Development of a multigenomic liquid biopsy (PROSTest) for prostate cancer in whole blood.

INTRODUCTION: We describe the development of a molecular assay from publicly available tumor tissue mRNA databases using machine learning and present preliminary evidence of functionality as a diagnostic and monitoring tool for prostate cancer (PCa) in whole blood. MATERIALS AND METHODS: We assessed 1055 PCas (public microarray data sets) to identify putative mRNA biomarkers. Specificity was confirmed against 32 different solid and hematological cancers from The Cancer Genome Atlas (n = 10,990). This defined a 27-gene panel which was validated by qPCR in 50 histologically confirmed PCa surgical specimens and matched blood. An ensemble classifier (Random Forest, Support Vector Machines, XGBoost) was trained in age-matched PCas (n = 294), and in 72 controls and 64 BPH. Classifier performance was validated in two independent sets (n = 263 PCas; n = 99 controls). We assessed the panel as a postoperative disease monitor in a radical prostatectomy cohort (RPC: n = 47). RESULTS: A PCa-specific 27-gene panel was identified. Matched blood and tumor gene expression levels were concordant (r = 0.72, p < 0.0001). The ensemble classifier ("PROSTest") was scaled 0%-100% and the industry-standard operating point of ≥50% used to define a PCa. Using this, the PROSTest exhibited an 85% sensitivity and 95% specificity for PCa versus controls. In two independent sets, the metrics were 92%-95% sensitivity and 100% specificity. In the RPCs (n = 47), PROSTest scores decreased from 72% ± 7% to 33% ± 16% (p < 0.0001, Mann-Whitney test). PROSTest was 26% ± 8% in 37 with normal postoperative PSA levels (<0.1 ng/mL). In 10 with elevated postoperative PSA, PROSTest was 60% ± 4%. CONCLUSION: A 27-gene whole blood signature for PCa is concordant with tissue mRNA levels. Measuring blood expression provides a minimally invasive genomic tool that may facilitate prostate cancer management.

Vascular persistence following precision micropuncture.

OBJECTIVE: The success of engineered tissues continues to be limited by time to vascularization and perfusion. Recently, we described a simple microsurgical approach, termed micropuncture (MP), which could be used to rapidly vascularize an adjacently placed scaffold from the recipient macrovasculature. Here we studied the long-term persistence of the MP-induced microvasculature. METHODS: Segmental 60 µm diameter MPs were created in the recipient rat femoral artery and vein followed by coverage with a simple Type 1 collagen scaffold. The recipient vasculature and scaffold were then wrapped en bloc with a silicone sheet to isolate intrinsic vascularization. Scaffolds were harvested at 28 days post-implantation for detailed analysis, including using a novel artificial intelligence (AI) approach. RESULTS: MP scaffolds demonstrated a sustained increase of vascular density compared to internal non-MP control scaffolds (p < 0.05) secondary to increases in both vessel diameters (p < 0.05) and branch counts (p < 0.05). MP scaffolds also demonstrated statistically significant increases in red blood cell (RBC) perfused lumens. CONCLUSIONS: This study further highlights that the intrinsic MP-induced vasculature continues to persist long-term. Its combination of rapid and stable angiogenesis represents a novel surgical platform for engineered scaffold and graft perfusion.

Accelerating Patterned Vascularization Using Granular Hydrogel Scaffolds and Surgical Micropuncture.

Bulk hydrogel scaffolds are common in reconstructive surgery. They allow for the staged repair of soft tissue loss by providing a base for revascularization. Unfortunately, they are limited by both slow and random vascularization, which may manifest as treatment failure or suboptimal repair. Rapidly inducing patterned vascularization within biomaterials has profound translational implications for current clinical treatment paradigms and the scaleup of regenerative engineering platforms. To address this long-standing challenge, a novel microsurgical approach and granular hydrogel scaffold (GHS) technology are co-developed to hasten and pattern microvascular network formation. In surgical micropuncture (MP), targeted recipient blood vessels are perforated using a microneedle to accelerate cell extravasation and angiogenic outgrowth. By combining MP with an adjacent GHS with precisely tailored void space architecture, microvascular pattern formation as assessed by density, diameter, length, and intercapillary distance is rapidly guided. This work opens new translational opportunities for microvascular engineering, advancing reconstructive surgery, and regenerative medicine.

