GPI-specific phospholipase D (GPI-PLD) is expressed during mouse development and is localized to the extracellular matrix of the developing mouse skeleton

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and has a well-characterized biochemistry; however, its physiological role is completely unknown. Previous investigations into GPI-PLD have focused on the adult animal or on in vitro systems and a putative role in development has been neither proposed nor investigated. We describe the first evidence of GPI-PLD expression during mouse embryonic ossification. GPI-PLD expression was detected predominantly at sites of skeletal development, increasing during the course of gestation. GPI-PLD was observed during both intramembraneous and endochondral ossification and localized predominantly to the extracellular matrix of chondrocytes and to primary trabeculae of the skeleton. In addition, the mouse chondrocyte cell line ATDC5 expressed GPI-PLD after experimental induction of differentiation. These results implicate GPI-PLD in the process of bone formation during mouse embryogenesis.

Early and Middle Holocene Hunter-Gatherer Occupations in Western Amazonia: The Hidden Shell Middens

We report on previously unknown early archaeological sites in the Bolivian lowlands, demonstrating for the first time early and middle Holocene human presence in western Amazonia. Multidisciplinary research in forest islands situated in seasonally-inundated savannahs has revealed stratified shell middens produced by human foragers as early as 10,000 years ago, making them the oldest archaeological sites in the region. The absence of stone resources and partial burial by recent alluvial sediments has meant that these kinds of deposits have, until now, remained unidentified. We conducted core sampling, archaeological excavations and an interdisciplinary study of the stratigraphy and recovered materials from three shell midden mounds. Based on multiple lines of evidence, including radiocarbon dating, sedimentary proxies (elements, steroids and black carbon), micromorphology and faunal analysis, we demonstrate the anthropogenic origin and antiquity of these sites. In a tropical and geomorphologically active landscape often considered challenging both for early human occupation and for the preservation of hunter-gatherer sites, the newly discovered shell middens provide evidence for early to middle Holocene occupation and illustrate the potential for identifying and interpreting early open-air archaeological sites in western Amazonia. The existence of early hunter-gatherer sites in the Bolivian lowlands sheds new light on the region’s past and offers a new context within which the late Holocene “Earthmovers” of the Llanos de Moxos could have emerged.

Treatment of multiple adjacent Miller class I and II gingival recessions with a Modified Coronally Advanced Tunnel (MCAT) technique and a collagen matrix or palatal connective tissue graft: a randomized, controlled clinical trial

BACKGROUND A newly developed collagen matrix (CM) of porcine origin has been shown to represent a potential alternative to palatal connective tissue grafts (CTG) for the treatment of single Miller Class I and II gingival recessions when used in conjunction with a coronally advanced flap (CAF). However, at present it remains unknown to what extent CM may represent a valuable alternative to CTG in the treatment of Miller Class I and II multiple adjacent gingival recessions (MAGR). The aim of this study was to compare the clinical outcomes following treatment of Miller Class I and II MAGR using the modified coronally advanced tunnel technique (MCAT) in conjunction with either CM or CTG. METHODS Twenty-two patients with a total of 156 Miller Class I and II gingival recessions were included in this study. Recessions were randomly treated according to a split-mouth design by means of MCAT + CM (test) or MCAT + CTG (control). The following measurements were recorded at baseline (i.e. prior to surgery) and at 12 months: Gingival Recession Depth (GRD), Probing Pocket Depth (PD), Clinical Attachment Level (CAL), Keratinized Tissue Width (KTW), Gingival Recession Width (GRW) and Gingival Thickness (GT). GT was measured 3-mm apical to the gingival margin. Patient acceptance was recorded using a Visual Analogue Scale (VAS). The primary outcome variable was Complete Root Coverage (CRC), secondary outcomes were Mean Root Coverage (MRC), change in KTW, GT, patient acceptance and duration of surgery. RESULTS Healing was uneventful in both groups. No adverse reactions at any of the sites were observed. At 12 months, both treatments resulted in statistically significant improvements of CRC, MRC, KTW and GT compared with baseline (p < 0.05). CRC was found at 42% of test sites and at 85% of control sites respectively (p < 0.05). MRC measured 71 ± 21% mm at test sites versus 90 ± 18% mm at control sites (p < 0.05). Mean KTW measured 2.4 ± 0.7 mm at test sites versus 2.7 ± 0.8 mm at control sites (p > 0.05). At test sites, GT values changed from 0.8 ± 0.2 to 1.0 ± 0.3 mm, and at control sites from 0.8 ± 0.3 to 1.3 ± 0.4 mm (p < 0.05). Duration of surgery and patient morbidity was statistically significantly lower in the test compared with the control group respectively (p < 0.05). CONCLUSIONS The present findings indicate that the use of CM may represent an alternative to CTG by reducing surgical time and patient morbidity, but yielded lower CRC than CTG in the treatment of Miller Class I and II MAGR when used in conjunction with MCAT.

