Ashwin Nathan

The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers

Aligned electrospun scaffolds are promising tools for engineering fibrous musculoskeletal tissues, as they reproduce the mechanical anisotropy of these tissues and can direct ordered neo-tissue formation. However, these scaffolds suffer from a slow cellular infiltration rate, likely due in part to their dense fiber packing. We hypothesized that cell ingress could be expedited in scaffolds by increasing porosity, while at the same time preserving overall scaffold anisotropy. To test this hypothesis, poly(epsilon-caprolactone) (a slow-degrading polyester) and poly(ethylene oxide) (a water-soluble polymer) were co-electrospun from two separate spinnerets to form dual-polymer composite fiber-aligned scaffolds. Adjusting fabrication parameters produced aligned scaffolds with a full range of sacrificial (PEO) fiber contents. Tensile properties of scaffolds were functions of the ratio of PCL to PEO in the composite scaffolds, and were altered in a predictable fashion with removal of the PEO component. When seeded with mesenchymal stem cells (MSCs), increases in the starting sacrificial fraction (and porosity) improved cell infiltration and distribution after three weeks in culture. In pure PCL scaffolds, cells lined the scaffold periphery, while scaffolds containing >50% sacrificial PEO content had cells present throughout the scaffold. These findings indicate that cell infiltration can be expedited in dense fibrous assemblies with the removal of sacrificial fibers. This strategy may enhance in vitro and in vivo formation and maturation of functional constructs for fibrous tissue engineering.

Tissue engineering with meniscus cells derived from surgical debris.

OBJECTIVE Injuries to the avascular regions of the meniscus fail to heal and so are treated by resection of the damaged tissue. This alleviates symptoms but fails to restore normal load transmission in the knee. Tissue engineering functional meniscus constructs for re-implantation may improve tissue repair. While numerous studies have developed scaffolds for meniscus repair, the most appropriate autologous cell source remains to be determined. In this study, we hypothesized that the debris generated from common meniscectomy procedures would possess cells with potential for forming replacement tissue. We also hypothesized that donor age and the disease status would influence the ability of derived cells to generate functional, fibrocartilaginous matrix. METHODS Meniscus derived cells (MDCs) were isolated from waste tissue of 10 human donors (seven partial meniscectomies and three total knee arthroplasties) ranging in age from 18 to 84 years. MDCs were expanded in monolayer culture through passage 2 and seeded onto fiber-aligned biodegradable nanofibrous scaffolds and cultured in a chemically defined media. Mechanical properties, biochemical content, and histological features were evaluated over 10 weeks of culture. RESULTS Results demonstrated that cells from every donor contributed to increasing biochemical content and mechanical properties of engineered constructs. Significant variability was observed in outcome parameters (cell infiltration, proteoglycan and collagen content, and mechanical properties) amongst donors, but these variations did not correlate with patient age or disease condition. Strong correlations were observed between the amount of collagen deposition within the construct and the tensile properties achieved. In scaffolds seeded with particularly robust cells, construct tensile moduli approached maxima of approximately 40 MPa over the 10-week culture period. CONCLUSIONS This study demonstrates that cells derived from surgical debris are a potent cell source for engineered meniscus constructs. Results further show that robust growth is possible in MDCs from middle-aged and elderly patients, highlighting the potential for therapeutic intervention using autologous cells.

