Assessment of Cell Viability of Fresh Osteochondral Allografts in N-Acetylcysteine-Enriched Medium.

OBJECTIVE: The purpose of this study was to evaluate the effect of N-acetylcysteine (NAC)-enriched storage medium on fresh osteochondral viability at 4°C. Our hypothesis was that the cell viability of chondrocytes obtained from human osteochondral tissue and stored at 4°C significantly improves in the presence of NAC. DESIGN: Controlled laboratory study. For this study, 8 samples of femoral condyle osteochondral tissue were obtained from patients undergoing total knee replacement. The samples were stored at either 4°C in phosphate-buffered saline (PBS) or at 3 different concentrations of NAC (NAC 1, 2, and 5 mM). Cell viability was analyzed at time 0 and 4 weeks by flow cytometry. The results of cell viability (median) were analyzed statistically using analysis of variance and Tukey's post hoc test. P values <0.05 were considered statistically significant. RESULTS: The viability at time 0 was 95.5% ± 3.7%. At 4 weeks, the cell viability was 56.8% ± 20.1% in the control group (PBS), 83.8% ± 11.9% in the group stored with NAC 1 mM, 73.4% ± 13.6% in the group stored with NAC 2 mM, and 66.4% ± 27.7% in the group stored with NAC 5 mM. A statistically significant difference from the baseline viability (time 0) was observed in the PBS control group (P = 0.0018) but not in the other groups. A statistically significant difference was observed in the NAC 1 mM group compared with the PBS group (P = 0.0255). CONCLUSION: The use of NAC at 1 mM concentration improves cell viability after 4 weeks of storage in chondrocytes obtained from human osteochondral tissue.

Assessment of cartilage regeneration on 3D collagen-polycaprolactone scaffolds: Evaluation of growth media in static and in perfusion bioreactor dynamic culture.

Efforts on bioengineering are directed towards the construction of biocompatible scaffolds and the determination of the most favorable microenvironment, which will better support cell proliferation and differentiation. Perfusion bioreactors are attracting growing attention as an effective, modern tool in tissue engineering. A natural biomaterial extensively used in regenerative medicine with outstanding biocompatibility, biodegradability and non-toxic characteristics, is collagen, a structural protein with undisputed beneficial characteristics. This is a study designed according to the above considerations. 3D printed polycaprolactone (PCL) scaffolds with rectangular pores were coated with collagen either as a coating on the scaffold's trabeculae, or as a gel-cell solution penetrating scaffolds' pores. We employed histological, molecular and imaging techniques to analyze colonization, proliferation and chondrogenic differentiation of Adipose Derived Mesenchymal Stem Cells (ADMSCs). Two different differentiation culture media were employed to test chondrogenic differentiation on gelated and non gelated PCL scaffolds in static and in perfusion bioreactors dynamic culture conditions. In dynamic culture, non gelated scaffolds combined with our in house TGF-ß2 based medium, augmented chondrogenic differentiation performance, which overall was significantly less favorable compared to StemPro™ propriety medium. The beneficial mechanical stimulus of dynamic culture, appears to outgrow the disadvantage of the "weaker" TGF-ß2 medium used for chondrogenic differentiation. Even though cells in static culture grew well on the scaffold, there was limited penetration inside the construct, so the purpose of the 3D culture was not fully served. In contrast dynamic culture achieved better penetration and uniform distribution of the cells within the scaffold.

European chondroitin sulfate and glucosamine food supplements: A systematic quality and quantity assessment compared to pharmaceuticals.

Chondroitin sulfate and glucosamine, commercialized as anti-osteoarthritis food supplements, do not undergo the strict quality controls of pharmaceuticals. In this paper a systematic multi-analytical approach was designed to analyse 25 food supplements from 8 European countries compared to 2 pharmaceuticals by using high performance anion-exchange chromatography with pulsed amperometric detection, size exclusion chromatography with triple detector array, capillary electrophoresis, mono and bi-dimensional NMR. Furthermore the biological activity was assessed on in vitro human synoviocyte and chondrocyte primary cell models. Most of the samples (over 19 out of 25) showed lower condroitin sulfate and glucosamine contents than the declared ones (up to -60.3%) while all of them showed a KS contamination (up to 47.1%). Mixed animal origin chondroitin sulfate and multiple molecular weight species were determined in more than 32% of the samples. Only 1 on 5 biologically screened samples had an effective action in vitro almost comparable to the pharmaceuticals.

