Mosaicplasty with photooxidized, mushroom shaped, bovine, osteochondral xenografts in experimental sheep.

The goal was to study the performance of mushroom shaped, photooxidized, osteochondral grafts in mosaicplasty focusing on graft stability and survival. Mushroom shaped, photooxidized grafts (6 mm for the cartilaginous head of the mushroom, 3 mm for the stem) were implanted in the medial femoral condyle of 10 sheep. Four transplants were inserted per condyle in an overlapping fashion using the pressfit technique (n=40 grafts in 10 condyles). The grafts were followed for 6 and 12 months. Semi-quantitative evaluation of graft performance was performed using a validated score system. All grafts were mechanically stable at 6 and 12 months with one exception, where the mushroom head broke off. The formation of cystic lesions in the subchondral bone area was minimal. Repopulation of the old photooxidized cartilage was noticed with cells invading the matrix from the subchondral bone area and also from the pannus on the surface. Fusion between host and graft cartilage was observed in some of the grafts at 12 months, while remodeling of the calcified cartilage zone and tidemark was noticed in all grafts. Results scored significantly better for the 6 months compared to the 12 months group if cartilage surface integrity was compared (p<0.05). In all other variables no significant differences were found between groups. Despite moderate graft recession in the 12 months group partial fusion of grafts and functional results were satisfactory. The photooxidized mushroom shaped osteochondral transplants may be a suitable type of graft for functional results in cartilage resurfacing if stable anchorage of the grafts can be achieved.

The use of photooxidized, mushroom-structured osteochondral grafts for cartilage resurfacing--a comparison to photooxidized cylindrical grafts in an experimental study in sheep.

OBJECTIVE: This article addresses the problem of structural design with osteochondral grafts used for cartilage resurfacing. METHODS: Photooxidized cylindrical or mushroom-shaped grafts were surgically implanted in the weight bearing area of the medial and lateral femoral condyles of eight sheep (condyles: N=8/group). Both types of photooxidized grafts contained no viable chondrocytes at the time of implantation. Results were evaluated at 2 and 6 months after surgical implantation of the grafts. Qualitative and quantitative evaluation of the subchondral bone area was performed using plastic embedded sections of non-decalcified bone and cartilage specimens and placing emphasis on graft anchorage, cyst-like lesions at the base of the cartilage junction and at the base of the graft in the subchondral bone region. Cartilage morphology was studied qualitatively focusing on viability of the graft and adjacent host cartilage, while a score system was developed for semi-quantitative evaluation of the overall articular cartilage performance. The semiquantitative scores and histomorphometrical measurements were subjected to statistical analysis using a factorial analysis of variance (ANOVA-test). RESULTS: The photooxidized mushroom-shaped grafts developed less fibrous tissue and cyst-like lesions in the subchondral bone area at 2 and 6 months compared to the cylindrical grafts. Areas of endochondral ossification and bone remodeling were noticeable in the mushroom structured grafts at 2 months, and also bone remodeling was more complete at 6 months than with the cylindrical grafts. Increased numbers of cells were seen in the basal remodeling zones of both graft types increased from the 2 months to the 6 months specimens, but mushroom structured grafts showed better results. In both graft types, however, the midzone of the cartilage matrix was still acellular at 6 months. Cells from the subchondral bone area started to penetrate the calcified cartilage zone and tide mark at 2 months and repopulated the old photooxidized cartilage matrix already at 6 months after implantation. Cartilage repopulation was dependent on a stable subchondral bone area in both types of grafts. Matrix degradation of the adjacent host cartilage was minimal at 2 and 6 months. At 6 months a junction between host and graft cartilage was already noticed in some of the mushroom-shaped grafts. CONCLUSION: This study confirmed the importance of the subchondral bone area for osteochondral graft survival. In addition it demonstrated that the structure of the graft influences considerably the architecture of the subchondral bone, and with this the possibility for the repopulation of the old cartilage matrix including the junction between the host and graft cartilage matrix.

Changes in subchondral bone in cartilage resurfacing--an experimental study in sheep using different types of osteochondral grafts.

