Large Animal Studies Research Articles

Transplanting human umbilical cord mesenchymal stem cells and hyaluronate hydrogel repairs cartilage of osteoarthritis in the minipig model.

Objectives:Osteoarthritis (OA) is a chronic disease of degenerative joints. Mesenchymal stem cells (MSCs) have been used for cartilage regeneration in OA. We investigated the therapeutic potential of human umbilical cord-derived MSCs (HUCMSCs) with hyaluronic acid (HA) hydrogel transplanted into a porcine OA preclinical model.Materials and Methods:The HUCMSCs were characterized with respect to morphology, surface markers, and differentiation capabilities. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to examine gene expressions in a HUCMSC–HA coculture. Two healthy female minipigs weighing 30–40 kg and aged approximately 4 months were used in this large animal study. A full-thickness chondral injury was created in the trochlear groove of each of the pig's rear knees. After 3 weeks, a second osteochondral defect was created. Then, 1.5 mL of a HUCMSC (5 × 106 cells) and HA composite (4%) was transplanted into the chondral-injured area in the right knee of each pig. Using the same surgical process, an osteochondral defect (untreated) was created in the left knee as a control. The pigs were sacrificed 12 weeks after transplantation. Macroscopic and microscopic histologies, qRT-PCR, and immunostaining evaluated the degree of chondral degradation.Results:The HUCMSCs exhibited typical MSC characteristics, including spindle morphology, expression of surface markers (positive for CD29, CD4, CD73, CD90, and human leukocyte antigen [HLA]-ABC; negative for CD34, CD45, and HLA-DR), and multipotent differentiation (adipogenesis, osteogenesis, and chondrogenesis). More extensive proliferation of HUCMSCs was noted with 4% and 25% of HA than without HA. Expression of COL2A1 and aggrecan in the HUCMSC-derived chondrocytes was increased when HA was included. The treated knees showed significant gross and histological improvements in hyaline cartilage regeneration when compared to the control knees. The International Cartilage Repair Society histological score was higher for the treated knees than the control knees.Conclusion:Our findings suggest that cartilage regeneration using a mixture of HUCMSCs and HA in a large animal model may be an effective treatment for OA, and this study is a stepping stone toward the future clinical trials.

Diffuse Correlation Spectroscopy Analysis Implemented on a Field Programmable Gate Array.

Diffusive correlation spectroscopy (DCS) is an emerging optical technique that measures blood perfusion in deep tissue. In a DCS measurement, temporal changes in the interference pattern of light, which has passed through tissue, are quantified by an autocorrelation function. This autocorrelation function is further parameterized through a non-linear curve fit to a solution to the diffusion equation for coherence transport. The computational load for this non-linear curve fitting is a barrier for deployment of DCS for clinical use, where real-time results, as well as instrument size and simplicity, are important considerations. We have mitigated this computational bottleneck through development of a hardware analyzer for DCS. This analyzer implements the DCS curving fitting algorithm on digital logic circuit using Field Programmable Gate Array (FPGA) technology. The FPGA analyzer is more efficient than a typical software analysis solution. The analyzer module can be easily duplicated for processing multiple channels of DCS data in real-time. We have demonstrated the utility of this analyzer in pre-clinical large animal studies of spinal cord ischemia. In combination with previously described FPGA implementations of auto-correlators, this hardware analyzer can provide a complete device-on-a-chip solution for DCS signal processing. Such a component will enable new DCS applications demanding mobility and real-time processing.

Mitochondrial Respiratory Capacity is Restored in Hibernating Cardiomyocytes Following Co-Culture with Mesenchymal Stem Cells.

Hibernating myocardium is a subset of ischemic cardiac disease characterized by viable but dysfunctional tissue. Standard treatment for hibernating myocardium is coronary artery bypass graft, which reduces arrhythmias and improves survival but does not fully restore function, presenting a gap in currently available treatments. Large animal studies of hibernating myocardium have identified impaired mitochondrial dynamics as a root cause of persistent cardiac dysfunction despite surgical revascularization. This study presents a novel in vitro model of hibernating myocardium cardiomyocytes to study active mitochondrial respiration in hibernating myocardium cells, and to test the paracrine effect of mesenchymal stem cells on impaired mitochondrial function. Exposure of cardiomyocytes to hypoxic conditions of 1% oxygen for 24 hours resulted in a phenotype consistent with hibernating myocardium cardiac tissue, including decreased respiratory capacity under high work states, decreased expression of mitochondrial proteins, and preserved cellular viability. Co-culture of hibernating myocardium cardiomyocytes with mesenchymal stem cells restored mitochondrial respiratory function, potentially via an increase in proliferator-activated receptor gamma coactivator 1-alpha-driven mitochondrial biogenesis. Co-culture treatment of hibernating myocardium cardiomyocytes with mesenchymal stem cells shows improvement in both mitochondrial function and ATP production, both of which are critical for effectively functioning cardiac tissue. These results suggest that mesenchymal stem cell therapy as an adjunct treatment to revascularization may address the current gap in treatment for hibernating myocardium patients.

