A functional tacrolimus-releasing nerve wrap for enhancing nerve regeneration following surgical nerve repair.

JOURNAL/nrgr/04.03/01300535-202501000-00036/figure1/v/2024-05-14T021156Z/r/image-tiff Axonal regeneration following surgical nerve repair is slow and often incomplete, resulting in poor functional recovery which sometimes contributes to lifelong disability. Currently, there are no FDA-approved therapies available to promote nerve regeneration. Tacrolimus accelerates axonal regeneration, but systemic side effects presently outweigh its potential benefits for peripheral nerve surgery. The authors describe herein a biodegradable polyurethane-based drug delivery system for the sustained local release of tacrolimus at the nerve repair site, with suitable properties for scalable production and clinical application, aiming to promote nerve regeneration and functional recovery with minimal systemic drug exposure. Tacrolimus is encapsulated into co-axially electrospun polycarbonate-urethane nanofibers to generate an implantable nerve wrap that releases therapeutic doses of bioactive tacrolimus over 31 days. Size and drug loading are adjustable for applications in small and large caliber nerves, and the wrap degrades within 120 days into biocompatible byproducts. Tacrolimus released from the nerve wrap promotes axon elongation in vitro and accelerates nerve regeneration and functional recovery in preclinical nerve repair models while off-target systemic drug exposure is reduced by 80% compared with systemic delivery. Given its surgical suitability and preclinical efficacy and safety, this system may provide a readily translatable approach to support axonal regeneration and recovery in patients undergoing nerve surgery.

BoneTape: A novel osteosynthetic device for the stabilization of zygomatic fractures.

BACKGROUND: The study aims to assess the safety and effectiveness of BoneTape™, a new resorbable bone fixation device, using a zygomatic fracture model in rabbits. METHODS: The study followed BoneTape™ samples and control (sham) groups over 2-, 6-, and 12-week periods post-zygomaticomaxillary (ZM) osteotomy and zygomaticofrontal (ZF) disarticulation. The osteotomized segments were analyzed for bone healing, inflammatory response, and tissue healing. µCT imaging and histological analysis were used to examine the axial alignment, offset, and quality of new bone formation. RESULTS: BoneTape™ samples demonstrated enhanced maintenance of the initial intraoperative positioning, reduced axial offset, and better alignment when compared with the control group, enabling stable bone healing under physiological loading conditions. Complete union was observed at 12-weeks in both groups. The BoneTape™ group experienced minimal immune and tissue reactions, classically associated with wound healing, and showed an increased number of giant cells at 6 and 12-weeks. CONCLUSION: BoneTape™ represents a promising advancement in osteosynthesis, demonstrating efficacy in maintaining stable zygomatic reconstruction and eliciting minimal immune response in a rabbit model. This study introduces BoneTape™ as a disruptive solution specifically designed for clinical application in cranio-maxillofacial fracture fixation, with the potential to eliminate the use of over-engineered solutions while offering benefits such as ease of application and fewer biologically disruptive steps.

Electrospun methacrylated natural/synthetic composite membranes for gingival tissue engineering.

New functional materials for engineering gingival tissue are still in the early stages of development. Materials for such applications must maintain volume and have advantageous mechanical and biological characteristics for tissue regeneration, to be an alternative to autografts, which are the current benchmark of care. In this work, methacrylated gelatin (GelMa) was photocrosslinked with synthetic immunomodulatory methacrylated divinyl urethanes and defined monomers to generate composite scaffolds. Using a factorial design, with the synthetic monomers of a degradable polar/hydrophobic/ionic polyurethane (D-PHI) and GelMa, composite materials were electrospun with polycarbonate urethane (PCNU) and light-cured in-flight. The materials had significantly different relative hydrophilicities, with unique biodegradation profiles associated with specific formulations, thereby providing good guidance to achieving desired mechanical characteristics and scaffold resorption for gingival tissue regeneration. In accelerated esterase/collagenase degradation models, the new materials exhibited an initial rapid weight loss followed by a more gradual rate of degradation. The degradation profile allowed for the early infiltration of human adipose-derived stromal/stem cells, while still enabling the graft's structural integrity to be maintained. In conclusion, the materials provide a promising candidate platform for the regeneration of oral soft tissues, addressing the requirement of viable tissue infiltration while maintaining volume and mechanical integrity. STATEMENT OF SIGNIFICANCE: There is a need for the development of more functional and efficacious materials for the treatment of gingival recession. To address significant limitations in current material formulations, we sought to investigate the development of methacrylated gelatin (GelMa) and oligo-urethane/methacrylate monomer composite materials. A factorial design was used to electrospin four new formulations containing four to five monomers. Synthetic immunomodulatory monomers were crosslinked with GelMa and electrospun with a polycarbonate urethane resulting in unique mechanical properties, and resorption rates which align with the original design criteria for gingival tissue engineering. The materials may have applications in tissue engineering and can be readily manufactured. The findings of this work may help better direct the efforts of tissue engineering and material manufacturing.

