This study employs an optimized and environmentally friendly method to extract and purify chondroitin sulfate (CS) from bovine nasal cartilage using enzymatic hydrolysis, ethanol precipitation, and DEAE Sepharose Fast Flow column chromatography. The extracted CS, representing 44.67 % ± 0.0016 of the cartilage, has a molecular weight of 7.62 kDa. Characterization through UV, FT-IR, NMR spectroscopy, and 2-aminoacridone derivatization HPLC revealed a high content of sulfated disaccharides, particularly ΔDi4S (73.59 %) and ΔDi6S (20.61 %). Interaction studies with bovine serum albumin (BSA) using fluorescence spectroscopy and molecular docking confirmed a high-affinity, static quenching interaction with a single binding site, primarily mediated by van der Waals forces and hydrogen bonding. The interaction did not significantly alter the polarity or hydrophobicity of BSA aromatic amino acids. These findings provide a strong foundation for exploring the application of CS in tissue engineering and drug delivery systems, leveraging its unique interaction with BSA for targeted delivery and enhanced efficacy.

Exosomes (Exos), natural nanovesicles released by various cell types, show potential as an effective drug delivery platform due to their intrinsic role as transporters of biomolecules between different cells. However, Exos functionalization with targeting ligands is a critical step to enhance their targeting capability, which could be challenging. In this study, Exos were modified to specifically bind to CD44-positive cells by anchoring chondroitin sulfate (CS) to their surface. Exo modification was facilitated with CS conjugation with alpha-tocopherol succinate (TOS) as an anchorage. The modified Exos were utilized for delivering curcumin (Cur) to pancreatic cancer (PC) cells. In vitro Cur release studies revealed that Exos play a crucial role in maintaining Cur within themselves, demonstrating their potential as effective carriers for drug delivery to targeted locations. Notably, Cur loaded into the modified Exos exhibited enhanced cytotoxicity compared to unmodified Exo-Cur. Meanwhile, Exo-Cur-TOS-CS induced apoptosis more effectively in AsPC-1 cells than unmodified Exos (70.2 % versus 56.9 %). It is worth mentioning that with CD44-mediated cancer-specific targeting, Exo-CS enabled increased intracellular accumulation in AsPC-1 cells, showing promise as a targeted platform for cancer therapy. These results confirm that Exo modification has a positive impact on enhancing the therapeutic efficacy and cytotoxicity of drugs.

The objective of the present study was to assess the effect of intra-articular Hyaluronic acid (HA) and Chondroitin sulfate (CS) supplementation (Hialurom® Hondro (HH)) on pain symptoms and joint mobility. In total, 60 mg/mL sodium hyaluronate and 90 mg/mL CS were administered to 21 patients (17 females and 4 males) respecting the in-force requirements, excluding patients with some specific comorbidities. In addition to the clinical study (where the pain intensity (severity) and joint mobility were assessed), rheological characterization was conducted evaluating the following parameters: elastic modulus (G\'), loss modulus (G″) oscillatory frequency (fc) at 0.5 Hz and 2.5 Hz, crossover frequency (fc), relaxation time (λ) where it was noticed that the addition of chondroitin sulfate (CS) to sodium hyaluronate (SH) significantly enhances and improves the viscoelastic properties, particularly at higher shear frequencies. A significant decrease in pain intensity felt by the subjects was found, from 7.48 (according to Wong-Baker scale)-pain close to 8 (the patient is unable to perform most activities), to more reduced values of 5.86-at 6 weeks after injection, 4.81-at 3 months after injection, and 5.24-at 6 months after injection, improvements in symptoms was fast and durable. Data related to the evolution of joint mobility show that at 6 weeks after injection, the mobility of joints increased by 17.8% and at 6 months by 35.61%. No serious adverse events were reported with undesired effects so that they would impose additional measures. Better resistance to enzymatic degradation and free radicals could be expected from the synergic combination of sodium hyaluronate and chondroitin sodium sulfate, this having a special importance for the patients, granting them the ability to perform more ample movements and reducing dependency on attendants, thus increasing quality of life.

