A decellularized nerve matrix scaffold inhibits neuroma formation in the stumps of transected peripheral nerve after peripheral nerve injury

Traumatic painful neuroma is an intractable clinical disease characterized by improper extracellular matrix(ECM)deposition around the injury site.Studies have shown that the microstructure of natural nerves provides a suitable microenvironment for the nerve end to avoid abnormal hyperplasia and neuroma formation.In this study,we used a decellularized nerve matrix scaffold(DNM-S)to prevent against the formation of painful neuroma after sciatic nerve transection in rats.Our results showed that the DNM-S effectively reduced abnormal deposition of ECM,guided the regeneration and orderly arrangement of axon,and decreased the density of regenerated axons.The epineurium-perilemma barrier prevented the invasion of vascular muscular scar tissue,greatly reduced the invasion ofα-smooth muscle actin-positive myofibroblasts into nerve stumps,effectively inhibited scar formation,which guided nerve stumps to gradually transform into a benign tissue and reduced pain and autotomy behaviors in animals.These findings suggest that DNM-S-optimized neuroma microenvironment by ECM remodeling may be a promising strategy to prevent painful traumatic neuromas.

Smart scaffolds in bone tissue engineering: A systematic review of literature

AIM: To improve osteogenic differentiation and attachment of cells.METHODS: An electronic search was conducted inPub Med from January 2004 to December 2013. Studies which performed smart modifications on conventional bone scaffold materials were included. Scaffolds with controlled release or encapsulation of bioactive molecules were not included. Experiments which did not investigate response of cells toward the scaffold(cell attachment, proliferation or osteoblastic differentiation) were excluded. RESULTS: Among 1458 studies, 38 met the inclusion and exclusion criteria. The main scaffold varied extensively among the included studies. Smart modifications included addition of growth factors(group Ⅰ-11 studies), extracellular matrix-like molecules(group Ⅱ-13 studies) and nanoparticles(nano-HA)(group Ⅲ-17 studies). In all groups, surface coating was the most commonly applied approach for smart modification of scaffolds. In group I, bone morphogenetic proteins were mainly used as growth factor stabilized on polycaprolactone(PCL). In group Ⅱ, collagen 1 in combination with PCL, hydroxyapatite(HA) and tricalcium phosphate were the most frequent scaffolds used. In the third group, nano-HA with PCL and chitosan were used the most. As variable methods were used, a thorough and comprehensible compare between the results and approaches was unattainable.CONCLUSION: Regarding the variability in methodology of these in vitro studies it was demonstrated that smart modification of scaffolds can improve tissue properties.

生物医用支架仿生设计及在组织工程中的应用

背景:基于解剖学对生物组织功能与结构的认识,对于具有恢复、维持或改善组织功能的生物活性材料仿生设计是目前再生医学领域研究的热点。目的:从生物医用支架的机械性能、三维空间结构和生化活性对细胞行为的影响进行讨论,并综述生物医用支架在组织工程领域的应用。方法:应用计算机检索中国知网、万方、Web of Science、PubMed数据库2003年1月至2023年4月发表的文献,中文检索词为“细胞外基质,组织工程,支架,生物材料,仿生结构,机械性能,三维结构,腱骨界面,骨软骨,神经导管,人造血管”;英文检索词为“extracellular matrix,tissue engineering,scaffolds,biomimetic structures,biomaterials,tendon bone interfaces,osteochondral,neural conduits,artificial blood vessels”。结果与结论:细胞处在一个复杂且动态变化的三维环境中,因此细胞外基质是生物材料模拟的最终目标,在设计生物医用支架的仿生结构时需要与其所处真实的微环境相似,让细胞可以正常地贴壁、生长和迁移,并保持其多样的生理功能。生物医用支架在机械性能、三维空间结构以及生物化学性质方面对细胞外基质的仿生设计可以对组织修复过程中的细胞起到决定性作用,从而影响组织修复最后的结果。进行仿生设计的生物医用支架在腱骨界面、骨软骨界面、神经、血管再生等领域已有广泛的应用,在临床上提供了一个有前途的新思路。