Periodontal healing after application of enamel matrix derivative in surgical supra/infrabony periodontal defects in rats with streptozotocin-induced diabetes

BACKGROUND AND OBJECTIVE Epidemiologic and clinical studies have indicated that diabetes is a risk factor for periodontal disease progression and healing. The aim of the present study was to evaluate short-term healing after enamel matrix derivative (EMD) application in combined supra/infrabony periodontal defects in diabetic rats. MATERIAL AND METHODS Thirty male Wistar rats were initially divided into two groups, one with streptozotocin-induced diabetes and another one with healthy (non-diabetic) animals. Bony defects were surgically created on the mesial root of the first maxillary molars. After root surface planing and EDTA conditioning, EMD was applied to the roots at one side of the maxillae, while those on the contralateral sides were left untreated. Animals were killed 3 wk after surgery, and block sections were prepared for histologic and histomorphometric analysis. RESULTS There was statistically significant more gingival recession in diabetic animals than in non-diabetic animals. The length of the junctional epithelium was significantly shorter in the EMD-treated sites in both diabetic and normoglycemic rats. Sulcus depth and length of supracrestal soft connective tissue showed no statistically significant differences between groups. In all animals, new bone formation was observed. Although new bone occurred more frequently in healthy animals, the extent of new bone was not significantly different between groups. In none of the teeth, a layer of new cementum was detectable. EMD had no influence on bone or cementum regeneration. Adverse reactions such as excessive inflammation due to bacterial root colonization, ankylosis and bone fractures were exclusively observed in diabetic animals, irrespective of EMD treatment. CONCLUSION Within the limits of the present study, it can be concluded that periodontal healing was impaired in streptozotocin-induced diabetic rats. EMD had no beneficial effects on new bone and cementum formation during short-term healing in this defect model and could not ameliorate the adverse effects in the systemically compromised animals.

Five-year results evaluating the effects of platelet-rich plasma on the healing of intrabony defects treated with enamel matrix derivative and natural bone mineral

BACKGROUND Regenerative periodontal surgery using the combination of enamel matrix derivative (EMD) and natural bone mineral (NBM) with and without addition of platelet-rich plasma (PRP) has been shown to result in substantial clinical improvements, but the long-term effects of this combination are unknown. METHODS The goal of this study was to evaluate the long-term (5-year) outcomes after regenerative surgery of deep intrabony defects with either EMD + NBM + PRP or EMD + NBM. Twenty-four patients were included. In each patient, one intrabony defect was randomly treated with either EMD + NBM + PRP or EMD + NBM. Clinical parameters were evaluated at baseline and 1 and 5 years after treatment. The primary outcome variable was clinical attachment level (CAL). RESULTS The sites treated with EMD + NBM + PRP demonstrated a mean CAL change from 10.5 ± 1.6 to 6.0 ± 1.7 mm (P <0.001) at 1 year and 6.2 ± 1.5 mm (P <0.001) at 5 years. EMD + NBM-treated defects showed a mean CAL change from 10.6 ± 1.7 to 6.1 ± 1.5 mm (P <0.001) at 1 year and 6.3 ± 1.4 mm (P <0.001) at 5 years. At 1 year, a CAL gain of ≥4 mm was measured in 83% (10 of 12) of the defects treated with EMD + NBM + PRP and in 100% (all 12) of the defects treated with EMD + NBM. Compared to baseline, in both groups at 5 years, a CAL gain of ≥4 mm was measured in 75% (nine of 12 in each group) of the defects. Four sites in the EMD + PRP + NBM group lost 1 mm of the CAL gained at 1 year. In the EMD + NBM group, one defect lost 2 mm and four other defects lost 1 mm of the CAL gained at 1 year. No statistically significant differences in any of the investigated parameters were observed between the two groups. CONCLUSIONS Within their limits, the present results indicate that: 1) the clinical outcomes obtained with both treatments can be maintained up to a period of 5 years; and 2) the use of PRP does not appear to improve the results obtained with EMD + NBM.

