**4. Preclinical and clinical applications of tissue engineering and tissue regeneration approaches**

The preclinical proof-of-principle of TEHVs as valve substitutes has been demonstrated in the lamb model already with the polymeric bioconctructs firstly produced *in vitro* [12]. Vascular cell-repopulated polyglycolic acid/polyglactin matrices were implanted in the pulmonary position up to 21 days. Function assessment by Doppler echocardiography demonstrated no stenosis or regurgitation signs, even if a substantial leaflet thickness was reported.

Each subsequent modification in scaffold or cell types, as introduced by the same group, was generally tested *in vivo*, validating progressive functional improvements in transplanted lambs or sheep [41, 73, 74].

Further TEHVs applications in preclinical models were substantially based on the use of P4HB/ PHA with few exceptions, as electrospun polydioxanone [88]. In combination with stem cells of various stromal origins, P4HB/PHA-formulated engineered tissues were evaluated in a long-term animal model, showing replacement of the exogenous matrix after nearly 8 weeks *in vivo* [16].

A MSC-engineered mesh of polyglycolic and polylactic acids was evaluated as autologous pulmonary valve replacement in juvenile sheep. The good performance of this *in vitro* generated construct could be appreciated in a long follow-up of 4 months with restoration of a native-like pulmonary heart valve [59].

Despite biomechanical stimulation induced optimal results in term of cell viability and differentiation almost independently from the cytotype utilised, combined polymer/cell-based efforts to obtain a valve substitute have usually failed in recreating the fibre arrangement of a native ECM. In fact, trilaminate distribution of collagens, GAGs and elastin has been reported only in few cases [88].

A finely organized ECM already exists in native heart valves and can be conserved after cell removal. After decellularization with trypsin/EDTA, heart valve conduits were seeded with ECs and myofibroblasts. Allogenically implanted in orthotopic position, they performed adequately. Ex vivo tissue analyses revealed surface endothelium reconstitution, myofibro‐ blasts-mediated repopulation and ECM synthesis with no signs of inflammation and calcifi‐ cation [89].

Sole ECs were used to obtain *in vitro* endothelium coverage of ovine acellular scaffolds. After 6 months of *in vivo* evaluation, explanted tissues presented no calcifications as assessed by atomic absorption spectrometry [90].

Equivalently promising results have been reported for the implantation of Triton X100/sodium cholate-decellularized allogeneic valves in the longest preclinical follow-up ever realized for a TGRHV. Evaluated in Vietnamese minipigs as RVOT replacements in heterotopic position, these acellular, alpha-gal-free certified valve substitutes have demonstrated good haemody‐ namic performance with low transvalvular gradients in a 15-month-long *in vivo* observation.

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**Figure 1.** *In vivo* tissue-regenerated heart valve after 12 months of implantation in Vietnamese minipig

created vasa vasorum. Moreover, re-innervation aspects were also identified [26, 27].

sheep model [103].

recipient 's cells was observed in both cases [104].

procedure for TGRHV production.

Acellular allologous conduits were also favourably approved as aortic valve substitutes in a

Few unfavourable outcomes with a TGRHV have been seldom disclosed. Two groups of heart valves, cryopreserved or decellularized and treated with an anticalcinosis devitalisation (digitonin and ethylenediaminetetraacetic acid), were tested in dogs by substituting an aorta fragment with the non-coronary sinus of the cusp allografts: albeit the lack of immune infiltrate in inserted decellularized specimens in contrast to cryopreserved ones, no engrafting of

The reasons for such different findings should first be searched in the used decellularization

Apart from animal studies, allologeneic TGR approaches were also applied in humans with optimal outcomes. Decellularized allogeneic valves, as obtained with the deoxycholic acid procedure, were evaluated in 68 patients in the medium term for RVOT reconstruction in Ross aortic valve substitution. Up to 4 years, Costa et al. observed very low mortality (1, 4 %), a

No calcification events could be appreciated within engrafted tissues by trans-thoracic echocardiography: these macroscopic observations found ex vivo confirmation by undetect‐ able calcific foci after von Kossa staining. In addition, no inflammatory or immunogenic cells could be observed. A progressive repopulating process occurred in implanted valves: most tissues were endothelialised and engrafted by rare stem cells and numerous myo/fibroblasts, both highly proliferating and suggesting the onset of a smooth muscle cell conversion. Provision of oxygen and nutrients was again established by a dense capillary network and re-

Lutter et al. coated stented pulmonary valves with small interstinal submucosa, both pigderived and decellularized. These scaffolds, dynamically seeded *in vitro* with ECs and myofibroblasts, were deployed in orthotopic position by means of transcatheter assistance. Valve performance and macroscopic appearance demonstrated to be normal during *in vivo* and post-mortem evaluation [91].

