Postnatal mechanical loading drives adaptation of tissues primarily through modulation of the non-collagenous matrix

Mature connective tissues demonstrate highly specialised properties, remarkably adapted to meet their functional requirements. Tissue adaptation to environmental cues can occur throughout life and poor adaptation commonly results in injury. However, the temporal nature and drivers of functional adaptation remain undefined. Here, we explore functional adaptation and specialisation of mechanically loaded tissues using tendon; a simple aligned biological composite, in which the collagen (fascicle) and surrounding predominantly non-collagenous matrix (interfascicular matrix) can be interrogated independently. Using an equine model of late development, we report the first phase-specific analysis of biomechanical, structural, and compositional changes seen in functional adaptation, demonstrating adaptation occurs postnatally, following mechanical loading, and is almost exclusively localised to the non-collagenous interfascicular matrix. These novel data redefine adaptation in connective tissue, highlighting the fundamental importance of non-collagenous matrix and suggesting that regenerative medicine strategies should change focus from the fibrous to the non-collagenous matrix of tissue.

Using this model, we investigate the process and drivers of functional adaptation, when 89 tendons transition from an absence of loading (foetal: mid to end (6 to 9 months) gestation, 90 and 0 days: full-term foetuses, and foals that did not weight-bear); through to weight-bearing 91 (0-1 month) and then to an increase in body weight and physical activity (3-6 months; and 1-92 2 years). We hypothesise that early in development during gestation, the fibre and matrix

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Mechanical adaptation is localised to the matrix phase 100 First, we determined how the mechanical properties of the fibre and matrix phase develop in 101 tendon, with a particular focus on the temporospatial nature of mechanical adaptation to 102 functional specialisation. Individual fascicles were dissected and tested as fibre samples, 103 while an isolated region of interfascicular matrix, matrix phase, was tested by shearing 104 fascicles apart. Samples were subjected to preconditioning followed by a pull to failure 105 (Supplementary Figure 1). The yield point of samples was identified, denoting the point at 106 which the sample became irreversibly damaged and was unable to recover from the applied 107 load, and the sample failure properties also recorded, highlighting the maximum stress and 108 strain the sample could withstand. for 10 preconditioning cycles for the SDFT and CDET fibre phase in the foetus and 1-2 years age 135 group. (b) Representative force-extension curves to failure for the SDFT and CDET fibre phase in the 136 same age groups. (c-i) Mean SDFT and CDET fibre phase biomechanical properties are presented 137 across development, with data grouped into age groups: foetus, 0 days (did not weight-bear), 0-1 138 month, 3-6 months, 1-2 years. ‡ significant interaction between tendon type and development, * 139 significant difference between tendons, a-b significant difference between age groups. Error bars 140 depict standard deviation. preconditioning cycles for the SDFT and CDET matrix phase in the foetus and 1-2 years age group.

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(b) Representative force-extension curves to failure for the SDFT and CDET matrix phase in the same 147 age groups. (c-i) Mean SDFT and CDET matrix phase biomechanical properties are presented across 148 development, with data grouped into age groups: foetus, 0 days (did not weight-bear), 0-1 month, 3-6 149 months, 1-2 years. (j-k) To visualise the extended low stiffness toe region in the SDFT matrix phase, 150 the amount of matrix phase extension at increasing percentages of failure force is presented, 151 comparing the SDFT and CDET in the foetus and 1-2 years age group. ‡ significant interaction 152 between tendon type and development, * significant difference between tendons, a-g significant 153 difference between age groups. Error bars depict standard deviation. 154 Structural adaptation is localised to the matrix phase 156 Having described mechanical adaptation of the matrix phase to meet functional demand, we 157 next performed a histological and immunohistochemical comparison of developing energy-158 storing and positional tendons to determine how temporospatial structural adaptation may 159 dictate this evolving mechanical behaviour.

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The energy-storing SDFT and positional CDET appeared histologically similar in the foetus,  Adaptation relies on evolution of matrix phase composition only 203 To explore these concepts in further detail and to scrutinise the capacity for ECM adaptation, 204 proteomic methodologies were adopted. With the mechanical and histological data 205 identifying that functional adaptation is particular to the energy-storing SDFT, mass 206 spectrometry analysis focused on a more detailed comparison of the matrix and fibre phase 207 development and adaptation in this tendon specifically. 208 Our results demonstrated that the proteomic profile of the matrix phase was more complex 209 (more identified proteins) and a higher percentage of matrix phase proteins were cellular,  (Table 1 and 2). Detailed consideration of protein changes also highlights that post loading 218 changes in the matrix phase appear more specific to proteoglycans and glycoproteins.

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Proteomic data also enable insight into the turnover of proteins in the different tendon phases, 220 through a comparison of the neopeptides produced by protein breakdown(Chavaunne T. 221 Thorpe, Peffers, et al., 2016). In the current study, we are able to profile the temporal nature 222 of fibre and matrix turnover, demonstrating that both phases display turnover during 223 development but that fibre phase turnover slows down towards the end of maturation, whilst 224 matrix phase turnover rates are maintained, suggesting structural and/or compositional 225 plasticity ( Figure 4b).

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Having identified the matrix phase as the location of functional adaptation, we next 227 investigated the regulation of this process, to detect targets for modulation in regeneration 0-1 month, 3-6 months, and 1-2 years SDFT matrix and fibre phases (p<0.05, fold change ≥2). 240 Heatmap colour scale ranges from blue to white to red with blue representing lower abundance and 241 red higher abundance. (b) Proteins with identified neopeptides and proteins showing differential total 242 neopeptide abundance across age groups. Graph of proteins showing differential total neopeptide 243 abundance in the SDFT fibre phase across development (p<0.05, fold change≥2, FDR 5%). No 244 proteins showed differential total neopeptide abundance in the matrix phase. (c) IPA networks for 245 TGFB1 in the foetus and at 3-6 months in the SDFT matrix phase. TGFB1 regulation in the matrix 246 phase is predicted to be inhibited in the foetus and activated at 3-6 months in the SDFT. Red nodes, 247 upregulated proteins, green nodes, downregulated proteins, intensity of colour is related to higher 248 fold-change, orange nodes, predicted upregulated proteins in the dataset, blue nodes, predicted 249 downregulated proteins. (d) Whole tendon relative mRNA expression for TGFB1 in the SDFT and 250 CDET during postnatal development. * significant difference between tendons, a significant 251 difference between age groups. Error bars depict standard deviation.  In this study, we describe the process and drivers of functional adaptation in tendon  Here, examining the fibre and matrix phase mechanical properties independently, we show 279 minimal distinction in fibre phase mechanics between functionally distinct tendons and,

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CTT assisted with study design, data analysis, and edited the manuscript. YAK assisted 602 with data collection and analysis. HLB, HRCS, PDC conceived the study, assisted with 603 data analysis, and edited the manuscript.

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Competing Interests statement 606 The authors declare that they have no competing interests.