Chronic osteoarthritis caused by Propionibacterium australiense infection in a captive sand gazelle.

Osteoarthritis is a common cause of morbidity and mortality in geriatric gazelles. Propionibacterium australiense has been reported as a cause of systemic granulomas in cattle, but there are no descriptions of this bacteria infecting other species nor causing osteoarthritis, to our knowledge. An 8-y-old, castrated male, sand gazelle (Gazella leptoceros leptoceros) was managed for chronic, intermittent, progressive osteoarthritis of the right tarsus. Serial biopsies revealed pyogranulomatous dermatitis with intralesional bacteria. Serial diagnostic imaging identified osseous and soft tissue proliferation with draining tracts. Treatments over 1 y included broad-spectrum antibiotics, anti-inflammatories, joint debridement, and infusion with platelet-rich plasma and stem cells. Despite therapy, lameness persisted, azotemia developed, and subsequently, the animal was euthanized. On postmortem examination, the periarticular tissue of the right tarsus was markedly expanded by pyogranulomas and fibrosis. Histologically, the synovium, joint capsule, and overlying soft tissues were markedly expanded by pyogranulomas and numerous gram-positive and acid-fast-negative filamentous bacteria surrounded by Splendore-Hoeppli material. Within the joint, there was regionally extensive cartilage ulceration, osteonecrosis, osteolysis, and pannus formation. PCR assay of affected formalin-fixed, paraffin-embedded tissue amplified segments of 16S rRNA and ß subunit of bacterial RNA polymerase (rpoB) genes with 99.7% and 95.6% identity to P. australiense. This bacterium should be considered a differential for chronic pyogranulomatous osteoarthritis in gazelles.

Curcumin as a potential therapeutic agent for treating neurodegenerative diseases.

Neurodegenerative diseases are characterized by the progressive loss of neuronal structure and function, posing a tremendous burden on health systems worldwide. Although the underlying pathological mechanisms for various neurodegenerative diseases are still unclear, a common pathological hallmark is the abundance of neuroinflammatory processes, which affect both disease onset and progression. In this review, we explore the pathways and role of neuroinflammation in various neurodegenerative diseases and further assess the potential use of curcumin, a natural spice with antioxidant and anti-inflammatory properties that has been extensively used worldwide as a traditional medicine and potential therapeutic agent. Following the examination of preclinical and clinical studies that assessed curcumin as a potential therapeutic agent, we highlight the bioavailability of curcumin in the body and discuss both the challenges and benefits of using curcumin as a therapeutic compound for treating neurodegeneration. Although elucidating the involvement of curcumin in aging and neurodegeneration has great potential for developing future CNS-related therapeutic targets, further research is required to elucidate the mechanisms by which Curcumin affects brain physiology, especially BBB integrity, under both physiological and disease conditions.

Macrophage phenotype impacts in vitro equine intrasynovial deep digital flexor tenocyte matrix metalloproteinase gene expression and secretion.

OBJECTIVE: To investigate matrix metalloproteinase (MMP) and their inhibitors tissue inhibitor matrix metalloproteinase (TIMP) gene expression and secretion during equine deep digital flexor tendon (DDFT) tenocyte and macrophage (undifferentiated, proinflammatory, and regulatory) co-culture. SAMPLE: Third passage DDF tenocytes and donor-matched macrophages differentiated from peripheral blood CD14+ monocytes from 5 healthy horses ages 9-11 years, euthanized for reasons unrelated to musculoskeletal conditions. METHODS: Passage 3 DDT tenocyte aggregate cultures were co-cultured with undifferentiated (control), proinflammatory (granulocyte-macrophage colony-stimulating factor; GM-CSF pretreated and lipopolysaccharide + interferon gamma-primed; LPS+IFN-γ) or regulatory (interleukin-4 and interleukin-10-primed; IL-4 + IL-10) macrophages in direct and transwell co-cultures for 72 hours. MMP-1, -2, -3, -9, -13, and TIMP -1, -2 mRNA were measured via real-time Polymerase Chain Reaction (rtPCR). Co-culture media MMP -3, -9, and TIMP -1, -2 concentrations were quantified via ELISA. RESULTS: Direct co-culture of DDF tenocytes with proinflammatory macrophages for 72 hours increased MMP-1, -3, and -13 mRNA levels whereas, MMP-9 mRNA levels decreased. Direct and transwell co-culture with proinflammatory and regulatory macrophages resulted in increased MMP-3 and decreased MMP-9 media concentrations. While direct co-culture with regulatory macrophages significantly increased TIMP-1 mRNA, overall, TIMP mRNA and culture media concentrations were largely unchanged. CLINICAL RELEVANCE: Cell-to-cell contact between DDF tenocytes and macrophages is not essential to induce MMP gene expression and secretion. Co-culture systems offer a viable in vitro platform to screen and evaluate immunomodulatory properties of therapies aimed at improving equine intrasynovial tendon healing.

