COVID-19 infection rates and mitigation strategies in orthodontic practices.

BACKGROUND: COVID-19 has impacted and increased risks for all populations, including orthodontic patients and providers. It also changes the practice management and infection control landscape in the practices. This study aimed to investigate the COVID-19 infection and vaccination status of orthodontic providers and mitigation approaches in orthodontic practices in the United States during 2021. METHODS: A validated 50-question research electronic data capture (REDCap) browser-based questionnaire was distributed to 12,393 orthodontists and pediatric dentists who reported actively providing orthodontic treatment. Questions were designed to collect demographic data of respondents, evaluate the COVID-19 mitigation approaches, and evaluate the history of COVID-19 infection and vaccination status of the orthodontic providers. Associations of demographic and the COVID-19 mitigation approaches were assessed using chi-square tests at the significance level of 0.05. RESULTS: Four hundred fifty-seven returned the survey (response rate 3.69%) for analysis. Most respondents were vaccinated, and increased infection control measures in response to the pandemic. Half of the respondents practiced teledentistry and switched to digital impression systems. Two-thirds reported difficulties in attaining PPEs due to the increased cost and scarcity of PPEs. About 6% of respondents reported a history of COVID-19 infection, and 68.9% of their staff had COVID-19 infection. Statistically significant associations were found between increased practice experience with difficulties in acquiring PPE (p = .010). There were no significant associations between races of respondents, geographic location, and years of practicing when cross-tabulated with vaccination status or COVID-19 infection rate (p > .05). CONCLUSION: Increased infection control strategies were employed in almost all orthodontic practices in addition to existing universal precaution. Most of the orthodontic providers and their staff members were vaccinated. While staff's infection rates were an issue, doctors' infection rates remained low.

Galloylated proanthocyanidins in dentin matrix exhibit biocompatibility and induce differentiation in dental stem cells.

Aim: Grape seed extract contains a complex mixture of proanthocyanidins (PACs), a plant biopolymer used as a biomaterial to improve reparative and preventive dental therapies. Co-polymerization of PACs with type I collagen mechanically reinforces the dentin extracellular matrix. This study assessed the biocompatibility of PACs from grape seed extract on dental pulp stem cells (DPSCs) in a model simulating leaching through dentin to the pulp cavity. The aim was to determine the type of PACs (galloylated vs. non-galloylated) within grape seed extract that are most compatible with dental pulp tissue. Methodology: Human demineralized dentin was treated with selectively-enriched dimeric PACs prepared from grape seed extract using liquid-liquid chromatography. DPSCs were cultured within a 2D matrix and exposed to PAC-treated dentin extracellular matrix. Cell proliferation was measured using the MTS assay and expression of odontoblastic genes was analyzed by qRT-PCR. Categorization of PACs leaching from dentin was performed using HPLC-MS. Results: Enriched dimeric fractions containing galloylated PACs increased the expression of certain odontoblastic genes in DPSCs, including Runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor (VEGF), bone morphogenetic protein 2 (BMP2), basic fibroblast growth factor (FGF2), dentin sialophosphoprotein (DSPP) and collagen, type I, alpha 1 (COLI). Galloylated dimeric PACs also exhibited minor effects on DPSC proliferation, resulting in a decrease compared to control after five days of treatment. The non-galloylated dimer fraction had no effect on these genes or on DPSC proliferation. Conclusions: Galloylated PACs are biocompatible with DPSCs and may exert a beneficial effect on cells within dental pulp tissue. The observed increase in odontoblastic genes induced by galloylated PACs together with a decrease in DPSC proliferation is suggestive of a shift toward cell differentiation. This data supports the use of dimeric PACs as a safe biomaterial, with galloylated dimeric PACs exhibiting potential benefits to odontoblasts supporting dentin regeneration.

Beneficial effect of sodium thiosulfate extends beyond myocardial tissue in isoproterenol model of infarction: Implication for nootropic effects.

