Objective To review the progress and clinical application of malleable bone paste/putty. MethodsRecent literature about malleable bone paste/putty was reviewed and analyzed. ResultsThe 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. ConclusionMalleable 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.
Objective To evaluate the effects of composite bone in strategy of tissue engineering on bone defect repair in rats. Methods Sixteen matured Wistar rats (male or female, weighing 250-300 g) were used to prepare platelet lysate (PL). PL/allogeneic decalcified bone granules (ADBG)/Col I (PAC) and ADBG/Col I (AC) were prepared by mixing Col Igel ADBG with or without PL. BMSCs of 8 Wistar rats (male or female, weighing 250-300 g) were isolated and cultured. The 5th passage of BMSCs were co-cultured with PAC at the density of 1 × 106 cells/mL to fabricate the tissue engineered composite PACB in vitro. Forty healthy Wistar rats were made bilateral bone defects in femoral condyles and divided into 4 groups (A, B, C and D, n=10). The defects were filled with equivalent PACB, PAC, AC and Col I in groups A, B, C and D respectively. At 4 weeks, the defect repair was evaluated with radiology, histology, ALP biochemical tests. Results At 4 weeks, the bone density measurement was (7.31 ± 0.54), (4.36 ± 0.67), (2.12 ± 0.47), and (1.09 ± 0.55) pixels in groups A, B, C, and D, respectively. The area of new bone formation in defect area under single view was (412.82 ± 22.31), (266.57 ± 17.22), (94.34 ± 20.22), and (26.12 ± 12.51) pixels in groups A, B, C and D respectively. The ALP contents in femoral condyles were (94.31 ± 7.54), (69.88 ± 4.12), (41.33 ± 3.46), and (21.03 ± 3.11) U/L, respectively. The above indexes of group A were significantly higher than those of groups B, C or D (P lt; 0.05). Three-color flow cytometry assay showed that the T lymphocyte subsets of CD3+CD4+CD8-, CD3+CD8+CD4-, and the ratio of CD4/CD8 displayed no significant difference among four groups (P gt; 0.05). Conclusion Tissue engineered bone PACB is capable to promote the bone defect repair.
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 × 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 (χ2=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 lt; 0.05), but no significant difference was found between the experimental group and positive control group (P gt; 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.
Objective To find out the recent progress in research of cl inical appl ication of fascia lata allograft. Methods The domestic and international articles were reviewed to summarize the princi pal 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 cl inical 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 withthe development of the biomedical techniques.
Objective To develop a deantigenated porcine bone graft through enzyme treatment and to study the biomechanical properties and osteoinductivity of the xenogeneic bone graft. Methods Deantigenated xenogeneic bone was prepared from porcine bone by a series treatment including α-galactosidase (α-Gal) treatment, freezedrying and irradiation at a does of 25 kGy. Samples were divided into 4 groups according to the treatment methods: fresh bone (group A); deantigenated bone (group B); deantigenated and freezedried bone (group C); deantigenated-freezedried-irradiated bone(group D). SEM observation to group B samples was performed and the diameters of the caves of the porcine cancellous bone were measured. The α-Gal contents of samples of groups A, B, D were measured by ELISA. The effects of different treatments on porcine bone mechanic properties were evaluated through compressive test of 4 groups. The prepared porcine cortical bone were demineral ized and ectopically implanted into muscle groups of hind l imbs of Wistar rats as experimental group, and the demineral ized cortical bone which were inacitviated by high temperature and pressure were implanted as control group. Histological observation was performed and ALP activity was tested at different time postoperatively to investigate the osteoinductivity of the xenogeneic bone implants. Results The porous structure of prepared porcine cancellous bone was similar to that of human cancellous bone. The diameters of the caves were between 150-600 μm. The A value of the groups A, B, D was 0.358 ± 0.027, 0.191 ± 0.011, 0.191 ± 0.009, respectively, with statistically differences between groups (P lt; 0.05). While the biomechanical properties among 4 groups had no statistically difference (P gt; 0.05). Histological observation showed mesenchymal cells immigrated into cartilage and converted into chondrocytes at 3 weeks postoperatively. Cartil iage was formed at 4 weeks and osteoid and more adult cartilage was formed at 6 weeks within the implants of experimental group, while there was no bone formation in control group. ALP activity of experimental group at different times were obviously higher than that of the control group (P lt; 0.05). Conclusion The main xenogeneic antigen of the porcine bone was removed while the mechanic properties and osteoinductivity remained, thus it may be a suitable bone substitute for cl inical use.
