended in a volume of a biologically compatible fluid such that the concentration of the NSTM is 10-20% w/v, wherein the suspension is operable to promote cellular growth within and through the NSTM.

2. The product of claim 1, wherein said biologically compatible fluid is selected from the group consisting of saline solution, a pharmaceutical solution, synovial fluid, blood serum, blood plasma, a chondrocyte suspension, a mesenchymal cell (MSC) suspension, and an adipose-derived adult stem (ADAS) cell suspension.

3. The product of claim 1, wherein said biologically compatible fluid is selected from the group consisting of collagen, gelatin, serum, fibrin, hyaluronic acid, proteoglycans and elastin.

4. The product of claim 1, wherein said NSTM is composed of at least one soft tissue selected from the group consisting of cartilage, meniscus, intervertebral disc, ligament, tendon, muscle, fascia, periosteum, perichondrium, skin, nerve, blood vessels and heart valves.

5. The product of claim 1, wherein said NSTM is composed of at least one soft tissue selected from the group consisting of organs such as kidney, liver, pancreas, thyroid, or thymus.

6. The product of claim 1, wherein said NSTM is composed of a soft tissue of the same type of tissue at the soft tissue site.

7. The product of claim 1, wherein said NSTM includes a combination of soft tissues obtained from multiple soft tissue sites.

8. The product of claim 1, wherein the soft tissue of the NSTM is devitalized.

9. The product of claim 1, wherein the soft tissue of said NSTM is morselized or pulverized to a particle size in the range of 1-100 μm.

10. The product of claim 1, further comprising cells combined with the NSTM.

11. The product of claim 10, wherein said cells are selected from the group consisting of autologous, allogeneic or xenogeneic cells.

12. The product of claim 10, wherein said cells are differentiated, phenotype-specific cells, committed progenitor cells, or toti-, pluri- or multi-potential stem cells.

13. The product of claim 1, further comprising bioactive molecules combined with said NSTM.

14. The product of claim 13, wherein said bioactive molecules include growth factors selected from the group consisting of TGF-β, IGF, bFGF, PDGF, VEGF and BMP.

15. The product of claim 13, wherein said bioactive molecules are selected from DNA and vectors capable of delivering therapeutic genes to the tissue site.

16. A product for introduction within a soft tissue site of the human body, comprising a native soft tissue matrix (NSTM) consisting of a morselized or pulverized substantially non-mineralized native soft tissue of the human body combined with a moldable biologic material, wherein the combination is operable to promote cellular growth within and through the NSTM.

17. The product of claim 16, wherein said moldable biologic material includes one or more of the following constituents: collagen, gelatin, fibrin, hyaluronic acid and elastin gels.

18. The product of claim 16, wherein said moldable biologic material is selected from the group consisting of alginate, agarose and chitosan gels.

19. The product of claim 16, wherein said moldable biologic material is bioresorbable or biodegradable.

20. The product of claim 16, wherein the soft tissue of the NSTM is devitalized.

Description

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0042] As examples, the following section describes methods of the present invention that can be used to create a devitalized native soft tissue matrix derived from cartilage (NSTM-CH) that can be provided to a point-of-care facility, or to processing or manufacturing facilities in various formulations. These formulations, including dry powder, dry morsels, frozen paste, or frozen slurry, can be reconstituted alone or in combination with cells, a carrier material, or bioactive molecules. The reconstituted NSTM-CH is used to repair cartilage focal defects or damage, or osteoarthritis-related lesions. It is understood that no limitation to the scope or the application of the methods of the current invention to cartilage repair is thereby intended. Rather, the methods of the current invention can be extended to include applications for repair of all soft tissues from which the NSTM can be derived including meniscus, intervertebral disc, ligament, tendon, muscle, fascia, periosteum, perichondrium, skin, nerve, blood vessels, bladder, lung, and heart valves. It is also understood that the NSTM derived from any of the above soft tissue and organs can be used to repair any of the above listed soft tissues and organs, as well as mineralized tissues such as bone.

