Abstract
Ligaments play a crucial role in the musculoskeletal system. When maximal and sub-maximal ligamentous injury occur, a cascading response is initiated, starting with platelet plug formation and ending with remodeling. A rodent model was used to examine the role of modulating the initial recruitment of platelets and their impact on biochemical, histological, functional, and mechanical material properties. Functional gait testing, Enzyme Linked Immunosorbent Assay (ELISA), histological evaluations, and material property testing were performed. Animals (n=150) were randomized to a no-treatment group, a platelet rich fibrin treatment group and, a Nonsteroidal Anti-inflammatory Drug (NSIAD) treatment group. Initial results show that antiinflammatory drugs slow gait functional recovery; whereas, PRF shows faster functional recovery. PRF shows a faster increase in VEGF, and NSAIDs slows down VEGF and reduced its intensity. Evaluating injury recovery and impact of treatment methods, in both occupational and sports settings, provides insights about the healing response.
Keywords
Introduction
In the US, musculoskeletal disorders (MSDs) account for 29–35% of all occupational injuries and illnesses as reflected in days away from work (Bhattacharya, 2014). The annual costs of Low Back Disorders (LBDs) exceeds $100 billion, and in the US alone an additional 13 million people will develop LBDs annually due to their occupation, making it the most prevalent musculoskeletal problem in the workplace (Fathallah, 2010; Marras, 2000; Brent L. Ulrey & Fathallah, 2011). For farm workers, cumulative musculoskeletal injuries in general, and LBDs in particular, pose a significant direct and indirect financial costs in addition to the associated human costs due to pain and discomfort (Bhattacharya, 2014; Davis & Kotowski, 2007).
LBDs result from direct stress to the facet joints, pressure on the annulus of the disc, or pressure on the longitudinal ligaments of the spine (Marras, 2000). About 600,000 workers in California perform work in stooped postures (bent forward and down at the waist and or mid-back while maintaining straight legs) increasing their risk of developing LBDs (B. L. Ulrey & Fathallah, 2013). During stooped posture, flexion relaxation phenomenon occur with an onset of silence period in muscle activity causing the longitudinal spinal ligaments to bear most of the load; thus, increasing the risk of ligamentous injury (Brent L. Ulrey & Fathallah, 2011). This exposure to sustained static loading is a major factor in spinal ligaments injury causation (Sbriccoli et al., 2004).
Ligaments play a crucial role in the musculoskeletal locomotion and form a flexible skeletal joint between bones. Their main function is to guide and restrain skeletal motion (Chamberlain, 2010). They are pale and have limited blood supply. When a ligament injury occurs, designing an efficacious treatment to heal and reconstruct poses a great challenge (Frank, 2004).
Despite the high incidence of ligament injuries in occupational and sports settings, a fundamental understanding of the tissue’s response to injury remains unclear. Current standard treatment methods rely on Non-Steroid AntiInflammatory Drugs (NSIADs) and physical therapy: Rest, Ice, Elevate and Compress (RICE). The treatment efficacy is questionable, produces inferior results that may lead to joint mechanical instability, and may increase the chances for subsequent injuries and joint diseases (Bleakley, McDonough, & MacAuley, 2004; van den Bekerom et al., 2012). New treatment methods using Platelet Rich Fibrin (PRF) show very promising results
(Amar et al., 2015; Baksh, Hannon, Murawski, Smyth, & Kennedy, 2013; Dolkart et al., 2014). Because of the uncertainty in the healing process after ligament injury, the proper therapeutic intervention and any prevention recommendation in occupational settings such as farming tasks with stooped postures will also be uncertain. Therefore, a basic understanding of the histological, biochemical, and morphological response to maximal and sub-maximal strain injury mimicking stooped work postures will help guide the formulation of occupational injury prevention efforts and treatment strategies.
The present study has characterized the ligament injury-healing response at the early stage of healing. Early events involve the creation of a platelet plug to maintain hemostasis, inflammation, and proliferation followed by a long-term remodeling phase. The study has measured biochemical and material property during healing to assess the impact of different treatment strategies.
