OBJECTIVE To investigate the methods to fabricate repair materials of tissue engineered peripheral nerve with bioactivity of Schwann cells (SC). METHODS 1. The materials were made by dry-wet spinning process to fabricate PLA hollow fiber canal with external diameter of 2.3 mm, internal diameter of 1.9 mm, thickness of 0.4 mm, pore size of 20 to 40 microns, pore ratio of 70% and non-spinning fiber net with pore size of 100 to 200 microns, pore ratio of 85%. 2. SC were implanted into excellular matrix (ECM) gel to observe the growth of SC. 3. SC/ECM complex were implanted into non-spinning PLA fiber net to observe the growth of SC. 4. SC, SC/ECM and SC/ECM/PLA were implanted into PLA hollow fiber canal to bridge 10 mm defect of rat sciatic nerve. RESULTS 1. SC were recovered bipolar shape at 1 day after implantation, and could be survived 14 days in ECM gel. 2. After SC/ECM complex was implanted into PLA net, most of SC were retained in the pore of PLA net with the formation of ECM gel. SC could be adhered and grown on PLA fiber. 3. Most of SC in ECM gel could be survived to 21 days after transplantation. Survival cell numbers of SC/ECM and SC/ECM/PLA groups were obviously higher than SC suspension group. CONCLUSION Non-spinning PLA porous biodegradable materials with ECM is benefit for SC to be adhered and grown.
Objective To determine whether the transforminggrowth factor β1 (TGF-β1) is a key regulatory molecule required for an increase or a balance of extracellular matrix (ECM) and DNA synthesis in the goat passaged nucleus pulposus (NP) cells. Methods The NP cells isolated from the goat intervertebral discs were cultured in vitro for a serial of passages and transfected with the replicationincompetent adenoviral vectors carrying the human TGF-β1 (hTGF-β1) or lacZ genes. Then, they were cultured in monolayer or alginate bead 3dimensional (3-D) systems for 10 days.The changes in the production and the molecular components of ECM that occurredin the NP cells transfected with Ad/hTGF-β1 or the controls were evaluated by Westernblot and absorbance of glycosaminoglycan (GAG)-Alcian Blue complexes. Differences of DNA synthesis in the variant cells and culture systems were assessed by fluorometric analysis of the DNA content. ResultsA quantitation in the variant culture systems indicated that in monolayers the NP cells at Passage 3 transfected with Ad/hTGF-β1 had a much higher cell viability and more DNA synthesis(P＜0.05); however, in the alginate 3-D culture system, the NP cells transfected with Ad/hTGF-β1 did not have any significant difference from the controls(P＞0.05). The Western blotting analysis ofthe protein sample isolated from the variant cells for TGF-β1, type Ⅱ collagen, and Aggrecan expression indicated that in the monolayers and alginate 3-D culture systems the NP cells at Passage 3 transfected with Ad/hTGF-β1 revealed much higher protein levels than the controls(P＜0.05)； whereas the type Ⅰcollagen content was much lower than the controls (P＜0.05), but a significatly increased ratio of type Ⅱ/type Ⅰ collagen was found in both of the cell culture systems(P＜0.05). The GAG quantification also showed a positive result in both the cell culture systems and the NP cells at Passage 3 transfected with Ad/hTGF-β1 had a much higher GAG content than the controls(P＜0.05). Conclusion To a greaterextent, hTGF-β1 can play a key role in maintaining the phenotype of the NP cells and can still have an effect of the phenotypic modulation after a serial of the cell passages. The NP cells that are genetically manipulated to express hTGF-β1 have a promising effect on the restoration of the intervertebral disc defects. The NP cells transfected with Ad/hTGF-β1 cultured in the 3-D alginate bead systems can show a nearly native phenotype.
ObjectiveTo review the properties of bio-derived hydrogels and their application and research progress in tissue engineering. MethodsThe literature concerning the biol-derived hydrogels was extensively reviewed and analyzed. ResultsBio-derived hydrogels can be divided into single-component hydrogels (collagen,hyaluronic acid,chitosan,alginate,silk fibroin,etc.) and multi-component hydrogels[Matrigel,the extract of extracellular matrix (ECM),and decellularized ECM].They have favorable biocompatibility and bioactivity because they are mostly extracted from the ECM of biological tissue.Among them,hydrogels derived from decellularized ECM,whose composition and structure are more in line with the requirements of bionics,have incomparable advantages and prospects.This kind of scaffold is the closest to the natural environment of the cell growth. ConclusionBio-derived hydrogels have been widely used in tissue engineering research.Although there still exist many problems,such as the poor mechanical properties,rapid degradation,the immunogenicity or safety,vascularization,sterilization methods,and so on,with the deep-going study of optimization mechanism,desirable bio-derived hydrogels could be obtained,and thus be applied to clinical application.