One of the common negative impacts in the management of acute myocardial infarction is cognitive decline. Using the rat model of isoproterenol (ISO)-induced myocardial infarction, we assessed the cardioprotective effect of sodium thiosulfate (STS) and its influence on cognition. STS treatment reduced the cardiac infarct size by 75%, injury markers (lactate dehydrogenase: 60%, creatine kinase-muscle/brain: 44%) release in the blood, maintain the heart rate within a normal range (365 ± 10 bpm) and minimize postinfarction hypertrophic changes in comparison with the ISO group. At the cellular level, the heart from these rats had reduced reactive oxygen species (ROS) (25%), caspase-9 (60%), and improved mitochondrial function (restored electron transport chain function and copy number) compared to ISO hearts. The brain of STS-treated rats also showed a reduction in ROS (45%), caspase-9 (37%), and improved mitochondrial function relative to the brain of the ISO group, particularly limited to the striatum region, and these rats showed improved cognitive ability. Predominantly, the STS treatment reduced the reference memory defects observed in comparison to rats challenged by ISO. Furthermore, elevated circulating mitochondrial DNA and ATP were found in ISO-challenged rats, which indicate the cardiac mitochondria linked damage-associated patterns were restored to the sham level when pretreated with STS. We found increased H2 S, a well-known metabolite of STS with a neuroprotective role in the brain after STS administration, hinting at a possible secondary defense mechanism. In conclusion, the STS mediated cardioprotection and its nootropic effects are primarily mediated via the improvement of mitochondrial function and reduction of oxidative stress.

Addressing the alterations in cerebral ischemia-reperfusion injury on the brain mitochondrial activity: A possible link to cognitive decline.

Studies suggest that different anatomical regions in a normal brain show distinct mitochondrial physiology. But the pathological role of mitochondria during ischemia-reperfusion injury (IRI) from these brain regions are yet to be addressed. The objective of this study is to identify mitochondrial perturbations from the brain regions of cortex and striatum, exposed to IRI and their correlation with cognition. A rat model of bilateral carotid artery occlusion was used to induce ischemia (15 min and 30 min) followed by reperfusion of varying time points (15 min, 30 min, 4 h and 24 h). It was evident from the results that ischemia (30 min) caused changes in cerebral histology which was aggravated upon reperfusion. Upon examining the mitochondria, ischemia significantly reduced the ETC enzyme activity (complex-I and II) and ATP level in the striatum. Reperfusion further aggravated this decline by 4 h. Following 24 h reperfusion, the functional activity of ETC recovered in the striatum but not in the cortex. The complex-I, II activity and ATP level significantly declined by 24 h reperfusion in the cortex. Cortical mitochondria showed significantly reduced antioxidant enzyme activities (catalase and GPx) by the end of 24 h reperfusion. The implication of cellular events was noted as a decline in the cortex related cognitive performance in radial arm maze and Morris water maze tests. The susceptibility of mitochondria to IRI is different across the brain regions (striatum > cortex). Hence the observed loss of mitochondrial energy metabolism might be a contributing factor for cognitive decline in IRI.

Preconditioning the rat heart with sodium thiosulfate preserved the mitochondria in response to ischemia-reperfusion injury.

Sodium thiosulfate preconditioning (SIPC) was recently reported to be cardioprotective due to its ability to inhibit caspase-3 activation, chelate calcium ions and scavenge free radicals. However, the rationale behind its ability to improve the contractility of isolated rat heart challenged with ischemia-reperfusion injury (IR) is not well understood. As mitochondrial preservation is implicated in cardioprotection against IR, the present study was conceived to identify whether the cardioprotective effects of SIPC is associated with mitochondrial preservation. Using the isolated Langendorff rat heart model, 1 mM sodium thiosulfate (STS) was used to precondition the rat heart before IR and was used to study its effect on cardiac mitochondria. The IR heart experienced a ventricular contractile dysfunction that was improved by SIPC. Upon assessing in-gel the ATP synthetic capacity of mitochondria from IR heart, there was a significant decline, while in SIPC it was well preserved close to sham. As a sustained flow of electrons through the ETC and well-integrated mitochondria are the prerequisites for ATP synthesis, SIPC improved the activities of ETC complex enzymes (I-IV), which was reflected from the preserved ultrastructure of the mitochondria as analyzed from electron-microscopy in the treated rat hearts. This observation was coherent with the elevated expression of PGC1α (20%), a critical regulator of ATP production, which increased the mitochondrial copy number as well in the STS treated heart compared to IR. In conclusion, mitochondria might be a critical target for SIPC mediated cardioprotection against IR.