Identification of a Metabolic, Transcriptomic, and Molecular Signature of Patatin-Like Phospholipase Domain Containing 3-Mediated Acceleration of Steatohepatitis.

BACKGROUND AND AIMS: The mechanisms by which the I148M mutant variant of the patatin-like phospholipase domain-containing 3 (PNPLA3I148M ) drives development of nonalcoholic steatohepatitis (NASH) are not known. The aim of this study was to obtain insights on mechanisms underlying PNPLA3I148M -induced acceleration of NASH. APPROACH AND RESULTS: Hepatocyte-specific overexpression of empty vector (luciferase), human wild-type PNPLA3, or PNPLA3I148M was achieved using adeno-associated virus 8 in a diet-induced mouse model of nonalcoholic fatty liver disease followed by chow diet or high-fat Western diet with ad libitum administration of sugar in drinking water (WDSW) for 8 weeks. Under WDSW, PNPLA3I148M overexpression accelerated steatohepatitis with increased steatosis, inflammation ballooning, and fibrosis (P < 0.001 versus other groups for all). Silencing PNPLA3I148M after its initial overexpression abrogated these findings. PNPLA3I148M caused 22:6n3 docosahexanoic acid depletion and increased ceramides under WDSW in addition to increasing triglycerides and diglycerides, especially enriched with unsaturated fatty acids. It also increased oxidative stress and endoplasmic reticulum stress. Increased total ceramides was associated with signature of transducer and activator of transcription 3 (STAT3) activation with downstream activation of multiple immune-inflammatory pathways at a transcriptomic level by network analyses. Silencing PNPLA3I148M reversed STAT3 activation. Conditioned media from HepG2 cells overexpressing PNPLA3I148M increased procollagen mRNA expression in LX2 cells; this was abrogated by hepatocyte STAT3 inhibition. CONCLUSIONS: Under WDSW, PNPLA3I148M overexpression promotes steatosis and NASH by metabolic reprogramming characterized by increased triglycerides and diglycerides, n3 polyunsaturated fatty acid depletion, and increased ceramides with resultant STAT3 phosphorylation and downstream inflammatory pathway activation driving increased stellate cell fibrogenic activity.

Induction of scaffold angiogenesis by recipient vasculature precision micropuncture.

The success of engineered tissues continues to be limited by time to vascularization and perfusion. Here, we studied the effects of precision injury to a recipient macrovasculature in promoting neovessel formation in an adjacently placed scaffold. Segmental 60 µm diameter micropunctures (MP) were created in the recipient rat femoral artery and vein followed by coverage with a simple collagen scaffold. Scaffolds were harvested at 24, 48, 72, and 96 h post-implantation for detailed analysis. Those placed on top of an MP segment showed an earlier and more robust cellular infiltration, including both endothelial cells (CD31) and macrophages (F4/80), compared to internal non-micropunctured control limbs (p < 0.05). At the 96-hour timepoint, MP scaffolds demonstrated an increase in physiologic perfusion (p < 0.003) and a 2.5-fold increase in capillary network formation (p < 0.001). These were attributed to an overall upsurge in small vessel quantity. Furthermore, MP positioned scaffolds demonstrated significant increases in many modulators of angiogenesis, including VEGFR2 and Tie-2 despite a decrease in HIF-1α at all timepoints. This study highlights a novel microsurgical approach that can be used to rapidly vascularize or inosculate contiguously placed scaffolds and grafts. Thereby, offering an easily translatable route towards the creation of thicker and more clinically relevant engineered tissues.

The contribution of cross-talk between the cell-surface proteins CD36 and CD47-TSP-1 in osteoclast formation and function.

