Objective To prepare a self-made compound, hemostatic jelly with polylactic acid(PLA), which has the hemostatic and absorbable effect on injured cancellous bone. Methods Two bone defects of 5 mm in diameter and 4 mm in depth were subjected on 20 health rabbits by drilling through their either outside plate of the iliac, and were filled with hemostatic jelly(group A), bone wax(group B) and blank(group C) respectively. Hemostasis were observed and recorded after 1 and 10 minutes. Five specimens were harvested at 2, 4, 8 and 12 weeks postoperatively for histological observation. Results ① Hemostatic effect: Bleeding of injured spongy bone stopped within 10 minutes after the treatment of hemostatic jelly and bone wax, but bleeding of balnk did not stop. Hemostatic jelly and bone wax adhered to bone defects firmly within 10 minutes was after the treatment. ② Absorbable effect: Hemostatic jelly and bone defects have not changed visibly in the first 2 weeks. With histological observation 4 to 8 weeks after the operation, hemastatic jelly was absorbed gradually and replaced by osteogenous tissue. It was absorbed completely after 8 to 12 weeks. Bone wax was not absorbed after 12 weeks, no new bone tissue was observed at bone wax area. The blank was replaced by connective tissue and osteogenous tissue partially after 12 weeks. Conclusion The compound hemostatic jelly manifests both hemostatic and absorbable effects on injured cancellous bone and may substitute for bone wax in clinical application.
ObjectiveTo investigate the influences of lactic acid (LA), the final degradation product of polylactic acid (PLA) on the prol iferation and osteoblastic phenotype of osteoblast-l ike cells so as to provide theoretical basis for bone tissue engineering.
MethodsRos17/2.8 osteoblast-l ike cells were harvested and divided into 3 groups. In groups A and B, the cells were cultured with the medium containing 4, 8, 16, 22, and 27 mmol/L L-LA and D, L-LA, respectively. In group C, the cells were cultured with normal medium (pH7.4). The cell prol iferation was determined with MTT method after 1, 3, and 5 days. The relative growth ratio (RGR) was calculated, and the cytotoxicity was evaluated according to national standard of China. In addition, the alkal ine phosphatase (ALP) activity of cells cultured with medium containing 4 mmol/L L-LA (group A), 4 mmol/ L D, L-LA (group B), and normal medium (group C) after 1 and 5 days were detected with ALP kits, and the relative ALP ratio (RAR) was calculated; after 21 days, the calcium nodules were tested with von Kossa staining method, and were quantitatively analyzed.
ResultsWhen LA concentration was 4 mmol/L, the mean RGR of both groups A and B were all above 80%, and the cytotoxic grades were grade 0 or 1, which meant non-cytotoxicity. When LA concentration was 8 mmol/L and 16 mmol/ L, groups A and B showed cytotoxicity after 5 days and 3 days, respectively. When LA concentration was above 22 mmol/L, cell prol iferations of groups A and B were inhibited evidently after 1-day culture. At each LA concentration, RGR of group A was significantly higher than that of group B at the same culture time (P<0.05) except those at 4 mmol/L after 1-day and 3-day culture. After 1 day, the RAR of group A was significantly higher than that of group B on 1 day (144.1%±3.2% vs. 115.2%±9.8%, P<0.05) and on 5 days (129.6%±9.8% vs. 78.2%±6.9%, P<0.05). The results of von Kossa staining showed that the black gobbets in group A were obviously more than those of groups B and C. The staining area of group A (91.2%±8.2%) was significantly higher than that of groups B (50.3%±7.9%) and C (54.2%±8.6%) (P<0.05).
ConclusionThe concentration and composition of LA have significant effects on the cell proliferation and osteoblastic phenotype of osteoblast-l ike cells.
Objective To establish an animal model for repairing the sciatic nerve defect with a biodegradable poly D,L-lactic acid/nerve growth factor (PDLLA/NGF) that can control the release conduit in rats and to observe an effect of the conduit on the sciatic nerve regeneration. Methods The PDLLA conduit and the PDLLA/NGF-controlled release conduit (NGF 450 U per conduit) were madewith the solvent-volatilixation method. Forty male SD rats were randomly and equally divided into 4 groups. The middle segments (10 mm) of the sciatic nerves of the rats were excised and were then repaired with the sciatic nerve autograft(Group A), with the PDLLA conduit (Group B), with the PDLLA conduit and an injection of NGF (30 U) into the conduit (Group C), and with the PDLLA/NGF controlled-release conduit (Group D), respectively, with the 10-mm nerve defect left behind. Three months after operation, the morphologic parameters of the nerve regeneration were observed and evaluated under light microscope and electron microscope, and the image analysis was also made. Results Three months after operation, porous adherence between the conduit and the surrounding tissues could be observed. The conduit presented a partial biodegradation but still remainedintact in the outline and the proximal nerve regenerated through the conduit cavity. Based on the histological observation, the quantity, uniformity, and maturity of the nerve fiber regeneration in Groups A and D were better than those in Groups B and C. The image analysis indicated that there were no significant differences in the nerve fiber diameter, axon diameter or myelin thickness between Group A and Group D (P＞0.05). However, all the parameters in Groups A and D were better than those in Groups B and C (P＜0.05). Conclusion The PDLLA/NGF-controlled release conduit can effectively promote the sciatic nerve regeneration of rats. Its morphological index is similar to that of the nerve autograft.
