Irreversible electroporation and electrochemotherapy in oncology: State of the art

Thermal tumor ablation techniques including radiofrequency, microwave, LASER, high-intensity focused ultrasound and cryoablation are routinely used to treated liver, kidney, bone, or lung tumors. However, all these techniques are thermal and can therefore be affected by heat sink effect, which can lead to incomplete ablation, and thermal injuries of non-targeted tissues are possible. Under certain conditions, high voltage pulsed electric field can induce formation of pores in the cell membrane. This phenomenon, called electropermeabilization, is also known as “electroporation”. Under certain conditions, electroporation can be irreversible, leading to cell death. Irreversible electroporation has demonstrated efficacy for the treatment of liver and prostate cancers, whereas data are scarce regarding pancreatic and renal cancers. During reversible electroporation, transient cell permeability can be used to introduce cytotoxic drugs into tumor cells (commonly bleomycin or cisplatin). Reversible electroporation used in conjunction with cytotoxic drugs shows promise in terms of oncological response, particularly for solid cutaneous and subcutaneous tumors such as melanoma. Irreversible and reversible electroporation are both not thermal ablation techniques and therefore open a new promising horizon for tumor ablation.     

Enhanced Bone Bonding of the Hydroxyapatite/β‐Tricalcium Phosphate Composite by Electrical Polarization in Rabbit Long Bone

A review of the osteogenic cell activity and new bone growth in the regions bordering negatively charged surfaces of polarized Hydroxyapatite/β‐tricalcium phosphate (HA/TCP) composites implanted in the long bone in rabbits was conducted. Polarized and non‐polarized HA/TCP specimens were implanted into the right and left femoral condyle, respectively (each n = 10). After 3 and 6 weeks, five rabbits were sacrificed in each group, and histological analysis was administered. Large cuboidal‐shaped osteoblastic cells were predominantly observed lining the newly formed bone on the negatively charged surface (N‐surface) in the polarized HA/TCP implants. The TRAP‐positive multinucleated cells were observed extensively in the newly formed bone on the N‐surfaces compared with the 0‐surface and adhered directly to the HA/TCP composite. The bone area (B.Ar) value, newly formed bone area contacting the implant, and contact length (C.Le) value, percentage length of newly formed bone directly attaching to the implant, on both the 0‐ and N‐surface increased significantly with time in each group. Both the B.Ar and C.Le value on the N‐surface were significantly greater than those on the 0‐surface after 3 and 6 weeks. The number of TRAP‐positive cells/total length value on the N‐surface was significantly greater than that on the 0‐surface after 3 and 6 weeks postoperatively. It is hypothesized that electrical charge acquired by electrical polarization treatment may modify the biochemical and biophysical processes of the osteogenic cells, resulting in enhanced new bone formation and direct bonding between the recipient bone and implants.     

The effect of low-frequency electrical fields on osteogenesis.

An in vivo animal model of disuse osteopenia was used to determine the osteogenic potential of specific components of electrical fields. The ability of a complex pulsed electrical field to inhibit loss of bone was compared with the remodeling response generated by extremely low-power, low-frequency (fifteen, seventy-five, and 150-hertz) sinusoidal electrical fields. The left ulnae of thirty adult male turkeys were functionally isolated by creation of distal and proximal epiphyseal osteotomies and then were exposed, for one hour each day, to an electrical field that had been induced exogenously by means of magnetic induction. After a fifty-six-day protocol, the remodeling response was quantified by a comparison of the cross-sectional area of the mid-part of the diaphysis of the functionally isolated ulna with that of the intact contralateral ulna. Disuse resulted in a 13 per cent mean loss of osseous tissue, which was not significantly different than the 10 per cent loss that was caused by disuse treated with inactive coils. Exposure to the pulsed electrical fields prevented this osteopenia and stimulated a 10 per cent mean increase in the bone area. The osteogenic influence of the sinusoidal electrical fields was strongly dependent on the frequency; the 150, seventy-five, and fifteen-hertz sinusoidal fields, respectively, generated a -3 per cent, + 5 per cent, and + 20 per cent mean change in the bone area. These results suggest a tissue sensitivity that is specific to very low-frequency sinusoidal electrical fields, and they imply that the induced electrical fields need not have complex waveforms to be osteogenic. Since the frequency and intensity range of the sinusoidal fields producing the greatest osteogenic response are similar to the levels produced intrinsically by normal functional activity, these results support the hypothesis that electricity plays a role in the retention of the normal remodeling balance within mature bone.     

