Tzong-Fu Kuo

Evaluation of articular cartilage repair using biodegradable nanofibrous scaffolds in a swine model: a pilot study

The aim of this study was to evaluate a cell‐seeded nanofibrous scaffold for cartilage repair in vivo. We used a biodegradable poly(ε‐caprolactone) (PCL) nanofibrous scaffold seeded with allogeneic chondrocytes or xenogeneic human mesenchymal stem cells (MSCs), or acellular PCL scaffolds, with no implant as a control to repair iatrogenic, 7 mm full‐thickness cartilage defects in a swine model. Six months after implantation, MSC‐seeded constructs showed the most complete repair in the defects compared to other groups. Macroscopically, the MSC‐seeded constructs regenerated hyaline cartilage‐like tissue and restored a smooth cartilage surface, while the chondrocyte‐seeded constructs produced mostly fibrocartilage‐like tissue with a discontinuous superficial cartilage contour. Incomplete repair containing fibrocartilage or fibrous tissue was found in the acellular constructs and the no‐implant control group. Quantitative histological evaluation showed overall higher scores for the chondrocyte‐ and MSC‐seeded constructs than the acellular construct and the no‐implant groups. Mechanical testing showed the highest equilibrium compressive stress of 1.5 MPa in the regenerated cartilage produced by the MSC‐seeded constructs, compared to 1.2 MPa in the chondrocyte‐seeded constructs, 1.0 MPa in the acellular constructs and 0.2 MPa in the no‐implant group. No evidence of immune reaction to the allogeneically‐ and xenogeneically‐derived regenerated cartilage was observed, possibly related to the immunosuppressive activities of MSCs, suggesting the feasibility of allogeneic or xenogeneic transplantation of MSCs for cell‐based therapy. Taken together, our results showed that biodegradable nanofibrous scaffolds seeded with MSCs effectively repair cartilage defects in vivo, and that the current approach is promising for cartilage repair. Copyright

Red mold rice ameliorates impairment of memory and learning ability in intracerebroventricular amyloid β-infused rat by repressing amyloid β accumulation

Amyloid β (Aβ) peptide related to the onset of Alzheimers disease (AD) damaged neurons and further resulted in dementia. Monascus‐fermented red mold rice (RMR), a traditional Chinese medicine as well as health food, includes monacolins (with the same function as statins) and multifunctional metabolites. In this study, ethanol extract of RMR (RE) was used to evaluate neuroprotection against Aβ40 neurotoxicity in PC12 cells. Furthermore, the effects of dietary administration of RMR on memory and learning abilities are confirmed in an animal model of AD rats infused with Aβ40 into the cerebral ventricle. During continuous Aβ40 infusion for 28 days, the rats of test groups were administered RMR or lovastatin. Memory and learning abilities were evaluated in the water maze and passive avoidance tasks. After sacrifice, cerebral cortex and hippocampus were collected for the examination of AD risk factors. The in vitro results clearly indicate that RE provides stronger neuroprotection in rescuing cell viability as well as repressing inflammatory response and oxidative stress. RMR administration potently reverses the memory deficit in the memory task. Aβ40 infusion increases acetylcholinesterase activity, reactive oxygen species, and lipid peroxidation and decreases total antioxidant status and superoxide dismutase activity in brain, but these damages were potently reversed by RMR administration, and the protection was more significant than that with lovastatin administration. The protection provided by RMR is able to prevent Aβ fibrils from being formed and deposited in hippocampus and further decrease Aβ40 accumulation, even though Aβ40 solution was infused into brain continuously.

Tissue engineering-based cartilage repair with mesenchymal stem cells in a porcine model†‡

This in vivo pilot study explored the use of mesenchymal stem cell (MSC) containing tissue engineering constructs in repair of osteochondral defects. Osteochondral defects were created in the medial condyles of both knees of 16 miniature pigs. One joint received a cell/collagen tissue engineering construct with or without pretreatment with transforming growth factor β (TGF‐β) and the other joint from the same pig received no treatment or the gel scaffold only. Six months after surgery, in knees with no treatment, all defects showed contracted craters; in those treated with the gel scaffold alone, six showed a smooth gross surface, one a hypertrophic surface, and one a contracted crater; in those with undifferentiated MSCs, five defects had smooth, fully repaired surfaces or partially repaired surfaces, and one defect poor repair; in those with TGF‐β‐induced differentiated MSCs, seven defects had smooth, fully repaired surfaces or partially repaired surfaces, and three defects showed poor repair. In Pineda score grading, the group with undifferentiated MSC, but not the group with TGF‐β‐induced differentiated MSCs, had significantly lower subchondral, cell morphology, and total scores than the groups with no or gel‐only treatment. The compressive stiffness was larger in cartilage without surgical treatment than the treated area within each group. In conclusion, this preliminary pilot study suggests that using undifferentiated MSCs might be a better approach than using TGF‐β‐induced differentiated MSCs for in vivo tissue engineered treatment of osteochondral defects.

