Spatiotemporal transcriptomic atlas of mouse organogenesis using DNA nanoball-patterned arrays.

Spatially resolved transcriptomic technologies are promising tools to study complex biological processes such as mammalian embryogenesis. However, the imbalance between resolution, gene capture, and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation embryos. Here, we combined DNA nanoball (DNB)-patterned arrays and in situ RNA capture to create spatial enhanced resolution omics-sequencing (Stereo-seq). We applied Stereo-seq to generate the mouse organogenesis spatiotemporal transcriptomic atlas (MOSTA), which maps with single-cell resolution and high sensitivity the kinetics and directionality of transcriptional variation during mouse organogenesis. We used this information to gain insight into the molecular basis of spatial cell heterogeneity and cell fate specification in developing tissues such as the dorsal midbrain. Our panoramic atlas will facilitate in-depth investigation of longstanding questions concerning normal and abnormal mammalian development.

Cell transcriptomic atlas of the non-human primate Macaca fascicularis.

Studying tissue composition and function in non-human primates (NHPs) is crucial to understand the nature of our own species. Here we present a large-scale cell transcriptomic atlas that encompasses over 1 million cells from 45 tissues of the adult NHP Macaca fascicularis. This dataset provides a vast annotated resource to study a species phylogenetically close to humans. To demonstrate the utility of the atlas, we have reconstructed the cell-cell interaction networks that drive Wnt signalling across the body, mapped the distribution of receptors and co-receptors for viruses causing human infectious diseases, and intersected our data with human genetic disease orthologues to establish potential clinical associations. Our M. fascicularis cell atlas constitutes an essential reference for future studies in humans and NHPs.

Fatty liver disease protective MTARC1 p.A165T variant reduces the protein stability of MTARC1.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of liver disease worldwide. MTARC1, encoded by the MTARC1 gene, is a mitochondrial outer membrane-anchored enzyme. Interestingly, the MTARC1 p.A165T (rs2642438) variant is associated with a decreased risk of NAFLD, indicating that MTARC1 might be an effective target. It has been reported that the rs2642438 variant does not have altered enzymatic activity so we reasoned that this variation may affect MTARC1 stability. In this study, MTARC1 mutants were generated and stability was assessed using a protein stability reporter system both in vitro and in vivo. We found that the MTARC1 p.A165T variant has dramatically reduced the stability of MTARC1, as assessed in several cell lines. In mice, the MTARC1 A168T mutant, the equivalent of human MTARC1 A165T, had diminished stability in mouse liver. Additionally, several MTARC1 A165 mutants, including A165S, A165 N, A165V, A165G, and A165D, had dramatically decreased stability as well, suggesting that the alanine residue of MTARC1 165 site is essential for MTARC1 protein stability. Collectively, our data indicates that the MTARC1 p.A165T variant (rs2642438) leads to reduced stability of MTARC1. Given that carriers of rs2642438 show a decreased risk of NAFLD, the findings herein support the notion that MTARC1 inhibition may be a therapeutic target to combat NAFLD.

Autophagy modulates the stability of Wee1 and cell cycle G2/M transition.

The mammalian cell cycle is divided into four sequential phases, namely G1 (Gap 1), S (synthesis), G2 (Gap 2), and M (mitosis). Wee1, whose turnover is tightly and finely regulated, is a well-known kinase serving as a gatekeeper for the G2/M transition. However, the mechanism underlying the turnover of Wee1 is not fully understood. Autophagy, a highly conserved cellular process, maintains cellular homeostasis by eliminating intracellular aggregations, damaged organelles, and individual proteins. In the present study, we found autophagy deficiency in mouse liver caused G2/M arrest in two mouse models, namely Fip200 and Atg7 liver-specific knockout mice. To uncover the link between autophagy deficiency and G2/M transition, we combined transcriptomic and proteomic analysis for liver samples from control and Atg7 liver-specific knockout mice. The data suggest that the inhibition of autophagy increases the protein level of Wee1 without any alteration of its mRNA abundance. Serum starvation, an autophagy stimulus, downregulates the protein level of Wee1 in vitro. In addition, the half-life of Wee1 is extended by the addition of chloroquine, an autophagy inhibitor. LC3, a central autophagic protein functioning in autophagy substrate selection and autophagosome biogenesis, interacts with Wee1 as assessed by co-immunoprecipitation assay. Furthermore, overexpression of Wee1 leads to G2/M arrest both in vitro and in vivo. Collectively, our data indicate that autophagy could degrade Wee1-a gatekeeper of the G2/M transition, whereas the inhibition of autophagy leads to the accumulation of Wee1 and causes G2/M arrest in mouse liver.

