Publications by authors named "Gabriela Ciapetti"

44 Publications

Osteonecrosis of the Femoral Head Safely Healed with Autologous, Expanded, Bone Marrow-Derived Mesenchymal Stromal Cells in a Multicentric Trial with Minimum 5 Years Follow-Up.

J Clin Med 2021 Feb 1;10(3). Epub 2021 Feb 1.

INSERM U957, Lab. Pathophysiology of Bone Resorption, Faculty of Medicine, University of Nantes, 44035 Nantes, France.

: Osteonecrosis (ON) of the femoral head represents a potentially severe disease of the hip where the lack of bone regeneration may lead to femoral head collapse and secondary osteoarthritis, with serious pain and disability. The aim of this European, multicentric clinical trial was to prove safety and early efficacy to heal early femoral head ON in patients through minimally invasive surgical implantation of autologous mesenchymal stromal cells (MSC) expanded from bone marrow (BM) under good manufacturing practices (GMP). : Twenty-two patients with femoral head ON (up to ARCO 2C) were recruited and surgically treated in France, Germany, Italy and Spain with BM-derived, expanded autologous MSC (total dose 140 million MSC in 7 mL). The investigational advanced therapy medicinal product (ATMP) was expanded from BM under the same protocol in all four countries and approved by each National Competent Authority. Patients were followed during two years for safety, based on adverse events, and for efficacy, based on clinical assessment (pain and hip score) and imaging (X-rays and MRIs). Patients were also reviewed after 5 to 6 years at latest follow-up for final outcome. : No severe adverse event was recalled as related to the ATMP. At 12 months, 16/20 per protocol and 16/22 under intention-to-treat (2 drop-out at 3 and 5 months) maintained head sphericity and showed bone regeneration. Of the 4 hips with ON progression, 3 required total hip replacement (THR). At 5 years, one patient (healed at 2 years visit) was not located, and 16/21 showed no progression or THR, 4/21 had received THR (all in the first year) and 1 had progressed one stage without THR. : Expanded MSCs implantation was safe. Early efficacy was confirmed in 80% of cases under protocol at 2 years. At 5 years, the overall results were maintained and 19% converted to THR, all in the first year.
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http://dx.doi.org/10.3390/jcm10030508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867148PMC
February 2021

Collagen Hybrid Formulations for the 3D Printing of Nanostructured Bone Scaffolds: An Optimized Genipin-Crosslinking Strategy.

Nanomaterials (Basel) 2020 Aug 27;10(9). Epub 2020 Aug 27.

Department of Applied Science and Technology, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy.

Bone-tissue regeneration induced by biomimetic bioactive materials is the most promising approach alternative to the clinical ones used to treat bone loss caused by trauma or diseases such as osteoporosis. The goal is to design nanostructured bioactive constructs able to reproduce the physiological environment: By mimicking the natural features of bone tissue, the cell behavior during the regeneration process may be addressed. At present, 3D-printing technologies are the only techniques able to design complex structures avoiding constraints of final shape and porosity. However, this type of biofabrication requires complex optimization of biomaterial formulations in terms of specific rheological and mechanical properties while preserving high biocompatibility. In this work, we combined nano-sized mesoporous bioactive glasses enriched with strontium ions with type I collagen, to formulate a bioactive ink for 3D-printing technologies. Moreover, to avoid the premature release of strontium ions within the crosslinking medium and to significantly increase the material mechanical and thermal stability, we applied an optimized chemical treatment using ethanol-dissolved genipin solutions. The high biocompatibility of the hybrid system was confirmed by using MG-63 and Saos-2 osteoblast-like cell lines, further highlighting the great potential of the innovative nanocomposite for the design of bone-like scaffolds.
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http://dx.doi.org/10.3390/nano10091681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7558137PMC
August 2020

Analysis of multiple protein detection methods in human osteoporotic bone extracellular matrix: From literature to practice.

Bone 2020 08 13;137:115363. Epub 2020 Apr 13.

Department of Clinical and Molecular Sciences (DISCLIMO), Università Politecnica delle Marche, Via Tronto 10/A, 60126 Ancona, Italy.

The punctual analysis of bone Extracellular Matrix (ECM) proteins represents a pivotal point for medical research in bone diseases like osteoporosis. Studies in this field, historically done to appreciate bone biology, were mainly conducted on animal samples and, up to today, only a few studies on protein detection in human bone are present. The challenges in bone ECM protein extraction and quantitation protocols are related to both the separation of proteins from the mineral content (i.e. hydroxyapatite) and the difficulty of avoiding protein denaturation during the extraction processes. The aim of the present work was to define appropriate protocol(s) for bone ECM protein extraction that could be applied to investigate both normal and pathological conditions. We compared and optimised some of the most used protocols present in the literature, modifying the protein precipitation method, the buffer used for resuspension and/or the volume of reagent used. Bradford and BCA assays and Western Blotting were used to evaluate the variations in the total protein recovery and the amount of selected proteins (Type I Collagen, TGF-β, IGF-1, Decorin, Osteopontin, Bone Sialoprotein-2 and Osteocalcin). Collectively, we were capable to draw-up two single-extract protocols with optimal recovery and ideal protein content, that can be used for a detailed analysis of ECM proteins in pathological bone samples. Time-consuming multi-extract procedures, optimised in their precipitation methods, are however crucial for a precise detection of specific proteins, like osteocalcin. As the matter of fact, also the demineralization processes, commonly suggested and performed in several protocols, could hinder an accurate protein detection, thus inherently affecting the study of a pathological bone ECM. This study represents a starting point for the definition of appropriate strategies in the study of bone extracellular matrix proteins involved in the onset and maintenance of bone diseases, as well as a tool for the development of customized scaffolds capable to modulate a proper feedback loop in bone remodelling, altered in case of diseases like osteoporosis.
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http://dx.doi.org/10.1016/j.bone.2020.115363DOI Listing
August 2020

Co-culture systems of osteoblasts and osteoclasts: Simulating in vitro bone remodeling in regenerative approaches.

Acta Biomater 2020 05 3;108:22-45. Epub 2020 Apr 3.

Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, IRCCS Istituto Ortopedico Rizzoli, Via di Barbiano 1/10, 40136 Bologna, Italy.

