Cellular Constructs Research Articles

Investment casting of porous Mg-alloy networks biomechanically tuned for bone implant applications

Manufacturing technology has been refined and described for the fabrication of honeycomb-based bioresorbable networks for temporal bone replacement applications. Two novel techniques, digital light processing and investment casting, were utilized to produce customized, shape-optimized cellular constructs with additional orifices promoting tissue ingrowth during osteo-regeneration. For this purpose, a conventional magnesium casting alloy (AZ91) was chosen. Numerical simulations were conducted to predict the compressive behavior of the proposed biodegradable lightweight scaffolds. Spatial castings were adjusted to possess mechanical properties comparable to the ones of cortical or trabecular bones. Two kinds of protective coatings (plasma electrolytic oxidation and organic ones based on natural polyphenols from tea extract) were deposited and characterized. They can be utilized to control the degradation rate during exploitation to achieve a predictable implant lifespan. The elaborated layers aim to mitigate the rapid corrosion of magnesium substrates by prolonging their bioresorption time and thus expanding their applicability in osseointegration. To evaluate this assumption, immersion tests in phosphate-buffered saline were performed, showing better chemical resistance of PEO coating and as-cast sample (for both mass gain by below 1%), and visible increase in mass of sample coated with organic coating (increase by almost 5%). Compressive strength results from numerical approach were further validated by experimental compression tests, showing that PEO coating deteriorated compressive strength by almost 3%, and organic coating improved it by over 9%. Results achieved in numerical approach were better than expected for stiffer sample, and slightly lower for the one with bigger pores.

Selection of Force Sensors for In Situ Measurement of Neotissue Microenvironments.

Mechanical forces are a critical stimulus in both native and engineered tissues. Direct measurement of these microenvironmental forces has been challenging, particularly for cell-dense models. To address this, we previously developed hydrogel-based force sensors that are approximately the size of a cell and can be imaged over time to computationally assess the forces exerted by surrounding cells and matrix. The goal of this project was to identify how the physical characteristics of force sensors impact measurements. Sensors were varied in size, elastic modulus, and surface coating before being included in stem cell suspensions that then spontaneously self-assembled into spheroidal neotissues. Using this model of early mesenchymal condensation, we hypothesized that larger, softer sensors would provide greater sensitivity and precision, whereas protein coatings would influence the directionality of applied forces (tensile vs. compressive). These experiments were conducted using a high-content imaging system that allowed analysis of over a thousand sensors to evaluate the various conditions. Results indicated that measurement fidelity was highest for force sensors that had a diameter >20 µm and modulus ∼0.2 kPa. Extremely soft sensors deformed too much, whereas stiffer sensors deformed too little. Collagen and N-cadherin coatings, which replicated cell-matrix or cell-cell binding, respectively, allowed for tensile forces to be exerted on the sensors, with greater forces being observed for N-cadherin sensors in these highly cellular neotissue constructs. Uncoated sensors were universally compressed due to the lack of cell-sensor adhesion. Disruption of the actin cytoskeleton lessened microenvironmental forces, whereas disruption of microtubules had no measurable effect. Potential future applications of the technology include studies of in situ forces in developing tissues as well as a real-time sensor for monitoring the growth of engineered constructs.

Effects of a bioengineered allogeneic cellular construct on burn-related macrophage phenotype.