MECHANO-TOPOGRAPHIC MODULATION OF STEM CELL NUCLEAR SHAPE ON NANOFIBROUS SCAFFOLDS

Stem cells transit along a variety of lineage-specific routes towards differentiated phenotypes. These fate decisions are dependent not just on the soluble chemical cues that are encountered or enforced in vivo and in vitro, but also on physical cues from the cellular microenvironment. These physical cues can consist of both nano- and micro-scale topographical features, as well as mechanical inputs provided passively (from the base properties of the materials to which they adhere) or actively (from extrinsic applied mechanical deformations). A suitable tool to investigate the coordination of these cues lies in nanofibrous scaffolds, which can both dictate cellular and cytoskeletal orientation and facilitate mechanical perturbation of seeded cells. Here, we demonstrate a coordinated influence of scaffold architecture (aligned vs. randomly organized fibers) and tensile deformation on nuclear shape and orientation. Sensitivity of nuclear morphology to scaffold architecture was more pronounced in stem cell populations than in terminally differentiated fibrochondrocytes. Tension applied to the scaffold elicited further alterations in nuclear morphology, greatest in stem cells, that were mediated by the filamentous actin cytoskeleton, but not the microtubule or intermediate filament network. Nuclear perturbations were time and direction dependent, suggesting that the modality and direction of loading influenced nuclear architecture. The present work may provide additional insight into the mechanisms by which the physical microenvironment influences cell fate decisions, and has specific application to the design of new materials for regenerative medicine applications with adult stem cells.

THE INFLUENCE OF AN ALIGNED NANOFIBROUS TOPOGRAPHY ON HUMAN MESENCHYMAL STEM CELL FIBROCHONDROGENESIS

Fibrocartilaginous tissues serve critical load-bearing functions in numerous joints throughout the body. As these structures are often injured, there exists great demand for engineered tissue for repair or replacement. This study assessed the ability of human marrow-derived mesenchymal stem cells (MSCs) to elaborate a mechanically functional, fibrocartilaginous matrix in a nanofibrous microenvironment. Nanofibrous scaffolds, composed of ultra-fine biodegradable polymer fibers, replicate the structural and mechanical anisotropy of native fibrous tissues and serve as a 3D micro-pattern for directing cell orientation and ordered matrix formation. MSCs were isolated from four osteoarthritic (OA) patients, along with meniscal fibrochondrocytes (FC) which have proven to be a potent cell source for engineering fibrocartilage. Cell-seeded nanofibrous scaffolds were cultured in a chemically-defined medium formulation and mechanical, biochemical, and histological features were evaluated over 9 weeks. Surprisingly, and contrary to previous studies with juvenile bovine cells, matrix assembly by adult human MSCs was dramatically hindered compared to donor-matched FCs cultured similarly. Unlike FCs, MSCs did not proliferate, resulting in sparsely colonized constructs. Increases in matrix content, and therefore changes in tensile properties, were modest in MSC-seeded constructs compared to FC counterparts, even when normalized to the lower cell number in these constructs. To rule out the influence of OA sourcing on MSC functional potential, constructs from healthy young donors were generated; these constructs matured no differently than those formed with OA MSCs. Importantly, there was no difference in matrix production of MSCs and FCs when cultured in pellet form, highlighting the sensitivity of human MSCs to their 3D microenvironment.

Adherence to Dual Antiplatelet Therapy After Coronary Stenting: A Systematic Review

Adherence to dual antiplatelet therapy (DAPT) is critical after coronary stenting. Although adherence rates are frequently assessed in clinical trials, adherence rates in the unselected population recommended for treatment but beyond clinical trials are largely unknown. Therefore, we performed a systematic review of published observational studies to describe rates of DAPT adherence, trends in DAPT use over time, and patient‐level factors associated with nonadherence.

