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Biomedical subjects

A I Caplan

Publications and source records attributed to A I Caplan.

At least 73 records · Page 4Linked to original sources
PubMed1996

The avian eggshell extracellular matrix as a model for biomineralization.

The avian eggshell is a complex, extracellularly assembled structure which contains both mineralized and non-mineralized regions. The composition of the hen eggshell organic matrix was examined by immunohistochemistry with antibodies to different extracellular matrix molecules. Type I collagen is found in the shell membranes, but only after treatment of the tissue sections with pepsin. When incomplete eggshells are removed from the oviduct and immunostained, type I collagen can be detected in the shell membranes without pepsin treatment. The shell membranes, which are non-mineralized, also contain type X collagen, and this immunostaining does not require pepsin treatment. The occurrence of type X collagen in the shell membranes is surprising, since this collagen has not been found in any tissue other than hypertrophic cartilage. Immunostaining for various glycosaminoglycans shows the presence of keratan sulfate and dermatan sulfate. Several different antibodies to keratan sulfate stain different regions of the eggshell; one keratan sulfate epitope is prominent in the calcium reserve assemblies. Dermatan sulfate staining is very intense in the palisade region. Demineralized matrix from the palisade region was extracted with guanidine and fractionated by ion exchange chromatography. A approximately 200-kDa dermatan sulfate proteoglycan is found in these extracts, along with a number of protein components. This preparation was tested for its ability to affect calcium carbonate crystal formation in vitro. Pieces of demineralized shell membranes were used as a substrate for crystal formation and various amounts of the palisade matrix dermatan sulfate proteoglycan preparation were added to the solution from which the crystals were formed. This material causes a concentration-dependent change in crystal morphology to one in which the crystals are smaller and more rounded, which more closely approximates the crystals normally observed in eggshells. These results suggest that the dermatan sulfate proteoglycans may be important in modulating crystal morphology in the hen eggshell and correlate with mineralization-modulating biomolecules from other calcified tissue, which are generally anionic.

Animals↗
PubMed1995

Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine.

The compound 5-azacytidine has been previously shown to convert cells of the rat embryonic fibroblastic cell line, C3H/10T1/2, into myoblasts, adipocytes, and chondrocytes. Rare, resident cells of bone marrow and periosteum, referred to as mesenchymal stem cells, have been shown to differentiate into a number of mesenchymal phenotypes including bone, cartilage, and adipocytes. Rat bone marrow-derived mesenchymal stem cells were exposed to 5-azacytidine beginning 24 h after seeding twice-passaged cells into culture dishes. After an exposure of 24 h, long, multinucleated myotubes were observed in some of the dishes 7-11 days later. Cells containing Sudan black-positive droplets in their cytoplasm were also observed. Thus, culture-propagated rat bone marrow mesenchymal stem cells appear to have the capacity to be induced to differentiate in vitro into myogenic and adipocytic phenotypes, although nonmesenchymal cells (rat brain fibroblasts) cannot be so induced. Taken together, these observations provide support for the suggestion that mesenchymal stem cells in the bone marrow of postnatal organisms may provide a source for myoprogenitor cells which could function in clinically relevant myogenic regeneration.

Adipocytes↗
PubMed1995

A chemically defined medium supports in vitro proliferation and maintains the osteochondral potential of rat marrow-derived mesenchymal stem cells.

Among the stromal elements in mammalian and avian bone marrow there exists a pluripotent subset of cells which we refer to as mesenchymal stem cells (MSCs). These cells can be isolated and will proliferate in culture. When such subcultured cells are introduced into porous tricalcium phosphate-hydroxyapatite ceramic cubes and implanted subcutaneously into syngeneic or immunocompromised hosts, the passaged MSCs are observed to differentiate into bone and cartilage. Heretofore, those assays have been conducted with MSCs which had been maintained in vitro in serum-containing medium. A serum-free medium (RDM-F), which consists of insulin, 5 micrograms/ml, linoleic acid-bovine serum albumin, 0.1%, platelet-derived growth factor-BB, 10 ng/ml, and basic fibroblast growth factor, 1 ng/ml in a base medium of 60% Dulbecco's modified Eagle's medium with low glucose and 40% MCDB-201, has been developed for rat marrow-derived MSCs. Proliferation rates of MSCs maintained in RDM-F equal those of cells maintained in serum-containing medium through Day 4 following subculturing and continue at up to 80% of the rate of the latter through Day 8 of subculture. When tested in the in vivo ceramic cube assay, MSCs cultured in RDM-F retain their osteochondral potential and differentiate into bone and cartilage in a manner indistinguishable from those cultivated in serum-containing medium. Utilization of this serum-free medium will facilitate analysis of the effects of other growth factors and cytokines on the proliferation and differentiation of MSCs, without the complexity of exogenous serum.

