Biocompatibility Studies of a New Biosynthetic Dermal Substitute Based on Collagen/Hyaluronan Conjugate

The use of glycosaminoglycans (GAGs) as the second macromolecular component with collagen for the membrane preparation is based on the idea to create a biodegradable scaffold for new tissue. A hybrid collagen/hyaluronan membrane with specific bubble macrostructure was designed to serve as a synthetic dermal substitute. The objectives were to explain the two questions: what is the local biological response to the implanted membrane, and what is its reconstruction rate in tissue. The histological study proved low irritability, good compatibility, ingrowth of autologous tissue starting on day 7 post implantation, and resorption within four weeks. The major part of the implant was replaced by autologous tissue at the end of three weeks post implantation. The overall local biological response to implant was very good.     

膜性高分子材料在兔眼部的生物相容性

目的评价聚羟基脂肪酸(polyhydroxyalkanoates,PHA)、聚乳酸(polylacetic acid,PLA)和聚己内酯(polycaprolactone,PCL)三种膜性高分子材料在兔眼部的生物相容性。方法将24只新西兰兔随机分为4组,每组6只。PHA、PLA、PCL为实验组,材料植入兔右眼结膜下。假手术组结膜下钝性分离,但不植入任何高分子材料。使用裂隙灯显微镜观察并记录植入后不同时间手术眼的反应并评分。裂隙灯下观察材料的吸收时间。术后4周和16周取眼球,行HE染色、Masson染色和天狼猩红染色分别定性观察组织结构和炎症细胞、胶原纤维和胶原纤维的亚型与排列方向。结果术眼刺激性评分等级各组均不高于"轻度刺激性"。结膜下吸收时间PHA、PLA和PCL组分别是16周,12周和大于16周。组织学观察术后4周PHA、PLA和PCL组均形成材料包裹囊腔,囊壁以纤维组织为主,伴有毛细血管形成和炎性细胞浸润,以中性粒细胞为主。胶原纤维染色与假手术组无明显差异,以Ⅰ型和Ⅲ型为主,大致呈平行排列。术后16周PHA和PLA组材料已不可查及,包裹囊腔结构不规则,而PCL材料整体可查及,包裹囊腔规则。各组未见毛细血管,偶见淋巴细胞浸润。胶原纤维与假手术组无明显差异,以Ⅰ型和Ⅲ型为主,仍大致呈平行排列。结论 PHA、PLA和PCL三种膜性高分子材料在兔眼具有较好的生物相容性,结膜下吸收时间分别是16周,12周和大于16周。     

Characterization of tissue response and in vivo degradation of cholecyst-derived extracellular matrix

A rat subcutaneous implantation model was used to evaluate the in vivo degradation and tissue response of cholecyst-derived extracellular matrix (CEM). This response was compared to that of glutaraldehyde (GA) cross-linked CEM and porcine heart valve (HV), which are designated as GAxCEM and GAxHV, respectively. Tissue composition, inflammatory cell distribution, and angiogenesis at the implant site were quantified using stereological parameters, thickness (Ta), volume fraction (Vv), surface density (Sv), length density (Lv), and radius of diffusion (Rdiff). CEM was completely infiltrated with host tissue at 21 days and resorbed by 63 days. GAxCEM was also infiltrated with host tissue, while GAxHV matrix was impermeable to host tissue infiltration. Both GAxCEM and GAxHV retained their scaffold integrity until 63 days with no apparent degradation. A fibrous tissue of thickness <52 mum, rich in collagen and vasculature, surrounded all scaffolds, and from 21 to 63 days the fibrous tissue showed maturation with a significant increase in their fibrocyte content. No signs of acute inflammatory response were observed in the study period for any of the scaffolds, while the chronic inflammatory response was predominated with macrophages for all scaffolds except for CEM at 63 days. A higher degree of giant cell formation was observed with GA cross-linked scaffolds. From 21 to 63 days, lymphocytic response decreased for CEM, while it increased significantly for GAxHV. Angiogenesis/neo-vascularization was uniform for CEM (reaching the core), significantly lower for GAxCEM within the implant area as compared to CEM, while restricted to the exterior of GAxHV matrix. In summary, CEM was a fast degrading scaffold that induced a transitional inflammatory response accompanied by gradual resorption and replacement by host connective tissue as compared to the very slow degrading GA cross-linked controls, GAxCEM and GAxHV, which caused a sustained chronic inflammatory response and remained at the site of implantation until the end of the study period of 63 days.     