Paracrine cross-talk between human adipose tissue-derived endothelial cells and perivascular cells accelerates the endothelialization of an electrospun ionomeric polyurethane scaffold.

The ex vivo endothelialization of small diameter vascular prostheses can prolong their patency. Here, we demonstrate that heterotypic interactions between human adipose tissue-derived endothelial cells and perivascular cells can be exploited to accelerate the endothelialization of an electrospun ionomeric polyurethane scaffold. The scaffold was used to physically separate endothelial cells from perivascular cells to prevent their diffuse neo-intimal hyperplasia and spontaneous tubulogenesis, yet enable their paracrine cross-talk to accelerate the integration of the endothelial cells into a temporally stable endothelial lining of a continuous, elongated, and aligned morphology. Perivascular cells stimulated endothelial basement membrane protein production and suppressed their angiogenic and inflammatory activation to accelerate this biomimetic morphogenesis of the endothelium. These findings demonstrate the feasibility and underscore the value of exploiting heterotypic interactions between endothelial cells and perivascular cells for the fabrication of an endothelial lining intended for small diameter arterial reconstruction. STATEMENT OF SIGNIFICANCE: Adipose tissue is an abundant, accessible, and uniquely dispensable source of endothelial cells and perivascular cells for vascular tissue engineering. While their spontaneous self-assembly into microvascular networks is routinely exploited for the vascularization of engineered tissues, it threatens the temporal stability of an endothelial lining intended for small diameter arterial reconstruction. Here, we demonstrate that an electrospun polyurethane scaffold can be used to physically separate endothelial cells from perivascular cells to prevent their spontaneous capillary morphogenesis, yet enable their cross-talk to promote the formation of a stable endothelium. Our findings demonstrate the feasibility of engineering an endothelial lining from human adipose tissue, poising it for the rapid ex vivo endothelialization of small diameter vascular prostheses in an autologous, patient-specific manner.

Matrix-Binding, N-Cadherin-Targeting Chimeric Peptide Inhibits Intimal Thickening but Not Endothelial Repair in Balloon-Injured Carotid Arteries.

BACKGROUND: Treatment of occluded vessels can involve angioplasty, stenting, and bypass grafting, which can be limited by restenosis and thrombosis. Drug-eluting stents attenuate restenosis, but the current drugs used are cytotoxic, causing smooth muscle cell (SMC) and endothelial cell (EC) death that may lead to late thrombosis. N-cadherin is a junctional protein expressed by SMCs, which promotes directional SMC migration contributing to restenosis. We propose that engaging N-cadherin with mimetic peptides can act as a cell type-selective therapeutic strategy to inhibit polarization and directional migration of SMCs without negatively impacting ECs. METHODS: We designed a novel N-cadherin-targeting chimeric peptide with a histidine-alanine-valine cadherin-binding motif, combined with a fibronectin-binding motif from Staphylococcus aureus. This peptide was tested in SMC and EC culture assays of migration, viability, and apoptosis. Rat carotid arteries were balloon injured and treated with the N-cadherin peptide. RESULTS: Treating scratch-wounded SMCs with the N-cadherin-targeting peptide inhibited migration and reduced polarization of wound-edge cells. The peptide colocalized with fibronectin. Importantly, EC junction, permeability, or migration was not impacted by peptide treatment in vitro. We also demonstrated that the chimeric peptide persisted for 24 hours after transient delivery in the balloon-injured rat carotid artery. Treatment with the N-cadherin-targeting chimeric peptide reduced intimal thickening in balloon-injured rat carotid arteries at 1 and 2 weeks after injury. Reendothelialization of injured vessels after 2 weeks was unimpaired by peptide treatment. CONCLUSIONS: These studies show that an N-cadherin-binding and fibronectin-binding chimeric peptide is effective in inhibiting SMC migration in vitro and in vivo and limiting neointimal hyperplasia after balloon angioplasty without affecting EC repair. These results establish the potential of an advantageous SMC-selective strategy for antirestenosis therapy.