Advances in three-dimensional bioprinted stem cell-based tissue engineering for cardiovascular regeneration.

Three-dimensional (3D) bioprinting of cellular or biological components are an emerging field to develop tissue structures that mimic the spatial, mechanochemical and temporal characteristics of cardiovascular tissues. 3D multi-cellular and multi-domain organotypic biological constructs can better recapitulate in vivo physiology and can be utilized in a variety of applications. Such applications include in vitro cellular studies, high-throughput drug screening, disease modeling, biocompatibility analysis, drug testing and regenerative medicine. A major challenge of 3D bioprinting strategies is the inability of matrix molecules to reconstitute the complexity of the extracellular matrix and the intrinsic cellular morphologies and functions. An important factor is the inclusion of a vascular network to facilitate oxygen and nutrient perfusion in scalable and patterned 3D bioprinted tissues to promote cell viability and functionality. In this review, we summarize the new generation of 3D bioprinting techniques, the kinds of bioinks and printing materials employed for 3D bioprinting, along with the current state-of-the-art in engineered cardiovascular tissue models. We also highlight the translational applications of 3D bioprinting in engineering the myocardium cardiac valves, and vascular grafts. Finally, we discuss current challenges and perspectives of designing effective 3D bioprinted constructs with native vasculature, architecture and functionality for clinical translation and cardiovascular regeneration.

High-resolution radioluminescence microscopy of FDG uptake in an engineered 3D tumor-stoma model.

PURPOSE: The increased glucose metabolism of cancer cells is the basis for 18F-fluorodeoxyglucose positron emission tomography (FDG-PET). However, due to its coarse image resolution, PET is unable to resolve the metabolic role of cancer-associated stroma, which often influences the metabolic reprogramming of a tumor. This study investigates the use of radioluminescence microscopy for imaging FDG uptake in engineered 3D tumor models with high resolution. METHOD: Multicellular tumor spheroids (A549 lung adenocarcinoma) were co-cultured with GFP-expressing human umbilical vein endothelial cells (HUVECs) within an artificial extracellular matrix to mimic a tumor and its surrounding stroma. The tumor model was constructed as a 200-µm-thin 3D layer over a transparent CdWO4 scintillator plate to allow high-resolution imaging of the cultured cells. After incubation with FDG, the radioluminescence signal was collected by a highly sensitive widefield microscope. Fluorescence microscopy was performed using the same instrument to localize endothelial and tumor cells. RESULTS: Simultaneous and co-localized brightfield, fluorescence, and radioluminescence imaging provided high-resolution information on the distribution of FDG in the engineered tissue. The microvascular stromal compartment as a whole took up a large fraction of the FDG, comparable to the uptake of the tumor spheroids. In vitro gamma counting confirmed that A549 and HUVEC cells were both highly glycolytic with rapid FDG uptake kinetics. Despite the relative thickness of the tissue constructs, an average spatial resolution of 64 ± 4 µm was achieved for imaging FDG. CONCLUSION: Our study demonstrates the feasibility of imaging the distribution of FDG uptake in engineered in vitro tumor models. With its high spatial resolution, the method can separately resolve tumor and stromal components. The approach could be extended to more advanced engineered cancer models but also to surgical tissue slices and tumor biopsies.

The ER-localized Ca2+-binding protein calreticulin couples ER stress to autophagy by associating with microtubule-associated protein 1A/1B light chain 3.

