Silica nanoparticles enhance interfacial self-adherence of a multi-layered extracellular matrix scaffold for vascular tissue regeneration.

PURPOSE: Based on the clinical need for grafts for vascular tissue regeneration, our group developed a customizable scaffold derived from the human amniotic membrane. Our approach consists of rolling the decellularized amniotic membrane around a mandrel to form a multilayered tubular scaffold with tunable diameter and wall thickness. Herein, we aimed to investigate if silica nanoparticles (SiNP) could enhance the adhesion of the amnion layers within these rolled grafts. METHODS: To test this, we assessed the structural integrity and mechanical properties of SiNP-treated scaffolds. Mechanical tests were repeated after six months to evaluate adhesion stability in aqueous environments. RESULTS: Our results showed that the rolled SiNP-treated scaffolds maintained their tubular shape upon hydration, while non-treated scaffolds collapsed. By scanning electron microscopy, SiNP-treated scaffolds presented more densely packed layers than untreated controls. Mechanical analysis showed that SiNP treatment increased the scaffold's tensile strength up to tenfold in relation to non-treated controls and changed the mechanism of failure from interfacial slipping to single-point fracture. The nanoparticles reinforced the scaffolds both at the interface between two distinct layers and within each layer of the extracellular matrix. Finally, SiNP-treated scaffolds significantly increased the suture pullout force in comparison to untreated controls. CONCLUSION: Our study demonstrated that SiNP prevents the unraveling of a multilayered extracellular matrix graft while improving the scaffolds' overall mechanical properties. In addition to the generation of a robust biomaterial for vascular tissue regeneration, this novel layering technology is a promising strategy for a number of bioengineering applications.

Silica nanoparticles enhance the cyto- and hemocompatibility of a multilayered extracellular matrix scaffold for vascular tissue regeneration.

PURPOSE: The limited availability of autologous vessels for vascular bypass surgeries is a major roadblock to treating severe cardiovascular diseases. Based on this clinical priority, our group has developed a novel engineered vascular graft by rolling human amniotic membranes into multilayered extracellular matrixes (ECM). When treated with silica nanoparticles (SiNP), these rolled scaffolds showed a significant improvement in their structural and mechanical properties, matching those from gold standard autologous grafts. However, it remained to be determined how cells respond to SiNP-treated materials. As a first step toward understanding the biocompatibility of SiNP-dosed biomaterials, we aimed to assess how endothelial cells and blood components interact with SiNP-treated ECM scaffolds. METHODS: To test this, we used established in vitro assays to study SiNP and SiNP-treated scaffolds' cyto and hemocompatibility. RESULTS: Our results showed that SiNP effects on cells were concentration-dependent with no adverse effects observed up to 10 µg/ml of SiNP, with higher concentrations inducing cytotoxic and hemolytic responses. The SiNP also enhanced the scaffold's hydrophobicity state, a feature known to inhibit platelet and immune cell adhesion. Accordingly, SiNP-treated scaffolds were also shown to support endothelial cell growth while preventing platelet and leukocyte adhesion. CONCLUSION: Our findings suggest that the addition of SiNP to human amniotic membrane extracellular matrixes improves the cyto- and hemocompatibility of rolled scaffolds and highlights this strategy as a robust mechanism to stabilize layered collagen scaffolds for vascular tissue regeneration.

Flow with variable pulse frequencies accelerates vascular recellularization and remodeling of a human bioscaffold.

Despite significant advances in vascular tissue engineering, the ideal graft has not yet been developed and autologous vessels remain the gold standard substitutes for small diameter bypass procedures. Here, we explore the use of a flow field with variable pulse frequencies over the regeneration of an ex vivo-derived human scaffold as vascular graft. Briefly, human umbilical veins were decellularized and used as scaffold for cellular repopulation with human smooth muscle cells (SMC) and endothelial cells (EC). Over graft development, the variable flow, which mimics the real-time cardiac output of an individual performing daily activities (e.g., resting vs. exercising), was implemented and compared to the commonly used constant pulse frequency. Results show marked differences on SMC and EC function, with changes at the molecular level reflecting on tissue scales. First, variable frequencies significantly increased SMC proliferation rate and glycosaminoglycan production. These results can be tied with the SMC gene expression that indicates a synthetic phenotype, with a significant downregulation of myosin heavy chain. Additionally and quite remarkably, the variable flow frequencies motivated the re-endothelialization of the grafts, with a quiescent-like structure observed after 10 days of conditioning, contrasting with the low surface coverage and unaligned EC observed under constant frequency (CF). Besides, the overall biomechanics of the generated grafts (conditioned with both pulsed and CFs) evidence a significant remodeling after 55 days of culture, depicted by high burst pressure and Young's modulus. These last results demonstrate the positive recellularization and remodeling of a human-derived scaffold toward an arterial vessel.

