Polysaccharide-based Scaffolds Research Articles

Delivery of antibacterial agents for wound healing applications using polysaccharide-based scaffolds

Delivery of antibacterial agents for wound healing applications using polysaccharide-based scaffolds

Polysaccharide-based biomaterials in a journey from 3D to 4D printing.

3D printing is a state-of-the-art technology for the fabrication of biomaterials with myriad applications in translational medicine. After stimuli-responsive properties were introduced to 3D printing (known as 4D printing), intelligent biomaterials with shape configuration time-dependent character have been developed. Polysaccharides are biodegradable polymers sensitive to several physical, chemical, and biological stimuli, suited for 3D and 4D printing. On the other hand, engineering of mechanical strength and printability of polysaccharide-based scaffolds along with their aneural, avascular, and poor metabolic characteristics need to be optimized varying printing parameters. Multiple disciplines such as biomedicine, chemistry, materials, and computer sciences should be integrated to achieve multipurpose printable biomaterials. In this work, 3D and 4D printing technologies are briefly compared, summarizing the literature on biomaterials engineering though printing techniques, and highlighting different challenges associated with 3D/4D printing, as well as the role of polysaccharides in the technological shift from 3D to 4D printing for translational medicine.

Nano-fibrillated cellulose-based scaffolds for enzyme (co)-immobilization: Application to natural product glycosylation by Leloir glycosyltransferases

Polysaccharide-based scaffolds are promising carriers for enzyme immobilization. Here, we demonstrate a porous scaffold prepared by direct-ink-writing 3D printing of an ink consisting of nanofibrillated cellulose, carboxymethyl cellulose and citric acid for immobilization application. Negative surface charge introduced by the components made the scaffold amenable for an affinity-like immobilization via the cationic protein module Zbasic2. Zbasic2 fusions of two sugar nucleotide-dependent glycosyltransferases (C-glycosyltransferase, Z-CGT; sucrose synthase, Z-SuSy) were immobilized individually, or co-immobilized, and applied to synthesize the natural C-glycoside nothofagin. The cascade reaction involved β-C-glycosylation of phloretin (10 mM, ~90 % conversion) from UDP-glucose, provided from sucrose and catalytic amounts of UDP (1.0 mM). Enzymes were co-immobilized at ~65 mg protein/g carrier to receive activities of 9.5 U/g (Z-CGT) and 4.5 U/g (Z-SuSy) in 22–33 % yield (protein) and an effectiveness of 23 % (Z-CGT) and 13 % (Z-SuSy). The scaffold-bound enzymes were recyclable for 5 consecutive reactions.

Biocompatibility and antioxidant activity of a novel carrageenan based injectable hydrogel scaffold incorporated with Cissus quadrangularis: an in vitro study

BackgroundOver the past years, polysaccharide-based scaffolds have emerged as the most promising material for tissue engineering. In the present study, carrageenan, an injectable scaffold has been used owing to its advantage and superior property. Cissus quadrangularis, a natural agent was incorporated into the carrageenan scaffold. Therefore, the present study aimed to assess the antioxidant activity and biocompatibility of this novel material.MethodsThe present in vitro study comprised of four study groups each constituting a sample of 15 with a total sample size of sixty (n = 60). The carrageenan hydrogel devoid of Cissus quadrangularis acted as the control group (Group-I). Based on the concentration of aqueous extract of Cissus quadrangularis (10% w/v, 20% w/v and 30% w/v) in carrageenan hydrogel, respective study groups namely II, III and IV were considered. Antioxidant activity was assessed using a 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay, whereas the biocompatibility test was performed using a brine shrimp lethality assay. The microstructure and surface morphology of the hydrogel samples containing different concentrations of Cissus quadrangularis aqueous extract was investigated using SEM. One-way ANOVA with the post hoc tukey test was performed using SPSS software v22.ResultsA significant difference (P < 0.05) in the antioxidant activity was observed among the study groups. Group III reported the highest activity, whereas the control group showed the least antioxidant activity. Additionally, a significant (P < 0.01) drop in the antioxidant activity was observed in group IV when compared with group III. While assessing the biocompatibility, a significant (P < 0.001) dose-dependent increase in biocompatibility was observed with the increasing concentration of aqueous extract of Cissus quadrangularis. SEM analysis in group III showed even distribution throughout the hydrogel although the particles are close and densely arranged. Reduced antioxidant activity in group IV was probably due to clumping of the particles, thus reducing the active surface area.ConclusionKeeping the limitations of in vitro study, it can be assumed that a carrageenan based injectable hydrogel scaffold incorporated with 20% w/v Cissus quadrangularis can provide a favourable micro-environment as it is biocompatible and possess better antioxidant property.

