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San Donato di Ninea, Italy

Lopa S.,Cell and Tissue Engineering Laboratory | Colombini A.,Galeazzi Orthopaedic Institute | Sansone V.,University of Milan | Preis F.W.B.,Galeazzi Orthopaedic Institute | Moretti M.,Galeazzi Orthopaedic Institute
International Journal of Immunopathology and Pharmacology | Year: 2013

Co-culture of mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) has been proposed for autologous cartilage cell-based therapies, to overcome the issues associated to limited availability of articular chondrocytes (ACs). To evaluate the potentiality of a co-culture approach in aged osteoarthritic patients, MSCs from infrapatellar fat pad (IFP-MSCs) and knee subcutaneous adipose tissue (ASCs) were co-cultured with donor-matched osteoarthritic, expanded and cryopreserved, ACs in a 75%/25% ratio. Co-cultures were prepared also from nasal chondrocytes (NCs) to evaluate their possible use as an alternative to ACs. Pellets were differentiated for 14 days, using mono-cultures of each cell type as reference. Chondrogenic genes SOX9, COL2A1, ACAN were less expressed in co-cultures compared to ACs and NCs. Total GAGs content in co-cultures did not differ significantly from values predicted as the sum of each cell type contribution corrected for the co-culture ratio, as confirmed by histology. No significant differences were observed for GAGs/DNA in mono-cultures, demonstrating a reduced chondrogenic potential of ACs and NCs. In conclusion, a small percentage of expanded and cryopreserved ACs and NCs did not lead to IFP-MSCs and ASCs chondro-induction. Our results suggest that chondrogenic potential and origin of chondrocytes may play a relevant role in the outcome of co-cultures, indicating a need for further investigations to demonstrate their clinical relevance in the treatment of aged osteoarthritic patients. © 2013 SAGE Publications.

Lopa S.,Cell and Tissue Engineering Laboratory | Mercuri D.,Limacorporate S.p.a. | Colombini A.,IRCCS Galeazzi Orthopaedic Institute | De Conti G.,Limacorporate S.p.a. | And 3 more authors.
Journal of Biomedical Materials Research - Part A | Year: 2013

Insufficient implant stability is an important determinant in the failure of cementless prostheses. To improve osseointegration, we aim at generating a bioactive implant combining a macroporous titanium (TT) with a biocompatible hydrogel to encapsulate osteo-inductive factors and osteoprogenitor cells. Amidation and cross-linking degree of an amidated carboxymethylcellulose hydrogel (CMCA) were characterized by FT-IR spectrometry and mechanical testing. Bone marrow mesenchymal stem cells (BMSCs) from osteoarthritic patients were cultured on CMCA hydrogels, TT, and TT loaded with CMCA (TT + CMCA) with an optimized concentration of SrCl2 to evaluate cell viability and osteo-differentiation. Amidation and cross-linking degree were homogeneous among independent CMCA batches. SrCl2 at 5 μg/mL significantly improved BMSCs osteo-differentiation increasing calcified matrix (P < 0.01), type I collagen expression (P < 0.05) and alkaline phosphatase activity. TT + CMCA samples better retained cells into the TT mesh, significantly improving cell seeding efficiency with respect to TT (P < 0.05). BMSCs on TT + CMCA underwent a more efficient osteo-differentiation with higher alkaline phosphatase (P < 0.05) and calcium levels compared to cells on TT. Based on these in vitro results, we envision the association of TT with strontium-enriched CMCA and BMSCs as a promising strategy to generate bioactive implants promoting bone neoformation at the implant site. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 3396-3403, 2013. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Lovati A.B.,Cell and Tissue Engineering Laboratory
Journal of biological regulators and homeostatic agents | Year: 2011

