In vitrothree-dimensional volumetric printing of vitreous body models using decellularized extracellular matrix bioink.

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.

Preparation, typical structural characteristics and relieving effects on osteoarthritis of squid cartilage type II collagen peptides.

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.

Structural analysis and anti-cancer activity of low-molecular-weight chondroitin sulfate from hybrid sturgeon cartilage.

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 [GlcUAß1-3GalNAc(6S)], 32.11% ΔDi-4S [GlcUAß1-3GalNAc(4S)], 16.05% ΔDi-2S,6S [GlcUA(2S)ß1-3GalNAc(6S)] and 3.0% ΔDi-0S [GlcUAß1-3GalNAc]. 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.

Effect of glutaraldehyde-crosslinked cartilage acellular matrix film on anti-adhesion and nerve regeneration in a rat sciatic nerve injury model.

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.

Type II collagen from squid cartilage mediated myogenic IGF-I and irisin to activate the Ihh/PThrp and Wnt/ß-catenin pathways to promote fracture healing in mice.

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-ß 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, ß-catenin and Runx2 expression in the callus by receptor αVß5. These results suggest that SCII can accelerate the process of endochondral osteogenesis and promote fracture healing through activating the Ihh/PThrp and Wnt/ß-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.

A novel chondroitin sulfate E from Dosidicus gigas cartilage and its antitumor metastatic activity.

Chondroitin sulfate (CS) chains containing GlcUAß1-3GalNAc(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.

Strategies to Control or Mimic Growth Factor Activity for Bone, Cartilage, and Osteochondral Tissue Engineering.

Growth factors play a critical role in tissue repair and regeneration. However, their clinical success is limited by their low stability, short half-life, and rapid diffusion from the delivery site. Supraphysiological growth factor concentrations are often required to demonstrate efficacy but can lead to adverse reactions, such as inflammatory complications and increased cancer risk. These issues have motivated the development of delivery systems that enable sustained release and controlled presentation of growth factors. This review specifically focuses on bioconjugation strategies to enhance growth factor activity for bone, cartilage, and osteochondral applications. We describe approaches to localize growth factors using noncovalent and covalent methods, bind growth factors via peptides, and mimic growth factor function with mimetic peptide sequences. We also discuss emerging and future directions to control spatiotemporal growth factor delivery to improve functional tissue repair and regeneration.

Enhancement of cartilage extracellular matrix synthesis in Poly(PCL-TMC)urethane scaffolds: a study of oriented dynamic flow in bioreactor.

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.

Three-Dimensional Printing Biologically Inspired DNA-Based Gradient Scaffolds for Cartilage Tissue Regeneration.

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.

Biobased pH-responsive and self-healing hydrogels prepared from O-carboxymethyl chitosan and a 3-dimensional dynamer as cartilage engineering scaffold.

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.

Biocompatible implant mimicking cartilage: A new horizon for reconstructive facial field.

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.

Spatiotemporal neocartilage growth in matrix-metalloproteinase-sensitive poly(ethylene glycol) hydrogels under dynamic compressive loading: an experimental and computational approach.

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.

Pulse Lavage Fails to Significantly Reduce Bone Marrow Content in Osteochondral Allografts: A Histological and DNA Quantification Study.

BACKGROUND: Current clinical practice calls for pulse lavage of fresh osteochondral allografts (OCAs) to reduce immunogenicity; however, there is limited evidence of its effectiveness in reducing allogenic bone marrow elements. PURPOSE: To evaluate the effectiveness of pulse lavage in removing marrow elements from trabecular bone in fresh OCA transplantation. STUDY DESIGN: Controlled laboratory study. METHODS: The authors evaluated 48 fresh OCA plugs with 4 different common sizes (14- and 24-mm diameter, 6- and 10-mm thickness). Within each size group, half of the samples underwent pulse lavage (n = 6) with saline solution and half were left untreated (no lavage; control group, n = 6). For each treatment and size group, 3 samples were analyzed for DNA content as an indicator of the number of residual nucleated cells; the other 3 samples were histologically analyzed to assess the presence and distribution of cells within subchondral bone pores in 3 specific locations within the plug: peripheral, intermediate, and core. RESULTS: Osteochondral plugs treated with pulse lavage did not show a significant decrease in DNA content in comparison with untreated plugs. Overall, histological analysis did not show a significant difference between the treated and untreated groups (P = .23). Subgroup analysis by size demonstrated decreased marrow content in treated versus untreated groups in the thinner plug sizes (14 × 6 mm and 24 × 6 mm). Histological evaluation by zone demonstrated a significant difference between groups only in the peripheral zone (P = .04). CONCLUSION: Pulse lavage has limited effectiveness in removing marrow elements, in particular in plugs that are larger in diameter and, more importantly, in thickness. Better techniques for subchondral bone treatment are required for more thorough removal of potentially immunogenic marrow elements. CLINICAL RELEVANCE: OCA transplantation has become an established treatment modality. Unfortunately, OCA is not without limitations, chiefly its mode of failure through inadequate integration of the allograft subchondral bone with subsequent collapse. In an effort to improve integration, current clinical practice calls for pulse lavage to remove allogenic bone marrow from the subchondral bone in hopes of decreasing the immunogenicity of the graft and facilitating revascularization.

