Use of ultra‐high molecular weight polycaprolactone scaffolds for ACL reconstruction

Previously, we reported on the implantation of electrospun polycaprolactone (PCL) grafts for use in ACL tissue engineering in a small animal model. In the present study, we hypothesized that grafts fabricated from ultra‐high molecular weight polycaprolactone (UHMWPCL) would have similarly favorable biologic properties but superior mechanical properties as compared to grafts fabricated from PCL. Two forms of polycaprolactone were obtained (UHMWPCL, MW = 500 kD, and PCL, MW = 80 kD) and electrospun into scaffolds that were used to perform ACL reconstruction in 7–8 week old male Lewis rats. The following groups were examined: UHMWPCL, PCL, flexor digitorum longus (FDL) allograft, native ACL, as well as sham surgery in which the ACL was transsected. At 16 weeks post‐operatively, biomechanical testing, histology, and immunohistochemistry (IHC) were performed. Analysis of cellularity indicated that there was no significant difference among the UHMWPCL, PCL, and FDL allograft groups. Quantification of birefringence from picrosirius red staining demonstrated significantly more aligned collagen fibers in the allograft than the PCL group, but no difference between the UHMWPCL and allograft groups. The peak load to failure of the UHMWPCL grafts was significantly higher than PCL, and not significantly different from FDL allograft. This in vivo study establishes the superiority of the higher molecular weight version of polycaprolactone over PCL as a scaffold material for ACL reconstruction. By 16 weeks after implantation, the UHMWPCL grafts were not significantly different from the FDL allografts in terms of cellularity, peak load to failure, stiffness, and collagen fiber alignment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:828–835, 2016.     

bFGF预载脱细胞支架构建小口径人工血管的研究

目的目前临床使用的小口径（〈5cm）人工血管因生物相容性差、远期通畅率低，效果不理想。拟通过在脱细胞血管支架表面预载bFGF，制备一种新型的小口径人工血管。方法采用去污剂一酶消化法制备犬颈动脉脱细胞支架，将bFGF预载在经肝素固化（肝素固化组）和未固化的（单脱细胞组）脱细胞支架表面，ELISA法检测结合的bFGF量及体外释放情况。通过与犬BMSCs体外复合培养1～5d，观察bFGF预载肝素固化脱细胞支架（bFGF预载组）和未固化的脱细胞支架（未预载组），以及各自空白对照组细胞生长情况。取8只杂交犬，切断并剪下颈总动脉造成约5cm缺损，随机选取一侧，将预载组支架行端端吻合于缺损中，作为实验侧；将未预载组支架以同样方法植入对侧，作为对照侧。术后8周取材行DSA、HE染色观察移植效果。结果犬颈动脉经脱细胞后大体形态完好、细胞基本去除、纤维结构完整。肝素固化组支架表面结合的bFGF量与bFGF反应浓度成正相关，与相同反应浓度下单脱细胞组比较差异有统计学意义（P〈0．05）；肝素固化组在浓度为100ng／mL下结合的bFGF可在体外持续释放20d。bFGF预载支架促进BMSCs增殖，MTT显示BMSCs在两组支架表面均可黏附生长，复合培养1、2d两组差异无统计学意义（P〉0．05）：3～5d，bFGF预载组支架表面细胞增殖活性明显高于未预载组（P〈0．01）。异体犬颈动脉移植后8周，实验侧支架均通畅，且有细胞覆盖内膜及浸润管壁，而对照侧通畅率仅为12．5％（1／8），闭塞的移植物腔内均为血栓形成，未见细胞覆盖。结论对同种异体血管脱细胞支架表面进行bFGF预载，初步获得一种具有良好生物相容性和通畅性的生物人工血管，其远期通畅率及生物安全性仍待进一步评价。     

