Sumrita Bhat

Supermacroprous chitosan–agarose–gelatin cryogels: in vitro characterization and in vivo assessment for cartilage tissue engineering

The study focuses on the synthesis of a novel polymeric scaffold having good porosity and mechanical characteristics synthesized by using natural polymers and their optimization for application in cartilage tissue engineering. The scaffolds were synthesized via cryogelation technology using an optimized ratio of the polymer solutions (chitosan, agarose and gelatin) and cross-linker followed by the incubation at sub-zero temperature (−12°C). Microstructure examination of the chitosan–agarose–gelatine (CAG) cryogels was done using scanning electron microscopy (SEM) and fluorescent microscopy. Mechanical analysis, such as the unconfined compression test, demonstrated that cryogels with varying chitosan concentrations, i.e. 0.5–1% have a high compression modulus. In addition, fatigue tests revealed that scaffolds are suitable for bioreactor studies where gels are subjected to continuous cyclic strain. In order to confirm the stability, cryogels were subjected to high frequency (5 Hz) with 30 per cent compression of their original length up to 1 × 105 cycles, gels did not show any significant changes in their mass and dimensions during the experiment. These cryogels have exhibited degradation capacity under aseptic conditions. CAG cryogels showed good cell adhesion of primary goat chondrocytes examined by SEM. Cytotoxicity of the material was checked by MTT assay and results confirmed the biocompatibility of the material. In vivo biocompatibility of the scaffolds was checked by the implantation of the scaffolds in laboratory animals. These results suggest the potential of CAG cryogels as a good three-dimensional scaffold for cartilage tissue engineering.

Biomaterials and bioengineering tomorrow's healthcare.

Biomaterials are being used for the healthcare applications from ancient times. But subsequent evolution has made them more versatile and has increased their utility. Biomaterials have revolutionized the areas like bioengineering and tissue engineering for the development of novel strategies to combat life threatening diseases. Together with biomaterials, stem cell technology is also being used to improve the existing healthcare facilities. These concepts and technologies are being used for the treatment of different diseases like cardiac failure, fractures, deep skin injuries, etc. Introduction of nanomaterials on the other hand is becoming a big hope for a better and an affordable healthcare. Technological advancements are underway for the development of continuous monitoring and regulating glucose levels by the implantation of sensor chips. Lab-on-a-chip technology is expected to modernize the diagnostics and make it more easy and regulated. Other area which can improve the tomorrow’s healthcare is drug delivery. Micro-needles have the potential to overcome the limitations of conventional needles and are being studied for the delivery of drugs at different location in human body. There is a huge advancement in the area of scaffold fabrication which has improved the potentiality of tissue engineering. Most emerging scaffolds for tissue engineering are hydrogels and cryogels. Dynamic hydrogels have huge application in tissue engineering and drug delivery. Furthermore, cryogels being supermacroporous allow the attachment and proliferation of most of the mammalian cell types and have shown application in tissue engineering and bioseparation. With further developments we expect these technologies to hit the market in near future which can immensely improve the healthcare facilities.

Cryogels: Freezing unveiled by thawing

Cryogels are interconnected supermacroporous gels prepared at sub-zero temperatures having applications in various research fields. The process of cryogelation is ideally thought to take place via following steps: phase separation with ice-crystal formation, cross-linking and polymerization followed by thawing of ice-crystals to form an interconnected porous cryogel network. This phenomenon mostly thought as a theoretical concept has now been revealed here in practical terms via data generated by micro-computed tomography (Micro CT). Micro CT is mainly used for characterizing the gel materials in terms of their physical properties like pore size, porosity, strut size, etc., whereas this work has pioneered its role in elucidating the process of cryogel formation.

Three-Dimensional Supermacroporous Carrageenan-Gelatin Cryogel Matrix for Tissue Engineering Applications

A tissue-engineered polymeric scaffold should provide suitable macroporous structure similar to that of extracellular matrix which can induce cellular activities and guide tissue regeneration. Cryogelation is a technique in which appropriate monomers or polymeric precursors frozen at sub-zero temperature leads to the formation of supermacroporous cryogel matrices. In this study carrageenan-gelatin (natural polymers) cryogels were synthesized by using glutaraldehyde and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride and N-hydroxysuccinimide (EDC-NHS) as crosslinking agent at optimum concentrations. Matrices showed large and interconnected pores which were in the range of 60–100 μm diameter. Unconfined compression analysis showed elasticity and physical integrity of all cryogels, as these matrices regained their original length after 90% compressing from the original size. Moreover Youngs modulus was found to be in the range of 4–11 kPa for the dry cryogel sections. These cryogels also exhibited good in vitro degradation capacity at 37 °C within 4 weeks of incubation. Supermacroporous carrageenan-gelatin cryogels showed efficient cell adherence and proliferation of Cos-7 cells which was examined by SEM. PI nuclear stain was used to observe cell-matrix interaction. Cytotoxicity of the scaffolds was checked by MTT assay which showed that cryogels are biocompatible and act as a potential material for tissue engineering and regenerative medicine.

