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The secretome of mesenchymal stem cells (MSCs) is being studied for its regenerative potential for the treatment of various disorders, including bone diseases. However, mimicking the physiological parameters of native bone could further improve MSCs' secretory profile. The proteomic analysis revealed that MSCs have a diverse secretory profile depending on the cell formats used to grow them, such as two-dimensional (2D) or three-dimensional (3D) microenvironments. Stem cells are given biochemical and biophysical stimuli in a 3D milieu that mimics

The skin is one of the most essential tissues in the human body, interacting with the outside environment and shielding the body from diseases and excessive water loss. Hydrogels, decellularized porcine dermal matrix, and lyophilized polymer scaffolds have all been used in studies of skin wound repair, wound dressing, and skin tissue engineering, however, these materials cannot replicate the nanofibrous architecture of the skin's native extracellular matrix (ECM). Electrospun nanofibers are a fascinating new form of nanomaterials with tremendous potential across a broad spectrum of applications in the biomedical field, including wound dressings, wound healing scaffolds, regenerative medicine, bioengineering of skin tissue, and multifaceted drug delivery. This article reviews recent in vitro and in vivo developments in multifunctional electrospun nanofibers (MENs) for wound healing. This review begins with an introduction to the electrospinning process, its principle, and the processing parameters which have a significant impact on the nanofiber properties. It then discusses the various geometries and advantages of MEN scaffolds produced by different innovative electrospinning techniques for wound healing applications when used in combination with stem cells. This review also discusses some of the possible future nanofiber-based models that could be used. Finally, we conclude with potential perspectives and conclusions in this area.

Cancer has recently been the second leading cause of death worldwide, trailing only cardiovascular disease. Cancer stem cells (CSCs), represented as tumor-initiating cells (TICs), are mainly liable for chemoresistance and disease relapse due to their self-renewal capability and differentiating capacity into different types of tumor cells. The intricate molecular mechanism is necessary to elucidate CSC's chemoresistance properties and cancer recurrence. Establishing efficient strategies for CSC maintenance and enrichment is essential to elucidate the mechanisms and properties of CSCs and CSC-related therapeutic measures. Current approaches are insufficient to mimic theiin vivo/ichemical and physical conditions for the maintenance and growth of CSC and yield unreliable research results. Biomaterials are now widely used for simulating the bone marrow microenvironment. Biomaterial-based three-dimensional (3D) approaches for the enrichment of CSC provide an excellent promise for future drug discovery and elucidation of molecular mechanisms. In the future, the biomaterial-based model will contribute to a more operative and predictive CSC model for cancer therapy. Design strategies for materials, physicochemical cues, and morphology will offer a new direction for future modification and new methods for studying the CSC microenvironment and its chemoresistance property. This review highlights the critical roles of the microenvironmental cues that regulate CSC function and endow them with drug resistance properties. This review also explores the latest advancement and challenges in biomaterial-based scaffold structure for therapeutic approaches against CSC chemoresistance. Since the recent entry of extracellular vesicles (EVs), cell-derived nanostructures, have opened new avenues of investigation into this field, which, together with other more conventionally studied signaling pathways, play an important role in cell-to-cell communication. Thus, this review further explores the subject of EVs in-depth. This review also discusses possible future biomaterial and biomaterial-EV-based models that could be used to study the tumor microenvironment (TME) and will provide possible therapeutic approaches. Finally, this review concludes with potential perspectives and conclusions in this area.

Strategies involving the inclusion of cell-instructive chemical and topographical cues to smart biomaterials in combination with a suitable physical stimulus may be beneficial to enhance nerve-regeneration rate. In this regard, we investigated the surface functionalization of poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV)-based electroconductive electrospun nanofibers coupled with externally applied electrical stimulus for accelerated neuronal growth potential. In addition, the voltage-dependent conductive mechanism of the nanofibers was studied in depth to interlink intrinsic conductive properties with electrically stimulated neuronal expressions. Surface functionalization was accomplished using 3-aminopropyltriethoxysilane (APTES) and 1,6-hexanediamine (HDA) as an alternative to costly biomolecule coating (e.g., collagen) for cell adhesion. The nanofibers were uniform, porous, electrically conductive, mechanically strong, and stable under physiological conditions. Surface amination boosted biocompatibility, 3T3 cell adhesion, and spreading, while the neuronal model rat PC12 cell line showed better differentiation on surface-functionalized mats compared to nonfunctionalized mats. When coupled with electrical stimulation (ES), these mats showed comparable or faster neurite formation and elongation than the collagen-coated mats with no-ES conditions. The findings indicate that surface amination in combination with ES may provide an improved strategy to faster nerve regeneration using MEH-PPV-based neural scaffolds.

