This work was partially supported by the American Society for Surgery of the Hand Pilot Award and NIH/NIAMS 1K01AR068386-01&#160;(to AEL) and NIAMS/NIH P30AR061307.

The University Committee on Animal Research at the University of Rochester approved all animal experiments. Ten-12 week old female C57BL/6J mice were used.
1. Preparation of Animals for Flexor Tendon Surgery (~15 min)
<ol>
	<li>Autoclave surgical instruments to sterilize, wear sterile gloves throughout, and maintain a sterile operating field.</li>
	<li>Anesthetize the mouse via intraperitoneal injection (i.p.) with a volume of ketamine (80 mg/kg) and xylazine (10 mg/kg) corresponding to body weight. Confirm depth of sedation via absence of toe-pinch reflex.</li>
	<li>Administer pre-emptive analgesia (buprenorphine, 0.05-0.1 mg/kg) via subcutaneous injection. Apply ophthalmic ointment to prevent eyes from drying out.</li>
	<li>Prepare the surgical site by clipping the fur on the entire hind limb. Sterilize the skin by scrubbing sequentially with povidone iodine, followed by 70% ethanol, and again with povidone iodine.</li>
</ol>
2. Murine Flexor Tendon Injury and Repair Surgery (~10 min)
<ol>
	<li>Find the flexor digitorum longus (FDL) tendon, seen superficially in the medial aspect of the calf. Using a scalpel, make a small 0.5-1 cm incision in the skin with micro-scissors to expose the tendon.</li>
	<li>Use forceps to separate the FDL tendon from surrounding tissue and trace it up to the myotendinous junction. Cut the tendon at this junction with spring scissors to release it, taking care to avoid the posterior tibial artery.</li>
	<li>Close the skin with 5-0 nylon sutures. 
	Note: The FDL transection and repair (steps 2.4) should be performed using a stereomicroscope.</li>
	<li>Make a 3 mm incision over the posterolateral aspect of the hind paw using spring micro-scissors. Gently retract the surrounding soft tissue and muscle with forceps, making sure to minimize tissue damage, and identify the FDL tendon.
	<ol>
		<li>Gently raise the FDL tendon and completely transect it using micro-scissors. Suture the ends of the FDL tendon together in a modified Kessler pattern with 8-0 sutures, avoiding drying of the tendon by periodically wetting it with saline.</li>
		<li>Replace soft tissue and muscle over the tendon, then close the incision with 5-0 nylon sutures.</li>
	</ol>
	</li>
	<li>Place mice on a slide warmer set to 37 &#176;C, to maintain body temperature until recovered from anesthesia.</li>
	<li>Monitor mice post-operatively for signs of impairment or infection including increased vocalizing and ruffled fur. Inject buprenorphine (50 &#181;l/mouse) as analgesic every 12 hr until mice no longer show signs of pain or distress.</li>
	<li>Allow mice to heal for a period of 10 days post-surgery before continuing to the next steps. 
	Note: Cells can be harvested at any time-point post-surgery. Here, 10 days are chosen for these representative experiments given the relatively high cellularity of the tissue at this time.</li>
</ol>
3. Labeling of Cycling Cells (~10 min)
<ol>
	<li>Prepare EdU (5-ethynyl-2&#39;deoxyuridine) solution (10 mg/ml in sterile Phosphate buffered saline [PBS] + 1% Dimethyl Sulfoxide [DMSO] to increase solubility).</li>
	<li>Inject mice with 100 &#181;l EdU (50 mg/kg) via i.p. injection 24 hr prior to sacrifice.</li>
</ol>
4. Harvest Cells for Cytology Centrifugation (2.5 hr, ~30 min Hands-on Time)
<ol>
	<li>Prepare 3 mg/ml collagenase I in PBS.</li>
	<li>Euthanize mouse via carbon dioxide asphyxiation at a flow rate of no more than 3 L/min, and perform cervical dislocation as a secondary measure.</li>
	<li>Locate the repaired tendon using step 2.4 above, and isolate tendon tissue by cutting about 2 mm on either side of the injury site with micro-scissors.
	<ol>
		<li>Mince tissue with scalpel in a petri dish with 1 ml of 3 mg/ml collagenase, then pass mixture through an 18 G needle several times to break up the tissue. Collect mixture into a 1.5 ml centrifuge tube.</li>
