Chitosan Glycerophosphate Research Articles

Intratympanic Diltiazem-Chitosan Hydrogel as an Otoprotectant Against Cisplatin-Induced Ototoxicity in a Mouse Model.

Local administration of the calcium-channel blocker (CCB), diltiazem, via intratympanic (IT) chitosan-glycerophosphate (CGP) hydrogel will protect against cisplatin-induced ototoxicity. Cisplatin induces calcium-mediated apoptosis of cochlear outer hair cells (OHCs). Previous work demonstrated otoprotection and reduced auditory brainstem response (ABR) threshold shifts in a cisplatin-induced ototoxicity mouse model treated with multiple doses of IT diltiazem given in solution. Here, we evaluated the role of a single dose of IT CGP-diltiazem as a novel otoprotectant against cisplatin-induced ototoxicity. Baseline pure-tone and click-evoked ABRs were performed in control (IT CGP-saline, n = 13) and treatment (IT CGP-diltiazem 2 mg/kg, n = 9) groups of female CBA/J mice. A single dose of IT CGP hydrogel was administered just before intraperitoneal injection of cisplatin (14 mg/kg). On Day 7 posttreatment, ABRs were performed and cochleae were harvested. Hair cells were quantified using anti-myosin VIIa immunostaining and inner hair cell ribbon synapses were quantified using Ctbp2 immunostaining. There was a statistically significant effect of treatment on click- and tone-evoked ABRs between groups. The mean threshold shifts were significantly reduced in both click- and tone-evoked ABRs on Day 7 in IT CGP-diltiazem treated mice compared with CGP-saline control mice. There were no significant differences in OHC counting between groups, but there appears to be an otoprotection against loss of synapses in the apical turn from IT CGP-diltiazem treated mice (p < 0.05). This preliminary work suggests that IT CGP-diltiazem reduces ABR threshold shifts with possible mechanisms of protecting ribbon synapses in the setting of cisplatin-induced ototoxicity. More work is necessary to determine the mechanism underlying this otoprotection.

A novel vehicle for local protein delivery to the inner ear: injectable and biodegradable thermosensitive hydrogel loaded with PLGA nanoparticles

Delivery of biomacromolecular drugs into the inner ear is challenging, mainly because of their inherent instability as well as physiological and anatomical barriers. Therefore, protein-friendly, hydrogel-based delivery systems following local administration are being developed for inner ear therapy. Herein, biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) containing interferon α-2 b (IFN α-2 b) were loaded in chitosan/glycerophosphate (CS/GP)-based thermosensitive hydrogel for IFN delivery by intratympanic injection. The injectable hydrogel possessed a physiological pH and formed semi-solid gel at 37 °C, with good swelling and deswelling properties. The CS/GP hydrogel could slowly degrade as visualized by scanning electron microscopy (SEM). The presence of NPs in CS/GP gel largely influenced in vitro drug release. In the guinea pig cochlea, a 1.5- to 3-fold increase in the drug exposure time of NPs-CS/GP was found than those of the solution, NPs and IFN-loaded hydrogel. Most importantly, a prolonged residence time was attained without obvious histological changes in the inner ear. This biodegradable, injectable, and thermosensitive NPs-CS/GP system may allow longer delivery of protein drugs to the inner ear, thus may be a potential novel vehicle for inner ear therapy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy &lt;em&gt;Versus&lt;/em&gt; Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures. Chitosan-glycerophosphate (CGP) and Ringer´s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear. The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

Rheological Study and Molecular Dynamics Simulation of Biopolymer Blend Thermogels of Tunable Strength.

