Cell and molecular strategies for massive bone repair/regeneration.
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Animals↗
Biomedical subjects
A I Caplan
Publications and source records attributed to A I Caplan.
Bone development.
The sequential cellular and molecular details of the initial embryonic formation of bone can be used to gain insight into the control of this process and subsequent bone physiology and repair. The functioning of osteogenic cells is governed by a complex balance between the intrinsic capacities of these cells in the context of extrinsic information and signalling. As with other mesenchymal tissues, the balance of intrinsic versus extrinsic capacities and influences is central to understanding both the sequence and consequence of bone development. It has been suggested that the cartilaginous model which forms at the centre of limbs is responsible for, and provides the scaffolding for, subsequent bone formation. Our recent studies of the embryonic chick tibia indicate that osteogenic progenitor cells are observed before the formation of the chondrogenic core. In particular, a layer of four to six cells, referred to as Stacked Cells, forms around a prechondrogenic core of undifferentiated cells. These osteoprogenitor cells give rise to all of the newly forming bone. Importantly, this newly forming bone arises outside and away from the chondrogenic core in a manner similar to the intramembranous bone formation seen in calvariae. Indeed, the cartilaginous core is replaced not by bone but by vascular and marrow tissues. The interplay between the osteogenic collar and the chondrogenic core provides an environment which stimulates the further differentiation of the cartilage core into hypertrophic cartilage and eventually renders this core replaceable by vascular and marrow tissue. There is an intimate relationship between the osteogenic cells and the vasculature which is obligatory for active bone formation. Bone formation in long bones, such as the tibia, as well as in the calvaria seems to proceed in a similar manner, with vascular tissue interaction being the most important aspect of successful osteogenesis, as opposed to the presence or interaction of cartilage. Our studies have focused on the development of long bones in aves, but detailed study of mouse and man indicates that many of the general features observed for birds apply to bone development in mammals. It is our current thesis that the general rules governing embryonic formation of long bones also apply to the formation of ectopic bone and are related to aspects of fracture repair.
Animals↗
Extravascular fluid dynamics of the embryonic chick wing bud.
While a number of models of positional information in the chick wing bud have involved the diffusion of morphogens to establish chemical gradients of morphogenetic activity, only recently have there been attempts to characterize the milieu in which such diffusion must take place. We report an analysis of the fluid dynamics of the extravascular (interstitial) spaces of stage 22-25 chick wing buds, into which aqueous aniline blue dye was microinjected as a visible, unreactive tracer. Six sites along the antero-posterior (A-P) and proximo-distal (P-D) axes were chosen for study. Injections of dye into the posterior half of the wing bud exhibited marked directionality of extravascular transport (mean of all posterior sites = 68%), while anterior injections showed little or no directionality (mean of all anterior sites = 13%). All instances of directed dye movement were disto-proximal, the same direction as the blood flow through the marginal veins. In embryos gassed in situ with CO2, which reversibly stopped the heartbeat and vascular flow, directionality was abolished, yet diffusion rates were unaffected. Posterior disto-proximal extravascular dye movement was correlated with the greater diameter, flow velocity, and volume flow rate of the posterior marginal vein, compared to the anterior marginal vein. Radial diffusion rates were measured, and posterior disto-proximal rates were corrected for measured disto-proximal directionality by the use of a simple diffusion-translation model. Three-way analysis of variance showed that directionality-uncorrected disto-proximal rates in posterior sites were not significantly different from anterior radial rates, but that directionality-corrected posterior rates did differ significantly (P less than 0.0001). A significant stage effect (P less than 0.005) and a significant interaction between the A-P axis and stage (P less than 0.05) were also found. We hypothesize that the spatial arrangement and flow patterns of the vasculature physically determine the fluid dynamics of the interstitium. Based on these observations, we also suggest that disto-proximal extravascular fluid movement in the posterior wing bud presents a barrier to the free diffusion of aqueous molecules, including morphogens originating in the "zone of polarizing activity."
