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Laboratory study using a rat T9 contusion model of spinal cord injury.This study aims to examine whether a combinatory treatment of Pioglitazone (PGZ) and granulocyte colony-stimulating factor (GCSF) can support neural stem/progenitor cells (NSPCs) directly and provide a sustainable microenvironment through immunomodulatory mechanisms.Neuroinflammation plays a crucial role in the progression of spinal cord injury (SCI) and hinders NSPC-mediated repair and regeneration. Broad acting drugs that mitigate inflammation and support NSPC proliferation have not been tested together in SCI research models.Isolated NSPCs were treated with vehicle control, PGZ, GCSF, or both PGZ and GSCF for 24 hours and stained with proliferation marker Ki67. Adult female Sprague-Dawley rats sustained moderate-to-severe contusion-based SCI at T9 and were administered either vehicle control, PGZ, GCSF, or both PGZ and GCSF treatments.Immunocytochemistry revealed that cultured NSPCs treated with both drugs produced higher numbers of actively proliferating cells and total cell numbers. ELISA on spinal cord tissue lysates at 1, 3, and 7 days post-injury (DPI) demonstrated that animals treated with PGZ, GCSF, or combination therapy showed significantly higher doublecortin levels at 7 DPI compared to control animals (P 0.05). Immunohistochemistry of injured tissue at 3, 7, and 14 DPI revealed no difference of ependymal NSPC proliferation between groups, but showed a significant decrease in lesion size with combination therapy compared to controls. Functional recovery was assessed by the Basso, Beattie, Bresnahan locomotor rating scale. Animals treated with both drugs had significantly higher levels of function at 1 (P 0.001), 3 (P 0.001), 7 (P 0.05), and 14 (P 0.05) DPI compared to controls.These results indicate that PGZ and GCSF treatment synergistically enhance NSPCs numbers and improve functional recovery after SCI. Our findings support an immunomodulatory strategy to recruit native NSPCs as a potential acute care intervention for SCI.Level of Evidence: N/A.

Due to the declining number of scientifically trained physicians and increasing demand for high-quality literature, our institution pioneered a seven-year Physician Scientist Training Program (PSTP) to provide research-oriented residents the knowledge and skills for a successful academic career. The present study sought to identify orthopaedic surgeons with MD/PhD degrees, residency programs with dedicated research tracks, and to assess the effectiveness of the novel seven-year program in training prospective academic orthopaedic surgeons. Surgeons with MD/PhD degrees account for 2.3% of all 3,408 orthopaedic faculty positions in U.S. residency programs. During the last 23 years, our PSTP residents produced 752 peer-reviewed publications and received $349,354 from 23 resident-authored extramural grants. Eleven of our seven-year alumni practice orthopaedic surgery in an academic setting. The seven-year PSTP successfully develops clinically trained surgeon scientists with refined skills in basic science and clinical experimental design, grant proposals, scientific presentations, and manuscript preparation. (Journal of Surgical Orthopaedic Advances 31(3):144-149, 2022).

Chemotherapy regimens that include 5-fluorouracil (5-FU) are central to colorectal cancer treatment; however, risk/benefit concerns limit 5-FU’s use, necessitating development of improved fluoropyrimidine (FP) drugs. In our study, we evaluated a second-generation nanoscale FP polymer, CF10, for improved antitumor activity. CF10 was more potent than the prototype FP polymer F10 and much more potent than 5-FU in multiple colorectal cancer cell lines including HCT-116, LS174T, SW480, and T84D. CF10 displayed improved stability to exonuclease degradation relative to F10 and reduced susceptibility to thymidine antagonism due to extension of the polymer with arabinosyl cytidine. In colorectal cancer cells, CF10 strongly inhibited thymidylate synthase (TS), induced Top1 cleavage complex formation and caused replication stress, while similar concentrations of 5-FU were ineffective. CF10 was well tolerated in vivo and invoked a reduced inflammatory response relative to 5-FU. Blood chemistry parameters in CF10-treated mice were within normal limits. In vivo, CF10 displayed antitumor activity in several colorectal cancer flank tumor models including HCT-116, HT-29, and CT-26. CF10’s antitumor activity was associated with increased plasma levels of FP deoxynucleotide metabolites relative to 5-FU. CF10 significantly reduced tumor growth and improved survival (84.5 days vs. 32 days; P < 0.0001) relative to 5-FU in an orthotopic HCT-116-luc colorectal cancer model that spontaneously metastasized to liver. Improved survival in the orthotopic model correlated with localization of a fluorescent CF10 conjugate to tumor. Together, our preclinical data support an early-phase clinical trial of CF10 for treatment of colorectal cancer.

Introduction Buprenorphine is a partial μ-opioid receptor agonist that, unlike full μ-opioid receptor agonists, has been shown to have a ceiling effect on respiratory depression. Buprenorphine buccal film (BBF) is approved by the US Food and Drug Administration for use in patients with chronic pain severe enough to require daily, around-the-clock, long-term opioid treatment and for whom alternative treatment options are inadequate. This study was conducted to compare the effects of BBF and immediate-release oral oxycodone hydrochloride administration on respiratory drive, as measured by the ventilatory response to hypercapnia (VRH) after drug administration. Methods Subjects (N = 19) were men and women, ages 27–41 years, self-identifying as recreational opioid users who were not physically dependent on opioids as determined via a Naloxone Challenge Test. Respiratory drive was evaluated by measuring VRH through the assessment of the maximum decrease in minute ventilation (Emax) after administration of each treatment. The treatments utilized in this study included 300, 600, and 900 μg BBF; 30 and 60 mg orally administered oxycodone; and placebo (each separated by a 7-day washout period). Effects on respiratory drive were assessed using a double-blind, double-dummy, six-treatment, six-period, placebo-controlled, randomized crossover design. Statistical analyses were performed using a linear mixed-effects model. Results The least squares mean differences in minute volume Emax (L/min, versus placebo) were as follows: 300 μg BBF (+ 1.24, P = 0.529), 600 μg BBF (+ 0.23, P = 0.908), 900 μg BBF (+ 0.93, P = 0.637), 30 mg oxycodone (− 0.79, P = 0.687), and 60 mg oxycodone (− 5.23, P = 0.010). Conclusions BBF did not significantly reduce respiratory drive at any dose compared with placebo, including at the maximum available prescription dose of 900 μg. Administration of oxycodone resulted in a significant dose-dependent decrease in respiratory drive. These data suggest that BBF may be a safer treatment option than full μ-opioid receptor agonists for patients with chronic pain. Trial Registration ClinicalTrials.gov identifier, NCT03996694. Electronic supplementary material The online version of this article (10.1007/s12325-020-01481-0) contains supplementary material, which is available to authorized users.

