Myelointegration of titanium implants: B lymphopoiesis and hemopoietic cell proliferation in mouse bone marrow exposed to titanium implants.

Multinucleated giant cells have been observed at interfaces between bone marrow and titanium implants in mouse femurs. This raises concern that macrophage-derived factors might perturb local lymphohemopoiesis, possibly even predisposing to neoplasia in the B lymphocyte lineage. It has been found that an implant-marrow interface with associated giant cells persists for at least 1.5 years. Precursor B cells show early increases in number and proliferative activity. At later intervals, however, they do not differ significantly from controls, and there are no perturbations in spatial localization of either B lineage cells or DNA-synthesizing hemopoietic cells. The results of this investigation in mice demonstrate that, following initial marrow regeneration and fluctuating precursor B cell activity, and despite the presence of giant cells, titanium implants apparently become well-tolerated by directly apposed bone marrow cells in a lasting state of "myelointegration."

Characterization of a 4.2-kb plasmid isolated from periodontopathic spirochetes.

Oral anaerobic treponemes are assoicated with active periodontal disease and may comprise up to 57% of the microbiota in periodontal pockets. Four treponeme strains (designated U2a, U2b, U9b, and U9c) isolated from clincial cases were found to harbor a new 4.2-kb plasmid when plasmid DNA was extracted and purified employing the Qiagen Plasmid Kit. This plasmid differs from the smaller plasmids (2.0-, 2.6-, and 2.7-Kb) reported previously by others in Treponema denticola. The newly discovered 4.2-kb plasmid was found to be the same in all four treponeme strains by restriction endonuclease analysis. It is a circular plasmid since restriction with PstI, Pvu II, Sma I, Xma I, Ava 1 or Bam HI produced a single band of the same size. Bacterial strain U2b was shown to be Treponema socranskii and U9c to be T. denticola. The plasmid is designated "pTS1". The presence of the same plasmid in different species of the treponemes isolated from the same patient suggests the possibility of a naturally occurring genetic transfer system within the oral spirochetes or their ability to take up and maintain mobilizable plasmids.

Enumeration of viable oral spirochetes from periodontal pockets.

We recently developed a successful method for quantifying oral anaerobic spirochetes in pure culture by a viable count. New oral spirochete medium was used with low temperature-gelling agarose in polystyrene tissue-culture flasks. We have extended the use of this method to determine the viable count of spirochetes from periodontal pockets. Sixteen subgingival plaque samples were obtained by insertion of sterile paper points into deep periodontal pockets. The points were placed into reduced transport medium at chairside, vortexed in the microbiology laboratory and aliquots of the medium inoculated into molten new oral spirochete-agarose medium (37 degrees C) containing rifampin (20 micrograms/ml) in a flask. Subsequent dilutions were made from this initial flask to other flasks containing selective medium in sequence. All flasks were incubated anaerobically. Most other subgingival bacteria were selectively inhibited by rifampin. Spirochete colonies were typically spherical and were either dense or cottony. Their identities were checked by darkfield examination. Counts of colony-forming units of cultivable spirochetes ranged from 12.5% to 28.2% of the total cultivable anaerobic flora by the method described.

Response of bone marrow to titanium implants: osseointegration and the establishment of a bone marrow-titanium interface in mice.

A method has been developed to examine the relationships between titanium implants in bone and the immune and hemopoietic cell populations in the adjacent bone marrow; it involves the use of miniaturized implants in the diaphysis of mouse femurs. After surgical placement of the implants, mice were sacrificed and the femurs were either embedded directly in Epon for preparation of ground sections or were decalcified in EDTA, embedded in Epon, subjected to a fracture technique to remove the implant, and sectioned for light and electron microscopy. Scanning electron microscopy revealed that the surfaces of implants removed in this way were virtually free of tissue remnants. Ground sections showed bone in direct contact with the implant surface, providing the first evidence of osseointegration in mice. In addition, an extensive surface of the implant interfaced directly with regenerated bone marrow, a condition that persisted for at least 18 weeks. Some bone marrow cells forming an incomplete layer in contact with the titanium interface had morphologic characteristics of macrophages and giant multinucleated cells. The results demonstrated a long-term integration between titanium implants and cellular elements of bone marrow and provide an experimental model to examine the possible implications of this interaction on the processes of lymphopoiesis and hemopoiesis.

Changes in the populations of null, NK1.1+, and Thy1lo lymphocytes in the bone marrow of tumor-bearing mice: effect of indomethacin treatment.

