Decellularized and Engineered Tendons as Biological Substitutes: A Critical Review

Table 2

In vivo studies of tendon tissue decellularization and implantation.

Tendon source; and size	Decellularization protocol(s)	Assessment of decellularization	Cell reseeding	Animal model	Implant site, size, and time point(s)	Experimental groups	Assessment of tendon repair	Results and comments	Reference
Rabbit; ST; Undefined size	Frozen −80°C (until use); 1% SDS RT (24 h)	—	Rabbit dermal fibroblasts; 2 × 10^6 cells/mL; Static culture by injection for 4 days	Rabbit	ACL replacement; 8 weeks	Autologous ST tendon; reseeded allogenic ST matrix; fresh rabbit ACL in knees (ctrl)	Histology; Mechanical testing	Before implantation; Stiffness → similar in autologous tendon and reseeded matrix; EM → higher in reseeded matrix than autologous tendon; After implantation; Reseeded matrix → weaker biomechanics than autologous tendons; necrosis and tissue remodeling higher than autologous tendon; inflammatory reaction; inhomogeneous cell repopulation	[16]
Rabbit; FDP; 3 cm; length	Frozen −80°C (until use); 0.05% Trypsin-EDTA RT (24 h); 0.5% Triton X100 RT (24 h)	—	Rabbit tenocytes (male); 2 × 10^6 cells/mL; Rotating culture for 24 h	Rabbit (female)	FDP replacement; 3, 6, 12, and 30 weeks	Acellularized FDP matrix; reseeded FDP matrix; fresh native FDP tendon (ctrl)	Histology; Fluorescent in situ hybridization (donor male tenocytes ≠ female tenocytes)	Reseeded matrix → good cell repopulation; similar to ctrl 6 weeks after implantation	[22]
Rabbit; FDP; 2 cm; length	Frozen −70°C (until use); 0.05% Trypsin/EDTA 37°C (24 h); 0.5% Triton X100 RT (24 h)	Histology	Rabbit tenocytes, Fs, BMSCs, and ASCs; 2 × 10^6 cells/mL; Static culture for 1, 4, and 8 weeks	Rabbit	FDP replacement; 4, 6, and 8 weeks	Autologous FDP tendon (ctrl); allogeneic FDP tendon; acellularized FDP matrix; reseeded FDP matrix	Histology	Reseeded matrix → viable cell distribution on surface, but not into the centre at 1, 4, and 8 weeks, good collagen architecture; Autologous, allogenic, and acellularized implants → mild inflammatory response	[21]
Rabbit; FDP; 2–2.5 cm; length	Frozen −70°C (until use); 0.05% Trypsin/EDTA 37°C (24 h); 0.5% Triton X100 RT (24 h)	—	Rabbit tenocytes, ASCs; 2 × 10^6 cells/mL; Static culture	Rabbit	FDP replacement; 2, 4, 10, and 20 weeks	Autologous FDP tendon (ctrl); acellularized FDP matrix; reseeded FDP matrix	Histology; Mechanical testing	Reseeded matrix → UTS similar to ctrl up to 4 weeks; weaker compared to ctrl at 10 weeks; greater in tendon reseeded matrix from 2 to 20 weeks; similar to acellularized matrix at 2, 4, and 20 weeks; cell penetration to the core of the grafts	[18]
Canine AT; 4 cm length; 0.4 cm; width; 0.3 mm thickness	Frozen −80°C/thaw (5 cycles); RNAse/DNAse 37°C (12 h)	—	—	Rabbit	IT full-thickness defect; 8 mm length; 4, 8, and 12 weeks	Acellularized AT matrix; void defect	Histology; Mechanical testing; MRI	Acellularized matrix → host cell ingrowth and tissue integration; developing of a tendon-like structure at 12 weeks; UTS similar to void defect at 4, 12 weeks; stiffness greater at 12 weeks than at 4, 8 weeks and compared to void defect at 12 weeks; MRI signal → similar in acellularized matrix and contralateral native tendon at 12 weeks; hypertrophy and scar tissue in void defects	[38]
Canine IT; 1 cm; length; 1 cm; width; 0.05 mm; thickness	Frozen −80°C (until use); Frozen liquid nitrogen/thaw (5 cycles) RNAse 37°C (12 h)	—	Rabbit BMSCs; 5 × 10^6 cells/mL; Static culture for 24 h	Rabbit	Patellar tendon defect; 10 mm length; 3 mm width; 2 weeks	Reseeded IT matrix; void defect	Histology; Gene expression; Cell fluorescent labeling	Reseeded matrix → cells aligned to fibrils before implantation; scattered after implantation; higher tenomodulin, collagen III, MMP3-13, and lower collagen I expressions compared to acellularized matrix after implantation; viable cells expressing tendon phenotype in vivo	[36]