The effect of cartilage extracellular matrix particle size on the chondrogenic differentiation of bone marrow mesenchymal stem cells.

Aim: To investigate the effect of cartilage extracellular matrix (ECM) particle size on the chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Materials & methods: BMSCs were seeded into the scaffolds fabricated by small particle ECM materials and large particle ECM materials. For the positive control, chondrogenically induced BMSCs were seeded into commercial poly-lactic-glycolic acid scaffolds. Macroscopic observation, histological and immunohistochemical staining, mechanical testing and biochemical analysis were performed to the cell-scaffold constructs. Results: BMSCs in small particle ECM materials and poly-lactic-glycolic acid scaffolds were induced to differentiate into chondrocytes, while BMSCs in the large particle ECM materials scaffold did not differentiate into chondrocytes. Conclusion: The small ECM particle materials improved the induction ability of the cartilage ECM-derived scaffold.

Effects of administration of glucosamine and chicken cartilage hydrolysate on rheumatoid arthritis in SKG mice.

Supplementation with cartilage constituents, such as glucosamine, chondroitin sulfate and collagen peptide, are believed to reduce pain associated with joint disorders, such as rheumatoid arthritis (RA). Here, we administered daily, 10 mg glucosamine or 100 mg chicken cartilage hydrolysate (CH) to SKG/Jcl mice, a model for spontaneous RA, for 5 weeks and evaluated their effects on RA development. In SKG mice, the administration of glucosamine had no reducing effect on RA score but suppressed the expression of Mmp13 and Col3a1 genes in articular cartilage. In contrast, administration of CH suppressed the RA score and levels of plasma interleukin-6 and interleukin-17 to half, although the differences were not significant. Mice administered with glucosamine also showed decreased bone strength of femur and these adverse effects could be eliminated when glucosamine was used in conjunction with CH. These results suggest that CH and glucosamine exert effects on different aspects in SKG mice.

Four Antioxidant Peptides from Protein Hydrolysate of Red Stingray (Dasyatis akajei) Cartilages: Isolation, Identification, and In Vitro Activity Evaluation.

In the work, water-soluble proteins of red stingray (Dasyatis akajei) cartilages were extracted by guanidine hydrochloride and hydrolyzed using trypsin. Subsequently, four antioxidant peptides (RSHP-A, RSHP-B, RSHP-C, and RSHP-D) were isolated from the water-soluble protein hydrolysate while using ultrafiltration and chromatographic techniques, and the amino acid sequences of RSHP-A, RSHP-B, RSHP-C, and RSHP-D were identified as Val-Pro-Arg (VPR), Ile-Glu-Pro-His (IEPH), Leu-Glu-Glu--Glu-Glu (LEEEE), and Ile-Glu-Glu-Glu-Gln (IEEEQ), with molecular weights of 370.46 Da, 494.55 Da, 647.64 Da, and 646.66 Da, respectively. VPR, IEPH, LEEEE, and IEEEQ exhibited good scavenging activities on the DPPH radical (EC50 values of 4.61, 1.90, 3.69, and 4.01 mg/mL, respectively), hydroxyl radical (EC50 values of 0.77, 0.46, 0.70, and 1.30 mg/mL, respectively), superoxide anion radical (EC50 values of 0.08, 0.17, 0.15, and 0.16 mg/mL, respectively), and ABTS cation radical (EC50 values of 0.15, 0.11, 0.19, and 0.18 mg/mL, respectively). Among the four isolated antioxidant peptides, IEPH showed the strongest reducing power and lipid peroxidation inhibition activity, but LEEEE showed the highest Fe2+-chelating ability. The present results suggested that VPR, IEPH, LEEEE, and IEEEQ might have the possibility of being an antioxidant additive that is used in functional food and pharmaceuticals.

Peptidomic analysis of cartilage and subchondral bone in OA patients.

BACKGROUND: The objective of this study was to develop a method for directly analysing osteochondral samples straight out of the operating room without cell culturing, thereby enabling identification of potential peptide biomarkers to better understand the mechanisms involved in the development of osteoarthritis and pain. MATERIAL AND METHODS: Osteochondral plugs from wounded and macroscopically nonwounded zones of the femur condyle were collected from six patients with manifest osteoarthritis (OA) undergoing total knee arthroplasty (TKA). The samples were demineralized and supernatant was collected and isotopically marked with Tandem Mass Tag (TMT) labelling and analysed using liquid chromatography coupled with tandem mass spectrometry LC-MS/MS. RESULTS: Using peptidomics, 6292 endogenous peptides were identified. Five hundred sixty-six peptides (8 identified endogenous peptides) differed significantly (P-value 0.10) from wounded zones compared to nonwounded zones. CONCLUSION: This pilot study shows promising results for enabling peptidomic analysis of cartilage and bone straight out of the operating room. With further refinement, peptidomics can potentially become a diagnostic tool for OA, and improve the knowledge of disease progression and genesis of pain.

