Organic-inorganic hybrid materials composed of interpenetrating molecular networks of polymer and silica are potential candidates for synthetic bone grafts as they can mimic the complex structure of a natural bone. Combining the properties of an elastic polymer and bioactive silica with molecular level interactions, the hybrids are expected to exhibit congruent degradation rate and tailored mechanical properties. Property control is primarily achieved through covalent bonds between the components, which can be achieved by functionalising the polymer with silane coupling agents that will then bond to the sol-gel silica. In this work we present new organic- inorganic hybrids of alginate and silica and compare the use of two different coupling agents: 3-glycidoxypropyl trimethoxysilane (GPTMS) or 3-aminopropyltriethoxysilane (APTES). The reaction between alginate and GPTMS involved nucleophilic opening of the GPTMS epoxy ring and formation of ester bonds with alginate carboxylate groups while the reaction with APTES was performed using carbodiimide chemistry. 1H solution state NMR analysis was used to monitor in situ the reaction between the alginate and the coupling agents. It revealed that within 72 hours of reaction most of GPTMS epoxy rings were unreacted or partially hydrolysed to diols indicating a slow functionalization process. 13C CP solid state NMR of aged gel samples did show formation of ester bonds between GPTMS molecules and alginate, showing that the aging step is necessary for covalent coupling to occur (confirmed by SIMS). The reaction between APTES and alginate readily occurred yielding elastic APTES - alginate hydrogels containing amide bonds, confirmed by FTIR, 1H NMR, 13C CP NMR and 15N CP NMR. Depending on the coupling molecule used in the synthesis the silica-alginate hybrids showed different degradation profiles and mechanical properties. GPTMS based hybrids showed faster dissolution in Tris buffer solution at physiological pH compared with APTES based hybrids, which had controllable dissolution behaviour relevant to their coupling degree. Scaffolds were produced by freeze drying APTES / alginate hybrids (pore size 150 µm) and compression tests showed that the scaffolds had an elastic behaviour suggesting that they would be appropriate for cartilage regeneration while the GPTMS based hybrids had improved compressive strength (150 MPa) and toughness comparable with the cortical bone and thus suitable for bone tissue application.

Designing artificial instructive coatings at the cell-biomaterial interface have become an appealing strategy to stimulate tissue regeneration and improve implanted material biocompatibility. Guiding cellular activities, such as adhesion, viability, and differentiation, on the biomaterial surface in a robust and cell-selective manner is often essential for both accelerated healing and the long term clinical success of the surgical intervention. In the biological milieu, however, stability of coatings on the material surface is restricted under the abrasive conditions, such as highly hydrated saline environment accompanied with constant mechanical wearing. Various organisms adapted to living in the seashores, or intertidal zones, suffer from the analogously harsh physical and chemical instabilities. To remain sessile, for example, mussels synthesize a highly complex, spatiotemporally evolving glue containing high amount of 3,4-dihydroxy-L-phenylalanine (DOPA). Herein, we imitate this mechanism by incorporating this chemical residue to self-assembling peptide amphiphile building blocks. Self-assembly of these molecules with complementary peptide amphiphiles carrying biofunctional ligands, such as REDV, DGEA, and KRSR, form nanofibrous networks, which closely mimic both structure and biochemical properties of the native extracellular matrix. Being capable of mediating both non-specific surface adhesion and cell type-specific behavioral guidance, we demonstrate general applicability of this supramolecular adhesive concept. We firstly apply this glue as bioactive cardiovascular stent coating, where endothelial cell adhesion and survival is favored over the smooth muscle cells, which is the main cause of re-closure of the arteries (restenosis) in the long term. We further develop an orthopedic/dental implant coating where mineral-depositing cells are selectively promoted over soft tissue forming fibroblasts. We also develop novel mussel-inspired nanofibers with biofunctional ligands that can synergistically deposit bone-like hydroxyapatite in body fluid mimetic conditions. This paves the way to develop a second generation orthopedic/dental implant, which efficiently mediates differentiation of human mesenchymal stem cells into functional osteoblasts. Altogether, we here concentrate on a mussel-mimetic, hybrid supramolecular adhesive interfaces for biofunctionalization of metal implant surfaces in order to direct the cellular behaviors at the molecular level for long-term success of the regenerating tissue.