Analysis of Variance↗
Chemotactic response of mesenchymal cells, fibroblasts and osteoblast-like cells to bone Gla protein.
Bone gla protein (BGP) and decarboxylated bone gla protein (dBGP) were tested for chemotactic activity against stage 24 chick limb bud mesenchymal cells, chick embryonic muscle-derived fibroblasts, murine Balb/C 3T3 cells, and two lines of rat osteosarcoma cells, ROS-17/2.8 and -25/1. Both BGP and dBGP were potent chemoattractants for all the cell types except 3T3 cells. The dose response curves were bell-shaped, with maximal chemotactic response ranging from 5 pg/ml for ROS 25/1 cells to 10 ng/ml for the stage 24 limb bud cells. dBGP was equally potent a chemoattractant as BGP for all cell types tested indicating that the gamma-carboxylation of the glutamic acid residues is not required for chemotactic activity. Given this chemotactic capability, it is possible that BGP acts in bone remodelling by attracting osteogenic cells to the sites of bone resorption where BGP may be liberated or exposed.
1-Carboxyglutamic Acid↗
Proteoglycans synthesized during the cartilage to bone transition in developing chick embryos.
Osteogenesis of the long bone involves a series of cellular and molecular events culminating in the fabrication of a mineralized matrix of the long bone. These studies focus upon the chemical identity and morphology of newly synthesized proteoglycans formed during the cartilage to bone transition in embryonic chick tibia development. As early as the onset of major ossification events (day 13; Hamburger-Hamilton Stage 39), synthesis of a small, highly 4-sulfated chondroitin sulfate proteoglycan was detected in the shaft of the tibia. The characterization of the chondroitin sulfate-rich proteoglycans from cartilage, hypertrophic cartilage, bone marrow, and new bone lead to the suggestion that these molecules can be used as distinctive markers for the cellular phenotypes involved in the cartilage to bone transition.
Animals↗
A water-soluble fraction from adult bone stimulates the differentiation of cartilage in explants of embryonic muscle.
A water-soluble fraction of a 4 M guanidine HCl extract of demineralized adult bovine bone stimulated the differentiation of cartilage in explants of minced skeletal muscle from embryonic chick legs; cartilage was also induced by a semipurified protein preparation. Cartilage could be identified in treated cultures at 1 week with muscle from day-9 embryos, not before 2 weeks with muscle from day-12 embryos, and not before 3 weeks with muscle from day-19 embryos. The ability to respond to this water-soluble fraction by exhibiting cartilage differentiation was dose-dependent, but not confined to any particular muscle region of the day-12 embryonic leg. These observations indicate that bone-derived soluble chondroinductive agents act on cells in minced embryonic muscle preparations. The induction of cartilage is dependent upon the accessibility of the responding cells to the agents, on the concentration of inductive agents, and on the developmental age of the responsive tissue.
Animals↗
Reinitiation of chondroitin sulphate proteoglycan synthesis in regenerating skeletal muscle.
Previous work from this laboratory involved the characterization of a large chondroitin sulphate proteoglycan unique to chick skeletal muscle. This proteoglycan is synthesized by embryonic skeletal muscle both in ovo and in culture but is not synthesized by adult muscle in vivo and myotubes in advanced cultures. Because regenerating skeletal muscle has been found to recapitulate synthesis of embryonic muscle protein isoforms, an analysis was performed to assess whether synthesis of chondroitin sulphate proteoglycans is reinitiated during muscle regeneration. Adult chicken pectoral and leg (gastrocnemius) muscle was injured by excision of a small piece of tissue or by cold injury; in the latter, the basement membrane has been reported to remain intact. At various times after injury, whole animals were exposed to [35S]sulphate and the proteoglycans were isolated by ion-exchange chromatography and analysed. Synthesis of only small proteoglycans, typical of normal adult skeletal muscle, is observed in the contralateral, uninjured muscle. In the regenerating muscle 4 days after injury, there is increased sulphate incorporation and abundant synthesis of chondroitin sulphate proteoglycans. This is observed in both pectoral and leg muscle irrespective of the type of injury, which suggests that the presence of basement membrane does not affect reinitiation of chondroitin sulphate proteoglycan synthesis. By 25 days after injury, synthesis of chondroitin sulphate proteoglycans is still detected but is significantly diminished. These data are consistent with the notion that skeletal muscle regeneration involves a recapitulation of embryonic events and give further credence to the hypothesis that skeletal muscle chondroitin sulphate proteoglycans play a role in some early aspect of myogenesis.
