Matrigel Group Research Articles

Dendritic epidermal T cell hydrogel induces the polarization of M2 macrophages to promote the healing of deep tissue pressure injury

Dendritic epidermal T cells (DETCs) have been shown to promote wound healing. However, the mechanisms involved need to be better understood. In the present study, we investigated the role and mechanism of DETCs in deep tissue pressure injury (DTPI). We established the DTPI model using C57BL/6 mice. Then, DTPI was evaluated and analyzed by histological staining, immunohistochemistry, real-time PCR, Western blotting, and flow cytometry in different treatment groups (DETCs, DETCs/gel, Matrigel, Saline, and Normal group). The results showed that insulin-like growth factor 1 and vascular endothelial growth factor-A expression increased after local DETCs and DETCs/gel implantation in DTPI on days 3 and 7. M1 (inducible nitric oxide synthas-marked) macrophages were predominant at 3 days after DTPI. At 7 days, M1 macrophages were decreased, and M2 (CD206-marked) macrophages were increased in the DETCs and DETCs/gel groups. In vitro, in the co-culture of DETCs and RAW264.7, CD206 expression was significantly increased in M2 macrophages. In addition, Interleukin-17A initially inhibited wound healing 1 day after injury. However, it promoted wound healing at 7, 14, and 21 days after treatment with DETCs and DETCs/gel, respectively. In conclusion, our data suggest that exogenous DETCs improve DTPI wound healing by regulating M1 to M2 macrophage polarization.

Chick Chorioallantoic Membrane as a Patient-Derived Xenograft Model for Uveal Melanoma: Imaging Modalities for Growth and Vascular Evaluation

Patient-derived tumor xenografts (PDXs) have emerged as valuable preclinical in vivo models in oncology as they largely retain the polygenomic architecture of the human tumors from which they originate. Although animal models are accompanied by cost and time constraints and a low engraftment rate, PDXs have primarily been established in immunodeficient rodent models for the in vivo assessment of tumor characteristics and of novel therapeutic cancer targets. The chick chorioallantoic membrane (CAM) assay represents an attractive alternative in vivo model that has long been used in the research of tumor biology and angiogenesis, and can overcome some of these limitations. In this study, we reviewed different technical approaches for the establishment and monitoring of a CAM-based uveal melanoma PDX model. Forty-six fresh tumor grafts were acquired after enucleation from six uveal melanoma patients and were implanted onto the CAM on ED7 with Matrigel and a ring (group 1), with Matrigel (group 2), or natively without Matrigel or a ring (group 3). Real-time imaging techniques, such as various ultrasound modalities, optical coherence tomography, infrared imaging, and imaging analyses with Image J for tumor growth and extension, as well as color doppler, optical coherence angiography, and fluorescein angiography for angiogenesis, were performed on ED18 as alternative monitoring instruments. The tumor samples were excised on ED18 for histological assessment. There were no significant differences between the three tested experimental groups regarding the length and width of the grafts during the development period. A statistically significant increase in volume (p = 0.0007) and weight (p = 0.0216) between ED7 and ED18 was only documented for tumor specimens of group 2. A significant correlation of the results for the cross-sectional area, largest basal diameter, and volume was documented between the different imaging and measurement techniques and the excised grafts. The formation of a vascular star around the tumor and of a vascular ring on the base of the tumor was observed for the majority of the viable developing grafts as a sign of successful engraftment. The establishment of a CAM-PDX uveal melanoma model could elucidate the biological growth patterns and the efficacy of new therapeutic options in vivo. The methodological novelty of this study, investigating different implanting techniques and exploiting advances in real-time imaging with multiple modalities, allows precise, quantitative assessment in the field of tumor experimentation, underlying the feasibility of CAM as an in vivo PDX model.

Towards Standardisation of a Diffuse Midline Glioma Patient-Derived Xenograft Mouse Model Based on Suspension Matrices for Preclinical Research.

Diffuse midline glioma (DMG) is an aggressive brain tumour with high mortality and limited clinical therapeutic options. Although in vitro research has shown the effectiveness of medication, successful translation to the clinic remains elusive. A literature search highlighted the high variability and lack of standardisation in protocols applied for establishing the commonly used HSJD-DIPG-007 patient-derived xenograft (PDX) model, based on animal host, injection location, number of cells inoculated, volume, and suspension matrices. This study evaluated the HSJD-DIPG-007 PDX model with respect to its ability to mimic human disease progression for therapeutic testing in vivo. The mice received intracranial injections of HSJD-DIPG-007 cells suspended in either PBS or Matrigel. Survival, tumour growth, and metastases were assessed to evaluate differences in the suspension matrix used. After cell implantation, no severe side effects were observed. Additionally, no differences were detected in terms of survival or tumour growth between the two suspension groups. We observed delayed metastases in the Matrigel group, with a significant difference compared to mice with PBS-suspended cells. In conclusion, using Matrigel as a suspension matrix is a reliable method for establishing a DMG PDX mouse model, with delayed metastases formation and is a step forward to obtaining a standardised in vivo PDX model.

