Search

OBJECTIVES Guided bone regeneration (GBR) aims to predictably restore missing bone that has been lost due to trauma, periodontal disease or a variety of systemic conditions. Critical to this procedure is the ability of a bone grafting material to predictably serve as a 3-dimensional scaffold capable of inducing cell and bone tissue in-growth at the material surface. Although all bone grafts are osteoconductive to bone-forming osteoblasts, only a small number of commercially available bone grafts with FDA approval are osteoinductive including demineralized freeze-dried bone allographs (DFDBA) and scaffolds containing bone morphogenetic proteins (BMPs). Recently, a class of synthetic bone grafts fabricated from biphasic calcium phosphate (BCP) sintered at a low temperature have been shown to form ectopic bone formation in non-skeletal sites without the use of growth factors. Therefore, the present study aimed to compare the osteoinductive potential of this group of synthetic BCP alloplasts with autografts, allografts and xenografts. MATERIALS AND METHODS In the present study, 4 types of bone grafting materials including autogenous bone harvested with a bone mill, DFDBA (LifeNet, USA), a xenograft derived from bovine bone mineral (NBM, BioOss, Geistlich, Switzerland) and a novel synthetic biphasic calcium phosphate (BCP, Straumman, Switzerland) were implanted into intramuscular pouches of 24 rats and analysed histologically for their ability to form ectopic bone formation around grafting particles. A semi-quantitative osteoinductive score was used to quantify the osteoinductive ability of each bone graft. RESULTS The results from the present study reveal that (1) autogenous bone resorbed rapidly in vivo, (2) the xenograft showed no potential to form ectopic bone formation and (3) both DFDBA and BCP were able to stimulate ectopic bone formation. CONCLUSION These studies demonstrate that these newly developed synthetic bone grafts have potential for inducing ectopic bone formation similar to DFDBA. Future clinical testing is necessary to reveal their bone-inducing properties in clinical scenarios including GBR procedures and in combination with implant dentistry. CLINICAL RELEVANCE Novel BCP scaffolds are able to induce ectopic bone formation without the use of osteoinductive growth factors such as BMP2 and thus demonstrate a large clinical possibility to further enhance bone formation for a variety of clinical procedures.

BACGROUND On June 5th, 2015 at Europerio 8, a group of leading experts were gathered to discuss what has now been 20 years of documented evidence supporting the clinical use of enamel matrix derivative (EMD). Original experiments led by Lars Hammarstrom demonstrated that enamel matrix proteins could serve as key regenerative proteins capable of promoting periodontal regeneration including new cementum, with functionally oriented inserting new periodontal ligament fibres, and new alveolar bone formation. This pioneering work and vision by Lars Hammarstrom has paved the way to an enormous amount of publications related to its biological basis and clinical use. Twenty years later, it is clear that all these studies have greatly contributed to our understanding of how biologics can act as mediators for periodontal regeneration and have provided additional clinical means to support tissue regeneration of the periodontium. AIMS This review article aims to: (1) provide the biological background necessary to understand the rational for the use of EMD for periodontal regeneration, (2) present animal and human histological evidence of periodontal regeneration following EMD application, (3) provide clinically relevant indications for the use of EMD and (4) discuss future avenues of research including key early findings leading to the development of Osteogain, a new carrier system for EMD specifically developed with better protein adsorption to bone grafting materials.

The aim of the study was to determine the effect of 10 sources of lactic acid bacteria (LAB) on dry matter digestibility (DM-D) and neutral detergent fiber digestibility (NDF-D), in various combinations with starch, in vitro. The soluble starch represented a concentrate feed, whereas silage represented feeding only roughage. The DM-D and NDF-D were determined after 24 and 48 h of incubation to represent effective (24 h) and potential (48 h) digestibility. Addition of LAB was both by direct application of the inoculants to rumen fluid (directly fed microbials) and by the use of preinoculated silages. For each feed combination, tubes without added LAB served as controls. The results indicate that, overall, some LAB inoculants applied at ensiling or added directly to the rumen fluid had the potential to increase the DM-D and NDF-D. The major significant inoculant effect on NDF-D was obtained after 24 h of incubation, whereas the effect after 48 h was mainly nonsignificant. The effective inoculants seemed to minimize the inhibitory effect of the starch on NDF-D within 24 h, perhaps by competition with lactate-producing rumen microorganisms.

OBJECTIVE Collagen membranes serve as barriers to separate bone grafts from soft tissues. Bone grafts harvested with a bone scraper release growth factors activating transforming growth factor-β (TGF-β) signaling in mesenchymal cells. The aim of the present pilot study was to determine whether collagen membranes adsorb molecules from bone-conditioned medium (BCM) with the capacity to provoke the expression of TGF-β target genes in vitro. MATERIALS AND METHODS Collagen membranes were soaked in aqueous extracts from fresh and demineralized bone chips placed in cell culture medium. Recombinant human TGF-β1 served as control. Gingival fibroblasts were seeded onto the washed collagen membranes and evaluated for the expression of adrenomedullin, pentraxin 3, interleukin 11, and proteoglycan 4. Cell viability and morphology with phalloidin staining were also determined. RESULTS Incubation of collagen membranes with BCM for at least one minute caused fibroblasts to decrease the expression of adrenomedullin and pentraxin 3, and to increase the expression of interleukin 11 and proteoglycan 4. Four different membrane treatments - incubated with recombinant TGF-β1, pre-wetted with saline solution, exposed to UV light, and dry out and stored one week at room temperature - also provoked significant changes in gene expression. Likewise, conditioned medium from demineralized bone chips caused gene expression changes. BCM did not alter the viability or morphology of gingival fibroblasts. CONCLUSIONS These findings demonstrate that collagen membranes rapidly adsorb the TGF-β activity released from bone chips, a molecular process that might contribute to guided bone regeneration.

BACKGROUND Recombinant bone morphogenetic protein two (rhBMP2) has been utilised for a variety of clinical applications in orthopaedic surgery and dental procedures. Despite its widespread use, concerns have been raised regarding its short half-life and transient bioactivity in vivo. Recent investigation aimed at developing rhBMP2 synthesized from a shorter polypeptide chain (108 amino acids) has been undertaken. METHODS The osteopromotive properties of BMP2 were investigated on cell behaviour. Five concentrations of rhBMP2_108 including 10, 50, 100, 200 and 500 ng/ml were compared to a commercially available rhBMP2 (100 ng/ml). Each of the working concentrations of rhBMP2_108 were investigated on MC3T3-E1 osteoblasts for their ability to induce osteoblast recruitment, proliferation and differentiation as assessed by alkaline phosphatase (ALP) staining, alizarin red staining, and real-time PCR for genes encoding ALP, osteocalcin (OCN), collagen-1 (COL-1) and Runx2. RESULTS The results demonstrate that all concentrations of rhBMP2_108 significantly improved cell recruitment and proliferation of osteoblasts at 5 days post seeding. Furthermore, rhBMP2_108 had the most pronounced effects on osteoblast differentiation. It was found that rhBMP2_108 had over a four fold significant increase in ALP activity at seven and 14 days post-seeding and the concentrations ranging from 50 to 200 ng/ml demonstrated the most pronounced effects. Analysis of real-time PCR for genes encoding ALP, OCN, COL-1 and Runx2 further confirmed dose-dependant increases at 14 days post-seeding. Furthermore, alizarin red staining demonstrated a concentration dependant increase in staining at 14 days. CONCLUSION The results from the present study demonstrate that this shorter polypeptide chain of rhBMP2_108 is equally as bioactive as commercially available rhBMP2 for the recruitment of progenitor cells by facilitating their differentiation towards the osteoblast lineage. Future in vivo study are necessary to investigate its bioactivity.

