PGD(2) modulates fibroblast-mediated native collagen gel contraction

Repair of tissues is a necessary step in restoring tissue function following injury consequent to inflammation. Many inflammatory mediators are capable of modulating not only the activity of "inflammatory cells" but also of modulating functions of parenchymal cells that may contribute to repair. Disordered repair is believed to contribute to tissue dysfunction in many inflammatory diseases, including bronchial asthma. The current study evaluated the ability of prostaglandin D(2) (PGD(2)) to modulate fibroblast repair using the in vitro contraction of three-dimensional native collagen gels as a model system. PGD(2) stimulated gel contraction in a concentration- and time-dependent manner. In contrast, the PGD(2) analog BW245C inhibited contraction. Both effects were blocked by a DP-receptor blocker (AH6809). Neither TP receptor blocker SQ29548 nor protein kinase (PK) A antagonist KT5720 hand an effect on PGD(2)-stimulated contraction, suggesting action through a novel prostaglandin D receptor. PKC inhibitor calphostin-C (10(-6) M) blocked the PGD(2) stimulation of gel contraction. A calcium-independent PKC-epsilon inhibitor (Ro31-8220), but not calcium-dependent PKC-alpha and -beta inhibitors, also blocked the PGD(2) effect on contraction, implying a role for a calcium-independent pathway. This study, therefore, supports a role for PGD(2) in tissue repair and remodeling. These effects of PGD(2) appear to be mediated through receptor-signal transduction pathways different from the cAMP-PKA pathways mediating the proinflammatory activity of PGD(2), creating the possibility for selective therapeutic manipulation.     

Ultrafine carbon black particles inhibit human lung fibroblast-mediated collagen gel contraction

Both acute and chronic exposure to particulates have been associated with increased mortality and morbidity from a number of causes, including chronic obstructive pulmonary disease and other chronic lung diseases. The current study evaluated the hypothesis that ultrafine carbon particles, a component of ambient particulates, could affect tissue repair. To assess this, the three-dimensional collagen gel contraction model was used. Ultrafine carbon black particles, but not fine carbon black, inhibited fibroblast-mediated collagen gel contraction. Although previous research has indicated that inflammatory effects of ultrafine carbon black particles are mediated by oxidant mechanisms, the current study suggests that ultrafine carbon black's inhibition of fibroblast gel contraction is mediated by the binding of both fibronectin and transforming growth factor (TGF)-beta to the ultrafine particles. Binding of TGF-beta was associated with a reduction in nuclear localization of Smads, indicative of inhibition of TGF-beta signal transduction. There was also a decrease in fibronectin mRNA, consistent with a decrease in TGF-beta-mediated response. Taken together, these results demonstrate the ability of ultrafine particles to contribute to altered tissue repair and extend the known mechanisms by which these biologically active particles exert their effects.     

Human T cells stimulate fibroblast-mediated degradation of extracellular matrix in vitro

Several chronic diseases are characterized by inflammation, T cell recruitment and tissue remodelling. We hypothesized that activated T cells may stimulate remodelling of extracellular matrix (ECM) in vitro. Total T cells (CD3+) as well as CD4+ and CD8+ subsets were isolated from peripheral blood and stimulated, after which conditioned media (CM) were obtained. CM was added to human lung fibroblasts in three-dimensional collagen gels and the area of gels was measured daily. Hydroxyproline was determined as a measure of collagen degradation in the gels. Matrix metalloproteinase (MMP) activity in the culture media was analysed by gelatine zymography. Cytokine secretion of stimulated CD4+ and CD8+ T cells was analysed. CD3+ CM augmented collagen gel contraction in a time- and dose-dependent manner (P < 0.0001). CD4+ T cell CM was more potent than CD8+ T cell CM (P < 0.001). CD3+ CM and CD4+ T cell CM, but not CD8+ T cell CM, stimulated fibroblast-mediated collagen degradation and MMP-9 activity. A broad-spectrum MMP-inhibitor added to the culture system inhibited both gel contraction and MMP activity. Activated CD4+ T cells secreted significantly more tumour necrosis factor (TNF) and interleukin (IL)-6 compared to CD8+ T cells. CD3+ CM from patients with chronic obstructive pulmonary disease stimulated fibroblast-mediated collagen gel contraction to the same magnitude as CD3+ CM from healthy controls. In conclusion, activated CD4+ T cells can stimulate fibroblast-mediated degradation of ECM in vitro. This could be a mechanism by which activated T cells stimulate degradation of lung tissue leading to pulmonary emphysema.     

