Characterizing the mechanism of action for mRNA therapeutics for the treatment of propionic acidemia, methylmalonic acidemia, and phenylketonuria.

Messenger RNA (mRNA) therapeutics delivered via lipid nanoparticles hold the potential to treat metabolic diseases caused by protein deficiency, including propionic acidemia (PA), methylmalonic acidemia (MMA), and phenylketonuria (PKU). Herein we report results from multiple independent preclinical studies of mRNA-3927 (an investigational treatment for PA), mRNA-3705 (an investigational treatment for MMA), and mRNA-3210 (an investigational treatment for PKU) in murine models of each disease. All 3 mRNA therapeutics exhibited pharmacokinetic/pharmacodynamic (PK/PD) responses in their respective murine model by driving mRNA, protein, and/or protein activity responses, as well as by decreasing levels of the relevant biomarker(s) when compared to control-treated animals. These preclinical data were then used to develop translational PK/PD models, which were scaled allometrically to humans to predict starting doses for first-in-human clinical studies for each disease. The predicted first-in-human doses for mRNA-3927, mRNA-3705, and mRNA-3210 were determined to be 0.3, 0.1, and 0.4 mg/kg, respectively.

mRNA therapy corrects defective glutathione metabolism and restores ureagenesis in preclinical argininosuccinic aciduria.

The urea cycle enzyme argininosuccinate lyase (ASL) enables the clearance of neurotoxic ammonia and the biosynthesis of arginine. Patients with ASL deficiency present with argininosuccinic aciduria, an inherited metabolic disease with hyperammonemia and a systemic phenotype coinciding with neurocognitive impairment and chronic liver disease. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-l-glutamate ([18F]FSPG). Up-regulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. To assess hepatic glutathione dysregulation and liver disease, we present [18F]FSPG PET as a noninvasive diagnostic tool to monitor therapeutic response in argininosuccinic aciduria. Human hASL mRNA encapsulated in lipid nanoparticles improved glutathione metabolism and chronic liver disease. In addition, hASL mRNA therapy corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis. These findings provide mechanistic insights in liver glutathione metabolism and support clinical translation of mRNA therapy for argininosuccinic aciduria.

The incidence of movement disorder increases with age and contrasts with subtle and limited neuroimaging abnormalities in argininosuccinic aciduria.

Argininosuccinate lyase (ASL) is integral to the urea cycle detoxifying neurotoxic ammonia and the nitric oxide (NO) biosynthesis cycle. Inherited ASL deficiency causes argininosuccinic aciduria (ASA), a rare disease with hyperammonemia and NO deficiency. Patients present with developmental delay, epilepsy and movement disorder, associated with NO-mediated downregulation of central catecholamine biosynthesis. A neurodegenerative phenotype has been proposed in ASA. To better characterise this neurodegenerative phenotype in ASA, we conducted a retrospective study in six paediatric and adult metabolic centres in the UK in 2022. We identified 60 patients and specifically looked for neurodegeneration-related symptoms: movement disorder such as ataxia, tremor and dystonia, hypotonia/fatigue and abnormal behaviour. We analysed neuroimaging with diffusion tensor imaging (DTI) magnetic resonance imaging (MRI) in an individual with ASA with movement disorders. We assessed conventional and DTI MRI alongside single photon emission computer tomography (SPECT) with dopamine analogue radionuclide 123 I-ioflupane, in Asl-deficient mice treated by hASL mRNA with normalised ureagenesis. Movement disorders in ASA appear in the second and third decades of life, becoming more prevalent with ageing and independent from the age of onset of hyperammonemia. Neuroimaging can show abnormal DTI features affecting both grey and white matter, preferentially basal ganglia. ASA mouse model with normalised ureagenesis did not recapitulate these DTI findings and showed normal 123 I-ioflupane SPECT and cerebral dopamine metabolomics. Altogether these findings support the pathophysiology of a late-onset movement disorder with cell-autonomous functional central catecholamine dysregulation but without or limited neurodegeneration of dopaminergic neurons, making these symptoms amenable to targeted therapy.

Lipid nanoparticle-mediated delivery of mRNA into the mouse and human retina and other ocular tissues.