Bone morphogenetic protein-2 (BMP2) has been successfully utilized in dentistry to promote new bone formation because of its osteoinductive ability to recruit mesenchymal progenitor cells and induce their differentiation to bone-forming osteoblasts. Recently, novel biphasic calcium phosphate scaffolds have been developed with similar osteoinductive properties capable of forming ectopic bone formation.The aim of the present study was to assess whether the combination of BMP2 with this novel Biphasic Calcium Phosphate (BCP) scaffold may additionally promote new bone regeneration.Cylindrical bone defects measuring 2.5 mm were created bilaterally in the femurs of 18 Wistar rats. After 4 weeks, the following six groups were assessed for new bone formation by micro-computed tomography (CT) as well as histological assessment: 1) collagen scaffolds + 20 μg of BMP2; 2) collagen scaffolds + 50 μg of BMP2; 3) collagen scaffolds + 100 μg of BMP2; 4) BCP scaffolds + 20 μg of BMP2; 5) BCP scaffolds + 50 μg of BMP2; and 6) BCP scaffolds + 100 μg of BMP2. Furthermore, tartrate-resistant acid phosphatase (TRAP) staining was utilized to assess osteoclast activity and osteoclast number. The release kinetics of BMP2 from both BCP and collagen scaffolds was investigated over a 14-day period.The results from present study demonstrate that BMP2 is able to promote new bone formation in a concentration dependant manner when loaded with either a collagen scaffolds or BCP scaffolds. Micro-CT analysis demonstrated significantly higher levels of new bone formation in groups containing BCP + BMP2 when compared with collagen scaffolds + BMP2. BMP2 had little effect on osteoclast activity; however, less TRAP staining and osteoclast number was observed in the defects receiving collagen scaffolds when compared with BCP scaffolds. The release of BMP2 over time was rapidly released after 1 day on BCP scaffolds whereas a gradually release over time was observed for collagen scaffolds up to 14 days.The osteoinductive properties of BMP2 may further be enhanced by its combination with a novel synthetically fabricated osteoinductive BCP scaffold. Future clinical testing is required to further assess these preliminary findings.

OBJECTIVE The aim of this study was to investigate the ability of Enamel Matrix Derivative (EMD) on vertical bone regeneration around dental implants placed in an extra-oral rabbit model. MATERIAL AND METHODS A total of 30 Straumann BL implants were partially embedded in transverse orientation into the posterior mandibles of 15 rabbits. Macro-structuring BiPhasicCaPST (BCPT1), micro-structuring BiPhasicCaPST (BCPT2), and deproteinized bovine bone mineral (DBBM) were placed around the implant and covered with a scaffold stabilizing "umbrella." EMD was incorporated within the scaffold for test sites, but not control sites. Histological analysis was performed on retrieved specimens after 10 weeks of healing to assess new bone formation. RESULTS All treatment groups displayed new supracrestal bone formation as determined by histomorphometric measurements, with mean values of new bone height ranging between 0.62 and 1.13 mm. Histological analysis revealed a higher mean bone formation (%) around the test sites where EMD (34.7, 95%CI: 27.1-39.4) was released from the scaffold, whereas the control group without EMD release (26.4, 95%CI: 16.3-31.9) (P = 0.069). The mean fBIC (%) in the BCPT2 group increased by the addition of EMD relative to no EMD (67.2, 95%CI: 48.6-84.1) and (54.7, 95%CI: 32.3-68.9), respectively). The BCPT2/EMD and DBBM/EMD interventions showed the greatest mean bone density (BA/TA), respectively, (12.8, 95%CI: 8.9-36.5) and (11.2, 6.3-16.4) in ROI 1. Values in ROI 2 were, similarly, (24.9, 95%CI: 17.2-31.7) and (27.7, 19.2-35.3). BA/TA in ROI 2 differences between the BCPT2 groups with and without EMD was statistically significant (P = 0.026), as well as the DBBM groups with and without EMD (P = 0.038). CONCLUSIONS A layer of new bone was formed in both test and controls. The release of EMD from BCPT2 and DBBM adjacent to a bone-level implant with an SLActive surface and scaffold retention umbrella consistently regenerated the greater fBIC and bone density along the length of the implant.

OBJECTIVES Bone replacement grafting materials play an important role in regenerative dentistry. Despite a large array of tested bone-grafting materials, little information is available comparing the effects of bone graft density on in vitro cell behavior. Therefore, the aim of the present study is to compare the effects of cells seeded on bone grafts at low and high density in vitro for osteoblast adhesion, proliferation, and differentiation. MATERIALS AND METHODS The response of osteoblasts to the presence of a growth factor (enamel matrix derivative, (EMD)) in combination with low (8 mg per well) or high (100 mg per well) bone grafts (BG; natural bone mineral, Bio-Oss®) density, was studied and compared for osteoblast cell adhesion, proliferation, and differentiation as assessed by real-time PCR. Standard tissue culture plastic was used as a control with and without EMD. RESULTS The present study demonstrates that in vitro testing of bone-grafting materials is largely influenced by bone graft seeding density. Osteoblast adhesion was up to 50 % lower when cells were seeded on high-density BG when compared to low-density BG and control tissue culture plastic. Furthermore, proliferation was affected in a similar manner whereby cell proliferation on high-density BG (100 mg/well) was significantly increased when compared to that on low-density BG (8 mg/well). In contrast, cell differentiation was significantly increased on high-density BG as assessed by real-time PCR for markers collagen 1 (Col 1), alkaline phosphatase (ALP), and osteocalcin (OC) as well as alizarin red staining. The effects of EMD on osteoblast adhesion, proliferation, and differentiation further demonstrated that the bone graft seeding density largely controls in vitro results. EMD significantly increased cell attachment only on high-density BG, whereas EMD was able to further stimulate cell proliferation and differentiation of osteoblasts on control culture plastic and low-density BG when compared to high-density BG. CONCLUSION The results from the present study demonstrate that the in vitro conditions largely influence cell behavior of osteoblasts seeded on bone grafts and in vitro testing. CLINICAL RELEVANCE These results also illustrate the necessity for careful selection of bone graft seeding density to optimize in vitro testing and provide the clinician with a more accurate description of the osteopromotive potential of bone grafts.

The health of peri-implant soft tissues is one of the most important aspects of osseointegration necessary for the long-term survival of dental implants.To review the process of soft tissue healing around osseointegrated implants and discuss the maintenance requirements as well as the possible short-comings of peri-implant soft tissue integration.Literature search on the process involved in osseointegration, soft tissue healing and currently available treatment modalities was performed and a brief description of each process was provided.The peri-implant interface has been shown to be less effective than natural teeth in resisting bacterial invasion because gingival fiber alignment and reduced vascular supply make it more vulnerable to subsequent peri-implant disease and future bone loss around implants. And we summarized common procedures which have been shown to be effective in preventing peri-implantitis disease progression as well as clinical techniques utilized to regenerate soft tissues with bone loss in advanced cases of peri-implantitis.Due to the difference between peri-implant interface and natural teeth, clinicians and patients should pay more attention in the maintenance and recovery of soft tissues around implants.

OBJECTIVES Enamel matrix derivative (EMD) has been successfully used for the regeneration of periodontal tissues including new cementum, periodontal ligament, and alveolar bone. Combination of EMD with bone grafting materials has however generated variable clinical results. Recently, we have demonstrated that a new formulation of EMD in a liquid carrier system (Osteogain®) has improved physicochemical properties for the adsorption of EMD to a bone grafting material. The aim of the present study was to investigate the regenerative potential of Osteogain®, in combination with a bone graft, on new bone formation in a rat femur defect model. MATERIALS AND METHODS Fifty-four critically sized femur defects (3 mm in diameter) were created bilaterally in 27 rats and treated following the group allocation: (1) drilled unfilled control, (2) a natural bone mineral (NBM), and (3) NBM + Osteogain®. All defects were histologically analyzed at 2, 4, and 8 weeks after surgical intervention. Micro-CT analysis, hematoxylin and eosin (H&E) staining, and Safranin O staining were performed to quantify new bone formation. RESULTS Significantly more new bone formation was observed in defects treated with NBM + Osteogain® at both 4 and 8 weeks when compared to NBM alone and the control unfilled defects (P

This study investigated the potential for applying an automatic milking system (AMS) to the management of high-yielding cows offered a total mixed ration (TMR). The null hypothesis was that it is desirable to maintain even in AMS, the TMR feeding management practice recommended for high-yielding cows and therefore it can be attained by ‘reducing the concentrate allocation in the robot without reducing the number of milkings’. Two feeding regimes were used: the ‘candy concept’, with only 1·2 kg of food concentrate – the minimum to attract the cow – provided at each visit to the milking robot; and the provision of a maximum of 7 kg of food concentrate per day. Approximately 100 cows were subjected to one or other of these two treatments. Although the cows in the first treatment consumed approximately 3·5 kg of concentrate per day and those in the second treatment approximately 5 kg per day, no significant differences were observed in the numbers of voluntary milkings.

