Nano-Sized Extracellular Matrix Particles Lead to Therapeutic Improvement for Cutaneous Wound and Hindlimb Ischemia.

Cell-derived matrix (CDM) has proven its therapeutic potential and been utilized as a promising resource in tissue regeneration. In this study, we prepared a human fibroblast-derived matrix (FDM) by decellularization of in vitro cultured cells and transformed the FDM into a nano-sized suspended formulation (sFDM) using ultrasonication. The sFDM was then homogeneously mixed with Pluronic F127 and hyaluronic acid (HA), to effectively administer sFDM into target sites. Both sFDM and sFDM containing hydrogel (PH/sFDM) were characterized via immunofluorescence, sol-gel transition, rheological analysis, and biochemical factors array. We found that PH/sFDM hydrogel has biocompatible, mechanically stable, injectable properties and can be easily administered into the external and internal target regions. sFDM itself holds diverse bioactive molecules. Interestingly, sFDM-containing serum-free media helped maintain the metabolic activity of endothelial cells significantly better than those in serum-free condition. PH/sFDM also promoted vascular endothelial growth factor (VEGF) secretion from monocytes in vitro. Moreover, when we evaluated therapeutic effects of PH/sFDM via the murine full-thickness skin wound model, regenerative potential of PH/sFDM was supported by epidermal thickness, significantly more neovessel formation, and enhanced mature collagen deposition. The hindlimb ischemia model also found some therapeutic improvements, as assessed by accelerated blood reperfusion and substantially diminished necrosis and fibrosis in the gastrocnemius and tibialis muscles. Together, based on sFDM holding a strong therapeutic potential, our engineered hydrogel (PH/sFDM) should be a promising candidate in tissue engineering and regenerative medicine.

Ischemic postconditioning ameliorates acute kidney injury induced by limb ischemia/reperfusion via transforming TLR4 and NF-κB signaling in rats.

BACKGROUND: The present study investigated the influence of ischemic postconditioning (I-postC) on the adjustment of renal injury after limb ischemia-reperfusion (I/R) injury, to elucidate the mechanisms of the Toll-like receptor 4 (TLR 4)/NF-κB signaling pathway using histopathological and immunohistochemical methods. METHODS: Male Sprague-Dawley rats were randomly assigned to five groups (numbered from 1 to 5): the sham group (Group 1, only the anesthesia procedure was conducted without limb I/R), the I/R group (Group 2, 4 h of reperfusion was conducted following 4 h limb ischemia under anesthesia), the I/R + I-postC group (Group 3, 4 h of ischemia and 4 h of reperfusion was conducted; before perfusion, 5 min of limb ischemia and 5 min of reperfusion were performed in the rats and repeated 3 times), the I/R + TAK group (Group 4, rats were injected with TLR4 antagonist TAK through the caudal vein before limb ischemia and reperfusion under anesthesia), the TAK group (Group 5, rats were injected with TAK, and the anesthesia procedure was conducted without limb I/R). Histological changes in the kidney in different groups were observed, and the extent of tubular injury was assessed. Changes in biochemical indexes and the expression of inflammatory factors, TLR4, and NF-κB were also evaluated. RESULTS: Compared with rats in the I/R group, the secretion of inflammatory factors and the expression levels of TLR4 and NF-κB were decreased in rats in the I/R + I-postC group. Histological analysis revealed renal injury, including inflammatory cell infiltration, dilatation of the tubuli lumen, congestion in glomerular capillaries, degeneration of tubuli epithelial cells, and necrosis was ameliorated by I-postC. Immunohistochemical studies showed that I/R-induced elevation in TLR4 and NF-κB expression was reduced by I-postC treatment. Moreover, the expression levels of TLR4, NF-κB, and inflammatory factors in rats in the I/R + TAK group were also decreased, and the renal pathological lesion was alleviated, which was similar to that in rats in the I/R + I-postC group. CONCLUSIONS: The present findings suggest that I-postC can reduce tissue injury and kidney inflammation induced by limb I/R injury, possibly via inhibition of the TLR4 and NF-κB pathways.

