Automated Assessment of Cell Infiltration and Removal in Decellularized Scaffolds - Experimental Study in Rabbits / Avaliação automatizada da infiltração e remoção celular em scaffolds descelularizados - Estudo experimental em coelhos

Abstract Objective Semiquantitative and automated measurement of nuclear material removal and cell infiltration in decellularized tendon scaffolds (DTSs). Method 16 pure New Zealand rabbits were used, and the gastrocnemius muscle tendon was collected bilaterally from half of these animals (16 tendons collected); 4 were kept as control and 12 were submitted to the decellularization protocol (DTS). Eight of the DTSs were used as an in vivo implant in the experimental rotator cuff tear (RCT) model, and the rest, as well as the controls, were used in the semiquantitative and automated evaluation of nuclear material removal. The eight additional rabbits were used to make the experimental model of RCT and subsequent evaluation of cellular infiltration after 2 or 8 weeks, within the DTS. Results The semiquantitative and automated analysis used demonstrated a removal of 79% of nuclear material (p< 0.001 and power > 99%) and a decrease of 88% (p < 0.001 and power >99%) in the area occupied by nuclear material after the decellularization protocol. On cell infiltration in DTS, an increase of 256% (p < 0.001 and power >99%) in the number of cells within the DTS was observed in the comparison between 2 and 8 weeks postoperatively. Conclusion The proposed semiquantitative and automated measurement method was able to objectively measure the removal of nuclear material and cell infiltration in DTS.

Resumo Objetivo Mensuração semiquantitativa e automatizada da remoção de material nuclear e da infiltração celular em scaffolds tendinosos descelularizados (STDs). Método Foram utilizados 16 coelhos Nova Zelândia puros, sendo o tendão do músculo gastrocnêmio coletado bilateralmente de metade destes animais (16 tendões coletados); 4 foram mantidos como controle e 12 foram submetidos ao protocolo de descelularização (STD). Dos STDs, 8 foram utilizados como implante in vivo no modelo experimental de lesão do manguito rotador (LMR) e os restantes, assim como os controles, foram utilizados na avaliação semiquantitativa e automatizada da remoção de material nuclear. Os oito coelhos adicionais foram utilizados na confecção do modelo experimental de LMR e posterior avaliação da infiltração celular após 2 ou 8 semanas, dentro do STD. Resultados A análise semiquantitativa e automatizada utilizada demonstrou uma remoção de 79% do material nuclear (p< 0,001 e poder > 99%) e uma diminuição de 88% (p< 0,001 e poder > 99%) na área ocupada por material nuclear após o protocolo de descelularização. Sobre a infiltração celular no STD, foi observado um aumento de 256% (p< 0,001 e poder > 99%) no número de células dentro do STD na comparação entre 2 e 8 semanas de pós-operatório. Conclusão O método de mensuração semiquantitativo e automatizado proposto foi capaz de mensurar objetivamente a remoção de material nuclear e a infiltração celular no STD.

Decellularized extracellular matrix mediates tissue construction and regeneration / 医学前沿

Contributing to organ formation and tissue regeneration, extracellular matrix (ECM) constituents provide tissue with three-dimensional (3D) structural integrity and cellular-function regulation. Containing the crucial traits of the cellular microenvironment, ECM substitutes mediate cell-matrix interactions to prompt stem-cell proliferation and differentiation for 3D organoid construction in vitro or tissue regeneration in vivo. However, these ECMs are often applied generically and have yet to be extensively developed for specific cell types in 3D cultures. Cultured cells also produce rich ECM, particularly stromal cells. Cellular ECM improves 3D culture development in vitro and tissue remodeling during wound healing after implantation into the host as well. Gaining better insight into ECM derived from either tissue or cells that regulate 3D tissue reconstruction or organ regeneration helps us to select, produce, and implant the most suitable ECM and thus promote 3D organoid culture and tissue remodeling for in vivo regeneration. Overall, the decellularization methodologies and tissue/cell-derived ECM as scaffolds or cellular-growth supplements used in cell propagation and differentiation for 3D tissue culture in vitro are discussed. Moreover, current preclinical applications by which ECM components modulate the wound-healing process are reviewed.

