3D printing of a biocompatible double network elastomer with digital control of mechanical properties.

The majority of 3D-printed biodegradable biomaterials are brittle, limiting their potential application to compliant tissues. Poly (glycerol sebacate) acrylate (PGSA) is a synthetic biodegradable and biocompatible elastomer, compatible with light-based 3D printing. In this work we employed digital-light-processing (DLP)-based 3D printing to create a complex PGSA network structure. Nature-inspired double network (DN) structures with two geometrically interconnected segments with different mechanical properties were printed from the same material in a single shot. Such capability has not been demonstrated by any other fabrication technique. The biocompatibility of PGSA after 3D printing was confirmed via cell-viability analysis. We used a finite element analysis (FEA) model to predict the failure of the DN structure under uniaxial tension. FEA confirmed the soft segments act as sacrificial elements while the hard segments retain structural integrity. The simulation demonstrated that the DN design absorbs 100% more energy before rupture than the network structure made by single exposure condition (SN), doubling the toughness of the overall structure. Using the FEA-informed design, a new DN structure was printed and the FEA predicted tensile test results agreed with tensile testing of the printed structure. This work demonstrated how geometrically-optimized material design can be easily and rapidly achieved by using DLP-based 3D printing, where well-defined patterns of different stiffnesses can be simultaneously formed using the same elastic biomaterial, and overall mechanical properties can be specifically optimized for different biomedical applications.

A sequential 3D bioprinting and orthogonal bioconjugation approach for precision tissue engineering.

Recent advances in 3D bioprinting have transformed the tissue engineering landscape by enabling the controlled placement of cells, biomaterials, and bioactive agents for the biofabrication of living tissues and organs. However, the application of 3D bioprinting is limited by the availability of cytocompatible and printable biomaterials that recapitulate properties of native tissues. Here, we developed an integrated 3D projection bioprinting and orthogonal photoconjugation platform for precision tissue engineering of tailored microenvironments. By using a photoreactive thiol-ene gelatin bioink, soft hydrogels can be bioprinted into complex geometries and photopatterned with bioactive moieties in a rapid and scalable manner via digital light projection (DLP) technology. This enables localized modulation of biophysical properties such as stiffness and microarchitecture as well as precise control over spatial distribution and concentration of immobilized functional groups. As such, well-defined properties can be directly incorporated using a single platform to produce desired tissue-specific functions within bioprinted constructs. We demonstrated high viability of encapsulated endothelial cells and human cardiomyocytes using our dual process and fabricated tissue constructs functionalized with VEGF peptide mimics to induce guided endothelial cell growth for programmable vascularization. This work represents a pivotal step in engineering multifunctional constructs with unprecedented control, precision, and versatility for the rational design of biomimetic tissues.

Controlled Growth Factor Release in 3D-Printed Hydrogels.

Growth factors (GFs) are critical components in governing cell fate during tissue regeneration. Their controlled delivery is challenging due to rapid turnover rates in vivo. Functionalized hydrogels, such as heparin-based hydrogels, have demonstrated great potential in regulating GF release. While the retention effects of various concentrations and molecular weights of heparin have been investigated, the role of geometry is unknown. In this work, 3D printing is used to fabricate GF-embedded heparin-based hydrogels with arbitrarily complex geometry (i.e., teabag, flower shapes). Simplified cylindrical core-shell structures with varied shell thickness are printed, and the rates of GF release are measured over the course of 28 days. Increasing the shell layers' thickness decreases the rate of GF release. Additionally, a mathematical model is developed, which is found capable of accurately predicting GF release kinetics in hydrogels with shell layers greater than 0.5 mm thick (R2 > 0.96). Finally, the sequential release is demonstrated by printing two GFs in alternating radial layers. By switching the spatial order, the delivery sequence of the GFs can be modulated. This study demonstrates how 3D printing can be utilized to fabricate user-defined structures with unique geometry in order to control the rate of GF release in hydrogels.

Hydroxyapatite mineral enhances malignant potential in a tissue-engineered model of ductal carcinoma in situ (DCIS).