Extremity trauma exacerbates acute kidney injury following prolonged hemorrhagic hypotension.

BACKGROUND: The incidence of and mortality due to acute kidney injury is high in patients with traumatic shock. However, it is unclear how hemorrhage and trauma synergistically affect renal function, especially when timely volume resuscitation is not available. METHOD: We hypothesized that trauma impairs renal tolerance to prolonged hemorrhagic hypotension. Sprague-Dawley rats were randomized into six groups: control, extremity trauma (ET), hemorrhage at 70 mm Hg (70-H), hemorrhage at 55 mm Hg (55-H), ET + 70 mm Hg (70-ETH), and ET + 55 mm Hg (55-ETH). Animals were anesthetized, and ET was induced via soft tissue injury and closed fibula fracture. Hemorrhage was performed via catheters 5 minutes after ET with target mean arterial pressure (MAP) clamped at 70 mm Hg or 55 mm Hg for up to 3 hours. Blood and urine samples were collected to analyze plasma creatinine (Cr), Cr clearance (CCr), renal oxygen delivery (DO2), urinary albumin, and kidney injury molecule-1 (KIM-1). RESULTS: Extremity trauma alone did not alter renal hemodynamics, DO2, or function. In 70-H, CCr was increased following hemorrhage, while Cr, renal vascular resistance (RVR), KIM-1, and albumin levels remained unchanged. Compared with 70-H, ET + 70 mm Hg exhibited increases in Cr and RVR with decreases in CCr and DO2. In addition, ET decreased the blood volume loss required to maintain MAP = 70 mm Hg by approximately 50%. Hemorrhage at 55 mm Hg and ET + 55 mm Hg exhibited a marked and similar decrease in CCr and increases in RVR, Cr, KIM-1, and albumin. However, ET greatly decreased the blood volume loss required to maintain MAP at 55 mm Hg and led to 50% mortality. CONCLUSION: These results suggest that ET impairs renal and systemic tolerance to prolonged hemorrhagic hypotension. Thus, traumatic injury should be considered as a critical component of experimental studies investigating outcomes and treatment following hemorrhagic shock. LEVEL OF EVIDENCE: This is an original article on basic science and does not require a level of evidence.

Hydrogen-rich saline reduces tissue injury and improves skin flap survival on a rat hindlimb degloving injury model.

BACKGROUND: Degloving injuries represent a challenge in plastic surgery. The aim of this study is to acknowledge the protective effects of hydrogen-rich saline (HRS) solution on a rat hindlimb degloved skin flap. METHODS: Twenty-one Sprague-Dawley rats were divided into three groups (control, saline and HRS). Degloving injury model was established, and flaps were sutured back following 5 min of ischemia. The control group did not receive any treatment. The saline group received intraperitoneal physiological saline (10 ml/kg) and the HRS group received intraperitoneal HRS solution (10 ml/kg) postoperatively and daily for 5 days after the operation. Skin samples were obtained for histological, immunohistochemical and biochemical evaluations. RESULTS: Inflammation was lower in the HRS compared with saline (p = 0.02) and control (p = 0.004) groups. Edema was lower in the HRS compared with saline (p = 0.02) and control (p = 0.001) groups. Malondialdehyde (MDA) level was lower in the HRS than the control group (p = 0.01). Total antioxidant level was higher in the HRS compared with saline (p = 0.009) and control (p = 0.03) groups. Total oxidant level was lower in the HRS than the control group (p = 0.02). Oxidative stress index was lower in the HRS compared with saline (p = 0.001) and control (p = 0.0001) groups`. Vascular proliferation was higher in the HRS compared with the control group (p = 0.01). CONCLUSION: Repeated HRS injections after trauma increased the viability of skin flap in rat degloving injury model by decreasing local tissue injury, due to its antioxidant, anti-inflammatory and angiogenic effects.

