Table 3.

Decellularization Methods for Peripheral Nervous System (PNS) Applications.

Application	Study model	Source Tissue	Additional Components	Post Processing	Decellularization Method				Notable Results	References
Application	Study model	Source Tissue	Additional Components	Post Processing	#	Step	Time	RPM	Notable Results	References
Transection and gap	In vitro analysis of decellularization	Rat sciatic nerve	N/A	N/A	Sondell et al. 1998 Method				Direct method comparison: Sondell’s method disrupted the nerve ECM more than the Hudson method. This disruption could be from shrinkage/swelling cycles when applying deoxycholate/water. Hudson’s method produced more demyelination and improved adhesive ratios.	[143,144,146,150-152,154]
						distilled water	7 h
					2x	3% Triton X-100	12 h
						4% Sodium deoxycholate distilled water	24 h	yes*
					Hudson et al. 2004 Method
						distilled water	7 h
						125 mM sulfobetaine-10	15 h	yes*
						50 mM phosphate and 100 mM sodium	15 m
						0.14% Triton X-200, 0.6 mM sulfobetaine-16, 10 mM phosphate and 50 mM sodium	24 h	yes*
					3x	50 mM phosphate and 100 mM sodium	5 m
						125 mM sulfobetaine-10	7 h	yes*
						50 mM phosphate and 100 mM sodium	15 m
						0.14% Triton X-200, 0.6 mM sulfobetaine-16, 10 mM phosphate and 50 mM sodium	15h
					3x	50 mM phosphate and 100 mM sodium	15 m
	1.5 cm rat sciatic nerve defect	Rat sciatic nerve	N/A	N/A		Distilled water	7 h	yes*	Decellularized similarly to Hudson’s method with increased ECM preservation. Outperformed Hudson’s method in nerve histomorphometry and muscle mass. Outperformed by autograft.	[158]
						1 M Sodium chloride	15 h	yes*
						2.5 nM Triton X-100	24 h	yes*
						1X PBS	15 m	yes*
						Distilled water	7 h	yes*
						1 M Sodium chloride	15h	yes*
						100 nM CHAPS	24 h	yes*
					5x	1X PBS	5 m	yes*
	In vitro characterization and effect on PC12 cells	Rat sciatic nerve	N/A	axially aligned channels	2x	distilled water	7 h		Decellularization significantly increased pore size and created axially aligned channels. In vitro work showed enhanced PC12 cell migration both in number and depth into the graft.	[147]
						3% Triton X-100	12 h	yes*
						2% deoxycholic acid in 50 nM Trizma base	24 h	yes*
						DNase and RNase (5 U/ml) in 10 nM magnesium chloride	12 h
						0.1% peracetic acid	1 h
	3.5 cm Rat sciatic nerve gap defect	Rat sciatic nerve	N/A	N/A		DMEM 10% FBS 2% pen/strep/amph PBS	2wks	yes*	Nerve autograft outperformed both the detergent free and detergent decellularized grafts. Only nerve autograft and detergent free grafts showed muscular recovery.	[186]
	3.5 cm Rat sciatic nerve gap defect	Rat sciatic nerve	N/A	N/A		DMEM 10% FBS 2% pen/strep/amph PBS	1wk	yes*		[186]
	In vitro characterization and subcutaneous implantation	Rat sciatic nerve	Apoptosis assisted	N/A		DMEM with camptothecin (5 or 10uM) 37 °C	1-3d	14	The apoptosis-assisted method maintained basal lamina microarchitecture with decreased cytotoxicity.	[171]
						4X PBS	24 h
						2X PBS	30 m
					3x	1X PBS	30 m		Also, inflammatory infiltrate and stromal remodeling decreased compared to autograft control.