Construction of Small-Caliber,Polydiaxanone Cyclohexanone Vascular Stents

Objective of this article is to construct and characterize a three-layered small-caliber, artificial vascular stent. The outer layer of the stent consisted of small intestine submucosa (SIS), the middle layer was the polydioxanone (PDS) vascular stents, and the inner layer. The SIS and PDS were attached with 8-0 PDS thread. Crosslinking with a 10% collagen/chondroitin sulfate solution and 0.020% glutaraldehyde secured the structure. The stent was implanted into the muscles on both sides of the canines’ spine at 2, 4, 12, and 24 weeks. A MTT assay using L-929 cells measured the cytotoxicity of the implant. Histological and microscopy analyses were employed to examine the degradation characteristics of the PDS stent. Biomechanical properties of the stent were tested and compared to those of normal physiological blood vessels. The PDS stent burst pressure (43.5 ± 8.3) kPa, rupture intensity (19.1 ± 1.56) N, strain ratio (42.88 ± 3.16)%, and radial compliance (5.96 ± 0.87)%/100 mmHg were similar to that of physiological vessels. The cytotoxicity test showed that the PDS stent complied with specifications for biological materials for medical applications, with a cell toxicity ranging from 0 to 1. After 12 weeks, SIS and collagen sponge were completely replaced by fibrous connective tissue. Although there was some degradation of PDS, inflammatory cell infiltration subsided. After 24 weeks, the scaffold material began to absorb the new fibers and became filled with inflammatory cells and macrophages. This artificial vascular stent met the requirements of transplant experiments and should be further investigated for future clinical applications.     

Tissue-engineered mesh for pelvic floor reconstruction fabricated from silk fibroin scaffold with adipose-derived mesenchymal stem cells

A tissue-engineered mesh fabricated with adipose-derived mesenchymal stem cells (AD-MSCs) cultured on a silk fibroin scaffold is evaluated for use in female pelvic reconstruction. Thirty-five female Sprague Dawley rats were divided into four groups. Group A (n?=?10) were implanted with polypropylene meshes, Group B (n?=?10) with silk fibroin scaffolds and Group C (n?=?10) with tissue-engineered meshes. Group D (n?=?5) acted as the tissue control. The tissue-engineered mesh was produced as follows. AD-MSCs were obtained from adipose tissue of rats designated to Group C. The cells were seeded onto a silk fibroin scaffold, cultured and then observed by scanning electron microscopy (SEM). Histological studies of these meshes were performed at 4 and 12 weeks after implantation and mechanical testing was carried out on all groups before implantation and at 12 weeks after implantation. AD-MSCs displayed fibroblast-like shapes and were able to differentiate into adipocytes or fibroblasts. SEM observation showed that AD-MSCs proliferated and secreted a matrix onto the silk fibroin scaffolds. After implantation of the scaffolds into rats, histological analysis revealed better organized newly formed tissue in Group C than in controls. Group C also had a similar failure force (2.67?±?0.15 vs 2.33?±?0.38 N) and a higher Young’s modulus (2.99?±?0.19 vs 1.68?±?0.20 MPa) than a normal vaginal wall, indicating the potential of this tissue-engineered approach. AD-MSCs were validated as seed cells for tissue engineering. The silk fibroin scaffold thus shows promise for application with AD-MSCs in the fabrication of tissue-engineered mesh with good biocompatibility and appropriate mechanical properties for pelvic floor reconstruction.     