Cardiac tissue-derived extracellular matrix scaffolds for myocardial repair:advantages and challenges

Decellularized extracellular matrix(dECM)derived from myocardium has been widely explored as a nature scaffold for cardiac tissue engineering applications.Cardiac dECM offers many unique advantages such as preservation of organ-specific ECM microstructure and composition,demonstration of tissue-mimetic mechanical properties and retention of biochemical cues in favor of subsequent recellularization.However,current processes of dECM decellularization and recellularization still face many challenges including the need for balance between cell removal and extracellular matrix preservation,efficient recellularization of dECM for obtaining homogenous cell distribution,tailoring material properties of dECM for enhancing bioactivity and prevascularization of thick dECM.This review summarizes the recent progresses of using dECM scaffold for cardiac repair and discusses its major advantages and challenges for producing biomimetic cardiac patch.

Neuron-fibrous scaffold interfaces in the peripheral nervous system: a perspective on the structural requirements

The nerves of the peripheral nervous system are not able to effectively regenerate in cases of severe neural injury.This can result in debilitating consequences,including morbidity and lifelong impairments affecting the quality of the patient’s life.Recent findings in neural tissue engineering have opened promising avenues to apply fibrous tissue-engineered scaffolds to promote tissue regeneration and functional recovery.These scaffolds,known as neural scaffolds,are able to improve neural regeneration by playing two major roles,namely,by being a carrier for transplanted peripheral nervous system cells or biological cues and by providing structural support to direct growing nerve fibers towards the target area.However,successful implementation of scaffold-based therapeutic approaches calls for an appropriate design of the neural scaffold structure that is capable of up-and down-regulation of neuron-scaffold interactions in the extracellular matrix environment.This review discusses the main challenges that need to be addressed to develop and apply fibrous tissue-engineered scaffolds in clinical practice.It describes some promising solutions that,so far,have shown to promote neural cell adhesion and growth and a potential to repair peripheral nervous system injuries.

Evaluation of an extracellular matrix-derived acellular biphasic scaffold/cell construct in the repair of a large articular high-load-bearing osteochondral defect in a canine model

Osteochondral lesion repair is a challenging area of orthopedic surgery. Here we aimed to develop an extracellular matrix-derived, integrated, biphasic scaffold and to investigate the regeneration potential of the scaffold loaded with chondrogenically-induced bone marrow-derived mesenchymal stem cells （BMSCs） in the repair of a large, high-load-bearing, osteochondral defect in a canine model. Methods The biphasic scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and characterized by scanning electron microscopy （SEM） and micro-computed tomography （micro-CT）. Osteochondral constructs were fabricated in vitro using chondrogenically-induced BMSCs and a biphasic scaffold, then assessed by SEM for cell attachment. Osteochondral defects （4.2 mm （diameter） ×6 mm （depth）） were created in canine femoral condyles and treated with a construct of the biphasic scaffold/chondrogenically-induced BMSCs or with a cell-free scaffold （control group）. The repaired defects were evaluated for gross morphology and by histological, biochemical, biomechanical and micro-CT analyses at 3 and 6 months post-implantation. Results The osteochondral defects of the experimental group showed better repair than those of the control group. Statistical analysis demonstrated that the macroscopic and histologic grading scores of the experimental group were always higher than those of the control group, and that the scores for the experimental group at 6 months were significantly higher than those at 3 months. The cartilage stiffness in the experimental group （6 months） was （6.95±0.79） N/mm, 70.77% of normal cartilage; osteochondral bone stiffness in the experimental group was （158.16±24.30） N/mm, 74.95% of normal tissue; glycosaminoglycan content of tissue-engineered neocartilage was （218±21.6） tJg/mg （dry weight）, 84.82% of native cartilage. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at 3 and 6 months, with no significant difference between the experimental and control groups. Conclusion The extracellular matrix-derived, integrated, biphasic scaffold shows potential for the repair of large, high-load-bearing osteochondral defects.

