[Preparation and bone repair capability of a new plastic bone filler material].

Objective: To prepare a new plastic bone filler material with adhesive carrier and matrix particles derived from human bone, and evaluate its safety and osteoinductive ability through animal tests. Methods: The human long bones donated voluntarily were prepared into decalcified bone matrix (DBM) by crushing, cleaning, and demineralization, and then the DBM was prepared into bone matrix gelatin (BMG) by warm bath method, and the BMG and DBM were mixed to prepare the experimental group's plastic bone filler material; DBM was used as control group. Fifteen healthy male thymus-free nude mice aged 6-9 weeks were used to prepare intermuscular space between gluteus medius and gluteus maximus muscles, and all of them were implanted with experimental group materials. The animals were sacrificed at 1, 4, and 6 weeks after operation, and the ectopic osteogenic effect was evaluated by HE staining. Eight 9-month-old Japanese large-ear rabbits were selected to prepare 6-mm-diameter defects at the condyles of both hind legs, and the left and right sides were filled with the materials of the experimental group and the control group respectively. The animals were sacrificed at 12 and 26 weeks after operation, and the effect of bone defect repair were evaluated by Micro-CT and HE staining. Results: In ectopic osteogenesis experiment, HE staining showed that a large number of chondrocytes could be observed at 1 week after operation, and obvious newly formed cartilage tissue could be observed at 4 and 6 weeks after operation. For the rabbit condyle bone filling experiment, HE staining showed that at 12 weeks after operation, part of the materials were absorbed, and new cartilage could be observed in both experimental and control groups; at 26 weeks after operation, the most of the materials were absorbed, and large amount of new bone could be observed in the 2 groups, while new bone unit structure could be observed in the experimental group. Micro-CT observation showed that the bone formation rate and area of the experimental group were better than those of the control group. The measurement of bone morphometric parameters showed that the parameters at 26 weeks after operation in both groups were significantly higher than those at 12 weeks after operation ( P<0.05). At 12 weeks after operation, the bone mineral density and bone volume fraction in the experimental group were significantly higher than those in the control group ( P<0.05), and there was no significant difference between the two groups in trabecular thickness ( P>0.05). At 26 weeks after operation, the bone mineral density of the experimental group was significantly higher than that of the control group ( P<0.05). There was no significant difference in bone volume fraction and trabecular thickness between the two groups ( P>0.05). Conclusion: The new plastic bone filler material is an excellent bone filler material with good biosafety and osteoinductive activity.

Theoretical Study on the Aggregation of Copper Clusters on a Liquid Surface.

The ground state structures of copper clusters with different sizes along with their aggregation have been systematic investigated using Amsterdam Density Functional (ADF) and Atomistix ToolKit (ATK) programs. On the basis of geometry optimization, some Cu clusters with more stable structures which were not reported previously have been revealed. In most cases, these Cu clusters prefer to adopt icosahedral structures which originate from the 13-atom icosahedron. It has also been demonstrated that the interaction between two Cu clusters is anisotropic, which is attributed to their charge distribution, especially the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of Cu clusters. Moreover, we have carried out the simulation of Cu clusters aggregation on the silicone oil substrate by means of Monte Carlo (MC) method, which shows good consistence with our previous experimental studies.

Impact of Graphene Work Function on the Electronic Structures at the Interface between Graphene and Organic Molecules.

The impact of graphene work function (WF) on the electronic structure at the graphene/organic interface has been investigated. WF manipulation of graphene is realized using self-assembled monolayers (SAMs) with different end groups. With this method, the upper surface of the functionalized graphene remains intact, and thus precludes changes of molecular orientation and packing structures of subsequently deposited active materials. The WF of NH2-SAM functionalized graphene is ~3.90 eV. On the other hand, the WF of graphene increases to ~5.38 eV on F-SAM. By tuning the WF of graphene, an upward band bending is found at the ZnPc/graphene interface on F-SAM. At the interface between C60 and NH2-SAM modified graphene, a downward band bending is observed.

Effects of FSH and the weather during induced ovulation and timed artificial insemination to increase jenny conception rates.

