为了正常的体验网站,请在浏览器设置里面开启Javascript功能!
首页 > 自体脂肪干细胞和脂肪基质修复海绵体神经

自体脂肪干细胞和脂肪基质修复海绵体神经

2012-02-09 8页 pdf 2MB 26阅读

用户头像

is_241833

暂无简介

举报
自体脂肪干细胞和脂肪基质修复海绵体神经 Basic and Translational Science C ir W A ri Gu rraz M F. OB ed m vern ME dece rix e an N in RE o ace occ k, 9 ogen 3 months after grafting into CN injury rats, approximately twice as many cells were found on seeded ADMT as on unseeded ADMT. The seeded ADMT als...
自体脂肪干细胞和脂肪基质修复海绵体神经
Basic and Translational Science C ir W A ri Gu rraz M F. OB ed m vern ME dece rix e an N in RE o ace occ k, 9 ogen 3 months after grafting into CN injury rats, approximately twice as many cells were found on seeded ADMT as on unseeded ADMT. The seeded ADMT also had various degrees of S100 and CO A ex ex sig mo wa On po Thi BK) Eur Belg F sity Leu Egy San Min R Cal E-m S © All neuronal nitric oxide synthase expression, suggesting CN axonal ingrowth. Rats grafted with seeded ADMT overall had the best erectile function recovery when compared with those grafted with unseeded ADMT and those ungrafted. However, as a result of large variations, the differences did not reach statistic significance (P � .07). NCLUSIONS Grafting of ADSC-seeded matrix resulted in a substantial recovery of erectile function and improvement of histology. However, further refinement of the matrix architecture is needed to improve the success rate. UROLOGY 77: 1509.e1–1509.e8, 2011. © 2011 Elsevier Inc. dipose tissue is unique in its ability to expand and regress throughout life. In developed and developing countries, the overall adipose tissue pansion phase outpaces regression, resulting in an ever- panding obese society. To shed the “extra pounds,” a nificant number of patients undergo surgeries to re- ve the unwanted fat. However, the resulting “medical stes” are potential therapeutic treasures for 2 reasons. e is that the adipose tissue is a rich source of multi- tent mesenchymal cells called adipose-derived stem cells (ADSCs),1,2 and the other is that the adipose extracellular matrix (ECM) can be fabricated into acellular scaffolds for tissue engineering.3-5 In regard to ADSCs, these cells have been shown to differentiate into Schwann cells that formed myelin sheath on axons6 and have been used to seed nerve conduits for peripheral nerve repair.7-9 In addition, we have shown that ADSCs secrete neurotrophic factors that promote cavernous nerve (CN) regeneration.10,11 In regard to adipose ECM, the fabricated scaffolds have been shown to support adipogenic differentiation of ADSCs.5 In the pres- ent study, we combined ADSCs’ neuroregenerative poten- tial and adipose ECMs’ scaffold potential to construct nerve grafts for the repair of peripheral nerves; in this case, CN. The CNs innervate penile erectile tissue and are es- sential for erection. Because of their proximity to the prostate, bladder, and rectum, these nerves are often damaged during surgeries on these organs, resulting in erectile dysfunction (ED).12-14 In particular, prostate cancer is the most prevalent malignancy in men and is often treated by radical prostatectomy, causing damage to the CN and subsequent ED.15 To repair the damaged s study was supported by a grant from the Department of Defense (PC030775 to . MA has received research grants from the Belgische Vereniging voor Urologie, opean Society of Surgical Oncology, Federico Foundation and Bayer Healthcare ium. MA is a fellow of the Research Foundation (FWO) Flanders. rom the Knuppe Molecular Urology Laboratory, Department of Urology, Univer- of California, San Francisco, CA; Department of Urology, University Hospitals, ven, Leuven, Belgium; Urology and Nephrology Center, Mansoura University, pt; Division of Plastic Surgery, Department of Surgery, University of California, Francisco, CA; and Department of Urologic Surgery, University of Minnesota, neapolis, MN eprint requests: Ching-Shwun Lin, M.D., Department of Urology, University of ifornia, San Francisco, 533 Parnassus Avenue, Box 0738, CA 94143-0738. ail: clin@urology.ucsf.edu ubmitted: November 11, 2010, accepted (with revisions): December 22, 2010 2011 Elsevier Inc. 0090-4295/11/$36.00 