Quantifying mechanical forces during vertebrate morphogenesis

Morphogenesis requires embryonic cells to generate forces and perform mechanical work to shape their tissues. Incorrect functioning of these force fields can lead to congenital malformations. Understanding these dynamic processes requires the quantification and profiling of three-dimensional mechanics during evolving vertebrate morphogenesis. Here we describe elastic spring-like force sensors with micrometre-level resolution, fabricated by intravital three-dimensional bioprinting directly in the closing neural tubes of growing chicken embryos. Integration of calibrated sensor read-outs with computational mechanical modelling allows direct quantification of the forces and work performed by the embryonic tissues. As they displace towards the embryonic midline, the two halves of the closing neural tube reach a compression of over a hundred nano-newtons during neural fold apposition. Pharmacological inhibition of Rho-associated kinase to decrease the pro-closure force shows the existence of active anti-closure forces, which progressively widen the neural tube and must be overcome to achieve neural tube closure. Overall, our approach and findings highlight the intricate interplay between mechanical forces and tissue morphogenesis

Engineering a 3D in vitro model of human skeletal muscle at the single fiber scale

The reproduction of reliable in vitro models of human skeletal muscle is made harder by the intrinsic 3D structural complexity of this tissue. Here we coupled engineered hydrogel with 3D structural cues and specific mechanical properties to derive human 3D muscle constructs ("myobundles") at the scale of single fibers, by using primary myoblasts or myoblasts derived from embryonic stem cells. To this aim, cell culture was performed in confined, laminin-coated micrometric channels obtained inside a 3D hydrogel characterized by the optimal stiffness for skeletal muscle myogenesis. Primary myoblasts cultured in our 3D culture system were able to undergo myotube differentiation and maturation, as demonstrated by the proper expression and localization of key components of the sarcomere and sarcolemma. Such approach allowed the generation of human myobundles of ~10 mm in length and ~120 \u3bcm in diameter, showing spontaneous contraction 7 days after cell seeding. Transcriptome analyses showed higher similarity between 3D myobundles and skeletal signature, compared to that found between 2D myotubes and skeletal muscle, mainly resulting from expression in 3D myobundles of categories of genes involved in skeletal muscle maturation, including extracellular matrix organization. Moreover, imaging analyses confirmed that structured 3D culture system was conducive to differentiation/maturation also when using myoblasts derived from embryonic stem cells. In conclusion, our structured 3D model is a promising tool for modelling human skeletal muscle in healthy and diseases conditions

Customized bioreactor enables the production of 3D diaphragmatic constructs influencing matrix remodeling and fibroblast overgrowth

The production of skeletal muscle constructs useful for replacing large defects in vivo, such as in congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents major limitations, such as hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the in vitro generation of diaphragmatic-like tissues as a proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A customized bioreactor for diaphragmatic muscle was designed to control mechanical stimulation and promote radial stretching during the construct engineering. In vitro tests demonstrated that both ECM remodeling and fibroblast overgrowth were positively influenced by the bioreactor culture. Mechanically stimulated constructs also increased tissue maturation, with the formation of new oriented and aligned muscle fibers. Moreover, after in vivo orthotopic implantation in a surgical CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers and hernia recurrence did not occur, suggesting the value of this approach for treating diaphragm defects

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape

Identification of a deep intronic mutation in the COL6A2 gene by a novel custom oligonucleotide CGH array designed to explore allelic and genetic heterogeneity in collagen VI-related myopathies

