Induction of fibroblast chemotaxis by fibronectin. Localization of the chemotactic region to a 140,000-molecular weight non-gelatin-binding fragment.

Plasma and cell-derived fibronectin are potent chemoattractants for human dermal fibroblasts in vitro. The chemotactic property of fibronectin resides in a major 140,000-mol wt non-gelatin-binding fragment of the native molecule. Human monocytes and neutrophils do not recognize fibronectin as a chemotactic stimulus. These findings suggest that fibronectin and perhaps certain fragments of fibronectin may function in vivo as a specific chemoattractant for fibroblasts and could, therefore, induce directional migration of fibroblasts to sites of tissue injury, remodeling or morphogenesis.

Subclass restriction of murine antibodies. III. Antigens that stimulate IgG3 in mice stimulate IgG2c in rats.

The IgG subclass distribution of rat antibodies to 13 different antigens was measured. Antibodies to protein and hapten-protein conjugates were predominantly IgG2a. Antigens labeled thymus-independent type 1, based upon responses in mice, stimulated both IgG2b and IgG2c antibodies, but little IgG2a. Polysaccharide and hapten-polysaccharide antigens (thymus-independent type 2) as well as phosphocholine-keyhole limpet hemocyanin, stimulated predominantly IgG2c antibodies. A division of antigens into essentially the same categories has been made on the basis of subclass restriction in mice. Antigens that stimulate IgG2c in rats stimulate IgG3 in mice. Thus, by comparing subclass preference with a variety of antigens, functional analogues among subclasses in different species can be identified.

Subclass restriction of murine antibodies. II. The IgG plaque-forming cell response to thymus-independent type 1 and type 2 antigens in normal mice and mice expressing an X-linked immunodeficiency.

Antigens have been classified previously into three categories, thymus-dependent (TD), thymus-independent type (TI) 1, and TI-2, based upon thymic dependence and ability to stimulate an immunodeficient strain of mouse, CBA/N. Here we demonstrate that the different antigen classes elicit IgG antibodies of different subclasses. TD antigens stimulate predominantly IgG1 antibodies, with smaller amounts of IgG2 and IgG3 being expressed. TI-1 antigens stimulate almost no IgG1 antibodies and equal amounts of IgG2 and IgG3. TI-2 antigens elicit predominantly IgG3 antibodies. Mice expressing the CBA/N phenotype are known to be nonresponsive to TI-2 antigens. This was confirmed in this study. In addition, we demonstrate that the IgG3 component of the response to TI-1 antigens is virtually absent in mice expressing the CBA/N phenotype, which supports our previous finding that the CBA/N defect may be restricted to a B-lymphocyte subpopulation containing most of the precursors of IgG3-secreting cells.

Effect of heparin on synthesis of short chain collagen by chondrocytes and smooth muscle cells.

The treatment of embryonic chick chondrocyte cultures with heparin results in a decrease in collagen synthesis. One of the collagens synthesized by hypertrophic chondrocytes, specifically type X collagen, may play an important role in cartilage mineralization and endochondral ossification. Recently a new short chain collagenous component was found in cultures of rat vascular smooth muscle cells (Majack, R. A., and P. Bornstein, 1985, J. Cell Biol., 100: 613-619). The present study was initiated to investigate heparin's effect on type X collagen in embryonic chick chondrocytes and to further evaluate the nature of the short chain component synthesized by rat vascular smooth muscle cells. Different tissues may respond differently to the administration of heparin. In chondrocyte cultures heparin decreased both total collagen synthesis as well as the synthesis of type X collagen. There was an accumulation of collagen precursors, found principally in the cell layer compartment, which appeared to be the result of heparin's inhibition of the NH2-terminal protease. In cultures of rat vascular smooth muscle cells heparin was found to increase the synthesis of a short chain collagenous component as previously reported. However, comparison with a type X collagen standard showed this to be different from type X. In all cases, the effect of heparin on collagen chain precursors, chondrocyte type X synthesis, and synthesis of a vascular smooth muscle short chain collagen was shown to be reversible. Similar effects were obtained by adding chondroitin sulfate to chondrocytes, suggesting a role for extracellular matrix components in the modulation of collagen synthesis. These findings are consistent with the concept of a group of short chain collagens with type X collagen being unique to hypertrophic chondrocytes.

