In vitro and in vivo evaluation of discogenic cells, an investigational cell therapy for disc degeneration.

BACKGROUND/CONTEXT: Disc degeneration (DD) is a significant driver of low back pain and few treatments exist to treat the pain and disability associated with the disease. PURPOSE: Our group has developed a method to generate therapeutic discogenic cells as a potential treatment for symptomatic DD. These cells are derived and modified from adult nucleus pulposus cells. In this study, we evaluated the characteristics, mode of action, and in vivo efficacy and safety of these cells prior to human clinical testing. STUDY DESIGN: Privately funded in vitro studies and in vivo preclinical models were used in this study. METHODS: Discogenic cells generated from different adult human donors were evaluated for surface marker expression profile, matrix deposition and tumorigenic potential. Discogenic cells were then injected subcutaneously into nude mice to assess cell survival and possible extracellular matrix production in vivo. Finally, a rabbit model of DD was used to evaluate the therapeutic potential of discogenic cells after disc injury. RESULTS: We found that discogenic cells have a consistent surface marker profile, are multipotent for mesenchymal lineages, and produce extracellular matrix consisting of aggrecan, collagen 1 and collagen 2. Cells did not show abnormal karyotype after culturing and did not form tumor-like aggregates in soft agar. After subcutaneous implantation in a nude mouse model, the human discogenic cells were found to have generated regions rich with extracellular matrix over the course of 4 months, with no signs of tumorigenicity. Intradiscal injection of human discogenic cells in a rabbit model of DD caused an increase in disc height and improvement of tissue architecture relative to control discs or injection of vehicle alone (no cells) with no signs of toxicity. CONCLUSIONS: This study demonstrates that intradiscal injection of discogenic cells may be a viable treatment for human degenerative disc disease. The cells produce extracellular matrix that may rebuild the depleting tissue within degenerating discs. Also, the cells do not pose any significant safety concerns. CLINICAL SIGNIFICANCE: Human clinical testing of discogenic cells combined with a sodium hyaluronate carrier is ongoing in multiple randomized, controlled, double-blinded studies in the United States (clinicaltrials.gov identifier NCT03347708) and Japan (clinicaltrials.gov identifier NCT03955315).

Tissue distribution of enrofloxacin in African clawed frogs (Xenopus laevis) after intramuscular and subcutaneous administration.

As part of an enrofloxacin pharmacokinetic study, concentrations of enrofloxacin and ciprofloxacin (metabolite) were measured in various tissues (brain, heart, kidney, liver, lung, and spleen) collected from treated (subcutaneous delivery, n = 3; intramuscular delivery, n = 3; untreated controls, n = 2) adult female Xenopus laevis by using HPLC. Enrofloxacin was rapidly absorbed after administration by either route and readily diffused into all sampled tissues. Enrofloxacin and ciprofloxacin were present in the tissue samples collected at 8 h. The highest average tissue concentrations for enrofloxacin were found in kidney, with the lowest concentrations in liver. Ciprofloxacin tissue concentrations paralleled but were always lower than those of enrofloxacin for all time points and tissues except brain and kidney. These results, together with previously published pharmacokinetic data and known minimal inhibitory concentrations of common pathogenic bacteria, provide a strong evidence-based rationale for choosing enrofloxacin to treat infectious diseases in X. laevis.

Analgesic effects of tramadol, tramadol-gabapentin, and buprenorphine in an incisional model of pain in rats (Rattus norvegicus).

Postoperative pain management in laboratory animals relies heavily on a limited number of drug classes, such as opioids and nonsteroidal antiinflammatory drugs. Here we evaluated the effects of saline, tramadol, tramadol with gabapentin, and buprenorphine (n = 6 per group) in a rat model of incisional pain by examining thermal hyperalgesia and weight-bearing daily for 6 d after surgery. All drugs were administered preemptively and continued for 2 consecutive days after surgery. Rats treated with saline or with tramadol only showed thermal hyperalgesia on days 1 through 4 and 1 through 3 after surgery, respectively. In contrast, buprenorphine-treated rats showed no thermal hyperalgesia on days 1 and 2 after surgery, and rats given tramadol with gabapentin showed reduced thermal hyperalgesia on days 2 and 4. For tests of weight-bearing, rats treated with saline or with tramadol only showed significantly less ipsilateral weight-bearing on day 1 after surgery, whereas rats given either buprenorphine or tramadol with gabapentin showed no significant change in ipsilateral weight-bearing after surgery. These data suggest that tramadol alone provides insufficient analgesia in this model of incisional pain; buprenorphine and, to a lesser extent, tramadol with gabapentin provide relief of thermal hyperalgesia and normalize weight-bearing.