Ultrasonographic diagnosis of closed pedal bone fractures in bovine claws: An ex-vivo study in slaughterhouse specimens.

Pedal bone fractures are one of the most common fracture locations in adult cattle and can be diagnosed by radiographs in two planes. Most bovine practitioners do not have access to such X-ray machines, but many use ultrasound units on a daily basis, primarily for reproductive medicine. For this reason, in this double-masked, randomized controlled study, we aimed to investigate the suitability of ultrasonographic examination using a 5 MHz linear transducer for diagnosing closed fractures of the pedal bone in cattle. A total of 54 hindlimb claws from slaughtered cattle were prepared and approximately 50% of the claws were artificially fractured. All claws were ultrasonographically examined twice by two examiners to determine the presence or absence of fractures and their locations. Ultrasound results were confirmed using radiographs of the claws as the reference standard method. All fracture locations as determined by ultrasonography were situated within ±2 mm of the radiographically-determined fracture zone. Ultrasound examination yielded a calculated sensitivity of 93%, a specificity of 91% and an inter-rater reliability of 0.77. The intra-rater reliability for the examiners were 0.96 and 0.88. Examiner experience with ultrasound examination and using ultrasound images for diagnosis could have influenced diagnostic accuracy. We conclude that artificially-created pedal bone fractures in ex-vivo bovine claws can be diagnosed using ultrasonography; similar results are expected in live animals. These results should encourage veterinarians to use ultrasonography for diagnosing pedal bone fractures in cattle.

Clinical and morphological justification of the treatment of purulonecrotic lesion in deep structures of limb distal segment in pig: case report / [Justificativa clínica e morfológica do tratamento de lesão purulonecrótica em estruturas profundas do segmento distal de membros em porco: relato de caso]

Surgical pathology of the musculoskeletal system, and in particular the diseases of the limb distal segment in pigs are quite common. Their significant spread leads to economic losses due to culling, compulsory slaughter, short-received pig production and pig crop. The purpose of this work was to reveal clinical and morphological features of limb distal segment diseases in pigs and to study the possibility of preserving their health and productivity. The conservative treatment of purulonecrotic lesions in the deep structures of the limb distal segment in pigs is not promising. It is shown that the best way to treat a given pathology is amputation of a sick limb. The technique of carrying out exarticulation of talus shin consists in separation of soft tissues, capsule and ligaments, ligation of vessels, formation of stump. The postoperative recovery period of the animal body is 25 days.(AU)

A patologia cirúrgica do sistema musculoesquelético e, em particular, as doenças do segmento distal dos membros em suínos são bastante comuns. A sua propagação significativa leva a perdas econômicas devido ao abate seletivo, abate obrigatório, produção de suínos pouco recebida e colheita de suínos. O objetivo deste trabalho foi revelar características clínicas e morfológicas das doenças do segmento distal de membros em suínos e estudar a possibilidade de preservar sua saúde e produtividade. O tratamento conservador das lesões purulonecróticas nas estruturas profundas do segmento distal do membro em porcos não é promissor. Fica estabelecido que a melhor forma de tratar uma determinada patologia é a amputação de um membro doente. A técnica de realizar a exarticulação da canela do talos consiste na separação dos tecidos moles, cápsula e ligamentos, ligadura dos vasos, formação do coto. O período de recuperação pós-operatória do corpo do animal é de 25 dias.(AU)

The Use of Bone Marrow-Derived Stem Cells in the Treatment of a Comminuted Tibiotarsal Fracture in a Juvenile Red-legged Seriema (Cariama cristata).

