Deep-freezing and decontamination strategy for a large autologous bone graft - with presentation of the osteogenic potential of resident osteoblasts

Sep 30, 2026

Musculoskeletal injuries from high-impact trauma can cause major bone defects requiring effective reconstruction, while contamination in large autologous bone grafts from open fractures creates a persistent clinical obstacle. The study established a decontamination protocol for massive autologous bone grafts and tested osteoblast viability and regenerative potential after deep freezing, including dimethyl sulfoxide (DMSO) effects. A combination of microbiological, histological, flow cytometric, molecular, and imaging analyses evaluated preserved cell function after processing.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages34

Reading Time1 h 26 min

AccessFree to download

Abstract

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Methods

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Clinical problem

Zekušić et al. Stem Cell Research & Therapy (2026) 17:13 [https://doi.org/10.1186/s13287-025-04839-9](https://doi.org/10.1186/s13287-025-04839-9) Stem Cell Research & Therapy RESEARCH Open Access Deep-freezing and decontamination strategy for a large autologous bone graft with presentation of the osteogenic potential of resident osteoblasts Marija Zekušić 1,2*†, Marija Skoko 1,3†, Kruno Vukušić4, Maja Ledinski 1, Marina Bujić Mihica 1, Dejan Blažević3,5,6, Zlata A. Sošić7, Mario Đura4, Jelena Martinčić4, Ivana Vrgoč Zimić 1, Ivanka Batarilo8, Denis Polančec9, Lucija Zenić9, Snježana Ramić 10, Patricija Sesar 10, Kristina Crkvenac Gornik 11,12, Slaven Babić3,5, Jure Pavešić5, Srećko Sabalić5,13, Iva M. Tolić4, Inga Urlić 14, Stanka Misir Šitum3,15, Tiha Vučemilo 1,3 and Dinko Vidović5,12,16 Abstract Background Musculoskeletal injuries resulting from high-impact trauma often lead to extensive bone defects that require effective reconstruction to prevent limb amputation. However, contamination of large autologous bone grafts in open fractures remains a major clinical challenge. This study aimed to establish a reliable protocol for decontamination of massive autologous bone graft and to assess the viability and regenerative potential of osteoblasts from frozen bone, including the effects of deep freezing and the cryoprotectant dimethyl sulfoxide (DMSO) . Methods A 28 cm femoral segment, extruded during a high-energy road traffic accident, was stored at − 80 °C for 15 days before microbiological analysis revealed contamination. The bone was thawed, decontaminated, and successfully reimplanted. To validate this protocol, femoral head fragments were experimentally contaminated with various microbial strains and treated using the same procedure. To evaluate how low-temperature storage (15 and 30 days, with or without DMSO) impacts osteoblasts, cells were isolated from both fresh and frozen bone and analyzed using a panel of cellular and molecular biology techniques, including histology, flow cytometry, immunofluorescence, gene expression analysis, and real-time imaging. Results The decontamination protocol effectively eliminated microbial contamination from both the clinical autograft and experimentally treated femoral head fragments, with minimal antibiotic residues. Histological analysis confirmed preservation of osteoblasts, osteocytes, and chondrocytes in frozen bone. Osteoblasts from frozen bone retained proliferative and differentiation capacities comparable to those from fresh bone, with no evidence of mitotic †Marija Zekušić and Marija Skoko have contributed equally to this work. *Correspondence: Marija Zekušić [marija.zekusic@kbcsm.hr](marija.zekusic@kbcsm.hr) Full list of author information is available at the end of the article © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit [http://creati](http://creati)[vecommons.org/l](vecommons.org/l)icenses/by-nc-nd/4.0/. Zekušić et al. Stem Cell Research & Therapy (2026) 17:13 Page 2 of 34 chromosomal instability. Cryopreser

FAQs

What problem does the study address in autologous bone graft reconstruction?

Contamination of large autologous bone grafts from open fractures is a major clinical challenge during reconstruction, especially after high-impact trauma causing extensive bone defects.

How was the decontamination and freezing protocol evaluated?

The protocol was applied to a massive autologous femoral segment that was frozen and later decontaminated and reimplanted, and it was further validated using experimentally contaminated femoral head fragments treated with the same procedure.

What role does DMSO play in the cryopreservation conditions tested?

Cells from fresh and frozen bone were analyzed across storage durations with or without DMSO to assess impacts on osteoblast viability and osteogenic potential, with results indicating no significant differences in osteogenic potential between DMSO and non-DMSO groups.

Sep 30, 2026

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