[0043] In one embodiment, a specific formulation of an allogeneic NSTM-CH is combined with cells, chondro-inductive growth factors, and a carrier material that can have the form of a hydrogel or a porous scaffold. In this embodiment, the NSTM-CH serves as the biological substrate onto which the cells can proliferate, differentiate, and synthesize competent cartilage matrix.

[0044] The cells used in this application are: 1) chondrocytes, which can be isolated from different autologous sources (such as articular cartilage, elastic cartilage, or epiphysial growth-plate) by enzymatic digestion (e.g., collagenase and pronase); 2) mesenchymal stem cells isolated and propagated from the patient's bone marrow; 3) adipose-derived adults stem cells isolated from fat tissue biopsies from the same patient; or 4) other sources of toti-, multi- or pluri-potential stem cells. The isolation and propagation of these cells might require the use of cell culture facilities remote from the point-of-care facility and in some cases extended processing time. In an alternate embodiment, bone marrow is directly used as a heterogeneous suspension of cells containing stem cells. A further alternative is to concentrate a specific population of cells in a density gradient in bone marrow and re-suspend them in a nutrient-rich medium at the point-of-care.

[0045] The carrier material can be a moldable biologic material or a porous scaffold. The carrier material provides a means for effective delivery, structural enforcement, and/or predetermined geometry guidance. In the preferred embodiment, the moldable biologic material is a hydrogel carrier material. Suitable hydrogel materials may include: 1) biological gels such as collagen, hyaluronic acid, fibrin, gelatin, elastin or other biological materials; 2) marine organism-derivatives such as, alginate, agarose, or chitosan gels; or 3) synthetic hydrogels such as Pluronic/F-12 and PEO/PEG gels. This carrier material is moldable so that the product can be configured to match the contours of the soft tissue site in which the product is to be implanted.

[0046] Alternately, the carrier material may be a porous, woven or non-woven, biodegradable or biocompatible (non-degradable) scaffold of predetermined geometry and dimensions made from biological or man-made polymers including but not limited to gelatin mesh, collagen mesh, polyglycolic acid, polylactic acid, polyorthoesters, polyglycolide-co-lactides, polyanhydrides, poly(amino acids), pluronic/F-12, PEO/PEG, polycaprolactones or other suitable synthetic hydrogels or mixtures thereof. In certain applications, a composite matrix made from a hydrogel and a porous scaffold may be appropriate.

[0047] In certain embodiments, the carrier material includes a non-resorbable and non-biodegradable constituent. The constituents are selected from the group consisting of acrylics, polycarbonates, polyesters, polyethers, poly(ether ketone), poly(ether, ether ketone), poly(aryl ether ketones), poly(ether ether ketone ether ketone), poly(ethylene terephthalate), poly(methyl (meth)acrylate), polyolefins, polysulfones, polyurethane, polyethylene, polypropylene, poly(vinyl chloride), fiber reinforced composites, or mixtures thereof.

[0048] The chondro-inductive factors may include growth factors with demonstrated chondro-inductive effects including but not limited to TGF-β, IGF, bFGF, and/or BMPs and combinations thereof. These growth factors can be obtained using recombinant technology from pharmaceutical suppliers or can be isolated and concentrated from the patient's own blood, serum, or bone marrow at the point-of-care.

[0049] The following specific examples are provided to illustrate the methods and materials of the present invention as they apply to cartilage. Other applications can be easily extrapolated using either identical or similar techniques to repair intervertebral disc, ligament, tendon, muscle, or any of the other tissues listed in this disclosure. Although derived from non-mineralized tissues, the NSTM may also be used to promote the repair of any mineralized tissues such as bone. Suitable modifications and adaptations of the variety of conditions and parameters normally encountered in surgical situations which are obvious to those skilled in the art are within the spirit and scope of the present invention. It is understood that all methods described within are to be performed in strict adherence to sterile cell culture techniques and good laboratory/manufacturing practices. All reagents and solutions used shall be sterile.

