新型旋转壁式生物反应器内三维组织工程骨的构建

利用微载体悬浮培养法将成骨细胞在旋转壁式生物反应器内进行大规模扩增,并检测细胞的组织形态和生物功能.然后以此作为种子细胞,分别以2×10⁶个/ml和1×10⁶个/ml两种密度接种到支架材料上,于旋转壁式生物反应器(RWV)内进行三维组织工程骨的构建.并将所构建的骨组织分别进行倒置显微镜(inverted microscope)、扫描电镜(SEM)、碱性磷酸酶(ALP)、矿化结构和AO/EB双重荧光染色等生物学性能检测,以及对培养过程的营养物质代谢情况进行监控和分析.结果表明,在RWV中培养的骨组织生长良好,分泌大量胶原纤维,并有矿化基质和新骨样组织形成. 由上述结果可断定,通过RWV内部流体对流所产生的应力刺激,可提高成骨细胞碱性磷酸酶的活性表达,并加速矿化结节的形成,从而完成成骨细胞的快速增殖与分化以及工程化组织的三维构建.     

组织工程的一般考虑

组织工程的最终目标是通过体外增减活细胞与其胞外环境相互作用而发育成具胡生物活性的组织或器官替代物,替换、修复组织或器官,或强化其生物学功能。本文主要讨论细胞移植和组织重建的影响因素和可能的方法。     

构建大段组织工程骨的新型生物反应器的设计与制造

目的:设计、制造一种新的灌注式生物反应器,专门用于高效地构建大体积、β-磷酸三钙组织工程骨。方法:在普通模式灌注生物反应器的灌流室内生成间断性低压环境(-0.01 mpa,0.5 Hz),用材料色素颗粒洗脱实验进行验证后,将复合兔骨髓间充质干细胞的大段、管状β-磷酸三钙材料分别在静态、反应器内常压灌注和间断低压灌注三种环境下培养4周。期间收集培养液检测葡萄糖日耗量、细胞活力(MTT比色法)、碱性磷酸酶比活性、骨桥蛋白水平,并进行硬组织切片检查。结果:色素颗粒洗脱实验证明,间断性低压可以改善低流量液流在材料内的分布;在培养2周和4周时,负压灌注组日均葡萄糖消耗量和细胞活力均显著高于常压灌注组:(t=20.254 P<0.05,t=64.794 P<0.05)及(t=17.586 P<0.05,t=7.583 P<0.05);碱性磷酸酶(ALP)比活性测定和骨桥蛋白水平(OPN)反映间断低压灌注组中骨髓间充质细胞向成骨细胞分化效率更高,但高峰相晚于常压灌注组和静态培养组;在间断低压灌注组中材料深部的占孔率最高,并且分布更均匀。结论:此新型灌注式生物反应器适用于构建大体积、特殊构型组织工程骨;其高效的促进细胞增殖效应可减少初始复合的种子细胞数量,缩短构建周期。     

以人胎盘来源干细胞为种子细胞构建组织工程软骨

通过胰酶消化法分离培养人胎盘来源干细胞（human placenta-derived stem cells,hPDSCs）,对其生物学性状进行检测,在一定条件下使其向软骨细胞诱导分化;将hPDSCs和制备的胶原海绵支架材料复合体外构建组织工程软骨组织,移植到裸鼠体内后观察其形成软骨组织的能力,为以hPDSCs作为种子细胞进行组织工程软骨组织的构建提供理论基础.研究发现hPDSCs具有间充质干细胞性状和良好的增殖能力,能连续培养30代以上保持未分化状态;将hPDSCs培养于软骨细胞诱导培养基中,可以分化形成具有生物学功能的软骨细胞.将hPDSCs与胶原海绵支架材料在诱导培养基中复合,7天后可以观察到有软骨样结构形成,Ⅱ型胶原表达阳性;裸鼠体内移植实验证实,hPDSCs与胶原海绵复合后可以在体内形成软骨组织,组织学观察软骨陷窝形成,并且Ⅱ型胶原阳性表达.研究结果表明hPDSCs具有向软骨细胞分化的潜能,与胶原海绵支架材料复合后可以构建形成具有生物学功能的软骨组织,为临床工作中软骨缺损的修复治疗提供了新的治疗策略,具有广阔的临床应用前景。     