Ectopic bone formation by electroporatic transfer of bone morphogenetic protein-4 gene

Orthopedic surgeons have long awaited the clinical application of bone morphogenetic proteins (BMPs) for bone regeneration. However, such possible applications involving proteins or genes transferred with virus vectors have encountered many problems, including high cost, immunological reactions, viral infection, etc. We adopted a new gene transfer system of in vivo electroporation with a plasmid expression vector. A solution of plasmid DNA containing mouse BMP-4 (pMiw-BMP4) was injected into the gastrocnemius of BALB/cA mice, and electric pulses were applied through paired-needle electrodes inserted percutaneously. As a control plasmid, LacZ-containing plasmid (pMiwZ) was transferred by electroporation. A control group in which pMiw-BMP4 was injected and not electroporated was also introduced. In these groups, the gastrocnemius was harvested at 7, 14, 21, and 28 days after electroporation (n = 6 in each). As nonplasmid controls, electroporation with saline injection (n = 6), electroporation without injection (n = 6), and saline injection only (n = 3) were prepared. In these groups, the mice were killed 7 days after experimentation. Ectopic calcification or ossification was examined by histology as well as soft X-ray. In all electroporated groups (pMiwZ, pMiw-BMP4, saline injection, and without injection), dystrophic calcification of muscle bundles and infiltration of mesenchymal cells were observed histologically. Ectopic bone formation was observed only in the pMiw-BMP4 electroporation group. At 7 days after pMiw-BMP4 electroporation, extracellular eosinophilic matrix in a collection of mesenchymal cells was observed. Between 14 and 28 days after electroporation, ectopic bone was observed in 44% of mice, and bone marrow-like cells observed in 22%. The newly formed bone was woven. Injection of pMiw-BMP4 or saline induced neither calcification nor ossification. Our findings indicate that BMP-4 transferred by electroporation can induce in vivo and in situ ectopic bone formation in skeletal muscle.     

Evaluation of osteogenic/chondrogenic cellular proliferation and differentiation in the xenogeneic periosteal graft

To determine whether grafted young periosteum can induce new bone formation in elderly patients, this preliminary study evaluated cell proliferation and differentiation in xenogeneic periosteal grafts in old rats radiographically, histologically, and immunohistochemically. Periosteum harvested from the tibia of young Japanese white rabbits were grafted into old Sprague-Dawley rats with or without administration of 1.0 mg per kilogram per day immunosuppressant FK506. Autogenous old periosteal tissue grafts were also evaluated as a control. Grafted tissue was extirpated after 7, 14, 21, and 45 days. In the xenogeneic group, proliferative cell nuclear antigen-positive cells were observed 7 days after surgery, which differentiated into chondroblasts with bone morphogenetic protein-2 expression and finally formed cartilage by 14 days. Endochondral ossification was observed at 21 days, and bone replacement was completed by 45 days. No osteogenic cell activity was observed in the two other groups. Xenogeneic young periosteum thus maintained its osteogenic/chondrogenic potentiality in older rats.     

Osteogenic activity of growth cartilage examined by implanting decalcified or devitalized ribs and costal cartilage zone, and living growth cartilage cells.

