Objective To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. Methods CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups (n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. Results The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 μm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes (P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers (P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B (P<0.05), and the level of TIMP-1 in group C was significantly higher (P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B (P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B (P<0.05).Conclusion CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.
ObjectiveTo investigate the mechanism of magnesium sulfate in protecting rabbit cartilage by initiating autophagy.MethodsTwenty-four adult female New Zealand rabbits were used to prepare post-traumatic osteoarthritis (PTOA) models by anterior cruciate ligament transection. Then, the PTOA models were randomly divided into PTOA group, distilled water group, and magnesium sulfate group, with 8 rabbits in each group. Immediately after operation, the distilled water group and the magnesium sulfate group were injected with 0.5 mL distilled water and 20 mmol/L magnesium sulfate solution in the joint cavity 3 times a week for 4 weeks, respectively. The PTOA group was not treated. The general condition of the animals was observed after operation. After 4 weeks, the expressions of tumor necrosis factor α (TNF-α) and collagen typeⅡ in the joint fluid and the expression of collagen type Ⅱ in venous blood were detected by ELISA assay. The protein expressions of transient receptor potential channel vanilloid 5 (TRPV5) and microtubule associated protein 1 light chain 3 (LC3; LC3-Ⅱ/LC3-Ⅰ) in femoral cartilage were detected by Western blot. The mRNA expressions of interleukin 1β (IL-1β), TNF-α, matrix metalloproteinases 3 (MMP-3) in synovial tissue and collagen type Ⅱ, Aggrecan (AGN), SOX9 in cartilage tissue were detected by real-time fluorescence quantitative PCR. Cartilage tissue sections were stained with HE staining, Masson staining, and Alcian blue staining and scored according to the modified histological osteoarthritis (OA) score.ResultsAll animals survived until the experiment was completed. Compared with the other two groups, the expression of TNF-α in joint effusion and collagen type Ⅱ in joint effusion and venous blood were decreased in magnesium sulfate group; the protein expression of TRPV5 decreased, and the ratio of LC3-Ⅱ/LC3-Ⅰ increased significantly; the mRNA expressions of IL-1β, TNF-α, and MMP-3 in synovial tissue were decreased, and the mRNA expressions of collagen type Ⅱ, AGN, and SOX9 in cartilage tissue were increased; OA scores also decreased significantly. All differences were statistically significant (P<0.05). There was no significant difference in the above indicators between the PTOA group and the distilled water group (P>0.05).ConclusionIntra-articular injection of magnesium sulfate can reduce intra-articular inflammation, reduce the loss of collagen type Ⅱ and AGN, and is beneficial to cartilage regeneration in rabbits. The mechanism may be related to the initiation of chondroautophagy by inhibiting the calcium channel TRPV5.
ObjectiveTo investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD+) levels.MethodsThe bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H2O2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H2O2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H2O2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD+ and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment.ResultsThe rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased (P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H2O2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD+ and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant (P<0.05).ConclusionNMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD+ levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.
ObjectiveTo explore the effect of icariin on early steroid-induced osteonecrosis of the femoral head in rabbits.MethodsFifty mature New Zealand rabbits (weighing, 2.5-3.0 kg) were randomly divided into control group (n=10), model group (n=20), and experimental group (n=20). The rabbits of model and experimental groups were injected with lipopolysaccharide and methylprednisolone to establish the animal model of early steroid-induced osteonecrosis of the femoral head. The rabbits of experimental group were feeded with icariin solution once a day for 6 weeks since the first injection of methylprednisolone, whereas the rabbits of control and model groups were given normal saline at the same time points. The left femoral heads were removed after 6 weeks and gross morphological features were evaluated. Micro-CT scan was performed to analyze the trabecular microstructure with the following parameters: trabecular bone volume to total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Tn), and trabecular separation (Tb.Sp). The Micro-CT scan was also converted to three-dimensional reconstruction images for observation. HE staining was applied to observe the trabecular