Amniotic fluid fibronectin. Characterization and synthesis by cells in culture.

A glycoprotein immunologically related to plasma cold-insoluble globulin (CIG) and fetal skin fibroblast fibronectin has been purified from second-trimester human amniotic fluid. This protein (amniotic fluid fibronectin) migrated more slowly than CIG on sodium dodecyl sulfate gel electrophoresis and showed greater polydispersity which could result, at least in part, from heterogeneity in glycosylation. Cloned human amniotic fluid epithelioid and fibroblastic cells synthesized and secreted a protein with similar properties into the culture medium. Fibronectin was shown to be associated with the pericellular and extracellular matrix of cultured amniotic fluid cells by immunofluorescence, lactoperoxidase-catalyzed iodination, and labeling with ferritin-conjugated antibodies. The kinetics of secretion of the protein were consistent with its role as a matrix protein. We anticipate that amniotic fluid fibronectin will prove to be the same protein which elsewhere in the body is incorporated into connective tissues and basement membranes. Amniotic fluid could, therefore, serve as a convenient source of in vivo synthesized fibronectin for biological and structural studies.

A 17-kd polypeptide, sensitive to bacterial collagenase, is synthesized by bone marrow macrophages.

A novel polypeptide with a molecular weight of 17 kd (17k protein) was identified in bone marrow cell cultures. The synthesis of 17k protein is elevated in cell cultures maintained under Dexter conditions, which support myelopoiesis. The predominance of macrophages in the stromal layer of these cultures and the observation that a mouse myelomonocytic cell line P388D1 is capable of synthesizing large amounts of 17k protein led us to the study of its synthesis by bone marrow macrophages. Metabolic labeling with [14C]proline and partial amino acid analysis of 17k protein demonstrated that the polypeptide contains relatively high amounts of proline and is also sensitive to degradation with bacterial collagenase. However, no hydroxyproline is detectable in 17k protein, and it is extensively degraded with bacterial collagenase. However, no hydroxyproline is detectable in 17k protein, and it is extensively degraded by proteolysis with pepsin, using conditions under which collagen triple helices are resistant to degradation, suggesting that collagen-like structures are not contained in 17k protein. This polypeptide is found predominantly in the cellular layers of bone marrow macrophage cultures. Incorporation of [14C]proline into 17k protein is diminished by increasing concentrations of colony-stimulating factor 1 (CSF-1). The 17k protein may be involved in macrophage proliferation because its synthesis is inhibited by CSF-1, which is required for the maintenance of bone marrow macrophages in vitro.

Distribution of fibronectin and laminin in basal cell epitheliomas.

The distribution of fibronectin (FN) and laminin (LM) in basal cell epithelioma was evaluated by indirect immunofluorescence. FN is a glycoprotein which promotes interaction between cells and the extracellular matrix, and is present at the dermal-epidermal junction (DEJ) and throughout the dermis, but absent in the normal epidermis. LM, a noncollagenous basement membrane protein, plays a role in epithelial adhesion to type IV collagen, and is normally present in the DEJ, but absent in the epidermis. The role of FN and LM in epithelial differentiation has not been established. Therefore, the distribution of FN and LM in a tumor of epithelial origin was studied by indirect immunofluorescence. Using affinity-purified antibodies to FN and LM, and the appropriate fluorescein-conjugated second antibodies, normal skin and 7 basal cell tumors were examined. By immunofluorescence, nests of malignant basal cells were surrounded by linear LM staining. FN immunofluorescence was intense throughout the connective tissue stroma of all tumors. Five tumors also showed FN staining within the nests of neoplastic cells, and 4 of these were also LM-positive in the tumor bulk. These immunofluorescent findings suggest that epidermal neoplasia can be associated with alterations in the distribution of FN and LM.

Organization of extracellular proteins on the connective tissue cell surface: relevance to cell-matrix interactions in vitro and in vivo.