Objective To explore the biocompatibility of poly(lacticacid/glycolic acid/asparagic acid-co-polyethylene glycol) biomaterials (PLGA-ASP-PEG) and biological behaviors of cultured marrow stroml stem cells (MSCs) combined with this new type of scaffold in tissue engineering. Methods The PLGA-ASP-PEG tri-block copolymers were obtained through bulk ringopening copolymerization method.MSCs were isolated from the bone marrow of 4 week old New Zealand rabbits. The 3rdgeneration MSCs were cultured combining with PLGA-ASP-PEG in vitro, while cells cultured in PLGA as control group. The cell adhesion rate and the adhesivepower were examined by conventional precipitation method and micropipette aspiration technique respectively. The morphological features were studied by scanning electron microscope. The proliferation behavior of the cells was analyzed by MTT assay. The cell cycle, proliferation index, DNA index and apoptosis of the cells were detected by flow cytometry. The synthesis of protein and collagen were examined by Coomassie Brilliant Blue dyes and 3H-Proline incorporation test. Results The MSCs adhered and grew well on the surface of the biomaterial PLGA-ASP-PEG. The powers of cell adhesion, proliferation and protein and collagen synthesis of the cells were all significantly higher than those of PLGA group (P＜0.05), but the apoptosis rate was significantly lower than that of PLGA group (P＜0.05). The DNA indexes showed the cells of both PLGA-ASP-PEG group and PLGAgroup were normal diploid cells. Conclusion PLGA-ASP-PEG showedgood biocompatibilityand the biological properties improved greatly compared with the PLGA scaffold materials. These results demonstrated that the promise of PLGAASPPEG canbe used as an ideal scaffold material for construction of tissue engineered bone to restore bone defects in bone tissue engineering．
Objective To study the result of using nerve conduit coated with chitin and filled with a guide-fiber to repair peripheral nerve defect. Methods Twenty-four female adult SD rats were made the model of 14 mm-gap on bilateral sciatic nerve under sterile condition. The rats were randomly divided into 4 groups(n=6),group A: polymer polyglycolic-lactic acid(PGLA) nerve conduit coated with chitin and filled with a guide-fiber as experimental group to repair 14 mm gap of rat sciatic nerve；group B: PGLA nerve conduit coated with chitin； group C: PGLA nerve conduit； group D: autograft (control group). The repair result was evaluated by normal observation, EMG testing and S-100 histological immunostaining analysis 4 and 12 weeks after operation.Results Four weeks after the operation，there were new regenerated immature fibers in groups A,B and C, 12 weeks after the operation, the regenerated nerve fibers were seen to have bridged the gap. There were myelinated fibers equably distributed and rarely newgenerated nerve fibers in distal parts of group D. The repair result of PGLA nerve conduit coated with a chitin and filled with guide-fiber was better than that of groups B and C（Plt;0.05）. There was significant difference of nerve fiber diameter，thickness of myelin sheath and fiber density in group D from those in groups A, B and C（Plt;0.05），but there were degenerative changes such as vacuoles insheaths and myelin separation in proximal and few new regenerated nerve fibers in distal parts of group D. Conclusion PGLA nerve conduit coated with chitin and filled with a guide-fiber offers a possible substitute for the repair of peripheral nerve defect.
ObjectiveTo evaluate the biocompatibility of poly lactic acid/bone matrix gelatin (PLA/BMG) composite biomaterial so as to lay a foundation for bone defect repair.
