Influence of in vitro maturation of engineered cartilage on the outcome of osteochondral repair in a goat model.

This study was designed to determine if the maturation stage of engineered cartilage implanted in a goat model of cartilage injury influences the repair outcome. Goat engineered cartilage was generated from autologous chondrocytes cultured in hyaluronic acid scaffolds using 2 d, 2 weeks or 6 weeks of pre-culture and implanted above hydroxyapatite/hyaluronic acid sponges into osteochondral defects. Control defects were left untreated or treated with cell-free scaffolds. The quality of repair tissues was assessed 8 weeks or 8 months post implantation by histological staining, modified O'Driscoll scoring and biochemical analyses. Increasing pre-culture time resulted in progressive maturation of the grafts in vitro. After 8 weeks in vivo, the quality of the repair was not improved by any treatment. After 8 months, O'Driscoll histology scores indicated poor cartilage architecture for untreated (29.7 ± 1.6) and cell-free treated groups (24.3 ± 5.8). The histology score was improved when cellular grafts were implanted, with best scores observed for grafts pre-cultured for 2 weeks (16.3 ± 5.8). As compared to shorter pre-culture times, grafts cultured for 6 weeks (histology score: 22.3 ± 6.4) displayed highest type II/I collagen ratios but also inferior architecture of the surface and within the defect, as well as lower integration with native cartilage. Thus, pre-culture of engineered cartilage for 2 weeks achieved a suitable compromise between tissue maturity and structural/integrative properties of the repair tissue. The data demonstrate that the stage of development of engineered cartilage is an important parameter to be considered in designing cartilage repair strategies.

Tissue engineered intervertebral disc repair in the pig using injectable polymers.

The intervertebral disc (IVD) has a central nucleus pulposus (NP) able to resist compressive loads and an outer annulus fibrosus which withstands tension and gives mechanical strength. The tissue engineering of a disc substitute represents a challenge from mechanical and biological (nutrition and transport) points of view. Two hyaluronan-derived polymeric substitute materials, HYAFF 120, an ester and HYADD 3, an amide were injected into the NP of the lumbar spine of female pigs (11.1 +/- 1.0 Kg) in which a nucleotomy had also been performed. Homologous bone marrow stem cells, obtained from the bone marrow three weeks before spinal surgery, were included in the HYADD 3 material (1x 10(6) cells/ml). Two lumbar discs were operated in each animal. Control discs received a nucleotomy only. The animals were killed after 6 weeks and the lumbar spines recovered for histopathological study. Nucleotomy resulted in loss of normal IVD structure with narrowing, fibrous tissue replacement and disruption of the bony end-plates (4/4). By contrast, both HYAFF 120 (4/4) and HYADD 3 (4/4) treatment prevented this change. The injected discs had a central NP-like region which had a close similarity to the normal biconvex structure and contained viable chondrocytes forming matrix like that of normal disc.

Adhesion reduction after knee surgery in a rabbit model by Hyaloglide, a hyaluronan derivative gel.

A randomized controlled experimental trial was performed in a rabbit model of surgical adhesions to investigate the anti-adhesive effects of Hyaloglide, a highly viscous hyaluronan derivative absorbable gel after knee surgery. Twenty New Zealand white rabbits were prepared and randomly divided into two groups of 10 animals each. An intra-articular fibro-adhesive scar was created in the right knee joint of the hind paw of each rabbit using a standardized surgical procedure, and Hyaloglide was administered into the joint cavity of the knee at the end of intervention in the animals belonging to the treatment group. No anti-adhesive treatment was applied in the control group. Additionally, immobilization using a Kirschner wire was applied in order to increase the risk of adhesions. Six weeks after surgery the animals were euthanized and after removal of the immobilization system, adhesions were evaluated both macroscopically and histologically. Results of gross observations using a specific adhesion scoring system showed a significant reduction (p<0.01) of both incidence and severity of adhesions in the hyaluronan-treated group compared to the control group. Histologically, adhesions in the treated group were thinner with less collagenic fibers. In conclusion, Hyaloglide may be considered as a promising absorbable barrier for prevention of post-operative fibrotic adhesions after knee surgery.

Metabolic and histological analysis of mesenchymal stem cells grown in 3-D hyaluronan-based scaffolds.

Sheep mesenchymal stem cells (MSCs) were isolated and expanded using the principle of plastic adherence. Their identity as progenitor cells was confirmed by induction along the osteoblastic lineage using osteogenic supplements and observation of calcific deposits by von Kossa staining. MSCs were seeded onto two types of hyaluronan-based cylindrical scaffolds in high concentrations and cultured for varying time points up to three weeks. Culture medium was supplied using the following conditions: statically, on a shaker, by stirring with a magnetic stirrer or by perfusion in a tubular flow circuit. Total cell metabolism was assessed by MTT assay and the quality of cell coverage and matrix formation observed by SEM and histological analysis of thin sections of the constructs. Perfusion culture was established as the most appropriate culturing conditions, with cell metabolism increasing by approximately 300% over three weeks. The coverage of the scaffold surface was very good and the deposition of collagenous matrix was superior in these conditions compared to the, static and other dynamic culture conditions.

Heterogeneity in proliferative potential of ovine mesenchymal stem cell colonies.

Bone marrow biopsies were taken from the iliac crest of 28 individual sheep from three different breeds, ranging in age from 4 months to 8 years and mesenchymal stem cells (MSCs) isolated using selection due to plastic adherence. Cells were cultured in medium that had been selected for its effect on observed MSC proliferation, until populations of greater than 50 million had been obtained from each biopsy. The identity of the isolated cell populations as progenitors of the mesenchymal lineage was verified by deriving both osteoblastic and chondrocytic phenotypes when cultured in osteogenic and chondrogenic medium supplements, respectively. The rate of cell proliferation for each marrow biopsy was measured at each passage and the number of initial stem cells in each sample estimated. There was no statistically significant correlation between the age of the sheep and MSC proliferative potential, or age and estimated initial MSC number. There was no apparent significant difference between proliferation rate and sheep breed and colonies established from frozen cells grew at similar rates to pre-frozen cells. Counter intuitively, there appeared to be a negatively correlated trend between proliferation rate and MSC concentration in the samples. It is concluded that no initial descriptive statistics of the marrow biopsies can assist in estimating the proliferative potential, and therefore the timing of future surgeries, of MSCs sampled for the purposes of tissue engineering.

Developmental differences in identifying and discriminating CV syllables.

The purpose of this experiment was to determine whether age-related differences would be observed for identification and discrimination of synthesized, five-formant CV syllables among listeners who showed equal performance scores on a standard clinical test of speech understanding. A second question concerned the relations between performance on identification and discrimination tasks as a function of age. Two 13-item continua that varied in the place of articulation feature ([ba, da, ga]) were used; they differed primarily in the presence of absence of a 5-ms noise burst at the consonant onset. Digitized natural speech syllables were also employed in one experimental task. Strong age effects were obtained for the three tasks--identification of syllables, adaptive estimation of "ba, da-" and "da, ga-" boundaries, and discrimination. With the exception of one condition for six-year-olds, only adults showed significant differences between boundaries and just noticeable differences. Minimal differences were obtained in responses to stimuli with and without initial bursts. Across ages there were no significant differences in the subjects' ability to label the synthesized syllables as compared to the natural speech stimuli. Possible explanations for the observed developmental effects are discussed.