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J Glowacki

Publications and source records attributed to J Glowacki.

At least 55 records · Page 3Linked to original sources
PubMed1996

Cellular reactions to bone-derived material.

Demineralized bone has been used as an acceptable alternative to fresh autogenous bone grafting in a variety of clinical reconstructive procedures. With the recent expansion of regional and national tissue banks, more implants are being used in routine applications. Questions raised about the bioactivity of human demineralized bone may be resolved by development of relevant assays for screening a lot of banked tissue. Such a bioassay could be developed based on the effects of osteoinductive materials on cells in vitro. Various effects of culture with demineralized bone or its components have been reported for cell lines or embryonic, fetal, or neonatal rodent and chick tissues and cells. In this study, human dermal fibroblasts and a variety of other cell types expressed features of chondroblastic phenotype when cultured with demineralized bone powder.

Biological Assay↗
PubMed1996

Osteogenic potential of murine osteosarcoma cells: comparison of bone-specific gene expression in in vitro and in vivo conditions.

Bone tissue formation and the expression of osteoblast-specific genes were compared in vitro and in vivo for two well characterized murine clonal osteosarcoma cell lines (K7 and K8). In vitro studies were carried out under conditions that promoted extracellular matrix morphogenesis and mineralization. The K8 cells showed 8-fold greater alkaline phosphatase activity and mineral accumulation than did K7 cells during 21 days of in vitro growth. The K8 cell line showed high levels of bone sialoprotein (BSP), collagen type I (COL I), and alkaline phosphatase (APase) mRNA expression throughout its growth in vitro. In contrast, K7 cells showed an almost complete absence of BSP and COL I and very low levels of APase throughout the culture period. In vitro, both cell lines expressed very low levels of osteocalcin (OC). For in vivo studies, we used a three-dimensional culture device that permitted analysis of tissue formation by the cells after their implantation into syngeneic mice. The K8 cells consistently generated extensive mineralized woven bone after their subcutaneous implantation. The striking features distinguishing the bone formed by the implanted cells from normal recipient bone were the complete absence of osteoclasts or matrix resorption, the absence of OC protein, and very low levels of OC mRNA expression in the tissues formed by these cell lines. BSP, APase, and COL I expressions were maintained at high levels in the K8-produced tissues. In contrast to their near absence in vitro, APase, BSP, and COL I were expressed by K7 cells and increased with time in vivo. These findings demonstrate that the K7 cells in vitro are less differentiated than K8 cells, but that K7 cells in vivo undergo osteogenic maturation. Thus, expression of bone-specific genes in these osteogenic cell lines was dependent on systemic or local factors in recipient animals and was distinct for these cell lines when grown under in vitro conditions. OC protein does not appear to be needed for the mineralization of the extracellular matrix but may be needed to provide the necessary signals for the resorption and remodeling of the tissue.

Alkaline Phosphatase↗
PubMed1994

Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene.

The parathyroid hormone-related peptide (PTHrP) gene was disrupted in murine embryonic stem cells by homologous recombination, and the null allele was introduced into the mouse germ line. Mice homozygous for the PTHrP null mutation died postnatally, probably from asphyxia, and exhibited widespread abnormalities of endochondral bone development. Histological examination revealed a diminution of chondrocyte proliferation, associated with premature maturation of chondrocytes and accelerated bone formation. Analysis of earlier developmental stages revealed that disturbance in cartilage growth preceded abnormal endochondral bone formation. There were no morphological abnormalities apparent in other tissues. These results provide direct evidence implicating PTHrP in normal skeletal development and serve to emphasize its potential involvement in human osteochondrodysplasias.

Animals↗
PubMed1994

Osteocalcin promotes differentiation of osteoclast progenitors from murine long-term bone marrow cultures.

Murine long-term bone marrow cultures (LTBMCs) were used to generate hematopoietic cells free from marrow stromal cells. These progenitor cells were treated with GM-CSF (5 U/ml) with or without rat bone osteocalcin or rat serum albumin in either alpha-MEM with 2% heat-inactivated horse serum alone (alpha) or supplemented with 10% L-cell-conditioned medium (as a source of M-CSF) (L10). Few substrate-attached cells survived in basal alpha medium, but when treated with L10 medium or GM-CSF, they survived and proliferated. Osteocalcin did not significantly affect survival or proliferation. Subcultures of cells treated with GM-CSF had large numbers of multinucleated cells, more than half of which were tartrate-resistant acid phosphatase-positive (TRAP). Osteocalcin further promoted the development of TRAP-positive multinucleated cells; a dose of 0.7 microgram/ml osteocalcin promoted osteoclastic differentiation by 60%. Using a novel microphotometric assay, we detected significantly more tartrate-resistant acid phosphatase activity in the osteocalcin plus GM-CSF group (75.6 +/- 14.2) than in GM-CSF alone (53.3 +/- 7.3). In the absence of M-CSF, GM-CSF stimulated tartrate-resistant acid phosphatase activity, but osteocalcin did not have an additional effect. These studies indicate that osteocalcin promotes osteoclastic differentiation of a stromal-free subpopulation of hematopoietic progenitors in the presence of GM-CSF and L-cell-conditioned medium. These results are consistent with the hypothesis that this bone-matrix constituent plays a role in bone resorption.

Acid Phosphatase↗
PubMed1994

Interleukin-1 beta-modulated gene expression in immortalized human chondrocytes.

