ObjectiveTo explore the morphological and functional features of tissue engineered composite constructed with bone mesenchymal stem cells (BMSCs) as seeding cells, thermosensitive collagen hydrogel (TCH) and poly-L-lactic acid (PLLA) as the extracellular matrix (ECM) scaffolds in the dynamic culture system.
MethodsBMSCs were separated from long bones of Fischer344 rat, and cultured; and BMSCs at the 3rd generation were seeded on the ECM scaffold constructed with braided PLLA fiber and TCH. The BMSCs-ECM scaffold composite was cultured in the dynamic culture system which was designed by using an oscillating device at a frequency of 0.5 Hz and at swing angle of 70° (experimental group), and in the static culture system (control group) for 7 days. The general observation and scanning electron microscopy (SEM) observation were performed; total DNA content was measured at 0, 1, 3, and 7 days.
ResultsPLLA was surrounded by collagen to form translucent gelatiniform in 2 groups; and compact membrane developed on the surface of PLLA. SEM observation showed that BMSCs had high viability and were fusiform in shape with microvilli on the surface of cells, and arranged in line; collagen and cells filled in the pores of PLLA fiber in the experimental group. The cells displayed a flat shape on the surface; there were less cells filling in the pores of PLLA fiber in the control group. At 1, 3, and 7 days, total DNA content in the experimental group was significantly higher than that in control group (P < 0.05). The total DNA content were increased gradually with time in 2 groups, showing significant difference between at 0 day and at 7 days (P < 0.05).
ConclusionThe ECM constructed with TCH and PLLA has good biocompatibility. The dynamic cultivation system can promote the cell proliferation, distribution, and alignment on the surface of the composite, so it can be used for tissue engineered composite in vitro.
ObjectiveTo summarize the research progress of tissue-engineered bile duct in recent years.
MethodsThe related literatures about the tissue-engineered bile duct were reviewed.
ResultsIn recent years, the research of tissue-engineered bile duct has made a breakthrough in scaffold materials, seed cells, growth factors etc. However, the tissue-engineered bile duct is still in the research stage of animal experiments, which can not be directly applied to clinical practice.
ConclusionsThe research of tissue-engineered bile duct becomes popular at present. With the rapid development of materials science and cell biology, the basic research and clinical application of tissue-engineered duct will be more in-depth research and extension, which might bring new ideas and therapeutic measures for patients with biliary defect or stenosis.
ObjectiveTo explore the feasibility of chitosan/allogeneic bone powder composite porous scaffold as scaffold material of bone tissue engineering in repairing bone defect.
MethodsThe composite porous scaffolds were prepared with chitosan and decalcified allogeneic bone powder at a ratio of 1∶5 by vacuum freeze-drying technique. Chitosan scaffold served as control. Ethanol alternative method was used to measure its porosity, and scanning electron microscopy (SEM) to measure pore size. The hole of 3.5 mm in diameter was made on the bilateral femoral condyles of 40 adult Sprague Dawley rats. The composite porous scaffolds and chitosan scaffolds were implanted into the hole of the left femoral condyle (experimental group) and the hole of the right femoral condyle (control group), respectively. At 2, 4, 8, and 12 weeks after implantation, the tissues were harvested for gross observation, histological observation, and immunohistochemical staining.
ResultsThe composite porous scaffold prepared by vacuum freeze-drying technique had yellowish color, and brittle and easily broken texture; pore size was mostly 200-300μm; and the porosity was 76.8%±1.1%, showing no significant difference when compared with the porosity of pure chitosan scaffold (78.4%±1.4%) (t=-2.10, P=0.09). The gross observation and histological observation showed that the defect area was filled with new bone with time, and new bone of the experimental group was significantly more than that of the control group. At 4, 8, and 12 weeks after implantation, the bone forming area of the experimental group was significantly larger than that of the control group (P < 0.05). The immunohistochemical staining results showed that osteoprotegerin (OPG) positive expression was found in the experimental group at different time points, and the positive expression level was significantly higher than that in the control group (P < 0.05).
ConclusionChitosan/allogeneic bone powder composite porous scaffold has suitable porosity and good osteogenic activity, so it is a good material for repairing bone defect, and its bone forming volume and bone formation rate are better than those of pure chitosan scaffold.
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.
ObjectiveTo summarize the research progress of several three-dimensional (3-D) printing scaffold materials in bone tissue engineering.
MethodThe recent domestic and international articles about 3-D printing scaffold materials were reviewed and summarized.
