Induction of osteogenic differentiation by the extracellular matrix of fetal bone tissues and adult cartilage.

Decellularized cortical bone powder derived from adult animals has been shown to induce bone remodeling. Furthermore, it is increasingly evident that the extracellular matrix (ECM) within decellularized tissues differs depending on the source tissue and the age of the animal, leading to distinct effects on cells. In this study, we prepared powders from decellularized fetal and adult porcine bone tissues and conducted biological analyses to determine if the decellularized tissue could induce adipose-derived stem cell differentiation. Decellularized fetal tissues and adult cortical bone were converted into powder by cryomilling, but decellularized adult bone marrow and cartilage were not powdered through this process. In vitro assessments revealed that decellularized fetal tissues, decellularized adult cartilage extract, and decellularized fetal cartilage powder can induce osteoblast differentiation. This study suggests that decellularized fetal bone tissues and adult cartilage contain ECM components that can induce osteoblast differentiation. Additionally, it highlights the utility of decellularized fetal cartilage powder for bone reconstruction.

Fabrication of a cell culture scaffold that mimics the composition and structure of bone marrow extracellular matrix.

Cell culture models that mimic tissue environments are useful for cell and extracellular matrix (ECM) function analysis. Decellularized tissues with tissue-specific ECM are expected to be applied as cell culture scaffolds, however, it is often difficult for seeded cells to permeate their structures. In this study, we evaluated the adhesion and proliferation of mouse fibroblasts seeded onto decellularized bone marrow scaffolds that we fabricated from adult and fetal porcine. Decellularized fetal bone marrow displays more cell attachment and faster cell proliferation than decellularized adult bone marrow. Our findings suggest that decellularized fetal bone marrow is useful as a cell culture scaffold with bone marrow ECM and structure.

Extraction and Biological Evaluation of Matrix-Bound Nanovesicles (MBVs) from High-Hydrostatic Pressure-Decellularized Tissues.

Decellularized tissues are widely used as promising materials in tissue engineering and regenerative medicine. Research on the microstructure and components of the extracellular matrix (ECM) was conducted to improve the current understanding of decellularized tissue functionality. The presence of matrix-bound nanovesicles (MBVs) embedded within the ECM was recently reported. Results of a previous experimental investigation revealed that decellularized tissues prepared using high hydrostatic pressure (HHP) exhibited good in vivo performance. In the current study, according to the hypothesis that MBVs are one of the functional components in HHP-decellularized tissue, we investigated the extraction of MBVs and the associated effects on vascular endothelial cells. Using nanoparticle tracking assay (NTA), transmission electron microscopy (TEM), and RNA analysis, nanosized (100-300 nm) and membranous particles containing small RNA were detected in MBVs derived from HHP-decellularized small intestinal submucosa (SIS), urinary bladder matrix (UBM), and liver. To evaluate the effect on the growth of vascular endothelial cells, which are important in the tissue regeneration process, isolated SIS-derived MBVs were exposed to vascular endothelial cells to induce cell proliferation. These results indicate that MBVs can be extracted from HHP-decellularized tissues and may play a significant role in tissue remodeling.

Cell Adhesion Behaviors on Spider Silk Fibers, Films, and Nanofibers.

Silk-based materials have garnered attention for use as medical supplies due to their mechanical toughness and low cytotoxicity. Silkworm silk has been applied as surgical sutures for decades. In contrast, the utilization of spider silk is limited mainly because of its scarcity. Although the biomimicry of spider silk has been developed using recombinant protein expression systems with the use of genetic engineering, the product often results in lower molecular weight and a lack of the N- or C-terminal regions. The incomplete sequence of the spider silk-like protein prevents the objective evaluation of the native spider silk as a medical application and retards the development of spider silk-inspired materials. Here, we reeled the native spider silk directly from live spiders and investigated the cell adhesion behavior based on three kinds of surface topography of spider silk-based substrates, namely, fibers, films, and non-woven fabrics. The cell adhesion behavior was largely influenced by the surface micro/nanostructure rather than the wettability of the surface. This study will contribute to promote the utilization of spider silk in the medical field as a candidate for promising bio-based fibers in the context of sustainable development goals.

4-Arm PEG-Functionalized Decellularized Pericardium for Effective Prevention of Postoperative Adhesion in Cardiac Surgery.

