Tissue stiffening promotes keratinocyte proliferation through activation of epidermal growth factor signaling.

Tissue biomechanics regulate a wide range of cellular functions, but the influences on epidermal homeostasis and repair remain unclear. Here, we examined the role of extracellular matrix stiffness on human keratinocyte behavior using elastomeric substrates with defined mechanical properties. Increased matrix stiffness beyond normal physiologic levels promoted keratinocyte proliferation but did not alter the ability to self-renew or terminally differentiate. Activation of epidermal growth factor (EGF) signaling mediated the proliferative response to matrix stiffness and depended on focal adhesion assembly and cytoskeletal tension. Comparison of normal skin with keloid scar tissue further revealed an upregulation of EGF signaling within the epidermis of stiffened scar tissue. We conclude that matrix stiffness regulates keratinocyte proliferation independently of changes in cell fate and is mediated by EGF signaling. These findings provide mechanistic insights into how keratinocytes sense and respond to their mechanical environment, and suggest that matrix biomechanics may play a role in the pathogenesis keloid scar formation.

Effect of radiation-induced damage of trabecular bone tissue evaluated using indentation and digital volume correlation.

Exposure to X-ray radiation for an extended amount of time can cause damage to the bone tissue and therefore affect its mechanical properties. Specifically, high-resolution X-ray Computed Tomography (XCT), in both synchrotron and lab-based systems, has been employed extensively for evaluating bone micro-to-nano architecture. However, to date, it is still unclear how long exposures to X-ray radiation affect the mechanical properties of trabecular bone, particularly in relation to lab-XCT systems. Indentation has been widely used to identify local mechanical properties such as hardness and elastic modulus of bone and other biological tissues. The purpose of this study is therefore, to use indentation and XCT-based investigative tools such as digital volume correlation (DVC) to assess the microdamage induced by long exposure of trabecular bone tissue to X-ray radiation and how this affects its local mechanical properties. Trabecular bone specimens were indented before and after X-ray exposures of 33 and 66 h, where variation of elastic modulus was evaluated at every stage. The resulting elastic modulus was decreased, and micro-cracks appeared in the specimens after the first long X-ray exposure and crack formation increased after the second exposure. High strain concentration around the damaged tissue exceeding 1% was also observed from DVC analysis. The outcomes of this study show the importance of designing appropriate XCT-based experiments in lab systems to avoid degradation of the bone tissue mechanical properties due to radiation and these results will help to inform future studies that require long X-ray exposure for in situ experiments or generation of reliable subject-specific computational models.

P2X7 Purinoceptor Affects Ectopic Calcification of Dystrophic Muscles.

Ectopic calcification (EC) of myofibers is a pathological feature of muscle damage in Duchenne muscular dystrophy (DMD). Mineralisation of muscle tissue occurs concomitantly with macrophage infiltration, suggesting a link between ectopic mineral deposition and inflammation. One potential link is the P2X7 purinoceptor, a key trigger of inflammation, which is expressed on macrophages but also up-regulated in dystrophic muscle cells. To investigate the role of P2X7 in dystrophic calcification, we utilised the Dmd mdx-ßgeo dystrophin-null mouse model of DMD crossed with a global P2X7 knockout (P2rx7 -/- ) or with our novel P2X7 knockin-knockout mouse (P2x7 KiKo ), which expresses P2X7 in macrophages but not muscle cells. Total loss of P2X7 increased EC, indicating that P2X7 overexpression is a protective mechanism against dystrophic mineralisation. Given that muscle-specific P2X7 ablation did not affect dystrophic EC, this underlined the role of P2X7 receptor expression on the inflammatory cells. Serum phosphate reflected dystrophic calcification, with the highest serum phosphate levels found in genotypes with the most ectopic mineral. To further investigate the underlying mechanisms, we measured phosphate release from cells in vitro, and found that dystrophic myoblasts released less phosphate than non-dystrophic cells. Treatment with P2X7 antagonists increased phosphate release from both dystrophic and control myoblasts indicating that muscle cells are a potential source of secreted phosphate while macrophages protect against ectopic mineralisation. Treatment of cells with high phosphate media engendered mineral deposition, which was decreased in the presence of the P2X7 agonist BzATP, particularly in cultures of dystrophic cells, further supporting a protective role for P2X7 against ectopic mineralisation in dystrophic muscle.

