|
| Tendon source/size | Decellularization protocol(s) | Assessment of decellularization | Cell reseeding | Reseeding assessment | Experimental groups | Results and comments | Reference |
|
Rabbit flexor FDP
3–5 cm length | Frozen −80°C (until use)
0.05% Trypsin/EDTA 37°C (24 h)
0.5% Triton X100 RT (24 h)
0.25% Trypsin 37°C (15′)
0.5% Collagenase 37°C (1 h) | Histology
Cell count | Rabbit tenocytes
2 × 106 cells/mL
Static culture for 4 days | Mechanical testing | Freeze-stored FDP tendon; acellularized FDP matrix; reseeded FDP matrix; fresh native FDP tendon (ctrl) | Acellularized matrix → absence of cells or morphological alteration; UTS/EM similar to ctrl
Reseeding matrix → cells present only on the surface; EM similar to ctrl | [12] |
|
Rabbit ST and FDP
2 cm
length | Stored in 0.02% EDTA 4°C (until use)
Protocol 1: 1% Triton X100 (24 h)
Protocol 2: 0.5% SDS (24 h)
Protocol 3: 1% TBP (24 h)
Protocol 4: 1% Triton X100 + 0.5% SDS (24 h)
Protocol 5: 1% TBP + 0.5% SDS (24 h)
Protocol 6: 1% TBP + 1% Triton X100 (24 h)
Followed by RNAse/DNAse 37°C (24 h)
0.02% EDTA 4°C (24 h) | Histology
Mechanical testing | — | — | Acellularized ST/FDP matrix; fresh native ST/FDP tendon (ctrl) | Protocol 4 → collagen ruptures
Protocol 5 → no impact on tendon structure and collagen content
Acellularized matrix → histology and biomechanics similar to ctrl | [13] |
|
Rabbit
FDP
5 cm
length | Frozen −80°C (until use)
0.05% Trypsin/EDTA 37°C (24 h)
0.5% Triton X100 RT (24 h) | Histology
Cell count | Rabbit tenocytes
2 × 106 cells/mL
Rotating reseeding (24 h)
Static or dynamic culture (LigaGen L30-4C) for 5 days | Histology
Mechanical testing | Freeze-stored acellularized FDP matrix (AC); acellularized FDP matrix + bioreactor loading (A+); acellularized FDP matrix w/o bioreactor loading (A−); reseeded FDP matrix + bioreactor loading (R+); reseeded FDP matrix w/o bioreactor loading (R−); fresh native FDP tendon (NC); freeze-stored FDP tendon (FC) | NC → highest UTS/EM
R+ → higher UTS/EM compared to FC, AC, R−, and A− | [14] |
|
Rabbit ST
Undefined size | Frozen −80°C (until use)
1% SDS (24 h) | — | Rabbit dermal fibroblasts
2 × 106 cells/mL
Static culture by injection for 4, 7, and 14 days | Histology
Mechanical testing
Immunohistochemistry | Acellularized ST matrix; reseeded ST matrix; fresh native ST tendon (ctrl) | Acellularized matrix → histology and immunohistochemistry similar to ctrl
Procollagen I → present in ctrl and reseeded matrix, absent in acellularized matrix
Collagens I-III-IV-VI, versican → similar among treatments
UTS/stiffness/elongation → higher in reseeded matrix | [15] |
|
Rabbit
FDP
5 cm
length | Frozen −80°C (until use)
0.05% Trypsin/EDTA 37°C (24 h)
0.5% Triton X100 RT (24 h) | Histology
Cell count | Rabbit ASCs and Fs
2 × 106 cells/mL
Rotating reseeding (24 h)
Static or dynamic culture (LigaGen L30-4C) for 5 days | Histology
Mechanical testing | Reseeded FDP matrix + bioreactor loading (ASCs+); reseeded FDP matrix w/o bioreactor loading (ASCs−); reseeded FDP matrix + bioreactor loading (Fs+); reseeded FDP matrix w/o bioreactor loading (Fs−); fresh native FDP tendon (ctrl) | UTS/EM → higher in ASCs+, Fs+ versus ASCs−, Fs−, ctrl
ASCs+, and Fs+ → cells oriented parallel to the direction of strain | [17] |
|
Rabbit
FDP
1.5 cm
length | Fresh samples
Protocol 1: 0.5% SDS (30′)
Protocol 2: 0.05% Trypsin/EDTA (24, 48, and 72 h)
Protocol 3: 0.05% Trypsin/EDTA (24 h); 0.5% Triton X100 (24 h)