Bioengineered allogeneic cellularised constructs (BACC) exert pro-healing effects in burn wounds and skew macrophage phenotype towards a predominately reparative phenotype. However, whether BACC can modulate the phenotype of dysregulated macrophages, like those present in burn wounds, is not known. To better understand the macrophage modulatory characteristics of the BACC, primary human macrophages were polarised to the M2b phenotype, an immunosuppressive phenotype relevant to burn wounds, by simultaneously exposing macrophages to polystyrene plate-coated immunoglobulin G and the endotoxin lipopolysaccharide (LPS). The resulting macrophage phenotype upregulated both inflammatory and reparative genes, and increased secretion of the M2b marker CCL1 compared to five different in vitro macrophage phenotypes. M2b macrophages were cultured with the BACC in the presence or absence of LPS to mimic infection, which is a common occurrence in burn wounds. The BACC caused up-regulation of reparative gene sets and down-regulation of pro-inflammatory gene sets, even when LPS was present in the cell culture media. Co-cultures were maintained for 1, 3, or 5 days in the presence of LPS, and by day 1 both non-activated macrophages and M2b macrophages exhibited signs of endotoxin tolerance, as demonstrated by a reduced secretion of tumour necrosis factor α (TNFα) in response to fresh LPS stimulus. The BACC was not able to prevent endotoxin tolerance, but reparative genes were upregulated in macrophages chronically exposed to LPS. These results suggest that the BACC can promote a reparative phenotype in dysregulated macrophages relevant to the pathophysiology of burns.

Cardiac Organoids: A 3D Technology for Disease Modeling and Drug Screening.

Cardiovascular diseases remain the leading cause of death worldwide; therefore, there is increasing attention to developing physiological-related in vitro cardiovascular tissue models suitable for personalized healthcare and preclinical test. Recently, more complex and powerful in vitro models have emerged for cardiac research. Human cardiac organoids (HCOs) are three-dimensional (3D) cellular constructs similar to in vivo organs. They are derived from pluripotent stem cells and can replicate the structure, function, and biogenetic information of primitive tissues. High-fidelity HCOs are closer to natural human myocardial tissue than animal and cell models to some extent, which helps to study better the development process of the heart and the occurrence of related diseases. In this review, we introduce the methods for constructing HCOs and the application of them, especially in cardiovascular disease modeling and cardiac drug screening. In addition, we propose the prospects and limitations of HCOs. In summary, we have introduced the research progress of HCOs and described their innovation and practicality of them in the biomedical field.

Embedded three-dimensional printing of thick pea-protein-enriched constructs for large, customized structured cell-based meat production

Recent 3D-printing research showed the potential of using plant-protein-enriched inks to fabricate cultivated meat (CM) via agar-based support baths. However, for fabricating large, customized, structured, thick cellular constructs and further cultivation, improved 3D-printing capabilities and diffusion limit circumvention are warranted. The presented study harnesses advanced printing and thick tissue engineering concepts for such purpose. By improving bath composition and altering printing design and execution, large-scale, marbled, 0.5-cm-thick rib-eye shaped constructs were obtained. The constructs featured stable fibrous architectures comparable to those of structured-meat products. Customized multi-cellular constructs with distinct regions were produced as well. Furthermore, sustainable 1-cm-thick cellular constructs were carefully designed and produced, which successfully maintained cell viability and activity for 3 weeks, through the combined effects of void-incorporation and dynamic culturing. As large, geometrically complex construct fabrication suitable for long-term cellular cultivation was demonstrated, these findings hold great promise for advancing structured CM research.

4D fabrication of shape-changing systems for tissue engineering: state of the art and perspectives

AbstractIn recent years, four-dimensional (4D) fabrication has emerged as a powerful technology capable of revolutionizing the field of tissue engineering. This technology represents a shift in perspective from traditional tissue engineering approaches, which generally rely on static—or passive—structures (e.g., scaffolds, constructs) unable of adapting to changes in biological environments. In contrast, 4D fabrication offers the unprecedented possibility of fabricating complex designs with spatiotemporal control over structure and function in response to environment stimuli, thus mimicking biological processes. In this review, an overview of the state of the art of 4D fabrication technology for the obtainment of cellularized constructs is presented, with a focus on shape-changing soft materials. First, the approaches to obtain cellularized constructs are introduced, also describing conventional and non-conventional fabrication techniques with their relative advantages and limitations. Next, the main families of shape-changing soft materials, namely shape-memory polymers and shape-memory hydrogels are discussed and their use in 4D fabrication in the field of tissue engineering is described. Ultimately, current challenges and proposed solutions are outlined, and valuable insights into future research directions of 4D fabrication for tissue engineering are provided to disclose its full potential.