New ligament healing model based on tissue‐engineered collagen scaffolds

The anterior cruciate ligament (ACL) is often the target of knee trauma. This ligament does not heal very well, leading to joint instability. Long‐term instability of the knee can lead to early arthritis and loss of function. To develop efficient strategies to stimulate posttraumatic ACL regeneration in vivo, a good healing model is needed in vitro. Such a model must remain as simple as possible, but should include key features to provide relevant answers to precise questions about the clinical problem addressed. Here, we report tissue‐engineered type I collagen scaffolds developed to establish an ACL healing model in vitro and a potential ACL substitute in vivo. Such scaffolds were used to evaluate ACL cell growth, migration, and the capacity to synthesize and assemble collagen fibers for up to 40 days in vitro and up to 180 days in vivo. They were anchored with two bone plugs to allow their static stretching in culture and to facilitate their surgical implantation in knee joints. Our results have shown that living ACL fibroblasts can attach, migrate, and colonize this type of scaffold. In vitro, the cells populated the scaffolds and expressed mRNAs coding for the prolyl‐4‐hydroxylase, involved in collagen fibers' assembly. In vivo, acellular implants were vascularized and populated with caprine cells that migrated from the osseous insertions toward the center of the grafts. This model is a very good tool to study ACL repair and identify the factors that could accelerate its healing postsurgery.     

Improvements in GORE-TEX vascular graft performance by Carmeda BioActive surface heparin immobilization.

OBJECTIVES: a performance improvement in small-diameter bypass grafts remains a clinical objective. The purpose of the present investigation was to evaluate the potential of enhancing the thromboresistance of ePTFE grafts using a bioactive heparinized graft luminal surface in a canine model. MATERIAL AND METHODS: this study investigated the utility of heparin immobilization onto expanded polytetrafluoroethylene using Carmeda BioActive Surface technology (CBAS-ePTFE) as a means of improving vascular graft thromboresistance. Graft luminal surfaces were covered uniformly with the stably bound, end-point immobilized heparin. RESULTS: acute canine (5 greyhounds) interposition experiments comparing CBAS-ePTFE grafts to control ePTFE grafts showed that CBAS-ePTFE grafts remained patent and had significantly greater thrombus-free luminal surface (p<0.05). In a chronic canine (16 greyhounds) interposition experiment, significantly improved patency (p<0.05) was observed with CBAS-ePTFE grafts compared to controls. Long-term in vivo heparin bioactivity was demonstrated on CBAS-ePTFE grafts explanted between 1 and 12 weeks. On all CBAS-ePTFE grafts, heparin activity levels ranged from 15-25pmol/cm(2) and did not differ significantly (p>0.05). DISCUSSION: these results support the conclusion that a stable, CBAS-ePTFE surface provides improved thromboresistance and improved patency in canine interposition models. Maintenance of heparin catalytic activity on the graft surface in vivo likely contributes to this outcome and holds promise for the utility of this graft surface for clinical applications.     

Basic fibroblast growth factor coating and endothelial cell seeding of a decellularized heparin-coated vascular graft

The objective of this study was to determine the effect of basic fibroblast growth factor (bFGF) coating on endothelial cell seeding and proliferation on a decellularized heparin coated vascular graft and to determine the retention of seeded cells on the graft under flow conditions. Disks of heparin coated decellularized grafts were incubated for 24 h as controls or with bFGF. Human microvascular endothelial cells (HMECs) or canine peripheral blood endothelial progenitor cells (CEPC) were seeded onto the disks and incubated for 96 h or 48 h, respectively. HMECs were also seeded onto the luminal surfaces of two heparin-coated decellularized grafts for 3 h. One graft was placed in a perfusion culture system and cultured for an additional 6 h with flow and pressure. After culturing, there were 4.7 +/- 1.4 cells/mm(2) HMECs on control grafts and 11.4 +/- 1.4 cells/mm(2) in bFGF treated grafts (P < 0.05). Likewise, with CEPCs, there were 14.8 +/- 4.8 cells/mm(2) in control grafts and 33.3 +/- 7.3 cells/mm(2) in bFGF treated grafts. After only 3 h of cell attachment, 60% of HMECs were retained in the intact graft exposed flow relative to the static control graft, which is an acceptable level. These data demonstrate that bFGF coating on the heparin bound decellularized grafts significantly increases both HMEC and dog EPC proliferation and that seeded cells are stable under perfusion conditions.     