Biomaterials play a critical role in the design of medical devices and the implementation of new therapies to improve patient care. Our laboratory pioneered the development of citrate-based polyesters, referred to as polydiolcitrates. Polydiolcitrates can be engineered to have properties that improve the function of medical devices and for applications in regenerative medicine. As an example, aberrant oxidative stress and inflammation play a significant role in the development of neointimal hyperplasia, a problem that eventually leads to the failure of small-diameter vascular grafts and stents. By incorporating antioxidant polydiolcitrates into the design of the device, one can control excess reactive oxygen species (ROS) contributing to oxidative stress and inflammation, reduce neointimal hyperplasia, and potentially increase the lifespan of the device. As for applications in regenerative medicine, when polydiolcitrates are combined with mesenchymal stem cells and hematopoietic stem cells one can create vascularized and innervated bladder tissue that may one day benefit patients with bladder cancer or bladder-related complications from spina bifida. In another example, the efficient healing of diabetic skin ulcers can be a significant challenge due to deacreased vascularization, chronic inflammation, high oxidative stress, and biofilm formation. To address this challenge we are investigating the use of thermoresponsive polydiolcitrate oligomers referred to as nanonets. Nanonets self-assemble into an elastic hydrogel with hierarchical nano- and microscale architecture within seconds upon contact with tissue above 30°C. The hydrogel can effectively entrap and slowly release bioactive compounds, proteins, and transgene products that modulate the wound healing response.

Age-related macular degeneration is a major ophthalmic disease that causes visual impairment and blindness. Although transplantation of autologous peripheral cells has been tested by injection of cell suspensions, limited visual improvement resulted due to the low viability of the injected cells in the subretinal tissue. As an alternative approach, there have been several reports on the development of natural and synthetic substrates for local delivery of retinal pigment epithelial (RPE) cells using collagen, poly(ethylene terephthalate) and poly(methyl methacrylate). However, these engineered substrates with micrometers in thickness (6 mu;m at thinnest) and several millimeters in size are not sufficiently flexible to be aspirated and injected through a conventional syringe needle into the narrow subretinal space. Thus, a large incision of the sclera and retinal tissue would be required for the injection of these rigid substrates. Such an incision might result in leakage of vitreous fluid and lead to post-surgical infection. Therefore, miniaturization of the substrates is an important approach to achieve minimally invasive delivery of the engineered tissue. In this study, we developed micropatterned polymeric nanosheets consisted of biodegradable poly(lactic-co-glycolic acid) and magnetic nanoparticles (MNPs, 10 nm in diameter) toward local delivery of the RPE cells. The micropatterned nanosheet encapsulating MNPs was fabricated by microcontact printing techniques. Owing to the magnetic property and flexible structure, the micropatterned nanosheet with 170 nm thick was manipulated remotely and delivered to the subretinal space of a swine eye. The micropatterned nanosheet also directed growth and morphogenesis of the RPE cells, and allowed for the injection of an engineered RPE monolayer through syringe needles flexibly without loss of cell viability. Such an ultra-thin flexible carrier has the promise of a minimally invasive delivery of organized cellular structures into narrow tissue spaces.

Introduction: Success in tissue engineering of the vocal fold lamina propria extracellular matrix (ECM) for treatment of chronic vocal fold scarring demands an understanding of how cells integrate the signals presented from the scar microenvironment, in combination with the signals from the biomaterial scaffold, to alter their response. To date, approaches to biomaterial development for scarred lamina propria treatment have examined the response of “normal” (non-scar) vocal fold fibroblasts (VFFs) to the biomaterial. As a result, they have failed to capture cellular responses, from activated macrophages and from resident scar-tissue VFFs (also known as myofibroblasts), from the in vivo implant environment which critically impact the quality and rate of VFF ECM production. In the present work, we conjugate cytokines, previously identified as anti-fibrotic and/or immunomodulatory, to biocompatible poly(ethylene glycol) [PEGDA] hydrogel formulations shown to have mechanical properties which preserve mucosal wave activity with low average phonation threshold pressures. We demonstrate these biomaterials influence macrophage polarization — shifting activated macrophages from a pro-inflammatory phenotype to a phenotype that is anti-inflammatory and pro-healing — and shift myofibroblasts to a “normal” or anti-fibrotic VFF phenotype.
Results and Discussion: Pro-inflammatory macrophages and/or myofibroblasts were encapsulated in PEG-bFGF gels. Following 3 days of culture in activation(pro-inflammatory, pro-fibrotic) media, expression of the fibrotic marker αSMA by myofibroblasts in PEG-bFGF gels was reduced over 7-fold relative to non-bFGF containing gels. Similarly, the expression of genes associated with matrix turnover (e.g. MMP1) were increased in response to bFGF. Activated macrophages exposed to bFGF gels displayed over a 50% reduction in inflammatory makers Nos2 and IL-12β relative to non-bFGF hydrogel controls. This reduction in fibrotic markers in myofibroblasts and inflammatory markers in macrophages in bFGF-containing gels occurred despite the continued presence of activating media and is consistent with anti-fibrotic and anti-inflammatory properties reported for bFGF. These results suggest that PEG-bFGF gels could be used to transition the activated cells present in chronic vocal fold scar to more phenotypes more conducive to healing.
Conclusions: The design of material environments to transition activated cells that are present in wound environments to more “normal” cell phenotypes would be desirable in the treatment of chronic scar and other chronic wounds. The present results represent an important step toward using tissue engineering principles to modulate cell phenotypes within an active wound environment.