EXAMPLE 1: Acellular NSTM-CH Slurry for Injection into Cartilage or Other Soft Tissue Lesions

Preparation of the NSTM-CH Dry Formulation:

[0050] Human cartilage from tissue banks or from trauma patients, obtained in accordance with standards and ethics appropriate for the handling of human tissue, is minced into small pieces (1-5 mm.sup.2) and then incubated at 37° C. in 50 ml test tubes containing phosphate buffered saline (“PBS”) and 10% of Penicillin/Streptomycin/Fungizone or similar antibiotic/antimycotic solutions for 1-2 hours. The cartilage pieces are then washed thoroughly in PBS, after which the PBS is removed and discarded, and the cartilage pieces are snap frozen in LN.sub.2. The frozen cartilage specimens are then crushed using a cryogenic tissue pulverizer, such as the Bio-Pulverizer™ of BioSpec Products, Inc., or similar tissue grinders/mills.

[0051] In a preferred embodiment of the invention, the tissue is pulverized to a particle size that is insufficient as a scaffold to be populated by native cells. Instead, the particle size is limited to a size effective to provide differentiation cues for the native cells of the soft tissue site in which the product of the present invention is implanted. In the preferred embodiment, the particle size is less than about 800 μm. In a more specific embodiment, the particle size is limited to a range of 1-100 μm.

[0052] The pulverized tissue is then placed in polypropylene tubes and suspended in an appropriate volume of distilled water (dH.sub.2O). The pulverized tissue suspension is then further homogenized using a tissue homogenizer. The homogenized tissue suspension is centrifuged, the supernatant removed, and the precipitate tissue particles are re-suspended and incubated at 37° C. in an enzymatic solution containing DNAase and RNAase to remove DNA and RNA proteins which can potentially induce an immunogenic response in the host joint. After treatment with DNAase and RNAase, the tissue suspension is centrifuged and washed several times in dH.sub.2O to remove traces of the enzymes. The tissue is then re-suspended in dH.sub.2O, mixed well, and then snap frozen in LN.sub.2. The frozen homogenized tissue tubes are then freeze dried or lyophilized to produce a dry form of the devitalized native cartilage matrix. The aseptically derived dried formulation may then be additionally sterilized by gamma irradiation or similar sterilization techniques. The dried formulation is provided to the surgeon at the point-of-care in a sterile vial as an off-the-shelf allogeneic devitalized biomatrix to be used in indications such as trauma-induced focal cartilage lesions, osteoarthritic lesions, and other cartilage pathologies.

Reconstitution and Surgical Injection of the NSTM-CH as Hydrated Slurry:

[0053] At the point-of-care, the surgeon reconstitutes the dry formulation of NSTM-CH in an appropriate volume of saline or a pharmaceutical solution of chondro-inductive growth factors such that the concentration of the reconstituted slurry is, for example, ˜20% (w/v). The surgeon then implants the hydrated slurry into the defect in a surgical repair procedure (See e.g., Brittberg et al., Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994; 331:889-95). During surgery, the surgeon may carefully remove the damaged tissue and prepare the lesion for the injection of the reconstituted NSTM-CH. A small patch of fascia obtained from the patient's joint capsule is sutured over the lesion and its edges sealed with biological glue (e.g., fibrin) to hold the viscous slurry in place. Alternately, a piece of periosteum obtained from the patient's bone can be used as a patch cover for the lesion. The hydrated slurry is then injected under the fascia patch into the lesion where the devitalized NSTM-CH can provide biological cues to attract and guide the differentiation of migrating cells from the host cartilage, subchondral bone, and/or bone marrow.

EXAMPLE 2: Synovial/NSTM-CH Slurry for Injection into Cartilage or Other

[0054] Soft Tissue Lesions

[0055] At the point-of-care, the surgeon reconstitutes the dry formulation of NSTM-CH in an appropriate volume of synovial fluid drawn from the patient's healthy joint, such that the concentration of the reconstituted slurry is, for example, ˜10-20% (w/v). Alternately, the NSTM-CH can be reconstituted in a pharmaceutical viscous synovial supplement (e.g., Synvisc® Hylan G-F 20 produced by Genzyme Corporation). The surgeon then implants the viscous slurry into the defect in a surgical repair procedure as described in Example 1.