The role of chondroitin sulfate in regulating hypertrophy during MSC chondrogenesis in a cartilage mimetic hydrogel under dynamic loading.

Mesenchymal stem cells (MSCs) are promising for cartilage regeneration, but readily undergo terminal differentiation. The aim of this study was two-fold: a) investigate physiochemical cues from a cartilage-mimetic hydrogel under dynamic compressive loading on MSC chondrogenesis and hypertrophy and b) identify whether Smad signaling and p38 MAPK signaling mediate hypertrophy during MSC chondrogenesis. Human MSCs were encapsulated in photoclickable poly(ethylene glycol) hydrogels containing chondroitin sulfate and RGD, cultured under dynamic compressive loading or free swelling for three weeks, and evaluated by qPCR and immunohistochemistry. Loading inhibited hypertrophy in the cartilage-mimetic hydrogel indicated by a reduction in pSmad 1/5/8, Runx2, and collagen X proteins, while maintaining chondrogenesis by pSmad 2/3 and collagen II proteins. Inhibiting pSmad 1/5/8 under free swelling culture significantly reduced collagen X protein, similar to the loading condition. Chondroitin sulfate was necessary for load-inhibited hypertrophy and correlated with enhanced S100A4 expression, which is downstream of the osmotic responsive transcription factor NFAT5. Inhibiting p38 MAPK under loading reduced S100A4 expression, and upregulated Runx2 and collagen X protein. Findings from this study indicate that chondroitin sulfate with dynamic loading create physiochemical cues that support MSC chondrogenesis and attenuate hypertrophy through Smad 1/5/8 inhibition and p38 MAPK upregulation.

Structural Features of Heparan Sulfate from Multiple Osteochondromas and Chondrosarcomas.

Multiple osteochondromas (MO) is a hereditary disorder associated with benign cartilaginous tumors, known to be characterized by absence or highly reduced amount of heparan sulfate (HS) in the extracellular matrix of growth plate cartilage, which alters proper signaling networks leading to improper bone growth. Although recent studies demonstrated accumulation of HS in the cytoplasm of MO chondrocytes, nothing is known on the structural alterations which prevent HS from undergoing its physiologic pathway. In this work, osteochondroma (OC), peripheral chondrosarcoma, and healthy cartilaginous human samples were processed following a procedure previously set up to structurally characterize and compare HS from pathologic and physiologic conditions, and to examine the phenotypic differences that arise in the presence of either exostosin 1 or 2 (EXT1 or EXT2) mutations. Our data suggest that HS chains from OCs are prevalently below 10 kDa and slightly more sulfated than healthy ones, whereas HS chains from peripheral chondrosarcomas (PCSs) are mostly higher than 10 kDa and remarkably more sulfated than all the other samples. Although deeper investigation is still necessary, the approach here applied pointed out, for the first time, structural differences among OC, PCS, and healthy HS chains extracted from human cartilaginous excisions, and could help in understanding how the structural features of HS are modulated in the presence of pathological situations also involving different tissues.

Isolation and Chemical Characterization of Chondroitin Sulfate from Cartilage By-Products of Blackmouth Catshark (Galeus melastomus).

Chondroitin sulfate (CS) is a glycosaminoglycan actively researched for pharmaceutical, nutraceutical and tissue engineering applications. CS extracted from marine animals displays different features from common terrestrial sources, resulting in distinct properties, such as anti-viral and anti-metastatic. Therefore, exploration of undescribed marine species holds potential to expand the possibilities of currently-known CS. Accordingly, we have studied for the first time the production and characterization of CS from blackmouth catshark (Galeus melastomus), a shark species commonly discarded as by-catch. The process of CS purification consists of cartilage hydrolysis with alcalase, followed by two different chemical treatments and ending with membrane purification. All steps were optimized by response surface methodology. According to this, the best conditions for cartilage proteolysis were established at 52.9 °C and pH = 7.31. Subsequent purification by either alkaline treatment or hydroalcoholic alkaline precipitation yielded CS with purities of 81.2%, 82.3% and 97.4% respectively, after 30-kDa membrane separation. The molecular weight of CS obtained ranges 53⁻66 kDa, depending on the conditions. Sulfation profiles were similar for all materials, with dominant CS-C (GlcA-GalNAc6S) units (55%), followed by 23⁻24% of CS-A (GlcA-GalNAc4S), a substantial amount (15⁻16%) of CS-D (GlcA2S-GalNAc6S) and less than 7% of other disulfated and unsulfated disaccharides.