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-ß1 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 © 2015 John Wiley & Sons, Ltd.

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.

Evaluation of three-dimensional chitosan-agarose-gelatin cryogel scaffold for the repair of subchondral cartilage defects: an in vivo study in a rabbit model.

In this study, the potential of a chitosan-agarose-gelatin (CAG) cryogel scaffold for the repair of subchondral cartilage defects was explored in female New Zealand white rabbits. Custom-made CAG cryogel scaffold was implanted in a surgically created subchondral defect (diameter of 4 mm, depth of 4 mm) in knee joint of rabbit. The repair of the subchondral defect was evaluated at regular time interval by both macroscopic as well as microscopic examinations. The gross evaluation of the scaffold-implanted site showed integration of the scaffold with the surrounding tissue. Scanning electron microscopy and histological staining of the remnants of implanted cryogel scaffold showed infiltration of the host cells. The repair of the subchondral defect along with well-integrated regenerated cartilage was confirmed by the histology analysis of the joint. Results showed significant cartilage regeneration by the fourth week until eighth week after implantation. Immunohistochemical analysis confirmed that regenerated tissue is hyaline cartilage and absence of hypertrophy marker was reported. In addition, the CAG scaffolds did not elicit any adverse immunological rejection as shown by hematological analysis. Enzyme-linked immunosorbent assay did not show any statistically significant change in the concentration of tumor necrosis factor-α in the serum, and remained in a nontoxic range. Rabbits with a surgically created defect but no scaffold did not show any cartilage regeneration throughout the experiment of 8 weeks. These results demonstrate that CAG cryogel scaffolds promote repair of an osteochondral defect at a load-bearing site in rabbits.

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 Young's 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.

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.

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.

Cell proliferation on three-dimensional chitosan-agarose-gelatin cryogel scaffolds for tissue engineering applications.

Tissue engineering is a potential approach for the repair of damaged tissues or organs like skin, cartilage, bone etc. Approach utilizes the scaffolds constructed from natural or synthetic polymers fabricated by the available fabrication technologies. This study focuses on the fabrication of the scaffolds using a novel technology called cryogelation, which synthesizes the scaffolds at sub-zero temperature. We have synthesized a novel scaffold from natural polymers like chitosan, agarose and gelatin in optimized ratio using the cryogelation technology. The elasticity of the scaffold was confirmed by rheological studies which supports the utility of the scaffolds for skin and cardiac tissue engineering. Proliferation of different cell types like fibroblast and cardiac cells was analysed by scanning electron microscopy (SEM) and fluorescent microscopy. Biocompatibility of the scaffolds was tested by MTT assay with specific cell type, which showed higher proliferation of the cells on the scaffolds when compared to the two dimensional culture system. Cell proliferation of C(2)C(12) and Cos 7 cells on these scaffolds was further analysed biochemically by alamar blue test and Hoechst test. Biochemical and microscopic analysis of the different cell types on these scaffolds gives an initial insight of these scaffolds towards their utility in skin and cardiac tissue engineering.

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 × 10(5) 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.