structure and morphological changes of osteocytes and marrow adipocytes. It was also used to determine whether the samples of femoral heads occurred osteonecrosis based on the criteria for pathological diagnosis, and calculate the rate of empty lacunae.ResultsSeven rabbits died during the study, and 9, 16, and 18 rabbits in the control, model, and experimental groups, respectively, enrolled the final analysis. Compared with control group, the femoral head collapse and trabecular breaks were more obvious, and the trabeculae were sparse with irregular arrangement in the model group according to the results of gross observation, Micro-CT scan, and three-dimensional reconstruction images. But in the experimental group, the surface of femoral head was slight shrinking without obvious collapse, and the degeneration of trabecular structure was mild. According to bone microstructures analysis, the Tb.N, Tb.Tn, and BV/TV of femoral head in model and experimental groups were lower than those in control group, while the Tb.Sp in the model and experimental groups were significantly higher. The Tb.N, Tb.Tn, and BV/TV of femoral head in experimental group were higher than those in model group, while the Tb.Sp in the experimental group was significantly lower. The differences between groups were all significant (P<0.05). In the model group, HE staining showed that the number of osteocytes reduced, the number of empty lacunae increased, and the marrow adipocytes piled up in the space between femoral trabeculae, some even mashed together like a cyst. In the experimental group, the trabecular structure was still relatively complete compared with model group, no obvious apoptosis of osteocytes was observed, the size and number of adipocytes were basically normal. None of the animals in control group occurred osteonecrosis of the femoral head based on the criteria for pathological diagnosis, and the incidence of osteonecrosis were 81.3% (13/16) in the model group and 66.7% (12/18) in the experimental group, and the difference was not significant (P=0.448). The rate of empty lacunae of osteonecrotic femoral heads in the model group was 33.1%±1.4%, which was higher than that in experimental group (18.9%±0.8%) and in control group (12.7%±1.5%), and the differences between groups were significant (P<0.05).ConclusionThe icariin has a protective effect on the early steroid-induced osteonecrosis of the femoral head in rabbits, which can decrease osteocytes apoptosis, improve the bone microstructure, and delay such disease processes.
Objective To explore the best centrifuge condition for preparing rabbit leukocyte-poor platelet-rich plasma (LP-PRP) by using single centrifugation method. Methods Sixteen healthy New Zealand rabbits, aged 3-4 months, were utilized in the investigation. A total of 15 mL anticoagulated blood was extracted from the central ear artery of each rabbit, with a repeat of the blood collection procedure after 1 and 2 months. The obtained blood specimens were individually subjected to centrifugation at a radius of 16.7 cm and speeds of 1 200, 1 300, 1 400, and 1 500 r/min (equivalent to centrifugal forces of 269×g, 315×g, 365×g, and 420×g) for durations of 2, 3, 4, and 5 minutes, resulting in a total of 16 groups. Following centrifugation, collect plasma from each group to a distance of 1.5 mL from the separation plane. The volumes, platelet enrichment coefficient, and platelet recovery rates of LP-PRP in each group, under varying centrifugation conditions, were methodically computed and subsequently compared. Results The volume of LP-PRP obtained under all centrifugation conditions ranged from 1.8 to 7.6 mL. At a consistent centrifugal speed, an extension of centrifugation time leaded to a significant increase in the volume of LP-PRP, accompanied by a declining trend in the platelet enrichment coefficient of LP-PRP. When centrifuged for 2 minutes, the volume of LP-PRP at speeds of 1 200 and 1 300 r/min was less than 2.0 mL, while the volume of LP-PRP obtained under other conditions was more than 2.0 mL. When centrifuged for 4 and 5 minutes, the volume of LP-PRP obtained at each speed was more than 4 mL. LP-PRP with a platelet enrichment coefficient more than 2.0 could be prepared by centrifuging at 1 200 r/min for each time group and 1 300 r/min for 2 and 3 minutes, and the highest LP-PRP platelet enrichment coefficient could be obtained by centrifugation for 2 minutes at a speed of 1 200 r/min. The platelet recovery rates of LP-PRP obtained by centrifugation at 1 200 r/min for 4 and 5 minutes, as well as centrifugation at 1 400 r/min for 5 minutes, were both greater than 60%. There was no significant difference between the groups when centrifuged at 1 200 r/min for 4 and 5 minutes (P>0.05). Conclusion In the process of preparing rabbit LP-PRP using a single centrifugation method, collecting 15 mL of blood and centrifuging at a radius of 16.7 cm and speed of 1 200 r/min for 4 minutes can prepare LP-PRP with a volume exceeding 2.0 mL, platelet enrichment coefficient exceeding 2.0, and platelet recovery rate exceeding 60%. This centrifugal condition can achieve the optimal LP-PRP action parameters in the shortest possible time.
