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±4.4 nm diameters, and CS microspheres possessed a rounded morphology and a diameter of 4.3±0.93 μm. Positively charged TGF-β1 demonstrated minimal release after loading in CS microspheres, while negatively charged TNF-α exhibited substantial release over the first 15 h, 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.     

Engineering a bioactive matrix by modifications of calcium sulfate

The goal of this study was to define the conditions for the fabrication of a bioactive matrix that induces and supports cell proliferation and tissue regeneration. The proposed hypothesis was that a composite graft could be engineered by the absorption of platelet-rich plasma (PRP) onto calcium sulfate (CS). Evaluation of the biological activity of the engineered grafts was based on osteoblast proliferation studies and scanning electron microscopy (SEM) analyses. Graft samples were created in a standard size and shape so that the surface available for attachment and cell proliferation was always identical. Proliferation data were expressed as counts per minute per group and differences among groups were statistically analyzed by analysis of variance followed by the Scheffé test (alpha = 0.1). SEM analysis showed that the combination of CS and PRP presents a preserved crystalline structure well integrated by organic matrix. This combination showed the highest cell proliferation levels (p < 0.001). Further evaluations demonstrated that PRP is activated when combined with CS. When tested as a possible carrier for biologically active molecules such as platelet-derived growth factor (PDGF), CS showed increased cell proliferation (p < 0.001). SEM revealed adherent osteoblasts with broad flattened edges on CS-PRP. This study proposes CS as an efficient carrier for PRP or PDGF and supports the use of these combinations as bioactive matrices in clinical or laboratory applications.     

Self-assembly of a swollen chitosan/chondroitin sulfate hydrogel by outward diffusion of the chondroitin sulfate chains

A hydrogel constituted of chitosan (CT) and chondroitin sulfate (CS) was synthesized. In previously reported works the stochiometric ratio has been used, but in this paper an excess of CS (40% CT and 60% CS) was used because the hydrogel could be applied as a CS carrier. The hydrogel properties were investigated by differential scanning calorimetry, wide-angle X-ray scattering (WAXS), scanning electron microscopy, and high-performance liquid chromatography. Changes in the pH of the gel-surrounding liquid had a considerable effect on both the release and the molecular reorganization of CS. Furthermore, the formed hydrogel exhibited interesting parameters for use in biotechnology, such as water affinity, thermal properties and morphology upon sequential pH variation. The protonation or deprotonation of the different groups that participate in the complex formation and the coiling or uncoiling of like or unlike chains concomitant to the release of CS are believed to be the main factors affecting the hydrogel properties. CS was released mainly at pH higher than 6.5, the value of pK_aCT, and the released CS maximum fraction was approximately 0.5. The WAXS data demonstrated that the CT/CS complex in the hydrogel presented macromolecular reorganization at pHs ranging from 6 to 12.     

Enhancing the transdermal delivery of rigid nanoparticles using the simultaneous application of ultrasound and sodium lauryl sulfate

The potential of rigid nanoparticles to serve as transdermal drug carriers can be greatly enhanced by improving their skin penetration. Therefore, the simultaneous application of ultrasound and sodium lauryl sulfate (referred to as US/SLS) was evaluated as a skin pre-treatment method for enhancing the passive transdermal delivery of nanoparticles. We utilized inductively coupled plasma mass spectrometry and an improved application of confocal microscopy to compare the delivery of 10- and 20-nm cationic, neutral, and anionic quantum dots (QDs) into US/SLS-treated and untreated pig split-thickness skin. Our findings include: (a) ～0.01% of the QDs penetrate the dermis of untreated skin (which we quantify for the first time), (b) the QDs fully permeate US/SLS-treated skin, (c) the two cationic QDs studied exhibit different extents of skin penetration and dermal clearance, and (d) the QD skin penetration is heterogeneous. We discuss routes of nanoparticle skin penetration and the application of the methods described herein to address conflicting literature reports on nanoparticle skin penetration. We conclude that US/SLS treatment significantly enhances QD transdermal penetration by 500-1300%. Our findings suggest that an optimum surface charge exists for nanoparticle skin penetration, and motivate the application of nanoparticle carriers to US/SLS-treated skin for enhanced transdermal drug delivery.     