Progress in replicative senescence of mesenchymal stem cells regulated by microenvironment / 生理学报

Mesenchymal stem cells (MSCs) are a class of pluripotent cells that can self-renew and differentiate. Numerous studies have shown that MSCs have important roles in areas such as regenerative medicine and tissue engineering. However, it is worth noting that MSCs will gradually age during long-term in vitro expansion with decreased stemness such as weakened migration ability, slowed proliferation rate and decreased differentiation potential, which greatly hinders the application of MSCs. Currently, the microenvironment for cell growth is recognized as one of the factors causing senescence in MSCs. Recent studies point out that the latest technologies such as exogenous administration, oxygen concentration regulation and extracellular matrix (ECM) construction can delay stem cell senescence by simulating or regulating the microenvironment. Here, we review the current knowledge of the characteristics and molecular mechanisms of senescent MSCs and microenvironment strategies to maintain MSCs stemness, which can provide a reference for future large-scale application of MSCs preparations in tissue engineering and clinical studies.

Effect of Notch1 on extracellular matrix deposition in the renal tubulointerstitium of diabetes / 生理学报

The aim of the present study was to observe the effects of Notch1 and autophagy on extracellular matrix deposition in renal tubulointerstitium of diabetes and to explore the mechanism. The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice). After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. Rat renal tubular epithelial cells NRK52E were cultured under normal glucose (NG) and high glucose (HG) respectively, and the expression of Notch1 and LC3 proteins were detected by Western blotting. Autophagosomes in NRK52E cells with overexpressed and knockdown Notch1 under NG and HG conditions were observed by confocal microscope, and the expression changes of Notch1, Collagen-I and III protein were detected by immunofluorescence. The results showed that the Notch1 and Collagen-III expressions were increased (P < 0.01) and the LC3 expression was decreased (P < 0.05) in db/db mice compared with db/m mice. In vitro, the Notch1 was increased (P < 0.01) and the LC3 expression was decreased significantly (P < 0.01) in NRK52E cells of HG group compared with NG group. There was no significant change of Notch1 and LC3 expression between the mannitol (MA) group and the NG group. Autophagy was decreased and extracellular matrix deposition was aggravated when Notch1 was overexpressed. In contrast, autophagy was increased and extracellular matrix deposition was relieved by knockdown of Notch1 under HG conditions. In conclusion, Notch1 protein expression was increased and autophagy was reduced in renal tissue of diabetes and renal tubular epithelial cells under HG. The extracellular matrix deposition in the renal tubulointerstitium was relieved by regulating autophagy after the knockdown of Notch1.

Effects of non-muscle myosin Ⅱ silenced bone marrow-derived mesenchymal stem cells transplantation on lung extracellular matrix in rats after endotoxin/lipopolysaccharide-induced acute lung injury / 中华烧伤杂志