In vitro cartilage production using an extracellular matrix-derived scaffold and bone marrow-derived mesenchymal stem cells

Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix （ECM）-derived scaffold for in vivo cartilage tissue engineering in nude mice.However,before these scaffolds can be used in clinical applications in vivo,the in vitro effects should be further explored.Methods We produced cartilage in vitro using a natural cartilage ECM-derived scaffold.The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy （SEM）,micro-computed tomography （micro-CT）,histological staining,cytotoxicity assay,biochemical and biomechanical analysis.After being chondrogenically induced,the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry.The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining.Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.Results SEM and micro-CT revealed a 3-D interconnected porous structure.The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris,and stained positive for safranin O and collagen Ⅱ.Viability staining indicated no cytotoxic effects of the scaffold.Biochemical analysis showed that collagen content was （708.2±44.7）μg/mg,with GAG （254.7±25.9） μg/mg.Mechanical testing showed the compression moduli （E） were （1.226±0.288） and （0.052±0.007） MPa in dry and wet conditions,respectively.Isolated canine bone marrow-derived stem cells （BMSCs） were induced down a chondrogenic pathway,labeled with PKH26,and seeded onto the scaffold.Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM.The cell-scaffold constructs contained pink,smooth and translucent cartilage-like tissue after 3 weeks of culture.We observed evenly distributed cartilage ECM proteoglycans and collagen type Ⅱ around seeded BMSCs on the surface and inside the pores throughout the scaffold.Conclusion This study stuggests that a cartilage ECM scaffold holds much promise for in vitro cartilage tissue engineering.

Crosslinking strategies for preparation of extracellular matrix-derived cardiovascular scaffolds

Heart valve and blood vessel replacement using artificial prostheses is an effective strategy for the treatment of cardiovascular disease at terminal stage.Natural extracellular matrix(ECM)-derived materials(decellularized allogeneic or xenogenic tissues)have received extensive attention as the cardiovascular scaffold.However,the bioprosthetic grafts usually far less durable and undergo calcification and progressive structural deterioration.Glutaraldehyde(GA)is a commonly used crosslinking agent for improving biocompatibility and durability of the natural scaffold materials.However,the nature ECM and GA-crosslinked materials may result in calcification and eventually lead to the transplant failure.Therefore,studies have been conducted to explore new crosslinking agents.In this review,we mainly focused on research progress of ECM-derived cardiovascular scaffolds and their crosslinking strategies.

Decellularized extracellular matrix particle-based biomaterials for cartilage repair applications

Cartilage Decellularized ExtraCellular Matrix(dECM)materials have shown promising cartilage regenera-tion capacity due to their chondrogenic bioactivity.However,the limited retention of ECM components and the reduced integrity of functional ECM molecules during traditional decellularization processes im-pair the biomimicry of these materials.The current study aims to fabricate biomimetic materials con-taining decellularized cartilage particles that have an intact molecular structure and native composition as biomaterial inks and hydrogels for cartilage repair.For this,we established a novel two-fraction de-cellularization strategy for the preparation of reconstituted dECM(rdECM)particles by mixing the two-fraction components,as well as a one-fraction decellularization strategy for the preparation of biomimetic dECM(bdECM)particles.Hyaluronic acid-tyramine(THA)hydrogels containing rdECM or bdECM particles were produced and characterized via rheological test,swelling and stability evaluation,and compression test.The results showed that our novel decellularization strategies preserved intact proteoglycans and collagen at a higher retention rate with adequate DNA removal compared to traditional methods of de-cellularization.The addition of rdECM or bdECM particles significantly increased the shear moduli of the THA bioinks while preserving their shear-thinning properties.bdECM particle-embedded THA hydrogels also achieved long-term stability with a swelling ratio of 70%and high retention of glycosaminoglycans and collagen after long-term incubation,while rdECM particle-embedded THA hydrogels showed unsat-isfactory stability as self-standing biomaterials.Compared to pure THA hydrogels,the addition of bdECM particles significantly enhanced the compression moduli.In summary,our decellularization methods are successful in the retention of functional and intact cartilage components with high yield.Both rdECM and bdECM particles can be supplemented in THA bioinks for biomimetic cartilage 3D printing.Hydro-gels with cartilage bdECM particles possess the functional structure and the natural composition of car-tilage ECM,long-term stability,and enhanced mechanical properties,and are promising biomaterials for cartilage repair.

