Hyaluronic Hydrogel Research Articles

Charge Matters: Modulating Secondary Interactions in Hyaluronan Hydrogels

Hyaluronic acid (HA) is a vital, functional component of the extracellular matrix (ECM). Following a synthetic biology approach, we designed polyelectrolyte hydrogels composed of HA as a polymeric backbone interconnected by short covalent crosslinkers. Utilizing a thiol-Michael addition, defined network structures are created, which can be modulated by the aromatic core and charge of the crosslinkers. With increasing negative network charge, varied both through the charge of the crosslinker and the degree of functionalization of the HA chain, the Young's modulus of the hydrogels decreases linearly as swelling ratios increase. Interestingly, secondary interactions with aromatic crosslinkers are stronger for elastin inspired crosslinkers with a pyridinium core compared to analogous triazolium crosslinkers. Based on the defined covalent interconnectivity of these HA-hydrogels, we were able to demonstrate the specific electrostatic and aromatic interactions induced by different short crosslinkers.

Intraperitoneal Delivery of Cisplatin via a Hyaluronan-Based Nanogel/in Situ Cross-Linkable Hydrogel Hybrid System for Peritoneal Dissemination of Gastric Cancer.

Intraperitoneal administration of chemotherapeutics is expected for the treatment of peritoneally disseminated gastric cancer because of poor migration of the drugs from the systemic circulation to the peritoneal cavity. In this study, for intraperitoneal delivery of cisplatin (CDDP), we developed a hyaluronan (HA)-based hybrid system in which CDDP-loaded HA nanogels were either physically encapsulated in or chemically conjugated to injectable HA hydrogels. Physical encapsulation enabled sustained release of HA nanogels from the HA hydrogel matrix for over a week. This was a longer release period than that of encapsulated free CDDP, which released 80% of the drug in 2 days. The longer release was attributed to delayed diffusion of HA nanogels from the hydrogel matrix network. The release profile could be tuned by modifying the chemical conjugation of HA nanogels to the HA hydrogel matrix, as well as the type of chelating ligands used to load CDDP to the nanogel. Furthermore, intraperitoneally administered hybrid had significant antitumor activity in a mouse model of peritoneally disseminated gastric cancer, especially for nodules smaller than 1.0 mm.

Abstract 5779: Building a novel 3D hyaluronan-based tri-culture model of the bone metastatic microenvironment for cancer studies

Abstract The high attrition rate of potential anti-cancer drugs entering clinical trials predicates a need for more predictive pre-clinical model systems. Three-dimensional (3D) culture systems provide cells a more physiological microenvironment than traditional cell culture methods—including relevant extracellular matrix (ECM) cues and cell-cell interactions—while allowing greater experimental control and higher-throughput studies than animal models. Prevalent cancers, including breast and prostate cancer, metastasize preferentially to bone, where they become incurable. The bone marrow microenvironment is a complex network of interacting bone homeostatic and hematopoietic niches that contribute to osteotropic cancer homing, proliferation, and treatment resistance. We created a 3D in vitro tri-culture model of the bone metastatic microenvironment to serve as a new platform to study the “trialogue” between bone metastatic cancer, bone marrow stroma, and bone marrow endothelial cells. This tool can be used to inform drug discovery and to combat treatment resistance. Within the present study, the utility of peptide-functionalized hyaluronan hydrogels for supporting the self-organization of 3D bone marrow microvascular networks was explored. Gel crosslinking density was varied to examine the effect of matrix porosity on cell migration and organization over time. Tubule length and diameter were quantified and the endothelial structures were assessed for indicators of mature vasculature, including lumen formation, basement membrane production, and the recruitment of supporting stromal cells. Breast/prostate cancer cell growth, survival, and physical association with bone marrow stromal and bone marrow microvascular endothelial cell networks was examined in tri-culture models. Ongoing studies seek to identify and disrupt key cell signaling pathways that mediate cell-cell interaction and promote cancer cell proliferation and survival, providing new tools to combat bone metastatic disease. Citation Format: Lindsey K. Sablatura, Daniel A. Harrington, Mary C. Farach-Carson. Building a novel 3D hyaluronan-based tri-culture model of the bone metastatic microenvironment for cancer studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5779. doi:10.1158/1538-7445.AM2017-5779

Bioimaging of botulinum toxin and hyaluronate hydrogels using zwitterionic near-infrared fluorophores

