The Research Of Tissue-engineered Blood Vessels Constructed By Human Gingival Fibroblasts

Part One Human gingival fibroblasts induced to differentiate vascular endothelial cells in vitroObjective:Human gingival fibroblasts were cultured and induced into vascular endothelial cells in vitro in order to confirm that human gingival fibroblasts with stem cell properties have the potential of differentiation into vascular endothelial cells.Methods:1 The obtainment and isolation of human gingivaHuman normal gingiva consisting of epithelium and connective tissue was obtained and isolated from patients in the clinic with an extraction of the mandibular third impact molars at the Department of Oral Maxillofacial Surgery, Hospital of Stomatology, Hebei Medical University.2 The culture of human gingival fibroblastsHuman normal gingiva was minced into 1×1 mm2 pieces in culture L-DMEM medium containing 15% fetal bovine serum. When 80% of the cells around the tissues were mixed together, the cells were digested and were passaged.3 The identification of human gingival fibroblasts3.1 The surface antigen of vimentin and CD31 in human gingival fibroblasts was detected by flow cytometry assay3.2 RT-PCR analysis of human gingival fibroblastsTotal RNA of the cells was isolated using TRIzol reagent. c DNA synthesis was carried out by using Revert Aid TM First Strand c DNA Synthesis Kit. PCR amplification of the template was carried out by using Dream Taq Green PCR Master Mix. The RT-PCR products were analyzed by 2% agarose gel electrophoresis. The expression of housekeeping gene GAPDH served as an internal control in all RT-PCR reaction.3.3 Immunocytochemical stain of human gingival fibroblastsImmunocytochemical stain was done to analyze the expression of vimentin protein and collagen III protein in human gingival fibroblasts.3.4 Immunofluorescence stain of human gingival fibroblastsImmunocytochemical stain was performed to analyze the expression of S-100A4 protein and ?-SMA protein in human gingival fibroblasts.4 The proliferation of human gingival fibroblasts by MTT assayOD490 of 1 ~ 6 generations of gingival fibroblasts from 1 to 7 days was determined by MTT assay.5 The expression of endothelial-related markers in differentiated human gingival fibroblastsWestern blot was used to quantitatively analyze the expression of vimentin, tie2 and VEGFR2 protein in different concentration VEGF165 groups and different induction period group.Real-time PCR was used to detect the relative expression of vimentin, tie2 and Ang2 gene m RNA in different concentration VEGF165 groups and different induction period group.6 The morphological observation in the process of inductionThe morphology of human gingival fibroblasts with 8 ng/ml VEGF165 induced for 0, 7, 14, 21, 28, 35, 42, and 50 days was observed by inverted microscope respectively.7 The identification of vascular endothelial-like cells induced7.1 Immunocytochemical stainAfter being cultured with 8ng/ml VEGF165 for 35 days, the cells were identified by immunocytochemical stain against CD34 and CD31 respectively.7.2 Immunofluorescence stainAfter being cultured with 8ng/ml VEGF165 for 35 days, the cells were identified by immunofluorescence stain against v WF and E-cadherin respectively.7.3 The expression of tie2 gene m RNA in vascular endothelial-like cells induced by RT-PCR analysis7.4 The ultrastructure of the vascular endothelial-like cells induced by Transmission electron microscope(TEM)8 Flow cytometry assay after inductionFlow cytometry assay was done to analyze the expression of the surface antigen of CD34 and CD31 in cells induced to evaluate the transformation efficiency from human gingival fibroblasts to vascular endothelial cells.9 Examination of tubular formation in vitroMatrigel assay was used to examine the tube-like structures of cells induced.Results:1 The culture of human gingival fibroblastsCells emigrated from the gingival tissue blocks after 7 days of the primary culture. The cells were tightly adhered, well-spread and spindle-shaped fibroblast-like in appearance.2 Identification of human gingival fibroblasts2.1 Flow cytometry analysis showed that fibroblast cells were positive for vimentin, but negative for CD31. Rate of fibroblast cells expressing vimentin was(97.13±0.62)%,but CD31 was(8.64±1.55)%.2.2 RT-PCR showed vimentin, S-100A4, and ?