Bioinspired Oligo-Urethane Nanoparticles for Delivering Exogenous C-Type Natriuretic Peptide: Synthetic Biomaterial Nanocarrier Complexes and Their Interactions with Cardiac Myofibroblasts.

In a healthy heart, cells naturally secrete C-type natriuretic peptide (CNP), a cytokine that protects against myofibroblast differentiation of cardiac fibroblasts and extracellular matrix deposition leading to fibrosis. CNP availability during myocardial remodeling is important to prevent cardiac fibrosis, but CNP is limited after an injury because of the loss of cardiomyocytes and the activation of cardiac fibroblasts to myofibroblasts. We hypothesized that the sustained release of exogenous CNP from oligo-urethane nanoparticles (NPs) would reduce differentiation of human cardiac fibroblasts toward a myofibrogenic phenotype. Our work used a modified form of a degradable polar hydrophobic ionic (D-PHI) oligo-urethane, which has shown the ability to self-assemble into NPs for the delivery of peptide and oligonucleotide biomolecules. The CNP-loaded NPs (NPCNP) were characterized for a diameter of 129 ± 1.4 nm and a ζ potential of -46 ± 7.8 mV. Treatment of cardiac fibroblasts with NPCNP increased cyclic guanosine-monophosphate (cGMP) synthesis, confirming that exogenous CNP delivered via oligo-urethane NPs is bioactive and can induce downstream signaling that has been implicated in antagonizing transforming growth factor-ß1 (TGF-ß1)-induced myofibrogenic differentiation. It is also shown that treatment with NPCNP attenuated contraction of collagen gels by cardiac myofibroblasts stimulated with TGF-ß1. Coating with heparin on the NPCNP (HEP-NPCNP) exemplified an approach to extend the release of CNP from the NPs. Both HEP-NPCNP and NPCNP show minimal cell toxicity, studied up to 0.25 × 1010 NPs/mL in culture media. These findings support further investigation of CNP delivery via NPs as a future therapy for suppressing cardiac fibrosis.

Less Is More: Developments in Nanotechnology for Antirestenosis Therapies.

Despite recent advancements in vascular disease treatments, thrombosis and poor long-term vessel patency remain significant barriers to effective endovascular intervention. Current balloon angioplasty and stenting techniques effectively restore acute blood flow in occluded vessels but have persistent limitations. Damage to the arterial endothelium caused by injury during catheter tracking triggers neointimal hyperplasia and the release of proinflammatory factors leading to increased risk of thrombosis and restenosis. Antirestenotic agents commonly delivered on angioplasty balloons and stents have lowered arterial restenosis rates, but the absence of cell type selectivity significantly delays critical endothelium repair. Targeted delivery of biomolecular therapeutics, coupled with engineered nanoscale excipients, has the potential to redefine cardiovascular interventions by improving long-term efficacy, limiting off-target effects, and reducing costs compared with conventional clinical standards of care. This review analyzes current forms of localized vascular drug delivery, emerging nanoscale therapeutic and excipient strategies, and provides recommendations for future areas of study to advance the treatment of vascular disease through innovations in nanotechnology.

Arrhenius-model-based degradable oligourethane hydrogels for controlled growth factor release.

