Porous Precision-Templated 40 µm Pore Scaffolds Promote Healing through Synergy in Macrophage Receptor with Collagenous Structure and Toll-Like Receptor Signaling.

Porous precision-templated scaffolds (PTS) with uniform, interconnected, 40 µm pores have shown favorable healing outcomes and a reduced foreign body reaction (FBR). Macrophage receptor with collagenous structure (MARCO) and toll-like receptors (TLRs) have been identified as key surface receptors in the initial inflammatory phase of wound healing. However, the role of MARCO and TLRs in modulating monocyte and macrophage phenotypes within PTS remains uncharacterized. In this study, we demonstrate a synergetic relationship between MARCO and TLR signaling in cells inhabiting PTS, where induction with TLR3 or TLR4 agonists to 40 µm scaffold-resident cells upregulates the transcription of MARCO. Upon deletion of MARCO, the prohealing phenotype within 40 µm PTS polarizes to a proinflammatory and profibrotic phenotype. Analysis of downstream TLR signaling shows that MARCO is required to attenuate nuclear factor kappa B (NF-κB) inflammation in 40 µm PTS by regulating the transcription of inhibitory NFKB inhibitor alpha (NFKBIA) and interleukin-1 receptor-associated kinase 3 (IRAK-M), primarily through a MyD88-dependent signaling pathway. Investigation of implant outcome in the absence of MARCO demonstrates an increase in collagen deposition within the scaffold and the development of tissue fibrosis. Overall, these results further our understanding of the molecular mechanisms underlying MARCO and TLR signaling within PTS. Impact statement Monocyte and macrophage phenotypes in the foreign body reaction (FBR) are essential for the development of a proinflammatory, prohealing, or profibrotic response to implanted biomaterials. Identification of key surface receptors and signaling mechanisms that give rise to these phenotypes remain to be elucidated. In this study, we report a synergistic relationship between macrophage receptor with collagenous structure (MARCO) and toll-like receptor (TLR) signaling in scaffold-resident cells inhabiting porous precision-templated 40 µm pore scaffolds through a MyD88-dependent pathway that promotes healing. These findings advance our understanding of the FBR and provide further evidence that suggests MARCO, TLRs, and fibrosis may be interconnected.

Vascular Grafts: Technology Success/Technology Failure.

Vascular prostheses (grafts) are widely used for hemodialysis blood access, trauma repair, aneurism repair, and cardiovascular reconstruction. However, smaller-diameter (≤4 mm) grafts that would be valuable for many reconstructions have not been achieved to date, although hundreds of papers on small-diameter vascular grafts have been published. This perspective article presents a hypothesis that may open new research avenues for the development of small-diameter vascular grafts. A historical review of the vascular graft literature and specific types of vascular grafts is presented focusing on observations important to the hypothesis to be presented. Considerations in critically reviewing the vascular graft literature are discussed. The hypothesis that perhaps the "biocompatible biomaterials" comprising our vascular grafts-biomaterials that generate dense, nonvascularized collagenous capsules upon implantation-may not be all that biocompatible is presented. Examples of materials that heal with tissue reconstruction and vascularity, in contrast to the fibrotic encapsulation, are offered. Such prohealing materials may lead the way to a new generation of vascular grafts suitable for small-diameter reconstructions.

Surface immobilized α-1 acid glycoprotein and collagen VI modulate mouse macrophage polarization and reduce the foreign body capsule.

Macrophages are widely recognized in modulating the foreign body response, and the manner in which they do so largely depends on their activation state, often referred to as their polarization. This preliminary study demonstrates that surface immobilized α-1 acid glycoprotein (AGP), as well as collagen VI (Col6) in conjunction with AGP, can direct macrophages towards the M2 polarization state in vitro and modify the foreign body response in vivo. AGP and Col6 are immobilized onto poly(2-hydroxyethyl methacrylate) (pHEMA) surfaces using carbonyl diimidazole chemistry. Mouse bone marrow derived macrophages are cultured on modified surfaces with or without lipopolysaccharide stimulation. Surface modified pHEMA discs are implanted subcutaneously into mice to observe differences in the foreign body response. After stimulation with lipopolysaccharide, macrophages cultured on AGP or Col6 modified surfaces showed a reduction in TNF-α expression compared to controls. Arg1 expression was also increased in macrophages cultured on modified surfaces. Explanted tissues showed that the foreign body capsule around implants with AGP or AGP and Col6 modification had reduced thickness, while also being more highly vascularized. These data demonstrate that α-1 acid glycoprotein and collagen VI could potentially be used for the surface modification of medical devices to influence macrophage polarization leading to a reduced and modulated foreign body response.

