Objective To observe the revascularization process of transplanted nerve after transplantation of long nerve and accompanying peri pheral vessels, to investigate its relationship with nerve regeneration. Methods The mediannerve defect models of the left forelimb (3 cm in length) were made in 60 New Zealand rabbits (aged 6-8 months, weighing 2.0-2.5 kg, and male or female), which were randomly divided into 2 groups (n=30). In situ anastomosis of the median nerves was performed in the control group; in situ anastomosis of the median nerves was made in parallel to the surrounding elbow veins, the transplanted epineurium and the adventitia were sutured with nerve anastomosis l ine in the experimental group. After operation, the gross observation, electrophysiological testing, and histopathology observation was performed at 1, 2, 4, 8, and 12 weeks, and transmission electron microscope at 12 weeks to observe the revascularization of nerve grafts, nerve fiber regeneration, and functional recovery. Results In the experimental group, revascularization was observed at 1 week after operation, and the degree of revascularization was significantly higher than that in the control group at 2, 4, 8, and 12 weeks. At 8 and 12 weeks, the nerve fiber regeneration speed, quality, and quantity in the experimental group were better than those in the control group. At 2, 4, 8, and 12 weeks, the nerve conduction velocities were (10.32 ± 0.94), (13.14 ± 1.22), (22.68 ± 1.16), and (24.09 ± 1.27) m/ s respectively in the experimental group, and were (9.18 ± 1.07), (11.12 ± 1.03), (19.81 ± 1.37), and (20.67 ± 1.19) m/s in the control group, showing significant difference at 12 weeks after operation (t=3.167, P=0.001). At 12 weeks in the experimental group, the myel in sheath had similar size, less sheath plate delamination, normal Schwann cells and rich organelles, in which normal microfilaments, microtubules and axonal mitochondria were observed; axonal mitochondria had clear crestfilm and no swelling and vacuolization, and the neurofibrils basically became normal. The myelinated nerve fibers area, myelin thickness, and axon diameter were (5.93 ± 0.94) mm2, (0.72 ± 0.12) μm, and (3.12 ± 0.12) μm respectively in the experimental group, and were (5.28 ± 0.72) mm2, (0.65 ± 0.09) μm, and (2.98 ± 0.16) μm respectively in the control group, all showing significant differences (t=3.736, P=0.002; t=3.271, P=0.002; t=4.533, P=0.001). Conclusion The transplanted nerves in parallel to large blood vessels can promote angiogenesis of the transplanted nerve, and accelerate the regeneration and functional recovery of the nerves.
Objective To study the preparation method of acellular vascular matrix and to evaluate its biocompatibil ity and safety so as to afford an ideal scaffold for tissue engineered blood vessel. Methods Fresh caprine carotids (length, 50 mm) were harvested and treated with repeated frozen (—80 )/thawing (37℃), cold isostatic pressing (506 MPa, 4 ), and 0.125% sodium dodecyl sulfate separately for preparation of acellular vascular matrix. Fluorescence staining and DNA remain test were used to assess the cell extracting results. Biological characteristics were compared with the raw caprine carotids using HE staining, Masson staining, scanning electron microscope (SEM), and mechanical test. Biocompatibil ity wasdetected using cell adhesion test, MTT assay, and subcutaneously embedding test. Ten SD rats were divided into 2 groups (n=5). In experimental group, acellular vascular matrix preserved by the combination of repeated frozen/thawing, ultrahigh pressure treatment and chemical detergent was subcutaneously embedded; and in control group, acellular vascular matrix preserved only by repeated frozen/thawing and ultrahigh pressure treatment was subcutaneously embedded. Results HE staining and Masson staining revealed that no nucleus was detected in the acellular vascular matrix. SEM demonstrated that a lot of collagen fibers were preserved which were beneficial for cell adhesion. Fluorescence staining and DNA remain test showed that the cells were removed completely. There was no significant difference in stress and strain under the maximum load between before and after treatment. Mechanical test revealed that the acellular vascular matrix reserved mechanical properties of the raw caprine carotids. Cell adhesion test and MTT assay confirmed that cytotoxicity was grade 0-1, and the acellular vascular matrix had good compatibil ity to endothel ial cells. After subcutaneously embedding for 8 weeks, negl igible lymphocyte infiltration was observed in experimental group but obvious lymphocyte infiltration in control group. Conclusion The acellular vascular matrix, which is well-preserved by the combination of repeated frozen/thawing, ultrahigh pressure treatment, and chemical detergent, is an ideal scaffold for tissue engineered blood vessel.