Cold blood perfusion for selective hypothermia in acute ischemic stroke.

Hypothermia is the most reliably effective neuroprotectant, and yet systemic complications limit application. A large body of animal data suggests that hypothermia is effective for focal cerebral ischemia, namely acute ischemic stroke. In order to apply hypothermia effectively, a selective approach is required to maximize the effect on the brain while minimizing systemic side effects. Due to poor transferability of promising findings in rodent models to human clinical trials for neuroprotection, the focus of this review is large animal gyrencephalic models. Unlike rodent data which favor mild hypothermia, the majority of large animal studies on selective hypothermia support moderate-to-deep hypothermia (<30°C). Cold blood perfusion produces the rapid rate of temperature reduction and depth of hypothermia required to produce meaningful neuroprotection. Further studies of selective hypothermia in acute ischemic stroke require attention to duration and rate of cooling to optimize the neuroprotection offered by this technique.

Intravenous and Intra-amniotic &lt;em&gt;In Utero&lt;/em&gt; Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Refinement, Reduction, and Replacement (3R) Strategies in Preclinical Testing of Medical Devices.

The U.S. Food and Drug Administration Center for Devices and Radiological Health (FDA/CDRH) has recently published several in vivo test guidance documents that mention refinements, reductions, or replacement animal testing strategies to facilitate the leveraging of data from large animal safety tests for conventional rodent testing. In response to the recently enacted Food and Drug Administration Safety and Innovation Act Section 907, which facilitates expedited access to novel therapies commonly described as Breakthrough Therapy Designation, FDA/CDRH has discussed efficient regulatory strategies for first-in-human investigation, including early feasibility study guidance. Large gains in humane care and translational research could also be attained by examples in FDA's Guidance for the Use of International Organization for Standardization 10993-1, which states that large animal safety studies may be considered as replacement rodent tests if the scientific principles, methods, and end points (SPME) are considered and applied. This article discusses SPME for the replacement of conventional rodent testing by the inclusion and integration of clinical, diagnostic, and pathologic data obtained from well-designed large animal studies. The recommendations include consideration for study designs that utilize methods for an overall more comprehensive interrogation of animal systems.

Corrigendum

International Wound JournalVolume 15, Issue 5 p. 855-855 CORRIGENDUMOpen Access Corrigendum This article corrects the following: Enhanced wound-healing performance of a phyto-polysaccharide-enriched dressing – a preclinical small and large animal study Chih-Hsin Wang, Shu-Jen Chang, Yuan-Sheng Tzeng, Yu-Jen Shih, Chang Adrienne, Shyi-Gen Chen, Tim-Mo Chen, Niann-Tzyy Dai, Juin-Hong Cherng, Volume 14Issue 6International Wound Journal pages: 1359-1369 First Published online: September 21, 2017 First published: 19 September 2018 https://doi.org/10.1111/iwj.12987AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In Wang et al.,1 an error was published in the author affiliations. ‘National Defense Medical Center’ was wrongly included in affiliation 2 instead of in affiliation 1. The correct affiliations are as follows: 1Department of Plastic and Reconstructive Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (R.O.C). 2Department of Dentistry, National Yang-Ming University, Taipei, Taiwan (R.O.C). We apologize for these errors. REFERENCE 1Wang C-H, Chang S-J, Tzeng Y-S, et al. Enhanced wound-healing performance of a phyto-polysaccharide-enriched dressing – a preclinical small and large animal study. Int Wound J. 2017; 14: 1359- 1369. Volume15, Issue5October 2018Pages 855-855 ReferencesRelatedInformation

Optimizing Livers for Transplantation Using Machine Perfusion versus Cold Storage in Large Animal Studies and Human Studies: A Systematic Review and Meta-Analysis.

