Analysis of cartilage differentiation from skeletal muscle grown on bone matrix

DEVELOPMENTAL

Analysis

BIOLOGY

78, 301-331

of Cartilage

(1980)

Differentiation Bone

from Matrix

I. Ultrastructural MARK Department

of Anatomy, Received

A. NATHANSON~ Harvard August

Medical

Skeletal

Muscle

Grown

on

Aspects

D.

AND ELIZABETH

School,

I, 1979; accepted

25 Shattuck in revised

Street, form

HAY

Boston,

January

3,

Massachusetts

02115

1980

Previous studies have demonstrated that embryonic skeletal muscle is competent to form hyaline cartilage when cultured in vitro on demineralized bone matrix (Nogami, H., and IJrist, M. R. (1970). Exp. Cell Res. 63.404-410; Nathanson, M. A., et al. (1978). Develop. Biol. 64,99-117). The present experiments were undertaken to determine the nature of the morphological alterations which attend this phenotypic transformation and to investigate the ultrastructural characteristics of the myoblasts and tibroblasts of skeletal muscle during the transformation. Nineteen-day embryonic rat limb muscles were minced and the tissue fragments explanted to bone matrix or collagen gels. The trauma of excision and mincing causes syncytial myotubes to degenerate and the nuclei of mononucleate cells to enter a heterochromatic “resting stage.” In culture, nuclei of mononucleate cells rapidly regain euchromasia. No myoblast or fibroblast cell death can be detected. On bone matrix, the entire mononucleate population transforms into fibroblast-like cells. Myoblasts are the major contributor to this population; they dissociate from the degenerate myotubes and begin to acquire endoplasmic reticulum by 24 h in vitro. The fibroblast-like morphology persists through 4 days in vitro. By 6 days in rjitro some of these fibroblast-like cells acquire the phenotypic characteristics of chondrocytes, and by 10 days masses of hyaline cartilage are found. In control explants of skeletal muscle onto collagen gels, the heterochromatic nuclei of the mononucleated cells expand after 24 hr in vitro, but the mononucleated cells remain as myoblasts and fibroblasts and begin to regenerate skeletal muscle by 4 days in uitro. No cartilage forms. The results indicate that both myoblasts and fibroblasts have chondrogenic potential when grown on demineralized bone. It is tempting to conclude that the embryonic mesenchymal cells which give rise to skeletal muscle, cartilage, and other connective tissue of the limb have similar developmental potentials and that local influences, rather than separate cell lineages, account for the final pattern of differentiation.

the means whereby these definitive cell types arise. One hypothesis suggests that early limb mesenchyme contains precursor cells which are already committed to either a myogenic or a chondrogenic fate (Holtzer et al., 1973; Abbott et al., 1974). The precursor cells in question, however, do not synthesize specific products and thus cannot be precisely identified. The evidence for this hypothesis lies in the observation (1) that limb mesenchyme contains low numbers of clonable muscle and cartilage cells prior to overt histogenesis of these tissues (Abbott et al., 1974; Dienstman et al., 1974) and (2) that transplanted quail

INTRODUCTION

The formation of chondrocytes from embryonic limb mesenchyme is not an isolated event, but is intimately linked with the differentiation of skeletal muscle and fibrous connective tissue. Both cell types arise from a pool of similar-appearing mesenchymal cells. Studies of the differentiation of skeletal muscle and cartilage in the developing avian and mammalian limb have led to several hypotheses concerning ’ Present address: Department of Anatomy, Jersey Medical College, 100 Bergen St., Newark, Jersey 07103.

New New 301

0012-1606/80/100301-31$02.00/O Copyright 0 1980 by Academic Press, All rights of reproduction in any form

Inc.

reserved.

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somites contribute to limb musculature rather than to cartilage in the avian limb (Chevalier et al., 1977). In the absence of a cellular contribution from somites, however, limb mesenchyme is still able to produce both muscle and cartilage (Chevalier et al., 1977). The hypothesis that all of the cells are predetermined does not explain this apparent adaptability. Another hypothesis contends that each mesenchymal cell is endowed with a potential for a limited number of alternative cellular programs and that its final position in the limb determines how this potential is realized (Wolpert, 1969, 1978a, b). For example, a program for “limb mesenchyme” may be expressed as skeletal muscle, fibrous connective tissue, or cartilage, depending on the location of a cell within the developing limb. There is some evidence for the view that limb mesenchymal cells are initially phenotypically unstable and become stabilized shortly after definitive myogenic and chondrogenic regions appear (Searls, 1967; Searls and Janners, 1969; Zwilling, 1966). A considerable body of data suggests that the immediate environment of a cell plays a large part in determining its ultimate fate (for review see Hall, 1970). Subsequent development of phenotypic stability has been noted both in vivo (Searls, 1965) and in vitro (Konigsberg, 1963; Coon, 1965) and is agreed upon by all investigators, the early mode of differentiation notwithstanding. Skeletal muscle cells and fibroblasts have been shown to be phenotypically unstable when presented with conditions which elicit cartilage or bone in viuo and in vitro (Urist, 1970; Nogami and Urist, 1970, 1974a,b; Reddi and Huggins, 1972; Anderson and Griner, 1977; Nathanson et al., 1978). When minced skeletal muscle is cultured upon demineralized bone (bone matrix) or in contact with similar bone powders, both the myogenic cells and the fibroblasts of skeletal muscle seem to alter their phenotype to that of cartilage. Indeed, cloned myoblasts

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have been shown in at least one instance to give rise to overt cartilage under these conditions (Nathanson et al., 1978). The fact that bone matrix consistently elicits chondrogenesis of nonchondrogenic cells adds weight to the hypothesis that environmental factors evoke this phenotype. These data also suggest that even though limb cells develop phenotypic stability, the broader early developmental potential is not lost, but held in abeyance for lack of an alternative stimulus. The major issue which we explore in this communication is the extent to which syncytial myofibers, mononucleate myoblasts, and tibroblasts participate in the chondrogenesis of skeletal muscle induced by bone matrix. While the general responsiveness of differentiated tissue to bone matrix has been amply demonstrated, little is known of the means whereby the component cells alter their complement of cellular organelles, and other aspects of their cytology, and become organized within the extracellular matrix of cartilage. Is it necessary for cells to acquire a common morphology before assuming another phenotype, or can they alter their differentiation so as to proceed directly from one phenotype to another? In the present study we have undertaken an electron microscopic investigation of chondrogenesis of skeletal muscle on bone matrix to answer questions such as these. In subsequent communications, we present the biochemical correlates of this process. MATERIALS

Preparation

AND

METHODS

of Embryonic Rat Muscle

Pregnant, Sprague-Dawley albino rats (Charles River Laboratories, Wilmington, Mass.) were killed by decapitation when their embryos reached 19 days of gestation. The embryos were immediately removed and placed into ice-cold Hanks’ balanced salt solution (HBSS), pH 7.4, for a short time until their upper arms and thighs had

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been removed. After isolation into a fresh aliquot of ice-cold HBSS the appendages were cleaned of skin and dermis. Skeletal muscle was then removed from the appendages and pooled into culture medium containing serum. With the aid of a dissecting microscope, the skeletal muscle was subsequently pulled apart with forceps into small pieces and vascular, nervous, and adherent skeletal elements were removed. The 8-12 embryos isolated from each pregnant rat yielded 32-48 appendages, which resulted in hundreds of muscle pieces which had to be mechanically cleaned of contaminant tissues. As a result, some of the excised muscle was held on ice for periods of up to 4 hr before being placed into organ culture. Immediately prior to organ culture, the tissue was finely minced with forceps, resulting in a final suspension of small fragments and pieces of teased skeletal muscle. Preparation

of Organ

Cultures

Bone matrix was prepared and used as described by Urist et al. (1973), Nogami and Urist (1970, 1974a,b), and Nathanson et al., (1978). After sequential extraction of the mineral and most of the proteinaceous material, the killed and demineralized bones were cut into cylindrical, diaphyseal fragments, frozen in liquid nitrogen, lyophilized to dryness, and stored at -20°C. Bone matrix was prepared for organ culture by rehydrating the cylinders at room temperature in culture medium containing serum. The hydrated cylinders were then split longitudinally to form hemicylinders and a narrow longitudinal segment was removed from each hemicylinder to serve as a tissue overlay. A series of transverse crevices was cut into each hemicylinder to provide increased surface area. Hemicylinders and tissue overlays were then coated with chicken plasma (frozen, not lyophilized; Grand Island Biological Co., Grand Island, N.Y.; GIBCO) to help cells attach to the matrix. Organ cultures were assembled by placing the hemicylinders onto an inverted

