Effect of amino acid levels on matrix vesicle formation by epiphyseal growth plate chondrocytes in primary culture

Abstract

The effect of varying the amino acid concentrations of the culture medium on matrix vesicle formation was studied in primary cultures of chicken epiphyseal growth plate chondrocytes grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS). Decreasing the levels of free amino acids in the culture medium to levels of one-half, one quarter, and one eighth of the values normally present in DME caused a progressive decline in matrix vesicle (MV) formation. Increasing the level in the culture medium of those amino acids that are enriched in extracellular fluid (ECF) of growth plate cartilage significantly increased formation of matrix vesicles (MV), as assayed by the alkaline phosphatase (AP) activities present in high-speed sediments from spent culture media. However, adjusting the levels of all amino acids to match those of the ECF produced the greatest stimulation of MV formation. Of the amino acids that are notably enriched in ECF, glutamate (GLU), alanine (ALA), serine (SER), asparagine (ASN), and taurine (TAU) individually enhanced MV production, whereas proline (PRO), glycine (GLY), and aspartate (ASP) had essentially no effect. The simple combination of ECF levels of ALA and GLU resulted in a stimulation of MV formation equal to that observed when the eight aforementioned amino acids were elevated to ECF levels. Other combinations of ASP and GLY, or of TAU, SER, and ASN showed some stimulation, but at a lower level. Increasing the amino acid concentrations, alone or in combination, also increased the levels of cellular AP, and to a lesser extent cellular protein. While increases in cellular AP were generally correlated with increased formation of AP-rich MV, this was not uniformly true. These results indicate that in addition to hormones and growth factors, nutritional factors such as the levels of amino acids are also critical for normal phenotypic expression, growth, and matrix formation by epiphyseal chondrocytes.

The role of amino acids in hydroxyapatite mineralization

“…In summary, this study revealed that the level of specific AAs in the cartilage ECF is not only critical for the growth of the chondrocytes, but also it is important for the formation of MV [70]. However, the local environment of cells in vivo varies significantly from cell to cell; therefore, this finding cannot be generalized to all cell types, and further studies are required to determine the composition of media in which the response of various cell types can be achieved [70]. AA intake can also increase bone mineral density (BMD) by increasing insulin-like growth factor 1 (IGF-1), which is involved in osteoblastic activity and the synthesis of collagen and muscle proteins [72].…”

“…More specifically, they showed that the concentrations of charged AAs, such as Arg, Asp and Glu, which are strongly involved in mineralization, were 0.44, 3.76 and 15.37 mM in chicken hypertophic cartilage extracellular fluid (ECF), whereas it was 0.37, 0.06 and 0.16 mM, respectively, in blood plasma [69]. They also revealed that increasing the concentration of eight AAs (Asp, Glu, Ser, Gly, Ala, Pro, taurine (Tau), and asparagine (Asn)) in the culture medium to the level present in the native cartilage ECF remarkably stimulated the formation of alkaline phosphatase (AP) and AP-rich matrix vesicles (MV) by chondrocytes [70]. Among these AAs, Pro and Gly showed the weakest effect on AP production while the combination of only two of the AAs, Ala and Glu, produced stimulatory effect on MV formation comparable to that seen in the presence of the eight AAs.…”

Effects of 24R,25‐ and 1α,25‐dihydroxyvitamin D3 on mineralizing growth plate chondrocytes

“…This culture medium, DATP5, fully supports proliferation, differentiation, and mineralization of primary cultures of GP chondrocytes isolated from rapidly growing broiler-strain chickens [Ishikawa et al, 1986;Wu et al, 1989Wu et al, , 1995Ishikawa and Wuthier, 1992]. Of special importance are supplemental ascorbate [Wu et al, 1989], specific amino acids [Ishikawa et al, 1986], and inorganic phosphate [Ishikawa and Wuthier, 1992], although other factors present in serum are also known to be important. This serum-containing medium consistently supports calcification in the absence of organic phosphate supplements.…”

Chondrocytes isolated from tibial dyschondroplasia lesions and articular cartilage revert to a growth plate‐like phenotype when cultured in vitro

“…The culture media were changed every 3-4 days with a fresh supplement of ascorbic acid (50 mg/ml) starting on day 3, for the duration of the experiments. The medium was later changed to DATP5 mineralization medium (Ishikawa et al, 1997), which is enriched with eight amino acids to levels normally present in growth plate extracellular fluid (Ishikawa et al, 1985(Ishikawa et al, , 1986, and contains insulin (5 mg/ml), transferrin (5 mg/ml), sodium selenite (5 ng/ ml), 5% defined FBS, and 1 mM Na 2 HPO 4 added to the DMEM basal medium . In this culture medium, inorganic phosphate (Pi) is also raised to the level present in normal growth plate extracellular fluid, which enables mineral formation.…”

“…2). The DATP5 medium used for these cells was designed to mimic the electrolyte (Wuthier, 1977) and amino acid (Ishikawa et al, 1985;Ishikawa et al, 1986) composition of normal growth plate extracellular fluid. We had used it extensively for Osteogenic protein-1 (OP-1) was administered at 15 ng/dish (2 ml of culture medium).…”