Objective: To evaluate the effects of low-intensity ultrasound (LIUS) stimulation on the anabolic state of human cartilage from patients with osteoarthritis (OA). Methods: Explant cultures of human OA cartilage were stimulated for 10 min every day for 7 consecutive days using continuous-wave sonication at a frequency of 1 MHz with spatial and temporal average intensities of 0 (control), 40, 200, 500, or 700 mW/cm 2 . The effects of LIUS on cell proliferation were evaluated by 3 H-thymidine incorporation. Proteoglycan synthesis was evaluated by the incorporation of 35 S-sulfate and by Safaranin O staining. Collagen synthesis was evaluated by 3 H-proline incorporation and immunohistochemistry. Results: At an intensity of 200 mW/cm 2 , LIUS treatment induced the expression of collagen type II and proteoglycan measured by the incorporation of radioactivity and specific staining of the cartilage explants. However, the expression decreased again at the higher intensities of 500 or 700 mW/cm 2 . Ultrasound had no stimulatory effect on cell proliferation at any intensity. Conclusion: LIUS has anabolic effects on human cartilage in explant cultures, indicating a potentially important method for the repair of osteoarthritic cartilage.Osteoarthritis (OA) is the most common joint disease and is characterized by progressive joint destruction, ultimately leading to the need to replace major joints. Although the precise pathological mechanism has not yet been defined, changes in the function of the chondrocytes are thought to be the main cause of the pathology of OA, particularly the imbalance between the anabolism and the catabolism of the extracellular matrix produced from chondrocytes. Methods to increase synthesis of the cartilage matrix and inhibit its degradation may therefore provide effective treatments for OA.Ultrasound (US) has been used extensively in diagnostic applications (1, 2) and there has recently been increasing interest in its therapeutic use for various musculoskeletal disorders. Several studies have shown that low-intensity US (LIUS) is effective in regenerating injured tissues, including muscles (3, 4) and bones (5-9). In particular, LIUS has been shown to enhance the healing of fractured bones by inducing chondrocyte proliferation (7,9). When applied to a monolayer culture of human chondrocytes, LIUS also increased cellular proliferation and matrix synthesis (10). In animal studies, LIUS stimulation in both bone fractures and cartilage resulted in increased expression of proteoglycan or type II collagen rather than that of bone formation factors such as transforming growth factor-b (TGF-b), osteocalcin, alkaline phosphatase, and a1(I)-procollagen, hence protecting the collagen matrix and preventing the destruction or degradation of cartilage (11-13). However, the effects of LIUS treatment on chondrocyte proliferation and the expression of matrix proteins such as proteoglycan and collagen type II have not been clearly shown in intact human cartilage.In this study, we evaluated the LIUS effects on the ana...
Novel chondroitin sulfate (CS) -poly(ethylene oxide) (PEO) hydrogel was synthesized and evaluated by a mechanism of self cross-linking of CS derivative with PEO with hexa-thiols (PEO-SH). A derivative of CS was synthesized by the sequential grafting of adipic acid dihydrazide (ADH) and acrylic acid: chemical grafting of ADH to the carboxylic acid in CS (CS-ADH) followed by grafting of the acrylic acid to the free amine groups in the CS-ADH (CS-ADH-Ac). The synthesis of CS-ADH-Ac molecules was confirmed by observing new acrylate peaks in CS-ADH-Ac by FTIR, ESCA, and NMR. The CS-PEO hydrogel was self cross-linked through a Michael type addition reaction between the acrylate end groups of CS-ADH-Ac and the thiol end groups of the PEO-SH. the gelation behavior of 10% CS-PEO was evaluated by rheological analyses from the changes in the solution properties, such as phase angles and visco-elasticities. Rheological analysis indicated that the gelation process was complete within 2 min after mixing two polymer solutions of CS-ADH-Ac and PEO-SH. The fabricated CS-PEO hydrogel was analyzed by measuring both its swelling under different water pHs and its mechanical strength against compression. The morphological shapes of both its surface and cross sections were also evaluated after the sequential processes of gel swelling to equilibrium followed by dehydration. Both the gelation time and swelling of the fabricated hydrogel were dependent on the pH of the polymer solutions and swelling medium, showing quicker gel formation and better swelling behaviors under basic conditions than under acidic conditions. The equilibrated gel showed different morphologies depending on its location, i.e. its cross sections demonstrated more homogeneous morphologies than the surfaces. While the dehydrated hydrogel demonstrated 8-10 µm pore sizes on its cross sections, the compression strength of the hydrogel ranged from 1.4 to 2.8 Pa depending on its gel concentration. Toluidine blue molecules as a model drug were released from the hydrogel over a period of more than 5 days. These hydrogel properties, such as formation of in situ gel, release behaviors of toluidine blue, and porous structures and mechanical properties of the fabricated gel, highlighted the potential of a hydrogel as a carrier for local drug delivery and a scaffold for tissue engineering.
