| ObjectiveIn bone tissue engineering a great deal of biodegraded and ab-sorbable material has been researched widely. The scaffold provides the base of cell adhesion, growth and differentiation, also is the structure that fixes growth factors in cell components and maintains three dimension spatial configuration to create new tissue. Although someone has narrated cell framework of acellular membrane, no ideal preparation of AECM or histomorphometry of bone lacunas have been reported in details. This experiment prepares ACEM by removing cell components and carries morphologic study to provide foundation for new natural supporting material of bone tissue engineering. Simultaneously, the components of AECM and remnant of detergent are determined.Methods1. Rabbit AECM preparation:After anesthetized by sodium pentobarbital, two lateral tibias ofrabbit were taken down. After have been incised into patches and gotten rid of periosteum, they were washed by PBS. Bone patches were put into jar containing Tris - HCI buffer and proteinase inhibitors. After at 4℃ constant temperature stirring, The samples were then immersed in Tris - HCI buffer containing 3% Triton x - 100 and proteinase inhibitors, After 4℃ constant temperature stirring, distilled water washed bone patches and the samples were digested by DNase and RNase at 31℃. Then the samples were immersed into Tris -HCl buffer containing 3% Triton x - 100 and washed by distilled water again. Finally the samples were reserved in Tris - HCl buffer at 4℃. When needed, AECM were put into Von Ebnel liquid for 30 days.2. HistologyFirst AECM was fixed by 10% formalin for 24 hours and paraffin embedded tissue, staining with hematoxylin and eosin, Mallory- Hei-denhain dyeing or Alcian blue. Under light microscope, AECM was examined3. Scanning electron microscopeAECM were prepared in 1. 25% glutaraldhyde and observed by Scanning electron microscope.4. Histomorphometry and parameters of bone lacunas Choosing 10 samples randomly and 10 eyeshot per sample staining by Mallory - Heidenhain rapid one - step dyeing, we measured density, perimeter, wide, length of bone lacunas and distance between lacunas under light microscope by LUZEX - F real - time image analyzing system.5.S-PAfter dimethylbenzene dewaxing AECM and dehydration through a graded series of alcohols, the expression of LN and FN were detec-ted by immunochemohistology. The samples of PBS exchanging antibodies were regarded blank comparison.6. Immuno - electron microscopic techniqueAfter S - P dyeing of AECM, fixation by osmic acid and hydra-tion through a graded series of acetone; Epon embedded samples; After ultrathin section the samples were observed under JEM - 1200EX Electron Microscope.7. Determination of Triton x - 100 by Rp - HPLCHigh - performance ehromatogram column ( Spherisorb c18 ) 5jxm,15mm x 6mm I. D. ; flow phase: acetonitrile /water( 1:1; v/v) ; flow - rate; Iml/min; room temperature: 40Tl; wave length: 280nm; sample-. 5 ul. Blank comparison was PBS. 0. 5ul,1ul,5ul Triton x -100 were put into 0. 5ml PBS buffer as comparison samples. Then join 0. 5ml methanol separately. Determine Triton x - 100 with Rp -HPLC. In 3min apex comes out. Weighing and rubbing AECM, PBS mixes with scraps. After centrifugation, take up - clear liquid and Determine Triton x - 100 with Rp - HPLCResults1. Morphologic studies and histomorphologic survey of AECM( 1 ) Bulk observationComparing with flesh tibia, appearance of AECM is more shallow and whiter. We also use different concentration of Triton x - 100 and time of cell extraction and Prove 30 days of cell extraction and 3% triton x -100 to be most effective. Cell component isnt found under light and electron microscope.( 2 ) HistologyHE, Mallory - Heidenhain rapid one - step dyeing,Alcian dyeing : Under light microscope, collagen fibers of arrange uniformly. It' s blankness in ellipse bone lacunas and there is no nucleus and other configuration.( 3 ) Osteocytes are in focus under Scanning... |
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