Properties of tableted high-amylose corn starch-pectin blend microparticles intended for controlled delivery of diclofenac sodium

This study reports the properties of tableted microparticles based on high-amylose corn starch (HACS)-pectin blend polymers as the controlled release system for diclofenac sodium (DS). HACS-pectin blend microparticles are prepared through a modified process by the spray drying technique, which is a widely used microencapsulation technique in the pharmaceutical industry. The mean particle size of various formulations of blend microparticles is in the range of 5.8-7.3 mum. Fourier transform infrared (FTIR) spectroscopy study reveals the absence of well-defined interaction between HACS-pectin and polymers-DS. The blend microparticles thus prepared were compressed into tablets using the directly compressible excipients. A cross-sectional view of the tablet reveals the presence of nearly spherical shaped particles in the tablet, suggesting that the system chosen is ideal for tableting. In vitro drug release study indicates that tableted microparticulate system is found to be suitable for the manipulation of release behavior for DS in the gastrointestinal tract. Release mechanism of the DS from tableted microparticles is by Fickian diffusion since the value of n approaches 0.5.     

Development of chondroitin sulfate encapsulated PLGA microsphere delivery systems with controllable multiple burst releases for treating osteoarthritis

The purpose of the study was to design and develop unique drug delivery systems with controllable multiple burst releases of drugs for treating osteoarthritis. Chondroitin sulfate (CS) was encapsulated into four types of PLGA materials, that is, PLGA 50:50, PLGA 65:35, PLGA 75:25, and PLGA 85:15. The effects of microsphere size and various combinations of blend PLGA microspheres on CS release were investigated. The cytotoxicity of the CS-encapsulated microspheres was investigated according to the ISO 10993 guideline. Our study showed that the encapsulation efficiency of CS into PLGA 50:50 microspheres varied with the size of microspheres; however, the encapsulation efficiencies of CS into PLGA microspheres were independent of the types of PLGA materials. The size of PLGA microspheres was shown to affect the rate of CS release. With the increase of microsphere size from 75-150 μm to 300-355 μm, the initial CS release decreased. Further increase in microsphere size led to an increase in the initial CS release. In addition, combination of different types of PLGA microspheres was shown to be capable of achieving multiple burst CS releases. Moreover, the CS encapsulated PLGA microspheres were shown to be non-cytotoxic. This study proved the concept of multiple burst drug releases that were achieved by encapsulating CS into different types of PLGA microspheres and delivering CS from systems consisting of mixed types of PLGA microspheres, which may be applied to treat osteoarthritis by mimicking multiple intra-joint injection of therapeutic agents.     

Opposing functions of chondroitin sulfate and heparan sulfate during early neuronal polarization

Axon-dendrite polarity of neurons is essential for information processing in the nervous system. Here we studied the functions of chondroitin sulfate (CS) and heparan sulfate (HS) in neuronal polarization using cultured dissociated hippocampal neurons. Immunohistochemical analyses of early cultured neurons indicated the distribution of these glycosaminoglycans to be quite different. While CS epitopes were accumulated in the focal contacts present in axons and cell bodies, those of HS were detected ubiquitously on the cell surface including on dendrites and axons. Treatment with chondroitinase (CHase) ABC, which degrades CS, and knockdown of a CS sulfotransferase, N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (4,6-ST), which is involved in the biosynthesis of oversulfated structures, induced the formation of multiple axons in hippocampal neurons. Time-lapse recordings revealed the multiple axons of CHase ABC-treated neurons to be highly unstable, extending and retracting, repeatedly. CHase ABC-treatments suggested that CS is involved in the formation of phosphorylated focal adhesion kinase-positive focal contacts. Thus, CS may enhance integrin signaling in the nascent axons, supporting axon specification. On the other hand, when neurons were treated with heparitinases that specifically degrade HS, neurons with a single axon increased. The axons of HSase-treated neurons extended steadily and showed almost no retraction. These results suggest that CS stabilizes and HS destabilizes the growth of axons in an opposing manner, contributing to early neuronal polarization.     

Chondroprotective action of chondroitin sulfate. Competitive action of chondroitin sulfate on the digestion of hyaluronan by bovine testicular hyaluronidase.