Effect of feeding pellets containing soy hulls (SH) as starchy grain substitute on milking performance was measured in Assaf lactating ewes. Eighty ewes were divided into two equal groups of forty animals each, and fed for eight weeks, one of the two experimental diets. Both diets contained 25% vetch hay and 10% wheat silage served at the feeding lane plus additive of 65% pellets served part on top of roughage in the feeding lane and part during milking. Diets differed only in the composition of the pelleted additive, being either high starch pellets in the control treatment or pellets containing 51% SH as starch replacer in the experimental group. In vitro organic matter digestibility of the two types of pellets was similar, whereas NDF digestibility and NDF content were higher in the SH pellets. The ewes fed the SH pellets demonstrated higher daily DM and NDF intake (2.71 and 1.19 kg/d, respectively) as compared with the control group (2.44 and 0.71 kg, respectively). This was reflected by significantly higher milk yield (2.41 kg/d versus 2.10 kg/d), milk fat content (6.05% versus 5.16%), and milk fat yield (0.15 kg/d versus 0.11 kg/d) in the experimental group as compared with the control ewes. Milk protein yields were similar in both treatments. Data suggest potential advantage of feeding the SH pellets for high producing lactating ewes.

The potential of soy hulls to replace barley grain in pellets supplemented to lactating cows was measured in automatic concentrate feeders (ACF). Thirty-six cows were divided into 2 equal groups and fed 1 of the 2 experimental pellet supplements individually for 7 wk. All cows were group-fed a basic mixture along the feeding lane (~64% of dietary DM) plus a pelleted additive containing 50% barley or soy hulls as barley replacer, fed individually to each cow via the ACF in 6 feeding windows. Extent and rate of in vitro DM digestibility were similar for both types of pellets; however, NDF content and digestibility were higher in the soy hulls pellets. Average number of rewarded cow visits at the ACF, pellets intake per meal, and accumulated intake of pellets (8.64 kg/d DM) were similar in the 2 experimental groups. Most pellets were consumed during day and night in the first 2 h after feeding windows were opened. Total visits per day in the ACF and the maximal interval between visits were similar for the 2 pellets, indicating similar attractiveness to the cow. Predicted intake of the basic mixture was similar in both groups (14.8 to 15.1 kg of DM/d). Higher milk fat content was observed in the soy hulls-fed cows, whereas higher milk protein content was found in the barley-fed cows. Milk yield was similar in both groups. Data suggest that replacement of barley pellets with soy hulls pellets may slightly enhance milk fat while reducing milk protein production for dairy herds using automatic milking systems.

Two total mixed rations (TMR) containing different proportions of roughage neutral detergent fiber (NDF) were fed to lactating cows under Israeli summer conditions, and the effects on feeding behavior and milk production were measured. Forty-two lactating cows were divided into 2 groups fed ad libitum a TMR containing either 18% NDF of roughage origin (control group) or only 12% roughage NDF, in which the corn silage component (16.5% of dry matter [DM]) was replaced with soy hulls (experiment group). This and additional adjustments in TMR were reflected in higher net energy for lactation and in vitro digestibility of the experimental TMR. Cow behavior was investigated at the feeding lane during June 2001; about 11,000 cow visits were analyzed. Feed intake per meal and average meal duration were significantly higher in the experiment group (1.51 kg of DM per meal and 12.1 min per meal, respectively) as compared with the control group (1.22 kg of DM per meal and 9.47 min per meal, respectively). However, number of meals per day recorded in the feeding lane was significantly higher in the control group (21.0 vs. 16.6 meals/d per cow). Distribution of meals and feed intake along the day depended more on management practices, such as milking and feed dispensing times, than on feed composition or weather conditions. These differences between groups were expressed in similar daily eating duration (approximately 200 min), and because the rate of feed consumption was similar for both treatments (approximately 127 g DM/min), the daily average DM intake was also similar (25.0 to 25.7 kg). However, NDF intake was higher in the experiment group. Consequently, the average milk yield was higher in the experimental group, and production of milk fat, 4% fat-corrected milk, and economically corrected milk were significantly higher in the experiment group than in the control group. Data imply that the experimental TMR containing only 12% NDF of roughage origin is more suitable for cows under hot climate conditions compared with the control TMR.

The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized. Subtractive enrichment procedures were used to isolate mutants of R. albus 8 that are defective in adhesion to cellulose. Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked. Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose. The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R. albus 8. One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A). Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B 2 (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum , respectively. Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex. Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus. Such X modules are also present in several other R. albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria.

The potential of soybean hulls (SH) to replace forage neutral detergent fiber (NDF) in total mixed rations (TMR) of lactating cows was examined. Forty lactating cows were divided into two groups and individually fed ad libitum one of two experimental diets for 8 weeks: (I) a TMR containing 18% forage NDF, corn silage (CS TMR); and (II) a similar TMR in which the corn silage component (16.5% of dry matter, DM) was replaced by SH (SH TMR), containing only 12% forage NDF. Total NDF content was 39 and 36% in the SH TMR and CS TMR, respectively. The SH group tended ( P =0.09) to eat more NDF than the CS group, but DM intake was not influenced. Average milk fat yield was higher ( P =0.04) in the SH group. Findings were supported by the extent of in vitro digestibility data showing higher digestibility ( P

Leukocyte and platelet-rich fibrin (PRF), a second-generation platelet concentrate has been the focus of intensive research endeavors over the last 2 decades. Over the years, numerous reports have however failed to accurately report g-force values which have caused considerable confusion in the field. These values have since been re-transcribed incorrectly in many studies moving forward, and this article aims to address this topic to avoid further confusion in the field. We address several reports in which PRF centrifugal g-forces have been calculated at the PRF clot (referred to as relative centrifugal force [RCF]-clot) as opposed to the international standard method described at the bottom of centrifugation tubes (RCF-max). We further highlight how RCF-clot is not only a deviation from the standard international method used to report g-force values, but one subject to significant error owing to centrifugation time, patient hematocrit levels, initial volume of blood collected, and other factors. For these reasons and those further reported throughout this article, we address this controversy in detail to avoid further confusion regarding the report of g-force values in future studies. Furthermore, we propose a standardization regarding the accurate report of g-force values in future studies investigating PRF at the RCF-max.