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stainless steel organ culture grid that had been previously placed into the center well of an organ culture dish (Falcon Plastics, Los Angeles, Calif.). After removing the excess chicken plasma, the minced skeletal muscle was placed into the hemicylinder and the tissue overlay put into position over the skeletal muscle (see Nogami and Urist, 1974b, for a diagram of the completed explant). For comparison, skeletal muscle, minced and cleaned in exactly the same manner as previously, was grown as an organ culture on cellulose ester filters (type HATF, pore size 0.45 pm; Millipore Corp., Bedford, Mass.) coated with gels of type I collagen. Type I collagen was prepared from the tail tendons of adult rats by the procedure of Elsdale and Bard (1972). Collagen was extracted from the tendons in 0.05 M acetic acid for 2-3 days. Gross insoluble material was then removed by filtration and the acid extract dialyzed for 24 hr against two lots of one-tenth strength F-12 culture medium (Grand Island Biological Co., Grand Island, N.Y.; GIBCO). The dialysate was sterilized by centrifugation for 15 hr at 37,300g. All steps were performed at 4°C and the clear, sterile solution was stored at 4°C (Elsdale and Bard, 1972). The gels were prepared by adjusting the pH of an aliquot of the collagen solution to pH 7.4 with 0.1 N sodium hydroxide and quickly spreading the resultant viscous mass onto the cellulose ester filters. Each gel received three such applications and each layer was allowed to dry onto the filter in between applications. After the final layer had dried, the filters were sterilized in 70% ethanol, dried, and either stored dry or rehydrated with culture medium containing serum. Explants onto collagen gels were prepared by transferring an amount of minced muscle, approximately equal in size to that placed onto bone matrix, directly onto the hydrated gels, with or without chicken plasma; the results were the same whether or not plasma was added. The explants were organ

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cultured on wire grids as were the explants onto bone matrix. Completed explants were fed on alternate days by changing one-half to three-fourths of the medium and maintained in a waterjacketed incubator (National Appliance Co., Portland, Ore.) at 37°C in an atmosphere of 5% CO* in air, at a gas flow rate equal to the volume of the incubator chamber per hour. Humidity was provided by saturating an absorbent pad in the outer well of the organ culture dishes with sterile water and by placing two trays of deionized water directly into the incubator chamber. At intervals, as described in the text, explants were removed from the dishes and fixed as described subsequently. Culture Medium The culture medium consisted of medium CMRL-1066 containing 15% heat-inactivated fetal calf serum (serum was heatinactivated for 45 min at 56°C) and penicillin-streptomycin (final concentration, 100 units/ml and 100 pg, respectively). All components of the culture medium were purchased from GIBCO (Grand Island Biological Co., Grand Island, N.Y.). Electron Microscopy Explants onto bone matrix and collagen gels were fixed for 30 min in an aldehyde fixative containing tritrophenol (picric acid). The fixative contained 2.5% formaldehyde (prepared from paraformaldehyde), 5.0% glutaraldehyde, 0.06% picric acid, and 0.06% calcium chloride in 0.1 M cacodylate buffer, pH 7.4 (Ito and Karnovsky, 1968). The explants were then washed in 0.1 M cacodylate buffer, pH 7.4, and postfixed for 60 min in 1.0% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.4. All steps were carried out at room temperature with the exception of the osmium postfixation, which was performed on ice. Intact skeletai muscle was fixed by immersing whole thighs from 19-day embryonic rats in the fixative for 60 min. After 60 min the tissue was hardened sufficiently to

VOLUME 78, 1980

prevent contraction of the individual muscles. The muscles were then dissected from the thighs, in fixative, and diced into segments of approximately 1 mm”. The diced tissue was subsequently fixed for an additional 30 min and washed and postfixed as before for the explants. After postfixation the fixed tissue was washed with water and stained en bloc for 30 min with 2.0% aqueous uranyl acetate. Dehydration was accomplished with a graded ethanol series and the explants and skeletal muscle were embedded in Spurr low viscosity embedding medium (Tousimis Research Corp., Rockville, Md.). Sections showing a gold interference color were cut with a DuPont diamond knife on a Sorvall MT-2B ultramicrotome and collected on uncoated 300-mesh grids. Sections were stained with lead citrate (Venable and Coggeshall, 1965) and examined with JEOL 1OOBand JEOL 100s electron microscopes. The content of the various samples was continuously monitored by viewing 0.5~pmthick sections. Thick sections were stained with a 1:l mixture of 1.0% azure II and 1.0% methylene blue in 1.0% borax. Representative sections were photographed with an Olympus Vanox light microscope. RESULTS

Our method of explanting skeletal muscle involves finely mincing the tissue into small pieces, placing aliquots of the mince onto hemicylindrical segments of demineralized bone (bone matrix), and overlaying the explanted tissue with an additional segment of bone matrix. As a result, the freshly excised skeletal muscle does not initially lie as a compact mass, but as a loosely arranged aggregate. The explanted skeletal muscle subsequently (l-2 days in uitro) appears as a solid massby light microscopy. Electron microscopic observations demonstrate that after 2 days in culture the explanted tissue contains randomly oriented myotubes, in various stages of necrosis, and numerous healthy mononucleate cells. Gross disorganization of the explanted skel-

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eta1 muscle, the presence of necrotic myotubes, and the presence of many mononucleate cells characterize the tissue during and after explantation. Subsequent changes in the organization of the tissue must be related to the surviving mononucleate cells. In order to determine the origin of the mononucleate cells in these cultures, we examined skeletal muscle fixed before excision from the embryonic thighs and at several periods prior to and after its being placed into organ culture.

Embryonic

Skeletal

Muscle in Situ

The near-term rat skeletal muscle used for these experiments consists largely of clusters of myotubes and myoblasts that are surrounded by an empty-appearing extracellular space which contains a few fibroblasts, nerves, and immature vessels (Figs. l-3). The loose arrangement differs from the morphology characteristic of adult skeletal muscle in which myotubes are bound together by endomysial connective tissue. The embryonic myotubes are surrounded instead by numerous mononucleate myoblasts. In cross section, it can be seen that the mononucleate cells and myotubes form distinct aggregates, each consisting of one or more myotubes in different stages of development, surrounded by several mononucleate cells (Fig. 3). Most of the embryonic myotubes contain well-developed myofibrils. The myofibrils do not fill the cytoplasmic compartment, but are separated by intracellular spaces which appear to contain a large amount of glycogen (gl, Figs. 1 and 2). As in adult skeletal muscle fibers, the organelle-rich cytoplasm (contrasted to myofilament-rich cytoplasm) occupies a small amount of the cellular volume mainly in the juxtanuclear and peripheral regions of the cell. Some myotubes give the impression of being recently formed and representing an even earlier stage. These myotubes appear relatively undifferentiated in that they contain fewer myofilaments, which in some cases

