Facile fabrication of Bi-layered perfusable hydrogel tubes as biomimetic 3D arterial construct.

Small-diameter arterial conduits with native physiological and biological equivalence continues to be a constant global demand posing critical challenges in fabrication. Advent of various strategies towards mimicking the structural hierarchy of a native blood vessel, often involve complex instrumentation and template-assistance with post-processing complications eventually compromising structural fidelity. In the present research, we report a template-free, facile strategy- '3D wet writing' by peripheral-core differential ionic gelation to fabricate perfusable customizable constructs of any dimension, thickness and length in <5 mins. Dual-crosslinking using di-diol complexation of borax with Alginate- poly (vinyl alcohol) was performed to enhance the stability of fabricated bi-layered tubular constructs (BLT). These fabricated BLTs demonstrated non-linear mechanical characteristics of native blood vessels in withstanding physiological (120/80 mmHg) hemodynamic loading conditions with cyclic strain (5.82 ± 0.88%). The BLTs also ensured adequate longitudinal (0.176 ± 0.03 MPa) & circumferential (0.29 ± 0.012 MPa) tensile strength and burst pressure strength of 353.875 ± 22.69 mmHg. Hemocompatible characteristics of BLT were clearly evident with lower hemolytic index (0.21 ± 0.03%) and maintenance of erythrocyte structural integrity under dynamic conditions. Further, non-thrombogenic and non-inflammatory characteristics of BLTs were confirmed by in-activated platelets and monocytes under dynamic conditions. The developed wet-writing technique exhibited facile integration of layer-specific cells concurrently with the BLT fabrication. The spatial cell-specific expressions of smooth muscle (α-SMA) and endothelial (CD-31) cells in BLT were comparable to native hierarchical cellular organization with the multi-layered medial and mono-layered intimal layers. Further,ex-vivodynamic studies on anastomotic interface between BLT and rat abdominal aorta clearly evidenced the functional efficacy of fabricated BLTs as physiologically relevant small-diameter vascular construct.

Self-Standing Photo-Crosslinked Hydrogel Construct: in vitro Microphysiological Vascular Model.

Modeling of the human vascular microphysiological system (MPS) has gained attention due to precise prediction of drug response and toxicity during drug screening process. Developing a physiologically equivalent vascular MPS still remains complex as it demands the recapitulation of dynamic structural and biological microenvironment similar to native vasculature. Hence, an ideal MPS would involve developing perfusable 3D in vitro models with multilayered human vascular cells encapsulated in a matrix to regulate the vascular tone resembling the native. Several attempts to model such anatomically accurate physiological and pathological blood vessels often fail to harmonize the essential vascular microenvironment. For instance, conventional microfluidic-based approaches employed for vascular MPS, though offering creation of perfusable channel, do not replicate the vascular hierarchical cellular arrangement due to planar geometry and confluent monolayered cell seeding. Also, recent advances with 3D biofabrication strategies are still limited by fabrication of small-diameter constructs and scalability besides post-processing techniques that indirectly distort the structural integrity of the hydrogel tubular constructs. These existing limitations toward fabricating a relevant vascular MPS demand a facile and mechanically stable construct. Hence, the present study is aimed toward developing a stable viable self-standing perfusable hydrogel construct by a rapid and scalable strategy toward vascular MPS application. The fabricated tubular constructs were found to be structurally stable with end-to-end perfusability exhibiting their potential as self-standing perfusable structures. Also, the construct exhibited nonhemolytic behavior with perfusion of red blood cells inside the luminal channel. The present study evidences creation of a dual-crosslinked stable, viable self-standing hydrogel construct with multilayered homogenous distribution of viable smooth muscle cells throughout the construct, thereby demonstrating its applicability as a promising 3D in vitro vascular microphysiological system.

Marigold-like tyrosinase-embedded nanostructures-a nano-in-micro system.

