Optimization of Flavonoid Extraction from Eucommia ulmoides pollen using Respond Surface Methodology and its biological activities.

Flavonoids, known for their abundance in Eucommia ulmoides pollen, possess diverse biological functions, including antioxidants, antibacterial agents, and anti-tumor properties. This study aims to establish effective parameters for flavonoid extraction from Eucommia ulmoides pollen using a microwave-assisted method, characterize the flavonoid composition of the extracted material, and explore its biological activities. Building upon the initial results from single-factor experiments, response surface methodology was employed to optimize the extraction parameters. The inhibitory effect of human breast cancer cells (MCF-7) was evaluated by CCK assay and Live/dead staining. Simultaneously, the extract's scavenging ability against DPPH free radicals and its antibacterial properties against Escherichia coli and Staphylococcus aureus were investigated. The results demonstrated that the flavonoid yield reached 3.28 g per 100 g of pollen, closely aligning with the predicted value. The IC50 for flavonoid-mediated DPPH radical scavenging was 0.04 mg/mL. The extract exhibited a robust inhibitory effect on both Escherichia coli and Staphylococcus aureus. Concurrently, the extract displayed a significant inhibitory effect on the growth and proliferation of MCF-7 cells in a dose-dependent and time-dependent manner. In addition, six kinds of flavonoids have been identified by UPLC-TOF-MS/MS technology, providing further support to the study on the anti-oxidation and anti-tumor mechanism of Eucommia ulmoides pollen extracts.

Controlled dual release of dihydrotestosterone and flutamide from polycaprolactone electrospun scaffolds accelerate burn wound healing.

Wound healing is a complex process involving multiple independent and overlapping sequential physiological mechanisms. In addition to cutaneous injury, a severe burn stimulates physiological derangements that induce a systemic hypermetabolic response resulting in impaired wound healing. Topical application of the anti-androgen drug, flutamide accelerates cutaneous wound healing, whereas paradoxically systemic dihydrotestosterone (DHT) improves burn wound healing. We developed and characterized a PCL scaffold that is capable of controlled release of androgen (DHT) and anti-androgen (F) individually or together. This study aims to investigate whether local modification of androgen actions has an impact on burn injury wound healing. In a full-thickness burn wound healing, mouse model, DHT/F-scaffold showed a significantly faster wound healing compared with F-scaffold or DHT-scaffold. Histology analysis confirmed that DHT/F-scaffold exhibited higher re-epithelization, cell proliferation, angiogenesis, and collagen deposition. Dual release of DHT and F from PCL scaffolds promoted cell proliferation of human keratinocytes and alters the keratinocyte cell cycle. Lastly, no adverse effects on androgen-dependent organs, spleen and liver were observed. In conclusion, we demonstrated DHT plus F load PCL scaffolds accelerated burn wound healing when loading alone did not. These findings point to a complex role of androgens in burn wound healing and open novel therapeutic avenues for treating severe burn patients.

The Role of E-Cadherin and microRNA on FAK Inhibitor Response in Malignant Pleural Mesothelioma (MPM).

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with limited effective treatment options. Focal adhesion kinase (FAK) inhibitors have been shown to efficiently suppress MPM cell growth initially, with limited utility in the current clinical setting. In this study, we utilised a large collection of MPM cell lines and MPM tissue samples to study the role of E-cadherin (CDH1) and microRNA on the efficacy of FAK inhibitors in MPM. The immunohistochemistry (IHC) results showed that the majority of MPM FFPE samples exhibited either the absence of, or very low, E-cadherin protein expression in MPM tissue. We showed that MPM cells with high CDH1 mRNA levels exhibited resistance to the FAK inhibitor PND-1186. In summary, MPM cells that did not express CDH1 mRNA were sensitive to PND-1186, and MPM cells that retained CDH1 mRNA were resistant. A cell cycle analysis showed that PND-1186 induced cell cycle disruption by inducing the G2/M arrest of MPM cells. A protein-protein interaction study showed that EGFR is linked to the FAK pathway, and a target scan of the microRNAs revealed that microRNAs (miR-17, miR221, miR-222, miR137, and miR148) interact with EGFR 3'UTR. Transfection of MPM cells with these microRNAs sensitised the CHD1-expressing FAK-inhibitor-resistant MPM cells to the FAK inhibitor.

