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Biomedical subjects

A I Caplan

Publications and source records attributed to A I Caplan.

At least 127 records · Page 7Linked to original sources
PubMed1990

Human keratinocytes contain carbohydrates that are recognized by keratan sulfate-specific monoclonal antibodies.

Monoclonal antibodies that recognize carbohydrate epitopes found in keratan sulfate glycosaminoglycan chains identified both intracytoplasmic and cell-surface carbohydrates of human keratinocytes. These carbohydrates were detected, using indirect immunoperoxidase methods, both in sections of paraffin-embedded tissues and in intact cultured keratinocytes. Of the seven anti-keratan sulfate monoclonal antibodies used in this study, five detected significant amounts of epitopes associated with keratinocytes. This indicates that only certain, specific types of keratan sulfate-like carbohydrates were expressed by these cells. The extent and localization of keratan sulfate-like carbohydrates appeared to be closely related to the differentiation status of cultured keratinocytes. These epitopes were very weakly expressed on surfaces of all monolayer keratinocytes, but flattened, suprabasal cells in high Ca++ cultures strongly expressed keratan sulfate-like carbohydrates on their surfaces. A much larger population of cultured keratinocytes expressed intracellular keratan sulfate-like carbohydrates identified by the same five antibodies that detected surface epitopes. In monolayer cells, keratan sulfate-like carbohydrates were predominantly found in a broad perinuclear zone. In addition, three of the five immunoreactive antibodies detected epitopes that appeared at cell boundaries, specifically at sites of close cell-to-cell contact. Thus, molecules bearing carbohydrates recognized by anti-keratan sulfate antibodies appear at developmentally important stages of keratinocyte differentiation, indicating that these carbohydrates may serve as markers for molecules important in the differentiation of human keratinocytes.

Antibodies, Monoclonal↗
PubMed1990

Stem cell delivery vehicle.

Bone marrow contains a rare class of stem cells capable of developing into a number of mesenchymal tissues. The delivery of these cells to appropriate skeletal sites will require uniquely compatible biomaterials. Porous calcium phosphate ceramics may provide such a vehicle for the repair of bone. The experimental basis for this proposal is discussed.

Animals↗
PubMed1990

In vivo osteochondrogenic potential of cultured cells derived from the periosteum.

Periosteal cells were isolated from young chicks, introduced into cell culture, subcultured, and then inoculated into athymic, nude mice to test the in vivo osteochondrogenic potential of cultured periosteal cells. In monolayer cultures, the adherent periosteal cells showed fibroblastlike morphology and overtly expressed neither osteogenic nor chondrogenic phenotypes. Cultured cells inoculated heterotopically into nude mice eventually gave rise to bone tissue at the subcutaneous injection site. The process of bone formation occurred through two different mechanisms: intramembranous bone formation at the peripheral portion of the inoculum early and endochondral bone formation in the central portion later. Frozen, preserved periosteal cells also formed bone after introduction into nude mice in the same temporal histologic sequence as the unfrozen cells. Cultured chick muscle fibroblasts from donors that were the same age as controls did not form bone or cartilage when inoculated under identical conditions to those of cultured periosteal cells. These results suggest that periosteum of young chicks contains subsets of progenitor cells that possess the potential to differentiate directly into osteoblasts or chondrocytes when inoculated in vivo. Importantly, this potential is retained after enzymatic isolation, cell culture, subculturing, and freeze preservation.

Animals↗
PubMed1990

Cartilage begets bone versus endochondral myelopoiesis.

Observations on the cellular events involved in embryonic long-bone formation, ectopic osteogenesis, fracture repair, and the analysis of mesenchymal stem-cell potential provide evidence of the intimate and direct relationship of osteogenesis to angiogenesis and vasculature. The sequence of cellular events is that cartilage is not replaced by bone but, rather, is a target for vascularization and myelopoiesis. With this in mind, it may be necessary to replace the term "endochondral bone formation" with "endochondral myelopoiesis."

Animals↗
PubMed1989

Ectopic induction of cartilage and bone by water-soluble proteins from bovine bone using a collagenous delivery vehicle.