A model has been developed that proposes a cell surface-associated protein meshwork, composed in part of fibronectin and collagen, for a connective tissue cell attached to a substratum. In support of this model are the observations that collagen and fibronectin interact and that these proteins are similarly distributed on the fibroblast cell surface. We suggest that this external meshwork interacts directly or indirectly with the internal cytoskeleton and with the extracellular matrix and thereby mediates several cellular properties, including adhesion, shape, and motility. Loss of cell surface fibronectin as a result of viral transformation, or due to treatment of normal cells with tunicamycin, an inhibitor of protein glycosylation, may contribute to the reduced adhesion and altered morphology observed in these circumstances. We therefore predict that the changes in these properties observed with virally transformed cells, mitotic cells, and cells treated with proteolytic enzymes are related to alterations in the external protein meshwork.

Intramedullary bone repair and ingrowth into porous coated implants in the adult chicken: a histologic study and biochemical analysis of collagens.

A new model was developed to study the histologic and biochemical events during intramedullary bone repair and ingrowth into porous coated implants. Adult chickens were used because of the availability of specific antibody probes. Repair in the metaphysis and diaphysis were compared. Entering through a medial arthrotomy, the distal tibiotarsus was reamed and either implanted with a double-ended porous coated rod or allowed to heal without implantation of a rod. Specimens analyzed histologically at 7, 14, 21, and 70 days postoperatively revealed direct formation of bone by osteoblasts with no evidence of a cartilaginous phase. At 70 days bony ingrowth was observed deep within the porous surface. Analysis of collagens with sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the synthesis of type I collagen predominated. Biosynthetic data coupled with quantitative immunologic analyses using antibodies to type II and type X collagen showed that neither of these two collagen types, which are characteristic of cartilage undergoing endochondral ossification, were produced during intramedullary bone repair. These results establish that the adult chicken is capable of bony ingrowth into porous coated implants and that this process is through direct bone deposition by osteoblasts without a cartilaginous intermediate.

Biosynthesis and processing of collagens in different cartilaginous tissues.

The distribution, structure, and biosynthesis of various collagen types have been studied in growth and structural cartilage from young rabbits. The major collagen of cartilage is alpha 1(II); however, all cartilage matrix also contains 1 alpha, 2 alpha, 3 alpha (Type Cm), as well as a high molecular weight disulfide-linked collagen (Type M). Cartilage fragments in organ culture demonstrate synthesis of precursors of collagen alpha chains and processing to their final forms. Although Type Cm collagen is present in the same proportion in the matrix of growth and structural cartilage, in vitro radiolabeling of rabbit cartilage showed that only growth cartilage is capable of actively synthesizing Type Cm, except in the newborn period when synthesis of Type Cm does occur in structural cartilage. A low molecular weight collagen (designated G collagen) is synthesized in vitro by growth cartilage but not by structural or articular cartilage. Preferential distribution of these minor collagens in growth cartilage suggests a role in development during normal cartilage growth.

Abnormality of cartilage collagen in a patient with unclassified chondrodystrophy.

We have described a previously unrecognized chondrodystrophy characterized by short-limbed dwarfism, blue sclera, severe cardiopulmonary problems, and failure of postnatal growth. The first of two siblings thus affected died at age 6 months following attempted correction of an atrial septal defect. Growth plate cartilage from multiple sites obtained at autopsy showed a marked abnormality of architecture on the light microscopic level. Biochemical studies demonstrated an absence of normal alpha 1(II) collagen in costochondral junction growth plate cartilage and an appearance of the major collagen in a band which comigrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 3 alpha collagen. Cartilage extracted from structural rib appeared to be normal.

Changes in the expression of type-X collagen in the fibrocartilage of rat Achilles tendon attachment during development.

