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A I Caplan

Publications and source records attributed to A I Caplan.

At least 163 records · Page 9Linked to original sources
PubMed1986

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.

Cartilage↗
PubMed1986

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.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

Partial isolation and characterization of a chemotactic factor from adult bovine bone for mesenchymal cells.

Demineralized adult bone matrix has the capacity to initiate de novo ectopic endochondral bone formation 2-3 weeks following intramuscular implantation into suitable hosts. An early step in this process is the migration of mesenchymal cells to the implant site; these cells later differentiate into cartilage and bone. Adult bone has been shown to contain a number of bioactive factors, such as chemotactic factors for various cell types, including osteoblasts. We have used embryonic chick limb bud mesenchymal cells to construct an in vitro assay for testing chemotactic activity derived from bone matrix extracts. With a modified Boyden chamber, water-soluble components from a 4 M guanidinium chloride extract of demineralized adult bovine bone matrix were found to stimulate the directional migration of these chick embryonic limb bud mesenchymal cells as well as embryonic muscle-derived fibroblasts and cells derived from embryonic skin. The chemotactic activity was destroyed by treatment with heat (100 degrees C) or trypsin. Partial purification by molecular sieve chromatography suggested that the chemotactic factor(s) has a molecular weight of between 50,000 and 90,000. This factor can be separated from bone matrix-derived chondrogenic stimulating activity by either ion exchange or molecular sieve chromatography. These observations confirm that bone matrix contains a chemoattractant for mesenchymal cells that may be important for in vivo recruitment of cells as part of the process of ectopic bone formation or in cases of bone repair.

Animals↗
PubMed1985

Isolation and characterization of proteoglycans synthesized in ovo by embryonic chick cartilage and new bone.

Cartilage proteoglycans have been well characterized in a number of developing systems, both in vitro and in vivo, but the newly synthesized molecules have been analyzed only from culture material. Because of potential culture artifacts, an attempt was made to characterize the proteoglycans newly synthesized in ovo in chick embryo sternum, tibial epiphysis, and tibial shaft. These in ovo synthesized proteoglycans share many structural features with chick proteoglycans synthesized by chondrocytes in culture including average monomer size, chondroitin sulfate chain size, keratan sulfate chain size, and the ability to aggregate with hyaluronic acid. Moreover, the newly synthesized in ovo proteoglycans, notably those of the tibial epiphysis, display reproducible changes in their structure as a function of embryonic age. These changes correlate with similar changes documented for chick cartilage proteoglycans synthesized in culture. Finally, the proteoglycans synthesized in ovo in the day 17 tibial shaft include, in addition to cartilage proteoglycans, one proteoglycan which seems to be characteristic of bone.

Animals↗
PubMed1985

Isolation and characterization of osteogenic cells derived from first bone of the embryonic tibia.

Osteogenesis in the embryonic long bone rudiment occurs initially within an outer periosteal membrane and subsequently inside the cartilaginous core as a consequence of the endochondral ossification process. In order to investigate the development of these two different mechanisms of bone formation, embryonic chick tibial cell isolates were prepared from sites of first periosteal bone formation and from the immediately underlying hypertrophic cartilaginous core region. Mid-diaphyseal periosteal collars and the corresponding cartilage core were microdissected free from Hamburger-Hamilton stage 35 (Day 9) chick tibias and separately digested with a trypsin-collagenase enzyme mixture. The released cell populations were cultivated in vitro and characterized by morphological analysis, histochemical localization of alkaline phosphatase, alizarin red S staining for mineral deposition, growth rate [( 3H]thymidine uptake), and proteoglycan content. Results of these studies showed that periosteal collar cell cultures form nodule-like structures that stain positive with alkaline phosphatase and alizarin red S. Light and electron microscopic observation revealed cell and matrix morphologies similar to that of intact periosteum. The nodules were composed of plump cell types embedded within a mineralized matrix surrounded by a fibroblastic cell layer. Core cartilage cell cultures displayed typical characteristics of the hypertrophic state in their visual appearance and proteoglycan composition. The formation of osseous-like structures in periosteal collar cell cultures but not in core chondrocyte cell cultures demonstrates the relatively autonomous nature of intramembranous ossification while emphasizing the dependence of the endochondral ossification process upon an intact vascularized environment present in the developing tibia.

