Genipin‐crosslinked gelatin–silk fibroin hydrogels for modulating the behaviour of pluripotent cells

Different hydrogel materials have been prepared to investigate the effects of culture substrate on the behaviour of pluripotent cells. In particular, genipin‐crosslinked gelatin–silk fibroin hydrogels of different compositions have been prepared, physically characterized and used as substrates for the culture of pluripotent cells. Pluripotent cells cultured on hydrogels remained viable and proliferated. Gelatin and silk fibroin promoted the proliferation of cells in the short and long term, respectively. Moreover, cells cultured on genipin‐crosslinked gelatin–silk fibroin blended hydrogels were induced to an epithelial ectodermal differentiation fate, instead of the neural ectodermal fate obtained by culturing on tissue culture plates. This work confirms that specific culture substrates can be used to modulate the behaviour of pluripotent cells and that our genipin‐crosslinked gelatin–silk fibroin blended hydrogels can induce pluripotent cells differentiation to an epithelial ectodermal fate. Copyright © 2014 John Wiley & Sons, Ltd.     

The fabrication and characterization of a multi‐laminate,angle‐ply collagen patch for annulus fibrosus repair

One major limitation of intervertebral disc (IVD) repair is that no ideal biomaterial has been developed that effectively mimics the angle‐ply collagen architecture and mechanical properties of the native annulus fibrosus (AF). Furthermore, it would be beneficial to devise a simple, scalable process by which to manufacture a biomimetic biomaterial that could function as a mechanical repair patch to be secured over a large defect in the outer AF that will support AF tissue regeneration. Such a biomaterial would: (1) enable the employment of early‐stage interventional strategies to treat IVD degeneration (i.e. nucleus pulposus arthroplasty); (2) prevent IVD re‐herniation in patients with large AF defects; and (3) serve as a platform to develop full‐thickness AF and whole IVD tissue engineering strategies. Due to the innate collagen fibre alignment and mechanical strength of pericardium, a procedure was developed to assemble multi‐laminate angle‐ply AF patches derived from decellularized pericardial tissue. Patches were subsequently assessed histologically to confirm angle‐ply microarchitecture, and mechanically assessed for biaxial burst strength and tensile properties. Additionally, patch cytocompatibility was evaluated following seeding with bovine AF cells. This study demonstrated the effective removal of porcine cell remnants from the pericardium, and the ability to reliably produce multi‐laminate patches with angle‐ply architecture using a simple assembly technique. Resultant patches demonstrated their inherent ability to resist biaxial burst pressures reminiscent of intradiscal pressures commonly borne by the AF, and exhibited tensile strength and modulus values reported for native human AF. Furthermore, the biomaterial supported AF cell viability, infiltration and proliferation. Copyright © 2016 John Wiley & Sons, Ltd.     

SIRT1‐dependent anti‐senescence effects of cell‐deposited matrix on human umbilical cord mesenchymal stem cells

Human umbilical cord‐derived mesenchymal stem cells (UC‐MSCs) are considered an attractive cell source for tissue regeneration. However, environmental oxidative stress can trigger premature senescence in MSCs and thus compromises their regenerative potential. Extracellular matrix (ECM) derived from MSCs has been shown to facilitate cell proliferation and multi‐lineage differentiation. This investigation evaluated the effect of cell‐deposited decellularized ECM (DECM) on oxidative stress‐induced premature senescence in UC‐MSCs. Sublethal dosages of H₂O₂, ranging from 50 μm to 200 μm , were used to induce senescence in MSCs. We found that DECM protected UC‐MSCs from oxidative stress‐induced premature senescence. When treated with H₂O₂ at the same concentration, cell proliferation of DECM‐cultured UC‐MSCs was twofold higher than those on standard tissue culture polystyrene (TCPS). After exposure to 100 μm H₂O₂, fewer senescence‐associated β‐galactosidase‐positive cells were observed on DECM than those on TCPS (17.6  ±  4.0% vs. 60.4  ±  6.2%). UC‐MSCs cultured on DECM also showed significantly lower levels of senescence‐related regulators, such as p16^INK4α and p21. Most importantly, DECM preserved the osteogenic differentiation potential of UC‐MSCs with premature senescence. The underlying molecular mechanisms involved the silent information regulator type 1 (SIRT1)‐dependent signalling pathway, confirmed by the fact that the SIRT1 inhibitor nicotinamide counteracted the DECM‐mediated anti‐senescent effect. Collagen type I, rather than fibronectin, partially contributed to the protective effect of decellularized matrix. These findings provide a new strategy of using stem cell‐deposited matrix to overcome the challenge of cellular senescence and to facilitate the clinical application of MSCs in regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.     

