Mechanical Stretching of Mouse Calvarial Osteoblasts In Vitro Models Changes in MMP-2 and MMP-9 Expression at the Bone-Implant Interface.

Bone to mechanical loading elicits a biological response that has clinical significance for several areas in dental medicine, including orthodontic tooth movement, tempromandibular joint disease, and endosseous dental implant osseointegration. Human orthopedic studies of failed hip implant sites have identified increased mRNA expression of several collagen-degrading matrix metalloproteinases (MMPs), while in vitro experiments have shown increases in MMP secretion after exposure to inflammatory mediators. This investigation evaluates the effects of mechanical deformation on in vitro osteoblasts by assessing changes in MMP gene expression and enzyme activity. We seeded mouse neonatal calvarial osteoblasts onto flexible 6-well plates and subjected to continuous cyclic mechanical stretching. The expression and activity of mRNA for several MMPs (2, 3, 9, and 10) was assessed. When subjected to mechanical stress in culture, only mRNA specific for MMP-9 was significantly increased compared to nonstretched controls (P < .005). Measurement of MMP activity by gelatin zymography demonstrated that none of the MMPs showed increased activity with stretching; however, MMP-2 activity decreased. Our results suggest that in response to stretch, MMP-2 responds rapidly by inhibiting conversion of a MMP-2 to the active form, while a slower up-regulation of MMP-9 may play a role in the long-term remodeling of extracellular matrix in response to continuous mechanical loading. This study suggests that the regulation of metalloproteinases at both the mRNA and protein level are important in the response of bone to mechanical stress.

Histologic Evaluation of Osseous Regeneration Following Combination Therapy With Platelet-Rich Plasma and Bio-Oss in a Rat Calvarial Critical-Size Defect Model.

Platelet-rich plasma (PRP) is an autogenous source of growth factors shown to facilitate human bone growth. Bio-Oss, an osteoconductive xenograft, is used clinically to regenerate periodontal defects, restore dental alveolar ridges, and facilitate sinus-lift procedures. The purpose of this study was to analyze whether a combination of PRP and Bio-Oss would enhance bone regeneration better than either material alone. PRP and/or Bio-Oss were administered in an 8-mm critical-size defect (CSD) rat calvarial model of bone defect between 2 polytetrafluoroethylene membranes to prevent soft tissue incursion. Eight weeks after the induction of the CSD, histologic sections were stained with hematoxylin and eosin stain and analyzed via light microscopy. Qualitative analyses revealed new bone regeneration in all 4 groups. The Bio-Oss and PRP plus Bio-Oss groups demonstrated greater areas of closure in the defects than the control or PRP-only groups because of the space-maintaining ability of Bio-Oss. The groups grafted with Bio-Oss showed close contact with new bone growth throughout the defects, suggesting a stronger graft. The use of PRP alone or in combination with Bio-Oss, however, did not appear to enhance osseous regeneration at 8 weeks. Areas grafted with Bio-Oss demonstrated greater space-maintaining capacity than controls, and PRP was an effective vehicle for placement of the Bio-Oss. However, at 8 weeks this study was unable to demonstrate a significant advantage of using PRP plus Bio-Oss over using Bio-Oss alone.

Influence of matrix-suspended demineralized bone on osseous repair using a critical-sized defect in the rat (Rattus norvegicus) calvarium.