Sodium Thiosulfate Preconditioning Ameliorates Ischemia/Reperfusion Injury in Rat Hearts Via Reduction of Oxidative Stress and Apoptosis.

PURPOSE: Sodium thiosulfate (STS) has of late been proven efficacious in models of urolithiasis and vascular calcification. However, its cardiovascular effects on ischemia reperfusion injury (IR) have not been revealed. Being an antioxidant and calcium chelator, it is assumed to play a vital role in IR as ROS production and calcium overload are major perpetrators of IR injury. METHODS: The cardioprotective effect of STS was evaluated in vitro using H9C2 cardiomyocytes and in vivo using both isolated rat heart and intact left anterior descending artery (LAD) occlusion models of ischemia reperfusion injury. Finally, in silico tools were utilized to establish its possible mode of action. Myocardial injury markers and expression of apoptotic proteins were studied along with myocardial histopathology. RESULTS: STS of 1 mM recovered H9C2 cells from glucose oxidase/catalase-induced apoptosis. The isolated rat heart treated with STS prior to IR injury improved its hemodynamics and reduced the infarct size to 9%. This was supported by the absence of derangement of cardiac fibers from H&E stained section of LAD-occluded rats. Plasma troponin levels decreased by 15% compared to IR and the myocardium showed diminished apoptotic proteins. An in silico docking analysis revealed higher binding affinity of STS for caspase-3 with a binding energy of - 60.523 kcal/mol for the complex. CONCLUSION: The effectiveness of STS as a cardioprotective agent is attributed to the reduction of apoptosis by binding to the active site of caspase-3 in silico, which was substantiated by the reduced expression of caspase-3 and poly ADP ribose polymerase levels.

Erythrocyte Membrane Bound ATPase and Antioxidant Enzyme Changes Associated with Vascular Calcification is Reduced by Sodium Thiosulfate.

Sodium thiosulfate (STS), a cyanide antidote has been reported to possess antioxidant and calcium chelation effects, useful for the treatment of renal failure due to vascular calcification and urolithiasis. The present study investigated the in vivo modulatory effects of STS on erythrocyte calcium, phosphorous levels, lipid peroxidation, antioxidant enzyme and membrane ATPase activities (Ca2+, Na+K+, Mg2+ and 5'' nucleotidase) in an adenine induced model of vascular calcification in rats. Adenine (0.75%) was supplemented through the diet for 28 days, which resulted in significantly (P < 0.05) increased circulating calcium and phosphorous product and oxidative stress within the RBCs, as measured from lipid peroxidation and reduced antioxidant enzymes. The membrane ATPase activities were altered (increased Ca2+, Na+K+ ATPase and decreased Mg+ ATPase, 5' nucleotidase) compared to the rats fed on normal diet. STS (400 mg/kg) given orally was effective in establishing a normalcy in the RBC alterations. This effect was more pronounced, when STS was given from day 28 to day 49 after induction of calcification, instead of day 0 to day 28. These findings may benefit to evaluate the effectiveness of STS therapy in patients with chronic renal failure associated with increased circulating calcium and phosphorous product that leads to stiffening of vascular smooth muscles of aorta, due to calcium deposition.

Multifunctional ECM proteins in bone and teeth.

The extracellular matrix (ECM) of all tissues and organs is a highly organized and complex structure unique to the specific organ type. The ECM contains structural and functional proteins that define cellular function, organization, behavior and ultimately organ characteristics and function. The ECM was initially thought to contain only a specific set of secretory proteins. However, our group and several other groups have shown that the ECM contains functional proteins that have been previously defined as solely intracellular. In the present review, we have focused on the ECM of mineralized tissues namely bone and dentin. We provide here, a brief review of some non-classical ECM proteins that have been shown to possess both intra and extracellular roles in the formation of these mineralized matrices.