EXAMPLE 3: Serum/NSTM-CH Slurry for Injection into Cartilage or Other Soft Tissue Lesions

[0056] At the point-of-care, the surgeon draws an appropriate volume of the patient's blood and collects the blood serum or blood plasma using established protocols. The surgeon then reconstitutes the dry formulation of NSTM-CH in an appropriate volume of serum or plasma, such that the concentration of the reconstituted slurry is, for example, ˜10-20% (w/v). The surgeon then implants the reconstituted slurry into the defect in a surgical repair procedure as described in Example 1.

EXAMPLE 4: Chondrocyte/NSTM-CH Suspension for Injection into Cartilage or Other Soft Tissue Lesions

[0057] A biopsy of hyaline, elastic, or fibro-cartilage is obtained from the patient from a variety of cartilage sources in the body including, but not limited, to non-load bearing articular cartilage, nasal cartilage, costal cartilage, auricular cartilage, physea (growth plate) cartilage, and tracheal cartilage. The cartilage biopsy is placed inside tubes containing culture media. The tubes are sealed sterile and sent on ice (˜4° C.) to the processing facility where cartilage is then sliced into small bits measuring approximately 2 mm in length and width. The cartilage is incubated at 37° C. in 50 ml test tubes containing Dulbecco's modified medium (“DMEM”) or other appropriate media and 10% of Penicillin/Streptomycin/Fungizone or similar antibiotic/antimycotic solutions for 30 minutes. The wash media is aspirated, and the minced tissue is incubated for an hour at 37° C. in a DMEM solution containing the nonspecific enzyme pronase (˜10,000 PUK/gram of tissue) with intermittent mixing. The pronase solution is then removed and the tissue is further incubated at 37° C. for 2-3 hours with a collagenase type-2 solution (˜0.4% (w/v)) in DMEM with intermittent mixing, until the cartilage bits have completely been digested. The isolated chondrocyte suspension is then passed through a 70-micron nylon mesh cell strainer to remove undigested bits of cartilage. The strained cell suspension is centrifuged, washed in PBS to remove traces of the enzyme, and prepared for use immediately or plated in appropriate culture conditions to increase cell yield. The viable cells are then shipped back to the point-of-care.

[0058] At the point-of-care, the chondrocytes are brought into suspension in a chondrogenic formulation of media, saline, a pharmaceutical solution of growth factors, serum, plasma, or synovial fluid or supplement. The cell suspension is them mixed with the dry formulation of NSTM-CH such that the concentration of the NSTM-CH component in the reconstituted suspension is, for example, ˜10-20% (w/v). The surgeon then implants the reconstituted cell suspension into the defect in a surgical repair procedure as described in Example 1.

[0059] As an alternative, fully differentiated phenotype specific cells for the specific tissue that is being replaced could be substituted in this example for the repair of other soft tissues. For instance, annular fibrosus cells or nucleus pulposus cells would be used if the targeted repair is located in the intervertebral disc. However, it should be noted that chondrocytes, or non-phenotypic cells may also be used to treat pathology in different soft tissues from which they are derived.

EXAMPLE 5: MSC/NSTM-CH Suspension for Injection into Cartilage or Other Soft Tissue Lesions

[0060] At the point-of-care, the surgeon obtains a bone marrow biopsy from the patient. A density gradient is used to eliminate unwanted cell types present in the marrow aspirate. The small percentage of mesenchymal cells (MSC) isolated using the density gradient are brought into suspension in a chondrogenic formulation of media, saline, a pharmaceutical solution of growth factors, serum, plasma, or synovial fluid or supplement. The cell suspension is them mixed with the dry formulation of NSTM-CH such that the concentration of the NSTM-CH component in the reconstituted suspension is ˜10-20% (w/v). The surgeon then implants the reconstituted cell suspension into the defect in a surgical repair procedure as described in Example 1.

EXAMPLE 6: ADAS/NSTM-CH Suspension for Injection into Cartilage or Other Soft Tissue Lesions

[0061] An aspirate of adipose tissue obtained from the patient using a liposuction procedure is sent to the processing/manufacturing facility. Adipose-derived adult stem (ADAS) cells are isolated from the stromal vascular fraction of the lipoaspirates using a collagenase digestion protocol. The cells are propagated in culture and then retrieved and shipped back to the point-of-care. The isolated cells are then shipped back to the point-of-care.