The temperature-induced gelation of chitosan/glycerophosphate (Chs/GP) systems through physical interactions has shown great potential for various biomedical applications. In the present work, hydroxyethyl cellulose (HEC) was added to the thermosensitive Chs/GP solution to improve the mechanical strength and gel properties of the incipient Chs/HEC/GP gel in comparison with the Chs/GP hydrogel at body temperature. The physical features of the macromolecular complexes formed by the synergistic interaction between chitosan and hydroxyethyl cellulose in the presence of β-glycerophosphate disodium salt solution have been studied essentially from a rheological point of view. The temperature and time sweep rheological characterizations of the thermogelling systems revealed that the sol-gel transition temperature of the Chs/HEC/GP blends is equal to 37 °C at neutral pH; with increasing HEC content in the solutions, more compact networks with considerably improved gel strength are formed without influencing the gelation time. The formed hydrogel matrix has enough mechanical integrity and adequate strength for using it as injectable in situ forming matrices for biomedical applications. The classical Winter-Chambon (W-C) and Fredrickson-Larson (F-L) theories were applied to determine the gel point. In view of the obtained results, it is shown that the F-L theory can be employed as a robust and less tedious method than the W-C approach to precisely determine the gel point in these systems. At the end, molecular simulation studies were conducted by using ab initio quantum mechanics (QM) calculations carried out on Chs and HEC models, and molecular dynamics (MD) simulations of solvated Chs/HEC blend systems showed the binding behavior of Chs/HEC polymers. Analyses of interaction energy, radial distribution function, and hydrogen bonding from simulation studies strongly supported the experimental results; they all disclosed that hydrogen-bond formation between Chs moieties with regard to HEC chains plays an important role for the stabilization of the complexes.

Injectable thermosensitive chitosan/glycerophosphate-based hydrogels for tissue engineering and drug delivery applications: a review.

Recently, great attention has been paid to in situ gel-forming chitosan/glycerophosphate (CS/Gp) formulation due to its high biocompatibility with incorporated cells and medical agents, biodegradability and sharp thermosensitive gelation. CS/Gp is in liquid state at room temperature and after minimally invasive administration into the desired tissue, it forms a solid-like gel as a response to temperature increase. The overview of various recently patented strategies on injectable delivery systems indicates the significance of this formulation in biomedical applications. This thermosensitive hydrogel has a great potential as scaffold material in tissue engineering, due to its good biocompatibility, minimal immune reaction, high antibacterial nature, good adhesion to cells and the ability to be molded in various geometries. Moreover, CS/Gp hydrogel has been utilized as a smart drug delivery system to increase patient compliance by maintaining the drug level in the therapeutic window for a long time while avoiding the need for frequent injections of the therapeutic agent. This review paper highlights the recent patents and investigations on different formulations of CS/Gp hydrogels as tissue engineering scaffolds and carriers for therapeutic agents. Additionally, the dominant mechanism of sol-gel transition in those systems as well as their physicochemical properties and biocompatibility are discussed in detail.

Beneficial effects of biomimetic nano-sized hydroxyapatite/antibiotic gentamicin enriched chitosan–glycerophosphate hydrogel on the performance of injectable polymethylmethacrylate

Preparation and characterization and injectable p-PMMA/CS-GP/nano-HA/GM cements.

Preparation andin vitrocytocompatibility of chitosan-siloxane hybrid hydrogels

Injectable systems can be used in minimally invasive surgical applications. Although chitosan-glycerophosphate hydrogel systems are biodegradable and biocompatible, the long periods of time required for their effective gelation have severely limited their clinical application. The challenges currently facing researchers in this field are therefore focused on shortening the gelation time and biocompatibility of these materials to develop hydrogels suitable for clinical application. Chitosan and γ-glycidoxypropyltrimethoxysilane (GPTMS) hybrids have recently demonstrated good cytocompatibility with respect to human osteoblastic cells (MG63) and human bone marrow cells. Although these precursor sols could form gels under physiological conditions, they required neutralization with a sodium hydroxide solution. In this study, the chitosan-GPTMS hybrid systems were neutralized with glycerophosphate to prepare injectable hydrogels. The results revealed that the gelation time of the hydrogels could be controlled by the amount of GPTMS in the precursor sols. The in vitro cytocompatibility of the hydrogels were evaluated in terms of the proliferation of MG63 cells cultured either directly onto the hydrogels or indirectly onto the cell culture plate under a hydrogel insert. In the former case, the cells showed good attachment and proliferated for up to 7 days. Similar results were observed in the in direct culture. These results suggest that this new chitosan-GPTMS hydrogel could potentially be used as an injectable biomaterial in clinical applications.