Objective To investigate the osteoblasts effect, compl ications and influencing factors in the appl ication of small freeze-drying allogeneic bone plots mixed autologous bone fragments in spinal surgery, and to compare with autogenous bone graft. Methods From January 2003 to January 2007, 515 cases of spinal injuries were treated. A total of 324 cases weretreated with small freeze-drying allogeneic bone plots mixed with autologous bone grafts (group A), including 211 males and 113 females with an average age of 36 years (18-83 years). There were 182 cases of thoracolumbar vertebra fracture, 68 cases of lumbar spondylol isthesis, 47 cases of lumbar vertebral canal stenosis, 17 cases of cervical disc herniation, 5 cases of cervical spine fracture-dislocation and 5 cases of thoracolumbar vertebra tumor. The weight of bone graft was 10-60 g (mean 30 g). A total of 191 cases were treated with autogenous bone grafting (group B), including 135 males and 56 females with an average age of 32 years (23-78 years). There were 109 cases of thoracolumbar vertebra fracture, 23 cases of lumbar spondylol isthesis, 17 cases of lumbar vertebral canal stenosis, 19 cases of cervical disc herniation, and 23 cases of cervical spine fracture-dislocation. The weight of bone graft was 10-50 g (mean 25 g). Results In group A, effusion of wound increased in 4 cases and the result of bacterial culture was negative; effusion was absorbed after 2 weeks of local irrigation, drainege and cortin management. In group B, no obvious effusion was observed. The follow-up time was 10-36 months (mean 17.4 months) in group A and 8-36 months (mean 16.8 months) in group B. The bone heal ing was achieved in 308 cases within 4-10 months (mean 8.1 months) and in 184 cases within 4-10 months (mean 5.8 months), and the bone fusion rates were 95.06% and 96.34% in groups Aand B, respectively. There was no significant difference in bone fusion rate between groups (P gt; 0.05). According to Mankin and Komender evaluation standard, the response rates were 95.06% and 96.34% in groups A and B, respectively, showing no significant difference (P gt; 0.05). Conclusion Mix-bone grafting has the same effective to autologous bone grafting in bone fusion rate. It could be used as the supplement of the autologous bone inadequacy.
Objective To investigate cl inical therapeutic effect on early stage femoral head necrosis managed with allogeneic cortical bone cage support combining with autologous cancellous bone grafting through core decompression tunnel, and to discuss its effect on preventing femoral head collapse and influence factors. Methods From January 2002 to December2005, 40 patients (42 hips) with femoral head necrosis underwent core decompression and an allogeneic threaded cortical bone supporting cage which was loaded with autologous cancellous bone inside. There were 26 males and 14 females, aging 27-45 years (mean 35.6 years). The disease course was 6-28 months (mean 18.3 months). All the cases underwent X-ray, CT and MRI examination to confirm the diagnosis and necrosis area. Twelve hips were at Ficat stage I, 29 hip at stage II, and 1 hips at stage III. Harris hip score system was used to evaluate the hip function pre- and post-operatively. X-ray films were taken regularly after operation. Results All the wound healed by first intention without any compl ications such as infection, fracture, nerve and vascular injury, and deep vein thrombosis. Thirty-six patients (38 hips) were followed up for 24-58 months with an average of 38 months. All the patients had different degrees of improvement in cl inical symptoms. According to Harris hip score system, the Harris score was 63.1 ± 6.4 before operation and 82.3 ± 16.5 at the last follow-up, showing significant difference (P lt; 0.001). The results were excellent in 24 hips, good in 11 hips, fair in 2 hips and poor in 1 hips. The X-ray films showed femoral head repairing and no advancement of osteonecrosis and collapsing in 23 patients (24 hips) 24 months after operation. Conclusion Allogeneic cortical bone cage support combining with autologous cancellous bone grafting is suitable for managing early stagefemoral head necrosis and its short- and middle-term effect is satisfactory.
