**6. Challenges and pitfalls**

*In situ* tissue engineering of heart valves represents a quick, cheap, and on-demand approach. Immunomodulatory scaffolds hold great promise for future application and commercializa‐ tion. However, some priority challenges remain to be addressed in the translation from bench to bed.

#### **6.1. ECM formation versus fibrosis**

**Figure 5.** Hypothesized role of the various macrophage polarization states throughout the process of inflammation

Mesenchymal stem cells (MSCs) proliferate during the healing phase, directed by cytokines secreted by nearby cells, e.g. activated platelets and macrophages, and by ECM components such as collagen peptides and fibronectin [29]. MSCs produce an immunoprivileged environment by preventing the activation and proliferation of DCs, T cells, macrophages, and PMNs through direct cell-cell interactions and paracrine signaling [8]. Cells derived from immunoprivileged regions have been delivered to promote cell engraftment and protect grafts against autoimmune and allogeneic rejection. These cells secrete a range of factors, eg. TGFβ and IL-10, inducing regulatory T-cell differentiation/expansion, which enhances immuno‐

Recruitment and adhesion of target cell types can be achieved by offering binding domains on the scaffold, for example using supramolecular building blocks with cell-specific peptide sequences [76,79]. When combined with anti-fouling materials, such as PEG, this results in

Independent of the biomaterial, the injury incurred during the implantation process will trigger an immune response, due to the disruption of host tissue and induction of cell damage. Besides substantial mortality and morbidity risks, invasive open heart surgery for heart valve

and tissue regeneration in response to scaffold implantation. Illustration by Anthal Smits.

protection [8].

234 Calcific Aortic Valve Disease

highly selective substrates.

**5.6. Minimally invasive implantation methods**

One of the main challenges for *in situ* tissue engineering is to stimulate functional ECM formation without inducing fibrosis. To maintain functionality of the valve, rapid ECM formation is required in order to overtake the load-bearing role of the degrading scaffold. However, cells and molecules that are stimulatory for ECM production have been designated as pro-fibrotic mediators. This poses a paradoxal challenge. Macrophage plasticity is a striking example. M2 macrophages have been identified as pro-wound healing cells, promoting ECM production by secretion of IL-4 and TGF-β. On the other hand, both IL-4 and TGF-β are strong inducers of fibrosis if not tightly regulated. Chemokines, such as MCP-1, have been identified as pro-fibrotic mediators by attracting fibrocytes and stimulation of M2 polarization [84,85]. On the other hand, MCP-1 inhibition leads to delayed or inhibited wound healing [86]. Fibrocytes are blood-borne mesenchymal stem cell progenitors with a fibroblast/myofibro‐ blast-like phenotype (CD34+ /CD45+ /collagen type I+ ) that similarly have been related to both ECM formation and fibrosis. The same holds for EndoMT-derived (myo-)fibroblasts. However, the local activation state of recruited myofibroblasts, rather than the source, determines their ECM remodeling activity. For example, TLR-signaling promotes fibroblasts to differentiate into collagen-producing myofibroblasts [84]. Valvular interstitial cells (VICs) in the adult valve have a quiescent myofibroblast-like phenotype. Regulating the activation state of colonizing myofibroblasts in the scaffold is pivotal in the prevention of fibrosis and obtaining a VIC-like population. The TGF-β pathway is one of the main players in this process. Furthermore, IL-10 has been shown to inhibit fibrosis in numerous animal models [84], underlining that timely resolution of inflammation is one of the main challenges for *in situ* tissue engineering.

#### **6.2. Hemodynamic environment**

In cardiovascular *in situ* tissue regeneration, the hemodynamic environment plays a key role by directing cell recruitment and cell differentiation. The mechanical load applied to the heart valves is a powerful regulator of cell phenotype, influencing many cell functions such as orientation, replication, growth factor production, and collagen synthesis [33]. In cardiovas‐ cular devices, apoptosis is often induced by shear stress arising from the blood flow [10]. Shear stress also has a significant effect on adhesion of circulating cells to the valve scaffold. Direct intimal binding of cells to the ventricular side of the leaflet is unlikely due to high shear forces during systole. In contrast, end-systolic and diastolic turbulations on the aortic side of the leaflet typically result in low shear stresses that allow for cell adhesion to the scaffold [4].

