3D bioprinting optimization of human mesenchymal stromal cell laden gelatin-alginate-collagen bioink.

3D bioprinting technology has gained increased attention in the regenerative medicine and tissue engineering communities over the past decade with their attempts to create functional living tissues and organsde novo. While tissues such as skin, bone, and cartilage have been successfully fabricated using 3D bioprinting, there are still many technical and process driven challenges that must be overcome before a complete tissue engineered solution is realized. Although there may never be a single adopted bioprinting process in the scientific community, adherence to optimized bioprinting protocols could reduce variability and improve precision with the goal of ensuring high quality printed constructs. Here, we report on the bioprinting of a gelatin-alginate-collagen bioink containing human mesenchymal stromal cells (hMSCs) which has been optimized to ensure printing consistency and reliability. The study consists of three phases: a pre-printing phase which focuses on bioink characterization; a printing phase which focuses on bioink extrudability/printability, construct stability, and printing accuracy; and a post-processing phase which focuses on the homogeneity and bioactivity of the encapsulated hMSC printed constructs. The results showed that eight identical constructs containing hMSCs could be reliably and accurately printed into stable cross-hatched structures with a single material preparation, and that batch-to-batch consistency was accurately maintained across all preparations. Analysis of the proliferation, morphology, and differentiation of encapsulated hMSCs within the printed constructs showed that cells were able to form large,interconnected colonies and were capable of robust adipogenic differentiation within 14 d of culturing.

Nutrition Intervention for Reduction of Cardiovascular Risk in African Americans Using the 2019 American College of Cardiology/American Heart Association Primary Prevention Guidelines.

INTRODUCTION: The 2019 American College of Cardiology/American Heart Association (ACC/AHA) Prevention Guidelines emphasize reduction in dietary sodium, cholesterol, refined carbohydrates, saturated fat and sweetened beverages. We hypothesized that implementing this dietary pattern could reduce cardiovascular risk in a cohort of volunteers in an urban African American (AA) community church, during a 5-week ACC/AHA-styled nutrition intervention, assessed by measuring risk markers and adherence, called HEART-LENS (Helping Everyone Assess Risk Today Lenten Nutrition Study). METHODS: The study population consisted of 53 volunteers who committed to eat only home-delivered non-dairy vegetarian meals (average daily calories 1155, sodium 1285 mg, cholesterol 0 mg; 58% carbohydrate, 17% protein, 25% fat). Body mass index (BMI) and fasting serum markers of cardiometabolic and risk factors were measured, with collection of any dietary deviation. RESULTS: Of 53 volunteers, 44 (mean age 60.2 years, 37 women) completed the trial (88%); 1 was intolerant of the meals, 1 completed both blood draws but did not eat delivered food, and 7 did not return for the tests. Adherence to the diet was reported at 93% in the remaining 44. Cardiometabolic risk factors improved significantly, highlighted by a marked reduction in serum insulin (-43%, p = 0.000), hemoglobin A1c (6.2% to 6.0%, p = 0.000), weight and BMI (-10.2 lbs, 33 to 31 kg/m2, p = 0.000), but with small reductions of fasting glucose (-6%, p = 0.405) and triglyceride levels (-4%, p = 0.408). Additionally, improved were trimethylamine-N-oxide (5.1 to 2.9 µmol/L, -43%, p = 0.001), small dense low-density lipoprotein cholesterol (LDL) (24.2 to 19.1 mg/dL, -21%, p = 0.000), LDL (121 to 104 mg/dL, -14%, p = 0.000), total cholesterol (TC) (190 to 168 mg/dL, -12%, p = 0.000), and lipoprotein (a) (LP(a)) (56 to 51 mg/dL, -11%, p = 0.000); high sensitivity C-reactive protein (hs-CRP) was widely variable but reduced by 16% (2.5 to 2.1 ng/mL, p = NS) in 40 subjects without inflammatory conditions. Soluble urokinase plasminogen activator (suPAR) levels were not significantly changed. The ACC/AHA pooled cohort atherosclerotic cardiovascular disease (ASCVD) risk scores were calculated for 41 and 36 volunteers, respectively, as the ASCVD risk could not be calculated for 3 subjects with low lipid fractions at baseline and 8 subjects after intervention (p = 0.184). In the remaining subjects, the mean 10-year risk was reduced from 10.8 to 8.7%, a 19.4% decrease (p = 0.006), primarily due to a 14% decrease in low-density lipoprotein cholesterol and a 10 mm Hg (6%) reduction in systolic blood pressure. CONCLUSIONS: In this prospective 5-week non-dairy vegetarian nutrition intervention with good adherence consistent with the 2019 ACC/AHA Guidelines in an at-risk AA population, markers of cardiovascular risk, cardiometabolism, and body weight were significantly reduced, including obesity, low-density lipoprotein cholesterol (LDLc) density, LP(a), inflammation, and ingestion of substrates mediating production of trimethylamine-N-oxide (TMAO). Albeit reduced, hs-CRP and suPAR, were not lowered consistently. This induced a significant decrease in the 10-year ASCVD risk in this AA cohort. If widely adopted, this could dramatically reduce and possibly eradicate, the racial disparity in ASCVD events and mortality, if 19% of the 21% increase is eliminated by this lifestyle change.