Decellularized tendon xenografts offer a promising alternative for reconstruction by using ubiquitously available material. This study compares static and centrifugal seeding of avian tendon scaffolds with NIH 3T3 fibroblasts. Incorporation of viable cells was achievable with both techniques, represented by DNA content. Proliferation rate and viability assay showed neither damage by centrifugal force nor superiority of the technique. Cell proliferation after 10 days of culture demonstrated that the scaffold did not hinder 3-D culturing. Confocal laser microscopy revealed structural details as formation of focal adhesions, to provide deeper insight into the process of cell attachment and growth in xenografts.

Study design Laboratory study using a rat T9 contusion model of spinal cord injury (SCI). Objective The purpose of this study was to evaluate which method of delivery of soluble keratin biomaterials would best support functional restoration through the macrophage polarization paradigm. Summary of background data SCI is a devastating neurologic event with complex pathophysiological mechanisms that currently has no cure. After injury, macrophages and resident microglia are key regulators of inflammation and tissue repair exhibiting phenotypic and functional plasticity. Keratin biomaterials have been demonstrated to influence macrophage polarization and promote the M2 anti-inflammatory phenotype that attenuates inflammatory responses. Methods Anesthetized female Lewis rats were subjected to moderate T9 contusion SCI and randomly divided into: no therapy (control group), an intrathecally injected keratin group, and a keratin-soaked sponge group (n = 11 in all groups). Functional recovery assessments were obtained at 3- and 6-weeks post-injury (WPI) using gait analysis performed with the DigiGait Imaging System treadmill and at 1, 3, 7, 14, 21, 28, 35, and 42 days post-injury by the Basso, Beattie, Bresnahan (BBB) locomotor rating scale. Histology and immunohistochemistry of serial spinal cord sections were performed to assess injury severity and treatment efficacy. Results Compared to control rats, applying keratin materials after injury improved functional recovery in certain gait parameters and overall trended toward significance in BBB scores; however, no significant differences were observed with tissue analysis between groups at 6 WPI. Conclusion Results suggest that keratin biomaterials support some locomotor functional recovery and may alter the acute inflammatory response by inducing macrophage polarization following SCI. This therapy warrants further investigation into treatment of SCI.Level of Evidence: N/A.

Described for the first time is the use of elemental analysis of diabetic toenails and machine learning techniques for the robust classification of type-2 diabetes. Aluminum, Cs, Ni, V and Zn concentrations in toenails were found to be significantly (p

Background Autograft (AG) is the gold standard bone graft due to biocompatibility, osteoconductivity, osteogenicity, and osteoinductivity. Alternatives include allografts and xenografts (XG). Methods We investigated the osseointegration and biocompatibility of a decellularized porcine XG within a critical defect animal model. We hypothesized that the XG will result in superior osseointegration compared to demineralized bone matrix (DBM) and equivalent immune response to AG. Critical defects were created in rat femurs and treated with XG, XG plus bone morphogenetic protein (BMP)-2, DBM, or AG. Interleukin (IL)-2 and IFN-gamma levels (inflammatory markers) were measured from animal blood draws at 1 week and 1 month post-operatively. At 1 month, samples underwent micro-positron-emission tomography (microPET) scans following 18-NaF injection. At 16 weeks, femurs were retrieved and sent for micro-computerized tomography (microCT) scans for blinded grading of osseointegration or were processed for histologic analysis with tartrate resistant acid phosphatase (TRAP) and pentachrome. Results Enzyme linked immunosorbent assay testing demonstrated greater IL-2 levels in the XG vs. AG 1 week post-op; which normalized by 28 days post-op. MicroPET scans showed increased uptake within the AG compared to all groups. XG and XG + BMP-2 showed a trend toward increased uptake compared with DBM. MicroCT scans demonstrated increased osseointegration in XG and XG + BMP groups compared to DBM. Pentachrome staining demonstrated angiogenesis and endochondral bone formation. Furthermore, positive TRAP staining in samples from all groups indicated bone remodeling. Conclusions These data suggest that decellularized and oxidized porcine XG is biocompatible and at least equivalent to DBM in the treatment of a critical defect in a rat femur model.

Nerve conduits are becoming increasingly popular for the repair of peripheral nerve injuries. Their ease of application and lack of donor site morbidity make them an attractive option for nerve repair in many situations. Today, there are many different conduits to choose in different sizes and materials, giving the reconstructive surgeon many options for any given clinical problem. However, to properly utilize these unique reconstructive tools, the peripheral nerve surgeon must be familiar not only with their standard indications but also with their functional limitations. In this review, the authors identify the common applications of nerve conduits, expected results, and shortcomings of current techniques. Furthermore, future directions for nerve conduit use are identified.