MethodsRats'MC3T3-E1 cells were cultured with leaching solution of PLA/BMG and PLA material respectively for 7 days. The cell proliferation rate was tested by MTT and cell toxicity grading was carried out everyday. The PLA/BMG and MC3T3-E1 cells were co-cultured, the cell shape and proliferation were observed by inverted phase contrast microscope at 1, 3, and 5 days and cell adhesion by scanning electron microscope at 5 days. The PLA and PLA/BMG were implanted subcutaneously in 15 Wistar rats. The histological observation was done, and the thickness of fibrous membrane, the number of inflammatory cells, and the vascularization area were measured at postoperative 2nd, 4th, and 8th week.
ResultsThe tests for cytotoxicity in vitro showed that the cell proliferation rates were over 100% and the cell cytotoxic grades were grade 0 at 1-7 days in PLA/BMG group. While in PLA group, the cell proliferation rates were less than 100% and the cell cytotoxic grades were grade 1 at 2, 4, and 7 days. After co-culture of PLA/BMG and MC3T3-E1 cells, cells grew on the surface and in the pores of PLA/BMG, and the cellular morphology was triangle or polygon with abundant microvillus on the surface. After subcutaneous implantation, the rats survived to the end of experiment, and incision healed well. PLA was wrapped by connective tissue where there were a lot of lymphocytes and neutrophilic granulocytes. The cells and tissue grew slowly in PLA. The PLA/BMG materials were wrapped by little connective tissue where there were a few inflammatory cells. The connective tissue ingrowth was observed in the center of PLA/BMG. There was no significant difference in the thickness of fibrous membrane between 2 groups at each time point (P>0.05). The number of inflammatory cells of PLA/BMG group were significantly less than those in PLA group at 2, 4, and 8 weeks (P<0.05); the vascularization area was significantly larger than that in PLA group (P<0.05).
ConclusionPLA/BMG composite biomaterials prepared by super critical-CO2 technique are good in cell and tissue biocompatibilty.
ObjectiveTo explore the effect of silk fibroin/poly(L-lactic acid-co-e-caprolactone) [SF/P(LLA-CL)] nanofibrous scaffold on tendon-bone healing of rabbits.MethodsSF/P(LLA-CL) nanofibrous scaffold was fabricated by electrospinning methods. The morphology of the scaffold was observed by scanning electron microscope (SEM). Pre-osteoblasts MC3T3-E1 cells were seeded on the scaffold and cultured for 1, 3, and 5 days. Cell adhesion and proliferation were also observed by SEM. Meanwhile, twenty-four New Zealand white rabbits were randomly divided into the autogenous tendon group (control group) and the autogenous tendon wrapped with SF/P(LLA-CL) scaffold group (experimental group), with twelve rabbits in each group. An extra-articular model was established, the effect was evaluated by histological examination and mechanical testing.ResultsThe morphology of SF/P(LLA-CL) nanofibrous scaffold was random, with a diameter of (219.4±66.5) nm. SEM showed that the MC3T3-E1 cells seeded on the scaffold were in the normal shape, growing well, and proliferating with time course. The results of histological examination showed that inflammatory cells infltrated into the graft-host bone interface at 6 weeks after operation in both groups. Besides, the width of interface showed no significant difference between groups. At 12 weeks after operation, protruding new bone tissue could be observed at the interface in the experimental group, while scar tissue but no new bone tissue could be seen at the interface in the control group. Mechanical testing showed that there was no significant difference in the failure load and the stiffness between groups at 6 weeks after operation (P>0.05). The failure load and the stiffness in the experimental group were significantly higher than those in the control group at 12 weeks after operation (P<0.05).ConclusionThe SF/P(LLA-CL) nanofibrous scaffold has good cell biocompatibility and can effectively promote tendon-bone healing, thus providing new method for modifying graft for ACL reconstruction in the clinical practice.