The selection of a scaffold material is both a critical and difficult choice that will determine the success of failure of any tissue engineering (TE) strategy. We believe that natural origin polymers are the best choice for many approaches. In addition, we have been developing an all range of processing methodologies to produce adequate scaffolds for different TE applications. Furthermore an adequate cell source should be selected. In many cases efficient cell isolation, expansion and differentiation methodologies should be developed and optimized. We have been using different human cell sources namely: mesenchymal stem cells from bone marrow, mesenchymal stem cells from human adipose tissue, human cells from amniotic fluids and membranes and cells obtained from human umbilical cords. The potential of each type of cells, to be used to develop novel useful regeneration therapies will be discussed. Their uses and their interactions with different natural origin degradable scaffolds and distinct nano and micro-carriers, and smart release systems, will be described. A great focus will be given to the different sources of stem cells, the isolation of distinct sub-populations, ways of differentiating them, as well as their interactions with different 3D architectures and materials for culturing them. The use of bioreactors to control cell differentiation, as well as the surface modification of the materials in order to control cell adhesion and proliferation will also be illustrated. Several biomimetic and nanotechnology based strategies to engineer mineralized tissues will be described.

Biomedical adhesive is important in clinical medicine and various surgical conditions require new and better polymeric adhesives. The biomedical adhesive should be wet adhesive, nontoxic, physiologically stable, rapidly crosslinkable and physically flexible. A new bio-inspired adhesive for medical applications was synthesized from three monomers, N-methacryloyl-3,4-dihydroxyl-L-phenylalanine, acrylic acid N-hydroxysuccinimide ester and acrylic acid. 3,4-dihydroxy-L-phenylalanine (DOPA) containing polymer segment has a function of strong wet adhesion properties. Poly(acrylic acid) is a water soluble segment and N-hydroxysuccinimide ester rapidly forms covalent bonds with thiols on thiol terminated 3-armed poly(ethylene glycol) cross-linking agents. The adhesive with DOPA incorporated demonstrated increased adhesion strength that is measured by lap shear strength tests on wet porcine skin mimicking human tissues. Crosslinking the polymers significantly enhanced the maximum adhesion strength. The novel adhesive shows viscosity drops at high shear rate, demonstrating its potential to be injected through syringe needles. The enhance moduli of crosslinked adhesive revealed that its robust stability after the adhesive attached on intended sites.
Recent developments in various invasive fetal diagnosis and fetal therapeutic technologies has been contributed to improve fetus&’ health, but the limited healing capability of fetal membrane may cause clinically dangerous Preterm Premature Rupture of Membranes (PPROM) from the medical device punctured site. This problem could be solved by the developed new bio-medical adhesives. The wet adhesive properties of the new terpolymer were evaluated in the context of sealing punctured fetal membranes. In order to perform realistic in vitro test on human fetal membrane, a new test device was designed and manufactured to mimic maternal uterus and amniotic sac. The new adhesive successfully sealed needle punctures on fetal membrane. Also the new terpolymer was directly compared to commercially available medical adhesives and was found to be more efficient in the presealing treatment of fetal membrane.

Microbial infections endanger public health unremittingly and cause a huge economic burden to our society. Numerous efforts have been made to develop molecules which are able to inhibit pathogens. Novel broad-spectrum antimicrobial materials were developed based on natural polysaccharide. Firstly, a group of antimicrobial materials were synthesized by quaternization and alkylation of chitosan.(1) An argon plasma-ultraviolet (UV) induced coating method for hydrogel surface immobilization was developed, which can be applied on diverse soft biomedical surfaces. A novel mechanism of these hydrogels based on “anion sponge” concept was proposed and proven. The optimized coating formulation and conditions show excellent antimicrobial potency. The in vitro and in vivo studies suggest this antimicrobial coating is biocompatible with mammalian cells. Secondly, a peptidopolysaccharide that mimics the bacterial peptidoglycan structure, which is a feature unique to bacterial membrane but absent in mammalian cells, was designed, synthesized and tested.(2) By the ring-opening polymerization of N-carboxyanhydrides (NCA), a polysaccharide backbone was copolymerized with cationic polylysine, and the resulting optimized peptidopolysacchride shows high selectivity to bacteria over mammalian cell. Preliminary results also suggest that the compound stimulates little or no inflammatory response. These two classes of polysaccharide based materials reveal new directions for the design of antimicrobial materials, and they have a promising prospect in further applications.
References:
1. Peng Li, et al. Nature Materials, 2011, 10(2), 149-156.
2. Peng Li, et al. Advanced Materials 2012, 24(30), 4130-4137.