This study histologically and immunohistochemically demonstrated developmental changes in cell morphology and expression of type-X collagen in the attachment of the Achilles tendon to the calcaneus in the rat. Although the site of attachment in the mature rat showed a well organized, direct insertion that was composed of tendon, fibrocartilage, calcified fibrocartilage, and bone, this four-zone structure was not observed in the immature 1-week-old rat. Formation of fibrocartilage was observed at 2 weeks, together with the hypertrophy of chondrocytes and the appearance of the secondary center of ossification. Type-X collagen was not detected either in chondrocytes in the attachment area at 1 week or in hypertrophic chondrocytes at the attachment at 2 weeks. In the 3-week-old rat, the secondary center of ossification extended to the area of attachment and type-X collagen was detected both in cartilage spicules within the secondary center of ossification and in cells found at the attachment adjacent to the secondary center of ossification. A four-zone structure had been established by 6 weeks and remained through 20 weeks. After 6 weeks, type-X collagen was identified both in the attachment of the tendon and beneath the calcaneal fibrocartilage. Type-X collagen is produced by cells in transitional zones between calcified and noncalcified tissue, such as the interface between articular cartilage and subchondral bone. In these areas, the expression of this protein persists through maturity and is not transient.

The use of demineralized bone matrix in the repair of segmental defects. Augmentation with extracted matrix proteins and a comparison with autologous grafts.

A soluble protein component of bone, bone morphogenetic protein, and decalcified bone matrix have been shown to induce the formation of bone in extraosseous tissue. Clinical and animal studies investigating the use of these materials as bone grafts have shown radiographic and histological evidence of formation of bone, but the clinical usefulness of these grafts remains unknown. This study compared the healing processes when plasma-coated demineralized bone matrix and autologous cancellous bone were used to graft segmental defects of bone. A standard procedure was used to make a two-centimeter defect bilaterally in the ulna of forty-eight skeletally mature New Zealand White rabbits. In each rabbit, one ulnar defect was grafted with autologous citrated plasma-coated demineralized bone matrix while the other defect served as a control and was grafted with either autologous cancellous bone from the iliac crest, demineralized bone matrix, or demineralized bone matrix augmented with bone proteins that had been extracted with guanidinium hydrochloride. The ulnar defect was stabilized by the intact radius, and no supplemental device was necessary for fixation. To examine spontaneous healing in this model, one group of rabbits had a control defect that was not grafted. The grafts were periodically evaluated by radiographs, and twelve weeks after surgery the grafts were harvested and tested to failure in a standard torsion-test machine. The mechanical parameters were calculated, and histological examination of major fragments of the grafts was performed. The results of the radiographic and histological evaluation showed that all of the grafted ulnae healed, with fusion of the graft to the cut ends of the defect and reformation of approximately normal anatomy. No ungrafted ulnar defects healed. The results from the mechanical tests were evaluated by comparing the defect that was grafted with plasma-coated demineralized bone matrix with the control graft in each animal. These data showed that: twelve weeks after grafting, the normal ulnae were significantly stronger than the ulnae that had been grafted with plasma-coated demineralized bone matrix; the ulnae that had been grafted with plasma-coated demineralized bone matrix and those that had been grafted with autologous bone were equivalent in strength; and twelve weeks after grafting, grafts of demineralized bone matrix that were augmented with extracted bone proteins were significantly stronger than those that had not been so augmented.

Steroid-induced adipogenesis in a pluripotential cell line from bone marrow.

UNLABELLED: We studied the effect of steroids on the differentiation of a pluripotential mesenchymal cell with use of a cell line (D1) from mouse bone-marrow stroma. The cells were treated with increasing (10[-9], 10(-8), and 10(-7)-molar) concentrations of dexamethasone for increasing durations ranging from forty-eight hours to twenty-one days. The appearance of triglyceride vesicles in the cells indicated that this treatment had induced the differentiation of the cell into adipocytes. The number of cells that contained the triglyceride vesicles and the expression of a fat-cell-specific gene, 422(aP2), increased with longer durations of exposure to dexamethasone and with higher concentrations of the steroid. Treatment with dexamethasone also diminished the expression of alpha1 type-I collagen mRNA and osteocalcin mRNA. The data indicate that dexamethasone stimulates the differentiation of cells in bone-marrow stroma into adipocytes as well as the accumulation of fat in the marrow at the expense of expression of type-I collagen and osteocalcin mRNA, thereby suppressing differentiation into osteoblasts. CLINICAL RELEVANCE: Steroid-induced adipogenesis by bone progenitor cells in marrow may influence the development of osteonecrosis. It is therefore important to consider the investigation of a treatment, such as the inhibition of the metabolism and accumulation of fat in marrow, that can prevent the onset of osteonecrosis.