A controlled-release delivery vehicle for water-soluble osteogenic proteins from demineralized bone matrix was constructed using purified type I collagen. The water-soluble proteins were isolated from a 4 M GdnHCl extract of bone matrix. Although the water-soluble proteins were capable of inducing cartilage formation in vitro, they were incapable of inducing cartilage or bone in vivo when implanted intramuscularly into mice in the absence of an appropriate delivery vehicle. The collagen-based delivery vehicle alone was also incapable of inducing osteogenesis in vivo. However, when the water-soluble proteins were incorporated into the delivery vehicle, the combination was capable of inducing cartilage and bone 76% of the time. These results demonstrate that it is possible to formulate a controlled-release delivery vehicles for soluble bioactive factors which upon release interact with local responsive cells.

Animals↗
PubMed1989

Histochemical analysis of newly synthesized and accumulated sulfated glycosaminoglycans during musculogenesis in the embryonic chick leg.

The leg musculature from 11, 14, and 17 day chick embryos was analyzed histochemically to investigate the temporal and spatial distribution of various types of sulfated glycosaminoglycans present during skeletal muscle development. Types of glycans were identified by selective degradation with specific glycosidases and nitrous acid coupled with Alcian blue staining procedures for sulfated polyanions and with [35S]sulfate autoradiography. On day 11, radiolabeled chondroitin sulfate glycosaminoglycans are localized extracellularly in both the myogenic and connective tissue cell populations. By day 17, incorporation of [35S]sulfate into chondroitin sulfate is substantially reduced, although Alcian blue-stained chondroitin sulfate molecules are still detectable. With increasing age and developmental state of the tissues, radiolabeled and stained dermatan sulfate and heparan sulfate progressively increase in relative quantity compared to chondroitin sulfate both in muscle and in associated connective tissue elements. These changes in glycosaminoglycans correlate well with similar changes previously determined biochemically and further document the alterations in extracellular matrix components during embryonic skeletal myogenesis.

Animals↗
PubMed1989

Heterotopic osteogenesis in porous ceramics induced by marrow cells.

When untreated porous calcium phosphate ceramics were transplanted into subcutaneous (s.c.) or intramuscular (i.m.) sites, fibrovascular tissue grew in the pore region without evidence of bone formation. However, when these same ceramics were combined with syngeneic marrow cells, osteogenesis was observed inside the pore region of the implanted ceramic. The osteogenesis began on the surface of the pore region at approximately 3 weeks postimplantation by a process of intramembranous bone formation, with the de novo bone tissue observed directly interfacing with the ceramic surface. Infrequently, small isolated areas showed cartilage formation with no noticeable endochondral ossification. At 4 weeks postimplantation of the ceramic with marrow cells, the osteogenesis in the ceramic accompanied an observed increase in compressive strength, rigidity, and energy absorption of the ceramic. These results suggest that a combination of porous ceramics and marrow cells may be useful for clinical problems requiring osseous reconstruction.

Animals↗
PubMed1989

Growth and differentiation of stage 24 limb mesenchyme cells in a serum-free chemically defined medium.

A serum-free defined medium which supports the differentiation of chick limb mesenchymal cells has been developed. In this medium, stage 24 embryonic limb mesenchymal cells which are plated at high density (5 x 10(6) cells/35-mm culture dish) differentiate into chondrocytes. Morphologically, these cultures appear only slightly different from those in which the cells are maintained in serum-containing medium. DNA levels and proline incorporation in cultures grown in defined medium are indistinguishable from control cultures. The rate of radiolabeled sulfate incorporation, a monitor of the rate of proteoglycan synthesis, in Day 8 high-density cultures maintained in defined medium is approximately 70-80% of control values. Additionally, growth and differentiation of intermediate-density (2 x 10(6) cells/35-mm culture dish) and low-density (1 x 10(6) cells/35-mm dish) cultures are also supported by this defined medium. The availability of this medium allows exploration of bioactive factors which affect or modulate mesenchymal cell differentiation and subsequent development.

Animals↗
PubMed1989

Altered cartilage proteoglycans synthesized by chick limb bud chondrocytes cultured in serum-free defined medium.