Complete Assessment of Multilineage Differentiation Potential of Human Skeletal Muscle-Derived Mesenchymal Stem/Stromal Cells.

The minimal criteria for mesenchymal stem/stromal cell (MSC) identification set by the International Society for Cellular Therapy include plastic adherence, presence and absence of a set of surface antigens and in vitro multilineage differentiation. This differentiation is assessed through stimulation of MSCs with defined combination and concentration of growth factors towards specific lineages and histological confirmation of the presence of differentiated cells. Here we provide protocols for multilineage differentiation, namely, osteogenesis, adipogenesis, chondrogenesis and myogenesis. We also provide their respective histological analyses.

Assessment of the effect of systemic delivery of sclerostin antibodies on Wnt signaling in distraction osteogenesis.

Sclerostin is a known inhibitor of the Wnt signaling pathway which is involved in osteogenesis and, when inactivated, stimulates bone formation. To our knowledge, this effect has not been studied in the context of distraction osteogenesis (DO). Tibial DO was conducted on a total of 24 wild-type mice, which were then divided into 2 groups-a saline injection group (control) and an anti-sclerostin (Scl-Ab) injection group (treatment). The mice in the treatment group received 100 mg/kg intravenous injections of the antibody weekly until killing. The 12 mice in each group were subdivided into four time points according to post-osteotomy time of killing-11 days (mid-distraction), 17 days (late distraction), 34 days (mid-consolidation) and 51 days (late consolidation), with 3 mice per subgroup. After killing, the tibia specimens were collected for immunohistochemical analysis. Our results show that the group injected with anti-sclerostin had an earlier peak (day 11) in the distraction phase of the osteogenic molecules involved in the Wnt signaling pathway in comparison to the placebo group. In addition, downregulation of the inhibitors of this pathway was noted in the treatment group when compared with the placebo group. Furthermore, LRP-5 showed a significant increase in expression in the treatment group. Sclerostin inhibition has a significant effect on the DO process through its effect on the Wnt pathway. This effect was evident through the decreased effect of sclerostin on LRP-5 and earlier upregulation of the osteogenic molecules involved in this pathway.

In vitro assessment of human chondrocyte viability after treatment with local anaesthetic, magnesium sulphate or normal saline.

PURPOSE: Local anaesthetic agents are often used as an intra-articular analgesic following arthroscopic procedures. However, there is increasing evidence of a potential toxic effect to chondrocytes within the articular cartilage. The aim of this study was to compare the effect on human chondrocyte viability of treatment with bupivacaine, levobupivacaine and ropivacaine. The second aim was to compare the effect on chondrocyte viability of the local anaesthetics with magnesium, a potential alternative analgesic agent. METHODS: Chondrocytes were exposed to one of the local anaesthetic agents (levobupivacaine 0.13, 0.25, 0.5%; bupivacaine 0.13, 0.25, 0.5%; ropivacaine 0.19, 0.38, 0.75%), normal saline or 10% magnesium sulphate for 15 min. Cells exposed to cell culture media served as controls. Twenty-four hours after exposure, cell viability was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. RESULTS: There was no significant difference in chondrocyte viability after treatment with either normal saline or magnesium sulphate. With the exception of 0.13% levobupivacine, all local anaesthetic treatment showed significantly greater toxic effects than either normal saline or magnesium sulphate. Statistically significant dose-dependent responses of decreasing cell viability were found with increasing local anaesthetic concentration. CONCLUSIONS: A dose-dependent reduction in chondrocyte viability after treatment with common local anaesthetic agents was confirmed. Local anaesthetic agents had a greater deleterious effect on chondrocytes than did 10% magnesium sulphate. These findings suggest the need for continuing caution with the use of intra-articular local anaesthetic. Magnesium sulphate is a potential alternative intra-articular analgesic agent.