ObjectiveTo investigate the biocompatibility and immunogenicity of the tracheal matrix decellularized by sodium perchlorate (NaClO4).MethodsBone marrow mesenchymal stem cells (BMSCs) were divided from 2-month-old New Zealand white rabbits. The trachea of 6-month-old New Zealand white rabbits were trimmed to a length of 1.5 cm and randomly divided into control group (group A1, n=5, just stripped the loose connective tissue outside the trachea) and experimental group (group B1, n=5, decellularized by improved NaClO4 immersion method). The cytotoxicity of the scaffold leaching solution was detected by MTT assay, and the major histocompatibility complex (MHC) expression was detected by immunohistochemical method. The 4th generation of BMSCs were seeded onto the scaffold of 2 groups, and the cell activity around the material was observed by inverted microscope after Giemsa staining at 48 hours, while the cells states on the scaffold were observed at 7 and 14 days after culturing by scanning electron microscope. Another 10 6-month-old New Zealand white rabbits were randomly divided into control group (group A2, n=5) and experimental group (group B2, n=5), which implanted the native trachea and decellularized tracheal matrix into the subcutaneous sac of the back neck, respectively. The serum immunoglobulin IgM and IgG contents were analysed at 5, 10, 15, 20, 25, and 30 days after operation, and HE staining observation was performed at 30 days after operation.ResultsMTT assay showed that the proliferation activity of BMSCs cultured in the leach liquor of group B1 was well, showing no significant difference when compared with group A1 and negative control group with pure culture medium (P>0.05). The immunohistochemical staining showed that the decellularized process could significantly reducing the antigenicity of matrix materials. Giemsa staining showed that BMSCs grew well around the two tracheal matrixs (groups A1 and B1) in vitro. Scanning electron microscope observation showed that the cells were attached to the outer wall of the tracheal material in group A1, which present a flat, round, oval shaped, tightly arranged cells and cluster distribution; and in group B1, the cells formed a single lamellar sheet cover the outer wall of the tracheal material, whose morphology was similar to that in group A1, and the growth trend was better. In vivo experimental results showed that the rejection of group B2 was lower than that of group A2. The contens of IgM and IgG in group A2 were significantly higher than those in group B2 at each time point after operation (P<0.05). HE staining showed no signs of rejection, macrophagocyte, or lymphocyte infiltration occurred, and the collagen fibers maintained their integrity in group B2.ConclusionThe decellularized matrix treated by NaClO4 has a fine biocompatibility, while its immunogenicity decreased, and it is suitable for the scaffold material for constructing of tissue engineered trachea.
ObjectiveTo observe and compare the effects of peptides on the repair of rabbit skull defects through two different binding modes of non-covalent and covalent, and the combination of carboxyl (-COOH) and amino (-NH2) groups with materials.MethodsTwenty-one 3-month-old male ordinary New Zealand white rabbits were numbered 1 to 42 on the left and right parietal bones. They were divided into 5 groups using a random number table, the control group (group A, 6 sides) and the material group 1, 2, 3, 4 (respectively group B, C, D, E, 9 sides in each group). All animals were prepared with 12-mm-diameter skull defect models, and bone morphogenetic protein 2 (BMP-2) non-covalently bound multiwalled carbon nanotubes (MWCNT)-COOH+poly (L-lactide) (PLLA), BMP-2 non-covalently bound MWCNT-NH2+PLLA, BMP-2 covalently bound MWCNT-COOH+PLLA, and BMP-2 covalently bound MWCNT-NH2+PLLA were implanted into the defects of groups B, C, D, and E, respectively. At 4, 8, and 12 weeks after operation, the samples were taken for CT scanning and three-dimensional reconstruction, the ratio of bone tissue regeneration volume to total volume and bone mineral density were measured, and the histological observation of HE staining and Masson trichrome staining were performed to quantitatively analyze the volume ratio of new bone tissue.ResultsCT scanning and three-dimensional reconstruction showed that with the extension of time, the defects in groups A-E were filled gradually, and the defect in group E was completely filled at 12 weeks after operation. HE staining and Masson trichrome staining showed that the volume of new bone tissue in each group gradually increased with time, and regenerated mature bone tissue appeared in groups D and E at 12 weeks after operation. Quantitative analysis showed that at 4, 8, and 12 weeks after operation, the ratio of bone tissue regeneration volume to total volume, bone mineral density, and the volume ratio of new bone tissue increased gradually over time; and at each time point, the above indexes increased gradually from group A to group E, and the differences between groups were significant (P<0.05).ConclusionThrough covalent binding and using -NH2 to bound peptides with materials, the best bone repair effect can be achieved.