TOXICITY ASSESSMENT OF MONTMORILLONITE AS A DRUG CARRIER FOR PHARMACEUTICAL APPLICATIONS: YEAST AND RATS MODEL

Our previous study indicated that montmorillonite (MMT for short) was pharmaceutically feasible to be used as a carrier of an anticancer drug 5-FU. Emphasis of this study is thus placed on the toxicity of MMT to address whether it is clinically safe for practical use. Hematological data of rats model showed that the rats administered with oral MMT had significant increases in hemoglobin (Hb) concentration, Hamatocrit and RBC count than those of oral PBS buffer (p 0.05). Hematological analysis for intravenous injection also showed no statistically significant differences between experimental and control group (p>0.05). Biochemical analysis pointed out that compared to oral PBS there was not only a significant decrease of sodium (Na+) and chloride (Cl−) ion (p MMTK-SA ≫ MMT-SA > MMT according to EC0 or EC50 line. These all suggested that MMT alone is much less toxic than Lannate and potassium hexacyanoferrate(III). Apparently, it was concluded that MMT alone could be considered non-toxic to S. cerevisiae and Wistar rat for myriads of applications.

Screening of salbutamol residues in swine meat and animal feed by an enzyme immunoassay in Taiwan.

An ELISA was developed for routine examination for extensive monitoring and screening programs for the residues of salbutamol in swine serum, animal feed, meat, and meat-related products destined for human consumption in Taiwan. Objectives of the study were to investigate the use of a new immunoassay for the detection of salbutamol residues in swine meat and animal feed samples, and to compare with a commercial kit in field test screens. A fast, simple and reliable sample preparation method for the determination of salbutamol was established. Field trials with 222 swine meat and 120 animal feed samples that were taken from local meat markets, auction markets and feed mills. The application and the results of two ELISA kits (a homemade and a commercial kit) for the screening of salbutamol were presented. Adopting 2microg kg(-1) salbutamol as a cut-off value for swine meat, the commercial beta-agonist ELISA had a sensitivity of 85.3% and a specificity of 95.2% versus GC-MS at a cut-off of 2microg kg(-1). The homemade salbutamol ELISA had a sensitivity of 100% and a specificity of 90.9% and gave no false-negative rate results. Furthermore, adopting 20microg kg(-1) salbutamol as a cut-off value for animal feed, both the commercial and homemade ELISA showed 100% sensitivity and 100% specificity of the assays. In conclusion, a sensitive, specific salbutamol polyclonal antibody-based ELISA has been developed that could serve as a rapid screening assay, and the detection of positive samples at the place of sampling can result in more effective control of the illegal use of beta-agonists.

Red Mold Rice Promotes Neuroprotective sAPPalpha Secretion Instead of Alzheimer’s Risk Factors and Amyloid Beta Expression in Hyperlipidemic Aβ40-Infused Rats

Amyloid beta (Abeta) peptide is closely related to the onset of Alzheimers disease (AD). A high-cholesterol or high-energy diet was demonstrated to stimulate Abeta formation and deposition in the amyloid precursor protein (APP) pathway and, oppositely, downregulate the secretion of the neuroprotective soluble APP alpha-fragment (sAPPalpha). Monascus-fermented red mold rice (RMR) including multiple cholesterol-lowering agents, antioxidants, and anti-inflammatory agents has been proven to ameliorate Abeta40 infusion-induced memory deficit in our previous study. In this study, the ethanol extract of RMR (RE) and natural RMR were respectively tested for their effect on the mediation of the proteolytic process of APP in cholesterol-treated human neuroblastoma IMR32 cell, as well as their effect on memory and learning ability and the expression of AD risk factors in intracerebroventricular Abeta40-infused hyperlipidemic rats. In the results, RE suppressed cholesterol-raised beta-secretase activity and further resulted in the increase of sAPPalpha secretion in the IMR32 cell. In the animal test, RMR potently reversed the memory deficit in the water maze and passive avoidance tasks. RMR administration could prevent against Abeta40 infusion plus the great damage caused by a high energy diet in hippocampus and cortex involved in the raise of thiobarbituric acid reactive substances and reactive oxygen species. The neuroprotection provided by RMR downregulates Abeta40 formation and deposition by suppressing the cholesterol-raised beta-secretase activity and apolipoprotein E expression, as well as mediates the proteolytic process of APP toward neuroprotective sAPPalpha secretion in hippocampus.

Development and fast screening of salbutamol residues in swine serum by an enzyme-linked immunosorbent assay in Taiwan.

