Stiffness-tunable biomaterials provide a good extracellular matrix environment for axon growth and regeneration

Neuronal growth, extension, branching, and formation of neural networks are markedly influenced by the extracellular matrix—a complex network composed of proteins and carbohydrates secreted by cells. In addition to providing physical support for cells, the extracellular matrix also conveys critical mechanical stiffness cues. During the development of the nervous system, extracellular matrix stiffness plays a central role in guiding neuronal growth, particularly in the context of axonal extension, which is crucial for the formation of neural networks. In neural tissue engineering, manipulation of biomaterial stiffness is a promising strategy to provide a permissive environment for the repair and regeneration of injured nervous tissue. Recent research has fine-tuned synthetic biomaterials to fabricate scaffolds that closely replicate the stiffness profiles observed in the nervous system. In this review, we highlight the molecular mechanisms by which extracellular matrix stiffness regulates axonal growth and regeneration. We highlight the progress made in the development of stiffness-tunable biomaterials to emulate in vivo extracellular matrix environments, with an emphasis on their application in neural repair and regeneration, along with a discussion of the current limitations and future prospects. The exploration and optimization of the stiffness-tunable biomaterials has the potential to markedly advance the development of neural tissue engineering.

Matrix Stiffness-Induced Transcriptome Alterations and Regulatory Mechanisms Revealed by RNA-Seq in Endothelial Cells

Changes in vascular stiffness are associated with the development and progression of many diseases, especially in cardiovascular disease. However, the effect of vascular stiffness on the endothelial cells (ECs) is not fully understood. Therefore, this study aims to determine the gene expression changes of ECs cultured on the matrices with different stiffness (1 kPa and 40 kPa, respectively) by RNA-seq, thereby broadening the knowledge between mechanics and biology. We obtained 1775 differentially expressed genes (DEGs) by RNA-seq, with 450 up-regulated and 1325 down-regulated DEGs in ECs cultured on soft matrix (1 kPa) compared to those cultured on stiff matrix (40 kPa). After that, we performed a series of functional enrichment analyses based on DEGs and found that DEGs were enriched in many signaling pathways like adhesion junction. Furthermore, transcription factor (TF) target gene prediction analysis and protein-protein interaction (PPI) analysis were also conducted. We found that mechanotransduction signaling related TFs such as BRD4 are involved in. And in the PPI analysis, some genes encoding extracellular matrix proteins such as fibronectin 1 (FN1) were identified as the hub genes. In order to confirm the RNA-seq results, we performed real-time qPCR analysis on the genes of interest, including FN1, collagen α2 (IV) chain, matrix metalloproteinase-14 and integrin α5, and found that the expression levels of all these genes were down-regulated on soft matrix, suggesting that soft matrix caused by pathological conditions may directly attenuate vascular barrier function. This study offers the insights about the effects of physical stimulation on cells, paving a way for vascular tissue engineering, regenerative medicine, disease modeling and therapies.

Biophysical Regulation of Cell Behavior-Cross Talk between Substrate Stiffness and Nanotopography

The stiffness and nanotopographical characteristics of the extracellular matrix （ECM） influence numerous developmental, physiological, and pathological processes in vivo. These biophysical cues have therefore been applied to modulate almost all aspects of cell behavior, from cell adhesion and spreading to proliferation and differentiation. Delineation of the biophysical modulation of cell behavior is critical to the rational design of new biomaterials, implants, and medical devices. The effects of stiffness and topographical cues on cell behavior have previously been reviewed, respectively; however, the interwoven effects of stiffness and nanotopographical cues on cell behavior have not been well described, despite similarities in phenotypic manifestations. Herein, we first review the effects of substrate stiffness and nanotopography on cell behavior, and then focus on intracellular transmission of the biophysical signals from integrins to nucleus. Attempts are made to connect extracellular regulation of cell behavior with the biophysical cues. We then discuss the challenges in dissecting the biophysical regulation of cell behavior and in translating the mechanistic understanding of these cues to tissue engineering and regenerative medicine.