脱细胞真皮基质水凝胶的制备及生物学评价

背景:最近脱细胞真皮基质水凝胶因其可注射性和能填充不规则空间,在组织填充与修复中有着广阔的应用前景,而细胞相容性和生物相容性是组织修复的关键,探究脱细胞真皮基质水凝胶的相容性具有重要意义。目的:构建一种脱细胞真皮基质水凝胶,用于组织填充和修复。方法:制备可注射型脱细胞真皮基质水凝胶,扫描电镜下观察其微观形貌。将质量浓度8,10 g/L的脱细胞真皮基质水凝胶分别与骨髓间充质干细胞共培养,检测细胞增殖与细胞相容性。将蛋白质量浓度0.25,0.5 g/L的脱细胞真皮基质水凝胶分别注射至同一SD大鼠背部皮下不同部位,术后2,4周,观察水凝胶降解情况。将蛋白质量浓度1,1.5,2 g/L的脱细胞真皮基质水凝胶分别注射至同一SD大鼠背部皮下不同部位,术后8,16周,观察水凝胶降解情况,并进行组织学分析。结果与结论:①扫描电镜下可见,脱细胞真皮基质水凝胶呈现一种不规则随机走向的多孔纤维结构,孔径大小不一。②共培养1,3,5 d后,两种浓度的水凝胶对骨髓间充质干细胞的增殖无影响;共培养1,2,3 d后的Live/Dead染色显示,两种浓度的水凝胶对骨髓间充质干细胞的增殖无影响,但水凝胶上的细胞形态发生了一定变化。③蛋白质量浓度0.25 g/L的水凝胶皮下注射2周后降解,0.5 g/L的水凝胶皮下注射4周后降解,1,1.5,2 g/L的水凝胶皮下注射16周均未降解,甚至可能保留更长时间。苏木精-伊红染色显示,皮下注射8周后,3种蛋白质量浓度水凝胶组织较为致密,出现少量的炎症浸润,2 g/L组有微血管生成;16周后,水凝胶组织变得松散,以1 g/L组较明显,炎症浸润减轻。免疫组化染色显示,随着注射时间的延长,水凝胶中的微血管数量增加;并且随着蛋白质量浓度的增加,水凝胶中的微血管数量增加。④结果表明,可注射脱细胞真皮基质水凝胶具有良好的细胞相容性和生物相容性。

脱细胞、病毒灭活与灭菌组合工艺对猪真皮基质理化性能的影响

背景:脱细胞、去抗原、病毒灭活与终端灭菌工艺的组合优化是制备符合临床要求组织再生材料的关键技术。目的:系统比较分析两种脱细胞、去抗原、病毒灭活与灭菌组合工艺对猪真皮基质理化性能的影响。方法:取新鲜猪皮,分两组工艺制备猪真皮基质:①方法A:采用1%Triton X-100、DNase和RNase、1%磷酸三丁脂进行脱细胞处理,1%过氧乙酸+25%乙醇病毒灭活,伽马射线辐照终端灭菌;②方法B:采用中性蛋白酶、0.5%Triton X-100和DNase进行脱细胞处理,α-半乳糖苷酶去除抗原,0.1%过氧乙酸病毒灭活,伽马射线辐照终端灭菌。对两种方法制备的猪真皮基质进行表征。结果与结论:①A组胶原蛋白含量低于B组,弹性蛋白、糖含量高于B组,材料硬度值高于B组;A组基质材料的悬垂性较差,B组基质材料的柔韧性较好;②A组脱细胞处理不影响基质材料的热稳定性,B组脱细胞略降低基质材料的热稳定性;伽马射线灭菌后,A组基质材料的热稳定性大幅度下降,B组基质材料的热稳定性下降很小;③与B组相比,A组基质材料的拉伸强度和弹性较小;体外酶降解实验显示,A组基质材料对胶原酶降解具有很强的抗性,对胰蛋白酶敏感、易降解,B组基质材料则相反;④扫描电镜显示,伽马射线灭菌后,A组基质材料基本看不到胶原三维结构,胶原纤维排列致密,胶原纤维之间存在非纤维结构性凝胶样物质;B组基质材料胶原纤维结构清晰,胶原纤维之间不存在非纤维结构性胶样物质。苏木精-伊红和三色染色显示,伽马射线灭菌后,A组基质材料可见少量细胞核,胶原纤维排列紧密,材料致密;B组基质材料无细胞核,保留了较好的胶原纤维三维空间结构;⑤结果表明,不同组合的制备工艺对脱细胞基质材料的理化性能影响极大,方法B在有效脱细胞和去抗原的同时较完整地保留了天然组织结构,方法A对材料的破坏性较大,制备的基质材料胶原成分含量、柔软度、热稳定性和力学性能均不如方法B。

