Marine Collagen Hydrolysates Downregulate the Synthesis of Pro-Catabolic and Pro-Inflammatory Markers of Osteoarthritis and Favor Collagen Production and Metabolic Activity in Equine Articular Chondrocyte Organoids.

Articular cartilage experiences mechanical constraints leading to chondral defects that inevitably evolve into osteoarthritis (OA), because cartilage has poor intrinsic repair capacity. Although OA is an incurable degenerative disease, several dietary supplements may help improve OA outcomes. In this study, we investigated the effects of Dielen® hydrolyzed fish collagens from skin (Promerim®30 and Promerim®60) and cartilage (Promerim®40) to analyze the phenotype and metabolism of equine articular chondrocytes (eACs) cultured as organoids. Here, our findings demonstrated the absence of cytotoxicity and the beneficial effect of Promerim® hydrolysates on eAC metabolic activity under physioxia; further, Promerim®30 also delayed eAC senescence. To assess the effect of Promerim® in a cartilage-like tissue, eACs were cultured as organoids under hypoxia with or without BMP-2 and/or IL-1ß. In some instances, alone or in the presence of IL-1ß, Promerim®30 and Promerim®40 increased protein synthesis of collagen types I and II, while decreasing transcript levels of proteases involved in OA pathogenesis, namely Htra1, and the metalloproteinases Mmp1-3, Adamts5, and Cox2. Both Promerim® hydrolysates also decreased Htra1 protein amounts, particularly in inflammatory conditions. The effect of Promerim® was enhanced under inflammatory conditions, possibly due to a decrease in the synthesis of inflammation-associated molecules. Finally, Promerim® favored in vitro repair in a scratch wound assay through an increase in cell proliferation or migration. Altogether, these data show that Promerim®30 and 40 hold promise as dietary supplements to relieve OA symptoms in patients and to delay OA progression.

Inhibition of cartilage degeneration and subchondral bone deterioration by Spinacia oleracea in human mimic of ACLT-induced osteoarthritis.

Osteoarthritis (OA) is an aging disorder characterized by degenerated cartilage and sub-chondral bone alteration in affected knee joints. Globally, millions of people suffer from this disease. However, there is a lack of safe and promising therapeutics, making the exploration and development of leads from natural sources urgent. Accordingly, food as medicine may be the most suitable approach for the treatment of this degenerative disease. Herein, we elucidated the protective role of Spinacia oleracea extract (SOE) in an anterior cruciate ligament transection (ACLT) model of osteoarthritis as a mimic of the human condition. ACL transection was done in the tibio-femoral joints of rats. SOE was orally administered at the dosage of 125 and 250 mg kg-1 day-1 for four weeks. It was shown that the animals with SOE treatment had better joint morphology than the ACLT animals, as evident by the shiny appearance of their cartilage. Hematoxylin and safranin-o staining showed that the number of chondrocytes was significantly reduced in the OA model, which was prevented with SOE treatment. The reduction in the cartilage thickness was well observed by toluidine blue staining. The reduced stain by safranin-o and toluidine blue, indicated proteoglycan loss in the ACLT-induced osteoarthritis model. The proteoglycan content and cartilage thickness were restored in the SOE group upon treatment at an SOE dosage of 125 and 250 mg kg-1 day-1. The micro-CT parameters of subchondral bone (SCB) and cartilage degradation markers in the serum corroborated our findings of the protective effects of SOE. In summary, our study suggests that SOE has therapeutic potential, which if taken regularly as a food supplement, can have beneficial effects.

Nanoliposomes from Agro-Resources as Promising Delivery Systems for Chondrocytes.

Investigations in cartilage biology have been hampered by the limited capacity of chondrocytes, especially in rats and humans, to be efficiently transfected. Liposomes are a promising delivery system due to their lipid bilayer structure similar to a biological membrane. Here we used natural rapeseed lecithin, which contains a high level of mono- and poly-unsaturated fatty acids, to evaluate the cytocompatibility of these phospholipids as future potential carriers of biomolecules in joint regenerative medicine. Results show that appropriate concentrations of nanoliposome rapeseed lecithin under 500 µg/mL were safe for chondrocytes and did not induce any alterations of their phenotype. Altogether, these results sustain that they could represent a novel natural carrier to deliver active substances into cartilage cells.

Allogeneic platelet rich plasma serves as a scaffold for articular cartilage derived chondroprogenitors.

