Extremely strong and tough hydrogels as prospective candidates for tissue repair – A review

Ideal candidates for the repair of robust biological tissues should exhibit diverse features such as biocompatibility, strength, toughness, self-healing ability and a well-defined structure. Among the available biomaterials, hydrogels, as highly hydrated 3D-crosslinked polymeric networks, are promising for Tissue Engineering purposes as result of their high resemblance with native extracellular matrix. However, these polymeric structures often exhibit a poor mechanical behavior, hampering their use in load-bearing applications. During the last years, several efforts have been made to create new strategies and concepts to fabricate strong and tough hydrogels. Although it is already possible to shape the mechanical properties of artificial hydrogels to mimic biotissues, critical issues regarding, for instance, their biocompatibility and hierarchical structure are often neglected. Therefore, this review covers the structural and mechanical characteristics of the developed methodologies to toughen hydrogels, highlighting some pioneering efforts employed to combine the aforementioned properties in natural-based hydrogels.

Biomaterials for cartilage tissue engineering under mechanical stimulus

Tese de Doutoramento em Ciências (Especialidade de Física)

Chondrogenic potential of injectable κ-carrageenan hydrogel with encapsulated adipose stem cells for cartilage tissue-engineering applications

Due to the limited self-repair capacity of cartilage, regenerative medicine therapies for the treatment of cartilage defects must use a significant amount of cells, preferably applied using a hydrogel system that can promise their delivery and functionality at the specific site. This paper discusses the potential use of k-carrageenan hydrogels for the delivery of stem cells obt ained from adipose tissue in the treatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropic gelation met hod and human adipose stem cells (hASCs) were encapsulated in 1.5% w/v k-carrageenan solution at a cell density of 5  10 6 cells/ml. The results from the analysis of the cell-encapsulating hydrogels, cultured for up to 21 days, indicated that k-carrageenan hydrogels support the viability, proliferation and chondrogenic differentiation of hASCs. Additionally, the mec hanical analysis demonstrated an increase in stiffness and viscoelastic properties of k-carrageenan gels with their encapsulated cells with increasing time in culture with chondrogenic medium. These results allowed the conclusion that k-carrageenan exhibits properties t hat enable the in vitro functionality of encapsulated hASCs and thus may provide the basis for new successful approaches for the treatment of cartilage defects.

Modulation of the secretome of hBMSCs by tailoring the macromolecular gradient in hydrogels to generate tissue-to-tissue interfaces

Tissue-to-tissue interfaces are commonly present in all tissues exhibiting structural, biological and chemical gradients serving a wide range of physiological functions. These interfaces are responsible for mediation of load transfer between two adjacent tissues. They are also important structures in sustaining the cellular communications to retain tissueâ s functional integration and homeostasis. [1] All cells have the capacity to sense and respond to physical and chemical stimulus and when cultured in three-dimensional (3D) environments they tend to perform their function better than in two-dimensional (2D) environments. Spatial and temporal 3D gradient hydrogels better resemble the natural environment of cells in mimicking their extracellular matrix. [2] In this study we hypothesize that differential functional properties can be engineered by modulation of macromolecule gradients in a cell seeded threedimensional hydrogel system. Specifically, differential paracrine secretory profiles can be engineered using human Bone Marrow Stem Cells (hBMSCâ s). Hence, the specific objectives of this study are to: assemble the macromolecular gradient hydrogels to evaluate the suitablity for hBMSCâ s encapsulation by cellular viability and biofunctionality by assessing the paracrine secretion of hBMSCâ s over time. The gradient hydrogels solutions were prepared by blend of macromolecules in one solution such as hyaluronic (HA) acid and collagen (Col) at different ratios. The gradient hydrogels were fabricated into cylindrical silicon moulds with higher ratio solutions assembled at the bottom of the mould and adding the two solutions consecutively on top of each other. The labelling of the macromolecules was performed to confirm the gradient through fluorescence microscopy. Additionally, AFM was conducted to assess the gradient hydrogels stiffness. Gradient hydrogels characterization was performed by HA and Col degradation assay, degree of crosslinking and stability. hBMSCâ s at P3 were encapsulated into each batch solution at 106 cells/ml solution and gradient hydrogels were produced as previously described. The hBMSCâ s were observed under confocal microscopy to assess viability by Live/Dead® staining. Cellular behaviour concerning proliferation and matrix deposition was also performed. Secretory cytokine measurement for pro-inflammatory and angiogenesis factors was carried out using ELISA. At genomic level, qPCR was carried out. The 3D gradient hydrogels platform made of different macromolecules showed to be a suitable environment for hBMSCâ s. The hBMSCâ s gradient hydrogels supported high cell survival and exhibited biofunctionality. Besides, the 3D gradient hydrogels demonstrated differentially secretion of pro-inflammatory and angiogenic factors by the encapsulated hBMSCâ s. References: 1. Mikos, AG. et al., Engineering complex tissues. Tissue Engineering 12,3307, 2006 2. Phillips, JE. et al., Proc Natl Acad Sci USA, 26:12170-5, 2008