Biodegradable hydrogels are growing in demand to enable the delivery of biomolecules (e.g. growth factors) for regenerative medicine. This research investigated the resorption of an oligourethane/polyacrylic acid hydrogel, a biodegradable hydrogel which supports tissue regeneration. The Arrhenius model was used to characterize the resorption of the polymeric gels in relevant in vitro conditions, and the Flory-Rehner equation was used to correlate the volumetric swelling ratio with the extent of degradation. The study found that the swelling rate of the hydrogel follows the Arrhenius model at elevated temperatures, estimating degradation time in saline solution at 37°C to be between 5 and 13 months, serving as a preliminary approximation of degradation in vivo. The degradation products had low cytotoxicity towards endothelial cells, and the hydrogel supported stromal cell proliferation. Additionally, the hydrogels were able to release growth factors and maintain the biomolecules' bioactivity towards cell proliferation. The study of the vascular endothelial growth factor (VEGF) release from the hydrogel used a diffusion process model, showing that the electrostatic attraction between VEGF and the anionic hydrogel allowed for controlled and sustained VEGF release over three weeks. In a rat subcutaneous implant model, a selected hydrogel with desired degradation rates exhibited minimal foreign body response and supported M2a macrophage phenotype, and vascularization. The low M1 and high M2a macrophage phenotypes within the implants were associated with tissue integration. This research supports the use of oligourethane/polyacrylic acid hydrogels as a promising material for delivering growth factors and supporting tissue regeneration. STATEMENT OF SIGNIFICANCE: There is a need for degradable elastomeric hydrogels that can support the formation of soft tissues and minimize long-term foreign body responses. An Arrhenius model was used to estimate the relative breakdown of hydrogels, in-vitro. The results demonstrate that hydrogels made from a combination of poly(acrylic acid) and oligo-urethane diacrylates can be designed to resorb over defined periods ranging from months to years depending on the chemical formulation prescribed by the model. The hydrogel formulations also provided for different release profiles of growth factors, relevant to tissue regeneration. In-vivo, these hydrogels had minimal inflammatory effects and showed evidence of integration into the surrounding tissue. The hydrogel approach can help the field design a broader range of biomaterials for tissue regeneration.

Serum- and xeno-free culture of human umbilical cord perivascular cells for pediatric heart valve tissue engineering.

BACKGROUND: Constructs currently used to repair or replace congenitally diseased pediatric heart valves lack a viable cell population capable of functional adaptation in situ, necessitating repeated surgical intervention. Heart valve tissue engineering (HVTE) can address these limitations by producing functional living tissue in vitro that holds the potential for somatic growth and remodelling upon implantation. However, clinical translation of HVTE strategies requires an appropriate source of autologous cells that can be non-invasively harvested from mesenchymal stem cell (MSC)-rich tissues and cultured under serum- and xeno-free conditions. To this end, we evaluated human umbilical cord perivascular cells (hUCPVCs) as a promising cell source for in vitro production of engineered heart valve tissue. METHODS: The proliferative, clonogenic, multilineage differentiation, and extracellular matrix (ECM) synthesis capacities of hUCPVCs were evaluated in a commercial serum- and xeno-free culture medium (StemMACS™) on tissue culture polystyrene and benchmarked to adult bone marrow-derived MSCs (BMMSCs). Additionally, the ECM synthesis potential of hUCPVCs was evaluated when cultured on polycarbonate polyurethane anisotropic electrospun scaffolds, a representative biomaterial for in vitro HVTE. RESULTS: hUCPVCs had greater proliferative and clonogenic potential than BMMSCs in StemMACS™ (p < 0.05), without differentiation to osteogenic and adipogenic phenotypes associated with valve pathology. Furthermore, hUCPVCs cultured with StemMACS™ on tissue culture plastic for 14 days synthesized significantly more total collagen, elastin, and sulphated glycosaminoglycans (p < 0.05), the ECM constituents of the native valve, than BMMSCs. Finally, hUCPVCs retained their ECM synthesizing capacity after 14 and 21 days in culture on anisotropic electrospun scaffolds. CONCLUSION: Overall, our findings establish an in vitro culture platform that uses hUCPVCs as a readily-available and non-invasively sourced autologous cell population and a commercial serum- and xeno-free culture medium to increase the translational potential of future pediatric HVTE strategies. This study evaluated the proliferative, differentiation and extracellular matrix (ECM) synthesis capacities of human umbilical cord perivascular cells (hUCPVCs) when cultured in serum- and xeno-free media (SFM) against conventionally used bone marrow-derived MSCs (BMMSCs) and serum-containing media (SCM). Our findings support the use of hUCPVCs and SFM for in vitro heart valve tissue engineering (HVTE) of autologous pediatric valve tissue. Figure created with BioRender.com.