Protocol 4: 3% Triton X100 (24, 48 h)
Protocol 5: 3% Triton X100 (24 h); 0.5% SDS (24 h)
Protocol 6: Frozen −70°C (until use); 0.05% Trypsin/EDTA (24 h); 0.5% Triton X100 (24 h) | — | Rabbit tenocytes (endotenon, epitenon)
1 × 106 cells/1.5 cm construct
Static culture for 1, 3, and 6 weeks | Histology
Cell fluorescent labeling | Reseeded FDP matrix with endotenon cells; with epitenon cells; and with mixed cells | Protocol 6 → best decellularization process
Epitenon/endotenon cells → attached to the periphery of the acellularized matrix
Endotenon cells → penetrated the matrix core by 3 weeks | [11] |
|
Rabbit PT
3 cm
length;
1.5 cm
width;
3 mm
thickness | Frozen −20°C (until use)
Protocol 1: 1% TBP RT (48, 72 h)
Protocol 2: 1% SDS RT (24, 48 h) | Histology
GAG content
Mechanical testing | Human HS68
2 × 105 cells/mL
Static culture for 4 h, 1, 3, 6, and 14 days | Histology
Cell count | Reseeded TBP-treated PT matrix; reseeded SDS-treated PT matrix; fresh native PT (ctrl) | SDS and TBP → 70–90% of cell removal
Reseeded matrix biomechanics → similar to ctrl
TBP-treated matrix → supported cell proliferation better than SDS-treated matrix and was histologically similar to ctrl | [19] |
|
Rabbit PT
1.5 cm length; 0.3 cm width;
1 mm
thickness | Frozen liquid nitrogen/thaw (5 cycles)
Frozen −80°C (until use) | — | Rabbit tendon fibroblasts
5 × 106 cells/mL
Static culture in collagen gel with or w/o anti-TGFβ1 antibody for 6 weeks | Mechanical testing
Cell fluorescent labeling | Acellularized PT matrix; reseeded PT matrix; reseeded PT matrix + anti-TGFβ1 antibody | Anti-TGFβ1 antibody decreased UTS/EM in reseeded matrix | [20] |
|
Canine FDP
2 cm
length | Frozen −80°C (until use)
0.05% Trypsin/EDTA 37°C (24 h)
0.5% Triton X100 RT (24 h) | Mechanical testing | Canine BMSCs
2 × 107 cells/mL
Static culture for 2 weeks | Histology
Cell/DNA content
LiveDead | Acellularized FDP matrix; acellularized FDP matrix, perforated with multiple slits (MS); acellularized FDP matrix, perforated with multiple slits + hyaluronic acid gelatin (MS-SM); reseeded FDP/MS matrix; fresh native FDP tendon (ctrl) | UTS → higher in acellularized FDP/MS matrix compared to ctrl
DNA content → lower in MS than ctrl; higher than in acellularized matrix with or w/o BMSCs
BMSCs were viable after 2 weeks | [34] |
|
Canine FDP
Undefined size | Frozen −80°C (until use)
0.05% Trypsin/EDTA 37°C (24 h)
0.5% Triton X100 RT (24 h) | Histology
SEM
Mechanical testing | — | — | Acellularized FDP matrix; acellularized FDP matrix + hyaluronic acid gelatin (GT); fresh native FDP tendon (ctrl) | UTS → higher in acellularized matrix than ctrl and GT
EM → similar among treatments
Matrix surfaces → smooth in ctrl and GT; rough in acellularized matrix | [33] |
|
Canine AT
4 cm
length;
0.3 mm
thickness | Frozen −80°C/thaw (5 cycles)
RNAse/DNAse 37°C (4, 8, and 12 h) | Histology
Cell/DNA content
SEM
Mechanical testing
Biochemical analysis | Murine fibroblasts (3T3)
1 × 106 cells/mL
Static culture for 4 days | Histology
SEM | Acellularized AT matrix (only freeze/thaw); acellularized AT matrix (freeze/thaw + nuclease treatments); fresh native AT (ctrl) | Complete cell removal → in repetitive freeze/thaw + nuclease treatment for 12 h
Acellularized matrix → ultrastructure, proteoglycans, and growth factors were preserved; UTS was retained in 85.62% of ctrl | [37] |
|
Canine IT
2.5 cm length;
1 cm
width;
0.05 mm
thickness | Frozen −80°C (until use)
