Potential mechanisms underlying ectodermal differentiation of Wharton's jelly mesenchymal stem cells.

Wharton's jelly mesenchymal stem cells (WJMSCs) are being increasingly recognized for their ectodermal differentiation potential. Previously, we demonstrated that when WJMSC were seeded onto an acellular matrix material derived from Wharton's jelly and cultured in osteogenic induction media, generated CK19 positive cells and hair-like structures indicative of ectodermal differentiation of WJMSCs. In this manuscript, we examine the underlying mechanism behind this observation using a variety of microscopy and molecular biology techniques such as western blotting and qPCR. We demonstrate that these hair-like structures are associated with live cells that are positive for epithelial and mesenchymal markers such as cytokeratin-19 and α-smooth muscle actin, respectively. We also show that up-regulation of ß-catenin and noggin, along with the expression of TGF-ß and SMAD and inhibition of BMP4 could be the mechanism behind this ectodermal differentiation and hair-like structure formation.

Calcium and phosphorus concentrations in native and decellularized semilunar valve tissues.

BACKGROUND AND AIM OF THE STUDY: Native, allograft, xenograft and bioprosthetic semilunar valves are all susceptible to calcific degeneration. However, intrinsic differences in baseline calcium and phosphorus tissue concentrations within mammalian normal valve structural components (e.g., cusps, sinus, vessel wall) additionally subdivided by tripartite regions (e.g., right-, left- and non-coronary leaflets) have never been systematically measured and reported. It was originally hypothesized that variations in normative tissue concentrations of calcium and phosphorus may correspond to subsequent clinical patterns of acquired dystrophic calcification; decellularization was also expected to reduce the tissue concentrations of these elements. METHODS: Native semilunar valves were freshly harvested from 12 juvenile sheep. Half of the valves were decellularized (six aortic and six pulmonary), while the other valves were flash-frozen at -80 degrees C within minutes of euthanasia as native valves. Elemental calcium and phosphorus concentrations were measured in the great vessels, sinus walls and cusps using inductively coupled plasma optical emission spectrometry (ICP-OES), and analyzed with non-parametric statistical tests. RESULTS: Calcium concentrations (microg/mg tissue; median (range) were similar in aortic native cusps (0.37 (0.21)), sinus walls (0.37 (0.09)) and aorta (0.37 (0.08)) (p = 0.8298). Pulmonary calcium concentrations were similar in cusps, but 10-25% higher in the native sinus (p = 0.0018) and pulmonary artery (p < 0.0001) compared to analogous aortic structures. All cusps had higher phosphorus concentrations than their respective conduit tissues. No tripartite regional variations were observed. Decellularization did not reduce the calcium content of cusps, but removed 50-55% of vessel and sinus wall calcium. However, up to 85% of phosphorus was removed from all valve tissues (p < 0.001). CONCLUSION: There were no significant differences in normal tissue concentrations of calcium between aortic valve functional structures, and no semilunar tripartite regional differences in either semilunar valve complex. Thus, the distribution of baseline tissue calcium content of healthy young valves is not inherently predictive of selective or asymmetric anatomical patterns of valve degenerative calcification. Native semilunar cusps contain the highest phosphorus concentrations. Decellularization reduces all elemental concentrations except for cuspal calcium.

Transesophageal Echocardiography in Healthy Young Adult Male Baboons (Papio hamadryas anubis): Normal Cardiac Anatomy and Function in Subhuman Primates Compared to Humans.

