Chick limb‐bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate. To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non‐mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific microarray. Based on replicate microarray experiments and K‐cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT‐PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co‐transporter, NaPi IIb, matrix metalloprotease 13. MMP‐13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed. J. Cell. Biochem. 112: 607–613, 2011. © 2010 Wiley‐Liss, Inc.

This study focuses on the development of a novel method of nonenzymatic glycation of fibrillar collagen gels. In contrast to previous studies in which type I collagen gels were glycated in the solid state, this study presents a method for glycation in solution. The type I collagen in solution or gels was exposed to a range of ribose concentrations from 0 to 250 mM. The binding of ribose to collagen was documented using Fourier transform infrared (FTIR) spectroscopy. formation of advanced glycation end products (AGEs) was quantified by fluorescence measurement. The bulk compressive modulus and viscoelastic time constant of processed gels were determined in stress relaxation studies. Both methods of glycation enhanced ribose addition and AGE formation in a dose-dependent manner, with glycation in the gel state being more efficient. Both methods enhanced mechanical properties similarly, with 250 mM ribose treatment resulting in a 10-fold increase in bulk modulus.

The murine mesenchymal cell line, C3H10T1/2 in micromass culture undergoes chondrogenic differentiation with the addition of BMP-2. This study compares the use of BMP-2 vs. insulin, transferrin, and sodium selenite (ITS) to create a chondrogenic micromass cell culture system that models cartilage calcification in the presence of 4mM inorganic phosphate. BMP-2 treated cultures showed more intense alcian blue staining for proteoglycans than ITS treated cultures at early time points. Both ITS and BMP-2 treated cultures showed similar mineral deposition in cultures treated with 4mM phosphate via von Kossa staining, however FTIR spectroscopy of cultures showed different matrix properties. ITS treated cultures produced matrix that more closely resembled mouse calcified cartilage by FTIR analysis. 45Ca uptake curves showed delayed onset of mineralization in cultures treated with BMP-2, however they had an increased rate of mineralization (initial slope of 45Ca uptake curve) when compared to the cultures treated with ITS. Immunohistochemistry showed the presence of both collagens type I and type II in BMP-2 and ITS treated control (1mM inorganic phosphate) and mineralizing cultures. BMP-2 treated mineralizing cultures displayed more intense staining for collagen type II than all other cultures. Collagen type X staining was detected at Day 9 only in mineralizing cultures treated with ITS. Western blotting of Day 9 cultures confirmed the presence of collagen type X in the mineralizing ITS cultures, and also showed very small amounts of collagen type X in BMP-2 treated cultures and control ITS cultures. By Day 16 all cultures stained positive for collagen type X. These data suggest that BMP-2 induces a more chondrogenic phenotype, while ITS treatment favors maturation and hypertrophy of the chondrocytes in the murine micromass cultures.

Chondrocyte apoptosis is thought to be an important step in the calcification of cartilage in vivo; however, there are conflicting reports as to whether or not this apoptosis is a necessary precursor to mineralization. The goal of this study was to determine whether or not apoptosis is necessary for mineralization in an in vitro murine micromass model of endochondral ossification. C3H10T1/2 murine mesenchymal stem cells were plated in micromass culture in the presence of 4 mM inorganic phosphate with the addition of the apoptogens, camptothecin, or staurosporine, to induce apoptosis. The rate and total accumulation of mineralization was measured with (45)Ca uptake. In these studies, both apoptogens increased the rate of mineralization, with staurosporine increasing (45)Ca accumulation by about 2.5 times that of controls and camptothecin increasing total amounts of mineralization about 1.5 times that of controls. Inhibiting cell apoptosis with the caspase inhibitor, ZVAD-fmk, to prevent apoptosis, caused slower rates of (45)Ca uptake; however, total amounts of (45)Ca accumulation reached the same values by day 30 of culture. FTIR data showed mineralization in all samples treated with 4 mM inorganic phosphate, with the highest mineral to matrix ratios in the camptothecin treated samples.

