A High-Throughput Mechanical Activator for Cartilage Engineering Enables Rapid Screening of in vitro Response of Tissue Models to Physiological and Supra-Physiological Loads.

Articular cartilage is crucially influenced by loading during development, health, and disease. However, our knowledge of the mechanical conditions that promote engineered cartilage maturation or tissue repair is still incomplete. Current in vitro models that allow precise control of the local mechanical environment have been dramatically limited by very low throughput, usually just a few specimens per experiment. To overcome this constraint, we have developed a new device for the high throughput compressive loading of tissue constructs: the High Throughput Mechanical Activator for Cartilage Engineering (HiT-MACE), which allows the mechanoactivation of 6 times more samples than current technologies. With HiT-MACE we were able to apply cyclic loads in the physiological (e.g., equivalent to walking and normal daily activity) and supra-physiological range (e.g., injurious impacts or extensive overloading) to up to 24 samples in one single run. In this report, we compared the early response of cartilage to physiological and supra-physiological mechanical loading to the response to IL-1ß exposure, a common but rudimentary in vitro model of cartilage osteoarthritis. Physiological loading rapidly upregulated gene expression of anabolic markers along the TGF-ß1 pathway. Notably, TGF-ß1 or serum was not included in the medium. Supra-physiological loading caused a mild catabolic response while IL-1ß exposure drove a rapid anabolic shift. This aligns well with recent findings suggesting that overloading is a more realistic and biomimetic model of cartilage degeneration. Taken together, these findings showed that the application of HiT-MACE allowed the use of larger number of samples to generate higher volume of data to effectively explore cartilage mechanobiology, which will enable the design of more effective repair and rehabilitation strategies for degenerative cartilage pathologies.

Pectin-GPTMS-Based Biomaterial: toward a Sustainable Bioprinting of 3D scaffolds for Tissue Engineering Application.

Developing green and nontoxic biomaterials, derived from renewable sources and processable through 3D bioprinting technologies, is an emerging challenge of sustainable tissue engineering. Here, pectin from citrus peels was cross-linked for the first time with (3-glycidyloxypropyl)trimethoxysilane (GPTMS) through a one-pot procedure. Freeze-dried porous pectin sponges, with tunable properties in terms of porosity, water uptake, and compressive modulus, were obtained by controlling GPTMS content. Cell experiments showed that GPTMS did not affect the cytocompatibility of pectin. The addition of GPTMS improved the printability of pectin due to an increase of viscosity and yield stress. Three-dimensional woodpile and complex anatomical-shaped scaffolds with interconnected micro- and macropores were, therefore, bioprinted without the use of any additional support material. These results show the great potential of using pectin cross-linked with GPTMS as biomaterial ink to fabricate patient-specific scaffolds, which could be used to promote tissue regeneration in vivo.

3D printing for tissue engineering in otolaryngology.

Airway and other head and neck disorders affect hundreds of thousands of patients each year and most require surgical intervention. Among these, congenital deformity that affects newborns is particularly serious and can be life-threatening. In these cases, reconstructive surgery is resolutive but bears significant limitations, including the donor site morbidity and limited available tissue. In this context, tissue engineering represents a promising alternative approach for the surgical treatment of otolaryngologic disorders. In particular, 3D printing coupled with advanced imaging technologies offers the unique opportunity to reproduce the complex anatomy of native ear, nose, and throat, with its import in terms of functionality as well as aesthetics and the associated patient well-being. In this review, we provide a general overview of the main ear, nose and throat disorders and focus on the most recent scientific literature on 3D printing and bioprinting for their treatment.

Design and validation of an osteochondral bioreactor for the screening of treatments for osteoarthritis.

