Healing of Tendon Defects Implanted with a Porous Collagen-GAG Matrix: Histological Evaluation

There is currently no method to restore normal function in tendon injuries that result in a gap. The objective of this study was to evaluate the early healing of tendon defects implanted with a porous collagen–glycosaminoglycan (CG) matrix, previously shown to facilitate the regeneration of dermis and peripheral nerve. A novel animal model that isolates the tendon defect site from surrounding tissue during healing was employed. This model used a silicone tube to entubulate the surgically produced tendon gap of 10 mm, allowing for the evaluation of the effects of the analog of extracellular matrix on healing of tendon, absent the influences of the external environment. The results showed that tendon stumps induced synthesis of a tissue cable inside the silicone tube in both the presence and absence of CG matrix. The presence of the CG matrix, however, altered the process of tendon healing. Tubes filled with CG matrix contained a significantly greater volume of tissue at the time periods of evaluation: 3, 6, and 12 weeks. Granulation tissue persisted for a longer period of time in the lesion site of CG-filled defects, and the amount of dense fibrous tissue increased continuously during the period of study in defects filled with CG matrix. In contrast, the amount of dense fibrous tissue decreased after 6 weeks in originally empty tubes. In tubes that did not contain the CG matrix, the new tissue consisted of dense aggregates of crimped fibers with a wavelength and fiber bundle thickness that were significantly shorter than those in normal tendon, and consistent with the type of scar that is the end result of repair of many connective tissues. Although, CG-filled tubes contained dense fibrous tissue by 12 weeks, the tissue had no crimp. The CG matrix may have prolonged the synthesis of granulation tissue and delayed or prevented the formation of scar.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63265/1/ten.1997.3.187.pd

Human adipose stem cells cell sheet constructs impact epidermal morphogenesis in full-thickness excisional wounds

Among the wide range of strategies to target skin repair/regeneration, tissue engineering (TE) with stem cells at the forefront, remains as the most promising route. Cell sheet (CS) engineering is herein proposed, taking advantage of particular cell-cell and cell-extracellular matrix (ECM) interactions and subsequent cellular milieu, to create 3D TE constructs to promote full-thickness skin wound regeneration. Human adipose derived stem cells (hASCs) CS were obtained within five days using both thermoresponsive and standard cell culture surfaces. hASCs-based constructs were then built by superimposing three CS and transplanted into full-thickness excisional mice skin wounds with delayed healing. Constructs obtained using thermoresponsive surfaces were more stable than the ones from standard cell culture surfaces due to the natural adhesive character of the respective CS. Both CS-generating strategies lead to prolonged hASCs engraftment, although no transdifferentiation phenomena were observed. Moreover, our findings suggest that the transplanted hASCs might be promoting neotissue vascularization and extensively influencing epidermal morphogenesis, mainly through paracrine actions with the resident cells. The thicker epidermis, with a higher degree of maturation characterized by the presence of rete ridges-like structures, as well as a significant number of hair follicles observed after transplantation of the constructs combining the CS obtained from the thermoresponsive surfaces, reinforced the assumptions of the influence of the transplanted hASCs and the importance of the higher stability of these constructs promoted by cohesive cell-cell and cell-ECM interactions. Overall, this study confirmed the potential of hASCs CS-based constructs to treat full-thickness excisional skin wounds and that their fabrication conditions impact different aspects of skin regeneration, such as neovascularisation, but mainly epidermal morphogenesis.We would like to thank Hospital da Prelada (Porto), in particular, to Dr. Paulo Costa for the lipoaspirates collection and for financial support by Skingineering (PTDC/SAU-OSM/099422/2008), Portuguese Foundation for Science and Technology (FCT) funded project. The research leading to these results has also received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under Grant Agreement No. REGPOT-CT2012-316331-POLARIS

Skin Regeneration in Adult Axolotls: A Blueprint for Scar-Free Healing in Vertebrates