BACKGROUND The use of an enamel matrix derivative (EMD) has been shown to enhance periodontal regeneration (e.g., formation of root cementum, periodontal ligament, and alveolar bone). However, in certain clinical situations, the use of EMD alone may not be sufficient to prevent flap collapse or provide sufficient stability of the blood clot. Data from clinical and preclinical studies have demonstrated controversial results after application of EMD combined with different types of bone grafting materials in periodontal regenerative procedures. The aim of the present study is to investigate the adsorption properties of enamel matrix proteins to bone grafts after surface coating with either EMD (as a liquid formulation) or EMD (as a gel formulation). METHODS Three different types of grafting materials, including a natural bone mineral (NBM), demineralized freeze-dried bone allograft (DFDBA), or a calcium phosphate (CaP), were coated with either EMD liquid or EMD gel. Samples were analyzed by scanning electron microscopy or transmission electron microscopy (TEM) using an immunostaining assay with gold-conjugated anti-EMD antibody. Total protein adsorption to bone grafting material was quantified using an enzyme-linked immunosorbent assay (ELISA) kit for amelogenin. RESULTS The adsorption of amelogenin to the surface of grafting material varied substantially based on the carrier system used. EMD gel adsorbed less protein to the surface of grafting particles, which easily dissociated from the graft surface after phosphate-buffered saline rinsing. Analyses by TEM revealed that adsorption of amelogenin proteins were significantly farther from the grafting material surface, likely a result of the thick polyglycolic acid gel carrier. ELISA protein quantification assay demonstrated that the combination of EMD liquid + NBM and EMD liquid + DFDBA adsorbed higher amounts of amelogenin than all other treatment modalities. Furthermore, amelogenin proteins delivered by EMD liquid were able to penetrate the porous surface structure of NBM and DFDBA and adsorb to the interior of bone grafting particles. Grafting materials coated with EMD gel adsorbed more frequently to the exterior of grafting particles with little interior penetration. CONCLUSIONS The present study demonstrates a large variability of adsorbed amelogenin to the surface of bone grafting materials when enamel matrix proteins were delivered in either a liquid formulation or gel carrier. Furthermore, differences in amelogenin adsorption were observed among NBM, DFDBA, and biphasic CaP particles. Thus, the potential for a liquid carrier system for EMD, used to coat EMD, may be advantageous for better surface coating.

OBJECTIVES Osteoporosis is a disease characterized by a reduction in bone mass, poor bone strength, and microarchitectural deterioration primarily in postmenopausal women. With respect to periodontal disease, osteoporosis is thought to contribute to pre-existing alveolar degeneration although the association between both diseases is not fully characterized. The aim of the present study was to observe the initial changes in mandibular alveolar bone for sham-operated and ovariectomized (OVX) rats in ligature-induced experimental periodontitis. MATERIALS AND METHODS A total of 64 Wistar rats (7 weeks of age, 180-200 g) were used in this study (32 control sham-operated animals + ligature placement, 32 OVX animals + ligature placement). Following an 8-week period to induce an OVX model, micro-CT analysis was performed to calculate vertical and furcation bone loss of mandibular first molars at time points 0, 3, 7, and 11 days following ligature placement (six animals per group per time point). Furthermore, histological analysis was performed to calculate the loss of alveolar bone crest height from the cemento-enamel junction, and tartrate-resistant acid phosphatase (TRAP) staining was utilized to calculate the number of osteoclasts. RESULTS The results from the present study demonstrate that OVX animals showed significant vertical bone loss at all time points when compared to control sham-operated animals. In the furcation area, no significant difference in bone loss was observed between sham-operated and OVX animals at 0, 3, and 7 days; however by 11 days, a significant decrease in bone volume/total volume and trabecular thickness was observed in the OVX group. The histological analysis also revealed that alveolar bone crest height was significantly reduced in OVX animals, and TRAP staining further revealed the greater number of multinucleated osteoclasts peaking at 3 days postligature placement. CONCLUSION The results from the present study demonstrate a direct correlation between the osteoporotic phenotype and the progression of periodontal breakdown in a diseased-induced animal model. CLINICAL RELEVANCE It may be suggested that an osteoporotic phenotype has the potential to speed periodontal breakdown and thus contributes to the overall degeneration of the periodontium in patients suffering from postmenopausal bone loss. Future research from human clinical studies are necessary to further understand the relationship between periodontal disease and osteoporosis.

Over 15 years have passed since an enamel matrix derivative (EMD) was introduced as a biologic agent capable of periodontal regeneration. Histologic and controlled clinical studies have provided evidence for periodontal regeneration and substantial clinical improvements following its use. The purpose of this review article was to perform a systematic review comparing the eff ect of EMD when used alone or in combination with various types of bone grafting material.A literature search was conducted on several medical databases including Medline, EMBASE, LILACS, and CENTRAL. For study inclusion, all studies that used EMD in combination with a bone graft were included. In the initial search, a total of 820 articles were found, 71 of which were selected for this review article. Studies were divided into in vitro, in vivo, and clinical studies. The clinical studies were subdivided into four subgroups to determine the eff ect of EMD in combination with autogenous bone, allografts, xenografts, and alloplasts.The analysis from the present study demonstrates that while EMD in combination with certain bone grafts is able to improve the regeneration of periodontal intrabony and furcation defects, direct evidence supporting the combination approach is still missing.Further controlled clinical trials are required to explain the large variability that exists amongst the conducted studies.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Aims: To compare the subcellular distribution of glycanase-related components between wild-type Ruminococcus albus SY3 and an adhesion-defective mutant, to identify their possible contribution to the adhesion process, and to determine their association with cellulosome-like complexes. Methods and Results: Cell fractionation revealed that most of the cellulases and xylanases were associated with capsular and cell-wall fractions. SDS-PAGE and gel filtration indicated that most of the bacterial enzyme activity was not integrated into cellulosome-like complexes. The adhesion-defective mutant produced significantly less (5- to 10-fold) overall glycanase activity, and the ‘true cellulase activity’ appeared to be entirely confined to the cell membrane fractions. Antibodies specific for the cellulosomal scaffoldin of Clostridium thermocellum recognized a single 240 kDa band in R. albus SY3. Conclusions: The adhesion-defective mutant appeared to be blocked in exocellular transport of enzymes involved in true cellulase activity. A potential cellulosomal scaffoldin candidate was identified in R. albus SY3. Significance and Impact of the Study: Several glycanase-related proteins and more than one mechanism appear to be involved in the adhesion of R. albus SY3 to cellulose.

Alfalfa was ensiled either by conventional wilting (WA) or as a mixture of fresh alfalfa plus SO 2 -treated wheat straw (A+SWS) at a dry matter (DM) ratio of 67:33 and the digestibility of carbohydrate constituents and some aspects of N-metabolism were examined in sheep. By adding SWS to alfalfa, the content of neutral detergent fiber (NDF) was increased from 34.8 to 42.6% and that of total N was decreased from 2.84 to 2.61%. Silage pH was 4.51 and 5.91 in A+SWS and WA silages, respectively. The major part of uronic acids (74–78%), rhamnose (65–68%) and arabinose (65–67%) was found in the neutral detergent soluble (NDS) fraction of the silages, implying pectin survival following silage fermentation. The digestibility values of the major sugars, glucose and xylose in whole silage were higher in A+SWS than in the WA. Consequently, whole tract carbohydrate digestibility was 71% in WA and 75% in A+SWS. The difference between treatments in carbohydrate digestibility originated from the digestibility pattern of NDF-monosaccharide components. The major difference was found with NDF-xylose, 31.0% versus 58.3% in WA and A+SWS silages, respectively. Total NDF-monosaccharide digestibility values were 61.4 and 69.9% in the WA and A+SWS silages, respectively. Rumen ammonia-N was 23.1 and 16.5 mg/100 ml in sheep fed the WA and A+TWS silages, respectively. Although, N-intake by the WA sheep was higher, the amount of non ammonia-N present at the duodenum was almost equal in both treatments.

Winter-annual ryegrass ( Lolium multiflorum , Lam.) was sown on a sandy soil in the southern Arava desert of Israel and watered by drip irrigation. The experimental design was that of a random block including four plots per treatment of the following three irrigation treatments. (I) Water desalinated by reversed osmosis, aimed at simulating a fresh water source, with a salinity expressed as electrical conductivity (EC) of 1.64 and 1.44 dS m −1 (dS: deci siemens). (II) Secondary effluent from the sewage treatment plant of the city of Eilat, EC=3.20 and 4.24 dS m −1 . (III) Salinated secondary effluent, produced by pumping a concentrated NaCl solution into a particular irrigation system of secondary effluent, EC 6.59 and 9.78, for the 1st and 3rd cuts, respectively. The middle 1-m wide stripe along the entire length of each plot was harvested, weighed and sampled. Freeze-dried ryegrass samples were ground and analyzed. Water-soluble carbohydrates (WSC) content was 3–6 times higher in the 1st cut forage than in the 3rd cut, and unlike neutral detergent fiber (NDF) concentration, responded positively to the increase in water salinity. During that period, the plants counteracted the high osmotic pressure of the salinated secondary effluent, by accumulating more WSC, on the expense of cell wall (CW) synthesis. The 3rd cut was apparently not sensitive to water quality with respect to WSC and NDF contents. During that period, osmotic accommodation was attained apparently by accumulating more sodium in the plants. Organic matter yield was negatively affected by salinity, but partly compensated by an increase in the in vitro organic matter digestibility from 70.9 to 83.2 and from 73.5 to 78.1 in the 1st and 3rd cuts, respectively. The NDF monosaccharide composition was unaffected by salinity treatment. The proportion of ferulates involved in bridging was ∼10% in the forage of the 1st cut and raised to 20–40% in that of the 3rd cut. Salinity effect on cinnamic acids content in the 1st cut, followed the pattern of NDF, and was negatively related to digestibility. Such relationship between cinnamic acids content and digestibility was not found in the 3rd cut. The lack of consistency in this regard, poses some doubt as to the role of cinnamic acids as CW degradation obstacles.

