Type VI collagen is a non-fibrillar collagen expressed in many connective tissues and implicated in extracellular matrix (ECM) organisation. 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 fibres 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-cellularised 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 behaviour and may play an important role in wound healing and tissue regeneration.Journal of Investigative Dermatology accepted article preview online, 09 September 2015. doi:10.1038/jid.2015.352.

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.

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.

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.

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.

Summary The regeneration of injured tendons and ligaments is challenging because the scaffolds needs proper mechanical properties and a biomimetic morphology. In particular, the morphological arrangement of scaffolds is a key point to drive the cells growth to properly regenerate the collagen extracellular matrix. Electrospinning is a promising technique to produce hierarchically structured nanofibrous scaffolds able to guide cells in the regeneration of the injured tissue. Moreover, the dynamic stretching in bioreactors of electrospun scaffolds had demonstrated to speed up cell shape modifications in vitro. The aim of the present study was to combine different imaging techniques such as high‐resolution X‐ray tomography (XCT), scanning electron microscopy (SEM), fluorescence microscopy and histology to investigate if hierarchically structured poly (L‐lactic acid) and collagen electrospun scaffolds can induce morphological modifications in human fibroblasts, while cultured in static and dynamic conditions. After 7 days of parallel cultures, the results assessed that fibroblasts had proliferated on the external nanofibrous sheath of the static scaffolds, elongating themselves circumferentially. The dynamic cultures revealed a preferential axial orientation of fibroblasts growth on the external sheath. The aligned nanofibre bundles inside the hierarchical scaffolds instead, allowed a physiological distribution of the fibroblasts along the nanofibre direction. Inside the dynamic scaffolds, cells appeared thinner compared with the static counterpart. This study had demonstrated that hierarchically structured electrospun scaffolds can induce different fibroblasts morphological modifications during static and dynamic conditions, modifying their shape in the direction of the applied loads. Lay Description To enhance the regeneration of injured tendons and ligaments cells need to growth on dedicated structures (scaffolds) with mechanical properties and a fibrous morphology similar to the natural tissue. In particular, the morphological organisation of scaffolds is fundamental in leading cells to colonise them, regenerating the collagen extracellular matrix. Electrospinning is a promising technique to produce fibres with a similar to the human collagen fibres, suitable to design complex scaffolds able to guide cells in the reconstruction of the natural tissue. Moreover, it is well established that the cyclic stretching of these scaffolds inside dedicated systems called bioreactors, can speed up cells growth and their shape modification. The aim of the present study was to investigate how hierarchically structured electrospun scaffolds, made of resorbable material such as poly(L‐lactic acid) and collagen, could induce morphological changes in human fibroblasts, while cultured during static and dynamic conditions. These scaffolds were composed by an external electrospun membrane that grouped inside it a ring‐shaped bundle, made of axially aligned nanofibres, resembling the morphological arrangement of tendon and ligament tis...

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 3rdlevel 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.

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.