		<li>Digest tendon tissue for 1 hour at 37 &#176;C with shaking.</li>
		<li>Spin tissue down (300 x g for 5 min), aspirate collagenase, then resuspend cells/tissue with 500 &#181;l 3% bovine serum albumin (BSA) in PBS by pipetting up and down several times.</li>
		<li>Filter cell suspension through a 70 &#181;m cell strainer to remove debris and large pieces of tendon, and set aside in a 37 &#176;C incubator.</li>
		<li>Place tendon tissue into another 1.5 ml centrifuge tube, and refresh with 1 ml collagenase solution, pipetting up and down to mix.</li>
		<li>Incubate tissue another hour at 37 &#176;C with shaking.</li>
		<li>Spin down digested tendon tissue (300 x g for 5 min), then resuspend in 500 &#181;l 3% BSA in PBS by pipetting up and down several times.</li>
		<li>Filter cell suspension through a 70 &#181;m cell strainer, combine with suspension isolated in 4.3.3, then count cells with a hemocytometer.</li>
		<li>Wash cells once more with 3% BSA in PBS, then resuspend to 3-6 x 104/100 &#181;l.</li>
	</ol>
	</li>
	<li>Attach a cytology funnel to a positively charged slide and lock into the slide carrier. Insert the entire apparatus into a cytology centrifuge and add 100 &#181;l cell suspension to the funnel. Centrifuge at 300 x g for 5 min to disperse the cells on to the slide.</li>
</ol>
5. Immunocytochemistry to Detect EdU (2 hr, ~45 min Hands-on Time)
Note: All incubation steps should be performed in the dark to limit photobleaching of fluorophores.
<ol>
	<li>Circle cells with a hydrophobic barrier pen to minimize solution needed for following steps, then immediately fix with 150 &#181;l 3% paraformaldehyde in PBS 15 min at room temperature (RT).
	<ol>
		<li>Wash twice for 5 min with 150 &#181;l 3% BSA in PBS.</li>
		<li>Permeabilize cells with 0.5% Triton-X 100 in PBS 20 min at RT.</li>
		<li>Repeat wash step as in 5.1.1.</li>
	</ol>
	</li>
	<li>Prepare EdU reaction cocktail according to kit instructions not more than 30 min before use.
	<ol>
		<li>Add 150 &#181;l cocktail to each slide, incubate 30 min at RT.</li>
		<li>Repeat wash step as in 5.1.1.</li>
	</ol>
	</li>
	<li>Incubate with 150 &#181;l nuclear counterstain Hoechst33342 diluted 1:2,000 in PBS 30 min at RT.
	<ol>
		<li>Wash twice for 5 min with 150 &#181;l 1x&#160;PBS.</li>
	</ol>
	</li>
	<li>Add 1-2 drops anti-fade mounting medium to cells, then slowly lower coverslip to prevent bubbles.
	<ol>
		<li>Allow mounting medium to cure overnight in the dark at RT.</li>
		<li>Use clear nail polish to seal the coverslip to the slide.</li>
	</ol>
	</li>
	<li>Image slides at 40X magnification using a fluorescent microscope equipped with excitation/emission filters capable of detecting DAPI (Hoeschst33342), and Texas Red (Alexa Fluor 594).</li>
</ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+EP4+Inhibition+Increases+Adhesion+Formation+in+a+Murine+Model+of+Flexor+Tendon+Repair.">Systemic EP4 Inhibition Increases Adhesion Formation in a Murine Model of Flexor Tendon Repair.</a> PloS one. 10, e0136351 (2015).</li><li>Loiselle, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+antisense+oligonucleotide+(ASO)+technology+against+Tgf-beta+signaling+to+prevent+scarring+during+flexor+tendon+repair.">Development of antisense oligonucleotide (ASO) technology against Tgf-beta signaling to prevent scarring during flexor tendon repair.</a> J Orthop Res. 33, 859-866 (2015).</li><li>Orner, C. A., Geary, M. B., Hammert, W. C., O'Keefe, R. J., Loiselle, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+and+short-duration+Matrix+Metalloproteinase+9+Inhibition+does+not+affect+adhesion+formation+during+murine+flexor+tendon+healing.">Low-dose and short-duration Matrix Metalloproteinase 9 Inhibition does not affect adhesion formation during murine flexor tendon healing.</a> Plast Reconstr Surg. , (2016).</li><li>Loiselle, A. 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The authors declare that they have no competing financial interests