Autophagy is of key importance for eliminating aggregated proteins during the maintenance of cellular proteostasis in response to endoplasmic reticulum (ER) stress. However, the upstream signaling that mediates autophagy activation in response to ER stress is incompletely understood. In this study, in vivo and in vitro approaches were utilized that include gain- and loss-of-function assays and mouse livers and human cell lines with tunicamycin-induced pharmacological ER stress. We report that calreticulin, a quality control chaperone that binds to misfolded glycoproteins for refolding in the ER, is induced under ER stress. Calreticulin overexpression stimulated the formation of autophagosomes and increased autophagic flux. Interestingly, calreticulin was sufficient for attenuating ER stress in tunicamycin- or thapsigargin-treated HeLa cells, whereas lentivirus-mediated shRNA calreticulin knockdown exacerbated ER stress. Mechanistically, we noted that calreticulin induces autophagy by interacting with microtubule-associated protein 1A/1B-light chain 3 (LC3). Confocal microscopy revealed that the colocalization of calreticulin and LC3 at the autophagosome was enhanced under ER stress conditions. Importantly, a conserved LC3-interacting region was necessary for calreticulin-mediated stimulation of autophagy and for reducing ER stress. These findings indicate a calreticulin-based mechanism that couples ER stress to autophagy activation, which, in turn, attenuates cellular stress, likely by alleviating the formation of aberrantly folded proteins. Pharmacological or genetic approaches that activate calreticulin-autophagy signaling may have potential for managing ER stress and related cellular disorders.

Sarcopenia as a predictor of mortality in women with breast cancer: a meta-analysis and systematic review.

BACKGROUND: Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide. Recently, studies have been published with inconsistent findings regarding whether sarcopenia is a risk factor for mortality in breast cancer patients. Therefore, the aim of this systematic review and meta-analysis was to systematically assess and quantify sarcopenia as a risk factor for mortality in breast cancer patients. METHODS: In a systematic literature review of PubMed, EMBASE, and the Cochrane CENTRAL Library, we searched for observational studies written in English (from database inception until April 30, 2019) that reported an association between sarcopenia and breast cancer in women who were 18 years or older. RESULTS: A total of six studies (5497 participants) were included in this meta-analysis. Breast cancer patients with sarcopenia were associated with a significantly higher risk of mortality, compared to breast cancer patients without sarcopenia (pooled HR-hazard ratio = 1.71, 95% CI: 1.25-2.33, I2 = 59.1%). In addition, the results of age subgroup analysis showed that participants younger than 55 years with sarcopenia had a lower risk of mortality than participants aged 55 years and older with sarcopenia (pooled HR = 1.46, 95% CI: 1.24-1.72 versus pooled HR = 1.99, 95% CI: 1.05-3.78), whereas both have an increased risk of mortality compared to non-sarcopenic patients. Subgroup analyses regarding stage at diagnosis revealed an increased risk of mortality in non-metastatic patients compared to participants without sarcopenia (pooled HR = 1.91, 95% CI: 1.32-2.78), whereas the association was not significant in metastatic breast cancer patients. Other subgroup analyses were performed using different follow-up periods (> 5 years versus ≤5 years) and the results were different (pooled HR = 1.81, 95% CI: 1.23-2.65 versus pooled HR = 1.70, 95% CI: 0.80-3.62). CONCLUSIONS: The present study found that sarcopenia is a risk factor for mortality among female early breast cancer patients. It is imperative that more research into specific interventions aimed at treating sarcopenia be conducted in the near future in order to provide evidence which could lead to decreased mortality rates in breast cancer patients.

Bursicon homodimers induce the innate immunity via Relish in Procambarus clarkii.

Bursicon (burs) is a neuropeptide hormone consisting of two cystine-knot proteins (burs α and burs ß), and burs α-ß is responsible for cuticle tanning in insects. Further studies show that burs homodimers induce prophylactic immunity. Here, we investigated the hypothesis that burs homodimers act in regulating immunity in the red swamp crayfish Procambarus clarkii. We found that burs α and burs ß are expressed in neural system of crayfish. Treating crayfish with recombinant burs-homodimer proteins led to up-regulation of several anti-microbial peptide (AMP) genes, and RNAi-mediated knockdown of burs led to decreased expression of AMP genes. The burs proteins also facilitated bacterial clearance and decreased crayfish mortality upon bacterial infection. Furthermore, burs proteins activated the transcriptional factor Relish, and knockdown of Relish abolished the influence of recombinant burs homodimers on AMP induction. We infer the burs homodimers induce expression of AMP genes via Relish in crayfish and this study extends this immune signaling pathway from insects to crustaceans.

The prevalence and influencing factors in anxiety in medical workers fighting COVID-19 in China: a cross-sectional survey.

