Objective To investigate the effect of homograft of marrow mesenchymal stem cells (MSCs) seeded onto poly-L-lactic acid (PLLA)/gelatin on repair of articular cartilage defects. Methods The MSCs derived from36 Qingzilan rabbits, aging 4 to 6 months and weighed 2.5-3.5 kg were cultured in vitroand seeded onto PLLA/gelatin. The MSCs/ PLLA/gelatin composite was cultured and transplanted into full thickness defects on intercondylar fossa. Thirty-six healthy Qingzilan rabbits were made models of cartilage defects in the intercondylar fossa. These rabbits were divided into 3 groups according to the repair materials with 12 in each group: group A, MSCs and PLLA/gelatin complex(MSCs/ PLLA/gelatin); group B, only PLLA/gelatin; and group C, nothing. At 4,8 and 12 weeks after operation, the gross, histological and immunohistochemical observations were made, and grading scales were evaluated. Results At 12 weeks after transplantation, defect was repaired and the structures of the cartilage surface and normal cartilage was in integrity. The defects in group A were repaired by the hylinelike tissue and defects in groups B and C were repaired by the fibrous tissues. Immunohistochemical staining showed that cells in the zones of repaired tissues were larger in size, arranged columnedly, riched in collagen Ⅱ matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in group A at 12 weeks postoperatively. In gross score, group A(2.75±0.89) was significantly better than group B (4.88±1.25) and group C (7.38±1.18) 12 weeks afteroperation, showing significant differences (P＜0.05); in histological score, group A (3.88±1.36) was better than group B (8.38±1.06) and group C (13.13±1.96), and group B was better than group C, showing significant differences (P＜0.05). Conclusion Transplantation of mesenchymal stem cells seeded onto PLLA/gelatin is a promising way for the treatment of cartilage defects.
Objective To evaluate the feasibility and the value of the layered cylindric collagenhydroxyapatite composite as a scaffold for the cartilage tissue engineering after an observation of how it absorbs the chondrocytes and affe cts the cell behaviors. Methods The chondrocytes were isolated and multiplied in vitro, and then the chondrocytes were seeded onto the porous collagen／h ydro xyapatite composite scaffold and were cultured in a three-dimensional environme n t for 3 weeks. The effects of the composite scaffold on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by the phase-contrast microscopy, histology, scanning electron micros copy, and immunohistochemistry. Results The pore diameter of the upper layer of the collagen-hydroxyapatite composite scaffold was about 147 μm. and the porosity was 89%; the pore diameter of the bottom layer was about 85 μm and the porosity was 85%. The layered cylindric collagenhydroxyapatite composite scaffold had good hydrophilia. The chondrocytes that adhered to the surface of the scaffold, proliferated and migrated into the scaffold after 24 hours. The chondrocytesattached to the wall of the microholes of the scaffold maintained a rounded morphology and could secrete the extracellular matrix on the porous scaffold. Conclusion The layered cylindric collagenhydroxyapatite composite scaffold has a good cellular compatibility, and it is ber in the mechanical property than the pure collagen. It will be an ideal scaffold for the cartilage tissue enginee ring.
Objective Mechanical stimulation and inductive factors are both crucial aspects in tissue engineered cartilage. To evaluate the effects of mechanical stimulation combined with inductive factors on the differentiation of tissue engineered cartilage. Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated from newborn porcine (aged7 days and weighing 3-6 kg) and expanded in vitro. The BMSCs at passage 2 were seeded onto a scaffold of poly (lactic-coglycol ic acid) (PLGA) in the concentration of 5 × 107/mL to prepare cell-scaffold composite. Cell-scaffold composites were cultivated in a medium with chondrocyte-inducted factors (group A), in a vessel with mechanic stimulating only (group B), or mechanic stimulating combined with chondrocyte-inducted factors (group C) (parameters of mechanics: 1 Hz, 0.5 MPa, and 4 hours/day). Cell-scaffold composite and auto-cartilage served as positive control (group D) and negative control (group E), respectively. After 4 weeks of cultivation, the thickness, elastic modulus, and glycosaminoglycan (GAG) content of composites were measured. Additionally, BMSCs chondrogenic differentiation was assessed via real-time fluorescent quantitative PCR, immunohistochemistry, and histological staining. Results The thickness, elastic modulus, and maximum load in group C were significantly higher than those in groups A and B (P lt; 0.05). In groups A, B, and C, cartilage lacuna formation, GAG expression, and positive results for collagen type II were obsersed through HE staining, Safranin-O staining, and immunohistochemistry staining. The dyeing depth was deeper in group A than in group B, and in group C than in groups A and B; group C was close to group E. The GAG content in group C was significantly higher than that in groups A and B (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that mRNA expressions of collagen type I, collagen type II, and GAG in group C were significantly higher than those in groups A and B (P lt; 0.05), and in group A than in group B (P lt; 0.05). Conclusion Mechanical stimulation combined with chondrocyte inductive factors can enhance the mechanical properties of the composite and induce higher expression of collagen and GAG of BMSCs.