Demineralized freeze-dried bone (DFDB) in matrix form must be rehydrated with a carrier medium which allows for easy manipulation during periodontal surgery. The purpose of this study was to evaluate how human DFDB suspended in a polyol matrix affects new bone formation in the rat calvarium critical-sized defect (CSD) model. Fifty-five adult male Harlan Sprague-Dawley rats were assigned to 1 of 5 treatment groups: polyol, 100% DFDB, 47% DFDB/polyol, 47% DFDB, or an unfilled control. They were then placed into 8-m calvarial CSDs. The bone donor source company for the DFDB and DFDB/polyol groups was the same. Calvaria were harvested 10 weeks after surgery and evaluated histomorphometrically. The diameter of bone particles from the 3 groups containing DFDB was measured by scanning electron microscopy. There was no statistically significant difference in the percentage of bone fill between any of the groups, although the 100% DFDB group exhibited the most bone fill. The 47% DFDB/polyol and 47% DFDB groups had similar amounts of bone formation. The average size of the demineralized bone particles from the 100% DFDB group was significantly smaller than that of the other 2 groups containing DFDB. Adding a polyol to DFDB produced similar osseous regeneration in the rat calvarium defect model vs DFDB alone. Yet from a clinical standpoint, the polyol enhanced graft handling and stability. Graft particle size may have an effect on bone fill.

An implant versus a conventional fixed prosthesis: a case report.

The success of endosseous implant dentistry is widely acknowledged. Clinical research and subsequent innovations with both surgical and restorative techniques--as well as improved surface features and restorative components--have made endosseous dental implants a routinely accepted dental treatment modality. Patients and referring clinicians often specifically request dental implant therapy. This article presents an example of such a patient; however, in this case, a surgical ridge augmentation and fabrication of a conventional fixed prosthesis was selected as the treatment modality. The authors believe that all therapeutic modalities should be considered when adapting appropriate treatment plans for individual patients.

The effect of residual calcium in decalcified freeze-dried bone allograft in a critical-sized defect in the Rattus norvegicus calvarium.

Demineralized freeze-dried bone allograft (DFDBA), a widely used graft material in periodontal regenerative procedures, is processed with hydrochloric acid in the attempt to expose proteins located within the bone matrixes that are capable of inducing new bone formation. However, the degree of DFDBA demineralization varies between tissue banks, which may have an effect on clinical regeneration. This study uses the critical-sized defect (CSD) model to evaluate the wound-healing response to the residual calcium of donor bone. If the percentage of residual calcium in a graft were demonstrated to significantly enhance wound healing, then periodontal patients may benefit from further standardization of human-allograft processing. Sixty adult, male, Harlan Sprague-Dawley rats (Rattus norvegicus) were randomly and equally divided into 4 test groups (ie, DFDBA at 1%, 2%, and 3% to 6% residual calcium levels and FDBA at 23% residual calcium) and a control group (no allograft). An 8-mm-diameter craniotomy was made in the rat calvarium, and polytetrafluoroethylene membranes with pore sizes of 0.50 microm were placed intracranially and ectocranially. Treatment materials were carefully placed into the CSD with a new sterilized dental amalgam carrier. Tetracycline hydrochloride was injected intraperitoneally for labeling new bone growth, and animals were euthanized 12 weeks postsurgery. As a result, histomorphometric bone fill at 12 weeks showed a statistically significant increase in the 2% DFDBA group as compared to all other groups. The authors conclude that a 2% residual calcium level in human DFDBA appears to significantly (P < or = .05) enhance osseous wound healing in the rat calvarium.

Effect of a hydroxyapatite tricalcium phosphate alloplast on osseous repair in the rat calvarium.

BACKGROUND: Bony defects caused by periodontitis are often treated by regenerative therapy using autografts and/or allografts. Alloplasts, such as hydroxyapatite or ceramics, are also used as osteoconductive materials that serve as a scaffold for new bony ingrowth. The purpose of this study was to determine the effect of hydroxyapatite tricalcium phosphate (HA-TCP) on osseous repair in the rat calvarium. METHODS: Forty-four adult male Harlan Sprague-Dawley rats were assigned to one of four treatment groups: HA-TCP macroporous disk, HA-TCP microporous disk, HA-TCP granules, and demineralized freeze-dried bone (DFDB). The materials were placed into 8-mm calvarial critical-size defects (CSD). Calvariae were harvested at 10 weeks post-surgery and evaluated histomorphometrically. RESULTS: The DFDB group had significantly (P <0.05) more new bone formation (47%) than any other group. The HA-TCP macroporous disk group had significantly (P <0.05) more new bone formation (19.7%) than the HA-TCP microporous disk (8.5%) or HA-TCP granule (6.9%) groups. CONCLUSIONS: The HA-TCP macroporous disk may elicit significant new bone formation due to its rigid space-maintaining scaffold and pore size for vascular ingrowth. It is well tolerated by host tissues and may be a suitable carrier for growth factors.

