Objective To study the vascularization of the compositeof bio-derived bone and marrow stromal stem cells(MSCs) in repairing goat tibial shaft defect.Methods Bio-derived bone was processed as scaffold material. MSCs were harvested and cultured in vitro. The multiplied and induced cells were seeded onto the scaffold to construct tissue engineered bone. A 20 mm segmental bone defect inlength was made in the middle of the tibia shaft in 20 mature goats and fixed with plate. The right tibia defect was repaired by tissue engineered bone (experimental side), and the left one was repaired by scaffold material (control side).The vascularization and osteogenesis of the implants were evaluated by transparent thick slide, image analysis of the vessels, and histology with Chinese ink perfusion 2, 4, 6, and 8 weeks after operation.Results More new vessels were found in control side than in experimental side 2 and 4 weeks after implantation (Plt;0.05). After 8 weeks, there was no significant difference in number of vessels between two sides(Pgt;0.05), and the implants were vascularized completely. New bone tissue was formed gradually as the time and the scaffold material degraded quickly after 6 and 8 weeks in the experimental side. However, no new bone tissue was formed andthe scaffold degraded slowly in control side 8 weeks after operation.Conclusion Bio-derived bone has good quality of vascularization. The ability of tissue-engineered bone to repair bone defect is better than that of bio-derived bone alone.
Objective To investigate the possible mechanism of the fibroblasts inducing the vascularization of dermal substitute. Methods Fibroblasts were seeded on the surface of acellular dermal matrix and cultivated in vitro to construct the living dermal substitute. The release of interleukin 8 (IL 8) and transfonming growth factor β 1(TGF β 1) in culture supernatants were assayed by enzyme linked immunosorbent assay, the mRNA expression of acid fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) were detected by RT-PCR. Then, the living substtute was sutured to fullth ickness excised wound on BALBouml;C m ice, and the fate of fibroblast w as observed by using in situ hybridizat ion. Results Fibroblasts cultured on acellular dermalmat rix p ro liferated and reached a single2layer confluence. Fibroblasts could secret IL 28 (192. 3±15. 9) pgouml;m l and TGF-B1 (1. 105±0. 051) pgouml;m l. There w as the mRNA exparession of aFGF and bFGF. Fibroblasts still survived and proliferated 3 weeks after graft ing. Conclusion Pept ides secreted by fibroblasts and its survival after graft ing may be relat ive to the vascularizat ion of the dermal subst itute.
Objective To investigate the effect of repairing bone defect with tissue engineered bone seeded with the autologous red bone marrow (ARBM) and wrapped by the pedicled fascial flap and provide experimental foundation for cl inicalappl ication. Methods Thirty-two New Zealand white rabbits (male and/or female) aged 4-5 months old and weighing2.0-2.5 kg were used to make the experimental model of bilateral 2 cm defect of the long bone and the periosteum in the radius. The tissue engineered bone was prepared by seeding the ARBM obtained from the rabbits on the osteoinductive absorbing material containing BMP. The left side of the experimental model underwent the implantation of autologous tissue engineered bone serving as the control group (group A). While the right side was designed as the experimental group (group B), one 5 cm × 3 cm fascial flap pedicled on the nameless blood vessel along with its capillary network adjacent to the bone defect was prepared using microsurgical technology, and the autologous tissue engineered bone wrapped by the fascial flap was used to fill the bone defect. At 4, 8, 12, and 16 weeks after operation, X-ray exam, absorbance (A) value test, gross morphology and histology observation, morphology quantitative analysis of bone in the reparative area, vascular image analysis on the boundary area were conducted. Results X-ray films, gross morphology observation, and histology observation: group B was superior to group A in terms of the growth of blood vessel into the implant, the quantity and the speed of the bone trabecula and the cartilage tissue formation, the development of mature bone structure, the remolding of shaft structure, the reopen of marrow cavity, and the absorbance and degradation of the implant. A value: there was significant difference between two groups 8, 12, and 16 weeks after operation (P lt; 0.05), and there were significant differences among those three time points in groups A and B (P lt; 0.05). For the ratio of neonatal trabecula area to the total reparative area, there were significant differences between two groups 4, 8, 12, and 16 weeks after operation (P lt; 0.05), and there were significant differences among those four time points in group B (P lt; 0.05).For the vascular regenerative area in per unit area of the junctional zone, group B was superior to group A 4, 8, 12, and 16 weeks after operation (P lt; 0.05). Conclusion Tissue engineered bone, seeded with the ARBM and wrapped by the pedicled fascial flap, has a sound reparative effect on bone defect due to its dual role of constructing vascularization and inducing membrane guided tissue regeneration.