Limited self-restorative ability of the cartilage has necessitated the use of cell and tissue engineering based therapies. Recent advances in the isolation, expansion and characterization of articular cartilage derived chondroprogenitors(CPs) has gained popularity in its role for cartilage repair. Platelet rich plasma (PRP) is a reliable biological scaffold for in-vitro and in-vivo studies with reported therapeutic applications in cartilage and bone pathologies. The aim of this study was to evaluate whether human allogeneic PRP could serve as a biological scaffold for chondroprogenitors (CPs) in cartilage repair. CPs were isolated from the superficial layer of three osteoarthritic knee joints by fibronectin adhesion assay and characterized using flow cytometric analysis. Allogeneic citrated blood was harvested from three subjects to obtain PRP. CPs at a concentration of one million cells per ml were gelled with PRP using calcium chloride. The PRP-CP scaffolds were subjected for adipogeneic, osteogenic, chondrogeneic differentiation and processed for post differentiation-staining studies (Oil Red O, Von Kossa, Alcian blue staining), immunofluorescence (collagen II) and live dead assays (Calcein AM-Ethidium Homodimer). We show that PRP was able to sustain CP cell viability and differentiate towards adipogenic, osteogenic and chondrogenic lineage under appropriate culture conditions. We also noted positive extracellular matrix production in PRP-CP scaffolds cultured without chondrogenic supplementation. Our results suggest that PRP could be a promising bio-active scaffold due to its synergistic effect in supporting cell proliferation, maintaining cell viability and favoring extracellular matrix production. PRP can be used as biological scaffold for the delivery of CPs in cartilage healing.

Effects of different omega-3 sources, fish oil, krill oil, and green-lipped mussel against cytokine-mediated canine cartilage degradation.

Our purpose was to evaluate the protective effect of three marine omega-3 sources, fish oil (FO), krill oil (KO), and green-lipped mussel (GLM) against cartilage degradation. Canine cartilage explants were stimulated with either 10 ng/mL interleukin-1ß (IL-1ß) or IL-1ß/oncostatin M (10 ng/mL each) and then treated with various concentrations of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA; 3 and 30 µg/mL), FO, KO, or GLM (250, 500, and 1000 µg/mL) for 28 days. Gene expression was then investigated in primary canine chondrocytes. Our results showed that DHA and EPA as well as omega-3 sources could suppress matrix degradation in cytokine-induced cartilage explants by significantly reducing the increase of sulfated glycosaminoglycans (s-GAGs) and preserving uronic acid and hydroxyproline content (except GLM). These agents were not able to reduce IL-1ß-induced IL1B and TNFA expression but were able to down-regulate the expression of the catabolic genes MMP1, MMP3, and MMP13 and up-regulate the anabolic genes AGG and COL2A1; FO and KO were especially effective. Our findings indicated that FO and KO were superior to GLM for their protective effect against proteoglycan and collagen degradation. Hence, FO and KO could serve as promising sources of chondroprotective agents.

Effects of microcurrent therapy on excisional elastic cartilage defects in young rats.

The effects of microcurrent application on the elastic cartilage defects in the outer ear of young animals were analyzed. Sixty male Wistar rats were divided into a control (CG) and a treated group (TG). An excisional lesion was created in the right outer ear of each animal. Daily treatment was started after 24h and consisted of the application of a low-intensity (20µA) continuous electrical current to the site of injury for 5min. The animals were euthanized after 7, 14 and 28 days of injury and the samples were submitted to analyses. In CG, areas of newly formed cartilage and intense basophilia were seen at 28 days, while in TG the same observations were made already at 14 days. The percentage of birefringent collagen fibers was higher in CG at 28 days. The number of connective tissue cells and granulocytes was significantly higher in TG. Ultrastructural analysis revealed the presence of chondrocytes in TG at 14 days, while these cells were observed in CG only at 28 days. Cuprolinic blue staining and the amount of glycosaminoglycans were significantly higher in TG at 14 days and 28 days. The amount of hydroxyproline was significantly higher in TG at all time points studied. The active isoform of MMP-2 was higher activity in TG at 14 days. Immunoblotting for type II collagen and decorin was positive in both groups and at all time points. The treatment stimulated the proliferation and differentiation of connective tissue cells, the deposition of glycosaminoglycans and collagen, and the structural reorganization of these elements during elastic cartilage repair.

The effect of light-emitting diode and laser on mandibular growth in rats.

OBJECTIVE: To evaluate the effect of a light-emitting diode (LED) and/or low-level laser (LLL) with or without the use of anterior bite jumping appliances (also known as functional appliances [FAs]) on mandibular growth in rats. MATERIALS AND METHODS: Thirty-six 8-week-old male Sprague-Dawley rats weighing 200 g were obtained from Charles River Canada (St. Constant, QC, Canada) and were divided into six groups of six animals each. Groups were as follows: group 1: LLL; group 2: LLL + FA; group 3: LED; group 4: LED + FA; group 5: FA; and group 6: control (no treatment). Mandibular growth was evaluated by histomorphometric and micro computed tomographic (microCT) analyses. RESULTS: The LED and LED + FA groups showed an increase in all condylar tissue parameters compared with other groups. CONCLUSION: The LED-treated groups showed more mandibular growth stimulation compared with the laser groups.

