San Donato di Ninea, Italy
San Donato di Ninea, Italy

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Bersini S.,Polytechnic of Milan | Bersini S.,IRCCS Instituto Ortopedico Galeazzi | Jeon J.S.,Massachusetts Institute of Technology | Dubini G.,Polytechnic of Milan | And 5 more authors.
Biomaterials | Year: 2014

Cancer metastases arise following extravasation of circulating tumor cells with certain tumors exhibiting high organ specificity. Here, we developed a 3D microfluidic model to analyze the specificity of human breast cancer metastases to bone, recreating a vascularized osteo-cell conditioned microenvironment with human osteo-differentiated bone marrow-derived mesenchymal stem cells and endothelial cells. The tri-culture system allowed us to study the transendothelial migration of highly metastatic breast cancer cells and to monitor their behavior within the bone-like matrix. Extravasation, quantified 24h after cancer cell injection, was significantly higher in the osteo-cell conditioned microenvironment compared to collagen gel-only matrices (77.5±3.7% vs. 37.6±7.3%), and the migration distance was also significantly greater (50.8±6.2μm vs. 31.8±5.0μm). Extravasated cells proliferated to form micrometastases of various sizes containing 4 to more than 60 cells by day 5. We demonstrated that the breast cancer cell receptor CXCR2 and the bone-secreted chemokine CXCL5 play a major role in the extravasation process, influencing extravasation rate and traveled distance. Our study provides novel 3D invitro quantitative data on extravasation and micrometastasis generation of breast cancer cells within a bone-like microenvironment and demonstrates the potential value of microfluidic systems to better understand cancer biology and screen for new therapeutics. © 2013 Elsevier Ltd.


PubMed | Korea University, Polytechnic of Milan, IRCCS Instituto Ortopedico Galeazzi, Massachusetts Institute of Technology and 2 more.
Type: Journal Article | Journal: Biomaterials | Year: 2014

Cancer metastases arise following extravasation of circulating tumor cells with certain tumors exhibiting high organ specificity. Here, we developed a 3D microfluidic model to analyze the specificity of human breast cancer metastases to bone, recreating a vascularized osteo-cell conditioned microenvironment with human osteo-differentiated bone marrow-derived mesenchymal stem cells and endothelial cells. The tri-culture system allowed us to study the transendothelial migration of highly metastatic breast cancer cells and to monitor their behavior within the bone-like matrix. Extravasation, quantified 24h after cancer cell injection, was significantly higher in the osteo-cell conditioned microenvironment compared to collagen gel-only matrices (77.53.7% vs. 37.67.3%), and the migration distance was also significantly greater (50.86.2m vs. 31.85.0m). Extravasated cells proliferated to form micrometastases of various sizes containing 4 to more than 60 cells by day 5. We demonstrated that the breast cancer cell receptor CXCR2 and the bone-secreted chemokine CXCL5 play a major role in the extravasation process, influencing extravasation rate and traveled distance. Our study provides novel 3D invitro quantitative data on extravasation and micrometastasis generation of breast cancer cells within a bone-like microenvironment and demonstrates the potential value of microfluidic systems to better understand cancer biology and screen for new therapeutics.


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.


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.


Lovati A.B.,Cell and Tissue Engineering Laboratory | Drago L.,IRCCS Galeazzi Orthopaedic Institute | Drago L.,University of Milan | Monti L.,IRCCS Galeazzi Orthopaedic Institute | And 5 more authors.
PLoS ONE | Year: 2013

Background:Periprosthetic bacterial infections represent one of the most challenging orthopaedic complications that often require implant removal and surgical debridement and carry high social and economical costs. Diabetes is one of the most relevant risk factors of implant-related infection and its clinical occurrence is growing worldwide. The aim of the present study was to test a model of implant-related infection in the diabetic mouse, with a view to allow further investigation on the relative efficacy of prevention and treatment options in diabetic and non-diabetic individuals.Methodology:A cohort of diabetic NOD/ShiLtJ mice was compared with non-diabetic CD1 mice as an in vivo model of S. aureus orthopaedic infection of bone and soft tissues after femur intramedullary pin implantation. We tested control and infected groups with 1×103 colony-forming units of S. aureus ATCC 25923 strain injected in the implant site. At 4 weeks post-inoculation, host response to infection, microbial biofilm formation, and bone damage were assessed by traditional diagnostic parameters (bacterial culture, C-reactive protein and white blood cell count), histological analysis and imaging techniques (micro computed tomography and scanning electron microscopy).Results:Unlike the controls and the CD1 mice, all the diabetic mice challenged with a single inoculum of S. aureus displayed severe osteomyelitic changes around the implant.Conclusions:Our findings demonstrate for the first time that the diabetic mouse can be successfully used in a model of orthopaedic implant-related infection. Furthermore, the same bacteria inoculum induced periprosthetic infection in all the diabetic mice but not in the controls. This animal model of implant-related infection in diabetes may be a useful tool to test in vivo treatments in diabetic and non-diabetic individuals. © 2013 Lovati et al.


