WO2022174077A1 - Methods and compositions for promoting regeneration of nucleus pulposus with highly negative glycosaminoglycan - Google Patents
Methods and compositions for promoting regeneration of nucleus pulposus with highly negative glycosaminoglycan Download PDFInfo
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Definitions
- compositions for promoting regeneration of nucleus pulposus with high osmolality, highly negative glycosaminoglycans e.g., chondroitin sulfate (CS) or chondroitin sulfate proteoglycan (CSPG).
- the compositions can be, for example, hydrogels, capsules, or powdered forms.
- IVD degeneration is one of the major independent risk factors [1]
- current surgical treatments for IVD degenerative diseases e.g., pathological disc excision and/or spinal fusion, result in the loss of some spinal function.
- regenerative therapy has been pursued; however, its success has been met with such obstacles as avascularity in IVD, lack of regenerative capability of resident cells, and incessant mechanical loading.
- the IVD has a complex structure, with the amorphous nucleus pulposus (NP) confined by the collagenous annulus fibrosus and cartilaginous endplates, supporting compressive loading and facilitating multidimensional spinal movement [2] Furthermore, the IVD is not only immune-privileged, but is also the largest avascular organ in the body [3], which places resident cells in an extremely harsh environment — low glucose, oxygen, and pH and high osmotic pressure (OP) and load repetition [4]
- NP amorphous nucleus pulposus
- OP osmotic pressure
- the methods comprise administering a therapeutically effective amount of a composition, preferably a hydrogel, comprising chondroitin sulfate (CS) and/or chondroitin sulfate proteoglycan (CSPG) at about 400-600 mOsm/kg H2O, preferably about 450 mOsm/kg H2O, to a subject in need thereof, wherein the composition is implanted in the subject at a site of intervertebral disc degeneration.
- a composition preferably a hydrogel, comprising chondroitin sulfate (CS) and/or chondroitin sulfate proteoglycan (CSPG) at about 400-600 mOsm/kg H2O, preferably about 450 mOsm/kg H2O
- compositions preferably hydrogels, comprising chondroitin sulfate (CS) and/or chondroitin sulfate proteoglycan (CSPG) at about 400-600 mOsm/kg H2O, preferably about 450 mOsm/kg H2O, for use in a method of treating intervertebral disc (IVD) disease in a subject (e.g., a mammal) in need thereof, wherein the composition is formulated to be implanted in the subject at a site of intervertebral disc degeneration.
- IVD intervertebral disc
- the subject has spinal stenosis and instability, radiculopathy, myelopathy, and/or disc herniation.
- the composition e.g., hydrogel
- the composition further comprises one or more of: cartilaginous cells, therapeutic molecules, or nucleic acids.
- the cartilaginous cells are autologous or allogeneic chondrocytes or stem cell-derived chondrogenic cells.
- the therapeutic molecules are anti-inflammatory agents, preferably corticosteroids.
- the nucleic acids comprise mRNA or DNA encoding chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACTl).
- compositions preferably hydrogels, comprising chondroitin sulfate (CS) and/or chondroitin sulfate proteoglycan (CSPG) at about 400-600 mOsm/kg H20, preferably about 450 mOsm/kg H20, and optionally one or more of: cartilaginous cells, therapeutic molecules, or nucleic acids.
- the chondrocytes are autologous or allogenic chondrocytes or stem cell-derived chondrogenic cells.
- the therapeutic molecules are anti-inflammatory agents, preferably corticosteroids.
- the nucleic acids comprise mRNA or DNA encoding chondroitin sulfate N- acetylgalactosaminyltransferase 1 (CSGALNACTl).
- CSGALNACTl chondroitin sulfate N- acetylgalactosaminyltransferase 1
- the hydrogel is formulated to be implanted in the subject at a site of intervertebral disc degeneration.
- the term “about” refers to a range of numerical values of up to ⁇ 10% of the recited value that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
- FIGs. 1 A-B The effects of augmenting extracellular matrix (ECM) on metabolism in bovine nucleus pulposus (bNP) cells under hydrostatic pressure (HP).
- Data are presented with box plots.
- FIGs. 2A-C The effects of augmenting extracellular matrix (ECM) on catabolic turnover in bovine nucleus pulposus (bNP) cells under hydrostatic pressure
- FIG. 3 Involvement of transient receptor potential vanilloid-4 (TRPV4) in the effects of augmenting extracellular matrix (ECM) on bovine nucleus pulposus (bNP) cells under hydrostatic pressure (HP).
- ECM extracellular matrix
- bNP bovine nucleus pulposus
- HP hydrostatic pressure
- FIGs. 4A-D Exemplary semi-permeable membrane pouch culture module and pressure/perfusion culture system.
- A Semi-permeable membrane pouches and a pressure-proof chamber.
- B Repetitive regimen of 2-day cyclic followed by 1-day constant hydrostatic pressure (HP).
- HP 1-day constant hydrostatic pressure
- C Macroscopic appearance of pressure/perfusion culture module and system.
- D Validation of bovine nucleus pulposus cell viability at 12 days. Calcein-AM indicates live cells and ethidium homodimer-1 (EthDl) indicates dead cells. The bar indicates 100 pm.
- FIGs. 5A-B Preparation of NP explant pieces and culture regimen.
- A NP explant harveseted from bovine tail and a representative example of uniform distribution of pieces and NP tissues.
- B A representative image of a piece of NP tissue with stainless steel wire protector and a nylon mesh bag and culture regimen.
- FIGs. 6A-B Osmolality of Bovine Chondroitin Sulfate Dissolved in Various Concentrations of Sodium Chloride.
- B Osmolality of Bovine Chondroitin Sulfate Dissolved in Various Concentrations of Sodium Chloride.
- ANOVA analysis of variance
- FIGs. 8A-B (A )Acan gene expression in human nucleus pulposus cells with chondroitin sulfate or sodium chloride at high osmotic pressure. (B ) Aeon expression in bovine nucleus pulposus cells with chondroitin sulfate and hydrostatic pressure. LOP: 290 mOsm, HOP 450 mOsm/kg H 2 0, CSPG: 450 mOsm/kg H2O.
- FIGs. 9A-E lmmunohistology for (A) keratan sulfate (KS), (B) Col-2, (C) Col-1, (D) MMP-13, and (E) TRPV4 stained with the relevant antibody.
- Low osmolality 290 mOsm/kg H2O.
- High Osmolality 450 mOsm/kg H2O.
- Closed arrows indicate KS gap.
- Open arrows indicate fibrous COL-2 networks. A square in each photo of upper panel is magnified shown in lower panel. Nuclei were counterstained with hematoxylen. A bar indicates 100 pm.
- the NP contains highly negatively charged extracellular matrix (ECM), which is capable of absorbing abundant interstitial fluid [3,5] and generating high osmotic pressure (OP) [6,7]
- ECM extracellular matrix
- OP high osmotic pressure
- HP hydrostatic pressure
- Our latest studies demonstrate that a repetitive regimen of cyclic HP followed by constant HP at high osmolality stimulated anabolic gene upregulation and dense accumulation of ECM in bovine NP (bNP) cells [10,11] Therefore, the combination of dynamic HP and intradiscal high OP is required in maintaining bNP-cell homeostasis [10,12]
- HA as candidate therapeutic materials, as they are the main components of disc and cartilage ECM. Both CSPG and HA attract interstitial water, contributing to the microenvironment and mechanical structure of the NP [14] Aggrecan, the primary
- CSPG in the IVD is characterized by its highly negative charge density, owing to sulfate chains [15,16]
- HA is a unique, non-sulfated glycosaminoglycan whose molecular weight reaches the millions [17] and has both anti-inflammatory and anabolic effects in the IVD [18]
- the present material-based therapeutic methods include implanting a composition, preferably a high osmolality hydrogel, comprising CS or CSPG into degenerated NP, which prevents progressive degeneration and ultimately promotes regeneration.
- a composition preferably a high osmolality hydrogel, comprising CS or CSPG into degenerated NP, which prevents progressive degeneration and ultimately promotes regeneration.
- CS Chondroitin sulfate
- CSPG chondroitin sulfate proteoglycan
- the primary CSPG in the IVD generally contains approximately 100 chains of chondroitin sulfate and 30 keratan sulfate chains [15,16]
- the negatively charged chondroitin sulfate chains contribute to the major function of aggrecan as a structural proteoglycan, holding large amounts of water in the ECM [15]
- the high OP including the capability to swell and resist compressive loads, is generated by the hydrated chondroitin sulfate chains [8,23]; meanwhile, HA has no sulfate chains, which is unique and distinct from other glycosaminoglycans [17,33]
- sulfate chains, generating OP is key.
- Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) composed of a chain of alternating sugars (D-glucuronic acid (GlcA) and N-acetyl-D- galactosamine (GalNAc)). Chondroitin sulfate includes 4 forms, two mono- and two di-sulfated:
- the chondroitin-4-sulfate and chondroitin-6-sulfate forms are used.
- the CS or CSPG comprises at least 40%, 60%, 80%, 90%, or more chondroitin-4-sulfate and/or chondroitin-6-sulfate.
- CS and CSPG can be obtained by extraction from of cartilaginous tissues in cows and pigs (e.g., cow trachea and pig ear and nose), but other sources such as shark, fish, and bird cartilage can also be used.
- the CS is at least 20,000 Dalton, e.g., 20,000 to 750,000 Dalton, e.g., at least 500,000 Dalton.
- the composition includes any of HA, dextran, alginate, poly(ethylene glycol) (PEG), pullulan, carboxymethyl pullulan-tyramine (CMP-TA), collagen, gelatin, heparin sulphate (HS), tetronic (a four-armed block copolymer of poly(ethylene oxide) and poly-(propylene oxide)), poly(L-glutamic acid, and/or chitosan (43).
- the composition does not include any HA.
- the composition does not include any of HA, dextran, alginate, poly(ethylene glycol) (PEG), pullulan, carboxymethyl pullulan-tyramine (CMP-TA), collagen, gelatin, heparin sulphate (HS), tetronic (a four-armed block copolymer of poly(ethylene oxide) and poly-(propylene oxide)), poly(L-glutamic acid, and/or chitosan.
