Postnatal mechanical loading drives adaptation of tissues primarily through modulation of the non-collagenous matrix
- Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, United Kingdom
- Comparative Biomedical Sciences, The Royal Veterinary College, Royal College Street, United Kingdom
- University College London, Department of Orthopaedics and Musculoskeletal Science, Stanmore Campus, Royal National Orthopaedic Hospital, United Kingdom
- Institute of Bioengineering, School of Engineering and Materials Science, Queen Mary University of London, United Kingdom
Figures
Figure 1 with 1 supplement
Fascicle response to mechanical testing shows increase in strength with development but few significant differences between tendon types, indicating that the fascicles show minimal structural specialisation in response to loading.
(a) Representative curves for 10 preconditioning cycles for the SDFT and CDET fascicles in the foetus and 1–2 years age group. (b) Representative force-extension curves to failure for the SDFT and …
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Figure 1—source data 1
Fascicle and IFM mechanical properties.
- https://cdn.elifesciences.org/articles/58075/elife-58075-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
SDFT and CDET in the equine forelimb, tendon structure, and schematic showing procedure for biomechanical testing.
(a) Schematic of the equine forelimb with the CDET and SDFT highlighted. (b) Tendon structure (partially reproduced from Figure 1, Spiesz et al., 2015), Journal of Orthopaedic Research, published …
Figure 2
Mechanical testing of the IFM shows an equivalent increase in failure properties between the SDFT and CDET with development, but development of an extended low stiffness toe region and more elastic behaviour in the SDFT.
(a) Representative curves for 10 preconditioning cycles for the SDFT and CDET IFM in the foetus and 1–2 years age group. (b) Representative force-extension curves to failure for the SDFT and CDET …
Figure 3 with 1 supplement
The SDFT and CDET are histologically similar at birth and differentiate with development especially in the IFM.
(a) Representative images of H and E sections of the SDFT and CDET demonstrate structural development: foetus, 0 days (did not weight-bear), 0–1 year, and 1–2 years, whilst (b) Radar plots enable …
Figure 3—figure supplement 1
Scoring of histologic variables for the IFM and fascicle in the SDFT and CDET through postnatal development.
* Significant difference between tendons, a-f significant difference between age groups. Error bars depict standard deviation.
Figure 4 with 1 supplement
Immunohistochemical assays show divergence of PGR4 (lubricin) and elastin with maturation between functionally distinct tendons.
IFM and fascicle staining scores are shown for decorin (DCN), fibromodulin (FMOD), lubricin (PRG4), and tenascin-C (TNC) in the SDFT and CDET, alongside representative images of immunohistochemical …
Figure 4—figure supplement 1
Scoring of ELN staining for the IFM and fascicle in the SDFT and CDET through postnatal development.
Error bars depict standard deviation.
Figure 5 with 2 supplements
The fascicle proteome remains the same during postnatal development and tendon loading whereas the IFM proteome starts changing following tendon loading in postnatal development.
(a) Heatmap of differentially abundant proteins in foetus, 0 days (did not weight-bear), 0–1 month, 3–6 months, and 1–2 years SDFT IFM and fascicles (p<0.05, fold change ≥2). Heatmap colour scale …
Figure 5—figure supplement 1
Classification of SDFT IFM and fascicle identified proteins and differentially abundant proteins (p<0.05, fold change ≥2) according to their associated location.
Figure 5—figure supplement 2
Relative mRNA expression of major ECM genes in whole tissue SDFT and CDET through postnatal development.
* Significant difference between tendons, a-e significant difference between age groups. Error bars depict standard deviation.
Figure 6
TGFB1 is predicted to be involved in compositional changes observed in the IFM.
(a) IPA networks for TGFB1 as an upstream regulator were generated for the foetus and 3–6 months SDFT IFM proteomic datasets. TGFB1 regulation in the IFM is predicted to be inhibited in the foetus …
Tables
Table 1
IFM differentially abundant matrisome and matrisome-associated proteins through development organised by highest mean condition (p<0.05, fold change ≥2).