The coronavirus disease 2019 (COVID-19) outbreak caused by the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2 virus) has been sustained in China since December 2019, and has become a pandemic. The mental health of frontline medical staff is a concern. In this study, we aimed to identify the factors influencing medical worker anxiety in China during the COVID-19 outbreak. We conducted a cross-sectional study to estimate the prevalence of anxiety among medical staff in China from 10 February 2020 to 20 February 2020 using the Zung Self-rating Anxiety Scale (SAS) to assess anxiety, with the criteria of normal (⩽49), mild (50-59), moderate (60-70) and severe anxiety (⩾70). We used multivariable linear regression to determine the factors (e.g. having direct contact when treating infected patients, being a medical staff worker from Hubei province, being a suspect case) for anxiety. We also used adjusted models to confirm independent factors for anxiety after adjusting for gender, age, education and marital status. Of 512 medical staff in China, 164 (32.03%) had had direct contact treating infected patients. The prevalence of anxiety was 12.5%, with 53 workers suffering from mild (10.35%), seven workers suffering from moderate (1.36%) and four workers suffering from severe anxiety (0.78%). After adjusting for sociodemographic characteristics (gender, age, education and marital status), medical staff who had had direct contact treating infected patients experienced higher anxiety scores than those who had not had direct contact (ß value = 2.33, confidence interval (CI) 0.65-4.00; P = 0.0068). A similar trend was observed in medical staff from Hubei province, compared with those from other parts of China (ß value = 3.67, CI 1.44-5.89; P = 0.0013). The most important variable was suspect cases with high anxiety scores, compared to non-suspect cases (ß value = 4.44, CI 1.55-7.33; P = 0.0028). In this survey of hospital medical workers during the COVID-19 outbreak in China, we found that study participants experienced anxiety symptoms, especially those who had direct clinical contact with infected patients; as did those in the worst affected areas, including Hubei province; and those who were suspect cases. Governments and healthcare authorities should proactively implement appropriate psychological intervention programmes, to prevent, alleviate or treat increased anxiety.

Association of sarcopenic obesity with the risk of all-cause mortality among adults over a broad range of different settings: a updated meta-analysis.

BACKGROUND: Previous cohort studies investigating the association between sarcopenic obesity (SO) and all-cause mortality among adult people have been inconsistent. We performed a meta-analysis to determine if SO is a predictor of all-cause mortality. METHODS: Prospective cohort studies that evaluated the association between SO and mortality in older people were identified via a systematic search of three electronic databases (PubMed, EMBASE, and the Cochrane Library). A random-effects model was applied to combine the results. We considered the methods recommeded by consensuses (dual X-ray absorptiometry,bio-impedancemetry, anthropometric measures or CT scan) to assess sarcopenic obesity. RESULTS: Of the 603 studies identified through the systematic review, 23 (Participants: 50866) were included in the meta-analysis. The mean age ranged from 50 to 82.5 years.SO was significantly associated with a higher risk of all-cause mortality among adult people (pooled HR = 1.21, 95% confidence interval [95% CI] = 1.10-1.32, p < 0.001, I2 = 64.3%). Furthermore, the subgroup analysis of participants showed that SO was associated with all-cause mortality (pooled HR = 1.14, 95% CI: 1.06-1.23) among community-dwelling adult people; similarly, this association was found in hospitalized patients (pooled HR = 1.65, 95% CI: 1.17-2.33). Moreover, the subgroup analysis demonstrated that SO was associated with all-cause mortality when using skeletal muscle mass (SMM) criteria, muscle strength criteria, and skeletal muscle index (SMI) criteria (HR = 1.12, 95% CI: 1.01-1.23; HR = 1.18, 95% CI: 1.05-1.33; and HR = 1.53, 95% CI: 1.13-2.07, respectively). In addition, we analyzed SO on the basis of obesity definition and demonstrated that participants with a SO diagnosis based on waist circumference (WC) (HR = 1.24, 95% CI: 1.09-1.40), body mass index (BMI) (HR = 1.29, 95% CI: 1.04-1.59), or visceral fat area (HR = 2.54, 95% CI: 1.83-3.53) have a significantly increase mortality risk compared with those without SO. CONCLUSION: Based on our update of existing scientific researches, SO is a significant predictor of all-cause mortality among older people, particularly hospitalized patients. Therefore, it is important to diagnose SO and to treat the condition to reduce mortality rates among older people.