An adult red-legged seriema (Cariama cristata) presented with a comminuted fracture of the tibiotarsus and fibula. Surgery was performed, and a type II external fixator, with 2 distal and 2 proximal pins, was used to stabilize the fracture. After a 10-day stabilization period, the bird developed a second fracture on the same bone, proximal to the first fracture site. Another surgery was performed on the seriema similar to the first one. However, in this second surgical procedure a single pin, instead of 2 perpendicular pins, was placed proximally to the fracture site. After the second surgical procedure, bone marrow stem cells (BMSCs) from the seriema's left ulna were collected. Twenty-seven days after the second surgery, the BMSCs were transplanted, into the fracture sites. Twenty-four days after the stem cells were injected into the fractures (51 days after the second surgical procedure), radiographic images revealed healing bone calluses at the fracture sites. The fracture healing was relatively long for this case (a total of 75 days). The addition of bone marrow stem cell therapy to the use of external fixation may have contributed to the healing observed radiographically 24 days after administration; therefore, bone marrow stem cell therapy, in addition to traditional surgical fracture reduction and stabilization, may be a promising therapeutic approach for avian cases with similar injuries and bone anatomy. However, as this is a single case, this therapeutic modality deserves further application and study. Moreover, we suggest modifications in the bone marrow stem cell collection and therapy, which may be useful for future studies and application involving birds.

A rat model of orthopedic injury-induced hypercoagulability and fibrinolytic shutdown.

BACKGROUND: Postinjury hypercoagulability occurs in >25% of injured patients, increasing risk of thromboembolic complications despite chemoprophylaxis. However, few clinically relevant animal models of posttraumatic hypercoagulability exist. We aimed to evaluate a rodent model of bilateral hindlimb injury as a preclinical model of postinjury hypercoagulability. METHODS: Forty Wistar rats were anesthetized with isoflurane: 20 underwent bilateral hindlimb fibula fracture, soft tissue and muscular crush injury, and bone homogenate injection intended to mimic the physiological severity of bilateral femur fracture. Twenty sham rats underwent anesthesia only. Terminal citrated blood samples were drawn at 0, 6, 12, and 24 hours (n = 5 per timed group) for analysis by native thromboelastography in the presence and absence of taurocholic acid to augment fibrinolysis. Plasminogen activator inhibitor 1 and α-2 antiplasmin levels in plasma were assessed via enzyme-linked immunosorbent assay. RESULTS: Injured rats became hypercoagulable relative to baseline by 6 hours based on thromboelastography maximal amplitude (MA) and G (p < 0.005); sham rats became hypercoagulable to a lesser degree by 24 hours (p < 0.005). Compared with sham animals, injured rats were hypercoagulable by MA and G within 6 hours of injury, remained hypercoagulable by MA and G through at least 24 hours (all p < 0.01), and showed impaired fibrinolysis by taurocholic acid LY30 at 12 hours (p = 0.019) and native LY30 at 24 hours (p = 0.045). In terms of antifibrinolytic mediators, α-2 antiplasmin was elevated in trauma animals at 24 hours (p = 0.009), and plasminogen activator inhibitor 1 was elevated in trauma animals at 6 hours (p = 0.004) and 12 hours (p < 0.001) when compared with sham. CONCLUSIONS: Orthopedic injury in rodents induced platelet and overall hypercoagulability within 6 hours and fibrinolytic impairment by 12 to 24 hours, mimicking postinjury hypercoagulability in injured patients. This rodent model of orthopedic injury may serve as a preclinical testing ground for potential therapies to mitigate hypercoagulability, maintain normal fibrinolysis, and prevent thromboembolic complications.

[Limb amputation in 2 small ruminants]. / Gliedmaßenamputation bei 2 kleinen Wiederkäuern.