Human Muscle–Derived Cells Are Capable of Tenogenic Differentiation and Contribution to Tendon Repair

Background: It has been reported that the harvested hamstring tendon for autograft could be regenerated with well-oriented fibers and uniformly distributed spindle-shaped cells after removal. However, which cell type might participate in the repair process remains unknown. Purpose: To investigate the tenogenic differentiation potential of human muscle–derived cells (MDCs) both in vitro and in vivo. Study Design: Controlled laboratory study. Methods: Primary human MDCs and tenocytes were isolated from discarded materials during a peroneus longus tendon–harvesting procedure. Expression of tenogenic genes were evaluated and compared among MDCs, MDCs with tenogenic induction, and tenocytes. RNA sequencing was performed to evaluate the expression profile of differentiated MDCs. Human MDCs were implanted in a tendon injury model to investigate the in vivo tenogenic differentiation potential. Histologic and functional analyses were performed to evaluate the function of MDCs for tendon repair. Results: The relative expression levels (in fold change) of tenogenic genes Col I, MKX, SCX, THBS4, and TNC in MDCs were significantly upregulated 11.5 ± 1.3, 957.1 ± 63.7, 19.1 ± 2.8, 61.9 ± 4.8, and 10.2 ± 2.8 after tenogenic induction, respectively. The expression profile of tenogenically differentiated MDCs was much closer to primary tenocytes. Activation of TGF-β/Smad3 signaling significantly promoted the tenogenic differentiation ability of MDCs. Transplanted human MDCs were identified in regenerated tendon and expressed tenogenic genes. As for biomechanical properties, the failure loads in the Matrigel, transplantation, and uninjured groups were 7.2 ± 0.5, 11.6 ± 0.3, and 13.9 ± 0.7 N, while the stiffness values were 4.4 ± 1.3 × 103, 7.6 ± 0.8 × 103, and 10.9 ± 1.1 × 103 N/m. Plantarflexion force, histologic morphology, and motor function were also significantly improved after MDC transplantation in a tendon injury model. Conclusion: There exist cells with tenogenic differentiation potential in human skeletal muscles. Activation of TGF-β/Smad3 signaling plays an important role in tenogenic differentiation for human MDCs. Human MDCs contribute to structural and functional repair for the injured tendon. MDCs are a potential cell source to participate in the repair process after tendon injury. Clinical Relevance: The MDCs could be a promising cell source to repair tendon injury.

An artificial LAMA2-GelMA hydrogel microenvironment for the development of pancreatic endocrine progenitors

The biomimetic pancreatic microenvironment improves the differentiation efficiency and function of human embryonic stem cell-derived β-cells (SC-β cells). Thus, a laminin subunit alpha 2-gelatin methacrylate (LAMA2-GelMA) hybrid hydrogel as a bionics carrier for the formation and maturation of endocrine lineage was developed in our research, based on pancreas proteomics analysis of postnatal mice. Pancreatic endocrine cells cultured on the hybrid hydrogel in vitro, which was composed of 0.5 μg/mL LAMA2 protein and 4% GelMA, the expression of transcription factors (TFs), including NKX6.1, NKX6.2, and NEUROD1 were upregulated. Single-cell transcriptomics was performed after LAMA2 knockdown during the early differentiation of pancreatic progenitor (PP) cells, a marked decrease in the forkhead box protein A2 (FOXA2+)/GATA-binding factor 6 (GATA6+) cluster was detected. Also, we clarified that as a receptor of LAMA2, integrin subunit alpha 7 (ITGA7) participated in Integrin-AKT signaling transduction and influenced the protein levels of FOXA2 and PDX1. In vivo experiments showed that, PP cells encapsulated in the LAMA2-GelMA hydrogel exhibited higher serum C-peptide levels compared to the GelMA and Matrigel groups in nude mice and reversed hyperglycemia more quickly in STZ-induced diabetic nude mice. Taken together, our findings highlighted the feasibility of constructing a pancreas-specific microenvironment based on proteomics and tissue engineering for the treatment of diabetes.

UC-MSCs promote frozen-thawed ovaries angiogenesis via activation of the Wnt/β-catenin pathway in vitro ovarian culture system

