Mechanical Stimulation Induces Vasa Vasorum Capillary Alignment in a Fibrin-Based Tunica Adventitia.

Generation of bioartificial blood vessels with a physiological three-layered wall architecture is a long pursued goal in vascular tissue engineering. While considerable advances have been made to resemble the physiological tunica intima and media morphology and function in bioartificial vessels, only very few studies have targeted the generation of a tunica adventitia, including its characteristic vascular network known as the vasa vasorum, which are essential for graft nutrition and integration. In healthy native blood vessels, capillary vasa vasorum are aligned longitudinally to the vessel axis. Thus, inducing longitudinal alignment of capillary tubes to generate a physiological tunica adventitia morphology and function may be advantageous in bioengineered vessels as well. In this study, we investigated the effect of two biomechanical stimulation parameters, longitudinal tension and physiological cyclic stretch, on tube alignment in capillary networks formed by self-assembly of human umbilical vein endothelial cells in tunica adventitia-equivalents of fibrin-based bioartificial blood vessels. Moreover, the effect of changes of the biomechanical environment on network remodeling after initial tube formation was analyzed. Both, longitudinal tension and cyclic stretch by pulsatile perfusion induced physiological capillary tube alignment parallel to the longitudinal vessel axis. This effect was even more pronounced when both biomechanical factors were applied simultaneously, which resulted in an alignment of 57.2 ± 5.2% within 5° of the main vessel axis. Opposed to that, a random tube orientation was observed in vessels incubated statically. Scanning electron microscopy showed that longitudinal tension also resulted in longitudinal alignment of fibrin fibrils, which may function as a guidance structure for directed capillary tube formation. Moreover, existing microvascular networks showed distinct remodeling in response to addition or withdrawal of mechanical stimulation with corresponding increase or decrease of the degree of alignment. With longitudinal tension and cyclic stretch, we identified two mechanical stimuli that facilitate the generation of a prevascularized tunica adventitia-equivalent with physiological tube alignment in bioartificial vascular grafts. Impact statement Fibrin-based bioartificial vessels represent a promising regenerative approach to generate vascular grafts with superior biocompatibility and hemocompatibility compared to currently available synthetic graft materials. Precapillarization of bioartificial vascular grafts may improve nutrition of the vessel wall and integration of the graft into the target organism's microvasculature. In native vessels, physiological vasa vasorum alignment is pivotal for proper function of the tunica adventitia. Thus, it is necessary to induce longitudinal capillary alignment in the tunica adventitia of bioengineered vessels as well to secure long-term graft patency and function. This alignment can be reliably achieved by controlled biomechanical stimulation in vitro.

An encapsulated fibrin-based bioartificial tissue construct with integrated macrovessels, microchannels, and capillary tubes.

Facilitating sufficient nutrient and oxygen supply in large-scale bioartificial constructs is a critical step in organ bioengineering. Immediate perfusion not only depends on a dense capillary network, but also requires integrated large-diameter vessels that allow vascular anastomoses during implantation. These requirements set high demands for matrix generation as well as for in vitro cultivation techniques and remain mostly unsolved challenges up until today. Additionally, bioartificial constructs must have sufficient biomechanical stability to withstand mechanical stresses during and after implantation. We developed a bioartificial tissue construct with a fibrin matrix containing human umbilical vein endothelial cells and adipose tissue-derived stem cells facilitating capillary-like network formation. This core matrix was surrounded by a dense acellular fibrin capsule providing biomechanical stability. Two fibrin-based macrovessels were integrated on each side of the construct and interconnected via four 1.2 mm thick microchannels penetrating the cellularized core matrix. After 4 days of perfusion in a custom-built bioreactor, homogeneous capillary-like network formation throughout the core matrix was observed. The fibrin capsule stabilized the core matrix and facilitated the generation of a self-supporting construct. Thus, the encapsulated fibrin tissue construct could provide a universal prevascularized matrix for seeding with different cell types in various tissue engineering approaches.

Establishment of a Modular Hemodynamic Simulator for Accurate In Vitro Simulation of Physiological and Pathological Pressure Waveforms in Native and Bioartificial Blood Vessels.