Collagen glycated with ribose (250 mM) in solution (pre-glycation) and as a gel (post-glycation) was seeded with chondrocytes and the effects of glycation on chondrocyte matrix assembly in culture were determined. Pre-glycation enhanced GAG accumulation significantly over controls at both 2 and 4 weeks (p < 0.05), although at both time points there were no statistical differences in cell number between pre-glycated and control gels. The increased proteoglycan accumulation was shown to be in part due to significantly increased GAG retention by the pre-glycated constructs (p < 0.05). Total collagen content in these pre-glycated gels was also significantly higher than unglycated gels at 4 weeks (p < 0.05). With post-glycation of collagen gels, chondrocyte number and GAG accumulation were all significantly lower than controls (p < 0.05). Post-glycation also inhibited GAG retention by the constructs (p < 0.05). Given these results, pre-glycation may be an improved processing method for collagen gels for tissue engineering techniques.

Protein phosphorylation and dephosphorylation are important regulators of cellular and extracellular events. The purpose of this study was to define how these events regulate cartilage matrix calcification in a cell culture system that mimics endochondral ossification. The presence of casein kinase II (CK2), an enzyme known to phosphorylate matrix proteins, was confirmed by immunohistochemistry. The importance of phosphoprotein phosphorylation and dephosphorylation was examined by comparing effects of inhibiting CK2 or phosphoprotein phosphatases on mineral accretion relative to untreated mineralizing controls. Specific inhibitors were added to differentiating chick limb-bud mesenchymal cell micromass cultures during the development of a mineralized matrix at the times of cell differentiation, proliferation, formation of the mineralized matrix, or proliferation of the mineral crystals. The mineralizing media for these cultures contained 4 mM inorganic phosphate and no organic-phosphate esters; control cultures had 1 mM inorganic phosphate. Mineralization was monitored based on (45)Ca uptake and infrared characterization of the mineral; cell viability was assessed by three independent methods. Treatments that caused cell toxicity were excluded from the analysis. Inhibition of CK2 activity with apigenin or CK2 inhibitor II reduced the rate of mineral deposition, but did not block mineral accretion. Effects were greatest during the time of mineralized matrix formation. Inhibition of phosphoprotein phosphatase activities with okadaic acid, calyculin A, and microcystin-LR, at early time points also markedly inhibited mineral accretion. Inhibition after mineralization had commenced increased the mineral yield. Levamisole, an alkaline phosphatase inhibitor, had no effect on mineral accretion in this system, suggesting the involvement of other phosphatases. Adding additional inorganic phosphate to the inhibited cultures after mineralization had started, but not earlier, reversed the inhibition indicating that the phosphatases were, in part, providing a source of inorganic phosphate. To characterize the roles of specific phosphoproteins blocking studies were performed. Blocking with anti-osteopontin antibody confirmed osteopontin's previously reported role as a mineralization inhibitor. Blocking antibodies to bone sialoprotein added from day 9 or on days 9 and 11 retarded mineralization, supporting its role as a mineralization nucleator. Antibodies to osteonectin slightly stimulated early mineralization, but had no effect after the time that initial mineral deposition occurs. Taken together, the results of this study demonstrate the importance of the phosphorylation state of extracellular matrix proteins in regulating mineralization in this culture system.