The effective control of microbial and metabolically derived biological toxins which negatively impact physical health remains a key challenge for the 21st century. 2-Dimensional graphene and MXene nanomaterials are relatively new additions to the field of biomedical materials with superior external surface areas suited to adsorptive remediation of biological toxins. However, relatively little is known about their physiological interactions with biological systems and, to date, no comparative biological studies have been done. This study compares titanium carbide MXene (Ti3C2Tx) in multilayered and delaminated forms with graphene variants to assess the impact of variable physical properties on cellular inflammatory response to endotoxin stimulus. No significant impact on cell metabolism or induction of inflammatory pathways leading to cell death was observed. No significant increase in markers of blood cell activation and haemolysis occurred. Whilst graphene nanoplatelets (GNP), graphene oxide (GO) and Ti3C2Tx showed insignificant antibacterial activity towards Escherichia coli, silver nanoparticle-modified GO (GO-Ag) induced bacterial cell death and at a lower dose than silver nanoparticles. All nanomaterials significantly reduced bacterial endotoxin induced THP-1 monocyte IL-8, IL-6 and TNF-α cytokine production by >99%, >99% and >80% respectively, compared to control groups. This study suggests the utility of these nanomaterials as adsorbents in blood contacting medical device applications for removal of inflammatory cytokines linked to poor outcome in patients with life-threatening infection.

Designing a temporary substrate to adhere, multiply and form new bone tissue for the bone-forming cells is a challenge in bone tissue engineering. In this article, a new biodegradable and composite...

Presently donor-derived platelets used in the clinic are associated with concerns about adequate availability, expense, risk of bacterial contamination and complications due to immunological reaction. To prevail over our dependence on transfusion of donor-derived platelets, efforts are being made to generate them in vitro. Development of biomaterials that support or mimic bone marrow niche micro-environmental cues could improve the in vitro production of platelets from megakaryocytes (MKs) derived from various stem cell sources. In spite of significant advances in the production of MKs from various stem cell sources using 2D as well as 3D culture approaches in vitro and the development of biomaterials-based platelet systems, yield and quality of these platelets remains unsuitable for clinical use. Thus, in vitro production of clinically useful platelets on a large scale remains an unmet target to date. This review summarizes the most frequently used 2D and 3D approaches to generate MKs and platelets in vitro, emphasizing the importance of mimicking in vivo micro-environment. Further, this review proposes the use of interpenetrating network (IPN) biomaterial-based approach as a promising strategy for improving the generation of MK and platelets in sufficient numbers in vitro. STATEMENT OF SIGNIFICANCE: Thrombocytopenia is one of the major global health and socio-economic problems. Transfusion with donor-derived platelets (PLTs) is the only effective treatment for this condition. However, this approach is limited by factors like short shelf-life of PLTs, PLT activation, alloimmunization, risk of bacterial contamination, infection etc. In vitro generated MKs and PLTs derived from non-donor-dependent sources may help to overcome the platelet transfusion concerns. Here we have reviewed various 2D and 3D strategies used for in vitro generation of MKs and PLTs, with special emphasis on various biomaterial platforms and different physico/chemical cues being used for the purpose. We have also proposed a biomaterial-based approach of using interpenetrating network (IPN) for generating clinically relevant numbers of MKs and PLTs.

This review discusses designing novel biomaterial-based hematopoietic stem cell (HSC) expansion strategies that would contribute to the field of hematopoiesis and also proposes possible approaches for HSC expansion using interpenetrating network hydrogels, emulsion templated polymers poly(HIPEs) (high internal phase emulsion templated polymers), and three-dimensional cell printing, which could provide optimal environment for HSC attachment, proliferation, and differentiation. These novel approaches could improve the efficacy of bone marrow transplantation and also offer new insights in the field of regenerative biology.

The efficacy of cell-based therapies as an alternative to autologous bone grafts requires biomaterials to localize cells at the defect and drive osteogenic differentiation. Hydrogels are ideal cell delivery vehicles that can provide instructional cues via their composition or mechanical properties but commonly lack osteoconductive components that nucleate mineral. To address this challenge, we entrapped mesenchymal stromal cells (MSCs) in a composite hydrogel based on two naturally-derived polymers (alginate and hyaluronate) containing biomineralized polymeric microspheres. Mechanical properties of the hydrogels were dependent upon composition. The presentation of the adhesive tripeptide Arginine-Glycine-Aspartic Acid (RGD) from both polymers induced greater osteogenic differentiation of ovine MSCs in vitro compared to gels formed of RGD-alginate or RGD-alginate/hyaluronate alone. We then evaluated the capacity of this construct to stimulate bone healing when transplanting autologous, culture-expanded MSCs into a surgical induced, critical-sized ovine iliac crest bone defect. At 12 weeks post-implantation, defects treated with MSCs transplanted in composite gels exhibited significant increases in blood vessel density, osteoid formation, and bone formation compared to acellular gels or untreated defects. These findings demonstrate the capacity of osteoconductive hydrogels to promote bone formation with autologous MSCs in a large animal bone defect model and provide a promising vehicle for cell-based therapies of bone healing.

The wearable artificial kidney can deliver continuous ambulatory dialysis for more than 3 million patients with end-stage renal disease. However, the efficient removal of urea is a key challenge in miniaturizing the device and making it light and small enough for practical use. Here, we show that two-dimensional titanium carbide (MXene) with the composition of Ti3C2Tx, where Tx represents surface termination groups such as −OH, −O–, and −F, can adsorb urea, reaching 99% removal efficiency from aqueous solution and 94% from dialysate at the initial urea concentration of 30 mg/dL, with the maximum urea adsorption capacity of 10.4 mg/g at room temperature. When tested at 37 °C, we achieved a 2-fold increase in urea removal efficiency from dialysate, with the maximum urea adsorption capacity of 21.7 mg/g. Ti3C2Tx showed good hemocompatibility; it did not induce cell apoptosis or reduce the metabolizing cell fraction, indicating no impact on cell viability at concentrations of up to 200 μg/mL. The biocompatibi...

