Preparation, structure characterization, and stability analysis of peptide-calcium complex derived from porcine nasal cartilage type II collagen.

BACKGROUND: Porcine nasal cartilage type II collagen-derived peptides (PNCPs) may be complexed with calcium to provide a highly bioavailable, low-cost, and effective calcium food supplement. However, the calcium-binding characteristics of PNCPs have not yet been investigated. In the present study, calcium-binding peptides were derived from porcine nasal cartilage type II collagen and the resulting PNCPs-Ca complex was characterized. RESULTS: The study reveals that the calcium-binding capacity of PNCPs is closely related to enzymatic hydrolysis conditions. The highest calcium-binding capacity of PNCPs was observed at a hydrolysis time of 4 h, temperature of 40 °C, enzyme dosage of 1%, and solid-to-liquid ratio of 1:10. Scanning electron microscopy and energy dispersive X-ray spectroscopy revealed that the PNCPs had a pronounced capacity for calcium binding, with the PNCPs-Ca complex exhibiting a clustered structure consisting of aggregated spherical particles. Fourier-transform infrared spectroscopy, fluorescence spectroscopy, X-ray diffraction, dynamic light scattering, amino acid composition, and molecular weight distribution analyses all indicated that the PNCPs and calcium complexed via the carboxyl oxygen and amino nitrogen atoms, leading to the formation of a ß-sheet structure during the chelation process. In addition, the stability of the PNCPs-Ca complex was maintained over a range of pH values consistent with those found in the human gastrointestinal tract, facilitating calcium absorption. CONCLUSION: These research findings suggest the feasibility of converting by-products from livestock processing into calcium-binding peptides, providing a scientific basis for the development of novel calcium supplements and the potential reduction of resource waste. © 2023 Society of Chemical Industry.

A compositional analysis of native and decellularized porcine nasal septum cartilage.

OBJECTIVES: Decellularization of porcine septum cartilage is necessary for its application as xenogenic replacement material. The aim of this study was to investigate spatial differences of structure and composition in the whole native and decellularized porcine nasal septum. Subsequently, the results shall be compared with studies of human nasal septum. METHODS: Ten porcine nasal septa were divided into six regions from caudal to cephalic and four regions from dorsal to ventral to create a grid of 24 approximately equal segments. All segments of five septal cartilages were decellularized separately by a wet chemical multistep procedure. The segments were analyzed to determine quantitative amounts of total collagen, chondrocytes, and sulfated glycosaminoglycans (sGAG). RESULTS: The distribution of cell number showed no significant differences between the individual regions. For the distribution of collagen and sGAG, no significant differences could be identified from caudal to cephalic, both in native and decellularized tissue. From dorsal to ventral, native and decellularized nasal septum showed significant differences between individual regions. In native septum, linear regression analysis indicated a decreasing collagen and an increasing sGAG content from dorsal to ventral. After decellularization, an increasing collagen and a decreasing sGAG content was detected. CONCLUSION: The results of this study showed slightly but significant differences in the distribution of collagen and sGAG from dorsal to ventral. From caudal to cephalic, no differences could be observed. Compared to human, nasal septum differences in cell, collagen, and sGAG content were detected. Despite this, human and porcine nasal septum showed similar distributions and a consistently inverse linearity of collagen and sGAG content. Nevertheless, the midcaudal and midcephalic regions showed the highest porosity and a high stability and thus offer the best conditions for the revitalization of porcine tissue by human cells.

A novel 3D histotypic cartilage construct engineered by supercritical carbon dioxide decellularized porcine nasal cartilage graft and chondrocytes exhibited chondrogenic capability in vitro.