Objective To sum up the experimental and clinical history as wellas latest development of repair of growth plate injury Methods Recent articles about repair of growth plate injury were extensively reviewed and major reparative methods were introduced, especially including tissue engineering research on growth plate.Results Repair of growth plate injury was a great difficulty inexperimental study and clinical treatment of pediatric orthopedics. Transplantation of free growth plate and cartilage were unfavorably used because of lack ofblood supplement. Although circulation problem was solved by transplantation ofvascularized growth plate, autografts of epiphyseal cartilage were involved in limitation of donor, and allografts of epiphyseal cartilage induced immunological reaction. Noncartilaginous tissue and material could only prevent formation of bony bridge in small defect of growth plate and lacked ability of regenerative repair. Transplantationof tissue engineered cartilage and chondrocytes might be a choice for repair ofgrowth plate injury Conclusion Owing to lack of safe and effective methods ofrepairing growth plate injury, research on chondrocyte and tissue engineered cartilage should be further done.
Objective To explore the effect of tissue engineered cartilage reconstructed by using sodium alginate hydrogel and SIS complex as scaffold material and chondrocyte as seed cell on the repair of full-thickness articular cartilage defects. Methods SIS was prepared by custom-made machine and detergent-enzyme treatment. Full-thickness articularcartilage of loading surface of the humeral head and the femoral condyle obtained from 8 New Zealand white rabbits (2-3weeks old) was used to culture chondrocytes in vitro. Rabbit chondrocytes at passage 4 cultured by conventional multipl ication method were diluted by sodium alginate to (5-7) × 107 cells/mL, and then were coated on SIS to prepare chondrocyte-sodium alginate hydrogel-SIS complex. Forty 6-month-old clean grade New Zealand white rabbits weighing 3.0-3.5 kg were randomized into two groups according to different operative methods (n=20 rabbits per group), and full-thickness cartilage defect model of the unilateral knee joint (right or left) was establ ished in every rabbit. In experimental group, the complex was implanted into the defect layer by layer to construct tissue engineered cartilage, and SIS membrane was coated on the surface to fill the defect completely. While in control group, the cartilage defect was filled by sodium alginate hydrogel and was sutured after being coated with SIS membrane without seeding of chondrocyte. General condition of the rabbits after operation was observed. The rabbits in two groups were killed 1, 3, 5, 7, and 9 months after operation, and underwent gross and histology observation. Results Eight rabbits were excluded due to anesthesia death, wound infection and diarrhea death. Sixteen rabbits per group were included in the experiment, and 3, 3, 3, 3, and 4 rabbits from each group were randomly selected and killed 1, 3, 5, 7, and 9 months after operation, respectively. Gross observation and histology Masson trichrome staining: in the experimental group, SIS on the surface of the implant was fused with the host tissue, and the inferface between them disappeared 1 month after operation; part of the implant was chondrified and the interface between the implant and the host tissue was fused 3 months after operation; the implant turned into fibrocartilage 5 months after operation; fiber arrangement of the cartilage in theimplant was close to that of the host tissue 7 months after operation; cartilage fiber in the implant arranged disorderly andactive cell metabol ism and prol iferation were evident 9 months after operation. While in the control group, no repair of thedefect was observed 9 months after operation. No obvious repair was evident in the defects of the control group within 9months after operation. Histomorphometric evaluation demonstrated that the staining intensity per unit area of the reparative tissue in the defect of the experimental group was significant higher than that of the control group at each time point (P lt; 0.05), the chondrification in the experimental group was increased gradually within 3, 5, and 7 months after operation (P lt; 0.05), and it was decreased 9 months after operation comparing with the value at 7 months after operation (P lt; 0.05). Conclusion Constructed by chondrocyte-sodium alginate hydrogel-SIS in complex with surficial suturing of SIS membrane, the tissue engineered cartilage can in-situ repair cartilage defect, promote the regeneration of cartilage tissue, and is in l ine with physiological repair process of articular cartilage.
