Recellularization of decellularized heart valves: Progress toward the tissue-engineered heart valve

Detergent valve decellularization

Of the various decellularization methods, the most common utilize detergents for the removal of cells. Ionic and non-ionic detergents are effective decellularization agents because they are able to solubilize cell membranes, lysing the cells and dissociating DNA. The anionic detergents sodium dodecyl sulfate (SDS) and sodium deoxycholate (SD) are often used in valve decellularization and have proven effective in removing cells and DNA from the ECM; however, prolonged chemical exposure during decellularization has been shown to disrupt collagen fiber structure in valve leaflets. ³⁶ The disruption in the ECM leads to an associated deterioration of mechanical properties, observed through increased areal strain and decreased flexural stiffness. ¹⁴ This is despite preservation of the primary structural proteins found in valves: glycosaminoglycans (GAGs), collagen, and elastin fibers (Figure 1(f)). SDS is also particularly difficult to completely remove from the tissue and residual detergents can adversely affect cell adhesion and repopulation. ⁴² Non-ionic detergents, such as Triton X-100, have also proven effective at cell and DNA removal. In addition to cell removal, Triton X-100 has been shown to delipidize tissue, reducing the concentration of GAGs (Figure 1(h)). GAGs are often attributed to the viscoelastic behavior of tissue and indeed, following decellularization with Triton X-100, a decrease in the stress relaxation of valve leaflet tissue has been observed.^12,15 Researchers have also successfully employed multi-detergent decellularization strategies. Studies using a combination of Triton X-100 and SD reported complete cell removal with less damage to the valve ECM, when compared to other methods.^39,48 However, neither study employed mechanical testing analysis of the decellularized leaflets and only described the ECM preservation using qualitative histologic methods. A combination of osmotic shock, Triton X-100, and the anionic detergent N-lauroylsarcosine sodium (NLS) salt has been used to successfully decellularize (>97% dsDNA removal) both pulmonary and aortic heart valves from a variety of species, including ovine, porcine, and human.^12,15,23 The elastin and collagen components of the ECM were preserved, yet a loss of GAGs associated with Triton X-100 was observed. Biaxial mechanical testing indicated increased areal strain, increased peak stretch ratios, and decreased stress relaxation for ovine pulmonary valves. ¹² Interestingly, the same trend was observed for ovine aortic valves, yet no change in the areal strain or peak stretch ratio was observed in human aortic valves. ¹⁵ The authors speculated that the species-specific effect of identical decellularization methods was the result of a loss of collagen crosslinking components and an initial higher cellularity in the ovine valves. ¹⁵

Enzyme valve decellularization

Enzymatic agents such as nucleases or trypsin have also been utilized in valve decellularization to breakdown biologic molecules and facilitate cell removal. Nucleases cleave nucleic acid sequences into shorter segments, expediting their removal from the ECM and the inclusion of nucleases, such as DNases or RNases, in a decellularization process is so commonplace their use was not specified in Table 1. Trypsin is another common enzyme used in valve decellularization. Trypsin, a serine protease, hydrolytically cleaves proteins and is used to digest cellular proteins in the decellularization process. However, the fibrous structural proteins of the valve ECM have limited resistance to trypsin cleavage and are often affected as well. Decellularization of heart valves using trypsin often results in visible histologic damage to the ECM as well as large alterations to valve mechanics (Figure 1(c)). Compared to detergents, trypsin decellularization is slower to remove cells, causes greater disruption to the elastin and collagen structure, but has a better preservation of GAGs.^14,43,45 Complete cell removal by trypsin alone actually requires lengthy decellularization protocols, and the prolonged exposure has been shown to disrupt the remaining ECM beyond practical use. ⁵⁰

Miscellaneous valve decellularization

Additional creative strategies for valve decellularization have been explored, with varying levels of success. Osmotic shock can be used to lyse cells; however, osmotic shock by itself is ineffective at removing the hydrophobic cell membranes and remnants and is therefore not recommended as the sole decellularization technique.^44,45 It is often used in combination with detergent or enzymatic-based methods as an initial step, reducing the required detergent/enzyme concentrations and/or exposure times.^12,41,45 Mechanical forces such as pressure gradients can also aid in decellularization, as perfusion of decellularizing solutions through the valve conduit has been shown to increase the removal of cellular material from the valve wall compared to immersion protocols. ⁵⁸ Glycol radiation, using a polyethylene glycol (PEG) detergent and gamma irradiation, has proven effective, resulting in >92% DNA removal in the valve cusps while not affecting ultimate tensile strength (UTS) and collagen content. ⁵³ Additionally, the gamma irradiation effectively removed the galactose-α(1,2)-galactose (α-Gal) epitope and the porcine endogenous retrovirus. ⁵³ Decellularization by sequential antigen solubilization is another effective method for the removal of cellular and antigenic material from xenogeneic tissues. ³⁵ Based on shared physiochemical properties, antigens are removed by sequential solubilization techniques targeting hydrophilic and lipophilic proteins.^54,55 Using this method, researchers have demonstrated the successful decellularization of bovine pericardium and porcine aortic heart valve leaflets.^54–56 In addition to the removal of cellular material, sequential antigen solubilization also successfully removes the α-Gal and major histocompatibility complex I (MHC I) antigens while preserving the structural and mechanical properties of the native, untreated tissue.^35,56 Another unique approach to decellularization utilizes supercritical fluid solution of carbon dioxide and ethanol as a cell extraction medium. ⁵⁷ The high permeability and high transfer rate of the supercritical fluid makes this an effective method, resulting in a visible lack of cell nuclei and a 90% reduction in phospholipids, the primary component of cell walls. However, a stiffening and dehydration of the ECM has also been observed, likely attributed to the high ethanol exposure. ⁵⁷