Human placenta-derived matrix with cancellous autograft and demineralized bone matrix for large segmental long-bone defects in two dogs with septic nonunion.

OBJECTIVE: To report the successful treatment of septic nonunion in two dogs with large segmental defects secondary to long-bone fractures by using a novel human placenta-derived matrix (hPM) as adjunct to fixation. ANIMALS: One 3-kg 9-year-old neutered male Yorkshire terrier with a distal antebrachial fracture and one 6-kg 4-year-old spayed female miniature pinscher with a distal humeral fracture. STUDY DESIGN: Short case series. METHODS: Both dogs presented for septic nonunion after internal fixation of Gustilo type II open diaphyseal fractures from dog bite injuries. During revision, debridement of nonviable bone resulted in segmental defects of 32% and 20% of the bone length for the antebrachial and humeral fractures, respectively. The antebrachial fracture was stabilized with a circular external fixator, and the humeral fracture was stabilized with biaxial bone plating. The fracture sites were not collapsed, and full length was maintained with the fixation. Autogenous cancellous bone graft and canine demineralized bone allograft were packed into the defects, and hPM was injected into the graft sites after closure. RESULTS: Radiographic union was documented at 8 weeks and 6 weeks for the antebrachial and humeral fractures, respectively. Both dogs became fully weight bearing on the affected limbs and returned to full activity. CONCLUSION: Augmenting fixation with grafts and hPM led to a relatively rapid union in both dogs reported here.

Biological characterization of dehydrated amniotic membrane allograft: Mechanisms of action and implications for wound care.

There is a growing clinical demand in the wound care market to treat chronic wounds such as diabetic foot ulcers. Advanced cell and tissue-based products (CTPs) are often used to address challenging chronic wounds where healing has stalled. These products contain active biologics such as growth factors and cytokines as well as structural components that support and stimulate cell growth and assist in tissue regeneration. This study addresses the in vitro biologic effects of a clinically available dehydrated amniotic membrane allograft (DAMA). The broad mechanism of action results from DAMA's biologic composition that leads to stimulation of cell migration cell proliferation, and reduction of pro-inflammatory cytokines. Results show that DAMA possesses growth factors and cytokines such as EGF, FGF, PDGFs, VEGF, TGF-ß, IL-8, and TIMPs 1 and 2. Furthermore, in vitro experiments demonstrate that DAMA stimulates cell proliferation, cell migration, secretion of collagen type I, and the reduction of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α. This study findings are consistent with the clinical benefits previously published for DAMA and other CTPs in chronic wounds suggesting that the introduction of DAMA to non-healing, complex wounds helps to improve the wound milieu by providing essential structural components, cytokines, and growth factors to create an appropriate environment for wound healing.

Sequential adaptation of perfusion and transport conditions significantly improves vascular construct recellularization and biomechanics.

Recellularization of ex vivo-derived scaffolds remains a significant hurdle primarily due to the scaffolds subcellular pore size that restricts initial cell seeding to the scaffolds periphery and inhibits migration over time. With the aim to improve cell migration, repopulation, and graft mechanics, the effects of a four-step culture approach were assessed. Using an ex vivo-derived vein as a model scaffold, human smooth muscle cells were first seeded onto its ablumen (Step 1: 3 hr) and an aggressive 0-100% nutrient gradient (lumenal flow under hypotensive pressure) was created to initiate cell migration across the scaffold (Step 2: Day 0 to 19). The effects of a prolonged aggressive nutrient gradient created by this single lumenal flow was then compared with a dual flow (lumenal and ablumenal) in Step 3 (Day 20 to 30). Analyses showed that a single lumenal flow maintained for 30 days resulted in a higher proportion of cells migrating across the scaffold toward the vessel lumen (nutrient source), with improved distribution. In Step 4 (Day 31 to 45), the transition from hypotensive pressure (12/8 mmHg) to normotensive (arterial-like) pressure (120/80 mmHg) was assessed. It demonstrated that recellularized scaffolds exposed to arterial pressures have increased glycosaminoglycan deposition, physiological modulus, and Young's modulus. By using this stepwise conditioning, the challenging recellularization of a vein-based scaffold and its positive remodeling toward arterial biomechanics were obtained.