Background Liver allograft preservation frequently involves static cold storage (CS) and machine perfusion (MP). With its increasing popularity, we investigated whether MP was superior to CS in terms of beneficial outcomes. Methods Human studies and large animal studies that optimized livers for transplantation using MP versus CS were assessed (PubMed/Medline/EMBASE). Meta-analyses were conducted for comparisons. Study quality was assessed according to the Newcastle-Ottawa quality assessment scale and SYRCLE's risk of bias tool. Results Nineteen studies were included. Among the large animal studies, lower levels of lactate dehydrogenase (SMD -3.16, 95% CI -5.14 to -1.18), alanine transferase (SMD -2.46, 95% CI -4.03 to -0.90), and hyaluronic acid (SMD -2.48, 95% CI -4.21 to -0.74) were observed in SNMP-preserved compared to CS-preserved livers. NMP-preserved livers showing lower level of hyaluronic acid (SMD -3.97, 95% CI -5.46 to -2.47) compared to CS-preserved livers. Biliary complications (RR 0.45, 95% CI 0.28 to 0.73) and early graft dysfunction (RR 0.56, 95% CI 0.34 to 0.92) also significantly reduced with HMP preservation in human studies. No evidence of publication bias was found. Conclusions MP preservation could improve short-term outcomes after transplantation compared to CS preservation. Additional randomized controlled trials (RCTs) are needed to develop clinical applications of MP preservation.

Squid Ring Teeth–coated Mesh Improves Abdominal Wall Repair

Hernia repair is a common surgical procedure with polypropylene (PP) mesh being the standard material for correction because of its durability. However, complications such as seroma and pain are common, and repair failures still approach 15% secondary to poor tissue integration. In an effort to enhance mesh integration, we evaluated the applicability of a squid ring teeth (SRT) protein coating for soft-tissue repair in an abdominal wall defect model. SRT is a biologically derived high-strength protein with strong mechanical properties. We assessed tissue integration, strength, and biocompatibility of a SRT-coated PP mesh in a first-time pilot animal study. PP mesh was coated with SRT (SRT-PP) and tested for mechanical strength against uncoated PP mesh. Cell proliferation and adhesion studies were performed in vitro using a 3T3 cell line. Rats underwent either PP (n = 3) or SRT-PP (n = 6) bridge mesh implantation in an anterior abdominal wall defect model. Repair was assessed clinically and radiographically, with integration evaluated by histology and mechanical testing at 60 days. Cell proliferation was enhanced on SRT-PP mesh. This was corroborated in vivo by abdominal wall histology, dramatically diminished craniocaudal mesh contraction, improved strength testing, and higher tissue failure strain. There was no increase in seroma or visceral adhesion formation. No foreign body reactions were noted on liver histology. SRT applied as a coating appears to augment mesh-tissue integration and improve abdominal wall stability following bridged repair. Further studies in larger animals will determine its applicability for hernia repair in patients.

HSP70iQ435A-Encoding DNA Repigments Vitiligo Lesions in Sinclair Swine

Human HSP70iQ435A carries a single amino-acid modification within the dendritic cell activating region and tolerizes dendritic cells invitro. The underlying DNA was used to prevent and treat disease in vitiligo mouse models through reduced dendritic cell activation and diminished skin T-cell infiltration, suggesting the same may be useful for patients. Physiologic differences between mouse and human skin then called for studies in large animals with human-like skin. We established the efficiency of DNA jet injection into swine skin before subcloning HSP70iQ435A into clinically suitable vector pUMVC3. Vitiligo lesions in Sinclair swine were treated with plasmid DNA to measure changes in depigmentation, T-cell infiltration, expression of HSP70i in skin, serum HSP70i, and anti-HSP70i serum titers. Remarkable repigmentation following HSP70iQ435A-encoding DNA treatment persisted throughout the 6-month follow-up period. Repigmentation was accompanied by an initial influx of T cells accompanied by increased CD4/CD8 ratios, waning by week 15. Melanocytes spanned the border of repigmenting skin, suggesting that melanocyte repopulation precedes skin melanization. Serum titer fluctuations were not treatment-associated. Importantly, treatment did not interfere with melanoma immunosurveillance. These data encourage clinical testing of HSP70iQ435A.