Alkaline Phosphatase↗
PubMed1985

Poly(ADP-ribose) synthetase and chick limb mesenchymal cell differentiation.

Poly(ADP-ribose) is a variably sized homopolymer synthesized from NAD as a covalent adduct to chromosomal proteins; its synthesis is catalyzed by the enzyme poly(ADP-ribose)synthetase (pADPRS). Using an assay to estimate the pADPRS levels during various phases of both in vivo and in vitro limb mesenchymal cell development, we report that the level of pADPRS undergoes a substantial change as limb cells differentiate into muscle or cartilage. This change involves a threefold transient increase in the level of pADPRS per unit DNA which is coincident with the initiation of specific phenotypic expression. These fluctuations are observed for both chondrogenic and for myogenic events. Such a transient increase in pADPRS levels seems to be characteristic of differentiating cells but is not observed in cells which have already differentiated. These observations establish a correlation between pADPRS levels and chick limb mesenchymal cell differentiation both in vivo and in vitro and suggest that previous quantification of in situ ADP-ribosylation activity reflects alterations in pADPRS levels. Based on the information reported here and by others, a speculative hypothesis is put forth to explain the role of poly(ADP-ribose)synthetase in developmental events.

Animals↗
PubMed1985

The relationship of nicotinamide adenine dinucleotide to the chondrogenic differentiation of limb mesenchymal cells.

The nicotinamide adenine dinucleotide (NAD) content of mesenchymal cells from the embryonic chick limb has been hypothesized to control the differentiation of these cells by modulation of ADP-ribosylations. To test this hypothesis, [35S]sulfate incorporation into proteoglycans was monitored as an estimate of the chondrogenic expression of cultured limb mesenchymal cells treated with nicotinamide and nicotinic acid to elevate cellular NAD levels or with nicotinamide and benzamide compounds to inhibit ADP-ribosylations. The results of this study indicated that serum component(s) modulate the interactions between these chemical agents and limb mesenchymal cells and, thus, complicate the interpretations of experiments performed in the presence of serum. With a chemically defined medium that promotes limb mesenchymal cell proliferation and differentiation in vitro, it was demonstrated that: (1) no clear correlation exists between cellular NAD content and the chondrogenic expression of cultured limb mesenchymal cells, (2) nicotinamide and benzamide compounds reduce cell proliferation and, at the higher doses tested, considerably reduce chondrogenesis in limb mesenchymal cell cultures, and (3) limb mesenchymal cells exhibit an enhanced susceptibility to benzamide compounds at a time very early in the culture period which temporally coincides with a transient increase in cellular ADP-ribosylation activity and initial chondrogenic differentiation. These results suggest that NAD does not control the differentiation of limb mesenchymal cells and that ADP-ribosylations are an integral, though not controlling, component of limb mesenchyme cytodifferentiation. A model is presented which proposes a role for ADP-ribosylations during the differentiation of limb mesenchymal cells.

Adenosine Diphosphate Ribose↗
PubMed1985

The vasculature and limb development.

The developing vascular pattern of the embryonic chick limb results from a combination of two properties: the intrinsic self-assembly and branching properties of the vascular cells and the extrinsic information associated with the expanding mitotic population of mesenchymal cells; and the inhibitory factors which restrict the entrance of vessels into particular domains and/or decrease the branching frequency of such vessels. It is hypothesized that an important component of limb pattern formation is the interplay between the dividing population of mesenchymal cells and the intrinsic properties of the vascular cells. It is further asserted that the presence of particular vascular elements may, indeed, be 'positional information'. Two examples are cited involving aspects of limb duplication to support this possibility; it is suggested that vascular vessel size of a host limb may dictate the polarity of duplication events. The presented hypothesis emphasizes that the interplay between the intrinsic properties of self-assembly into tissues and extrinsic factors which establish boundaries and morphologies is involved in both vascular and limb pattern formation.

