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Biomedical subjects

J Glowacki

Publications and source records attributed to J Glowacki.

At least 19 recordsLinked to original sources

Hand reconstruction with allograft demineralized bone: twenty-six implants in twelve patients.

A long-term study of 26 phalangeal or metacarpal defects that were reconstructed with allogeneic demineralized bone implants demonstrates healing comparable to that which follows autogenous bone grafting. Average follow-up was 54 months. Five patients had multiple enchondromas (Ollier's syndrome), five children had congenital hand deformities, and all of these had previously had bone grafts harvested for associated craniofacial reconstructions. With the use of demineralized bone implants, tourniquet and operative times were significantly reduced and potential donor site morbidity was eliminated. Further, regional anesthesia was used more frequently and hospitalization time was reduced. There were no postoperative complications. Demineralized bone implants have been particularly useful in patients who previously had refused bone grafting.

Adolescent

Differential effects of glucocorticoid on recruitment and activity of osteoclasts induced by normal and osteocalcin-deficient bone implanted in rats.

Prolonged glucocorticoid excess is associated with bone loss. Among the contributory factors are glucocorticoids' suppression of bone formation and stimulation of bone resorption. In this study, the effects of glucocorticoids on bone resorption were evaluated in a rodent model. Subcutaneous implants of devitalized mineralized bone particles (BPs) elicit the recruitment of progenitor cells and their differentiation to osteoclasts which resorb the BPs. The effects of glucocorticoids on both the recruitment and the activity of cells induced by normal BPs were distinguished based upon when treatment was initiated. When treatment with hydrocortisone or dexamethasone was initiated at the time of BP implantation, the recruitment of bone-resorbing cells was impaired and a subsequent decrease in BP resorption was found. On the other hand, when treatment was initiated on day 7, glucocorticoids increased osteoclastic resorption and tartrate-resistant acid phosphatase activity. We also tested hydrocortisone's effect to stimulate the activity of cells associated with osteocalcin-deficient BPs. As previously reported, BPs deficient in osteocalcin were poorly resorbed as a result of decreased formation and activity of osteoclasts. Hydrocortisone had an even more pronounced effect in stimulating the low level resorption of the osteocalcin-deficient BP implants than of the normal BP implants. These findings show differential effects of glucocorticoids on two aspects of bone resorption: they inhibit the recruitment and/or differentiation of bone-resorbing cells, but they stimulate the activity of existing osteoclastic cells. The ability of glucocorticoids to increase resorption of normal bone and to overcome resistance to resorption of osteocalcin-deficient bone suggests an important regulatory effect of glucocorticoids in the activation of osteoclasts to increase bone resorption.

Acid Phosphatase

Origin of stromal cells associated with osteoclast recruitment in s.c. implants of bone particles in chimeric mice.

Subcutaneous implantation of devitalized bone particles (BPs) in mice elicits a fibrovascular response with subsequent differentiation of multinucleated osteoclast-like cells. In bone marrow, stromal cells are known to play important roles in controlling hematopoiesis. Similarly, the stromal cells in the initial reaction to BPs may take part in supporting subsequent osteoclast recruitment and differentiation within the implants. Cross-gender chimeric mice were used to allow determination of whether these stromal cells were derived from local tissue or from hematopoietic stem cells. In radiation-chimeric mice, there was a 7-day delay in stromal recruitment and osteoclastic differentiation. Therefore cultures were established from the stromal tissue elicited 11 days after implantation, prior to osteoclastogenesis. Analysis of Y-chromatin DNA from these lines demonstrated that the majority (97%) of the lines were of recipient origin. It is possible that these fibroblast-like cells migrate to the site of BP implantation and play a role in the initiation of osteoclast development. This model can be used to define cellular interactions in osteoclastogenesis.

Animals

A role for osteocalcin in osteoclast differentiation.

