FETAL DECELLULARIZED NUCLEUS PULPOSUS MATERIAL AND METHODS FOR OBTAINING PHARMACEUTIC COMPOSITIONS TO BE USED IN THE TREATMENT OF INTERVERTEBRAL DISC DEGENERATION AND BACK PAIN

Abstract

A fetal-origin decellularized nucleus pulposus (NP) allogenic material to regenerate a host's Intervertebral Disc (IVD). The decellularized NP material, obtained from a vertebrate fetus and characterized by comprising high levels of collagen 12 and 14, is used in a pharmacological composition for the treatment of IVD degeneration. The advance is based on the increased ability of the fetal decellularized NP material to stimulate the host constituent cell's to increase the expression of collagen 2 and aggrecan, promoting intrinsic IVD regeneration. A related method includes preparing the pharmaceutical compositions of fetal decellularized material in the form of fragments/microparticles and hydrogel for an injectable mode of administration. The involved material, pharmaceutical compositions and methods may be advantageously used for the prevention and treatment of IVD degeneration and back pain in human and veterinary settings.

Claims

1. A biomaterial characterized by, comprising a fetal decellularized nucleus pulposus (NP) of the intervertebral disc (IVD) of the fetus of a vertebrate animal.

2. The biomaterial according to claim 1 characterized by, comprising a quantity of collagen type XII (COL12A1) higher than 1.000.000 intensity-Based Absolute Quantification (iBAQ) units, defined by the sum of all peptide intensities divided by the number of theoretically observable tryptic peptides of a protein obtained by gel-free proteomics, most preferably a quantity higher than 10.000.000 COL12A1 iBAQ units and a ratio between Collagen type XII and total protein higher than 4 in comparison to young decellularized NP.

3. The biomaterial according to claim 1 characterized by, comprising a quantity of collagen type XIV (COL14A1) higher than 1.400.000 iBAQ units, most preferably higher than 10.000.000 COL14A1 iBAQ units and a ratio between Collagen type XIV and total protein higher than 10 in comparison to young decellularized NP.

4. The biomaterial according to claim 1 in which the said fetus of a vertebrate animal comprises bovine fetus, porcine fetus, sheep fetus, horse fetus, donkey fetus, kangaroo fetus and other non-limiting examples of vertebrate fetus.

5. A pharmaceutical composition for use in IVD regeneration characterized by, comprising the said biomaterial, as described in claim 1.

6. A pharmaceutical composition for use in IVD regeneration according to claim 5 characterized by, comprising the biomaterial in combination with other components, such as proteins, antibiotics, fungicides, preservation or culture medium, hydrogels, excipients, vehicle diluents, adjuvants, and combinations thereof.

7. A pharmaceutical composition for use in IVD regeneration according to claim 5 characterized by, comprising the said fetal decellularized biomaterial in the form of an implantable graft, for example an IVD graft.

8. A pharmaceutical composition for use in IVD regeneration according to claim 5 characterized by, comprising the said fetal decellularized biomaterial in an injectable form, for example in the form of microparticles.

9. A pharmaceutical composition for use in IVD regeneration according to claim 5 characterized by, comprising the said fetal decellularized biomaterial in an injectable form, for example in the form of a hydrogel.

10. A pharmaceutical composition for use in IVD regeneration according to claim 5 characterized by, further comprising other materials and cell component, non-limiting examples include cells, mesenchymal stem cells and exosomes.

11. A pharmaceutical composition for use in IVD regeneration characterized by, comprising COL12A1 and/or COL14A1 and combinations thereof, obtained from other natural or synthetic sources.

