{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T22:35:12Z","timestamp":1765233312221,"version":"build-2065373602"},"reference-count":43,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2022,5,26]],"date-time":"2022-05-26T00:00:00Z","timestamp":1653523200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Ministry of Health\u2014Ricerca Corrente"},{"name":"5 \u00d7 1000 Anno 2018 project"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Background: Bisphosphonates are widely employed drugs for the treatment of pathologies with high bone resorption, such as osteoporosis, and display a great affinity for calcium ions and apatitic substrates. Here, we aimed to investigate the potentiality of zoledronate functionalized hydroxyapatite nanocrystals (HAZOL) to promote bone regeneration by stimulating adhesion, viability, metabolic activity and osteogenic commitment of human bone marrow derived mesenchymal stromal cells (hMSCs). Methods: we adopted an advanced three-dimensional (3D) in vitro fracture healing model to study porous scaffolds: hMSCs were seeded onto the scaffolds that, after three days, were cut in halves and unseeded scaffolds were placed between the two halves. Scaffold characterization by X-ray diffraction, transmission and scanning electron microscopy analyses and cell morphology, viability, osteogenic differentiation and extracellular matrix deposition were evaluated after 3, 7 and 10 days of culture. Results: Electron microscopy showed a porous and interconnected structure and a uniform cell layer spread onto scaffolds. Scaffolds were able to support cell growth and cells progressively colonized the whole inserts in absence of cytotoxic effects. Osteogenic commitment and gene expression of hMSCs were enhanced with higher expressions of ALPL, COL1A1, BGLAP, RUNX2 and Osterix genes. Conclusion: Although some limitations affect the present study (e.g., the lack of longer experimental times, of mechanical stimulus or pathological microenvironment), the obtained results with the adopted experimental setup suggested that zoledronate functionalized scaffolds (GHAZOL) might sustain not only cell proliferation, but positively influence osteogenic differentiation and activity if employed in bone fracture healing.<\/jats:p>","DOI":"10.3390\/ijms23115988","type":"journal-article","created":{"date-parts":[[2022,5,27]],"date-time":"2022-05-27T07:05:07Z","timestamp":1653635107000},"page":"5988","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Antiosteoporotic Nanohydroxyapatite Zoledronate Scaffold Seeded with Bone Marrow Mesenchymal Stromal Cells for Bone Regeneration: A 3D In Vitro Model"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6241-3669","authenticated-orcid":false,"given":"Matilde","family":"Tschon","sequence":"first","affiliation":[{"name":"Complex Structure Surgical Sciences and Technologies, IRCCS Istituto Ortopedico Rizzoli, Via di Barbiano 1\/10, 40136 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3754-0273","authenticated-orcid":false,"given":"Elisa","family":"Boanini","sequence":"additional","affiliation":[{"name":"Department of Chemistry \u2018\u2018Giacomo Ciamician\u201d, University of Bologna, Via Selmi 2, 40126 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2123-9032","authenticated-orcid":false,"given":"Maria","family":"Sartori","sequence":"additional","affiliation":[{"name":"Complex Structure Surgical Sciences and Technologies, IRCCS Istituto Ortopedico Rizzoli, Via di Barbiano 1\/10, 40136 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7402-4054","authenticated-orcid":false,"given":"Francesca","family":"Salamanna","sequence":"additional","affiliation":[{"name":"Complex Structure Surgical Sciences and Technologies, IRCCS Istituto Ortopedico Rizzoli, Via di Barbiano 1\/10, 40136 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Silvia","family":"Panzavolta","sequence":"additional","affiliation":[{"name":"Department of Chemistry \u2018\u2018Giacomo Ciamician\u201d, University of Bologna, Via Selmi 2, 40126 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2668-8627","authenticated-orcid":false,"given":"Adriana","family":"Bigi","sequence":"additional","affiliation":[{"name":"Department of Chemistry \u2018\u2018Giacomo Ciamician\u201d, University of Bologna, Via Selmi 