PURPOSE: In vitro stimulation of native and bioartificial vessels in perfusable systems simulating natural mechanical environments of the human vasculature represents an emerging approach in cardiovascular research. Promising results have been achieved for applications in both regenerative medicine and etiopathogenetic investigations. However, accurate and reliable simulation of the wide variety of physiological and pathological pressure environments observed in different vessels still remains an unmet challenge. METHODS: We established a modular hemodynamic simulator (MHS) with interchangeable and modifiable components suitable for the perfusion of native porcine-(i.e. the aorta, brachial and radial arteries and the inferior vena cava) and bioartificial fibrin-based vessels with anatomical site specific pressure curves. Additionally, different pathological pressure waveforms associated with cardiovascular diseases including hyper- and hypotension, tachy- and bradycardia, aortic valve stenosis and insufficiency, heart failure, obstructive cardiomyopathy and arterial stiffening were simulated. Pressure curves, cyclic distension and shear stress were measured for each vessel and compared to ideal clinical pressure waveforms. RESULTS: The pressure waveforms obtained in the MHS showed high similarity to the ideal anatomical site specific pressure curves of different vessel types. Moreover, the system facilitated accurate emulation of physiological and different pathological pressure conditions in small diameter fibrin-based vessels. CONCLUSION: The MHS serves as a variable in vitro platform for accurate emulation of physiological and pathological pressure environments in biological probes. Potential applications of the system include bioartificial vessel maturation in cardiovascular tissue engineering approaches as well as etiopathogenetic investigations of various cardiovascular pathologies.

Pressure-compacted and spider silk-reinforced fibrin demonstrates sufficient biomechanical stability as cardiac patch in vitro.

OBJECTIVE: The generation of bio-/hemocompatible cardiovascular patches with sufficient stability and regenerative potential remains an unmet goal. Thus, the aim of this study was the generation and in vitro biomechanical evaluation of a novel cardiovascular patch composed of pressure-compacted fibrin with embedded spider silk cocoons. METHODS: Fibrin-based patches were cast in a customized circular mold. One cocoon of Nephila odulis spider silk was embedded per patch during the casting process. After polymerization, the fibrin clot was compacted by 2 kg weight for 30 min resulting in thickness reduction from up to 2 cm to <1 mm. Tensile strength and burst pressure was determined after 0 weeks and 14 weeks of storage. A sewing strength test and a long-term load test were performed using a customized device to exert physiological pulsatile stretching of a silicon surface on which the patch had been sutured. RESULTS: Fibrin patches resisted supraphysiological pressures of well over 2000 mmHg. Embedding of spider silk increased tensile force 1.8-fold and tensile strength 1.45-fold (p < .001), resulting in a final strength of 1.07 MPa and increased sewing strength. Storage for 14 weeks decreased tensile strength, but not significantly and suturing properties of the spider silk patches were satisfactory. The long-term load test indicated that the patches were stable for 4 weeks although slight reduction in patch material was observed. CONCLUSION: The combination of compacted fibrin matrices and spider silk cocoons may represent a feasible concept to generate stable and biocompatible cardiovascular patches with regenerative potential.

Transluminal compression increases mechanical stability, stiffness and endothelialization capacity of fibrin-based bioartificial blood vessels.

Fibrin is used successfully as a biological matrix in various bioengineering approaches. Its unique combination of autologous availability, hemocompatibility and biological activity makes it an almost ideal matrix material for vascular tissue engineering. However, clinical application of fibrin-based bioartificial blood vessels is still limited due to insufficient mechanical stability and stiffness of fibrin matrices. Biomechanical properties of fibrin-based constructs can potentially be modified by adjusting matrix density. Thus, as an attempt to optimize strength and elasticity of fibrin matrices for vascular tissue engineering applications, we developed a simple and reproducible method for transluminal compression of small-diameter fibrin-based vessels: After initial polymerization of high-concentration fibrin matrices in a vascular mold, vessels were compressed using an intraluminal angioplasty balloon. Vessels compacted with different pressures were compared for ultimate strength, elastic and structural properties and cellularization capacity. Transluminal compression increased fibrin network density and facilitated rapid production of homogenous vessels with a length of 10 cm. Compared to non-compressed controls, compacted fibrin vessels showed superior maximal burst pressure (199.8 mmHg vs. 94.0 mmHg), physiological elastic properties similar to the elastic behavior of natural arteries and higher luminal endothelial cell coverage (98.6% vs. 34.6%). Thus, transluminal compaction represents a suitable technique to enhance biomechanical properties of fibrin-based bioartificial vessels while preserving the biological advantages of this promising biomaterial.