Marigold-like tyrosinase-entrenched nanostructures were developed by a facile method using a metal cofactor to overcome the limitations of conventional enzyme immobilization techniques. The protein-copper complex promotes the hierarchical self-assembly of nanopetals into marigold-like microstructures through a sequential germination process. Nanopetals, which originated from bead-like tiny projections, showed budding over the surface and promoted the anisotropic growth of copper phosphate nanocrystals upon co-ordination with the active functional groups in protein. This organic-inorganic hybrid showed excellent re-usability, comparable catalytic efficiency, faster reaction rate, improved storage, and thermal stability without affecting the enzyme activity.

Additive manufacturing of biodegradable porous orthopaedic screw.

Advent of additive manufacturing in biomedical field has nurtured fabrication of complex, customizable and reproducible orthopaedic implants. Layer-by-layer deposition of biodegradable polymer employed in development of porous orthopaedic screws promises gradual dissolution and complete metabolic resorption thereby overcoming the limitations of conventional metallic screws. In the present study, screws with different pore sizes (916 × 918 µm to 254 × 146 µm) were 3D printed at 200 µm layer height by varying printing parameters such as print speed, fill density and travel speed to augment the bone ingrowth. Micro-CT analysis and scanning electron micrographs of screws with 45% fill density confirmed porous interconnections (40.1%) and optimal pore size (259 × 207 × 200 µm) without compromising the mechanical strength (24.58 ± 1.36 MPa). Due to the open pore structure, the 3D printed screws showed increased weight gain due to the deposition of calcium when incubated in simulated body fluid. Osteoblast-like cells attached on screw and infiltrated into the pores over 14 days of in vitro culture. Further, the screws also supported greater human mesenchymal stem cell adhesion, proliferation and mineralized matrix synthesis over a period of 21 days in vitro culture as compared to non-porous screws. These porous screws showed significantly increased vascularization in a rat subcutaneous implantation as compared to control screws. Porous screws produced by additive manufacturing may promote better osteointegration due to enhanced mineralization and vascularization.

Surface topography of polylactic acid nanofibrous mats: influence on blood compatibility.

Fabricating nanofibrous scaffolds with robust blood compatibility remains an unmet challenge for cardiovascular applications since anti-thrombogenic surface coatings did not withstand physiological shear force. Hence, the present study envisages the influence of smooth and porous topographies of poly(lactic acid) (PLA) nanofibers on hemocompatibility as it could offer time-independent blood compatibility. Further, recent studies have evolved to integrate various contrasting agents for augmenting the prognostic properties of tissue engineered scaffolds; an attempt was also made to synthesize Curcumin-superparamagnetic iron oxide nanoparticle complex (Cur-SPION) as a contrasting agent and impregnated into PLA nanofibers for evaluating the blood compatibility. Herein, electrospun nanofibers of PLA with different topographies (smooth and porous) were fabricated and characterized for surface morphology, zeta potential, fluorescence, and crystallinity. The scaffolds with smooth, porous and rough surface topographies were thoroughly investigated for its hemocompatibility by evaluating hemolysis percentage, platelet adhesion, in vitro kinetic clotting time, serum protein adsorption, plasma recalcification time (PRT), capture and release of erythrocytes. Although the nanofibers of all three groups showed acceptable hemolytic percentage (HP < 5%), the adhered RBCs on Cur-SPION based fibers undergo morphological transformation from biconcave discocytes to echinocytes with cube-like protrusions. On the contrary, no morphological changes were observed in RBCs cultured on smooth and porous nanofibers. Porous fibers exhibited excellent anti-thrombogenic property and adhered lesser platelets and maintained the discoidal morphology of native platelets. Cur-SPION integrated PLA nanofibers showed inactivated platelets with anti-thrombogenic activity compared to smooth nanofibers. In conclusion, PLA nanofibers porous topography did not affect the RBC membrane integrity and maintained discoidal morphology of platelets with superior anti-thrombogenic activity. However, smooth and Cur-SPION integrated PLA nanofibers were found to activate the platelets and deform the RBC membrane integrity, respectively. Hence, the nanofibers with porous structures provide an ideal topography for time-independent hemocompatibility.

Gradient nano-engineered in situ forming composite hydrogel for osteochondral regeneration.