Ingrowth of aorta wall into stent grafts impregnated with basic fibroblast growth factor: a porcine in vivo study of blood vessel prosthesis healing

OBJECTIVE: Endovascular aneurysm repair is an alternative treatment of abdominal aortic aneurysm. The procedure is less invasive, and morbidity and most probably mortality are reduced. However, some problems, such as endoleakage, are yet to be resolved. Endoleakage can occur after graft migration, as a result of insufficient fixation of the stent graft. One cause is deficient healing between the aortic neck and the stent graft. We hypothesize that better healing, achieved by induction of vascular cell ingrowth into the graft material, results in better graft fixation. Previously we demonstrated ingrowth of neointima into the graft material if the stent graft is impregnated with a coat of basic fibroblast growth factor (bFGF), heparin, and collagen. In this study we evaluated healing with bFGF-heparin-collagen-coated stent grafts in vivo. METHODS: In 4 pigs, 32 endovascular stent grafts, manufactured from standard Dacron and Gianturco Z-stents, were placed in the aorta. The stent grafts were impregnated with either bFGF-heparin containing collagen (n=16) or control collagen (n=16). After 4 and 8 weeks animals were killed, and ingrowth and healing of the stent grafts were macroscopically and electron microscopically evaluated. RESULTS: After 8 weeks all bFGF-impregnated stent grafts demonstrated ingrowth of tissue and healing between the graft and the aorta, whereas the control nonimpregnated stent grafts showed no ingrowth. Microscopic evaluation demonstrated alpha-smooth muscle actin-positive cells, most probably smooth muscle cells or myofibroblasts, growing from the vascular wall through the graft material. CONCLUSION: A Dacron prosthesis impregnated with collagen, heparin, and bFGF induced graft healing in an in vivo pig model, in contrast to nonimpregnated stent grafts. This in vivo study confirms our previous findings in vitro. These results indicate that healing between Dacron and the aorta can be achieved, and suggest that type I endoleakage may be resolved by inducing healing between the aortic wall and the prosthesis with graft material containing growth factor.     

Promotion of osteogenesis in tissue‐engineered bone by pre‐seeding endothelial progenitor cells‐derived endothelial cells

In addition to a biocompatible scaffold and an osteogenic cell population, tissue‐engineered bone requires an appropriate vascular bed to overcome the obstacle of nutrient and oxygen transport in the 3D structure. We hypothesized that the addition of endothelial cells (ECs) may improve osteogenesis and prevent necrosis of engineered bone via effective neovascularization. Osteoblasts and ECs were differentiated from bone marrow of BALB/c mice, and their phenotypes were confirmed prior to implantation. Cylindrical porous polycaprolactone (PCL)‐hydroxyapatite (HA) scaffolds were synthesized. ECs were seeded on scaffolds followed by seeding of osteoblasts in the EC‐OB group. In the OB group, scaffolds were only seeded with osteoblasts. The cell‐free scaffolds were denoted as control group. A 0.4‐cm‐long segmental femur defect was established and replaced with the grafts. The grafts were evaluated histologically at 6 weeks postimplantation. In comparison with the OB group, the EC‐OB group resulted in a widely distributed capillary network, osteoid generated by osteoblasts and absent ischemic necroses. Pre‐seeding scaffold with ECs effectively promoted neovascularization in grafts, prevented the ischemic necrosis, and improved osteogenesis. The integration of bone marrow‐derived ECs and osteoblasts in porous scaffold is a useful strategy to achieve engineered bone. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1147–1152, 2008     

Effects of ACL graft placement on in vivo knee function and cartilage thickness distributions

Injuries to the anterior cruciate ligament (ACL) frequently lead to early‐onset osteoarthritis. Despite advancement in surgical techniques, ACL reconstruction has a limited ability to prevent these degenerative changes. While previous studies have investigated knee function after ACL reconstruction, in vivo investigations of the effects of graft placement on in vivo joint function and cartilage health are limited. This review presents a series of studies that used novel imaging and 3D modeling techniques to determine the in vivo placement of the ACL graft on the femur using two different ACL reconstruction techniques. These techniques resulted in two distinct graft placement groups: one where the ACL was placed anatomically near the center of the native ACL footprint and another where the graft was placed anteroproximally on the femur, centered outside the ACL footprint. We quantified the effects of graft placement on graft deformation during in vivo loading and how these variables affected knee motion. Finally, we quantified whether femoral placement of the graft affected cartilage thickness. Our results demonstrate that achieving anatomic graft placement on the femur is critical to restoring native ACL function and normal knee kinematics. Knees with grafts that more closely restored normal ACL function, and thus knee motion, experienced less focal cartilage thinning than did those that experienced abnormal knee motion. These results suggest that achieving anatomic graft placement is a critical factor in restoring normal knee motion and potentially slowing the development of degenerative changes after ACL reconstruction. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1160–1170, 2017.