成骨细胞的骨形成调控机制

成骨细胞在骨的形成、发育、改建、修复过程中发挥重要作用,多种生长因子可影响成骨细胞的增殖、分化及其合成细胞外基质的作用。多种生长因子对成骨细胞的作用既有相同之处,也有不同点。寻找一种或联合应用几种不同的生长因子达到既能促进成骨细胞增殖又保持其成骨活性将是今后研究的热点。     

组织器官三维构建及原位组织工程概念——组织工程连载之四

组织器官三维构建就是把种子细胞和支架材料结合而获得设计的组织或器官,属于组织工程的核心内容,也最能体现组织工程的技术水平,如血管、气管的构建。由于传统组织工程存在缺陷,Shimizu于1998年首先提出了原位组织工程的概念,它是运用组织工程学基本原理,通过各种方法诱导移植的外源性的种子细胞或内源性的缺损组织局部细胞发生迁移、增殖、分化形成新生组织修复缺损。原位组织工程最大的特点是不依赖体外的细胞培养装置--生物反应器。原位组织工程是传统离体组织工程的有益补充。离体组织工程仍具有广阔的发展前景。     

组织工程中应力对骨髓间充质干细胞分化为成骨细胞的影响

骨髓间充质干细胞具有自我复制、未分化的特点,并可在不同条件下分化为中胚层起源的多种细胞,是一种成体多能干细胞。就组织工程而言,良好的种子细胞是组织工程技术的关键,骨髓间充质干细胞的性质决定了其在骨组织工程领域中的重要地位。此外,骨骼系统属于机体的运动系统,承担体重是骨骼的重要功能之一;而且,人体内几乎所有的细胞都会受到力学因素的影响,故有必要研究力学因素对骨髓间充质干细胞诱导分化为成骨细胞的作用,为骨髓间充质干细胞的体外扩增、诱导分化及培养提供一种新途径。     

软骨基质来源定向结构支架复合软骨细胞体外构建组织工程软骨的研究

目的：探讨采用软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下生成组织工程软骨的可能性。方法：制备牛关节软骨细胞外基质材料,利用温度梯度热诱导相分离技术构建具备垂直定向孔道结构的软骨支架,同时采用传统冷冻干燥方法制备非定向支架,检测两组支架的力学性能;提取兔关节软骨细胞,分别接种两组支架,体外静态培养2周及4周后取材,对构建的组织工程软骨进行组织切片染色、生物化学分析及生物力学检测。结果：定向软骨支架的压缩弹性模量数值明显高于非定向软骨支架,体外培养时定向支架上种子细胞在3-9d内增殖高于非定向支架,差异有统计学意义（P〈0．05）;体外静态培养4周后形成的两组新生组织工程软骨进行软骨特异性染色均呈阳性,在定向组新生软骨切片中在垂直方向上可见大量呈规则平行排列的粗大胶原纤维,两组新生软骨的生物化学检测包括总DNA、总GAG及总胶原含量差异无统计学意义（P〉0．05）。定向组织工程软骨压缩弹性模量在2周及4周时均高于非定向组织工程软骨,差异有统计学意义（P〈0．05）。但两组组织工程软骨上述指标均显著低于正常关节软骨（P〈0．05）。结论：软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞,在体外静态培养条件下能够成功生成具有定向纤维结构的组织工程软骨,并可以有效促进新生软骨组织力学性能的提升,在软骨组织工程中具有良好的应用前景。     

以HaCaT细胞为种子细胞构建新型人组织工程皮肤

目的:评估以人永生化角朊细胞HaCaT为种子细胞构建新型人组织工程皮肤的可行性。方法:对HaCaT进行无血清培养(DK-SFM)及传代培养;取处于对数生长期的细胞进行接种;利用物理化学方法,采用SD大鼠网状层真皮制备(Acelluar dermal matrix,ADM);应用MTT比色法作生长曲线观察HaCaT细胞长满真皮支架所需时间。HE染色法观察脱细胞前后真皮支架和细胞单层及初步形成的细胞多层。结果:脱细胞真皮的细胞成分消失,组织疏松,为理想的组织皮肤支架;将HaCaT细胞按2×10~5/cm~2密度接种到ADM上,细胞长满支架的时间为5天。结论:在SD大鼠脱细胞真皮支架上以HaCaT细胞为种子细胞可以构建新型人组织工程皮肤     

