脂肪组织工程种子细胞筛选的体外实验研究
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摘要
目的 培养人骨髓间充质干细胞(bone marrow mesenchymal stem cells,MSCs)和皮下脂肪来源前脂肪细胞(preadipocyte,Pra);筛选适合于脂肪组织工程的MSCs诱导成脂条件;观察和分析人MSCs成脂诱导分化过程;识别、鉴定由人MSCs诱导的前脂肪细胞(differentiated bone marrow mesenchymal stem cells,DBM);比较人Pra与DBM的增殖及成脂分化能力,从中为脂肪组织工程筛选出比较理想的种子细胞。并进一步分析筛选出的种子细胞在体外扩增阶段,不同代次细胞的老化程度,为脂肪组织工程选择合适代次的种子细胞提供部分实验依据。方法 选拟行矫形手术的成人(18～40ys)志愿者,于术中取切口皮下脂肪组织及髂嵴骨髓组织,体外进行人MSCs和Pra原代培养及传代扩增。结合成骨诱导分化(DMEM+10%胎牛血清+10~(-8)mol/L地塞米松+10mmol/LΒ—甘油磷酸钠+50ug/ml Vit—C,持续诱导3周)及免疫荧光流式细胞术(CD44、CD34抗体),对人MSCs进行鉴定。通过诱导成脂分化及S-100、脂肪酸结合蛋白(Fatty acid-binding protein,aP2)的免疫细胞化学方法鉴定人Pra。以DMEM、地塞米松、3-异丁基-1-甲基黄嘌呤、胰岛素、吲哚美辛复合诱导剂,体外诱导人MSCs成脂分化,观察分化过程形态学变化,有、无血清及细胞融合程度对成脂分化的影响。从形态学变化方面初步判定人MSCs诱导进入前
Objectives TO isolate and identify normal adults bone marrow mesenchymal stem cells(MSCs) as well as preadipocytes derived from adipose tissue subcutaneously(Pra). Investigate the adipogenetic effects of inducing conditions besides adipogenetic drugs(inducers),including serum-content or serum-free, cells in confluent or nonconfluent situation.Observe the morphological change during the course of human MSCs adipogenetic inducing, try to recognize a stage that MSCs has been induced into preadipocytes phase (differentiated bone marrow mesenchymal stem cells,DBM ) but not into terminal differentiation by morphological change preliminary ; identify DBM further at protein and molecular levels.In order to select more promising seeding cells of adipse tissue engineering,we compare DBM with Pra in proliferative activity and adipogenetic potentials. Evaluate the senescent ratio among the cells in different passages of selected seeding cells in vitro to get a proper passages for adipse tissue engineering. Methods Pra and MSCs isolated from the adults(18~40ys) volunteers who need orthopaedic operation without inflammatory or metabolic disease as well as tumor.Cells were proliferated in vitro. MSCs are
identified with FCM(CD44 and CD34) and osteogenetic differentiation potential by detect mineral nodule formation, alkaline phosphatase and collagen type I expression. Pra is identified with adipogenetic potential and expression of fatty acid binding protein(aP2).As adipogensis are concered, MSCs are induced with mixture inducers including dexamesasone, 3-isobutyl-l-methylxanine,insulin and indomethacin.We observe the morphological change during MSCs adipogensis differentiatiation under microscope, intracellular lipid formation confirmed by oil-red O staining. Evaluate the effect of serum and confluent extent on induced adipogensis by counting adipogenetic ratio and observing cells survival situation.Through morphological change ,we recognized preadipocyte stage (DBM)during MSCs adipogensis differention, confirmed by aP2 immunocytochemistry and A2COL6 RT-PCR semiquantitative analysis(both aP2 and A2COL6 are specific to preadipocyte according to reported literature). Then ,we compare the proliferative activity of DBM and Pra combination with MTT;cell cycle and apoptosis analysis with flow cytometry(FCM) ;and adipogenetic potentials through induced adipogenesis with inducing mixture showed above, in addition with serum(10% fetal bovine serum,FBS),and cells in a nonconfluent state for 10 days.The effort of adipogenesis evaluated with counting differentiated ratio and lipid quantity containing in the differentiated cells analysis through detect the
