Bibhas K. Bhunia

Silk fiber reinforcement modulates in vitro chondrogenesis in 3D composite scaffolds

The limited self-regenerative capacity of adult cartilage has steered the upsurge in tissue engineered replacements to combat the problem of osteoarthritis. In the present study, the potential of fiber-reinforced silk composites from mulberry (Bombyx mori) and non-mulberry (Antheraea assamensis) silk has been investigated for cartilage tissue engineering. The fabricated composites were physico-chemically characterized and analyzed for cellular viability, proliferation, extracellular matrix formation and immunocompatibility. Both mulberry and non-mulberry silk composites showed effective swelling (25%-30%) and degradation (10%-30%) behavior, owing to their interconnected porous nature. The non-mulberry fiber-reinforced composite scaffolds showed slower degradation (∼90% mass remaining) than mulberry silk over a period of 28 days. The reinforcement of silk fibers within silk solution resulted in an increased compressive modulus and stiffness (nearly eight-fold). The biochemical analysis revealed significant increase in DNA content, sulphated glycosaminoglycan (sGAG) (∼1.5 fold) and collagen (∼1.4 fold) in reinforced composites as compared to pure solution scaffolds (p ≤ 0.01). Histological and immunohistochemical (IHC) staining corroborated enhanced deposition of sGAG and localization of collagen type II in fiber-reinforced composites. This was further substantiated by real time polymerase chain reaction studies, which indicated an up-regulation (∼1.5 fold) of cartilage-specific gene markers namely collagen type II, sox-9 and aggrecan. The minimal secretion of tumor necrosis factor-α (TNF-α) by murine macrophages further demonstrated in vitro immunocompatibility of the scaffolds. Taken together, the results signified the potential of silk fiber-reinforced composite (particularly non-mulberry, A. assamensis) scaffolds as viable alternative biomaterial for cartilage tissue engineering.

Strategic Formulation of Graphene Oxide Sheets for Flexible Monoliths and Robust Polymeric Coatings Embedded with Durable Bioinspired Wettability

Artificial bioinspired superhydrophobicity, which is generally developed through appropriate optimization of chemistry and hierarchical topography, is being recognized for its immense prospective applications related to environment and healthcare. Nevertheless, the weak interfacial interactions that are associated with the fabrication of such special interfaces often provide delicate biomimicked wettability, and the embedded antifouling property collapses on exposure to harsh and complex aqueous phases and also after regular physical deformations, including bending, creasing, etc. Eventually, such materials with potential antifouling property became less relevant for practical applications. Here, a facile, catalyst-free, and robust 1,4-conjugate addition reaction has been strategically exploited for appropriate covalent integration of modified graphene oxide to developing polymeric materials with (1) tunable mechanical properties and (2) durable antifouling property, which are capable of performing both in air and under oil. Furthermore, this approach provided a facile basis for (3) engineering a superhydrophobic monolith into arbitrary free-standing shapes and (4) decorating various flexible (metal, synthetic plastic, etc.) and rigid (glass, wood, etc.) substrates with thick and durable three-dimensional superhydrophobic coatings. The synthesized superhydrophobic monoliths and polymeric coatings with controlled mechanical properties are appropriate to withstand different physical insults, including twisting, creasing, and even physical erosion of the material, without compromising the embedded antiwetting property. The materials are also equally resistant to various harsh chemical environments, and the embedded antifouling property remained unperturbed even after continuous exposure to extremes of pH (pH 1 and pH 11), artificial sea water for a minimum of 30 days. These flexible and formable free-standing monoliths and stable polymeric coatings that are extremely water-repellent both in air and under oil, are of utmost importance owing to their suitability in practical circumstances and robust nature.

Silk-based multilayered angle-ply annulus fibrosus construct to recapitulate form and function of the intervertebral disc

Significance In this study we have developed a fabrication procedure for a silk-based bioartificial disc adopting a directional freezing technique. The fabricated biodisc mimicked the internal intricacy of the native disc as evaluated by electron microscopy. The mechanical properties of these biodiscs were similar to those of the native ones. The fabricated biodiscs supported primary annulus fibrosus or human mesenchymal stem cell proliferation, differentiation, and deposition of a sufficient amount of specific ECM. A small unit of the construct was implanted subcutaneously to show its negligible immune response. The success here means that the silk-based bioartificial disc can be a promising strategy for future direction toward disc replacement therapy. Recapitulation of the form and function of complex tissue organization using appropriate biomaterials impacts success in tissue engineering endeavors. The annulus fibrosus (AF) represents a complex, multilamellar, hierarchical structure consisting of collagen, proteoglycans, and elastic fibers. To mimic the intricacy of AF anatomy, a silk protein-based multilayered, disc-like angle-ply construct was fabricated, consisting of concentric layers of lamellar sheets. Scanning electron microscopy and fluorescence image analysis revealed cross-aligned and lamellar characteristics of the construct, mimicking the native hierarchical architecture of the AF. Induction of secondary structure in the silk constructs was confirmed by infrared spectroscopy and X-ray diffraction. The constructs showed a compressive modulus of 499.18 ± 86.45 kPa. Constructs seeded with porcine AF cells and human mesenchymal stem cells (hMSCs) showed ∼2.2-fold and ∼1.7-fold increases in proliferation on day 14, respectively, compared with initial seeding. Biochemical analysis, histology, and immunohistochemistry results showed the deposition of AF-specific extracellular matrix (sulfated glycosaminoglycan and collagen type I), indicating a favorable environment for both cell types, which was further validated by the expression of AF tissue-specific genes. The constructs seeded with porcine AF cells showed ∼11-, ∼5.1-, and ∼6.7-fold increases in col Iα 1, sox 9, and aggrecan genes, respectively. The differentiation of hMSCs to AF-like tissue was evident from the enhanced expression of the AF-specific genes. Overall, the constructs supported cell proliferation, differentiation, and ECM deposition resulting in AF-like tissue features based on ECM deposition and morphology, indicating potential for future studies related to intervertebral disc replacement therapy.