A regulated delivery system for inner ear drug application

ObjectiveWe have recently developed a novel inner ear drug delivery system using chitosan glycerophosphate (CGP) hydrogel loaded with drugs commonly used for treatment of inner ear diseases, significantly improving the drugs' sustained delivery. The goal of this study is to evaluate the effectiveness of chitosanase as a “switch off” mechanism for this drug delivery system when side effects and potential ototoxicities appear during treatment. To evaluate this effect, we tested gentamicin (GENT) in the inner ear following CGP delivery with/without regulation. MethodsPurified chitosanase was obtained and used for regulating the CGP delivery system. In vitro studies were performed to evaluate the effect of the interaction between chitosanase and CGP-hydrogel loaded with GENT or Texas Red-labeled GENT (GTTR). In vivo studies were performed using our mouse model to investigate the regulatory effect of chitosanase application on the delivery of GENT to the inner ear. To assess the potential drug rerouting regulatory effect of chitosanase the GTTR fluorescence intensity was evaluated at the round window niche (RWN) and the Eustachian tube (ET). To further characterize this regulatory effect, GENT concentration in the perilymph of the inner ear was analyzed by chromatographic tandem mass spectrometry (LC–MS/MS), and the uptake in the inner ear cells was measured using fluorescence microscopy following CGP delivery with/without chitosanase application. ResultsThe chitosanase effectively digested the CGP-hydrogel, quickly releasing GENT and GTTR from the system in vitro. When reacted with GENT alone chitosanase did not produce any reducing sugars and did not affect GENT's antimicrobial activity. In vivo GTTR was effectively rerouted from the RWN to the ET, limiting its uptake in inner ear hair cells. Concurrent with these findings, GENT concentration in the inner ear perilymph was significantly decreased after chitosanase application. ConclusionOur study findings suggest that, for the first time, sustained and controlled inner ear drug delivery can be successfully regulated enhancing its translation potential for clinical application. The use of chitosanase to digest the CGP-hydrogel results in the rerouting of the loaded drug away from the RWN, effectively downregulating its delivery to the inner ear. This important modification to our drug delivery system has the ability to deliver therapy to the inner ear until desired effect is achieved and to stop this process when side effects or treatment-related ototoxicities start to occur, providing a novel and salient approach for safe and effective delivery to the inner ear.

Thermosensitive hydrogel made of ferulic acid-gelatin and chitosan glycerophosphate

Reactive oxygen species-induced oxidative stress is involved in apoptosis of nucleus pulposus (NP) cells that can alter cellular phenotype and accelerate disc degeneration. Ferulic acid (FA) possesses an excellent antioxidant and anti-inflammatory properties. In the study, we developed the thermosensitive FA–gelatin/chitosan/glycerol phosphate (FA–G/C/GP) hydrogel which was applied as a sustained release system of FA to treat NP cells from the damage caused by oxidative stress. The gelation temperature of the FA–G/C/GP hydrogel was 32.17°C. NP cells submitted to oxidative stress promoted by H2O2, and post-treated with FA–G/C/GP exhibited down-regulation of MMP-3 and up-regulation aggrecan and type II collagen in mRNA level. The sulfated-glycosaminoglycan production was increased and the apoptosis was inhibited in the post-treatment group. The results suggest that the thermosensitive FA–G/C/GP hydrogel can treat NP cells from the damage caused by oxidative stress and may apply in minimally invasive surgery for NP regeneration.

