Stem cell-based therapies have evolved to become a key component of regenerative medicine approaches to human pathologies. Exogenous stem cell transplantation takes advantage of the potential of stem cells to self-renew, differentiate, home to sites of injury, and sufficiently evade the immune system to remain viable for the release of anti-inflammatory cytokines, chemokines, and growth factors. Common to many pathologies is the exacerbation of inflammation at the injury site by proinflammatory macrophages. An increasing body of evidence has demonstrated that mesenchymal stromal cells (MSCs) can influence the immunophenotype and function of myeloid lineage cells to promote therapeutic effects. Understanding the degree to which MSCs can modulate the phenotype of macrophages within an inflammatory environment is of interest when considering strategies for targeted cell therapies. There is a critical need for potency assays to elucidate these intercellular interactions in vitro and provide insight into potential mechanisms of action attributable to the immunomodulatory and polarizing capacities of MSCs, as well as other cells with immunomodulatory potential. However, the complexity of the responses, in terms of cell phenotypes and characteristics, timing of these interactions, and the degree to which cell contact is involved, have made the study of these interactions challenging. To provide a research tool to study the direct interactions between MSCs and macrophages, we developed a potency assay that directly co-cultures MSCs with naïve macrophages under proinflammatory conditions. Using this assay, we demonstrated changes in the macrophage secretome and phenotype, which can be used to evaluate the abilities of the cell samples to influence the cell microenvironment. These results suggest the immunomodulatory effects of MSCs on macrophages while revealing key cytokines and phenotypic changes that may inform their efficacy as potential cellular therapies. Key features • The protocol uses monocytes differentiated into naïve macrophages, which are loosely adherent, have a relatively homogeneous genetic background, and resemble peripheral blood mononuclear cells-derived macrophages. • The protocol requires a plate reader and a flow cytometer with the ability to detect six fluorophores. • The protocol provides a quantitative measurement of co-culture conditions by the addition of a fixed number of freshly thawed or culture-rescued MSCs to macrophages. • This protocol uses assessment of the secretome and cell harvest to independently verify the nature of the interactions between macrophages and MSCs.

Streamlined procedures for processing and cryopreservation of cell therapies using good laboratory practices are integral to biomanufacturing process development and clinical applications. The protocol herein begins with the preparation of human cell types cultured as adherent (i.e., mesenchymal stromal cells, MSCs) or suspension cells (i.e., peripheral blood mononuclear cells, PBMCs) to comprehensively demonstrate procedures that are applicable to commonly used primary cell cultures. Cell processing steps consist of preparing high yields of cells for cryopreservation using instruments routinely used in cell manufacturing, including the Finia® Fill and Finish System and a controlled-rate freezer. The final steps comprise the storage of cells at subzero temperatures in liquid nitrogen vapor phase followed by the analysis of cell phenotypes before and after processing and cryopreservation, along with cell quality metrics for validation. Additionally, the protocol includes important considerations for the implementation of quality control measures for equipment operation and cell handling, as well as Good Laboratory Practices for cell manufacturing, which are essential for the translational use of cell therapies. Key features • The protocol applies to small- or large-scale manufacturing of cell therapy products. • It includes streamlined procedures for processing and cryopreservation of cells cultured as adherent cells (MSCs) and suspension cells (PBMCs). • Provides temperature control and rapid partitioning of sample in cryopreservation solution to maintain high viability of a range of cell types throughout the procedures. • This protocol employs the Finia® Fill and Finish System and a controlled-rate freezer. Graphical overview.

453Three-dimensional bioprinting (3DBP) is an additive manufacturing technique that has emerged as a promising strategy for the fabrication of scaffolds, which can successfully recapitulate the architectural, biochemical, and physical cues of target tissues. More importantly, 3DBP offers fine spatiotemporal control and high submicron scale resolution, which can be leveraged for the incorporation and directional gradient release of single or multiple biomimetic cues, including cell-derived exosomes (EXOs). EXOs are extracellular vesicles that originate from the endosomal compartment of various cell types, with sizes ranging from 30 to120 nm. They act as cell mediators and contain discrete cell constituents, including growth factors, cytokines, lipid moieties, nucleic acids, metabolites, and cell surface markers, depending on the cell type. Essentially, owing to their therapeutic potential, EXOs derived from mesenchymal stem cells (MSCs) have been recently investigated in several clinical trials for the treatment of various conditions, including cancer, diabetes, dry eyes, periodontitis, and acute ischemic stroke. The 3DBP strategy of EXOs is especially useful in tissue engineering and regenerative medicine applications, as tissues can be biofabricated to closely mimic the complex microarchitecture and developmental profiles of native heterogeneous tissues for restoring biological functions. Moreover, EXOs can be manipulated to carry exogenous cargo such as genes or proteins of therapeutic interest, confer multifunctional attributes, and further enhance their tissue regenerative potential. However, significant challenges, including the selection of appropriate bioink, pattern resolution, engineering-defined exosomal gradient, spatial presentation and modulation of EXO release kinetics, as well as EXO stability and storage conditions, must be addressed for the successful translation of therapeutic grade EXOs to clinical settings. In this review, we highlight the recent advances and offer future perspectives on the bioprinting of EXOs as regenerative biotherapeutics for the fabrication of complex heterogeneous tissues that are suitable for clinical transplantation.