Animals↗
Initial characterization of small proteoglycans synthesized by embryonic chick leg muscle-associated connective tissues.
Muscle development in the embryonic chick involves the interfacing of two different cell types: myogenic cells forming the contractile network and connective tissue cells which wrap this network into discrete morphologies. Previous reports from this laboratory detailed the characterization of large extracellular matrix proteoglycans synthesized by skeletal muscle. This report describes the initial characterization of small muscle-associated connective tissue proteoglycans synthesized in embryonic chick leg in ovo and by embryonic leg muscle-associated secondary fibroblasts in vitro by CsCl equilibrium density gradient centrifugation. Analysis of the D1 gradient fractions from the in ovo and in vitro material revealed that the proteoglycans were of relatively small size. These molecules eluted from Sepharose CL-2B through a Kav range of 0.6-0.8. Analysis of alkaline borohydride-released glycosaminoglycan chains by Sepharose CL-6B, Sephadex G-25, and thin layer chromatography demonstrated a mixture of three proteoglycan families composed of chondroitin sulfate, dermatan sulfate, and heparan sulfate glycosaminoglycans.
Animals↗
Chemotactic response of embryonic limb bud mesenchymal cells and muscle-derived fibroblasts to transforming growth factor-beta.
Transforming growth factor beta (TGF-beta) was tested for its ability to stimulate a chemotactic response in Stage 24 embryonic chick limb bud mesenchymal cells and muscle-derived fibroblasts. TGF-beta stimulated dose-dependent chemotaxis in both cell populations. Maximal chemotaxis was achieved with a concentration of 5 ng/ml for limb bud cells and as low as 15 pg/ml for muscle-derived fibroblasts. TGF-beta was not chemokinetic at these levels. Several other proteins found in bone, namely fibronectin, type I collagen, and osteonectin, were not chemotactic. However, both Bone Gla-protein and basic-FGF were found to be chemotactic but less effective than TGF-beta. Comparison with extracts of adult bone indicates that while TGF-beta is a potent chemoattractant, it does not account for all the chemotactic activity found in adult bone.
Animals↗
Characterization of the core protein of the large chondroitin sulfate proteoglycan synthesized by chondrocytes in chick limb bud cell cultures.
The core protein of high buoyant density proteoglycans synthesized by chondrocytes in stage 24 chick limb bud mesenchymal cell cultures was cleaved with cyanogen bromide to produce 17 resolvable peptides on sodium dodecyl sulfate-polyacrylamide slab gels. Of these peptides, 10 appear to originate from the chondroitin sulfate-rich region, 2 appear to be derived from the keratan sulfate-rich region, and 5 seem to be derived from the hyaluronic acid-binding region. The peptides from the chondroitin sulfate-rich region are almost all large (200 to 64 kDa). In contrast, the peptides from the keratan sulfate-rich and hyaluronic acid-binding regions are relatively small (47 to 12 kDa). One peptide from the hyaluronic acid-binding region appears to contain mannose-rich N-linked oligosaccharides as inferred from its observed binding by concanavalin A. A different hyaluronic acid-binding region peptide and one of the keratan sulfate-rich peptides were shown to contain disulfide bonds and therefore may be involved in contributing to the tertiary structure of the hyaluronic acid-binding region. Based on these observations, a map of the chick chondrocyte proteoglycan core protein has been constructed.
