Advances in Tendon and Ligament Tissue Engineering: Materials Perspective
Table 1
Summary of the literature related to the applications of hybrid biomaterial in tendon tissue engineering. indicates that approximate numbers were extracted from supplied figures since no exact reference values are mentioned in the text. PDS: poly-dioxanone; SCX, scleraxis; TGFβ, transforming growth factor beta; hECS-MSCs, mesenchymal stem cells derived from human embryonic stem cells; rhSDF1, recombinant human stromal cell-derived factor 1; PGA, poly-(lactide-co-glycolide); PCL, poly-caprolactone; bFGF, basic fibroblast growth factor.
After implantation in Achilles tendon, Tensile strength: 4.88±8.07 MPa No report of other mechanical properties.
(i) Grossly, the implanted cell-seeded scaffold was integrated with the native tissue interference, with a smooth surface cord-like shape with less noticeable remaining material after 45 weeks. (ii) Tissue adhesions, described grossly to be less compared to control. (iii) Parallel and more mature collagen fibers and longitudinally aligned cells are presents than control.
Young’s modulus Nonseeded about 2.2 MPa. Cell seeded about 3 MPa. Tensile strength Nonseeded about 3 MPa. Cell seeded about 4.5 MPa.
(i) Significantly higher cell proliferation in the nanoyarn scaffold compared to other scaffolds and control. (ii) SEM showed spindle-shaped cell in both nanoyarn and aligned nanofiber scaffold, while polygonal and random pattern cells are found in the random oriented fibers. (iii) Expression of tendon specific ECM (type I collagen, type III collagen, decorin, tenascin-C, and biglycan) was significantly higher at 14 days in the nanoyarn group.
Mechanical stress: Dynamic group was 59.58 ± 7.81 MPa Static group was 43.18± 6.58 MPa Control group was 32.43± 5.27% MPa Young’s modulus: Dynamic group was 51.99±7.16 MPa Static group was 34.76 ± 4.75 MPa. Control group was 23.30 ± 3.83 MPa
TDSCs were used in scaffold seeding with both dynamic and static culture conditions. In vitro: (i) TDSCs showed elongated fibroblast-like morphology with a significant increase in cell count in dynamic group at 14 days. (ii) More cell infiltration and dense matrix in dynamic group. (iii) PCR confirmed Tendon related mRNA expression more in dynamic group. (iv) Western blotting showed significant increase in the protein expression levels of Collagens I and III, and tenascin-C in the dynamic group. In-vivo: (i) Significantly lower number of cells at 12 weeks with greater matrix deposition and longitudinal spindle-shaped cells in dynamic group compared to others. (ii) Collagen content was highest in dynamic group reaching 77.76 ± 6.82% of normal rabbit patellar tendon (174.31 ± 13.89 μg/mg). (iii) Collagen I expression was Significantly higher in dynamic group.
(i) Both produced scaffolds supported fibroblasts metabolic activity and proliferation till final assessment point (72 hrs). (ii) In vivo assessment showed lack of any local or systemic inflammatory response using N-acetylglucosaminidase (NAG) and nitric oxide (NO) serum levels. (iii) No associated hepatotoxicity in histologic assessment. (iv) Histologic assessment of explanted scaffold showed formation of thin capsule around the implant with homogenous granulation tissue.
Tensile strength was 0.5058 ± 0.2130 MPa Maximum strain was 18.49% ± 8.210 Young’s modulus was 7.339 ± 2.131 MPa
(i) Statistically higher viability of both myoblast and fibroblasts in all regions of the scaffold. (ii) Scaffold could support the formation of myotubes that is essential for normal muscle-tendon junction formation.
Maximum Force to break For uncoated 2 and 3 layers scaffold: 7.89±1.5N and 7.45±0.3N, For gel coated 2 and 3 layers scaffolds: 4.76 ± 0.23N and 6.49 ± 0.09N. No report of other mechanical properties.
(i) Alginate coating was associated with significantly less attached proteins than control. (ii) Both coated and uncoated scaffold maintain 50% of their substance after incubation with PBS containing 104 units/ml lysozyme solution. (iii) Alamar blue and DNA concentration assessment showed high cellular viability, metabolic activity, and proliferation up to 7 days after seeding. (iv) Seeded scaffolds were shown to support cellular alignment.