Postoperative adhesions are a very common and serious complication in cardiac surgery, and the development of an effective anti-adhesion membrane showing resistance to the physical stimulus generated by the pulsation of the heart is desirable. In this study, an anti-adhesion material was developed through amine coupling between decellularized bovine pericardia (dBPCs) and 4-arm poly(ethylene glycol) succinimidyl glutarate (4-arm PEG-NHS) for the postoperative care of cardiac surgical patients. The efficacy of the 4-arm PEG-functionalized dBPCs in the prevention of adhesions after cardiac surgery was investigated in a rabbit heart adhesion model. The dBPCs meet the requirements for biocompatibility, flexibility, and sufficient suturable strength, and the 4-arm PEG moieties provide an anti-adhesion effect by the high excluded volume interactions of the PEG chains with proteins. The 4-arm PEG-functionalized dBPCs had a significantly greater anti-adhesion effect than the other materials tested and showed re-establishment of the mesothelial monolayer. These results suggested that the 4-arm PEG-functionalized dBPCs are a favorable material for an anti-adhesion membrane.

All-in-One Silk Fibroin Sponge as the Vitrification Cryodevice of Rat Pancreatic Islets and the VEGF-Embedded Scaffold for Subrenal Transplantation.

Islet transplantation is a promising option for the clinical treatment of insulin-dependent diabetes, but a reliable islet cryopreservation/transplantation protocol should be established to overcome the donor shortage. The current study reports that a silk fibroin (SF) sponge disk can be used as a cryodevice for vitrification of large quantity pancreatic islets and the scaffold for subsequent subrenal transplantation in a rat model. The marginal islet mass (550 islet equivalents [IEQs]) on an SF sponge disk was vitrified-warmed and transplanted beneath the kidney capsule of a streptozotocin-induced diabetic rat with or without vascular endothelial growth factor (VEGF). Subrenal transplantation (no scaffold) of 550 IEQ fresh islets and post-warm islets vitrified on a nylon mesh device resulted in achieving euglycemia of recipient rats at 60% and 0%, respectively. Transplantation of 550 IEQ islets vitrified-warmed on an SF sponge disk failed to achieve euglycemia of recipient rats (0%), but the VEGF inclusion in the SF sponge disk contributed to acquiring the euglycemic recipients (33%). All cured recipient rats regained hyperglycemia after nephrectomy, and the histopathologic analysis exhibited a well-developing blood vessel network into the islet engrafts. Thus, an SF sponge disc was successively available as the cryodevice for islet vitrification, the transporter of the angiogenic VEGF, and the scaffold for subrenal transplantation in the rat model.

Acellular matrix derived from rat liver improves the functionality of rat pancreatic islets before or after vitrification.

Cryopreservation of pancreatic islets can overcome the severe shortage of islet donors in clinical islet transplantation, but the impaired quality of post-warm islets need improvement. This present study was conducted to investigate whether the pre- or post-treatment of rat islets with liver decellularized matrix (LDM) for vitrification can improve the viability (FDA/PI double staining) and the functionality (glucose-stimulated insulin secretion [GSIS] assay). Rat LDM was prepared by high-hydrostatic pressure, lyophilization, and re-suspension in saline. Co-culturing of isolated islets with 0 (control), 30, 60, or 90 µg/ml LDM for 24 h resulted in the comparable viability among the 4 groups (98.7-99.6%) and the higher insulin secretion potential in 30 and 60 µg/ml LDM treatment groups than the control group (stimulation index [SI]: 12.1 and 12.7, respectively, vs. 6.5 in the control group, P < 0.05). When the islets co-cultured with 60 µg/ml LDM were vitrified-warmed on a nylon mesh cryodevice, the viability and the GSIS of the post-warm islets were not improved. Post-treatment of vitrified-warmed islets with 60 µg/ml LDM during the recovery culture for 12 h resulted in the comparable clearance of degenerating cell debris from the post-warm islets, while their insulin secretion potential was improved (SI: 5.0 vs. 3.5 in the control group, P < 0.05). These findings indicate that the components in LDM can enhance the insulin secretion potential of rat islets suffering damage by enzymatic stress during the islet isolation process or by cryoinjuries during the vitrification-warming process.