Background: Schwann cells are integral to the regenerative capacity of the peripheral nervous system, which declines after adolescence. The mechanisms underlying this decline are poorly understood. This study sought to compare the protein expression of Notch, c-Jun, and Krox-20 after nerve crush injury in adolescent and young adult rats. We hypothesized that these Schwann cell myelinating regulatory factors are down-regulated after nerve injury in an age-dependent fashion. Methods: Adolescent (2 months old) and young adult (12 months old) rats (n = 48) underwent sciatic nerve crush injury. Protein expression of Notch, c-Jun, and Krox-20 was quantified by Western blot analysis at 1, 3, and 7 days post-injury. Functional recovery was assessed in a separate group of animals (n = 8) by gait analysis (sciatic functional index) and electromyography (compound motor action potential) over an 8-week post-injury period. Results: Young adult rats demonstrated a trend of delayed onset of the dedifferentiating regulatory factors, Notch and c-Jun, corresponding to the delayed functional recovery observed in young adult rats compared to adolescent rats. Compound motor action potential area was significantly greater in adolescent rats relative to young adult rats, while amplitude and velocity trended toward statistical significance. Conclusions: The process of Schwann cell dedifferentiation following peripheral nerve injury shows different trends with age. These trends of delayed onset of key regulatory factors responsible for Schwann cell myelination may be one of many possible factors mediating the significant differences in functional recovery between adolescent and young adult rats following peripheral nerve injury.

To evaluate the biological, immunological, and biomechanical properties of a scaffold derived by architectural modification of a fresh-frozen porcine patella tendon using a decellularization protocol that combines physical, chemical, and enzymatic modalities.Porcine patellar tendons were processed using a decellularization and oxidation protocol that combines physical, chemical, and enzymatic modalities. Scaffolds (n = 88) were compared with native tendons (n = 70) using histologic, structural (scanning electron microscopy, porosimetry, and tensile testing), biochemical (mass spectrometry, peracetic acid reduction, DNA quantification, alpha-galactosidase [α-gal] content), as well as in vitro immunologic (cytocompatibility, cytokine induction) and in vivo immunologic nonhuman primate analyses.A decrease in cellularity based on histology and a significant decrease in DNA content were observed in the scaffolds compared with the native tendon (P.001). Porosity and pore size were increased significantly (P.001). Scaffolds were cytocompatible in vitro. There was no difference between native tendons and scaffolds when comparing ultimate tensile load, stiffness, and elastic modulus. The α-gal xenoantigen level was significantly lower in the decellularized scaffold group compared with fresh-frozen, nondecellularized tissue (P.001). The in vivo immunological response to implanted scaffolds measured by tumor necrosis factor-α and interleukin-6 levels was significantly (P.001) reduced compared with untreated controls in vitro. These results were confirmed by an attenuated response to scaffolds in vivo after implantation in a nonhuman primate model.Porcine tendon was processed via a method of decellularization and oxidation to produce a scaffold that possessed significantly less inflammatory potential than a native tendon, was biocompatible in vitro, of increased porosity, and with significantly reduced amounts of α-gal epitope while retaining tensile properties.Porcine-derived scaffolds may provide a readily available source of material for musculoskeletal reconstruction and repair while eliminating concerns regarding disease transmission and the morbidity of autologous harvest.

The long-term adaptations to microgravity and other spaceflight challenges within the confines of a spacecraft, and readaptations to weight-bearing upon reaching a destination, are unclear. While post-flight gait change in astronauts have been well documented and reflect multi-system deficits, no data from rodents have been collected. Thus, the purpose of this study was to evaluate gait changes in response to spaceflight. A prospective collection of gait data was collected on 3 groups of mice: those who spent~35 days in orbit (FLIGHT) aboard the International Space Station (ISS); a ground-based control with the same habitat conditions as ISS (Ground Control; GC); and a vivarium control with typical rodent housing conditions (VIV). Pre-flight and post-flight gait measurements were conducted utilizing an optimized and portable gait analysis system (DigiGait, Mouse Specifics, Inc). The total data acquisition time for gait patterns of FLIGHT and control mice was 1.5–5 min/mouse, allowing all 20 mice per group to be assessed in less than an hour. Patterns of longitudinal gait changes were observed in the hind limbs and the forelimbs of the FLIGHT mice after ~35 days in orbit; few differences were observed in gait characteristics within the GC and VIV controls from the initial to the final gait assessment, and between groups. For FLIGHT mice, 12 out of 18 of the evaluated gait characteristics in the hind limbs were significantly changed, including: stride width variability; stride length and variance; stride, swing, and stance duration; paw angle and area at peak stance; and step angle, among others. Gait characteristics that decreased included stride frequency, and others. Moreover, numerous forelimb gait characteristics in the FLIGHT mice were changed at post-flight measures relative to pre-flight. This rapid DigiGait gait measurement tool and customized spaceflight protocol is useful for providing preliminary insight into how spaceflight could affect multiple systems in rodents in which deficits are reflected by altered gait characteristics.

Ascorbic acid (vitamin C) is an essential micronutrient with evidence supporting its role in bone formation, tissue repair, and collagen production. Its clinical importance to the field of orthopaedic surgery has yet to be fully defined. Several observational studies have shown improved bone density and reduced hip fracture risk with supplementation. Its effect on bone fracture and soft tissue injury has been promising in animal models, but is not adequately studied in human trials. Results have been mixed concerning its role in chondroprotection and osteoarthritis treatment. Evidence suggesting reduced incidence of complex regional pain syndrome following distal radius fracture when treated with adjuvant ascorbic acid has prompted much debate but has received an endorsement of moderate support from the American Academy of Orthopaedic Surgeons. Given its potential benefits, low cost, and safety profile, ascorbic acid supplementation warrants consideration by orthopaedic surgeons in the treatment of a variety of musculoskeletal injuries (Journal of Surgical Orthopaedic Advances 27(4):261-268, 2018).