Electrospun collagen I nanofiber/collagen microfiber [32]
In vitro (fibroblast) In vivo (rabbit)
Before implantation: Maximum load was 28.33 ± 2.19 N. Maximum stress was 2.69 ± 0.47 MPa Maximum strain was 61.34 ± 4.71 % Young’s modulus was 43.81 ± 4.19 KPa. 60 days after implantation to Achilles tendon: Maximum load was about 63.72 N. Maximum stress was about 9.84 N/mm. Maximum strain was about 16.35% Young’s modulus was about 0.62 N/mm.
In vitro: (i) Significantly higher cell viability in the aligned hybrid scaffold compared to others. (ii) SEM and immunofluorescence proven superior alignment of fibroblasts together with cell proliferation with close cell-to-cell contact in the aligned hybrid scaffold compared to others. In vivo: (i) Significant increase in the number, density, and alignment of the collagen deposition with mature elastic fibers. (ii) Significant increase in the number and maturity of tenoblasts and tenocytes. (iii) Significantly lower peritendinous adhesions, muscle fibrosis, and atrophy and inflammatory cells found in the treated tendons compared to controls.
Electrospun collagen I nanofiber/collagen microfiber/ PDS sheets. [33]
In vivo (rabbit)
After 60 days of implantation to Achilles tendon: Maximum load was about 74.02 N Maximum stress was about 11.37 MPa Young’s modulus was about 0.754 MPa
(i) Significantly higher fibrillogenesis after PDS treatment. (ii) Significantly higher collagen fibrils were found in the PDS treated scaffolds compared to the regular one. (iii) Significant increase in the load to failure and load to yield point with PDS treatment. (iv) Significantly improved peritendinous adhesions, muscle fibrosis, and atrophy scores that are comparable in the PDS treated and nontreated collagen scaffolds.
Core-shell Collagen type I/ Glycosaminoglycan. [34]
In vitro (Tenocytes)
Tensile modulus for dry membranes was about 636 ± 47 MPa to 693 ± 20 MPa Tensile modulus for hydrated membranes was 30 MPa. Tensile modulus for dry aligned core scaffold ranges from 833 ± 236 to 829 ± 165 KPa
(i) Significant increase in the number of cells at day 1 after seeding in the core-shell composite compared to core alone scaffolds. (ii) Higher metabolic activity observed at day 7 in the core alone scaffold that is statistically significant. (iii) No significant difference in the metabolic activity and cell number between the two groups at 14-day period.
Maximum stress was 34.63 ± 9.75 MPa Maximum strain was 0.21 ± 0.04 MPa Young’s modulus was 49.48 ± 10.71 MPa
(i) Human adult fibroblasts were used in the assessment process. (ii) The authors showed that addition of resilin and the use of poly(ethylene glycol) either tetrasuccinimidyl glutarate did compromise the cellular activity. (iii) The scaffold produced managed to support cellular proliferation and 100% cellular alignment after 7 days of seeding compared to 80% in collagen control.
poly--caprolactone (PCL) and methacrylated gelatin (mGLT) [37]
In vitro (ADSCs)
Maximum load was about 0.25 N. No report of other mechanical properties.
(i) Tenogenic differentiation was induced through differentiation medium having DMEM, 2% FBS, P/S, and 10 ng/ml TGF-β3 for 7 days after seeding. (ii) Seeded constructs were shown to exhibit elongated morphology as well as expression of tenogenic markers (scleraxis and Tenascin-C). (iii) Stacked scaffold was shown to have adequate porosity for cell diffusion and differentiation.
