Gelatinized Copper–Capillary Alginate Gel Functions as an Injectable Tissue Scaffolding System for Stem Cell Transplants

In severe hypoxic–ischemic brain injury, cellular components such as neurons and astrocytes are injured or destroyed along with the supporting extracellular matrix. This presents a challenge to the field of regenerative medicine since the lack of extracellular matrix and supporting structures makes the transplant milieu inhospitable to the transplanted cells. A potential solution to this problem is the use of a biomaterial to provide the extracellular components needed to keep cells localized in cystic brain regions, allowing the cells to form connections and repair lost brain tissue. Ideally, this biomaterial would be combined with stem cells, which have been proven to have therapeutic potentials, and could be delivered via an injection. To study this approach, we derived a hydrogel biomaterial tissue scaffold from oligomeric gelatin and copper–capillary alginate gel (GCCAG). We then demonstrated that our multipotent astrocytic stem cells (MASCs) could be maintained in GCCAG scaffolds for up to 2 weeks in vitro and that the cells retained their multipotency. We next performed a pilot transplant study in which GCCAG was mixed with MASCs and injected into the brain of a neonatal rat pup. After a week in vivo, our results showed that: the GCCAG biomaterial did not cause a significant reactive gliosis; viable cells were retained within the injected scaffolds; and some delivered cells migrated into the surrounding brain tissue. Therefore, GCCAG tissue scaffolds are a promising, novel injectable system for transplantation of stem cells to the brain.     

A Micro-Scale Surface-Structured PCL Scaffold Fabricated by a 3D Plotter and a Chemical Blowing Agent

To study cell responses, polymeric scaffolds with a controllable pore size and porosity have been fabricated using rapid-prototyping methods. However, the scaffolds fabricated by rapid prototyping have very smooth surfaces, which tend to discourage initial cell attachment. Initial cell attachment, migration, differentiation and proliferation are strongly dependent on the chemical and physical characteristics of the scaffold surface. In this study, we propose a three-dimensional (3D) plotting method supplemented with a chemical blowing agent to produce a surface-modified 3D scaffold in which the surface is inscribed with nano- and micro-sized pores. The chemically-blown 3D polymeric scaffold exhibited positive qualities, including the compressive modulus, hydrophilicity and initial cell adhesion. Cell cultures on the scaffolds demonstrated that chondrocytes interacted better with the surface-modified scaffold than with a normal 3D scaffold.     

Preparation and characterization of nano-hydroxyapatite/silk fibroin porous scaffolds

Novel tissue engineering scaffold materials of nano-hydroxyapatite (nHA)/silk fibroin (SF) biocomposite were prepared by freeze-drying. The needle-like nHA crystals of about 10 nm in diameter by 50–80 nm in length, which were uniformly distributed in the porous nHA/SF scaffolds, were prepared by a co-precipitation method with a size. The as-prepared nHA/SF scaffolds showed good homogeneity, interconnected pores and high porosity. XRD and FT-IR analysis suggested that the silk fibroin was in β-sheet structure, which usually provides outstanding mechanical properties for silk materials. In this work, composite scaffolds containing as high as 70% (w/w) nHA were prepared, which had excellent compressive modulus and strength, higher than the scaffolds at low nHA content level and other porous biodegradable polymeric scaffolds often considered in bone-related tissue engineering reported previously. The cell compatibility of composite scaffolds was evaluated through cell viability by MTT assay. All these results indicated that these nHA/SF scaffold materials may be a promising biomaterial for bone tissue engineering.     

Electrospinning versus knitting: two scaffolds for tissue engineering of the aortic valve

Two types of scaffolds were developed for tissue engineering of the aortic valve; an electrospun valvular scaffold and a knitted valvular scaffold. These scaffolds were compared in a physiologic flow system and in a tissue-engineering process. In fibrin gel enclosed human myofibroblasts were seeded onto both types of scaffolds and cultured for 23 days under continuous medium perfusion. Tissue formation was evaluated by confocal laser scanning microscopy, histology and DNA quantification. Collagen formation was quantified by a hydroxyproline assay. When subjected to physiologic flow, the spun scaffold tore within 6 h, whereas the knitted scaffold remained intact. Cells proliferated well on both types of scaffolds, although the cellular penetration into the spun scaffold was poor. Collagen production, normalized to DNA content, was not significantly different for the two types of scaffolds, but seeding efficiency was higher for the spun scaffold, because it acted as a cell impermeable filter. The knitted tissue constructs showed complete cellular in-growth into the pores. An optimal scaffold seems to be a combination of the strength of the knitted structure and the cell-filtering ability of the spun structure.     

