Repair of superficial osteochondral defects with an autologous scaffold-free cartilage construct in a caprine model: implantation method and short-term results.

OBJECTIVE: To compare four different implantation modalities for the repair of superficial osteochondral defects in a caprine model using autologous, scaffold-free, engineered cartilage constructs, and to describe the short-term outcome of successfully implanted constructs. METHODS: Scaffold-free, autologous cartilage constructs were implanted within superficial osteochondral defects created in the stifle joints of nine adult goats. The implants were distributed between four 6-mm-diameter superficial osteochondral defects created in the trochlea femoris and secured in the defect using a covering periosteal flap (PF) alone or in combination with adhesives (platelet-rich plasma (PRP) or fibrin), or using PRP alone. Eight weeks after implantation surgery, the animals were killed. The defect sites were excised and subjected to macroscopic and histopathologic analyses. RESULTS: At 8 weeks, implants that had been held in place exclusively with a PF were well integrated both laterally and basally. The repair tissue manifested an architecture similar to that of hyaline articular cartilage. However, most of the implants that had been glued in place in the absence of a PF were lost during the initial 4-week phase of restricted joint movement. The use of human fibrin glue (FG) led to massive cell infiltration of the subchondral bone. CONCLUSIONS: The implantation of autologous, scaffold-free, engineered cartilage constructs might best be performed beneath a PF without the use of tissue adhesives. Successfully implanted constructs showed hyaline-like characteristics in adult goats within 2 months. Long-term animal studies and pilot clinical trials are now needed to evaluate the efficacy of this treatment strategy.

A static, closed and scaffold-free bioreactor system that permits chondrogenesis in vitro.

OBJECTIVE: To characterise in vitro engineered cartilaginous constructs made employing a novel static, scaffold-free and closed chamber system. DESIGN: Chondrocytes derived from slaughter age pigs (3-6 months) were seeded at high density (20 x 10(6)) into cylindrical chambers (1.0 x 0.5cm) and were maintained between an upper and a lower membrane (100 kDa) for 21 days and subsequently cultured in open culture for 7 additional days. RESULTS: Viable constructs produced were approximately 10 mmx2mm in size and were stable enough to be handled by surgical pincers. Histology and electron microscopy evaluations revealed a neo-cartilage structure of high cell density with a comprehensive extracellular matrix. Predominately collagen type II and negligible amounts of collagen types I and X were detected using RT-PCR and SDS-PAGE analyses. CONCLUSIONS: In this study, we provide evidence of a scaffold-free system that can produce immature hyaline-like cartilaginous constructs suitable for in vivo implantation, or that may be useful for in vitro studies of events related to the process of chondrogenesis.

Articular cartilage repair using a tissue-engineered cartilage-like implant: an animal study.

OBJECTIVE: Because articular cartilage has limited ability to repair itself, treatment of (osteo)chondral lesions remains a clinical challenge. We aimed to evaluate how well a tissue-engineered cartilage-like implant, derived from chondrocytes cultured in a novel patented, scaffold-free bioreactor system, would perform in minipig knees with chondral, superficial osteochondral, and full-thickness articular defects. DESIGN: For in vitro implant preparation, we used full-thickness porcine articular cartilage and digested chondrocytes. Bioreactors were seeded with 20x10(6) cells and incubated for 3 weeks. Subsequent to culture, tissue cartilage-like implants were divided for assessment of viability, formaldehyde-fixed and processed by standard histological methods. Some samples were also prepared for electron microscopy (TEM). Proteoglycans and collagens were identified and quantified by SDS-PAGE gels. For in vivo studies in adult minipigs, medial parapatellar arthrotomy was performed unilaterally. Three types of defects were created mechanically in the patellar groove of the femoral condyle. Tissue-engineered cartilage-like implants were placed using press-fit fixation, without supplementary fixation devices. Control defects were not grafted. Animals could bear full weight with an unlimited range of motion. At 4 and 24 weeks postsurgery, explanted knees were assessed using the modified ICRS classification for cartilage repair. RESULTS: After 3-4 weeks of bioreactor incubation, cultured chondrocytes developed a 700-microm- to 1-mm-thick cartilage-like tissue. Cell density was similar to that of fetal cartilage, and cells stained strongly for Alcian blue and safranin O. The percentage of viable cells remained nearly constant (approximately 90%). Collagen content was similar to that of articular cartilage, as shown by SDS-PAGE. At explantation, the gross morphological appearance of grafted defects appeared like normal cartilage, whereas controls showed irregular fibrous tissue covering the defect. Improved histologic appearance was maintained for 6 months postoperatively. Although defects were not always perfectly level upon implantation at explanation the implant level matched native cartilage levels with no tissue hypertrophy. Once in place, implants remodelled to tissues with decreased cell density and a columnar organization. CONCLUSIONS: Repair of cartilage defects with a tissue-engineered implant yielded a consistent gross cartilage repair with a matrix predominantly composed of type II collagen up to 6 months after implantation. This initial result holds promise for the use of this unique bioreactor/tissue-engineered implant in humans.

Tissue engineering in space.

The purpose of this paper is to present the status of that part of the [Microgravity Application Program] project related to the study of cartilage formation from pig chondrocytes. The work carried out so far followed two lines: (i) chondrocytes were incubated for up to three weeks in the RPM; (ii) a module developed for in-vitro cartilage formation will be tested in a sounding rocket flight (MASER 9, November 2001).

Immediate shear stress resistance of endothelial cell monolayers seeded in vitro on fibrin glue-coated ePTFE prostheses.

The shear stress resistance of endothelial cells (EC) previously seeded onto ePTFE grafts was assessed by morphometric determination of the number of cells per cm2 of graft surface before and after exposure of 6 h of arterial blood flow interposed in the canine femoral artery. Autologous venous endothelial cells (AVEC) were harvested from the extrajugular veins of five dogs. The AVEC were cultured in vitro and seeded at a density of 150 x 10(3) cells per cm2 onto 4 mm ID ePTFE grafts precoated with fibrin glue and human fibronectin. Subsequently, the AVEC monolayers on the grafts were cultured for 8 days using a perfusion system and then implanted end-to-end in the femoral artery. All grafts remained patent (5/5). Scanning electron microscopy demonstrated complete, thrombus-free monolayers of AVEC after 6 h of arterial blood flow. The cell densities were 124 +/- 14 and 129 +/- 7 x 10(3) cells per cm2 respectively before and after implantation. It is concluded that in vitro lining of 4 mm ePTFE vascular prostheses is feasible and results in EC monolayers on the graft surface which are shear stress resistant and athrombogenic.

Effect of vitamin E substitution in very low birth weight infants. Active and inactive forms of vitamin E in plasma and red cells after moderate intramuscular supplementation.

Vitamin E supplementation was investigated in 38 very low birth weight infants after intramuscular substitution with two doses of 25 mg given on the 1st and 3rd day of life. Plasma and red cell concentrations of the biologically inactive tocopherol acetate and the active tocopherol were followed during the first month of life. Plasma levels of this substitution regime were compared with recent clinical studies. Although the applied dose was low in comparison to other studies, the levels obtained exceed the critical concentration of 12 microM which was reported to prevent retinopathy. Concentrations associated with side effects (84 microM) were found in nearly 50% of the babies but only for short duration (24 h). In more than 80% of the infants, biologically inactive tocopherol acetate was detected in plasma up to 1 day after substitution but not in red cells.