2, 40126 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Milena","family":"Fini","sequence":"additional","affiliation":[{"name":"Complex Structure Surgical Sciences and Technologies, IRCCS Istituto Ortopedico Rizzoli, Via di Barbiano 1\/10, 40136 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,5,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"S56","DOI":"10.1016\/j.injury.2011.06.011","article-title":"The use of bone-graft substitutes in large bone defects: Any specific needs?","volume":"42","author":"Calori","year":"2011","journal-title":"Injury"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2445","DOI":"10.1007\/s10856-014-5240-2","article-title":"Bone substitutes in orthopaedic surgery: From basic science to clinical practice","volume":"25","author":"Campana","year":"2014","journal-title":"J. Mater. Sci. Mater. Med."},{"key":"ref_3","first-page":"2041731418776819","article-title":"Bone substitutes: A review of their characteristics, clinical use, and perspectives for large bone defects management","volume":"9","author":"Keller","year":"2018","journal-title":"J. Tissue Eng."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"625","DOI":"10.14336\/AD.2020.0904","article-title":"The Impact of Frailty on Spine Surgery: Systematic Review on 10 years Clinical Studies","volume":"12","author":"Veronesi","year":"2021","journal-title":"Aging Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1007\/s11657-013-0136-1","article-title":"Osteoporosis in the European Union: Medical management, epidemiology and economic burden. A report prepared in collaboration with the International Osteoporosis Foundation (IOF) and the European Federation of Pharmaceutical Industry Associations (EFPIA)","volume":"8","author":"Hernlund","year":"2013","journal-title":"Arch. Osteoporos"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Pfeiffenberger, M., Damerau, A., Lang, A., Buttgereit, F., Hoff, P., and Gaber, T. (2021). Fracture Healing Research-Shift towards In Vitro Modeling?. Biomedicines, 9.","DOI":"10.3390\/biomedicines9070748"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1726","DOI":"10.1007\/s00198-006-0172-4","article-title":"An estimate of the worldwide prevalence and disability associated with osteoporotic fractures","volume":"17","author":"Johnell","year":"2006","journal-title":"Osteoporos. Int."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Salamanna, F., Gambardella, A., Contartese, D., Visani, A., and Fini, M. (2021). Nano-Based Biomaterials as Drug Delivery Systems Against Osteoporosis: A Systematic Review of Preclinical and Clinical Evidence. Nanomaterials, 11.","DOI":"10.3390\/nano11020530"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1002\/jbmr.3877","article-title":"Secondary Fracture Prevention: Consensus Clinical Recommendations from a Multistakeholder Coalition","volume":"35","author":"Conley","year":"2020","journal-title":"J. Bone Miner Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1921","DOI":"10.1007\/s00198-021-05957-9","article-title":"How can the orthopedic surgeon ensure optimal vitamin D status in patients operated for an osteoporotic fracture?","volume":"32","author":"Chevalley","year":"2021","journal-title":"Osteoporos Int."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"898","DOI":"10.1097\/EDE.0000000000000729","article-title":"Thirty-five-year trends in first-time hospitalization for hip fracture, 1-year mortality, and the prognostic impact of comorbidity: A Danish nationwide cohort study, 1980\u20132014","volume":"28","author":"Pedersen","year":"2017","journal-title":"Epidemiology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1007\/s11657-020-0706-y","article-title":"International Osteoporosis Foundation. Fragility fractures in Europe: Burden, management and opportunities","volume":"15","author":"Karlsson","year":"2020","journal-title":"Arch. Osteoporos."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"837","DOI":"10.1039\/C5CP05650K","article-title":"Simulations reveal the role of composition into the atomic-level flexibility of bioactive glass cements","volume":"18","author":"Tian","year":"2015","journal-title":"Phys. Chem. Chem. Phys."