Low-intensity pulsed ultrasound stimulation of condylar growth in rats.

OBJECTIVE: To test the hypothesis that low-intensity pulsed ultrasound (LIPUS) stimulation does not histologically affect the growth of mandibular condylar cartilage. MATERIALS AND METHODS: Thirty-five 20-day-old Sprague-Dawley rats were assigned to experimental and control groups. Experimental rats were stimulated with LIPUS in the temporomandibular joint (TMJ) region unilaterally, for 10 or 20 minutes for 20 days. After euthanasia, histological specimens were analyzed qualitatively and histomorphometrically at the anterior and posterior aspects of the mandibular condyle, including the condylar cartilage and the area and perimeter of subchondral bony trabeculae. RESULTS: LIPUS stimulation may alter the histological arrangement of the condylar bone and cartilage, showing qualitative differences on specimens treated for 10 or 20 minutes daily compared with controls. Cartilaginous layer thickness was not affected by LIPUS stimulation to a significant level, but was modified at the relative layer thickness within the cartilage at the anterior aspect of the condyle (P < .05). At the subchondral bone level, 20-minute stimulation significantly increases trabecular perimeter (P = .01). CONCLUSIONS: LIPUS application may affect mandibular growth pattern in rats acting at the cartilage and bone level. The effect of LIPUS on the growing condyle is expressed through a variation in trabecular shape and perimeter. A greater response is achieved when stimulated for 20 minutes instead of 10 minutes daily.

Effect of different ascorbate supplementations on in vitro cartilage formation in porcine high-density pellet cultures.

Articular cartilage has only very limited potential for self-repair and regeneration. For this reason, various tissue engineering approaches have been developed to generate cartilage tissue in vitro. Usually, most strategies require ascorbate supplementation to promote matrix formation by isolated chondrocytes. In this study, we evaluate and compare the effect of different ascorbate forms and concentrations on in vitro cartilage formation in porcine chondrocyte high-density pellet cultures. l-ascorbate, sodium l-ascorbate, and l-ascorbate-2-phosphate were administered in 100 microM, 200 microM, and 400 microM in the culture medium over 16 days. Pellet thickness increased independently from the supplemented ascorbate form and concentration. Hydroxyproline content increased as well, but here, medium concentration of AsAP and low concentration of AsA showed a more pronounced effect. Proteoglycan and collagen formation were evaluated histologically and could be proven in all supplemented cultures. Non-supplemented cultures, however, showed no stable matrix formation at all. Effects on the gene expression pattern of cartilage marker genes (type I and type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP)) were studied by real-time RT-PCR and compared to non-supplemented control cultures. Expression level of cartilage marker genes was elevated in all cultures showing that dedifferentiation of chondrocytes could be prevented. Again, all supplementations caused a similar effect except for low concentration of AsA, which resulted in an even higher expression level of all marker genes. Besides that, we could not detect a pronounced difference between ascorbate and its derivates as well as between the different concentrations.

Effects of gel concentration, human fibronectin, and cation supplement on the tissue-engineered cartilage.

Cultivation of bovine knee chondrocytes (BKCs) in various cationic additives was studied using chitosan-gelatin scaffolds, whose surfaces were modified by human fibronectin (HFN). Here, the genipin-crosslinked scaffolds were fabricated by the freezing/lyophilization method with various concentrations of the precursory gels. The experimental results indicated that a lower freezing temperature led to higher moisture content, porosity, and specific surface area of a scaffold. The higher the precursor concentration, the larger the moisture content of a scaffold. A fast biodegradation of scaffold matrix was generated by a high porosity with BKCs. A higher concentration of HFN coated on scaffold surfaces yielded a faster rate of BKC attachment from the culture medium. The amounts of BKCs, glycosaminoglycans, and collagen over 28-day cultivation increased with the scaffold porosity, the coating concentration of HFN, the seeding density of BKCs, and the calcium concentration in medium.

Mechanisms of cartilage growth: modulation of balance between proteoglycan and collagen in vitro using chondroitinase ABC.