Kappa-carrageenan/chitosan/gelatin scaffolds enriched with potassium chloride for bone tissue engineering

Kappa-carrageenan is a biocompatible natural polysaccharide able to form hydrogels for tissue regeneration. In bone tissue engineering, achieving a bioactive microenvironment with appropriate mechanical properties in polysaccharide-based scaffolds remains a challenge. This study aims to fabricate 3D scaffolds comprising kappa-carrageenan, chitosan and gelatin, crosslinked with KCl, and evaluate their mechanical and biological properties for bone tissue engineering. The produced scaffolds include kappa-carrageenan/chitosan (KC), kappa-carrageenan/chitosan/gelatin (KCG), kappa-carrageenan/chitosan/gelatin enriched with KCl (KCG-KCl), and chitosan/gelatin (CG). All scaffolds present degradation rates ranging from 30% weight loss on day 21, pore size distribution in the range of 100–160 μm and porosity above 80%. The Young modulus values range from 9 to 256 kPa, with the KCl-containing KCG scaffolds demonstrating the highest values, validating the role of KCl in the coil to helix transition of kappa-carrageenan leading to firmer structures. In vitro biological evaluation indicates that pre-osteoblasts proliferate significantly from day 3 up to day 14 on all scaffold compositions. The alkaline phosphatase activity shows a significant increase up to day 14. The calcium production displays a constant increase from day 14 up to day 28, proving that all scaffold compositions support the osteogenic differentiation potential.

Mussel-inspired extracellular matrix-mimicking hydrogel scaffold with high cell affinity and immunomodulation ability for growth factor-free cartilage regeneration.

BackgroundInjury to articular cartilage cause certain degree of disability due to poor self-repair ability of cartilage tissue. To promote cartilage regeneration, it is essential to develop a scaffold that properly mimics the native cartilage extracellular matrix (ECM) in the aspect of compositions and functions.MethodsA mussel-inspired strategy was developed to construct an ECM-mimicking hydrogel scaffold by incorporating polydopamine-modified hyaluronic acid (PDA/HA) complex into a dual-crosslinked collagen (Col) matrix for growth factor-free cartilage regeneration. The adhesion, proliferation, and chondrogenic differentiation of cells on the scaffold were examined. A well-established full-thickness cartilage defect model of the knee in rabbits was used to evaluated the efficacy and functionality of the engineered Col/PDA/HA hydrogel scaffold.ResultsThe PDA/HA complex incorporated-hydrogel scaffold with catechol moieties exhibited better cell affinity than bare negatively-charged HA incorporated hydrogel scaffold. In addition, the PDA/HA complex endowed the scaffold with immunomodulation ability, which suppressed the expression of inflammatory cytokines and effectively activated the polarization of macrophages toward M2 phenotypes. The in vivo results revealed that the mussel-inspired Col/PDA/HA hydrogel scaffold showed strong cartilage inducing ability to promote cartilage regeneration.ConclusionsThe PDA/HA complex-incorporated hydrogel scaffold overcame the cell repellency of negatively-charged polysaccharide-based scaffolds, which facilitated the adhesion and clustering of cells on the scaffold, and therefore enhanced cell-HA interactions for efficient chondrogenic differentiation. Moreover, the hydrogel scaffold modulated immune microenvironment, and created a regenerative microenvironment to enhance cartilage regeneration.The translational potential of this articleThis study gives insight into the mussel-inspired approach to construct the tissue-inducing hydrogel scaffold in a growth-factor-free manner, which show great advantage in the clinical treatment. The hydrogel scaffold composed of collagen and hyaluronic acid as the major component, providing cartilage ECM-mimicking environment, is promising for cartilage defect repair.