Chemically induced hypoxia by dimethyloxalylglycine (DMOG)-loaded nanoporous silica nanoparticles supports endothelial tube formation by sustained VEGF release from adipose tissue-derived stem cells.

Inadequate vascularization leading to insufficient oxygen and nutrient supply in deeper layers of bioartificial tissues remains a limitation in current tissue engineering approaches to which pre-vascularization offers a promising solution. Hypoxia triggering pre-vascularization by enhanced vascular endothelial growth factor (VEGF) expression can be induced chemically by dimethyloxalylglycine (DMOG). Nanoporous silica nanoparticles (NPSNPs, or mesoporous silica nanoparticles, MSNs) enable sustained delivery of molecules and potentially release DMOG allowing a durable capillarization of a construct. Here we evaluated the effects of soluble DMOG and DMOG-loaded NPSNPs on VEGF secretion of adipose tissue-derived stem cells (ASC) and on tube formation by human umbilical vein endothelial cells (HUVEC)-ASC co-cultures. Repeated doses of 100 µM and 500 µM soluble DMOG on ASC resulted in 3- to 7-fold increased VEGF levels on day 9 (P < 0.0001). Same doses of DMOG-NPSNPs enhanced VEGF secretion 7.7-fold (P < 0.0001) which could be maintained until day 12 with 500 µM DMOG-NPSNPs. In fibrin-based tube formation assays, 100 µM DMOG-NPSNPs had inhibitory effects whereas 50 µM significantly increased tube length, area and number of junctions transiently for 4 days. Thus, DMOG-NPSNPs supported endothelial tube formation by upregulated VEGF secretion from ASC and thus display a promising tool for pre-vascularization of tissue-engineered constructs. Further studies will evaluate their effect in hydrogels under perfusion.

A 3-Layered Bioartificial Blood Vessel with Physiological Wall Architecture Generated by Mechanical Stimulation.

The generation of cellularized bioartificial blood vessels resembling all three layers of the natural vessel wall with physiological morphology and cell alignment is a long pursued goal in vascular tissue engineering. Simultaneous culture of all three layers under physiological mechanical conditions requires highly sophisticated perfusion techniques and still today remains a key challenge. Here, three-layered bioartificial vessels based on fibrin matrices were generated using a stepwise molding technique. Adipose-derived stem cells (ASC) were differentiated to smooth muscle cells (SMC) and integrated in a compacted tubular fibrin matrix to resemble the tunica media. The tunica adventitia-equivalent containing human umbilical vein endothelial cells (HUVEC) and ASC in a low concentration fibrin matrix was molded around it. Luminal seeding with HUVEC resembled the tunica intima. Subsequently, constructs were exposed to physiological mechanical stimulation in a pulsatile bioreactor for 72 h. Compared to statically incubated controls, mechanical stimulation induced physiological cell alignment in each layer: Luminal endothelial cells showed longitudinal alignment, cells in the media-layer were aligned circumferentially and expressed characteristic SMC marker proteins. HUVEC in the adventitia-layer formed longitudinally aligned microvascular tubes resembling vasa vasorum capillaries. Thus, physiologically organized three-layered bioartificial vessels were successfully manufactured by stepwise fibrin molding with subsequent mechanical stimulation.

Perfusion promotes endothelialized pore formation in high concentration fibrin gels otherwise unsuitable for tube development.