The formation of cartilage prior to bone in an endochondral bone formation process suggests that some osteogenic factors exist in the cartilage. This osteogenic activity of cartilage or cartilage cells was examined by implanting ribs and costal cartilage zone into a subcutaneous pocket of abdominal wall, or the growth cartilage (GC) cells into a peritoneal cavity. Rib segments of young (four-week-old) rabbits and rats were decalcified (DCed) in 0.6 N HCl for 24 h at 4 degrees C, or devitalized (DVed) by freezing/thawing and submerged in water for 48 h at 4 degrees C. These specimens were implanted into the subcutaneous pocket of abdominal wall of eight-week-old rabbits and rats. Both the DCed ribs and the DVed ones formed bone at costal GC zone after four to six weeks. Costal cartilage zone of young rabbits and rats was DCed or DVed as stated above, and implanted into the subcutaneous pocket of abdominal wall of eight-week-old rabbits and rats. The DVed costal cartilage zone specimens formed bone after three to four weeks, but the DCed ones did not, even after eight weeks. GC cells were dissociated enzymatically from rat costal GC zone, centrifuged to be made into pellets (5 x 10(5) cells/pellet), and implanted into the peritoneal cavity of eight-week-old syngeneic rats. After four weeks, implanted GC cells formed bone. These results suggest that there are some osteogenic factors in the GC zone produced by GC cells, whose activity is lost by decalcification, and which are different from known BMPs.     

Rapamycin as an inhibitor of osteogenic differentiation in bone marrow-derived mesenchymal stem cells

Background An autograft of cultured bone marrow-derived mesenchymal stem cells has already been used in clinical practice. In those patients whose bone marrow cannot be used, a cell allograft with the use of immunosuppressant drugs will be an option in the future. However, little is known about the effects of immunosuppressant drugs on mesenchymal stem cells. This study assessed the effects of immunosuppressant drugs on osteogenic differentiation of mesenchymal stem cells and analyzed the manner in which immunosuppressant drugs modulate the osteogenic effect of dexamethasone. Methods Rat bone marrow cells were cultured with or without dexamethasone as an osteogenic supplement. In each experimental group, one of three immunosuppressants (rapamycin, cyclosporine A, or FK506) was added. As a control, cells were cultured without immunosuppressants. Histologically, mineralization was assessed by alizarin red S staining and phase-contrast microscopy. Biochemically, alkaline phosphatase activity, calcium content, and osteocalcin content were assessed. Results On histological analysis, no mineralized nodules were seen on alizarin red S staining or phase-contrast microscopy in the groups not treated with dexamethasone, except in the group that was treated with FK506. Mineralized nodules were seen in the groups treated with dexamethasone, except in the group that was treated with rapamycin. On biochemical analysis, it was found that, compared to the control group, rapamycin reduced alkaline phosphatase activity and the calcium content of mesenchymal stem cells; FK506 increased alkaline phosphatase activity, calcium content, and osteocalcin content; and cyclosporine A had negligible effects. Dexamethasone increased alkaline phosphatase activity, calcium content, and osteocalcin content, but these effects were decreased by rapamycin. Conclusions Rapamycin did not have an osteogenic effect on mesenchymal stem cells, but inhibited the effect of osteogenic differentiation induced by dexamethasone. In contrast, FK506 had an osteogenic effect on mesenchymal stem cells. Therefore, FK506 might be more useful than rapamycin in allogeneic transplantation of mesenchymal stem cells.     

Osteopromotion for cranioplasty. An experimental study in rats using a membrane technique

Various techniques for treatment of large cranial defects have been reported, but the use of alloplastic materials still seems to predominate. The authors have applied and explored a new approach for bone repair which appears promising, even for use in less osteogenic environments such as the adult calvaria. Seventy-two adult Sprague-Dawley rats each received bilateral 8-mm trephine defects in the temporoparietal area; this defect size precludes spontaneous osseous healing during the lifetime of the animal. Five surgical procedures, employing various alternatives of biologically inert expanded polytetrafluoroethylene membrane positioning and intramembranous bone-chip implantation, were performed and compared to control defects. Slight improvement of bone regeneration was demonstrated with subperiosteal ectocranial and endocranial membranes, alone or in combination, and with bone chips alone or in combination with an outer or inner membrane. Virtually complete bone healing was observed in animals receiving both an outer and an inner membrane with interpositioned bone chips. The latter appeared to function primarily as space-holders by keeping the membranes separated throughout the defect. Consequently, this technique seems to significantly promote bone repair by excluding soft-tissue components from the bone-healing site.     

