Charged Chondroitin Sulfate Research Articles

Cell Surface Modification-Mediated Primary Intestinal Epithelial Cell Culture Platforms for Assessing Host-Microbiota Interactions.

Background: Intestinal epithelial cells (IECs) play a crucial role in regulating the symbiotic relationship between the host and the gut microbiota, thereby allowing them to modulate barrier function, mucus production, and aberrant inflammation. Despite their importance, establishing an effective exvivo culture method for supporting the prolonged survival and function of primary IECs remains challenging. Here, we aim to develop a novel strategy to support the long-term survival and function of primary IECs in response to gut microbiota by employing mild reduction of disulfides on the IEC surface proteins with tris(2-carboxyethyl)phosphine. Methods: Recognizing the crucial role of fibroblast-IEC crosstalk, we employed a cell surface modification strategy, establishing layer-to-layer contacts between fibroblasts and IECs. This involved combining negatively charged chondroitin sulfate on cell surfaces with a positively charged chitosan thin film between cells, enabling direct intercellular transfer. Validation included assessments of cell viability, efficiency of dye transfer, and IEC function upon lipopolysaccharide (LPS) treatment. Results: Our findings revealed that the layer-by-layer co-culture platform effectively facilitates the transfer of small molecules through gap junctions, providing vital support for the viability and function of primary IECs from both the small intestine and colon for up to 5 days, as evident by the expression of E-cadherin and Villin. Upon LPS treatment, these IECs exhibited a down-regulation of Villin and tight junction genes, such as E-cadherin and Zonula Occludens-1, when compared to their nontreated counterparts. Furthermore, the transcription level of Lysozyme exhibited an increase, while Mucin 2 showed a decrease in response to LPS, indicating responsiveness to bacterial molecules. Conclusions: Our study provides a layer-by-layer-based co-culture platform to support the prolonged survival of primary IECs and their features, which is important for understanding IEC function in response to the gut microbiota.

Nano-elemental selenium particle developed via supramolecular self-assembly of chondroitin sulfate A and Na2SeO3 to repair cartilage lesions

Cartilage repair is a significant clinical issue due to its restricted ability to regenerate and self-heal after cartilage lesions or degenerative disease. Herein, a nano-elemental selenium particle (chondroitin sulfate A‑selenium nanoparticle, CSA-SeNP) is developed by the supramolecular self-assembly of Na2SeO3 and negatively charged chondroitin sulfate A (CSA) via electrostatic interactions or hydrogen bonds followed by in-situ reducing of l-ascorbic acid for cartilage lesions repair. The constructed micelle exhibits a hydrodynamic particle size of 171.50 ± 2.40 nm and an exceptionally high selenium loading capacity (9.05 ± 0.03 %) and can promote chondrocyte proliferation, increase cartilage thickness, and improve the ultrastructure of chondrocytes and organelles. It mainly enhances the sulfation modification of chondroitin sulfate by up-regulating the expression of chondroitin sulfate 4-O sulfotransferase-1, −2, −3, which in turn promotes the expression of aggrecan to repair articular and epiphyseal-plate cartilage lesions. The micelles combine the bio-activity of CSA with selenium nanoparticles (SeNPs), which are less toxic than Na2SeO3, and low doses of CSA-SeNP are even superior to inorganic selenium in repairing cartilage lesions in rats. Thus, the developed CSA-SeNP is anticipated to be a promising selenium supplementation preparation in clinical application to address the difficulty of healing cartilage lesions with outstanding repair effects.

Neural Tissue Homeostasis and Repair Is Regulated via CS and DS Proteoglycan Motifs.