Bioreactors are systems that can be used to monitor the response of tissues and cells to candidate drugs. Building on the experience developed in the creation of an osteochondral bioreactor, we have designed a new 3D printed system, which allows optical access to the cells throughout testing for in line monitoring. Because of the use of 3D printing, the fluidics could be developed in the third dimension, thus maintaining the footprint of a single well of a typical 96 well plate. This new design was optimized to achieve the maximum fluid transport through the central chamber, which corresponds to optimal nutrient or drug exposure. This optimization was achieved by altering each dimension of the bioreactor fluid path. A physical model for optimized drug exposure was then created and tested.

Correction to: Design and validation of an osteochondral bioreactor for the screening of treatments for osteoarthritis.

The original version of this article unfortunately contained a mistake.

Rapidly dissociated autologous meniscus tissue enhances meniscus healing: An in vitro study.

PURPOSE: Treatment of meniscus tears is a persistent challenge in orthopedics. Although cell therapies have shown promise in promoting fibrocartilage formation in in vitro and preclinical studies, clinical application has been limited by the paucity of autologous tissue and the need for ex vivo cell expansion. Rapid dissociation of the free edges of the anterior and posterior meniscus with subsequent implantation in a meniscus lesion may overcome these limitations. The purpose of this study was to explore the effect of rapidly dissociated meniscus tissue in enhancing neotissue formation in a radial meniscus tear, as simulated in an in vitro explant model. MATERIALS AND METHODS: All experiments in this study, performed at minimum with biological triplicates, utilized meniscal tissues from hind limbs of young cows. The effect of varying collagenase concentration (0.1%, 0.2% and 0.5% w/v) and treatment duration (overnight and 30 minutes) on meniscus cell viability, organization of the extracellular matrix (ECM), and gene expression was assessed through a cell metabolism assay, microscopic examination, and quantitative real-time reverse transcription polymerase chain reaction analysis, respectively. Thereafter, an explant model of a radial meniscus tear was used to evaluate the effect of a fibrin gel seeded with one of the following: (1) fibrin alone, (2) isolated and passaged (P2) meniscus cells, (3) overnight digested tissue, and (4) rapidly dissociated tissue. The quality of in vitro healing was determined through histological analysis and derivation of an adhesion index. RESULTS: Rapid dissociation in 0.2% collagenase yielded cells with higher levels of metabolism than either 0.1% or 0.5% collagenase. When seeded in a three-dimensional fibrin hydrogel, both overnight digested and rapidly dissociated cells expressed greater levels of collagens type I and II than P2 meniscal cells at 1 week. At 4 and 8 weeks, collagen type II expression remained elevated only in the rapid dissociation group. Histological examination revealed enhanced healing in all cell-seeded treatment groups over cell-free fibrin controls at weeks 1, 4, and 8, but there were no significant differences across the treatment groups. CONCLUSIONS: Rapid dissociation of meniscus tissue may provide a single-step approach to augment regenerative healing of meniscus repairs.

A quantitative interpretation of the response of articular cartilage to atomic force microscopy-based dynamic nanoindentation tests.

In this paper, a quantitative interpretation for atomic force microscopy-based dynamic nanoindentation (AFM-DN) tests on the superficial layers of bovine articular cartilage (AC) is provided. The relevant constitutive parameters of the tissue are estimated by fitting experimental results with a finite element model in the frequency domain. Such model comprises a poroelastic stress-strain relationship for a fibril reinforced tissue constitution, assuming a continuous distribution of the collagen network orientations. The identification procedure was first validated using a simplified transversely isotropic constitutive relationship; then, the experimental data were manually fitted by using the continuous distribution fibril model. Tissue permeability is derived from the maximum value of the phase shift between the input harmonic loading and the harmonic tissue response. Tissue parameters related to the stiffness are obtained from the frequency response of the experimental storage modulus and phase shift. With this procedure, an axial to transverse stiffness ratio (anisotropy ratio) of about 0.15 is estimated.

Stem cell-based microphysiological osteochondral system to model tissue response to interleukin-1ß.