双喷头静电纺丝法制备载软骨脱细胞基质的复合纳米纤维支架

背景:软骨脱细胞基质是一种理想的用于构建软骨组织工程支架的生物材料,可以用于软骨缺损的修复。目的:以聚乙烯醇为脱细胞基质的负载材料,3-羟基丁酸酯与4-羟基丁酸酯共聚物为框架材料,通过双喷头静电纺丝构建复合纳米纤维支架,并初步探索其体外生物活性。方法:通过酶、化学与超声振荡清洗结合的方法去除软骨组织中的细胞成分,制成软骨脱细胞基质。用双喷头静电纺丝的方法分别制备3-羟基丁酸酯与4-羟基丁酸酯共聚物(记为P34HB)、3-羟基丁酸酯与4-羟基丁酸酯共聚物-聚乙烯醇(记为P34HB-PVA)、3-羟基丁酸酯与4-羟基丁酸酯共聚物-聚乙烯醇-脱细胞基质(记为P34HB-PVA-dECM)纳米纤维支架,表征支架的纤维形貌、组成成分、亲水性能和力学性能。将人骨髓间充质干细胞与3种支架共培养,通过Live/Dead染色来检测细胞在支架的活力,扫描电镜观察细胞在支架上的黏附形态,阿尔玛蓝试剂盒检测细胞在支架上的增殖性能,Ⅱ型胶原免疫荧光评估细胞在支架上的成软骨分化。结果与结论:(1)扫描电镜下可见,各组支架纤维呈随机分布,纤维之间互相连接呈现多孔结构,其中P34HB-PVA-dECM支架的纤维直径最小;P34HB-PVA-dECM支架的水接触角小于P34HB、P34HB-PVA支架(P<0.05),吸水率高于P34HB、P34HB-PVA支架(P<0.05);P34HB-PVAdECM支架的弹性模量高于P34HB、P34HB-PVA支架(P<0.05);(2)Live/Dead染色结果显示,大多数骨髓间充质干细胞在3组支架上均能够存活;扫描电镜观察显示,骨髓间充质干细胞在P34HB-PVA-dECM支架上的伪足伸展更充分,与支架的融合更好;阿尔玛蓝染色显示,P34HB-PVA-dECM支架上的骨髓间充质干细胞增殖速度快于P34HB、P34HB-PVA支架(P<0.05);免疫荧光染色显示,P34HB-PVA-dECM支架上骨髓间充质干细胞生成的Ⅱ型胶原量多于P34HB、P34HB-PVA支架(P<0.05);(3)结果表明,P34HB-PVA-dECM支架具有更细的纤维结构、更好亲水性和力学性能,更适合细胞黏附、增殖,有利于骨髓间充质干细胞向成软骨方向分化。