Animals↗
PubMed1995

Crystallization studies on avian eggshell membranes: implications for the molecular factors controlling eggshell formation.

The avian eggshell is a natural biopolymer and mineral composite. It is a very useful model for biomimetic mineralization, since it is among the fastest forming hard tissues known. Isolated eggshell membranes, which were demineralized in vitro, were used to investigate the in vitro modulation of CaCO3 crystal deposition by organic matrix materials. Crystallization on the demineralized eggshell membrane occurred almost exclusively at the peripheries of residual calcium reserve assemblies, which contain a high concentration of sulfur. Similar structures are observed for eggshell membranes after natural demineralization. The characteristic rhombohedral crystal morphologies of the calcite crystals grown in this in vitro system are much less regular when grown in the presence of organic matrix or partially purified dermatan sulfate proteoglycans obtained from the eggshell. The effect of these macromolecules on the morphology and size of CaCO3 crystals is concentration-dependent. These studies indicate the complexity of the molecular and ionic interactions involved in the initiation and formation of the eggshell, with the focus on the role of the organic matrix.

Animals↗
PubMed1995

Osteogenesis imperfecta, rehabilitation medicine, fundamental research and mesenchymal stem cells.

A speculative suggestion for a curative approach to osteogenesis imperfecta is based on the experimental observation that a pluripotent stem cell for mesenchymatous tissues can be isolated from marrow or periosteum. This cell, referred to as a Mesenchymal Stem Cell (MSC) could provide a vehicle for gene therapy based on that fact that many mesenchymal tissues have reasonably high cellular turnover rates. If autologous MSCs with the "proper" gene construct can be transplanted into affected patients, it may be that the progeny of the gene-corrected MSC can replace the defective mature phenotypes and bring about curative events associated with normal cell turnover.

Animals↗
PubMed1995

Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use.

We report a phase I trial to determine the feasibility of collection, ex vivo culture-expansion and intravneous infusion of human bone marrow-derived progenitor stromal cells (mesenchymal progenitor cells (MPCs)). Ten milliliter bone marrow samples were obtained from 23 patients with hematologic malignancies in complete remission. Bone marrow mononuclear cells were separated and adherent cells were culture-expanded in vitro for 4-7 weeks. Autologous MPCs were reinfused intravenously and a bone marrow examination repeated 2 weeks later for histologic assessment and in vitro hematopoietic cultures. Patient age ranged from 18 to 68 years and 12 subjects previously had undergone an autologous or syngeneic bone marrow transplant 4-52 months prior to collection of MPCs. A median of 364 x 10(6) nucleated bone marrow cells (range: 103 to 1004 x 10(6)) were used for ex vivo expansion. Median number of MPCs which were obtained after ex vivo culture expansion was 59.0 (range: 1.1 to 347 x 10(6)) representing a median cell doubling of 16,000-fold (13 doublings). Fifteen of 23 patients completed the ex vivo expansion and underwent MPC infusion. Time to infusion of MPCs after collection ranged from 28 to 49 days. Five patients in each of three groups were given 1, 10 and 50 x 10(6) MPCs. No adverse reactions were observed with the infusion of the MPCs. MPCs obtained from cancer patients can be collected, expanded in vitro and infused intravenously without toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Identity of the core proteins of the large chondroitin sulphate proteoglycans synthesized by skeletal muscle and prechondrogenic mesenchyme.

