Direct 3D Printing of Seashell Precursor toward Engineering a Multiphasic Calcium Phosphate Bone Graft.

Multiphasic calcium phosphate (Ca-P) has widely been explored for bone graft replacement. This study represents a simple method of developing osteoinductive scaffolds by direct printing of seashell resources. The process demonstrates a coagulation-assisted extrusion-based three-dimensional (3D) printing process for rapid fabrication of multiphasic calcium phosphate-incorporated 3D scaffolds. These scaffolds demonstrated an interconnected open porous architecture with improved compressive strength and higher surface area. Multiphasic calcium phosphate (Ca-P) and hydroxyapatite present in the multi-scalar naturally resourced scaffold displayed differential protein adsorption, thus facilitating cell adhesion, migration, and differentiation, resulting in enhanced deposition of the extracellular matrix. The microstructural and physicochemical attributes of the scaffolds also lead to enhanced stem cell differentiation as witnessed from gene and protein expression analysis. Furthermore, the histological study of subcutaneous implantation evidently portrays promising biocompatibility without foreign body reaction. Neo-tissue in-growth was manifested with abundant blood vessels, thus indicative of excellent vascularization. Notably, cartilaginous and proteoglycan-rich tissue deposition indicated ectopic bone formation via an endochondral ossification pathway. The hierarchical interconnected porous architectural tribology accompanied with multiphasic calcium phosphate composition manifests its successful implication in enhancing stem cell differentiation and promoting excellent tissue in-growth, thus making it a plausible alternative in bone tissue engineering applications.

Carbon Nanodots Doped Super-paramagnetic Iron Oxide Nanoparticles for Multimodal Bioimaging and Osteochondral Tissue Regeneration via External Magnetic Actuation.

Super-paramagnetic iron oxide nanoparticles (SPIONs) have multiple theranostics applications such as T2 contrast agent in magnetic resonance imaging (MRI) and electromagnetic manipulations in biomedical devices, sensors, and regenerative medicines. However, SPIONs suffer from the limitation of free radical generation, and this has a certain limitation in its applicability in tissue imaging and regeneration applications. In the current study, we developed a simple hydrothermal method to prepare carbon quantum dots (CD) doped SPIONs (FeCD) from easily available precursors. The nanoparticles are observed to be cytocompatible, hemocompatible, and capable of scavenging free radicals in vitro. They also have been observed to be useful for bimodal imaging (fluorescence and MRI). Further, 3D printed gelatin-FeCD nanocomposite nanoparticles were prepared and used for tissue engineering using static magnetic actuation. Wharton's jelly derived mesenchymal stem cells (MSCs) were cultured on them with magnetic actuation and implanted at the subcutaneous region. The tissues obtained have shown features of both osteogenic and chondrogenic differentiation of the stem cells in vivo. In vitro, PCR studies show MSCs express gene expression of both bone and cartilage-specific markers, suggesting FeCDs under magnetic actuation can lead MSCs to go through differentiating into an endochondral ossification route.

Doping of carbon nanodots for saving cells from silver nanotoxicity: A study on recovering osteogenic differentiation potential.

Silver nanoparticles are explored for many advanced biological applications including the development of antimicrobial surfaces on implants, SERS imaging, nanotherapeutics, biosensing and much more. However, recent research findings suggest silver nanoparticles provide blockade of differentiation of mesenchymal stem cells (MSCs), especially into osteogenic developmental pathway via generation of reactive oxygen species. These studies suggest that the application of silver nanoparticles in medical implants should be prohibited. In the current study, carbon nanodots (CND) supported silver clusters (AgC) is explored as a remedy to this problem. The nanostructure was synthesized in microwave irradiation induced rapid method and characterization was conducted via UV-Vis spectroscopy, fluorescence spectroscopy, HRTEM, XRD, FTIR, Raman spectroscopy, DLS, AFM, and XPS. Fluorescence spectrum showed a quantum yield of 0.25 while Raman spectroscopy showed rapid amplification of CND specific peaks implicating significant SERS property. Further in vitro biocompatibility (MTT) and bio-imaging capability was assessed culturing Wharton's Jelly-derived MSCs. In this study, its efficacy as in-situ cellular oxidative stress scavenger is also studied using NBT and DCFH-DA assay. Via ALP assay, alizarin red staining, cell membrane nanoindentation studies, PCR analysis and immunocytochemistry for osteoblast-like gene expression it was confirmed that AgCs can control silver nanoparticle-induced inhibition of osteogenic differentiation in vitro. Thus, AgCs (Carbon nanodots supported silver clusters) are not only considered to be a dual-mode bio-imaging nanoprobe but also a remedy to the silver-induced ROS generation and osteogenic differentiation blockade of MSCs.

