Creating an Extraordinary Outcome With Ordinary Digital Planning.

A patient was dissatisfied with her previous extensive dental treatment and wanted a comfortable bite with less gingival display for a more attractive smile. This article describes a systematic approach that was used in diagnosis, planning, and treatment sequencing to effectively manage the esthetic, functional, and biomechanical concerns involved in the case while minimizing risks. Digital planning enhanced interdisciplinary communication making the outcome more efficient and predictable.

A Pro-Regenerative Supramolecular Prodrug Protects Against and Repairs Colon Damage in Experimental Colitis.

Structural repair of the intestinal epithelium is strongly correlated with disease remission in inflammatory bowel disease (IBD); however, ulcer healing is not addressed by existing therapies. To address this need, this study reports the use of a small molecule prolyl hydroxylase (PHD) inhibitor (DPCA) to upregulate hypoxia-inducible factor one-alpha (HIF-1α) and induce mammalian regeneration. Sustained delivery of DPCA is achieved through subcutaneous injections of a supramolecular hydrogel, formed through the self-assembly of PEG-DPCA conjugates. Pre-treatment of mice with PEG-DPCA is shown to protect mice from epithelial erosion and symptoms of dextran sodium sulfate (DSS)-induced colitis. Surprisingly, a single subcutaneous dose of PEG-DPCA, administered after disease onset, leads to accelerated weight gain and complete restoration of healthy tissue architecture in colitic mice. Rapid DPCA-induced restoration of the intestinal barrier is likely orchestrated by increased expression of HIF-1α and associated targets leading to an epithelial-to-mesenchymal transition. Further investigation of DPCA as a potential adjunctive or stand-alone restorative treatment to combat active IBD is warranted.

Epithelial-mesenchymal transition: an organizing principle of mammalian regeneration.

Introduction: The MRL mouse strain is one of the few examples of a mammal capable of healing appendage wounds by regeneration, a process that begins with the formation of a blastema, a structure containing de-differentiating mesenchymal cells. HIF-1α expression in the nascent MRL wound site blastema is one of the earliest identified events and is sufficient to initiate the complete regenerative program. However, HIF-1α regulates many cellular processes modulating the expression of hundreds of genes. A later signal event is the absence of a functional G1 checkpoint, leading to G2 cell cycle arrest with increased cellular DNA but little cell division observed in the blastema. This lack of mitosis in MRL blastema cells is also a hallmark of regeneration in classical invertebrate and vertebrate regenerators such as planaria, hydra, and newt. Results and discussion: Here, we explore the cellular events occurring between HIF-1α upregulation and its regulation of the genes involved in G2 arrest (EVI-5, γH3, Wnt5a, and ROR2), and identify epithelial-mesenchymal transition (EMT) (Twist and Slug) and chromatin remodeling (EZH-2 and H3K27me3) as key intermediary processes. The locus of these cellular events is highly regionalized within the blastema, occurring in the same cells as determined by double staining by immunohistochemistry and FACS analysis, and appears as EMT and chromatin remodeling, followed by G2 arrest determined by kinetic expression studies.

Ridge Augmentation in a Site of a Previous Implant Failure Using Tenting Screws With Allograft and Collagen Membrane.

As digital workflows become increasingly common in dentistry, clinicians are able to quickly demonstrate esthetic possibilities to their patients and easily implement the digitally designed outcome. In this case presentation of a young patient, it was critical to maintain maximum tooth structure and alveolar bone when treating her esthetic concerns. Chairside confirmation of the proposed treatment plan, coupled with careful multidisciplinary consultations, led to improved oral health and an excellent esthetic outcome without increasing the patient's risk for future disease.

Prolyl-hydroxylase inhibitor-induced regeneration of alveolar bone and soft tissue in a mouse model of periodontitis through metabolic reprogramming.