1509.e1 avernous Nerve Repa ith Allogenic Adipose utologous Adipose-de iting Lin, Maarten Albersen, Ahmed M. Ha ary H. McGrath, Badrinath R. Konety, Tom JECTIVES To investigate whether adipose-deriv can facilitate the repair of injured ca THODS Human and rat adipose tissues were adipose tissue-derived acellular mat transplanted into subcutaneous spac ADMTs were then used to repair C erectile function. SULTS Adipose tissue can be fabricated int threads and sheets. Seeding of ADMT covered with ADSC and within 1 wee into the subcutaneous space of an all Rights Reserved Matrix and ved Stem Cells , Thomas M. Fandel, Maurice Garcia, Lue, and Ching-Shwun Lin atrix seeded with adipose-derived stem cells (ADSC) ous nerves (CNs). llularized and fabricated into various forms, including thread (ADMT). ADMT seeded with ADSC were d examined for signs of inflammation. ADSC-seeded jury in rats, followed by assessment of histology and llular matrices of various shapes and sizes, including urred rapidly: within 24 hours, 55% of the surface was 0% was covered. Transplantation of the seeded ADMT ic host showed no signs of inflammatory reaction. At doi:10.1016/j.urology.2010.12.076 CN, autologous nerve grafting with sural nerves has ini- tially been shown to result in an erectile function recov- ery ne lab ha bid rep fun ex do ica low vo an rat see va suc M Pr Ad rou acc on epi (C car tio De Th liq 37 an rpm the an inc aci slo inc RT 1% Pr Th int exa thr tiss exa pla of ing tiss Seeding of Acellular Matrix Thread with Adipose- Derived Stem Cells AD pla des �L add in 3 m add tor wit mi flu Su Ap and for 3 t the see wh wa inc tan Te exa Th Ed and sta exa an rey Gr Inj Th int W ma tai exp fro res lay the sur usi De Th rat W an 23 cav ne val IC pu 15 rate of 43%.16 However, the harvest of the sural rves causes donor site morbidity and requires the col- orative support of plastic surgeons. By contrast, the rvest of the genitofemoral nerves causes minimal mor- ity and can be done by the urological surgeon. CN air with genitofemoral nerve grafting has an erectile ction recovery rate of approximately 50%.17,18 On an perimental basis, grafting with decellularized CN from nor rats has also been attempted.19 However, the clin- l applicability of such a treatment procedure is rather because of the need to harvest the CN from human lunteers or cadavers. In the present study, we processed lipectomized human d rat adipose tissues into acellular matrices, seeded the matrix with allogenic rat ADSCs, and grafted the ded matrix into rats with transected CN. We observed riable but substantial recovery of erectile function after h grafting. ATERIAL AND METHODS ocurement of Adipose Tissues ipose tissue samples were obtained from patients during tine abdominoplasty after informed patient consent and ording to the guidelines set by our institution’s Committee Human Research. Rat adipose tissue was obtained from the didymal fat pad of 2-month-old male Sprague-Dawley rats harles River Laboratories, Wilmington, MA). All animal e, treatments, and procedures were approved by our Institu- nal Animal Care and Use Committee. cellularization of Adipose Tissue e adipose tissue in a 50-mL conical tube was dipped into uid nitrogen for 5 minutes and then immediately placed in a °C water bath for 10 minutes. After repeating this freeze- d-thaw step 2 more times, the tissue was centrifuged at 1500 for 10 minutes at room temperature (RT). After removing liquid fatty portion, the tissue was washed in 70% ethanol d phosphate-buffered saline (PBS) 3 times each. It was then ubated in 0.05% trypsin, 0.05% ethylenediaminetetraacetic d, 20 ng/mL DNase I, and 20 ng/mL RNase for 4 hours with w rotation at RT. After washing twice in PBS, the tissue was ubated in 0.1% sodium dodecyl sulfate (SDS) for 12 hours at . After washing 3 times in PBS, the tissue was incubated in penicillin and streptomycin for 12 hours at 4°C. eparation of Acellular Matrix e decellularized tissue just described was further processed o various forms of acellular matrices as follows. In one mple, the decellularized tissue was cut to�0.5� 1� 8–mm