Nanofibrous Scaffold Containing Osteoblast-Derived Extracellular Matrix for the Proliferation of Bone Marrow Mesenchymal Stem Cells

Extracellular matrix( ECM) plays a prominent role in establishing and maintaining an appropriate microenvironment for tissue regeneration. The aims of this study were to construct a tissue engineered scaffold by reconstituting osteoblast cell-derived ECM( O-ECM) on the electrospun nanofibrous scaffold,and further to evaluate its subsequent application for promoting the proliferation of bone marrow mesenchymal stem cells( BMSCs). To engineer a biomimetic scaffold, calvarial osteoblasts and electrospun poly-llactic acid( PLLA) nanofibers were prepared and subjected to decellularize for O-ECM deposition. To evaluate and characterize the O-ECM/PLLA scaffold, the morphology was examined and several specific mark proteins of osteoblasts matrix were evaluated.Furthermore,the cell counting kit-8( CCK-8) assay was used to detect the proliferation of the BMSCs cultivated on the O-ECM/PLLA scaffold. The results indicated O-ECM/PLLA scaffold was loaded with Collagen I, Fibronectin, and Laminin, as the composition of the marrow ECM. After decellularization,O-ECM deposition was observed in O-ECM/PLLA scaffold. Moreover,the O-ECM/PLLA scaffold could significantly enhance the proliferation of BMSCs,suggesting better cytocompatibility compared to the other groups tested. Taken together,a biomimetic scaffold based on the joint use of O-ECM and PLLA biomaterials,which represents a promising approach to bone tissue engineering, facilitates the expansion of BMSCs in vitro.

Extracellular matrix based biomaterials for central nervous system tissue repair: the benefits and drawbacks

Functional repair of injured tissue in the adult central nervous system （CNS） still remains a big challenge for current biomed- ical research and its upcoming clinical translation. The axonal regeneration of the adult CNS is generally low, and it is addi- tionally restricted after injury by the presence of inhibitory mol- ecules, generated by the glial scar.

Neurodegenerative diseases are a function of matrix breakdown:how to rebuild extracellular matrix and intracellular matrix

Matrix within cells,the cytoskeleton,and that which surrounds cells,the extracellular matrix（ECM）,are connected to one another through a number of receptors including those in primary cilia,serving as an important chemical and physical signaling system：Mechanical forces generated through the matrix play a critical role in determining the form and function of tissues（Hughes et al.,2018）.

Neuronal growth cones and regeneration: gridlock within th extracellular matrix

The extracellular matrix is a diverse composition of glycoproteins and proteoglycans found in all cellular systems. The extracellular matrix, abundant in the mammalian central nervous system, is temporally and spatially regulated and is a dynamic ＂living＂ entity that is reshaped and redesigned on a continuous basis in response to changing needs. Some modifications are adaptive and some are maladaptive. It is the maladaptive responses that pose a significant threat to successful axonal regeneration and/or sprouting following traumatic and spinal cord injuries, and has been the focus of a myriad of research laboratories for many years. This review focuses largely on the extracellular matrix component, chondroitin sulfate proteoglycans, with certain comparisons to heparan sulfate proteoglycans, which tend to serve opposite functions in the central nervous system. Although about equally as well characterized as some of the other proteoglycans such as hyaluronan and dermatan sulfate proteoglycan, chondroitin sulfate proteoglycans are the most widely researched and discussed proteoglycans in the field of axonal injury and regeneration. Four laboratories discuss various aspects of chondroitin sulfate proteoglycans and proteoglycans in general with respect to their structure and function （Beller and Snow）, the recent discovery of specific chondroitin sulfate proteoglycan receptors and what this may mean the field （Shen）, extracellular for increased advancements in matrix degradation by matrix metalloproteinases, which sculpt and resculpt to provide support for outgrowth, synapse formation, and synapse stability （Phillips et al.）, and the perilesion microenvironment with respect to immune system function in response to proteoglycans and central nervous system injuries （Jakeman et al.）.