Photografting of poly(hydroxylethyl acrylate) onto porous polyurethane scaffolds to improve their endothelial cell compatibility

In order to improve the cytocompatibility of polyurethane (PU) porous scaffolds obtained by thermally-induced phase separation, poly(hydroxylethyl acrylate) (PHEA) was covalently immobilized by grafting copolymerization of HEA by photo-oxidation of the scaffolds and initiation of UV light. FT-IR-ATR spectroscopy and X-ray photoelectron spectroscopy (XPS) characterizations confirmed the occurrence of the grafting co-polymerization of HEA on the porous PU scaffolds. The measurement of water absorption demonstrated the improvement of the hydrophilicity after grafting with PHEA. The results obtained in a human umbilical vein endothelial cell (HUVEC) culture proved that the porous PU scaffold modified with the hydrophilic PHEA had better cytocompatibility than the control. The influence of surface pore size on the HUVEC growth behavior was assessed regarding cell anchorage, proliferation and viability, as well as morphology. An overall increase of cell number and viability with the decrease of the surface pore size was found.     

The influence of molecular weight of chitosan on the physical and biological properties of collagen/chitosan scaffolds

Biopolymer blends between collagen and chitosan have the potential to produce cell scaffolds with biocompatible properties. However, the relationship between the molecular weight of chitosan and its effect on physical and biological properties of collagen/chitosan scaffolds has not been elucidated yet. Porous scaffolds were fabricated by freeze-drying the solution of collagen and chitosan, followed by cross-linking by dehydrothermal treatment. Various types of scaffolds were prepared using chitosan with various molecular weights and blending ratios. Fourier transform infrared spectroscopy proved that collagen and chitosan scaffolds at all blending ratios contained mainly electrostatic interactions at the molecular level. The compressive modulus decreased with increasing the concentration of chitosan. Equilibrium swelling ratios of approximately 6–8, determined in phosphate-buffered saline at physiological pH (7.4), were found in case of collagen-dominated scaffolds. The lysozyme biodegradation test demonstrated that the presence of chitosan, especially the high-molecular-weight species, could significantly prolong the biodegradation of collagen/chitosan scaffolds. In vitro culture of L929 mouse connective tissue fibroblast evidenced that low-molecular-weight chitosan was more effective to promote and accelerate cell proliferation, particularly for scaffolds containing 30 wt% chitosan. The results elucidated that the blends of collagen with low-molecular-weight chitosan have a high potential to be applied as new materials for skin-tissue engineering.     

Cartilaginous tissue formation using a mechano-active scaffold and dynamic compressive stimulation

It is known that complex loading is involved in the development and maintenance of articular cartilage in the body. It means the compressive mechanical stimulation is a very important factor for formation of articular cartilage using a tissue-engineering technique. The objective of this study is to engineer cartilaginous constructs with mechano-active scaffolds and to evaluate the effect of dynamic compression for regeneration of cartilage. The mechano-active scaffolds were prepared from a very elastic poly(L-lactide-co-ε-caprolactone) (PLCL) with 85% porosity and 300–500 μm pore size using a gel-pressing method. The scaffold was seeded with 2 × 10⁶ chondrocytes and the continuous compressive deformation of 5% strain was applied with 0.1 Hz for 10 days and 24 days, respectively. Then, the chondrocytes-seeded constructs were implanted subcutaneously into nude mice. Mechano-active scaffolds with complete rubber-like elasticity showed almost complete (over 97%) recovery at an applied strain of up to 500%. The amount of chondral extracellular matrix was increased significantly by mechanical stimulation on the highly elastic mechano-active scaffolds. Histological analysis showed the mechanically stimulated implants formed mature and well-developed cartilaginous tissue, as evidenced by the chondrocytes within lacunae and the abundant accumulation of sulfated GAGs. However, unhealthy lacunae shapes and hypertrophy forms were observed in the implants stimulated mechanically for 24 days, compared with those stimulated for 10 days. In conclusion, the proper periodical application of dynamic compression can encourage chondrocytes to maintain their phenotypes and enhance the production of GAGs, which would improve the quality of cartilaginous tissue formed both in vitro and in vivo.     