Chick high-density culture chondrocytes synthesize cartilage-specific proteoglycans with much structural similarity to the proteoglycans made by cartilage in vivo. Such cultures can be maintained in a defined medium formulated in this laboratory in which chondrogenesis occurs without the addition of serum. The proteoglycans synthesized by the chondrocytes in the presence of defined medium are of a cartilage-specific structure but differ in some aspects from the proteoglycans made in serum-containing medium. While their buoyant density, ability to aggregate with hyaluronic acid, and keratan sulfate chain size are unchanged, the proteoglycans synthesized in defined medium have altered chondroitin sulfate chains. This chondroitin sulfate is of significantly larger size and has a different sulfation pattern relative to that produced in serum-containing medium. The larger size of the chondroitin sulfate results in a larger monomer size of the defined medium proteoglycans. These differences have implications about the regulation of the structure of chondroitin sulfate proteoglycans.

Animals↗
PubMed1989

First bone formation and the dissection of an osteogenic lineage in the embryonic chick tibia is revealed by monoclonal antibodies against osteoblasts.

Detailed studies of the origin and differentiation of osteogenic cells can be facilitated by cell-specific markers. To this end, we immunized mice with a heterogeneous population of chick embryonic bone cells and subsequently generated and selected for monoclonal antibodies against cell surface determinants. Supernatants from growing hybridoma colonies were screened immunohistochemically against frozen sections of embryonic stage 35 (day 9.5) chick tibiae. Three cell lines, SB-1, SB-2, and SB-3, which each secrete a different antibody against osteogenic cells, have been successfully cloned, stabilized, and immortalized. Antibody SB-1 reacts with a family of cells in embryonic bone, liver, kidney, and intestine, which are identically stained by the histochemical stain for alkaline phosphatase. The SB-2 antigen is present only on osteoblasts, while the SB-3 antigen is expressed on the surface of osteoblasts, ependymal cells and ventricular myoblasts. Studies on the developmental progression of osteoblasts in the embryonic tibia indicate that the determinants recognized by SB-1, SB-2, and SB-3 are temporally coupled to the appearance of the pre-osteoblast marker alkaline phosphatase. Detailed morphologic analyses reveal that SB-1 reacts with a large family of osteogenic cells residing between the surface of newly formed bone and the overlying periosteal osteoprogenitor cells. By contrast, SB-2 and SB-3 appear to react with those mature osteoblasts involved in the secretion and mineralization of osteoid. Cells which are imprisoned within bone matrix (osteocytes) of the developing tibia are not recognized by these antibodies, but are immunostained by an osteocyte-specific monoclonal antibody which does not react with SB-1, SB-2 or SB-3 positive cells. The results reported here suggest the existence of an osteogenic cell lineage which is characterized by a series of discrete cell states prior to the overt expression of the Secretory Osteoblastic phenotype. We propose that the emergence and abatement of phenotypically distinct osteogenic cell surface antigens follows a precise spatial and temporal sequence which reflects the position of cells within the osteogenic lineage.

Alkaline Phosphatase↗
PubMed1989

Comparison of fixatives for maximal retention of radiolabeled glycoconjugates for autoradiography, including use of sodium sulfate to release unincorporated [35S]-sulfate.

Previous studies have used [35S]-sulfate as a specific marker to autoradiographically localize sulfated glycosaminoglycans, proteoglycans, and glycoproteins. Embryonic chicks were labeled with [35S]-sulfate, followed by previously reported routine fixation and processing techniques. Subsequent processing revealed loss of radiolabeled macromolecules and retention of unincorporated label in the tissue, using these procedures. Biochemical analysis after various fixation and processing procedures demonstrated that an additional agent, such as cetylpyridinium chloride, was necessary in the fixative to retain the highly aqueous soluble sulfated macromolecular components. Molecular sieve chromatography was used to monitor digestate solutions for the identity of glycosaminoglycans and proteoglycans as indicated by selective enzymatic removal. Retained unincorporated [35S]-sulfate could be completely removed by rinsing the tissue in dehydration solutions containing exogenous sodium sulfate. This new procedure ensures the quantitative retention of sulfate labeled macromolecules in fixed tissue with the complete removal of unincorporated radiotracer, both of which are necessary for meaningful autoradiography.