Experiments were conducted to improve jenny conception rates through induced ovulation and timed insemination. Jennies in control, hCG and LH groups were injected intramuscularly with saline alone or saline containing hCG or LH, respectively, when the dominant follicle diameter reached 35 mm. Then, follicle development was checked every 8 h until the dominant follicle ovulated. While 76% of the hCG-treated jennies ovulated between 24 and 48 h, and 84% of the LH-treated ovulated between 24 and 40 h after injection, ovulations in control jennies scattered over an extended period after injection. Conception rates after insemination were significantly higher in LH- or hCG-treated jennies than in the conventionally-bred jennies. The LH preparation used in this study contained more FSH than the hCG preparation did, and supplementing the hCG treatment with FSH significantly improved ovulation synchronization. Ovulations in jennies treated on rainy days were significantly postponed and less synchronized compared to those in jennies treated on sunny days. Together, the results suggested that jenny conception could be significantly improved by inducing ovulation with LH or hCG treatment followed by timed insemination and that FSH and the weather during treatment had profound effects on ovulation induction of jennies.

[Biocompatibility research of true bone ceramics].

Objective: To investigate the biocompatibility of true bone ceramic (TBC) and provide experimental basis for clinic application. Methods: TBC was prepared from healthy adult bovine cancellous bone by deproteinization and high temperature calcinations. Mouse fibroblast cell line (L929 cells) were cultured with the leaching liquor of TBC in vitro, and the cytotoxicity was evaluated at 2nd, 4th, and 7th days. L929 cells were inoculated into the TBC and cultured for 4 days. The cell adhesion and proliferation on the surface of the TBC were observed by scanning electron microscopy, and evaluated the cell compatibility of TBC. Ten New Zealand white rabbits were divided into 2 groups, and drilled holes at the tibia of both hind limbs. TBC and hydroxyapatite (HA) were implanted into the left side (experimental group) and the right side (control group), respectively. And the biocompatibility of TBC was evaluated by general observation and histological observation at 4 and 26 weeks after implantation. Results: Cytotoxicity test showed that the cytotoxicity level of leaching liquor of TBC was grade 0-1. Cell compatibility experiments showed that the L929 cells adhered well on the surface of TBC and migrated into the pores. The implantation test in vivo showed that experimental group and control group both had mild or moderate inflammatory response at 4 weeks, and new bone formation occurred. At 26 weeks, there was no inflammatory reaction observed in both groups, and new bone formation was observed in varying degrees. Conclusion: TBC have good biocompatibility and can be used to repair bone defect in clinic.

GS/DBM/PLA porous composite biomaterial for the treatment of infective femoral condyle defect in rats.

A bone defect resulting from open bone trauma may easily become infected; however, the administration of efficacious systemic antibiotics cannot be performed at safe levels. Previous studies have investigated anti-infective biomaterials that incorporate into bone and facilitate the direct application of high-concentration local antibiotics. In the present study, the effect of a novel porous composite with gentamicin sulfate (GS) in treating infected femoral condyle defects was investigated using a rat model. A novel porous composite biomaterial was prepared based on a supercritical carbon dioxide fluid technique that combined GS, demineralized bone matrix (DBM) and polylactic acid (PLA). A rat femoral condyle fracture model of infection was established. The GS/DBM/PLA composite biomaterial was implanted and its physicochemical characteristics, biocompatibility and ability to facilitate repair of infected bone defect were assessed. The GS/DBM/PLA composite biomaterial maintained the antibiotic activity of GS, with good anti-compression strength, porosity and biocompatibility. The results of the animal experiments indicated that the GS/DBM/PLA composite biomaterial exerted marked anti-infective effects and facilitated bone defect repair, while simultaneously controlling infection. Porous GS/DBM/PLA is therefore a promising composite biomaterial for use in bone tissue engineering.

Tendon allograft sterilized by peracetic acid/ethanol combined with gamma irradiation.