eads and dried for 12 hours for the preparation of adipose ue–derived acellular matrix thread (ADMT). In another mple, the decellularized tissue was homogenized in PBS, ced into a 10-cm plastic dish, and dried for the preparation adipose-derived acellular matrix sheet (ADMS). The result- ADMT or ADMS was further ultraviolet-sterilized inside a ue culture hood. 09.e2 MT was washed 3 times in PBS, dried at RT for 2 hours, and ced in a 6-well cell culture dish. ADSCs were isolated as cribed previously.20 Approximately 1 � 104 ADSCs in 200 of Dulbecco modified Eagle medium (DMEM) was then ed evenly to the ADMT. The culture dish was then placed a humidified 37°C incubator with 5% CO2. Four hours later, L of DMEM supplemented with 10% fetal bovine serum was ed to the ADMT, and the dish was returned to the incuba- . At 24 hours and 1 week, the seeded ADMT was stained h 1 �g/mL calcein AM (Invitrogen, Carlsbad, CA) for 10 nutes at 37°C and examined with a Nikon Eclipse E600 orescence microscope (Tokyo, Japan). bcutaneous Transplantation of Seeded Matrix proximately 5 � 104 ADSCs were grown to 60% confluence then labeled with 10 �M of 5-ethynyl-2-deoxyuridine (EdU) 24 h as previously described.21 The cells were then washed imes in PBS and then seeded onto an allogenic ADMT by aforementioned procedure. Forty-eight hours later, the ded ADMT was transplanted into an autologous host (from ich the ADSC was isolated). The transplantation procedure s as follows. With the rat under inhalant anesthesia, an ision was made in the lower abdomen to expose the subcu- eous space, into which the seeded ADMT was transplanted. n days later, the rat was sacrificed and the transplanted tissue mined by hematoxylin and eosin staining and microscopy. e transplanted ADMT was also retrieved and stained with an U detection cocktail that contained Alexa-594 (Invitrogen) with 4=,6-diamidino-2-phenylindole (DAPI, for nuclear ining, 1 �g/mL, Sigma-Aldrich). The stained matrix was mined with a Nikon Eclipse E600 fluorescence microscope d photographed with a Retiga 1300 Q-imaging camera (Sur- , BC, Canada). afting of Unseeded and Seeded Matrix in Nerve ury Rats irty 2-month-old male Sprague-Dawley rats were randomized o 3 equal groups: control, ADMT, and ADMT�ADSC. ith the rats under inhalant anesthesia, a midline incision was de in the lower abdomen and the periprostatic space con- ning the major pelvic ganglion (MPG) and the CNs were osed. A 5-mm–long nerve segment, starting 5 mm distal m the origin of the CN at the MPG was isolated and ected. In the control group, the abdomen was then closed in ers without further treatment. In the treatment groups, ei- r acellular ADMT or ADMT�ADSC construct was micro- gically interposed and fixed against the prostatic capsule ng 10-0 nylon sutures to bridge the nerve gap. termination of Erectile Function ree months after CN injury with and without grafting, all s were examined for erectile function by standard protocol.10 ith the rats under ketamine-midazolam anesthesia, the MPG d CN were exposed bilaterally via a midline laparotomy. A G butterfly needle was inserted into the proximal left corpus ernosum, filled with 250 U/mL heparin solution, and con- cted to a pressure transducer (Utah Medical Products, Mid- e, UT) for intracavernous pressure (ICP) measurement. The P was recorded at a rate of 10 samples/second using a com- ter with LabVIEW 6.0 software (National Instruments, Aus- UROLOGY 77 (6), 2011 tin, TX). A bipolar stainless-steel hook electrode was used to stimulate the origin of the CN at the MPG proximally from the ne me ato cur wit an per wa cal sys ne tio Im St Tis sat ho 30 Co sto at Sci im no inc 30 inc exp Cr Ba tio con ora inc gen sta res Q- St Da Di Tu po �. RE De De lat are un tra tra an ad ori represented approximately 35% of the original tissue mass. After drying under ambient conditions, the decel- lul sha niz arc Se It SC rip AD tri als mo all sho gra an fou to sur the see spa sig his tha lay all rem Gr To un rec see the sac tha wi de un an bo ma ces bu de ma its we As Ere by wh UR rve gap (control group) or the interposed construct (treat- nt groups). The electrode was connected to a signal gener- r (National Instruments) and a custom-built, constant- rent amplifier generating monophasic rectangular pulses h stimulus parameters of 1.5 mA, 20 Hz, pulse width 0.2 ms, d duration 50 seconds. Three stimulations were conducted side and the erection with the maximum increase in ICP s included for statistical analysis in each animal. For the culation of ICP increase/mean arterial pressure (MAP) ratio, temic blood pressure was recorded using a 23G butterfly edle inserted into the aorta at the level of the iliac bifurca- n. munohistochemical and Immunofluorescence aining sue samples were fixed in cold 2% formaldehyde and 0.002% urated picric acid in 0.1-M phosphate buffer, pH 8.0, for 4 urs followed by overnight immersion in buffer containing % sucrose. The specimens were then embedded in OCT mpound (Sakura Finetec United States, Torrance, CA) and red at –70°C until use. Fixed frozen tissue specimens were cut 7 �m, mounted onto SuperFrost-plus charged slides (Fisher entific, Pittsburgh, PA), and air dried for 5 minutes. For munostaining, the slides were placed in 0.3% H2O2/metha- l for 10 minutes, washed twice in PBS for 5 minutes, and ubated with 3% horse serum in PBS/0.3% Triton x-100 for minutes at RT. After draining this solution, the slides were ubated at RT with anti- neuronal nitric oxide synthase ression (nNOS) antibody (Santa Cruz Biotechnology, Santa uz, CA) or anti-S100 antibody (Leica Microsystems, Inc., nnockburn, IL) for 1.5 hours. After rinses in PBS, the sec- ns were incubated with fluorescein isothiocyanate– jugated secondary antibody (Jackson ImmunoResearch Lab- tories, West Grove, PA). After rinses in PBS, the slides were ubated with freshly made EdU detection cocktail (Invitro- ) for 30 minutes at RT followed by staining with DAPI. The ined tissue was examined with a Nikon Eclipse E600 fluo- cence microscope and photographed with a Retiga 1300 imaging camera. atistical Analysis ta were analyzed with Prism 4 (GraphPad Software, Inc., San ego, CA) using one-way analysis of variance followed by key-Kramer test for post hoc comparisons. All data are re- rted as mean � standard deviation. Significance was set at P 05. SULTS cellularization and Molding cellularization was performed with adipose tissue iso- ed from both human and rat. Data shown in Figure 1 for a tissue sample obtained from a patient who derwent elective abdominoplasty. After a 5-day ex- ction procedure, the original tissue (Fig. 1A) was nsformed into a loose matrix that was devoid of cells d cell debris (Fig. 1B). Qualitatively, the decellularized ipose tissue had similar dimensions and shape as the ginal tissue (Fig. 1A, B). Quantitatively, the matrix OLOGY 77 (6), 2011 arized adipose tissue could be cut into various sizes and pes, e.g., threads (Fig. 1C). It could also be homoge- ed and then molded into various three-dimensional hitectures; in this case, a sheet (Fig. 1D). eding of Matrix with ADSCs has been shown that nerve conduits seeded with AD- s produced better results than unseeded ones for pe- heral nerve repair.7-9,22 We thus examined how well SCs could attach and grow on acellular adipose ma- x; in this case, threads to be used as nerve conduits. We o considered that, for future clinical application, the st ideal matrix-based nerve conduit should combine ogenic matrix with autologous ADSCs. Thus, data wn in Figure 2 are for a nerve conduit that will be fted into a rat host, and it was constructed by seeding allogenic matrix with autologous ADSCs. Twenty- r hours after seeding, the autologous ADSCs adhered the allogenic matrix and covered about 55% of the face. One week later, the cells covered about 90% of matrix surface (Fig. 2A–C). As a pregrafting test, the ded matrix was transplanted into the subcutaneous ce of an allogenic host, and during a 10-day course, no n of inflammatory reaction was observed. Furthermore, tologic examination of the transplanted tissue showed t the matrix remained intact and was covered with a er of cells (Fig. 2D). Thus, autologous ADSC-seeded ogenic adipose matrix was