Hari S. Prasad

Enhanced Osteoclastogenesis Causes Osteopenia in Twisted Gastrulation-Deficient Mice Through Increased BMP Signaling†‡

The uncoupling of osteoblastic and osteoclastic activity is central to disorders such as osteoporosis, osteolytic malignancies, and periodontitis. Numerous studies have shown explicit functions for bone morphogenetic proteins (BMPs) in skeletogenesis. Their signaling activity has been shown in various contexts to be regulated by extracellular proteins, including Twisted gastrulation (TWSG1). However, experimental paradigms determining the effects of BMP regulators on bone remodeling are limited. In this study, we assessed the role of TWSG1 in postnatal bone homeostasis. Twsg1‐deficient (Twsg1−/−) mice developed osteopenia that could not be explained by defective osteoblast function, because mineral apposition rate and differentiation markers were not significantly different compared with wildtype (WT) mice. Instead, we discovered a striking enhancement of osteoclastogenesis in Twsg1−/− mice, leading to increased bone resorption with resultant osteopenia. Enhanced osteoclastogenesis in Twsg1−/− mice was caused by increased cell fusion, differentiation, and function of osteoclasts. Furthermore, RANKL‐mediated osteoclastogenesis and phosphorylated Smad1/5/8 levels were enhanced when WT osteoclasts were treated with recombinant BMP2, suggesting direct regulation of osteoclast differentiation by BMPs. Increase in detectable levels of phosphorylated Smad 1/5/8 was noted in osteoclasts from Twsg1−/− mice compared with WT mice. Furthermore, the enhanced osteoclastogenesis in Twsg1−/− mice was reversed in vitro in a dose‐dependent manner with exposure to Noggin, a BMP antagonist, strongly suggesting that the enhanced osteoclastogenesis in Twsg1 mutants is attributable to increased BMP signaling. Thus, we present a novel and previously uncharacterized role for TWSG1 in inhibiting osteoclastogenesis through regulation of BMP activity.

Dental Magnetic Resonance Imaging: Making the Invisible Visible

INTRODUCTION Clinical dentistry is in need of noninvasive and accurate diagnostic methods to better evaluate dental pathosis. The purpose of this work was to assess the feasibility of a recently developed magnetic resonance imaging (MRI) technique, called SWeep Imaging with Fourier Transform (SWIFT), to visualize dental tissues. METHODS Three in vitro teeth, representing a limited range of clinical conditions of interest, imaged using a 9.4T system with scanning times ranging from 100 seconds to 25 minutes. In vivo imaging of a subject was performed using a 4T system with a 10-minute scanning time. SWIFT images were compared with traditional two-dimensional radiographs, three-dimensional cone-beam computed tomography (CBCT) scanning, gradient-echo MRI technique, and histological sections. RESULTS A resolution of 100 μm was obtained from in vitro teeth. SWIFT also identified the presence and extent of dental caries and fine structures of the teeth, including cracks and accessory canals, which are not visible with existing clinical radiography techniques. Intraoral positioning of the radiofrequency coil produced initial images of multiple adjacent teeth at a resolution of 400 μm. CONCLUSIONS SWIFT MRI offers simultaneous three-dimensional hard- and soft-tissue imaging of teeth without the use of ionizing radiation. Furthermore, it has the potential to image minute dental structures within clinically relevant scanning times. This technology has implications for endodontists because it offers a potential method to longitudinally evaluate teeth where pulp and root structures have been regenerated.