BACKGROUND: Research and clinical applications have demonstrated that the effects of tendon allografts are comparable to those of autografts when reconstructing injured tendons or ligaments, but allograft safety remains problematic. Sterilisation could eliminate or decrease the possibility of disease transmission, but current methods seldom achieve satisfactory sterilisation without affecting the mechanical properties of the tendon. HYPOTHESIS: Peracetic acid-ethanol in combination with low-dose gamma irradiation (PE-R) would inactivate potential deleterious microorganisms without affecting mechanical and biocompatible properties of tendon allograft. STUDY DESIGN: Controlled laboratory design. METHODS: HIV, PPV, PRV and BVDV inactivation was evaluated. After verifying viral inactivation, the treated tendon allografts were characterised by optical microscopy, scanning electron microscopy and tensile testing, and the cytocompatibility was assessed with an MTT assay and by subcutaneous implantation. RESULTS: Effective and efficient inactivation of HIV, PPV, PRV and BVDV was observed. Histological structure and ultrastructure were unchanged in the treated tendon allograft, which also exhibited comparable biomechanical properties and good biocompatibility. CONCLUSION: The preliminary results confirmed our hypothesis and demonstrated that the PE-R tendon allograft has significant potential as an alternative to ligament/tendon reconstruction. CLINICAL RELEVANCE: Tendon allografts have been extensively used in ligament reconstruction and tendon repair. However, current sterilisation methods have various shortcomings, so PE-R has been proposed. This study suggests that PE-R tendon allograft has great potential as an alternative for ligament/tendon reconstruction. WHAT IS KNOWN ABOUT THIS SUBJECT: Sterilisation has been a great concern for tendon allografts. However, most sterilisation methods cannot inactivate viruses and bacteria without impairing the mechanical properties of the tendon allograft. WHAT THIS STUDY ADDS TO EXISTING KNOWLEDGE: Peracetic acid/ethanol with gamma irradiation can effectively inactivate viruses and bacteria. Meanwhile, tendon allografts sterilised by this method maintain their physiological tendon structure, biomechanical integrity and good compatibility.

The effects of different crossing-linking conditions of genipin on type I collagen scaffolds: an in vitro evaluation.

The purpose of this paper is to analyze the properties of fabricating rat tail type I collagen scaffolds cross-linked with genipin under different conditions. The porous genipin cross-linked scaffolds are obtained through a two step freeze-drying process. To find out the optimal cross-link condition, we used different genipin concentrations and various cross-linked temperatures to prepare the scaffolds in this study. The morphologies of the scaffolds were characterized by scanning electron microscope, and the mechanical properties of the scaffolds were evaluated under dynamic compression. Additionally, the cross-linking degree was assessed by ninhydrin assay. To investigate the swelling ratio and the in vitro degradation of the collagen scaffold, the tests were also carried out by immersion of the scaffolds in a PBS solution or digestion in a type I collagenase respectively. The morphologies of the non-cross-linked scaffolds presented a lattice-like structure while the cross-linked ones displayed a sheet-like framework. The morphology of the genipin cross-linked scaffolds could be significantly changed by either increasing genipin concentration or the temperature. The swelling ratio of each cross-linked scaffold was much lower than that of the control (non-cross-linked).The ninhydrin assay demonstrated that the higher temperature and genipin concentration could obviously increase the cross-linking efficiency. The in vitro degradation studies indicated that genipin cross-linking can effectively elevate the biostability of the scaffolds. The biocompatibility and cytotoxicity of the scaffolds was evaluated by culturing rat chondrocytes on the scaffold in vitro and by MTT. The results of MTT and the fact that the chondrocytes adhered well to the scaffolds demonstrated that genipin cross-linked scaffolds possessed an excellent biocompatibility and low cytotoxicity. Based on these results, 0.3 % genipin concentrations and 37 °C cross-linked temperatures are recommended.

Rapid development of tissue bank achieved by International Atomic Energy Agency (IAEA) Tissue Banking Programme in China.

Before 1986, the development of tissue banking in China has been slow and relatively uncoordinated. Under the support of International Atomic Energy Agency (IAEA), Tissue Banking in China experienced rapid development. In this period, China Institute for Radiation Protection tissue bank mastered systematic and modern tissue banking technique by IAEA training course and gradually developed the first regional tissue bank (Shanxi Provincial Tissue Bank, SPTB) to provide tissue allograft. Benefit from training course, SPTB promoted the development of tissue transplantation by ways of training, brochure, advertisement and meeting. Tissue allograft transplantation acquired recognition from clinic and supervision and administration from government. Quality system gradually is developing and perfecting. Tissue allograft transplantation and tissue bank are developing rapidly and healthy.

Porcine bone grafts defatted by lipase: efficacy of defatting and assessment of cytocompatibility.

Defatting is an important procedure for the preparation of bone grafts because lipids in bone grafts strongly influence the osteointegration. Lipases have been widely used in different fields. However, study on the application to defatting process for bone grafts preparation has never been found so far. In this study, bone samples were treated respectively by lipase, NaHCO(3)/Na(2)CO(3), acetone and deionized water. The lipids content of processed bone grafts was calculated in Soxhlet extractor method. Surface morphology of the bone grafts was observed under scanning electron microscope (SEM). DNA content of processed bone grafts was measured. Cytocompatibility was evaluated by co-culturing mouse preosteoblasts (MC3T3-E1) on defatted bone cubes. Proliferation rates of MC3T3-E1 were examined by cell counting kit-8 (CCK-8) assay. No statistically significant difference was found between lipids amount of bone processed by lipase (0.46 ± 0.16 %) and acetone (1.11 ± 0.13 %) (P > 0.05). Both of them were significantly lower than that in groups processed by Na(2)CO(3)/NaHCO(3) (3.46 ± 0.69 %) and deionized water (8.88 ± 0.18 %) (P = 0.000). Only cell debris were discovered over the surface of bone processed by lipase or acetone, while lipid droplets were observed on bone processed by Na(2)CO(3)/NaHCO(3) or water by SEM. The difference of DNA concentration between the bone processed by lipase (3.16 ± 0.81 ng/µl) and acetone (4.14 ± 0.40 ng/µl) is not statistically significant (P > 0.05). Both of them are significantly lower than that groups processed by Na(2)CO(3)/NaHCO(3) (5.22 ± 0.38 ng/µl) and water (7.88 ± 0.55 ng/µl) (P < 0.05). MC3T3-E1 cells maintained their characteristic spreading on the trabecular surfaces of bone processed by lipase. There were no statistically significant differences among absorbance of lipase, acetone groups in CCK-8 assay. The application of lipase to bone tissue defatting appears to be a very promising technique for bone grafts preparation.

The effect of non-growth factors on chondrogenic differentiation of mesenchymal stem cells.

Chondrogenic differentiation of mesenchymal stem cells (MSCs) in vitro usually requires the presence of growth factors in the culture condition. But many cost-effect methods can successfully fulfill this without addition of these cytokines. This article focuses upon the effect of non-growth factors on the chondrogenic differentiation of MSCs and the concise introduction of the potential mechanism of these methods.

[Recent progress of small intestinal submucosa in application research of tissue repair and reconstruction].

OBJECTIVE: To review the recent progress of the small intestinal submucosa (SIS) in application research of tissue repair and reconstruction. METHODS: The domestic and international articles on the SIS were reviewed and summarized. RESULTS: As a natural extracellular matrix, SIS has outstanding biological advantages, such as good mechanical property, tissue compatibility, and lower immunogenicity. SIS has been used to repair and reconstruct various types of tissue defects in animal models and clinical application, especially in the treatment of hernia, urinary system disease, and refractory skin trauma. The development of the tissue engineering technology expands the field of SIS repair and reconstruction and promotes the intensive study of SIS. However, the long-term effect of SIS in tissue repair and reconstruction still remains to be further observation, while the cell/SIS material construction by tissue engineering technology also needs more studies. CONCLUSION: SIS has a widely promising application future in the tissue repair and reconstruction.

First-principles study on stability and magnetism of Ni m Al n (m = 1-3, n = 1-9) clusters.

The investigation on the structures, stabilities, and magnetism of Ni(m) Al(n) (m = 1-3, n = 1-9) clusters has been made by using first principles. We found some new ground-state structures which had not been found before. These mixed species prefer to adopt three-dimensional (3D) structures starting from four atoms. All the ground-state structures for the Ni-Al clusters are different from those of the corresponding pure Al clusters with the same number of atoms except for three atoms. The Mulliken population analysis shows that some charges transfer from the Al atoms to the Ni atoms. NiAl n (n = odd number) cations, Ni(2)Al(6) neutral, Ni(2)Al(1) and Ni(3)Al cations and anions, and Ni(3)Al(5) anion have the magnetic moments of 2 µ B. The magnetic moments of NiAl(4) and NiAl(6) cluster neutrals and cations are 2 µ B and 3 µ B, respectively. All the other cluster neutrals and ions do not have any nontrivial magnetic moments. The 3d electrons in Ni atoms are mainly responsible for the magnetism of the mixed Ni-Al clusters.

[Experimental study on repair of skin defects by tissue engineered skin with isogeneic cells].

OBJECTIVE: To evaluate the effect of tissue engineered skin with isogeneic cells on repairing skin defects in inbred rat model so as to provide relevant evidences for the clinical application. METHODS: The skins of newborn inbred F344 rats were harvested and treated with Dispase trypsin to isolate the epidermal cells. The skins of adult Sprague Dawley rats were obtained and treated with hypertonic sodium-SDS-trypsin to prepare the acellular dermal matrix. The tissue engineered skin was reconstructed by submerging culturing and air-liquid interface culturing in vitro. The full-thickness skin defects of 1.5 cm x 1.5 cm in size were prepared along the dorsal both sides of 36 adult inbred F344 rats, and 72 defects were repaired with tissue engineered skin in experimental group (n=24), with allogeneic acellular dermal matrix in negative control group (n=24), and with autologous full-thickness skin in positive control group (n=24). Finally the gross observation, the survival rate, wound contraction rate, and histological observation were used to evaluate the effect. RESULTS: The wound healed by first intension at 4 weeks postoperatively in the experimental group; the grafts connected with the adjacent tissue tightly and had normal appearance. At 4 weeks after operation, the survival rate of the graft was 0 in the negative control group; the survival rates were 62.5% (15/24) in the experimental group and 91.7% (22/24) in the positive control group, showing significant difference between 2 groups (chi2=5.779, P=0.016). The wound contraction rates of the experimental group and positive control group were significantly lower than that of the negative control group (P < 0.05), but no significant difference was found between the experimental group and positive control group (P > 0.05). Histological observation showed that slight inflammation reaction appeared at 1 week postoperatively in the experimental group; the regeneration of the blood vessel and the proliferation of the fibroblasts in dermis and the gradual maturation of epidermis were observed at 2 weeks, and new collagen deposition and collagen remodeling in the dermis of the graft were found at 4 weeks postoperatively. CONCLUSION: The tissue engineered skin is able to repair full-thickness skin defect of rats effectively, it has similar effect to the autologous full-thickness skin in preventing the wound contraction and promoting the wound healing, which provides experimental evidences for the clinical application.

[Progress of malleable bone paste/putty].

OBJECTIVE: To review the progress and clinical application of malleable bone paste/putty. METHODS: Recent literature about malleable bone paste/putty was reviewed and analyzed. RESULTS: The preparation and clinical application of malleable bone paste/putty have become increasingly mature. Many kinds of malleable bone paste/putty have been applied extensively and the good clinical results have been achieved in the treatment of the irregular bone defects. The materials and methods for preparing malleable bone paste/putty are different. Then they have different bone repair abilities. CONCLUSION: Malleable bone paste/putty provides effective method to treat irregular bone defects. But the malleable bone paste/putty still has some shortage, so further researches should be carried out.

[Progress in research of clinical applications of fascia lata allograft].

OBJECTIVE: To find out the recent progress in research of clinical application of fascia lata allograft. METHODS: The domestic and international articles were reviewed to summarize the principal properties, processing techniques, and various uses of fascia lata allograft. RESULTS: Histologically fascia lata is composed of parallel and compact bundles of collagen fibers with few cells and immunologically it is low-antigenic. After varied tissue processing and storage techniques, fascia lata, as the scaffold only with the extracellular matrix, has been used in clinical practice and achieved good results, such as ophthalmology, urology, and orthopaedics. CONCLUSION: Because of these unique properites in repairing defects and reconstructing functions, fascia lata allograft, as a natural biomaterial, is promising to be used in more aspects with the development of the biomedical techniques.

[Porcine acellular dermal matrix for repair of abdominal wall defects in rabbit model].