Short collagen fibers provide control of contraction and permeability in fibroblast-seeded collagen gels

Tissue engineering may allow for the reconstruction of breast, facial, skin, and other soft tissue defects in the human body. Cell-seeded collagen gels are a logical choice for creating soft tissues because they are biodegradable, mimic the natural tissue, and provide a three-dimensional environment for the cells. The main drawback associated with this approach, however, is the subsequent contraction of the gel by the constituent cells, which severely reduces permeability, initiates apoptosis, and precludes control of the resulting shape and size of the construct. In this study, type I collagen gels were seeded with fibroblasts and cast either with or without the addition of short collagen fibers. Gel contraction was monitored and permeability was assessed after 7 and 14 days in culture. The addition of short collagen fibers both significantly limited contraction and increased permeability of fibroblast-seeded collagen gels. The addition of short collagen fibers had no detrimental effect on cell proliferation, and there were a high number of viable fibroblasts in gels with fibers and gels without fibers. Gels containing short collagen fibers demonstrated permeabilities that were 100 to 1000 times greater than controls and also closely maintained their casting dimensions (never less than 96% of original). By limiting contraction and maintaining permeability, the incorporation of short collagen fibers should enable the creation of larger constructs by allowing for greater nutrient diffusion, and permit the creation of more complicated shapes during gel casting.     

Heterogeneity in dermatosparaxis is shown by contraction of collagen gels

Dermal fibroblasts from sheep exhibiting a mild form of dermatosparaxis were able to contract reconstituted, fibrillar collagen gels at the same rate as control dermal fibroblasts, indicating a normal interaction between the cells and a collagenous matrix. An extract from dermatosparactic skin was shown, after partial purification, to have N-proteinase activity, although the level of activity was much lower than found in normal skin. These data show that dermatosparaxis is a heterogeneous disease, since in the severe forms of the disease the defect has been characterized as an absence of N-proteinase and an inability of the cells to interact with and contract collagen gels.     

The mechanisms of fibroblast-mediated compaction of collagen gels and the mechanical niche around individual fibroblasts

Fibroblast-mediated compaction of collagen gels attracts extensive attention in studies of wound healing, cellular fate processes, and regenerative medicine. However, the underlying mechanism and the cellular mechanical niche still remain obscure. This study examines the mechanical behaviour of collagen fibrils during the process of compaction from an alternative perspective on the primary mechanical interaction, providing a new viewpoint on the behaviour of populated fibroblasts. We classify the collagen fibrils into three types – bent, stretched, and adherent – and deduce the respective equations governing the mechanical behaviour of each type; in particular, from a putative principle based on the stationary state of the instantaneous Hamiltonian of the mechanotransduction system, we originally quantify the stretching force exerted on each stretched fibrils. Via careful verification of a structural elementary model based on this classification, we demonstrate a clear physical picture of the compaction process, quantitatively elucidate the panorama of the micro mechanical niche and reveal an intrinsic biphasic relationship between cellular traction force and matrix elasticity. Our results also infer the underlying mechanism of tensional homoeostasis and stress shielding of fibroblasts. With this study, and sequel investigations on the putative principle proposed herein, we anticipate a refocus of the research on cellular mechanobiology, in vitro and in vivo.     