12. A method to produce the biomaterial and the pharmaceutical composition in the form of injectable microparticles according to claim 7 characterized by, comprising the steps of: a) Obtaining a vertebrate fetus, most preferably a bovine fetus tail, most preferably male, most preferably 8 months of gestation. b) Cleaning with ethanol 70%. c) Removing excess fascia and muscle with a scalpel. d) Cutting as close as possible to the vertebral body above and underneath to obtain the intervertebral disc. e) Washing with phosphate buffered saline (PBS), most preferably supplemented with 10% Penicillin/Streptomycin and 1% Fungizone, for 15 minutes, under orbital agitation at 100 rpm. f) Punching, most preferably using a 4 mm puncher, to obtain nucleus pulposus from the central zone of the disc. g) Contacting the nucleus pulposus punches with a hypotonic buffer most preferably comprising 10 mM Tris-Base, 0.1% EDTA, 0.1% Gentamicin, 1% Penicillin/Streptomycin of and 0.5% of Fungizone at pH 7.8, most preferably for 18 h under orbital agitation at 165 rpm, at room temperature. h) Removing hypotonic buffer and wash three times with PBS, most preferably for 1 hour under orbital agitation at 165 rpm, at room temperature. i) Treating the punches most preferably for 1 hour with 0.1% SDS in 10 mM Tris-Base and 0.1% Gentamicin, 1% Penicillin/Streptomycin and 0.5% Fungizone at pH 7.8 under orbital agitation at 165 rpm, at room temperature. j) Washing most preferably with 0.1% SDS in 10 mM Tris-Base and 0.1% Gentamicin, 1% Penicillin/Streptomycin and 0.5% Fungizone at pH 7.8, under orbital agitation at 165 rpm, at room temperature, for three times for 20 minutes each. k) Performing a DNAse treatment, most preferably with a 20 mM Tris-Base, 2 mM MgCl.sub.2, 0.1% Gentamicin, 1% Penicillin/Streptomycin and 0.5% Fungizone solution with, most preferably, 50 U/mL of DNAse, most preferably for 3 hours under orbital agitation at 165 rpm, at 37° C. l) Washing with PBS 1×, most preferably 3 times, 20 minutes each, under orbital agitation (165 rpm), at room temperature. m) Cutting decellularized IVD samples into pieces of 0.1-5 mm, most preferably 1 mm. n) Submerging the pieces in an appropriate amount of an excipient or a vehicle solution, for example sterile saline. o) Grinding the pieces with a tissue homogenizer, for example (Bertin Precellys 24, from Bertin Technologies) at 1000-10.000 rpms, preferably 6000 rpm for 5-60 seconds, preferably 30 seconds for 1-50 circles, preferably 20 circles at a temperature of 0-25° C., preferably 4° C. p) Controlling the size of the decellularized fetal IVD-derived microparticles to be between 10-500 μm, most preferably 200 μm by filtering the suspension for example through an 80-mesh sample sieve (for 200 μm). q) Adjusting the concentration of the microparticles suspension to 1-500 mg/ml, most preferably 50 mg/ml, with an appropriate amount of an excipient or a vehicle solution, for example sterile saline.

13. A method to produce the biomaterial and the pharmaceutical composition in the form of a hydrogel according to claim 8 characterized by, comprising the steps of: a) Isolating and decellularizing fetal nucleus pulposus as described previously in steps a)-l). b) Lyophilizing. c) Cutting small pieces of 0.1-5 mm, most preferably 1 mm. d) Solubilizing, most preferably to a concentration of 20 mg/mL, most preferably in 1 mg/mL pepsin in 3% acetic acid, most preferably at room temperature, most preferably for 72 hours. e) Neutralizing to pH 7.4, most preferably using 0.1M sodium hydroxide. f) Buffering with 10% of 10×PBS. g) Maintaining the gels stable by submerging in 1×PBS.

14. A biomaterial and pharmaceutical composition as described in claim 1 for use as in vitro coating and scaffolds for repopulating, expanding and culturing cells, and extracellular matrix models.

15. A biomaterial and pharmaceutical composition as described in claim 1 for use in the prevention and treatment of pathologic and age-related degenerative disc disease and back pain, including neck, cervical and back pain, in vertebrate animals including dogs and humans.

16. A biomaterial and pharmaceutical composition as described in claim 1 for use in the prevention and treatment of other degenerative conditions of cartilage tissues in animals, such as rheumatoid arthritis, osteoarthritis, cartilage rupture or detachment, achondroplasia, costochondritis, and polychondritis.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0116] FIG. 1: Collagen 12 and 14 composition of fetal and young age decellularized nucleus pulposus material.

[0117] (A) Intensity-Based Absolute Quantification (iBAQ) units are defined by the sum of all peptide intensities divided by the number of theoretically observable tryptic peptides of a protein obtained by intensity-Based Absolute Quantification (iBAQ) units, defined by the sum of all peptide intensities divided by the number of theoretically observable tryptic peptides of a protein obtained by proteomic gel-free proteomics. The absolute quantity of collagen 12 and 14 in decellularized bovine IVD scaffolds is 19 and 15-fold higher, respectively, in fetal NP relative to young-derived NP. Western blotting for Collagen type XII (B) and Collagen type XIV (C) of fetus (F) native and decellularized NPs and compared to the young (Y) native NPs (negative control). Graphs represents the average of three to four independent experiments obtained by band quantification. Protein expression levels were normalized by the total protein loading. Data are expressed as mean±SEM. Kruskal Wallis test followed by Dunn's multiple comparison test. As observed from the graphs, ratio between Collagen type XII and total protein in fetal decellularized is higher than 4 and the ratio between Collagen type XIV and total protein is higher than 10, compared to young.