					75 U/mL DNase	36 h
					2x	1X PBS	30 m
	In vitro characterization	Rat sciatic nerve	N/A	N/A	2x	10 mM TRIS-HCl; pH 8.0		yes*	Reduced DNA content by greater than 95% while preserving collagen, laminin, and fibronectin.	[126]
						0.1% SDS with 10 kIU/mL		yes*
					3x	1X PBS	30 m	yes*
						1 U/mL Benzonase	3 h	yes*
						1.5 M NaCl		yes*
						0.1% peracetic acid in PBS	3 h	yes*
					3x	1X PBS at 4C	30 m	yes*
						1X PBS at 4C	48 h	yes*
	1 cm and 1.5 cm rat sciatic nerve defect models	Rat sciatic nerve and rabbit median nerve	N/A	N/A		1X PBS			Allogeneic and xenogeneic tissue sources were used and compared in vivo to isograft control up to 24 weeks. Many axons and Schwann cells were shown migrating into the bridge, with few macrophages present. Both tissue sources were inferior to isograft control, contradicting previous results from the group in Wakimura et al. 2015.	[124,162]
						1% SDS	24 h	yes*
						Distilled water	30 m
						1% Triton X-100	1 h	yes*
						Distilled water	30 m
						100 U/mL penicillin-G, 100 ug/mL streptomycin, 0.25 ug/mL amphotericin B	7d
	In vitro characterization	Rat sciatic nerve	N/A	N/A	2x	50 nM Tris buffer	12 h		Comparisons were made to Sondell’s and Hudson’s methods. The proposed method showed a better removal of DNA content and preservation of ECM components.	[146,150,151,159,160,187,188]
						1% Triton X-100 in 50 nM Tris buffer at 4 °C	24 h
						HBSS	24 h	24
					2x	40,000 U/L DNase, 20 mg/L RNase, 100 mg/L Trypsin at 37C	45 m	yes*
						1% Triton X-100 in 50 nM Tris buffer at 4 °C	12 h
					3x	HBSS at 4 °C
	1.5 cm rat sciatic nerve defect	Porcine sciatic nerve	N/A	Processed into an ECM hydrogel		distilled water	7 h		Resembling Sondell’s method, this study reduced washing steps to preserve ECM needed for gelation. The hydrogel was found to be superior using the altered method, but inferior to autograft control.	[189]
						3% Triton X-100	12 h
						4% Sodium deoxycholate	24 h	yes*
					3x	distilled water	15 m	yes*
						Ethanol:Dichloromethane (1:2) distilled water	24 h
	1.5 cm rat sciatic nerve defect model	Porcine sciatic nerve	N/A	Processed into an ECM hydrogel		Distilled water	14 h	300	The decellularized nerve ECM hydrogel was used as a lumen filler for a rat model of nerve gap injury. It showed increased regenerative bridge formation followed by enhanced M2 macrophage and Schwann cell migration and involvement.	[145]
						0.02% Trypsin/0.05% EDTA at 37 °C	1 h	300
						3% Triton X-100	1 h	300
						Distilled water		300
1 M Sucrose						15 m	300
4% Sodium deoxycholate						1 h	300
0.1% Peracetic acid/4% ethanol						2 h	300
1X PBS						15 m	300
2x					Distilled water	15 m	300
2x					1X PBS	15 m	300
1.5 cm rat sciatic nerve defect model	Rat sciatic nerve	N/A	N/A		Distilled water	6 h		Method altered detergent combinations from Hudson’s method to reduce residual myelin. Comparing these two methods to the nerve autograft, the modified method had reduced immunogenicity and remyelination more comparable to autograft controls. However, morphologic assessment of axon regrowth showed no difference between the two methods.	[142]
					125 mM sulfobetaine-10	12 h	yes*
				3x	1X PBS	10m
					0.14% Triton X-200, 0.6 mM sulfobetaine-16	24 h	yes*
					1X PBS	10 m
					Distilled water	10 m
					125 mM sulfobetaine-10	12 h	yes*
					4% Sodium deoxycholate	24 h	yes*
				3x	1X PBS	2 h
				3x	0.1% Peracetic acid	3 h