Adipose tissue‐derived extracellular matrix hydrogels as a release platform for secreted paracrine factors

Fat grafting is an established clinical intervention to promote tissue repair. The role of the fat's extracellular matrix (ECM) in regeneration is largely neglected. We investigated in vitro the use of human adipose tissue‐derived ECM hydrogels as release platform for factors secreted by adipose‐derived stromal cells (ASCs). Lipoaspirates from nondiabetic and diabetic donors were decellularized. Finely powdered acellular ECM was evaluated for cell remainders and DNA content. Acellular ECM was digested, and hydrogels were formed at 37°C and their viscoelastic relaxation properties investigated. Release of ASC‐released factors from hydrogels was immune assessed, and bio‐activity was determined by fibroblast proliferation and migration and endothelial angiogenesis. Acellular ECM contained no detectable cell remainders and negligible DNA contents. Viscoelastic relaxation measurements yielded no data for diabetic‐derived hydrogels due to gel instability. Hydrogels released several ASC‐released factors concurrently in a sustained fashion. Functionally, released factors stimulated fibroblast proliferation and migration as well as angiogenesis. No difference between nondiabetic and diabetic hydrogels in release of factors was measured. Adipose ECM hydrogels incubated with released factors by ASC are a promising new therapeutic modality to promote several important wound healing‐related processes by releasing factors in a controlled way.     

Mobilization effects of G‐CSF,GM‐CSF,and darbepoetin‐α for allogeneic peripheral blood stem cell transplantation

The effects of GM‐/G‐CSF and darbepoetin‐α on stem cell mobilization were investigated. From February 2005 to March 2007, 30 allogeneic sibling donors were randomly assigned to a G‐CSF group (5 μg/kg/day for 5–7 days) or triple group (GM‐CSF 10 μg/kg/day on 1st and 2nd day, G‐CSF 5 μg/kg/day for 5–7 days, and darbepoetin‐α 40 mg on 1st day). The MNCs and CD34⁺ cells were not different between the two groups, although the doses (×10⁸/kg of recipient body weight) of CD3⁺ cells (3.64 ± 1.75 vs. 2.63 ± 1.36, P = 0.089) and CD8⁺ cells (1.07 ± 0.53 vs. 0.60 ± 0.30, P = 0.006) were lower in the triple group. The engraftments, frequency of RBC transfusions, and hemoglobin recovery were not different between the two groups. The cumulative incidence of overall and Grades II–IV aGVHD was 64.3% vs. 61.1% and 25.9% vs. 27.1% in the G‐CSF and triple regimen group, respectively, whereas the cumulative incidence of cGVHD was 20.8 ± 1.3% and 24.4 ± 1.7%, respectively. In conclusion, the triple regimen did not seem to be superior to G‐CSF alone in terms of the CD34+ cell dose, hemoglobin recovery, and GVHD. However, the CD8+ cell count was significantly lower in the triple regimen group. The role of a lower CD8+ cell count in the graft may need to be elucidated in the future. J. Clin. Apheresis, 2009. © 2009 Wiley‐Liss, Inc.     

Circulating nucleated peripheral blood cells contribute to early‐phase meniscal healing

The purpose of this study was to assess how peripheral blood cells (PBCs) contribute to meniscus repair, using a parabiotic rat model. Wild‐type (WT) and green fluorescent protein (GFP) transgenic rats were conjoined at the torso. After 4 weeks, the anterior part of the medial meniscus of both groups of rats was removed. At 1, 2, 4, 8 and 12 weeks post‐meniscectomy, repaired tissue was evaluated using stereomicroscopy, histology with toluidine blue staining, and immunofluorescence microscopy. Stereomicroscopic observations and confocal laser microscopy revealed that a high number of GFP‐positive cells were present in the repaired meniscus of WT rats 1 week post‐meniscectomy, and the number of GFP‐positive cells decreased over time. Based on blood chimerism, the ratios of PBCs in the repaired meniscus were 20.5 ± 2.3% at 1 week, 8.3 ± 0.9% at 2 weeks, 4.4 ± 0.9% at 4 weeks, 2.1 ± 0.9% at 8 weeks, and 0.5 ± 0.4% at 12 weeks, post‐meniscectomy. Histologically, fibrochondrocytes were observed in the repaired meniscus of WT rats after 4 weeks, some of which were GFP‐positive. The chondrogenic marker, type II collagen, was merged within the PBCs in the repaired tissue. However, type‐II‐collagen‐positive cell ratio and metachromasia in the repaired meniscus were not equivalent in normal meniscal tissue. This indicated that PBCs were present within the repaired meniscus at an early phase, replacing the excised meniscal cells, suggesting PBCs contributed to meniscal healing. The tissue repair contribution by these cells decreased at later phases. Copyright © 2014 John Wiley & Sons, Ltd.     

