Matrix stiffness determines the fate of nucleus pulposus-derived stem cells.

Intervertebral disc (IVD) degeneration and consequent low-back pain present a major medical challenge. Nucleus pulposus-derived stem cells (NP-SCs) may lead to a novel therapy for this severe disease. It was recently shown that survival and function of mature NP cells are regulated in part by tissue stiffness. We hypothesized that modification of matrix stiffness will influence the ability of cultured NP-SCs to proliferate, survive, and differentiate into mature NP cells. NP-SCs were subcultured in three-dimensional matrices of varying degrees of stiffness as measured by the material's shear storage modulus. Cell survival, activity, and rate of differentiation toward the chondrogenic or osteogenic lineage were analyzed. NP-SCs were found to proliferate and differentiate in all matrices, irrespective of matrix stiffness. However, matrices with a low shear storage modulus (G' = 1 kPa) promoted significantly more proliferation and chondrogenic differentiation, whereas matrices with a high modulus (G' = 2 kPa) promoted osteogenic differentiation. Imaging performed via confocal and scanning electron microscopes validated cell survival and highlighted stiffness-dependent cell-matrix interactions. These results underscore the effect of the matrix modulus on the fate of NP-SCs. This research may facilitate elucidation of the complex cross-talk between NP-SCs and their surrounding matrix in healthy as well as pathological conditions.

Synthesis and fabrication of a degradable poly(N-isopropyl acrylamide) scaffold for tissue engineering applications.

Biodegradable poly(N-isopropyl acrylamide) (polyNIPAM) hydrogels with controlled molecular weight of the parent polymer and its degradation products were synthesized by atom transfer radical polymerization in the presence of a polycaprolactone-based di-chlorinated macroinitiator and polycaprolactone dimethacrylate. The phase transition temperature, swelling, hydrolytic degradability, and mechanical properties at 25 and 37°C were explored. A cytocompatibility study showed good NIH3T3 cell response over 5 days culture on the surface of the hydrogels, demonstrated by a consistent increase in cell proliferation detected by an Alamar Blue assay. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] results suggested that the hydrogels and their degradation products in the concentration range of 1-25 mg/mL were not cytotoxic to NIH3T3 cells. A sphere-templating technique was utilized to fabricate biodegradable polyNIPAM scaffolds with monodisperse, pore size. Scaffolds with pore diameter of 48 ± 6 µm were loaded with A-10 smooth muscle cells and then warmed to 37°C entrapping cells in pores approximately 40 µm in diameter, a size we have found to be optimal for angiogenesis and biointegration. Due to their degradable nature, tunable molecular weight, highly interconnected morphology, thermally controlled monodisperse pore size, and temperature-induced volume expansion-contraction, the polyNIPAM-based scaffolds developed in this work will be valuable in tissue engineering.

Histological study of graft failure in AlphaCor transplantation.

To report clinical and histopathologic findings in a case of a failed AlphaCor artificial cornea explanted due to corneal stromal melting. We describe the case of a 77-year-old man who received multiple penetrating keratoplasties (PKPs) and subsequent placement of an AlphaCor artificial cornea. Examination showed total corneal infiltration as well as an AlphaCor that was partially dehisced from the host cornea. After explantation, the implant and adjacent host tissue underwent hematoxylin and eosin staining and high-resolution scanning electron microscopy (HR-SEM). Histopathologic analysis of the specimens revealed infiltration of the skirt pores by reactive corneal fibroblasts. Although the AlphaCor implant is an established method of treating multiple failed PKPs, in this case, HR-SEM imaging strongly suggests that the strength of the interface between the implant and corneal tissue is highly dependent on collagen deposition between the pores found in the implant skirt. Collagen deposition then increases the mechanical strength of the cornea-skirt interface.

Sustained antibiotic release from an intraocular lens-hydrogel assembly for cataract surgery.