The use of cell culture to characterize bone and tooth mineralization has blossomed thanks to the development of genetically engineered animals with altered bone and tooth phenotypes 1,2, the tissue engineering of bones, teeth, and cartilage 3,4, and genetic profiling of mineralized tissue development 5-8. There is little uniformity in the conditions used in these cultures, and where there is uniformity the chemical basis for the reagents used is sometimes questionable. Moreover, while it is well established that histochemical staining is a poor substitute for physicochemical assays of the nature of the mineral formed 9, the majority of both recent and classical papers in the literature rely on histochemistry to report the presence of bone- or tooth-like mineral. The goal of this review is to place cell culture methodologies in a chemical context, to review the different types of cell culture systems that have been used to study the deposition of mineral in bones and teeth, discuss their limitations, and suggest some guidelines for future studies. 1.1. The Cells and Tissues of Bones and Teeth Cell culture systems generally seek to recapitulate the events in the development of the tissue in situ. While some culture systems start with preformed substrates and others rely on the differentiation, proliferation, and maturation of cells, the ultimate goal is the formation of an analogue of the naturally occurring tissue. Questions that arise during these studies may include whether the cells in question have an altered ability to form this tissue, what the role of the genetic modification might be, or whether the material formed can be used to repair and replace native tissue. To understand the cell culture methodologies, it is necessary to first review the composition of these tissues and the cells involved in their formation. 1.1.1. The Mineral and Extracellular Matrix of Bones and Teeth The mineral phase that is found in bones and teeth as a highly substituted analogue of the geologic mineral, hydroxyapatite (Ca10(PO4)6(OH)210. In bone, cementum, and dentin, the plate-like OH-deficient, CO3-substituted apatite nano-crystals are oriented with their long axis parallel to the axis of the collagen fibrils. The insoluble fibrillar protein, collagen, is the major component of the organic matrix. Other noncollagenous proteins are important for the maintenance of the cell-matrix interactions, cell-signaling, regulation of cell metabolism, and control of the mineralization process11,12. In mature enamel, the hydroxyapatite crystals are also carbonate-substituted, but they are larger, and there is little (<3%) organic matrix. There is no collagen in enamel; the major protein in mature enamel is amelogenin, but proteins account for a very small amount of the mature enamel’s composition. During early enamel formation there is a greater proportion of proteins, and these include enamel specific proteins such as amelogenin, enamelin, tuftelins, and amelin, and some non-specific proteins such as phosphophoryn 12,13.

A large deflection elasticity model was used to describe the tensile elastic modulus of cartilage tissues during three point bending tests. Native and engineered cartilage were compared for both auricular and costal cartilage types.

Abstract The current literature presents many techniques for analysis of the mechanical properties of articular cartilage [1]. By comparison few techniques are available for analysis of auricular cartilage which experiences a different mechanical environment than articular cartilage. Specifically, large deflection bending is a deformation mode that is most relevant to auricular cartilage, but has not been studied thoroughly in soft tissues. The goals of this study were to: 1) apply an elasticity model to three point bending data from normal and engineered auricular and costal cartilage; and 2) use this model to determine the tensile elastic moduli.

Using a custom galvanotaxis chamber and time-lapse digital video microscopy, we report the novel observation that cultured chondrocytes exhibit cathodal migration when subjected to applied direct current (DC) electric fields as low as 0.8 V/cm. The response was dose-dependent for field strengths greater than 4 V/cm. Cell migration appeared to be an active process with extension of cytoplasmic processes in the direction of movement. In some cells, field application for greater than an hour induced elongation of initially round cells accompanied by perpendicular alignment of the long axis with respect to the applied field. Antagonists of the inositol phospholipid pathway, U-73122 and neomycin, were able to inhibit cathodal migration. Cell migration toward the cathode did not require the presence of serum during field application. However, the directed velocity was nearly threefold greater in studies performed with serum. Studies performed at physiologic temperatures (approximately 37 degrees C) revealed a twofold enhancement in migration speed compared to similar studies at room temperature (approximately 25 degrees C). Findings from the present study may help to elucidate basic mechanisms that mediate chondrocyte migration and substrate attachment. Since chondrocyte migration has been implicated in cartilage healing, the ability to direct chondrocyte movement has the potential to impact strategies for addressing cartilage healing/repair and for development of cartilage substitutes.

Cultured chondrocytes exhibit cathodal migration when subjected to applied direct current (DC) electric fields [1]. Our studies showed a 2-fold enhancement in migratory velocity at physiologic temperature (∼37°C) compared to room temperature (∼25°C). The inositol phosphate pathway antagonists, U-73122 and neomycin, were found to inhibit the directed response, indicating that it may be involved in chondrocyte migration. Further understanding of the molecular mechanisms underlying chondrocyte migration and galvanotaxis may contribute to the development of new strategies for cartilage repair.