Objective To explore the effect of NaOH on the surface morphology of three-dimensional (3D) printed poly-L-lactic acid (PLLA) mesh scaffolds. Methods The 3D printed PLLA mesh scaffolds were prepared by fused deposition molding technology, then the scaffold surfaces were etched with the NaOH solution. The concentrations of NaOH solution were 0.01, 0.1, 0.5, 1.0, and 3.0 mol/L, and the treatment time was 1, 3, 6, 9, and 12 hours, respectively. There were a total of 25 concentration and time combinations. After treatment, the microstructure, energy spectrum, roughness, hydrophilicity, compressive strength, as well as cell adhesion and proliferation of the scaffolds were observed. The untreated scaffolds were used as a normal control. Results 3D printed PLLA mesh scaffolds were successfully prepared by using fused deposition molding technology. After NaOH etching treatment, a rough or micro porous structure was constructed on the surface of the scaffold, and with the increase of NaOH concentration and treatment time, the size and density of the pores increased. The characterization of the scaffolds by energy dispersive spectroscopy showed that the crystal contains two elements, Na and O. The surface roughness of NaOH treated scaffolds significantly increased (P<0.05) and the contact angle significantly decreased (P<0.05) compared to untreated scaffolds. There was no significant difference in compressive strength between the untreated scaffolds and treated scaffolds under conditions of 0.1 mol/L/12 h and 1.0 mol/L/3 h (P>0.05), while the compression strength of the other treated scaffolds were significantly lower than that of the untreated scaffolds (P<0.05). After co-culturing the cells with the scaffold, NaOH treatment resulted in an increase in the number of cells on the surface of the scaffold and the spreading area of individual cells, and more synapses extending from adherent cells. Conclusion NaOH treatment is beneficial for increasing the surface hydrophilicity and cell adhesion of 3D printed PLLA mesh scaffolds.
OBJECTIVE To confirm membrane-guided tissue regeneration in the healing course of segmental bone defects and study the mechanism. METHODS Segmental, 1 cm osteoperiosteal defects were produced in both radii of 12 rabbits. One side was covered with hydroxyapatite/polylactic acid(HA/PLA) membrane encapsulated as a tube. The contralateral side served as an untreated control. Healing courses were detected by radiographic and histologic examinations. RESULTS All control sides showed nonunion, whereas there were consistent healing pattern in test sides. CONCLUSION Membrane technique can promote bone regeneration.
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.
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.
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 fabricate a novel porous bioactivecomposite biomaterial consisting of poly lactic acid (PLA)bone matrix gelatin(BMG) by using the supercritical carbon dioxide fluid technique (SC-CO2) and to evaluate its osteoinductive activity. Methods The cortical bones selected from healthy adult donors were processed into BMG by the defatting, demineralizing, and deproteinizing processes. PLA and BMG were mixed at a volume radio of 3∶1; then, the PLA-BMG mixed material and the pure PLA material were respectively placed in the supercritical carbon dioxide reaction kettles, and were respectively added by the NaCl particles 100200 μm in diameter for theporosity of the materials so that the porous PLA-BMG composite material and the porous PLA composite material could be formed. The mouse osteoblastlike MC3T3-E1 cells were cultured in the dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum. Then, 20 μl of the MC3T3E1 cell suspensions containing 2 ×106 cells /ml were delivered into the culturing plate (24 wells/plate) made of the different materials, which were co-cultured for 2 weeks. In the PLA-BMG group, 100 μg of the crushed PLA-BMG material was contained in each well; in the PLA group, 100 μg of the crushed PLA material was containedin each well; and in the DMEM group, only DMEM was contained, which served as the control group. There were 6 wells in each group. The quantitative analysis onthe calcification area was performed by the staining of the alizarin red S. Theco-cultured cells were harvested and lysated in 1 ml of 0.2% Nonidet P-40 by the ultrasonic lysating technique. Then, the ALP activity and the Ca content were measured according to the illuminations of the reagent kits. Results The porous PLABMG composite material showed a good homological porosity with a pore diameter of 50-150 μm and a good connectivity between the pores. The ALP activity, the Ca content, and the calcification area were significantly greater in the PLABMG group than in the PLA group and the control group (325.59±70.40 U/gprot, 3.51±1.64 mmol/gprot, 42.98±4.44% vs. 63.62±30.01 U/gprot, 1.04±0.21 mmol/gprot, 9.55±1.94%, and 2.40±1.47 U/gprot, 0.70±0.24 mmol/gprot, 0.86±0.41%; Plt;0.05). Meanwhile, there was a statistically significant difference between the PLA group and the control group in the ALP activity and the calcification area (Plt;0.05). Conclusion The porous PLABMG composite material prepared by the use of SC-CO2 has a good steoinductive activity and can be used as a promising bone biomaterial and a bone tissue engineered scaffold.
Objective To investigate the effect of homograft of marrow mesenchymal stem cells (MSCs) seeded onto poly-L-lactic acid (PLLA)/gelatin on repair of articular cartilage defects. Methods The MSCs derived from36 Qingzilan rabbits, aging 4 to 6 months and weighed 2.5-3.5 kg were cultured in vitroand seeded onto PLLA/gelatin. The MSCs/ PLLA/gelatin composite was cultured and transplanted into full thickness defects on intercondylar fossa. Thirty-six healthy Qingzilan rabbits were made models of cartilage defects in the intercondylar fossa. These rabbits were divided into 3 groups according to the repair materials with 12 in each group: group A, MSCs and PLLA/gelatin complex(MSCs/ PLLA/gelatin); group B, only PLLA/gelatin; and group C, nothing. At 4,8 and 12 weeks after operation, the gross, histological and immunohistochemical observations were made, and grading scales were evaluated. Results At 12 weeks after transplantation, defect was repaired and the structures of the cartilage surface and normal cartilage was in integrity. The defects in group A were repaired by the hylinelike tissue and defects in groups B and C were repaired by the fibrous tissues. Immunohistochemical staining showed that cells in the zones of repaired tissues were larger in size, arranged columnedly, riched in collagen Ⅱ matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in group A at 12 weeks postoperatively. In gross score, group A(2.75±0.89) was significantly better than group B (4.88±1.25) and group C (7.38±1.18) 12 weeks afteroperation, showing significant differences (P＜0.05); in histological score, group A (3.88±1.36) was better than group B (8.38±1.06) and group C (13.13±1.96), and group B was better than group C, showing significant differences (P＜0.05). Conclusion Transplantation of mesenchymal stem cells seeded onto PLLA/gelatin is a promising way for the treatment of cartilage defects.
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.
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.
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.