Implantable, viable tissue engineered cardiovascular constructs are rapidly approaching clinical translation. Species typically utilized as preclinical large animal models are food stock ungulates for which cross species biological and genomic differences with humans are great. Multiple authorities have recommended developing subhuman primate models for testing regenerative surgical strategies to mitigate xenotransplant inflammation. However, there is a lack of specific quantitative cardiac imaging comparisons between humans and the genomically similar baboons (Papio hamadryas anubis). This study was undertaken to translate to baboons transesophageal echocardiographic functional and dimensional criteria defined as necessary for defining cardiac anatomy and function in the perioperative setting. Seventeen young, healthy baboons (approximately 30 kg, similar to 5 year old children) were studied to determine whether the requisite 11 views and 52 measurement parameters could be reliably acquired by transesophageal echocardiography (TEE). The obtained measurements were compared to human adult normative literature values and to a large relational database of pediatric "normal heart" echo measurements. Comparisons to humans, when normalized to BSA, revealed a trend in baboons toward larger mitral and aortic valve effective orifice areas and much larger left ventricular muscle mass and wall thickness, but similar pulmonary and tricuspid valves. By modifying probe positioning relative to human techniques, all recommended TEE views except transgastric could be replicated. To supplement, two transthoracic apical views were discovered that in baboons could reliably replace the transgastric TEE view. Thus, all requisite echo views could be obtained for a complete cardiac evaluation in Papio hamadryas anubis to noninvasively quantify cardiac structural anatomy, physiology, and dimensions. Despite similarities between the species, there are subtle and important physiologic and anatomic differences when compared to human.

Determining cell seeding dosages for tissue engineering human pulmonary valves.

BACKGROUND: This study examines in vitro seeding of decellularized human pulmonary valves (hPVs) with human valve interstitial cells (hVICs) isolated from unrelated donor aortic valve leaflets. An assay was developed to assess seeding using precut uniform sized biopsies from whole hPVs for sequential evaluation of seeding efficiency, proliferation, and migration. MATERIALS AND METHODS: Scaffolds for seeding were created from decellularized hPVs using a reciprocating osmolality, double detergent, enzyme, multiple solvent protocol. hVICs seeded decellularized leaflet and sinus wall scaffolds were incubated in either static or cyclic pressure bioreactors. Low, medium, and high initial cell seeding "dosing" densities were assayed at subsequent three time points, using eight replicates each (n = 576 biopsies including manufactured scaffold controls). Metabolically viable seeded cells were quantified by MTT assay. Histology defined cell locations and morphology. RESULTS: After 24 h of static seeding with 2.5 × 10(5) cells (medium dose), 100 ± 13 cells/mm(2) (2.5%) attached to leaflets, compared with 193 ± 21 cells/mm(2) (8%) for sinuses. Subsequent 4 d in static culture yielded 894 ± 84 and 838 ± 50 cells/mm(2)versus pulsatile culture yielding 80 ± 12 and 79 ± 12 cells/mm(2) for leaflet and sinus, respectively. However, 76.0% ± 12.2% of cells in leaflets in the pulsatile bioreactor were subsurface as compared to 21.4% ± 3.9% in statically cultured leaflets (P < 0.001). CONCLUSION: Different seeding modes suggest a tradeoff between surface proliferation resulting in higher absolute cell numbers for static seeding versus fewer cells in a cyclic pressure bioreactor but with a greater percentage having migrated into the matrix. The medium seeding dose determined to be optimal is actually feasible for tissue engineering heart valves, and can be achieved by fairly traditional cell amplification methods.

Pediatric cardiopulmonary bypass adaptations for long-term survival of baboons undergoing pulmonary artery replacement.

Cardiopulmonary bypass (CPB) protocols of the baboon (Papio cynocephalus anubis) are limited to obtaining experimental data without concern for long-term survival. In the evaluation of pulmonary artery tissue engineered heart valves (TEHVs), pediatric CPB methods are adapted to accommodate the animals' unique physiology enabling survival up to 6 months until elective sacrifice. Aortic access was by a 14F arterial cannula and atrial access by a single 24F venous cannula.The CPB circuit includes a 3.3 L/min flow rated oxygenator, 1/4" x %" arterial-venous loop, 3/8" raceway, and bubble trap. The prime contains 700 mL Plasma-Lyte, 700 units heparin, 5 mL of 50% dextrose, and 20 mg amiodarone. Heparinization (200 u/kg) targets an activated clotting time of 350 seconds. Normothermic CPB was initiated at a 2.5 L/m2/min cardiac index with a mean arterial pressure of 55-80 mmHg. Weaning was monitored with transesophageal echocardiogram. Post-CPB circuit blood was re-infused. Chest tubes were removed with cessation of bleeding. Extubation was performed upon spontaneous breathing. The animals were conscious and upright 3 hours post-CPB. Bioprosthetic valves or TEHVs were implanted as pulmonary replacements in 20 baboons: weight = 27.5 +/- 5.6 kg, height = 73 +/- 7 cm, body surface area = 0.77 m2 +/- 0.08, mean blood flow = 1.973 +/- .254 L/min, core temperature = 37.1 +/- .1 degree C, and CPB time = 60 +/- 40 minutes. No acidosis accompanied CPB. Sixteen animals survived, four expired. Three died of right ventricular failure and one of an anaphylactoid reaction. Surviving animals had normally functioning replacement valves and ventricles. Baboon CPB requires modifications to include high systemic blood pressure for adequate perfusion into small coronary arteries, careful CPB weaning to prevent ventricular distention, and drug and fluid interventions to abate variable venous return related to a muscularized spleno-splanchnic venous capacity.