				3x	1X PBS	1 h
Rabbit tibial nerve	Rabbit tibial and femoral nerves	N/A	N/A	Method 1: Hudson Method (control)				These methods limit hands-on time to prevent contamination and necessity of sterilization. Methods 2 and 3 were less harsh than Hudson’s method with small amount of DNA fragments and Schwann cells still present. Method 2 produced uneven decellularization towards the center of the graft. Method 3 did remove most of the myelin, axons, and DNA residue to a satisfactory degree with ECM preservation.	[121]
				2x	125mM SB-10, 10 mM phosphate, 50 mM sodium	15 h	yes*
					0.14% Triton X-200, 0.6 mM SB-16, 10 mM phosphate, 50 mM sodium	24 h	yes*
				Method 2: Triton X-100 based Method
					125 mM SB-10, 0.2% Triton X-100, 1% penicillin–streptomycin	48 h	yes*
					Sonication at 40 Hz	2m
				Method 3: Triton X-100 and SDS method
					125 mM SB-10, 0.2% Triton X-100, 1% penicillin–streptomycin freeze–thaw cycle	48 h
				3x	1X PBS	30 m
					0.25% SDS and sonicated for 5 m every 30 m at 40 Hz	3 h
rat trigeminal, infraorbital sciatic nerve transection model	Porcine fetal urinary bladder matrix	N/A	Pressed and lyophilized into a sheet. ETO sterilized	8x	Nanopure water	30 m		The method uniquely included a chamber that cycled between ambient pressure and 0 psi at a rate of 1 min/cycle. The wrap enhanced epi- and endoneurial organization and increased angiogenesis; however, the number of axons and myelination were unchanged.	[161]
					3% Triton X-100	30 m
					3 M NaCl	30 m
					DNase	1 h
					0.1% Peracetic acid/4% ethanol 1X PBS	1 h
				3x	Nanopure water
Rat sciatic nerve gap model looking at immunogenicity and mechanical properties	Rat sciatic nerve	N/A	N/A	Method 1: Sondell method (control)				The freeze-thaw method combined with enzymatic degradation showed the lowest immunogenicity of all methods tested. Also, fewer inflammatory cells were present and less necrosis seen at the downstream muscle with less MHC-II presentation compared to all other methods and native tissue. All decellularization techniques had similar mechanical properties to the native nerve.	[138]
					distilled water	12 h	yes
				2x	3% Triton X-100	12 h
					4% Sodium deoxycholate	24 h
				Method 2: Low detergent method
					ultrapure water	1 h	yes
					0.5% Triton X-100	48 h	yes
					ultrapure water	48 h
					DNase and RNase at 37 °C	12 h
				Method 3: Freeze-thaw method
					hypotonic solution at 4 °C	12 h
					hypotonic solution at −80 °C	6 h
					hypotonic solution at 37 °C	30 m
					0.05% pancreatin	6 h
					DNase and RNase at 37 °C	12 h
1.5 cm rat sciatic nerve defect model	Rat cauda equina	N/A	Decellularized cauda equina allograft embedded within chitosan conduit	3x	1X PBS			Cauda equina source tissue used to test less harsh method because it lacks epineurium. This tissue required half the time (12 h) in SDS to achieve similar decellularization results to a sciatic nerve control (24 h). Cauda equina grafts performed better than other groups and similarly to autograft.	[123]
					0.5% SDS at 16 °C penicillin (100 U/mL) and streptomycin (0.1 mg/mL) in PBS	12 h	40
				10x		12 h	40
	Rat sciatic nerve				1X PBS	12 h
				10x	1X PBS
In vitro analysis	Porcine cauda equina	N/A	Homogenized cauda equina ECM with PLGA to form an electrospun scaffold		distilled water			Method incorporated insoluble proteins of the cauda equina ECM into an aligned, electrospun PLGA material. This incorporation of ECM proteins enhanced neurite outgrowth of dorsal root ganglion cells compared to PLGA material alone.	[148]
