A novel extrusion-based 3D bioprinting system for skeletal muscle tissue engineering.

Three-dimensional (3D) bioprinting is an emerging technology, which turned out to be an optimal tool for tissue engineering approaches. To date, different printing systems have been developed. Among them, the extrusion-based approach demonstrated to be the most suitable for skeletal muscle tissue engineering, due to its ability to produce and deposit printing fibers in a parallel pattern that well mimic the native skeletal muscle tissue architecture. In tissue bioengineering, a key role is played by biomaterials, which must possess the key requisite of 'printability'. Nevertheless, this feature is not often well correlated with cell requirements, such as motives for cellular adhesion and/or absorbability. To overcome this hurdle, several efforts have been made to obtain an effective bioink by combining two different biomaterials in order to reach a good printability besides a suitable biological activity. However, despite being efficient, this strategy reveals several outcomes limitations. We report here the development and characterization of a novel extrusion-based 3D bioprinting system, and its application for correction of volumetric muscle loss (VML) injury in a mouse model. The developed bioprinting system is based on the use of PEG-Fibrinogen, a unique biomaterial with excellent biocompatibility, well-suited for skeletal muscle tissue engineering. With this approach, we obtained highly organized 3D constructs, in which murine muscle progenitors were able to differentiate into muscle fibers arranged in aligned bundles and capable of spontaneously contracting when culturedin vitro. Furthermore, to evaluate the potential of the developed system in future regenerative medicine applications, bioprinted constructs laden with either murine or human muscle progenitors were transplanted to regenerate theTibialis Anteriormuscle of a VML murine model, one month after grafting.

PlGF-MMP9-engineered iPS cells supported on a PEG-fibrinogen hydrogel scaffold possess an enhanced capacity to repair damaged myocardium.

Cell-based regenerative therapies are significantly improved by engineering allografts to express factors that increase vascularization and engraftment, such as placental growth factor (PlGF) and matrix metalloproteinase 9 (MMP9). Moreover, the seeding of therapeutic cells onto a suitable scaffold is of utmost importance for tissue regeneration. On these premises, we sought to assess the reparative potential of induced pluripotent stem (iPS) cells bioengineered to secrete PlGF or MMP9 and delivered to infarcted myocardium upon a poly(ethylene glycol)-fibrinogen scaffold. When assessing optimal stiffness of the PEG-fibrinogen (PF) scaffold, we found that the appearance of contracting cells after cardiogenic induction was accelerated on the support designed with an intermediate stiffness. Revascularization and hemodynamic parameters of infarcted mouse heart were significantly improved by injection into the infarct of this optimized PF scaffold seeded with both MiPS (iPS cells engineered to secrete MMP9) and PiPS (iPS cells engineered to secrete PlGF) cells as compared with nonengineered cells or PF alone. Importantly, allograft-derived cells and host myocardium were functionally integrated. Therefore, survival and integration of allografts in the ischemic heart can be significantly improved with the use of therapeutic cells bioengineered to secrete MMP9 and PlGF and encapsulated within an injectable PF hydrogel having an optimized stiffness.

MMP-2 sensitive, VEGF-bearing bioactive hydrogels for promotion of vascular healing.

We sought to develop bioactive hydrogels to facilitate arterial healing, e.g., after balloon angioplasty. Toward this end, we developed a new class of proteolytically sensitive, biologically active polyethylene glycol (PEG)-peptide hydrogels that can be formed in situ to temporarily protect the arterial injury from blood contact. Furthermore, we incorporated endothelial cell-specific biological signals with the goal of enhancing arterial reendothelialization. Here we demonstrate efficient endothelial cell anchorage and activation on PEG hydrogel matrices modified by conjugation with both the cell adhesive peptide motif RGD and an engineered variant of vascular endothelial growth factor (VEGF). By crosslinking peptide sequences for cleavage by MMP-2 into the polymer backbone, the hydrogels became sensitive to proteolytic degradation by cell-derived matrix metalloproteinases (MMPs). Analysis of molecular hallmarks associated with endothelial cell activation by VEGF-RGD hydrogel matrices revealed a 70% increase in production of the latent MMP-2 zymogen compared with PEG-peptide hydrogels lacking VEGF. By additional provision of transforming growth factor beta1 (TGF-beta1) within the PEG-peptide hydrogel, conversion of the latent MMP zymogen into its active form was demonstrated. As a result of MMP-2 activation, strongly enhanced hydrogel degradation by activated endothelial cells was observed. Our data illustrate the critical importance of growth factor activities for remodeling of synthetic biomaterials into native tissue, as it is desired in many applications of regenerative medicine. Functionalized PEG-peptide hydrogels could help restore the native vessel wall and improve the performance of angioplasty procedures.