Met-Flow, a strategy for single-cell metabolic analysis highlights dynamic changes in immune subpopulations.

A complex interaction of anabolic and catabolic metabolism underpins the ability of leukocytes to mount an immune response. Their capacity to respond to changing environments by metabolic reprogramming is crucial to effector function. However, current methods lack the ability to interrogate this network of metabolic pathways at single-cell level within a heterogeneous population. We present Met-Flow, a flow cytometry-based method capturing the metabolic state of immune cells by targeting key proteins and rate-limiting enzymes across multiple pathways. We demonstrate the ability to simultaneously measure divergent metabolic profiles and dynamic remodeling in human peripheral blood mononuclear cells. Using Met-Flow, we discovered that glucose restriction and metabolic remodeling drive the expansion of an inflammatory central memory T cell subset. This method captures the complex metabolic state of any cell as it relates to phenotype and function, leading to a greater understanding of the role of metabolic heterogeneity in immune responses.

Dominant-negative NFKBIA mutation promotes IL-1ß production causing hepatic disease with severe immunodeficiency.

Although IKK-ß has previously been shown as a negative regulator of IL-1ß secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1ß expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1ß secretion was elevated in the patient's stimulated leukocytes, in her induced pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hypersecretion of IL-1ß correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1ß release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1ß secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.

Non-physiologic Bioreactor Processing Conditions for Heart Valve Tissue Engineering.

PURPOSE: Conventional methods of seeding decellularized heart valves for heart valve tissue engineering have led to inconsistent results in interstitial cellular repopulation, particularly of the distal valve leaflet, and notably distinct from documented re-endothelialization. The use of bioreactor conditioning mimicking physiologic parameters has been well explored but cellular infiltration remains challenging. Non-characteristic, non-physiologic conditioning parameters within a bioreactor, such as hypoxia and cyclic chamber pressure, may be used to increase the cellular infiltration leading to increased recellularization. METHODS: To investigate the effects of novel and perhaps non-intuitive bioreactor conditioning parameters, ovine aortic heart valves were seeded with mesenchymal stem cells and cultured in one of four environments: hypoxia and high cyclic pressures (120 mmHg), normoxia and high cyclic pressures, hypoxia and negative cyclic pressures (- 20 mmHg), and normoxia and negative cyclic pressures. Analysis included measurements of cellular density, cell phenotype, and biochemical concentrations. RESULTS: The results revealed that the bioreactor conditioning parameters influenced the degree of recellularization. Groups that implemented hypoxic conditioning exhibited increased cellular infiltration into the valve leaflet tissue compared to normoxic conditioning, while pressure conditioning did not have a significant effect of recellularization. Protein expression across all groups was similar, exhibiting a stem cell and valve interstitial cell phenotype. Biochemical analysis of the extracellular matrix was similar between all groups. CONCLUSION: These results suggest the use of non-physiologic bioreactor conditioning parameters can increase in vitro recellularization of tissue engineered heart valve leaflets. Particularly, hypoxic culture was found to increase the cellular infiltration. Therefore, bioreactor conditioning of tissue engineered constructs need not always mimic physiologic conditions, and it is worth investigating novel or uncharacteristic culture conditions as they may benefit aspects of tissue culture.