Serum clinical biochemical and hematologic reference ranges of laboratory-reared and wild-caught Xenopus laevis.

The South African clawed frogs Xenopus laevis and X. tropicalis are fully aquatic amphibians and well-established animal models. Because genetically engineered laboratory Xenopus are now being produced, the establishment of normal reference ranges for serum biochemical and hematologic parameters is essential for phenotyping and as a diagnostic aide. We determined normal reference ranges for hematologic values from 3 populations of X. laevis: wild-caught frogs (n = 43) and frogs from 2 commercial sources (A, n = 166; B, n = 109). For serum biochemistry, we determined normal reference ranges for frogs from source A and wild-caught frogs divided by sex and season. Significant differences across populations were found in WBC and RBC counts, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin concentration, and mean corpuscular volume. Among serum biochemical analytes, significant differences were found for albumin:globulin ratio, anion gap, and concentrations of albumin, globulin, total protein, lipase, alanine transaminase, γ-glutamyl transpeptidase; creatine phosphokinase; indirect, direct, and total bilirubin; cholesterol, low-density lipoprotein lipase, carbon dioxide, glucose, lactacte dehydrogenase, calcium, chloride, and sodium. We hypothesize that these differences can be attributed to differences in water quality, habitat, ambient temperature, diet, sex, recent transport or shipment, and genetic background. However, testing that hypothesis is beyond the scope of the current study. In addition, clinical chemistry and hematologic reference range values Xenopus laevis are quite distinct from those for other species and are most consistent with the only values published for another fully aquatic amphibian, the Eastern hellbender (Cryptobranchus alleganiensis).

Comparison of Gastrografin to barium sulfate as a gastrointestinal contrast agent in red-eared slider turtles (Trachemys scripta elegans).

Red-eared slider turtles (Trachemys scripta elegans) commonly develop intestinal obstruction. The gastrointestinal transit time in turtles tends to be longer than in other animals, making a rapid diagnosis of obstruction difficult. Fifteen red-eared sliders were given either Gastrografin or 30% w/v barium sulfate orally to compare ease of administration, transit time, and image quality. Each contrast medium was easy to administer but barium sulfate had to be administered more slowly (mean = 40s) than Gastrografin (mean = 20s) to prevent regurgitation. The mean transit and emptying time of Gastrografin was at least 9 h faster than barium sulfate at all time points except gastric transit. Both contrast media had a smooth, uniform appearance that outlined the mucosa with well-defined margins within the stomach and proximal small intestine. Dilution of Gastrografin occurred as it progressed through the intestines, resulting in decreased opacity in the distal small intestine and colon. Pre-administration packed cell volume and total serum protein levels of four turtles receiving Gastrografin were compared with levels at 24-, 96-, and 168-hours postadministration as well as to four control turtles not receiving contrast medium. Packed cell volume and total serum protein levels did not significantly differ among the Gastrografin and control group. From a clinical perspective, administration of Gastrografin allows for quicker results with only minor hematologic changes in red-eared sliders, but visualization of this contrast medium in the lower gastrointestinal tract may be insufficient for an accurate diagnosis.

The pharmacokinetics of enrofloxacin in adult African clawed frogs (Xenopus laevis).

Pharmacokinetics of enrofloxacin, a fluoroquinolone antibiotic, was determined in adult female Xenopus laevis after single-dose administration (10 mg/kg) by intramuscular or subcutaneous injection. Frogs were evaluated at various time points until 8 h after injection. Plasma was analyzed for antibiotic concentration levels by HPLC. We computed pharmacokinetic parameters by using noncompartmental analysis of the pooled concentrations (naive pooled samples). After intramuscular administration of enrofloxacin, the half-life was 5.32 h, concentration maximum was 10.85 µg/mL, distribution volume was 841.96 mL/kg, and area under the time-concentration curve was 57.59 µg×h/mL; after subcutaneous administration these parameters were 4.08 h, 9.76 µg/mL, 915.85 mL/kg, and 47.42 µg×h/mL, respectively. According to plasma pharmacokinetics, Xenopus seem to metabolize enrofloxacin in a manner similar to mammals: low levels of the enrofloxacin metabolite, ciprofloxacin, were detected in the frogs' habitat water and plasma. At necropsy, there were no gross or histologic signs of toxicity after single-dose administration; toxicity was not evaluated for repeated dosing. The plasma concentrations reached levels considered effective against common aquatic pathogens and suggest that a single, once-daily dose would be a reasonable regimen to consider when treating sick frogs. The treatment of sick frogs should be based on specific microbiologic identification of the pathogen and on antibiotic susceptibility testing.