Name: a rat tibial growth plate injury model to characterize repair mechanisms and evaluate growth plate regeneration strategies
Uploaded: 2017-07-04T12:30:00
Description: Watch this Scientific Journal Video about A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies at JoVE.com

The authors acknowledge funding support from the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIH) under award number R03AR068087, the Academic Enrichment Fund of the University of Colorado School of Medicine, and the Gates Center for Regenerative Medicine. This work was also supported by NIH/NCATS Colorado CTSA Grant Number UL1 TR001082. The contents are the authors' sole responsibility and do not necessarily represent official NIH views.

All animal procedures must be approved by the local Institutional Animal Care and Use Committee (IACUC). The animal protocol for the following procedure was approved by the University of Colorado Denver IACUC.
1. Obtain Rats
NOTE: Unless genetically modified animals are desired, 6-week-old, skeletally immature Sprague-Dawley rats are needed at the time of surgery. Other strains could potentially be used; however, the majority of published studies have been performed o...

<ol>
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A growth plate injury animal model greatly adds to our understanding of the biological mechanisms of this injury, thus potentially leading to more effective therapeutic interventions for children suffering from growth plate injuries. To successfully create a bony bar and to study its formation in vivo using the model presented in this work, it is critical to disrupt the growth plate by drilling to a sufficient depth, without disrupting the articular cartilage. Variation in surgical implementation among animals a...

Growth plate injuries account for 30% of all pediatric fractures and can result in impaired bone growth1. In addition to fractures, growth plate injuries may be caused by other etiologies, including osteomyelitis2, primary bone tumors3, radiation and chemotherapy4, and iatrogenic damage5. The growth plate (or physis) is a cartilage region at the end of children&#39;s long bones that is responsible for longitudinal bone growth. It drives bone elongation through endochondral ossification; chondrocytes undergo proliferation and hypertrophy and are then remodeled by incoming osteoblasts to form trabecular bone6. The growth plate is also a weak area of the developing skeleton, making it prone to injury. The major concern with growth plate fractures or injuries is that the damaged cartilage tissue within the growth plate can be replaced with unwanted bony repair tissue, also known as a &#34;bony bar.&#34; Depending on its size and location within the growth plate, the bony bar can lead to angular deformities or complete growth arrest, a devastating sequela for young children that have not yet reached their full height7.
There is currently no treatment that can fully repair an injured growth plate. Once the bony bar forms, the clinician must decide whether or not to surgically remove it8. Patients with at least 2 years or 2 cm of skeletal growth remaining and with a bony bar that spans less than 50% of the growth plate area are usually candidates for bony bar resection8. Surgical removal of the bony bar is often followed by interposition of an autologous fat graft to prevent reformation of the bony tissue and to allow the surrounding uninjured growth plate to restore growth. However, these techniques are problematic and often fail, leading to bony bar recurrence and continued negative effect on growth9. There is a critical need to develop effective treatments that not only prevent bony bar formation, but also regenerate the growth plate cartilage, thus restoring normal bone elongation.
The molecular mechanisms underlying bony bar formation have yet to be fully elucidated. A greater understanding of these biological mechanisms could lead to more effective therapeutic interventions for children suffering from growth plate injuries. Since studying these mechanisms in humans is difficult, animal models have been used, especially the rat model of growth plate injury10,11,12,13,14,15,16. The method presented in this paper describes how a drill-hole defect in the rat tibial growth plate leads to predictable and reproducible repair tissue that begins ossification as early as 7 days after injury and forms a fully mature bony bar with remodeling at 28 days after injury10. This provides a small animal in vivo model in which to study the biological mechanisms of bony bar formation, as well as to evaluate novel therapies that could prevent the bony bar and/or regenerate the growth plate cartilage. For example, this model can be used to test chondrogenic biomaterials that can regenerate growth plate cartilage and offer valuable treatment for children suffering from growth plate injuries. The techniques presented in this paper will describe the surgical methods used to produce the growth plate injury and the subsequent delivery of biomaterials to the injury site. We will also discuss methods to assess bony bar formation and repair tissue.