电穿孔介导的基因治疗对下颌骨牵引新骨生成影响的组织形态计量学研究

目的：探索电穿孔介导的基因治疗对下颌骨牵引成骨（distraction osteogenesis,DO）过程中牵引间隙新骨生成的影响。方法：采用新西兰大白兔50只,双侧下颌骨截骨安装牵引器后3天开始牵引,每天0.8mm,连续牵引7天后,将实验动物分为5组,每组10只：A组：在牵引区注射2μg（0.1μg/μl）pIRES-hVEGF165-hBMP2;B组：在牵引区注射2μg（0.1μg/μl）pIRES-hBMP2。C组：在牵引区注射2μg（0.1μg/μl）pIRES-hVEGF165;D组：在牵引区注射2μg（0.1μg/μl）空质粒pIRES;E组：在牵引区注射相同剂量的生理盐水。5组实验动物均施加电穿孔刺激。各组分别于固定期第2、4、8周取材行组织学检查和形态计量学分析,测定牵引区新生骨量和新生骨小梁宽度。结果：A、B、C组新生骨量及新生骨小梁宽度明显高于D、E组（P〈0.01）。且A组明显高于B、C组,但B、C组,D、E组间无显著性差异。结论：电脉冲介导的pIRES-hVEGF165-hBMP2重组质粒体内转染可能实现成骨与血供的联合重建,使单一生长因子的效应放大,促进牵引区新骨的生成。     

家兔作为胎龄期骨髓间充质干细胞动物模型的实验研究

目的建立以家兔为对象的胎龄期BMSC研究动物模型。方法通过人工授精方法,获得孕期3周的孕兔4只,行剖腹产术取出胎兔20只,冲取胎兔骨髓,以贴壁培养法进行体外扩增培养。测量第3代胎兔BMSC的生长曲线,克隆形成率,并进行成骨、成脂及成软骨诱导分化。另外,将原代细胞冻存30 d后复苏,测量其第3代生长曲线,对冻存前后增殖能力的变化进行观察。扫描电镜观察胎兔BMSC与β-TCP形成细胞材料复合物的体外形态。将BMSCs-β-TCP复合物植入裸鼠皮下,于术后1、3、6个月分别取材,行HE、VG、Masson染色观察。结果胎兔来源的BMSC于倒置相差显微镜下观察,细胞饱满均匀,呈梭形或倒三角形状;传代后各代细胞形态未发生明显变化,生长曲线相差不大;成骨、成脂以及成软骨诱导观察到钙结节、脂肪空泡、黏多糖。冻存30 d后复苏,其第3代生长曲线与冻存前相比未见明显变化。电镜下观察,与β-TCP复合7 d后,细胞能均匀紧密贴合于材料上,伸展良好分布均匀。植入裸鼠皮不同时间点取材行HE、VG、Masson染色,均显示有新生骨组织生成。结论以家兔为研究动物模型,可以在胎龄期获取并分离得到BMSC,并可在异位构建组织工程骨,是间充质干细胞动物研究的新选择。     