Electrocardiographic features of arrhythmogenic right ventricular cardiomyopathy in school-aged children.

It can be difficult to distinguish children with early-stage arrhythmogenic right ventricular cardiomyopathy (ARVC) from those with benign premature ventricular contraction (PVC). We retrospectively evaluated six school-aged children with ARVC and compared with those of 20 with benign PVC. The median age at initial presentation was 11.4 and 10.2 years in ARVC and benign PVC, respectively. None of the ARVC patients fulfilled the diagnostic criteria of ARVC at initial presentation. At ARVC diagnosis, the treadmill exercise test and Holter monitoring showed provoked PVC during exercise and pleomorphic PVC in all ARVC cases, respectively. During the observation period, terminal activation duration (TAD) was prolonged in all ARVC patients. In addition, ΔTAD (5.5 [3-10] ms) were significantly longer than those with benign PVC (p < 0.001). A new notched S-wave in V1 appeared in four (67%) ARVC patients, who had myocardial abnormalities in the right ventricle, and in zero benign PVC. Our electrocardiographic findings, such as provoked PVC during exercise, pleomorphic PVC, prolonged TAD, and a new notched S-wave in V1 could contribute to the early detection of ARVC in school-aged children.

A large-scale observational study to investigate the current status of diabetic complications and their prevention in Japan (JDCP study 6): baseline dental and oral findings.

Japan Diabetes Complication and Prevention prospective (JDCP) study was conducted to examine the association between glycemic control and oral conditions in a large database of Japanese patients with diabetes. It included a total of 6099 patients with diabetes (range, 40-75 years) who had been treated as outpatients between 2007 and 2009. The mean number of present teeth at baseline was 19.8 and women with type 2 diabetes had fewer teeth than men with type 2 diabetes. Within the previous year, 17% of all patients had lost teeth. At baseline, 32% had experienced gingival swelling, 69% had brushed more than twice a day, 37% had used interdental cleaning aids, and 43% had undergone regular dental checkups. Multiple logistic regression analysis indicated that type 1 patients with HbA1c ≥ 7.0% were at higher risk of having fewer than 20 teeth (odds ratio [OR] 2.38; 95% confidence interval [CI] 1.25-4.78), and type 2 patients with HbA1c ≥ 8.0% also were at high risk of having fewer than 20 teeth (OR 1.16; 95% CI 1.00-1.34), after adjustment for nine possible confounding factors. In conclusion, patients with diabetes were found to be at high risk of tooth loss, and the poorer the glycemic control, the higher the risk of tooth loss in these patients.

Transplantation of rat pancreatic islets vitrified-warmed on the nylon mesh device and the silk fibroin sponge disc.

We report the adaptability of rat islets vitrified-warmed on nylon mesh (NM) device or silk fibroin (SF) sponge disc for the normalization of the blood glucose level in rat models of diabetes. One-hundred rat islets were cryopreserved according to a minimum volume cooling protocol on an NM device or a solid surface vitrification protocol on an SF sponge disc. The recovery rate (97.1% vs. 93.8%), the viability (77.9% vs. 74.4%), and the stimulation index (4.7 vs. 4.2) in glucose-stimulated insulin secretion (GSIS) assay of the post-warm islets were comparable between the NM vitrification and the SF vitrification groups. The viability and the stimulation index of the fresh control islets were identified to be 97.5% and 6.5, respectively. Eight hundred islets from the NM or the SF vitrification group or the fresh control group were transplanted beneath the kidney capsule of a streptozotocin-induced diabetic rat (blood glucose level > 350 mg/dl). Within 3 weeks after transplantation, the acquisition of euglycemia (< 200 mg/dl) was observed in recipient rats (80.0-83.3%). An intraperitoneal glucose tolerance test on Day-30 and Day-60 showed similar 2-h responses to the glucose uptake of cured rats among the compared groups. Moreover, the successful engraftment of transplants was confirmed by the Day-70 nephrectomy through the subsequent diabetes reversal and histological evaluation. Thus, large quantities of rat islets vitrified-warmed on an NM device or an SF sponge disc were proven to be fully functional both in vitro and in vivo, due to the GSIS and syngeneic transplantation, respectively.

Introduction of Cells into Porous Poly-l-Lactic Acid Scaffolds Using Impregnation Techniques.

