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The promoting effects of collagen and its derivatives on bone health have been uncovered. However, the structure and effects of type II collagen peptides from squid cartilage (SCIIP) on osteoarthritis still need to be clarified. In this study, SCIIP was prepared from squid throat cartilage with pretreatment by 0.2 mol/L NaOH at a liquid-solid ratio of 10:1 for 18 h and hydrolyzation using alkaline protease and flavourzyme at 50 °C for 4 h. The structure of SCIIP was characterized as a molecular weight lower than 5 kDa (accounting for 87.7 %), a high glycine level of 35.0 %, typical FTIR and CD features of collagen peptides, and a repetitive sequence of Gly-X-Y. GP(Hyp)GPD and GPAGP(Hyp)GD were separated and identified from SCIIP, and their binding energies with TLR4/MD-2 were - 8.4 and - 8.0 kcal/mol, respectively. SCIIP effectively inhibited NO production in RAW264.7 macrophages and alleviated osteoarthritis in rats through the TLR4/NF-κB pathway. Therefore, SCIIP exhibited the potential for application as an anti-osteoarthritis supplement., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The interactions between glycosaminoglycans (GAGs) and proteins are essential in numerous biochemical processes that involve ion-pair interactions. However, there is no evidence of direct and specific interactions between GAGs and collagen proteins in native cartilage. The resolution of solid-state NMR (ssNMR) can offer such information but the detection of GAG interactions in cartilage is limited by the sensitivity of the experiments when 13 C and 15 N isotopes are at natural abundance. In this communication, this limitation is overcome by taking advantage of dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) experiments to obtain two-dimensional (2D) 15 N- 13 C and 13 C- 13 C correlations on native samples at natural abundance. These experiments unveiled inter-residue correlations in the aliphatic regions of the collagen protein previously unobserved. Additionally, our findings provide direct evidence of charge-pair salt-bridge interactions between negatively charged GAGs and positively charged arginine (Arg) residues of collagen protein. We also identified potential hydrogen bonding interactions between hydroxyproline (Hyp) and GAGs, offering atomic insights into the biochemical interactions within the extracellular matrix of native cartilage. Our approach may provide a new avenue for the structural characterization of other native systems.

Hyalocytes, which are considered to originate from the monocyte/macrophage lineage, play active roles in vitreous collagen and hyaluronic acid synthesis. Obtaining a hyalocyte-compatible bioink during the 3D bioprinting of eye models is challenging. In this study, we investigated the suitability of a cartilage-decellularized extracellular matrix (dECM)-based bioink for printing a vitreous body model. Given that achieving a 3D structure and environment identical to those of the vitreous body necessitates good printability and biocompatibility, we examined the mechanical and biological properties of the developed dECM-based bioink. Furthermore, we proposed a 3D bioprinting strategy for volumetric vitreous body fabrication that supports cell viability, transparency, and self-sustainability. The construction of a 3D structure composed of bioink microfibers resulted in improved transparency and hyalocyte-like macrophage activity in volumetric vitreous mimetics, mimicking real vitreous bodies. The results indicate that our 3D structure could serve as a platform for drug testing in disease models and demonstrate that the proposed printing technology, utilizing a dECM-based bioink and volumetric vitreous body, has the potential to facilitate the development of advanced eye models for future studies on floater formation and visual disorders., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

Type II collagen (Col II) and chondroitin sulfate (CS) are the main macromolecules in the extracellular matrix. This study investigated the characteristics of Col II and CS obtained from chicken sternal cartilage (CSC) via enzymatic hydrolysis for various treatment times. For Col II and CS, the highest efficiency of enzymatic hydrolysis was achieved after 24 and 6 h of treatment, respectively. The average molecular weights were α1 chain-130 kDa, β chain-270 kDa for Col II, and 80.27 kDa for CS. Fourier transform infrared spectroscopy revealed that the Col II samples maintained their triple-helical structure and that the predominant type of CS was chondroitin-4-sulfate. Scanning electron microscopy revealed that the Col II and CS samples possessed fibrillar and clustered structures, respectively. This study suggests that collagen and CS obtained from CSC can be used as promising molecules for application in food and pharmaceutical industries., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sebnem Tavman reports financial support was provided by Ege University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Proteoglycans are hierarchically organized structures that play an important role in the hydration and the compression resistance of cartilage matrix. In this study, the static and dynamic properties relevant to the biomechanical function of cartilage are determined at different levels of the hierarchical structure, using complementary osmotic pressure, neutron scattering (SANS) and light scattering (DLS) measurements. In cartilage proteoglycans (PGs), two levels of bottlebrush structures can be distinguished: the aggrecan monomer, which consists of a core protein to which are tethered charged glycosaminoglycan (GAG) chains, and complexes formed of the aggrecan monomers attached around a linear hyaluronic acid backbone. The principal component of GAG, chondroitin sulfate (CS), is used as a baseline in this comparison. The osmotic modulus, measured as a function of the proteoglycan concentration, follows the order CS < aggrecan < aggrecan-HA complex. This order underlines the benefit of the increasing complexity at each level of the molecular architecture. The hierarchical bottlebrush configuration, which prevents interpenetration among the bristles of the aggrecan monomers, enhances both the mechanical properties and the osmotic resistance. The osmotic pressure of the collagen solution is notably smaller than in the proteoglycan systems. This is consistent with its known primary role to provide tensile strength to the cartilage and to confine the aggrecan-HA complexes, as opposed to load bearing. The collective diffusion coefficient D governs the rate of recovery of biological tissue after compressive load. In CS solutions the diffusion process is fast, D ≈ 3 × 10 -6 cm 2 s -1 at concentrations comparable with that of the GAG chains inside the aggrecan molecule. In CS solutions D is a weakly decreasing function of calcium ion concentration, while in aggrecan and its complexes with HA, the relaxation rate is insensitive to the presence of calcium.

The utilization of non-animal-derived materials to imitate cartilage is critical for the advancement of plant-based simulated meat. In this study, gellan gum (GG), konjac glucomannan (KGM), and wheat fiber (WF) were used to construct hydrogel, and the mechanical strength, water properties, and microstructure were regulated by constructing Ca 2+ cross-links and moisture control. The hardness, chewiness, resilience, shear force, and shear energy of the Ca 2+ cross-linked samples were significantly improved. Extrusion dehydration further changes the related mechanical properties of the hydrogel and results in a tighter microstructure. The findings suggest that the establishment of Ca 2+ cross-links and water regulation are efficacious techniques for modifying the texture of the GG/KGM/WF composite hydrogel. Correlation analysis and sensory evaluation showed that the test indexes and sensory scores of the samples with Ca 2+ crosslinking and 80 % moisture content were similar to chicken breast cartilage, and the samples with Ca 2+ crosslinking and 70 % moisture content were similar to pig crescent bone. This study presents a framework for designing edible cartilage simulators using polysaccharide hydrogels, with implications for enhancing the resemblance of plant-based meat products to real meat and expanding the range of vegetarian offerings available., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hydrogels are considered as a potential cartilage replacement material based on their structure being similar to natural cartilage, which are of great significance in repairing cartilage defects. However, it is difficult for the existing hydrogels to combine the high load bearing and low friction properties (37 °C) of cartilage through sample methods. Herein, we report a facile and new fabrication strategy to construct the PNIPAm/EYL hydrogel by using the macrophase separation of supersaturated N -isopropylacrylamide (NIPAm) monomer solution to promote the formation of liposomes from egg yolk lecithin (EYL) and asymmetric template method. The PNIPAm/EYL hydrogels possess a relatively high compressive strength (more than 12 MPa), fracture energy (9820 J/m 2 ), good fatigue resistance, lubricating properties, and excellent biocompatibility. Compared with the PNIPAm hydrogel, the friction coefficient (COF 0.046) of PNIPAm/EYL hydrogel is reduced by 50%. More importantly, the COF (0.056) of PNIPAm/EYL hydrogel above lower critical solution temperature (LCST) does not increase significantly, exhibiting heat-tolerant lubricity. The finite element analysis further proves that PNIPAm/EYL hydrogel can effectively disperse the applied stress and dissipate energy under load conditions. This work not only provides new insights for the design of high-strength lubricating hydrogels but also lays a foundation for the treatment of cartilage injury as a substitute material.