Bone injuries and fractures reliably heal through a process of regeneration with restoration to original structure and function when the gap between adjacent sides of a fracture site is small. However, when there is significant volumetric loss of bone, bone regeneration usually does not occur. In the present studies, we explore a particular case of volumetric bone loss in a mouse model of human periodontal disease (PD) in which alveolar bone surrounding teeth is permanently lost and not replaced. This model employs the placement a ligature around the upper second molar for 10 days leading to inflammation and bone breakdown and faithfully replicates the bacterially-induced inflammatory etiology of human PD to induce bone degeneration. After ligature removal, mice are treated with a timed-release formulation of a small molecule inhibitor of prolylhydroxylases (PHDi; 1,4-DPCA) previously shown to induce epimorphic regeneration of soft tissue in non-regenerating mice. This PHDi induces high expression of HIF-1α and is able to shift the metabolic state from OXPHOS to aerobic glycolysis, an energetic state used by stem cells and embryonic tissue. This regenerative response was completely blocked by siHIF1a. In these studies, we show that timed-release 1,4-DPCA rapidly and completely restores PD-affected bone and soft tissue with normal anatomic fidelity and with increased stem cell markers due to site-specific stem cell migration and/or de-differentiation of local tissue, periodontal ligament (PDL) cell proliferation, and increased vascularization. In-vitro studies using gingival tissue show that 1,4-DPCA indeed induces de-differentiation and the expression of stem cell markers but does not exclude the role of migrating stem cells. Evidence of metabolic reprogramming is seen by the expression of not only HIF-1a, its gene targets, and resultant de-differentiation markers, but also the metabolic genes Glut-1, Gapdh, Pdk1, Pgk1 and Ldh-a in jaw periodontal tissue.

Unlocking mammalian regeneration through hypoxia inducible factor one alpha signaling.

Historically, the field of regenerative medicine has aimed to heal damaged tissue through the use of biomaterials scaffolds or delivery of foreign progenitor cells. Despite 30 years of research, however, translation and commercialization of these techniques has been limited. To enable mammalian regeneration, a more practical approach may instead be to develop therapies that evoke endogenous processes reminiscent of those seen in innate regenerators. Recently, investigations into tadpole tail regrowth, zebrafish limb restoration, and the super-healing Murphy Roths Large (MRL) mouse strain, have identified ancient oxygen-sensing pathways as a possible target to achieve this goal. Specifically, upregulation of the transcription factor, hypoxia-inducible factor one alpha (HIF-1α) has been shown to modulate cell metabolism and plasticity, as well as inflammation and tissue remodeling, possibly priming injuries for regeneration. Since HIF-1α signaling is conserved across species, environmental or pharmacological manipulation of oxygen-dependent pathways may elicit a regenerative response in non-healing mammals. In this review, we will explore the emerging role of HIF-1α in mammalian healing and regeneration, as well as attempts to modulate protein stability through hyperbaric oxygen treatment, intermittent hypoxia therapy, and pharmacological targeting. We believe that these therapies could breathe new life into the field of regenerative medicine.

An injectable hydrogel-formulated inhibitor of prolyl-4-hydroxylase promotes T regulatory cell recruitment and enhances alveolar bone regeneration during resolution of experimental periodontitis.

The hypoxia-inducible factor 1α (HIF-1α) is critically involved in tissue regeneration. Hence, the pharmacological prevention of HIF-1α degradation by prolyl hydroxylase (PHD) under normoxic conditions is emerging as a promising option in regenerative medicine. Using a mouse model of ligature-induced periodontitis and resolution, we tested the ability of an injectable hydrogel-formulated PHD inhibitor, 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA/hydrogel), to promote regeneration of alveolar bone lost owing to experimental periodontitis. Mice injected subcutaneously with 1,4-DPCA/hydrogel at the onset of periodontitis resolution displayed significantly increased gingival HIF-1α protein levels and bone regeneration, as compared to mice treated with vehicle control. The 1,4-DPCA/hydrogel-induced increase in bone regeneration was associated with elevated expression of osteogenic genes, decreased expression of pro-inflammatory cytokine genes, and increased abundance of FOXP3+ T regulatory (Treg) cells in the periodontal tissue. The enhancing effect of 1,4-DPCA/hydrogel on Treg cell accumulation and bone regeneration was reversed by AMD3100, an antagonist of the chemokine receptor CXCR4 that mediates Treg cell recruitment. In conclusion, the administration of 1,4-DPCA/hydrogel at the onset of periodontitis resolution promotes CXCR4-dependent accumulation of Treg cells and alveolar bone regeneration, suggesting a novel approach for regaining bone lost due to periodontitis.