Severe localized periodontal destruction associated with cervical cemental separation.

Cemental separations and tears are secondary etiological factors that may lead to rapid periodontal destruction if the lesion communicates with the oral cavity and allows bacterial invasion. Although many cemental tears that occur on proximal surfaces can be diagnosed radiographically, separations on the facial or lingual surface that cannot be detected on radiographs may present a diagnostic dilemma. This article describes a case of lingual cemental separation on a maxillary incisor, with subsequent successful surgical correction utilizing an osseous graft.

In vivo effectiveness of a glycerol-compounded demineralized freeze-dried bone xenograft in the rat calvarium.

BACKGROUND: Demineralized freeze-dried bone (DFDB) is commonly hydrated with sterile water into a paste-like consistency for improved clinical handling or reconstituted with biodegradable barriers, such as glycerol, to promote handling and wound stability following human periodontal surgery. The purpose of this study was to evaluate the in vivo effects of glycerol-compounded human DFDB on bone formation in the rat calvarial critical-sized defect (CSD) model. METHODS: Forty-eight adult male Harland Sprague-Dawley rats were assigned to one of four treatment groups: glycerol, DFDB, DFDB plus glycerol, or a non-grafted control, and placed into 8 mm calvarial CSDs. DFDB (particle size 0.106 to 0.5 mm), glycerol, and their combination were from identical sources. Calvaria were harvested at 8 weeks postsurgery and evaluated histomorphometrically. RESULTS: A statistically significant increased percentage of total bone fill was detected in the glycerol plus DFDB group and DFDB group as compared to glycerol group or the control. However, no significant difference was noted between the DFDB plus glycerol group and the DFDB group. CONCLUSION: The addition of glycerol to DFDB results in comparable osseous regeneration in the rat calvarium defect model versus DFDB alone; however, based upon clinical judgment, handling characteristics of DFDB were greatly improved.

Nicotine stimulates osteoclast resorption in a porcine marrow cell model.

BACKGROUND: Combustible tobacco use is generally linked with accelerated periodontal bone loss and diminished post-surgical wound healing; however, the pathogenesis of this process at the cellular level remains unclear. Nicotine is known to affect human gingival fibroblast orientation, attachment, and beta1 integrin expression, yet little is known about its effects on osteoclasts, the cells most responsible for bone resorption. The purpose of this study was to determine the effects of physiologically relevant nicotine levels on porcine osteoclast function as measured by resorption of calcium phosphate. METHODS: Pure nicotine was diluted in medium to the following concentrations: 0.03 microM, 0.15 microM, 0.30 microM, 0.60 microM, and 1.50 microM. Porcine osteoclasts were seeded onto calcium phosphate multi-test slides and incubated at 37 degrees C with half media changes every 24 hours. Cells received 0, 0.15, 0.30, 0.60, and 1.50 microM nicotine, or 25 nM parathyroid hormone (PTH). Osteoclast resorption was quantified by measuring the resorbed surface area of the calcium phosphate substrate. RESULTS: Osteoclast cultures resorbed bone slices and calcium phosphate substrate. All nicotine concentrations and PTH resulted in statistically significantly greater mean percent resorptions than the control group (P < 0.05). However, no statistical difference was found between the various nicotine doses or PTH. The number of osteoclasts increased in a linear relationship to the increasing nicotine concentrations; however, no correlation was found between osteoclast number and the amount of resorption. CONCLUSIONS: Nicotine is non-toxic to osteoclasts at the clinically relevant levels tested. Nicotine appears to stimulate osteoclast differentiation and resorption of calcium phosphate, the major component of bone. Nicotine-modulated osteoclast stimulation may, in part, explain the increased rapidity of periodontal bone loss and refractory disease incidence in smokers.