OBJECTIVE: This article addresses the subchondral bone integrity in cartilage resurfacing by comparing fresh, untreated auto-, xeno-, and photooxidized osteochondral allo- and xenografts. Photooxidation was expected to improve mechanical stability of the osteochondral grafts through an improved linkage of the collagen fibers within the bone matrix. DESIGN: Untreated auto- and xenografts and with photooxidation pretreated allo- and xenografts were surgically implanted in femoral condyles of sheep (n=40). After 2, 6, 12 and 18 months results were evaluated histologically using non-decalcified bone embedded in acrylic resin. Qualitative evaluation was performed with emphasis on bone matrix, biomechanical stability of graft anchorage, formation of cystic lesions, and bone resorption and formation. Quantitative evaluation of the total subchondral bone area was conducted histomorphometrically. Statistical analysis (factorial ANOVA test) was used to compare differences between groups with respect to the percentage of bone matrix and fibrous tissue per section. RESULTS: Subchondral bone resorption was fastest in untreated, fresh autografts, followed by photooxidized allografts, untreated, fresh xenografts and last pretreated photooxidized xenografts. Cystic lesions were seen in all types of grafts, but were most pronounced at 6 months in autografts and least in photooxidized grafts. Cyst-like lesions had subsided substantially in the untreated auto- and photooxidized xenografts, if no graft dislocation occurred during the healing period. Mononuclear cell infiltration and an increase in the presence of multinuclear cells were observed at 2 months, mostly in untreated autografts, followed by photooxidized allo- and untreated xenografts. They were much higher in numbers compared to photooxidized grafts, at least in the early specimens at 2 months. Graft stability was linked to the rate of bone resorption. CONCLUSION: Substantial resorption of the subchondral bone, involving the development of cyst-like lesions, lead to dislocation and finally to cartilage matrix degradation of the grafts. The process of photooxidation decreased the speed of bone resorption in osteochondral grafts and, thus, improved graft stability and cartilage survival. These results suggest that the remodeling of the subchondral bone of the host and the graft within the first 6 months is an important factor in graft stability and overall results of cartilage resurfacing.

In vitro studies of a photo-oxidized bovine articular cartilage.

Bovine articular cartilage was photo-oxidized and cultured with native articular bovine cartilage and synovial membrane to study the interaction between these tissues mimicking the physiological situation in the joint. The photo-oxidation was applied as a pretreatment of cartilage for future use in cartilage resurfacing procedures in joints. Properties of the transplant were assessed by testing the production of local mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and neutral metalloproteinase activities under normal conditions and after stimulation with various stimulants representative of inflammatory changes in pathophysiological conditions. Unlike normal cartilage photo-oxidized cartilage did not release significant amounts of NO and PGE2 and showed less gelatinolytic and caseinolytic activity compared to native bovine articular cartilage. Enzyme activity of the combined cultures was at a level intermediate between that of photo-oxidized cartilage and native cartilage cultures alone. In contrast to normal cartilage, living chondrocytes were not visible in photo-oxidized cartilage using live/dead staining. These results indicate, that the photo-oxidized cartilage may have a beneficial effect on adjacent native host cartilage and therefore be a suitable transplant for use in in vivo experiments.

Long term in-vivo studies of a photo-oxidized bovine osteochondral transplant in sheep.

BACKGROUND: Articular cartilage has limited capacity to repair. Defects greater than 3 mm heal with formation of inferior fibrous cartilage. Therefore, many attempts have been made to find the ideal graft for larger cartilage lesions. Different grafts, such as untreated or cryopreserved osteochondral transplants, have been used with variable success. METHODS: Photo-oxidized osteochondral grafts were implanted in both femoral condyles of one ovine knee. Untreated xenogeneic and autogeneic grafts served as controls. Three groups of 8 sheep each were formed and they were sacrificed 6, 12 or 18 months after surgery. RESULTS: The macroscopic evaluation of the condyle and graft showed a well-maintained cartilage surface in most grafts at all time points. However, the host cartilage matrix deteriorated considerably in all xenogeneic, most autogeneic and fewer of the photo-oxidized grafts at 12 and 18 months, respectively. The blue colour of the photo-oxidized grafts resulting from the process of photo-oxidation was visible in all grafts at 6 months, had diminished at 12 months and had completely disappeared at 18 months after surgery. Histologically a loss of matrix staining was almost never noticed in untreated xenografts, transiently at 6 months in photo-oxidized grafts and increased at 12 and 18 months. Fusion between graft and host cartilage could be seen in photo-oxidized grafts at 12 and 18 months, but was never seen in autografts and xenografts. CONCLUSIONS: The photo-oxidation of osteochondral grafts and its use as transplant appears to have a beneficial effect on cartilage and bone remodelling. Osteochondral grafts pre-treated with photo-oxidation may be considered for articular cartilage replacement and therefore may delay artificial joint replacements in human patients.

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).

[Long-term results in the dog carotid artery with small lumen vascular prostheses with microvascular endothelial cells]. / Langzeitresultate in der Hundekarotis mit kleinlumigen Gefässprothesen mit mikrovaskulären Endothelzellen.