Decellularized Wharton jelly matrix: a biomimetic scaffold for ex vivo hematopoietic stem cell culture.

Hematopoietic stem progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic "niche," a special 3-dimensional (3D) microenvironment that regulates HSPC self-renewal and multipotency. In this study, we evaluated a novel 3D in vitro culture system that uses components of the BM hematopoietic niche to expand umbilical cord blood (UCB) CD34+ cells. We developed this model using decellularized Wharton jelly matrix (DWJM) as an extracellular matrix (ECM) scaffold and human BM mesenchymal stromal cells (MSCs) as supporting niche cells. To assess the efficacy of this model in expanding CD34+ cells, we analyzed UCB CD34+ cells, following culture in DWJM, for proliferation, viability, self-renewal, multilineage differentiation, and transmigration capability. We found that DWJM significantly expanded UCB HSPC subset. It promoted UCB CD34+ cell quiescence, while maintaining their viability, differentiation potential with megakaryocytic differentiation bias, and clonogenic capacity. DWJM induced an increase in the frequency of c-kit+ cells, a population with enhanced self-renewal ability, and in CXCR4 expression in CD34+ cells, which enhanced their transmigration capability. The presence of BM MSCs in DWJM, however, impaired UCB CD34+ cell transmigration and suppressed CXCR4 expression. Transcriptome analysis indicated that DWJM upregulates a set of genes that are specifically involved in megakaryocytic differentiation, cell mobility, and BM homing. Collectively, our results indicate that the DWJM-based 3D culture system is a novel in vitro model that supports the proliferation of UCB CD34+ cells with enhanced transmigration potential, while maintaining their differentiation potential. Our findings shed light on the interplay between DWJM and BM MSCs in supporting the ex vivo culture of human UCB CD34+ cells for use in clinical transplantation.

A novel extracellular matrix-based leukemia model supports leukemia cells with stem cell-like characteristics.

Acute myeloid leukemia (AML) relapse results from the survival of chemotherapy-resistant and quiescent leukemia stem cells (LSC). These LSCs reside in the bone marrow microenvironment, comprised of other cells and extracellular matrix (ECM), which facilitates LSC quiescence through expression of cell adhesion molecules. We used decellularized Wharton's jelly matrix (DWJM), the gelatinous material in the umbilical cord, as a scaffolding material to culture leukemia cells, because it contains many components of the bone marrow extracellular matrix, including collagen, fibronectin, lumican, and hyaluronic acid (HA). Leukemia cells cultured in DWJM demonstrated decreased proliferation without undergoing significant differentiation. After culture in DWJM, these cells also exhibited changes in morphology, acquiring a spindle-shaped appearance, and an increase in the ALDH+ cell population. When treated with a high-dose of doxorubicin, leukemia cells in DWJM demonstrated less apoptosis compared with cells in suspension. Serial colony forming unit (CFU) assays indicated that leukemia cells cultured in DWJM showed increased colony-forming ability after both primary and secondary plating. Leukemia cell culture in DWJM was associated with increased N-cadherin expression by flow cytometry. Our data suggest that DWJM could serve as an ECM-based model to study AML stem cell-like cell behavior and chemotherapy sensitivity.

Wharton's Jelly Matrix Decellularization for Tissue Engineering Applications.

Scaffolds, both natural and synthetic, used in tissue engineering provide mechanical support to cells. Tissue decellularization has been used to provide natural extracellular matrix scaffolds for tissue engineering purposes. In this chapter we focus on describing the methodology used to decellularize Wharton's jelly matrix, the mucous connective tissue that surrounds umbilical cord vessels, to obtain decellularized Wharton's jelly matrix (DWJM); an extracellular matrix that can be used for tissue engineering purposes. We also, briefly, describe our experience with processing DWJM for cell seeding and recellularization.

Synergistic effects of cyclic tension and transforming growth factor-beta1 on the aortic valve myofibroblast.