Background Xenografts are an attractive alternative to traditional bone grafts because of the large supply from donors with predictable morphology and biology as well as minimal risk of human disease transmission. Clinical series involving xenograft bone transplantation, most commonly from bovine sources, have reported poor results with frequent graft rejection and failure to integrate with host tissue. Failures have been attributed to residual alpha-Gal epitope in the xenograft which humans produce natural antibody against. To the authors' knowledge, there is currently no xenograft-derived bone graft substitute that has been adopted by orthopedic surgeons for routine clinical use. Methods In the current study, a bone scaffold intended to serve as a bone graft substitute was derived from porcine cancellous bone using a tissue decellularization and chemical oxidation protocol. In vitro cytocompatibility, pathogen clearance, and alpha-Gal quantification tests were used to assess the safety of the bone scaffold intended for human use. Results In vitro studies showed the scaffold was free of processing chemicals and biocompatible with mouse and human cell lines. When bacterial and viral pathogens were purposefully added to porcine donor tissue, processing successfully removed these pathogens to comply with sterility assurance levels established by allograft tissue providers. Critically, 98.5% of the alpha-Gal epitope was removed from donor tissue after decellularization as shown by ELISA inhibition assay and immunohistochemical staining. Conclusions The current investigation supports the biologic safety of bone scaffolds derived from porcine donors using a decellularization protocol that meets current sterility assurance standards. The majority of the highly immunogenic xenograft carbohydrate was removed from donor tissue, and these findings support further in vivo investigation of xenograft-derived bone tissue for orthopedic clinical application.

1.AbstractBone grafting is the second most common tissue transplantation procedure worldwide. The gold standard for bone grafting is the autograft; however, due to morbidity and limited supply, new alternatives, including allograft and tissue-engineered bone substitutes, are needed to satisfy long-term demand. One of the most desired properties of tissue-engineered bone substitutes is osteoinductivity, defined as the ability to stimulate primitive cells to differentiate into a bone forming lineage. In the current study, we treated porcine bone with a decellularization protocol to produce a bone scaffold. We examined whether the scaffold possessed osteoinductive potential and could be used to create a tissue-engineered bone microenvironment. To test if the bone scaffold was a viable host, pre-osteoblasts were seeded, incubated in vitro, and analyzed for markers of osteogenic differentiation. To assess these properties in vivo, scaffolds with and without pre-osteoblasts pre-seeded were subcutaneously implanted in mice for four weeks. The scaffolds underwent micro-computed tomography (microCT) scanning before implantation. After retrieval, the scaffolds were analyzed for osteogenic differentiation or re-scanned by microCT to assess new bone formation with the subsequent histological assessment. The osteoinductive potential was observed in vitro with similar osteogenic markers being expressed as observed in demineralized bone matrix and significantly greater expression of these markers than controls. By microCT, paired t-tests demonstrated significantly increased bone volume:total volume (BV/TV) and trabecular thickness (Tb.Th) after explantation in all groups. Pentachrome staining demonstrated osteogenesis within the scaffold, and angiogenesis in the scaffold was confirmed by CD31 staining for blood vessels. These results demonstrate that porcine bone maintains its osteoinductive properties after the application of a novel decellularization and oxidation protocol. Future work must be performed to definitively prove osteogenesis of human mesenchymal stem cells, biocompatibility in large animal models, and osteoinduction/osseointegration in a relevant clinical model in vivo. The ability to create a functional bone microenvironment using decellularized xenografts will impact regenerative medicine, orthopaedic reconstruction, and could be used in the research of multiple diseases.

INTRODUCTION Neurotrophin receptors, such as p75(NTR) , direct neuronal response to injury. Insulin-like growth factor-1 receptor (IGF-1R) mediates the increase in p75(NTR) during aging. The aim of this study was to examine the effect of aging and insulin-like growth factor-1 (IGF-1) treatment on recovery after peripheral nerve injury. METHODS Young and aged rats underwent tibial nerve transection with either local saline or IGF-1 treatment. Neurotrophin receptor mRNA and protein expression were quantified. RESULTS Aged rats expressed elevated baseline IGF-1R (34% higher, P = 0.01) and p75(NTR) (68% higher, P

Traumatic spinal cord injury (SCI) is typically the result of direct mechanical impact to the spine, leading to fracture and/or dislocation of the vertebrae along with damage to the surrounding soft tissues. Injury to the spinal cord results in disruption of axonal transmission of signals. This primary trauma causes secondary injuries that produce immunological responses such as neuroinflammation, which perpetuates neurodegeneration and cytotoxicity within the injured spinal cord. To date there is no FDA-approved pharmacological agent to prevent the development of secondary SCI and induce regenerative processes aimed at healing the spinal cord and restoring neurological function. An alternative method to electrically activate spinal circuits is the application of a noninvasive electromagnetic field (EMF) over intact vertebrae. The EMF method of modulating molecular signaling of inflammatory cells emitted in the extra-low frequency range of

Study design A laboratory study using a rabbit annular puncture model of intervertebral disc degeneration (IDD). Objective The aims of this study were to assess whether an amniotic suspension allograft (ASA) containing particulated human amnion and amniotic fluid derived cells regains intervertebral disc height and morphology and improves histologic scoring in a rabbit model of IDD. Summary of background data In contrast to current surgical interventions for IDD, in which the primary goal is to relieve symptomatic pain, one novel strategy involves the direct injection of anabolic cytokines. Current therapies for IDD are limited by both the short half-life of therapeutic proteins and general decline in anabolic cell populations. Methods Intervertebral discs in New Zealand white rabbits were punctured using 18-gauge needle under fluoroscopic guidance. Four weeks post-puncture, two groups of rabbits were injected with either ASA or a vehicle/sham control, while a third group was untreated. Weekly radiographs were obtained for 12 weeks to assess disc height index (DHI). Magnetic resonance imaging (MRI) T2 relaxation time was evaluated at weeks 4 and 12 to assess morphological changes. Histologic sections were evaluated on a semi-quantitative grading scale. Results Before treatment at week 4, DHIs and normalized T2 relaxation times between the three groups were not significantly different. At week 12, ASA-treated rabbits exhibited significantly greater DHIs and MRI T2 relaxation times than vehicle and untreated control groups. The ASA group had higher mean histologic score than the vehicle group, which demonstrated extensive fiber disorganization and delamination with reduced proteoglycan staining on histology. Conclusion Minimally invasive intervention with intradiscal injection of ASA was successful in reducing IDD in a reproducible rabbit model, with significant improvement in disc height and morphology when compared with vehicle and untreated control groups on radiographic and MRI analyses. Level of evidence N/A.