OD510 of the extraction from the intracellular stained oil-red (that reflect the lipid containing intracellular indirectly according to the literature) .Senescence of different passages of seeding cells detected with senescent associated (3- galactoside staining(SA-P-Gal). Mean senescent percents of different passages of seeding cells are evaluated under microscopy through counting SA-p-Gal positive staining cells among all cells in 5 visual fields(100X).Results FCM detect show that the cultured MSCs are negative for CD34 (<1%) , positive for CD44 (>90%).After inducing with dexamethasone,vit-C and disodium P-glycerophosphate for 3 weeks, MSCs show osteogenesis confirmed by expression of collagen type I , alkaline phosphatase and mineral noduleformation. During the regular culture ,MSCs have no adipogenesis spontaneously. Pra express aP2 intensively; S-100 weakly. Though Pra spontaneous adipogenesis is rare (<0.01%) , even be so in over confluent condition; adipogenetic inducing with mixture inducer mentioned above (serum containing, nonconfluent) results in almost thoroughly differentiation(>95%)o The growth speed of Pra cultured in vitro is faster than MSCs for average 4~6 passages precession. Serum-free in addition to mixture inducers results in majority MSCs dead. Adipogenetic inducers incorporate with 10%FBS and cells in confluent results in a low differentiation percent(17.59% ± 4.72%),while nonconfluent(50%~70% confluence) results in a very high percent(95.81%±2.15%). Morphological change start at 48~72 hours after inducing with cellular body broadening and dipolar shortening, but without intracellular change under microscopy. After inducing 3 ~ 6 days, there are some particles emerging under
cytomembrane around nuclei .These particles are proved to be lipid by oil-red Ostaining.After inducing for 6 days, particles in partial cells confluent into round lipid droplet with sparkling visualization.Within 6 ~ 10 days continuous inducing, most cells (>95% in nonconfluent ) contain size uniform, round lipid droplet.From 10th days to 3 weeks, either continue inducing or just feeding with ordinary DMEM medium(containing 10%FBS) results in further differentiation with lipid droplet confluent into large drop, or even lipid vacuole gradually ; thoroughly differentiated cells show a large round or lobulated shape, and easy detaching from culture bottle bottom and float on medium .Differentiation tend to be at terminal stage. Cells adipogenetic inducing from MSCs for 3,6 days all express aP2;while A2COL6 mRNA express intensively in the six-day inducing cells, weakly in three-day inducing cells,analysed semiquantatively according to GAPDH expression(quantity rate of A2COL6/ GAPDH is 1.09 in the former,0.48 in the latter).These results hint that MSCs induced adipogenticly for 6 days could processed into the preadipocyte stage.We call it temporally DBM. Pra have a more powerfully proliferative potential than MSCs and DBM from comparing the OD value of 570nm from MTT method. Cell cycle and apoptosis analysis from FCM show that the proportion of cells in quiescent stage (G0+G1) is 25.6% in Pra, 55.8% in MSCs, 70.4% in DBM;and proportion in proliferative stage ( S + G2 + M ) is respectively 74.4%,44.2%,29.6%; apoptosis is respectively 1.3%,1.5%,8.7% ? After 10-day adipogenetic inducing ,the mean differentiation ratio of DBM and Pra is respectively 96.93% + 3.0% and 94.15% ± 8.9% (statistics comparing ,p>0.05 ) .Quantity of lipid containing in the
differentiated cells analysis through detecting the mean OD510 of the extraction from the intracellular oil-red is 0.56 + 0.07 in DBM, 0.54 + 0.17 in Pra (statistics comparing ,p>0.05) . Senescent analysis with SA-P-Gal staining show that the mean senescent proportion of cells within 10 passages are not more than 0.01%;while the 10th, 12th ,15thand 18th passages are respectively 6.9%±0.43%, 9.1%±1.4%,16.7%±2.3%,and 43.9%±6.3%( statistics comparing respectively,p<0.05). The confluent time in half-quantity passage of Pra in passage 1,4,7,10,12 are 10~15,2~3,3~5,4~7,4~7 days respectively; while the 15th more than 15days,and the 18th passages have no intention to grow into confluent during our two-month culture. It is necessary to emphasize that cells in confluent may have a high positive(>80%) in SA-P-Gal staining even in low passages(<10 passages).Conclusions Human MSCs and Pra are easy to culture in vitro. Identify MSCs with fluorescent antibody CD34 and CD44 through FCM is an convenient and feasible method. Human MSCs under the regular culture have no spontaneous adipogenesis; while in proper inducing condition, resulting in almost thoroughly differentiation(>95%).Pra have a very low spontaneous adipogenesis,, confluent or over confluent condition can't improve spontaneous adipogenesis; while in proper inducing condition, resulting in almost thoroughly differentiation(>95%). MSCs in nonconfluent condition are easy to be induced adipogenesis. Serum-free in coordination with large dose adipogenetic inducers results in majority MSCs death. Human MSCs induced with adipogenetic inducers for 3 ~ 6 days could make the differentiation processing into preadipocyte(DBM). Pra have a more
powerful proliferation potential than MSCs and DBM; DBM show somehow negative proliferation phenomenon. Pra and DBM(even with MSCs)have the similar adipogenetic potential. Once MSCs adipogeneytic orientation, even in preadipocyte stage controlled by adipogenetic inducers in vitro ,will results in deprivation of proliferative potential. As the adipose tissue engineering is concerned, Pra still is a more proper seeding cells as to be considered its proliferative and adipogenetic potential than DBM and MSCs. The method of SA-(3-Gal staining is not suitable for evaluate the senescence cells in confluence. Pra with the 10th passages have a very low senescent proportion; while beyond 10-15 passages have a gradually increased senescent proportion. So, if one choose Pra as seeding cells for adipose tissue engineering ,of which passages should not beyond 10~15,as the senescence(indirectly reflect the proliferation) only is concerned.
Objectives TO isolate and identify normal adults bone marrow mesenchymal stem cells(MSCs) as well as preadipocytes derived from adipose tissue subcutaneously(Pra). Investigate the adipogenetic effects of inducing conditions besides adipogenetic drugs(inducers),including serum-content or serum-free, cells in confluent or nonconfluent situation.Observe the morphological change during the course of human MSCs adipogenetic inducing, try to recognize a stage that MSCs has been induced into preadipocytes phase (differentiated bone marrow mesenchymal stem cells,DBM ) but not into terminal differentiation by morphological change preliminary ; identify DBM further at protein and molecular levels.In order to select more promising seeding cells of adipse tissue engineering,we compare DBM with Pra in proliferative activity and adipogenetic potentials. Evaluate the senescent ratio among the cells in different passages of selected seeding cells in vitro to get a proper passages for adipse tissue engineering. Methods Pra and MSCs isolated from the adults(18~40ys) volunteers who need orthopaedic operation without inflammatory or metabolic disease as well as tumor.Cells were proliferated in vitro. MSCs are
identified with FCM(CD44 and CD34) and osteogenetic differentiation potential by detect mineral nodule formation, alkaline phosphatase and collagen type I expression. Pra is identified with adipogenetic potential and expression of fatty acid binding protein(aP2).As adipogensis are concered, MSCs are induced with mixture inducers including dexamesasone, 3-isobutyl-l-methylxanine,insulin and indomethacin.We observe the morphological change during MSCs adipogensis differentiatiation under microscope, intracellular lipid formation confirmed by oil-red O staining. Evaluate the effect of serum and confluent extent on induced adipogensis by counting adipogenetic ratio and observing cells survival situation.Through morphological change ,we recognized preadipocyte stage (DBM)during MSCs adipogensis differention, confirmed by aP2 immunocytochemistry and A2COL6 RT-PCR semiquantitative analysis(both aP2 and A2COL6 are specific to preadipocyte according to reported literature). Then ,we compare the proliferative activity of DBM and Pra combination with MTT;cell cycle and apoptosis analysis with flow cytometry(FCM) ;and adipogenetic potentials through induced adipogenesis with inducing mixture showed above, in addition with serum(10% fetal bovine serum,FBS),and cells in a nonconfluent state for 10 days.The effort of adipogenesis evaluated with counting differentiated ratio and lipid quantity containing in the differentiated cells analysis through detect the
OD510 of the extraction from the intracellular stained oil-red (that reflect the lipid containing intracellular indirectly according to the literature) .Senescence of different passages of seeding cells detected with senescent associated (3- galactoside staining(SA-P-Gal). Mean senescent percents of different passages of seeding cells are evaluated under microscopy through counting SA-p-Gal positive staining cells among all cells in 5 visual fields(100X).Results FCM detect show that the cultured MSCs are negative for CD34 (<1%) , positive for CD44 (>90%).After inducing with dexamethasone,vit-C and disodium P-glycerophosphate for 3 weeks, MSCs show osteogenesis confirmed by expression of collagen type I , alkaline phosphatase and mineral noduleformation. During the regular culture ,MSCs have no adipogenesis spontaneously. Pra express aP2 intensively; S-100 weakly. Though Pra spontaneous adipogenesis is rare (<0.01%) , even be so in over confluent condition; adipogenetic inducing with mixture inducer mentioned above (serum containing, nonconfluent) results in almost thoroughly differentiation(>95%)o The growth speed of Pra cultured in vitro is faster than MSCs for average 4~6 passages precession. Serum-free in addition to mixture inducers results in majority MSCs dead. Adipogenetic inducers incorporate with 10%FBS and cells in confluent results in a low differentiation percent(17.59% ± 4.72%),while nonconfluent(50%~70% confluence) results in a very high percent(95.81%±2.15%). Morphological change start at 48~72 hours after inducing with cellular body broadening and dipolar shortening, but without intracellular change under microscopy. After inducing 3 ~ 6 days, there are some particles emerging under
cytomembrane around nuclei .These particles are proved to be lipid by oil-red Ostaining.After inducing for 6 days, particles in partial cells confluent into round lipid droplet with sparkling visualization.Within 6 ~ 10 days continuous inducing, most cells (>95% in nonconfluent ) contain size uniform, round lipid droplet.From 10th days to 3 weeks, either continue inducing or just feeding with ordinary DMEM medium(containing 10%FBS) results in further differentiation with lipid droplet confluent into large drop, or even lipid vacuole gradually ; thoroughly differentiated cells show a large round or lobulated shape, and easy detaching from culture bottle bottom and float on medium .Differentiation tend to be at terminal stage. Cells adipogenetic inducing from MSCs for 3,6 days all express aP2;while A2COL6 mRNA express intensively in the six-day inducing cells, weakly in three-day inducing cells,analysed semiquantatively according to GAPDH expression(quantity rate of A2COL6/ GAPDH is 1.09 in the former,0.48 in the latter).These results hint that MSCs induced adipogenticly for 6 days could processed into the preadipocyte stage.We call it temporally DBM. Pra have a more powerfully proliferative potential than MSCs and DBM from comparing the OD value of 570nm from MTT method. Cell cycle and apoptosis analysis from FCM show that the proportion of cells in quiescent stage (G0+G1) is 25.6% in Pra, 55.8% in MSCs, 70.4% in DBM;and proportion in proliferative stage ( S + G2 + M ) is respectively 74.4%,44.2%,29.6%; apoptosis is respectively 1.3%,1.5%,8.7% ? After 10-day adipogenetic inducing ,the mean differentiation ratio of DBM and Pra is respectively 96.93% + 3.0% and 94.15% ± 8.9% (statistics comparing ,p>0.05 ) .Quantity of lipid containing in the