The surgical procedure for a murine model of complete transection and repair of the flexor digitorum longus tendon is presented in this study. In addition a novel application of concentrating small cell populations with cytology centrifuge is demonstrated, allowing for quantitative immunocytochemical analysis of the cellular environment during flexor tendon healing. This model of flexor tendon repair demonstrates a reproducible healing response, which can be used to assess changes in the healing process using knockout models or with pharmacological intervention. Following surgery, an inflammatory reaction begins within 24 hr, during which cells are recruited to the injury site and a callus of fibrous tissue begins to form. A proliferative phase follows at about day four, involving greatly increased cellularity and abundant deposition of disorganized extracellular matrix at the repair site, leading to adhesion formation between 14-21 days. Tissue remodeling continues through day 35, at which time continuity and organization of the flexor tendon is restored, and cellularity is reduced 18.
Currently, there are several murine models of flexor tendon repair in addition to the one presented here. Tsubone et al., have described an in vitro model of flexor tendon healing 19. While this model can assess changes in gene expression over time, it cannot account for the recruitment of extrinsic cells or changes in the inflammatory/cytokine milieu that occurs in vivo. In vivo, two models of partial transection have been developed using either incisional or excisional defects 20,21. More recently, Wong et al., characterized a Zone II model of flexor tendon injury in a murine model 22. This is an important development, as it allows the contribution of synovial sheath-derived cells to the healing process to be defined. However, no quantifiable changes in gliding function have been described using this model. Each model has its own benefits and limitations that must be considered, including technical ease, clinical relevance and anatomy. A main limitation of the model described here is that the healing tendon may rupture prior to evaluation, rendering it unusable for further analysis. Harvesting sufficient cells is made unfeasible under these circumstances, and any histochemical analysis is not viable, as a ruptured tendon does not follow the standard healing process described above. Releasing the FDL tendon proximally to decrease strain on the healing tendon is therefore a critical step for prevention, despite the fact that it is not clinically relevant. Conducting the surgery unilaterally also lowers the rate of rupture. Another limitation is the inability to track the contribution of peri-tendinous sheath cells, as the synovial sheath does not surround the segment of FDL tendon that is injured in this model.
An important function of this flexor tendon repair model is that it allows evaluation of the cells at the healing site. Immunofluorescence/immunohistochemistry (IHC) is a common biological technique that can be used to characterize these cells. While IHC is uniquely suited for determination of spatial information, it is largely a qualitative method. Changes in tissue or cell composition throughout the depth of the tissue can be missed, and quantification of populations is difficult. Flow cytometry represents an ideal approach to define, quantify and potentially isolate cell populations during healing. However, isolation and digestion of healing tendon tissue from this model yields very low cell numbers for flow cytometric analysis (1-2 x 105 on average from each mouse), and fixation/permeabilization steps for detection of intracellular markers reduces this number further. Using cytology centrifugation to concentrate the isolated cells, followed by immunocytochemistry, provides a way to characterize these small cell populations. A significant advantage of this technique is that there is no need to pool samples together to obtain enough cells; therefore each mouse represents an individual sample for analysis. An important consideration in generating these cell preparations is maintaining consistency in the tissue harvested for analysis. Surrounding muscle and fascia may adhere to the granulation tissue and must be carefully dissected away. Since this method involves minimal processing of the cells prior to fixation on slides, it retains a majority of isolated cells for further analysis. In this way, nearly the entire population of cells making up the adhesion tissue at the repair site can be quantitatively assessed via immunocytochemistry. In the present study, EdU incorporation by proliferating cells is used to demonstrate the application of this cytology technique, however, it can also be used for more complex and in-depth characterization of the heterogeneous cell populations involved in flexor tendon healing. Future applications of this technique may lead to more in depth characterization of the cell populations involved in tendon healing, as it allows for determining co-localization of cellular markers in the cells associated with the granulation tissue in healing tendon.