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			<td>McKesson</td>
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			<td>Needle holder</td>
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			<td>Adson tissue forceps</td>
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			<td>Dental Burs USA</td>
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			<td>Simpex Medical</td>
			<td>T-078</td>
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			<td>Hair clippers</td>
			<td>Wahl&#160;</td>
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			<td>Sterile gauze 2x2&#34;</td>
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			<td>Povidone Iodine</td>
			<td>McKesson</td>
			<td>922-00801</td>
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			<td>Sterile saline</td>
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			<td>10 ml luer lock syringe</td>
			<td>Becton Dickinson</td>
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			<td>23 gauge needle</td>
			<td>Becton Dickinson</td>
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			<td>Isopropyl alcohol pads</td>
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			<td>1113</td>
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			<td>Isoflurane</td>
			<td>IsoFlo</td>
			<td>30125-2</td>
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			<td>Caliper</td>
			<td>Mitutoyo</td>
			<td>500-196-30</td>
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			<td>27180</td>
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			<td>Buprenorphine</td>
			<td>Par Pharmaceuticals Inc</td>
			<td>NDC 42023-179</td>
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			<td>Fenestrated Surgical Drape</td>
			<td>McKesson</td>
			<td>25-517</td>
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			<td>Uline</td>
			<td>S-20204</td>
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			<td>9mm wound clips</td>
			<td>Fine Science Tools</td>
			<td>12032-09</td>
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			<td>Reflex clip applier</td>
			<td>World Precision Instruments</td>
			<td>500345</td>
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			<td>McKesson</td>
			<td>MON 43723110</td>
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			<td>Tec 3 Iso Vaporizer&#160;</td>
			<td>VetEquip</td>
			<td>911103&#160;</td>
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			<td>Germinator 500</td>
			<td>Braintree Scientific</td>
			<td>GER&#160;5287-120V</td>
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			<td>Warm water recirculator</td>
			<td>Kent Scientific</td>
			<td>TP-700</td>
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			<td>Absorbent Underpads</td>
			<td>Medline Industries</td>
			<td>MSC281230</td>
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a rat tibial growth plate injury model to characterize repair mechanisms and evaluate growth plate regeneration strategies

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found at the end of all long bones in children that is responsible for longitudinal bone growth. Once damaged, cartilage tissue within the growth plate can undergo premature ossification and lead to unwanted bony repair tissue, which forms a "bony bar." In some cases, this bony bar can result in bone growth deformities, such as angular deformities, or it can completely halt longitudinal bone growth. There is currently no clinical treatment that can fully repair an injured growth plate. Using an animal model of growth plate injury to better understand the mechanisms underlying bony bar formation and to identify ways to inhibit it is a great opportunity to develop better treatments for growth plate injuries. This protocol describes how to disrupt the rat proximal tibial growth plate using a drill-hole defect. This small animal model reliably produces a bony bar and can result in growth deformities similar to those seen in children. This model allows for investigation into the molecular mechanisms of bony bar formation and serves as a means to test potential treatment options for growth plate injuries.

Successful growth plate injury using this method involves the disruption of the center of the tibial growth plate without disrupting the articular cartilage surface. Bony repair tissue has been reported to begin at approximately 7 days post-injury and becomes fully developed by 28 days post-injury13, as visualized by micro computed tomography (micro CT) (Figure 2). Although these timepoints were chosen here to display the beginning and maturation o...

Watch this Scientific Journal Video about A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies at JoVE.com

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

The growth plate is a cartilaginous region in children's long bones where longitudinal growth occurs. When injured, bony tissue can form and impair growth. We describe a rat model of growth plate injury that leads to bony repair tissue, allowing the study of repair mechanisms and growth plate regeneration strategies.

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found ...