组织工程的基本科学问题

疾病和创伤引起的组织、器官的缺损或功能障碍是人类健康所面临的主要危害之一,也是导致人类死亡的最主要原因。如何克服自体或异体组织、器官移植中存在的“以创伤修复创伤”、供体来源不足等缺陷,从根本上解决组织、器官缺损修复和功能重建等问题,己成为生命科学领域的国际性前沿课题。组织工程的提出、建立和发展,为解决这一问题提供了新的策略,     

Antigenic Profile of Osteoblasts Present in Human Bone Tissue Sections

The antigenic profile of human osteoblasts was previously analyzed by our group using primary cultures as study samples. These studies suggested a novel functional approach to this cell population. Osteoblasts have a characteristic antigenic profile and share antigens in common with other cell populations that also originate in the bone marrow. Some of the detected antigens are constitutively expressed, while others are modulated by different factors and/or cytokines. The aim of the present study was to analyze the antigens present in osteoblasts in vivo, since the presence of certain biomolecules in fetal bovine serum may modulate the antigenic expression, compromising the results. For this purpose, human bone tissue sections were analyzed with a wide panel of mAbs and using the immunoperoxidase technique. CD10, CD44 and alkaline phosphatase antigens and IL-12, IL-18 and IFNγ cytokines were detected in osteoblasts in the bone tissue. However, CD80 and HLA-DR antigens were not found in all samples and when present their expression was weak. The expression of CD54 antigen was moderate or weak. These results allow data obtained by the primary culture of osteoblast-like cells to be endorsed.     

Repair of Segmental Bone Defect Using Totally Vitalized Tissue Engineered Bone Graft by a Combined Perfusion Seeding and Culture System

Background

The basic strategy to construct tissue engineered bone graft (TEBG) is to combine osteoblastic cells with three dimensional (3D) scaffold. Based on this strategy, we proposed the “Totally Vitalized TEBG” (TV-TEBG) which was characterized by abundant and homogenously distributed cells with enhanced cell proliferation and differentiation and further investigated its biological performance in repairing segmental bone defect.

Methods

In this study, we constructed the TV-TEBG with the combination of customized flow perfusion seeding/culture system and β-tricalcium phosphate (β-TCP) scaffold fabricated by Rapid Prototyping (RP) technique. We systemically compared three kinds of TEBG constructed by perfusion seeding and perfusion culture (PSPC) method, static seeding and perfusion culture (SSPC) method, and static seeding and static culture (SSSC) method for their in vitro performance and bone defect healing efficacy with a rabbit model.

Results

Our study has demonstrated that TEBG constructed by PSPC method exhibited better biological properties with higher daily D-glucose consumption, increased cell proliferation and differentiation, and better cell distribution, indicating the successful construction of TV-TEBG. After implanted into rabbit radius defects for 12 weeks, PSPC group exerted higher X-ray score close to autograft, much greater mechanical property evidenced by the biomechanical testing and significantly higher new bone formation as shown by histological analysis compared with the other two groups, and eventually obtained favorable healing efficacy of the segmental bone defect that was the closest to autograft transplantation.

Conclusion

This study demonstrated the feasibility of TV-TEBG construction with combination of perfusion seeding, perfusion culture and RP technique which exerted excellent biological properties. The application of TV-TEBG may become a preferred candidate for segmental bone defect repair in orthopedic and maxillofacial fields.     

Engineered Tissue Folding by Mechanical Compaction of the Mesenchyme

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Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child''s small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols^{[2, 3]}. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel™, and NaHCO₃. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size^{[1, 4, 5]}. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures^[6]. Once the tissue has solidified at 37°C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.Open in a separate windowClick here to view.^{(86M, flv)}     

Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PP_i). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2P_i). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PP_i-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.     

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5^th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.Download video file.^{(95M, mp4)}     

组织工程皮肤生物反应器的研究与设计

目的设计一套生物反应器,能针对不同支架材料———细胞复合物进行构建组织工程皮肤。方法根据皮肤的自身生长特点和不同支架材料-细胞复合物的特性,模拟皮肤的生长环境和力学环境,通过生物反应器解决组织工程皮肤构建中支架的装夹和气液界面问题。结果生物反应器由控制系统和生物反应器主体两部分构成,能提供对多种皮肤细胞复合物的动态培养。结论皮肤生物反应器能够满足不同组织工程皮肤产品的需要。能够形成气液界面和模拟生物力学的刺激。     

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.