ObjectiveTo preliminarily investigate morghological changes of rabbits reshaping ear cartilage assisted by microdissection needle and explore feasibility of new therapy for ear deformity.MethodsThe bilateral ears of 5 male New Zealand rabbits (aged, 5-6 months) were fixed maintaining the curvature and randomly divided into 2 groups (5 ears in each group). The ears were stimulated by microdissection needle in experimental group and were not treated with stimulation in control group. The skin reaction in the experimental group was observed immediately and at 4 weeks after stimulation. Then, the fixtures were removed at 4 weeks, and the shapes of the ears were observed. The cartilages were harvested from the ears to examined morphological changes after HE staining, and measured the chondrocyte layer thickness.ResultsAll rabbits survived until the end of the experiment. The skin has healed completely after 4 weeks in experimental group. After removing fixtures, the ears in the two groups all maintained certain forms momentarily; while 24 hours later, the ears in the control group mostly recovered original form, and the ears in the experimental group still maintained certain molding form until 8 weeks. HE staining showed there were smooth cartilage and uniform distribution of cells in the control group; the matrix staining was basically consistent; and the skin was normal appearance with epidermis, dermis, and cartilage of normal aspect. But the proliferation of chondrocyte with more layers of cells were observed in the experimental group. In addition, there were degeneration and injury of cartilage cells and connective tissue with necrotic cells and inflammatory cells at needle insertion sites. The chondrocyte layer thickness was (385.714±2.027) μm in the control group and (1 594.732±1.872) μm in the experimental group, there was significant difference between the two groups (t=–759.059, P=0.000).ConclusionRabbit ear cartilage can be effectively reshaped by microdissection needle. Proliferation of chondrocyte and changes in matrix can be found during the reshaping process.
Objective To explore the best flexion angle of the transplantation tendon for fixing joint in simultaneously reconstructing of the anterior cruciate l igament (ACL) and posterior cruciate l igament (PCL) using semitendinosus tendon as autologous graft. Methods Twenty-four clean level New Zealand White rabbits [(aged 6-8 months, male or female, and weighing (2.5 ± 0.2) kg] were selected and divided randomly into 3 groups (n=8) according to fixation angle of the reconstructed l igaments. The bilateral semitendinosus tendons of hind legs were used to reconstruct the PCL and ACL of right hind leg, and the reconstructed l igaments were fixed at knee flexion angles of 90° (group A), 60° (group B), and 30° (group A). The rabbit general situation was observed after operation, and the specimens of the knee joints (including 10 cmdistal end and 10 cm proximal end) were harvested for testing extension and flexion, displacement, and internal and external rotation at 3 months after operation. Results All the rabbits survived to the end of experiment. There was no significant difference in maximal displacements of ACL and PCL among 3 groups (P gt; 0.05). The anterior and posterior displacements of shift in 3 groups were less than 1 mm, suggesting good stabil ity. The anterior displacement and the posterior displacement at 30° flexion and 90° flexion in group A were significantly larger than those in group C (P lt; 0.05). There were significant differences in internal rotation angle and external rotation angle between group A and group C (P lt; 0.05), and there was no significant difference among other groups (P gt; 0.05). Conclusion When simultaneously reconstructing ACL and PCL, the knee flexion angle of 60° for fixing the reconstructed l igaments can achieve the best effect.
Objective To explore the feasibility of establishing a rabbit model of flail chest. Methods Flail chest model was eatablished in 12 New Zealand white rabbits after anesthesia and sterile surgery. The paradoxical movement of chest wall was recorded by the biological signal acquisition system, arterial blood was collected for blood gas analysis, the vital signs were recorded by electrocardiogram (ECG) and the lung tissue was taken for the pathological analysis at the end of the experiment. The effect of flail chest on the respiratory function of experimental animals was analyzed to evaluate the feasibility of establishing flail chest model. Results All surgeries were successful without mortality. The operation time was 41.42±7.08 min. Duration of endotracheal intubation was 79.33±12.21 min. Statistical results showed that the pH, partial pressure of arterial carbon dioxide (PaCO2) and base excess (BE) increased; while partial pressure of oxygen (PaO2) and oxygen saturation (SaO2) reduced. Pathological results showed that flail chest not intervented for a long period would lead to organic lesions. Conclusion The rabbit model of flail chest is feasible, safe, repeatable, easy and simple to handle. The animal is easy to access which is the foundation to study the disease process, recovery procedure and the efficacy after intervention.