Genomic Deletion of BAP1 and CDKN2A Are Useful Markers for Quality Control of Malignant Pleural Mesothelioma (MPM) Primary Cultures.

Malignant pleural mesothelioma (MPM) is a deadly cancer that is caused by asbestos exposure and that has limited treatment options. The current standard of MPM diagnosis requires the testing of multiple immunohistochemical (IHC) markers on formalin-fixed paraffin-embedded tissue to differentiate MPM from other lung malignancies. To date, no single biomarker exists for definitive diagnosis of MPM due to the lack of specificity and sensitivity; therefore, there is ongoing research and development in order to identify alternative biomarkers for this purpose. In this study, we utilized primary MPM cell lines and tested the expression of clinically used biomarker panels, including CK8/18, Calretinin, CK 5/6, CD141, HBME-1, WT-1, D2-40, EMA, CEA, TAG72, BG8, CD15, TTF-1, BAP1, and Ber-Ep4. The genomic alteration of CDNK2A and BAP1 is common in MPM and has potential diagnostic value. Changes in CDKN2A and BAP1 genomic expression were confirmed in MPM samples in the current study using Fluorescence In situ Hybridization (FISH) analysis or copy number variation (CNV) analysis with digital droplet PCR (ddPCR). To determine whether MPM tissue and cell lines were comparable in terms of molecular alterations, IHC marker expression was analyzed in both sample types. The percentage of MPM biomarker levels showed variation between original tissue and matched cells established in culture. Genomic deletions of BAP1 and CDKN2A, however, showed consistent levels between the two. The data from this study suggest that genomic deletion analysis may provide more accurate biomarker options for MPM diagnosis.

KCa1.1, a calcium-activated potassium channel subunit alpha 1, is targeted by miR-17-5p and modulates cell migration in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive, locally invasive, cancer elicited by asbestos exposure and almost invariably a fatal diagnosis. To date, we are one of the leading laboratory that compared microRNA expression profiles in MPM and normal mesothelium samples in order to identify dysregulated microRNAs with functional roles in mesothelioma. We interrogated a significant collection of MPM tumors and normal pleural samples in our biobank in search for novel therapeutic targets. METHODS: Utilizing mRNA-microRNA correlations based on differential gene expression using Gene Set Enrichment Analysis (GSEA), we systematically combined publicly available gene expression datasets with our own MPM data in order to identify candidate targets for MPM therapy. RESULTS: We identified enrichment of target binding sites for the miR-17 and miR-30 families in both MPM tumors and cell lines. RT-qPCR revealed that members of both families were significantly downregulated in MPM tumors and cell lines. Interestingly, lower expression of miR-17-5p (P = 0.022) and miR-20a-5p (P = 0.026) was clearly associated with epithelioid histology. We interrogated the predicted targets of these differentially expressed microRNA families in MPM cell lines, and identified KCa1.1, a calcium-activated potassium channel subunit alpha 1 encoded by the KCNMA1 gene, as a target of miR-17-5p. KCa1.1 was overexpressed in MPM cells compared to the (normal) mesothelial line MeT-5A, and was also upregulated in patient tumor samples compared to normal mesothelium. Transfection of MPM cells with a miR-17-5p mimic or KCNMA1-specific siRNAs reduced mRNA expression of KCa1.1 and inhibited MPM cell migration. Similarly, treatment with paxilline, a small molecule inhibitor of KCa1.1, resulted in suppression of MPM cell migration. CONCLUSION: These functional data implicating KCa1.1 in MPM cell migration support our integrative approach using MPM gene expression datasets to identify novel and potentially druggable targets.

Fibulin-3 levels in malignant pleural mesothelioma are associated with prognosis but not diagnosis.