Flexor tendons in the hand work in concert with the flexor muscles of the forearm and digital sheaths to enable flexion of the digits and grasping function of the hand. Flexor tendons run along the palmar aspect of the hand; this relatively superficial location often results in injuries to the flexor tendons during trauma to the hand. Tendons heal through a scar tissue response rather than regeneration of normal tendon tissue 1. While this scar tissue provides continuity to the tendon, function is dramatically decreased relative to healthy tendon. Tendon-scar tissue composites are characterized by impaired mechanical properties 1, rendering the repaired tendons more likely to rupture. In addition, scar tissue lacks the organization of the native tendon collagen fiber structure, resulting in an increase in tendon size and bulk. Given the anatomic constraints of the tendon-sheath unit, even a modest increase in tendon size can drastically reduce the gliding function of the tendon, and therefore digit range of motion and hand function.
Prior to the 1960&#39;s injuries to the flexor tendons, particularly those in Zone II of the hand, were not routinely repaired due to the severe complications in healing that arose with these repairs 2. This area of the hand was referred to as &#39;no man&#39;s land&#39; 3. However, improvements in surgical techniques, suture patterns and physical therapy rehabilitation protocols have dramatically improved outcomes of flexor tendon repairs 2. Despite these advances, up to 40% of repairs result in sufficient adhesion formation to impede hand function 4. Therefore, a biological approach is required to improve healing. Unfortunately, very little is known about the tendon healing process at the cellular and molecular level. Thus, the goal was to develop a murine model that could be used to improve the fundamental understanding of the cellular and molecular components of flexor tendon healing and the scar formation response, as a means to identify novel therapeutic targets to improve healing.
Larger animal models have been instrumental in furthering understanding of the flexor tendon healing process. Canine and rabbit studies have demonstrated both the intrinsic and extrinsic healing ability of flexor tendons 5,6, the importance of early controlled passive motion in minimizing adhesion formation relative to immobilization 7, as well as the effects of different suture patterns on the healing process 8,9. In addition, the canine model has been useful in testing translational tissue-engineering approaches to improve healing 10. However, there are several important advantages in using a murine model relative to a large animal model, including the relative cost, availability of murine specific reagents, and the ease of generating global knock-outs or tissue-specific deletion/overexpression constructs. Moreover, the functional similarities between human and mice with respect to flexor tendons 11 indicate the potential utility in developing a murine model.
Development of a murine model of flexor tendon transection and repair mimics many aspects of clinical healing, including the formation of abundant scar tissue and impaired mechanical properties. The model described here is not a true recapitulation of clinical practice due to transection of the FDL at the myotendinous junction in order to protect the repair site. Furthermore, this model does not account for the contribution of synovial sheath cells to the healing response, as there is no synovial sheath covering the mid-portion of the tendon where the repair occurs. Despite these limitations, this model has the advantage of generating range of motion-limiting adhesions, which has yet to be demonstrated in murine models that more closely approximate the clinical scenario. This model has been used to assess knock-out mouse models 12,13, and to test different pharmacological approaches to improve healing 14-17. Histological analyses of this model, using immunohistochemistry and in situ hybridization, can provide important insights in to the localization of key genes and proteins during healing. However, histology provides only a cross-sectional spatial analysis and does not permit quantification throughout the entire tissue. Flow cytometry represents a more quantitative approach, but only a very limited number of cells can be isolated from the healing tendon tissue in the mouse model, and this number is further decreased during fixation, permeabilization, and washing steps. Taking this in to account, flow cytometry becomes an unfeasible approach due to the number of animals that would be required. An alternative method is necessary to preserve the majority of this small cell population in order to further characterize the healing milieu. The method used to accomplish this, shown here, involves concentration of the isolated cells via cytology centrifugation onto a glass slide, followed by immunocytochemistry. In the present study EdU (5-ethynyl-2&#39;deoxyuridine, a thymidine analog) incorporation and subsequent labeling was used to determine the relative proliferative state of cells at the healing site. This approach can be applied to test the efficacy of pharmacological treatments on cell proliferation, gene knock-out or overexpression, or to identify and quantify different cell populations.

