Surface modification of zirconia ceramics through cold plasma treatment and the graft polymerization of biomolecules.

Background/purpose: Although zirconia ceramics were highly versatile as dental implants, their long-term presence in the human body may slow down healing and impede cell growth in the past. To enhance the cytocompatibility of zirconia ceramics, surface activation modification was used to immobilize biopolymers such that a biomimetic environment was created. Materials and methods: Hexamethyldisilazane thin films were deposited onto the surface of inorganic zirconia through cold plasma treatment under various power and deposition time settings to form an organosilane interface layer. Next, oxygen plasma treatment was performed to activate the free radicals on the surface. Subsequently, ultraviolet light was employed to graft and polymerize acrylic acid for generating carboxyl groups on the surface. This was followed by a condensation reaction with biopolymers (chitosan, chitosan/poly-γ-glutamic acid, and gelatin). Results: Under a 20-min deposition time at 40 W and 150 mTorr, the thin films had a maximum graft density of 2.1 mg/cm2. MG-63 cells (human osteosarcoma cells) were employed to evaluate cell compatibility. Chitosan and chitosan/poly-γ-glutamic acid promoted the compatibility of MG-63 cells (a human osteosarcoma cell line) with zirconia ceramics, whereas gelatin reduced this compatibility. Conclusion: The findings confirm that cold plasma treatment and graft polymerization can promote the immobilization of biomolecules and improve the biocompatibility of zirconia ceramics. This approach can be applied to the modification of zirconia ceramic implants.

Hyaluronic Acid Supplement as a Chondrogenic Adjuvant in Promoting the Therapeutic Efficacy of Stem Cell Therapy in Cartilage Healing.

The main aim of this study is to investigate the therapeutic efficacy of direct intra-articular injection of bone-marrow-derived stem/stromal cells (BMSCs) and the adjuvant role of hyaluronic acid (HA) in facilitating rabbit articular cartilage repair. First, rabbit BMSCs were treated with a medium containing different concentrations of HA. Later, HA's influence on BMSCs' CD44 expression, cell viability, extracellular glycosaminoglycan (GAG) synthesis, and chondrogenic gene expression was evaluated during seven-day cultivation. For the in vivo experiment, 24 rabbits were used for animal experiments and 6 rabbits were randomly allocated to each group. Briefly, chondral defects were created at the medial femoral condyle; group 1 was left untreated, group 2 was injected with HA, group 3 was transplanted with 3 × 106 BMSCs, and group 4 was transplanted with 3 × 106 BMSCs suspended in HA. Twelve weeks post-treatment, the repair outcome in each group was assessed and compared both macroscopically and microscopically. Results showed that HA treatment can promote cellular CD44 expression. However, the proliferation rate of BMSCs was downregulated when treated with 1 mg/mL (3.26 ± 0.03, p = 0.0002) and 2 mg/mL (2.61 ± 0.04, p = 0.0001) of HA compared to the control group (3.49 ± 0.05). In contrast, 2 mg/mL (2.86 ± 0.3) of HA treatment successfully promoted normalized GAG expression compared to the control group (1.88 ± 0.06) (p = 0.0009). The type II collagen gene expression of cultured BMSCs was significantly higher in BMSCs treated with 2 mg/mL of HA (p = 0.0077). In the in vivo experiment, chondral defects treated with combined BMSC and HA injection demonstrated better healing outcomes than BMSC or HA treatment alone in terms of gross grading and histological scores. In conclusion, this study helps delineate the role of HA as a chondrogenic adjuvant in augmenting the effectiveness of stem-cell-based injection therapy for in vivo cartilage repair. From a translational perspective, the combination of HA and BMSCs is a convenient, ready-to-use, and effective formulation that can improve the therapeutic efficacy of stem-cell-based therapies.

Novel transplant of combined platelet-rich fibrin Releasate and bone marrow stem cells prevent bone loss in Ovariectomized osteoporotic mice.