Matrix bound vesicles and miRNA cargoes are bioactive factors within extracellular matrix bioscaffolds

Injury to central nervous system （CNS） tissues in adult mam- mals often leads to neuronal loss, scarring, and permanently lost neurologic functions, and this default healing response is increasingly linked to a pro-inflammatory innate immune response. Extracellular matrix （ECM） technology can reduce inflammation, while increasing functional tissue remodeling in various tissues and organs, including the CNS.

Analyzing the role of extracellular matrix during nervous system development to advance new regenerative strategies

Regeneration in the central nervous system （CNS） is limited, and CNS damage often leads to cognitive impairment or permanent functional motor and sensory loss. Impaired regenerative capacity is multifactorial and includes inflammation, loss of the blood-brain barrier, and alteration in the extracellular matrix （ECM）. One of the main problems is the formation of a glial scar and the production of inhibitory ECM, such as proteoglycans, that generates a physical and mechanical barrier, impeding axonal regrowth （Figure 1A）.

Association between Gene Polymorphisms and SNP-SNP Interactions of the Matrix Metalloproteinase 2 Signaling Pathway and the Risk of Vascular Senescence

Objective This study aimed to explore the association of single nucleotide polymorphisms(SNP)in the matrix metalloproteinase 2(MMP-2)signaling pathway and the risk of vascular senescence(VS).Methods In this cross-sectional study,between May and November 2022,peripheral venous blood of151 VS patients(case group)and 233 volunteers(control group)were collected.Fourteen SNPs were identified in five genes encoding the components of the MMP-2 signaling pathway,assessed through carotid-femoral pulse wave velocity(cf PWV),and analyzed using multivariate logistic regression.The multigene influence on the risk of VS was assessed using multifactor dimensionality reduction(MDR)and generalized multifactor dimensionality regression(GMDR)modeling.Results Within the multivariate logistic regression models,four SNPs were screened to have significant associations with VS:chemokine(C-C motif)ligand 2(CCL2)rs4586,MMP2 rs14070,MMP2rs7201,and MMP2 rs1053605.Carriers of the T/C genotype of MMP2 rs14070 had a 2.17-fold increased risk of developing VS compared with those of the C/C genotype,and those of the T/T genotype had a19.375-fold increased risk.CCL2 rs4586 and MMP-2 rs14070 exhibited the most significant interactions.Conclusion CCL2 rs4586,MMP-2 rs14070,MMP-2 rs7201,and MMP-2 rs1053605 polymorphisms were significantly associated with the risk of VS.

ECM重塑下肿瘤淋巴管生成模型的定性分析与数值模拟

肿瘤转移是肿瘤发展过程中的重要环节,也是导致癌症恶化和治疗失败的主要原因之一。以肿瘤转移为背景,本文研究基于肿瘤与细胞外基质(Extracellular Matrix,ECM)相互作用的肿瘤淋巴管生成模型。首先用数学语言梳理肿瘤淋巴管生成的生物原理,其次做出假设,建立数学模型并进行定性分析。主要通过逼近方法、偏微分方程定性理论和Banach不动点定理证明模型局部解的存在唯一性,以及借助局部解的正则性估计和嵌入不等式证明模型整体解的存在唯一性。最后利用差分数值方法进行数值模拟来说明模型的可靠性与准确性。本文对深入理解肿瘤转移机制、指导癌症治疗以及推动相关研究具有重要意义。

ECM修复兔桡骨缺损的实验研究

[目的]研究自制的兔耳廓脱细胞软骨基质(extracellular cartilage matrix,ECM)修复骨缺损的特点及机理。[方法]采用新西兰大白兔共40只,随机分A、B两组,在其两侧桡骨干中段制作5 mm的骨缺损模型。右侧作为实验侧,缺损区A组植入脱细胞软骨基质复合自体培养骨髓干细胞,B组仅植入脱细胞软骨基质,左侧为空白对照。分别于2、4、6、10周处死动物,标本行放射学及组织学检查。[结果]X线及组织切片均证实复合体在6周时已有致密骨组织形成,10周时已有新生髓腔生成。[结论]表明兔耳廓脱细胞软骨基质与自体骨髓干细胞复合体有良好的成骨能力,有引导组织再生、防止骨不连作用。

一类脂蛋白沉积症家系ECM1基因突变检测

目的:报道1例类脂蛋白沉积症家系,并对其家系成员的细胞外基质蛋白1(ECM1)基因突变进行分析。方法:PCR,DNA直接测序以及RFLP对患者的ECM1编码区进行了基因突变分析。结果:先证者及其胞姐在ECM1基因6号染色体上均发现纯合性单核苷酸颠换c.658T>G,产生纯合错义突变p.C220G。该家系中父母二人均为此突变的杂合子,该突变在100个非相关对照中未被检测出。结论:p.C220G突变是引起该家系临床病变的特异突变,不是多态性变化。

细胞外基质硬度对肿瘤进展的影响及治疗策略

背景:细胞外基质是一个复杂的网络结构,不仅为组织构建提供了物理支撑,还在细胞的生存、增殖、分化、死亡等过程中发挥着重要调控作用。细胞外基质生物化学和生物力学性质的异常改变会显著影响肿瘤细胞的增殖、迁移、免疫逃逸及治疗抵抗等行为。硬度是细胞外基质的重要力学特性,基质硬度的异常与肿瘤进展紧密相关。目的:通过梳理近年来关于细胞外基质硬化的机制、高硬度基质对肿瘤进展的影响以及降低基质硬度治疗癌症等方面的最新研究进展,深化对细胞外基质力学特性的理解,提升对肿瘤进展复杂机制的认识,为肿瘤治疗提供新的思路和方向。方法:以“细胞外基质功能,细胞外基质硬度,胶原蛋白沉积交联,细胞外基质僵硬治疗,免疫治疗”为中文检索词,以“extracellular matrix function,extracellular matrix stiffness,collagen deposition cross-linking,extracellular matrix stiffness therapy,immunotherapy”为英文检索词,检索中国知网、PubMed数据库、万方数据库2016年1月至2024年6月发表的相关文献,最终纳入80篇文章进行综述。结果与结论:①细胞外基质中胶原蛋白和弹性蛋白的沉积和过度交联导致基质重塑,进而增加基质硬度,这种硬化激活了cyclin-D1、Rho/ROCK、p-PXN-Rac1-YAP、STAT3/p-STAT3等促癌信号通路,促进了癌细胞的增殖、转移、肿瘤微血管生成及免疫逃逸等恶性行为,加速了肿瘤的进展;②减少基质蛋白的沉积和交联可以降低基质硬度,不仅可以抑制多种促癌信号通路的活化,还能够增强药物在肿瘤部位的渗透和递送,是癌症治疗的新策略;③目前,基于基质降解以降低肿瘤硬度的药物正在研发中,少数药物已进入临床试验阶段,有望为肿瘤治疗提供新的有力武器。

维生素E对CC1_4作用下HSC增殖和ECM影响

目的观察维生素E(VE)对四氯化碳(CC14)作用下大鼠肝星状细胞(HSC)增殖和细胞外基质(ECM)产生的影响。方法采用原位灌注方法分离大鼠HSC。结果CC14作用下大鼠HSC3H-脯氨酸(3H-Pro)的掺入显著增加,Ⅲ型前胶原(PCⅢ)、透明质酸(HA)、板层素(LN)及纤维连接素(FN)的分泌水平明显提高。200nmol/LVE预处理4h可有效抑制CC14作用下大鼠HSC3H-胸腺密啶(3H-TdR)、3H-Pro的掺入,VE保护能使CC14作用下HSC分泌PCⅢ、HA、LN和FN减少与HSC产生丙二醛(MDA)下降,二者呈正相关。结论VE可抑制CC14作用下大鼠HSC增殖和通过抗氧化损伤抑制HSCECM的分泌。