We divided the adhesion process of the predominant cellulolytic rumen bacteria Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus into four phases: 1) transport of the nonmotile bacteria to the substrate; 2) initial nonspecific adhesion of bacteria to unprotected sites of the substrate that is dominated by constitutive elements of bacterial glycocalyx; 3) specific adhesion via adhesins or ligands formation with the substrate, which can be dominated by several bacterial organelles including cellulosome complexes, fimbriae connections, glycosylated epitopes of cellulose-binding protein (CBP) or glycocalyx, and cellulose-binding domain (CBD) of enzymes; 4) proliferation of the attached bacteria on potentially digestible tissues of the substrate. Each of the phases and its significance in the adhesion process are described. Factors affecting bacterial adhesion are described including: 1) factors related to bacterial age, glycocalyx condition, and microbial competition; 2) factors related to the nature of substrate including, cuticle protection, surface area, hydration, and ionic charge; and 3) environmental factors including pH, temperature, and presence of cations and soluble carbohydrate. Based on the information available from the literature, it appears that each of the predominant rumen bacteria--F. succinogenes, R. flavefaciens, and R. albus--has a specific mechanism of adhesion to cellulose. In F. succinogenes, both the glycosidic residues of the outer membrane CBP and especially of the 180-kDa CBP, and the distinct CBD of EG2 EGF and Cl-stimulated cellobiosidase, may play a role in the adhesion to cellulose. No direct evidence, except scanning electron microscopy observations, yet supports the existence of either cellulosome complex or fimbriae structures involved in the adhesion mechanism of F. succinogenes. At least two mechanisms, cellulosome-like complexes and carbohydrate epitopes of the glycocalyx layer are involved in the specific adhesion of R. flavefaciens to cellulose. Ruminococcus albus possesses at least two mechanisms for specific adhesion to cellulose: a cellulosomal-like mechanism, and a CbpC (Pil)-protein mechanism that probably involves the production of fimbrial-like structures. Indirect and direct studies suggested that carbohydrate epitopes of CBPs and CBD epitope of cellulases may also be involved mostly in the nonspecific phase of adhesion of R. albus.

An obligatory step in cellulose degradation by anaerobic bacteria is the adhesion of the bacterium to the polysaccharide. In many anaerobic bacteria the adhesion protein, and the enzymes required for extensive polysaccharide hydrolysis, are organized into a complex and interesting structure called the cellulosome. The Gram-positive anaerobe Ruminococcus albus also produces a cellulosome-like complex, but the bacterium appears to possess other mechanism(s) for adhesion to plant surfaces and genes encoding functions relevant to growth on cellulose are conditionally expressed, as suggested by a combination of functional proteomics, differential display reverse-transcriptase PCR, and mutational analysis. A novel form of cellulose-binding protein has been identified and shown to belong to the Pil-protein family, being most similar to the type 4 fimbrial proteins of Gram-negative, pathogenic bacteria. These studies have provided new insights into the adhesion of bacteria to plant surfaces, and call attention to the likely existence of genetically analogous adhesion determinants in both pathogenic and non-pathogenic bacteria.

The effectiveness of new diagnostic tools for suspected pulmonary embolism (PE), such as clinical probability assessment, plasma D-dimer (DD) measurement and lower limb venous compression ultrasonography (US), has not been specifically studied in patients with a suspected PE occurring during hospital stay. This study applied a sequential, decision analysis-based strategy adding these instruments to a ventilation/perfusion lung scan in a cohort of 114 consecutive inpatients clinically suspected of PE in order to establish in how many patients a pulmonary angiogram could thereby be avoided. A definitive diagnosis could be established by the noninvasive protocol in 61% of these patients: normal/near-normal lung scan, 14%; high probability lung scan, 19%; clinical probability combined with lung scan result, 18%; and US, 8%. Specificity of DD was only 7% and contributed to the exclusion of PE in only two patients. Pulmonary angiography was required in 39% of patients. The 3-month thromboembolic risk in patients in whom PE was excluded by the diagnostic process was 0% (95% confidence interval 0-4.9%). In conclusion, a noninvasive work-up for suspected pulmonary embolism is effective in hospitalized patients, allowing to forego angiography in 61% of them, and it appears to be safe, although this should be further investigated. In contrast to outpatients, D-dimer measurement appears to be useless in hospitalized patients.

We designed a simple and integrated diagnostic algorithm for acute venous thromboembolism based on clinical probability assessment of deep-vein thrombosis (DVT) or pulmonary embolism (PE), plasma D-dimer measurement, lower-limb venous compression ultrasonography, and lung scan to reduce the need for phlebography and pulmonary angiography.918 consecutive patients presenting at the emergency ward of the Geneva University Hospital, Geneva, Switzerland, and Hôpital Saint-Luc, Montreal, Canada, with clinically suspected venous thromboembolism were entered into a sequential diagnostic protocol. Patients in whom venous thromboembolism was deemed absent were not given anticoagulants and were followed up for 3 months.A normal D-dimer concentration (500 microg/L by a rapid ELISA) ruled out venous thromboembolism in 286 (31%) members of the study cohort, whereas DVT by ultrasonography established the diagnosis in 157 (17%). Lung scan was diagnostic in 80 (9%) of the remaining patients. Venous thromboembolism was also deemed absent in patients with low to intermediate clinical probability of DVT and a normal venous ultrasonography (236 [26%] patients), and in patients with a low clinical probability of PE and a non-diagnostic result on lung scan (107 [12%] patients). Pulmonary angiography and phlebography were done in only 50 (5%) and 2 (1%) of the patients, respectively. Hence, a non-invasive diagnosis was possible in 866 (94%) members of the entire cohort. The 3-month thromboembolic risk in patients not given anticoagulants, based on the results of the diagnostic protocol, was 1.8% (95% CI 0.9-3.1).A diagnostic strategy combining clinical assessment, D-dimer, ultrasonography, and lung scan gave a non-invasive diagnosis in the vast majority of outpatients with suspected venous thromboembolism, and appeared to be safe.

Replacement bone grafting materials are used clinically for a variety of clinical procedures to augment and replace lost or missing bone. Little information is available regarding their degradation properties.The aim of the present study was to investigate the degradation rate and modes of degradation of four commonly used bone grafting materials.A natural bone mineral (NBM) of bovine origin, NBM in combination with enamel matrix derivative (EMD), LifeNet demineralized freeze-dried bone allograft (DFDBA), and Osteotech DFDBA were analyzed for particle degradation over time in 3 mm femur defects created in female Wistar rats. At 2, 4, and 8 weeks postimplantation, femur defects were assigned to histological analysis. Hematoxylin and eosin, Safranin O, tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor kappa B ligand (RANKL), and matrix metalloproteinase-2 (MMP-2) staining were performed to determine the rate of particle degradation, number of osteoclasts around particles, and intensity and localization of TRAP, RANKL, and MMP-2 staining.In the present study, NBM particles demonstrated little signs of degradation. The combination of NBM with EMD significantly increased the number of osteoclasts around NBM particles and increased expression of RANKL and MMP-2 specifically around particle surface. Only minor resorption was observed. Both DFDBA particles showed much faster degradation of particles. Interestingly, fewer osteoclasts were found on their surface when compared with NBM particles, specifically on Osteotech DFDBA particles, suggesting an alternative mode of degradation. Osteotech DFDBA particles demonstrated significantly faster degradation when compared with all other bone grafts. No obvious increase in TRAP, RANKL, or MMP2 was observed to validate this fast rate of degradation.The results from the present study demonstrate a wide range of particle degradation between various commonly commercially available bone grafts. Further research to determine the precise mechanisms that influence particle degradation is necessary.

