Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jan 28;23(3):1530.
doi: 10.3390/ijms23031530.

Validation of a Cleanroom Compliant Sonication-Based Decellularization Technique: A New Concept in Nerve Allograft Production

Federico Bolognesi  1   2 , Nicola Fazio  3 , Filippo Boriani  4 , Viscardo Paolo Fabbri  2   5 , Davide Gravina  3 , Francesca Alice Pedrini  6   7 , Nicoletta Zini  8   9 , Michelina Greco  3 , Michela Paolucci  10 , Maria Carla Re  10 , Sofia Asioli  2   5 , Maria Pia Foschini  2   5 , Antonietta D'Errico  11 , Nicola Baldini  2   3 , Claudio Marchetti  1   2
Affiliations

Validation of a Cleanroom Compliant Sonication-Based Decellularization Technique: A New Concept in Nerve Allograft Production

Federico Bolognesi et al. Int J Mol Sci. .

Abstract

Defects of the peripheral nervous system are extremely frequent in trauma and surgeries and have high socioeconomic costs. If the direct suture of a lesion is not possible, i.e., nerve gap > 2 cm, it is necessary to use grafts. While the gold standard is the autograft, it has disadvantages related to its harvesting, with an inevitable functional deficit and further morbidity. An alternative to autografting is represented by the acellular nerve allograft (ANA), which avoids disadvantages of autograft harvesting and fresh allograft rejection. In this research, the authors intend to transfer to human nerves a novel technique, previously implemented in animal models, to decellularize nerves. The new method is based on soaking the nerve tissues in decellularizing solutions while associating ultrasounds and freeze-thaw cycles. It is performed without interrupting the sterility chain, so that the new graft may not require post-production γ-ray irradiation, which is suspected to affect the structural and functional quality of tissues. The new method is rapid, safe, and inexpensive if compared with available commercial ANAs. Histology and immunohistochemistry have been adopted to evaluate the new decellularized nerves. The study shows that the new method can be applied to human nerve samples, obtaining similar, and, sometimes better, results compared with the chosen control method, the Hudson technique.

