Two-Photon Fluorescence Microscopy and Applications in Angiogenesis and Related Molecular Events.

The role of angiogenesis in health and disease have gained considerable momentum in recent years. Visualizing angiogenic patterns and associated events of surrounding vascular beds in response to therapeutic and laboratory-grade biomolecules has become a commonplace in regenerative medicine and the biosciences. To achieve high-quality imaging for elucidating the molecular mechanisms of angiogenesis, the two-photon excitation fluorescence (2PEF) microscopy, or multiphoton fluorescence microscopy is increasingly utilized in scientific investigations. The 2PEF microscope confers several distinct imaging advantages over other fluorescence excitation microscopy techniques-for the observation of in-depth, three-dimensional vascularity in a variety of tissue formats, including fixed tissue specimens and in vivo vasculature in live specimens. Understanding morphological and subcellular changes that occur in cells and tissues during angiogenesis will provide insights to behavioral responses in diseased states, advance the engineering of physiologically relevant tissue models, and provide biochemical clues for the design of therapeutic strategies. We review the applicability and limitations of the 2PEF microscope on the biophysical and molecular-level signatures of angiogenesis in various tissue models. Imaging techniques and strategies for best practices in 2PEF microscopy will be reviewed. Impact Statement Deep live tissue imaging provides unique opportunities to study angiogenesis and associated events in real-time. In contrast to cross-sectional data provided by conventional methods, two-photon microscopy enables high-resolution tissue imaging, data acquisition over time, real-time visualization of angiogenic events, and reduces the number of animal models used in scientific research. This review provides insights on different two-photon microscopy methods and its application in live and deep tissue imaging of angiogenesis on in vitro and in vivo tissues. We believe that the current trends in imaging can transform the investigation of angiogenesis, cancer research, and biofabrication of vascularized tissues.

3D bioprinting and microscale organization of vascularized tissue constructs using collagen-based bioink.

Bioprinting three-dimensional (3D) tissue equivalents have progressed tremendously over the last decade. 3D bioprinting is currently being employed to develop larger and more physiologic tissues, and it is of particular interest to generate vasculature in biofabricated tissues to aid better perfusion and transport of nutrition. Having an advantage over manual culture systems by bringing together biological scaffold materials and cells in precise 3D spatial orientation, bioprinting could assist in placing endothelial cells in specific spatial locations within a 3D matrix to promote vessel formation at these predefined areas. Hence, in the present study, we investigated the use of bioprinting to generate tissue-level capillary-like networks in biofabricated tissue constructs. First, we developed a bioink using collagen type-1 supplemented with xanthan gum (XG) as a thickening agent. Using a commercial extrusion-based multi-head bioprinter and collagen-XG bioink, the component cells were spatially assembled, wherein the endothelial cells were bioprinted in a lattice pattern and sandwiched between bioprinted fibroblasts layers. 3D bioprinted constructs thus generated were stable, and maintained structural shape and form. Post-print culture of the bioprinted tissues resulted in endothelial sprouting and formation of interconnected capillary-like networks within the lattice pattern and between the fibroblast layers. Bioprinter-assisted spatial placement of endothelial cells resulted in fabrication of patterned prevascularized constructs that enable potential regenerative applications in the future.

Peripheral sensory neurons promote angiogenesis in neurovascular models derived from hESCs.

In the adult tissues, blood vessels traverse the body with neurons side by side; and share common signaling molecules. Developmental studies on animal models have shown that peripheral sensory neurons (PSNs) secrete angiogenic factors and endothelial cells (ECs) secrete neurotrophic factors which contribute to their coexistence, thereby forming the peripheral neurovascular (PNV) unit. Despite the large number of studies showing that innervation and vascularization complement each other, the interaction between human PSNs and ECs is still largely unknown. To study this interaction and to evaluate if PSNs affect angiogenesis, we derived both PSNs and ECs from human embryonic stem cells (hESCs) and developed a co-culture system. Seeding the two cell types together showed that PSNs induced endothelial morphogenesis with formation of vessel-like structures (VLSs). The PSN precursors, neural crest stem cells also induced VLS formation in the co-culture system; however, to a lesser extent. This sheds new light on the in vitro angiogenic potential of these cell types. PSNs derived from hESCs are powerful tools for studying development and disease as human PSNs are inaccessible for in vitro assays. Our novel approach, with optimized media condition allowed for integrating hESC-derived PSNs with hESC-derived ECs in three-dimensional (3D) collagen gel for creating a completely humanised PNV model. This preliminary model showed that innervation improves the development of vascularized channels in vitro, and provides insight to the development of innervated 3D models in future.

3D-Printed PCL/PPy Conductive Scaffolds as Three-Dimensional Porous Nerve Guide Conduits (NGCs) for Peripheral Nerve Injury Repair.