Bone is an extremely dynamic tissue, undergoing continuous remodeling for its whole lifetime, but its regeneration or augmentation due to bone loss or defects are not always easy to obtain. Bone tissue engineering (BTE) is a promising approach, and its success often relies on a "smart" scaffold, as a support to host and guide bone formation through bone cell precursors. Bone homeostasis is maintained by osteoblasts (OBs) and osteoclasts (OCs) within the basic multicellular unit, in a consecutive cycle of resorption and formation. Therefore, a functional scaffold should allow the best possible OB/OC cooperation for bone remodeling, as happens within the bone extracellular matrix in the body. In the present work OB/OC co-culture models, with and without scaffolds, are reviewed. These experimental systems are intended for different targets, including bone remodeling simulation, drug testing and the assessment of biomaterials and 3D scaffolds for BTE. As a consequence, several parameters, such as cell type, cell ratio, culture medium and inducers, culture times and setpoints, assay methods, etc. vary greatly. This review identifies and systematically reports the in vitro methods explored up to now, which, as they allow cellular communication, more closely resemble bone remodeling and/or the regeneration process in the framework of BTE. STATEMENT OF SIGNIFICANCE: Bone is a dynamic tissue under continuous remodeling, but spontaneous healing may fail in the case of excessive bone loss which often requires valid alternatives to conventional treatments to restore bone integrity, like bone tissue engineering (BTE). Pre-clinical evaluation of scaffolds for BTE requires in vitro testing where co-cultures combining innovative materials with osteoblasts (OBs) and osteoclasts (OCs) closely mimic the in vivo repair process. This review considers the direct and indirect OB/OC co-cultures relevant to BTE, from the early mouse-cell models to the recent bone regenerative systems. The co-culture modeling of bone microenvironment provides reliable information on bone cell cross-talk. Starting from improved knowledge on bone remodeling, bone disease mechanisms may be understood and new BTE solutions are designed.
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http://dx.doi.org/10.1016/j.actbio.2020.03.043DOI Listing
May 2020

Early efficacy evaluation of mesenchymal stromal cells (MSC) combined to biomaterials to treat long bone non-unions.

Injury 2020 Apr 26;51 Suppl 1:S63-S73. Epub 2020 Feb 26.

INSERM U957, Lab. Pathophysiology of bone resorption, Faculty of Medicine, University of Nantes, Nantes, France.

Background And Study Aim: Advanced therapy medicinal products (ATMP) frequently lack of clinical data on efficacy to substantiate a future clinical use. This study aims to evaluate the efficacy to heal long bone delayed unions and non-unions, as secondary objective of the EudraCT 2011-005441-13 clinical trial, through clinical and radiological bone consolidation at 3, 6 and 12 months of follow-up, with subgroup analysis of affected bone, gender, tobacco use, and time since the original fracture.

Patients And Methods: Twenty-eight patients were recruited and surgically treated with autologous bone marrow derived mesenchymal stromal cells expanded under Good Manufacturing Practices, combined to bioceramics in the surgical room before implantation. Mean age was 39 ± 13 years, 57% were males, and mean Body Mass Index 27 ± 7. Thirteen (46%) were active smokers. There were 11 femoral, 4 humeral, and 13 tibial non-unions. Initial fracture occurred at a mean ± SD of 27.9 ± 31.2 months before recruitment. Efficacy results were expressed by clinical consolidation (no or mild pain if values under 30 in VAS scale), and by radiological consolidation with a REBORNE score over 11/16 points (value of or above 0.6875). Means were statistically compared and mixed models for repeated measurements estimated the mean and confidence intervals (95%) of the REBORNE Bone Healing scale. Clinical and radiological consolidation were analyzed in the subgroups with Spearman correlation tests (adjusted by Bonferroni).

Results: Clinical consolidation was earlier confirmed, while radiological consolidation at 3 months was 25.0% (7/28 cases), at 6 months 67.8% (19/28 cases), and at 12 months, 92.8% (26/28 cases including the drop-out extrapolation of two failures). Bone biopsies confirmed bone formation surrounding the bioceramic granules. All locations showed similar consolidation, although this was delayed in tibial non-unions. No significant gender difference was found in 12-month consolidation (95% confidence). Higher consolidation scale values were seen in non-smoking patients at 6 (p = 0.012, t-test) and 12 months (p = 0.011, t-test). Longer time elapsed after the initial fracture did not preclude the occurrence of consolidation.

Conclusion: Bone consolidation was efficaciously obtained with the studied expanded hBM-MSCs combined to biomaterials, by clinical and radiological evaluation, and confirmed by bone biopsies, with lower consolidation scores in smokers.
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http://dx.doi.org/10.1016/j.injury.2020.02.070DOI Listing
April 2020

Development and Biocompatibility of Collagen-Based Composites Enriched with Nanoparticles of Strontium Containing Mesoporous Glass.

Materials (Basel) 2019 Nov 11;12(22). Epub 2019 Nov 11.

Applied Science and Technology Department, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy.

In the last years bone tissue engineering has been increasingly indicated as a valid solution to meet the challenging requirements for a healthy bone regeneration in case of bone loss or fracture. In such a context, bioactive glasses have already proved their great potential in promoting the regeneration of new bone tissue due to their high bioactivity. In addition, their composition and structure enable us to incorporate and subsequently release therapeutic ions such as strontium, enhancing the osteogenic properties of the material. The incorporation of these inorganic systems in polymeric matrices enables the formulation of composite systems suitable for the design of bone scaffolds or delivery platforms. Among the natural polymers, type I collagen represents the main organic phase of bone and thus is a good candidate to develop biomimetic bioactive systems for bone tissue regeneration. However, alongside the specific composition and structure, the key factor in the design of new biosystems is creating a suitable interaction with cells and the host tissue. In this scenario, the presented study aimed at combining nano-sized mesoporous bioactive glasses produced by means of a sol-gel route with type I collagen in order to develop a bioactive hybrid formulation suitable for bone tissue engineering applications. The designed system has been fully characterized in terms of physico-chemical and morphological analyses and the ability to release Sr ions has been studied observing a more sustained profile in presence of the collagenous matrix. With the aim to improve the mechanical and thermal stability of the resulting hybrid system, a chemical crosslinking approach using 4-star poly (ethylene glycol) ether tetrasuccinimidyl glutarate (4-StarPEG) has been explored. The biocompatibility of both non-crosslinked and 4-StarPEG crosslinked systems was evaluated by in vitro tests with human osteoblast-like MG-63 cells. Collected results confirmed the high biocompatibility of composites, showing a good viability and adhesion of cells when cultured onto the biomaterial samples.
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http://dx.doi.org/10.3390/ma12223719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888293PMC
November 2019

Osteoporosis-related variations of trabecular bone properties of proximal human humeral heads at different scale lengths.

J Mech Behav Biomed Mater 2019 12 26;100:103373. Epub 2019 Jul 26.

Politecnico di Torino, Department of Applied Science and Technology, Corso Duca degli Abruzzi 24, 10129, Torino, Italy. Electronic address:

Osteoporosis (OP) is a skeletal disorder responsible for the weakening of the bone structure and, consequently, for an increased fracture risk in the elderly population. In the past, bone mineral density (BMD) variation was considered the best OP indicator, but recently the focus has shifted toward the variation of microstructural bone parameters. This work is based on the characterisation of 8-mm cylindrical biopsies harvested from proximal humeral heads belonging to healthy and osteoporotic patients, in order to assess the OP-related variations of bone properties at different scale lengths. In particular, bone biopsies underwent micro-computed tomography analysis to study the most relevant features of bone architecture and extrapolate the tissue mineral density (TMD) value of bone trabeculae. Compression tests and nanoindentations were performed to investigate the macro- and micromechanical properties of bone biopsies, respectively. In addition, XRD analyses were performed to obtain the mean hydroxyapatite (HA) crystallite size, while Raman spectroscopy investigated the collagen secondary structure. Thermogravimetric analysis was performed to evaluate the ratio between organic and inorganic phases. From the obtained results, OP samples showed a more anisotropic and less interconnected structure responsible for reduced compression strength. From this, it can be supposed that OP caused an alteration of bone structure that led to inferior macroscopic mechanical properties. Furthermore, OP samples possessed higher TMD and bigger HA crystals that are correlated to an increase of the hardness value obtained by means of nanoindentation. This less controlled HA crystal growth is probably due to an alteration of the organic matrix structure, as revealed by the increase of the random coil contribution in the Raman spectra of the OP bone. This higher crystal content led to an increase in trabecular density and hardness. In conclusion, the obtained data showed that OP affects bone properties at different scale lengths causing an alteration of its morphological, structural and mechanical features.
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http://dx.doi.org/10.1016/j.jmbbm.2019.103373DOI Listing
December 2019

Biomarkers of bone healing induced by a regenerative approach based on expanded bone marrow-derived mesenchymal stromal cells.

Cytotherapy 2019 08 1;21(8):870-885. Epub 2019 Jul 1.

SSD Fisiopatologia Ortopedica e Medicina Rigenerativa, IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy; Dipartimento di Scienze Biomediche e Neuromotorie, Università degli Studi di Bologna, Bologna, Italy.

Background: Safety and feasibility of a regenerative strategy based on the use of culture-expanded mesenchymal stromal cells (MSCs) have been investigated in phase 2 trials for the treatment of nonunion and osteonecrosis of the femoral head (ONFH). As part of the clinical study, we aimed to evaluate if bone turnover markers (BTMs) could be useful for predicting the regenerative ability of the cell therapy product.

Materials And Methods: The bone defects of 39 patients (nonunion: n = 26; ONFH: n = 13) were treated with bone marrow-derived MSCs, expanded using a clinical-grade protocol and combined with biphasic calcium phosphate before implantation. Bone formation markers, bone-resorption markers and osteoclast regulatory proteins were measured before treatment (baseline) and after 12 and 24 weeks from surgery. At the same time-points, clinical and radiological controls were performed to evaluate the bone-healing progression.

Results: We found that C-Propeptide of Type I Procollagen (CICP) and C-terminal telopeptide of type-I collagen (CTX) varied significantly, not only over time, but also according to clinical results. In patients with a good outcome, CICP increased and CTX decreased, and this trend was observed in both nonunion and ONFH. Moreover, collagen biomarkers were able to discriminate healed patients from non-responsive patients with a good diagnostic accuracy.

Discussion: CICP and CTX could be valuable biomarkers for monitoring and predicting the regenerative ability of cell products used to stimulate the repair of refractory bone diseases. To be translated in a clinical setting, these results are under validation in a currently ongoing phase 3 clinical trial.
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http://dx.doi.org/10.1016/j.jcyt.2019.06.002DOI Listing
August 2019

Effect of calcium phosphate heparinization on the in vitro inflammatory response and osteoclastogenesis of human blood precursor cells.

J Tissue Eng Regen Med 2019 07 31;13(7):1217-1229. Epub 2019 May 31.

Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Universitat Politècnica de Catalunya (UPC), Barcelona, Spain.

The immobilization of natural molecules on synthetic bone grafts stands as a strategy to enhance their biological interactions. During the early stages of healing, immune cells and osteoclasts (OC) modulate the inflammatory response and resorb the biomaterial, respectively. In this study, heparin, a naturally occurring molecule in the bone extracellular matrix, was covalently immobilized on biomimetic calcium-deficient hydroxyapatite (CDHA). The effect of heparin-functionalized CDHA on inflammation and osteoclastogenesis was investigated using primary human cells and compared with pristine CDHA and beta-tricalcium phosphate (β-TCP). Biomimetic substrates led to lower oxidative stresses by neutrophils and monocytes than sintered β-TCP, even though no further reduction was induced by the presence of heparin. In contrast, heparinized CDHA fostered osteoclastogenesis. Optical images of stained TRAP positive cells showed an earlier and higher presence of multinucleated cells, compatible with OC at 14 days, while pristine CDHA and β-TCP present OC at 21-28 days. Although no statistically significant differences were found in the OC activity, microscopy images evidenced early stages of degradation on heparinized CDHA, compatible with osteoclastic resorption. Overall, the results suggest that the functionalization with heparin fostered the formation and activity of OC, thus offering a promising strategy to integrate biomaterials in the bone remodelling cycle by increasing their OC-mediated resorption.
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http://dx.doi.org/10.1002/term.2872DOI Listing
July 2019

Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial.

Biomaterials 2019 03 19;196:100-108. Epub 2018 Mar 19.

INSERM U957, Lab. Pathophysiology of Bone Resorption, Faculty of Medicine, University of Nantes, Nantes, France.

Background: ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis.

Methods: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5-10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority.

Findings: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging).

Interpretation: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic.

Funding: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876).
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http://dx.doi.org/10.1016/j.biomaterials.2018.03.033DOI Listing
March 2019

A Multicentric, Open-Label, Randomized, Comparative Clinical Trial of Two Different Doses of Expanded hBM-MSCs Plus Biomaterial versus Iliac Crest Autograft, for Bone Healing in Nonunions after Long Bone Fractures: Study Protocol.

Stem Cells Int 2018 22;2018:6025918. Epub 2018 Feb 22.

Reparto di Ortopedia e Traumatologia, Azienda Ospedaliero-Universitaria di Modena, Largo del Pozzo 71, 41125 Modena, Italy.

ORTHOUNION is a multicentre, open, comparative, three-arm, randomized clinical trial (EudraCT number 2015-000431-32) to compare the efficacy, at one and two years, of autologous human bone marrow-derived expanded mesenchymal stromal cell (hBM-MSC) treatments versus iliac crest autograft (ICA) to enhance bone healing in patients with diaphyseal and/or metaphysodiaphyseal fracture (femur, tibia, and humerus) status of atrophic or oligotrophic nonunion (more than 9 months after the acute fracture, including recalcitrant cases after failed treatments). The primary objective is to determine if the treatment with hBM-MSCs combined with biomaterial is superior to ICA in obtaining bone healing. If confirmed, a secondary objective is set to determine if the dose of 100 × 10 hBM-MSCs is noninferior to that of 200 × 10 hBM-MSCs. The participants ( = 108) will be randomly assigned to either the experimental low dose ( = 36), the experimental high dose ( = 36), or the comparator arm ( = 36) using a central randomization service. The trial will be conducted in 20 clinical centres in Spain, France, Germany, and Italy under the same clinical protocol. The confirmation of superiority for the proposed ATMP in nonunions may foster the future of bone regenerative medicine in this indication. On the contrary, absence of superiority may underline its limitations in clinical use.
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http://dx.doi.org/10.1155/2018/6025918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842679PMC
February 2018

Biological effects of metal degradation in hip arthroplasties.

Crit Rev Toxicol 2018 02 13;48(2):170-193. Epub 2017 Nov 13.

a Orthopedic Pathophysiology and Regenerative Medicine Unit , Rizzoli Orthopedic Institute , Bologna , Italy.

Metals and metal alloys are the most used materials in orthopedic implants. The focus is on total hip arthroplasty (THA) that, though well tolerated, may be associated with local and remote adverse effects in the medium-long term. This review aims to summarize data on the biological consequences of the metal implant degradation that have been attributed predominantly to metal-on-metal (MoM) THA. Local responses to metals consist of a broad clinical spectrum ranging from small asymptomatic tissue lesions to severe destruction of bone and soft tissues, which are designated as metallosis, adverse reactions to metal debris (ARMD), aseptic lymphocytic vasculitis associated lesion (ALVAL), and pseudotumors. In addition, the dissemination of metal particles and ions throughout the body has been associated with systemic adverse effects, including organ toxicity, cancerogenesis, teratogenicity, and immunotoxicity. As proved by the multitude of studies in this field, metal degradation may increase safety issues associated with THA, especially with MoM hip systems. Data collection regarding local, systemic and long-term effects plays an essential role to better define any safety risks and to generate scientifically based recommendations.
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http://dx.doi.org/10.1080/10408444.2017.1392927DOI Listing
February 2018

Changes of Bone Turnover Markers in Long Bone Nonunions Treated with a Regenerative Approach.

Stem Cells Int 2017 20;2017:3674045. Epub 2017 Jun 20.

Orthopedic Pathophysiology and Regenerative Medicine Unit, Rizzoli Orthopedic Institute, Bologna, Italy.

In this clinical trial, we investigated if biochemical bone turnover markers (BTM) changed according to the progression of bone healing induced by autologous expanded MSC combined with a biphasic calcium phosphate in patients with delayed union or nonunion of long bone fractures. Bone formation markers, bone resorption markers, and osteoclast regulatory proteins were measured by enzymatic immunoassay before surgery and after 6, 12, and 24 weeks. A satisfactory bone healing was obtained in 23 out of 24 patients. Nine subjects reached a good consolidation already at 12 weeks, and they were considered as the "early consolidation" group. We found that bone-specific alkaline phosphatase (BAP), C-terminal propeptide of type I procollagen (PICP), and beta crosslaps collagen (CTX) changed after the regenerative treatment, BAP and CTX correlated to the imaging results collected at 12 and 24 weeks, and BAP variation along the healing course differed in patients who had an "early consolidation." A remarkable decrease in BAP and PICP was observed at all time points in a single patient who experienced a treatment failure, but the predictive value of BTM changes cannot be determined. Our findings suggest that BTM are promising tools for monitoring cell therapy efficacy in bone nonunions, but studies with larger patient numbers are required to confirm these preliminary results.
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http://dx.doi.org/10.1155/2017/3674045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506673PMC
June 2017

Focus Ion Beam/Scanning Electron Microscopy Characterization of Osteoclastic Resorption of Calcium Phosphate Substrates.

Tissue Eng Part C Methods 2017 02 3;23(2):118-124. Epub 2017 Feb 3.

1 Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Technical University of Catalonia (UPC) , Barcelona, Spain .

This article presents the application of dual focused ion beam/scanning electron microscopy (FIB-SEM) imaging for preclinical testing of calcium phosphates with osteoclast precursor cells and how this high-resolution imaging technique is able to reveal microstructural changes at a level of detail previously not possible. Calcium phosphate substrates, having similar compositions but different microstructures, were produced using low- and high-temperature processes (biomimetic calcium-deficient hydroxyapatite [CDHA] and stoichiometric sintered hydroxyapatite, respectively). Human osteoclast precursor cells were cultured for 21 days before evaluating their resorptive potential on varying microstructural features. Alternative to classical morphological evaluation of osteoclasts (OC), FIB-SEM was used to observe the subjacent microstructure by transversally sectioning cells and observing both the cells and the substrates. Resorption pits, indicating OC activity, were visible on the smoother surface of high-temperature sintered hydroxyapatite. FIB-SEM analysis revealed signs of acidic degradation on the grain surface under the cells, as well as intergranular dissolution. No resorption pits were evident on the surface of the rough CDHA substrates. However, whereas no degradation was detected by FIB sections in the material underlying some of the cells, early stages of OC-mediated acidic degradation were observed under cells with more spread morphology. Collectively, these results highlight the potential of FIB to evaluate the resorptive activity of OC, even in rough, irregular, or coarse surfaces where degradation pits are otherwise difficult to visualize.
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http://dx.doi.org/10.1089/ten.TEC.2016.0361DOI Listing
February 2017

Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates.

Acta Biomater 2017 03 8;50:102-113. Epub 2016 Dec 8.

Orthopaedic Pathophysiology and Regenerative Medicine Unit, Istituto Ortopedico Rizzoli, Bologna, Italy; Department of Biomedical and Neuromotor Sciences, University of Bologna, Italy. Electronic address:

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca) and phosphorus (P) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium.

Statement Of Significance: The novelty of the paper is the differentiation of human blood-derived osteoclast precursors, instead of mouse-derived macrophages as used in most studies, directly on biomimetic micro-nano structured HA-based surfaces, as triggered by osteoblast-produced factors (RANKL/OPG), and influenced by chemistry and topography of the substrate(s). Biomimetic HA-surfaces, like those obtained in calcium phosphate cements, are very different from the conventional calcium phosphate ceramics, both in terms of topography and ion exchange. The role of these factors in modulating precursors' differentiation and activity is analysed. The system is closely reproducing the physiological process of attachment of host cells and further maturation to osteoclasts toward resorption of the substrate, which occurs in vivo after filling bone defects with the calcium phosphate grafts.
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http://dx.doi.org/10.1016/j.actbio.2016.12.013DOI Listing
March 2017

Effects of hypoxia on osteogenic differentiation of mesenchymal stromal cells used as a cell therapy for avascular necrosis of the femoral head.

Cytotherapy 2016 09 12;18(9):1087-99. Epub 2016 Jul 12.

Orthopedic Pathophysiology and Regenerative Medicine Unit, Istituto Ortopedico Rizzoli, Bologna, Italy; Department of Biomedical and Neuromotor Sciences, University of Bologna, Italy.

Background Aims: Avascular necrosis of the femoral head (AVN) occurs as common result of various conditions or develops as a primary entity, with a high freqency in young adults. Because of its tendency toward osteoarthritis requiring total hip arthroplasty, alternative treatments are being advocated, including cell therapy with mesenchymal stromal cells (MSCs). Because osteonecrotic bone is a severely hypoxic tissue, with a 1-3% oxygen tension, the survival and function of multipotent cells is questionable.