Objective: To investigate the effects of non-muscle myosin Ⅱ (NMⅡ) gene silenced bone marrow-derived mesenchymal stem cells (BMMSCs) on pulmonary extracellular matrix (ECM) and fibrosis in rats with acute lung injury (ALI) induced by endotoxin/lipopolysaccharide (LPS). Methods: The experimental research methods were adopted. Cells from femur and tibial bone marrow cavity of four one-week-old male Sprague-Dawley rats were identified as BMMSCs by flow cytometry, and the third passage of BMMSCs were used in the following experiments. The cells were divided into NMⅡ silenced group transfected with pHBLV-U6-ZsGreen-Puro plasmid containing small interference RNA sequence of NMⅡ gene, vector group transfected with empty plasmid, and blank control group without any treatment, and the protein expression of NMⅡ at 72 h after intervention was detected by Western blotting (n=3). The morphology of cells was observed by an inverted phase contrast microscope and cells labeled with chloromethylbenzoine (CM-DiⅠ) in vitro were observed by an inverted fluorescence microscope. Twenty 4-week-old male Sprague-Dawley rats were divided into blank control group, ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group according to the random number table, with 5 rats in each group. Rats in blank control group were not treated, and rats in the other 3 groups were given LPS to induce ALI. Immediately after modeling, rats in ALI alone group were injected with 1 mL normal saline via tail vein, rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were injected with 1×107/mL BMMSCs and NMⅡ gene silenced BMMSCs of 1 mL labelled with CM-DiⅠ via tail vein, and rats in blank control group were injected with 1 mL normal saline via tail vein at the same time point, respectively. At 24 h after intervention, the lung tissue was collected to observe intrapulmonary homing of the BMMSCs by an inverted fluorescence microscope. Lung tissue was collected at 24 h, in 1 week, and in 2 weeks after intervention to observe pulmonary inflammation by hematoxylin eosin staining and to observe pulmonary fibrosis by Masson staining, and the pulmonary fibrosis in 2 weeks after intervention was scored by modified Ashcroft score (n=5). The content of α-smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), and MMP-9 was detected by immunohistochemistry in 2 weeks after intervention (n=3), the activity of superoxide dismutase (SOD), malondialdehyde, myeloperoxidase (MPO) was detected by enzyme-linked immunosorbent assay at 24 h after intervention (n=3), and the protein expressions of CD11b and epidermal growth factor like module containing mucin like hormone receptor 1 (EMR1) in 1 week after intervention were detected by immunofluorescence staining (n=3). Data were statistically analyzed with one-way analysis of variance, Bonferroni method, and Kruskal-Wallis H test. Results: At 72 h after intervention, the NMⅡprotein expression of cells in NMⅡ silenced group was significantly lower than those in blank control group and vector group (with P values <0.01). BMMSCs were in long spindle shape and grew in cluster shaped like vortexes, which were labelled with CM-DiⅠ successfully in vitro. At 24 h after intervention, cell homing in lung of rats in ALI+NMⅡ silenced BMMSC group was more pronounced than that in ALI+BMMSC group, while no CM-DiⅠ-labelled BMMSCs were observed in lung of rats in blank control group and ALI alone group. There was no obvious inflammatory cell infiltration in lung tissue of rats in blank control group at all time points, while inflammatory cell infiltration in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly less than that in ALI alone group at 24 h after intervention, and alveolar wall turned to be thinner and a small amount of congestion in local lung tissue appeared in rats of the two groups in 1 week and 2 weeks after intervention. In 1 week and 2 weeks after intervention, collagen fiber deposition in lung tissue of rats in ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group was significantly aggravated compared with that in blank control group, while collagen fiber deposition in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly improved compared with that in ALI alone group. In 2 weeks after intervention, modified Ashcroft scores for pulmonary fibrosis of rats in ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group were 2.36±0.22, 1.62±0.16, 1.06±0.26, respectively, significantly higher than 0.30±0.21 in blank control group (P<0.01). Modified Ashcroft scores for pulmonary fibrosis of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were significantly lower than that in ALI alone group (P<0.01), and modified Ashcroft score for pulmonary fibrosis of rats in ALI+NMⅡ silenced BMMSC group was significantly lower than that in ALI+BMMSC group (P<0.01). In 2 weeks after intervention, the content of α-SMA in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were significantly decreased compared with that in ALI alone group (P<0.05 or P<0.01). The content of MMP-2 in lung tissue of rats in the 4 groups was similar (P>0.05). The content of MMP-9 in lung tissue of rats in ALI alone group was significantly increased compared with that in blank control group (P<0.01), and the content of MMP-9 in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI alone group (P<0.01). At 24 h after intervention, the activity of malondialdehyde, SOD, and MPO in lung tissue of rats in ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group were significantly increased compared with that in blank control group (P<0.01), the activity of malondialdehyde in lung tissue of rats in ALI+NMⅡ silenced BMMSC group and the activity of SOD in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were significantly increased compared with that in ALI alone group (P<0.05 or P<0.01), and the activity of SOD in lung tissue of rats in ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI+BMMSC group (P<0.01). The activity of MPO in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI alone group (P<0.01), and the activity of MPO in lung tissue of rats in ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI+BMMSC group (P<0.01). In 1 week after intervention, the protein expression of CD11b in lung tissue of rats in ALI+NMⅡ silenced BMMSC group was significantly increased compared with those in the other three groups (P<0.05 or P<0.01), while the protein expressions of EMR1 in lung tissue of rats in the four groups were similar (P>0.05). Conclusions: Transplantation of NMⅡ gene silenced BMMSCs can significantly improve the activity of ECM components in the lung tissue in LPS-induced ALI rats, remodel its integrity, and enhance its antioxidant capacity, and alleviate lung injury and pulmonary fibrosis.

Research advances on application of botulinum toxin type A in scar prevention and treatment / 中华烧伤杂志

The wound healing time, tension of wound edge, proliferation of fibroblast, and extracellular matrix deposition are the important factors of scar formation, and botulinum toxin type A can regulate the above. Prevention and treatment of scar with botulinum toxin type A is one of the hot topics of clinical research in recent years. This paper briefly reviews researches by scholars at home and abroad on the mechanism, clinical application, complications, and adverse effects of botulinum toxin type A in scar prevention and treatment.