Ten-year results following treatment of intrabony defects with an enamel matrix protein derivative combined with either a natural bone mineral or a β-tricalcium phosphate

BACKGROUND The purpose of the present study is to evaluate the 10-year results following treatment of intrabony defects treated with an enamel matrix protein derivative (EMD) combined with either a natural bone mineral (NBM) or β-tricalcium phosphate (β-TCP). METHODS Twenty-two patients with advanced chronic periodontitis and displaying one deep intrabony defect were randomly treated with a combination of either EMD + NBM or EMD + β-TCP. Clinical evaluations were performed at baseline and at 1 and 10 years. The following parameters were evaluated: plaque index, bleeding on probing, probing depth, gingival recession, and clinical attachment level (CAL). The primary outcome variable was CAL. RESULTS The defects treated with EMD + NBM demonstrated a mean CAL change from 8.9 ± 1.5 mm to 5.3 ± 0.9 mm (P <0.001) and to 5.8 ± 1.1 mm (P <0.001) at 1 and 10 years, respectively. The sites treated with EMD + β-TCP showed a mean CAL change from 9.1 ± 1.6 mm to 5.4 ± 1.1 mm (P <0.001) at 1 year and 6.1 ± 1.4 mm (P <0.001) at 10 years. At 10 years two defects in the EMD + NBM group had lost 2 mm, whereas two other defects had lost 1 mm of the CAL gained at 1 year. In the EMD + β-TCP group three defects had lost 2 mm, whereas two other defects had lost 1 mm of the CAL gained at 1 year. Compared with baseline, at 10 years, a CAL gain of ≥3 mm was measured in 64% (i.e., seven of 11) of the defects in the EMD + NBM group and in 82% (i.e., nine of 11) of the defects in the EMD + β-TCP group. No statistically significant differences were found between the 1- and 10-year values in either of the two groups. Between the treatment groups, no statistically significant differences in any of the investigated parameters were observed at 1 and 10 years. CONCLUSION Within their limitations, the present findings indicate that the clinical improvements obtained with regenerative surgery using EMD + NBM or EMD + β-TCP can be maintained over a period of 10 years.

Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2(-/-)/MMP9(-/-) mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2(-/-)/MMP9(-/-) allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

Cell and matrix morphology in articular cartilage from adult human knee and ankle joints suggests depth-associated adaptations to biomechanical and anatomical roles

OBJECTIVE Marked differences exist between human knee and ankle joints regarding risks and progression of osteoarthritis (OA). Pathomechanisms of degenerative joint disease may therefore differ in these joints, due to differences in tissue structure and function. Focussing on structural issues which are design goals for tissue engineering, we compared cell and matrix morphologies in different anatomical sites of adult human knee and ankle joints. METHODS Osteochondral explants were acquired from knee and ankle joints of deceased persons aged 20 to 40 years and analyzed for cell, matrix and tissue morphology using confocal and electron microscopy and unbiased stereological methods. Variations associated with joint (knee versus ankle) and biomechanical role (convex versus concave articular surfaces) were identified by 2-way analysis of variance and post-hoc analysis. RESULTS Knee cartilage exhibited higher cell densities in the superficial zone than ankle cartilage. In the transitional zone, higher cell densities were observed in association with convex versus concave articular surfaces, without significant differences between knee and ankle cartilage. Highly uniform cell and matrix morphologies were evident throughout the radial zone in the knee and ankle, regardless of tissue biomechanical role. Throughout the knee and ankle cartilage sampled, chondron density was remarkably constant at approximately 4.2×10(6) chondrons/cm(3). CONCLUSION Variation of cartilage cell and matrix morphologies with changing joint and biomechanical environments suggests that tissue structural adaptations are performed primarily by the superficial and transitional zones. Data may aid the development of site-specific cartilage tissue engineering, and help identify conditions where OA is likely to occur.

Characterization of dermatan sulfate proteoglycan 3 (DSPG3) and cartilage oligomeric matrix protein (COMP)

This dissertation describes the identification and characterization of human dermatan sulfate proteoglycan 3 (DSPG3) and the characterization of the transcriptional regulation of human cartilage oligomeric matrix protein (COMP) in cartilage, ligament, and tendon cells. DSPG3 and COMP are two extracellular matrix proteins. The function of these ECM proteins is unknown.^ DSPG3 was cloned, sequenced, and shown to be expressed in cartilage, ligament, and placenta. DSPG3 was mapped to human chromosome 12q21, and the genomic structure was identified. 1.6 kb of the promoter region has been sequenced, and several putative SOX9 sites were identified as well as 3 TATA sites. Furthermore, an evolutionary tree of the SLRP gene family, which includes DSPG3, is presented.^ The promoter region of COMP was cloned and sequenced. Several putative transcription factor binding sites were identified including multiple AP2 and SP1 sites. Three transcription start sites were found to be located directly downstream of one of the SP1 sites. In addition, the expression of COMP was demonstrated to be higher in tendon than in cartilage and ligament by both Northern and Western blot analysis, and several regions of the COMP promoter were shown to contain cell-specific regulatory elements. Analysis of the proximal 370bp region of the COMP promoter has also identified distinct patterns of nuclear protein binding for the three tissues, and two SP1 sites may play a role in the tissue-specific expression of COMP. ^