In the present study, a conducting polymer, poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) along with a biodegradable polymer poly(ε-caprolactone) (PCL) was used to prepare an electrically conductive, biocompatible, bioactive, and biodegradable nanofibrous scaffold for possible use in neural tissue engineering applications. Core-sheath electrospun nanofibers of PCL as the core and MEH-PPV as the sheath, were surface-functionalized with (3-aminopropyl) triethoxysilane (APTES) and 1,6-hexanediamine to obtain amine-functionalized surface to facilitate cell-biomaterial interactions with the aim of replacing the costly biomolecules such as collagen, fibronectin, laminin, and arginyl-glycyl-aspartic acid (RGD) peptide for surface modification. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) confirmed the formation of core-sheath morphology of the electrospun nanofibers, whereas Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) revealed successful incorporation of amine functionality after surface functionalization. Adhesion, spreading, and proliferation of 3T3 fibroblasts were enhanced on the surface-functionalized electrospun meshes, whereas the neuronal model rat pheochromocytoma 12 (PC12) cells also adhered and differentiated into sympathetic neurons on these meshes. Under a constant electric field of 500 mV for 2 h/day for 3 consecutive days, the PC12 cells displayed remarkable improvement in the neurite formation and outgrowth on the surface-functionalized meshes that was comparable to those on the collagen-coated meshes under no electrical signal. Electrical stimulation studies further demonstrated that electrically stimulated PC12 cells cultured on collagen I coated meshes yielded more and longer neurites than those of the unstimulated cells on the same scaffolds. The enhanced neurite growth and differentiation suggest the potential use of these scaffolds for neural tissue engineering applications.

Whilst various remedial human monoclonal antibodies have been developed to treat the potentially life-threatening systemic complications associated with anthrax infection, an optimal and universally effective administration route has yet to be established. In the later stages of infection when antibody administration by injection is more likely to fail one possible route to improve outcome is via the use of an antibody-bound, adsorbent haemoperfusion device. We report here the development of an adsorbent macroporous polymer column containing immobilised B. anthracis exotoxin-specific antibodies, PANG (a non-glycosylated, version of a plant-produced human monoclonal antibody) and Valortim (a fully human monoclonal N-linked glycosylated antibody), for removal of anthrax protective antigen (PA) from freshly frozen human plasma and human whole blood. In addition, we have demonstrated that continuous extracorporeal blood recirculation through a Valortim-bound haemoperfusion column significantly reduced the blood plasma concentration of anthrax PA over 2 hours using an in vivo PA rat infusion model. This work provides proof-of-concept evidence to support the development of such alternative detoxification platforms.

Hydrogels are attractive for tissue engineering applications due to their incredible versatility, but their use is limited by inadequate mechanical strength and poor biocompatibility. In this study, to better mimic the mineral component and the mechanical strength of natural bone, two biocompatible materials, 2-hydroxyethyl agarose and poly(ethylene glycol) diacrylate, were combined with nanocrystalline hydroxyapatite (nHAp)-coated poly(lactic-co-glycolic acid) (PLGA) microspheres. A novel composite interpenetrating network (IPN) hydrogel scaffold was created to investigate its mechanical and osteoconductive performance for bone tissue engineering-related applications. The inclusion of nHAp-coated PLGA microspheres in an IPN hydrogel led to an increase in compressive modulus. In the absence of nHAp-coated microspheres, cell viability dropped to 59·1% at 3 weeks post-encapsulation. However, by incorporating nHAp-coated microspheres, cell viability improved to 80·6%. The capacity of composite IPN hydrogels to promote bone formation in cell culture was assessed. In the presence of mineralised microspheres, a composite IPN gel showed a significant increase in alkaline phosphatase activity and calcium (Ca) deposition following 3 weeks of incubation when compared with plain IPNs. This technology may be also applied to other cell-based applications where the improved mechanical integrity and osteoconductivity of cell-containing IPN hydrogels may be used to mimic bone tissue replacement.

The conventional treatments for critical-sized bone defect repairs, such as autograft or allograft of bone are expensive procedures. Hence, developing better and economically feasible biomaterial-based alternatives for bone tissue engineering (BTE) becomes imperative. The mesenchymal stem cells (MSCs) are used for injured or damaged bone repairs and regeneration, due to their ability to proliferate, self-renew and differentiate into several cell lineages. This regenerative potential of MSCs happens through paracrine mode via their secretome comprising both, extracellular vesicles (EVs) and soluble factors. The recent trend shows that the cells could be replaced by MSC-derived EVs, including microvesicles and exosomes, as a “cell-free therapy”. Using EVs as a cell-free biologic could reduce complications associated with MSC transplantation. To retain at the site of injury, EVs could be encapsulated in suitable biomaterials and effectively used for bone repair and regeneration, as the EV-functionalized biomaterials provide a sustained delivery of EVs at the site of injury throughout the regenerative process. This review compiles the most frequently used biomaterial-based 3D approaches [β-tricalcium phosphate (β-TCP)-modified scaffolds, EV-encapsulated hydrogels, 3D-bioprinting, PEI-engineered-EVs coated PLA scaffold, injectable chitosan hydrogels] for bone tissue engineering and angiogenesis using MSC-derived EVs. In addition, this review highlighs the importance of imitating the bone tissue micro-environment in vivo and discusses the promising future of EVs as a cell-free therapy in bone tissue regeneration in defect/fractures resulting from accidental injuries and diseases like osteoporosis and osteoarthritis. Accumulating evidence suggests that EV-based cell-free therapy could be a convincing alternative to the use of MSCs themselves because of its advantages over the respective MSCs. These EVs-based cell-free approaches could form a cutting-edge science, however, they are still in their preliminary stages and suffer from lack of evidence from clinical studies. Further, this review suggests the use of the biomaterial-based interpenetrating network (IPN) hydrogel-based approach, comprising both, natural and synthetic polymer networks which are physically interlocked, as a promising technique as this system provides superior mechanical strength and biological acceptability for augmenting encapsulation performance and the delivery of EVs.