PURPOSE: To develop a simple, novel polymeric drug-delivery device for prevention of postoperative bacterial infection after cataract surgery in the developing world. METHODS: A poly(2-hydroxyethyl-methacrylate) (pHEMA) hydrogel was developed to achieve sustained release characteristics of antibiotics. The in vitro antibiotic release kinetics and efficacy of antibiotic function were tested using a silicone biofilm model. In vivo feasibility was investigated using a rabbit model. The control group of rabbits underwent standard cataract surgery with intraocular lens (IOL) implant and postoperative topical antibiotic and steroid. The experimental group received the polymeric device inserted with standard three-piece IOL at the time of surgery and received only topical steroids postoperatively. In vivo intraocular antibiotic levels and outcomes after cataract surgery were evaluated. RESULTS: The in vitro studies demonstrate the antibiotic release kinetics can be controlled by optimization of the surface coating. The in vivo results showed sustained sufficient antibiotic concentration (above minimum inhibitory concentration for most common bacteria related to endophthalmitis) for >4 weeks. There was minimum toxicity observed in vivo. The device was effective in treating induced intraocular infection after cataract surgery. CONCLUSIONS: The initial findings of the polymeric drug-delivery device demonstrate the feasibility delivering sufficient antibiotic in the anterior chamber for the immediate postoperative period in a rabbit model. The device is simple to produce and may help alleviate the potential postsurgical infections in the developing nations.

Peptide-modified "smart" hydrogels and genetically engineered stem cells for skeletal tissue engineering.

Stimuli responsive or "smart" hydrogels are of interest for tissue-engineering applications, featuring the advantages of minimally invasive application. Currently, these materials have yet to be used as a biological replacement in restoring the function of damaged tissues or organs. The aim of this study was to demonstrate the advantages of thermoresponsive, peptide-containing hydrogels as a supportive matrix for genetically engineered stem cells. We used injectable hydrogels, enabling cell delivery to the desired site and providing adequate scaffolding postimplantation. Thermoresponsive hydrogels were developed based on amphiphilic block copolymers of polyethylene-oxide and polypropylene-oxide end-capped with methacrylate or maleimide entities and further reacted with RGD-containing peptides. Cell metabolic activity and survival within those hydrogels was studied, illustrating that the stable peptide-polymer conjugate is required for prolonged cell support. The unique polymer characteristics, combined with its enhanced cell interactions, suggest the potential use of these biomaterials in various tissue engineering applications.

Sustained release of antibiotic from poly(2-hydroxyethyl methacrylate) to prevent blinding infections after cataract surgery.

Intraocular lens implantation after opacified natural lens removal is the primary treatment for cataracts in developed countries. Cataract surgery is generally considered safe, but entails significant risks in countries where sophisticated sterile operating theaters are not widely available. Post-operative infection (endophthalmitis) is a potential blinding complication. Infection often results from bacterial colonization of the new lens implant and subsequent antibiotic-tolerant biofilm formation. To combat this risk, we developed a polymeric hydrogel system that can deliver effective levels of antibiotic over an extended period of time within the globe of the eye. Norfloxacin antibiotic was loaded into cross-linked poly(2-hydroxyethyl methacrylate) (pHEMA) gels, which were subsequently surface-modified with octadecyl isocyanate to produce a hydrophobic rate-limiting barrier controlling norfloxacin release. Octadecyl surface modification was characterized using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). A 15-min modification leads to a uniform surface coating and near zero order release of norfloxacin from the matrix. Norfloxacin released from coated pHEMA kills Staphylococcus epidermidis in suspension and on a simulated medical implant surface. With these data, we demonstrate a new and effective system for sustained drug release from a hydrogel matrix with specific application for intraocular lens surgery.

An essential role for the C-terminal domain of a dragline spider silk protein in directing fiber formation.