Sabatino M.A.,University of Basel | Sabatino M.A.,Cell and Tissue Engineering Laboratory | Santoro R.,University of Basel | Gueven S.,University of Basel | And 5 more authors.
Journal of Tissue Engineering and Regenerative Medicine | Year: 2015

Co-culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small-scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold-based constructs generated by combining primary human ACs with human bone marrow MSCs (BM-MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM-MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co-culture of ACs (25%) with BM-MSCs (75%) in scaffolds resulted in up to 1.4-fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8-fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM-MSCs with a relatively low fraction of non-expanded ACs in porous scaffolds. The proposed co-culture strategy is directly relevant towards a single-stage surgical procedure for cartilage repair. © 2015 John Wiley & Sons, Ltd.


Bersini S.,IRCCS Instituto Ortopedico Galeazzi | Gilardi M.,IRCCS Instituto Ortopedico Galeazzi | Gilardi M.,University of Milan Bicocca | Arrigoni C.,Cell and Tissue Engineering Laboratory | And 5 more authors.
Biomaterials | Year: 2016

The generation of functional, vascularized tissues is a key challenge for both tissue engineering applications and the development of advanced in vitro models analyzing interactions among circulating cells, endothelium and organ-specific microenvironments. Since vascularization is a complex process guided by multiple synergic factors, it is critical to analyze the specific role that different experimental parameters play in the generation of physiological tissues. Our goals were to design a novel meso-scale model bridging the gap between microfluidic and macro-scale studies, and high-throughput screen the effects of multiple variables on the vascularization of bone-mimicking tissues. We investigated the influence of endothelial cell (EC) density (3-5 Mcells/ml), cell ratio among ECs, mesenchymal stem cells (MSCs) and osteo-differentiated MSCs (1:1:0, 10:1:0, 10:1:1), culture medium (endothelial, endothelial + angiopoietin-1, 1:1 endothelial/osteo), hydrogel type (100%fibrin, 60%fibrin+40%collagen), tissue geometry (2 × 2 × 2, 2 × 2 × 5 mm3). We optimized the geometry and oxygen gradient inside hydrogels through computational simulations and we analyzed microvascular network features including total network length/area and vascular branch number/length. Particularly, we employed the "Design of Experiment" statistical approach to identify key differences among experimental conditions. We combined the generation of 3D functional tissue units with the fine control over the local microenvironment (e.g. oxygen gradients), and developed an effective strategy to enable the high-throughput screening of multiple experimental parameters. Our approach allowed to identify synergic correlations among critical parameters driving microvascular network development within a bone-mimicking environment and could be translated to any vascularized tissue. © 2015 Elsevier Ltd.


De Girolamo L.,IRCCS Instituto Ortopedico Galeazzi | Stanco D.,IRCCS Instituto Ortopedico Galeazzi | Galliera E.,University of Milan | Galliera E.,IRCCS Instituto Ortopedico Galeazzi | And 6 more authors.
Ultrasound in Medicine and Biology | Year: 2014

Focused extracorporeal shock waves have been found to upregulate the expression of collagen and to initiate cell proliferation in healthy tenocytes and to positively affect the metabolism of tendons, promoting the healing process. Recently, soft-focused extracorporeal shock waves have also been found to have a significant effect on tissue regeneration. However, very few invitro reports have dealt with the application of this type of shock wave to cells, and in particular, no previous studies have investigated the response of tendon cells to this impulse. We devised an original model to investigate the invitro effects of soft-focused shock waves on a heterogeneous population of human resident tendon cells in adherent monolayer culture. Our results indicate that soft-focused extracorporeal shock wave treatment (0.17mJ/mm2) is able to induce positive modulation of cell viability, proliferation and tendon-specific marker expression, as well as release of anti-inflammatory cytokines. This could prefigure a new rationale for routine employment of soft-focused shock waves to treat the failed healing status that distinguishes tendinopathies. © 2014 World Federation for Ultrasound in Medicine & Biology.


PubMed | Cell and Tissue Engineering Laboratory
Type: Journal Article | Journal: 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.


PubMed | Cell and Tissue Engineering Laboratory
Type: Journal Article | Journal: PloS one | Year: 2013

Periprosthetic bacterial infections represent one of the most challenging orthopaedic complications that often require implant removal and surgical debridement and carry high social and economical costs. Diabetes is one of the most relevant risk factors of implant-related infection and its clinical occurrence is growing worldwide. The aim of the present study was to test a model of implant-related infection in the diabetic mouse, with a view to allow further investigation on the relative efficacy of prevention and treatment options in diabetic and non-diabetic individuals.A cohort of diabetic NOD/ShiLtJ mice was compared with non-diabetic CD1 mice as an in vivo model of S. aureus orthopaedic infection of bone and soft tissues after femur intramedullary pin implantation. We tested control and infected groups with 110(3) colony-forming units of S. aureus ATCC 25923 strain injected in the implant site. At 4 weeks post-inoculation, host response to infection, microbial biofilm formation, and bone damage were assessed by traditional diagnostic parameters (bacterial culture, C-reactive protein and white blood cell count), histological analysis and imaging techniques (micro computed tomography and scanning electron microscopy).Unlike the controls and the CD1 mice, all the diabetic mice challenged with a single inoculum of S. aureus displayed severe osteomyelitic changes around the implant.Our findings demonstrate for the first time that the diabetic mouse can be successfully used in a model of orthopaedic implant-related infection. Furthermore, the same bacteria inoculum induced periprosthetic infection in all the diabetic mice but not in the controls. This animal model of implant-related infection in diabetes may be a useful tool to test in vivo treatments in diabetic and non-diabetic individuals.

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