A green and economic approach to synthesize magnetic Lagenaria siceraria biochar (γ-Fe2O3-LSB) for methylene blue removal from aqueous solution.

In the printing and textile industries, methylene blue (a cationic azo dye) is commonly used. MB is a well-known carcinogen, and another major issue is its high content in industrial discharge. There are numerous removal methodologies that have been employed to remove it from industrial discharge; however, these current modalities have one or more limitations. In this research, a novel magnetized biochar (γ-Fe2O3-LSB) was synthesized using Lagenaria siceraria peels which were further magnetized via the co-precipitation method. The synthesized γ-Fe2O3-LSB was characterized using FTIR, X-ray diffraction, Raman, SEM-EDX, BET, and vibrating sample magnetometry (VSM) for the analysis of magnetic properties. γ-Fe2O3-LSB showed a reversible type IV isotherm, which is a primary characteristic of mesoporous materials. γ-Fe2O3-LSB had a specific surface area (SBET = 135.30 m2/g) which is greater than that of LSB (SBET = 11.54 m2/g). γ-Fe2O3-LSB exhibits a saturation magnetization value (Ms) of 3.72 emu/g which shows its superparamagnetic nature. The batch adsorption process was performed to analyze the adsorptive removal of MB dye using γ-Fe2O3-LSB. The adsorption efficiency of γ-Fe2O3-LSB for MB was analyzed by varying parameters like the initial concentration of adsorbate (MB), γ-Fe2O3-LSB dose, pH effect, contact time, and temperature. Adsorption isotherm, kinetic, and thermodynamics were also studied after optimizing the protocol. The non-linear Langmuir model fitted the best to explain the adsorption isotherm mechanism and resulting adsorption capacity ( q e =54.55 mg/g). The thermodynamics study showed the spontaneous and endothermic nature, and pseudo-second-order rate kinetics was followed during the adsorption process. Regeneration study showed that γ-Fe2O3-LSB can be used up to four cycles. In laboratory setup, the cost of γ-Fe2O3-LSB synthesis comes out to be 162.75 INR/kg which is low as compared to commercially available adsorbents. The results obtained suggest that magnetic Lagenaria siceraria biochar, which is economical and efficient, can be used as a potential biochar material for industrial applications in the treatment of wastewater.

Advances in additive manufacturing of polycaprolactone based scaffolds for bone regeneration.

Critical sized bone defects are difficult to manage and currently available clinical/surgical strategies for treatment are not completely successful. Polycaprolactone (PCL) which is a biodegradable and biocompatible thermoplastic can be 3D printed using medical images into patient specific bone implants. The excellent mechanical properties and low immunogenicity of PCL makes it an ideal biomaterial candidate for 3D printing of bone implants. Though PCL suffers from the limitation of being bio-inert. Here we describe the use of PCL as a biomaterial for 3D printing for bone regeneration, and advances made in the field. The specific focus is on the different 3D printing techniques used for this purpose and various modification that can enhance bone regeneration following the development pathways. We further describe the effect of various scaffold characteristics on bone regeneration both in vitro and the translational assessment of these 3D printed PCL scaffolds in animal studies. The generated knowledge will help understand cell-material interactions of 3D printed PCL scaffolds, to further improve scaffold chemistry and design that can replicate bone developmental processes and can be translated clinically.

Alginate based hydrogel inks for 3D bioprinting of engineered orthopedic tissues.

3D printed hydrogels have emerged as a novel tissue engineering and regeneration platform due to their ability to provide a suitable environment for cell growth. To obtain a well-defined scaffold with good post-printing shape fidelity, a proper hydrogel ink formulation plays a crucial role. In this regard, alginate has received booming interest owing to its biocompatibility, biodegradability, easy functionalization, and fast gelling behavior. Hence, this review highlights the significance of alginate-based hydrogel inks for fabricating 3D printed scaffolds for bone and cartilage regeneration. Herein, we discuss the fundamentals of direct extrusion 3D bioprinting method and provide a comprehensive overview of various alginate-based hydrogel ink formulations that have been used so far. We also summarize the requirements of hydrogel inks and 3D printed scaffolds to achieve similarity to the native tissue environment. Finally, we discuss the challenges, and research directions relevant for future clinical translation.

Direct 3D printing of decellularized matrix embedded composite polycaprolactone scaffolds for cartilage regeneration.