Reactive nitrogen species augment fibroblast-mediated collagen gel contraction, mediator production, and chemotaxis

Reactive nitrogen species (RNS) such as peroxynitrite cause cellular injury and tissue inflammation. Excessive production of nitrotyrosine, which is a footprint of RNS, has been observed in the airways of patients with asthma and chronic obstructive pulmonary disease, disorders characterized by tissue remodeling. The aim of this study was to evaluate whether RNS can affect tissue remodeling through direct effects on fibroblasts, and to determine if these effects depend on production of transforming growth factor-beta (TGF-beta). To accomplish this, human fetal lung fibroblasts (HFL-1) were used to assess fibroblast-mediated contraction of floating gels and chemotaxis toward fibronectin. In addition, the ability of fibroblasts to release TGF-beta1, fibronectin, and vascular endothelial growth factor (VEGF) was assessed by enzyme-linked immunosorbent assay. Authentic peroxynitrite significantly augmented gel contraction (P < 0.01) and chemotaxis (P < 0.01) compared with control in a concentration-dependent manner. Similarly, the peroxynitrite donor 3-morpholynosidenonimine hydrochloride (SIN-1) also augmented gel contraction (P < 0.01). RNS also significantly increased TGF-beta1 (P < 0.01), fibronectin (P < 0.01), and VEGF (P < 0.01) release into the media in both 3D gel and monolayer culture. Anti-TGF-beta antibody reversed RNS-augmented gel contraction (P < 0.01) and mediator production (P < 0.01). Anti-TGF-beta antibody also partially, but significantly, reversed RNS-augmented chemotaxis toward fibronectin (P < 0.01). Finally, peroxynitrite enhanced expression of alpha5beta1 integrin, which is a receptor for fibronectin (P < 0.01), and neutralizing anti-TGF-beta antibody suppressed peroxynitrite-augmented alpha5beta1 expression (P < 0.01). These results suggest that RNS can affect the tissue repair process by modulating TGF-beta1.     

弹力蛋白酶和基质金属蛋白酶在肺组织破坏中协同作用的实验研究

目的：采用肺成纤维细胞三维立体培养技术，直接观察弹力蛋白酶和基质金属蛋白酶对肺的主要成分之一胶原组织的降解作用。方法： 用明胶酶谱法和Western blotting法测定MMP-1、2、 3、9和TIMP-1和2 (金属蛋白酶抑制因子), 同时测定胶原含量。结果： TNF-α和IL-1β诱导培养在立体胶原内的肺成纤维细胞产生MMP-1、3和9, 但是只引起少量胶原降解（2.8%±1.5%, P＞0.05），同时加入中性粒细胞弹力蛋白酶(NE), 结果导致胶原完全降解（Ｐ＜0.01）。NE 使MMP-1、2、3和9由非活化的形式转化为较小分子量的活化分子，同时，清除TIMP-1和2。胶原降解后基质发生强烈收缩。结论： 基质金属蛋白酶可直接降解肺内胶原和其它间质组织，导致肺组织破坏，中性粒细胞弹力蛋白酶通过活化基质金属蛋白酶引起或加速细胞外基质的破坏。基质金属蛋白酶与弹力蛋白酶的协同作用可能是肺气肿等肺组织破坏的机理。     

Factors related to contraction and mechanical strength of collagen gels seeded with canine endotenon cells

Fibroblasts can construct a hydrated collagen lattice to a tissue-like structure that is greatly influenced by initial culture conditions. The purpose of this study was to investigate the effects of cell concentration and collagen concentration on the contraction kinetics and mechanical properties of resultant endotenon-derived fibroblast-seeded collagen lattice. The experiment was designed to evaluate the effect of cell concentration (0, 0.25, 0.5, and 1.0 x10(6) cells/mL) and collagen concentration (0.5, 1.0, 1.5, and 2.0 mg/mL). Collagen lattice contraction was recorded for 42 days, after which time the lattices were mechanically tested. The collagen lattices seeded with higher initial cell concentration had a shorter contraction lag phase (p < 0.01), and exhibited a higher ultimate stress (p < 0.01) and instantaneous and equilibrium modulus (p < 0.01) than those seeded with a lower initial cell concentration. The collagen lattices cultured with a lower initial collagen concentration also had a shorter contraction lag phase, and exhibited greater instantaneous and equilibrium modulus (p < 0.01) than those cultured with higher initial collagen concentration. The collagen lattices of initial 0.5 mg/mL collagen concentration had the highest value of ultimate stress (p < 0.03).     