In this study we evaluated the long-term results of microvascular endothelial cell seeding of small diameter Dacron grafts with omentally derived cells in a canine model. 6 cm long and 4 mm I.D. seeded Dacron prostheses were implanted end-to-end in the carotid position in 12 dogs for 6 to 12 months. Microvascular endothelial cells were enzymatically harvested from omentum prior to implantation and seeded onto Dacron grafts with a seeding density of 1.5 x 10(6) cells/cm2 of the graft. The antiplatelet therapy (Aspirin, Dipyridamol) was administered for 4 weeks postoperatively. All seeded grafts were patent throughout the study. The thrombus-free surface area for seeded grafts was 99.6 +/- 0.8% and 99.6 +/- 0.9% at 6 months and one year, respectively. Scanning electron microscopy revealed a confluent endothelial layer. We concluded that endothelial cell seeding of smaller-diameter prosthetic vascular grafts with omentally derived endothelial cells obtained excellent long-term patency rate in the canine model.

[Development of a small-lumen vascular prosthesis coated with autologous endothelial cells]. / Entwicklung einer kleinlumigen mit autologen Endothelzellen beschichteten Gefässprothese.

A new compliant prosthesis with a monolayer of autologous endothelial cells (ENC) has been developed. It consists of a porous polyurethane-siloxane-copolymer reinforced by a polyester network to prevent excessive dilatation. On the inner surface an ENC monolayer is established before implantation by a cell culture procedure. The prosthesis displays compliance (13.2 +/- 3.0 x 10(-4) mmHg-1) comparable to native arteries. It is non-kinkable (minimal radius of curvature less than 5 mm). Burst resistance, remaining deformation, cut out force and tensile strength are superior to standard values. ENC-coverage in excess of 95% of the inner surface was produced in vitro using a lining procedure. The monolayer of confluent cells was demonstrated to consist of endothelial cells by their characteristic cobblestone morphology, the expression of factor VIII related antigen and the specific uptake of Dil-Ac-LDL. The unstimulated prostacyclin production was similar both in native veins as well as in lined prostheses. Antithrombogenicity of the endothelial cell lining was demonstrated in 24 h animal implants.

A compliant small-diameter vascular prosthesis lined with functional venous endothelial cells.

The long-term patency of small-diameter vascular grafts is still unsatisfactory. In contrast to native arteries, they are inelastic and lack active antithrombogenicity. To improve long-term patency, a new 4 mm internal diameter prosthesis was developed which is compliant and lined with functional endothelial cells (ENC). The wall of this prosthesis consists of a microporous polyurethane-siloxane copolymer reinforced with a polyester network. It displays compliance (13.2 x 10(-4) mmHg-1) comparable to native arteries, is nonkinkable (minimum radius of curvature = 5 mm), burst resistant, and easily suturable. Using a lining procedure, coverage of prostheses by ENC was in excess of 95%. The ENC populations were found to be highly pure (by factor VIII-related antigen, DilAcLDL uptake) and to produce about 0.3 ng prostacyclin per cm2. In vitro tests of shear stress resistance demonstrated that ENC monolayers on the new elastic prosthesis remain intact for 3 hr in physiologically pulsating culture medium (Vmax = 50 cm/sec). Lined prostheses implanted for 24 hours in mongrel dogs as an arteriovenous shunt demonstrated the antithrombogenicity of the cultured ENC. The results suggest that small-diameter vascular prostheses which are compliant, porous, and actively antithrombogenic are feasible.

[Development of small-diameter vascular prostheses covered with human endothelial cells: Possibilities and limits of in vitro-tests]

There is an increasing demand for small-diameter vascular prostheses for the replacement of arteriosclerotic coronary arteries. They may be replaced by autologous blood vessels, usually parts of the saphenous vein. Prostheses of synthetic materials and an inner diameter of less than 4 to 6 mm are unsatisfactory and, therefore, not implanted for coronary arteries. A substantial improvement is, however, expected for prostheses covered with human autologous endothelial cells. It has to be proved that this new type of vascular prostheses is an adequate replacement for small arteries. Tests of the new prosthesis should comprise cell and tissue compatibility of the synthetic materials as well as normal function of the endothelial cells. The aim of the present paper was to reduce the number of animal experiments in this development by establishing new in vitro tests for endothelial cell compatibility of synthetic materials and for the adherence of endothelial cell on the prosthesis. Physiologically haemodynamic streaming conditions are in vitro produced by self-constructed circulatory systems. First results demonstrate that physiologic shear stress is achieved. Limits and relevance of the in vitro tests are discussed in relation to animal experiments and clinical studies.