Aggrecans↗
Thermal and solvent stability of proteoglycan aggregates by quasielastic laser light-scattering.
The dissociation behavior of several species of proteoglycan aggregates (PGA) has been studied quantitatively by monitoring the changes in particle size by dynamic laser light-scattering. Firstly, studies of the thermal dissociation of reconstituted PGA from bovine nasal septum and bovine fetal epiphysus are described. The effect of link protein in stabilizing reconstituted PGA against changes in temperature has been demonstrated. It was also confirmed that, upon prolonged heating at 70 degrees, the dissociation of link-containing, reconstituted PGA is effectively irreversible. Secondly, the dissociation characteristics of native PGA isolated from chick chondrocyte and rat chondrosarcoma cell-cultures were investigated in aqueous solvents containing increasing concentrations of guanidine hydrochloride. It was found that the more densely packed, native, aggregate structure has a higher susceptibility to thermal disruption than the reconstituted, link-containing material. Considerable loss of subunits from the native aggregates occurs at temperature where the link protein retains its activity. The dissociation of native PGA is irreversible, in the sense that reconstituted PGA always exhibits smaller sizes than the native PGA, reflecting a smaller degree of aggregation of the subunits.
Animals↗
An appreciation. Dame Honor B. Fell, F.R.S. (1900-1986).
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Bibliographies as Topic↗
In vitro chondrogenesis stimulated by extracts of gall bladder epithelium.
Living adult gall bladder epithelium has previously been shown to initiate in vivo ectopic cartilage and/or bone formation. In the present study, we demonstrate that solubilized extracts of gall bladder, which have never been inductive when tested in vivo, do in fact contain a factor that stimulates chondrogenesis in stage 24 chick limb bud mesenchymal cell cultures. These 4 M guanidinium chloride soluble, cold water soluble protein fractions initiate a dose-dependent increase in the amount of cartilage formed following an 8-10 day continuous exposure as evidenced by the presence of greater numbers of chondrocytes when viewed with phase optics, more intense toluidine blue metachromatic staining in fixed plates and elevated 35S-sulfate incorporation into the cell layer (as a maker for proteoglycan biosynthesis). The factor(s) responsible for this activity binds to DEAE anion-exchange resins, is stable to heat (100 degrees C for 10 min) and 2-mercaptoethanol reduction but is labile to trypsin digestion. These results emphasize the importance of in vitro test systems to identify bioactive agents which may otherwise go undetected using an in vivo implantation assay.
Animals↗
Calibration of the relative molecular mass of proteoglycan subunit by column chromatography on Sepharose CL-2B.
Calibration relationships were derived for cartilage proteoglycan subunit (PGS) that relate the inverse z-average hydrodynamic radius (Rs) and the weight-average Mr (Mw) to the partition coefficient (Kav.) for PGS when chromatographed on a Sepharose CL-2B column. PGS isolated from chick limb-bud chondrocyte cell cultures was fractionated chromatographically into eight pools, for which Mw and Rs were determined by total-intensity and dynamic light-scattering measurements. These data were found to be related to Kav. through the following empirical equations: log Mw = -(1.65 +/- 0.27)Kav. +(6.58 +/- 0.08); log Rs = -(0.69 +/- 0.04)Kav. +(2.75 +/- 0.01). Application of these relationships to the chromatographic data led to Mw = 1.48 X 10(6) and Rs = 38.7 nm (387 A) for the unfractionated specimens compared with values of Mw = 1.46 X 10(6) and Rs = 38.2 nm (382 A) determined by light-scattering. Our results were found to be consistent with previously proposed phenomenological models for the gel-filtration mechanism. Application of these calibration relationships to Kav. for several unfractionated specimens led to predicted values of Mw and Rs that are accurate to within 10%.
Animals↗
The extracellular matrix is instructive.
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Animals↗
Structure of native proteoglycan aggregates from chick limb-bud chondrocytes.
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Animals↗
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.
Actin Cytoskeleton↗