Porous materials containing cells-prepared via cell seeding on scaffolds or gelation of cell-containing solutions-have been widely studied to investigate tissue regeneration and three-dimensional cultures. However, these methods cannot introduce cells into porous materials that have low water absorption or scaffolds that require cytotoxic solvents or processes for their production. In this study, first, three different impregnation treatments conditions (vacuum, pressure, and vacuum pressure impregnation: VPI) were applied to cell suspensions to evaluate the effect of each treatment on cells. Following all three treatments, fibroblasts adhered to the cell culture dish and proliferated in the same manner as untreated cells, which confirmed that the three impregnation treatments did not affect cell function. Second, cells were introduced into a poly-l-lactic acid (PLA) scaffold, which has low water absorption, using the same impregnation treatments. The PLA scaffolds subjected to the three impregnation treatments that exhibited a significantly greater amount of DNA than those subjected to immersion treatments and showed increasing amounts of DNA in the order vacuum treatment > VPI treatment > pressure treatment. Furthermore, the amount of DNA in the vacuum-treated and VPI-treated PLA scaffolds increased on the first, third, and fifth days of culture, and it was confirmed that the cells introduced into the PLA scaffolds proliferated. These results suggest that vacuum and VPI treatments may be useful methods for introducing cells into porous materials.

Impact of delayed ventricular wall area ratio on pathophysiology of mechanical dyssynchrony: implication from single-ventricle physiology and 0D modeling.

Electrical disparity can induce inefficient cardiac performance, representing an uncoordinated wall motion at an earlier activated ventricular wall: an early shortening followed by a systolic rebound stretch. Although regional contractility and distensibility modulate this pathological motion, the effect of a morphological factor has not been emphasized. Our strain analysis in 62 patients with single ventricle revealed that those with an activation delay in 60-70% of ventricular wall area suffered from cardiac dysfunction and mechanical discoordination along with prolonged QRS duration. A computational simulation with a two-compartment ventricular model also suggested that the ventricle with an activation delay in 70% of the total volume was most vulnerable to a large activation delay, accompanied by an uncoordinated motion at an earlier activated wall. Taken together, the ratio of the delayed ventricular wall has a significant impact on the pathophysiology due to an activation delay, potentially highlighting an indicator of cardiac dysfunction.

Etiology of atrial fibrillation in patients with complex congenital heart disease - for a better treatment strategy.

BACKGROUND: The demographics of patients with congenital heart disease (CHD) and atrial fibrillation (AF) differ significantly from the general population. The etiology and treatment strategy for AF in CHD patients have been investigated but are to date inconclusive. METHODS: To determine the etiology of AF in CHD and to seek a better treatment strategy, we retrospectively evaluated the atrial overload in 42 complex CHD cases with normal atrial arrangements and AF (age 25; range, 9-66 years) and the impact of a reduction in the atrial overload on the atrial rhythm. RESULTS: Cardiac defect diagnoses varied, with 17% of the patients having a persistent left superior vena cava (PLSVC). In regard to the volume overload, the frequencies of an overload in the right atrium (RA), left atrium (LA), or both, were 50 %, 23%, and 10%, respectively (p = 0.015). Other sustained supraventricular tachycardias were observed in 29 patients (69%) before and after the onset of AF. Among these 29 patients, 26 had intra-atrial reentrant tachycardia. Fifteen patients (36%), 10 of whom had chronic AF, died during the follow-up including 3 with arrhythmias and 10 because of heart failure. Fourteen (33%) patients had no AF at the last follow-up due to medical interventions, 8 of which underwent solely an RA-sided catheter ablation and/or surgical RA overload reduction. CONCLUSIONS: AF in complex CHD with a normal atrial arrangement correlates with a higher RA-sided overload than an LA-sided and exhibits a high incidence of PLSVCs, high comorbidity of intra-atrial reentrant tachycardias, and high mortality rate. In a substantial number of patients, RA-sided interventions were effective in controlling AF. To effectively manage AF in complex CHD it is essential to understand each individual's hemodynamics and consider hemodynamic interventions.

Effect of Stiffened and Dilated Ascending Aorta on Aerobic Exercise Capacity in Repaired Patients With Complex Congenital Heart Disease.