Vascularization of tissue engineered implants is crucial for their survival and integration in the recipient's body. Pre-vascularized, fibrin-based implants offer a solution since low concentration fibrin hydrogels (1 mg/mL) have been shown to promote tube formation of endothelial cells in co-culture with adipogenic stem cells. However, higher fibrinogen concentrations (> 20 mg/mL) enabling the fabrication of stable implants are necessary.We here characterized fibrin gels of 1-30 mg/mL for their rheological properties and whether they support tube formation of endothelial cell-adipogenic stem cell co-cultures for up to 7 days. Moreover, 20 mg/mL gels containing preformed channels and endothelial cell-adipogenic stem cell co-culture were perfused continuously in a customized flow chamber with 3.9 dyn/cm2 for 12 days and analyzed for capillary formation.Rheology of fibrin gels showed increasing stability proportional to fibrinogen concentration with 20 mg/mL gels having a storage module of 465 Pa. Complex tube networks stable for 7 days were observed at 1-5 mg/mL gels whereas higher concentrations showed initial sprouting only. However, perfusion of 20 mg/mL fibrin gels resulted in endothelialized pore formation in several layers of the gel with endothelial cell-adipogenic stem cell co-culture.Thus, perfusion supports the formation of capillary-like structures in fibrin gels that are too dense for spontaneous tube formation under static conditions. Future studies are necessary to further increase pore density and to investigate proper nutrition of tissue-specific target cells in the scaffold.

Complete Myogenic Differentiation of Adipogenic Stem Cells Requires Both Biochemical and Mechanical Stimulation.

Vascular tissue engineering of the middle layer of natural arteries requires contractile smooth muscle cells (SMC) which can be differentiated from adipose-derived mesenchymal stem cells (ASC) by treatment with transforming growth factor-ß, sphingosylphosphorylcholine and bone morphogenetic protein-4 (TSB). Since mechanical stimulation may support or replace TSB-driven differentiation, we investigated its effect plus TSB-treatment on SMC orientation and contractile protein expression. Tubular fibrin scaffolds with incorporated ASC or pre-differentiated SMC were exposed to pulsatile perfusion for 10 days with or without TSB. Statically incubated scaffolds served as controls. Pulsatile incubation resulted in collagen-I expression and orientation of either cell type circumferentially around the lumen as shown by alpha smooth muscle actin (αSMA), calponin and smoothelin staining as early, intermediate and late marker proteins. Semi-quantitative Westernblot analyses revealed strongly increased αSMA and calponin expression by either pulsatile (12.48-fold; p < 0.01 and 38.15-fold; p = 0.07) or static incubation plus TSB pre-treatment (8.91-fold; p < 0.05 and 37.69-fold; p < 0.05). In contrast, contractility and smoothelin expression required both mechanical and TSB stimulation since it was 2.57-fold increased (p < 0.05) only by combining pulsatile perfusion and TSB. Moreover, pre-differentiation of ASC prior to pulsatile perfusion was not necessary since it could not further increase the expression level of any marker.

Decellularized equine carotid artery layers as matrix for regenerated neurites of spiral ganglion neurons.

Today's best solution in compensating for sensorineural hearing loss is the cochlear implant, which electrically stimulates the spiral ganglion neurons in the inner ear. An optimum hearing impression is not ensured due to, among other reasons, a remaining anatomical gap between the spiral ganglion neurons and the implant electrodes. The gap could be bridged via pharmacologically triggered neurite growth toward the electrodes if biomaterials for neurite guidance could be provided. For this, we investigated the suitability of decellularized tissue. We compared three different layers (tunica adventitia, tunica media, and tunica intima) of decellularized equine carotid arteries in a preliminary approach. Rat spiral ganglia explants were cultured on decellularized equine carotid artery layers and neurite sprouting was assessed quantitatively. Generally, neurite outgrowth was possible and it was most prominent on the intima (in average 83 neurites per spiral ganglia explants, followed by the adventitia (62 neurites) and the lowest growth on the media (20 neurites). Thus, decellularized equine carotid arteries showed promising effects on neurite regeneration and can be developed further as efficient biomaterials for neural implants in hearing research.

Biochemical Myogenic Differentiation of Adipogenic Stem Cells Is Donor Dependent and Requires Sound Characterization.