Lysosomal hyaluronidase is responsible for the degradation of hyaluronan, a component of the extracellular matrix, in degenerative disorders of the joints. It has been hypothesized that the administration of chondroitin sulfate (both a component of the extracellular matrix and a substrate for hyaluronidase) could compete for this enzyme and reduce the degradation process. The present study shows that a mixture of chondroitin 4-sulfate and chondroitin 6-sulfate is a good competitor of hyaluronan for hyaluronidase. The digestion of hyaluronan is reduced in proportion to the amount of competing chondroitin. The competitive ability is dependent on the 4-sulfate, 6-sulfate composition of the chondroitin mixture. Mixtures richer in the 4-sulfate isomer are more effective. The enzymatic reactions have been monitored by HPLC and PAGE.     

Binding of interleukin-8 to heparan sulfate and chondroitin sulfate in lung tissue

Interleukin (IL)-8, a member of the CXC chemokine family, is a potent neutrophil chemotactic factor. Mechanisms that regulate the activity of chemokines in tissue are not clear. The goal of this study was to determine whether IL-8-glycosaminoglycan interactions are responsible for the binding of IL-8 in lung tissue. Experiments were performed with a quantitative tissue-binding assay to measure the amount of 125I-IL-8 binding and an in situ tissue-binding assay to characterize the location of IL-8 binding in lung tissue. Confocal microscopy demonstrated IL-8 binding to specific anatomic locations such as cell surfaces and extracellular matrix that were enriched with heparan sulfate and chondroitin sulfate. Removal of heparan sulfate or chondroitin sulfate from lung tissue significantly decreased the binding of 125I-IL-8. Two forms of IL-8 with single amino acid mutations in the glycosaminoglycan-binding domain showed decreased binding. In addition, studies with normal and monomeric IL-8 showed that dimerization increased the binding of 125I-IL-8 in lung tissue. These findings suggest that IL-8-glycosaminoglycan interactions determine the location where IL-8 binds in lung tissue and provides a site for the dimerization of IL-8, which increases the local concentration of IL-8 in the lungs.     

Predicting protein instability in sustained protein delivery systems using spectral-phase interference

Biodegradable and non-biodegradable polymers represent promising materials for sustained protein delivery systems. However, structural protein instabilities due to interactions with the polymer surface are often observed. Aim of the present study was to analyze and predict these instabilities by determination of adsorption pattern and extent via biomolecular interaction analysis. A new optical method based on spectral-phase interference successfully demonstrated its suitability for this new application scope. It was characterized in terms of sensitivity, reproducibility and dynamic range using bovine serum albumin (BSA) as model compound. For protein-polymer interaction studies, materials with different wettabilities and zeta potential were selected and successfully applied on the sensor chip: Glass, poly(styrene), poly(lactic acid), poly(lactic-co-glycolic acid), and poly(ethylene carbonate). Concentration dependent adsorption curves revealed two principal adsorption patterns based on the connection between BSA spreading and supply rate. This connection was stronger influenced by polymer hydrophobicity than surface charge. Association, dissociation and binding rate constants in the range from 0.15 to 34.19 × 10⁻⁶ M were obtained. Atomic force microscopy images of the films before and after adsorption confirmed the previous elaborated model. Poly(ethylene carbonate) emerged as highly promising biomaterial for protein delivery due to its favorable adsorption behavior based on low polymer-protein interactions.     

A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin,hyaluronic acid and chondroitin sulfate

The development of hydrogels tailored for cartilage tissue engineering has been a research and clinical goal for over a decade. Directing cells towards a chondrogenic phenotype and promoting new matrix formation are significant challenges that must be overcome for the successful application of hydrogels in cartilage tissue therapies. Gelatin–methacrylamide (Gel-MA) hydrogels have shown promise for the repair of some tissues, but have not been extensively investigated for cartilage tissue engineering. We encapsulated human chondrocytes in Gel-MA-based hydrogels, and show that with the incorporation of small quantities of photocrosslinkable hyaluronic acid methacrylate (HA-MA), and to a lesser extent chondroitin sulfate methacrylate (CS-MA), chondrogenesis and mechanical properties can be enhanced. The addition of HA-MA to Gel-MA constructs resulted in more rounded cell morphologies, enhanced chondrogenesis as assessed by gene expression and immunofluorescence, and increased quantity and distribution of the newly synthesized extracellular matrix (ECM) throughout the construct. Consequently, while the compressive moduli of control Gel-MA constructs increased by 26 kPa after 8 weeks culture, constructs with HA-MA and CS-MA increased by 114 kPa. The enhanced chondrogenic differentiation, distribution of ECM, and improved mechanical properties make these materials potential candidates for cartilage tissue engineering applications.     