天然血管基质/聚己内酯复合血管支架的制备与性能表征

将脱细胞血管基质(decellularized vascular matrix,DVM)与聚己内酯(Polycaprolactone,PCL)相复合,得到的复合支架能保留天然血管基质的血管诱导再生特性,克服降解过快的不足,扩大其在组织工程领域的应用范围。将新鲜的猪主动脉经脱细胞、脱脂等一系列处理后得到DVM粉末,再将其经酶解得到脱细胞血管基质凝胶(decellularized vascular matrix gel,DVMG),将用冰醋酸配置的PCL溶液与DVMG按照不同比例混合,将混合溶液反复包裹模具,室温中冷却后得到不同组的复合血管支架。共制备出4组DVMG/PCL的支架材料,通过扫描电子显微镜(scanning electron microscope,SEM)观察发现支架表面较为均匀,截面结构有明显孔隙,利于细胞黏附与迁移。通过力学拉伸实验检测PCL支架、DVMG∶PCL分别为1∶1,1.5∶1,5∶1的各组支架的拉伸模量值依次为49.00±9.52 MPa,46.29±3.08 MPa,38.77±2.07 MPa和31.18±2.80 MPa。体外降解实验显示:4组支架材料均有一定的降解能力,降解速率与DVMG的含量有关,说明改变DVMG与PCL的比例可以制备出不同力学性能、降解性能的复合血管支架。相较于单纯的PCL支架,复合支架中DVMG的含量越高,支架的刚度越低,降解速率越快,DVMG/PCL支架的整体性能得到一定改善。研究结果对于进一步扩大DVM和DVMG的应用范围,改善高分子材料的生物学响应性能具有参考价值。

脱细胞基质复合支架在组织再生中的应用

背景:目前的脱细胞方法不可避免地会对脱细胞基质支架造成损伤,为更好地发挥其作为组织工程支架的优势,对脱细胞基质支架进行修饰以改善性能显得尤为重要。目的:综述脱细胞基质复合支架在组织再生中的应用进展。方法:以“decellularized extracellular matrix,tissue engineering,crosslinking,Electrospun nanofibers,3D bioprinting technology,tissue regeneration;脱细胞基质,组织工程,交联,静电纺丝纳米纤维,三维生物打印技术,组织再生”等作为关键词,在PubMed数据库、万方数据库、中国知网数据库进行检索,文献的语种限定为中文和英文,检索时限为2009-2022年。共检索到文献142余篇,最终纳入79篇进行综述。结果与结论:采用化学、物理及生物方法对组织或器官去除细胞的过程,会对脱细胞基质支架的超微结构造成损伤,导致支架的机械性能差与不可控的降解等。通过交联、静电纺丝技术、三维生物打印技术、纳米颗粒、甲氧基聚乙二醇及生长因子修饰构建复合支架,可优化脱细胞基质支架的性能,其中三维生物打印技术可将修饰后的脱细胞基质生物墨水打印出更加稳定、精准化的复合支架,对于个性化、精准化的组织再生显示出巨大潜力,但要实现功能和结构的组织再生及临床转化仍需不断探索和研究。

去细胞全肾生物支架的制备与鉴定

目的通过灌注加浸泡法制备全肾细胞外基质支架,并对该生物支架进行鉴定。方法健康成年SD大鼠20只,取肾,行肾动脉留置针插管,灌注压3.6mmHg,恒温37℃依次浸泡灌注肝素化PBS溶液、0.05%胰蛋白酶溶液、1%十二烷基硫酸钠(SDS)溶液、1%Trition X-100溶液以及含青、链霉素的PBS溶液。处理后,行DNA定量与定性分析,透射电镜、HE染色及免疫荧光观察残留细胞核成分,进一步丙烯腈-丁二烯-苯乙烯树脂(ABS)铸型观察肾内血管分布情况。结果去细胞组DNA含量较对照组下降了97%,琼脂糖凝胶电泳未见明显DNA条带;透射电镜、HE染色及免疫荧光观察去细胞组保留了大量细胞外基质,未见明显细胞核成分残留;铸型标本显示去细胞组血管较稀疏,分支完整、清晰。结论联合酶消化法与去垢剂洗涤法,经灌注加浸泡法处理的全肾,可有效清除肾内所有细胞成分,较好地保留细胞外基质支架和血管网络结构,是一种简单易行且较为理想的制备组织工程肾生物支架的方法。