Objective To study the vascularization of the compositeof bone morphogenetic protein 2 (BMP-2) gene transfected marrow mesenchymal stem cells (MSCs) and biodegradable scaffolds in repairing bone defect. Methods Adenovirus vector carrying BMP-2 (Ad-BMP-2) gene transfected MSCs and gene modified tissue engineered bone was constructed. The 1.5 cm radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly(n=15, 30 sides). Different materials were used in 4 groups: Ad-BMP-2 transfected MSCs plus PLA/PCL (group A), AdLacz transfected MSCs plus PLA/PCL (group B), MSCs plus PLA/PCL (group C) and only PLA/PCL scaffolds (group D). The X-ray, capillary vessel ink infusion, histology, TEM, VEGF expression and microvacular density counting(MVD) were made 4, 8, and 12 weeks after operation. Results In group A after 4 weeks, foliated formed bones image was observed in the transplanted bones, new vessels grew into the bones, the pores of scaffolds were filled with cartilage callus, osteoblasts with active function grew around the microvessels, and VEGF expression and the number of microvessels were significantly superior to those of other groups, showing statistically significant difference (Plt;0.01); after 8 weeks, increasingly more new bones grew in the transplanted bones, microvessels distended and connected with each other, cartilage callus changed into trabecular bones; after 12 weeks, lamellar bone became successive, marrow cavity recanalized, microvessels showed orderly longitudinal arrangement. In groups B and C, the capability of bone formation was weak, the regeneration of blood vessels was slow, after 12 weeks, defects were mostly repaired, microvessels grew among the new trabecular bones. In group D, few new vessels were observed at each time, after 12 weeks, broken ends became hardened, the defectedarea was filled with fibrous tissue. Conclusion BMP-2 gene therapy, by -upregulating VEGF expression, indirectly induces vascularization ofgrafts,promotes the living of seed cells, and thus accelerates new bone formation.
The engineered heart tissues (EHTs) present a promising alternative to current materials for native myocardial tissue due to the unique characteristics. However, until now, the clinical application of EHTs is limited by a serial of practical problems yet. Generally, the challenges need to further optimize include biomaterials, cell sources, and strategies of revascularization or establishment of EHTs. This review focuses on the newly progress on these aspects to encourage the emergence of novel EHTs that can meet clinic requirement properly.
【Abstract】 Objective To investigate the impact of dermal papillary cells on vascularization of tissue engineered skinsubstitutes consisting of epidermal stem cells and allogeneic acellular dermal matrix. Methods Human foreskins from routinecircumcisions were collected to separate epidermal cells by using dispase with trypsogen. Collagen type IV was used to isolateepidermal stem cells from the 2nd and 3rd passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenaseI from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seedingepidermal stem cells on the papillary side of allogeneic acellular dermis with (the experimental group) or without (the controlgroup) seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skindefects (1 cm × 1 cm in size) on the back of the BALB/C-nu nude mice (n=30). The grafting survival rate was recorded 2 weeks aftergrafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate themicrovessel density at 2 and 4 weeks after grafting. Results Those adhesion cells by collagen type IV coexpressed Keratin 19 andβ1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papillary cells were identified by expressinghigh levels of α-smooth muscle actin. The grafting survival rate was significantly higher in experimental group (28/30, 93.3%), thanthat in control group (24/30, 80.0%). HE staining showed that the epithelial layer in experimental group was 12-layered with largeepithelial cells in the grafted composite skin, and that the epithelial layer in control group was 4-6-layered with small epithelial cells.At 2 and 4 weeks after grafting, the microvessel density was (38.56 ± 2.49)/mm2 and (49.12 ± 2.39)/mm2 in experimental group andwas (25.16 ± 3.73)/mm2 and (36.26 ± 3.24)/mm2 in control group respectively, showing significant differences between 2 groups(P lt; 0.01). Conclusion Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization,which promotes epidermis formation and improves the grafting survival rate.
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 study the effect of platelet-rich plasma (PRP) on the survival and quality of fat grafts in the nude mice so as to provide a method and the experimental basis for clinical practice.
Methods
Fat tissue was harvested from the lateral thigh of a 25-year-old healthy woman and the fat was purified by using saline. The venous blood was taken from the same donor. PRP was prepared by centrifugation (200 × g for 10 minutes twice) and activated by 10% calcium chloride (10 : 1). Then 24 female nude mice [weighing (20 ± 3) g, 5-week-old] were allocated randomly to the experimental group and the control group (12 mice per group). Each subcutaneous layer of two sides of the back (experimental group) was infiltrated with 0.8 mL fat tissue-activated PRP mixtures (10 : 2); the control group was infiltrated with 0.8 mL fat tissue-saline mixtures (10 : 2); 0.14 mL activated PRP and 0.14 mL saline were injected into the experimental group and the control group respectively at 5 and 10 days after the first operation. At 15, 30, 90, and 180 days after the first operation, the samples were harvested for gross and histological observations.
Results
All nude mice survived to the end of the experiment. No inflammation and abscess formation of the graft were observed. Experimental group was better than control group in angiogenesis, liquefaction, and necrosis. The grafted fat weight and volume in the experimental group were significantly larger than those in the control group at 15, 30, and 90 days (P lt; 0.05); but there was no significant difference between the 2 groups at 180 days (P gt; 0.05). Histological observation showed good morphological and well-distributed adipocytes, increasing vacuoles, few necrosis and calcification in the experimental group; but disordered distribution, obvious necrosis, and calcification in the control group. The necrosis area ratio of the experimental group was significantly lower than that of the control group (P lt; 0.05), and the number of micro-vessels was significantly higher in the experimental group than in the control group at 15 and 180 days (P lt; 0.05).