Sepsis is a complex clinical syndrome that features excessive release of cytokines and other inflammatory mediators that could lead to organ dysfunction. Despite different treatment and management options, sepsis associated high morbidity and mortality rates remain. This has prompted intensive research into alternative therapeutic approaches such as targeted removal of sepsis related molecules using extracorporeal hemoperfusion. In this study, we explore the use of graphene nanoplatelets (GNP) as low-cost alternative hemosorbents for rapid removal of a broad spectrum of proinflammatory cytokine markers. Firstly, the physical characteristics, cytotoxicity, and cytokine marker adsorption profile of GNP were assessed. The results not only confirmed the surface characteristics of GNP and their ability to rapidly remove cytokine markers, but also indicated a low cytotoxicity towards the hepatic cell line HepG2. GNP were then incorporated into a freestanding flexible GNP-poly(tetrafluoroethylene) film with preserved surface characteristics and cytokine adsorption profile for potential use in hemoperfusion applications.

Nanoporous adsorbents are promising materials to augment the efficacy of haemodialysis for the treatment of end stage renal disease where mortality rates remain unacceptably high despite improvements in membrane technology. Complications are linked in part to inefficient removal of protein bound and high molecular weight uremic toxins including key marker molecules albumin bound indoxyl sulphate (IS) and p-cresyl sulphate (PCS) and large inflammatory cytokines such as IL-6. The following study describes the assessment of a nanoporous activated carbon monolith produced using a novel binder synthesis route for scale up as an in line device to augment haemodialysis through adsorption of these toxins. Small and large monoliths were synthesised using an optimised ratio of lignin binder to porous resin of 1 in 4. Small monoliths showing combined significant IS, p-CS and IL-6 adsorption were used to measure haemocompatibility in an ex vivo healthy donor blood perfusion model, assessing coagulation, platelet, granulocyte, t cell and complement activation, haemolysis, adsorption of electrolytes and plasma proteins. The small monoliths were tested in a niave rat model and showed stable blood gas values, blood pressure, blood biochemistry and the absence of coagulopathies. These monoliths were scaled up to a clinically relevant size and were able to maintain adsorption of protein bound uremic toxins IS, PCS and high molecular weight cytokines TNF and IL-6 over 60 minutes using a flow rate of 300 mL/min without platelet activation. The nanoporous monoliths where haemocompatible and retained adsorptive efficacy on scale up with negligible pressure drop across the system indicating potential for use as an in-line device to improve haemodialysis efficacy by adsorption of otherwise poorly removed uraemic toxins.

Our previous reports of interpenetrating networks (IPNs) have demonstrated drastic improvements in mechanical performance relative to individual constituent networks while maintaining viability of encapsulated cells. The current study investigated whether covalent linkage of RGD to the poly(ethylene glycol) diacrylate (PEGDA) network could improve upon cell viability and performance of agarose-PEGDA IPNs compared to unmodified IPNs (control) and to IPNs with different concentrations of physically entrapped aggrecan, providing a point of comparison to previous work. The inclusion of RGD or aggrecan generally did not adversely affect mechanical performance, and significantly improved chondrocyte viability and performance. Although both 4 and 100 μ g/mL of aggrecan improved cell viability, only 100 μ g/mL aggrecan was clearly beneficial to improving biosynthesis, whereas 100 μg/mL of RGD was beneficial to both chondrocyte viability and biosynthesis. Interestingly, clustering of cells within the IPNs with RGD and the higher aggrecan concentration were observed, likely indicating cell migration and/or preferred regional proliferation. This clustering resulted in a clearly visible enhancement of matrix production compared to the other IPNs. With this cell migration, we also observed significant cell proliferation and matrix synthesis beyond the periphery of the IPN, which could have important implications in facilitating integration with surrounding cartilage in vivo. With RGD and aggrecan (at its higher concentration) providing substantial and comparable improvements in cell performance, RGD would be the recommended bioactive signal for this particular IPN formulation and cell source given the significant cost savings and potentially more straightforward regulatory pathway in commercialization.

