Differential effects of bone graft substitutes on regeneration of bone marrow.

BACKGROUND: This study used a rat tibial marrow ablation model to test the hypothesis that bone remodeling within the medullary canal varies with bone graft materials of different chemical compositions and structural properties, impacting marrow cavity restoration. Bone graft materials were selected based on their relative resorption or degradation in vivo and their osteogenic properties. METHODS: Following ablation of the right tibial marrow in male Sabra-strain rats, materials were implanted in the proximal marrow cavity: poly-D,L-lactide-co-glycolide 75 : 25 (PLGA); coralline-hydroxyapatite (HA), calcium-sulfate (CaSO4), collagen-HA-tricalcium phosphate granules, anorganic bovine bone mineral, demineralized bone matrix (DBM), 45S5 Bioglass (BG), PLGA with BG 50 : 50, PLGA : BG 80 : 20, and PLGA and PLGA:BG 50 : 50 plus bone marrow (BM). Control tibias were ablated but received no implants. At 2 (endosteal bone healing), 4 (marrow cavity remodeling) and 8 weeks (marrow restoration), six to eight animals per group were euthanized and tibias processed for histomorphometry of proximal and distal medullary canals. RESULTS: Control tibias showed primary bone in proximal and distal medullary canals at 2 weeks, with trabeculae surrounded by cellular marrow. At 4 and 8 weeks, control trabeculae were thinned and marrow had more fat cells. In the treated tibias, trabecular bone volume (TBV) varied with time and was material specific. Most implants supported comparable TBV at 2 weeks. Sites with CaSO4 or DBM exhibited decreased TBV with time whereas trabecular bone was retained in proximal tibias containing other materials, closely juxtaposed to the implants. TBV did not always correlate directly with implant volume, but changes in BM volume were correlated inversely with TBV. Addition of BM increased marrow restoration in sites containing PLGA; however, BM reduced restoration of marrow when added to PLGA : BG. Although the presence of implants in the proximal tibia resulted in retention of trabecular bone, there was a time-dependent reduction in TBV in distal canals; the rate and extent of the distal TBV reduction were implant dependent. CONCLUSIONS: Thus, although many materials can support bone formation in the marrow cavity, bone quality, quantity, and physical relationship to the implant, and its rate of resorption differ in a material-dependent manner, resulting in differences in the restoration of marrow. CLINICAL RELEVANCE: Bone graft materials should be selected not only for their ability to support new bone formation but also for their impact on the remodeling phase of bone healing.

Osteoinductivity of demineralized bone matrix in immunocompromised mice and rats is decreased by ovariectomy and restored by estrogen replacement.

The osteoinduction potential of human demineralized bone matrix (DBM) in females with low estrogen (E2) is unknown. Moreover, the osteoinductivity of commercial human DBM is tested in male athymic rats and mice, but DBM performance in these animals may not reflect performance in female animals or provide information on E2's role in the process. To gain insight, human DBM was implanted bilaterally in the gastrocnemius of twenty-four athymic female mice (10 mg/implant) and twenty-four athymic female rats (15 mg/implant). Eight animals in each group were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized with E2-replacement (OVX+E2) via subcutaneous slow release capsules of 17beta-estradiol. OVX and OVX+E2 animals were pair-fed to SHAM animals. Four animals from each group were euthanized at 35 days and four at 56 days. Animal weight, uterine weight, and blood estrogen levels confirmed that pair feeding, ovariectomy, and E2 replacement were successful. Histological sections of implanted tissues were evaluated qualitatively for absence or presence of DBM, ossicle formation, and new bone or cartilage using a previously developed qualitative scoring system (QS) and by histomorphometry to obtain a quantitative assessment of osteoinduction. OVX mice had a small but significant QS decrease at 35 days compared to SHAM mice, confirmed by quantitative measurement of ossicle, marrow space, and new bone areas. The QS in rats was not affected by OVX but histomorphometry showed decreased new bone in OVX rats, which was restored by E2. The QS indicated that the number of new bone sites was not reduced by OVX in rats or mice at 56 days, but the relative amount of new bone v. marrow space was affected and differed with animal species. Residual DBM was less in OVX animals, indicating that DBM resorption was affected. Cartilage was present in rats but not in mice, suggesting that endochondral ossification was slower and indicating that bone graft studies in these species are not necessarily comparable. These results show the importance of E2 in human DBM-induced bone formation and suggest that E2 may be needed for clinical effectiveness in post-menopausal women.

Subcellular localization of glutaraldehyde.