去细胞处理对牛颈静脉带瓣管道细胞外基质骨架的影响

目的:探讨去细胞处理对牛颈静脉带瓣管道细胞外基质骨架成分及组织稳定性的影响,为确立构建组织工程化的人工血管技术提供依据。方法:采集新鲜牛颈静脉,筛选瓣膜功能良好的血管,采用曲那通X-100、胰蛋白酶/EDTA和DNase-I/RNase-A三步法去细胞处理,组织化学染色和透射电镜观察细胞外基质骨架成分(胶原和弹力蛋白)的变化。对新鲜和去细胞处理的牛颈静脉血管壁(n=10)分别进行弹力蛋白(Fastin弹性蛋白法)和羟脯氨酸(碱水解、分光光度法)含量测定;并测定处理前后的热皱缩温度和抗张强度以比较组织稳定性。结果:去细胞处理牛颈静脉血管壁光镜和透射电镜检测提示细胞及其成分完全去除,组织间隙增宽,但胶原成分较好保留,弹力蛋白部分减少及断裂。与新鲜组织比较,去细胞组羟脯氨酸含量相对增加[(25.73±2.97)mg/gvs.(29.25±2.99)mg/g,P<0.05],弹力蛋白含量相对减少[(159.71±21.06)mg/gvs.(134.91±35.40)mg/g,P<0.05],热皱缩温度降低[(72.50±0.53)℃vs.(69.75±0.54)℃,P<0.05],抗张强度也下降明显[(5.19±0.65)MPavs.(3.13±0.94)MPa,P<0.05]。结论:去细胞处理对牛颈静脉细胞外基质骨架有一定损害,牛颈静脉的组织稳定性有所下降,得到的细胞外基质基本成分需经进一步交联处理才可以作为组织工程支架材料。

离体大鼠胰腺去细胞生物支架的制备与鉴定

目的通过离体灌注加浸泡的方法制备大鼠胰腺去细胞生物支架(PDBC),为胰岛和胰腺的组织工程研究提供新型天然生物支架。方法健康成年大鼠20只,以物理冻融及灌注洗脱法(脱氧胆酸钠+DNAase),采用胆管、胰管联合逆向在体灌流法获取胰腺去细胞生物支架。并通过基因组DNA定量定性分析,组织化学染色、免疫荧光组织化学染色、荧光积分吸光度分析、透射电镜观察等进一步测定细胞残留以及观察去细胞支架,如胶原IV、纤维连接蛋白和层黏连蛋白等成分的保留。结果 DNA定性分析显示,实验组未见明显DNA条带,对照组DNA条带可达100bp,定量检测实验组DNA残留不足对照组的5%;组织化学染色和透射电镜观察均表明,去细胞支架无细胞残留,细胞外支架连续性完好,脉管支架保存完整;免疫荧光结果表明,胶原蛋白、弹性蛋白等细胞外支架成分保留较完整,未见明显细胞核成分残留。结论运用冻融加浸泡灌注制备的胰腺去细胞生物支架,细胞去除彻底,细胞外支架保留完好。

灌注法制备离体大鼠心脏脱细胞基质

目的探索制备离体大鼠心脏脱细胞生物支架材料的新方法,为心脏组织工程研究提供三维立体天然支架。方法取30只成年SD大鼠心脏,运用冻融加化学萃取的组织工程学方法(胰蛋白酶、十二烷基硫酸钠和曲拉通X-100)处理离体大鼠心脏,同时观察心脏大体形态及颜色变化,并对脱细胞支架进行基因组DNA分析;HE染色,免疫荧光法,扫描和透射电镜进一步检测鉴定脱细胞支架的生物学特征。结果心脏脱细胞支架外观透明,包膜完整,维持心脏三维立体结构,肉眼可见心脏内脉管系统;脱细胞支架DNA残留量不及对照组的3%;HE染色、扫描和透射电镜结果显示,心脏脱细胞生物支架去细胞彻底,细胞外基质网状结构保留完整;免疫荧光结果表明,胶原、弹性蛋白等细胞外支架成分保留较完整,未见明显细胞核成分残留。结论运用冻融加化学萃取法所制备的离体心脏脱细胞生物支架去细胞彻底,细胞外基质保留较完整,是较为理想的心脏三维立体生物支架材料。