Red blood cells stimulate human lung fibroblasts to secrete interleukin-8

Following lung injury, red blood cells (RBC) may interact with extracellular matrix (ECM). Fibroblasts, the resident cell in the ECM, have the capacity to produce and secrete a variety of mediators including interleukin-8 (IL-8). In the present study we hypothesized that RBC, or soluble factors released from them, may stimulate IL-8 production by fibroblasts. Fibroblasts were cultured in a three-dimensional collagen gel culture system in the presence or absence of RBC or conditioned medium from RBC (RBC-CM). IL-8 release from fibroblasts was significantly increased when cultured with RBC or RBC-CM and both tumor necrosis factor-alpha (TNF-) and interleukin-1 (IL-1) further stimulated this IL-8 secretion. The enhanced production of IL-8 within fibroblasts was accompanied by increased IL-8 mRNA expression. To evaluate whether RBC-fibroblast interaction may lead to recruitment of neutrophils, a functional migration assay was performed. RBC and RBC-CM, in the presence of IL-1 and TNF-, increased the transmigration of neutrophils. Our results indicate that RBC, when interacting with ECM, may participate in the recruitment of inflammatory cells by stimulating fibroblasts to secrete IL-8. This might be an important mechanism regulating tissue repair after injury.     

Red blood cells inhibit proliferation and stimulate apoptosis in human lung fibroblasts in vitro

Cell proliferation and apoptosis are both important mechanisms for the regulation of tissue homeostasis. For instance, proliferation is crucial in wound repair, whereas apoptosis is important for removal of damaged cells and resolution of inflammation. Imbalance between cell proliferation and apoptosis can therefore lead to pathological conditions and disease. In inflammatory and fibrotic lung disorders, red blood cells (RBCs) can interact with fibroblasts and connective tissue. In the present study, we therefore hypothesized that the presence of RBCs can affect fibroblast proliferation and apoptosis. Human foetal lung fibroblasts (HFL-1) were cultured in the presence or absence of purified whole RBCs and RBC-conditioned media. RBC significantly decreased fibroblast proliferation as determined both by DNA content analysis (Hoechst 33258 staining, P < 0.01; WST-1, P < 0.001) and BrdU incorporation. After treatment with staurosporine (STS) for 48 h, apoptosis was determined by TUNEL and propidium iodide staining followed by flow cytometry analysis. RBCs augmented STS-induced apoptosis (median: 46.4%; range 12.0-90.4) compared to control cells (median 26.2%; range 7.1-45.5). Thus, our data indicate that the presence of RBCs affects both fibroblast proliferation and susceptibility to undergo apoptosis. Our findings therefore suggest a role for RBCs in regulating fibroblast homeostasis after tissue injury.     

Prostacyclin analogs inhibit fibroblast contraction of collagen gels through the cAMP-PKA pathway

Prostacyclin is an arachidonic acid metabolite that modulates vascular tone within the lung. The current study evaluated the hypothesis that prostacyclin can also modulate tissue remodeling by affecting fibroblast-mediated contraction of extracellular matrix. To accomplish this, fibroblasts were cultured in three-dimensional native type I collagen gels in the presence of prostacyclin analogs: carbaprostacyclin, iloprost, and beraprost. All three analogs significantly inhibited contraction of the three-dimensional collagen gels mediated by three different fibroblasts. All three analogs significantly inhibited fibronectin release and reduced fibroblast fibronectin mRNA expression. Addition of exogenous fibronectin restored the contractile activity to fibroblasts incubated in the presence of all three analogs. Iloprost and beraprost significantly activated cAMP-dependent protein kinase-A (PKA), and an action through this pathway was confirmed by blockade of the inhibitory effect on contraction and fibronectin release with the PKA inhibitor KT-5720. In contrast, carbaprostacyclin, which is not as selective for the prostacyclin (IP) receptor, did not activate PKA, and its effects on contraction and fibronectin release were not fully blocked by KT-5720. Finally, the cAMP analogs N(6)-Benzoyl- (6-Bnz-) cAMP and dibutyryl-cAMP inhibited contraction, and this contrasted with the activity of an Epac selective agonist 8-pCPT-2'-O-Me-cAMP, which had no effect. Taken together, these results indicate that prostacyclin, acting through the IP receptor and by activating PKA, can lead to inhibition of fibronectin release and can subsequently inhibit fibroblast-mediated collagen gel contraction. The ability of prostacyclin to modulate fibroblast function suggests that prostacyclin can contribute to tissue remodeling.     