Overwork Affects Extracellular Matrix of Arterial Vessel Wall in Rats / 中国医学科学院学报

Objective To explore the effect of overwork (OW) on extracellular matrix of arterial vessel wall in rats. Methods Random number grouping method was employed to assign 18 Sprague-Dawley rats into three groups(n=6):the control group(no special treatment),group OW(forced swimming twice a day for 15 days),and sleep deficiency(SD)+OW group(in addition to forced swimming twice a day,the rats were put on the platforms in water to limit sleep for 15 days).On the 16th day,the abdominal aorta and common carotid artery were collected after blood sampling from heart under deep anesthesia.A part of the abdominal aorta sample was taken for Masson staining of collagen fiber,and Verhoeff-Van Gieson staining was carried out for the elastic fiber of common carotid artery.Image J was employed for the quantitative analysis of collagen fiber and elastic fiber content.The expression of collagen 1(Col-1) protein was quantified by immunohistochemistry and the ultrastructure of vascular matrix was examined by transmission electron microscopy.The other part of the abdominal aorta sample was used to determine the mRNA levels of matrix metalloproteinase(MMP)-1,MMP-2,MMP-9,tissue inhibitor of metalloproteinases-1(TIMP-1),and Col-1 by quantitative real-time polymerase chain reaction. Results Compared with that in control group,the content of collagen fiber in groups OW and SD+OW had no significant change(all P>0.05);the content of elastic fiber in groups OW and SD+OW decreased(all P<0.001) and had no significant difference between each other(P>0.05).The vascular vessel wall of group OW showed slight fiber breakage,while that of group SD+OW presented wormhole-like or spongy fiber fragmentation.The mRNA levels of MMP-1 and MMP-2 in groups OW and SD+OW had no significant difference between each other(P>0.05) but were higher than that in control group(all P<0.001).The mRNA levels of MMP-9 and TIMP-1 had no significant difference among the three groups(all P>0.05).Groups OW and SD+OW had lower mRNA level(all P<0.001) and protein level(all P<0.001) of Col-1 than control group,while the mRNA and protein levels of Col-1 had no significant difference between groups OW and SD+OW(P>0.05). Conclusion OW can reduce the content of Col-1 and elastic fibers in the extracellular matrix of arterial vessels,destroy the elastic lamina of vascular wall,up-regulate the expression of MMP-1 and MMP-2,thereby injuring arterial vessels.

Application of microcarrier technology in cartilage repairing: a review / 生物工程学报

Cartilage has poor self-recovery because of its characteristics of no blood vessels and high extracellular matrix. In clinical treatment, physical therapy or drug therapy is usually used for mild cartilage defects, and surgical treatment is needed for severe ones. In recent years, cartilage tissue engineering technology provides a new way for the treatment of cartilage defects. Compared with the traditional surgical treatment, cartilage tissue engineering technology has the advantages of small wound and good recovery. The application of microcarrier technology in the design of tissue engineering scaffolds further expands the function of scaffolds and promotes cartilage regeneration. This review summarized the main preparation methods and development of microcarrier technology in recent years. Subsequently, the properties and specific application scenarios of microcarriers with different materials and functions were introduced according to the materials and functions of microcarriers used in cartilage repair. Based on our research on osteochondral integrated layered scaffolds, we proposed an idea of optimizing the performance of layered scaffolds through microcarriers, which is expected to prepare bionic scaffolds that are more suitable for the structural characteristics of natural cartilage.

Research advances on biomaterials for the delivery of growth factors to regulate wound repair / 中华烧伤杂志

Wound repair is a highly coordinated and mutually regulated complex process involving various kinds of cells, extracellular matrices and cytokines. A variety of growth factors play an important regulatory role in wound healing, and it is critical to achieve effective delivery and sustained function of growth factors. In recent years, the application of biomaterials in tissue engineering has shown great potential, and the effective delivery of growth factors by biomaterials has attracted increasing attention. Based on this, this paper introduces the mechanism of related growth factors in the process of wound healing, focusing on the recent progress of biomaterial delivery of growth factors to accelerate wound healing, in order to provide new enlightenment for clinical wound treatment.

Increased Expression of Type-I Collagen in Malignant Colorectal Lesion and Positive Correlations with Clinical Factors / Expressão aumentada do colágeno tipo I na lesão colorretal maligna e correlações positivas com fatores clínicos

Abstract Objective Type-I collagen (Col-I) is one of the main macromolecules of the extracellular matrix, and it is involved in the desmoplastic stromal reaction, an indicator of worse prognosis in cases of colorectal cancer (CRC). The purpose of the present study was to investigate Col-I expression in cases of CRC and adenoma and to correlate with the clinical data and the data regarding the lifestyle of the patients. Methods A retrospective study including 22 patients with adenoma and 15 with CRC treated at a coloproctology service. The clinical and lifestyle data were obtained through medical records, and Col-I expression was investigated by immunohistochemistry. Results Women represented most cases of adenoma (63.64%), whereas CRC was found mainly in men (73.33%) (p=0.0448). Immunoexpression of Col-I showed a basement membrane thickening in areas of lining of epithelium and around the glands in both lesions. The cases of CRC had a quite evident fibrosis process in the stroma. The quantitative analysis demonstrated a higher protein expression in CRCs compared to adenomas (p=0.0109), as well as in female patients (p=0.0214), patients aged ≥ 50 years (p=0.0400), and in those with a positive family history of colorectal disease (p=0.0292). These results suggested a remodeling of the microenvironment of the Worked developed at the Department of Morphology, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, ES, Brazil. Conclusion The immunohistochemical analysis encourages the performance of more comprehensive studies to ascertain if our results could be a tool for the diagnosis and monitoring of the patients.