Immortalized human chondrocytes were established by transfection of primary cultures of juvenile costal chondrocytes with vectors encoding simian virus 40 large T antigen and selection in suspension culture over agarose. Stable cell lines were generated that exhibited chondrocyte morphology, continuous proliferative capacity (> 80 passages) in monolayer culture in serum-containing medium, and expression of mRNAs encoding chondrocyte-specific collagens II, IX, and XI and proteoglycans in an insulin-containing serum substitute. They did not express type X collagen or versican mRNA. These cells synthesized and secreted extracellular matrix molecules that were reactive with monoclonal antibodies against type II collagen, large proteoglycan (PG-H, aggrecan), and chondroitin-4- and chondroitin-6-sulfate. Interleukin-1 beta (IL-1 beta) decreased the levels of type II collagen mRNA and increased the levels of mRNAs for collagenase, stromelysin, and immediate early genes (egr-1, c-fos, c-jun, and jun-B). These cell lines also expressed reporter gene constructs containing regulatory sequences (-577/+3,428 bp) of the type II collagen gene (COL2A1) in transient transfection experiments, and IL-1 beta suppressed this expression by 50-80%. These results show that immortalized human chondrocytes displaying cartilage-specific modulation by IL-1 beta can be used as a model for studying normal and pathological repair mechanisms.

Antigens, Viral, Tumor↗
PubMed1994

Stromal cell-mediated stimulation of osteoclastogenesis.

In the bone marrow microenvironment, stromal cells or their products are known to regulate proliferation and differentiation of hematopoietic stem cells. The purpose of this investigation was to characterize stroma-mediated effects of differentiation-inducing factors on osteoclastogenesis in defined murine cultures. Hematopoietic progenitors (derived from long-term bone marrow cultures, LTBMCs) were cocultured with cloned stromal cell lines to demonstrate the indirect effects of various differentiation-inducing factors. Osteoclastogenesis was compared in three murine marrow systems (whole bone marrow, progenitors cultured alone, and cocultures of progenitors with stromal cell lines) by analysis of multinuclearity and tartrateresistant acid phosphatase (TRAP) activity. The cultures were treated for two weeks with murine recombinant GM-CSF (5 U/ml), 1,25-dihydroxyvitamin D3 (10(-8) M), or parathyroid hormone (PTH, 10(-8) M). In whole bone marrow cultures, osteoclast differentiation was stimulated by GM-CSF, PTH and 1,25-dihydroxyvitamin D3. With progenitors alone, only GM-CSF promoted osteoclastogenesis. Each agent stimulated osteoclastogenesis in cocultures of progenitors with a stromal cell line (GBLneo'). Thus, the coculture system is a partially defined model for whole bone marrow cultures. In contrast, progenitors that were cocultured with a stromal cell line derived from an osteopetrotic op/op mouse failed to differentiate in the presence of PTH or 1,25-dihydroxyvitamin D3. These results indicate that stimulation of osteoclastogenesis by PTH or 1,25-dihydroxyvitamin D3 is mediated indirectly through factors present in normal marrow stromal cells and that an osteopetrotic stromal cell line failed to support differentiation.

Animals↗
PubMed1994

The roles of revascularization and resorption on endurance of craniofacial onlay bone grafts in the rabbit.

A total of 32 New Zealand white rabbits underwent subperiosteal implantation of fresh autogenous unicortical calvarial and iliac crest grafts on their snouts with microscrew rigid fixation. After 3 and 10 days, vascularity was assessed by latex casting, and osteoclastic activity was determined by histochemical staining for tartrate-resistant acid phosphatase. After 70 days, volumetric analysis and tartrate-resistant acid phosphatase staining were performed on six animals. The calvarial grafts demonstrated greater volume maintenance than the iliac bone (72 percent versus 32 percent, p < 0.025). There were significantly greater osteoclastic activity and revascularization in the cancellous portion of calvarial and iliac crest bone grafts by the 10th day of onlay grafting. Minimal activities were present at the cortical bone. Because calvarial grafts contain more cortical bone, its superior volume maintenance can be understood by the architectural influence on revascularization and resorption.

Animals↗
PubMed1993

Effects of macrophage colony stimulating factor and granulocyte-macrophage colony stimulating factor on osteoclastic differentiation of hematopoietic progenitor cells.

Although the hematopoietic origin of the osteoclast is generally accepted, the precise phenotype of the progenitor and the regulation of its differentiation are unclear. This study compares proliferation and differentiation of progenitors in response to macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Nonadherent progenitor cells from murine long-term bone marrow cultures (LTBMC) (as a source of osteoclast progenitors) demonstrated a significant proliferative response to M-CSF. In addition, M-CSF increased the number of multinucleated cells, only a small percent of which (14-16%) were tartrate-resistant, acid phosphatase (TRAP)-positive. In contrast, cells cultured with GM-CSF generated more TRAP-positive multinucleated cells even at concentrations less stimulatory of proliferation than M-CSF. The osteoclast phenotype of these multinucleated cells was also assessed by ultrastructural characterization of ruffled borders in association with bone fragments. The bone-active hormone 1,25-dihydroxyvitamin D3 inhibited the proliferation of this subset of progenitor cells in the presence of M-CSF or GM-CSF. All of these results show effects on progenitors in the absence of the stromal cell microenvironment in this system. These results provide evidence for a divergence in the biological responsiveness of osteoclast progenitor cells to M-CSF compared with GM-CSF; they support the notion that M-CSF has a "priming" effect on osteoclast progenitors whose subsequent differentiation to osteoclastic multinucleated cells is promoted by GM-CSF.

Animals↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1990

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1988

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↗