Rat; AT; Undefined	Frozen −80°C (until use); 0.05% Trypsin/EDTA 37°C (24 h); 0.5% Triton X100 RT (24 h)	Histology	Rat tenocytes; 1 × 10^6 cells/mL; Static culture for 48 h	Rat	Achilles tendon defect; Mid 1/3 portion	Autologous AT (ctrl); acellularized AT matrix; reseeded AT matrix	Histology; Mechanical testing	Reseeded matrix → preserved collagen at 12 weeks; best histological score and biomechanics at 24 weeks and better organized ECM compared to ctrl and acellularized matrix; Acellularized matrix → less inflammatory cells than ctrl and reseeded matrix	[24]
Human; FDP; FDS; FPL; 8–10 cm; length	Frozen −70°C (until use); 0.1% EDTA RT (4 h) 0.1% SDS + 0.1% EDTA RT (24 h); 5% PAA (4 h)	Histology; Cell/DNA content; Immunohistochemistry; TEM; Mechanical testing	Human ASCs-luc2-eGFP; 1 × 10^6 cells/mL; Rotating seeding (6 h); Static culture for 2 weeks	Athymic rat	Subcutaneous pocket; 3 cm length; 4 weeks	Surface scored matrix (S); rehydrated matrix (F); surface scored + rehydrated matrix (S + F); fresh human tendons (ctrl)	Cell fluorescent labeling	Reseeded S and S + F → greater viable cell attachment and penetration compared to reseeded F and ctrl; reduced apoptosis, persistent procollagen production, and similar ultrastructure to ctrl; Rehydration of F and S + F did not improve reseeding; UTS/EM → greater in reseeded matrix than acellularized matrix	[28]
Human; FDP; 5 cm; length	Frozen −70°C (until use); 0.1% EDTA + 0.1% SDS RT (24 h); 5% PAA (6 h)	Immunohistochemistry	—	Rat	Subcutaneous pocket; 2, 4 weeks	Acellularized FDP matrix; fresh native FDP tendon (ctrl)	Histology; Immunohistochemistry; Mechanical testing	Acellularized matrix → cell and MHC-1 complex removal; preserved collagen structure and increased porosity; UTS/EM greater compared to ctrl; lower inflammatory response and abnormal collagen architecture compared to ctrl; B-cell infiltration → detected in ctrl compared to acellularized matrix after implantation; Macrophage infiltration → absent after implantation	[30]
Human; FDP; 4 cm; length	Frozen −70°C (until use); 0.1% EDTA (4 h); 0.1% SDS + 0.1 EDTA (24 h); 5% PAA (4 h); Manually scored; Dehydrated	Histology; DNA content	Human ASCs-luc2; 1-2 × 10^6 cells/mL; Static culture for 30 h	Rat	Subcutaneous pocket; 7, 14, 21, and 28 days	Acellularized FDP matrix; reseeded FDP matrix	Histology; Immunohistochemistry; Cell fluorescent labeling; Mechanical testing	Reseeded matrix → viable cells during implantation up to 4 weeks; Host cell invasion, proliferation, and vascularization were observed after implantation; UTS → similar between acellularized and reseeded matrixes; Collagen I > III → consistent in reseeded matrix after implantation	[32]
Porcine tendon; 1 cm; length; 1 cm; width; 0.08 mm; thickness	Frozen liquid nitrogen/thaw (5 cycles) DNAse 4°C (14 h)	Histology; Cell/DNA/collagen; SEM; Gene expression	Human TSPCs; 11.5 × 10^5 cells/cm^2; Static culture for 1, 7 days	Athymic mouse; Rat	Subcutaneous pocket; 4 weeks; AT defect (6 mm length); 2, 4 weeks	Acellularized matrix; reseeded matrix	Histology; Collagen content; TEM; Mechanical testing	Reseeded matrix → good cell adhesion and proliferation, promoting a tendon phenotype (scleraxis); mature structure, larger collagen fibrils, and stronger mechanical properties compared to acellularized matrix after implantation; No osteogenic behavior was observed in reseeded matrix after implantation	[44]
Chicken; FDP; 6 cm; length; 0.3 cm; width; 1 mm; thickness	Stored 4°C (until use); 0.05% Trypsin/EDTA 37°C (1 h); 2% Triton X100 + 1.5% PAA 37°C (4 h)	Histology; SEM; DNA content; Mechanical testing	—	Mouse	Subcutaneous pocket; 2 mm length; 2 mm width; 1 mm thickness; 3, 7, 14, and 21 days	Acellularized FDP matrix; fresh native FDP tendon (ctrl)	Histology	Acellularized matrix → decreased or absent DNA content compared to ctrl; UTS/EM 76–78% of that observed in ctrl; No host-cell-mediated foreign-body immune response was observed after implantation	[25]

FDS = flexor digitorum superficialis; FPL = flexor pollicis longus; GFP = green fluorescent protein; luc2-GFP = luciferase 2-green fluorescent protein; ACL = anterior cruciate ligament; MRI = magnetic resonance imaging; MHC-1 = major histocompatibility complex-1.