Methods: In this study, the proliferative, immunophenotypic and osteogenic properties of bone marrow (BM)-derived MSCs from a clinical series of patients with AVN were evaluated under in vitro conditions mimicking the hypoxic milieu of AVN to verify the rationale for cell therapy. MSCs retrieved from the iliac crest (BM-MSC) were isolated, expanded and induced to osteogenic differentiation under a 2% pO2 atmosphere (hypoxia) in comparison with the standard 21% pO2 (normoxia) that is routinely used in cell culture assays.

Results: Both proliferation and colony-forming ability were significantly enhanced in hypoxia-exposed BM-MSCs compared with BM-MSCs under normoxia. The expression of bone-related genes, including alkaline phosphatase, Type I collagen, and osteocalcin was significantly increased under hypoxia. Moreover, mineral deposition after osteogenic induction was not hampered, but in some cases even enhanced under low oxygen tension.

Conclusions: These findings support autologous cell therapy as an effective treatment to stimulate bone healing in the hypoxic microenvironment of AVN.
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http://dx.doi.org/10.1016/j.jcyt.2016.06.005DOI Listing
September 2016

Hyaluronan-based pericellular matrix: substrate electrostatic charges and early cell adhesion events.

Eur Cell Mater 2013 Sep 20;26:133-49; discussion 149. Epub 2013 Sep 20.

SSD Laboratory of Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, via di Barbiano 1/10, 40136 Bologna,

Cells are surrounded by a hyaluronan-rich coat called 'pericellular matrix' (PCM), mainly constituted by hyaluronan, a long-chain linear polysaccharide which is secreted and resorbed by the cell, depending on its activity. Cell attachment to a surface is mediated by PCM before integrins and focal adhesions are involved. As hyaluronan is known to bear a negative charge at physiological pH, the relevance of its electrical properties in driving the early cell adhesion steps has been studied, exploring how PCM mediates cell adhesion to charged surfaces, such as polyelectrolyte multilayer (PEM) films. Poly(ethylene imine) (PEI) and poly(sodium 4-styrene sulphonate) (PSS), assembled as PEI/PSS and PEI/PSS/PEI layers, were used. The nanoscale morphology of such layers was analysed by atomic force microscopy, and the detailed surface structure was analysed by X-ray photoemission spectroscopy. PCM-coated and PCM-depleted MG63 osteoblast-like cells were used, and cell density, morphology and adhesive structures were analysed during early steps of cell attachment to the PEM surfaces (1-6 h). The present study demonstrates that the pericellular matrix is involved in cell adhesion to material surfaces, and its arrangement depends on the cell interaction with the surface. Moreover, the PCM/surface interaction is not simply driven by electrostatic effects, as the cell response may be affected by specific chemical groups at the material surface. In the development of biomimetic surfaces promoting cell adhesion and function, the role of this unrecognised outer cell structure has to be taken into account.
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http://dx.doi.org/10.22203/ecm.v026a10DOI Listing
September 2013

Enhancing osteoconduction of PLLA-based nanocomposite scaffolds for bone regeneration using different biomimetic signals to MSCs.

Int J Mol Sci 2012 22;13(2):2439-58. Epub 2012 Feb 22.

Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto OrtopedicoRizzoli, Bologna 40136, Italy; E-Mails: (D.G.); (V.D.); (S.R.B.); (E.L.); (N.B.).

In bone engineering, the adhesion, proliferation and differentiation of mesenchymal stromal cells rely on signaling from chemico-physical structure of the substrate, therefore prompting the design of mimetic "extracellular matrix"-like scaffolds. In this study, three-dimensional porous poly-L-lactic acid (PLLA)-based scaffolds have been mixed with different components, including single walled carbon nanotubes (CNT), micro-hydroxyapatite particles (HA), and BMP2, and treated with plasma (PT), to obtain four different nanocomposites: PLLA + CNT, PLLA + CNTHA, PLLA + CNT + HA + BMP2 and PLLA + CNT + HA + PT. Adult bone marrow mesenchymal stromal cells (MSCs) were derived from the femur of orthopaedic patients, seeded on the scaffolds and cultured under osteogenic induction up to differentiation and mineralization. The release of specific metabolites and temporal gene expression profiles of marrow-derived osteoprogenitors were analyzed at definite time points, relevant to in vitro culture as well as in vivo differentiation. As a result, the role of the different biomimetic components added to the PLLA matrix was deciphered, with BMP2-added scaffolds showing the highest biomimetic activity on cells differentiating to mature osteoblasts. The modification of a polymeric scaffold with reinforcing components which also work as biomimetic cues for cells can effectively direct osteoprogenitor cells differentiation, so as to shorten the time required for mineralization.
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http://dx.doi.org/10.3390/ijms13022439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292032PMC
August 2015

Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs.

Eur Spine J 2012 May 7;21 Suppl 1:S10-9. Epub 2012 Mar 7.

Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, via di Barbiano 1/10, 40136 Bologna, Italy.

Purpose: Disc degeneration, and associated low back pain, are a primary cause of disability. Disc degeneration is characterized by dysfunctional cells and loss of proteoglycans: since intervertebral tissue has a limited capacity to regenerate, this process is at present considered irreversible. Recently, cell therapy has been suggested to provide more successful treatment of IVD degeneration. To understand the potential of cells to restore IVD structure/function, tissue samples from degenerated IVD versus healthy discs have been compared.

Methods: Discal tissue from 27 patients (40.17 ± 11 years) undergoing surgery for degenerative disc disease (DDD), DDD + herniation and congenital scoliosis, as controls, was investigated. Cells and matrix in the nucleus pulposus (NP) and annulus fibrosus (AF) were characterized by histology. AF- and NP-derived cells were isolated, expanded and characterized for senescence and gene expression. Three-dimensional NP pellets were cultured and stained for glycosaminoglycan formation.

Results: Phenotypical markers of degeneration, such as cell clusters, chondrons, and collagen disorganization were seen in the degenerate samples. In severe degeneration, granulation tissue and peripheral vascularization were observed. No correlation was found between the Pfirrmann clinical score and the extent of degeneration.

Conclusion: The tissue disorganization in degenerate discs and the paucity of cells out of cluster/chondron association, make the IVD-derived cells an unreliable option for disc regeneration.
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http://dx.doi.org/10.1007/s00586-012-2234-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325381PMC
May 2012

The combined use of mesenchymal stromal cells and scaffolds for bone repair.

Curr Pharm Des 2012 ;18(13):1796-820

Istituto Ortopedico Rizzoli, Bologna, Italy.