Animal science meets agricultural practice: Preliminary results of an innovative technical approach for exhaled breath analysis in cattle under field conditions

In human and veterinary medicine, the search for exhaled volatiles as potential markers for disease detection has gained increasing attention because exhaled breath analysis bears the advantages of being continuous, non-invasive and reveals instantly available results. The aim of this study was to introduce a technical set-up for breath analysis in bovines. The technical set-up allows (i) CO2-controlled sampling and preconcentration of alveolar gas and (ii) direct breath gas analysis via proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) in parallel. While alveolar gas sampling enables the analysis by gas chromatography-mass spectrometry for unequivocal substance identification, PTR-ToF-MS ensures real-time identification of confounding signals within one measurement. Applicability of the system to conscious cattle and reliability of the signals obtained under field conditions were tested at eight dairy farms. This first testing phase in veterinary practice confirmed that the set-up was well tolerated by each of the cows and that reliable measured values (i.e., raw data) were gained at different respiratory rates. Inspiration could be clearly distinguished from expiration, thus providing the basis for excluding ambient air contamination. Consecutive measurements under standardised conditions in two bovines aged 5–6 months revealed effects of time of day, food intake and handling that need to be evaluated in-depth in future studies with larger groups of animals. As result of these proof-of-concept trials, the introduced technical set-up can be recommended for future studies evaluating exhaled markers in cattle. Breath gas analysis offers a new research area in veterinary medicine and is one step forward towards refining large animal studies, even under field conditions.

Amniotic Epithelial Stem Cells: Salient Features and Possible Therapeutic Role.

This is a study of amniotic epithelial cells, which form the innermost layer of the amniotic membrane. These cells can be easily isolated and display peculiar and unique properties, such as plasticity and differentiation potential toward the 3 germinal layers, that may aid regeneration and/or repair of damaged or diseased tissues and organs. A robust literature based on in vitro, experimental, and clinical studies in large animals demonstrates that these cells can enhance the regeneration of tendons, bone, and articular cartilage. On the basis of these considerations, allotransplantation of human amniotic epithelial cells could be proposed for clinical trials in human orthopedic conditions.

Online monitoring of carbon dioxide and oxygen in exhaled mouse breath via substrate-integrated hollow waveguide Fourier-transform infrared-luminescence spectroscopy

Exhaled breath offers monitoring bio markers, as well as diagnosing diseases and therapeutic interventions. In addition, vital functions may be non-invasively monitored online. Animal models are frequently used in research for determining novel therapeutic approaches and/or for investigating biological pathways. The exhaled carbon dioxide concentration, exhaled and inhaled oxygen concentration, and the subsequent respiratory quotient (RQ) offer insight into metabolic activity. One may adapt breath sampling systems and equipment designed for human applications to large animal studies. However, such adaptations are usually impossible for small animals due to their minuscule breath volume. Here, we present a system for the online monitoring of exhaled breath in a ‘mouse intensive care unit’ (MICU) based on a modified Fourier-transform infrared spectrometer equipped with a substrate-integrated hollow waveguide gas cell, and a luminescence-based oxygen flow-through sensor integrated into the respiratory equipment of the MICU. Thereby, per-minute resolution of O2 consumption and CO2 production was obtained, and the 95% confidence range of the determined RQ was ±0.04 or approximately ±5% of the nominal value. Changes in the RQ value caused by intervention in either the metabolic or respiratory system may therefore reliably be detected.

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species.

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

Normothermic Ex-vivo Liver Perfusion and the Clinical Implications for Liver Transplantation.

Despite significant improvements in outcomes after liver transplantation, many patients continue to die on the waiting list, while awaiting an available organ for transplantation. Organ shortage is not only due to an inadequate number of available organs, but also the inability to adequately assess and evaluate these organs prior to transplantation. Over the last few decades, ex-vivo perfusion of the liver has emerged as a useful technique for both improved organ preservation and assessment of organs prior to transplantation. Large animal studies have shown the superiority of ex-vivo perfusion over cold static storage. However, these studies have not, necessarily, been translatable to human livers. Small animal studies have been essential in understanding and improving this technology. Similarly, these results have yet to be translated into clinical use. A few Phase 1 clinical trials have shown promise and confirmed the viability of this technology. However, more robust studies are needed before ex-vivo liver perfusion can be widely accepted as the new clinical standard of organ preservation. Here, we aimed to review all relevant large and small animal research, as well as human liver studies on normothermic ex-vivo perfusion, and to identify areas of deficiency and opportunities for future research endeavors.