Purpose: Various animal models have been proposed to mimic the pathophysiologic process of intervertebral disc degeneration, a leading cause of back pain. The purpose of this study is to describe a minimally invasive technique via percutaneous needle puncture of the annulus fibrosus in New Zealand white rabbits. Methods: Under fluoroscopic guidance, an 18-gauge spinal needle was inserted 2 cm lateral to the midline spinous process. The needle was slowly advanced at approximately 45° angle until it was adjacent to the L5/L6 disc space. Lateral and anteroposterior views were used to verify correct needle position before advancing into the nucleus pulposus. The rabbits underwent weekly X-rays for 4 weeks to assess disc height index. MRI T2 relaxation was evaluated at week four to assess morphological changes. Discs were histologically graded on a 12-point scale to assess degeneration and compared to discs obtained from uninjured rabbits. Results: There were no complications associated with the percutaneous needle puncture procedure. All animals survived the duration of the experiment. Four weeks after injury, the disc height had progressively narrowed to approximately 50% of baseline. MRI assessment at the 4-week time point demonstrated a mean T2 relaxation time at the L5/L6 level that was 20.9% of the T2 relaxation time at the uninjured L4/L5 disc level ( p < 0.001). Histological analysis demonstrated lamellar disorganization of the annulus and decreased cellularity and proteoglycan content within the injured nucleus compared to uninjured control discs. Conclusion: The present study demonstrated a reliable technique of inducing an annular tear via a percutaneous needle puncture. Compared to open surgical approaches, the percutaneous model produces similar progressive disc degeneration while minimizing harm to the animal subjects. Clinical Relevance: The present study establishes a technique for the introduction of novel therapeutic agents to treat disc degeneration that may translate to future clinical trials.

Background: Bone grafts are used in approximately one half of all musculoskeletal surgeries. Autograft bone is the historic gold standard but is limited in supply and its harvest imparts significant morbidity to the patient. Alternative sources of bone graft include allografts, synthetics and, less commonly, xenografts which are taken from animal species. Xenografts are available in unlimited supply from healthy animal donors with controlled biology, avoiding the risk of human disease transmission, and may satisfy current demand for bone graft products. Methods: In the current study, cancellous bone was harvested from porcine femurs and subjected to a novel decellularization protocol to derive a bone scaffold. Results: The scaffold was devoid of donor cellular material on histology and DNA sampling (p &lt, 0.01). Microarchitectural properties important for osteoconductive potential were preserved after decellularization as shown by high resolution imaging modalities. Proteomics data demonstrated similar profiles when comparing the porcine bone scaffold against commercially available human demineralized bone matrix approved for clinical use. Conclusion: We are unaware of any porcine-derived bone graft products currently used in orthopaedic surgery practice. Results from the current study suggest that porcine-derived bone scaffolds warrant further consideration to serve as a potential bone graft substitute.

Langerhans cell sarcoma of the skin is a rare tumor with aggressive behavior. There are reports of Langerhans cell sarcoma involving the skin in patients with underlying systemic Langerhans cell histiocytosis. However, to our knowledge, sarcomatous transformation of skin Langerhans cell histiocytosis, has not been previously described. We report a case of Langerhans cell sarcoma of the skin representing a probable transformation from Langerhans cell histiocytosis. The lesion had a clinical and pathological biphasic pattern, presenting as a plaque and nodule in the lower leg of a 66 year-old female. The plaque area showed a superficial epidermotropic proliferation of atypical Langerhans cells. The associated deep nodule had an overtly sarcomatous Langerhans cell proliferation. The immunohistochemistry profile was characteristic of Langerhans cells in both components. We consider this case a unique example of deep nodular Langerhans cell sarcoma of the skin, in which the associated superficial Langerhans cell proliferation is consistent with the precursor lesion.

Premature musculoskeletal joint failure is a major source of morbidity among childhood cancer survivors. Radiation effects on synovial joint tissues of the skeleton are poorly understood. Our goal was to assess long-term changes in the knee joint from skeletally mature rats that received total-body irradiation while skeletal growth was ongoing.14 week-old rats were irradiated with 1, 3 or 7 Gy total-body doses of 18 MV X-rays. At 53 weeks of age, structural and compositional changes in knee joint tissues (articular cartilage, subchondral bone, and trabecular bone) were characterized using 7T MRI, nanocomputed tomography (nanoCT), microcomputed tomography (microCT), and histology.T2 relaxation times of the articular cartilage were lower after exposure to all doses. Likewise, calcifications were observed in the articular cartilage. Trabecular bone microarchitecture was compromised in the tibial metaphysis at 7 Gy. Mild to moderate cartilage erosion was scored in the 3 and 7 Gy rats.Late degenerative changes in articular cartilage and bone were observed after total-body irradiation in adult rats exposed prior to skeletal maturity. 7T MRI, microCT, nanoCT, and histology identified potential prognostic indicators of late radiation-induced joint damage.

To understand the role of Angiotensin-(1-7) (Ang-(1-7)) in vasculature of pregnant women, we compared cardiac output (CO), total peripheral resistance (TPR) and forearm blood flow (FBF) responses to Ang-(1-7) infusion between normotensive pregnant women in their third trimester and healthy age matched non-pregnant women. The responses of skin microcirculation to Ang-(1-7) were tested in preeclamptic, normotensive pregnant and non-pregnant women. Responses to Angiotensin II (Ang II) were also determined.Non-invasive methods for systemic (bioimpedance and VascuMAP), FBF (venous occlusion strain gauge plethysmography), and skin (laser Doppler) hemodynamics assessments were used.Compared to non-pregnant women, systemic infusion of Ang-(1-7) (2000 pmol/min) resulted in a greater increase in CO (9.4 ± 6.4 versus -3.3 ± 2.1%, n = 9-10) in normotensive pregnant women. Brachial local infusion of Ang-(1-7) had no effect on FBF in either group. In non-pregnant and normotensive pregnant women, local Ang II induced a dose-dependent decrease in FBF and increase in forearm resistance at 50 and 100 pmol/min (p 0.05 versus corresponding baseline, n = 7-10). Following iontophoretic application of 5 mmol/l dose of Ang-(1-7), the change in skin flow was higher in normotensive pregnant versus preeclamptic women (182.5 ± 93 versus 15.76 ± 19.46%, n = 14-15). Skin flow was lower in normotensive pregnant versus preeclamptic women (-46.5 ± 48.7 versus 108.7 ± 49.1%, n = 14-15) following Ang II infusion at 1.0 pmol/min.In the third trimester of pregnancy, Ang-(1-7) induces alterations in CO and differentially regulates micro- and macro-circulations, depending on the dose. Dysregulation in skin vasculature may contribute to the development of vascular dysfunction and hypertension in preeclampsia.