IMPACT STATEMENT: We here showed that even under optimized conditions for biochemical differentiation of adipose-derived stem cells (with respect to a pronounced marker protein expression for a reasonable period of time) it was not possible to obtain functional smooth muscle cells from all donors. Moreover, an underestimated role may play the effect of the scaffold material on smooth muscle cell functionality. Both aspects are crucial for the successful tissue engineering of the vascular medial layer combining autologous cells with a suitable scaffold material and thus should be thoroughly addressed in each individualized therapeutic approach.

Biocompatibility and Immunogenicity of Decellularized Allogeneic Aorta in the Orthotopic Rat Model.

IMPACT STATEMENT: The generation of a small-caliber arterial graft, utilizing a large vessel of a small animal, such as the aorta of the rat or rabbit, for clinical use in the peripheral arterial tree, can widen the options for arterial prostheses. This in vivo study demonstrated the ability of the decellularization protocol that was used to produce a noncytotoxic acellular small-caliber arterial graft, with sufficient biomechanical and biological integrity to withstand the demanding flow and pressure environment of the rat aorta. This work also demonstrated the superiority of the decellularized homograft over its intact counterpart, in terms of lower immunogenicity.

Dehydration improves biomechanical strength of bioartificial vascular graft material and allows its long-term storage.

INTRODUCTION: We have recently reported about a novel technique for the generation of bioartificial vascular grafts based on the use of a compacted fibrin matrix. In this study, we evaluated the effects of a dehydration process on the biomechanical properties of compacted fibrin tubes and whether it allows for their long-term storage. MATERIALS AND METHODS: Fibrin was precipitated from fresh frozen plasma by means of cryoprecipitation and simultaneously with a thrombin solution applied in a high-speed rotating casting mold. Subsequent dehydration of the fibrin tubes (29/38) was performed in dry air with a dilator inside the tube to prevent the collapse of the lumen. Dehydrated fibrin tubes were stored for six (n=9) and 12 months (n=10) at room temperature. Comparative analysis was done on initially generated and dehydrated fibrin tubes before and after storage to evaluate the effects of the dehydration process and storage on the biomechanical properties and structure of the tubes. RESULTS: Thirty-eight fibrin tubes were generated by high-speed rotation-molding from 142±3 mg fibrinogen with an inner diameter of 5.8±0.1 mm and a length of 100 mm. A centrifugal force of nearly 900×g compacted applied fibrin, while fluid was pressed out of the matrix and drained from the mold via holes resulting in a 16-fold compaction of the fibrin matrix. Dehydration was characterized by shrinkage of the tubes to a diameter of 3.2±0.2 mm, while the length remained at 100 mm equivalent to a further two-fold compaction. The biomechanical strength of the dehydrated fibrin tubes significantly increased to values comparable to that of native ovine carotid arteries and maintained during the first 6 months of storage. After 12 months of storage, only five of 10 tubes were intact, and only one showed maintained biomechanical strength. DISCUSSION: Compaction of a fibrin matrix in high-speed rotation-moulding and subsequent dehydration enables for the construction of small-caliber fibrin grafts. Over and above, the dehydration process allows their storage and stockpiling as a prerequisite for clinical use.

Biocompatibility of Intensified Decellularized Equine Carotid Arteries in a Rat Subcutaneous Implantation Model and in a Human In Vitro Model.

Limited biocompatibility of decellularized scaffolds is an ongoing challenge in tissue engineering. We recently demonstrated that intensified detergent-based decellularization of equine carotid artery (dEACintens) removed residual cellular molecules from the scaffold more efficiently than a conventional decellularization (dEACcon), although this approach did not eliminate its immunogenicity entirely. CCN1 has been shown to improve biocompatibility of dEACcon in a sheep model. In this study, we tested the biocompatibility of dEACintens and dEACcon with or without CCN1 coating after subcutaneous implantation in rats for up to 12 weeks. Explants were assessed by conventional histopathology and immunostaining for infiltrating M2 macrophages. Moreover, human macrophages derived from monocytes (MDM) or THP-1 cells (THP-derived macrophages [TDM]) were seeded onto dEACcon and dEACintens, and activation was assessed either by cytokine expression or matrix metalloprotease 2 and 7 staining. dEACintens showed a significantly reduced inflammatory infiltration (52%; p < 0.0001), as well as an earlier and denser neovascularization (1.4-fold, p < 0.0001) independent of CCN1 coating, which, however, reduced fibrosis exclusively with dEACintens (26-53%; p < 0.05). Human MDM seeded for 48 h onto dEACintens showed higher transcript levels for anti-inflammatory IL-10 (2.3-fold), proinflammatory TNFα (2.2-fold), and macrophage/monocyte recruiting MIP1α (3.5-fold; all p < 0.05) and MCP (2.7-fold; p < 0.01), whereas 1.92-fold more TDM on dEACintens showed staining for MMP2 (p > 0.001). Thus, although being advantageous in regard to fibrosis, CCN1 coating of dEACintens does not appear to be necessary for further improving dEACintens excellent biocompatibility in rats. In humans, the unspecific cellular immune response toward dEACintens seemed to be more complex, but generally comparable to the mild acute inflammatory tissue reaction with high remodeling activity as observed after rat subcutaneous implantation.