Despite rare reports of deleterious therapeutic effects associated to TEHVs' implantation in humans [92], clinical application of these substitutes, attained by combination of acellular scaffolds and ECs or EPCs, reached already more than 10 years of experience with proved function and absence of calcifications [93, 94].

Another modality of heart valve tissue engineering has been more recently proposed. It is realized by means of a one-phase intraoperative approach. The rational of such a strategy rises from the necessity of a ready-to-apply TEHV, when the surgical therapy has to be promptly adopted with no time for *in vitro* cell seeding and bioreactor conditioning. Weber and collea‐ gues implanted such prepared TEHVs in the RVOT of non-human primates through minimally invasive, transapical procedures. These polymeric trileaflet heart valves have been just seeded with unselected autologous bone marrow cells before the crimping necessary for valve insertion. After one month, the completely remodelled valves were still functioning [95]. Similarly conceived TEHVs demonstrated patency also in the aortic position, being able to sustain the higher pressure regimen of the systemic circulation [96,97]. Another *in situ* TEHV delivery has been applied by Vincentelli et al, by injecting mesenchymal stem cells into a just deployed decellularized heart valve. As element of comparison, they used acellular scaffolds: these ones showed equal performance and reconstructed tissue [98]. However, these are no more TE-, but TGRHVs.

The first attempt of tissue-guided heart valve regeneration has been challenged by the extensive work of Konertz and colleagues, who, moving from the classical paradigm of tissue engineering, compared the two methods. Common for each approach is only the application of the same decellularizing detergent, deoxy-cholic acid. By using an allogeneic decellularized valve for the reconstruction of the right ventricular outflow tract in sheep, they ascertained there was no need to seed the scaffolds prior to implantation, after the good repopulation observed at six months [99]. Follow-up of the valve function revealed increase in the annulus diameter in response to animal growth [100]. As further step to the clinic, they developed a xenogeneic model again with substitution of the autologous pulmonary valve, transferred in aortic position during Ross intervention. They tested porcine decellularized valves, called Matrix P, in a pig-to-sheep interaction. By comparison to sheep cryopreserved allografts, decellularized porcine valves demonstrated better valvular performance, decreased calcific potential and feasible tissue regeneration [101]. Another group compared the haemodynamic function of valve allografts, either cryopreserved and/or decellularized, verifying a reduced calcification tendency in the sheep implanted with decellularized matrices [102].

Equivalently promising results have been reported for the implantation of Triton X100/sodium cholate-decellularized allogeneic valves in the longest preclinical follow-up ever realized for a TGRHV. Evaluated in Vietnamese minipigs as RVOT replacements in heterotopic position, these acellular, alpha-gal-free certified valve substitutes have demonstrated good haemody‐ namic performance with low transvalvular gradients in a 15-month-long *in vivo* observation.

Sole ECs were used to obtain *in vitro* endothelium coverage of ovine acellular scaffolds. After 6 months of *in vivo* evaluation, explanted tissues presented no calcifications as assessed by

Lutter et al. coated stented pulmonary valves with small interstinal submucosa, both pigderived and decellularized. These scaffolds, dynamically seeded *in vitro* with ECs and myofibroblasts, were deployed in orthotopic position by means of transcatheter assistance. Valve performance and macroscopic appearance demonstrated to be normal during *in vivo*

Despite rare reports of deleterious therapeutic effects associated to TEHVs' implantation in humans [92], clinical application of these substitutes, attained by combination of acellular scaffolds and ECs or EPCs, reached already more than 10 years of experience with proved

