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.

Altered expression of bone sialoproteins in vitamin D-deficient rBSP2.7Luc transgenic mice.

Bone sialoprotein (BSP) and osteopontin (OPN) are two major noncollagenous matrix proteins in mineralized connective tissue that have discrete roles in bone matrix formation, mineralization, and remodeling. The osteotropic secosteroid, 1,25-dihydroxyvitamin D3, a potent regulator of bone remodeling required for normal bone development, has been shown to exert differential effects on OPN and BSP expression by bone cells in vitro. To investigate these effects in vivo, we induced vitamin D3 deficiency in a transgenic mouse line (rBSP2.7Luc) that has a 2.7 kb rat BSP promoter linked to a luciferase reporter gene in its genome. Pregnant rBSP2.7Luc mice were fed vitamin D3-deficient food and demineralized water for 6 weeks. Their offspring were weaned at 3 weeks of age and then fed vitamin D-deficient food for an additional week. The control group were fed normal rodent pellets and water during the entire experimental procedure. Bone tissues from 40, 4-week-old offspring in each group were analyzed for BSP, OPN and luciferase expression. Vitamin D3-deficient mice displayed a rachitic phenotype that included reduced size and malformation of bones. Assays of the BSP promoter transgene in calvariae, mandibles, and tibiae of the rachitic mice showed increases in luciferase activity of 3.1-, 1.9-, and 4.6-fold, respectively, when compared with control littermates. Semiquantitative reverse transcriptase polymerase chain reaction assays of BSP mRNA revealed increases of 7-, 74-, and 66-fold, respectively, in the same rachitic bones, while OPN mRNA was reduced 12.5-fold in calvariae and 2-fold in tibiae and mandibles. In situ hybridization using mouse cRNA probes revealed that the increased BSP expression and decreased OPN expression in the vitamin D3-deficient mice was primarily in osteoblastic cells on the surface of calvariae and endosteal spaces of alveolar bone, on newly formed epiphyseal bone, and in cementoblasts and in hypertrophic chondrocytes. These studies are the first to show that BSP and OPN are differentially regulated by vitamin D3 in vivo, reflecting the diverse roles of these protein in bone remodeling. Moreover, the increased expression of the BSP transgene in the rachitic mice demonstrates that vitamin D3 regulation of BSP expression is mediated, in part, by element(s) within the 2.7 kb promoter region.

Osteoblast response to bioactive glasses in vitro correlates with inorganic phosphate content.

Inorganic phosphate (Pi) is a physiological regulator of osteoblasts and chondrocytes, suggesting that phosphate may contribute to the biological response of these cells to bioactive glasses like Bioglass 45S5, which is composed of 45% SiO2, 24.5% CaO, 24.5% Na2O, and 6% P2O5. We investigated the effect of varying the Pi content of bioactive glass disks (0%, 3%, 6% and 12% P2O5) using human osteoblast-like MG63 cells as the model. Cell number on 6% Pi disks was comparable to cultures on tissue culture plastic, but was reduced at higher and lower Pi concentrations. Alkaline phosphatase specific activity of isolated cells and cell layer lysates, as well as PGE2, TGF-beta1 and NO levels in conditioned media, were elevated in cultures grown on bioactive glass and varied with the Pi content. The greatest effects were observed in cultures grown on disks with the lowest Pi concentrations. Thus, growth on the bioactive glasses enhances cell function in comparison with tissue culture plastic and lower Pi content favors osteoblast differentiation.

The effects of formocresol on rat sponge implant tissue: a biochemical study.

An investigation was conducted on the effect of formocresol treatment of the aqueous extractability and enzymatic susceptibility of excised implant tissue. The treated tissue demonstrated a decreased solubility and a diminished digestibility by trypsin, pepsin and collagenase. However, its reactivity toward hyaluronidase showed little alteration.

A biochemical study of two bifunctional reagents as alternatives to formocresol.

Two bifunctional reagents, glutaraldehyde and dimethylsuberimidate, were compared to formaldehyde with respect to fixation and modification of protein. Glutaraldehyde proved the superior fixative based on cross-linking and enzyme degradation assays. An immunoprecipitation assay suggests that the reaction products of glutaraldehyde may have altered antigenicity.

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.

Distribution, metabolism, and excretion of [14C] glutaraldehyde.

