Chin J Trauma, Ap ril 2006, Vol. 22, No. 4

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1 Chin J Trauma, Ap ril 2006, Vol. 22, No ,, (DBM ), 3,, DNA (ALP),,,, DNA ALP, 2, ; ; ; Exper im en ta l study on in vitro ma tura tion of tissue2eng ineered bone fabr ica ted w ith b ipha sic see2 d ing LΒ Ren2fa, ZHOU Q iang, XU J ian2zhong, LUO Fei, ZEN G L ing. D epartm ent of O rthopedics, Southw est Hospital, Third M ilitary M edical U niversity, Chongqing , China Corresponding author: ZHOU Q iang, Tel: AbstractO bjective To observe the effect of the tissue2engineered bone fabricated by biphasic seeding method on p roliferation, differentiation and osteogenic exp ression of osteoblast that is derived from human bone mesenchymal stem cells (MSCs) and seeded onto dem ineralized bone matrix (DBM ) scaf2 fold. M ethods Bone marrow was obtained from healthy adult volunteers and by using density gradient centrifugation, to isolate mesenchymal stem cells that were then cultured in vitro. The tissue2engineered bone fabricated with biphasic seeding ( experimental group) or normal seeding ( control group) in combi2 nation with DBM was in vitro cultured for three days to count the number of the seeding cells. The cell density, form and matrix secretion of the MSCs2DBM comp lex was observed with inverted phase contrast m icroscope and scanning electron m icroscope ( SEM ). A t days 5, 7, 10, 14 and 21 during culture, sam2 p les were exam ined for DNA content, alkaline phosphatase (ALP) activity, calcium content. Results Compared with the control group, biphasic seeding method obtained inoculation rate of nearly 100%. Lots of refractive sp indle cells could be seen in the pores of scaffold under the inverted phase contrast m icro2 scope in experimental group. Fissure could be seen in collagen matrix on account of cell contraction. Un2 der SEM, the tissue block fabricated by biphasic seeding method had smooth surface and small fissures, with cells embedded in collagen; while the tissue block fabricated by normal seeding showed cells, with less extracellular matrix. DNA content and ALP activity of the M SCs2DBM comp lex in experimental group were higher significantly from 5 days of culture compared with that of the M SCs2DBM comp lex in control group. Calcium measurements showed significant difference after two weeks of culture. Conclusion s B iphasic seeding is an effective method for fabricating tissue2engineered bone, can imp rove the seeding ef2 ficiency and facilitate in vitro maturation of the tissue2engineered bone. Key words Cell culture; Tissue culture; Stem cells; Tissue engineering :(2003AA205020) ; ( ) : , (,, ) :, :

2 Chin J Trauma, Ap ril 2006, Vol. 22, No. 4,,,,,, DMEM /F 12 ( Hyclone ), - C ( Sigma ),( TBD ), ( dem ineralized bone matrix, DBM ),( ortho - cresolphthalein com2 p lexone, OCPC) ( Sigma ) (mesenchymal stem cells,mscs) : Stute [ 1 ], 15 m l, 1MSCs, 50 m l, DMEM /F 12, /m l -, 8 10 m l5 m l 6 DBM, ,4 m l 50 l, MSCs,, r/m in 20 m in,,, 6DBM, 50 l, 20 m l DMEM /F m l % CO r/m in 5 m in,, 5 m in - DBM DMEM /F 12,,, 10 m in 10%, / cm 2 8 m l 15% DMEM /F m l, 37 5% CO 2 24 h MSCs, 48 h, 1 2 d 1, 10 80% 90%,, 25 g/l 4 m l 5 m in, 4 m l 15% DMEM /F 12, 50 m l, r/m in 5 m in,, DMEM /F 12, /cm m l MSCs: 3 MSCs, /cm 2 15 m l 10% mol/l 10 mmol/l - 50 mg/lc 250 m l, 37 5% CO 2,,5,80%, DBM : DBM 6 4 mm 4 mm 4 mm,,,37 DBM : 5 d 3,, 2 50 m l1 50 m l, 4 m l 5 g/l 0. 5 m l 10 DMEM /F m l,, ph 7. 4, ;2 h, 4 h, 8 m l,, :, : ( PBS) 3 30 g/l 48 h,,,