Another modality of heart valve tissue engineering has been more recently proposed. It is realized by means of a one-phase intraoperative approach. The rational of such a strategy rises from the necessity of a ready-to-apply TEHV, when the surgical therapy has to be promptly adopted with no time for *in vitro* cell seeding and bioreactor conditioning. Weber and collea‐ gues implanted such prepared TEHVs in the RVOT of non-human primates through minimally invasive, transapical procedures. These polymeric trileaflet heart valves have been just seeded with unselected autologous bone marrow cells before the crimping necessary for valve insertion. After one month, the completely remodelled valves were still functioning [95]. Similarly conceived TEHVs demonstrated patency also in the aortic position, being able to sustain the higher pressure regimen of the systemic circulation [96,97]. Another *in situ* TEHV delivery has been applied by Vincentelli et al, by injecting mesenchymal stem cells into a just deployed decellularized heart valve. As element of comparison, they used acellular scaffolds: these ones showed equal performance and reconstructed tissue [98]. However, these are no

The first attempt of tissue-guided heart valve regeneration has been challenged by the extensive work of Konertz and colleagues, who, moving from the classical paradigm of tissue engineering, compared the two methods. Common for each approach is only the application of the same decellularizing detergent, deoxy-cholic acid. By using an allogeneic decellularized valve for the reconstruction of the right ventricular outflow tract in sheep, they ascertained there was no need to seed the scaffolds prior to implantation, after the good repopulation observed at six months [99]. Follow-up of the valve function revealed increase in the annulus diameter in response to animal growth [100]. As further step to the clinic, they developed a xenogeneic model again with substitution of the autologous pulmonary valve, transferred in aortic position during Ross intervention. They tested porcine decellularized valves, called Matrix P, in a pig-to-sheep interaction. By comparison to sheep cryopreserved allografts, decellularized porcine valves demonstrated better valvular performance, decreased calcific potential and feasible tissue regeneration [101]. Another group compared the haemodynamic function of valve allografts, either cryopreserved and/or decellularized, verifying a reduced

calcification tendency in the sheep implanted with decellularized matrices [102].

atomic absorption spectrometry [90].

256 Calcific Aortic Valve Disease

and post-mortem evaluation [91].

more TE-, but TGRHVs.

function and absence of calcifications [93, 94].

**Figure 1.** *In vivo* tissue-regenerated heart valve after 12 months of implantation in Vietnamese minipig

No calcification events could be appreciated within engrafted tissues by trans-thoracic echocardiography: these macroscopic observations found ex vivo confirmation by undetect‐ able calcific foci after von Kossa staining. In addition, no inflammatory or immunogenic cells could be observed. A progressive repopulating process occurred in implanted valves: most tissues were endothelialised and engrafted by rare stem cells and numerous myo/fibroblasts, both highly proliferating and suggesting the onset of a smooth muscle cell conversion. Provision of oxygen and nutrients was again established by a dense capillary network and recreated vasa vasorum. Moreover, re-innervation aspects were also identified [26, 27].

Acellular allologous conduits were also favourably approved as aortic valve substitutes in a sheep model [103].

Few unfavourable outcomes with a TGRHV have been seldom disclosed. Two groups of heart valves, cryopreserved or decellularized and treated with an anticalcinosis devitalisation (digitonin and ethylenediaminetetraacetic acid), were tested in dogs by substituting an aorta fragment with the non-coronary sinus of the cusp allografts: albeit the lack of immune infiltrate in inserted decellularized specimens in contrast to cryopreserved ones, no engrafting of recipient 's cells was observed in both cases [104].

The reasons for such different findings should first be searched in the used decellularization procedure for TGRHV production.

Apart from animal studies, allologeneic TGR approaches were also applied in humans with optimal outcomes. Decellularized allogeneic valves, as obtained with the deoxycholic acid procedure, were evaluated in 68 patients in the medium term for RVOT reconstruction in Ross aortic valve substitution. Up to 4 years, Costa et al. observed very low mortality (1, 4 %), a good valve function, both comparable with the cryopreserved allografts used as control, and a progressive engraftment – even if discontinuous [105].

controlled fabrication procedures. So far, animal tissues fixed in glutaraldehyde have been the reserve to exploit at the time of clinical need. However, this should be no longer considered as viable route in accordance to vitality maintenance, above all for those organs that cannot perform a correct function in absence of a proper cell physiology. The use of non-human sources is accompanied by a major raising concern for a broad clinical application, i.e. the immunological barrier. For a donor-receiver mismatched allocombination, the most serious medical issue is the inability of accommodation and therefore the onset of chronic rejection, whose main manifestation, in the case of heart transplant, is graft vascular disease. This allotransplantation drawback is characterized by an unchanged gravity and entity in respect to 40 years ago, when this research line started to be investigated as possible treatment of end-