Purpose: Lipid nanoparticles (LNPs) show promise in their ability to introduce mRNA to drive protein expression in specific cell types of the mammalian eye. Here, we examined the ability of mRNA encapsulated in lipid nanoparticles (LNPs) with two distinct formulations to drive gene expression in mouse and human retina and other ocular tissues. Methods: We introduced mRNA carrying LNPs into two biological systems. Intravitreal injections were tested to deliver LNPs into the mouse eye. Human retinal pigment epithelium (RPE) and retinal explants were used to assess mRNA expression in human tissue. We analyzed specificity of expression using histology, immunofluorescence, and imaging. Results: In mice, mRNAs encoding GFP and ciliary neurotrophic factor (CNTF) were specifically expressed by Müller glia and retinal pigment epithelium (RPE). Acute inflammatory changes measured by microglia distribution (Iba-1) or interleukin-6 (IL-6) expression were not observed 6 hours post-injection. Human RPE also expressed high levels of GFP. Human retinal explants expressed GFP in cells with apical and basal processes consistent with Müller glia and in perivascular cells consistent with macrophages. Conclusions: We demonstrated the ability to reliably transfect subpopulations of retinal cells in mice eye tissues in vivo and in human ocular tissues. Of significance, intravitreal injections were sufficient to transfect the RPE in mice. To our knowledge we demonstrate delivery of mRNA using LNPs in human ocular tissues for the first time.

Ex vivo precision-cut liver slices model disease phenotype and monitor therapeutic response for liver monogenic diseases.

Background: In academic research and the pharmaceutical industry, in vitro cell lines and in vivo animal models are considered as gold standards in modelling diseases and assessing therapeutic efficacy. However, both models have intrinsic limitations, whilst the use of precision-cut tissue slices can bridge the gap between these mainstream models. Precision-cut tissue slices combine the advantage of high reproducibility, studying all cell sub-types whilst preserving the tissue matrix and extracellular architecture, thereby closely mimicking a mini-organ. This approach can be used to replicate the biological phenotype of liver monogenic diseases using mouse models. Methods: Here, we describe an optimised and easy-to-implement protocol for the culture of sections from mouse livers, enabling its use as a reliable ex-vivo model to assess the therapeutic screening of inherited metabolic diseases. Results: We show that precision-cut liver sections can be a reliable model for recapitulating the biological phenotype of inherited metabolic diseases, exemplified by common urea cycle defects such as citrullinemia type 1 and argininosuccinic aciduria, caused by argininosuccinic synthase (ASS1) and argininosuccinic lyase (ASL) deficiencies respectively. Conclusions: Therapeutic response to gene therapy such as messenger RNA replacement delivered via lipid nanoparticles can be monitored, demonstrating that precision-cut liver sections can be used as a preclinical screening tool to assess therapeutic response and toxicity in monogenic liver diseases.

Ultrasonic Instrument Effects on Different Implant Surfaces: Profilometry, Energy-Dispersive X-ray Spectroscopy, and Microbiology In Vitro Study.

PURPOSE: To assess the effects of grade IV titanium ultrasonic tip instrumentation on different grade IV titanium implant surfaces and compare the decontamination of different implant surfaces using chlorhexidine, blue laser, or ozone. MATERIALS AND METHODS: Profilometry and energy-dispersive x-ray spectroscopy (EDS) analyses were performed on smooth, laser-micropatterned, and sandblasted grade IV titanium sample disks before (t0) and after (t1) ultrasonic instrumentation with an ultrasonic grade IV titanium tip. Samples were also incubated with a Streptococcus sanguinis culture. Each surface type was then treated with chlorhexidine, blue laser, or ozone (three test groups + control group). Scanning electron microscopy (SEM) images were taken after bacterial growth and after decontamination. RESULTS: After ultrasonic instrumentation, surface roughness (Ra) decreased on sandblasted and micropatterned surfaces, whereas it remained substantially unvaried on the smooth surface. SEM images revealed that the laser-micropatterned structure remained substantially unvaried after instrumentation. EDS revealed a minimal quantity of carbon and iron, found in the laser-treated and sandblasted group at t0. A minimal quantity of aluminum and oxygen was found on the sandblasted surface at t0 and t1. Ozone therapy achieved the highest decontaminating effect, regardless of implant surface topography. CONCLUSION: Among the alternative therapies to ultrasonic instrumentation with titanium tips, ozone appears to be effective regardless of the type of implant surface; it can be used for the decontamination treatment of implants without altering the surface structure.

mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease.