Scaffold‐free 3D cellulose acetate membrane‐based cultures form large cartilaginous constructs

Scaffold‐free three‐dimensional (3D) cultures provide clinical potential in cartilage regeneration. The purpose of this study was to characterize a scaffold‐free 3D membrane‐based culture system, in which human articular chondrocytes were cultivated on a cellulose acetate membrane filter, and compare it to pellet and monolayer cultures. Chondrocytes were expanded in monolayer culture for up to 5 passages, transferred to membrane‐based or pellet cultures and harvested after 7 or 21 days. The chondrogenic potential was assessed by histology (toluidine blue, safranin‐O), immunohistochemistry for collagen type II and quantitative analysis of collagen type II α₁ (COL2A1). Membrane‐based cultures (P1) formed flexible disc‐like constructs (diameter 4000 µm, thickness 150 µm) with a large smooth surface after 7 days. Positive safranin‐O and collagen type II staining was found in membrane‐based and pellet cultures at P1–3. Expression of COL2A1 after 7 days was increased in both culture systems compared to monolayer culture up to P3, whereas cells from monolayer > P3 did not redifferentiate. The best results for COL2A1 expression were obtained from membrane‐based cultures at P1. After 21 days the membrane‐based cultures did not express COL2A1. We concluded that membrane‐based and pellet cultures showed the ability to promote redifferentiation of chondrocytes expanded in monolayer culture. The number of cell passages had an impact on the chondrogenic potential of cells. Membrane‐based cultures provided the highest COL2A1 expression and a large, smooth and cartilage‐like surface. As these are appropriate features for clinical applications, we assume that membrane‐based cultures might be of use in cartilage regeneration if they displayed similar results in vivo. Copyright © 2010 John Wiley & Sons, Ltd.     

Efficacy of thermoresponsive,photocrosslinkable hydrogels derived from decellularized tendon and cartilage extracellular matrix for cartilage tissue engineering

Tissue engineering using adult mesenchymal stem cells (MSCs), a promising approach for cartilage repair, is highly dependent on the nature of the matrix scaffold. Thermoresponsive, photocrosslinkable hydrogels were fabricated by functionalizing pepsin‐soluble decellularized tendon and cartilage extracellular matrices (ECM) with methacrylate groups. Methacrylated gelatin hydrogels served as controls. When seeded with human bone marrow MSCs and cultured in chondrogenic medium, methacrylated ECM hydrogels experienced less cell‐mediated contraction, as compared against non‐methacrylated ECM hydrogels. However, methacrylation slowed or diminished chondrogenic differentiation of seeded MSCs, as determined through analyses of gene expression, biochemical composition and histology. In particular, methacrylated cartilage hydrogels supported minimal due to chondrogenesis over 42 weeks, as hydrogel disintegration beginning at day 14 presumably compromised cell–matrix interactions. As compared against methacrylated gelatin hydrogels, MSCs cultured in non‐methacrylated ECM hydrogels exhibited comparable expression of chondrogenic genes (Sox9, Aggrecan and collagen type II) but increased collagen type I expression. Non‐methacrylated cartilage hydrogels did not promote chondrogenesis to a greater extent than either non‐methacrylated or methacrylated tendon hydrogels. Whereas methacrylated gelatin hydrogels supported relatively homogeneous increases in proteoglycan and collagen type II deposition throughout the construct over 42 days, ECM hydrogels possessed greater heterogeneity of staining intensity and construct morphology. These results do not support the utility of pepsin‐solubilized cartilage and tendon hydrogels for cartilage tissue engineering over methacrylated gelatin hydrogels. Methacrylation of tendon and cartilage ECM hydrogels permits thermal‐ and light‐induced polymerization but compromises chondrogenic differentiation of seeded MSCs.     