Assessment of growth factor treatment on fibrochondrocyte and chondrocyte co-cultures for TMJ fibrocartilage engineering.

Treatments for patients suffering from severe temporomandibular joint (TMJ) dysfunction are limited, motivating the development of strategies for tissue regeneration. In this study, co-cultures of fibrochondrocytes (FCs) and articular chondrocytes (ACs) were seeded in agarose wells, and supplemented with growth factors, to engineer tissue with biomechanical properties and extracellular matrix composition similar to native TMJ fibrocartilage. In the first phase, growth factors were applied alone and in combination, in the presence or absence of serum, while in the second phase, the best overall treatment was applied at intermittent dosing. Continuous treatment of AC/FC co-cultures with TGF-ß1 in serum-free medium resulted in constructs with glycosaminoglycan/wet weight ratios (12.2%), instantaneous compressive moduli (790 kPa), relaxed compressive moduli (120 kPa) and Young's moduli (1.87 MPa) that overlap with native TMJ disc values. Among co-culture groups, TGF-ß1 treatment increased collagen deposition ∼20%, compressive stiffness ∼130% and Young's modulus ∼170% relative to controls without growth factor. Serum supplementation, though generally detrimental to functional properties, was identified as a powerful mediator of FC construct morphology. Finally, both intermittent and continuous TGF-ß1 treatment showed positive effects, though continuous treatment resulted in greater enhancement of construct functional properties. This work proposes a strategy for regeneration of TMJ fibrocartilage and its future application will be realized through translation of these findings to clinically viable cell sources.

C-type natriuretic peptide as a new regulator of food intake and energy expenditure.

The physiological implication of C-type natriuretic peptide (CNP) including energy metabolism has not been elucidated, because of markedly short stature in CNP-null mice. In the present study we analyzed food intake and energy expenditure of CNP-null mice with chondrocyte-targeted CNP expression (CNP-Tg/Nppc(-/-) mice), in which marked skeletal dysplasia was rescued, to investigate the significance of CNP under minimal influences of skeletal phenotypes. In CNP-Tg/Nppc(-/-) mice, body weight and body fat ratio were reduced by 24% and 32%, respectively, at 20 wk of age, and decreases of blood glucose levels during insulin tolerance tests were 2-fold exaggerated at 17 wk of age, as compared with CNP-Tg/Nppc(+/+) mice. Urinary noradrenalin excretion of CNP-Tg/Nppc(-/-) mice was greater than that of CNP-Tg/Nppc(+/+) mice by 28%. In CNP-Tg/Nppc(-/-) mice, rectal temperature at 1600 h was higher by 1.1 C, and uncoupling protein-1 mRNA expression in the brown adipose tissue was 2-fold increased, which was canceled by propranolol administration, as compared with CNP-Tg/Nppc(+/+) mice. Oxygen consumption was significantly increased in CNP-Tg/Nppc(-/-) mice compared with that in CNP-Tg/Nppc(+/+) mice. Food intake of CNP-Tg/Nppc(-/-) mice upon ad libitum feeding and refeeding after 48 h starvation were reduced by 21% and 61%, respectively, as compared with CNP-Tg/Nppc(+/+) mice. This study unveiled a new aspect of CNP as a molecule regulating food intake and energy expenditure. Further analyses on precise mechanisms of CNP actions would lead to the better understanding of the significance of the CNP/guanylyl cyclase-B system in food intake and energy expenditure.

Mechano-functional assessment of human mesenchymal stem cells grown in three-dimensional hyaluronan-based scaffolds for cartilage tissue engineering.