[0118] FIG. 2. Decellularization efficiency of several experimental methods using bovine nucleus pulposus from young and fetal donors. Chemical detergents investigated for bovine nucleus pulposus decellularization. Sodium dodecyl sulfate (SDS) and Triton X-100 (Triton) treatments were explored at different concentration and time point (A). PicoGreen DNA quantification (B) and Blyscan sulfate GAGs quantification (C) of native and decellularized fetal and young NPs from two to three independent experiments. Data were normalized by wet weight (ng/mg for DNA and μg/mg for GAGs). Data are expressed as mean±SEM. Kruskal Wallis test followed by Dunn's multiple comparison test. Data from each decellularization treatment and age group were compared to the correspondent control (native). *p<0.05; **p<0.01. D—Schematic representation of optimal chemical, mechanical and enzymatic decellularization treatments.

[0119] FIG. 3: Molecular evaluation of bovine nucleus pulposus from different ages after repopulation. Constituent cells from young adult IVDs were isolated through a method comprising the following steps: [0120] 1. Obtain young adult bovine tails (male ˜12 months old) within 2-3 h after animal sacrifice, from the local abattoir and dissect aseptically. Wash the tails with EtOH 70% before put inside the flow chamber. [0121] 2. Remove excess fascia and muscle with a scalpel. [0122] 3. By using a sterile scalpel cut through the intervertebral disc (IVD) as close as possible to the vertebral body above and underneath the disc to obtain the disc as complete as possible. [0123] 4. By using a scalpel blade, separate the nucleus pulposus from the annulus fibrosus. [0124] 5. Weight the isolated nucleus pulposus. While dissecting keep the discs hydrated with isolation media on a sterile Petri dish. [0125] 6. Cut the isolated nucleus pulposus into approximately 2×2 mm segments with blade. [0126] 7. Use 10% of the nucleus pulposus wet weight as a volume of digestion media. [0127] 8. Weight collagenase I (0.5 mg/mL) and add the corresponding volume of digestion media. Add also DNAse I and filter the digestion media. [0128] 9. Transfer the tissue in the falcon with the digestion media and a sterile magnet. [0129] 10. Incubate the tissue overnight at 37° C. on a magnetic agitator (gentle agitation) in hypoxia incubator. [0130] 11. After digestion, filter through a 40/70 μm cell strainer to remove undigested ECM and produce a single cell suspension. [0131] 12. Centrifuge the filtrate at 400 g for 15 minutes in a 15 mL falcon to get a cell pellet and clear suspension. [0132] 13. Remove the supernatant and re-suspend the cells in 5 mL of IVD-medium. [0133] 14. Remove 10 μL of the suspension (homogenize well) for cell counting and mix with 10 μL of trypan blue. Count total cells using a microscope and haemocytometer to give an estimate of total cell number in 5 mL. [0134] 15. Freeze some aliquot of fresh cells in Trizol to use for RNA extraction (eventually as a control for gene expression). [0135] 16. Maintain in 2D culture other fresh cells in hypoxia incubator with IVD media to use for RNA extraction (as a control for gene expression).

[0136] Afterwards, the isolated cells were used for repopulation of decellularized nucleus pulposus material from different ages, including fetal and young, employing the following steps: [0137] 1. After equilibration of decellularized nucleus pulposus and isolation of adult bovine nucleus pulposus cells, start with cell seeding by dropping and scaffold turnover. [0138] 2. Resuspend 1×10.sup.5 cells in 10 ul of IVD media (5 ul each side). [0139] 3. Drop 5 ul of cell suspension in one side of the decellularized matrix and incubate for 2 hours in hypoxia atmosphere without IVD media. [0140] 4. After 2 hours, turn the nucleus pulposus and drop the other 5 ul of cell suspension. Incubate in hypoxia atmosphere for 2 hours without IVD media. [0141] 5. After these 4 hours, add IVD media and maintain the scaffolds in ex vivo culture for 7 days in hypoxia atmosphere. [0142] 6. Use as a control decellularized nucleus pulposus from different ages without cell seeding, cultured for 7 days as the repopulated matrices. [0143] 7. Collect and store conditioned media every 2 days. [0144] 8. Measure metabolic activity by Resazurin assay 24 h, 3 days, 7 days after cell seeding.