Augmentative and reconstructive rhinoplasty surgical procedures use autologous tissue grafts or synthetic grafts to repair the nasal defect and aesthetic reconstruction. Donor site trauma and morbidity are common in autologous grafts. The desperate need for the production of grafted 3D cartilage tissues as rhinoplasty grafts without the adverse effect is the need of the hour. In the present study, we developed a bioactive 3D histotypic construct engineered with the various ratio of adipose-derived stem cells (ADSC) and chondrocytes together with decellularized porcine nasal cartilage graft (dPNCG). We decellularized porcine nasal cartilage using supercritical carbon dioxide (SCCO2) extraction technology. dPNCG was characterized by H&E, DAPI, alcian blue staining, scanning electron microscopy and residual DNA content, which demonstrated complete decellularization. 3D histotypic constructs were engineered using dPNCG, rat ADSC and chondrocytes with different percentage of cells and cultured for 21 days. dPNCG together with 100% chondrocytes produced a solid mass of 3D histotypic cartilage with significant production of glycosaminoglycans. H&E and alcian blue staining showed an intact mass, with cartilage granules bound to one another by extracellular matrix and proteoglycan, to form a 3D structure. Besides, the expression of chondrogenic markers, type II collagen, aggrecan and SOX-9 were elevated indicating chondrocytes cultured on dPNCG substrate facilitates the synthesis of type II collagen along with extracellular matrix to produce 3D histotypic cartilage. To conclude, dPNCG is an excellent substrate scaffold that might offer a suitable environment for chondrocytes to produce 3D histotypic cartilage. This engineered 3D construct might serve as a promising future candidate for cartilage tissue engineering in rhinoplasty.

Preparation of chondroitin sulfates with different molecular weights from bovine nasal cartilage and their antioxidant activities.

Biological functions of chondroitin sulfate, including anti-oxidation and anti-inflammation, are associated with its molecular weight. This study aimed to evaluate the correlation between antioxidant activity and molecular weights of chondroitin sulfate derived from bovine nasal cartilage (BCS). BCS extracted by compound enzymatic method was further purified via DEAE-cellulose column separation to obtain BCS-II (129.4 kDa), which was further degraded by H2O2-Vc to obtain four subfractions: BCS-II-1 (92.7 kDa), BCS-II-2 (54.1 kDa), BCS-II-3 (26.3 kDa), and BCS-II-4 (19.7 kDa). Changes in the physicochemical properties of BCS-II before and after degradation were compared via FT-IR, NMR and monosaccharide composition analysis. Finally, antioxidant activities of BCS-II and its subfractions BCS-II-1-4 were compared. Our results showed that the H2O2-Vc system did not disrupt the primary functional group of BCS-II, with no significant change in sulfate content between BCS-II and its degraded fractions; however, uronic acid levels increased in degraded fractions when compared with BCS-II. In vitro, BCS-II-4 displayed the lowest molecular weight and had the strongest antioxidant activity. Therefore, the antioxidant activity of chondroitin sulfate in vitro is robustly associated with its molecular weight, and low-molecular-weight chondroitin sulfate can be used as an antioxidant in the food and pharmaceutical industries and other sectors.

Strategies to Mitigate Variability in Engineering Human Nasal Cartilage.

Skin cancer and its associated treitments can have devastating consequences for survivors; this is particularly true when cancer occurs on the nose. Recent work has applied cell-based tissue engineering (TE) strategies to develop nasal cartilage constructs for reconstruction of the nose. In this study, we have generated human nasal cartilage on a clinically approved collagen scaffold to investigate the donor-to-donor variability of TE cartilage and evaluated strategies to mitigate it. We also evaluated the gene expression of the family of fibroblast growth factor receptors (FGFR1-4) and their association with tissue quality. FGFR 1 was significantly positively correlated with GAG/DNA; a measure of chondrogenic capacity. We implemented two strategies: hypoxic culture and co-culture with mesenchymal stromal cells (MSCs) to increase tissue quality. Total glycosaminoglycan (GAG) content varied significantly between donors initially, with >10-fold difference between the best and worst donor tissue. Our co-culture strategy was able to increase TE construct quality from poor quality donor tissue while supressing hypertrophy relative to MSCs alone. However, no differences were observed with the use of hypoxic culture. Tissues generated using co-culture with MSCs became vascularized and calcified in vivo, demonstrating a non-stable cartilage phenotype in co-culture and MSCs cartilage constructs.

Salmon nasal cartilage proteoglycan enhances growth of normal human dermal fibroblast through Erk1/2 phosphorylation.

