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.

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.

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.

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.

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.

Recent progress in Glinus oppositifolius research.

CONTEXT: Glinus oppositifolius (L.) Aug. DC. (Molluginaceae), a perennial subshrubs herb, grows at low altitudes in the southern part of Taiwan, and is used in traditional Chinese medicine for herpes zoster and herpangina. OBJECTIVE: This study describes nutritional and therapeutic potential of Glinus oppositifolius and summarizes scientific evidence that supports traditional claims; recent progress in research for this plant is reviewed herein. MATERIALS AND METHODS: The literature has been retrieved from the web-based online systems including PubMed, Medline, and Google Scholar. The articles related to phytochemistry, pharmaceutical biology and ethnopharmacology have been excluded. RESULTS AND DISCUSSION: In clinical practice, the plant has been extensively investigated in a broad range of studies to provide scientific evidence for folklore claims or to find new therapeutic uses. The present review may arouse related research and make a more valid display for Taiwanese native medicinal plants.

Establishment of a competitive ELISA for detection of florfenicol antibiotic in food of animal origin.

Florfenicol (FF) is a synthetic antibiotic with a broad antibacterial spectrum and the high therapeutic effectiveness that has been developed specifically for veterinary use. Obviously, FF adulterated in animal supplies is one of essential global concerns. A competitive ELISA for the detection of florfenicol in food of animal origin (swine, chicken, and fish) is described. Influence of immunoconjugate structure on the assay sensitivity and specificity was investigated. The new ELISA showed much lower than the MRPLs for FF at 100-3,000 mg kg(-1) in the European Communities and the sensitivity of our ELISA method was superior to that described in other reports. According to the test preparation record, the limit of detection of the developed ELISA performed on meat species was 0.3 µg kg(-1) (IC50 value 1.9 µg kg(-1)). The method developed permits FF concentrations to be determined in the range 0.3-24.3 µg kg(-1). A low cross-reactivity with florfenicol amine (FFA), thiamphenicol (TAP), and chloramphenicol (CAP) was displayed (16.2%, 9.5%, and 9.4%, respectively). Recovery in different food samples (swine, chicken, and fish) averages between 87-115%. The method can be applied for inspection of animal supplies for trace florfenicol residues.

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.

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.

In vitro studies of composite bone filler based on poly(propylene fumarate) and biphasic α-tricalcium phosphate/hydroxyapatite ceramic powder.

While many different filler materials have been applied in vertebral augmentation procedures, none is perfect in all biomechanical and biological characteristics. To minimize possible shortages, we synthesized a new biodegradable, injectable, and premixed composite made from poly(propylene fumarate) (PPF) and biphasic α-tricalcium phosphate (α-TCP)/hydroxyapatite (HAP) ceramics powder and evaluated the material properties of the compound in vitro. We mixed the PPF cross-linked by N-vinyl pyrrolidinone and biphasic α-TCP/HAP powder in different ratios with benzoyl peroxide as an initiator. The setting time and temperature were recorded, although they could be manipulated by modulating the concentrations of hydroquinone and N,N-dimethyl-p-toluidine. Degradation, cytocompatibility, mechanical properties, and radiopacity were analyzed after the composites were cured by a cylindrical shape. We also compared the study materials with poly(methyl methacrylate) (PMMA) and PPF with pure HAP particles. Results showed that lower temperature during curing process (38-44°C), sufficient initial mechanical compressive fracture strength (61.1±3.7MPa), and gradual degradation were observed in the newly developed bone filler. Radiopacity in Hounsfield units was similar to PMMA as determined by computed tomography scan. Both pH value variation and cytotoxicity were within biological tolerable limits based on the biocompatibility tests. Mixtures with 70% α-TCP/HAP powder were superior to other groups. This study indicated that a composite of PPF and biphasic α-TCP/HAP powder is a promising, premixed, injectable biodegradable filler and that a mixture containing 70% α-TCP/HAP exhibits the best properties.

Bone marrow combined with dental bud cells promotes tooth regeneration in miniature pig model.

Growth factors and morphogens secreted by bone marrow mesenchymal stem cells (BMSCs) of bone marrow fluid may promote tooth regeneration. Accordingly, a tissue engineering approach was utilized to develop an economical strategy for obtaining the growth factors and morphogens from BMSCs. Unerupted second molar tooth buds harvested from miniature pigs were cultured in vitro to obtain dental bud cells (DBCs). Bone marrow fluid, which contains BMSCs, was collected from the porcine mandible before operation. DBCs suspended in bone marrow fluid were seeded into a gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer scaffold (GCHT scaffold). The DBCs/bone marrow fluid/GCHT scaffold was autografted into the original alveolar sockets of the pigs. Radiographic and histological examinations were applied to identify the structure of regenerated tooth at 40 weeks postimplantation. The present results showed that one pig developed a complete tooth with crown, root, pulp, enamel, dentin, odontoblast, cementum, blood vessel, and periodontal ligament in indiscriminate shape. Three animals had an unerupted tooth that expressed dentin matrix protein-1, vascular endothelial growth factor, and osteopontin; and two other pigs also had dental-like structure with dentin tubules. This study reveals that DBCs adding bone marrow fluid and a suitable scaffold can promote the tooth regeneration in autogenic cell transplantation.

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.

Metabolism of dicentrine: identification of the phase I and phase II metabolites in miniature pig urine.