A general principle of stem cell therapy is to exploit the natural ability of the human body to heal through the process of regeneration. Here, we review the current status of cell therapy based on adult mesenchymal stem cells (MSC) with emphasis on therapeutic application in bone-related diseases. The main issues for an effective bone engineering strategy include: - A sufficient number of bone-forming cells, where cell yield, separation, expansion, commitment, as well as patient age, are all variables to be considered; - An ECM-like scaffold conductive for and informative to cells, where structural/physico-chemical/mechanical parameters, administration form (injectable or free-form), and degradation rate have to be tuned according to the clinical application; - Biochemical signals, such as growth factors/cytokines to induce osteogenic differentiation, where the choice between autogenous or exogenous sourcing, dose, timing, etc. are critical; - An adequate blood supply, provided by angiogenetic factors, pre-vascularization, pre-implant co-culture of vessel and bone progenitors. We also discuss the safety and efficacy of different approaches, as well as bottlenecks hampering rapid translation of adult MSC therapy from the laboratories to the clinics. A central paradigm for the effective regeneration of bone tissue is the re-creation at the site of injury of a microenvironment as close as possible to the natural MSC repository in the body. This would allow adult MSC to serve as cellular factories, i.e. to express paracrine activity in situ by secretion of inflammatory and reparative cytokines and to cooperate with other cells. The results from a wide array of in vitro and in vivo studies, as well as from some clinical trials, are expanding the range of clinical protocols for bone repair, that is the ultimate goal of orthopaedics.
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http://dx.doi.org/10.2174/138161212799859648DOI Listing
July 2012

Effects of osteogenic differentiation inducers on in vitro expanded adult mesenchymal stromal cells.

Int J Artif Organs 2011 Oct;34(10):998-1011

Unit of Orthopedic Pathophysiology and Regenerative Medicine, Rizzoli Orthopedic Institute, Bologna, Italy.

Purpose: For bone regeneration therapy using stem cells, well-defined ex vivo protocols to expand mesenchymal stromal cells (MSC), as well as assays to show their potential differentiation into the osteogenic lineage, are needed. Aim of this study was to analyze the role of the biochemical osteogenic inducers, i.e. ascorbic acid, dexamethasone, and ß-glycerophosphate, employed in the current protocols for osteogenic differentiation of MSC in vitro, to address the requirements for reliable differentiation systems.

Methods: MSC were isolated from the bone marrow of donors (46-73 years of age) undergoing total hip replacement, and expanded in vitro. At confluence, MSC were cultured under four different conditions: α-MEM plus serum (basal medium or C1), basal medium plus ascorbate (C2), basal medium plus ascorbate and dexamethasone (C3), or basal medium plus ascorbate, dexamethasone and ß-glycerophosphate (C4). Morphology, proliferation, mineralization, alkaline phosphatase, collagen and expression of bone-related genes of MSC under the different media were analyzed at fixed time points.

Results: MSC proliferation and the number of colony forming units were increased by ascorbic acid, whereas dexamethasone enhanced the proportion of ALP-positive CFU and was critical for mineral deposition. Runx-2 and type I collagen gene expression decreased along with additive-induced MSC differentiation, i.e. from C1 to C4, while ALP and osteocalcin were differently regulated.

Conclusion: Our findings support the role of different inducers on the sequential stages of MSC expansion and osteogenic differentiation in vitro, suggesting the addition of DEX following proliferation to ensure mineralization, as an index of in vivo osteogenic potency of human mesenchymal cells.
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http://dx.doi.org/10.5301/ijao.5000001DOI Listing
October 2011

Development of the foremost light-curable calcium-silicate MTA cement as root-end in oral surgery. Chemical-physical properties, bioactivity and biological behavior.

Dent Mater 2011 Jul 6;27(7):e134-57. Epub 2011 May 6.

Laboratory of Biomaterials and Oral Pathology, Department of Odontostomatological Sciences, University of Bologna, Via San Vitale 59, 40125 Bologna, Italy.

Aim: An innovative light-curable calcium-silicate cement containing a HEMA-TEGDMA-based resin (lc-MTA) was designed to obtain a bioactive fast setting root-end filling and root repair material.

Methods: lc-MTA was tested for setting time, solubility, water absorption, calcium release, alkalinizing activity (pH of soaking water), bioactivity (apatite-forming ability) and cell growth-proliferation. The apatite-forming ability was investigated by micro-Raman, ATR-FTIR and ESEM/EDX after immersion at 37°C for 1-28 days in DPBS or DMEM+FBS. The marginal adaptation of cement in root-end cavities of extracted teeth was assessed by ESEM/EDX, and the viability of Saos-2 cell on cements was evaluated.

Results: lc-MTA demonstrated a rapid setting time (2min), low solubility, high calcium release (150-200ppm) and alkalinizing power (pH 10-12). lc-MTA proved the formation of bone-like apatite spherulites just after 1 day. Apatite precipitates completely filled the interface porosities and created a perfect marginal adaptation. lc-MTA allowed Saos-2 cell viability and growth and no compromising toxicity was exerted.

Significance: HEMA-TEGDMA creates a polymeric network able to stabilize the outer surface of the cement and a hydrophilic matrix permeable enough to allow water absorption. SiO(-)/Si-OH groups from the mineral particles induce heterogeneous nucleation of apatite by sorption of calcium and phosphate ions. Oxygen-containing groups from poly-HEMA-TEGDMA provide additional apatite nucleating sites through the formation of calcium chelates. The strong novelty was that the combination of a hydraulic calcium-silicate powder and a poly-HEMA-TEGDMA hydrophilic resin creates the conditions (calcium release and functional groups able to chelate Ca ions) for a bioactive fast setting light-curable material for clinical applications in dental and maxillofacial surgery. The first and unique/exclusive light-curable calcium-silicate MTA cement for endodontics and root-end application was created, with a potential strong impact on surgical procedures.
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http://dx.doi.org/10.1016/j.dental.2011.03.011DOI Listing
July 2011

Biomimetic calcium-silicate cements aged in simulated body solutions. Osteoblast response and analyses of apatite coating.

J Appl Biomater Biomech 2009 Sep-Dec;7(3):160-70

Department of Odontostomatological Sciences, Endodontic Clinical Section, Laboratory of Endodontic Materials - Alma Mater Studiorum, University of Bologna and Department of Earth Sciences - Alma Mater Studiorum, University of Bologna, Bologna - Italy.

Purpose: Calcium-silicate cements have been recently proposed for application in dentistry as root-end filling and root-perforation repair materials. The aim of this study was to investigate the effect of ageing of experimental calcium-silicate cements on the chemistry, morphology and in vitro bioactivity of the surface, as well as on osteoblast viability and proliferation.

Methods: Two experimental cements (wTC-Bi, containing bismuth oxide and wTC), mainly based on dicalcium-silicate and tricalcium-silicate, were prepared and tested for their bioactivity after soaking in Dulbecco's phosphate buffered saline (DPBS), used as simulated body fluid. Human marrow stromal cells (HMSC) were seeded on the cements maintained in DPBS for 5 hr (non-aged group), 14 and 28 days (aged group). Cell viability was assessed by the Alamar blueTM test and morphology by scanning electron microscopy (SEM) at different time endpoints. The surface of the soaked cements was analyzed by environmental scanning electron microscopy or SEM coupled with energy dispersive X-ray microanalysis (ESEM/EDX or SEM/EDX respectively) and the micro-Raman technique.