Antioxidant Effects of N-Acetylcysteine Prevent Programmed Metabolic Disease in Mice.

An adverse maternal in utero and lactation environment can program offspring for increased risk for metabolic disease. The aim of this study was to determine whether N-acetylcysteine (NAC), an anti-inflammatory antioxidant, attenuates programmed susceptibility to obesity and insulin resistance in offspring of mothers on a high-fat diet (HFD) during pregnancy. CD1 female mice were acutely fed a standard breeding chow or HFD. NAC was added to the drinking water (1 g/kg) of the treatment cohorts from embryonic day 0.5 until the end of lactation. NAC treatment normalized HFD-induced maternal weight gain and oxidative stress, improved the maternal lipidome, and prevented maternal leptin resistance. These favorable changes in the in utero environment normalized postnatal growth, decreased white adipose tissue (WAT) and hepatic fat, improved glucose and insulin tolerance and antioxidant capacity, reduced leptin and insulin, and increased adiponectin in HFD offspring. The lifelong metabolic improvements in the offspring were accompanied by reductions in proinflammatory gene expression in liver and WAT and increased thermogenic gene expression in brown adipose tissue. These results, for the first time, provide a mechanistic rationale for how NAC can prevent the onset of metabolic disease in the offspring of mothers who consume a typical Western HFD.

Supramolecular Polymer Hydrogels for Drug-Induced Tissue Regeneration.

Supramolecular polymers self-assemble into nanofibers, micelles, and other nanostructures through weak noncovalent interactions between subunits. Such systems possess attractive properties for use in a variety of practical settings such as energy, sustainability, and healthcare. In regenerative medicine, a common approach involves implanting a supramolecular material containing cell and growth factor binding motifs directly into a diseased or traumatized tissue defect, whereupon it interacts with and/or recruits components of the biological system to induce tissue healing. Here we introduce a supramolecular therapeutic in which tissue regeneration is orchestrated by a supramolecular polymer prodrug implanted subcutaneously in a remote tissue. Our approach exploits a hydrophobic small-molecule inhibitor of prolyl hydroxylase enzyme as both a regeneration-inducing therapeutic and a structure-directing agent in a supramolecular polymer that forms shear-thinning nanofiber hydrogels. Subcutaneous injection of the supramolecular hydrogel in the back of mice wounded with a critical-sized defect in the ear led to transient upregulation of hypoxia inducible factor-1α and regeneration of ear tissue in a manner reminiscent of epimorphic regeneration. This drug-induced regeneration strategy utilizes a simple and translatable supramolecular design, eliminates the need for delivery of biologics ( e. g., growth factors, cells), and avoids implantation of a foreign material directly in a tissue defect.

Drug delivery and epimorphic salamander-type mouse regeneration: A full parts and labor plan.

The capacity to regenerate entire body parts, tissues, and organs had generally been thought to be lost in evolution with very few exceptions (e.g. the liver) surviving in mammals. The discovery of the MRL mouse and the elucidation of the underlying molecular pathway centering around hypoxia inducible factor, HIF-1α, has allowed a drug and materials approach to regeneration in mice and hopefully humans. The HIF-1α pathway is ancient and permitted the transition from unicellular to multicellular organisms. Furthermore, HIF-1α and its regulation by PHDs, important oxygen sensors in the cell, provides a perfect drug target. We review the historical background of regeneration biology, the discovery of the MRL mouse, and its underlying biology, and novel approaches to drugs, targets, and delivery systems (see Fig. 1).