Characterization of tissue from the bone-polymethylmethacrylate interface in a rat experimental model. Demonstration of collagen-degrading activity and bone-resorbing potential.

In previous studies, we described a layer of tissue that formed around methylmethacrylate cement that had been implanted into the posterior cervical spine of dogs. We are now reporting on a rat model in which we induced, in the interface between the bone of the posterior elements of the dorsal spine and methylmethacrylate, the formation of a layer of tissue that was morphologically similar to the tissue that had been produced in the dogs. As in the dogs, we noted macrophages and giant cells and we demonstrated that the interface tissue synthesized several basement-membrane components (type-IV collagen, laminin, and fibronectin). In addition, we demonstrated the synthesis of an additional extracellular-matrix protein--type-VI collagen. We also showed that extracts of organ cultures of tissue from the rat model degraded type-I collagen into three-quarter and one-quarter-length fragments. Such enzymatic activity is characterized of mammalian collagenase, an enzyme that is known to play a critical role in the resorption of bone.

Two cell lines from bone marrow that differ in terms of collagen synthesis, osteogenic characteristics, and matrix mineralization.

Two cloned cell lines were isolated from cultures of mouse bone-marrow cells. One of the lines, D1, exhibited osteogenic properties and synthesized type-I collagen (alpha 1)2 alpha 2. The second cell line, D2, was not osteogenic and produced a collagen homotrimer (alpha 1)3. Whereas the extracellular matrix of the D1 cell cultures contained striated collagen fibrils, presumably composed of type-I collagen, the homotrimer-producing D2 cells did not demonstrate striated collagen fibrils. Instead, they had thin filaments without detectable striations. Sodium ascorbate stimulated collagen synthesis at the transcriptional level in both the D1 and the D2 cells. The bone-producing characteristics of D1 in vitro included high levels of alkaline phosphatase, increased cyclic adenosine monophosphate on treatment with parathyroid hormone, and expression of osteocalcin mRNA. The D1 cells, unlike the D2 cells, produced a mineralized matrix in vitro. Mineralization in the cultures of the D1 cells occurred in nodules of increased cell density, which also contained the cells with the highest concentrations of collagen mRNA, as shown by in situ hybridization. When the D1 cells were implanted in a diffusion chamber in vivo, a mixture of both osteogenic and adipogenic tissues was formed. This indicates that the D1 cell line is derived from an early marrow stromal precursor that is multipotential.

Induction and characterization of an interface tissue by implantation of methylmethacrylate cement into the posterior part of the cervical spine of the dog.

After the implantation of methylmethacrylate cement into the posterior part of the cervical spine of the dog, a thick layer of connective tissue forms at the bone-cement interface. The tissue is six to eight millimeters thick and in all animals it surrounds the dorsal and lateral aspects of the masses of implanted cement, grows between the undersurface of the cement and the bone of the posterior elements, and completely covers that bone. This tissue was examined by light and electron microscopy and its collagenous components were extracted and analyzed biochemically by gel electrophoresis. Specific extracellular matrix proteins in the tissue at the bone-cement interface were also localized by immunohistochemistry. The tissue at the host-cement interface contained zones of fibrocytes and plump and teardrop-shaped cells within a collagenous matrix. Type-I, Type-III, and Type-V collagen were extracted and were identified by gel electrophoresis. Type-V collagen and fibronectin were localized predominantly around the plump and teardrop-shaped cells. Type-IV collagen and laminin were localized predominantly in an area just beneath the teardrop-shaped cells at the surface of the tissue overlying the cement, suggesting that a basement-membrane-like tissue had formed in this area.

Comparison of lumbar spine fusion using mixed and cloned marrow cells.