Corneal diseases are the third most prevalent cause of blindness after cataract and glaucoma. It is estimated that about 5 million people in the world are affected by bilateral corneal blindness with an additional 23 million with unilateral blindness. Cornea transplantation is the standard practice for the management of various cornea related pathologies like fibrosis, ulcers, keratitis, etc. The high transplant cost, increased risk of graft failure/rejection, and long waiting list due to limited availability of good quality donor cornea imposes a huge clinical burden. Recently, biofabrication technologies are gaining a lot of attention because of their potential to direct hierarchical assembly of three-dimensional (3D) biological structures for tissue construction for various biomedical and clinical applications. In this regard, 3D bioprinting, which involves layer-by-layer deposition of acellular or cell-laden bioink in a specific pattern corresponding to the organotypic morphology of tissues/organs, has been extensively investigated for the fabrication of corneal substitutes. In addition to this methodology, novel biofabrication techniques have been explored for the fabrication of corneal tissues using bioinks with optical and mechanical performances comparable to native cornea tissue. In this review, we highlight the recent advances and offer future perspectives in the fabrication of corneal tissue equivalents that can be potentially employed for effective clinical repair, reconstruction, and regeneration of the cornea.

Functionally graded materials (FGMs) composed of a polymer matrix embedded with calcium phosphate particles are preferred for bone tissue engineering, as they can mimic the hierarchical and gradient structure of bones. In this study, we report the design and development of a FGM based on thiolated poly(vinyl alcohol) (TPVA) and nano-hydroxyapatite (nano-HA) with graded bioactivity, cell compatibility, and degradability properties that are conducive for bone regeneration. The polymer matrix comprises crosslinked poly(vinyl alcohol) with ester and thioether linkages formed via the thiol-ene click reaction, avoiding undesired additives and byproducts. Freshly precipitated and spray-dried HA was mixed with the TPVA hydrogel, and layers of varying concentrations were cast. Upon lyophilization, the hydrogel structure yielded porous sheets of the graded composite of TPVA and nano-HA. The new FGM showed higher values of tensile strength and degradation in phosphate buffer saline (PBS) in vitro, compared to bare TPVA. The bioactive nature of the FGM was confirmed through bioactivity studies in simulated body fluid (SBF), while cytocompatibility was demonstrated with human periodontal ligament cells in vitro. Cumulatively, our results indicate that based on the composition, mechanical properties, bioactivity, and cytocompatibility, the fabricated TPVA-HA composites can find potential use as guided bone regeneration (GBR) membranes.

Biological materials derived from extracellular matrix (ECM) proteins have garnered interest as their composition is very similar to that of native tissue. Herein, we report the use of human cornea derived decellularized ECM (dECM) microparticles dispersed in human fibrin sealant as an accessible therapeutic alternative for corneal anterior stromal reconstruction. dECM microparticles had good particle size distribution (≤10 µm) and retained the majority of corneal ECM components found in native tissue. Fibrin–dECM hydrogels exhibited compressive modulus of 70.83 ± 9.17 kPa matching that of native tissue, maximum burst pressure of 34.3 ± 3.7 kPa, and demonstrated a short crosslinking time of ~17 min. The fibrin–dECM hydrogels were found to be biodegradable, cytocompatible, non-mutagenic, non-sensitive, non-irritant, and supported the growth and maintained the phenotype of encapsulated human corneal stem cells (hCSCs) in vitro. In a rabbit model of anterior lamellar keratectomy, fibrin–dECM bio-adhesives promoted corneal re-epithelialization within 14 days, induced stromal tissue repair, and displayed integration with corneal tissues in vivo. Overall, our results suggest that the incorporation of cornea tissue-derived ECM microparticles in fibrin hydrogels is non-toxic, safe, and shows tremendous promise as a minimally invasive therapeutic approach for the treatment of superficial corneal epithelial wounds and anterior stromal injuries.