differentiated cells analysis through detecting the mean OD510 of the extraction from the intracellular oil-red is 0.56 + 0.07 in DBM, 0.54 + 0.17 in Pra (statistics comparing ,p>0.05) . Senescent analysis with SA-P-Gal staining show that the mean senescent proportion of cells within 10 passages are not more than 0.01%;while the 10th, 12th ,15thand 18th passages are respectively 6.9%±0.43%, 9.1%±1.4%,16.7%±2.3%,and 43.9%±6.3%( statistics comparing respectively,p<0.05). The confluent time in half-quantity passage of Pra in passage 1,4,7,10,12 are 10~15,2~3,3~5,4~7,4~7 days respectively; while the 15th more than 15days,and the 18th passages have no intention to grow into confluent during our two-month culture. It is necessary to emphasize that cells in confluent may have a high positive(>80%) in SA-P-Gal staining even in low passages(<10 passages).Conclusions Human MSCs and Pra are easy to culture in vitro. Identify MSCs with fluorescent antibody CD34 and CD44 through FCM is an convenient and feasible method. Human MSCs under the regular culture have no spontaneous adipogenesis; while in proper inducing condition, resulting in almost thoroughly differentiation(>95%).Pra have a very low spontaneous adipogenesis,, confluent or over confluent condition can't improve spontaneous adipogenesis; while in proper inducing condition, resulting in almost thoroughly differentiation(>95%). MSCs in nonconfluent condition are easy to be induced adipogenesis. Serum-free in coordination with large dose adipogenetic inducers results in majority MSCs death. Human MSCs induced with adipogenetic inducers for 3 ~ 6 days could make the differentiation processing into preadipocyte(DBM). Pra have a more
powerful proliferation potential than MSCs and DBM; DBM show somehow negative proliferation phenomenon. Pra and DBM(even with MSCs)have the similar adipogenetic potential. Once MSCs adipogeneytic orientation, even in preadipocyte stage controlled by adipogenetic inducers in vitro ,will results in deprivation of proliferative potential. As the adipose tissue engineering is concerned, Pra still is a more proper seeding cells as to be considered its proliferative and adipogenetic potential than DBM and MSCs. The method of SA-(3-Gal staining is not suitable for evaluate the senescence cells in confluence. Pra with the 10th passages have a very low senescent proportion; while beyond 10-15 passages have a gradually increased senescent proportion. So, if one choose Pra as seeding cells for adipose tissue engineering ,of which passages should not beyond 10~15,as the senescence(indirectly reflect the proliferation) only is concerned.
引文
1. Robb GL. (2000). Breast reconstruction. Internt Site: YourDoctor. com.
2. Bostwick J, Jones G. Why I chose autogenous tissue in breast reconstruction. Clin Plast Surg, 1994; 21: 165.
3. Fish J, Hammond D.The combination of expanders with autogenous tissue in breast reconstruction. Clin Plast Surg, 1994; 21: 309.
4. Katz AJ, Llull R, Hedrick MH, Futrell JW. Emerging approaches to the tissue engineering of fat Clin Plast Surg, 1999; 26: 587～603.
5. Ersek RA. Transplant of purified autologous fat: A 3-year follow-up disappointing. Plast Reconstr Surg, 1991; 87: 219～227.
6.孙庆仲,闫红艳,李正维,等.前脂肪细胞凝胶植入预防腰椎术后硬膜外腔瘢痕粘连的实验研究.中国矫形外科杂志,2003;11(14):970-972.
7. Ozgenta HE, Shenaq S, Spira M. Prefabrication of a secondary TRAM flap. Plast Reconstr Surg, 1995; 95: 441～449.
8. Billings E.Jr, May J. W. Jr. Historical review and present status of free fat graft autotransplantation in plastic and reconstructive surgery. Plast Reconstr Surg, 1989; 83: 368-381.