Resumo Objetivo O colágeno tipo I (Col-I) é uma das principais macromoléculas da matriz extracelular, e está envolvido na reação desmoplástica estromal, um indicador de pior prognóstico em casos de câncer colorretal (CCR). O objetivo foi investigar a expressão do Col-I emcasos de CCR e adenoma, e correlacioná-la comdados clínicos e de estilo de vida dos pacientes. Metodologia Foi realizado umestudoretrospectivo com22pacientes comadenoma e 15 comCCR tratadosemumserviço de coloproctologia.Os dados dos pacientes foramobtidos dos prontuários médicos, e a expressão do Col-I foi investigada por imunohistoquímica. Resultados As mulheres representaram a maioria dos casos de adenomas (63,64%), enquanto o CCR (73,33%) (p=0,0448) foi mais comum entre os homens. A imunoexpressão de Col-I mostrou espessamento da membrana basal em áreas de revestimento do epitélio e em volta de glândulas em ambas as lesões. O CCR apresentou fibrose no estroma. As análises quantitativas demonstraram maior expressão proteica no CCR (p=0,0109), assim como em mulheres (p=0,0214), pacientes com idade ≥ 50 anos (p=0,0400), e em pacientes com histórico positivo de doença colorretal na família (p=0,0292). Estes resultados sugerem a remodelação do microambiente tumoral na carcinogênese do CCR. As correlações clínico-patológicas positivas mostram uma ligação plausível entre o perfil do paciente e os achados imunohistoquímcos, o que indica uma possível forma de estratificação dos pacientes. Conclusão As análises imunohistoquímicas estimulam a execução de estudos mais abrangentes para confirmar se nossos resultados poderão ser uma ferramenta para o diagnóstico e o monitoramento dos pacientes.

Alterações miogênicas em células musculares humanas infectadas pelo vírus zika: susceptibilidade de mioblastos e resistência de miotubos / Myogenic alterations in human muscle cells infected by zika virus: myoblast susceptibility and myotube resistance

O músculo esquelético é responsável pelo movimento e manutenção da postura, e é um órgão produtor de miocinas e altamente metabólico, onde alterações em sua fisiologia podem ter consequências sistêmicas. Esse tecido é alvo para diferentes arboviroses, e mialgia é um sintoma frequentemente relatado. O músculo esquelético é composto majoritariamente por fibras musculares, e uma pequena população de células progenitoras denominadas células satélites (SC), que em caso de lesão podem ser ativadas, proliferam e se diferenciam, sendo capazes de regenerar o tecido muscular. Recentemente nosso grupo demonstrou que SC em proliferação (mioblastos) são infectadas pelo vírus ZIKA (ZIKV), enquanto células diferenciadas e fusionadas (miotubos) não apresentam proteínas virais. O presente trabalho avaliou alterações miogênicas e o perfil transcricional de mioblastos e miotubos humanos após tratamento com ZIKV, com o objetivo de identificar fatores e mecanismos envolvidos na susceptibilidade e resistência destas células à infecção. Confirmamos infecção produtiva do ZIKV nos mioblastos, que apresentaram uma redução no número de células expressando a molécula KI67 em altas concentrações (característico de células em mitose). A análise de sequenciamento mostrou perturbação das vias do ciclo celular em mioblastos infectados, que ainda apresentaram enriquecimento de vias relacionadas à morte celular. Também confirmamos a ausência de infecção produtiva nos miotubos. Interessantemente, verificamos que o ZIKV entra nas células diferenciadas, mas não consegue replicar o RNA viral, e a análise do transcriptoma identificou um enriquecimento de vias e modulação de genes antivirais maior ou exclusivamente nas células diferenciadas em comparação aos mioblastos infectados. Além disso, miotubos expostos ao ZIKV aparentam ter aumento de fusão/hipertrofia. Ao contrário dos mioblastos, miotubos apresentaram enriquecimento de vias relacionadas a organização da matriz extracelular. Dados preliminares do nosso grupo mostraram que o cultivo de mioblastos sobre a isoforma de laminina 511 levou à redução da infecção pelo ZIKV. Contudo, em nossos ensaios, a infecção pelo ZIKV não modulou a expressão de receptores para LM e o bloqueio do receptor de LM, a integrina α6, não reduziu a infecção pelo ZIKV em mioblastos. Nosso trabalho mostrou que a infecção pelo ZIKV induz resposta imune e antiviral, que foram enriquecidos em mioblastos e miotubos, sendo que estes apresentaram uma assinatura única de vias e genes antivirais, que poderiam explicar a resistência frente ao ZIKV.

