The effect of matrix bound parathyroid hormone on bone regeneration

INTRODUCTION: Autogenous bone is the most successful bone-grafting material; however, multiple disadvantages continue to drive developments of improved methods for bone regeneration. AIM: The aim of the present study was to test the hypothesis that an arginine-glycine-aspartic acid (RGD) modified polyethylene glycol-based matrix (PEG) containing covalently bound peptides of the parathyroid hormone (PTH(1-34)) enhances bone regeneration to a degree similar to autogenous bone. MATERIAL AND METHODS: Six American foxhounds received a total of 48 cylindrical titanium implants placed in the mandible between the first premolar and the second molar. Five, respectively, 7 months following tooth extraction, implants were placed into the center of surgically created defects. This resulted in a circumferential bone defect simulating an alveolar defect with a circular gap of 1.5 mm. Four treatment modalities were randomly allocated to the four defects per side: (1) PEG-matrix containing 20 microg/ml of PTH(1-34), and 350 microg/ml cys-RGD peptide, (2) PEG alone, (3) autogenous bone and (4) empty defects. Histomorphometric analysis was performed 4 and 12 weeks after implantation. The area fraction of newly formed bone was determined within the former defect and the degree of bone-to-implant contact (BIC) was evaluated both in the defect region and in the apical region of the implant. For statistical analysis ANOVA and subsequent pairwise Student's t-test were applied. RESULTS: Healing was uneventful and all implants were histologically integrated. Histomorphometric analysis after 4 weeks showed an average area fraction of newly formed bone of 41.7+/-1.8% for matrix-PTH, 26.6+/-4.1% for PEG alone, 43.9+/-4.5% for autogenous bone, and 28.9+/-1.5% for empty defects. After 12 weeks, the respective values were 49.4+/-7.0% for matrix-PTH, 39.3+/-5.7% for PEG alone, 50.5+/-3.4% for autogenous bone and 38.7+/-1.9% for empty defects. Statistical analysis after 4 and 12 weeks revealed significantly more newly formed bone in the PTH(1-34) group compared with PEG alone or empty defects, whereas no difference could be detected against autogenous bone. Regarding BIC no significant difference was observed between the four treatment groups neither at 4 nor at 12 weeks. CONCLUSION: It is concluded that an RGD-modified PEG hydrogel containing PTH(1-34) is an effective matrix system to obtain bone regeneration.

Targeted cell replacement with bone marrow cells for airway epithelial regeneration

It has been suggested that some adult bone marrow cells (BMC) can localize to the lung and develop tissue-specific characteristics including those of pulmonary epithelial cells. Here, we show that the combination of mild airway injury (naphthalene-induced) as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype. Confocal analysis of airway and alveolar-localized BMC (fluorescently labeled) with epithelial markers shows expression of the pulmonary epithelial proteins, Clara cell secretory protein, and surfactant protein C. To confirm epithelial gene expression by BMC, we generated transgenic mice expressing green fluorescent protein (GFP) driven by the epithelial-specific cytokeratin-18 promoter and injected BMC from these mice transtracheally into wild-type recipients after naphthalene-induced airway injury. BMC retention in the lung was observed for at least 120 days following cell delivery with increasing GFP transgene expression over time. Some BMC cultured in vitro over time also expressed GFP transgene, suggesting epithelial transdifferentiation of the BMC. The results indicate that targeted delivery of BMC can promote airway regeneration.

Early implant placement with simultaneous guided bone regeneration following single-tooth extraction in the esthetic zone: a cross-sectional, retrospective study in 45 subjects with a 2- to 4-year follow-up

BACKGROUND: The concept of early implant placement is a treatment option in postextraction sites of single teeth in the anterior maxilla. Implant placement is performed after a soft tissue healing period of 4 to 8 weeks. Implant placement in a correct three-dimensional position is combined with a simultaneous guided bone regeneration procedure to rebuild esthetic facial hard and soft tissue contours. METHODS: In this retrospective, cross-sectional study, 45 patients with an implant-borne single crown in function for 2 to 4 years were recalled for examination. Clinical and radiologic parameters, routinely used in implant studies, were assessed. RESULTS: All 45 implants were clinically successful according to strict success criteria. The implants demonstrated ankylotic stability without signs of a peri-implant infection. The peri-implant soft tissues were clinically healthy as indicated by low mean plaque (0.42) and sulcus bleeding index (0.51) values. None of the implants revealed a mucosal recession on the facial aspect as confirmed by a clearly submucosal position of all implant shoulders. The mean distance from the mucosal margin to the implant shoulder was -1.93 mm on the facial aspect. The periapical radiographs showed stable peri-implant bone levels, with a mean distance between the implant shoulder and the first bone-implant contact of 2.18 mm. CONCLUSIONS: This retrospective study demonstrated successful treatment outcomes for all 45 implants examined. The mid-term follow-up of 2 to 4 years also showed that the risk for mucosal recession was low with this treatment concept. Prospective clinical studies are required to confirm these encouraging results.

Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels

BACKGROUND: Despite a large body of clinical and histological data demonstrating beneficial effects of enamel matrix proteins (EMPs) for regenerative periodontal therapy, it is less clear how the available biological data can explain the mechanisms underlying the supportive effects of EMPs. OBJECTIVE: To analyse all available biological data of EMPs at the cellular and molecular levels that are relevant in the context of periodontal wound healing and tissue formation. METHODS: A stringent systematic approach was applied using the key words "enamel matrix proteins" OR "enamel matrix derivative" OR "emdogain" OR "amelogenin". The literature search was performed separately for epithelial cells, gingival fibroblasts, periodontal ligament cells, cementoblasts, osteogenic/chondrogenic/bone marrow cells, wound healing, and bacteria. RESULTS: A total of 103 papers met the inclusion criteria. EMPs affect many different cell types. Overall, the available data show that EMPs have effects on: (1) cell attachment, spreading, and chemotaxis; (2) cell proliferation and survival; (3) expression of transcription factors; (4) expression of growth factors, cytokines, extracellular matrix constituents, and other macromolecules; and (5) expression of molecules involved in the regulation of bone remodelling. CONCLUSION: All together, the data analysis provides strong evidence for EMPs to support wound healing and new periodontal tissue formation.

Patch selection by wintering white-tailed deer : consequences for eastern hemlock regeneration at local and landscape scales

Tsuga canadensis (eastern hemlock) is a highly shade-tolerant, late-successional, and long-lived conifer species found throughout eastern North America. It is most often found in pure or nearly pure stands, because highly acidic and nutrient poor forest floor conditions are thought to favor T. canadensis regeneration while simultaneously limiting the establishment of some hardwood species with greater nutrient requirements. Once a common species, T. canadensis is currently experiencing widescale declines across its range. The hemlock woolly adelgid (Adelges tsugae) is decimating the population across its eastern distribution. Across the Upper Great Lakes region, where the adelgid is currently being held at bay by cold winter temperatures, T. canadensis has been experiencing failures in regeneration attributed, in part, to herbivory by white-tailed deer (Odocoileus virginianus). Deer utilize T. canadensis stands as winter habitat in areas of high snow depth. Tsuga canadensis, once a major component of these forests, currently exists at just a fraction of its pre-settlement abundance due to historic logging and contemporary forest management practices, and what remains is found in small remnant patches surrounded by second- and third-growth deciduous forests. The deer population across the region, however, is likely double that of pre-European settlement times. In this dissertation I explore the relationship between white-tailed deer use of T. canadensis as winter habitat and the effect this use is having on regeneration and forest succession. For this research I quantified stand composition and structure and abiotic variables of elevation and snow depth in 39 randomly selected T. canadensis stands from across the western Upper Peninsula of Michigan. I also quantified composition and the configuration of the landscapes surrounding these stands. I measured relative deer use of T. canadensis stands as pellet group piles deposited in each stand during each of three consecutive winters, 2005-06, 2006-07, and 2007-08. The results of this research suggest that deer use of T. canadensis stands as winter habitat is influenced primarily by snow depth, elevation, and the composition and configuration of the greater landscapes surrounding these stands. Specifically, stands with more heterogeneous landscapes surrounding them (i.e., a patchy mosaic of conifer, deciduous, and open cover) had higher relative deer use than stands surrounded by homogenous deciduous forest cover. Additionally, the intensity of use and the number of stands used was greater in years with higher average snow depth. Tsuga canadensis regeneration in these stands was negatively associated with deer use and Acer saccharum (sugar maple) basal area. Of the 39 stands, 17 and 22 stands had no T. canadensis regeneration in small and large sapling categories, respectively. Acer saccharum was the most common understory tree species, and the importance of A. saccharum in the understory (stems < 10 cm dbh) of the stands was positively associated with overstory A. saccharum dominance. Tsuga canadensis establishment was associated with high-decay coarse woody debris and moss, and deciduous leaf litter inputs in these stands may be limiting access to these important microsites. Furthermore, A. saccharum is more tolerant to the effects of deer herbivory than T. canadensis, giving A. saccharum a competitive advantage in stands being utilized as winter habitat by deer. My research suggests that limited microsite availability, in conjunction with deer herbivory, may be leading to an erosion in T. canadensis patch stability and an altered successional trajectory toward one of A. saccharum dominance, an alternately stable climax species.

