Histological structure of the trabecular meshwork in the eyeball: challenging the traditional concept and preliminary findings in rabbits, rats and mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To verify that the trabecular meshwork (TM) in the wall of the eyeball consists of smooth muscle fibers instead of collagen fibers or endothelial cells.</p><p><b>METHODS</b>Eighteen fresh eyeballs from 3 rabbits, 3 SD rats and 3 mice were sectioned along the sagittal plane and sliced after paraffin embedding for HE staining, VG staining, Masson staining, α-SMA immunohistochemistry or CD31 immunohistochemistry. These slices were observed under microscope and the structure of the TM was compared with those of scleral collagen fibers, ciliary muscles and endothelial cells.</p><p><b>RESULTS</b>HE staining of the eyeball slices from the 3 animal species resulted in purplish red staining of the TM, which was highly consistent with ciliary muscle fibers. The cell?like structures on the surface of the TM were not clearly outlined, with flat nuclei showing a dark purple staining; these structures did not show obvious boundaries from the TM. Ciliary muscle fibers, which were smooth muscle cells in nature, were aligned in bundles in various directions. The longitudinally sectioned cells were flat and contained purplish cytoplasm and highly flattened nuclei. Scleral collagen fibers were stained dark red with a few fibroblasts sandwiched among them. The long axis of the fibroblasts was in parallel with that of the collagen fibers. The outline of the fibroblast was not clear and the nucleus was flat in dark blue. The vascular endothelial cells presented with different morphologies and contained light purplish cytoplasm and dark nuclei, protruding into the vascular cavity. VG staining of the TM revealed a pale red filamentous structure, and the collagen fibers were stained bright red. Masson staining of the TM showed a reticular structure consisting mainly of dark red fibers intermingled with thin green fibers. Scleral collagen fibers presented with a cord?like green wavy structure. The endothelial cells were green and flat, while the ciliary smooth muscle fibers were purple. In immunohistochemistry for α?SMA, the TM and the ciliary smooth muscle fibers showed a strong positivity in the cytoplasm, while the scleral collagen fibers and vascular endothelial cells showed negative staining; immunohistochemistry for CD31 showed no obvious positive staining in the TM, collagen fibers or ciliary smooth muscle cells from all the animals in spite of slight differences among them.</p><p><b>CONCLUSION</b>The TM consists mainly of smooth muscle fibers with a thin layer of peripheral endomysium without endothelial cells.</p>

A two-step protocol for induction of rat adipose tissue-derived stem cells into neuron-like cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the differentiation potential of rat adipose tissue-derived cells (ADSCs) into neuron-like cells in vitro using a two-step induction protocol.</p><p><b>METHODS</b>ADSCs isolated from the epididymal fat pads in male SD rats by means of differential attachment were cultured in vitro and subjected to adipogenic induction. After flow cytometric identification of the cell surface antigens CD106, CD11b, CD45, CD49d, CD90 and CD29, the third-passage ADSCs were induced to transdifferentiate into neural stem cell (NSC)-like cells in DMEM/F12 medium containing 10 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF) and 2% B27. The resultant NSC-like cells were then induced to differentiate into neuron-like cells in the neurobasal medium containing 10 ng/ml brain-derived neurotrophic factor (BDNF), 10 ng/ml glial cell line-derived neurotrophic factor (GDNF) and 1 µmol/L retinoic acid (RA). Immunocytochemistry was employed to identify the expression of the cell surface markers nestin, MAP2 and NeuN.</p><p><b>RESULTS</b>The isolated ADSCs were positive for CD90 and CD29, and oil red O staining of the induced adipose-like cells yielded positive results. The third-passage ADSCs induced for 7 days aggregated as floating cell spheres positive for NSC surface antigen nestin. Further induction in neurobasal medium for 4 h resulted in adhesion of the cell spheres and the formation of cell processes extending from some peripheral cells, suggesting a morphological resemblance to neurons. Most of the cells showed positivity for MAP2 and NeuN.</p><p><b>CONCLUSION</b>ADSCs can be induced to differentiate into neuron-like cells in vitro under appropriate conditions.</p>

