Asymmetrical interactions between nanoparticles and proteins arising from deformation upon adsorption to surfaces.

Drug release from polymeric nanoparticles (NPs) is governed by their adsorption onto cell membranes and transmigration across cell walls. These steps are influenced by their interactions with proteins near the cells. These interactions were investigated by studying the sequential adsorption of plasma proteins, albumin (Alb) and fibrinogen (Fg), and micellar NPs using quartz crystal microbalance with dissipation (QCMD), X-ray photoelectron spectroscopy (XPS), and small-angle X-ray scattering (SAXS). The three NPs in the study all have poly(ethylene glycol) (PEG) shells but different cores: amorphous poly(propylene oxide) (PPO), crystalline polycaprolactone (PCL), and poly(desaminotyrosyl-tyrosine octyl ester-co-suberic acid) (DTO-SA). None of the NPs adsorbed onto a pre-adsorbed Fg layer. On the other hand, when the deposition sequence was reversed, Fg was adsorbed onto DTO-SA NP and PCL NP surfaces, but not onto the PPO NP surface. The interactions with Alb were different: DTO-SA did not adsorb onto Alb and vice versa; PPO NP adsorbed onto an Alb layer, but Alb did not adsorb onto the PPO NP layer; and PCL NP reversibly adsorbed onto Alb, but Alb displaced pre-adsorbed PCL NP. Thus, in most instances, the adsorption behavior was asymmetric in that it was dependent on the order of arrival of the adsorbates at the substrate. SAXS data did not show evidence for complex formation in solution. Thus, the solution behavior appears not to be a predictor of the interaction of proteins and the NPs near surfaces. Differing strengths of pairwise interactions of proteins, NPs and substrates account for this adsorption behavior. These differences in interactions could be the results of deformation of the adsorbates immobilized at the surface and the different degrees of surface remodeling that occur upon adsorption. Deformation could lead to disassembly of the NPs that has implications on their ability to release their payload of drugs upon adsorption onto tissue surfaces.

Examining polymer-protein biophysical interactions with small-angle x-ray scattering and quartz crystal microbalance with dissipation.

Polymer-protein hybrids can be deployed to improve protein solubility and stability in denaturing environments. While previous work used robotics and active machine learning to inform new designs, further biophysical information is required to ascertain structure-function behavior. Here, we show the value of tandem small-angle x-ray scattering (SAXS) and quartz crystal microbalance with dissipation (QCMD) experiments to reveal detailed polymer-protein interactions with horseradish peroxidase (HRP) as a test case. Of particular interest was the process of polymer-protein complex formation under thermal stress whereby SAXS monitors formation in solution while QCMD follows these dynamics at an interface. The radius of gyration (Rg ) of the protein as measured by SAXS does not change significantly in the presence of polymer under denaturing conditions, but thickness and dissipation changes were observed in QCMD data. SAXS data with and without thermal stress were utilized to create bead models of the potential complexes and denatured enzyme, and each model fit provided insight into the degree of interactions. Additionally, QCMD data demonstrated that HRP deforms by spreading upon surface adsorption at low concentration as shown by longer adsorption times and smaller frequency shifts. In contrast, thermally stressed and highly inactive HRP had faster adsorption kinetics. The combination of SAXS and QCMD serves as a framework for biophysical characterization of interactions between proteins and polymers which could be useful in designing polymer-protein hybrids.

Biomimetic Strategies for Peripheral Nerve Injury Repair: An Exploration of Microarchitecture and Cellularization.

Injuries to the nervous system present formidable challenges to scientists, clinicians, and patients. While regeneration within the central nervous system is minimal, peripheral nerves can regenerate, albeit with limitations. The regenerative mechanisms of the peripheral nervous system thus provide fertile ground for clinical and scientific advancement, and opportunities to learn fundamental lessons regarding nerve behavior in the context of regeneration, particularly the relationship of axons to their support cells and the extracellular matrix environment. However, few current interventions adequately address peripheral nerve injuries. This article aims to elucidate areas in which progress might be made toward developing better interventions, particularly using synthetic nerve grafts. The article first provides a thorough review of peripheral nerve anatomy, physiology, and the regenerative mechanisms that occur in response to injury. This is followed by a discussion of currently available interventions for peripheral nerve injuries. Promising biomaterial fabrication techniques which aim to recapitulate nerve architecture, along with approaches to enhancing these biomaterial scaffolds with growth factors and cellular components, are then described. The final section elucidates specific considerations when developing nerve grafts, including utilizing induced pluripotent stem cells, Schwann cells, nerve growth factors, and multilayered structures that mimic the architectures of the natural nerve.