A dual crosslinking strategy to tailor rheological properties of gelatin methacryloyl

3D bioprinting is an emerging technology that enables the fabrication of three-dimensional organised cellular constructs. One of the major challenges in 3D bioprinting is to develop a material to meet the harsh requirements (cell-compatibility, printability, structural stability post-printing and bio-functionality to regulate cell behaviours) suitable for printing. Gelatin methacryloyl (GelMA) has recently emerged as an attractive biomaterial in tissue engineering because it satisfies the requirements of bio-functionality and mechanical tunability. However, poor rheological property such as low viscosity at body temperature inhibits its application in 3D bioprinting. In this work, an enzymatic crosslinking method triggered by Ca2+-independent microbial transglutaminase (MTGase) was introduced to catalyse isopeptide bonds formation between chains of GelMA, which could improve its rheological behaviours, specifically its viscosity. By combining enzymatic crosslinking and photo crosslinking, it is possible to tune the solution viscosity and quickly stabilize the gelatin macromolecules at the same time. The results showed that the enzymatic crosslinking can increase the solution viscosity. Subsequent photo crosslinking could aid in fast stabilization of the structure and make handling easy.

Abstract Mo089: Fontan-Associated Liver Disease On A Chip

Introduction: The Fontan procedure is performed in children with single ventricle physiology, and has led to excellent short-term palliation and survival rates but at the cost of long-term complications. Fontan-associated liver disease (FALD) results from hemodynamic changes following the Fontan procedure, leading to elevated central venous pressures, chronic alterations in hepatic perfusion, and resultant hepatic fibrosis. Improved understanding of FALD pathophysiology, which may provide insights for prevention and treatment is challenged by the lack of experimental models. Current in vitro liver models, mostly 2D or suspension hepatic cell cultures, lack biomimicry. This study presents a 3D bioprinted perfusable human liver model for in vitro study of hepatic fibrosis in response to altered flow conditions in FALD patients. Methods: Vascular 3D model of hepatic sinusoid was biofabricated using hybrid bioinks in two distinct peripheral layers and a central lumen. The outer layer was cast with hepatic (HepG2) cells encapsulated in a mixture of gelatin methacrylate (GelMA) and collagen type I. The inner layer consisted of hepatic stellate cells (HSCs) in GelMA. Human endothelial cells (ECs) were seeded onto the central lumen. 3D cellular constructs were cultured in vitro for three weeks in static or dynamic conditions, by circulating the culture media at varying flow conditions to simulate sinusoid pressure characteristic of healthy vs. disease state. Using Volcano system in cath lab, we invasively measured the pressure in mid-channel. Cell viability, proliferation, maturation into functional liver tissue, and tissue stiffness were evaluated via microindentation, flow hemodynamics assays, metabolomic assays, and immunohistochemistry analyses. Results: Bioengineered liver constructs cultured under static and dynamic conditions demonstrated high cell viability and growth and full endothelialization of the sinusoid channel. HepG2 cells exhibited long-term function and maturation, forming clusters. Under FALD flow conditions, there were notable changes in EC-HepG2 cell morphology compared to dynamic flow in the healthy range, accompanied by reduced levels of functional markers. Conclusion: This proof-of-principle study introduces an innovative and highly biomimetic 3D model of hepatic sinusoid, which can serve as a robust platform for in vitro investigations of complex cell-microenvironment interactions in the context of FALD as well as other hepatic disorders.