Human mesenchymal stem cells (hMSCs) are an alternative cell source in bioconstruct production for cartilage regeneration, and hyaluronic acid (HA) is a widely-used bioabsorbable scaffold material used for cartilage regeneration. In this work, the aims were to evaluate the mechanical competency of hMSC-seeded HA scaffolds compared with native intact human articular cartilage, and in relation to its cellular properties. Human MSCs were grown under static conditions in HA scaffolds and then tested, in stepwise, stress-relaxation indentation, 7, 14, and 21 days later. Scaffolds at days 14 and 21 showed a significant increase in mechanical measures when compared with day 7 and unseeded scaffold material, but did not achieve the same levels as human cartilage. There was consistent stiffness within the scaffold, with a decreased stiffness around the edge. In vitro culture of hMSC-seeded HA scaffolds over 3 weeks produces a white, solid tissue compared with unseeded constructs. Increased cell proliferation and collagen type II expression were also seen over this period of time. These results demonstrate the competency of the neo-formed cartilage-like tissue in relation to its mechanical and cellular properties, and further, the importance, for future clinical use, of implanting this construct after 14 days of culture.

Growth and growth factor production by human nasal septal chondrocytes in serum-free media.

BACKGROUND: Tissue-engineered human cartilage offers vast possibilities as a source of graft implant material for reconstructive surgery. Serum-supplemented growth media is successful in supporting chondrocyte proliferation in vitro. Serum, however, contains exogenous growth factors that hamper the identification and quantification of growth factors autogenously produced by chondrocytes. We explore the possibility of using a commercially available serum-free medium UltraCULTURE as an alternative to modified Webber's medium (MWM), the standard media used in chondrocyte cell culture. METHODS: Human nasal septal chondrocytes were grown in UltraCULTURE containing various concentrations of basic fibroblast growth factor (bFGF; 0, 1, 10, and 100 ng/mL) with or without insulin-like growth factor and compared with chondrocytes grown in MWM. Growth curves and transforming growth factor (TGF) beta 1 production were analyzed. RESULTS: We found no differences in the ability to sustain cell viability in culture between the two base media types. We also found no statistically significant differences in TGF-beta 1 production by chondrocytes grown in either system. Finally, there were no statistically significant differences in chondrocyte proliferation between cultures supplemented with bFGF at 10 and 100 ng/mL. CONCLUSION: UltraCULTURE media is a cost-effective, serum-free alternative to standard media with compatible growth characteristics. It offers specific advantages over standard serum-containing media for the precise measurement of autogenous growth factor production by cultured chondrocytes. Furthermore, UltraCULTURE's serum-free environment would be ideal for safely producing tissue-engineered cartilage grafts.

Three-dimensional culture of human disc cells within agarose or a collagen sponge: assessment of proteoglycan production.

The objective of the present study was to assess proteoglycan production by human intervertebral disc cells cultured in vitro in selected cell carriers. Based on previous studies which evaluated disc cells seeded into collagen sponge, collagen gel, agarose, alginate or fibrin gel three-dimensional (3D) cell carriers, collagen sponge and agarose were found to provide superior microenvironments for formation of extracellular matrix (ECM). A standardized test design was used to evaluate ECM formed after 14 days of culture using the 1,9-dimethylmethylene blue (DMB) assay to assess sulfated glycosaminoglycan (S-GAG) production. Although agarose culture showed higher S-GAG levels compared to collagen sponge (2.94+/-2.20 (19) microg/ml S-GAG (mean+/-S.D. (n)) vs. 0.94+/-0.77 (22), respectively, p=0.0003), this is off-set by the significantly lower proliferation rate associated with culture of disc cells in agarose.

Degeneration of intervertebral discs due to smoking: experimental assessment in a rat-smoking model.

We have investigated the intervertebral discs of rat-smoking models to demonstrate that smoking is a cause of degenerative intervertebral disc disease. A smoking box was developed for this study. We exposed 8-week-old rats to indirect tobacco smoke inhalation. Each rat was forced to inhale the smoke from one cigarette per hour. The mean blood nicotine level of rodents exposed to cigarette smoke corresponds to about twice that of ordinary human smokers. Histological and immunological studies were then performed to assess the effects of smoking for varying periods of time. After 8 weeks, the chondrocytes in the disordered annulus fibrosus layer tended to grow larger and attain a rounder form than normal chondrocytes. The interleukin-1beta level in the 8-week smoking group was significantly higher than that of the control group. Tobacco smoke inhalation increased local production and release of inflammatory cytokines and resultant decomposition of chondrocyte activity.