In Vivo MRI Tracking of Degradable Polyurethane Hydrogel Degradation In Situ Using a Manganese Porphyrin Contrast Agent.

BACKGROUND: A noninvasive method to track implanted biomaterials is desirable for real-time monitoring of material interactions with host tissues and assessment of efficacy and safety. PURPOSE: To explore quantitative in vivo tracking of polyurethane implants using a manganese porphyrin (MnP) contrast agent containing a covalent binding site for pairing to polymers. STUDY TYPE: Prospective, longitudinal. ANIMAL MODEL: Rodent model of dorsal subcutaneous implants (10 female Sprague Dawley rats). FIELD STRENGTH/SEQUENCE: A 3-T; two-dimensional (2D) T1-weighted spin-echo (SE), T2-weighted turbo SE, three-dimensional (3D) spoiled gradient-echo T1 mapping with variable flip angles. ASSESSMENT: A new MnP-vinyl contrast agent to covalently label polyurethane hydrogels was synthesized and chemically characterized. Stability of binding was assessed in vitro. MRI was performed in vitro on unlabeled hydrogels and hydrogels labeled at different concentrations, and in vivo on rats with unlabeled and labeled hydrogels implanted dorsally. In vivo MRI was performed at 1, 3, 5, and 7 weeks postimplantation. Implants were easily identified on T1-weighted SE, and fluid accumulation from inflammation was distinguished on T2-weighted turbo SE. Implants were segmented on contiguous T1-weighted SPGR slices using a threshold of 1.8 times the background muscle signal intensity; implant volume and mean T1 values were then calculated at each timepoint. Histopathology was performed on implants in the same plane as MRI and compared to imaging results. STATISTICAL TESTS: Unpaired t-tests and one-way analysis of variance (ANOVA) were used for comparisons. A P value <0.05 was considered to be statistically significant. RESULTS: Hydrogel labeling with MnP resulted in a significant T1 reduction in vitro (T1 = 517 ± 36 msec vs. 879 ± 147 msec unlabeled). Mean T1 values of labeled implants in rats increased significantly by 23% over time, from 1 to 7 weeks postimplantation (651 ± 49 msec to 801 ± 72 msec), indicating decreasing implant density. DATA CONCLUSION: Polymer-binding MnP enables in vivo tracking of vinyl-group coupling polymers. EVIDENCE LEVEL: 1. TECHNICAL EFFICACY: Stage 1.

A Polypyrrole-Polycarbonate Polyurethane Elastomer Alleviates Cardiac Arrhythmias via Improving Bio-Conductivity.

Myocardial fibrosis, resulting from myocardial infarction (MI), significantly alters cardiac electrophysiological properties. As fibrotic scar tissue forms, its resistance to incoming action potentials increases, leading to cardiac arrhythmia, and eventually sudden cardiac death or heart failure. Biomaterials are gaining increasing attention as an approach for addressing post-MI arrhythmias. The current study investigates the hypothesis that a bio-conductive epicardial patch can electrically synchronize isolated cardiomyocytes in vitro and rescue arrhythmic hearts in vivo. A new conceived biocompatible, conductive, and elastic polyurethane composite bio-membrane, referred to as polypyrrole-polycarbonate polyurethane (PPy-PCNU), is developed, in which solid-state conductive PPy nanoparticles are distributed throughout an electrospun aliphatic PCNU nanofiber patch in a controlled manner. Compared to PCNU alone, the resulting biocompatible patch demonstrates up to six times less impedance, with no conductivity loss over time, as well as being able to influence cellular alignment. Furthermore, PPy-PCNU promotes synchronous contraction of isolated neonatal rat cardiomyocytes and alleviates atrial fibrillation in rat hearts upon epicardial implantation. Taken together, epicardially-implanted PPy-PCNU could potentially serve as a novel alternative approach for the treatment of cardiac arrhythmias.

An efficient and cost-effective purification protocol for Staphylococcus aureus Cas9 nuclease.