Extrusion-Based Bioprinting in a Cost-Effective Bioprinter

Three-dimensional (3D) bioprinting has emerged as a revolutionary approach in the life sciences, combining multiple disciplines such as computer engineering, materials science, robotics, and biomedical engineering. This innovative technology enables the production of cellular constructs using bio-inks, and differs from conventional 3D printing by incorporating living cells. The present work addresses the conversion of a commercial thermoplastic 3D printer into a low-cost bioprinter. The modification addresses the challenges of the high cost of commercial bioprinters, limited adaptability, and specialized personnel requirements. This modification uses an extrusion-based bioprinting method that is particularly popular in research due to its viscosity tolerance and versatility. The individual steps, including replacing the extruder with a syringe pump, rebuilding the electronic motherboard, and configuring the firmware, are explained in detail. The work aims at providing access to bioprinting technology so that laboratories with modest resources can take advantage of the immense potential of this technology. This modification resulted in improved resolution, allowing submicron movements, which is comparable to some of the commercially available bioprinters. The accuracy of the modified printer was validated using hydrogel bioprinting tests, suggesting that it is suitable for broader applications in regenerative medicine.

The search for an optimal tissue-engineered urethra model for clinical application based on preclinical trials in male animals: A systematic review and meta-analysis.

Tissue engineering has emerged as a promising avenue for reconstructive urology, though only a limited number of tissue-engineered urethral constructs have advanced to clinical testing. Presently, there exists a dearth of agreement regarding the most promising constructs deserving of implementation in clinical practice. The objective of this review was to provide a comprehensive analysis of preclinical trials findings of a tissue-engineered urethra and to identify the most promising constructs for future translation into clinical practice. A systematic search of the Pubmed, Scopus, and PMC databases was conducted in accordance with the PRISMA statement. Manuscripts published in English between 2015 and 2022, reporting on the methodology for creating a tissue-engineered urethra, assessing the regenerative potential of the scaffold in a male animal model, and evaluating the clinical and histological outcomes of treatment, were included. A total of 48 manuscripts met the inclusion criteria, with 12 being eligible for meta-analysis. Meta-analysis revealed no significant benefit of any matrix type in terms of complication rates. However, acellular matrices demonstrated significant advantage over cellular matrices in case of no postoperative stricture formation (odds ratio = 0.06 [95% CI 0.01; 0.23], p < 0.01). Among all subgroups (animal models and scaffold types), the usage of acellular matrices resulted in advantageous effects. The meta-regression analysis did not show a significant impact of defect length (β1 = -0.02 [-0.28; 0.23], p = 0.86). We found that decellularized materials may carry less relevance for urethral reconstruction due to unfavorable preclinical outcomes. Natural polymers, used independently or with synthetic materials, resulted in better postoperative outcomes in animals compared to purely synthetic constructs. Acellular scaffolds showed promising outcomes, matching or exceeding cellular constructs. However, more studies are needed to confirm their clinical effectiveness.

Rapid prototyping of perfusion cell culture devices for three-dimensional imaging of mesenchymal stem cell deposition and proliferation

Perfusion of porous scaffolds transports cells to the surface to yield cellular constructs for 3D models of disease and for tissue engineering applications. While ceramic scaffolds mimic the structure and composition of trabecular bone, their opacity and tortuous pores limit the penetration of light into the interior. Scaffolds that are both perfusable and amenable to fluorescence microscopy are therefore needed to visualize the spatiotemporal dynamics of cells in the bone microenvironment. In this study, a hybrid injection molding approach was designed to enable rapid prototyping of collector arrays with variable configurations that are amenable to longitudinal imaging of attached human mesenchymal stem cells (hMSCs) using fluorescence microscopy. Cylindrical collectors were arranged in an array that is permeable to perfusion in the xy-plane and to light in the z-direction for imaging from below. The effects of the collector radius, number, and spacing on the collection efficiency of perfused hMSCs was simulated using computational fluid dynamics (CFD) and measured experimentally using fluorescence microscopy. The effect of collector diameter on simulated and experimental cell collection efficiencies followed a trend similar to that predicted by interception theory corrected for intermolecular and hydrodynamic forces for the arrays with constant collector spacing. In contrast, arrays designed with constant collector number yielded collection efficiencies that poorly fit the trend with collector radius predicted by interception theory. CFD simulations of collection efficiency agreed with experimental measurements within a factor of two. These findings highlight the utility of CFD simulations and hybrid injection molding for rapid prototyping of collector arrays to optimize the longitudinal imaging of cells without the need for expensive and time-consuming tooling.