Normothermic Ex Vivo Machine Perfusion for Liver Grafts Recovered from Donors after Circulatory Death: A Systematic Review and Meta-Analysis

As a result of donation after circulatory death liver grafts' poor tolerance to cold storage, there has been increasing research interest in normothermic machine perfusion. This study aims to systematically review the current literature comparing normothermic perfusion to cold storage in donation after circulatory death liver grafts and complete a meta-analysis of published large animal and human studies. A total of nine porcine studies comparing cold storage to normothermic machine perfusion for donation after circulatory death grafts were included for analysis. There was a significant reduction in AST (mean difference −2291 U/L, CI (−3019, −1563); P ≤ 0.00001) and ALT (mean difference −175 U/L, CI (−266, −85); P = 0.0001), for normothermic perfusion relative to static cold storage, with moderate (I2 = 61%) and high (I2 = 96%) heterogeneity, respectively. Total bile production was also significantly higher (mean difference = 174 ml, CI (155, 193); P ≤ 0.00001). Further research focusing on standardization, performance of this technology following periods of cold storage, economic implications, and clinical trial data focused on donation after circulatory death grafts will be helpful to advance this technology toward routine clinical utilization for these grafts.

Emerging Innovations in Liver Preservation and Resuscitation

The availability of marginal liver grafts represents a potential resource of orthotopic liver transplantation (OLT) in the era of organ shortage. However, an increased susceptibility to ischemia-reperfusion injury results in the increased risk for primary nonfunction and biliary complications. Novel methods of organ preservation and resuscitation are under investigation to improve outcomes of OLT with the use of marginal grafts. Machine perfusion (MP) in OLT is categorized based on the temperature setting. Hypothermic MP minimizes oxygen demand and uses single- or dual-vessel infusion with or without oxygenation. Normothermic MP increases the metabolic rate to the physiologic level, and requires dual-vessel infusion with a higher blood flow rate with oxygenation typically by blood as an oxygen carrier. Midthermic or subnormothermic MP uses medium temperature settings to allow metabolic activity to maintain cellular integrity as well as decrease oxygen demand. Since 2010, a total of 9 clinical trials have investigated the safety and feasibility of hypothermic and normothermic MP. The results suggest that the incidence of primary nonfunction was minimal even when marginal grafts were used. Organ preservation time could be reasonably increased. MP offers an opportunity to assess the viability and time to apply therapeutic interventions. This review highlights the history of MP, mechanisms of different temperature settings in MP, and recent large animal studies and clinical trials in OLT.

Post-Irradiation Treatment with a Superoxide Dismutase Mimic, MnTnHex-2-PyP5+, Mitigates Radiation Injury in the Lungs of Non-Human Primates after Whole-Thorax Exposure to Ionizing Radiation.

Radiation injury to the lung is the result of acute and chronic free radical formation, and there are currently few effective means of mitigating such injury. Studies in rodents indicate that superoxide dismutase mimetics may be effective in this regard; however, studies in humans or large animals are lacking. We hypothesized that post-exposure treatment with the lipophilic mitochondrial superoxide dismutase mimetic, MnTnHex-2-PyP5+ (hexyl), would reduce radiation-induced pneumonitis and fibrosis in the lungs of nonhuman primates. Rhesus monkeys (Macaca mulatta) received 10 Gy whole thorax irradiation, 10 Gy + hexyl treatment, sham irradiation, or sham irradiation + hexyl. Hexyl was given twice daily, subcutaneously, at 0.05 mg/kg, for 2 months. Animals were monitored daily, and respiratory rates, pulse oximetry, hematology and serum chemistry panels were performed weekly. Computed tomography scans were performed at 0, 2, and 4 months after irradiation. Supportive fluid therapy, corticosteroids, analgesics, and antibiotics were given as needed. All animals were humanely euthanized 4.5 months after irradiation, and pathologic assessments were made. Multifocal, progressive lung lesions were seen at 2 and 4 months in both irradiated groups. Hexyl treatment delayed the onset of radiation-induced lung lesions, reduced elevations of respiratory rate, and reduced pathologic increases in lung weight. No adverse effects of hexyl treatment were found. These results demonstrate (1) development of a nonhuman primate model of radiation-induced lung injury, (2) a significant mitigating effect of hexyl treatment on lung pathology in this model, and (3) no evidence for toxicity of hexyl at the dose studied.

The challenges of promoting osteogenesis in segmental bone defects and osteoporosis.