Guided tissue regeneration

The most obvious, and most attempted, approach to in situ recellularization has been the implantation of stand-alone decellularized valve grafts without any further chemical or mechanical modification. This approach has come to be known as Guided tissue regeneration. As mentioned previously, decellularized valve grafts have been implanted in animal and clinical applications with varying results in terms of survival and growth (see section “Clinical use of decellularized heart valves”). In this section, the focus is the in situ recellularization of the implanted valve scaffolds (Table 2). A more thorough review on the guided tissue regeneration of heart valves is available in Iop and Gerosa. ⁸⁰

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Table 2.

Summary of in situ results for the implantation of non-conditioned decellularized valve scaffolds in various animal models and clinical trials.

Recell method	Tissue	Conditioning	Implant model	Details	Results
In situ—no conditioning	mPV 81	None	Mouse PV	Decell valve attached to donor heart and implanted in another mouse	Leaflets were thickened with many αSMA+ cells present early, though less αSMA+ cells present later
	rbAV 82 pAV 83	None	Canine PV, 83 aorta 82	Decell xenogeneic valves implanted in canines	Rabbit valve leaflets degenerated; porcine valves re-endothelialized with minimal cell infiltration near leaflet surface
	oAV, 60 pAV,84,85 oPV,6,24,86 pPV 86	None	Ovine AV, 60 PV,6,24,85,86 aorta 84	Decell xenogeneic and allogeneic valves implanted in sheep	Re-endothelialization; recell of valve wall; minimal recell of leaflet; xenograft comparable to allograft
	pAV80,85,87	None or stented 87	Porcine AV, 87 PV, 79 aorta 85	Decell allogeneic valves implanted in pigs	Aorta implants led to loss of leaflets; PV implants led to good recell of surface and interior of leaflet; AV implants showed recell of conduit wall only
	hPV,88,89 hAV, 75 pPV17,37,63,64,67,73,74,90,91	None	Human PV, AV 75	Decell allogeneic and xenogeneic valves implanted in humans	Allogeneic performed better than xenogeneic; recell of valve wall and endothelialization evident; no evidence of leaflet recell unless by inflammatory cells

αSMA: alpha-activated smooth muscle actin.

The results are overall observations and the outcome of individual studies may vary. Lower case letters in acronyms denote species (o = ovine, p = porcine, h = human, m = mouse, rb = rabbit).

Currently, the in situ autologous recellularization of implanted decellularized valves is limited and variable. As mentioned previously, many studies have observed complete re-endothelialization and recellularization of the valve wall up to the leaflet base, yet the distal subsurface leaflet remains mostly acellular. This prevents tissue growth and remodeling in the valve leaflets, ultimately leading to valve degeneration. James et al. ⁸¹ reported an exception to this limited recellularization using a mouse model. At 3 months of implant, they observed a thickening of the decellularized mouse leaflets and repopulation by αSMA⁺ cells, although by 6 months the leaflets appeared normal and the number of αSMA⁺ cells decreased. ⁸¹ This may be the first study demonstrating a state of valve remodeling after implant transitioning into a quiescent, healthy state. While the study by James et al. ⁸¹ is significant, the implant model presents challenges for clinical translation and the recellularization success is likely due to the thinness of the mouse leaflets, expediting cell infiltration. Additionally, it appears that leaflet recellularization typically decreases as higher order animals are used as implant models (Table 2). Furthermore, it appears that if the decellularized replacement valve is more robust than the original, native valve, the recellularization of the leaflet is better. For example, porcine valves implanted in canines led to good recellularization, as did porcine aortic valves implanted in a porcine pulmonary valve model.^79,83

