Acellular Porcine Cornea Produced by Supercritical Carbon Dioxide Extraction: A Potential Substitute for Human Corneal Regeneration.

PURPOSE: The aim of this study was to develop a non-cytotoxic, biocompatible innovative acellular porcine cornea (APC) for corneal wound healing and corneal blindness treatment. METHODS: APC was produced by using supercritical carbon dioxide (SCCO2) to decellularize the porcine cornea. Decellularization of the porcine cornea was examined by hematoxylin and eosin staining and 4,6-diamidino-2-phenylindole, dihydrochloride staining. The residual DNA content of APC was analyzed in comparison with the native porcine cornea. Virus inactivation up to at least 6 log10 was confirmed for the stepwise process of APC for 4 different model viruses. In addition, a series of in vitro and in vivo tests in accordance with ISO-10993 biocompatibility assay and animal performance tests were performed. RESULTS: APC produced by the SCCO2 process revealed complete decellularization, without any residual non-collagenous proteins. The scanning electron microscopy structural features of the decellularized cornea were similar to those of human. APC was found to be nontoxic and exhibited excellent biocompatibility in both in vitro and in vivo studies. The animal performance test proved that APC exerted excellent adaptability on the cornea and no sign of irritation and good compatibility in lamellar corneal transplantation. CONCLUSIONS: APC manufactured by SCCO2 technology revealed complete cells and non-collagenous protein removal compared with the Triton-sodium dodecyl sulfate decellularization process. APC showed excellent biocompatibility in rabbit lamellar corneal transplantation with a follow-up to 1 year. APC can be a potential substitute for human-donated cornea for corneal transplantation in the near future.

Evaluating the bone-regenerative role of the decellularized porcine bone xenograft in a canine extraction socket model.

OBJECTIVE: To evaluate the efficacy of a novel decellularized porcine bone xenograft, produced by supercritical carbon dioxide extraction technology, on alveolar socket healing after tooth extraction compared to a commercially available deproteinized bovine bone (Bio-Oss®). MATERIALS AND METHODS: Nine dogs (about 18 months old and weighing between 20 kg and 30 kg) underwent extractions of lower second to fourth premolars, bilaterally. The dogs were randomly selected and allocated to the following groups: Group 1: control unfilled socket; Group 2: socket filled with decellularized porcine bone xenograft (ABCcolla®) and covered by a commercially available porcine collagen membrane (Bio-Gide®); Group 3: socket filled with Bio-Oss® and covered by Bio-Gide® membrane. One dogs from each group was sacrificed at 4-, 12-, and 24-week to evaluate the socket healing after tooth extraction. The mandible bone blocks were processed without decalcification and specimens were embedded in methyl methacrylate and subjected to histopathology analyses to evaluate the bone regeneration in the extraction sockets. RESULTS: At 24-week after socket healing, ABCcolla® treated defects demonstrated significantly higher histopathology score in new bone formation and bone bridging, but significantly lower score in fluorescent labeling than those of the Bio-Oss®. In the microphotographic examination, decellularized porcine bone xenograft showed similar characteristics of new bone formation to that of Bio-Oss®. However, there was significantly less remnant implant materials in the decellularized porcine bone xenograft compared to the Bio-Oss® group at 24-week. Thus, the decellularized porcine bone graft seems to have promising bone regeneration properties similar to that of Bio-Oss® with less remnant grafted material in a canine tooth extraction socket model. CONCLUSIONS: Within the limits of the study, we concluded that ABCcolla® treated defects demonstrated significantly more new bone formation and better bone bridging, but less amount of fluorescent labeling than those of the Bio-Oss® group. However, clinical studies in humans are recommended to confirm these findings.

Attenuating Spinal Cord Injury by Conditioned Medium from Bone Marrow Mesenchymal Stem Cells.

Spinal cord injury (SCI) is a devastating neurological condition and might even result in death. However, current treatments are not sufficient to repair such damage. Bone marrow mesenchymal stem cells (BM-MSC) are ideal transplantable cells which have been shown to modulate the injury cascade of SCI mostly through paracrine effects. The present study investigates whether systemic administration of conditioned medium from MSCs (MSCcm) has the potential to be efficacious as an alternative to cell-based therapy for SCI. In neuron-glial cultures, MSC coculture effectively promoted neuronal connection and reduced oxygen glucose deprivation-induced cell damage. The protection was elicited even if neuron-glial culture was used to expose MSCcm, suggesting the effects possibly from released fractions of MSC. In vivo, intravenous administration of MSCcm to SCI rats significantly improved behavioral recovery from spinal cord injury, and there were increased densities of axons in the lesion site of MSCcm-treated rats compared to SCI rats. At early days postinjury, MSCcm treatment upregulated the protein levels of Olig 2 and HSP70 and also increased autophage-related proteins in the injured spinal cords. Together, these findings suggest that MSCcm treatment promotes spinal cord repair and functional recovery, possibly via activation of autophagy and enhancement of survival-related proteins.