Evaluation of a novel alloplast for osseous regeneration in the rat calvarial model.

BACKGROUND: Alloplasts are inert foreign bodies acting as osteoconductive space maintainers during osseous wound healing. They may also function as carriers for growth factors that are known to enhance neovascularization and osteoinduction; human recombinant transforming growth factor beta (rhTGF-beta1) is one such factor. The purpose of this study was to evaluate di-vinyl styrene beads (DVSb) and rhTGF-beta1 effects on osseous regeneration in the rat calvaria critical-sized defect model. METHODS: Di-vinyl styrene beads (DVSb) with and without rhTGF-beta1 were placed between gelfilm membranes in a critical-sized defect in the rat (Rattus norvegicus) calvaria. Actual bone fill; percentage bone fill; bone formation rate (BFR); and mineralization at 3, 6, and 12 weeks postsurgery were measured using densitometry, histomorphometry, scanning electron microscopy (SEM), and vital staining with tetracycline-HCl. RESULTS: Mean radiographic density and percentage fill were statistically greater for DVSb treatment groups as compared with controls (P < or = 0.05). BFR was consistently between 3 and 7 microm per day for all groups; alloplast group BFR was significantly greater than controls or the rhTGF-beta1 groups at 6 weeks (P < or = 0.05); however, at 3 and 12 weeks, the control BFR was greater than treatment groups (P < or = 0.05). Membranes often collapsed and little bone fill or mineralization occurred in defects without DVSb. Mineralization appeared to occur adjacent to the alloplast by 12 weeks in the histologic and SEM sections. While DVSb fibrous attachment occurred in some specimens, there was no evidence of an inflammatory response. CONCLUSION: Di-vinyl styrene beads, with or without rhTGF-beta1, significantly enhance bone regeneration in the rat calvaria defect model.

Enhancement of osteoblast proliferation in vitro by selective enrichment of demineralized freeze-dried bone allograft with specific growth factors.

Decalcified freeze-dried bone allograft (DFDBA), believed to serve as a matrix for new bone growth and to contain various bone-inducing growth factors, is currently used to regenerate periodontal defects and to restore and maintain dental alveolar ridges. Growth factors within DFDBA are extracted during the demineralization process, thus rendering the allograft incapable of spontaneous osteogenesis; however, exogenous growth factor addition to DFDBA may enhance the osteogenic capacity of native osteoblasts. This study's purpose is to evaluate murine osteoblast proliferation in the presence of various exogenous soluble growth factors as measured by fluorescence units. Osteoblasts harvested from mouse pup calvaria were cultured with 2% residual calcium-DFDBA and supplemented by one of the following growth factors or combinations of these factors: transforming growth factor-beta (TGF-beta), insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), fibroblast growth factors basic (bFGF), or vascular endothelial growth factors (VEGF). Osteoblast proliferation rates indicate that the in vitro supplementation of 2% residual calcium-DFDBA with the combination of IGF and TGF-beta, IGF and PDGF, and PDGF and TGF-beta significantly (P < or = .05) enhance murine osteoblast activity and proliferation at 7 days compared with the control containing no exogenous growth factors.

External root resorption following partial-thickness connective tissue graft placement: a case report.

We report an unusual case of external root resorption (ERR) that developed in a 37-year-old black male approximately 1 year following routine partial thickness connective tissue graft surgery. The lesion was accessed via flap surgery, thoroughly root planed, and the mucoperiosteal flap replaced. The site healed uneventfully and the patient has been closely observed for over 1 year without symptoms or recurrence of the resorptive lesion and the affected tooth remained vital. Clinicians performing partial-thickness connective tissue grafts should be alert to the possible occurrence of root resorption over extended periods of time. The authors speculate that retention of the donor periosteum with placement on the recipient dentin and root biomodification may limit the resorptive response following connective tissue graft procedures to treat tooth root recession.