Keywords: allografting; decellularization; nerve regeneration; orthopedic and maxillofacial surgery; reconstructive surgery; tissue transplantation.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Light and electron microscopy of human nerve sample n.1. (A–C) Untreated nerve; (D–F) control decellularization method (Hudson method); (G–I) innovative decellularization method. (A,D,G) LM 40× of semithin cross-sections (toluidine blue, bar: 10 μm); (B,E,H) TEM 3000× of ultrathin cross-sections (bar: 2 μm); (C,F,I) TEM 12,000× at higher magnification (bar: 0.5 μm). Arrows indicate the preserved basal lamina.
Figure 2
Figure 2
Light and electron microscopy of human nerve sample n.2. (A–C) Untreated nerve; (D–F) control decellularization method (Hudson method); (G–I) innovative decellularization method. (A,D,G) LM 40× of semithin cross-sections (toluidine blue, bar: 10 μm); (B,E,H) TEM 3000× of ultrathin cross-sections (bar: 2 μm); (C,F,I) TEM 12,000× at higher magnification (bar: 0.5 μm). Arrows indicate the preserved basal lamina.
Figure 3
Figure 3
Fascicle morphology (A,C,E) (hematoxylin–eosin) and S-100 immunohistochemical analysis (B,D,F) of human nerve sample n.1. (A,B) Untreated nerve; (C,D) control decellularization method (Hudson method); (E,F) innovative decellularization method. Original magnification 20× (A–F).
Figure 4
Figure 4
Fascicle morphology (A,C,E) (hematoxylin-eosin) and S-100 immunohistochemical analysis (B,D,F) of human nerve sample n.2. (A,B) Untreated nerve; (C,D) control decellularization method (Hudson method); (E,F) innovative decellularization method. Original magnification 20× (A–F).
Figure 5
Figure 5
Light and electron microscopy of human nerve sample n.3. (A–C) Untreated nerve; (D–F) control decellularization method (Hudson method); (G–I) innovative decellularization method. (A,D,G) LM 40× of semithin cross-sections (toluidine blue, bar: 10 μm); (B,E,H) TEM 3000× of ultrathin cross-sections (bar: 2 μm); (C,F,I) TEM 12,000× at higher magnification (bar: 0.5 μm. Arrows indicate the preserved basal lamina.
Figure 6
Figure 6
Light and electron microscopy of human nerve sample n.6. (A–C) Untreated nerve; (D–F) control decellularization method (Hudson method); (G–I) innovative decellularization method. (A,D,G) LM 40× of semithin cross-sections (toluidine blue, bar: 10 μm); (B,E,H) TEM 3000× of ultrathin cross-sections (bar: 2 μm); (C,F,I) TEM 12,000× at higher magnification (bar: 0.5 μm). Arrows indicate the preserved basal lamina.
Figure 7
Figure 7
Light and electron microscopy of human nerve sample n.7. (A–C) Untreated nerve; (D–F) control decellularization method (Hudson method); (G–I) innovative decellularization method. (A,D,G) LM 40× of semithin cross-sections (toluidine blue, bar: 10 μm); (B,E,H) TEM 3000× of ultrathin cross-sections (bar: 2 μm); (C,F,I) TEM 12,000× at higher magnification (bar: 0.5 μm). Arrows indicate the preserved basal lamina.
Figure 8
Figure 8
Example of structural and cellular comparison between the nerves before (A–C) and after decellularization: innovative method (D–F) and Hudson method (G–I). Note the absence of well-preserved nuclei in (D) and the presence of nuclear debris in (G).
Figure 9
Figure 9
Example of axonal and ECM preservation in nerves before (A–C) and after decellularization: innovative method (D–F) and Hudson method (G–I). Note the increasing density and the different distribution of the ECM after decellularization.
See this image and copyright information in PMC

References

    1. López-Cebral R., Silva-Correia J., Reis R.L., Silva T.H., Oliveira J.M. Peripheral nerve injury: Current challenges, conventional treatment approaches, and new trends in biomaterials-based regenerative strategies. ACS Biomater. Sci. Eng. 2017;3:3098–3122. doi: 10.1021/acsbiomaterials.7b00655. - DOI - PubMed
    1. Huckhagel T., Nüchtern J., Regelsberger J., Lefering R. Nerve injury in severe trauma with upper extremity involvement: Evaluation of 49,382 patients from the TraumaRegister DGU® between 2002 and 2015. Scand. J. Trauma Resusc. Emerg. Med. 2018;26:76. doi: 10.1186/s13049-018-0546-6. - DOI - PMC - PubMed
    1. Biglioli F., Allevi F., Rabbiosi D., Cupello S., Battista V.M.A., Saibene A.M., Colletti G. Triple innervation for re-animation of recent facial paralysis. J. Craniomaxillofac. Surg. 2018;46:851–857. doi: 10.1016/j.jcms.2018.02.014. - DOI - PubMed
    1. Fogagnolo P., Giannaccare G., Bolognesi F., Digiuni M., Tranchina L., Rossetti L., Dipinto A., Allevi F., Lozza A., Rabbiosi D., et al. Direct versus indirect corneal neurotization for the treatment of neurotrophic keratopathy: A multicenter prospective comparative study. Am. J. Ophthalmol. 2020;220:203–214. doi: 10.1016/j.ajo.2020.07.003. - DOI - PubMed
    1. Giannaccare G., Bolognesi F., Pellegrini M., Spena R., Allevi F., Marchetti C., Scorcia V., Biglioli F. Corneal neurotization: A game-changing surgical procedure for neurotrophic keratopathy. Cornea. 2021 doi: 10.1097/ICO.0000000000002746. - DOI - PubMed

LinkOut - more resources