QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 June 2003
doi: 10.1242/jcs.00527

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Muraglia, A. Articles by Quarto, R. Search for Related Content PubMed PubMed Citation Articles by Muraglia, A. Articles by Quarto, R. Social Bookmarking                         What's this?

Formation of a chondro-osseous rudiment in micromass cultures of human bone-marrow stromal cells

¹ Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
² Dipartimento di Medicina Sperimentale, Universita' dell'Aquila, Italy
³ Dipartimento di Medicina Sperimentale e Patologia, Universita' La Sapienza, Roma, Italy
⁴ Dipartimento di Oncologia, Biologia e Genetica, Universita' di Genova, Italy
⁵ Parco Scientifico Biomedico San Raffaele Roma, Italy

View larger version (172K): [in a new window]   Fig. 1. Chondrogenesis in pellet cultures. Cartilage beads formed by BMSCs cultured under chondrogenic conditions for 1 (a,b), 2 (c,d) and 3 (e,f) weeks. (a,c,e) Toluidine-blue staining. (b,d,f) Alcian-blue staining. Overall morphology, alcianophilia and metachromatic staining with toluidine blue are typical of cartilage. Bar, 100 µm.

View larger version (66K): [in a new window]   Fig. 2. Chondrogenesis in pellet cultures. (a,b) Immunoreactivity for type II collagen at 7 days (a) and 14 days (b) of culture. Notice the diffuse staining, which spares a thin rim of matrix at the periphery of the tissue `bead'. (c) Growth in size of the tissue bead over time. A plateau is reached at day 14. The arrow indicates the switch to medium conducive to in vitro mineralization. Each point represents the mean of the cross-sectional diameter measured in three samples per time point. Bar, 125 µm.

View larger version (95K): [in a new window]   Fig. 3. Osteogenesis in pellet cultures. BMSCs were cultured under chondrogenic conditions for 4 weeks and subsequently in mineralization-inducing medium for 1 week. Histological sections of the cultured tissue harvested at 35 days of culture. (a) Toluidine-blue staining. Notice the absence of metachromatic staining (purple) in a peripheral rind of tissue. (b) Alizarin-red-S staining of a comparable section shows that the peripheral rind of non-chondroid tissue has accumulated calcium, which indicates mineralization. (c) Haematoxylin and eosin staining of another section, demonstrating a central core of histologically typical hyaline cartilage (hc) surrounded by a transition zone (tz) where basophilia (blue staining) fades out and an outermost layer of mineralized bone-like tissue (b) containing individual cells encased in lacunar spaces (d). Bar: a,b, 100 µm; c, 50 µm; d, 12.5 µm.

View larger version (149K): [in a new window]   Fig. 4. Electron microscopic analysis of cartilage and bone tissues formed in pellet culture. The culture was harvested at 35 days (28 days of culture under chondrogenic conditions and 7 days of culture in mineralization-inducing medium). (a) Azur II /methylene blue stained semithin section demonstrating the relative position of the electron-microscopy fields imaged in b-e. Mineralized tissue is on the left and hyaline cartilage on the right. (b) Bone-like matrix containing thick, periodically banded collagen fibrils and areas of mineral deposition (arrows) (undecalcified section, original magnification 28500x). (c) Transition zone containing alternate thick, periodically banded, and thin, non-banded collagen fibrils. Arrow indicates mineral clusters. No proteoglycan granules are seen (undecalcified section, original magnification 28500x). (d) Mineralizing cartilage. Arrows indicate mineral clusters. The remaining matrix has typical features of hyaline cartilage (thin, randomly oriented collagen fibrils and proteoglycan granules) (decalcified section, original magnification 11500x). (e) Hyaline cartilage (decalcified section, original magnification 11500x). Bar: a, 25 µm; b,c, 6 µm; d,e, 2.3 µm.

View larger version (71K): [in a new window]   Fig. 5. Immunoreactivity for type I procollagen N-terminal propeptide at 7 (a), 14 (b) and 28 (c) days of culture under chondrogenic conditions. Notice the growing peripheral region of type I collagen production (double arrows). Extracellular immunoreactivity, denoting the initial deposition (id) of collagen, marks the boundary of cartilage. Intracellular labelling (arrows) is restricted to a peripheral rind of tissue where immunoreactivity is lost in the extracellular matrix, consistent with zonally defined matrix maturation events (m). Bar, 50 µm.

View larger version (137K): [in a new window]   Fig. 6. Spatial and temporal expression of BSP and osteocalcin in BMSC pellet cultures. Haematoxylin and eosin stained section (a-d), BSP immunolabelling (e,f) and osteocalcin immunolabelling (g-l) at 7, 14 and 28 days of culture (chondrogenic conditions), and at 35 days of culture (28 days under chondrogenic conditions and 7 days under mineralization-permissive conditions). Concurrent with a morphological change in the outer layer of tissue (a-d), BSP production is restricted to a progressively thicker rind of tissue during the chondrogenic phase (e-g). Deposition of extracellular BSP (h, arrows) and osteocalcin (l) is only observed when mineralization has occurred (h, arrows). Notice the similar patterns of production of BSP and type I procollagen (Fig. 5), including the apparent centripetal shift of the cellular production of either protein between day 14 and 28 (f,g, double arrows for BSP). Bar, 50 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter