Mesenchymal stem cell interaction with a non-woven hyaluronan-based scaffold suitable for tissue repair (original) (raw)
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Biomaterials, 2011
We have created hyaluronic acid (HA)-based, cell-adhesive hydrogels that direct the initial attachment and the subsequent differentiation of human mesenchymal stem cells (MSCs) into pre-osteoblasts without osteogenic supplements. HA-based hydrogel particles (HGPs) with an average diameter of 5e6 mm containing an estimated 2.2 wt% gelatin (gHGPs) were synthesized by covalent immobilization of gelatin to HA HGPs prepared via an inverse emulsion polymerization technique. Separately, a photocrosslinkable HA macromer (HAGMA) was synthesized by chemical modification of HA with glycidyl methacrylate (GMA). Doubly crosslinked networks (DXNs) were engineered by embedding gHGPs in a secondary network established by HAGMA at a particle concentration of 2.5 wt%. The resultant composite gels, designated as HA-gHGP, have an average compressive modulus of 21 kPa, and are nontoxic to the cultured MSCs. MSCs readily attached to these gels, exhibiting an early stage of stress fiber assembly 3 h post seeding. By day 7, stellate-shaped cells with extended filopodia were found on HA-gHGP gels. Moreover, cells had migrated deep into the matrix, forming a three dimensional, branched and interconnected cell community. Conversely, MSCs on the control gels lacking gelatin moieties formed isolated spheroids with rounded cell morphology. After 28 days of culture on HA-gHGP, Type I collagen production and mineral deposition were detected in the absence of osteogenic supplements, suggesting induction of osteogenic differentiation. In contrast, cells on the control gels expressed markers for adipogenesis. Overall, the HA-gHGP composite matrix has great promise for directing the osteogenic differentiation of MSCs by providing an adaptable environment through the spatial presentation of cell-adhesive modules.
Journal of Materials Science: Materials in Medicine, 2004
Sheep mesenchymal stem cells (MSCs) were isolated and expanded using the principle of plastic adherence. Their identity as progenitor cells was con®rmed by induction along the osteoblastic lineage using osteogenic supplements and observation of calci®c deposits by von Kossa staining. MSCs were seeded onto two types of hyaluronan-based cylindrical scaffolds in high concentrations and cultured for varying time points up to three weeks. Culture medium was supplied using the following conditions: statically, on a shaker, by stirring with a magnetic stirrer or by perfusion in a tubular¯ow circuit. Total cell metabolism was assessed by MTT assay and the quality of cell coverage and matrix formation observed by SEM and histological analysis of thin sections of the constructs. Perfusion culture was established as the most appropriate culturing conditions, with cell metabolism increasing by approximately 300% over three weeks. The coverage of the scaffold surface was very good and the deposition of collagenous matrix was superior in these conditions compared to the static and other dynamic culture conditions.
Properties of Biologic Scaffolds and Their Response to Mesenchymal Stem Cells
Arthroscopy: The Journal of Arthroscopic & Related Surgery, 2014
The purpose of this study was to examine, in vitro, the cellular response of human mesenchymal stem cells (MSCs) to sample types of commercially available scaffolds in comparison with control, native tendon tissue (fresh-frozen rotator cuff tendon allograft). Methods: MSCs were defined by (1) colony-forming potential; (2) ability to differentiate into tendon, cartilage, bone, and fat tissue; and (3) fluorescence-activated cell sorting analysis (CD73, CD90, CD45). Samples were taken from fresh-frozen human rotator cuff tendon (allograft), human highly cross-linked collagen membrane (Arthroflex; LifeNet Health, Virginia Beach, VA), porcine nonecross-linked collagen membrane (Mucograft; Geistlich Pharma, Lucerne, Switzerland), a human platelet-rich fibrin matrix (PRF-M), and a fibrin matrix based on platelet-rich plasma (ViscoGel; Arthrex, Naples, FL). Cells were counted for adhesion (24 hours), thymidine assay for cell proliferation (96 hours), and live/dead stain for viability (168 hours). Histologic analysis was performed after 21 days, and the unloaded scaffolds were scanned with electron microscopy. Results: MSCs were successfully differentiated into all cell lines. A significantly greater number of cells adhered to both the nonecross-linked porcine collagen scaffold and PRF-M. Cell activity (proliferation) was significantly higher in the nonecrosslinked porcine collagen scaffold compared with PRF-M and fibrin matrix based on platelet-rich plasma. There were no significant differences found in the results of the live/dead assay. Conclusions: Significant differences in the response of human MSCs to biologic scaffolds existed. MSC adhesion, proliferation, and scaffold morphology evaluated by histologic analysis and electron microscopy varied throughout the evaluated types of scaffolds. Nonecross-linked porcine collagen scaffolds showed superior results for cell adhesion and proliferation, as well as on histologic evaluation. Clinical Relevance: This study enables the clinician and scientist to choose scaffold materials according to their specific interaction with MSCs.