<table><tbody><tr><td>&lt;strong&gt;Surgical preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C57BL/6J mice&amp;nbsp;</td><td>Jackson Laboratories</td><td>000664</td><td></td></tr><tr><td>Ketamine</td><td>Hospira</td><td>NDC# 0409-2051-05</td><td></td></tr><tr><td>Xylazine</td><td>Lloyd Inc.</td><td>NDC# 61311-482-10</td><td></td></tr><tr><td>Buprenorphine</td><td>Par Pharmaceutical Inc.</td><td>NDC# 42023-179-10</td><td></td></tr><tr><td>0.9% sodium chloride irrigation</td><td>Hospira</td><td>NDC# 0409-6138-03</td><td>For preparation of ketamine/xylazine and buprenorphine solutions</td></tr><tr><td>1 ml syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>30 G needle</td><td>BD</td><td>305106</td><td></td></tr><tr><td>Povidone-Iodine solution</td><td>Aplicare</td><td>82-226</td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>Puralube vet opthalmic ointment</td><td>Dechra Veterinary Products</td><td>NDC# 17033-211-38</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Surgical tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Portable balance 200 g</td><td>Ohaus</td><td>SP202</td><td></td></tr><tr><td>Spring scissors</td><td>Fine Science Tools</td><td>15124-12</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Needle holders</td><td>Fine Science Tools</td><td>91201-13</td><td></td></tr><tr><td>Micro spring scissors</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Micro needle holders</td><td>Fine Science Tools</td><td>12061-02</td><td></td></tr><tr><td>5-0 nylon sutures</td><td>Ethicon</td><td>668G</td><td></td></tr><tr><td>8-0 microsurgery nylon sutures</td><td>Ethicon</td><td>2808G</td><td></td></tr><tr><td>Lab-Line histology slide warmer</td><td>Barnstead International</td><td>26025</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cytospin method&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Collagenase Type I, lyophilized</td><td>Life Technologies&amp;nbsp;</td><td>1700-017</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Cell Signaling Technologies</td><td>9998S</td><td></td></tr><tr><td>1x PBS</td><td>Thermo Fisher</td><td>10010-023</td><td></td></tr><tr><td>Cytology funnels</td><td>Fisher HealthCare</td><td>10-354</td><td></td></tr><tr><td>HistoBond+ microscope slides</td><td>VWR</td><td>16005-110</td><td></td></tr><tr><td>Cytospin 2 centrifuge</td><td>Shandon</td><td>SH-CYTO2</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immunocytochemistry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Slide staining tray with black lid</td><td>IHC World</td><td>M920-2</td><td></td></tr><tr><td>Click-iT Plus EdU Imaging Kit</td><td>Life Technologies&amp;nbsp;</td><td>C10639</td><td>Includes EdU and&amp;nbsp; Hoeschst 33342</td></tr><tr><td>Immedge hydrophobic barrier pen</td><td>Vector Laboratories</td><td>H-4000</td><td></td></tr><tr><td>ProLong Diamond mounting medium</td><td>Thermo Fisher</td><td>P36970</td><td></td></tr><tr><td>Glass coverslips 24 x 50 mm #1.5</td><td></td><td></td><td></td></tr><tr><td>Clear nail polish</td><td></td><td></td><td></td></tr></tbody></table>

murine flexor tendon injury and repair surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

