Objective The tendon-bone heal ing is the key point to ensure the success of the anterior cruciate l igament (ACL) reconstruction. To observe the histological change in the tendon-bone heal ing after ACL reconstruction by different concentrations of platelet-rich plasma (PRP) combined with deproteinized bone (DPB) of calf as bone tunnel infill ing and to investigate the active effect of the complex on tendon-bone heal ing and to define the optimal concentration of PRP. Methods Eight mL blood was drawn from central artery of New Zealand rabbit ears; PRP was prepared by Landesbergmethod, and l iquid supernatant was used as thinner to prepare different concentrations of PRP (30%, 60%, and 100%). Fresh osteoepiphysis spongy bone was harvested from lower end of femur of newborn calf to prepare DPB by way of 30% H2O2 and ether alternating soaking for 24 hours continuous 6 times. DPB was soaked in different concentrations of PRP and mixed with activator to prepare the PRP/DPB complex. A total of 54 New Zealand white rabbits, aging 8-12 months, weighing (2.5 ± 0.4) kg, were divided randomly into 3 groups: group A (30%PRP/DPB complex, n=18), group B (60%PRP/DPB complex, n=18), and group C (100%PRP/DPB complex, n=18). The legs of the rabbits were randomly divided into experimental side and the control side; ACL was reconstructed by semitendinosus and PRP/DPB complex in bone tunnel in the experimental side, and only by semitendinosus in the control side. The general conditions of the rabbits were observed postoperatively and HE staining was used to observe the tendon-bone heal ing, then I-IV levels of semi-quantitative analysis of the tendon-bone heal ing were evaluated according to Demirag standard at 3, 6, and 12 weeks. Results General observation: Synovial fluid sl ightly increased in the specimens and no bony tissue was found in inner of femoral tunnel at 3 weeks; there was no synovial fluid in all the specimens and scar tissue was discovered in inner of femoral tunnel at 6 weeks; and there was no synovial fluid and the tendons became tighter with fibrous tissue at 12 weeks. Histological observation: New granulation tissue formed in the tendon-bone interface of group A experimental sides at 3 weeks; there was various widths of Sharpey type textile fiber in the tendon-bone interface at 6 weeks; Sharpey type textile fiber arranged regularly, which formed an irregular and blur “tidal l ine” at 12 weeks. Group B experimental sides were better than any other group at 3, 6, and 12 weeks; chondrocyte-l ike arranged regularly in the tendonboneinterface at 3 weeks; the number of chondrocyte-l ike per unit area was more than that of the other groups at 6 weeks;and chondrocyte-l ike prol iferated and matured in the tendon-bone interface, Sharpey type textile fiber became tighter andordered. Group C experimental sides were similar to both sides of group A at 3 weeks, however, the prol iferation of relatively mature dense connective tissue was worse than that of other groups at 6 and 12 weeks. According to Demirag grading, there were significant differences in tendon-bone heal ing between the experimental sides and the control sides of group B at 3 and 6 weeks, and between group B experimental sides and group C experimental sides at 12 weeks (P lt; 0.05). Conclusion The mixture of PRP/PRP has good biocompatibil ity and bone induction, so it can enhance tendon-bone heal ing after ACL reconstruction when the concentration of PRP is 60%.
Objective To explore a new method of treating early avascular necrosis of femoral head (AVNFH). Methods Sixty-nine New Zealand adult rabbitswith a mean weight of 2.8 kg after AVNFH presenting were randomly divided into three groups. In group A, deproteinized bone(DPB) combined with the recombinant plasmid pcDNA3.1/vascular endothelial growth factor 165(VEGF165) was implanted in the drilled channel of the necrotic femoral head. In group B, only DPB was implanted. In group C, channel was drilled without DPB or plasmid implanted. Femoral head specimens were obtained 3 days, 1, 2, 4, 8 and 16 weeks after operation. The expression of VEGF165 was examined by RT-PCR, Western blot and immunohistochemical techniques. X-ray testedbone formation generally. Angiogenesis and repair of the femoral head were observed by histological and histomorphometric analysis. Results In group A, the expressions of VEGF165 mRNA and protein were detected 3 days postoperatively, reached apex 1 week and lasted more than 3 weeks after implantation. The ratios of IOD of collagen type Ⅰ were 0.29±0.11, 0.55±0.13 and 0.67±0.10 IOD/μm2 respectively at 2, 4 and 8 weeks postoperatively and the ratios of IOD of new capillary vessels were 0.33±0.10and 0.57±0.16 IOD/μm2 respectively at 2, 4 weeks postoperatively in group A, showing statistically significant difference (Plt;0.01) when compared with groups B and D. X-ray test indicated much bone callus formed early. Conclusion Transfection of the VEGF165 gene can enhance local angiogenesis at early stage andDPBVEGF165 compound can improve bone formation. Deproteinized bone combined with VEGF165 gene provides a potential method for therapy of osteonecrosis.