Evaluation of pluronic polyols as carriers for grafting materials: study in rat calvaria defects.

BACKGROUND: Pluronic polyols are a family of non-ionic surfactants currently used as drug carriers for antibiotic, anti-inflammatory, and anti-neoplastic agents. Therapeutic administration of non-ionic surface-active agents is known to facilitate early collagen synthesis and microcirculation, thus promoting wound healing. The purpose of this study was to determine the in vivo effects of pluronic polyols combined with either an allograft or an alloplast on the healing of critical-sized calvarial defects. METHODS: One hundred fifty (150) adult (95 to 105 days old) male Sprague-Dawley rats weighing between 375 and 425 g were randomly and evenly assigned to each of 15 separate treatment groups and anesthetized, and 8 mm calvarial critical-sized defects were created. Pluronic F-68 (F-68) or pluronic F-127 (F-127) was administered either topically or systemically and in conjuction with demineralized bone powder (DBP), tricalcium phosphate (TCP), or non-grafted controls. Pluronic polyols are easily mixed with either DBP or TCP to improve handling ease. Calvaria were harvested at 12 weeks postsurgery and evaluated histomorphometrically, by contact radiography with subsequent densitometric analysis, through energy spectrometry utilizing a scanning electron microscope, and by fluorescent microscopy. RESULTS: There was a significant difference in the percentage of bone fill among the control, TCP, and DBP only groups, P <0.05. The only significant difference within any of these groups was between the TCP control and TCP plus systemic F-127, P<0.05. CONCLUSIONS: Although there were isolated differences, the overall trend was that the pluronic polyol and the mode of administration did not result in a significant change in bone wound healing as measured by the percentage of bone fill. Pluronic polyols may be considered as carriers for osseous graft materials.

The effects of varying degrees of allograft decalcification on cultured porcine osteoclast cells.

BACKGROUND: Demineralized freeze-dried bone allograft (DFDBA) is widely used in periodontal therapy as a scaffold for new bone formation in periodontal defects. It is demineralized, theoretically, to expose osteoinductive or osteoconductive bone matrix proteins that should facilitate osteogenesis. The degree of DFDBA demineralization varies between tissue banks and may affect clinical regeneration. A 2% residual calcium level in DFDBA has been shown to result in the highest alkaline phosphatase activity levels in cultured human periosteal cells and is optimally osteoinductive or osteoconductive for new bone formation. The purpose of this study was to evaluate the effect of 4 different residual calcium levels in commercially available DFDBA samples on porcine osteoclast activity as measured by resorption on calcium phosphate-coated disks. METHODS: Bone marrow was harvested from the femurs of 3-week-old farm pigs and cultured for 3 weeks. Hematopoietic stem cells were allowed to differentiate into mature active polykaryons displaying genuine osteoclast characteristics. The osteoclast cells displayed a dense actin band inside the margins of the cytoplasm under light microscopy. Culture media was decanted and collagenase added to free the attached cells. Equal cell samples were pipetted onto calcium phosphate-coated disks in 24-well plates. DFDBA samples with 1.44%, 2.41%, and 5.29% residual calcium; FDBA (30% residual calcium); and control cultures without allograft samples were prepared and all samples incubated for 1 week. Cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), Oregon Green 488-phalloidin, a stain for cytoskeletal proteins, and counterstained with propidium iodide. Specimens were examined by light and fluorescence microscopy using epi-illumination. Calcium phosphate disks were then rinsed in 5% sodium hypochlorite to remove adherent osteoclasts, and substrate surface changes were measured by white light interferometry and image analysis. RESULTS: A higher yield of TRAP-positive cells was produced without DFDBA; however, resorptive activity appears to be significantly increased in the presence of 2.41% residual calcium as compared to all other experimental groups (P<0.0065). CONCLUSION: In this in vitro model, porcine osteoclasts show significantly more resorptive activity as measured on calcium phosphate-coated disks in the presence of 2.41% residual calcium in DFDBA than in other DFDBA residual calcium levels.