Glycogen Storage Disease 1a (GSD1a) is a rare, inherited metabolic disorder caused by deficiency of glucose 6-phosphatase (G6Pase-α). G6Pase-α is critical for maintaining interprandial euglycemia. GSD1a patients exhibit life-threatening hypoglycemia and long-term liver complications including hepatocellular adenomas (HCAs) and carcinomas (HCCs). There is no treatment for GSD1a and the current standard-of-care for managing hypoglycemia (Glycosade®/modified cornstarch) fails to prevent HCA/HCC risk. Therapeutic modalities such as enzyme replacement therapy and gene therapy are not ideal options for patients due to challenges in drug-delivery, efficacy, and safety. To develop a new treatment for GSD1a capable of addressing both the life-threatening hypoglycemia and HCA/HCC risk, we encapsulated engineered mRNAs encoding human G6Pase-α in lipid nanoparticles. We demonstrate the efficacy and safety of our approach in a preclinical murine model that phenotypically resembles the human condition, thus presenting a potential therapy that could have a significant therapeutic impact on the treatment of GSD1a.

Synthetic human ABCB4 mRNA therapy rescues severe liver disease phenotype in a BALB/c.Abcb4-/- mouse model of PFIC3.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare lethal autosomal recessive liver disorder caused by loss-of-function variations of the ABCB4 gene, encoding a phosphatidylcholine transporter (ABCB4/MDR3). Currently, no effective treatment exists for PFIC3 outside of liver transplantation. METHODS: We have produced and screened chemically and genetically modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles (LNPs). We examined their pharmacological effects in a cell-based model and in a new in vivo mouse model resembling human PFIC3 as a result of homozygous disruption of the Abcb4 gene in fibrosis-susceptible BALB/c.Abcb4-/- mice. RESULTS: We show that treatment with liver-targeted hABCB4 mRNA resulted in de novo expression of functional hABCB4 protein and restored phospholipid transport in cultured cells and in PFIC3 mouse livers. Importantly, repeated injections of the hABCB4 mRNA effectively rescued the severe disease phenotype in young Abcb4-/- mice, with rapid and dramatic normalisation of all clinically relevant parameters such as inflammation, ductular reaction, and liver fibrosis. Synthetic mRNA therapy also promoted favourable hepatocyte-driven liver regeneration to restore normal homeostasis, including liver weight, body weight, liver enzymes, and portal vein blood pressure. CONCLUSIONS: Our data provide strong preclinical proof-of-concept for hABCB4 mRNA therapy as a potential treatment option for patients with PFIC3. LAY SUMMARY: This report describes the development of an innovative mRNA therapy as a potential treatment for PFIC3, a devastating rare paediatric liver disease with no treatment options except liver transplantation. We show that administration of our mRNA construct completely rescues severe liver disease in a genetic model of PFIC3 in mice.

Dual mRNA therapy restores metabolic function in long-term studies in mice with propionic acidemia.

Propionic acidemia/aciduria (PA) is an ultra-rare, life-threatening, inherited metabolic disorder caused by deficiency of the mitochondrial enzyme, propionyl-CoA carboxylase (PCC) composed of six alpha (PCCA) and six beta (PCCB) subunits. We herein report an enzyme replacement approach to treat PA using a combination of two messenger RNAs (mRNAs) (dual mRNAs) encoding both human PCCA (hPCCA) and PCCB (hPCCB) encapsulated in biodegradable lipid nanoparticles (LNPs) to produce functional PCC enzyme in liver. In patient fibroblasts, dual mRNAs encoded proteins localize in mitochondria and produce higher PCC enzyme activity vs. single (PCCA or PCCB) mRNA alone. In a hypomorphic murine model of PA, dual mRNAs normalize ammonia similarly to carglumic acid, a drug approved in Europe for the treatment of hyperammonemia due to PA. Dual mRNAs additionally restore functional PCC enzyme in liver and thus reduce primary disease-associated toxins in a dose-dependent manner in long-term 3- and 6-month repeat-dose studies in PA mice. Dual mRNAs are well-tolerated in these studies with no adverse findings. These studies demonstrate the potential of mRNA technology to chronically administer multiple mRNAs to produce large complex enzymes, with applicability to other genetic disorders.