Several studies have reported aortic dilation and increased stiffness of the ascending aorta in patients after repair of congenital heart disease (CHD), which may be a predominant cardiovascular risk. However, the clinical significance has not been described in detail. In this retrospective study, 175 repaired patients with complex CHD achieving biventricular circulation and age-matched 39 control subjects were reviewed (median age: 14.9 and 15.7 years, respectively). We measured the diameters of the ascending aorta and descending aorta from catheterization angiograms to yield Z-scores and stiffness indexes (ß) using diameter fluctuations corresponding to pulsatile pressures. Clinical profile, peak oxygen uptake during the cardiopulmonary exercise test, and incidence of unscheduled hospitalization during follow-up was also reviewed. Compared with controls, patients with complex CHD, except for those with aortic coarctation, exhibited significant dilation and increased stiffness of the aortic root and ascending aorta, but not of the descending aorta. In this CHD population (n = 147, including 112 conotruncal anomalies), exercise capacities correlated independently with the diameter Z-score and stiffness index of the ascending aorta along with the history of repetitive thoracotomies, reduced forced vital capacity, and right ventricular hypertension. During a follow-up period (median 15.6 years), either dilation (Z-score >3.5) or increased stiffness (ß >6.0) of the ascending aorta stratified morbidity, but no synergistic impact was detected. In conclusion, in repaired patients with complex CHD, a stiffened and dilated ascending aorta was frequently found, exerting significant adverse impacts on diminished exercise capacity and morbidity.

Progressive stiffening and relatively slow growth of the dilated ascending aorta in long-term Fontan survivors-Serial assessment for 15 years.

BACKGROUND: A stiffened, dilated ascending aorta may represent an important predictor of cardiovascular mortality, and has been reported in patients with congenital heart disease, including single ventricle. However, the serial conformational changes and determinants of reduced distensibility in ascending aorta have not been clarified. METHODS: This retrospective study investigated 115 postoperative Fontan survivors (median age at Fontan: 3.7 years). All patients underwent cardiac catheterization before and 1, 5, 10, and 15 years after the Fontan operation. We measured Z-scores for diameters and stiffness indexes (ß) of the ascending aorta and descending aorta from angiograms. We also reviewed the clinical profiles, hemodynamic parameters, and exercise capacities of patients and compared them with results from 47 control subjects. RESULTS: Fontan survivors displayed significantly larger Z-score and ß of the ascending aorta from before to 15 years after surgery than controls, whereas values for the descending aorta were comparable. Z-score for the ascending aorta was decreased, but ß was elevated significantly according to the trend test. In multivariable analysis, ß of the ascending aorta at 15 years after Fontan operation and its increasing trend were associated with older age at Fontan operation and elevated ventricular end-diastolic pressure. Reduced exercise capacity also correlated with stiffening of the ascending aorta. CONCLUSIONS: Fontan survivors showed progressive stiffening and relatively slow growth of the dilated ascending aorta. Progressive stiffening of the ascending aorta may be coupled to diastolic dysfunction and reduced exercise capacity, suggesting the importance of lifelong management of subclinical Fontan pathophysiology.

Septal Flash-like Motion of the Earlier Activated Ventricular Wall Represents the Pathophysiology of Mechanical Dyssynchrony in Single-Ventricle Anatomy.

BACKGROUND: In biventricular physiology, abnormal septal motion is a hallmark of mechanical dyssynchrony in the left bundle branch block. However, in single-ventricle (SV) physiology, morphologic variations in systemic ventricles pose a challenge in evaluating the negative impact of mechanical dyssynchrony. The present study aimed to characterize the pathologic dyssynchronous contraction patterns in patients with SV. METHODS: In this retrospective study, 70 consecutive postoperative patients with SV anatomy with prolonged QRS duration (25 female patients; median age, 14 years) were enrolled. We divided each SV into two regions and analyzed independent strains using two-dimensional speckle-tracking echocardiography. From an earlier activated ventricular wall, we calculated the strain ratio (Rstrains) of two values (%) during the QRS period and the ejection period: (100 + Strainejection)/(100 + StrainQRS). We reviewed the clinical profiles, B-type natriuretic peptide plasma levels, exercise capacity, and morbidity. Six patients who underwent cardiac resynchronization therapy (CRT) were analyzed regarding changes in strain patterns and ventricular volume. RESULTS: Higher Rstrains, indicating a preceding contraction and subsequent dyskinetic dilation of the earlier activated ventricular wall, was associated with increased B-type natriuretic peptide, reduced exercise capacity, and poor outcome. However, delayed contraction of the later activated ventricular wall was not associated with the effects. Decreases in Rstrains and ventricular volume reductions were observed in all patients after CRT. CONCLUSIONS: A specific strain pattern in an earlier activated ventricular wall indicates mechanical dyssynchrony in patients with SV. This pattern is very similar to the septal flash in adult patients with left bundle branch block. This strategy might be a promising approach for selecting appropriate candidates for CRT in patients with SV.