BackgroundOvarian tissue cryopreservation and transplantation are novel therapeutic approaches for fertility preservation. However, follicle loss caused by ischemic and hypoxic damage is one of the issues after frozen-thawed ovarian tissue transplantation. Promoting angiogenesis in grafts is the key to restore cryopreserved ovarian function. Mesenchymal stem cells (MSCs) have been reported to facilitate angiogenesis in the cryopreserved ovarian tissue transplantation. However, the risk of embolization, immunogenic effect and tumorigenesis hinders the clinical application of MSCs to human organ transplantation. In this study, we established an in vitro ovarian culture system to restore frozen-thawed ovarian function before transplantation with the application of umbilical cord mesenchymal stem cells (UC-MSCs), and explored the effects of UC-MSCs on frozen-thawed ovaries in vitro ovarian culture system and the mechanisms of UC-MSCs on the angiogenesis of frozen-thawed ovaries.MethodsA simple in vitro three dimensional (3D) ovarian culture system using Matrigel was established to support to an ideal niche, and ovary was alone cultured in the 24-well plate as a control. We also evaluated the effects of UC-MSCs treatment on ovarian function with or without Matrigel support. All thawed ovaries were randomly divided into control group (Matrigel−/UC-MSCs−), Matrigel group (Matrigel+/UC-MSCs−), UC-MSCs group (Matrigel−/UC-MSCs+) and UC-MSCs + Matrigel group (Matrigel+/UC-MSCs+). HE staining was used to detect the histological structure of follicles and TUNEL staining was used to detect cell apoptosis. The number of microvessels was counted to evaluate neovascularization. The mRNA expression of VEGFA, IGF1 and ANGPT2 were detected by RT-PCR. Western blotting was used to measure the expression of GSK-3β, β-catenin and p-β-catenin.ResultsIn the absence of UC-MSCs, 3D culture system supported by Matrigel showed significantly improved follicular development and microvascular number. Additionally, UC-MSCs were also found to effectively improve follicular development and microvascular number regardless of the culture condition used. However, alleviated follicular apoptosis, increased mRNA expression of angiogenesis-related gene and activated Wnt/β-catenin pathway occurred only in the UC-MSCs + Matrigel group. Besides, with the application of IWP-2 in UC-MSCs + Matrigel group, Wnt//β-catenin pathway could be blocked by IWP-2 serving as one of Wnt/β-catenin pathway inhibitors.ConclusionsThis in vitro study showed the beneficial effects of UC-MSCs on thawed ovaries and explored a potential mechanism inducing angiogenesis. In particular, 3D ovarian culture system supported by Matrigel further improved UC-MSCs treatment. The in vitro culture system using Matrigel and UC-MSCs may provide a potential treatment strategy for improving the success rate of thawed ovaries transplantation.

Decellularized Periosteum-Derived Hydrogels Promote the Proliferation, Migration and Osteogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells.

Human umbilical cord mesenchymal stem cells (hUCMSCs) are promising for bone tissue engineering, which have a non-invasive harvesting process, high cell yield, favorable proliferation capacity, and low immunogenicity. However, the osteogenic efficacy of hUCMSCs is relatively lower than that of bone marrow mesenchymal stem cells (BMSCs). Hydrogels from decellularized extracellular matrix (dECM) preserve the biological compositions and functions of natural ECM, which can provide tissue-specific cues to regulate phenotypic expression and cell fate. It is unknown, however, whether hydrogels from periosteum can serve as pro-osteogenic carriers of hUCMSCs. Herein, a decellularized periosteum-derived hydrogel (dPH) was fabricated to reveal the effects of periosteum-specific cues on the bioactivities of hUCMSCs. A widely used non-bone/periosteum-derived ECM hydrogel product, Matrigel, was used as the control group. After decellularization, the absence of nuclei in the histological analysis indicated a successful removal of cellular components, which was also confirmed by DNA content quantification. The storage modulus of dPH increased (from 164.49 ± 29.92 Pa to 855.20 ± 20.67 Pa) with increasing concentration (from 0.5% to 1%). With a highly porous, fibrous microstructure, dPH had a more hydrophilic surface than Matrigel, of which the water contact angle reduced 62.62 ± 0.04%. Furthermore, dPH prominently promoted the initial cellular spreading with a significantly higher cell surface area (1.47-fold), cell spreading length (1.45-fold) and proliferation (approximately 1.05–1.13-fold) of hUCMSCs than those of Matrigel. Additionally, dPH was conducive to cell migration, whereas no cells migrated to Matrigel in the Transwell model. Compared with those of the Matrigel group, the osteogenesis-related genes expression levels (runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN)) and mineralized matrix formation (9.74-fold) of the hUCMSCs significantly increased in the dPH group. Our study indicated that dPH could provide a pro-osteogenic microenvironment for hUCMSCs, thereby revealing a promising application potential to repair bone defects.

Melatonin attenuates radiation-induced cortical bone-derived stem cells injury and enhances bone repair in postradiation femoral defect model