Specific cellular interactions with components of the extracellular matrix can influence cellular differentiation and development of many tissues. The extracellular matrix of bone is composed of organic constituents and a solid phase of calcium and inorganic phosphate (apatite). When implanted subcutaneously in rats, particles of bone matrix (BPs) recruit progenitors that differentiate into multinucleated cells with osteoclastic features. Because BPs deficient in osteocalcin, a bone matrix protein, were less efficient at promoting osteoclast formation than were normal BPs, we directly examined the influence of osteocalcin on osteoclast differentiation. We evaluated tissue responses to particles of synthetic crystalline apatite alone (Ap), having many of the features of native apatite of mature bone, or to apatite prepared with osteocalcin (Ap/OC), bovine serum albumin (Ap/BSA) or rat bone collagen (Ap/Col). Twelve days after subcutaneous implantation in normal rats, Ap, Ap/BSA, and Ap/Col particles generated a mild foreign body reaction with multinucleated cells in direct contact with the particles; these cells were negative for tartrate-resistant acid phosphatase (TRAP) activity and lacked ruffled borders. In contrast, Ap particles containing approximately 0.1% osteocalcin were partially resorbed and they generated more multinucleated cells that were TRAP-positive, were immunoreactive with an antibody against tartrate-resistant purple acid phosphatase, and displayed ultrastructural features of active osteoclasts including ruffled borders and clear zones. These data support the hypothesis that osteocalcin may function as a matrix signal in the recruitment and differentiation of bone-resorbing cells.

Acid Phosphatase

Normal bone particles are preferentially resorbed in the presence of osteocalcin-deficient bone particles in vivo.

In an in vivo model of osteoclastic bone resorption, we previously showed that osteocalcin-deficient bone particles (BPs), derived from warfarin-treated rats, were resorbed 50% as well as normal BPs and that they recruited fewer osteoclastic cells with decreased tartrate-resistant acid phosphatase (TRAP) activity. In order to determine the specificity of the resorption response, we evaluated the fate of implanted mixtures of normal and osteocalcin-deficient BPs. Normal and warfarin-treated donor rats were prelabeled in vivo with oxytetracycline to permit identification of BPs from either source. Normal, osteocalcin-deficient, and 50:50 mixtures of BPs (either labeled or unlabeled) were implanted into normal rats and recovered 12 days later for enzymatic (TRAP) and nondecalcified histomorphometric analyses. The incorporated oxytetracycline had no significant effect on resorption of bone particles. The recovered osteocalcin-deficient BPs were surrounded by fewer osteoclastic cells, were resorbed less, and contained less extractable TRAP activity than normal BPs. In mixed BP implants with normal and osteocalcin-deficient BPs, each type of bone particle elicited the same tissue response as when implanted separately. Remarkably, the different particles evoked dissimilar osteoclastic responses and were resorbed to different extents, even when adjacent within the same implant. These data suggest that osteocalcin may act as a substrate signal for resorption and that osteocalcin in the normal BPs does not influence the cellular response to adjacent osteocalcin-deficient BPs.

Acid Phosphatase

Tissue response to composite ceramic hydroxyapatite/demineralized bone implants.

This study evaluated the tissue reactions to two materials: ceramic hydroxyapatite (CHA), and a composite material of demineralized bone powder (DBP) and CHA (ratio of 4:1) in a collagen vehicle. The materials were tested in a subcutaneous pocket, a mandibular onlay, and in a calvarial onlay model. Specimens were evaluated histologically at 7, 10, 14, and 21 days postimplantation. Ceramic hydroxyapatite, implanted subcutaneously, elicited a fibrous response with minimal inflammation, but did not induce bone formation. In specimens of subcutaneously implanted composite material, induced bone was evident in association with the DBP. In CHA onlay specimens, there was a small amount of reactive bone extending from the host bone into the implant. In composite onlays, bone filled the entire body of the implant. The results of this study indicate that CHA particles were not osteoinductive in heterotopic sites and that osteoconductive ingrowth was minimal in onlays. Bone was induced by DBP even when mixed with CHA particles and implanted in subcutaneous and intraosseous sites. It was concluded that composite implants may provide a means of combining the osteoinductive properties of DBP with the bulk and structural support of osteoconductive CHA particles.

Alveolar Process

Impaired osteoclast differentiation in subcutaneous implants of bone particles in osteopetrotic mutants.

Because of its synchrony and relative homogeneity, the subcutaneous model of the resorption of mineral-containing, devitalized bone particles (BPs) is useful to evaluate the recruitment, differentiation, and activity of bone-resorbing, osteoclastic cells. Bone particles were prepared from normal rats or mice and were implanted in normal and osteopetrotic rats (ia, tl, op strains) or mice (mi strain). In addition, particles of microcrystalline hydroxyapatite or polymethylmethacrylate were implanted into tl and op mutants and their unaffected littermates. Non-decalcified histomorphometry of elicited tissues after 12 days revealed significantly less resorption in each mutant. Enzyme histochemical assays revealed that only normal animals showed tartrate-resistant acid phosphatase-positive cells around the BPs. In agreement with this, only normal animals showed ruffled borders against the BPs. op and tl strains were tested for generation of foreign body giant cells in response to particulate hydroxyapatite or polymethylmethacrylate and no differences were found between mutant and normal animals. These mutants appear to have intact fusion of mononuclear progenitors. These data show impaired recruitment of osteoclasts by BP implants in several rodent strains of osteopetrotic mutants.