     

Electrospun small-diameter polyurethane vascular grafts: ingrowth and differentiation of vascular-specific host cells

No small-diameter synthetic graft has yet shown comparable performance to autologous vessels. Synthetic conduits fail due to their inherent surface thrombogenicity and the development of intimal hyperplasia. In addressing these shortcomings, electrospinning offers an interesting alternative to other nanostructured, cardiovascular substitutes because of the close match of electrospun materials to the biomechanical and structural properties of native vessels. In this study, we investigated the in vivo behavior of electrospun, small-diameter conduits in a rat model. Vascular grafts composed of polyurethane were fabricated by electrospinning. Prostheses were implanted into the abdominal aorta in 40 rats for either 7 days, 4 weeks, 3 months, or 6 months. Retrieved specimens were evaluated by histology, immunohistochemical staining, confocal laser scanning microscopy, and scanning electron microscopy. At all time points, we found no evidence of foreign body reaction or graft degradation. The overall patency rate of the intravascular implants was 95%. Within 7 days, grafts revealed ingrowth of host cells. CD34+ cells increased significantly from 7 days up to 6 months of implantation (P < 0.05). Myofibroblasts and myocytes showed increasing cell numbers up to 3 months (P < 0.05). Ki67 staining indicated unaltered cell proliferation during the whole follow-up period. Besides biomechanical benefits, electrospun polyurethane grafts exhibit excellent biocompatibility in vivo. Cell immigration and differentiation seems to be promoted by the nanostructured artificial matrix.     

Nonthrombogenic small-caliber human umbilical vein vascular prosthesis

The present investigation demonstrates covalent binding of heparin with carbodiimide to ammonium hydroxide-treated collagenous surfaces. Human umbilical vein grafts (HUVG) outperform carotid arteries of goat, porcine, and canine origin in both heparin loading and stability of the immobilized heparin preparation. The average heparin loading on the untreated carotid arteries and HUVGs and ammonium hydroxide-treated HUVGs were 18, 27, and 31.5 micrograms/cm2, respectively. There was negligible loss of heparin activity under in vitro and in vivo conditions. In vitro studies demonstrate that heparin-bound HUVGs discourage platelet adhesion and subsequent fibrin clot formation. In vivo studies with heparin-bound HUVGs show a significant increase in thrombus-free surface compared with control grafts. Heparin-bound HUVGs also show an enhanced patency rate in the two sets of protocols tested--one lasting for 3 in vivo days (seven grafts) and the other lasting for 7 (15 grafts) in vivo days. The studies conducted so far demonstrate the promise of developing a nonthrombogenic small-caliber HUVG prosthesis.     

Innervation of an engineered muscle graft for reconstruction of muscle defects

Autologous muscle flaps are commonly used to reconstruct defects that involve muscle impairment. To maintain viability and functionality of these flaps, they must be properly vascularized and innervated. Tissue‐engineered muscles could potentially replace autologous muscle tissue, but still require establishment of sufficient innervation to ensure functionality. In this study, we explored the possibility of innervating engineered muscle grafts transplanted to an abdominal wall defect in mice, by transferring the native femoral nerve to the graft. Six weeks posttransplantation, nerve conduction studies and electromyography demonstrated increased innervation in engineered grafts neurotized with the femoral nerve, as compared to non‐neurotized grafts. Histologic assessments revealed axonal penetration and formation of neuromuscular junctions within the grafts. The innervation process described here may advance the fabrication of a fully functional engineered muscle graft that will be of utility in clinical settings.     