This study presents new insights into the potential role of polyelectrolyte interfaces in regulating low friction and interstitial fluid pressurization of cartilage. Polymer brushes composed of hydrophilic 3-sulfopropyl methacrylate potassium salt (SPMK) tethered to a PEEK substrate (SPMK- g -PEEK) are a compelling biomimetic solution for interfacing with cartilage, inspired by the natural lubricating biopolyelectrolyte constituents of synovial fluid. These SPMK- g -PEEK surfaces exhibit a hydrated compliant layer approximately 5 μm thick, demonstrating the ability to maintain low friction coefficients (μ ∼ 0.01) across a wide speed range (0.1-200 mm/s) under physiological loads (0.75-1.2 MPa). A novel polyelectrolyte-enhanced tribological rehydration mechanism is elucidated, capable of recovering up to ∼12% cartilage strain and subsequently facilitating cartilage interstitial fluid recovery, under loads ranging from 0.25 to 2.21 MPa. This is attributed to the combined effects of fluid confinement within the contact gap and the enhanced elastohydrodynamic behavior of polymer brushes. Contrary to conventional theories that emphasize interstitial fluid pressurization in regulating cartilage lubrication, this work demonstrates that SPMK- g -PEEK's frictional behavior with cartilage is independent of these factors and provides unabating aqueous lubrication. Polyelectrolyte-enhanced tribological rehydration can occur within a static contact area and operates independently of known mechanisms of cartilage interstitial fluid recovery established for converging or migrating cartilage contacts. These findings challenge existing paradigms, proposing a novel polyelectrolyte-cartilage tribological mechanism not exclusively reliant on interstitial fluid pressurization or cartilage contact geometry. The implications of this research extend to a broader understanding of synovial joint lubrication, offering insights into the development of joint replacement materials that more accurately replicate the natural functionality of cartilage.

Background: In the ageing population, issues with bone and joint health are highly prevalent. Both beneficial and potential risks of dairy products on bone and joint health are reported in epidemiological studies. Furthermore, the phosphorus (P) load from dairy could potentially lead to unfavorable changes in P metabolism., Objective: To investigate the effect of dairy intake on markers of bone and joint metabolism and P metabolism in an intervention study with high and low dairy intake., Methods: In a post hoc analysis of a randomized cross-over trial with overweight adults, the effect of a standardized high dairy intake [HDI (5-6 dairy portions per day) versus low dairy intake (LDI, ≤ 1 dairy portion/day)] for 6 weeks on markers of bone and joint health was assessed using enzyme-linked immunosorbent assays and electrochemiluminescence immunoassays. Markers indicative for cartilage breakdown, including urinary CTX-II, serum COMP and 4-hydroxyproline, and markers indicative for bone remodeling, such as serum CTX-I, PTH, 25(OH)D, osteocalcin, P1NP and FGF23, were investigated using linear mixed models. Furthermore, changes in P metabolism, including the main phosphate-regulating hormone FGF23 were explored., Results: This study was completed by 46 adults (57% female, age 59 ± 4 years, BMI 28 ± 2 kg/m 2 ). Following HDI, markers such as urinary CTX-II excretion, COMP, 25(OH)D, PTH and CTX-I were significantly lower after HDI, as compared to LDI. For example, CTX-II excretion was 1688 ng/24 h at HDI, while it was 2050 ng/24 h at LDI (p < 0.001). Concurrently, P intake was higher at HDI than at LDI (2090 vs 1313 mg/day, p < 0.001). While plasma P levels did not differ (1.03 vs 1.04 mmol/L in LDI, p = 0.36), urinary P excretion was higher at HDI than at LDI (31 vs 28 mmol/L, p = 0.04). FGF23 levels tended to be higher at HDI than at LDI (76.3 vs. 72.9 RU/mL, p = 0.07)., Conclusions: HDI, as compared to LDI, reduced markers that are indicative for joint and bone resorption and bone turnover. No changes in P metabolism were observed., Clinical Trial Registry: This trial was registered at https://trialsearch.who.int/Trial2.aspx?TrialID=NTR4899 as NTR4899., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Photo-cross-linked hydrogels, which respond to light and induce structural or morphological transitions, form a microenvironment that mimics the extracellular matrix of native tissue. In the last decades, photo-cross-linked hydrogels have been widely used in cartilage and osteochondral tissue engineering due to their good biocompatibility, ease of fabrication, rapid in situ gel-forming ability, and tunable mechanical and degradable properties. In this review, we systemically summarize the different types and physicochemical properties of photo-cross-linked hydrogels (including the materials and photoinitiators) and explore the biological properties modulated through the incorporation of additives, including cells, biomolecules, genes, and nanomaterials, into photo-cross-linked hydrogels. Subsequently, we compile the applications of photo-cross-linked hydrogels with a specific focus on cartilage and osteochondral repair. Finally, current limitations and future perspectives of photo-cross-linked hydrogels are also discussed.

Objectives: This study aimed to compare the extracellular matrix of primary cartilage with the secondary cartilage of chicks using immunohistochemical analyses in order to understand the features of chick secondary chondrogenesis., Methods: Immunohistochemical analysis was performed on the extracellular matrix of quadrate (primary), squamosal, surangular, and anterior pterygoid secondary cartilages using various antibodies targeting the extracellular matrix of cartilage and bone., Results: The localization of collagen types I, II, and X, versican, aggrecan, hyaluronan, link protein, and tenascin-C was identified in the quadrate cartilage, with variations within and between the regions. Newly formed squamosal and surangular secondary cartilages showed simultaneous immunoreactivity for all molecules investigated. However, collagen type X immunoreactivity was not observed, and there was weak immunoreactivity for versican and aggrecan in the anterior pterygoid secondary cartilage., Conclusions: The immunohistochemical localization of extracellular matrix in the quadrate (primary) cartilage was comparable to that of long bone (primary) cartilage in mammals. The fibrocartilaginous nature and rapid differentiation into hypertrophic chondrocytes, which are known structural features of secondary cartilage, were confirmed in the extracellular matrix of squamosal and surangular secondary cartilages. Furthermore, these tissues appear to undergo developmental processes similar to those in mammals. However, the anterior pterygoid secondary cartilage exhibited unique features that differed from primary and other secondary cartilages, suggesting it is formed through a distinct developmental process., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Abnormal N -glycosylation has been shown to play an important role in the pathogenesis of multiple diseases. However, little is known about the relationship between N -glycosylation and knee osteoarthritis (KOA) progression at the tissue level. Thus, the aim of this study was to quantify the cartilage histomorphometric changes in formalin-fixed paraffin-embedded (FFPE) tissue collected from the lateral and medial compartments of the tibial plateau KOA patients ( n = 8). Subsequently, N -glycans were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) followed by in situ MS/MS fragmentation. Overall, the Osteoarthritis Research Society International (OARSI) histological grade and cartilage surface fibrillation index were significantly higher, and chondrocyte size in the superficial zone was much larger, for the medial high-loaded cartilage compared to the lateral less-loaded cartilage. Among 92 putative N -glycans observed by MALDI-MSI, 3 complex-type N -glycans, (Hex) 4 (HexNAc) 3 , (Hex) 4 (HexNAc) 4 , and (Hex) 5 (HexNAc) 4 , and 1 oligomannose-type N -glycan, (Hex) 9 (HexNAc) 2 , were significantly higher in intensity in the medial cartilage compared to the lateral cartilage, whereas 2 tetra-antennary fucosylated-type N -glycans, (Hex) 3 (HexNAc) 6 (Fuc) 2 and (Hex) 3 (HexNAc) 6 (Fuc) 3 , were significantly higher in intensity in the lateral cartilage than the medial cartilage. Our findings indicate that complex-type N -glycans are associated with higher severity of cartilage degeneration and may influence the cellular processes of KOA.