Systemic modified messenger RNA for replacement therapy in alpha 1-antitrypsin deficiency.

Alpha 1-antitrypsin (AAT) deficiency arises from an inherited mutation in the SERPINA1 gene. The disease causes damage in the liver where the majority of the AAT protein is produced. Lack of functioning circulating AAT protein also causes uninhibited elastolytic activity in the lungs leading to AAT deficiency-related emphysema. The only therapy apart from liver transplantation is augmentation with human AAT protein pooled from sera, which is only reserved for patients with advanced lung disease caused by severe AAT deficiency. We tested modified mRNA encoding human AAT in primary human hepatocytes in culture, including hepatocytes from AAT deficient patients. Both expression and functional activity were investigated. Secreted AAT protein increased from 1,14 to 3,43 µg/ml in media from primary human hepatocytes following mRNA treatment as investigated by ELISA and western blot. The translated protein showed activity and protease inhibitory function as measured by elastase activity assay. Also, mRNA formulation in lipid nanoparticles was assessed for systemic delivery in both wild type mice and the NSG-PiZ transgenic mouse model of AAT deficiency. Systemic intravenous delivery of modified mRNA led to hepatic uptake and translation into a functioning protein in mice. These data support the use of systemic mRNA therapy as a potential treatment for AAT deficiency.

Long-term efficacy and safety of mRNA therapy in two murine models of methylmalonic acidemia.

BACKGROUND: Isolated methylmalonic acidemia/aciduria (MMA) is an ultra-rare, serious, inherited metabolic disorder with significant morbidity and mortality. Exogenously delivered mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, is a potential therapy to produce functional MUT enzyme in liver. METHODS: Two 12-week repeat-dose studies were conducted to evaluate the efficacy and safety of intravenously-administered hMUT mRNA encapsulated in lipid nanoparticles in two murine models of MMA. FINDINGS: In MMA hypomorphic mice, hMUT mRNA treatment resulted in dose-dependent and reproducible biomarker responses after each dose. Enzymatically-active MUT protein was produced in liver in a dose-dependent manner. hMUT mRNA was well-tolerated with no adverse effects, as indicated by the lack of clinical observations, minimal changes in clinical chemistry parameters, and histopathology examination across all tissues. In severe MMA mice, hMUT mRNA led to substantially improved survival and growth and ameliorated biochemical abnormalities, all of which are cardinal clinical manifestations in severely affected patients. INTERPRETATION: These data demonstrate durable functional benefit of hMUT mRNA and support development of this new class of therapy for a devastating, pediatric disorder. FUND: This work was funded by Moderna, Inc.

mRNA Therapy Improves Metabolic and Behavioral Abnormalities in a Murine Model of Citrin Deficiency.

Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.

Systemic mRNA Therapy for the Treatment of Fabry Disease: Preclinical Studies in Wild-Type Mice, Fabry Mouse Model, and Wild-Type Non-human Primates.

Fabry disease is an X-linked lysosomal storage disease caused by loss of alpha galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of globotriaosylceramide and its analogs in all cells and tissues. Although enzyme replacement therapy (ERT) is considered standard of care, the long-term effects of ERT on renal and cardiac manifestations remain uncertain and thus novel therapies are desirable. We herein report preclinical studies evaluating systemic messenger RNA (mRNA) encoding human α-Gal A in wild-type (WT) mice, α-Gal A-deficient mice, and WT non-human primates (NHPs). The pharmacokinetics and distribution of h-α-Gal A mRNA encoded protein in WT mice demonstrated prolonged half-lives of α-Gal A in tissues and plasma. Single intravenous administration of h-α-Gal A mRNA to Gla-deficient mice showed dose-dependent protein activity and substrate reduction. Moreover, long duration (up to 6 weeks) of substrate reductions in tissues and plasma were observed after a single injection. Furthermore, repeat i.v. administration of h-α-Gal A mRNA showed a sustained pharmacodynamic response and efficacy in Fabry mice model. Lastly, multiple administrations to non-human primates confirmed safety and translatability. Taken together, these studies across species demonstrate preclinical proof-of-concept of systemic mRNA therapy for the treatment of Fabry disease and this approach may be useful for other lysosomal storage disorders.

Systemic messenger RNA as an etiological treatment for acute intermittent porphyria.