Approaches to improve integration and regeneration of an ex vivo derived temporomandibular joint disc scaffold with variable matrix composition.

Due to their natural biochemical and biomechanical characteristics, using ex vivo tissues as platforms for guided tissue regeneration has become widely accepted, however subsequent attachment and integration of these constructs in vivo is often overlooked. A decellularized porcine temporomandibular joint (TMJ) disc has shown promise as a scaffold to guide disc regeneration and preliminary work has shown the efficacy of surfactant (SDS) treatment within the fibrocartilaginous disc to remove cellular components. The majority of studies focus on the intermediate region of the disc (or disc proper). Using this approach, inherent attachment tissues can be maintained to improve construct stability and integration within the joint. Unlike human disc attachment tissue, the porcine attachment tissues have high lipid content which would require a different processing approach to remove immunogenic components. In order to examine the effect of delipidation on the attachment tissue properties, SDS and two organic solvent mixtures (acetone/ethanol and chloroform/methanol) were compared. Lipid and cellular solubilization, ECM alteration, and seeded human mesenchymal stem cell (MSC) morphology and viability were assessed. Quantitative analysis showed SDS treatments did not effectively delipidate the attachment tissues and cytotoxicity was noted toward MSC in these regions. Acetone/ethanol removed cellular material but not all lipids, while chloroform/methanol removed all visible lipid deposits but residual porcine cells were observed in histological sections. When a combination of approaches was used, no residual lipid or cytotoxicity was noted. Preparing a whole TMJ graft with a combined approach has the potential to improve disc integration within the native joint environment.

Human Perinatal-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.

Pilot assessment of a human extracellular matrix-based vascular graft in a rabbit model.

BACKGROUND: Herein we describe a small-diameter vascular graft constructed from rolled human amniotic membrane (hAM), with in vitro evaluation and subsequent in vivo assessment of its mechanical and initial biologic viability in the early postimplantation period. This approach for graft construction allows customization of graft dimensions, with wide-ranging potential clinical applicability as a nonautologous, allogeneic, cell-free graft material. METHODS: Acellular hAMs were rolled into layered conduits (3.2-mm diameter) that were bound with fibrin and lyophilized. Constructs were seeded with human smooth muscle cells and cultured under controlled arterial hemodynamic conditions in vitro. Additionally, the acellular hAM conduits were surgically implanted as arterial interposition grafts into the carotid arteries of immunocompetent rabbits. RESULTS: On in vitro analysis, smooth muscle cells were shown to adhere to, proliferate within, and remodel the scaffold during a 4-week culture period. At the end of the culture period, there was histologic and biomechanical evidence of graft wall layer coalescence. In vivo analysis demonstrated graft patency after 4 weeks (n = 3), with no hyperacute rejection or thrombotic occlusion. Explants displayed histologic evidence of active cellular remodeling, with endogenous cell repopulation of the graft wall concurrent with degradation of initial graft material. Cells were shown to align circumferentially to resemble a vascular medial layer. CONCLUSIONS: The vascular grafts were shown to provide a supportive scaffold allowing cellular infiltration and remodeling by host cell populations in vivo. By use of this approach, "off-the-shelf" vascular grafts can be created with specified diameters and wall thicknesses to satisfy specific anatomic requirements in diverse populations of patients.

Controlled release of a heterogeneous human placental matrix from PLGA microparticles to modulate angiogenesis.