Osteoarthritis (OA) is a chronic degenerative disease of the articular joint that involves both bone and cartilage degenerative changes. An engineered osteochondral tissue within physiological conditions will be of significant utility in understanding the pathogenesis of OA and testing the efficacy of potential disease-modifying OA drugs (DMOADs). In this study, a multichamber bioreactor was fabricated and fitted into a microfluidic base. When the osteochondral construct is inserted, two chambers are formed on either side of the construct (top, chondral; bottom, osseous) that is supplied by different medium streams. These medium conduits are critical to create tissue-specific microenvironments in which chondral and osseous tissues will develop and mature. Human bone marrow stem cell (hBMSCs)-derived constructs were fabricated in situ and cultured within the bioreactor and induced to undergo spatially defined chondrogenic and osteogenic differentiation for 4 weeks in tissue-specific media. We observed tissue specific gene expression and matrix production as well as a basophilic interface suggesting a developing tidemark. Introduction of interleukin-1ß (IL-1ß) to either the chondral or osseous medium stream induced stronger degradative responses locally as well as in the opposing tissue type. For example, IL-1ß treatment of the osseous compartment resulted in a strong catabolic response in the chondral layer as indicated by increased matrix metalloproteinase (MMP) expression and activity, and tissue-specific gene expression. This induction was greater than that seen with IL-1ß application to the chondral component directly, indicative of active biochemical communication between the two tissue layers and supporting the osteochondral nature of OA. The microtissue culture system developed here offers novel capabilities for investigating the physiology of osteochondral tissue and pathogenic mechanisms of OA and serving as a high-throughput platform to test potential DMOADS.

A Pilot Study of Decellularized Cartilage for Laryngotracheal Reconstruction in a Neonatal Pig Model.

OBJECTIVE: Severe subglottic stenosis develops as a response to intubation in 1% of the >200,000 neonatal intensive care unit infants per year and may require laryngotracheal reconstruction (LTR) with autologous hyaline cartilage. Although effective, LTR is limited by comorbidities, severity of stenosis, and graft integration. In children, there is a significant incidence of restenosis requiring revision surgery. Tissue engineering has been proposed to develop alterative grafting options to improve outcomes and eliminate donor-site morbidity. Our objective is to engineer a decellularized, channel-laden xenogeneic cartilage graft, that we deployed in a proof-of-concept, neonatal porcine LTR model. METHODS: Meniscal porcine cartilage was freeze-thawed and washed with pepsin/elastase to decellularize and create microchannels. A 6 × 10-mm decellularized cartilage graft was then implanted in 4 infant pigs in an anterior cricoid split. Airway patency and host response were monitored endoscopically until sacrifice at 12 weeks, when the construct phenotype, cricoid expansion, mechanics, and histomorphometry were evaluated. RESULTS: The selective digestion of meniscal components yielded decellularized cartilage with cell-size channels. After LTR with decellularized meniscus, neonatal pigs were monitored via periodic endoscopy observing re-epithelization, integration, and neocartilage formation. At 12 weeks, the graft appeared integrated and exhibited airway expansion of 4 mm in micro-CT and endoscopy. Micro-CT revealed a larger lumen compared with age-matched controls. Finally, histology showed significant neocartilage formation. CONCLUSION: Our neonatal porcine LTR model with a decellularized cartilage graft is a novel approach to tissue engineered pediatric LTR. This pilot study sets the stage for "off-the-shelf" graft procurement and future optimization of MEND for LTR. LEVEL OF EVIDENCE: NA Laryngoscope, 134:807-814, 2024.

Azithromycin Prevents Subglottic Stenosis in Mice.