Translational Aspects of 3D and 4D Printing and Bioprinting.

Three-dimensional (3D) printed medical devices include orthopedic and craniofacial implants, surgical tools, and external prosthetics that have been directly used in patients. While the advances of additive manufacturing techniques in the production of medical devices have been on the rise, clinical translation of living cellular constructs face significant limitations in terms of regulatory affairs, process technology, and materials development. In this perspective, the current status-quo of 3D and four-dimensional (4D) (bio)printing is summarized, current advancements are discussed and the challenges that need to be addressed for improved industrial translation and clinical applications of bioprinting are highlighted. It is focused on a multidisciplinary approach in discussing the key translational considerations, from the perspective of industry, regulatory bodies, funding strategies, and future directions.

Correlation of CT-based radiomics analysis with pathological cellular infiltration in fibrosing interstitial lung diseases

PurposeWe aimed to identify computed tomography (CT) radiomics features that are associated with cellular infiltration and construct CT radiomics models predictive of cellular infiltration in patients with fibrotic ILD.Materials and methodsCT images of patients with ILD who underwent surgical lung biopsy (SLB) were analyzed. Radiomics features were extracted using artificial intelligence-based software and PyRadiomics. We constructed a model predicting cell counts in histological specimens, and another model predicting two classifications of higher or lower cellularity. We tested these models using external validation.ResultsOverall, 100 patients (mean age: 62 ± 8.9 [standard deviation] years; 61 men) were included. The CT radiomics model used to predict cell count in 140 histological specimens predicted the actual cell count in 59 external validation specimens (root-mean-square error: 0.797). The two-classification model’s accuracy was 70% and the F1 score was 0.73 in the external validation dataset including 30 patients.ConclusionThe CT radiomics-based model developed in this study provided useful information regarding the cellular infiltration in the ILD with good correlation with SLB specimens.

A 3D-printable gelatin/alginate/ε-poly-l-lysine hydrogel scaffold to enable porcine muscle stem cells expansion and differentiation for cultured meat development

The mass proliferation of seed cells and imitation of meat structures remain challenging for cell-cultured meat production. With excellent biocompatibility, high water content and porosity, hydrogels are frequently-studied materials for anchorage-dependent cell scaffolds in biotechnology applications. Herein, a scaffold based on gelatin/alginate/ε-Poly-l-lysine (GAL) hydrogel is developed for skeletal muscle cells, which has a great prospect in cell-cultured meat production. In this work, the hydrogel GAL-4:1, composed of gelatin (5 %, w/v), alginate (5 %, w/v) and ε-Poly-l-lysine (molar ratio vs. alginate: 4:1) is selected as cell scaffold based on Young's modulus of 11.29 ± 1.94 kPa, satisfactory shear-thinning property and suitable porous organized structure. The commercially available C2C12 mouse skeletal myoblasts and porcine muscle stem cells (PMuSCs), are cultured in the 3D-printed scaffold. The cells show strong ability of attachment, proliferation and differentiation after induction, showing high biocompatibility. Furthermore, the cellular bioprinting is performed with GAL-4:1 hydrogel and freshly extracted PMuSCs. The extracted PMuSCs exhibit high viability and display early myogenesis (desmin) on the 3D scaffold, suggesting the great potential of GAL hydrogel as 3D cellular constructs scaffolds. Overall, we develop a novel GAL hydrogel as a 3D-printed bioactive platform for cultured meat research.

Shape-Versatile Fixed Cellular Materials for Multiple Target Immunomodulation.