OBJECTIVE: To examine the cartilage growth-associated effects of a disruption in the balance between the swelling pressure of glycosaminoglycans (GAGs) and the restraining function of the collagen network, by diminishing GAG content prior to culture using enzymatic treatment with chondroitinase ABC. METHODS: Immature bovine articular cartilage explants from the superficial and middle layers were analyzed immediately or after incubation in serum-supplemented medium for 13 days. Other explants were treated with chondroitinase ABC to deplete tissue GAG and also either analyzed immediately or after incubation in serum-supplemented medium for 13 days. Treatment- and incubation-associated variations in tissue volume, contents of proteoglycan and collagen network components, and tensile mechanical properties were assessed. RESULTS: Incubation in serum-supplemented medium resulted in expansive growth with a marked increase in tissue volume that was associated with a diminution of tensile integrity. In contrast, chondroitinase ABC treatment on day 0 led to a marked reduction of GAG content and enhancement of tensile integrity, and subsequent incubation led to maturational growth with minimal changes in tissue volume and maintenance of tensile integrity at the enhanced levels. CONCLUSION: The data demonstrate that a manipulation of GAG content in articular cartilage explants can distinctly alter the growth phenotype of cartilage. This may have practical utility for tissue engineering and cartilage repair. For example, the expansive growth phenotype may be useful to fill cartilage defects, while the maturational growth phenotype may be useful to induce matrix stabilization after filling defect spaces.

[Comparative yearly growth rate of children with mucoviscidosis treated and not treated with ciprofloxacin:clinicomorphological comparisons]. / Cravnitel'naiia godovaia skorost' rosta u detei s mukovistsidozom, poluchavshikh i nepolychavshikh tsiprofloksatsin:klinicomorfologicheskie copostavleniia.

The results of the prospective and comparative investigation of the linear growth of children at the age of 4 to 16 years with mucoviscidosis treated with ciprofloxacin in combination with a cephalosporin or an aminoglycoside in the main group and a cephalosporin or an aminoglycoside alone in the control group are presented. The children were observed for 3 and 5 years. It was shown that in spite of the treatment term with ciprofloxacin the yearly growth rate in the children in the main and control groups did not significantly differ. The morphological investigation did not reveal any injury of the armicular cartilage and growth zone. The hyperplastic reaction in the tegmental cartilage was states and considered as a physiological one in response to the presence of ciprofloxacin and therefore reversible. No chondrotoxicity of the fluoroquinolones and ciprofloxacin, particularly in the children, is explained.

Differences in fatty acid composition of immature and mature articular cartilage in humans and sheep.

Chondrocytes are imbedded in an avascular, highly charged extracellular matrix which could form a barrier to the transfer of dietary essential fatty acids (EFA) to chondrocytes. A study was designed to assess the composition of immature and mature joint cartilage with respect to essential and nonessential fatty acids relevant to EFA deficiency. Cartilage and muscle samples were obtained from human fetus, infant and adult cadavers, and from fetal and mature sheep. Lipid extracts were prepared and the fatty acid composition determined. In human and sheep joint cartilage, linoleic acid (LA; 18:2n-6) content was lower, and n-9 eicosatrienoic acid (ETrA; 20:3n-9) and arachidonic acid (AA; 20:4n-6) were higher in fetuses compared to mature subjects. An intermediate pattern was seen in infant cartilage. n-3 Fatty acids tended to be higher in fetal than in mature cartilage in humans and in sheep. In human muscle (and in other noncartilaginous comparison tissues), similar differences between fetuses and adults were seen in LA and AA, but not in ETrA. In fetal sheep muscle, very low LA, reduced AA and raised ETrA levels compared to mature sheep muscle were seen. However, although the pattern is characteristic of EFA deficiency, the abundance of n-6 EFA in liver and spleen of human fetuses and of n-3 EFA in liver and spleen of fetal sheep suggests that placental transfer of EFA is not likely to be limiting. During fetal development, the metabolism of fatty acids is distinctive and differs between the species. ETrA appears to be a readily measurable component of some tissues at certain stages of development when its presence in tissues does not indicate EFA deficiency.

Effects of pulsing electromagnetic fields on bone growth and articular cartilage.

Observations made during treatment of juvenile pseudarthrosis by pulsing electromagnetic fields (PEMF) suggested that bone growth might be altered. PEMF applied to immature rabbits under conditions of continuous stimulation (24 hours/day for 8 weeks) produced no major changes in bone growth. Continuous stimulation by PEMF induced a statistically significant increase (22%) in femoral articular cartilage glycosaminoglycan. Intermittent PEMF stimulation (12 hours with stimulation/12 hours without stimulation) for 18 weeks produced no significant change in bone growth or time of epiphyseal plate closure. No significant changes in the physical characteristics of growing bone were observed with any treatment.