The flexor digitorum longus (FDL) muscle, located in the calf, acts to flex the digits of the mouse hind paw via the flexor tendon (outlined in blue in Figure 1A, and shown histologically in Figure 2A), which runs proximally from the myotendinous junction and terminates in the distal phalanges. In this model of flexor tendon healing, the FDL tendon is transected and repaired at the mid-foot, proximal to the bifurcation in to the digits of the hind paw (red arrows, Figure 1A). To prevent rupture of the repair, which would preclude experimental analysis, the tendon (and adjacent muscle) is cut, or released, at the myotendinous junction (yellow arrow in Figure 1B, release shown in Figure 1C). While this step is not representative of the clinical scenario, it is critical to protect the repair by decreasing strain on the injury site. Figure 2D is a histological example of a ruptured flexor tendon repair, compared to a successful repair shown in Figure 2C. In the latter, the granulation tissue displays greatly increased cellularity as compared to the uninjured tendon (Figure 2B), and this cell population can then be analyzed further via cytology centrifugation onto a slide followed by immunocytochemistry. Figure 3 outlines the experimental procedure used to evaluate the proliferative state of the cells collected from healing tendons 10 days post-repair via EdU incorporation into newly synthesized DNA. EdU labeling therefore shows only those cells that actively proliferated during the 24 hr period following the EdU pulse in live mice, as seen in Figure 4. A quantitative assessment of proliferation in the granulation tissue of the healing tendon can then be determined by point-counting of the entire cell preparation. In this way, a baseline of proliferation can be established at each time point, and any changes due to genetic or pharmacological interventions may be assessed.
<img alt="Figure 1" src="/files/ftp_upload/54433/54433fig1.jpg" /> 
Figure 1:. Murine Hind Paw Anatomy and Identification of the Flexor Digitorum Longus Tendon. The flexor digitorum longus (FDL) flexes the digits in the mouse hind paw and runs along the palmar aspect of the hind paw (FDL outlined in blue) where it bifurcates into the digits (red arrows) (Figure 1A). Proximally, the FDL tendon runs through the tarsal tunnel (black arrow), into the calf, ending at the myotendinous junction (yellow arrow) (Figure 1B). The release of the tendon at the myotendinous junction to prevent rupture is shown in Figure 1C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54433/54433fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54433/54433fig2.jpg" /> 
Figure 2: Representative Histological Images of Un-injured, Normal Healing and Ruptured Flexor Tendon Repairs. The anatomy of the FDL tendon in the murine hind paw is shown in Figure 2A. Scale bar represents 1 mm. In Figures 2B-D, the tendon is magnified at 8X to compare uninjured tendon tissue (Figure 2B) to an intact and ruptured repair (Figures 2C &#38; 2D, respectively). Whole hind paw samples were fixed in neutral buffered formalin, embedded in paraffin, cut in to 3 &#181;m sections, and stained with Alcian Blue/Hematoxylin/Orange G as previously described 13. Native tendon is outlined in blue, tendon/granulation tissue composite is outlined in green, and black arrows indicate sutures. Scale bars represent 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54433/54433fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54433/54433fig3.jpg" /> 
Figure 3: Experimental Procedure to Perform EdU Click-it Chemistry on Cell Preparations. On day 10 post-surgery healing FDL tendons were dissected from mouse hind paws. The tendons were minced then digested for two hours in collagenase I to dissociate the cells. The cell suspension was strained to remove debris, and ~6 x 104 cells were spun into a monolayer on positively charged glass slides using a cytology centrifuge. The cells were circled with a hydrophobic barrier pen and then fixed immediately with paraformaldehyde. After permeabilization with Triton X-100, the cells were treated with an EdU detection cocktail, and a click chemistry reaction bound Alexa Fluor 594 to incorporated EdU. Hoechst 33342 was used as a nuclear stain, and the cells were analyzed at 10-40X with a fluorescent microscope. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54433/54433fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54433/54433fig4.jpg" /> 
Figure 4: Immunocytochemistry Analysis of Cycling EdU-labeled Cells During Flexor Tendon Healing. Healing FDL tendons were harvested from the hind paws of mice 10 days post-surgery and digested with collagenase to obtain a cell suspension. Centrifugation was used to disperse these cells into a monolayer on slides, followed by immunocytochemistry to image EdU incorporation. Figure 4A and Figure 4B are images taken at 10X and 40X respectively of the cells following immunocytochemical analysis. Nuclear stain Hoechst 33342 is blue, and cells that have incorporated EdU are dotted with red. Scale bars represent 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54433/54433fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Flexor Tendon Injury and Repair Surgery at JoVE.com

Murine Flexor Tendon Injury and Repair Surgery

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

murine-flexor-tendon-injury-and-repair-surgery

Nanyang Technological University

Research

JoVE Journal

Medicine

13.0K Views.  University of Rochester Medical Center.  Flexorpezen in de hand worden vaak gewond, wat leidt tot een verminderde handfunctie. Echter, het littekenweefsel genezende reactie niet goed gekarakteriseerd. Een muizenmodel van flexorpees genezing is hier gedemonstreerd. Dit model kan algemeen begrip van het genezingsproces te verbeteren en therapeutische benaderingen om de genezing te verbeteren beoordelen. 

Video: Murine Flexor Tendon Injury and Repair Surgery

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to restrict knee joint mobility is a widely accepted model to generate experimental contracture. However, implanting application will inevitably cause surgical trauma to the animals. Aiming to develop a less invasive approach, we combined a muscle-gap separation modus with a previously reported mini-incision skill during the surgical procedure: Two mini skin incisions were made on the lateral thigh and leg, followed by performing muscle-gap separation to expose the bone surface. The rat knee joint was gradually immobilized by a preconstructed internal fixation at approximately 135&#176; knee flexion without interfering essential nerves or blood vessels. As expected, this simple technique permits rapid postoperative rehabilitation in animals. The correct position of the internal fixation was confirmed by an x-ray or micro-CT scanning analysis. The range of motion was significantly restricted in the immobilized knee joint than that observed in the contralateral knee joint demonstrating the effectiveness of this model. Besides, histological analysis revealed the development of fibrous deposition and adhesion in the posterior-superior knee joint capsule over time. Thus, this mini-invasive model may be suitable for mimicking the development of immobilized knee joint contracture.

Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to ...