Dentin Matrix Proteins in Bone Tissue Engineering.

Dentin and bone are mineralized tissue matrices comprised of collagen fibrils and reinforced with oriented crystalline hydroxyapatite. Although both tissues perform different functionalities, they are assembled and orchestrated by mesenchymal cells that synthesize both collagenous and noncollagenous proteins albeit in different proportions. The dentin matrix proteins (DMPs) have been studied in great detail in recent years due to its inherent calcium binding properties in the extracellular matrix resulting in tissue calcification. Recent studies have shown that these proteins can serve both as intracellular signaling proteins leading to induction of stem cell differentiation and also function as nucleating proteins in the extracellular matrix. These properties make the DMPs attractive candidates for bone and dentin tissue regeneration. This chapter will provide an overview of the DMPs, their functionality and their proven and possible applications with respect to bone tissue engineering.

Acidic domain in dentin phosphophoryn facilitates cellular uptake: implications in targeted protein delivery.

Dentin phosphophoryn is nature's most acidic protein found predominantly in the dentin extracellular matrix. Its unique amino acid composition containing Asp-Ser (DS)-rich repeats makes it highly anionic. It has a low isoelectric point (pI 1.1) and, therefore, tends to be negatively charged at physiological pH. Phosphophoryn is normally associated with matrix mineralization as it can bind avidly to Ca(2+). It is well known that several macromolecules present in the extracellular matrix can be internalized and localized to specific intracellular compartments. In this study we demonstrate that dentin phosphophoryn (DPP) is internalized by several cell types via a non-conventional endocytic process. Utilizing a DSS polypeptide derived from DPP, we demonstrate the repetitive DSS-rich domain facilitates that endocytosis. As a proof-of-concept, we further demonstrate the use of this polypeptide as a protein delivery vehicle by delivering the osteoblast transcription factor Runx2 to the nucleus of mesenchymal cells. The functionality of the endocytosed Runx2 protein was demonstrated by performing gene expression analysis of Runx2 target genes. Nuclear localization was also demonstrated with the fusion protein DSS-Runx2 conjugated to quantum dots in two- and three-dimensional culture models in vitro and in vivo. Overall, we demonstrate that the DSS domain of DPP functions as a novel cell-penetrating peptide, and these findings demonstrate new opportunities for intracellular delivery of therapeutic proteins and cell tracking in vivo.

Dentin phosphophoryn activates Smad protein signaling through Ca2+-calmodulin-dependent protein kinase II in undifferentiated mesenchymal cells.

Dentin phosphophoryn (DPP) is a major noncollagenous protein in the dentin matrix. In this study, we demonstrate that pluripotent stem cells such as C3H10T1/2 and human bone marrow cells can be committed to the osteogenic lineage by DPP. Treatment with DPP can stimulate the release of intracellular Ca(2+). This calcium flux triggered the activation of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII induced the phosphorylation of Smad1 and promoted nuclear translocation of p-Smad1. Inhibition of store Ca(2+) depletion by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or down-regulation of CaMKII by KN-62, a selective cell-permeable pharmacological inhibitor or a dominant negative plasmid of CaMKII, blocked DPP-mediated Smad1 phosphorylation. Activation of Smad1 resulted in the expression of osteogenic markers such as Runx2, Osterix, DMP1, Bone sialoprotein, Osteocalcin, NFATc1, and Schnurri-2, which have been implicated in osteoblast differentiation. These findings suggest that DPP is capable of triggering commitment of pluripotent stem cells to the osteogenic lineage.

Bone-specific overexpression of DMP1 influences osteogenic gene expression during endochondral and intramembranous ossification.