Bioactivity effects of extracellular matrix proteins on apical papilla cells

Abstract Potent signaling agents stimulate and guide pulp tissue regeneration, especially in endodontic treatment of teeth with incomplete root formation. Objective This study evaluated the bioactive properties of low concentrations of extracellular matrix proteins on human apical papilla cells (hAPCs). Methodology Different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96-well plates. Non-treated and pre-treated wells were used as negative (NC) and positive (PC) controls. After seeding the hAPCs (5×103 cells/well) on the different substrates, we assessed the following parameters: adhesion, proliferation, spreading, total collagen/type I collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) (ANOVA/Tukey; α=0.05). Results We observed greater attachment potential for cells on the FN substrate, with the effect depending on concentration. Concentrations of 5 and 10 µg/mL of FN yielded the highest cell proliferation, spreading and collagen synthesis values with 10 µg/mL concentration increasing the ITGA5, ITGAV, and COL1A1 expression compared with PC. LM (5 and 10 µg/mL) showed higher bioactivity values than NC, but those were lower than PC, and COL showed no bioactivity at all. Conclusion We conclude that FN at 10 µg/mL concentration exerted the most intense bioactive effects on hAPCs.

Extracellular matrix metalloproteinase expression in endometrial tissue after arterial embolization of myomas

OBJECTIVES: Arterial embolization of myomas (AEM) is controversial because of the changes that occur in the extracellular matrix (ECM) of the endometrium and its effect on gestational success in infertile patients desiring reproductive capability. Therefore, we performed this study on the expression of genes in the ECM of the endometrium, such as those coding metalloproteinases (MMP), before and 6 months after embolization of the uterine arteries. METHODS: Seven women with leiomyomas were evaluated, and MMP3 and MMP10 levels were measured. The women underwent pelvic nuclear magnetic resonance (NMR), examination, and endometrial biopsy between the 20th and 24th day of the menstrual cycle, and pre- and post-AEM (after 6 months). For data analysis, the Cq comparative method, also known as the 2-ΔΔCT method, was used to calculate the relative quantities of MMP gene expression among the samples collected. RESULTS: There was a significant decrease by 9.52 times in the expression of MMP3 (p=0.007), and a non-significant change in the expression of MMP10 (p=0.22) in post-AEM-treated women than pre-AEM-treated women. CONCLUSIONS: The results suggest that ECM continues to undergo tissue remodeling 6 months after AEM, at least with regard to MMP3 expression, suggesting that AEM affects the ECM for at least 6 months after the procedure.

Effects of two common acellular methods on the physicochemical properties of dermal acellular matrix / 生物医学工程学杂志

At present, acellular matrix is an effective replacement material for the treatment of skin damage, but there are few systematic evaluation studies on its performance. The experimental group of this study used two decellularization methods to prepare the matrix: one was the acellular matrix which sterilized with peracetic acid first (0.2% PAA/4% ethanol solution) and then treated with hypertonic saline (group A), the other was 0.05% trypsin/EDTA decellularization after γ irradiation (group B); and the control group was soaked in PBS (Group C). Then physical properties and chemical composition of the three groups were detected. Hematoxylin eosin (HE) staining showed that the acellular effect of group B was good. The porosity of group A and B were both above 84.9%. In group A, the compressive modulus of elasticity was (9.94 ± 3.81) MPa, and the compressive modulus of elasticity was (12.59 ± 5.50) MPa in group B. There was no significant difference between group A or B and group C. The total content of collagen in acellular matrix of group A and B was significantly lower than that of group C (1. 662 ± 0.229) mg/g, but there was no significant difference in the ratio of collagen Ⅰ/Ⅲ between group B and group C. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that there was no significant difference in microstructure. Qualitative detection of fibronectin and elastin in each group was basically consistent with that in group C. Therefore, acellular matrix of group B had better performance as scaffold material. The experimental results show that the acellular matrix prepared by γ-ray sterilization and decellularization of 0.05% Trypsin enzyme/EDTA could be used for the construction of tissue-engineered skin. It could also provide reference for the preparation and mounting of heterogeneous dermal acellular matrix. It was also could be used for electrostatic spinning or three-dimensional printed tissue engineered skin scaffold which could provide physical and chemical parameters for it.