Exercise Oscillatory Ventilation in Patients With Fontan Physiology

Background—Exercise oscillatory ventilation (EOV) refers to regular oscillations in minute ventilation (VE) during exercise. Its presence correlates with heart failure severity and worse prognosis in adults with acquired heart failure. We evaluated the prevalence and predictive value of EOV in patients with single ventricle Fontan physiology. Methods and Results—We performed a cross-sectional analysis and prospective survival analysis of patients who had undergone a Fontan procedure and subsequent cardiopulmonary exercise test. Data were reviewed for baseline characteristics and incident mortality, heart transplant, or nonelective cardiovascular hospitalization. EOV was defined as regular oscillations for >60% of exercise duration with amplitude >15% of average VE. Survival analysis was performed using Cox regression. Among 253 subjects, EOV was present in 37.5%. Patients with EOV were younger (18.8±9.0 versus 21.7±10.1 years; P=0.02). EOV was associated with higher New York Heart Association functional class (P=0.02) and VE/VCO2 slope (36.8±6.9 versus 33.7±5.7; P=0.0002), but not with peak VO2 (59.7±14.3 versus 61.0±16.0% predicted; P=0.52) or noninvasive measures of cardiac function. The presence of EOV was associated with slightly lower mean cardiac index but other invasive hemodynamic variables were similar. During a median follow-up of 5.5 years, 22 patients underwent transplant or died (n=19 primary deaths, 3 transplants with 2 subsequent deaths). EOV was associated with increased risk of death or transplant (hazard ratio, 3.9; 95% confidence interval, 1.5–10.0; P=0.002) and also predicted the combined outcome of death, transplant, or nonelective cardiovascular hospitalization after adjusting for New York Heart Association functional class, peak VO2, and other covariates (multivariable hazard ratio, 2.0; 95% confidence interval, 1.2–3.6; P=0.01). Conclusions—EOV is common in the Fontan population and strongly predicts lower transplant-free survival.

Impact of Optimal Medical Therapy in the Dual Antiplatelet Therapy Study

Background: Continued dual antiplatelet therapy and optimal medical therapy (OMT) improve outcomes in selected patient populations with established coronary heart disease, but whether OMT modifies the treatment effect of dual antiplatelet therapy is unknown. Methods: The DAPT (Dual Antiplatelet Therapy) Study, a double-blind trial, randomly assigned 11 648 patients who had undergone coronary stenting and completed 1 year of dual antiplatelet therapy without major bleeding or ischemic events to an additional 18 months of continued thienopyridine or placebo. OMT was defined as a combination of statin, &bgr;-blocker, and angiotensin-converting enzyme inhibitor/angiotensin receptor blocker use in patients with an American College of Cardiology/American Heart Association class I indication for each medication. Per protocol, all patients were treated with 75 to 325 mg aspirin daily. End points included myocardial infarction, major adverse cardiovascular and cerebrovascular events, and Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Arteries moderate or severe bleeding events. Results: Of 11 643 randomly assigned patients with complete medication data, 63% were on OMT. Between 12 and 30 months, continued thienopyridine reduced myocardial infarction in comparison with placebo in both groups (on OMT 2.1% versus 3.3%, hazard ratio [HR], 0.64; 95% confidence interval [CI], 0.48–0.86; P=0.003; off OMT 2.2% versus 5.2%, HR, 0.41; CI, 0.29–0.58; P<0.001; interaction P=0.103). Comparing continued thienopyridine versus placebo, rates of major adverse cardiovascular and cerebrovascular events were 4.2% versus 5.0% among patients on OMT (HR, 0.82; CI, 0.66–1.02; P=0.077) and 4.5% versus 7.0% among those off OMT (HR, 0.63; CI, 0.49–0.82; P<0.001; interaction P=0.250); rates of bleeding for thienopyridine versus placebo in patients on OMT were 2.2% versus 1.0% (HR, 2.13; CI, 1.43–3.17; P<0.001), and in patients off OMT were 2.8% versus 2.2% (HR, 1.30; CI, 0.88–1.92; P=0.189; interaction P=0.073). Overall, patients on OMT had lower rates of myocardial infarction (2.7% versus 3.7%, P=0.003), major adverse cardiovascular and cerebrovascular events (4.6% versus 5.7%, P=0.007), and bleeding (1.6% versus 2.5%, P<0.001) in comparison with patients off OMT. Rates of stent thrombosis (0.8% versus 1.0%, P=0.171) and death (1.6% versus 1.9%, P=0.155) did not differ. Conclusions: Continued thienopyridine therapy reduced the rate of myocardial infarction regardless of OMT status and had consistent effects on reduction in major adverse cardiovascular and cerebrovascular events and increased bleeding. Clinical Trial Registration: URL: http://clinicaltrials.gov. Unique identifier: NCT00977938.