BackgroundThe injection of botulinum toxin (BTX) to reduce facial wrinkles is one of the most frequently performed plastic surgery procedures. The biocompatible hydrogels are injected with BTX for effective tissue augmentation. However, it is difficult to determine the interval of injection for effective tissue augmentation.MethodBTX and hyaluronate (HA) hydrogels were labeled with zwitterionic (ZW) near-infrared (NIR) fluorophores and visualized for 3 weeks after injection to BALB/c nude mice.ResultsBTX-ZW conjugates and diaminohexane (DAH)-HA-ZW hydrogels were successfully prepared by the conventional EDC/NHS chemistry. Using the NIR fluorescence imaging, we confirmed that approximately 10% of BTX-ZW conjugates and 50% of DAH-HA-ZW hydrogels remained 3 weeks post-injection.ConclusionThis bioimaging technique using invisible NIR fluorescence light can be exploited for various biomedical applications.

Hyaluronan hydrogels with a low degree of modification as scaffolds for cartilage engineering

In the field of cartilage engineering, continuing efforts have focused on fabricating scaffolds that favor maintenance of the chondrocytic phenotype and matrix formation, in addition to providing a permeable, hydrated, microporous structure and mechanical support. The potential of hyaluronan-based hydrogels has been well established, but the ideal matrix remains to be developed.This study describes the development of hyaluronan sponges-based scaffolds obtained by lysine methyl-ester crosslinking. The reaction conditions are optimized with minimal chemical modifications to obtain materials that closely resemble elements in physiological cellular environments. Three hydrogels with different amounts of crosslinkers were produced that show morphological, water-uptake, mechanical, and stability properties comparable or superior to those of currently available hyaluronan-scaffolds, but with significantly fewer hyaluronan modifications. Primary human chondrocytes cultured with the most promising hydrogel were viable and maintained lineage identity for 3 weeks. They also secreted cartilage-specific matrix proteins. These scaffolds represent promising candidates for cartilage engineering.

The stiffness of a crosslinked hyaluronan hydrogel affects its chondro-induction activity on hADSCs.

Matrix stiffness plays an important role in stem cell differentiation. This study reports the synthesis of methacrylated hyaluronan (MeHA) with different degrees of methacrylation, ranging from 15 to 140% per disaccharide unit, which corresponds to a matrix stiffness ranging from 1.5 to 8 KPa. The swelling ratio was inversely proportional to the matrix stiffness, but the water content remained constant at >97% of the hydrogel mass. A fibril-like surface morphology and larger pore size were observed in lyophilized MeHA hydrogel with a lower stiffness. The matrix stiffness also affected the degradability of the MeHA hydrogel, where softer MeHA hydrogels (MeHA15 and MeHA30 ) were completely degraded within 6 days and a stiffer MeHA hydrogel (MeHA140 ) was able to retain ∼25% of its initial mass after 30 days. Subsequently, the crosslinked MeHA hydrogel was used as a scaffold to encapsulate human adipose-derived stem cells (hADSCs). The embedded cells remained viable and expressed ∼11-fold higher levels of aggrecan and 42-fold higher levels of collagen type II in MeHA140 compared with ADSCs cultured in HA-coated wells. In addition, cells grown in MeHA140 exhibited the highest rates of glycosaminoglycan and collagen type II synthesis of ∼5 ng/DNA and 0.4 ng/DNA, respectively. Immunofluorescence staining showed an increase of collagen type II synthesis in MeHA65 , MeHA85 and MeHA140 . This study showed that the matrix stiffness of a hydrogel can be modulated by the degree of methacrylation, thus affecting the efficacy of chondrogenesis in hADSCs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 808-816, 2018.

Efficacy of two different thiol-modified crosslinked hyaluronate formulations as vitreous replacement compared to silicone oil in a model of retinal detachment.