Journal of Biomedical Materials Research Part A, 2005
Ligaments are complex structures that maintain the mechanical stability of the joint. Healing of injured ligaments involves the interactions of different cell types, local cellular environment, and the use of devices. To gain new information on the complex interactions between mesenchymal stem cells (MSCs) and a specific hyaluronan-based prototype scaffold (HYAFF), useful for ligament tissue engineering, short time-course experiments were performed to analyze the proliferation, vitality, and phenotype of MSCs grown on the scaffold. MSC proliferation was analyzed using the MTT test, during the early time points (2, 4, 6, days). Viability was assessed using calcein/acetyloxymethylester immunofluorescence dye and confocal microscopy analysis. Hyaluronic acid receptor (CD44), typical matrix ligament proteins (collagen type I, type III, laminin, fibronectin, actin), and chondrogenic/osteogenic markers (collagen type II and bone sialoprotein) were evaluated by immunohistochemistry. Our data demonstrated that MSC growth and viability were cell density-dependent. MSCs completely wrapped the fibers of the scaffold, expressed CD44, collagen type I, type III, laminin, fibronectin, and actin, and were negative to collagen type II and bone sialoprotein. These data demonstrate that MSCs survive well in the hyaluronan-based prototype ligament scaffold, as assessed after 2 days from seeding, and express CD44, a receptor important for scaffold interaction, and proteins responsible for the functional characteristics of the ligaments.
Neoarteries grown in vivo using a tissue-engineered hyaluronan-based scaffold
Faseb Journal, 2008
Vascular tissue engineering has emerged as a promising technology for the design of an ideal, responsive, living conduit with properties similar to that of native tissue. The missing link in tissue-engineered blood vessels is elastin biosynthesis. Several biomaterials are currently used but few support elastin biosynthesis in a 3-D array. In previous studies, we demonstrated that a hyaluronan-based scaffold (HYAFF-11 ™ ) grafted in the infrarenal rat aorta successfully guided the complete regeneration of a well-functioning smalldiameter (2 mm) neoartery. The aim of the present study was to test the ability of HYAFF-11 biodegradable grafts to develop into neovessels of larger size (4 mm) in a porcine model, focusing on extracellular matrix (ECM) remodeling and elastin biosynthesis. HYAFF-11 tubes (diameter 4 mm, length 5 cm) were implanted in an end-to-end fashion in the common carotid artery. Grafts were analyzed for patency with a Duplex scan every 15 days. ECM components were evaluated by histological and molecular biological methods. All the animals survived the observation period without complications. Intimal hyperplasia (initiating at the anastomotic site) and graft thrombosis led to 3 cases of partial or complete occlusion, as demonstrated by histological examination. There were no signs of stenoses or aneurysms in the remaining grafts. After 5 months, the biomaterial was almost completely degraded and replaced by a neoartery segment composed of mature smooth muscle cells, collagen, and elastin fibers organized in layers and was completely covered on the luminal surface by endothelial cells (vWF ؉ ). Whereas in previous small animal studies, patency rates were not optimal, those obtained in the present study using hyaluronan-based grafts of larger size confirmed the ability of these constructs to guide the development of a well-functioning neoartery, with the remarkable additional attribute of facilitating the formation of organized layers of elastin fibers.-Zavan, B., Vindigni, V., Lepidi, S., Iacopetti, I., Avruscio, G., Abatangelo, G., Cortivo, R. Neoarteries grown in vivo using a tissueengineered hyaluronan-based scaffold. FASEB J. 22, 2853-2861 (2008)
Role of Biological Scaffolds, Hydro Gels and Stem Cells in Tissue Regeneration Therapy
Advances in Tissue Engineering & Regenerative Medicine: Open Access, 2017
Present review article emphasizes role of biological scaffolds, hydrogels and stem cells in tissue engineering mainly in regeneration or repairing of damaged tissues. Highly porous scaffold biomaterials are developed which act as templates for tissue regeneration and potentially guide the growth of new tissue. Present article also describes de-cellularization, cell printing, vascularization, integration of cross linking of methods for scaffolding of biomaterials to reproduce and recapitulate complexity in engineered tissues. It also explains different scaffold types, polymer hydrogels which are necessary for formation of microstructure, cell attachment, differentiation, tissue vascularization and integration. It also explains use of induced pluripotent stem cells (iPSCs) and engineered MSCs as new diagnostic and potential therapeutic tools to remove sustained damage and complications from organ failures. These engineered MSCs assist in making self-assembling supramolecular hydrogels, which have larger applications in cell therapy of intractable diseases and tissue regeneration. No doubt development of more advanced biomaterials, growth factors and stem cell derived products/factors and tissue transplantation methods based on cell regeneration programming will revolutionize the clinical therapeutics. This article suggests identification of new biomaterials/biomolecules such as growth factors, scaffolds, integration, adhesion and regulatory molecules and cell secreted factors which can induce major metabolic and signaling pathways during phase of tissue repairing and induction of regeneration. This innovative research area needs many conceptual improvements in organ therapies, methods and technological advancement to scale more services to the human society.