Animals↗
PubMed1985

The anatomy, ultrastructure and fluid dynamics of the developing vasculature of the embryonic chick wing bud.

The spatiotemporal sequence of vascular pattern development in the embryonic chick wing bud and surrounding shoulder, flank and belly regions is detailed for Hamburger-Hamilton (1951) stages 20-25. Vasculature was microinjected with an unreactive aqueous tracer (aniline blue), and major traffic patterns were visualized. Formation of extensive avascular regions and the emergence of chondrogenic phenotypes are correlated with the retreat of the vasculature from the wing core. Ultrastructural studies of vascular cells show that vessels remain monolayered and undifferentiated until stage 25, after the adult vascular pattern has been laid down. Vascular cytodifferentiation occurs only in the cells of the brachial artery until stage 35, with the veins and capillaries retaining an 'early' morphology. This vascular pattern may be an important component reflecting or directing limb pattern development.

Animals↗
PubMed1985

Electron microscopic characterization of chick embryonic skeletal muscle proteoglycans.

In this article, proteoglycans from embryonic chick leg muscle are quantitatively and qualitatively compared with day 8 high density cell culture cartilage proteoglycans by electron microscopy of proteoglycan-cytochrome c monolayers. The visualized proteoglycan profiles were separated into four categories according to shape, size, and complexity. The two major categories were further characterized by lengths of core proteins, lengths of side projections, and distance between side projections. Two large proteoglycans are identifiable in spread leg muscle preparations. One group has a core protein (mean length of 205 nm) from which extend long thin side projections that we interpret to be groups of chondroitin sulfate glycosaminoglycans with a mean length of 79 nm. This large chondroitin sulfate proteoglycan is the only type found in muscle cultures as determined both biochemically in the past and now by electron microscopy and is referred to as muscle proteoglycan. The second large proteoglycan has a mean core protein length of 250 nm and side projections that are visibly shorter (mean length of 38 nm) and thicker than those of the muscle proteoglycan. This group is referred to as the mesenchymal proteoglycan since its biosynthetic origin is still uncertain. We compare these two profiles with the chick cartilage chondroitin sulfate proteoglycan that has a mean core protein length of 202 nm and side projections with a mean length of 50 nm. The data presented here substantiate the earlier biochemical characterization of these noncartilage proteoglycans and establish the unique structural features of the muscle proteoglycan as compared with the similar profiles of the cartilage and mesenchymal proteoglycans.

Animals↗
PubMed1985

The in vitro chondrogenic response of limb-bud mesenchyme to a water-soluble fraction prepared from demineralized bone matrix.

Demineralized adult bone matrix initiates de novo ectopic endochondral ossification 2-3 weeks following its intramuscular implantation into adult animals. This phenomenon appears to be similar, in some ways, to inductive cell-matrix interactions which regulate cartilage and bone formation during development. In the present study, we used embryonic chick limb-bud mesenchymal-cell cultures to bioassay extracts of demineralized bone matrix for chondrogenic activity. Guanidinium-chloride (4 M) extracts of demineralized bovine bone were dialyzed against buffers of decreasing ionic strength and then cold water. The cold-water-soluble fraction was found to stimulate chondrogenesis in intermediate-density limb-bud cell cultures (2.2 X 10(6) cells per 35-mm dish), as revealed by visual inspection with phase optics, toluidine-blue staining of fixed plates, and [35S] sulfate incorporation in the cell layer. Further fractionation of this material by anion-exchange, carbohydrate-affinity, and molecular-sieve chromotography produced a semipurified preparation possessing chondrogenic-stimulating activity at doses ranging from 3 to 10 micrograms/ml. The in vitro chondrogenic response of limb-bud mesenchymal cells was dose-dependent, required a minimal initial plating density of 2.08 X 10(5) cells/mm2 of culture dish, and developed gradually over 8-10 days. At an optimal dose of extract, a continuous exposure period of at least 2-3 days was necessary to produce detectable chondrogenic stimulation. In addition, the amount of cartilage formed following an 8-day exposure was markedly influenced by the culture 'age' of the mesenchymal cells (i.e., the time between plating and the start of treatment).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Isolation and partial characterization of high-buoyant-density proteoglycans synthesized in ovo by embryonic chick skeletal muscle and heart.