Tuning Physicochemical Properties of a Macroporous Polysaccharide-Based Scaffold for 3D Neuronal Culture.

Central nervous system (CNS) lesions are a leading cause of death and disability worldwide. Three-dimensional neural cultures in biomaterials offer more physiologically relevant models for disease studies, toxicity screenings or in vivo transplantations. Herein, we describe the development and use of pullulan/dextran polysaccharide-based scaffolds for 3D neuronal culture. We first assessed scaffolding properties upon variation of the concentration (1%, 1.5%, 3% w/w) of the cross-linking agent, sodium trimetaphosphate (STMP). The lower STMP concentration (1%) allowed us to generate scaffolds with higher porosity (59.9 ± 4.6%), faster degradation rate (5.11 ± 0.14 mg/min) and lower elastic modulus (384 ± 26 Pa) compared with 3% STMP scaffolds (47 ± 2.1%, 1.39 ± 0.03 mg/min, 916 ± 44 Pa, respectively). Using primary cultures of embryonic neurons from PGKCre, Rosa26tdTomato embryos, we observed that in 3D culture, embryonic neurons remained in aggregates within the scaffolds and did not attach, spread or differentiate. To enhance neuronal adhesion and neurite outgrowth, we then functionalized the 1% STMP scaffolds with laminin. We found that treatment of the scaffold with a 100 μg/mL solution of laminin, combined with a subsequent freeze-drying step, created a laminin mesh network that significantly enhanced embryonic neuron adhesion, neurite outgrowth and survival. Such scaffold therefore constitutes a promising neuron-compatible and biodegradable biomaterial.

Engineering Polysaccharides for Tissue Repair and Regeneration.

The success of repair or regeneration depends greatly on the architecture of 3D scaffolds that finely mimic natural extracellular matrix to support cell growth and assembly. Polysaccharides have excellent biocompatibility with intrinsic biological cues and they have been extensively investigated as scaffolds for tissue engineering and regenerative medicine (TERM). The physical and biochemical structures of natural polysaccharides, however, can barely meet all the requirements of tissue-engineered scaffolds. To take advantage of their inherent properties, many innovative approaches including chemical, physical, or joint modifications have been employed to improve their properties. Recent advancement in molecular and material building technology facilitates the fabrication of advanced 3D structures with desirable properties. This review focuses on the latest progress of polysaccharide-based scaffolds for TERM, especially those that construct advanced architectures for tissue regeneration.

Development of 3D Hepatic Constructs Within Polysaccharide-Based Scaffolds with Tunable Properties.

Organoids production is a key tool for in vitro studies of physiopathological conditions, drug-induced toxicity assays, and for a potential use in regenerative medicine. Hence, it prompted studies on hepatic organoids and liver regeneration. Numerous attempts to produce hepatic constructs had often limited success due to a lack of viability or functionality. Moreover, most products could not be translated for clinical studies. The aim of this study was to develop functional and viable hepatic constructs using a 3D porous scaffold with an adjustable structure, devoid of any animal component, that could also be used as an in vivo implantable system. We used a combination of pharmaceutical grade pullulan and dextran with different porogen formulations to form crosslinked scaffolds with macroporosity ranging from 30 µm to several hundreds of microns. Polysaccharide scaffolds were easy to prepare and to handle, and allowed confocal observations thanks to their transparency. A simple seeding method allowed a rapid impregnation of the scaffolds with HepG2 cells and a homogeneous cell distribution within the scaffolds. Cells were viable over seven days and form spheroids of various geometries and sizes. Cells in 3D express hepatic markers albumin, HNF4α and CYP3A4, start to polarize and were sensitive to acetaminophen in a concentration-dependant manner. Therefore, this study depicts a proof of concept for organoid production in 3D scaffolds that could be prepared under GMP conditions for reliable drug-induced toxicity studies and for liver tissue engineering.