The Ganesh-Grewal cystitome maker - A step in standardizing cataract surgery.

A well-centered, adequately sized continuous curvilinear capsulorhexis (CCC) is a prerequisite for successful cataract surgery. A perfect capsulorhexis ensures safe and effective performance of various steps of surgery as well as a correctly positioned intraocular lens (IOL) with optimal rotational stability. Ganesh and Grewal (GG) cystitome maker is a step toward standardizing the creation of a cystitome to reduce variations and complications associated with the crucial step of CCC in cataract surgery. We conducted a study to measure the repeatability and precision of cystitomes made by the GG cystitome maker versus those made manually with a needle holder. The results showed that the cystitomes made with GG cystitome maker had a lesser degree of variation. This indicates a more repeatable cystitome, which will inadvertently help in reducing the error caused by the instrument in making a good CCC during cataract surgery.

Astrocytic K+ clearance during disease progression in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder in which patients lose motor functions due to progressive loss of motor neurons in the cortex, brainstem, and spinal cord. Whilst the loss of neurons is central to the disease, it is becoming clear that glia, specifically astrocytes, contribute to the onset and progression of neurodegeneration. Astrocytes play an important role in maintaining ion homeostasis in the extracellular milieu and regulate multiple brain functions by altering their extracellular concentrations. In this study, we have investigated the ability of astrocytes to maintain K+ homeostasis in the brain via direct measurement of the astrocytic K+ clearance rate in the motor and somatosensory cortices of an ALS mouse model (SOD1G93A ). Using electrophysiological recordings from acute brain slices, we show region-specific alterations in the K+ clearance rate, which was significantly reduced in the primary motor cortex but not the somatosensory cortex. This decrease was accompanied by significant changes in astrocytic morphology, impaired conductivity via Kir4.1 channels and low coupling ratio in astrocytic networks in the motor cortex, which affected their ability to form the K+ gradient needed to disperse K+ through the astrocytic syncytium. These findings indicate that the supportive function astrocytes typically provide to motoneurons is diminished during disease progression and provides a potential explanation for the increased vulnerability of motoneurons in ALS.

Astrocytic modulation of neuronal signalling.

Neuronal signalling is a key element in neuronal communication and is essential for the proper functioning of the CNS. Astrocytes, the most prominent glia in the brain play a key role in modulating neuronal signalling at the molecular, synaptic, cellular, and network levels. Over the past few decades, our knowledge about astrocytes and their functioning has evolved from considering them as merely a brain glue that provides structural support to neurons, to key communication elements. Astrocytes can regulate the activity of neurons by controlling the concentrations of ions and neurotransmitters in the extracellular milieu, as well as releasing chemicals and gliotransmitters that modulate neuronal activity. The aim of this review is to summarise the main processes through which astrocytes are modulating brain function. We will systematically distinguish between direct and indirect pathways in which astrocytes affect neuronal signalling at all levels. Lastly, we will summarize pathological conditions that arise once these signalling pathways are impaired focusing on neurodegeneration.

Basal and inducible Osterix expression reflect equine mesenchymal progenitor cell osteogenic capacity.

Introduction: Mesenchymal stem cells are characterized by their capacities for extensive proliferation through multiple passages and, classically, tri-lineage differentiation along osteogenic, chondrogenic and adipogenic lineages. This study was carried out to compare osteogenesis in equine bone marrow-, synovium- and adipose-derived cells, and to determine whether osteogenic capacity is reflected in the basal expression of the critical osteogenic transcription factors Runx2 and Osterix. Methods: Bone marrow, synovium and adipose tissue was collected from six healthy 2-year-old horses. Cells were isolated from these sources and expanded through two passages. Basal expression of Runx2 and Osterix was assessed in undifferentiated third passage cells, along with their response to osteogenic culture conditions. Results: Bone marrow-derived cells had significantly higher basal expression of Osterix, but not Runx2. In osteogenic medium, bone-marrow cells rapidly developed dense, multicellular aggregates that stained strongly for mineral and alkaline phosphatase activity. Synovial and adipose cell cultures showed far less matrix mineralization. Bone marrow cells significantly up-regulated alkaline phosphatase mRNA expression and enzymatic activity at 7 and 14 days. Alkaline phosphatase expression and activity were increased in adipose cultures after 14 days, although these values were less than in bone marrow cultures. There was no change in alkaline phosphatase in synovial cultures. In osteogenic medium, bone marrow cultures increased both Runx2 and Osterix mRNA expression significantly at 7 and 14 days. Expression of both transcription factors did not change in synovial or adipose cultures. Discussion: These results demonstrate that basal Osterix expression differs significantly in progenitor cells derived from different tissue sources and reflects the osteogenic potential of the cell populations.

