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The lack of a functional endothelium on small-diameter vascular grafts leads to intimal hyperplasia and thrombotic occlusion. Shear stress conditioning through controlled hydrodynamics within in vitroperfusion bioreactors has shown promise as a mechanism to drive endothelial cell (EC) phenotype from an activated, pro-inflammatory wound state toward a quiescent functional state that inhibits responses that lead to occlusive failure. As part of an overall design strategy to engineer functional vascular grafts, we present a novel two-phase shear conditioning approach to improve graft endothelialization. Axial rotation was first used to seed uniform EC monolayers onto the lumenal surface of decellularized scaffolds derived from the human umbilical vein. Using computer-controlled perfusion circuits, a flow-ramping paradigm was applied to adapt endothelia to arterial levels of fluid shear stress and pressure without graft denudation. The effects of constant pulse frequencies (CF) on EC quiescence were then compared with pulse frequencies modeled from temporal fluctuations in blood flow observed in vivo, termed physiologically modeled pulse dynamics (PMPD). Constructs exposed to PMPD for 72 h expressed a more functional transcriptional profile, lower metabolic activity (39.8%±8.4% vs. 62.5%±11.5% reduction, p=0.012), and higher nitric oxide production (80.42±23.93 vs. 48.75±6.93 nmol/105cells, p=0.028) than those exposed to CF. By manipulating in vitroflow conditions to mimic natural physiology, endothelialized vascular grafts can be stimulated to express a nonactivated phenotype that would better inhibit peripheral cell adhesion and smooth muscle cell hyperplasia, conditions that typically lead to occlusive failure. Development of robust, functional endothelia on vascular grafts by modulation of environmental conditions within perfusion bioreactors may ultimately improve clinical outcomes in vascular bypass grafting.

The temporomandibular joint (TMJ) disc is susceptible to numerous pathologies that may lead to structural degradation and jaw dysfunction. The limited treatment options and debilitating nature of severe temporomandibular disorders has been the primary driving force for the introduction and development of TMJ disc tissue engineering as an approach to alleviate this important clinical issue. This study aimed to evaluate the efficacy of laser micropatterning (LMP) ex vivo-derived TMJ disc scaffolds to enhance cellular integration, a major limitation to the development of whole tissue implant technology. LMP was incorporated into the decellularized extracellular matrix scaffold structure using a 40 W CO2laser ablation system to drill an 8×16 pattern with a bore diameter of 120 μm through the scaffold thickness. Disc scaffolds were seeded with human neonatal-derived umbilical cord mesenchymal stem cells differentiated into chondrocytes at a density of 900 cells per mm2and then assessed on days 1, 7, 14, and 21 of culture. Results derived from histology, PicoGreen DNA quantification, and cellular metabolism assays indicate that the LMP scaffolds improve cellular remodeling compared to the unworked scaffold over the 21-day culture period. Mechanical analysis further supports the use of the LMP showing the compressive properties of the LMP constructs closely represent native disc mechanics. The addition of an artificial path of infiltration by LMP culminated in improved chondrocyte adhesion, dispersion, and migration after extended culture aiding in recapitulating the native TMJ disc characteristics.

In the development of engineered vascular grafts, assessing the material's interactive properties with peripheral blood cells and its capacity to endothelialize are important for predicting in vivograft behavior. Current in vitrotechniques used for characterizing cell adhesion at the surface of engineered scaffolds under flow only facilitate a terminal quantification of cell/surface interactions. Here, we present the design of an innovative flow chamber for real-time analysis of blood–biomaterial interactions under controllable hemodynamic conditions. Decellularized human umbilical veins (dHUV) were used as model vascular allografts to characterize platelet, leukocyte, and endothelial cell (EC) adhesion dynamics. Confluent EC monolayers adhered to the lumenal surface of the grafting material were flow conditioned to resist arterial shear stress levels (up to 24 dynes/cm2) over a 48 h period, and shown to maintain viability over the 1 week assessment period. The basement membrane was imaged while whole blood/neutrophil suspensions were perfused across the HUV surface to quantify cell accumulation. This novel method facilitates live visualization of dynamic events, including cell adhesion, migration, and morphological adaptation at the blood–graft interface on opaque materials, and it can be used for preliminary assessment of clinically relevant biomaterials before implantation.

Whereas functionally different, both organogenesis and wound-healing processes create zones or regions of hypoxia that persist until capillary networks are formed to facilitate oxygen and nutrient delivery. Similarly, regenerative processes within in vitroengineered tissues experience the same hypoxic regions, but without the capacity to form functional capillaries resulting in a major limitation in developing full-thickness organs and tissues. Due to the importance of oxygen in wound healing and tissue regeneration, we hypothesize that directed oxygen gradients can be used to modulate cell function and promote more effective tissue regeneration. The effect of controlled oxygen gradients on human smooth muscle cells (SMCs) was assessed using dual chambered perfusion bioreactors to regulate transport conditions occurring in a model vascular construct. SMCs were seeded onto the ablumenal surface of the scaffold and cultured for 21 days under 3 independent gas environments: (1) 21% oxygen, (2) 11% oxygen, or (3) an ablumen to lumen oxygen gradient from 11% to 21%. When compared to 21% oxygen and 11% oxygen conditions, the directed 11%–21% oxygen gradient resulted in a raised metabolic activity and significantly improved cell migration. After 21 days from seeding, cells were shown to migrate entirely across the scaffold to the vessel lumen (>450 μm). Concomitant with a more uniform cell dispersion, scaffold mechanics were significantly enhanced with increased stiffness and tensile strength. Native oxygen gradients are known to play a pivotal role during organ development; these results show that directed oxygen gradients within in vitrosystems can be used to facilitate early remodeling leading to significantly enhanced cell migration and scaffold biomechanics.