Adjusting the stiffness of a cell-free hydrogel system based on tissue-specific extracellular matrix to optimize adipose tissue regeneration

Background:Large-area soft tissue defects are challenging to reconstruct.Clinical treatment methods are hampered by problems associated with injury to the donor site and the requirement for multiple surgical procedures.Although the advent of decellularized adipose tissue(DAT)offers a new solution to these problems,optimal tissue regeneration efficiency cannot be achieved because the stiffness of DAT cannot be altered in vivo by adjusting its concentration.This study aimed to improve the efficiency of adipose regeneration by physically altering the stiffness of DAT to better repair large-volume soft tissue defects.Methods:In this study,we formed three different cell-free hydrogel systems by physically crosslinking DAT with different concentrations of methyl cellulose(MC;0.05,0.075 and 0.10 g/ml).The stiffness of the cell-free hydrogel system could be regulated by altering the concentration of MC,and all three cell-free hydrogel systems were injectable and moldable.Subsequently,the cell-free hydrogel systems were grafted on the backs of nude mice.Histological,immunofluorescence and gene expression analyses of adipogenesis of the grafts were performed on days 3,7,10,14,21 and 30.Results:The migration of adipose-derived stem cells(ASCs)and vascularization were higher in the 0.10 g/ml group than in the 0.05 and 0.075 g/ml groups on days 7,14 and 30.Notably,on days 7,14 and 30,the adipogenesis of ASCs and adipose regeneration were significantly higher in the 0.075 g/ml group than in the 0.05 g/ml group(p<0.01 or p<0.001)and 0.10 g/ml group(p<0.05 or p<0.001).Conclusion:Adjusting the stiffness of DAT via physical cross-linking with MC can effectively promote adipose regeneration,which is of great significance to the development of methods for the effective repair and reconstruction of large-volume soft tissue defects.

EXPRESSION OF MATRIX METALLOPROTEINASE-9 IN HUMANABDOMINAL AORTIC ANEURYSMAL TISSUES

Objective To study the effects of MMP-9 (Matrix Metalloproteinase-9, MMP-9) in the pathogenesis of abdominal aortic aneurysms (AAAs) by localizing the expression of MMP-9 in the aneurysmal tissues. Methods By means of immunohistochemistry, the frozen sections(5 μ m) with aneurysmal tissues (n=10) were incubated with MMP-9 antibody-added agents, then the sections were stained and observed under the microscope to localize the expression of MMP-9, which displayed a brown precipitate within the arterial walls. The normal arterial wall tissues(n=10) and the diseased arterial wall tissues from the arterial occlusive diseases (AODs) (n=15) were also immunized exactly the same way as control. Results A quantity of positive granules which appeared within the aortic media showed the strong expression of MMP-9 in the AAAs, with the positive rate reaching 95%(19/20), while no expression of MMP-9 was observed in the normal artery. However, the scattered distributed positive granules were seen within the arterial wall of some cases of the AODs, implying the weak positive expression of MMP-9 in this disease with the positive rate of 26.7%(4/15). There was a significant difference of the expression of MMP-9 within the arterial wall between the AAAs and AODs(P<0.01). Conclusion High expression of MMP-9 within the aortic media faciliatates the degradation of collagen and elastin fibres and subsequent dilation of the aortic artery ,thus playing an important role in the pathogenesis of AAAs. To refrain MMP-9 from enhanced expressing within the aortic wall is of clinical significance in the prevention and treatment of AAAs.