Conventional clinical management of complex bone healing scenarios continues to result in 5-10% of fractures forming non-unions. Additionally, the aging population and prevalence of osteoporosis-related fractures necessitate the further exploration of novel ways to augment osteogenesis in this special population. This review focuses on the current clinical modalities available, and the ongoing clinical and pre-clinical research to promote osteogenesis in segmental bone defects, delayed unions, and osteoporosis. In summary, animal models of fracture repair are often small animals as historically significant large animal models, like the dog, continue to gain favor as companion animals. Small rodents have well-documented limitations in comparing to fracture repair in humans, and few similarities exist. Study design, number of studies, and availability of funding continue to limit large animal studies. Osteoinduction with rhBMP-2 results in robust bone formation, although long-term quality is scrutinized due to poor bone mineral quality. PTH 1-34 is the only FDA approved osteo-anabolic treatment to prevent osteoporotic fractures. Limited to 2 years of clinical use, PTH 1-34 has further been plagued by dose-related ambiguities and inconsistent results when applied to pathologic fractures in systematic human clinical studies. There is limited animal data of PTH 1-34 applied locally to bone defects. Gene therapy continues to gain popularity among researchers to augment bone healing. Non-integrating viral vectors and targeted apoptosis of genetically modified therapeutic cells is an ongoing area of research. Finally, progenitor cell therapies and the content variation of patient-side treatments (e.g., PRP and BMAC) are being studied. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1559-1572, 2018.

Is NS-EDTA Effective in Clearing Bacteria From Infected Wounds in a Rat Model?

BackgroundIrrigation is one of the key procedures in open fracture management to eliminate pathogens and prevent infection. Metal ion deprivation could inhibit bacterial adhesins and weaken adhesion to the host tissue. EDTA in solution can competitively bind to a metal ion and thus might be able to inhibit bacterial adhesins.Questions/purposes(1) Is normal saline-EDTA toxic to fibroblasts and endothelial cells? (2) In a contaminated wound rat model, does irrigation with normal saline-EDTA solution decrease the risk of positive bacterial cultures and infection when compared with normal saline and soap solutions? (3) In an infected wound rat model, are fewer surgical débridements and irrigations with normal saline-EDTA solution required to obtain culture-free wounds when compared with normal saline and soap controls?MethodsNormal saline-EDTA solution refers to 1 mmol/L EDTA dissolved in normal saline (pH adjusted to 7.4). Normal saline and soap solutions acted as controls. The toxicity of these solutions to fibroblasts and endothelial cells was assessed in vitro by Annexin V/propidium iodide staining and flow cytometer counting (a well-established method to quantitatively measure the number of dead cells). We established contaminated and infected wound models (bone-exposed or not) with either Staphylococcus aureus or Escherichia coli in rats to investigate the efficacy of normal saline-EDTA solution (n = 30 for the contaminated model and n = 50 for the infected model). For contaminated wounds, the proportion of positive bacterial cultures and infections was compared after irrigation and débridement among the three groups. For infected wounds, we performed irrigation and débridement every 48 hours until the cultures were negative and compared the number of débridements required to achieve a negative culture with survival analysis.ResultsNormal saline-EDTA showed no additional toxicity to fibroblasts and endothelial cells when compared with normal saline (normal saline [97%] versus EDTA [98%] on fibroblasts, p = 0.654; normal saline [97%] versus EDTA [98%] on endothelial cells, p = 0.711). When bone was exposed in the contaminated models, EDTA irrigation resulted in fewer positive bacterial cultures with S aureus (EDTA: 23%, normal saline: 67%, soap: 40%, p = 0.003) and with E coli (EDTA: 27%, normal saline: 57%, soap: 30%, p = 0.032); however, infection risk was only lower with EDTA irrigation (S aureus with EDTA: 10%, normal saline: 33%, soap: 37%, p = 0.039; E coli with EDTA: 3%, normal saline: 27%, soap: 23%, p = 0.038). In the infected wound model, EDTA irrigation resulted in earlier culture-negative wounds (fewer surgical sessions) compared with normal saline and soap solutions (nonbone-exposed wounds infected by S aureus: p = 0.003, infected by E coli: p = 0.001; bone-exposed wounds infected by S aureus: p = 0.012, infected by E coli: p = 0.022).ConclusionsAfter in vitro assessment of toxicity and in vivo evaluation of efficacy, we concluded that normal saline-EDTA is superior to normal saline and soap solution in our laboratory models.Clinical RelevanceThe use of normal-saline EDTA as an irrigation solution may reduce the infection rate of wounds. Future studies in large animals and humans might prove our observation in rat models that normal saline-EDTA has an advantage over normal saline as an irrigation solution.