BACKGROUND: Osteoporosis is a metabolic bone disorder characterized by deterioration in the quantity and quality of bone tissue, with a consequent increase susceptibility to fracture. METHODS: In this study, we sought to determine the efficacy of platelet-rich fibrin releasates (PRFr) in augmenting the therapeutic effects of stem cell-based therapy in treating osteoporotic bone disorder. An osteoporosis mouse model was established through bilateral ovariectomy on 12-week-old female ICR (Institute of Cancer Research) mice. Eight weeks postoperatively, the ovariectomized (OVX) mice were left untreated (control) or injected with PRFr, bone marrow stem cells (BMSCs), or the combination of BMSCs and PRFr. Two different injection (single versus quadruple) dosages were tested to investigate the accumulative effects of BMSCS and PRFr on bone quality. Eight weeks after injection, the changes in tibial microstructural profiles included the percentage of bone volume versus total tissue volume (BV/TV, %), bone mineral density (BMD, g/cm3), trabecular number (Tb.N, number/mm), and trabecular separation (Tb.Sp, mm) and bony histology were analyzed. RESULTS: Postmenopausal osteoporosis model was successfully established in OVX mice, evidenced by reduced BMD, decreased BV/TV, lower Tb.N but increased Tb.Sp. Eight weeks after injection, there was no significant change to BMD and bone trabeculae could be detected in mice that received single-injection regimen. In contrast, in mice which received 4 doses of combined PRFr and BMSCs, the BMD, BV/TV, and TB.N increased, and the TB.Sp decreased significantly compared to untreated OVX mice. Moreover, the histological analysis showed the trabecular spacing become narrower in OVX-mice treated with quadruple injection of BMSCs and combined PRFr and BMSCs than untreated control. CONCLUSION: The systemic administration of combined BMSCs and PRFr protected against OVX-induced bone mass loss in mice. Moreover, the improvement of bony profile scores in quadruple-injection group is better than the single-injection group, probably through the increase in effect size of cells and growth factors. Our data also revealed the combination therapy of BMSCs and PRFr has better effect in enhancing osteogenesis, which may provide insight for the development of a novel therapeutic strategy in osteoporosis treatment.

The gastroprotective effect of the foxtail millet and adlay processing product against stress-induced gastric mucosal lesions in rats.

Foxtail millet (Setaria italica (L.) P. Beauv.) and adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) seeds have substantial benefits possesses remarkable edible and nutritive values, and ease of processing and food manufacturing. They have nutraceutical properties in the form of antioxidants which prevent deterioration of human health and have long been used in traditional Chinese medicine as a remedy for many diseases. The present study is designed to investigate the gastroprotective effect of foxtail millet and adlay processing product (APP) diet on water immersion restraint stress (WIRS) induced ulceration in rats. We examined the effects of intake of AIN-93G diet containing either foxtail millet (10, 20 and 40%, 4 weeks) or APP (15 and 30%, 5 weeks) on macroscopic ulcer index (UI), plasma calcium level, lipid peroxidation products (estimated by the thiobarbituric acid reactive substances; TBARS), non-protein sulfhydryl (NPSH), digestive enzyme activities, and histopathology were determined. The results showed that pretreatment with millet and adlay diets significantly prevented the gastric mucosal lesion development. In addition, ulcerated rats showed depletion of NPSH levels whereas treatment with millet and adlay reverted this decline in stress-induced rats. Histological studies confirmed the results. The finding suggests that millet and adlay diets promote ulcer protection by the decrease in ulcer index, TBARS values and increase NPSH concentrations. Millet and adlay diets retain the advantage of being a natural product which may protect the gastric mucosa against ulceration.

Enhanced Bone Formation in Osteoporotic Mice by a Novel Transplant Combined with Adipose-derived Stem Cells and Platelet-rich Fibrin Releasates.