Induced differentiation of rat adipose-derived stem cells into Schwann-like cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the differentiation potential of rat adipose-derived stem cells (ADSCs) into Schwann-like cells in vitro.</p><p><b>METHODS</b>ADSCs isolated from adult SD rats were cultured in vitro and identified with the cell surface antigens CD44, CD49d and CD106 by immunocytochemistry. The ADSCs of the sixth to eighth passages were inoculated in polylysine-coated culture plate and cultured for 12 days in DMEM/F12 culture medium containing 10% fetal bovine serum, 5 ng/ml platelet-derived growth factor, 10 ng/ml basic fibroblast growth factor, 14 micromol/L Forskolin and 200 ng/ml Heregulin to induce their differentiation in vitro. Immunocytochemistry was performed to identify the expression of the cell surface markers nestin, glial fibrillary acidic protein (GFAP), S-100, and P75.</p><p><b>RESULTS</b>The isolated and purified ADSCs were positive for CD44 and CD49d expressions but negative for CD106. After 12 days of culture in the conditional culture medium, most of the cells showed positive expressions of GFAP, S-100, and P75, the specific protein markers of Schwann cells.</p><p><b>CONCLUSION</b>Adult rat ADSCs are confirmed to have potentials of neuroglial differentiation and capable of differentiating into Schwann-like cells in vitro.</p>

Biological dressing with human hair keratin-collagen sponge-poly 2-hydroxyethyl methacrylate composite promotes burn wound healing in SD rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a composite material containing human hair keratin (HHK), collagen sponge (inner layer) and poly 2-hydroxyethyl methacrylate (PHEMA) film that allows sustained release of polydatin and test its effect as a biological dressing in promoting burn wound healing in SD rats.</p><p><b>METHODS</b>Three HHK materials with fast, moderate, and low degradation rates were mixed at the ratio of 4:3:3 to prepare a reticular structure, which was processed into a composite material with bovine tendon-derived collagen sponge, and further complexed with HEMA film containing PD prepared by polymerization. Degree II burn wound was induced in SD rats by scalding and within postburn day 2-5, the wounds were cleansed and covered with the composite material or with glutaraldehyde-treated porcine skin (positive control). At week 1, 2, 4, 6 and 8 following wound dressing, 6 full-thickness skin samples were harvested from the wounds for histological observation and immunohistochemical detection of collagen and elastic fibers, and the wound healing time and healing rate were recorded.</p><p><b>RESULTS</b>The prepared collagen sponge film was transparent and porous (50-300 microm in diameter) and allowed sustained PD release into normal saline within 48 h. Compared with the porcine skin, the composite material reduced exudation and maintained ideal moisture of the wound, and significantly shortened the wound healing time (P=0.000). On day 7, 14, and 21 following dressing, the composite material and porcine skin significantly increased the wound healing rate as compared with the negative control group (P=0.000), and on day 14, the composite achieved significantly greater healing rate than the porcine skin (P<0.05).</p><p><b>CONCLUSION</b>HHK-collagen sponge-PHEMA/PD composite as a dressing material promotes burn wound healing in rats by allowing in vivo construction of tissue engineered epidermis. PHEMA is feasible for sustained drug delivery in this composite.</p>

Preliminary study of chicken calamus conduit as a scaffold material for tissue engineering / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the unique structural features of chicken calamus keratin (CCK) conduit as a candidate scaffold material for tissue engineering and its in vivo degradation and histocompatibility after its implantation into living tissues.</p><p><b>METHODS</b>Chicken calami were taken from healthy chickens and treated through sequential, controllable physical and biochemical procedures for preparation of three types of CCK conduits, namely CCK-I (mildly treated), CCK-II (moderately treated) and CCK-III (intensely treated). Light microscopy (LM) and scanning electron microscopy (SEM) were performed for morphological observation. Each of these three types of CCK pieces (experimental group) and the untreated ones (control group) was implanted into the dorsal muscular tissue on both sides of SD rats, respectively. Routine tissue sectioning and HE stain were performed to identify the morphological changes under light microscope. Each of the CCK threads (experimental group) and the untreated chicken calamus threads (control group) was also grafted within the sciatic nerve bundles of SD rats, respectively.</p><p><b>RESULTS</b>The wall of the chicken calamus was composed of 4 compact parts from inside to outside on cross sections, namely the innermost basophilic homogenous coarse line, 3-5 layers of acidophilic corneum, 60-100 layers of circular keratin tracts containing massive pigment granules, and 10-20 outmost layers of keratin tracts with only a few pigment granules. The three-dimensional surface features of chicken calamus identified by SEM, as compared with untreated chicken calamus, was characterized by loose arrangement containing horizontal and vertical keratins with obvious pores of different sizes and depths on its surface. At 8 weeks after implantation into the muscular tissue in experimental groups, the CCK grafts were degraded into thin filaments or/and dispersed pieces and fine granules with the appearance of blood vessels, which facilitated the absorption of the degradation products; at 12 weeks, the grafts were markedly degraded into tiny fragments. In the control group, in contrast, the grafts remains intact throughout the experiment. After implantation of the material into the nerve bundles, similar cell infiltration and tissue responses to the grafts were observed as compared to those occur in intramuscular grafting. The degradation products did not seem to cause nerve tissue degeneration or necrosis.</p><p><b>CONCLUSIONS</b>Fresh chicken calamus is a natural tube composed of multi-layered compact keratin tracts with pigment granules and small amount of matrix, and is non-absorbable in vivo, and therefore does not favor the purpose for use directly as a candidate biological scaffold. After proper treatment, the chicken calamus becomes loosely arranged porous material, and can be degraded and absorbed in vivo without resulting in tissue degradation or necrosis, suggesting its potential for applications in tissue engineering.</p>