STUDY DESIGN: Prospective study on lumbar spine fusion using cloned and mixed marrow cells. OBJECTIVE: To analyze the effectiveness of cloned osteoprogenitor cells in spine fusion and their differentiation in vivo using a traceable gene. SUMMARY OF BACKGROUND DATA: Although autografts are currently the standard for stable spine fusion, supply is limited. Alternative graft materials need to be developed and evaluated. METHODS: An osteoprogenitor cell, D1-BAG, cloned from mouse bone marrow and transduced with LacZ and neomycin resistance genes, and mixed marrow stromal cells from marrow blowouts were used in athymic rats to establish posterior spinal fusion; 2 x 10(6) cells in 100 microL Matrigel were implanted into the lumbar fusion bed in 36 animals, whereas Matrigel without cells was used in 16 animals as control. Rats were killed at 2, 3, 6, and 9 weeks, and the spines were evaluated by manual palpation, radiographs, and histology. RESULTS: Two weeks after surgery radiopaque tissue was seen at transplantation sites with D1-BAG cells but not at sites with mixed marrow stromal cells. Successful spine fusion at 6 and 9 weeks was observed in 8 of 8 (100%) animals receiving DI-BAG cells, 4 of 8 (50%) in mixed marrow stromal cells, and 0 of 8 (0%) in control animals. CONCLUSIONS: Compared with mixed marrow stromal cells, cloned osteoprogenitor cells can produce a larger amount of mature osseous tissue at an earlier time point during spine fusion.

T-lymphocyte activation by immunogenic determinants.

Synthetic antigens have been of great value in elucidating the relationships between antigen structure and lymphocyte activation. The compound RAT behaves as a monofunctional antigen in guinea pigs and mice, inducing T-lymphocyte responses without appreciable circulating antibody, although the ABA-specific B cell population is expanded by immunization with the monovalent molecule. On the other hand, bifunctional antigens composed of one RAT moiety serving as a carrier and a second chemical group, either identical to or different from RAT, serving as a hapten, induced antibody responses. In such responses, T cell specificity was always directed against the RAT component. Using symmetrical bifunctional antigens with rigid or flexible spacers between the two determinants, marked differences in structural requirements for cell triggering, assessed by antigen-induced lymphocyte proliferation, and for cell cooperation, determined by antibody formation, were found. Rigidly spaced bifunctional antigens serve admirably for cooperation but poorly for T cell activation, underscoring the advantage of two-point binding for the latter.

Marrow stromal cells embedded in alginate for repair of osteochondral defects.

Articular cartilage defects of sufficient size ultimately degenerate with time, leading to arthritic changes. Numerous strategies have been used to address full-thickness cartilage defects, yet none thus far has been successful in restoring the articular surface to its preinjury state. We compared the effects of agarose, alginate, and type I collagen gels on the expression of cartilage-specific markers from rabbit marrow stromal cells and then assessed the in vivo effects of cells seeded in alginate beads on the repair of full-thickness osteochondral defects in the rabbit model. Marrow aspirates from rabbits were cultured and the stromal population selected. Marrow stromal cells were then placed in either 1.2% w/v alginate, type I collagen gels (3 mg/mL), or 0.5% agarose suspension culture. After 2, 5, 10, and 20 days in culture, the RNA was extracted and analyzed by reverse transcription polymerase chain reaction for the cartilage-specific markers aggrecan and type II collagen. The strongest increase in aggrecan and type II collagen gene expression was found in the agarose suspension followed by alginate; type I collagen gels induced the lowest levels. Alginate beads were chondrogenic and maintained their size and consistency over time in culture, whereas the cell-seeded collagen gels invariably contracted. Full-thickness defects measuring 3 x 6 mm x 3 mm deep were then created in the medial femoral condyles of rabbit knees and filled with alginate beads, alginate beads seeded with stromal cells, or left empty. Alginate beads containing stromal cells remained within the defects and progressively filled the defects with regenerate tissue. Histologic analysis showed viable, phenotypically chondrogenic cells in the defects. The matrix stained positive with safranin O, indicating proteoglycan synthesis, and bonding between the regenerate and host tissue was excellent. We have shown quantitative differences in the chondrogenic effects of the biomaterials tested. Alginate induces the chondrogenic phenotype in marrow stromal cells in vitro, and possesses the necessary physical characteristics and handling properties to support cells and serve as a carrier to fill full-thickness osteochondral defects in vivo.