Iodination of PEGylated Polymers Counteracts the Inhibition of Fibrinogen Adsorption by PEG.

Poly(ethylene glycol), PEG, known to inhibit protein adsorption, is widely used on the surfaces of biomedical devices when biofilm formation is undesirable. Poly(desaminotyrosyl-tyrosine ethyl ester carbonate), PDTEC, PC for short, has been a promising coating polymer for insertion devices, and it has been anticipated that PEG plays a similar role if it is copolymerized with PC. Earlier studies show that no fibrinogen (Fg) is adsorbed onto PC polymers with PEG beyond the threshold weight percentage. This is attributed to the phase separation of PEG. Further, iodination of the PC units in the PC polymer, (I2PC), has been found to counteract this Fg-repulsive effect by PEG. In this study, we employ surface-sensitive X-ray techniques to demonstrate the surface affinity of Fg toward the air-water interface, particularly in the presence of self-assembled PC-based film, in which its constituent polymer units are assumed to be much more mobile as a free-standing film. Fg is found to form a Gibbs monolayer with its long axis parallel to the aqueous surface, thus maximizing its interactions with hydrophobic interfaces. It influences the amount of insoluble, surface-bound I2PC likely due to the desorption of the formed Fg-I2PC complex and/or the penetration of Fg onto the I2PC film. The results show that the phase behavior at the liquid-polymer interface shall be taken into account for the surface behavior of bulk polymers surrounded by tissue. The ability of PEG units rearranging into a protein-blocking layer, rather than its mere presence in the polymer, is the key to antifouling characteristics desired for polymeric coating on insertion devices.

Axonal Tract Reconstruction Using a Tissue-Engineered Nigrostriatal Pathway in a Rat Model of Parkinson's Disease.

Parkinson's disease (PD) affects 1-2% of people over 65, causing significant morbidity across a progressive disease course. The classic PD motor deficits are caused by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), resulting in the loss of their long-distance axonal projections that modulate striatal output. While contemporary treatments temporarily alleviate symptoms of this disconnection, there is no approach able to replace the nigrostriatal pathway. We applied microtissue engineering techniques to create a living, implantable tissue-engineered nigrostriatal pathway (TE-NSP) that mimics the architecture and function of the native pathway. TE-NSPs comprise a discrete population of dopaminergic neurons extending long, bundled axonal tracts within the lumen of hydrogel micro-columns. Neurons were isolated from the ventral mesencephalon of transgenic rats selectively expressing the green fluorescent protein in dopaminergic neurons with subsequent fluorescent-activated cell sorting to enrich a population to 60% purity. The lumen extracellular matrix and growth factors were varied to optimize cytoarchitecture and neurite length, while immunocytochemistry and fast-scan cyclic voltammetry (FSCV) revealed that TE-NSP axons released dopamine and integrated with striatal neurons in vitro. Finally, TE-NSPs were implanted to span the nigrostriatal pathway in a rat PD model with a unilateral 6-hydroxydopamine SNpc lesion. Immunohistochemistry and FSCV established that transplanted TE-NSPs survived, maintained their axonal tract projections, extended dopaminergic neurites into host tissue, and released dopamine in the striatum. This work showed proof of concept that TE-NSPs can reconstruct the nigrostriatal pathway, providing motivation for future studies evaluating potential functional benefits and long-term durability of this strategy. This pathway reconstruction strategy may ultimately replace lost neuroarchitecture and alleviate the cause of motor symptoms for PD patients.

Machine Learning on a Robotic Platform for the Design of Polymer-Protein Hybrids.

