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 investigate the protocols of combined culture of human placenta-derived mesenchymal stem cells (HPMSCs) and human umbilical vein endothelial cells (HUVECs) from the same and different individuals on collagen material, to provide the. Methods Under voluntary contributions, HPMSCs were isolated and purified from human full-term placenta using collagenase IV digestion and lymphocyte separation medium, and confirmed by morphology methods and flow cytometry, and then passage 2 cells were cultured under condition of osteogenic induction. HUVECs were isolated from fresh human umbilical vein by collagenase I digestion and subcultured to purification, and cells were confirmed by immunocytochemical staining of von Willebrand factor (vWF). There were 2 groups for experiment. Passage 3 osteoblastic induced HPMSCs were co-cultured with HUVECs (1 ∶ 1) from different individuals in group A and with HUVECs from the same individual in group B on collagen hydrogel. Confocal laser scanning microscope was used to observe the cellular behavior of the cell-collagen composites at 1, 3, 5, and 7 days after culturing. Results Flow cytometry showed that HPMSCs were bly positive for CD90 and CD29, but negative for CD31, CD45, and CD34. After induction, alizarin red, alkaline phosphatase, and collagenase I staining were positive. HUVECs displayed cobble-stone morphology and stained positively for endothelial cell marker vWF. The immunofluorescent staining of CD31 showed that HUVECs in the cell-collagen composite of group B had richer layers, adhered and extended faster and better in three-dimension space than that of group A. At 7 days, the class-like microvessel lengths and the network point numbers were (6.68 ± 0.35) mm/mm2 and (17.10 ± 1.10)/mm2 in group A, and were (8.11 ± 0.62) mm/mm2 and (21.30 ± 1.41)/mm2 in group B, showing significant differences between the 2 groups (t=0.894, P=0.000; t=0.732, P=0.000). Conclusion Composite implant HPMSCs and HUVECs from the same individual on collagen hydrogel is better than HPMSCs and HUVECs from different individuals in integrity and continuity of the network and angiogenesis.
【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.
【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 explore the osteogenesis and angiogenesis effect of bone marrow mesenchymal stem cells (BMSCs) derived osteoblasts and endothelial cells compound with chitosan/hydroxyapatite (CS/HA) scaffold in repairing radialdefect in rats. Methods The BMSCs were isolated from Sprague Dawley rats and the 3rd generation of BMSCs were induced into osteoblasts and endothelial cells. The endothelial cells, osteoblasts, and mixed osteoblasts and endothelial cells (1 ∶ 1) were compound with CS/HA scaffold in groups A, B, and C respectively to prepare the cell-scaffold composites. The cell proliferation was detected by MTT. The rat radial segmental defect model was made and the 3 cell-scaffolds were implanted, respectively. At 4, 8, and 12 weeks after transplantation, the graft was harvested to perform HE staining and CD34 immunohistochemistry staining. The mRNA expressions of osteopontin (OPN) and osteoprotegerin (OPG) were detected by RT-PCR. Results Alkal ine phosphatase staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days after osteogenic induction and the nuclei were stained red. CD34 immunocytochemical staining of the endothelial cells showed that there were brown grains in the cytoplasm at 14 days after angiogenesis induction. MTT test showed that the proliferation level of the cells in 3 groups increased with the time. HE staining showed that no obvious osteoid formation, denser microvessel, and more fibrous tissue were seen at 12 weeks in group A; homogeneous osteoid which distributed with cord or island, and many osteoblast-l ike cells were seen in groups B and C. The microvessel density was significantly higher in groups A and C than group B at 3 time points (P lt; 0.05), and in group A than in group C at 12 weeks (P lt; 0.05). The OPN and OPG mRNA expressions of group A were significantly lower than those of groups B and C at 3 time points (P lt; 0.05). In groups B and C, the OPN mRNA expressions reached peak t8 and 12 weeks, respectively, and OPG mRNA expressions reached peak at 4 weeks. Conclusion BMSCs derived steoblasts and endothelial cells (1 ∶ 1) compound with CS/HA porous scaffold can promote bone formation and vascularization in bone defect and accelerate the healing of bone defect.
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 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 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.
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 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.