重建端粒酶活性的正常人成纤维细胞的成骨潜能研究

目的　通过重建端粒酶活性延长人成纤维细胞寿命 ,并对其成骨潜能进行研究 ,为解决组织工程骨修复种子细胞老化问题提供实验依据。方法　将人端粒酶催化亚基 (h TERT)基因用电穿孔法导入正常人原代成纤维细胞 ,用 TRAP- PCR检测细胞端粒酶活性 ,用β-半乳糖苷酶活性测定评价细胞衰老情况。在此基础上用骨形成蛋白(BMP- 2 )和肿瘤坏死因子 -α(TNF-α)联合诱导已重建端粒酶活性的成纤维细胞在体外培养条件下成骨 ,用四环素活体标记和茜素红染色显示钙盐结节形成。结果　转染 h TERT的人成纤维细胞能稳定表达端粒酶活性 ,培养超过 5 0代后细胞仍保持β-半乳糖苷酶阴性。经 BMP- 2和 TNF-α诱导后 ,转染后传 80代的人成纤维细胞仍可形成四环素标记和茜素红染色均为阳性的钙盐结节。结论　重建端粒酶活性、寿命延长的人成纤维细胞仍然维持了其本身所具有的成骨潜能     

Dynamic expression of DKK1 protein in the process whereby Epimedium‐derived flavonoids up‐regulate osteogenic and down‐regulate adipogenic differentiation of bone marrow stromal cells in ovariectomized rats

Objective: To observe the dynamic expression of DKK1 protein in the process whereby Epimedium‐derived flavonoids (EFs) regulate the balance between osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats, and to provide experimental evidence for the mechanism of EFs in the treatment of postmenopausal osteoporosis. Methods: Bone marrow stromal cells from ovariectomized rats were separated and cultivated in osteoinductive or liquid medium for 15 days in vitro. EFs (10 µg/mL) were applied to both cultures. Alkaline phosphatase (ALP) staining, ALP activity determination, Oil Red O staining and fluorescence quantitative polymerase chain reaction were used to determine the influence of EFs on osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats. Moreover, in order to explore the exact mechanism of EFs on osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats, enzyme linked immunosorbent assay was used to determine the dynamic expression of DKK1 protein in this process. Results: EFs increased activity of ALP and mRNA expression of Runx2 (early osteoblast differentiation factor) and decreased mRNA expression of PPARγ‐2 (key factor of fat generation). Importantly, EFs down‐regulated expression of DKK1 protein in an osteogenic induction medium and inhibited up‐regulation of DKK1 protein in an adipogenic induction medium. Conclusion: EFs regulate the balance between osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats by down‐regulating expression of DKK1 protein. This may be an important molecular mechanism of EFs in the context of treatment of postmenopausal osteoporosis.     

Inhibition of primate spinothalamic tract cells by TENS

Transcutaneous electrical nerve stimulation (TENS) was applied in an experimental animal model to investigate the underlying mechanisms of this treatment. Recordings were made from identified spinothalamic tract (STT) neurons in the lumbosacral spinal cords of seven anesthetized monkeys. The STT cells were activated by stimulating the common peroneal nerve at a suprathreshold intensity for C-fibers. Evoked responses of C-fibers were compared before, during, and after application of TENS for 5 minutes from a commercially available TENS unit. The current delivered by the TENS unit was monitored. In 14 STT cells, some degree of inhibition of C-fiber evoked responses occurred only when the intensity of TENS exceeded the threshold of A delta fibers. At a given stimulus intensity, bursts of pulses repeated at a low rate were more effective than high-rate pulses. When TENS was applied to an area of the skin within a cell's receptive field, it was more effective than when it was applied outside the receptive field. The C-fiber volley recorded from a peripheral nerve was not reduced in size, and there were no substantial changes in its latency due to TENS. The inhibition of the activity of STT cells was not altered appreciably after intravenous injection of naloxone hydrochloride. These results suggest that TENS produces central nervous system inhibition by activating A delta afferent fibers. The inhibitory effects of TENS on STT cells appear to be due to a mechanism that does not involve release of endogenous opioid substances.     