Novel HALO® image analysis to determine cell phenotype in porous precision-templated scaffolds.

Image analysis platforms have gained increasing popularity in the last decade for the ability to automate and conduct high-throughput, multiplex, and quantitative analyses of a broad range of pathological tissues. However, imaging tissues with unique morphology or tissues containing implanted biomaterial scaffolds remain a challenge. Using HALO®, an image analysis platform specialized in quantitative tissue analysis, we have developed a novel method to determine multiple cell phenotypes in porous precision-templated scaffolds (PTS). PTS with uniform spherical pores between 30 and 40 µm in diameter have previously exhibited a specific immunomodulation of macrophages toward a pro-healing phenotype and an overall diminished foreign body response (FBR) compared to PTS with larger or smaller pore sizes. However, signaling pathways orchestrating this pro-healing in 40 µm PTS remain unclear. Here, we use HALO® to phenotype PTS resident cells and found a decrease in pro-inflammatory CD86 and an increase in pro-healing CD206 expression in 40 µm PTS compared to 100 µm PTS. To understand the mechanisms that drive these outcomes, we investigated the role of myeloid-differentiation-primary-response gene 88 (MyD88) in regulating the pro-healing phenomenon observed only in 40 µm PTS. When subcutaneously implanted in MyD88KO mice, 40 µm PTS reduced the expression of CD206, and the scaffold resident cells displayed an average larger nuclear size compared to 40 µm PTS implanted in mice expressing MyD88. Overall, this study demonstrates a novel image analysis method for phenotyping cells within PTS and identifies MyD88 as a critical mediator in the pore-size-dependent regenerative healing and host immune response to PTS.

Dialysate regeneration via urea photodecomposition with TiO2 nanowires at therapeutic rates.

BACKGROUND: The standard weekly treatment for end-stage renal disease patients is three 4-h-long hemodialysis sessions with each session c'onsuming over 120 L of clean dialysate, which prevents the development of portable or continuous ambulatory dialysis treatments. The regeneration of a small (~1 L) amount of dialysate would enable treatments that give conditions close to continuous hemostasis and improve patient quality of life through mobility. METHODS: Small-scale studies have shown that nanowires of TiO2 are highly efficient at photodecomposing urea into CO2 and N2 when using an applied bias and an air permeable cathode. To enable the demonstration of a dialysate regeneration system at therapeutically useful rates, a scalable microwave hydrothermal synthesis of single crystal TiO2 nanowires grown directly from conductive substrates was developed. These were incorporated into 1810 cm2 flow channel arrays. The regenerated dialysate samples were treated with activated carbon (2 min at 0.2 g/mL). RESULTS: The photodecomposition system achieved the therapeutic target of 14.2 g urea removal in 24 h. TiO2 electrode had a high urea removal photocurrent efficiency of 91%, with less than 1% of the decomposed urea generating NH4 + (1.04 µg/h/cm2 ), 3% generating NO3 - and 0.5% generating chlorine species. Activated carbon treatment could reduce total chlorine concentration from 0.15 to <0.02 mg/L. The regenerated dialysate showed significant cytotoxicity which could be removed by treatment with activated carbon. Additionally, a forward osmosis membrane with sufficient urea flux can cut off the mass transfer of the by-products back into the dialysate. CONCLUSION: Urea could be removed from spent dialysate at a therapeutic rate using a TiO2 based photooxidation unit, which can enable portable dialysis systems.

