Growth and differentiation of stage 24 limb mesenchyme cells in a serum-free chemically defined medium.

A serum-free defined medium which supports the differentiation of chick limb mesenchymal cells has been developed. In this medium, stage 24 embryonic limb mesenchymal cells which are plated at high density (5 x 10(6) cells/35-mm culture dish) differentiate into chondrocytes. Morphologically, these cultures appear only slightly different from those in which the cells are maintained in serum-containing medium. DNA levels and proline incorporation in cultures grown in defined medium are indistinguishable from control cultures. The rate of radiolabeled sulfate incorporation, a monitor of the rate of proteoglycan synthesis, in Day 8 high-density cultures maintained in defined medium is approximately 70-80% of control values. Additionally, growth and differentiation of intermediate-density (2 x 10(6) cells/35-mm culture dish) and low-density (1 x 10(6) cells/35-mm dish) cultures are also supported by this defined medium. The availability of this medium allows exploration of bioactive factors which affect or modulate mesenchymal cell differentiation and subsequent development.

Altered cartilage proteoglycans synthesized by chick limb bud chondrocytes cultured in serum-free defined medium.

Chick high-density culture chondrocytes synthesize cartilage-specific proteoglycans with much structural similarity to the proteoglycans made by cartilage in vivo. Such cultures can be maintained in a defined medium formulated in this laboratory in which chondrogenesis occurs without the addition of serum. The proteoglycans synthesized by the chondrocytes in the presence of defined medium are of a cartilage-specific structure but differ in some aspects from the proteoglycans made in serum-containing medium. While their buoyant density, ability to aggregate with hyaluronic acid, and keratan sulfate chain size are unchanged, the proteoglycans synthesized in defined medium have altered chondroitin sulfate chains. This chondroitin sulfate is of significantly larger size and has a different sulfation pattern relative to that produced in serum-containing medium. The larger size of the chondroitin sulfate results in a larger monomer size of the defined medium proteoglycans. These differences have implications about the regulation of the structure of chondroitin sulfate proteoglycans.

Association of the C-propeptide of type II collagen with mineralization of embryonic chick long bone and sternal development.

Several proteins may play a role in bone formation. The C-propeptide of type II collagen is intimately associated with endochondral bone formation in bovine growth plate. We have used an antibody against this peptide to determine its immunofluorescent distribution in early stages of embryonic chick limb development with emphasis on first bone formation which occurs in the mid-diaphyseal region. The C-propeptide II is first evident by immunofluorescent localization at stage 27 (day 5-6) of embryonic tibia development with chondrocytes in the central mid-diaphysis. In subsequent stages, there is an increase in the number of chondrocytes in which it is localized in discrete vacuoles. Up to stage 30, immunofluorescence is observed intracellularly, after which it appears in the matrix. The released C-propeptide II appears to remain only transiently associated with the cartilage matrix and becomes concentrated in the calcifying periosteum, the region outside of the cartilage core where bone formation first occurs in a sequence of events comparable to intramembranous bone formation. These observations can be reproduced in cultures of stage 35 hypertrophic chondrocytes (core cells) and periosteum cells (collar cells). Core cells contain intensely stained intracellular vacuoles while collar cells are negative, although the collar cell osteogenic matrix concentrates exogenously added C-propeptide II. Double label immuno-staining shows that the C-propeptide II, unlike type II collagen and proteoglycan, which are secreted and incorporated into extracellular sites, is initially stored in intracellular vacuoles. The matrix localization of the C-propeptide II during the transition from cartilage to bone indicates a close association with the initiation of mineralization events of cartilage and bone and its specific origin in chondrocytes and not osteoblasts. These observations suggest that the C-propeptide II made by chondrocytes is associated with the formation of bone.

Hyaluronic acid bonded to cell-culture surfaces stimulates chondrogenesis in stage 24 limb mesenchyme cell cultures.