Oxygen, Metabolism, and Regeneration: Lessons from Mice.

The discovery that the Murphy Roths Large (MRL) mouse strain is a fully competent, epimorphic tissue regenerator, proved that the machinery of regeneration was preserved through evolution from hydra, to salamanders, to mammals. Such concepts have allowed translation of the biology of amphibians, and their ability to regenerate, to a mammalian context. We identified the ancient hypoxia-inducible factor (HIF)-1α pathway, operating through prolyl hydroxylase domain proteins (PHDs), as a central player in mouse regeneration. Thus, the possibility of targeting PHDs or other HIF-1α modifiers to effectively recreate the amphibian regenerative state has emerged. We posit that these regenerative pathways are critical in mammals. Moreover, the current approved use of PHD inhibitors in the clinic should allow fast-track translation from mouse studies to drug-based regenerative therapy in humans.

In Utero Exposure to a High-Fat Diet Programs Hepatic Hypermethylation and Gene Dysregulation and Development of Metabolic Syndrome in Male Mice.

Exposure to a high-fat (HF) diet in utero is associated with increased incidence of cardiovascular disease, diabetes, and metabolic syndrome later in life. However, the molecular basis of this enhanced susceptibility for metabolic disease is poorly understood. Gene expression microarray and genome-wide DNA methylation analyses of mouse liver revealed that exposure to a maternal HF milieu activated genes of immune response, inflammation, and hepatic dysfunction. DNA methylation analysis revealed 3360 differentially methylated loci, most of which (76%) were hypermethylated and distributed preferentially to hotspots on chromosomes 4 [atherosclerosis susceptibility quantitative trait loci (QTLs) 1] and 18 (insulin-dependent susceptibility QTLs 21). Interestingly, we found six differentially methylated genes within these hotspot QTLs associated with metabolic disease that maintain altered gene expression into adulthood (Arhgef19, Epha2, Zbtb17/Miz-1, Camta1 downregulated; and Ccdc11 and Txnl4a upregulated). Most of the hypermethylated genes in these hotspots are associated with cardiovascular system development and function. There were 140 differentially methylated genes that showed a 1.5-fold increase or decrease in messenger RNA levels. Many of these genes play a role in cell signaling pathways associated with metabolic disease. Of these, metalloproteinase 9, whose dysregulation plays a key role in diabetes, obesity, and cardiovascular disease, was upregulated 1.75-fold and hypermethylated in the gene body. In summary, exposure to a maternal HF diet causes DNA hypermethylation, which is associated with long-term gene expression changes in the liver of exposed offspring, potentially contributing to programmed development of metabolic disease later in life.

A High Fat Diet During Pregnancy and Lactation Induces Cardiac and Renal Abnormalities in GLUT4 +/- Male Mice.

BACKGROUND/AIMS: Altered nutrients during the in utero (IU) and/or lactation (L) period predispose offspring to cardio-renal diseases in adulthood. This study investigates the effect of a high fat diet (HFD) fed to female mice during IU/L on gene expression patterns associated with heart and kidney failure and hypertension in male offspring. METHODS: Female wild type (WT) mice were fed either a HFD or control chow (C) prior to mating with males with a genetic heterozygous deletion of GLUT4 (G4+/-, a model of peripheral insulin resistance and hypertension) and throughout IU/L. After weaning male offspring were placed on a standard rodent chow until 24 weeks of age. RESULTS: All offspring exposed to a maternal HFD showed increased heart and kidney weight and reduced cardiac insulin responsiveness. G4+/- offspring on a HFD displayed early hypertension associated with increased renal gene expression of renin and the AT1- receptors compared to G4+/- on a C diet. This group showed decreased cardiac expression of key genes involved in fatty acid oxidation compared to WT on a C diet. CONCLUSIONS: These results indicate an interaction between a HFD diet and genotype during early life development that can enhance susceptibility to cardio-renal diseases later in life.