OBJECTIVE: Pediatric subglottic stenosis (SGS) is characterized by subglottic narrowing which occurs when pathological fibroblasts deposit extracellular matrix that reduces airway patency. Recent clinical observations have suggested that azithromycin may have favorable impacts on SGS reduction while treating airway infections; furthermore, our recent work in mice demonstrated that the airway microbiome influences SGS. In this work, we characterize the protective effect of azithromycin as an immunomodulatory and antibacterial therapeutic against subglottic stenosis. METHODS: Immunomodulatory and antifibrotic effects of azithromycin were assessed on TGF-ß1-stimulated airway fibroblasts at 10 µg/mL for 5 days. Changes in gene expression were quantified by RT-qPCR and myofibroblast differentiation by α-SMA immunostaining. Murine airways were pretreated (2-weeks) with intranasal azithromycin before SGS injury by a twisted wire brush. Disease severity and immune response were characterized by histology and immunostaining for immune cells. RESULTS: In vitro, azithromycin treatment of TGF-ß1-stimulated fibroblasts exhibited strong reductions in extracellular matrix (COL1A1, LOX) and myofibroblast-related gene expression (ACTA2). Notably, there was a significant reduction in pro-fibrotic expression, which was observed with 10 µg/mL azithromycin. Immunostaining of fibroblasts for α-SMA revealed strong reductions in the number of positive-staining cells and the intensity of each positive cell. In vivo, azithromycin exhibited a significant decrease in lamina propria thickness indicative of reduced stenosis with associated changes in T-cell infiltration. CONCLUSIONS: Overall, we show azithromycin prevents pro-fibrotic gene expression and myofibroblast differentiation and can help protect mice from developing SGS. This introduces azithromycin as a potential treatment for SGS. LEVEL OF EVIDENCE: NA Laryngoscope, 2024.

Modulated Fibrosis and Mechanosensing of Fibroblasts by SB525334 in Pediatric Subglottic Stenosis.

OBJECTIVE: Subglottic stenosis (SGS) may result from prolonged intubation where fibrotic scar tissue narrows the airway. The scar forms by differentiated myofibroblasts secreting excessive extracellular matrix (ECM). TGF-ß1 is widely accepted as a regulator of fibrosis; however, it is unclear how biomechanical pathways co-regulate fibrosis. Therefore, we phenotyped fibroblasts from pediatric patients with SGS to explore how key signaling pathways, TGF-ß and Hippo, impact scarring and assess the impact of inhibiting these pathways with potential therapeutic small molecules SB525334 and DRD1 agonist dihydrexidine hydrochloride (DHX). METHODS: Laryngeal fibroblasts isolated from subglottic as well as distal control biopsies of patients with evolving and maturing subglottic stenosis were assessed by α-smooth muscle actin immunostaining and gene expression for α-SMA, FN, HGF, and CTGF markers. TGF-ß and Hippo signaling pathways were modulated during TGF-ß1-induced fibrosis using the inhibitor SB525334 or DHX and analyzed by RT-qPCR for differential gene expression and atomic force microscopy for ECM stiffness. RESULTS: SGS fibroblasts exhibited higher α-SMA staining and greater inflammatory cytokine and fibrotic marker expression upon TGF-ß1 stimulation (p < 0.05). SB525334 restored levels to baseline by reducing SMAD2/3 nuclear translocation (p < 0.0001) and pro-fibrotic gene expression (p < 0.05). ECM stiffness of stenotic fibroblasts was greater than healthy fibroblasts and was restored to baseline by Hippo pathway modulation using SB525334 and DHX (p < 0.01). CONCLUSION: We demonstrate that distinct fibroblast phenotypes from diseased and healthy regions of pediatric SGS patients respond differently to TGF-ß1 stimulation, and SB525334 has the superior potential for subglottic stenosis treatment by simultaneously modulating TGF-ß and Hippo signaling pathways. LEVEL OF EVIDENCE: NA Laryngoscope, 134:287-296, 2024.

Cyr61 delivery promotes angiogenesis during bone fracture repair.

