Using type III recombinant human collagen to construct a series of highly porous scaffolds for tissue regeneration.

As an alternative biopolymer material without the risks of the use of animal-derived collagens in soft tissue engineering applications, recombinant human collagen polypeptide (RHC) was used to construct three-dimensional porous scaffolds. RHC and RHC-chitosan (RHC-CHI) porous scaffolds were fabricated using a freeze-drying method to create highly porous internal structures and then cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). All scaffolds had interconnected porous structures with high porosity (90%), and pore size that ranged from 111 µm to 159 µm. The swelling ability and in vitro degradation of the prepared scaffolds were investigated. The mechanical properties could be tailored to meet the requirements of end-use application by adjusting the concentrations of the polymer or cross-linking agent, and the resulting mechanical strengths were comparable to those of biological soft tissues. The cytocompatibility of the fabricated porous scaffolds was investigated by seeding 3T3 fibroblasts into the porous structures, and then cell proliferation, distribution, morphology, and synthesis of extra cellular matrix-associated proteins were assessed. The results indicated that RHC-based porous scaffolds were non-cytotoxic and promoted the attachment and proliferation of the seeded cells. Finally, the in vivo study proved these porous scaffolds were able to accelerate the cell infiltration and collagen deposition that promoted the wound closure. Overall, the results indicate that RHC-based porous scaffolds show promise for use in soft tissue engineering due to their excellent in vitro cytocompatibility and adjustable mechanical properties.

Recombinant human collagen/chitosan-based soft hydrogels as biomaterials for soft tissue engineering.

Animal-derived collagen may contain viruses, and its impurity can cause immunological reactions. Chitosan, always required a neutralization step in fabricating it into the biocompatible tissue engineering scaffolds. To avoid these risks and simplify the production process, a series of recombinant human collagen/carboxylated chitosan (RHC-CHI) based soft hydrogel scaffolds were prepared by crosslinking-induced gelation and then investigated their feasibilities for use as soft tissue engineering scaffolds. The gelation time was optimized by modulating the biopolymer concentration or reaction temperature. The hydrogel swelling, degradation rate, and mechanical properties were also investigated. The results showed that these parameters could be tuned by adjusting either the RHC-to-chitosan ratio or the total polymer concentration. The mechanical properties of the hydrogels were improved by adding chitosan, but excess chitosan reduced the hydrogel mechanical strength and accelerated the degradation speed. Cytotoxicity tests showed that all fabricated soft hydrogels were biocompatible and displayed no cytotoxicity. Cytocompatibility tests and qRT-PCR studies indicated that the hydrogel system promoted the adhesion and proliferation of NIH-3T3 cells, and cellular activities were directly up-regulated by RHC. Finally, our in vivo study proved these hydrogels were able to accelerate the cell infiltration and wound closure. These results show that the soft RHC-CHI hydrogels show promise in soft-tissue engineering.

A cost-effective, analytical method for measuring metabolic load of mitochondria.

Analysis of cellular energetics is central to understanding metabolic diseases including diabetes and cancer. The two most commonly used methods to monitor cellular respiration are the Seahorse-XF system, and Glo™ assays, which are considered "gold standards". These commercial methods measure energetics indirectly and require considerable financial investment. Here we describe an alternative assay that enables accurate quantification of NADH turnover and that is affordable. This method measures resazurin reduction to resorufin at rising concentrations in the presence of purified mitochondrial extracts until NADH becomes a rate-limiting factor. This indicates the maximal level of NADH turnover in each sample and therefore infers metabolic activity. Here we compare MRC5, MCF7 and MDA231 cell lines which have differing metabolic profiles.

The use of decellularised animal tissue to study disseminating cancer cells.

Since the establishment of cell culture, common practice has been to grow adherent cells in 2D monolayers. Although cells behave completely differently when grown under these artificial conditions, the ease of 2D culturing has meant that this practice still prevails, and adopting conditions that more closely reflect the natural microenvironment has been met with substantial inertia. The alternative, animal models that mimic natural human physiology, are less accessible, strictly regulated and require licences and expensive facilities. Although transition from 2D to 3D cell culturing is gathering momentum, there is a clear need for alternative culturing methods that more closely resemble in vivo conditions. Here, we show that decellularised organs gleaned from discarded animal carcasses are ideal biomimetic scaffolds to support secondary tumour initiation in vitro Further, we describe how to decellularise tissue and perform basic histochemistry and immunofluorescence procedures for cell and matrix detection. Cancer cell behaviour on this matrix is followed by way of an example. Because integration into the traditional work flow is easy and inexpensive, we hope this article will encourage other researchers to adopt this approach.

Mechanical Behavior Optimization of Chitosan Extracted from Shrimp Shells as a Sustainable Material for Shopping Bags.