Osteoporotic fracture is the main complication of osteoporosis (OP) and accounts for millions of injuries annually. Local intervention by intra-marrow injection has been a good option for preventing osteoporotic bone loss when the osteoporotic femoral fracture has been treated. In this study, tail vein transplantations were examined to evaluate the cell-based therapeutic approach for treating OP with adipose-derived stem cells (ADSCs) and platelet-rich fibrin releasates (PRFr) in an ovariectomized (OVX) mice model. Thirty-six 12-wk-old female ICR mice were randomly divided into six groups: untreated control; sham-operated; OVX-control; OVX-ADSCs; OVX-PRFr; and OVX-ADSCs+PRFr. Starting 8 wk after ovariectomy, the OVX mice received tail vein injections once each week for four consecutive weeks, then were evaluated radiographically and histopathologically 8 wk after the first injection. We also assessed changes to bone trabeculae in the proximal tibial growth plate. In OVX mice treated with ADSCs or PRFr alone, or with a combination of ADSCs and PRFr, the trabecular bone mineral density (BMD), bone volume ratios (BV/TV), and numbers (Tb.N) in the proximal tibia areas were significantly higher than that in the OVX-control group. Significant differences between OVX-treated mice and OVX controls were found for trabecular separation, but not for trabecular thickness. These results indicate that ADSCs or PRFr treatment enhances bone microarchitecture in OP. The treatment of bone loss of OVX mice with ADSCs+PRFr induced greater bone consolidation with bone tissue production (P < 0.01) when compared to the others. Thus, we conclude that the transplantation of ADSCs combined with PRFr might provide an alternative strategy for the treatment of various bone disorders in OP with an unlimited source of cells and releasates.

Regenerative Potential of Platelet-Rich Fibrin Releasate Combined with Adipose Tissue-Derived Stem Cells in a Rat Sciatic Nerve Injury Model.

Sciatic nerve injuries, not uncommon in trauma with a limited degree of functional recovery, are considered a persistent clinical, social, and economic problem worldwide. Accumulating evidence suggests that stem cells can promote the tissue regeneration through various mechanisms. The aim of the present study was to investigate the role of adipose tissue-derived stem cells (ADSCs) and combine with platelet-rich fibrin releasate (PRFr) in the regeneration of sciatic nerve injury in rats. Twenty-four Sprague-Dawley rats were randomly assigned to four groups, a blade was used to transect the left hindlimb sciatic nerve, and silicon tubes containing one of the following (by injection) were used to bridge the nerve proximal and distal ends (10-mm gap): group 1: untreated controls; group 2: PRFr alone; group 3: ADSCs alone; group 4: PRFr + ADSCs-treated. Walking function was assessed in horizontal rung ladder apparatus to compare the demands of the tasks and test sensitivity at 1-mo interval for a total of 3 mo. The gross inspection and histological examination was performed at 3 mo post transplantation. Overall, PRFr + ADSCs-treated performed better compared with PRFr or ADSCs injections alone. Significant group differences of neurological function were observed in ladder rung walking tests in all treated groups compared to that of untreated controls (P < 0.05). This injection approach may provide a successfully employed technique to target sciatic nerve defects in vivo, and the combined strategy of ADSCs with PRFr appears to have a superior effect on nerve repair.

Four New Iridoid Metabolites Have Been Isolated from the Stems of Neonauclea reticulata (Havil.) Merr. with Anti-Inflammatory Activities on LPS-Induced RAW264.7 Cells.

One new iridoid, namely neonanin C (1) one monocyclic iridoid ring-opened derivative namely neonanin D (2), two new bis-iridoid derivatives namely reticunin A (3) and reticunin B (4) with sixteen known compounds (5-20) were isolated from the stems of Neonauclea reticulata (Havil.) Merr. These new structures were determined by the detailed analysis of spectroscopic data and comparison with the data of known analogues. Compounds 1-20 were evaluated for inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages cell line. The results showed that all compounds exhibited no obvious cytotoxicity compared to the control group and five compounds including isoboonein (7), syringaresinol (10), (+)-medioresinol (12), protocatechuic acid (14) and trans-caffeic acid (15) exhibited inhibitory activities with IC50 values at 86.27 ± 3.45; 9.18 ± 1.90; 76.18 ± 2.42; 72.91 ± 4.97 and 95.16 ± 1.20 µg/mL, respectively.