Electroporation-mediated muscarinic M3 receptor gene transfer into rat urinary bladder

BACKGROUND: Muscarinic M3 (M3) receptor has been recognized as a major muscarinic receptor for smooth muscle contractions of the urinary bladder. Under the hypothesis that overexpression of M3 receptor in the urinary bladder would enhance urinary bladder contractions, we have transferred the M3 receptor gene into rat bladders using electroporation (EP) and evaluated the functional expression of the transferred gene. METHODS: Plasmids expressing luciferase, a green fluorescence protein and M3 receptor were injected into the rat bladder and square-wave electric pulses were immediately applied. Two days after gene transfer, we analyzed gene expression. Immunohistochemical staining was performed and the contractile responses from isolated bladder strips, which were induced KCl, carbachol and electrical field stimulation (EFS), were evaluated. RESULTS: The optimal conditions of electroporation were 8 pulses, 45 voltages, 50 milliseconds/pulses and 1 Hz. Under these conditions, luciferase gene expression was enhanced approximately 300-fold, compared to an injection of DNA only. Regarding immunohistochemistry with an anti-M3 receptor, an increase in immunoactivity was observed in the M3 receptor gene transferred rat bladder, compared to the bladder of the control rat. In rats with the transferred M3 receptor gene, carbachol- and EFS-induced maximum contractile responses of bladder smooth muscle strips significantly increased. CONCLUSIONS: These findings suggest that an in vivo EP procedure is an useful method for gene transfer into the bladder and that an overexpression of M3 receptor in the rat bladder enhances bladder contractility. This technique may become a new treatment modality for detrusor underactivity.     

Autologous platelet-rich plasma induces bone formation of tissue-engineered bone with bone marrow mesenchymal stem cells on beta-tricalcium phosphate ceramics

Background

The purpose of the study is to investigate whether autologous platelet-rich plasma (PRP) can serve as bone-inducing factors to provide osteoinduction and improve bone regeneration for tissue-engineered bones fabricated with bone marrow mesenchymal stem cells (MSCs) and beta-tricalcium phosphate (β-TCP) ceramics. The current study will give more insight into the contradictory osteogenic capacity of PRP.

Methods

The concentration of platelets, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-β1 (TGF-β1) were measured in PRP and whole blood. Tissue-engineered bones using MSCs on β-TCP scaffolds in combination with autologous PRP were fabricated (PRP group). Controls were established without the use of autologous PRP (non-PRP group). In vitro, the proliferation and osteogenic differentiation of MSCs on fabricated constructs from six rabbits were evaluated with MTT assay, alkaline phosphatase (ALP) activity, and osteocalcin (OC) content measurement after 1, 7, and 14 days of culture. For in vivo study, the segmental defects of radial diaphyses of 12 rabbits from each group were repaired by fabricated constructs. Bone-forming capacity of the implanted constructs was determined by radiographic and histological analysis at 4 and 8 weeks postoperatively.

Results

PRP produced significantly higher concentration of platelets, PDGF-AB, and TGF-β1 than whole blood. In vitro study, MTT assay demonstrated that the MSCs in the presence of autologous PRP exhibited excellent proliferation at each time point. The results of osteogenic capacity detection showed significantly higher levels of synthesis of ALP and OC by the MSCs in combination with autologous PRP after 7 and 14 days of culture. In vivo study, radiographic observation showed that the PRP group produced significantly higher score than the non-PRP group at each time point. For histological evaluation, significantly higher volume of regenerated bone was found in the PRP group when compared with the non-PRP group at each time point.

Conclusions

Our study findings support the osteogenic capacity of autologous PRP. The results indicate that the use of autologous PRP is a simple and effective way to provide osteoinduction and improve bone regeneration for tissue-engineered bone reconstruction.