The in situ recellularization of decellularized valves in clinical use has been less successful than in pre-clinical studies. Decellularized allografts have functioned better than decellularized xenografts, as previously mentioned; however, no evidence has been presented demonstrating successful in situ autologous recellularization. Biopsies of the valve conduit taken from decellularized allografts during non-valve related reoperation in humans have shown repopulation of the conduit, although such recellularization is in accordance with similar animal studies.^18,75 During the FDA panel meeting in 2014 to re-evaluate decellularized heart valve classification, CryoLife, Inc. ⁷¹ presented results of their explanted decellularized allografts. The explanted valves had partial recellularization of the distal conduit but an absence of cellularity in the decellularized leaflet up to 11 years in vivo. ⁷¹ Leaflet repopulation was demonstrated during clinical use of the Matrix P^TM line of valves, a decellularized xenograft; however, the repopulating cells appear to be inflammatory rather than phenotypically appropriate valve cells.^63,64,90,91

Chemical conditioning for in situ recellularization

As autologous recellularization of decellularized valves is limited, a number of studies have investigated the use of chemical conditioning, or other chemical modifications, before implantation to increase valve recellularization (Table 3). This approach applies cytokine or signaling molecules to modulate the healing response of the host. Specifically, conditioning can be applied that is targeted at increasing cell attachment/migration, decreasing the immune response, increasing biologic activity, or increasing mechanical integrity. One approach designed to increase cell attachment has been the pre-implant conditioning of decellularized valves with fibronectin (FN), either alone or in combination with another growth factor.^92–94 In all examples, the valves treated with FN performed better than the respective decellularized or cryopreserved control valve groups.^92–94 FN treatment alone led to increased recellularization of the luminal side of the treated graft in a small animal model, although no information was given on specific leaflet recellularization. ⁹² FN in combination with stromal cell–derived factor 1α (SDF-1α) increased the surface and interstitial leaflet recellularization of valves implanted in sheep, as well as decreased the calcification, pannus formation, and immune response. ⁹³ FN plus hepatocyte growth factor (HGF) was used to achieve good recellularization in situ. ⁹⁴ Ota et al. ⁹⁴ treated decellularized porcine aortic valves with FN + HGF, and after 1 month of implantation in canines they observed great recellularization of the valve cusps with gene expression of vimentin (VIM) comparable to native valves.

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Table 3.

Summary of methods for in situ recellularization of decellularized valve scaffolds with chemical conditioning.

Recell method	Tissue	Conditioning	Implant model	Details	Results
In situ—chemical conditioning	rAV, 92 oAV, 93 pAV 94	Valves treated with FN, 92 FN + SDF-1α, 93 or FN + HGF 94	rIVC, 92 oPV, 93 cPV 94	FN-treated valves implanted	FN alone led to luminal recell; FN + SDF-1α led to moderate leaflet recell; FN + HGF led to great recell of the entire leaflet
	pPV95,96	CD133 conjugated to valve surface	oPV	Valves conjugated with CD133 and implanted	Early endothelial layer and leaflet interstitial recell with αSMA+ cells; MMP proteins present
	bPV, 97 hPV, 97 oPV23,98	Valves treated with collagen conditioning solution	bPV, 97 oPV23,98	Valves treated in conditioning solution before implant	Treated valves re-endothelialized but no distal leaflet recell; treated valves decreased antibody production in baboons

FN: fibronectin; SDF-1α: stromal cell–derived factor 1α; HGF: hepatocyte growth factor; αSMA: alpha-activated smooth muscle actin; MMP: matrix metallopeptidase.

The results are overall observations and the outcome of individual studies may vary. Lower case letters in acronyms denote species (o = ovine, p = porcine, h = human, r = rat, c = canine, b = baboon).

Another approach using chemical conditioning and in situ recellularization has investigated the conjugation of antibodies onto decellularized valve scaffolds to encourage in situ cell attachment. Antibody conjugation is an interesting method because it allows for selective attachment for the cell phenotype of interest. One group has explored conjugating CD133 antibodies onto decellularized valve scaffolds with the purpose of selectively targeting hematopoietic stem cells and endothelial progenitor cells (EPCs) from circulating blood.^95,96 After 3 months of implantation in the right ventricular outflow tract (RVOT) of sheep, the antibody-conjugated valves showed increased recellularization throughout the leaflet compared to non-conjugated and cell-seeded controls, including a von Willebrand factor (vWF)⁺ surface cell layer and an αSMA⁺ cell population in the leaflet interstitial tissue. ⁹⁵ Additionally, explanted valves showed an increase in collagen, GAG, and matrix metallopeptidase 9 (MMP-9) concentrations compared to non-conjugated valves, indicating both matrix production and remodeling. ⁹⁵ A follow-up study found that CD133⁺ cells were present by 3 days of implant, but from 30 to 90 days most cells were CD133⁻, αSMA⁺, and VIM⁺, demonstrating the transition and differentiation of the cell population from progenitor cell adhesion to mature valve-like cells (Figure 3). ⁹⁶ However, the increased expression of αSMA⁺ cells, compared to native controls, is noteworthy since αSMA indicates an aVIC phenotype that may lead to valve disease. ⁹⁶

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Figure 3.