[0062] The ADAS cells are brought into suspension in a chondrogenic formulation of media, saline, a pharmaceutical solution of growth factors, serum, plasma, or synovial fluid or supplement. The cell suspension is them mixed with the dry formulation of NSTM-CH such that the concentration the NSTM-CH component in the reconstituted suspension is, for example, ˜10-20% (w/v). The surgeon then implants the reconstituted cell suspension into the defect in a surgical repair procedure as described in Example 1.

EXAMPLE 7: Cell/Hydrogel/NSTM-CH Suspension for Injection into Cartilage or Other Soft Tissue Lesions

[0063] At the point-of-care, cells (which could be chondrocytes, MSCs, ADAS cells, or other chondroprogenitor cells) are brought into suspension in a chondrogenic formulation of media, saline, a pharmaceutical solution of growth factors, serum, plasma, or synovial fluid or supplement. The cell suspension is them mixed with the dry formulation of NSTM-CH such that the concentration of the NSTM-CH component in the reconstituted suspension is, for example, ˜10-20% (w/v). The cell/NSTM-CH suspension is mixed with a pharmaceutical grade hydrogel at an appropriate concentration. The preferred form of the hydrogel is an in situ cross-linkable material which can be mixed with the cell/NSTM-CH suspension, injected into the lesion as described in Example 1, and cross-linked by means of a change in temperature (e.g., agarose, pluronic F-127), pH (e.g., collagen), ionic bond formation (e.g., alginate) or enzymatic cross-linking pathway (e.g., fibrin).

EXAMPLE 8: Porous Scaffold/NSTM-CH for Implantation into Cartilage or Other Soft Tissue Lesions

[0064] Porous, woven or non-woven, synthetic or biological polymer scaffolds are used in this example to provide structural support and geometrical control and guidance. The scaffold is preferably degradable and could be made from gelatin mesh, collagen mesh, polyglycolic acid, polylactic acid, glycolide-co-lactide, polyanhydride, polycaprolactone scaffolds, or other biological or synthetic polymers. At the point-of-care, cells (which could be chondrocytes, MSCs, ADAS cells, or other chondroprogenitor cells) are brought into suspension in a chondrogenic formulation of media, saline, a pharmaceutical solution of growth factors, serum, plasma, or synovial fluid or supplement. The cell suspension is them mixed with the dry formulation of NSTM-CH such that the concentration the NSTM-CH component in the reconstituted suspension is, for example, ˜10-20% (w/v). The cell/NSTM-CH suspension is seeded onto the porous scaffold.

[0065] The surgeon then prepares the defect bed to match the geometry and dimensions of the scaffold and the cell/NSTM-CH-laden scaffold is implanted into the bed. A small patch of fascia obtained from the patient's joint capsule is sutured over the implant and its edges sealed with biological glue (e.g., fibrin) to prevent leakage of the cell/NSTM-CH suspension. In this embodiment, the NSTM-CH serves to provide biological cues to guide cell differentiation and neomatrix formation whereas the porous scaffold provides structural support, geometrical control and growth guidance.

[0066] Alternately, the cells, NSTM-CH, and scaffold can be coaxed into a tissue-engineered (TE) cartilage implant at a remote processing/manufacturing facility. The TE cartilage implant is cultured in a closed-system bioreactor by which physical and chemical chondrogenic factors are controlled to produce a consolidated TE cartilage implant, which can then be shipped to the point-of-care for implantation.

[0067] While the invention has been illustrated and described in detail in the foregoing description, the same should be considered as illustrative and not restrictive in character. It is understood that only the preferred embodiments have been presented and that all changes, modifications and further applications that come within the spirit of the invention are desired to be protected.

[0068] For example, other applications can be extrapolated using either identical or similar techniques to repair intervertebral disc, ligament, tendon, muscle are any of the other soft, non-mineralized tissues listed in this disclosure. Suitable modifications and adaptations of the variety of conditions and parameters normally encountered in surgical situations which are obvious to those skilled in the art are within the spirit and scope of the present invention