Radiolabeled glutaraldehyde (GA) was infused into rats in order to determine its distribution between cellular and humoral fractions of the blood, its potential metabolism by RBC's, its rate of excretion, and the nature of its urinary products. This study demonstrated that [14C]GA was distributed between the RBC's and plasma at a ratio greater than 1. Although the absolute counts of both fractions dropped 80% over 3 days, the percentage bound or incorporated increased over time. Despite the extensive uptake by RBC's these cells were unable to metabolize GA to CO2. Urinary excretion of the radiolabel was rapid; the predominant form in the urine was less than 1 kDa in size. All evidence suggested that it was not native GA. We conclude that the RBC's can incorporate GA, but can not metabolize it completely to CO2. Nevertheless, much of the infused GA was rapidly converted to nonreactive metabolites and eliminated by the kidney.

Osteocalcin expression in young and aged dental pulps as determined by RT-PCR.

Dental pulps were obtained from third molars of young adults (17-25 yr) or from molar teeth of individuals > 50 yr of age and examined for the expression of osteocalcin (OC) mRNA by RT-PCR. OC was selected as a determinant of pulp vitality, because it has long been associated with the production of hard tissue matrix in teeth and bone. For comparative purposes, the expression of OC in each pulp was normalized relative to its housekeeping gene-product GAPDH by the establishment of a OC/GAPDH ratio. This study demonstrated that OC expression, presumably by cells of odontoblast lineage, does not diminish relative to the extant cell population. Our findings suggest, despite a reduction in volume and cell numbers, that the pulps of aging teeth retain a capacity for dentin deposition and a potential for caries and trauma resistance.

Mandibular implants and the growing patient.

The dynamic relation of the anteroposterior and rotational growth of the mandible to the transverse arch width and dental height changes must be understood before placing endosseous implants in actively growing patients. Research models demonstrate that osseointegrated implants lack the compensatory growth mechanism of the natural dentition. Remodeling associated with skeletal growth in the region of the implant placement site could cause the implant to either become unsupported by bone or submerged within it. Implants placed after age 15 in girls and 18 in boys have the most predictable prognosis. When placed in the growing patient, dental implants should be closely monitored and carefully restored with implant prostheses designed to accommodate growth and development.

Maxillary implants and the growing patient.

Maxillary skeletal and dental growth results in dramatic changes in all three dimensions during active growth. Experimental evidence and the behavior of ankylosed teeth suggest that an osseointegrated object remains stationary in the bone surrounding it and does not move or adapt to bone remodeling. Growth changes may result in the burying or loss of implants depending on the placement site. Hence, implants placed in the early mixed dentition have a poor prognosis of continued usefulness through puberty. When placed early, implants may disturb growth or have to be replaced. Implants placed during late puberty or early adulthood have the best change for long-term usefulness.

Current and potential pulp therapies for primary and young permanent teeth.

OBJECTIVES: This paper aims to alert the dental practitioner to the rapidly evolving therapies for treating the pulps of primary and young permanent teeth. DATA SOURCES: Experimental research on animals, clinical studies and case reports. STUDY SELECTION: Indirect pulp capping, direct pulp capping, pulpotomies, and pulpectomies are standard procedures for treating primary teeth. However, direct pulp capping, heretofore not very successful, is being revisited. Based on studies in animals and clinical findings in humans, there has been a movement in pediatric dentistry to find alternatives to formocresol and calcium hydroxide for pulpotomy therapy. Venues range from eradication by cautery to the possibility of healing with growth factors. New studies with iodoform paste for pulpectomies are confirming the success rates of previous publications. The new dental adhesives are being tested as agents for direct pulp capping, as well as partial and complete pulpotomy protocols. CONCLUSIONS: More thought is being given by clinicians to preserving pulp, either through more ambitious indirect pulp therapy or partial pulpotomy. Formocresol and calcium hydroxide pulpotomies, while still popular, may soon be challenged by other chemical treatments, electrocautery or stimulation of reparative dentine by growth factors. Iodoform pastes are promising easier and more successful pulpectomy therapy. Total etch direct bonding materials could soon transform direct pulp capping, as well as partial and complete pulpotomy protocols.

Reviewing pulp treatment for primary teeth.

Deficiencies of pulpotomy and pulpectomy treatment of primary teeth have promoted considerable laboratory and clinical research. Some of these findings and their implications are reviewed.

Craniofacial growth.