ObjectiveTo evaluate the effect of tissue engineered periosteum on the repair of large diaphysis defect in rabbit radius, and the effect of deproteinized bone (DPB) as supporting scaffolds of tissue engineering periosteum. MethodsBone marrow mesenchymal stem cells (BMSCs) were cultured from 1-month-old New Zealand Rabbit and osteogenetically induced into osteoblasts. Porcine small intestinal submucosa (SIS) scaffold was produced by decellular and a series mechanical and physiochemical procedures. Then tissue engineered periosteum was constructed by combining osteogenic BMSCs and SIS, and then the adhesion of cells to scaffolds was observed by scanning electron microscope (SEM). Fresh allogeneic bone was drilled and deproteinized as DPB scaffold. Tissue engineered periosteum/DPB complex was constructed by tissue engineered periosteum and DPB. Tissue engineered periosteum was "coat-like" package the DPB, and bundled with absorbable sutures. Forty-eight New Zealand white rabbits (4-month-old) were randomly divided into 4 groups (groups A, B, C, and D, n=12). The bone defect model of 3.5 cm in length in the left radius was created. Defect was repaired with tissue engineered periosteum in group A, with DPB in group B, with tissue engineered periosteum/DPB in group C; defect was untreated in group D. At 4, 8, and 12 weeks after operation, 4 rabbits in each group were observed by X-ray. At 8 weeks after operation, 4 rabbits of each group were randomly sacrificed for histological examination. ResultsSEM observation showed that abundant seeding cells adhered to tissue engineered periosteum. At 4, 8, and 12 weeks after operation, X-ray films showed the newly formed bone was much more in groups A and C than groups B and D. The X-ray film score were significantly higher in groups A and C than in groups B and D, in group A than in group C, and in group B than in group D (P<0.05). Histological staining indicated that there was a lot of newly formed bone in the defect space in group A, with abundant newly formed vessels and medullary cavity. While in group B, the defect space filled with the DPB, the degradation of DPB was not obvious. In group C, there was a lot of newly formed bone in the defect space, island-like DPB and obvious DPB degradation were seen in newly formed bone. In group D, the defect space only replaced by some connective tissue. ConclusionTissue engineered periosteum constructed by SIS and BMSCs has the feasibility to repair the large diaphysis defect in rabbit. DPB isn't an ideal support scaffold of tissue engineering periosteum, the supporting scaffolds of tissue engineered periosteum need further exploration.
ObjectiveTo investigate the effects of different concentrations of osteoprotegerin (OPG) combined with deproteinized bone (DPB) on the bone tunnel after the anterior cruciate ligament (ACL) reconstruction. MethodsThe femoral epiphyseal side was harvested from newborn calf, and allogenic DPB were prepared by hydrogen peroxide-chloroform/methanol method. Then, DPB were immersed in 3 concentrations levels of OPG (30, 60, 100 μg/mL) and 3 concentration ratios (30%, 60%, 100%) of the gel complex were prepared. Sixty healthy New Zealand white rabbits, male or female, weighing (2.7±0.4) kg, were divided randomly into 4 groups (n=15):control group (group A), 30% (group B), 60% (group C), and 100% (group D) OPG/DPB gel complex. The ACL reconstruction models were established by autologous Achilles tendon. Different ratios of OPG/DPB gel complex were implanted in the femoral and tibial bone tunnel of groups B, C, and D, but group A was not treated. The pathology observation (including the percentage of the femoral bone tunnel enlargement) and histological observation were performed and the biomechanical properties were measured at 4, 8, and 12 weeks after operation. ResultsOne rabbit died of infection in groups A and D, 2 rabbits in groups B and C respectively, and were added. General pathology observation showed that the internal orifices of the femoral and tibia tunnels were covered by a little of scar tissue at 4 weeks in all groups. At 8 weeks, white chondroid tissues were observed around the internal orifices of the femoral and tibia tunnels, especially in groups C and D. At 12 weeks, the internal orifices of the femoral and tibia tunnels enlarged in groups A, B, and C, but it was completely closed in group D. At each time point, the rates of the femoral bone tunnel enlargement in groups B, C, and D were significantly lower than that in group A, and group D was significantly lower than groups B and C (P<0.05); group C was significantly lower than group B at 8 weeks, but no significant difference was found at 4 and 12 weeks (P<0.05). Hisological observation showed that fresh fibrous connective tissue was observed in 4 groups at 4 weeks; there was various arrangements of Sharpey fiber in all groups at 8 weeks and the atypical 4-layer structure of bone was seen in group D; at 12 weeks, Sharpey fiber arranged regularly in all groups, with typical 4-layer structure of bone in groups B, C, and D, and an irregular "tidal line" formed, especially in group D. Biomechanics measurement showed that the maximum tensile load in group D was significantly higher than that in groups A and B at 4 weeks (P<0.05), but no significant difference was shown among groups A, B, and C, and between groups C and D (P>0.05); at 8 weeks, it was significantly higher in groups C and group D than group A, and in group D than group B (P<0.05), but there was no significant difference between groups A, C and group B (P>0.05); at 12 weeks, it was significantly higher in groups C and D than groups A and B, and in group D than group C (P<0.05), but difference was not significant between groups A and B (P>0.05). ConclusionDifferent concentrations ratios of OPG/DPB gel complexes have different effects on the bone tunnel after ACL reconstruction. 100% OPG/DPB gel complex has significant effects to prevent the enlargement of bone tunnel and to enhance tendon bone healing.