Advances about perineuronal nets in the repair of nerve function after spinal cord injury / 中国骨伤

Perineuronal nets (PNNs) is a complex network composed of highly condensed extracellular matrix molecules surrounding neurons. It plays an important role in maintaining the performance of neurons and protecting them from harmful substances. However, after spinal cord injury, PNNs forms a physical barrier that surrounds the neuron and limits neuroplasticity, impedes axonal regeneration and myelin formation, and promotes local neuroinflammatory uptake. This paper mainly describes the composition and function of PNNs of neurons and its regulatory effects on axonal regeneration, myelin formation and neuroinflammation after spinal cord injury.

Preparation and application of decellularized extracellular matrix bioink: a review / 生物工程学报

Decellularized extracellular matrix (dECM), which contains many proteins and growth factors, can provide three-dimensional scaffolds for cells and regulate cell regeneration. 3D bioprinting can print the combination of dECM and autologous cells layer by layer to construct the tissue structure of carrier cells. In this paper, the preparation methods of tissue and organ dECM bioink from different sources, including decellularization, crosslinking, and the application of dECM bioink in bioprinting are reviewed, with future applications prospected.

Interactions between cells and biomaterials in tissue engineering: a review / 生物工程学报

Seed cells, biomaterials and growth factors are three important aspects in tissue engineering. Biomaterials mimic extra cellular matrix in vivo, providing a sound environment for cells to grow and attach, so as to maintain cell viability and function. The physicochemical properties and modification molecules of material surface mediate cell behaviors like cell adhesion, proliferation, migration and differentiation, which in turn affect cellular function and tissue regeneration efficacy. Furthermore, the modification molecules of material surface are the direct contact point for cell adhesion and growth. Therefore, the interactions between cells and surface modification molecules are the key to tissue engineering. This review summarizes the effects of surface modification molecules on cell phenotypes and functions.

New insights into vascular mechanobiology: roles of matrix mechanics in regulating smooth muscle cell function / 生理学报

Vascular smooth muscle cell (vSMC) is the predominant cell type in the blood vessel wall and is constantly subjected to a complex extracellular microenvironment. Mechanical forces that are conveyed by changes in stiffness/elasticity, geometry and topology of the extracellular matrix have been indicated by experimental studies to affect the phenotype and function of vSMCs. vSMCs perceive the mechanical stimuli from matrix via specialized mechanosensors, translate these stimuli into biochemical signals controlling gene expression and activation, with the consequent modulation in controlling various aspects of SMC behaviors. Changes in vSMC behaviors may further cause disruption of vascular homeostasis and then lead to vascular remodeling. A better understanding of how SMC senses and transduces mechanical forces and how the extracellular mechano-microenvironments regulate SMC phenotype and function may contribute to the development of new therapeutics for vascular diseases.

Biocompatibility of extracellular matrix hydrogel with human iPSCs differentiated cardiomyocytes / 中华心血管病杂志