Objective To investigate the feasibility of the complex of the fibrin sealant (FS) and the bone marrow mesenchymal stem cells（MSCs） to createanew cartilage in the nude mice by the issue engineering technique. Methods T he MSCs were isolated from healthy humans and were expanded in vitro. And then the MSCs were induced by the defined medium containing the transforming growth factor β1 (TGF-β1), dexamethasone, and ascorbic acid. The biomechanical properties of the chondrocytes were investigated at 7 and 14 days. The MSCs induced for 7days were collected and mixed with FS. Then, the FSMSCs mixture was injectedby a needle into the dorsum of the nude mice in the experimental group. In the tw o control groups, only FS or MSCs were injected respectively. The specimens were harvested at 6 and 12 weeks，and the ability of chondrogenesis in vivo was inve stigated by the gross observation, HE, Alcian Blue staining, and type Ⅱ collagen immunohistochemistry. Results The MSCs changed from a spindlel ike fibroblastic appearance to a polygonal shape when transferred to the defined medium, and couldbe induced to express the chondrocyte matrix. After an injection of the mixture , the cartilage-like tissue mass was formed, and the specimens were harvested from the mass at 6 and 12 weeks in the experimental group. The tissue mass at 6 we eks was smaller and relatively firm in texture, which had a distinct lacuna structure. And glycosaminoglycan (GAG) and Type II Collagen expressions were detecte d. The tissue mass at 12 weeks was bigger, firmer and glossier with the mature c hondrocytes lying in the lacuna structure. The positive Alcian blue and Collagen II immunohistochemistry stainings were ber at 12 weeks than at 6 weeks. But there was no cartilage-like tissue mass formed in the two control groups. Conclusion This study demonstrates that the fibrin sealant and the bone marrow mesenchymal stem cells can be successfully used in a constructing technique for the tissue engineered injectable cartilage.
Objective To study the influence of different mechanical environments on repair cartilage defect with marrow mesenchymal stem cells as seed cells. Methods The rabbit marrow mesenchymal stem cells were isolated and cultured. The cartilage defects were repaired by autologous tissue engineered cartilage with the marrow mesenchymal stem cells as seed cells. Fifteen rabbits with cartilage defect were divided into 3 groups: dislocation group with cell-free scaffold(controlgroup), dislocation group with cartilaginous construct and normal mechanical environment group with cartilaginous construct. The repaired tissue was harvested and examined 6 weeks postoperatively. Results The repair tissue in normal mechanical environment group with cartilaginous construct showed cartilage-like tissue in superficial layer and subchondral bone tissue in deep layer 6 weeks postoperatively. The defect was filled with bone tissue in dislocation group with cartilaginous construct 6 weeks postoperatively. The surrounding normal cartilage tissue showed vascular invasion from subchondral area and the concomitant thinningof the normal cartilage layer. The cartilaginous construct left in the femoral trochlea groove formed hyaline cartilage-like tissue. The defect was repaired byfibrous tissue in control group. Conclusion The repaired tissue by tissue engineered cartilage with marrow mesenchymal stem cells as seed cells showed the best result in normal mechanical environment group, which indicates that it will be essential for the formation and maintenance of tissue engineered cartilage to keep the normal mechanical stress stimulus.