Dentin matrix protein 1 (DMP1) is a key regulator of biomineralization within the extracellular matrix (ECM) of bone and plays a role in regulating osteogenic gene expression. Osteocalcin (OCN) is one of the most abundantly expressed non-collagenous proteins by osteoblasts. In the present study, we generated a mouse model (OC-DMP1) that overexpresses full-length DMP1 utilizing the mouse OCN promoter. Expression of genes encoding osteogenic transcription factors and ECM proteins during early post-natal development in male OC-DMP1 and wild type (WT) mice was evaluated in femurs and calvaria. Bones were dissected from n = 4 animals at 15, 30, 60 and 90-d of age. Total RNA was isolated, reverse transcribed, and real-time PCR analysis was performed. Results confirmed a difference (p < 0.05) in osteogenic gene expression between OC-DMP1 and WT mice at the specified time points. Additionally, distinctive osteogenic gene expression profiles for calvaria and femur, representing intramembranous and endochondral bone formation, were identified. These data suggest that bone-specific DMP1 overexpression changes the pattern in osteogenic gene expression pattern thereby influencing bone development. This animal model presented here provides new opportunities for analysis of in vivo roles of DMP1 in bone.

Evaluation of nanoscale versus hybrid micro/nano surface topographies for endosseous implants.

We examined the effect of a nanoscale titanium surface topography (D) versus two hybrid micro/nanoscale topographies (B and OS) on adherent mesenchymal stem cells (MSCs) and bone marrow derived macrophages (BMMs) function in cell culture and in vivo. In the in vitro study, compared to OS and B surfaces, D surface induced earlier and greater cell spreading, and earlier and profound mRNA expression of RUNX2, Osterix and BMP2 in MSCs. D surface induced earlier and higher expression of RUNX2 and BMP2 and lower expression of inflammatory genes in implant adherent cells in vivo. Measurement of osteogenesis at implant surfaces showed greater bone-to-implant contact at D versus OS surfaces after 21 days. We explored the cell population on the D and OS implant surfaces 24 h after placement using single-cell RNA sequencing and identified distinct cell clusters including macrophages, neutrophils and B cells. D surface induced lower expression and earlier reduction of inflammatory genes expression in BMMs in vitro. BMMs on D, B and OS surfaces demonstrated a marked increase of BMP2 expression after 1 and 3 days, and this increase was significantly higher on D surface at day 3. Our data implicates a dynamic process that may be influenced by nanotopography at multiple stages of osseointegration including initial immunomodulation, recruitment of MSCs and later osteoblastic differentiation leading to bone matrix production and mineralization. The results suggest that a nanoscale topography (D) favorably modulates adherent macrophage polarization toward anti-inflammatory and regenerative phenotypes and promotes the osteoinductive phenotype of adherent mesenchymal stem cells. STATEMENT OF SIGNIFICANCE: Our manuscript contains original data developed to define effects of a novel nanotopography on the process of osseointegration at the cell and tissue level.  Few studies have compared the effects of a nanoscale surface versus the more typical hybrid micro/nano-scale surfaces used today. We have utilized single-cell RNA sequencing for the first time to identify earliest cell populations on implant surfaces in vivo. We provide data indicating that the nanoscale surface acts upon both osteoprogenitor and immune cell (macrophages) to alter the process of bone formation in a surface-specific manner. This work represents new observations regarding osseointegration and immunomodulation.

Application of sodium triple-quantum coherence NMR spectroscopy for the study of growth dynamics in cartilage tissue engineering.

We studied the tissue growth dynamics of tissue-engineered cartilage at an early growth stage after cell seeding for four weeks using sodium triple-quantum coherence NMR spectroscopy. The following tissue-engineering constructs were studied: 1) bovine chondrocytes cultured in alginate beads; 2) bovine chondrocytes cultured as pellets (scaffold-free chondrocyte pellets); and 3) human marrow stromal cells (HMSCs) seeded in collagen/chitosan based biomimetic scaffolds. We found that the sodium triple-quantum coherence spectroscopy could differentiate between different tissue-engineered constructs and native tissues based on the fast and slow components of relaxation rate as well as on the average quadrupolar coupling. Both fast (Tf ) and slow (Ts ) relaxation times were found to be longer in chondrocyte pellets and biomimetic scaffolds compared to chondrocytes suspended in alginate beads and human articular cartilage tissues. In all cases, it was found that relaxation rates and motion of sodium ions measured from correlation times were dependent on the amount of macromolecules, high cell density and anisotropy of the cartilage tissue-engineered constructs. Average quadrupolar couplings were found to be lower in the engineered tissue compared to native tissue, presumably due to the lack of order in collagen accumulated in the engineered tissue. These results support the use of sodium triple-quantum coherence spectroscopy as a tool to investigate anisotropy and growth dynamics of cartilage tissue-engineered constructs in a simple and reliable way.