XPS and ToF-SIMS Characterization of New Biodegradable Poly(Peptide-Urethane-Urea) Block Copolymers.

New, linear, segmented poly(peptide-urethane-urea) (PPUU) block copolymers are synthesized and their surface compositions are characterized with angle dependent X-ray photoelectron spectroscopy (ADXPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). These new PPUU block copolymers contain three types of segments. The soft segment (SS) is poly(caprolactone diol) (PCL). The hard segment is lysine diisocyanate with a hydrazine chain extender. The oligopeptide segment (OPS) contains three types of amino acids (proline, hydroxyproline, and glycine). Incorporation of the OPS into the polyurethane backbone is done to provide a synthetic polymer material with controllable biodegradation properties. As biodegradation processes normally are initiated at the interface between the biomaterial and the living tissue, it is important to characterize the surface composition of biomaterials. ADXPS and ToF-SIMS results show that the surfaces of all four polymers are enriched with the PCL SS, the most hydrophobic component of the three polymer segments.

Monocytes contribute to a pro-healing response in 40 µm diameter uniform-pore, precision-templated scaffolds.

Porous precision-templated scaffolds (PTS) with uniformly distributed 40 µm spherical pores have shown a remarkable ability in immunomodulating resident cells for tissue regeneration. While the pore size mediated pro-healing response observed only in 40 µm pore PTS has been attributed to selective macrophage polarization, monocyte recruitment and phenotype have largely been uncharacterized in regulating implant outcome. Here, we employ a double transgenic mouse model for myeloid characterization and a multifaceted phenotyping approach to quantify monocyte dynamics within subcutaneously implanted PTS. Within 40 µm PTS, myeloid cells were found to preferentially infiltrate into the scaffold. Additionally, macrophage receptor with collagenous structure (MARCO), an innate activation marker, was significantly upregulated within 40 µm PTS. When 40 µm PTS were implanted in monocyte-depleted mice, the transcription of MARCO was significantly decreased and an increase in pro-inflammatory inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNFα) were observed. Typical of a foreign body response (FBR), 100 µm PTS significantly upregulated pro-inflammatory iNOS, secreted higher amounts of TNFα, and displayed a pore size dependent morphology compared to 40 µm PTS. Overall, these results identify a pore size dependent modulation of circulating monocytes and implicates MARCO expression as a defining subset of monocytes that appears to be responsible for regulating a pro-healing host response.

Precision-porous polyurethane elastomers engineered for application in pro-healing vascular grafts: Synthesis, fabrication and detailed biocompatibility assessment.

Unmet needs for small diameter, non-biologic vascular grafts and the less-than-ideal performance of medium diameter grafts suggest opportunities for major improvements. Biomaterials that are mechanically matched to native blood vessels, reduce the foreign body capsule (FBC) and demonstrate improved integration and healing are expected to improve graft performance. In this study, we developed biostable, crosslinked polyurethane formulations and used them to fabricate scaffolds with precision-engineered 40 µm pores. We matched the scaffold mechanical properties with those of native blood vessels by optimizing the polyurethane compositions. We hypothesized that such scaffolds promote healing and mitigate the FBC. To test our hypothesis, polyurethanes with 40 µm pores, 100 µm pores, and non-porous slabs were implanted subcutaneously in mice for 3 weeks, and then were examined histologically. Our results show that 40 µm porous scaffolds elicit the highest level of angiogenesis, cellularization, and the least severe foreign body capsule (based on a refined assessment method). This study presents the first biomaterial with tuned mechanical properties and a precision engineered porous structure optimized for healing, thus can be ideal for pro-healing vascular grafts and in situ vascular engineering. In addition, these scaffolds may have wide applications in tissue engineering, drug delivery, and implantable device.

Biocompatibility Evolves: Phenomenology to Toxicology to Regeneration.