BACKGROUND: Molecular characterization of collagen-VI related myopathies currently relies on standard sequencing, which yields a detection rate approximating 75-79% in Ullrich congenital muscular dystrophy (UCMD) and 60-65% in Bethlem myopathy (BM) patients as PCR-based techniques tend to miss gross genomic rearrangements as well as copy number variations (CNVs) in both the coding sequence and intronic regions. METHODS: We have designed a custom oligonucleotide CGH array in order to investigate the presence of CNVs in the coding and non-coding regions of COL6A1, A2, A3, A5 and A6 genes and a group of genes functionally related to collagen VI. A cohort of 12 patients with UCMD/BM negative at sequencing analysis and 2 subjects carrying a single COL6 mutation whose clinical phenotype was not explicable by inheritance were selected and the occurrence of allelic and genetic heterogeneity explored. RESULTS: A deletion within intron 1A of the COL6A2 gene, occurring in compound heterozygosity with a small deletion in exon 28, previously detected by routine sequencing, was identified in a BM patient. RNA studies showed monoallelic transcription of the COL6A2 gene, thus elucidating the functional effect of the intronic deletion. No pathogenic mutations were identified in the remaining analyzed patients, either within COL6A genes, or in genes functionally related to collagen VI. CONCLUSIONS: Our custom CGH array may represent a useful complementary diagnostic tool, especially in recessive forms of the disease, when only one mutant allele is detected by standard sequencing. The intronic deletion we identified represents the first example of a pure intronic mutation in COL6A genes

Autophagy is defective in collagen VI muscular dystrophies and its reactivation rescues myofiber degeneration

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Role of collagen VI in skeletal muscle homeostasis

Collagen VI (ColVI) is an extracellular matrix protein forming a microfilamentous network in various tissues, and composed by three chains, α1(VI), α2(VI) and α3(VI), encoded by separate genes. Mutations of ColVI genes in humans cause various muscle diseases, including Bethlem Myopathy and Ullrich Congenital Muscular Dystrophy (UCMD). Mice lacking ColVI (Col6a1-/-) display a myopathic phenotype with mitochondrial dysfunction and spontaneous apoptosis of muscle fibers. Analysis of muscle biopsies and primary cultures of UCMD patients revealed a similar phenotype, which could be normalized by treatment with cyclosporin A or its non-immunosuppressive derivatives. During the first two years of my PhD work, I focused on studies aimed at elucidating collagen VI pathomolecular defects in human disorders, characterizing three novel ColVI chains in humans and mice and understanding molecular pathways underlying the phenotype of Col6a1-/- mice. Considering the remarkable apoptotic phenotype displayed by Col6a1-/- and UCMD myoblasts, and persuaded by the regenerative effect of cyclosporin A treatment in UCMD patients, during the second half of my PhD work I started investigating muscle regeneration and satellite cell (SC) activity in the Col6a1-/- mouse model. SCs are an adult stem cell population of skeletal muscle, representing the main player in skeletal muscle regeneration. Under physiological condition, I found that Col6a1-/- mice display an altered regenerative activity when compared to wild-type mice. In order to investigate further muscle regeneration capability of Col6a1-/- mice, I analyzed the regenerative response after muscle injury, induced either by cardiotoxin injection or by muscle training by voluntary exercise on running wheels. Light microscopy showed that after repeated cardiotoxin injury Col6a1-/- mice lose the ability to properly regenerate muscles, compared to wild type mice. Further studies, using Pax7 as a marker for SCs, revealed that in Col6a1-/- muscles SCs are able to complete muscle regeneration, but are unable to expand and maintain their pool. Interestingly, during the first stages of regeneration wild-type muscles showed a marked increase of ColVI deposition in the regenerating area, where Pax7-positive cells were found to proliferate extensively. In addition, almost all Pax7-positive cells were found in close contact with ColVI, and some of these cells were totally surrounded by the protein. Moreover, apoptotic nuclei were found to be strongly increased in Col6a1–/– muscles during starting events of regeneration, when compared to both untreated Col6a1–/– and cardiotoxin-treated wild-type muscles. In order to perform further studies on SC activity, I set up an in vitro experimental system with single myofiber cultures derived from wild-type and Col6a1–/– EDL muscles. These in vitro studies showed that wild-type SCs doubled during myofiber culture and gave origin both to