Enhancement of bone healing using non-glycosylated rhBMP-2 released from a fibrin matrix in dogs and cats

OBJECTIVES To test a non-glycosylated recombinant human bone morphogenetic protein-2 (ngly-rhBMP-2)/fibrin composite, which has been shown experimentally to enhance healing of bone defects in rodents, in a clinical case series of dogs and cats undergoing treatment for fracture non-unions and arthrodesis. METHODS A ngly-rhBMP-2/fibrin composite was applied in 41 sites in 38 dogs and cats for which a cancellous bone autograft was indicated, replacing the graft. RESULTS Bridging of the bone defect with functional bone healing was achieved in 90 per cent of the arthrodesis and fracture nonunions treated in this manner. CLINICAL SIGNIFICANCE This prospective clinical study demonstrates the beneficial effects of ngly-rhBMP-2 in a specially designed fibrin matrix on the treatment of bone defects, and validates the use of this composite as an alternative to bone autografts in dogs and cats.

Clinical outcomes following regenerative therapy of non-contained intrabony defects using a deproteinized bovine bone mineral combined with either enamel matrix derivative or collagen membrane

BACKGROUND The purpose of this study is to compare clinical outcomes in the treatment of deep non-contained intrabony defects (i.e., with ≥70% 1-wall component and a residual 2- to 3-wall component in the most apical part) using deproteinized bovine bone mineral (DBBM) combined with either enamel matrix protein derivative (EMD) or collagen membrane (CM). METHODS Forty patients with multiple intrabony defects were enrolled. Only one non-contained defect per patient with an intrabony depth ≥3 mm located in the interproximal area of single- and multirooted teeth was randomly assigned to the treatment with either EMD + DBBM (test: n = 20) or CM + DBBM (control: n = 20). At baseline and after 12 months, clinical parameters including probing depth (PD) and clinical attachment level (CAL) were recorded. The primary outcome variable was the change in CAL between baseline and 12 months. RESULTS At baseline, the intrabony component of the defects amounted to 6.1 ± 1.9 mm for EMD + DBBM and 6.0 ± 1.9 mm for CM + DBBM sites (P = 0.81). The mean CAL gain at sites treated with EMD + DBBM was not statistically significantly different (P = 0.82) compared with CM + DBBM (3.8 ± 1.5 versus 3.7 ± 1.2 mm). No statistically significant difference (P = 0.62) was observed comparing the frequency of CAL gain ≥4 mm between EMD + DBBM (60%) and CM + DBBM (50%) or comparing the frequency of residual PD ≥6 mm between EMD + DBBM (5%) and CM + DBBM (15%) (P = 0.21). CONCLUSION Within the limitations of the present study, regenerative therapy using either EMD + DBBM or CM + DBBM yielded comparable clinical outcomes in deep non-contained intrabony defects after 12 months.

Enamel matrix derivative and bone grafts for periodontal regeneration of intrabony defects. A systematic review and meta-analysis

OBJECTIVE The aim of the present systematic review and meta-analysis was to assess the clinical efficacy of regenerative periodontal surgery of intrabony defects using a combination of enamel matrix derivative (EMD) and bone graft compared with that of EMD alone. MATERIALS AND METHODS The Cochrane Oral Health Group specialist trials, MEDLINE, and EMBASE databases were searched for entries up to February 2014. The primary outcome was gain of clinical attachment (CAL). Weighted means and forest plots were calculated for CAL gain, probing depth (PD), and gingival recession (REC). RESULTS Twelve studies reporting on 434 patients and 548 intrabony defects were selected for the analysis. Mean CAL gain amounted to 3.76 ± 1.07 mm (median 3.63 95 % CI 3.51-3.75) following treatment with a combination of EMD and bone graft and to 3.32 ± 1.04 mm (median 3.40; 95 % CI 3.28-3.52) following treatment with EMD alone. Mean PD reduction measured 4.22 ± 1.20 mm (median 4.10; 95 % CI 3.96-4.24) at sites treated with EMD and bone graft and yielded 4.12 ± 1.07 mm (median 4.00; 95 % CI 3.88-4.12) at sites treated with EMD alone. Mean REC increase amounted to 0.76 ± 0.42 mm (median 0.63; 95 % CI 0.58-0.68) at sites treated with EMD and bone graft and to 0.91 ± 0.26 mm (median 0.90; 95 % CI 0.87-0.93) at sites treated with EMD alone. CONCLUSIONS Within their limits, the present results indicate that the combination of EMD and bone grafts may result in additional clinical improvements in terms of CAL gain and PD reduction compared with those obtained with EMD alone. The potential influence of the chosen graft material or of the surgical procedure (i.e., flap design) on the clinical outcomes is unclear. CLINICAL RELEVANCE The present findings support the use of EMD and bone grafts for the treatment of intrabony periodontal defects.