OBJECTIVE: To research the effect of porcine acellular dermal matrix in the reconstruction of abdominal wall defects in rabbits, and to investigate the application feasibility of xeno-transplantation of acellular dermal matrix. METHODS: The porcine acellular dermal matrix was prepared from a health white pig. Twenty-six Japanese white rabbits (weighing 2.2-2.3 kg, female or male) were randomly assigned to 2 groups: the control group (n=6) and the experimental group (n=20). In the control group, the full-thickness abdominal wall defect of 5.0 cm x 0.5 cm was made, and the defect was sutured directly; in the experimental group, the full-thickness abdominal wall defect of 5.0 cm x 2.5 cm was made, and the defect was repaired with porcine acellular dermal matrix patch at the same size as the defect. At 5 weeks after surgery, the incidence of hernia and the intra-abdominal adhesions were observed and the wound breaking strength was compared between the patch-fascia interface and the fascia-fascia interface. The graft vascularization was evaluated through histological analysis at 6 months after surgery in the experimental group. RESULTS: No hernia occurred in all rabbits of 2 groups. At 5 weeks after surgery, healing was observed between patch and the muscular fascia; the vascularization was seen in the porcine acellular dermal matrix patch. There was no significant difference in the adhesion grade (Z= -0.798, P=0.425) between the experimental group (grade 2 in 1 rabbit, grade 1 in 5, and grade 0 in 12) and the control group (grade 1 in 1 and grade 0 in 5). No significant difference was found (t= -0.410, P=0.683) in the breaking strength between the patch-fascia interface in the experimental group [(13.0 +/- 5.5) N] and the fascia-fascia interface in control group [(13.6 +/- 4.0) N]. In the experimental group, the small vessels and the infiltration of inflammatory cells were observed in the porcine acellular dermal matrix patch after 5 weeks through histological observations. The junctions of the patch-fascia interface healed with fibrous connective tissue. At 6 months after surgery, the inflammation was subsided and the collagen fiber of the patch was reconstructed. CONCLUSION: The porcine acellular dermal matrix patch has good results in repairing full-thickness abdominal wall defect. The patch-fascia interface has similar breaking strength to the fascia-fascia interface. The collagen fibers of the patch are reconstructed.

[Study on cytotoxicity of microdoses peracetic acid in vitro].

OBJECTIVE: To evaluate the cytotoxicity of microdoses peracetic acid (PAA) so as to provide the evidence for making residual limit of PAA sterilization. METHODS: Mouse fibroblasts (L929 cell line) cultured in vitro were observed to evaluate the influence of microdoses PAA including 1 x 10(-6), 2 x 10(-6), 3 x 10(-6), 4 x 10(-6), 5 x 10(-6), and 10 x 10(-6) (V/V). The proliferation of cells was determined by MTT assay at 2, 4, and 7 days of culture. The growth curve and the relative growth rate (RGR) were obtained. The cytotoxicity of PAA at different concentrations was evaluated according to RGR. RESULTS: At 2, 4, and 7 days after culture, fibroblasts of 1 x 10(-6) group grew with normal morphology analogous to control group, while the cell growth of other groups were poor. With the increase of PAA concentration, the absorbance (A) values decreased, which suggested that there was a significant negative correlation between cell proliferation and PAA concentration. And the correlation coefficient was -1.000 at 2 and 4 days, - 0.964 at 7 days. There was no significant difference in A value between 1 x 10(-6) group and the control group (P > 0.05), while there were significant differences in A value between the control group and other concentration groups (P < 0.05). The growth curve of 1 x 10(-6) group was similar to that of the control group, both had obvious phase of exponential growth. The growth curves of other groups had no obvious phase of exponential growth. The cytotoxicity of 1 x 10(-6) group was classified as level 1, 2 x 10(-6) group as level 2, 3 x 10(-6) group as level 3, 4 x 10(-6) group as level 3-4, 5 x 10(-6) group and 10 x 10(-6) group as level 4. CONCLUSION: PAA of 1 x 10(-6) had no obvious cytotoxicity. The residual limit of PAA less than 1 x 10(-6) was recommended.

[Experimental study on porcine acellular dermal matrix and split-thickness skin grafts to repair full-thickness skin defects].