Microcarrier culture systems offer an attractive method for cell amplification and as delivery vehicle. At the same time, super paramagnetic iron oxide (SPIO) nanoparticles represent a unique in vivo tracking system, already approved for clinical use. In our study, we tested the combination of clinically approved microcarriers and SPIO nanoparticles for cell-construct delivery and subsequent tracking after implantation. In order to mimic better a clinical setting, biodegradable macroporous microcarriers were employed as an alternative approach to expand human primary chondrocytes in a dynamic culture system for subsequent direct transplantation. In addition, cellseeded microcarriers were labeled with SPIO nanoparticles to evaluate the benefits of cell-constructs tracking with magnetic resonance. In vivo subcutaneous implants were monitored for up to 3 weeks and orthotopic implantation was simulated and monitored in ex vivo osteochondral defects.

Occhetta P.,Polytechnic of Milan | Sadr N.,Gruppo Ospedaliero San Donato Foundation | Piraino F.,Polytechnic of Milan | Redaelli A.,Polytechnic of Milan | And 2 more authors.
Biofabrication | Year: 2013

Native tissues are characterized by spatially organized three-dimensional (3D) microscaled units which functionally define cells-cells and cells-extracellular matrix interactions. The ability to engineer biomimetic constructs mimicking these 3D microarchitectures is subject to the control over cell distribution and organization. In the present study we introduce a novel protocol to generate 3D cell laden hydrogel micropatterns with defined size and shape. The method, named photo-mold patterning (PMP), combines hydrogel micromolding within polydimethylsiloxane (PDMS) stamps and photopolymerization through a recently introduced biocompatible ultraviolet (UVA) activated photoinitiator (VA-086). Exploiting PDMS micromolds as geometrical constraints for two methacrylated prepolymers (polyethylene glycol diacrylate and gelatin methacrylate), micrometrically resolved structures were obtained within a 3 min exposure to a low cost and commercially available UVA LED. The PMP was validated both on a continuous cell line (human umbilical vein endothelial cells expressing green fluorescent protein, HUVEC GFP) and on primary human bone marrow stromal cells (BMSCs). HUVEC GFP and BMSCs were exposed to 1.5% w/v VA-086 and UVA light (1 W, 385 nm, distance from sample = 5 cm). Photocrosslinking conditions applied during the PMP did not negatively affect cells viability or specific metabolic activity. Quantitative analyses demonstrated the potentiality of PMP to uniformly embed viable cells within 3D microgels, creating biocompatible and favorable environments for cell proliferation and spreading during a seven days' culture. PMP can thus be considered as a promising and cost effective tool for designing spatially accurate in vitro models and, in perspective, functional constructs. © 2013 IOP Publishing Ltd.

Kakegawa T.,University of Tsukuba | Mochizuki N.,University of Tsukuba | Sadr N.,Cell and Tissue Engineering Laboratory | Suzuki H.,University of Tsukuba | Fukuda J.,University of Tsukuba
Tissue Engineering - Part A | Year: 2013

In this study, we describe the development of oligopeptide-modified cell culture surfaces from which adherent cells can be rapidly detached by application of an electrical stimulus. An oligopeptide, CGGGKEKEKEK, was designed with a terminal cysteine residue to mediate binding to a gold surface via a gold-thiolate bond. The peptide forms a self-assembled monolayer through the electrostatic force between the sequence of alternating charged glutamic acid (E) and lysine (K) residues. The dense and electrically neutral oligopeptide zwitterionic layer of the modified surface was resistant to nonspecific adsorption of proteins and adhesion of cells, while the surface was altered to cell adhesive by the addition of a second oligopeptide (CGGGKEKEKEKGRGDSP) containing the RGD cell adhesion motif. Application of a negative electrical potential to this gold surface cleaved the gold-thiolate bond, leading to desorption of the oligopeptide layer, and rapid (within 2 min) detachment of virtually all cells. This approach was applicable not only to detachment of cell sheets but also for transfer of cell micropatterns to a hydrogel. This electrochemical approach of cell detachment may be a useful tool for tissue-engineering applications. © Mary Ann Liebert, Inc.

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