Bioprosthetic heart valve (BHV) replacement has been the predominant treatment for severe heart valve diseases over decades. Most clinically available BHVs are crosslinked by glutaraldehyde (GLUT), while the high toxicity of residual GLUT could initiate calcification, severe thrombosis, and delayed endothelialization. Here, we construed a mechanically integrating robust hydrogel-tissue hybrid to improve the performance of BHVs. In particular, recombinant humanized collagen type III (rhCOLIII), which was precisely customized with anti-coagulant and pro-endothelialization bioactivity, was first incorporated into the polyvinyl alcohol (PVA)-based hydrogel via hydrogen bond interactions. Then, tannic acid was introduced to enhance the mechanical performance of PVA-based hydrogel and interfacial bonding between the hydrogel layer and bio-derived tissue due to the strong affinity for a wide range of substrates. In vitro and in vivo experimental results confirmed that the GLUT-crosslinked BHVs modified by the robust PVA-based hydrogel embedded rhCOLIII and TA possessed long-term anti-coagulant, accelerated endothelialization, mild inflammatory response and anti-calcification properties. Therefore, our mechanically integrating robust hydrogel-tissue hybrid strategy showed the potential to enhance the service function and prolong the service life of the BHVs after implantation.

The number of patients with valvular heart disease is increasing yearly, and valve replacement is the most effective treatment, during which bioprosthetic heart valves (BHVs) are the most widely used. Commercial BHVs are mainly prepared with glutaraldehyde (Glut) cross-linked bovine pericardial or porcine aortic valves, but the residual free aldehyde groups in these tissues can cause calcification and cytotoxicity. Moreover, insufficient glycosaminoglycans (GAGs) in tissues can further reduce biocompatibility and durability. However, the anti-calcification performance and biocompatibility might be improved by blocking the free aldehyde groups and increasing the GAGs content in Glut-crosslinked tissues. In our study, adipic dihydrazide (ADH) was used to neutralize the residual free aldehyde groups in tissues and provide sites to blind with oligohyaluronan (OHA) to increase the content of GAGs in tissues. The modified bovine pericardium was evaluated for its content of residual aldehyde groups, the amount of OHA loaded, physical/chemical characteristics, biomechanical properties, biocompatibility, and in vivo anticalcification assay and endothelialization effects in juvenile Sprague-Dawley rats. The results showed that ADH could completely neutralize the free aldehyde groups in the Glut-crosslinked bovine pericardium, the amount of OHA loaded increased and the cytotoxicity was reduced. Moreover, the in vivo results also showed that the level of calcification and inflammatory response in the modified pericardial tissue was significantly reduced in a rat subcutaneous implantation model, and the results from the rat abdominal aorta vascular patch repair model further demonstrated the improved capability of the modified pericardial tissues for endothelialization. Furthermore, more α-SMA+ smooth muscle cells and fewer CD68+ macrophages infiltrated in the neointima of the modified pericardial patch. In summary, blocking free-aldehydes and loading OHA improved the anti-calcification, anti-inflammation and endothelialization properties of Glut-crosslinked BHVs and in particularly, this modified strategy may be a promising candidate for the next-generation of BHVs.