The prevalence of cardiovascular disease and the limited availability of suitable autologous transplant vessels for coronary and peripheral bypass surgeries is a significant clinical problem. A great deal of progress has been made over recent years to develop biodegradable materials with the potential to remodel and regenerate vascular tissues. However, the creation of functional biological scaffolds capable of withstanding vascular stress within a clinically relevant time frame has proved to be a challenging proposition. As an alternative approach, we report the use of a multilaminate rolling approach using the human amnion to generate a tubular construct for blood vessel regeneration. The human amniotic membrane was decellularized by agitation in 0.03% (w/v) sodium dodecyl sulfate to generate an immune compliant material. The adhesion of human umbilical vein endothelial cells (EC) and human vascular smooth muscle cells (SMC) was assessed to determine initial binding and biocompatibility (monocultures). Extended cultures were either assessed as flat membranes, or rolled to form concentric multilayered conduits. Results showed positive EC adhesion and a progressive repopulation by SMC. Functional changes in SMC gene expression and the constructs' bulk mechanical properties were concomitant with vessel remodeling as assessed over a 40-day culture period. A significant advantage with this approach is the ability to rapidly produce a cell-dense construct with an extracellular matrix similar in architecture and composition to natural vessels. The capacity to control physical parameters such as vessel diameter, wall thickness, shape, and length are critical to match vessel compliance and tailor vessel specifications to distinct anatomical locations. As such, this approach opens new avenues in a range of tissue regenerative applications that may have a much wider clinical impact.

Background:Little attention has been given to understanding the variation in biomechanical behavior of oral soft tissues, and this represents an obstacle for the development of biomaterials that perform with appropriate biomechanical characteristics. With this as our motivation, a uniaxial mechanical analysis was performed on lingual and buccal aspects of the attached gingiva, alveolar mucosa, and buccal mucosa to gain insight into human tissue performance and site‐specific mechanical variation. Methods:A discrete quantitative mechanical evaluation of each soft tissue region using tensile, dynamic compression, and stress relaxation analysis was conducted to correlate tissue structure with function as assessed histologically. Results:Results confirm the keratinized gingiva to have increased tensile strength (3.94 ± 1.19 MPa) and stiffness (Young modulus of 19.75 ± 6.20 MPa) relative to non‐keratinized mucosal regions, where densely arranged elastin fibers contribute to a tissue with increased viscoelastic properties. Dynamic compression analysis indicated the instantaneous modulus (Eint), steady modulus (Es), and peak stress increased with loading frequency and strain amplitude, with the highest values found in the buccal attached gingiva. Conclusion:These investigations quantify the biomechanical properties of oral soft tissues and show region‐to‐region variation that details structure–function relationships and provides key parameters to aid development of biomaterials that perform with appropriate biomechanical properties.

The initial seeding density is a critical variable in functional tissue engineering. A sufficient number of cells uniformly distributed throughout the scaffold is a key requirement to achieve homogeneous extracellular matrix deposition in vitro. However, high initial seeding densities might have negative repercussions on nutrient availability, cellular metabolism, and cell viability. In the current study, our aim was to understand the implications of using high seeding densities (3, 5, and 10 million cells/mL) in a human umbilical vein (HUV) tendon model subjected to 1 h of cyclic stretching per day at 2% strain and a frequency of 0.0167 Hz in a mechanostimulating bioreactor, on nutrient availability, cell viability and metabolism, and construct properties. Mechanostimulated constructs seeded with 3 million cells/mL had significantly higher cell number than the static controls and resulted in a 20-fold increase in proliferation rates and a 3-fold increase in tensile strength values after 1 week of culture in the bioreactor. However, higher seeding densities resulted in cell death, degraded extracellular matrix, and poorer mechanical properties. Nutrient and growth factor mass transport limitations are implicated in the inability of the decellularized HUV to support high cell numbers. The effective diffusion coefficient for glucose was measured to be 0.21±0.04 cm2/day. In the absence of convective flow, proteins and growth factors with a molecular radius larger than 4.9 nm could not diffuse through the HUV. Cells seeded in the HUV consumed 10.5±0.5 ng/cell/day of glucose. Glucose diffusion coefficient and glucose consumption rates in the HUV indicated the presence of glucose mass transport limitations when cell seeding densities exceed 3 million cells/mL.