Sericin extracted knitted silk/cross-linked collagen type I microsponges [38]
In vitro (hESC-MSCs) In vivo (mouse)
4 weeks after implantation to Achilles tendon of cell-seeded scaffolds: Maximum load was 65.24 ± 9.58 N Maximum stress was 6.72 ± 0.90 MPa Stiffness was 28.26 ± 2.95 N/mm Young’s modulus was 34.91 ± 5.08 MPa
In vitro: (i) Good cell attachment, proliferation, and spreading at 14 days' period. (ii) Scx., collagen I, and collagen III expression were significantly higher in dynamic group than the control. In vivo assessment after 4 weeks of implantation: (i) Grossly, well-integrated construct with native tendons. (ii) Viable cells are presents showing spindle-shaped morphology. (iii) Significantly higher collagen I, III, Vα1, Vα2, and TGFβ1 in cell-seeded scaffold compared to cell free scaffold. (iv) Neocollagen was replacing exogenous one with more content, and mature morphology in cell-seeded scaffold compared to cell free scaffold.
Sericin extracted knitted silk/cross-linked collagen type I microsponges/rhSDF-1 alpha [39]
In vitro (fibroblast) In vivo (rat)
4 weeks after implantation in Achilles tendon: Maximum load was 68.5 ± 18 N Maximum stress was 7.02 ± 1.7 MPa Stiffness was 39.0 ± 6.6 N/mm Young’s modulus was 45.3 ± 10.4 MPa
In vivo assessment after 4 weeks of implantation: (i) More fibroblasts-like cells and less inflammatory cells are found early after implantation (4 days, 1 week). (ii) More organized continuous collagen fibers are found with a higher concentration of collagen type I. (iii) Good vascular components with less inflammatory cells are present. (iv) Significantly higher expression of Collagens I and III, decorin in the SDF-1 scaffolds at all assessment periods. (v) Large fibrils of Achilles tendons formed in the SDF-1 scaffolds (41.6 ± 5.5 nm) compared to control (37.1 ± 2.9 nm).
Sericin extracted knitted silk/cross-linked collagen type I microsponges/SCX engineered cells [40]
In vitro (hESC-MSCs) In vivo (mouse)
4 weeks after implantation: Young’s modulus was 60.63 ± 17.6 MPa Maximum stress was 8.73 ± 2.15 MPa 8 weeks after implantation: Young’s modulus was 71.3 ± 12.4 MPa Maximum stress was 10.1 ± 2.2 MPa
In vitro: (i) SCX treatment increases collagen I expression with enhanced cell-sheet formation. (ii) Decreased capacity of adipogenic, chondrogenic, and osteogenic differentiation of the cells with more tenogenic differentiation. In vivo: (i) Good proliferation and early matrix deposition in the SCX cells compared to control. (ii) Increased Collagen Ia1, Ia2, and tendon related transcription factor Eya2 in SCX cells compared to control. (iii) More fibroblast-like spindle-shaped cells and fewer immunogenic cell infiltrates in the SCX cells group. (iv) Higher expression of biglycan in the SCX cells group, indicating more mature endogenous collagen.
Maximum stress was 13.5 ±1.5 MPa Young’s modulus was 21.7±4 MPa
(i) Human fibroblasts were seeded on composite scaffold to test for viability. (ii) Assessment was made through Alamar Blue assay and showed that samples with higher collagen content had higher cellular viability profile (COL75/PU25) than other groups.
Silk coated with Polycaprolactone (PCL) or Poly(3-hydroxybutyrate) (P3HB) nanofibers [42]
In vitro (Fibroblast)
Maximum load 97.6±11.4 N for silk fibroin/P3HB 110.5±6.6 N for silk fibroin/PCL No report of other mechanical properties.
(i) Human fibroblasts were seeded on composite scaffold and showed good cellular viability and no toxicity (assessment for 3 days).
Maximum load for dynamic culture condition Aligned scaffold 144.44 ± 5.03 N (7 days) 172.08 ± 6.28 N (14 days) Random Scaffold 122.35 ± 3.67 N (7 days) 138.67 ± 9.22 N (14 days) Stiffness Aligned scaffold 24.33 ± 1.40 N/mm (7 days) 26.93 ± 2.40 N/mm (14 days) Random Scaffold 17.48 ± 0.93 N/mm (7 days) 23.07 ± 2.54 N/mm (14 days) No report of other mechanical properties.