Therapeutic cells are usually administered as living agents, despite the risks of undesired cell migration and acquisition of unpredictable phenotypes. Additionally, most cell-based therapies rely on the administration of single cells, often associated with rapid in vivo clearance. 3D cellular materials may be useful to prolong the effect of cellular therapies and offer the possibility of creating structural volumetric constructs. Here, the manufacturing of shape-versatile fixed cell-based materials with immunomodulatory properties is reported. Living cell aggregates with different shapes (spheres and centimeter-long fibers) are fixed using a method compatible with maintenance of structural integrity, robustness, and flexibility of 3D constructs.The biological properties of living cells can be modulated before fixation, renderingan in vitroanti-inflammatory effect toward human macrophages, in line with a decreased activation of the nuclear factor kappa B (NF-κB) pathway that preponderantly correlated with the surface area of the materials. These findings are further corroborated in vivo in mouse skin wounds. Contact with fixed materials also reduces the proliferation ofactivated primary T lymphocytes, while promotingregulatory populations. The fixation of cellular constructs is proposed as a versatile phenotypic stabilization method that can be easily implemented to prepare immunomodulatory materials with therapeutic potential.

Enrichment using a selectable CD34t marker enhances TCR/CAR transduced T cell functionality and transgene expression in clinically relevant receptors

Abstract Gene-modified TCR transduced T cells and CAR T cells are used clinically and display a range of efficacy across multiple cancers. Many therapies have successfully controlled patient’s tumors; however, some have shown off-target adverse events which are harmful to patients’ overall outcomes. T cell-based therapies have high variation in patient outcome due to multiple factors, such as transduction efficiency and T cell phenotypes. To reduce product variability, our lab developed a selectable marker gene for use in cellular constructs. This selectable marker is a truncated, non-signaling CD34 molecule (CD34t), which can be used to remove the non-transduced T cells that function as bystanders. Our clinical trials have used two TCRs (TIL 1383I and HERV-E) and one CAR (CD19 CAR). Following transduction with these CD34t constructs, we determined transduction efficiency by expression of CD34t. CD34t expression correlates with transgene, allowing us to select high CD34t expressors, and thus high transgene expressors. The selected population produces higher levels of inflammatory cytokines and CD107a expression compared to the nonselected. This improved functionality is likely due to the selected population having higher levels of transgene across the population, whereas the nonselected has a broader range of transgene expressions with fewer expressors total. This selectable marker contributes to a more functional product, which may in turn improve patient response clinically.

Effects of a Bioengineered Allogeneic Cellularized Construct (BACC) on Primary Human Macrophage Phenotype.

The mechanisms behind the pro-healing effects of multicellular, bioengineered allogeneic cellularized constructs (BACC) are not known. Macrophages are key regulators of every phase of the wound healing process and the primary cells that mediate the response to biomaterials. It is hypothesized that cells within the BACC modulate macrophage behavior, which may contribute to the mechanism by which BACC promotes healing. To probe the influence of cells within the BACC compared to effects of the underlying collagen substrate, primary human macrophages are cultured in direct or indirect contact with BACC or with the same collagen substrate used in the BACC manufacturing. Macrophage phenotype is characterized over time via multiplex gene expression, protein secretion, multidimensional flow cytometry, and functional assays with fibroblasts and endothelial cells. The BACC causes macrophages to exhibit a predominately reparative phenotype over time compared to relevant collagen substrate controls, with multiple subpopulations expressing both pro-inflammatory and reparative markers. Conditioned media from macrophage-BACC co-cultures causes distinct effects on fibroblast and endothelial cell proliferation, migration, and network formation. Given the critical role of the reparative macrophage phenotype in wound healing, these results suggest that modulation of macrophage phenotype may be a critical part of the mechanisms behind BACC's pro-healing effects.

Cost effectiveness analysis for commonly used human cell and tissue products in the management of diabetic foot ulcers.