ResultsCompared with conventional manufacturing methods, 3-D printing has distinctive advantages, such as enhancing the controllability of the structure and increasing the productivity. In addition to the traditional metal and ceramic scaffolds, 3-D printing scaffolds carrying seeding cells and tissue factors as well as scaffolds filling particular drugs for special need have been paid more and more attention.
ConclusionsThe development of 3-D printing porous scaffolds have revealed new perspectives in bone repairing. But it is still at the initial stage, more basic and clinical researches are still needed.
Objective To review the research status of the neovascularization of adi pose tissue engineering in the past decade so as to provide theoretical references for the development of the rapid revascularization of tissue engineered adi pose. Methods The l iterature about the revascularization of adi pose tissue engineering was extensively reviewed andanalyzed, centering on 5 elements: specificity of histological structures and blood supply, revascularization mechanism, coculture of different seed cells, modification of scaffold, and microenvironment. Results Adi pose tissue engineering offers a new solution for soft tissue defects. However, there is still the unfulfilled need in the size of engineered adipose tissue (less than 1 mL), which was determined by the degree of neovascularization in engineered tissue. Overall, rapid neovascularization in engineering tissue is a key l ink of experimental study changing into cl inical appl ication. Conclusion Providing a sufficient supply with nutrients and oxygen by means of a sufficient and rapid neovascularization will be at the heart of any attempts to obtain bigger tissue engineered adipose to meet the demand of repairing large soft tissue defect.
Objective To explore the method of preparing spongy and porous scaffold materials with swine articular cartilage acellular matrix and to investigate its appl icabil ity for tissue engineered articular cartilage scaffold. Methods Fresh swine articular cartilage was freeze-dried and freeze-ground into microparticles. The microparticles with diameter of less than 90 μm were sieved and treated sequentially with TNE, pepsin and hypotonic solution for decellularization at cryogenic temperatures. Colloidal suspension with a mass/volume ratio of 2% was prepared by dissolving the microparticles into 1.5% HAc, and then was lyophil ized for molding and cross-l inked by UV radiation to prepare the decellularized cartilage matrix sponge. Physicochemical property detection was performed to identify aperture, porosity and water absorption rate. Histology and scanning electron microscope observations were conducted. The prepared acellular cartilage matrix sponge was implanted into the bilateral area of spine in 24 SD rats subcutaneously (experimental group), and the implantation of Col I sponge served as control group. The rats were killed 1, 2, 4, and 8 weeks after operation to receive histology observation, and the absorption and degeneration conditions of the sponge in vivo were analyzed. BMSCsobtained from femoral marrow of 1-week-old New Zealand white rabbits were cultured. The cells at passage 3 were cultured with acellular cartilage matrix sponge l ixivium at 50% (group A), acellular cartilage matrix sponge l ixivium at 100% (group B), and DMEM culture medium (group C), respectively. Cell prol iferation was detected by MTT method 2, 4, and 6 days after culture. Results The prepared acellular cartilage matrix sponge was white and porous. Histology observation suggested that the sponge scaffold consisted primarily of collagen without chondrocyte fragments. Scanning electron microscope demonstrated that the scaffold had porous and honeycomb-shaped structure, the pores were interconnected and even in size. The water absorption rate was 20.29% ± 25.30%, the aperture was (90.66 ± 21.26) μm, and the porosity of the scaffold was 90.10% ± 2.42%. The tissue grew into the scaffold after the subcutaneous implantation of scaffold into the SD rats, angiogenesis was observed, inflammatory reaction was mild compared with the control group, and the scaffold was degraded and absorbed at a certain rate. MTT detection suggested that there were no significant differences among three groups in terms of absorbance (A) value 2 and 4 days after culturing with the l ixivium (P gt; 0.05), but significant differences were evident among three groups 6 days after culturing with the l ixivium (P lt; 0.05). Conclusion With modified treatment and processing, the cartilage acellular matrix sponge scaffold reserves the main components of cartilage extracellular matrix after thorough decellularization, has appropriate aperture and porosity, and provides even distribution of pores and good biocompatibil ity without cytotoxicity. It can be used as an ideal scaffold for cartilage tissue engineering.
Objective To introduce the research advances of scaffold materials of intervertebral disc tissue engineering. Methods The recent original articlesabout the scaffolds in intervertebral disc tissue engineering were extensively reviewed. Results At present, agarose, alginate gel, collagentype Ⅰ, PLA, PGAare still major scaffold materials for intervertebral disc tissue engineering because of their good biocompatibility. Conclusion It is one of the popular studies on current intervertebral disc tissue engineering to explore the ideal scaffold materials.