Micro RNA based MSC EV engineering: Targeting the BMP2 cascade for bone repair.

Mesenchymal stem cell derived extracellular vesicles (MSC EVs) possess excellent immunomodulatory and therapeutic properties. While beneficial, from a translational perspective, extracellular vesicles with consistent functionality and target specificity are required to achieve the goals of precision medicine and tissue engineering. Prior research has identified that the miRNA composition of mesenchymal stem cell derived extracellular vesicles contributes significantly towards extracellular vesicles functionality. In this study, we hypothesized that mesenchymal stem cell derived extracellular vesicle functionality can be rendered pathway-specific using a miRNA-based extracellular vesicles engineering approach. To test this hypothesis, we utilized bone repair as a model system and the BMP2 signaling cascade as the targeted pathway. We engineered mesenchymal stem cell extracellular vesicles to possess increased levels of miR-424, a potentiator of the BMP2 signaling cascade. We evaluated the physical and functional characteristics of these extracellular vesicles and their enhanced ability to trigger the osteogenic differentiation of naïve mesenchymal stem cell in vitro and facilitate bone repair in vivo. Results indicated that the engineered extracellular vesicles retained their extracellular vesicles characteristics and endocytic functionality and demonstrated enhanced osteoinductive function by activating SMAD1/5/8 phosphorylation and mesenchymal stem cell differentiation in vitro and enhanced bone repair in vivo. Furthermore, the inherent immunomodulatory properties of the mesenchymal stem cell derived extracellular vesicles remained unaltered. These results serve as a proof-of-concept for miRNA-based extracellular vesicles engineering approaches for regenerative medicine applications.

Evaluation of prophylactic efficacy of sodium thiosulfate in combating I/R injury in rat brain: exploring its efficiency further in vascular calcified brain slice model.

Cerebral ischemia reperfusion injury (CIR) is one of the clinical manifestations encountered during the management of stroke. High prevalence of intracranial arterial calcification is reported in stroke patients. However, the impact of vascular calcification (VC) in the outcome of CIR and the efficacy of mechanical preconditioning (IPC) and pharmacological conditioning with sodium thiosulphate (STS) in ameliorating IR remains unclear. Two experimental models namely carotid artery occlusion (n = 36) and brain slice models (n = 18) were used to evaluate the efficacy of STS in male Wistar rats. IR was inflicted in rat by occluding carotid artery for 30 min followed by 24-h reperfusion after STS (100 mg/kg) administration. Brain slice model was used to reconfirm the results to account blood brain barrier permeability. Further, brain slice tissue was utilised to evaluate the efficacy of STS in VC rat brain by measuring the histological alterations and biochemical parameters. Pre-treatment of STS prior to CIR in intact animal significantly reduced the IR-associated histopathological alterations in brain, declined oxidative stress and improved the mitochondrial function found to be similar to IPC. Brain slice model data also confirmed the neuroprotective effect of STS similar to IPC in IR challenged tissue slice. Higher tissue injury was noted in VC brain IR tissue than normal IR tissue. Therapeutic efficacy of STS was evident in VC rat brain tissues and normal tissues subjected to IR. On the other hand, IPC-mediated protection was noted only in IR normal and adenine-induced VC brain tissues not in high-fat diet (HFD) induced VC brain tissues. Based on the results, we concluded that similar to IPC, STS was effective in attenuating IR injury in CIR rat brain. Vascular calcification adversely affected the recovery protocol of brain tissues from ischemic insult. STS was found to be an effective agent in ameliorating the IR injury in both adenine and HFD induced vascular calcified rat brain, but IPC-mediated neuroprotection was absent in HFD-induced VC brain tissues.