Conclusion
The method of repeatedly using the PRP within 180 days in assisting fat grafts can obviously improve the survival and quality.
Objective To compare the effect between vascularization osteogenesis and membrane guided osteogenesis in the bone repair by the tissue engineered bone with pedicled fascial flap packing autologous red bone marrow (ARBM), so as to provide a reference for the bone defect repair in cl inic. Methods The tissue engineered bone was constructed with ARBM and the osteoinductive absorbing recombinant human materials with recombinant human bone morphogenetic protein 2. Sixty New Zealand rabbits (aged 4-5 months, weighing 2.0-2.5 kg) were randomly divided into group A (n=16), group B (n=22), and group C (n=22). The complete periosteum defect model of 1.5 cm in length was prepared in right ulnar bone, then the tissue engineered bone was implanted in the bone defect area in group A, the tissue engineered bonewith free fascial flap in group B, and the tissue engineered bone with pedicled fascial flap in group C. At 4, 8, 12, and 16 weeks, the tissue of bone defect area was harvested from 4 rabbits of each group for the general, histological, and immunohistochemical staining observations; at 8, 12, and 16 weeks, 2 rabbits of groups B and C, respectively were selected to perform ink perfusion experiment by axillary artery. Results The general observation showed that the periosteum-l ike tissues formed in the fascial flap of groups B and C, chondroid tissues formed in group B, new bone formed in group C, and the fibrous and connective tissues in group A at 4 and 8 weeks; a few porosis was seen in group A, more new bone in group B, and bone stump formation in group C at 12 and 16 weeks. Histological observation showed that there were few new blood vessels and new bone trabeculae in groups A and B, while there were large amounts of new blood vessels and mature bone trabeculae in group C at 4 and 8 weeks. There were a few new blood vessels and new bone trabeculae in group A; more blood vessels, significantly increased mature trabeculae, and the medullary cavity formation in group B; and gradually decreased blood vessels, the mature bone structure formation, and the re-opened medullary cavity in group C at 12 and 16 weeks. The immunohistochemical staining observation showed that the levels of CD105, CD34, and factor VIII were higher in group C than in groups A and B at different time points.The bone morphometry analysis showed that the trabecular volume increased gradually with time in 3 groups after operation; the trabecular volume in group C was significantly more than those in groups A and B at different time points (P lt; 0.05); and there was significant difference between groups A and B (P lt; 0.05) except the volume at 4 weeks (P gt; 0.05). The vascular image analysis showed that the vascular regenerative area ratio in group C was significantly higher than those in groups A and B at different time points (P lt; 0.05). The ink perfusion experiment showed that the osteogenic zone had sparse ink area with no obvious change in group B, while the osteogenic zone had more intensive ink area and reached the peak at 8 weeks, then decreased in group C. Conclusion The tissue engineered bone with pedicled fascial flap packing ARBM has the vascularization osteogenesis effect at early stage, but the effect disappears at late stage gradually when the membrane guided osteogenesis is main.
【Abstract】 Objective To discuss the impact of adi pose-derived stem cells (ADSCs) combined with vascular bundle implantation on vascularized tissue engineering scaffolds in vivo so as to provide a theoretical basis for the repair ofavascular necrosis of the femoral head. Methods ADSCs were isolated from 4-month-old Sprague Dawley (SD) rats andcultured, then were induced to osteogenesis and identified. ADSCs at the 3rd passage were seeded on the nano-hydroxyapatide/ polyamide-66 (nHA/PA66) to prepare the composite scaffolds. The compound condition of cells and scaffold materials were observed under scanning electronic microscope (SEM). Twenty-four 4-month-old SD rats (weighing 350-400 g) were randomly divided into 3 groups (n=8). In group A and group B, the inferior epigastric artery and vein of rats were implanted into composite scaffold cultured for 10 days or simple nHA/PA66 scaffold, respectively. In group C, two composite scaffolds cultured for 10 days were embedded into quadriceps femoris muscle of both thighs, respectively. After 2 and 4 weeks of operation, angiogenesis was observed by HE staining and CD34 immunohistochemical staining. Results Cells isolated from adi pose were identified as ADSCs. SEM showed that the number of cells increased after being cultured for 10 days, cell morphology stretched fully with a shape of long spindle. HE staining and immunohistochemical staining showed that a large number of angiogenesis was observed around the implanted artery and vein in group A, which was superior to groups B and C in the number of blood vessels and the maturity of blood vessel wall. After 2 and 4 weeks of operation, the blood vessel density and blood vessel diameter were significantly higher in group A than in group B and group C, and in group B than in group C (P lt; 0.05). Conclusion Combined application of ADSCs and vascular bundle implantation can promote the degree of vascularization, which could make the scaffold vascularization rel iable.