Polymer-protein hybrids are intriguing materials that can bolster protein stability in non-native environments, thereby enhancing their utility in diverse medicinal, commercial, and industrial applications. One stabilization strategy involves designing synthetic random copolymers with compositions attuned to the protein surface, but rational design is complicated by the vast chemical and composition space. Here, a strategy is reported to design protein-stabilizing copolymers based on active machine learning, facilitated by automated material synthesis and characterization platforms. The versatility and robustness of the approach is demonstrated by the successful identification of copolymers that preserve, or even enhance, the activity of three chemically distinct enzymes following exposure to thermal denaturing conditions. Although systematic screening results in mixed success, active learning appropriately identifies unique and effective copolymer chemistries for the stabilization of each enzyme. Overall, this work broadens the capabilities to design fit-for-purpose synthetic copolymers that promote or otherwise manipulate protein activity, with extensions toward the design of robust polymer-protein hybrid materials.

Thermal Processing of a Degradable Carboxylic Acid-Functionalized Polycarbonate into Scaffolds for Tissue Engineering.

Degradable polymers are often desirable for the fabrication of medical implants, but thermal processing of these polymers is a challenge. We describe here how these problems can be addressed by discussing the extrusion of fibers and injection molding of bone pins from a hydrolytically degradable tyrosine-derived polycarbonate. Our initial attempts produced fibers and pins with bubbles, voids, and discoloration, and resulted in the formation of large polymer plugs that seized screws and blocked extruder dies. The material and process parameters that contribute to these issues were investigated by studying the physical and chemical changes that occur during processing. Differential scanning calorimetry (DSC) scans and thermogravimetric analysis combined with IR (TGA-IR) analysis revealed the role of residual moisture and residual solvents that in conjunction with heat cause degradation and crosslinking as indicated by gel permeation chromatography (GPC). Rheology and melt-flow index measurements were useful in characterizing the extent of dependence of polymer viscosity on temperature and molecular weight. With these insights, we could process our polymer into fibers and rods by controlling residual moisture, time and temperature, and by adjusting processing parameters in real-time. The systematic approach described here is applicable to other degradable polymers that are difficult to process.

A multilayered scaffold for regeneration of smooth muscle and connective tissue layers.

Tissue regeneration often requires recruitment of different cell types and rebuilding of two or more tissue layers to restore function. Here, we describe the creation of a novel multilayered scaffold with distinct fiber organizations-aligned to unaligned and dense to porous-to template common architectures found in adjacent tissue layers. Electrospun scaffolds were fabricated using a biodegradable, tyrosine-derived terpolymer, yielding densely-packed, aligned fibers that transition into randomly-oriented fibers of increasing diameter and porosity. We demonstrate that differently-oriented scaffold fibers direct cell and extracellular matrix (ECM) organization, and that scaffold fibers and ECM protein networks are maintained after decellularization. Smooth muscle and connective tissue layers are frequently adjacent in vivo; we show that within a single scaffold, the architecture supports alignment of contractile smooth muscle cells and deposition by fibroblasts of a meshwork of ECM fibrils. We rolled a flat scaffold into a tubular construct and, after culture, showed cell viability, orientation, and tissue-specific protein expression in the tube were similar to the flat-sheet scaffold. This scaffold design not only has translational potential for reparation of flat and tubular tissue layers but can also be customized for alternative applications by introducing two or more cell types in different combinations.

Tyrosine-derived polycarbonate nerve guidance tubes elicit proregenerative extracellular matrix deposition when used to bridge segmental nerve defects in swine.