Interleukin-1ß and methylprednisolone acetate demonstrate differential effects on equine deep digital flexor tendon and navicular bone fibrocartilage cells in vitro.

OBJECTIVE: To investigate the effects of interleukin-1ß (IL-1ß) and methylprednisolone acetate (MPA) on equine intrabursal deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) cells in vitro. SAMPLE: Third passage DDFT and NBF cells from 5 healthy donor horses ages 11-17 years euthanized for reasons unrelated to musculoskeletal conditions. PROCEDURES: Aggregate cultures were incubated with culture medium alone (control), 10 ng/mL IL-1ß, 10 ng/mL IL-1ß + 0.05 mg/mL MPA, or 10 ng/mL IL-1ß + 0.5 mg/mL MPA for 24 hours. Extracellular matrix (ECM) gene expressions were assessed via real-time polymerase chain reaction (rtPCR). Culture media matrix metalloproteinase (MMP) -3 and -13 concentrations were quantified via ELISA. Total glycosaminoglycan (GAG) content in the cell pellets and culture media was also assessed. RESULTS: IL-1ß and IL-1ß combined with MPA significantly downregulated ECM gene expression to a greater extent in NBF cells compared with DDFT cells. IL-1ß and IL-1ß combined with MPA significantly upregulated MMP-3 culture media concentrations in DDFT cells only, and MMP-13 culture media concentrations to a greater extent in NBF cells compared with DDFT cells. CLINICAL RELEVANCE: NBF cells were more susceptible to IL-1ß and MPA-mediated ECM gene expression downregulation in vitro. These results serve as a first step for future work to determine intrabursal corticosteroid regimens that limits or resolve the inflammation as well as take into consideration NBF cell biosynthesis in horses with navicular disease, for which currently no information exists.

Equine peripheral blood CD14+ monocyte-derived macrophage in-vitro characteristics after GM-CSF pretreatment and LPS+IFN-γ or IL-4+IL-10 differentiation.

Macrophages are a heterogeneous population of immune cells that exhibit dynamic plasticity, polarize into inflammatory or regulatory/pro-resolving macrophages, and influence the healing tissue microenvironment. This study evaluated the in-vitro morphological, proliferative, cell surface marker expression and cytokine/soluble factor secretion characteristics of control, GM-CSF pretreated and inflammatory (LPS+IFN-γ) and regulatory (IL-4 + IL-10) differentiated equine CD14+ monocyte-derived macrophages. Phase contrast microscopy demonstrated that LPS+IFN-γ-primed macrophages exhibited a rounded, granular morphology, whereas IL-4 +IL-10-primed macrophages were elongated with a spindle-shaped morphology. GM-CSF enhanced the proliferation rate of monocytes/macrophages during adherent in-vitro culture. Flow cytometry analysis showed that GM-CSF alone and GM-CSF pretreatment with LPS+IFN-γ or IL-4 +IL-10 priming increased CD86 immunopositivity by 2-fold (p = 0.6); and CD206 immunopositivity remained unchanged. GM-CSF pretreatment and subsequent priming with LPS and IFN-γ yielded inflammatory macrophages that secrete significantly increased quantities of IL-1ß compared to control (p = 0.012) and IL-4 +IL-10-primed (p = 0.0047) macrophages. GM-CSF pretreatment followed by both LPS + IFN-γ and IL-4 + IL-10 priming significantly increased IL-1Ra secretion by 6-fold (p < 0.05). There were no differences in TGFß-1 secretion among control, LPS+IFN-γ or IL-4 + IL-10 primed macrophages (p = 0.85). All groups contained an average of 643 ± 51.5 pg/mL of TGFß1. Among the culture conditions evaluated, IL-4 +IL-10 priming for 24 h after 6 days of adherent culture yielded macrophages that were the least inflammatory compared to GM-CSF pretreated and LPS+IFN-γ or IL-4 +IL-10-primed macrophages. These results provide a basis for subsequent in-vitro and in-vivo studies that investigate macrophage-tissue cell interactions and related biological mechanisms relevant to the field of immunomodulatory approaches for enhancing tissue healing.