The word "biocompatibility," is inconsistent with the observations of healing for so-called biocompatible biomaterials. The vast majority of the millions of medical implants in humans today, presumably "biocompatible," are walled off by a dense, avascular, crosslinked collagen capsule, hardly suggestive of life or compatibility. In contrast, one is now seeing examples of implant biomaterials that lead to a vascularized reconstruction of localized tissue, a biological reaction different from traditional biocompatible materials that generate a foreign body capsule. Both the encapsulated biomaterials and the reconstructive biomaterials qualify as "biocompatible" by present day measurements of biocompatibility. Yet, this new generation of materials would seem to heal "compatibly" with the living organism, where older biomaterials are isolated from the living organism by the dense capsule. This review/perspective article will explore this biocompatibility etymological conundrum by reviewing the history of the concepts around biocompatibility, today's standard methods for assessing biocompatibility, a contemporary view of the foreign body reaction and finally, a compendium of new biomaterials that heal without the foreign body capsule. A new definition of biocompatibility is offered here to address advances in biomaterials design leading to biomaterials that heal into the body in a facile manner.

Uniform 40-µm-pore diameter precision templated scaffolds promote a pro-healing host response by extracellular vesicle immune communication.

Implanted porous precision templated scaffolds (PTS) with 40-µm spherical pores reduce inflammation and foreign body reaction (FBR) while increasing vascular density upon implantation. Larger or smaller pores, however, promote chronic inflammation and FBR. While macrophage (MØ) recruitment and polarization participates in perpetuating this pore-size-mediated phenomenon, the driving mechanism of this unique pro-healing response is poorly characterized. We hypothesized that the primarily myeloid PTS resident cells release small extracellular vesicles (sEVs) that induce pore-size-dependent pro-healing effects in surrounding T cells. Upon profiling resident immune cells and their sEVs from explanted 40-µm- (pro-healing) and 100-µm-pore diameter (inflammatory) PTS, we found that PTS pore size did not affect PTS resident immune cell population ratios or the proportion of myeloid sEVs generated from explanted PTS. However, quantitative transcriptomic assessment indicated cell and sEV phenotype were pore size dependent. In vitro experiments demonstrated the ability of PTS cell-derived sEVs to stimulate T cells transcriptionally and proliferatively. Specifically, sEVs isolated from cells inhabiting explanted 100 µm PTS significantly upregulated Th1 inflammatory gene expression in immortalized T cells. sEVs isolated from cell inhabiting both 40- and 100-µm PTS upregulated essential Treg transcriptional markers in both primary and immortalized T cells. Finally, we investigated the effects of Treg depletion on explanted PTS resident cells. FoxP3+ cell depletion suggests Tregs play a unique role in balancing T cell subset ratios, thus driving host response in 40-µm PTS. These results indicate that predominantly 40-µm PTS myeloid cell-derived sEVs affect T cells through a distinct, pore-size-mediated modality.

Plasma Polymerized HMDSO Coatings For Syringes To Minimize Protein Adsorption.

Current parenteral containers used for the storage and delivery of protein-based drugs, contain silicone oil which may seep into the protein solution and can result in adsorption, aggregation and denaturation of the protein. Tightly adherent surface coatings prepared by radio frequency glow-discharge (RFGD) plasma polymerization are described in this paper. Using this robust technique, methacrylic acid (MA) (hydrophilic), hexamethyldisiloxane (HMDSO) (hydrophobic), tetraglyme (TG) (hydrophilic) were plasma polymerized onto glass. In addition, HMDSO and MA were copolymerized to create a plasma polymerized HMDSO-MA (hydrophobic) surface. Untreated glass and glass dip-coated in PDMS were used as controls. TG and MA plasma coatings adsorbed the least amount of protein in all pH conditions. Interestingly HMDSO-MA retained significantly lesser protein compared to HMDSO and dip-coated PDMS samples. In the presence of Polysorbate 80 (PS80) all plasma polymerized coatings adsorbed and retained negligible amounts of protein, compared to controls. Furthermore, the peak glide force of plasma coated syringes did not significantly increase compared to syringes without plasma coating. Due to the versatility of RFGD plasma, this process is scalable and could potentially be used for the treatment of hypodermic syringes used for the storage and delivery of protein-based therapeutics.

Polymer Surface Analysis: The Leadership and Contributions of David Briggs.