In Vitro Neo-Cartilage Formation on a Three-Dimensional Composite Polymeric Cryogel Matrix

Limited blood supply and the avascular nature of articular cartilage restricts its self repair capacity, frequently leading to osteoarthritis. This work focuses on scaffolds for tissue repair from natural polymers, for example gelatin, chitosan, and agarose in the form of composite. A novel way of fabrication, known as cryogelation, is presented, in which matrices are synthesized at sub-zero temperature. Cell seeded scaffolds incubated under appropriate conditions result in the accumulation of matrix components on the surface of the gel in the form of neo-cartilage. Neo-cartilage exhibits similarity to native cartilage with respect to its physical, mechanical and biochemical properties. Based on the similarities of neo-cartilage to the native cartilage, it can provide a new approach for the treatment of localised joint injuries.

Efficacy of supermacroporous poly(ethylene glycol)-gelatin cryogel matrix for soft tissue engineering applications.

Three dimensional scaffolds synthesized using natural or synthetic polymers act as an artificial niche for cell adherence and proliferation. In this study, we have fabricated cryogels employing blend of poly (ethylene glycol) (PEG) and gelatin using two different crosslinkers like, glutaraldehyde and EDC-NHS by cryogelation technique. Synthesized matrices possessed interconnected porous structure in the range of 60-100 μm diameter and regained their original length after 90% compression without deformation. Visco-elastic behavior was studied by rheology and unconfined compression analysis, elastic modulus of these cryogels was observed to be >10(5)Pa which showed their elasticity and mechanical strength. TGA and DSC also showed the stability of these cryogels at different temperatures. In vitro degradation capacity was analyzed for 4 weeks at 37°C. IMR-32, C2C12 and Cos-7 cells proliferation and ECM secretion on PEG-gelatin cryogels were observed by SEM and fluorescent analysis. In vitro biocompatibility was analyzed by MTT assay for the period of 15 days. Furthermore, cell proliferation efficiency, metabolic activity and functionality of IMR-32 cells were analyzed by neurotransmitter assay and DNA quantification. The cell-matrix interaction, elasticity, mechanical strength, stability at different temperatures, biocompatible, degradable nature showed the potentiality of these cryogels towards soft tissue engineering such as neural, cardiac and skin.

Study of Different Delivery Modes of Chondroitin Sulfate Using Microspheres and Cryogel Scaffold for Application in Cartilage Tissue Engineering

This study focuses on the development of an efficient delivery modes designed for chondroitin sulfate (CS) for application in cartilage tissue engineering. Novel three-dimensional (3-D) scaffold fabricated from natural polymers such as chitosan and gelatin blended with chondroitin sulfate (CGC) were synthesized using cryogelation technology. Other methods to deliver CS were also tried, which included incorporation into microparticles for sustained release and embedding the CS loaded microparticles in CG (chitosan-gelatin) cryogel scaffold. Novel CGC scaffolds were characterized by rheology, scanning electron microscopy (SEM), and mechanical assay. Scaffolds exhibited compression modulus of 50 KPa confirming the utility of these scaffolds for cartilage tissue engineering. Primary goat chondrocytes were used for the in vitro testing of all the delivery modes. So this study shows that CS microparticles when given freely with matrix (chitosan–gelatin) or embedded into scaffold has potential to enhance chondrocyte proliferation together with improved matrix production than in control without microspheres.

