Dynamic loading leads to increased metabolic activity and spatial redistribution of viable cell density in nucleus pulposus tissue

Background: Nucleus pulposus (NP) cell density is orchestrated by an interplay between nutrient supply and metabolite accumulation. Physiological loading is essential for tissue homeostasis. However, dynamic loading is also believed to increase metabolic activity and could thereby interfere with cell density regulation and regenerative strategies. The aim of this study was to determine whether dynamic loading could reduce the NP cell density by interacting with its energy metabolism. Methods: Bovine NP explants were cultured in a novel NP bioreactor with and without dynamic loading in milieus mimicking the pathophysiological or physiological NP environment. The extracellular content was evaluated biochemically and by Alcian Blue staining. Metabolic activity was determined by measuring glucose and lactate in tissue and medium supernatants. A lactate-dehydrogenase staining was performed to determine the viable cell density (VCD) in the peripheral and core regions of the NP. Results: The histological appearance and tissue composition of NP explants did not change in any of the groups. Glucose levels in the tissue reached critical values for cell survival (≤0.5 mM) in all groups. Lactate released into the medium was increased in the dynamically loaded compared to the unloaded groups. While the VCD was unchanged on Day 2 in all regions, it was significantly reduced in the dynamically loaded groups on Day 7 (p ≤ 0.01) in the NP core, which led to a gradient formation of VCD in the group with degenerated NP milieu and dynamic loading (p ≤ 0.05). Conclusion: It was demonstrated that dynamic loading in a nutrient deprived environment similar to that during IVD degeneration can increase cell metabolism to the extent that it was associated with changes in cell viability leading to a new equilibrium in the NP core. This should be considered for cell injections and therapies that lead to cell proliferation for treatment of IVD degeneration.</p

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach—the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis—are discussed in detail

Layer-specific cell differentiation in bi-layered vascular grafts under flow perfusion

Bioengineered grafts have the potential to overcome the limitations of autologous and non-resorbable synthetic vessels as vascular substitutes. However, one of the challenges in creating these living grafts is to induce and maintain multiple cell phenotypes with a biomimetic organization. Our biomimetic grafts with heterotypic design hold promises for functional neovessel regeneration by guiding the layered cellular and tissue organization into a native-like structure. In this study, a perfusable two-compartment bioreactor chamber was designed for the further maturation of these vascular grafts, with a compartmentalized exposure of the graft's luminal and outer layer to cell-specific media. We used the system for a co-culture of endothelial colony forming cells and multipotent mesenchymal stromal cells (MSCs) in the vascular grafts, produced by combining electrospinning and melt electrowriting. It was demonstrated that the targeted cell phenotypes (i.e., endothelial cells (ECs) and vascular smooth muscle cells (vSMCs), respectively) could be induced and maintained during flow perfusion. The confluent luminal layer of ECs showed flow responsiveness, as indicated by the upregulation of COX-2, KLF2, and eNOS, as well as through stress fiber remodeling and cell elongation. In the outer layer, the circumferentially oriented, multi-layered structure of MSCs could be successfully differentiated into vSM-like cells using TGFβ, as indicated by the upregulation of αSMA, calponin, collagen IV, and (tropo)elastin, without affecting the endothelial monolayer. The cellular layers inhibited diffusion between the outer and the inner medium reservoirs. This implies tightly sealed cellular layers in the constructs, resulting in truly separated bioreactor compartments, ensuring the exposure of the inner endothelium and the outer smooth muscle-like layer to cell-specific media. In conclusion, using this system, we successfully induced layer-specific cell differentiation with a native-like cell organization. This co-culture system enables the creation of biomimetic neovessels, and as such can be exploited to investigate and improve bioengineered vascular grafts

Decoupling the effect of shear stress and stretch on tissue growth and remodeling in a vascular graft

The success of cardiovascular tissue engineering (TE) strategies largely depends on the mechanical environment in which cells develop a neotissue through growth and remodeling processes. This mechanical environment is defined by the local scaffold architecture to which cells adhere, that is, the microenvironment, and by external mechanical cues to which cells respond, that is, hemodynamic loading. The hemodynamic environment of early developing blood vessels consists of both shear stress (due to blood flow) and circumferential stretch (due to blood pressure). Experimental platforms that recapitulate this mechanical environment in a controlled and tunable manner are thus critical for investigating cardiovascular TE. In traditional perfusion bioreactors, however, shear stress and stretch are coupled, hampering a clear delineation of their effects on cell and tissue response. In this study, we uniquely designed a bioreactor that independently combines these two types of mechanical cues in eight parallel vascular grafts. The system is computationally and experimentally validated, through finite element analysis and culture of tissue constructs, respectively, to distinguish various levels of shear stress (up to 5 Pa) and cyclic stretch (up to 1.10). To illustrate the usefulness of the system, we investigated the relative contribution of cyclic stretch (1.05 at 0.5 Hz) and shear stress (1 Pa) to tissue development. Both types of hemodynamic loading contributed to cell alignment, but the contribution of shear stress overruled stretch-induced cell proliferation and matrix (i.e., collagen and glycosaminoglycan) production. At a macroscopic level, cyclic stretching led to the most linear stress-stretch response, which was not related to the presence of shear stress. In conclusion, we have developed a bioreactor that is particularly suited to further unravel the interplay between hemodynamics and in situ TE processes. Using the new system, this work highlights the importance of hemodynamic loading to the study of developing vascular tissues

Layer-specific cell differentiation in bi-layered vascular grafts under flow perfusion