Norfloxacin-Loaded Electrospun Scaffolds: Montmorillonite Nanocomposite vs. Free Drug.

Infections in nonhealing wounds remain one of the major challenges. Recently, nanomedicine approach seems a valid option to overcome the antibiotic resistance mechanisms. The aim of this study was the development of three types of polysaccharide-based scaffolds (chitosan-based (CH), chitosan/chondroitin sulfate-based (CH/CS), chitosan/hyaluronic acid-based (CH/HA)), as dermal substitutes, to be loaded with norfloxacin, intended for the treatment of infected wounds. The scaffolds have been loaded with norfloxacin as a free drug (N scaffolds) or in montmorillonite nanocomposite (H—hybrid-scaffolds). Chitosan/glycosaminoglycan (chondroitin sulfate or hyaluronic acid) scaffolds were prepared by means of electrospinning with a simple, one-step process. The scaffolds were characterized by 500 nm diameter fibers with homogeneous structures when norfloxacin was loaded as a free drug. On the contrary, the presence of nanocomposite caused a certain degree of surface roughness, with fibers having 1000 nm diameters. The presence of norfloxacin–montmorillonite nanocomposite (1%) caused higher deformability (90–120%) and lower elasticity (5–10 mN/cm2), decreasing the mechanical resistance of the systems. All the scaffolds were proven to be degraded via lysozyme (this should ensure scaffold resorption) and this sustained the drug release (from 50% to 100% in 3 days, depending on system composition), especially when the drug was loaded in the scaffolds as a nanocomposite. Moreover, the scaffolds were able to decrease the bioburden at least 100-fold, proving that drug loading in the scaffolds did not impair the antimicrobial activity of norfloxacin. Chondroitin sulfate and montmorillonite in the scaffolds are proven to possess a synergic performance, enhancing the fibroblast proliferation without impairing norfloxacin’s antimicrobial properties. The scaffold based on chondroitin sulfate, containing 1% norfloxacin in the nanocomposite, demonstrated adequate stiffness to sustain fibroblast proliferation and the capability to sustain antimicrobial properties to prevent/treat nonhealing wound infection during the healing process.

Magnetic Resonance Imaging of Single Cells.

This chapter discusses a methodology for simultaneously imaging stem cells and endothelial cells within polysaccharide-based scaffolds for tissue engineering. These scaffolds were then implanted into nude mice. Human mesenchymal stem cells (HMSCs) were labeled with the T1-marker Gd(III)-DOTAGA-functionalized polysiloxane nanoparticles (GdNPs), whereas endothelial umbilical vein cells (HUVECs) were labeled with citrate-stabilized maghemite nanoparticles (IONPs), which predominantly shorten the T2-relaxation times of the water molecules in scaffolds and tissue. Dual cell detection was achieved by performing T1- and T2-weighted MRI in both tissue scaffolds and in vivo.

Chitosan/gellan gum ratio content into blends modulates the scaffolding capacity of hydrogels on bone mesenchymal stem cells

Here, we have demonstrated the production and characterization of hydrogel scaffolds based on chitosan/gellan gum (CS/GG) assemblies, without any covalent and metallic crosslinking agents, conventionally used to yield non-soluble polysaccharide-based materials. The polyelectrolyte complexes (physical hydrogels called as PECs) are characterized by Fourier-transform infrared spectroscopy, wide-angle X-ray scattering, and scanning electron microscopy. Hydrogels containing chitosan (CS) excesses (ranging from 60 to 80 wt%) were created. Durable polysaccharide-based scaffolds with structural homogeneity and interconnecting pore networks are developed by modulating the CS/GG weight ratio. The CS/GG hydrogel prepared at 80/20 CS/GG weight ratio (sample CS/GG80-20) is cytocompatible, supporting the attachment, growth, and spreading of bone marrow mesenchymal stem cells (BMSCs) after nine days of cell culture. The cytocompatibility is correlated to the swelling capacity of the PEC in PBS buffer (pH 7.4). By controlling the CS content, we can tune the water uptake of the material, enhancing the capacity to serve as a three-dimensional cell scaffold for BMSCs. This work presents for the first time that CS/GG hydrogels can be applied as scaffolds for tissue engineering purposes.