Treatment options for large osteochondral injuries (OCIs) are limited by donor tissue scarcity, morbidity, and anatomic mismatch. 3D printing technology can produce patient-specific scaffolds to address these large defects. Thermoplastics like polycaprolactone (PCL) offer necessary mechanical properties, but lack bioactivity. We fabricated 3D printed PCL scaffolds embedded with polylactic acid microspheres containing decellularized cartilage matrix (DM). DM incorporation within polylactic acid microspheres prevented its thermal degradation during the 3D printing process. The scaffolds replicated the mechanical properties of native cartilage and demonstrated controlled release of DM proteins. Human mesenchymal stem cells (hMSCs) seeded on the composite scaffolds with DM and cultured in basal media self-assembled into aggregates mimicking mesenchymal condensates during embryonic development. The DM composite scaffolds also induced higher expression of biochemical markers of cartilage development than controls, providing evidence for their translational application in the treatment of OCIs. The present study demonstrates the potential of direct incorporation of DM with thermoplastics for 3D printing of patient-specific scaffolds for osteochondral regeneration.

Tissue Engineering Strategies for Treating Avascular Necrosis of the Femoral Head.

Avascular necrosis (AVN) of the femoral head commonly leads to symptomatic osteoarthritis of the hip. In older patients, hip replacement is a viable option that restores the hip biomechanics and improves pain but in pediatric, adolescent, and young adult patients hip replacements impose significant activity limitations and the need for multiple revision surgeries with increasing risk of complication. Early detection of AVN requires a high level of suspicion as diagnostic techniques such as X-rays are not sensitive in the early stages of the disease. There are multiple etiologies that can lead to this disease. In the pediatric and adolescent population, trauma is a commonly recognized cause of AVN. The understanding of the pathophysiology of the disease is limited, adding to the challenge of devising a clinically effective treatment strategy. Surgical techniques to prevent progression of the disease and avoid total hip replacement include core decompression, vascular grafts, and use of bone-marrow derived stem cells with or without adjuncts, such as bisphosphonates and bone morphogenetic protein (BMP), all of which are partially effective only in the very early stages of the disease. Further, these strategies often only improve pain and range of motion in the short-term in some patients and do not predictably prevent progression of the disease. Tissue engineering strategies with the combined use of biomaterials, stem cells and growth factors offer a potential strategy to avoid metallic implants and surgery. Structural, bioactive biomaterial platforms could help in stabilizing the femoral head while inducing osteogenic differentiation to regenerate bone and provide angiogenic cues to concomitantly recover vasculature in the femoral head. Moreover, injectable systems that can be delivered using a minimal invasive procedure and provide mechanical support the collapsing femoral head could potentially alleviate the need for surgical interventions in the future. The present review describes the limitations of existing surgical methods and the recent advances in tissue engineering that are leading in the direction of a clinically effective, translational solution for AVN in future.

Fibrinogen activates focal adhesion kinase (FAK) promoting colorectal adenocarcinoma growth.

BACKGROUND: We previously showed that fibrinogen is a major determinant of the growth of a murine model of colorectal cancer (CRC). OBJECTIVE: Our aim was to define the mechanisms coupling fibrin(ogen) to CRC growth. RESULTS: CRC tumors transplanted into the dorsal subcutis of Fib- mice were less proliferative and demonstrated increased senescence relative to those grown in Fib+ mice. RNA-seq analyses of Fib+ and Fib- tumors revealed 213 differentially regulated genes. One gene highly upregulated in tumors from Fib- mice was stratifin, encoding 14-3-3σ, a master regulator of proliferation/senescence. In a separate cohort, we observed significantly increased protein levels of 14-3-3σ and its upstream and downstream targets (i.e., p53 and p21) in tumors from Fib- mice. In vitro analyses demonstrated increased tumor cell proliferation in a fibrin printed three-dimensional environment compared with controls, suggesting that fibrin(ogen) in the tumor microenvironment promotes tumor growth in this context via a tumor cell intrinsic mechanism. In vivo analyses showed diminished activation of focal adhesion kinase (FAK), a key negative regulator of p53, in Fib- tumors. Furthermore, nuclear magnetic resonance-based metabolomics demonstrated significantly reduced metabolic activity in tumors from Fib- relative to Fib+ mice. Together, these findings suggest that fibrin(ogen)-mediated engagement of colon cancer cells activates FAK, which inhibits p53 and its downstream targets including 14-3-3σ and p21, thereby promoting cellular proliferation and preventing senescence. CONCLUSIONS: These studies suggest that fibrin(ogen) is an important component of the colon cancer microenvironment and may be exploited as a potential therapeutic target.