The efficacy of two novel artificial vitreous body substitutes (VBS) consisting of highly biocompatible thiolated cross-linked hyaluronic acid (HA)-based hydrogels in comparison to silicone oil in a model of retinal detachment was investigated. Pars plana vitrectomy (23G) was performed in the right eye of 24 pigmented rabbits. Retinal detachment of two quadrants was induced by creating a small retinotomy near the vascular arcade and injecting balanced salt solution (BSS) subretinally. The retina was reattached by injecting air, which was followed by increasing the infusion pressure, and the retinal tear was treated by endolaser photocoagulation. At the end of the procedure, the eye was filled either with 5000-cs silicone oil (after fluid air exchange) or the respective hydrogel (with two different viscosities). Follow-up examination included slit lamp examination, funduscopy, intraocular pressure measurements (IOP), optical coherence tomography (OCT) and electroretinogram (ERG) measurements. After a maximum follow-up of four weeks both eyes were removed, examined macroscopically, photographed, and prepared for histology. Of the eight rabbits that received silicone oil, seven (87.5%) developed a recurrent retinal detachment with pronounced proliferative vitreoretinopathy within the first two weeks after surgery. In contrast, in the hydrogel treated eyes, the retina stayed attached in the majority of the cases (73.3%). IOP and retinal morphology were normal as long as the retina remained re-attached. In conclusions, this model of retinal detachment, both thiolated crosslinked hyaluronate hydrogels showed superior efficacy when compared to silicone oil. These hydrogels have a promising potential as novel vitreous body substitutes.

A hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo expansion of human corneal epithelial stem cells.

PurposeTo develop a hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo cultivation of human corneal epithelial stem cells (CESCs).Patients and MethodsCESCs were cultivated from donor limbal explants on the HyStem-C Hydrogel bio-scaffold in 12-well plates for 3 weeks. Group A used the traditional supplemented hormonal epidermal medium (SHEM) and group B used the defined SHEM (without fetal bovine serum and toxin A, adding 20% serum replacement). The growth and morphology of the cultured cells were assessed by phase contrast microscope. The expressions of specific cell markers were assessed by immunofluorescence staining and quantitative real-time PCR (qRT-PCR).ResultsSuccessful cultures of CESCs were obtained in both groups, resulting in multilayered stratified epithelia. Comparing to group A, the cells in group B was grown slightly slower and formed less cellular layers at the end of culture. The corneal specific cytokeratin (K) 12 and differentiation markers, involucrin, and connexin 43, were mainly expressed in the superficial cellular layers in both groups. Interestingly, certain basal cells were immune-positive to proposed stem cell markers such as K19, ABCG2, and integrin β1 in both groups. There was no significant difference between the two groups with regard to the gene expression levels of all these selected corneal markers (all P>0.05).ConclusionsThe hyaluronan hydrogel scaffold-based xeno-free culture system may support the expansion of regenerative CESCs without the risk of xeno component contamination. The regenerated epithelium maintains similar characteristics of native corneal epithelium.

Photocrosslinked tyramine-substituted hyaluronate hydrogels with tunable mechanical properties improve immediate tissue-hydrogel interfacial strength in articular cartilage

Articular cartilage lacks the ability to self-repair and a permanent solution for cartilage repair remains elusive. Hydrogel implantation is a promising technique for cartilage repair; however for the technique to be successful hydrogels must interface with the surrounding tissue. The objective of this study was to investigate the tunability of mechanical properties in a hydrogel system using a phenol-substituted polymer, tyramine-substituted hyaluronate (TA-HA), and to determine if the hydrogels could form an interface with cartilage. We hypothesized that tyramine moieties on hyaluronate could crosslink to aromatic amino acids in the cartilage extracellular matrix. Ultraviolet (UV) light and a riboflavin photosensitizer were used to create a hydrogel by tyramine self-crosslinking. The gel mechanical properties were tuned by varying riboflavin concentration, TA-HA concentration, and UV exposure time. Hydrogels formed with a minimum of 2.5 min of UV exposure. The compressive modulus varied from 5 to 16 kPa. Fluorescence spectroscopy analysis found differences in dityramine content. Cyanine-3 labelled tyramide reactivity at the surface of cartilage was dependent on the presence of riboflavin and UV exposure time. Hydrogels fabricated within articular cartilage defects had increasing peak interfacial shear stress at the cartilage-hydrogel interface with increasing UV exposure time, reaching a maximum shear stress 3.5× greater than a press-fit control. Our results found that phenol-substituted polymer/riboflavin systems can be used to fabricate hydrogels with tunable mechanical properties and can interface with the surface tissue, such as articular cartilage.

