A Library of Elastin-like Proteins with Tunable Matrix Ligands for In Vitro 3D Neural Cell Culture.

Hydrogels with encapsulated cells have widespread biomedical applications, both as tissue-mimetic 3D cultures in vitro and as tissue-engineered therapies in vivo. Within these hydrogels, the presentation of cell-instructive extracellular matrix (ECM)-derived ligands and matrix stiffness are critical factors known to influence numerous cell behaviors. While individual ECM biopolymers can be blended together to alter the presentation of cell-instructive ligands, this typically results in hydrogels with a range of mechanical properties. Synthetic systems that allow for the facile incorporation and modulation of multiple ligands without modification of matrix mechanics are highly desirable. In the present work, we leverage protein engineering to design a family of xeno-free hydrogels (i.e., devoid of animal-derived components) consisting of recombinant hyaluronan and recombinant elastin-like proteins (ELPs), cross-linked together with dynamic covalent bonds. The ELP components incorporate cell-instructive peptide ligands derived from ECM proteins, including fibronectin (RGD), laminin (IKVAV and YIGSR), collagen (DGEA), and tenascin-C (PLAEIDGIELTY and VFDNFVL). By carefully designing the protein primary sequence, we form 3D hydrogels with defined and tunable concentrations of cell-instructive ligands that have similar matrix mechanics. Utilizing this system, we demonstrate that neurite outgrowth from encapsulated embryonic dorsal root ganglion (DRG) cultures is significantly modified by cell-instructive ligand content. Thus, this library of protein-engineered hydrogels is a cell-compatible system to systematically study cell responses to matrix-derived ligands.

[Vena cava thrombectomy in kidney cancer. Report of 32 nephrectomies]. / Nefrectomía radical con trombectomía de vena cava: 20 años de cirugías por tumor renal.

BACKGROUND: Vena cava (VC) involvement in kidney tumors occurs in 4 to 10% of cases, and is associated with a higher mortality. Nephrectomy with thrombectomy of the VC, performed by a multidisciplinary team, improves survival. AIM: To report a series of consecutive nephrectomies with caval thrombectomy performed in an academic center. PATIENTS AND METHODS: We report 32 patients with cT3b and 3c renal tumors, who underwent radical nephrectomy with VC thrombectomy between 2001 and 2021. A descriptive analysis of clinical, surgical and pathological variables was performed. Overall survival (OS) and cancer-specific survival (CSS) was calculated using Kaplan-Meier curves. RESULTS: The mean tumor size was 9.7 cm. According to Mayo classification 3/32 (9%) patients had a type I thrombus, 10/32 (31%) had a type II thrombus, 8/32 (25%) had a type III thrombus, and 5/32 (16%) had a type IV thrombus. The mean bleeding was 2000 cc. There was one intraoperative death. Nineteen percent of patients had complications >= 3 according to Clavien-Dindo classification. Reoperations occurred in 9%. Pre and postoperative creatinine levels were 1.17 and 1.91 mg/dl respectively (p < 0.01). Pre and postoperative Hematocrit levels were 47.9 and 31% respectively (p = 0.02). Sixty six percent of tumors were clear cell renal cancer, 9% were papillary and 3% were chromophobic. Mean OS was 10 months. Two-year SCE was 40%. CONCLUSIONS: Our results are similar to those reported elsewhere. Despite being an unusual pathology, the surgical technique has been improving, thanks to the multidisciplinary work of urologists and surgeons.

3D printing microporous scaffolds from modular bioinks containing sacrificial, cell-encapsulating microgels.

Microgel-based biomaterials have inherent porosity and are often extrudable, making them well-suited for 3D bioprinting applications. Cells are commonly introduced into these granular inks post-printing using cell infiltration. However, due to slow cell migration speeds, this strategy struggles to achieve depth-independent cell distributions within thick 3D printed geometries. To address this, we leverage granular ink modularity by combining two microgels with distinct functions: (1) structural, UV-crosslinkable microgels made from gelatin methacryloyl (GelMA) and (2) sacrificial, cell-laden microgels made from oxidized alginate (AlgOx). We hypothesize that encapsulating cells within sacrificial AlgOx microgels would enable the simultaneous introduction of void space and release of cells at depths unachievable through cell infiltration alone. Blending the microgels in different ratios produces a family of highly printable GelMA : AlgOx microgel inks with void fractions ranging from 0.03 to 0.35. As expected, void fraction influences the morphology of human umbilical vein endothelial cells (HUVEC) within GelMA : AlgOx inks. Crucially, void fraction does not alter the ideal HUVEC distribution seen throughout the depth of 3D printed samples. This work presents a strategy for fabricating constructs with tunable porosity and depth-independent cell distribution, highlighting the promise of microgel-based inks for 3D bioprinting.