Bioengineered grafts have the potential to overcome the limitations of autologous and non-resorbable synthetic vessels as vascular substitutes. However, one of the challenges in creating these living grafts is to induce and maintain multiple cell phenotypes with a biomimetic organization. Our biomimetic grafts with heterotypic design hold promises for functional neovessel regeneration by guiding the layered cellular and tissue organization into a native-like structure. In this study, a perfusable two-compartment bioreactor chamber was designed for the further maturation of these vascular grafts, with a compartmentalized exposure of the graft's luminal and outer layer to cell-specific media. We used the system for a co-culture of endothelial colony forming cells and multipotent mesenchymal stromal cells (MSCs) in the vascular grafts, produced by combining electrospinning and melt electrowriting. It was demonstrated that the targeted cell phenotypes (i.e. endothelial cells (ECs) and vascular smooth muscle cells (vSMCs), respectively) could be induced and maintained during flow perfusion. The confluent luminal layer of ECs showed flow responsiveness, as indicated by the upregulation of COX-2, KLF2, and eNOS, as well as through stress fiber remodeling and cell elongation. In the outer layer, the circumferentially oriented, multi-layered structure of MSCs could be successfully differentiated into vSM-like cells using TGFβ, as indicated by the upregulation of αSMA, calponin, collagen IV, and (tropo)elastin, without affecting the endothelial monolayer. The cellular layers inhibited diffusion between the outer and the inner medium reservoirs. This implies tightly sealed cellular layers in the constructs, resulting in truly separated bioreactor compartments, ensuring the exposure of the inner endothelium and the outer smooth muscle-like layer to cell-specific media. In conclusion, using this system, we successfully induced layer-specific cell differentiation with a native-like cell organization. This co-culture system enables the creation of biomimetic neovessels, and as such can be exploited to investigate and improve bioengineered vascular grafts

Layer-specific cell differentiation in bi-layered vascular grafts under flow perfusion

\u3cp\u3eBioengineered grafts have the potential to overcome the limitations of autologous and non-resorbable synthetic vessels as vascular substitutes. However, one of the challenges in creating these living grafts is to induce and maintain multiple cell phenotypes with a biomimetic organization. Our biomimetic grafts with heterotypic design hold promises for functional neovessel regeneration by guiding the layered cellular and tissue organization into a native-like structure. In this study, a perfusable two-compartment bioreactor chamber was designed for the further maturation of these vascular grafts, with a compartmentalized exposure of the graft's luminal and outer layer to cell-specific media. We used the system for a co-culture of endothelial colony forming cells and multipotent mesenchymal stromal cells (MSCs) in the vascular grafts, produced by combining electrospinning and melt electrowriting. It was demonstrated that the targeted cell phenotypes (i.e., endothelial cells (ECs) and vascular smooth muscle cells (vSMCs), respectively) could be induced and maintained during flow perfusion. The confluent luminal layer of ECs showed flow responsiveness, as indicated by the upregulation of COX-2, KLF2, and eNOS, as well as through stress fiber remodeling and cell elongation. In the outer layer, the circumferentially oriented, multi-layered structure of MSCs could be successfully differentiated into vSM-like cells using TGFβ, as indicated by the upregulation of αSMA, calponin, collagen IV, and (tropo)elastin, without affecting the endothelial monolayer. The cellular layers inhibited diffusion between the outer and the inner medium reservoirs. This implies tightly sealed cellular layers in the constructs, resulting in truly separated bioreactor compartments, ensuring the exposure of the inner endothelium and the outer smooth muscle-like layer to cell-specific media. In conclusion, using this system, we successfully induced layer-specific cell differentiation with a native-like cell organization. This co-culture system enables the creation of biomimetic neovessels, and as such can be exploited to investigate and improve bioengineered vascular grafts.\u3c/p\u3

Decoupling the effect of shear stress and stretch on tissue growth and remodeling in a vascular graft

\u3cp\u3eThe success of cardiovascular tissue engineering (TE) strategies largely depends on the mechanical environment in which cells develop a neotissue through growth and remodeling processes. This mechanical environment is defined by the local scaffold architecture to which cells adhere, that is, the microenvironment, and by external mechanical cues to which cells respond, that is, hemodynamic loading. The hemodynamic environment of early developing blood vessels consists of both shear stress (due to blood flow) and circumferential stretch (due to blood pressure). Experimental platforms that recapitulate this mechanical environment in a controlled and tunable manner are thus critical for investigating cardiovascular TE. In traditional perfusion bioreactors, however, shear stress and stretch are coupled, hampering a clear delineation of their effects on cell and tissue response. In this study, we uniquely designed a bioreactor that independently combines these two types of mechanical cues in eight parallel vascular grafts. The system is computationally and experimentally validated, through finite element analysis and culture of tissue constructs, respectively, to distinguish various levels of shear stress (up to 5 Pa) and cyclic stretch (up to 1.10). To illustrate the usefulness of the system, we investigated the relative contribution of cyclic stretch (1.05 at 0.5 Hz) and shear stress (1 Pa) to tissue development. Both types of hemodynamic loading contributed to cell alignment, but the contribution of shear stress overruled stretch-induced cell proliferation and matrix (i.e., collagen and glycosaminoglycan) production. At a macroscopic level, cyclic stretching led to the most linear stress-stretch response, which was not related to the presence of shear stress. In conclusion, we have developed a bioreactor that is particularly suited to further unravel the interplay between hemodynamics and in situ TE processes. Using the new system, this work highlights the importance of hemodynamic loading to the study of developing vascular tissues.\u3c/p\u3