Immunological staining demonstrating successful recellularization of leaflets from pulmonary valves conjugated with CD133 and implanted in the pulmonary position in sheep. Texas Red–labeled secondary antibodies show αSMA (top row) and vimentin (bottom row), and the nuclei are DAPI counterstained. Percentage values are the percent of cells with positive expression compared to the total number of cells which represent the mean calculated from all three leaflets. Note the high αSMA expression in the tissue-engineered leaflets compared to native leaflets. L denotes lumen. * indicates p < 0.05. Scale bars are 100 µm.

Another chemical conditioning strategy has been employed that aims to restore the mechanical properties of the decellularized scaffold and to make the ECM more hospitable for recellularization. This chemical treatment was accomplished by normalizing tissue pH, rehydrating the collagen helix moisture envelope, compacting collagen fibrils, and restoring soluble proteins by treating decellularized valves in a conditioning solution composed of citric acid, hyaluronic acid, lauryl alcohol, and species-specific protein.^12,23,97,98 Biomechanical evaluation revealed that the conditioning solution restored the stress relaxation behavior toward that of the native valve properties. ¹² In an ovine allograft valve implant model, the conditioning process led to better hemodynamic performance, compared to cryopreserved and decellularized valves implanted without conditioning.^23,98 Improved re-endothelialization and matrix recellularization were observed following conditioning, although the interstitial subsurface repopulation was limited to the proximal portion of the leaflet.^23,98 Similar results were observed using a baboon model and conditioned human valves to simulate human paradigms. ⁹⁷ Re-endothelialization and cusp base recellularization were observed in conditioned human valves after 26 weeks, as well as a decrease in class I and class II antibody production compared to cryopreserved valves, indicating reduced antigenicity. ⁹⁷ In all the above-mentioned animal studies, the conditioned valves performed better than the decellularized and cryopreserved valve controls, indicating they may function better clinically than the current standards of care. However, because the distal portions of the leaflet do not completely recellularize, the potential for a lifelong valve replacement without full leaflet active ECM remodeling is likely limited.

In vitro seeding without conditioning

Early work in the field of in vitro cell seeding of heart valves investigated the effect of seeding under static culture conditions without added mechanical or chemical conditioning (Table 4). Some of these first efforts studied the potential for leaflet repopulation by isolating the decellularized leaflets and statically seeding cells in culture flasks or well plates. A primary objective of this work was re-creating a healthy endothelial layer; therefore, seeded ECs were often used in these studies.^43,100,101 Endothelial cell seeding resulted in a confluent monolayer of cells on the leaflet surface that stained positive for vWF, but lead to little to no cell penetration into the leaflet. Interestingly, the study by Rieder et al. ¹⁰¹ found that the decellularization protocol and chemicals used greatly affected the re-endothelialization of decellularized leaflets, with SDS having cytotoxic effects and Triton X-100 leading to the best endothelial coverage. The value of ex vivo (and especially under static conditions) endothelial seeding seems low since proper decellularization and pre-implant conditioning seems to encourage autologous re-endothelialization (presumably effected by circulating EPCs). In addition, without an interstitial cell population, in vitro EC layers typically delaminate and are rapidly lost under physiologic flow conditions.

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Table 4.

Summary of in vitro recellularization methods of decellularized valve scaffolds with no conditioning steps applied.

Recell method	Cell source	Conditioning	Implant model	Details	Results
In vitro—no conditioning	ECs43,100–102	None	None	Leaflets seeded statically	EC coverage; no internal leaflet repopulation
	Fibroblast-like cells103–105	None	None	Leaflets seeded statically	Mild cell infiltration in leaflet interior; cells αSMA+ and VIM+
	MFs and ECs106,107	None	cPV 106 oPV 107	Valve seeded statically—no conditioning	Complete surface coverage and partial interstitial repopulation
	MNC or MSC 108	None	oPV	Cells injected into valve and implanted	Complete cell surface coverage for both groups; MNC leaflets were damaged; MSC leaflets were healthy with αSMA+ cells

EC: endothelial cell; αSMA: alpha-activated smooth muscle actin; VIM: vimentin; MF: myofibroblast; MNC: mononuclear cell; MSC: mesenchymal stem cell.

The results are overall observations and the outcome of individual studies may vary. Lower case letters in acronyms denote species (o = ovine, c = canine).