A significant hurdle limiting musculoskeletal tissue regeneration is the inability to develop effective vascular networks to support cellular development within engineered constructs. Due to the inherent complexity of angiogenesis, where multiple biochemical pathways induce and control vessel formation, our laboratory has taken an alternate approach using a matrix material containing angiogenic and osteogenic proteins derived from human placental tissues. Single bolus administrations of the human placental matrix (hPM) have been shown to initiate angiogenesis but vascular networks deteriorated over time. Controlled/sustained delivery was therefore hypothesized to stabilize and extend network formation. To test this hypothesis, hPM was encapsulated in degradable poly(lactic-co-glycolic acid) (PLGA) microparticles to extend the release period. Microparticle preparation including loading, size, encapsulation efficiency, and release profile was optimized for hPM. The angiogenic cellular response to the hPM/PLGA-loaded microparticles was assessed in 3D alginate hydrogel matrices seeded with primary human endothelial cells. Results show an average microparticle diameter of 91.82 ± 2.92 µm, with an encapsulation efficiency of 75%, and a release profile extending over 30 days. Three-dimensional angiogenic assays with hPM-loaded PLGA microparticles showed initial stimulation of angiogenic tubules after 14 days and further defined network formations after 21 days of culture. Although additional optimization is necessary, these studies confirm the effectiveness of a novel controlled multi-protein release approach to induce and maintain capillary networks within alginate tissue scaffolds.

Mechanical Integrity of a Decellularized and Laser Drilled Medial Meniscus.

Since the meniscus has limited capacity to self-repair, creating a long-lasting meniscus replacement may help reduce the incidence of osteoarthritis (OA) after meniscus damage. As a first step toward this goal, this study evaluated the mechanical integrity of a decellularized, laser drilled (LD) meniscus as a potential scaffold for meniscal engineering. To evaluate the decellularization process, 24 porcine menisci were processed such that one half remained native tissue, while the other half was decellularized in sodium dodecyl sulphate (SDS). To evaluate the laser drilling process, 24 additional menisci were decellularized, with one half remaining intact while the other half was LD. Decellularization did not affect the tensile properties, but had significant effects on the cyclic compressive hysteresis and unconfined compressive stress relaxation. Laser drilling decreased the Young's modulus and instantaneous stress during unconfined stress relaxation and the circumferential ultimate strength during tensile testing. However, the losses in mechanical integrity in the LD menisci were generally smaller than the variance observed between samples, and thus, the material properties for the LD tissue remained within a physiological range. In the future, optimization of laser drilling patterns may improve these material properties. Moreover, reseeding the construct with cells may further improve the mechanical properties prior to implantation. As such, this work serves as a proof of concept for generating decellularized, LD menisci scaffolds for the purposes of meniscal engineering.

Biomechanical and biochemical outcomes of porcine temporomandibular joint disc deformation.

OBJECTIVE: The structure-function relationship in the healthy temporomandibular joint (TMJ) disc has been well established, however the changes in dysfunctional joints has yet to be systematically evaluated. Due to the poor understanding of the etiology of temporomandibular disorders (TMDs) this study evaluated naturally occurring degenerative remodeling in aged female porcine temporomandibular joint (TMJ) discs in order to gain insight into the progression and effects on possible treatment strategies of TMDs. DESIGN: Surface and regional biomechanical and biochemical properties of discal tissues were determined in grossly deformed (≥Wilkes Stage 3) and morphologically normal (≤Wilkes Stage 2) TMJ discs. RESULTS: Compared to normal disc structure the deformed discs lacked a smooth biconcave shape and characteristic ECM organization. Reduction in tensile biomechanical integrity and increased compressive stiffness and cellularity was found in deformed discs. Regionally, the posterior and intermediate zones of the disc were most frequently affected along with the inferior surface. CONCLUSIONS: The frequency of degeneration observed on the inferior surface of the disc (predominantly posterior), suggests that a disruption in the disc-condyle relationship likely contributes to the progression of joint dysfunction more than the temporodiscal relationship. As such, the inferior joint space may be an important consideration in early clinical diagnosis and treatment of TMDs, as it is overlooked in techniques performed in the upper joint space, including arthroscopy and arthrocentesis. Furthermore, permanent damage to the disc mechanical properties would limit the ability to successfully reposition deformed discs, highlighting the importance of emerging therapies such as tissue engineering.

Comparing the mechanical properties of the porcine knee meniscus when hydrated in saline versus synovial fluid.