- CSPG extracts can also include minor molecules e.g., keratan sulfate.
- the composition is a hydrogel, powder, or capsule comprising encapsulated powder; in some embodiments, the composition is a hydrogel that includes only CS and/or CSPG, salts, and water.
- the composition e.g., hydrogel
- the hydrogels are about 400-600 mOsm/kg H2O, e.g., 400-500 mOsm/kg H2O, e.g., 450 mOsm/kg H2O.
- compositions e.g., hydrogels
- the compositions can also include cells, e.g., cartilaginous cells such as chondrocytes (preferably autologous chondrocytes) or stem cell-derived chondrogenic cells (see, e.g., Craft et ah, Nat Biotechnol 33:638-645, 2015), or therapeutic molecules such as anti inflammatory agents, e.g., corticosteroids, or growth factors (e.g., Transforming growth factor-beta, insulin-like growth factor).
- compositions can also include nucleic acids, e.g., mRNA or DNA encoding Chondroitin sulfate N- acetylgalactosaminyltransferase 1 (CSGALNACTl).
- nucleic acids e.g., mRNA or DNA encoding Chondroitin sulfate N- acetylgalactosaminyltransferase 1 (CSGALNACTl).
- CSGALNACTl Chondroitin sulfate N- acetylgalactosaminyltransferase 1
- NP OO 1123990.1 An exemplary nucleic acid sequence encoding human CSGALNACTl protein can be found in GenBank at RefSeq. Acc. No.
- intervertebral disc disease which generally refers to the presence of damaged intervertebral disc tissue caused by trauma or a disc degenerative condition.
- Disc degeneration can occur naturally, e.g., with ageing, and can manifest in many clinical conditions including spinal stenosis and instability, radiculopathy, myelopathy and disc herniation.
- Disc degeneration is generally but not always associated with back pain.
- Subjects who can be treated by this method include mammals, e.g., humans and non-human veterinary subjects, e.g., horses, cows, pigs, goats, dogs, and cats.
- the methods include administering a therapeutically effective amount of a composition, e.g., a hydrogel, powder, or capsule, as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.
- a composition e.g., a hydrogel, capsule, or powder
- the composition can be implanted around the circumference of the disc, or implanted in the spine between an affected disc and an adjacent vertebra.
- the composition can be implanted by any suitable means, for example by injection using a suitable syringe or by surgical implantation.
- a composition for surgical implantation can have a higher viscosity than a composition for injection, for example, can be solid or semi-solid (such as a plug or capsule).
- MR images can be used to determine a approximate volume of degenerated disc (nucleus pulposus).
- the volume of CS or CSPG e.g., the dry weight of CS powder needed, can be estimated by the volume of space (dry weight/volume of space).
- to “treat” means to ameliorate at least one symptom of the disorder associated with IVD degeneration.
- the methods improve one or more symptoms of IVD degeneration, e.g., discogenic pain.
- Example 1 Augmented Chondroitin Sulfate Proteoglycan Has Therapeutic Potential for Intervertebral Disc Degeneration by Stimulating Anabolic Turnover in Bovine Nucleus Pulposus Cells under Changes in Hydrostatic Pressure
- CLS-1 collagenase
- F- 12 F12 nutrient mixture
- bNP cells were collected, rinsed with Dulbecco’s phosphate- buffered saline (D-PBS), seeded onto 1.5% cell-culture-grade agarose-coated 6-well plates, and incubated in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 (1 : 1) supplemented with 10% fetal bovine serum (FBS) and 100-units/mL penicillin/100 pg/mL streptomycin at 37°C, 5% CO2 for 2-3 days. After this preincubation, bNP cells/clusters were collected with a pipette under a dissection microscope.
- D-PBS Dulbecco’s phosphate- buffered saline
- FBS fetal bovine serum
- semipermeable membrane pouches were prepared as previously described [11] Briefly, hollow fiber tubing (1 mm in diameter, polyvinylidene fluoride, 500 kD molecular weight cut-off) was cut into 35 mm lengths, immersed in ethyl alcohol (200 proof) for 30 min, and autoclaved in D-PBS at 121°C for 15 min.
- the pouches were incubated under two different sets of culture conditions based on our previous study [10,11]: (1) no HP, in which pouches were placed in culture medium under atmospheric pressure; and (2) HP, in which 2-day cyclic HP (0.2-0.7 MPa, 0.5 Hz) followed by 1-day constant HP (0.3 MPa) was repeated 4 times over 12 days (Figure 4B). Both sets of conditions also included 37°C, 5% CO2, and 3% O2 to stimulate a physiologic hypoxic IVD environment [3,4] High-osmolality medium at 450 mOsm/kg H2O was made using 4.6-g/L sodium chloride and the osmolality was confirmed with a freeze-point osmometer (MICRO-OSMETTETM).
- MICRO-OSMETTETM freeze-point osmometer
- the pouches were suspended within a stainless-steel mesh basket held in 100 mL medium with a stirrer to maintain sufficient mass transfer through the pouches.
- the pouches were placed in a culture chamber filled with the medium to load cyclic or constant HP with medium replenishment at 0.1 mL/min using a pressure/perfusion culture system (TEP-2; Figure 4C).
- TEP-2 pressure/perfusion culture system
- cell viability of the bNP cells/clusters within the pouches was validated with calcein-AM for live cells and ethidium homodimer-1 (EthDl) for dead cells (LIVE/DEADTM Viability/Cytotoxicity Kit) according to the manufacturer’s instruction, which demonstrated predominant population of live cells (Figure 4D).
- RNA Isolation and Real-Time Reverse Transcription Polymerase Chain Reaction (RT PCR)
- RNA messenger RNA
- mRNA messenger RNA
- Collaf Col2af Has2,Mmpl3 and Timp2 relative to glyceraldehyde 3-phosphate dehydrogenase ( Gapdh ) as an endogenous control were evaluated in quadruplicate by real-time RT-PCR using TaqManTM gene expression master mix and fluorescent-labeled specific primers.
- the commercially available validated primers used were as follows: Acan, Bt03212189_ml; Collal, Bt03225358_gl; Col2al, Bt03251837_mH; Has2, Bt03212694_gl; wp73, Bt03214051_ml; Timp2 , Bt03231007_ml; Gapdh , Bt03210919_gl (TaqManTM probes). Measurements were performed using the QuantStudio 5 Real-Time PCR System. Relative mRNA expression was analyzed by the 2 AACt method using ExpressionSuite Software vl.0.4 [35] The value of the no material control sample with D-PBS under no HP at 3 days was set at 1.0.
- the bNP cells/clusters were fixed with 2% paraformaldehyde/0.1 M cacodylate buffer (pH 7.4) at 4 °C, embedded in paraffin, and cut into 7 pm sections. Dewaxed sections were incubated with primary antibodies against KS (1:500), MMP13 (1:50), and TRPV4 (1:100) overnight. The sections were then rinsed and incubated with a biotinylated secondary antibody (VECTASTAIN ® Elite ABC-HRP kit) for 30 min. The color was developed with 3,3'- diaminobenzidine and nickel (DAB substrate kit). Counterstaining was performed with Harris’s hematoxylin for KS, and with Contrast Red for MMP13 and TRPV4.
- the number of positive cells was counted in four random high-power fields (x400) using the ImageJ software (imagej.nih.gov/ij/, accessed on 4 October 2018).
- the positive cell percentage for MMP13 and TRPV4 was calculated relative to the total number of hematoxylin- or Contrast Red-positive cells.
- Immunohistology showed MMP 13 to be denser within the bNP cells/clusters under no HP than HP in each material.
- bNP cells/clusters with HA showed denser MMP 13 than the no-material control and CSPG regardless of HP at each time point.
- the bNP cells were exposed to cyclic and constant HP applied using our hydrostatic pressure culture system.
- Intense TRPV4 staining was found under HP at 3 days with all materials, localized at the surface of bNP cells/clusters; staining was weaker without HP; intense staining was diminished by 12 days.
- ECM-based materials such as hyaluronan-based, alginate-based, and collagen -based ones, appear to stimulate IVD-cell metabolism, although their clinical and therapeutic strategies have not been fully addressed [13]
- augmenting ECM specifically with CSPG promoted regenerative turnover in bNP cells through restoring the native ECM microenvironment.
- the significant Acan upregulation and Collal downregulation with CSPG under no HP at 3 days demonstrates the anabolic and anti-fibrotic effects of CSPG early in incubation.
- Augmenting HA has an anabolic and anti-inflammatory effect on human chondrocytes [25], and intra-articular administration of HA is an established treatment for such joint diseases as knee osteoarthritis [26]
- HAS activity appears to be an important modulator, and the anabolic and anti-catabolic effects of extracellular HA without activating HAS could be limited in vitro [27]
- the augmented HA used in our experiments is commercially available as a cell scaffold for in vitro cell culture, careful interpretation is required in evaluating the clinical relevance of our results.
- HP at physiological range increases ECM synthesis in bovine, dog, and rabbit NP cells in vitro [28-30], while higher HP (>2.5 MPa) induced a catabolic trend, increasing MMP3 activity and decreasing matrix synthesis [31,32]
- HP at physiological range
- the OP is generated by water content absorbed within the tissue [6,7] and influences bNP cell metabolism [10,11] Therefore, to reproduce the microenvironment in IVD homeostasis, we chose CSPG and HA in this study, both of which are capable of absorbing abundant interstitial fluid. Despite the hydrophilic properties of both materials, our results for bNP-cell anabolism and catabolism were opposite between these materials.
- the primary CSPG in the IVD generally contains approximately 100 chains of chondroitin sulfate and 30 keratan sulfate chains [15,16]
- the negatively charged chondroitin sulfate chains contribute to the major function of aggrecan as a structural proteoglycan, holding large amounts of water in the ECM [15]
- the high OP including the capability to swell and resist compressive loads, is generated by the hydrated chondroitin sulfate chains [8,23]; meanwhile, HA has no sulfate chains, which is unique and distinct from other glycosaminoglycans [17,33]
- the augmented CSPG provides OP to the bNP cells and stimulates ECM synthesis early in culture, and then newly synthesized ECM would maintain OP and the microenvironment within the semi-permeable membrane pouch during later phases.