Promising biomaterials should be tested in appropriate large animal models that recapitulate human inflammatory and regenerative responses. Previous studies have shown tyrosine-derived polycarbonates (TyrPC) are versatile biomaterials with a wide range of applications across multiple disciplines. The library of TyrPC has been well studied and consists of thousands of polymer compositions with tunable mechanical characteristics and degradation and resorption rates that are useful for nerve guidance tubes (NGTs). NGTs made of different TyrPCs have been used in segmental nerve defect models in small animals. The current study is an extension of this work and evaluates NGTs made using two different TyrPC compositions in a 1 cm porcine peripheral nerve repair model. We first evaluated a nondegradable TyrPC formulation, demonstrating proof-of-concept chronic regenerative efficacy up to 6 months with similar nerve/muscle electrophysiology and morphometry to the autograft repair control. Next, we characterized the acute regenerative response using a degradable TyrPC formulation. After 2 weeks in vivo, TyrPC NGT promoted greater deposition of pro-regenerative extracellular matrix (ECM) constituents (in particular collagen I, collagen III, collagen IV, laminin, and fibronectin) compared to commercially available collagen-based NGTs. This corresponded with dense Schwann cell infiltration and axon extension across the lumen. These findings confirmed results reported previously in a mouse model and reveal that TyrPC NGTs were well tolerated in swine and facilitated host axon regeneration and Schwann cell infiltration in the acute phase across segmental defects - likely by eliciting a favorable neurotrophic ECM milieu. This regenerative response ultimately can contribute to functional recovery.

Calcium phosphate enriched synthetic tyrosine-derived polycarbonate - dicalcium phosphate dihydrate polymer scaffolds for enhanced bone regeneration.

Optimal repair of large craniomaxillofacial (CMF) defects caused by trauma or disease requires the development of new, synthetic osteoconductive materials in combination with cell-based therapies, to overcome the limitations of traditionally used bone graft substitutes. In this study, tyrosine-derived polycarbonate, E1001(1k) scaffolds were fabricated to incorporate the osteoinductive coating, Dicalcium phosphate dihydrate (DCPD). The biocompatibility of E1001(1k)-DCPD, E1001(1k)-ßTCP and E1001(1k) scaffolds was compared using in vitro culture with human dental pulp stem cells (hDPSCs). We found that the DCPD coating was converted to carbonated hydroxyapatite over time in in vitro culture in Osteogenic Media, while the ßTCP did not. hDPSCs exhibited slow initial attachment and proliferation on DCPD E1001(1k) scaffolds, but subsequently improved over time in culture, and promoted osteogenic differentiation. To the best of our knowledge, this study highlights for the first time the effects of Osteogenic Media on phase changes of DCPD, and on DCPD scaffold cytocompatibility with hDPSCs. DCPD showed similar hDPSC biocompatibility and osteoconductivity as compared to ßTCP, and osteogenic differentiation of seeded hDPSCs. These studies suggest that E1001(1k)-DCPD scaffolds are a superior tool for craniofacial bone regeneration and provide the foundation for future in vivo testing.

Disassembly of Nanospheres with a PEG Shell upon Adsorption onto PEGylated Substrates.

Polymeric nanospheres have the ability to encapsulate drugs and are therefore widely used in drug delivery applications. Structural transformations that affect drug release from nanospheres are governed by the surrounding environment. To understand these effects, we investigated the adsorption behavior of three types of nanospheres onto model surfaces using quartz crystal microbalance with dissipation (QCM-D) and by atomic force microscopy (AFM). Substrates were prepared from polymers with different degrees of PEGylation (0, 1, and 15%). Nanospheres were prepared via self-assembly of block copolymers. Tyrosine-derived nanospheres are A-B-A triblock copolymers with methoxy poly(ethylene glycol) (PEG) as the A-blocks and an alternating copolymer of desaminotyrosyl-tyrosine octyl ester and suberic acid oligo(DTO-SA) as the B-block. On non-PEGylated substrates, these nanospheres assembled into a close-packed structure; on PEGylated substrates, the adsorbed nanospheres formed a continuous film, thinner than the size of the nanospheres suggesting unraveling of the PEG corona and disassembly of the nanospheres. Also, the adsorption was concentration-dependent, the final thickness being attained at exponentially longer times at lower concentrations. Such substrate- and concentration-dependent behavior was not observed with Pluronic F-127 and PEG-poly(caprolactone) (PCL) nanospheres. Since the essential difference among the three nanospheres is the composition of the core, we conclude that the core influences the adsorption characteristics of the nanospheres as a consequence of their disassembly upon adsorption. These results are expected to be useful in designing nanospheres for their efficient transport across vascular barriers and for delivering drugs to their targets.

A step toward engineering thick tissues: Distributing microfibers within 3D printed frames.