玉米醇溶蛋白动物体内植入后的免疫学分析方法初探

目的：为了考察玉米醇溶蛋白作为生物医用材料的应用前景,需要检测玉米醇溶蛋白植入后的免疫反应。本实验中我们将玉米醇溶蛋白管植入SD（Sprague Dawley）大鼠皮下,流式细胞仪检测植入后大鼠外周血中CD4^＋和CD8^＋T淋巴细胞含量的变化;对不同部位植入材料的组织学切片方法进行改进,用于后续机体免疫反应分析。方法：雄性SD大鼠八只,随机分成两组：空白对照组、材料植入组。分别于植入后一、二和四周对实验大鼠进行眼眶取血,并用流式细胞仪检测外周血中CD4^＋和CD8^＋T淋巴细胞的含量。皮下或肌袋植入材料四周后,将玉米醇溶蛋白管或三维多孔支架连同周围组织一起取出,用改进的石蜡切片方法制备组织切片,HE染色后观察。结果：玉米醇溶蛋白管植入后一周,蛋白管植入组CD4^＋（T—test,P〈0．01）和CD8^＋（T-test,P〈0．05）T淋巴细胞的含量下降。植入后两周和四周,蛋白管植入组CD4^＋和CD8^＋T淋巴细胞的含量（T—test,P〉0．05）均无明显变化。植入后一、二和四周,与空白对照组相比,蛋白管植入组CD4^＋／CD8^＋的比值（T—Test,P〉0．05）均无显著差异。改进后的切片方法可以制备出完整的、并且耐HE（hematoxylin—eosin）和免疫组化染色处理的组织学切片。结论：利用流式细胞仪检测出了大鼠皮下植入玉米蛋白管后外周血中CD4^＋、CD8^＋T淋巴细胞的含量变化。并且,克服了切片制备过程中材料的脆性问题和贴片不牢固问题,可以进行HE和免疫组化染色处理,为进一步深入地分析玉米醇溶蛋白植入机体后的免疫反应打下了基础。     

rhBMP-2对壳聚糖复合成骨细胞后的成骨活性影响

将重组人骨形态发生蛋白2(rhBMP-2)与壳聚糖为主要基质的支架材料相复合,γ射线辐射灭菌后接种上原代培养的大鼠成骨细胞。体外培养3天后扫描电镜观察细胞与材料的黏附情况,可见成骨细胞紧密黏附于材料孔隙内并保持良好的生长状态。将接种有成骨细胞的复合材料植入裸鼠背部皮下,植入8周后X-ray摄片、组织学染色观察植入部位骨形成及支架材料降解情况。X-ray下可见与植入物大小、位置相符的高密度影,组织学染色证实材料的降解及孔隙内硬骨的生成。     

The Graft of Autologous Adipose-Derived Stem Cells in the Corneal Stromal after Mechanic Damage

This study was designed to explore the feasibility of using autologous rabbit adipose derived stem cells (rASCs) as seed cells and polylactic-co-glycolic acid (PLGA) as a scaffold for repairing corneal stromal defects. rASCs isolated from rabbit nape adipose tissue were expanded and seeded on a PLGA scaffold to fabricate cell-scaffold constructs. After 1 week of cultivation in vitro, the cell-scaffold complexes were transplanted into corneal stromal defects in rabbits. In vivo, the autologous rASCs-PLGA constructed corneal stroma gradually became transparent without corneal neovascularization after 12 weeks. Hematoxylin and eosin staining and transmission electron microscopy examination revealed that their histological structure and collagen fibril distribution at 24 weeks after implantation were similar to native counterparts. As to the defect treated with PLGA alone, the stromal defects remained. And scar tissue was observed in the untreated-group. The implanted autologous ASCs survived up to 24 weeks post-transplantation and differentiated into functional keratocytes, as assessed by the expression of aldehyde-3-dehydrogenase1A1 (ALDH1A1) and cornea-specific proteoglycan keratocan. Our results revealed that autologous rASCs could be one of the cell sources for corneal stromal restoration in diseased corneas or for tissue engineering of a corneal equivalent.     