Bone and cartilage differentiation of a single stem cell population driven by material interface

Adult stem cells, such as mesenchymal stem cells, are a multipotent cell source able to differentiate towards multiple cell types. While used widely in tissue engineering and biomaterials research, they present inherent donor variability and functionalities. In addition, their potential to form multiple tissues is rarely exploited. Here, we combine an osteogenic nanotopography and a chondrogenic hyaluronan hydrogel with the hypothesis that we can make a complex tissue from a single multipotent cell source with the exemplar of creating a three-dimensional bone–cartilage boundary environment. Marrow stromal cells were seeded onto the topographical surface and the temperature gelling hydrogel laid on top. Cells that remained on the nanotopography spread and formed osteoblast-like cells, while those that were seeded into or migrated into the gel remained rounded and expressed chondrogenic markers. This novel, simple interfacial environment provides a platform for anisotropic differentiation of cells from a single source, which could ultimately be exploited to sort osteogenic and chondrogenic progenitor cells from a marrow stromal cell population and to develop a tissue engineered interface.

Fabrication of cell-compatible hyaluronan hydrogels with a wide range of biophysical properties through high tyramine functionalization.

Enzymatically mediated crosslinked hyaluronan-tyramine hydrogels (HA-Tyr) are promising matrices for tissue engineering and regenerative medicine. However, due to relatively low tyramine modifications of the hyaluronan backbone achieved, HA-Tyr matrices have a weak and narrow range of mechanical properties. The iterative use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling agent increased the yields of tyramine functionalization, which was reflected by a two-fold increase in Young's modulus of HA-Tyr hydrogels. The accurate control over hydrogel degradation was also facilitated. Viable encapsulation of human mesenchymal stem cells, with 85-98% over 6 days, was achieved in all hydrogels and distinct cellular spreading was observed in the absence of additional binding cues. The biophysical properties of the tunable HA-Tyr hydrogels are improved to study a wide range of cellular behaviors.

Factor XIII Cross-Linked Hyaluronan Hydrogels for Cartilage Tissue Engineering.

In this study, transglutaminase-cross-linked hyaluronan (HA-TG) hydrogels are investigated for their potential to treat cartilage lesions. We show the hydrogels fulfill key requirements: they are simultaneously injectable, fast-gelling, biocompatible with encapsulated cells, mitogenic, chondroinductive, and form a stable and strongly adhesive bond to native cartilage. Human chondroprogenitors encapsulated in HA-TG gels simultaneously show good growth and chondrogenesis. Strikingly, within soft gels (∼1 kPa), chondroprogenitors proliferate and deposit extracellular matrix to the extent that the hydrogels reach a modulus (∼0.3 MPa) approaching that of native cartilage (∼1 MPa) within 3 weeks. The combination of such an off-the-shelf human chondroprogenitor cell source with HA-TG hydrogels lays the foundation for a cell-based treatment for cartilage lesions which is based on a minimally invasive one-step procedure, with improved reproducibility due to the defined cells and with improved integration with the surrounding tissue due to the new hydrogel chemistry.

Synthesis and characterization of a dually crosslinked heparin-conjugated HA hydrogel for BMP-2 sustained delivery

ABSTRACTA dually crosslinked heparin-conjugated hyaluronan hydrogel (DCH) was synthesized for reinforcement and sustainably delivering growth factors in tissue engineering. Heparin-conjugated hyaluronan microgels after glycidyl methacrylation as reinforcing phase crosslinked with glycidyl-methacrylated hyaluronan as matrix phase by ultraviolet radiation. The morphology, water swelling ratio, rheological property, degradation, bone morphogenetic protein-2 (BMP-2) loading and delivery, and cytotoxicity were characterized. Increasing heparin content enhanced the loading and sustainable delivery of BMP-2, but partly attenuated the mechanical property of DCH that presented a much higher elastic modulus than bulk gel. The incorporation of heparin and dually crosslinked network was harmless to the good cytocompatibility of hyaluronan hydrogel.