Chondroitin sulfate (CS) is the most abundant and widely distributed glycosaminoglycan (GAG) in the human body. As a component of proteoglycans (PGs) it has numerous roles in matrix stabilization and cellular regulation. This chapter highlights the roles of CS and CS-PGs in the central and peripheral nervous systems (CNS/PNS). CS has specific cell regulatory roles that control tissue function and homeostasis. The CNS/PNS contains a diverse range of CS-PGs which direct the development of embryonic neural axonal networks, and the responses of neural cell populations in mature tissues to traumatic injury. Following brain trauma and spinal cord injury, a stabilizing CS-PG-rich scar tissue is laid down at the defect site to protect neural tissues, which are amongst the softest tissues of the human body. Unfortunately, the CS concentrated in gliotic scars also inhibits neural outgrowth and functional recovery. CS has well known inhibitory properties over neural behavior, and animal models of CNS/PNS injury have demonstrated that selective degradation of CS using chondroitinase improves neuronal functional recovery. CS-PGs are present diffusely in the CNS but also form denser regions of extracellular matrix termed perineuronal nets which surround neurons. Hyaluronan is immobilized in hyalectan CS-PG aggregates in these perineural structures, which provide neural protection, synapse, and neural plasticity, and have roles in memory and cognitive learning. Despite the generally inhibitory cues delivered by CS-A and CS-C, some CS-PGs containing highly charged CS disaccharides (CS-D, CS-E) or dermatan sulfate (DS) disaccharides that promote neural outgrowth and functional recovery. CS/DS thus has varied cell regulatory properties and structural ECM supportive roles in the CNS/PNS depending on the glycoform present and its location in tissue niches and specific cellular contexts. Studies on the fruit fly, Drosophila melanogaster and the nematode Caenorhabditis elegans have provided insightful information on neural interconnectivity and the role of the ECM and its PGs in neural development and in tissue morphogenesis in a whole organism environment.

Chondroitin sulfate-mediated albumin corona nanoparticles for the treatment of breast cancer

Chondroitin sulfate-mediated albumin corona nanoparticles were readily prepared without any chemical reaction, and their active tumor targeting and therapeutic effects were examined. Negatively charged chondroitin sulfate (CS) and positively charged doxorubicin (DOX) self-assembled into nanoparticles (CS-DOX-NPs) via electrostatic interactions. Bovine serum albumin (BSA) was then adsorbed on the surface of CS-DOX-NPs to form albumin corona nanoparticles (BC-DOX-NPs) protected from endogenous proteins. Due to the dual effect of BSA and CS, BC-DOX-NPs interacted with the gp60, SPARC and CD44 receptors on tumor cells, facilitating their rapid and efficient transcytosis and improving their accumulation and uptake within tumor tissues. The simultaneous presence of BSA and CS also allowed BC-DOX-NPs to target CD44 efficiently, leading to greater cellular uptake and cytotoxicity against 4T1 cells than CS-DOX-NPs or free DOX. Intravenous injection of BC-DOX-NPs into orthotopic 4T1 tumor-bearing mice led to greater drug accumulation at the tumor site than with CS-DOX-NPs or free DOX, resulting in significant inhibition of tumor growth and lower exposure of major organs to the drug.

Polysaccharide dual coating of yeast capsules for stabilization of anthocyanins

The dual coated yeast capsules for anthocyanin encapsulation and stabilization were fabricated. Anthocyanins were preloaded in hollow yeast capsules, and then the dual coating was performed by deposition of opposite charged polysaccharides using layer-by-layer technique. The combination of positively charged chitosan and negatively charged chondroitin sulfate was found to confer the yeast capsules with the highest encapsulation efficiency and retention rate of anthocyanins. Additionally, the coated yeast capsules featured high tolerance to environmental stresses (i.e., oxygen, ascorbic acid, and heat) and therefore effectively inhibited the degradation of anthocyanins. These stabilizing effects were related to the formation of high penetration barrier provided by the double layers of polysaccharides, as well as the enhanced hydrophobic microenvironment in the capsules. Further development of the polysaccharide-coated yeast capsules may hold promise for the controlled delivery of other water-soluble bioactive components.