A cross-linking spacer molecule in biomaterials is an important factor in controlling their biocompatibility. The effects of various cross-linking spacers on the biocompatibility of chitosan-spacer-poly(ethylene oxide) (PEO) hydrogel were evaluated by various cytotoxicity assays and by targeting to specific cell organs. The chitosanspacer-PEO hydrogel was synthesized using a Michael type addition reaction by employing chitosan-acrylate and PEO-thiols. Chitosan-acrylates have been synthesized employing three different cross-linking spacers such as 2-carboxyethyl acrylate, linoleic acid and oleic acid. This study verified the grafting of the spacer molecules to the amine groups of chitosan side chains with FTIR by comparing the peaks of the chitosan-acrylates with those of the unmodified chitosan. After evaluating the cytotoxicity of the chitosan-acrylates in solution with a live a dead assay, the cytotoxicity of the chitosan-spacer-PEO gels was compared with each other by testing them with CCK-8 for their effects on cell proliferation, with a MTT assay for their effects on mitochondria damage, with BrdU assay for their effects on DNA damages and with the neutral red assay for their effects on lysosome damages. The degrees of cytotoxicity of the chitosan-spacer-PEO hydrogels were compared with those of Teflon and Latex, positive and negative control, respectively, using neural cells, such as PC-12 cells. The chitosan-2-carboxyethyl acrylate-PEO hydrogels demonstrated the best cell compatibility among the hydrogels employed in this study.
Autologous chondrocyte implantation (ACI) is used to treat some articular cartilage defects. However, the fate of the cultured chondrocytes after in-vivo transplantation and their role in cartilage regeneration remains unclear. To monitor the survival and fate of such cells in vivo, the chondrocytes were labelled with a lipophilic dye and the resultant regenerated tissue in dogs examined. It was found that, 4 weeks after implantation, the osteochondral defects were filled with regenerative tissue that resembled hyaline cartilage. Fluorescence microscopy of frozen sections of the regenerated tissue revealed that the majority of cells were derived from the DiI-labelled implanted chondrocytes. From these results, it was concluded that a large population of implanted autologous chondrocytes can survive at least 4 weeks after implantation and play a direct role in cartilage regeneration. However, it remains unknown whether other cells, such as periosteal cells or bone marrow stromal stem cells, are involved in the regeneration of cartilage after ACI.
753ryopreserved patellar tendon allografts are often recommended for reconstruction of anterior cruciate ligaments (ACLs) because living donor fibroblasts are thought to promote repair. Animal studies, however, indicate that ligaments regenerate from recipient rather than donor cells. If applicable to man, these observations suggest that allograft cell viability is unimportant. We therefore used short tandem repeat analysis with polymerase chain reaction (PCR) amplification to determine the source of cells in nine human ACLs reconstructed with cryopreserved patellar tendon allografts. PCR amplification of donor and recipient DNA obtained before operation and DNA from the graft obtained two to ten months after transplantation revealed the genotype of cells and showed only recipient cells in the graft area. Rather than preserve the viability of donor cells, a technique is required which will facilitate the introduction of recipient cells into patellar tendon allografts.
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