Ofer Moses

In vitro Effects of Enamel Matrix Proteins on Rat Bone Marrow Cells and Gingival Fibroblasts

Emdogain® (EMD), a formulation of Enamel Matrix Proteins (EMP), is used clinically for periodontal regeneration, where it stimulates cementum formation and promotes gingival healing. In this study, we investigated the in vitro effects of EMD on rat bone marrow stromal cells (BMSC) and gingival fibroblasts (GF). EMD (at 25 μg/mL) increased the osteogenic capacity of bone marrow, as evidenced by ~ three-fold increase in BMSC cell number and ~ two-fold increase in alkaline phosphatase (ALP) activity and mineralized nodule formation. The presence of EMD in the initial stages (first 48 hrs) of the culture was crucial for this effect. In contrast, EMD did not induce osteoblastic differentiation of GF (evidenced by lack of mineralization or ALP activity) but increased up to two-fold both their number and the amount of matrix produced. These in vitro data on BMSC and GF could explain the promotive effect of EMD on bone formation and connective tissue regeneration, respectively.

Mucosal considerations for osseointegrated implants

Tissue resistance is determined by the nature of cells and intercellular contacts irrespective of the presence or absence of keratinization, masticatory mucosa, or skin. However, these tissues are more easily maintained and less vulnerable to inflammation when in contact with dental implants. Lack of masticatory mucosa and the presence of alveolar mucosa embracing the implant are often associated with plaque, which can induce inflammation resulting in subsequent peri-implant destruction. To facilitate proper mechanical oral hygiene maintenance, transplantation of autogenous masticatory mucosal grafts at the implant sites was performed in patients without attached gingiva, unfavorable vestibulum with submucosal muscular activity, and uncontrolled peri-implant mucositis. The rationale for having attached mucosa around osseointegrated implants and illustration of possible methods of mucosal management in the different phases of implant rehabilitation are presented.

Long‐term bio‐degradation of cross‐linked and non‐cross‐linked collagen barriers in human guided bone regeneration

OBJECTIVE This double-blind study clinically and histologically evaluated long-term barrier bio-durability of cross-linked and non-cross-linked collagen membranes (CLM and NCLM) in sites treated by guided bone regeneration procedures. MATERIALS AND METHODS In 52 patients, 52 bony defects were randomly assigned to treatment with either a CLM or a NCLM. Post-surgical spontaneous membrane exposures were recorded. Before implant placement, full-thickness standard soft tissue discs were retrieved wherever suitable for histologic examination. RESULTS Spontaneous membrane exposure was observed in 13 (50%) CLM sites and in six (23.1%) NCLM sites (P<0.05). Clinical healing at exposed sites lasted 2-4 weeks. CLM were histologically intact in all non-perforated sites, were interrupted in five perforated sites, and undetected in four. NCLMs were undetected in all 18 specimens examined. In three non-perforated CLM sites, bone apposition and ossification at or within the membrane was observed. CONCLUSIONS CLMs were more resistant to tissue degradation than NCLMs, and maintained integrity during the study. Neither membrane was resistant to degradation when exposed to the oral environment. CLMs were associated with a higher incidence of tissue perforations. In non-perforated sites, CLM ossification at or within the membrane was occasionally observed.

Bio-degradation of a resorbable collagen membrane (Bio-Gide) applied in a double-layer technique in rats.

OBJECTIVE The aim of this study was to evaluate histologically the bio-degradation of two layers of Bio-Gide((R)) (BG) membrane, as compared with that of a single layer. MATERIAL AND METHODS Two circular calvarial bony defects, 5 mm in diameter, were made in 24 Wistar rats. BG membrane, labeled with biotin, was cut into 5-mm-diameter disks, and placed in defects either as a mono-layer membrane (MLM) or as a double-layer membrane (DLM). Rats were sacrificed after 4 or 9 weeks and histology was performed. Membranes were stained with horseradish peroxidase-conjugated streptavidin and aminoethyl carbazole as a substrate for detection of biotinylated collagen. The area of collagen and thickness of the residual membranes were measured by image analysis software. Statistical analysis was performed using the non-parametric Wilcoxons signed-ranks test. RESULTS At 4-week collagen area per measurement window within the DLM sites (0.09+/-0.05 mm(2)) was significantly greater (P<0.01) than that in the MLM sites (0.047+/-0.034 mm(2)). At 9 weeks, the collagen area was also greater in the DLM sites (0.037+/-0.026 mm(2)) compared with that of the MLM sites (0.025+/-0.016 mm(2)); however, this difference did not reach statistical significance. The rate of membrane degradation, calculated as percent membrane lost compared with baseline, was similar for the DLM and MLM at both time points ( approximately 60% at 4 weeks and approximately 80% at 9 weeks). In addition, the residual DLM thickness at 4 weeks (475.5+/-73.77 mum) was significantly (P<0.01) greater than that of MLM (262.38+/-48.01 mum). At 9 weeks, membrane thickness was also greater in the DLM sites (318.22+/-70.45 mum) compared with that of the MLM sites (183.32+/-26.72 mum); however, this difference did not reach statistical significance. The reduction in thickness between 4 and 9 weeks was 30% for MLM and 33% for DLM. DISCUSSION The use of a double layer of BG membrane results in a barrier of increased collagen area and thickness, compared with application of a single layer.