Microfiber mats for tissue engineering scaffolds support cell growth, but are limited by poor cell infiltration and nutrient transport. Three-dimensional printing, specifically fused deposition modeling (FDM), can rapidly produce customized constructs, but macroscopic porosity resulting from low resolution reduces cell seeding efficiency and prevents the formation of continuous cell networks. Here we describe the fabrication of hierarchical scaffolds that integrate a fibrous microenvironment with the open macropore structure of FDM. Biodegradable tyrosine-derived polycarbonate microfibers were airbrushed iteratively between layers of 3D printed support structure following optimization. Confocal imaging showed layers of airbrushed fiber mats supported human dermal fibroblast growth and extracellular matrix development throughout the scaffold. When implanted subcutaneously, hierarchical scaffolds facilitated greater cell infiltration and tissue formation than airbrushed fiber mats. Fibronectin matrix assembled in vitro throughout the hierarchical scaffold survived decellularization and provided a hybrid substrate for recellularization with mesenchymal stromal cells. These results demonstrate that by combining FDM and airbrushing techniques we can engineer customizable hierarchical scaffolds for thick tissues that support increased cell growth and infiltration.

Temperature-Activated PEG Surface Segregation Controls the Protein Repellency of Polymers.

Poly(ethylene glycol) (PEG) is widely used to modulate the hydration states of biomaterials and is often applied to produce nonfouling surfaces. Here, we present X-ray scattering data, which show that it is the surface segregation of PEG, not just its presence in the bulk, that makes this happen by influencing the hydrophilicity of PEG-containing substrates. We demonstrate a temperature-dependent trigger that transforms a PEG-containing substrate from a protein-adsorbing to a protein-repelling state. On films of poly(desaminotyrosyl-tyrosine-co-PEG carbonate) with high (20 wt %) PEG content, in which very little protein adsorption is expected, quartz crystal microbalance data showed significant adsorption of fibrinogen and bovine serum albumin at 8 °C. The surface became protein-repellent at 37.5 °C. When the same polymer was iodinated, the polymer was protein-adsorbent, even when 37 wt % PEG was incorporated into the polymer backbone. This demonstrates that high PEG content by itself is not sufficient to repel proteins. By inhibiting phase separation either with iodine or by lowering the temperature, we show that PEG must phase-separate and bloom to the surface to create an antifouling surface. These results suggest an opportunity to design materials with high PEG content that can be switched from a protein-attractant to a protein-repellent state by inducing phase separation through brief exposure to temperatures above their glass transition temperature.

Adsorption of Fibrinogen and Fibronectin on Elastomeric Poly(butylene succinate) Copolyesters.

Proteins adsorbed onto biomaterial surfaces facilitate cell-material interactions, including adhesion and migration. Of particular importance are provisional matrix components, fibrinogen (Fg) and fibronectin (Fn), which play an important role in the wound-healing process. Here, to assess the potential of a series of elastomeric poly(butylene succinate) (PBS) copolymers for soft tissue engineering and regenerative medicine applications, we examined the adsorption of Fg and Fn. We prepared spin-coated thin films of the poly(butylene succinate) homopolymer and a series of elastomeric poly(butylene succinate) copolymers with butylene succinate (PBS, hard segment) to succinate-dimer linoleic diol unit (dilinoleic succinate (DLS), soft segments) weight ratios of 70:30, 60:40, and 50:50. X-ray diffraction was used to assess crystallinity, whereas the obtained thin films were characterized using a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy. Protein adsorption was assessed using QCM-D, followed by data analysis using viscoelastic modeling. On all three copolymers, we observed robust adsorption of both key provisional matrix proteins. Importantly, for both proteins, viscoelastic modeling determined that the adlayers were 30-40 nm thick and had low shear modulus values (<25 kPa), thus indicating soft orientations (end-on for Fg) or conformations (open for Fn) of the hydrated proteins. Overall, our results are very encouraging, as they predict excellent cell adhesion and migration, key features enabling tissue integration of potential PBS-DLS scaffolds.

Achieving molecular orientation in thermally extruded 3D printed objects.