Controlling the spatial organization of cells with well-defined architecture is a vital aspect of tissue engineering. Odontoblasts are the cells that synthesize dentin. They are densely packed and line the pulpal wall while anchored into dentin through their processes. Recreating the odontoblast layer and morphology may facilitate dentin development in-vitro. Objective: To craft an in-vitro construct that is able to position cells in a configuration that mimics the highly organized layer of odontoblasts in teeth using micro porous membranes and promote secretion of a collagenous matrix that mineralizes. Method: A two-step lithographic process was used to generate a master-mold on a silicon wafer. Polycaprolactone (PCL) was spin cast onto the mold to create a membrane with 1-3µm pores, and pillars with 10µm heights surrounding each pore. Commercial cell culture inserts with 1 or 3mu;m pores and homemade porous PCL membrane were used to enable high-density, single-layer cell accumulation and formation of cell protrusion. MC3T3 cells were seeded at confluent densities onto membranes and centrifuged at 700 rpm to position. Cells were allowed to mineralize under a chemical gradient for up to 21 days. Results: MC3T3 cells formed a monolayer of cells on membrane after centrifugation and produced collagenous matrix, which appeared continuous over large areas. Chemical gradient created with serum, led to the formation of cell protrusions extending through pores at day 21. However, it was observed that MC3T3 cells lose its 3D organization on the porous membranes within 24hr with the absence of pillar structures. Presence of pillars on PCL membrane may prolong cellular organization for a longer time and enable organized matrix production. Conclusion: A porous membranes engineered with micropatterning technology can be used to confine one cell to a single pore while inducing cell protrusion successfully, providing us the possibility of recreating the odontoblastic morphology in-vitro.

Collagen is a naturally occurring biopolymer and its&’ architecture is important in tissue development and repair. We have explored the nucleation, assembly and the 3-D architecture in collagen hydrogels prepared under the conditions commonly employed in biomedical, tissue engineering and medical device research. Employing in situ multi-photon optical microscopy that combines nonlinear optical phenomena of second harmonic generation (SHG) and two-photon fluorescence (TPF) signals we showed that drastically different microstructures are prepared by changing collagen solid content, incubation temperature or ions and moreover that the non-zero cross-linkers for example genipin significantly alter the originally assembled 37 C architectures. Specifically, genipin induces formation of aggregated fluorescent fibers concomitantly with a 3.5 fold drop in SHG contrast (after 24-hour reaction at 37 C) suggesting that this modification disaggregates initial collagen microstructure within the materials at the expense of forming the new fluorescent features. The 800 nm excitation wavelength generates complex TPF contrast within genipin-modified materials. It is centered within the blue-green (~ 490 nm center) as well as red (~ 615 nm center) spectral regions and spatially distinct. The ratio of the intensity of 615 nm emission band to the intensity of 490 nm emission band is 5.3 (after 24-hour reaction at 37 C). There is a slight bathochromic (red) shift when the excitation wavelength is varied between 760 nm and 900 nm. The findings will be discussed within the context of our ongoing tissue engineering and wound repair research.

In the recent years, there is an alarming increase in obesity worldwide. Obesity associated health concerns as well as stigma has driven affected people for reconstructive surgeries. With this, the amount of adipose tissue discarded as a clinical waste has increased proportionally. Literature suggests different protocols that decellularize adipose tissue. These protocols rely on combinations of physical, chemical and enzymatic agents to remove cells and lipids. The end-product of decellularization yields extracellular matrix (ECM) that supports the cells in an organ; both structurally and functionally. Although the previously designed protocols have been successful in the decellularization, they span a number of days. Our study involves a purely enzymatic, chemical or physical process of decellularization. Further, we distinguish them based on their ability to remove lipid and cells. We then tested the capability of these processes in preserving the essential biomolecules such as collagen, Glucoseaminoglyxcans (GAG), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Additionally, we tested the ability of ECMs generated by the enzymatic (ECM-E), chemical (ECM-C) and physical (ECM-P) to support various cell types. We report that the physical method of decellularization is advantageous due to its shorter time; efficient lipid and cell removal; and collagen, GAG, VEGF and bFGF conservation. Therefore, we recommend the use of ECM-P for scaffolds and coats to apply in tissue engineering.