Design Parameters for Injectable Biopolymeric Hydrogels with Dynamic Covalent Chemistry Crosslinks.

Dynamic covalent chemistry (DCC) crosslinks can form hydrogels with tunable mechanical properties permissive to injectability and self-healing. However, not all hydrogels with transient crosslinks are easily extrudable. For this reason, two additional design parameters must be considered when formulating DCC-crosslinked hydrogels: 1) degree of functionalization (DoF) and 2) polymer molecular weight (MW). To investigate these parameters, hydrogels comprised of two recombinant biopolymers: 1) a hyaluronic acid (HA) modified with benzaldehyde and 2) an elastin-like protein (ELP) modified with hydrazine (ELP-HYD), are formulated. Several hydrogel families are synthesized with distinct HA MW and DoF while keeping the ELP-HYD component constant. The resulting hydrogels have a range of stiffnesses, G' ≈ 10-1000 Pa, and extrudability, which is attributed to the combined effects of DCC crosslinks and polymer entanglements. In general, lower MW formulations require lower forces for injectability, regardless of stiffness. Higher DoF formulations exhibit more rapid self-healing. Gel extrusion through a cannula (2 m length, 0.25 mm diameter) demonstrates the potential for minimally invasive delivery for future biomedical applications. In summary, this work highlights additional parameters that influence the injectability and network formation of DCC-crosslinked hydrogels and aims to guide future design of injectable hydrogels.

3D bioprinting of dynamic hydrogel bioinks enabled by small molecule modulators.

Three-dimensional bioprinting has emerged as a promising tool for spatially patterning cells to fabricate models of human tissue. Here, we present an engineered bioink material designed to have viscoelastic mechanical behavior, similar to that of living tissue. This viscoelastic bioink is cross-linked through dynamic covalent bonds, a reversible bond type that allows for cellular remodeling over time. Viscoelastic materials are challenging to use as inks, as one must tune the kinetics of the dynamic cross-links to allow for both extrudability and long-term stability. We overcome this challenge through the use of small molecule catalysts and competitors that temporarily modulate the cross-linking kinetics and degree of network formation. These inks were then used to print a model of breast cancer cell invasion, where the inclusion of dynamic cross-links was found to be required for the formation of invasive protrusions. Together, we demonstrate the power of engineered, dynamic bioinks to recapitulate the native cellular microenvironment for disease modeling.

Cell Microencapsulation Within Engineered Hyaluronan Elastin-Like Protein (HELP) Hydrogels.

Three-dimensional cell encapsulation has rendered itself a staple in the tissue engineering field. Using recombinantly engineered, biopolymer-based hydrogels to encapsulate cells is especially promising due to the enhanced control and tunability it affords. Here, we describe in detail the synthesis of our hyaluronan (i.e., hyaluronic acid) and elastin-like protein (HELP) hydrogel system. In addition to validating the efficacy of our synthetic process, we also demonstrate the modularity of the HELP system. Finally, we show that cells can be encapsulated within HELP gels over a range of stiffnesses, exhibit strong viability, and respond to stiffness cues. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Elastin-like protein modification with hydrazine Basic Protocol 2: Nuclear magnetic resonance quantification of elastin-like protein modification with hydrazine Basic Protocol 3: Hyaluronic acid-benzaldehyde synthesis Basic Protocol 4: Nuclear magnetic resonance quantification of hyaluronic acid-benzaldehyde Basic Protocol 5: 3D cell encapsulation in hyaluronan elastin-like protein gels.

Tunable hydrogel viscoelasticity modulates human neural maturation.

Human-induced pluripotent stem cells (hiPSCs) have emerged as a promising in vitro model system for studying neurodevelopment. However, current models remain limited in their ability to incorporate tunable biomechanical signaling cues imparted by the extracellular matrix (ECM). The native brain ECM is viscoelastic and stress-relaxing, exhibiting a time-dependent response to an applied force. To recapitulate the remodelability of the neural ECM, we developed a family of protein-engineered hydrogels that exhibit tunable stress relaxation rates. hiPSC-derived neural progenitor cells (NPCs) encapsulated within these gels underwent relaxation rate-dependent maturation. Specifically, NPCs within hydrogels with faster stress relaxation rates extended longer, more complex neuritic projections, exhibited decreased metabolic activity, and expressed higher levels of genes associated with neural maturation. By inhibiting actin polymerization, we observed decreased neuritic projections and a concomitant decrease in neural maturation gene expression. Together, these results suggest that microenvironmental viscoelasticity is sufficient to bias human NPC maturation.