Developmental Biology

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Bioengineering

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

Understanding Tendon Impingement &#8211; Murine Explant Model for Studying Mechanobiology

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

Author Spotlight: Unraveling the Mechanobiology of Tendon Impingement &#8211; A Multiaxial Murine Hind Limb Explant Model 

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

Using Q Suture to Enhance Resistance to Gap Formation and Tensile Strength of Repaired Flexor Tendons

Peripheral epitendinous sutures are believed to enhance core suture strength in tendon repair and decrease the risk of gapping between tendon ends. Here Q suture, an alternative to peripheral sutures, is presented for the use in tendon repair. Its effects on gap formation and tensile strength of the repaired tendons were compared with conventional running peripheral sutures. Three 2-strand sutures and three 4-strand sutures were used in repairing porcine tendons. The time required for performing 2Q and running sutures were recorded. The repaired tendons were subjected to a cyclic loading test, and the cycle number, during which a 2-mm gap was formed, was determined. After the cyclic loading, the gap size at the tendon ends and the ultimate strength of the repaired tendons were measured. Augmentation with the Q sutures reduced the number of tendons showing 2-mm gaps at tendon ends during cyclic loading. With addition of Q sutures 2-strand sutures significantly increased the ultimate strength of the repaired tendons and 4-strand sutures decreased the gap distance at the repair site of tendons. The time required for performing 2Q sutures was significantly less than that for running sutures. Therefore, we conclude that the Q suture is efficient in enhancing the tensile resistance and tendon repair strength and can be an alternative to conventional peripheral sutures.

Here, we present a &#8220;Q&#8221; suture technique that can be performed in tendon repair and its effects on the gap formation and tensile strength of the repaired tendons. Q suture is shown to be efficient in enhancing the tensile resistance and tendon repair strength.

Peripheral epitendinous sutures are believed to enhance core suture strength in tendon repair and decrease the risk of gapping between tendon ends. Here Q ...

Polytetrafluoroethylene (PTFE) as a Suture Material in Tendon Surgery

With the evolution of suture materials, there has been a change in paradigms in primary and secondary tendon repair. Improved mechanical properties allow more aggressive rehabilitation and earlier recovery. However, for the repair to hold against higher mechanical demands, more advanced suturing and knotting techniques must be assessed in combination with those materials. In this protocol, the use of polytetrafluoroethylene (PTFE) as a suture material in combination with different repair techniques was investigated. In the first part of the protocol, both linear tension strength and elongation of knotted against not-knotted strands of three different materials used in flexor tendon repair were evaluated. The three different materials are polypropylene (PPL), ultra-high molecular weight polyethylene with a braided jacket of polyester (UHMWPE), and polytetrafluoroethylene (PTFE). In the next part (ex vivo experiments with cadaveric flexor tendons), the behavior of PTFE using different suture techniques was assessed and compared with PPL and UHMWPE.
This experiment is comprised of four steps: harvesting of the flexor tendons from fresh cadaveric hands, transection of the tendons in a standardized manner, tendon repair by four different techniques, mounting, and measurement of the tendon repairs on a standard linear dynamometer. The UHMWPE and PTFE showed comparable mechanical properties and were significantly superior to PPL in terms of linear traction strength. Repairs with four- and six-strand techniques proved stronger than two-strand techniques. Handling and knotting of PTFE are a challenge due to very low surface friction but fastening of the four- or six-strand repair is comparatively easy to achieve. Surgeons routinely use PTFE suture material in cardiovascular surgery and breast surgery. The PTFE strands are suitable for use in tendon surgery, providing a robust tendon repair so that early active motion regimens for rehabilitation can be applied.

Polytetrafluoroethylene PTFE as a Suture Material in Tendon Surgery

The present protocol illustrates a method for assessing the biophysical properties of tendon repairs ex vivo. A polytetrafluoroethylene (PTFE) suture material was evaluated by this method and compared to other materials under different conditions.

With the evolution of suture materials, there has been a change in paradigms in primary and secondary tendon repair. Improved mechanical properties allow ...

A1 Pulley Reconstruction for Severe Trigger Finger

The aim of this study was to evaluate the effectiveness of A1 pulley reconstruction in treating severe trigger fingers and to compare its outcomes with those of the traditional A1 pulley release technique. A total of 43 patients participated in the study, divided into two groups: 22 patients underwent A1 pulley reconstruction, while the remaining 21 patients received the standard A1 pulley release procedure. The outcomes were assessed using the Michigan Hand Outcomes Questionnaire (MHQ) 1 month post-surgery. The results demonstrated that patients who received the A1 pulley reconstruction reported significantly better outcomes. These included enhanced hand function and quality of life, reduced levels of pain, improved aesthetic appearances, and higher overall satisfaction when compared to the traditional release group. The findings suggest that A1 pulley reconstruction offers superior benefits over the standard release procedure for individuals suffering from severe trigger fingers, highlighting its potential as a more effective surgical intervention.