Load-bearing tissues, such as muscle and cartilage, exhibit high elasticity, high toughness and fast recovery, but have different stiffness (with cartilage being significantly stiffer than muscle) 1-8 . Muscle achieves its toughness through finely controlled forced domain unfolding-refolding in the muscle protein titin, whereas articular cartilage achieves its high stiffness and toughness through an entangled network comprising collagen and proteoglycans. Advancements in protein mechanics and engineering have made it possible to engineer titin-mimetic elastomeric proteins and soft protein biomaterials thereof to mimic the passive elasticity of muscle 9-11 . However, it is more challenging to engineer highly stiff and tough protein biomaterials to mimic stiff tissues such as cartilage, or develop stiff synthetic matrices for cartilage stem and progenitor cell differentiation 12 . Here we report the use of chain entanglements to significantly stiffen protein-based hydrogels without compromising their toughness. By introducing chain entanglements 13 into the hydrogel network made of folded elastomeric proteins, we are able to engineer highly stiff and tough protein hydrogels, which seamlessly combine mutually incompatible mechanical properties, including high stiffness, high toughness, fast recovery and ultrahigh compressive strength, effectively converting soft protein biomaterials into stiff and tough materials exhibiting mechanical properties close to those of cartilage. Our study provides a general route towards engineering protein-based, stiff and tough biomaterials, which will find applications in biomedical engineering, such as osteochondral defect repair, and material sciences and engineering., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Numerous studies have shown that type II collagen (CII) has a potential role in the treatment of rheumatoid arthritis. However, most of the current studies have used terrestrial animal cartilage as a source of CII extraction, with fewer studies involving marine organisms. Based on this background, collagen (BSCII) was isolated from blue shark ( Prionace glauca ) cartilage by pepsin hydrolysis and its biochemical properties including protein pattern, total sugar content, microstructure, amino acid composition, spectral characteristics and thermal stability were further investigated in the present study. The SDS-PAGE results confirmed the typical characteristic of CII, comprising three identical α 1 chains and its dimeric β chain. BSCII had the fibrous microstructure typical of collagen and an amino acid composition represented by high glycine content. BSCII had the typical UV and FTIR spectral characteristics of collagen. Further analysis revealed that BSCII had a high purity, while its secondary structure comprised 26.98% of β-sheet, 35.60% of β-turn, 37.41% of the random coil and no α-helix. CD spectra showed the triple helical structure of BSCII. The total sugar content, denaturation temperature and melting temperature of BSCII were (4.20 ± 0.03)%, 42 °C and 49 °C, respectively. SEM and AFM images confirmed a fibrillar and porous structure of collagen and denser fibrous bundles formed at higher concentrations. Overall, CII was successfully extracted from blue shark cartilage in the present study, and its molecular structure was intact. Therefore, blue shark cartilage could serve as a potential source for CII extraction with applications in biomedicine.

N-Glycan alterations contribute to the pathophysiology and progression of various diseases. However, the involvement of N-glycans in knee osteoarthritis (KOA) progression at the tissue level, especially within articular cartilage, is still poorly understood. Thus, the aim of this study was to spatially map and identify KOA-specific N-glycans from formalin-fixed paraffin-embedded (FFPE) osteochondral tissue of the tibial plateau relative to cadaveric control (CTL) tissues. Human FFPE osteochondral tissues from end-stage KOA patients (n=3) and CTL individuals (n=3), aged >55 years old, were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Overall, it was revealed that 22 N-glycans were found in the cartilage region of KOA and CTL tissue. Of those, 15 N-glycans were more prominent in KOA cartilage than CTL cartilage. We then compared sub-regions of KOA and CTL tissues based on the Osteoarthritis Research Society International (OARSI) histopathological grade (1 to 6), where 1 is an intact cartilage surface and 6 is cartilage surface deformation. Interestingly, three specific complex-type N-glycans, (Hex) 4 (HexNAc) 3 , (Hex) 4 (HexNAc) 4 , and (Hex) 5 (HexNAc) 4 , were found to be localized to the superficial fibrillated zone of degraded cartilage (KOA OARSI 2.5-4), compared to adjacent cartilage with less degradation (KOA OARSI 1-2) or relatively healthy cartilage (CTL OARSI 1-2). Our results demonstrate that N-glycans specific to degraded cartilage in KOA patients have been identified at the tissue level for the first time. The presence of these N-glycans could further be evaluated as potential diagnostic and prognostic biomarkers., (© 2022. The Author(s).)

In this paper, a combination of hot-pressure, enzymatic hydrolysis and membrane separation process is used for efficiently and environmentally friendly extraction of chondroitin sulfate (CS) from large hybrid sturgeon cartilage, namely, HPCS. The recovery and yield of CS were 93.68% and 36.47% under the optimized conditions. Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography (HPLC) indicated that the HPCS was composed of monosulfated disaccharides in position 6 and 4 of the N-acetyl-D-galactosamine (58.38% and 27.34%, respectively) and nonsulfated disaccharide (14.29%), which was similar to the composition of CS extracted by dilute alkali-enzymatic hydrolysis-chemical precipitation from large hybrid sturgeon cartilage (SCS). The wound healing results indicated that HPCS could promote cell migration and proliferation, alleviate inflammation and facilitate angiogenesis, which results in its excellent wound treatment activity. These results provide theoretical and practical significance for the production and application of chondroitin sulfate., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In the present study, a selenium-chondroitin sulfate (SeCS) was synthesized by the sodium selenite (Na 2 SeO 3 ) and ascorbic acid (Vc) redox reaction using chondroitin sulfate derived from shark cartilage as a template, and characterized by SEM, SEM-EDS, FTIR and XRD. Meanwhile, its stability was investigated at different conditions of pH and temperatures. Besides, its antioxidant activity was further determined by the DPPH and ABTS assays. The results showed the SeCS with the smallest particle size of 131.3 ± 4.4 nm and selenium content of 33.18% was obtained under the optimal condition (CS concentration of 0.1 mg/mL, mass ratio of Na 2 SeO 3 to Vc of 1:8, the reaction time of 3 h, and the reaction temperature of 25 °C). SEM image showed the SeCS was an individual and spherical nanostructure and its structure was evidenced by FTIR and XRD. Meanwhile, SeCS remained stable at an alkaline pH and possessed good storage stability at 4 °C for 28 days. The results on scavenging free radical levels showed that SeCS exhibited significantly higher antioxidant activity than SeNPs and CS, indicating that SeCS had a potential antioxidant effect.