BACKGROUND: Fibulin-3 (FBLN3) was recently presented as a promising novel biomarker for malignant pleural mesothelioma (MPM), warranting independent validation studies. METHODS: ELISA was used to measure cellular and secreted FBLN3 in cell lines, in plasma of xenograft tumour-bearing mice, in plasma from two independent series of MPM and non-MPM patients and in pleural fluid from a third series. Diagnostic and prognostic potential of FBLN3 was assessed by receiver operating characteristics curve analysis and Kaplan-Meier method, respectively. RESULTS: FBLN3 was expressed in all MPM and benign mesothelial cell lines tested, and a correlation was observed between cellular protein expression and secreted levels. Human FBLN3 was detectable in plasma of tumour-bearing mice, suggesting that MPM cells contribute to levels of circulating FBLN3. Plasma FBLN3 was significantly elevated in MPM patients from the Sydney cohort, but not the Vienna cohort, but the diagnostic accuracy was low (63%, (95% CI: 50.1-76.4) and 56% (95% CI: 41.5-71.0), respectively). Although FBLN3 levels in pleural effusions were not significantly different between cases and controls, FBLN3 levels in pleural effusion fluid were found to be independently associated with prognosis (hazard ratio of 9.92 (95% CI: 2.14-45.93)). CONCLUSIONS: These data confirm the potential prognostic value of pleural effusion FBLN3, but question the diagnostic value of this protein in MPM patients.

Small Extracellular Vesicle-Derived Circular RNA hsa_circ_0007386 as a Biomarker for the Diagnosis of Pleural Mesothelioma.

Pleural mesothelioma (PM) is a highly aggressive tumor that is caused by asbestos exposure and lacks effective therapeutic regimens. Current procedures for PM diagnosis are invasive and can take a long time to reach a definitive result. Small extracellular vesicles (sEVs) have been identified as important communicators between tumor cells and their microenvironment via their cargo including circular RNAs (circRNAs). CircRNAs are thermodynamically stable, highly conserved, and have been found to be dysregulated in cancer. This study aimed to identify potential biomarkers for PM diagnosis by investigating the expression of specific circRNA gene pattern (hsa_circ_0007386) in cells and sEVs using digital polymerase chain reaction (dPCR). For this reason, 5 PM, 14 non-PM, and one normal mesothelial cell line were cultured. The sEV was isolated from the cells using the gold standard ultracentrifuge method. The RNA was extracted from both cells and sEVs, cDNA was synthesized, and dPCR was run. Results showed that hsa_circ_0007386 was significantly overexpressed in PM cell lines and sEVs compared to non-PM and normal mesothelial cell lines (p < 0.0001). The upregulation of hsa_circ_0007386 in PM highlights its potential as a diagnostic biomarker. This study underscores the importance and potential of circRNAs and sEVs as cancer diagnostic tools.

Three-dimensional printing and decellularized-extracellular-matrix based methods for advances in artificial blood vessel fabrication: A review.

Blood vessels are the tubes through which blood flows and are divided into three types: millimeter-scale arteries, veins, and capillaries as well as micrometer-scale capillaries. Arteries and veins are the conduits that carry blood, while capillaries are where blood exchanges substances with tissues. Blood vessels are mainly composed of collagen fibers, elastic fibers, glycosaminoglycans and other macromolecular substances. There are about 19 feet of blood vessels per square inch of skin in the human body, which shows how important blood vessels are to the human body. Because cardiovascular disease and vascular trauma are common in the population, a great number of researches have been carried out in recent years by simulating the structures and functions of the person's own blood vessels to create different levels of tissue-engineered blood vessels that can replace damaged blood vessels in the human body. However, due to the lack of effective oxygen and nutrient delivery mechanisms, these tissue-engineered vessels have not been used clinically. Therefore, in order to achieve better vascularization of engineered vascular tissue, researchers have widely explored the design methods of vascular systems of various sizes. In the near future, these carefully designed and constructed tissue engineered blood vessels are expected to have practical clinical applications. Exploring how to form multi-scale vascular networks and improve their compatibility with the host vascular system will be very beneficial in achieving this goal. Among them, 3D printing has the advantages of high precision and design flexibility, and the decellularized matrix retains active ingredients such as collagen, elastin, and glycosaminoglycan, while removing the immunogenic substance DNA. In this review, technologies and advances in 3D printing and decellularization-based artificial blood vessel manufacturing methods are systematically discussed. Recent examples of vascular systems designed are introduced in details, the main problems and challenges in the clinical application of vascular tissue restriction are discussed and pointed out, and the future development trends in the field of tissue engineered blood vessels are also prospected.