Absorbable collagen sponges (ACS) are used clinically as carriers of recombinant human bone morphogenetic protein 2 (rhBMP-2) to promote bone regeneration. ACS exhibit ectopic bone growth due to delivery of supraphysiological levels of rhBMP-2, which is particularly problematic in craniofacial bone injuries for both functional and esthetic reasons. We hypothesized that hydrogels from the reduced form of keratin proteins (kerateine) would serve as a suitable alternative to ACS carriers of rhBMP-2. The rationale for this hypothesis is that keratin biomaterials degrade slowly in vivo, have modifiable material properties, and have demonstrated capacity to deliver therapeutic agents. We investigated kerateine hydrogels and freeze-dried scaffolds as rhBMP-2 carriers in a critically-sized rat mandibular defect model. ACS, kerateine hydrogels, and kerateine scaffolds loaded with rhBMP-2 achieved bridging in animals by 8 weeks as indicated by micro-computed tomography. Kerateine scaffolds achieved statistically increased bone mineral density compared to ACS and kerateine hydrogels, with levels reaching those of native bone. Importantly, both kerateine hydrogels and kerateine scaffolds had significantly less ectopic bone growth than ACS sponges at both 8 and 16 weeks post-operatively. These studies demonstrate the suitability of keratins as rhBMP-2 carriers due to equal regenerative capacity with reduced ectopic growth compared to ACS.

The influence of age on rotator cuff function and muscle structure remains poorly understood. We hypothesize that normal aging influences rotator cuff function, muscle structure, and regulatory protein expression in an established rat model of aging.Seventeen rats were obtained from the National Institute on Aging. The supraspinatus muscles in 11 middle-aged (12 months old) and 6 old (28 months old) rats were studied for age-related changes in rotator cuff neuromuscular function by in vivo muscle force testing and electromyography (EMG). Changes in muscle structure and molecular changes were assessed with quantitative immunohistochemistry for myogenic determination factor 1 (MyoD) and myogenic factor 5 (Myf5) expression.Old animals revealed significantly decreased peak tetanic muscle force at 0.5 N and 0.7 N preload tension (P.05). The age of the animal accounted for 20.9% of variance and significantly influenced muscle force (P = .026). Preload tension significantly influenced muscle force production (P.001) and accounted for 12.7% of total variance. There was regional heterogeneity in maximal compound motor action potential (CMAP) amplitude in the supraspinatus muscle; the proximal portion had a significantly higher CMAP than the middle and distal portions (P.05). The expression of muscle regulatory factors MyoD and Myf5 was significantly decreased in old animals compared with middle-aged animals (P.05).The normal aging process in this rat model significantly influenced contractile strength of the supraspinatus muscle and led to decreased expression of muscle regulatory factors. High preload tensions led to a significant decrease in force production in both middle-aged and old animals.

Peripheral nerve injuries requiring surgery can be repaired by autograft, the clinical "gold standard", allograft, or nerve conduits. Most published clinical studies show the effectiveness of nerve conduits in small size defects in sensory nerves. Many preclinical studies suggest that peripheral nerve regeneration through conduits can be enhanced and repair lengths increased with the use of a biomaterial filler in the conduit lumen. We have previously shown that a luminal hydrogel filler derived from human hair keratin (HHK) can improve electrophysiological and histological outcomes in mouse, rabbit, and non-human primate nerve injury models, but insight into potential mechanisms has been lacking. Based on the premise that a keratin biomaterial (KOS) hydrogel provides an instantaneous structural matrix within the lumen, the current study compares the cellular behavior elicited by KOS hydrogel to Matrigel (MAT) and saline (SAL) conduit fillers in a 1 cm rat sciatic nerve injury model at early stages of regeneration. While there was little difference in initial cellular influx, the KOS group showed earlier migration of dedifferentiated Schwann cells (SC) from the proximal nerve end compared to the other groups. The KOS group also showed faster SC dedifferentiation and myelin debris clearance, and decreased macrophage infiltration during Wallerian degeneration of the distal nerve tissue.

Nonhuman primates have similar shoulder anatomy and physiology compared to humans, and may represent a previously underutilized model for shoulder research. This study sought to identify naturally occurring bony and muscular degeneration in the shoulder of nonhuman primates and to assess relationships between structural and functional aspects of the shoulder and measures of physical function of the animals. We hypothesized that age-related degenerative changes in the shoulders of nonhuman primates would resemble those observed in aging humans.Middle-aged (n = 5; ages 9.4-11.8 years) and elderly (n = 6; ages 19.8-26.4 years) female vervet monkeys were studied for changes in mobility and shoulder function, and radiographic and histologic signs of age-related degeneration.Four out of 6 (4/6) elderly animals had degenerative changes of the glenoid compared to 0/5 of the middle-aged animals (P = .005). Elderly animals had glenoid retroversion, decreased joint space, walked slower, and spent less time climbing and hanging than middle-aged vervets (P .05). Physical mobility and shoulder function correlated with glenoid version angle (P .05). Supraspinatus muscles of elderly animals were less dense (P = .001), had decreased fiber cross-sectional area (P .001), but similar amounts of nuclear material (P = .085). Degenerative rotator cuff tears were not observed in any of the eleven animals.The vervet monkey naturally undergoes age-related functional, radiographic and histological changes of the shoulder, and may qualify as an animal model for selected translational research of shoulder osteoarthritis.

