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.
To explore the method of inducing axial vascularization in a processed bovine cancellous bone scaffold by using an arteriovenous loop, and to evaluate its effect of vascularization. Methods Custom-made processed bovine cancellous bone discs were processed into cyl inder with circular grooves. Thirty male SD rats weighing 300-350 g (3-4 months old) were randomly divided into 2 groups (n=15 per group): experimental group in which the femoral veins in the groin of rats were separated and transplanted to the contralateral femoral artery and vein stump, the processed bovine cancellousbone scaffold was inserted into the arteriovenous loop, which was placed into the annular groove. Control group, in which the blood vessels in the groin of rats were cut, no anastomosis was conducted, and the processed bovine cancellous bone scaffold was planted. At 2, 4 and 8 weeks after operation, gross observation, ink infusion histology observation and microvessel bulk density detection were conducted. Results At each postoperative time point, the samples in the experimental group were fresh red, the circulation of blood vessels were smooth bidirectionally, while the samples in the control group were dark red soft, and flexible. Ink infusion histology observation showed the processed bovine cancellous bone scaffold in the experimental group had obvious vascularization, the blood vessels tended to be mature and integrated into network, and neovascular sprouts originated from arteriovenous loop were evident, especially at 8 weeks after operation; while there was no vascularization in the control group. At 2, 4 and 8 weeks after operation, the bulk density of the microvessels in the experimental group was (3.59 ± 1.84), (16.61 ± 10.23) and (39.04 ± 13.46) μm3/μm3, respectively, and it was (2.43 ± 0.97), (6.79 ± 2.92) and (25.31 ± 10.98) μm3/μm3, respectively, in the control group. Significant differences was noted between two groups at 4 and 8 weeks after operation (P lt; 0.05), and no significant difference was evident at 2 weeks after operation (P gt; 0.05). Conclusion Inducing vascularization in a rocessed bovine cancellous bone using an arteriovenous loop is a new strategy of prevascularization and may provide valuable clues for the preparation of functional artificial bone
Objective Rapid and effective vascularization of scaffolds used for bone tissue engineering is critical to bony repair. To study the cooperative and promotion effects of enhanced bioactive glass/collagen composite scaffold on vascularization for searching for a kind of el igible vascularized scaffold to repair bone defect. Methods The human umbil ical vein endothel ial cells (HUVECs) were collected from human umbil ical core, and identified through von Willebrandfactor (vWF) and CD34 immunofluorescence. The 1st passage of HUVECs were suspensed and seeded into the scaffold. The attachment and prol iferation of HUVECs on the scaffold were observed through scanning electron microscope (SEM). HUVECs were seeded on the scaffold as the experimental group, and on 96-well plate as the control group. The growth rate of HUVECs was detected through alarmarBlue at 1, 3, 5, 7, 9, and 11 days. Meanwhile, the mRNA expression levels of VEGF, fms-related tyrosine kinase 1 (Flt-1), and kinase insert domain receptor (Kdr) were detected through real-time fluorescence quantitative PCR. Twelve scaffolds were embedded subcutaneouly into 6 Sprague-Dawley rats. The enhanced scaffolds were used and the arteria and vein saphena bundle were embedded straightly through the central slot of scaffold in experimental group, and the common scaffolds were used in control group. Frozen section and HE staining of scaffolds were performed at 5 days and 10 days to observe the vascularization of embedded scaffold. Results HUVECs were identified through morphology, vWF and CD34 immunofluorescence. SEM results showed HUVECs could attach to the scaffold tightly and viably. HUVECs prol iferated actively on the scaffold in experimental group; the growth rate in experimental group was higher than that in control group at 3-11 days, showing significant differences within 5-11 days (P lt; 0.05). The real-time fluorescence quantitative PCR results showed thatthe mRNA expression levels of VEGF, Flt-1, and Kdr in experimental group were higher than those in control group at 3 days, showing significant differences (P lt; 0.05). Frozen section and HE staining of the scaffolds in experimental group showed that the embedded vessel bundle were still patency at 5 days and 10 days, that many new vessels were observed around the embedded vessel bundle and increased with time, host vessels infiltrated in the surrounding area of scaffold and fewer neo-vessels at the distant area. But there was only some fibrous tissue appeared in control group, and at 10 days, the common scaffold degradated, so few normal tissue appeared at the embedded area. Conclusion Enhanced bioactive glass/collagen composite scaffold can promote vascularization in vitro and in vivo, and may be used in bone tissue engineering.