Increasing studies focus on depolymerization of chondroitin sulfate (CS) to enhance its biological activities. In the present study, low-molecular-weight chondroitin sulfate (LMWCS)‑iron complexes were obtained by photocatalysis-Fenton reaction. After degradation with the optimal condition of 0.25 % (w/v) TiO2, 10 mM FeSO4, and 400 mM H2O2 for 0, 15, and 60 min, the average relative molecular weights of CS were reduced to 4.77, 2.47, and 1.21 kDa, respectively. Electron paramagnetic resonance and free radical capture test identified •OH, •O2-, and h+ in the photocatalysis-Fenton system, among them h+ was the major contributor for CS degradation. The structures of degradation products were analyzed by UV, CD, XRD, SEM-EDS, and NMR, and the results indicated that CS chelated iron with its carboxyl and sulfate groups, leading to changes in conformation and microtopography. Then 10 oligosaccharides were identified in the degradation products using HPLC-MSn and the depolymerization mechanism was proposed. Furthermore, iron release was observed in simulated gastrointestinal digestion of LMWCS‑iron complexes. Notably, the everted gut sac experiment demonstrated that LMWCS‑iron complex possessed 3.75 times higher iron absorption than FeSO4 (p < 0.01) and 12.60 times higher CS absorption than original CS (p < 0.0001). In addition, LMWCS‑iron exhibited stronger in vitro antioxidant activity than CS.

BACKGROUND: Cartilage is an avascular and alymphatic tissue that lacks the intrinsic ability to undergo spontaneous repair and regeneration in the event of significant injury. The efficacy of conventional therapies for invasive cartilage injuries is limited, thereby prompting the emergence of cartilage tissue engineering as a possible alternative. In this study, we fabricated three-dimensional hydrogel films utilizing sodium alginate (SA), gelatin (Gel), and chondroitin sulfate (CS). These films were included with Wharton\'s jelly mesenchymal stem cells (WJ-MSCs) and intended for cartilage tissue regeneration.
METHODS: The hydrogel film that were prepared underwent evaluation using various techniques including scanning electron microscope (SEM), Fourier transform infrared (FTIR) spectroscopy, assessment of the degree of swelling, degradation analysis, determination of water vapor transmission rate (WVTR), measurement of water contact angle (WCA), evaluation of mechanical strength, and assessment of biocompatibility. The rabbit ear cartilage regeneration by hydrogel films with and without of WJ-MSCs was studied by histopathological investigations during 15, 30, and 60 days.
RESULTS: The hydrogel films containing CS exhibited superior metrics compared to other nanocomposites such as better mechanical strength (12.87 MPa in SA/Gel compared to 15.56 in SA/Gel/CS), stability, hydrophilicity, WVTR (3103.33 g/m2/day in SA/Gel compared to 2646.67 in nanocomposites containing CS), and swelling ratio (6.97 to 12.11% in SA/Gel composite compared to 5.03 to 10.90% in SA/Gel/CS). Histopathological studies showed the presence of chondrocyte cells in the lacunae on the 30th day and the complete restoration of the cartilage tissue on the 60th day following the injury in the group of SA/Gel/CS hydrogel containing WJ-MSCs.
CONCLUSIONS: We successfully fabricated a scaffold composed of alginate, gelatin, and chondroitin sulfate. This scaffold was further enhanced by the incorporation of Wharton\'s jelly mesenchymal stem cells. Our findings demonstrate that this composite scaffold has remarkable biocompatibility and mechanical characteristics. The present study successfully demonstrated the therapeutic potential of the SA-Gel-CS hydrogel containing WJ-MSCs for cartilage regeneration in rabbits.