Determination of chondroitin sulfate in synovial fluid and drug by ratiometric fluorescence strategy based on carbon dots quenched FAM-labeled ssDNA

Chondroitin sulfate (CS) plays an increasingly important role in clinical settings and pharmacy quality control. However, sensitive and simple methods for CS detection remain limited. In this work, positively charged nitrogen doped carbon dots (P-NCDs) with internal luminescence and quenching property to FAM-labeled random-sequence ssDNA (F-ssDNA) were prepared by a simple heating method. P-NCDs attached and quenched F-ssDNA through electrostatic interaction to form the system of P-NCDs and F-ssDNA (P-NCDs/F-ssDNA) with retained fluorescence intensity of P-NCDs. The highly negatively charged CS reacted electrostatically with P-NCDs and then replaced F-ssDNA in P-NCDs/F-ssDNA to recover the fluorescence intensity of the original quenched F-ssDNA while retaining the internal fluorescence intensity of P-NCDs. Thus, by using restored F-ssDNA as the signal controlled by adding CS to P-NCDs/F-ssDNA, a ratiometric fluorescence strategy based on the retained fluorescence of P-NCDs as reference signal was fabricated through synchronous fluorescence spectrometry for the sensitive detection of CS. Under the optimal experimental conditions, a linear equation for CS was obtained for CS concentration within the range of 0.05–2.0 μg/mL. The method was also successfully applied for the accurate determination of CS in joint fluid samples of arthritic patients, chondroitin sulfate tablets, and chondroitin sulfate eye drops, suggesting its appreciable application potential in the clinic.

Balancing Neuroprotection with Functional Recovery: The Potential Role of the PNN in Preventing Excitotoxicity after SCI

In spinal cord injury (SCI), initial mechanical trauma causes debilitating primary damage to neural cells and blood vessels. Following this, secondary cascades of downstream events occur, including inflammation, ischemia, and excitotoxicity — an increase in intracellular Ca2+ concentration from overactive glutamate (Glu) receptors leading to cell death. Additionally, there is an upregulation of the perineuronal net (PNN), a lattice‐like structure of the extracellular matrix (ECM) which modulates neural communication and homeostasis. The PNN is partially composed of negatively charged chondroitin sulfate proteoglycans (CSPGs). While CSPGs can stabilize plasticity and neuronal growth during development, these molecules become inhibitory to regeneration, sprouting and plasticity after injury, as well as contribute greatly to the glial scar. However, administration of the bacterial enzyme chondroitinase ABC (ChABC) can digest the PNN and CSPGs, ultimately promoting functional recovery. What remains unknown are the other impacts of removing the PNN at very acute stages of injury. We hypothesize that the PNN and its negatively charged CSPGs are upregulated after SCI as a neuroprotective response that attenuates excitotoxicity by acting as a buffer for Ca2+. To test our hypothesis, we induced excitotoxicity by injecting rats with a threshold dose of NMDA with or without ChABC utilizing the well defined spinal respiratory motor system. 59% of SCI occurs at the cervical level, and mechanical ventilation is the leading cause of death and restriction of independence in these cases. Therefore, we administered the dose instraspinally at the C4 level and paired treatment with intrapleural injection of cholera toxin‐B to retrogradely label the phrenic motor neuron pool, which innervates the diaphragm. Preliminary findings suggest that animals lacking the PNN have exacerbated cell death versus control. This implies that, following SCI, the body's main focus is to survive and not necessarily to preserve function. PNN upregulation after injury could be a conserved mechanism which promotes survival and CNS tissue preservation at the expense of plasticity and functional regeneration. The next steps are to detail the spread of ChABC and how long it takes for the PNN to regenerate. Future directions will investigate the optimal timing of ChABC administration to balance preserving cell survival with promoting functional recovery.Support or Funding InformationNINDS R01NS101105 (WJA)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aggrecan-like biomimetic proteoglycans (BPGs) composed of natural chondroitin sulfate bristles grafted onto a poly(acrylic acid) core for molecular engineering of the extracellular matrix