嗅鞘细胞/细胞外基质夹层支架的制备和形态学观察

目的研究嗅粘膜嗅鞘细胞在细胞外基质夹层支架内的生长特性,为治疗神经系统损伤寻找新的移植供体。方法嗅鞘细胞/细胞外基质夹层支架由四层毯状细胞外基质支架和三层嗅粘膜嗅鞘细胞相间叠加构成。纤维蛋白原、层粘连蛋白和纤粘连蛋白按一定比例混合后,在新鲜大鼠血浆促凝作用下,构建单层毯状细胞外基质支架,在支架表面种植嗅粘膜来源的嗅鞘细胞。于倒置显微镜下观察嗅鞘细胞在支架上/内的生长过程,培养3d后,在细胞表面再次滴加上述细胞外基质混合胶以构建第二层毯状支架,依次重复两次。夹层支架制成组织切片和超薄切片后,用光镜和电镜观察和分析夹层支架的内部结构以及嗅鞘细胞在支架内的生长情况。结果由顶层至底层,支架内的嗅鞘细胞数量逐渐增多,细胞突起逐渐延长,细胞内分泌颗粒逐渐增多及其电子密度逐渐增高;细胞可在支架之间迁移,其水平排列方向具有一致性;嗅鞘细胞的细胞膜可与支架紧密粘附,支架内局部区域出现组织间隙。结论嗅粘膜嗅鞘细胞可在细胞外基质夹层支架内正常增殖和分化,细胞与支架具有良好的组织相容性。该夹层支架可作为嗅鞘细胞三维生长的载体,用于移植治疗神经损伤。

在体制备大鼠全肾去细胞支架的程序性分析

目的通过在体灌注Triton X-100、SDS溶液法制备大鼠全肾去细胞生物支架,并对支架制备过程中的关键步骤进行程序性检测、分析,为制备科学合理的实验用大鼠全肾去细胞生物支架提供基础。方法 SD大鼠40只,随机分为4组,每组10只。肝素化后直接切取肾脏作为对照组(control组);肝素PBS灌注组(H组);肝素PBS、Triton X-100灌注组(HT组);肝素PBS、Triton X-100、十二烷基硫酸钠(SDS)溶液灌注组(HTS组)。HE、Masson、PAS染色及透射电镜观察各组肾组织病理及超微结构改变,免疫荧光结合4,6-二脒基-2-苯基吲哚(DAPI)观察各组胶原蛋白Ⅳ(collagenⅣ)、层黏连蛋白(LN)、纤维连接蛋白(FN)、硫酸乙酰肝素蛋白多糖2(HSPG2)、弹性蛋白(elastin)及细胞核的表达情况,总DNA测定各组DNA残留浓度。结果 Triton X-100、SDS灌注6h左右制备大鼠肾脏去细胞生物支架。在灌注过程中,肾内细胞和细胞碎片逐渐被清洗,最终变成半透明状。HE、Masson、PAS染色及透射电镜显示连续分布、形态排列类似肾小球、肾小管轮廓结构的网状结构,基膜连续完整。免疫荧光结合DAPI染色结果表明,去细胞过程中细胞外基质中的重要蛋白collagen IV、LN、FN、HSPG2、elastin都较好的得到了保存,细胞核在灌注过程中逐渐减少,直至完全消失;最终残留组织的DNA为4.90μg/g。结论通过合适浓度和灌注比例的Triton X-100、SDS制备大鼠肾脏去细胞生物支架,并对其进行程序性分析,发现能有效清除大鼠肾内所有细胞成分,较完整的保留网络状肾及肾血管细胞外基质结构和成分,是一种简单易行且较为理想的制备实验用全肾生物支架的方法。