Development of photoreactive demineralized bone matrix 3D printing colloidal inks for bone tissue engineering.

Demineralized bone matrix (DBM) has been widely used clinically for dental, craniofacial and skeletal bone repair, as an osteoinductive and osteoconductive material. 3D printing (3DP) enables the creation of bone tissue engineering scaffolds with complex geometries and porosity. Photoreactive methacryloylated gelatin nanoparticles (GNP-MAs) 3DP inks have been developed, which display gel-like behavior for high print fidelity and are capable of post-printing photocrosslinking for control of scaffold swelling and degradation. Here, novel DBM nanoparticles (DBM-NPs, ∼400 nm) were fabricated and characterized prior to incorporation in 3DP inks. The objectives of this study were to determine how these DBM-NPs would influence the printability of composite colloidal 3DP inks, assess the impact of ultraviolet (UV) crosslinking on 3DP scaffold swelling and degradation and evaluate the osteogenic potential of DBM-NP-containing composite colloidal scaffolds. The addition of methacryloylated DBM-NPs (DBM-NP-MAs) to composite colloidal inks (100:0, 95:5 and 75:25 GNP-MA:DBM-NP-MA) did not significantly impact the rheological properties associated with printability, such as viscosity and shear recovery or photocrosslinking. UV crosslinking with a UV dosage of 3 J/cm2 directly impacted the rate of 3DP scaffold swelling for all GNP-MA:DBM-NP-MA ratios with an ∼40% greater increase in scaffold area and pore area in uncrosslinked versus photocrosslinked scaffolds over 21 days in phosphate-buffered saline (PBS). Likewise, degradation (hydrolytic and enzymatic) over 21 days for all DBM-NP-MA content groups was significantly decreased, ∼45% less in PBS and collagenase-containing PBS, in UV-crosslinked versus uncrosslinked groups. The incorporation of DBM-NP-MAs into scaffolds decreased mass loss compared to GNP-MA-only scaffolds during collagenase degradation. An in vitro osteogenic study with bone marrow-derived mesenchymal stem cells demonstrated osteoconductive properties of 3DP scaffolds for the DBM-NP-MA contents examined. The creation of photoreactive DBM-NP-MAs and their application in 3DP provide a platform for the development of ECM-derived colloidal materials and tailored control of biochemical cue presentation with broad tissue engineering applications.

3D Printed Modeling of the Mitral Valve for Catheter-Based Structural Interventions.

As catheter-based structural heart interventions become increasingly complex, the ability to effectively model patient-specific valve geometry as well as the potential interaction of an implanted device within that geometry will become increasingly important. Our aim with this investigation was to combine the technologies of high-spatial resolution cardiac imaging, image processing software, and fused multi-material 3D printing, to demonstrate that patient-specific models of the mitral valve apparatus could be created to facilitate functional evaluation of novel trans-catheter mitral valve repair strategies. Clinical 3D transesophageal echocardiography and computed tomography images were acquired for three patients being evaluated for a catheter-based mitral valve repair. Target anatomies were identified, segmented and reconstructed into 3D patient-specific digital models. For each patient, the mitral valve apparatus was digitally reconstructed from a single or fused imaging data set. Using multi-material 3D printing methods, patient-specific anatomic replicas of the mitral valve were created. 3D print materials were selected based on the mechanical testing of elastomeric TangoPlus materials (Stratasys, Eden Prairie, Minnesota, USA) and were compared to freshly harvested porcine leaflet tissue. The effective bending modulus of healthy porcine MV tissue was significantly less than the bending modulus of TangoPlus (p < 0.01). All TangoPlus varieties were less stiff than the maximum tensile elastic modulus of mitral valve tissue (3697.2 ± 385.8 kPa anterior leaflet; 2582.1 ± 374.2 kPa posterior leaflet) (p < 0.01). However, the slopes of the stress-strain toe regions of the mitral valve tissues (532.8 ± 281.9 kPa anterior leaflet; 389.0 ± 156.9 kPa posterior leaflet) were not different than those of the Shore 27, Shore 35, and Shore 27 with Shore 35 blend TangoPlus material (p > 0.95). We have demonstrated that patient-specific mitral valve models can be reconstructed from multi-modality imaging datasets and fabricated using the multi-material 3D printing technology and we provide two examples to show how catheter-based repair devices could be evaluated within specific patient 3D printed valve geometry. However, we recognize that the use of 3D printed models for the development of new therapies, or for specific procedural training has yet to be defined.

Emerging Trends in Heart Valve Engineering: Part IV. Computational Modeling and Experimental Studies.

In this final portion of an extensive review of heart valve engineering, we focus on the computational methods and experimental studies related to heart valves. The discussion begins with a thorough review of computational modeling and the governing equations of fluid and structural interaction. We then move onto multiscale and disease specific modeling. Finally, advanced methods related to in vitro testing of the heart valves are reviewed. This section of the review series is intended to illustrate application of computational methods and experimental studies and their interrelation for studying heart valves.

Design and physical characterization of a synchronous multivalve aortic valve culture system.

For many tissues, cyclic mechanical stimulation is considered necessary to maintain the normal morphology in vitro. The aim of this study was to design and evaluate a simple bioreactor system capable of medium-term (more than 2 weeks) culture of native and engineered aortic valves. The system consists of three pistons in separate cylindrical chambers that are simultaneously driven through the culture medium by a crank and cam assembly. The faces of these pistons have unidirectional valves mounted in opposing orientations that permit flow from one side of the face to the other. A custom designed stent was employed to secure either native or engineered tri-leaflet valves to the pistons. Computational fluid dynamics and finite element modeling was used to assist selection of materials and components in the system. Finally, sterility testing using base culture medium was performed to verify the ability of the system to retain sterile conditions. The current design permits the cyclic opening and closing of three aortic valves, however this device can be modified to accommodate up to 12 valves simultaneously. This new bioreactor system has applications not only for development of tissue-engineered valves, but for also studying disease models in the aortic valve.

Influence of strain on proteoglycan synthesis by valvular interstitial cells in three-dimensional culture.

Differently loaded regions of the mitral valve contain distinct amounts and types of proteoglycans (PGs); these PG profiles are altered in abnormal loading and disease conditions. We developed an in vitro three-dimensional model to analyze PGs secreted by valvular interstitial cells (VICs) isolated from distinct regions of porcine mitral valves (leaflet or chordae) and subjected to either biaxial or uniaxial mechanical constraints. In addition, the PGs, DNA and collagen content of the collagen gels was monitored over time. All three PGs previously found in heart valves (decorin, biglycan and versican) were present in the collagen gels and the conditioned medium. Compared to unconstrained gels, the constrained collagen gels (whether biaxially or uniaxially loaded) retained more decorin and biglycan but less versican. However, the conditioned medium from constrained collagen gels contained higher amounts of all three PGs than did medium from unconstrained gels. Constrained collagen gels containing leaflet cells retained more decorin and biglycan than did those containing chordal cells. DNA content was maintained early in the culture period but was reduced by 55-80% after 7 days, whereas PG synthesis increased over time. At the end of the culture period, the cell density was highest in the biaxial region of gels seeded with leaflet cells. In contrast, collagen content in both constrained and unconstrained gels remained consistent over culture duration. This study provides valuable information about the role of applied loading on proteoglycan segregation, which should aid in tissue engineering applications and for understanding valve biology and pathology.