Preparation of acellular scaffold for corneal tissue engineering by supercritical carbon dioxide extraction technology.

In this study, we developed a novel method using supercritical carbon dioxide (SCCO2) to prepare acellular porcine cornea (APC). Under gentle extraction conditions using SCCO2 technology, hematoxylin and eosin staining showed that cells were completely lysed, and cell debris, including nuclei, was efficiently removed from the porcine cornea. The SCCO2-treated corneas exhibited intact stromal structures and appropriate mechanical properties. Moreover, no immunological reactions and neovascularization were observed after lamellar keratoplasty in rabbits. All transplanted grafts and animals survived without complications. The transplanted APCs were opaque after the operation but became transparent within 2weeks. Complete re-epithelialization of the transplanted APCs was observed within 4weeks. In conclusion, APCs produced by SCCO2 extraction technology could be an ideal and useful scaffold for corneal tissue engineering. STATEMENT OF SIGNIFICANCE: We decellularized the porcine cornea using SCCO2 extraction technology and investigated the characteristics, mechanical properties, and biocompatibility of the decellularized porcine cornea by lamellar keratoplasty in rabbits. To the best of our knowledge, this is the first report describing the use of SCCO2 extraction technology for preparation of acellular corneal scaffold. We proved that the cellular components of porcine corneas had been efficiently removed, and the biomechanical properties of the scaffold were well preserved by SCCO2 extraction technology. SCCO2-treated corneas maintained optical transparency and exhibited appropriate strength to withstand surgical procedures. In vivo, the transplanted corneas showed no evidence of immunological reactions and exhibited good biocompatibility and long-term stability. Our results suggested that the APCs developed by SCCO2 extraction technology could be an ideal and useful scaffold for corneal replacement and corneal tissue engineering.

Cytoprotective and anti-inflammatory effects of PAL31 overexpression in glial cells.

BACKGROUND: Acute spinal cord injury (SCI) leads to a series of reactive changes and causes severe neurological deficits. A pronounced inflammation contributes to secondary pathology after SCI. Astroglia respond to SCI by proliferating, migrating, and altering phenotype. The impact of reactive gliosis on the pathogenesis of SCI is not fully understood. Our previous study has identified an inflammatory modulating protein, proliferation related acidic leucine-rich protein (PAL31) which is upregulated in the microglia/macrophage of injured cords. Because PAL31 participates in cell cycle progression and reactive astroglia often appears in the injured cord, we aim to examine whether PAL31 is involved in glial modulation after injury. RESULTS: Enhanced PAL31 expression was shown not only in microglia/macrophages but also in spinal astroglia after SCI. Cell culture study reveal that overexpression of PAL31 in mixed glial cells or in C6 astroglia significantly reduced LPS/IFNγ stimulation. Further, enhanced PAL31 expression in C6 astroglia protected cells from H2O2 toxicity; however, this did not affect its proliferative activity. The inhibiting effect of PAL31 on LPS/IFNγ stimulation was observed in glia or C6 after co-culture with neuronal cells. The results demonstrated that the overexpressed PAL31 in glial cells protected neuronal damages through inhibiting NF-kB signaling and iNOS. CONCLUSIONS: Our data suggest that PAL31upregulation might be beneficial after spinal cord injury. Reactive gliosis might become a good target for future therapeutic interventions.

Comparative effects of bone marrow mesenchymal stem cells on lipopolysaccharide-induced microglial activation.

After injury to the CNS, microglia are rapidly activated and concentrated and trigger inflammatory reaction at the sites of injury. Bone marrow mesenchymal stem cells (BMMSC) represent attractive cell sources for treating CNS injury. Although anti-inflammatory and paracrine effects of grafted BMMSC have been shown, direct modulation of BMMSC on microglia in situ remains unclear. The present work employs in vitro transwell assay to characterize the effects of BMMSC on LPS-stimulated microglia. BMMSC are cultivated in serum and serum-free (sf) conditions, namely, BMMSC and BMMSC-sf. Both cultures express major surface markers specific for mesenchymal stem cells. However, the BMMSC-sf exhibit sphere-like structure with reduced expression of two adherent cell markers, CD29 and CD90. Compared to BMMSC-sf, BMMSC are fibroblast like and have faster differentiation potential into neural-like cells. Furthermore, BMMSC release significant levels of TIMP-1 and VEGF, regardless of being alone or in coculture. The downregulated MMP-9 mRNA may be caused by TIMP-1 secretion from BMMSC. Our cell culture system provides a powerful tool for investigating the molecular and cellular changes in microglia-BMMSC cocultures.

Is the corrected-creatinine model for end-stage liver disease a feasible strategy to adjust gender difference in organ allocation for liver transplantation?