Animals

Effects of bone matrix components on osteoclast differentiation.

When implanted subcutaneously in rats, devitalized bone particles (BP) elicit the differentiation of osteoclastic cells. Those cells can be distinguished from foreign body giant cells that form in response to particulate plastics. Osteoclast features include resorption of the bone substrate, ruffled borders, calcitonin receptors, tartrate-resistant acid phosphatase activity, and modulation by bone active agents. To determine whether expression of these features depends on specific components of the matrix, we characterized the multinucleated cells that developed in response to osteocalcin-deficient BPs, particulate microcrystalline hydroxyapatite (HA), and HA containing 0.1% osteocalcin, collagen, or bovine serum albumin. Only those particles that contained mineral and osteocalcin were associated with osteoclastic cells. These studies support the hypothesis that osteocalcin may function as a matrix signal in the differentiation of osteoclasts.

Animals

Multinucleated cells elicited in response to implants of devitalized bone particles possess receptors for calcitonin.

The introduction into soft tissues of particulate materials resistant to digestion results in the induction of a "foreign-body giant-cell reaction." We have examined the relation between osteoclasts and foreign-body giant cells by comparing the tissue responses elicited by subcutaneous implants of devitalized, mineral-containing bone particles (BP), nonresorbable plastics such as polymethylmethacrylate (PMMA), or both. Implantation of BP results in the recruitment of multinucleated cells with features of in osso osteoclasts including tartrate-resistant acid phosphatase activity, contact-mediated resorption of BP, membrane specializations (ruffled borders and clear zones), and inhibition of resorption by calcitonin treatment of animals. In the present study, an autoradiographic technique employing 125I-salmon calcitonin was used to demonstrate the presence of receptors for this hormone on multinucleated cells from BP implants. In contrast, outgrowth cells from PMMA implants lacked calcitonin receptors. Demonstration of features of the osteoclastic phenotype in multinucleated cells elicited in response to BP supports the hypothesis that the mineralized matrix of bone may be a requirement for acquisition of the osteoclast phenotype.

Animals

The effect of deficiencies of manganese and copper on osteoinduction and on resorption of bone particles in rats.

Subcutaneous implantation of devitalized demineralized bone powers (DBP) and mineral-containing bone particles (BP) into rats raised on either a control (C), low manganese and low copper (L), or manganese-deplete (D) diet, allowed the separate evaluation of bone formation and of bone resorption, respectively. DBP failed to induce chondrogenesis or osteogenesis in D rats. Cartilage formation was delayed in the L rats compared to C rats. There was significantly less resorption of BP by L and D rats than C rats. These results show multiple cellular effects of long-term manganese (Mn) and copper (Cu) deficiencies on bone metabolism including decreased osteogenesis and a decrease in osteoclast activity.

Aging

Expression of differentiated function by mineralizing cultures of chicken osteoblasts.

This report documents osteoblast differentiation in vitro, as demonstrated by the 50-100X increase of proteins which are known markers of the osteoblast phenotype. Collagen type I and osteocalcin synthesis and accumulation, alkaline phosphatase activity, and matrix calcification show similar temporal relationships that are analogous to those seen during in vivo bone development. Chicken embryonic osteoblast progenitor cells were selected by initial growth at low densities in minimal medium. Upon subcultivation into nutrient-enriched medium at higher cell densities, near homogeneous populations of osteoblasts were obtained as demonstrated by the greater than 80% enrichment of cells positive for alkaline phosphatase activity. A comparison was made between cells grown in the presence or absence of 10 mM beta-glycerolphosphate (beta-GPO4), a chemical stimulant of matrix calcification, as a function of time. Cultures treated with beta-GPO4 showed visible calcification at Day 12 when culture monolayers became confluent. By Day 30, numerous large foci of calcification were visible and a 20-fold increase in calcium (Ca) content was observed. In contrast, untreated cultures had only a 3-fold increase in Ca content with many smaller diffuse areas of calcification. DNA, RNA, and total protein levels were nearly identical between the two cultures, indicating that beta-GPO4 had no marked effect on either cell proliferation or transcriptional activity. The major collagen type produced by either culture was type I, with no detectable type III as determined by CNBr peptide mapping and delayed reduction analysis. Alkaline phosphatase activity showed a rapid approximately 50-fold induction by Day 18 and remained elevated in control cultures. However, cultures treated with beta-GPO4 demonstrated a rapid 80% decline of enzyme activity after 18 days. In contrast, total osteocalcin levels showed a 100-fold induction by Day 18 and remained elevated in both control and beta-GPO4-treated cultures throughout the time period examined. While the overall levels of osteocalcin were the same in beta-GPO4-treated and untreated cultures, 2- to 5-fold more osteocalcin was associated with the more mineralized matrices of the beta-GPO4-treated cultures. In order to confirm the association of osteocalcin with areas of mineralization, co-localization of mineral to osteocalcin and collagen was carried out by combining vital labeling with tetracycline and immunofluorescent staining with anti-osteocalcin and anti-collagen antibodies. Both collagen and osteocalcin showed strong localization with areas of mineralization.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaline Phosphatase