Three-dimensional breast cancer tumor models based on natural hydrogels: a review. / åŸºäºŽå¤©ç„¶æ°´å‡èƒ¶çš„ä¸‰ç»´ä¹³è ºç™Œè‚¿ç˜¤æ¨¡åž‹ç ”ç©¶è¿›å±•.

Breast cancer is the most common cancer in women and one of the deadliest cancers worldwide. According to the distribution of tumor tissue, breast cancer can be divided into invasive and non-invasive forms. The cancer cells in invasive breast cancer pass through the breast and through the immune system or systemic circulation to different parts of the body, forming metastatic breast cancer. Drug resistance and distant metastasis are the main causes of death from breast cancer. Research on breast cancer has attracted extensive attention from researchers. In vitro construction of tumor models by tissue engineering methods is a common tool for studying cancer mechanisms and anticancer drug screening. The tumor microenvironment consists of cancer cells and various types of stromal cells, including fibroblasts, endothelial cells, mesenchymal cells, and immune cells embedded in the extracellular matrix. The extracellular matrix contains fibrin proteins (such as types I, II, III, IV, VI, and X collagen and elastin) and glycoproteins (such as proteoglycan, laminin, and fibronectin), which are involved in cell signaling and binding of growth factors. The current traditional two-dimensional (2D) tumor models are limited by the growth environment and often cannot accurately reproduce the heterogeneity and complexity of tumor tissues in vivo. Therefore, in recent years, research on three-dimensional (3D) tumor models has gradually increased, especially 3D bioprinting models with high precision and repeatability. Compared with a 2D model, the 3D environment can better simulate the complex extracellular matrix components and structures in the tumor microenvironment. Three-dimensional models are often used as a bridge between 2D cellular level experiments and animal experiments. Acellular matrix, gelatin, sodium alginate, and other natural materials are widely used in the construction of tumor models because of their excellent biocompatibility and non-immune rejection. Here, we review various natural scaffold materials and construction methods involved in 3D tissue-engineered tumor models, as a reference for research in the field of breast cancer.

A capsule-based scaffold incorporating decellularized extracellular matrix and curcumin for islet beta cell therapy in type 1 diabetes mellitus.

The transplantation of islet beta cells offers an alternative to heterotopic islet transplantation for treating type 1 diabetes mellitus (T1DM). However, the use of systemic immunosuppressive drugs in islet transplantation poses significant risks to the body. To address this issue, we constructed an encapsulated hybrid scaffold loaded with islet beta cells. This article focuses on the preparation of the encapsulated structure using 3D printing, which incorporates porcine pancreas decellularized extracellular matrix (dECM) to the core scaffold. The improved decellularization method successfully preserved a substantial proportion of protein (such as Collagen I and Laminins) architecture and glycosaminoglycans in the dECM hydrogel, while effectively removing most of the DNA. The inclusion of dECM enhanced the physical and chemical properties of the scaffold, resulting in a porosity of 83.62% ± 1.09% and a tensile stress of 1.85 ± 0.16 MPa. In teams of biological activity, dECM demonstrated enhanced proliferation, differentiation, and expression of transcription factors such as Ki67, PDX1, and NKX6.1, leading to improved insulin secretion function in MIN-6 pancreatic beta cells. In the glucose-stimulated insulin secretion experiment on day 21, the maximum insulin secretion from the encapsulated structure reached 1.96 ± 0.08 mIU ml-1, representing a 44% increase compared to the control group. Furthermore, conventional capsule scaffolds leaverage the compatibility of natural biomaterials with macrophages to mitigate immune rejection. Here, incorporating curcumin into the capsule scaffold significantly reduced the secretion of pro-inflammatory cytokine (IL-1ß, IL-6, TNF-α, IFN-γ) secretion by RAW264.7 macrophages and T cells in T1DM mice. This approach protected pancreatic islet cells against immune cell infiltration mediated by inflammatory factors and prevented insulitis. Overall, the encapsulated scaffold developed in this study shows promise as a natural platform for clinical treatment of T1DM.

