Objective To establish a scaffold model from heterogeneoussmall blood vessels. Methods Caudal arteries from 34 Wistar rats( average length 12.08±1.69 cm) were made into acellular blood vessel scaffolds. Some scaffoldswere observed by electron microscope, and others were transplanted to the cut ends of ear central arteries of male Japanese big ear white rabbits. Results Average external diameter was 0.74±0.08 mm in proximal, and 0.55±0.08 mm in distal end of rat caudal arteries. The small blood vessel scaffolds had shin wall whichwas white and soft, composed of fibrous tissues without cells. On the intima surface the fibrous tissues were arrayed densely in a grid-like pattern. After transplantation, the blood flow was reserved, and kept flowing freely in 24 hours. The pulsation of the transplanted artery was accessible and no blood leakage wasfound.Conclusion The natural scaffolds are composed of fibrous tissues, and can sustain the artery pulse pressure for 24 hours. It is better to suture the blood vessels by sleeve anastomosis.
Objective To study the influence of in vitro force-vascularization on in vivo vascularization of porous polylactic glycolic acid copolymer(PLGA) scaffolds with internal network channels (PPSINC). Methods After the in vitro forcevascula ization of PPSINCs covered with microvessel endothelial cells (MVEC) of mice, they were divided into two groups: the force-vascularization group (group A) and the control group with only PSINCs (group B). All the PPSINCs were planted in the mesentery of 12 mice for 2 and 4 weeks, the PPSINCs were cut out, the vascular ization of PPSINCs was investigated by histology and immunohistochemistry, and the vascularization area of the histologic section of the PPSINCswas measured with the computer-assistant image analysis system. Result After the in vitro forcevascularization of PPSINCs, the MVEC of the mice sticking on the channel wall could be seen. After the scaffold was im planted into the mice for 2 weeks, the vascularization area of the histologic section of PPSINCs (VA) in group A (2 260.91±242.35 μm2) was compared with that in group B (823.64±81.29 μm2)，and the difference was sig nificant in statistics（P＜0.01).The VA for 4 weeks in group A (17 284.36 ±72.67 μm2) was compared with that in group B (17 041.14±81.51 μm2), and the difference was not significant in statistics（P＞0.05）.The area of the actin positivestaining (AA) in the histologi c section of PPSINCs for 2 weeks’ implantation in group A (565.22±60.58 μm2) was compared with that in group B (205.91±16.25 μm2), and the difference was signi ficant in statistics（P＜0.01). After the implantation for 4 weeks, the VA in group A (4 321.09±19.82 μm2) was compared with group B (4 260.28±27.17 μm2), and the difference was not significant in statistics（P＞0.05）. Conclusion The PPSINC is a good simple scaffold model of vasculariazation. The in vitro force-vascularization can increase the in vivo vascularization of PPSINCs in the early stage.
Objective To evaluate the feasibility and validity of chondrogenic differentiation of marrow clot after microfracture of bone marrow stimulation combined with bone marrow mesenchymal stem cells (BMSCs)-derived extracellular matrix (ECM) scaffold in vitro. Methods BMSCs were obtained and isolated from 20 New Zealand white rabbits (5-6 months old). The 3rd passage cells were cultured and induced to osteoblasts, chondrocytes, and adipocytes in vitro, respectively. ECM scaffold was manufactured using the 3rd passage cells via a freeze-dying method. Microstructure was observed by scanning electron microscope (SEM). A full-thickness cartilage defect (6 mm in diameter) was established and 5 microholes (1 mm in diameter and 3 mm in depth) were created with a syringe needle in the trochlear groove of the femur of rabbits to get the marrow clots. Another 20 rabbits which were not punctured were randomly divided into groups A (n=10) and B (n=10): culture of the marrow clot alone (group A) and culture of the marrow clot with transforming growth factor β3 (TGF-β3) (group B). Twenty rabbits which were punctured were randomly divided into groups C (n=10) and D (n=10): culture of the ECM scaffold and marrow clot composite (group C) and culture of the ECM scaffold and marrow clot composite with TGF-β3 (group D). The cultured tissues were observed and evaluated by gross morphology, histology, immunohistochemistry, and biochemical composition at 1, 2, 4, and 8 weeks after culture. Results Cells were successfully induced into osteoblasts, chondrocytes, and adipocytes in vitro. Highly porous microstructure of the ECM scaffold was observed by SEM. The cultured tissue gradually reduced in size with time and disappeared at 8 weeks in group A. Soft and loose structure developed in group C during culturing. Chondroid tissue with smooth surface developed in groups B and D with time. The cultured tissue size of groups C and D were significantly larger than that of group B at 4 and 8 weeks (P lt; 0.05); group D was significantly larger than group C in size (P lt; 0.05). Few cells were seen, and no glycosaminoglycan (GAG) and collagen type II accumulated in groups A and C; many cartilage lacunas containing cells were observed and more GAG and collagen type II were synthesized in groups B and D. The contents of GAG and collagen increased gradually with time in groups B and D, especially in group D, and significant difference was found between groups B and D at 4 and 8 weeks (P lt; 0.05). Conclusion The BMSCs-derived ECM scaffold combined with the marrow clot after microfracture of bone marrow stimulation is effective in TGF-β3-induced chondrogenic differentiation in vitro.