Elucidation of bio-inspired hydroxyapatie crystallization on oxygen-plasma modified 3D printed poly-caprolactone scaffolds.

Bioapatite formation in bones is a slow process starting with deposition of calcium phosphate and then its nucleation and crystallization into hydroxyapatite crystals. If the same process can be replicated on tissue engineered scaffolds, it will result in the formation of biomimetic bone constructs that will have comparable mechanical properties to native tissue. To mimic the same process on 3D printed polycaprolactone (PCL) scaffolds oxygen plasma treatment was performed to modify their surface chemistry. The attenuated total reflectance-fourier transform infrared (ATR-FTIR) analysis showed formation of carboxyl groups on the PCL surface with corresponding increase in roughness as analyzed by atomic force microscope (AFM) studies. A biomimetic acellular mineralization procedure was then utilized to deposit calcium minerals on these scaffolds. Though amorphous calcium phosphate was deposited on all the scaffolds with highest amount on PCL scaffolds with tricalcium phosphate (TCP), biomimetic hydroxyapatite crystals were only formed on oxygen plasma treated scaffolds, as shown by X-ray diffraction (XRD) analysis. The COOH groups on the plasma treated scaffolds acted as nucleation sites for amorphous calcium phosphate and the crystal growth was observed in the (211) plane simulating the crystal growth in developing bones. The ATR-FTIR study demonstrated the carbonated nature of these hydroxyapatite crystals mimicking that of bioapatite. The electronegative COOH groups mimic the negative amino acid side chains in collagen Type I present in bone tissue and the carbonated environment helps in creating bioapatite like deposits. The present study demonstrated the important role of PCL surface chemistry in mimicking a bone like mineralization process in vitro. This work details novel insights regarding improved mineralization of 3D printed PCL scaffolds useful for the development of more biomimetic bone constructs with improved mechanical properties.

Modulation of Macrophage Phenotype, Maturation, and Graft Integration through Chondroitin Sulfate Cross-Linking to Decellularized Cornea.

Decellularized corneas obtained from other species have gained intense popularity in the field of tissue engineering due to its role to serve as an alternative to the limited availability of high-quality donor tissues. However, the decellularized cornea is found to evoke an immune response inspite of the removal of the cellular contents and antigens due to the distortion of the collagen fibrils that exposes certain antigenic sites, which often lead to graft rejection. Therefore, in this study we tested the hypothesis that cross-linking the decellularized corneas with chondroitin sulfate may help in restoring the distorted conformationation changes of fibrous matrix and thus help in reducing the occurrence of graft rejection. Cross-linking of the decellularized cornea with oxidized chondroitin sulfate was validated by ATR-FTIR analysis. An in vitro immune response study involving healthy monocytes and differentiated macrophages with their surface marker analysis by pHrodo red, Lysotracker red, ER tracker, and CD63, LAMP-2 antibodies confirmed that the cross-linked decellularized matrices elicited the least immune response compared to the decellularized ones. We implanted three sets of corneal scaffolds obtained from goat, i.e., native, decellularized, and decellularized corneas conjugated with chondroitin sulfate into the rabbit stroma. Histology analysis, three months after implantation into the rabbit corneal stromal region, confirmed the restoration of the collagen fibril conformation and the migration of cells to the implanted constructs, affirming proper graft integration. Hence we conclude that the chondroitin sulfate cross-linked decellularized corneal matrix may serve as an efficient alternative to the allograft and human cadaveric corneas.

Effect of visco-elastic silk-chitosan microcomposite scaffolds on matrix deposition and biomechanical functionality for cartilage tissue engineering.

Commonly used polymer-based scaffolds often lack visco-elastic properties to serve as a replacement for cartilage tissue. This study explores the effect of reinforcement of silk matrix with chitosan microparticles to create a visco-elastic matrix that could support the redifferentiation of expanded chondrocytes. Goat chondrocytes produced collagen type II and glycosaminoglycan (GAG)-enriched matrix on all the scaffolds (silk:chitosan 1:1, 1:2 and 2:1). The control group of silk-only constructs suffered from leaching out of GAG molecules into the medium. Chitosan-reinforced scaffolds retained a statistically significant (p < 0.02) higher amount of GAG, which in turn significantly increased (p < 0.005) the aggregate modulus (as compared to silk-only controls) of the construct akin to that of native tissue. Furthermore, the microcomposite constructs demonstrated highly pronounced hysteresis at 4% strain up to 400 cycles, mimicking the visco-elastic properties of native cartilage tissue. These results demonstrated a step towards optimizing the design of biomaterial scaffolds used for cartilage tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

Impact of osmoregulatory agents on the recovery of collagen conformation in decellularized corneas.