The aim of the study was to optimize preprocessing of sparse infrared spectral data. The sparse data were obtained by reducing broadband Fourier transform infrared attenuated total reflectance spectra of bovine and human cartilage, as well as of simulated spectral data, comprising several thousand spectral variables into datasets comprising only seven spectral variables. Different preprocessing approaches were compared, including simple baseline correction and normalization procedures, and model-based preprocessing, such as multiplicative signal correction (MSC). The optimal preprocessing was selected based on the quality of classification models established by partial least squares discriminant analysis for discriminating healthy and damaged cartilage samples. The best results for the sparse data were obtained by preprocessing using a baseline offset correction at 1800 cm -1 , followed by peak normalization at 850 cm -1 and preprocessing by MSC.

Low-molecular-weight chondroitin sulfate (CS) has attracted widespread attention due to its better bioavailability and bioactivity than native CS. In this study, a low-molecular-weight CS (named SCS-F2) was prepared from hybrid sturgeon (Acipenser schrenckii × Huso dauricus) cartilage by enzymatic depolymerization with high in vitro absorption and anti-cancer activity. The structure of SCS-F2 was characterized and the in vivo biodistribution and colorectal cancer prevention effect was investigated. The results revealed that SCS-F2 consisted of 48.84% ΔDi-6S [GlcUAβ1-3GalNAc(6S)], 32.11% ΔDi-4S [GlcUAβ1-3GalNAc(4S)], 16.05% ΔDi-2S,6S [GlcUA(2S)β1-3GalNAc(6S)] and 3.0% ΔDi-0S [GlcUAβ1-3GalNAc]. Animal study showed that the SCS-F2 could be effectively absorbed and delivered to the tumor site and significantly prevented the growth of HT-29 xenograft by inhibiting cell proliferation and inducing apoptosis without showing any negative effect to normal tissues. Therefore, SCS-F2 could be developed as a potential nutraceutical to protect against colorectal cancer., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We consider the longitudinal quadrupole relaxation rate enhancement (QRE) of a 1 H nucleus due to the time fluctuations of the local dipolar magnetic field created by a close quadrupole 14 N nucleus, the electric-field gradient (EFG) Hamiltonian of which changes with time because of vibrations/distortions of its chemical environment. The QRE is analytically expressed as a linear combination of the cosine Fourier transforms of the three quantum time auto-correlation functions G AA (t) of the 14 N spin components along the principal axes A = X, Y, and Z of the mean (time-averaged) EFG Hamiltonian. Denoting the three transition frequencies between the energy levels of this mean Hamiltonian by ν A , the functions G AA (t) oscillate at frequencies ν A + s A /(2π) with mono-exponential decays of relaxation times τ A , where the frequency dynamic shifts s A and the relaxation times τ A are closed expressions of the magnitude of the fluctuations of the instantaneous EFG Hamiltonian about its mean and of the characteristic fluctuation time. Thus, the theoretical QRE is the sum of three Lorentzian peaks centered at ν A + s A /(2π) with full widths at half maxima 1/(πτ A ). The predicted peak widths are nearly equal. The predicted dynamic shifts of the peaks are much smaller than their widths and amazingly keep proportional to the transition frequencies ν A for reasonably fast EFG fluctuations. The theory is further improved by correcting the transition frequencies by the 14 N Zeeman effects of second order. It is successfully applied to reinterpret the QRE pattern measured by Broche, Ashcroft, and Lurie [Magn. Reson. Med. 68, 358 (2012)] in normal cartilage.

Decellularized extra-cellular matrix (ECM) has been studied as an alternative to anti-adhesive biomaterials and cartilage acellular matrix (CAM) has been shown to inhibit postoperative adhesion in several organs. This study aimed to evaluate the suitability of glutaraldehyde (GA) crosslinked CAM-films as anti-adhesion barriers for peripheral nerve injury. The films were successfully fabricated and showed improved physical properties such as mechanical strength, swelling ratio, and lengthened degradation period while maintaining the microstructure and chemical composition after GA crosslinking. In the in vitro study of CAM-film, the dsDNA content met the recommended limit of decellularization and more than 70% of the major ECM components were preserved after decellularization. The adhesion and proliferation of seeded human umbilical vein endothelial cells and fibroblasts were significantly lower in CAM-film than in control, but similar with Seprafilm. However, the CAM-film extract did not show cytotoxicity. In the in vivo study, the peri-neural fibrosis was thicker, adhesion score higher, and peri-neural collagen fibers more abundant in the control group than in the CAM-film group. The total number of myelinated axons was significantly higher in the CAM-film group than in the control group. The inflammatory marker decreased with time in the CAM-film group compared to that in the control group, whereas the nerve regenerative marker expression was maintained. Moreover, the ankle angles at contracture and toe-off were higher in the CAM film-treated rats than in the control rats. GA-crosslinked CAM films may be used during peripheral nerve surgery to prevent peri-neural adhesion and enhance nerve functional recovery., (© 2021 John Wiley & Sons Ltd.)

Here, engineered cartilage-like scaffold using an extracellular matrix (ECM) from sturgeon fish cartilage provided a chondroinductive environment to stimulate cartilaginous matrix synthesis in human adipose stem cells (hASCs). Three dimensional porous and degradable fish cartilage ECM-derived scaffold (FCS) was produced using a protocol containing chemical decellularization, enzymatic solubilization, freeze-drying and EDC-crosslinking treatments and the effect of different ECM concentrations (10, 20, 30, and 40 mg/ml) on prepared scaffolds was investigated through physical, mechanical and biological analysis. The histological and scanning electron microscopy analysis revealed the elimination of the cell fragments and a 3-D interconnected porous structure, respectively. Cell viability assay displayed no cytotoxic effects. The prepared porous constructs of fish cartilage ECM were seeded with hASCs for 21 days and compared to collagen (Col) and collagen-10% hyaluronic acid (Col-HA) scaffolds. Cell culture results evidenced that the fabricated scaffolds could provide a proper 3-D structure to support the adhesion, proliferation and chondrogenic differentiation of hASCs considering the synthesis of specific proteins of cartilage, collagen type II (Col II) and aggrecan (ACAN). Based on the results of the present study, it can be concluded that the porous scaffold derived from fish cartilage ECM possesses an excellent potential for cartilage tissue engineering., (© 2021 Wiley Periodicals LLC.)

Two-dimensional NMR spectroscopies are one of the most frequently used techniques for the structural determination of carbohydrates. However, the data analysis is challenging because of the signal overlap in the 1 H homonuclear correlation spectra. We attempted to explore a general strategy for the structural determination of carbohydrates by combined multi-dimensional spectroscopies. The strategy was applied to a human milk oligosaccharide lacto-N-difucohexaose I, that has been previously studied by conventional two-dimensional NMR spectroscopy. Assignment of the intra-residue resonances of the hexasaccharide using the three-dimensional spectrum was straightforward. Consequently, data analysis of the multi-dimensional spectra was significantly simplified, leading to a quicker determination of the intra- and inter-residue connections in the hexasaccharide. Application of the NMR strategy to chondroitin sulfate from bovine cartilage revealed two repeating disaccharide regions of the A and C units of chondroitin sulfate, indicating the high potential of this technique for the structural determination of complex polysaccharides., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The in situ gelation of injectable precursors is desirable in the field of tissue regeneration, especially in the context of irregular defect filling. The current driving forces for fast gelation include the phase-transition of thermally sensitive copolymers, click chemical reactions with tissue components, and metal coordination effect. However, the rapid formation of tough hydrogels remains a challenge. Inspired by aerobic metabolism, we herein propose a tissue-fluid-triggered cascade enzymatic polymerization process catalyzed by glucose oxidase and ferrous glycinate for the ultrafast gelation of acryloylated chondroitin sulfates and acrylamides. The highly efficient production of carbon radicals and macromolecules contribute to rapid polymerization for soft tissue augmentation in bone defects. The copolymer hydrogel demonstrated the regeneration-promoting capacity of cartilage. As the first example of using artificial enzyme complexes for in situ polymerization, this work offers a biomimetic approach to the design of strength-adjustable hydrogels for bio-implanting and bio-printing applications., (© 2021 Wiley-VCH GmbH.)