Artificial thymus: a tissue engineering strategy

The thymus is the central organ responsible for the generation of T lymphocytes (1). Various diseases cause the thymus to produce in- sufficient T cells, which can lead to immune-suppression (2). Since T cells are essential for the protection against pathogens, it is crucial to promote de novo differentiation of T cells on diseased individuals. The available clinical solutions are: 1) one protocol involving the transplant of thymic stroma from unrelated children only applicable for athymic children (3); 2) for patients with severe peripheral T cell depletion and reduced thymic activity, the administration of stimu- lating molecules stimulating the activity of the endogenous thymus (4). A scaffold (CellFoam) was suggested to support thymus regen- eration in vivo (5), although this research was discontinued. Herein, we propose an innovative strategy to generate a bioartificial thymus. We use a polycaprolactone nanofiber mesh (PCL-NFM) seeded and cultured with human thymic epithelial cells (hTECs). The cells were obtained from infant thymus collected during pediatric cardio-tho- racic surgeries. We report new data on the isolation and characterization of those cells and their interaction with PCL-NFM, by expanding hTECs into relevant numbers and by optimizing cell seeding methods.

PHB-HV 3D Scaffold supports osteoblast growth

Bone tissue engineering requires a biocompatible scaffold that supports cell growth and enhances the native repair process. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV) is a biodegradable 3D scaffold with 88.1 â 0.3% porosity and pore size of 163.5 â 0.1 mm. Previous studies demonstrated the potential of PHB-HV as a scaffold in spinal cord repair. The aim of this study was to evaluate PHB-HV as a scaffold for bone regeneration by assessing the cytocompatability of this scaffold.

Stromal vascular fraction cell sheets angiogenic potential for tissue engineering and regenerative medicine applications

One of the biggest concerns in the Tissue Engineering field is the correct vascularization of engineered constructs. Strategies involving the use of endothelial cells are promising but adequate cell sourcing and neo-vessels stability are enduring challenges. In this work, we propose the hypoxic pre-conditioning of the stromal vascular fraction (SVF) of human adipose tissue to obtain highly angiogenic cell sheets (CS). For that, SVF was isolated after enzymatic dissociation of adipose tissue and cultured until CS formation in normoxic (pO2=21%) and hypoxic (pO2=5%) conditions for 5 and 8 days, in basal medium. Immunocytochemistry against CD31 and CD146 revealed the presence of highly branched capillary-like structures, which were far more complex for hypoxia. ELISA quantification showed increased VEGF and TIMP-1 secretion in hypoxia for 8 days of culture. In a Matrigel assay, the formation of capillary-like structures by endothelial cells was more prominent when cultured in conditioned medium recovered from the cultures in hypoxia. The same conditioned medium increased the migration of adipose stromal cells in a scratch assay, when compared with the medium from normoxia. Histological analysis after implantation of 8 days normoxic- and hypoxic-conditioned SVF CS in a hindlimb ischemia murine model showed improved formation of neo-blood vessels. Furthermore, Laser Doppler results demonstrated that the blood perfusion of the injured limb after 30 days was enhanced for the hypoxic CS group. Overall, these results suggest that SVF CS created under hypoxia can be used as functional vascularization units for tissue engineering and regenerative medicine.

Xenogeneic M2-polarization of Macrophages by undifferentiated and differentiated adipose tissue-derived stem cells

Mesenchymal stem cells (MSCs) are considered to be â â immunologically privileged.â â In a previous work when human adipose tissue-derived stem cells (hASCs) subcutaneously implanted in mice we did not identify an adverse host response1. Recently, it was shown that tissue regeneration could benefit from the polarization of M2 macrophages subpopulations 2. In this study we hypothesised that undifferentiated hASCs and derived osteoblasts and chondrocytes are able to switch murine bone marrow-derived macrophages (mBMMÃ s) into M2 phenotype, aiding tissue regeneration. Murine BMMÃ s were plated in direct contact with undifferentiated and osteo or chondro-differentiated hASCs for 4 h, 10 h, 24 h and 72 h. The cytokine profile was analysed by qRT-PCR and the surface markers were detected by flow cytometry. The direct interaction of both cell types was observed by time lapse microscopy. The results showed that mBMMÃ s polarized after contacting tissue culture polystyrene. This M2 phenotype was maintained along the experiment in direct contact with both undifferentiated and osteo or chondro-differentiated hASCs. This was confirmed by the expression of IL-1, IL-10, IL-4, TNF-a and IFN-g (genetic profile) and surface markers (CD206 + + , CD336 + + , MHC II + and CD86 + + ) detection. These data suggest the potential of hASCs in contemporary xenogenic tissue engineering and regenerative medicine strategies, as well as host immune system modulation in autoimmune diseases. 