Elastin-like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function.

Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell-applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin-like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine-modified ELP (ELP-HYD) and aldehyde/benzaldehyde-modified polyethylene glycol (PEG-ALD/PEG-BZA). The reversible DCC crosslinks in ELP-PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast-relaxing or slow-relaxing hydrogels with a range of stiffness (500-3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two-dimensional substrates, on which ECs exhibited greater cell spreading on fast-relaxing hydrogels up through 3 days, compared with slow-relaxing hydrogels at the same stiffness. In three-dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast-relaxing, low-stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast-relaxing, low-stiffness hydrogel produced significantly more vascularization compared with the slow-relaxing, low-stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast-relaxing, low-stiffness hydrogels supported the highest capillary density in vivo.

[Age of initiation of treatment for hip dysplasia with Pavlik harness and residual dysplasia]. / Edad de inicio del tratamiento de la displasia de caderas con correas de Pavlik y displasia residual.

The successful treatment of hip dysplasia consists of achieving a concentric reduction and avoiding residual dysplasia. One of the essential factors is early diagnosis and treatment. OBJECTIVE: Evaluate the relationship between the age at initiation of hip dysplasia treatment and the presence of residual dysplasia at one year of age. PATIENTS AND METHOD: Prognostic retrospective study. Patients with hip dysplasia treated with Pavlik harness in a tertiary healthcare center were selected. Residual dyspla sia was defined as an acetabular index greater than 28 degrees at one year of age. An association of residual dysplasia with the age at treatment initiation, bilaterality, and acetabular index more than 36 degrees was determined. The T-Student, Chi-Square, and Youden index tests were used. A p- value < 0.05 was considered significant. The STATA v.16 software was used. RESULTS: 153 patients (262 hips) were included, 84.3% (129) were females, and 71.2% (109) presented bilateral dysplasia. Fifty-nine hips (22.52%) presented residual dysplasia, finding a significant association with the age at treatment initiation (p = 0.03), bilateral dysplasia (p < 0.01), and acetabular index greater than 36 degrees (p = 0.01). Starting treatment after 4.5 months increases the risk of residual dysplasia by 2.5 times (95% CI 1.25-5.03). CONCLUSION: An increase in residual dysplasia was observed at the start of treatment after 4.5 months. It is relevant to consider this result in local clinical guidelines to achieve a successful diagnosis and treatment.

Tuning Polymer Hydrophilicity to Regulate Gel Mechanics and Encapsulated Cell Morphology.

Mechanically tunable hydrogels are attractive platforms for 3D cell culture, as hydrogel stiffness plays an important role in cell behavior. Traditionally, hydrogel stiffness has been controlled through altering either the polymer concentration or the stoichiometry between crosslinker reactive groups. Here, an alternative strategy based upon tuning the hydrophilicity of an elastin-like protein (ELP) is presented. ELPs undergo a phase transition that leads to protein aggregation at increasing temperatures. It is hypothesized that increasing this transition temperature through bioconjugation with azide-containing molecules of increasing hydrophilicity will allow direct control of the resulting gel stiffness by making the crosslinking groups more accessible. These azide-modified ELPs are crosslinked into hydrogels with bicyclononyne-modified hyaluronic acid (HA-BCN) using bioorthogonal, click chemistry, resulting in hydrogels with tunable storage moduli (100-1000 Pa). Human mesenchymal stromal cells (hMSCs), human umbilical vein endothelial cells (HUVECs), and human neural progenitor cells (hNPCs) are all observed to alter their cell morphology when encapsulated within hydrogels of varying stiffness. Taken together, the use of protein hydrophilicity as a lever to tune hydrogel mechanical properties is demonstrated. These hydrogels have tunable moduli over a stiffness range relevant to soft tissues, support the viability of encapsulated cells, and modify cell spreading as a consequence of gel stiffness.

Is Uro-oncological Surgery Safe During the COVID-19 Pandemic? Comparative Morbidity and Mortality in Patients Undergoing Surgery 2019-2020.