Predisposition to Achilles tendon (AT) ruptures in middle-aged individuals may be associated with age-related changes to inherent passive biomechanical properties of the gastrocnemius-Achilles (GC-AT) muscle-tendon unit, due to known muscle-tendon structural changes in normal aging. The goal of this study was to determine whether the passive biomechanical response of the GC-AT muscle-tendon unit was altered with age in 6 young (8 months) and 6 middle-aged (24 months) F344xBN hybrid rats from the National Institute on Aging colony. Fung's quasilinear viscoelastic (QLV) model was used to determine in vivo history and time-dependent load-relaxation response of the GC-AT. Effective stiffness and modulus were also estimated using linear regression analysis. Fung's QLV revealed a significantly decreased magnitude of the relaxation response (parameter C, p=0.026) in middle-aged animals compared to young animals (0.108±0.007 vs. 0.144±0.015), with similar time-dependent viscous GC-AT properties (τ(1), τ(2)). The product of elastic parameters (A*B), which represents the initial slope of the elastic response, was significantly increased by 50% in middle-aged rats (p=0.014). Estimated GC-AT stiffness increased 28% at peak tensions in middle-aged rats (2.7±0.2 N/mm) compared to young rats (1.9±0.2 N/mm; p=0.036). While the limitations of this animal model must be considered, the changes we describe could be associated with the observation that GC-AT pathology and injury is more common in middle-aged individuals. Further studies are necessary to characterize the load-to-failure behavior of AT in middle-aged compared to young animals.

Aim: To evaluate the potential use of zinc chelation for prostate cancer therapy using a new liposomal formulation of the zinc chelator, N,N,N’,N’-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Materials & methods: TPEN was encapsulated in nontargeted liposomes or liposomes displaying an aptamer to target prostate cancer cells overexpression prostate-specific membrane antigen. The prostate cancer selectivity and therapeutic efficacy of liposomal (targeted and nontargeted) and free TPEN were evaluated in vitro and in tumor-bearing mice. Results & conclusion: TPEN chelates zinc and results in reactive oxygen species imbalance leading to cell death. Delivery of TPEN using aptamer-targeted liposomes results in specific delivery to targeted cells. In vivo experiments show that TPEN-loaded, aptamer-targeted liposomes reduce tumor growth in a human prostate cancer xenograft model.

Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) are often avoided by orthopaedic surgeons because of their possible influence on bone-healing. This belief stems from multiple studies, in particular animal studies, that show delayed bone-healing or nonunions associated with NSAID exposure. The purpose of this review was to critically analyze the quality of published literature that evaluates the impact of NSAIDs on clinical bone-healing. Methods: A MEDLINE and Embase search was conducted to identify all articles relating to bone and fracture-healing and the utilization of NSAIDs. All human studies, including review articles, were identified for further analysis. Non-English-language manuscripts and in vitro and animal studies were excluded. A total of twelve clinical articles and twenty-four literature reviews were selected for analysis. The quality of the clinical studies was assessed with a modified Coleman Methodology Score with emphasis on the NSAID utilization. Review articles were analyzed with regard to variability in the cited literature and final conclusions. Results: The mean modified Coleman Methodology Score (and standard deviation) was significantly lower (p = 0.032) in clinical studies that demonstrated a negative effect of NSAIDs on bone-healing (40.0 ± 14.3 points) compared with those that concluded that NSAIDs were safe (58.8 ± 10.3 points). Review articles also demonstrated substantial variability in the number of cited clinical studies and overall conclusions. There were only two meta-analyses and twenty-two narrative reviews. The mean number (and standard deviation) of clinical studies cited was significantly greater (p = 0.008) for reviews that concluded that NSAIDs were safe (8.0 ± 4.8) compared with those that recommended avoiding them (2.1 ± 2.1). Unanimously, all reviews admitted to the need for prospective randomized controlled trials to help clarify the effects of NSAIDs on bone-healing. Conclusions: This systematic literature review highlights the great variability in the interpretation of the literature addressing the impact of NSAIDs on bone-healing. Unfortunately, there is no consensus regarding the safety of NSAIDs following orthopaedic procedures, and future studies should aim for appropriate methodological designs to help to clarify existing discrepancies to improve the quality of care for orthopaedic patients. Clinical Relevance: This systematic review highlights the limitations in the current understanding of the effects of NSAIDs on bone healing. Thus, withholding these medications does not have any proven scientific benefit to patients and may even cause harm by increasing narcotic requirements in cases in which they could be beneficial for pain management. This review should encourage further basic-science and clinical studies to clarify the risks and benefits of anti-inflammatory medications in the postoperative period, with the aim of improving patient outcomes.