BackgroundThe healing of bone defects can be challenging for clinicians to manage, especially after exposure to ionizing radiation. In this regard, radiation therapy and accidental exposure to gamma (γ)-ray radiation have been shown to inhibit bone formation and increase the risk of fractures. Cortical bone-derived stem cells (CBSCs) are reportedly essential for osteogenic lineages, bone maintenance and repair. This study aimed to investigate the effects of melatonin on postradiation CBSCs and bone defect healing.MethodsCBSCs were extracted from C57BL/6 mice and were identified by flow cytometry. Then CBSCs were subjected to 6 Gy γ-ray radiation followed by treatment with various concentrations of melatonin. The effects of exogenous melatonin on the self-renewal and osteogenic capacity of postradiation CBSCs in vitro were analyzed. The underlying mechanisms involved in genomic stability, apoptosis and oxidative stress-related signaling were further analyzed by Western blotting, flow cytometry and immunofluorescence assays. Moreover, postradiation femoral defect models were established and treated with Matrigel and melatonin. The effects of melatonin on postradiation bone healing in vivo were evaluated by micro-CT and pathological analysis.ResultsThe decrease in radiation-induced self-renewal and osteogenic capacity were partially reversed in postradiation CBSCs treated with melatonin (P < 0.05). Melatonin maintained genomic stability, reduced postradiation CBSC apoptosis and intracellular oxidative stress, and enhanced expression of antioxidant-related enzymes (P < 0.05). Western blotting validated the anti-inflammatory effects of melatonin by downregulating interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels via the extracellular regulated kinase (ERK)/nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase-1 (HO-1) signaling pathway. Melatonin was also found to exhibit antioxidant effects via NRF2 signaling. In vivo experiments demonstrated that the newly formed bone in the melatonin plus Matrigel group had higher trabecular bone volume per tissue volume (BV/TV) and bone mineral density values with lower IL-6 and TNF-α levels than in the irradiation and the Matrigel groups (P < 0.05).ConclusionThis study suggested that melatonin could protect CBSCs against γ-ray radiation and assist in the healing of postradiation bone defects.

Therapeutic potential of small extracellular vesicles derived from lipoma tissue in adipose tissue regeneration\u2014an in vitro and in vivo study

ObjectiveTo explore the adipogenic effects of the small extracellular vesicles derived from the lipoma tissues (sEV-LT), and to find a new cell-free therapeutic approach for adipose tissue regeneration.MethodsAdipose tissue-derived stem cells (ADSCs) and small extracellular vesicles derived from the adipose tissues (sEV-AT) were isolated from human adipose tissue, while sEV-LT were isolated from human lipomatous tissue. ADSCs were characterized by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. sEV was identified by electron microscopy, nanoparticle tracking, and western blotting. ADSCs were treated with sEV-LT and sEV-AT, respectively. Fluorescence confocal microscopy was used to investigate whether sEV-LT and sEV-AT could be taken by ADSCs. The proliferation and migration abilities and adipogenic differentiation assay of ADSCs were evaluated by CCK-8 assays, scratch test, and oil red O staining test, and the expression levels of adipogenic-related genes C/EBP-δ, PPARγ2, and Adiponectin in ADSCs were assessed by real-time quantitative PCR (RT-PCR). The sEV-LT and sEV-AT transplantation tubes were implanted subcutaneously in SD rats, and the neotissues were qualitatively and histologically evaluated at 2, 4, 8, and 12 weeks after transplantation. Hematoxylin and eosin (H&E) staining was subsequently used to observe and compare the adipogenesis and angiogenesis in neotissues, while immunohistochemistry was used to examine the expression and the distribution of C/EBP-α, PPARγ, Adiponectin, and CD31 at the 4th week.ResultsThe in vitro experiments showed that both sEV-LT and sEV-AT could be taken up by ADSCs via endocytosis. The scratch experiment and CCK-8 experiment showed that the migration area and proliferation number of ADSCs in sEV-LT group and sEV-AT group were significantly higher than those in the non-sEV group (p < 0.05). Compared with sEV-AT group, sEV-LT group had larger migration area and proliferation number of ADSCs (p < 0.05). Oil red O staining and RT-PCR experiments showed that, compared with the non-sEVs group, the lipid droplets and the mRNA expression levels of adipogenesis-related genes PPARγ2 and Adiponectin of ADSCs in sEV-LT group and sEV-AT group were significantly upregulated (p < 0.05); however, there was no statistical significance in the expression level of C/EBP-δ (p > 0.05). In addition, no significant difference in the amount of lipid droplets and adipogenesis-related genes between the sEV-LT groups and sEV-AT was seen (p > 0.05). At 2, 4, 8, and 12 weeks, the adipocyte area and the number of capillaries in neotissues in the sEV-LT groups and sEV-AT groups were significantly increased compared with the Matrigel group (p < 0.05); however, there was no dramatic difference between sEV-LT groups and sEV-AT groups (p > 0.05). At the 4th week, neotissues in the sEV-LT groups and sEV-AT groups all showed upregulated expression of C/EBP-α, PPARγ, Adiponectin, and CD31 protein, while neotissues in the Matrigel group only showed positive expression of CD31 protein.ConclusionsThis study demonstrated that sEV-LT exerted promotion effects on adipose tissue regeneration by accelerating the proliferation, migration, and adipogenic differentiation of ADSCs in vitro and recruiting adipocytes and promoting angiogenesis in vivo. The sEV-LT could serve as an alternative cell-free therapeutic strategy for generating adipose tissue, thus providing a promising application prospect in tissue engineering.