Preparation of Thermosensitive Lignocellulose Hollow Fiber Membrane Grafted With PNIPAAm and Its Application as a Cell Culture Carrier in a RSOC Dynamic Culture.

Currently, the cells, which are urgently required for large-scale application in biomedical-related fields, harvested by traditional trypsin digestion are usually subject to repeated digestion, leading to a reduction of cell activity. In this study, poly (N-isopropylacrylamide) (PNIPAAm) was grafted onto the lignocellulose hollow fiber membranes (HFMs) with cerium ammonium nitrate (CAN) as the initiator to prepare thermosensitive HFMs, which was combined with a rotation system of culture (RSOC) to achieve dynamic culture and non-destructive harvesting of cells from the HFMs. The results of ATR-FTIR, elemental analysis, and SEM confirmed the successful preparation of PNIPAAm-grafted-HFMs, which also showed good biocompatibility to apply for cell culture carriers. In cooling detachment, the HFMs-0.01 group could completely detach the cells within 1 h with a cell separation efficiency of more than 90%. The laminin (LN) and fibronectin (FN) harvested by cooling detachment of P8 generation PC12 cells reached 0.0531 ± 0.0032 and 2.5045 ± 0.0001 pg/cell, respectively, which were significantly higher than that by trypsin digestion. In addition, the cells on the thermosensitive HFMs proliferated fastest in RSOC at 30 rpm with higher glucose consumption and lactate metabolism than in static conditions. Moreover, the cells that had dynamic detachment at 20 rpm had the highest cell density and activity. Therefore, the thermosensitive HFMs could be applied as cell culture carriers in RSOC for cell culturing at 30 rpm and harvesting at 20 rpm, which would provide considerable potential for large-scale cell culture in vitro.

Electromagnetic field-mediated chitosan/gelatin/nano-hydroxyapatite and bone-derived scaffolds regulate the osteoblastic and chondrogenic phenotypes of adipose-derived stem cells to construct osteochondral tissue engineering niche in vitro.

It is critical to explore the effects of electromagnetic field (EMF) on the construction of functional osteochondral tissue, which has shown certain clinical significance for the treatment of osteochondral injury. At present, there are few studies on the effect of the direction of EMF on cells. This study aimed to investigate the effects of EMF coupling on different parameters to control adipose-derived stem cells (ADSCs) proliferation and specific chondrogenic and osteogenic differentiation at 2D level and 3D level. The proliferation and differentiation of EMF-induced ADSCs are jointly regulated by EMF and space structure. In this study, Cs7/Gel3/nHAP scaffolds were prepared with good degradation rate (86.75 ± 4.96 %) and absorb water (1100 %), and the pore size was 195.63 ± 54.72 µm. The bone-derived scaffold with a pore size of 267.17 ± 129.18 µm was obtained and its main component was hydroxyapatite. Cs7/Gel3/nHAP scaffolds and bone-derived scaffolds are suitable as 3D level materials. The optimal EMF intensity was 2 mT for chondrogenic differentiation and proliferation and 1 mT for osteogenic differentiation and proliferation. It is noteworthy that EMF has a negative correlation with ADSCs proliferation in the vertical direction at 2D level, while it has a positive correlation with ADSCs proliferation at 3D level. EMF mediated 3D osteochondral scaffold provide good strategy for osteochondral tissue engineering construction.

Nutrient-delivery and metabolism reactivation therapy for melanoma.

To fulfil the demands of rapid proliferation, tumour cells undergo significant metabolic alterations. Suppression of hyperactivated metabolism has been proven to counteract tumour growth. However, whether the reactivation of downregulated metabolic pathways has therapeutic effects remains unexplored. Here we report a nutrient-based metabolic reactivation strategy for effective melanoma treatment. L-Tyrosine-oleylamine nanomicelles (MTyr-OANPs) were constructed for targeted supplementation of tyrosine to reactivate melanogenesis in melanoma cells. We found that reactivation of melanogenesis using MTyr-OANPs significantly impeded the proliferation of melanoma cells, primarily through the inhibition of glycolysis. Furthermore, leveraging melanin as a natural photothermal reagent for photothermal therapy, we demonstrated the complete eradication of tumours in B16F10 melanoma-bearing mice through treatment with MTyr-OANPs and photothermal therapy. Our strategy for metabolism activation-based tumour treatment suggests specific nutrients as potent activators of metabolic pathways.