Comparative Osteogenesis of Three Clinical Bone Graft Materials: An In Vivo Study.

PURPOSE: To investigate bone regeneration among three different bone graft materials in a rat calvarum model. MATERIALS AND METHODS: A total of 24 rats had two 5-mm defects placed per calvarial. Rats were divided into four groups: bovine xenograft (XG), demineralized bone matrix (DBM), mineralized bone graft (MBG), and collagen membrane control (CC). Within each group, samples were collected at two time points: 4 weeks (T4) and 8 weeks (T8). Bone regeneration was assessed by microcomputed tomography (micro-CT) imaging and was analyzed using MATLAB software. Additionally, the fixed samples were subsequently demineralized for immunohistochemistry and histomorphometry. Slides were mounted and stained with hematoxylin and eosin (H&E) stain as well as bone morphogenetic protein 2 (BMP-2) and runt-related transcription factor 2 (RUNX2) markers. The numbers of positive cells/area were calculated for each group and analyzed. RESULTS: At 4 weeks, DBM showed low mineral density (7.7%) compared to the control (25.2%), but increased dramatically at 8 weeks (DBM, T8 = 27.6%; CC, T8 = 27.2%). Xenograft material showed an increase in mineral desnity between T4 and T8 (XG, T4 = 25.0%; XG, T8 = 32.3%). MBG remained consistent over the 8-week trial period (MBG, T4 = 30.4%; MBG, T8 = 30.4%). BMP-2 expression was present in cells adherent to all graft materials. RUNX2 expression was also observed in cells adherent to all graft materials, indicating that during the 4- to 8-week healing period, all materials supported osteogenesis. CONCLUSIONS: Compared to other materials, the DBM had high osteoinductive properties during the 4- to 8-week time period based on increased mineral content. All materials were associated with immunohistologic evidence of osteogenesis in the rat calvarial defect model.

NG2/CSPG4 attenuates motility in mandibular fibrochondrocytes under serum starvation conditions.

The migration of mandibular fibrochondrocytes is important for the development of the mandible, the homeostasis of the mandibular cartilage, and for the capacity of the tissue to respond to injury. Mandibular fibrochondrocytes have to overcome formidable obstacles during migration including a dense and heterogeneous three-dimensional matrix. Guiding the direction of cell migration and commitment to a migratory phenotype in this microenvironment necessitates a multivalent response to chemotactic and extracellular matrix-mediated stimuli. One of the key matrix components in the cartilage of the temporomandibular joint is type VI collagen. Neuron/glial antigen 2 (NG2/CSPG4) is a transmembrane proteoglycan that binds with collagen VI and has been implicated in a wide range of cell behaviors including cell migration, motility, adhesion, and proliferation. While NG2/CSPG4 has been shown to be a key regulator of mandibular cartilage homeostasis, its role in the migration of mandibular fibrochondrocytes during normal and cell stress conditions has yet to be resolved. Here, we address this gap in knowledge by characterizing NG2/CSPG4-dependent migration in mandibular fibrochondrocytes using primary mandibular fibrochondrocytes isolated from control and full length NG2/CSPG4 knockout mice, in primary mandibular fibrochondrocytes isolated from NG2|DsRed reporter mice and in an immortalized mandibular fibrochondrocyte cell line with a mutated NG2/CSPG4 ectodomain. All three cells demonstrate similar results, with loss of the full length or truncated NG2/CSPG4 increasing the rate of cell migration in serum starvation/cell stress conditions. These findings clearly implicate NG2/CSPG4 as a key molecule in the regulation of cell migration in mandibular fibrochondrocytes in normal and cell stress conditions, underscoring the role of NG2/CSPG4 as a mechanosensitive signaling hub in the mandibular cartilage.

MicroRNA-based engineering of mesenchymal stem cell extracellular vesicles for treatment of retinal ischemic disorders: Engineered extracellular vesiclesand retinal ischemia.