BACKGROUND: Phenotypically, aortic valve interstitial cells are dynamic myofibroblasts, appearing contractile and activated in times of development, disease, and remodeling. The precise mechanism of phenotypic modulation is unclear, but it is speculated that both biomechanical and biochemical factors are influential. Therefore, we hypothesized that isolated and combined treatments of cyclic tension and transforming growth factor-beta1 would alter the phenotype and subsequent collagen biosynthesis of aortic valve interstitial cells in situ. METHODS AND RESULTS: Porcine aortic valve leaflets received 7- and 14-day treatments of 15% cyclic stretch (Tension); 0.5 ng/ml transforming growth factor-beta1 (TGF); 15% cyclic stretch and 0.5 ng/ml transforming growth factor-beta1 (Tension+TGF); or neither mechanical nor cytokine stimuli (Null). Tissues were homogenized and assayed for aortic valve interstitial cell phenotype (smooth muscle alpha-actin) and collagen biosynthesis (via heat shock protein 47, which was further verified with type I collagen C-terminal propeptide). At both 7 and 14 days, smooth muscle alpha-actin, heat shock protein 47, and type I collagen C-terminal propeptide quantities were significantly greater (P<.001) in the Tension+TGF group than in all other groups. Additionally, Tension alone appeared to maintain smooth muscle alpha-actin and heat shock protein 47 levels that were measured on Day 0, while TGF alone elicited an increase in smooth muscle alpha-actin and heat shock protein 47 compared to Day 0 levels. Null treatment revealed diminished proteins at both time points. CONCLUSIONS: Elevated transforming growth factor-beta1 levels, in the presence of cyclic mechanical tension, resulted in synergistic increases in contractile and biosynthetic proteins in aortic valve interstitial cells. Since cyclic mechanical stimuli can never be relieved in vivo, the presence of transforming growth factor-beta1 (possibly from infiltrating macrophages) may result in overly biosynthetic aortic valve interstitial cells, leading to altered extracellular matrix architecture, compromised valve function, and, ultimately, degenerative valvular disease.

Non-bacterial thrombotic endocarditis in an adult 14 months after cryopreserved aortic allograft valve implantation.

Cryopreserved aortic allograft tissue is used to correct aortic valve disease in adults and to reconstruct the right ventricular outflow tract in children with congenital heart disease. In adults, allograft durability is regarded as comparable to or better than that of manufactured bioprostheses, with failure usually due to slow fibrocalcific degeneration. Normally, allograft semilunar valves have excellent hemodynamics and low rates of infectious endocarditis and thromboembolism. The role of immune-mediated inflammation in post-implant allograft valve performance is slow in onset, and variable. Herein is presented the case of a male adult with rapid deterioration of the aortic valve homograft wall, without loss of the valve leaflets, resulting in severe aortic regurgitation. The pathological findings were consistent with classical marantic (sterile) endocarditis with acute and chronic inflammatory changes associated with advanced atherosclerotic lesions in the allograft aortic wall tissue resulting in thrombosis and subsequent cerebral embolization.

Recellularization of decellularized heart valves: Progress toward the tissue-engineered heart valve.

The tissue-engineered heart valve portends a new era in the field of valve replacement. Decellularized heart valves are of great interest as a scaffold for the tissue-engineered heart valve due to their naturally bioactive composition, clinical relevance as a stand-alone implant, and partial recellularization in vivo. However, a significant challenge remains in realizing the tissue-engineered heart valve: assuring consistent recellularization of the entire valve leaflets by phenotypically appropriate cells. Many creative strategies have pursued complete biological valve recellularization; however, identifying the optimal recellularization method, including in situ or in vitro recellularization and chemical and/or mechanical conditioning, has proven difficult. Furthermore, while many studies have focused on individual parameters for increasing valve interstitial recellularization, a general understanding of the interacting dynamics is likely necessary to achieve success. Therefore, the purpose of this review is to explore and compare the various processing strategies used for the decellularization and subsequent recellularization of tissue-engineered heart valves.

Species-specific effects of aortic valve decellularization.