Neoarteries grownin vivousing a tissue‐engineered hyaluronan‐based scaffold
The FASEB Journal, 2008
Vascular tissue engineering has emerged as a promising technology for the design of an ideal, responsive, living conduit with properties similar to that of native tissue. The missing link in tissue-engineered blood vessels is elastin biosynthesis. Several biomaterials are currently used but few support elastin biosynthesis in a 3-D array. In previous studies, we demonstrated that a hyaluronan-based scaffold (HYAFF-11 ™) grafted in the infrarenal rat aorta successfully guided the complete regeneration of a well-functioning smalldiameter (2 mm) neoartery. The aim of the present study was to test the ability of HYAFF-11 biodegradable grafts to develop into neovessels of larger size (4 mm) in a porcine model, focusing on extracellular matrix (ECM) remodeling and elastin biosynthesis. HYAFF-11 tubes (diameter 4 mm, length 5 cm) were implanted in an end-to-end fashion in the common carotid artery. Grafts were analyzed for patency with a Duplex scan every 15 days. ECM components were evaluated by histological and molecular biological methods. All the animals survived the observation period without complications. Intimal hyperplasia (initiating at the anastomotic site) and graft thrombosis led to 3 cases of partial or complete occlusion, as demonstrated by histological examination. There were no signs of stenoses or aneurysms in the remaining grafts. After 5 months, the biomaterial was almost completely degraded and replaced by a neoartery segment composed of mature smooth muscle cells, collagen, and elastin fibers organized in layers and was completely covered on the luminal surface by endothelial cells (vWF ؉). Whereas in previous small animal studies, patency rates were not optimal, those obtained in the present study using hyaluronan-based grafts of larger size confirmed the ability of these constructs to guide the development of a well-functioning neoartery, with the remarkable additional attribute of facilitating the formation of organized layers of elastin fibers.
Journal of Tissue Engineering and Regenerative Medicine, 2014
In this study we investigated the potential of artificial extracellular matrix (aECM) coatings containing collagen II and two types of glycosaminoglycan (GAGs) with different degrees of sulphation to promote human bone formation in biomedical applications. To this end their impact on growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assessed. The cell proliferation was found to be significantly retarded in the first 14 days of culture on surfaces coated with collagen II and GAGs (coll-II/GAG) as compared to tissue culture polystyrol (TCPS) and those coated with collagen II. At later time points it only tended to be retarded on coll-II/sHya3.1. Heat-inactivation of the serum significantly reduced cell numbers on collagen II and coll-II/sHya3.1. Alkaline phosphatase (ALP) activity and calcium deposition, on the other hand, were higher for coatings containing sHya3.1 and were not significantly changed by heat-inactivation of the serum. Expression levels of the bone matrix proteins bone sialoprotein (BSP-II) and osteopontin (OP) were also increased on aECM coatings as compared to TCPS, which further validated the differentiation of hMSCs towards the osteogenic lineage. These observations reveal that aECM coatings, in particular those containing sHya3.1, are suitable to promote the osteogenic differentiation of hMSCs.
Osteoarthritis Treated with Mesenchymal Stem Cells on Hyaluronan-Based Scaffold in Rabbit
Tissue Engineering Part C: Methods, 2009
Objective: Osteoarthritis (OA) is a disease that limits the mobility of patients and is of considerable economical importance. Up to now, despite the increasing number of patients with OA, treatments to manage the disease remain symptomatic, designed to control pain, and improve function and quality of life limiting adverse events. With the aim to explore a new approach to treat OA patients suffering from early degenerative lesions of hyaline cartilage, we transplanted in an experimental animal model of OA a hyaluronan-based scaffold (Hyaff Ò 11) seeded with mesenchymal stem cells (MSCs) obtained from bone marrow and expanded in culture. Design: Rabbit knee joints were bilaterally subjected to anterior cruciate ligament transection to surgically induce OA. After 8 weeks, the time necessary to the development of cartilage surface damage, animals were treated with MSCs seeded onto Hyaff-11 scaffold in the left condyle and unseeded Hyaff-11 in the controlateral knee. Untreated rabbits were used as controls. All the animals were sacrificed at 3 and 6 months after surgery. Histological, histomorphometric, and immunohistological evaluations were performed. Results: OA changes developed in all animals subjected to anterior cruciate ligament transection. The predominant macroscopically observed OA changes were mild (lateral femoral condyle) or moderate (medial femoral condyle) ulcerations. Statistically significant differences in the quality of the regenerated tissue were found between the implants with scaffolds carrying MSCs compared to the scaffold alone or controls in particular at 6 months. Conclusions: From the observations, it is possible to demonstrate that Hyaff-11, a hyaluronan-based scaffold, has potential for MSC implantation and that may have application for the treatment of early OA in humans.