Mouse bone marrow produces many "null" lymphocytes which lack B and T lineage markers (B220-Thy1-). A subset of these cells expresses the natural killer (NK) cell marker, NK1.1. In addition, some rapidly renewed bone marrow lymphocytes express low intensities of Thy1 (Thy1lo). In view of their possible implication in tumor-host interactions these various cell populations have now been examined in mice injected with either the nonmetastatic Ehrlich ascites (EA) tumor or the Lewis lung carcinoma (LLc), a highly metastatic solid tumor. In each case, the number of null lymphocytes, as defined by a lack of radioautographic labeling of either B220 glycoprotein or Thy1, increased markedly in both the bone marrow and spleen. Treatment with the prostaglandin inhibitor, indomethacin, enhanced the increase in null cells in the bone marrow and spleen of LLc-bearing mice. The number of null small lymphocytes expressing NK1.1, as detected by combined radioautographic and immunoperoxidase techniques, increased almost 30-fold in LLc-bearing mice. The number of Thy1lo small lymphocytes increased in parallel with null cells during EA tumor growth. The findings accord with the hypothesis that the null lymphocyte population produced in mouse bone marrow includes newly formed NK lineage cells which sequentially express NK1.1 and Thy1lo. The present work demonstrates that the populations of null, NK1.1+, and Thy1lo lymphocytes in mouse bone marrow expand rapidly during the early growth of transplanted tumors, the initial increase in null lymphocytes apparently being curtailed by prostaglandin production. The results suggest that the production of null lymphocytes in mouse bone marrow is responsive to tumor development, possibly providing cells to be involved in tumor-host interactions.

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.

Heterogeneity of bone marrow lymphocytes: radioautographic detection of pre-B cells bearing cytoplasmic mu chains, and of B and T lymphocytes, and characterization of null lymphoid cells.

A radioautographic immunolabeling technique has been developed to detect pre-B cells bearing cytoplasmic mu chains among populations of bone marrow lymphoid cells identified by conventional hematologic stains. 125I-Anti-mu antibody was applied either to fixed marrow smears, labeling total mu chains both in the cytoplasm (c mu) and at the cell surface (s mu), or to cell suspensions, labeling s mu alone. In stained radioautographs the incidence of c mu+ s mu- pre-B cells was derived both indirectly by subtracting values for s mu+ cells from those for total mu+ cells of various sizes in normal mice and directly by the total mu chain labeling in mice depleted of s mu+ cells by anti-IgM treatment in vivo. Binding specificity was demonstrated by the displacement of labeling by nonradioactive anti-mu antibody. The c mu+ s mu- cells showed a bimodal size distribution. They accounted for 40% of the large lymphoid cells and 30% of the small lymphocytes in the marrow. A further 50% of the small lymphocytes were B lymphocytes (s mu+) and 8% were T lymphocytes (Thy 1.2+). Thus, the technique demonstrates the presence of c mu+ s mu- pre-B cells among both proliferating large lymphoid cells and nondividing small lymphocytes, as classically defined in marrow smears. In addition, the results reveal a broad size distribution of mu- lymphoid cells, including a subset of small lymphocytes which lack c mu, s mu, and Thy 1.2 and thus cannot be assigned to either B or T lineage by these criteria. The findings suggest that in addition to B cells the marrow may produce other types of lymphoid cells, yet to be defined.

Maturation of bone marrow lymphocytes. IV. Kinetics of maturation and renewal of lymphocytes expressing Ia and H-2K antigens.

As part of a series of studies on bone marrow lymphocyte differentiation, I region-associated (Ia) and H-2K antigens have been quantified on small lymphocytes in mouse bone marrow and spleen, and correlated with cell renewal. Ia antigens were revealed radioautographically on some marrow small lymphocytes, the antigen density being lower than on spleen cells. Kinetic studies, combining surface rosetting and [3H]-thymidine labelling, showed that most Ia antigen-bearing lymphocytes were newly formed cells, rapidly renewed in the marrow. Ia antigens were first expressed after a post-mitotic lag period and increased in density with time. A minority of Ia antigen-bearing small lymphocytes in the marrow and a large majority of those in the spleen remained unlabelled by [3H]-thymidine, infused for 3-5 days. Some putative progenitor cells in the marrow showed surface Ia antigens of medium density. In neonatal mice the incidence and density of Ia antigens were low in the marrow and spleen, reaching adult values by 4 and 10 weeks of age, respectively. In contrast, H-2K antigens were expressed in high density on all lymphoid cells in both marrow and spleen from early postnatal life onwards. Thus, Ia antigens, but not H-2K antigens, behave as differentiation markers in the maturation of small lymphocytes produced in the bone marrow. In addition, some Ia antigen-bearing small lymphocytes in adult bone marrow are slowly renewed cells, putative long-lived immigrants from the recirculating pool.