-SMA specifically expressed in these fibroblast cells.2.3 Immunocytochemistry showed cells positive for vimentin and collagen III.2.4 Immunofluorescence stain showed cells positive for ?-SMA and S-100A4.3 The proliferation of human gingival fibroblastsMTT assay showed that the second or the third generation of human gingival fibroblasts presented optimal proliferation.4 The expression of endothelial-related markers in differentiated human gingival fibroblastsWestern blot showed that the expression of tie2 and VEGFR2 protein was highest when 8 ng/ml VEGF165 was used. Real-time PCR showed that the m RNA relative expression of tie2 and Angiopoietin2(Ang2) gene was highest when 8 ng/ml VEGF165 was used. Western blot showed that the expression of tie2 and VEGFR2 protein was highest when the cells were induced for 35 days. Real-time PCR showed that the m RNA relative expression of tie2 and Angiopoietin2(Ang2) gene was highest when the cells were induced for 35 days.5 Effect of VEGF165 on cell morphology of vascular endothelial-like cells inducedThe spindle-shaped cells turned into cube-shaped, and formed cobblestone-like pattern, or arranged in vascular lumen-like structures after culturing in 8ng/ml VEGF165 for 35 days.6 Immunocytochemical stain of the cells inducedImmunocytochemical stain showed cells induced positive for CD34 and CD31.7 Immunofluorescence stain of the cells inducedImmunocytochemical stain showed cells induced positive for v WF and E-cadherin.8 The expression of tie2 gene in the cells inducedThe RT-PCR result demonstrated that tie2 was specifically expressed in the cells induced.9 Effect of VEGF165 on cell ultrastructure of vascular endothelial cells inducedTEM showed microvillus on the surface of vascular endothelial cells induced. Lysosomes and Weibel-Palade bodies were found in the cytoplasm of vascular endothelial cells induced.10 Flow cytometry analysis of the transformation efficiency from human gingival fibroblasts to vascular endothelial cellsFlow cytometry assay showed that higher rate of cells expressing either CD34 or CD31 in induction group than that in control group(P<0.05).11 The formation of tube-like structures in matrigel of human gingival fibroblasts with optimal induction mediumTube-like structures were observed when cells induced were embedded in matrigel for 24 h. It was remarkably higher rate of tube-like structures in induction group than that in control group(P<0.05). Part Two Human gingival fibroblasts induced to differentiate vascular smooth muscle cells in vitroObjective:Human gingival fibroblasts were cultured and induced into vascular smooth muscle cells in vitro in order to confirm that human gingival fibroblasts have the potential of differentiation into vascular smooth muscle cells.Methods:1 The isolation and culture of human gingival fibroblasts were the same as that in Part One.2 The identification of human gingival fibroblasts was the same as that in Part One.2.1 RT-PCR analysis of human gingival fibroblasts was the same as that in Part One.2.2 Immunocytochemical stain of human gingival fibroblasts was the same as that in Part One.2.3 Immunofluorescence stain of human gingival fibroblasts was the same as that in Part One.3 MTT assay analysis of the proliferation of human gingival fibroblasts was the same as that in Part One.4 The expression of smooth muscle cell-related markers in differentiated human gingival fibroblastsWestern blot was used to quantitative analyze vimentin, ?-SMA and SM-MHC protein expressing in different concentration PDGF-BB(including 2 ng/ml TGF-?1) groups and different induction period group.Real-time PCR was used to detect the relative expression of vimentin, ?