The metabolic profile of dicentrine, a selective α(1)-adrenoceptor antagonist with potent antiarrhythmic and antihypertensive activities, in miniature pig urine via oral administration was investigated for the first time. The urine, collected after a single oral administration of dicentrine, was pretreated using solvent extraction and column chromatographic methods to identify the metabolites containing fractions. Twenty-four metabolites (MI-1-9 and MII-1-15), of which 21 compounds are new, were identified by mass spectrometry and high-performance liquid chromatography-diode array detector solid-phase extraction-NMR techniques. Of these, 14 metabolites (MI-5, MII-1 and 2, and MII-5-15) were further isolated for structure confirmation. The phase I metabolic transformations of dicentrine were found to be N-demethylation, N-oxidation, O-demethylation (9,10-OMe), O,O-demethylenation (1-OCH(2)O-2), and hydroxylation at the benzylic (C-4) and the aromatic (C-3) positions, whereas those for the phase II were O-glucuronidation and O-glucosylation of the phenolic group of the phase I metabolites.

Chicken model of steroid-induced bone marrow adipogenesis using proteome analysis: a preliminary study.

BACKGROUND: Steroid-induced adipogenesis increases fat-cell volume and pressure in bone marrow. This may be a contributing factor in some forms of osteonecrosis. In this observational study, we aimed to determine the protein expression relating to steroid-induced adipogenesis of femoral bone marrow with use of a chicken model. We compared the histologic features of the femoral marrow of eight methylprednisolone (MP)-treated chickens with those of three control chickens and assessed differential proteins with 2-dimensional gel electrophoresis and differential proteins were identified by MALDI-TOF MS. RESULTS: One MP-induced chicken died of overdose anesthesia. Methylprednisolone-induced proliferation of adipose tissue and new bone formation were found on histologic examination. In our study, 13 proteins in the control and MP-induced groups were differently expressed and nine protein spots showed marked threefold downregulation after 19 weeks of MP treatment. These were serum amyloid P-component precursor, zinc finger protein 28, endothelial zinc finger protein 71, T-box transcription factor 3, cyclin-dependent kinase inhibitor 1, myosin 1D, dimethylaniline monooxygenase, and two uncharacterized proteins. CONCLUSIONS: Proteomic profiling can be a useful dynamic approach for detecting protein expression in MP-induced adipogenesis of the femur in chickens.

Preventive effects of Chinese herb chai-hu-gui-zhi-tang extract on water immersion restraint stress-induced acute gastric ulceration in rats.

It is well known that maintenance therapy using Chai-hu-gui-zhi-tang (CHGZT), a traditional Chinese medicine, has been proven to prevent the recurrence of peptic ulcers. However, little is known as to whether or not it has protective effects against acute gastric injury. In the present study, we investigated the preventive effects of pretreatment with CHGZT extract on the development of water immersion restraint stress-induced acute gastric ulceration in male Wistar rats. The CHGZT extract (50, 250, 500 mg/kg b.w., p.o.) was given to rats before they were exposed to 2 or 4 hr of water immersion restraint stress; they were then were sacrificed immediately after stress exposure. Gastric mucosal lesions were evaluated macroscopically, and the gastric mucosal and hepatic non-protein sulfhydryls (NP-SH) were measured simultaneously. The results indicate that exposure to water immersion restraint stress resulted in the development of acute gastric stress erosions. Pretreatment with CHGZT extract caused a significant reduction of stress lesions and an increase in the gastric mucosal NP-SH and hepatic NP-SH concentrations. We conclude that the anti-ulcer response and extensive antioxidant effect of Chai-hu-gui-zhi-tang may be valuable in prevention of experimental gastric mucosal lesions in rats because it possesses preventive and gastroprotective effects.

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.

Chitosan/gelatin hydrogel as immunoisolative matrix for injectable bioartificial pancreas.

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. METHODS: Chitosan/gelatin solution with glycerol 2-phosphate disodium salt hydrate was prepared and utilized as a cell carrier. The biocompatibility of the chitosan/gelatin hydrogel was first established by using a mouse insulinoma cell line, NIT-1. Insulinoma cells were encapsulated in agarose as microspheres and then macroencapsulated in chitosan/gelatin hydrogel. In vitro cell activity, material-mediated cytotoxicity, cytokine-mediated cytotoxicity assay, and insulin secreting profiles of insulinoma/agarose microspheres macroencapsulated in chitosan/gelatin hydrogel were analyzed. For in vivo study, insulinoma/agarose microspheres with chitosan/gelatin solution was applied as an injectable bioartificial pancreas (BAPs). Insulinoma/agarose microspheres suspended in phosphate-buffered saline or in chitosan/gelatin solution was injected into the subcutaneous layer of diabetic rats. Non-fasting blood glucose (NFBG) concentration of rat was measured perioperatively. After pre-determined intervals, the chitosan/gelatin hydrogel containing insulinoma/agarose microspheres was retrieved for histologic examinations. RESULTS: Insulinoma/agarose microspheres macroencapsulated in hydrogel revealed functional activity and secreted insulin continually for 60 days in vitro. Chitosan/gelatin hydrogel was not cytotoxic to islet cells, and in contrast, the hydrogel showed cytoprotective effects against cytokine-mediated cytotoxicity. The NFBG of diabetic rats transplanted with free insulinoma/agarose microspheres was decreased to euglycemia but restored to hyperglycemia in 15 days. Contrarily, the NFBG of rats transplanted with insulinoma/agarose microspheres with hydrogel remained euglycemic for 42 days. Histologic sections revealed that the fibrous tissue envelopment and the infiltrated immune-related cells contributed to the dysfunction of BAPs. CONCLUSIONS: This study indicates that using chitosan/gelatin hydrogel as a cell carrier is feasible and can provide an additional protection for the microencapsulated islet cells during xenotransplantation.