Methods
Animals
One hundred and fifty (150) 2-month-old male Wistar rats (The Jackson Laboratory, Sacramento, CA, USA), ranging in weight from 300 g to 350 g, were used for this study with the approval of the Institutional Animal Care and Use Committee (IACUC). All animals received humane care in compliance with the “Guide for the care and use of laboratory animals” prepared by the National Institute of Health (NIH publication No 86–23, revised 1996). Animals received standard rat chow and tap water ad libitum and were cared for under the supervision of a veterinarian.
Surgical method
Rodent survival surgery was conducted on animals per approved protocol by the UC Davis IACUC.
MCL Transection Injury
MCL injury was surgically induced. Rats were anesthetized using isoflurane inhalation (1-4% in O2 (2L/min). Hair on the medial side of the knee was removed with application of hair remover cream and then washed with betadine solution and isopropyl alcohol. Under sterile conditions, a small incision (10 mm) is made in their skin on the medial side of the knee joint of the right hind limb at the site of the MCL. The overlying connective tissue is dissected to expose the MCL, and a 1-mm gap in the mid-substance is surgically created with scalpel and the gap is left without suturing and allowed to heal spontaneously.
Sham Injury
A sham injury for the biochemical and histological experiments on the contralateral MCL was performed by following the same procedure with the exception of transecting the MCL. The MCL was tugged gently using forceps without inducing injury. For ELISA and histology experiments the sham injury served as an internal contralateral control within the same animal by inducing the same injury conditions without transecting the MCL.
The skin incision is closed using sutures and animals were given buprenorphine subcutaneously for pain suppression. After surgery, the rats were allowed free cage activities and were monitored for 7 days for infections and complications. After surgery, animals were allowed free cage activity and monitored for infections and complications. A second dose of buprenorphine was given within 24 hours of surgery.
Contralateral Control
For gait testing, injury was induced on one knee leaving the other knee intact serving as an internal or ‘Contralateral’ MCL Control.
PRF Preparation
A group of rats (n=6) donated the plasma to prepare the platelet rich fibrin (PRF). Rats were anesthetized with Isoflurane inhalation. A syringe with no anticoagulant equipped with an 18-gauge needle was used to exsanguinate by cardiac puncture about 10 ml blood from each anesthetized rat. Immediately after collection, blood is aliquoted into 2 ml microcentrifuge tubes. Blood is then immediately and before coagulating (within 60 seconds of blood collection) centrifuged at 1,500 rpm (250g) for 10 min resulting in separation of red blood cells and a fibrin gel-like supernatant rich in activated platelets. The PRF was then stored at -80C until use.
Treatment Groups
The animals were randomized to one of three treatment methods with 50 animals in each group. The study groups are: no-treatment, Platelet Rich Fibrin (PRF), and COX-2 inhibitor; Meloxicam (MX). Functional gait testing (n=30), analytical biochemical testing (n=60) with enzyme linked immunoassay (ELISA) for Vascular Endothelial Growth Factor (VEGF), and histological evaluation with a Modified Bonar Scoring technique (n=60) were evaluated.
No treatment controls
Study groups undergone survival surgery with MCL transection and allowed to heal spontaneously with no intervention.
PRF treatment
Immediately after MCL transection and prior to closure of incision, animals received the PRF preparation at the injury site and the PRF was secured at the injury site with internal absorbable sutures that ran through the PRF and closed the pocket at the MCL site by approximating subcutaneous tissue and fascia.
Meloxicam Treatment
Immediately after surgery and again in 24 hours, Meloxicam, a selective COX-2 inhibitor, was injected at the injury site at a dose of 3 mg/kg body weight using an insulin syringe.
Experimental Methods
Gait Analysis
A gait test followed the rodent’s locomotion during a walk along a straight path towards a target enclosure. Eight parameters were measured: stride lengths, front and back paws stride widths, paws overlap, mean stride length, toe spread, and inner toe spread (Carter, Morton, & Dunnett, 2001). Difference in gait patterns were used characterize injury-healing response ((Klapdor, Dulfer, Hammann, & Van der Staay, 1997; Kloefkorn, Jacobs, Loye, & Allen, 2015; Varejão et al., 2001)).