Phytochemical Investigation of Tradescantia Albiflora and Anti-Inflammatory Butenolide Derivatives.

Phytochemical investigation of the whole plant of Tradescantia albiflora Kunth led to the isolation and characterization of a butanolide, rosmarinosin B (1), that was isolated from natural sources for the first time, a new butenolide, 5-O-acetyl bracteanolide A (2), and a new apocarotenoid, 2ß-hydroxyisololiolide (11), together with 25 known compounds (compounds 3-10 and 12-28). The structures of the new compounds were elucidated by analysis of their spectroscopic data, including MS, 1D, and 2D NMR experiments, and comparison with literature data of known compounds. Furthermore, four butenolides 4a-4d were synthesized as novel derivatives of bracteanolide A. The isolates and the synthesized derivatives were evaluated for their preliminary anti-inflammatory activity against lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in RAW 264.7 cells. Among them, the synthesized butenolide derivative n-butyl bracteanolide A (4d) showed enhanced NO inhibitory activity compared to the original compound, with an IC50 value of 4.32 ± 0.09 µg/mL.

The effect of adipose-derived mesenchymal stem cells and chondrocytes with platelet-rich fibrin releasates augmentation by intra-articular injection on acute osteochondral defects in a rabbit model.

BACKGROUND: This study aimed to investigate the efficacy of adipose-derived mesenchymal stem cells (ADSCs), platelet-rich fibrin releasates (PRFr), and chondrocyte transplantation in rabbit acute osteochondral defects. METHODS: Thirty rabbits were randomly assigned to five groups: untreated controls; ADSCs alone; PRFr alone; PRFr + ADSCs; and PRFr + chondrocytes. The critical size osteochondral defects in right knee femoral condyles were injected intra-articularly according to the groups, as listed. The experimental rabbits received treatments once a week for two weeks postoperatively. All evaluations were conducted for 14 weeks following surgery, and the regenerated cartilages were assessed by gross inspection and histological examination. RESULTS: There were no complications encountered in any of the rabbits. The size of the defect decreased and the volume of repaired cartilage increased in the medial femoral condyles of the PRFr + ADSCs group. Relative to the ADSCs or PRFr group, histological examination demonstrated that the PRFr + ADSCs group had thicker hyaline cartilage-specific extracellular matrix. Grading scores revealed that PRFr + ADSCs injection had better matrix, cell distribution, and surface indices than other groups (P < 0.05). However, the histological scores reported for PRFr + chondrocytes on cartilage repair were similar to those of PRFr, and there were no significant between-group differences. CONCLUSIONS: These findings showed that intra-articular injections of PRFr + ADSCs into the knee can reduce cartilage defects by regenerating hyaline-like cartilage without complications. This approach may provide an alternative method for functional reconstruction of acute osteochondral defects with an unlimited source of cells and releasates.

Platelet-Rich Fibrin Facilitates Rabbit Meniscal Repair by Promoting Meniscocytes Proliferation, Migration, and Extracellular Matrix Synthesis.

Although platelet-rich fibrin (PRF) has been used in clinical practice for some time, to date, few studies reveal its role as a bioactive scaffold in facilitating meniscal repair. Here, the positive anabolic effects of PRF on meniscocytes harvested from the primary culture of a rabbit meniscus were revealed. The rabbit meniscocytes were cultured with different concentrations of PRF-conditioned medium, and were evaluated for their ability to stimulate cell migration, proliferation, and extracellular matrix formation. In vivo, meniscal defects were created via an established rabbit animal model and were evaluated by a histology-based four-stage scoring system to validate the treatment outcome three months postoperatively. The in vitro results showed that PRF could induce cellular migration and promote proliferation and meniscocyte extracellular matrix (ECM) synthesis of cultured meniscocytes. In addition, PRF increased the formation and deposition of cartilaginous matrix produced by cultured meniscocytes. Morphological and histological evaluations demonstrated that PRF could facilitate rabbit meniscal repair. The data highlight the potential utility of using PRF in augmenting the healing of meniscal injuries. These advantages would benefit clinical translation, and are a potential new treatment strategy for meniscal repair.