Objective To investigate the cellular compatibil ity of polyvinyl alcohol (PVA)/wild antheraea pernyisilk fibroin (WSF), and to explore the feasibil ity for tendon tissue engineering scaffold in vitro. Methods The solutions of WSF (11%), PVA (11%), and PVA/WSF (11%) were prepared with 98% formic acid (mass fraction) at a mass ratio of 9 : 1. The electrospinning membranes of WSF, PVA, and PVA/WSF were prepared by electrostatic spinning apparatus. The morphologies of scaffolds were evaluated using scanning electronic microscope (SEM). The tendon cells were isolated from tail tendon of 3-dayold Sprague Dawley rats in vitro. The experiment was performed using the 3rd generation cells. The tendon cells (1 × 106/mL) were cocultured with PVA and PVA/WSF electrospinning film, respectively, and MTT test was used to assess the cell adhesion rate 4, 12 hours after coculture. The tendon cells were cultured in PVA and PVA/WSF extraction medium of different concentration (1, 1/2, and 1/4), respectively; and the absorbance (A) values were detected at 1, 3, 5, and 7 days to evaluate the cytotoxicity. The composite of tendon cells and the PVA or PVA/WSF scaffold were observed by HE staining at 7 days and characterized by SEM at 1,3, 5, and 7 days. Results The solution of WSF could not be used to electrospin; and the solution of PVA and PVA/WSF could be electrospun. After coculture of tendon and PVA or PVA/WSF electrospinning membranes, the cell adhesion rates were 26.9% ±0.4% and 87.0% ± 1.0%, respectively for 4 hours, showing significant difference (t=100.400, P=0.000); the cell adhesion rates were 35.2% ± 0.6% and 110.0% ± 1.7%, respectively for 12 hours, showing significant difference (t=42.500, P=0.000). The cytotoxicity of PVA/WSF was less significantly than that of PVA (P lt; 0.05) and significant difference was observed between 1/2 PVA and 1/4PVA (P lt; 0.05). HE staining and SEM images showed that the tendon cells could adhere to PVA and PVA/WSF scaffolds, but that the cells grew better in PVA/WSF scaffold than in PVA scaffold in vitro. Conclusion PVA/WSF electrospinning membrane scaffold has good cell compatibility, and it is expected to be an ideal scaffold of tendon tissue engineering.
Objective To observe the biocompatibility of the acellular corneal stroma materials prepared by three different methods. Methods Three different serial digestion methods were used to produce the acellular corneal stroma materials. The biocompatibility of the materials was investigated by the cell seeding and the materials were implanted into the rabbit corneal stroma layer. Results The cells in the materials 1 and 2 were not decellularized completely. The rabbit corneal fibroblasts died on the materials 1 and 2 after the cell seeding for 3-4 days. An obvious rejection could be observed after the implantation. The cells in material 3 were decellularized completely and the collagen fibers or elastic fibers were reserved integrally，showing a typical three-dimensional net work. The rabbit corneal fibroblasts could expand on the materials in vitro. No obvious rejection could be observed and the materials were gradually absorbed. Conclusion The acellular porcine cornea stroma materials prepared by trypsin-Dnase-Rnase are suitable for reconstruction of the tissue engineered cornea.
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.