摘要:目的:研究生物降解聚DL乳酸(PDLLA)自鎖式捆綁帶固定骨折的生物力學性能。方法:80只新西蘭大白兔隨機分為兩組,建立股骨干非負重骨折動物模型,應用生物降解自鎖式捆綁帶固定骨折為實驗組,鋼絲固定骨折為對照組,分別于術后1、4、8、12周行生物力學檢查進行比較。結果:捆綁帶組在術后4、8、12周均比鋼絲組的彎曲強度高,但4周、12周時Pgt;005,無統計學差異,8周時Plt;005,提示有統計學差異。離體同種固定物不同時間段抗拉強度自身比較:鋼絲固定術后4階段抗拉強度比較Pgt;005,任何兩兩比較都沒有統計學差異,抗拉強度未隨術后時間延長發生明顯下降。捆綁帶固定術后4周與術后1周比較Pgt;005,抗拉強度無明顯降低,但術后8周和術后12周時Plt;005,抗拉強度明顯下降。結論:生物降解自鎖式捆綁帶在非負重骨折治療中可發揮良好的固定作用。生物降解自鎖式捆綁帶降解時,應力傳導促進了骨折的愈合。Abstract: Objective: To study the biomechanics function of selflocking cerclage band made of biodegradable material polyDLlactic acid (PDLLA) in the fixation of fractures. Methods: Eighty rabbits were divided into two groups. Femur fracture models were made. Fractures were fixed using biodegradable selflocking cerclage band in experimental group and metal fixation material in control group. The biomechanics was analyzed and compared after 1, 4, 8 and 12 weeks respectively． Results: The bending strength of experimental group is more ber than that of control group after 4, 8 and 12 weeks, but it was not statistically significant at 4 and 12 weeks (Pgt;005). It was statistically significant at 8 weeks (Plt;005). The tensile strength of the same cerclage instrument was compared at different stage in vitro, and the result of the control group was not statistically significant at the four stage (〖WTBX〗P〖WTBZ〗gt;005). Regarding the changes of tensile strength of the cerclage instrument at different stage, the result of the experimental group was not statistically significant after 1 and 4 weeks (Pgt;005). However, the decrease of tensile strength was statistically significant after 8 and 12 weeks (Plt;005). Conculsion: Biodegradable selflocking cerclage band could be used in thetreatment of nonweightbearing fractures. The stress force conducting promotes healing of fracture when the selflocking biodegradable cerclage band degrades.
ObjectiveTo investigate the effect of electrospun chitosan/polylactic acid (ch/PLA) nerve conduit for repairing peripheral nerve defect in rats.
MethodsNerve conducts loaded with ch/PLA was made by the way of electrospun. The mechanical property, hydrophility, biocompatibility were tested, and the scanning electron microscope was used to observe the ultrastructure. The same experiments were also performed on pure PLA nerve conducts as a comparison. Then, 54 Sprague Dawley rats were divided into 3 groups randomly, 18 rats in each group. Firstly, the 10 mm defects in the right sciatic nerves were made in the rats and were respectively repaired with ch/PLA (group A), autografts (group B), and no implant (group C). At 4, 8, and 12 weeks after operation, general observations, sciatic functional index (SFI), electrophysiological evaluation, wet weight of gastrocnemius and soleus muscles, histological examination, immunohistological analysis, and transmission electron microscopy were performed to evaluate the effects.
ResultsCompared with pure PLA nerve conducts, the addition of chitosan could improve the mechanical property, hydrophility, biocompatibility, and ultrastructure of the nerve conducts. At 4 weeks postoperatively, the regenerated nerve bridged the nerve defect in group A. The SFI improved gradually in both group A and group B, showing no significant difference (P>0.05). Compound muscle action potentials and nerve conduction velocity could be detected in both group A and group B at 8 and 12 weeks after operation, and significant improvements were shown in both groups (P<0.05). The wet weight and myocyte cross section of gastrocnemius and soleus muscles showed no significant difference between group A and group B (P>0.05), but there was significant difference when compared with group C (P<0.05) at 12 weeks postoperatively. Immunohistological analysis revealed that S-100 positive Schwann cells migrated in both group A and group B, and axon also regenerated by immunohistological staining for growth associated protein 43 and neurofilaments 160. Transmission electron microscopy showed no significant difference in the diameter of nerve fiber between group A and group B (P>0.05), but the thickness of myelin sheath in group A was significantly larger than that in group B (P<0.05).
ConclusionThe electrospun ch/PLA nerve conduits can effectively promote the peripheral nerve regeneration, and may promise an alternative to nerve autograft for repairing peripheral nerve defect.
Objective To study the mechanism of ectopic osteogenesis of nacre/Polylactic acid (N/P) artificial bone combined with allogenic osteoblasts, and to explore the possibility as a scaffold material of bone tissue engineering. Methods The allogenic- osteoblasts seeded onto N/P artificial bone were co-cultured in vivo 1 week.The N/P artificial bone with allogenic osteoblasts were implanted subcutaneously into the left back sites of the New Zealand white rabbits in the experimental group and the simple N/P artificial bone into the right ones in the control group. The complexes were harvested and examined by gross observation, histologic analysis and immunohistochemical investigation 2, 4 and 8 weeks after implantation respectively.Results In experimental group, the osteoid formed after 4 weeks, and the mature bone tissue withbone medullary cavities formed after 8 weeks; but in control group there was nonew bone formation instead of abundant fibrous tissue after 4 weeks, and more fibrous tissue after 8 weeks.Conclusion N/P artificial bone can be used as an optical scaffold material of bone tissue engineering.