Proteoglycan (PG) is a heavily glycosylated protein, localized to cell surface and extracellular matrix, and has various functions. Recently, it has been gradually revealed that PG interacts with various growth factors and morphogens and regulates cellular functions. Although salmon nasal cartilage PG (Salmon-PG) increases proliferation of immortalized cells, its mechanism remains unclear. In this study, we confirmed the effect of Salmon-PG on normal human dermal fibroblast (NHDF) and investigated the mechanism of PG action on NHDF. Salmon-PG dose- and time-dependently increased NHDF proliferation. Receptor tyrosine kinase array revealed that Salmon-PG increased only Erk1/2 signaling. Erk1/2 phosphorylation was significantly increased by Salmon-PG in a time-(10 min) and dose-(400 or 800 µg/mL) dependent manner. MEK inhibitor suppressed the enhancement of NHDF proliferation by Salmon-PG. The overall findings indicate that Salmon-PG plays a role as a growth factor in NHDF via Erk1/2 activation, suggesting that Salmon-PG contributes to the maintenance of skin homeostasis.

Preparation of proteoglycan from salmon nasal cartilage under nondenaturing conditions.

Salmon nasal cartilage was micronized in ethanol using a rotor-stator homogenizer for the high yield of proteoglycan extraction. This procedure also brought about depressing the degradation of proteoglycan and the contamination of collagens. Proteoglycan was extracted by 4 M magnesium chloride and isolated by anion-exchange chromatography. The gel filtration HPLC and the antibody reactivity showed that the core protein was intact.

Effects of Salmon Nasal Cartilage Proteoglycan on Plasma Glucose Concentration and Active Glucose Transport in the Small Intestine.

Recently, proteoglycan was purified from the nasal cartilage of salmon. Although several physiological effects have been reported, the effect of salmon nasal cartilage proteoglycan (salmon PG) on glucose metabolism remains unclear. We studied the effect of salmon PG on rat plasma glucose levels. Oral administration of 1% salmon PG significantly attenuated the increase in portal plasma glucose levels following an oral glucose tolerance test (OGTT). Additionally 1% salmon PG delayed the increase in peripheral glucose concentration induced by the OGTT. Mucosal administration of 1% salmon PG significantly decreased active glucose transport using the everted jejunal sac method. Furthermore, transmural potential difference (ΔPD) measurements using the everted jejunum revealed that 1% salmon PG significantly decreased glucose-dependent and phlorhizin (inhibitor of sodium-glucose co-transporter 1; SGLT1)-sensitive ΔPD. These results suggest that salmon PG decreases glucose absorption via SGLT1 in the jejunum, thereby attenuating the increase in portal and peripheral plasma glucose levels in rats.

Proteoglycan from salmon nasal cartridge [corrected] promotes in vitro wound healing of fibroblast monolayers via the CD44 receptor.

Proteoglycans (PGs) are involved in various cellular functions including cell growth, adhesion, and differentiation; however, their physiological roles are not fully understood. In this study, we examined the effect of PG purified from salmon nasal cartilage (SNC-PG) on wound closure using tissue-cultured cell monolayers, an in vitro wound-healing assay. The results indicated that SNC-PG significantly promoted wound closure in NIH/3T3 cell monolayers by stimulating both cell proliferation and cell migration. SNC-PG was effective in concentrations from 0.1 to 10µg/ml, but showed much less effect at higher concentrations (100-1000µg/ml). The effect of SNC-PG was abolished by chondroitinase ABC, indicating that chondroitin sulfates (CSs), a major component of glycosaminoglycans (GAGs) in SNC-PG, are crucial for the SNC-PG effect. Furthermore, chondroitin 6-sulfate (C-6-S), a major CS of SNC-PG GAGs, could partially reproduce the SNC-PG effect and partially inhibit the binding of SNC-PG to cells, suggesting that SNC-PG exerts its effect through an interaction between the GAGs in SNC-PG and the cell surface. Neutralization by anti-CD44 antibodies or CD44 knockdown abolished SNC-PG binding to the cells and the SNC-PG effect on wound closure. These results suggest that interactions between CS-rich GAG-chains of SNC-PG and CD44 on the cell surface are responsible for the SNC-PG effect on wound closure.