Vascularized Bone-Mimetic Hydrogel Constructs by 3D Bioprinting to Promote Osteogenesis and Angiogenesis.

Bone is a highly vascularized tissue with a unique and complex structure. Long bone consists of a peripheral cortical shell containing a network of channels for vascular penetration and an inner highly vascularized bone marrow space. Bioprinting is a powerful tool to enable rapid and precise spatial patterning of cells and biomaterials. Here we developed a two-step digital light processing technique to fabricate a bone-mimetic 3D hydrogel construct based on octacalcium phosphate (OCP), spheroids of human umbilical vein endothelial cells (HUVEC), and gelatin methacrylate (GelMA) hydrogels. The bone-mimetic 3D hydrogel construct was designed to consist of a peripheral OCP-containing GelMA ring to mimic the cortical shell, and a central GelMA ring containing HUVEC spheroids to mimic the bone marrow space. We further demonstrate that OCP, which is evenly embedded in the GelMA, stimulates the osteoblastic differentiation of mesenchymal stem cells. We refined the design of a spheroid culture device to facilitate the rapid formation of a large number of HUVEC spheroids, which were embedded into different concentrations of GelMA hydrogels. It is shown that the concentration of GelMA modulates the extent of formation of the capillary-like structures originating from the HUVEC spheroids. This cell-loaded hydrogel-based bone construct with a biomimetic dual ring structure can be potentially used for bone tissue engineering.

Oxidative Stress Induces Neuronal Apoptosis Through Suppressing Transcription Factor EB Phosphorylation at Ser467.

BACKGROUND/AIMS: This study determined the role and mechanism of action of transcription factor EB (TFEB) in H2O2-induced neuronal apoptosis. METHODS: SH-SY5Y cells were treated with Akt inhibitor/activator and different concentrations of H2O2. Cell apoptosis was detected by flow cytometric analysis. Akt and TFEB phosphorylation and PARP cleavage were determined by Western blotting. HEK293T cells were transfected with different truncated TFEB mutants and HA-Akt-WT; SH-SY5Y cells were transfected with Flag-vector, Flag-TFEB, Flag-TFEB-S467A or Flag-TFEB-S467D; and TFEB interaction with Akt was determined by co-immunoprecipitation and GST pull-down assays. RESULTS: A low concentration of H2O2 induces TFEB phosphorylation at Ser467 and nuclear translocation, facilitating neuronal survival, whereas a high concentration of H2O2 promotes SH-SY5Y cell apoptosis via suppressing TFEB Ser467 phosphorylation and nuclear translocation. The TFEB-S467D mutant is more easily translocated into the nucleus than the non-phosphorylated TFEB-S467A mutant. Further, Akt physically binds to TFEB via its C-terminal tail interaction with the HLH domain of TFEB and phosphorylates TFEB at Ser467. Mutation of TFEB-Ser467 can prevent the phosphorylation of TFEB by Akt, preventing inhibition of oxidative stress-induced apoptosis. CONCLUSIONS: Oxidative stress induces neuronal apoptosis through suppressing TFEB phosphorylation at Ser467 by Akt, providing a novel therapeutic strategy for neurodegenerative diseases.

Functionally Graded, Bone- and Tendon-Like Polyurethane for Rotator Cuff Repair.

Critical considerations in engineering biomaterials for rotator cuff repair include bone-tendon-like mechanical properties to support physiological loading and biophysicochemical attributes that stabilize the repair site over the long-term. In this study, UV-crosslinkable polyurethane based on quadrol (Q), hexamethylene diisocyante (H), and methacrylic anhydride (M; QHM polymers), which are free of solvent, catalyst, and photoinitiator, is developed. Mechanical characterization studies demonstrate that QHM polymers possesses phototunable bone- and tendon-like tensile and compressive properties (12-74 MPa tensile strength, 0.6-2.7 GPa tensile modulus, 58-121 MPa compressive strength, and 1.5-3.0 GPa compressive modulus), including the capability to withstand 10 000 cycles of physiological tensile loading and reduce stress concentrations via stiffness gradients. Biophysicochemical studies demonstrate that QHM polymers have clinically favorable attributes vital to rotator cuff repair stability, including slow degradation profiles (5-30% mass loss after 8 weeks) with little-to-no cytotoxicity in vitro, exceptional suture retention ex vivo (2.79-3.56-fold less suture migration relative to a clinically available graft), and competent tensile properties (similar ultimate load but higher normalized tensile stiffness relative to a clinically available graft) as well as good biocompatibility for augmenting rat supraspinatus tendon repair in vivo. This work demonstrates functionally graded, bone-tendon-like biomaterials for interfacial tissue engineering.