Biomimetic proteoglycans (BPGs) were designed to mimic the three-dimensional (3D) bottlebrush architecture of natural extracellular matrix (ECM) proteoglycans, such as aggrecan. BPGs were synthesized by grafting native chondroitin sulfate bristles onto a synthetic poly(acrylic acid) core to form BPGs at a molecular weight of approximately ∼1.6 MDa. The aggrecan mimics were characterized chemically, physically, and structurally, confirming the 3D bottlebrush architecture as well as a level of water uptake, which is greater than that of the natural proteoglycan, aggrecan. Aggrecan mimics were cytocompatible at physiological concentrations. Fluorescently labeled BPGs were injected into the nucleus pulposus of the intervertebral disc ex vivo and were retained in tissue before and after static loading and equilibrium conditioning. BPGs infiltrated the tissue, distributed and integrated with the ECM on a molecular scale, in the absence of a bolus, thus demonstrating a new molecular approach to tissue repair: molecular matrix engineering. Molecular matrix engineering may compliment or offer an acellular alternative to current regenerative medicine strategies. Statement of SignificanceAggrecan is a natural biomolecule that is essential for connective tissue hydration and mechanics. Aggrecan is composed of negatively charged chondroitin sulfate bristles attached to a protein core in a bottlebrush configuration. With age and degeneration, enzymatic degradation of aggrecan outpaces cellular synthesis resulting in a loss of this important molecule. We demonstrate a novel biomimetic molecule composed of natural chondroitin sulfate bristles grafted onto an enzymatically-resistant synthetic core. Our molecule mimics a 3D architecture and charge density of the natural aggrecan, can be delivered via a simple injection and is retained in tissue after equilibrium conditioning and loading. This novel material can serve as a platform for molecular repair, drug delivery and tissue engineering in regenerative medicine approaches.

The Role of Negative Charge in the Delivery of Quantum Dots to Neurons.

Despite our extensive knowledge of the structure of negatively charged cell surface proteoglycans and sialoglycoconjugates in the brain, we have little understanding of how their negative charge contributes to brain function. We have previously shown that intensely photoluminescent 9-nm diameter quantum dots (QDs) with a CdSe core, a ZnS shell, and a negatively charged compact molecular ligand coating (CL4) selectively target neurons rather than glia. We now provide an explanation for this selective neuronal delivery. In this study, we compared three zwitterionic QD coatings differing only in their regions of positive or negative charge, as well as a positively charged (NH2) polyethylene glycol (PEG) coat, for their ability to deliver the cell-membrane-penetrating chaperone lipopeptide JB577 (WG(Palmitoyl)VKIKKP9G2H6) to individual cells in neonatal rat hippocampal slices. We confirm both that preferential uptake in neurons, and the lack of uptake in glia, is strongly associated with having a region of greater negative charge on the QD coating. In addition, the role of negatively charged chondroitin sulfate of the extracellular matrix (ECM) in restricting uptake was further suggested by digesting neonatal rat hippocampal slices with chondroitinase ABC and showing increased uptake of QDs by oligodendrocytes. Treatment still did not affect uptake in astrocytes or microglia. Finally, the future potential of using QDs as vehicles for trafficking proteins into cells continues to show promise, as we show that by administering a histidine-tagged green fluorescent protein (eGFP-His6) to hippocampal slices, we can observe neuronal uptake of GFP.

Multi-positively charged dendrimeric nanoparticles induced fluorescence quenching of graphene quantum dots for heparin and chondroitin sulfate detection.