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are disorganized and may show poor sarcomere organization (mf, Fig. 1). In addition, the nuclei and myofilaments of these myotubes are surrounded by a cytoplasm rich in free ribosomes which occupies much of the volume of the cell. The embryonic nature of the myotubes is further reinforced by the presence of central, as well as peripherally located, nuclei. Centrally located nuclei are typically associated with young myotubes and often display a more highly convoluted morphology than peripheral nuclei, especially if the cell is rich in glycogen. The nuclei of myotubes are typically euchromatic (N4, Fig. 1) and contain prominent nucleoli (Figs. 2 and 3). The basal lamina at this stage of skeletal muscle morphogenesis is not well developed and appears as a thin surface coat of little electron density. Around some of the aggregates a basal lamina-like coat can be seen which consists of flocculent extracellular material associated with a few weakly striated collagen fibrils. A typical basal lamina is a continuous sheet of extracellular material, 200-500 nm in diameter, that runs parallel to the plasmalemma and is separated from it by a space of approximately 100 nm (Farquhar, 1978). That most of the basal lamina-like material in embryonic muscle represents little more than a surface coat is demonstrated by its thickness (
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FIGS. 1-3. Nineteen-day-old embryonic rat muscle is characterized in situ by numerous myotubes which in logitudinal section can be seen to contain organized striated myotibrils (Figs. 1 and 2) and myofilaments in the process of assembly (mf, Fig. 1). The myotubes are surrounded by mononucleate cells with the morphology of myoblasts (Figs. 1 and 2), and in cross section it can be seen that the myogenic cells are arranged in clusters (Fig. 3). The extracellular space is relatively empty in appearance, containing a few fibroblasts (f, Fig. 3). vessels, and nerves. In the electron micrograph (Fig. 1). two myoblasts can be seen with typical, slightly heterochromatic nuclei (N2, N3), ribosome-rich cytoplasm (r), mitochondria. and a few profiles of granular endoplasmic reticulum. The cell whose nucleus is labeled Nl appears to be in a state of transition from the myoblast heterochromatic pattern (N3) to the euchromatic pattern characteristic of myotube nuclei fN4). This cell (Nl) is probably in the process of fusing with the adjacent myotube (mt). The myoblasts divide by mitosis (mi) and are the source of the myotube nuclei. Their nuclei contain nucleoli (nut, Fig. 1) as do the nuclei of myotubes (Figs. 2 and 3). The myotube cytoplasm may contain glycogen deposits (gl, Figs. 1 and 2). Figure 1. x 8750. Figure 2 and 3 (light micrographs), x 790 and x 845.

mononucleate cells as myoblasts depends on the following criteria. Myoblasts are characterized by a rounded or fusiform morphology, the presence of free ribosomes and polysomes, and relatively little granular endoplasmic reticulum (Hay, 1963; Lipton, 1977). In addition to abundant free cytoplasmic ribosomes (r, Fig. 5), mitochondria, and a few profiles of endoplasmic reticulum and Golgi elements, (not shown), myoblasts display a larger nuclear to cytoplasmic area than fibroblasts and are most often found in close proximity to the myotube aggregates described previously, rather than free within the extracellular space. Myoblast nuclei (Figs. 4 and 5) contain significantly more heterochromatin than fibroblast nuclei (Fig. 6) and nucleoli

are prominent. Nuclei can also be seen (Nl, Fig. 1) which are intermediate in morphology between those of typical myoblasts (N2, N3, Fig. 1) and myotubes (N4, Fig. 1); these cells seem to be myoblasts that have recently fused with myofibers. Mitotic figures are common (Fig. 1). No cells were observed with the very dense heterochromatic nuclei and scanty cytoplasm said to be typical of satellite cells (Muir, 1970). While myoblasts in this embryonic skeletal muscle contain more heterochromatin than myotube nuclei, they are not as heterochromatic as satellite cells and are not enclosed by a well developed basal lamina. Fibroblasts are characterized by a flattened or stellate shape, elongate euchromatic nuclei, and moderately abundant cy-

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toplasm containing well-developed granular endoplasmic reticulum (Fig. 6). Golgi complexes and nucleoli are prominent. Minced

Skeletal

Muscle

After excision, skeletal muscles contract into rounded masses, myoiibril organization is lost, and individual myotubes traverse a wavy course through the tissue, rather than the straight course so commonly observed in intact muscle. Minced skeletal muscle was routinely held in the culture medium, on ice, for varying periods of time up to 4 hr so that we could accumulate sufficient material for each experiment. To assess the morphological alterations that may have taken place during the preparative period, we fixed the minced muscle at hourly intervals following its excision from the embryonic rats. Within 2 hr after mincing, contracted myotube fragments can be seen to possess a highly convoluted plasmalemma and supercontracted myofibrils (Fig. 7). The convolutions are quite large and contain ribosomes, mitochondria, and filamentous material. The filamentous material appears to be in a state of degeneration. Actin and myosin are no longer visible as interdigitating filaments, having been replaced by broad condensations containing alternating light and dark bands that probably derived from previously existing “A” and “I” bands (df, Fig. 7). Z lines are absent. Normally composed of a finely granular nucleoplasm,

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myonuclei after mincing become heterochromatic (Nl, N2, Fig. 7), pycnotic, or highly vesicular. They occur within a highly disorganized, vacuolated cytoplasmic compartment (Fig. 7). Other myotubes, which probably suffered greater injury, display these same characteristics, but to a degree than can be described as overt degeneration. Myofibrils become very condensed and often lack any striated pattern (df, Fig. 9). The cytoplasm becomes a mass of membranous profiles and most nuclei are pycnotic. Morphological alterations are also displayed by myoblasts and fibroblasts in response to mincing. Nuclei of myoblasts (N3, Fig. 7) and of fibroblasts (not shown) take on a dense heterochromatic pattern reminiscent of the “checkerboard” appearance sometimes seen in plasma cells. The ribosome-rich cytoplasm becomes more electron dense (cyt, Fig. 8). The appearance of the myoblast is that of a small dark cell containing a large heterochromatic nucleus with a prominent nucleolus (nut, Fig. 8). Contraction of the excised tissue not only causes the myotubes to collapse, but also causes the myoblasts to occupy a smaller area. As a consequence, these cells appear more concentrated than previously noted in intact skeletal muscle. We paid careful attention to a possible relative increase in cell number of fibroblasts. Again, however, relatively few fibroblasts were found, confirming our earlier finding that the tissue

FIGS. 4-6. Additional characteristics of 19-day-old rat muscle in situ are illustrated in these electron micrographs. In Fig. 4, the extreme in the extent of condensation of heterochromatin in a myoblast is illustrated, but this nucleus is not condensed as that of the “satellite cell” in more mature muscle. The adjacent myotube contains myotibrils (mfb) in various states of organization. Myoblasts are surrounded by tufts of surface extracellular material. The tufts of surface extracellular material (t, Fig. 5) may run from myoblast to myoblast and probably are the forerunner of the basal lamina. A somewhat more developed extracellular surface coat (t, inset, Fig. 6) surrounds some myotubes. The myoblasts depicted in Fig. 5 have ribosome-rich cytoplasm (r), slightly heterochromatic nuclei, nucleoli (one of which is labeled nut), mitochondria. and a few profiles of granular endoplasmic reticulum and Golgi elements (not shown). Figure 6 illustrates the typical morphology of a muscle fibroblast in situ, with its euchromatic nucleus (Nl), nucleoli (not shown), and abundant cytoplasm containing well-developed granular endoplasmic reticulum (er), a Golgi complex (not shown), and other organelles. Small groups of collagen fibrils (cf. Fig. 6) occur near the fibroblasts. Myotubes have peripheral (N2) or centrally located nuclei (N3), which are euchromatic, but exhibit a denser nucleoplasm than fibroblast nuclei (compare N2 and N3 with Nl). Figure 4, X 10,400. Figure 5. x 23,400. Figure 6, x 10,409; inset, x 29,950.