The process of decellularization of the cornea leads to the removal of cells and antigens. However, during decellularization the ultrastructure of the corneal matrix is usually damaged and a secondary conformation of the collagen fibrils is modulated resulting in altered transparency and physical properties. The strategy for recovering modulation in collagen conformation may help to attain the native physical properties and transparency of the cornea. Decellularized corneas were treated with varied concentrations of glycerol and dextran, and the collagen conformation was monitored by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), x-ray diffraction (XRD) and Raman spectroscopic analysis. The peak at ~4 Å in XRD established the presence of transitional conformations that decreased with the application of osmoregulatory agents, but could not be completely eliminated. This was validated by the results of ATR-FTIR and Raman analysis. Importantly, the mechanism of this loss and the regaining of transparency has been proposed on the basis of the detachment of decorin molecules from the collagen triple helices, due to the change in collagen conformation during decellularization, and the subsequent partial reversal due to the desiccation effect of the osmoregulatory agents on collagen molecules. Taken together, collagen conformational transition can be considered as an indexing tool for the development of improved decellularization techniques.

Establishment of an in vitro monolayer model of macular corneal dystrophy.

Macular corneal dystrophy (MCD) is characterized by multiple punctate gray-white opacities in the corneal stromal region, due to the accumulation of abnormally sulfated keratan sulfates. We attempted to develop an in vitro model of MCD by simulating the sulfation inhibition using sodium chlorate, a chemical inhibitor of 3'-phosphoadenosine-5'-phosphosulfate (PAPs). The SEM and micro-Raman spectroscopy results showed the hallmark feature of MCD. Further the gene expression studies elucidated the direct effect of sulfation inhibition on the WNT pathway, that in turn downregulated production of matrix metalloproteinases (MMPs), which causes abnormal matrix deposits leading to loss of transparency in vivo. It also resulted in downregulation of integrin and cadherin complexation that leads to disruption of the epithelial layer in the MCD affected corneas. This study offers a promising initial step toward establishing a relevant in vitro MCD disease model, to assess signaling transduction pathways and devise potential treatment strategies based on MMP administration to the MCD affected corneas.

Osteogenic signaling on silk-based matrices.

Bone tissue engineering has mainly focused on generating 3D grafts to repair bone defects. However, the underlying signaling mechanisms responsible for development of such 3D bone equivalents have largely been ignored. Here we describe the crucial aspects of embryonic osteogenesis and bone development including cell sources and general signaling cascades that guide mesenchymal progenitors towards osteogenic lineage. Drawing from the knowledge of developmental biology, we then review how silk biomaterial can regulate osteogenic signaling by focusing on the expression of cell surface markers, functional genomic information (mRNA) of stem cells cultured on silk matrices. In an attempt to recapitulate exact in vivo microenvironment of osteogenesis, role of scaffold architecture and material chemistry in regulating cellular differentiation is elaborated. The generated knowledge will not only improve our understanding of cell-material interactions but reveal newer strategies beyond a conventional tissue engineering paradigm and open new prospects for developing silk-based therapies against clinically relevant bone disorders.

Role of chondroitin sulphate tethered silk scaffold in cartilaginous disc tissue regeneration.

Strategies for tissue engineering focus on scaffolds with tunable structure and morphology as well as optimum surface chemistry to simulate the anatomy and functionality of the target tissue. Silk fibroin has demonstrated its potential in supporting cartilaginous tissue formation both in vitro and in vivo. In this study, we investigate the role of controlled lamellar organization and chemical composition of biofunctionalized silk scaffolds in replicating the structural properties of the annulus region of an intervertebral disc using articular chondrocytes. Covalent attachment of chondroitin sulfate (CS) to silk is characterized. CS-conjugated silk constructs demonstrate enhanced cellular metabolic activity and chondrogenic redifferentiation potential with significantly improved mechanical properties over silk-only constructs. A matrix-assisted laser desorption ionization-time of flight analysis and protein-protein interaction studies help to generate insights into how CS conjugation can facilitate the production of disc associated matrix proteins, compared to a silk-only based construct. An in-depth understanding of the interplay between such extra cellular matrix associated proteins should help in designing more rational scaffolds for cartilaginous disc regeneration needs.