Polysaccharide-based Scaffolds for Bone Marrow Regeneration: Recent Work and Commercial Utility (Patent)

Biochemical applications of polysaccharide are often demonstrated in various drug delivery systems and bone tissue engineering. Perhaps they are similar in biochemical properties with human components of the extracellular matrix, the body recognized them easily. In this manuscript, the polysaccharides, such as chitosan chitin, carrageenan, chondroitin sulfate,and cellulose used as scaffolds for bone regeneration, are discussed. Scaffolds have a porous structure which is extremely interconnected &amp; permits cell penetration. They provide a 3-dimensional environment for bone regeneration. Polysaccharides such as chitosan have great mechanical characteristics and biocompatibility. Present manuscript deals with the polysaccharides based scaffolds that should possess osteoconductivity, biocompatibility,and mechanical strength like property during the tissue repairing process.They also show decreased degradation rate which means that they are present for tissue regeneration for an extended period of time. So it can be concluded that the polysaccharide-based scaffolds have good mechanical strength and stimulate the natural extracellular of bone for the regeneration process. In this manuscript, various patentsbased on applications of polysaccharide in bone marrow and tissue regeneration is also included.

Assessment of Hedgehog Signaling Pathway Activation for Craniofacial Bone Regeneration in a Critical-Sized Rat Mandibular Defect.

Osseous craniofacial defects are currently reconstructed with bone grafting, rigid fixation, free tissue transfer, and/or recombinant human bone morphogenetic protein 2. Although these treatment options often have good outcomes, they are associated with substantial morbidity, and many patients are not candidates for free tissue transfer. To assess whether polysaccharide-based scaffold (PS) constructs that are cross-linked with smoothened agonist (SAG), vascular endothelial growth factor (VEGF), and bone morphogenetic protein 6 (BMP-6) would substantially increase bone regeneration. This animal model study was conducted at the University of Virginia School of Medicine Cui Laboratory from March 1, 2017, to June 30, 2017. Thirty-three 10-week-old female Lewis rats were acquired for the study. Bilateral nonsegmental critical-sized defects were created in the angle of rat mandibles. The defects were either left untreated or filled with 1 of the 9 PSs. The rats were killed after 8 weeks, and bone regeneration was evaluated using microcomputed tomographic imaging and mechanical testing. Analysis of variance testing was used to compare the treatment groups. Blinded analysis and computer analysis of the microcomputed tomographic images were used to assess bone regeneration. In the 33 female Lewis rats, minimal healing was observed in the untreated mandibles. Addition of SAG was associated with increases in bone regeneration and bone density in all treatment groups, and maximum bone healing was seen in the group with BMP-6, VEGF, and SAG cross-linked to PS. For each of the 5 no scaffold group vs BMP-6, VEGF, and SAG cross-linked to PS group comparisons, mean defect bone regeneration was 4.14% (95% CI, 0.94%-7.33%) vs 66.19% (95% CI, 54.47%-77.90%); mean bone volume, 14.52 mm3 (95% CI, 13.07-15.97 mm3) vs 20.87 mm3 (95% CI, 14.73- 27.01 mm3); mean bone surface, 68.97 mm2 (95% CI, 60.08-77.85 mm2) vs 96.77 mm2 (95% CI, 76.11-117.43 mm2); mean ratio of bone volume to total volume, 0.11 (95% CI, 0.10-0.11) vs 0.15 (95% CI, 0.10-0.19); and mean connectivity density 0.03 (95% CI, 0.02-0.05) vs 0.32 (95% CI, 0.25-0.38). On mechanical testing, mandibles with untreated defects broke with less force than control mandibles in which no defect was made, although this force did not reach statistical significance. No significant difference in force to fracture was observed among the treatment groups. In this rat model study, activation of the hedgehog signaling pathway using smoothened agonist was associated with increased craniofacial bone regeneration compared with growth factors alone, including US Food and Drug Administration-approved recombinant human bone morphogenetic protein 2. Pharmaceuticals that target this pathway may offer a new reconstructive option for bony craniofacial defects as well as nonunion and delayed healing fractures. NA.