Drug-induced regeneration in adult mice.

Whereas amphibians regenerate lost appendages spontaneously, mammals generally form scars over the injury site through the process of wound repair. The MRL mouse strain is an exception among mammals because it shows a spontaneous regenerative healing trait and so can be used to investigate proregenerative interventions in mammals. We report that hypoxia-inducible factor 1α (HIF-1α) is a central molecule in the process of regeneration in adult MRL mice. The degradation of HIF-1α protein, which occurs under normoxic conditions, is mediated by prolyl hydroxylases (PHDs). We used the drug 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), a PHD inhibitor, to stabilize constitutive expression of HIF-1α protein. A locally injectable hydrogel containing 1,4-DPCA was designed to achieve controlled delivery of the drug over 4 to 10 days. Subcutaneous injection of the 1,4-DPCA/hydrogel into Swiss Webster mice that do not show a regenerative phenotype increased stable expression of HIF-1α protein over 5 days, providing a functional measure of drug release in vivo. Multiple peripheral subcutaneous injections of the 1,4-DPCA/hydrogel over a 10-day period led to regenerative wound healing in Swiss Webster mice after ear hole punch injury. Increased expression of the HIF-1α protein may provide a starting point for future studies on regeneration in mammals.

From Immunity and Vaccines to Mammalian Regeneration.

Our current understanding of major histocompatibility complex (MHC)-mediated antigen presentation in self and nonself immune recognition was derived from immunological studies of autoimmunity and virus-host interactions, respectively. The trimolecular complex of the MHC molecule, antigen, and T-cell receptor accounts for the phenomena of immunodominance and MHC degeneracy in both types of responses and constrains vaccine development. Out of such considerations, we developed a simple peptide vaccine construct that obviates immunodominance, resulting in a broadly protective T-cell response in the absence of antibody. In the course of autoimmunity studies, we identified the MRL mouse strain as a mammalian model of amphibian-like regeneration. A significant level of DNA damage in the cells from this mouse pointed to the role of the cell cycle checkpoint gene CDKN1a, or p21(cip1/waf1). The MRL mouse has highly reduced levels of this molecule, and a genetic knockout of this single gene in otherwise nonregenerating strains led to an MRL-type regenerative response, indicating that the ability to regenerate has not been lost during evolution.

Tendon regeneration and scar formation: The concept of scarless healing.

Tendon healing is characterized by the formation of fibrovascular scar tissue, as tendon has very little intrinsic regenerative capacity. This creates a substantial clinical challenge in the setting of large, chronic tears seen clinically. Interest in regenerative healing seen in amphibians and certain strains of mice has arisen in response to the biological behavior of tendon tissue. Bone is also a model of tissue regeneration as healing bone will achieve the mechanical and histologic characteristics of the original tissue. The ultimate goal of the study of genes and mechanisms that contribute to true tissue regeneration is to ultimately attempt to manipulate the expression of those genes and activate these mechanisms in the setting of tendon injury and repair. Clearly, further research is needed to bring this to the forefront, however, study of scarless healing has potential to have meaningful application to tendon healing.

Inflammation and Its Correlates in Regenerative Wound Healing: An Alternate Perspective.

Objective: The wound healing response may be viewed as partially overlapping sets of two physiological processes, regeneration and wound repair with the former overrepresented in some lower species such as newts and the latter more typical of mammals. A robust and quantitative model of regenerative healing has been described in Murphy Roths Large (MRL) mice in which through-and-through ear hole wounds in the ear pinna leads to scarless healing and replacement of all tissue through blastema formation and including cartilage. Since these mice are naturally autoimmune and display many aspects of an enhanced inflammatory response, we chose to examine the inflammatory status during regenerative ear hole closure and observed that inflammation has a clear positive effect on regenerative healing. Approach: The inflammatory gene expression patterns (Illumina microarrays) of early healing ear tissue from regenerative MRL and nonregenerative C57BL/6 (B6) strains are presented along with a survey of innate inflammatory cells found in this tissue type pre and postinjury. The role of inflammation on healing is tested using a COX-2 inhibitor. Innovation and Conclusion: We conclude that (1) enhanced inflammation is consistent with, and probably necessary, for a full regenerative response and (2) the inflammatory gene expression and cell distribution patterns suggest a novel mast cell population with markers found in both immature and mature mast cells that may be a key component of regeneration.