While ductal carcinoma in situ (DCIS) is known as a precursor lesion to most invasive breast carcinomas, the mechanisms underlying this transition remain enigmatic. DCIS is typically diagnosed by the mammographic detection of microcalcifications (MC). MCs consisting of non-stoichiometric hydroxyapatite (HA) mineral are frequently associated with malignant disease, yet it is unclear whether HA can actively promote malignancy. To investigate this outstanding question, we compared phenotypic outcomes of breast cancer cells cultured in control or HA-containing poly(lactide-co-glycolide) (PLG) scaffolds. Exposure to HA mineral in scaffolds increased the expression of pro-tumorigenic interleukin-8 (IL-8) among transformed but not benign cells. Notably, MCF10DCIS.com cells cultured in HA scaffolds adopted morphological changes associated with increased invasiveness and exhibited increased motility that were dependent on IL-8 signaling. Moreover, MCF10DCIS.com xenografts in HA scaffolds displayed evidence of enhanced malignant progression relative to xenografts in control scaffolds. These experimental findings were supported by a pathological analysis of clinical DCIS specimens, which correlated the presence of MCs with increased IL-8 staining and ductal proliferation. Collectively, our work suggests that HA mineral may stimulate malignancy in preinvasive DCIS cells and validate PLG scaffolds as useful tools to study cell-mineral interactions.

Correlative imaging reveals physiochemical heterogeneity of microcalcifications in human breast carcinomas.

Microcalcifications (MCs) are routinely used to detect breast cancer in mammography. Little is known, however, about their materials properties and associated organic matrix, or their correlation to breast cancer prognosis. We combine histopathology, Raman microscopy, and electron microscopy to image MCs within snap-frozen human breast tissue and generate micron-scale resolution correlative maps of crystalline phase, trace metals, particle morphology, and organic matrix chemical signatures within high grade ductal carcinoma in situ (DCIS) and invasive cancer. We reveal the heterogeneity of mineral-matrix pairings, including punctate apatitic particles (<2 µm) with associated trace elements (e.g., F, Na, and unexpectedly Al) distributed within the necrotic cores of DCIS, and both apatite and spheroidal whitlockite particles in invasive cancer within a matrix containing spectroscopic signatures of collagen, non-collagen proteins, cholesterol, carotenoids, and DNA. Among the three DCIS samples, we identify key similarities in MC morphology and distribution, supporting a dystrophic mineralization pathway. This multimodal methodology lays the groundwork for establishing MC heterogeneity in the context of breast cancer biology, and could dramatically improve current prognostic models.

Multiscale characterization of the mineral phase at skeletal sites of breast cancer metastasis.

Skeletal metastases, the leading cause of death in advanced breast cancer patients, depend on tumor cell interactions with the mineralized bone extracellular matrix. Bone mineral is largely composed of hydroxyapatite (HA) nanocrystals with physicochemical properties that vary significantly by anatomical location, age, and pathology. However, it remains unclear whether bone regions typically targeted by metastatic breast cancer feature distinct HA materials properties. Here we combined high-resolution X-ray scattering analysis with large-area Raman imaging, backscattered electron microscopy, histopathology, and microcomputed tomography to characterize HA in mouse models of advanced breast cancer in relevant skeletal locations. The proximal tibial metaphysis served as a common metastatic site in our studies; we identified that in disease-free bones this skeletal region contained smaller and less-oriented HA nanocrystals relative to ones that constitute the diaphysis. We further observed that osteolytic bone metastasis led to a decrease in HA nanocrystal size and perfection in remnant metaphyseal trabecular bone. Interestingly, in a model of localized breast cancer, metaphyseal HA nanocrystals were also smaller and less perfect than in corresponding bone in disease-free controls. Collectively, these results suggest that skeletal sites prone to tumor cell dissemination contain less-mature HA (i.e., smaller, less-perfect, and less-oriented crystals) and that primary tumors can further increase HA immaturity even before secondary tumor formation, mimicking alterations present during tibial metastasis. Engineered tumor models recapitulating these spatiotemporal dynamics will permit assessing the functional relevance of the detected changes to the progression and treatment of breast cancer bone metastasis.

Stability and reliability of error-related electromyography over the corrugator supercilii with increasing trials.