Effect of Honeycomb β-TCP Geometrical Structure on Bone Tissue Regeneration in Skull Defect.

The effect of the geometric structure of artificial biomaterials on skull regeneration remains unclear. In a previous study, we succeeded in developing honeycomb β-tricalcium phosphate (β-TCP), which has through-and-through holes and is able to provide the optimum bone microenvironment for bone tissue regeneration. We demonstrated that β-TCP with 300-μm hole diameters induced vigorous bone formation. In the present study, we investigated how differences in hole directions of honeycomb β-TCP (horizontal or vertical holes) influence bone tissue regeneration in skull defects. Honeycomb β-TCP with vertical and horizontal holes was loaded with BMP-2 using Matrigel and Collagen gel as carriers, and transplanted into skull bone defect model rats. The results showed that in each four groups (Collagen alone group, Matrigel alone group, Collagen + BMP group and Matrigel + BMP-2), vigorous bone formation was observed on the vertical β-TCP compared with horizontal β-TCP. The osteogenic area was larger in the Matrigel groups (with and without BMP-2) than in the Collagen group (with and without BMP-2) in both vertical β-TCP and horizontal β-TCP. However, when BMP-2 was added, the bone formation area was not significantly different between the Collagen group and the Matrigel group in the vertical β-TCP. Histological finding showed that, in vertical honeycomb β-TCP, new bone formation extended to the upper part of the holes and was observed from the dura side to the periosteum side as added to the inner walls of the holes. Therefore, we can control efficient bone formation by creating a bone microenvironment provided by vertical honeycomb β-TCP. Vertical honeycomb β-TCP has the potential to be an excellent biomaterial for bone tissue regeneration in skull defects and is expected to have clinical applications.

850-P: Continued Monitoring of the Foreign-Body Response to Improve Long-Term Success of Macroencapsulation Devices for Type 1 Diabetes: A Proof-of-Concept Study

Over 463 million people are living with diabetes mellitus (DM). Type 1 DM results from an autoimmune destruction of pancreatic β-cells and accounts for 5-10% of cases. Macroencapsulation devices are a promising therapy for the treatment of T1DM as they improve insulin producing cell retention and viability. However they have had limited success due to a complex cascade of events, causing fibrous encapsulation and ultimately device failure (the foreign body response (FBR)). The FBR is a critical hurdle associated with the success of implantable devices. Here, we aim to develop a biosensing membrane that monitors the FBR in real time; ensuring steps can be taken to modulate the host’s response to the implant. A biosensing membrane was developed consisting of a conductive layer of nonmetallic, highly conductive acrylic adhesive sandwiched between two layers of 0.08 mm thermoplastic polyurethane (TPU). Thereafter the membrane was incorporated into a refillable macroencapsulation device with soft robotic capabilities. In vitro experiments were carried out using electrical impedance spectroscopy. Baseline impedance measurement were taken for porous and non-porous devices using a buffered saline (PBS). Various concentrations of Matrigel (30%, 40%, 50% and 60%) were left to solidify on the membrane, as it contains extracellular matrix proteins, making it analogous to fibrotic tissue components. A significant increase of impedance was seen across the Matrigel groups when compared to PBS alone (p&amp;lt;0.001). A significant difference was also evident between the varying concentrations of Matrigel along the spectra, an example of which is 30% vs. 50% Matrigel at 1.2649 Hz (p&amp;lt;0.001). We have developed a novel approach to measure the membrane permeability in vitro. Monitoring the FBR in vivo, will provide feedback on the functionality of implanted devices, to improve their longevity for the delivery of insulin producing cells. Disclosure R.M. Beatty: None. E.B. Dolan: None. S.T. Robinson: None. R. OConnor: None. R. Wylie: None. G.P. Duffy: None.

Bone marrow-derived mesenchymal stem cells versus adipose-derived mesenchymal stem cells for peripheral nerve regeneration.

Studies have confirmed that bone marrow-derived mesenchymal stem cells (MSCs) can be used for treatment of several nervous system diseases. However, isolation of bone marrow-derived MSCs (BMSCs) is an invasive and painful process and the yield is very low. Therefore, there is a need to search for other alterative stem cell sources. Adipose-derived MSCs (ADSCs) have phenotypic and gene expression profiles similar to those of BMSCs. The production of ADSCs is greater than that of BMSCs, and ADSCs proliferate faster than BMSCs. To compare the effects of venous grafts containing BMSCs or ADSCs on sciatic nerve injury, in this study, rats were randomly divided into four groups: sham (only sciatic nerve exposed), Matrigel (MG; sciatic nerve injury + intravenous transplantation of MG vehicle), ADSCs (sciatic nerve injury + intravenous MG containing ADSCs), and BMSCs (sciatic nerve injury + intravenous MG containing BMSCs) groups. Sciatic functional index was calculated to evaluate the function of injured sciatic nerve. Morphologic characteristics of nerves distal to the lesion were observed by toluidine blue staining. Spinal motor neurons labeled with Fluoro-Gold were quantitatively assessed. Compared with sham-operated rats, sciatic functional index was lower, the density of small-diameter fibers was significantly increased, and the number of motor neurons significantly decreased in rats with sciatic nerve injury. Neither ADSCs nor BMSCs significantly improved the sciatic nerve function of rats with sciatic nerve injury, increased fiber density, fiber diameters, axonal diameters, myelin sheath thickness, and G ratios (axonal diameter/fiber diameter ratios) in the sciatic nerve distal to the lesion site. There was no significant difference in the number of spinal motor neurons among ADSCs, BMSCs and MG groups. These results suggest that neither BMSCs nor ADSCs provide satisfactory results for peripheral nerve repair when using MG as the conductor for engraftment.