Direct real-time measurement of nitric oxide (NO) in living cells has proven quite challenging, owing in part to the lack of tools that are selective and sensitive to measure intracellular concentrations of NO. Herein we report the synthesis and characterization of polyvinyl alcohol (PVA) based nanosensors for fluorescence imaging of cytosolic NO using an o-phenylenediamine-rhodamine (OPD-RhB) platform. More specifically, thiol-functionalized PVA incorporating RhB conjugated OPD was disulfide crosslinked to yield NO-responsive nanosensors. The polymeric nanosensors were anionic, averaged 170 nm in hydrodynamic size, and exhibited linear increases in fluorescence intensity (FLI) to micro- and nanomolar concentrations of NO in a sodium nitroprusside (SNP) concentration-dependent manner. In the presence of SNP, the engineered nanosensors demonstrated physical stability at extracellular glutathione (GSH) conditions, while favoring NO detection at cytoplasmic GSH conditions. In addition, the PVA-based nanosensors were non-cytotoxic, cell membrane-permeable and demonstrated hydrogen peroxide-dependent FL increases upon incubation with activated synoviocytes in vitro. Most notably, NO-induced cell FLIs correlated strongly with total nitrite/nitrate content of conventional Griess assays with Pearson correlation coefficients of 0.96. Comprehensively, our results show that OPD-RhB-conjugated PVA nanosensors offer real-time imaging of NO with high sensitivity in living cells that can be employed for direct quantification of NO.

Biomedical research in health innovation and product development encompasses convergent technologies that primarily integrate biomaterials science and engineering at its core. Particularly, research in this area is instrumental for the implementation of biomedical devices (BMDs) that offer innovative solutions to help maintain and improve quality of life of patients worldwide. Despite achieving extraordinary success, implantable BMDs are still confronted with complex engineering and biological challenges that need to addressed for augmenting device performance and prolonging lifetime in vivo. Biofabrication of tissue constructs, designing novel biomaterials and employing rational biomaterial design approaches, surface engineering of implants, point of care diagnostics and micro/nano-based biosensors, smart drug delivery systems, and noninvasive imaging methodologies are among strategies exploited for improving clinical performance of implantable BMDs. In India, advances in biomedical technologies have dramatically advanced health care over the last few decades and the country is well-positioned to identify opportunities and translate emerging solutions. In this article, we attempt to capture the recent advances in biomedical research and development progressing across the country and highlight the significant research work accomplished in the areas of biomaterials science and engineering.

We report the preparation of protein encapsulated amino acid derived redox-responsive nanoparticles (NPs) as effective nanocarriers for intracellular delivery of proteins. More specifically, acryloyl derivatives of lysine, ornithine, cystine and cystamine, were employed as monomers and disulfide crosslinkers for non-covalent encapsulation of model protein bovine serum albumin (BSA) and were interfacially crosslinked via free radical polymerization to form redox-responsive protein NPs. Notably, prepared NPs exhibited high protein loading content between 37 and 45%, averaged similar to 400 nm in hydrodynamic size and possessed a mean surface charge of -15 mV. Furthermore, blank polymeric NPs displayed exceptional cytocompatibility with cell viability exceeding 92% at concentrations as high as 4 mg/mL, while redox-responsive protein NPs displayed glutathione (GSH)-dependent BSA release behavior in vitro. Additionally, cellular uptake studies confirmed that protein NPs entered MDA-MA-231 cells predominantly via the endocytic pathway. Upon cellular internalization, redox-responsive NPs delivered protein into cytosol of cells within 60 min demonstrating intrinsic endosomolytic characteristics and efficient protein release under cytoplasmic high GSH conditions. Most importantly, insulin analog-loaded NPs significantly increased glucose consumption in HepG2 cultures confirming protein stability and retention of protein function. Cumulatively, our approach presents a simple yet effective strategy for intracellular delivery of biologically active proteins for various biomedical applications. (C) 2018 Elsevier B.V. All rights reserved.

Biomaterial-associated aseptic inflammation is a major cause for limiting integration, biological performance, and reduced lifespan of implanted medical devices in vivo. Implantation of biomaterial elicits a strong foreign body response (FBR) which is associated with recruitment of macrophages and neutrophils at the implant site. Activated proinflammatory cells, such as macrophages and neutrophils, secrete a wide gamut of inflammatory mediators including cytokines, chemokines, matrix metalloproteases and extracellular matrix proteins, reactive oxygen species, and signaling molecules that promote and sustain host inflammatory responses towards implanted biomaterials. Currently, assessment of tissue responses to biomaterial implants primarily rely on endpoint analyses such as histology, immunohistochemistry and biochemical assays which are inherently invasive, destructive and time-consuming by nature. Hence, there is a great need for developing fast, reliable, non-invasive, longitudinal imaging methodologies for monitoring implant-associated inflammation in vivo. Such imaging methodologies could greatly improve the diagnosis and augment therapeutic approaches to mitigate the severity of device-associated inflammation. In this chapter, the various preclinical imaging modalities that are currently being explored for non-invasive imaging of aseptic biomaterial-associated inflammation are detailed. Particularly, imaging methodologies that target diverse aspects of implant-associated FBR including inflammatory cell imaging, macromolecular protein imaging, and small molecule imaging are reviewed.