A 2-month-old goat and a 5-year-old sheep were referred to the clinic due to severe lameness. The goat had a 5-week-old infected comminuted, left metacarpal fracture with extended osteomyelitis of the distal metacarpal bones. The sheep presented with an open oblique fracture of the left tibia and a concurrent, perforating wound of the fetlock joint in the same hindlimb. In both cases, limb amputation was performed at the level of the shoulder joint and the proximal femur, respectively, under general anesthesia combined with blocks of the large proximal limb nerves. The surgical procedure was successful in both cases and the animals showed good postoperative mobility and quality of life. Limb amputations in small ruminants are rarely described, but may offer a valuable salvage procedure instead of euthanasia.

Can Intra-articular 1α, 25-Dihydroxyvitamin D3 Administration Be Therapeutical in Joint Cartilage Damage?

INTRODUCTION Vitamin D-deficiency is known to cause nerve conduction impairments, cancer and chronic diseases, as well as the pathogenesis of osteoarthritis. Our goal with this study is to evaluate the cartilage healing by applying intraarticular 1α, 25 (OH) 2D3 at different doses in rats with normal vitamin D levels and metabolism, which we made focal chondral damage model in the knee joint. MATERIAL AND METHODS 35 male Sprague-Dawley rats aged 20-24 weeks were used in our study. Both knees of rats were cartilage defected surgically on day 0. Joint injections performed at 06:00 am on 0th and 2nd days and after second injection others performed on days 9-16 and 23 following a weekly period. RESULTS In the fourth week, hematoxylin eosin staining measurements showed statistically significant difference according to the groups (p < 0.01) Metalloproteinase-13 (MMP-13) in histological staining for evaluating cartilage healing and healing levels showed statistically significant differences between the groups at first week and fourth week (p < 0.05). DISCUSSION Vitamin D, which affects many tissues through its receptors, is believed to be chondroprotective and neuroprotective by decreasing the expression of MMP in cartilage fibroblast, macrophage, lymphocyte through its intracellular receptors. To the best of our knowledge, this is the first study known to be intraarticular use of 1α, 25-dihydroxyvitamin D3. Our study has been found to be safe and successful in terms of weight, systemic PTH and 1α, 25-dihydroxyvitamin D3 levels in rats during treatment as well as better healing of cartilage damage. Key words: vitamin D3 receptor, articular cartilage, orthopedics, nerve conduction.

Neonatal Injury Evokes Persistent Deficits in Dynorphin Inhibitory Circuits within the Adult Mouse Superficial Dorsal Horn.

Neonatal tissue damage induces long-term deficits in inhibitory synaptic transmission within the spinal superficial dorsal horn (SDH) that include a reduction in primary afferent-evoked, feedforward inhibition onto adult projection neurons. However, the subpopulations of mature GABAergic interneurons which are compromised by early-life injury have yet to be identified. The present research illuminates the persistent effects of neonatal surgical injury on the function of inhibitory SDH interneurons derived from the prodynorphin (DYN) lineage, a population that synapses directly onto lamina I spinoparabrachial neurons and is known to suppress mechanical pain and itch in adults. The results demonstrate that hindpaw incision at postnatal day 3 (P3) significantly decreased the strength of primary afferent-evoked glutamatergic drive onto DYN neurons within the adult mouse SDH while increasing the appearance of afferent-evoked inhibition onto the same population. Neonatal injury also dampened the intrinsic membrane excitability of mature DYN neurons, and reduced their action potential discharge in response to sensory input, compared with naive littermate controls. Furthermore, P3 incision decreased the efficacy of inhibitory DYN synapses onto adult spinoparabrachial neurons, which reflected a prolonged reduction in the probability of GABA release. Collectively, the data suggest that early-life tissue damage may persistently constrain the ability of spinal DYN interneurons to limit ascending nociceptive transmission to the adult brain. This is predicted to contribute to the loss of feedforward inhibition onto mature projection neurons, and the "priming" of nociceptive circuits in the developing spinal cord, following injuries during the neonatal period.SIGNIFICANCE STATEMENT Neonatal injury has lasting effects on pain processing in the adult CNS, including a reduction in feedforward inhibition onto ascending projection neurons in the spinal dorsal horn. While it is clear that spinal GABAergic interneurons are comprised of multiple subpopulations that play distinct roles in somatosensation, the identity of those interneurons which are compromised by tissue damage during early life remains unknown. Here we document persistent deficits in spinal inhibitory circuits involving dynorphin-lineage interneurons previously implicated in gating mechanical pain and itch. Notably, neonatal injury reduced the strength of dynorphin-lineage inhibitory synapses onto mature lamina I spinoparabrachial neurons, a major output of the spinal nociceptive network, which could contribute to the priming of pain pathways by early tissue damage.

Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration.

Mesenchymal stem cells (MSCs) encapsulation by three-dimensionally (3D) printed matrices were believed to provide a biomimetic microenvironment to drive differentiation into tissue-specific progeny, which made them a great therapeutic potential for regenerative medicine. Despite this potential, the underlying mechanisms of controlling cell fate in 3D microenvironments remained relatively unexplored. Here, we bioprinted a sweat gland (SG)-like matrix to direct the conversion of MSC into functional SGs and facilitated SGs recovery in mice. By extracellular matrix differential protein expression analysis, we identified that CTHRC1 was a critical biochemical regulator for SG specification. Our findings showed that Hmox1 could respond to the 3D structure activation and also be involved in MSC differentiation. Using inhibition and activation assay, CTHRC1 and Hmox1 synergistically boosted SG gene expression profile. Together, these findings indicated that biochemical and structural cues served as two critical impacts of 3D-printed matrix on MSC fate decision into the glandular lineage and functional SG recovery.

Changes in BMP-2 expression and mechanical properties during treatment of rats with osteoporotic hindlimb fracture with strontium ranelate.

OBJECTIVES: This study aims to investigate the changes in bone morphogenetic protein-2 (BMP-2) expression and mechanical properties in the healing process of rats with osteoporotic hindlimb fracture. METHODS: 120 rat models of osteoporotic hindlimb fracture were established and randomly divided into experimental group and control group. Quantitative real-time polymerase chain reaction (PCR) used to detect the BMP-2 expression in the rat's callus tissue on the fractured side. The mechanical properties of rat's hindlimb skeleton were examined using a universal material mechanics testing machine. RESULTS: The BMP-2 expression in the experimental group was higher than that in the control group (p<0.05). The linear correlation analysis showed that the BMP-2 was positively correlated with healing time (r=0.87, p<0.05). The mechanical properties were markedly improved at T2, T3 and T4, which peaked at T4 (p<0.05). However, the mechanical properties in the rats in the experimental group were notably superior to those in the control group at T2, T3, and T4 (p<0.05). CONCLUSIONS: The treatment with strontium ranelate can effectively improve the BMP-2 and bone mechanical properties of the rats with osteoporotic hindlimb fracture in the healing stage and accelerate the healing progress, which could be proved to be an efficacious means in treating osteoporotic fracture.

Methods for the reliable induction of heterotopic ossification in the conditional Alk2Q207D mouse.

OBJECTIVES: Conditional Alk2Q207D-floxed (caALK2fl) mice have previously been used as a model of heterotopic ossification (HO). However, HO formation in this model can be highly variable, and it is unclear which methods reliably induce HO. Hence, these studies report validated methods for reproducibly inducing HO in caALK2fl mice. METHODS: Varying doses of Adex-cre and cardiotoxin (CTX) were injected into the calf muscles of 9, 14, or 28-day-old caALK2fl/- or caALK2fl/fl mice. HO was measured by planar radiography or microCT at 14-28 days post-injury. RESULTS: In 9-day-old caALK2fl/- or caALK2fl/fl mice, single injections of 109 PFU Adex-cre and 0.3 µg of CTX were sufficient to induce extensive HO within 14 days post-injury. In 28-day-old mice, the doses were increased to 5 x 109 PFU Adex-cre and 3.0 µg of CTX to achieve similar consistency, but at a slower rate versus younger mice. Using a crush injury, instead of CTX, also provided consistent induction of HO. Finally, the Type 1 BMPR inhibitor, DMH1, significantly reduced HO formation in 28-day-old caALK2fl/fl mice. CONCLUSIONS: These data illustrate multiple methods for reliable induction of localized HO in the caALK2flmouse that can serve as a starting point for new laboratories utilizing this model.