Establishing guidelines for obtaining optimal cell sources to use in tendon tissue engineering strategies

Cell-based approaches in tissue engineering (TE) have been barely explored for the treatment of tendon and ligament (T/L) tissues, requiring the establishment of a widely available cell source with tenogenic potential. As T/L cells are scarce, stem cells may provide a good alternative. Understanding how resident cells behave in vitro, might be useful for recapitulating the tenogenic potential of stem cells for tendon TE applications. Therefore, we propose to isolate and characterize human T/L-derived cells (hTDCs and hLDCs) and compare their regenerative potential with stem cells from adipose tissue (hASCs) and amniotic fluid (hAFSCs)(1). T/L cells were isolated using different procedures and stem cells isolated as described elsewhere(1). Moreover, T/L cells were stimu- lated into the three mesenchymal lineages, using standard differentia- tion media. Cells were characterized for the typical stem cell markers as well as T/L related markers, namely tenascin-C, collagen I and III, decorin and scleraxis, using different complementary techniques such as real time RT-PCR, immunocytochemistry and flow cytometry. No differences were observed between T/L in gene expression and protein deposition. T/L cells were mostly positive for stem ness markers (CD73/CD90/CD105), and have the potential to differentiate towards osteogenesis, chondrogenesis and adipogenesis, demonstrated by the positive staining for AlizarinRed, SafraninO, ToluidineBlue and OilRed. hASCs and hAFSCs exhibit positive expression of all tenogenic mark- ers, although at lower levels than hTDCs and hLDCs. Nevertheless, stem cells availability is key factor in TE strategies, despite that it’s still required optimization to direct their tenogenic phenotype.

Saloplastic membranes as green devices for soft tissue regeneration

Implantable devices must exhibit mechanical properties similar to native tissues to promote appropriate cellular behavior and regeneration. Herein, we report a new membrane manufacture method based on the synthesis of polyelectrolyte complexes (PECs) that exhibit saloplasticity, i.e. variable physical-chemistry using salt as a plasticizer. This is a Green Chemistry approach, as PECs generate structures that are stabilized solely by reversible electrostatic interactions, avoiding the use of harmful crosslinkers completely. Furthermore, natural polyelectrolytes - chitosan and alginate - were used. Upon mixing them, membranes were obtained by drying the PECs at 37ºC, yielding compact PECs without resorting to organicsolvents. The plasticizing effect of salt after synthesis was shown by measuring tensile mechanical properties, which were lower when samples were immersed in high ionic strength solutions.Salt was also used during membrane synthesis in different quan- tities (0 M, 0.15 M and 0.5 M in NaCl) yielding structures with no significant differences in morphology and degradation (around 15% after 3 months in lysozyme). However, swelling was higher (about 10x) when synthesized in the presence of salt. In vitro cell studies using L929 fibroblasts showed that cells adhered and proliferated preferentially in membranes fabricated in the presence of salt (i.e. the membranes with lower tensile strength). Structures with physical-chemical properties controlled with precision open a path to tissue engineering strategies depending on fine tuning mechanical properties and cellular adhesion simply by changing ionic strength during membrane manufacture

Release of insulin from PLGA-alginate dressing stimulates regenerative healing of burn wounds in rats

Burn wound healing involves a complex set of overlapping processes in an environment conducive to ischemia, inflammation, and infection costing $7.5 billion/year in the US alone, in addition to the morbidity and mortality that occur when the burns are extensive. We previously showed that insulin, when topically applied to skin excision wounds, accelerates re-epithelialization, and stimulates angiogenesis. More recently, we developed an alginate sponge dressing (ASD) containing insulin encapsulated in PLGA microparticles that provides a sustained release of bioactive insulin for >20days in a moist and protective environment. We hypothesized that insulin-containing ASD accelerates burn healing and stimulates a more regenerative, less scarring, healing. Using a heat-induced burn injury in rats, we show that burns treated with dressings containing 0.04mg insulin/cm2, every three days for 9 days, have faster closure, faster rate of disintegration of dead tissue, and decreased oxidative stress.In addition, in insulin-treated wounds the pattern of neutrophil inflammatory response suggests faster clearing of the burn dead tissue. We also observe faster resolution of the pro-inflammatory macrophages. We also found that insulin stimulates collagen deposition and maturation with the fibers organized more like a basket weave (normal skin) than aligned and crosslinked (scar tissue). In summary , application of ASD-containing insulin-loaded PLGA particles on burns every three days stimulates faster and more regenerative healing. These results suggest insulin as a potential therapeutic agent in burn healing and, because of its long history of safe use in humans, insulin could become one of the treatments of choice when repair and regeneration are critical for proper tissue function.