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A peculiar atherosclerotic process interests the heart transplant with few calcifications, but increased cellularity and extracellular matrix deposition at the entire intimal level with a

A similar event occurs in the xenotransplantation approach, where an even more severe expression is attended. Furthermore, in addition to a chronic response, antibody-mediated hyperacute rejection represents a dramatic hurdle to early-term xenograft survival, when a trans-species interaction has to be considered. By developing in a time period from minutes to hours in the pig-to-primate combination, hyperacute rejection, commonly defined as HAR, is a typical humoral immune response in vascularized organs with deposition of xenoreactive natural antibodies and complement activation [118]. Pig-to-primate xenotransplantation has properly enabled to discover the progression of delayed immunological answer to the cardiac graft (DXR): besides a strong humoral activity, acute cellular infiltrates and endothelium

Not well known - and in the last years very debated- is the real immunological trigger able to cause the complete loss of the xenograft during time. Probably the prompts of this phenom‐ enon are not to be found in a unique opponent, but in more factors, which alone or in coop‐ eration provoke it. One of the most powerful antigens is definitely Galα1-3Galβ1-4GlcNAc, commonly identified as α-Gal. This oligosaccharide is a component of the glycoproteins and glycolipids, displayed on the surface of vascular endothelial cells in all mammals except apes, Old World monkeys and humans, unable to metabolize it for evolutionary gene silencing of the related enzyme α1-3-Galactosyltransferase [120, 121]. Reaching an expression concentra‐

specific pattern soon after birth similarly to ABO antibodies. In fact, microorganisms coloniz‐ ing or transiting through the intestinal flora express it on their surface and due also to the dietary use of animal-derived nourishment, 1% of serum circulating IgGs are specifically directed against this epitope with a quite pure protective role against parasite and viral attacks [122, 123]. Already at the end of the previous century, a restricted but well developed body of evidence considered alpha-gal as an immunogenic suspect, but it was only more recent the full demonstration of its causative role in HAR, by studying the pig-to-primate interaction

epitopes per pig cell, alpha-gal is recognized by human cells in a highly

activation seriously compromise the function of newly transplanted organ [119].

stage pathologies [116].

concentric distribution [117].

*5.1.1. Alpha-gal and other xenoantigens*

tion of at least 107

TGR has been further practised as an alternative RVOT replacement strategy for human paediatric and young patients with confident results in a follow-up of more than 5 years. Freedom from re-intervention, lower transvalvular gradients and adaptive dimensional modifications in response to somatic growth have been reported from *in vivo* early-term comparison with pulmonary allografts [106]. In a just slighter observational window, allogeneic pulmonary valves were evaluated in a multicentre study with 342 patients undergoing RVOT reconstruction: improvement in haemodynamic function was regis‐ tered for implanted valves and suggested to be related to decreased tissue antigenicity [107]. After a 5-year follow-up in 48 patients, Burch et al. put the accent on the relevance of the economic burden, related to the use of decellularized cryopreserved allografts in respect to their untreated counterparts [108].

Although good reported outcomes open the route for a promising treatment of heart valve failure, it will be imperative to reconsider therapeutic effects in a longer clinical evaluation, by taking into account also socio-economic considerations.

A debated note is, however, represented by the clinical application of unseeded decellularized xenogeneic tissues as valvular replacement solutions. *In vivo* infiltration of tissue-engineered Matrix P heart valves by human cells, not related to inflammatory or immune system, was observed in some explanted specimens [109]. These relevancies were at the basis of the pure Matrix P adoption in the clinical arena. Although favourable performance and lack of xeno‐ geneic tissue-mediated immune reactions have been demonstrated by the same valvemanufacturing group in respect to current RVOT substitutes [110, 111], controversial issues were evidenced after implantation of the same Matrix P valves in other studies [112, 113].

These reports, together with the dramatic results of the early failed Synergraft decellularized valves [114], should lower the speed in the human application of xenogeneic tissues-derived devices, moving a step backward to more robust human-like preclinical trials, as non-human primate animal models.