- the semi-confined pouch not only holds both augmented CSPG and synthesized ECM around the cells but also helps the generated OP directly affect the cells.
- TRPV4 staining was more intense with HP than without HP, both at 3 and 12 days; and it diminished by 12 days under HP although the difference was statistically significant only with CSPG.
- TRPV4 activation is implicated in transmitting the effects of HP to the bNP cell metabolism.
- ECM was newly synthesized and accumulated around bNP cells over time, as shown by immunohistology against KS. The accumulated ECM likely altered membrane characteristics and subsequently decelerated TRPV4 activation.
- TRPV4- positive cell percentages were not significantly different among the groups, the accumulation of TRPV4 in HA was not as robust as in the control and CSPG, suggesting that the presence of HA might interrupt TRPV4 activation.
- TRPV4 transient receptor potential cation channel vanilloid-4
- Bovine tails from 2- to 3-years-old cows were purchased from a local slaughterhouse (Adams Farm, Athol, MA) certified by United States Department of Agriculture (USDA).
- Caudal NP tissues were aseptically harvested from the proximal two intact discs within 6 h after slaughter. The cows are skeletally mature, and their NP-cell phenotypes are relevant to human NP cells. Consequently, a total of 24 NP explants were obtained from 12 tails and five tails were used for each experiment. Each NP explant was cut into 6 pieces in the proximal two segments (approximately 30-40 mg) (Fig. 5A). To eliminate anatomic difference in cell metabolism and tissue composition, each different regions and levels was distributed randomly among the three different experimental conditions.
- NP explant pieces were suspended in a sterile nylon mesh bag (Cancer Diagnostics, Durham, NC) with a stainless wire frame (Fig. 5B) to avoid tissue deformation from the pieces contacting one another, then assigned to one of three culture medium conditions as follows: 1) Low osmolality medium (LOP, Physiological 290 mOsm/kg H2O); 2) High osmolality medium prepared from NaCl (HOPNa, 450 mOsm/kg H2O); 3) High osmolarity prepared from CSPGs (HOPCS, 450 mOsm/kg H2O).
- LOP Low osmolality medium
- HOPNa High osmolality medium prepared from NaCl
- HOPCS High osmolarity prepared from CSPGs
- LOP medium was prepared from Dulbecco’s Modified Eagle Medium/Ham’s F-12 nutrient mixture (1:1) (DMEM/F12, Gibco, Waltham, MA) supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin, and 100 pg/mL streptomycin (Gibco).
- HOPNa at 450 mOsm/kg H2O was prepared from supplemental sodium chloride 4.6 g/L into the LOP medium validated with a freezing point osmometer (MICRO-OSMETTETM, Precision System, Natick, MA).
- HOPCS medium was prepared from CSPG 65 mg/ml (C4384, Sigma- Aldrich, St.
- the NP explant pieces were harvested, minced, and digested in 2-mg/mL pronase (Merck Millipore, Burlington, MA) and 1-mg/mL hyaluronidase (Sigma- Aldrich) dissolved in DMEM/F12 on a vibration shaker at 37°C for 1 hour. Five ml of this enzyme cocktail was added to 100 mg of wet weight of the NP explant pieces. To stop enzyme activity, fetal bovine serum (0.5 ml per 5 ml of the enzyme cocktail) was added to the sample.
- 2-mg/mL pronase Merck Millipore, Burlington, MA
- 1-mg/mL hyaluronidase Sigma- Aldrich
- RNA isolation was conducted according to the manufacture’s protocol. Precipitation was performed with 0.25-mL isopropanol and 0.25 mL high salt precipitation solution.
- mRNA Messenger RNA expression levels of aggrecan core protein ( Acan ), collagen types I and II ( Collal and CoUal ), Brachyury, matrix metalloproteinase 13 ( Mmpl3 ), and tissue inhibitor of metalloproteinases 2 ( Timp2 ) relative to glyceraldehyde 3 -phosphate dehydrogenase (' Gapdh ) as an endogenous control were quantified in quadruplicate by RT-qPCR using fluorescent-labeled specific primers and gene expression master mix (Applied Biosystems).
- Acan aggrecan core protein
- collagen types I and II Collal and CoUal
- Brachyury matrix metalloproteinase 13
- Mmpl3 matrix metalloproteinase 13
- Timp2 tissue inhibitor of metalloproteinases 2
- Timp2 tissue inhibitor of metalloproteinases 2
- the NP explants were harvested, fixed in 2% paraformaldehyde/10 mM PBS (pH 7.4) at 4°C, embedded in paraffin, and cut into 7- pm sections.
- the sections were dewaxed in xylene, rehydrated in graded concentrations of ethanol, and rinsed in PBS 3 times.
- the sections were incubated in blocking solution, 3% normal horse serum (Vector Laboratories, Burlingame, CA) at room temperature for 20 min and then the sections were incubated with the primary antibodies at 4°C overnight.
- the chosen primary antibodies were collagen type II (COL2, 1:100, MBS397123, MyBiosource, San Diego, CA), collagen type I (COL1, 1:100, Abeam, Cambridge, MA), MMP13 (1:100, LS-B3168, LifeSpan BioScience, Seattle, WA), TRPV4 (1:100, LS-A8583, LifeSpan BioScience), and keratan sulfate (KS, 1:500, sc-73518, Santa Cruz Biotechnology, Santa Cruz, CA) for supporting gene expression results. Only for KS, the sections were incubated with 0.05 unit/ml chondroitinase ABC (Millipore-Sigma) at 37°C for 1 hour to expose epitopes.
- the section incubated with the first antibody were rinsed in PBS 3 times, then incubated in biotinylated secondary antibody solution at room temperature for 30 min followed by a manufacture’s instruction (VECTASTAIN ® Elite ABC-HRP kit, Vector Laboratories). After rinsing the section with PBS 3 times, the sections were incubated with ABC solution at room temperature for 30 min. Lastly, the color was developed with 3,3’-diaminobenzidine and nickel (DAB substrate kit, SK4100, Vector Laboratory).
- DAB substrate kit 3,3’-diaminobenzidine and nickel
- Lectin labeling was performed as previously described (40-41) with slight modifications to capture glycosylation, indicating proteoglycan distribution.
- the dewaxed sections followed by rehydration were rinsed in 20 mM Tris-HCl buffered saline supplemented with 100 mM NaCl, 1 mM CaCh, and 1 mM MgCh (TBS+ buffer) at pH 7.2.
- the sections were then blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich) at room temperature for 1 hour to reduce non-specific binding.
- BSA bovine serum albumin
- the sections were washed in TBS+ buffer 3 times and incubated with fluorescently- labelled-lectin (Wisteria floribunda lectin, FL-1351-2, Vector Laboratories) diluted in TBS+ (10 pg/mL, 1 :200) at room temperature for 1 h.
- the sections were rinsed in TBS+ buffer 3 times, then applied with SlowFadeTM Gold Antifade mount with 4', 6- diamidino-2-phenylindole (DAPI) (Invitrogen, S36938).
- Lectin histochemical staining was captured using the Leica DMi8 microscope (Leica Camera, Wetzlar, Germany). From eight random middle-power fields (x200) of each slide, fluorescent area fraction was calculating by quantifying the positively stained matrix component divided by the total area and converting into a percentage (%) using the ImageJ software (imagej.nih.gov/ij/).
- Chondroitin sulfate with high osmolality in culture medium was used for these experiments because there is no 450 mOsm osmolality fluid in the body. Chondroitin sulfate can create high osmotic pressure locally in the body such as nucleus pulposus and cartilage.
- FIGs. 6A the osmolality in chondroitin sulfate dissolved in various concentration of sodium chloride solution increased dose dependent manner.
- FIG. 6B shows that adding chondroitin sulfate can alter the osmolality of a solution compared to other biological molecules e.g., bovine serum albumin.
- chondroitin sulfate can be injected into degenerated disc space instead of injecting other high osmotic fluids.
- NP tissues were harvested from bovine caudal intervertebral discs (Figs. 5A-B).
- the NP tissues were incubated in regular culture medium at physiological osmotic pressure (290 mOsm/kg H20), in modified culture medium supplemented with NaCl at high osmotic pressure (450 mOsm/H20), and in modified culture medium with supplemented with chondroitin sulfate at high osmotic pressure (450 mOsm/kg H20).
- the nucleus pulposus tissues were incubated for 6 days.
- the tissues were harvested for molecular evaluation using RT-qPCR (gene expression of key molecules) and immunohistology (with corresponding proteins).
- Catabolic molecules suppression is desirable.
- Tissue inhibitor for matrix metalloproteinase-2 Timp2
- Bovine nucleus pulposus were incubated with chondroitin sulfate at high osmolality (450 mOsm/kg H20) and with/without hydrostatic pressure at 0.5 MPa, 0.5 Hz for 6 days.
- hydrostatic pressure Acan gene expression showed the trend of greater upregulation compared to without pressure. The results are shown in FIG. 8B.
- Hyaluronan syn-thase 2 (HAS2) overexpression diminishes the procatabolic activity of chondrocytes by a mechanism independent of extra-cellular hyaluronan. J. Biol. Chem. 2019, 294, 13562-13579
- TRPV4 mediates cell damage induced by hyperphysiological compression and regulates COX2/PGE2 in intervertebral discs. 4 (3):ell49
- TRPV4 transient receptor potential vanilloid 4
Abstract
Described herein are methods and compositions for promoting regeneration of nucleus pulposus with high osmolality, highly negative glycosaminoglycans, e.g., chondroitin sulfate (CS) or chondroitin sulfate proteoglycan (CSPG). The compositions can be, for example, hydrogels.
Description
Methods and Compositions for Promoting Regeneration of Nucleus Pulposus with Highly Negative Glycosaminoglycan
CLAIM OF PRIORITY
This application claims the benefit of U.S. Provisional Application Serial No. 63/148,537, filed on February 11, 2021. The entire contents of the foregoing are incorporated herein by reference.