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consists mainly of myotubes and myoblasts. The increased nuclear heterochromaticity affects fibroblasts to the same degree as myoblasts and their organelle-rich cytoplasm also becomes dense, demonstrating that all mononucleate cells within the excised tissue respond in a similar way to the trauma imposed by the procedure. Skeletal muscle 4 hr after mincing displays few additional changes, but myotube degeneration progresses in magnitude. Myotubes, which displayed vacuolated cytoplasm, degenerating nuclei, and disorganized myofilaments at 2 hr, now become more necrotic. Cytoplasm is replaced by membranous and filamentous inclusions and myelin figures, and nuclei finally transform into empty, vesicular structures (Nl, N2, Fig. 9). While most myotubes present a necrotic appearance, it was possible at 4 hr to find a few intact myotubes with dense cytoplasm (mt, Fig. 9) and heterochromatic nuclei (N3, N4, Fig. 9). The heterochromatin pattern is the checkerboard type observed in viable mononucleate cells (Fig. 8). Such myotubes may have been short enough to escape being cut into fragments and thus remain alive. After 4 hr the mononucleate population consists mainly of myoblasts, although, as before, a few fibroblasts can be found. Probably due to the continued contraction of the tissue, myoblasts seem even more numerous at 4 hr than 2 hr after mincing. The myoblast cytoplasm continues to be electron dense and nuclei remain quite heterochromatic. The fibroblasts that are present also have heterochromatic nuclei and elec-

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tron-dens. cytoplasm, being distinguished from the myoblasts by their more abundant granular endoplasmic reticulum. None of the myoblasts or fibroblasts show evidence of cell death (vesicular nuclei, vacuolated cytoplasm). These studies demonstrate that minced myotubes explanted into organ culture undergo necrosis. However, the mononucleate population survives mincing and largely consists of myoblasts with heterochromatic nuclei. Explants

to Bone Matrix

The nuclei of all of the mononucleate cells become euchromatic within 24 hr after being explanted onto bone matrix. The transition back to euchromasia seems as rapid as the initial transition to heterochromasia and there is no evidence of myoblast or fibroblast cell death. Although the explanted muscle was originally composed almost entirely of surviving myoblasts, all of the mononucleate cells explanted onto bone matrix gradually take on the characteristics of fibroblasts. Transformations from myoblast-like cytoplasm (rich in free ribosomes) to fibroblast-like cytoplasm (rich in granular endoplasmic reticulum) could readily be found in l- to 2-day cultures (Figs. 10-12). The cytoplasm of these cells contains a variable amount of granular endoplasmic reticulum, numerous mitochondria, and prominent Golgi apparati (Fig. 11). Some of the cells still contain many free ribosomes and sparse granular endoplasmic reticulum and most probably represent myo-

FIGS. 7-9. Electron micrographs showing the appearance of the excised muscle 2 hr (Figs. 7 and 8) and 4 hr (Fig. 9) after mincing, but before being placed in organ culture. The nuclei of the myotubes become diffusely heterochromatic (Nl, N2, Fig. 7) and then washed-out or vesicular in appearance (Nl, N2, Fig. 9). Some become pycnotic and it seems clear that they degenerate, along with the myofibrils (df, Fig. 9) and cytoplasm of the injured syncytial tubes. The mononucleate cells of the minced explant do not degenerate, The nuclei of the myoblasts acquire a unique, checkerboard pattern of condensed chromatin (N3, Fig. 7; NIL4; Fig. 8) and the cytoplasm (cyt, Fig. 8) becomes dense but retains its ribosome complement, mitochondria, and profiles of endoplasmic reticulum. Nucleoli can still be identified (nut, Fig. 8). Fibroblast nuclei acquire similar heterochromatin after mincing of the muscle. Even the nuclei (N3, N4. Fig. 9) of surviving immature myofibers (mt, Fig. 9) acquire the checkerboard nuclear pattern in response to the injury. The nuclear pattern reverts to normal as soon as the tissue is placed in organ culture. Figure 7, x 5200. Figure 8, x 5720. Figure 9, x 5460.

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blasts whose nuclei have become euchromatic, but whose cytoplasm has not yet developed fibroblast-like amounts of endoplasmic reticulum (Fig. 10). The nucleolus of this myoblast-in-transition changes significantly. Whereas myoblast nucleoli are condensed and granular in appearance (nut, Fig. 5), these cells acquire nucleoli which display a dispersed pars granulosa and pars fibrosa (nut, Fig. 11). This is not to suggest that the transition, which is rapid, occurs in all heterochromatic myoblasts simultaneously. In addition to myoblasts which contain a typical myoblast-like cytoplasm (mb, Fig. 12), myoblasts can be found which also possessfibroblast-like cytoplasm (er, Fig. 12). Similarly, not all myoblasts are equally heterochromatic. These morphological variations most probably represent corresponding variations in the metabolic state of the mononucleate cells in vivo and following the mincing procedure. The recovering fibroblasts also show variations in their morphology. Since it is impossible to follow the same cells throughout their life history on bone matrix, we must ask whether it is possible that authentic survivor fibroblasts give rise to all of the fibroblast-like cells in the l- to a-day period. Assuming that surviving cells could have gone through at most two population doublings in 2 days, it would be impossible for fibroblasts present in excised skeletal muscle to overgrow the myoblasts in this time period. Moreover, the myoblasts-in-transition are located next to the

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myotubes (Fig. 12), and this characteristic location considered together with the morphological changes noted previously makes the conclusion that the myoblasts revert to a mesenchymal cell type inescapable. The explant at l-2 days still contains myotube remnants and degenerating myofibrils (df, Figs. 10 and 11). Whereas the extracellular space previously appeared empty, it now contains myofibrillar remnants and a large amount of flocculent extracellular material (inset, Fig. 12). The flocculent material is not randomly dispersed throughout the cultures, but concentrated in the vicinity of the fibroblast-like cells. As viewed in the light microscope, it gives a metachromatic staining reaction. This material is not amorphous, but delicately fibrillar in appearance as viewed by electron microscope. In regions which are devoid of myotube debris, it is found to extend around all of the fibroblast-like cells. Surface material (t, Fig. 10) reminiscent of basal lamina gradually disappears, but tufts of extracellular matrix material (t, Figs. 13 and 14) remain on the cell surfaces of fibroblast-like cells. After 4 days on bone matrix the explanted skeletal muscle consists of a population of fibroblast-like mononucleate cells amid necrotic myotubes. The abundant granular endoplasmic reticulum of fibroblast-like cells in 4- to 6-day-old cultures is composed of dilated sacs containing electron-dense material (Figs. 13 and 14). The secretory organelles are more highly devel-

FIGS. 10-12. Electron micrographs showing stages in the recovery of the mononucleate cells in minced muscle cultured on bone matrix for 2 days. A typical myoblast is illustrated in Fig. IO, with its slightly heterochromatic nucleus, ribosome-rich cytoplasm (r), a few profiles of endoplasmic reticulum (er). and a few tufts (t) of surface material. The cells gradually become fibroblast-like. The fibroblast-like cells (Fig. 11) have more euchromatin, more dispersed nucleoli (nut), and more prominent endoplasmic reticulum (er) and Golgi elements (pa) than do myoblasts. The location next to degenerating myofilaments (df, Figs. 10 and 11). together with the transitions that can be observed in their morphology, indicates that myoblasts transform into tibroblastlike cells. Cells in transition (Fig. 12) may retain myoblast-like nuclei (Nl, N2) and ribosome-rich myoblast-like cytoplasm (mb) while acquiring amounts of endoplasmic reticulum (er) characteristic of fibroblasts. Nucleoli become more dispersed (nut 1 and 2) when the nuclei become euchromatic (N3). The fibroblast-like cells, some of which derive from authentic muscle fibroblasts, move away from the myotube and begin to secrete a metachromatic matrix with a finely fibrillar, reticulated ultrastructure (inset, Fig. 12). Figure 10, x 15,300. Figure 11, X 13,870. Figure 12, X 9590; inset, X 12,550.