Simultaneous hydrothermal bioactivation with nano-topographic modulation of porous titanium alloys towards enhanced osteogenic and antimicrobial responses.

Post-implantation failure associated with insufficient host tissue integration at the bone-implant interface and aseptic loosening is a major concern in orthopaedics as well as in dentistry. To overcome the failure in early stages of implantation, prosthetic design combining the mechanisms of porosity guided bone ingrowth along with topographic manipulation of osteogenic cells over bacterial colonization would be an ideal choice, although achieving such a goal is highly challenging. In this study, facile rapid hydrothermal synthesis of nanostructures with simultaneous deposition of hydroxyapatite on the titanium alloy surface was demonstrated by using an aqueous sodium tripolyphosphate and calcium hydroxide mixture. Nanostructures with wire-like morphology exhibited significantly higher osteogenic related gene expression (COL I, OPN, and OCN) through differentiation of adipose derived mesenchymal stem cells as well as the bactericidal response against S. aureus and E. coli as compared to other nanotopographic features. The same also exhibited elongated cell morphology with the highest expression of paxillin towards cell boundaries as compared to the polished surface with flattened cell morphology and localized expression of paxillin around the nucleus. Implantation of treated porous Ti6Al4V samples representing a multiscalar hierarchy with wire-like nanostructures accelerated osteochondral healing in rabbits without any major signs of infection. Also, significantly higher bone formation was observed within the defects implanted with treated porous Ti6Al4V (44.0%) as compared to that of untreated porous samples (36.9%) as well as empty defects (19.6%).

Surface Modification of Eggshell Membrane with Electrospun Chitosan/Polycaprolactone Nanofibers for Enhanced Dermal Wound Healing.

Eggshell membrane (ESM), a naturally occurring microfibrous biopolymer network comprising collagen I, V, and X, GAGs, and other significant proteins, is responsible for guided tissue regeneration. The extraction methodology of ESM and surface topography of the microfibers impede its extensive usage in skin tissue engineering. Herein we deploy a unique route of ESM surface modification utilizing chitosan/polycaprolactone (CS/PCL) nanofibers to fabricate a bilayered scaffold for wound healing application. Microstructural and surface topographic analysis of the construct confirms the bilayered structure of the composite with smooth nanofibers of CS/PCL decorated on ESM. The two layers were cross-linked by carbodiimide chemistry as confirmed by XPS and FTIR analysis. Cytocompatibility of the scaffolds was evaluated with human dermal fibroblast (HDF) cells culture study. The biomimetic architecture and composition of modified ESM facilitated extensive cell adhesion, migration, and proliferation while an impeded cell adhesion was observed on the natural tissue. Moreover, owing to the presence of ESM, the scaffolds adhered naturally to the wound bed while implanted on a full-thickness wound in a rat model. Further, the nanofiber modified ESM group showed extensive host cell migration and proliferation thus leading to faster re-epithelization and dermal regeneration with high collagen deposition in comparison to natural ESM. The above in vitro and in vivo results substantiate the effect of nanofiber functionalization on the ESM surface thus making the bilayered construct a potential dermal substitute.

Nano-/Microfibrous Cotton-Wool-Like 3D Scaffold with Core-Shell Architecture by Emulsion Electrospinning for Skin Tissue Regeneration.