Acute intermittent porphyria (AIP) results from haploinsufficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis pathway. Patients with AIP have neurovisceral attacks associated with increased hepatic heme demand. Phenobarbital-challenged mice with AIP recapitulate the biochemical and clinical characteristics of patients with AIP, including hepatic overproduction of the potentially neurotoxic porphyrin precursors. Here we show that intravenous administration of human PBGD (hPBGD) mRNA (encoded by the gene HMBS) encapsulated in lipid nanoparticles induces dose-dependent protein expression in mouse hepatocytes, rapidly normalizing urine porphyrin precursor excretion in ongoing attacks. Furthermore, hPBGD mRNA protected against mitochondrial dysfunction, hypertension, pain and motor impairment. Repeat dosing in AIP mice showed sustained efficacy and therapeutic improvement without evidence of hepatotoxicity. Finally, multiple administrations to nonhuman primates confirmed safety and translatability. These data provide proof-of-concept for systemic hPBGD mRNA as a potential therapy for AIP.

Release of ICTP and CTX telopeptides from demineralized dentin matrices: Effect of time, mass and surface area.

OBJECTIVE: The present study evaluated the influence of time, mass and surface area of demineralized dentin collagen matrices on telopeptides release. The hypotheses tested were that the rates of ICTP and CTX release by matrix bound endogenous proteases are 1) not time-dependent, 2) unrelated to specimen mass, 3) unrelated to specimen surface area. METHODS: Non-carious human molars (N=24) were collected and randomly assigned to three groups. Dentin slabs with three different thicknesses: 0.37mm, 0.75mm, and 1.50mm were completely demineralized and stored in artificial saliva for one week. Collagen degradation was evaluated by sampling storage media for ICTP and CTX telopeptidases. Activity of MMPs in the aging medium was evaluated using fluorometric activity assay kit. RESULTS: A statistically significant (p<0.05) decrease in the release of both ICTP and CTX fragments over time was observed irrespective of the specimen thickness. When data were normalized by the specimen mass, no significant differences were observed. Releases of ICTP and CTX were significantly related to the aging time as a function of surface area for the first 12h. Total MMP activity, mainly related to MMP-2 and -9, decreased with time (p<0.05). SIGNIFICANCE: Because the release of collagen fragments was influenced by specimen storage time and surface area, it is likely that cleaved collagen fragments closer to the specimen surface diffuse into the incubation medium; those further away from the exposed surface are still entrapped within the demineralized dentin matrix. Bound MMPs can only degrade the substrate within the limited zone of their molecular mobility.

Systemic Messenger RNA Therapy as a Treatment for Methylmalonic Acidemia.

Isolated methylmalonic acidemia/aciduria (MMA) is a devastating metabolic disorder with poor outcomes despite current medical treatments. Like other mitochondrial enzymopathies, enzyme replacement therapy (ERT) is not available, and although promising, AAV gene therapy can be limited by pre-existing immunity and has been associated with genotoxicity in mice. To develop a new class of therapy for MMA, we generated a pseudoU-modified codon-optimized mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, and encapsulated it into biodegradable lipid nanoparticles (LNPs). Intravenous (i.v.) administration of hMUT mRNA in two different mouse models of MMA resulted in a 75%-85% reduction in plasma methylmalonic acid and was associated with increased hMUT protein expression and activity in liver. Repeat dosing of hMUT mRNA reduced circulating metabolites and dramatically improved survival and weight gain. Additionally, repeat i.v. dosing did not increase markers of liver toxicity or inflammation in heterozygote MMA mice.

The 1st Baltic Osseointegration Academy and Lithuanian University of Health Sciences Consensus Conference 2016. Summary and Consensus Statements: Group I - Peri-Implantitis Aetiology, Risk Factors and Pathogenesis.