The analysis of salbutamol in swine serum is the more practical basis for large scale surveillance programs in Taiwan. Objectives of the study were to develop a new assay and to compare with a commercially available kit in field test screens. A simple and reliable enzyme-linked immunosorbent assay (ELISA) to monitor the presence of beta-agonist, salbutamol, in 1,358 field samples of swine serum that were collected from local meat markets was described. The method proved to be suitable and sensitive for the detection of beta-agonist residues caused by growth promoting dosage. The limit of detection of the developed ELISA directly performed on diluted serum was 0.25 ng/mL. The application and the results of two ELISA kits (homemade and commercially available) for the screening of salbutamol were presented. For further confirmation, all samples that showed to be ELISA positive for salbutamol residues were analyzed by GC-MS. Adopting 1 ng/mL salbutamol as a cutoff value, the commercial beta-agonist ELISA had a sensitivity of 89.2% and a specificity of 86.7% versus GC-MS at a cutoff of 1 ng/mL. The homemade salbutamol ELISA had a sensitivity of 81.1% and a specificity of 98.6% and gave a low proportion of false-positive rate results. The reliability of the developed kit in terms of the percentage of false-positive rate results is evaluated. In conclusion, a sensitive, specific salbutamol ELISA has been developed that could serve as a rapid screening assay, and the detection of positive samples at the place of sampling can result in more effective control of the illegal use of beta-agonists.

Chitosan/gelatin hydrogel as immunoisolative matrix for injectable bioartificial pancreas

Abstract:  Background:  The feasibility of using chitosan/gelatin hydrogel as immunoisolative matrix to provide an additional protection to the microencapsulated islet cells was demonstrated in this study. We hope that the use of hydrogel can extend the functional longevity of microencapsulated islet cells during xenotransplantation.

Magnetic cryopreservation for dental pulp stem cells

Magnetic cryopreservation has been successfully used for tooth banking with satisfactory implantation outcomes, suggesting that the method preserves human periodontal ligament cells and dental pulp stem cells (DPSCs). Therefore, magnetic cryopreservation may be applied for the preservation of DPSCs; however, this method has not been evaluated yet. A reliable cryopreservation method for live-cell preservation is important for the clinical applications of regenerative medicine. The conventional slow-freezing procedure with 10% dimethylsulfoxide (DMSO) may not be appropriate for stem cell-based therapies because DMSO is cytotoxic. The objective of this study was to investigate whether magnetic cryopreservation can be applied for DPSC cryopreservation. Cells isolated from human dental pulp were subjected to magnetic cryopreservation. Postthawing cell viability, adhesion, proliferation, expression of markers for mesenchymal stem cells (MSCs), differentiation ability of magnetically cryopreserved DPSCs and DNA stability were compared to those of cells subjected to the conventional slow-freezing method. The results indicated that a serum-free cryopreservation medium (SFM) containing 3% DMSO is optimal for magnetic cryopreservation. Post-thaw magnetically cryopreserved DPSCs express MSC markers, and perform osteogenesis and adipogenesis after induction similarly to fresh MSCs. No significant DNA damage was found in magnetically cryopreserved DPSCs. Magnetic cryopreservation is thus a reliable and effective method for storage of DPSCs. The smaller amount of DMSO required in SFM for cryopreservation is beneficial for the clinical applications of post-thaw cells in regenerative medicine.

Fibrin glue mixed with platelet-rich fibrin as a scaffold seeded with dental bud cells for tooth regeneration

Odontogenesis is a complex process with a series of epithelial‐mesenchymal interactions and odontogenic molecular cascades. In tissue engineering of teeth from stem cells, platelet‐rich fibrin (PRF), which is rich in growth factors and cytokines, may improve regeneration. Accordingly, PRF was added into fibrin glue to enrich the microenvironment with growth factors. Unerupted second molar tooth buds were harvested from miniature swine and cultured in vitro for 3 weeks to obtain dental bud cells (DBCs). Whole blood was collected for the preparation of PRF and fibrin glue before surgery. DBCs were suspended in fibrin glue and then enclosed with PRF, and the DBC‐fibrin glue‐PRF composite was autografted back into the original alveolar sockets. Radiographic and histological examinations were used to identify the regenerated tooth structure 36 weeks after implantation. Immunohistochemical staining was used to detect proteins specific to tooth regeneration. One pig developed a complete tooth with crown, root, pulp, enamel, dentin, odontoblast, cementum, blood vessels, and periodontal ligaments in indiscriminate shape. Another animal had an unerupted tooth that expressed cytokeratin 14, dentin matrix protein‐1, vascular endothelial growth factor, and osteopontin. This study demonstrated, using autogenic cell transplantation in a porcine model, that DBCs seeded into fibrin glue‐PRF could regenerate a complete tooth. Copyright