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oped than in either the myoblast or the fibroblast of origin. The transition is most marked near the bone matrix (Fig. 13), but is not limited to juxtamatrix regions. In addition, the cytoplasm of the mononucleate cells contains numerous subplasmalemma1 and cytoplasmic filaments (f, Figs. 13 and 14) and many of the cells have the morphological characteristic of migratory cells. The extracellular space still contains myofibrillar remnants and the flocculent material. Striated collagen fibrils are found in increasing numbers within this flocculent material. Mononucleate cells containing cellular debris can be found more commonly at 4 days than at earlier stages, especially among the necrotic myotubes. Some of these cells may be multinucleate and their cytoplasm contains abundant secretory organelles. These macrophage-like cells are very large and difficult to view at the electron microscopic level. Urist (1970) described multinucleate cells (“matrixclasts”) at the light microscopic level which are presumed to excavate chambers within the bone matrix substratum and subsequently give rise to the chondrocytes which later populate these chambers. While we cannot rule out the possibility that these cells have a matrix-lytic activity, their location suggests that they function to remove cellular debris remaining from the necrotic myotubes. After 6 days on bone matrix the dominant cell type is the mononucleate cell with the morphology of the fibroblast described previously. A large amount of necrotic ma-

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terial has disappeared by this stage, although some myofibrillar remnants are present in the extracellular matrix. In many areas, the extracellular space contains dense fibrillar material, which seems to represent newly synthesized extracellular matrix. Morphologically, this extracellular material is similar to the flocculent material noted earlier. The fibroblast-like cells occur within this material and it seems likely that they are the source of it. The first recognizable chondrocytes appear at 6 days on bone matrix. The chondrocytes are round or fusiform in shape and extend numerous fine processes out into the surrounding matrix. The extracellular matrix surrounding chondrocytes consists of a sea of delicate fibrils (inset, Fig. 16), in contrast to the amorphous fibrillar material around the fibroblasts. At the light microscope level, the cartilage matrix is typically metachromatic and hyaline in appearance; it is well developed in crevices of the bone matrix (Anderson and Griner, 1977; Nathanson et al., 1978). However, chondrocytes appear simultaneously at a distance from the bone matrix surface, from which they are separated by other chondrocytes with whom they make contact. Myotube nuclei, membranes, and cytoplasmic components disappear by 6 to 8 days on bone matrix, leaving behind patches of extracellular myofibrillar debris. Scattered among the debris are numerous fibroblast-like cells. Fibroblasts seem to wander freely among the debris that has not been completely removed by the macrophage-like cells.

F~cs. 13 AND 14. Electron micrographs showing the fibroblast-like morphology of the mononucleate cells in minced muscle cultured 4 days on bone matrix. Nuclei contain prominent nucleoli (nut. fig. 13) and are highly euchromatic. Granular endoplasmic reticulum is well-developed (er, Fig. 13) and the cisternae are often dilated due to the accumulation of moderately electron-dense secretory products (er, Fig. 14). The Golgi apparatus (ga) is large and is rich in vesicles, vacuoles, and stacked cisternae. Mitochondria are prominent and cytoplasmic inclusions, including lipid (L, Fig. 14), are present. The cytoplasm also contains bundles of small filaments, mainly in the periphery of the cell (0. The fibroblast-like cells contact each other by broad cell processes and by filopodia (arrows). Tufts (t) of amorphous material are associated with the cell surface. The cells next to the bone matrix (matrix, Fig. 13) are similar in morphology to cells that are several cell layers distant from the substratum. Figure 13, x 23,710. Figure 14. x 15,600.

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The interval from 6 to 8 days is characterized by the appearance of increasing numbers of chondrocytes, although fibroblast-like cells are still present in large numbers. The chondrocytes contain more granular endoplasmic reticulum after 8 days than at 6 days, and more of the previously elongate (fibroblast-like) cells are rounded in their appearance. Close inspection reveals that chondrocytes and fibroblast-like cells intermingle and form intercellular contacts with each other (arrows Figs. 15 and 16). Indeed, the distinction between a rounded fibroblast-like cell and a chondrocyte is not always apparent (Fig. 15). The transition from fibroblast to chondrocyte morphology seems to begin with the rounding up of the cell and the acquisition of a larger Golgi apparatus (ga, Fig. 15). The surface of the rounded cells simultaneously becomes more irregular (compare cells a and b, Fig. 15) and many cell processes are trapped in the expanding matrix (cp, Figs. 15 and 16). The cytoplasmic ground substance increases in amount and density, and at the same time the profiles of the endoplasmic reticulum become narrower and more widely separated (compare Figs. 13 and 14 with Figs. 15 and 16). Nucleoli remain prominent and the euchromatic nuclei become round and regular in outline (Fig. 16). Other organelles, such as mitochondria, are similar in appearance in the fibroblast-like cells and chondrocytes. By 10 days in vitro the morphology of the explants to bone matrix has changed from mixtures of chondrocytes and fibroblast-like cells to primarily cartilage and

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the myofibrillar remnants are gradually disSulfated glycosaminoglycan appearing. synthesis has increased dramatically (Nathanson and Hay, 1980). Chondrocytes appear as large, rounded cells containing numerous sacs of rough endoplasmic reticulum, which may be dilated at this time, and extensive Golgi apparati (Fig. 17). Glycogen and lipid inclusions occur within the cytoplasm of many of the chondrocytes at this stage. The chondrocytes are surrounded by abundant extracellular matrix containing weakly banded collagen fibrils, lo-20 nm in diameter, typical of cartilage. Many of the collagen fibrils have a beaded appearance indicating the presence of proteoglycan granules. Chondrocytes, proteoglycan granules, and faintly striated collagen fibrils have been previously well illustrated in the cartilaginous matrix that appears in 14-day cultures of muscle on bone matrix (Anderson and Griner, 1977). The majority of the chondrocytes occupy positions near the bone matrix surface (Fig. 17). However, the tissue extends some distance away from the bone matrix and it is likely that many of the fibroblast-like cells which transform into chondrocytes never come in contact with the matrix. Close examination reveals that chondrocyte organelles are not polarized with respect to the bone matrix surface. Higher-magnification electron micrographs reveal no surface specializations at the chondrocyte-bone matrix interface. Cell processes contact the demineralized collagen fibrils of the bone matrix (inset, Fig. 17). The entire surface of the chondrocytes bears cytoplasmic extensions,

FIGS. 15 AND 16. Electron micrographs showing stages in the differentiation of chondrocytes from fibroblastlike cells, 8 days after minced muscle was placed onto bone matrix. The cell at a (Fig. 15) has the flattened shape of a fibroblast, whereas cell b is beginning to round up and contains a very prominent Golgi apparatus (ga, Fig. 15) and the more irregular cell surface characteristic of the chondrocyte. This cell and adjacent chondrogenic cells extend numerous cell processes (cp) into the cartilage-like matrix that is accumulating around them. In regions of transformation of fibroblast-like cells to chondrocytes, nuclei are usually euchromatic (N2. N3) rather than heterochromatic (Nl). In fully developed active chondrocytes (Fig. 161, nuclei are euchromatic, regular in shape with a moderately prominent nucleoli (nut) and abundant nucleoplasm. The Golgi apparatus (ga) and endoplasmic reticulum are well-developed, as is the ground cytoplasm which separates the membranous cisternae. There is considerable intracellular contact (arrows). Typical cartilage matrix contains very fine collagen fibrils (inset, Fig. If?). Figure 15, x 8840. Figure 16, x 10,080; inset, x 20,800.

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however, and they are no more numerous near the bone matrix than away from it (Fig. 17). After 20 days on bone matrix, the dominant cell type is the chondrocyte (Fig. 18). Whereas fibroblast-like cells were previously found to be widely scattered throughout the cultures, chondrocytes replace them even among the myofibril remnants at this stage. The extracellular matrix becomes more dense than at 10 days and contains a high concentration of cartilage-like collagen fibrils. These newly synthesized collagen fibrils can easily be distinguished from preexisting bone collagen because they are thin and poorly striated (inset, Fig. 18), as compared to the large, cross-banded tibrils of bone collagen (inset, Fig. 17). Of some interest in the 20-day cultures on bone matrix is the appearance of rounded, multinucleate cells (Fig. 19), which display no myofibrillar specializations and are not likely to have derived from myotubes that have degenerated. Some of these multinucleate cells contain inclusion bodies (db, Fig. 19) and so they may have descended from the macrophagelike cells observed earlier. The multinucleate cells are not found on the bone matrix surface, but near to the myofibrillar remains. Nuclei are euchromatic, nucleoli are prominent, and the cytoplasm is rich in ground substance, mitochondria, and endoplasmic reticulum (Fig. 19). Muscle

Explants

onto Collagen

Gels

While we have chosen to describe immediate as well as late-occuring morphological changes as part of the response of skel-