Antibody-mediated blockade of cluster of differentiation 47 (CD47)-thrombospondin-1 (TSP-1) interactions blocks osteoclast formation in vitro and attenuates parathyroid hormone (PTH)-induced hypercalcemia in vivo in mice. Hypercalcemia in this model reflects increased bone resorption. TSP-1 has two cell-associated binding partners, CD47 and CD36. The roles of these two molecules in mediating the effects of TSP1 in osteoclasts are unclear. Osteoclast formation was attenuated but not absent when preosteoclasts isolated from CD47-/- mice were cocultured with WT osteoblasts. Suppressing CD36 in osteoclast progenitors also attenuated osteoclast formation. The hypercalcemic response to a PTH infusion was blunted in CD47-/-/CD36-/- (double knockout (DKO)) female mice but not CD47-/- mice or CD36-/- animals, supporting a role for both CD47 and CD36 in mediating this effect. Consistent with this, DKO osteoclasts had impaired resorptive activity when analyzed in vitro Inhibition of nitric oxide (NO) signaling is known to promote osteoclastogenesis, and TSP-1 suppresses NO production and signaling. An anti-TSP-1 antibody increased NO production in osteoclasts, and the inhibitory effect of anti-TSP-1 on osteoclastogenesis was completely rescued by l-nitroarginine methyl ester (l-NAME), a competitive NO synthase inhibitor. Supportive of an important role for CD36 in mediating the pro-osteoclastogenic effects of TSP-1, engaging CD36 with a synthetic agonist, p907, suppressed NO production in anti-TSP-1-treated cultures, allowing osteoclast maturation to occur. These results establish that CD36 and CD47 both participate in mediating the actions of TSP-1 in osteoclasts and establish a physiologically relevant cross-talk in bone tissue between these two molecules.

Transplantation of Bioprinted Tissues and Organs: Technical and Clinical Challenges and Future Perspectives.

: Three-dimensional (3D) bioprinting is a revolutionary technology in building living tissues and organs with precise anatomic control and cellular composition. Despite the great progress in bioprinting research, there has yet to be any clinical translation due to current limitations in building human-scale constructs, which are vascularized and readily implantable. In this article, we review the current limitations and challenges in 3D bioprinting, including in situ techniques, which are one of several clinical translational models to facilitate the application of this technology from bench to bedside. A detailed discussion is made on the technical barriers in the fabrication of scalable constructs that are vascularized, autologous, functional, implantable, cost-effective, and ethically feasible. Clinical considerations for implantable bioprinted tissues are further expounded toward the correction of end-stage organ dysfunction and composite tissue deficits.

Clinical and pharmacodynamic analysis of pomalidomide dosing strategies in myeloma: impact of immune activation and cereblon targets.

In preclinical studies, pomalidomide mediated both direct antitumor effects and immune activation by binding cereblon. However, the impact of drug-induced immune activation and cereblon/ikaros in antitumor effects of pomalidomide in vivo is unknown. Here we evaluated the clinical and pharmacodynamic effects of continuous or intermittent dosing strategies of pomalidomide/dexamethasone in lenalidomide-refractory myeloma in a randomized trial. Intermittent dosing led to greater tumor reduction at the cost of more frequent adverse events. Both cohorts experienced similar event-free and overall survival. Both regimens led to a distinct pattern but similar degree of mid-cycle immune activation, manifested as increased expression of cytokines and lytic genes in T and natural killer (NK) cells. Pomalidomide induced poly-functional T-cell activation, with increased proportion of coinhibitory receptor BTLA(+) T cells and Tim-3(+) NK cells. Baseline levels of ikaros and aiolos protein in tumor cells did not correlate with response or survival. Pomalidomide led to rapid decline in Ikaros in T and NK cells in vivo, and therapy-induced activation of CD8(+) T cells correlated with clinical response. These data demonstrate that pomalidomide leads to strong and rapid immunomodulatory effects involving both innate and adaptive immunity, even in heavily pretreated multiple myeloma, which correlates with clinical antitumor effects. This trial was registered at www.clinicaltrials.gov as #NCT01319422.