Development of Biodegradable Polyurethane Scaffolds Using Amino Acid and Dipeptide-Based Chain Extenders for Soft Tissue Engineering

The inherent flexibility of polyurethane (PU) chemistry allows the incorporation of specific chemical moieties into the backbone structure conferring a unique biological function to these synthetic polymers. We describe here the synthesis and characterization of a PU containing a Gly–Leu linkage, the cleavage site of several matrix metalloproteinases. A Gly–Leu dipeptide was introduced into the chain extender of the polyurethane through the reaction with 1,4-cyclohexane dimethanol. PUs synthesized with the Gly–Leu-based chain extender had a high weight-average molecular weight (M _w > 125 × 10³) and were phase segregated, semi-crystalline polymers with a low soft-segment glass-transition temperature (T _g < –50°C). Uniaxial tensile testing of PU films indicated that the polymer could withstand high ultimate tensile strengths (approx. 13 MPa) and were flexible with breaking strains of approx. 900%. The Gly–Leu PU had a significantly higher initial modulus, yield stress and ultimate stress compared to a PU previously developed in our laboratory containing a phenylalanine-based chain extender (Phe PU). The Gly–Leu-based chain extender allowed for better hard segment packing and hydrogen bonding leading to enhanced mechanical properties. Electrospinning was used to form scaffolds with randomly organized fibers and an average fiber diameter of approx. 3.6 μm for both the Gly–Leu and Phe PUs. Mouse embryonic fibroblasts were successfully cultured on the PU scaffolds out to 28 days. Further investigations into cell-mediated polymer degradation will help to identify the suitability of this new biomaterial as scaffolds for soft tissue applications.     

Covalent RGD Modification of the Inner Pore Surface of Polycaprolactone Scaffolds

Scaffold production for tissue engineering was demonstrated by means of a hot compression molding technique and subsequent particulate leaching. The utilization of spherical salt particles as the pore-forming agent ensured complete interconnectivity of the porous structure. This method obviated the use of potentially toxic organic solvents. To overcome the inherent non-cell-adhesive properties of the hydrophobic polymer polycaprolactone (PCL) surface activation with a diamine was performed, followed by the covalent immobilization of the adhesion-promoting RGD-peptide. The wet-chemical approach was performed to guarantee modification throughout the entire scaffold structure. The treatment was characterized by means of chemical and physical methods with respect to an exclusive surface modification without altering the bulk properties of the polymer. RGD-modified scaffolds were tested in cell-culture experiments to investigate the initial attachment and the proliferation of three different cell types.     

Neural Progenitor Cells Survival and Neuronal Differentiation in Peptide-Based Hydrogels

We designed nanofibrous hydrogels as 2-D and 3-D scaffolds for anchorage-dependent cells. The IKVAV-containing peptide amphiphile molecules spontaneously self-assembled into higher-order nanofiber hydrogels under cell-containing media. Neural progenitor cells (NPCs) were incubated in peptide-based hydrogels. Effects of self-assembling hydrogels on survival and neural differentiation of NPCs were observed. Peptide was synthesized using a solid-phase method. TEM study of the hydrogel revealed a network of nanofibers. Phase-contrast light micrographs showed that the described hydrogel had no observable cytotoxicity to NPCs. Additionally this hydrogel could induce cells to differentiate into neuron-like cells and glial-like cells. Moreover, the cells encapsulated within hydrogel had a higher neuronal differentiation rate than in the surface of the hydrogel. This self-assembled hydrogel might serve as nerve tissue-engineering scaffold.