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To address many of these limitations, researchers have developed tape-stabilized cryosectioning techniques to maintain section morphology of mineralized tissues. Tape-stabilized cryosectioning was first introduced with the CryoJane system originally from Instrumedics Hackensack, NJ , but now Leica Biosystems Deer Park, IL , which uses an adhesive tape that can be adhered to the frozen tissue block on the cryostat [ 1 ]. The tape serves to maintain tissue quality during the sectioning process.
The section is then transferred from the tape to special adhesive-coated slides i. This technique was transformational, in that it allowed researchers to maintain biological features within the tissue while not sacrificing section quality. However, the CryoJane system had a few challenges. First, to purchase the system there was a substantial upfront equipment cost to purchase the system.
Additionally, there were some complications associated with the tape transfer process requiring fine temperature control to successfully transfer the complete tissue section from the cryotape to the slide. Since the Kawamoto cryofilm is transparent and minimally autofluorescent, unlike the tape in the CryoJane system, it can be glued to a glass slide with the tissue facing upward , followed by staining and imaging. With the cryofilm-tissue units glued to the slides, the coverslips can be easily removed without dislodging the section.
Hence, the Kawamoto cryofilm allows for several rounds of staining and imaging of the same section. Within the field of orthopaedic research, tape-stabilized cryohistology is particularly useful for reinforcing soft tissue-to-bone interfaces with incongruous material properties [ 6 , 8 , 9 ]. Tissue interfaces are commonly investigated in tissue injury and repair studies [ 10—12 ], but notoriously challenging to cryosection. These challenges are exacerbated as tissue size increases.
Tissues harvested from larger species e. Despite the utility of tape-stabilized cryohistology for large animal samples, only a few studies have applied this method to section such tissues [ 13—15 ].
In addition to improving section quality, tape-stabilized cryohistology could greatly benefit the large animal research community because this method maximizes data output while minimizing sample utilization. Due to housing and budget constraints, sample number is often limited in large animal studies, and samples are carefully divided between outcome measures [ 16 ].
A single tape-stabilized cryosection can be repeatedly stained, imaged, and washed, permitting the overlay of images from multiple staining rounds, as previously established with rodent tissues [ 3 ]. The overlaid images provide insight into the co-localization or lack thereof of deposited minerals, aligned collagen, matrix elements, and specific cell populations [ 6 ], which can be quantified [ 3 , 4 , 14 , 15 ].
To enable the widespread adoption of multiplexed tape-stabilized cryohistology within the large animal research community, this work establishes a detailed pipeline for mineralized sample harvest, preparation, sectioning, staining, and imaging Fig. The established workflow is validated using vertebral body, femoral head, and temporomandibular joint samples from adult Yucatan minipigs. Prior to euthanasia, these animals received intravenous injections of xylenol orange and calcein green bone fluorochrome labels, administered two weeks apart [ 14 ].
The developed protocol enables the overlay of the incorporated mineral labels, aligned collagen polarized light , tartrate resistant phosphatase TRAP , alkaline phosphatase AP , and toluidine blue staining on a single histological section. Most importantly, this protocol provides step-by-step instructions to create tear-free histological sections from large, mineralized samples, and perform sequential staining to maximize data output from these cost and time intensive large animal studies.
Figure 1 Overview of multiplexed tape-stabilized cryohistology. A Tissues are prepared for embedding and sectioning. B Trimmed tissues are embedded in cryo-embedding medium and sectioned using cryofilm. The following sections are glued to glass slides. C Sequential staining and imaging are performed using a slide scanner. Smaller samples will be easier to section and handle. Figure 2 Representative harvested bones, and smaller trimmed bony units from one year old Yucatan minipigs.
The smaller bony units were embedded for sectioning. NOTE: Reduce the trim thickness and sectioning speed if the sample is difficult to cut.
Figure 3 Tape-stabilized cryosectioning of mineralized large animal specimens. B Cryofilm applied to the block face. Since the Gaussian effect washes out the image corners, go ahead and smooth them.
Click to select the Blur Tool in the Tools palette and drag over the image corners. Click OK to apply the effect. Subsequent mineralization of the larval skeleton during metamorphosis proceeded normally as well, and the first subtle differences in skull shape only became apparent at juvenile age. The gradually worsening phenotype observed in humans, mice and zebrafish further suggests that loss of MMP14 disrupts later stages of skeletal remodeling.