David Briggs was a surface analysis pioneer. Starting in 1970 and continuing throughout his career, Dave used his expertise, vision and ability to quickly master new surface analysis methods and solve important industrial problems. It certainly helped that he was an outstanding fund raiser in both industrial and academic settings, which ensured he always had an impressive array of the latest, most advanced surface analysis instrumentation at his disposal. He insisted on doing surface analysis correctly and through his publications, databases and books he provided the community with the needed guidelines and methods to do so. In the 1970s Dave's research was largely focused on x-ray photoelectron spectroscopy (XPS, also known as electron spectroscopy for chemical analysis (ESCA)) characterization of polymers and catalysts. He added secondary ion mass spectrometry (SIMS) to his instrumentation arsenal in the 1980s and provided many of the key, pioneering publications that described how to use this method to characterize polymer surfaces. He also did some of the first surface analysis imaging experiments in the 1980s. In the 1990s he continued his XPS and SIMS research on polymers and advanced the surface analysis community's ability to properly interpret surface analysis data through data bases and advanced data processing methods. Dave continued to publish polymer and catalysis surface analysis papers in the 2000s, but also expanded his surface analysis studies to several other topics.

Photoreactive Carboxybetaine Copolymers Impart Biocompatibility and Inhibit Plasticizer Leaching on Polyvinyl Chloride.

Protein and cell interactions on implanted, blood-contacting medical device surfaces can lead to adverse biological reactions. Medical-grade poly(vinyl chloride) (PVC) materials have been used for decades, particularly as blood-contacting tubes and containers. However, there are numerous concerns with their performance including platelet activation, complement activation, and thrombin generation and also leaching of plasticizers, particularly in clinical applications. Here, we report a surface modification method that can dramatically prevent blood protein adsorption, human platelet activation, and complement activation on commercial medical-grade PVC materials under various test conditions. The surface modification can be accomplished through simple dip-coating followed by light illumination utilizing biocompatible polymers comprising zwitterionic carboxybetaine (CB) moieties and photosensitive cross-linking moieties. This surface treatment can be manufactured routinely at small or large scales and can impart to commercial PVC materials superhydrophilicity and nonfouling capability. Furthermore, the polymer effectively prevented leaching of plasticizers out from commercial medical-grade PVC materials. This coating technique is readily applicable to many other polymers and medical devices requiring surfaces that will enhance performance in clinical settings.

Surface-Treated Pellethanes: Comparative Quantification of Encrustation in Artificial Urine Solution.

Introduction: Encrustation of implanted urinary tract devices is associated with significant morbidity. Pellethane® is a polyether-based compound noted for its strength, porosity, and resistance to solvents. We assessed Pellethane thermoplastic polyurethane (TPU) with and without surface coatings 2-hydroxyethyl methacrylate (HEMA) and tetraethylene glycol dimethyl ether (TETRA) for the potential to resist encrustation in an artificial urine environment. Materials and Methods: Samples of Pellethane TPU, HEMA Pellethane TPU, TETRA Pellethane TPU, and hydrogel-coated ureteral stent (Cook®) were suspended in a batch-flow model with an artificial urine solution (AUS). Every 48 hours for 90 days, 40% of the solution was replaced with fresh AUS. All samples were stored in a 37°C incubator. Subsequently, the samples were thoroughly dried for 48 hours before weighing. Scanning electron microscopy was used to assess the degree of encrustation. Nu-Attom Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used to determine the precise compositions of the encrustation specifically with regard to calcium, magnesium, and phosphate. Results: At the conclusion of the 90-day trial, the samples were analyzed, and the average mass changes were as follows: stent 63.78%, uncoated Pellethane TPU 11.50%, HEMA-coated Pellethane TPU 2.90%, and TETRA-coated Pellethane TPU 0.60%. Pellethane TPU products, and specifically those coated with HEMA and TETRA, exhibited less average mass increase and a lesser propensity to form encrustation than the traditional urinary tract stent. The mass increases noted on coated Pellethane devices were primarily ionic, whereas that of the stent was not. Conclusion: Pellethane, particularly with an HEMA-based preventative coating, may serve as a favorable alternative to traditional urinary stent material, providing its improved resistance to encrustation.