In vitro and in vivo assessment of chitosan modified urocanic acid as gene carrier.

Chitosan nanoparticles modified with 10 and 30% urocanic acid (CUA) via carbodiimide crosslinking were examined for an efficient gene delivery carrier. The CUA gene carrier was characterized by FTIR, TEM, SEM and the in vitro transfection efficiency CUA polyplex was tested with HeLa and 3T3 cells. The loading efficiency of CUA complexes with DNA was assessed at different N/P ratio of 1, 2, 4, 6, 8, and 10. The DNA loading efficiency was found be to >85% for chitosan, CUA10 and CUA30% and the DNA protection ability of CUA10 and CUA30 nanoparticle complexes was confirmed upon incubation with NheI and HindIII. The cell toxicity and cell viability results have supported the non-toxic nature of CUA10 and CUA30 nanoparticles. In vitro transfection efficiency of CUA10 and CUA30 polyplex was tested for EGFP expression in 3T3 and HeLa cells and a relative maximum % transfection of about 10% was confirmed by CUA10 and CUA30 after 96h transfection. The feasibility and biocompatibility of CUA gene carrier in transgenic chickens was also demonstrated. The in vitro transfection and in vivo embryonic viability studies further confirmed the CUA as promising gene carrier because of the improved biocompatibility and DNA protection ability.

Intra-articular Injection of platelet-rich fibrin releasates in combination with bone marrow-derived mesenchymal stem cells in the treatment of articular cartilage defects: An in vivo study in rabbits.

The use of mesenchymal stem cells (MSCs), which can be differentiated into chondrocytes under specific conditions, has been proposed for the treatment of cartilage defects. Blood-derived platelet-rich fibrin releasate (PRFr), which is rich in growth factors and cytokines, may improve cartilage regeneration. In this study, the therapeutic effects of PRFr in combination with bone marrow-derived MSCs for articular cartilage regeneration were evaluated in a rabbit model. Critical osteochondral defects were surgically created in the femoral condyle of the rabbits, and 3 × 106 of MSCs, 0.8 mL of PRFr, or a combination of MSCs and PRFr were injected intra-articularly and one week after first administration. The animals were sacrificed 12 weeks postoperatively, and the regenerated cartilages were assessed by gross inspection and histological examination. No treatment-related adverse events were noted in any of the rabbits. The size of the defect decreased and the volume of regenerated cartilage increased in the medial femoral condyles of the MSCs + PRFr group. Relative to the MSCs or PRFr group, histological examination demonstrated that the MSCs + PRFr group had thicker hyaline-like cartilaginous tissue with normal glycosaminoglycan production. Grading scores revealed that MSCs + PRFr injection had better matrix, cell distribution, and surface indices than other groups. The results showed that intra-articular injections of MSCs + PRFr into the knee can reduce cartilage defects by regenerating hyaline-like cartilage without adverse events. This approach may provide an alternative method of autologous chondrocyte implantation to repair cartilage defects with an unlimited source of cells and releasate. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1536-1543, 2017.

Evaluation of Xanthine Oxidase Inhibitory Potential and In vivo Hypouricemic Activity of Dimocarpus longan Lour. Extracts.