Modeling of diesel particulate filter filtration and regeneration for transient driving schedules

Due to their high thermal efficiency, diesel engines have excellent fuel economy and have been widely used as a power source for many vehicles. Diesel engines emit less greenhouse gases (carbon dioxide) compared with gasoline engines. However, diesel engines emit large amounts of particulate matter (PM) which can imperil human health. The best way to reduce the particulate matter is by using the Diesel Particulate Filter (DPF) system which consists of a wall-flow monolith which can trap particulates, and the DPF can be periodically regenerated to remove the collected particulates. The estimation of the PM mass accumulated in the DPF and total pressure drop across the filter are very important in order to determine when to carry out the active regeneration for the DPF. In this project, by developing a filtration model and a pressure drop model, we can estimate the PM mass and the total pressure drop, then, these two models can be linked with a regeneration model which has been developed previously to predict when to regenerate the filter. There results of this project were: 1 Reproduce a filtration model and simulate the processes of filtration. By studying the deep bed filtration and cake filtration, stages and quantity of mass accumulated in the DPF can be estimated. It was found that the filtration efficiency increases faster during the deep-bed filtration than that during the cake filtration. A “unit collector” theory was used in our filtration model which can explain the mechanism of the filtration very well. 2 Perform a parametric study on the pressure drop model for changes in engine exhaust flow rate, deposit layer thickness, and inlet temperature. It was found that there are five primary variables impacting the pressure drop in the DPF which are temperature gradient along the channel, deposit layer thickness, deposit layer permeability, wall thickness, and wall permeability. 3 Link the filtration model and the pressure drop model with the regeneration model to determine the time to carry out the regeneration of the DPF. It was found that the regeneration should be initiated when the cake layer is at a certain thickness, since a cake layer with either too big or too small an amount of particulates will need more thermal energy to reach a higher regeneration efficiency. 4 Formulate diesel particulate trap regeneration strategies for real world driving conditions to find out the best desirable conditions for DPF regeneration. It was found that the regeneration should be initiated when the vehicle’s speed is high and during which there should not be any stops from the vehicle. Moreover, the regeneration duration is about 120 seconds and the inlet temperature for the regeneration is 710K.

Regulated catalysis of extracellular nucleotides by vascular CD39/ENTPD1 is required for liver regeneration

BACKGROUND & AIMS: Little is known about how endothelial cells respond to injury, regulate hepatocyte turnover and reconstitute the hepatic vasculature. We aimed to determine the effects of the vascular ectonucleotidase CD39 on sinusoidal endothelial cell responses following partial hepatectomy and to dissect purinergic and growth factor interactions in this model. METHODS: Parameters of liver injury and regeneration, as well as the kinetics of hepatocellular and sinusoidal endothelial cell proliferation, were assessed following partial hepatectomy in mice that do not express CD39, that do not express ATP/UTP receptor P2Y2, and in controls. The effects of extracellular ATP on vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-6 responses were determined in vivo and in vitro. Phosphorylation of the endothelial VEGF receptor in response to extracellular nucleotides and growth factors was assessed in vitro. RESULTS: After partial hepatectomy, expression of the vascular ectonucleotidase CD39 increased on sinusoidal endothelial cells. Targeted disruption of CD39 impaired hepatocellular regeneration, reduced angiogenesis, and increased hepatic injury, resulting in pronounced vascular endothelial apoptosis, and decreased survival. Decreased HGF release by sinusoidal endothelial cells, despite high levels of VEGF, reduced paracrine stimulation of hepatocytes. Failure of VEGF receptor-2/KDR transactivation by extracellular nucleotides on CD39-null endothelial cells was associated with P2Y2 receptor desensitization. CONCLUSIONS: Regulated phosphohydrolysis of extracellular nucleotides by CD39 coordinates both hepatocyte and endothelial cell proliferation following partial hepatectomy. Lack of CD39 activity is associated with decreased hepatic regeneration and failure of vascular reconstitution.