Mechanically-enhanced polysaccharide-based scaffolds for tissue engineering of soft tissues

Collagen-based materials are probably among the most used class of biomaterials in tissue engineering and regenerative medicine. Although collagen is often privileged for providing a suitable substrate on which cells can be cultured or a matrix in which cells can be dispersed, its mechanical properties represent a major limitation for clinical translation and even for handling of the obtained regenerated tissue. In this work, the combination of polysaccharides chitosan (Ch) and xanthan gum (X) was investigated as an alternative for scaffolds for soft tissue engineering. Moreover, in an attempt to reach a compromise between obtaining highly porous biomaterials while maintaining appropriate mechanical properties, a surfactant (Kolliphor® P188, K) was added to Ch-X matrices to generate pores, while silicone rubber (Silpuran® 2130A/B, S) was used to balance their mechanical properties. Addition of K (10 or 25% w/w) increased the porosity and pore-dimensions, while addition of S improved by up to 156% and 85% the elastic moduli and the elastic behavior of Ch-X-based scaffolds, under both compressive and tensile loads, respectively, at 50% strain. Relaxation tests confirmed that these materials do have a viscoelastic behavior. The presence of S increased thickness and microscale surface roughness and did not affect liquid uptake and stability, thrombogenicity, biodegradation and cytotoxicity of polysaccharide-based scaffolds. In conclusion, this work shows that Ch-X-S porous blends constitute suitable scaffolds for soft tissue engineering.

Exploiting natural polysaccharides to enhance in vitro bio-constructs of primary neurons and progenitor cells

Current strategies in Central Nervous System (CNS) repair focus on the engineering of artificial scaffolds for guiding and promoting neuronal tissue regrowth. Ideally, one should combine such synthetic structures with stem cell therapies, encapsulating progenitor cells and instructing their differentiation and growth. We used developments in the design, synthesis, and characterization of polysaccharide-based bioactive polymeric materials for testing the ideal composite supporting neuronal network growth, synapse formation and stem cell differentiation into neurons and motor neurons. Moreover, we investigated the feasibility of combining these approaches with engineered mesenchymal stem cells able to release neurotrophic factors. We show here that composite bio-constructs made of Chitlac, a Chitosan derivative, favor hippocampal neuronal growth, synapse formation and the differentiation of progenitors into the proper neuronal lineage, that can be improved by local and continuous delivery of neurotrophins. Statement of SignificanceIn our work, we characterized polysaccharide-based bioactive platforms as biocompatible materials for nerve tissue engineering. We show that Chitlac-thick substrates are able to promote neuronal growth, differentiation, maturation and formation of active synapses. These observations support this new material as a promising candidate for the development of complex bio-constructs promoting central nervous system regeneration. Our novel findings sustain the exploitation of polysaccharide-based scaffolds able to favour neuronal network reconstruction. Our study shows that Chitlac-thick may be an ideal candidate for the design of biomaterial scaffolds enriched with stem cell therapies as an innovative approach for central nervous system repair.