Regulated Chitosan‐Based Inner‐Ear Drug Delivery

Objective: 1) To evaluate a novel system for regulating the chitosan-glycero-phosphate (CGP)-based hydrogel drug delivery to the inner ear. 2) To understand the distribution of gentamicin in the inner ear following CGP drug delivery with and without regulation. Method: Chitosanase, specifically targeting CGP, was identified and purified for its use as a CGP regulation system. In vitro and in vivo studies were conducted to evaluate the effect of the chitosanase on the inner ear drug delivery and distribution. Gentamicin-Texas Red (GTTR) was used for tracking the drug distribution. Results: Purified chitosanase was confirmed with Western blot. The in vitro study showed that the chitosanase-based regulation system effectively controlled the gentamicin releasing. The in vivo data concur with in vitro findings and demonstrated that the gentamicin concentration in the inner ear can be successfully regulated by applying the chitosanase. Moreover, the gentamicin inner ear distribution can also be regulated by changing its inner ear concentration. Conclusion: Our study suggests that the chitosanase-based regulation system can be effectively used for controlled inner ear drug delivery. This system has a potential to stop inner ear drug application when side effects or drug-related toxicities start to occur and should be used for safe drug application in the inner ear.

Distribution of Gentamicin in Inner Ear after Local Administration via a Chitosan Glycerophosphate Hydrogel Delivery System

We examined the distribution of gentamicin sulfate in the inner ear after delivery via a chitosan glycerophosphate (CGP) hydrogel system and examined the change in morphology of the hair cells so as to determine how gentamicin affected the function of the inner ear. A matrix of CGP hydrogel loaded with gentamicin conjugated with Texas Red (GTTR) was injected into the round window niche of the left ear of C57/BL6 mice. The mice were painlessly killed on day 1 or day 7 after injection. Confocal fluorescence microscopy was used to locate the gentamicin in the cochlear and vestibular systems. In the vestibule, the intensity of GTTR staining in the hair cells of the macula of the saccule on day 1 was significantly stronger than that on day 7, and the number of hair cell bundles on top of the cuticular plate on day 7 was obviously decreased in comparison to that on day 1. In the cochlea, the intensity of GTTR staining in the basal turn was significantly stronger than that in the medial turn on both day 1 and day 7. Negligible fluorescence was observed in the apical turn on both day 1 and day 7. Less-intense GTTR staining was detected on day 7 than on day 1 in both the basal turn and the medial turn. There was some outer hair cell loss in the basal turn on day 7, and no hair cell loss in the medial and apical turns at any time point. Gentamicin is distributed in the inner ear via the CGP hydrogel delivery system in a time-dependent and basal-to-apical manner. This finding implies that the vestibule and the basal turn may retain more gentamicin for a longer period than do other sites in the inner ear. These two characteristics may account for the high-frequency hearing loss and vestibular dysfunction seen with use of this system to deliver gentamicin into the inner ear.

Preparation of alginate coated chitosan hydrogel beads by thermosensitive internal gelation technique

Chitosan hydrogel beads were successfully prepared by the method of thermosensitive internal gelation technique. The prepared beads were spherical, smooth-surfaced and non-aggregated with a diameter of 1.7–2.1 mm. The diameters of beads can be controlled and have a correlation with the initial drop size, the concentration of CaCl2, alginate and the time of solidification. The bead is comprised of three parts, which are chitosan/glycerophosphate (CS/GP) hydrogel core, chitosan-alginate (CS/SA) gel layer in the middle and calcium-alginate gelatin capsules in outer layer. Swelling studies indicate that the beads can be stable in simulated gastric fluid. But the beads shrink sharply when removed to simulated intestinal fluid. Drug release behavior showed that release of ornidazole in the beads is much slower than in the CS/GP hydrogel.