Analysis of the cellular infiltration of benzyl-esterified hyaluronan sponges implanted in rats

Unseeded sponges of benzyl-esterified hyaluronan (HYAFF11) and HYAFF11 coated with unmodified hyaluronan were implanted subcutaneously and intramuscularly in adult rats for 1, 2, 4, 8, 12, and 26 weeks. Explanted samples were stained tincturally using Van Geison, von Kossa, and hematoxylin and eosin, enzyme histochemically by chloroacetate esterase, and by immunohistochemistry for the specific identification of cell types and subpopulations, targeting immature (ED1) and mature macrophages (ED2), MHC-I subset, MHC-II subset, CD54, T-cell alpha-beta receptor, T-cell gamma-delta receptor, CD2, CD4, CD8, natural killer cells, B-cells, vimentin, and TGFbeta. Little or no fibrous tissue formation was observed in any sample in either sponge type at any implantation site. Little degradation was observed in either location until 26 weeks. Little neovascularization occurred at early time periods but was in evidence at 26 weeks. Complete cellular infiltration was observed after 4 weeks, with some mature adipocytes observed within the center of the subcutaneous implants, but these cells were mainly observed around the periphery of the sponges. At 26 weeks, cells were mostly macrophages, with small numbers of T-lymphocytes present. No natural killer cells, B-cells, helper/inducer, or cytotoxic/suppressor T-cells were observed in any sample. Most infiltrating cells were MHC-II positive, and discrete pockets of TGFbeta protein were observed within the sponges. While a sustained inflammatory response was observed within both sponge types at 26 weeks, it was relatively benign and nonspecific immunologically, and inflammatory markers such as MHC-II were declining after 12 weeks. No fibrous capsule was observed, and sponge degradation was only observed at 26 weeks, an event essential for induction of neovasculargenesis. At 26 weeks, there was significant staining for vimentin and ED2 on macrophages. Taken with the pattern of other macrophage activation markers, angiogenic environment and absence of inhibitory matrix proteins, the conditions were consistent with the onset of neoadipogenesis, although this would need to be confirmed by longer term studies. For the generation of neoadipose tissue for clinical therapy, we hypothesize that macrophages require an inflammatory stimulus for infiltration, then a reduction in proinflammatory cytokine secretion simultaneous with angiogenic conditions allowing macrophage differentiation into adipocytes.     

Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats

OBJECTIVE: The aim of this study was to develop a feasible approach to promote bone healing in osteoporotic rats using autogenous bone tissue-engineering and gene transfection of human bone morphogenetic protein 2 (hBMP-2). METHODS: Bone marrow stromal cells (BMSCs) from the left tibia of osteoporotic rats were transfected with the hBMP-2 gene in vitro which was confirmed by immunohistochemistry, in situ hybridization and Western blotting. Autogenous transfected or untransfected BMSCs were seeded on macroporous coral hydroxyapatite (CHA) scaffolds. Each cell-scaffold construct was implanted into a defect site which was created in the ramus of the mandible of osteoporotic rats. Four or eight weeks after implantation in situ hybridization was performed in BMSCs transfected with hBMP-2, X-ray examinations, histological and histomorphological analyses were used to evaluate the effect of tissue-engineered bone on osseous defect repair. RESULTS: Newly formed bone was observed at the margin of the defect 4 weeks after implantation with BMSCs transfected with BMP-2. Mature bone was observed 8 weeks after treatment. In the control group there was considerably less new bone and some adipose tissue was observed at the defect margins 8 weeks after implantation. CONCLUSIONS: Autogenous cells transfected with hBMP-2 promote bone formation in osteoporotic rats. BMSC-mediated BMP-2 gene therapy used in conjunction with bone tissue engineering may be used to successfully treat bone defects in osteoporotic rats. This method provides a powerful tool for bone regeneration and other tissue engineering.     

Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications

Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.     