Cellulose based micro- nano structured scaffolds for bone regeneration

Event Abstract Back to Event Cellulose based micro- nano structured scaffolds for bone regeneration Aja Aravamudhan1, 2 and Sangamesh G. Kumbar1, 2, 3, 4 1 University of Connecticut Health Center, Institute for Regenerative Engineering, United States 2 University of Connecticut Health Center, Department of Orthopeadic Surgery, United States 3 University of Connecticut Health Center, Department of Materials Science and Engineering, United States 4 University of Connecticut Health Center, Department of Biomedical Engineering, United States Introduction: Several polymer based scaffold systems have been used as an alternative to traditional autografts and allografts to repair non-healing defects of the bone. These materials fail to provide required osteoconductive, osteoinductive, and osteointegrative properties in line with autografts. Natural polymers have the advantage of being similar to biological macromolecules and elicit favorable tissue healing responses, in contrast to synthetic polymers that are used conventionally in scaffolds for bone regeneration. In this study, we present the characterization of cellulose acetate based scaffolds in comparison to synthetic poly(lactic-co-glycolic acid) (PLGA) scaffolds with respect to their ability to promote hMSC’s progression into osteoblastic lineage in vitro, biocompatibility in a rat subcutaneous implantation model and bone healing capability in a mouse calvarial defect model. Materials and Methods: Cellulose acetate (CA) (Sigma-Aldrich, 30kD) and (PLGA) (P) microparticles were prepared using emulsion solvent evaporation method. Solvent sintering technique[1],[2] was employed to prepare CA scaffolds and heat sintering was used for PLGA. Composite scaffolds of CA (CAc) PLAGA (Pc) were prepared by coating 0.1 % collagen solution on the scaffolds. In-vitro studies, using hMSCs in basal media (BM) and osteogenic media (OM), were conducted by seeding 500,000 cells per tablet type scaffold. Osteoblastic differentiation was monitored by alkaline phosphate activity assay and mineralization along with change in osteogenic gene expression (RUNX2, Col1, Col3, and BSP after 21 days). Further, CA, CAc and PLGA scaffolds were implanted subcutaneously in rats to determine the biocompatibility of the materials by contrasting the immune responses between the groups using histological staining. The scaffolds seeded with bone marrow stomal cells from donor mice (Col 3.6 cyan+) were implanted in critical sized (3.5mm dia) calvarial defect in mice (Col 3.6 GFP+) and its ability to heal bone was evaluated by histological staining. Results and Discussion: Osteogenic genes such as RUNX2, collagen1 and BSP showed greater expression on the CA and CAc groups in contrast to the PLGA groups (Figure 1.a.). The CA and CAc scaffolds showed greater progression of osteogenic phenotype as seen by greater protein levels of Col1 and BSP (Figure 1.b.) and mineralization of osteoinduced hMSCs on these scaffolds (Figure 1.c.). Among the scaffolds implanted subcutaneously (Figure 2.a.), the foreign body response (FBR) reduced significantly over time on the CA groups, while the FBR response increased over time on PLGA (Figure 2.b.). These results indicate the long-term biocompatibility of CA groups over synthetic PLGA. Finally, CA scaffolds seeded with MSCs showed uniform bone formation in contrast to PLGA scaffolds that had islands of new bone formation (Figure 2.c.). The greater hydrophilic and biomimetic nature of CA may be attributed to these differences. Conclusions: The CA and CAc scaffolds induced and maintained osteoblastic differentiation of the seeded hMSCs. The biocompatibility and cellularity of CA increased over time, while that of PLGA decreased over time. The stable and more hydrophilic properties of CA could be attributed to these results. Finally the bone formation by CA scaffolds was uniformly well distributed through the scaffold than PLGA, reflecting the superior osteogenic performace of the polysaccharide-based scaffolds. Hence CA based polysaccharide platform may be a viable alternative to synthetic polymers like PLGA in bone tissue engineering. Coulter Foundation, National Science Foundation (IIP-1355327, IIP- 1311907 and EFRI-1332329); Raymond and Beverly Sackler Center

High-Resolution Cellular MRI: Gadolinium and Iron Oxide Nanoparticles for in-Depth Dual-Cell Imaging of Engineered Tissue Constructs