ELISA
Sandwich Enzyme Linked Immunosorbent Assay (ELISA) methods was used. After necropsy, tissue was stored at -80 degrees Celsius. Tissue was then homogenized with a cell lysis buffer and aliquoted into smaller volumes for future testing. Due to low protein density in ligament tissue samples, ELISA is performed with no sample dilutions. The ELISA kits used are pre-coated with target protein antibodies (My Bio Source, San Diego, CA). Vascular endothelia growth factor (VEGF) (González-Martínez, Puchades, & Maquieira, 2018) was assayed. ELISA was performed on days 1, 3, 5, and 7.
Histology
Histological analysis will characterize collagen organization, vascularization, and cellularity. After necropsy, ligaments were placed in 4% paraformaldehyde for 48 hours and for up to 7 days in 70% ethanol until further histological processing to embed tissue into paraffin blocks. Sectioning was performed on a Leica microtome with 5-micron sections. Orientation of tissue was preserved during all steps using optimized techniques to maintain the skin and joint facing sides on microscope slides. Hematoxylin & Eosin (H&E) stains were used to visualize tissue samples and nucleated cellular components. Modified Bonar Scoring technique was used to quantify tendinopathy by characterizing three factors; (1) Collagen organization, (2) Vascularization and, (3) Cellularity (Maffulli, Longo, Franceschi, Rabitti, & Denaro, 2008). Histological classification was performed on tissue at days 1, 2, 3, 5, and 7 post injury.
Histomorphology and Mechanical Testing
Histomorphology and mechanical testing are currently underway. Using Hematoxylin and Eosin stains on tissue sections, we will measure total ligament section thickness in the frontal plane of ligament using ImageJ (NIH Software). Morphometric measurements will be performed on days 1, 2, 3, 5, and 7 post injury.
Material testing will be performed on the Femur-MCL-Tibia segments to ensure proper attachment to the Material Testing System (MTS) with a system force and displacement resolutions of 0.01 N and 0.001 mm, respectively. The segments will be pulled to tensile failure at a strain rate of 10%/s (.0.6-0.8 mm/s) (Provenzano, Heisey, Hayashi, Lakes, & Ray Vanderby, 2002). Four parameters will be obtained from the MTS; load to failure, displacement, stiffness, and energy to failure (Amar et al., 2015). We will conduct mechanical testing at days 15 and then at day 30.
Results
For this paper, results from the gait analysis and ELISA are presented.
Gait results
Initial evaluation of the various gait factors showed that Inner Toe Spread (ITS) was the only factor that showed a significant difference between injured and non-injured contralateral controls. Therefore, analysis was performed using ITS as a functional recovery indicator. In control animals, functional recovery was realized 14 days following injury, in Meloxicam treated animals after 7 days whereas PRF treated animals showed no difference in ITS after day 1 (Figure 1).
ITS Results for Control (C), Meloxicam (MX), and Platelet Rich Fibrin (PRF).
ELISA Results
No-treatment animals showed a VEGF concentration peak in day 5. PRF treated animals showed a peak VEGF concentration on day 3 with a two-fold increase in concentration compared to notreatment animals.
Discussion
Following injury, ligaments undergo a series of events to restore tissue function. The results show that functional and biochemical responses can be used to examine the healing response as well as evaluate the impact of treatment methods. Restoration of function was evaluated using gait testing. Animals treated with PRF showed a function restoration in 7 days. Non-treated animals and Meloxicam treated animals required much longer to reach the same level of functional recovery.
VEGF ELISA showed that PRF treated animals have an increased concentration of VEGF at an earlier time point than untreated animals. This implies a more intense repair response as VEGF is a potent growth factor for the formation of new vascular tissue responsible for the delivery of oxygen, nutrients, and other cells needed for to carry out ligament repair.
Conclusion
Ligaments play a major functional role in the musculoskeletal system. When injured, the standard treatment methods are focused on pain management and return to function as appose to promoting time to recovery and healing of tissue to preinjury properties. The goal of this study was to provide a basis to discriminate between treatment methods and to characterize tissue’s response in animal models. Learning from this study may help improve treatment and intervention methods and examination of other injury mechanisms.
Footnotes
Acknowledgements
This research was partially funded by the NIOSH Western Center for Agricultural Health and Safety, and NIOSH training grant T42-OH008429.