Electrospun nanofibrous scaffold has long been studied as skin substitutes for their structural resemblance to the dermal extracellular matrix. However, packed fibrous architecture with small pore size restricts cellular infiltration into nanofibrous mat. In this article, we report highly porous, nano-/microfibrous 3D structure using polycaprolactone-chitosan emulsion and its application in skin regeneration. Under the influence of electric field, the emulsion containing encapsulated charged chitosan droplets enhances charge of the spinning solution and residual charge in the core of the deposited fiber, thereby creating core-shell, cotton-like fluffy structure with average pore size 62 µm, fiber diameter ∼1.62 µm, contact angle of 72° and 80% water uptake capacity of the scaffold. Further, differential stirring period of the specific emulsion developed compact nanofibrous membrane with nanometer ranged pore size emphasizing the role played by emulsion droplet size and the charge carried thereafter. Presence of nanofibers with high-interconnected porosity promoted efficient cellular infiltration and proliferation from initial days of cell seeding. The scaffold supported extracellular matrix protein expression and stratified epithelialization in vitro. Effective integration and attachment of scaffold with margins of a full-thickness excision wound created in a rat model with accelerated healing within 3 weeks proved the efficiency of the scaffold as skin substitute. Additionally, gradual and prolong release of acidic chitosan from the core section benefitted wound healing by lowering the pH of wound environment. Simple technique with inexpensive raw materials endorsed the scaffold as a promising off-the-shelf matrix for skin tissue regeneration.

Bilayered nanofibrous 3D hierarchy as skin rudiment by emulsion electrospinning for burn wound management.

Mimicking skin extracellular matrix hierarchy, the present work aims to develop a bilayer skin graft comprising a porous cotton-wool-like 3D layer with membranous structure of PCL-chitosan nanofibers. Emulsion electrospinning with differential stirring periods of PCL-chitosan emulsion results in development of a bilayer 3D structure with varied morphology. The electrospun membrane has fiber diameter ∼274 nm and pore size ∼1.16 µm while fluffy 3D layer has fiber diameter ∼1.62 µm and pore size ∼62 µm. The 3D layer was further coated with collagen I isolated from Cirrhinus cirrhosus fish scales to improve biofunctionality. Surface coating with collagen I resulted in bundling the fibers together, thereby increasing their average diameter to 2.80 µm and decreasing pore size to ∼45 µm. The architecture and composition of the scaffold promotes efficient cellular activity where interconnected porosity with ECM resembling collagen I coating assists cellular adhesion, infiltration, and proliferation from initial days of fibroblast seeding, while keratinocytes migrate on the surface only without infiltrating in the membranous nanofiber layer. Anatomy of the scaffold arising due to variation in pore size distribution at different layers thereby facilitates compartmentalization and prevents initial cellular transmigration. The scaffold also assists in extracellular matrix protein synthesis and keratinocyte stratification in vitro. Further, the scaffold effectively integrates and attaches with third-degree burn wound margins created in rat models and accelerates healing in comparison to standard Tegaderm dressing™. The bilayer scaffold is thus a promising, readily available, cost-effective, off-the-shelf matrix as a skin substitute.

Influence of Porosity and Pore-Size Distribution in Ti6Al4 V Foam on Physicomechanical Properties, Osteogenesis, and Quantitative Validation of Bone Ingrowth by Micro-Computed Tomography.