Mesenchymal stem cell (MSCs)-derived extracellular vesicles (EVs) are emerging therapeutic tools. Hypoxic pre-conditioning (HPC) of MSCs altered the production of microRNAs (miRNAs) in EVs, and enhanced the cytoprotective, anti-inflammatory, and neuroprotective properties of their derivative EVs in retinal cells. EV miRNAs were identified as the primary contributors of these EV functions. Through miRNA seq analyses, miRNA-424 was identified as a candidate for the retina to overexpress in EVs for enhancing cytoprotection and anti-inflammatory effects. FEEs (functionally engineered EVs) overexpressing miR424 (FEE424) significantly enhanced neuroprotection and anti-inflammatory activities in vitro in retinal cells. FEE424 functioned by reducing inflammatory cytokine production in retinal microglia, and attenuating oxygen free radicals in retinal Muller cells and microvascular endothelial cells, providing a multi-pronged approach to enhancing recovery after retinal ischemic insult. In an in vivo model of retinal ischemia, native, HPC, and FEE424 MSC EVs robustly and similarly restored function to close to baseline, and prevented loss of retinal ganglion cells, but HPC EVs provided the most effective attenuation of apoptosis-related and inflammatory cytokine gene expression. These results indicate the potential for EV engineering to produce ameliorative effects for retinal diseases with a significant inflammatory component. STATEMENT OF SIGNIFICANCE: We show that functionally engineered extracellular vesicles (FEEs) from mesenchymal stem cells (MSCs) provide cytoprotection in rat retina subjected to ischemia. FEEs overexpressing microRNA 424 (FEE424) function by reducing inflammatory cytokine production in retinal microglia, and attenuating oxygen free radicals in Muller cells and microvascular endothelial cells, providing a multi-pronged approach to enhancing recovery. In an in vivo model of retinal ischemia in rats, native, hypoxic-preconditioned (HPC), and FEE424 MSC EVs robustly and similarly restored function, and prevented loss of retinal ganglion cells, but HPC EVs provided the most effective attenuation of apoptosis-related and inflammatory cytokine gene expression. The results indicate the potential for EV engineering to produce ameliorative effects for retinal diseases with a significant inflammatory component.

CABG Patients Develop Global DNA Hypermethylation, That Negatively Affect the Mitochondrial Function and Promote Post-Surgical Cognitive Decline: A Proof of Concept in Small Cohort.

Global DNA hypermethylation and mitochondrial dysfunction are reported to be associated with the development of mild cognitive decline (MCI). The present study aims to generate preliminary data that connect the above association with post-surgical coronary artery bypass grafting (CABG) cognitive decline in patients. Data were collected from 70 CABG patients and 25 age-matched controls. Cognitive function was assessed using the Montreal Cognitive Assessment (MOCA) test on day 1 (before surgery) and on the day of discharge. Similarly, blood was collected before and one day after the CABG procedure for mitochondrial functional analysis and expression of DNA methylation genes. Test analysis score suggested 31 (44%) patients had MCI before discharge. These patients showed a significant decrease in complex I activity and an increase in malondialdehyde levels (p < 0.001) from the control blood samples. Post-surgical samples showed a significant reduction in blood MT-ND1 mRNA expression from control and from pre-surgical samples (p < 0.005), along with elevated DNMT1 gene expression (p < 0.047), with an insignificant increase in TET1 and TET3 gene expression. Correlation analysis showed a significant positive relation between cognitive decline and elevated blood DNMT1 and declined blood complex I activity, signifying that cognitive decline experienced by post-surgical CABG patients is associated with increased DNMT1 expression and declined complex I activity. Based on the data, we conclude that both DNA hypermethylation and mitochondrial dysfunction are associated with post-CABG MCI, where the former is negatively correlated, and the latter is positively correlated with post-surgical MCI in CABG cases. Additionally, a multimarker approach that comprises MOCA, DNA methylation, DNMT, and NQR activities can be utilized to stratify the population that is sensitive to developing post-CABG MCI.