[0145] After 7 days of culture samples were processed for histology and for gene expression, according to the following procedures: [0146] 1. Histology: fix the scaffolds in formalin overnight at 4 degree. The day after mount the cassettes with the sample and leave in PBS until the use for Tissue Processor and Embedding. [0147] 2. Gene expression: cut the repopulated nucleus pulposus by the help of blades and digest with Pronase at 37 degree under magnetic agitation for 1 hour. Neutralize the enzyme activity with FBS and wash tissue with cold PBS. Freeze the tissue with liquid nitrogen and store at −80 degree until further use. Afterwards proceed with RNA extraction and collagen 2 mRNA quantification by real-time PCR.

[0148] mRNA expression level of aggrecan (A) and collagen type II (B) by quantitative real-time PCR, of fetus, young and old repopulated NPs, compared to 2D bovine NP cells, after 7 days of ex vivo culture. mRNA values were interpolated in a calibration curve (mRNA level of 2D bovine NP cells at different concentrations) and normalized by GAPDH, an internal control (mRNA level of 2D bovine NP cells) and native bovine NP (mRNA level of organ culture: 8 mm punched NP cultured ex vivo for 7 days). Data are represented as box and whiskers plots. Error bars on box-and-whiskers plots indicate the minimum and maximum values. Kruskal-Wallis Test followed by Dunn's multiple comparison test.

[0149] Collagent type II composition of bovine nucleus pulposus from different ages after repopulation: Expression of collagen by immunofluorescence (C) in fetus (F+cells) and young (Y+cells) repopulated NPs, compared to the correspondent controls (decellularized matrices; ctrl). Representative images of four to six independent experiments. Collagen type II: magnification 20× and scale bar 100 μm. Collagen type II quantification by IntensityStatisticsMask Software (D). Data are represented as dot plots. Error bars plots indicate the minimum and maximum values. Graphs corresponds to the mean with SEM of the technical replicates. Wilcoxon test was used in comparisons.

[0150] Aggrecan content of bovine nucleus pulposus from different ages after repopulation: Expression of aggrecan by immunoistochemistry (E) in fetus (F+cells) and young (Y+cells) repopulated NPs, compared to the correspondent controls (decellularized matrices; ctrl). Representative images of four to six independent experiments. Aggrecan: magnification 20×. Quantification by ImageJ Software (F). Data are represented as dot plots. Error bars plots indicate the minimum and maximum values. Graphs corresponds to the mean with SEM of the technical replicates. Wilcoxon test was used in comparisons.

[0151] FIG. 4. Structural and biochemical composition of bovine nucleus pulposus from fetal and young decellularized NPs with the optimal procedure.

[0152] A. Biomechanical characterization of bovine nucleus pulposus from different ages decellularized with the optimal procedure (SDS 0.1% 1h). Complex shear modulus (G*) values (at 5% of strain), retrieved from the linear viscoelastic region (0.04-1 Hz) of the frequency sweep, performed by rheology, of fetus and young native and decellularized NPs. Graphs represents the average of three independent experiments (three to four NPs tissue from the same animal donor for each native and decellularized condition). Data are expressed as mean±SEM. Kruskal-Wallis test followed by Dunn's multiple comparison test.

[0153] FIG. 5. Picrosirius red staining followed by polarized light microscopy to evaluate collagen organization (A). Graph (B) represents the average ratio of green to red fibers of four to eight NPs for each native and decellularized condition. Data are expressed as mean±SEM. Mann-Whitney test was used in comparisons.

[0154] FIG. 6—The graph shows the percentage of water lost per sample following freeze drying.

[0155] FIG. 7—Turbidimetric gelation kinetics. Representative curves of the different NP-derived hydrogel compositions tested, as well as of the controls. Neutralized and buffered pre-gel solutions were added to 96-well plates at 37° C. to induce gelation. The absorbance was measured every 2 minutes at 405 nm.

[0156] FIG. 8—The graph presents the values of the mean G* for each age expressed as mean±SEM.

OTHER EXAMPLES

[0157] Examples of other forms of the present invention comprise the use of nucleus pulposus material derived from the fetus of other vertebrates, including, but not limited to, decellularized nucleus pulposus material from porcine fetus, decellularized nucleus pulposus material from sheep fetus, decellularized nucleus pulposus material from horse fetus, decellularized nucleus pulposus material from donkey fetus, decellularized nucleus pulposus material from kangaroo fetus etc. [0158] Lisbon, 14 Dec. 2020