Cell factory‐derived bioactive molecules with polymeric cryogel scaffold enhance the repair of subchondral cartilage defect in rabbits

We have explored the potential of cell factory‐derived bioactive molecules, isolated from conditioned media of primary goat chondrocytes, for the repair of subchondral cartilage defects. Enzyme‐linked immunosorbent assay (ELISA) confirms the presence of transforming growth factor‐β1 in an isolated protein fraction (12.56 ± 1.15 ng/mg protein fraction). These bioactive molecules were used alone or with chitosan–agarose–gelatin cryogel scaffolds, with and without chondrocytes, to check whether combined approaches further enhance cartilage repair. To evaluate this, an in vivo study was conducted on New Zealand rabbits in which a subchondral defect (4.5 mm wide × 4.5 mm deep) was surgically created. Starting after the operation, bioactive molecules were injected at the defect site at regular intervals of 14 days. Histopathological analysis showed that rabbits treated with bioactive molecules alone had cartilage regeneration after 4 weeks. However, rabbits treated with bioactive molecules along with scaffolds, with or without cells, showed cartilage formation after 3 weeks; 6 weeks after surgery, the cartilage regenerated in rabbits treated with either bioactive molecules alone or in combinations showed morphological similarities to native cartilage. No systemic cytotoxicity or inflammatory response was induced by any of the treatments. Further, ELISA was done to determine systemic toxicity, which showed no difference in concentration of tumour necrosis factor‐α in blood serum, before or after surgery. In conclusion, intra‐articular injection with bioactive molecules alone may be used for the repair of subchondral cartilage defects, and bioactive molecules along with chondrocyte‐seeded scaffolds further enhance the repair. Copyright