A label-free fluorescence assay for rapid and sensitive detection of heparin (Hep) or chondroitin sulfate (CS) was developed by guanidine-terminated poly (amidoanime) (PAMAM-Gu(+)) dendrimers induced aggregation of graphene quantum dots (GQDs). The fluorescence of GQDs was obviously quenched after mixing with PAMAM-Gu(+). However, the addition of highly negatively charged Hep or CS into the fluorescence sensing system resulted in the fluorescence recovery. Because the multi-positively charged PAMAM-Gu(+) would prefer to bind with highly negatively charged Hep or CS, resulting in the deaggregation of GQDs. Under the optimized experimental conditions, the recovery of fluorescence intensity ratio I/I0 (I0 and I were the fluorescence intensity of the sensing system in the absence or presence of target analytes, respectively) was proportional to the concentration of target analytes in the range of 0.04-1.6 μg mL(-1) for Hep and 0.1-2.5 μg mL(-1) for CS. In addition, this method afforded high sensitivity with the detection limit as low as 0.02 μg mL(-1) and 0.05 μg mL(-1) for Hep and CS, respectively. All results suggested that the fluorescence turn-on method could be successfully employed for sensitive and selective detection of heparin analogs.

Effect of brevican deficiency on neuroplasticity mediating molecules

Extracellular matrix (ECM) forms the pericellular part of the tissue. Due to the ECM’s constitution it defines the biophysical and biochemical properties of the tissue. In vertebrates’ brain the neuronal ECM mainly consists of negatively charged chondroitin sulfate proteoglycans (CSPG), hyaluronan and glycoproteins. CSPG are supposed to be involved in cell adhesion, axonal pathfinding and receptor binding. Therefore they are considered to play a role in neural development and plasticity as well as in several neurological and psychiatric disorders (e.g. schizophrenia and depression). One member of the CSPG family is brevican which was previously demonstrated by Blosa et al. (2013) to occur adjacent to the active zone of synapses. Accessorily, deficiency in brevican was reported to lead to a reduction of hippocampal LTP and recruitment of local plasticity. In this study we therefore focused on the effect of brevican on the neuronal cell adhesion molecule NCAM and its polysialylated form PSA-NCAM. Both of them are known to be involved in the establishment and modulation of neuroplasticity which are essential for memory formation. Furthermore we analysed the expression of the prolyl endopeptidase PREP, the matrix metalloproteinase 9 (MMP9) and tissue inhibitors of MMPs (TIMP). Immunohistochemical and western blot analyses of hippocampus tissue of brevican knockout mice compared with wild type littermates did not reveal changes in the expression of MMP9, PREP, NCAM nor PSA-NCAM, but showed a reduction of TIMP1 and 3 by trend. Consequently, changes of LTP and local plasticity in brevican deficient mice do not seem be mediated by an alteration of NCAM and PSA-NCAM, but might be supported by different expression levels of TIMP 1 and 3 modulating several MMPs’ activity.

Ionic osmolytes and intracellular calcium regulate tissue production in chondrocytes cultured in a 3D charged hydrogel

The goal of this study was to investigate the role of fixed negative charges in regulating cartilage-like tissue production by chondrocytes under static and dynamic three-dimensional culture, and to determine whether intracellular calcium ([Ca2+]i) is involved in mediating this response. Initial experiments using the 3D neutral hydrogel were conducted in static isotonic culture with ionic and non-ionic osmolytes added to the culture medium. Tissue production by bovine chondrocytes with non-ionic osmolytes was 1.9-fold greater than with ionic osmolytes, suggesting that the ionic nature of the osmolyte is an important regulator of tissue production. To investigate fixed negative charges, a 3D culture system containing encapsulated chondrocytes was employed based on a synthetic and neutral hydrogel platform within which negatively charged chondroitin sulfate was incorporated in a controlled manner. Incorporation of negative charges did not affect the mechanical properties of the hydrogel; however, intracellular ion concentration was elevated from the culture medium (330mOsm) and estimated to be similar to that in ~400mOsm culture medium. With dynamic loading, GAG synthesis decreased by 26% in neutral hydrogels cultured in 400mOsm medium, and increased by 26% in charged gels cultured in 330mOsm. Treatment of chondrocyte-seeded hydrogels with the Ca2+ chelator BAPTA-AM decreased GAG synthesis by 32–46% and was similar among all conditions, suggesting multiple roles for Ca2+ mediated tissue production including with ionic osmolytes. In conclusion, findings from this study suggest that a dynamic ionic environment regulates tissue synthesis and points to [Ca2+]i signaling as a potential mediator.