Objective To study the ectopic osteogenesis and vascularization ofthe tissue engineered bone promoted by an artificial bone composite that consists of coral hydroxyapatite (CHA), 1,25-(OH)2 D3, human marrow stromal osteoblast (hMSO), and human umbilical vein endothelial cell (hUVEC).Methods After the isolation and the culture in vitro, hMSO and hUVEC were obtained. Then, hMSO (5×105/ml) and hUVEC (2.5×105/ml) were seeded at a ratio of 2∶1 onto the CHA scaffolds coated with 1,25-(OH)2 D3 (the experimental group) or onto the CHA scaffolds without 1,25-(OH)2 D3 (the control group). The scaffolds were culturedin vitro for 3 days, and then the scaffolds were implanted into the pockets that had beenmade on the backs of 18 nude mice. Then, 6 of the mice were implanted with one experimental engineered bone bilaterally; another 6 mice were implanted with onecontrol engineered bone bilaterally; the remaining 6 mice were implanted with one experimental engineered bone and one control engineered bone on each side. At4, 8 and 12 weeks after operation, the retrieved scaffolds and cells were examined by the nake eye and histology as well as by the scanning electron microscopy. The quantitative assessment of the newly-formed bone and the quantitative analysis of the newly-formed blood vessels were performed. Results The evaluationsby the histology revealed that at 4 weeks the original bone tissues grew into the scaffolds in all the groups, but significantly more newly-formed bone tissuesand newly-formed blood vessels were found in the experimental group. At 12 weeks the newly-formed bone tissues were found in all the groups, but there was a typical bone unit found in the experimental group. There was a significantly smaller amount of capillary vessels in the control group than in the experimental group at all the time points. The evaluations by the scanning electron microscopy revealed that at 4 weeks in the experimental group there were great amounts of extracelluar matrix that embedded the cells, and plenty of capillary vessels were found on the surface of the implanted bone materials and some of them grew into the materials; however, in the control group there was a smaller amount of capillary vessels although much extracelluar matrix was still found there. At 8 weeks sarciniform osteoids were found on some of the implanted materials, with much extracelluar matrix and many newly-formed capillary vessels in the experimental group; however, in the control group there were fewer capillary vessels and lower degrees of the bone maturity. The quantitative assessment of the newly-formed bone showed that the newformed bones were 3.1±0.52 in the experimental group but2.30±0.59 in the control group at 8 weeks (Plt;0.05), and 4.63±0.55 vs. 3.53±0.62 at 12 weeks. There was a significant difference at these two time points between the two groups (Plt;0.05). The quantitative analysis of the newly-formed blood vessels showed that the vascular areas were 28.74％±7.81％i n the experimental group but 19.52％±4.57% in the control group at 4 weeks (Plt;0.05), and 24.66％±7.38％ vs. 1784%±5.22% at 12 weeks. There was a significant difference at these two time points between the two groups (Plt;0.05). Conclusion 1,25-(OH)2 D3 as an active factor can increase the interaction between hMSO and hUVEC, and thus promote the ectopic osteogenesis and vascularization in the tissue engineered bone.
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 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.
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.
【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 Vascular bundle and sensory nerve bundle implantation can promote the osteogenesis of tissue engineered bone. To investigate whether vascular bundle and sensory nerve bundle implantation will affect the expressions of neurokinin 1 receptor (NK1R) and vasoactive intestinal peptide type 1 receptor (VIPR1). Methods Fifty-four 5-montholdNew Zealand rabbits were selected. Autologous bone marrow was aspirated from the posterior il iac spine of rabbits, and the bone marrow mesenchymal stem cells (BMSCs) were prol iferated in vitro. At the 3rd passage, the BMSCs were cultured in the osteogenic culture medium for 7 days. The tissue engineered bone was prepared by the combined culture of these osteoblastic induced BMSCs and β tricalcium phosphate scaffold material. A 1.5 cm segmental bone defect was created at the right femur of rabbits. After the plate fixation, defects were repaired with sensory nerve bundle plus tissue engineered bone (group A, n=18), with vascular bundle plus tissue engineered bone (group B, n=18), and tissue engineered bone only (group C, n=18). X-ray examination was used to evaluate the degree of the ossification. The expression levels of NK1R and VIPR1 were measured by the immuohistochemistry analysis and the mRNA expression of NK1R and VIPR1 by real-time PCR at 4, 8, and 12 weeks after operation. Results The better osteogenesis could be observed in group A and group B than in group C at all time points. X-ray scores were significantly higher in group B than in groups A and C (P lt; 0.05) at 4 weeks, and in groups A and B than in groupC (P lt; 0.05) at 8 and 12 weeks. The mRNA expressions of NK1R and VIPR1 were highest at 8 weeks in groups A and B and gradually decreased at 12 weeks (P lt; 0.05); the expressions were higher in groups A and B than that in group C (P lt; 0.05), and in group B than group A (P lt; 0.05). Immunohistochemistry analysis showed that the expressions of NK1R and VIPR1 were highest at 8 weeks in 3 groups, and the expressions were higher in groups A and B than in group C. Conclusion Implanting vascular bundles into the tissue engineered bone can significantly improve the expression levels of NK1R and VIPR1. It is an ideal method to reconstruct composite tissue engineered bone.