1/2000 - Společnost pro pojivové tkáně

Proteins in effluent were monitored at performed under sterile conditions. All238 nm, to determine glycosaminoglycans animals tolerated the procedures well andin the effluent single fractions were no perioperative death occured. Lateralcollected each 20 min. and then their parapatellar approach was used to harvestcontent was measured according to cartilage explants from the lateral condyleFarndale et al. (4).in the region of femoro-patelar joint of the2.1.3. Tripeptide GHK: (Gly-His-Lys) 2. right knee. After an interval of 4 -5 weeksC u . 2 H 2 O . 2 N a C l ( B i o c h e m . second surgery was performed. AnFeinchemikalien AG, Bubendorf, artificial osteochondral defect performedSwitzerland) was put into culture with a trephine (diameter 4 mm) in themedium to make its concentration 0.06 intercondylic region of the left knee wasmg/g collagen.filled up with construct prepared from2.1.4. Chondrocytes: were isolated using autologous chondrocytes. Routine closureocollagenase digestion (0.25%, 37.0 C) of subcutaneous tissue, and skin was(Sigma, St.Louis, USA) from the performed. Parenteral Penicillinexplants of minipig articular cartilage (Pendepon, Léčiva, Prague, Czech Rep.)according to Green (6). They were was administered intraoperatively and4 -2inoculated (8 x 10 cells .cm ) onto plastic repeated after surgery every week up to the2flasks (25cm Falcon Dickinson Benelux) end of the experiment.covered with cartilage collagens and 2.5. Evaluation of the implantationcultured to the 1st passage in MEM 2.5.1. X-ray and CT examination: aftersupplemented with antibiotics animals were sacrified, knee joints with(streptomycine 100ug/ml, penicillin 200 implanted chondrocytes were removedU/ml (SEVAC, Prague, Czech Rep.), and subjected to X-ray and CT10% fetal calf serum (Veterinary faculty examination.oBrno, Czech Rep.) in a 37 C, 5% CO 2 2.5.2. Histology: part of explantedatmosphere.newly formed tissues filling cartilage2.2. Construct: contained chondrocytes defects was fixed in 4% calcium(120 000 per 1.0 ml medium), cartilage formalin and bisected sagittally forcollagens (1.2 mg per 1.0 ml), aggrecan histologic analysis. The explants were(0.60 mg per 1.0 ml) and GHK (0.06mg/g divided into two equal parts: a) One partcoll). Diagram of implant preparation of explants fixed in formalin was used forsee on the Fig 1.cryostat sections, which were stained2.3. Experimental animals: 22 female with Toluidin blue. b) The second oneor castrated male skeletally mature was sectioned into well-defined piecesminipigs (Anlab Prague, Czech Rep) were suitable for embedding into epoxy resin.used for evaluation. The animals were These sections were washed up inscreened for systemic disease and phosphate buffer (pH 7.4), briefly fixed inconditioned for at least 8 months before 2.5% glutaraldehyd (diluted with the sameinclusion in this study. They were buffer) and then in 1.0% osmium tetroxide.sacrificed 9 weeks after surgery and the After dehydration in acetone, sectionesrespective tissue underwent further were embedded into Durcupan (Flukaexaminations.Chemie AG, Buchs, Switzerland).2.4. Surgical procedure : after general Semithin sections were stained, type ofanesthesia (5% Narcamon, Léčiva staining is described with legend to eachPrague, Czech Rep.) minipig was placed picture.prone on operating table. The surgery was 2 . 5 . 3 . P o l y a c r y l a m i d e g e l36LOCOMOTOR SYSTEM VOL. 7, 2000, No.1