As research progresses to find a suitable knee meniscus replacement, accurate in vitro testing becomes critical for feasibility and comparison studies of mechanical integrity. Within the knee, the meniscus is bathed in synovial fluid, yet the most common hydration fluid in laboratory testing is phosphate buffered saline (PBS). PBS is a relatively simple salt solution, while synovial fluid is a complex non-Newtonian fluid with multiple lubricating factors. As such, PBS may interact with meniscal tissue differently than synovial fluid, and thus, the hydration fluid may be an important factor in obtaining accurate results during in vitro testing. To evaluate these effects, medial porcine menisci were used to evaluate tissue mechanics in tension (n=11) and compression (n=15). In all tests, two samples from the same meniscus were taken, where one sample was hydrated in PBS and the other was hydrated in synovial fluid. Statistical analysis revealed no significant differences between the mean mechanical properties of samples tested in PBS compared to synovial fluid; however, compressive testing revealed the variability between samples was significantly reduced if samples were tested in synovial fluid. For example, the compressive Young׳s Modulus was 12.69±7.49MPa in PBS versus 12.34±4.27MPa in synovial fluid. These results indicate testing meniscal tissue in PBS will largely not affect the mean value of the mechanical properties, but performing compression testing in synovial fluid may provide more consistent results between samples and assist in reducing sample numbers in some experiments.

Variation in Cardiac Pulse Frequencies Modulates vSMC Phenotype Switching During Vascular Remodeling.

In vitro perfusion systems have exposed vascular constructs to mechanical conditions that emulate physiological pulse pressure and found significant improvements in graft development. However, current models maintain constant, or set pulse/shear mechanics that do not account for the natural temporal variation in frequency. With an aim to develop clinically relevant small diameter vascular grafts, these investigations detail a perfusion culture model that incorporates temporal pulse pressure variation. Our objective was to test the hypothesis that short-term variation in heart rate, such as changes in respiratory activity, plays a significant role in vascular remodeling and graft development. The pulse rate of a healthy volunteer was logged to model the effect of daily activities on heart rate. Vascular bioreactors were used to deliver perfusion conditions based on modeled frequencies of temporal pulse variability, termed Physiologically Modeled Pulse Dynamics (PMPD). Acellular scaffolds derived from the human umbilical vein were seeded with human vascular smooth muscle cells and perfused under defined pulsatile conditions. vSMC exposed to constant pulse frequencies expressed a contractile phenotype, while exposure to PMPD drove cells to a synthetic state with continued cell proliferation, increased tensile strength and stiffness as well as diminished vasoactivity. Results show the temporal variation associated with normal heart physiology to have a profound effect on vascular remodeling and vasoactive function. While these models are representative of vascular regeneration further investigation is required to understanding these and other key regulators in vSMC phenotype switching in non-pathological or wound healing states. This understanding has important clinical implications that may lead to improved treatments that enhance vessel regeneration.

The consequence of biologic graft processing on blood interface biocompatibility and mechanics.

Processing ex vivo derived tissues to reduce immunogenicity is an effective approach to create biologically complex materials for vascular reconstruction. Due to the sensitivity of small diameter vascular grafts to occlusive events, the effect of graft processing on critical parameters for graft patency, such as peripheral cell adhesion and wall mechanics, requires detailed analysis. Isolated human umbilical vein sections were used as model allogenic vascular scaffolds that were processed with either: 1. sodium dodecyl sulfate (SDS), 2. ethanol/acetone (EtAc), or 3. glutaraldehyde (Glu). Changes in material mechanics were assessed via uniaxial tensile testing. Peripheral cell adhesion to the opaque grafting material was evaluated using an innovative flow chamber that allows direct observation of the blood-graft interface under physiological shear conditions. All treatments modified the grafts tensile strain and stiffness properties, with physiological modulus values decreasing from Glu 240±12 kPa to SDS 210±6 kPa and EtAc 140±3 kPa, P<.001. Relative to glutaraldehyde treatments, neutrophil adhesion to the decellularized grafts increased, with no statistical difference observed between SDS or EtAc treatments. Early platelet adhesion (% surface coverage) showed no statistical difference between the three treatments; however, quantification of platelet aggregates was significantly higher on SDS scaffolds compared to EtAc or Glu. Tissue processing strategies applied to the umbilical vein scaffold were shown to modify structural mechanics and cell adhesion properties, with the EtAc treatment reducing thrombotic events relative to SDS treated samples. This approach allows time and cost effective prescreening of clinically relevant grafting materials to assess initial cell reactivity.

Physiologically Modeled Pulse Dynamics to Improve Function in In Vitro-Endothelialized Small-Diameter Vascular Grafts.