The overall goal of this surgical procedure is to create a drill-hole growth plate injury for the study of repair tissue formation in the evaluation of growth plate repair strategies in a small animal model. This method can help answer key questions related to growth plate injuries, specifically why the bony repair tissue forms after injury and also allows us to test novel treatments that could potentially regenerate the growth plate cartilage. The main advantages of this technique are the relative ease of implementation and the reproducibility of creating bony repair tissue that mimics clinical injuries.The implications of this technique extend towards the treatment of growth plate injuries as successful outcomes using this model can be translated to similar injuries in the clinic. Generally, individuals new to this technique will struggle with the difficulty of measuring and estimating appropriate drill depth necessary to disrupt the growth plate but without disrupting the articular surface. Before beginning the procedure, use an electric shaver to remove the hair from the entire hind leg of an anesthetized rat from the medial malleolus to the pelvis.Using calipers, measure and record the tibial length from the anterior tibial plateau to the inferior side of the medial malleolus and use alcohol swabs and povidone iodine soaked gauze to disinfect the entire leg, the abdomen, and the genitalia. Administration of IACUC approved analgesics can be done at this point. Wearing sterile surgical gloves, place a fenestrated sterile surgical drape over the animal, leaving the leg exposed through the central fenestration.Holding the leg firmly with one hand while pulling the skin tight against the bone, use a scalpel in the other hand to make a one centimeter incision through the skin from the distal end of the medial femoral condyle along the medial anterior aspect of the proximal tibia. Take note of the important anatomical markers, including the growth plate, the growth plate angle, the knee capsule, and the semitendinosus insertion. Then use a scalpel to make 0.5 centimeter incision through the fascia and soft tissues on the medial anterior aspect of the proximal tibia from the growth plate to the bottom of the skin incision and gently scrape away the fascia and soft tissues from the bone.When all of the tissue has been removed, grasp the leg firmly with one hand and hold a drill equipped with a Steinmann pin perpendicular to the tibial diaphysis. Keeping a firm hold on the leg with the other hand, slowly drill through the tibial cortical bone at the diaphysis to create a two millimeter cortical window that aligns with the distal semitendinosus insertion without drilling through to the other side of the bone. Then dab the finished cortical window with gauze as light bleeding is expected.To create the growth plate injury, align the end of a 1.8 millimeter dental burr with the proximal tibia where the semitendinosus crosses the knee capsule. To determine the appropriate depth for the burr to fully disrupt the growth plate without disrupting the articular surface, place the end of the dental burr at the knee capsule and follow the burr shaft along the semitendinosus insertion taking note of where the burr aligns with the cortical window. To attain the appropriate drill angle, hold the rotary tool at a less than 30 degree angle with respect to the tibial diaphysis and visualize a line along the dental burr to the growth plate angle.Now turn on the rotary tool at 10, 000 RPM and enter the cortical window, pushing the tool until until the burr marker aligns with the cortical window and removing the burr once the proper depth has been attained. Dab the cortical window with gauze for about 30 seconds and confirm the appropriate depth of the injury by reinserting the burr into the drill track with the rotary tool off and aligning the burr with the cortical window. Bleeding will interfere with visualization of the dental burr while making the injury so it is important to remeasure the burr to ensure that appropriate depth has been attained.If the depth is inadequate, push the burr to the desired depth with the tool on. Then use a 10 milliliter syringe equipped with a 23 gauge needle to rinse the drill track with three milliliters of sterile saline and dry the wound with gauze. Inject a biomaterial into the injury site as experimentally appropriate.Then use 3-0 polyglycolic acid sutures to close the wound and close the skin incision with wound clips. Bony repair tissue has been reported to begin at approximately seven days post-injury, becoming fully developed by 28 days post-injury as visualized by microcomputed tomography. Alcian blue hematoxylin with Orange G-eosin counterstain can also be used to histologically evaluate a variety of repair tissues at different stages of bony bar formation allowing the visualization of different types of repair tissue including mesenchymal, cartilaginous, bony trabeculae, and bone marrow.Further, the injection of a chitosan microgel into the growth plate injury site can be clearly observed at the injury site allowing subsequent analysis of the effects of the biomaterial on repair tissue composition, limb length, or growth plate measurements. Disruption of the articular cartilage surface creates a larger injury that can introduce articular cartilage into the growth plate injury site complicating the healing process. Disrupting the growth plate at an inappropriate angle or direction results in a non-central injury that results in bony bar formation that is lateral or medial to the desired location.Once mastered this technique can be completed on both limbs in about 25 minutes. While attempting this procedure it's important to have a surgical assistant to help maintain aseptic surgical technique. After watching this video you should have a good understanding of how to create a drill-hole growth plate injury in a rat model.

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Research

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Medicine

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

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.

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 ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...