Dental magnetic resonance imaging

INTRODUCTION Clinical dentistry is in need of noninvasive and accurate diagnostic methods to better evaluate dental pathosis. The purpose of this work was to assess the feasibility of a recently developed magnetic resonance imaging (MRI) technique, called SWeep Imaging with Fourier Transform (SWIFT), to visualize dental tissues. METHODS Three in vitro teeth, representing a limited range of clinical conditions of interest, imaged using a 9.4T system with scanning times ranging from 100 seconds to 25 minutes. In vivo imaging of a subject was performed using a 4T system with a 10-minute scanning time. SWIFT images were compared with traditional two-dimensional radiographs, three-dimensional cone-beam computed tomography (CBCT) scanning, gradient-echo MRI technique, and histological sections. RESULTS A resolution of 100 μm was obtained from in vitro teeth. SWIFT also identified the presence and extent of dental caries and fine structures of the teeth, including cracks and accessory canals, which are not visible with existing clinical radiography techniques. Intraoral positioning of the radiofrequency coil produced initial images of multiple adjacent teeth at a resolution of 400 μm. CONCLUSIONS SWIFT MRI offers simultaneous three-dimensional hard- and soft-tissue imaging of teeth without the use of ionizing radiation. Furthermore, it has the potential to image minute dental structures within clinically relevant scanning times. This technology has implications for endodontists because it offers a potential method to longitudinally evaluate teeth where pulp and root structures have been regenerated.

Clinical, Histologic, and Histomorphometric Evaluation of Mineralized Solvent-dehydrated Bone Allograft (Puros) in Human Maxillary Sinus Grafts

Demineralized freeze-dried bone allografts (DFDBA) have been successfully used alone or in composite grafts for many decades. Little research has been done on the effect of retaining the mineral content of bone allografts. This study histologically and histomorphometrically evaluated a new mineralized bone allograft material placed in human atrophic maxillary sinuses. Seven partially edentulous patients requiring sinus grafts before implant placement were selected for this study Their age range was 56 to 81 years (mean 67.7 years). Test grafts consisted of a mineralized solvent-dehydrated cancellous bone allograft, and control grafts were a composite of DFDBA and deproteinized bovine bone xenograft (1:1). Bilateral cases (n = 3) received both test and control grafts on opposite sides, and unilateral cases received either a test (n = 3) or control (n = 1) graft only. At 10 months, core biopsies were taken from each graft site, and dental implants were placed into the augmented bone. All bone grafts resulted in new bone formation and all implants osseointegrated. Test grafts resorbed and were replaced by newly formed bone significantly faster and in greater quantities than were control grafts. No complications with grafts or implants were noted. Both test and control grafts achieved excellent results. The faster bone formation observed with the test graft may be due, in part, to its smaller particle size compared with the bovine portion of the control graft. Test grafts were either replaced by new bone or displayed new bone-to-particle surface contact in higher percentages than did control grafts. No differences in osseointegration or graft stability were noted 2 years after the study.

Bone formation at recombinant human bone morphogenetic protein-2-coated titanium implants in the posterior maxilla (Type IV bone) in non-human primates

BACKGROUND Studies using ectopic rodent and orthotopic canine models (Type II bone) have shown that titanium porous oxide (TPO) surface implants adsorbed with recombinant human bone morphogenetic protein-2 (rhBMP-2) induce local bone formation including osseointegration. The objective of this study was to evaluate local bone formation and osseointegration at such implants placed into Type IV bone. MATERIAL AND METHODS rhBMP-2-coated implants were installed into the edentulated posterior maxilla in eight young adult Cynomolgus monkeys: four animals each received three TPO implants adsorbed with rhBMP-2 (2.0 mg/ml) and four animals each received three TPO implants adsorbed with rhBMP-2 (0.2 mg/ml). Contra-lateral jaw quadrants received three TPO implants without rhBMP-2 (control). Treatments were alternated between left and right jaw quadrants. Mucosal flaps were advanced and sutured to submerge the implants. The animals received fluorescent bone markers at weeks 2, 3, 4, and at week 16 when they were euthanized for histologic analysis. RESULTS Clinical healing was uneventful. Extensive local bone formation was observed in animals receiving implants adsorbed with rhBMP-2 (2.0 mg/ml). The newly formed bone exhibited a specific pinpoint bone-implant contact pattern regardless of rhBMP-2 concentration resulting in significant osseointegration; rhBMP-2 (2.0 mg/ml): 43% and rhBMP-2 (0.2 mg/ml): 37%. Control implants exhibited a thin layer of bone covering a relatively larger portion of the implant threads. Thus, TPO control implants bone exhibited significantly greater bone-implant contact ( approximately 75%; p<0.05). There were no statistically significant differences between rhBMP-2-coated and control implants relative to any other parameter including peri-implant and intra-thread bone density. CONCLUSION rhBMP-2-coated TPO implants enhanced/accelerated local bone formation in Type IV bone in a dose-dependent fashion in non-human primates resulting in significant osseointegration. rhBMP-2-induced de novo bone formation did not reach the level of osseointegration observed in native resident bone within the 16-week interval.