TECHNICAL FIELD
Described are methods and compositions for promoting regeneration of nucleus pulposus with high osmolality, highly negative glycosaminoglycans, e.g., chondroitin sulfate (CS) or chondroitin sulfate proteoglycan (CSPG). The compositions can be, for example, hydrogels, capsules, or powdered forms.
BACKGROUND
Back pain is a global health problem with a considerable socioeconomic burden, and intervertebral disc (IVD) degeneration is one of the major independent risk factors [1] However, current surgical treatments for IVD degenerative diseases, e.g., pathological disc excision and/or spinal fusion, result in the loss of some spinal function. Thus, the development of regenerative therapy has been pursued; however, its success has been met with such obstacles as avascularity in IVD, lack of regenerative capability of resident cells, and incessant mechanical loading. The IVD has a complex structure, with the amorphous nucleus pulposus (NP) confined by the collagenous annulus fibrosus and cartilaginous endplates, supporting compressive loading and facilitating multidimensional spinal movement [2] Furthermore, the IVD is not only immune-privileged, but is also the largest avascular organ in the body [3], which places resident cells in an extremely harsh environment — low glucose, oxygen, and pH and high osmotic pressure (OP) and load repetition [4]
SUMMARY
Provided herein are methods for treating intervertebral disc (IVD) disease. The methods comprise administering a therapeutically effective amount of a composition, preferably a hydrogel, comprising chondroitin sulfate (CS) and/or chondroitin sulfate proteoglycan (CSPG) at about 400-600 mOsm/kg H2O, preferably about 450
mOsm/kg H2O, to a subject in need thereof, wherein the composition is implanted in the subject at a site of intervertebral disc degeneration. Also provided herein are compositions, preferably hydrogels, comprising chondroitin sulfate (CS) and/or chondroitin sulfate proteoglycan (CSPG) at about 400-600 mOsm/kg H2O, preferably about 450 mOsm/kg H2O, for use in a method of treating intervertebral disc (IVD) disease in a subject (e.g., a mammal) in need thereof, wherein the composition is formulated to be implanted in the subject at a site of intervertebral disc degeneration.
In some embodiments, the subject has spinal stenosis and instability, radiculopathy, myelopathy, and/or disc herniation. In some embodiments, the composition (e.g., hydrogel) further comprises one or more of: cartilaginous cells, therapeutic molecules, or nucleic acids. In some embodiments, the cartilaginous cells are autologous or allogeneic chondrocytes or stem cell-derived chondrogenic cells. In some embodiments, the therapeutic molecules are anti-inflammatory agents, preferably corticosteroids. In some embodiments, the nucleic acids comprise mRNA or DNA encoding chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACTl).
Also provided herein are compositions, preferably hydrogels, comprising chondroitin sulfate (CS) and/or chondroitin sulfate proteoglycan (CSPG) at about 400-600 mOsm/kg H20, preferably about 450 mOsm/kg H20, and optionally one or more of: cartilaginous cells, therapeutic molecules, or nucleic acids. In some embodiments, the chondrocytes are autologous or allogenic chondrocytes or stem cell-derived chondrogenic cells. In some embodiments, the therapeutic molecules are anti-inflammatory agents, preferably corticosteroids. In some embodiments, the nucleic acids comprise mRNA or DNA encoding chondroitin sulfate N- acetylgalactosaminyltransferase 1 (CSGALNACTl). In some embodiments, the hydrogel is formulated to be implanted in the subject at a site of intervertebral disc degeneration.
As used herein, the term “about” refers to a range of numerical values of up to ± 10% of the recited value that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in
the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
DESCRIPTION OF DRAWINGS
FIGs. 1 A-B. The effects of augmenting extracellular matrix (ECM) on metabolism in bovine nucleus pulposus (bNP) cells under hydrostatic pressure (HP). (A) Gene expression of aggrecan core protein (Acan), collagen type II and I (Col2al and Collal), hyaluronan synthase 2 (Has2) with the no-material control, chondroitin sulfate proteoglycan (CSPG), and hyaluronan (HA) under no HP and HP at 3 and 12 days relative to the control under no HP at 3 days (n = 6 for each). Data are presented with box plots. Multi-way repeated measures analysis of variance (ANOVA) with the Bonferroni post hoc test was used. * p < 0.05 and ** p < 0.01. Gapdh, glyceraldehyde 3-phosphate dehydrogenase. RQ, relative quantity. (B) The accumulation of keratan sulfate (KS) in brown counterstained with hematoxylin. Arrows indicate intense accumulation, each section is 7 pm thick, and the bar indicates 50 pm.
FIGs. 2A-C. The effects of augmenting extracellular matrix (ECM) on catabolic turnover in bovine nucleus pulposus (bNP) cells under hydrostatic pressure
(HP). (A) Gene expressions of catabolic matrix metalloproteinase 13 (Mmpl3) and anti-catabolic tissue inhibitor of metalloproteinases 2 (Timp2) with the no-material control, chondroitin sulfate proteoglycan (CSPG), and hyaluronan (HA) under no HP and HP at 3 and 12 days relative to the control under no HP at 3 days (n = 6 for each).
Data are presented with box plots. Multi-way repeated measures analysis of variance
(ANOVA) with the Bonferroni post hoc test was used. * p < 0.05 and ** p < 0.01.
Gapdh, glyceraldehyde 3 -phosphate dehydrogenase. RQ, relative quantity. (B) The correlation between Mmpl3 and Timp2 expression with the control, CSPG, and HA under no HP and HP at 12 days relative to the control under no HP at 3 days (n = 6 for each). Pearson correlation analysis was used. (C) The accumulation of MMP13 in black counterstained with Contrast Red and positive cell percentage of MMP13 (n = 6
for each). Arrows indicate intense accumulation, each section is 7 mih thick, and the bar indicates 50 mih. Data are presented with box plots. Multi-way repeated measures ANOVA with the Bonferroni post hoc test was used. * p < 0.05 and ** p < 0.01.
FIG. 3. Involvement of transient receptor potential vanilloid-4 (TRPV4) in the effects of augmenting extracellular matrix (ECM) on bovine nucleus pulposus (bNP) cells under hydrostatic pressure (HP). The accumulation of TRPV4 in black counterstained with Contrast Red and positive cell percentage of TRPV4 are shown (n = 6 for each). Arrows indicate intense accumulation, each section is 7 pm thick, and the bar indicates 50 pm. Data are presented with box plots. Multi-way repeated measures analysis of variance (ANOVA) with the Bonferroni post hoc test was used. * p < 0.05 and ** p < 0.01.
FIGs. 4A-D. Exemplary semi-permeable membrane pouch culture module and pressure/perfusion culture system. (A) Semi-permeable membrane pouches and a pressure-proof chamber. (B) Repetitive regimen of 2-day cyclic followed by 1-day constant hydrostatic pressure (HP). (C) Macroscopic appearance of pressure/perfusion culture module and system. (D) Validation of bovine nucleus pulposus cell viability at 12 days. Calcein-AM indicates live cells and ethidium homodimer-1 (EthDl) indicates dead cells. The bar indicates 100 pm.
FIGs. 5A-B. Preparation of NP explant pieces and culture regimen. (A) NP explant harveseted from bovine tail and a representative example of uniform distribution of pieces and NP tissues. (B) A representative image of a piece of NP tissue with stainless steel wire protector and a nylon mesh bag and culture regimen.
FIGs. 6A-B. (A) Osmolality of Bovine Chondroitin Sulfate Dissolved in Various Concentrations of Sodium Chloride. (B)
FIG. 7. Gene expression of aggrecan core protein (Acan), collagen type II and I (Col2al and Collal), matrix metalloproteinase 13 (Mmpl3), tissue inhibiter of metalloproteinases 2 (Timp2), and transient receptor potential cation channel subfamily V member4 (Trpv4) with low osmolality, high osmolality by sodium chloride (NaCl), and high osmolality by chondroitin sulfate proteoglycan (CSPG) at 3 and 6 days relative to the low osmolality at 3 days (n = 6). Data are presented as mean + standard deviation. Two-way analysis of variance (ANOVA) with the Bonferroni post hoc test was used. *P < 0.05 and **P < 0.01. Gapdh, glyceraldehyde 3-phosphate dehydrogenase. RQ, relative quantity.
FIGs. 8A-B. (A )Acan gene expression in human nucleus pulposus cells with chondroitin sulfate or sodium chloride at high osmotic pressure. (B ) Aeon expression in bovine nucleus pulposus cells with chondroitin sulfate and hydrostatic pressure. LOP: 290 mOsm, HOP 450 mOsm/kg H20, CSPG: 450 mOsm/kg H2O.
FIGs. 9A-E. lmmunohistology for (A) keratan sulfate (KS), (B) Col-2, (C) Col-1, (D) MMP-13, and (E) TRPV4 stained with the relevant antibody. Low osmolality : 290 mOsm/kg H2O. High Osmolality: 450 mOsm/kg H2O. Closed arrows indicate KS gap. Open arrows indicate fibrous COL-2 networks. A square in each photo of upper panel is magnified shown in lower panel. Nuclei were counterstained with hematoxylen. A bar indicates 100 pm.