Objective To investigate the application potential of alginate-strontium (Sr) hydrogel as an injectable scaffold material in bone tissue engineering. Methods The alginate-Sr/-calcium (Ca) hydrogel beads were fabricated by adding 2.0wt% alginate sodium to 0.2 mol/L SrCl2/CaCl2 solution dropwise. Microstructure, modulus of compression, swelling rate, and degradability of alginate-Sr/-Ca hydrogels were tested. Bone marrow mesenchymal stem cells (BMSCs) were isolated from femoral bones of rabbits by flushing of marrow cavity. BMSCs at passage 5 were seeded onto the alginate-Sr hydrogel (experimental group) and alginate-Ca hydrogel (control group), and the viability and proliferation of BMSCs in 2 alginate hydrogels were assessed. The osteogenic differentiation of cells embeded in 2 alginate hydrogels was evaluated by alkaline phosphate (ALP) activity, osteoblast specific gene [Osterix (OSX), collagen type I, and Runx2] expression level and calcium deposition by fluorescent quantitative RT-PCR and alizarin red staining, Von Kossa staining. The BMSCs which were embeded in alginate-Ca hydrogel and cultured with common growth medium were harvested as blank control group. Results The micromorphology of alginate-Sr hydrogel was similar to that of the alginate-Ca hydrogel, with homogeneous pore structure; the modulus of compression of alginate-Sr hydrogel and alginate-Ca hydrogel was (186.53 ± 8.37) and (152.14 ± 7.45) kPa respectively, showing significant difference (t=6.853, P=0.002); there was no significant difference (t=0.737, P=0.502) in swelling rate between alginate-Sr hydrogel (14.32% ± 1.53%) and alginate-Ca hydrogel (15.25% ± 1.64%). The degradabilities of 2 alginate hydrogels were good; the degradation rate of alginate-Sr hydrogel was significantly lower than that of alginate-Ca hydrogel on the 20th, 25th, and 30th days (P lt; 0.05). At 1-4 days, the morphology of cells on 2 alginate hydrogels was spherical and then the shape was spindle or stellate. When three-dimensional cultured for 21 days, the DNA content of BMSCs in experimental group [(4.38 ± 0.24) g] was significantly higher than that in control group [(3.25 ± 0.21) g ] (t=8.108, P=0.001). On the 12th day after osteogenic differentiation, the ALP activity in experimental group was (15.28 ± 1.26) U/L, which was significantly higher than that in control group [(12.07 ± 1.12) U/L] (P lt; 0.05). Likewise, the mRNA expressions of OSX, collagen type I, and Runx2 in experimental group were significantly higher than those in control group (P lt; 0.05). On the 21th day after osteogenic differentiation, alizarin red staining and Von Kossa staining showed calcium deposition in 2 groups; the calcium nodules and phosphate deposition in experimental group were significantly higher than those in control group (P lt; 0.05). Conclusion Alginate-Sr hydrogel has good physicochemical properties and can promote the proliferation and osteogenic differentiation of BMSCs, so it is an excellent injectable scaffold material for bone tissue engineering.
Objective To fabricate a nanohydroxyapatite-chitosan(nano-HA-CS) scaffold with high porosity by a simple and effective technique and to evaluate the physical and chemical properties and the cytocompatibility of the composite scaffold. Methods The threedimensional nano-HA-CS scaffolds with high porosity were prepared by the in situ hybridization-freeze-drying method. The microscopic morphology and components of the composite scaffolds were analyzed by the scanning electron microscopy (SEM), the transmission electron microscopy（TEM）, the X-ray diffraction（XRD）examination, and the Fourier transformed infrared spectroscopy（FTIR）. The calvarial osteoblasts were isolated from the neonatal Wistar rats. The serial subcultured cells (3rd passage) were respectively seeded onto the nanoHACS scaffold and the CS scaffold, and then were cocultured for 2, 4, 6 and 8 hours. At each time point,four specimens from each matrix were taken to determine the celladhesion rate. The cell morphology was observed by the histological staining and SEM. Results The macroporous nanoHACS scaffolds had a feature of high porosity with a pore diameter from 100 to 500 μm (mostly 400500 μm). The scaffolds had a high interval porosity; however, the interval porosity was obviously decreased and the scaffold density was increased with an increase in the contents of CS and HA. The SEM and TEM results showed that the nanosized HA was synthesized and was distributed on the pore walls homogeneously and continuously. The XRD and FTIR results showed that the HA crystals were carbonatesubstituded and not wellcrystallized. The cytocompatibility test showed that the seeded osteoblasts could adhere the scaffolds, proliferating and producing the extracellular matrix on the scaffolds. The adherence rate for the nanoHACS scaffolds was obviously higher than that for the pure CS scaffolds. Conclusion The nano-HA-CS scaffolds fabricated by the in situ hybridization-freeze-drying method have a good physical and chemical properties and a good cytocompatibility; therefore, this kind of scaffolds may be successfully used in the bone tissue engineering.
ObjectiveTo review the research progress of the tissue engineering technique in the esophageal defect repair and reconstruction. MethodsThe recently published clinical and experimental literature at home and abroad on the scaffold materials and the seeding cells used in the tissue engineered esophageal reconstruction was consulted and summarized. ResultsA large number of basic researches and clinical applications show that the effect of the tissue engineered esophagus is close to the autologous structure and function of the esophagus and it could be used for the repair of the esophageal defect. However, those techniques have a long distance from the clinical application and need an acknowledged rule of technology. ConclusionTissue engineering technique could provide an innovative theory for the esophageal defect reconstruction, but its clinical application need further research.
ObjectiveTo review the research progress of tissue engineered ligament. MethodsThe literature in recent years on tissue engineered ligament in repair of anterior cruciate ligament (ACL) injury was extensively reviewed, including cell sources, scaffold materials, growth factors, and mechanical stimulation in tissue engineered ligament. ResultsTissue engineered ligament constructed by mesenchymal stem cells and ACL fibroblasts has been successfully used in animal experiments. It is crucial for qualified tissue engineered ligament to choose appropriate seed cells, scaffold, mechanical stimulation, and essential cytokines. To further optimize culture condition and how to realize the tissue engineered ligament in vivo better survival and prognosis need to be further studied. ConclusionEnormous progress has been made in tissue engineered ligament for repair and regeneration of ACL. With the development of biochemistry and scaffold materials, tissue engineered ligament will be used in clinic in the near future.