Polyvinyl alcohol (PVA) hydrogel has gained interest in cartilage repair because of its highly swollen, porosity, and viscoelastic properties. However, PVA has some deficiencies, such as its poor biocompatibility and microstructure. This research aimed to design novel hydroxyapatite (HA)-collagen (COL)-PVA hydrogels. COL was added to improve cell biocompatibility, and the microstructure of the hydrogels was controlled by fused deposition modeling (FDM). The feasibility of the COL-HA-PVA hydrogels in cartilage repair was evaluated by in vitro and in vivo experiments. The scanning electron microscopy results showed that the hybrid hydrogels had interconnected macropore structures that contained a COL reticular scaffold. The diameter of the macropore was 1.08-1.85 mm, which corresponds to the diameter of the denatured PVA column. The chondrocytes were then seeded in hydrogels to assess the cell viability and formation of the cartilage matrix. The in vitro results revealed excellent cellular biocompatibility. Osteochondral defects (8 mm in diameter and 8 mm in depth) were created in the femoral trochlear of goats, and the defects were implanted with cell-seeded hydrogels, cell-free hydrogels, or a blank control. The in vivo results showed that the COL-HA-PVA hydrogels effectively repaired cartilage defects, especially the conditions inoculated with chondrocyte in advance. This research suggests that the COL-HA-PVA hydrogels have promising application in cartilage repair., (© 2021. The Author(s).)

Fractures are the most common large-organ, traumatic injury in humans. The fracture healing stage includes the inflammatory stage (0-5d), cartilage callus stage (5-14d) and hard callus stage (14-21d). All mice underwent open tibial fracture surgery and were treated with saline, Glu or SCII for 21d. Calluses were harvested 5d, 10d and 21d after fracture. Compared with the model group, SCII significantly decreased TNF-α and increased aggrecan serum levels by 5d. H&E results showed that fibrous calluses were already formed in the SCII group and that chondrocytes had begun to proliferate. By 10d, the chondrocytes in the SCII group became hypertrophic and mineralized, and the serum TGF-β and Col-Iα levels were significantly increased, which indicated that the mice with SCII treatment rapidly passed the cartilage repair period and new bone formation was accelerated. Skeletal muscle repaired bones through muscle paracrine factors. IGF-1 and irisin are the two major secretory cytokines. The results showed that the content of muscle homogenate IGF-1 in the SCII group reached the peak at 10d, followed by the up-regulation of Ihh, Patched, Gli1 and Col10α in the callus through the bone surface receptor IGF-1R. Besides, SCII also significantly elevated the muscle irisin level (10 and 21d), and then increased Wnt10b, LRP5, β-catenin and Runx2 expression in the callus by receptor αVβ5. These results suggest that SCII can accelerate the process of endochondral osteogenesis and promote fracture healing through activating the Ihh/PThrp and Wnt/β-catenin pathways by regulating muscle paracrine factors. To our knowledge, this is the first study to investigate the effect of marine-derived collagen on fracture healing. This study may provide a theoretical basis for the high-value application of the laryngeal cartilage of squid in the future.

Chondroitin sulfate (CS) chains containing GlcUAβ1-3GalNAc(4S,6S) (E unit) have been shown to be involved in various physiological and pathological processes. However, commercial E unit-rich CS (CS-E) is difficult to produce on a large scale due to expensive and limited squid cartilage resources. In this study, a novel CS-E (CS-nE) was isolated from the cheap and abundant cartilage of the giant squid Dosidicus gigas. The CS-nE has a surprisingly large molecular mass of 696 kDa and a relatively high E unit proportion (44.5 %). It can interact with various growth factors, including HGF, bFGF, pleiotrophin, and HB-EGF, with high affinity, and exhibits dose-dependent anti-metastatic activity. Furthermore, the E unit-rich decasaccharide selectively prepared from CS-nE has been shown to be the minimal functional domain with the strongest antitumor metastatic activity. Taken together, CS-nE will be a very promising candidate for the development of CS-E-based pharmaceutical products., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Various hydrogel systems have been developed as biomaterial inks for bioprinting, including natural and synthetic polymers. However, the available biomaterial inks, which allow printability, cell viability, and user-defined customization, remains limited. Incorporation of biological extracellular matrix materials into tunable synthetic polymers can merge the benefits of both systems towards versatile materials for biofabrication. The aim of this study was to develop novel, cell compatible dual-component biomaterial inks and bioinks based on poly(vinyl alcohol) (PVA) and solubilized decellularized cartilage matrix (SDCM) hydrogels that can be utilized for cartilage bioprinting. In a first approach, PVA was modified with amine groups (PVA-A), and mixed with SDCM. The printability of the PVA-A/SDCM formulations cross-linked by genipin was evaluated. On the second approach, the PVA was functionalized with cis-5-norbornene-endo-2,3-dicarboxylic anhydride (PVA-Nb) to allow an ultrafast light-curing thiol-ene cross-linking. Comprehensive experiments were conducted to evaluate the influence of the SDCM ratio in mechanical properties, water uptake, swelling, cell viability, and printability of the PVA-based formulations. The studies performed with the PVA-A/SDCM formulations cross-linked by genipin showed printability, but poor shape retention due to slow cross-linking kinetics. On the other hand, the PVA-Nb/SDCM showed good printability. The results showed that incorporation of SDCM into PVA-Nb reduces the compression modulus, enhance cell viability, and bioprintability and modulate the swelling ratio of the resulted hydrogels. Results indicated that PVA-Nb hydrogels containing SDCM could be considered as versatile bioinks for cartilage bioprinting.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) has been successfully used to elucidate the relative abundance and spatial mapping of analytes in situ. Currently, sample preparation workflows for soft formalin-fixed paraffin-embedded (FFPE) tissues, such as brain, liver, kidney, and heart, have been successfully developed. However, hard tissues, such as cartilage-bone, tooth, and whole mouse body, have resulted in the loss of morphology or tissue during the heat-induced epitope retrieval (HIER) step on commercially available conductive indium tin oxide (ITO) slides. Therefore, we have successfully developed a novel and cost-effective sample preparation workflow in which commercial conductive ITO slides are pre-coated with gelatin and chromium potassium sulfate dodecahydrate to improve the adherence of FFPE human osteoarthritic cartilage-bone tissue sections. Gelatin-coated ITO slides also resulted in overall higher N-glycan signal intensity for not only FFPE osteoarthritic cartilage-bone tissue but also for FFPE hard-boiled egg white used as a quality control to assess the quality of sample preparation and MALDI-MSI acquisition. In summary, we present a novel straightforward workflow to improve slide adherence and morphological preservation of FFPE cartilage-bone tissue sections during HIER while improving the signal intensity of N-glycans spatially mapped from the same tissue sections by MALDI-MSI.