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etal muscle to bone matrix, these changes might not necessarily all be caused by bone matrix. Transformation to a fibroblast-like morphology might occur as a result of excision (and thus relate to tissue damage) and occur in tissue explanted onto any in vitro substratum. As a comparison, therefore, we have carefully examined the morphology of the minced tissue explanted onto Millipore filters coated with gels of type I collagen. Explants onto collagen gels consisted of aliquots of minced muscle, similar in size to those explanted onto bone matrix, and they were organ cultured in an identical fashion to those explanted onto bone matrix. Minced skeletal muscle grown in vitro on collagen gels for 1-2 days displays a morphology similar to that of uncultured, excised skeletal muscle in that myoblasts are the predominant cell type and myotubes are undergoing necrosis. As in intact skeletal muscle, relatively few fibroblasts are present. The response of the heterochromatic myoblasts to bone matrix was shown previously to consist of a rapid transformation into fibroblast-like cells with euchromatic nuclei. In contrast, after 24 hr on collagen gels the myoblasts maintain a rounded morphology, “myogenic” cytoplasm containing many free ribosomes, and a high nuclear to cytoplasmic ratio (Fig. 20). Considerable heterogeneity among the nuclear and cytoplasmic compartments was found. Many myoblasts display a high nuclear to cytoplasmic area, but others display a lowered nuclear to cytoplasmic area after l-2 days on collagen gels and contain granular endoplasmic reticulum in addition to

FIGS. 17 AND 18. Electron micrographs showing chondrocytes in IO-day (Fig. 17) and 20-day (Fig. 18) cultures of muscle on bone matrix. The basal surface of the chondrocyte in relation to the bone matrix substratum (matrix, Fig. 17) is not specialized. Both broad cell processes and filopodia (inset, Fig. 17) touch the collagen fibrils on the cut surface of the bone matrix, but do not penetrate it. The cells also contact each other via filopodia (arrows, Fig. 17). The Golgi apparatus (pa) and endoplasmic reticulum of the chondrocytes continue to be highly developed at 10 and 20 days; the cisternae of the endoplasmic reticulum may be dilated (Fig. 17) or compact (Fig. 18), as at earlier stages. Chondrocyte nuclei are typically round and regular in shape (Fig. 17). The cartilage matrix contains small collagen fibrils (inset, Fig. 18) and, in the light microscope, gives a metachromatic staining reaction. Figure 17, x 8320; inset, x 16,640. Figure 18, x 10,090; inset, x 20,590.

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FIG. 19. Electron micrograph of a large, multinucleated cell with prominent nucleoli (nut) in a 20-day-old bone matrix culture. The cell is in the vicinity of the remainder of the degenerating myotibrils (df) and may contribute to the removal of the debris. It contains dense inclusion bodies (db), grandular endoplasmic reticulum (er), mitochondria, and dense ground cytoplasm. It seems to correspond to the cell termed “matrixclast” by others. x 6600.

abundant free ribosomes (Fig. 22). These cells with their increased amount of endoplasmic reticulum resemble the myoblastsin-transition seen on bone matrix. The nuclei of all of the mononucleated cells transform from the intensely heterochromatic

state evoked by mincing into a more euchromatic state shortly after the beginning of the culture period. Myoblast nuclei, however, tend to regain the peripheral and diffuse heterochromatin that characterized them in ho.

FIGS. 20-22. Electron micrographs showing the appearance of mononucleated cells in minced muscle explants grown on collagen gels for 1 day (Figs. 20 and 21) and 2 days (Fig. 22). As in the case of cultures on bone matrix, the nuclei of myoblasta resume their typical slightly heterochromatic pattern and their cytoplasm contains a few profiles of granular endoplasmic reticulum (er, Fig. 20). Fibroblasta are characterized by euchromatic nuclei and more extensive endoplasmic reticulum (Fig. 21). There is considerable intercellular contact (arrows, Figs. 20 and 21). In developing myotubes, myoblast nuclei change from slightly heterochromatic (N2, Fig. 22) to euchromatic (Nl, Fig. 22). At 2 days, myoblasts contain somewhat more granular endoplasmic reticulum (er, Fig. 22) than did the cells of origin, but they are still characterized by ribosome-rich cytoplasm. The same degenerative changes are noted in myofibrils (df) of the injured myofibers as in cultures on bone matrix. nut, nucleolus. Figure 20, x 14,920. Figure 21, x 10,920. Figure 22, x 7440.

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The persisting fibroblasts may be distinguished from myoblasts on the basis of their nuclear characteristics, which include a large vesicular nucleus, a lack of peripheral heterochromatin, and a compact nucleolus (Fig. 21). The cytoplasm of fibroblasts is richer in endoplasmic reticulum (Fig. 21) than that of the “transitional” myoblast (Fig. 22). Both cell types occur in close proximity to one another and to the myotube remnants (df, Fig. 22). After 2 days on collagen gels, we have the impression that more of the mononucleate cells have the morphology of myoblasts than at 1 day. That is to say, there seems to be a tendency on the part of the myoblasts to acquire fibroblast-like characteristics in response to injury and culture, but they soon revert to myogenic (as opposed to secretory) morphology. On bone matrix, in contrast, the fibroblast-like cells derived from myoblasts are not a temporary phenotypic modulation and after 2-4 days in vitro they comprise most of the mononucleate population. The 2-day explants onto collagen gels also contain a few multinucleate cells. The nuclei of these cells are convoluted, as are true myotube nuclei, although myofilaments are lacking. Because the myotubes that survive mincing are immature, it is impossible to distinguish these multinucleate cells as either newly formed myotubes or short myotubes which survived the mincing procedure. It should be noted, however, that no myotubes were observed at 2 days on bone matrix. The explanted tissue on collagen gels remains relatively undifferentiated at 2 days and consists of myoblasts, a few fibroblasts, multinucleate cells, and the necrotic myotubes. After 4 days on collagen gels, the explanted tissue consists mainly of skeletal muscle in different stages of organization (Figs. 23 and 24). Both mononucleate and

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multinucleate cells are present, the mononucleate cells being mainly of myoblast morphology. Necrotic myotubes were still present at 4 days. It is obvious that the tissue must have regenerated via the surviving myoblasts. Not only did the starting material contain few surviving myotubes, but the regenerating myotubes were found to be branched in many cases, a phenomenon which has been reported to occur only in vitro (Shimada, 1971). Moreover, stages in myofibril regeneration are easy to find (Fig. 23). Regenerated myotubes contain a well-formed contractile apparatus and large, euchromatic nuclei with prominent nucleoli. The sarcomeres display well-developed A and I bands (Fig. 24). In addition to elongate myotubes, multinucleate cells without a well-formed contractile apparatus are present. However, scattered patches of myofilaments are found in their cytoplasm. Clearly, the viable minced tissue, which consisted largely of heterochromatic myoblasts, has engaged in a process of muscle regeneration during this 4-day interval. After 10 days on collagen gels, most of the regenerating myotubes have begun to fill their cytoplasmic compartment with myofilaments. Cells with the morphology of matrixclasts can be seen (Fig. 25). The tissue contains myoblasts as well as fibroblasts. The myotubes course randomly throughout the tissue mass and may be surrounded by a scant basal lamina. While the remains of necrotic myotubes are still present, they are packed together with viable tissue. The explant no longer contains much open space (Fig. 25). The speed with which the myoblasts acquired and lost secretory organelles (l-2 days) contrasts markedly with the length of time required to develop a contractile apparatus. Even at 20 days, many myotubes contain an admixture of organelle-rich and myofilament-rich

FIGS. 23 AND 24. Electron micrographs showing the onset of active myogenesis in minced muscle explants grown for 4 days on collagen gels. Young myotubes are characterized by centrally located euchromatic nuclei, ribosome-rich cytoplasm, and disorganized-appearing myofibrils (mfb, Fig. 23). The more advanced regenerating myotubes contain well-organized myofilbrils (Fig. 24). Figure 23. X 7180. Figure 24, x 28,080.