Assessment of the Efficacy and Safety of a Novel, Low-Cost, Junctional Tourniquet in a Porcine Model of Hemorrhagic Shock.

INTRODUCTION: Commercially available junctional tourniquets (JTQs) have several drawbacks. We developed a low-cost, compact, easy to apply JTQ. The aim of this study was to assess the tourniquets' safety and efficacy in a swine model of controlled hemorrhage. MATERIALS AND METHODS: Five pigs were subjected to controlled bleeding of 35% of their blood volume. Subsequently, the JTQ was applied to the inguinal area for 180 minutes. Afterwards, the tourniquet was removed for additional 60 minutes of follow up. During the study, blood flow to both hind limbs and blood samples for tissue damage markers were repeatedly assessed. Following sacrifice, injury to both inguinal areas was evaluated microscopically and macroscopically. RESULTS: Angiography demonstrated complete occlusion of femoral artery flow, which was restored following removal of the tourniquet. No gross signs of tissue damage were noticed. Histological analysis revealed mild necrosis and infiltration of inflammatory cells. Blood tests showed a mild increase in potassium and lactic acid levels throughout the protocol. CONCLUSIONS: The tourniquet achieved effective arterial occlusion with minimal tissue damage, similar to reports of other JTQs. Subjected to further human trials, the tourniquet might be a suitable candidate for widespread frontline deployment because of its versatility, compactness, and affordable design.

Minimally Invasive Osteosynthesis Techniques of the Femur.

A thorough working knowledge of the anatomic landmarks of the femur facilitates anatomic alignment during minimally invasive osteosynthesis (MIO). A variety of fixation techniques, including plate, plate-rod, and interlocking nail, are well suited for stabilization of femoral shaft fractures with MIO techniques. Axis and torsional alignment can be assessed with various intraoperative techniques to ensure that anatomic alignment is obtained.

Knockout of beta-2 microglobulin reduces stem cell-induced immune rejection and enhances ischaemic hindlimb repair via exosome/miR-24/Bim pathway.

Generating universal human umbilical mesenchymal stem cells (UMSCs) without immune rejection is desirable for clinical application. Here we developed an innovative strategy using CRISPR/Cas9 to generate B2M- UMSCs in which human leucocyte antigen (HLA) light chain ß2-microglobulin (B2M) was deleted. The therapeutic potential of B2M- UMSCs was examined in a mouse ischaemic hindlimb model. We show that B2M- UMSCs facilitated perfusion recovery and enhanced running capability, without inducing immune rejection. The beneficial effect was mediated by exosomes. Mechanistically, microRNA (miR) sequencing identified miR-24 as a major component of the exosomes originating from B2M- UMSCs. We identified Bim as a potential target of miR-24 through bioinformatics analysis, which was further confirmed by loss-of-function and gain-of-function approaches. Taken together, our data revealed that knockout of B2M is a convenient and efficient strategy to prevent UMSCs-induced immune rejection, and it provides a universal clinical-scale cell source for tissue repair and regeneration without the need for HLA matching in the future.

Minimally Invasive Fracture Repair of the Tibia and Fibula.

Fractures of the tibia and fibula are common in dogs and cats and occur most commonly as a result of substantial trauma. Tibial fractures are particularly amenable to treatment using minimally invasive fracture repair (MIFR) techniques that preserve blood supply to comminuted fracture fragments, accelerating bone callus production and speeding fracture healing. Treatment of tibial fractures using MIFR techniques has been found to reduce surgical time, reduce the time for fracture healing, and to decrease patient morbidity, while at the same time reducing complications compared with traditional open reduction and internal fixation.