While considerable progress has been made towards understanding the complex processes and pathways that regulate human wound healing, regenerative medicine has been unable to develop therapies that coax the natural wound environment to heal scar-free. The inability to induce perfect skin regeneration stems partly from our limited understanding of how scar-free healing occurs in a natural setting. Here we have investigated the wound repair process in adult axolotls and demonstrate that they are capable of perfectly repairing full thickness excisional wounds made on the flank. In the context of mammalian wound repair, our findings reveal a substantial reduction in hemostasis, reduced neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM) during scar-free healing. Additionally, we test the hypothesis that metamorphosis leads to scarring and instead show that terrestrial axolotls also heal scar-free, albeit at a slower rate. Analysis of newly forming dermal ECM suggests that low levels of fibronectin and high levels of tenascin-C promote regeneration in lieu of scarring. Lastly, a genetic analysis during wound healing comparing epidermis between aquatic and terrestrial axolotls suggests that matrix metalloproteinases may regulate the fibrotic response. Our findings outline a blueprint to understand the cellular and molecular mechanisms coordinating scar-free healing that will be useful towards elucidating new regenerative therapies targeting fibrosis and wound repair

A Cellular Potts Model simulating cell migration on and in matrix environments

Cell migration on and through extracellular matrix plays a critical role in a wide variety of physiological and pathological phenomena, and in scaffold-based tissue engineering. Migration is regulated by a number of extracellular matrix- or cell-derived biophysical parameters, such as matrix fiber orientation, gap size, and elasticity, or cell deformation, proteolysis, and adhesion. We here present an extended Cellular Potts Model (CPM) able to qualitatively and quantitatively describe cell migratory phenotype on both two-dimensional substrates and within three-dimensional environments, in a close comparison with experimental evidence. As distinct features of our approach, the cells are represented by compartmentalized discrete objects, differentiated in the nucleus and in the cytosolic region, while the extracellular matrix is composed of a fibrous mesh and of a homogeneous fluid. Our model provides a strong correlation of the directionality of migration with the topological ECM distribution and, further, a biphasic dependence of migration on the matrix density, and in part adhesion, in both two-dimensional and three-dimensional settings. Moreover, we demonstrate that the directional component of cell movement is strongly correlated with the topological distribution of the ECM fibrous network. In the three-dimensional networks, we also investigate the effects of the matrix mechanical microstructure, observing that, at a given distribution of fibers, cell motility has a subtle bimodal relation with the elasticity of the scaffold. Finally, cell locomotion requires deformation of the cell's nucleus and/or cell-derived proteolysis of steric fibrillar obstacles within rather rigid matrices characterized by small pores, not, however, for sufficiently large pores. In conclusion, we here propose a mathematical modeling approach that serves to characterize cell migration as a biological phenomen in health, disease and tissue engineering applications. The research that led to the present paper was partially supported by a grant of the group GNFM of INdA

Living Bacterial Sacrificial Porogens to Engineer Decellularized Porous Scaffolds

Decellularization and cellularization of organs have emerged as disruptive methods in tissue engineering and regenerative medicine. Porous hydrogel scaffolds have widespread applications in tissue engineering, regenerative medicine and drug discovery as viable tissue mimics. However, the existing hydrogel fabrication techniques suffer from limited control over pore interconnectivity, density and size, which leads to inefficient nutrient and oxygen transport to cells embedded in the scaffolds. Here, we demonstrated an innovative approach to develop a new platform for tissue engineered constructs using live bacteria as sacrificial porogens. E.coli were patterned and cultured in an interconnected three-dimensional (3D) hydrogel network. The growing bacteria created interconnected micropores and microchannels. Then, the scafold was decellularized, and bacteria were eliminated from the scaffold through lysing and washing steps. This 3D porous network method combined with bioprinting has the potential to be broadly applicable and compatible with tissue specific applications allowing seeding of stem cells and other cell types

3D printing strategies for peripheral nerve regeneration

After many decades of biomaterials research for peripheral nerve regeneration, a clinical product (the nerve guide), is emerging as a proven alternative for relatively short injury gaps. This review identifies aspects where 3D printing can assist in improving long-distance nerve guide regeneration strategies. These include (1) 3D printing of the customizable nerve guides, (2) fabrication of scaffolds that fill nerve guides, (3) 3D bioprinting of cells within a matrix/bioink into the nerve guide lumen and the (4) establishment of growth factor gradients along the length a nerve guide. The improving resolution of 3D printing technologies will be an important factor for peripheral nerve regeneration, as fascicular-like guiding structures provide one path to improved nerve guidance. The capability of 3D printing to manufacture complex structures from patient data based on existing medical imaging technologies is an exciting aspect that could eventually be applied to treating peripheral nerve injury. Ultimately, the goal of 3D printing in peripheral nerve regeneration is the automated fabrication, potentially customized for the patient, of structures within the nerve guide that significantly outperform the nerve autograft over large gap injuries.Griffith Health, School of MedicineNo Full Tex