Antibiotic Eluting Chitosan Glycerophosphate Implant in the Setting of Acute Bacterial Sinusitis: A Rabbit Model

Topical therapy offers the potential for treatment of sinonasal disease with minimal systemic side effects. Chitosan glycerophosphate (CGP) is a mucoadhesive polymer that can be used as an antibiotic eluting sinonasal implant in the treatment of sinusitis. The purpose of this study was to assess the potential for CGP as an antibiotic impregnated implant in a rabbit model of acute bacterial sinusitis. The Institutional Animal Care and Use Committee approved study of acute bacterial sinusitis in 12 New Zealand white rabbits using either Pseudomonas aeruginosa (n = 6) or Staphylococcus aureus (n = 6). CGP impregnated with 50 mg of either gentamicin or vancomycin was bilaterally implanted in two rabbits in each arm, respectively. The sinuses were irrigated with saline for 4 days and the lavage was collected for colony-forming unit (CFU) determination. Within each group, the CFU log reduction in the lavage was compared with that of rabbits receiving saline alone (n = 2) or a daily 80-microgram/mL gentamicin or vancomycin irrigation (n = 2) and analyzed using a Student's t-test. Within the S. aureus group, the CFU log reduction using CGP + vancomycin (-2.57 +/- 0.21) was greater than vancomycin irrigation (-1.66 +/- 0.5; p = NS) and significantly greater than saline alone (2.46 +/- 0.97; p = 0.018). Within the P. aeruginosa group, the CFU log reduction using the CGP + gentamicin (-4.62 +/- 0.74) was greater than gentamicin irrigation (-4.09 +/- 0.70) and saline alone (-1.90 +/- 0.90); however, the results were not significant. In all rabbits receiving the CGP + antibiotic implant, no viable bacteria were present in the lavage by day 4. Placement of a single antibiotic impregnated CGP implant in the setting of an acute Gram-positive or Gram-negative bacterial sinusitis resulted in a greater log reduction of CFU than daily antibiotic irrigation and led to complete sterilization of the lavage within 4 days.

Effect of organic and inorganic acids on chitosan/glycerophosphate thermosensitive hydrogel

The influence of organic and inorganic acids on chitosan/glycerophosphate (CS/GP) hydrogel has been investigated by dissolving chitosan in different acids. The results of gelation showed that all of the chitosan dissolved in monovalent acid solutions (i.e., formic, acetic, propionic, butyric, isobutyric, lactic, nitric, hydrochloric, and chloroacetic acid), when neutralized by GP solution, could transform into hydrogel after 2–5 min at 37 °C, while those dissolved in multivalent acids failed in gel formation. The inner structures of CS/GP hydrogels prepared with monovalent acids depended on the ionic strength and chain length of acids. Morphological examination by scanning electron microphotography demonstrated that large pores occurred during the gel-forming process, and the aperture size was also related to different acids. The cytotoxicity studies indicated that CS/GP systems prepared by dissolving chitosan in tested acids except chloroacetic acid were nontoxic to mouse embryonic fibroblasts and Hela cells.

Chitosan Glycerophosphate-Based Semirigid Dexamethasone Eluting Biodegradable Stent

Chitosan is a polysaccharide derived from chitin deacetylation, which can form a biodegradable matrix capable of reversibly binding dexamethasone. The purpose of this study was to optimize the chitosan formulation to produce a pliable sheet, to assess the innate longevity and inflammatory potential of the polymer, and to study the in vivo rate of dexamethasone release over time. Chitosan glycerophosphate (CGP) sheets were produced with varying degrees of deacetylation (70-100%) and analyzed for structural integrity. Two-squared centimeter sheets of 91.7% deacetylated CGP were mixed with dexamethasone and implanted in 12 rabbit maxillary sinuses. Nasal lavage and peripheral blood samples were tested for dexamethasone levels by enzyme-linked immunosorbent assay (ELISA) over 15 days. Sinuses were examined histologically on postoperative days 3, 7, and 15 for persistence of the stent and degree of inflammation when compared with CGP alone. The 91.7% deacetylated CGP formulation was found to have optimal mechanical properties and remained present with moderate degradation and negligible inflammation through postoperative day 15. Dexamethasone levels were detectable in nasal lavage and blood samples through postoperative day 15 and decayed over time (lavage: day 0, 7.70 +/- 0.97 ng/mL, and day 15, 2.53 +/- 1.71 ng/mL; blood: day 3, 2.51 +/- 0.14 ng/mL, and day 15, 1.70 +/- 0.36 ng/mL). CGP may be used to create a semirigid sheet that is malleable, inert, and capable of eluting steroid over 15 days when implanted intranasally. This material may be used to create a pharmacologically active stent that spontaneously degrades over time.