This study considers the cost-effectiveness of commonly used cellular, acellular, and matrix‑like products (CAMPs) of human origin also known as human cell and tissue products (HCT/Ps) in the management of diabetic foot ulcers. We developed a 1-year economic model assessing six CAMPs [cryopreserved placental membrane with viable cells (vCPM), bioengineered bilayered living cellular construct (BLCC), human fibroblast dermal substitute (hFDS), dehydrated human amnion chorion membrane (dHACM), hypothermically stored amniotic membrane (HSAM) and human amnion membrane allograft (HAMA) which had randomized controlled trial evidence compared with standard of care (SoC). CAMPs were compared indirectly and ranked in order of cost-effectiveness using SoC as the baseline, from a CMS/Medicare's perspective. The mean cost, healed wounds (hw) and QALYs per patient for vCPM is $10,907 (0.914 hw, 0.783 QALYs), for HAMA $11,470 (0.903 hw, 0.780 QALYs), for dHACM $15,862 (0.828 hw, 0.764 QALYs), for BLCC $18,430 (0.816 hw, 0.763 QALYs), for hFDS $19,498 (0.775 hw, 0.757 QALYs), for SoC $19,862 (0.601 hw, 0.732 QALYs) and $24, 214 (0.829, 0.763 QALYs) for HSAM respectively. Over 1 year, vCPM results in cheaper costs overall and better clinical outcomes compared to other CAMPs. Following probabilistic sensitivity analysis, vCPM has a 60%, HAMA 40% probability of being cost-effective then dHACM, hFDS, BLCC, and lastly HSAM using a $100,000/healed wound or QALY threshold. All CAMPs were shown to be cost-effective when compared to SoC in managing DFUs. However, vCPM appears to be the most cost-effective CAMP over the modelled 52 weeks followed by HAMA, dHACM, hFDS, BLCC, and HSAM. We urge caution in interpreting the results because we currently lack head-to-head evidence comparing all these CAMPs and therefore suggest that this analysis be updated when more direct evidence of CAMPs becomes available.

Real-world data analysis of bilayered living cellular construct and fetal bovine collagen dressing treatment for pressure injuries: a comparative effectiveness study.

Aim: To determine the effectiveness of bilayered living cellular construct (BLCC) versus a fetal bovine collagen dressing (FBCD) in pressure injuries (PRIs). Methods: A real-world data study was conducted on 1352 PRIs analyzed digitally. 1046 and 306 PRIs were treated with BLCC and FBCD, respectively. Results: Cox healing for BLCC (n=1046) was significantly greater (p<0.0001) at week 4 (13 vs 7%), 8 (29 vs 17%), 12 (42 vs 27%), 24 (64 vs 45%), and 36 (73 vs 56%). The probability of healing increased by 66%, (hazard ratio=1.66 [95% CI (1.38, 2.00)]; p<0.0001. Time to healing was 162days for FBCD and 103days for BLCC showing a 36% reduction in time to healing with BLCC; (p<0.0001). Conclusion: BLCC significantly improved healing of PRIs versus FBCD.

Single-neuronal elements of speech production in humans

Humans are capable of generating extraordinarily diverse articulatory movement combinations to produce meaningful speech. This ability to orchestrate specific phonetic sequences, and their syllabification and inflection over subsecond timescales allows us to produce thousands of word sounds and is a core component of language1,2. The fundamental cellular units and constructs by which we plan and produce words during speech, however, remain largely unknown. Here, using acute ultrahigh-density Neuropixels recordings capable of sampling across the cortical column in humans, we discover neurons in the language-dominant prefrontal cortex that encoded detailed information about the phonetic arrangement and composition of planned words during the production of natural speech. These neurons represented the specific order and structure of articulatory events before utterance and reflected the segmentation of phonetic sequences into distinct syllables. They also accurately predicted the phonetic, syllabic and morphological components of upcoming words and showed a temporally ordered dynamic. Collectively, we show how these mixtures of cells are broadly organized along the cortical column and how their activity patterns transition from articulation planning to production. We also demonstrate how these cells reliably track the detailed composition of consonant and vowel sounds during perception and how they distinguish processes specifically related to speaking from those related to listening. Together, these findings reveal a remarkably structured organization and encoding cascade of phonetic representations by prefrontal neurons in humans and demonstrate a cellular process that can support the production of speech.