Objective To evaluate an effect of the vascularendothelial growth factor (VEGF) geneactivated matrix (GAM) on repair of the sciatic nerve defect in rats. Methods The peripheral nerve extracellular matrix（ECM） was harvested by the chemical extraction from 30 SD rats. The VEGF-GAM comprised of ECM and the plasmids encoding VEGF. Thirty adult Wistar rats were made as a model of the asciatic nerve defect and were randomly divided into the following 3 groups(n=10): Group A (VEGF-GAM conduits), Group B (ECM conduits),and Group C (autografts). At 12 weeks, the rats from each groupwere subjected to an inspection for the walking tract analysis and electrophysiological and histomorphological studies.Results The VEGF DNA could be retained in GAM, promoting the transgene expressing in the sciatic nerve, and more importantly, in the axotomized neurons in the spinal cord for 12 weeks. The motor neuron recovery rate in Group A (79.13％±2.53％) was similar to that in Group C (75.26％±4.48％, Pgt;0.05), but significantly better than that in Group B (56.09％±1.89％, Plt;0.01). The number of the regenerationaxons in the distal sciatic nerve in Group A (13 463±794/mm2) was significantly lower than that in Group C (16 809±680/mm2, Plt; 0.01), but significantly higher than that in Group B (10 260±1 117/mm2,Plt;0.01). The motor nerve conduction velocity in Group A (16.44±1.65 m/s) was significantly lowerthan that in Group C (23.79±2.75 m/s, Plt;0.01), but significantly higherthan that in Group B (12.8 ±1.42 m/s, Plt;0.01). The recovery rate of thegastrocnemius muscle wet weight in Group A (71.40％±3.05%) was significantlylower than that in Group C (87.00％±1.87％,Plt;0.01), but significantly higher than that in Group B (50.00％±4.90％, Plt;0.01). The sciatic nerve function index in Group A （39.37%±4.81%） was significantly lower 〖KG6〗than that in Group C (26.27%±2.71%, Plt;0.01), but significantly higher than that in Group B (4693％±296％, Plt;0.01). Conclusion The results indicate that VEGF-GAM as a bridge can promote the functional recovery of the defected sciatic nerve in rats, but the effect is not so good as that by autografts.
Aortic aneurysm and dissection are critical cardiovascular diseases that threaten human life and health seriously. No pharmacological treatment can effectively prevent disease progression. The imbalance of aortic wall cells and non-cellular components leads to structural or functional degeneration of the aorta, which is a prerequisite for disease occurrence. As the important non-cellular component, extracellular matrix (ECM) is crucial to maintain the aortic structure, function, and homeostasis. Abnormal production of elastin and collagen, destruction of cross-linking between elastic fibers and collagen fibers, and the imbalance of metalloproteinase and inhibitors leads to excessive degradation of ECM proteins, all of which have destroyed the structure and function of aorta. It will provide more ideas for disease prevention and treatment by learning ECM proteins and their metabolic mechanism. Here, we focus on the ECM proteins that have been reported to be involved in aortic aneurysm and dissection, and discuss the regulatory mechanism of metalloproteinase and inhibitors.
This paper reviewed the main achievements in the research on tissue engineering tendon, focusing on major problems concerning the substitute for extracellular matrix (ECM) of tendon, biological characteristics of tendon cells, and tendon cells compounding with ECM substitute. It was concluded the important problems in the study of the tissue engineering having specific reparative functions could be: to prepare the ECM materials suitable for the tendon cells to attach, grow, and function; to establish the tendon cell line whose growth, proliferation, and immunological antigenicity could be modulated and controlled, and simulating the mechanical environment of tendon in vivo, to adopt three-dimensional tendon cell culture method.
ObjectiveTo investigate the effect of hydrogel from acellular porcine adipose tissue (HAPA) on the survival of transplanted adipose tissue.MethodsFor in vitro study, adipose tissue and HAPA-adipose tissue complex were cultured in normoxia and hypoxia atmospheres for 24 and 72 hours. TUNEL and Perilipin immunofluorescence staining were performed to observe the effect of HAPA on apoptosis and survival of adipocities. For in vivo study, 42 healthy male nude mice (4-6 weeks old) weighing 15-18 g were randomly divided into adipose group (group A), 10%HAPA group (group B), 20%HAPA group (group C), 30%HAPA group (group D), 40%HAPA group (group E), and 50%HAPA group (group F) according to different HAPA/adipose tissue volume ratio (n=7). For each group, 1 mL adipose tissue or HAPA-adipose tissue complex was injected subcutaneously into the dorsum of the nude mice. At 4 weeks after transplantation, 7 nude mice in each group were sacrificed and grafts were harvested, gross observation, volume measurement, ultrasound examination, and histologic staining (HE staining, CD31 and Perilipin immunofluorescence stainings) were applied.ResultsHypoxia showed a tendency of promoting adipose tissue necrosis and apoptosis, while HAPA exhibited an obvious effect of inhibiting cell apoptosis in vitro study (P<0.05). For in vivo study, grafts of all groups had intact fibrocapsule. No obvious signs of infection and necrosis were observed at 4 weeks. Volume shrinkage was observed in all groups, however, the groups A-D had significantly higher volume retention rate than groups E and F (P<0.05). Ultrasound examination showed that there were no significant difference in the number and volume of liquify area of the grafts in each group (P>0.05). With the increase of HAPA’s volume ratio, HE staining proved an improved fat integrity while a gradually decreased vacuoles and fibrosis. CD31 immunohistochemical staining showed that the number of neo-vascularisation in groups E and F were significantly higher than those in groups A-D (P<0.05). Perilipin immunofluorescence staining showed that with the increase of HAPA volume ratio, the number of living adipocytes increased gradually, and more new adipocytes could be seen in the field of vision.ConclusionAs the volume ratio of HAPA gradually increased, the survival of transplanted adipose tissue also increased, but the volume retention rate decreased gradually. 30%HAPA was considered the relative optimal volume ratio for its superior adipose tissue survival and volume retation rate.
