A novel technique for quantitative detection of mRNA expression in human bone derived cells cultured on biomaterials.
A nonisotopic and quantitative in situ hybridization technique was adapted to investigate the effect of biomaterials on the cellular expression of mRNA from human bone derived cells (HBD cells). HBD cells were cultured for 24 or 48 h on tissue culture plastic, alumina, and ion modified alumina. Osteocalcin, osteopontin, alkaline phosphatase, type I collagen alpha 1, and type I collagen alpha 2 mRNAs were quantified. Protein expression for collagen types I, III, and V, and for anti-human macrophages CD68 (DAKO-CD68, KP1) and CD68 (PG-M1), and anti-human myeloid/histiocyte antigen (DAKO-MAC 387) were determined immunohistochemically using monoclonal antibodies. At 24 and 48 h, levels of mRNA for alkaline phosphatase and osteonectin were greater than mRNA levels for osteopontin, osteocalcin, collagen type I alpha 1, and collagen type I alpha 2 for cells grown on the three substrata. However, at 48 h mRNA levels for alkaline phosphatase and osteonectin were significantly higher on the modified ceramic substrata relative to the native alumina. HBD cells appear to express CD68-KP1 when cultured for 24 h. The techniques provide a sensitive and reproducible assay to evaluate gene and protein expression of cells grown on different substrata.
Zreiqat
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Bone Biomaterial Unit, School of Pathology, Prince of Wales Hospital, University of New South Wales, Sydney, Australia.
Markovic
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Walsh
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Howlett
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D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000537:Aluminum Oxide; D015703:Antigens, CD / Q000032:analysis / Q000096:biosynthesis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis / Q000096:biosynthesis; D001672:Biocompatible Materials; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D003094:Collagen / Q000032:analysis / Q000096:biosynthesis; D000090602:Collagen Type I, alpha 1 Chain; D046508:Culture Techniques / Q000295:instrumentation / Q000379:methods; D015342:DNA Probes; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D015675:Osteocalcin / Q000096:biosynthesis; D053495:Osteopontin; D012333:RNA, Messenger / Q000096:biosynthesis*; D012795:Sialoglycoproteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D000097382:CD68 Molecule
D000469:Alkaline Phosphatase; D000537:Aluminum Oxide; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D001672:Biocompatible Materials; D003094:Collagen; D000090602:Collagen Type I, alpha 1 Chain; D015342:DNA Probes; D015675:Osteocalcin; D053495:Osteopontin; D012333:RNA, Messenger; D012795:Sialoglycoproteins; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C000716127:COL1A1 protein, human; C506895:SPP1 protein, human