Large, chondroitin sulphate-containing proteoglycans are synthesized by three prominent tissue in the embryonic chick limb. One of these proteoglycans is aggrecan, the phenotype-specific proteoglycan of cartilage. Another, PG-M, is produced by prechondrogenic mesenchymal cells. The third, M-CSPG, is made by developing skeletal muscle cells. While the carbohydrate components of PG-M and M-CSPG share some similarities, both of these proteoglycans clearly have different carbohydrate moieties from those of aggrecan. To compare these three proteoglycans at another level, their core protein structures were analysed in three ways: by the presence or absence of monoclonal antibody epitopes, by one-dimensional peptide display of the cyanogen bromide-cleaved core proteins and by electron microscopic imaging of the molecules. Monoclonal antibodies whose epitopes are present in aggrecan core protein were tested with core protein preparations from M-CSPG and PG-M. One of these, 7D1, recognizes both PG-M and M-CSPG, while another, 1C6, shows no reactivity for the non-cartilage proteoglycans. The absence of 1C6 reactivity is of interest, as its epitope is in a region of the aggrecan core protein known to have a functional homologue in the core proteins of PG-M and M-CSPG. The cyanogen bromide-fragmented peptide pattern of M-CSPG is the same as that of PG-M, and both are different from that of aggrecan. The aggrecan pattern has one prominent large band (molecular mass 130 kDa), some less prominent large bands (molecular mass 70-100 kDa) and several smaller bands. In contrast, the PG-M and M-CSPG patterns show no bands with molecular masses > 73 kDa, and the smaller bands (molecular mass < 40 kDa) have a different pattern to that of the smaller bands from aggrecan. The electron microscopic images of aggrecan show a core protein with one end having two globular regions separated by a short linear segment; adjacent to this is a long linear segment, which sometimes contains a third globular region at the end of the core protein opposite the end with the double-globe structure. M-CSPG and PG-M core proteins never show images with the double-globe structure. Instead, one end of the molecule has a single globular domain, and a second globular region is variably present at the opposite end of the core protein. Thus, by all three methods, the core proteins of PG-M and M-CSPG appear to be the same and both differ from the core protein of aggrecan.

Animals↗
PubMed1994

Transient expression of type II collagen and tissue mobilization during development of the scleral ossicle, a membranous bone, in the chick embryo.

Development of the chick scleral ossicle was studied with respect to expression of various collagen types, cartilage matrix molecules, and osteoblastic cell surface antigens. The extra-cellular matrix of the scleral ossicle primordium of stage 35.5 chick sclera and the mesenchyme beneath the conjunctival epithelium was immunoreactive with anti-type II collagen antibody, giving the impression that certain materials and/or cell clusters surrounded by reactive matrix were descending from the epithelial-mesenchymal interface to the scleral ossicle primordium. In stage 37 embryos, type II collagen immunoreactivity was restricted to the bone matrix of the scleral ossicles, and persisted through stage 39. However, at stage 41, virtually no type II collagen was detected. In contrast, strong immunostaining of type I collagen was first detected in the developing scleral ossicle at stage 37, coinciding with the formation of mineralized bone matrix. Following the extensive accumulation of type I collagen in bone matrix, type XII collagen was detected at the surface of the bone; both type I and type XII collagen immunostainings then remained. By stage 37, immunoreactivity with a pre-osteoblastic cell surface marker was detected on cells of the scleral ossicle, and typical osteocytes were subsequently identified by both morphological and specific immunostaining techniques. Antibodies other than for type II collagen, specific to chondrogenic mesenchyme or cartilage matrix, never reacted with the scleral ossicle and its primordium during development. Taken together, these observations indicate that the scleral ossicle is a membranous bone, whose development may not require overt chondrogenesis. Implications of type II collagen distribution during the positioning of scleral ossicles and their early bone matrix formation are discussed with respect to the origin and evolution of endoskeletons in vertebrate animals.

Animals↗
PubMed1994

Bone morphogenetic protein 2 stimulates osteogenesis but does not affect chondrogenesis in osteochondrogenic differentiation of periosteum-derived cells.

The effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) on osteochondrogenesis were examined in high-density cultures of periosteum-derived cells, which have the potential to differentiate into bone and hypertrophic cartilage in vitro. Proliferation of these cells was inhibited by treatment with rhBMP-2. The time course for alkaline phosphatase (ALP) expression was shortened and the mineralization of the culture was increased by supplementation with rhBMP-2. These stimulatory effects of rhBMP-2 were observed at doses of 10-100 ng/m. Bone Gla protein (BGP) was immunocytochemically detectable earlier in the culture treated with rhBMP-2, and the BGP-positive layer of the rhBMP-2-treated cultures was thicker than that of the control cultures. On the other hand, there was no difference in uronic acid content or the time course of alpha 1(II) collagen mRNA expression between the rhBMP-2-treated and the control cultures. These results indicate that rhBMP-2 shortens the time course of osteogenesis and increases the amount of bone formation, whereas chondrogenesis remains unaffected.

Alkaline Phosphatase↗
PubMed1994

Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy.

