Encapsulating therapeutic cells in RGD-modified agarose microcapsules.

Current cell-based strategies for repairing damaged tissue often show limited efficacy due to low cell retention at the site of injury. Encapsulation of cells within hydrogel microcapsules demonstrably increases cell retention but benefits can be limited due to premature cell escape from the hydrogel microcapsules and subsequent clearance from the targeted tissue. We propose a method of encapsulating cells in agarose microcapsules that have been modified to increase cell retention by providing cell attachment domains within the agarose hydrogel allowing cells to adhere to the microcapsules. We covalently modified agarose with the addition of the cell adhesion peptide, RGD (arginine, glycine, aspartic acid). We then used a microfluidic platform to encapsulate single cells within 50 µm agarose microcapsules. We tracked encapsulated cells for cell viability, egress from microcapsules and attachment to microcapsules at 2 h, 24 h, and 48 h after encapsulation. Many encapsulated cells eventually egress their microcapsule. Those that were encapsulated using RGD-modified agarose adhered to the outer surface of the microcapsule following egress. NIH 3T3 cells showed nearly 45% of egressed cells attached to the outside of RGD modified agarose microcapsules, while minimal cellular adhesion was observed when using unmodified agarose. Similarly, human umbilical vein endothelial cells had up to 33% of egressed cells attached and explant-derived cardiac cells showed up to 20% attachment with the presence of RGD binding domains within the agarose microcapsules.

Enhanced Collagen-like Protein for Facile Biomaterial Fabrication.

We present a collagen-mimetic protein of bacterial origin based upon a modified subdomain of the collagen-like Sc12 protein from Streptococcus pyogenes, as an alternative collagen-like biomaterial platform that is highly soluble, forms stable, homogeneous, fluid-like solutions at elevated concentrations, and that can be efficiently fabricated into hydrogel materials over a broad range of pH conditions. This extended bacterial collagen-like (eBCL) protein is expressed in a bacterial host and purified as a trimeric assembly exhibiting a triple helical secondary structure in its collagen-like subdomain that is stable near physiological solution conditions (neutral pH and 37 °C), as well as over a broad range of pH conditions. We also show how this sequence can be modified to include biofunctional attributes, in particular, the Arg-Gly-Asp (RGD) sequence to elicit integrin-specific cell binding, without loss of structural function. Furthermore, through the use of EDC-NHS chemistry, we demonstrate that members of this eBCL protein system can be covalently cross-linked to fabricate transparent hydrogels with high protein concentrations (at least to 20% w/w). These hydrogels are shown to possess material properties and resistance to enzymatic degradation that are comparable or superior to a type I collagen control. Moreover, such hydrogels containing the constructs with the RGD integrin-binding sequence are shown to promote the adhesion, spreading, and proliferation of C2C12 and 3T3 cells in vitro. Due to its enhanced solubility, structural stability, fluidity at elevated concentrations, ease of modification, and facility of cross-linking, this eBCL collagen-mimetic system has potential for numerous biomedical material applications, where the ease of processing and fabrication and the facility to tailor the sequence for specific biological functionality are desired.

A biomimetic scaffold for culturing limbal stem cells: a promising alternative for clinical transplantation.

Limbal tissues can be cultured on various types of scaffolds to create a sheet of limbal-corneal epithelium for research as well as clinical transplantation. An optically clear, biocompatible, biomimetic scaffold would be an ideal replacement graft for transplanting limbal stem cells. In this study, we evaluated the physical and culture characteristics of the recombinant human cross-linked collagen scaffold (RHC-III scaffold) and compared it with denuded human amniotic membrane (HAM). Optical/mechanical properties and microbial susceptibility were measured for the scaffolds. With the approval of the institutional review board, 2 mm fresh human limbal tissues were cultured on 2.5 x 2.5 cm(2) scaffolds in a medium containing autologous serum in a feeder cell-free submerged system. The cultured cell systems were characterized by morphology and immunohistochemistry for putative stem cells and differentiated cell markers. The refractive index (RI) and tensile strength of the RHC-III scaffold were comparable to human cornea, with delayed in vitro degradation compared to HAM. RHC-III scaffolds were 10-fold less susceptible to microbial growth. Cultures were initiated on day 1, expanded to form a monolayer by day 3 and covered the entire growth surface in 10 days. Stratified epithelium on the scaffolds was visualized by transmission electron microscopy. The cultured cells showed p63 and ABCG2 positivity in the basal layer and were immunoreactive for cytokeratin K3 and K12 in the suprabasal layers. RHC-III scaffold supports and retains the growth and stemness of limbal stem cells, in addition to resembling human cornea; thus, it could be a good replacement scaffold for growing cells for clinical transplantation.