3 Chin J Trauma, Ap ril 2006, Vol. 22, No , OCPC 5 m l mol/l / (ALP) : m l, ( [ 2 ] : NaCl 800 mg, KCl 20 mg, NaH 2 PO 4 H 2 O 5 mg 10 m l, 137 mg, 50 mg, ph 7. 2, 100 m l, ) 1 m l, 37 6 h,, 5, 6 s/, 30 s1 000 r/m in 5 m in, ALP, 50 l a, b, c, (A ) ALP: ALP ( /100 m l) = / ( mg) 100 m l/0. 05 m l DNA : 0. 2 m l 1. 3 m l 200 ng/m l 33258, 455 nm, 350 nm (Da2 ta ) DNA, DNA :5, 7, 10, 14, 214, PBS 2 10 m l, 1. 6 m l 0. 5 mol/l, OCPC : 80 mg OCPC 75 m l 0. 5 m l 1 mol/l KOH 0. 5 m l 0. 5 mol/l : 5 m l (ph 11) 2 m l 8 - (5 g 100 m l 95% ) 88 m l 300 l 10 l, 96, 10 m in, 540 nm g/m l CaCl gx s, SPSS t P < m in,,, 4 h, 8 h,, 2, ( 1) 8 h, 2 ( 2) 2. 2,,, ( 3) ;,, ( 4) 1 14 d, d, 1003,,, 504,, ALP 15, ALP ( P < 0. 01),ALP, ALP DNA ( P < 0. 01),

4 Chin J Trauma, Ap ril 2006, Vol. 22, No. 4 DNA ( 6),,,,DNA, DNA, ALP 6 DNA DBM ( ) g/m l, 1,57 DBM, 10,, ( P > 0. 05), 2( P < 0. 01) 3,, S, [ 3 ],, 10% [ 4 ], [ 5 ],, -, - -, ( biphasic seeding) 100%, [ 6 ] MSCs DBM,( ) %, ( ) %,, Hase [ 7 ] 2, Dunn [ 8 ], 42 d,,,,,,,,,, 5ALP 14, ALP5 ALP,DNA,, DNA,, ALPDNA, ; ;, MSCs M izuno [ 9 ], MSCs,

5 Chin J Trauma, Ap ril 2006, Vol. 22, No ( g/m l, gx s) ( d) t P DGEA MSCs 2 1 MSCs 10, 2 MSCs,,, 4 1 Stute N, Holtz K, Bubenheim M, et al. Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use. Hematol, 2004, 32: ,.. 1. :, Exp 3 L i Y, Ma T, KnissDA, et al. Effects of filtration seeding on cell den2 sity, spatial distribution, and p roliferation in nonwoven fibrous matri2 ces. B iotechnol Prog, 2001, 17: ,,,.., 2004, 26: ,,,.., 2002, 22: ,,,.., 2005, 27: Hase T. A hybrid artificial liver system composed of p rimary cultured canine hepatocytes. Jpn J Surg, 1990, 91: Dunn JC, Yarmush ML, Koebe HG, et al. Hepatocytes function and extracellular matrix geometry: long - term culture in a sandwich con2 figuration. FASEB J, 1989, 3: M izuno M, Kuboki Y. O steoblast - related gene exp ression of bone marrow cells during the osteoblastic differentiation induced by type I collagen. J B iochem ( Tokoyo), 2001, 129: ( : ) ( : ) ( ) ( ESW T) ; ( 2), 8 ; (3) , : ( 1 ) ESW T ; (2) ESW T ; (3) ESW T ; (4) ESW T ; (5) ESW T ; ( 6) ES2 W T ; ( 7 ) ESW T; ( 8) ESW T ; (9) ESW T : (1) ESW T,; ( 4) 100 :800 /, ; (5) ( ),, : : 69 ;: : ; : ; : 010 ; ( 10) ESW T, , ,