The development of bulk, three-dimensional (3D), macroporous polymers with high permeability, large surface area and large volume is highly desirable for a range of applications in the biomedical, biotechnological and environmental areas. The experimental techniques currently used are limited to the production of small size and volume cryogel material. In this work we propose a novel, versatile, simple and reproducible method for the synthesis of large volume porous polymer hydrogels by cryogelation. By controlling the freezing process of the reagent/polymer solution, large-scale 3D macroporous gels with wide interconnected pores (up to 200 μm in diameter) and large accessible surface area have been synthesized. For the first time, macroporous gels (of up to 400 ml bulk volume) with controlled porous structure were manufactured, with potential for scale up to much larger gel dimensions. This method can be used for production of novel 3D multi-component macroporous composite materials with a uniform distribution of embedded particles. The proposed method provides better control of freezing conditions and thus overcomes existing drawbacks limiting production of large gel-based devices and matrices. The proposed method could serve as a new design concept for functional 3D macroporous gels and composites preparation for biomedical, biotechnological and environmental applications.

Porous epoxy-activated copolymer beads were synthesized as support for the covalent immobilization of Aspergillus melleus l -aminoacylase. Here, a series of copolymer bead were synthesized using either glycidyl methacrylate (GMA) or allyl glycidyl ether (AGE) as monomer units and ethylene glycol dimethacrylate (EGDM) as cross-linking agent. The effect of monomer used and the effect of amount of cross-linking agent on covalent immobilization of aminoacylase were studied. Furthermore, the effect of porogen on immobilization of aminoacylase was also evaluated. AGE-co-EGDM copolymer beads gave higher binding of aminoacylase than GMA-co-EGDM copolymer beads. AGE-co-EGDM copolymer beads synthesized with lauryl alcohol as porogen and having 150% cross-linked density (i.e. AGE-(L)-150) gave maximum enzyme binding. Under optimum conditions, AGE-(L)-150 copolymer beads gave about 130 U/g of aminoacylase activity which corresponds to 72.24% of activity yield. Immobilized aminoacylase showed a broader pH, higher temperature and extended storage stability.

We recently introduced agarose-poly(ethylene glycol) diacrylate (PEGDA) interpenetrating network (IPN) hydrogels to cartilage tissue engineering that were able to encapsulate viable cells and provide a significant improvement in mechanical performance relative to its two constituent hydrogels. The goal of the current study was to develop a novel synthesis protocol to incorporate methacrylated chondroitin sulfate (MCS) into the IPN design hypothesized to improve cell viability and biosynthesis. The IPN was formed by encapsulating porcine chondrocytes in agarose, soaking the construct in a solution of 1:10 MCS:PEGDA, which was then photopolymerized to form a copolymer network as the second network. The IPN with incorporated CS (CS-IPN) (~0.5 wt%) resulted in a 4- to 5-fold increase in the compressive elastic modulus relative to either the PEGDA or agarose gels. After 6 weeks of in vitro culture, more than 50% of the encapsulated chondrocytes remained viable within the CS-modified IPN, in contrast to 35% viability observed in the unmodified. At week 6, the CS-IPN had significantly higher normalized GAG contents (347 ± 34 µg/µg) than unmodified IPNs (158 ± 27 µg/µg, P < 0.05). Overall, the approach of incorporating biopolymers such as CS from native tissue may provide favorable micro-environment and beneficial signals to cells to enhance their overall performance in IPNs.

A new method for encapsulating cells in interpenetrating network (IPN) hydrogels of superior mechanical integrity was developed. In this study, two biocompatible materials—agarose and poly(ethylene glycol) (PEG) diacrylate—were combined to create a new IPN hydrogel with greatly enhanced mechanical performance. Unconfined compression of hydrogel samples revealed that the IPN displayed a fourfold increase in shear modulus relative to a pure PEG-diacrylate network (39.9 vs. 9.9 kPa) and a 4.9-fold increase relative to a pure agarose network (8.2 kPa). PEG and IPN compressive failure strains were found to be 71% ± 17% and 74% ± 17%, respectively, while pure agarose gels failed around 15% strain. Similar mechanical property improvements were seen when IPNs-encapsulated chondrocytes, and LIVE/DEAD cell viability assays demonstrated that cells survived the IPN encapsulation process. The majority of IPN-encapsulated chondrocytes remained viable 1 week postencapsulation, and chondrocytes exhibited glycosaminoglycan synthesis comparable to that of agarose-encapsulated chondrocytes at 3 weeks postencapsulation. The introduction of a new method for encapsulating cells in a hydrogel with enhanced mechanical performance is a promising step toward cartilage defect repair. This method can be applied to fabricate a broad variety of cell-based IPNs by varying monomers and polymers in type and concentration and by adding functional groups such as degradable sequences or cell adhesion groups. Further, this technology may be applicable in other cell-based applications where mechanical integrity of cell-containing hydrogels is of great importance.

The purpose of this research work was to explore an application of uncoated porous drug carrier prepared by single-step drug adsorption for a delivery system based on integration of floating and pulsatile principles intended for chronotherapy. This objective was achieved by utilizing 3(2) factorial design, solvent volume (X(1)) and drug amount (X(2)) as selected variables, for drug adsorption using solvents, methanol, and dichloromethane (DCM), of varying polarity. Nitrogen adsorption (N(2)), scanning electron microscopy of cross-sections, and atomic force microscopy were done to study adsorption patterns and their effect on release pattern. Drug release study was customized by performing for 6 h in acidic environment to mimic gastroretention followed by basic environment akin to transit phase. Correlation between porous data from mercury and N(2) adsorption was probably studied for the first time. Observed regression analysis values for pore volume, surface area, and drug release indicated the influence of selected variables. Total release range in acidic medium was 12.77-24.57% for methanol, 8.79-15.26% for DCM, and final release of 69.45-92.23% for methanol, and 60.16-99.99% for DCM influenced by varying internal geometries was observed. Present form of drug delivery system devoid of any additives/excipients influencing drug release shows distinct behavior from other approaches/technologies in chronotherapy by (a) observing desired low drug release (8%) in acidic medium, (b) overcoming the limitations of process variables caused by multiple formulation steps and different characteristic polymers, (c) reducing time consumption due to single step process, and (d) extending as controlled/extended release.