Objective To investigate the feasibil ity of preparing the porous extracellular matrix (ECM) by use of some chemicals and enzymes to decellularize the porcine carotid artery. Methods The porcine carotid artery was procured, and warm ischemia time was less than 30 minunts. The porcine carotid artery was decellularized with 1% sodium dodecyl sulfate (SDS) for 60 hours to prepare common ECM; then common ECM was treated with 0.25% trypsin (for 6 hours) and 0.3 U/ mL collagenase (for 24 hours) to prepare porous ECM. The common ECM and porous ECM were stained with HE,Masson’s trichrome, and Orcein to evaluate the histological features. Then the mechanical property, cytotoxicity, and pore size of ECMs were determined. After 4 weeks of subcutaneous implantation in dogs, the histological examination was used for the study. Results Histological observation confirmed that 2 kinds of ECMs were decellularized completely and more porous structure was observed in porous ECM. Scanning electron microscope showed the pores in porous ECM were greater and the length of shorter axis in porous ECM ranged from 5 to 30 μm, the length of longer axis from 40 to 100 μm. The porosity of porous ECM (99.25%) was greater than that of common ECM (91.50%). The burst pressure of porous ECM decreased when compared with common ECM, showing significant difference [(0.154 3 ± 0.012 7) MPa vs [0.305 2 ± 0.015 7) MPa, P lt; 0.05]. There was no significant difference in suture retention strength between 2 kinds of ECMs (P gt; 0.05). The cytotoxicity test showed no obvious cytotoxicity in 2 kinds of ECMs. In vivo implantation test showed that the deeper host cells infiltration and more neo-microvessels in porous ECM were observed than in common ECM. Conclusion SDS and some enzymes can be used to prepare porous ECM as the scaffold for tissue engineered blood vessels.
Objective To observe the adhesion and prol iferation of late endothel ial progenitor cells (EPCs) planted on nanoporous PLLA scaffold in vitro and to provide a new approach that optimizes tissue engineered material. Methods Male and female New Zealand rabbits (weight 2.5-3.0 kg) were used. Isolated late EPCs from rabbit peri pheral blood were cultured. Electrostatic spinning technique was adopted to prepare misal igned nanofibers, al igned nanofibers and super-al igned nanofibers, and low temperature plasma technique was appl ied to prepare misal igned membrane, al igned membrane and super-al igned membrane. After being divided into group A (cells only), B (misal igned membrane), C (normal membrane), D (al igned membrane) and E (super-al igned membrane), the primary late EPCs (1 × 105/mL) werecultured on scaffolds and MTT method was used to detect cell prol iferation abil ity at 3, 5, 7, 9, 11, 13, 15 and 17 days afterculture. After being divided into group A (misal igned membrane), B (normal membrane), C (al igned membrane) and D (superal igned membrane), precipitation method was appl ied to detect cell adhesion rate at 4, 12 and 24 hours after compound culture, and the morphologic changes of cells were observed at 4, 24 and 72 hours after compound culture. Results Fiber diameters in nanofibrous PLLA scaffolds were 300-400 nm, with a porosity