Knowledge, attitude, and practice of ophthalmic manifestations in COVID-19 patients at a tertiary care center.

Purpose: Since the start of the COVID-19 pandemic, various manifestations have been reported, including ophthalmic symptoms, especially with the different mutations and variants that have occurred over the last few years. In view of this, our study was conducted to gauge the knowledge, attitude, and practices of patients toward the ophthalmic manifestations of COVID-19. Methods: This was a hospital-based, cross-sectional, observational study. Patients who had tested positive for COVID-19 were asked to answer a detailed questionnaire about their knowledge of COVID-19 ophthalmic symptoms, their experience with the symptoms, and their attitude and practice toward the same. The data collected was analyzed using Microsoft Excel, and the Chi-squared test was used to determine significant differences in the results among different demographic profiles. Results: Our study found that 82 (39%) of the 210 participants were aware that COVID-19 could present with symptoms in the eyes. A total of 47 participants had experienced eye symptoms of COVID-19. Among them, only 15 (31.91%) consulted and received treatment from an ophthalmologist or general physician for the same. Most of them (59.57%) did not seek any treatment, and 8.5% self-medicated or used non-allopathic forms of medicine. The most common symptom was redness of the eyes, reported by 57.44% of those who had eye symptoms. Conclusion: Most people were unaware of ocular manifestations of COVID-19 and most of those who were aware were medical professionals. Amongst those who developed symptoms, only a minority sought medical treatment.

A perspective on molecular signalling dysfunction, its clinical relevance and therapeutics in autism spectrum disorder.

Intellectual disability (ID) and autism spectrum disorder (ASD) are neurodevelopmental disorders that have become a primary clinical and social concern, with a prevalence of 2-3% in the population. Neuronal function and behaviour undergo significant malleability during the critical period of development that is found to be impaired in ID/ASD. Human genome sequencing studies have revealed many genetic variations associated with ASD/ID that are further verified by many approaches, including many mouse and other models. These models have facilitated the identification of fundamental mechanisms underlying the pathogenesis of ASD/ID, and several studies have proposed converging molecular pathways in ASD/ID. However, linking the mechanisms of the pathogenic genes and their molecular characteristics that lead to ID/ASD has progressed slowly, hampering the development of potential therapeutic strategies. This review discusses the possibility of recognising the common molecular causes for most ASD/ID based on studies from the available models that may enable a better therapeutic strategy to treat ID/ASD. We also reviewed the potential biomarkers to detect ASD/ID at early stages that may aid in diagnosis and initiating medical treatment, the concerns with drug failure in clinical trials, and developing therapeutic strategies that can be applied beyond a particular mutation associated with ASD/ID.

Autologous platelet-rich plasma effects on Staphylococcus aureus-induced chondrocyte death in an in vitro bovine septic arthritis model.

OBJECTIVE: To investigate the chondroprotective effects of autologous platelet-rich plasma (PRP), ampicillin-sulbactam (AmpS), or PRP combined with AmpS (PRP+AmpS) in an in vitro chondrocyte explant model of bovine Staphylococcus aureus-induced septic arthritis. SAMPLE: Autologous PRP and cartilage explants obtained from 6 healthy, adult, nonlactating Jersey-crossbred cows. PROCEDURES: Autologous PRP was prepared prior to euthanasia using an optimized double centrifugation protocol. Cartilage explants collected from grossly normal stifle joints were incubated in synovial fluid (SF) alone, S aureus-inoculated SF (SA), or SA supplemented with PRP (25% culture medium volume), AmpS (2 mg/mL), or both PRP (25% culture medium volume) and AmpS (2 mg/mL; PRP+AmpS) for 24 hours. The metabolic activity, percentage of dead cells, and glycosaminoglycan content of cartilage explants were measured with a resazurin-based assay, live-dead cell staining, and dimethylmethylene blue assay, respectively. Treatment effects were assessed relative to the findings for cartilage explants incubated in SF alone. RESULTS: Application of PRP, AmpS, and PRP+AmpS treatments significantly reduced S aureus-induced chondrocyte death (ie, increased metabolic activity and cell viability staining) in cartilage explants, compared with untreated controls. There were no significant differences in chondrocyte death among explants treated with PRP, AmpS, or PRP+AmpS. CLINICAL RELEVANCE: In this in vitro explant model of S aureus-induced septic arthritis, PRP, AmpS, and PRP+AmpS treatments mitigated chondrocyte death. Additional work to confirm the efficacy of PRP with bacteria commonly associated with clinical septic arthritis in cattle as well as in vivo evaluation is warranted.