With the aging of the population in worldwide, valvular heart disease has become one of the most prominent life-threatening diseases in human health, and heart valve replacement surgery is one of the therapeutic methods for valvular heart disease. Currently, commercial bioprosthetic heart valves (BHVs) for clinical application are prepared with xenograft heart valves or pericardium crosslinked by glutaraldehyde. Due to the residual cell toxicity from glutaraldehyde, heterologous antigens, and immune response, there are still some drawbacks related to the limited lifespan of bioprosthetic heart valves, such as thrombosis, calcification, degeneration, and defectiveness of re-endothelialization. Therefore, the problems of calcification, defectiveness of re-endothelialization, and poor biocompatibility from the use of bioprosthetic heart valve need to be solved. In this study, hydrogel hybrid heart valves with improved anti-calcification and re-endothelialization were prepared by taking decellularized porcine heart valves as scaffolds following grafting with double bonds. Then, the anti-biofouling zwitterionic monomers 2-methacryloyloxyethyl phosphorylcholine (MPC) and vascular endothelial growth factor (VEGF) were utilized to obtain a hydrogel-coated hybrid heart valve (PEGDA-MPC-DHVs@VEGF). The results showed that fewer platelets and thrombi were observed on the surface of the PEGDA-MPC-DHVs@VEGF. Additionally, the PEGDA-MPC-DHVs@VEGF exhibited excellent collagen stability, biocompatibility and re-endothelialization potential. Moreover, less calcification deposition and a lower immune response were observed in the PEGDA-MPC-DHVs@VEGF compared to the glutaraldehyde-crosslinked DHVs (Glu-DHVs) after subcutaneous implantation in rats for 30 days. These studies demonstrated that the strategy of zwitterionic hydrogels loaded with VEGF may be an effective approach to improving the biocompatibility, anti-calcification and re-endothelialization of bioprosthetic heart valves.

The bioprosthetic heart valves (BHVs) are the best option for the treatment of valvular heart disease. Glutaraldehyde (Glut) is commonly used as the golden standard reagent for the crosslinking of BHVs. However, the obvious defects of Glut, including residual aldehyde toxicity, degradation and calcification, increase the probability of valve failure in vivo and motivated the exploration of alternatives. Thus, the aim of this study is to develop a non-glutaraldehyde hybrid cross-linking method composed of Neomycin Trisulfate, Polyethylene glycol diglycidyl ether and Tannic acid as a substitute for Glut, which was proven to reduce calcification, degradation, inflammation of the biomaterial. Evaluations of the crosslinked bovine pericardial included histological and ultrastructural characterization, biomechanical performance, biocompatibility and structural stability test, and in vivo anti-inflammation and anti-calcification assay by subcutaneous implantation in juvenile Sprague Dawley rats. The results revealed that the hybrid crosslinked bovine pericardial were superior to Glut crosslinked biomaterial in terms of better hydrophilicity, thermodynamics stability, hemocompatibility and cytocompatibility, higher Young\'s Modulus, better stability and resistance to enzymatic hydrolysis, and lower inflammation, degradation and calcification levels in subcutaneous implants. Considering all above performances, it indicates that the hybrid cross-linking method is appropriate to replace Glut as the method for BHV preparation, and particularly this hybrid crosslinked biomaterials may be a promising candidate for next-generation BHVs.

Bioprosthetic heart valves (BHVs) used in clinics are fabricated via glutaraldehyde (GLUT) crosslinking, which results in cytotoxicity and causes eventual valve calcification after implantation into the human body; therefore, the average lifetime and application of BHVs are limited. To address these issues, the most commonly used method is modification with amino acids, such as glycine (GLY), which is proven to effectively reduce toxicity and calcification. In this study, we used the l-glutathione (GSH) in a new modification treatment based on GLUT-crosslinked bovine pericardium (BP) as the GLUT + GSH group, BPs crosslinked with GLUT as GLUT-BP (control group), and GLY modification based on GLUT-BP as the GLUT + GLY group. We evaluated the characteristics of BPs in different treatment groups in terms of biomechanical properties, cell compatibility, aldehyde group content detection, and the calcification content. Aldehyde group detection tests showed that the GSH can completely neutralize the residual aldehyde group of GLUT-BP. Compared with that of GLUT-BP, the endothelial cell proliferation rate of the GLUT + GSH group increased, while its hemolysis rate and the inflammatory response after implantation into the SD rat were reduced. The results show that GSH can effectively improve the cytocompatibility of the GLUT-BP tissue. In addition, the results of the uniaxial tensile test, thermal shrinkage temperature, histological and SEM evaluation, and enzyme digestion experiments proved that GSH did not affect the ECM stability and biomechanics of the GLUT-BP. The calcification level of GLUT-BP modified using GSH technology decreased by 80%, indicating that GSH can improve the anti-calcification performance of GLUT-BP. Compared with GLUT-GLY, GLUT + GSH yielded a higher cell proliferation rate and lower inflammatory response and calcification level. GSH can be used as a new type of anti-calcification agent in GLUT crosslinking biomaterials and is expected to expand the application domain for BHVs in the future.