Critical periods of increased fetal vulnerability to a maternal high fat diet.

BACKGROUND: Fetal adaptations to high fat (HF) diet in utero (IU) that may predispose to Metabolic Syndrome (MetS) in adulthood include changes in fetal hepatic gene expression. Studies were performed to determine whether maternal exposure to HF diet at different stages during pregnancy had different effects on the fetus, including hepatic gene expression. METHODS: Female wild type mice were fed either a HF or breeding chow (C) for 2 wks prior to mating. The experimental groups were composed of embryonic day (e) 18.5 fetuses obtained from WT female mice that were fed HF (HF, 35.5% fat) or breeding chow (C, 9.5% fat) for 2 wk before mating until e9.5 of pregnancy (periconception-midpregnancy). At e9.5 dams were switched to the opposite diet (C-HF or HF-C). RESULTS: Exposure to HF diet throughout pregnancy reduced maternal weight gain compared to C diet (p < 0.02 HF vs. C). HF-C dams had significantly decreased adiponectin levels and litter size when compared to C-HF (p < 0.02 HF-C vs C-HF). Independent of the timing of exposure to HF, fetal weight and length were significantly decreased when compared to C diet (HF, C-HF and HF-C vs. C p < 0.02). HF diet during the second half of pregnancy increased expression of genes in the fetal liver associated with fetal growth (C-HF vs C p < 0.001), glucose production (C-HF vs C p < 0.04), oxidative stress and inflammation (C-HF vs C p < 0.01) compared to C diet. CONCLUSIONS: This model defines that there are critical periods during gestation in which the fetus is actively shaped by the environment. Early exposure to a HF diet determines litter size while exposure to HF during the second half of pregnancy leads to dysregulation of expression of key genes responsible for fetal growth, hepatic glucose production and oxidative stress. These findings underscore the importance of future studies designed to clarify how these critical periods may influence future risk of developing MetS later in life.

Effects of genetics and in utero diet on murine pancreatic development.

Intrauterine (IU) malnutrition could alter pancreatic development. In this study, we describe the effects of high-fat diet (HFD) during pregnancy on fetal growth and pancreatic morphology in an 'at risk' animal model of metabolic disease, the glucose transporter 4 (GLUT4) heterozygous mouse (G4+/-). WT female mice mated with G4+/- males were fed HFD or control diet (CD) for 2 weeks before mating and throughout pregnancy. At embryonic day 18.5, fetuses were killed and pancreata isolated for analysis of morphology and expression of genes involved in insulin (INS) cell development, proliferation, apoptosis, glucose transport and function. Compared with WT CD, WT HFD fetal pancreata had a 2.4-fold increase in the number of glucagon (GLU) cells (P=0.023). HFD also increased GLU cell size by 18% in WT pancreata compared with WT CD. Compared with WT CD, G4+/- CD had an increased number of INS cells and decreased INS and GLU cell size. Compared with G4+/- CD, G4+/- HFD fetuses had increased pancreatic gene expression of Igf2, a mitogen and inhibitor of apoptosis. The expression of genes involved in proliferation, apoptosis, glucose transport, and INS secretion was not altered in WT HFD compared with G4+/- HFD pancreata. In contrast to WT HFD pancreata, HFD exposure did not alter pancreatic islet morphology in fetuses with GLUT4 haploinsufficiency; this may be mediated in part by increased Igf2 expression. Thus, interactions between IU diet and fetal genetics may play a critical role in the developmental origins of health and disease.