PDE4 inhibitors attenuate fibroblast chemotaxis and contraction of native collagen gels

Therapies that mitigate the fibrotic process may be able to slow progressive loss of function in many lung diseases. Because cyclic adenosine monophosphate is known to regulate fibroblasts, the current study was designed to evaluate the activity of selective phosphodiesterase (PDE) inhibitors on two in vitro fibroblast responses: chemotaxis and contraction of three-dimensional collagen gels. Selective PDE4 inhibitors, rolipram and cilomilast, each inhibited the chemotaxis of human fetal lung fibroblasts (HFL-1) toward fibronectin in the blindwell assay system (control: 100% versus cilomilast [10 microM]: 40.5 +/- 7.3% versus rolipram: [10 microM] 32.1 +/- 2.7% cells/5 high-power fields; P < 0.05, both comparisons). These PDE4 inhibitors also inhibited contraction of three-dimensional collagen gels (control: 100% versus cilomilast: 167.7 +/- 6.9% versus rolipram: 129.9 +/- 1.9% of initial size; P < 0.05, both comparisons). Amrinone, a PDE3 inhibitor, and zaprinast, a PDE5 inhibitor, had no effect in either system. Prostaglandin E(2) (PGE(2)) inhibited both chemotaxis and gel contraction, and the PDE4 inhibitors shifted the PGE(2) concentration-dependence curve to the left in both systems. The inhibition of endogenous PGE(2) production by indomethacin diminished the effects of the PDE4 inhibitors in both chemotaxis and gel contraction, consistent with the concept that the PDE4 inhibitory effects on fibroblasts are related to the presence of cyclic adenosine monophosphate in the cells. In summary, these in vitro results suggest that PDE4 inhibitors may be able to suppress fibroblast activity and, thus, have the potential to block the development of progressive fibrosis.     

Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis

BACKGROUND: Mast cells, the key cells of immediate hypersensitivity type reactions, have also been postulated to have a central role in influencing tissue remodelling and fibrosis occurring in the skin. OBJECTIVE: Our aim was to investigate the direct role of human mast cells (HMC) in skin fibrotic processes, by assessing the effects of the addition of the human mast cell line HMC-1 to human skin fibroblasts, and to identify the responsible mediators. METHODS: HMC-1 sonicates were added to human skin fibroblasts and the following parameters were evaluated: proliferation ([3H]-thymidine), collagen synthesis ([3H] proline), activity of matrix metalloproteinases (MMPs) (zymography) and tissue inhibitors of metalloproteinases (TIMPs) (reverse zymography), and collagen gel contraction. RESULTS: HMC-1 sonicate increased significantly both proliferation and collagen production in the human skin fibroblasts and these properties were not affected by heating of the sonicate (56 degrees C, 30 min, or 100 degrees C, 3 min). Two main mast cell mediators, histamine and tryptase, were found to be responsible for the increase in fibroblast proliferation and collagen production. HMC-1 sonicate did not display any pre-formed gelatinase activity, and its addition to the fibroblasts did not change their pro-MMP-2 and MMP-2 activity. On the other hand, HMC-1 were found to possess TIMP-1 and TIMP-2. Addition of HMC-1 had no effect on fibroblasts TIMP-1 but induced a dose-dependent increase of TIMP-2 activity. In addition, HMC-1 sonicate seeded together with the fibroblasts in tri-dimensional collagen gel significantly enhanced their contraction. CONCLUSION: We have shown that human mast cells, by granule-stored and therefore quickly releasable mediators, increase human skin fibroblast proliferation, collagen synthesis, TIMP-2 and collagen gel contraction. Therefore, mast cells have a direct and potentiating role in skin remodelling and fibrosis.     

