Letter to the editor UC-II® Undenatured type II collagen: update to analytical methods.

Supplementation with UC-II® undenatured type II collagen has been shown to provide joint health benefits for both healthy adults and adults with knee osteoarthritis in controlled clinical trials. These trials used UC-II® materials with undenatured type II collagen characterized by an ELISA method that utilizes a monoclonal antibody specific for epitopes expressed by undenatured type II collagen protein only. In 2014, we modified the sample preparation part of the ELISA method in order to reduce the amount of time devoted to this procedure. We undertook these modifications in order to provide commercial manufacturers with a streamlined assay methodology better aligned with their product testing requirements. In doing so, it altered the percent of undenatured collagen now reported for UC-II® undenatured type II collagen. The intent of this letter is to clarify that the UC-II® materials used in the published clinical research cited herein, and the commercially available ingredient, remain identical and to describe the rationale for the change in the extraction method.

Beneficial effects of Ankaferd Blood Stopper on dermal wound healing: an experimental study.

Ankaferd Blood Stopper(®) (ABS) is a folkloric medicinal plant extract used as a haemostatic agent in traditional Turkish medicine. The aim of this study was to investigate the efficacy of ABS on the healing of dermal wounds in a rat model. Twenty Wistar albino rats were divided into two groups. Standard full-thickness skin defects were created on the back of the rats. In the control group (group 1), dressings moisturised with saline were changed daily. In the study group (group 2), the wounds were cleaned daily with saline, Ankaferd solution was applied, then the wounds were covered with moisturised dressings. The contraction percentage of wound areas were calculated on the 3rd, 7th, 10th and 14th days using a planimetric programme. On day 14, the wound areas were excised for histopathological examination, inflammatory scoring and evaluation of collagen deposition. The study group was superior to the control group in terms of inflammatory scoring, type I/type III collagen ratio and wound contraction rates. ABS(®) may be used effectively and safely on full-thickness wounds as a natural product.

Supplementation with platelet-rich plasma improves the in vitro formation of tissue-engineered cartilage with enhanced mechanical properties.

PURPOSE: This study aimed to determine the effects of platelet-rich plasma (PRP) on the histologic, biochemical, and biomechanical properties of tissue-engineered cartilage. METHODS: Chondrocytes isolated from bovine metacarpal-phalangeal articular cartilage were seeded on top of a porous ceramic substrate (calcium polyphosphate [CPP]). Cultures were supplemented with fetal bovine serum (FBS), PRP, or platelet-poor plasma (PPP) at 5%. On day 5, the concentration was increased to 20%. PRP and PPP were obtained through centrifugation of whole blood withdrawn from a mature cow. After 2 weeks, samples (n = 8) were analyzed histologically, biochemically, and biomechanically. Data were analyzed using the Wilcoxon test (significance, P < .05). RESULTS: Chondrocytes cultured in 20% PRP formed thicker cartilage tissue (1.6 ± 0.2 mm) than did cells grown in 20% FBS (0.7 ± 0.008 mm; P = .002) and 20% PPP (0.8 ± 0.2 mm; P = .03). Cartilage tissue generated in the presence of 20% PRP had a greater equilibrium modulus of 38.1 ± 3.6 kPa versus 15.6 ± 1.5 kPa (P = .0002) for 20% PPP and 20.4 ± 3.5 kPa (P = .007) for 20% FBS. Glycosaminoglycan (GAG) content was increased in tissues formed in 20% PRP (176 ± 18.8 µg GAG/mg) compared with those grown in 20% FBS (112 ± 10.6 µg GAG/mg; P = .01) or 20% PPP (131.5 ± 14.8 µg GAG/mg; P = .11). Hydroxyproline content was similar whether the media was supplemented with 20% PRP (8.7 ± 0.9 µg/mg), 20% FBS (7.6 ± 0.9 µg/mg; P = .37), or 20% PPP (6.4 ± 1 µg/mg; P = .28). DNA content was similar in all tissues whether formed in 20% PRP (11.9 ± 3.5 µg/mg), 20% FBS (9.3 ± 2.5 µg/mg; P = .99), or 20% PPP (7.2 ± 1.3 µg/mg; P = .78). Immunostained samples showed prevalence of type II collagen in tissues formed in the presence of 20% PRP. CONCLUSIONS: The presence of PRP in the culture media enhances the in vitro formation of cartilage, with increased GAG content and greater compressive mechanical properties, while maintaining characteristics of hyaline phenotype. CLINICAL RELEVANCE: Understanding the in vitro effects of PRP on tissue-engineered cartilage may lead to the creation of engineered cartilage tissue with enhanced properties suitable for cartilage repair.