Decline in Peak Oxygen Consumption over Time Predicts Death or Transplantation in Adults with a Fontan Circulation

Background Peak oxygen consumption (pVO2) measured by cardiopulmonary exercise test (CPET) predicts mortality in adults with a Fontan circulation. The purpose of this study was to assess the additive prognostic value of change in pVO2 over time. Methods We analyzed a cohort of adults (≥18 years old) with a Fontan circulation who underwent at least 2 maximal CPETs separated by 6‐30 months at Boston Childrens Hospital between 2000 and 2015. Survival analysis was performed to determine whether changes in CPET variables, including pVO2 between consecutive tests, were associated with subsequent clinical events. The primary outcome was transplant‐free survival. Results The study included 130 patients with 287 CPET test pairs. Average age was 26.6 ± 9.5 years. Baseline pVO2 averaged 22.0 ± 5.7 mL/kg/min or 60.9% ± 13.7% predicted. In the cohort overall, there was no change in mean pVO2 between sequential CPETs. Eleven patients died and 2 underwent transplant. On average, pVO2 declined for patients who subsequently died or underwent transplant but remained stable among those who did not (−9.8% ± 14.6% vs 0.0 ± 13.0%, P < .01). Those with a decline in pVO2 between CPETs were at greater risk of death or transplantation (per 10% decrease in pVO2: HR = 2.0, 95% CI 1.2‐3.1, P = .004). Change in pVO2 remained a significant predictor of death or transplant after adjusting for pVO2 at first CPET (per 10% decline in pVO2: HR = 2.5, 95% CI 1.5‐4.2, P < .001). Conclusions A decline in pVO2 between consecutive CPETs predicts increased risk for death or transplant in adults with a Fontan circulation independent of baseline pVO2. These results support the additive clinical value of serial CPET in this population.

Serial Classic and Inverted Pattern Takotsubo Cardiomyopathy in a Middle-Aged Woman

We report the case of a 56-year-old woman with no significant medical history who was diagnosed with recurrent Takotsubo cardiomyopathy with variations in ventricular regional involvement including the classic and inverted patterns. She presented on 3 separate occasions with these findings; emotional stressors provoked all presentations. We present echocardiography, cardiac catheterization, and magnetic resonance images from her consecutive presentations. This case of emotional stress repeatedly eliciting classic and inverted forms of Takotsubo cardiomyopathy within the same patient highlights the importance of elucidating the pathological mechanisms of regional ventricular dysfunction.

MULTI-LAMELLAR AND MULTI-AXIAL MATURATION OF CELL-SEEDED FIBER- REINFORCED TISSUE ENGINEERED CONSTRUCTS

The architecture of load-bearing fibrous tissues is optimized to enable a specific set of mechanical functions. This organization arises from a complex process of cell patterning, matrix deposition, and functional maturation [1]. In their mature state, these tissues span multiple length scales, encompassing nanoscale interactions of cells with extracellular matrix to the centimeter length scales of the anatomic tissue volume and shape. Two structures that typify dense fibrous tissues are the meniscus of the knee and the annulus fibrosus (AF) of the intervertebral disc (IVD). The mechanical function of the wedge-shaped knee meniscus is based on its stiff prevailing circumferential collagen architecture that resists tensile deformation [2,3]. Adding to its complexity, radial tie fibers and sheets are interwoven amongst these fibers, increasing stiffness in the transverse direction and binding the tissue together [4]. In the annulus fibrosus, multiple anisotropic lamellae are stacked in concentric rings with their prevailing fiber directions alternating above and below the horizontal axis in adjacent layers [5]. The high circumferential tensile properties of this laminate structure allow it to resist bulging of the nucleus pulposus with compressive loading of the spine. Given their structural properties, unique form, and demanding mechanical environments, the knee meniscus and the AF region of the IVD represent two of the most challenging tissues to consider for functional tissue engineering.Copyright