Fabrication of anisotropic osteochondral-mimetic scaffold with mineralized subchondral zone and gradient interface remains challenging. We have developed an injectable semi-interpenetrating network hydrogel construct with chondroitin sulfate nanoparticles (ChS-NPs) and nanohydroxyapatite (nHA) (∼30-90 nm) in chondral and subchondral hydrogel zones respectively. Mineralized subchondral hydrogel exhibited significantly higher osteoblast proliferation and alkaline phosphatase activity (p < 0.05). Osteochondral hydrogel exhibited interconnected porous structure and spatial variation with gradient interface of nHA and ChS-NPs. Microcomputed tomography (µCT) demonstrated nHA gradation while rheology showed predominant elastic modulus (∼930 Pa) at the interface. Co-culture of osteoblasts and chondrocytes in gradient hydrogels showed layer-specific retention of cells and cell-cell interaction at the interface. In vivo osteochondral regeneration by biphasic (nHA or ChS) and gradient (nHA + ChS) hydrogels was compared with control using rabbit osteochondral defect after 3 and 8 weeks. Complete closure of defect was observed in gradient (8 weeks) while defect remained in other groups. Histology demonstrated collagen and glycosaminoglycan deposition in neo-matrix and presence of hyaline cartilage-characteristic matrix, chondrocytes and osteoblasts. µCT showed mineralized neo-tissue formation, which was confined within the defect with higher bone mineral density in gradient (chondral: 0.42 ± 0.07 g/cc, osteal: 0.64 ± 0.08 g/cc) group. Further, biomechanical push-out studies showed significantly higher load for gradient group (378 ± 56 N) compared to others. Thus, the developed nano-engineered gradient hydrogel enhanced hyaline cartilage regeneration with subchondral bone formation and lateral host-tissue integration.

Responsive Nanomicellar Theranostic Cages for Metastatic Breast Cancer.

Precluding the progression of metastasis with early diagnosis of triple-negative breast cancer remains challenging due to lack of targeting specificity with poor diagnostic potential. Herein, an amphipathic chitosan-based targeted nanomicellar theranostics (30-45 nm) comprising doxorubicin-superparamagnetic iron oxide nanoparticles complexes (89.23%) with lower critical micelle concentration (0.1 µg/mL) were developed. Micelles exhibit concentration-based contrast enhancement in MRI (r2 6.27 mM-1 s-1) and hyperthermia rather than thermal-ablation. This theranostics delivers doxorubicin under alternating magnetic field (480 kHz) and at endosomal pH (pH 5.2) while showing stability at pH 7.4. Anti-αvß3 integrin antibody conjugation onto PEGylated micelles (62.3%) enhances micellar internalization into drug-resistant MDA-MB-231 after 1 h and magnetizes the cells after 6 h over that with nonconjugated micelles. Immigration of MDA-MB-231 and 4T1 cells retards after 24 h, while significant reduction of mitochondrial membrane potential is observed under hyperthermia. Intratumoral administration of nanomicelles in 4T1 orthotopic spontaneous metastasis model demonstrated antitumor and fibrosis mediated caging effect with simultaneous enhancement of MRI-T2 contrast.

Development of nanotheranostics against metastatic breast cancer--A focus on the biology & mechanistic approaches.

Treatment for metastatic breast cancer still remains to be a challenge since the currently available diagnostic and treatment strategies fail to detect the micro-metastasis resulting in higher mortality rate. Moreover, the lack of specificity to target circulating tumor cells is also a factor. In addition, currently available imaging modalities to identify the secondaries vary with respect to various metastatic anatomic areas and size of the tumor. The drawbacks associated with the existing clinical management of the metastatic breast cancer demands the requirement of multifunctional nanotheranostics, which could diagnose at macro- and microscopic level, target the solid as well as circulating tumor cells and control further progression with the simultaneous evaluation of treatment response in a single platform. However, without the understanding of the biology as well as preferential homing ability of circulating tumor cells at distant organs, it is quite impossible to address the existing challenges in the present diagnostics and therapeutics against the breast cancer metastasis. Hence this review outlines the severity of the problem, basic biology and organ specificity with the sequential steps for the secondary progression of disease followed by the various mechanistic approaches in diagnosis and therapy at different stages.