Compromised vascular supply and insufficient neovascularization impede bone repair, increasing risk of non-union. Cyr61, Cysteine-rich angiogenic inducer of 61kD (also known as CCN1), is a matricellular growth factor that is regulated by mechanical cues during fracture repair. Here, we map the distribution of endogenous Cyr61 during bone repair and evaluate the effects of recombinant Cyr61 delivery on vascularized bone regeneration. In vitro, Cyr61 treatment did not alter chondrogenesis or osteogenic gene expression, but significantly enhanced angiogenesis. In a mouse femoral fracture model, Cyr61 delivery did not alter cartilage or bone formation, but accelerated neovascularization during fracture repair. Early initiation of ambulatory mechanical loading disrupted Cyr61-induced neovascularization. Together, these data indicate that Cyr61 delivery can enhance angiogenesis during bone repair, particularly for fractures with stable fixation, and may have therapeutic potential for fractures with limited blood vessel supply.

YAP and TAZ couple osteoblast precursor mobilization to angiogenesis and mechanoregulation in murine bone development.

Fetal bone development occurs through the conversion of avascular cartilage to vascularized bone at the growth plate. This requires coordinated mobilization of osteoblast precursors with blood vessels. In adult bone, vessel-adjacent osteoblast precursors are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Here, we show that the mechanoresponsive transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) spatially couple osteoblast precursor mobilization to angiogenesis, regulate vascular morphogenesis to control cartilage remodeling, and mediate mechanoregulation of embryonic murine osteogenesis. Mechanistically, YAP and TAZ regulate a subset of osteoblast-lineage cells, identified by single-cell RNA sequencing as vessel-associated osteoblast precursors, which regulate transcriptional programs that direct blood vessel invasion through collagen-integrin interactions and Cxcl12. Functionally, in 3D human cell co-culture, CXCL12 treatment rescues angiogenesis impaired by stromal cell YAP/TAZ depletion. Together, these data establish functions of the vessel-associated osteoblast precursors in bone development.

Carbon nanotubes as a novel tool for vaccination against infectious diseases and cancer.

Due to their unusual properties, carbon nanotubes have been extensively employed in electronics, nanotechnology and optics, amongst other. More recently, they have also been used as vehicles for drug and antigen delivery, the latter being a novel immunization strategy against infectious diseases and cancer. Here we discuss the potential of carbon nanotubes as an antigen delivery tool and suggest further directions in the field of vaccination.

"Zero-dimensional" single-walled carbon nanotubes.

The shorter, the more dispersible: An iterative, emulsion-based shortening technique has been used to reduce the length of single-walled carbon nanotubes (SWNTs) to the same order of magnitude as their diameter (ca. 1 nm), thus achieving an effectively "zero-dimensional" structure with improved dispersibility and, after hydroxylation, long-term water solubility. Finally, zero-dimensional SWNTs were positively identified using mass spectrometry for the first time.

Breathing room: Toward next-generation tracheal engineering.

The creation of an engineered trachea with robust phenotype and sufficient mechanical properties for clinical application remains a challenge. In their work, Tang et al.1 propose a stacked approach of alternating cartilaginous and fibrous rings to form a tracheal segment, which integrated and retain patency in rabbits for 8 weeks.

4D bioprinted self-folding scaffolds enhance cartilage formation in the engineering of trachea.

Trachea defects that required surgical interventions are increasing in number in the recent years, especially for pediatric patients. However, current gold standards, such as biological grafts and synthetic prothesis, do not represent an effective solution, due to the lack of mimicry and regeneration capability. Bioprinting is a cutting-edge approach for the fabrication of biomimetic scaffold to empower tissue engineering toward trachea replacement. In this study, we developed a self-folding gelatin-based bilayer scaffold for trachea engineering, exploiting the 4D bioprinting approach, namely the fabrication of dynamic scaffolds, able to shape morph in a predefined way after the application of an environmental stimulus. Indeed, starting form a 2D flat position, upon hydration, this scaffold forms a closed tubular structure. An analytical model, based on Timoshenko's beam thermostats, was developed, and validated to predict the radius of curvature of the scaffold according to the material properties and the scaffold geometry. The 4D bioprinted structure was tested with airway fibroblast, lung endothelial cells and ear chondral progenitor cells (eCPCs) toward the development of a tissue engineered trachea. Cells were seeded on the scaffold in its initial flat position, maintained their position after the scaffold actuation and proliferated over or inside it. The ability of eCPCs to differentiate towards mature cartialge was evaluated. Interestingly, real-time PCR revealed that differentiating eCPCs on the 4D bioprinted scaffold promote healthy cartilage formation, if compared with eCPCs cultured on 2D static scaffold. Thus, eCPCs can perceive scaffold folding and its final curvature and to react to it, towards the formation of mature cartilage for the airway.