9. Cohen BE, Casso D, Whetstone M. Analysis of risk and aesthetics in breast reconstruction. Plast Reconstr Surg, 1992; 89: 840～843.
10. Kral AJ, Grandall MH. Development of a human adipocyte synthetic polymer scaffold. Plast Reconstr Surg, 1999; 104: 1732～1738.
11. Geloen A, Roy P E, Bukowecki L J, et al. Regression of white adipose tissue in diabetic rats. Am J physiol, 1989, 257: E547～553.
12. Aiehaud P, Grimaldi R, Negnel. Cellular and molecular aspects of adipose tissue development. Annu Rev Nutr 1992, 12: 207～233.
13. SC Butterwith. Molecular events in adipocyte development. Pharm Ther, 1994, 61: 399～411.
14. Solchaga LA, Dennis JE, Goldberg VM, et al. Hyaluronic acid-based polymers as cell carriers for tissue engineered repair of bone and cartilage. J Orthop Res, 1999.
15. Von-Heimbury Q, Zachariah S, Low A, et al. Influence of different biodegradable carriers in the in vivo behavior of human adipose precursor cells.Plast Reconstr Surg. 2001. 08(2): 411～420
16. Dani C, Doglio A, Bardon S, et al. Cloning and regulation of a mRNA specifically expressed in the preadipose state. J Biol Chem, 1989, 264: 10119～10125.
17. Green H, Kehinde O. Formation of normally differentiated subcutaneous fat pads by an established preadipose cell line. J Cell Physiol. 1979, 101: 169～172.
18. Von-Heimburg D.Zachariah S,Heschel I,et al.Human preadipocytcs seeded on freeze-dried couagen scaffolds investigated in vitro and in vivo. Biomaterials, 2001, 22: 429-438.
19. Patrick C,Chauvin P,Hobley I,et al.Preadipocyte Seeded PLGA Scaffolds for adipose tissue engineering.Tissue Eng. 1999. 5(2): 139-151.
20. Pitterenger MF, Mackay AM, Beck SC ,et al . Multilineage potential of adult hunan mesenchymal stem cell. Science 1999 , 284(5411):143~147.
21. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissue. Science 1997 , 276:71~74.
22. Seshi B, Kumar S, Sellers D. Human bone marrow stromal cell: coexpression of markers specific for multiple mesenchymal cell lineages. Blood Cells Mol Dis. 2000, 26(3): 234-246.
23. 23 Reddi A. Morphogensis and tissue engineering of bone and cartilage: inductive signals,stem cells, and biomimetic sbiomaterals. Tissue-Eng, 2000:6(4):351~359.
24. Nguyen A, Pasyk KA, Bouvier TN, et al. Comparative study of survival of autologous adipose tissue taken and transplanted by different techniches. Plast Reconstr Surg, 1990; 85:378-386.
25. Kononas TC, Bucky LP, Hurley C, et al. The fates of suctioned and surgically removed fat after reimplantation for
soft-tissue augmentation: A volumetric and histological study in the rabbit. Plast Reconstr Surg,1993;91.:763~768.
26. Hang-Fu L, Marmolya G, Feiglin DH. Liposuction fat-fillant implant for breast augmentation and reconstruction. Aesthetic Plast surg, 1995;19:427~437.
2. Bostwick J, Jones G. Why I chose autogenous tissue in breast reconstruction. Clin Plast Surg, 1994; 21: 165.
3. Fish J, Hammond D.The combination of expanders with autogenous tissue in breast reconstruction. Clin Plast Surg, 1994; 21: 309.
4. Katz AJ, Llull R, Hedrick MH, Futrell JW. Emerging approaches to the tissue engineering of fat Clin Plast Surg, 1999; 26: 587～603.