INTRODUCTION: The task of Group 1 was to review and update the existing data concerning aetiology, risk factors and pathogenesis of peri-implantitis. Previous history of periodontitis, poor oral hygiene, smoking and presence of general diseases have been considered among the aetiological risk factors for the onset of peri-implant pathologies, while late dental implant failures are commonly associated with peri-implantitis and/or with the application of incorrect biomechanical forces. Special interest was paid to the bone cells dynamics as part of the pathogenesis of peri-implantitis. MATERIAL AND METHODS: The main areas indagated by this group were as follows: influence of smoking, history of periodontitis and general diseases on peri-implantitis development, bio-mechanics of implant loading and its influence on peri-implant bone and cellular dynamics related to the pathogenesis of peri-implantitis. The systematic reviews and/or meta-analyses were registered in PROSPERO, an international prospective register of systematic reviews: http://www.crd.york.ac.uk/PROSPERO/. The literature in the corresponding areas of interest was screened and reported following the PRISMA (Preferred Reporting Item for Systematic Review and Meta-Analysis) Statement: http://www.prisma-statement.org/. Method of preparation of the systematic reviews, based on comprehensive search strategies, was discussed and standardized. The summary of the materials and methods employed by the authors in preparing the systematic reviews and/or meta-analyses is presented in Preface chapter. RESULTS: The results and conclusions of the review process are presented in the respective papers. One systematic review with meta-analysis, three systematic reviews and one theoretical analysis were performed. The group's general commentaries, consensus statements, clinical recommendations and implications for research are presented in this article.

Risk Factors for Peri-Implantitis: Effect of History of Periodontal Disease and Smoking Habits. A Systematic Review and Meta-Analysis.

OBJECTIVES: The purpose of this review was to evaluate whether history of periodontitis and smoking habits could represent a risk factor for peri-implantitis and implant loss. MATERIAL AND METHODS: This systematic review followed PRISMA guidelines and was registered at the PROSPERO database [registration numbers CRD42016034160 (effect of history of periodontitis) and CRD42016033676 (effect of smoking)]. Broad electronic (MEDLINE) and manual searches were conducted among articles published from January 1st 1990 up to December 31st 2015, resulting in 49332 records for history of periodontitis and 3199 for smoking habits. Selection criteria included prospective studies comparing two cohorts of patients, with and without the investigated risk factor, with a minimum follow-up period of three years, and reporting data on peri-implantitis and implant loss occurrence. Considering that only prospective studies were included, dichotomous data were expressed as risk ratios and 95% confidence intervals. RESULTS: Three studies evaluating history of periodontitis (on which quantitative analysis was performed) and one study on smoking effect were included. Both implant and patient-based meta-analyses revealed a significantly higher risk of developing peri-implantitis in patients with a history of periodontitis compared with periodontally healthy subjects, but not a statistically significant increased risk for implant loss. CONCLUSIONS: The outcomes of this systematic review indicate history of periodontitis as a possible risk factor for peri-implantitis, while insufficient data are present in literature to evaluate the role of smoking. However, available evidence is still weak and immature, and sound epidemiological studies are needed to analyse the specific contribution of these potential risk factors.

Evaluation of the In Vitro Effects of Cervical Marginal Relocation Using Composite Resins on the Marginal Quality of CAD/CAM Crowns.

PURPOSE: To evaluate the effect of cervical margin relocation (CMR) for crowns designed using CAD/CAM technology, and made of pre-cured resin or lithium disilicate, before and after thermomechanical loading. The test hypothesis was that the marginal quality of the crowns would not be influenced by the CMR with composite resins before or after thermomechanical loading. MATERIALS AND METHODS: Standard crown preparations were created in 40 human molars. The margins were located in enamel, except for the mesial proximal box, where the cervical margin was 2.0 mm below the cementoenamel junction, with 4.0 mm in the buccolingual and 2.0 mm in the mesiodistal dimension. For the CMR technique, a 2-mm layer of conventional or flowable composite resin was applied to the mesial box. Using the Cerec CAD/CAM system, 40 standard crowns were prepared, and restorations were luted using a dual-curing adhesive cement. SEM analysis was performed using epoxy resin replicas before and after loading to assess the marginal quality of interfaces of the mesial proximal box with CMR/crown and the distal face of the tooth without CMR. Statistical differences between groups were analyzed using the Kruskal-Wallis test and Bonferroni's post-hoc test. RESULTS: The null hypothesis was accepted, since no statistically significant differences were found in marginal quality before and after thermomechanical cycling (p > 0.05). CONCLUSION: The implementation of CMR before and after thermomechanical cycling had no effect on the quality of cervical margins. To establish whether CMR is a suitable procedure for the adhesive luting of composite resin crowns in deep proximal boxes, additional studies are required.