Influence of sample matrix on the alkaline extraction of Cr(VI) in soils and industrial materials

An accurate and efficient determination of the highly toxic Cr(VI) in solid materials is important to determine the total Cr(VI) inventory of contaminated sites and the Cr(VI) release potential from such sites into the environment. Most commonly, total Cr(VI) is extracted from solid materials following a hot alkaline extraction procedure (US EPA method 3060A) where a complete release of water-extractable and sparingly soluble Cr(VI) phase is achieved. This work presents an evaluation of matrix effects that may occur during the hot alkaline extraction and in the determination of the total Cr(VI) inventory of variably composed contaminated soils and industrial materials (cement, fly ash) and is compared to water-extractable Cr(VI) results. Method validation including multiple extractions and matrix spiking along with chemical and mineralogical characterization showed satisfying results for total Cr(VI) contents for most of the tested materials. However, unreliable results were obtained by applying method 3060A to anoxic soils due to the degradation of organic material and/or reactions with Fe2+-bearing mineral phases. In addition, in certain samples discrepant spike recoveries have to be also attributed to sample heterogeneity. Separation of possible extracted Cr(III) by applying cation-exchange cartridges prior to solution analysis further shows that under the hot alkaline extraction conditions only Cr(VI) is present in solution in measurable amounts, whereas Cr(III) gets precipitated as amorphous Cr(OH)3(am). It is concluded that prior to routine application of method 3060A to a new material type, spiking tests are recommended for the identification of matrix effects. In addition, the mass of extracted solid material should to be well adjusted to the heterogeneity of the Cr(VI) distribution in the material in question.

Interactions and structural analysis of the zinc -binding motif in decorin, a small leucine rich proteoglycan in extracellular matrix

Decorin, a dermatan/chondroitin sulfate proteoglycan, is ubiquitously distributed in the extracellular matrix (ECM) of mammals. Decorin belongs to the small leucine rich proteoglycan (SLRP) family, a proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. The decorin core protein appears to mediate the binding of decorin to ECM molecules, such as collagens and fibronectin. It is believed that the interactions of decorin with these ECM molecules contribute to the regulation of ECM assembly, cell adhesions, and cell proliferation. These basic biological processes play critical roles during embryonic development and wound healing and are altered in pathological conditions such as fibrosis and tumorgenesis. ^ In this dissertation, we discover that decorin core protein can bind to Zn2+ ions with high affinity. Zinc is an essential trace element in mammals. Zn2+ ions play a catalytic role in the activation of many enzymes and a structural role in the stabilization of protein conformation. By examining purified recombinant decorin and its core protein fragments for Zn2+ binding activity using Zn2+-chelating column chromatography and Zn2+-equilibrium dialysis approaches, we have located the Zn2+ binding domain to the N-terminal sequence of the decorin core protein. The decorin N-terminal domain appears to contain two Zn2+ binding sites with similar high binding affinity. The sequence of the decorin N-terminal domain does not resemble any other reported zinc-binding motifs and, therefore, represents a novel Zn 2+ binding motif. By investigating the influence of Zn2+ ions on decorin binding interactions, we found a novel Zn2+ dependent interaction with fibrinogen, the major plasma protein in blood clots. Furthermore, a recombinant peptide (MD4) consisting of a 41 amino acid sequence of mouse decorin N-terminal domain can prolong thrombin induced fibrinogen/fibrin clot formation. This suggests that in the presence of Zn2+ the decorin N-terminal domain has an anticoagulation activity. The changed Zn2+-binding activities of the truncated MD4 peptides and site-directed mutagenesis generated mutant peptides revealed that the functional MD4 peptide might contain both a structural zinc-binding site in the cysteine cluster region and a catalytic zinc site that could be created by the flanking sequences of the cysteine cluster region. A model of a loop-like structure for MD4 peptide is proposed. ^