Perfusion-based co-culture model system for bone tissue engineering.

In this work, we report on a perfusion-based co-culture system that could be used for bone tissue engineering applications. The model system is created using a combination of Primary Human Umbilical Vein Endothelial Cells (HUVECs) and osteoblast-like Saos-2 cells encapsulated within a Gelatin Methacrylate (GelMA)-collagen hydrogel blend contained within 3D printed, perfusable constructs. The constructs contain dual channels, within a custom-built bioreactor, that were perfused with osteogenic media for up to two weeks in order to induce mineral deposition. Mineral deposition in constructs containing only HUVECs, only Saos-2 cells, or a combination thereof was quantified by microCT to determine if the combination of endothelial cells and bone-like cells increased mineral deposition. Histological and fluorescent staining was used to verify mineral deposition and cellular function both along and between the perfused channels. While there was not a quantifiable difference in the amount of mineral deposited in Saos-2 only versus Saos-2 plus HUVEC samples, the location of the deposited mineral differed dramatically between the groups and indicated that the addition of HUVECs within the GelMA matrix allowed Saos-2 cells, in diffusion limited regions of the construct, to deposit bone mineral. This work serves as a model on how to create perfusable bone tissue engineering constructs using a combination of 3D printing and cellular co-cultures.

Measuring Changes in Electrical Impedance During Cell-Mediated Mineralization.

Background: The fundamental electrical properties of bone have been attributed to the organic collagen and the inorganic mineral component; however, contributions of individual components within bone tissue toward the measured electrical properties are not known. In our study, we investigated the electrical properties of cell-mediated mineral deposition process and compared our results with cell-free mineralization. Materials and Methods: Saos-2 cells encapsulated within gelatin methacrylate (GelMA) hydrogels were chemically stimulated in osteogenic medium for a period of 4 weeks. The morphology, composition, and mechanical properties of the mineralized constructs were characterized using bright-field imaging, scanning electron microscopy (SEM) energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy (FITR), nuclear magnetic resonance spectroscopy (NMR), micro-CT, immunostaining, and mechanical compression tests. In parallel, a custom-made device was used to measure the electrical impedance of mineralized constructs. All results were compared with cell-free GelMA hydrogels mineralized through the simulated body fluid approach. Results: Results demonstrate a decrease in the electrical impedance of deposited mineral in both cell-mineralized and cell-free mineralized samples. Conclusions: This study establishes a model system to investigate in vivo and in vitro mineralization processes.

Perfusion directed 3D mineral formation within cell-laden hydrogels.