Amelioration of Subglottic Stenosis by Antimicrobial Peptide Eluting Endotracheal Tubes.

Introduction: Pediatric subglottic stenosis (SGS) results from prolonged intubation where scar tissue leads to airway narrowing that requires invasive surgery. We have recently discovered that modulating the laryngotracheal microbiome can prevent SGS. Herein, we show how our patent-pending antimicrobial peptide-eluting endotracheal tube (AMP-ET) effectively modulates the local airway microbiota resulting in reduced inflammation and stenosis resolution. Materials and Methods: We fabricated mouse-sized ETs coated with a polymeric AMP-eluting layer, quantified AMP release over 10 days, and validated bactericidal activity for both planktonic and biofilm-resident bacteria against Staphylococcus aureus and Pseudomonas aeruginosa. Ex vivo testing: we inserted AMP-ETs and ET controls into excised laryngotracheal complexes (LTCs) of C57BL/6 mice and assessed biofilm formation after 24 h. In vivo testing: AMP-ETs and ET controls were inserted in sham or SGS-induced LTCs, which were then implanted subcutaneously in receptor mice, and assessed for immune response and SGS severity after 7 days. Results: We achieved reproducible, linear AMP release at 1.16 µg/day resulting in strong bacterial inhibition in vitro and ex vivo. In vivo, SGS-induced LTCs exhibited a thickened scar tissue typical of stenosis, while the use of AMP-ETs abrogated stenosis. Notably, SGS airways exhibited high infiltration of T cells and macrophages, which was reversed with AMP-ET treatment. This suggests that by modulating the microbiome, AMP-ETs reduce macrophage activation and antigen specific T cell responses resolving stenosis progression. Conclusion: We developed an AMP-ET platform that reduces T cell and macrophage responses and reduces SGS in vivo via airway microbiome modulation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00769-9.

YAP and TAZ couple osteoblast precursor mobilization to angiogenesis and mechanoregulated bone development.

Endochondral ossification requires coordinated mobilization of osteoblast precursors with blood vessels. During adult bone homeostasis, vessel adjacent osteoblast precursors respond to and are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Previously, we found that deletion of the mechanoresponsive transcriptional regulators, YAP and TAZ, from Osterix-expressing osteoblast precursors and their progeny caused perinatal lethality. Here, we show that embryonic YAP/TAZ signaling couples vessel-associated osteoblast precursor mobilization to angiogenesis in developing long bones. Osterix-conditional YAP/TAZ deletion impaired endochondral ossification in the primary ossification center but not intramembranous osteogenesis in the bone collar. Single-cell RNA sequencing revealed YAP/TAZ regulation of the angiogenic chemokine, Cxcl12, which was expressed uniquely in vessel-associated osteoblast precursors. YAP/TAZ signaling spatially coupled osteoblast precursors to blood vessels and regulated vascular morphogenesis and vessel barrier function. Further, YAP/TAZ signaling regulated vascular loop morphogenesis at the chondro-osseous junction to control hypertrophic growth plate remodeling. In human cells, mesenchymal stromal cell co-culture promoted 3D vascular network formation, which was impaired by stromal cell YAP/TAZ depletion, but rescued by recombinant CXCL12 treatment. Lastly, YAP and TAZ mediated mechanotransduction for load-induced osteogenesis in embryonic bone.