Glutaraldehyde (GA) has been proposed as an alternative to formocresol for pulpotomies in primary teeth and as an irrigant in root canal therapy. These studies were undertaken to determine if GA can associate with the nucleus of living cells, thereby posing a mutagenic threat. Rats were infused IV with 14C-GA and killed 5 min and 1 h later. The cystolic, membrane, and nuclear fractions of harvested liver cells were separated and analyzed for radioactivity. We determined that significant radioactivity was located in the cytosol and membrane fractions, but not in the nuclear fraction. In an in vitro experiment, liver slices were incubated with 14C-GA in sealed vials in which 14C-CO2 was captured. After 1 h the nucleic acids of the liver slices were isolated and counted. In vitro the liver cells incorporated and metabolized GA to CO2 but no significant label could be detected in the isolated nucleic acids. We concluded from these experiments that GA which was incorporated into liver cells did not reach the nucleus to a significant extent, and that its potential for mutagenicity in the context of pulp treatment was nil.

Assessment of the systemic distribution and toxicity of formaldehyde following pulpotomy treatment: Part two.

The active constituents of formocresol, formaldehyde and cresol, are known toxic agents. The authors administered increments of formaldehyde until systemic morbidity was demonstrated so that tissue damage could be equated with the number of concurrent pulpotomies required to achieve a toxic body load.

Assessment of the systemic distribution and toxicity of formaldehyde following pulpotomy treatment: Part one.

This report, the first of a two-part study, was undertaken to quantitate the systemic distribution of formaldehyde from a pulpotomy site, and to compare this level to doses that elicit overt systemic pathology. Maxillary first molars of rats were pulpotomized and treated with 14C-labeled formaldehyde, for 5 minutes. Additionally, four groups of rats were infused with 10, 20, 30, or 50 percent of the first quantity applied to the site. The data show that approximately 30 percent of the 14C-formaldehyde placed in the pulp chamber was distributed systemically; 50 percent to 59 percent was expired as CO2; and 2 percent was excreted.

The effect of alternatives to formocresol on antigenicity of proteins.

As part of an overall evaluation of possible substitutes for the pulpotomy agent formocresol, this study was initiated to compare the antigenicity of the reaction products of protein with formaldehyde, glutaraldehyde, or dimethylsuberimidate (DMS). Rabbits were injected with rabbit serum albumin (RSA) which had been treated with one of the following solutions: phosphate-buffered saline, 2% glutaraldehyde, 4% formaldehyde, or 2% DMS. The antisera from the rabbits were analyzed for elicited antibodies by the enzyme-linked immunosorbent assay (ELISA) and in a horseradish peroxidase (HRP) assay using a spot technique on nitrocellulose paper. These assays demonstrated that DMS-treated RSA was the most antigenic of the reaction products tested. The least provocative was the glutaraldehyde-treated RSA; the reaction product of formaldehyde was intermediate. Our findings suggest that if non-immunogenicity of a pulpotomy agent is a desirable property, then DMS does not meet the criteria of an alternative pulp fixative. In contrast, the relatively low antigenicity of glutaraldehyde reinforces other favorable findings which support its use clinically.

The effect of formocresol and glutaraldehyde on certain enzymes in bovine dental pulp.

The effect of a 2-hour formocresol and glutaraldehyde treatment on two enzymes of bovine pulp was measured. Lactic dehydrogenase, a respiratory enzyme, was sharply affected by 0.5 percent and one percent glutaraldehyde and a 1:5 dilution of formocresol, exhibiting 7-, 71-, and 40-fold decreases in activity, respectively. Alkaline phosphatase was much less responsive to these same agents, giving only 4.5-, 17-, and 2.5-fold reductions after treatment, respectively. These findings support histochemical studies which have suggested the sensitivity of respiratory enzymes of the pulp to fixative medicaments.

Biochemical effects of formocresol on bovine pulp tissue.

Calf pulp was treated with full-strength formocresol, diluted formocresol, or saline for 4 hours. After washing and homogenization, the water extractable supernates were analyzed for total amino acid, carbohydrate, and hydroxyproline content. Additional samples were tested against trypsin, pepsin, collagenase, and hyaluronidase. Other tissue samples treated with 1/5, 1/10, and 1/25 dilutions of formocresol were subjected to trypsin and collagenase. The control tissue gave 50 per cent more extractable material, which contained over 300 per cent more total amino acids and hydroxyproline but only slightly more carbohydrate than the treated tissue. Formocresol treatment produced an 80 to 90 per cent reduction in reactivity to trypsin, pepsin, and collagenase but little change from hyaluronidase action. The increase in reactivity of the tissue to enzyme hydrolysis paralleled the increase in dilution of formocresol. These results indicate a profound effect on the protein fraction of pulp exposed to full-strength formocresol.