Here, we present a protocol to compare A1 pulley reconstruction with traditional release surgery in treating a trigger finger, revealing enhanced outcomes and patient satisfaction through the Michigan Hand Outcomes Questionnaire.

The aim of this study was to evaluate the effectiveness of A1 pulley reconstruction in treating severe trigger fingers and to compare its outcomes with ...

A Mouse Model of Ankle-Subtalar Complex Joint Instability

Ankle sprains are perhaps the most common sports injuries in daily life, often resulting in instability of the ankle-subtalar complex joint (ASCJ), and can eventually lead to post-traumatic osteoarthritis (PTOA) in the long term. However, due to the complexity of the injury mechanism and the clinical manifestations, such as ecchymosis, hematoma, or tenderness in the lateral foot, there is no clinical consensus on diagnosing and treating ASCJ instability. Since the musculoskeletal structure of the bones and ligaments of the mouse hindfoot is comparable to that of humans, an animal model of ASCJ instability in mice was established by the transection of ligaments around the ASCJ. The model was well-validated through a series of behavioral tests and histological analyses, including a balance beam test, a footprint analysis (an assessment of exercise level and balance ability in mice), a thermal nociception assessment (an assessment of foot sensory function in mice), micro-computed tomography (CT) scanning, and section staining of the articular cartilage (an assessment of articular cartilage damage and degeneration in mice). The successful establishment of a mouse model of ASCJ instability will provide a valuable reference for clinical research on the injury mechanism and result in better treatment options for ankle sprain.

The ankle-subtalar complex joint (ASCJ) is the core of the foot and plays a key role in balance control in daily activities. Sports injuries often lead to instability in this joint. Here, we describe a mouse model of ligament transection-induced instability of the ASCJ.

Ankle sprains are perhaps the most common sports injuries in daily life, often resulting in instability of the ankle-subtalar complex joint (ASCJ), and ...

Biology

Development of a Rabbit Chronic-Like Rotator Cuff Injury Model for Study of Fibrosis and Muscular Fatty Degeneration

Rabbit rotator cuff (RC) pathophysiology can lead to progressive and highly degenerative changes in its associated musculature and tendons, which negatively influences clinically relevant parameters, such as strength and retraction of the muscle-tendon/myotendinous unit, ultimately causing loss of shoulder function and negatively affecting RC repair outcomes. Animal models that mimic aspects of human RC anatomy and pathophysiology are crucial for advancing the conceptual understanding of injury progression and developing effective tissue engineering and regenerative medicine-based therapeutics.
Within this context, a rabbit subscapularis (SSC) model is suitable due to (i) its anatomical similarity to the human supraspinatus (SSP) bone-tendon-muscle unit, which is the most frequently injured RC site; (ii) its pathophysiological similarity to humans in terms of fibrosis and muscle fatty degeneration (FD); and (iii) its amenability to surgical procedures. Therefore, the goal of this study is to describe the surgical techniques for inducing SSC RC injury. Briefly, the procedure involves the isolation of the SSC by identifying the coracobrachialis muscle followed by a full-thickness transection at the muscle-tendon junction and wrapping the free end of the muscle-tendon junction with a silicone-based penrose tubing to prevent spontaneous reattachment. Histologic evaluations are performed to monitor the progression of muscle FD at 4 weeks post-surgery using hematoxylin and eosin (H&#38;E) as well as Masson&#39;s trichrome staining.
Loss of muscle and FD were evident 4 weeks after transection of the SSC muscle-tendon junction, similar to human RC pathophysiological conditions. This protocol demonstrates the steps for successfully establishing a chronic-like rabbit SSC RC injury model, which can serve as a powerful tool to study skeletal muscle changes associated with RC pathophysiology and aid the development of novel therapeutic strategies for chronic-like RC tears.

This study details procedures for establishing a chronic-like rabbit rotator cuff (RC) injury. Specifically, the injury is created in the subscapularis (SSC) muscle-tendon/myotendinous unit to mimic human RC anatomy and pathophysiology, including severe muscle fatty degeneration (FD). This protocol can be applied to study RC injuries and assess regenerative therapies.

Murine Flexor Tendon Injury and Repair Surgery

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