Most of the conditions involving cartilaginous tissues are irreversible and involve degenerative processes. The aim of the present study was to fabricate a biocompatible fibrous and film scaffolds using electrospinning and casting techniques to induce chondrogenic differentiation for possible application in cartilaginous tissue regeneration. Polycaprolactone (PCL) electrospun nanofibrous scaffolds and PCL film were fabricated and incorporated with multi-walled carbon nanotubes (MWCNTs). Thereafter, coating of chondroitin sulfate (CS) on the fibrous and film structures was applied to promote chondrogenic differentiation of human dental pulp stem cells (hDPSCs). First, the morphology, hydrophilicity and mechanical properties of the scaffolds were characterized by scanning electron microscopy (SEM), spectroscopic characterization, water contact angle measurements and tensile strength testing. Subsequently, the effects of the fabricated scaffolds on stimulating the proliferation of human dental pulp stem cells (hDPSCs) and inducing their chondrogenic differentiation were evaluated via electron microscopy, flow cytometry and RT‒PCR. The results of the study demonstrated that the different forms of the fabricated PCL-MWCNTs scaffolds analyzed demonstrated biocompatibility. The nanofilm structures demonstrated a higher rate of cellular proliferation, while the nanofibrous architecture of the scaffolds supported the cellular attachment and differentiation capacity of hDPSCs and was further enhanced with CS addition. In conclusion, the results of the present investigation highlighted the significance of this combination of parameters on the viability, proliferation and chondrogenic differentiation capacity of hDPSCs seeded on PCL-MWCNT scaffolds. This approach may be applied when designing PCL-based scaffolds for future cell-based therapeutic approaches developed for chondrogenic diseases.

Cryogels represent a valid strategy as scaffolds for tissue engineering. In order to adequately support adhesion and proliferation of anchorage-dependent cells, different polymers need to be combined within the same scaffold trying to mimic the complex features of a natural extracellular matrix (ECM). For this reason, in this work, gelatin (Gel) and chondroitin sulfate (CS), both functionalized with methacrylic groups to produce CSMA and GelMA derivatives, were selected to prepare cryogel networks. Both homopolymer and heteropolymer cryogels were produced, via radical crosslinking reactions carried out at -12 °C for 2 h. All the scaffolds were characterized for their mechanical, swelling and morphological properties, before and after autoclave sterilization. Moreover, they were evaluated for their biocompatibility and ability to support the adhesion of human gingival fibroblasts and tenocytes. GelMA-based homopolymer networks better withstood the autoclave sterilization process, compared to CSMA cryogels. Indeed, GelMA cryogels showed a decrease in stiffness of approximately 30%, whereas CSMA cryogels of approximately 80%. When GelMA and CSMA were blended in the same network, an intermediate outcome was observed. However, the hybrid scaffolds showed a general worsening of the biological performance. Indeed, despite their ability to withstand autoclave sterilization with limited modification of the mechanical and morphological properties, the hybrid cryogels exhibited poor cell adhesion and high LDH leakage. Therefore, not only do network components need to be properly selected, but also their combination and ability to withstand effective sterilization process should be carefully evaluated for the development of efficient scaffolds designed for tissue engineering purposes.

Perineuronal nets (PNNs) are a condensed subtype of extracellular matrix that form a net-like coverings around certain neurons in the brain. PNNs are primarily composed of chondroitin sulfate (CS) proteoglycans from the lectican family that consist of CS-glycosaminoglycan side chains attached to a core protein. CS disaccharides can exist in various isoforms with different sulfation patterns. Literature suggests that CS disaccharide sulfation patterns can influence the function of PNNs as well as their labeling. This study was conducted to characterize such interregional CS disaccharide sulfation pattern differences in adult human (n = 81) and mouse (n = 19) brains. Liquid chromatography tandem mass spectrometry was used to quantify five different CS disaccharide sulfation patterns, which were then compared to immunolabeling of PNNs using Wisteria Floribunda Lectin (WFL) to identify CS-glycosaminoglycans and anti-aggrecan to identify CS proteoglycans. In healthy brains, significant regional and species-specific differences in CS disaccharide sulfation and single versus double-labeling pattern were identified. A secondary analysis to investigate how early-life stress impacts these PNN features discovered that although early-life stress increases WFL+ PNN density, the CS-glycosaminoglycan sulfation code and single versus double PNN-labeling distributions remained unaffected in both species. These results underscore PNN complexity in traditional research, emphasizing the need to consider their heterogeneity in future experiments.