OBJECTIVE: To compare the effect of the composite skin graft consisting of split-thickness skin grafts (STSGs) and porcine acellular dermal matrix (PADM) with STSGs only, and to histologically observe the turnover of the PADM in rats. METHODS: Twenty female Sprague-Dawley rats, weighing 200-225 g, were included. The size of 4.0 cm x 2.5 cm PADM was implanted into hypoderm of the left side of Sprague-Dawley rats' back. After 10-14 days, the size of 4.0 cm x 2.5 cm full-thickness skin defects were made on the left to expose the PADM under the skin and the same size of full-thickness skin defects were made on the right of the rats' back. The excised full-thickness skin was made to STSGs about 0.2 mm by drum dermatome. The defects were grafted with composite skin (STSGs on the PADM, experimental group) and STSGs only (control group). The survival rate, the construction degree of grafts, and the histological change in grafts area were observed at 2, 4, 8, and 20 weeks after operation. RESULTS: At 2 weeks after STSGs (0.2 mm) placed on vascularized PADM, STSGs and PADM adhered together and the composite skin had a good survival. The control group also had a good survival. Histological observations showed that STSGs and PADM grew together, neutrophilic granulocytes and lymphocytes infiltrated in the PADM and some macrophages around the PADM. Fibrous connective tissues were filled under the STSGs in control group. At 4-8 weeks after transplantation, the composite skin had a good survival and the composite skin was thick, soft, and elastic. STSGs survived almost totally in control group, but the grafts were thin. Histological observations showed that inflammatory reactions of PADM faded gradually in experimental group; scar tissues formed under the STSGs in control group. At 20 weeks after transplantation, composite skin was flat, thick, and elastic in experimental group, but the STSGs were thinner and less elastic in control group. Histological observations showed that histological structures of the PADM were similar to the dermal matrix of rats, and the results showed that the collagen matrix of PADM was gradually replaced by the rats' collagen matrix. Scar tissues were filled under the STSGs in control group. Wound healing rates of experimental group were lower than those of control group at 4 and 8 weeks (P < 0.05); wound contraction rates of experimental group had lower tendency than those of control group, but showing no significant differences (P > 0.05). CONCLUSION: Coverage wound with composite skin which composed of STSGs and PADM could improve wound healing quality; the composite skin is thicker and better elastic than STSGs only. The collagen matrix of PADM is gradually replaced by rats' collagen matrix.

[Application of technique of labeling BMSCs with PKH26 to tissue engineered bone construction].

OBJECTIVE: To explore the feasibility of using PKH26 as a cell tracer to construct tissue engineered bone. METHODS: BMSCs isolated from the bone marrow of 1-week-old New Zealand white rabbit were cultured. The BMSCs at passage 3 were labeled with PKH26 and were observed under fluorescence microscope. The percentage of the labeled cells was detected by Flow cytometer. The labeled cells were induced to differentiate into osteoblasts in vitro and the morphology of the cells after induction was observed under inverted phase contrast microscope. The osteogenic induction was evaluated by ALP staining and Alizarin red staining. The cells labeled with PKH26 were seeded on the bio-derived bone to construct tissue engineered bone in vitro. Then the compound of cells and material were observed under fluorescence microscope. The compound of labeled cells and material were implanted into the rabbit thigh muscle, and the transformation of the labeled cells was observed by fluorescence microscope 14 and 28 days later. RESULTS: Fluorescence microscope observation: the BMSCs labeled by PKH26 were spherical and presented with red and uniform-distributed fluorescence, and the contour of the cells were clearly observed when they were adherent 24 hours after culture. Flow cytometric detection revealed that the percentage of labeled cells was 97.2%. After osteogenic induction, the morphology of the cells changed from long-fusiform to polygon-shape or cube-shape, more ECM was secreted, and the ALP and the Alizarin red staining were positive. At 48 hours after culturing the PKH26 labeled BMSCs with bio-derived bone, the fluorescence microscope observation showed that there was red fluorescence on the surface and inside of the material. At 14 days after implantation, the labeled cells with red and light fluorescence were evident in the implantation area; while at 28 days, the cells with red fluorescence were still evident but less in quantity and weaker in fluorescence strength. CONCLUSION: PKH26 can be used as BMSCs label for the construction of tissue engineered bone in vitro and the short-term tracing in vivo.