Modulation of Self-Assembly Process of Fibroin: An Insight for Regulating the Conformation of Silk Biomaterials.

Controlling the mechanism of self-assembly in proteins has emerged as a potent tool for various biomedical applications. Silk fibroin self-assembly consists of gradual conformational transition from random coil to ß-sheet structure. In this work we elucidated the intermediate secondary conformation in the presence of Ca(2+) ions during fibroin self-assembly. The interaction of fibroin and calcium ions resulted in a predominantly α-helical intermediate conformation, which was maintained to certain extent even in the final conformation as illustrated by circular dichroism and attenuated total reflectance-Fourier transform infrared spectroscopy. Further, to elucidate the mechanism behind this interaction molecular modeling of the N-terminal region of fibroin with Ca(2+) ions was performed. Negatively charged glutamate and aspartate amino acids play a key role in the electrostatic interaction with positively charged calcium ions. Therefore, insights about modulation of self-assembly mechanism of fibroin could potentially be utilized to develop silk-based biomaterials consisting of the desired secondary conformation.

Glucosamine loaded injectable silk-in-silk integrated system modulate mechanical properties in bovine ex-vivo degenerated intervertebral disc model.

Injectable hydrogels offer a tremendous potential for treatment of degenerated intervertebral disc due to their ability to withstand complex loading, conforming precisely to the defect spaces and eliminating the need for invasive surgical procedures. We have developed an injectable hydrogel platform of N-acetyl-glucosamine (GlcNAc) loaded silk hollow spheres embedded in silk hydrogel for in situ therapeutic release and enhanced mechanical strength. The assembled silk hydrogel provided adequate structural support to the ex vivo degenerated disc model in a cyclic compression test at par with the native tissue. Spatiotemporal release of GlcNAc in a controlled manner from the silk hollow microspheres trigger enhanced proteoglycan production from ADSCs embedded in the composite system. Role of MAPK and SMAD pathways in increasing proteoglycan production have been explored by immunohistological analysis as a result of the action of GlcNAc on the cells, elucidating the potential of injectable silk microsphere-in-silk hydrogel for the regeneration of degenerated disc tissue.

Tissue engineering strategies to study cartilage development, degeneration and regeneration.

Cartilage tissue engineering has primarily focused on the generation of grafts to repair cartilage defects due to traumatic injury and disease. However engineered cartilage tissues have also a strong scientific value as advanced 3D culture models. Here we first describe key aspects of embryonic chondrogenesis and possible cell sources/culture systems for in vitro cartilage generation. We then review how a tissue engineering approach has been and could be further exploited to investigate different aspects of cartilage development and degeneration. The generated knowledge is expected to inform new cartilage regeneration strategies, beyond a classical tissue engineering paradigm.

Matrix-embedded cytokines to simulate osteoarthritis-like cartilage microenvironments.

In vivo, cytokines noncovalently bind to the extracellular matrix (ECM), to facilitate intimate interactions with cellular receptors and potentiate biological activity. Development of a biomaterial that simulates this type of physiological binding and function is an exciting proposition for designing controlled advanced delivery systems for simulating in vivo conditions in vitro. We have decorated silk protein with sulfonated moieties through diazonium coupling reactions to noncovalently immobilize pro-inflammatory cytokines interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) in such a biomimetic manner. After adsorption of the cytokines to the diazonium-modified silk matrix, constant release of cytokines up to at least 3 days was demonstrated, as an initial step to simulate an osteoarthritic (OA) microenvironment in vitro. Matrix-embedded cytokines induced the formation of multiple elongated processes in chondrocytes in vitro, akin to what is seen in OA cartilage in vivo. Gene expression profiles with this in vitro tissue model of OA showed significant similarities to profiles from explanted OA cartilage tissues collected from patients who underwent total knee replacement surgery. The common markers of OA, including COL, MMP, TIMP, ADAMTS, and metallothioneins, were upregulated at least 35-fold in the in vitro model when compared to the control-non-OA in vitro generated tissue-engineered cartilage. The microarray data were validated by reverse transcriptase-polymerase chain reaction. Mechanistically, protein interaction studies indicated that TNF-α and IL-1ß synergistically controlled the equilibrium between MMPs and their inhibitors, TIMPs, resulting in ECM degradation through the MAPK pathway. This study offers a promising initial step toward establishing a relevant in vitro OA disease model, which can be further modified to assess signaling mechanisms, responses to cell or drug treatments and patient-specific features.