RGD‐mimic polyamidoamine–montmorillonite composites with tunable stiffness as scaffolds for bone tissue‐engineering applications

This paper reports on the development of montmorillonite (MMT)‐reinforced hydrogels, based on a peptidomimetic polyamidoamine carrying guanidine pendants (AGMA1), as substrates for the osteo‐induction of osteoblast precursor cells. AGMA1 hydrogels of various degrees of crosslinking responded favourably to MMT reinforcement, giving rise to composite hydrogels with shear storage modulus G′, when fully swollen in water, up to 200 kPa, i.e. 20 times higher than the virgin hydrogels and of the same order or higher than other hydrogel‐based composites proposed for orthopaedic applications. This significant improvement was ascribed to the effective interpenetration between the polymer matrix and the inorganic filler. AGMA1–MMT hydrogels, when evaluated as scaffolds for the osteogenic differentiation of mouse calvaria‐derived pre‐osteoblastic MC3T3‐E1 cells, proved able to support cell adhesion and proliferation and clearly induced differentiation towards the osteoblastic phenotype, as indicated by different markers. In addition, AGMA1–MMT hydrogels proved completely degradable in aqueous media at pH 7.4 and did not provide any evidence of cytotoxicity. The experimental evidence suggests that AGMA1–MMT composites definitely warrant potential as scaffolds for osteoblast culture and bone grafts. Copyright © 2016 John Wiley & Sons, Ltd.     

In vitro evaluation of Ficoll‐enriched and genipin‐stabilised collagen scaffolds

Polysaccharides are frequently incorporated into scaffolds for tissue engineering applications to improve mechanical and biological properties. We evaluated the influence of a Ficoll® scaffold on collagen films, a scaffold that is extensively used for soft and hard tissue repair. To avoid cytotoxicity issues associated with chemical reagents, the influence of genipin, a naturally occurring crosslinking agent, was assessed. Ultra‐structural level collagen films formed with and without Ficoll showed a fine fibrillar structure whereas genipin crosslinked films showed a coarse fibrillar and partially nodular structure. In contrast, glutaraldehyde crosslinked films lost their fibrillar pattern. Crosslinking significantly increased denaturation temperature (p < 0.001), stress (p < 0.0001) and force (p < 0.0001) at break. Collagen/Ficoll and collagen/Ficoll/genipin films showed the highest WI38 fibroblast attachment than any other scaffold (p < 0.003) and significantly greater WI38 fibroblast metabolic activity than other scaffolds (p < 0.001). By day 6. collagen/Ficoll/genipin films also induced higher and more aligned fibronectin matrix deposition than other scaffolds. Overall, this study indicates the suitability of collagen/Ficoll/genipin for tissue engineering applications. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of endothelium‐denuded human umbilical veins as living scaffolds for tissue‐engineered small‐calibre vascular grafts

Tissue‐engineered small‐calibre vessel grafts may help to alleviate the lack of graft material for coronary and peripheral bypass grafting in an increasing number of patients. This study explored the use of endothelium‐denuded human umbilical veins (HUVs) as scaffolds for vascular tissue engineering in a perfusion bioreactor. Vessel diameter (1.2 ± 0.4 mm), wall thickness (0.38 ± 0.09 mm), uniaxial ultimate failure stress (8029 ± 1714 kPa) and burst pressure (48.4 ± 20.2 kPa, range 28.4–83.9 kPa) were determined in native samples. The effects of endothelium removal from HUVs by enzymatic digestion, hypotonic lysis and dehydration were assessed. Dehydration did not significantly affect contractile function, tetrazolium dye reduction, mechanical strength and vessel structure, whereas the other methods failed in at least one of these parameters. Denudation by dehydration retained laminin, fibronectin, collagen and elastic fibres. Denuded HUVs were seeded in a perfusion bioreactor with either allogeneic HUVs endothelial cells or with saphenous vein endothelial cells harvested from patients with coronary artery disease. Seeding in a perfusion bioreactor resulted in a confluent monolayer of endothelial cells from both sources, as judged by histology and scanning electron microscopy. Seeded cells contained von Willebrand factor and CD31. In conclusion, denuded HUVs should be considered an alternative to decellularized blood vessels, as the process keeps the smooth muscle layer intact and functional, retains proteins relevant for biomechanic properties and for cell attachment and provides a suitable scaffold for seeding an autologous and flow‐resistant endothelium. Copyright © 2012 John Wiley & Sons, Ltd.     