国产多孔钽材料修复兔胫骨缺损的实验研究

摘要 目的：研究国产多孔钽材料能否在兔胫骨缺损模型中顺利实现骨长入,用于修复胫骨缺损。方法：在36只新西兰大白兔双侧胫骨骨干处建立骨缺损模型,每只动物左右侧缺损随机分组,分别进入实验组(植入多孔坦材料)和对照组（不植入多孔坦材料）。植入后4周、8周和12周取材,通过X线检测以及硬组织切片苏木精伊红染色,检测多孔钽材料与骨界面的骨整合情况。采用推出实验检测多孔钽材料与骨界面的结合强度。结果：将术后不同时间点取得的胫骨标本作X射线拍片分析,4周时,骨缺损端与材料结合部位有骨质生成,在8周时材料表面有骨形成现象,逐渐完全覆盖材料表面,在12周时骨量继续增加,形成覆盖材料并桥接骨缺损断端的骨痂。样本行硬组织切片并行HE染色后检测,植入4周后实验组材料两端被新生骨所覆盖,材料深部的孔隙中也可见少量骨组织长入;植入8周后发现实验组材料与骨组织生长良好,多孔钽材料表面和两端材料孔隙内均有骨组织长入,材料孔隙与组织紧密连接,有骨小梁长入;植入12周时两端骨组织长入深度没有明显变化,但材料表面骨组织继续长入,并完全嵌入圆柱体材料内。材料植入后4周与8周比较差异无统计学意义（P＞0.05）,材料植入后8周与12周比较差异有统计学意义（P<0.05）。将植入4周、8周和12周后含材料样本置于动态疲劳试验机上进行推出实验,随时间延长所需推出力明显增加,植入后4周和8周相比,虽然后者所需推力较大,但两者比较差异无统计学意义（P＞0.05）,而8周和12周比较则差异有统计学意义（P<0.05）。结论：国产多孔坦材料能在胫骨缺损中实现与骨整合,能用于皮质骨缺损修复。     

The restoration of full-thickness cartilage defects with mesenchymal stem cells (MSCs) loaded and cross-linked bilayer collagen scaffolds on rabbit model

Cartilage has a limited self-repair capability and the repair of large cartilage defects remains a challenge in clinic. This study aimed to investigate the effect of mesenchymal stem cells (MSCs) loaded three-dimensional bilayer collagen scaffold for cartilage repair. Cross-linked three-dimensional bilayer collagen scaffolds seeded with or without MSCs were implanted into large cartilage defects (4 mm in diameter; 3 mm in depth) in rabbit knees. The untreated cartilage defects served as control. The tissue response was evaluated at 6 and 12 weeks after implantation by general histology and semi-quantitative histological grading systems. In addition, the repaired tissues were evaluated by mechanical test at 12 weeks after implantation. The MSCs-loaded collagen scaffold group showed the most hyaline cartilage, highest histological scores and compressive modulus. Moreover, it showed a good integration with the subchondral bone and adjacent cartilage. The structure of the novel bilayer collagen scaffolds provided architectural support for the differentiation of MSCs and demonstrated successful induction of in vivo chondrogenesis. These findings suggested that MSCs-loaded bilayer collagen scaffold could be an appealing candidate to be used for cartilage regeneration.     

Flexible polyimide microelectrode array for in vivo recordings and current source density analysis

This work presents implantable, flexible polymer-based probes with embedded microelectrodes for acute and chronic neural recordings in vivo, as tested on rodents. Acute recordings using this array were done in mice under urethane anesthesia and compared to those made using silicon-based probes manufactured at the Center for Neural Communication Technology, University of Michigan. The two electrode arrays yielded similar results. Recordings with chronically implanted polymer-based electrodes were performed for 60 days post-surgically in awake, behaving rats. The microelectrodes were used to monitor local field potentials and capture laminar differences in function of cortex and hippocampus, and produced response waveforms of undiminished amplitude and signal-to-noise ratios 8 weeks after chronic implantation. The polymer-based electrodes could also be connected to a lesion current to mark specific locations in the tissue. Current source density (CSD) analysis from the recordings depicted a source - sink-composition. Tissue response was assessed 8 weeks after insertion by immunochemical labeling with glial fibrillary acidic protein (GFAP) to identify astrocytes, and histological analysis showed minimal tissue reaction to the implanted structures.     