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"104627","DOI":"10.1016\/j.tiv.2019.104627","article-title":"Cell physiological effects of glass ionomer cements on fibroblast cells","volume":"61","author":"Lang","year":"2019","journal-title":"Toxicol. Vitr."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1064","DOI":"10.1002\/jbm.a.36882","article-title":"Electrospun poly(d,l-lactide)\/gelatin\/glass-ceramics tricomponent nanofibrous scaffold for bone tissue engineering","volume":"108","author":"Bochicchio","year":"2020","journal-title":"J. Biomed. Mater. Res. A"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"119366","DOI":"10.1016\/j.biomaterials.2019.119366","article-title":"Bioinspired surface modification of orthopedic implants for bone tissue engineering","volume":"219","author":"Hu","year":"2019","journal-title":"Biomaterials"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Codrea, C.I., Croitoru, A.-M., Baciu, C.C., Melinescu, A., Ficai, D., Fruth, V., and Ficai, A. (2021). Advances in Osteoporotic Bone Tissue Engineering. J. Clin. Med., 10.","DOI":"10.3390\/jcm10020253"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Bigi, A., and Boanini, E. (2018). Calcium Phosphates as Delivery Systems for Bisphosphonates. J. Funct. Biomater., 9.","DOI":"10.3390\/jfb9010006"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1016\/j.bone.2005.05.003","article-title":"Novel insights into actions of bisphosphonates on bone: Differences in interactions with hydroxyapatite","volume":"38","author":"Nancollas","year":"2006","journal-title":"Bone"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1002\/jbm.b.31500","article-title":"Differences between bisphosphonates in binding affinities for hydroxyapatite","volume":"92B","author":"Lawson","year":"2009","journal-title":"J. Biomed. Mater. Res. Part B Appl. Biomater."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"5619","DOI":"10.1016\/j.biomaterials.2014.03.053","article-title":"Combined effect of strontium and zoledronate on hydroxyapatite structure and bone cell responses","volume":"35","author":"Boanini","year":"2014","journal-title":"Biomaterials"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3560","DOI":"10.1002\/jbm.a.34662","article-title":"3D interconnected porous biomimetic scaffolds: In vitro cell response","volume":"101","author":"Panzavolta","year":"2013","journal-title":"J. Biomed. Mater. Res. A"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"6695","DOI":"10.1016\/j.biomaterials.2013.05.040","article-title":"Bioelectric modulation of wound healing in a 3D in vitro model of tissue-engineered bone","volume":"34","author":"Sundelacruz","year":"2013","journal-title":"Biomaterials"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Di Filippo, M.F., Amadori, S., Casolari, S., Bigi, A., Dolci, L.S., and Panzavolta, S. (2019). Cylindrical Layered Bone Scaffolds with Anisotropic Mechanical Properties as Potential Drug Delivery Systems. Molecules, 24.","DOI":"10.3390\/molecules24101931"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1101","DOI":"10.1038\/nprot.2008.73","article-title":"Analyzing real-time PCR data by the comparative C(T) method","volume":"3","author":"Schmittgen","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1215","DOI":"10.1016\/S0731-7085(02)00021-3","article-title":"Spectrophotometric determination of alendronate in pharmaceutical formulations via complex formation with Fe(III) ions","volume":"28","author":"Kuljanin","year":"2002","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1016\/j.biomaterials.2018.01.040","article-title":"Harnessing macrophage-mediated degradation of gelatin microspheres for spatiotemporal control of BMP2 release","volume":"161","author":"Annamalai","year":"2018","journal-title":"Biomaterials"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1038\/s41419-017-0126-0","article-title":"IL-6 potentiates BMP-2-induced osteogenesis and adipogenesis via two different BMPR1A-mediated pathways","volume":"9","author":"Huang","year":"2018","journal-title":"Cell Death Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.biocel.2016.08.003","article-title":"Cell-specific paracrine actions of IL-6 family cytokines from bone, marrow and muscle that control bone formation and resorption","volume":"79","author":"Sims","year":"2016","journal-title":"Int. J. Biochem. Cell Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1700612","DOI":"10.1002\/adhm.201700612","article-title":"Biomimetic Materials and Fabrication Approaches for Bone Tissue Engineering","volume":"6","author":"Kim","year":"2017","journal-title":"Adv. Healthc. Mater."