Luminescence response of an osmium(II) complex to macromolecular polyanions for the detection of heparin and chondroitin sulfate in biomedical preparations

Heparin, dextran sulfate (DS), chondroitin sulfate (CS), and carrageenan are found to enhance the luminescence intensity of an osmium(II) carbonyl complex with phenanthroline (phen) and 4-phenylpyridine (4-phpy) ligands in aqueous and ethanol solutions. The enhancing effect of the polyanions on the luminescence of the complex is heavily dependent on the sulfate content and other factors such as structure, solubility, and counter ions of the polyanion. The highly sulfated dextran and ι-carrageenan have the most profound effect, while the low charged κ-carrageenan and CS have the least response in aqueous solution. All polyanions exhibited enhanced luminescence intensity of the complex in ethanol solutions, and even the low charged CS and κ-carrageenan enhanced the luminescence more than 4 times. DS contamination of the sodium heparin at 5% can show a significant increase in luminescence response. The osmium complex is found to be highly successful in the fast and sensitive detection of heparin in commercial injectable samples with various backgrounds as well as the detection of CS in over the counter food supplement tablets.

A sulfate polysaccharide/TNF-related apoptosis-inducing ligand (TRAIL) complex for the long-term delivery of TRAIL in poly(lactic-co-glycolic acid) (PLGA) microspheres

The aim was to develop a long-term delivery system for Apo2 ligand/tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) without chemical modification (such as pegylation). A nanocomplex system between the positively charged TRAIL and the negatively charged chondroitin sulfate (CS) (CS/TRAIL) was designed and applied in poly(lactide-co-glycolide) (PLGA) microspheres (MSs). A nanocomplex of approximately 200 nm was easily formed in a weight ratio of 2 TRAIL to CS (TC2) at pH 5.0. The cytotoxicity of CS/TRAIL against HeLa cells was similar to that of native TRAIL. The complex also had higher loading efficiency (above 95%) in PLGA MSs prepared by the multi-emulsion method than that of native TRAIL. The release behaviour of TRAIL from the PLGA MSs was monitored. Although the release of TRAIL from native TRAIL-loaded PLGA MSs (TMS0) was almost complete after 3 days, TC2-loaded PLGA MSs (TMS2) showed sustained TRAIL release without an initial burst for 10 days. The released TRAIL from TMS2 led to cytotoxicity accompanied by massive apoptosis of cancer cells. TMS2 significantly inhibited tumour growth in an in-vivo xenograft model in mice, without any loss of body weight after treatment. From the results, we concluded that TC-loaded PLGA MSs have the potential for long-term delivery of TRAIL without side effects.

Effect of introduction of chondroitin sulfate into polymer-peptide conjugate responding to intracellular signals

We recently developed a novel tumor-targeted gene delivery system responding to hyperactivated intracellular signals. Polymeric carrier for gene delivery consists of hydrophilic neutral polymer as main chains and cationic peptide substrate for target enzyme as side chains, and was named polymer-peptide conjugate (PPC). Introduction of chondroitin sulfate (CS), which induces receptor-medicated endocytosis, into polymers mainly with a high cationic charge density such as polyethylenimine can increase tumor-targeted gene delivery. In the present study, we examined whether introduction of CS into PPC containing five cationic amino acids can increase gene expression in tumor cells. Size and zeta potential of plasmid DNA (pDNA)/PPC/CS complex were <200 nm and between -10 and -15 mV, respectively. In tumor cell experiments, pDNA/PPC/CS complex showed lower stability and gene regulation, compared with that of pDNA/PPC. Moreover, no difference in gene expression was identified between positive and negative polymer. These results were caused by fast disintegration of pDNA/PPC/CS complexes in the presence of serum. Thus, we suggest that introduction of negatively charged CS into polymers with a low charge density may lead to low stability and gene regulation of complexes.