Low Immunogenic Endothelial Cells Maintain Morphological and Functional Properties Required for Vascular Tissue Engineering.

The limited availability of native vessels suitable for the application as hemodialysis shunts or bypass material demands new strategies in cardiovascular surgery. Tissue-engineered vascular grafts containing autologous cells are considered ideal vessel replacements due to the low risk of rejection. However, endothelial cells (EC), which are central components of natural blood vessels, are difficult to obtain from elderly patients of poor health. Umbilical cord blood represents a promising alternative source for EC, but their allogeneic origin corresponds with the risk of rejection after allotransplantation. To reduce this risk, the human leukocyte antigen class I (HLA I) complex was stably silenced by lentiviral vector-mediated RNA interference (RNAi) in EC from peripheral blood and umbilical cord blood and vein. EC from all three sources were transduced by 93.1% ± 4.8% and effectively, HLA I-silenced by up to 67% compared to nontransduced (NT) cells or transduced with a nonspecific short hairpin RNA, respectively. Silenced EC remained capable to express characteristic endothelial surface markers such as CD31 and vascular endothelial cadherin important for constructing a tight barrier, as well as von Willebrand factor and endothelial nitric oxide synthase important for blood coagulation and vessel tone regulation. Moreover, HLA I-silenced EC were still able to align under unidirectional flow, to take up acetylated low-density lipoprotein, and to form capillary-like tube structures in three-dimensional fibrin gels similar to NT cells. In particular, addition of adipose tissue-derived mesenchymal stem cells significantly improved tube formation capability of HLA I-silenced EC toward long and widely branched vascular networks necessary for prevascularizing vascular grafts. Thus, silencing HLA I by RNAi represents a promising technique to reduce the immunogenic potential of EC from three different sources without interfering with EC-specific morphological and functional properties required for vascular tissue engineering. This extends the spectrum of available cell sources from autologous to allogeneic sources, thereby accelerating the generation of tissue-engineered vascular grafts in acute clinical cases.

Antibody formation towards porcine tissue in patients implanted with crosslinked heart valves is directed to antigenic tissue proteins and αGal epitopes and is reduced in healthy vegetarian subjects.

BACKGROUND: Glutaraldehyde-fixed porcine heart valves (ga-pV) are one of the most frequently used substitutes for insufficient aortic and pulmonary heart valves which, however, degenerate after 10-15 years. Yet, xeno-immunogenicity of ga-pV in humans including identification of immunogens still needs to be investigated. We here determined the immunogenicity of ga-pV in patients with respect to antibody formation, identity of immunogens and potential options to reduce antibody levels. METHODS: Levels of tissue-specific and anti-αGal antibodies were determined retrospectively in patients who received ga-pV for 51 months (n=4), 25 months (n=6) or 5 months (n=4) and compared to age-matched untreated subjects (n=10) or younger subjects with or without vegetarian diet (n=12/15). Immunogenic proteins were investigated by Western blot approaches. RESULTS: Tissue-specific antibodies in patients were elevated after 5 (1.73-fold) and 25 (1.46-fold, both P<.0001) months but not after 51 months, whereas anti-Gal antibodies were induced 4.75-fold and 3.66-fold after 5 and 25 months (both P<.0001) and still were significantly elevated after 51 months (2.85-fold, P<.05). Western blots of porcine valve extracts with and without enzymatic deglycosylation revealed strong specific staining at ≈65 and ≈140 kDa by patient sera in either group which were identified by 2D Western blots and mass spectrometry as serum albumin and collagen 6A1. Vegetarian diet reduced significantly (0.63-fold, P<.01) the level of pre-formed αGal but not of tissue-specific antibodies. CONCLUSION: Immune response in patients towards ga-pV is induced by the porcine proteins albumin and collagen 6A1 as well as αGal epitopes, which seemed to be more sustained. In contrast, in healthy young subjects pre-formed anti-Gal antibodies were reduced by a meat-free nutrition.