Despite the promise of stem cell engineering and the new advances in bioprinting technologies, one of the major challenges in the manufacturing of large scale bone tissue scaffolds is the inability to perfuse nutrients throughout thick constructs. Here, we report a scalable method to create thick, perfusable bone constructs using a combination of cell-laden hydrogels and a 3D printed sacrificial polymer. Osteoblast-like Saos-2 cells were encapsulated within a gelatin methacrylate (GelMA) hydrogel and 3D printed polyvinyl alcohol pipes were used to create perfusable channels. A custom-built bioreactor was used to perfuse osteogenic media directly through the channels in order to induce mineral deposition which was subsequently quantified via micro-CT. Histological staining was used to verify mineral deposition around the perfused channels, while COMSOL modeling was used to simulate oxygen diffusion between adjacent channels. This information was used to design a scaled-up construct containing a 3D array of perfusable channels within cell-laden GelMA. Progressive matrix mineralization was observed by cells surrounding perfused channels as opposed to random mineral deposition in static constructs. Micro-CT confirmed that there was a direct relationship between channel mineralization within perfused constructs and time within the bioreactor. Furthermore, the scalable method presented in this work serves as a model on how large-scale bone tissue replacement constructs could be made using commonly available 3D printers, sacrificial materials, and hydrogels.

Fabrication of conductive gelatin methacrylate-polyaniline hydrogels.

Hydrogels with inherently conductive properties have been recently developed for tissue engineering applications, to serve as bioactive scaffolds to electrically stimulate cells and modulate their function. In this work, we have used interfacial polymerization of aniline monomers within gelatin methacrylate (GelMA) to develop a conductive hybrid composite. We demonstrate that as compared to pure GelMA, GelMA-polyaniline (GelMA-Pani) composite has similar swelling properties and compressive modulus, comparable cell adhesion and spreading responses, and superior electrical properties. Additionally, we demonstrate that GelMA-Pani composite can be printed in complex user-defined geometries using digital projection stereolithography, and will be useful in developing next-generation bioelectrical interfaces. STATEMENT OF SIGNIFICANCE: We report the fabrication of a conductive hydrogel using naturally-derived gelatin methyacrylate (GelMA) and inherently conductive polyaniline (Pani). This work is significant, as GelMA-Pani composite has superior electrical properties as compared to pure Gelma, all the while maintaining biomimetic physical and biocompatible properties. Moreover, the ability to fabricate conductive-GelMA in complex user-defined micro-geometries, address the significant processing challenges associated with all inherently conductive polymers including Pani. The methodology described in this work can be extended to several conductive polymers and hydrogels, to develop new biocompatible electrically active interfaces.

Krüppel-like transcription factors KLF1 and KLF2 have unique and coordinate roles in regulating embryonic erythroid precursor maturation.

The Krüppel-like transcription factors KLF1 and KLF2 are essential for embryonic erythropoiesis. They can partially compensate for each other during mouse development, and coordinately regulate numerous erythroid genes, including the ß-like globins. Simultaneous ablation of KLF1 and KLF2 results in earlier embryonic lethality and severe anemia. In this study, we determine that this anemia is caused by a paucity of blood cells, and exacerbated by diminished ß-like globin gene expression. The anemia phenotype is dose-dependent, and, interestingly, can be ameliorated by a single copy of the KLF2, but not the KLF1 gene. The roles of KLF1 and KLF2 in maintaining normal peripheral blood cell numbers and globin mRNA amounts are erythroid cell-specific. Mechanistic studies led to the discovery that KLF2 has an essential function in erythroid precursor maintenance. KLF1 can partially compensate for KLF2 in this role, but is uniquely crucial for erythroid precursor proliferation through its regulation of G1- to S-phase cell cycle transition. A more drastic impairment of primitive erythroid colony formation from embryonic progenitor cells occurs with simultaneous loss of KLF1 and KLF2 than with loss of a single factor. KLF1 and KLF2 coordinately regulate several proliferation-associated genes, including Foxm1. Differential expression of FoxM1, in particular, correlates with the observed KLF1 and KLF2 gene dosage effects on anemia. Furthermore, KLF1 binds to the FoxM1 gene promoter in blood cells. Thus KLF1 and KLF2 coordinately regulate embryonic erythroid precursor maturation through the regulation of multiple homeostasis-associated genes, and KLF2 has a novel and essential role in this process.

Erythropoietin-induced phosphorylation/degradation of BIM contributes to survival of erythroid cells.