Macroporous polymer particles containing surface epoxy groups were synthesized for immobilization of Candida rugosa lipase (CRL). The effect of incorporation of two different sets of monomers [allyl glycidyl ether (AGE) and glycidyl methacrylate (GMA)] and the effect of crosslinking density on immobilization of lipase were studied. AGE-co-EGDM polymers gave higher binding and expression of lipase than GMA-co-EGDM polymers. Optimization of immobilization parameters was done with respect to immobilization time and enzyme loading. Amongst AGE-co-EGDM polymer series, AGE-150 polymer found to give maximum lipase activity yield and therefore evaluated for temperature, pH and storage stability. Under optimum conditions, AGE-150 polymer gave 78.40% of activity yield. Immobilized lipase on AGE-150 showed a broader pH, higher temperature and excellent storage stability.

Lipase isolated from Arthrobacter sp. (bacterial strain, MTCC No. 5125) at RRL Jammu, being used for various process development. Arthrobacter sp. lipase (ABL) now has been immobilized on synthetic polymers and reused many a times. In this investigation number of various synthetic macroporous alkylated glycidyl epoxy copolymers with varying hydrophobicity, pore volume and surface area were prepared and used for this study. Among all the polymers prepared and used only two epoxy polymers GMA-EGDM 75-20(I) and GMA-EGDM 75-30(I) with particle size in the range of 150–450 nm, epoxy groups 80 and 70%, tertiary amino groups 20 and 30% was found suitable for immobilization of lipase (ABL). Dibutyl amine (DBA) incorporation created an internal pore radii 20–50 nm and hydrophobic microenvironment in both the polymers for binding the enzyme, which led to improvement in stability and enatioselectivity in racemic resolution process especially by binding to one of the isomers. The optimal ABL binding capacity of polymer GMA-EGDM 75-20(I) was 60 units, 34 mg protein and GMA-EGDM 75-30(I) was 36 units, 21 mg protein/g polymer. The immobilized lipase matrices displayed enhanced pH, thermal, organic solvent and long-term storage stability. Both the immobilized enzyme matrices were tested firstly for the hydrolysis of triglycerides using tributyrin as substrate. After testing, both the matrices were reused for racemic resolution of ethyl-3-hydroxy-3-phenyl propanoate (fluoxetine intermediate, an antidepressant drug) and racemic chiral auxiliary, acetyl-1-phenyl ethanol (intermediate of many chiral drugs) for 15 cycles. These immobilized lipase matrices have shown very high stability on recycling, high-enantioselectivity, high conversion and faster recovery of product compare to free enzyme, therefore these matrices may find use in kinetic resolution process developments.

Functional, macroporous, beaded copolymers containing epoxy groups were synthesized for immobilization of polyphenol oxidase (PPO) from edible mushroom (Agaricus bisporus). The effect of incorporation of two different sets of monomers such as glycidyl methacrylate (GMA) and allyl glycidyl ether (AGE) and the effect of cross-linking agent ethylene glycol dimethacrylate (EGDM) with varying cross-link densities on binding and expression of mushroom PPO activity were studied. The effect of porogen viz. cyclohexanol and hexanol on PPO immobilization was studied. AGE copolymers with hexanol as a porogen were found to give higher binding and expression of PPO activity than GE polymers. Cross-linking of amino groups of enzyme with 5% glutaraldehyde for 6 h gave a stable binding of PPO on AGE-75(Hex) polymer with storage half-life of approximately 25 days. Under optimum conditions, AGE-75(Hex) polymer gave 70.3% of activity yield while percent retention of PPO activity was found to be 83.5%. Immobilized PPO showed a broader pH, higher temperature and excellent storage stability.

Chronotherapy, a new approach for treating pathological conditions, is based on circadian rhythm. Present work conceptualizes a specific technology, based on combining floating and pulsatile principles to develop drug delivery system, intended for chronotherapy in arthritis. This approach was achieved by using low density microporous polypropylene, Accurel MP 1000 ® , as a multiparticulate carrier along with drug of choice ibuprofen. Carrier amount and solvent volume was kept invariant in designing this simple system by adsorbing drug via melting or solvent evaporation using different carrier: drug ratios. In solvent evaporation, methanol (M) and dichloromethane (DCM) were used. Drug loaded multiparticulate system was subjected to various characterization and evaluation parameters showing influence of adsorption process. Drug release study was performed in acidic environment using pH 1.2 HCl IP medium for 6 h to mimic gastric condition for evaluating gastroretention followed by basic environment using appropriate medium as phosphate buffer pH 7.2 IP for 3 h resembling transit. The release pattern showed influence of drug adsorption methods characterized by ever changing pore geometry with total release ranges in acidic medium as 10.7–27.6% and final release as 55.6–88.6%. Present drug delivery system devoid of any additives/excipients influencing drug release show distinct behaviour from other approaches/technologies in chronotherapy by (a) observing desired low drug release (11%) in acidic medium (b) overcoming the limitations of process variables caused by multiple formulation steps (c) reducing time consumption due to single step process (d) can be extended to controlled release also.