BACKGROUND: Longan is a fruit tree known to contain many phenolic components, which are capable of protecting people from oxidative damage through an anti-inflammatory mechanism. It may be also worthwhile to study the effect on lowering uric acid activity. MATERIALS AND METHODS: This study investigates the lowering of uric acid using longan extracts, including flowers, pericarps, seeds, leaves, and twigs, on potassium-oxonate-induced hyperuricemia mice and its inhibitory actions against xanthine oxidase (XO) activities. RESULTS: The findings revealed that ethyl acetate fraction of longan extracts exhibited strong XO-inhibitory activity, and the flower extracts (IC50 = 115.8 µg/mL) revealed more potent XO-inhibitory activity to those of pericarps (118.9 µg/mL), twigs (125.3 µg/mL), seeds (262.5 µg/mL), and leaves (331.1 µg/mL) in vitro. In addition, different dosages of longan extract (50-100 mg/kg) were administered to hyperuricemic mice. The lowering effect of longan extracts on uric acid at 75 mg/kg markedly reduced plasma uric acid levels in decreasing order: Flowers (80%) > seeds (72%) > pericarps (64%) > twigs (59%) > leaves (41%), compared with allopurinol (89%). Finally, 10 isolated phytochemicals from longan flowers were then examined in vitro. The results indicated that proanthocyanidin A2 and acetonylgeraniin A significantly inhibited XO activity in vitro. This is the first report providing new insights into the urate-reducing effect of phenolic dimer and hydrolyzable tannin, which can be developed to potential hypouricemic agents. SUMMARY: Longan flower extracts possess more potent XO-inhibitory activity than pericarps, twigs, seeds, and leaves in vitroThe lowering effect of longan flowers and seeds extracts markedly reduced plasma uric acid levels as compared to allopurinol in vivoThe extract proanthocyanidin A2 and acetonylgeraniin A were demonstrated potent XO inhibitory activity in vitro Abbreviations used: PO: Potassium-oxonate, XO: xanthine oxidase, HE: n-hexane, EA: ethyl acetate, i.p.: intraperitoneal, PBS: phosphate-buffered saline, AP: allopurinol, PUA: plasma uric acid.

Phytochemicals from Tradescantia albiflora Kunth Extracts Reduce Serum Uric Acid Levels in Oxonate-induced Rats.

BACKGROUND: Tradescantia albiflora (TA) Kunth (Commelinaceae) has been used for treating gout and hyperuricemia as folklore remedies in Taiwan. Therefore, it is worthwhile to study the effect of TA extracts on lowering uric acid activity. The hypouricemic effects of TA extracts on potassium oxonate (PO)-induced acute hyperuricemia were investigated for the first time. MATERIALS AND METHODS: All treatments at the same volume (1 ml) were orally administered to the abdominal cavity of PO-induced hyperuricemic rats. One milliliter of TA extract in n-hexane (HE), ethyl acetate (EA), n-butanol (BuOH), and water fractions has 0.28, 0.21, 0.28, and 1.03 mg TA, respectively; and the plasma uric acid (PUA) level was measured for a consecutive 4 h after administration. RESULTS: All four fractions' extracts derived from TA were observed to significantly reduce PUA compared with the PO group. The EA-soluble fraction (TA-EA) exhibited the best xanthine oxidase (XO) inhibitory activity. Following column chromatography, 12 phytochemicals were isolated and identified from the EA fraction. The IC50 values of isolated phytochemicals indicated that bracteanolide A (AR11) showed the remarkable XO inhibitory effect (IC50 value of 76.4 µg/ml). These findings showed that the in vivo hypouricemic effect in hyperuricemic rats was consistent with in vitro XO inhibitory activity, indicating that TA extracts and derived phytochemicals could be potential candidates as hypouricemic agents. SUMMARY: Tradescantia albiflora extracts possess in vivo hypouricemic action in hyperuricemic ratsT. albiflora extracts exhibited strong inhibitory activity against xanthine oxidase (XO)Butenolide may play an important role in XO inhibitionThe extract bracteanolide A was demonstrated potent XO inhibitory activity in vitro. Abbreviations used: TA: Tradescantia albiflora, PO: potassium oxonate, HE: n-hexane, EA: ethyl acetate, BuOH: n-butanol, PUA: plasma uric acid, XO: xanthine oxidase, MeOH: methanol, IP: intraperitoneal.