Novel enzymatically cross-linked hyaluronan hydrogels support the formation of 3D neuronal networks

Hyaluronan (HA) is an essential component of the central nervous system's extracellular matrix and its high molecular weight (MW) form has anti-inflammatory and anti-fibrotic properties relevant for regenerative medicine. Here, we introduce a new hydrogel based on high MW HA which is cross-linked using the transglutaminase (TG) activity of the activated blood coagulation factor XIII (FXIIIa). These HA-TG gels have significant advantages for neural tissue engineering compared to previous HA gels. Due to their chemical inertness in the absence of FXIIIa, the material can be stored long-term, is stable in solution, and shows no cytotoxicity. The gelation is completely cell-friendly due to the specificity of the enzyme and the gelation rate can be tuned from seconds to hours at physiological pH and independently of stiffness. The gels are injectable, and attach covalently to fibrinogen and fibrin, two common bioactive components in in vitro tissue engineering, as well as proteins present in vivo, allowing the gels to covalently bind to brain or spinal cord defects. These optimal chemical and bioactive properties of HA-TG gels enabled the formation of 3D neuronal cultures of unprecedented performance, showing fast neurite outgrowth, axonal and dendritic speciation, strong synaptic connectivity in 3D networks, and rapidly-occurring and long-lasting coordinated electrical activity.

Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds.

Advanced tissue engineered heart valves must be constructed from multiple materials to better mimic the heterogeneity found in the native valve. The trilayered structure of aortic valves provides the ability to open and close consistently over a full human lifetime, with each layer performing specific mechanical functions. The middle spongiosa layer consists primarily of proteoglycans and glycosaminoglycans, providing lubrication and dampening functions as the valve leaflet flexes open and closed. In this study, hyaluronan hydrogels were tuned to perform the mechanical functions of the spongiosa layer, provide a biomimetic scaffold in which valve cells were encapsulated in 3D for tissue engineering applications, and gain insight into how valve cells maintain hyaluronan homeostasis within heart valves. Expression of the HAS1 isoform of hyaluronan synthase was significantly higher in hyaluronan hydrogels compared to blank-slate poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Hyaluronidase and matrix metalloproteinase enzyme activity was similar between hyaluronan and PEGDA hydrogels, even though these scaffold materials were each specifically susceptible to degradation by different enzyme types. KIAA1199 was expressed by valve cells and may play a role in the regulation of hyaluronan in heart valves. Cross-linked hyaluronan hydrogels maintained healthy phenotype of valve cells in 3D culture and were tuned to approximate the mechanical properties of the valve spongiosa layer. Therefore, hyaluronan can be used as an appropriate material for the spongiosa layer of a proposed laminate tissue engineered heart valve scaffold.

Promotion of In Vitro Chondrogenesis of Mesenchymal Stem Cells Using In Situ Hyaluronic Hydrogel Functionalized with Rod-Like Viral Nanoparticles.

This study focuses on the development of injectable hydrogels to mimic the cartilage microenvironment using hyaluronic acid (HA) derivatives as starting materials. Cysteine-inserted Tobacco mosaic virus (TMV) mutants (TMV1cys) could be cross-linked to methacrylated hyaluronic acid (MeHA) polymers by thiol-ene "click" chemistry and form hydrogels under physiological condition. The resulting hydrogels could promote in vitro chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) significantly higher than that in the TMV-free HA hydrogels by upregulating bone morphogenetic protein-2 (BMP-2) expression and enhancing collagen accumulation.

Evaluation of an injectable thermoresponsive hyaluronan hydrogel in a rabbit osteochondral defect model.

Articular cartilage displays very little self-healing capabilities, generating a major clinical need. Here, we introduce a thermoresponsive hyaluronan hydrogel for cartilage repair obtained by covalently grafting poly(N-isopropylacrylamide) to hyaluronan, to give a brush co-polymer HpN. The gel is fluid at room temperature and becomes gel at body temperature. In this pilot study HpN safety and repair response were evaluated in an osteochondral defect model in rabbit. Follow-up was of 1 week and 12 weeks and the empty defect served as a control, for a total of four experimental groups. At 12 weeks the defect sites were evaluated macroscopically and histologically. Local lymph nodes, spleen, liver, and kidneys were analyzed for histopathological evaluation. HpN could be easily injected and remained into the defect throughout the study. The macroscopic score was statistically superior for HpN versus empty. Histological score gave opposite trend but not statistically significant. A slight tissue reaction was observed around HpN, however, vascularization and subchondral bone formation were not impeded. An upper proteoglycans rich fibro-cartilaginous tissue with fairly good continuity and lateral integration into the existing articular cartilage was observed in all cases. No signs of local or systemic acute or subacute toxicity were observed. In conclusion, HpN is easily injectable, remains into an osteochondral defect within a moving synovial joint, is biocompatible and does not interfere with the intrinsic healing response of osteochondral defects in a rabbit model. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1469-1478, 2016.