New development of biomaterial scaffolds remains a prominent issue for the regeneration of lost or fractured bone. Of these scaffolds, a number of bioactive polymers have been synthesized and fabricated for diverse biological roles. Although recent evidence has demonstrated that composite scaffolds such as HA/PLLA have improved properties when compared to either HA or PLLA alone, recent investigations have demonstrated that the phase compatibility between HA and PLLA layers is weak preventing optimal enhancement of the mechanical properties and making the composites prone to breakdown. In the present study, poly (γ-benzyl-L-glutamate) modified hydroxyapatite/(poly (L-lactic acid)) (PBLG-g-HA/PLLA) composite scaffolds were fabricated with improved phase compatibility and tested for their osteogenic properties in 18 Wistar female rats by analyzing new bone formation in 3 mm bilateral femur defects in vivo. At time points, 2, 4 and 8 weeks post surgery, bone formation was evaluated by µ-CT and histological analysis by comparing 4 treatment groups; 1) blank defect, 2) PLLA, 3) HA/PLLA and 4) PBLG-g-HA/PLLA scaffolds. The in vivo analysis demonstrated that new bone formation was much more prominent in HA/PLLA and PBLG-g-HA/PLLA groups as depicted by µ-CT, H&E staining and immunohistochemistry for collagen I. TRAP staining was also utilized to determine the influence of osteoclast cell number and staining intensity to the various scaffolds. No significant differences in either staining intensity or osteoclast numbers between all treatment modalities was observed, however blank defects did contain a higher number of osteoclast-like cells. The results from the present study illustrate the potential of PBLG-g-HA/PLLA scaffolds for bone tissue engineering applications by demonstrating favorable osteogenic properties.

BACKGROUND Findings from animal and human studies have indicated that an oily calcium hydroxide suspension (OCHS) may improve early wound healing in the treatment of periodontitis. Calcium hydroxide as the main component is well known for its antimicrobial activity, however at present the effect of OCHS on the influence of periodontal wound healing/regeneration is still very limited. The purpose of this in vitro study was to investigate the effect of OCHS on periodontopathogenic bacteria as well as on the attachment and proliferation of osteoblasts and periodontal ligament fibroblasts. METHODS Human alveolar osteoblasts (HAO) and periodontal ligament (PDL) fibroblasts were cultured on 3 concentrations of OCHS (2.5, 5 and 7.5 mg). Adhesion and proliferation were counted up to 48 h and mineralization was assayed after 1 and 2 weeks. Furthermore potential growth inhibitory activity on microorganisms associated with periodontal disease (e.g. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans) as well as the influence of periodontopathogens and OCHS on the HAO and PDL fibroblasts counts were determined. RESULTS More than a 2-fold increase in adherent HAO cells was observed at 4 h following application of OCHS when compared to the control group (p = 0.007 for 2.5 mg). Proliferation of HAO cells at 48 h was stimulated by moderate concentrations (2.5 mg; 5 mg) of OCHS (each p

OBJECTIVES Previously, the use of enamel matrix derivative (EMD) in combination with a natural bone mineral (NBM) was able to stimulate periodontal ligament cell and osteoblast proliferation and differentiation. Despite widespread use of EMD for periodontal applications, the effects of EMD on bone regeneration are not well understood. The aim of the present study was to test the ability of EMD on bone regeneration in a rat femur defect model in combination with NBM. MATERIALS AND METHODS Twenty-seven rats were treated with either NBM or NBM + EMD and assigned to histological analysis at 2, 4, and 8 weeks. Defect morphology and mineralized bone were assessed by μCT. For descriptive histology, hematoxylin and eosin staining and Safranin O staining were performed. RESULTS Significantly more newly formed trabecular bone was observed at 4 weeks around the NBM particles precoated with EMD when compared with NBM particles alone. The drilled control group, in contrast, achieved minimal bone regeneration at all three time points (P < 0.05). CONCLUSIONS The present results may suggest that EMD has the ability to enhance the speed of new bone formation when combined with NBM particles in rat osseous defects. CLINICAL RELEVANCE These findings may provide additional clinical support for the combination of EMD with bone graft for the repair of osseous and periodontal intrabony defects.

BACKGROUND Despite the worldwide increased prevalence of osteoporosis, no data are available evaluating the effect of an enamel matrix derivative (EMD) on the healing of periodontal defects in patients with osteoporosis. This study aims to evaluate whether the regenerative potential of EMD may be suitable for osteoporosis-related periodontal defects. METHODS Forty female Wistar rats (mean body weight: 200 g) were used for this study. An osteoporosis animal model was carried out by bilateral ovariectomy (OVX) in 20 animals. Ten weeks after OVX, bilateral fenestration defects were created at the buccal aspect of the first mandibular molar. Animals were randomly assigned to four groups of 10 animals per group: 1) control animals with unfilled periodontal defects; 2) control animals with EMD-treated defects; 3) OVX animals with unfilled defects; and 4) OVX animals with EMD-treated defects. The animals were euthanized 28 days later, and the percentage of defect fill and thickness of newly formed bone and cementum were assessed by histomorphometry and microcomputed tomography (micro-CT) analysis. The number of osteoclasts was determined by tartrate-resistant acid phosphatase (TRAP), and angiogenesis was assessed by analyzing formation of blood vessels. RESULTS OVX animals demonstrated significantly reduced bone volume in unfilled defects compared with control defects (18.9% for OVX animals versus 27.2% for control animals) as assessed by micro-CT. The addition of EMD in both OVX and control animals resulted in significantly higher bone density (52.4% and 69.2%, respectively) and bone width (134 versus 165μm) compared with untreated defects; however, the healing in OVX animals treated with EMD was significantly lower than that in control animals treated with EMD. Animals treated with EMD also demonstrated significantly higher cementum formation in both control and OVX animals. The number of TRAP-positive osteoclasts did not vary between untreated and EMD-treated animals; however, a significant increase was observed in all OVX animals. The number of blood vessels and percentage of new vessel formation was significantly higher in EMD-treated samples. CONCLUSIONS The results from the present study suggest that: 1) an osteoporotic phenotype may decrease periodontal regeneration; and 2) EMD may support greater periodontal regeneration in patients suffering from the disease. Additional clinical studies are necessary to fully elucidate the possible beneficial effect of EMD for periodontal regeneration in patients suffering from osteoporosis.

Recent studies demonstrate that the rate of periodontal breakdown significantly increased in patients compromised from both periodontal disease and osteoporosis. One pharmacological agent used for their treatment is strontium renalate due to its simultaneous ability to increase bone formation and halt bone resorption. The aim of the present study was to achieve periodontal regeneration of strontium-incorporated mesoporous bioactive glass (Sr-MBG) scaffolds in an osteoporotic animal model carried out by bilateral ovariectomy (OVX). 15 female Wistar rats were randomly assigned to three groups: control unfilled periodontal defects, 2) MBG alone and 3) Sr-MBG scaffolds. 10 weeks after OVX, bilateral fenestration defects were created at the buccal aspect of the first mandibular molar and assessed by micro-CT and histomorphometric analysis after 28 days. Periodontal fenestration defects treated with Sr-MBG scaffolds showed greater new bone formation (46.67%) when compared to MBG scaffolds (39.33%) and control unfilled samples (17.50%). The number of TRAP-positive osteoclasts was also significantly reduced in defects receiving Sr-MBG scaffolds. The results from the present study suggest that Sr-MBG scaffolds may provide greater periondontal regeneration. Clinical studies are required to fully characterize the possible beneficial effect of Sr-releasing scaffolds for patients suffering from a combination of both periodontal disease and osteoporosis.