Biochemical and atomic force microscopic characterization of salmon nasal cartilage proteoglycan.

Biological activities of salmon nasal cartilage proteoglycan fractions are known, however, structural information is lacking. Recently, the major proteoglycan of this cartilage was identified as aggrecan. In this study, global molecular images and glycosaminoglycan structure of salmon nasal cartilage aggrecan purified from 4M guanidine hydrochloride extract were analyzed using HPLCs and atomic force microscopy with bovine tracheal cartilage aggrecan as a control. The estimated numbers of sulfates per disaccharide unit of chondroitin sulfate chains of salmon and bovine aggrecans were similar (approximately 0.85). However, the disaccharide composition showed a higher proportion of chondroitin 6-sulfate units in salmon aggrecan, 60%, compared to 40% in bovine. Gel filtration HPLC and monosaccharide analysis showed the salmon aggrecan had a lower number (approximately one-third), but 1.5-3.3 times longer chondroitin sulfate chains than the bovine aggrecan. Atomic force microscopic molecular images of aggrecan supported the images predicted by biochemical analyses.

A compositional analysis of cadaveric human nasal septal cartilage.

OBJECTIVES/HYPOTHESIS: To localize quantitatively the major biochemical constituents of native adult human septal cartilage across whole septa. STUDY DESIGN: Prospective, basic science. METHODS: The nasal septa from seven cadavers were partitioned into 24 separate regions: six from caudal to cephalic and four from dorsal to ventral. Biochemical assays were used to determine the quantities, relative to wet weight, of the major constituents of cartilage: chondrocytes, collagen, and sulfated glycosaminoglycan. RESULTS: On average, each milligram of wet cartilage contained 24,900 cells, 73.9 µg collagen, and 17.1 µg sulfated glycosaminoglycan. Cell number showed no significant variation across the septa. In contrast, the caudal regions of the septa were associated with higher levels of collagen, the ventral regions correlated with higher levels of sulfated glycosaminoglycan, and the dorsal regions were associated with an elevated ratio of collagen to sulfated glycosaminoglycan. CONCLUSIONS: This study represents the first characterization of the biochemical composition of native human septal cartilage across whole septa. Quantities of collagen and sulfated glycosaminoglycan showed region-specific variation across the septum. The localized pattern of collagen and sulfated glycosaminoglycan deposition are consistent with the significance of preserving the L-strut during rhinoplasty and other nasal reconstructive procedures. In addition, it may assist in defining design goals for tissue-engineered septal neocartilage constructs to meet specific reconstructive needs in the future.

Epiphycan from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan.

Chum salmon (Oncorhynchus keta) nasal cartilage was examined by next-generation DNA sequencing and mass spectrometric analyses, and 14 types of proteoglycans including epiphycan (EPY) were found. A cDNA encoding EPY was cloned and sequenced. The cDNA encoded 589 amino acids comprised a glycosaminoglycan (GAG) domain containing 55 potential GAG-modified sites (Ser-Gly and/or Gly-Ser), a cysteine cluster and 6 leucine-rich repeats. EPY was purified from salmon nasal cartilage and the structure of the GAG was characterized. As a result of unsaturated disaccharide analysis, GAG was found to be composed of chondroitin 6-sulfate (58.0%), chondroitin 4-sulfate (26.5%) and non-sulfated chondroitin (15.3%). The average molecular weight of GAG was estimated to be 3.0 × 10(4). Ser-100 and Ser-103 were identified as serine residues substituted by GAG chains by chemical modification and mass spectrometric analysis. More than 50 serine residues were assumed to be substituted by GAG chains. EPY is heavily substituted by chondroitin sulfate, giving an overall molecular weight of just under 2 × 10(6). EPY from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan.

Dependencies of multi-component T2 and T1ρ relaxation on the anisotropy of collagen fibrils in bovine nasal cartilage.

