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Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_14 | Pages 16 - 16
1 Dec 2022
Ragni E Orfei CP Colombini A Viganò M De Luca P Libonati F de Girolamo L
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In the context of regenerative medicine for the treatment of musculoskeletal pathologies mesenchymal stromal cells (MSCs) have shown good results thanks to secretion of therapeutic factors, both free and conveyed within the extracellular vesicles (EV), which in their totality constitute the “secretome”. The portfolio and biological activity of these molecules can be modulated by both in vitro and in vivo conditions, thus making the analysis of these activities very complex. A deep knowledge of the targets regulated by the secretome has become a matter of fundamental importance and a homogeneous and complete molecular characterization is still lacking in the field of applications for the musculoskeletal system. Therefore, the aim of this work was to characterize the secretome obtained from adipose-derived MSCs (ASCs), and its modulation after pre-conditioning of the ASCs. Pre-conditioning was done by culturing cells in the presence of i) high levels of IFNγ, as proposed for the production of clinical grade secretome with enhanced regenerative potential, ii) low levels of inflammatory stimuli, mimicking conditions found in the osteoarthritis (OA) synovial fluid. Furthermore, EVs ability to migrate within cartilage, chondrocyte and synoviocytes obtained from OA patients was evaluated.

The data showed that more than 50 cytokines / chemokines and more than 200 EV-microRNAs are detectable at various intensity levels in ASCs secretomes. The majority of the most abundantly present molecules are involved in the remodelling of the extracellular matrix and in the homeostasis and chemotaxis of inflammatory cells including macrophages, which in OA are often characterized by an M1 inflammatory polarization, promoting their transition to an M2 anti-inflammatory phenotype. Inflammatory priming with IFNγ and synovial fluid-like conditions were able to further increase the ability of the secretome to interact with inflammatory cells and modulate their migration. Finally, the penetration of the EVs in the cartilage explants resulted a rapid process, which begins a few minutes after administration of the EVs that are able to reach a depth of 30-40 μm in 5 hours. The same capacity for interaction was also verified in chondrocytes and synoviocytes isolated from the cartilage and synovial membrane of OA patients.

Thanks to the soluble factors and EV-microRNAs, the ASCs secretome has shown a strong propensity to modulate the inflammatory and degenerative processes that characterize OA. The inflammatory pre-conditioning through high concentrations of inflammatory molecules or in conditions similar to the synovial fluid of OA patients was able to increase this capacity by increasing their chemotactic power. The microscopy data also support the hypothesis of the ability of MSC-EVs to influence the chondrocytes residing in the ECM of the cartilage and the synovial cells of the synovial membrane through active interaction and the release of their therapeutic content.


Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_14 | Pages 18 - 18
1 Dec 2022
Marmotti A Coco M Orso F Mangiavini L de Girolamo L Bellato E Agati G Peretti GM Taverna D Castoldi F
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Summary:

Hamstring tendons (HT) represent a widely used autograft for ACL reconstruction. Harvesting, processing and pretensioning procedures together with the time out of the joint could theoretically hamper tendon cells (TCs) viability. The authors hypothesize that HT cells are not impaired at the end of the surgical procedures and their tenogenic phenotype may be strongly improved by exposure to PEMF.

Methods

Remnants of semitendinosus and gracilis tendons were collected at the end of the surgical procedures before skin closure from 15 healthy donors who underwent ACL reconstruction with autologous hamstring tendons. To isolate TCs, the tendon was minced and digested with 0.3 % type I collagenase and the nucleated cells were plated at a density 5x10E3 cells/cm2 and cultured in chamber slides in differentiation medium composed of DMEM + 5ng/ml basic fibroblast growth factor (b-FGF) for 7, 14, 21 days

The following cell cultures were set up:

TCs cultured with differentiation medium + exposure to PEMF 8 h/day (PEMF generator system IGEA, intensity of magnetic field = 1.5 mT, frequency = 75 Hz)

TCs cultured with differentiation medium without exposure to PEMF

At day 0, day 7, day 14 and day 21, immunofluorescence analysis was performed to evaluate the expression of collagen type I, collagen type VI, scleraxis and PCNA (proliferative marker)

Subsequently, tendon explant cultures were set up to verify, at day 21, explant viability and the expression of collagen type I, collagen type VI, beta-catenin and PCNA


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_13 | Pages 85 - 85
1 Nov 2021
Viganò M Ragni E Torretta E Colombini A Orfei CP De Luca P Libonati F Gelfi C de Girolamo L
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Introduction and Objective

The use of microfragmented adipose tissue (mFAT) for the treatment of musculoskeletal disorders, especially osteoarthritis, is gaining popularity following the positive results reported in recent case series and clinical trials. The purpose of this study is to characterize mFAT in terms of structure, cell content and secretome (i.e. protein and microvescicles released as paracrine mediators), and to compare it with unprocessed lipoaspirate tissue, in order to understand the possible mechanisms of action and the benefit derived from tissue processing.