An experimental model of proliferative vitretinopathy(PVR) induced by macrophages was used for the evaluation of drug efficacy of daunomycin encapsulated in liposomes in the treatment of PVR.Five mu;g daunomycin(n=40),10mu;g daunomycin-liposome(DL,n=30)and 0.1 ml saline or empty liposomes(n=40,as controls)were injected into the rabbit vitreous after macrophage injection.Retinal detachment developed in 77.5% of the control eyes on day 28,compared to 33.3% of the eyes treated with DL(P＜0.01)and 50% of the daunomycin-treated eyes(P＜0.05).The results suggest that encapsulation in liposomes of cytotoxic agents can enhance drug efficacy.The phasic course of development of PVR is important in the selection of particular drugs. （Chin J Ocul Fundus Dis,1993,9:77-80）
Objective To investigate the effect of ultrasonic irradiation time on enhanced green fluorescent protein (EGFP) gene transfection efficiency and local tissue in bone defects using ultrasound-mediated microbubble destruction. Methods Thirty 3-month-old New Zealand rabbits (2.5-3.0 kg in weight) were randomly divided into 5 groups (n=6) and bone defect models were made on the right ulna. At 10 days after modeling, suspension of microbubbles and EGFP plasmids were locally injected (0.3 mL/kg) and then ultrasound was performed on defect at a frequency of 1 MHz, a intensity of 0.5 W/cm2, and a duty ratio of 20% for 1, 2, 3, 4, and 5 minutes respectively (in 1, 2, 3, 4, and 5 minutes groups respectively). The survival condition was observed. Rabbits were sacrificed for gross observation at 7 days after transfer. The gene expression was observed by fluorescence staining. HE staining and transmission electron microscopy were used to observe the local tissue damage. Results The animals all survived. New soft tissue formed in bone defects area at 1 week after transfer, the surrounding muscle tissue was partly filled in it. Green fluorescence expression was observed in all rabbits. The expression was the strongest in 2 minutes group, and was the weakest in 1 minute group. The absorbance (A) value showed significant differences when compared 1 minute and 2 minutes groups with other groups (P<0.05), but no significant difference was found between 3, 4, and 5 minutes groups (P>0.05). Tissue damage was observed in all groups and it was aggravated with the increase of irradiation time. Conclusion EGFP transfection efficiency in bone defect by ultrasound-mediated microbubble destruction is related to irradiation time. EGFP gene can be efficiently transfected without obvious toxicity at 1 MHz, 0.5W/cm2, and duty ratio of 20% for 2 minutes in bone defects of rabbits.
ObjectiveTo study the effect of intraarticular injection of crosslinked-chitosan in the treatment of knee osteoarthritis in rabbits.MethodsThirty-two New Zealand white rabbits were randomly divided into 4 groups (groups A, B, C, and D; 8 rabbits in each group). The knee osteoarthritis models were prepared by anterior cruciate ligament transection in the left hind in groups A, B, and C. At 4 weeks after operation, the rabbits were received intraarticular injection of 0.6 mL crosslinked-chitosan in group A, 0.3 mL chitosan (once per 2 weeks, for twice) in group B, and 0.3 mL saline (once per 2 weeks, for twice) in group C. The rabbits in group D were treated with sham operation in the left hind, and received intraarticular injection of 0.3 mL saline (once per 2 weeks, for twice). At 8 weeks, the macroscopic observation, histological examination (HE staining, Safranin-fast green double staining, and Mankin score), scanning electron microscopy (SEM) observation, and immunohistochemical staining of collagen type Ⅱ were performed.ResultsMacroscopic and SEM observations showed that the cartilage in group D was basically the same as normal and better than that in groups A and B, and the abrasion of cartilage in group C was the most serious. The histological observation results in groups A and B were slightly similar and better than those in group C, but not up to the structure of group D. The macroscopic score and Mankin score of groups B and C were significantly higher than those of group D (P<0.05), and there was no significant difference between group A and group B (P>0.05). Immunohistochemical staining results showed that the collagen type Ⅱ positive percentage of chondrocytes was significantly higher in group D than that in groups B and C, and no significant difference was found between group A and group B (P>0.05). ConclusionThe crosslinked-chitosan can significantly improve the osteoarthritis of the rabbit knee, delay the pathological changes of osteoarthritis, and decrease the frequency of injection.