Rho蛋白激酶抑制剂对体外ECM介导的牙周膜细胞功能的作用

目的探究Rho蛋白激酶抑制剂在细胞外基质(ECM)介导的牙周膜细胞(PDL细胞)增殖、迁移、愈合和成骨分化过程中的作用。方法收集西北妇女儿童医院口腔科的5名牙周组织健康提供者的牙周膜组织样本,通过消化获取PDL细胞,并分为4组:无ECM包被的PDL细胞对照组(PDL组),ECM包被的PDL细胞组(PDL+ECM组),ECM包被的PDL细胞含盐酸土根碱(EmD)组(PDL+ECM+EmD组),无ECM包被的PDL细胞含EmD组(PDL+EmD组)。通过细胞增殖检测(MTS)分析PDL细胞活力及增殖能力;通过ALP试剂盒测定PDL细胞中碱性磷酸酶(ALP)的活性;通过实时RT-PCR测定ALP的mRNA表达水平;免疫荧光和蛋白质印迹实验分析PDL细胞的Ⅰ型胶原和纤连蛋白的免疫原性及表达水平;Transwell实验和伤口愈合实验分析PDL细胞迁移和愈合能力。结果MTS检测结果表明,10μmol/L EmD对PDL细胞的活力无显著影响。与PDL组相比,PDL+EmD组PDL细胞中ALP的mRNA表达水平和活性均显著降低(P<0.05)。免疫荧光分析结果表明,与PDL组相比,PDL+EmD组细胞出现更细的肌动蛋白丝,Procollagen-Ⅰ和Fibronectin蛋白的免疫反应性和表达水平均显著降低(P<0.05)。与PDL组相比,PDL+ECM组ALP活性、细胞增殖、迁移及愈合能力显著升高(P<0.01)。与PDL+ECM组相比,PDL+ECM+EmD组ALP阳性菌落及活性、细胞增殖、迁移及愈合能力显著降低(P<0.05)。结论牙周ECM可能通过依赖于Rho蛋白激酶(ROCK)并影响ECM相关基因表达的机制来介导PDL细胞的成骨分化,并保证细胞正常的增殖及愈合。

富含半胱氨酸的酸性分泌蛋白在肿瘤中的研究进展

富含半胱氨酸的酸性分泌蛋白(secreted protein acidic and rich in cysteine, SPARC)是肿瘤微环境中重要的基质成分,它作为正常细胞恶变和肿瘤发生发展过程中一个重要的分子,能够调节多种肿瘤相关细胞因子及其受体的相互作用,并调节多种信号通路以及蛋白水解酶的表达水平。由于它抑制和促进肿瘤进展的能力取决于细胞类型、肿瘤分期等,因此在多种类型的肿瘤中差异表达,并可能成为肿瘤早期诊断的新型生物标记物以及治疗的新靶点。本文就近年来SPARC在肿瘤研究中的进展作一综述。

颅缝发育与颅缝早闭的分子机制

颅骨的发育依赖于颅缝(cranial suture)正常的骨供给,期间颅缝的间充质干细胞经历系统地增殖和成骨分化以保证骨的正常生长。遗传因素或外界干扰则可能引起干细胞的过度成骨分化,导致颅骨的过早闭合,称为颅缝早闭。颅缝细胞与细胞外基质、骨膜以及脑膜共同构成颅缝微环境(niche)。它们相互调节以保证颅缝的正常发育。临床研究发现,绝大多数颅缝早闭(craniosynostosis)患者都伴有硬脑膜和细胞外基质(extracellular matrixc,ECM)的异常。许多研究也发现,作用于颅缝的异常外界机械刺激也可能导致颅缝发育的异常。考虑到颅缝发育受多方面因素影响,本篇综述将介绍近年来有关颅缝微环境异常,以及机械力在调控颅缝发育中的研究进展,总结颅缝早闭发生的分子机制和目前研究方向,旨在为该病的诊断治疗提供新的思路。