Materials and Methods

Unprocessed lipoaspirate (LA) and mFAT were obtained from 7 donors. Each tissue sample was divided in four aliquots: A) fixed in formalin for histological evaluation; B) enzymatically digested to harvest cells with the exclusion of adipocytes; C) cultured for 24 hours in serum-free DMEM to harvest secretome; D) freshly frozen for proteomic evaluation. Hematoxylin and eosin staning, as well as immunohistochemistry for CD31, CD90, CD146 were performed on aliquot A. Cell count, viability, senescence and immunophenotype were assessed on aliquot B. Culture medium from aliquot C was collected and used for proteomic analysis and micro-RNA extraction and quantitation from extracellular vesicles. Aliquot D was lysed, protein were extracted and analyzed using a high-throughput proteomic approach.


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_13 | Pages 150 - 150
1 Nov 2021
Ragni E Viganò M Orfei CP Colombini A De Luca P Libonati F de Girolamo L
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Introduction and Objective

Hyaluronic acid (HA) is an effective option for the treatment of osteoarthritis (OA) patients due to several properties such as normalization of the mechanical and rheological properties of the synovial fluid and amelioration of OA symptoms and joints function by promoting cartilage nutrition. Since OA progression is also significantly related to oxidative stress and reactive oxygen species (ROS), sodium succinate (SS) is envisioned as a promising compound for cartilage treatment by providing antioxidant defense able to normalize intracellular metabolism and tissue respiration via mitochondrial mechanism of action. The scope of this study was to investigate on an in vitro inflammatory model the efficacy of Diart® product, a combination of HA and SS.

Materials and Methods

Donor-matched chondrocytes and synoviocytes were obtained from KL 3–4 OA patients undergoing total knee replacement. At passage 4, inflammation was promoted with 1 ng/ml IL-1B for 48 hours in absence and presence of Diart® at 1:3 dilution rate. Nitric oxide (NO) from cell culture supernatant was measured by Griess reaction. Mitochondrial and cytoplasmatic ROS evaluation was assessed by flow cytometry with MitoSox and dichlorodihydrofluorescein diacetate (DCFDA) assays. Gene expression of inflammation/oxidative stress-related transcripts (MMP1/MMP3/INOS/COX2) was evaluated by qRT-PCR using TBP as reference.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_16 | Pages 41 - 41
1 Nov 2018
Lugano G Orfei CP Talò G Perteghella S Viganò M Fontana FF Ragni E Moretti M de Girolamo L
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Among the innovative therapeutic techniques in orthopedics, a considerable interest arose around Mesenchymal Stem Cells (MSCs) - based therapies for one-step clinical applications. In order to achieve a better cell targeting at the injury site, these applications would need a specific cell delivery system. Hence, in this study a protocol for an efficient cell delivery based on the rapid cell adhesion on the surface of lyophilized fibroin-coated alginate microcarriers (L-FAMs) was optimized by the Design of Experiment (DoE) method in accordance with the minimum requirements for one-step clinical application. Specific parameters (seeding time, intermittent or not dynamic culture, stirring speed and volume of cell suspension) were combined in 13 different protocols, tested on human Adipose derived stem cells - ASCs (n=3). Cell adhesion rate in term of DNA quantification and metabolic activity of cells adhered on L-FAMs, and their qualitative observations by Calcein Staining were evaluated. The data showed that a suspension of 3.75 × 105 cells/ml and 10 mg/ml of FAMs, 12.3 rpm of stirring speed and 85.6 minutes of seeding time are the most performing combination of parameters. The final protocol was then tested and validated on both hASCs (n=3) and human bone marrow derived stem cells - BMSCs (n=3). The results confirmed a high adhesion rate of cells, homogenously arranged on the surface of L-FAMs without cell cluster formation. Even though further optimizations are still needed, the present protocol may represent the proof of concept for the introduction of L-FAMs as carriers in one-step intraoperative applications.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_15 | Pages 4 - 4
1 Nov 2018
Orfei CP Lovati A Lugano G Viganò M Bottagisio M D'Arrigo D Setti S de Girolamo L
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Tendon-related pathologies such as tendinopathy represent a relevant clinical and socioeconomic issue. The most innovative and conservative therapeutic approaches are meant to stimulate the intrinsic healing capability of the tissue. In this study, the use of pulsed electromagnetic fields (PEMFs) was investigated in a rat model of Achilles tendinopathy as a potential therapy. Achilles tendinopathy was chemically induced in eighty-six Sprague Dawley rats by injecting collagenase Type I within the tendon fibers. Fifty-six of them were stimulated with PEMFs (8 hours/day, 1.5 ± 0.2 mT; 75 Hz), divided in different experimental groups basing on the starting-time of PEMFs exposure (after 0, 7, 15 after Collagenase injection) and its duration (7, 15 or 30 days). Thirty animals were left unstimulated (CTRL group). According to the different time points, explanted tendons were evaluated through histological and immunohistochemical analyses in term of matrix deposition, fiber re-organization, neovascularization and inflammatory reaction. The most effective PEMF stimulation was demonstrated in the 15 days of treatment. However, when PEMF were applied immediately after the collagenase injection, no significant therapeutic results were found. On the contrary, when PEMF were applied after 7 and 15 days from the collagenase injection, they promoted the deposition of extracellular matrix and tendon fiber re-organization, reducing both the inflammatory reaction and vascularization, with significant differences compared to the CTRL group (p<0.05). Therefore, these results suggest an effective activity of PEMFs stimulation that provides a satisfying restoration of the damaged tissue, although the most performing protocol of application still needs to be identified.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_2 | Pages 17 - 17
1 Jan 2017
Viganò M Orfei CP Lovati A Stanco D Bottagisio M Di Giancamillo A Setti S de Girolamo L
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Tendinopathies represent the 45% of the musculoskeletal lesions and they are a big burden in clinics. Indeed, despite the relevant social impact, both the pathogenesis and the development of the tendinopathy are still under-investigated, thus limiting the therapeutic advancement in this field. Indeed, current treatment for tendinopathy are mainly symptomatic, and they present a high rate of pathology re-occurrence. In this contest, the development of an efficient in vivo model of acute tendinopathy, focused on the choice of the most appropriate species and strategy to induce the disease, would allow a better understanding of the pathology progression throughout its phases.