Results: The ESEM/EDX results showed a uniform surface composed of CSH hydrogel (mainly derived from the hydration of belite and alite) on both non-aged cements. Micro-Raman spectroscopy revealed the presence of calcium carbonate, anhydrite, ettringite, alite and belite. The SEM/EDX data showed an irregular calcium-phosphate multi-layered biocoating with many sharp and protruding crystals on both the aged cements. Micro-Raman spectroscopy revealed crystalline apatite and calcite. The osteoblast response results showed that both the experimental cements exerted no acute toxicity in the cell assay systems. The non-aged samples promoted greater cell growth. SEM showed cells well spread and adherent to the non-aged materials. A reduced number of attached cells was noticed on the aged cements. Bismuth oxide-containing cement allowed a reduced cell viability suggesting some cytotoxic effects. However, the thick biocoating formed on the 28-day aged samples lowered the deleterious effect of bismuth oxide on cell growth. Actually, micro-Raman spectroscopy revealed progressive bismuth oxide depletion on the wTC-Bi surface, due to the increased thickness of the apatite deposit.

Conclusions: The study demonstrated that (1) these materials support osteogenic cells growth and may induce early bone formation, (2) the ageing in DPBS reduced the growth of HMSC, but eliminated the deleterious effect of the bismuth oxide on cell growth. In conclusion, the experimental cements have adequate biological properties to be used as root-end/root repair filling materials or pulp capping materials.
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October 2012

Apatite formation on bioactive calcium-silicate cements for dentistry affects surface topography and human marrow stromal cells proliferation.

Dent Mater 2010 Oct 23;26(10):974-92. Epub 2010 Jul 23.

Laboratory of Biomaterials and Oral Pathology, Dept. of Odontostomatological Sciences, University of Bologna, Via S. Vitale 59, Bologna, Italy.

The effect of ageing in phosphate-containing solution of bioactive calcium-silicate cements on the chemistry, morphology and topography of the surface, as well as on in vitro human marrow stromal cells viability and proliferation was investigated. A calcium-silicate cement (wTC) mainly based on dicalcium-silicate and tricalcium-silicate was prepared. Alpha-TCP was added to wTC to obtain wTC-TCP. Bismuth oxide was inserted in wTC to prepare a radiopaque cement (wTC-Bi). A commercial calcium-silicate cement (ProRoot MTA) was tested as control. Cement disks were aged in DPBS for 5 h ('fresh samples'), 14 and 28 days, and analyzed by ESEM/EDX, SEM/EDX, ATR-FTIR, micro-Raman techniques and scanning white-light interferometry. Proliferation, LDH release, ALP activity and collagen production of human marrow stromal cells (MSC) seeded for 1-28 days on the cements were evaluated. Fresh samples exposed a surface mainly composed of calcium-silicate hydrates CSH (from the hydration of belite and alite), calcium hydroxide, calcium carbonate, and ettringite. Apatite nano-spherulites rapidly precipitated on cement surfaces within 5 h. On wTC-TCP the Ca-P deposits appeared thicker than on the other cements. Aged cements showed an irregular porous calcium-phosphate (Ca-P) coating, formed by aggregated apatite spherulites with interspersed calcite crystals. All the experimental cements exerted no acute toxicity in the cell assay system and allowed cell growth. Using biochemical results, the scores were: fresh cements>aged cements for cell proliferation and ALP activity (except for wTC-Bi), whereas fresh cements
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http://dx.doi.org/10.1016/j.dental.2010.06.002DOI Listing
October 2010

Resorbable glass-ceramic phosphate-based scaffolds for bone tissue engineering: synthesis, properties, and in vitro effects on human marrow stromal cells.

J Biomater Appl 2011 Nov 21;26(4):465-89. Epub 2010 Jun 21.

Department of Materials Science and Chemical Engineering, Politecnico di Torino, Corso Duca degli Abruzzi 24, Torino 10129, Italy.

Highly porous bioresorbable glass-ceramic scaffolds were prepared via sponge replication method by using an open-cell polyurethane foam as a template and phosphate-based glass powders. The glass, belonging to the P2O5-SiO2-CaO-MgO-Na2O-K2O system, was synthesized by a melting-quenching route, ground, and sieved to obtain powders with a grain size of less than 30 μm. A slurry containing glass powders, polyvinyl alcohol, and water was prepared to coat the polymeric template. The removal of the polymer and the sintering of the glass powders were performed by a thermal treatment, in order to obtain an inorganic replica of the template structure. The structure and properties of the scaffold were investigated from structural, morphological, and mechanical viewpoints by means of X-ray diffraction, scanning electron microscopy, density measurements, image analysis, and compressive tests. The scaffolds exhibited a trabecular architecture that closely mimics the structure of a natural spongy bone. The solubility of the porous structures was assessed by soaking the samples in acellular simulated body fluid (SBF) and Tris-HCl for different time frames and then by assessing the scaffold weight loss. As far as the test in SBF is concerned, the nucleation of hydroxyapatite on the scaffold trabeculae demonstrates the bioactivity of the material. Biological tests were carried out using human bone marrow stromal cells to test the osteoconductivity of the material. The cells adhered to the scaffold struts and were metabolically active; it was found that cell differentiation over proliferation occurred. Therefore, the produced scaffolds, being biocompatible, bioactive, resorbable, and structurally similar to a spongy bone, can be proposed as interesting candidates for bone grafting.
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http://dx.doi.org/10.1177/0885328210372149DOI Listing
November 2011

Osteogenic properties of late adherent subpopulations of human bone marrow stromal cells.

Histochem Cell Biol 2009 Nov 27;132(5):547-57. Epub 2009 Aug 27.

Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Via di Barbiano 1/10, 40136, Bologna, Italy,

The nonadherent (NA) population of bone-marrow-derived mononuclear cells (MNC) has been demonstrated to be a source of osteogenic precursors in addition to the plastic-adherent mesenchymal stromal cells (MSC). In the current study, two subpopulations of late adherent (LA) osteoprogenitors were obtained by subsequent replating of NA cells, and their phenotypic, functional, and molecular properties were compared with those of early adherent (EA) MSC. Approximately 35% of MNC were LA cells, and they acquired a homogeneous expression of MSC antigens later than EA cells. In EA-MSC, the alkaline phosphatase (ALP) activity increased significantly from time of seeding to the first confluence, whereas in LA cells it raised later, after the addition of mineralization medium. All subpopulations were able to produce type I collagen and to deposit extracellular matrix with organized collagen fibrils. The proportion of large colonies with more than 50% of ALP positive cells as well as the calcium content was higher in LA than in EA cells. Molecular analysis highlighted the upregulation of bone-related genes in LA-MSC, especially after the addition of mineralization medium. Our results confirm that bone marrow contains LA osteoprogenitors which exhibit a delay in the differentiation process, despite an osteogenic potential similar to or better than EA-MSC. LA cells represent a reservoir of osteoprogenitors to be recruited to gain an adequate bone tissue repair and regeneration when a depletion of the most differentiated component occurs. Bone tissue engineering and cell therapy strategies could take advantage of LA cells, since an adequate amount of osteogenic MSCs may be obtained while avoiding bone marrow manipulation and cell culture expansion.
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http://dx.doi.org/10.1007/s00418-009-0633-xDOI Listing
November 2009

Gene expression patterns related to osteogenic differentiation of bone marrow-derived mesenchymal stem cells during ex vivo expansion.