Frozen liquid nitrogen/thaw (5 cycles) RNAse/DNAse 37°C (12 h) | — | Canine BMSCs
5 × 106 cells/mL
Static culture for 2, 7, and 14 days | Histology
Cell fluorescent labeling
Gene expression
Mechanical testing | Acellularized IT matrix; reseeded IT matrix; fresh native IT (ctrl) | Reseeded matrix → viable cells aligned to the collagen fibers; higher tenomodulin, MMP13 and lower collagen I than in BMSCs before seeding Biomechanics → similar between acellularized and reseeded matrixes | [35] |
|
Canine AT
4 cm
length;
0.3 mm
thickness | Frozen liquid nitrogen/thaw (5 cycles) RNAse/DNAse 37°C (12 h) | SEM
Mechanical testing | Rat BMSCs and TSPCs
2 × 105 cells/cm2
Static culture for 6 h and 1, 3 days | LiveDead
Cell proliferation
SEM | Acellularized AT matrix; reseeded AT matrix; fresh native AT (ctrl) | Acellularized matrix → preserved ECM and EM similar to ctrl
Reseeded matrix → homogenous cell distribution, alignment, and tenogenic differentiation of seeded cells | [57] |
|
Canine AT
4 cm
length;
0.3 mm
thickness | Frozen liquid nitrogen/thaw (5 cycles) RNAse/DNAse 37°C (12 h) | — | Canine BMSCs
5 × 105 cells/mL
Dynamic culture for 1, 3, and 7 days | Histology
Cell fluorescent labeling
SEM
Mechanical testing | Acellularized AT matrix; reseeded AT matrix; fresh native AT (ctrl) | Reseeded matrix → homogenous cell distribution at day 7; tenogenic differentiation of BMSCs; UTS similar to ctrl | [39] |
|
Porcine
AT
1 cm
length;
0.5 cm
width;
3 mm
thickness | Frozen −20°C (until use)
Protocol 1: 3% Triton X100 37°C (24 h)
Protocol 2: 1% SDS + 0.2% natrium acid + 5 mM EDTA RT (24 h)
Protocol 3: 0.05% Trypsin/EDTA RT (24 h) | — | Human tenocytes
4 × 106 cells/cm2
Centrifuging reseeding + static culture
Rotating culture
Reseeding by injection + rotating culture for 1, 3 weeks | Histology
Cell fluorescent labeling
DNA/GAGs content | Acellularized porcine AT matrix; reseeded porcine AT matrix; fresh native human/porcine AT (ctrl) | Porcine ctrl → higher cell/GAGs content; more compact and wavy pattern than human ctrl
Acellularized matrix → lower GAGs/DNA content
Reseeded porcine matrix → greater cell/GAGs content compared to ctrl; inhomogeneous, superficial, and not aligned tenocyte distribution
Rotating culture → greater cell/GAGs content compared to centrifuging-based seeding, lower cell/GAGs content compared to ctrl
Cell injection → poor cell distribution; no GAGs deposition | [40] |
|
Porcine
ATT
12 cm
length;
1 cm
width | Frozen −70°C (until use)
0.25% Trypsin/collagenase type I 37°C (4 h)
NaCl washing 4°C (3 cycles) (1 h)
Ultrasonication (5′) | Histology
DNA/GAG/collagen
SEM
Mechanical testing | Dynamic culture w/o cells in bioreactor (110% tension + 90° torsion) for 1, 3, and 7 days | — | Acellularized ATT matrix; acellularized ATT matrix cultured in bioreactor; fresh native ATT (ctrl) | Acellularized matrix → lower DNA content; lower UTS compared to ctrl
Acellularized matrix treated in bioreactor → greater UTS than ctrl | [41] |
|
Porcine DT
Undefined size | Stored at 4°C (until use)
Protocol 1: 0.1% PAA + 4% EtOH stirring RT (16 h)
Protocol 2: 1% Triton X100 RT (24 h)
Protocol 3: 1% Triton X100 + 1% TBP RT (24 h)
Protocol 4: 2% TBP RT (24 h)
Protocol 5: 1% TBP RT (24 h)
Protocol 6: 1% SDS RT (24 h)
Protocol 7: 0.5% SDS RT (24 h) | Histology
Cell viability
SEM
Mechanical testing
Hydroxyproline | — | — | Acellularized DT matrix; stored native DT (ctrl) | Protocol 5 → good cell removal; tissue structure and composition were preserved