Then, the purpose of this study was to evaluate the dose-dependent and time-related tissue-level changes occurring in a collagenase-induced tendinopathy in rat Achilles tendons, in order to establish a standardized model for future pre-clinical studies.

40 Sprague Dawley rats were randomly divided into two groups, treated by injection of collagenase type I within the Achilles tendon at 1 mg/mL (low dose, LD) or 3 mg/mL (high dose, HD). Tendon explants were histologically evaluated at 3, 7, 15, 30 and 45 days by H&E staining.

Our results showed that both the collagenase doses induced a disorganization of collagen fibers and increased the number of rounded resident cells. In particular, the high dose treatment determined a greater fatty degeneration and neovascularization with respect to the lower dose. These changes are time-dependent, thus resembling the tendinopathy development in humans. Indeed, the acute phase occurred from day 3 to day 15, while from day 15 to 45 it progressed towards the proliferative phase, displaying a degenerative appearance associated with a precocious remodeling process.

The model represents a good balance between feasibility, in terms of reproducibility and costs, and similarity with the human disease. Moreover, the present model contributes to improve the knowledge about tendinopathy development, and then it could be useful to design further pre-clinical studies, in particular in order to test innovative treatments for tendinopathy.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 23 - 23
1 Jul 2014
Viganò M Stanco D Setti S Galliera E Sansone V de Girolamo L
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Summary

In an in vitro tendon cell model, the tendon-specific gene expression up-regulation induced by PEMF negatively correlates with field intensity; moreover repeated lower-intensity PEMF treatments (1.5 mT) provokes a higher release of anti-inflammatory cytokines respect to the single treatment.

Introduction

Tendon disorders represent a diagnostic and therapeutic challenge for physicians. Traditional treatments are characterised by a long recovery time and a high occurrence of injury relapses. Despite the growing clinical interest in pulsed electromagnetic fields (PEMFs) few studies on their effect on tendons and ligaments have been conducted. Tendon resident cells (TCs) are a mixed population, made up mostly by tenocytes and tendon stem/progenitor cells, which are responsible of the tissue homeostasis. Since studies on the effect of PEMFs on this cell population are conflicting, we evaluated the possible relation between PEMFs dosage and TCs’ response. In particular, we compared the in vitro effect of low and high PEMFs on TCs (PEMF-1.5 mT; PEMF-3 mT); moreover we assessed the results of repeated treatments (R-PEMF-1.5mT).


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 64 - 64
1 Jul 2014
Lopa S Colombini A Stanco D de Girolamo L Sansone V Moretti M
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Summary

The donor-matched comparison between mesenchymal stem cells from knee infrapatellar and subcutaneous adipose tissue revealed their preferential commitment towards the chondrogenic and osteogenic lineage, respectively. These peculiarities could be relevant for the development of successful bone and cartilage cell-based applications.

Introduction

Mesenchymal stem cells (MSCs) have been proposed in bone and cartilage tissue engineering applications as an alternative to terminally differentiated cells. In the present study we characterised and performed a donor-matched comparison between MSCs resident within the infrapatellar fat pad (IFP-MSCs) and the knee subcutaneous adipose tissue (ASCs) of osteoarthritic patients. These two fat depots, indeed, can be considered appealing candidates for orthopaedic cell-based therapies since they are highly accessible during knee surgery.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 261 - 261
1 Jul 2014
Stanco D Viganò M Thiebat G de Girolamo L
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Summary

Mesenchymal stem cells from human semitendinosus and gracilis tendons (TSPCs) could be a promising MSCs resource for tissue-engineering application. In comparison to adipose-derived stem cells, TSPCs possess similar stem-cells properties and a higher chondrogenic differentiation potential.

Introduction

Mesenchymal stem cells (MSCs) isolated from bone marrow (BMSCs) or adipose tissue (ASCs) have been deeply characterised for their usefulness in musculoskeletal tissue regeneration. However, other potentially valuable MSCs sources have been recently proposed. The goal of this study was to isolate MSCs from human semitendinosus and gracilis tendons (TSPCs, tendon stem progenitor cells) and to compare their features with that of human ASCs.