Prostaglandin E2 inhibits the proliferation of human gingival fibroblasts via the EP2 receptor and Epac

Elevated levels of prostaglandins such as PGE2 in inflamed gingiva play a significant role in the tissue destruction caused by periodontitis, partly by targeting local fibroblasts. Only very few studies have shown that PGE2 inhibits the proliferation of a gingival fibroblast (GF) cell line, and we expanded this research by using primary human GFs (hGFs) and looking into the mechanisms of the PGE2 effect. GFs derived from healthy human gingiva were treated with PGE2 and proliferation was assessed by measuring cell number and DNA synthesis and potential signaling pathways were investigated using selective activators or inhibitors. PGE2 inhibited the proliferation of hGFs dose‐dependently. The effect was mimicked by forskolin (adenylate cyclase stimulator) and augmented by IBMX (a cAMP‐breakdown inhibitor), pointing to involvement of cAMP. Indeed, PGE2 and forskolin induced cAMP generation in these cells. Using selective EP receptor agonists we found that the anti‐proliferative effect of PGE2 is mediated via the EP2 receptor (which is coupled to adenylate cyclase activation). We also found that the effect of PGE2 involved activation of Epac (exchange protein directly activated by cAMP), an intracellular cAMP sensor, and not PKA. While serum increased the amount of phospho‐ERK in hGFs by ∼300%, PGE2 decreased it by ∼50%. Finally, the PGE2 effect does not require endogenous production of prostaglandins since it was not abrogated by two COX‐inhibitors. In conclusion, in human gingival fibroblasts PGE2 activates the EP2—cAMP—Epac pathway, reducing ERK phosphorylation and inhibiting proliferation. This effect could hamper periodontal healing and provide further insights into the pathogenesis of inflammatory periodontal disease. J. Cell. Biochem. 108: 207–215, 2009.

Long-Term Results of Implants Immediately Placed Into Extraction Sockets Grafted With β-Tricalcium Phosphate: A Retrospective Study

PURPOSE The aim of this 10 year retrospective study was to evaluate the crestal bone loss around immediate implant placed in tricalcium phosphate (TCP) grafted extraction sockets MATERIALS AND METHODS Data were collected from files of 58 patients (33 females, 25 males, average age 54.78 years) undergoing immediate implant placement into fresh extraction socket with or without the use of TCP (Cerasorb, Curasan AG, Kleinostheim, Germany) grafting. After implant placement, horizontal gaps larger than 1.5 mm between the implant surface and the bony plate were grafted with TCP without the use of a membrane, while smaller gaps were not grafted. Two hundred fifty-four implants were inserted: 79 were placed immediately with the use of β-TCP as grafting material (group A), 175 were placed in healed extraction sites, with 61 implants placed with the use of β-TCP graft material (group B), and 114 implants were placed without any grafting material (group C). Bone loss recordings were performed using periapical radiography. Measurements were performed from the neck of the implant to level of the surrounding bone in the vertical dimension. RESULTS No implant was lost during the follow-up period. Statistical analysis showed no correlation between implant placement timing (delayed or immediate), the use of bone graft, and extent of bone loss. CONCLUSION The use of TCP (Cerasorb) as a grafting material during immediate implant placement allowed no bone loss in 72.1% of the implants, which was very similar to the nongrafted cases for which implants were placed in favorable conditions.

Streptozotocin-induced diabetes in rats diminishes the size of the osteoprogenitor pool in bone marrow.

AIMS Bone formation is reduced in animals and humans with type 1 diabetes, leading to lower bone mass and inferior osseous healing. Since bone formation greatly depends on the recruitment of osteoblasts from their bone marrow precursors, we tested whether experimental type 1 diabetes in rats diminishes the number of bone marrow osteoprogenitors. METHODS Diabetes was induced by 65 mg/kg streptozotocin and after 4 weeks, femoral bone marrow cells were extracted and cultured. Tibia and femur were frozen for further analysis. RESULTS The size of the osteoprogenitor pool in bone marrow of diabetic rats was significantly reduced, as evidenced by (1) lower (~35 %) fraction of adherent stromal cells (at 24h of culture); (2) lower (20-25%) alkaline phosphatase activity at 10 days of culture; and (3) lower (~40 %) mineralized nodule formation at 21 days of culture. Administration of insulin to hyperglycemic rats normalized glycemia and abrogated most of the decline in ex vivo mineralized nodule formation. Apoptotic cells in tibial bone marrow were more numerous in hyperglycemic rats. Also, the levels of malondialdehyde (indicator of oxidative stress) were significantly elevated in bone marrow of diabetic animals. CONCLUSIONS Experimental type 1 diabetes diminishes the osteoprogenitor population in bone marrow, possibly due to increased apoptosis via Oxidative Stress. Reduced number of osteoprogenitors is likely to impair osteoblastogenesis, bone formation, and bone healing in diabetic animals.