Craniofacial growth represents a complex enlargement and differentiation of hard and soft tissues, characterized by cephalocaudal and allometric patterns. The cranial base matures earlier than the face and is used cephalometrically as a relatively stable reference area. The maxilla, although intimately associated with the cranium, exhibits some independent growth, particularly in the vertical dimension. The mandible, hanging in a sling of muscles and articulating only at the temporomandibular fossae and with the opposing dentition, is purportedly able to adapt its shape and position in space to some extent as a function of condylar growth.

Pulpotomy therapy in primary teeth: new modalities for old rationales.

Pulpotomy therapy for the primary dentition has developed along three lines: devitalization, preservation, and regeneration. Devitalization, where the intent is to destroy vital tissue, is typified by formocresol and electrocautery. Preservation, the retention of maximum vital tissue with no induction of reparative dentin, is exemplified by glutaraldehyde and ferric sulfate treatment. Regeneration, the stimulation of a dentin bridge, has long been associated with calcium hydroxide. Of the three categories, regeneration is expected to develop the most rapidly in the coming years. Advances in the field of bone morphogenetic protein (BMP) have opened new vistas in pulp therapy. Human BMPs with dentinogenic properties are becoming available through recombinant technology. We are now entering an era of pulpotomy therapy with healing as the guiding principle.

Assessment of the systemic distribution and toxicity of glutaraldehyde as a pulpotomy agent.

The systemic distribution of glutaraldehyde (GA) from a pulpotomized tooth of a rat was estimated to be 40 nanomoles or 25% of the applied dose. Metabolic studies disclosed that GA was eliminated in urine and expired gases; 90% was cleared from body tissues in 3 days. To evaluate the toxicity of GA, doses 500x greater than that systemically distributed from a pulpotomy site were infused into the jugular veins of rats. Twenty-four hours postinfusion the rats were evaluated in vivo for physiologic changes or sacrificed for biochemical and histologic evaluation of harvested tissues. Only one of the assays, a physiologic parameter, was altered by the 500x dose. Considering the relatively large dose administered and the limited effects, we conclude that GA would not be toxic when used as a pulpotomy agent.

Resorption of a calcium hydroxide/iodoform paste (Vitapex) in root canal therapy for primary teeth: a case report.

This case report presents a clinical and radiographic follow-up (38 months) of pulpectomy treatment performed on maxillary primary anterior teeth using Vitapex. Vitapex was resorbed extraradicularly and intraradicularly without apparent ill effect, and proved to be clinically and radiographically successful. The present case report illustrates that even if the paste resorbs within the canals, the clinical and radiographical outcome is excellent. A longer follow-up is recommended to evaluate if there is any effect on the permanent succedaneous tooth.

Early orofacial development.

Early orofacial development is a period of very rapid allometric growth and specialization of tissue. During the first three years of life, the brain achieves approximately 90% of its growth and the face reaches almost 65% of its adult size. Between birth and three years of age the oral cavity changes form and function, replacing gum pads with a dentition composed of twenty primary teeth. By three years of age, all succedaneous teeth are developing as well as the first permanent molars. Involvement with neonates and infants by neonatologists, pediatricians, and pediatric dentists begs a better appreciation of this rapidly changing period.

Effect of dentin adhesives on sealant bond strength.

This study evaluated the effect of four dentin adhesives on sealant shear bond strength. Five groups of 15 human extracted teeth (75 in total) were prepared to receive the following treatments: group 1, sealant only; group 2, sealant plus gluma primer; group 3, sealant plus gluma primer and sealer, group 4, sealant plus C & B Metabond. Each tooth had a bonding site prepared for each material by grinding a flat plane on the enamel with 600 grit. The enamel site was etched for 30 seconds, washed for 20 seconds and dried. Each material was mixed according to the instructions of the manufacturer. The sealant (Concise) was placed in a nylon cylinder, light-cured, stored in distilled water for 24 hours, thermocycled (100 cycles at 5 to 55 degrees C) and shear bond strength determined with a knife-edge blade in an Instron running at a crosshead speed of 0.5 mm/min. The results in MPa were: group 1, 11.8 +/- 4.5; group 2, 12.3 +/- 5.0; group 3, 10.3 +/- 1.9; group 4, 12.5 +/- 8.6; and group 5, 15.8 +/- 5.2. Statistically (ANOVA one way) there was no significant difference in the groups.