Bone formation on carbon nanotube composite

The effects of a layer-by-layer assembled carbon nanotube composite (CNT-comp) on osteoblasts in vitro and bone tissue in vivo in rats were studied. The effects of CNT-comp on osteoblasts were compared against the effects by commercially pure titanium (cpTi) and tissue culture dishes. Cell proliferation on the CNT-comp and cpTi were similar. However, cell differentiation, measured by alkaline phosphatase activity and matrix mineralization, was better on the CNT-comp. When implanted in critical-sized rat calvarial defect, the CNT-comp permitted bone formation and bone repair without signs of rejection or inflammation. These data indicate that CNT-comp may be a promising substrate for use as a bone implant or as a scaffold for tissue engineering.

Bone block allograft impregnated with bone marrow aspirate

Purpose:To evaluate the influence of bone marrow aspirate when added to bone block allograft to repair osseous defects. Background:Bone-marrow aspirate has been combined with xenograft and allograft particulate material and has produced a significant quantity of new bone growth. However, the use of allograft bone blocks has advantages in some clinical situations. This article discusses cell-based therapies by means of in vivo transplantation of stem cells derived from bone-marrow aspirate and incorporated into allograft corticocancellous bone block for bone regeneration. Materials:A technique for combining bone-marrow aspirate with block allografts was developed. To evaluate its influence in repairing osseous defects, a maximum of 3 to 4 mL of bone marrow was aspirated from the anterior iliac crest of 5 patients who had severely atrophic maxillary and mandibular ridges. Five sites were grafted with allograft bone blocks saturated with bone-marrow aspirate and secured with bone screws (ACE Surgical Supply Company, Inc. Brockton, MA). At one of the sites a core specimen was taken 4 months after implant placement and submitted for standard histologic and histomorphometric analysis. Results:After 4 to 8 months of healing, all the grafts had integrated into the recipient bone. Implants were placed at all 5 sites and osseointegrated successfully. Examination of the bone core showed the graft to be well-integrated, with 54% of the core consisting of bone and 46% of marrow. Eighty-nine percent of the bone was vital. Conclusion:Impregnation of bone-marrow aspirate into allograft bone block activates the bodys ability to form new bone. The bone-marrow aspiration technique is less invasive than harvesting autogenous bone from a second surgical site, offers predictable results, and is cost effective.

Comparison of Bone Grafting Materials in Human Extraction Sockets: Clinical, Histologic, and Histomorphometric Evaluations

Purpose:Although there are a number of bone replacement graft materials that are currently available for clinical use, there are few studies that directly compare efficacy among graft treatments before implant placement. The purpose of this report was to compare 3 bone replacement graft materials (PepGen P-15 228 FLOW [DENTSPLY Friadent CeraMed, Lakewood, CO], Puros® [Zimmer Dental, Carlsbad, CA], and C-Graft 228 [Clinician’s Preference, Golden, CO]) for bone formation by clinical, histologic, and histomorphometric evaluation. Materials and Methods:In this prospective, intraoral pilot study, 13 maxillary sockets in 2 patients (both smokers) were grafted immediately after tooth extraction with C-Graft 228, Puros®, or PepGen P-15 228 FLOW (containing additional PepGen P-15 228 particles; FLOW PUTTY). After 4 months, bone cores were retrieved and analyzed histologically. Results:PepGen P-15 228 FLOW PUTTY produced a significantly (P <0.01) higher amount of vital bone than C-Graft 228 or Puros®. The amount of vital bone for FLOW PUTTY was 12-fold higher than for C-Graft 228 and 4-fold higher than Puros®. Of 7 FLOW PUTTY treated sites, 7 showed >14% vital bone versus 0 of 3 C-Graft 228 and 0 of 3 Puros® treated sites. FLOW PUTTY treated sites showed new vital bone between particles of residual graft. C-Graft 228 treated sites showed residual particles in a background of connective tissue with very little bone. Puros® treated sites showed nonvital bone particles in a background of connective tissue, with some new vital bone forming around the nonvital bone. Conclusion:PepGen P-15 228 FLOW PUTTY produced significantly greater vital bone as compared to Puros® and C-Graft 228 after 4 months. A larger clinical study is required to confirm these results.