Decellularized heart valves have great potential as a stand-alone valve replacement or as a scaffold for tissue engineering heart valves. Before decellularized valves can be widely used clinically, regulatory standards require pre-clinical testing in an animal model, often sheep. Numerous decellularization protocols have been applied to both human and ovine valves; however, the ways in which a specific process may affect valves of these species differently have not been reported. In the current study, the comparative effects of decellularization were evaluated for human and ovine aortic valves by measuring mechanical and biochemical properties. Cell removal was equally effective for both species. The initial cell density of the ovine valve leaflets (2036±673cells/mm2) was almost triple the cell density of human leaflets (760±386cells/mm2; p<0.001). Interestingly, post-decellularization ovine leaflets exhibited significant increases in biaxial areal strain (p<0.001) and circumferential peak stretch (p<0.001); however, this effect was not observed in the human counterparts (p>0.10). This species-dependent difference in the effect of decellularization was likely due to the higher initial cellularity in ovine valves, as well as a significant decrease in collagen crosslinking following the decellularization of ovine leaflets that was not observed in the human leaflet. Decellularization also caused a significant decrease in the circumferential relaxation of ovine leaflets (p<0.05), but not human leaflets (p>0.30), which was credited to a greater reduction of glycosaminoglycans in the ovine tissue post-decellularization. These results indicate that an identical decellularization process can have differing species-specific effects on heart valves. STATEMENT OF SIGNIFICANCE: The decellularized heart valve offers potential as an improved heart valve substitute and as a scaffold for the tissue engineered heart valve; however, the consequences of processing must be fully characterized. To date, the effects of decellularization on donor valves from different species have not been evaluated in such a way that permits direct comparison between species. In this manuscript, we report species-dependent variation in the biochemical and biomechanical properties of human and ovine aortic heart valve leaflets following decellularization. This is of clinical significance, as current regulatory guidelines required pre-clinical use of the ovine model to evaluate candidate heart valve substitutes.

Design and efficacy of a single-use bioreactor for heart valve tissue engineering.

Heart valve tissue engineering offers the promise of improved treatments for congenital heart disorders; however, widespread clinical availability of a tissue engineered heart valve (TEHV) has been hindered by scientific and regulatory concerns, including the lack of a disposable, bioreactor system for nondestructive valve seeding and mechanical conditioning. Here we report the design for manufacture and the production of full scale, functional prototypes of such a system. To evaluate the efficacy of this bioreactor as a tool for seeding, ovine aortic valves were decellularized and subjected to seeding with human mesenchymal stem cells (hMSC). The effects of pulsatile conditioning using cyclic waveforms tuned to various negative and positive chamber pressures were evaluated, with respect to the seeding of cells on the decellularized leaflet and the infiltration of seeded cells into the interstitium of the leaflet. Infiltration of hMSCs into the aortic valve leaflet was observed following 72 h of conditioning under negative chamber pressure. Additional conditioning under positive pressure improved cellular infiltration, while retaining gene expression within the MSC-valve interstitial cell phenotype lineage. This protocol resulted in a subsurface pilot population of cells, not full tissue recellularization. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 249-259, 2017.

Decellularized Wharton's Jelly from human umbilical cord as a novel 3D scaffolding material for tissue engineering applications.

In tissue engineering, an ideal scaffold attracts and supports cells thus providing them with the necessary mechanical support and architecture as they reconstruct new tissue in vitro and in vivo. This manuscript details a novel matrix derived from decellularized Wharton's jelly (WJ) obtained from human umbilical cord for use as a scaffold for tissue engineering application. This decellularized Wharton's jelly matrix (DWJM) contained 0.66 ± 0.12 µg/mg sulfated glycosaminoglycans (GAGs), and was abundant in hyaluronic acid, and completely devoid of cells. Mass spectroscopy revealed the presence of collagen types II, VI and XII, fibronectin-I, and lumican I. When seeded onto DWJM, WJ mesenchymal stem cells (WJMSCs), successfully attached to, and penetrated the porous matrix resulting in a slower rate of cell proliferation. Gene expression analysis of WJ and bone marrow (BM) MSCs cultured on DWJM demonstrated decreased expression of proliferation genes with no clear pattern of differentiation. When this matrix was implanted into a murine calvarial defect model with, green fluorescent protein (GFP) labeled osteocytes, the osteocytes were observed to migrate into the matrix as early as 24 hours. They were also identified in the matrix up to 14 days after transplantation. Together with these findings, we conclude that DWJM can be used as a 3D porous, bioactive and biocompatible scaffold for tissue engineering and regenerative medicine applications.