Functional materials of 3D bioprinting for wound dressings and skin tissue engineering applications: A review.

The skin plays an important role in vitamin D synthesis, humoral balance, temperature regulation, and waste excretion. Due to the complexity of the skin, fluids loss, bacterial infection, and other life-threatening secondary complications caused by skin defects often lead to the damage of skin functions. 3D bioprinting technology, as a customized and precise biomanufacturing platform, can manufacture dressings and tissue engineering scaffolds that accurately simulate tissue structure, which is more conducive to wound healing. In recent years, with the development of emerging technologies, an increasing number of 3D-bioprinted wound dressings and skin tissue engineering scaffolds with multiple functions, such as antibacterial, antiinflammatory, antioxidant, hemostatic, and antitumor properties, have significantly improved wound healing and skin treatment. In this article, we review the process of wound healing and summarize the classification of 3D bioprinting technology. Following this, we shift our focus on the functional materials for wound dressing and skin tissue engineering, and also highlight the research progress and development direction of 3D-bioprinted multifunctional wound healing materials.

Fabrication and detection of a novel hybrid conductive scaffold based on alginate/gelatin/carboxylated carbon nanotubes (Alg/Gel/mMWCNTs) for neural tissue engineering.

Carbon nanotubes (CNTs), as kinds of conductive carbon nanomaterials, were widely applied in neural tissue engineering due to their excellent electrical conductivity and good biocompatibility. In this study, the carboxyl-modified multi-walled carbon nanotubes (mMWCNTs) were introduced into sodium alginate/gelatin (Alg/Gel) scaffolds to optimize the function of the hybrid scaffolds. The Alg/Gel/mMWCNTs conductive scaffolds with mMWCNTs content of 1%, 3%, and 5% were prepared by freeze-drying, respectively. Following this, the physicochemical properties and biocompatibility of the hybrid scaffolds at different magnetic field intensities were evaluated. The conductive scaffolds were characterized by Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). In general, the mMWCNTs addition improved the hydrophilic, electrical conductivity and mechanical properties of the composite scaffold, and PC12 cells showed a trend of gradual increase over culture time. Particularly, the Alg/Gel-1%C scaffold exhibited the best cell proliferation behavior. Briefly, the surface contact angle decreased from 74 ± 1° to 60 ± 3°, the electrical conductivity and compressive modulus increased to 1.32 × 10-3 ± 2.1 × 10-4 S/cm and 1.40 ± 0.076 Mpa, the G1 phase from 55.67 ± 1.86% to 59.77 ± 0.94% and the G2 phase from 10.32 ± 0.35% to 13.93 ± 1.26%，respectively. In the SEM images, PC12 cells were well-shaped and densely distributed. Therefore, the Alg/Gel/mMWCNTs conductive scaffold has potential as a tissue engineering scaffold in nerve regeneration.

A 3D bioprinted tumor model fabricated with gelatin/sodium alginate/decellularized extracellular matrix bioink.

109Tissue-engineered scaffolds are more commonly used to construct three-dimensional (3D) tumor models for in vitro studies when compared to the conventional two-dimensional (2D) cell culture because the microenvironments provided by the 3D tumor models closely resemble the in vivo system and could achieve higher success rate when the scaffolds are translated for use in pre-clinical animal model. Physical properties, heterogeneity, and cell behaviors of the model could be regulated to simulate different tumors by changing the components and concentrations of materials. In this study, a novel 3D breast tumor model was fabricated by bioprinting using a bioink that consists of porcine liver-derived decellularized extracellular matrix (dECM) with different concentrations of gelatin and sodium alginate. Primary cells were removed while extracellular matrix components of porcine liver were preserved. The rheological properties of biomimetic bioinks and the physical properties of hybrid scaffolds were investigated, and we found that the addition of gelatin increased hydrophilia and viscoelasticity, while the addition of alginate increased mechanical properties and porosity. The swelling ratio, compression modulus, and porosity could reach 835.43 ± 130.61%, 9.64 ± 0.41 kPa, and 76.62 ± 4.43%, respectively. L929 cells and the mouse breast tumor cells 4T1 were subsequently inoculated to evaluate biocompatibility of the scaffolds and to form the 3D models. The results showed that all scaffolds exhibited good biocompatibility, and the average diameter of tumor spheres could reach 148.52 ± 8.02 µm on 7 d. These findings suggest that the 3D breast tumor model could serve as an effective platform for anticancer drug screening and cancer research in vitro.