Advances in vibrational spectroscopy have propelled new insights into the molecular composition and structure of biological tissues. In this review, we discuss common modalities and techniques of vibrational spectroscopy, and present key examples to illustrate how they have been applied to enrich the assessment of connective tissues. In particular, we focus on applications of Fourier transform infrared (FTIR), near infrared (NIR) and Raman spectroscopy to assess cartilage and bone properties. We present strengths and limitations of each approach and discuss how the combination of spectrometers with microscopes (hyperspectral imaging) and fiber optic probes have greatly advanced their biomedical applications. We show how these modalities may be used to evaluate virtually any type of sample (ex vivo, in situ or in vivo) and how "spectral fingerprints" can be interpreted to quantify outcomes related to tissue composition and quality. We highlight the unparalleled advantage of vibrational spectroscopy as a label-free and often nondestructive approach to assess properties of the extracellular matrix (ECM) associated with normal, developing, aging, pathological and treated tissues. We believe this review will assist readers not only in better understanding applications of FTIR, NIR and Raman spectroscopy, but also in implementing these approaches for their own research projects.

Immunostaining is the process of identifying proteins in tissue sections by incubating the sample with antibodies specific to the protein of interest, then visualizing the bound antibody using a chromogen (immunohistochemistry or IHC) or fluorescence (immunofluorescence or IF). Unlike in situ hybridization, which identifies gene transcripts in cells, immunostaining identifies the products themselves and provides information about their localization within cells (nuclear, cytoplasmic, or membrane) or extracellular matrix. This can be particularly important in the context of bone and cartilage because they contain many cell types as well as matrix components, each with distinct protein expression patterns. As the number of antibodies continues to grow, this technique has become vital for research laboratories studying the skeleton. Here, we describe a detailed protocol for antibody-based in situ analysis of bone and associated tissues, addressing specific issues associated with staining of hard and matrix-rich tissues.

With the worldwide increase of fisheries, fish wastes have had a similar increase, alternatively they can be seen as a source of novel substances for the improvement of society's wellbeing. Elasmobranchs are a subclass fished in high amounts, with some species being mainly bycatch. They possess an endoskeleton composed mainly by cartilage, from which chondroitin sulfate is currently obtained. Their use as a viable source for extraction of type II collagen has been hypothesized with the envisaging of a biomedical application, namely in biomaterials production. In the present work, raw cartilage from shark ( Prionace glauca ) and ray ( Zeachara chilensis and Bathyraja brachyurops ) was obtained from a fish processing company and submitted to acidic and enzymatic extractions, to produce acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC). From all the extractions, P. glauca PSC had the highest yield (3.5%), followed by ray ASC (0.92%), ray PSC (0.50%), and P. glauca ASC (0.15%). All the extracts showed similar properties, with the SDS-PAGE profiles being compatible with the presence of both type I and type II collagens. Moreover, the collagen extracts exhibited the competence to maintain their conformation at human basal temperature, presenting a denaturation temperature higher than 37 °C. Hydrogels were produced using P. glauca PSC combined with shark chondroitin sulfate, with the objective of mimicking the human cartilage extracellular matrix. These hydrogels were cohesive and structurally-stable at 37 °C, with rheological measurements exhibiting a conformation of an elastic solid when submitted to shear strain with a frequency up to 4 Hz. This work revealed a sustainable strategy for the valorization of fisheries' by-products, within the concept of a circular economy, consisting of the use of P. glauca , Z. chilensis, and B. brachyurops cartilage for the extraction of collagen, which would be further employed in the development of hydrogels as a proof of concept of its biotechnological potential, ultimately envisaging its use in marine biomaterials to regenerate damaged cartilaginous tissues.

The development of new technologies to produce three-dimensional and biocompatible scaffolds associated with high-end cell culture techniques have shown to be promising for the regeneration of tissues and organs. Some biomedical devices, as meniscus prosthesis, require high flexibility and tenacity and such features are found in polyurethanes which represent a promising alternative. The Poly(PCL-TMC)urethane here presented, combines the mechanical properties of PCL with the elasticity attributed by TMC and presents great potential as a cellular carrier in cartilage repair. Scanning electron microscopy showed the presence of interconnected pores in the three-dimensional structure of the material. The scaffolds were submitted to proliferation and cell differentiation assays by culturing mesenchymal stem cells in bioreactor. The tests were performed in dynamic flow mode at the rate of 0.4 mL/min. Laser scanning confocal microscopy analysis showed that the flow rate promoted cell growth and cartilage ECM synthesis of aggrecan and type II collagen within the Poly(PCL-TMC)urethane scaffolds. This study demonstrated the applicability of the polymer as a cellular carrier in tissue engineering, as well as the ECM was incremented only when under oriented flow rate stimuli. Therefore, our results may also provide data on how oriented flow rate in dynamic bioreactors culture can influence cell activity towards cartilage ECM synthesis even when specific molecular stimuli are not present. This work addresses new perspectives for future clinical applications in cartilage tissue engineering when the molecular factors resources could be scarce for assorted reasons.

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted and purified from the cartilages of skate and sturgeon. Their typical structure and physicochemical properties were evaluated by circular dichroism (CD), X-ray diffraction (XRD), and so on. Results showed that the extracted collagen was likely identified as collagen-II composed of three α-chains (135 kDa), with the typical peptide sequence of Gly-X-Y. It showed the collagen retained the native and intact triple helical structure, and its intensity ratio of the positive and negative absorption peaks (Rpn) was 0.19-0.25. In addition, the extracted collagen exhibited obvious self-assembly behavior with the concentration above 0.3 mg/mL, the adjustment of pH 7.4-7.6 and the NaCl concentration of 120 mmol/L. The critical aggregate mass concentrations of pepsin-soluble collagens from skate and sturgeon were 0.93 and 0.86 g/L, respectively. Therefore, collagens from skate and sturgeon cartilages have potential commercial application., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5' and 3' end, with >99% having one of two seed sequences (5' bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3'UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140 -null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR., (© 2020 Woods et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Cartilage is avascular with limited to no regenerative capacity, so its loss could be a challenge for reconstructive surgery. Current treatment options for damaged cartilage are also limited. In this aspect there is a tremendous need to develop an ideal cartilage-mimicking biomaterial that could repair maxillofacial defects. Considering this fact in this study we have prepared twelve silicone-based materials (using Silicone 40, 60, and 80) reinforced with hydroxyapatite, tri-calcium phosphate, and titanium dioxide which itself has proven their efficacy in several studies and able to complement the shortcomings of using silicones. Among the mechanical properties (Young's modulus, tensile strength, percent elongation, and hardness), hardness of Silicone-40 showed similarities with goat ear (P > .05). Silicone peaks have been detected in FTIR. Both AFM morphology and SEM images of the samples confirmed more roughed surfaces. All the materials were nonhemolytic in hemocompatibility tests, but among the twelve materials S2, S3, S5, and S6 showed the least hemolysis. For all tested bacterial strains, adherence was lower on each material than that grown on the plain industrial silicone material which was used as a positive control. S2, S3, S5, and S6 samples were selected as the best based on mechanical characterizations, surface characterizations, in vitro hemocompatibility tests and bacterial adherence activity. So, outcomes of this present study would be promising when developing ideal cartilage-mimicking biocomposites and their emerging applications to treat maxillofacial defects due to cartilage damage., (© 2020 International Center for Artificial Organs and Transplantation and Wiley Periodicals LLC.)

Polycaprolactone (PCL) electrospun scaffolds have been widely investigated for cartilage repair application. However, their hydrophobicity and small pore size has been known to prevent cell attachment, proliferation and migration. Here, PCL was blended with gelatin (GEL) combining the favorable biological properties of GEL with the good mechanical performance of the former. Also, polyethylene glycol (PEG) particles were introduced during the electrospinning of the polymers blend by simultaneous electrospraying. These particles were subsequently removed resulting in fibrous scaffolds with enlarged pore size. PCL, GEL and PEG scaffolds formulations were developed and extensively structural and biologically characterized. GEL incorporation on the PCL scaffolds led to a considerably improved cell attachment and proliferation. A substantial pore size and interconnectivity increase was obtained, allowing cell infiltration through the porogenic scaffolds. All together these results suggest that this combined approach may provide a potentially clinically viable strategy for cartilage regeneration.