Conductivity is a desirable property of an ideal nerve guide conduit (NGC) that is being considered for peripheral nerve regeneration. Most of the conductive polymers reported in use for fabrication of tissue engineering scaffolds such as polypyrrole (PPy), polyaniline, polythiophene, and poly(3,4-ethylenedioxythiophene) are non-biodegradable and possess weak mechanical properties to be fabricated into 3D structures. In this study, a biodegradable and conductive block copolymer of PPy and Polycaprolactone (PPy-b-PCL) was used to fabricate 3D porous NGCs using a novel electrohydrodynamic jet 3D printing process which offers superior control over fiber diameter, pore size, porosity, and fiber alignment. PCL/PPy scaffolds with three different concentrations of PPy-b-PCL (0.5, 1, and 2% v/v) were fabricated as a mesh (pore size 125 ± 15 µm) and the effect of incorporation of PPy-b-PCL on mechanical properties, biodegradability, and conductivity of the NGCs were studied. The mechanical properties of the scaffolds decreased with the addition of PPy-b-PCL which aided the ability to fabricate softer scaffolds that are closer to the properties of the native human peripheral nerve. With increasing concentrations of PPy-b-PCL, the scaffolds displayed a marked increase in conductivity (ranging from 0.28 to 1.15 mS/cm depending on concentration of PPy). Human embryonic stem cell-derived neural crest stem cells (hESC-NCSCs) were used to investigate the impact of PPy-b-PCL based conductive scaffolds on the growth and differentiation to peripheral neuronal cells. The hESC-NCSCs were able to attach and differentiate to peripheral neurons on PCL and PCL/PPy scaffolds, in particular the PCL/PPy (1% v/v) scaffolds supported higher growth of neural cells and a stronger maturation of hESC-NCSCs to peripheral neuronal cells. Overall, these results suggest that PPy-based conductive scaffolds have potential clinical value as cell-free or cell-laden NGCs for peripheral neuronal regeneration.

Application of platelet rich fibrin for management of an electrosurge induced osteonecrosis involving maxillary alveolus.

BACKGROUND: Application of principles of electrocautery for hemostasis dates back to prehistoric times. Its modern implementation in various fields of general and head and neck surgeries have been well documented. However its usage in minor oral surgical procedures has gained popularity only recently. Complications associated with electro-surgery in the dental field are relatively rare and there is insufficient literature on its management. CASE REPORT: We present a case report on management of an electrosurgery induced osteonecrosis involving maxillary alveolus of left premolars. DISCUSSION: Inadvertent contact of the electrosurgery tip on bone can result in necrosis making it necessary to remove the sequestrum and graft the defect. Platelet rich fibrin in combination with bone grafts have been well documented to provide successful periodontal regeneration. CLINICAL IMPLICATIONS: Our aim of presenting this report is to create awareness among the health care providers regarding electrosurgical injuries. To our knowledge, this is the first time platelet rich fibrin has been used in the management of intraoral electrosurgical injury. Combining bone grafts with platelet rich fibrin is a good alternative as it can be done with relative ease and predictable outcome.

Multiple bony overgrowths in the mouth - report of two cases.

Tori and exostoses are benign bony protuberances that arise from bone surfaces in the oral cavity. The etiology of these growths has been implicated as multifactorial, but no consensus has been reached so far. These painless overgrowths seldom present as a complaint in the dental office unless functional or esthetic complications set in, and there is a fear for cancer. Here we discuss two rare cases where bony overgrowths present in the mouth were extensive and multiple.

Sleep bruxism associated with nocturnal enuresis in a 6-year-old child.

BACKGROUND: Bruxism is the involuntary grinding of teeth that occurs during sleep or wake time. The prevalence of bruxism in children and adolescents is high when compared to the adult population. CLINICAL PRESENTATION: This article presents a case report of sleep bruxism in a 6-year-old child. The interesting finding here was its association with nocturnal enuresis (NE)/bedwetting. CONCLUSION/CLINICAL RELEVANCE: NE could be a predisposing factor for sleep bruxism in children, as it causes emotional stress and has a negative impact on a child's self esteem. Psychological support and techniques for overcoming NE, like a scheduled waking program at night helps children with these problems by alleviating the associated stress. In addition, NE and bruxism are often associated with other sleep disorders, such as obstructive sleep apnea syndrome, and hence, child bruxers require careful medical evaluation to rule out such potential interactions.

Radiographic assessment of the prevalence of pulp stones in Malaysians.

INTRODUCTION: The aim of this study was to determine the prevalence of pulp stones in the Malaysian population using radiographs, and to assess the association of pulp stones with gender, age, tooth type, dental arch and tooth status. Occurrence of pulp stones among the three races in Malaysia (Malay, Chinese and Indians) was also studied. METHODS: A retrospective study was performed from a random sample of 361 dental records in AIMST Dental Centre, Faculty of Dentistry, AIMST University, Kedah, Malaysia. Data were collected from patient files and 507 intraoral periapical radiographs. All radiographs were examined by an oral radiologist to identify pulp stones and associated factors. Statistical analysis was performed using the Pearson chi-square test. RESULTS: Of the 361 patients, 205 were female and 156 were male. Pulp stones were identified in 162 (44.9%) subjects in 1 or more teeth and in 280 (15.7%) teeth of the total 1779 teeth examined. Pulp stones were found significantly more in molars and teeth that were not intact (carious/restored/both carious and restored). There was no significant correlation with sex, increasing age, dental arches, and ethnic races. CONCLUSIONS: The prevalence of pulp stones in the Malaysian population studied was 44.9%. Pulp stones were more often seen in teeth that were restored or affected with caries. These findings require further investigation.

Thermal burn of palate in an elderly diabetic patient.

BACKGROUND: Burns of the oral mucosa may be caused by thermal, mechanical, chemical, electrical or radiation injury. Clinically, these burns can produce localised or diffuse areas of tissue damage depending on the severity and extent of the insult. Most oral thermal burns produce erosions or ulcers on the palate or tongue. MATERIALS AND METHODS: A case of palatal burn in a 66-year-old diabetic patient caused by drinking hot cereal is presented. The role of diabetes in causing oral mucosal dysesthesia that predisposed the occurrence of this burn is also discussed. CONCLUSION: Insensate palatal burn as a rare complication of diabetes mellitus is reported here. With the disease being more widespread now, its potential oral complications will be seen with increasing frequency.