activated cells (Pax7+MyoD+) and cell returned to the quiescence state (Pax7+MyoD-). Conversely, the number of quiescent Pax7-positive cells per fiber was strongly reduced in Col6a1–/– cultures compared to both wild-type cultures and freshly isolated Col6a1–/– myofibers. Altogether, these in vivo and in vitro studies strongly suggest a new role for ColVI in skeletal muscle regeneration and indicate that in Col6a1–/– muscles SCs have an impaired ability to expand and maintain the stem cell pool. We are currently investigating the mechanism through which ColVI signals are transduced in SCs in vivo and in vitro.Il collagene Vi è una proteina della matrice extracellulare presente in diversi tessuti e costituito da tre catene proteiche α1(VI), α2(VI) and α3(VI), codificate da tre geni distinti. Mutazioni a carico dei geni codificanti il collagene VI causano nell'uomo una serie di disordini muscolari, tra cui la miopatia di Bethlem (BM) e la distrofia congenita muscolare di Ullrich (UCMD). I topi privi di collagene VI (Col6a1-/-) presentano un fenotipo miopatico associato a disfunzione mitocondriale ed apoptosi spontanea delle fibre muscolari. L'analisi delle biopsie muscolari e delle colture primarie di pazienti affetti da UCMD rivela un fenotipo simile a quello osservato nei topi Col6a1-/-. Il trattamento farmacologico con ciclosporina A e i suoi derivati non immunosoppressivi permette il recupero del fenotipo mitocondriale ed apoptotico. Durante i primi due anni del mio lavoro di dottorato mi sono occupata di una serie di progetti con lo scopo di chiarire i difetti patomolecolari del collagene VI nelle patologie umane, di caratterizzare tre nuove catene del collagene VI nell'uomo e nel topo e di chiarire gli eventi molecolari legati al fenotipo riscontrato nei topi privi di collagene VI. Durante l'ultimo anno del mio dottorato ho avviato uno studio sulla rigenerazione muscolare e l'attività delle cellule satelliti nei topi Col6a1-/-. In condizioni fisiologiche i topi privi di collagene VI presentano un'alterata attività rigenerativa dei muscoli scheletrici. A seguito di danno acuto, mediante cardiotossina, o cronico, dopo esercizio volontario, i topi Col6a1-/- hanno evidenziato una riduzione della capacità rigenerativa dei muscoli TA ed EDL. Inoltre, le analisi di immunofluorescenza per Pax7, noto marcatore delle cellule satelliti, ha evidenziato l'incapacità di espansione e mantenimento delle cellule Pax7-positive nei topi Col6a1 a seguito dei danni. Durante le prime fasi di rigenerazione, nei muscoli dei topi selvatici il collagene VI appare maggiormente depositato in matrice ed in stretto contatto con le cellule Pax7-positive. Inoltre, l'analisi dell'apoptosi ha mostrato un forte incremento delle cellule apoptotiche nei muscoli in fase rigenerativa dei topi Col6a1-/-, sia rispetto ai muscoli sevatici in fase di rigenerazione che ai muscoli Col6a1-/- in condizioni fisiologiche. Con lo scopo di studiare più in dettaglio l'attività delle cellule satelliti, ho allestito colture di singole fibre isolate da EDL di topi selvatici e privi di collagene VI. Le analisi condotte hanno evidenziato che, dopo 48 ore di coltura, il numero delle cellule satelliti quiescenti per fibra dei topi Col6a1-/- risulta inferiore rispetto a quello selvatico. In conclusione, questi studi suggeriscono che il collagene VI svolga un ruolo importante durante la rigenerazione muscolare e che le cellule satelliti in assenza della proteina possiedono una minore abilità al mantenimento del pool staminale

Decellularized Tissue for Muscle Regeneration

Several acquired or congenital pathological conditions can affect skeletal muscle leading to volumetric muscle loss (VML), i.e., an irreversible loss of muscle mass and function. Decellularized tissues are natural scaffolds derived from tissues or organs, in which the cellular and nuclear contents are eliminated, but the tridimensional (3D) structure and composition of the extracellular matrix (ECM) are preserved. Such scaffolds retain biological activity, are biocompatible and do not show immune rejection upon allogeneic or xenogeneic transplantation. An increase number of reports suggest that decellularized tissues/organs are promising candidates for clinical application in patients affected by VML. Here we explore the different strategies used to generate decellularized matrix and their therapeutic outcome when applied to treat VML conditions, both in patients and in animal models. The wide variety of VML models, source of tissue and methods of decellularization have led to discrepant results. Our review study evaluates the biological and clinical significance of reported studies, with the final aim to clarify the main aspects that should be taken into consideration for the future application of decellularized tissues in the treatment of VML conditions