A straightforward method to produce decellularized dermis‐based matrices for tumour cell cultures

Decellularized matrices are steadily gaining popularity to study the biology of cells and tissues, as they represent a biomimetic environment in which cells can recapitulate certain behaviours that share similarities with those observed in vivo. Basically, biochemistry, microstructure and mechanics of the decellularized matrices are the most valuable properties that differentiate these culturing systems from conventional bidimensional models. Several procedures to decellularize tissues have been proposed so far, with the common aim to preserve the tissue chemical/physical properties of the original tissue. However, these processes are complex, time‐consuming and expensive. In this work, we propose a cost‐effective, easy‐to‐produce decellularized dermal matrix, derived from animal skin. The chemical/physical processes to obtain the matrices proved to not alter matrix structure and did not induce cytotoxicity issues. To test the validity of the decellularized matrices as a model to study the behaviour of tumour cells in vitro, we performed microstructural and mechanical investigations as well as cell proliferation assays. In particular, three different tumour cell lines were used, which proliferated and invaded the matrix with no additional treatments. Decellularized skin scaffold, presented in this work, could be a strong competitor for conventional 3D systems like synthetic porous scaffolds or hydrogels. Copyright © 2016 John Wiley & Sons, Ltd.     

Re‐endothelialization of rat lung scaffolds through passive,gravity‐driven seeding of segment‐specific pulmonary endothelial cells

Effective re‐endothelialization is critical for the use of decellularized scaffolds for ex vivo lung engineering. Current approaches yield insufficiently re‐endothelialized scaffolds that haemorrhage and become thrombogenic upon implantation. Herein, gravity‐driven seeding coupled with bioreactor culture facilitated widespread distribution and engraftment of endothelial cells throughout rat lung scaffolds. Initially, human umbilical vein endothelial cells were seeded into the pulmonary artery by either gravity‐driven, variable flow perfusion seeding or pump‐driven, pulsatile flow perfusion seeding. Gravity seeding evenly distributed cells and supported cell survival and re‐lining of the vascular walls while perfusion pump‐driven seeding led to increased cell fragmentation and death. Using gravity seeding, rat pulmonary artery endothelial cells and rat pulmonary vein endothelial cells attached in intermediate and large vessels, while rat pulmonary microvascular endothelial cells deposited mostly in microvessels. Combination seeding of these cells led to positive vascular endothelial cadherin staining. In addition, combination seeding improved barrier function as assessed by serum albumin extravasation; however, leakage was observed in the distal portions of the re‐endothelialized tissue suggesting that recellularization of the alveoli is necessary to complete barrier function of the capillary–alveolar network. Overall, these data indicate that vascular recellularization of rat lung scaffolds is achieved through gravity seeding. Copyright © 2016 John Wiley & Sons, Ltd.     

Chondrogenic potential of injectable κ‐carrageenan hydrogel with encapsulated adipose stem cells for cartilage tissue‐engineering applications

Due to the limited self‐repair capacity of cartilage, regenerative medicine therapies for the treatment of cartilage defects must use a significant amount of cells, preferably applied using a hydrogel system that can promise their delivery and functionality at the specific site. This paper discusses the potential use of κ‐carrageenan hydrogels for the delivery of stem cells obtained from adipose tissue in the treatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropic gelation method and human adipose stem cells (hASCs) were encapsulated in 1.5% w/v κ‐carrageenan solution at a cell density of 5 × 10⁶ cells/ml. The results from the analysis of the cell‐encapsulating hydrogels, cultured for up to 21 days, indicated that κ‐carrageenan hydrogels support the viability, proliferation and chondrogenic differentiation of hASCs. Additionally, the mechanical analysis demonstrated an increase in stiffness and viscoelastic properties of κ‐carrageenan gels with their encapsulated cells with increasing time in culture with chondrogenic medium. These results allowed the conclusion that κ‐carrageenan exhibits properties that enable the in vitro functionality of encapsulated hASCs and thus may provide the basis for new successful approaches for the treatment of cartilage defects. Copyright © 2013 John Wiley & Sons, Ltd.     