Extracellular matrix protein-coated scaffolds promote the reversal of diabetes after extrahepatic islet transplantation

BACKGROUND: The survival and function of transplanted pancreatic islets is limited, owing in part to disruption of islet-matrix attachments during the isolation procedure. Using polymer scaffolds as a platform for islet transplantation, we investigated the hypothesis that replacement of key extracellular matrix components known to surround islets in vivo would improve graft function at an extrahepatic implantation site. METHODS: Microporous polymer scaffolds fabricated from copolymers of lactide and glycolide were adsorbed with collagen IV, fibronectin, laminin-332 or serum proteins before seeding with 125 mouse islets. Islet-seeded scaffolds were then implanted onto the epididymal fat pad of syngeneic mice with streptozotocin-induced diabetes. Nonfasting glucose levels, weight gain, response to glucose challenges, and histology were used to assess graft function for 10 months after transplantation. RESULTS: Mice transplanted with islets seeded onto scaffolds adsorbed with collagen IV achieved euglycemia fastest and their response to glucose challenge was similar to normal mice. Fibronectin and laminin similarly promoted euglycemia, yet required more time than collagen IV and less time than serum. Histopathological assessment of retrieved grafts demonstrated that coating scaffolds with specific extracellular matrix proteins increased total islet area in the sections and vessel density within the transplanted islets, relative to controls. CONCLUSIONS: Extracellular matrix proteins adsorbed to microporous scaffolds can enhance the function of transplanted islets, with collagen IV maximizing graft function relative to the other proteins tested. These scaffolds enable the creation of well-defined microenvironments that promote graft efficacy at extrahepatic sites.     

Sternal repair with bone grafts engineered from amniotic mesenchymal stem cells

Purpose

We aimed at determining whether osseous grafts engineered from amniotic mesenchymal stem cells (aMSCs) could be used in postnatal sternal repair.

Methods

Leporine aMSCs were isolated, identified, transfected with green fluorescent protein (GFP), expanded, and seeded onto biodegradable electrospun nanofibrous scaffolds (n = 6). Constructs were dynamically maintained in an osteogenic medium and equally divided into 2 groups with respect to time in vitro as follows: 14.6 or 33.9 weeks. They were then used to repair full-thickness sternal defects spanning 2 to 3 intercostal spaces in allogeneic kits (n = 6). Grafts were submitted to multiple analyses 2 months thereafter.

Results

Chest roentgenograms showed defect closure in all animals, confirmed at necropsy. Graft density as assessed by microcomputed tomographic scans increased significantly in vivo, yet there were no differences in mineralization by extracellular calcium measurements preimplantation and postimplantation. There was a borderline increase in alkaline phosphatase activity in vivo, suggesting ongoing graft remodeling. Histologically, implants contained GFP-positive cells and few mononuclear infiltrates. There were no differences between the 2 construct groups in any comparison.

Conclusions

Engineered osseous grafts derived from amniotic mesenchymal stem cells may become a viable alternative for sternal repair. The amniotic fluid can be a practical cell source for engineered chest wall reconstruction.     

Preparation and evaluation of polycaprolactone/chitosan/Jaft biocompatible nanofibers as a burn wound dressing

Tissue engineering is an emerging method for replacing damaged tissues. In this study, the potential application of electrospun polycaprolactone/chitosan/ the internal layer of oak fruit (Jaft) as skin scaffolds was investigated. A combination of Polycaprolactone (PCL), chitosan (CH), and the internal layer of oak fruit (Jaft) was used to incorporate mechanical properties of synthetic polymers, biological properties of natural polymers, and antibacterial activity of Jaft. Physical and morphological characteristics of prepared scaffolds were investigated using a scanning electron microscope (SEM), mechanical analysis, swelling ratio, and contact angle. Moreover, chemical and biological properties were evaluated by Fourier-transform infrared spectroscopy (FTIR), chromatography, flow cytometry, DAPI staining, MTT assay, and trypan blue exclusion assay. Obtained results demonstrated that the fabricated scaffolds have good mechanical properties. Moreover, the addition of chitosan and Jaft to the PCL scaffolds improved their water absorption capacity as well as surface hydrophilicity. MTT results showed the fabricated nanofibrous scaffolds have adequate cell viability, which is higher than the cell culture plate at each time point of culture. Furthermore, SEM images of cultured scaffolds, trypan blue exclusion assay, and DAPI staining confirmed that fibroblast cells could be well-attached and proliferate on the PCL/CH/Jaft scaffolds. Results have proven that this novel bioactive scaffold has promising mechanical properties, suitable biocompatibility in vitro, and in vivo. Consequently, it could be a promising candidate for skin tissue engineering applications.     