rate of above 90%. At 3, 5, 7, 9, 11, 13, 15 and 17 days after culture, A value of each group was increased with time and the cells in each group grew well, showing there was no significant difference between group A and group B at each time point (P gt; 0.05 ); during the period of 7-15 days after culture, the difference between groups C, D and E and groups A and B was significant (P lt; 0.05). At 4 hours after compound culture, the adhesion rate of group A was superior to that of groups B, C and D (P lt; 0.05); at 12 and 24 hours after compound culture, the adhesion rate of groups B, C and D was remarkably higher than that of group A (P lt; 0.05); significant difference was noted in each group between the time point of 4 hours and the time point of 12 and 24 hours after compound culture (P lt; 0.05), but no significant difference between 12 hours and 24 hours was detected (P gt; 0.05). Morphology observation demonstrated that cells grew well on the scaffolds, the cells in groups A and B grew sporadically and disorderly, while the cells in groups C and D attached and al igned along fiber and prol iferated, with an excretion of ECM. Group D was better at maintaining cell morphology. Conclusion Al igned and superal igned nanofibers of PLLA scaffold can promote the adhesion and prol iferation of seed cells on the scaffold and maintain good cell morphology, which is an appropriate candidate scaffold material for blood vessel tissue engineering. Late EPCs is an ideal cell source for blood vessel tissue engineering.
【Abstract】 Objective To build nano-biomimetic tissue engineered blood vessel (NBTEBV) with nanotopology by using electrospinning (ELSP) technology. Methods Cony vascular endothel ial cell(VEC) on tubiform tooting in vitro was cultured. NBTEBV was built by use of multi-row nozzle with the suspension of cony vascular smooth muscle cell (VSMC) and mimic ECM (MECM) solution. NBTEBV was cultured with bioreactor in vitro . VEC and VSMC viabil ity and prol iferation were observed with MTT; and HE staining, scanning electron microscopy(SEM) observation and biomechanical test were carried out after 24 hours of static culture and 7 days of dynamic culture. Results After 7 days of culture, the length of NBTEBV was 57 mm, the external diameter was 4 mm and the thickness of wall was 0.4 mm. The NBTEBV’s color was white and the texture was even and flexible. MTT results indicated the viabil ity of cells cultured on NBTEBV for 7 days was normal(8.9 × 106 /mg, 3.5 ×105/mg for 24 hours). SEM and HE staining indicated that the topologic character of NBTEBV was similar to that of the naturalblood vessel. The NBTEBV showed a network scaffolds structure with 100 nm thick fiber and 600 nm aperture. The HE stainingresult showed that the NBTEBV was composed of VEC and VSMC by layer. Vascular mechanical results showed that the NBTEBVultimate hydrostatic pressure was 950 mmHg, the compl iance of the NBTEBV under physio-pressure (110/70 mmHg) was 3.0%; the ultimate tensile strength of 20 mm × 5 mm tissue sl ice was 18.5 MPa. Conclusion The technology of ELSP can use VSMC and MECM scaffold simultaneously to build tissue engineered blood vessel with nanotopology mimic native blood vessel.