INTRODUCTION: The SARS-CoV-2 infection has resulted in an unprecedented pandemic. Patients undergoing surgery are a group at risk of exposure. Also, patients with ongoing infection undergoing surgery may be more susceptible to developing complications. There is no significant data on surgical safety in the pandemic period. MATERIAL AND METHODS: Observational study based in a prospective database of urological oncological surgery. Data were obtained during the 2020 mandatory confinement period compared to the same period in 2019. The records were reviewed 45 days post-surgery. The objective was to compare surgical morbidity and mortality during the pandemic versus an average year in urological cancer surgery. RESULTS: During confinement period (2020), 85 patients underwent uro-oncology surgery, while in 2019, during the same period, 165. The Clavien-Dindo morbidity ≥3 in 2020 was 2.3% (n=2), and in 2019, it reached 6% (n=10). In 2020, 9 patients were readmitted (10.5%). One patient (1.1%) was re-interfered, with a perioperative mortality of 1.1%. In 2019, 21 patients (12.7%) were readmitted. Seventeen patients (10.3%) were re-interfered, with a perioperative mortality of 1.8%. The median number of days hospitalized was 2 (IQR=2) in 2020 and 3 (IQR=3) in 2019. No significant differences were found in population or morbimortality, except for reoperation in a normal year. CONCLUSION: Postoperative morbidity and mortality reported are lower than those shown in the literature concerning COVID-19 and similar to that historically reported by our centers. This study suggests that it is safe to operate patients with urological cancer following the appropriate protocols during a pandemic.

Biomimetic tubular scaffold with heparin conjugation for rapid degradation in in situ regeneration of a small diameter neoartery.

To address the clinical need for readily available small diameter vascular grafts, biomimetic tubular scaffolds were developed for rapid in situ blood vessel regeneration. The tubular scaffolds were designed to have an inner layer that is porous, interconnected, and with a nanofibrous architecture, which provided an excellent microenvironment for host cell invasion and proliferation. Through the synthesis of poly(spirolactic-co-lactic acid) (PSLA), a highly functional polymer with a norbornene substituting a methyl group in poly(l-lactic acid) (PLLA), we were able to covalently attach biomolecules onto the polymer backbone via thiol-ene click chemistry to impart desirable functionalities to the tubular scaffolds. Specifically, heparin was conjugated on the scaffolds in order to prevent thrombosis when implanted in situ. By controlling the amount of covalently attached heparin we were able to modulate the physical properties of the tubular scaffold, resulting in tunable wettability and degradation rate while retaining the porous and nanofibrous morphology. The scaffolds were successfully tested as rat abdominal aortic replacements. Patency and viability were confirmed through dynamic ultrasound and histological analysis of the regenerated tissue. The harvested tissue showed excellent vascular cellular infiltration, proliferation, and migration with laminar cellular arrangement. Furthermore, we achieved both complete reendothelialization of the vessel lumen and native-like media extracellular matrix. No signs of aneurysm or hyperplasia were observed after 3 months of vessel replacement. Taken together, we have developed an effective vascular graft able to generate small diameter blood vessels that can function in a rat model.

Nefrectomía radical con trombectomía de vena cava: 20 años de cirugías por tumor renal / Vena cava thrombectomy in kidney cancer. Report of 32 nephrectomies

BACKGROUND: Vena cava (VC) involvement in kidney tumors occurs in 4 to 10% of cases, and is associated with a higher mortality. Nephrectomy with thrombectomy of the VC, performed by a multidisciplinary team, improves survival. Aim: To report a series of consecutive nephrectomies with caval thrombectomy performed in an academic center. PATIENTS AND METHODS: We report 32 patients with cT3b and 3c renal tumors, who underwent radical nephrectomy with VC thrombectomy between 2001 and 2021. A descriptive analysis of clinical, surgical and pathological variables was performed. Overall survival (OS) and cancer-specific survival (CSS) was calculated using Kaplan-Meier curves. Results: The mean tumor size was 9.7 cm. According to Mayo classification 3/32 (9%) patients had a type I thrombus, 10/32 (31%) had a type II thrombus, 8/32 (25%) had a type III thrombus, and 5/32 (16%) had a type IV thrombus. The mean bleeding was 2000 cc. There was one intraoperative death. Nineteen percent of patients had complications >= 3 according to Clavien-Dindo classification. Reoperations occurred in 9%. Pre and postoperative creatinine levels were 1.17 and 1.91 mg/dl respectively (p < 0.01). Pre and postoperative Hematocrit levels were 47.9 and 31% respectively (p = 0.02). Sixty six percent of tumors were clear cell renal cancer, 9% were papillary and 3% were chromophobic. Mean OS was 10 months. Two-year SCE was 40%. CONCLUSIONS: Our results are similar to those reported elsewhere. Despite being an unusual pathology, the surgical technique has been improving, thanks to the multidisciplinary work of urologists and surgeons.