Zonal characterization and differential trilineage potentials of equine intrasynovial deep digital flexor tendon-derived cells.

BACKGROUND: Intrasynovial deep digital flexor tendon (DDFT) injuries occur frequently and are often implicated in cases of navicular disease with poor outcomes and reinjuries. Cell-based approaches to tendon healing are gaining traction in veterinary medicine and ultimately may contribute to improved DDFT healing in horses. However, a better understanding of the innate cellular characteristics of equine DDFT is necessary for developing improved therapeutic strategies. Additionally, fibrocartilaginous, intrasynovial tendons like the DDFT are common sites of injury and share a poor prognosis across species, offering translational applications of this research. The objective of this study is to isolate and characterize tendon-derived cells (TDC) from intrasynovial DDFT harvested from within the equine forelimb podotrochlear bursa. TDC from the fibrocartilaginous and tendinous zones are separately isolated and assessed. Flow cytometry is performed for mesenchymal stem cell (MSC) surface markers (CD 29, CD 44, CD 90). Basal tenogenic, osteogenic and chondrogenic markers are assessed via quantitative real time-PCR, and standard trilineage differentiation is performed with third passage TDC from the fibrocartilaginous (fTDC) and tendinous (tTDC) zones of DDFT. RESULTS: Low-density plating isolated homogenous TDC populations from both zones. During monolayer passage, both TDC subpopulations exhibited clonogenicity, high in vitro proliferation rate, and fibroblast-like morphology. fTDC and tTDC were positive for MSC surface markers CD90 and CD29 and negative for CD44. There were no significant differences in basal tenogenic, osteogenic or chondrogenic marker expression between zones. While fTDC were largely restricted to chondrogenic differentiation, tTDC underwent osteogenic and chondrogenic differentiation. Both TDC subpopulations displayed weak adipogenic differentiation potentials. CONCLUSIONS: TDC at the level of the podotrochlear bursa, that potentially could be targeted for enhancing DDFT injury healing in horses were identified and characterized. Pending further investigation, promoting chondrogenic properties in cells administered exogenously into the intrasynovial space may be beneficial for intrasynovial tendon regeneration.

Comparative molecular docking and simulation analysis of molnupiravir and remdesivir with SARS-CoV-2 RNA dependent RNA polymerase (RdRp).

Treatment of SARS-CoV-2 targeting its RNA dependent RNA polymerase (RdRp) is of current interest. Remdesivir has been approved for the treatment of COVID-19 around the world. However, the drug has been linked with pharmacological limitations like adverse effects and reduced efficiency. Nevertheless, recent advancements have depicted molnupiravir as an effective therapeutic agent to target the SARS-CoV-2 RdRp. The drug has cleared both in vitro and in vivo screening. It is in phase-III clinical trial. Nonetheless, there are no data on themolecular binding interaction of molnupiravir with RdRp. Therefore, it is of interest to report the binding interaction of molnupiravir using molecular docking. It is also of interest to show its stability during interaction using molecular dynamics and binding free energy calculations along with drug likeliness and pharmacokinetic properties in comparison with remdesivir.

In vitro Effects of Methylprednisolone Acetate on Equine Deep Digital Flexor Tendon-Derived Cells.

