Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes.

BACKGROUND: Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% ) of N. METHODS: In the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes) cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13) and molecules implicated in cell division (cyclin B2). Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes) and nitrogen-containing cation polystyrene (Primaria®), were also investigated for comparison. RESULTS: Results showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels. CONCLUSION: Hypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving.

Role of calsequestrin evaluated from changes in free and total calcium concentrations in the sarcoplasmic reticulum of frog cut skeletal muscle fibres.

Calsequestrin is a large-capacity Ca-binding protein located in the terminal cisternae of sarcoplasmic reticulum (SR) suggesting a role as a buffer of the concentration of free Ca in the SR ([Ca2+](SR)) serving to maintain the driving force for SR Ca2+ release. Essentially all of the functional studies on calsequestrin to date have been carried out on purified calsequestrin or on disrupted muscle preparations such as terminal cisternae vesicles. To obtain information about calsequestrin's properties during physiological SR Ca2+ release, experiments were carried out on frog cut skeletal muscle fibres using two optical methods. One - the EGTA-phenol red method - monitored the content of total Ca in the SR ([Ca(T)](SR)) and the other used the low affinity Ca indicator tetramethylmurexide (TMX) to monitor the concentration of free Ca in the SR. Both methods relied on a large concentration of the Ca buffer EGTA (20 mM), in the latter case to greatly reduce the increase in myoplasmic [Ca2+] caused by SR Ca2+ release thereby almost eliminating the myoplasmic component of the TMX signal. By releasing almost all of the SR Ca, these optical signals provided information about [Ca(T)](SR) versus [Ca2+](SR) as [Ca2+](SR) varied from its resting level ([Ca2+](SR,R)) to near zero. Since almost all of the Ca in the SR is bound to calsequestrin, this information closely resembles the binding curve of the Ca-calsequestrin reaction. Calcium binding to calsequestrin was found to be cooperative (estimated Hill coefficient = 2.95) and to have a very high capacity (at the start of Ca2+ release, 23 times more Ca was estimated to initiate from calsequestrin as opposed to the pool of free Ca in the SR). The latter result contrasts with an earlier report that only approximately 25% of released Ca2+ comes from calsequestrin and approximately 75% comes from the free pool. The value of [Ca2+](SR,R) was close to the K(D) for calsequestrin, which has a value near 1 mm in in vitro studies. Other evidence indicates that [Ca2+](SR,R) is near 1 mM in cut fibres. These results along with the known rapid kinetics of the Ca-calsequestrin binding reaction indicate that calsequestrin's properties are optimized to buffer [Ca2+](SR) during rapid, physiological SR Ca2+ release. Although the results do not entirely rule out a more active role in the excitation-contraction coupling process, they do indicate that passive buffering of [Ca2+](SR) is a very important function of calsequestrin.

Limitations of using aggrecan and type X collagen as markers of chondrogenesis in mesenchymal stem cell differentiation.

The study was initially designed to differentiate human bone marrow-derived mesenchymal stem cells (MSC) into chondrocyte-like cells, for use in tissue engineering. We cultured MSCs in defined chondrogenic medium as pellet cultures supplemented with transforming growth factor (TGF)-beta1 or -beta3 and dexamethazone, as they are commonly used to promote in vitro chondrogenesis. Markers of chondrogenesis used were type II collagen and aggrecan, with type X collagen being used as a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification). Our results show that aggrecan is constitutively expressed by MSCs and that type X collagen is expressed as an early event. Furthermore, we found that type X collagen was expressed before type II collagen in some cases. This is surprising because it is understood that stem cells have to be differentiated into chondrocytes before they can become hypertrophic. Thus, caution must be exercised when using aggrecan and type X collagen as markers for chondrogenesis and chondrocyte hypertrophy, respectively, in association with stem cell differentiation from this source.