DETAILED DESCRIPTION
To develop therapeutic strategies for IVD diseases, we have been focusing on the physiological microenvironment and homeostasis within NP. The NP contains highly negatively charged extracellular matrix (ECM), which is capable of absorbing abundant interstitial fluid [3,5] and generating high osmotic pressure (OP) [6,7] The NP is also exposed to changes in hydrostatic pressure (HP) in daily cycles owing to weight bearing in the upright position and off-loading in the recumbent position [8,9] Our latest studies demonstrate that a repetitive regimen of cyclic HP followed by constant HP at high osmolality stimulated anabolic gene upregulation and dense accumulation of ECM in bovine NP (bNP) cells [10,11] Therefore, the combination of dynamic HP and intradiscal high OP is required in maintaining bNP-cell homeostasis [10,12]
In addition to the aforementioned cellular responses to changes in HP at high
OP, approaches using biomaterials and exploiting endogenous cell populations to restore cellular properties and stimulate ECM production have gained increased attention in the field of IVD regeneration [13] While no biomaterials are clinically approved for IVD repair, mainly due to safety concerns and unclarified repair mechanisms, we selected chondroitin sulfate proteoglycan (CSPG) and hyaluronan
(HA) as candidate therapeutic materials, as they are the main components of disc and cartilage ECM. Both CSPG and HA attract interstitial water, contributing to the microenvironment and mechanical structure of the NP [14] Aggrecan, the primary
CSPG in the IVD, is characterized by its highly negative charge density, owing to sulfate chains [15,16] HA is a unique, non-sulfated glycosaminoglycan whose
molecular weight reaches the millions [17] and has both anti-inflammatory and anabolic effects in the IVD [18]
The present material-based therapeutic methods include implanting a composition, preferably a high osmolality hydrogel, comprising CS or CSPG into degenerated NP, which prevents progressive degeneration and ultimately promotes regeneration.
Chondroitin sulfate (CS) / chondroitin sulfate proteoglycan (CSPG)
Aggrecan, the primary CSPG in the IVD, generally contains approximately 100 chains of chondroitin sulfate and 30 keratan sulfate chains [15,16] The negatively charged chondroitin sulfate chains contribute to the major function of aggrecan as a structural proteoglycan, holding large amounts of water in the ECM [15] The high OP, including the capability to swell and resist compressive loads, is generated by the hydrated chondroitin sulfate chains [8,23]; meanwhile, HA has no sulfate chains, which is unique and distinct from other glycosaminoglycans [17,33] Without wishing to be bound by theory, it is believed that the presence of sulfate chains, generating OP, is key.
Thus, provided herein are methods that use compositions comprising CS or CSPG. Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) composed of a chain of alternating sugars (D-glucuronic acid (GlcA) and N-acetyl-D- galactosamine (GalNAc)). Chondroitin sulfate includes 4 forms, two mono- and two di-sulfated:
Preferably the chondroitin-4-sulfate and chondroitin-6-sulfate forms are used. In some embodiments, the CS or CSPG comprises at least 40%, 60%, 80%, 90%, or more chondroitin-4-sulfate and/or chondroitin-6-sulfate. CS and CSPG can be obtained by extraction from of cartilaginous tissues in cows and pigs (e.g., cow trachea and pig ear and nose), but other sources such as shark, fish, and bird cartilage can also be used. Preferably, the CS is at least 20,000 Dalton, e.g., 20,000 to 750,000 Dalton, e.g., at least 500,000 Dalton.
In some embodiments, the composition includes any of HA, dextran, alginate, poly(ethylene glycol) (PEG), pullulan, carboxymethyl pullulan-tyramine (CMP-TA), collagen, gelatin, heparin sulphate (HS), tetronic (a four-armed block copolymer of poly(ethylene oxide) and poly-(propylene oxide)), poly(L-glutamic acid, and/or chitosan (43). In some embodiments, the composition does not include any HA. In some embodiments, the composition does not include any of HA, dextran, alginate, poly(ethylene glycol) (PEG), pullulan, carboxymethyl pullulan-tyramine (CMP-TA), collagen, gelatin, heparin sulphate (HS), tetronic (a four-armed block copolymer of poly(ethylene oxide) and poly-(propylene oxide)), poly(L-glutamic acid, and/or chitosan. In some embodiments, CSPG extracts can also include minor molecules e.g., keratan sulfate. In some embodiments, the composition is a hydrogel, powder, or capsule comprising encapsulated powder; in some embodiments, the composition is a hydrogel that includes only CS and/or CSPG, salts, and water.
The composition, e.g., hydrogel, should have high osmolality, i.e., osmolality above physiological osmolality (290-300 mOsm). In preferred embodiments, the hydrogels are about 400-600 mOsm/kg H2O, e.g., 400-500 mOsm/kg H2O, e.g., 450 mOsm/kg H2O.
Supplemental Ingredients
In addition to the CS/CSPG, the compositions (e.g., hydrogels) described herein can also include cells, e.g., cartilaginous cells such as chondrocytes (preferably autologous chondrocytes) or stem cell-derived chondrogenic cells (see, e.g., Craft et ah, Nat Biotechnol 33:638-645, 2015), or therapeutic molecules such as anti inflammatory agents, e.g., corticosteroids, or growth factors (e.g., Transforming growth factor-beta, insulin-like growth factor). The compositions can also include nucleic acids, e.g., mRNA or DNA encoding Chondroitin sulfate N- acetylgalactosaminyltransferase 1 (CSGALNACTl). An exemplary sequence for human CSGALNACTl protein can be found in GenBank at RefSeq. Acc. No.
NP OO 1123990.1. An exemplary nucleic acid sequence encoding human CSGALNACTl protein can be found in GenBank at RefSeq. Acc. No.
NM 001130518.2.
Methods of Treatment
Provided herein are methods for treating intervertebral disc disease, which generally refers to the presence of damaged intervertebral disc tissue caused by trauma or a disc degenerative condition. Disc degeneration can occur naturally, e.g., with ageing, and can manifest in many clinical conditions including spinal stenosis and instability, radiculopathy, myelopathy and disc herniation. Disc degeneration is generally but not always associated with back pain. Subjects who can be treated by this method include mammals, e.g., humans and non-human veterinary subjects, e.g., horses, cows, pigs, goats, dogs, and cats.
Generally, the methods include administering a therapeutically effective amount of a composition, e.g., a hydrogel, powder, or capsule, as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. The composition, e.g., a hydrogel, capsule, or powder, can be implanted around the circumference of the disc, or implanted in the spine between an affected disc and an adjacent vertebra. The composition can be implanted by any suitable means, for example by injection using a suitable syringe or by surgical implantation.
A composition for surgical implantation can have a higher viscosity than a composition for injection, for example, can be solid or semi-solid (such as a plug or capsule). In some embodiments, e.g., e.g., where powder is used, MR images can be used to determine a approximate volume of degenerated disc (nucleus pulposus). The volume of CS or CSPG, e.g., the dry weight of CS powder needed, can be estimated by the volume of space (dry weight/volume of space).
As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with IVD degeneration. In some embodiments, the methods improve one or more symptoms of IVD degeneration, e.g., discogenic pain.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1. Augmented Chondroitin Sulfate Proteoglycan Has Therapeutic Potential for Intervertebral Disc Degeneration by Stimulating
Anabolic Turnover in Bovine Nucleus Pulposus Cells under Changes in Hydrostatic Pressure
It was hypothesized that augmentation of CSPG stimulates the anabolic capability of bNP cells under repetitive changes in HP at high osmolality. To test that hypothesis, we incubated isolated bNP cells with CSPG or HA under a repetitive regimen of cyclic HP at 0.2-0.7 MPa, 0.5 Hz for 2 days, followed by constant HP at 0.3 MPa for 1 day at high osmolality (450 mOsm/kg H2O) for up to 12 days, and compared the gene expression and immunohistology of metabolic markers in the bNP cells. We also sought to clarify the involvement of mechanoreceptor of transient receptor potential vanilloid-4 (TRPV4) activation in bNP-cell metabolism [19,20]
Materials and Methods
The following materials and methods were used in this Example.
Isolation and Pre-Culture of bNP Cells
Bovine tails (from cows 1-2 years old) were purchased from a local slaughterhouse certified by United States Department of Agriculture (USD A), and caudal NP tissues were harvested. The cows are skeletally mature, and their NP-cell phenotypes are relevant to human NP cells [34] Five tails were used for one set of experiments, which was repeated six times in = 6). The harvested bNP tissues were digested in 0.10% collagenase (CLS-1), dissolved in Ham’s F12 nutrient mixture (F- 12), and supplemented with 100-units/mL penicillin and 100 pg/mL streptomycin at 37°C overnight. Then bNP cells were collected, rinsed with Dulbecco’s phosphate- buffered saline (D-PBS), seeded onto 1.5% cell-culture-grade agarose-coated 6-well plates, and incubated in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 (1 : 1) supplemented with 10% fetal bovine serum (FBS) and 100-units/mL penicillin/100 pg/mL streptomycin at 37°C, 5% CO2 for 2-3 days. After this preincubation, bNP cells/clusters were collected with a pipette under a dissection microscope.
Cell Seeding
For the bNP tissue model, semipermeable membrane pouches were prepared as previously described [11] Briefly, hollow fiber tubing (1 mm in diameter, polyvinylidene fluoride, 500 kD molecular weight cut-off) was cut into 35 mm lengths, immersed in ethyl alcohol (200 proof) for 30 min, and autoclaved in D-PBS at 121°C for 15 min. After carefully collecting the bNP cells from each well in the 6-
well plate, 1.0 c 105 bNP cells (the DNA equivalent) with D-PBS as a no-material control, with 2 mg/mL CSPG (aggrecan from bovine articular cartilage), or with 10 mg/mL HA-based uncross-linked hydrogel (HyStem™), were injected into the pieces of tubing aseptically using a 200 pL pipette, and both ends of the tubing were closed with stainless steel clips (Figure 4A).
Culture Regimen of HP and Medium Osmolality /Incubation Conditions
The pouches were incubated under two different sets of culture conditions based on our previous study [10,11]: (1) no HP, in which pouches were placed in culture medium under atmospheric pressure; and (2) HP, in which 2-day cyclic HP (0.2-0.7 MPa, 0.5 Hz) followed by 1-day constant HP (0.3 MPa) was repeated 4 times over 12 days (Figure 4B). Both sets of conditions also included 37°C, 5% CO2, and 3% O2 to stimulate a physiologic hypoxic IVD environment [3,4] High-osmolality medium at 450 mOsm/kg H2O was made using 4.6-g/L sodium chloride and the osmolality was confirmed with a freeze-point osmometer (MICRO-OSMETTE™). Under no HP, the pouches were suspended within a stainless-steel mesh basket held in 100 mL medium with a stirrer to maintain sufficient mass transfer through the pouches. Under HP, the pouches were placed in a culture chamber filled with the medium to load cyclic or constant HP with medium replenishment at 0.1 mL/min using a pressure/perfusion culture system (TEP-2; Figure 4C). At 12 days, cell viability of the bNP cells/clusters within the pouches was validated with calcein-AM for live cells and ethidium homodimer-1 (EthDl) for dead cells (LIVE/DEAD™ Viability/Cytotoxicity Kit) according to the manufacturer’s instruction, which demonstrated predominant population of live cells (Figure 4D).