Primary deep digital flexor tendon (DDFT) pathologies and those accompanying degenerative changes of navicular bone fibrocartilage are major causes of lameness associated with navicular disease. Intrasynovial corticosteroids are mainstay in the treatment due to the anti-inflammatory effects, but their effect on DDFT cell biosynthesis are unknown. The objective of this in-vitro study was to investigate the effects of methylprednisolone acetate (MPA) on cells isolated from the dorsal fibrocartilaginous region of forelimb DDFTs (DDFT-derived cells) of 5 horses (aged 11-17 years). Non-adherent aggregate cultures were established from third passage cells over a 72 to 96-h duration prior to treating with medium containing 0 (control), 0.05 and 0.5 mg/mL MPA for 24 h. Tendon and cartilage extracellular matrix (ECM) related gene expression, cell aggregate and culture medium GAG contents, culture medium collagen and MMP-3 and-13 concentrations were measured. After 24 h of treatment, only the higher MPA concentration (0.5 mg/mL) significantly down-regulated tendon ECM related genes; whereas, both MPA doses significantly down-regulated cartilage ECM related genes. MPA treatment did not affect the total GAG content of DDFT-derived cells or total GAG, soluble collagen and MMP-3 and-13 contents in culture medium compared to untreated controls. Future studies to determine the response of DDFT-derived cells with longer exposure times to corticosteroids and in the presence of inflammatory cytokines are necessary. These results are a first step in assessing the effects of intrasynovial medications on equine DDFT, for which currently no information exists.

Percutaneous ultrasonic tenotomy effectively debrides tendons of the extensor mechanism of the knee: A technical note.

BACKGROUND: Percutaneous ultrasonic tenotomy (PUT) is a minimally-invasive method of treating patellar tendinosis, but its immediate effect on tendon structure has never been studied. Given the crucial nature of the extensor mechanism of the knee, it is important to understand the procedure's effect on tendon structure prior to clinical implementation. The aim of this study was to analyze the tendon structure of the extensor mechanism of the knee after PUT in a cadaveric model. METHODS: Four fresh-frozen cadaveric specimens (two patellar and two quadriceps tendons) underwent PUT. The tendons were then sectioned and stained with hematoxilin & eosin (H&E). The sections were analyzed for a clear area of debridement. The area of debridement was calculated as an average of three measurements. RESULTS: All four tendons demonstrated a clear area of debridement limited to the treatment area without damaging any surrounding tissue. The area of debridement for the patellar and quadriceps tendons treated was 2.89 mm2, 1.5 mm2, 2.98 mm2 and 7.29 mm2, respectively. CONCLUSIONS: Percutaneous ultrasonic tenotomy effectively debrided the treatment area in all tendons without damaging surrounding tissue. Further work is needed to report clinical outcomes, assess the risk of post-procedure tendon rupture and define return-to-sport progression.

Barriers to Outpatient Pediatric Chronic Pain Clinic Participation Among Referred Patients.

Initial clinic evaluation among referred patients and factors limiting treatment initiation are not well characterized. We conducted a retrospective review of referrals to our outpatient pain clinic to identify intake visits and factors associated with treatment initiation among adolescents with chronic pain. We identified adolescents aged 13 to 18 years at the time of referral to clinic (2010-2016). Factors associated with completion of visits were evaluated using logistic regression. Patients who completed visits more frequently had private insurance than public or no insurance (P = .053). The most common reasons for caregiver decision not to attend the pain clinic included use of another pain clinic, that services were not wanted or no longer needed, and that their child was undergoing further testing. The current study demonstrated that patients with head pain were more likely to complete an intake visit, while there was a trend showing that lack of private insurance decreased this likelihood.

Investigation of a novel prosthesis technique for extracapsular stabilization of cranial cruciate ligament-deficient stifle joints in adult cattle.

OBJECTIVE: To evaluate a novel prosthesis technique for extracapsular stabilization of cranial cruciate ligament (CCL)-deficient stifle joints in adult cattle. SAMPLE: 13 cadaveric bovine stifle joint specimens. PROCEDURES: In the first of 3 study phases, the most isometric points on the distal aspect of the femur (distal femur) and proximal aspect of the tibia (proximal tibia) were determined from measurements obtained from lateromedial radiographs of a stifle joint specimen maintained at angles of 135°, 90°, 65°, and 35°. During phase 2, 800-lb-test monofilament nylon leader line was cut into 73-cm-long segments. Each segment was secured in a loop by use of 2, 3, or 4 crimping sleeves such that there were 12 replicates for each construct. Each loop was distracted to failure at a constant rate of 1 mm/s. Mean force at failure and elongation and mode of failure were compared among the 3 constructs. During phase 3, bone tunnels were created in the distal femur and proximal tibia at the isometric points identified during phase 1 in each of 12 CCL-deficient stifle joint specimens. The 3-sleeve construct was applied to each specimen. Specimens were distracted to failure at a constant rate of 1 mm/s. RESULTS: Among the 3 constructs evaluated, the 3-sleeve construct was considered optimal in terms of strength and amount of foreign material. In phase 3, all replicates failed because of suture slippage. CONCLUSIONS AND CLINICAL RELEVANCE: Use of 800-lb-test monofilament nylon leader line as a prosthesis might be a viable alternative for extracapsular stabilization of CCL-deficient stifle joints in adult cattle. Further in vivo studies are necessary.