OBJECTIVE: A proapoptotic BH3-only protein BIM (BCL-2 interacting mediator of cell death) can link cytokine receptor signaling with the apoptotic machinery in hematopoietic cells. We investigated here the role of BIM in erythropoietin (EPO)-mediated survival in erythroid cells. MATERIALS AND METHODS: We downregulated BIM in EPO-dependent HCD57 erythroid cells with short hairpin RNA (shRNA), and used real-time polymerase chain reaction, Western blots, and flow cytometry to characterize BIM expression and apoptosis. Hematologic analyses of BIM-deficient (Bim(-/-)) mice were conducted. RESULTS: BIM expression increases in primary murine erythroid cells and HCD57 cells deprived of EPO. Whereas Bim mRNA increased less than twofold, BIM protein increased more than 10-fold after EPO withdrawal, suggesting posttranscriptional regulation of BIM. EPO treatment resulted in rapid phosphorylation of BIM at Serine 65 and phosphorylation correlated with degradation of BIM. Inhibition of extracellular signal-regulated kinase (ERK) by a MEK/ERK inhibitor, U0126, blocked both phosphorylation and degradation of BIM, resulting in apoptosis. Treatment with a proteasome inhibitor, MG-132, also blocked degradation of phosphorylated BIM. Downregulation of BIM with the shRNA resulted in HCD57 cells more resistant to apoptosis induced by either EPO withdrawal or ERK inhibition. Although we observed no significant changes in the number of erythrocytes or reticulocytes in the circulation of Bim(-/-) mice, erythroid progenitors from bone marrow in Bim(-/-) mice were reduced in number and more resistant to apoptosis induced by U0126 MEK/ERK inhibitor. CONCLUSION: EPO protects erythroid cells from apoptosis in part through ERK-mediated phosphorylation followed by proteasomal degradation of BIM.

Erythropoietin fails to interfere with the antiproliferative and cytotoxic effects of antitumor drugs.

PURPOSE: Erythropoietin (EPO) therapy is widely used for the prevention and treatment of anemia resulting from cancer chemotherapy. Native EPO regulates erythropoiesis, at least in part, by protecting erythroid progenitor cells from apoptotic cell death. The recent discovery of the EPO receptor (EPOR) on cancer cells raises the concern that EPO therapy might stimulate tumor growth and/or protect cancer cells from drug-induced apoptosis. Therefore, the capacity of EPO to interfere with the effects of conventional chemotherapeutic drugs on proliferation, apoptosis, and the induction of senescence was investigated in MCF-7 and MDA-MB231 breast tumor cells, which express the EPOR as well as in F-MEL erythroleukemia cells. EXPERIMENTAL DESIGN: Breast cancer cells and F-MEL leukemic cells were cultured in the presence or absence of EPO and then exposed to antitumor drugs. Cell proliferation was assessed by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction assay 72 hours after drug exposure. Cytotoxicity was monitored by clonogenic survival. Apoptosis was evaluated either by the terminal deoxyribonucleotide transferase-mediated nick-end labeling assay or fluorescence-activated cell sorting analysis, and senescence was monitored by beta-galactosidase staining. EPO signaling was assessed by monitoring the phosphorylation/activation of specific signaling proteins. RESULTS: EPO failed to stimulate the proliferation of MCF-7 or MDA-MB231 breast tumor cells or F-MEL leukemic cells. EPO treatment also failed to interfere with the antiproliferative and/or cytotoxic effects of Adriamycin, Taxol, and tamoxifen in breast tumor cells (or of cytarabine and daunorubicin in F-MEL cells). EPO failed to prevent apoptosis induced by Taxol or senescence induced by Adriamycin in MCF-7 cells. EPO stimulated the activation of extracellular signal-regulated kinase, p38, and c-Jun-NH(2)-kinase in MCF-7 cells but did not activate Akt or signal transducers and activators of transcription 5 (STAT5). EPO failed to activate any of these signaling pathways in MDA-MB231 cells. Cytarabine and daunorubicin interfered with EPO signaling in F-MEL cells. CONCLUSIONS: These findings suggest that EPO is unlikely to directly counteract the effectiveness of cancer chemotherapeutic drugs. This may be a consequence of either ineffective signaling through the EPOR or drug-mediated suppression of EPO signaling.

