Most contemporary biomaterial designs for osteochondral regeneration utilize monolithic, biphasic, or even multiphasic constructs. We have introduced a microsphere-based approach to create a continuous gradient in both material composition and encapsulated growth factors. The gradients were fabricated by filling a cylindrical mold with opposing gradients of two different types of poly(D,L-lactic-co-glycolic acid) microspheres. The chondrogenic microspheres were loaded with transforming growth factor-β1, whereas the osteogenic microspheres contained bone morphogenetic protein-2 with or without nanophase hydroxyapatite. The gradient scaffolds (material gradient only, signal gradient only, or material/signal gradient combination) or blank control scaffolds were implanted in 3.5 mm-diameter defects in rabbit knees for 6 or 12 weeks. This is the first in vivo evaluation of these novel gradient scaffolds in the knee. The gross morphology, MRI, and histology indicated that the greatest extent of regeneration was achieved when both signal and material gradients were included together. This combination resulted in complete bone ingrowth, with an overlying cartilage layer with high glycosaminoglycan content, appropriate thickness, and integration with the surrounding cartilage and underlying bone. The results suggest that osteochondral regeneration may benefit from biomaterials that integrate a continuous gradient in both material composition and encapsulated growth factors.

Context Clinical efforts to repair damaged articular cartilage (AC) currently face major obstacles due to limited intrinsic repair capacity of the tissue and unsuccessful biological interventions. This highlights a need for better therapeutic strategies. Evidence Acquisition Relevant articles were identified through a search of the PubMed database from January 1956 to August 2014 using the following keywords: articular cartilage repair, stem cell, cartilage tissue-engineering, synovium, and NFAT. Evidence Synthesis In both animals and humans, AC defects that penetrate into the subchondral bone marrow are mainly filled with fibrocartilaginous tissue through the differentiation of bone marrow mesenchymal stem cells (MSCs), followed by degeneration of repaired cartilage and osteoarthritis. Cell therapy and tissue engineering techniques using culture-expanded chondrocytes, bone marrow MSCs, or pluripotent stem cells with chondroinductive growth factors may generate cartilaginous tissue in AC defects but do not form hyaline cartilage-based articular surface because repair cells often lose chondrogenic activity or result in chondrocyte hypertrophy. The new evidence that AC and synovium develop from the same pool of precursors with similar gene profiles and that synovium-derived chondrocytes have stable chondrogenic activity has promoted use of synovium as a new cell source for AC repair. The recent finding that NFAT1 and NFAT2 transcription factors inhibit chondrocyte hypertrophy and maintain metabolic balance in AC is a significant advance in the field of AC repair. Conclusions The use of synovial MSCs and discovery of upstream transcriptional regulators that help maintain the AC phenotype have opened new avenues to improve the outcome of AC regeneration.

molecules by a cell-based high-throughput screening (HTS) in human chondrocytes. Methods: To induce cellular senescence, immortalized human chondrocytes (TC28a2) were seeded (5000 cells/well) in 384 well plates, and treated with IL-6 (10 ng/ml) for 24 hours to induce cellular senescence or defective autophagy. Then, chondrocytes were incubated with Prestwick Chemical Library (1280 approved drugs with chemical and pharmacological diversity, as well as bioavailability and safety in humans) at 10 mM for 48 hours. Chondrocytes were incubated for 20 min with 4% formaldehyde and washed 3 times with PBS. Then, nuclei was stained with Hoechst 33342 (10 mM), while b-galactosidase subcellular structures and autophagy vacuoles were stained by using Imagene Green C12FDG substrate (10 mM) and Cyto-ID® (1 mM), respectively. Plates were imaged by using Operetta® High Content Screening (HCS) system in non-confocal mode using the 20x WD objective. For each well, 4 fields and 4 planes of bright field, Hoechst and fluorescein channels were obtained. Image analysis was performed by Harmony software. Relative intensity of C12FDG in cytoplasm and number of autophagosomes per area of cytoplasm were determined to quantitate b-galactosidase activity and autophagy flux respectively. Low signal of senescence was found for chloroquine (30 mM) and used as reference for anti-senescence activity. High signal of autophagy flux was found for Rapamycin (5 mM) and used as reference for proautophagy activity. Results: A primary screening was performed to identify anti-senescence compounds by measurement of senescence-associated bgalactosidase activity. From the total number of 1280 small molecules, 299 compounds with anti-senescence effects were identified by HTS. A secondary screening with the 299 compounds was performed for "cherry picking". Finally, 216 compounds were confirmed to have antisenescence activity. The anti-senescence compounds were analyzed by monitoring autophagy flux. 38 compounds with both anti-senescence and pro-autophagy effects were selected. For compound validation, a reporter cell line was generated by lentiviral transfection of pBABE-mCherry-EGFP-LC3 plasmid in TC28a2 chondrocytes. LC3 reporter chondrocytes can be used to determine autophagy activation by color changes upon stimulation. The preliminary results indicate a subset of compounds selectively induced LC3-associated autophagy activation. Conclusions: These observations provide a unique opportunity to study cartilage aging with the objective to explore the therapeutic potential of pharmacological prevention of chondrocyte senescence and autophagy as a strategy to slow or reverse aging-associated changes, prevent the onset of OA and provide benefits for its clinical management.