RNA Isolation and Real-Time Reverse Transcription Polymerase Chain Reaction (RT PCR)
The bNP cells/clusters were harvested at 3 and 12 days. Total RNA was extracted using the RNeasy® mini kit, and 0.3 pg RNA was reverse-transcribed with random primers (High-Capacity cDNA Reverse Transcription Kit). Messenger RNA (mRNA) expression levels of A can, Collaf Col2af Has2,Mmpl3 , and Timp2 relative to glyceraldehyde 3-phosphate dehydrogenase ( Gapdh ) as an endogenous control were evaluated in quadruplicate by real-time RT-PCR using TaqMan™ gene expression master mix and fluorescent-labeled specific primers. The
commercially available validated primers used were as follows: Acan, Bt03212189_ml; Collal, Bt03225358_gl; Col2al, Bt03251837_mH; Has2, Bt03212694_gl; wp73, Bt03214051_ml; Timp2 , Bt03231007_ml; Gapdh , Bt03210919_gl (TaqMan™ probes). Measurements were performed using the QuantStudio 5 Real-Time PCR System. Relative mRNA expression was analyzed by the 2 AACt method using ExpressionSuite Software vl.0.4 [35] The value of the no material control sample with D-PBS under no HP at 3 days was set at 1.0.
Immunohistology
At 3 and 12 days, the bNP cells/clusters were fixed with 2% paraformaldehyde/0.1 M cacodylate buffer (pH 7.4) at 4 °C, embedded in paraffin, and cut into 7 pm sections. Dewaxed sections were incubated with primary antibodies against KS (1:500), MMP13 (1:50), and TRPV4 (1:100) overnight. The sections were then rinsed and incubated with a biotinylated secondary antibody (VECTASTAIN® Elite ABC-HRP kit) for 30 min. The color was developed with 3,3'- diaminobenzidine and nickel (DAB substrate kit). Counterstaining was performed with Harris’s hematoxylin for KS, and with Contrast Red for MMP13 and TRPV4. The number of positive cells was counted in four random high-power fields (x400) using the ImageJ software (imagej.nih.gov/ij/, accessed on 4 October 2018). The positive cell percentage for MMP13 and TRPV4 was calculated relative to the total number of hematoxylin- or Contrast Red-positive cells.
Statistical Analysis
Data are expressed as box plots in the graphs. Multi-way repeated measures analysis of variance (ANOVA) with the Bonferroni post hoc test was used in real time RT-PCR (relative values to the no-material control sample under no HP at 3 days) and immunohistology. Pearson correlation analysis was performed to assess the correlation between Mmpl2 and Timp2 expression at 12 days. The / values < 0.05 were regarded as statistically significant using IBM SPSS Statistics 23.0 (IBM, Armonk, NY, USA).
Results
The Effects of Augmenting ECM on Metabolism in bNP Cells under HP We compared the expression of ECM-related genes in bNP cells augmented with CSPG or HA and incubated under HP loading or no HP. We also assessed the
histological characteristics of accumulated keratan sulfate (KS) as the specific glycosaminoglycan chain of aggrecan to support the gene expression of aggrecan core protein ( Acan ).
The gene expression of Acan in bNP cells with CSPG under no HP was significantly upregulated compared to the no-material control under no HP at 3 days (p = 0.03). Under HP, Acan expression with CSPG was significantly higher than the control (p = 0.007) at 12 days. In addition, Acan expression with CSPG at 12 days was also significantly greater under HP than no HP (p < 0.001). On the other hand, Acan expression with HA (augmented as another main ECM component) under HP was significantly downregulated compared to CSPG under HP at 12 days (p < 0.001) (Figure 1 A). The expression of collagen type II {CoUal) in bNP cells with CSPG under HP was significantly upregulated compared to no HP at 12 days (p = 0.02). With HA, however, CoUal expression was significantly lower than in the no-material control, with or without HP, at 12 days (p = 0.01 and p = 0.02, respectively) (Figure 1 A). The expression of collagen type I {Collal) in bNP cells with CSPG under no HP was significantly downregulated compared to the no-material control under no HP at 3 days (p = 0.02). With HA under HP, Collal expression was significantly higher at 12 days than 3 days (p = 0.006) (Figure 1A). The expression of hyaluronan synthase 2 ( Has2 ) in bNP cells with CSPG under HP was significantly upregulated compared to the no-material control under HP at 12 days (p = 0.002). TheHas2 expression with HA under HP was significantly lower than that of CSPG under HP at 12 days (p = 0.001) (Figure 1A).
Immunohistological staining showed KS accumulation within the bNP cells/clusters in the no-material control and CSPG, in which accumulation was particularly dense at 12 days. Meanwhile, relatively little accumulation of KS was found in the bNP cells/clusters with HA, which was denser at 12 days than at 3 days (Figure IB).
These results indicate that augmenting CSPG enhances anabolic turnover and suppresses gene expression of fibrotic molecules in bNP cells at early time points. In addition, CSPG is suggested to have a synergistic effect with dynamic HP on ECM synthesis at later time points (12 days). However, augmenting HA does not appear as beneficial as CSPG for anabolic turnover in bNP cells.
The Effects of Augmenting ECM on Catabolic Turnover in bNP Cells under HP
We compared the expression of catabolic and anti-catabolic genes in bNP cells augmented with CSPG or HA, as well as between the loading conditions. We also performed immunohistological staining against matrix metalloproteinase 13 (MMP13).
The gene expression oiMmpl3 in bNP cells was downregulated under HP compared to no HP in the control, with CSPG, and with HA both at 3 and 12 days, although only HA produced a statistically significant difference (3 days, » = 0.004; 12 days,/» < 0.001). The Mmp 13 expression with HA was significantly upregulated compared to the no-material control regardless of HP at each time point (no HP at 3 days,/» = 0.02; HP at 3 days,/» = 0.02; no HP at 12 days,/» = 0.001; HP at 12 days,/» = 0.03) and compared to CSPG (no HP at 3 days, p = 0.02; HP at 3 days, p = 0.02; no HP at 12 days,/» = 0.001; HP at 12 days,/» = 0.04) (Figure 2A). The expression of tissue inhibitor of metalloproteinases 2 ( Timp2 ) in bNP cells with CSPG under HP was significantly higher at 12 days than at 3 days (p = 0.01) (Figure 2A).
We performed a Pearson analysis to assess the correlation between Mmpl3 and Timp2 gene expression with each material at 12 days. The value in the no material control under no HP at 3 days was set as 1.0. The scatter plot demonstrated Mmpl3 suppression under HP compared to no HP with each material, while Timp2 expression did not differ between no HP and HP. In bNP cells with the control and CSPG, positive correlations were found between Mmp 13 and Timp2 under HP at 12 days ( R = 0.61 and A = 0.52, respectively) (Figure 2B).
Immunohistology showed MMP 13 to be denser within the bNP cells/clusters under no HP than HP in each material. In addition, bNP cells/clusters with HA showed denser MMP 13 than the no-material control and CSPG regardless of HP at each time point. The percentage of cells positive for MMP 13 was significantly higher under no HP than HP with all materials, both at 3 and 12 days (control, 3 days, p = 0.03; 12 days,/» = 0.001; CSPG, 3 days,/» = 0.03; 12 days,/» = 0.001; HA, 3 days,/»
= 0.03; 12 days,/» = 0.003), and significantly higher with HA than the control regardless of HP (no HP at 3 days,/» = 0.03; HP at 3 days,/» = 0.02; no HP at 12 days,/» = 0.02; HP at 12 days,/» = 0.004) and then CSPG under HP (3 days,/» = 0.04; 12 days,/» = 0.01) (Figure 2C).
These results demonstrate that repetitive changes in HP elicit catabolic Mmpl3 downregulation in the no-material control, with CSPG, and with HA and anti- catabolic Timp2 upregulation in response to slight MmpI3 upregulation with the no material control and CSPG. Conversely, HA augmentation promotes catabolic turnover regardless of the presence of HP.
Involvement of TRPV4 in the Effects of Augmenting ECM on bNP Cells under HP
The bNP cells were exposed to cyclic and constant HP applied using our hydrostatic pressure culture system. We stained bNP cells/clusters immunohistologically against TRPV4, a mechanosensitive calcium-permeable channel [19,20], in an attempt to clarify the possible mechanisms of HP effects on bNP cells.
Intense TRPV4 staining was found under HP at 3 days with all materials, localized at the surface of bNP cells/clusters; staining was weaker without HP; intense staining was diminished by 12 days. The percentage of cells positive for TRPV4 was significantly higher under HP than no HP with all materials both at 3 and 12 days (control, 3 days, » < 0.001; 12 days,/» = 0.03; CSPG, 3 days,/» < 0.001; 12 days,/» = 0.002; HA, 3 days,/» < 0.001; 12 days,/» = 0.02). Immunopositivity was lower at 12 days than 3 days under HP with all materials, although this difference reached statistical significance only with CSPG (p = 0.02) (Figure 3). These results indicate that the activation of TRPV4 is involved in the cellular responses to dynamic HP, especially at early phases of culture.
Effects of Augmenting ECM on bNP -Cell Metabolism
Several ECM-based materials, such as hyaluronan-based, alginate-based, and collagen -based ones, appear to stimulate IVD-cell metabolism, although their clinical and therapeutic strategies have not been fully addressed [13] We managed to shed light on the effects of augmenting CSPG and HA on metabolic turnover in bNP-cells under alternating cyclic and constant HP, mimicking diurnal spinal motion. As expected, we found that augmenting ECM specifically with CSPG promoted regenerative turnover in bNP cells through restoring the native ECM microenvironment. The significant Acan upregulation and Collal downregulation with CSPG under no HP at 3 days demonstrates the anabolic and anti-fibrotic effects
of CSPG early in incubation. Furthermore, the significant upregulation of Acan and CoUal with CSPG under HP at 12 days indicates the synergy of augmenting CSPG with dynamic HP at a later time point. The Has2 expression and Acan expression showed a similar tendency, which is consistent with the work of Holmes [21], Roughley [22], and Sivan [23] reporting the close association between HA and aggrecan turnover both in the articular cartilage and the IVD.