Cementless fixation for orthopedic implants aims to obviate challenges associated with bone cement, providing long-term stability of bone prostheses after implantation. The application of porous titanium and its alloy-based implants is emerging for load-bearing applications due to their high specific strength, low stiffness, corrosion resistance, and superior osteoconductivity. In this study, coagulant-assisted foaming was utilized for the fabrication of porous Ti6Al4 V using egg-white foam. Samples with three different porosities of 68.3%, 75.4%, and 83.1% and average pore sizes of 92, 178, and 297 µm, respectively, were prepared and subsequently characterized for mechanical properties, osteogenesis, and tissue ingrowth. A microstructure-mechanical properties relationship study revealed that an increase of porosity from 68.3 to 83.1% increased the average pore size from 92 to 297 µm with the subsequent reduction of compresive strength by 85% and modulus by 90%. Samples with 75.4% porosity and a 178 µm average pore size produced signifcant osteogenic effects on human mesenchymal stem cells, which was further supported by immunocytochemistry and real-time polymerase chain reaction data. Quantitative assessment of bone ingrowth by micro-computed tomography revealed that there was an approximately 52% higher bone formation and more than 90% higher bone penetration at the center of femoral defects in rabbit when implanted with Ti6Al4 V foam (75.4% porosity) compared to the empty defects after 12 weeks. Hematoxylin and eosin (H&E) and Masson trichrome (MT) staining along with energy-dispersive X-ray mapping on the sections obtained from the retrieved bone samples support bone ingrowth into the implanted region.

Accelerating full thickness wound healing using collagen sponge of mrigal fish (Cirrhinus cirrhosus) scale origin.

The potentiality of collagen sponge as a skin substitute, derived from mrigal (Cirrhinus cirrhosus) scale has been explored in this study. Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the scale of mrigal were isolated and characterized. The yields of ASC and PSC were ∼3% and ∼7% based on the dry weight of scale while the hydroxyproline content was ∼90mg/g. Scanning electron microscope revealed progressive demineralization with EDTA on time dependent scale. Further, the D-Spacing in fibril bundles were calculated to be ∼67nm. Fourier transform infrared and circular dichroism spectra confirmed extracted protein to be collagen I, where both ASC and PSC comprised of two different α-chains (α1 and α2). The denaturation temperature (Td) of the collagen solution was 35°C closer to Td of mammalian collagen. In vitro cell culture studies on the extracted collagen sponge showed efficient cell growth and proliferation. Additionally, co-culture with fibroblast and keratinocyte cells showed development of stratified epidermal layer in vitro. Faster wound healing potential of the extracted collagen in a rat model proved its applicability as a dermal substitute.

Carbon nanodots from date molasses: new nanolights for the in vitro scavenging of reactive oxygen species.

Most of the nanoimaging tools like quantum dots and metallic nanoparticles are shown to have different levels of cytotoxicity via various mechanisms. However carbon nanodots (CNDs) are a new group of ultra small nano structures (average 4-6 nm) which is potential candidate of next generation optical imaging. Being carbonaceous in origin, CNDs possess excellent luminescence and photostability with significantly less cytotoxicity. In present study, we have synthesized carbon nano-dots from date molasses by microwave irradiation at ∼pH 11. The synthesized carbon nanodots were characterized using UV-Vis spectroscopy, fluorescence spectroscopy, TEM, XRD analysis, FTIR study and Zeta potential measurement. The average sizes of the dots were found to be 5-7 nm. A clear band emission was visible around 480 nm when an excitation beam of 415 nm was incident. For biological applicability, MTT assay and hemocompatibility studies were performed. The results exhibited the material to be highly cytocompatible within the application limit. Upon immediate exposure to CNDs, no significant changes to cellular surface morphology were observed via AFM imaging. Significant hemolysis or blood cell aggregation was not observed after incubation of CNDs with blood. After labelling with CNDs, MG-63 cells were found to be unbleached up to several hours even on exposure to light. We are reporting first time in this study the free radical scavenging property of CNDs in ex vivo and in vitro models. Antioxidant activity was measured ex vivo via potassium permanganate assay and DPPH assay. In vitro superoxide inhibition activity was measured both by spectroscopy and under microscope by NBT reduction assay. Hydroxyl free radical inhibition activity was measured via DCFH-DA Assay. The results were comparable with scavenging activity of standard antioxidant molecules (BHT and l-ascorbic acid). A novel assay for quantitative analysis of cellular oxidative stress was also proposed. Therefore, this material could be useful for long-term live cell imaging and cell tracking in a scaffold with minimal cytotoxicity and oxidative stress.