Dental cementum virtual histology of Neanderthal teeth from Krapina (Croatia, 130-120 kyr): an informed estimate of age, sex and adult stressors.

The evolution of modern human reproductive scheduling is an aspect of our life history that remains vastly uncomprehended. The present work aims to address this gap by validating a non-destructive cutting-edge methodology to infer adult life-history events on modern teeth with known life history and then applying it to fossil specimens. We use phase-contrast synchrotron X-ray microtomography to visualize the dental cementum of 21 specimens: nine contemporary humans; 10 Neanderthals from Krapina (Croatia, 130-120 kyr); one Neolithic Homo sapiens from Ajmana (Serbia); and one Mesolithic H. sapiens from Vlasac (Serbia). We were able to correctly detect and time (root mean square error = 2.1 years; R2 = 0.98) all reproductive (menarche, parturition, menopause) and other physiologically impactful events in the modern sample. Nonetheless, we could not distinguish between the causes of the events detected. For the fossil specimens, we estimated age at death and age at occurrence of biologically significant events. Finally, we performed an exploratory analysis regarding possible sexual dimorphism in dental cementum microstructure, which allowed us to correctly infer the sex of the Neolithic specimen, for which the true value was known via DNA analysis.

Biomimetic generation of the strongest known biomaterial found in limpet tooth.

The biomaterial with the highest known tensile strength is a unique composite of chitin and goethite (α-FeO(OH)) present in teeth from the Common Limpet (Patella vulgata). A biomimetic based on limpet tooth, with corresponding high-performance mechanical properties is highly desirable. Here we report on the replication of limpet tooth developmental processes ex vivo, where isolated limpet tissue and cells in culture generate new biomimetic structures. Transcriptomic analysis of each developmental stage of the radula, the organ from which limpet teeth originate, identifies sequential changes in expression of genes related to chitin and iron processing. We quantify iron and chitin metabolic processes in the radula and grow isolated radula cells in vitro. Bioinspired material can be developed with electrospun chitin mineralised by conditioned media from cultured radula cells. Our results inform molecular processes behind the generation of limpet tooth and establish a platform for development of a novel biomimetic with comparable properties.

Micromechanical evaluation of cortical bone using in situ XCT indentation and digital volume correlation.

The overall mechanical behaviour of cortical bone is strongly dependant on its microstructure. X-ray computed tomography (XCT) has been widely used to identify the microstructural morphology of cortical tissue (i.e. pore network, Haversian and Volkmann's canals). However, the connection between microstructure and mechanics of cortical bone during plastic deformation is unclear. Hence, the purpose of this study is to provide an in-depth evaluation of the interplay of plastic strain building up in relation to changes in the canal network for cortical bone tissue. In situ step-wise XCT indentation was used to introduce a localised load on the surface of the tissue and digital volume correlation (DVC) was employed to assess the three-dimensional (3D) full-field plastic strain distribution in proximity of the indent. It was observed that regions adjacent to the imprint were under tensile strain, whereas the volume underneath experienced compressive strain. Canal loss and disruption was detected in regions of higher compressive strains exceeding -20000 µÎµ and crack formation occurred in specimens where Haversian canals were running parallel to the indentation tip. The results of this study outline the relationship between the micromechanical and structural behaviour of cortical bone during plastic deformation, providing information on cortical tissue fracture pathways.

Time-resolved in situ synchrotron-microCT: 4D deformation of bone and bone analogues using digital volume correlation.

Digital volume correlation (DVC) in combination with high-resolution micro-computed tomography (microCT) imaging and in situ mechanical testing is gaining popularity for quantifying 3D full-field strains in bone and biomaterials. However, traditional in situ time-lapsed (i.e., interrupted) mechanical testing cannot fully capture the dynamic strain mechanisms in viscoelastic biological materials. The aim of this study was to investigate the time-resolved deformation of bone structures and analogues via continuous in situ synchrotron-radiation microCT (SR-microCT) compression and DVC to gain a better insight into their structure-function relationships. Fast SR-microCT imaging enabled the deformation behaviour to be captured with high temporal and spatial resolution. Time-resolved DVC highlighted the relationship between local strains and damage initiation and progression in the different biostructures undergoing plastic deformation, bending and/or buckling of their main microstructural elements. The results showed that SR-microCT continuous mechanical testing complemented and enhanced the information obtained from time-lapsed testing, which may underestimate the 3D strain magnitudes as a result of the stress relaxation occurring in between steps before image acquisition in porous biomaterials. Altogether, the findings of this study highlight the importance of time-resolved in situ experiments to fully characterise the time-dependent mechanical behaviour of biological tissues and biomaterials and to further explore their micromechanics under physiologically relevant conditions. STATEMENT OF SIGNIFICANCE: Time-resolved synchrotron X-ray tomography in combination with in situ mechanical testing provided the first four-dimensional analysis of the mechanical deformation of bone and bone analogues. To unravel the interplay of damage initiation and progression with local deformation, digital volume correlation was used to map the local strain field while microstructural changes were tracked with high temporal and spatial resolution. The results highlighted the importance of fast imaging and time-resolved in situ experiments to capture the real deformation of complex porous materials to fully characterize the local strain-damage relationship. The findings are notably improving the understanding of time-dependent mechanical behaviour of bone tissue, with the potential to be extend to highly viscoelastic biomaterials and soft tissues.

Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly.

BACKGROUND: Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as "coatopathies". METHODS: Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (ß-COP). To investigate Family 1's splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2's missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2's mutation. RESULTS: We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between ß-COP and ß'-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant ß-COP, with the mutant protein being retarded in the Golgi. CONCLUSIONS: This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.

Low-cycle full-field residual strains in cortical bone and their influence on tissue fracture evaluated via in situ stepwise and continuous X-ray computed tomography.

As a composite material, the mechanical properties of bone are highly dependent on its hierarchical organisation, thus, macroscopic mechanical properties are dictated by local phenomena, such as microdamage resulting from repetitive cyclic loading of daily activities. Such microdamage is associated with plastic deformation and appears as a gradual accumulation of residual strains. The aim of this study is to investigate local residual strains in cortical bone tissue following compressive cyclic loading, using in situ X-ray computed tomography (XCT) and digital volume correlation (DVC) to provide a deeper insight on the three-dimensional (3D) relationship between residual strain accumulation, cortical bone microstructure and failure patterns. Through a progressive in situ XCT loading-unloading scheme, localisation of local residual strains was observed in highly compressed regions. In addition, a multi-scale in situ XCT cyclic test highlighted the differences on residual strain distribution at the microscale and tissue level, where high strains were observed in regions with the thinnest vascular canals and predicted the failure location following overloading. Finally, through a continuous in situ XCT compression test of cycled specimens, the full-field strain evolution and failure pattern indicated the reduced ability of bone to plastically deform after damage accumulation due to high number of cyclic loads. Altogether, the novel experimental methods employed in this study, combining high-resolution in situ XCT mechanics and DVC, showed a great potential to investigate 3D full-field residual strain development under repetitive loading and its complex interaction with bone microstructure, microdamage and fracture.

Evaluation of X-ray tomography contrast agents: A review of production, protocols, and biological applications.

X-ray computed tomography is a strong tool that finds many applications both in medical applications and in the investigation of biological and nonbiological samples. In the clinics, X-ray tomography is widely used for diagnostic purposes whose three-dimensional imaging in high resolution helps physicians to obtain detailed image of investigated regions. Researchers in biological sciences and engineering use X-ray tomography because it is a nondestructive method to assess the structure of their samples. In both medical and biological applications, visualization of soft tissues and structures requires special treatment, in which special contrast agents are used. In this detailed report, molecule-based and nanoparticle-based contrast agents used in biological applications to enhance the image quality were compiled and reported. Special contrast agent applications and protocols to enhance the contrast for the biological applications and works to develop nanoparticle contrast agents to enhance the contrast for targeted drug delivery and general imaging applications were also assessed and listed.

In vivo delivery of VEGF RNA and protein to increase osteogenesis and intraosseous angiogenesis.

Deficient bone vasculature is a key component in pathological conditions ranging from developmental skeletal abnormalities to impaired bone repair. Vascularisation is dependent upon vascular endothelial growth factor (VEGF), which drives both angiogenesis and osteogenesis. The aim of this study was to examine the efficacy of blood vessel and bone formation following transfection with VEGF RNA or delivery of recombinant human VEGF165 protein (rhVEGF165) across in vitro and in vivo model systems. To quantify blood vessels within bone, an innovative approach was developed using high-resolution X-ray computed tomography (XCT) to generate quantifiable three-dimensional reconstructions. Application of rhVEGF165 enhanced osteogenesis, as evidenced by increased human osteoblast-like MG-63 cell proliferation in vitro and calvarial bone thickness following in vivo administration. In contrast, transfection with VEGF RNA triggered angiogenic effects by promoting VEGF protein secretion from MG-63VEGF165 cells in vitro, which resulted in significantly increased angiogenesis in the chorioallantoic (CAM) assay in ovo. Furthermore, direct transfection of bone with VEGF RNA in vivo increased intraosseous vascular branching. This study demonstrates the importance of continuous supply as opposed to a single high dose of VEGF on angiogenesis and osteogenesis and, illustrates the potential of XCT in delineating in 3D, blood vessel connectivity in bone.