Topical vanadate enhances the repair of median laparotomy incisions.

BACKGROUND: There are over two million laparotomies performed in the United States each year with an incisional hernia rate between 2% and 11%. A total of 100,000 ventral hernia repairs are undertaken each year with recurrences as high as 50%. MATERIALS AND METHODS: Full thickness midline fascia incisions from the xiphoid to the pubic symphysis were made in rats. The fascia and/or muscular layer was sutured closed and a gel with 300 µM of sodium orthovanadate or saline was placed over the suture line with the skin closed over it. On day 10, 1-cm strips from the superior, middle, and inferior regions of the abdominal wall were tested for breaking strength and processed for histology. RESULTS: The mean wound breaking strength of vanadate-treated wounds was 18.6 ± 2.7 N compared with 9.4 ± 3.6 N for controls (P < 0.0001). Similar quantities of granulation tissue were deposited in treated and control wounds. Fine green birefringence patterns, characteristic of immature connective tissue, were seen in control samples viewed with polarized light. In contrast, vanadate-treated wounds showed thick yellow-orange birefringence patterns characteristics of more mature connective tissue. Using α-smooth muscle actin immunostaining, myofibroblasts were prominent in control incisions, but few were identified in vanadate-treated incisions. CONCLUSIONS: In rat laparotomy wounds, a single application of vanadate increases wound breaking strength, through enhanced connective tissue organization. These combined data suggest topical application of vanadate immediately after fascial closure will increase wound strength, possibly reducing hernia recurrences in the repaired abdominal wall.

A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer.

BACKGROUND & AIMS: The lack of a preclinical model of progressive non-alcoholic steatohepatitis (NASH) that recapitulates human disease is a barrier to therapeutic development. METHODS: A stable isogenic cross between C57BL/6J (B6) and 129S1/SvImJ (S129) mice were fed a high fat diet with ad libitum consumption of glucose and fructose in physiologically relevant concentrations and compared to mice fed a chow diet and also to both parent strains. RESULTS: Following initiation of the obesogenic diet, B6/129 mice developed obesity, insulin resistance, hypertriglyceridemia and increased LDL-cholesterol. They sequentially also developed steatosis (4-8weeks), steatohepatitis (16-24weeks), progressive fibrosis (16weeks onwards) and spontaneous hepatocellular cancer (HCC). There was a strong concordance between the pattern of pathway activation at a transcriptomic level between humans and mice with similar histological phenotypes (FDR 0.02 for early and 0.08 for late time points). Lipogenic, inflammatory and apoptotic signaling pathways activated in human NASH were also activated in these mice. The HCC gene signature resembled the S1 and S2 human subclasses of HCC (FDR 0.01 for both). Only the B6/129 mouse but not the parent strains recapitulated all of these aspects of human NAFLD. CONCLUSIONS: We here describe a diet-induced animal model of non-alcoholic fatty liver disease (DIAMOND) that recapitulates the key physiological, metabolic, histologic, transcriptomic and cell-signaling changes seen in humans with progressive NASH. LAY SUMMARY: We have developed a diet-induced mouse model of non-alcoholic steatohepatitis (NASH) and hepatic cancers in a cross between two mouse strains (129S1/SvImJ and C57Bl/6J). This model mimics all the physiological, metabolic, histological, transcriptomic gene signature and clinical endpoints of human NASH and can facilitate preclinical development of therapeutic targets for NASH.

Dendritic cell-mediated activation-induced cytidine deaminase (AID)-dependent induction of genomic instability in human myeloma.

