Supercritical carbon dioxide decellularized porcine cartilage graft with PRP attenuated OA progression and regenerated articular cartilage in ACLT-induced OA rats.

Knee osteoarthritis (OA) is a common degenerative articular disorder and considered one of the primary causes of pain and functional disability. Knee OA is prevalent in 10% of men and 13% of women aged 60 years above. The study aims to use cartilage tissue engineering that combines the triads of decellularized porcine cartilage graft as "scaffold," plasma rich platelet (PRP) as "signal" and chondrocytes from rat as "cell" to attenuate ACLT-induced OA progression and regenerate the knee cartilage in rats. Decellularization of the porcine cartilage was characterized by hematoxylin and eosin, 4,6-Diamidino-2-phenylindole staining, scanning electron microscopy and residual DNA quantification. The protective effect of decellularized porcine cartilage graft (dPCG) was evaluated by intra-articular administration in surgically induced anterior cruciate ligament transection (ACLT) rat osteoarthritis (OA) model. Supercritical carbon dioxide technology completely decellularized the porcine cartilage. Intra-articular administration of dPCG with or without PRP significantly reduced the ACLT-induced OA symptoms and attenuated the OA progression. Pain-relief by dPCG with or without PRP was assessed by capacitance meter and improved articular cartilage damage in the rat knee was characterized by X-ray and micro-CT. Besides, the histological analysis depicted cartilage protection by dPCG with or without PRP. The repairation and attenuation effect by dPCG with or without PRP in the articular knee cartilage damage were also explored by safranin-O, type II collagen, aggrecan and SOX-9 immuno-staining. To conclude, intra-articular administration of dPCG with or without PRP is efficient in repairing the damaged cartilage in the experimental OA model.

A novel 3D histotypic cartilage construct engineered by supercritical carbon dioxide decellularized porcine nasal cartilage graft and chondrocytes exhibited chondrogenic capability in vitro.

Augmentative and reconstructive rhinoplasty surgical procedures use autologous tissue grafts or synthetic grafts to repair the nasal defect and aesthetic reconstruction. Donor site trauma and morbidity are common in autologous grafts. The desperate need for the production of grafted 3D cartilage tissues as rhinoplasty grafts without the adverse effect is the need of the hour. In the present study, we developed a bioactive 3D histotypic construct engineered with the various ratio of adipose-derived stem cells (ADSC) and chondrocytes together with decellularized porcine nasal cartilage graft (dPNCG). We decellularized porcine nasal cartilage using supercritical carbon dioxide (SCCO2) extraction technology. dPNCG was characterized by H&E, DAPI, alcian blue staining, scanning electron microscopy and residual DNA content, which demonstrated complete decellularization. 3D histotypic constructs were engineered using dPNCG, rat ADSC and chondrocytes with different percentage of cells and cultured for 21 days. dPNCG together with 100% chondrocytes produced a solid mass of 3D histotypic cartilage with significant production of glycosaminoglycans. H&E and alcian blue staining showed an intact mass, with cartilage granules bound to one another by extracellular matrix and proteoglycan, to form a 3D structure. Besides, the expression of chondrogenic markers, type II collagen, aggrecan and SOX-9 were elevated indicating chondrocytes cultured on dPNCG substrate facilitates the synthesis of type II collagen along with extracellular matrix to produce 3D histotypic cartilage. To conclude, dPNCG is an excellent substrate scaffold that might offer a suitable environment for chondrocytes to produce 3D histotypic cartilage. This engineered 3D construct might serve as a promising future candidate for cartilage tissue engineering in rhinoplasty.

3D composite engineered using supercritical CO2 decellularized porcine cartilage scaffold, chondrocytes, and PRP: Role in articular cartilage regeneration.

At present, no definitive treatment for articular cartilage defects has been perfected. Most of the previous treatments involved multiple drilling and microfracture over defect sites with repair-related substances, which poses a limited therapeutic effect. End-stage therapy includes artificial knee joint replacement. In this study, we prepared a novel decellularized natural cartilage scaffold from porcine articular cartilage by supercritical CO2 extraction technology and three-dimensional (3D) composites made using decellularized porcine cartilage graft (dPCG) as scaffolds, platelet-rich plasma (PRP), thrombin as signals and chondrocytes as cells for the treatment of articular cartilage defects. In this study, in vitro and in vivo cartilage regeneration and the expression of chondrogenic markers were examined. Decellularized cartilage graft (dPCG) was evaluated for the extent of cell and DNA removal. Residual cartilage ECM structure was confirmed to be type II collagen by SDS PAGE and immunostaining. The new 3D composite with dPCG (100 mg and 2 × 106 chondrocytes) scaffold promotes chondrogenic marker expression in vitro. We found that the in vivo 3D composite implanted cartilage defect showed significant regeneration relative to the blank and control implant. Immunohistochemical staining showed increase of expression including Collagen type II and aggrecan in 3D composite both in vitro and in vivo studies. In this study, the bioengineered 3D composite by combining dPCG scaffold, chondrocytes, and PRP facilitated the chondrogenic marker expression in both in vitro and in vivo models with accelerated cartilage regeneration. This might serve the purpose of clinical treatment of large focal articular cartilage defects in humans in the near future.

Hypoxia suppresses myocardial survival pathway through HIF-1α-IGFBP-3-dependent signaling and enhances cardiomyocyte autophagic and apoptotic effects mainly via FoxO3a-induced BNIP3 expression.