					3% Triton X-100	2 h	yes
					4% Sodium deoxycholate 1X PBS rinse Homogenized Centrifugation steps to isolate ECM precipitate	2 h	yes
In vitro analysis	Rat sciatic nerves	N/A	Nerve ECM coated onto PCL based conduits		distilled water	15 m		Polydopamine (PDA) was coated onto polycaprolactone (PCL) conduits to successfully increase attachment of peripheral nerve ECM as a surface modification. This was shown to have potentially beneficial changes in Schwann cell behavior when cultured on the material in vitro.	[122]
					0.5% Triton X-100 in 1 M NaCl distilled water	2 h	yes
					1% SDS in 1 M NaCl distilled water, solution changed daily	2d	yes
					0.1% peracetic acid and 4% EtOH in 1 M NaCl	4 h
					distilled water lyophilized	4 h
1.0 cm rat sciatic nerve defect model	Rat sciatic nerve	Acellular nerve graft augmented with additional NGF or GDNF	N/A	3x	Liquid nitrogen	2m		Comparison of chondroitinase ABC treated acellular nerve grafts to NGF or GDNF imbibed grafts. NGF significantly enhanced motor axon outgrowth and number of myelinated axons. Sensory nerve outgrowth was significantly stunted. Dorsal root ganglion outgrowth in the presence of chondroitinase ABC and NGF showed no effect on sensory neurite outgrowth.	[172,173]
					Thawed in water bath at 37 °C	2 m
					2 U/mL chondroitinase ABC in PBS at 37 °C	16 h
					Incubated with NGF (10ug/mL) or GDNF (20 ug/mL) solution Rinsed in lactated Ringer solution	1 h
				2x	Rinsed in lactated Ringer solution
Clinical study of digital nerve gaps > 2.5 cm	Human peripheral nerve	N/A	gamma irradiated		distilled water	7 h		Applying the Avance decellularized nerve graft from Axogen, 86% of patients recovered meaningful function, matching autograft repair (60–80%). When studied in large diameter repairs, Avance decellularized graft was not limited in this regard and presented similar motor and sensory recovery as the gold-standard. In sensory repair applications, patients regained sensory function in 15 out of 16 cases with the Avance graft.	[149,153,155-157]
					125 mM sulfobetaine-10	15 h	yes*
					50 mM phosphate and 100 mM sodium	15 m
Clinical study of large diameter cable repairs					0.14% Triton X-200, 0.6 mM sulfobetaine-16, 10 mM phosphate and 50 mM sodium	24 h	yes*
				3x	50 mM phosphate and 100 mM sodium	5 m
					125 mM sulfobetaine-10	7 h	yes*
Clinical study of trigeminal nerve repair					50 mM phosphate and 100 mM sodium	15 m
					0.14% Triton X-200, 0.6 mM sulfobetaine-16, 10 mM phosphate and 50 mM sodium	15 h
				3x	50 mM phosphate and 100 mM sodium Chondroitinase ABC at 37 °C	15 m
Crush	rabbit sciatic nerve crush model, clinical model of recurrent cubital tunnel syndrome	Porcine Small intestinal submucosa	N/A	Pressed and lyophilized into a sheet. ETO sterilized		Distilled water Delaminate tunica mucosa and tunica muscularis externa			Scaffold promoted vascularization and remodeling at implantation site. The product, AxoGuard Nerve Protector from AxoGen, showed enhanced grip and pinch strength as well as reduced pain levels for patients with chronic cubital tunnel syndrome.	[169,170]
Crush		Porcine Small intestinal submucosa	N/A	Pressed and lyophilized into a sheet. ETO sterilized		0.1% Peracetic acid/4% ethanol Distilled water	2 h			[169,170]