Glutaraldehyde (GA) was conventionally used to crosslink bovine pericardium to prepare bioprosthetic heart valves (BHVs), which usually fail within 10 years because of valve deterioration and calcification. To overcome the high cytotoxicity and severe calcification of GA-crosslinked BHVs, a quaternary ammonium salt of epoxy chitosan (epoxy group-modified 3-chlorine-2-hydroxypropyl trimethyl chitosan, abbreviated as \"eHTCC\") was developed to modify the acellular bovine pericardium to substitute GA and improve its anti-calcification and biocompatible properties. Mechanical test, enzymatic stability test, blood compatibility assay, and cytocompatibility assay were used to investigate its mechanical property and biocompatibility. The anti-calcification effect of the eHTCC-modified bovine pericardium (eHTCC-BP) was assessed by in vitro assay and rat subcutaneous implantation assay. The results showed that eHTCC-BP could improve the mechanical properties and anti-enzymolysis ability of BP, as well as retain the original three-dimensional structure, compared with the uncrosslinked-BP group. Moreover, the in vivo calcification level of the eHTCC-BP group was much lower than that of the GA-BP group, which was 5.1% (2 weeks), 2.3% (4 weeks), and 0.8% (8 weeks) of the GA-BP group. In summary, this study demonstrated that eHTCC could be a potential crosslinking agent for the extracellular matrix for its favorable crosslinking effects, anti-enzymolysis, anti-calcification, and biocompatibility.

Objective: Sevelamer hydrochloride is a phosphate binder used to treat hyperphosphatemia in chronic kidney disease (CKD) patients that can reduce valvular and vascular calcification. The aim of this study was to examine the effects of sevelamer treatment on calcification in bioprosthetic heart valves (BHVs). Methods: Wister rats were randomly divided into three groups according to sevelamer intake and implantation (sham-sham operation; implant-implantation and normal diet, implant+S implantation, and sevelamer diet). Two kinds of BHVs-bovine pericardium treated with glutaraldehyde (GLUT) or non-GLUT techniques-were implanted in rat dorsal subcutis at 4 weeks. After implantation, sevelamer was administered to the implant+S group. The animals were executed at days 0 (immediately after implantation), 7, 14, 28, and 56. Calcium levels were determined by atomic absorption spectroscopy and von Kossa staining. Serum biochemistry analysis, Western blotting, real-time quantitative polymerase chain reaction, alkaline phosphatase activity measurement, histopathologic analysis, immunohistochemistry, and enzyme-linked immunosorbent assay were conducted to identify the anti-calcification mechanism of sevelamer. Results: Non-GLUT crosslinking attenuates BHV calcification. Serum phosphate and calcium remained unreactive to sevelamer after a 14-day treatment. However, the mean calcium level in the implant+S group was significantly decreased after 56 days. In addition, the PTH level, inflammatory cell infiltration, system and local inflammation, and expression of Bmp2, Runx2, Alp, IL-1β, IL-6, and TNF-α were significantly reduced in the implant+S group. Conclusion: Sevelamer treatment significantly attenuated the calcification of BHVs and had anti-inflammation effects that were independent from serum calcium and phosphate regulation. Thus, sevelamer treatment might be helpful to improve the longevity of BHVs.