Effects of T-2 toxin and selenium on chondrocyte expression of matrix metalloproteinases (MMP-1, MMP-13), α2-macroglobulin (α2M) and TIMPs.

T-2 toxin is regarded as an important etiological factor of Kashin-Beck disease, and supplementation of selenium-salt partly prevents Kashin-Beck disease. The present study investigated the effects of T-2 toxin on the degradation of type II collagen in human chondrocytes in vitro. Human chondrocytes were isolated and cultured on bone matrix gelatin to form an artificial cartilage model in vitro with or without T-2 toxin and selenium. Immunohistochemistry analyses showed that T-2 toxin decreased type II collagen staining and selenium appeared to prevent the decrease in type II collagen induced by T-2 toxin in engineered cartilage. Then, Western blot and RT-PCR analyses showed that an increase in MMP-13 and MMP-1 expressions, and a decrease in the expression of the general endoproteinase inhibitor (α(2)M) were induced by T-2 toxin. Gelatin reverse zymography showed that TIMP-1 and TIMP-2 levels were decreased in a dose-dependent manner after exposure of T-2 toxin. Selenium had a protective role by increasing the level of type II collagen protein through down-regulation of MMP-13 protein and mRNA expression and up-regulation of TIMP-1 and TIMP-2 expressions. These data suggest T-2 toxin induces cartilage matrix degradation by the up-regulation of MMP-13 and TIMP-1, and down-regulation of TIMP-2 and α(2)M expressions.

Effect of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose on elastase and hyaluronidase activities and its type II collagen expression.

In the current era, natural products are gaining prime attention in the fields of cosmeceuticals and pharmaceuticals due to higher safety margins and biological functions, as they have a considerable amount of potential in treating different ailments. Thus, to find effective elastase and hyaluronidase inhibitors from natural resources, fifty Korean plants were screened, and the fruit of Terminalia chebula RETZIUS (Combretaceae) was selected for further structural isolation due to its potent efficacy. The methanol crude extract of the fruits showed 80% elastase and 87% hyaluronidase enzyme inhibition activities at 1 mg/mL. The crude extract, upon bioassay-directed fractionation, led to the isolation of compound 1, whose structure was found by spectral analysis to be 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG). PGG displayed significant elastase and hyaluronidase inhibitory activities with IC50, values of 57 microg/mL and 0.86 mg/mL, respectively; also, treatment of PGG on rabbit articular chondrocytes significantly induced the type II collagen expression. Based on elastase and hyaluronidase inhibitions, and type II collagen expression, it could be suggested that PGG might have an influence on skin conditions when used cosmetically as an active anti-aging ingredient with no cytotoxicity; also, it might be beneficial in relieving painful joint conditions, and thus have relevance for treating arthritis. Therefore, it can be concluded that PGG may prove to be an active ingredient in cosmeceutical and pharmaceutical formulations, and that it definitely merits further in vivo investigations.

Prevention of colonic fibrosis by Boswellia and Scutellaria extracts in rats with colitis induced by 2,4,5-trinitrobenzene sulphonic acid.