-SMA, SM-MHC, Cnn1 and SM 22? gene m RNA in different concentration PDGF-BB groups and different induction period group.5 The morphological observation in the process of inductionThe morphology of human gingival fibroblasts with 10 ng/ml PDGF-BB(including 2 ng/ml TGF-?1) induced for 0, 7, 14, 21, 28, and 35 days was observed by inverted microscope respectively.6 The identification of the cells induced.6.1 Immunocytochemical stainAfter being cultured with 10 ng/ml PDGF-BB(including 2 ng/ml TGF-?1) for 28 days, the cells were identified by immunocytochemical stain against ?-SMA and SM-MHC respectively.6.2 Immunofluorescence stainAfter being cultured with 10 ng/ml PDGF-BB(including 2 ng/ml TGF-?1) for 28 days, the cells were identified by Immunofluorescence stain against Cnn1 and SM 22? respectively.6.3 The expression of ?-SMA and SM-MHC genes m RNA in the cells induced by RT-PCR analysis6.4 The ultrastructure of the cells induced by Transmission electron microscope(TEM)Results:1 The culture of human gingival fibroblasts was the same as that in Part One.2 Identification of human gingival fibroblasts2.1 RT-PCR showed that vimentin and S-100A4 was specifically expressed, ?-SMA was weekly expressed, and SM-MHC was not expressed in these fibroblast cells.2.2 Immunocytochemistry showed cells positive for vimentin and collagen III.2.3 Immunofluorescence stain showed cells positive for ?-SMA and S-100A4.3 The proliferation of human gingival fibroblasts was the same as that in Part One.4 The expression of smooth muscle cell-related markers in differentiated human gingival fibroblastsWestern blot showed that the expression of ?-SMA and SM-MHC protein was highest when 10 ng/ml PDGF-BB was used. Real-time PCR showed that the m RNA relative expression of ?-SMA, SM-MHC, Cnn1 and SM 22? gene was highest when 10 ng/ml PDGF-BB was used. Western blot show that the expression of ?-SMA and SM-MHC protein was highest when cells were induced for 28 days. Real-time PCR showed that the m RNA relative expression of ?-SMA, SM-MHC, Cnn1 and SM 22? gene was highest when cells were induced for 28 days.5 Effect of PDGF-BB(including 2 ng/ml TGF-?1) on cell morphology of the cells inducedAfter induction in 10 ng/ml PDGF-BB(including 2 ng/ml TGF-?1) for 7 days, most cells still present plexiform; From 7 to 21 days, a part of cells present apoptosis and reduced state, but still present plexiform arrangement; From 21 to 28 days, cells joined together and arranged as "hill and valley" structures slightly; From 28 to 35 days, most of cells arranged as typical "hill and valley" structures.6 Immunocytochemical stain of the cells inducedImmunocytochemical stain showed the cells induced positive for ?-SMA and SM-MHC. The control group was positive for ?-SMA, but negative for SM-MHC.7 Immunofluorescence stain of the cells inducedImmunocytochemical stain showed the cells induced positive for SM 22? and Cnn1.8 The expression of ?-SMA and SM-MHC gene in the cells inducedRT-PCR results showed that the m RNA expression of ?-SMA and SM-MHC genes were remarkably higher in induction group than that in the control group(P<0.05).9 The ultrastructure of the cells inducedTEM showed that Golgi complex, mitochondria, rough endoplasmic reticulum, a large number of myofilament-like structures and high electronic density of dense body-like structure were in the cytoplasm of the cells induced. Part Three Biological characteristics and biocompatibility of ACVM-0.25% HLC-I scaffolds for tissue engineeringObjective:Freeze-drying technology was used to combine the Human-like collagen I(HLC-I) with acellular vascular matrix(ACVM) to obtain ACVM-0.25% HLC-I scaffolds as matrices for the construction of human tissues to discuss the biological characteristics and biocompatibility.Methods:1 Preparation of ACVM scaffoldBlood vessels of rabbit were acquired and washed by normal saline. Then, the blood vessel was soaked in PBS contained 1% benzalkonium bromide for 1hour, PBS for 5 min, 0.1% trypsin for 6 hours, PBS for 5min, 1% Triton-100 for 72 hours. Finally, blood vessel were removed into sterile PBS solution in 4 oC for later use.2 HE stain of ACVM scaffold3 Masson stain of ACVM scaffold4 Bioactivation of ACVM scaffold crosslink with HLC-IAcrylic acid was used to pretreatment ACVM scaffold. ACVM-acrylic acid was exposed to UV radiation for 30 min, and then washed with distilled water to remove the excess homopolymer