Aortic annuloplasty with aortic root reconstruction to prevent patient-prosthesis mismatch.

Part of the ongoing argument concerning patient-prosthesis mismatch (PPM) following aortic valve replacement (AVR) is due to the perception that aortic annulus enlargement procedures increase the risk and technical difficulty of aortic valve surgery. Here, an aortic root reconstruction that involves enlargement of the annulus and tailoring of the aortic root to accommodate larger stented prostheses is presented that has been personally performed in 196 patients with no technique-related surgical deaths or complications, and thus can be carried out without additional risk. This aortic root enlargement aortoplasty and annuloplasty method can be calibrated to all AVRs involving stented manufactured prostheses when these are deemed the prosthesis of choice for the patient with a relatively small annulus and/or aortic root, severe left ventricular hypertrophy, compromised LV function or a very active lifestyle, to achieve predicted EOA values > or = 1.00 cm2/m2.

Pulse Duplicator Hydrodynamic Testing of Bioengineered Biological Heart Valves.

There are many heart valve replacements currently available on the market; however, these devices are not ideal for pediatric patients with congenital heart valve defects. Decellularized valve substitutes offer potential for improved clinical outcomes and require pre-clinical testing guidelines and testing systems suitable for non-crosslinked, biological heart valves. The objective of this study was to assess the hydrodynamic performance of intact, bioengineered pulmonary valves using a custom pulse duplicator capable of testing intact biological valved conduits. The mechanical behavior of valve associated sinus and arterial tissue was also evaluated under biaxial loading. Cryopreserved, decellularized, extracellular matrix (ECM) conditioned and glutaraldehyde fixed valves showed reduced pressure gradients and increased effective orifice area for decellularized and ECM conditioned valves. ECM conditioning resulted in increased elastic modulus but decreased stretch in circumferential and longitudinal directions under biaxial loading. Overall, decellularization and ECM conditioning did not compromise the scaffolds, which exhibited satisfactory bench top performance.

Chondroinduction from Naturally Derived Cartilage Matrix: A Comparison Between Devitalized and Decellularized Cartilage Encapsulated in Hydrogel Pastes.

Hydrogel precursors are liquid solutions that are prone to leaking after surgical placement. This problem was overcome by incorporating either decellularized cartilage (DCC) or devitalized cartilage (DVC) microparticles into traditional photocrosslinkable hydrogel precursors in an effort to achieve a paste-like hydrogel precursor. DCC and DVC were selected specifically for their potential to induce chondrogenesis of stem cells, given that materials that are chondroinductive on their own without growth factors are a revolutionary goal in orthopedic medicine. We hypothesized that DVC, lacking the additional chemical processing steps in DCC to remove cell content, would lead to a more chondroinductive hydrogel with rat bone marrow-derived mesenchymal stem cells. Hydrogels composed of methacrylated hyaluronic acid (MeHA) and either DCC or DVC microparticles were tested with and without exposure to transforming growth factor (TGF)-ß3 over a 6 week culture period, where swelling, mechanical analysis, and gene expression were observed. For collagen II, Sox-9, and aggrecan expression, MeHA precursors containing DVC consistently outperformed the DCC-containing groups, even when the DCC groups were exposed to TGF-ß3. DVC consistently outperformed all TGF-ß3-exposed groups in aggrecan and collagen II gene expression as well. In addition, when the same concentrations of MeHA with DCC or DVC microparticles were evaluated for yield stress, the yield stress with the DVC microparticles was 2.7 times greater. Furthermore, the only MeHA-containing group that exhibited shape retention was the group containing DVC microparticles. DVC appeared to be superior to DCC in both chondroinductivity and rheological performance of hydrogel precursors, and therefore DVC microparticles may hold translational potential for cartilage regeneration.