The HIF-1α transcriptional factor and the BH-3 only protein BNIP3 are known to play fundamental roles in response to hypoxia. The objective of this research is to investigate the molecular mechanisms and the correlation of HIF-1α, BNIP3 and IGFBP-3 in hypoxia-induced cardiomyocytes injuries. Heart-derived H9c2 cells and neonatal rat ventricular myocytes (NRVMs) were incubated in normoxic or hypoxic conditions. Hypoxia increased HIF-1α expression and activated the downstream BNIP3 and IGFBP-3 thereby triggered mitochondria-dependent apoptosis. Moreover, IGF1R/PI3K/Akt signaling was attenuated by HIF-1α-dependent IGFBP-3 expression to enhance hypoxia-induced apoptosis. Autophagy suppression with 3-methyladenine or siATG5 or siBeclin-1 significantly decreased myocardial apoptosis under hypoxia. Knockdown of FoxO3a or BNIP3 significantly abrogated hypoxia-induced autophagy and mitochondria-dependent apoptosis. Moreover, prolonged-hypoxia induced HIF-1α stimulated BNIP3 and enhanced IGFBP-3 activation to inhibit IGF1R/PI3K/Akt survival pathway and mediate mitochondria-dependent cardiomyocyte apoptosis. HIF-1α and FoxO3a blockage are sufficient to annul the change of excessive hypoxia of hearts.

17ß-Estradiol and/or Estrogen Receptor ß Attenuate the Autophagic and Apoptotic Effects Induced by Prolonged Hypoxia Through HIF-1α-Mediated BNIP3 and IGFBP-3 Signaling Blockage.

BACKGROUND/AIMS: The risk of heart disease is higher in males than in females. However, this advantage of females declines with increasing age, presumably a consequence of decreased estrogen secretion and malfunctioning of the estrogen receptor. We previously demonstrated that 17ß-estradiol (E2) prevents cardiomyocyte hypertrophy, autophagy and apoptosis via estrogen receptor α (ERα), but the effects of ERß on myocardial injury remained elusive. The present paper thus, investigated the cardioprotective effects of estrogen (E2) and ERß against hypoxia-induced cell death. METHODS: Transient transfection of Tet-On ERß gene construct was used to overexpress ERß in hypoxia-treated H9c2 cardiomyoblast cells. RESULTS: Our data revealed that IGF1R, Akt phosphorylation and Bcl-2 expression are enhanced by ERß in H9c2 cells. Moreover, ERß overexpression reduced accumulation of hypoxia-related proteins, autophagy-related proteins and mitochondria-apoptotic proteins and enhanced the protein levels of Bcl-2, pAkt and Bad under hypoxic condition. In neonatal rat ventricular myocytes (NRVMs), we observed that hypoxia induced cell apoptosis as measured by TUNEL staining, and E2 and/or ERß could totally abolish hypoxia-induced apoptosis. The suppressive effects of E2 and/or ERß in hypoxia-treated NRVMs were totally reversed by ER antagonist, ICI. Taken together, E2 and/or ERß exert the protective effect through repressed hypoxia-inducible HIF-1α, BNIP3 and IGFBP-3 levels to restrain the hypoxia-induced autophagy and apoptosis effects in H9c2 cardiomyoblast cells. CONCLUSION: The results suggest that females probably could tolerate better prolonged hypoxia condition than males, and E2/ERß treatment could be a potential therapy to prevent hypoxia-induced heart damage."

[An experience applying the transtheoretical model to assist a highly educated case to quit smoking].

Although smokers who are highly educated understand the risks involved in smoking, they tend to be highly influenced by the opinions of others and thus subject to severe social pressures. This is a case report on the application of the transtheoretical model of behavioral change to a an individual with a PhD in biotechnology who had been smoking for 20 years. The effects of the developed smoking cessation intervention were investigated and data were collected through personal meetings, telephone interviews, and e-mail during a smoking cessation counseling program that lasted from October 18th, 2012, to April 15th, 2013. The results of the assessment indicated that the main problem encountered by the case when quitting was a lack of confidence in attempting to quit smoking after experiencing previous failed attempts. In this report, strategies related to the transtheoretical model were used and counseling was provided during the smoking cessation process. Individualized strategic methods were developed to be effective in highly educated people who smoke in order to achieve successfully cessation of smoking. This report serves as a reference for using the transtheoretical model of behavioral change in developing smoking cessation programs.

Up-regulation of connective tissue growth factor in hyperoxia-induced lung fibrosis.

Pulmonary oxygen toxicity plays an important role in the lung injury process that leads to the development of bronchopulmonary dysplasia. Connective tissue growth factor (CTGF) is a fibroblast mitogen and promoter of collagen deposition. We investigated the effects of postnatal hyperoxia on lung collagen and CTGF expression in rats. Rat pups were exposed to 7 d of >95% O2 and a further 3 wk of 60% O2. CTGF mRNA and protein expression increased after hyperoxia treatment, and the values were significantly higher in hyperoxia-exposed rats on postnatal d 7 and 14. Lung collagen levels increased as rats aged, and the values were comparable between room air-exposed and hyperoxia-exposed rats on postnatal d 7 and 14 and were significantly higher in hyperoxia-exposed rats on postnatal d 21 and 28. Increases in CTGF mRNA and protein expressions preceded the onset of increased lung collagen. These data demonstrate that CTGF is up-regulated at time points preceding the fibrotic phase of the lung injury adding credence to the hypothesis that CTGF seems to be involved in the pathogenesis of hyperoxia-induced lung fibrosis and an anti-CTGF strategy might attenuate hyperoxia-induced lung fibrosis.