Objective: To observe the biocompatibility of porcine omental derived extracellular matrix (ECM) hydrogel with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and the feasibility of ECM hydrogel as a delivery vector of cell transplantation. Methods: A series of chemical, physical and enzymatic methods were applied to acellularize the porcine omentum. Subsequently, the extracted ECM was prepared into thermosensitive hydrogel. The biochemical composition of the hydrogel was identified by histological staining. The microstructure was observed by scanning electron microscopy. The hydrogel was then injected into the myocardium of mice to observe its in situ gelation ability. Differentiation of human induced pluripotent stem cells into cardiomyocytes was achieved by small molecule induction, and then the obtained hiPSC-CMs were cultured. hiPSC-CMs cultured onto the prepared hydrogel were defined as the hydrogel group, while conventionally cultured hiPSC-CMs were defined as the control group. Cardiomyocyte viability and growth patterns were detected using live/dead staining, CCK-8 and phalloidin staining. Immunofluorescence staining and Western blot of cardiomyocytes were used to determine the survival and phenotypic maintenance markers of cardiomyocytes in materials. Results: The results of HE staining, oil red O staining and DAPI fluorescence staining showed that there was no significant cell debris, nucleus and lipid residue in the prepared ECM hydrogel. The Sirius red staining and Alcian blue staining showed that the hydrogel retained collagen and glycolaminoglycan, which were the main components of ECM. The prepared hydrogel behaves as a viscous liquid at 4 ℃ and as a gel state at 37 ℃. Scanning electron microscope results showed that the microstructure of the hydrogel was composed of irregular fibers and pores of different sizes. Under the guidance of ultrasound, the prepared ECM hydrogel could be successfully injected into the myocardium of mice. Immediately after the injection, the hyperechoic signal could be observed under ultrasound, suggesting that the hydrogel remained in the myocardium. HE staining of myocardial tissue evidenced that there was lump of gel in the injection area. The differentiated hiPSC-CMs were co-cultured with the prepared ECM hydrogel, and the results of live/dead staining showed that most of the hiPSC-CMs in the hydrogel group and the control group were alive, dead cells were scanty. The results of CCK-8 test showed that the absorbance values of the two groups were similar (P>0.05). The results of phalloidin staining showed that hiPSC-CMs could extend normally when co-cultured with ECM hydrogel. The cell morphology of the hydrogel group was similar with that of the control group, and there was no statistically significant difference in the F-actin coverage area per cell between the two groups (P>0.05). Immunofluorescence staining of cardiomyocyte markers showed that there was no significant difference in the coverage area of α-actinin and connexin-43 (Cx-43) per field between the hydrogel group and the control group (both P>0.05), the quantitative results of DAPI staining showed that there was no statistically significant difference in the number of cells between the two groups (P>0.05). Meanwhile, the results of Western blot showed that the expression levels of α-actinin and Cx-43 in cardiomyocytes in the hydrogel group were similar as those in the control group (both P>0.05). Conclusions: These results show that preparation of the ECM hydrogel from porcine omentum is successful. The hydrogel has good biocompatibility and no obvious cytotoxicity. Besides, the hydrogel can support the survival of hiPSC-CMs in vitro and maintain its phenotype. These properties make it a promising injectable cardiac tissue engineering material.

Expression of enamel proteins in human dental pulp stem cells by the effect of extracellular matrix / Expresión de proteínas de esmalte en células madre de pulpa dental humana por el efecto de matriz extracelular

SUMMARY: Mesenchymal stem cells are present in adult tissues such as the human dental pulp. They are pluripotent and can differentiate into various specialized cell types in vitro through appropriate stimuli. Ameloblasts produce human tooth enamel only during embryonic development before tooth eruption, so endogenous regeneration is not possible. Various efforts have been aimed at generating natural or artificial substitutes for dental enamel with properties similar to the specific components of said tissue. The purpose of this study was to induce human dental pulp stem cells to produce enamel proteins using extracellular matrix derived from the rat tail tendon and pigskin. Primary cultures of human dental pulp stem cells were established and characterized by RT-PCR and immunofluorescence, using mesenchymal cell markers such as CD14, CD40, CD44, CD105, and STRO-1. The cells were then incubated with the extracellular matrix for fourteen days and labeled with specific antibodies to detect the expression of dental enamel proteins such as amelogenin, ameloblastin, enamelisin, tuftelin, and parvalbumin, characteristics of the phenotype of ameloblasts. This work demonstrated a positive effect of the extracellular matrix to induce the expression of enamel proteins in the stem cells of the human dental pulp.

RESUMEN: Las células madre mesenquimales están presentes en los tejidos adultos como la pulpa dental humana. Son pluripotentes y pueden diferenciarse en varios tipos de células especializadas in vitro a través de estímulos adecuados. Los ameloblastos producen esmalte dental humano sólo durante el desarrollo embrionario antes de la erupción dental, por lo que no es posible su regeneración endógena. Varios esfuerzos se han orientado a generar sustitutos naturales o artificiales de esmalte dental con propiedades similares a los componentes específicos de este tejido. El propósito de este estudio fue inducir células madre de pulpa dental humana para producir proteínas del esmalte dental a través del estímulo de matriz extracelular derivada del tendón de la cola de rata y piel de cerdo. Se establecieron cultivos primarios de células madre de pulpa dental humana y se caracterizaron por RT-PCR e inmunofluorescencia utilizando marcadores de células mesenquimales como CD14, CD40, CD44, CD105 y STRO-1. Posteriormente, las células se incubaron con matriz extracelular durante un período de catorce días y se marcaron con anticuerpos específicos para detectar la expresión de proteínas de esmalte dental como amelogenina, ameloblastina, enamelisina, tuftelina y parvalbúmina, las cuales son características del fenotipo de ameloblastos. Este trabajo demostró el efecto positivo que tiene el empleo de la matriz extracelular para inducir la expresión de proteínas de esmalte en las células pluripotenciales de la pulpa dental humana.