The vascular ectonucleotidase CD39 is permissive for sinusoidal endothelial cell proliferation during liver regeneration: evidence for synergism between growth factors and extracellular nucleotides

Crosstalk between elements of the sinusoidal vasculature, platelets and hepatic parenchymal cells influences regenerative responses to liver injury and/or resection. Such paracrine interactions include hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), IL-6 and small molecules such as serotonin and nucleotides. CD39 (nucleoside triphosphate diphosphohydrolase-1) is the dominant vascular ectonucleotidase expressed on the luminal surface of endothelial cells and modulates extracellular nucleotide signaling. We have previously shown that integrity of P2-receptors, as maintained by CD39, is required for angiogenesis in Matrigel plugs in vivo and that there is synergism between nucleotide P2-receptor- and growth factor-mediated cell proliferation in vitro. We have now explored effects of CD39 on liver regeneration and vascular endothelial growth factor responses in a standard small animal model of partial hepatectomy. The expression of CD39 on liver sinusoidal endothelial cells (LSEC) is substantially boosted during liver regeneration. This transcriptional upregulation precedes maximal sinusoidal endothelial cell proliferation, noted at day 5-8 in C57BL6 wild type mice. In matched mutant mice null for CD39 (n=14), overall survival is decreased to 71% by day 10. Increased lethality occurs as a consequence of extensive LSEC apoptosis, decreased endothelial proliferation and failure of angiogenesis leading to hepatic infarcts and regenerative failure in mutant mice. This aberrant vascular remodeling is associated with biochemical liver injury, elevated serum levels of VEGF (113.9 vs. 65.5pg/ml, p=0.013), and decreased circulating HGF (0.89 vs. 1.43 ng/ml, p=0.001) in mice null for CD39. In agreement with these observations, wild type LSEC but not CD39 null cultures upregulate HGF expression and secretion in response to exogenous VEGF in vitro. CD39 null LSEC cultures show poor proliferation responses and heightened levels of apoptosis when contrasted to wild type LSEC where agonists of P2Y receptors augment cell proliferation in the presence of growth factors. These observations are associated with features of P2Y-desensitization, normal levels of the receptor tyrosine kinase VEGFR-1 (Flt-1) and decreased expression of VEGFR-2 (FLK/KDR) in CD39 null LSEC cultures. We provide evidence that CD39 and extracellular nucleotides impact upon growth factor responses and tyrosine receptor kinases during LSEC proliferation. We propose that CD39 expression by LSEC might co-ordinate angiogenesis-independent liver protection by facilitating VEGF-induced paracrine release of HGF to promote vascular remodeling in liver regeneration.

Granulocyte colony stimulating factor supports liver regeneration in a surgical small for size liver remnant mouse model

ntense liver regeneration and almost 100% survival follows partial hepatectomy of up to 70% of liver mass in rodents. More extensive resections of 70 to 80% have an increased mortality and partial hepatectomies of >80% constantly lead to acute hepatic failure and death in mice. The aim of the study was to determine the effect of systemically administered granulocyte colony stimulating factor (G-CSF) on animal survival and liver regeneration in a small for size liver remnant mouse model after 83% partial hepatectomy (liver weight <0.8% of mouse body weight). Methods: Male Balb C mice (n=80, 20-24g) were preconditioned daily for five days with 5μg G-CSF subcutaneously or sham injected (aqua ad inj). Subsequently 83% hepatic resection was performed and daily sham or G-CSF injection continued. Survival was determined in both groups (G-CSF n=35; Sham: n=33). In a second series BrdU was injected (50mg/kg Body weight) two hours prior to tissue harvest and animals euthanized 36 and 48 hours after 83% liver resection (n=3 each group). To measure hepatic regeneration the BrdU labeling index and Ki67 expression were determined by immunohistochemistry by two independent observers. Harvested liver tissue was dried to constant weight at 65 deg C for 48 hours. Results: Survival was 0% in the sham group on day 3 postoperatively and significantly better (26.2% on day 7 and thereafter) in the G-CSF group (Log rank test: p<0.0001). Dry liver weight was increased in the G-CSF group (T-test: p<0.05) 36 hours after 83% partial hepatectomy. Ki67 expression was elevated in the G-CSF group at 36 hours (2.8±2.6% (Standard deviation) vs 0.03±0.2%; Rank sum test: p<0.0001) and at 48 hours (45.1±34.6% vs 0.7±1.0%; Rank sum test: p<0.0001) after 83% liver resection. BrdU labeling at 48 hours was 0.1±0.3% in the sham and 35.2±34.2% in the G-CSF group (Rank sum test: p<0.0001) Conclusions: The surgical 83% resection mouse model is suitable to test hepatic supportive regimens in the setting of small for size liver remnants. Administration of G-CSF supports hepatic regeneration after microsurgical 83% partial hepatectomy and leads to improved long-term survival in the mouse. G-CSF might prove to be a clinically valuable supportive substance in small for size liver remnants in humans after major hepatic resections due to primary or secondary liver tumors or in the setting of living related liver donation.