Other studies investigating cell seeding on isolated leaflets have attempted to repopulate the leaflet interstitium using fibroblast-like cells such as cardiac stromal cells (CStCs) and neonatal dermal fibroblasts with moderate success.^103–105 Dainese et al. ¹⁰³ seeded isolated leaflets with CStCs and observed up to 90% of native cellularity within 50 µm of the leaflet edge, yet they observed only 30% of native cellularity in the inner leaflet regions. These studies demonstrate the efficacy of introducing cells onto decellularized leaflet samples and they led to the seeding of intact decellularized valves. Kim et al. ¹⁰⁶ and Steinhoff et al. ¹⁰⁷ both seeded MFs and ECs onto decellularized pulmonary valves and subsequently implanted them into animal models without further in vitro conditioning. Kim et al. ¹⁰⁶ used an allogeneic model and seeded canine MFs and ECs onto decellularized canine pulmonary valves and implanted them in the pulmonary position of dogs. Steinhoff et al. ¹⁰⁷ used a xenogeneic animal model and seeded ovine MFs and ECs onto decellularized porcine pulmonary valves before implanting them in the pulmonary position of sheep. Neither group used chemical or mechanical conditioning before implantation, but allowed 6–7 days of static culture after seeding for cell adhesion and proliferation.^106,107 Following the longest implant periods evaluated, both studies demonstrated complete endothelialization and partial recellularization of the leaflet interior, although notably less than compared to native leaflet tissue.^106,107

An interesting study by Vincentelli et al. ¹⁰⁸ compared the efficacy of MSCs and MNCs for heart valve tissue engineering by directly injecting the cells into the arterial wall and annulus of decellularized valve scaffold, followed immediately by implantation in lambs. After 7 days in vivo, the injected cells in both groups had scattered throughout the matrix and host cells were also observed. At 4 months, both cell groups showed re-endothelialization but had markedly different recellularization responses. The MNC-seeded valves had thickened and retracted leaflets with calcified nodules and a high presence of CD68⁺ cells. The MSC-seeded valves had thin leaflets partially recellularized with αSMA⁺ cells and showed no signs of calcification. While MSC seeding elicited a more favorable in vivo response in this study, it is worth noting that the applicability of MNCs toward heart valve tissue engineering has been explored more recently using polymeric scaffolds in which seeded MNCs induced an inflammation-mediated recellularization response in vivo.^109,110 Considering the potential for regenerative paracrine signaling by seeded MNCs, it is not entirely clear whether the CD68⁺ inflammatory cells present in the MNC-seeded valves in the study by Vincentelli et al. ¹⁰⁸ were exhibiting an M1 or M2 inflammation response, or whether they would eventually be replaced by VIC-like cells. However, the condition of the leaflets and the presence of calcified nodules in the MNC-seeded valves were not encouraging.

In vitro seeding and mechanical conditioning

Mechanical conditioning of TEHVs utilizes bioreactors and mechanical stimulation with the goal of achieving a mature cell population in vitro (Table 5). Physiologic conditions of high fluid shear and pressure have been shown to encourage the VEC and VIC phenotypes, support cell proliferation, and encourage ECM remodeling.^111–117 Mechanical conditioning in bioreactors can simulate the physiological forces of heart valves and drive the differentiation and proliferation of seeded cells down the appropriate pathways.

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Table 5.

Various methods for in vitro recellularization of decellularized valve scaffolds with mechanical conditioning.

Recell method	Cell source	Conditioning	Implant model	Details	Results
In vitro—mechanical conditioning	MFs and ECs118–120	Cultured at pulmonary pressure and flow	None	Valves seeded with EC or MF then EC and then cultured	EC seeding led to surface coverage; MF and EC seeding led to great recell with appropriate phenotype
	MSC 121	Cultured at static, negative, or negative then positive pressure	None	Seeded valves cultured under various pressures	Negative and positive pressures led to EC coverage and moderate cell infiltration of HSP47, VIM+, and αSMA+ cells
	oMSC, 122 oEC 123	Cultured in pulsatile flow bioreactor then implanted	Ovine aorta 122 oAV 123	Seeded valves conditioned before implant	TE valves showed complete endothelium at explant; partial recellularization of leaflets
	hMNCs 88	Cultured in perfusion bioreactor	Human PV	Seeded valves conditioned before implant in two patients	In vitro seeding led to complete EC monolayer; both patients showed somatic growth, valve growth, and no valve degradation at 3.5 years

MF: myofibroblast; EC: endothelial cell; MSC: mesenchymal stem cell; HSP47: heat shock protein 47; VIM: vimentin; αSMA: alpha-activated smooth muscle actin; TE: tissue-engineered; MNC: mononuclear cell.

The results are overall observations and the outcome of individual studies may vary. Lower case letters in acronyms denote species (o = ovine, h = human).