Preparation and Characterization of Polylactic Acid/Nano Hydroxyapatite/Nano Hydroxyapatite/Human Acellular Amniotic Membrane (PLA/nHAp/HAAM) Hybrid Scaffold for Bone Tissue Defect Repair.

Bone tissue engineering is a novel and efficient repair method for bone tissue defects, and the key step of the bone tissue engineering repair strategy is to prepare non-toxic, metabolizable, biocompatible, bone-induced tissue engineering scaffolds of suitable mechanical strength. Human acellular amniotic membrane (HAAM) is mainly composed of collagen and mucopolysaccharide; it has a natural three-dimensional structure and no immunogenicity. In this study, a polylactic acid (PLA)/Hydroxyapatite (nHAp)/Human acellular amniotic membrane (HAAM) composite scaffold was prepared and the porosity, water absorption and elastic modulus of the composite scaffold were characterized. After that, the cell-scaffold composite was constructed using newborn Sprague Dawley (SD) rat osteoblasts to characterize the biological properties of the composite. In conclusion, the scaffolds have a composite structure of large and small holes with a large pore diameter of 200 µm and a small pore diameter of 30 µm. After adding HAAM, the contact angle of the composite decreases to 38.7°, and the water absorption reaches 249.7%. The addition of nHAp can improve the scaffold's mechanical strength. The degradation rate of the PLA+nHAp+HAAM group was the highest, reaching 39.48% after 12 weeks. Fluorescence staining showed that the cells were evenly distributed and had good activity on the composite scaffold; the PLA+nHAp+HAAM scaffold has the highest cell viability. The adhesion rate to HAAM was the highest, and the addition of nHAp and HAAM could promote the rapid adhesion of cells to scaffolds. The addition of HAAM and nHAp can significantly promote the secretion of ALP. Therefore, the PLA/nHAp/HAAM composite scaffold can support the adhesion, proliferation and differentiation of osteoblasts in vitro which provide sufficient space for cell proliferation, and is suitable for the formation and development of solid bone tissue.

3D bioprinting of dECM/Gel/QCS/nHAp hybrid scaffolds laden with mesenchymal stem cell-derived exosomes to improve angiogenesis and osteogenesis.

Craniofacial bone regeneration is a coupled process of angiogenesis and osteogenesis, which, associated with infection, still remains a challenge in bone defects after trauma or tumor resection. 3D tissue engineering scaffolds with multifunctional-therapeutic properties can offer many advantages for the angiogenesis and osteogenesis of infected bone defects. Hence, in the present study, a microchannel networks-enriched 3D hybrid scaffold composed of decellularized extracellular matrix (dECM), gelatin (Gel), quaterinized chitosan (QCS) and nano-hydroxyapatite (nHAp) (dGQH) was fabricated by an extrusion 3D bioprinting technology. And enlightened by the characteristics of natural bone microstructure and the demands of vascularized bone regeneration, the exosomes (Exos) isolated from human adipose derived stem cells as angiogenic and osteogenic factors were then co-loaded into the desired dGQH20hybrid scaffold based on an electrostatic interaction. The results of the hybrid scaffolds performance characterization showed that these hybrid scaffolds exhibited an interconnected pore structure and appropriate degradability (>61% after 8 weeks of treatment), and the dGQH20hybrid scaffold displayed the highest porosity (83.93 ± 7.38%) and mechanical properties (tensile modulus: 62.68 ± 10.29 MPa, compressive modulus: 16.22 ± 3.61 MPa) among the dGQH hybrid scaffolds. Moreover, the dGQH20hybrid scaffold presented good antibacterial activities (against 94.90 ± 2.44% ofEscherichia coliand 95.41 ± 2.65% ofStaphylococcus aureus, respectively) as well as excellent hemocompatibility and biocompatibility. Furthermore, the results of applying the Exos to the dGQH20hybrid scaffold showed that the Exo promoted the cell attachment and proliferation on the scaffold, and also showed a significant increase in osteogenesis and vascularity regeneration in the dGQH@Exo scaffoldsin vitroandin vivo. Overall, this novel dECM/Gel/QCS/nHAp hybrid scaffold laden with Exo has a considerable potential application in reservation of craniofacial bone defects.