The effect of initial graft tension on the biomechanical properties of a healing ACL replacement graft: a study in goats.

While a number of in vitro studies have shown that the tension on an anterior cruciate ligament (ACL) replacement graft at the time of fixation has an affect on joint stability, most in vivo studies have reported little or no long-term difference in outcome. The objectives of this study were to (1) establish a large animal model in which differences in knee stability are present at time-zero after ACL reconstruction with grafts fixed at a low (5 N) and high (35 N) initial tension and to (2) quantitatively determine if these initial effects remain after six weeks of healing and if the tensile properties of an ACL replacement graft are influenced by initial graft tension. Seventeen skeletally mature female Saanan breed goats were used. Using the robotic/UFS testing system, the knee kinematics and in situ forces in the replacement graft in response to an externally applied 67 N anterior-posterior (A-P) tibial load were evaluated at time-zero and after six weeks of healing. Afterward, the femur-ACL graft-tibia complexes (FGTCs) from the six-week group were tested under uniaxial tension so that the stress relaxation and structural properties of the FGTC were obtained.At time-zero, knees fixed with a high initial graft tension could better reproduce the A-P translation of the intact knee in response to the 67 N A-P tibial load. Further, in situ forces in these grafts were also closer to those in the intact ACL under the same external loading condition. After six weeks of healing, the A-P translation of the knee and in situ forces in the replacement grafts became similar for the low and high tension groups, while both were significantly different from controls. Further, the percentage of stress relaxation as well as the stiffness, ultimate load at failure, ultimate elongation at failure, and energy absorbed of the FGTCs for both reconstruction groups were not significantly different from each other, but were significantly different from controls. These results demonstrate that while the high initial graft tension could better replicate the normal knee kinematics at time-zero, these effects may diminish during the early graft healing process.     

In vivo retention of endothelial cells adenovirally transduced with tissue-type plasminogen activator and seeded onto expanded polytetrafluoroethylene

PURPOSE: Seeding a prosthetic graft with genetically engineered vascular endothelial cells (ECs) has the potential to enhance the graft's antithrombotic properties. However, it has been reported that ECs transduced with tissue-type plasminogen activator (tPA) have very low levels of retention on grafts, probably because of increased proteolytic activity. We examined the retention of human tPA (htPA)-transduced ECs after the cells were seeded onto expanded polytetrafluoroethylene (ePTFE) grafts and implanted into dogs. We also examined the function of secreted htPA in this model.Methods and Results: Canine jugular venous ECs were transduced with adenoviral vectors encoding htPA (Adex1CAhtPA) and beta-galactosidase (Adex1CALacZ). There was a positive relationship between the percentage of X-gal ECs staining and the multiplicity of infection (MOI) of Adex1CALacZ. The level of htPA production in vitro increased with the increasing MOI of Adex1CAhtPA, but decreased gradually 4 days after infection. ECs coinfected with Adex1CAhtPA and Adex1CALacZ (htPAEC) or ECs infected with Adex1CALacZ alone (LacZEC) were seeded onto ePTFE grafts at densities equivalent to confluence to visualize retained ECs in an in vivo flow study. The grafts were implanted into canine carotid arteries and harvested after 5 hours of exposure to blood flow. The harvested grafts showed patchy defects in ECs, most of which were covered with mural thrombi. There was no significant difference in retention between htPAEC (29.3% +/- 8.7%) and LacZEC (19.5% +/- 3. 6%). There was a significant negative correlation between the in vivo EC retention on the grafts and the in vitro cellular passage level of ECs (P =.041; r = -.40). htPAEC produced 210.3 +/- 22.2 ng htPA antigen/10(6) cells per 6 hours in vitro and continued to secrete htPA on the harvested graft. CONCLUSIONS: We demonstrated that a large amount of functional htPA was produced by adenovirally modified canine ECs. The results of the in vivo study may suggest that overexpression of tPA has little effect on the short-term retention of early passage ECs seeded onto ePTFE grafts.     