Expandable Scaffold Improves Integration of Tissue-Engineered Cartilage: An In Vivo Study in a Rabbit Model.

One of the major limitations of tissue-engineered cartilage is poor integration of chondrocytes and scaffold structures with recipient tissue. To overcome this limitation, an expandable scaffold with a honeycomb-like structure has been developed using microfluidic technology. In this study, we evaluated the performance of this expandable gelatin scaffold seeded with rabbit chondrocytes in vivo. The chondrocyte/scaffold constructs were implanted into regions of surgically introduced cylindrical osteochondral defects in rabbit femoral condyles. At 2, 4, and 6 months postsurgery, the implanted constructs were evaluated by gross and histological examinations. As expected, the osteochondral defects, which were untreated or transplanted with blank scaffolds, showed no signs of repair, whereas the defects transplanted with chondrocyte/scaffold constructs showed significant cartilage regeneration. Furthermore, the expandable scaffolds seeded with chondrocytes had more regenerated cartilage tissue and better integration with the recipient tissue than autologous chondrocyte implantation. Biomechanical tests revealed that the chondrocyte/scaffold group had the highest compressive strength among all groups at all three time points and endured a similar compressive force to normal cartilage after 6 months of implantation. Histological examinations revealed that the chondrocytes were distributed uniformly within the scaffolds, maintained a normal phenotype, and secreted functional components of the extracellular matrix. Histomorphometric assessment showed a remarkable total interface of up to 87% integration of the expandable scaffolds with the host tissue at 6 months postoperation. In conclusion, the expandable scaffolds improved chondrocyte/scaffold construct integration with the host tissue and were beneficial for cartilage repair.

Tooth Germ-Like Construct Transplantation for Whole-Tooth Regeneration: An In Vivo Study in the Miniature Pig.

The purpose of this study was to demonstrate the feasibility of whole-tooth regeneration using a tooth germ-like construct. Dental pulp from upper incisors, canines, premolars, and molars were extracted from sexually mature miniature pigs. Pulp tissues were cultured and expanded in vitro to obtain dental pulp stem cells (DPSCs), and cells were differentiated into odontoblasts and osteoblasts. Epithelial cells were isolated from gingival epithelium. The epithelial cells, odontoblasts, and osteoblasts were seeded onto the surface, upper, and lower layers, respectively, of a bioactive scaffold. The lower first and second molar tooth germs were removed bilaterally and the layered cell/scaffold constructs were transplanted to the mandibular alveolar socket of a pig. At 13.5 months postimplantation, seven of eight pigs developed two teeth with crown, root, and pulp structures. Enamel-like tissues, dentin, cementum, odontoblasts, and periodontal tissues were found upon histological inspection. The regenerated tooth expressed dentin matrix protein-1 and osteopontin. All pigs had regenerated molar teeth regardless of the original tooth used to procure the DPSCs. Pigs that had tooth germs removed or who received empty scaffolds did not develop teeth. Although periodontal ligaments were generated, ankylosis was found in some animals. This study revealed that implantation of a tooth germ-like structure generated a complete tooth with a high success rate. The implant location may influence the morphology of the regenerated tooth.

Enhanced Bone Tissue Regeneration by Porous Gelatin Composites Loaded with the Chinese Herbal Decoction Danggui Buxue Tang.

Danggui Buxue Tang (DBT) is a traditional Chinese herbal decoction containing Radix Astragali and Radix Angelicae sinensis. Pharmacological results indicate that DBT can stimulate bone cell proliferation and differentiation. The aim of the study was to investigate the efficacy of adding DBT to bone substitutes on bone regeneration following bone injury. DBT was incorporated into porous composites (GGT) made from genipin-crosslinked gelatin and ß-triclacium phosphates as bone substitutes (GGTDBT). The biological response of mouse calvarial bone to these composites was evaluated by in vivo imaging systems (IVIS), micro-computed tomography (micro-CT), and histology analysis. IVIS images revealed a stronger fluorescent signal in GGTDBT-treated defect than in GGT-treated defect at 8 weeks after implantation. Micro-CT analysis demonstrated that the level of repair from week 4 to 8 increased from 42.1% to 71.2% at the sites treated with GGTDBT, while that increased from 33.2% to 54.1% at GGT-treated sites. These findings suggest that the GGTDBT stimulates the innate regenerative capacity of bone, supporting their use in bone tissue regeneration.