Using a dynamic bioreactor, Lichtenberg et al.^119,120 explored the effects of mechanical conditioning after seeding pulmonary heart valves with vascular ECs. They demonstrated that culturing seeded valves under physiological pulmonic conditions (2.0 L/min, 60 bpm, 25 mmHg mean system pressure) can lead to complete re-endothelialization; however, care should be taken when introducing the seeded valve scaffolds to increased shear stress. ¹²⁰ Rapid increases in the pulsatile bioreactor flow (0.1–2.0 L/min; 0.35 L/min increases) led to significant interruptions of the endothelium. ¹¹⁹ Conversely, a stepwise, gradual increase in pulsatile flow (0.1–0.5 L/min; 0.1 L/min increases) resulted in a nearly complete endothelial layer. ¹¹⁹ These studies showed no recellularization of the leaflet interior, although it was not expected since they seeded ECs. A study by Schenke-Layland et al. ¹¹⁸ also investigated physiologic mechanical conditioning on valve recellularization. MFs were seeded onto trypsin decellularized porcine pulmonary valves and cultured under pulmonic conditions (3.0 L/min, 60 bpm, 60/40 mmHg), followed by subsequent seeding with ECs. After culture, the tissue-engineered leaflets were very well recellularized, to an extent comparable with native valves. Cells repopulating the leaflet interior were αSMA⁺ and VIM⁺ while the cells on the leaflet surface were vWF⁺. After recellularization and conditioning, tissue-engineered valves were also mechanically more similar to native valves than their decellularized-only counterparts. Indeed, the only noticeable difference between the tissue-engineered valves and native valves was the presence of αSMA⁺ cells throughout the tissue-engineered leaflets, likely due to the seeded myofibroblasts. While these results are promising, the high αSMA expression is indicative of aVICs, which are responsible for tissue remodeling but their prolonged activation can also lead to valve disease. ⁷⁸

A different approach using mechanical conditioning under non-physiological conditions has been investigated as a means of repopulating the interstitium of the decellularized leaflet. Converse et al. ¹²¹ studied the effects of cyclic negative and positive pressures on the recellularization of decellularized valves seeded with bone marrow–derived MSCs. After seeding with MSCs, the valves were cultured in a static bioreactor for 24 h, either conditioned in negative cyclic pressure (5 to −20 mmHg) for 72 h or conditioned at the same negative cyclic pressure for 72 h followed by an additional 10 days conditioning at positive pressure up to 50 mmHg. Static cultured resulted in clumping of the cells on the valve surface and minimal cell infiltration. Mechanical conditioning under negative pressures resulted in a more even distribution of cell coverage on the surface of the leaflet and limited infiltration of cells into the interstitium of the leaflet. Valves subjected to a combination of negative and positive pressures conditioning exhibited further improved cell coverage and displayed moderate cell infiltration, although still less than native. The repopulated interior cells stained positive for CD90, CD29, heat shock protein 47 (HSP47), VIM, and αSMA. Biaxial mechanical testing also revealed that increased culture under positive pressure resulted in mechanical properties more similar to cryopreserved valves. ¹²¹

Despite encouraging bench-top studies, mechanically conditioned, in vitro seeded valves implanted in animal and pediatric models have only seen moderate recellularization success.^122,123 Tudorache et al. ¹²³ seeded ovine aortic valves with ovine ECs followed by pulsatile flow conditioning up to 1.0 L/min over the course of a week. After conditioning, the tissue-engineered valves showed a complete endothelial layer on the leaflet surface although no interstitial cells were present. The conditioned valves were implanted in the descending aorta of sheep for 3 months, at which time all valves exhibited normal function. A complete endothelial layer was observed, as expected; however, there was very minimal leaflet interstitial recellularization. ¹²³ In this study, mechanical conditioning resulted in endothelialization in the bioreactor, yet the benefit remains unclear since implanted decellularized valves will re-endothelialize autologously, especially in sheep.^23,24 One case study investigated pediatric clinical use of mechanically conditioned tissue-engineered valves. Cebotari et al. ⁸⁸ implanted tissue-engineered valves in two pediatric patients using decellularized human pulmonary valve scaffolds seeded with autologous MNCs isolated from peripheral blood. After seeding, the valves were cultured in a continuous perfusion bioreactor using a very low flow rate (15 mL/min) for 21 days. ⁸⁸ Pre-implant histology revealed a confluent monolayer of cells along the leaflet surface with markers positive for EC types. At 3.5 years of follow-up, both patients had recovered normally and had experienced somatic growth, valve annulus growth, and no signs of degradation, stenosis, or cusp thickening. These are highly encouraging results; however, the sample size is very limited and no further follow-up data have been provided so the long-term outcome remains unclear.

In vitro seeding and chemical modification

Another common approach to increase the recellularization of in vitro seeded TEHVs has been to chemically modify the decellularized valve scaffolds prior to seeding (Table 6). Chemical modification utilizes cytokines, chemokines, growth factors, antibodies, and polymers to support phenotype-specific cellular attachment, bolster valve mechanics, and drive cell differentiation.

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Table 6.