Hierarchically structured seamless silk scaffolds for osteochondral interface tissue engineering

The osteochondral healthcare market is driven by the increasing demand for affordable and biomimetic scaffolds. To meet this demand, silk fibroin (SF) from Bombyx mori and Antheraea assamensis is used to fabricate a biphasic scaffold, with fiber-free and fiber-reinforced phases, stimulating cartilage and bone revival. The fabrication is a facile reproducible process using single polymer (SF), for both phases, designed in a continuous and integrated manner. Physicochemical and mechanical scaffold characterization, display interconnected pores with differential swelling and tunable degradation. The compressive modulus values, extend to 40 kPa and 25%, for tensile strain, at elongation. The scaffold support, for growth and proliferation of chondrocytes and osteoblasts, for respective cartilage and bone regeneration, is verified from in vitro assessment. Up-regulation of alkaline phosphatase (ALP) activity, extracellular matrix secretion and gene expression are significant; with acceptable in vitro immune response. Upon implantation in rabbit osteochondral defects for 8 weeks, the histological and micro-CT examinations show biphasic scaffolds significantly enhance regeneration of cartilage and subchondral bone tissues, as compared to monophasic scaffolds. The regenerated bone mineral density (BMD) ranges from 600-700 mg hydroxyapatite (HA) per cm3. The results, therefore, showcase the critically positive characteristics of in vitro ECM deposition, and in vivo regeneration of osteochondral tissue by this hierarchically structured biphasic scaffold.

Alkali metal-ion assisted Michael addition reaction in controlled tailoring of topography in a superhydrophobic polymeric monolith

In general, lotus leaf and rose petal-inspired wettabilities are artificially developed through the integration of hierarchical topography and essential chemical functionality. However, a fundamental and important aspect of this nature inspired wettability is not yet addressed; between the hierarchical topography and essential chemical modulation, which is the more sensitive parameter for this nature inspired special wettability? The design of a common approach for tailoring both the hierarchical topography and chemical functionalities is highly essential for investigating such a relevant fundamental aspect; however the synthesis of such a material is extremely difficult in reality. In the current computational study, the Michael addition reaction between unsaturated ester and primary amine groups was found to be facilitated in the presence of alkali metal ions. Interestingly, the mixture of branched polyethyleneimine (BPEI) and dipentaerythritol pentaacrylate (5Acl) in ethanol transformed into a chemically reactive polymeric monolith with tailored hierarchical features—based on the selection of appropriate alkali metal ions in the reaction mixture. The current study investigates the impact of the change in hierarchy in the topography on the nature inspired super-wettability—keeping the chemical functionality of the hierarchical topography intact. On the other hand, the inherent chemical reactivity of the hierarchical interfaces allowed us to examine the change in the chemical modulation—independently and precisely. The post-covalent modification of the hierarchical topography with longer and shorter hydrocarbon tails has a significant impact in controlling the metastable trapped air in the artificially biomimicked interfaces, and eventually controls the Wenzel, Cassie–Baxter and Cassie–Wenzel transition states. This current approach also allows us to modulate other relevant physical properties in the material—including the shrinkage of the material after the removal of the reaction solvent and compressive modulus. The materials with optimized physical properties were successfully exploited in the separation and collection of different forms of oil spills through both selective absorption process and gravity driven filtration process, even under practically relevant severe settings. Such a facile and general approach for tailoring both the chemical functionality and topography could be of potential interest for developing various functional and smart materials—including tissue engineering, patterned interfaces, etc.

Synthesis and characterization of a non-cytotoxic and biocompatible acrylamide grafted pullulan – Application in pH responsive controlled drug delivery

The aim of present study was to develop a pH responsive rate controlling polymer by acrylamide grafting onto pullulan. Grafting was performed using free radical induced microwave assisted irradiation technique using ceric ammonium nitrate as free radical inducer. Acrylamide grafted pullulan (Aam-g-pull) was characterized by Fourier transform infrared spectroscopy, solid state 13C nuclear magnetic resonance and field emission scanning electron microscopy. In vitro enzymatic degradation of Aam-g-pull showed degradation of 22.45% after 8 h with degradation rate constant (k) of 0.019 min-1. In vitro cytotoxicity test did not show cell viability below 80% on HepG2 cell line. Pirfenidone tablets were prepared by utilizing wet granulation method using Aam-g-pull as the only rate controlling polymer. The tablets were characterized in terms of in-process quality control parameters like weight variation, hardness, assay, and in vitro dissolution study. The dissolution study showed that the cumulative drug release in phosphate buffer pH 6.8 (rel3 h = 44.12 ± 0.56%) got a significant jump as compared to the release in 0.1 N hydrochloric acid (rel2 h = 26.78 ± 0.23%), confirming the material to be pH responsive. Aam-g-pull can be used as pH responsive rate controlling polymer.