A novel modified physiologically relevant model for cardiac angiogenesis

Angiogenesis assays are important tools for studying both the mechanisms of cardiac angiogenesis and the potential development of therapeutic strategies to ischemic heart diseases. Currently, various assays have been used to quantitate cardiac tubule formation, yet no consensus has been reached regarding a suitable assay for evaluating the efficacy of angiogenic stimulants or inhibitors. Most in vivo angiogenesis assays are complex and difficult to interpret, whereas traditional in vitro angiogenesis models measure only one aspect of this process. To bridge the gap between in vivo and in vitro angiogenesis assays, here, we have developed a novel modified cardiac explants matrigel assay. We observed the morphology of vascular sprouts formed in three forms of cardiac angiogenesis assays then used quantitative image analyses to further compare the morphological features of vascular sprouts formed in two cardiac explants angiogenesis assays. Vascular sprouts formed in the fibronectin group were less and short, whereas those formed in the matrigel group were significantly longer, consisting of more area and branch points. Moreover, we found the benefits of this matrigel model by observing the ability of cardiac explants to form vascular sprouts under normoxia or hypoxia condition in the presence of angiogenic stimulant and inhibitor, VEGF and PEDF. In summary, the above analyses revealed that the morphology of vascular sprouts formed in this model appears more representative of myocardial capillary formation in vivo, and this accessible, reliable angiogenic assay is a more physiologically relevant assay which allows further assessment of pharmacologic compounds on cardiac angiogenesis.

Protective and Heat Retention Effects of Thermo-sensitive Basement Membrane Extract (Matrigel) in Hepatic Radiofrequency Ablation in an Experimental Animal Study.

To evaluate the protective effect of using thermo-sensitive basement membrane extract (Matrigel) for hydrodissection to minimize thermal injury to nearby structures and to evaluate its heat sink effect on the ablation zone in radiofrequency ablation (RFA) of the liver. First, the viscosity profile and heat sink effect of Matrigel were assessed during RFA in vitro and ex vivo. Fresh pig liver tissue was used, and the temperature changes in Matrigel and in 5% dextrose in water (D5W) during RFA were recorded. Then, the size of the ablation zone in the peripheral liver after RFA was measured. Second, in an in vivo study, 45 Sprague-Dawley rats were divided into three groups of 15 rats each (Matrigel, D5W and control). In the experimental groups, artificial ascites with 10ml of Matrigel or D5W were injected using ultrasound guidance prior to RFA. The frequency of thermal injury to the nearby organs was compared among the three groups, with assessments of several locations: near the diaphragm, the abdominal wall and the gastrointestinal (GI) tract. Finally, the biological degradation of Matrigel by ultrasound was evaluated over 60days. First, Matrigel produced a greater heat retention (less heat sink) effect than D5W during ex vivo ablation (63±9 vs. 26±6°C at 1min on the surface of the liver, P<0.001). Hepatic ablation zone volume did not differ between the two groups. Second, thermal injury to the nearby structures was found in 14 of 15 cases (93.3%) in the control group, 8 of 15 cases (53.3%) in the D5W group, and 1 of 15 cases (6.7%) in the Matrigel group. Significant differences in the thermal injury rates for nearby structures were detected among the three groups (P<0.001). The most significant difference in the thermal injury rate was found in locations near the GI tract (P=0.003). Finally, Matrigel that was injected in vivo was gradually degraded during the following 60days. Using thermo-sensitive Matrigel as a hydrodissection material might help reduce the frequency of collateral thermal injury to nearby structures, especially in locations close to the GI tract, compared to conventional D5W. Additionally, Matrigel did not increase the heat sink effect on the ablation zone during ablation and was degraded over time in vivo.