Identification of the metabolites of TCM prescription Sinisan, found in miniature pig urine via intragastric administration.

The metabolic profile of the traditional Chinese medicine, Sinisan, in miniature pig urine via intragastric administration was investigated. In total, 50 compounds, including 10 unchanged parent glycosides, which were not found from Sinisan's metabolic profile in rats' urine, were identified. Among these, 36 compounds were characterized by HPLC-SPE-NMR coupled with HPLC-HRESIMS, five of which are new and nine are endogenous metabolites of miniature pig. Most of phase I and phase II metabolites are hydrolytic products of parent glycosides and glucuronide conjugates, respectively, the latter having been reported as sulfate conjugates while the experimental animal is rat. Benzoic acid, obtained from hydrolysis of albiflorin and paeoniflorin, and phenylpropenoic acids, obtained from oxidative cleavage of flavones, formed phase II glycine conjugates.

Residue Depletion Study of Danofloxacin in Cultured Tilapia (Oreochromis mossambicus).

Danofloxacin is an antibacterial drug of the fluoroquinolone group developed for therapeutic purposes in veterinary medicine. The studies described here include investigations of the residues following a single dose or multiple doses of danofloxacin. Residue depletion studies were performed to determine residues in plasma and tissues of saltwater tilapia fish (Oreochromis mossambicus) after a single oral administration of danofloxacin at the dose of 10 mg/kg body weight and also after daily dose of 10 mg/kg body weight for five consecutive days. Danofloxacin residues were analyzed by HPLC with fluorescence detection. Following a single oral dose, danofloxacin residues in 6 h postdosing tilapia were at a maximum of 1.44, 12.48, and 13.18 µg/g in serum, liver, and kidney samples, respectively, while a peak muscle concentration of 2.15 µg/g was reached at 12 h. From single-dose data, the concentration of danofloxacin in serum, muscle, liver, and kidney samples declined with half-lives of 29, 34, 49, and 44 h, respectively. Based on the maximum residue level (MRL) of 0.1 µg/g in edible tissue for fin fish, the withdrawal times of danofloxacin in muscle were estimated to fall below the MRL after a withdrawal period of 21 days following the multiple-dose administration. These results may be helpful to regulatory agencies as they determine what tissues should be monitored to ensure that the established residue safety tolerance levels are not exceeded.

An in vivo swine study for xeno-grafts of calcium sulfate-based bone grafts with human dental pulp stem cells (hDPSCs).

The purpose of this in vivo study was to evaluate the effect of human dental pulp stem cells (hDPSCs) on various resorbable calcium sulfate/calcium phosphate bone grafts in bone regeneration. Granular particles of calcium sulfate dehydrate (CSD), α-calcium sulfate hemihydrate/amorphous calcium phosphate (α-CSH/ACP), and CSD/ß-tricalcium phosphates (ß-TCP) were prepared for in vitro dissolution and implantation test. The chemical compositions of specimen residues after dissolution test were characterized by XRD. The ratios of new bone formation for implanted grafts/hDPSCs were evaluated using mandible bony defect model of Lanyu pig. All the graft systems exhibited a similar two-stage dissolution behavior and phase transformation of poor crystalline HAp. Eight weeks post-operation, the addition of hDPSCs to various graft systems showed statistically significant increasing in the ratio of new bone formation (p<0.05). Null hypothesis of hDPSCs showing no scaffold dependence in bone regeneration was rejected. The results suggest that the addition of hDPSCs to calcium sulfate based xenografts could enhance the bone regeneration in the bony defect.