Alveolar ridge augmentation using implants coated with recombinant human growth/ differentiation factor-5: histologic observations

OBJECTIVES In vitro and in vivo preclinical studies suggest that growth/differentiation factor-5 (GDF-5) may induce local bone formation. The objective of this study was to evaluate the potential of recombinant human GDF-5 (rhGDF-5) coated onto an oral implant with a purpose-designed titanium porous oxide surface to stimulate local bone formation including osseointegration and vertical augmentation of the alveolar ridge. MATERIALS AND METHODS Bilateral, critical-size, 5 mm, supraalveolar peri-implant defects were created in 12 young adult Hound Labrador mongrel dogs. Six animals received implants coated with 30 or 60 microg rhGDF-5, and six animals received implants coated with 120 microg rhGDF-5 or left uncoated (control). Treatments were alternated between jaw quadrants. The mucoperiosteal flaps were advanced, adapted, and sutured to submerge the implants for primary intention healing. The animals received fluorescent bone markers at weeks 3, 4, 7, and 8 post-surgery when they were euthanized for histologic evaluation. RESULTS The clinical examination showed no noteworthy differences between implants coated with rhGDF-5. The cover screw and implant body were visible/palpable through the alveolar mucosa for both rhGDF-5-coated and control implants. There was a small increase in induced bone height for implants coated with rhGDF-5 compared with the control, induced bone height averaging (+/-SD) 1.6+/-0.6 mm for implants coated with 120 microg rhGDF-5 versus 1.2+/-0.5, 1.2+/-0.6, and 0.6+/-0.2 mm for implants coated with 60 microg rhGDF-5, 30 microg rhGDF-5, or left uncoated, respectively (p<0.05). Bone formation was predominant at the lingual aspect of the implants. Narrow yellow and orange fluorescent markers throughout the newly formed bone indicate relatively slow new bone formation within 3-4 weeks. Implants coated with rhGDF-5 displayed limited peri-implant bone remodelling in the resident bone; the 120 microg dose exhibiting more advanced remodelling than the 60 and 30 microg doses. All treatment groups exhibited clinically relevant osseointegration. CONCLUSIONS rhGDF-5-coated oral implants display a dose-dependent osteoinductive and/or osteoconductive effect, bone formation apparently benefiting from local factors. Application of rhGDF-5 appears to be safe as it is associated with limited, if any, adverse effects.

Root and pulp response after intentional injury from miniscrew placement

INTRODUCTION The purpose of this study was to evaluate the effects on the pulp and supporting tissues when miniscrews severely abrade the root surface. METHODS Sixty self-drilling and self-tapping miniscrews were placed between the premolars and molars of 3 beagles (20 miniscrews per dog). The miniscrews were placed according to the protocol suggested by the manufacturer, but with the intention of inflicting root damage. Radiographs were taken of all 60 interdental alveolar sites, 5 in each quadrant, and sites that evidenced root proximity were identified. The animals were killed at 12 weeks, and 20 of the most probable injury sites were selected for histologic analysis. Serial nondecalcified sections and microcomputed tomography scans were obtained with the implants in place. RESULTS There was no histologic evidence of inflammatory response either at the root surface or in the pulp. Pulp necrosis, external resorption, and ankylosis were not found, but reparative cementum was seen at each injury site. The presence of woven bone along miniscrew threads lends evidence to support the osseointegration of miniscrews. CONCLUSIONS Permanent damage to the pulp and supporting tissues is not a regular occurrence when miniscrews abrade or even enter the root surface.