Acellularized matrix → UTS, EM, and hydroxyproline release were similar to ctrl | [42] |
|
Porcine PT
6 cm
length;
1.5 cm
width;
5 mm
thickness | Protocol 1: 0.1% EDTA + aprotinin RT (24 h); 0.1% SDS RT (24 h); RNAse/DNAse 37°C (3 h)
Protocol 2: 0.1% EDTA + aprotinin RT (24 h); 0.1% SDS RT (24 h); RNAse/DNAse 37°C (3 h) | Histology
Immunohistochemistry
Hydroxyproline
Collagen/GAGs
Mechanical testing | Human tenocytes
1 × 105 cells/mL
Static culture for 3 weeks | Histology
LiveDead | Acellularized PT matrix; acellularized + sonicated PT matrix; reseeded PT matrix; reseeded + sonicated PT matrix; fresh native PT (ctrl) | Ultrasonication treatment → optimal treatment; collagen and GAGs were similar to ctrl
Reseeded + sonicated PT matrix → centrally colonized by unviable cells | [43] |
|
Human FDP
2 cm
length;
0.25 cm
width | 0.1% EDTA + protease inhibitor 37°C (24 h)
0.1% SDS 37°C (5 h)
DNAse RT (1 h) | — | Human ASCs
1 × 106 cells/0.5 mL
Static culture by injection for 7 days | Histology
Cell/DNA content
Immunohistochemistry | Acellularized FDP matrix; reseeded FDP matrix; reseeded FDP matrix + collagen solution; fresh native FDP tendon | Acellularized matrix → complete cell removal, absence of DNA content, and no changes in ECM structure
Reseeded matrix → cell distribution on surface
Reseeded matrix + collagen → deeper penetration of collagen, COMP positive, and viable cells aligned to collagen fibers | [26] |
|
Human FDP
1.5 cm length | Frozen −70°C (until use)
Protocol 1: 0.1% EDTA RT (4 h); 0.1% SDS + 0.1% EDTA RT (24 h)
Protocol 2: 0.1% EDTA RT (4 h); 0.1% SDS + 0.1% EDTA RT (24 h); 2, 5, and 10% PAA (4, 20 h) | Histology | Human dermal fibroblasts
1 × 106 cells/mL
Rotating culture for 5 days | Cell/GAGs/collagen content
LiveDead
Mechanical testing | Acellularized FDP matrix with 5% PAA; reseeded FDP matrix | Protocol 2 → increased porosity, improved cell penetration, and migration
Acellularized matrix → similar collagen/GAG content when treated with or w/o PAA; UTS/EM lower than in reseeded matrix | [27] |
|
Human FDP
1 cm
length | 0.1% SDS + 0.1% EDTA RT (24 h)
5% PAA (6 h)
frozen −80°C (until use) | — | Human fibroblasts, tenocytes, and ASCs
5 × 105 cells/mL
Rotating culture with or w/o growth factors for 5 days | Histology
Cell count
Immunohistochemistry | Acellularized FDP matrix; reseeded FDP matrix; fresh native FDP tendon (ctrl) | Enhanced cell proliferation using 5 ng/mL bFGF, 50 ng/mL IGF-1, and 50 ng/mL PDGF-BB
Reseeded matrix + growth factors → improved reseeding compared to ctrl w/o growth factors | [29] |
|
Human FDP
1 cm
length | Frozen −70°C (until use)
0.1% EDTA (4 h)
Protocol 1: 1% Triton X100 (24 h)
Protocol 2: 1% TBP (24 h)
Protocol 3: 1% SDS (24 h)
Protocol 4: 0.1% SDS (24 h) | Histology
DNA/GAGs/collagen
Mechanical testing | Human dermal fibroblasts
0.5, 1, 2 × 106 cells/mL
Static culture for 6, 24 h | Histology
Cell count
LiveDead | Acellularized FDP matrix; reseeded FDP matrix; fresh native FDP tendon (ctrl) | Protocols3/4 → lower DNA content compared to ctrl
Acellularized matrix → good collagen/GAG content
UTS was similar to ctrl (especially Protocol 4)
Reseeded matrix → cell distribution on surface | [31] |
|
Equine SDFT and DDFT
3 cm
length;
1 cm
width;
2 mm
thickness | Protocol 1: frozen liquid nitrogen/thaw (5 cycles); 1% Triton X100 RT (48 h)