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 vitro perfusion 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/10(5) cells, p = 0.028) than those exposed to CF. By manipulating in vitro flow 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.

Novel human-derived extracellular matrix induces in vitro and in vivo vascularization and inhibits fibrosis.

The inability to vascularize engineered organs and revascularize areas of infarction has been a major roadblock to delivering successful regenerative medicine therapies to the clinic. These investigations detail an isolated human extracellular matrix derived from the placenta (hPM) that induces vasculogenesis in vitro and angiogenesis in vivo within bioengineered tissues, with significant immune reductive properties. Compositional analysis showed ECM components (fibrinogen, laminin), angiogenic cytokines (angiogenin, FGF), and immune-related cytokines (annexins, DEFA1) in near physiological ratios. Gene expression profiles of endothelial cells seeded onto the matrix displayed upregulation of angiogenic genes (TGFB1, VEGFA), remodeling genes (MMP9, LAMA5) and vascular development genes (HAND2, LECT1). Angiogenic networks displayed a time dependent stability in comparison to current in vitro approaches that degrade rapidly. In vivo, matrix-dosed bioscaffolds showed enhanced angiogenesis and significantly reduced fibrosis in comparison to current angiogenic biomaterials. Implementation of this human placenta derived extracellular matrix provides an alternative to Matrigel and, due to its human derivation, its development may have significant clinical applications leading to advances in therapeutic angiogenesis techniques and tissue engineering.

Mechanobiological Assessment of TMJ Disc Surfaces: Nanoindentation and Transmission Electron Microscopy.

OBJECTIVES: Temporomandibular disc is a mechanically robust fibrocartilage tissue exhibiting highly elastic compressive, shear, and tensile moduli with structurally dense extracellular matrix that supports functional loading of the joint. The aim of this study was to illustrate structural complexities of the superior and inferior disc surfaces, to demonstrate the robust mechanical ability of the disc as a whole may be due to depth-dependent regional/layered variation, and also to provide characterization data imperative for future tissue engineering efforts focused on restoring function to the joint. MATERIAL AND METHODS: Nanoindentation was used to assess tissue zones in conjunction with detailed Transmission Electron Microscopy to define structural attributes that influence the temporomandibular disc function. RESULTS: The disc architecture adjacent to the superior surface was shown to have three distinct regional segments within the interface layer: 1-a surface peripheral layer; 2-subsurface region; and 3-a layer of helical matrix bundles. The inferior surface displayed an interface layer (20 µm) that showed limited cell populations with little depth-dependent structural variation, a stiffer elastic modulus and reduced energy dissipation compared to the superior surface. These data indicate that the primary function of the inferior surface is resistance to compression rather than load distribution during joint motion. CONCLUSIONS: These are the first works that demonstrate that the superior central surface of the he temporomandibular disc is structured in depth-dependent isometric layers, each of which provides different mechanical function supporting the bulk tissue's properties. From a clinical perspective these data have potential to define regions susceptible to fatigue that may translate to diagnostic criteria to better define the stages of dysfunction.

Engineered microporosity: enhancing the early regenerative potential of decellularized temporomandibular joint discs.

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 CO2 laser 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 mm(2) and 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.

The effect of terminal sterilization on structural and biophysical properties of a decellularized collagen-based scaffold; implications for stem cell adhesion.

Terminal sterilization induces physical and chemical changes in the extracellular matrix (ECM) of ex vivo-derived biomaterials due to their aggressive mechanism of action. Prior studies have focused on how sterilization affects the mechanical integrity of tissue-based biomaterials but have rarely characterized effects on early cellular interaction, which is indicative of the biological response. Using a model fibrocartilage disc scaffold, these investigations compare the effect of three common sterilization methods [peracetic acid (PAA), gamma irradiation (GI), and ethylene oxide (EtO)] on a range of material properties and characterized early cellular interactions. GI and EtO produced unfavorable structural damage that contributed to inferior cell adhesion. Conversely, exposure to PAA resulted in limited structural alterations while inducing chemical modifications that favored cell attachment. Results suggest that the sterilization approach can be selected to modulate biomaterial properties to favor cellular adhesion and has relevance in tissue engineering and regenerative medicine applications. Furthermore, the study of cellular interactions with modified biomaterials in vitro provides information of how materials may react in subsequent clinical applications.