5. Ersek RA. Transplant of purified autologous fat: A 3-year follow-up disappointing. Plast Reconstr Surg, 1991; 87: 219～227.
6.孙庆仲,闫红艳,李正维,等.前脂肪细胞凝胶植入预防腰椎术后硬膜外腔瘢痕粘连的实验研究.中国矫形外科杂志,2003;11(14):970-972.
7. Ozgenta HE, Shenaq S, Spira M. Prefabrication of a secondary TRAM flap. Plast Reconstr Surg, 1995; 95: 441～449.
8. Billings E.Jr, May J. W. Jr. Historical review and present status of free fat graft autotransplantation in plastic and reconstructive surgery. Plast Reconstr Surg, 1989; 83: 368-381.
9. Cohen BE, Casso D, Whetstone M. Analysis of risk and aesthetics in breast reconstruction. Plast Reconstr Surg, 1992; 89: 840～843.
10. Kral AJ, Grandall MH. Development of a human adipocyte synthetic polymer scaffold. Plast Reconstr Surg, 1999; 104: 1732～1738.
11. Geloen A, Roy P E, Bukowecki L J, et al. Regression of white adipose tissue in diabetic rats. Am J physiol, 1989, 257: E547～553.
12. Aiehaud P, Grimaldi R, Negnel. Cellular and molecular aspects of adipose tissue development. Annu Rev Nutr 1992, 12: 207～233.
13. SC Butterwith. Molecular events in adipocyte development. Pharm Ther, 1994, 61: 399～411.
14. Solchaga LA, Dennis JE, Goldberg VM, et al. Hyaluronic acid-based polymers as cell carriers for tissue engineered repair of bone and cartilage. J Orthop Res, 1999.
15. Von-Heimbury Q, Zachariah S, Low A, et al. Influence of different biodegradable carriers in the in vivo behavior of human adipose precursor cells.Plast Reconstr Surg. 2001. 08(2): 411～420
16. Dani C, Doglio A, Bardon S, et al. Cloning and regulation of a mRNA specifically expressed in the preadipose state. J Biol Chem, 1989, 264: 10119～10125.
17. Green H, Kehinde O. Formation of normally differentiated subcutaneous fat pads by an established preadipose cell line. J Cell Physiol. 1979, 101: 169～172.
18. Von-Heimburg D.Zachariah S,Heschel I,et al.Human preadipocytcs seeded on freeze-dried couagen scaffolds investigated in vitro and in vivo. Biomaterials, 2001, 22: 429-438.
19. Patrick C,Chauvin P,Hobley I,et al.Preadipocyte Seeded PLGA Scaffolds for adipose tissue engineering.Tissue Eng. 1999. 5(2): 139-151.
20. Pitterenger MF, Mackay AM, Beck SC ,et al . Multilineage potential of adult hunan mesenchymal stem cell. Science 1999 , 284(5411):143~147.
21. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissue. Science 1997 , 276:71~74.
22. Seshi B, Kumar S, Sellers D. Human bone marrow stromal cell: coexpression of markers specific for multiple mesenchymal cell lineages. Blood Cells Mol Dis. 2000, 26(3): 234-246.
23. 23 Reddi A. Morphogensis and tissue engineering of bone and cartilage: inductive signals,stem cells, and biomimetic sbiomaterals. Tissue-Eng, 2000:6(4):351~359.
24. Nguyen A, Pasyk KA, Bouvier TN, et al. Comparative study of survival of autologous adipose tissue taken and transplanted by different techniches. Plast Reconstr Surg, 1990; 85:378-386.
25. Kononas TC, Bucky LP, Hurley C, et al. The fates of suctioned and surgically removed fat after reimplantation for
soft-tissue augmentation: A volumetric and histological study in the rabbit. Plast Reconstr Surg,1993;91.:763~768.
26. Hang-Fu L, Marmolya G, Feiglin DH. Liposuction fat-fillant implant for breast augmentation and reconstruction. Aesthetic Plast surg, 1995;19:427~437.