Tumor microenvironment (TME) is commonly implicated in regulating the growth of tumors, but whether it can directly alter the genetics of tumors is not known. Genomic instability and dendritic cell (DC) infiltration are common features of several cancers, including multiple myeloma (MM). Mechanisms underlying genomic instability in MM are largely unknown. Here, we show that interaction between myeloma and DCs, but not monocytes, leads to rapid induction of the genomic mutator activation-induced cytidine deaminase (AID) and AID-dependent DNA double-strand breaks (DSBs) in myeloma cell lines as well as primary MM cells. Both myeloid as well as plasmacytoid DCs have the capacity to induce AID in tumor cells. The induction of AID and DSBs in tumor cells by DCs requires DC-tumor contact and is inhibited by blockade of receptor activator of NF-κB/receptor activator of NF-κB ligand (RANKL) interactions. AID-mediated genomic damage led to altered tumorigenicity and indolent behavior of tumor cells in vivo. These data show a novel pathway for the capacity of DCs in the TME to regulate genomic integrity. DC-mediated induction of AID and resultant genomic damage may therefore serve as a double-edged sword and be targeted by approaches such as RANKL inhibition already in the clinic.

Posttranscriptional Modification to Modulate Progenitor Differentiation on Heterotypic Spheroids.

Cell aggregates are widely used to study heterotypic cellular interactions during the development of vascularization in vitro. In this study, we examined heterotypic cellular spheroids made of adipose-derived stem cells and CD34+/CD31- endothelial progenitor cells induced by the transfection of miR-148b mimic for de novo induction of osteogenic differentiation and miR-210 mimic for de novo induction of endotheliogenesis, respectively. The effect of the microRNA (miRs) mimic treatment group and induction time on codifferentiation was assessed in spheroids formed of transfected cells over the course of a 4-week culture. Based on gene and protein markers of osteogenic and endotheliogenic differentiation, as well as mineralization assays, our results showed that miRs directed cell differentiation and that progenitor maturity influenced the development of heterotypic cellular regions in aggregates. Overall, the success of coculture to create a prevascularized bone model is dependent on a number of factors, particularly the induction time of differentiation before combining the multiple cell types in aggregates. The approach that has been proposed could be valuable in creating vascularized bone tissue by employing spheroids as the building blocks of more complex issues through the use of cutting-edge methods such as 3D bioprinting.

AATF inhibition exerts antiangiogenic effects against human hepatocellular carcinoma.

Background and aims: Angiogenesis is a key factor in the growth and metastasis of hepatic tumors and thus a potential therapeutic target in hepatocellular carcinoma (HCC). In this study, we aim to identify the key role of apoptosis antagonizing transcription factor (AATF) in tumor angiogenesis and its underlying mechanisms in HCC. Methods: HCC tissues were analyzed for AATF expression by qRT-PCR and immunohistochemistry. Stable clones of control and AATF knockdown (KD) were established in human HCC cells. The effect of AATF inhibition on the angiogenic processes was determined by proliferation, invasion, migration, chick chorioallantoic membrane (CAM) assay, zymography, and immunoblotting techniques. Results: We identified high levels of AATF in human HCC tissues compared to adjacent normal liver tissues, and the expression was found to be correlated with the stages and tumor grades of HCC. Inhibiting AATF in QGY-7703 cells resulted in higher levels of pigment epithelium-derived factor (PEDF) than controls due to decreased matric metalloproteinase activity. Conditioned media from AATF KD cells inhibited the proliferation, migration, and invasion of human umbilical vein endothelial cells as well as the vascularization of the chick chorioallantoic membrane. Furthermore, the VEGF-mediated downstream signaling pathway responsible for endothelial cell survival and vascular permeability, cell proliferation, and migration favoring angiogenesis was suppressed by AATF inhibition. Notably, PEDF inhibition effectively reversed the anti-angiogenic effect of AATF KD. Conclusion: Our study reports the first evidence that the therapeutic strategy based on the inhibition of AATF to disrupt tumor angiogenesis may serve as a promising approach for HCC treatment.

Mesenchymal Stem Cell-Derived Extracellular Vesicles for Therapeutic Use and in Bioengineering Applications.