Electrocardiographic changes and long-term prognosis of children diagnosed with hypertrophic cardiomyopathy by the school screening program for heart disease in Japan.

BACKGROUND: In Japan, the school screening program for heart disease (SS) has been performed since 1973. However, little has been reported on the electrocardiogram (ECG) changes and long-term prognosis in patients with hypertrophic cardiomyopathy (HCM) detected by the SS. METHODS: All 44 consecutive pediatric HCM patients (10.1 ±â€¯3.0 years old), who had been originally consulted by the SS before the diagnosis of HCM from April 1981 to April 2017, were reviewed retrospectively. RESULTS: At the SS, all patients showed mild or no symptoms. All patients showed ECG abnormalities, and 75 % had a high proposed ECG risk score (≧6). However, 30 % of them had no echocardiogram finding of myocardial hypertrophy. During the follow-up period (14.8 ±â€¯10.0 years), life-threatening events (LTE) occurred in 11 (25 %) patients, and the first LTE occurred during exercise in 8 (18 %). The estimated LTE and heart failure death-free survival rate at 10 years was 64.9 %. The LTE-free survival rate was lower in patients without than in those with myocardial hypertrophy at the SS. CONCLUSIONS: The SS was useful in detecting patients with HCM with mild or no symptoms at the early stage. However, our study indicated that early detection of HCM is not associated with improvement in the prognosis of the patients. Further studies are needed.

PLA-Collagen Composite Scaffold Fabrication by Vacuum Pressure Impregnation.

Composite scaffolds are made by various methods, such as copolymerization, freeze gelation, and thermally induced phase separation, which can compound materials with different properties using solvents and heat. However, it is difficult to compound solvents and heat-sensitive materials such as natural polymers. In this study, we investigated a vacuum pressure impregnation (VPI) method for creating a composite of natural polymers. A collagen solution could not be introduced into a poly (l-lactide) (PLA) porous material using an immersing treatment, but it is possible using the VPI method. The resulting PLA-collagen composite scaffold had greater water adsorption and degradation than a PLA scaffold. These results indicate that VPI may be a promising method for creating composites of natural materials. Impact Statement This is the development of a method for introducing cells into a completed porous material in a short time. This technology is expected to be applied to tissue regeneration and 3D culture.

Extremely short setting of optimal sensed atrioventricular interval in patients after Fontan procedure with implanted dual-chamber pacemaker.

BACKGROUND: Atrioventricular interval optimisation is important in patients with dual-chamber pacing, especially with heart failure. In patients with CHD, especially in those with Fontan circulation, the systemic atrial contraction is supposed to be more important than in patients without structural heart disease. METHODS: We retrospectively evaluated two patients after Fontan procedure with dual-chamber pacemaker with a unique setting of optimal sensed atrioventricular interval. RESULTS: The optimal sensed atrioventricular interval determined by echocardiogram was extremely short sensed atrioventricular interval at 25 and 30 ms in both cases; however, the actual P wave and ventricular pacing interval showed 180 and 140 ms, respectively. In both cases, the atrial epicardial leads were implanted on the opposite site of the origin of their own atrial rhythm. The time differences between sensed atrioventricular interval and actual P wave and ventricular pacing interval occurred because of the site of the epicardial atrial pacing leads and the intra-atrial conduction delay. CONCLUSION: We need to consider the origin of the atrial rhythm, the site of the epicardial atrial lead, and the atrial conduction delay by using electrocardiogram and X-ray when we set the optimal sensed atrioventricular interval in complicated CHD.