Animals↗
PubMed1989

Association of the C-propeptide of type II collagen with mineralization of embryonic chick long bone and sternal development.

Several proteins may play a role in bone formation. The C-propeptide of type II collagen is intimately associated with endochondral bone formation in bovine growth plate. We have used an antibody against this peptide to determine its immunofluorescent distribution in early stages of embryonic chick limb development with emphasis on first bone formation which occurs in the mid-diaphyseal region. The C-propeptide II is first evident by immunofluorescent localization at stage 27 (day 5-6) of embryonic tibia development with chondrocytes in the central mid-diaphysis. In subsequent stages, there is an increase in the number of chondrocytes in which it is localized in discrete vacuoles. Up to stage 30, immunofluorescence is observed intracellularly, after which it appears in the matrix. The released C-propeptide II appears to remain only transiently associated with the cartilage matrix and becomes concentrated in the calcifying periosteum, the region outside of the cartilage core where bone formation first occurs in a sequence of events comparable to intramembranous bone formation. These observations can be reproduced in cultures of stage 35 hypertrophic chondrocytes (core cells) and periosteum cells (collar cells). Core cells contain intensely stained intracellular vacuoles while collar cells are negative, although the collar cell osteogenic matrix concentrates exogenously added C-propeptide II. Double label immuno-staining shows that the C-propeptide II, unlike type II collagen and proteoglycan, which are secreted and incorporated into extracellular sites, is initially stored in intracellular vacuoles. The matrix localization of the C-propeptide II during the transition from cartilage to bone indicates a close association with the initiation of mineralization events of cartilage and bone and its specific origin in chondrocytes and not osteoblasts. These observations suggest that the C-propeptide II made by chondrocytes is associated with the formation of bone.

Animals↗
PubMed1989

Structural characterization of chick embryonic skeletal muscle chondroitin sulfate proteoglycan.

Embryonic chick skeletal muscle has been shown to synthesize a distinct proteoglycan of large size with relatively large, highly 6-sulfated chondroitin sulfate glycosaminoglycans. Further analysis of these proteoglycans indicates that tryptic digestion gives rise to fragments with an average of two chondroitin sulfate chains per peptide. The skeletal muscle chondroitin sulfate proteoglycan also contains oligosaccharides whose characteristics suggest the presence of both O-linked and N-linked oligosaccharides. These characteristics include the average hydrodynamic size of the oligosaccharides as well as their localization. Approximately 10% of the putative O-linked oligosaccharides reside on the same tryptic fragments which contain the chondroitin sulfate chains, while the presumptive N-linked oligosaccharides appear to be present at sites distant from the chondroitin sulfate. Further support for this identification comes from radioisotopic labeling with [3H]mannose, which is incorporated exclusively into the putative N-linked oligosaccharides. Some of the O-linked oligosaccharides which are not in close apposition to the chondroitin sulfate seem to occur in clusters. The skeletal muscle chondroitin sulfate proteoglycan has the ability to interact in a link protein-stabilized fashion with hyaluronic acid. This ability as well as the estimated number of chondroitin sulfate chains per cluster and the estimated number of oligosaccharides per chondroitin sulfate chain have implications about the structure of the core protein of the skeletal muscle proteoglycan. The information presented is used to construct a model of these molecules; with this detailed model, attention can now be directed at other aspects of the skeletal muscle chondroitin sulfate proteoglycan, such as its role in myogenesis.

Animals↗
PubMed1989

Cellular and molecular events during embryonic bone development.