Lipase isolated from Arthrobacter sp. (RRLJ-1, MTCC No. 5125, named ABL), is effective in resolving a wide range of racemic drug intermediates. In this study, ABL was immobilized on a series of synthetic macroporous epoxy copolymers beads with varying pore sizes, surface area and hydrophobicity. Poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads, with 75% crosslink density and 10% of epoxy groups modified with dibutyl amine [GMA-EGDM-75 (10% DBA)] had a pore volume of 0.77 mL/g and a surface area of 86.05 m 2/g; these beads were optimally suitable for ABL immobilization. The covalent binding of the lipase was optimized by varying the ionic strength, buffers, pH, temperature and time. The optimal binding was achieved in 100 mM phosphate buffer at 4°C, pH 7.0 in three hours. Under these conditions the polymer retained 34 units and 12 mg of ABL per gram. Immobilized ABL displayed enhanced thermal, organic solvent and pH stability compared to the free enzyme. The immobilized enzyme was used repeatedly (fifteen cycles) to resolve the fluoxitine intermediate (racemic ethyl-3-hydroxy-3-phenyl propanoate) without any loss in stereospecificity. The resolution time of fluoxitine intermediate was reduced to almost half (from 84 to 48 hours) by using the immobilized enzyme.

Low density porous carriers are widely used in the pharmaceutical applications. Response surface methodology, using 3(2) factorial design was used to study drug adsorption on and its release patterns from microporous polypropylene (Accurel MP 1000) in the absence of additives. Ibuprofen, as model drug, was adsorbed on the polymer by solvent evaporation using two organic solvents methanol (M) and dichloromethane (DCM). The amount of carrier (100 mg) and its particle size range (250-350 microm) were kept invariant while solvent volume (X1) and drug amount (X2) were taken as variables. Drug adsorption pattern depended on the type and amount of solvent used. DSC, XRD, FTIR and TGA, predict crystalline nature and physical form of adsorption. SEM showed the penetration and adsorption of the drug in and on the microporous polymer. Accurel MP 1000 had a pore volume of 1.992 g/cm3 and surface area of 55.9855 m2/g as detected by mercury porosimetery. On drug adsorption, pore volume ranged from 0.413 to 1.198 g/cm3 for methanol and 0.280-0.759 g/cm3 for DCM. Similarly surface area was in the range 38.445-25.497 m2/g for methanol and 18.710-32.528m2/g for DCM. The drug release was investigated in phosphate buffer pH 7.2. All batches showed excellent in vitro floating property. Drug release was partial with recovery to complete dependent on type and volume of solvent. R2 values relating to bulk density, pore volume, surface area and drug release at 60, 120 and 180 min were estimated. Effect of solvent properties shows a positive influence on drug adsorption and release. Release profiles of some batches could be considered as gastroretentive drug delivery system.

Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim®), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim® attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim® antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p

Recombinant proteins and cytokines are under broad preclinical and clinical investigation to promote angiogenesis, but their success is limited by ineffective delivery, lack of long-term stability, and excessive cost. Mesenchymal stem/stromal cells (MSC) secrete bioactive trophic factors, and thus, may provide an effective alternative to address these challenges. Glycine-Histidine-Lysine (GHK) is a peptide fragment of osteonectin (SPARC), a matricellular protein with reported proangiogenic potential. We examined the capacity of GHK to upregulate secretion of proangiogenic factors from human MSC in culture and when covalently coupled to alginate hydrogels. GHK had no apparent cytotoxic effects on MSC in culture over a wide range of concentrations. We detected a dose-dependent increase in vascular endothelial growth factor (VEGF) concentration in media conditioned by GHK-treated MSC, which increased endothelial cell proliferation, migration, and tubule formation. We covalently coupled GHK to alginate using carbodiimide chemistry, and human MSC were entrapped in alginate hydrogels to assess VEGF secretion. Similar to monolayer culture, MSC responded to GHK-modified gels by secreting increased concentrations of VEGF and basic fibroblast growth factor (bFGF) compared to unmodified gels. The pre-treatment of MSC with antibodies to α6 and β1 integrins prior to entrapment in GHK-modified gels abrogated VEGF secretion, suggesting that the proangiogenic response of MSC was integrin-mediated. These data demonstrate that the proangiogenic potential of MSC can be significantly increased by the presentation of GHK with a biodegradable carrier, therefore increasing their clinical potential when used for tissue repair.

Polymeric nanofibers have potential as tissue engineering scaffolds, as they mimic the nanoscale properties and structural characteristics of native extracellular matrix (ECM). Nanofibers composed of natural and synthetic polymers, biomimetic composites, ceramics, and metals have been fabricated by electrospinning for various tissue engineering applications. The inherent advantages of electrospinning nanofibers include the generation of substrata with high surface area-to-volume ratios, the capacity to precisely control material and mechanical properties, and a tendency for cellular in-growth due to interconnectivity within the pores. Furthermore, the electrospinning process affords the opportunity to engineer scaffolds with micro- to nanoscale topography similar to the natural ECM. This review describes the fundamental aspects of the electrospinning process when applied to spinnable natural and synthetic polymers; particularly, those parameters that influence fiber geometry, morphology, mesh porosity, and scaffold mechanical properties. We describe cellular responses to fiber morphology achieved by varying processing parameters and highlight successful applications of electrospun nanofibrous scaffolds when used to tissue engineer bone, skin, and vascular grafts.