Objective To develop a novel porous three-dimensional scaffold and to investigate its physico-chemical properties for tissue engineering cartilage.Methods Refined 88% deacetylation degree chitosan was prepared and dissolved in 0.2 mol/L acetate acid and fully mixed with highly purified porcine type Ⅱcollagen in 0.5 mol/L acetate acid solution in a ratio of 4 to 1 (wt/wt). Freeze-drying process was employed to fabricate the composite scaffold. The construct wascross-linked by use of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and Nhydroxysuccinimide (NHS). A mechanical tester was utilized to determine the tensilestrength change before and after cross-linking. The microstructure was observed via scanning electron microscopy (SEM). The lysozyme degradation was performedto evaluate the degradability of the scaffold in vitro. Results A bulk scaffold with desired configuration was obtained. The mechanical test showed that the crosslinking treatment could enhance the mechanical strength of the scaffold. The SEM results revealed that the two constituents evenly distributed in the scaffold and that the matrix was porous, sponge-like with interconnected pore sizing 100250 μm. In vitro lysozyme degradation indicated that crosslinked or uncross-linked composite scaffolds had faster degradation rate than the chitosan matrix. Conclusion Chitosan and typeⅡcollagen can be developed into a porous three-dimensional scaffold. The related physico-chemical tests suggest that the composite socaffold meets requirements for tissue engineered scaffold and may serve as an alternative cellcarrier for tissue engineering cartilage.
Objective To develop a scaffold material containing collagen Ⅰ and sodium hyaluronate for the cartilage tissue engineering and to evaluate its biocompatibility by using the rabbit chondrocytes derived from amandibular condylar process. Methods The porous matrices containing collagen Ⅰ and sodium hyaluronate were fabricated by the freezedrying technique and were crosslinked by using 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC). The microstructure of the scaffold was observed under thescanning electron microscope (SEM), and the enzymatic degradation test was performed to compare the ability of the scaffold resistance to collagenase before and after the crosslinking. The chondrocytes from the rabbits’ condylar process were isolated and cultured before they were seeded into the scaffold, and cell attachment and proliferation were measured by the cell count 1, 3, 5, 7 and 10 daysafter the cell being seeded; then, the biocompatibility of the scaffold was evaluated by the light microscopic examination, histological examination, and the SEM exmination. Results The porous structure of the scaffold facilitated the penetration and attachment of the seeded cells. The porosity was 83.7% and the pore size was 100-120 μm. The cell number increased from 3.7×104 per scaffold 1 day after the cell being seeded to 8.2×104 per scaffold 10 days after the cell being seeded. The crosslinking treatment could significantly enhance the scaffold resistance to the collagenase activity. The examinations under the light microscope and SEM indicated that the chondrocyte adhered and spread well on the scaffold, and the extracellular matrices were also observed around the chondrocytes. Conclusion The porous scaffold composed of collagen Ⅰ and hyaluronan has anappropriate structure and a good biocompatibility for the attachment and proliferation of the chondrocytes, which can facilitate it to become a useful scaffold in the cartilage tissue engineering.
Objective To evaluate collagen（Col）hyaluronan (HA) chondroitin sulfate (CS) tri-copolymer as a new biomimetic biodegradable polymer scaffold for application of the articular cartilage tissue engineering. Methods The Col-HACS tricopolymer was prepared by freezing and lyophilization and was cross-linked by 1-ethyl-3(3-dimethy inaminoproyl) carbodiimide (EDC). The morpholog icalcharacteristics of the matrices were evaluated by the SME and HE stainings. The rabbit chondrocytes were isolated and seeded in the tricopolymer scaffold. Morphology, proliferation and differentiation of glycosaminoglycan (GAG), and phenotypic expression of the rabbit articular chondrocytes cultured within the tricopolymer scaffold were indicated by the histological examination, SEM, biochemica l analysis, and reverse transcriptase PCR for collagen typeⅡ（ColⅡ）. Results The chondrocytes proliferated and differentiated well, and th ey preserved the phenotypic expression of ColⅡ in the Col-HA-CS scaffold. After the 21day cell culture within the Col-HA-CS scaffolds, the cartilage-specific morphologyand the structural characteristics such as lacunae appeared，and DNA and GAG conten ts increased with the time. In addition, DNA and GAG contents were significantly higher in the Col-HA-CS matrix than in the collagen matrix alone (Plt;0.05 ). Conclusion These results show that the Col-HA-CS tri-copolymer matrices can provide an appropriate environment for the generation of cartilage-like tissues and have a potential application in the cartilage tissue engineering scaffold field.