Novel dynamic hydrogels were prepared from O-carboxymethyl chitosan (CMCS) and a water soluble dynamer Dy via crosslinking by imine bond formation using an environmentally friendly method. Dy was synthesized by reaction of Benzene-1,3,5-tricarbaldehyde with Jeffamine. The resulting soft hydrogels exhibit a porous and interconnected morphology, storage modulus up to 1400 Pa, and excellent pH-sensitive swelling properties. The swelling ratio is relatively low at acidic pH due to electrostatic attraction, and becomes exceptionally high up to 7000 % at pH 8 due to electrostatic repulsion. Moreover, hydrogels present outstanding self-healing properties as evidenced by closure of split pieces and rheological measurements. This study opens up a new horizon in the preparation of dynamic hydrogels with great potential for applications in drug delivery, wound dressing, and in particular in tissue engineering as the hydrogels present excellent cytocompatibility., (Copyright © 2020. Published by Elsevier Ltd.)

Hyaluronic acid (HA) and chondroitin sulfate (CS) are valuable bioactive polysaccharides that have been highly used in biomedical and pharmaceutical applications. Extensive research was done to ensure their efficient extraction from marine and terrestrial by-products at a high yield and purity, using specific techniques to isolate and purify them. In general, the cartilage is the most common source for CS, while the vitreous humor is main used source of HA. The developed methods were based in general on tissue hydrolysis, removal of proteins and purification of the target biopolymers. They differ in the extraction conditions, enzymes and/or solvents used and the purification technique. This leads to specific purity, molecular weight and sulfation pattern of the isolated HA and CS. This review focuses on the analysis and comparison of different extraction and purification methods developed to isolate these valuable biopolymers from marine and terrestrial animal by-products., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study was designed to isolate (isolation yield of 36.51 %) and characterise chondroitin sulphate (CS) from skate (Raja porosa) cartilage. Gel permeation chromatography demonstrated that the Raja porosa chondroitin sulphate (RPCS) obtained was a relatively uniform polysaccharide with a molecular weight of 40,752 Da and a purity of 94.0 %. Fourier-transform infrared, nuclear magnetic resonance spectroscopy and strong anion-exchange high-performance liquid chromatography indicated that the content of the predominant GlcA-GalNAc6S unit in RPCS was 65.84 %, which was higher than that in shark CS (53.93 %). Furthermore, RPCS displayed more effective free radical scavenging effects than did shark CS, indicating the potential of RPCS to promote oxidative stress resistance and to act as an antioxidant agent. Skate cartilage could be exploited as a sufficient and sustainable source for the preparation of CS with higher 6S-sulphation, which in turn could be scaled up for use in the pharmaceutical industry., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cartilage damage caused by aging, repeated overloading, trauma, and diseases can result in chronic pain, inflammation, stiffness, and even disability. Unlike other types of tissues (bone, skin, muscle, etc.), cartilage tissue has an extremely weak regenerative capacity. Currently, the gold standard surgical treatment for repairing cartilage damage includes autografts and allografts. However, these procedures are limited by insufficient donor sources and the potential for immunological rejection. After years of development, engineered tissue now provides a valuable artificial replacement for tissue regeneration purposes. Three-dimensional (3D) bioprinting technologies can print customizable hierarchical structures with cells. The objective of the current work was to prepare a 3D-printed three-layer gradient scaffold with lysine-functionalized rosette nanotubes (RNTK) for improving the chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs). Specifically, biologically inspired RNTKs were utilized in our work because they have unique surface chemistry and biomimetic nanostructure to improve cell adhesion and growth. Different ratios of gelatin methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA) were printed into a three-layer GelMA-PEGDA gradient scaffold using a stereolithography-based printer, followed by coating with RNTKs. The pores and channels (∼500 μm) were observed in the scaffold. It was found that the population of ADSCs on the GelMA-PEGDA-RNTK scaffold increased by 34% compared to the GelMA-PEGDA scaffold (control). Moreover, after 3 weeks of chondrogenic differentiation, collagen II, glycosaminoglycan, and total collagen synthesis on the GelMA-PEGDA-RNTK scaffold significantly respectively increased by 59%, 71%, and 60%, as compared to the control scaffold. Gene expression of collagen II α1, SOX 9, and aggrecan in the ADSCs growing on the GelMA-PEGDA-RNTK scaffold increased by 79%, 52%, and 47% after 3 weeks, compared to the controls, respectively. These results indicated that RNTKs are a promising type of nanotubes for promoting chondrogenic differentiation, and the present 3D-printed three-layer gradient GelMA-PEGDA-RNTK scaffold shows considerable promise for future cartilage repair and regeneration.

Hydration lubrication has emerged as a new paradigm for lubrication in aqueous and biological media, accounting especially for the extremely low friction (friction coefficients down to 0.001) of articular cartilage lubrication in joints. Among the ensemble of molecules acting in the joint, phosphatidylcholine (PC) lipids have been proposed as the key molecules forming, in a complex with other molecules including hyaluronic acid (HA), a robust layer on the outer surface of the cartilage. HA, ubiquitous in synovial joints, is not in itself a good boundary lubricant, but binds the PC lipids at the cartilage surface; these, in turn, massively reduce the friction via hydration lubrication at their exposed, highly hydrated phosphocholine headgroups. An important unresolved issue in this scenario is why the free HA molecules in the synovial fluid do not suppress the lubricity by adsorbing simultaneously to the opposing lipid layers, i.e., forming an adhesive, dissipative bridge between them, as they slide past each other during joint articulation. To address this question, we directly examined the friction between two hydrogenated soy PC (HSPC) lipid layers (in the form of liposomes) immersed in HA solution or two palmitoyl-oleoyl PC (POPC) lipid layers across HA-POPC solution using a surface force balance (SFB). The results show, clearly and surprisingly, that HA addition does not affect the outstanding lubrication provided by the PC lipid layers. A possible mechanism indicated by our data that may account for this is that multiple lipid layers form on each cartilage surface, so that the slip plane may move from the midplane between the opposing surfaces, which is bridged by the HA, to an HA-free interface within a multilayer, where hydration lubrication is freely active. Another possibility suggested by our model experiments is that lipids in synovial fluid may complex with HA, thereby inhibiting the HA molecules from adhering to the lipids on the cartilage surfaces.

According to the Chinese medicine, magnoflorine exerted significant anti-inflammatory effects and potentially promoted synthesis of proteoglycans in chondrocytes to reverse the progression of rheumatoid arthritis. However, the latent beneficial effect of magnoflorine for the treatment of traumatic osteoarthritis (OA) is still unknown. Therefore, we aim to demonstrate the efficacy of magnoflorine combined with HA-gel in attenuating cartilage degeneration in anterior cruciate ligament transection (ACLT) induced OA rat model. We found that the histological results showed the elevated cartilage matrix, chondrogenic signals and chondroprogenitor cells in HA-gel + magnoflorine treatment. HA-gel + magnoflorine treatment resulted in a decreased modified Mankin's score, and a higher volume ratio of hyaline cartilage (HC)/calcified cartilage (CC) and HC/Sum (whole cartilage), compared to ACLT and HA-gel groups. Furthermore, both the volume ratios of HC/Sum and HC/CC were negatively correlated with modified Mankin's scores. Finally, HA-gel + magnoflorine could significantly increase the BV/TV, Tb.Th, and decrease the Tb.Pf, Po(tot), Conn.Dn and Tb.Sp. In vitro, 50 μg/ml magnoflorine treatment could significantly increase the viability, S-phase, migration rate and chondrogenesis of chondroprogenitor cells. There were significant downregulations of MAPK/NF-κB signaling, and upregulations of chondrogenic signals in 50 μg/ml magnoflorine treatment. There were significant downregulations of proinflammatory cytokines and upregulation of IL-10 in HA-gel + magnoflorine treated group. Therefore, our study elucidated a protective effect of HA-gel + magnoflorine on attenuating cartilage degradation and maintaining SCB stabilization in ACLT induced OA., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2019. Published by Elsevier Masson SAS.)