Differential effects of exogenous and endogenous hyaluronan on contraction and strength of collagen gels

The addition of exogenous hyaluronan to biomaterial scaffolds has been an important area of investigation for many decades. The ability to manipulate endogenous production of hyaluronan via the hyaluronan syntheses has offered another mechanism to study the effect of hyaluronan. While the literature suggests that exogenously added hyaluronan and endogenously produced hyaluronan will have varying impacts on extracellular matrix organization and function, no studies have directly shown this phenomenon. In this investigation, we demonstrate that the addition of exogenous high molecular weight (～1 MDa) hyaluronan and hyaluronan oligosaccharides have a distinct impact on both contraction and strength of smooth muscle cell-seeded collagen gels when compared to the effects of hyaluronan that is endogenously produced by the hyaluronan synthases. More specifically, the addition of exogenous high molecular weight hyaluronan resulted in more compact collagen gels with a higher ultimate tensile strength, whereas the endogenous overproduction of hyaluronan resulted in the opposite effect. We suggest that the addition of exogenous HA to collagen gels represents a model for the therapeutic administration of HA, whereas the addition of excess HA to a tissue via the endogenous overexpression of has represents a model for the pathological accumulation of HA.     

Invasion of collagen gels by mouse lymphoid cells.

Small mouse lymphocytes from lymph nodes rapidly invaded three-dimensional collagen gels (in the absence of any added chemical attractant). In short-term assays (2-8 hr) this property was restricted to 20-25% of the cell population. Invasion was an active process involving cell locomotion. Time-lapse cinematography revealed that movement was erratic with frequent changes in cell speed. Tracks of cell paths within collagen gels demonstrated that lymphocytes made narrow angles of turn and thus showed a 'persistent random-walk' similar to other cell types moving on plane substrata. Analysis of lymphocyte movement within aligned collagen gels demonstrated that locomotion was biased in the axis of fibre alignment, i.e. lymphocytes showed contact guidance. Separated B lymphocytes invaded collagen gels at a slower rate than unseparated lymph node cells, as also did T cells purified by filtration through nylon wool columns. This latter anomaly implied that nylon wool filtration selectively depleted cells with invasive characteristics from a heterogeneous lymphocyte population. A comparison of Peyer's patch and lymph node lymphocytes showed that both populations invaded at the same rate but the latter cell type did this in greater numbers. This difference may reflect the different proportions of B and T lymphocytes in the two tissues. Lymphocytes from oxazolone-stimulated lymph nodes showed greatly increased movement into collagen matrices compared to unstimulated control lymph node lymphocytes. This increase was demonstrated to be a property of the blast cell population by separating the cells on Percoll gradients into lymphoblast-enriched and -depleted populations.     

Three-dimensional culture of rat exocrine pancreatic cells using collagen gels

Rat pancreatic cells were dissociated using a combined enzyme and EDTA method, grown on a plastic surface and then overlayed with collagen gel. Our studies have shown that exocrine pancreatic cells grown in this way have the ability to rearrange themselves into a three-dimensional organoid structure in which well defined epithelial lumina have been identified by ultrastructural and light microscopic examination. This in vitro system has advantages in examining the cytodifferentiation of pancreatic cells and may be exploited in studying pancreatic carcinogenesis.     