The response of endothelial cells to fluid shear stress using a co-culture model of the arterial wall.

An endothelial cell (EC) smooth muscle cell (SMC) co-culture model of the arterial wall was used to study the effect of fluid shear stress on EC behavior. This model, in addition to being a more realistic tissue analogue, is a valuable research tool for studying the effects of mechanical stimulation upon the behavior of both SMCs and ECs. In the present study, a 10% cyclic strain was used to alter the characteristics of an SMC-seeded collagen gel. This form of strain preconditioning resulted in a rearrangement of the vessel wall that yielded circumferentially oriented cells and collagen fibrils. The preconditioned collagen gel was subsequently seeded with ECs and exposed to fluid-induced shear stress (10 dynes/cm2) for 48 hr. In the absence of flow, ECs seeded on slab constructs were oriented with the underlying collagen fibrils. Sheared constructs exhibited ECs oriented in the flow direction. Shear stress also affected EC proliferation, reducing the total number of dividing ECs by as much as 48 percent compared to unsheared constructs. The shear-induced reduction in proliferation was further enhanced when constructs were first strain-preconditioned (64% reduction). Moreover, conditioned media from shear stress experiments inhibited proliferation of ECs seeded on tissue culture plastic. These results suggest that EC response to fluid shear stress in a collagen co-culture model is influenced by the underlying substrate, and one that in this study is modified by strain preconditioning.

Vascular tissue engineering.

The development of a tissue-engineered blood vessel substitute has motivated much of the research in the area of cardiovascular tissue engineering over the past 20 years. Several methodologies have emerged for constructing blood vessel replacements with biological functionality. These include cell-seeded collagen gels, cell-seeded biodegradable synthetic polymer scaffolds, cell self-assembly, and acellular techniques. This review details the most recent developments, with a focus on core technologies and construct development. Specific examples are discussed to illustrate both the benefits and shortcomings of each methodology, as well as to underline common themes. Finally, a brief perspective on challenges for the future is presented.

The role of matrix metalloproteinase-2 in the remodeling of cell-seeded vascular constructs subjected to cyclic strain.

Tissue engineering offers the opportunity to develop vascular substitutes that mimic the responsive nature of native arteries. A good blood vessel substitute should be able to remodel its matrix in response to mechanical stimulation, as imposed by the hemodynamic environment. We have developed a novel method of studying the influence of mechanical strain on the remodeling of cell-seeded collagen gel blood vessel analogs. We assessed the remodeling capacity by examining the effect of mechanical conditioning upon the expression of enzymes which remodel the extracellular matrix, called matrix metalloproteinases (MMPs), and upon the mechanical properties of the constructs. We found that subjecting collagen constructs to a 10% cyclic radial distention, over a course of 4 days, resulted in an overall increase in the production of MMP-2. Cyclic mechanical strain also stimulated enzymatic activation of latent MMP-2. We found that cyclic strain also significantly increased the mechanical strength and material modulus, as indicated by an increase in circumferential tensile properties of the constructs. These observations suggested that MMP-2-dependent remodeling affects the material properties of vascular tissue analogs. To further investigate this possible connection we examined the effects of dynamic conditioning in the presence of two nonspecific inhibitors of MMP activity. Interestingly, we found that nonspecific inhibition of MMP ablated the benefits of mechanical conditioning upon mechanical properties. Our observations suggest that a better understanding of the complex relation between mechanical stimulation and construct remodeling is key for the proper design of tissue-engineered blood vessel substitutes.

Dynamic mechanical conditioning of collagen-gel blood vessel constructs induces remodeling in vitro.

Dynamic mechanical conditioning is investigated as a means of improving the mechanical properties of tissue-engineered blood vessel constructs composed of living cells embedded in a collagen-gel scaffold. This approach attempts to elicit a unique response from the embedded cells so as to reorganize their surrounding matrix, thus improving the overall mechanical stability of the constructs. Mechanical conditioning, in the form of cyclic strain, was applied to the tubular constructs at a frequency of 1 Hz for 4 and 8 days. The response to conditioning thus evinced involved increased contraction and mechanical strength, as compared to statically cultured controls. Significant increases in ultimate stress and material modulus were seen over an 8 day culture period. Accompanying morphological changes showed increased circumferential orientation in response to the cyclic stimulus. We conclude that dynamic mechanical conditioning during tissue culture leads to an improvement in the properties of tissue-engineered blood vessel constructs in terms of mechanical strength and histological organization. This concept, in conjunction with a proper biochemical environment, could present a better model for engineering vascular constructs.