Abstract B30: Ex vivo culture of breast cancer patient-derived xenografts in modified 3-D hyaluronan hydrogels

Abstract The high attrition rate of potential anti-cancer drugs entering clinical trials indicates the need for more predictive pre-clinical model systems. Standard compound screening is high-throughput and relatively inexpensive, but conducted with non-representative cells in a non-physiological environment. Three-dimensional (3D) culture systems provide cells a more physiological microenvironment including relevant extracellular matrix (ECM) cues and cell-cell interactions. Patient-derived xenograft (PDX) culture preserves tumor heterogeneity while mitigating harsh selective pressures induced by traditional 2D culture methods. However, in vivo mouse models are slow and expensive, often with low “take” rates that limit the numbers of drugs and drug combinations that can be tested. Within the present study, we established the direct culture of breast cancer PDX cells in a hyaluronan-based hydrogel matrix that is amenable to automated dispensing. Washington University Human in Mouse (WHIM) breast cancer PDX tumors were dissociated and PDX cell were encapsulated in 3D hydrogels for in vitro culture. In vitro cultured PDX cells showed high viability over 7 days, as assessed by standard live/dead assay. Cell morphology and marker retention were also examined using immunofluorescence staining for markers including ER, EpCAM, and vimentin, and were found to be subtype-consistent. Ongoing studies seek to expand the PDX tumors in hydrogel matrices such that they may be useful for a variety of applications including drug screening, proteome and kinome activation analysis, and other applications that will enable the development of personalized therapies suited to individual breast cancer patients presenting with various disease subtypes. Citation Format: Lindsey K. Sablatura, MeghaShyam Kavuri, Priya Richards, Daniel A. Harrington, Matthew J. Ellis, Mary C. Farach-Carson. Ex vivo culture of breast cancer patient-derived xenografts in modified 3-D hyaluronan hydrogels. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B30.

Topologically controlled hyaluronan-based gel coatings of hydrophobic grid-like scaffolds to modulate drug delivery

Scaffolds based on poly(ethyl acrylate) having interwoven channels were coated with a hyaluronan (HA) hydrogel to be used in tissue engineering applications. Controlled typologies of coatings evolving from isolated aggregates to continuous layers, which eventually clog the channels, were obtained by using hyaluronan solutions of different concentrations. The efficiency of the HA loading was determined using gravimetric and thermogravimetric methods, and the hydrogel loss during the subsequent crosslinking process was quantified, seeming to depend on the mass fraction of hyaluronan initially incorporated to the pores. The effect of the topologically different coatings on the scaffolds, in terms of mechanical properties and swelling at equilibrium under different conditions was evaluated and correlated with the hyaluronan mass fraction. The potential of these hydrogel coatings as vehicle for controlled drug release from the scaffolds was validated using a protein model.

Dynamic loading, matrix maintenance and cell injection therapy of human intervertebral discs cultured in a bioreactor.

Low back pain originating from intervertebral disc (IVD) degeneration affects the quality of life for millions of people, and it is a major contributor to global healthcare costs. Long-term culture of intact IVDs is necessary to develop ex vivo models of human IVD degeneration and repair, where the relationship between mechanobiology, disc matrix composition and metabolism can be better understood. A bioreactor was developed that facilitates culture of intact human IVDs in a controlled, dynamically loaded environment. Tissue integrity and cell viability was evaluated under 3 different loading conditions: low 0.1-0.3, medium 0.1-0.3 and high 0.1-1.2MPa. Cell viability was maintained >80% throughout the disc at low and medium loads, whereas it dropped to approximately 70% (NP) and 50% (AF) under high loads. Although cell viability was affected at high loads, there was no evidence of sGAG loss, changes in newly synthesised collagen type II or chondroadherin fragmentation. Sulphated GAG content remained at a stable level of approximately 50µg sGAG/mg tissue in all loading protocols. To evaluate the feasibility of tissue repair strategies with cell supplementation, human NP cells were transplanted into discs within a thermoreversible hyaluronan hydrogel. The discs were loaded under medium loads, and the injected cells remained largely localised to the NP region. This study demonstrates the feasibility of culturing human IVDs for 14 days under cyclic dynamic loading conditions. The system allows the determination a safe range-of-loading and presents a platform to evaluate cell therapies and help to elucidate the effect of load following cell-based therapies.