Impaired recruitment and differentiation of osteoclast progenitors by osteocalcin-deplete bone implants.

This is a report of an experimental system to study differentiation of bone-resorbing osteoclasts and demonstrates that osteocalcin, an extracellular bone-specific component, is necessary for the recruitment of osteoclast progenitor cells. The subcutaneous implantation of devitalized bone particles (BPs) elicits the recruitment and differentiation of osteoclasts that resorb the BPs. In a previous study, we showed by histomorphometric analysis that BPs that were deficient in osteocalcin were resorbed only 60% as well as normal BPs. In this study, the mechanism of this difference was investigated by measurements of recruitment, differentiation and activity of bone resorbing cells by normal and osteocalcin-deficient BP. Mononuclear cells were attracted to control BPs soon after implantation. In dramatic contrast, cellularity was depressed around osteocalcin-deficient BPs with very few mononuclear cells within the implant on day 5 (35% of control cellularity). In implants of normal BPs, tartrate-resistant acid phosphatase-positive multinucleated cells were evident by day 5; very few appeared in implants of osteocalcin-deplete BPs even by day 12. The amount of tartrate-resistant acid phosphatase activity in homogenates of the osteocalcin-deficient bone particle specimens not only lagged behind controls but never reached the maximum activity of control BP specimens. These data support the hypothesis that osteocalcin may function as a matrix signal in the recruitment and/or activation of cells for bone resorption.

Acid Phosphatase

Engraftment of a clonal bone marrow stromal cell line in vivo stimulates hematopoietic recovery from total body irradiation.

Whether bone marrow stromal cells of donors contribute physiologically to hematopoietic stem cell reconstitution after marrow transplantation is unknown. To determine the transplantability of nonhematopoietic marrow stromal cells, stable clonal stromal cell line (GB1/6) expressing the a isoenzyme of glucose-6-phosphate isomerase (Glu6PI-a, D-glucose-6-phosphate ketol-isomerase; EC 5.3.1.9) was derived from murine long-term bone marrow cultures and made resistant to neomycin analogue G418 by retroviral gene transfer. GB1/6 cells were fibronectin+, laminin+, and collagen-type IV+ and collagen type I-; these GB1/6 cells supported in vitro growth of hematopoietic stem cells forming colony-forming units of spleen cells (CFU-S) and of granulocytes, erythrocytes, and macrophage/megakarocytes (CFU-GEMM) in the absence of detectable growth factors interleukin 3 (multi-colony-stimulating factor), granulocyte/macrophage colony-stimulating factor, granulocyte-stimulating factor, or their poly(A)+ mRNAs. The GB1/6 cells produced macrophage colony-stimulating factor constitutively. Recipient C57BL/6J (glucose-6-phosphate isomerase b) mice that received 3-Gy total-body irradiation and 13 Gy to the right hind limb were injected i.v. with GB1/6 cells. Engrafted mice demonstrated donor-originating Glu6PI-a+ stromal cells in marrow sinuses in situ 2 mo after transplantation and a significantly enhanced hematopoietic recovery compared with control irradiated nontransplanted mice. Continuous (over numerous passages) marrow cultures derived from transplanted mice demonstrated G418-resistant, Glu6PI-a+ stromal colony-forming cells and greater cumulative production of multipotential stem cells of recipient origin compared with cultures established from irradiated, nontransplanted control mice. These data are evidence for physiological function in vivo of a transplanted bone marrow stromal cell line.

Animals

Comparison of multinucleated cells elicited in rats by particulate bone, polyethylene, or polymethylmethacrylate.