In addition, repetitive changes in HP also elicited catabolic Mmpl2 downregulation and anti-catabolic Timp2 upregulation in response to slight MmpI3 upregulation even without any material augmentation. Since disc-ECM catabolism indicated by MMPs is balanced by the inhibitory effects of TIMPs [24], dynamic HP stimulates anti -catabolic turnover of bNP cells and suppresses catabolic changes. In this study, HA inhibited ECM synthesis and strongly promoted catabolic turnover with or without HP. Augmenting HA has an anabolic and anti-inflammatory effect on human chondrocytes [25], and intra-articular administration of HA is an established treatment for such joint diseases as knee osteoarthritis [26] However, HAS activity appears to be an important modulator, and the anabolic and anti-catabolic effects of extracellular HA without activating HAS could be limited in vitro [27] Because the augmented HA used in our experiments is commercially available as a cell scaffold for in vitro cell culture, careful interpretation is required in evaluating the clinical relevance of our results.
The Relationship between the Augmented Materials and OP
The HP at physiological range (0.3-1.0 MPa) increases ECM synthesis in bovine, dog, and rabbit NP cells in vitro [28-30], while higher HP (>2.5 MPa) induced a catabolic trend, increasing MMP3 activity and decreasing matrix synthesis [31,32] We developed and applied a repetitive regimen of cyclic HP followed by constant HP at high osmolality to mimic physiological changes in intradiscal pressure and to reproduce the microenvironment in disc homeostasis [11]
The OP is generated by water content absorbed within the tissue [6,7] and influences bNP cell metabolism [10,11] Therefore, to reproduce the microenvironment in IVD homeostasis, we chose CSPG and HA in this study, both of which are capable of absorbing abundant interstitial fluid. Despite the hydrophilic properties of both materials, our results for bNP-cell anabolism and catabolism were opposite between these materials. Aggrecan, the primary CSPG in the IVD, generally
contains approximately 100 chains of chondroitin sulfate and 30 keratan sulfate chains [15,16] The negatively charged chondroitin sulfate chains contribute to the major function of aggrecan as a structural proteoglycan, holding large amounts of water in the ECM [15] The high OP, including the capability to swell and resist compressive loads, is generated by the hydrated chondroitin sulfate chains [8,23]; meanwhile, HA has no sulfate chains, which is unique and distinct from other glycosaminoglycans [17,33] We regard the presence of sulfate chains, generating OP, as the biggest factor in the different responses of the bNP cells between CSPG and HA in the current study. The augmented CSPG provides OP to the bNP cells and stimulates ECM synthesis early in culture, and then newly synthesized ECM would maintain OP and the microenvironment within the semi-permeable membrane pouch during later phases. The semi-confined pouch not only holds both augmented CSPG and synthesized ECM around the cells but also helps the generated OP directly affect the cells. Based on our findings, we consider ECM-associated OP, generated by augmented or synthesized ECM, a key player in NP cell metabolism.
HP Mechanotransduction via TRPV4
The TRPV4 staining was more intense with HP than without HP, both at 3 and 12 days; and it diminished by 12 days under HP although the difference was statistically significant only with CSPG. Thus, TRPV4 activation is implicated in transmitting the effects of HP to the bNP cell metabolism. Simultaneously, ECM was newly synthesized and accumulated around bNP cells over time, as shown by immunohistology against KS. The accumulated ECM likely altered membrane characteristics and subsequently decelerated TRPV4 activation. Although TRPV4- positive cell percentages were not significantly different among the groups, the accumulation of TRPV4 in HA was not as robust as in the control and CSPG, suggesting that the presence of HA might interrupt TRPV4 activation. This is another reason why we regard the physicochemical properties of HA as a possible cause of unfavorable response to dynamic HP, whereas CSPG did not diminish TRPV4 activation. We also hypothesize that dynamic HP stimulates metabolic turnover via TRPV4 activation early in incubation and involves another signal pathway with longer incubation due to ECM accumulation [19,20], which remains to be investigated in future studies.
Example 2. High Osmolality with Supplemented Chondroitin Sulfate Stimulates Anabolism and Suppresses Catabolism in Bovine Nucleus Pulposus Explants
It was hypothesized that physiologically balancing the osmotic pressure (OP) of NP explants by CSPGs further enhance anabolic turnover and prevent progression of degeneration due to biologically apart condition from the native NP environment.
We tested this hypothesis using bovine caudal NP explants in three different medium conditions; 290 mOsm/kg H2O (Low osmolality), 450 mOsm/kg H2O balanced by NaCl (High osmolality by NaCl), 450 mOsm/kg H2O balanced by CSPGs (High osmolality by CSPGs). We compared the gene expression and immunohistology of ECM metabolic markers in the bovine NP explants. In addition, to clarify the involvement of mechanotransduction by these OP difference, we evaluated transient receptor potential cation channel vanilloid-4 (TRPV4), which is a major mechanoreceptor in response to changes in osmolarity in the NP (36-38).
Materials and Methods
The following materials and methods were used in Example 2.
Preparation of Free Swelled Bovine Caudal NP Explants
Bovine tails (from 2- to 3-years-old cows) were purchased from a local slaughterhouse (Adams Farm, Athol, MA) certified by United States Department of Agriculture (USDA). Caudal NP tissues were aseptically harvested from the proximal two intact discs within 6 h after slaughter. The cows are skeletally mature, and their NP-cell phenotypes are relevant to human NP cells. Consequently, a total of 24 NP explants were obtained from 12 tails and five tails were used for each experiment. Each NP explant was cut into 6 pieces in the proximal two segments (approximately 30-40 mg) (Fig. 5A). To eliminate anatomic difference in cell metabolism and tissue composition, each different regions and levels was distributed randomly among the three different experimental conditions.
Incubation of NP Explants
NP explant pieces were suspended in a sterile nylon mesh bag (Cancer Diagnostics, Durham, NC) with a stainless wire frame (Fig. 5B) to avoid tissue deformation from the pieces contacting one another, then assigned to one of three culture medium conditions as follows: 1) Low osmolality medium (LOP,
Physiological 290 mOsm/kg H2O); 2) High osmolality medium prepared from NaCl (HOPNa, 450 mOsm/kg H2O); 3) High osmolarity prepared from CSPGs (HOPCS, 450 mOsm/kg H2O). LOP medium was prepared from Dulbecco’s Modified Eagle Medium/Ham’s F-12 nutrient mixture (1:1) (DMEM/F12, Gibco, Waltham, MA) supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin, and 100 pg/mL streptomycin (Gibco). HOPNa at 450 mOsm/kg H2O was prepared from supplemental sodium chloride 4.6 g/L into the LOP medium validated with a freezing point osmometer (MICRO-OSMETTE™, Precision System, Natick, MA). HOPCS medium was prepared from CSPG 65 mg/ml (C4384, Sigma- Aldrich, St. Louis, MO) validated with the osmometer. Since CSPG solution generates high viscosity interfering osmotic measurement, the HOPCS medium was diluted into 4 times. All media were sterilized using a filter (0.45 pm, Fisher Scientific). The NP explant pieces were incubated in defined OP of each culture medium for 6 days in 6-well plate at 37°C, 3% O2, 5% CO2 to stimulate a physiologic hypoxic NP environment. The explants were harvested at 3 and 6 days for molecular and immunohistological evaluation. Identical experiment was conducted six times.
Gene Expression
At 3 and 6 days, the NP explant pieces were harvested, minced, and digested in 2-mg/mL pronase (Merck Millipore, Burlington, MA) and 1-mg/mL hyaluronidase (Sigma- Aldrich) dissolved in DMEM/F12 on a vibration shaker at 37°C for 1 hour. Five ml of this enzyme cocktail was added to 100 mg of wet weight of the NP explant pieces. To stop enzyme activity, fetal bovine serum (0.5 ml per 5 ml of the enzyme cocktail) was added to the sample. The samples were centrifuged at 300xg- for 3 minutes and rinsed twice with Dulbecco’s phosphate-balanced saline (DPBS, Gibco). Then the samples were transferred to 1.5 mL tubes containing 1 mL TRIzol® reagent (Invitrogen, Carlsbad, CA). RNA isolation was conducted according to the manufacture’s protocol. Precipitation was performed with 0.25-mL isopropanol and 0.25 mL high salt precipitation solution. The amount and purity of isolated total RNA was measured using an optical densitometer (Nanodrop, ThermoFisher) and then 0.3- pg RNA was reverse-transcribed with random primers (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster City, CA). Messenger RNA (mRNA) expression levels of aggrecan core protein ( Acan ), collagen types I and II ( Collal and CoUal ), Brachyury, matrix metalloproteinase 13 ( Mmpl3 ), and tissue inhibitor of
metalloproteinases 2 ( Timp2 ) relative to glyceraldehyde 3 -phosphate dehydrogenase (' Gapdh ) as an endogenous control were quantified in quadruplicate by RT-qPCR using fluorescent-labeled specific primers and gene expression master mix (Applied Biosystems). The commercially available validated probes (TaqMan™, Life Technologym CA) were used as follows: Aeon , Bt03212189_ml; Collal , Bt03225358_gl; Col2al, Bt03251837_ml; Mmpl3, Bt03214051_ml; Timp2 , Bt03231007_ml; Gapdh , Bt03210919_gl. Relative quantity (RQ) of mRNA expression was analyzed using QuantStudio 5 Real-Time PCR Systems (Applied Biosystems) and ExpressionSuite Software vl.0.4 (Applied Biosystems) (39). The RQ value of the sample in low osmolarity control medium at 3 days was set as 1.0. Immunohistology and Lectin staining
At 3 and 6 days, the NP explants were harvested, fixed in 2% paraformaldehyde/10 mM PBS (pH 7.4) at 4°C, embedded in paraffin, and cut into 7- pm sections. The sections were dewaxed in xylene, rehydrated in graded concentrations of ethanol, and rinsed in PBS 3 times. For immunohistology, the sections were incubated in blocking solution, 3% normal horse serum (Vector Laboratories, Burlingame, CA) at room temperature for 20 min and then the sections were incubated with the primary antibodies at 4°C overnight. The chosen primary antibodies were collagen type II (COL2, 1:100, MBS397123, MyBiosource, San Diego, CA), collagen type I (COL1, 1:100, Abeam, Cambridge, MA), MMP13 (1:100, LS-B3168, LifeSpan BioScience, Seattle, WA), TRPV4 (1:100, LS-A8583, LifeSpan BioScience), and keratan sulfate (KS, 1:500, sc-73518, Santa Cruz Biotechnology, Santa Cruz, CA) for supporting gene expression results. Only for KS, the sections were incubated with 0.05 unit/ml chondroitinase ABC (Millipore-Sigma) at 37°C for 1 hour to expose epitopes. The section incubated with the first antibody were rinsed in PBS 3 times, then incubated in biotinylated secondary antibody solution at room temperature for 30 min followed by a manufacture’s instruction (VECTASTAIN® Elite ABC-HRP kit, Vector Laboratories). After rinsing the section with PBS 3 times, the sections were incubated with ABC solution at room temperature for 30 min. Lastly, the color was developed with 3,3’-diaminobenzidine and nickel (DAB substrate kit, SK4100, Vector Laboratory). Counterstaining was performed with Harris's hematoxylin (HHS16, Sigma- Aldrich) for KS and COL2, and with Contrast Red (5540-0001, Sera care, Milford, MA) for COL1, MMP13, and
TRPV4. The photomicrographs of each slide were acquired using a microscope (Zeiss) and digital camera (SPOT Imaging, Sterling Heights, MI).