and placed to dry in a vacuum desiccator. ACVM-acrylic acid was immersed into PBS containing 5 mg/ml carbodiimide water-soluble at 4oC for 1 hour. The incorporation of HLC-I in concentration of 0.10 mg/ml, 0.25 mg/ml, 0.50 mg/ml, 0.75 mg/ml and 1.00 mg/ml for 12 hours was carried out respectively. The ACVM-HLC-I scaffold were washed in PBS and dried under reduced pressure, and stored at 4oC.5 The culture and identification of human gingival fibroblasts was the same as that in Part One.6 The optimal concentration of HLC-I crosslink with ACVM scaffoldACVM-HLC-I scaffolds were seeded with HGFs. Absorbance at 490 nm of each ACVM-HLC-I scaffold was determined by MTT assay after being cultured for 1, 4 and 7 days to determine the optimal concentration of HLC-I crosslink with ACVM scaffold.7 Water absorption assayThe water absorption capacities of ACVM-0.25% HLC-I scaffold and ACVM scaffold were determined by swelling in PBS at room temperature. The percentage of water absorption of the scaffolds was calculated using the equation: W1-W0/W0×100%, where W1 represents the wet weight of the scaffolds after 24 hours and W0 is the initial weight of the scaffolds.8 Mechanical properties assayZwick/Roell Z020 was used to evaluate the tensile strength,breaking strength and elongation at break of ACVM-0.25% HLC-I scaffold, ACVM scaffold and normal blood vessel. The stress-strain curve was performed.9 Burst pressure assayPressure gun with normal saline was used to perform burst pressure assay on ACVM-0.25% HLC-I scaffold, ACVM scaffold and normal blood vessel.10 Scanning electron microscopy(SEM) assayThe surface and innerface of ACVM scaffold and ACVM-0.25% HLC-I scaffold were observed using SEM. Normal blood vessel was as the control group.11 CCK-8 kits analysis the cytotoxicity of ACVM-0.25% HLC-I scaffoldCCK-8 was used to evaluate grow kinetics of HGFs on the ACVM scaffold and ACVM-0.25% HLC-I scaffold. Absorbance of each well at 450 nm was immediately measured with a microplate reader on 24, 48, and 72 hours after cell seeding. Cytotoxic activity, the relative growth rate was used to evaluate cytotoxicity of ACVM-0.25% HLC-I scaffold in vitro.Results:1 The general observation of ACVM scaffoldACVM scaffold presents as ivory-white, translucent and nonelastic vascular wall with folded and dilapidated lumen.2 HE staining of ACVM scaffoldResults showed that ACVM scaffold presented as red reticular fiber and collagen fiber without cell nucleus, cell debris and media layer smooth muscle cells. The inner membrance was mainly distributed in reticular fiber. The gap between collagen fibers was large.3 Masson staining of ACVM scaffoldResults showed that vascular endothelial cells, vascular smooth muscle cells and most of muscle fibers of ACVM scaffold were decellularized. These thin fiber structures were green collagen fibers and red muscle fibers.4 The culture and identification of human gingival fibroblasts was the same as that in Part One.5 The optimal concentration of HLC-I crosslink with ACVM scaffoldResults showed that ACVM scaffold coated with 0.25mg/ml HLC-I presented the highest in cell adhesion after 1, 4 and 7 days cell culture on different concentration of HLC-I crosslink with ACVM scaffold.6 Water absorption capacities of ACVM-0.25% HLC-I scaffoldResults showed that no significant difference between ACVM-0.25% HLC-I scaffold and ACVM scaffold(P>0.05).7 Mechanical properties of ACVM-0.25% HLC-I scaffoldThe stress and strain of ACVM-0.25% HLC-I scaffold, similar with rabbit blood vessels, were greater than ACVM scaffold; the breaking strength and elongation at break of ACVM-0.25% HLC-I were similar to rabbit blood vessels; the breaking strength of ACVM scaffold was lower than rabbit blood vessels, but the elongation at break of ACVM scaffold was greater than rabbit blood vessels.8 Burst pressure of ACVM-0.25% HLC-I scaffoldResults showed that burst pressure of ACVM-0.25% HLC-I scaffold was greater than ACVM scaffold(P<0.05), and was similar as normal rabbit blood vessel(P>0.05).9 SEM images of the ultrastructure of ACVM-0.25% HLC-I scaffoldSEM images showed that the fibers of ACVM-0.25% HLC-I