Changes of Smoothened expression during retinofugal pathway development in mouse embryos / 南方医科大学学报

<p><b>OBJECTIVE</b>To examine the change of Smoothened (Smo) expression in the retinofugal pathway and in the growth cones during the period of embryonic day 13 (E13) to E15.</p><p><b>METHODS</b>Smo expression in the chiasm and growth cones was observed by fluorescent immunostaining and retinal explant culture.</p><p><b>RESULTS</b>On E13 and E14, Smo was expressed moderately in the retina and optic disc, and in the corner of the retina, Smo expression was especially dense. On E13, Smo expression was detected in the optic nerves and ventral diencephalon, but only in the superficial region of the optic tract on E14. Smo was also detected in the stem and filopodia of the growth cones in the retinal explant culture during this period.</p><p><b>CONCLUSION</b>Smo expression changes in different developmental phases, suggesting that Smo might play a role in signal optic axon growth during the development of the retinofugal pathway.</p>

From meridians and acupoints to self-supervision and control system: a hypothesis of the 10th functional system based on anatomical studies of digitized virtual human / 南方医科大学学报

Chinese acupuncture and moxibustion has been widely accepted as a useful therapeutics all over the world, but its mechanism has not been fully defined. For this purpose, a reticular framework of whole-body fascia and connective tissues has been established by means of digitized virtual human technique. The virtual acupoints represented three-dimensionally were compared with the sites for stimulation in practice of traditional Chinese medicine (TCM) acupuncture therapy. The results showed that the fascial network constituted by the connective tissues may be the anatomical basis for acupuncture therapy. We found that the acupoints were mainly located where thick connective tissues were present. In this fascial network, sensitive nerve endings, active cells and lymphatic vessels abounded in the sites with thick connective tissue, and needling at these sites induced definite biological effects. In light of biological phylogeny and embryo development, we believe that the connective tissue network may constitute a new functional system in the human body, the Self-supervision and control system (Fasciology), which provides a theoretical base for acupuncture therapy.

Collagen gel coating or cyclosporine A for improving histocompatibility of chicken calamus keratin / 南方医科大学学报

<p><b>OBJECTIVE</b>To improve the histocompatibility of chicken calamus keratin (CCK) graft by collagen-gel coating or using of cyclosporine A (CsA).</p><p><b>METHODS</b>Thirty SD rats were equally randomized into 5 groups, and in 4 of them, CCK implantation into the bilateral erector spinae was performed on different treatment protocols. In group A, the rats received daily intraperitoneal injection of CsA (5 mg/kg) for two consecutive weeks after CCK implantation; in group B, CCK was soaked in CsA (2.5 mg/ml) solution at 4 degrees Celsius; for 48 h before grafting; in group C, CCK coated with collagen gel was grafted; and in group D, only CCK was implanted. Rats in the fifth group received only cutaneous incision as well as muscular dissection to serve as the blank control. CCK degradation and its effect on the surrounding tissues were observed at 2, 4 and 8 weeks after grafting. Immunohistochemistry was performed to identify T lymphocyte infiltration in the host tissues.</p><p><b>RESULTS</b>All the rats survived the operation. Numerous macrophages, especially multinucleated giant cells occurred on the peripheral of the CCK grafts, and small degraded CCK pieces were observed in their cytoplasm. Only a few inflammatory cells were seen in the host tissues. At 2, 4 and 8 weeks after CCK implantation, only a few CD3-positive cells were found in all the groups, and in group A and B, the density of T lymphocytes was significantly lower than that in group D, and there was no significant difference between group A and the blank control group.</p><p><b>CONCLUSIONS</b>CsA significantly improves the histocompatibility of CCK material, and short-term systemic CsA administration achieves the best results. Macrophages, especially multinucleated giant cells participate in CCK degradation in vivo.</p>