Background: Radiation-induced fibrosis (RIF) of musculoskeletal tissue is a common complication of radiation therapy for extremity soft-tissue sarcoma, with no standardized strategy for prevention and treatment. Angiotensin-(1-7) (Ang-[1-7]), a well-tolerated endogenous heptapeptide hormone with antitumor and antifibrotic properties, was tested as a radioprotectant for RIF and stiffening of irradiated muscles. Methods: Male CD-1 mice were randomized to one of three treatment groups: control, simulated sarcoma radiation therapy to the gastrocnemius and soleus muscles, or radiation therapy along with continuous Ang-(1-7) delivery initiated three days before radiation therapy. The biologically equivalent dose of radiation (∼100.3 Gy) absorbed by normal musculature during the course of radiation therapy for extremity sarcoma was delivered by means of four dose fractions of 7.3 Gy over two weeks. Fibrosis (n = 5 per group) and mechanical properties (n = 4 to 6 per group) of the muscles were measured at six weeks and four months after radiation therapy, and the intramuscular concentration of the profibrotic cytokines transforming growth factor-beta (TGF-β) and connective tissue growth factor (CTGF) (n = 8 to 10 per group) were measured at six weeks. Results: Interstitial (p < 0.01) and perivascular (p < 0.05) fibrosis increased significantly in the muscles treated with radiation therapy alone versus the nonirradiated controls at both six weeks (interstitial, +89%; perivascular, +112%) and four months (interstitial, +154%; perivascular, +88%). The muscles treated with radiation alone also exhibited increased tension (p < 0.01) versus nonirradiated controls at both six weeks (+779%) and four months (+1761%) when placed under 5% strain, and at four months (+1390%; p < 0.001) under 10% strain. At four months, muscle stiffness had increased in the mice treated with radiation therapy alone (+90%; p = 0.002) compared with nonirradiated controls. TGF-β production was also greater in this group at six weeks (+37%; p = 0.06) versus control. Ang-(1-7) administration prevented RIF and stiffening, with no differences observed for any other outcome between those receiving radiation therapy with Ang-(1-7) and the nonirradiated controls. Likewise, Ang-(1-7) mitigated the increase in TGF-β and CTGF concentration from radiation therapy. Conclusions: Ang-(1-7) attenuated RIF, stiffening, and production of profibrotic cytokines that were elevated in mouse skeletal muscles after simulated radiation therapy for extremity sarcoma. Clinical Relevance: Ang-(1-7) may serve as a potential therapy for the prevention of RIF in patients who require radiation therapy as adjuvant treatment for soft-tissue sarcoma.

Botulinum Neurotoxin A (BoNT-A) injections have been used for the treatment of muscle contractures and spasticity. This study assessed the influence of (BoNT-A) injections on passive biomechanical properties of the muscle-tendon unit. Mouse gastrocnemius muscle (GC) was injected with BoNT-A (n = 18) or normal saline (n = 18) and passive, non-destructive, in vivo load relaxation experimentation was performed to examine how the muscle-tendon unit behaves after chemical denervation with BoNT-A. Injection of BoNT-A impaired passive muscle recovery (15% vs. 35% recovery to pre-stretching baseline, p < 0.05) and decreased GC stiffness (0.531 ± 0.061 N/mm vs. 0.780 ± 0.037 N/mm, p < 0.05) compared to saline controls. The successful use of BoNT-A injections as an adjunct to physical therapy may be in part attributed to the disruption of the stretch reflex; thereby modulating in vivo passive muscle properties. However, it is also possible that BoNT-A injection may alter the structure of skeletal muscle; thus modulating the in vivo passive biomechanical properties of the muscle-tendon unit.

The purpose of this article is to review basic science studies using various animal models for rotator cuff research and to describe structural, biomechanical, and functional changes to muscle following rotator cuff tears. The use of computational simulations to translate the findings from animal models to human scale is further detailed. A comprehensive review was performed of the basic science literature describing the use of animal models and simulation analysis to examine muscle function following rotator cuff injury and repair in the ageing population. The findings from various studies of rotator cuff pathology emphasize the importance of preventing permanent muscular changes with detrimental results. In vivo muscle function, electromyography, and passive muscle–tendon unit properties were studied before and after supraspinatus tenotomy in a rodent rotator cuff injury model (acute vs chronic). Then, a series of simulation experiments were conducted using a validated computational human musculoskeletal shoulder model to assess both passive and active tension of rotator cuff repairs based on surgical positioning. Outcomes of rotator cuff repair may be improved by earlier surgical intervention, with lower surgical repair tensions and fewer electromyographic neuromuscular changes. An integrated approach of animal experiments, computer simulation analyses, and clinical studies may allow us to gain a fundamental understanding of the underlying pathology and interpret the results for clinical translation.

Our series on stopping drugs provides practical advice on why, when and how to withdraw drug therapy. Here the authors consider the challenges associated with discontinuing drugs in the elderly and frail.

BACKGROUND Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, heptapeptide hormone with anti-proliferative and anti-angiogenic properties. The primary objective of this study was to determine whether Ang-(1-7) effectively reduces prostate cancer metastasis in mice. METHODS Human PC3 prostate cancer cells were injected into the aortic arch via the carotid artery of SCID mice pre-treated with Ang-(1-7) or injected into the tibia of athymic mice, administered Ang-(1-7) for 5 weeks beginning 2 weeks post-injection. Tumor growth and volume were determined by bioluminescent and magnetic resonance imaging. The presence of tumors was confirmed by hematoxylin and eosin staining; TRAP histochemistry was used to identify osteolytic lesions. The effect of Ang-(1-7) on osteoclastogenesis was assessed in differentiated bone marrow cells. RESULTS Pre-treatment with Ang-(1-7) prevented metastatic tumor formation following intra-aortic injection of PC3 cells, while 83% of untreated mice developed tumors in metastatic sites. Circulating VEGF was significantly higher in control mice compared to mice administered Ang-(1-7). A 5-week regimen of the heptapeptide hormone attenuated intra-tibial tumor growth; Ang-(1-7) was significantly higher in the tibia of treated mice than in control animals. Osteoclastogenesis was reduced by 50% in bone marrow cells differentiated in the presence of Ang-(1-7), suggesting that the heptapeptide hormone prevents the formation of osteolytic lesions to reduce tumor survival in the bone microenvironment. CONCLUSIONS These findings suggest that Ang-(1-7) may serve as an anti-angiogenic and anti-metastatic agent for advanced prostate cancer. By extension, the heptapeptide hormone may provide effective therapy for bone metastasis produced from primary tumors of the lung and breast. Prostate 73: 71–82, 2013. © 2012 Wiley Periodicals, Inc.

After a series of powerful tornadoes struck several states in April and May of last year, the Federal Emergency Management Agency dispatched a team to assess the performance of residential and commercial buildings and such critical facilities as hospitals and emergency operations centers. The team’s observations formed the basis of several publications issued by the agency referred to as tornado recovery advisories.