Proteoglycans, a major component of the extracellular matrix, are produced in many tissues. A report from this laboratory describes the proteoglycans synthesized in culture by chick embryonic skeletal muscle myotubes. To extend this study to in vivo conditions, chick embryos were radiolabeled in ovo and the newly synthesized high-buoyant-density proteoglycans from skeletal muscle analyzed. In both leg muscle and pectoral muscle, three major high-density proteoglycans are synthesized. One is small and is similar to the proteoglycans synthesized in culture by muscle fibroblasts. The other two proteoglycans are large. The larger of these shares structural features with the proteoglycan synthesized by skeletal muscle cells in culture. It has large chondroitin sulfate chains (estimated molecular weight of 70,000) with a high proportion of chondroitin 6-sulfate (approximately 90%). The smaller of the two large proteoglycans is distinct (chondroitin sulfate of estimated molecular weight 24,000 and approximately 60% 6-sulfated disaccharides) and is not detected in muscle cultures; evidence suggests it is not made by myoblasts. Whole hearts synthesize proteoglycans with some structural similarities, and also differences, to those made in skeletal muscle. These data indicate that the proteoglycans synthesized in muscle cultures are likewise made in developing muscle in ovo but that another distinct strictly in ovo proteoglycan is also produced.

Animals↗
PubMed1984

An osteo-inductive bone matrix extract stimulates the in vitro conversion of mesenchyme into chondrocytes.

Urea and guanidine extracts of demineralized beef and rabbit bone matrix were assayed both in vivo and in vitro. One month following intramuscular implantation into mouse thighs, these extracts induced ectopic cartilage and bone. Seven days following continuous in vitro exposure to the same extracts, stage 24 chick limb bud mesenchymal cells in cultures had differentiated into greater numbers of chondrocytes than controls. These results suggest the feasibility of using limb bud mesenchymal cell cultures as an in vitro assay for bone matrix derived, extractable bioactive factors which effect the conversion of mesenchymal cells into chondrocytes as a requisite step in in vivo osteogenesis.

Animals↗
PubMed1984

A fraction from extracts of demineralized adult bone stimulates the conversion of mesenchymal cells into chondrocytes.

Demineralized adult bone contains factors which stimulate nonskeletal mesenchymal cells to undergo a developmental progression resulting in de novo endochondral ossification. In this study, isolated embryonic stage 24 chick limb bud mesenchymal cells maintained in culture were utilized as an in vitro assay system for detection of specific bioactive components solubilized from adult chicken bone matrix. Guanidinium chloride extracts (4 M) of demineralized-defatted bone were fractionated and tested in limb mesenchymal cell cultures for possible effects upon growth and chondrogenesis. Two low-molecular-weight fractions were found to be active in these cultures. A cold water-insoluble, but warm Tris-buffered saline-soluble fraction provoked a dose-dependent increase in the amount of cartilage formed after 7 days of continuous exposure as evidenced by an increased number of chondrocytes observed in living cultures, elevated cell-layer-associated 35S incorporation per microgram DNA, and greater numbers of toluidine blue-staining foci (i.e., cartilage nodules). Growth inhibitory substances were detected in a low-molecular-weight, water-soluble fraction; 7 days of continuous exposure to this material resulted in less cartilage formation and reduced cell numbers (accumulated DNA) on each plate. These observations demonstrate the usefulness of stage 24 chick limb bud cell cultures for identifying bioactive factors extracted from adult bone matrix. In addition, the action of these factors on mesenchymal cells may now be studied in a cell culture system.

Animals↗
PubMed1984

Cartilage.

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Aging↗