Three-dimensional (3D) printing is used to fabricate tissue scaffolds. Polymer chains in these objects are typically unoriented. The mechanical properties of these scaffolds can be significantly enhanced by proper alignment of polymer chains. However, post-processing routes to increase orientation can be limited by the geometry of the printed object. Here, we show that it is possible to orient polymer chains during printing by optimizing printing parameters to take advantage of the flow characteristics of the polymer. This is demonstrated by printing a polymeric scaffold for meniscus regeneration using poly(desaminotyrosyl-tyrosine dodecyl dodecanedioate), poly(DTD DD). Alignment of polymer chains was achieved by translating the printhead at sufficiently high speeds when the polymer was still in a semi-solid state as it cooled from the fluid state at the tip of the nozzle using a critical combination of nozzle diameter, extrusion pressure, and temperature. The degree of orientation as evaluated by x-ray diffraction and thermal shrinkage, was greater than that of drawn fibers. Significant orientation and defect-free printing was achieved even for scaffolds with complex geometries. The ability to orient polymers during 3D printing has the potential to combine the advantages of 3D printing with the superior mechanical performance of more conventional polymer processing methods, such as drawing.

Structure of intermediate filament assembly in hair deduced from hydration studies using small-angle neutron scattering.

Intermediate filaments (IFs) are ubiquitous in biological structures including hair. Small-angle neutron scattering (SANS) data from hydrated samples were used in this study to investigate the distribution of water in hair, and model the structure of the IF assembly. A main diffraction peak at a d-spacing of ∼90 Å, and two weaker reflections show that IFs are arranged in a ∼105 Šquasi-hexagonal lattice. Changes in the diffraction peaks show that only a small fraction of the water absorbed by hair enters between the IFs, and little water diffuses into the core of the IFs. The amount of water in the IF assembly increases rapidly up to 10% relative humidity (RH), and then slowly with further increase in RH. Most of the water appears to reside outside the IF assembly, in the voids and at the interfaces, and contribute to the central diffuse scattering. The IF assembly in the decuticled hair absorbs more water and is more ordered than that the native hair. This suggests that cuticle acts as a barrier, and might constrain the structure by compressing the cortex radially. Treatments with oils that are hydrophobic, heat treatment, and reduction of the S-S linkages that opens up the matrix by disulfide bond cleavage, all affect structure and water permeability. Coconut oil was found to impede hydration more than the soybean oil because of its ability to penetrate deeper into hair. A new model for the IF assembly that is sterically more favorable than the previous models is proposed.

A method to deliver patterned electrical impulses to Schwann cells cultured on an artificial axon.

Information from the brain travels back and forth along peripheral nerves in the form of electrical impulses generated by neurons and these impulses have repetitive patterns. Schwann cells in peripheral nerves receive molecular signals from axons to coordinate the process of myelination. There is evidence, however, that non-molecular signals play an important role in myelination in the form of patterned electrical impulses generated by neuronal activity. The role of patterned electrical impulses has been investigated in the literature using co-cultures of neurons and myelinating cells. The co-culturing method, however, prevents the uncoupling of the direct effect of patterned electrical impulses on myelinating cells from the indirect effect mediated by neurons. To uncouple these effects and focus on the direct response of Schwann cells, we developed an in vitro model where an electroconductive carbon fiber acts as an artificial axon. The fiber provides only the biophysical characteristics of an axon but does not contribute any molecular signaling. In our "suspended wire model", the carbon fiber is suspended in a liquid media supported by a 3D printed scaffold. Patterned electrical impulses are generated by an Arduino 101 microcontroller. In this study, we describe the technology needed to set-up and eventually replicate this model. We also report on our initial in vitro tests where we were able to document the adherence and ensheath of human Schwann cells to the carbon fiber in the presence of patterned electrical impulses (hSCs were purchased from ScienCell Research Laboratories, Carlsbad, CA, USA; ScienCell fulfills the ethic requirements, including donor's consent). This technology will likely make feasible to investigate the response of Schwann cells to patterned electrical impulses in the future.

PET-RAFT and SAXS: High Throughput Tools to Study Compactness and Flexibility of Single-Chain Polymer Nanoparticles.