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FIG. 25. Light micrograph of a lo-day-old culture of minced muscle grown collagen gel. Well-organized myotubes, as well as myotubes in the process of differentiation, can be seen. A multinucleated cell resembling a “matrixclast” is present on the surface of this culture (arrow). x 630.

cytoplasm. The appearance at 20 days is similar to that at 10 days (Fig. 25); differentiated muscle fibers, myoblasts, and tibroblasts are present and the cultures continue to be healthy. In all cases, cultures grown on collagen gels fail to produce cartilage. We have examined serial-sectioned, paraftin-embedded material at the light microscopic level, as well as sections at the electron microscopic level, with uniform results. All of the explants on collagen gels were comprised of regenerating myotubes, myoblasts, and fibroblasts, with no suggestion of chondrogenesis. Our data demonstrate that chondrogenesis is limited to those cultures grown upon bone matrix and that this transition is not dependent upon the direct transformation of myoblasts to chondrocytes, but takes place through a fibroblastic interme-

diate. The transformation does not involve the myotubes, but rather the surviving mononucleate cells (myoblasts and fibroblasts). DISCUSSION

The results presented here demonstrate that cartilage arises from the mononucleate population of cells within embryonic rat skeletal muscle cultured on bone matrix. Examination of fixed tissue showed that mincing of the skeletal muscle is a trauma that myotubes cannot survive. Following injury, nuclei within myotubes become pycnotic and then degenerate along with the sarcoplasm. The mononucleate population of the muscle of origin is composed mainly of myoblasts next to myotubes, with a few fibroblasts scattered between the groups of myogenic cells. The present experiments indicate that both myoblasts and tibro-

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blasts survive injury. No celI death was noted within the mononucleate population and it is possible to detect stages in the transformation of myoblasts on bone matrix during which they develop a morphology similar to that of authentic fibroblasts. Later, the majority of these fibroblast-like cells t,ransform into cartilage. These observations confirm and extend previous evidence that either cloned myoblasts or cloned fibroblasts can transform into cartilage when grown on bone matrix (Nathanson et al., 1978). It is likely that the embryonic mesenchymal cells which gave rise to the limb had similar developmental potentials and that local influences determine the final pattern of differentiation into muscle and cartilage (see Introduction). In the Discussion, we consider the significance of the morphological changes which occur in the muscle from the time of its excision from the embryo to the completion of chondrogenesis in uitro. Initial

Effects

of Mincing

and Culture

After isolation and mincing of the tissue, the nuclei of the myoblasts and fibroblasts enter a heterochromatic state, but within 24 hr in vitro, the nuclei of the mononucleate cells become euchromatic again. The pattern of heterochromatin induced by the isolation procedure is quite distinct and is the same in myoblasts and fibroblasts. Dense strands of chromatin line the nuclear envelope, enclose the nucleolus, and coarse through the nucleus in a checkerboard fashion reminiscent of the chromatin pattern observed in plasma cells and other relatively “inert” nuclei. The ground cytoplasm appears dense, but the cell organelles seem healthy. It is tempting to conclude that the nuclei have entered a metabolic “resting” state (Hay and Revel, 1963). When they become euchromatic again under favorable culture conditions, fibroblast nuclei contain little heterochromatin, whereas myoblast nuclei are initially characterized by a diffusely clumped chromatin pattern similar to that which they exhibit in ho.

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In contrast, the nuclei of the injured myotubes exhibit a diffuse heterochromatin pattern and then either swell or become highly compacted (pycnotic). The swollen, vesicular nuclei disintegrate and the pycnotic nuclei are probably eventually phagocytosed. The cytoplasm begins to degenerate immediately following mincing, probably because the fibers are cut, exposing the cytoplasm to the culture medium. Indeed, a few of the more immature myotubes that were too short to be cut survive, and their nuclei, interestingly enough, temporarily take on the checkerboard pattern characteristic of the resting mononucleated cells. Here again, we conclude that this heterochromatin pattern is a temporary response to trauma, for these immature myotubes appear to survive on collagen gels. During the first day in culture, not only do the nuclei of all of the mononucleate cells recover, but also their cytoplasm begins to expand. The amount of granular endoplasmic reticulum increases initially in myoblasts and fibroblasts on both collagen gels and bone matrix. However, a differential effect of the bone matrix is noticeable after 48 hr in culture. After this interval most of the cells explanted onto bone matrix are fibroblast-like, whereas on collagen gels myoblasts resume a rounded morphology and contain abundant free ribosomes in spite of the initial increase in granular endoplasmic reticulum. We would like to suggest that the early increase in granular endoplasmic reticulum is a regeneration response which is common to all of the explanted cells and is transitory in its appearance. After 4 days in culture the differences between the two types of culture are marked, with the cells on bone matrix appearing entirely fibroblast-like while those on collagen gels are differentiating into skeletal muscle. The nature of this so-called regenerative response in vitro is not fully understood, but it is possible that the mescnchymal cells are stepping up their production of hyaluronate and extracellular glycoproteins, such

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as fibronectin (Hynes, 1976). The acquisition of endoplasmic reticulum by the cells attests to a secretory function and, in addition, new metachromatic materials are seen in the extracellular matrix. One is reminded of the events which occur after amputation of a salamander limb (Hay, 1974). Here again, cells released from the muscle and connective tissues acquire endoplasmic reticulum and assume the morphology of mesenchymal cells. At the same time hyaluronate is produced and collagen is removed (Toole and Gross, 1971). The cells remain fibroblast-like throughout the blastema stage and, interestingly enough, the first tissue they produce is cartilage, a sequence of events not unlike those we observed to occur in bone matrix explants. Differentiation

of Muscle on Collagen Gels

In contrast, the mononucleated cells in minced muscle explanted to collagen gels do not form a “blastema” of fibroblast-like cells. In the 2- to 4-day period in vitro, fibroblast-like myoblasts revert to a more typical myoblast morphology, fuse, synthesize actin and myosin, and quickly convert the tissue to a morphology not unlike that of the muscle of origin. This result demonstrates that the culture conditions per se are adequate to maintain myogenesis and that this in vitro environment does not elicit chondrogenesis without the presence of bone matrix. In comparing the effect of bone matrix and collagen gels on the muscle explants, it is interesting to note that both substrata contain type I collagen and both are relatively poor in proteoglycan. Collagen gels are known to support myogenesis, even of cloned myoblasts (Hauschka and Konigsberg, 1966), and they also support and promote the continued differentiation in vitro of corneal epithelium (Meier and Hay, 1974) and sclerotome (Kosher and Church, 1975). They do not induce a new pattern of differentiation. The effect of bone matrix, however, must be classified as metaplastic,

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even as applied to the component fibroblasts. Neither the fibroblasts nor the myoblasts of the 19-day-old embryonic rat muscle would be expected to undergo chondrogenesis under any other conditions. We will return to discuss the mode of action of the bone matrix below. Let us examine first the morphological steps in the transformation to cartilage. Steps in the Differentiation Bone Matrix

of Cartilage on

The formation of cartilage in this system occurs in three phases. During the first phase (l-3 days) the explanted cells assume the morphology of fibroblast-like cells, and during the second phase (4-5 days) the secretory morphology of the cells is augmented. A good deal of new, amorphous appearing, metachromatic matrix is produced by the fibroblast-like cells cultured on bone matrix. During the third phase (610 days) these libroblast-like cells respond to the bone matrix by assuming a chondrocytic morphology and producing morphologically typical cartilage matrix, which we show in the second paper of this series to be also biochemically typical of cartilage (Nathanson and Hay, 1980). The initial effect (1-3 days in uitro) can, as we discussed previously, be likened to a “regenerative” response. The fact that the cells did not develop directly into chondrocytes, but grew as fibroblasts for the next 4- to E&day period, is undoubtedly of significance. All of the mononucleate cells appear healthy and it seems highly unlikely that one population overgrew another during this period. Rather, after gearing up their secretory machinery to produce what seems to be a transitory “embryonic” matrix, the cells gradually acquire the characteristics of chondrocytes. The cellular and nuclear shape changes and cytoplasmic organelles become dispersed in a characteristic chondrocyte pattern. The cells begin to produce cartilage proteoglycans (Reddi et al., 1978) and type II collagen (Reddi et al., 1977;

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von der Mark, 1979), indicating change has taken place in their machinery. Mode of Action of the Substratum Vitro Chondrogenesis