Recent advances in cell therapy and tissue engineering opened new windows for regenerative medicine, but still necessitate innovative noninvasive imaging technologies. We demonstrate that high-resolution magnetic resonance imaging (MRI) allows combining cellular-scale resolution with the ability to detect two cell types simultaneously at any tissue depth. Two contrast agents, based on iron oxide and gadolinium oxide rigid nanoplatforms, were used to "tattoo" endothelial cells and stem cells, respectively, with no impact on cell functions, including their capacity for differentiation. The labeled cells' contrast properties were optimized for simultaneous MRI detection: endothelial cells and stem cells seeded together in a polysaccharide-based scaffold material for tissue engineering appeared respectively in black and white and could be tracked, at the cellular level, both in vitro and in vivo. In addition, endothelial cells labeled with iron oxide nanoparticles could be remotely manipulated by applying a magnetic field, allowing the creation of vessel substitutes with in-depth detection of individual cellular components.

Cell interactions between human progenitor-derived endothelial cells and human mesenchymal stem cells in a three-dimensional macroporous polysaccharide-based scaffold promote osteogenesis

Several studies have reported the benefits of mesenchymal stem cells (MSCs) for bone tissue engineering. However, vascularization remains one of the main obstacles that must be overcome to reconstruct large bone defects. In vitro prevascularization of the three-dimensional (3-D) constructs using co-cultures of human progenitor-derived endothelial cells (PDECs) with human bone marrow mesenchymal stem cells (HBMSCs) appeared as a potential strategy. However, the crosstalk between the two lineages has been studied in two-dimensional (2-D), but remains unknown in 3-D. The aim of this study is to investigate the cell interactions between PDECs and HBMSCs in a porous matrix composed of polysaccharides. This biodegradable scaffold promotes cell interactions by inducing multicellular aggregates composed of HBMSCs surrounded by PDECs. Cell aggregation contributes to the formation of junctional proteins composed of Connexin43 (Cx43) and VE-cadherin, and an activation of osteoblastic differentiation of HBMSCs stimulated by the presence of PDECs. Inhibition of Cx43 by mimetic peptide 43GAP27 induced a decrease in mRNA levels of Cx43 and all the bone-specific markers. Finally, subcutaneous implantations for 3 and 8weeks in NOG mice revealed an increase in osteoid formation with the tissue-engineered constructs seeded with HBMSCs/PDECs compared with those loaded with HBMSCs alone. Taking together, these results demonstrate that this 3-D microenvironment favored cell communication, osteogenesis and bone formation.

Mesenchymal Stem Cell Delivery into Rat Infarcted Myocardium Using a Porous Polysaccharide-Based Scaffold: A Quantitative Comparison With Endocardial Injection

The use of mesenchymal stem cells (MSCs) for tissue regeneration is often hampered by modest engraftment in host tissue. This study was designed to quantitatively compare MSCs engraftment rates after delivery using a polysaccharide-based porous scaffold or endocardial (EC) injection in a rat myocardial infarction model. Cellular engraftment was measured by quantitative reverse transcription-polymerase chain reaction using MSCs previously transduced with a lentiviral vector that expresses green fluorescent protein (GFP). The use of a scaffold promoted local cellular engraftment and survival. The number of residual GFP(+) cells was greater with the scaffold than after EC injection (9.7% vs. 5.1% at 1 month and 16.3% vs. 6.1% at 2 months, respectively [n=5]). This concurred with a significant increase in mRNA vascular endothelial growth factor level in the scaffold group (p<0.05). Clusters of GFP+ cells were detected in the peri-infarct area, mainly phenotypically consistent with immature MSCs. Functional assessment by echocardiography at 2 months postinfarct also showed a trend toward a lower left ventricular dilatation and a reduced fibrosis in the scaffold group in comparison to direct injection group (n=10). These findings demonstrate that using a porous biodegradable scaffold is a promising method to improve cell delivery and engraftment into damaged myocardium.