Various methods for in vitro recellularization of decellularized valve scaffolds with chemical conditioning.

Recell method	Cell source	Conditioning	Implant model	Details	Results
In vitro—chemical conditioning	rMSC, 124 mMF, 125 hMF, 125 hEC 125	Valves coated with P3/4HB before seeding	None	Hybrid valves seeded in vitro	Hybrid valves had increased mechanics; in vitro seeding led to cell coverage, but no infiltration
	rMF, 126 hECs,127,128 hMF 129	Leaflets modified with PEG plus TGF-β1, VEGF, or RGD peptides	None	PEG-peptide-modified leaflets seeded with cells	PEGylation increased mechanics and cell surface density, regardless of additional peptide; no cellular infiltration
	gMSC 130	Encapsulation of cells in PEG before seeding	Goat aorta	PEG encapsulated cells seeded on decell scaffold and then implanted in goats	PEG cell seeding increased tensile strength, increased ratio of endothelial cells, and decreased thrombosis
	rMSC, 131 hEPC132,133	Polyelectrolyte layers of heparin and SDF-1α/chitosan/VEGF adsorbed onto valve	None,132,133 rAorta 131	Valve scaffolds coated with polyelectrolyte multilayers and then seeded with cells	Treated valves led to reduced platelet activation; chitosan and VEGF increased EC adherence and proliferation in vitro; SDF-1α had increased endothelial layer after implant in vivo
	oECs 134	Valves coated in CCN1	oPV	Valves coated in CCN1 and before implant	Treated valves had good recell in vivo; cell coverage higher on ventricularis with mild infiltration of VIM+ and αSMA+ cells
	rMSCs 135	Valves conjugated with CD90 antibody	None	Treated leaflets cultured in shear flow chamber with MSCs	Treated leaflets had increased cell attachment distributed across surface
	pVIC, 136 hMSC 11	Valves treated with fibronectin before seeding	None	Treated leaflets seeded statically	Leaflet surface coverage and mild cell infiltration by appropriate phenotypes
	oECs 137	Valves treated with fibronectin before seeding	oPV	Treated valves seeded and then implanted	Treated valves had complete EC layer and good interstitial repopulation
	hECs19–21	Valve treated with ProNectin F before seeding	hPV	Treated valves seeded and implanted in 11 patients	100% survival at 10 years 3 months biopsy of valve wall showed endothelialization and partial recell by fibroblast cells

MSC: mesenchymal stem cell; MF: myofibroblast; EC: endothelial cell; P3/4HB: poly(3-hydroxybutyrate-co-4-hydroxybutyrate); PEG: polyethylene glycol; TGF-β1: transforming growth factor beta 1; VEGF: vascular endothelial growth factor; EPC: endothelial progenitor cell; SDF-1α: stromal cell–derived factor 1α; VIM: vimentin; αSMA: alpha-smooth muscle actin; VIC: valvular interstitial cell.

The results are overall observations and the outcome of individual studies may vary. Lower case letters in acronyms denote species (o = ovine, p = porcine, h = human, m = mouse, r = rat, g = goat).

As discussed previously, decellularization can have deleterious effects on the mechanics of the valve leaflet ECM. Chemical crosslinking has been explored as a means of improving the structural and mechanical integrity of the decellularized ECM. Conventional crosslinking methods using glutaraldehyde block immunogenic antigens, yet glutaraldehyde is also cytotoxic preventing cellular ingrowth and therefore is not appropriate for recellularizing valve scaffolds. Other crosslinking methods have been explored using procyanidin, quercetin, and nordihydroguaiaretic acid (NDGA), which have been found to improve the mechanics of the valve leaflets and are not cytotoxic at low concentrations.^138–140 After crosslinking, the treated leaflets had increased UTS and elastic modulus compared to untreated decellularized leaflets and in some cases greater than comparable fresh leaflets or glutaraldehyde-treated leaflets.^138–140 The recellularization potential after leaflet crosslinking remains largely unknown, however, as only NDGA-treated valves have been briefly seeded with ECs for 24 h, although in that time ECs showed adhesion and proliferation on the crosslinked leaflets. ¹³⁸