Effect of Intensified Decellularization of Equine Carotid Arteries on Scaffold Biomechanics and Cytotoxicity.

Decellularized equine carotid arteries (dEAC) are suggested to represent an alternative for alloplastic vascular grafts in haemodialysis patients to achieve vascular access. Recently it was shown that intensified detergent treatment completely removed cellular components from dEAC and thereby significantly reduced matrix immunogenicity. However, detergents may also affect matrix composition and stability and render scaffolds cytotoxic. Therefore, intensively decellularized carotids (int-dEAC) were now evaluated for their biomechanical characteristics (suture retention strength, burst pressure and circumferential compliance at arterial and venous systolic and diastolic pressure), matrix components (collagen and glycosaminoglycan content) and indirect and direct cytotoxicity (WST-8 assay and endothelial cell seeding) and compared with native (n-EAC) and conventionally decellularized carotids (con-dEAC). Both decellularization protocols comparably reduced matrix compliance (venous pressure compliance: 32.2 and 27.4% of n-EAC; p < 0.01 and arterial pressure compliance: 26.8 and 23.7% of n-EAC, p < 0.01) but had no effect on suture retention strength and burst pressure. Matrix characterization revealed unchanged collagen contents but a 39.0% (con-dEAC) and 26.4% (int-dEAC, p < 0.01) reduction of glycosaminoglycans, respectively. Cytotoxicity was not observed in either dEAC matrix which was also displayed by an intact endothelial lining after seeding. Thus, even intensified decellularization generates matrix scaffolds highly suitable for vascular tissue engineering purposes, e.g., the generation of haemodialysis shunts.

The immune response to crosslinked tissue is reduced in decellularized xenogeneic and absent in decellularized allogeneic heart valves.

BACKGROUND: The degeneration and failure of xenogeneic heart valves, such as the Matrix P Plus valve (MP-V) consisting of decellularized porcine valves (dec-pV) and equine glutaraldehyde-fixed conduits (ga-eC) have been linked to tissue immunogenicity accompanied by antibody formation. In contrast, decellularized allograft valves (dec-aV) are well-tolerated. Here, we determined tissue-specific antibody levels in patients after implantation of MP-V or dec-aV and related them to valve failure or time period after implantation. METHODS AND RESULTS: Specific antibodies toward whole tissue-homogenates or alphaGal were determined retrospectively by ELISA analyses from patients who received MP-V with an uneventful course of 56.1 ± 5.1 months (n = 15), or with valve failure after 25.3 ± 14.6 months (n = 3), dec-aV for various times from 4 to 46 months (n = 14, uneventful) and from healthy controls (n = 4). All explanted valves were assessed histopathologically.MP-V induced antibodies toward both tissue components with significantly higher levels toward ga-eC than toward dec-pV (68.7 and 26.65 µg/ml IgG). In patients with valve failure, levels were not significantly higher and were related to inflammatory tissue infiltration. Anti-Gal antibodies in MP-V patients were significantly increased in both, the uneventful and the failure group. In contrast, in dec-aV patients only a slight tissue-specific antibody formation was observed after 4 months (6.24 µg/ml) that normalized to control levels after 1 year. CONCLUSIONS: The strong humoral immune response to glutaraldehyde-fixed tissues is reduced in decellularized xenogeneic valves and almost absent in decellularized allogeneic tissue up to 4.5 years after implantation.