Interpenetrating network (IPN) hydrogels were recently introduced to the cartilage tissue engineering literature, with the approach of encapsulating cells in thermally gelling agarose that is then soaked in a poly(ethylene glycol) diacrylate (PEGDA) solution, which is then photopolymerized. These IPNs possess significantly enhanced mechanical performance desirable for cartilage regeneration, potentially allowing patients to return to weight-bearing activities quickly after surgical implantation. In an effort to improve cell viability and performance, inspiration was drawn from previous studies that have elicited positive chondrogenic responses to aggrecan, the proteoglycan largely responsible for the compressive stiffness of cartilage. Aggrecan was incorporated into the IPNs in conservative concentrations (40 μg/mL), and its effect was contrasted with the incorporation of chondroitin sulfate (CS), the primary glycosaminoglycan associated with aggrecan. Aggrecan was incorporated by physical entrapment within agarose and methacrylated CS was incorporated by copolymerization with PEGDA. The IPNs incorporating aggrecan or CS exhibited over 50% viability with encapsulated chondrocytes after 6 weeks. Both aggrecan and CS improved cell viability by 15.6% and 20%, respectively, relative to pure IPNs at 6 weeks culture time. In summary, we have introduced the novel approach of including a raw material from cartilage, namely aggrecan, to serve as a bioactive signal to cells encapsulated in IPN hydrogels for cartilage tissue engineering, which led to improved performance of encapsulated chondrocytes.

Effects of changes in hydrophobicity of polymeric support on structure and activity of alpha-chymotrypsin (E.C. 3.4.21.1) have been studied with copolymers of allyl glycidyl ether (AGE) and ethylene glycol dimethacrylate (EGDM) with increasing molar ratio of EGDM to AGE (cross-link density 0.05 to 1.5). The enzyme is readily adsorbed from aqueous buffer at room temperature following Langmuir adsorption isotherms in unexpectedly large amounts (25% w/w). Relative hydrophobicity of the copolymers has been assessed by studying adsorption of naphthalene and Fmoc-methionine by the series of copolymers from aqueous solutions. Polymer hydrophobicity appears to increase linearly on increasing cross-link density from 0.05 to 0.25. Further increase in cross-link density causes a decrease in naphthalene binding but has little effect on binding of Fmoc-Met. Binding of alpha-chymotrypsin to these copolymers follow the trend for Fmoc-methionine binding, rather than naphthalene binding, indicating involvement of polar interactions along with hydrophobic interactions during binding of protein to the polymer. The adsorbed enzyme undergoes extensive denaturation (ca. 80%) with loss of both tertiary and secondary structure on contact with the copolymers as revealed by fluorescence, CD and Raman spectra of the adsorbed protein. Comparison of enzyme adsorption behavior with Eupergit C, macroporous Amberlite XAD-2, and XAD-7 suggests that polar interactions of the EGDM ester functional groups with the protein play a significant role in enzyme denaturation.

The aim of the present study was to design a novel/conceptual delivery system using ibuprofen, anticipated for chronotherapy in arthritis with porous material to overcome the formulation limits (multiple steps, polymers, excipients) and to optimize drug loading for a desired release profile suitable for in vitro investigations. The objective of this delivery system lies in the availability of maximum drug amount for absorption in the wee hours as recommended. Drug loading using 3(2) factorial design on porous carrier, synthesized by high internal phase emulsion technique using styrene and divinylbenzene, was done via solvent evaporation using methanol and dichloromethane. The system was evaluated in vitro for drug loading, encapsulation efficiency, and surface characterization by scanning electron, atomic force microscopy, and customized drug release study. This study examined critical parameters such as solvent volume, drug amount, and solvent polarity on investigations related to drug adsorption and release mostly favoring low-polarity solvent dichloromethane. Overall release in all batches ranged 0.98-52% in acidic medium and 71-94% in basic medium. These results exhibit uniqueness in achieving the least drug release of 0.98%, an ideal one, without using any release modifiers, making it distinct from other approaches/technologies for time and controlled release and for chronotherapy.

A series of telechelic dimethacrylic ether-esters (MEE) were prepared by solventless reaction of alpha,omega-dihydroxy poly(oxytetramethylene) (polytetrahydrofuran, PTHF) with phthalic anhydride and glycidyl methacrylate (GMA). MEE was polymerized with GMA as well as GMA-ethylene dimethacrylate (EGDM) to form porous beads. The terpolymer beads were observed using optical microscopy and SEM and characterized for internal pore volume, equilibrium volume-swelling ratio and dimethyl formamide and aqueous buffer regain. The morphology of the beads was dictated by the mole fraction and molecular weight of MEE in the feed. Porosity was found to increase with increase in molecular weight of MEE. Thus, porosity as high as 49%, 50% and 55% was observed with MEE of molecular weights 1580, 2580 and 3480, respectively. At a specific terpolymerization feed ratio of monomers, the terpolymers formed transform from gel like structure into a macroporous one, with increase in molecular weight of MEE. (c) 2006