Proteins are arranged into colour-coded divisions and categories. Bar graphs profile the relative abundance of each protein at each development stage, a foetus, b 0 days, c 0–1 month, d 3–6 months, …
| Protein | Division | Category | Highest mean cond. | a b c d e |
|---|---|---|---|---|
| SERPINH1 | Matrisome-associated | ECM Regulators | Foetus |  |
| COL14A1 | Core matrisome | Collagens | 0–1 month |  |
| ASPN | Core matrisome | Proteoglycans | 0–1 month |  |
| FMOD | Core matrisome | Proteoglycans | 0–1 month |  |
| KERA | Core matrisome | Proteoglycans | 0–1 month |  |
| FBLN5 | Core matrisome | ECM Glycoproteins | 0–1 month |  |
| FGB | Core matrisome | ECM Glycoproteins | 0–1 month |  |
| FGG | Core matrisome | ECM Glycoproteins | 0–1 month |  |
| COL1A2 | Core matrisome | Collagens | 3–6 months |  |
| COL2A1 | Core matrisome | Collagens | 3–6 months |  |
| COL4A1 | Core matrisome | Collagens | 3–6 months |  |
| COL4A2 | Core matrisome | Collagens | 3–6 months |  |
| COL6A3 | Core matrisome | Collagens | 3–6 months |  |
| BGN | Core matrisome | Proteoglycans | 3–6 months |  |
| HSPG2 | Core matrisome | Proteoglycans | 3–6 months |  |
| ADIPOQ | Core matrisome | ECM Glycoproteins | 3–6 months |  |
| FBN1 | Core matrisome | ECM Glycoproteins | 3–6 months |  |
| FN1 | Core matrisome | ECM Glycoproteins | 3–6 months |  |
| LAMB2 | Core matrisome | ECM Glycoproteins | 3–6 months |  |
| LAMC1 | Core matrisome | ECM Glycoproteins | 3–6 months |  |
| NID1 | Core matrisome | ECM Glycoproteins | 3–6 months |  |
| ANXA4 | Matrisome-associated | ECM-affiliated | 3–6 months |  |
| S100A4 | Matrisome-associated | Secreted Factors | 3–6 months |  |
| COL21A1 | Core matrisome | Collagens | 1–2 years |  |
| COL3A1 | Core matrisome | Collagens | 1–2 years |  |
| COL5A1 | Core matrisome | Collagens | 1–2 years |  |
| COL5A2 | Core matrisome | Collagens | 1–2 years |  |
| COL6A1 | Core matrisome | Collagens | 1–2 years |  |
| COL6A2 | Core matrisome | Collagens | 1–2 years |  |
| DCN | Core matrisome | Proteoglycans | 1–2 years |  |
| LUM | Core matrisome | Proteoglycans | 1–2 years |  |
| OGN | Core matrisome | Proteoglycans | 1–2 years |  |
| PRELP | Core matrisome | Proteoglycans | 1–2 years |  |
| COMP | Core matrisome | ECM Glycoproteins | 1–2 years |  |
| DPT | Core matrisome | ECM Glycoproteins | 1–2 years |  |
| TGFBI | Core matrisome | ECM Glycoproteins | 1–2 years |  |
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Table 1—source data 1
IFM and fascicle matrisome proteins intensity.
- https://cdn.elifesciences.org/articles/58075/elife-58075-table1-data1-v2.xlsx
Table 2
Fascicle differentially abundant matrisome and matrisome-associated proteins through development organised by highest mean condition (p<0.05, fold change ≥2).
Proteins are arranged into colour-coded divisions and categories. Bar graphs on the right profile the relative abundance of each protein at each development stage, a foetus, b 0 days, c 0–1 month, d …
| Protein | Division | Category | Highest mean cond. | A B C D e |
|---|---|---|---|---|
| COL11A1 | Core matrisome | Collagens | Foetus |  |
| DCN | Core matrisome | Proteoglycans | Foetus |  |
| FMOD | Core matrisome | Proteoglycans | Foetus |  |
| KERA | Core matrisome | Proteoglycans | Foetus |  |
| PCOLCE | Core matrisome | ECM Glycoproteins | Foetus |  |
| SERPINF1 | Matrisome-associated | ECM Regulators | Foetus |  |
| ANXA1 | Matrisome-associated | ECM-affiliated Proteins | Foetus |  |
| ANXA2 | Matrisome-associated | ECM-affiliated Proteins | Foetus |  |
| ANXA5 | Matrisome-associated | ECM-affiliated Proteins | Foetus |  |
| LGALS1 | Matrisome-associated | ECM-affiliated Proteins | Foetus |  |
| COL12A1 | Core matrisome | Collagens | 0 days |  |
| COL3A1 | Core matrisome | Collagens | 1–2 years |  |
| PRELP | Core matrisome | Proteoglycans | 1–2 years |  |
| COMP | Core matrisome | ECM Glycoproteins | 1–2 years |  |
| FN1 | Core matrisome | ECM Glycoproteins | 1–2 years |  |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Equus caballus) | Superficial digital flexor tendon and common digital extensor tendon | Equine practices and commercial abattoir | Foetus-2 years old | |