Here, we describe a protocol for purifying functional clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Staphylococcus aureus within 24 h and over 90% purity. SaCas9 purification begins with immobilized metal affinity chromatography, followed by cation exchange chromatography, and ended with centrifugal concentrators. The simplicity, cost-effectiveness, and reproducibility of such protocols will enable general labs to produce a sizable amount of Cas9 proteins, further accelerating CRISPR research.

Streptococcus mutans Proteases Degrade Dentinal Collagen.

Here, we explored the role of S. mutans's whole cell and discrete fractions in the degradation of type I collagen and dentinal collagen. Type I collagen gels and human demineralized dentin slabs (DS) were incubated in media alone or with one of the following: overnight (O/N) or newly inoculated (NEW) cultures of S. mutans UA159; intracellular proteins, supernatant or bacterial membranes of O/N cultures. Media from all groups were analyzed for protease-mediated release of the collagen-specific imino acid hydroxyproline. Images of type I collagen and DS were analyzed, respectively. Type I collagen degradation was highest for the supernatant (p < 0.05) fractions, followed by intracellular components and O/N cultures. Collagen degradation for DS samples was highest for O/N samples, followed by supernatant, and intracellular components (p < 0.05). There was lower detectable degradation for both type I collagen and DS from NEW culture samples (p < 0.05), and there was no type I collagen or DS degradation detected for bacterial membrane samples. Structural changes to type I collagen gel and dentinal collagen were observed, respectively, following incubation with S. mutans cultures (O/N and NEW), intracellular components, and supernatant. This study demonstrates that intracellular and extracellular proteolytic activities from S. mutans enable this cariogenic bacterium to degrade type I and dentinal collagen in a growth-phase dependent manner, potentially contributing to the progression of dental caries.

Towards the development of biostable dental resin systems - design criteria and constraints beyond ester-free chemistries.

OBJECTIVE: The objective of this review article is to summarize the current literature on dental resin-based restorative (RBR) materials specifically from the perspective of emerging resin technologies, and to provide researchers with structured design criteria enabling the effective screening of new RBR developments. METHODS: The continued failure of newly introduced RBRs to address biostability without compromising function, over the last decade, are presented as a rationale to support different resin-based concepts. Several developments in the field, aimed at addressing the issues facing modern resin-based systems are summarized and their limitations discussed. A design workflow is proposed for evaluating new RBR, considering resource needs. RESULTS: While several alternative resin chemistries have been suggested over the past decade, all have shown serious limitations in replacing MA-based materials, including their limited physical and mechanical properties, and curing kinetics. Additionally, a broad and inconsistent range of laboratory methods have been used to validate these developments, leading to results that are difficult to compare across studies. A design workflow was conceptualized to facilitate the screening of novel RBRs from both a clinical and research perspective. SIGNIFICANCE: While several alternative chemistries have shown some degree of potential in emulating material property aspects of MA-based resins, a complete restorative system that is resistant to biochemical reactions in saliva has yet to achieve broad clinical adoption. To further spur development, it would be useful to have a more systematic design workflow, that may be used to easily screen new resin technologies effectively early in the design phase, so as to mitigate potential performance failures in the clinic.

Harnessing conserved signaling and metabolic pathways to enhance the maturation of functional engineered tissues.

The development of induced-pluripotent stem cell (iPSC)-derived cell types offers promise for basic science, drug testing, disease modeling, personalized medicine, and translatable cell therapies across many tissue types. However, in practice many iPSC-derived cells have presented as immature in physiological function, and despite efforts to recapitulate adult maturity, most have yet to meet the necessary benchmarks for the intended tissues. Here, we summarize the available state of knowledge surrounding the physiological mechanisms underlying cell maturation in several key tissues. Common signaling consolidators, as well as potential synergies between critical signaling pathways are explored. Finally, current practices in physiologically relevant tissue engineering and experimental design are critically examined, with the goal of integrating greater decision paradigms and frameworks towards achieving efficient maturation strategies, which in turn may produce higher-valued iPSC-derived tissues.

Compatibility and function of human induced pluripotent stem cell derived cardiomyocytes on an electrospun nanofibrous scaffold, generated from an ionomeric polyurethane composite.