Deconvolution of the evolved isobutene data obtained from temperature-programmed, low-pressure steady-state conversion of isobutane over HZSM-5 has yielded apparent activation energies for isobutane dehydrogenation, isobutene dimerization and desorption. Intrinsic activation energies and associated isobutane collision frequencies are also estimated. A combination of wavelet shrinkage denoising, followed by time-varying flexible least squares of the evolved mass-spectral abundance data over the temperature range 150 to 450 °C, provides accurate, temperature-dependent, apparent rate parameters. Intrinsic activation energies for isobutane dehydrogenation range from 86 to 235.2 kJ mol −1 (average = 150 ± 42 kJ mol −1 ) for isobutene dimerization from 48.3 to 267 kJ mol −1 (average = 112 ± 74 kJ mol −1 ) and for isobutene desorption from 64.4 to 97.8 kJ mol −1 (average = 77 ± 12 kJ mol −1 ). These wide ranges reflect the heterogeneity and acidity of the zeolite surface and structure. Seven distinct locations and sites, including Lewis and Bronsted acid sites can be identified in the profiles. Isobutane collision frequencies range from 10 −0.4 to 10 22.2 s −1 and are proportional to the accessibility of active sites, within the HZSM-5 micropores or on the external surface.

SUMMARY: Recently, the use of the pharmacological agent strontium ranelate has come to prominence for the treatment of osteoporosis. While much investigation is focused on preventing disease progression, here we fabricate strontium-containing scaffolds and show that they enhance bone defect healing in the femurs of rats induced by ovariectomy. INTRODUCTION: Recently, the use of the pharmacological agent strontium ranelate has come to prominence for the treatment of osteoporosis due to its ability to prevent bone loss in osteoporotic patients. Although much emphasis has been placed on using pharmacological agents for the prevention of disease, much less attention has been placed on the construction of biomaterials following osteoporotic-related fracture. The aim of the present study was to incorporate bioactive strontium (Sr) trace element into mesoporous bioactive glass (MBG) scaffolds and to investigate their in vivo efficacy for bone defect healing in the femurs of rats induced by ovariectomy. METHODS: In total, 30 animals were divided into five groups as follows: (1) empty defect (control), (2) empty defects with estrogen replacement therapy, (3) defects filled with MBG scaffolds alone, (4) defects filled with MBG + estrogen replacement therapy, and (5) defects filled with strontium-incorporated mesopore-bioglass (Sr-MBG) scaffolds. RESULTS: The two groups demonstrating the highest levels of new bone formation were the defects treated with MBG + estrogen replacement therapy and the defects receiving Sr-MBG scaffolds as assessed by μ-CT and histological analysis. Furthermore, Sr scaffolds had a reduced number of tartrate-resistant acid phosphatase-positive cells when compared to other modalities. CONCLUSION: The results from the present study demonstrate that the local release of Sr from bone scaffolds may improve fracture repair. Future large animal models are necessary to investigate the future relationship of Sr incorporation into biomaterials.

The purpose of this investigation was to compare the osteoinductive and osteopromotive potential of two widely used demineralized freeze-dried bone allografts (DFDBA) (Osteotech® DFDBA and LifeNet® DFDBA).Twenty-seven male Wistar rats (mean body weight 200 g) were treated with either DFDBA from Osteotech and LifeNet or control for femoral and intramuscular defects and assigned to histological analysis at 2, 4, and 8 weeks postimplantation. Hematoxylin and eosin (HE), Safranin-O, tartrate-resistant acid phosphatase (TRAP), and osteopontin (OPN) staining were performed. Quantitative analysis of mineralized new bone to total volume (BV/TV) was assessed by micro-computed tomography.Both allografts demonstrated osteoinductive potential at 2 weeks as assessed by intramuscular bone formation. LifeNet DFDBA displayed continual new bone formation at 4 and 8 weeks, whereas Osteotech particles were fully resorbed by 4 weeks postimplantation. Femur defects demonstrated significantly greater BV/TV at 4 and 8 weeks with higher expression of OPN staining around LifeNet DFDBA particles. TRAP-positive cells were visible in and around both allograft materials.The results from the present study indicate that variability among allografts exists. In the present, LifeNet DFDBA supported more new bone formation. Further larger animal models or clinical trials are required to validate these findings.

Cryptosporidium parvum is considered one of the most common enteropathogens, responsible for the high incidence of diarrhea and deleterious implications on immunity and health in neonatal calves. The pomegranate is well known for its health-promoting properties. Two experiments were designed to test the antiparasitical and antidiarrheal effects of concentrated pomegranate extract (CPE) supplement in milk in neonatal Holstein calves. Forty-one calves were randomly divided into control (n=20) and treatment (n=21) groups. For the first experiment, the treatment group was supplemented with 3.75% CPE in the daily milk ration, between 3 and 14 d of age, whereas the control group received only milk. Fecal samples were collected between d 5 and 13 to quantify Cryptosporidium oocysts, and the duration and intensity of diarrhea were evaluated. Reduced fecal oocyst count and diarrhea intensity and duration were revealed in the 3.75% CPE calves. No difference was noted in average daily gain between groups. In a second experiment, which was designed to test the effect of a lower CPE concentration (0.6% of daily milk allocation), no effects on fecal oocyst count and average daily gain were observed. However, compared with control, the lower CPE group was characterized by a shorter duration of diarrhea and higher weight gain among males at 14 d of age. These results suggest that the CPE supplement-to-calf milk ratio may potentially alleviate intestinal morbidity caused by Cryptosporidium.

Five rumen- and abomasum-cannulated cows in mid-lactation were fed ad libitum, for two periods of 21 d each, two isonitrogenous (17% crude protein) total mixed rations (TMR) composed of 45% sorghum-wheat combination, 38% corn silage, 12% soybean meal, urea, buffers and minerals. The two TMRs differed in their sorghum (S): wheat (W) ratio, which was 7030 in S and 3070 in W treatments. Dry matter intake was 17.2 and 16.6 kg d−1 in S and W, respectively. Effect of treatments on the digestibility of carbohydrate constituents and N metabolism was examined. Neutral detergent fiber (NDF)-polysaccharides digestibility in stomachs and the entire gastrointestinal tract (GIT) was significantly higher in S than in W. The digestibility of neutral detergent soluble (NDS)-α-glucans was significantly higher in W than in S. More of NDS-α-glucans escaping ruminal fermentation was digested in the intestine of the S cows as compared with the W cows. The digestibility in entire GIT of total NDS-monosaccharide residues was 93% in W and 86% in S. Digestibility values of total carbohydrates in stomachs (49% and 52%) and entire GIT (79% and 80%) of S and W cows were similar. S and W cows were similar in rumen pH (6.15 and 6.13), rumen volatile fatty acids concentrations (133 and 136 mM) and rumen NH3 concentration (21 and 23 mg per 100 ml). Non-ammonia-N flow through the abomasum (410 and 403 g d−1) and N absorption in the intestine (67%) were also similar in S and W.