Development of nano- and microscale chondroitin sulfate particles for controlled growth factor delivery

Size scale plays an important role in the release properties and cellular presentation of drug delivery vehicles. Because negatively charged chondroitin sulfate (CS) is capable of electrostatically sequestering positively charged growth factors, CS-derived nanoscale micelles and microscale spheroids were synthesized as potential growth factor carriers to enhance differentiation of stem cells. Particles were characterized for morphology, size distribution, surface charge and cytocompatibility, as well as release of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α). CS micelles were spherical and negatively charged with a bimodal distribution of 324.1±8.5 and 73.2±4.4nm diameters, and CS microspheres possessed a rounded morphology and a diameter of 4.3±0.93μm. Positively charged TGF-β1 demonstrated minimal release after loading in CS microspheres, while negatively charged TNF-α exhibited substantial release over the first 15h, suggesting that TGF-β1 electrostatically complexed with CS. The micelles and microparticles were found to be cytocompatible at moderate concentrations with marrow stromal cell monolayers and within embryonic stem cell embryoid bodies. These synthesis techniques, which allow the formation of CS-based carriers over a variety of nano- and microscale sizes, offer versatility for tailored release of positively charged growth factors and controlled CS presentation for a variety of stem cell-based applications in tissue engineering and regenerative medicine.

Micro- and Nanomechanical Analysis of Articular Cartilage by Indentation-Type Atomic Force Microscopy: Validation with a Gel-Microfiber Composite

As documented previously, articular cartilage exhibits a scale-dependent dynamic stiffness when probed by indentation-type atomic force microscopy (IT-AFM). In this study, a micrometer-size spherical tip revealed an unimodal stiffness distribution (which we refer to as microstiffness), whereas probing articular cartilage with a nanometer-size pyramidal tip resulted in a bimodal nanostiffness distribution. We concluded that indentation of the cartilage's soft proteoglycan (PG) gel gave rise to the lower nanostiffness peak, whereas deformation of its collagen fibrils yielded the higher nanostiffness peak. To test our hypothesis, we produced a gel-microfiber composite consisting of a chondroitin sulfate-containing agarose gel and a fibrillar poly(ethylene glycol)-terephthalate/poly(butylene)-terephthalate block copolymer. In striking analogy to articular cartilage, the microstiffness distribution of the synthetic composite was unimodal, whereas its nanostiffness exhibited a bimodal distribution. Also, similar to the case with cartilage, addition of the negatively charged chondroitin sulfate rendered the gel-microfiber composite's water content responsive to salt. When the ionic strength of the surrounding buffer solution increased from 0.15 to 2 M NaCl, the cartilage's microstiffness increased by 21%, whereas that of the synthetic biomaterial went up by 31%. When the nanostiffness was measured after the ionic strength was raised by the same amount, the cartilage's lower peak increased by 28%, whereas that of the synthetic biomaterial went up by 34%. Of interest, the higher peak values remained unchanged for both materials. Taken together, these results demonstrate that the nanoscale lower peak is a measure of the soft PG gel, and the nanoscale higher peak measures collagen fibril stiffness. In contrast, the micrometer-scale measurements fail to resolve separate stiffness values for the PG and collagen fibril moieties. Therefore, we propose to use nanostiffness as a new biomarker to analyze structure-function relationships in normal, diseased, and engineered cartilage.