BACKGROUND: Currently, no effective preventive measures or medical therapies are available for intestinal fibrosis and, thus, surgery remains the only available strategy in the management of fibrostenotic enteropathies, especially Crohn's disease. The aim of this study was to evaluate the efficacy of a combined therapy of anti-inflammatory Boswellia and antifibrotic Scutellaria extracts on the development of colonic fibrosis in rats. MATERIALS AND METHODS: Chronic colonic inflammation-associated fibrosis was induced in rats by intracolonic administration of 2,4,5-trinitrobenzene sulphonic acid (TNBS). Sixty-four healthy male Sprague-Dawley rats were assigned to five groups: 8 controls, 14 TNBS, 14 TNBS orally treated with Boswellia extracts (50 mg kg(-1) day(-1)), 14 TNBS orally treated with Scutellaria extracts (150 mg kg(-1) day(-1)), and 14 TNBS orally treated with both Boswellia (50 mg kg(-1) day(-1)) and Scutellaria extracts (150 mg kg(-1) day(-1)). The colon was removed after 21 days of treatment and assessed by macroscopic, histological, morphometric and immunohistochemical analyses. For immunohistochemical analysis, alpha-smooth muscle actin (alpha-SMA), collagen types I-III, connective tissue growth factor (CTGF), transforming growth factor-beta1 (TGF-beta1), Smad3, Smad7 and CD3 antibodies were used. RESULTS: Combined oral administration of Boswellia and Scutellaria significantly improved the course and macroscopic findings of TNBS-induced chronic colitis assessed by disease activity index, colon weight, length, adhesions, strictures, dilatation, thickness, oedema, ulcerations and extension of damage. The histological severity of the colonic fibrosis was also notably improved by the treatment and associated with a significant reduction in the colonic expression of alpha-SMA, collagen I-III, CTGF, TGF-beta1, Smad3, and Smad7. CONCLUSIONS: These data demonstrate that the prophylactic administration of anti-inflammatory Boswellia and antifibrotic Scutellaria extracts is effective in preventing colonic fibrosis in TNBS-induced colitis. Their antifibrotic mechanism of action seems to be mediated by the inhibition of TGF-beta1/Smad3 pathway.

Human serum for tissue engineering of human nasal septal cartilage.

OBJECTIVE: To compare the chondrogenic and proliferative effects of pooled human serum (HS) and fetal bovine serum (FBS) on tissue-engineered human nasal septal chondrocytes. STUDY DESIGN AND SETTING: Human chondrocytes were expanded for one passage in monolayer in medium supplemented with 10% FBS, 2% HS, 10% HS, or 20% HS. Cells were then suspended in alginate beads for 3D culture for 2 weeks with 10% FBS, 2% HS, 10% HS, or 20% HS. RESULTS: Monolayer cell yields were greater with HS than FBS. In alginate, cellular proliferation, glycosaminoglycan production per cell, and type II collagen were significantly higher with 10% HS compared to 10% FBS controls. CONCLUSION: HS results in increased proliferation and production of cartilaginous extracellular matrix by tissue-engineered human nasal septal chondrocytes, compared to FBS controls. SIGNIFICANCE: Culture with human serum may facilitate creation of neocartilage constructs that more closely resemble native tissue.

[Major constituent proteins in donkey hide and their interaction].

OBJECTIVE: To analyze the constituent proteins in donkey hide, the key ingredient for Ejiao, an important traditional Chinese medicine for the blood-related conditions, in hope to eventually decipher the biochemical mechanism behind Ejiao's prominent medicinal efficacy. METHOD: Two methods were employed to extract proteins in donkey skin. One used TriPure isolation reagent to extract the total proteins in donkey skin. Another used 1% sodium dodecyl sulfate (SDS) to heat the sample at 100 degrees C overnight. And then sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and capillary HPLC were used to analyze the component of proteins. RESULT: There are not only collagen alpha1 (I) and collagen alpha2 (I), but also serum albumin in donkey skin. The content is over 25% in total proteins with the method of TriPure isolation reagent. The content of donkey serum albumin is up to 20% with the method of 1% SDS heating. And two bands, molecular weight are nearly 200 kDa,were found on 7.5% SDS-PAGE. Extracted these proteins to analyze with capillary HPLC, they were found to be the complex products of collagen and serum albumin of donkey. CONCLUSION: Donkey serum albumin is a main protein component in the hide, which is a clue to expose is the effect of Ejiao on blood.

The effect of irradiation and hyperbaric oxygenation (HBO) on extracellular matrix of the condylar cartilage after mandibular distraction osteogenesis in the rabbit.