Investigating the effects of preinduction on human adipose-derived precursor cells in an athymic rat model

The osteogenic potential of human adipose-derived precursor cells seeded on medical-grade polycaprolactone-tricalcium phosphate scaffolds was investigated in this in vivo study. Three study groups were investigated: (1) induced--stimulated with osteogenic factors only after seeding into scaffold; (2) preinduced--induced for 2 weeks before seeding into scaffolds; and (3) uninduced--cells without any introduced induction. For all groups, scaffolds were implanted subcutaneously into the dorsum of athymic rats. The scaffold/cell constructs were harvested at the end of 6 or 12 weeks and analyzed for osteogenesis. Gross morphological examination using scanning electron microscopy indicated good integration of host tissue with scaffold/cell constructs and extensive tissue infiltration into the scaffold interior. Alizarin Red histology and immunostaining showed a heightened level of mineralization and an increase in osteonectin, osteopontin, and collagen type I protein expression in both the induced and preinduced groups compared with the uninduced groups. However, no significant differences were observed in these indicators when compared between the induced and preinduced groups.     

Ectopic Osteogenesis and Scaffold Biodegradation of Nano-Hydroxyapatite-Chitosan in a Rat Model

The bone-formation and scaffold-biodegradation processes have not been fully characterized. This study aimed to determine the osteogenic ability of nHA-CS osteo-induced bone marrow mesenchymal stem cell (BMSC) composites and to explore the relationship between bone formation and scaffold biodegradation. The nHA-CS osteo-induced BMSC composites (nHA-CS+cells group) and the nHA-CS scaffolds (nHA-CS group) were implanted into the femoral spatium intermusculare of SD rats. At 2, 4, 6, 8, and 12 weeks post-implantation, the rat femurs were scanned using computerized tomography (CT), and the CT values of the implants were measured and comparatively analyzed. The implants were then harvested and subjected to hematoxylin and eosin (HE) and Masson''s trichrome staining, and the percentages of bone area, scaffold area and collagen area were compared between the two groups. The CT values of the implants were higher in the nHA-CS+cells group than the nHA-CS group at the same time points (P < 0.05). Histological analysis revealed that de novo bone and collagen formation in the pores of the scaffolds gradually increased from 2 weeks post-implantation in both groups and that the scaffold gradually degraded as bone formation proceeded. However, more de novo bone and collagen formation and scaffold degradation occurred in the nHA-CS+cells group than in the nHA-CS group at the same time points (P < 0.05). In conclusion, nHA-CS osteo-induced BMSC composites are promising bone tissue engineering substitutes, and osteo-induced BMSCs can significantly enhance the osteogenic ability and play an active role in the degradation of nHA-CS scaffolds on par with bone formation.     

Durable engraftment of genetically modified FVIII‐secreting autologous bone marrow stromal cells in the intramedullary microenvironment

Genetically modified FVIII‐expressing autologous bone marrow‐derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture‐expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII‐secreting BMSC in vivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture‐expanded primary canine BMSCs. FVIII‐secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implanted × FVIII activity secreted/million BMSCs/24 hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti‐human FVIII antibody having negligible cross‐reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48 weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti‐human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII‐secreting BMSCs.     

A novel small-diameter vascular graft: in vivo behavior of biodegradable three-layered tubular scaffolds

Small-diameter vascular grafts are potential substitutes for damaged vessels in patients, but most biodegradable grafts available now are not strong enough. The present study examined the burst strength, radial compliance, suture retention strength for a novel biodegradable tubular scaffold and investigated its behavior in vivo. The tubular scaffold (6-mm i.d., 4 cm long) has three layers including porous polylacticglycolic- acid in both inner and outer layers, a compact polyurethanes layer in midst. Bone marrow stromal cells (bMSCs) were seeded on the scaffolds and cultured for 7 days in vitro to construct tissue engineered vascular grafts which were then implanted in canine abdominal aorta. After 1, 3, 6, 12 and 24 weeks, the grafts were retrieved and evaluated histologically, angiographically and immunohistochemically. The biodegradable tubular scaffolds showed wall thickness of 0.295 mm to 0.432 mm; radial compliance of 3.80%/100 mmHg approximately 0.57%/100 mmHg, burst strength of 160 kPa approximately 183 kPa, and suture retention strength of 1959 N/cm(2) approximately 3228N/cm(2). The implanted grafts were fully patent without any signs of dilation or obstruction after 3 months' implantation. Scanning electron microscopy revealed a confluence endothelial cell layer spreading on the inner surface of the grafts. Immunohistochemistry of the retrieved grafts showed that vWF-stainin, alphaSMA-staining were positive in the inner and medium layer respectively. Masson's trichrome staining showed that amount of collagen fibers existed in the grafts wall. Overall, these novel three-layered scaffolds exhibited favourable mechanical strength, long term patency and good remodeling in vivo.     