Systematic characterization of 3D-printed PCL/ß-TCP scaffolds for biomedical devices and bone tissue engineering: influence of composition and porosity.

This work aims at providing guidance through systematic experimental characterization, for the design of 3D printed scaffolds for potential orthopaedic applications, focusing on fused deposition modeling (FDM) with a composite of clinically available polycaprolactone (PCL) and ß-tricalcium phosphate (ß-TCP). First, we studied the effect of the chemical composition (0% to 60% ß-TCP/PCL) on the scaffold's properties. We showed that surface roughness and contact angle were respectively proportional and inversely proportional to the amount of ß-TCP, and that degradation rate increased with the amount of ceramic. Biologically, the addition of ß-TCP enhanced proliferation and osteogenic differentiation of C3H10. Secondly, we systematically investigated the effect of the composition and the porosity on the 3D printed scaffold mechanical properties. Both an increasing amount of ß-TCP and a decreasing porosity augmented the apparent Young's modulus of the 3D printed scaffolds. Third, as a proof-of-concept, a novel multi-material biomimetic implant was designed and fabricated for potential disk replacement.

Sarcopenia as a predictor of hospitalization among older people: a systematic review and meta-analysis.

BACKGROUND: Previous cohort studies investigating the association between sarcopenia and the risk of hospitalization have been inconsistent. We performed a meta-analysis to determine if sarcopenia is a predictor of hospitalization. METHODS: Prospective cohort studies that evaluated the association between sarcopenia and hospitalization in older people were identified via a systematic search of four electronic databases (PubMed, EMBASE, Science Citation Index, and the Cochrane Library). A random-effect model was applied to combine the results according to the heterogeneity of the included studies. RESULTS: Five studies (2832 participants) were included in this meta-analysis. Pooled results demonstrated that older people with sarcopenia were at an increased risk of hospitalization (pooled hazards ratio [HR] = 1.57, 95% confidence interval [CI] = 1.26, 1.94, I2 = 4.5%, P = 0.000) compared to those without sarcopenia. Results of subgroup analyses showed that hospitalized patients with sarcopenia had a higher rate of hospitalization (HR = 2.01, 95% CI = 1.41, 2.88, p = 0.000) versus patients without sarcopenia. A similar result was also found in community-dwelling older people with sarcopenia versus those without sarcopenia (HR = 1.40, 95% CI = 1.05, 1.88, p = 0.023). In addition, the subgroup analysis for length of follow-up showed that studies with a follow-up period of 3 years or more (pooled HR = 1.52, 95% CI = 1.19, 1.94, P = 0.001) reported a significantly higher rate of hospitalization among individuals with sarcopenia compared to those without sarcopenia. However, this association was not found in the studies with a follow-up period of less than 3 years (pooled HR = 1.76, 95% CI = 0.90, 3.44, P = 0.099). CONCLUSIONS: Sarcopenia is a significant predictor of hospitalization among older individuals, and the association may not be significantly affected by the characteristics of the population or the definition of sarcopenia.

Treatment of critical-sized bone defects: clinical and tissue engineering perspectives.

Critical-sized bone defects are defined as those that will not heal spontaneously within a patient's lifetime. Current treatment options include vascularized bone grafts, distraction osteogenesis, and the induced membrane technique. The induced membrane technique is an increasingly utilized method with favorable results including high rates of union. Tissue engineering holds promise in the treatment of large bone defects due to advancement of stem cell biology, novel biomaterials, and 3D bioprinting. In this review, we provide an overview of the current operative treatment strategies of critical-sized bone defects as well as the current state of tissue engineering for such defects.

No systemic exposure of transtympanic heparin-binding epidermal growth factor like growth factor.