These investigations describe the development of a novel ex vivothree-dimensional scaffold derived from the human umbilical vein (HUV), and its potential as a regenerative matrix for tissue regeneration. Unique properties associated with the vascular wall have shown potential to function as a surgical barrier for guided tissue regeneration, particularly with the regeneration of periodontal tissues. HUV was isolated from umbilical cords using a semiautomated machining technology, decellularized using 1% sodium dodecyl sulfate, and then opened longitudinally to form tissue sheets. Uniaxial tensile testing, stress relaxation, and suture retention tests were performed on the acellular matrix to evaluate the HUV's biomechanical properties, followed by an evaluation of cellular interactions by seeding human gingival fibroblasts to assess adhesion, metabolic function, and proliferation on the scaffold. The scaffold's biomechanical properties were shown to display anisotropic behavior, which is attributed to the ex vivomaterial's composite structure. Detailed results indicated that the ultimate tensile strength of the longitudinal strips was significantly higher than that of the circumferential strips (p < 0.001). The HUV also exhibited significantly higher stress relaxation response in the longitudinal direction than in the circumferential orientation (p < 0.05). The ablumenal and lumenal surfaces of the material were also shown to differentially influence cell proliferation and metabolic activity, with both cellular functions significantly increased on the ablumenal surface (p < 0.05). Human gingival fibroblast migration into the scaffold was also influenced by the organization of extracellular matrix components, where the lumenal surface inhibits cell migration, acting as a barrier, while the ablumenal surface, which is proposed to interface with the wound site, promotes cellular invasion. These results show the HUV bioscaffold to be a promising naturally derived surgical barrier that may function well as a resorbable guided tissue regeneration membrane as well as in other clinical applications.

This study investigated the potential of the decellularized human umbilical vein (HUV) as an allogeneic, acellular extracellular matrix (ECM) scaffold for engineering the vocal fold lamina propria in vitro. HUV specimens with Wharton's jelly on the abluminal surface were uniformly dissected from native umbilical cords using an automated procedure and subjected to a novel saline-based decellularization treatment for removal of potentially antigenic epitopes. Human vocal fold fibroblasts from primary culture were seeded onto the resulting acellular constructs and cultured for 21 days. The structures of decellularized and fibroblast-repopulated HUV constructs and the attachment, proliferation, and infiltration of fibroblasts were examined with light microscopy and scanning electron microscopy. Changes in the relative densities of collagen in the constructs associated with decellularization and recellularization were quantified using digital image analysis. In addition, fibroblasts infiltrating the scaffolds were released by cell recovery and quantified by counting. Viscoelastic properties of the scaffolds were measured using a linear, simple-shear rheometer at phonatory frequencies. Results showed that an acellular ECM construct with an intact three-dimensional structure of Wharton's jelly was fabricated. Vocal fold fibroblasts readily attached on the abluminal surface of the construct with high viability, with significant cellular infiltration up to approximately 600 μm deep into the construct. A significant increase in collagen expression was observed with recellularization. The elastic modulus and dynamic viscosity of the fibroblast-repopulated scaffolds were comparable to those of the human vocal fold lamina propria. These findings supported the potential of the construct as a possible surgical allograft for vocal fold restoration and reconstruction.

With advantageous biomechanical properties, materials derived from ex vivotissues are being actively investigated as scaffolds for tissue engineering applications. However, decellularization treatments are required before implantation to reduce the materials immune impact. The aim of these investigations was to assess a convective flow model as an enhanced methodology to decellularize ex vivotissue. Isolated human umbilical veins were decellularized using two methods: rotary agitation at 100 rpm on orbital shaker plates, and convective flow run at 5, 50, and 150 mmHg within perfusion bioreactors. Extracted phospholipids and total soluble protein were assessed over time. Histology, SEM, and uniaxial tensile testing analysis were carried out to evaluate variation in the tissues. After 72 h, samples exposed to traditional rotary agitation showed retention of whole cells and cellular components, whereas pressure-based systems showed no visual sign of cells. The convective flow method was significantly more effective at removing phospholipid and total protein than the agitation model. High transmembrane pressure (150 mmHg) resulted in higher phospholipids extraction. However, a more efficient protein extraction occurred at 50 mmHg. Variation in extraction rates was dependent on tissue permeability, which varied as pressure increased. Collectively, these findings show significant improvements in decellularization efficiency that may lead to more immune compliant ex vivo–derived biomaterials.

Human perinatal tissues have been used for over a century as allogeneic biomaterials. Due to their advantageous properties including angiogenecity, anti‐inflammation, anti‐microbial, and immune privilege, these tissues are being utilized for novel applications across wide‐ranging medical disciplines. Given continued clinical success, increased adoption of perinatal tissues as a disruptive technology platform has allowed for significant penetration into the multi‐billion dollar biologics market. Here, we review current progress and future applications of perinatal biomaterials, as well as associated regulatory issues. Biomaterials derived from human perinatal tissueshave had widespread clinical success across multiple medical disciplines. This progress report provides an overview of their biological and mechanical properties as well as their current and future potential clinical applications. In addition, we review the current state of regulatory issues associated with perinatal biomaterial usage.