A papain‐induced disc degeneration model for the assessment of thermo‐reversible hydrogel–cells therapeutic approach

Nucleus pulposus (NP) regeneration by the application of injectable cell‐embedded hydrogels is an appealing approach for tissue engineering. We investigated a thermo‐reversible hydrogel (TR‐HG), based on a modified polysaccharide with a thermo‐reversible polyamide [poly(N‐isopropylacrylamide), pNIPAM], which is made to behave as a liquid at room temperature and hardens at > 32 °C. In order to test the hydrogel, a papain‐induced bovine caudal disc degeneration model (PDDM), creating a cavity in the NP, was employed. Human mesenchymal stem cells (hMSCs) or autologous bovine NP cells (bNPCs) were seeded in TR‐HG; hMSCs were additionally preconditioned with rhGDF‐5 for 7 days. Then, TR‐HG was reversed to a fluid and the cell suspension injected into the PDDM and kept under static loading for 7 days. Experimental design was: (D1) fresh disc control + PBS injection; (D2) PDDM + PBS injection; (D3) PDDM + TR‐HG (material control); (D4) PDDM + TR‐HG + bNPCs; (D5) PDDM + TR‐HG + hMSCs. Magnetic resonance imaging performed before and after loading, on days 9 and 16, allowed imaging of the hydrogel‐filled PDDM and assessment of disc height and volume changes. In gel‐injected discs the NP region showed a major drop in volume and disc height during culture under static load. The RT–PCR results of injected hMSCs showed significant upregulation of ACAN, COL2A1, VCAN and SOX9 during culture in the disc cavity, whereas the gene expression profile of NP cells remained unchanged. The cell viability of injected cells (NPCs or hMSCs) was maintained at over 86% in 3D culture and dropped to ~72% after organ culture. Our results underline the need for load‐bearing hydrogels that are also cyto‐compatible. Copyright © 2013 John Wiley & Sons, Ltd.     

Osteogenesis of adipose‐derived stem cells on polycaprolactone–β‐tricalcium phosphate scaffold fabricated via selective laser sintering and surface coating with collagen type I

The current study aimed to fabricate three‐dimensional (3D) polycaprolactone (PCL), polycaprolactone and β‐tricalcium phosphate (PCL–TCP) scaffolds via a selective laser‐sintering technique (SLS). Collagen type I was further coated onto PCL–TCP scaffolds to form PCL–TCP–COL scaffolds. The physical characters of these three scaffolds were analysed. The osteogenic potential of porcine adipose‐derived stem cells (pASCs) was compared among these three scaffolds in order to find an optimal scaffold for bone tissue engineering. The experimental results showed no significant differences in pore size and porosity among the three scaffolds; the porosity was ca. 75–77% and the pore size was ca. 300–500 µm in all three. The compressive modulus was increased from 6.77 ± 0.19 to 13.66 ± 0.19 MPa by adding 30% β‐TCP into a 70% PCL scaffold. No significant increase of mechanical strength was found by surface‐coating with collagen type I. Hydrophilicity and swelling ratios showed statistical elevation (p < 0.05) after collagen type I was coated onto the PCL–TCP scaffolds. The in vitro study demonstrated that pASCs had the best osteogenic differentiation on PCL–TCP–COL group scaffolds, due to the highest ALP activity, osteocalcin mRNA expression and mineralization. A nude mice experiment showed better woven bone and vascular tissue formation in the PCL–TCP–COL group than in the PCL group. In conclusion, the study demonstrated the ability to fabricate 3D, porous PCL–TCP composite scaffolds (PCL:TCP = 70:30 by weight) via an in‐house‐built SLS technique. In addition, the osteogenic ability of pASCs was found to be enhanced by coating COL onto the PCL–TCP scaffolds, both in vitro and in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Post‐ischaemic angiogenic therapy using in vivo prevascularized ascorbic acid‐enriched myocardial artificial grafts improves heart function in a rat model