Evaluation of a Commercially Available Hyaluronic Acid Hydrogel (Restylane) as Injectable Scaffold for Dental Pulp Regeneration: An In Vitro Evaluation

IntroductionRegenerative endodontic procedures (REPs) are viable alternatives for treating immature teeth, yet these procedures do not predictably lead to pulp-dentin regeneration. A true bioengineering approach for dental pulp regeneration requires the incorporation of a scaffold conducive with the regeneration of the pulp-dentin complex. Several materials have been proposed as scaffolds for REPs; nonetheless, the majority are not eligible for immediate clinical chairside use. Thus, the aim of this study was to evaluate Restylane, a Food and Drug Administration-approved hyaluronic acid–based gel, as possible scaffold for REPs. MethodsStem cells of the apical papilla (SCAP) were cultured either alone or in mixtures with either Restylane or Matrigel scaffolds. Groups were cultured in basal culture medium for 6, 24, and 72 hours, and cell viability was assessed. For the mineralizing differentiation experiments, groups were cultured in differentiation medium either for 7 days and processed for alkaline phosphatase activity or for 14 days and processed for gene expression by using quantitative reverse-transcription polymerase chain reaction. SCAP in basal medium served as control. ResultsCell encapsulation in either Restylane or Matrigel demonstrated reduced cell viability compared with control. Nonetheless, cell viability significantly increased in the Restylane group in the course of 3 days, whereas it decreased significantly in the Matrigel group. Restylane promoted significantly greater alkaline phosphatase activity and upregulation of dentin sialophosphoprotein, dentin matrix acidic phosphoprotein-1, and matrix extracellular phosphoglycoprotein, compared with control. ConclusionsA Food and Drug Administration-approved hyaluronic acid–based injectable gel promoted SCAP survival, mineralization, and differentiation into an odontoblastic phenotype and may be a promising scaffold material for REPs.

Effects of extracellular matrix on the growth characteristics of human dermal papillae cells in vitro.

In humans, dermal papillae cells (DPCs) are surrounded by basal membranes. However, conventional in vitro culture conditions for DPCs do not include extracellular matrix (ECM). To investigate the effects of different ECM types on the characteristics of cultured human DPCs. Freshly isolated human DPCs were inoculated onto plates precoated with type IV collagen, fibronectin, Matrigel or hyaluronate. Cell migration, growth and viability were measured. Real-time quantitative (q)PCR and western blotting were used to detect the influence of different ECM types on the expression of DPC-specific biomarkers. In planar culture, type IV collagen and fibronectin had a beneficial effect on the migration, aggregation and viability of DPCs, in contrast to Matrigel and hyaluronate. Real-time qPCR revealed that significantly increased expression of mRNA was seen only for neural cell adhesion molecule (NCAM) and α-smooth muscle actin (α-SMA) in the Matrigel group, whereas versican, NCAM and α-SMA all decreased significantly in the hyaluronate group. In the type IV collagen and fibronectin groups, mRNA expression of the above genes did not change significantly. Western blotting indicated that there were no significant differences in expression of the above proteins between the four groups and the control group. In planar culture, exogenous ECM types (Matrigel, type IV collagen, hyaluronate and fibronectin) changed the growth characteristics of DPCs, but they did not affect the characteristics associated with the biological functions of DPCs.

SEMA3A suspended in matrigel improves titanium implant fixation in ovariectomized rats.

The aim of this study was to evaluate the effect of SEMA3A released from matrigel on implant fixation in ovariectomized (OVX) rats. Sixty female rats were subjected to bilateral ovariectomy. Twelve weeks later, rats were randomly divided into three groups according to implants they accepted: (1) Control, implants with distilled water; (2) Matrigel, implants with matrigel coating; (3) Matrigel + SEMA3A, implants with coating of SEMA3A suspended in matrigel. Implants were inserted in metaphysis of proximal tibiae in all animas bilaterally. In vitro release of SEMA3A was tested using enzyme linked immunosorbent assay. In vitro release of SEMA3A was detectable during the first 10 days, and a burst release of was observed during the first 3 days. No significant difference was observed between Control and Matrigel group. The protective effects of SEMA3A in matrigel on peri-implant bone, implant osseointegration and fixation was confirmed. Compared to matrigel alone, SEMA3A suspended in matrigel increased percent bone volume by 88.7% and 83.3% (p < 0.01), bone-to-implant contact ratio by 148.9% (p < 0.01), and 24.8% (p < 0.05), the maximal push-out force by 149.3% and 209.2% (p < 0.01) at 4 and 8 weeks after implant insertion, respectively. Surface modification with SEMA3A suspended in matrigel improved implant osseointegration and fixation in the proximal tibiae of OVX rats. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2060-2065, 2017.

Effect of different concentration of ulinastatin on the proliferation, apoptosis, migration, adhesion of the human umbilical vein endothelial cells