Construction and properties detection of 3D micro-structure scaffolds base on decellularized sheep kidney before and after crosslinking.

Decellularized extracellular matrix is one form of natural material in tissue engineering. The process of dECM retains the tissue microstructure, provides good cell adhesion sites, maintains most of biological signals that promotes the survival and differentiation ability of cells. In this study, sheep kidney was decellularized followed by histochemical staining, elemental analysis and scanning electron microscopy characterizations. The dECM scaffold was prepared with different sequences of freeze drying technology, crosslinking and the water absorption, porosity, mechanical strength with subsequent thermogravimetric analysis, Infrared spectroscopy and biocompatibility tests. Our results indicated that these decellularized treatments of sheep kidney can effectively remove DNA and retain uniform pore size distribution. After crosslinking the scaffold's water absorption decreased from 987.56 ± 40.21% to 934.39 ± 39.61%, the porosity decreased from 89.64 ± 3.2% to 85.09 ± 17.63%, and the compression modulus increased from 304.32 ± 25.43 kPa to 459.53 ± 38.92 kPa, with thermal process the percentage of weight loss decreased from 66.57% to 44.731%, in addition, the composition didn't change significantly, crosslinking could also promote the stability. In terms of biocompatibility, the number of viable cells increased significantly with the days. In conclusion, the crosslinked decellularized sheep kidney extracellular matrix scaffold reduced water absorption and porosity slightly, but has a significant increase in mechanical properties, and presented excellent biocompatibility which are beneficial to cell adhesion, growth and differentiation.

Investigation on repairing diabetic foot ulcer based on 3D bio-printing Gel/dECM/Qcs composite scaffolds.

Diabetic foot ulcers are one of the most serious of the numerous complications of diabetes mellitus, causing great physical trauma and financial stress to patients, and accelerating wound healing in diabetic patients remains one of the major clinical challenges. Exosomes from adipose-derived stem cells can directly and indirectly promote wound healing. However, due to the low retention rate of exosomes in the wound, exosome treatment is difficult to achieve the expected effect. Therefore, it is of great significance to synthesize a composite scaffold that can stably load exosomes and has antibacterial properties. In this study, fresh pig skin was decellularized to obtain decellularized matrix (dECM). Secondly, quaternized chitosan (Qcs) was modified with quaternary ammonium salt to make it soluble in water after quaternization. Finally, Gel-dECM-Qcs (GDQ) bioink was prepared by adding acellular matrix and quaternized chitosan with temperature sensitive gelatin (Gel) as carrier. Tissue engineered composite scaffolds were then prepared by extrusion 3D printing technology. Subsequently, the physicochemical properties, biocompatibility and antimicrobial capacity of the composite scaffolds were determined, and the data showed that the composite scaffolds had good mechanical properties, biocompatibility and antimicrobial capacity, and the maximum stress of the composite scaffolds was 1.16 ± 0.05 MPa, the composite scaffolds were able to proliferate and adhered to the L929 cells, and the kill rates of composite scaffolds against E. coli and S. aureus after incubation for 24 h were 93.24 ± 1.22 % and 97.34 ± 0.23 %, respectively. Overall, the GDQ composite scaffolds have good mechanical properties adapted to skin bending, its good biocompatibility can promote the growth and migration of fibroblasts, reshape injured tissues, accelerate the wound healing, and excellent antimicrobial ability can inhibit the growth of E. coli and S. aureus, reducing the impact of bacterial infections on wounds. Moreover, the composite scaffolds have the potential to be used as exosom-loaded hydrogel dressings, which provides a basis for the subsequent research on the repair of diabetic foot ulcers.