CONTEXT: Heparin-binding epidermal growth factor like growth factor (HB-EGF) is an emerging therapeutic for the regeneration of the tympanic membrane (TM). OBJECTIVE: Our aim was to determine whether the doses of HB-EGF delivered in a sustained release hydrogel into a middle ear mouse model, would be measurable in the systemic circulation. We also aimed to observe, in the scenario that the intended dose was absorbed directly into the circulation, whether these levels could be measured above the background levels of HB-EGF in the circulation. METHODS: A total of 12 mice had transtympanic injections of 5 µg/ml of HB-EGF contained within a previously described novel hydrogel vehicle, while another 12 mice had intravenous delivery of 10 µg/kg of HB-EGF. Intravenous blood samples were collected at 0-, 3-, 24-, 168-, 288- and 720-h post-injection. A double-antibody sandwich one-step process enzyme-linked immunosorbent assay (ELISA) was used to determine the level of HB-EGF in the serum. RESULTS: No mice in the transtympanic administration group and no mice in the intravenous administration group were found to have blood level measured above that in the controls. DISCUSSION: The inability of the positive control to measure levels above background, suggest the total dose used in our studies, even if 100% absorbed into the system circulation is insignificant. CONCLUSIONS: HB-EGF at the doses and delivery method proposed for treatment of chronic TM perforation in a mouse model are likely to have no measurable systemic effect.

Graded porous ß-tricalcium phosphate scaffolds enhance bone regeneration in mandible augmentation.

Bone augmentation requires scaffold to promote forming of natural bone structure. Currently, most of the reported bone scaffolds are porous solids with uniform pores. The aim of the current study is to evaluate the effect of a graded porous ß-tricalcium phosphate scaffolds on alveolar bone augmentation. Three groups of scaffolds were fabricated by a template-casting method: (1) graded porous scaffolds with large pores in the center and small pores at the periphery, (2) scaffolds with large uniform pores, and (3) scaffolds with small uniform pores. Bone augmentation on rabbit mandible was investigated by microcomputed tomography, sequential fluorescent labeling, and histologic examination 3 months after implantation.The result presents that all the scaffold groups maintain their augmented bone height after 3-month observation, whereas the autografting group presents an obvious bone resorption. Microcomputed tomography reveals that the graded porous group has significantly greater volume of new bone (P < 0.05) and similar bone density compared with the uniform pores groups. Bone substance distributes unevenly in all the 3 experimental groups. Greater bone volume can be observed in the area closer to the bone bed. The sequential fluorescent labeling observation reveals robust bone regeneration in the first month and faster bone growth in the graded porous scaffold group than that in the large porous scaffold group. Histologic examinations confirm bone structure in the aspect of distribution, activity, and maturity. We conclude that graded porous designed biodegradable ß-tricalcium phosphate scaffolds are beneficial to promote bone augmentation in the aspect of bone volume.

Deletion of the transforming growth factor ß receptor type II gene in articular chondrocytes leads to a progressive osteoarthritis-like phenotype in mice.

OBJECTIVE: While transforming growth factor ß (TGFß) signaling plays a critical role in chondrocyte metabolism, the TGFß signaling pathways and target genes involved in cartilage homeostasis and the development of osteoarthritis (OA) remain unclear. Using an in vitro cell culture method and an in vivo mouse genetic approach, we undertook this study to investigate TGFß signaling in chondrocytes and to determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFß signaling. METHODS: TGFß receptor type II (TGFßRII)-conditional knockout (KO) (TGFßRII(Col2ER)) mice were generated by breeding TGFßRII(flox/flox) mice with Col2-CreER-transgenic mice. Histologic, histomorphometric, and gene expression analyses were performed. In vitro TGFß signaling studies were performed using chondrogenic rat chondrosarcoma cells. To determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFß signaling, TGFßRII/matrix metalloproteinase 13 (MMP-13)- and TGFßRII/ADAMTS-5-double-KO mice were generated and analyzed. RESULTS: Inhibition of TGFß signaling (deletion of the Tgfbr2 gene in chondrocytes) resulted in up-regulation of Runx2, Mmp13, and Adamts5 expression in articular cartilage tissue and progressive OA development in TGFßRII(Col2ER) mice. Deletion of the Mmp13 or Adamts5 gene significantly ameliorated the OA-like phenotype induced by the loss of TGFß signaling. Treatment of TGFßRII(Col2ER) mice with an MMP-13 inhibitor also slowed OA progression. CONCLUSION: Mmp13 and Adamts5 are critical downstream target genes involved in the TGFß signaling pathway during the development of OA.