Dynamic loading stimulates chondrocyte biosynthesis when encapsulated in charged hydrogels prepared from poly(ethylene glycol) and chondroitin sulfate

This study aimed to elucidate the role of charge in mediating chondrocyte response to loading by employing synthetic 3D hydrogels. Specifically, neutral poly(ethylene glycol) (PEG) hydrogels were employed where negatively charged chondroitin sulfate (ChS), one of the main extracellular matrix components of cartilage, was systematically incorporated into the PEG network at 0%, 20% or 40% to control the fixed charge density. PEG hydrogels were employed as a control environment for extracellular events which occur as a result of loading, but which are not associated with a charged matrix (e.g., cell deformation and fluid flow). Freshly isolated bovine articular chondrocytes were embedded in the hydrogels and subject to dynamic mechanical stimulation (0.3 Hz, 15% amplitude strains, 6 h) and assayed for nitric oxide production, cell proliferation, proteoglycan synthesis, and collagen deposition. In the absence of loading, incorporation of charge inhibited cell proliferation by ~ 75%, proteoglycan synthesis by ~ 22–50% depending on ChS content, but had no affect on collagen deposition. Dynamic loading had no effect on cellular responses in PEG hydrogels. However, dynamically loading 20% ChS gels inhibited nitrite production by 50%, cell proliferation by 40%, but stimulated proteoglycan and collagen deposition by 162% and 565%, respectively. Dynamic loading of 40% ChS hydrogels stimulated nitrite production by 62% and proteoglycan synthesis by 123%, but inhibited cell proliferation by 54% and collagen deposition by 52%. Upon removing the load and culturing under free-swelling conditions for 36 h, the enhanced matrix synthesis observed in the 20% ChS gels was not maintained suggesting that loading is necessary to stimulate matrix production. In conclusion, extracellular events associated with a charged matrix have a dramatic affect on how chondrocytes respond to mechanical stimulation within these artificial 3D matrices suggesting that streaming potentials and/or dynamic changes in osmolarity may be important regulators of chondrocytes while cell deformation and fluid flow appear to have less of an effect.

Layer‐by‐layer assembly of biomacromolecules on poly(ethylene terephthalate) films and fiber fabrics to promote endothelial cell growth

Poly(ethylene terephthalate) (PET) film and PET vascular graft were modified by layer-by-layer (LBL) assembly of collagen and chondroitin sulfate (CS) to mimic the natural composition and structure of extracellular matrix. PET materials were first hydrolyzed to endow the surfaces with negative charge. Alternative assembly of positively charged collagen and negatively charged CS was then conducted in an LBL manner to produce biological multilayers. X-ray photoelectron spectroscopy confirmed the occurrence of hydrolysis and existence of collagen and CS. The stepwise layer growth on the PET film was monitored by UV-vis spectroscopy. The multilayers had sufficient stability to pass through the cell culture period (8 days). In vitro endothelial cell culture revealed that the incorporated biomacromolecules could significantly improve the cell attachment, proliferation, and viability. Cells showed more spreading morphology with preserved phenotype, as illustrated by scanning electron microscopy and factor VIII-related antigen immunocytochemistry, respectively.

Surface modification of poly(ethylene terephthalate) via hydrolysis and layer-by-layer assembly of chitosan and chondroitin sulfate to construct cytocompatible layer for human endothelial cells

Surface modification of poly(ethylene terephthalate) (PET) film was performed by surface hydrolysis and layer-by-layer (LBL) assembly followed a mechanism of electrostatic adsorption of oppositely charged polymers, exemplified with chitosan and chondroitin sulfate (CS). Hydrolysis of PET in concentrated alkaline solution produced a carboxyl-enriched surface. The changes of weight loss and surface chemistry, morphology and wettability were monitored and verified by UV–vis spectroscopy, atomic force microscopy (AFM) and water contact angle. Assembly of positively charged chitosan and negatively charged CS was then conducted in a LBL manner to create multilayers on the hydrolyzed PET film. The process of layer growth and oscillation of surface wettability were monitored by UV–vis spectroscopy and water contact angle measurement, respectively. In vitro cell culture revealed that the adherence of endothelial cells was significantly enhanced on the biomacromolecules-modified PET film with preserved endothelial cell function, in particular on those assembled with larger number of chitosan/CS layers. However, with regard to cell proliferation and viability properties after cultured for 4 days, minor difference was determined between the modified and the unmodified PET films.