Any chronic disease which affects the cardio-respiratory system may adversely affect the supply of oxygen and other nutrients required for wound healing. Although hypoxia is one of the chemoattractants for neutrophils and macrophages, oxygen is needed for their optimal function and to allow phagocytosis. Oxygen is also essential for collagen deposition as it acts as a substrate in the hydroxylation of proline and lysine residues. Smoking affects oxygen partial pressures and causes more wound healing complications and it is likely that smoking

The Immune Response in *In Situ* Tissue Engineering of Aortic Heart Valves

http://dx.doi.org/10.5772/54354

237

The use of diseased cells or tissues in humanized animal models or *in vitro* model systems [89]

*In vivo* remodeling of tissue engineered heart valves displays considerable variability among patients, owing to biological heterogeneity among individuals in physiological tissue remodeling potential [5]. This heterogeneity could be a result of mutations or polymorphisms in key proteins central to ECM synthesis and remodeling [2]. The goal is to understand and potentially control human variation in different facets of biomaterial-tissue interaction and the

To cope with patient-to-patient heterogeneity, an important issue in tissue engineering of aortic heart valves will be the real-time noninvasive and non-destructive assessment of mechanical properties both *in vitro* and *in vivo* to ensure tissue quality and function [5]. The challenge here is to find appropriate methodologies to evaluate the evolving structural remodeling and functionality, especially in a noninvasive manner so that the valve can be followed over time [5]. One way of approaching this issue is developing imaging modalities and discovering new biomarkers of inflammation which would help further understanding of inflammatory diseases and discerning events related to inflammation in heart valve tissueengineered implants [12]. When applied to engineered heart valves, developed biomarkers should correlate directly with success and failure in order to generate outcome measurements, such as laboratory assays or imaging results that substitute for and reflect the mechanism of a significant clinical event or characteristic, e.g. stenosis, calcification, or infection [5]. An important consideration is whether calcification, the major pathologic process in valve degeneration, will be problematic. Evidence suggests that calcification may not be a major problem as long as the scaffold is ultimately resorbed and/or not intrinsically mineralizable,

For the translation from bench to bed, there must be understanding of the mechanisms involved and development of biomarkers, assays and tools for the assessment of valve regeneration. Surrogate and true endpoints must be defined to characterize and assure the quality of the tissue constructs, and predict outcomes as early as possible [5]. Key targets for characterizing tissue-engineered constructs include tissue composition, cellular gene expression and phenotype, ECM, and other key effectors of tissue remodeling and tissue

may aid in gaining insight in the effects of comorbidities on valve regeneration.

healing process by developing robust or even patient-tailored scaffolds.

the interstitial cells are viable, and the ECM is capable of remodeling [5].

may also affect immune function and collagen deposition [90].

**6.4. Patient heterogeneity**

quality [5].

So far, the exact mechanism behind cell population of heart valve replacements with host cells remains elusive. For blood vessels, animal studies have identified trans-anastomotic ingrowth as the main source of host tissue cells in the scaffold [87]. However, this is most likely an animal model-dependent phenomenon, as it is known that trans-anastomotic ingrowth is very limited in humans [88]. Therefore, the use of humanized animal models or *in vitro* model systems [89] is indispensible in evaluating scaffold performance for future clinical applications.

#### **6.3. Comorbidity and impaired wound healing**

Little is known about the effect of the pathological status of a tissue, organ, or patient on the fate of a tissue engineered heart valve. It is reasonable to believe that the pre-existing pathology or existing risk factors would influence wound healing and long-term outcomes of valve implantation. One of the most complicated aspects of designing a replacement scaffold for diseased tissue would be the incorporation of measures which prevent the device from succumbing to the same fate as the diseased tissue it is replacing [12].

Impaired wound healing conditions include advanced age, diabetes mellitus (insulin resistance), vascular diseases (e.g. atherosclerosis), and obesity, in which adipose tissue functions as initiator of the chronic inflammatory response. Diabetic patients have significantly impaired wound healing as they are relatively immunocompromised and have higher blood glucose levels affecting leukocyte function [90]. Diabetes and advanced age are associated with delayed or impaired wound healing through a reduced ability to transition from an M1 to an M2 macrophage phenotype [52]. Malnutrition adversely affects wound healing by prolonging inflammation, inhibiting fibroblast function, and reducing angiogenesis and collagen deposition. For example, carbohydrates are needed for collagen synthesis, and ω-3-fatty acids are needed for modulation of the arachidonic acid pathway, resolving inflammation [90].

The patient's regenerative potential is dependent on age. The concentration of progenitor cells in human blood decreases with age [4]. Furthermore, aging typically leads to impaired angiogenesis and local immunity is altered due to lack of growth factors, increased neutrophil invasion and higher number of mature macrophages. Levels of TGF-β in wounds of elderly are, like fetal, markedly reduced, which is possibly related to reduced scarring with age [35]. Next to regeneration potential, the rate at which the scaffold degrades may also be age-specific due to variations in cell availability.

Any chronic disease which affects the cardio-respiratory system may adversely affect the supply of oxygen and other nutrients required for wound healing. Although hypoxia is one of the chemoattractants for neutrophils and macrophages, oxygen is needed for their optimal function and to allow phagocytosis. Oxygen is also essential for collagen deposition as it acts as a substrate in the hydroxylation of proline and lysine residues. Smoking affects oxygen partial pressures and causes more wound healing complications and it is likely that smoking may also affect immune function and collagen deposition [90].

The use of diseased cells or tissues in humanized animal models or *in vitro* model systems [89] may aid in gaining insight in the effects of comorbidities on valve regeneration.