Other chemical modifications that can bolster the mechanical integrity of valve leaflets are the incorporation of polymers into the decellularized valves to create hybrid valve scaffolds. These hybrid valves have improved mechanical properties compared to decellularized valves, and the polymer additions can act as a transport mechanism for drug delivery. The biodegradable polymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB) has been applied to decellularized porcine aortic valves through impregnation into the tissue or electrospinning onto the valve surface.^124,125 Hybrid valves created through P3/4HB electrospinning followed by seeding with MSCs under static conditions had a greater maximum load carrying capacity, UTS, and elastic modulus than their decellularized-only counterparts; however, recellularization was similar between groups and limited to the leaflet surface. ¹²⁴ Impregnated P3/4HB hybrid valves also had increased biomechanics and could support the culture of mouse MFs, human MFs, and human ECs, but only on the valve surface. ¹²⁵ A more common polymer for creating hybrid valves is PEG because it is highly hydrophilic, water soluble, and has active functional groups that facilitate peptide conjugation.^126–130 Two groups have investigated modifying decellularized porcine aortic valve leaflets with PEG conjugated with transforming growth factor beta 1 (TGF-β1), vascular endothelial growth factor (VEGF), and/or RGD.^126–129 They found that PEGylation of decellularized valves resulted in mechanical properties similar to native valves and that conjugation of various peptides increased surface recellularization after seeding with rat MFs or human umbilical cord vascular ECs.^126–128 In fact, in all studies the PEG-peptide-modified valves showed greater surface cell density than valves treated with PEG only or decellularized control valves; however, none of the PEG-peptide-modified valves resulted in repopulation of the leaflet interstitium.^126–128

Another group has explored chemical modification of decellularized valves by applying polyelectrolyte multilayers (PEMs) composed of heparin plus VEGF, chitosan, or SDF-1α.^131–133 Decellularized porcine aortic valves treated with PEM of heparin–chitosan or heparin–VEGF were subsequently seeded with EPCs, and it was observed that both PEM coatings improved hemocompatibility, showing decreased platelet adhesion and activation.^132,133 Although seeding with EPCs did not result in leaflet interstitial recellularization, valves modified with heparin–chitosan were able to support an EC population while valves modified with heparin–VEGF actually had better adhesion, proliferation, and migration of ECs compared to decellularized control valves.^132,133 Zhou et al. ¹³¹ applied a PEM of heparin–SDF-1α to decellularized rat aortic valves prior to seeding with rat bone marrow MSCs and observed increased proliferation and migration of MSCs on the modified scaffolds. Zhou et al. ¹³¹ also implanted unseeded, heparin–SDF-1α-modified valves in rats and observed luminal re-endothelialization of the aortic wall; however, there was no indication of leaflet recellularization.

Other efforts involving chemical modification of in vitro seeded decellularized valve scaffolds have aimed at increasing the adhesion of both the seeded cells and host cells in vivo. Theodoridis et al. ¹³⁴ coated decellularized ovine pulmonary valves in CCN1, a matricellular protein associated with cell adhesion, proliferation, and differentiation, before seeding them with ovine ECs and implanting them in sheep. At explant, the modified valves had greater cell surface coverage and partial interstitial recellularization by VIM⁺ and αSMA⁺ cells. ¹³⁴ Similarly, Ye et al. ¹³⁵ conjugated decellularized porcine aortic valve leaflets with an anti-human CD90 antibody and then cultured them in a shear flow bioreactor with media containing an MSC population. The modified leaflets had a greater cell population distributed evenly across the surface than the un-modified control samples. ¹³⁵ These approaches demonstrate that conjugation of peptides and antibodies can increase the adhesion of cells onto valve surfaces. However, this approach has not yet resulted in the complete repopulation of the leaflet interior. Increasing surface cell density through chemical modification may encourage cell infiltration to a small degree, but other chemokines may be required to repopulate the entire leaflet.

Decellularized valves have also been treated with FN before in vitro cell seeding. FN has been shown to promote differentiation into a VIC phenotype by human MSCs and to reduce the formation of calcific nodules by VICs, particularly in combination with VEGF.^141,142 Individual decellularized leaflets treated with FN showed only moderate recellularization when seeded with VICs or MSCs, although immunohistochemistry revealed appropriate differentiation into the correct cell phenotypes (surface cells were vWF⁺ and interstitial cells were αSMA⁺ and VIM⁺).^11,136 Dohmen et al. ¹³⁷ treated decellularized porcine pulmonary valves with FN, seeded them with autologous ovine ECs, and implanted them in the pulmonary position of sheep for 6 months. At explant, FN-treated/EC-seeded valves had a complete EC monolayer throughout the valve and good internal recellularization of the leaflet, while valves treated with FN but not seeded had only a partial EC layer and minimal leaflet recellularization. ¹³⁷ The same group also performed a small clinical study using the same in vitro seeding and FN treatment approach. Between 2000 and 2002, 11 patients underwent pulmonary valve replacement with a tissue-engineered pulmonary heart valve treated with ProNectin F (a synthetic FN alternative) and seeded with autologous ECs.^19–21 At 10 years of follow-up, there was a 100% survival rate and transthoracic echocardiography showed patent and functioning valves in all patients. ²⁰ At 3 months, one patient required a non-related reoperation, during which a small biopsy was taken from the conduit of the TEHV. ¹⁸ Histology of the biopsy revealed repopulation of the conduit wall by CD31⁺ and vWF⁺ surface cells and fibroblast-like interstitial cells. ¹⁸