To study the feasibil ity of human adipose derived stem cells (ADSCs) in monolayer culture induced into smooth muscle cells in vitro as seeding cells in vascular tissue engineering. Methods The mononuclear cells in human adipose were separated by collagenase treatment and seeded on culture dishes with the density of 5 × 105/cm2. Cellswere cultured in M-199 plus 10% FBS. When reaching confluence, the cells were subcultured by 0.1% trypsin and 0.02%EDTA treatment, PDGF-BB (50 ng/mL) and TGF-β1 (5 ng/mL) were added at the passage 1 to enhance the smooth muscle cells’ phenotype. Cells were cultured under the inducing medium for 14 days. The morphology of induced cells was observed under the microscope. Cellular immunofluorescence and RT-PCR were used to determine the expression of smooth muscle cell markers of the post-induced cells. Flow cytometry (FACs) was used to examine the positive rate of induced team. Results Cocultured in M-199 media including TGF-β1 and PDGF-BB, the prol iferating capabil ity of the induced cells was significantly downregulated compared with the uninduced cells(P lt; 0.01). The induced cells exhibited “Hill and Valley” morphology, while the uninduced cells were similar to ADSCs of P0 which had the fibroblast-l ike morphology. The results of immunofluorescence indicated that the induced cells expressed smooth muscle (SM) cell- specific markers including α-smooth muscle actin (α-SMA), SM-myosin heavy chain (SM-MHC) and Calponin. The results of RT-PCR revealed that the induced cells also expressed α-SMA, SM-MHC, Calponin and SM-22α.The positive rates of α-SMA, SM-MHC and Calponin in FACs were 3.26% ± 1.31%, 3.55% ± 1.6% and 4.02% ± 1.81%, respectively, before the cells were induced. However, 14 days after the cell induction, the positive rates were 48.13% ± 8.31%, 45.33% ± 10.68% and 39.13% ± 9.42%, respectively. The positive rates in induced cells were remarkably higher than those in uninduced cells(P lt; 0.01). Conclusion The human ADSCs can be induced to express vascular smooth muscle markers, and they are a new potential source of vascular tissue engineering.
Objective To develop an experimental model of abdominal aorta transplantation with nano-biomimetictissue engineered blood vessel (NBTEBV) and to investige the change of histomorphology in evolutionary process of degradation and remodel ing. Methods Twenty 6-month-old New Zealand rabbits were included, weighing 2-3 kg, male or female. The autologous seed cells of rabbits were harvested to build NBTEBV in vitro. After the branch of abdominal aorta under kidney was l igated, about 10 mm abdominal aorta was cut and replaced by NBTEBV; the anastomotic stoma was marked by Ti cl ips. NBTEBV’s evolutionary processes of degradation and rebuilding were observed. Twelve weeks after operation, DSA and color Doppler examinations were made. At 1, 4 and 12 weeks after operation, the gross and histological observations were made and 14C binding in PLGA was detected with X-ray photon spectroscopy. Results Of 20 rabbits, 17 showed that the NBTEBV was patency; 3 died from NBTEBV occlusion 36 or 72 hours after operation. The results of DAS and color Doppler showed the blood flow was patency, the blood flow rate was normal and there was no angiectasis. The lumen of transplanted blood vessel was covered with monolayer endothel ial cells. At 1 week, smooth muscle cells (SMCs) arranged regularly and much PLGA distributed in the EMCs. At 4 weeks, SMCs arranged in a layer, ECM was forming, mimic ECM degraded partly; PLGA decreased obviously. At 12 weeks, the SMCs arranged regularly, ECM formed, mimic ECM degraded, no PLGA was seen in the wall, the shape of graft was similar to the natural vessel. The decreasing crest value of 14C in specimen showed the degradation of PLGA. Conclusion NBTEBV has a good surgical maneuverabil ity and histocompatibil ity, its remodel ing evolutionary process fits in with tissue engineering specification. Building NBTEBV with ELSP is feasible.
Objective To explore the influence of different stress environmentson the growth of tissue engineering blood vessels in vivo． Methods The engineering vascular scaffolds were prepared with the porcine small intestinal submucosa(SIS) wrapping vascular endothelial cells and smooth muscle cells，which were implanted into the subcutaneous tissue(subcutaneous group), the femoral quadriceps(intramuscular group), and sheathed the femoral artery(perivascular group) respectively. Four weeks postoperatively, these cultured tissues were harvested, and evaluated by macroscopic observation and histology detection. Results The cultivated tissues in different stress environments had obvious difference in respectof the tubular configuration, cellular proliferation and tissue shape. In subcutaneous group, the wall structure integrity, seed cell proliferation and SIS scaffold decomposition were poor, lumen surface was covered without endothelial cells; in intramuscular group, integrity tubular structure had formed, seed cell proliferation was found to a certain extent, lumen surface was covered with sparseendothelial cells, and a little SIS scaffold was found, cellular and fiber structured arranged irregularly; in perivascular group, vascular-like structure formed, the seed cell growth and proliferation were good, the lumen surface was completely covered with endothelial cells, the smooth muscle cells were in good morphologicaldistribution, the antihydrostatic pressure was 247.0±35 kPa，showingsignificant differences when compared with subcutaneous group(67.0±5.8 kPa) and intramuscular group(104.0±7.6 kPa) (Plt;0.01).The total scoring of tissue engineering blood vessel formation in subcutaneous group, intramuscular group and perivascular group were 5.529±0.272，8.875±0.248 and 14.824±0.253 respectively, and the differences among them were significant (P lt; 0.05). Conclusion Stress excitation has a great influence on the cellular proliferation and the growth of tissue engineering blood vessel in vivo.