HAY

that a genetic in in

Chondrogenesis is limited to those explants growing upon bone matrix. It is reasonable, therefore, to conclude that bone matrix has a direct or indirect influence on the nuclear machinery of the cells, leading to the expression of the cartilage phenotype. A great deal of effort has been expended to identify factors within the bone matrix that induce chondrogenesis (Strates et al., 1971; Urist, 1972; Iwata and Urist, 1972; Urist and Iwata, 1973; Mikulski and Urist, 1975; Urist et al., 1975, 1977b) and some evidence exists that bone matrix contains a diffusible inductor (Buring and Urist, 1967; Nogami and Urist, 1975; Nakagawa and Urist, 1977; Urist et al., 1977a), which may be a noncollagenous glycoprotein (Urist et al., 1979). The concentration of any putative inductor should be greatest at or near to its source, and thus chondrogenesis in this system might be expected to occur earliest in the vicinity of the bone matrix. While chondrocytes are found along the bone matrix surface, they have also been found at some distance from it. As noted above, the appearance of chondrocytes is preceded by the presence of mesenchymal cells that are surrounded by an amorphous metachromatic matrix. Chondrocytes differentiate throughout these regions. Since the emergence of chondrocytes from skeletal muscle explants does not appear to be correlated with their position relative to the bone matrix surface, it is unlikely that an inductor is concentrated at the bone matrix-skeletal muscle interface. Our electron microscopic observations are consistent with this conclusion in the sense that cellular specializations are not observed at the interface with bone matrix. Chondrocytes characteristically extend filopodia into the extracellular space. In the

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cultures on bone matrix, the chondrocytes probe the extracellular space and the bone matrix surface with similar-appearing filopodia. The basal surface lacked obvious concentrations of secretory vesicles and no digestion of the adjacent bone matrix was noted. The fact that the fibroblast-like cells and the chondrocytes exhibit intercellular contacts may be of significance, however, because an effect exerted on cells in contact with bone matrix could conceivably be propagated from cell to cell. A recent report hypothesizes that osteocyte debris found within the bone matrix may participate in cartilage induction; contacts between cell debris and chondrocytes were illustrated (Anderson and Griner, 1977). We have also found osteocyte debris in bone matrix, but more often than not it bears no direct relation to explanted cells and, in fact, some of the most dramatic metaplasias are at some distance from the bone matrix. The occurrence of osteocyte debris, rather than intact cells, demonstrates that the extractants used to prepare the bone matrix (chloroform-methanol, hydrochloric acid, calcium chloride, EDTA, lithium chloride) do penetrate the entire bone and destroy the cellular architecture. Other investigators utilize demineralized bone powders, which might contain cell debris, to induce chondrogenesis (Reddi and Huggins, 1972,1973; Reddi and Anderson, 1976). Further studies are necessary to evaluate the role of cell debris in the induction process. Absence of Satellite Origin

Cells in the Muscle

of

The textbook appearance of mature skeletal muscle as a compact tissue containing satellite cells and endomysial connective tissue was not found to characterize rat limb muscle at 19 days of embryonic development. Even though parturition occurs at 21 days, this skeletal muscle retains an immature character in that the groups of myotubes surrounded by myoblasts are well spaced from one another and the extracel-

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lular space is largely devoid of fibroblasts and collagen. Satellite cells, by definition, are inactive-appearing mononucleate cells that lie enclosed by the basal lamina of the myotube (Muir, 1970). Embryonic rat skeletal muscle at 19 days is enclosed by only a diffuse surface coat of extracellular material, which does not resemble a true basal lamina. The matter of the presence or absence of satellite cells in the muscle of origin is of some interest because of their postulated “reserve cell” role (Muir, 1970). In addition to their location under the myotube basal lamina, satellite cells are said to have very dense nuclei and scanty cytoplasm. No such cells are present in prenatal rat limb muscle. We have, however, observed numerous myoblasts with a slightly heterochromatic nuclear pattern and ribosome-rich cytoplasm in close proximity to myofibers. Neonatal skeletal muscle lacking a distinct basal lamina has been reported to contain satellite cells as judged by dense nuclei (Ontell, 1977), but nuclear morphology alone seemsan inadequate definition. Some neonatal muscles display well-formed basal laminae (Aloisi, 1970) or display them in some cases and not in others (Ishikawa, 1970). These muscles are probably in a transitional state. Most embryonic muscles probably contain free myoblasts rather than satellite cells (Hauschka, 1972; Nathanson, 1979). Satellite cells do occur with increasing frequency in adult and neonatal muscle and probably arise from preexisting free myoblasts (Hay, 1974; Schmalbruch and Hellammer, 1977; Popiela, 1976). Our observations demonstrate that satellite cells do not develop until birth or shortly before in the rat and thus cannot be postulated to play the role of an embryonic reserve cell in this system. Relations between Fibroblasts blasts

and Myo-

The morphology of fibroblasts and myoblasts has been found to differ enough that

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criteria for the differential recognition of these cell types can be defined (Hay, 1963; see also Lipton, 1977). In addition to the well-known bipolar morphology of myoblasts in vitro (Konigsberg, 1963), typical myoblasts contain slightly heterochromatic nuclei, nucleoli, abundant free ribosomes, and relatively little granular endoplasmic reticulum and few Golgi elements. In contrast, fibroblasts are stellate and flattened and contain an abundance of granular endoplasmic reticulum and Golgi apparati. These morphological criteria are based upon observations of tissue in situ or tissue which has been cultured in vitro under conditions which are known to elicit a “classical” phenotype. In vitro, however, myogenic cells are exposed to an artificial, quasi-normal environment which may alter their appearance and interaction with one another (Konigsberg, 1963; Richler and Yaffe, 1970; Hauschka, 1972, 1974; Ramierez and Aleman, 1972; Abbott et al., 1974; Lipton, 1977). In each case, phenotypic variations were observed within a population of myogenic cells. Not only can the in vitro environment elicit a range of phenotypes from skeletal muscle, running from myogenic to fibrogenic, but ischemic conditions have been observed to elicit an adipose-like morphology (Lipton, 1977). While these variations reflect the influence of the culture environment on the cells, they also have implications regarding the developmental potential of the embryonic mesenthyme which gave rise to a range of phenotypes of which skeletal muscle is but one example. During normal limb myogenesis the cells that specialize in myofibril production characteristically produce less extracellular matrix than those that become fibroblasts, but the specialization is not absolute. For example, skeletal muscle has been shown to synthesize glycosaminoglycans (Schubert et al., 1973;Ahrens et al., 1977) and collagen (Kelley et al., 1976), and nonmuscle cells synthesize significant amounts of actin and

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myosin (Pollard and Weihing, 1974; Goldman et al., 1976). However, even though skeletal muscle and fibrous connective tissue synthesize and secrete glycosaminoglycans and collagen, they usually do not produce cartilage-typical products, such as type II collagen (von der Mark, 1979). It is evident that while there is considerable biochemical similarity among muscle and nonmuscle cells, there are also more important differences which regulate the phenotype that is expressed. Actin, myosin, collagen, and glycosaminoglycans are comprised of multiple forms, each of which may be characteristic of a particular tissue. Even different fibroblasts produce different amounts of several glycosaminoglycans (Conrad et al., 1977). It is not surprising to find myoblasts and fibroblasts secreting extracellular matrix; the differences among myoblasts, fibroblasts, and chondroblasts may have their basis in the means by which cells control relative amounts and types of extracellular and intracellular proteins synthesized, and thus transformation from one cell type to another may not involve the initiation of completely de novo pathways. The formation of fibroblasts may-be considered a phenotypic alteration concomitant to the production of increased amounts and types of collagen and glycosaminoglycans, invoked by injury and sustained by bone matrix. The factors in bone matrix which further affect the genetic program of the cells, leading to the production of cartilage-specific proteoglycans and collagen, remain to be determined. The interconvertibility of the cell types under culture conditions such as those used here emphasizes the common origin of the cells and raises fundamental questions regarding the nature of the so-called commitment to phenotype in vivo. The authors wish to express their sincere appreciation to Mr. Wayne B. Colin for his expert technical assistance. This work was supported by United States Public Health Service Fellowship F32-AM-05481 to M.A.N. and N.I.H. Grant HD-00143 to E.D.H.

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