The influence on the differentiation of stage 24 chick limb mesenchymal cells of hyaluronic acid (HA) covalently bonded onto plastic substrates has been examined. Under control conditions, stage 24 cells express phenotypes related to the initial plating density: When plated at high density (5 X 10(6) cells/35-mm culture dish), these cells express a chondrogenic phenotype collectively visualized as a mound or nodule of cartilage. Cartilage nodules are not found in cultures plated at intermediate or low densities, 2 X 10(6) and 1 X 10(6) cells/35-mm dish, respectively. However, when cells are plated onto HA surfaces, expression of the cartilage phenotype occurs at all three plating densities in roughly comparable frequencies. This increase in cartilage nodule formation does not appear to be due to an increased plating efficiency or increased replication rate. The observed effect is dependent on HA concentration; with an increase in bound HA, an increase in the number of cartilage nodules is observed. Digestion of HA substrates with hyaluronidase abolishes the stimulation in chondrogenesis, while no effect is observed if the HA substrates are treated with either trypsin or alkaline borohydride. No other glycosaminoglycan, except for the HA analog, unsulfated chondroitin, exhibits this unique stimulation of chondrogenic expression. While the rate of radiolabeled sulfate incorporation is dramatically increased with cells plated onto HA substrates, the protein biosynthetic rate, as evidenced by radiolabeled proline incorporation, remains unaffected. This dramatic increase in chondrogenic expression is considered in contrast to the previously reported inhibitory effect of HA substrates on myogenesis. These observations suggest that HA may have a regulatory role in the chondrogenic differentiation of chick limb mesenchymal cells.

Substrate-bonded hyaluronic acid exhibits a size-dependent stimulation of chondrogenic differentiation of stage 24 limb mesenchymal cells in culture.

Extracellular matrix molecules including glycosaminoglycans have been implicated in several differentiative and morphogenetic processes including cell aggregation and migration. Previous reports have shown that plating of stage 24 limb mesenchyme cells onto hyaluronic acid (HA) bonded to the culture substrate causes an increase in the number of cells exhibiting chondrogenesis. This increased chondrogenesis is now shown to be dependent upon the source of the HA. When limb mesenchymal cells are plated onto HA from bovine vitreous humor, human umbilical cord, or large molecular weight HA (Healon), increased chondrogenesis is observed only on the bovine vitreous humor HA. Unsulfated chondroitin, which has a structure and charge density similar to those of HA, is capable of enhancing chondrogenesis, while cells plated onto sulfated glycosaminoglycan substrates are indistinguishable from controls. The evidence in this report suggests that the differentiation response is related to the molecular size of the HA bound to the culture substrate. Healon and human umbilical cord HA are ineffective because their molecular weight is too large, while smaller HA derived from these larger molecules or normally present in bovine vitreous humor preparations stimulates the chondrogenic differentiation of stage 24 limb mesenchymal cells in culture. The most active size class of HA elutes from a Sepharose CL-2B column with a Kav between 0.6 and 0.7 and, thus, has a molecular weight of approximately 200,000-400,000. These observations reinforce the hypothesis that local cues have an informational effect on the differentiation of chick limb mesenchymal cells.

Morphological and histochemical events during first bone formation in embryonic chick limbs.

Staged embryos from White Leghorn chicken eggs were used to assemble a detailed morphological, cellular and molecular picture of the complex events of first-bone formation. To provide these details, light and electron microscopic, histochemical and immunocytochemical techniques were used to establish a temporal sequence for long bone development in chick wing and leg from Hamburger-Hamilton stage 29 through stage 35. Three distinctive cell regions can be morphologically identified by stage 28 (leg) or 29 (wing) at the mid-diaphysis. These regions are: 1. an outer grouping of loose mesenchymal and myogenic cells, 2. an osteoprogenitor layer which will later divide to maintain this progenitor layer in a brickwork or stacked configuration and to produce round, tightly packed osteoblasts, and 3. a core (rod) of cartilage. First bone is laid down just outside the cartilage core, initially as a layer of Type I collagen-rich osteoid which later becomes mineralized. Vascular elements then come to reside above this mineral layer, and osteoid is laid down between vascular elements and eventually above them to form a second layer of trabecular bone. As this radial formation of layers of bone is progressing, so too is the proximal and distal expansion of the first bone forming process. A model is presented which considers that chondrogenic and osteogenic cell commitment occur simultaneously in early limb development and that it is the expression of the osteogenic phenotype which governs the boundaries of cartilage development. Importantly, the vasculature plays a key role in the patterning of bone formation well before it enters the cartilaginous core at stage 35 and participates in the erosion of the core. While this report is restricted to events occurring through stage 35, it relies on data presented in a companion report detailing later bone development and remodeling (Pechak et al; Bone 1986) and emphasizes that the cartilage model does not provide the scaffolding for bone but rather defines the marrow space.