     

电穿孔介导的pIRES-hVEGF165-EGFP转染对牵引成骨过程中早期血管生成的影响

目的 探讨电穿孔介导的基因治疗对下领骨牵引成骨过程中早期血管生成的影响.方法 32只新西兰大白兔随机分为4组:质粒+电穿孔组(A组),质粒组(B组),生理盐水+电穿孔组(C组),空白对照组(D组).各组动物分别于注射后1、3、7、14 d处死,取牵引区组织进行组织学检查、电镜观察、CD34免疫组织化学染色及微血管密度检测.结果 A、B组血管内皮细胞呈增殖活跃状态;C、D组多数血管内皮细胞部分呈现退变及凋亡早期改变.免疫组化染色发现,转染后第1天血管壁内皮细胞浆CD34表达较弱;第3、7、14天,牵引区肉芽组织血管内皮细胞均出现CD34阳性表达.A组CD34阳性表达较B组强,A、B组的CD34表达持续阳性且呈上升趋势;C、D组表达最弱,CD34阳性表达维持在第1天水平上平稳波动.结论 电穿孔介导的pIRES-hVEGF165-EGFP重组质粒体内转染能够促进牵引区早期微血管的生成,使局部血管增生、渗入,增加骨断端的血流量.对调节和促进骨的生长和修复过程具有重要作用.     

电穿孔介导的pIRES-hVEGF165-EGFP转染对牵引成骨过程中早期血管生成的影响

目的 探讨电穿孔介导的基因治疗对下领骨牵引成骨过程中早期血管生成的影响.方法 32只新西兰大白兔随机分为4组：质粒＋电穿孔组（A组）,质粒组（B组）,生理盐水＋电穿孔组（C组）,空白对照组（D组）.各组动物分别于注射后1、3、7、14 d处死,取牵引区组织进行组织学检查、电镜观察、CD34免疫组织化学染色及微血管密度检测.结果 A、B组血管内皮细胞呈增殖活跃状态;C、D组多数血管内皮细胞部分呈现退变及凋亡早期改变.免疫组化染色发现,转染后第1天血管壁内皮细胞浆CD34表达较弱;第3、7、14天,牵引区肉芽组织血管内皮细胞均出现CD34阳性表达.A组CD34阳性表达较B组强,A、B组的CD34表达持续阳性且呈上升趋势;C、D组表达最弱,CD34阳性表达维持在第1天水平上平稳波动.结论 电穿孔介导的pIRES-hVEGF165-EGFP重组质粒体内转染能够促进牵引区早期微血管的生成,使局部血管增生、渗入,增加骨断端的血流量.对调节和促进骨的生长和修复过程具有重要作用.     

Comparison of Osteogenic Potentials of BMP4 Transduced Stem Cells from Autologous Bone Marrow and Fat Tissue in a Rabbit Model of Calvarial Defects

We compared bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) of adult rabbits under identical conditions in terms of their culture characteristics, proliferation capacity, osteogenic differentiation potentials induced by adenovirus-containing bone morphogenetic protein 4 (Ad-BMP4) in vitro, and capacity to repair calvarial defects in the rabbit model by autologous transplantation ex vivo. According to the results of growth curve, cell cycle, and telomerase activity analysis, ADSCs possess a higher proliferation potential. Both of the Ad-BMP4 transduced MSCs expressed BMP4 mRNA and protein and underwent osteogenic differentiation. Up-regulated mRNA expression of all osteogenic genes was observed in differentiated BMSCs and ADSCs, but with different patterns confirmed by real-time RT-PCR. Deposition of calcified extracellular matrix was significantly greater in differentiated ADSCs compared with differentiated BMSCs. X-ray and histological examination indicated significant bone regeneration in the calvarial defects transplanted with Ad-BMP4 transduced autologous MSCs compared to the control groups. There was no significant difference in new bone formation in Ad-BMP4 transduced MSCs based on quantitative digital analysis of histological sections. The use of ADSCs often resulted in the growth of fat tissue structures in the control groups, and the fat tissue structures were not seen with BMSC cells. Our data demonstrate that BMP4 can be potently osteoinductive in vivo, resulting in bone repair. ADSCs may be an attractive alternative to BMSCs for bone tissue engineering under appropriate stimuli. But the easy adipogenic differentiation needs to be considered when choosing adipose tissue for specific clinical application.