The assessment of xenogeneic bone immunotoxicity and risk management study.

BACKGROUND: Xenogeneic bone has been widely used in a variety of clinical bone-related disease to promote bone healing and restore bone defects. However, the adverse effects of immune system limit its application in the clinic. The aim of this study was to evaluate xenogeneic bone safety of immunotoxicity and explore the methods for immune risk supervision. RESULTS: Xenogeneic bone, which is freeze-dried bovine cancellous bone, was implanted into the muscle of mice. On day 7, 14 and 28, the effects of xenogeneic bone were examined on humoral immunity and cellular immunity, including the levels of IgG, IgM, C3, inflammatory factors (TNF-α, IL-6), alkaline phosphatase (ALP) and the lymphocyte phenotype. The data showed that xenogeneic bone implantation had no potential to induce immune responses not only in humoral immunity but also in cellular immunity. To reveal the risk of immunogenicity, the residual DNA and the clearance of α-gal epitope were analyzed in 2 different bones (bone 1 is deproteinized bone, bone 2 is acellular and defatted bone). It was suggested that DNA of xenogeneic bone can be limited to < 50 ng per mg dry weight for the repair or regeneration with the acceptable immune risk. And α-gal clearance of xenogeneic bone could be an effective risk factor for improving xenograft quality management. CONCLUSIONS: Through the detection of xenogeneic bone immunotoxicity, our findings indicated that the supervisions of risk factors could contribute to reduce the immune risk. And the risk factors under the acceptable limitation could decrease or replace animal experiment. However, it still needs to be studied on the limitation of α-gal epitope to predict rejection of xenogeneic bone more accurately.

Mechanism of remodeling and local effects in vivo of a new injectable cosmetic filler.

By studying the local effects of a new type of injectable cosmetic filler implanted into the animal to explore the mechanism of remodeling and cosmetic effect of this kind of product. Take 12 rabbits and select 4 implantation points on both sides of the spine, respectively, and implant the test sample (PLLA) and negative control sample (HDPE) into the subcutaneous tissues on both sides. In the same way, take another 12 rabbits and implant the marketing control sample (cross-linked sodium hyaluronate) and negative control sample (HDPE) into the subcutaneous tissues on both sides. The animals were executed at 1 week, 4 weeks, 13 weeks and 52 weeks respectively, and Hematoxylin-Eosin staining, Masson trichrome staining and immunofluorescence staining were performed to characterize the local effects in vivo and the expression of type I collagen (Col. I), type III collagen (Col.III) and matrix metalloproteinase 9 (MMP-9). Good histocompatibility of the test sample and the marketing control sample were found. The foreign body reaction of marketing control sample was more intense than that of the test sample after 13 weeks. The foreign body reaction of testing sample was more intense after 52 weeks, while that of the marketing control sample was more stable. With the process of tissue repair, the collagen fibers of test samples and marketing control samples gradually increased after implantation. Type I collagen was mainly found inside the fiber capsule, while type III collagen was mainly found outside. The positive expression of matrix metalloproteinase 9 gradually increased, the positive expression of test samples increased significantly after 52 weeks, while that of marketing control samples did not change significantly. Good histocompatibility of PLLA filler is found. Matrix metalloproteinase 9 participates in foreign body reaction and collagen formation, which can reflect the process of tissue remodeling.

[Introduce a new vitro replacement method of skin irritation test].

A series of new replacement methods of skin irritation test such as EpiSkin, EpiDermSIT (updated) and SkinEthicRHE have been validated by ECVAM. Due to it, animals are protected to the full extent. These provide more methods for biological evaluation of medical devices.

Dose- and time-dependent systemic adverse reactions of sodium carboxy methyl cellulose after intraperitoneal application in rats.

Sodium carboxy methyl cellulose (SCMC) is an important absorbable biomaterial for anti-adhesion and hemostasis medical devices used in the abdominal cavity. However, the systemic toxicity of SCMC following intraperitoneal route has not been revealed sufficiently. Three SCMC solutions with gradient concentrations were intraperitoneally injected into 3 groups of rats with the doses of 50 mg/kg, 320 mg/kg and 2000 mg/kg respectively all at once to observe the dose-dependence of systemic reactions of SCMC and 10 rats (5 rats per sex) of each group were sacrificed 3 days, 7 days, 28 days and 90 days after injection to evaluate the time-dependence of the reactions. A range of adverse effects were shown in rats of the high-dose group which were found varied with time extending and virtually disappeared 90 days after injection. Slight reactions were observed in the medium-dose group while negligible effects were found in the low-dose group. The intraperitoneal application of SCMC can induce reversible systemic adverse effects to rats at the dose higher than 320 mg/kg and it is essential to take both dose- and time-dependent effects into account while designing a systemic toxicity study for absorbable biomaterials.

[The subchronic toxicity study on the biological amnion].

As the new type cornea ulcer renovation material, the biological amnion is to be implanted into the human body for a long time, a subchronic toxicity study in rats is made to evaluate its possibility of subchronic toxicity. The study is based on the requirements of "Biological Evaluation of Medical Devices, Part 11: Tests for systemic toxicity and Part 6: Tests for local effects after implantation". After the implantation of examples to be tested, animals were observed daily for mortality and 92 days later the possible subchronic toxicity was evaluated. And a necropsy was conducted and the selected organs were excised, weighed, and processed histologically. Body weights, organ weights, organ/body weight ratios, hematology values and clinical chemistry values were analyzed statistically. Results show that daily clinical observation, body weights, necropsy findings, organ weights and organ/body weight ratios were within acceptable limits in test and control treatment groups. There were no obvious changes in histopathology, hematology values or clinical chemistry values in either male or female rats and no notable differences between the biological amnion and the control amnion. This study proves that, the cornea ulcer renovation material, the biological amnion does not induce subchronic toxicity.

Biological evaluation of the copper/low-density polyethylene nanocomposite intrauterine device.

Devices and materials intended for clinical applications as medical and implant devices should be evaluated to determine their biocompatibility in physiological systems. This article presents results from cytotoxicity assay of L929 mouse fibroblasts culture, tests for skin irritation, intracutaneous reactivity and sensitization, and material implantation tests for the novel copper/low-density polyethylene nanocomposite intrauterine device (nano-Cu/LDPE IUD) with potential for future clinical utilization. Cytotoxicity test in vitro was conducted to evaluate the change in morphology, growth and proliferation of cultured L929 mouse fibroblasts, which in vivo examination for skin irritation (n = 6) and intracutaneous reactivity (n = 6) were carried out to explore the irritant behavior in New Zealand White rabbits. Skin sensitization was implemented to evaluate the potential skin sensitizing in Hartley guinea pigs (n = 35). The materials were implanted into the spinal muscle of rabbits (n = 9). The cytotoxicity grade of the nano-Cu/LDPE IUD was 0-1, suggested that the composite was nontoxic or mildly cytotoxic; no irritation reaction and skin sensitization were identified in any animals of specific extracts prepared from the material under test; similarly to the control sides, the inflammatory reaction was observed in the rabbits living tissue of the implanted material in intramuscular implantation assay. They indicated that the novel composite intrauterine device presented potential for this type of application because they meet the requirements of the standard practices recommended for evaluating the biological reactivity. The nano-Cu/LDPE IUD has good biocompatibility, which is biologically safe for the clinical research as a novel contraceptive device.