Coating decellularized equine carotid arteries with CCN1 improves cellular repopulation, local biocompatibility, and immune response in sheep.

Decellularized equine carotid arteries (dEAC) are potential alternatives to alloplastic vascular grafts although there are certain limitations in biocompatibility and immunogenicity. Here, dEAC were coated with the matricellular protein CCN1 and evaluated in vitro for its cytotoxic and angiogenic effects and in vivo for cellular repopulation, local biocompatibility, neovascularization, and immunogenicity in a sheep model. CCN1 coating resulted in nontoxic matrices not compromising viability of L929 fibroblasts and endothelial cells (ECs) assessed by WST-8 assay. Functionality of CCN1 was maintained as it induced typical changes in fibroblast morphology and MMP3 secretion. For in vivo testing, dEAC±CCN1 (n=3 each) and polytetrafluoroethylene (PTFE) protheses serving as controls (n=6) were implanted as cervical arteriovenous shunts. After 14 weeks, grafts were harvested and evaluated immunohistologically. PTFE grafts showed a patency rate of only 33% and lacked cellular repopulation. Both groups of bioartificial grafts were completely patent and repopulated with ECs and smooth muscle cells (SMCs). However, whereas dEAC contained only patch-like aggregates of SMCs and a partial luminal lining with ECs, CCN1-coated grafts showed multiple layers of SMCs and a complete endothelialization. Likewise, CCN1 coating reduced leukocyte infiltration and fibrosis and supported neovascularization. In addition, in a three-dimensional assay, CCN1 coating increased vascular tube formation in apposition to the matrix 1.6-fold. Graft-specific serum antibodies were increased by CCN1 up to 6 weeks after implantation (0.89±0.03 vs. 1.08±0.04), but were significantly reduced after 14 weeks (0.85±0.04 vs. 0.69±0.02). Likewise, restimulated lymphocyte proliferation was significantly lower after 14 weeks (1.78±0.09 vs. 1.32±0.09-fold of unstimulated). Thus, CCN1 coating of biological scaffolds improves local biocompatibility and accelerates scaffold remodeling by enhancing cellular repopulation and immunologic tolerance, making it a promising tool for generation of bioartificial vascular prostheses.

Cytotoxic effects of polyhexanide on cellular repopulation and calcification of decellularized equine carotids in vitro and in vivo.

PURPOSE: Disinfection of biological implants is indispensable for clinical safety. Here, decellularized equine carotid arteries (dECAs) were disinfected by polyhexanide (PHX), an effective, well-tolerated and nontoxic wound disinfectant and evaluated as vascular grafts for their repopulation and local biocompatibility in vivo. 
 METHODS: dECAs were terminally disinfected by a combination of 0.1% PHX and 70% ethanol (dECA_PHX-ET) or exclusively ethanol (dECA-ET) and subsequently implanted as arteriovenous shunts in sheep for 14 weeks. Repopulation was determined by immunohistochemistry for endothelial- (ECs) or smooth muscle cells (SMCs) using antibodies against CD31 and smooth muscle actin. Histological evaluation was performed on HE-stained sections. Cytotoxicity of dECAs was measured directly by seeding the scaffolds with L-929 fibroblasts, which were visualized by calcein staining. Indirect cytotoxicity was determined by WST-8 viability assay by incubation of L-929 with dECA extracts. 
 RESULTS: dECA_PHX-ET completely lacked repopulation with ECs and SMCs, showed leukocyte infiltration, strong calcification and poor neovascularization indicating insufficient biocompatibility and inflammatory graft degeneration. PHX-treatment reduced cell viability to 33.2 ± 12.6% and disturbed cell growth at direct contact. In contrast, dECA_ET had no direct cytotoxic effect and only slightly influenced cell viability (82.9 ± 12.5%), showed a substantial repopulation by ECs and SMCs including neovascularization, and were only slightly calcified. 
 CONCLUSION: The disinfectant polyhexanide seems to exert severe cytotoxic effects when used for the processing of decellularized matrices and may result in degenerative graft deterioration. In contrast, dECAs exclusively disinfected with ethanol were well integrated. Thus, ethanol seems to be a more suitable tool for graft processing than polyhexanide.