【Abstract】 Objective To explore the interventional effect of platelet lysate (PL) on osteogenic differentiation ofBMSCs by induction in rats in vitro. Methods Twenty-four clean-grade adult Wistar rats, weighing from 250 g to 300 g, maleor female, were included in this study. PL was obtained through three times of centrifugation and repeated freeze-thaw for the blood aspirated from cardiac cavities in 16 Wistar rats. ELISA assay was conducted to detect the concentration of growth factors PDGF, TGF-β1, IGF-1 and VEGF in PL. The BMSCs harvested by flushing femurs of 8 adult Wistar rats were isolated, cultivated and expanded in vitro. The cells at the 4 passage were performed for osteogenic differentiation by induction in three groups of A (5% PL of final concentration in basic induction medium), B (1% PL of final concentration in basic induction medium), and C (no presence of PL in basic induction medium as a control). The morphological changes of the cells were dynamically observed with inverted phase contrast microscope during the whole period. At different time-points, ALP staining (7 days) and ALP/TP (2, 8, 12 days) of the cells were detected to evaluate ALP activity, and the mineral formation in extracellular martrix was examined with Al izarin red staining which provided quantitative analysis of mineral deposits. Results ELISA assay showed that the content of PDGF, TGF-β1, IGF-1 and VEGF in PL reached (300 ± 30), (140 ± 25), (80 ± 35), (70 ± 20) pg/mL, respectively. Morphological observation displayed BMSCs in group A or B gradually turned from spindle-shape to square- or polygon-shape as the morphorlogical type of osteoblast-l ike cells at 7 days. The cells in group A showed slower shape changesbut higher prol iferation than that in group B or C. Moreover, at the 20 days, the cells in group A still displayed dense gro wth and produced obviously decreased amount of mineral deposits in ECM when compared with group B or C. At the 7 days, the cells ofgroup A showed smaller amount of granules positive for ALP staining in cytoplasm when compared with groups B and C, and displayed marked reduction in ALP activity assay at the 2, 8, and 10 days compared with that of groups B and C (P lt; 0.05). At the 20 days, Al izarin red staining showed the number of mineral deposits in groups A, B and C were 7.67 ± 1.10, 12.87 ± 0.81 and 15.59 ± 0.25, respectively, while the area of mineral deposits were (161 778.70 ± 44 550.80), (337 349.70 ± 56 083.24), and (415 921.70 ± 71 725.39) pixels, respectively. The number of mineral deposits and the area of mineral deposits in group A were smaller than those in groups B and C (P lt;0.05). But there was no statistically significant difference between groups B and C (P gt; 0.05). Conclusion PL is a kind of system carrying various growth factors. Exposure of PL inhibits both ALP activity and mineral formation of BMCs in a dose-dependent way under the osteogenic induction environment.
Objective To evaluate the effect of implantation of the complex of high viscous chitosan/glycerol phosphate with demineral ized bone matrix (HV-C/GP-DBM) in repairing cartilage defects of rabbits. Methods HV-C/ GPDBM was prepared by compounding HV-C/GP and DBM by 2:1 (W/W). Twenty-four 34-week-old New Zealand white adult rabbits, weighing 3.5-4.5 kg, were included. A bit with the diameter of 3.5 mm was used to drill 3-cm-deep holes in both sides of femoral condyle to make cartilage defects. The complex of HV-C/GP-DBM was then injected into the right holes as the experimental group and the left ones serve as the control group. The rabbits were killed at 4, 8 and 16 weeks after theoperation, respectively. The obtained specimens were observed macroscopically, microscopically and histologically. According to the International Cartilage Repair Society Histological Scoring (ICRS), the effect of cartilage repair was assessed at 16 weeks postoperatively. Results At 4-8 weeks postoperatively, in the experimental group, the defects were filled with hyal ine cartilage-l ike tissues; the majority of chitosan degradated; and the DBM particles were partly absorbed. However, in the control group, there were small quantities of discontinuous fibrous tissues and maldistributed chondrocytes at the border and the bottom of the defects. At 16 weeks postoperatively, 6 joints in the experimental group had smooth surface, and the defects were basically repaired by hyal ine cartilage-l ike tissues. The newly-formed tissues integrated well with the surrounding area. Under the cartilage, the new bone formation was still active and some DBM particles could be seen. However, the defects in the control group were repaired by fibrous tissues. The result of histological scoring of the specimens at 16 weeks showed that a total of 6 aspects including formation of chondrocytes and integration with the surrounding cartilages were superior in the experimental group to those in the control group, and there were significant differences between the two groups (P lt; 0.05). Conclusion The biodegradable and injectable complex of HV-C/GP-DBM with good histocompatibil ity and non-toxic side effects can repair cartilage defects and is a promising biomaterial for cartilage defect repair.
Objective To explore the feasibil ity 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 wasdetected 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, andthe 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 l ight 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.