Objective This study aims to research the effect of different concentration of ulinastatin on the proliferation, apoptosis, migration, adhesion of the human umbilical vein endothelial cells. Methods First, cultivate the primary human umbilical vein endothelial cell (HUVEC) until the fourth generation. Divide the cells into the control group (U) and concentration respectively are 625, 2 500, 5 000, 10 000 U/ml (group A, B, C, D). And then the CellTrace experiment, TdT-mediated dUTP nick end labeling (TUNEL) experiment, scratch assay, cell adhesion assay were respectively applied to detecte the proliferation, apoptosis, migration, adhesion of HUVEC. Results Wound healing assay: The 6 hours' migration rates of group U, A, B, C, D are 4.08%, 2.61%, 5.24%, 3.99%, 2.27%, the 24 hours' migration rates of group U, A, B, C, D are 12.53%, 13.04%, 20.65%, 21.37%, 30.10%, the 48 hours' migration rates of group U, A, B, C, D are 20.18%, 25.00%, 37.92%, 49.22%, 49.01%. Cell proliferation assay: The rates of prolification of group U, A, B, C, D are 60.1%, 70.3%, 77.2%, 82.1%, 88.5%. Cell apoptosis assay: The rates of apoptosis of group U, A, B, C, D are 3.1%, 2.5%, 2.1%, 2.3%, 35.3%. Cell adhesion assay: The percentages of cell attachment of group U, A, B, C, D on BSA group are 38.0%, 39.0%, 41.0%, 43.0%, 43.0%, the percentages of cell attachment of group U, A, B, C, D on matrigel group are 50.0%, 51.0%, 54.0%, 61.0%, 66.0%, the percentages of cell attachment of group U, A, B, C, D on FN group are 60.0%, 61.0%, 64.0%, 73.0%, 81.0%. Conclusion In this study, ulinastatin can accelerate the proliferation, migration and adhesion of the HUVEC cultured in vitro; but high concentraction of ulinastatin can lead to cell apoptosis. Key words: Ulinastatin; Human umbilical vein endothelial cells; Proliferation; Apoptosis; Migration; Adhesion

Effect of thermo-sensitive Matrigel on minimization of thermal injury to the nearby structures in radiofrequency ablation of subcapsular hepatic tumors in a rat model

To evaluate the effect and safety of using thermo-sensitive Matrigel as artificial ascites on minimizing thermal injury to the nearby structures in a rat model of radiofrequency (RF) ablation of the liver. In this research, the morphological characterization of Matrigel was observed at different temperatures in vitro. In vivo study was conducted by using 42 SD rats in Matrigel, Saline and control groups of 14 rats each. Artificial ascites with 10 ml of Matrigel or saline was produced under ultrasound guidance before RF in the experimental groups. Using a 0.7 cm exposed 17 G RF electrode for 5 minutes (90 ± 2) ℃, 41 hepatic ablations was performed abutting the diaphragm, abdominal wall and stomach. The 41 rats were sacrificed at 24 h, and necropsy was performed.Gross and histopathologic examinations were performed to compare the frequency and extent of thermal injury to the nearby organs.The animal status was followed up to achieve long term safety evaluation of Matrigel. Matrigel was in liquid formation at 0-4 ℃, but was not moving in vials at temperature 37 ℃ in 1 min, presenting gel formation.In vivo experiment, the artificial fluid thickness of Matrigel in site was significant larger than that of saline group, especially at 5 min of ablation ((8.1 ± 1.1) mm vs (2.4 ± 0.8)mm, P<0.01). Thermal injury to the nearby structures was found in 12 of 13 cases in control group, 8 of 14 cases (P=0.037) in saline group, and 1 of 14 cases (P<0.01) in Matrigel group. The sizes of the ablation zone of the liver did not differ among the three groups. There was no difference in the incidence of operation related complications among the three groups. There was no significant difference in body weight and health related parameters between Matrigel and saline group during 60 days of follow up. Using thermo-sensitive Matrigel as artificial ascites may be a safe and useful technique and help to further reduce the frequency and severity of collateral thermal injury to the nearby structures compare to conventional saline, during RF ablation of subcapsular hepatic tumors.

Extracellular Matrix and Growth Factors Improve the Efficacy of Intramuscular Islet Transplantation.

BackgroundThe efficacy of intramuscular islet transplantation is poor despite being technically simple, safe, and associated with reduced rates of severe complications. We evaluated the efficacy of combined treatment with extracellular matrix (ECM) and growth factors in intramuscular islet transplantation.MethodsMale BALB/C mice were used for the in vitro and transplantation studies. The following three groups were evaluated: islets without treatment (islets-only group), islets embedded in ECM with growth factors (Matrigel group), and islets embedded in ECM without growth factors [growth factor-reduced (GFR) Matrigel group]. The viability and insulin-releasing function of islets cultured for 96 h were significantly improved in Matrigel and GFR Matrigel groups compared with the islets-only group.ResultsBlood glucose and serum insulin levels immediately following transplantation were significantly improved in the Matrigel and GFR Matrigel groups and remained significantly improved in the Matrigel group at postoperative day (POD) 28. On histological examination, significantly decreased numbers of TdT-mediated deoxyuridine triphosphate-biotin nick end labeling-positive islet cells and significantly increased numbers of Ki67-positive cells were observed in the Matrigel and GFR Matrigel groups at POD 3. Peri-islet revascularization was most prominent in the Matrigel group at POD 14.ConclusionsThe efficacy of intramuscular islet transplantation was improved by combination treatment with ECM and growth factors through the inhibition of apoptosis, increased proliferation of islet cells, and promotion of revascularization.