Dynamic expression of a native chondroitin sulfate epitope reveals microheterogeneity of extracellular matrix organization in the embryonic chick heart

TC2 is a novel monoclonal antibody produced by in vitro immunization of splenocytes with a peanut agglutinin‐positive fraction from extracts of prechondrogenic micromass cultures of chick limb mesenchyme. ELISA results demonstrated TC2 reactivity with a native epitope on a glycosaminoglycan (GAG) enriched in chondroitin‐4‐sulfate and with multiple intact proteoglycans, but not with other GAGs tested. TC2 immunohistochemical reactivity was abolished by pretreatment of sections with chondroitinase AC or preadsorption with chondroitin‐4‐sulfate GAG. Strong TC2 localization occurred throughout the developing heart at stage 9. As looping ensued, a graded reactivity was observed from lowest in the atrium to highest in the conotruncus that correlated well with versican localization. The superior atrioventricular cushion stained preferentially with TC2 as compared to the inferior cushion at stages 16–18. At these later stages TC2 patterns did not agree completely with anti‐versican reactivity. By stage 23 there was a marked reduction in TC2 localization in the heart, however, strong reactivity remained at certain sites, including the conotruncus and in subcompartments of both atrioventricular cushions. A heterogeneous distribution of other native chondroitin sulfate glycosaminoglycan epitopes recognized by monoclonal antibodies d1C4 and CS‐56 was observed as well. The distribution of the TC2 epitope usually did not overlap with d1C4 or CS‐56 localization at the stages examined. Overall, the spatiotemporal characteristics of TC2 reactivity in the developing chick heart appear to correlate with subdomains of the endocardial cushions as well as with trabecular and atrial septal formation. Anat Rec 254:181–195, 1999. © 1999 Wiley‐Liss, Inc.     

The potential of chondroitin sulfate as a therapeutic agent

Chondroitin sulfate (CS) is an omnipresent glycosaminoglycan with significant biologic roles. Chondroitin sulfate has not one structure but its polysaccharide backbone is modified to a smaller or higher degree according to the cell, tissue, species localization, and/or physiopathological stimuli. The potential of chondroitin sulfate for the therapy of osteoarthritis has been under investigation in several clinical trials, which have shown that it is safe and well tolerated. However, there are many issues still unresolved, such as the structure-modifying effects of CS in osteoarthritis, symptom-modifying efficacy in certain groups of patients, structure-activity-pharmacokinetic relationships, knowledge of mechanism of action, and better quality control of the preparations. Furthermore, ongoing basic research on its biologic role will probably show other therapeutic applications.     

Controlled immobilization of chondroitin sulfate in polyacrylic acid networks

Novel semi-interpenetrating polymer networks (semi-IPNs) of chondroitin sulfate (ChS) and acrylic acid (AA) were prepared with the aim of obtaining a hydrogel for use as a colon-specific drug carrier. By controlling the concentrations of cross-linking agent, diethylene glycol dimethacrylate (DEGDA), as well as the reaction solvent, high swelling percentages were obtained (approx. 1600%). However, the highest sol percent obtained for these hydrogels was approx. 70%, and most of the chondroitin sulfate remained soluble and could be extracted. Therefore, an alternative approach was adopted: methacrylate-grafted ChS (ChSMA) was synthesized and then co-polymerized with acrylic acid (AA) at a molar ratio of 1:5 with various concentrations of AA. The sol content of these ChSMA-AA hydrogels was reduced to approx. 20%, and the cross-linking densities were almost 100-fold higher than those of the semi-IPNs. FT-IR spectra showed that the H-bonding interactions between ChS and PAA and the spectra of the semi-IPNs were similar to that of PAA itself after sol extraction. In contrast, the FT-IR spectra of ChSMA-AA remained intact after sol extraction. Ketoprofen was used as a model drug to test the sustained release behavior of these hydrogels.     