Objective To explore the method of preparing the electrospinning of synthesized triblock copolymers of ε-caprolactone and L-lactide (PCLA) for the biodegradable vascular tissue engineering scaffold and to investigateits biocompatibil ity in vitro. Methods The biodegradable vascular tissue engineering scaffold was made by the electrospinning process of PCLA. A series of biocompatibil ity tests were performed. Cytotoxicity test: the L929 cells were cultured in 96-wellflat-bottomed plates with extraction media of PCLA in the experimental group and with the complete DMEM in control group, and MTT method was used to detect absorbance (A) value (570 nm) every day after culture. Acute general toxicity test: the extraction media and sal ine were injected into the mice’s abdominal cavity of experimental and control groups, respectively, and the toxicity effects on the mice were observed within 72 hours. Hemolysis test: anticoagulated blood of rabbit was added into the extracting solution, sal ine, and distilled water in 3 groups, and MTT method was used to detect A value in 3 groups. Cell attachment test: the L929 cells were seeded on the PCLA material and scanning electron microscope (SEM) observation was performed 4 hours and 3 days after culture. Subcutaneous implantation test: the PCLA material was implanted subcutaneously in rats and the histology observation was performed at 1 and 8 weeks. Results Scaffolds had the characteristics of white color, uniform texture, good elasticity, and tenacity. The SEM showed that the PCLA ultrafine fibers had a smooth surface and proper porosity; the fiber diameter was 1-5 μm and the pore diameter was in the range of 10-30 μm. MTT detection suggested that there was no significant difference in A value among 3 groups every day after culturing (P gt; 0.05). The mice in 2 groups were in good physical condition and had no respiratory depression, paralysis, convulsion, and death. The hemolysis rate was 1.18% and was lower than the normal level (5%). The SEM showed a large number of attached L929 cells were visible on the surface of the PCLA material at 4 hours after implantation and the cells grew well after 3 days. The PCLA material was infiltrated by the inflammatory cells after 1 week. The inflammatory cells reduced significantly and the fiber began abruption after 8 weeks. Conclusion The biodegradable vascular tissue engineering scaffold material made by the electrospinning process of PCLA has good microstructure without cytotoxicity and has good biocompatibil ity. It can be used as an ideal scaffold for vascular tissue engineering.
ObjectiveTo study the preparation and cytocompatibility of bone tissue engineering scaffolds by combining low temperature three dimensional (3D) printing and vacuum freeze-drying techniques.
MethodsCollagen (COL)and silk fibroin (SF) were manufactured from fresh bovine tendon and silkworm silk. SolidWorks2014 was adopted to design bone tissue engineering scaffold models with the size of 9 mm×9 mm×3 mm and pore diameter of 500μm. According to the behavior of composite materials that low temperature 3D printing equipment required, COL, SF, and nano-hydroxyapatite (nHA)at a ratio of 9:3:2 and low temperature 3D printing in combination with vacuum freeze-drying techniques were accepted to build COL/SF/nHA composite scaffolds. Gross observation and scanning electron microscope (SEM) were applied to observe the morphology and surface structures of composite scaffolds. Meanwhile, compression displacement, compression stress, and elasticity modulus were measured by mechanics machine to analyze mechanical properties of composite scaffolds. The growth and proliferation of MC3T3-E1 cells were evaluated using SEM, inverted microscope, and MTT assay after cultured for 1, 7, 14, and 21 days on the composite scaffolds. The RT-PCR and Western blot techniques were adopted to detect the gene and protein expressions of COL I, alkaline phosphatase (ALP), and osteocalcin (OCN) in MC3T3-E1 cells after 21 days.
ResultsCOL/SF/nHA composite scaffolds were successfully prepared by low temperature 3D printing technology and vacuum freeze-drying techniques; the SEM results showed that the bionic bone scaffolds were 3D polyporous structures with macropores and micropores. The mechanical performance showed that the elasticity modulus was (344.783 07±40.728 55) kPa; compression displacement was (0.958 41±0.000 84) mm; and compression stress was (0.062 15±0.007 15) MPa. The results of inverted microscope, SEM, and MTT method showed that a large number of cells adhered to the surface with full extension and good cells growth inside the macropores, which demonstrated a satisfactory proliferation rate of the MC3T3-E1 cells on the composite scaffolds. The RT-PCR and Western blot electrophoresis revealed gene expressions and protein synthesis of COL I, ALP, and OCN in MC3T3-E1 cells.
ConclusionLow temperature 3D printing in combination with vacuum freeze-drying techniques could realize multi-aperture coexisted bionic bone tissue engineering scaffolds and control the microstructures of composite scaffolds precisely that possess good cytocompatibility. It was expected to be a bone defect repair material, which lays a foundation for further research of bone defect.