#### **6.4. Patient heterogeneity**

**6.2. Hemodynamic environment**

236 Calcific Aortic Valve Disease

In cardiovascular *in situ* tissue regeneration, the hemodynamic environment plays a key role by directing cell recruitment and cell differentiation. The mechanical load applied to the heart valves is a powerful regulator of cell phenotype, influencing many cell functions such as orientation, replication, growth factor production, and collagen synthesis [33]. In cardiovas‐ cular devices, apoptosis is often induced by shear stress arising from the blood flow [10]. Shear stress also has a significant effect on adhesion of circulating cells to the valve scaffold. Direct intimal binding of cells to the ventricular side of the leaflet is unlikely due to high shear forces during systole. In contrast, end-systolic and diastolic turbulations on the aortic side of the leaflet typically result in low shear stresses that allow for cell adhesion to the scaffold [4].

So far, the exact mechanism behind cell population of heart valve replacements with host cells remains elusive. For blood vessels, animal studies have identified trans-anastomotic ingrowth as the main source of host tissue cells in the scaffold [87]. However, this is most likely an animal model-dependent phenomenon, as it is known that trans-anastomotic ingrowth is very limited in humans [88]. Therefore, the use of humanized animal models or *in vitro* model systems [89]

Little is known about the effect of the pathological status of a tissue, organ, or patient on the fate of a tissue engineered heart valve. It is reasonable to believe that the pre-existing pathology or existing risk factors would influence wound healing and long-term outcomes of valve implantation. One of the most complicated aspects of designing a replacement scaffold for diseased tissue would be the incorporation of measures which prevent the device from

Impaired wound healing conditions include advanced age, diabetes mellitus (insulin resistance), vascular diseases (e.g. atherosclerosis), and obesity, in which adipose tissue functions as initiator of the chronic inflammatory response. Diabetic patients have significantly impaired wound healing as they are relatively immunocompromised and have higher blood glucose levels affecting leukocyte function [90]. Diabetes and advanced age are associated with delayed or impaired wound healing through a reduced ability to transition from an M1 to an M2 macrophage phenotype [52]. Malnutrition adversely affects wound healing by prolonging inflammation, inhibiting fibroblast function, and reducing angiogenesis and collagen deposition. For example, carbohydrates are needed for collagen synthesis, and ω-3-fatty acids are needed for modulation of the arachidonic acid pathway, resolving inflammation [90].

The patient's regenerative potential is dependent on age. The concentration of progenitor cells in human blood decreases with age [4]. Furthermore, aging typically leads to impaired angiogenesis and local immunity is altered due to lack of growth factors, increased neutrophil invasion and higher number of mature macrophages. Levels of TGF-β in wounds of elderly are, like fetal, markedly reduced, which is possibly related to reduced scarring with age [35]. Next to regeneration potential, the rate at which the scaffold degrades may also be age-specific

is indispensible in evaluating scaffold performance for future clinical applications.

succumbing to the same fate as the diseased tissue it is replacing [12].

**6.3. Comorbidity and impaired wound healing**

due to variations in cell availability.

*In vivo* remodeling of tissue engineered heart valves displays considerable variability among patients, owing to biological heterogeneity among individuals in physiological tissue remodeling potential [5]. This heterogeneity could be a result of mutations or polymorphisms in key proteins central to ECM synthesis and remodeling [2]. The goal is to understand and potentially control human variation in different facets of biomaterial-tissue interaction and the healing process by developing robust or even patient-tailored scaffolds.

To cope with patient-to-patient heterogeneity, an important issue in tissue engineering of aortic heart valves will be the real-time noninvasive and non-destructive assessment of mechanical properties both *in vitro* and *in vivo* to ensure tissue quality and function [5]. The challenge here is to find appropriate methodologies to evaluate the evolving structural remodeling and functionality, especially in a noninvasive manner so that the valve can be followed over time [5]. One way of approaching this issue is developing imaging modalities and discovering new biomarkers of inflammation which would help further understanding of inflammatory diseases and discerning events related to inflammation in heart valve tissueengineered implants [12]. When applied to engineered heart valves, developed biomarkers should correlate directly with success and failure in order to generate outcome measurements, such as laboratory assays or imaging results that substitute for and reflect the mechanism of a significant clinical event or characteristic, e.g. stenosis, calcification, or infection [5]. An important consideration is whether calcification, the major pathologic process in valve degeneration, will be problematic. Evidence suggests that calcification may not be a major problem as long as the scaffold is ultimately resorbed and/or not intrinsically mineralizable, the interstitial cells are viable, and the ECM is capable of remodeling [5].

For the translation from bench to bed, there must be understanding of the mechanisms involved and development of biomarkers, assays and tools for the assessment of valve regeneration. Surrogate and true endpoints must be defined to characterize and assure the quality of the tissue constructs, and predict outcomes as early as possible [5]. Key targets for characterizing tissue-engineered constructs include tissue composition, cellular gene expression and phenotype, ECM, and other key effectors of tissue remodeling and tissue quality [5].
