Marilyn Krukowski

Inductive specificity of mineralized bone matrix in ectopic osteoclast differentiation

SummaryThe present report describes the first in a series of studies designed to identify the factor or factors responsible for eliciting osteoclast differentiation. Particles of mineralized and demineralized bone, hydroxyapatite (HA), and eggshell were grafted onto the chorioallantoic membranes (CAMs) of chick embryos. After 3 or 6 days, portions of CAMs with associated grafts were harvested, processed for light and electron microscopy, and examined for the presence of multinucleated giant cells with the morphological characteristics of osteoclasts. Light microscopic examination revealed that, within only 3 days, many particles of mineralized materials had become surrounded or engulfed by multinucleated giant cells. Ultrastructurally, all such cells possessed a vacuolated and mitochondriaenriched cytoplasm, but they differed in the nature of the contacts formed at the cell-particle interface. With eggshell, the cells developed filopodia but lacked clear zones and ruffled membranes. With HA, clear zones were evident but cytoplasmic extensions and membrane ruffling were absent. Implants of mineralized bone, however, elicited the formation of giant cells with prominent clear zones and ruffling of the plasma membrane like that observed in bonafide osteoclasts. In contrast, grafts of demineralized bone did not evoke giant cell formation but rather recruited two cell types morphologically akin to either fibroblasts or macrophages. We conclude that the factor(s) responsible for osteoclast differentiation resides specifically within bone matrix and is intimately associated with the mineral phase. Further, in response to such a factor(s), osteoclast differentiation can occur ectopically, outside of the developing vertebrate body.

Enhanced healing of cutaneous wounds in rats using beads with positively charged surfaces.

The efficacy of electrical fields in soft-tissue repair is unclear. Materials with a charged surface provide a localized charged environment. We examined the effects of surface-charged particles in wound healing in rats with paired dorsal incisions with one side serving as a control. Tensiometry demonstrated that after 10 days, wounds with positively charged particles were 53 percent stronger (p less than 0.001) than controls (10 rats, 30 wound strips), whereas differences with negatively charged (6 rats, 15 strips) or uncharged beads (11 rats, 33 strips) were insignificant. Histologically, wounds with positively charged particles were characterized by large quantities of collagen-rich connective tissue and by prominent bead-associated giant cells. At 94 days, no differences in wound strength were noted. This method of creating charged local environments has potential clinical implications and may add insights into the behavior of cells in response to charged stimuli.

Osteoclast precursor cells are present in the blood of preossification chick embryos

Abstract In an attempt to ascertain the time of appearance of circulating osteoclast precursor cells, we have transplanted quail bone rudiments into or onto the extraembryonic membranes of variously aged chick embryos. We observed that osteoclast precursor cells (1) are present in the embryonic circulation prior to the onset of osteogenesis, (2) differentiate precociously in response to a factor or factors present in developing bone rudiments, and (3) increase in number until about midway in embryonic life.

Enhancement of repair in experimental calvarial bone defects using calcium sulfate and dextran beads

Granular medical-grade calcium sulfate hemihydrate (MGCSH) and negatively charged dextran beads (DB) were evaluated both separately and in combination in cranial defects. Thirty-six rats received bilateral parietal inlay reconstructions using MGCSH, MGCSH plus DB, or DB alone or the defects were left unfilled. Postoperative evaluation at 45, 90, and 135 days was done by visual inspection, caliper measurements of defect thickness, and histologic examination. The best fibro-osseous repair of the defects occurred with the use of MGCSH in combination with DB. Repair tissue, which was confluent with the adjacent cranium, was noted to have good thickness, and a high bone content. By contrast, defects repaired with either DB or MGCSH alone had poorer quality repair tissue, with concavities, voids, bead migration, decreased thickness, and minimal bony replacement/ingrowth. Unfilled control defects showed only a thin, friable, translucent connective tissue layer of repair. No method produced complete bony closure.

Osteoclast recruitment in response to human bone matrix is age related

The effect of bone matrix age on the recruitment and differentiation of osteoclast precursors was studied using the chick chorioallantoic membrane (CAM) implant system. Devitalized mineralized bone particles (75-250 microns) were prepared from human femoral cortical bone obtained postmortem from 8 men (age range: 18-72 years). The particles were implanted onto the CAM and 8 days later implants were harvested and processed for light microscopic, morphometric or immunohistochemical analysis. Histomorphometric analysis was performed on samples representing each donor age. The analysis was grouped into three categories consisting of bone from young adults (18-20 years), adults (34-53 years) and aged individuals (67 years and older). Total osteoclast number, osteoclast number per bone particle, cell area, cell size, number of nuclei per cell profile, nucleocytoplasmic ratio, and the presence of a distinctive osteoclast antigen defined by monoclonal anti-body 121F were determined. Bone matrix from older individuals, and therefore the oldest age group (67 years and older), elicited significantly fewer multinucleated cells when compared to bone matrix from younger donors. The number of nuclei per cell profile was highest in the adult population (34-53 years), and there was a continuous increase in cell area with aging. As a consequence, the nucleocytoplasmic ratio decreased from the youngest to the oldest age group. These findings indicate that, relative to factors that affect the recruitment and differentiation of osteoclast precursor cells, bone matrix of older individuals is changed in quality and/or quantity compared to bone matrix from younger individuals. It is hypothesized that this decline in osteoclast formation in response to older bone matrix may contribute to the impaired bone remodeling associated with aging.