Extracellular vesicles (EVs) are small lipid bilayer-delimited particles that are naturally released from cells into body fluids, and therefore can travel and convey regulatory functions in the distal parts of the body. EVs can transmit paracrine signaling by carrying over cytokines, chemokines, growth factors, interleukins (ILs), transcription factors, and nucleic acids such as DNA, mRNAs, microRNAs, piRNAs, lncRNAs, sn/snoRNAs, mtRNAs and circRNAs; these EVs travel to predecided destinations to perform their functions. While mesenchymal stem cells (MSCs) have been shown to improve healing and facilitate treatments of various diseases, the allogenic use of these cells is often accompanied by serious adverse effects after transplantation. MSC-produced EVs are less immunogenic and can serve as an alternative to cellular therapies by transmitting signaling or delivering biomaterials to diseased areas of the body. This review article is focused on understanding the properties of EVs derived from different types of MSCs and MSC-EV-based therapeutic options. The potential of modern technologies such as 3D bioprinting to advance EV-based therapies is also discussed.

Regenerative Engineering: Current Applications and Future Perspectives.

Many pathologies, congenital defects, and traumatic injuries are untreatable by conventional pharmacologic or surgical interventions. Regenerative engineering represents an ever-growing interdisciplinary field aimed at creating biological replacements for injured tissues and dysfunctional organs. The need for bioengineered replacement parts is ubiquitous among all surgical disciplines. However, to date, clinical translation has been limited to thin, small, and/or acellular structures. Development of thicker tissues continues to be limited by vascularization and other impediments. Nevertheless, currently available materials, methods, and technologies serve as robust platforms for more complex tissue fabrication in the future. This review article highlights the current methodologies, clinical achievements, tenacious barriers, and future perspectives of regenerative engineering.

Navigating the Genomic Landscape of Human Adipose Stem Cell-Derived ß-Cells.

Diabetes is a pandemic manifested through glucose dysregulation mediated by inadequate insulin secretion by beta cells. A beta cell replacement strategy would transform the treatment paradigm from pharmacologic glucose modulation to a genuine cure. Stem cells have emerged as a potential source for beta cell (ß-cell) engineering. The detailed generation of functional ß-cells from both embryonic and induced pluripotent stem cells has recently been described. Adult stem cells, including adipose derived, may also offer a therapeutic approach, but remain ill defined. In our study, we performed an in-depth assessment of insulin-producing beta cells generated from human adipose, irrespective of donor patient age, gender, and health status. Cellular transformation was confirmed using flow cytometry and single-cell imaging. Insulin secretion was observed with glucose stimulation and abrogated following palmitate exposure, a common free fatty acid implicated in human beta cell dysfunction. We used next-generation sequencing to explore gene expression changes before and after differentiation of patient-matched samples, which revealed more than 5,000 genes enriched. Adipose-derived beta cells displayed comparable gene expression to native ß-cells. Pathway analysis demonstrated relevance to stem cell differentiation and pancreatic developmental processes, which are vital to cellular function, structural development, and regulation. We conclude that the functions associated with adipose derived beta cells are mediated through relevant changes in the transcriptome, which resemble those seen in native ß-cell morphogenesis and maturation. Therefore, they may represent a viable option for the clinical translation of stem cell-based therapies in diabetes.

Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer.

The tumor necrosis factor (TNF) receptor super family comprises of members that induce two distinct signaling cascades, leading to either cell survival or apoptosis. However, in prostate cancer (PCa), TNF-mediated prosurvival signaling is the predominant pathway that leads to cell survival and resistance to therapy. Although inhibition of TNF signaling by pharmacological agents or monoclonal antibodies has gained importance in the field of cancer therapy, toxicity to normal cells has impaired their extensive use for cancer treatment. We previously identified a natural, nontoxic compound psoralidin that inhibited viability and induced apoptosis in androgen independent prostate cancer (AIPC) cells. Thus, the goal of our study is to investigate whether psoralidin inhibits TNF-mediated prosurvival signaling in AIPC cells. Our results suggest that psoralidin inhibits constitutive and TNF-induced expression of TNF-alpha and its downstream prosurvival signaling molecules such as NF-kappaB and Bcl-2 in AIPC cells. On the other hand, psoralidin simultaneously induces the death receptor (DR)-mediated apoptotic signaling eventually causing the activation of caspase cascade and resultant induction of apoptosis. Oral administration of psoralidin inhibits expression of TNF-alpha and NF-kappaB/p65 in tumor sections, resulting in tumor regression in PC-3 xenografts. Our results suggest that psoralidin inhibits TNF-mediated survival signaling in AIPC and thus is a potent therapeutic agent for prostate cancer.