We report a new prodrug micelle-based approach in which a model hydrophobic non-steroidal anti-inflammatory drug (NSAID), ibuprofen (Ibu), is tethered to amphiphilic methoxy polyethylene glycol-polypropylene fumarate (mPEG-PPF) diblock copolymer via hydrolytic anhydride linkages for potential controlled release applications of NSAIDs. Synthesized mPEG-PPF-Ibu polymer drug conjugates (PDCs) demonstrated high drug conjugation efficiency (∼90%) and self-assembled to form micellar nanostructures in aqueous medium with critical micelle concentrations ranging between 16 and 30μg/mL. The entrapment efficiency of Ibu in prepared PDC micelles was as high as 18% (w/w). Crosslinking of prodrug micelles with N,N′-dimethylaminoethyl methacrylate conferred pH-responsive characteristics. pH-responsive PDC micelles averaged 100nm in size at pH 7.4 and exhibited concomitant changes in size upon incubation in physiologically relevant mildly acidic conditions. Ibu release was observed to increase with increasing acidic conditions and could be controlled by varying the amount of crosslinker used. Furthermore, the prepared mPEG-PPF-based micelles demonstrated excellent cytocompatibility and cellular internalization in vitro. More importantly, PDC micelles exerted anti-inflammatory effects by significantly decreasing monosodium urate crystal-induced prostaglandin E2 levels in rabbit synoviocyte cultures in vitro. Cumulatively, our results indicate that this new prodrug micelle approach is promising for NSAID-based therapies in the treatment of arthritis and cancer.

Injectable in situ crosslinking hydrogels offer unique advantages over conventional prefabricated hydrogel methodologies. Herein, we synthesize poly(xylitol-co-maleate-co-PEG) (pXMP) macromers and evaluate their performance as injectable cell carriers for tissue engineering applications. The designed pXMP elastomers were non-toxic and water-soluble with viscosity values permissible for subcutaneous injectable systems. pXMP-based hydrogels prepared via free radical polymerization with acrylic acid as crosslinker possessed high crosslink density and exhibited a broad range of compressive moduli that could match the natural mechanical environment of various native tissues. The hydrogels displayed controlled degradability and exhibited gradual increase in matrix porosity upon degradation. The hydrophobic hydrogel surfaces preferentially adsorbed albumin and promoted cell adhesion and growth in vitro. Actin staining on cells cultured on thin hydrogel films revealed subconfluent cell monolayers composed of strong, adherent cells. Furthermore, fabricated 3D pXMP cell-hydrogel constructs promoted cell survival and proliferation in vitro. Cumulatively, our results demonstrate that injectable xylitol-PEG-based hydrogels possess excellent physical characteristics and exhibit exceptional cytocompatibility in vitro. Consequently, they show great promise as injectable hydrogel systems for in situ tissue repair and regeneration. (C) 2014 Elsevier B.V. All rights reserved.

Composite materials based on bioceramics and polymers offer excellent opportunities in the quest for developing optimal bone grafts for bone tissue engineering. Herein, we have functionalized nano hydroxyapatite with citric acid and subsequently cross-linked with poly(propylene fumarate) and poly(ethylene glycol) to afford a composite with better interfacial bonding properties. This study involved two biomimetic composites, 3CP-VP and 5CP-VP, prepared by varying the concentration of hydroxyapatite. Uniform homogenous distribution of hydroxyapatite was identified through Raman spectral imaging in both the composite matrices. The compressive moduli of the biomimetic composites after 4-week immersion in phosphate-buffered saline ranged between 100 and 300 MPa, which falls well within the accepted values reported for human trabecular bone. Moreover, biodegradation studies revealed only an average weight loss of 10%–17% during the 7-week time period. Furthermore, apatite mineralization was evaluated using scanning electron microscopy and energy dispersive X-ray analysis, and contact angle measurements revealed hydrophobic surfaces with preferential adsorption to albumin. More importantly, blood compatibility studies demonstrated no significant hemolysis and no visible red blood cell aggregation, while cytotoxicity evaluation via direct contact, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and live–dead assays on human osteoblast sarcoma cell line exhibited good biocompatibility with negligible cytotoxicity. In addition, in vitro drug release studies with gentamycin-loaded composites demonstrated a controlled and sustained release profile with about 35% of drug released over a period of 2 weeks. These findings show that these composites could be developed into stand-alone bone substitutes for bone tissue engineering coupled with drug delivery applications.