Bioresorbable Collagen Membranes for Guided Bone Regeneration

Localized lack of bone volume in the jaws may be due to congenital, post-traumatic, postsurgical defects or different disease processes. Increasing the bone volume has long been an attractive field of basic and clinical research. The introduction of implant therapy, and the proven relationship between long-term prognosis of dental implants and adequate bone volume at the implant site (Lekholm et al. 1986), dramatically increased the interest of both clinicians and scientists in this field, making augmentation procedures an important part of contemporary implant therapy. Basically, four methods have been described to augment bone volume: a. osteoinduction, using appropriate growth factors (Reddi 1981; Urist 1965); b. osteoconduction, using grafting materials that serve as scaffolds for new bone growth (Buch et al. 1986; Reddi et al. 1987); c. distraction osteogenesis, by which a surgically induced bone fracture enables slow controlled pulling apart of the separated bone fragments (Ilizarov 1989a,b); d. guided bone regeneration, which allows selective bone tissue growth into a space maintained by tissue barriers (Dahlin et al. 1988, 1991a; Kostopoulos & Karring 1994; Nyman & Lang 1994). Among the different methods, guided bone regeneration (GBR) is the most popular and best documented for the treatment of localized bone defects in the jaws, probably due to its relative simplicity of use while allowing the placement of endosseous implants in areas of the jaw with bony defects and/or insufficient bone volume. Highly predictable success rates can be achieved using GBR; in fact, it has been shown that success rates of implants placed at GBR treated sites and sites without bone augmentation are comparable (Hammerle et al. 2002).

Heat generation in 1-piece implants during abutment preparations with high-speed cutting instruments.

Objectives:Evaluation of heat generation in 1-piece implants according to 3 variables: preparation time, bur type, and preparation environment. Materials and Methods:Study implants were 1-piece designs with the same endosseous dimensions and surface microtexture, but with abutment sections that were either conical in shape, which required clinical preparations to shape and establish a restorative finish line (test), or with a pre-machined shape and restorative margin, which required minimal preparations (control) to accommodate a cemented crown. Burs were either carbide (group 1) or diamond (group 2), and the preparation environment was either ambient air or under water spray. An infrared camera was used to measure temperature changes in the exposed endosseous implant threads during grinding procedures. Three endosseous zones of the implant body were defined for heat measurements: crestal bone region (SP01), middle of the threaded region (SPO2), and apical region (SPO3). Grinding was performed in either ambient air or under water spray. The abutment was reduced to a 2.0 mm height, and one side was ground down to a 30 degree angle. Results:Highest heat elevations were concentrated in SP01, followed by SP02 and SP03. Average temperature changes in SP01 showed that preparation time and environment significantly affected heat generation but not bur type. Lowest temperatures were exhibited by control implants prepared under water spray. Bur type (carbide or diamond) did not affect temperature changes. Conclusion:Intraoral implant abutment preparations can transfer heat to the bone capable of impairing osseointegration.

Opposing Effects of Diabetes and Tetracycline on the Degradation of Collagen Membranes in Rats

BACKGROUND Increased collagenolytic activity, characteristic of uncontrolled diabetes, may compromise collagen membrane (CM) survival. Tetracycline (TCN) possesses anticollagenolytic properties and delays CM degradation in healthy animals. This study evaluates the degradation of TCN--immersed and -non-immersed CMs in rats with diabetes compared to those with normoglycemia. METHODS Diabetes was induced in 15 12-week-old male Wistar rats by injection of 65 mg/kg streptozotocin. The control group consisted of 15 rats with normoglycemia. Sixty bilayered CM disks were labeled before implantation with aminohexanoyl-biotin-N-hydroxy-succinimide ester, of which 30 were immersed in 50 mg/mL TCN solution (experimental) or phosphate-buffered saline (PBS) (control). In each animal, two disks (control and experimental) were implanted in two midsagittal calvarial defects in the parietal bone. Similar non-implanted disks served as baseline. After 3 weeks, animals were euthanized, and the calvaria and overlying soft tissues were processed for demineralized histologic analysis. Horseradish peroxidase-conjugated streptavidin was used to detect the biotinylated collagen. The area of residual collagen within the membrane disks was measured and analyzed with a digital image analysis system. Several slides from each specimen were also stained with hematoxylin and eosin. Statistical analysis consisted of paired and unpaired t tests. RESULTS The amount of residual collagen in PBS-immersed disks was lower in rats with diabetes compared to rats with normoglycemia (69% of baseline versus 93%, respectively, P <0.001). TCN immersion increased the amount of residual collagen contents in both diabetic (83% of baseline) and healthy (97.5% of baseline) animals (P <0.0001). CONCLUSION Diabetes increases CM degradation, whereas immersion in 50 mg/mL TCN solution before implantation presents an opposite effect.