外周血单个核细胞与HepG2.215细胞共培养体系的研究

目的建立外周血单个核细胞与HepG2.215细胞共培养体系,探讨不同培养基及效靶比对共培养体系的影响。方法分离正常人外周血单个核细胞,加入植物血凝素（PHA）,置于不同的培养基（DMEM、RPMI1640）中进行培养,采用不同效靶比（5∶1、10∶1、20∶1、40∶1）构建PBMCs与HepG2.215细胞共培养体系。用倒置显微镜观察细胞的形态及生长情况,台盼蓝拒染法检测PBMCs与HepG2.215细胞的细胞活力,cck-8法检测HepG2.215细胞的增殖活性。结果 DMEM培养基培养的HepG2.215细胞的细胞活力比RPMI1640培养基培养的细胞高;而两种培养基培养的PBMCs的细胞活力无明显变化。共培养条件下,PBMCs对HepG2.215细胞增殖活性的抑制作用随效靶比的不同而有所差别,效靶比为20∶1时抑制作用最强。结论在PBMCs与HepG2.215细胞共培养体系中,细胞培养基和效靶比对HepG2.215细胞的生长有影响。     

Three-dimensional culture of rat exocrine pancreatic cells using collagen gels.

Rat pancreatic cells were dissociated using a combined enzyme and EDTA method, grown on a plastic surface and then overlayed with collagen gel. Our studies have shown that exocrine pancreatic cells grown in this way have the ability to rearrange themselves into a three-dimensional organoid structure in which well defined epithelial lumina have been identified by ultrastructural and light microscopic examination. This in vitro system has advantages in examining the cytodifferentiation of pancreatic cells and may be exploited in studying pancreatic carcinogenesis.     

Morphogenesis of precursor subpopulations of chicken limb mesenchyme in three dimensional collagen gel culture

Although homogeneous in appearance, several lines of evidence suggest early (stage 17-19) limb mesenchymal cells are committed to particular cell lineages, e.g., myogenic or chondrogenic. However, subsequent expression of cell or tissue phenotype in the developing limb does not occur in a randomized process but rather in a spatially specific pattern. The potential regulatory mechanisms controlling the "patterned" expression of tissue phenotype in the limb have not been resolved. The purpose of this study was to determine if, prior to the formation of an apical ectodermal ridge, nondissociated limb mesenchyme has inherent morphogenetic potential to form nonrandomized patterns of tissue organization. The hypotheses to be tested were that, if provided a spatially permissive culture environment, 1) mesenchymal cells committed to a particular lineage would segregate into precursor (sub)populations prior to overt expression of phenotype and 2) the ultimate expression of a tissue phenotype may be regulated, in part, by histogenic interactions between the precursor cell groups. For these studies, mesoblasts (intact mesenchyme minus ectoderm) from stage 17-19 hindlimb buds were explanted intact to the surface of a 1-3 mm thick hydrated lattice of repolymerized type I collagen and incubated for 2-11 days. Examination of cultures at variable intervals revealed three distinct temporal sequences (periods) which were arbitrarily termed early morphogenesis (0-3 days), cytodifferentiation (3-5.5 days), and primitive tissue formation (5.5-11 days) based on similarities to in situ limb development. By the end of the first period, the mesenchymal cells had sorted into three distinct precursor populations: 1) an epithelial-like outgrowth of premyogenic and prefibrogenic cells at the surface of the gel lattice (termed the "surface subset") which circumscribed, 2) a centrally positioned prechondrogenic condensate ("central subset"), and overlaid 3) a dispersed, population of free cells that invaded the collagen lattice ("seeded subset"). Subsequent cytodifferentiation led to the appearance of multinucleated myotubes within the surface subset and chondrification of the central subset. Cells of the seeded subset remained dispersed within the collagen lattice. Primitive histogenic events were initiated during the final period of development including 1) at sites where surface cells established boundaries with the central subset, collectives or "bundles" of variable sized myotubes were formed which became partially ensheathed by the attenuated processes of fibroblastlike cells; and 2) a secondary site of chondrogenic activity was initiated within the gel lattice at the boundary between the central and seeded cell populations. Transformation of seeded fibroblasts into chondroblasts accompanied expansion of the secondary chondrogenic element within the gel lattice.(ABSTRACT TRUNCATED AT 400 WORDS)