Erythropoietin-dependent autocrine secretion of tumor necrosis factor-alpha in hematopoietic cells modulates proliferation via MAP kinase--ERK-1/2 and does not require tyrosine docking sites in the EPO receptor.

Primary erythroid cells and erythroid cell lines may synthesize and secrete tumor necrosis factor-alpha (TNF-alpha) following stimulation with erythropoietin (EPO). The effect of triggering TNF-alpha synthesis and secretion was investigated in erythroleukemia and myeloid cell lines: HCD57, DA3-EPOR, and BAF3-EPOR. The EPO-induced, membrane-bound form of autocrine TNF-alpha seemed to enhance proliferation of HCD57 and DA3-EPOR cells; however, the concentration of secreted autocrine/paracrine TNF-alpha was never sufficient to have an effect. Autocrine TNF-alpha acts through TNFRII receptors to stimulate proliferation. Modulation of mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK-1/2) activity by the membrane-bound form of autocrine TNF-alpha apparently played a central role in the control of EPO-dependent proliferation of HCD57 and DA3-EPOR cells. Primary erythroid cells and DA3-EPOR cells were found to express similar, high levels of both TNFRI and TNFRII, showing that differential expression of TNF-alpha receptors does not explain why primary cells are inhibited and DA3-EPOR cells are stimulated by autocrine TNF-alpha. BAF3 cells expressing a mutant EPOR with no cytoplasmic tyrosine residues were capable of triggering EPO-dependent TNF-alpha synthesis and secretion, indicating that tyrosine-docking sites in the EPOR were not required for EPO-dependent TNF-alpha secretion.

Jun N-terminal kinase promotes proliferation of immature erythroid cells and erythropoietin-dependent cell lines.

Erythropoietin (EPO) is the hormone necessary for development of erythrocytes from immature erythroid cells. EPO activates Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family in the EPO-dependent murine erythroid HCD57 cells. Therefore, we tested if JNK activity supported proliferation and/or survival of these cells. Treatment with the JNK inhibitor SP600125 inhibited JNK activity and EPO-dependent proliferation of HCD57 cells and the human EPO-dependent cell lines TF-1 and UT7-EPO. SP600125 also increased the fraction of cells in G2/M. Introduction of a dominant-negative form of JNK1 inhibited EPO-dependent proliferation in HCD57 cells but did not increase the fraction of cells in G2/M. Constitutive JNK activity was observed in primary murine erythroid progenitors. Treatment of primary mouse bone marrow cells with the SP600125 inhibitor reduced the number of erythroid burst-forming units (BFU-e's) but not the more differentiated erythroid colony-forming units (CFU-e's), and SP600125 protected the BFU-e's from apoptosis induced by cytosine arabinoside, demonstrating that the SP600125 inhibited proliferation of the BFU-e's. Therefore, JNK activity appears to be an important regulator of proliferation in immature, primary erythroid cells and 3 erythroid cell lines but may not be required for the survival or proliferation of CFU-e's or proerythroblasts.

Targeted molecular mechanisms of epoetin alfa.

Despite therapeutic improvements and ongoing efforts to develop more efficacious therapies, the majority of lung cancer patients face a poor prognosis. Therefore, the primary goal of current treatment is palliation, improvement and maintenance of quality of life (QOL), and (modest) prolongation of survival. Anemia frequently occurs in lung cancer patients and has been associated with decreased QOL, impaired treatment outcomes, and shortened survival time. Furthermore, anemia is a causative factor of tumor hypoxia, which compromises the efficacy of chemotherapy and radiotherapy. Thus, correction of even mild anemia seems to have a beneficial effect on QOL and cancer treatment outcomes. The current article describes the basis and mechanism for the use of recombinant human erythropoietin (rHuEPO, epoetin alfa), a molecular targeted therapy, for the treatment of cancer-related anemia, with a focus on lung cancer. Epoetin alfa has proven efficacy and safety in correcting anemia and improving QOL based on numerous clinical studies and over a decade of clinical practice. In addition, emerging data show that epoetin alfa may offer potential benefits beyond treating anemia, specifically in terms of treatment outcomes and cognitive function. Future research needs to be conducted to explore the potential for epoetin alfa to improve survival time in lung cancer patients.