Hyaluronic acid bonded to cell culture surfaces inhibits the program of myogenesis.

Primary isolates of chick leg muscle myoblasts cultured on hyaluronic acid substrates have been examined by transmission electron microscopy for evidence of myoblast fusion and subsequent differentiation. Even though these cells form close contacts, no evidence of multinucleated myotubes is found in these cultures. Two-dimensional SDS-polyacrylamide gel electrophoresis shows that the muscle macromolecular biosynthetic program is not initiated in these hyaluronic acid fusion-blocked cells. Further, these fusion-blocked myoblasts continue replicating while cultured on hyaluronic acid surfaces. The inhibition of both fusion and the myogenic expressional program is reversed by replating these myoblasts onto a denatured collagen (gelatin) substrate; both the synthesis of muscle-specific proteins and the formation of multinucleated myotubes are observed when these subcultured cells are introduced onto gelatin substrates. These observations indicate that the hyaluronic acid inhibition of fusion is not permanent and is manifested in a way different from other fusion blockers in that hyaluronic acid inhibits both fusion and the myogenic expressional program.

Morphology of bone development and bone remodeling in embryonic chick limbs.

Staged embryos from White Leghorn chicken eggs were used to assemble a detailed morphological sequence of events occurring in long bone development from Hamburger-Hamilton stage 32 through stage 44 and 2 days post hatching. The detailed patterning of osteoblasts, osteoid, mineral, and vasculature were observed at the mid-diaphysis of the tibia. At stage 32, the cartilage core is composed of hypertrophic chondrocytes and is surrounded by a continuous ring of mineralized osteoid on which osteoblasts and vasculature reside. At stage 35, the vasculature and associated cell types invade the cartilage core region. By stage 37, marrow occupies the entire cartilage core region at the mid-diaphysis. Anastamosing channels, containing vasculature, interconnect with each other and the marrow region to the inside and the periosteal region to the outside. Clearly, the cartilage is replaced by marrow, not bone. Mineral deposition at the periosteal surface continues through stage 44 as does mineral resorption on the endosteal surface, although the rate of mineral deposition and resorption varies at different developmental stages. Vasculature plays an important role in the pattern formation of the trabeculae and their channels as can be seen in the developmental sequence within one bone (the tibia) or comparisons between two bones (the tibia and fibula). A model is presented which considers the possibility that osteoprogenitor cells are formed as early as the chondroprogenitor cells. This model also emphasizes the observation that cartilage is not replaced by bone but is replaced by marrow.

Culturing chick muscle cells on glycosaminoglycan substrates: attachment and differentiation.

The effects of different glycosaminoglycans (GAGs) on myogenesis were tested by culturing embryonic chick myoblasts on tissue culture dishes to which either hyaluronic acid (HA) or chondroitin sulfate (ChS) was covalently bound. Both in cell number and in apparent cell type distribution, the population of cells bound to GAGs is similar to that on gelatin and significantly different from that observed with uncoated dishes. When plated on ChS, myoblasts proliferate, align, and fuse at a rate similar to cells plated on gelatin. The final fused cells appear as sheets rather than long, thin myotubes. On HA, the cells proliferate but are inhibited from differentiation. The extent of inhibition is dependent on the amount of HA present. The inhibition of myogenesis is maintained through four subcultures on HA, but can be reversed at any time by culturing cells on gelatin. These experiments indicate that different GAGs have different effects on myogenesis and that HA can actively inhibit the process.