Synthetic scaffolds are needed for generating organized neo-myocardium constructs to promote functional tissue repair. This study investigated the biocompatibility of an elastomeric electrospun degradable polar/hydrophobic/ionic polyurethane (D-PHI) composite scaffold with human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The composite material was electrospun to generate scaffolds, with nanofibres oriented in aligned or random directions. These features enabled the authors to evaluate the effect of characteristic elements which mimic that of the native extracellular matrix (alignment, chemical heterogeneity, and fiber topography) on hiPSC-CMs activity. The functional nature of the hiPSC-CM cultured on gelatin and Matrigel-coated scaffolds were assessed, investigating the influence of protein interactions with the synthetic substrate on subsequent cell phenotype. After 7 days of culture, high hiPSC-CM viability was observed on the scaffolds. The cells on the aligned scaffold were elongated and demonstrated aligned sarcomeres that oriented parallel to the direction of the fibers, while the cells on random scaffolds and a tissue culture polystyrene (TCPS) control did not exhibit such an organized morphology. The hiPSC-CMs cultured on the scaffolds and TCPS expressed similar levels of cardiac troponin-T, but there was a higher expression of ventricular myosin light chain-2 on the D-PHI composite scaffolds versus TCPS, indicating a higher proportion of hiPSC-CM exhibiting a ventricular cardiomyocyte like phenotype. Within 7 days, the hiPSC-CMs on aligned scaffolds and TCPS beat synchronously and had similar conductive velocities. These preliminary results show that aligned D-PHI elastomeric scaffolds allow hiPSC-CMs to demonstrate important cardiomyocytes characteristics, critical to enabling their future potential use for cardiac tissue regeneration.

The Structure and Function of Next-Generation Gingival Graft Substitutes-A Perspective on Multilayer Electrospun Constructs with Consideration of Vascularization.

There is a shortage of suitable tissue-engineered solutions for gingival recession, a soft tissue defect of the oral cavity. Autologous tissue grafts lead to an increase in morbidity due to complications at the donor site. Although material substitutes are available on the market, their development is early, and work to produce more functional material substitutes is underway. The latter materials along with newly conceived tissue-engineered substitutes must maintain volumetric form over time and have advantageous mechanical and biological characteristics facilitating the regeneration of functional gingival tissue. This review conveys a comprehensive and timely perspective to provide insight towards future work in the field, by linking the structure (specifically multilayered systems) and function of electrospun material-based approaches for gingival tissue engineering and regeneration. Electrospun material composites are reviewed alongside existing commercial material substitutes', looking at current advantages and disadvantages. The importance of implementing physiologically relevant degradation profiles and mechanical properties into the design of material substitutes is presented and discussed. Further, given that the broader tissue engineering field has moved towards the use of pre-seeded scaffolds, a review of promising cell options, for generating tissue-engineered autologous gingival grafts from electrospun scaffolds is presented and their potential utility and limitations are discussed.

Design of a Mechanobioreactor to Apply Anisotropic, Biaxial Strain to Large Thin Biomaterials for Tissue Engineered Heart Valve Applications.