Toxicity evaluation of chicken calamus keratin conduit as a tissue-engineering scaffold biomaterial / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the toxicity of chicken calamus keratin (CCK) conduit as a tissue-engineered scaffold material.</p><p><b>METHODS</b>The chemical composition of the leaching solution of CCK was determined by means of ultraviolet spectrometry, and the toxic effects of the solution was evaluated by skin sensitization test in rats, intracutaneous stimulation test in rabbits, acute systemic toxicity test in mice, and cytotoxicity test in L929 cells.</p><p><b>RESULTS</b>The leaching solution of CCK consisted mainly of middle-molecular-weight peptides with a small quantity of macromolecular proteins. Skin sensitization test in rats showed that application of the CCK leaching solution caused no obvious skin reddening, regional edema, or skin necrosis. Intracutaneous injection of the leaching solution in rabbits did not induce obvious skin stimulation manifested by intradermal erythema or edema. In acute systemic toxic test, administration of the leaching solution in mice caused no death, organ dysfunction, cyanosis, tremor, severe peritoneal irritation, ptosis, or dyspnoea. In vitro cytotoxicity test indicated that the cell toxicity of the CCK leaching solution was approximately at 0 level.</p><p><b>CONCLUSION</b>CCK contained in the treated chicken calamus easily undergoes hydrolysis to release mainly some peptides which do not induce obvious toxic effects, suggesting the safe potential applications of CCK conduit as a tissue-engineering biomaterial.</p>

Preparation and bioactivity of human hair keratin-collagen sponge, a new type of dermal analogue / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a three-dimensional porous film of human hair keratin (HHK)-collagen sponge complex for use as a dermal substitute.</p><p><b>METHODS</b>The three components F, B, and Z derived from healthy human hair were weaved into a meshwork and integrated with purified soluble type I collagen extracted from bovine tendons to prepare a highly porous film with vacuum freeze-drying followed by secondary cross-linking with glutaraldehyde. The film was grafted beneath the dorsal skin in 21 SD rats (experimental group), with simple collagen sponge serving as the negative control. The rats receiving surgical operation but without graft served as the blank control. The graft and its surrounding tissue were harvested on days 3, 7 and at weeks 2, 4, 6, 8, 12 after implantation for evaluation of tissue compatibility, vascularization and degradation.</p><p><b>RESULTS</b>The prepared collagen sponge film was semitransparent and porous. Three to 7 days after grafting, inflammatory reaction was relieved gradually, and several fibroblasts and blood vessels were found adherent to the grafts in the experimental groups. At week 4, the wounds healed in the experimental groups, and the fibroblasts were actively secreting collagen and the film degraded obviously with the appearance of elastic fibers. At weeks 6 and 8, new collagen fibers thickened and assumed regular arrangement, and the collagen sponge films disappeared completely. In the control groups, the changes were less obvious and total HHK degradation occurred till week 12.</p><p><b>CONCLUSION</b>The degradable and absorbable HHK-collagen sponge film has relatively satisfactory tissue compatibility and can accelerate wound healing by stimulating cell proliferation and vascularization, showing the potential as an optimal dermal substitute.</p>

Changes of retinofugal pathway development in mouse embryos after Sonic hedgehog antibody perturbation / 南方医科大学学报

<p><b>OBJECTIVE</b>To understand the function of Sonic hedgehog in chiasm development in mouse embryos of embryonic day 13 (E13) to E15.</p><p><b>METHODS</b>Brain slices of E13-E15 mouse embryos containing the optic pathway from the eyes to the optic tract were prepared and cultured in DMEM/F12 in the presence of 10% fetal bovine serum at 37 degrees in a rolling incubator for 5 h. The antibody to Shh was added into the culture medium of the slices in the treatment group, while no additional chemical or only normal mouse IgG was added in the control groups. After culture, the brain slices were fixed and a DiI granule was inserted into the optic disc in one eye. Seven days later, the tissue overlying the chiasm was removed to expose the DiI-labeled chiasm for observation under confocal microscope, and the images were analyzed by METAMORPH software.</p><p><b>RESULTS</b>Shh antibody treatment produced a reduction of crossing of the earliest retinal axons at the midline of E13 chiasm, and the uncrossed axons were also influenced by Shh antibody at E15.</p><p><b>CONCLUSION</b>Shh executes a transient but important function in axon decussation in the early stage of mouse optic chiasm development and signals axon turning in the later stage.</p>

Ooplasmic transfer: problems and prospects / 南方医科大学学报

Cytoplasmic transfer between human oocytes, which represents a complete cytoplasmic exchange, has been performed recently as a means to improve the outcome of assisted reproduction and becomes a hotspot of researches. Many studies have indicated that mitochondria in the oocytoplasm obviously affect fertilization of the oocytes and early embryo development. However, ooplasmic transfer can lead to mitochondrial DNA heteroplasmy and the prospect of mitochondrial heteroplasmy and its potential problems necessitate further studies. The authors reviews the ooplasmic transfer, the relation between ooplasm and fertilization and embryo development, and the mitochondrial heteroplasmy. The authors also propose a new theory of "reverse cloning technique".