We have employed baculovirus-mediated expression of the recombinant A. diadematus spider dragline silk fibroin rADF-4 to explore the role of the evolutionary conserved C-terminal domain in self-assembly of the protein into fiber. In this unique system, polymerization of monomers occurs in the cytoplasm of living cells, giving rise to superfibers, which resemble some properties of the native dragline fibers that are synthesized by the spider using mechanical spinning. While the C-terminal containing rADF-4 self-assembled to create intricate fibers in the host insect cells, a C-terminal deleted form of the protein (rADF-4-DeltaC) self-assembled to create aggregates, which preserved the chemical stability of dragline fibers, yet lacked their shape. Interestingly, ultrastructural analysis showed that the rADF-4-DeltaC monomers did form rudimentary nanofibers, but these were short and crude as compared to those of rADF-4, thus not supporting formation of the highly compact and oriented "superfiber" typical to the rADF-4 form. In addition, using thermal analysis, we show evidence that the rADF-4 fibers but not the rADF-4-DeltaC aggregates contain crystalline domains, further establishing the former as a veritable model of authentic dragline fibers. Thus, we conclude that the conserved C-terminal domain of dragline silk is important for the correct structure of the basic nanofibers, which assemble in an oriented fashion to form the final intricate natural-like dragline silk fiber.

PEO-PPO-PEO-based poly(ether ester urethane)s as degradable reverse thermo-responsive multiblock copolymers.

Aiming at developing biodegradable thermo-responsive polymers that display enhanced rheological properties, a family of PEO-PPO-PEO based poly(ether ester urethane)s, was developed. The materials were produced following a two-step synthetic pathway. The PEO-PPO-PEO triblocks were first end-capped with LA or CL oligo(ester)s whereby pentablocks were produced. Then, the different precursors were chain extended using hexamethylene diisocyanate to create the respective polymers. The length and type of the ester block influenced the behavior of the molecules in water, especially their viscosity versus temperature response. The gelation temperature increased from 23 degrees C for a 20wt% F127 solution to 26 and 31 degrees C for pentablocks with 4.4 and 7.5 lactoyl units, respectively. Materials containing longer LA units failed to show any reverse thermo-responsiveness. The presence of the oligo(ester) blocks also reduced the viscosity of the gel at 37 degrees C. While F127 displayed a viscosity of around 28,000Pas, pentablocks containing 4.4 and 7.5 LA units showed values of 15,400 and 12,600Pas. Also, the viscosity at 37 degrees C as well as the gelation temperature decreased as the molecular weight of the oligo(ester)s increased. Finally, the degradation process of the gels was studied by monitoring their viscosity at body temperature and determining the molecular weight of the polymers, over time. Polymers were tailored so to combine high initial viscosity values with diverse degradation rates, as a function of the length and type of the oligo(ester) present along the polymeric backbone.

Smart hydrogels for in situ generated implants.

The objective of this study was to explore the use of reverse thermo-responsive (RTG) polymers for generating implants at their site of performance, following minimally invasive surgical procedures. Aiming at combining syringability and enhanced mechanical properties, a new family of injectable RTG-displaying polymers that exhibit improved mechanical properties was created, following two different strategies: (1) to synthesize high-molecular-weight polymers by covalenty joining poly(ethylene glycol) and poly(propylene glycol) chains using phosgene as the coupling molecule and (2) to cross-link poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO triblocks after end-capping them with triethoxysilane or methacrylate reactive groups. While the methacrylates cross-linked rapidly, the triethoxysilane groups enabled the system to cross-link gradually over time. The chain-extended PEO/PPO copolymers had molecular weights in the 39 000-54 000 interval and exhibited improved mechanical properties. Reverse thermo-responsive systems displaying gradually increasing mechanical properties were generated by cross-linking triethoxysilane-capped (EO)(99)-(PO)(67)-(EO)(99) (F127) triblocks. Over time, the ethoxysilane groups hydrolyzed and created silanol moieties that subsequently condensated. With the aim of further improving their mechanical behavior, F127 triblocks were reacted with methacryloyl chloride and the resulting dimethacrylate was subsequently cross-linked in an aqueous solution at 37 degrees C. The effect of the concentration of the F127 dimethacrylate on the mechanical properties and the porous structure of the cross-linked matrixes produced was assessed. Rheometric studies revealed that the cross-linked hydrogels attained remarkable mechanical properties and allowed the engineering of robust macroscopic constructs, such as large tubular structures. The microporosity of the matrixes produced was studied by scanning electron microscopy. Monolayered conduits as well as structures comprising two and three layers were engineered in vitro, and their compliance and burst strength were determined.