再造组织工程尿路移行上皮组织的实验研究

目的采用组织工程技术，将培养扩增的膀胱移行上皮细胞与可降解的聚乳酸／聚羟基乙酸共聚物（polylactical／glycolic acid copolymer，PLGA）支架材料复合并植入裸鼠体内进行培育，探讨构建尿路移行上皮组织的可行性。方法取幼兔膀胱，机械分离与酶消化法获取膀胱移行上皮细胞，并在体外行原代培养与传代扩增，将第4～5代扩增的上皮细胞接种至8个PLGA聚合材料的表面作为实验组，4个单纯支架材料作为对照组。实验组：细胞材料复合体培育4周和8周各4个，对照组：单纯支架培育4周和8周各2个，分别植入各组裸鼠体内进行培育，按各时间点取出标本后进行大体观察、组织学及免疫组织化学检测。结果实验组标本经HE和马森染色，4周显示在材料表面形成数层上皮细胞；8周移行上皮细胞进一步增殖形成上皮组织，抗角蛋白免疫组织化学染色，胞浆呈棕黄色阳性。对照组4周和8周经HE染色材料表面见少量成纤维细胞沉积，马森染色呈蓝色，经抗角蛋白免疫组织化学染色为阴性。结论采用组织工程技术可再造人尿路移行上皮组织，为尿路组织工程进一步实验研究奠定了基础。     

Infrainguinal ePTFE vascular graft with bioactive surface heparin bonding. First clinical results

AIM: The ultimate aim of improved expanded polytetrafluoroethylene (ePTFE) vascular graft design is to achieve patency rates in femoropopliteal bypass grafting comparable with autologous saphenous vein grafts. Enhanced thromboresistance of the ePTFE material by bioactive surface heparin bonding is one conceivable path toward this goal. This retrospective study was performed to collect the first clinical data for a new ePTFE graft with bioactive surface heparin immobilization. METHODS: Between March 2003 and February 2004, 43 femoropopliteal or femorocrural ePTFE vascular prostheses with bioactive end-point immobilized heparin (Gore-Tex Propaten Vascular Graft), using the Car-meda BioActive Surface technology, were implanted in 40 patients. Twelve prostheses were implanted in above-knee and 31 in below-knee position. The indication for bypass grafting was limb-threatening ischemia in 88% of the patients. The mean follow-up was 16.6 months. RESULTS: The primary 1-year patency was 91% for above-knee bypass grafts and 92% for below-knee bypass grafts. The 2-year primary patency rate for above-knee bypass grafts was 68% and 81% for below-knee bypass grafts. Limb salvage was achieved in 98%. The perioperative mortality was 0%, but during follow-up 22% of the patients died with patent bypass grafts. CONCLUSIONS: While conventional ePTFE grafts, particularly in the below-knee position, have shown poor results even in the short-term, the bioactive heparinized ePTFE graft evaluated in this study provides patency rates comparable with autologous vein grafts. Because the influence of luminal heparin bonding is not only limited to thromboresistance but has impact on, amongst other elements, protein adsorption (thereby improving hemocompatibility), a continuous effect for long-term patency could also be expected.     

Cellular interactions with bacterial cellulose: Polycaprolactone nanofibrous scaffolds produced by a portable electrohydrodynamic gun for point‐of‐need wound dressing

Electrospun nanofibrous scaffolds are promising regenerative wound dressing options but have yet to be widely used in practice. The challenge is that nanofibre productions rely on bench‐top apparatuses, and the delicate product integrity is hard to preserve before reaching the point of need. Timing is critically important to wound healing. The purpose of this investigation is to produce novel nanofibrous scaffolds using a portable, hand‐held “gun”, which enables production at the wound site in a time‐dependent fashion, thereby preserving product integrity. We select bacterial cellulose, a natural hydrophilic biopolymer, and polycaprolactone, a synthetic hydrophobic polymer, to generate composite nanofibres that can tune the scaffold hydrophilicity, which strongly affects cell proliferation. Composite scaffolds made of 8 different ratios of bacterial cellulose and polycaprolactone were successfully electrospun. The morphological features and cell–scaffold interactions were analysed using scanning electron microscopy. The biocompatibility was studied using Saos‐2 cell viability test. The scaffolds were found to show good biocompatibility and allow different proliferation rates that varied with the composition of the scaffolds. A nanofibrous dressing that can be accurately moulded and standardised via the portable technique is advantageous for wound healing in practicality and in its consistency through mass production.