Angiogenesis plays a key role in post‐ischaemic myocardial repair. We hypothesized that epicardial implantation of an ascorbic acid (AA)‐enriched myocardial artificial graft (MAG), which has been prevascularized in the recipients' own body, promotes restoration of the ischaemic heart. Gelatin patches were seeded with GFP–luciferase‐expressing rat cardiomyoblasts and enriched with 5 μm AA. Grafts were prevascularized in vivo for 3 days, using a renal pouch model in rats. The MAG patch was then implanted into the same rat's ischaemic heart following myocardial infarction (MI). MAG‐treated animals (MAG group, n = 6) were compared to untreated infarcted animals as injury controls (MI group, n = 6) and sham‐operated rats as healthy controls (healthy group, n = 7). In vivo bioluminescence imaging indicated a decrease in donor cell survival by 83% during the first week post‐implantation. Echocardiographic and haemodynamic assessment 4 weeks after MI revealed that MAG treatment attenuated left ventricular (LV) remodelling (LV end‐systolic volume, 0.31 ± 0.13 vs 0.81 ± 0.01 ml, p < 0.05; LV end‐diastolic volume 0.79 ± 0.33 vs 1.83 ± 0.26 ml, p < 0.076) and preserved LV wall thickness (0.21 ± 0.03 vs 0.09 ± 0.005 cm, p < 0.05) compared to the MI group. Cardiac output was higher in MAG than MI (51.59 ± 6.5 vs 25.06 ± 4.24 ml/min, p < 0.01) and comparable to healthy rats (47.08 ± 1.9 ml/min). Histology showed decreased fibrosis, and a seven‐fold increase in blood vessel density in the scar area of MAG compared to MI group (15.3 ± 1.1 vs 2.1 ± 0.3 blood vessels/hpf, p < 0.0001). Implantation of AA‐enriched prevascularized grafts enhanced vascularity in ischaemic rat hearts, attenuated LV remodelling and preserved LV function. Copyright © 2011 John Wiley & Sons, Ltd.     

Development of decellularized scaffolds for stem cell‐driven tissue engineering

Organ transplantation is an effective treatment for chronic organ dysfunctioning conditions. However, a dearth of available donor organs for transplantation leads to the death of numerous patients waiting for a suitable organ donor. The potential of decellularized scaffolds, derived from native tissues or organs in the form of scaffolds has been evolved as a promising approach in tissue‐regenerative medicine for translating functional organ replacements. In recent years, donor organs, such as heart, liver, lung and kidneys, have been reported to provide acellular extracellular matrix (ECM)‐based scaffolds through the process called ‘decellularization’ and proved to show the potential of recellularization with selected cell populations, particularly with stem cells. In fact, decellularized stem cell matrix (DSCM) has also emerged as a potent biological scaffold for controlling stem cell fate and function during tissue organization. Despite the proven potential of decellularized scaffolds in tissue engineering, the molecular mechanism responsible for stem cell interactions with decellularized scaffolds is still unclear. Stem cells interact with, and respond to, various signals/cues emanating from their ECM. The ability to harness the regenerative potential of stem cells via decellularized ECM‐based scaffolds has promising implications for tissue‐regenerative medicine. Keeping these points in view, this article reviews the current status of decellularized scaffolds for stem cells, with particular focus on: (a) concept and various methods of decellularization; (b) interaction of stem cells with decellularized scaffolds; (c) current recellularization strategies, with associated challenges; and (iv) applications of the decellularized scaffolds in stem cell‐driven tissue engineering and regenerative medicine. Copyright © 2015 John Wiley & Sons, Ltd.     

快速心室起搏犬心房颤动模型研究

目的建立快速起搏心室致心力衰竭犬房颤模型,研究其电生理及心房结构和功能改变。方法 15只健康杂种犬分两组:对照组6只,实验组9只[240次/min心室起搏(25±3)d]。超声心动图测定起搏前后心房面积、面积缩小分数及左心室功能,利用心内电极测定心房有效不应期、传导速度及房颤诱发情况。结果实验组7只犬完成了实验。快速心室起搏(25±3)d后,犬的收缩末期和舒张末期左、右心房面积显著增大(与起搏前比较,P<0.01),左、右心房面积缩小分数显著减小(左心房:(35.7±1.9)%和(20.7±2.7)%,P<0.01;右心房:(35.0±2.3)%和(18.0±2.3)%,P<0.01),左室射血分数从(65.3±2.1)%降至(31.6±2.8)%(P<0.01)。实验组犬左、右心房有效不应期显著延长,心房内传导速率较对照组减慢。实验组有5只犬诱发出超过30 min的房颤,平均房颤持续时间较对照组显著延长(687±290)s和(13±9)s,P<0.01)。实验组平均房颤持续时间与左、右心房面积及面积缩小分数相关(P<0.05)。结论 快速心室起搏致心衰模型能稳定地诱发出房颤,房颤持续时间与心衰引起的显著心房结构和功能异常相关。