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1002\/jcp.25875","article-title":"An advanced tri-culture model to evaluate the dynamic interplay among osteoblasts, osteoclasts, and endothelial cells","volume":"233","author":"Pagani","year":"2018","journal-title":"J. Cell Physiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1016\/j.actbio.2017.02.040","article-title":"Antiresorptive and anti-angiogenetic octacalcium phosphate functionalized with bisphosphonates: An in vitro tri-culture study","volume":"54","author":"Forte","year":"2017","journal-title":"Acta Biomater."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"93","DOI":"10.3389\/fbioe.2020.00093","article-title":"Use of Computational Modeling to Study Joint Degeneration: A Review","volume":"8","author":"Mukherjee","year":"2020","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Barh, D., Chaitankar, V., Yiannakopoulou, E.C., Salawu, E.O., Chowbina, S., Ghosh, P., and Azevedo, V. (2014). In Silico Models: From Simple Networks to Complex Diseases. Animal Biotechnology, Academic Press.","DOI":"10.1016\/B978-0-12-416002-6.00021-3"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1002\/wsbm.1356","article-title":"In silico bone mechanobiology: Modeling a multifaceted biological system","volume":"8","author":"Giorgi","year":"2016","journal-title":"WIREs Syst. Biol. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.bone.2014.07.010","article-title":"The rational use of animal models in the evaluation of novel bone regenerative therapies","volume":"70","author":"Peric","year":"2015","journal-title":"Bone"},{"key":"ref_37","unstructured":"Russell, W.M.S., and Burch, R.L. (1959). The Principles of Humane Experimental Technique. Universities Federation for Animal Welfare Wheathampstead, Methuen & Co. Ltd.. (Reprinted in 1992)."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"S21","DOI":"10.1016\/S0020-1383(16)47004-X","article-title":"Fracture healing in osteoporotic bone","volume":"47","author":"Cheung","year":"2016","journal-title":"Injury"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1615\/CritRevEukarGeneExpr.v21.i4.60","article-title":"Mesenchymal stem cells in the aging and osteoporotic population","volume":"21","author":"Veronesi","year":"2011","journal-title":"Crit. Rev. Eukaryot. Gene Expr."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1177\/039139880402700803","article-title":"In vitro models to test orthopedic biomaterials in view of their clinical application in osteoporotic bone","volume":"27","author":"Torricelli","year":"2004","journal-title":"Int. J. Artif. Organs"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3429","DOI":"10.1021\/acsbiomaterials.9b00795","article-title":"Multifunctionalization Modulates Hydroxyapatite Surface Interaction with Bisphosphonate: Antiosteoporotic and Antioxidative Stress Materials","volume":"5","author":"Forte","year":"2019","journal-title":"ACS Biomater. Sci. Eng."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"20046","DOI":"10.1002\/jcp.28601","article-title":"Effect of strontium substituted \u00df-TCP associated to mesenchymal stem cells from bone marrow and adipose tissue on spinal fusion in healthy and ovariectomized rat","volume":"234","author":"Salamanna","year":"2019","journal-title":"J. Cell. Physiol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2079","DOI":"10.1007\/s10856-011-4379-3","article-title":"Osteopenic bone cell response to strontium-substituted hydroxyapatite","volume":"22","author":"Boanini","year":"2011","journal-title":"J. Mater. Sci. Mater. Med."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/11\/5988\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:19:21Z","timestamp":1760138361000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/11\/5988"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,5,26]]},"references-count":43,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2022,6]]}},"alternative-id":["ijms23115988"],"URL":"https:\/\/doi.org\/10.3390\/ijms23115988","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2022,5,26]]}}}