Protocol 2: frozen liquid nitrogen/thaw (5 cycles); 1% SDS RT (48 h)
Protocol 3: 1% Triton X100 RT (48 h)
Protocol 4: 1% SDS RT (48 h) | Histology
Cell/DNA content
TEM | Equine ASCs
1.3 × 105 cells/cm2
Static culture for 7, 14 days | Histology
MRI
LiveDead | Acellularized SDFT/DDFT matrix; reseeded SDFT/DDFT matrix; fresh native SDFT/DDFT (ctrl) | Acellularized matrix → lower DNA content in Protocols 1/2 (1% residual nuclei; 20% residual DNA) compared to Protocols3/4 (20% residual nuclei; 40% residual DNA); absence of morphological ECM alterations
Reseeded matrix → best cell distribution in Protocol 1 | [45] |
|
Equine SDFT
4 cm
length;
1 cm
width;
0.4 mm
thickness | Frozen −80°C (until use)
Protocol 1: 1% TBP 4°C (48 h)
Protocol 2: 1% SDS + 0.5% Triton X100 4°C (48 h)
Protocol 3: 1% SDS 4°C (48 h)
Protocol 4: 2% SDS 4°C (48 h)
Followed by
0.05% Trypsin/EDTA (10′)
DNAse (30′) | Histology
DNA/GAGs/collagen
LiveDead
SEM
Mechanical testing | Equine BMSCs
2 × 104 cells/cm2
Static culture for 11 days | Histology
Cell count
LiveDead | Acellularized SDFT matrix; reseeded SDFT matrix; fresh native SDFT (ctrl) | Protocols2, 3, and 4 → lower DNA/GAGs content compared to ctrl (especially Protocol 4); preserved collagen content; UTS/EM were similar to ctrl
Reseeded matrix → higher cell proliferation/integration in Protocol 4 | [46] |
|
Equine SDFT
4.5 cm
length;
0.4 mm
thickness | Frozen/thaw (4 cycles)
2% SDS 4°C (48 h)
0.05% Trypsin/EDTA (10′)
DNAse (30′) | Histology
DNA/GAGs/collagen
Mechanical testing | Equine BMSCs
2 × 104 cells/cm2
Static and dynamic culture for 11 days | Histology
DNA/GAGs/collagen
Mechanical testing | Acellularized SDFT matrix; reseeded SDFT matrix; fresh native SDFT (ctrl) | Acellularized matrix → GAGs lost
Reseeded matrix → best dynamic protocol for BMSCs integration and tenogenic differentiation was cyclic strain of 3% at 0.33 Hz; UTS/EM greater than acellularized matrix; GAGs similar to ctrl | [48] |
|
Equine SDFT
1 cm
length;
1 cm
width;
0.5 mm
thickness | Frozen −80°C/thaw (4 cycles) | — | Equine BMSCs and TSPCs
1.25 × 105 cells/cm2
Static culture for 7 days | Histology
Cell/GAGs/collagen
Gene expression | Acellularized SDFT matrix; reseeded SDFT matrix (BMSCs); reseeded SDFT matrix (BMSCs + IGF1); reseeded SDFT matrix (TSPCs); reseeded SDFT matrix (TSPCs + IGF1) | Acellularized matrix → cell numbers 1.6- to 2.8-fold higher for TSPCs than for BMSCs and 0.8- to 1.7-fold higher for IGF-I-treated than for untreated cells
TSPCs IGF1-treated cell → greatest collagen/GAGs content
Collagens I, III, COMP → similar among groups | [47] |
|
Bovine AT
2 cm
length;
2 cm
width; 2 mm
thickness | Frozen (until use)
1% SDS 4°C (48 h)
0.1 mM EDTA (48 h) | SEM
Mechanical testing | — | — | Acellularized matrix; acellularized matrix crosslinked with 0.1, 0.5, 1, and 2.5% of glutaraldehyde | Crosslinked acellularized matrix → UTS/EM greater than acellularized matrix | [49] |
|
Rat tail tendon
2-3 mm Ø | Frozen −20°C (until use)
Protocol 1: 1% Triton X100 (24 h)
Protocol 2: 1% Triton X100 + 1% TBP (24 h)
Protocol 3: 1% TBP (12, 24, and 48 h)
Protocol 4: 2% TBP (24 h)
Protocol 5: 0.5% SDS (24 h)
Protocol 6: 1% SDS (12, 24 h) | Histology
Mechanical testing | — | — | Acellularized tail tendon matrix; fresh native rat tail tendon (ctrl) | Protocol 1 → collagen fiber disruption; no cell removal
Protocol 3 (48 h) or Protocol 6 (24 h) → good acellularized matrix, normal structure and mechanical properties | [23] |
|