Electromyographic activity over the corrugator supercilii (cEMG), the primary facial muscle involved in negative emotions, is increased during the commission of errors on speeded reaction-time tasks. In the present paper, data from two previously published studies were reanalyzed to investigate the reliability and stability of error-related, correct-related, and difference cEMG across increasing numbers of trials. For a modified go/no-go and a flanker task, we found that error-related cEMG was highly stable and reliable in 14 trials, and correct-related cEMG between 56 and 82 trials, respectively. Given the typical number of trials used in studies of cognitive control, these findings suggest that many investigations of error monitoring are already sufficient to obtain acceptable error- and correct-related cEMG signals. Error-related cEMG activity is relatively easy to measure and, as such, it shows great promise for future research investigating the cognitive and affective mechanisms of error monitoring.

No Evidence That Gratitude Enhances Neural Performance Monitoring or Conflict-Driven Control.

It has recently been suggested that gratitude can benefit self-regulation by reducing impulsivity during economic decision making. We tested if comparable benefits of gratitude are observed for neural performance monitoring and conflict-driven self-control. In a pre-post design, 61 participants were randomly assigned to either a gratitude or happiness condition, and then performed a pre-induction flanker task. Subsequently, participants recalled an autobiographical event where they had felt grateful or happy, followed by a post-induction flanker task. Despite closely following existing protocols, participants in the gratitude condition did not report elevated gratefulness compared to the happy group. In regard to self-control, we found no association between gratitude--operationalized by experimental condition or as a continuous predictor--and any control metric, including flanker interference, post-error adjustments, or neural monitoring (the error-related negativity, ERN). Thus, while gratitude might increase economic patience, such benefits may not generalize to conflict-driven control processes.

Distribution characteristics of meridian sinew (jingjin) syndrome in 313 cases of whiplash-associated disorders.

OBJECTIVE: To investigate and analyze the characteristics of Meridian Sinew (Jingjin) syndrome in patients with whiplash-associated disorders (WAD). METHODS: From August 2010 to September 2011, 313 WAD cases from New York and California states were collected. The survey mostly collects the information of "Sinew Knotted Points" and symptoms of four types of Meridian Sinew differentiation-Taiyang, Shaoyin, Shaoyang and Yangming. RESULTS: Among the cases which are on the average of medium injury level, the higher frequency of "Sinew Knotted Points" tenderness were found on Jianwaishu (SI 14), Jianzhongshu (SI 15), Tianchuang (SI 16), C3-6 Spinous Process, Dazhui (GV 14), Fengchi (GB 20), Tianliao (SJ 15) and Tianding (LI 17). The most commonly presented symptoms were widespread spasm and tenderness in the neck (Taiyang), difficulty in lateral flexion (Shaoyang), problems of extension and flexion (Taiyang), and stiffness and pain during neck movement (Yangming). Among the cases, 237 cases (75.72%) were related to Taiyang Meridian Sinew syndrome, 82 cases (26.20%) to Shaoyin syndrome and 175 (55.91%) and 176 (56.23%) cases to Shaoyang and Yangming syndrome respectively. The most of cases presented in a combination format. The syndrome distribution under Grade I, II and III reflected that more combination of the Meridian Sinew syndromes in the whiplash injury patients which is resulted from more severity of injury. CONCLUSION: It is practical to identify the location of abnormality through Meridian Sinew differentiation, considering both "Sinew Knotted Points" tenderness and corresponding symptoms, for the local neck symptoms of WAD.

Mitogen-inducible gene-6 is a multifunctional adaptor protein with tumor suppressor-like activity in papillary thyroid cancer.

