Maturation of Tissue Engineered Cartilage Implanted in Injured and Osteoarthritic Human Knees

Abstract

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.

Lumican inhibits collagen deposition in tissue engineered cartilage

“…The samples were then solubilised with trypsin and processed for complete biochemical analysis as recently described (Dickinson et al, 2005;Hollander et al, 2006). Briefly, samples were digested with 2.0 mg/ml TPCK-treated bovine pancreatic trypsin containing 1 mM iodoacetamide, 1 mM EDTA and 10 µg/ml pepstatin A.…”

“…All samples were boiled for 15 min at the end of incubation to destroy any remaining enzyme activity. The extracts were assayed by inhibition ELISA using a mouse IgG monoclonal antibody to denatured type II collagen, COL2-3/4 m, as previously described (Dickinson et al, 2005;Hollander et al, 2006). Peptide CB11B (CGKVGPSG-AP[OH] GEDGRP[OH]GPP[OH]GPQY) was synthesized using 9-fluorenyl-methoxycarbonyl chemistry, by Dr. A. Moir (Krebs Institute, Sheffield University, UK) and was used as a standard in all of the immunoassays.…”

“…Peptide CB11B (CGKVGPSG-AP[OH] GEDGRP[OH]GPP[OH]GPQY) was synthesized using 9-fluorenyl-methoxycarbonyl chemistry, by Dr. A. Moir (Krebs Institute, Sheffield University, UK) and was used as a standard in all of the immunoassays. Proteoglycan in the digests was measured as sulfated glycos-aminoglycan by colorimetric assay using dimethylmethylene blue (Sigma-Aldrich) as previously described (Dickinson et al, 2005;Hollander et al, 2006). Hydroxypoline as well as mature and immature collagen crosslinks were measured by amino acid analysis, as previously described (Dickinson et al, 2005;Hollander et al, 2006).…”

“…Proteoglycan in the digests was measured as sulfated glycos-aminoglycan by colorimetric assay using dimethylmethylene blue (Sigma-Aldrich) as previously described (Dickinson et al, 2005;Hollander et al, 2006). Hydroxypoline as well as mature and immature collagen crosslinks were measured by amino acid analysis, as previously described (Dickinson et al, 2005;Hollander et al, 2006). Briefly, samples were reduced with sodium borohydride to stabilize collagen crosslinks to heat and acid and then hydrolyzed in constant boiling hydrochloric acid.…”

An anti-inflammatory cell-free collagen/resveratrol scaffold for repairing osteochondral defects in rabbits

“…Then, an acute trauma defect in the rabbit joint was created to evaluate the anti-inflammatory effect of the Coll/PAA-Res scaffold. The acute trauma and foreign body implants may cause inflammation of the host in the defect region to elicit the gathering of polymorphonuclear cells, catabolic enzymes and IL-1b and TNF-a [43]. After in vivo implantation into the trauma defect, both Coll and Coll/Res scaffold can down-regulate inflammatory factors, such as the IL-1b, MMP-13 and COX-2, as reported by others [12,13,18].…”