From protein science, it is well understood that ordered folding and 3D structure mainly arises from balanced and noncovalent polar and nonpolar interactions, such as hydrogen bonding. Similarly, it is understood that single-chain polymer nanoparticles (SCNPs) will also compact and become more rigid with greater hydrophobicity and intrachain hydrogen bonding. Here, we couple high throughput photoinduced electron/energy transfer reversible addition-fragmentation chain-transfer (PET-RAFT) polymerization with high throughput small-angle X-ray scattering (SAXS) to characterize a large combinatorial library (>450) of several homopolymers, random heteropolymers, block copolymers, PEG-conjugated polymers, and other polymer-functionalized polymers. Coupling these two high throughput tools enables us to study the major influence(s) for compactness and flexibility in higher breadth than ever before possible. Not surprisingly, we found that many were either highly disordered in solution, in the case of a highly hydrophilic polymer, or insoluble if too hydrophobic. Remarkably, we also found a small group (9/457) of PEG-functionalized random heteropolymers and block copolymers that exhibited compactness and flexibility similar to that of bovine serum albumin (BSA) by dynamic light scattering (DLS), NMR, and SAXS. In general, we found that describing a rough association between compactness and flexibility parameters (R g /R h and Porod Exponent, respectively) with logP, a quantity that describes hydrophobicity, helps to demonstrate and predict material parameters that lead to SCNPs with greater compactness, rigidity, and stability. Future implementation of this combinatorial and high throughput approach for characterizing SCNPs will allow for the creation of detailed design parameters for well-defined macromolecular chemistry.

"Ruffled border" formation on a CaP-free substrate: A first step towards osteoclast-recruiting bone-grafts materials able to re-establish bone turn-over.

Osteoclasts are large multinucleated giant cells that actively resorb bone during the physiological bone turnover (BTO), which is the continuous cycle of bone resorption (by osteoclasts) followed by new bone formation (by osteoblasts). Osteoclasts secrete chemotactic signals to recruit cells for regeneration of vasculature and bone. We hypothesize that a biomaterial that attracts osteoclasts and re-establishes BTO will induce a better healing response than currently used bone graft materials. While the majority of bone regeneration efforts have focused on maximizing bone deposition, the novelty in this approach is the focus on stimulating osteoclastic resorption as the starter for BTO and its concurrent new vascularized bone formation. A biodegradable tyrosine-derived polycarbonate, E1001(1k), was chosen as the polymer base due to its ability to support bone regeneration in vivo. The polymer was functionalized with a RGD peptide or collagen I, or blended with ß-tricalcium phosphate. Osteoclast attachment and early stages of active resorption were observed on all substrates. The transparency of E1001(1k) in combination with high resolution confocal imaging enabled visualization of morphological features of osteoclast activation such as the formation of the "actin ring" and the "ruffled border", which previously required destructive forms of imaging such as transmission electron microscopy. The significance of these results is twofold: (1) E1001(1k) is suitable for osteoclast attachment and supports osteoclast maturation, making it a base polymer that can be further modified to optimize stimulation of BTO and (2) the transparency of this polymer makes it a suitable analytical tool for studying osteoclast behavior.

Nonsolvent-induced morphological changes and nanoporosity in poly(l-lactide) films.

The role of a nonsolvent in controlling the crystallization and morphology of solvent-crystallized poly(l-lactide) (PLLA) films was investigated using various microscopy techniques and small- and wide-angle X-ray scattering (SAXS/WAXS). PLLA films crystallized in THF and acetone had 40-80 µm spherulites. When water was present in the solvent, a completely different morphology was observed with nanosized voids and the surfaces of the films were smooth. In contrast, SEM studies revealed that the films crystallized in acetone and THF which had macroporous structures, had larger voids and film surfaces were rough because of the presence of globular structures. Voids appeared within the spherulites in the THF/water treated film, whereas crystals nucleated at the surface of the nanosized voids in acetone/water treated PLLA films. The formation of such voids is attributed to the interface-enhanced crystal nucleation in a solvent/nonsolvent system where the nonsolvent increases the polymer crystal nucleation and the subsequent evaporation of the nonsolvent. The method described in this work can be extended to other polymers to control the morphologies of polymer films during solvent-induced crystallization.