Bone formation in the embryo, and during adult fracture repair and remodeling, involves the progeny of a small number of cells called mesenchymal stem cells (MSCs). These cells continuously replicate themselves, while a portion become committed to mesenchymal cell lineages such as bone, cartilage, tendon, ligament, and muscle. The differentiation of these cells, within each lineage, is a complex multistep pathway involving discrete cellular transitions much like that which occurs during hematopoiesis. Progression from one stage to the next depends on the presence of specific bioactive factors, nutrients, and other environmental cues whose exquisitely controlled contributions orchestrate the entire differentiation phenomenon. An understanding of the cellular and molecular events of osteogenic differentiation of MSCs provides the foundation for the emergence of a new therapeutic technology for cell therapy. The isolation and in vitro mitotic expansion of autologous human MSCs will support the development of novel protocols for the treatment of many clinically challenging conditions. For example, local bone defects can be repaired through site-directed delivery of MSCs in an appropriate carrier vehicle. Generalized conditions, such as osteoporosis, may be treatable by systemic administration of culture-expanded autologous MSCs or through biopharmaceutical regimens based on the discovery of critical regulatory molecules in the differentiation process. With this in mind, we can begin to explore therapeutic options that have never before been available.

Animals↗
PubMed1994

The effects of beta-D-xyloside on the synthesis of proteoglycans by skeletal muscle: lack of effect on decorin and differential polymerization of core protein-bound and xyloside-linked chondroitin sulfate.

Developing skeletal muscle cells, as both myoblasts and myotubules, synthesize a distinctive large chondroitin sulfate proteoglycan. To probe the role of this proteoglycan in myogenesis, chick embryonic muscle cells in culture were treated with beta-D-xyloside, a compound which interferes with proteoglycan synthesis by acting as an artificial acceptor for glycosaminoglycan synthesis and thereby competing with the proteoglycan core protein. Analysis of the proteoglycans indicates that with increasing concentrations of beta-D-xyloside, synthesis of the chondroitin sulfate proteoglycan is inhibited, with concomitant massive synthesis of xyloside-linked chondroitin sulfate glycosaminoglycans. Xyloside does not appear to inhibit glycosaminoglycan attachment onto the small heparan sulfate and dermatan sulfate proteoglycans which are synthesized in the muscle cultures, even though, because of the mechanism of action of beta-xyloside, these proteoglycans should be affected. At submaximal concentrations of beta-xyloside, there is synthesis of both large chondroitin sulfate proteoglycans and xyloside-linked chondroitin sulfate. The xyloside-linked chondroitin sulfate chains have the same sulfation pattern as the core protein-bound skeletal muscle chondroitin sulfate (90% 6-sulfated isomer), but are much smaller (24,000 vs. 65,000 in molecular weight). The discrepancy in size but identify of sulfation indicates that, although sulfation takes place normally on either the core protein or the xyloside acceptor, termination of glycosylation occurs earlier for xyloside-initiated chondroitin sulfate. In spite of these dramatic effects on chondroitin sulfate proteoglycan synthesis, beta-xyloside elicits no observable effects on in vitro myogenesis. This suggests that the function served by the large chondroitin sulfate proteoglycan is not required in the more simplified environment of the muscle cultures.

Animals↗
PubMed1994

Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage.

UNLABELLED: Osteochondral progenitor cells were used to repair large, full-thickness defects of the articular cartilage that had been created in the knees of rabbits. Adherent cells from bone marrow, or cells from the periosteum that had been liberated from connective tissue by collagenase digestion, were grown in culture, dispersed in a type-I collagen gel, and transplanted into a large (three-by-six-millimeter), full-thickness (three-millimeter) defect in the weight-bearing surface of the medial femoral condyle. The contralateral knee served as a control: either the defect in that knee was left empty or a cell-free collagen gel was implanted. The periosteal and the bone-marrow-derived cells showed similar patterns of differentiation into articular cartilage and subchondral bone. Specimens of reparative tissue were analyzed with use of a semiquantitative histological grading system and by mechanical testing with employment of a porous indenter to measure the compliance of the tissue at intervals until twenty-four weeks after the operation. There was no apparent difference between the results obtained with the cells from the bone marrow and those from the periosteum. As early as two weeks after transplantation, the autologous osteochondral progenitor cells had uniformly differentiated into chondrocytes throughout the defects. This repair cartilage was subsequently replaced with bone in a proximal-to-distal direction, until, at twenty-four weeks after transplantation, the subchondral bone was completely repaired, without loss of overlying articular cartilage. The mechanical testing data were a useful index of the quality of the long-term repair. Twenty-four weeks after transplantation, the reparative tissue of both the bone-marrow and the periosteal cells was stiffer and less compliant than the tissue derived from the empty defects but less stiff and more compliant than normal cartilage. CLINICAL RELEVANCE: The current modalities for the repair of defects of the articular cartilage have many disadvantages. The transplantation of progenitor cells that will form cartilage and bone offers a possible alternative to these methods. As demonstrated in this report, autologous, bone-marrow-derived, osteochondral progenitor cells can be isolated and grown in vitro without the loss of their capacity to differentiate into cartilage or bone. Sufficient autologous cells can be generated to initiate the repair of articular cartilage and the reformation of subchondral bone. The repair tissues appear to undergo the same developmental transitions that originally led to the formation of articular tissue in the embryo.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Biological resurfacing: an alternative to total joint arthroplasty.