Valve replacement surgery is the golden standard for end-stage valvular disease due to the lack of self-repair ability. Currently, bioprosthetic heart valves (BHVs) crosslinked by glutaraldehyde (GA) have been the most popular choice in clinic, especially after the emerge of transcatheter aortic valve replacement (TAVR). Nevertheless, the lifespan of BHVs is limited due to severe calcification and deterioration. In this study, to improve the anti-calcification property of BHVs, decellularized heart valves were modified by methacrylic anhydride to introduce double bonds (MADHVs), and a hybrid hydrogel made of sulfobetaine methacrylate (SBMA) and methacrylated hyaluronic acid (MAHA) was then coated onto the surface of MADHVs. Followed by grafting of Arg-Glu-Asp-Val (REDV), an endothelial cell-affinity peptide, the BHVs with improved affinity to endothelial cell (SMHVs-REDV) was obtained. SMHVs-REDV exhibited excellent collagen stability, reliable mechanical property and superior hemocompatibility. Moreover, enhanced biocompatibility and endothelialization potential compared with GA-crosslinked BHVs were achieved. After subcutaneous implantation for 30 days, SMHVs-REDV showed significantly reduced immune response and calcification compared with GA-crosslinked BHVs. Overall, simultaneous endothelialization and anti-calcification can be realized by this strategy, which was supposed to be benefit for improving the main drawbacks for available commercial BHVs products.

In this study, electrospun scaffolds were fabricated by blending poly(l-lactide-co-ε-caprolactone) (PLCL) and silk fibroin (SF) with different ratios, and further the feasibility of electrospun PLCL/SF scaffolds were evaluated for application of tissue engineered heart valve (TEHV). Scanning electron microscopy (SEM) results showed that the surface of PLCL/SF electrospun scaffolds was smooth and uniform while the mechanical properties were appropriate as valve prosthesis. In vitro cytocompatibility evaluation results demonstrated that all of the PLCL/SF electrospun scaffolds were cytocompatible and valvular interstitial cells (VICs) cultured on PLCL/SF scaffolds of 80/20 & 70/30 ratios exhibited the best cytocompatibility. The in vitro osteogenic differentiation of VICs including alkaline phosphatase (ALP) activity and quantitative polymerase chain reaction (qPCR) assays indicated that PLCL/SF scaffolds of 80/20 & 90/10 ratios behaved better anti-calcification ability. In the in vivo calcification evaluation model of rat subdermal implantation, PLCL/SF scaffolds of 80/20 & 90/10 ratios presented better anti-calcification ability, which was consistent with the in vitro results. Moreover, PLCL/SF scaffolds of 80/20 & 70/30 ratios showed significantly enhanced cell infiltration and M2 macrophage with higher CD206+/CD68+ ratio. Collectively, our data demonstrated that electrospun scaffolds with the PLCL/SF ratio of 80/20 hold great potential as TEHV materials.

In the past decade, balloon-expandable percutaneous pulmonary valves have been developed and applied in clinical practice. However, all the existing products of pulmonary artery interventional valves in the market have a straight structure design, and they require a preset support frame and balloon expansion. This shape design of the valve limits the application range. In addition, the age of the population with pulmonary artery disease is generally low, and the existing products cannot meet the needs of anti-calcification properties and valve material durability. In this study, through optimization of the support frame and leaflet design, a self-expanding pulmonary valve product with a double bell-shaped frame was designed to improve the match of the valve and the implantation site. A loading and deployment study showed that the biomaterial of the valve was not damaged after being compressed. Pulsatile flow and fatigue in vitro tests showed that the fabricated pulmonary valve met the hydrodynamic requirements after 2 × 108 accelerated fatigue cycles. The safety and efficacy of the pulmonary valve product were demonstrated in studies of pulmonary valve implantation in 11 pigs. Angiography and echocardiography showed that the pulmonary valves were implanted in a good position, and they had normal closure and acceptable valvular regurgitation. The 180 days\' implantation results showed that the calcium content was 0.31-1.39 mg/g in the anti-calcification treatment group, which was significantly lower than that in the control valve without anti-calcification treatment (16.69 mg/g). Our new interventional pulmonary valve product was ready for clinical trials and product registration.