The use of biodegradable materials for shopping bag production, and other products made from plastics, has recently been an object of intense research-with the aim of reducing the environmental burdens given by conventional materials. Chitosan is a potential material because of its biocompatibility, degradability, and non-toxicity. It is a semi-natural biopolymeric material produced by the deacetylation of chitin, which is the second most abundant natural biopolymer (after cellulose). Chitin is found in the exoskeleton of insects, marine crustaceans, and the cell walls of certain fungi and algae. The raw materials most abundantly available are the shells of crab, shrimp, and prawn. Hence, in this study chitosan was selected as one of the main components of biodegradable materials used for shopping bag production. Firstly, chitin was extracted from shrimp shell waste and then converted to chitosan. The chitosan was next ground to a powder. Although, currently, polyethylene bags are prepared by blown extrusion, in this preliminary research the chitosan powder was dissolved in a solvent and the films were cast. Composite films with several fillers were used as a reinforcement at different dosages to optimize mechanical properties, which have been assessed using tensile tests. These results were compared with those of conventional polyethylene bags used in Egypt. Overall, the chitosan films were found to have a lower ductility but appeared to be strong enough to fulfill shopping bag functions. The addition of fillers, such as chitin whiskers and rice straw, enhanced the mechanical properties of chitosan films, while the addition of chitin worsened overall mechanical behavior.

Comparison of glutaraldehyde and procyanidin cross-linked scaffolds for soft tissue engineering.

Soft tissue injuries are among the most difficult orthopaedic conditions to treat, and regenerative medicine holds the promise of better treatments of these injuries. There is therefore a requirement for substrates and porous scaffolds which provide an appropriate chemical and mechanical environment for cell attachment, growth, proliferation and differentiation. In this study, cross-linked porous gelatin-chitosan (Gel/Chi) scaffolds with high porosity (>90%) were fabricated and their internal morphology, pore sizes and porosities were characterized using scanning electron microscopy (SEM), micro computed tomography (micro-CT) and mercury intrusion porosimetry. The cross-linking agents chosen for this study were Procyanidin (PA), chosen for its biocompatibility, and glutaraldehyde (GA), chosen for comparison as a highly effective cross-linker. Concentrations of these cross-linkers varied from 0.1% to 1% (w/v) and controls had the same gelatin-chitosan blend but were untreated. It was found that the water absorption of cross-linked scaffolds decreased as the cross-linker concentration increased and in vitro collagenase degradation test showed both cross-linkers increased the biostability of the scaffolds. Scaffolds were also tested under compressive load to investigate their resistance to deformation. The results indicated that both cross-linkers increase the stiffness of the scaffolds both initially and at higher strains, but GA cross-linked scaffolds had a higher compressive stiffness than scaffolds cross-linked with PA for a given concentration. Results from cyclic compression and stress relaxation tests showed that PA cross-linked scaffolds recover more rapidly after deformation. 3T3 fibroblasts were cultured on the scaffolds to assess cytotoxicity and biocompatibility. The results indicated that PA was non-cytotoxic and promoted the attachment and proliferation of the seeded cells, while fewer cells were seen on GA cross-linked scaffolds, indicating that the GA had conferred some cytotoxicity. PA cross-linked Gel/Chi porous scaffolds show promise as three dimentional porous scaffolds in tissue engineering, as porous substrates for biomimetic culture environments, and for regenerative medicine applications, due to their excellent biocompatibility and easily adaptable mechanical properties, as well as their lower cost compared to collagen and fibrin based substrates.

Mechanical properties of epidermal cells of whole living roots of Arabidopsis thaliana: an atomic force microscopy study.

The knowledge of mechanical properties of root cell walls is vital to understand how these properties interact with relevant genetic and physiological processes to bring about growth. Expansion of cell walls is an essential component of growth, and the regulation of cell wall expansion is one of the ways in which the mechanics of growth is controlled, managed and directed. In this study, the inherent surface mechanical properties of living Arabidopsis thaliana whole-root epidermal cells were studied at the nanoscale using the technique of atomic force microscopy (AFM). A novel methodology was successfully developed to adapt AFM to live plant roots. Force-Indentation (F-I) experiments were conducted to investigate the mechanical properties along the length of the root. F-I curves for epidermal cells of roots were also generated by varying turgor pressure. The F-I curves displayed a variety of features due to the heterogeneity of the surface. Hysteresis is observed. Application of conventional models to living biological systems such as cell walls in nanometer regimes tends to increase error margins to a large extent. Hence information from the F-I curves were used in a preliminary semiquantitative analysis to infer material properties and calculate two parameters. The work done in the loading and unloading phases (hysteresis) of the force measurements were determined separately and were expressed in terms of "Index of Plasticity" (η), which characterized the elasticity properties of roots as a viscoelastic response. Scaling approaches were used to find the ratio of hardness to reduced modulus (H/E(*)).

Zonal release of proteins within tissue engineering scaffolds.

The manufacture of a scaffold for tissue engineering applications that can control the location and timing of growth factor release is described. The scaffold is formed by the sintering of poly(DL-lactic acid) (P(DL)LA) microparticles, plasticized with poly(ethylene glycol) (PEG), although the method can be used for many other polymer types. The microparticles were loaded with model proteins, trypsin and horseradish peroxidase (HRP), or recombinant human bone morphogenetic protein-2 (rhBMP-2). Entrapment efficiencies above 75% were achieved using a solid-in-oil-in-water system. Controlled release of active protein was achieved for at least 30 days. Microparticles were built into protein-loaded or protein-free layers and release of the protein was restricted to zones within the scaffold. Cell response to rhBMP-2 was tuneable by changing the dose of the rhBMP-2 released by varying the ratio of protein-loaded and protein-free microparticles within scaffolds. Zonal activity of rhBMP-2 on C2C12 cells was demonstrated. The scaffolds may find utility in applications where gradients of growth factors within 3D templates are required or where zonation of tissue growth is required.