CONTEXT: Low tumoral expression of mitogen-inducible gene-6 (Mig-6) is associated with papillary thyroid cancer (PTC) recurrence after thyroidectomy. OBJECTIVE: We hypothesize that Mig-6 behaves as a tumor suppressor in PTC. DESIGN: Mig-6 expression and promoter methylation status were compared in 31 PTC specimens with matched normal thyroid tissue from the same patient. The impact of Mig-6 loss and gain of function on nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation, global tyrosine kinase phosphorylation, and cellular invasion was determined in vitro. RESULTS: Mig-6 protein was abundant in all normal thyroid specimens, whereas 77% of PTC had low Mig-6 expression. Mig-6 promoter methylation was found in 79% of PTC with low Mig-6 expression. Low Mig-6 expression in PTC specimens was associated with low NF-κB activity but high levels of epidermal growth factor receptor (EGFR) and ERK phosphorylation. Mig-6 expression inversely correlated with PTC size but had no association with other clinicopathological variables including age, extrathyroidal extension, lymphovascular invasion, or histological subtype. Mig-6 knockdown in thyroid cancer cell lines resulted in EGFR phosphorylation and diminished NF-κB activity, whereas Mig-6 overexpression had the opposite effects. Mig-6 knockdown activated ErbB2, Met, and Src phosphorylation. Furthermore, Mig-6 regulated ERK phosphorylation independent from its effects on EGFR. Mig-6 knockdown promoted cellular proliferation, as determined by clonogenic survival. Lastly, Mig-6 knockdown increased matrix metalloproteinase-2 and -9 activities and increased cellular invasion. CONCLUSIONS: Mig-6 has tumor suppressor-like activity in PTC. In vivo studies are required to confirm that Mig-6 is a putative tumor suppressor in PTC, and future studies should investigate the utility of Mig-6 as a diagnostic marker.

Amplification of CRKL induces transformation and epidermal growth factor receptor inhibitor resistance in human non-small cell lung cancers.

UNLABELLED: We previously identified a region of recurrent amplification on chromosome 22q11.21 in a subset of primary lung adenocarcinomas. Here we show that CRKL, encoding for an adaptor protein, is amplified and overexpressed in non-small cell lung cancer (NSCLC) cells that harbor 22q11.21 amplifications. Overexpression of CRKL in immortalized human airway epithelial cells promoted anchorage-independent growth and tumorigenicity. Oncogenic CRKL activates the SOS1-RAS-RAF-ERK and SRC-C3G-RAP1 pathways. Suppression of CRKL in NSCLC cells that harbor CRKL amplifications induced cell death. Overexpression of CRKL in epidermal growth factor receptor (EGFR)-mutant cells induces resistance to gefitinib by activating extracellular signal-regulated kinase and AKT signaling. We identified CRKL amplification in an EGFR inhibitor-treated lung adenocarcinoma that was not present before treatment. These observations demonstrate that CRKL overexpression induces cell transformation, credential CRKL as a therapeutic target for a subset of NSCLC that harbor CRKL amplifications, and implicate CRKL as an additional mechanism of resistance to EGFR-directed therapy. SIGNIFICANCE: These studies credential CRKL as an oncogene in a subset of NSCLC. Overexpression of CRKL induces cell transformation and resistance to epidermal growth factor receptor inhibitor treatment and suggest that therapeutic interventions targeting CRKL may confer a clinical benefit in a defined subset of NSCLCs.

Autophagy induction with RAD001 enhances chemosensitivity and radiosensitivity through Met inhibition in papillary thyroid cancer.

Although autophagy is generally considered a prosurvival mechanism that preserves viability, there is evidence that it could drive an alternative programmed cell death pathway in cells with defects in apoptosis. Because the inhibition of autophagic activity promotes resistance to both chemotherapy and external beam radiation in papillary thyroid cancer (PTC), we determined if RAD001, a potent activator of autophagy, improves the efficacy of either therapy. We found that RAD001 increased the expression level of light chain 3-II, a marker for autophagy, as well as autophagosome formation in cell lines and in human PTC ex vivo. RAD001 sensitized PTC to doxorubicin and external beam radiation in a synergistic fashion, suggesting that combination therapy could improve therapeutic response at less toxic concentrations. The effects of RAD001 were abrogated by RNAi knockdown of the autophagy-related gene 5, suggesting that RAD001 acts, in part, by enhancing autophagy. Because the synergistic activity of RAD001 with doxorubicin and external radiation suggests distinct and complementary mechanisms of action, we characterized how autophagy modulates signaling pathways in PTC. To do so, we performed kinome profiling and discovered that autophagic activation resulted in Src phosphorylation and Met dephosphorylation. Src inhibition did not reverse the effects of RAD001, whereas Met inhibition reversed the effects of autophagy blockade on chemosensitivity. These results suggest that the anticancer effects of autophagic activation are mediated largely through Met. We conclude that RAD001 induces autophagy, which enhances the therapeutic response to cytotoxic chemotherapy and external beam radiation in PTC.