Mesenchymal stromal cells from dermal and adipose tissues induce macrophage polarization to a pro-repair phenotype and improve skin wound healing.

The process of wound healing restores skin homeostasis but not full functionality; thus, novel therapeutic strategies are needed to accelerate wound closure and improve the quality of healing. In this context, tissue engineering and cellular therapies are promising approaches. Although sharing essential characteristics, mesenchymal stromal cells (MSCs) isolated from different tissues might have distinct properties. Therefore, the aim of this study was to comparatively investigate, by a mouse model in vivo assay, the potential use of dermal-derived MSCs (DSCs) and adipose tissue-derived MSCs (ASCs) in improving skin wound healing. Human DSCs and ASCs were delivered to full-thickness mouse wounds by a collagen-based scaffold (Integra Matrix). We found that the association of both DSCs and ASCs with the Integra accelerated wound closure in mice compared with the biomaterial only (control). Both types of MSCs stimulated angiogenesis and extracellular matrix remodeling, leading to better quality scars. However, the DSCs showed smaller scar size,superior extracellular matrix deposition, and greater number of cutaneous appendages. Besides, DSCs and ASCs reduced inflammation by induction of macrophage polarization from a pro-inflammatory (M1) to a pro-repair (M2) phenotype. In conclusion, both DSCs and ASCs were able to accelerate the healing of mice skin wounds and promote repair with scars of better quality and more similar to healthy skin than the empty scaffold. DSCs associated with Integra induced superior overall results than the Integra alone, whereas scaffolds with ASCs showed an intermediate effect, often not significantly better than the empty biomaterial.

Highly-reactive haloester surface initiators for ARGET ATRP readily prepared by radio frequency glow discharge plasma.

New surface initiators for ARGET ATRP (activators regenerated by electron transfer atomic transfer radical polymerization) have been prepared by the plasma deposition of haloester monomers. Specifically, methyl 3-bromopropionate (M3BP), methyl 2-chloropropionate, and ethyl 2-fluoropropionate (E2FP) were plasma deposited onto glass discs using RF glow discharge plasma. This technique creates surface coatings that are resistant to delamination and rich in halogen species making them good candidates for surface initiators for ARGET ATRP. Of all the plasma polymerized surface coatings, M3BP showed the highest halogen content and was able to grow 2-hydroxyethyl methacrylate (HEMA) polymer brushes on its surface via ARGET ATRP in as little as 15 min as confirmed by XPS. Surprisingly, E2FP, a fluoroester, was also able to grow HEMA polymer brushes despite fluorine being a poor leaving group for ARGET ATRP. The versatility of RF glow discharge plasma offers a clear advantage over other techniques previously used to immobilize ARGET ATRP surface initiators.

Trifluoromethyl-functionalized poly(lactic acid): a fluoropolyester designed for blood contact applications.

Fluorinated polymers are strong candidates for development of new cardiovascular medical devices, due to their lower thrombogenicity as compared to other polymers used for cardiovascular implants. Few studies have reported the development of fluorinated polyesters and their potential in blood contact applications has never been examined. In this study, we developed a versatile method for preparing trifluoromethyl-functionalized poly(lactic acid) that can be potentially extended to prepare a new class of polyesters with various halogen or halocarbon substitutions. The resulting fluorinated polymer was hydrophobic relative to poly(lactic acid) and extracts from this polymer showed no in vitro cytotoxicity to NIH-3T3 mouse fibroblast cells. A preliminary consideration of the blood interactions of the CF3-functionalized polyester was evaluated by measuring the amount of the adsorbed albumin and fibrinogen from human blood plasma. The fluorinated polyester adsorbed and retained higher amounts of albumin and fibrinogen with a higher albumin/fibrinogen ratio as compared to poly(lactic acid), suggesting enhanced hemocompatibility. Plasma protein adsorption is the first event that occurs seconds after device implantation and controlling the adsorbed proteins will dictate the performance of medical implants.