Multiscale hierarchical bioresorbable scaffolds for the regeneration of tendons and ligaments.

Lesions of tendons and ligaments account for over 40% of the musculoskeletal lesions. Surgical techniques and materials for repair and regeneration are currently not satisfactory. The high rate of post-operative complications and failures mainly relates to the technical difficulties in replicating the complex multiscale hierarchical structure and the mechanical properties of the native tendons and ligaments. With the aim of overcoming the limitations of non-biomimetic devices, we developed a hierarchical structure replicating the organization of tendons and ligaments. The scaffold consists of multiple bundles made of resorbable electrospun nanofibers of Poly-L-Lactic acid (PLLA) having tailored dimensions, wrapped in a sheath of nanofibers able to compact the construct. The bundles in turn consist of electrospun nanofibers with a preferential direction. High-resolution x-ray tomographic investigation at nanometer resolution confirmed that the morphology of the single bundles and of the entire scaffold replicated the hierarchical arrangement in the natural tendons and ligaments. To confirm that these structures could adequately restore tendons and ligaments, we measured the tensile stiffness, strength and toughness. The mechanical properties were in the range required to replace and repair tendons and ligaments. Furthermore, human fibroblasts were able to attach to the scaffolds and showed an increase in cell number, indicated by an increase in metabolic activity over time. Fibroblasts were preferentially aligned along the electrospun nanofibers. These encouraging in vitro results open the way for the next steps towards in vivo regeneration of tendons and ligaments.

Morphologically bioinspired hierarchical nylon 6,6 electrospun assembly recreating the structure and performance of tendons and ligaments.

Reconstructions of ruptured tendons and ligaments currently have dissatisfactory failure rate. Failures are mainly due to the mechanical mismatch of commercial implants with respect to the host tissue. In fact, it is crucial to replicate the morphology (hierarchical in nature) and mechanical response (highly-nonlinear) of natural tendons and ligaments. The aim of this study was to develop morphologically bioinspired hierarchical Nylon 6,6 electrospun assemblies recreating the structure and performance of tendons and ligaments. First, we built different electrospun bundles to find the optimal orientation of the nanofibers. A 2nd-level hierarchical assembly was fabricated with a dedicated process that allowed tightly joining the bundles one next to the other with an electrospun sheath, so as to improve the mechanical performance. Finally, a further hierarchical 3rd-level assembly was constructed by grouping several 2nd-level assemblies. The morphology of the different structures was assessed with scanning electron microscopy and high-resolution X-ray tomography, which allowed measuring the directionality of the nanofibers in the bundles and in the sheaths. The mechanical properties of the single bundles and of the 2nd-level assemblies were measured with tensile tests. The single bundles and the hierarchical assemblies showed morphology and directionality of the nanofibers similar to the tendons and ligaments. The strength and stiffness were comparable to that of tendons and ligaments. In conclusion, this work showed an innovative electrospinning production process to build nanofibrous Nylon 6,6 hierarchical assemblies which are suitable as future implantable devices and able to mimic the multiscale morphology and the biomechanical properties of tendons and ligaments.

Preservation of Bone Tissue Integrity with Temperature Control for In Situ SR-MicroCT Experiments.

Digital volume correlation (DVC), combined with in situ synchrotron microcomputed tomography (SR-microCT) mechanics, allows for 3D full-field strain measurement in bone at the tissue level. However, long exposures to SR radiation are known to induce bone damage, and reliable experimental protocols able to preserve tissue properties are still lacking. This study aims to propose a proof-of-concept methodology to retain bone tissue integrity, based on residual strain determination using DVC, by decreasing the environmental temperature during in situ SR-microCT testing. Compact and trabecular bone specimens underwent five consecutive full tomographic data collections either at room temperature or 0 °C. Lowering the temperature seemed to reduce microdamage in trabecular bone but had minimal effect on compact bone. A consistent temperature gradient was measured at each exposure period, and its prolonged effect over time may induce localised collagen denaturation and subsequent damage. DVC provided useful information on irradiation-induced microcrack initiation and propagation. Future work is necessary to apply these findings to in situ SR-microCT mechanical tests, and to establish protocols aiming to minimise the SR irradiation-induced damage of bone.