Both NMR spectroscopy and MRI were used to investigate the dependencies of multi-component T2 and T1ρ relaxation on the anisotropy of bovine nasal cartilage (BNC). The non-negative least square (NNLS) method and the multi-exponential fitting method were used to analyze all experimental data. When the collagen fibrils in nasal cartilage were oriented at the magic angle (55°) to the magnetic field B0, both T2 and T1ρ were single component, regardless of the spin-lock field strength or the echo spacing time in the pulse sequences. When the collagen fibrils in nasal cartilage were oriented at 0° to B0, both T2 and T1ρ at a spin-lock field of 500 Hz had two components. When the spin-lock field was increased to 1000 Hz or higher, T1ρ relaxation in nasal cartilage became a single component, even when the specimen orientation was 0°. These results demonstrate that the specimen orientation must be considered for any multi-component analysis, even for nasal cartilage that is commonly considered homogenously structured. Since the rapidly and slowly relaxing components can be attributed to different portions of the water population in tissue, the ability to resolve different relaxation components could be used to quantitatively examine individual molecular components in connective tissues.

Anti-aging effects of extracts prepared from salmon nasal cartilage in hairless mice.

The skin anti-aging effects of orally administered salmon nasal cartilage extract (SNCE), which includes abundant proteoglycan, were investigated using a hairless mouse skin-aging model, in which aging was caused by repetitive ultraviolet B (UV-B) irradiation. Physiological analysis of the skin surface following repetitive UV-B irradiation of 8 weeks revealed inhibition of erythema levels and reduction of transepidermal water loss (TEWL) due to oral administration of SNCE. Similarly, inhibitory actions of epidermal and dermal hypertrophy were revealed by hematoxylin and eosin staining. Furthermore, effects on the hydration level of the skin surface by SNCE administration were indicated at 4 weeks of UV-B irradiation, but greater effects were not apparent. These results indicate that SNCE may serve as an anti-aging agent for healthy skin.

Identification of proteoglycan from salmon nasal cartilage.

There has been no structural information about the core protein of salmon nasal cartilage proteoglycan although its physiological activities have been investigated. Internal amino acid sequencing using nano-LC/MS/MS revealed that the salmon proteoglycan was aggrecan. Primer walk sequencing based on the amino acid information determined that the salmon aggrecan cDNA is comprised of 4207bp nucleotides predicted to encode 1324 amino acids with a molecular mass of 143,276. It exhibited significant similarities to predicted pufferfish aggrecan, zebrafish similar to aggrecan, zebrafish aggrecan, bovine aggrecan and human aggrecan isoform 2 precursor; whose amino acid identities were 56%, 55%, 49%, 31% and 30%, respectively. Salmon cartilage aggrecan had globular domains G1, G2 and G3 as in mammalian aggrecans. Neither the putative keratan sulfate attachment domain enriched with serine, glutamic acid and proline, nor the putative chondroitin sulfate attachment domain with repeating amino acid sequence containing serine-glycine, found in mammalian aggrecans were observed in salmon, however, random serine-glycine (or glycine-serine) sequences predicted to the sugar chain attachment sites were observed. Based on cDNA analysis and amino acid analysis after ß-elimination, the ratio of serine attached to sugar chains was calculated to be approximately 37.7% of total serine, that is, 46 of 123 serine residues.

Macromolecular concentrations in bovine nasal cartilage by Fourier transform infrared imaging and principal component regression.

Fourier transform infrared imaging (FT-IRI) and principal component regression (PCR) were used to quantitatively determine collagen and proteoglycan concentrations in bovine nasal cartilage (BNC). An infrared spectral library was first established by obtaining eleven infrared spectra from a series of collagen and chondroitin 6-sulfate mixed in different ratios. FT-IR images were obtained from 6-µm-thick sections of BNC specimens at 6.25-µm pixel size. The spectra from the FT-IR images were imported into a PCR program to obtain the relative concentrations of collagen and proteoglycan in BNC, based on the spectral library of pure chemicals. These PCR-determined concentrations agreed with the molecular concentrations determined biochemically using an enzyme digestion assay. Use of the imaging approach revealed that proteoglycan loss in the specimens occurs first at the surface of the tissue block when compared with the middle portion of the tissue block. The quantitative correlation of collagen and proteoglycan revealed that their infrared absorption peak areas at 1338 and 1072-855 cm(-1) can only be used as qualitative indicators of the molecular contents. The use of PCR with FT-IRI offers an accurate tool to spatially determine the distributions of macromolecular concentration in cartilage.