Decellularized cartilage scaffold (DCS) is an emerging substitute for cartilage defect application. However, it is hard to preserve an intact structure of such scaffolds derived from terrestrial animals, because of their dense and compact constitution. In contrast, squid (Dosidicus gigas) cranial cartilage, which possesses a relatively loose structure, could be easily decellularized using mild conditions and it retains the original microstructures of extracellular matrix (ECM). Herein, decellularized squid cranial cartilage scaffold (DSCS) was fabricated successfully after substantially removing the cells, which contained abundant ECM components (proteoglycans and type II collagen). Microscopic structure results showed that the DSCS possesses a relatively smooth and dense surface with a favorable interconnected inner porous structure for cell growth. DSCS exhibited excellent biomechanics and hydrophilicity. In vitro experiments indicated that the scaffold extracts were not toxic to cells, and were amenable to chondrocyte migration. Meanwhile, chondrocytes seeded in DSCS could maintain a favorable viability and present a spreading morphology. Furthermore, the in vivo experiments revealed that both cell-free scaffold and cell-laden scaffold exert promotive effects for the regeneration of cartilage in a full-thickness rabbit cartilage defect model. Taken together, these results suggested that DSCS presents a novel and promising cell-free therapeutic choice for cartilage tissue engineering.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Enzyme-sensitive hydrogels containing encapsulated chondrocytes are a promising platform for cartilage tissue engineering. However, the growth of neotissue is closely coupled to the degradation of the hydrogel and is further complicated due to the encapsulated cells serving as the enzyme source for hydrogel degradation. To better understand these coupled processes, this study combined experimental and computational methods to analyze the transition from hydrogel to neotissue in a biomimetic MMP-sensitive poly(ethylene glycol) (PEG) hydrogel with encapsulated chondrocytes. A physics-based computational model that describes spatial heterogeneities in cell distribution was used. Experimentally, cell-laden hydrogels were cultured for six weeks under free swelling or subjected daily to one-hour of dynamic compressive loading. Extracellular matrix (ECM) synthesis rates were used as model inputs, and the model was fit to the experimentally determined construct modulus over time for the free swelling condition. Experimentally, ECM accumulation comprising collagen II and aggrecan increased over time concomitant with hydrogel degradation observed by a loss in PEG. Simulations demonstrated rapid degradation in regions of high cell density (i.e., cell clusters) reaching complete degradation by day 13, which facilitated localized ECM growth. Regions of low cell density degraded more slowly, had limited ECM, and led to the decrease in construct modulus during the first two weeks. The primary difference between the two culture environments was greater ECM accumulation in the clusters under free swelling, which facilitated a faster recovery in construct modulus. By 6 weeks the compressive modulus increased 2.5-fold to 107 kPa under free swelling, but dropped 1.6-fold to 26 kPa under loading. In summary, this biomimetic MMP-sensitive hydrogel supports neocartilage growth by facilitating rapid ECM growth within cell clusters, which was followed by slower growth in the rest of the hydrogel. Subtle temporal differences in hydrogel degradation and ECM accumulation, however, had a significant impact on the evolving mechanical properties.

Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1 . Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol.

Differentiation of stem cells into chondrocytes has been studied for the engineering of cartilage tissue. However, stem cells cultured two-dimensionally have limited ability to differentiate into chondrocytes, which led to the development of three-dimensional culture systems. A recently developed microtechnological method uses microwells as a tool to form uniformly sized spheroids. In this study, we fabricated an array (10 × 10) of goblet-shaped microwells based on polydimethylsiloxane for spheroid culture. A central processing unit (CPU) was used to form holes, and metallic beads were used to form hemispherical microwell geometry. The holes were filled with Pluronic F-127 to prevent cells from sinking through the holes and allowing the cells to form spheroids. Viability and chondrogenic differentiation of human adipose-derived stem cells were assessed. The fabrication method using a micro-pin mold and metallic beads is easy and cost-effective. Our three-dimensional spheroid culture system optimizes the efficient differentiation of cells and has various applications, such as drug delivery, cell therapy, and tissue engineering.

Supramolecular and dynamic covalent crosslinking (DCC) hydrogels not only display unique physicochemical properties that can mimic the dynamic extracellular matrix (ECM), but also have the capabilities of shear-thinning, self-healing and even shape memorizing. Specifically, through the breaking and reforming of the reversible linkage, cells can be readily encapsulated in the matrix and can well maintain their differentiation potentials. The dynamic shear-thinning and self-healing hydrogels can also be explored as cell-compatible bio-inks for the design of complex multicellular structures. These distinctive properties of dynamic hydrogels have attracted increasing interests in cell retention as well as cartilage tissue engineering. The biophysical and biochemical cues of hydrogel matrices have significant effects on cell fate. The studies on the relationship of cell response and the critical properties of hydrogels, such as mechanical strength, elasticity, ligand chemistry and degradation, are helpful in advancing dynamic hydrogels for cell retention and delivery. In this review, we highlight the most recent progress in the gelation strategies of biomedical supramolecular and DCC hydrogels and then focus on their applications for enhancing cell retention and cartilage/osteochondral regeneration. Furthermore, the challenges and future perspectives of supramolecular and DCC hydrogels in cell retention and cartilage regeneration are also discussed.

Structural and associated biomechanical gradients within biological tissues are important for tissue functionality and preventing damaging interfacial stress concentrations. Articular cartilage possesses an inhomogeneous structure throughout its thickness, driving the associated variation in the biomechanical strain profile within the tissue under physiological compressive loading. However, little is known experimentally about the nanostructural mechanical role of the collagen fibrils and how this varies with depth. Utilising a high-brilliance synchrotron X-ray source, we have measured the depth-wise nanostructural parameters of the collagen network in terms of the periodic fibrillar banding (D-period) and associated parameters. We show that there is a depth dependent variation in D-period reflecting the pre-strain and concurrent with changes in the level of intrafibrillar order. Further, prolonged static compression leads to fibrillar changes mirroring those caused by removal of extrafibrillar proteoglycans (as may occur in aging or disease). We suggest that fibrillar D-period is a sensitive indicator of localised changes to the mechanical environment at the nanoscale in soft connective tissues. STATEMENT OF SIGNIFICANCE: Collagen plays a significant role in both the structural and mechanical integrity of articular cartilage, allowing the tissue to withstand highly repetitive loading. However, the fibrillar mechanics of the collagen network in cartilage are not clear. Here we find that cartilage has a spatial gradient in the nanostructural collagen fibril pre-strain, with an increase in the fibrillar pre-strain with depth. Further, the fibrillar gradient changes similarly under compression when compared to an enzymatically degraded tissue which mimics age-related changes. Given that the fibrils potentially have a finite capacity to mechanically respond and alter their configuration, these findings are significant in understanding how collagen may alter in structure and gradient in diseased cartilage, and in informing the design of cartilage replacements., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)