| Biological sample (Equus caballus) | Primary superficial digital flexor tendon tenocytes | Commercial abattoir | P3 from adult specimens | |
| Antibody | Anti-decorin (mouse IgG) | Other | (1:1500), Prof. Caterson, Cardiff University, UK | |
| Antibody | Anti-proteoglycan 4 (mouse IgG) | Other | (1:200), Prof. Caterson, Cardiff University, UK | |
| Antibody | Anti-fibromodulin (rabbit IgG) | Other | (1:400), Prof. Roughley, McGill University, Canada | |
| Antibody | Anti-tenascin C (mouse IgG) | Santa Cruz Biotechnology | RRID:AB_785991 | (1:250) |
| Antibody | Anti-elastin (mouse IgG) | Abcam | RRID:AB_2099589 | (1:250) |
| Antibody | Zytochem Plus HRP polymer anti-mouse | Zytomed systems | RRID:AB_2868565 | (75 µL) |
| Antibody | Zytochem Plus HRP polymer anti-rabbit | Zytomed systems | RRID:AB_2868566 | (75 µL) |
| Sequenced-based reagent | Equus caballus TGFB1 Accell SMARTpool | Dharmacon, Horizon Discovery | https://horizondiscovery.com/en/products/tools/Custom-SMARTpool | (1 µM) |
| Sequenced-based reagent | Equus caballus Accell Non-targeting siRNA | Dharmacon, Horizon Discovery | https://horizondiscovery.com/en/products/tools/Custom-SMARTpool | (1 µM) |
| Peptide, recombinant protein | Recombinant Human TGF-β1 | Peprotech | 100–21 | (10 ng/mL) |
| Commercial assay or kit | FASTIN Elastin Assay | Biocolor | https://www.biocolor.co.uk/product/fastin-elastin-assay/ | |
| Chemical compound, drug | RapiGest SF | Waters | https://www.waters.com/waters/en_GB/RapiGest-SF-Surfactant/ | (0.1% w/v) |
| Software, algorithm | HistoQuest Analysis Software | Tissuegnostics | RRID:SCR_014823 | |
| Software, algorithm | Adobe Photoshop CS3 | Adobe | RRID:SCR_014199 | |
| Software, algorithm | Peaks Studio v8.5 | Bioinformatics Solutions | www.bioinfor.com/peaks-studio | |
| Software, algorithm | Ingenuity Pathway Analysis | Qiagen | RRID:SCR_008653 | |
| Software, algorithm | Matrisome | PMID:2197732 | http://matrisomeproject.mit.edu | |
| Software, algorithm | Mascot | Matrix Science | RRID:SCR_014322 | |
| Software, algorithm | Neopeptide Analyser | PMID:28503667 | https://github.com/PGB-LIV/neo-pep-tool/releases/ | |
| Software, algorithm | SigmaPlot | Systat Software Inc | RRID:SCR_003210 | |
| Software, algorithm | GProX | PMID:21602510 | RRID:SCR_000273 | |
| Other | Chondroitinase ABC from Proteus vulgaris | Merck | C2509 | (0.2 U/mL) |
| Other | Hyaluronidase from bovine testes | Merck | H3506 | (4800 U/mL) |
Additional files
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Supplementary file 1
Histologic variables used in the H and E scoring of the SDFT and CDET sections and the analysis method and reporting criteria adopted.
- https://cdn.elifesciences.org/articles/58075/elife-58075-supp1-v2.docx
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Supplementary file 2
Gene primer sequences used in relative mRNA expression analysis.
- https://cdn.elifesciences.org/articles/58075/elife-58075-supp2-v2.docx
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Supplementary file 3
Samples used for analysis along with statistical test used for analysis.
- https://cdn.elifesciences.org/articles/58075/elife-58075-supp3-v2.docx
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Supplementary file 4
Collagens and proteoglycans identified in SDFT IFM and fascicle.
- https://cdn.elifesciences.org/articles/58075/elife-58075-supp4-v2.docx
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Supplementary file 5
Correlation analysis of IFM protein abundance and mechanical properties across development.
- https://cdn.elifesciences.org/articles/58075/elife-58075-supp5-v2.docx
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Supplementary file 6
Correlation analysis of TGFB1 whole tendon mRNA expression and IFM protein abundance across development.
- https://cdn.elifesciences.org/articles/58075/elife-58075-supp6-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/58075/elife-58075-transrepform-v2.pdf