BACKGROUND: The Model for End-stage Liver Disease (MELD) scoring system is used for organ allocation in liver transplantation. Female cirrhotic patients have lower glomerular filtration rates (GFR) than males for the same creatinine (Cr) level. Correcting the Cr in females for the same GFR as in males shows that females have lower MELD scores and therefore a lower priority for liver transplantation; however, there has been no outcome data that justifies this modification. METHODS: We investigated 472 cirrhotic patients, comparing the mortality rate between males and females in relation to MELD and corrected-Cr MELD. RESULTS: Compared to females, male patients had a higher MELD (14.5+/-5.5 vs. 13.8+/-5.7) and significantly higher GFR (61.7+/-21.4 vs. 54.7+/-25.6 mlLmin/1.73 m, P=0.0002) because their Cr value was higher (1.4+/-0.4 vs. 1.3+/-0.5 mg/dL, P=0.0002). The corrected-Cr MELD score in females was higher (15.7+/-6.3) compared to the MELD in their original counterpart (P<0.0001) and the males (P=0.060). Female and male patients had a similar 3-month mortality rate (6.7% vs. 6.3%) and MELD (21.9+/-8.6 vs. 21.7+/-8.9) among deceased patients. At 6 months, female patients tended to have a lower mortality (12.5% vs. 14.7%) and a lower MELD (18.9+/-7.7 vs. 19.4+/-8.5) in deceased patients. However, at 9 and 12 months, females had a consistently higher mortality (25% vs. 21.2% and 37.5% vs. 31.3%, respectively) but lower MELD scores than males by 0.3-1 point. CONCLUSIONS: Using corrected-Cr MELD, which would prioritize female patients for liver transplantation, may only be justified in predicting intermediate-term (9- and 12-month), but not short-term (3- and 6-month) mortality.

Mutant Bik gene transferred by cationic liposome inhibits peritoneal disseminated murine colon cancer.

Peritoneal carcinomatosis of intraabdominal malignancies, such as pancreatic, ovarian, gastric, and colorectal cancers, represents an unmet medical need as conventional cancer treatments rarely eliminate these tumors. Satisfactory treatment for either peritoneally disseminated tumors or prevention of local recurrence after surgery is yet to be developed. To improve the efficacy of novel strategies against peritoneal metastasis, a sensitive, and less invasive model is needed to scrutinize the in vivo tumor growth and response to experimental therapeutics. To study this we intraperitoneally inoculated CT-26 stably expressing luciferase (CT-26-Luc) to mimic tumor spreading within the abdomen. Bioluminescent signals emitted from the living experimental mice correlate well with the injected cell numbers as well as the weights of dissected tumors. Since a nonviral cationic liposome coupled mutant pro-apoptotic gene, Bik(T33D/S35D) (BikDD), was previously shown to have potent anti-cancer effects on an orthotopic breast cancer animal model (Li et al., Cancer Res 63(22):7630-7633, 2003), we evaluated the inhibitory effect of BikDD on the growth kinetics of intraperitoneally inoculated CT-26-Luc. We found that intraperitoneal (i.p.) injection of liposome coupled BikDD suppressed the expansion of CT-26-Luc and prolonged life span of experimental mice. These results suggest a therapeutic effect of BikDD gene therapy on peritoneal carcinomatosis of colon cancer.

Modulation of 5-fluorouracil cytotoxicity through thymidylate synthase and NF-kappaB down-regulation and its application on the radiolabelled iododeoxyuridine therapy on human hepatoma cell.

The inhibition of thymidylate synthase (TS) by 5-fluorouracil (5-FU) was known to increase the incorporation of radiolabelled iododeoxyuridine (IdUrd) into DNA. The relatively non-toxic compounds such as thiol-containing antioxidant pyrrolidinodithiocarbamte (PDTC) or aromatic fatty acid phenylbutyrate (PB) had been reported to enhance the cytotoxic efficacy of 5-FU. We designed a novel strategy through triplet combination of PB, PDTC and 5-FU to increase the radiolabelled IdUrd uptake and investigated the underlying mechanisms. The growth inhibition and [(125)I]IdUrd-DNA incorporation by PB, PDTC, 5-FU in different combinations were tested on parent or p21(Waf1) transfected Hep3B cells. The combination of PB and PDTC was more effective in enhancing 5-FU cytotoxicity than either drug alone. The combination of PB/PDTC and 5-FU blocked cells in S-phase and resulted in 8.5-fold increase of radiolabelled IdUrd-DNA incorporation. The transfection of p21(Waf1) did not change the general pattern of enhancement. Intriguingly, the combination of PB and PDTC effectively down-regulated NF-kappaB and TS and prevented their up-regulation from 5-FU treatment than either drug alone through a p21(Waf1)-independent mechanism. Based on this strategy, the 3-drug combination offered potential for improved radiolabelled IdUrd molecular radiotherapy for hepatoma treatment.