Decellularized extracellular matrix (dECM) has been widely used as a scaffold for regenerative medicine due to its high biomimetic and excellent biocompatibility. As a functional polymer material with high water content and controlled fluidity, hydrogel is very promising for some minimally invasive surgery in clinical practice. In recent years, with the rapid development of hydrogel theory and technology, dECM hydrogel has gradually become a research hotspot in the field of regenerative medicine. In this paper, the related researches in recent years are reviewed regarding the preparation of dECM hydrogel and its preclinical application. The future clinical use is also prospected.
Objective To review the decellularized methods for obtaining extracellular matrix (ECM) and the applications of decellularized ECM scaffold in tissue engineering. Methods Recent and related literature was extensively and comprehensively reviewed. The decellularized methods were summarized and classified. The effects of different sterilization methods on decellularized scaffolds were analyzed; the evaluation criterion of extent of decellularization was put forward; and the application of decellularized ECM scaffold in different tissues and organs engineering field was summarized. Results The decellularized methods mainly include physical methods, chemical methods, and biological methods, and different decellularization methods have different effects on the extent of cell removal and ECM composition and structure. Therefore, the best decellularization method will be chosen according to the characteristics of the tissues and decellularization methods to achieve the ideal result. Conclusion It is very important to choose the appropriate decellularized method for preparing the biological materials desired by tissue engineering. The biological scaffolds prepared by decellularized methods will play an important role in tissue engineering and regenerative medicine.
Objective To evaluate tissue regeneration, body reaction, and biological safety of xenogeneous bladder acellular matrix (BAM) that can be used to repair rabbit bladder. Methods Porcine BAM was prepared through physical, chemical, and enzymatic methods, and the effects of acellularization and the structure were observed with HE staining and scanning electron microscope (SEM). Eighteen New Zealand white rabbits (weighing, 2.5-3.0 kg) undergoing partial cystectomy were randomly divided into 2 groups. After partial (about 30%) cystectomy, the porcine BAM was used to replace partial rabbit bladder in the experimental group (n=12), and the incision was directly sutured as control group (n=6). The survival condition of animals was observed after operation. At 15 days, 1, 2, 3, and 6 months after operation, the blood routine, renal function, and electrolyte were tested by collecting the blood samples. At 1, 2, 3, and 6 months after operation, maximum bladder capacity, bladder leak point pressure, and bladder compliance were measured through urodynamic studies. Then gross observation was performed for regeneration of bladder, and the specimens of the bladder were harvested for HE staining and immunohistochemical staining. The surrounding organs and local lymphoid tissues were harvested for gross observation and HE staining. Results Cell components were completely removed in the porcine BAM, showing three-dimensional porous structure under SEM. All the animals survived during the experiment. At 15 days after operation, white blood cell count increased, and then returned to normal level in 2 groups, showing no significant difference between 2 groups (P gt; 0.05). The tests of renal function and electrolyte suggested no significant difference between 2 groups (P gt; 0.05). The level of serum creatinine showed a tendency of increase, but it remained within normal range at 6 months after operation. The maximum bladder capacity and compliance in experimental group were significantly higher than those in control group at 3 and 6 months after operation (P lt; 0.05), but no significant difference in bladder leak point pressure at each time point between 2 groups (P gt; 0.05). The urothelial regeneration, smooth muscle regeneration, and blood vessel regeneration were seen by histological observation in 2 groups. In the 2 groups, chronic inflammatory cells infiltration could be observed at 1 month postoperatively, and then chronic inflammatory cells decreased significantly (P lt; 0.05), until complete disappearance. There was no significant difference in score of chronic inflammatory cell infiltration between 2 groups at 3 and 6 months after operation (P gt; 0.05). The α-smooth muscle actin expression was significantly increased with time passing in 2 groups (P lt; 0.05), and it was significantly higher in control group than in experimental group at each time point (P lt; 0.05). In addition, gross and HE staining observations showed no abnormalities in surrounding organs and local lymphoid tissues. Conclusion No immune rejection response occurs when porcine BAM is used for xenotransplantation. It is indicated that porcine BAM is relative safety for xenotransplantation.