scaffold were more density than ACVM scaffold either on the surface or in the cross-section.10 CCK-8 kits analysis the cytotoxicity of ACVM-0.25% HLC-I scaffoldRelative growth rate of ACVM-0.25% HLC-I scaffold and ACVM scaffold was over 75% at 24, 48 and 72 hours. With time increasing(24, 48 and 72 hours), the proliferation of HGFs on both scaffolds presented the tendency of rising. Part Four The construction of tissue-engineered blood vessel in vitro anddynamic test in nude miceObjective:To explore the feasibility of combining human gingival fibroblasts with ACVM-0.25% HLC-I scaffolds in constructing of tissue-engineered blood vessels, and the influence of dynamic on tissue-engineered blood vessels in vivo. Vascular smooth muscle cells and endothelial cells differentiated of human gingival fibroblasts were seeded into ACVM-0.25% HLC-I scaffold to construct of tissue-engineered blood vessels, which were implanted in the subcutaneous tissue of nude mice to perform the dynamic test.Methods:1 Preparation of ACVM-0.25% HLC-I scaffold was the same as that in Part Three.2 The disinfection of ACVM-0.25% HLC-I scaffold3 Ed U Apollo488 marked vascular smooth muscle cells and endothelial cells4 Ed U Apollo488 marked vascular smooth muscle cells implanted into ACVM-0.25% HLC-I scaffoldMultiple precipitation was used to implant Ed U Apollo488 marked vascular smooth muscle cells on ACVM-0.25% HLC-I scaffold for 7 days to construct the tissue-engineered tunica media.5 DAPI marked vascular endothelial cells6 Gel Methods was performed to seed vascular endothelial cells inducedDAPI marked vascular endothelial cells with Matrigel(4:1) was implanted onto tissue-engineered tunica media. After 12 hours, 1640 and L-DMEM(1:1) containing 15 % FBS was used to culture for 3 days.7 HE stain of ACVM-0.25% HLC-I scaffold seeded with cells8 Immunocytochemical stain of ACVM-0.25% HLC-I scaffold seeded with cellsVascular smooth muscle cells on ACVM-0.25% HLC-I scaffold were identified by immunocytochemical stain against ?-SMA and SM-MHC antibody respectively. Vascular endothelial cells on ACVM-0.25% HLC-I scaffold were identified by immunocytochemical stain against CD34 and CD31 antibody respectively.9 Immunofluorescence stain of ACVM-0.25% HLC-I scaffold seeded with cells9.1 Single-labelling immunofluorescenceVascular smooth muscle cells on ACVM-0.25% HLC-I scaffold were identified by immunofluorescence stain against Cnn1 and SM 22? antibody respectively, and FITC was used as the second antibody. Vascular endothelial cells on ACVM-0.25% HLC-I scaffold were identified by immunofluorescence stain against v WF and E-cadherin antibody respectively, and Rhodamine was used as the second antibody.9.2 Double-labelling immunofluorescenceVascular smooth muscle cells and vascular endothelial cells on ACVM-0.25% HLC-I scaffold were identified by immunofluorescence stain against SM 22? and v WF antibody, against Cnn1 and E-cadherin antibody, against ?-SMA and CD34 antibody, or against SM-MHC and CD31 antibody.10 SEM images of ACVM-0.25% HLC-I scaffold seeded with cells11 Dynamic assay in nude miceACVM-0.25% HLC-I scaffolds and tissue-engineered vessels were implanted in the subcutaneous tissue of nude mice to perform histological observation, fluorescence staining and burst pressure assay after operation at week 3, 6 and 9 respectively.11.1 Histological observation11.2 Cells tracking by Ed U Apollo488 and DAPI in nude mice11.3 Burst pressure assayPressure gun with normal saline was used to perform burst pressure assay on ACVM-0.25% HLC-I scaffolds, tissue-engineered vessels and normal blood vessels implanted in the subcutaneous tissue of nude mice.Results:1 Labeling rate of cell trackingMore than 90% of vascular smooth muscle cells induced and vascular endothelial cells induced marked by Ed U Apollo488. More than 90% of vascular endothelial cells induced were marked by DAPI.2 The construction of tissue-engineered vessel in nude mice2.1 HE stain observationVascular smooth muscle cells induced in ACVM-0.25% HLC-I scaffold were uniform spindle-shaped in appearance. Vascular endothelial cells induced in ACVM-0.25% HLC-I scaffold were monolayer distribution in appearance. tissue-engineered intima, tunica media and adventitia were observed.2.2 Immunocytochemical stainVascular smooth muscle cells in ACVM-0.25% HLC-I scaffolds were positive for ?