原子力显微镜对硫酸软骨素分形结构的研究

自组装纳米结构一直是化学科学中的前沿课题。本文运用原子力显微镜(AFM)对硫酸软骨素溶液在Ca2+试剂作用下的自组装纳米结构随时间的形态学变化进行了研究,实验结果显示当硫酸软骨素的浓度大于1.0mg/mL时,该聚合物在水溶液中可以形成纳米尺寸的类似于胶束结构的自组装聚集体,且这种类似胶束结构体现了分形学上所说的自相似性,聚合物凝集的过程实际上就是分形生长的过程。该分形结构会因为Ca2+的作用而随时间而发生形态学改变。本文探讨了这种分形生长的机理,并对这种分形结构随时间变化的现象给予了初步解释,为高分子自组装为纳米分散粒子,特别是功能化的聚合物在水中形成稳定的纳米粒子的研究提供了一定的实验依据。     

Identification and immunolocalization of chondroitin sulfate proteoglycans in tooth cementum

Proteoglycans (PGs) display a great diversity in their core proteins as well as carbohydrate structures and are thought to be involved in many biological functions. Recently we have identified and immunolocalized two keratan sulfate PGs, fibromodulin and lumican, in bovine tooth cementum (Cheng et al., Connect. Tissue Res. 34: 87-96, 1996). The objectives of this study were to identify and characterize chondroitin sulfate (CS) PGs in cementum. In order to explore their potential association with mineral, bovine cementum matrix molecules were fractionated into mineral-unbound and -bound matrices by sequential extraction. Both fractions were subjected to DEAE anion exchange column chromatography and the eluate collected was assayed for C4S and C6S isomers by dot blot immunoassay with specific monoclonal antibodies, 2-B-6 and 3-B-3, respectively. Two families of CSPGs were identified mainly in the mineral-unbound fraction. One contained only C4S glycosaminoglycan and the other both C6S and C4S. By biochemical and immunochemical analyses, decorin and biglycan were identified in the former and versican in the latter. The ratio of C6S to C4S isomers of cementum versican was approximately 7:1. Furthermore, these PGs were immunolocalized in and around tooth cementum using antibodies generated against the respective core proteins. Intensive immunostaining for versican was found almost exclusively in the lacunae housing cementocytes in cementum and osteocytes in alveolar bone, respectively. Immunostaining for decorin was mainly associated with collagen fibers in the periodontal ligament and slightly in cementum matrix, while the one for biglycan was mainly in cementoblasts/precementum. These differential tissue distributions of the CSPGs suggest that they may play distinct roles in cementogenesis.     

Collageneous matrix coatings on titanium implants modified with decorin and chondroitin sulfate: characterization and influence on osteoblastic cells

Studies in developmental and cell biology have established the fact that responses of cells are influenced to a large degree by morphology and composition of the extracellular matrix. Goal of this work is to use this basic principle to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM), utilizing the natural self-assembly potential of collagen in combination with further ECM components in close analogy to the situation in vivo. Aiming at load-bearing applications in bone contact, collagen type I in combination with the proteoglycan decorin and the glycosaminoglycan chondroitin sulfate (CS) was used; fibrillogenesis, fibril morphology, and adsorption of differently composed fibrils onto titanium were assessed. Both decorin and CS could be integrated into the fibrils during fibrillogenesis, the amount bound respectively desorbed depending on the ionic strength of fibrillogenesis buffer. Including decorin always resulted in a significant decrease of fibril diameter, CS in only a slight decrease or even increase, depending on the collagen preparation used. No significant changes in adsorption to titanium could be detected. Osteoblastic cells showed different reactions for cytoskeletal arrangement and osteopontin expression depending on the composition of the ECM, with CS enhancing the osteoblast phenotype.     

Effect of chondroitin sulfate on murine splenocytes sensitized with ovalbumin

Precursors for Thy-1(+) dendritic epidermal T cells (DETC) develop as Vgamma3(+) T cells in the fetal thymus and become distributed in the adult skin. DETC are variably distributed from site to site and from strain to strain. To elucidate the basis of strain variation, we first compared the density of DETC in the ear epidermis among different mouse strains. In the ear epidermis, we detected the highest level of DETC in C57BL/6 mice, intermediate levels in C3H and CBA/J mice, and the lowest levels in other strains including BALB/c and 129 mice. Although BALB/c and 129+Ter/Sv mice showed higher levels of DETC in the abdomen than in the ear, the levels were significantly lower than C57BL/6 mice. Furthermore, in neonatal abdominal epidermis we detected considerably lower numbers of DETC in BALB/c and 129+Ter/Sv mice than in C57BL/6 mice. In contrast, Vgamma3(+) DETC precursors in the fetal thymus are rather increased in 129+Ter/Sv mice. These results suggest that fewer DETC precursors are seeded in the neonatal skin of BALB/c and 129+Ter/Sv mice and that their expansion in the skin during neonatal to adult stages does not reach the levels in C57BL/6 mice.