In order to repair cartilage defect in joint with transplantation of cryopreserved homologous embryonic periosteum, 30 rabbits were used and divided into two groups. A 4 mm x 7 mm whole thickness cartilage defect was made in the patellar groove of femur of each rabbit. The homologous embryonic rabbit skull periosteum (ERSP), preserved in two-step freezing schedule, was transplanted onto the cartilage defect of joints of one group and autogenous periosteal graft was done in the joint defect of the other group. The knees were not immobilized, following operation and 16 weeks later, the newly formed tissue in the defects were assessed by gross observation, histochemical examination and biochemical analysis. The results showed that new hyaline-like cartilage was formed in the cryopreserved ERSP grafted knee, and had no significant difference from that of the knee receiving autogenous periosteal graft, but had significant difference from that of the fresh ERSP grafted knee and the non-grafted knee. Furthermore, the new hyaline-like cartilage had the biochemical characteristics of a fibrous cartilage. The conclusion was that this method might be feasible to repair articular cartilage defects.
We compared the sensitivities of human embryo conjtmctival fihroblasts(HECF)and rabbit conjunctival fibroblasts(RCF)m five anlineoplasties in vitro.When the concentration of vincrismum and doxorubicin was 0.001~10mg/L.5-FU was 1~1000mg/L and cisplatin was 0.01~10mg/L,the sensitivities of HECF to tile drugs were lower than that of RCF (Plt;0.01).while the difference of the sensitivilics be;ween HECF and RCF to VP-16 was not significant (P＜0.05). The results suggested that the selection of therapeutic agents for intraocular proliferative disease wilh'hunmn conjunctiva fihroblasts may be more valuable than that with RCF. （Chin J Ocul Fundus Dis,1994,10:223-225）
Objective To investigate the performance of loading naringin composite scaffolds and its effects on repair of osteochondral defects. Methods The loading naringin and unloading naringin sustained release microspheres were prepared by W/O/W method; with the materials of the attpulgite and the collagen type I, the loading naringin, unloading naringin, and loading transforming growth factor β1 (TGF-β1) osteochondral composite scaffolds were constructed respectively by " 3 layers sandwich method”. The effect of sustained-release of loading naringin microspheres, the morphology of the composite scaffolds, and the biocompatibility were evaluated respectively by releasingin vitro, scanning electron microscope, and cell counting kit 8. Forty Japanese white rabbits were randomly divided into groups A, B, C, and D, 10 rabbits each group. After a osteochondral defect of 4.5 mm in diameter and 4 mm in depth was made in the intercondylar fossa of two femurs. Defect was not repaired in group A (blank control), and defect was repaired with unloading naringin composite scaffolds (negative control group), loading naringin composite scaffolds (experimental group), and loading TGF-β1 composite scaffolds (positive control group) in groups B, C, and D respectively. At 3 and 6 months after repair, the intercondylar fossa was harvested for the general, HE staining, and toluidine blue staining to observe the repair effect. Western blot was used to detect the expression of collagen type II in the new cartilage. Results Loading naringin microspheres had good effect of sustained-release; the osteochondral composite scaffolds had good porosity; the cell proliferation rate on loading naringin composite scaffold was increased significantly when compared with unloading naringin scaffold (P<0.05). General observation revealed that defect range of groups C and D was reduced significantly when compared with groups A and B at 3 months after repair; at 6 months after repair, defects of group C were covered by new cartilage, and new cartilage well integrated with the adjacent cartilage in group D. The results of histological staining revealed that defects were filled with a small amount of fibrous tissue in groups A and B, and a small amount of new cartilage in groups C and D at 3 months after repair; new cartilage of groups C and D was similar to normal cartilage, but defects were filled with a large amount of fibrous tissue in groups A and B at 6 months after repair. The expression of collagen type II in groups C and D was significantly higher than that in groups A and B (P<0.05), but no significant difference was found between groups C and D (P>0.05). Conclusion Loading naringin composite scaffolds have good biocompatibility and effect in repair of rabbit articular osteochondral defects.