Consistent with our in vitro data, these observations argue against a major role for MMP14 supporting cellular migratory and invasive behavior in vivo. During these types of ossification, bone forms within, respectively in close association with, a cartilage template 39 , It was shown in Mmp14 KO mice that these cartilage templates are not properly replaced, respectively removed, owing to impaired MMPdependent collagen remodeling 32 , 33 , Taken together, our in vitro and in vivo studies highlight the significance of impaired proteolytic MMP14 activity underlying the WS skeletal phenotype.
This observation has therapeutic implications. Attempts have been made in the past to treat WS and related disorders with bisphosphonates, with limited success 45 , Bisphosphonates exert their therapeutic effect mostly by inhibiting osteoclasts Whereas our observations as well as previously published work do support a role for osteoclasts in the WS bone phenotype, the major problem seems to be defective collagen remodeling.
Thus, we would propose that any treatment of WS should seek to address this fundamental issue. Our zebrafish model could be used to develop and test such treatments, which might also be able to address osteoporosis and other conditions with decreased bone density. MRC-5V1 immortalized human fetal lung fibroblasts were provided by Prof. Four expression vectors encoding either WT or mutant mmp14a or mmp14b with a C-terminal HA tag were generated.
The amplicon was gel-extracted and cloned into the pCR2. Cells were seeded on No. Cells were transfected with 1. Non-transfected cells were seeded 18 h prior to fixation. The investigator taking and analyzing images was blinded for the transfection of the cells. Raw images were processed with Fiji software U. For endogenous MMP14 and analysis of double-tagged MMP14, cells were seeded at a density of cells per well of a six-well plate.
Protein concentration was determined by Bradford protein assay Sigma. An appropriate volume of Laemmli sample buffer was added to equal amounts of total protein lysate followed by boiling for 5 min. Media was refreshed 6 h post-transfection.
Protein concentration was determined by Bradford protein assay. Samples were mixed with Laemmli sample buffer and boiled as described above. Gelatin zymography was performed as previously described Finally, the gel was stained with Coomassie Blue R Thermo; 0. To quantify the number of transfected cells N in a given field, a find-object macro was created in Fiji.
Threshold was manually adjusted to cover the entire degradation area A that was measured. For qualitative analysis, fluorescent cells were identified 2 h post-seeding and z-stacks recorded ever 15 min for 5 h with a spinning disk Yokogawa, Tokyo, Japan; CSU confocal microscope Nikon, Tokyo, Japan; Nikon Ti inverted equipped with the Nikon Perfect Focus System.
A maximum intensity projection was performed in MetaMorph 7. Migration of the transfected cells was recorded by time-lapse imaging starting 2 h post-seeding with a spinning disk confocal microscope.
Time-lapse images were then subjected to customize cell-tracking analysis in Imaris Image Analysis Software 8. Danio rerio larvae of the AB strain were grown in E3 medium 0. From 4 weeks of age onwards, fish were kept at Three-month-old F0 mosaic mutants were intercrossed and their F1 offspring genotyped at 2 months of age to identify fish with heterozygous frameshift mutations in mmp14a or mmp14b.
Primers listed in Supplementary Material , Table S4 were tested at different concentrations 1. Generation of a single amplicon was verified by direct Sanger sequencing. Images were stitched together in Pixelmator 3. The images were reconstructed using a Feldkamp cone-beam algorithm. Images were exported as TIFF files and extracorporeal skeletal elements of previously imaged fish were manually removed with Pixelmator software. Three-month-old fish were euthanized and fixed as described above.
Fish were dehydrated through graded ethanol into xylene Chemtech Trading before paraffin Leica; infiltration. The sections were deparaffinized and rehydrated through graded ethanol into water. Stained sections were dehydrated through graded ethanol into xylene and a cover slip added.
Slides were imaged with an automated slide scanner equipped with Zeiss AxioImager Z. Images were viewed with VSViewer V2. Larvae were stained for cartilage and calcified bone according to a protocol adapted from Walker et al.
Larvae were stained with either 0. All larvae were bleached with 1. The system was operated with AxioVision version 4. The system was operated with Leica Application Suite software version 2.
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