Tumor necrosis factor-alpha expressed constitutively in erythroid cells or induced by erythropoietin has negative and stimulatory roles in normal erythropoiesis and erythroleukemia.

Binding of erythropoietin (EPO) to its receptor (EPOR) on erythroid cells induces the activation of numerous signal transduction pathways, including the mitogen-activated protein kinase Jun-N-terminal kinase (JNK). In an effort to understand the regulation of EPO-induced proliferation and JNK activation, we have examined the role of potential autocrine factors in the proliferation of the murine erythroleukemia cell line HCD57. We report here that treatment of these cells with EPO induced the expression and secretion of tumor necrosis factor alpha (TNF-alpha). EPO-dependent proliferation was reduced by the addition of neutralizing antibodies to TNF-alpha, and exogenously added TNF-alpha induced proliferation of HCD57 cells. EPO also could induce TNF-alpha expression in BAF3 and DA3 myeloid cells ectopically expressing EPOR. Addition of TNF-alpha activated JNK in HCD57 cells, and the activity of JNK was partially inhibited by addition of a TNF-alpha neutralizing antibody. Primary human and murine erythroid progenitors expressed TNF-alpha in either an EPO-dependent or constitutive manner. However, TNF-alpha had an inhibitory effect on both immature primary human and murine cells, suggestive that the proliferative effects of TNF-alpha may be limited to erythroleukemic cells. This study suggests a novel role for autocrine TNF-alpha expression in the proliferation of erythroleukemia cells that is distinct from the effect of TNF-alpha in normal erythropoiesis.

New insights into erythropoiesis.

Commitment of hematopoietic cells to the erythroid lineage involves the actions of several transcription factors, including TAL1, LMO2, and GATA-2. The differentiation of committed erythroid progenitor cells involves other transcription factors, including NF-E2 and EKLF. Upon binding erythropoietin, the principal regulator of erythropoiesis, cell surface erythropoietin receptors dimerize and activate specific intracellular kinases, including Janus family tyrosine protein kinase 2, phosphoinositol-3 kinase, and mitogen-activated protein kinase. Important substrates of these kinases are tyrosines in the erythropoietin receptors themselves and the signal transducer and transcription activator proteins. Erythropoietin prevents erythroid cell apoptosis. Some of the apoptotic tendency of erythroid cells can be attributed to proapoptotic molecules produced by hematopoietic cells, macrophages, and stromal cells. Cell divisions accompanying terminal erythroid differentiation are finely controlled by cell cycle regulators, and disruption of these terminal divisions causes erythroid cell apoptosis. In reticulocyte maturation, regulated degradation of internal organelles involves a lipoxygenase, whereas survival requires the antiapoptotic protein Bcl-x.

Role of JunB in erythroid differentiation.

The role of junB as a regulator of erythroid cell survival, proliferation, and differentiation was tested by controlled expression of JunB in the erythropoietin (EPO)-dependent erythroleukemia cell line HCD57. JunB induced erythroid differentiation as evidenced by increased expression of the erythroid-specific proteins beta-globin, spectrin-alpha, and TER-119. Expression of JunB for at least 48 h was required for the differentiated phenotype to emerge. Differentiation was accompanied by a slower rate of proliferation and an increase in the expression of the cell cycle inhibitory protein p27. p27 protein expression increased due to reduced turnover without changes in transcription, indicating global changes in cell physiology following JunB induction. JunB expression was also studied in mouse and human primary erythroid cells. JunB expression increased immediately in both primary mouse cells and HCD57 cells treated with EPO and quickly returned to base-line levels, followed by a secondary rise in JunB in primary erythroid cells, but not in HCD57 cells, 36-48 h later. This result suggested that the initial EPO-dependent JunB induction was not sufficient to induce differentiation, but that the late EPO-independent JunB expression in primary erythroid cells was necessary for differentiation. This study suggests that JunB is an important regulator of erythroid differentiation.