Molecular level and microstructural characterisation of thermally sensitive chitosan hydrogels

Chitosan–glycerophosphate (GP) is a physiological pH solution at room temperature which forms a physical hydrogel upon heating to 37 °C. The hydrogel is suitable for biomedical applications and in particular as a biodegradable scaffold for tissue engineering. Since the structure of the hydrogel is critical to cell–material interactions, small angle neutron scattering (SANS) and ultra SANS (USANS) were used to examine the molecular conformation of chitosan chains and the larger scale microstructure. On the nanoscale, the hydrogel is described in terms of a static component (characteristic length, Ξ) which accounts for the physical cross-links, and a dynamic component (correlation length, ξ) which accounts for solution-like properties. Over the range 0.25–1.5 w/v% chitosan concentration the molecular structure is dependent on concentration with the characteristic length decreasing from ∼450 Å to ∼300 Å, and the correlation length increasing from ∼110 Å to ∼130 Å. The chitosan chains were arranged at a larger scale into polymer-rich aggregates of 1–2 µm diameter which decreased in size as the chitosan concentration increased. Such structural information is important for tailoring the hydrogel for specific applications.

A Novel Controlled Local Drug Delivery System for Inner Ear Disease

Our goal is to develop a novel drug delivery system that can potentially improve clinical outcomes compared to current methods of dosing drugs such as dexamethasone or gentamicin. This system focuses on a single local application to the inner ear via the round window membrane. A chitosan-glycerophosphate (CGP)-hydrogel based drug delivery system can be engineered to provide local and sustained drug release to the inner ear. In vitro: drug release and (CGP)-hydrogel matrix degradation were characterized using dexamethasone as a model drug. In vivo: dexamethasone laden CGP-hydrogel was placed in the round window niche of mice. Perilymph samples were obtained from the oval window and analyzed for dexamethasone. The impact of CGP-hydrogel on auditory function was evaluated. In vitro: A CGP-hydrogel was designed to release 92% of the dexamethasone load over 4 consecutive days with concurrent degradation of the hydrogel matrix. In vivo: After surgical placement of CGP-hydrogel to the round window niche, we detected elevated levels of dexamethasone in perilymph for 5 days. Auditory function testing revealed a temporary hearing loss in the immediate postoperative period, which resolved by the 10th postoperative day. We report the development of CGP-hydrogel, a biodegradable matrix that achieves local, sustained delivery of dexamethasone to the inner ear. There were no significant complications resulting from the surgical procedure or the administration of CGP-hydrogel to our murine model.

Human mesenchymal stem cell differentiation to NP-like cells in chitosan–glycerophosphate hydrogels

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, interest in methods focused on biological repair has increased. Several tissue engineering approaches using different cell types and hydrogels/scaffolds have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering attention has focused on the use of mesenchymal stem cells (MSCs). Additionally, while rigid scaffolds have been demonstrated to allow MSC differentiation to the chondrocyte-like cells of the IVD, hydrogels are being increasingly studied as they allow minimally invasive implantation without extensive damage to the IVD. Here, we have studied the temperature-sensitive hydrogel chitosan–glycerophosphate (C/Gp), seeded with human MSCs and cultured for 4 weeks in standard medium. We have analysed the gene and protein expression profile of the MSCs and compared it to that of both nucleus pulposus (NP) cells and articular chondrocytes cultured in C/Gp. Gene expression analysis for chondrocytic-cell marker genes demonstrated differentiation of MSCs to a phenotype which showed similarities to both articular chondrocytes and NP cells. Conventional PCR demonstrated a lack of expression of osteogenic marker genes and the hypertrophic marker gene type X collagen. MSCs also secreted both proteoglycans and collagens in a ratio, which more closely resembled that of NP cells than articular chondrocytes. These results therefore suggest that MSC-seeded C/Gp gels could be used clinically for the regeneration of the degenerate human IVD.