tolerated by the host and ained intact for an extended period in vivo. afting of Seeded and Unseeded Matrix simulate postprostatectomy nerve injury, male rats derwent bilateral CN transection. These rats then eived no graft, unseeded adipose matrix, or ADSC- ded adipose matrix (Fig. 3A). Three months later, se rats were examined for erectile function and then rificed. Histologic examination of the grafts showed t both unseeded and seeded matrices were covered th numerous cells (Fig. 3B–F). On average, the cell nsity was nearly twice as high on seeded matrix as on seeded matrix (Fig. 3B). S100, a general nerve marker, d nNOS, an erectile nerve–specific nerve marker, were th absent and present on the unseeded and seeded trices, respectively (Fig. 3C–F). On the seeded matri- , S100 and nNOS were expressed at various degrees t mostly colocalized near the MPG, indicating various grees of CN axonal extension. One of the seeded trices contained an S100/nNOS-positive nerve across entire length, and several EdU-positive cells (ADSCs) re found along this nerve fiber (Fig. 3E, F). sessment of Erectile Function ctile function of the aforementioned rats was assessed measuring the ICP after electrostimulation of CN, ich was normalized against mean MAP. The results 1509.e3 sho tio be a c AD AD sig CO Au fun gra mo the tra ma sid no to are ma cau the Fig the his ho 15 wed that the ADMT group had better erectile func- n than the control, and the ADMT�ADSC group was tter than the ADMT group (Fig. 4). However, despite lear trend toward functional recovery, especially in the MT�ADSC group, the difference (P � .07 between MT�ADSC and control) did not reach statistical nificance because of large variations. MMENT tologous nerve grafting inevitably requires sacrificing a ctional nerve; in addition, the procurement of such a ft often requires a secondary surgery and causes donor site rbidity. By contrast, xenografting materials do not have se problems but carry the risks of host rejection and nsmitting zoonotic diseases.23 Thus, allogenic grafting y have the best potential but still requires careful con- eration. For example, allogenic graft procurement should t be harmful to the living donor and should be agreeable the family of the deceased donor. However, these criteria difficult to fulfill because few tissues are graft-quality terial that can be removed from a living person without sing harm. In addition, cultural confines may prevent wide adoption of cadaveric tissues. ure 1. Decellularization and fabrication of adipose tissue. protocol shown in Materials and Methods and transform tologic images of the adipose tissue before and after dec mogenized and then molded into a sheet (D). 09.e4 Adipose tissue is one of the rare tissues that can be rtially removed from a living person without causing rm. Its superficial location makes it more accessible than st other tissues, and its removal is an intervention desired many patients. As the volume of surgeries for obesity and st–weight loss contouring continues to increase, adipose ue is removed in an increasing rate. Thus, there is no ubt that there is an abundance of adipose tissue, and a ent study by Flynn5 has demonstrated its potential as an llular matrix for adipose tissue reconstruction. Specifi- ly, scanning electron microscopic examination of the llular adipose matrix identified regions of network-type llagen, consistent with the rich basement membrane mponent in adipose tissue. Thus, the decellularized adi- se tissue appears to be an ideal material for tissue recon- uction. The present study was initiated more than 1 year fore the publication of the aforementioned study by nn. Therefore, both the idea of developing an acellu- adipose matrix and the formulation of the decellular- tion protocol were conceived independently. In fact, h
/
本文档为【自体脂肪干细胞和脂肪基质修复海绵体神经】,请使用软件OFFICE或WPS软件打开。作品中的文字与图均可以修改和编辑, 图片更改请在作品中右键图片并更换,文字修改请直接点击文字进行修改,也可以新增和删除文档中的内容。
[版权声明] 本站所有资料为用户分享产生,若发现您的权利被侵害,请联系客服邮件isharekefu@iask.cn,我们尽快处理。 本作品所展示的图片、画像、字体、音乐的版权可能需版权方额外授权,请谨慎使用。 网站提供的党政主题相关内容(国旗、国徽、党徽..)目的在于配合国家政策宣传,仅限个人学习分享使用,禁止用于任何广告和商用目的。

历史搜索

    清空历史搜索