Cellular Components and Growth Factor Content of Platelet-Rich Plasma With a Customizable Commercial System.

BACKGROUND: Platelet-rich plasma (PRP) is an autologous orthobiologic treatment option for musculoskeletal conditions with favorable results in a limited number of high-quality clinical trials. Because different blood-processing methods result in PRP with varying cellular and growth factor content, it is critical that clinicians understand the content of the specific PRP being used in clinical practice. One adjustable system, the Angel System, has few independent laboratory reports on the specific composition of its PRP. The goal of this study was to quantify the cellular and growth factor composition of PRP produced by this system at its lowest hematocrit settings. HYPOTHESIS: The authors hypothesized that the system would significantly concentrate platelets over baseline and, at the lowest hematocrit settings, would reduce leukocytes to produce leukocyte-poor PRP. STUDY DESIGN: Descriptive laboratory study. METHODS: Ten healthy male volunteers donated 150 mL of whole blood for processing. Three separate processing cycles were performed for each sample at the 0%, 1%, and 2% hematocrit settings. The resultant PRP from each cycle was sent for complete blood counts and enzyme-linked immunosorbent assay to quantify the following growth factors: platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF). RESULTS: The system consistently concentrated platelets 5-fold over baseline, with no significant differences among settings. Leukocytes were concentrated at all settings, between 2 and 5 times over baseline. The 0% and 1% settings had significantly lower leukocyte concentrations than the 2% setting. Lymphocytes made up >89% of the leukocyte differential, while neutrophils represented <11% of the differential at each setting. There was a significant increase in PDGF and bFGF, a significant decrease in IGF-1, and no change in VEGF, with no difference among settings. CONCLUSION: The system consistently concentrated platelets 5 times but was unable to reduce leukocytes, therefore resulting in leukocyte-rich PRP at each setting tested. Leukocytes had a differential composition of >89% lymphocytes and <11% neutrophils. For all settings, PDGF and bFGF were concentrated; IGF-1 was reduced; and VEGF was not significantly different from baseline. CLINICAL RELEVANCE: These data can serve to guide clinicians considering using this particular PRP system. It consistently yielded leukocyte-rich PRP with a lymphocyte-predominant/neutrophil-reduced profile. Further research is needed to better understand how to apply this specific PRP in clinical practice.

Insulin Enhances the In Vitro Osteogenic Capacity of Flexor Tendon-Derived Progenitor Cells.

There is increased incidence of tendon disorders and decreased tendon healing capacity in people with diabetes mellitus (DM). Recent studies have also suggested pathological ossification in repair tendon of people with DM. Therefore, the objective of this study is to investigate the effects of insulin supplementation, an important pathophysiologic stimulus of DM, on tendon progenitor cell (TPC) proliferation and in vitro osteogenic capacity. Passage 3 TPCs were isolated from collagenase-digested, equine superficial digital flexor tendons. TPC proliferation was measured via MTT assay after 3 days of monolayer culture in medium supplemented with 0, 0.007, 0.07, and 0.7 nM insulin. In vitro osteogenic capacity of TPCs (Alizarin Red staining, osteogenic mRNA expression, and alkaline phosphatase bioactivity) was assessed with 0, 0.07, and 0.7 nM insulin-supplemented osteogenic induction medium. Insulin (0.7 nM) significantly increased TPC proliferation after 3 days of monolayer culture. Alizarin Red staining of insulin-treated TPC osteogenic cultures demonstrated robust cell aggregation and mineralized matrix secretion, in a dose-dependent manner. Runx2, alkaline phosphatase, and Osteonectin mRNA and alkaline phosphatase bioactivity of insulin-treated TPC cultures were significantly higher at day 14 of osteogenesis compared to untreated controls. Addition of picropodophyllin (PPP), a selective IGF-I receptor inhibitor, mitigated the increased osteogenic capacity of TPCs, indicating that IGF-I signaling plays an important role. Our findings indicate that hyperinsulinemia may alter TPC phenotype and subsequently impact the quality of repair tendon tissue.