(i) Highly significant cell viability in the aligned scaffold compared to the random one at both dynamic and static conditions. (ii) Consistent cellular proliferation in both aligned (dynamic and static) and dynamic random scaffold. (iii) Higher collagen deposition in the dynamic aligned scaffold compared to the static one and the random scaffold groups. (iv) Dynamic culture improved the histological assessment of both aligned and random scaffold with more cell elongation and matrix deposition. (v) Higher expression level of collagen I, tenascin-C, and tenomodulin in the aligned dynamic scaffolds compared to others.
Knitted PLGA-PLLA / coating with (1) PCL. (2) PLGA nanofiber. Type I collagen [44]
In vitro (BMSCs)
Maximum load Uncoated 68.4 ± 5.37 N. PCL coated 63.1 ± 4.52 N. PLGA coated 56.3 ± 6.61 N. Collagen coated 59.5 ± 8.3 N. Stiffness Uncoated 9.1 ± 2.38 N/mm. PCL coated 4.3 ± 0.91 N/mm. PLGA coated 5.8 ± 0.70 N/mm. Collagen coated 5.7 ± 0.48 N/mm. No report of other mechanical properties.
(i) Efficient cell seeding on PLGA nanofiber coated and collagen-coated scaffolds. (80-89% and 61-69%, respectively). (ii) Significantly higher cell proliferation between and culture days of PLGA nanofiber coated knitted PLGA scaffolds.
Maximum load of unseeded scaffolds 75.3 ± 4.79 N on day 0, and 61.5 ± 3.43 N on day 21 Maximum load of rolled seeded scaffolds 68.2 ± 6.72 N on day 21 Stiffness of unseeded scaffolds 4.8 ± 0.52 N/mm on day 0, and 5.9 ± 0.54 N/mm on day 21 Stiffness of rolled seeded scaffolds 5.5 ± 0.30 N/mm on day 21 No report of other mechanical properties.
(i) Dual surface seeded scaffolds showed significantly higher cell proliferation rates compared to single surface seeding. (ii) Increased cell proliferation between 14 days and 21 days after culture. (iii) Rolled-up scaffolds had nonsignificantly lower cell proliferation rates.
3 weeks after culture of rolled scaffolds: Maximum load Unseeded scaffolds were about 61.5 N Seeded bFGF-free scaffolds were about 68.2 N Seeded bFGF scaffolds were about 82.7 N Stiffness Unseeded scaffolds were about 5.92 N/mm Seeded bFGF-free scaffolds were about 5.53 N/mm Seeded bFGF scaffolds were about 6.97 N/mm
(i) Significantly higher cell viability in the bFGF scaffolds compared to bFGF-free scaffolds. (ii) Significantly higher collagens I and III, fibronectin, and biglycan 14 days after culture in the bFGF scaffolds compared to bFGF free scaffolds. (iii) Significantly higher collagen content in the bFGF scaffolds compared to bFGF free scaffolds by week after culture.
Tensile strength was 235.2 ± 8.5 MPa Maximum strain was 12.3 ± 0.3 %
(i) Significantly lower number of unattached cells in alginate–chitosan group compared to polyglactin and alginate alone. (ii) Fibroblasts were spread on the polymer fibers. (iii) Prominent collagen type I production 14 days after culture was more on the scaffold surface by immune staining with no clear visualization of both types II and III collagen.
(i) In vitro: Tensile strength: Before seeding was 213.3 ± 10 MPa 2 hrs after seeding was 60 ± 6.7 MPa 14 days after seeding was 66.7 ± 6.8 MPa 28 days after seeding was 65.1 ± 6.6 MPa Maximum strain: Before seeding was 3.2 ± 0.6 % (ii) In vivo tendon model: Tangent modulus for cell-seeded scaffold: 4 weeks after implantation: about 58 ± MPa 12 weeks after implantation: about 85 ± MPa In vivo ligament model: Maximum load for cell-seeded scaffold: 12 weeks after implantation: about 110 ± 10 N
In vitro up to 28 days after cell seeding: (i) Gross observation of ECM production by light microscopy. (ii) Prominent collagen type I production 14 days after culture more on the scaffold surface. In vivo tendon model: (i) Type I collagen is seen only in cell-seeded scaffold. In vivo ligament model: None.