Fluorogenic naked-eye sensing and live-cell imaging of cyanide by a hydrazine-functionalized CAU-10 metal–organic framework

A hydrazine-functionalized, highly stable Al(III) based metal–organic framework (MOF) with CAU-10 (CAU = Christian-Albrechts-University) framework topology namely CAU-10-N2H3 (1) was specifically designed to detect lethal CN− ions in aqueous medium. The MOF was characterized by X-ray powder diffraction, infrared spectroscopy, gas sorption and thermogravimetric analyses. The isophthalate ligand in CAU-10 was functionalized by the hydrazine group to make the acidic –NH protons easily available to the CN− ions. Indeed, the activated compound (1′) showed highly selective and sensitive responses to CN− ions over other common anions with a detection limit of 0.48 μM. A rapid fluorescence enhancement was observed for 1′ with a large spectral shift (Δλ = 30 nm) in the presence of CN− ions in aqueous solution with the development of visible green fluorescence under a UV lamp. Due to the excellent detection performance to CN− by 1′ in aqueous medium, the material was further used for CN− detection in real water samples. The fluorescence increment with a large blue shift is attributed to the cyanide-induced deprotonation of the –NH group, which was confirmed by 1H NMR titration measurements. The initial photo-induced electron transfer (PET) process is prohibited by this deprotonation, which causes the fluorescence enhancement. Time-resolved fluorescence lifetime measurements suggest that 1′ can also act as a lifetime based CN− sensor. Finally, the CN− sensing ability of 1′ inside living RAW 264.7 macrophages was demonstrated through live-cell imaging investigations.

In vitro and in vivo evaluation of pirfenidone loaded acrylamide grafted pullulan-poly(vinyl alcohol) interpenetrating polymer networks

The aim of present study was to develop controlled release formulation of pirfenidone using acrylamide grafted pullulan. Interpenetrating polymer network (IPN) microspheres were prepared using acrylamide grafted pullulan and PVA utilizing glutaraldehyde assisted water-in-oil emulsion crosslinking method. IPN microspheres were characterized by FTIR, solid state 13C NMR and XRD spectroscopy. In vitro enzymatic degradation study showed 34.30% degradation after 24 h with degradation rate constant of 0.0088 min-1. In vitro biocompatibility test showed no changes in cellular morphology and cell adherence to microspheres, indicating its biocompatible nature. The release exponent value of all formulations was less than 0.45, indicating the release mechanism to be Fickian diffusion. Finally, in vivo pharmacokinetic study showed longer Tmax (1.16 h) and greater AUC value (10037.76 ng h/mL,) as compared to Pirfenex® (Tmax = 0.5 h; AUC = 4310.45 ng h/mL,). The results indicated that the prepared formulation could successfully control the drug release for prolonged time period.

Therapeutically Effective Controlled Release Formulation of Pirfenidone from Nontoxic Biocompatible Carboxymethyl Pullulan-Poly(vinyl alcohol) Interpenetrating Polymer Networks

The present study was conducted to develop therapeutically effective controlled release formulation of pirfenidone (PFD) and explore the possibility to reduce the total administered dose and dosing regimen. For this purpose, pH-sensitive biomaterial was prepared by inducing carboxymethyl group on pullulan by Williamson ether synthesis reaction, and further, interpenetrating polymeric network microspheres were prepared by glutaraldehyde-assisted water-in-oil (w/o) emulsion cross-linking method, which showed higher swelling ratio in acidic and basic pH. The formation of microspheres was confirmed by different spectral characterization techniques, and thermal kinetic study indicated the formation of thermally stable microspheres. Cell viability and biocompatibility studies on hepatocellular carcinoma (HepG2) cell showed the polymeric matrix to be biocompatible. In vitro dissolution of optimized formulation (F5) showed releases of 54.09 and 76.37% in 0.1 N HCl after 2 h and phosphate buffer (pH 6.8) up to 8 h, respectively. In vivo performances of prepared microsphere and marketed product of PFD were compared in rabbit. Tmax (time taken to reach peak plasma concentration) was found to be achieved at 0.83 h, compared to 0.5 h for Pirfenex with no significant difference complementing the immediate action, while area under curve was significantly greater for optimized formulation (9768 ± 1300 ng h/mL) compared to Pirfenex (4311 ± 110 ng h/mL), complementing the sustained action. In vivo pharmacokinetic study suggested that the prepared microsphere could be a potential candidate for therapeutically effective controlled delivery of PFD used in dyspnea and cough management due to idiopathic pulmonary fibrosis.