Objective To evaluate which is better method zymogen or low temperature frozen in removing vascular endothelial cell so as to lay a foundation for creating a kind of brace which is not to be rejected and the same as own blood vessel. Methods Fresh and not damaged umbilical blood vessel was collected from natural labour women, human umbilical blood vessel was remove carefully from normal foetus, then was put into disinfectant at 37℃ for 24 hours. They were divided into 3 groups:normal group(NG),zymogen group(ZG) and low temperature frozen group(LG). ZG: 0.1% collagenⅡ enzyme was addedin umbilical blood vessel and closed the both sides and the vascular endothelialcell was removed in 37℃ water. LG:Umbilical blood vessel was put into liquidnitrogen for 24 hours after frozened step by step, and then it was put into 37℃ water for 30-60 s and the vascular endothelial cells were washed away by normal saline. NG:Umbilical blood vessel was kept into 4℃ Kerb’s liquid. The bacteria were culturedin each group. The samples were stained by HE,elastic fiber and collagen fiberwere observed by light and scanning electron microscope. The difference of compliance was compared. Human leukocyte antigen ABC(HLA-ABC) and HLA-DR were observed by immunohistochemical method and the expression of antigen of umbilical blood vessel was analysed. Results In LG, umbilical vascular endothelial cells were removed completely; artery showed vertical smooth muscle and vein showed elastic membrane. InZG, umbilical vascular endothelial cells were removed completely after 20 minutes;artery showed vertical smooth muscle cells and vein showed lower endothelial layer. The vascular compliance in LG was higher than that in NG, and the latter was also higher than that in ZG,but showing no significant differences (Pgt;0.05). The compliance of umbilical vein was 2-3 times as much asthat of umbilical artery.The expression of HLA-ABC and HLA-DR in LG andZG were lower than that in NG, showing significant differences (Plt;0.01). Conclusion Low temperature frozen methodand zymogen method(0.1% collagen Ⅱ enzyme for 20 min) can remove vascular endothelial cells of human umbilical blood vessel completely.Low temperature frozenmethod was better than zymogen method.
Objective To evaluate repair and reconstructionof the femoral pseudoaneurysm caused by drug injection. Methods From May 2000 to May 2005, 15 cases of femoral pseudoaneurysm caused by drug injection underwent operation treatment. All patients were male, aging 20-36 years. The disease course was 18-52 days(mean 35 days) and the course of druginjection was 3-17 months. The locations were the left side in 5 cases and theright side in 10 cases. After having been bandaged with pressure and supportedwith nutrition, they had been all operated. One case received fistula repair, and 14 cases received vascular grafting with ePTFE man-made blood vessel. Results The wounds healed by the first intention in 14 cases. All limbs survived. The complexion, temperature and response of involved leg were in gear. The postoperative color ultrasound Doppler detection showed that all the vascular grafts were of patency. The function of the involved limbs restored to normal. Conclusion Complete debridement, vascular reconstruction and better microsurgery skill were the key factors of treating successfullythe femoral pseudoaneurysm caused by drug injection.