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.

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.

Bioelectrical investigations have long shown that surfaces of bone formation and resorption are negatively and positively charged respectively. We also know that in a number of experimental situations [1], implants of negatively-charged ion exchange resin (NCR= Sephadex, CM)are osteotropic, and that implants of positively-charged resin (PCR= Sephadex DEAE) strongly inhibit bone formation [2]. While the cellular mechanism of action for NCR is thought to involve the local production of transforming growth factor beta [3], the mechanics of PCR action is an unknown. Our laboratory has shown that PCR stunts the in vitro growth of medullary osteoprogenitor cells, normal and transformed osteoblasts, and a number of tumor cell lines [4], PCR was also able to strongly inhibit hamster pancreatic cell engraftment and the growth of established pancreatic cell tumors.

The osteogenic response to subperiosteal injection of negatively charged ion exchange resins was compared in the tibiae of one-month and 16- to 22-month-old rats. The resins were administered either in the form of beads (CM Sephadex) or as particles (CM cellulose, carboxymethylcellulose), and the animals were killed at two weeks and at one month after injection. Histologically, the resins did not produce an inflammatory response. Periosteal bone formation was observed wherever resin was in contact with bone, and in the resin bed the connective tissues that invested the charged materials ossified within the first month. Marrow spaces commonly formed where periosteal growth was most rapid. The osteogenic effect was independent of resin conformation, and it was more pronounced in the younger rats.

We have examined the effects of modulating nitric oxide (NO) levels on osteoclast-mediated bone resorption in vitro and the effects of nitric oxide synthase (NOS) inhibitors on bone mineral density in vivo. Diaphorase-based histochemical staining for NOS activity of bone sections or highly enriched osteoclast cultures suggested that osteoclasts exhibit substantial NOS activity that may account for basal NO production. Chicken osteoclasts were cultured for 36 hr on bovine bone slices in the presence or absence of the NO-generating agent sodium nitroprusside or the NOS inhibitors N-nitro-L-arginine methyl ester and aminoguanidine. Nitroprusside markedly decreased the number of bone pits and the average pit area in comparison with control cultures. On the other hand, NOS inhibition by N-nitro-L-arginine methyl ester or aminoguanidine dramatically increased the number of bone pits and the average resorption area per pit. In a model of osteoporosis, aminoguanidine potentiated the loss of bone mineral density in ovariectomized rats. Aminoguanidine also caused a loss of bone mineral density in the sham-operated rats. Inhibition of NOS activity in vitro and in vivo resulted in an apparent potentiation of osteoclast activity. These findings suggest that endogenous NO production in osteoclast cultures may regulate resorption activity. The modulation of NOS and NO levels by cells within the bone microenvironment may be a sensitive mechanism for local control of osteoclast bone resorption.

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.

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 μm) 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. Is is hypothesized that this decline in osteoclast formation in response to older bone matrix may contribute to the impaired bone remodeling associated with aging.

Little 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.

To test for their osteogenic stimulating capacity, charged beads were implanted into cranial or mandibular defects, used as an onlay on the nasal bone surface, or injected into femoral medullary cavities of young adult rats. One month later, negatively charged beads were found to have stimulated extensive bone formation resulting in closure of craniofacial defects, a new layer of bone on the nasal bone surface, and a bead-bone lattice within marrow cavities of long bones. Positively charged beads were nonosteogenic, but elicited a pronounced fibroblastic response in the craniofacial skeleton. Positively charged beads were found associated with multinucleated giant cells at all implantation sites. Uncharged beads failed to elicit formation of new bone and were associated with connective tissue that was less cellular and less organized than was seen with positively charged beads. It was concluded that beads that have ben chemically treated to confer either a negative or positive surface charge, when placed in contact with bone, evoke osteogenesis or formation of dense connective tissue, the response depending on the surface charge of the bead. The mechanism(s) by which the charged beads foster the osteogenic or fibroblastic response is not clear. The use, however, of alloplastic materials with charged surfaces in repair and augmentation of bone, and in wound repair, warrants further investigation.