Lectin labeling was performed as previously described (40-41) with slight modifications to capture glycosylation, indicating proteoglycan distribution. The dewaxed sections followed by rehydration were rinsed in 20 mM Tris-HCl buffered saline supplemented with 100 mM NaCl, 1 mM CaCh, and 1 mM MgCh (TBS+ buffer) at pH 7.2. The sections were then blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich) at room temperature for 1 hour to reduce non-specific binding. The sections were washed in TBS+ buffer 3 times and incubated with fluorescently- labelled-lectin (Wisteria floribunda lectin, FL-1351-2, Vector Laboratories) diluted in TBS+ (10 pg/mL, 1 :200) at room temperature for 1 h. The sections were rinsed in TBS+ buffer 3 times, then applied with SlowFade™ Gold Antifade mount with 4', 6- diamidino-2-phenylindole (DAPI) (Invitrogen, S36938). Lectin histochemical staining was captured using the Leica DMi8 microscope (Leica Camera, Wetzlar, Germany). From eight random middle-power fields (x200) of each slide, fluorescent area fraction was calculating by quantifying the positively stained matrix component divided by the total area and converting into a percentage (%) using the ImageJ software (imagej.nih.gov/ij/).
Statistical analysis
Data were expressed as mean ± SD in the text and the figures. Two-factorial ANOVA with the Bonferroni post-hoc test was used in real-time RT-PCR (relative values of the sample in low osmolarity at 3 days). The <0.05 were regarded as statistically significant using IBM SPSS Statistics 23.0 (IBM, Armonk, NY).
Results
Effects of altering osmotic pressure with chondroitin sulfate
We demonstrated the effects of hydrostatic pressure and osmotic pressure on anabolic turnover in bovine and human nucleus cells and tissues using the culture system described in Example 1. High osmotic pressure was achieved with supplemental sodium chloride (NaCl: 450 mOsm/kg H2O).
Chondroitin sulfate with high osmolality in culture medium was used for these experiments because there is no 450 mOsm osmolality fluid in the body. Chondroitin
sulfate can create high osmotic pressure locally in the body such as nucleus pulposus and cartilage.
As shown in FIGs. 6A, the osmolality in chondroitin sulfate dissolved in various concentration of sodium chloride solution increased dose dependent manner. FIG. 6B shows that adding chondroitin sulfate can alter the osmolality of a solution compared to other biological molecules e.g., bovine serum albumin. Thus, chondroitin sulfate can be injected into degenerated disc space instead of injecting other high osmotic fluids.
Effects of chondroitin sulfate-based high osmolality on metabolic turnover in bovine nucleus pulposus tissue
Normal nucleus pulposus tissues were harvested from bovine caudal intervertebral discs (Figs. 5A-B). The NP tissues were incubated in regular culture medium at physiological osmotic pressure (290 mOsm/kg H20), in modified culture medium supplemented with NaCl at high osmotic pressure (450 mOsm/H20), and in modified culture medium with supplemented with chondroitin sulfate at high osmotic pressure (450 mOsm/kg H20). The nucleus pulposus tissues were incubated for 6 days. The tissues were harvested for molecular evaluation using RT-qPCR (gene expression of key molecules) and immunohistology (with corresponding proteins).
The following were evaluated:
Anabolic molecules: Upregulation is desirable.
• Aggrecan: Acan
• Chondroitin sulfate N-acetylgalactosaminyltransferase 1 : Csgalnact 1
• Collagen type-2: CoUal
Undesired molecules: minimal expression or decline is desirable.
• Collagen type- 1: Collal
Catabolic molecules: suppression is desirable.
• Matrix metalloproteinase- 13 : Mmp-13
Desired molecules: Upregulation is desirable
• Tissue inhibitor for matrix metalloproteinase-2: Timp2
The results, presented in FIG. 7, showed that with chondroitin sulfate, Acan was significantly upregulated at 6 days. Csgalnact 1 showed the trend of upregulation at 6 days. CoUal showed the trend of upregulation at 6 days. Collal was significantly suppressed at 6 days. Mmpl3 was significantly declined at 6 days, and
Timp2 was significantly upregulated at 3 and 6 days. These results indicated that Chondroitin sulfate at high osmotic pressure (450 mOsm/kg H2O, normal in the NP) stimulates anabolic turnover, suppresses catabolic turnover and minimizes undesirable molecules. Human nucleus pulposus isolated from Pfirrmann grade 2-3 were incubated at high osmolality (450 mOsm/kg H20) for 6 days. With chondroitin sulfate, Acan gene expression showed the trend of greater upregulation compared to sodium chloride and low osmolality. The results (FIG. 8 A) showed that Acan was upregulated in the presence of chondroitin sulfate at high osmotic pressure (450 mOsm/kg H2O). Immunohistology was used to detect levels of keratan sulfate (representing
Acan ) and collagen type-2 (representing Col2al). The results, shown in FIGs. 9A-E, demonstrated denser accumulation of KS and Col-2 in NP with CS than that without CS.
In addition, Bovine nucleus pulposus were incubated with chondroitin sulfate at high osmolality (450 mOsm/kg H20) and with/without hydrostatic pressure at 0.5 MPa, 0.5 Hz for 6 days. With hydrostatic pressure, Acan gene expression showed the trend of greater upregulation compared to without pressure. The results are shown in FIG. 8B.
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OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Claims
1. A method of treating intervertebral disc (IVD) disease, the method comprising administering a therapeutically effective amount of a composition, preferably a hydrogel, comprising chondroitin sulfate (CS) and/or chondroitin sulfate proteoglycan (CSPG) at about 400-600 mOsm/kg H2O, preferably about 450 mOsm/kg H2O, to a subject in need thereof, wherein the composition is implanted in the subject at a site of intervertebral disc degeneration.
2. The method of claim 1, wherein the subject has spinal stenosis and instability, radiculopathy, myelopathy, and/or disc herniation.
3. The method of claim 1, wherein the composition further comprises one or more of: cartilaginous cells, therapeutic molecules, or nucleic acids.
4. The method of claim 3, wherein the cartilaginous cells are autologous or allogeneic chondrocytes or stem cell-derived chondrogenic cells.
5. The method of claim 3, wherein the therapeutic molecules are anti-inflammatory agents, preferably corticosteroids.
6. The method of claim 3, wherein the nucleic acids comprise mRNA or DNA encoding chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1).
7. A composition, preferably a hydrogel, comprising chondroitin sulfate (CS) and/or chondroitin sulfate proteoglycan (CSPG) at about 400-600 mOsm/kg H2O, preferably about 450 mOsm/kg H2O, for use in a method of treating intervertebral disc (IVD) disease in a subject in need thereof, wherein the composition is formulated to be implanted in the subject at a site of intervertebral disc degeneration.
8. The composition for the use of claim 7, wherein the subject has spinal stenosis and instability, radiculopathy, myelopathy, and/or disc herniation.
9. The composition for the use of claim 7, wherein the composition further comprises one or more of: cartilaginous cells, therapeutic molecules, or nucleic acids.
10. The composition for the use of claim 9, wherein the cartilaginous cells are autologous or allogenic chondrocytes or stem cell-derived chondrogenic cells.
11. The composition for the use of claim 9, wherein the therapeutic molecules are anti-inflammatory agents, preferably corticosteroids.
12. The composition for the use of claim 9, wherein the nucleic acids comprise mRNA or DNA encoding chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACTl).
13. A hydrogel comprising chondroitin sulfate (CS) and/or chondroitin sulfate proteoglycan (CSPG) at about 400-600 mOsm/kg H2O, preferably about 450 mOsm/kg H20, and optionally one or more of: cartilaginous cells, therapeutic molecules, or nucleic acids.
14. The hydrogel of claim 13, wherein the chondrocytes are autologous or allogenic chondrocytes or stem cell-derived chondrogenic cells.
15. The hydrogel of claim 13, wherein the therapeutic molecules are anti inflammatory agents, preferably corticosteroids.
16. The hydrogel of claim 13, wherein the nucleic acids comprise mRNA or DNA encoding chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACTl).
17. The hydrogel of claims 13-16, wherein the hydrogel is formulated to be implanted in the subject at a site of intervertebral disc degeneration.
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