Advancing tendon regeneration through the development of bio-stimulating tissue engineering approaches

Tendon tissue engineering (TE) requires tailoring scaffolds designs and properties to the anatomical and functional requirements of tendons located in different regions of the body. Cell sourcing is also of utmost importance as tendon cells are scarce. Recently, we have found that it is possible to direct the tenogenic differentiation of Amniotic fluid and Adipose tissue derived stem cells (hAFSCs and hASCs), and also that there are hASCs subpopulations that might be more prone to tenogenic differentiation. Nevertheless, biochemical stimulation may not be enough to develop functional TE substitutes for a tissue that is known to be highly dependent on mechanical loading.

Compact saloplastic membranes of natural polysaccharides for soft tissue engineering

The regeneration of soft biological tissues requires new substitutes that exhibit mechanical properties similar to the native tissue. Herein, thin saloplastic membranes with tunable physical properties are prepared by complexation of chitosan and alginate solutions containing different concentrations of sodium chloride. Polyelectrolyte complexes (PECs) are transferred to flat Petri dishes for compaction into membrane shapes by sedimentation and solvent evaporation. All membranes are resistant to degradation by lysozyme and are stable in solutions with pH values between 1 and 13. Immersing the different membranes in new doping solutions of increasing salt concentrations triggers the typical saloplastic behavior, with a high water absorption and decrease of the rigidity and ultimate tensile strength. The range of such variations is tuned by the sodium chloride amount used in the synthesis: high salt concentrations increase water uptake and tensile moduli, while decreasing the ultimate strength. Cellular assays demonstrate high proliferation rates and viability of L929 fibroblasts seeded onto the most rigid membranes. The results validate the use of saloplastic membranes as soft tissue substitutes for future biomedical applications.

Tensile properties of polymer repair materials - effect of test parameters

In this research, five types of polymer repair materials were selected for investigation of the influence of sample shape, deformation rate and test temperature on the mechanical properties determined with an uniaxial tensile test. The results showed the clear effect of measurement conditions on tensile strength, elongation and modulus of elasticity. The highest tensile strength and modulus of elasticity were exhibited by epoxy resin for the filling of concrete cracks, which achieved 1% elongation. The lowest coefficient of dispersion characterized the results of tensile test carried out using dumbbell samples at a deformation rate of 50 mm/min. The effect of temperature varied with the material type.

Poly-N-acetyl glucosamine expression in Staphylococcus epidermidis biofilm cells affects the immune response and promotes tissue pathology in infected hosts

Staphylococcus epidermidis is a biofilm - forming bacterium and a leading etiological agent of nosocomial infections. The ability to establish biofilms on indwelling medical devices is a key virulence factor for this bacterium. Still, the influence of poly - N - acetyl glucosamine (PNAG), the major component of the extracellular biofilm matrix, in the host immune response has been scarcely studied. Here, t h is influence was assessed in mice challenged i.p. with PNAG - p roducing (WT) and isogenic - mutant lacking PNAG (M10) bacteria grown in biofilm - inducing conditions. Faster bacterial clearance was observed in the mice infected with WT bacteria than in M10 - infected counterparts , which w as accompanied by earlier neutrophil recruitment and higher IL - 6 production. Interestingly, in the WT - infected mice, but not in those infected with M10 , elevated serum IL - 10 was detected . To further study the effe ct of PNAG in the immune response, mice were primed with WT or M10 biofilm bacteria and subsequently infected with WT biofilm - released cells. WT - primed mice presented a higher frequency of splenic IFN - γ + and IL - 17 + CD4 + T cells, and more severe liver patho logy than M10 - primed counterparts. Nevertheless, T reg cells obtained from the WT - primed mice presented a higher suppressive function than those obtained from M10 - primed mice. This effect was abrogated when IL - 10 - deficient mice were similarly primed and infected indicating that PNAG promotes the differentiati on of highly suppressive T reg cells by a mechanism dependent on IL - 10. Altogether, these results provide evidence help ing explain ing the coexistence of inflammation and bacterial persistence often observed in biofilm - originated S. epidermidis infections