Repair and replacement solutions for congenitally diseased heart valves capable of post-surgery growth and adaptation have remained elusive. Tissue engineered heart valves (TEHVs) offer a potential biological solution that addresses the drawbacks of existing valve replacements. Typically, TEHVs are made from thin, fibrous biomaterials that either become cell populated in vitro or in situ. Often, TEHV designs poorly mimic the anisotropic mechanical properties of healthy native valves leading to inadequate biomechanical function. Mechanical conditioning of engineered tissues with anisotropic strain application can induce extracellular matrix remodelling to alter the anisotropic mechanical properties of a construct, but implementation has been limited to small-scale set-ups. To address this limitation for TEHV applications, we designed and built a mechanobioreactor capable of modulating biaxial strain anisotropy applied to large, thin, biomaterial sheets in vitro. The bioreactor can independently control two orthogonal stretch axes to modulate applied strain anisotropy on biomaterial sheets from 13 × 13 mm2 to 70 × 40 mm2. A design of experiments was performed using experimentally validated finite element (FE) models and demonstrated that biaxial strain was applied uniformly over a larger percentage of the cell seeded area for larger sheets (13 × 13 mm2: 58% of sheet area vs. 52 × 31 mm2: 86% of sheet area). Furthermore, bioreactor prototypes demonstrated that over 70% of the cell seeding area remained uniformly strained under different prescribed protocols: equibiaxial amplitudes between 5 to 40%, cyclic frequencies between 0.1 to 2.5 Hz and anisotropic strain ratios between 0:1 (constrained uniaxial) to 2:1. Lastly, proof-of-concept experiments were conducted where we applied equibiaxial (εx = εy = 8.75%) and anisotropic (εx = 12.5%, εy = 5%) strain protocols to cell-seeded, electrospun scaffolds. Cell nuclei and F-actin aligned to the vector-sum strain direction of each prescribed protocol (nuclei alignment: equibiaxial: 43.2° ± 1.8°, anisotropic: 17.5° ± 1.7°; p < 0.001). The abilities of this bioreactor to prescribe different strain amplitude, frequency and strain anisotropy protocols to cell-seeded scaffolds will enable future studies into the effects of anisotropic loading protocols on mechanically conditioned TEHVs and other engineered planar connective tissues.

Vascular tissue engineering from human adipose tissue: fundamental phenotype of its resident microvascular endothelial cells and stromal/stem cells.

Adipose tissue is an abundant, accessible, and uniquely dispensable source of cells for vascular tissue engineering. Despite its intrinsic endothelial cells, considerable effort is directed at deriving endothelium from its resident stem and progenitor cells. Here, we investigate the composition of human adipose tissue and characterize the phenotypes of its constituent cells in order to help ascertain their potential utility for vascular tissue engineering. Unsupervised clustering based on cell-surface protein signatures failed to detect CD45-CD31-VEGFR2+ endothelial progenitor cells within adipose tissue, but supported further investigation of its resident CD45-CD31+ microvascular endothelial cells (HAMVECs) and CD45-CD31- stromal/stem cells (ASCs). The endothelial differentiation of ASCs altered their proteome, but it remained distinct from that of primary endothelial cell controls - as well as HAMVECs - regardless of their arterial-venous specification or macrovascular-microvascular origin. Rather, ASCs retained a proteome indicative of a perivascular phenotype, which was supported by their ability to facilitate the capillary morphogenesis of HAMVECs. This study supports the use of HAMVECs for the generation of endothelium. It suggests that the utility of ASCs for vascular tissue engineering lies in their capacity to remodel the extracellular matrix and to function as mural cells.

Self-Assembled Oligo-Urethane Nanoparticles: Their Characterization and Use for the Delivery of Active Biomolecules into Mammalian Cells.

Developing safe and effective strategies to deliver biomolecules such as oligonucleotides and proteins into cells has grown in importance over recent years, with an increasing demand for non-viral methods that enable clinical translation. Here, we investigate uniquely configured oligo-urethane nanoparticles based on synthetic chemistries that minimize the release of pro-inflammatory biomarkers from immune cells, show low cytotoxicity in a broad range of cells, and efficiently deliver oligonucleotides and proteins into mammalian cells. The mechanism of cell uptake for the self-assembled oligo-urethane nanoparticles was shown to be directed by caveolae-dependent endocytosis in murine myoblasts (C2C12) cells. Inhibiting caveolae functions with genistein and methyl-ß-cyclodextrin limited nanoparticle internalization. The nanoparticles showed a very high delivery efficiency for the genetic material (a 47-base oligonucleotide) (∼80% incorporation into cells) as well as the purified protein (full length firefly luciferase, 67 kDa) into human embryonic kidney (HEK293T) cells. Luciferase enzyme activity in HEK293T cells demonstrated that intact and functional proteins could be delivered and showed a significant extension of activity retention up to 24 h, well beyond the 2 h half-life of the free enzyme. This study introduces a novel self-assembled oligo-urethane nanoparticle delivery platform with very low associated production costs, enabled by their scalable chemistry (the benchwork cost is $ 0.152/mg vs $ 974.6/mg for typical lipid carriers) that has potential to deliver both oligonucleotides and proteins for biomedical purposes.