These preliminary observations suggest that the functional repair of adult hyaline cartilage is controlled by the entrance and lineage progression of uncommitted MSCs into chondrocytes under the direction of specific biological and mechanical cues which represent a recapitulation of embryonic events. This concept implies that to some extent the regeneration of destroyed articular cartilage is limited by an inadequate supply of MSCs from the host and their inefficient interaction with the appropriate factors at the local site. Finally, the use of autologous cell tissue engineering could provide the basis of an important application for the repair of deficient joint surfaces as a result of trauma or osteoarthritis.

Animals↗
PubMed1994

The mesengenic process.

The genesis of mesodermal tissues, such as bone, cartilage, muscle, marrow stoma, tendon, fat, dermis, and connective tissues, in either embryos or adult organisms is referred to as the mesengenic process. The process is involved in the continual rejuvenation of each mesenchymal tissue and provides a logic for understanding the rapid repair of tissue injuries. The isolation and identification of mesenchymal stem cells and their quantity and distribution allow a better understanding of the turnover and repair motifs used in adult organisms. Mesenchymal stem cells undergo proliferative and commitment events, and their progeny enter discrete lineages that result in the ultimate terminal differentiation of definitive phenotypes such as osteoblasts, chondrocytes, or myoblasts. The molecular and cellular control of the mesengenic process provides a logic for designing cell therapies for the reconstruction of mesenchymal tissues.

Adolescent↗
PubMed1993

Clonal analysis for developmental potential of chick periosteum-derived cells: agar gel culture system.

The developmental potential of periosteum-derived cells was clonally assessed with an agar gel culture system. Morphologically, two types of colonies were predominantly observed. By immunocytochemical observation with antibodies against aggrecan or bone Gla protein, one type of colony was judged to be chondrogenic, and the other osteogenic. By chronological observation, each type of colony did not convert to the other. Supplementation with transforming growth factor (TGF)-beta 1 shortened the time course of chondrogenesis and also increased colony forming efficiency of chondrogenic colonies. On the other hand, colony forming efficiency of osteogenic colonies decreased with TGF-beta 1 treatment, whereas the time course of osteogenesis remained unaffected. These observations suggest that there are both committed osteoprogenitor and chondroprogenitor cells present in the periosteal cell population, and TGF-beta 1 stimulates proliferation and differentiation of chondrogenic cell population by its targeted action.

Agar↗
PubMed1993

Human bone marrow stromal cells express an osteoblastic phenotype in culture.

This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two "clonings" and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [3H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D3 by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of beta-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by "budding" structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with 45Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.

Adult↗
PubMed1993

Structural domains in chondroitin sulfate identified by anti-chondroitin sulfate monoclonal antibodies. Immunosequencing of chondroitin sulfates.

Monoclonal antibodies have been developed that recognize epitopes in native chondroitin sulfate chains. One of these antibodies, CS-56, reportedly recognizes chondroitin 4- and 6-sulfates. However, this antibody, and four other anti-chondroitin sulfate antibodies, 4C3, 4D3, 6C3 and 7D4, do not recognize epitopes in chondroitin sulfate chains from Swarm rat chondrosarcoma proteoglycan, an indication that native chondroitin sulfate epitopes are more structurally complex than the standard 0-, 4-, and 6-sulfated disaccharide repeats that constitute the backbone of chondroitin sulfate chains. A series of limited chondroitinase digestions was performed on the large aggregating proteoglycan monomer extracted from embryonic chick chondrocyte cultures to identify the digestion parameters required to release the different native chondroitin sulfate epitopes. Some epitopes were more accessible to enzymatic digestion than other epitopes. The approximate location of epitopes was determined by measuring the size of undigested oligosaccharides retained on the core protein following a limited digestion, and correlating this with the level of immunoreactivity for the different antibodies. These analyses identified the locations of three different antigenic domains. Domain 1 resides at the linkage region and contains epitopes for two of the five antibodies, and a portion of the epitopes for a third antibody. Domain 2 lies in the interior of the chain and contains epitopes for three of the five antibodies. Domain 3 resides at the non-reducing terminus and does not contain epitopes for any of the anti-chondroitin sulfate antibodies used in this study. These results indicate that specific native chondroitin sulfate epitopes are non-randomly distributed within the linear framework of chondroitin sulfate chains.

Animals↗