Tendon Fascicle-Inspired Nanofibrous Scaffold of Polylactic acid/Collagen with Enhanced 3D-Structure and Biomechanical Properties.

Surgical treatment of tendon lesions still yields unsatisfactory clinical outcomes. The use of bioresorbable scaffolds represents a way forward to improve tissue repair. Scaffolds for tendon reconstruction should have a structure mimicking that of the natural tendon, while providing adequate mechanical strength and stiffness. In this paper, electrospun nanofibers of two crosslinked PLLA/Collagen blends (PLLA/Coll-75/25, PLLA/Coll-50/50) were developed and then wrapped in bundles, where the nanofibers are predominantly aligned along the bundles. Bundle morphology was assessed via SEM and high-resolution x-ray computed tomography (XCT). The 0.4-micron resolution in XCT demonstrated a biomimetic morphology of the bundles for all compositions, with a predominant nanofiber alignment and some scatter (50-60% were within 12° from the axis of the bundle), similar to the tendon microstructure. Human fibroblasts seeded on the bundles had increased metabolic activity from day 7 to day 21 of culture. The stiffness, strength and toughness of the bundles are comparable to tendon fascicles, both in the as-spun condition and after crosslinking, with moderate loss of mechanical properties after ageing in PBS (7 and 14 days). PLLA/Coll-75/25 has more desirable mechanical properties such as stiffness and ductility, compared to the PLLA/Coll-50/50. This study confirms the potential to bioengineer tendon fascicles with enhanced 3D structure and biomechanical properties.

3D nanomechanical evaluations of dermal structures in skin.

Skin is a multilayered multiscale composite material with a range of mechanical and biochemical functions. The mechanical properties of dermis are important to understand in order to improve and compare on-going in vitro experiments to physiological conditions, especially as the mechanical properties of the dermis can play a crucial role in determining cell behaviour. Spatial and isotropy variations in dermal mechanics are thus critical in such understanding of complex skin structures. Atomic force microscopy (AFM) based indentation was used in this study to quantify the three dimensional mechanical properties of skin at nanoscale resolution over micrometre length scales. A range of preparation methods was examined and a mechanically non-evasive freeze sectioning followed by thawing method was found to be suitable for the AFM studies. Subsequent mechanical evaluations established macroscale isotropy of the dermis with the ground substance of the dermis dominating the mechanical response. Mechanical analysis was extended to show significant variation in the elastic modulus of the dermis between anatomical locations that suggest changes in the physiological environment influence local mechanical properties. Our results highlight dependence between an isotropic mechanical response of the dermal microenvironment at the nanoscale and anatomical location that define the variable mechanical behaviour of the dermis.

Type VI Collagen Regulates Dermal Matrix Assembly and Fibroblast Motility.

Type VI collagen is a nonfibrillar collagen expressed in many connective tissues and implicated in extracellular matrix (ECM) organization. We hypothesized that type VI collagen regulates matrix assembly and cell function within the dermis of the skin. In the present study we examined the expression pattern of type VI collagen in normal and wounded skin and investigated its specific function in new matrix deposition by human dermal fibroblasts. Type VI collagen was expressed throughout the dermis of intact human skin, at the expanding margins of human keloid samples, and in the granulation tissue of newly deposited ECM in a mouse model of wound healing. Generation of cell-derived matrices (CDMs) by human dermal fibroblasts with stable knockdown of COL6A1 revealed that type VI collagen-deficient matrices were significantly thinner and contained more aligned, thicker, and widely spaced fibers than CDMs produced by normal fibroblasts. In addition, there was significantly less total collagen and sulfated proteoglycans present in the type VI collagen-depleted matrices. Normal fibroblasts cultured on de-cellularized CDMs lacking type VI collagen displayed increased cell spreading, migration speed, and persistence. Taken together, these findings indicate that type VI collagen is a key regulator of dermal matrix assembly, composition, and fibroblast behavior and may play an important role in wound healing and tissue regeneration.