-SMA and SM-MHC, and vascular endothelial cells on Matrigel-ACVM-0.25% HLC-I scaffolds were positive for CD34 and CD31.2.3 Immunofluorescence stain2.3.1 Single-labelling immunofluorescenceVascular smooth muscle cells in ACVM-0.25% HLC-I scaffolds were positive for Cnn1 and SM 22?, which presented green fluorescence. Vascular endothelial cells on Matrigel-ACVM-0.25% HLC-I scaffolds were positive for v WF and E-cadherin, which presented red fluorescence.2.3.2 Double-labelling immunofluorescenceVascular smooth muscle cells in ACVM-0.25% HLC-I scaffolds were positive for SM 22?,Cnn1,?-SMA and SM-MHC, which presented green fluorescence. Parts of cells on the surface of ACVM-0.25% HLC-I scaffolds were positive for v WF, E-cadherin, CD34 and CD31, which presented red fluorescence.2.4 SEM observationResults showed that the double-layer of cross-section of scaffold could be observed. Cells on the surface of scaffold arranged in cobblestone-like pattern.3 Dynamic assay in nude mice3.1 The general observation of specimen implanted in subcutaneous tissue of nude miceAfter 3 weeks, ACVM-0.25% HLC-I scaffold or tissue-engineered vessel implanted in subcutaneous tissue was surrounding by a very thin tissue of nude mice.After 6 weeks, tissues of nude mice were gradually growing into ACVM-0.25% HLC-I scaffold or tissue-engineered vessel.After 9 weeks, tubular structures of ACVM-0.25% HLC-I scaffold or tissue-engineered vessel remains intact with thick tissues of nude mice ingrowth.3.2 Histological observationAfter 3 weeks, ACVM-0.25% HLC-I scaffold implanted in subcutaneous tissue was surrounding by a small amount of fiber and inflammatory cells infiltration of nude mice. Tissue-engineered vessel implanted was surrounding by a small amount of fiber and massive inflammatory cells infiltration.After 6 weeks, ACVM-0.25% HLC-I scaffold implanted with few degradation and nude mice cells ingrowth was surrounding by a small amount of inflammatory cells infiltration. Tissue-engineered vessel implanted with plenty of cells was surrounding by a small amount of fiber and inflammatory cells infiltration.After 9 weeks, ACVM-0.25% HLC-I scaffold implanted with bulk degradation was surrounding by a small amount of inflammatory cells infiltration and massive fiber. Tissue-engineered vessel implanted with plenty of cells was surrounding by a small amount of fiber.3.3 Cell tracking by Ed U Apollo488 and DAPI in nude miceAfter 9 weeks, vascular smooth muscle cells induced seeded in ACVM-0.25% HLC-I scaffolds in nude mice were marked by Ed U Apollo488, and presented green fluorescence. Vascular endothelial cells induced were marked by Ed U Apollo488 and DAPI, and presented green fluorescence and red fluorescence respectively.3.4 Burst pressure assayResults showed that burst pressure of tissue-engineered vessel was greater than ACVM-0.25% HLC-I scaffold(P<0.05), and was similar as normal rabbit blood vessel(P>0.05).Conclusion:1. Human gingival fibroblasts have the potentiality to differentiate into vascular endothelial cells.2. Human gingival fibroblasts have the potentiality to differentiate into vascular smooth muscle cells.3. ACVM-0.25% HLC-I scaffold, combining 0.25% HLC-I with ACVM scaffold, are significant superior to ACVM scaffold as matrices for the construction of tissue-engineered vessels with good abilities of cell adhesion, cell proliferation, cell migration, mechanical and biomechanical properties.4. Vascular smooth muscle cells and endothelial cells differentiated from human gingival fibroblasts seeded into ACVM-0.25% HLC-I scaffolds have been confirmed to construct of tissue-engineered blood vessels in vitro successfully.5. Dynamic culture in nude mice have been confirmed the integrity of combining vascular smooth muscle cells and endothelial cells differentiated of human gingival fibroblasts with ACVM-0.25% HLC-I scaffolds after 9 weeks in constructing of tissue-engineered blood vessels in nude mice.