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 evaluate the effect of bone morphogenetic protein 7 (BMP-7)/poly (lactide-co-glycolide) (PLGA) microspheres on in vitro proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs).MethodsBMP-7/PLGA microspheres were fabricated by double emulsion-drying in liquid method. After mixing BMP-7/PLGA microspheres with the chondrogenic differentiation medium, the supernatant was collected on the 1st, 3rd, 7th, 14th, and 21st day as the releasing solution. The BMSCs were isolated from the bilateral femurs and tibias of 3-5 days old New Zealand rabbits, and the 3rd generation BMSCs were divided into 2 groups: microspheres group and control group. The BMSCs in microspheres group were cultured by 200 μL BMP-7/PLGA microspheres releasing solution in the process of changing liquid every 2-3 days, while in control group were cultured by chondrogenic medium. The cell proliferation (by MTT assay) and the glycosaminoglycan (GAG) contents (by Alician blue staining) were detected after chondrogenic cultured for 1, 3, 7, 14, and 21 days. The chondrogenic differentiation of BMSCs was observed by safranine O staining, toluidine blue staining, and collagen type Ⅱ immunohistochemistry staining at 21 days.ResultsMTT test showed that BMSCs proliferated rapidly in 2 groups at 1, 3, and 7 days; after 7 days, the proliferation of BMSCs in the control group was slow and the BMSCs in microspheres group continued to proliferate rapidly. There was no significant difference of the absorbance (A) value at 1, 3, and 7 days between 2 groups (P>0.05), but theA value at 14 and 21 days in microspheres group was significantly higher than that in control group (P<0.05). Compared with control group at 21 days, in microsphere group, almost all nuclei were dyed bright red by safranine O staining, almost all the nuclei appeared metachromatic purple red by toluidine blue staining, and the most nuclei were yellow or brown by immunohistochemical staining of collagen type Ⅱ. Alcian blue staining showed that the content of GAG in 2 groups increased continuously at different time points; after 7 days, the increasing trend of the control group was slow and the microspheres group continued hypersecretion. There was no significant difference of the GAG content at 1, 3, and 7 days between 2 groups (P>0.05), but the GAG content at 14 and 21 days in microspheres group was significantly higher than that in control group (P<0.05).ConclusionBMP-7/PLGA microspheres prepared by double emulsion-drying in liquid method in vitro can promote proliferation and chondrogenic differentiation of rabbit BMSCs.
ObjectiveTo evaluate the in vivo biological safety of porous zinc oxide (ZnO)/hydroxyapatite (HA) composite materials.MethodsThe porous ZnO/HA composite materials and porous HA materials were prepared by the spark plasma sintering technology. First, the materials were characterized, including scanning electron microscopy to observe the material structure, in vitro degradation experiments to detect the degradation rate of the materials, and inductively coupled plasma emission spectrometer to detect the concentration of Zn2+ dissolved out of the composite material degradation. Then the two kinds of material extracts were prepared for acute systemic toxicity test. Fifteen male Kunming mice were randomly divided into groups A, B, and C (n=5) and injected intraperitoneally with normal saline, HA extracts, and ZnO/HA extracts, respectively. The body mass of the mice was recorded before injection and at 24, 48, and 72 hours after injection. The liver and kidney tissues were taken at 72 hours for HE staining to evaluate the safety of the composite material. Finally, the biological safety of the material in vivo was evaluated by implantation experiment. The eighteen male New Zealand white rabbits were randomly divided into HA group and ZnO/HA group (n=9); a bilateral radius defect model (1 cm) was established, and the right forelimbs of the two groups were implanted with porous HA materials and porous ZnO/HA composite materials, respectively; the left untreated as a blank control. The general condition of the animals were observed after operation. The rabbit blood was collected at 1 day before operation and at 1 day, 1 week, 4 weeks, and 8 weeks after operation for routine blood test (inflammation-related indicators) and blood biochemistry (liver and kidney function-related indicators). X-ray films were taken at 4, 8, and 12 weeks after operation to observe the repair of bone defects.ResultsMaterial characterization showed that porous ZnO/HA composite materials had interconnected large and small pore structures with a pore size between 50 and 500 μm, which degraded faster than porous HA materials, and continuously and slowly dissolved Zn2+. The acute systemic toxicity test showed that the mice in each group had no abnormal performance after injection, and the body mass increased (P<0.05). HE staining showed that the cells shape and structure of liver and kidney tissue were normal. Animal implantation experiments showed that all rabbits survived until the experiment was completed; routine blood tests showed inflammation in each group (neutrophils, monocytes, and lymphocytes increased) at 1 day after operation, and all returned to normal at 8 weeks (P>0.05); compared with 1 day before operation, the content of inflammatory cells in the HA group increased at 1 day, 1 week, and 4 weeks after operation (P<0.05), and the ZnO/HA group increased at 1 day after operation (P<0.05); blood biochemistry showed that the liver and kidney function indexes were in the normal range; X-ray films showed that the ZnO/HA group had better osseointegration than the HA group at 4 weeks after operation.ConclusionThe porous ZnO/HA composite material has good in vivo biological safety and good bone repair ability, which is a potential bone repair material.
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.