仿生聚己内酯支架用于乳房组织工程的可行性研究

目的探讨聚己内酯（PCL）乳房形态支架用于组织工程乳房的构建的可能性。 方法通过熔融沉积3D打印制备形态仿生的PCL支架,测量其机械性能,并使用新西兰大白兔动物模型,皮下植入该PCL支架12周和18周后,利用核磁共振成像（MRI）观察支架内部新生组织分布情况,在组织学（HE、Masson及EVG染色）上评估支架内部的脂肪、纤维及血管的分布情况,并进一步使用qRT-PCR检测了12周时PCL支架内部组织的成脂相关基因（PPAR-γ、C/EBP-β、AP-2）、炎症相关基因TNF-α及巨噬细胞标记物F4-80的表达情况,同时使用凝胶渗透色谱法分析了PCL植入体内后平均分子量的变化。2组间均数比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验,配对设计的均数比较采用配对t检验。 结果制备的PCL支架孔隙率为（85.30±1.12）%,压缩模量为（8.18±1.39）MPa,植入新西兰大白兔动物模型皮下12周后,MRI影像学显示脂肪组织已由支架周围向内部侵入,HE、Masson及EVG染色同样在该支架边缘观察到部分新生脂肪组织及血管,而支架内部则以疏松排列的纤维组织为主;与原生脂肪比较,12周PCL支架内组织的基因表达分析成脂相关基因C/EBPβ表达水平（2.32±0.28比1.00±0.02）升高,而巨噬细胞标记物F4/80表达（0.80±0.12比1.00±0.03）降低（P均< 0.01）;18周后,HE染色证实支架内部已充满脂肪组织。基因表达证实,与原生脂肪比较,支架内部组织C/EBP-β （3.30±0.63比1.00±0.02）,PPAR-γ （1.81±0.71比0.99±0.02）及AP-2表达水平（1.38±0.16比1.01±0.01）升高（P均< 0.01）;而TNF-α（0.50±0.15比1.00±0.01）及F4/80表达水平（0.52±0.09比1.00±0.03）均降低（P均< 0.001）。而植入体内PCL支架的分子量（M_n）在18周内变化不大[（65.04±2.24）kDa比（64.20±4.09） kDa]。 结论PCL支架具有较好的生物相容性,可用于组织工程乳房的构建,该新西兰大白兔动物模型的建立有利于乳房组织工程的进一步临床转化。     

Kinetics of in vivo bone deposition by bone marrow stromal cells within a resorbable porous calcium phosphate scaffold: an X-ray computed microtomography study

Resorbable ceramic scaffolds based on Silicon stabilized tricalcium phosphate (Si-TCP) were seeded with bone marrow stromal cells (BMSC) and ectopically implanted for 2, 4, and 6 months in immunodeficient mice. Qualitative and quantitative evaluation of the scaffold material was performed by X-ray synchrotron radiation computed microtomography (microCT) with a spatial resolution lower than 5 microm. Unique to these experiments was that microCT data were first collected on the scaffolds before implantation and then on the same scaffolds after they were seeded with BMSC, implanted in the mice and rescued after different times. Volume fraction, mean thickness and thickness distribution were evaluated for both new bone and scaffold phases as a function of the implantation time. New bone thickness increased from week 8 to week 16. Data for the implanted scaffolds were compared with those derived from the analysis of the same scaffolds prior to implantation and with data derived from 100% hydroxyapatite (HA) scaffold treated and analyzed in the same way. At variance with findings with the 100% HA scaffolds a significant variation in the density of the different Si-TCP scaffold regions in the pre- and post-implantation samples was observed. In particular a post-implantation decrease in the density of the scaffolds, together with major changes in the scaffold phase composition, was noticeable in areas adjacent to newly formed bone. Histology confirmed a better integration between new bone and scaffold in the Si-TCP composites in comparison to 100% HA composites where new bone and scaffold phases remained well distinct.