Objectives: Bioresorbable collagen membranes are routinely utilized in guided bone regeneration to selectively direct the growth and repopulation of bone cells in areas of insufficient volume. However, the exact nature by which alveolar osteoblasts react to barrier membranes as well as the effects following the addition of growth factors to the membranes are still poorly understood. The objective of the present study was therefore to investigate the effect of a bioresorbable collagen membrane soak-loaded in growth factors bone morphogenetic protein 2 (BMP2) or transforming growth factor β1 (TGFβ1) on osteoblast adhesion, proliferation, and differentiation. Material and methods: Prior to experimental seeding, membranes were soaked in either BMP2 or TGFβ1 at a concentration of 10ng/ml for 5min. Results: Human osteoblasts adhered to all soak-loaded membranes as assessed by scanning electron microscopy. Growth factors BMP2 and TGFβ1 increased osteoblast proliferation at 3 or 5days post-seeding when compared to control collagen membranes. Analysis of real-time PCR revealed that administration of BMP2 increased osteoblast differentiation markers such as osterix, collagen I, and osteocalcin. BMP2 also increased mineralization of primary osteoblasts as demonstrated by alizarin red staining when compared to control and TGFβ1 soak-loaded membranes. Conclusion: The combination of a collagen barrier membrane with growth factors TGFβ1 and BMP2 significantly influenced adhesion, proliferation, and differentiation of primary human osteoblasts. Clinical relevance: The described in vitro effects following the combination of collagen barrier membranes with growth factors TGFβ1 and BMP2 provide further biologic support for the clinical application of this treatment strategy in guided bone regeneration procedures

Enamel matrix derivative (EMD), a porcine extract harvested from developing porcine teeth, has been shown to promote formation of new cementum, periodontal ligament and alveolar bone. Despite its widespread use, an incredibly large variability among in vitro studies has been observed. The aim of the present study was to determine the influence of EMD on cells at different maturation stages of osteoblast differentiation by testing 6 cell types to determine if cell phenotype plays a role in cell behaviour following treatment with EMD. Six cell types including MC3T3-E1 pre-osteoblasts, rat calvarial osteoblasts, human periodontal ligament (PDL) cells, ROS cells, MG63 cells and human alveolar osteoblasts were cultured in the presence or absence of EMD and proliferation rates were quantified by an MTS assay. Gene expression of collagen1(COL1), alkaline phosphate(ALP) and osteocalcin(OC) were investigated by real-time PCR. While EMD significantly increased cell proliferation of all cell types, its effect on osteoblast differentiation was more variable. EMD significantly up-regulated gene expression of COL1, ALP and OC in cells early in their differentiation process when compared to osteoblasts at later stages of maturation. Furthermore, the effect of cell passaging of primary human PDL cells (passage 2 to 15) was tested in response to treatment with EMD. EMD significantly increased cell proliferation and differentiation of cells at passages 2-5 however had completely lost their ability to respond to EMD by passages 10+. The results from the present study suggest that cell stimulation with EMD has a more pronounced effect on cells earlier in their differentiation process and may partially explain why treatment with EMD primarily favors regeneration of periodontal defects (where the periodontal ligament contains a higher number of undifferentiated progenitor cells) over regeneration of pure alveolar bone defects containing no periodontal ligament and a more limited number of osteoprogenitor cells.

Alfalfa (A) was ensiled either wilted (35% dry matter (DM)) (WA) or as a mixture of 55% fresh alfalfa + 45% conoxyltreated cotton gin trash (A + CT-CGT) (DM basis), and the effects of feeding these silages to growing lambs were examined. Lactic acid concentration was higher in A + CT-CGT than in WA silage, but levels of TCA-N (g per 100 g N) were similar, pointing to equal ability of the two silages to preserve protein. Neutral detergent fiber (NDF) glucose level was unchanged, while neutral detergent soluble (NDS) glucose was extensively fermented in the WA silage. Conversely, in the A + CT-CGT silage there was a drop in NDF glucose and a slight increase in NDS glucose concentration. NDF and NDS arabinose and galactose in both silages were slightly reduced during ensilage. Source materials and corresponding silages had similar in vitro digestibility values, but in vitro digestibility of monosaccharides in WA was 10% higher than that of the A + CT-CGT silage. Twenty lambs of the Assaf breed (average body weight (BW) 29 kg) were divided into two equal groups and fed rations composed of 50% concentrate, 25% poultry litter and 25% of either WA silage or A + CT-CGT silage. The diets were fed ad libitum for 64–67 days until the lambs reached 47–48 kg BW. Voluntary feed intake was 5% higher in the A + CT-CGT group than in the WA group. The average daily gains (ADG) were 284 g and 288 g in the WA and A + CT-CGT groups, respectively, which are considered as the low range of intensive growth. Slaughter data of the two dietary groups were comparable. Therefore CT-CGT can be used successfully as a silage additive for direct ensilage of fresh alfalfa. Intensive growth of lambs can be attained on a ration consisting of agricultural wastes including poultry litter, and CT-CGT used as a silage additive.

Lucerne was ensiled either by the conventional wilting process (WL) or as a mixture of 80% fresh lucerne + 20% ozonated cotton stalks (L + OCS), and the effect of feeding these silages to dairy cows on performance and on the digestibility of ration carbohydrate constituents was examined. Ten cows in mid-lactation were divided into two equal groups with similar performance data at the onset of the experiment, and fed ad libitum for 28 days total mixed rations composed of 70% concentrate, 15% lucerne hay and 15% of either WL silage or L + OCS silage. Average daily milk yields during the experimental period were 27.1 and 27.6 kg in the WL and L + OCS groups, respectively, and the L + OCS cows gained higher body weight. Milk composition was similar in both treatments. The WL silage had a lower content of neutral detergent soluble (NDS) glucose, neutral detergent fibre (NDF) glucose and NDF xylose, and a higher content of arabinose, galactose and mannose than L + OCS silage. Carbohydrate digestibility was 83 and 85% in the WL and L + OCS rations, respectively. NDF digestibility was similar (45.0%) in both rations; however, digestion of NDF glucose, xylose and total polysaccharides was slightly higher in the L + OCS group than in the WL ration. In both rations, NDF glucose was more digestible than NDF xylose and NDF uronic acid and less digestible than NDF arabinose, galactose and mannose. Digestibility of total NDS carbohydrate was 89% in WL ration and 91% in L + OCS ration, and in both groups, digestion of NDS mannose and galactose was lower than that of NDS glucose, arabinose and pectin. Data of this study indicate that a direct-cut and ensiled lucerne silage made of L + OCS is equivalent to lucerne haylage as a component in lactating cow rations.

Tissue engineering generally requires three basic elements; stem/progenitor cells, inductive agents and a biomaterial scaffold; the latter is one of the key components which directly influences cellular activity and matrix formation. Commonly used scaffolds to repair defects in general do not induce stem cell recruitment, which is an essential element to tissue regeneration. In this study, fabrication of a scaffold which is capable of restoring damaged tissue through the recruitment of mesenchymal stem cells (MSCs) by gene therapy of the gene encoding platelet-derived growth factor-B (PDGF-B) was investigated. PDGF-B adenovirus (AdPDGF) was combined into novel mesoporous bioglass-silk fibrin scaffolds, which were characterized for their controlled release and sustained bioactivity. Our results demonstrate that these scaffolds can release PDGF-B adenovirus for up to 3 weeks and increase MSC recruitment, both in vitro and following subcutaneous implantation in mice. Osseous calvarial defects in mice further demonstrate the ability of these scaffolds to enhance tissue regeneration through stem cell homing. This study demonstrates the potent ability of host stem cells to regenerate tissue defects through recruitment of MSCs via gene therapy. Copyright © 2013 John WileySons, Ltd.

Autogenous bone grafts obtained by different harvesting techniques behave differently during the process of graft consolidation; the underlying reasons are however not fully understood. One theory is that harvesting techniques have an impact on the number and activity of the transplanted cells which contribute to the process of graft consolidation.To test this assumption, porcine bone grafts were harvested with four different surgical procedures: bone mill, piezosurgery, bone drilling (bone slurry), and bone scraper. After determining cell viability, the release of molecules affecting bone formation and resorption was assessed by reverse transcription polymerase chain reaction and immunoassay. The mitogenic and osteogenic activity of the conditioned media was evaluated in a bioassay with isolated bone cells.Cell viability and the release of molecules affecting bone formation were higher in samples harvested by bone mill and bone scraper when compared with samples prepared by bone drilling and piezosurgery. The harvesting procedure also affected gene expression, for example, bone mill and bone scraper samples revealed significantly higher expression of growth factors such as bone morphogenetic protein-2 and vascular endothelial growth factor compared with the two other modalities. Receptor activator of nuclear factor kappa B ligand expression was lowest in bone scraper samples.These data can provide a scientific basis to better understand the impact of harvesting techniques on the number and activity of transplanted cells, which might contribute to the therapeutic outcome of the augmentation procedure.