Cryogels: Novel matrices for the repair of cartilage lesions

Zvi Nevo serves as a Professor Emeritus in the Department of Human Molecular Genetics and Biochemistry at the Sackler School of Medicine, Tel Aviv University. He runs an active laboratory, directing 3 Ph.D. students and 3 students for Master Degree, supported by grants from the Israeli Academy of Science and GIF German Israeli Binational Foundation. His field of expertise is in general developmental, maturation and aging of connective tissues in health and disease, with special emphasis on cartilage and cartilage repair with composite implants. Prof. Nevo serves as a member of the Editorial Board of the American Journal of Cell Transplantation, Cartilage Section. He sponsored 15 Ph.D. students, 12 students for Master Degree and 25 MD for basic science training projects. He has one hundred scientific articles, ten chapters in books and 23 patents. Elongating the survival of hyaluronan (HA) molecules: Employing HA cross-linkers, modified HA or HA hybridsC treatments have advanced in recent years, yet current models for drug discovery and metastasis exploration poorly mimic human cancer in vivo. Research generally relies on animal systems and two-dimensional (2-D) cultures, both of which suffer from inadequacies. Alternatively, 3-D systems of human-derived cells can promote cell-cell and cell-matrix interactions, resembling in vivo conditions, but unfortunately are largely underutilized for cancer research. Here we present multiple host tissue-tumor systems for in vitro modeling of colon carcinoma metastasis with implications in exploring cancer biology and the potential to streamline the drug development pipeline. Aleksander Skardal, J Tissue Sci Eng 2013, 4:2 http://dx.doi.org/10.4172/2157-7552.S1.010F repair tissue is found to be formed after the repair process of cartilage defects even after chondrocyte transplantation. The type I collagen compound of fibrocartilage can not fulfil the mechanical properties of hyaline cartilage which consists of mostly type 2 collagen. Gene therapy during the chondrocyte culturation stage may work on leading the cells to form a hyaline cartilage in-vivo. The ideal timing of the transfection in post transcriptional gene silencing of cultured chondrocytes must be displayed first. The standard chondrocyte cultures were preperared with this purpose. The cells were seeded in 6 main groups and also each main groups were taken in to the study in 3 sub-groups. Invert microscopic evaluation was performed after the passage of the cells on 0, 24th, 48th, and 72th hours. Non-targeting siRNA (p.GFP) ve GFP si RNA transfections were achieved. The efficiency of the transfection was determined with RNA isolation on protein acquisition, on 0, 48th and 72th hours. Expression of mRNA was evaluated with Bradford and semi-dry Western Blot analysis using anti-GFP and anti GAPDH anti-bodies. The ideal time, which resulted in better results, was reported by evaluating the expression on electrophoretic bands. One way ANOVA and Newman-Keuls multiple comparison tests were performed in the stastistical analysis of the comparison of the tranfection efficiency between each group. The discrepancies between groups were determined with Student’s T Test. The most efficient silencing was obtained on 48th hour (P<0.05); The GFP expression decresed to 11.4% in post-transcriptional gene silencing performed group, while it was 59.7% in none silenced group, on 48th hour. The efficient gene silencing was achieved in 77% of the cells. Nevzat Selim Gokay et al., J Tissue Sci Eng 2013, 4:2 http://dx.doi.org/10.4172/2157-7552.S1.010T scaffold materials for musculoskeletal tissue engineering are either hydrogel-based or solid-based (e.g., polymeric, ceramic, or metallic). We have developed an entirely new class of biomaterials for tissue regeneration based on colloidal gel technology. Colloidal gels are paste-like, shear thinning materials made of nanoparticles that interact through electrostatic forces, van der Waals attraction, and steric hindrance. These materials are thus malleable, they ‘set’ after placement, and they may facilitate resorption, tissue integration, and rapid tissue remodeling. Thus, colloidal gels possess unique advantages to traditional scaffolds as hydrogels are prone to leaking from the defect site prior to polymerization and because solid scaffolds cannot be molded into an irregularly-shaped defect. Although these materials can be utilized by any application where a moldable material is desirable, our team is exploring their use in cranial defects. We established proof-of-concept of these materials first by exploring oppositely charged poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, where shear thinning and recovery properties were observed. Additionally, the PLGA particles were implanted in rat cranial defects, where enhanced cranial defect regeneration was observed. We are now investigating natural materials, found within the extracellular matrix of tissues that have been modified to perform as colloidal gels. Currently, we are characterizing the shear thinning and recovery properties of different colloidal gel component ratios. In addition, we are also exploring the use of colloids in traditional hydrogels to allow for surgical placement prior to polymerization. Emily Beck, J Tissue Sci Eng 2013, 4:2 http://dx.doi.org/10.4172/2157-7552.S1.010Introduction. Valvular heart disease is a common diagnosis worldwide, with many of these patients eventually needing an operative intervention. There are significant limitations to existing heart valve replacements, including their inability to grow with patients, a life-time anticoagulation therapy with mechanical valves, and progressive tissue degeneration with bioscaffolds. Tissue-engineering techniques may play a prominent future role in the development of heart valve replacement therapy. More recent laboratory efforts have focused on identifying appropriate scaffold materials, progenitor cell sources, and seeding/conditioning techniques. However, this involves the time-consuming task of isolating and culturing cells before adding them to a valve construct and then conditioning the resulting construct in vitro, a process that can take many weeks or even months. As an alternative, we proposed to develop a heart valve bioscaffold with antibody-directed reseeding properties. The goal is to develop a heart valve construct that is easy to fabricate, has short preparation time (hours), and the ability to remodel and grow with the patient.In nature, molecules spontaneously self-assemble to form stable assemblies, creating for example, proteins and lipid structures in cells. In recent years, this process has been exploited to produce hydrogels based on amino acids, the building blocks of proteins. These supramolecular hydrogels can be based on β-sheets, α-helices and random coils. With 20 natural amino acids to use as the building blocks, and an infinite