Aplysinopsin analogs: Synthesis and anti-proliferative activity of substituted (Z)-5-(N-benzylindol-3-ylmethylene)imidazolidine-2,4-diones.

A series of substituted (Z)-5-(N-benzylindol-3-ylmethylene)imidazolidine-2,4-dione (3) analogs structurally related to aplysinopsin, and that incorporate a variety of substituents in both the indole and N-benzyl moieties have been synthesized under microwave irradiation and conventional heating methods These analogs were evaluated for their anti-proliferative activity against MCF-7 and MDA-231 breast cancer cell lines, and A549 and H460 lung cancer cell lines. Two analogs, 3f and 3j had IC(50) values of 4.4 and 5.2microM, respectively, compared to 5-fluorouracil (IC(50)=15.2microM) against MCF-7 cells.

Differentiation of Adipose Tissue-Derived CD34+/CD31- Cells into Endothelial Cells In Vitro.

In this study, CD34+/CD31- progenitor cells were isolated from the stromal vascular fraction (SVF) of adipose tissue using magnetic activated cell sorting. The endothelial differentiation capability of these cells in vitro was evaluated by culturing them in vascular endothelial growth factor (VEGF) induced medium for 14 days. Viability, proliferation, differentiation and tube formation of these cells were evaluated. Cell viability study revealed that both undifferentiated and endothelial differentiated cells remained healthy for 14 days. However, the proliferation rate was higher in undifferentiated cells compared to endothelial differentiated ones. Upregulation of endothelial characteristic genes (Von Willebrand Factor (vWF) and VE Cadherin) was observed in 2D culture. However, PECAM (CD31) was only found to be upregulated after the cells had formed tube-like structures in 3D Matrigel culture. These results indicate that adipose derived CD34+/CD31- cells when cultured in VEGF induced medium, are capable differentiation into endothelial-like lineages. Tube formation of the cells started 3h after seeding the cells on Matrigel and formed more stable and connected network 24 h post seeding in presence of VEGF.

Hybrid Bioprinting of Zonally Stratified Human Articular Cartilage Using Scaffold-Free Tissue Strands as Building Blocks.

The heterogeneous and anisotropic articular cartilage is generally studied as a layered structure of "zones" with unique composition and architecture, which is difficult to recapitulate using current approaches. A novel hybrid bioprinting strategy is presented here to generate zonally stratified cartilage. Scaffold-free tissue strands (TSs) are made of human adipose-derived stem cells (ADSCs) or predifferentiated ADSCs. Cartilage TSs with predifferentiated ADSCs exhibit improved mechanical properties and upregulated expression of cartilage-specific markers at both transcription and protein levels as compared to TSs with ADSCs being differentiated in the form of strands and TSs of nontransfected ADSCs. Using the novel hybrid approach integrating new aspiration-assisted and extrusion-based bioprinting techniques, the bioprinting of zonally stratified cartilage with vertically aligned TSs at the bottom zone and horizontally aligned TSs at the superficial zone is demonstrated, in which collagen fibers are aligned with designated orientation in each zone imitating the anatomical regions and matrix orientation of native articular cartilage. In addition, mechanical testing study reveals a compression modulus of ≈1.1 MPa, which is similar to that of human articular cartilage. The prominent findings highlight the potential of this novel bioprinting approach for building biologically, mechanically, and histologically relevant cartilage for tissue engineering purposes.