Osteoclast formation is related to bone matrix age

SummaryLittle is known about the relationship between the age of the skeleton and the development of multinucleated bone-resorbing cells, osteoclasts. It has been shown that mineralized bone implanted onto the chick chorioallantoic membrane (CAM) is effective in the recruitment and differentiation of osteoclast precursors. In studies reported here we used the CAM system to examine the influence of bone matrix age on osteoclast formation. Devitalized mineralized bone particles (75–250 μm) were prepared from rats of various ages (2, 4, 9, 12, and 16 months). The particles were implanted onto the chick chorioallantoic membrane and 8 days later implants were harvested and processed for morphometric or immunohistochemical analysis. Osteoclast number, cell area, nucleocytoplasmic ratio, and the presence of a distinctive osteoclast antigen, defined by the 121F monoclonal antibody, were determined. Bone particles of each age group resulted in the formation of osteoclast-like giant cells. Compared with multinucleated cells that formed in response to bone particles obtained from 2-month-old rats, matrix from the oldest age group (16 months) elicited significantly fewer and smaller cells which contained a smaller number of nuclei. These data suggest that with aging, bone undergoes qualitative and/or quantitative changes that affect the recruitment and differentiation of osteoclast precursor cells.

Efficacy of positively charged ion exchange beads in radiation-impaired models of wound healing

We have previously demonstrated in a rat surgical incision model that charged dextran beads enhance wound repair in a charge-specific manner: positively charged beads increased wound breaking strength (WBS), whereas neutral or negatively charged beads were ineffective. The present work extends these observations into two models of radiation-impaired healing. One hundred five rats were divided into three groups: group 1, controls, no irradiation; group 2, total body irradiation; group 3, surface irradiation. Three days after irradiation, 0.1 ml of a suspension of either positively charged beads, neutral beads, or vehicle alone was applied to paired 6-cm incisions on the dorsum of the rats. Ten days later, wounds were excised and sample strips were harvested for determination of WBS and for histological analysis. Following total body irradiation, positively charged beads enhanced WBS 84% compared to beadless controls. When results with positively charged beads were compared to those with neutral beads, a 39% increase in WBS was noted. Healing, as measured by WBS, was more impaired in surface-irradiated animals, but when results with positively charged beads were compared with those obtained with neutral beads, a similar degree of healing (+38%) was noted. Neutral beads proved ineffective. Histology revealed that neutral beads evoked a modest foreign body response, yet there was a consistently greater clustering of giant cells around positively charged beads in wound sites. This paralleled in vitro experiments demonstrating increased monocyte aggregation around positively charged beads in tissue culture. The results suggest that positively charged ion exchangers, such as these positively charged beads, perhaps through their recruitment and/or activation of wound macrophages, may have a potential clinical role in the treatment of impaired wounds.

Acidosis, Hypercalcemia, and Hyperphosphatemia in Rat Fetuses near Term and Effects of Maternal Acid/Base Loading

Summary In the rat fetus, under normal conditions, blood calcium, phosphorus, and pCO2 rise, and pH declines, during the last 6 days of gestation. Little or no correlation is seen between changes noted in the maternal organism and those occurring in her unborn offspring. Oral administration of NH4Cl to pregnant females results in a lowering of maternal blood pH, with no change in blood calcium, phosphorus, or respiratory gases. NaHCO3 administration does not alter any of these parameters. In contrast, profound changes are observed in the fetuses of both acid- and alkali-treated dams. With NH4Cl feeding, fetal pH falls while blood calcium, phosphorus, and pCO2 rise; with NaHCO3 administration, fetal blood pH increases and serum calcium, phosphorus, and pCO2 decline. These observations point strongly to an association between fetal blood pH and circulating levels of calcium and phosphorus, and suggest that the levels of these elements may be, in part, determined by the acid/base status of the organism in utero. The authors wish to express their appreciation to Dr. Carl H. Smith and Mrs. Donna Breidenbach for their help and for making available facilities at Childrens Hospital where all pH and blood gas determinations were done.

Positively charged dextran resin inhibits trabecular bone repair in the rabbit tibial physis

Because exposure to positively charged dextran resin (PCDR) inhibits the growth of cultured rat and human bone cells, we tested the hypothesis that PCDR might inhibit bone repair in vivo. Central physeal defects were created by drilling 3.0-mm holes from the proximal tibial plateau into the metaphysis. The defects in left tibiae were packed with neutral resin (control); those in right tibiae were filled with PCDR. At the end of the 1st, 2nd, 3rd, and 10th postoperative weeks, the outcomes were quantitated by documenting the percent trabecular bone volume within the defect. The PCDR-filled defects showed a significant decrease in trabecular bone formation as early as the 2nd week. By the 10th postoperative week, formation of trabeculae had been reduced by nearly 40%. The inhibition conferred by PCDR suggests that the resin could be used as a suppressive interpositional material.

Antitumor effect of positively charged resin in the hamster cheek pouch model

Following the signal observation that contact with positively charged dextran resin (PCDR) inhibited the growth of cultured mammary (Hs578T and MDA-MB-231), pancreatic (H2T), and myeloma (RR-658) tumor cell lines, studies were developed in the hamster cheek pouch model using hamster H2T pancreatic tumor cells to determine if the antiproliferative effect of PCDR could inhibit tumorigenesis. In these studies, the control population represented groups injected with H2T cells alone or in combination with either neutral or negatively charged resin. When cells (5 x 10(2) to 1 x 10(5)) and PCDR were administered simultaneously, the tumor incidence (percent engraftment) and growth of tumors that already had been established were significantly reduced. When PCDR was injected into already established 1-35-mm2 H2T tumors (engraftment for 21 days = 96%), the resin suppressed the growth of the smallest tumors (< 10 mm2). In none of these trials was the somatic growth of the host hamsters affected. PCDR contact with H2T cells in vitro for 4 days or used to treat growing solid tumors for 72 days significantly reduced cellular ornithine decarboxylase activity. While the mechanism of PCDR action has not been established, the observations have implications for in vivo tumor therapeutic models.