The cellular events underlying the processes of bone maintenance, remodeling, and repair have their basis in the embryonic production of bone. Although it has been suggested that the cartilaginous model which resides in the center of developing limbs provides the scaffolding for new bone formation, our detailed morphological and histochemical studies indicate otherwise. In particular, there exists a layer of four to six cells, referred to as Stacked Cells, which surrounds a prechondrogenic core of undifferentiated cells in the limb. These Stacked Cells give rise to the lineage of osteogenic cells responsible for the formation of all structural bone. This bone is fabricated outside of the cartilage core, and the core is not replaced by bone, but rather, is replaced by marrow and vascular elements. Several generalizations arise from analysis of the cellular and molecular events of embryonic osteogenesis, and they are as follows: 1) osteogenesis and chondrogenesis are independent events which are programmed early in development, 2) bone formation is a vascular driven phenomenon which is characterized by the directional nature of osteoid secretion, and 3) the fundamental events of bone formation are consistent in chick, rat, and human long bones.

Animals↗
PubMed1989

Discrete stages within the osteogenic lineage are revealed by alterations in the cell surface architecture of embryonic bone cells.

Specific markers for cells of the osteogenic lineage would be valuable in studies on the differentiation and maintenance of skeletal tissue. In order to better characterize the lineage of cells responsible for the formation and remodeling of bone, we immunized mice with a heterogeneous population of chick embryonic bone cells and subsequently generated and selected for monoclonal antibodies against cell surface determinants. We report here on the generation of three cell lines, SB-1, SB-2, and SB-3, which each secrete a unique antibody against a subset of differentiating osteogenic cells. Differences in immunoreactivity of osteogenic cells at precise stages of embryonic tibia development suggest the existence of an osteogenic cell lineage which is characterized by a series of discrete functional cell states progressing from osteoprogenitor cell to secretory osteoblast.

Animals↗
PubMed1989

Proteoglycan synthesis in vitamin D-deficient cartilage: recovery from vitamin D deficiency.

Vitamin D appears to be required for mineralization of skeletal elements. There is also evidence that cartilage proteoglycans may be involved in the regulation of mineralization. Previous studies have shown an alteration in the structure of the proteoglycans of the epiphyseal growth cartilage as a result of the decrease in serum calcium related to deficiency of dietary vitamin D. Vitamin D deficiency also induces a thickening of the epiphyseal growth plate presumably because of the inhibition of maturation of the growth plate chondrocytes. In order to compare the effect on proteoglycan structure with that on growth plate morphology, the proteoglycans of healing epiphyseal cartilage were characterized. The results indicate that, consistent with previous data, in vitamin D-deficient hatching chicks, the proteoglycans of the growth cartilage, but not of the articular cartilage, are smaller in monomer size with slightly smaller chondroitin sulfate chains whose sulfation pattern is unaltered. Sternal cartilage proteoglycans are unaffected. During recovery from vitamin D deficiency, the proteoglycans isolated from the growth cartilage are still not completely normal one day after supplementation with vitamin D, but are indistinguishable from normal by four days. In addition, the results conflict with those of a previous study in which only growth cartilage of hatchling chicks, not sternal or articular cartilage, was reported to synthesize large proteoglycans. Instead, all of these cartilages in the normal chicken have been found in this study to produce large proteoglycans of a size typical for mammalian cartilage and embryonic chick cartilage.

24,25-Dihydroxyvitamin D 3↗
PubMed1989

Repair of bone defects with marrow cells and porous ceramic. Experiments in rats.

We studied the role of bone-marrow reconstituted porous ceramics in enhancing healing of a 5-mm femoral diaphyseal defect fixed with a rigid polyethylene plate in rats. Osseous repair was evaluated by histologic scoring. When blocks of porous calcium phosphate ceramics alone were introduced into the defects, most cases showed fibrous tissue interposition at the host bone-ceramic junction 1 month after implantation, and only four of 12 defects developed osseous or osteochondral union at both the proximal and distal junctions 2 months after surgery. However, when the ceramic was combined with syngeneic viable marrow cells, new bone formation occurred in isolated pore regions of the ceramic at 1 month, and extensive bone formation was seen in most pore regions 2 months after implantation. Out of 12 implants, complete bone union was seen in eight, and one showed osseous or osteochondral union at both junctions 2 months after surgery. Our results indicated that composite grafts of porous calcium phosphate ceramics and marrow cells may be clinically applicable to enhance osteogenesis and osteoconduction.

Animals↗