Dynamic modeling of active regeneration in catalyzed and non-catalyzed diesel particulate filters

Particulate matter (PM) emissions standards set by the US Environmental Protection Agency (EPA) have become increasingly stringent over the years. The EPA regulation for PM in heavy duty diesel engines has been reduced to 0.01 g/bhp-hr for the year 2010. Heavy duty diesel engines make use of an aftertreatment filtration device, the Diesel Particulate Filter (DPF). DPFs are highly efficient in filtering PM (known as soot) and are an integral part of 2010 heavy duty diesel aftertreatment system. PM is accumulated in the DPF as the exhaust gas flows through it. This PM needs to be removed by oxidation periodically for the efficient functioning of the filter. This oxidation process is also known as regeneration. There are 2 types of regeneration processes, namely active regeneration (oxidation of PM by external means) and passive oxidation (oxidation of PM by internal means). Active regeneration occurs typically in high temperature regions, about 500 - 600 °C, which is much higher than normal diesel exhaust temperatures. Thus, the exhaust temperature has to be raised with the help of external devices like a Diesel Oxidation Catalyst (DOC) or a fuel burner. The O2 oxidizes PM producing CO2 as oxidation product. In passive oxidation, one way of regeneration is by the use of NO2. NO2 oxidizes the PM producing NO and CO2 as oxidation products. The passive oxidation process occurs at lower temperatures (200 - 400 °C) in comparison to the active regeneration temperatures. Generally, DPF substrate walls are washcoated with catalyst material to speed up the rate of PM oxidation. The catalyst washcoat is observed to increase the rate of PM oxidation. The goal of this research is to develop a simple mathematical model to simulate the PM depletion during the active regeneration process in a DPF (catalyzed and non-catalyzed). A simple, zero-dimensional kinetic model was developed in MATLAB. Experimental data required for calibration was obtained by active regeneration experiments performed on PM loaded mini DPFs in an automated flow reactor. The DPFs were loaded with PM from the exhaust of a commercial heavy duty diesel engine. The model was calibrated to the data obtained from active regeneration experiments. Numerical gradient based optimization techniques were used to estimate the kinetic parameters of the model.

Granulocyte colony-stimulating factor increases hepatic sinusoidal perfusion during liver regeneration in mice

Conditioning with granulocyte colony-stimulating factor (G-CSF) promotes liver regeneration in an experimental small-for-size liver remnant mouse model. The mechanisms involved in this extraordinary G-CSF effect are unknown. The aim of this study was to investigate the influence of G-CSF on the hepatic microvasculature in the regenerating liver. The hepatic sinusoidal microvasculature and microarchitecture of the regenerating liver were evaluated by intravital microscopy in mice. Three experimental groups were compared: (1) unoperated unconditioned animals (control; n = 5), (2) animals conditioned with G-CSF 48 h after 60% partial hepatectomy (G-CSF-PH; n = 6), and (3) animals sham conditioned 48 h after 60% PH (sham-PH; n = 6). PH led to hepatocyte hypertrophy and increased hepatic sinusoidal velocity in the sham-PH and G-CSF-PH groups. Increased sinusoidal diameter and increased hepatic blood flow were observed in the G-CSF-PH group compared to the sham-PH and control groups. Furthermore, there was a strong positive correlation between spleen weight and hepatic sinusoidal diameter in the G-CSF-PH group. The increased hepatic blood flow could explain the observed benefit of G-CSF conditioning during liver regeneration. These results elucidate an unexplored aspect of pharmacological modulation of liver regeneration and motivate further experiments.