The effects of irradiation and hyperbaric oxygenation (HBO) on the extracellular matrix of condylar cartilage after mandibular distraction were evaluated. Unilateral distraction was performed on 19 rabbits. Five study groups were included: control, low- and high-dose irradiation, and low- and high-dose irradiation groups with HBO. Additionally, four temporomandibular joints (TMJ) were used as control material. The high-dose irradiated animals were given in the TMJ 22.4 Gy/4 fractions irradiation (equivalent to 50 Gy/25 fractions). Low-dose irradiation group received a 2.2 Gy dosage. Two groups were also given preoperatively HBO 18 x 2.5ATA x 90 min. After a two-week distraction period (14 mm lengthening) and four-week consolidation period the TMJs were removed. Proteoglycan (PG) distribution of the extracellular matrix was evaluated using safranin O staining and collagen I and II using immunohistochemistry. The organization of fibrillar network was studied by polarized light microscopy. On the operated side of the control group and on the unoperated side in all, except for high-dose irradiated group, PG distribution and fibrillar network were normal appearing. In the irradiated groups, with or without HBO, the cartilaginous layer was partially or totally devoid of PG and the network structure was severely damaged. In conclusion, irradiation in conjunction with the pressure applied by distraction causes severe damage to extracellular matrix of condylar cartilage.

Bone-constructing cells from ethmoid bone may have multilineage differentiation potential: preliminary report.

OBJECTIVE: In order to investigate multilineage differentiation in human cultured cells from ethmoid bone, we conducted a morphological study to examine adipogenic and chondrogenic differentiation. MATERIAL AND METHODS: After reaching confluence, cells underwent terminal adipogenic differentiation by treatment with 100 microM indomethacin, 0.5 mM 1-methyl-3-isobutylxanthine, 1 microM dexamethasone (DEX), 10 microg/ml insulin and 0.3% dimethylsulfoxide in a medium supplemented with 10% fetal bovine serum. Chondrogenic differentiation was attempted by centrifuging a pelleted micromass using transforming growth factor-beta3 (TGF-beta3), DEX, ascorbic acid (AA), pyruvate acid, proline, glucose and (ITS)-plus. RESULTS: The cultured cells displayed adipocyte but not chondrogenic lineage under these conditions. Considering the possibility that some differentiation potential may be lost with in vitro culture but maintained using another chondrogenic differentiation medium containing TGF-beta1, it is possible that cultured cells may have multilineage potential, including chondrogenic differentiation ability. CONCLUSIONS: These morphological abilities of human cultured cells may indicate the possibility of the existence of mesenchymal stem cells in sinus bone. If mesenchymal stem cells exist in ethmoid bone, they may play an important role in future research on the regulation mechanisms of human bone tissue.

[Mesenchymal stem cells--a new pathway for tissue engineering in reconstructive surgery]. / Adulte mesenchymale Stammzellen--neue Möglichkeiten der Gewebezüchtung für die plastisch-rekonstruktive Chirurgie. andreas.naumann@hno.med.uni-muenchen.de

INTRODUCTION: Mesenchymal stem cells (MSC) have the capacity to differentiate into chondrocytes with the synthesis of cartilage. This report presents the use of human adult bone marrow derived mesenchymal stem cells for tissue engineering of autologous cartilage grafts. METHODS: Human bone marrow aspirates were obtained from the iliac crest and fractionated on a Percoll gradient. The isolated hMSC were plated at 20 x 10 (6) cells per 100 mm (2) culture dish. After 21 days in culture at 37 degrees C with 5 % CO 2, the adherent multiplied MSC were trypsinized, counted, and tested for viability by trypan blue assay. The hMSCs were loaded into a sterile 15 ml polypropylene tube (0.5 Mio cells/ml) and centrifuged on the bottom of the tube at 500 g for 5 minutes. The MSC were cultivated for 3 weeks in vitro in a specific chondrogenetic medium composed of Dulbecco's Modified Eagles Medium-High Glucose supplemented with 10 ng/ml transforming growth factor-beta 1, 1 % ITS-Premix medium, 80 micro M ascorbic acid, and 100 nM dexamethasone. RESULTS: Histological and immunohistochemical studies performed after 3 weeks in three dimensional culture demonstrated the expression of cartilage specific collagen type II and X as well as proteoglycans. CONCLUSION: Human adult mesenchymal stem cells derived from bone marrow aspirates have the ability to differentiate into chondrocytes under specific culture conditions by growth factors. The use of adult mesenchymal stem cells may be a promising tool for tissue engineering of autologous cartilage grafts in reconstructive surgery in the future.