Tissue Eng Part C Methods 2010 Jun;16(3):511-24

Rizzoli Orthopaedic Institute, Bologna, Italy.

Bone marrow is commonly used as a source of adult multipotent mesenchymal stem cells (MSCs), defined for their ability to differentiate in vitro into multiple lineages. The ex vivo-expanded MSCs are currently being evaluated as a strategy for the restoration of function in damaged skeletal tissue, both in cell therapy and tissue engineering applications. The aim of this study was to define gene expression patterns underlying the differentiation of MSCs into mature osteoblasts during the expansion in vitro, and to explore a variety of cell functions that cannot be easily evaluated using morphological, cytochemical, and biochemical assays. Cell cultures were obtained from bone marrow samples of six individuals undergoing total hip replacement, and a large-scale transcriptome analysis, using Affymetrix HG-U133A Plus 2.0 array (Affymetrix((R)), Santa Clara, CA), was performed at the occurrence of specific events, including the appearance of MSC surface markers, formation of colonies, and deposition of mineral nodules. We focused our attention on 213 differentially upregulated genes, some belonging to well-known pathways and some having one or more Gene Ontology annotations related to bone cell biology, including angiogenesis, bone-related genes, cell communication, development and morphogenesis, transforming growth factor-beta signaling, and Wnt signaling. Twenty-nine genes, whose role in bone cell pathophysiology has not been described yet, were found. In conclusion, gene expression patterns that characterize the early, intermediate, and late phases of the osteogenic differentiation process of ex vivo-expanded MSCs were defined. These signatures represent a useful tool to monitor the osteogenic process, and to analyze a broad spectrum of functions of MSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration.
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http://dx.doi.org/10.1089/ten.TEC.2009.0405DOI Listing
June 2010

The influence of hydroxyapatite particles on in vitro degradation behavior of poly epsilon-caprolactone-based composite scaffolds.

Tissue Eng Part A 2009 Nov;15(11):3655-68

Institute of Composite and Biomedical Materials, National Research Council, Naples, Italy.

The design of composite scaffolds with slow degradation kinetics imposes the assessment of the time-course of degradation to predict the long-term in vitro behavior. In this work, the effect of hydroxyapatite (HA) particles on the hydrolytic degradation of poly epsilon-caprolactone composite scaffold was investigated. The study of accelerated degradation mechanisms in alkaline medium enabled analysing comparable degradation profiles at different times. The accurate qualitative and quantitative study of morphology by scanning electron microscopy supported by image analysis demonstrated only a negligible effect on the structural porosity, to be ascribed to the addition of micrometric HA as a filler. Moreover, by comparing the Raman spectra with thermal analysis(thermogravimetry and differential scanning calorimetry) the role of HA on the composite degradation mechanism was defined, by separately quantifying the contribution of HA particles in the bulk and on the surface, on the bone formation as a function of modifications induced in the pore morphology, as well as physical and chemical properties of the polymer matrix. Indeed, HA particles alter the poly epsilon-caprolactone crystallinity inducing a "shielding" effect of the polymer matrix. Meanwhile, the slight reduction of pore size as a function of the increasing HA content and the improvement of the effective hydrophilicity of the scaffolds also influence the degradation by faster mechanisms. Finally, it has been proven that the presence of HA enhances the scaffold bioactivity and human osteoblast cell response, remarking the active role of bioactive signals on the promotion of the surface mineralization and, as a consequence, on the cell-material interaction.
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http://dx.doi.org/10.1089/ten.TEA.2008.0543DOI Listing
November 2009

Is wear debris responsible for failure in alumina-on-alumina implants?

Acta Orthop 2009 Apr;80(2):162-7

Laboratory for Pathophysiology of Orthopaedic Implant, Bologna, Italy.

Background And Purpose: Ceramic-on-ceramic articulation is an attractive alternative to metal-on-polyethylene (PE) bearings, but little is known about the in vivo effects induced by dissemination of alumina wear debris in the periprosthetic tissues. We hypothesized that wear debris is not the main factor responsible for loosening and failure of the implant but that mechanical problems caused by incorrect surgical technique, prosthetic design, or trauma, may cause instability of the implants and result in production of wear debris.

Patients And Methods: Clinical, radiographic, laboratory, and microbiological data from 30 consecutive patients with failed alumina-on-alumina arthroplasties, 19 with screwed socket and 11 with press-fit socket, were systematically collected and evaluated. Retrieved peri-implant tissues and prosthesis wear were also analyzed.

Results And Interpretation: Loosening was due to malpositioning, primary mechanical instability, trauma, or infection. Bone stock was generally preserved, even if screwed implants showed higher levels of osteolysis. Variable implant wear and tissue macrophage reaction were present but activation of giant cells/osteoclasts was not induced, and no correlation between histocytic reaction and the level of osteolysis was found. These findings indicate that, in contrast to the situation with metal-on-PE bearings, wear debris and occasional osteolysis were the effect rather than the cause of failure of ceramic-on-ceramic implants, and that press-fit socket fixation was the socket fixation design of preference.
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http://dx.doi.org/10.3109/17453670902876730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2823169PMC
April 2009

Endothelial cells incubated with platelet-rich plasma express PDGF-B and ICAM-1 and induce bone marrow stromal cell migration.

J Orthop Res 2009 Nov;27(11):1493-8

Laboratory for Pathophysiology of Orthopaedic Implants, Istituto Ortopedico Rizzoli, via di Barbiano 1/10, 40136 Bologna, Italy.

Platelet-rich plasma (PRP) is used to accelerate bone repair through the growth factors released by platelets. The purpose of this study was to evaluate if PRP induce human umbilical vein endothelial cells (HUVEC) to express mRNA for osteogenic growth factors and stimulate the migration of bone marrow stromal cell (BMSC). The effects of PRP were compared to those induced by vascular endothelial growth factor-A (VEGF-A) or, as a negative control, by platelet poor plasma (PPP). After incubation with PRP, but not with PPP, HUVEC showed an increased expression of mRNA for platelet derived growth factor-B (PDGF-B), and this effect was not inhibited by an anti-VEGF-A antibody. The migration of BMSC was more stimulated by HUVEC incubated with PRP than by HUVEC incubated with low serum medium or PPP. Besides, PRP increased the expression of intercellular adhesion molecule-1 (ICAM-1) and osteoprotegerin, but did not affect the expression either of the receptor activator for nuclear factor kappaB ligand (RANKL) or of RANK. These findings support the hypothesis that PRP contribute to bone repair by favoring the pro-osteogenic function of endothelial cells, including the recruitment of osteoblast precursors and the expression of adhesion molecules for monocyte/macrophages, while inhibiting their pro-osteolytic properties.
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http://dx.doi.org/10.1002/jor.20896DOI Listing
November 2009