Prostaglandin D2 signaling is not involved in the recovery of rat hind limb tendons from injury.

Injured tendons heal through the formation of a fibrovascular scar that has inferior mechanical properties compared to native tendon tissue. Reducing inflammation that occurs as a result of the injury could limit scar formation and improve functional recovery of tendons. Prostaglandin D2 (PGD2 ) plays an important role in promoting inflammation in some injury responses and chronic disease processes, and the inhibition of PGD2 has improved healing and reduced disease burden in animal models and early clinical trials. Based on these findings, we sought to determine the role of PGD2 signaling in the healing of injured tendon tissue. We tested the hypothesis that a potent and specific inhibitor of hematopoietic PGD synthase (HPGDS), GSK2894631A, would improve the recovery of tendons of adult male rats following an acute tenotomy and repair. To test this hypothesis, we performed a full-thickness plantaris tendon tenotomy followed by immediate repair and treated rats twice daily with either 0, 2, or 6 mg/kg of GSK2894631A. Tendons were collected either 7 or 21 days after surgical repair, and mechanical properties of tendons were assessed along with RNA sequencing and histology. While there were some differences in gene expression across groups, the targeted inhibition of HPGDS did not impact the functional repair of tendons after injury, as HPGDS expression was surprisingly low in injured tendons. These results indicate that PGD2 signaling does not appear to be important in modulating the repair of injured tendon tissue.

Duration of extremity tourniquet application profoundly impacts soft-tissue antibiotic exposure in a rat model of ischemia-reperfusion injury.

INTRODUCTION: Extremity tourniquet (TNK) application is an effective means of achieving compressible hemorrhage control in the emergency prehospital and clinical trauma setting. Modern United States military medical doctrine recommends TNK use to prevent lethal hemorrhage from extremity injury, followed by systemic prophylactic antibiotics to prevent wound infection. Because tissue pharmacokinetics of prophylactic antimicrobials during and after TNK-induced limb ischemia are largely unknown, this study was conducted to empirically determine the relationship between TNK application time and soft tissue antibiotic exposure in order to guide medical personnel in the management of extremity trauma. MATERIALS AND METHODS: Hind limbs of anesthetized male Sprague Dawley rats were exsanguinated, and ischemia maintained by a pneumatic cuff placed at the level of the mid femur on one limb; the non-ischemic contralateral limb served as comparison tissue. Systemic prophylactic antibiotics (cefazolin, moxifloxacin, or ertapenem) were administered intravenously before or after TNK release following 2 or 4 h of ischemia with subsequent re-dosing every 12 h for 3 days. Free antibiotic in the interstitial fluid (ISF) of the tibialis anterior muscle of both hind limbs was recovered via microdialysis during ischemia and over three periods during reperfusion: immediately following TNK release, at 24 h post TNK release, and at 72 h post TNK release. Plasma and ISF free drug concentrations were determined by high-performance liquid chromatography. RESULTS: Tourniquet application prevented delivery of prophylactic antibiotics into distal soft tissue for the duration of ischemia, and caused a profound reduction in skeletal muscle drug exposure for up to 72 h following TNK release. A progressive decline in tissue antibiotic exposure during reperfusion was observed as TNK times increased from 2 h to 4 h. The timing and severity of reduced drug distribution in post-ischemic skeletal muscle varied substantially among the three antibiotic classes evaluated. CONCLUSIONS: Prolonged tourniquet application can significantly reduce distribution of prophylactic antibiotics into soft tissue during and after ischemia, potentially impairing prophylaxis of extremity wound infection. Our findings support the examination of alternative approaches to wound infection prophylaxis under conditions of delayed casualty evacuation when occlusive hemorrhage control measures are utilized.