The regeneration of damaged organs requires that engineered tissues mature when implanted at sites of injury or disease. We have used new analytic techniques to determine the extent of tissue regeneration after treatment of knee injury patients with a novel cartilage tissue engineering therapy and the effect of pre-existing osteoarthritis on the regeneration process. We treated 23 patients, with a mean age of 35.6 years, presenting with knee articular cartilage defects 1.5 cm2 to 11.25 cm2 (mean, 5.0 cm2) in area. Nine of the patients had X-ray evidence of osteoarthritis. Chondrocytes were isolated from healthy cartilage removed at arthroscopy. The cells were cultured for 14 days, seeded onto esterified hyaluronic acid scaffolds (Hyalograft C), and grown for a further 14 days before implantation. A second-look biopsy was taken from each patient after 6 to 30 months (mean, 16 months). After standard histological analysis, uncut tissue was further analyzed using a newly developed biochemical protocol involving digestion with trypsin and specific, quantitative assays for type II collagen, type I collagen, and proteoglycan, as well as mature and immature collagen crosslinks. Cartilage regeneration was observed as early as 11 months after implantation and in 10 out of 23 patients. Tissue regeneration was found even when implants were placed in joints that had already progressed to osteoarthrosis. Cartilage injuries can be effectively repaired using tissue engineering, and osteoarthritis does not inhibit the regeneration process.

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Reliable and reproducible outcome measures are essential to assess the efficacy of competing and novel tissue-engineering techniques. The aim of this study was to compare traditional histological analyses with newly developed quantitative biochemical outcome measures for the repair of articular cartilage. The production of a new anti-peptide antibody and the development and validation of a novel method for the extraction and immunoassay of type I collagen are described. The assay was used, in conjunction with existing assays for type II collagen and proteoglycans, to measure levels of the matrix components in repair tissue biopsies obtained from patients treated with the new tissue-engineering therapy Hyalograft C. Frozen sections cut from the same biopsies were stained for proteoglycans, using safranin O, and immunohistochemical analysis was used to assess type I and II collagen staining. Although there was general agreement between the extent of staining and the amounts of the three matrix components, there was a large degree of overlap in biochemical content between biopsies classified histologically on the basis of low, moderate, or abundant staining. The results demonstrate that histological grading of matrix protein abundance to classify repair cartilage as hyaline or fibrocartilagenous is often misleading. In addition, we demonstrate for the first time the ability to measure collagen cross-links in repair tissue biopsies and show that it can be used as a surrogate marker for tissue maturity. Our new range of biochemical techniques provides a standardized method to assess the quality of both engineered cartilage produced in vitro and repair tissue biopsies obtained after in vivo implantation.

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The use of CNS cultures for detection and quantification of neuronotrophic activity in the CNS has been analyzed. In particular the development, i.e., neurotransmitter uptake characteristics, and survival of dopaminergic and GABAergic neurons in fetal mouse (E13)- dissociated mesencephalic cells cultured in serum-free, hormone- supplemented medium have been assessed as a function of culture time and cell density. At all times, more than 98% of the cells were classified as neurons on the basis of immunocytochemical criteria. Results indicate that the increase of cell density in vitro significantly enhances specific high-affinity dopamine uptake per dopaminergic cell and cell survival. This effect is not limited to the dopaminergic cells and suggests that the development of neurotransmitter-related traits and cell survival are influenced by cell density-derived trophic signals. The above-mentioned cultures and parameters have also been used to detect neuronotrophic activity in adult mammalian brain extracts or more purified preparations. In particular, bovine striatal extracts contain activity capable of increasing high-affinity neurotransmitter uptake parameters and cell survival of at least the dopaminergic and GABAergic neurons present in the culture system. The neuronotrophic activity from bovine striatum has been partially purified and is associated with a fraction whose main component is a basic protein of approximately 14 kDa.