Optical characteristics of cartilage at a wavelength of 1560 nm and their dynamic behavior under laser heating conditions.

A double-integrating-sphere system was used to measure the diffuse transmittance, diffuse reflectance, and collimated transmittance of cartilage and polyacrylamide hydrogel samples as a function of temperature under 1560-nm laser heating conditions. The dynamic behavior of the absorption and scattering coefficients and scattering anisotropy of the biomaterials was calculated by the inverse Monte Carlo method. The absorption coefficient of the cartilage and hydrogel samples proved to be linear in temperature. Raising the temperature of the cartilage samples to 80°C caused their absorption coefficient to decrease by some 25%. The temperature-induced change of the absorption spectrum of the interstitial water was found to be responsible for the clarification of the cartilage tissue observed to occur under 1560-nm laser heating conditions. The temperature field produced in the tissue by the laser energy deposited therein was calculated using a bioheat transfer equation with temperature-dependent parameters. The calculation results demonstrated that the temperature-induced changes of the optical parameters of biological tissues should be taken into account to make their 1560-nm laser treatment effective and safe.

Effects of proteoglycan extracted from nasal cartilage of salmon heads on maturation of dendritic cells derived from human peripheral blood monocytes.

Dendritic cells (DCs) play an essential role in the immune system. The transition from immature DC (iDCs) to mature DCs (mDCs) requires appropriate stimuli such as pro-inflammatory cytokines. Proteoglycans (PGs) are one of the main components of extracellular matrix, and some types of PGs are known to induce maturation of murine DCs. Recent studies have investigated the potential benefits of PG from nasal cartilage of salmon head (S-PG). This study investigated the effects of S-PG on maturation of human monocyte-derived DCs. iDCs were prepared from human monocytes using the appropriate cytokines and then stimulated by S-PG alone. In another experiment, iDCs were stimulated by a combination of pro-inflammatory cytokines (MIX) plus S-PG. Although the stimulation of S-PG alone did not induce phenotypic maturation from iDCs, CD40 expression on DCs stimulated by S-PG alone was lower than that of iDCs. In contrast, the phenotypic and functional characteristics of DCs stimulated by MIX+S-PG were similar to those of DCs stimulated by MIX alone. As a result, S-PG did not demonstrate a significant effect with regard to maturation of human monocyte-derived DCs.

Characterization of the quantitative relationship between signal-to-noise (S/N) ratio and sample amount on-target by MALDI-TOF MS: Determination of chondroitin sulfate subsequent to enzymatic digestion.

Chondroitin sulfate (CS) is an important glycosaminoglycan of the extracellular matrix and its quantitative detection is of interest in different pathologies. Although there are already methods of quantitative CS determination, many of them are laborious, require time-consuming sample workup and/or suffer from low sensitivity. It will be shown here that the CS content of biological samples can be easily assessed in the picomole range subsequent to enzymatic digestion. MALDI-TOF MS (matrix-assisted laser desorption and ionization time-of-flight mass spectrometry) was used to determine the concentrations of the unsaturated disaccharide of CS obtained by enzymatic digestion of native CS with chondroitin ABC lyase. The signal-to-noise (S/N) ratio can be used as a quantitative measure: amounts of CS (measured as the disaccharide) down to at least 500fmol could be detected and there is a direct correlation between the S/N ratio and the amount of CS between about 2 and 200pmol although the curve per se is sigmoidal. The influence of different parameters such as the used matrix, the applied laser intensity and different methods of data analysis were also tested. Advantages and drawbacks of this approach are critically discussed. Finally, the method was validated by the determination of the CS content in samples of known concentration as well as in enzymatically digested bovine nasal cartilage and compared with two further established methods of CS determination (Carbazole and Alcian Blue method).