Preparation of human decellularized peripheral nerve allograft using amphoteric detergent and nuclease

Abstract

Animal studies have shown that amphoteric detergent and nuclease (DNase I and ribonuclease A) is the most reliable decellularization method of the peripheral nerve. However, the optimal combination of chemical reagents for decellularization of human nerve allograft needs further investigation. To find the optimal protocol to remove the immunogenic cellular components of the nerve tissue and preserve the basal lamina and extracellular matrix and whether the optimal protocol can be applied to larger-diameter human peripheral nerves, in this study, we decellularized the median and sural nerves from the cadavers with two different methods: nonionic and anionic detergents (Triton X-100 and sodium deoxycholate) and amphoteric detergent and nuclease (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), deoxyribonuclease I, and ribonuclease A). All cellular components were successfully removed from the median and sural nerves by amphoteric detergent and nuclease. Not all cellular components were removed from the median nerve by nonionic and anionic detergent. Both median and sural nerves treated with amphoteric detergent and nuclease maintained a completely intact extracellular matrix. Treatment with nonionic and anionic detergent decreased collagen content in both median and sural nerves, while the amphoteric detergent and nuclease treatment did not reduce collagen content. In addition, a contact cytotoxicity assay revealed that the nerves decellularized by amphoteric detergent and nuclease was biocompatible. Strength failure testing demonstrated that the biomechanical properties of nerves decellularized with amphoteric detergent and nuclease were comparable to those of fresh controls. Decellularization with amphoteric detergent and nuclease better remove cellular components and better preserve extracellular matrix than decellularization with nonionic and anionic detergents, even in large-diameter human peripheral nerves. In Korea, cadaveric studies are not yet legally subject to Institutional Review Board review.

Keywords: detergent; human decellularized nerve graft; median nerve; nuclease; sural nerve.

Figure 1

Preparation of the median and sural nerves and of nerve fragments for staining. (A) Harvested median nerve. (B) Harvested sural nerve. (C) Schematic diagram of grouping. The number of segments per group is as follows: n = 8 segments each for groups A–D and n = 12 segments each for groups E and F. (D) Section diagram of a single nerve showing histological analysis. CHAPS: 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate; DAPI: 4’,6-diamidino-2-phenylindole; DR: Dnase + Rnase; H&E: hematoxylin and eosin; SD: sodium deoxycholate; Tx: triton X-100.

Figure 2

Hematoxylin-eosin staining results of median and sural nerves decellularized by two different methods. (A–F) Both non-ionic and anionic detergents showed satisfactory cell removal in the median (Group B) and sural (Group E) nerves. The amphoteric detergent and nuclease showed almost complete cell removal in both the median (Group C) and sural (Group F) nerves. Groups A and D refer to fresh median and sural nerves, respectively. Original magnification, 100×; scale bars: 100 μm. (G) Quantification of cellular components. Data are presented as the mean ± SD, n = 5. **P < 0.01, ***P < 0.001 (Student’s t-test). H&E: Hematoxylin and eosin; SD: standard deviation.

Figure 3

Comparison of DAPI staining of median and sural nerves between two decellularization methods. (A–F) Treatment with amphoteric detergent and nuclease showed almost complete cell removal in both the median (Group C) and sural (Group F) nerves. Additionally, in the context of the use of nonionic and anionic detergents, the sural nerve showed almost complete cell removal (Group E), whereas a significantly higher number of cells remained in the median nerve (Group B). Blue color indicates remnant cellular components. Groups A and D refer to fresh median and sural nerves, respectively. Original magnification, 100×; scale bars: 100 μm. (G) DAPI-positive counts. Data are presented as the mean ± SD, n = 5. ***P < 0.001 (Student’s t-test). DAPI: 4’,6-Diamidino-2-phenylindole.

Figure 4

DNA content and S-100 immunohistochemistry of median and sural nerves decellularized by two different methods. (A) Comparison of the DNA content of sural and median nerve between two decellularization methods. Median and sural nerves decellularized using amphoteric detergent and nuclease showed significantly lower DNA contents (Group C and F) than median and sural nerves decellularized using nonionic and anionic detergent (Groups B and E). Data are presented as the mean ± SD, n = 5. *P < 0.05, ***P < 0.001 (Student’s t-test). (B–G) S-100 immunohistochemistry of the sural and median nerves decellularized by two different methods. Schwann cells were removed almost completely in the median and sural nerves treated with amphoteric detergent and nuclease. Original magnification, 100×, scale bars: 100 μm.

Figure 5

Immunostaining against laminin of median nerves and sural nerves decellularized by two different methods. (A–F) Groups A–C: Median nerve; Groups D–F: sural nerve; Groups A and D: fresh nerve tissues; Groups B and E: nonionic and anionic detergent treatment; Groups C and F; amphoteric detergent and nuclease treatment. The median nerve (Group C) and sural nerve (Group F) treated with amphoteric detergent and nuclease showed better preservation of the structural integrity than those treated with nonionic and anionic detergents (Groups B and E). Original magnification, 200×. Scale bars: 50 μm. (G) Quantification of laminin expression. Data are presented as the mean ± SD, n = 5. ***P < 0.0001 (Student’s t-test).

Figure 6

Comparison of collagen content on median and sural nerves decellularized using two different methods after collagen myelin staining. (A) Comparison of collagen content of sural and median nerves (hydroxyproline assay). Groups A-C: median nerve; Groups D-F: sural nerve; Groups A and D: Fresh nerve tissue; Groups B and E: nonionic and anionic detergent; Groups C and F; amphoteric detergent and nuclease. Data are expressed as the mean ± SD, n = 7. *P < 0.05, **P < 0.01 (Student’s t-test). (B–G) Masson’s trichrome staining. Median (D) and sural (G) nerves treated with amphoteric detergent and nuclease show stronger blue staining (collagen) than those decellularized with nonionic (C) and anionic detergents (F). (H–M) Luxol fast blue-cresyl violet staining. The median and sural nerves treated with amphoteric detergent and nuclease (J, M) showed significantly reduced myelin content (blue-green color) than those decellularized with nonionic and anionic detergents (I, L). Original magnification, 100×. Scale bars: 100 μm.

Figure 7

Contact cytotoxicity assay of sural nerve decellularized with amphoteric detergent and nuclease. (A) Cyanoacrylate glue. (B) Collagen gel. (C) Fresh surface nerve segment. (D) Sural nerve segment decellularized by amphoteric detergent and nuclease. Original magnification, 40×. Scale bars: 1 mm. The images show that the decellularized nerves (D) are harmless to normal human fibroblasts.