Data description: Deprivation of loading during early healing of rat Achilles tendons affects extracellular matrix composition and structure, and reduces cell density and cell alignment

Data description: Deprivation of loading during early healing of rat Achilles tendons affects extracellular matrix composition and structure, and reduces cell density and cell alignment

Malin Hammerman, Maria Pierantoni, Hanna Isaksson *, Pernilla Eliasson *

* joint last authors

This dataset contains microscope images obtained from sections of healing and intact rat Achilles tendons undergoing different in vivo loading protocols and different time points post-transection. The data presented are the full resolution microscope images available in lower resolution in the accompanying manuscript’s Supplementary Figures 4-6.

Each zipped folders contain images (tif-files) from all time-points for each respective staining and loading group.   

Col1: Sections stained with Collagen 1 antibodies

Col3: Sections stained with Collagen 3 antibodies

Elastin: Sections stained with Elastin antibodies

Full_loading: Free cage activity

Reduced_loading: Paralysis of the calf muscle with Botox

Minimal_loading: Botox combined with joint fixation using a steel-orthosis

Intact_reference: Contralateral uninjured Achilles tendons, used as reference

More description of the datasets inside the zipped files are available below and in the file 'Data Description.pdf'

Brief re-cap of methods

Histological analysis was performed on healing Achilles tendons from Female Sprague-Dawley rats, specific-pathogen free (11-12 weeks, weight 299 ± 15 g), that had undergone full transection [13] of the right Achilles tendon, and been exposed to different levels of loading. Altered loading was imposed through two mechanisms. Reduced loading involved intramuscular Botox injections in the right calf muscles to induce plantar flexor muscle paralysis [24]. Additionally, the rats in the minimal loading group received a steel-orthosis around their right hindlimb directly after surgery [24].

Snap frozen tendons in OCT were sectioned longitudinally (7 μm thickness) and stained with immunofluorescent staining for collagen 1, collagen 3, or elastin. Sections were counterstained with DAPI followed by mounting. The tissue sections were imaged under a microscope (DMi8, Leica Microsystems, Wetzlar, Germany, with a Hamamatsu Orca LT Flash sCMOS camera) where fluorescence was detected at 550 nm (secondary antibody Alexa Fluor 594), 470 nm (secondary antibody Alexa Fluor 488) and 385 nm (DAPI), and exposure time was held constant for each color channel regarding magnification and staining.

Mapping images of the entire tendon were obtained for one section per group (n=1 per healing time, loading group and ECM matrix protein). All images were adjusted to the negative control, where the primary antibody was omitted, to correct for unspecific antibody detection.

Microscope images and description of file-names

All data is presented in the form of .tif files. Please refer to the scale bars in the images. All image-files are named using the following abbreviations, as described below. As an example, the file name “Tendon_col1_FL_1W_col1.tif” refers to a tendon section stained for collagen 1 from a rat exposed to full loading for a period of 1 week after tendon transection, where only the channel for collagen 1 is shown, whereas “Tendon_col1_FL_1W_merged.tif” includes the channels for both staining for collagen 1 and DAPI of the same section.

Col1: Sections stained with Collagen 1 antibodiesCol3: Sections stained with Collagen 3 antibodiesElastin: Sections stained with Elastin antibodiesdapi: Sections stained with 4',6-Diamidino-2-Phenylindole Dihydrochloride.FL:   Full loading (free cage activity),RL:   Reduced loading (paralysis of the calf muscle with Botox),ML:  Minimal loading (Botox combined with joint fixation using a steel-orthosis)IT:    Intact contralateral Achilles tendons, used as reference.

1W: Healing time point 1 week after transection2W: Healing time point 2 weeks after transection3W: Healing time point 3 weeks after transection20W: Healing time point 20 weeks after transection

Settings for brightness and contrast

Collagen 11w FL 2000-12 000, UL 4000-10 000, ML 4000-12 0002w FL 2500-10 000, UL 4000-10 000, ML 5000-12 0003w FL 2000-12 000, UL 3500-13 000, ML 3500-14 00012w FL 3000-12 00020w FL 3000-11 000IT 2000-8 000

Collagen 31w FL 3000-12 000, UL 4000-10 000, ML 4000-13 0002w FL 2000 - 7 000, UL 2500-12 000, ML 2000-12 0003w FL 2000-12 000, UL 3500-13 000, ML 3500-14 00012w FL 3000-12 00020w FL 2000-12 000IT 3000-12 000

Elastin1w FL 4000-10 000, UL 5000 - 8000, ML 3500-12 0002w FL 3000-12 000, UL 3000-12 000, ML 3000-12 0003w FL 2500-12 000, UL 2000-12 000, ML 2500-12 000,12w FL 3500-12 00020w FL 3500-12 000IT 2000-12 000