Osteoarthritis (OA) is a degenerative disease potentially exacerbated due to inflammation, cartilage degeneration, and increased friction. Both mesenchymal stem cells (MSCs) and pro-inflammatory macrophages play important roles in OA. A promising approach to treating OA is to modify multi-functional hydrogel microspheres to target the OA microenvironment and structure. Arginyl-glycyl-aspartic acid (RGD) is a peptide widely used in bioengineering owing to its cell adhesion properties, which can recruit BMSCs and macrophages. We developed TLC-R, a microsphere loaded with TGF-β1-containing liposomes. The recruitment effect of TLC-R on macrophages and BMSCs was verified by in vitro experiments, along with its function of promoting chondrogenic differentiation of BMSCs. And we evaluated the effect of TLC-R in balancing OA metabolism in vitro and in vivo. When TLC-R was co-cultured with BMSCs and lipopolysaccharide (LPS)-treated macrophages, it showed the ability to recruit both cells in substantial numbers. As the microspheres degraded, TGF-β1 and chondroitin sulfate (ChS) were released to promote chondrogenic differentiation of the recruited BMSCs, modulate chondrocyte metabolism and inhibit inflammation induced by the macrophages. Furthermore, in vivo analysis showed that TLC-R restored the narrowed space, reduced osteophyte volume, and improved cartilage metabolic homeostasis in OA rats. Altogether, TLC-R provides a comprehensive and novel solution for OA treatment by dual-modulating inflammatory and chondrocyte metabolism.

Presently, the clinical treatment of intervertebral disc degeneration (IVDD) remains challenging, but the strategy of simultaneously overcoming the overactive inflammation and restoring the anabolic/catabolic balance of the extracellular matrix (ECM) in the nucleus pulposus (NP) has become an effective way to alleviate IVDD. IL-1ra, a natural antagonist against IL-1β, can mitigate inflammation and promote regeneration in IVDD. Chondroitin sulfate (CS), an important component of the NP, can promote ECM synthesis and delay IVDD. Thus, these were chosen and integrated into functionalized microspheres to achieve their synergistic effects. First, CS-functionalized microspheres (GelMA-CS) with porous microstructure, good monodispersion, and about 200 µm diameter were efficiently and productively fabricated using microfluidic technology. After lyophilization, the microspheres with good local injection and tissue retention served as the loading platform for IL-1ra and achieved sustained release. In in vitro experiments, the IL-1ra-loaded microspheres exhibited good cytocompatibility and efficacy in inhibiting the inflammatory response of NP cells induced by lipopolysaccharide (LPS) and promoting the secretion of ECM. In in vivo experiments, the microspheres showed good histocompatibility, and local, minimally invasive injection of the IL-1ra-loaded microspheres could reduce inflammation, maintain the height of the intervertebral disc (IVD) and the water content of NP close to about 70 % in the sham group, and retain the integrated IVD structure. In summary, the GelMA-CS microspheres served as an effective loading platform for IL-1ra, eliminated inflammation through the controlled release of IL-1ra, and promoted ECM synthesis via CS to delay IVDD, thereby providing a promising intervention strategy for IVDD. STATEMENT OF SIGNIFICANCE: The strategy of simultaneously overcoming the overactive inflammation and restoring the anabolic/catabolic balance of the extracellular matrix (ECM) in nucleus pulposus (NP) has shown great potential prospects for alleviating intervertebral disc degeneration (IVDD). From the perspective of clinical translation, this study developed chondroitin sulfate functionalized microspheres to act as the effective delivery platform of IL-1ra, a natural antagonist of interleukin-1β. The IL-1ra loading microspheres (GelMA-CS-IL-1ra) showed good biocompatibility, good injection with tissue retention, and synergistic effects of inhibiting the inflammatory response induced by lipopolysaccharide and promoting the secretion of ECM in NPCs. In vivo, they also showed the beneficial effect of reducing the inflammatory response, maintaining the height of the intervertebral disc and the water content of the NP, and preserving the integrity of the intervertebral disc structure after only one injection. All demonstrated that the GelMA-CS-IL-1ra microspheres would have great promise for the minimally invasive treatment of IVDD.