number of synthetic analogues, with various properties, a wide range of gels with varying physical and chemical properties can be produced. The high water content, porosity, potential for cellular remodeling and the nanofibrous architecture has resulted in peptide hydrogels being labelled as extracellular matrix (ECM) mimics, with the potential for numerous biological applications. We have produced nanofibrous peptide hydrogels based on tripeptide and octapeptide systems for skin, cartilage and intervertebral disc cell culture and tissue engineering. Cell viability, proliferation and matrix production suggest these gels can maintain viable environments for use as 3D cell culture systems and tissue engineering applications.T structure of hydrogels resembles extracellular matrix (ECM) in vivo. Thus, hydrogels are attractive scaffold materials for three-dimensional (3D) cell culturing. Various hydrogels, either synthetic or from natural origin, have been used as 3D cell culture scaffolds for different biomedical applications. From immunological point of view, hydrogel scaffolds without human or animal products are highly preferred. Here we show that plant-derived nanofibrillar cellulose (NFC) hydrogel promotes 3D culture of liver cells. NFC composes of fibrillar glucan chains whose diameter is in nanometer range and length in micrometer range. NFC hydrogel serves as a biocompatible culture scaffold without added growth factors. Human hepatic cell lines HepaRG and HepG2 form 3D multicellular spheroids in NFC hydrogel. Both cell lines secret human albumin confirming the liverspecific functionality of these cells. HepaRG cells polarize in 3D NFC hydrogel culture as revealed in filamentous actin (F-actin) immunostaining. Accumulation of F-actin in apical cell membrane domain demonstrates the formation of bile canalicularlike constructions. In conclusion, NFC hydrogel is a promising 3D scaffold for cell culturing. It provides ECM mimicking 3D environment, free of human and animal products, and it could be applied in organotypic culture systems for drug discovery and tissue engineering. Liisa Kanninen et al., J Tissue Sci Eng 2013, 4:2 http://dx.doi.org/10.4172/2157-7552.S1.010Here we developed bioactive self-assembling peptide nanofiber hydrogel (RADA-SP and KLD-SP) which could recruit mesenchymal stem cells. The self-assembling peptide forms fibers (5 to 10nm) and assembles into a 3D scaffold at physiological solution. Substance P(SP) is an injury-inducible factor that acts early in the wound healing process to induce CD29+ stromal-like cell mobilization. To investigate the inducible ability of them, we implanted KLD-SP into subcutaneous of nude mice. And then, we injected NIR-labeled hMSCs into tail vein. The migration of injected cells was tracked using multi spectrum imaging system in real time. By applying these bioactive peptides on ischemic hind limb models and osteoarthritis models, the abilities of stem cell recruitment and angiogenesis were evaluated. Limb ischemia was produced in athymic mice and peptides were injected into ischemic sites. In RADA-SP group, it was shown that many mesenchymal stem cells were recruited into injected sites compared to other groups. Moreover, TUNEL+ cell density was 7 times lower than ischemia group. In Masson’s trichrome staining, injection of RADA-SP could prevent fibrosis. Osteoarthritis was produced in rats and 0.5% (wt/vol) peptides were injected into osteoarthritis sites 3weeks after surgery. And the tissues were harvested 6weeks after injection for analysis. In micro CT datas, it was shown that articular cartilage of KLD-SP group has smooth surface. In histological staining, injection of KLD-SP could regenerate cartilage tissue. In conclusion, SP coupled with self-assembling peptide nanofiber is effective to recruit mesenchymal stem cells and that leads to protect limb ischemia and regenerate articular cartilage.Polymeric gels are widely used for regenerative medicine to control the functions of cells and drug delivery applications. Molecular structure and corresponding 3-dimensional architecture of the gels play a major role in regulating these functions in regenerative applications. Molecularly, most polymeric gel materials are homogenous in nature and forms gel like structure in presence of suitable solvent, most commonly water. As a result, these gels are often limited to exert differential signal at the molecular level. To address this, biphasic polymeric gel materials are developed by coupling segments with different physicochemical characteristics. Specifically, these biphasic gels are designed from polyethylene glycol and urethane segments and through molecular engineering of different segments. Depending on the segmental structure, these biphasic gels exhibits molecular properties which can be adapted to encapsulate therapeutic molecules, controlled release kinetics, and cellular functions. Preliminary analysis shows that biphasic gels developed from biocompatible segments are capable to encapsulate and release model drug molecules and biological macromolecules. Additionally, these materials were able to control adult stem cells structure and functions which can be used for tissue regeneration. In general, the versatile character of biphasic gel materials represents a novel class of biological material which can be adaptable to different conditions and applications.Xiaohua Liu completed his Ph.D. at the age of 28 years from Tsinghua University and postdoctoral studies from University of Michigan. He is an Assistant Professor at the Biomedical Sciences Department, Texas A&M University Baylor College of Dentistry. His research focuses on design and synthesizes biomimetic materials and control drug delivery system for translational medicine. He has published more than 35 papers in reputed journals including Nature Materials, Biomaterials, and Tissue Engineering. His work has been cited by others for over 1000 times and has an H-index of 17. He serves as a reviewer for more than 15 journals. Injectable microcarrier with sustained growth factor delivery for bone regenerationC patterning commonly employs photolithographic methods for the micro-fabrication of structures on silicon chip. This requires expensive photo-mask development and complex photolithographic processing. Laser based patterning for cell printing has been previously employed a low cost option to avoid such processing steps. Separate to this, laser ablation of polymers is an active area of research promising high aspect ratios. This seminar reports the first application of laser ablation as a means of ablating the biocompatible polymer parylene-C from SiO2 substrates for the patterning of human brain cells, hence, removing the expensive photolithographic steps. The results demonstrate how this method provides a low cost, rapid prototyping option and high cell yield solution. Charles P. Unsworth, J Tissue Sci Eng 2013, 4:2 http://dx.doi.org/10.4172/2157-7552.S1.010