Osteoclasts, the multinucleated resorbing cells of bone, are identified by their characteristic morphology, unique cell membrane specializations, and more recently by the presence of cell surface antigens recognized by monoclonal antibodies. They are derived from mononuclear precursor cells of hematogenous origin. The precise relationship between osteoclasts and other types of tissue giant cells is unknown. This study was designed to examine factors involved in the recruitment and differentiation of multinucleated cells and to investigate the relationship between so-called foreign body giant cells and bone-resorbing osteoclasts. Particles of various materials were implanted into subcutaneous pockets in rats. Histological, histochemical, and electron microscopic evaluations were made of specimens harvested 12 days after implantation. Large, foamy multinucleated cells were evident around particles of devitalized bone, polyethylene, and polymethylmethacrylate. Bone particles showed scalloped surfaces and were partially resorbed. Those cells adjacent to the bone particles stained positive for tartrate-resistant acid phosphatase, in contrast to the multinucleated cells adjacent to the other materials. All the cell types had extensive rough endoplasmic reticulum, abundant mitochondria, cytoplasmic vacuoles and dense bodies, giant centrospheres, and areas of fusion of plasma membranes. Cells in lacunae on the surface of the bone particles showed a clear zone of attachment to the bone substrate and ruffled borders, a feature characteristic of in osso osteoclasts. Although the giant cells occasionally displayed an extensive clear zone of attachment to the polyethylene and polymethylmethacrylate particles, no ruffled borders were detected. The results of these studies show that the multinucleated cells elicited in response to different materials, although sharing many common features, do demonstrate certain features that are substrate specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase

Osteoclastic features of cells that resorb bone implants in rats.

The subcutaneous implantation of devitalized, mineral-containing bone particles in rats elicits the recruitment and differentiation of multinucleated cells and the rapid resorption of the bone. Cytochemical and ultrastructural characteristics were studied 12 days after implantation of bone. Tartrate-resistant acid phosphatase activity was demonstrated in these cells, especially at the cell/bone interface. Mast cells were seen vicinal to, but not in contact with the osteoclasts and the bone particles. Electron microscopic evaluation revealed that many multinucleated cells in lacunae on the surfaces of the bone particles displayed features characteristic of in osso osteoclasts, including clear zones of attachment to the bone substrate, and ruffled borders overlying bone with frayed collagen fibers. Many cells contained multiple giant centrospheres; this finding suggests that fusion can occur between multinucleated cells. This model may be useful to characterize the differentiation and regulation of bone-resorbing cells.

Acid Phosphatase

An ultrastructural study of mast cell interactions in hemangiomas.

Hemangiomas, the most common tumors of infancy, are characterized by a postnatal period of rapid growth, followed by a phase of gradual involution. The proliferative phase is characterized by increased numbers of endothelial and mast cells and thickened basement membrane. Ultrastructural analysis of hemangiomas in the late proliferative phase showed that mast cells had numerous fingerlike processes aligned parallel to the outer lamina of the thickened basement membranes surrounding the vessels and to the surfaces of opposing cell membranes. We found evidence of different types of interactions between mast cells and adjacent connective tissue cells (fibroblasts, macrophages, multinucleated giant cells, and plasma cells) in the perivascular regions of the lesions. Intercellular contacts were observed in areas where these mast cell processes were in close association to the opposing cell membrane. Areas of membrane fusions were seen between cell types. Coated vesicles and pinocytotic vesicles were present along the periphery of cells adjacent to mast cells. Occasionally, cytoplasmic bridges were found between mast cells and fibroblasts. These ultrastructural findings suggest the proliferation and involution of hemangiomas are determined by interactions between the various types of cells found in the lesions.

Cell Communication

Calcitonin produces hypercalcemia in leopard sharks.

Calcitonin was detected by RIA in sera from four marine species, leopard sharks (Triakis semifasciata), horn sharks (Heterodontus francisci), thornback rays (Platyrhinoides triseriata), and kelp bass (Paralabrax clathratus). These animals have levels of calcitonin and calcium higher than freshwater and terrestrial species have. The administration of salmon calcitonin to bass (4 micrograms/kg BW) produced hypocalcemia and hypophosphatemia as has been reported for other bony vertebrates. In marked contrast, calcitonin produced a prompt hypercalcemia in sharks; the average was 9.8% increase in serum calcium in nine animals with no attendant change in phosphorus. These findings demonstrate that calcitonin can increase serum calcium in sharks. Because shark skeleton is composed of cartilage, this hypercalcemic effect of calcitonin does not require a bony skeleton.

Animals