Objective To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 (BMP-2) gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells (BMSCs) osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering. Methods BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines (weighing, 15 kg), and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection (MOI) of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group (BMP-2 vector group); BMSCs transfected by empty vector (empty vector group), and non-transfected BMSCs (non-transfection group) were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase (ALP) staining, ALP activities, and Alizarin red staining. Results The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentiviral vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed. Conclusion The porcine BMP- 2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.
Objective To evaluate the effect of weight-bearing time on micro-fracture therapy for small sized osteochondral lesion of the talus (OLT) by comparing early weight-bearing and postponed weight-bearing. Methods Between March 2010 and September 2011, 43 patients with small sized OLT (lt; 2 cm2) scheduled for arthroscopic micro-fracture therapy were randomly divided into early weight-bearing group (n=22) and postponed weight-bearing group (n=21). There was no significant difference in gender, age, body mass index, disease duration, disease cause, preoperative visual analogue scale (VAS) score, and preoperative American Orthopaedic Foot and Ankle Society (AOFAS) score between 2 groups (P gt; 0.05). All patients of 2 groups received micro-fracture treatment under arthroscopy. Full weight bearing began under the protection of “8” figure shaped splint at immediately after operation in early weight-bearing group, and weight bearing began at 6 weeks after operation in postponed weight-bearing group. Results The size of cartilage injury was (1.24 ± 0.35) cm2 in early weight-bearing group and was (1.25 ± 0.42) cm2 in postponed weight-bearing group by arthroscopy measurement, showing no significant difference between 2 groups (t=0.09, P=0.93); and there was no significant difference in cartilage injury grading between 2 groups (Z= — 1.45, P=0.15). The follow-up time was 12-18 months (mean, 14.5 months) in 2 groups. VAS and AOFAS scores of each group at each time point after operation were all significantly improved when compared with preoperative scores (P lt; 0.05), but no significant difference was found between 2 groups at 3, 6, and 12 months after operation (P gt; 0.05). The time of returning to work in early weight-bearing group [(6.35 ± 1.93) months] was significantly shorter than that in postponed weight-bearing group [(8.75 ± 1.48) months] (t= — 4.10, P=0.00). Conclusion For patients with small sized OLT, early weight-bearing and postponed weight-bearing after micro-fracture therapy under arthroscopy have similar short-term results. But patients undergoing early weight-bearing can earlier return to work than patients undergoing postponed weight-bearing.
Objective To prepare collagen-chitosan /nano-hydroxyapatite-collagen-polylactic acid (Col-CS/ nHAC-PLA) biomimetic scaffold and to examine its biocompatibility so as to lay the foundation for its application on the treatment of osteochondral defect. Methods PLA was dissolved in dioxane for getting final concentration of 8%, and the nHAC power was added at a weight ratio of nHAC to PLA, 1 ∶ 1. The solution was poured into a mold and frozen. CS and Col were dissolved in 2% acetum for getting the final concentrations of 2% and 1% respectively, then compounded at a weight ratio of CS to Col, 20 ∶ 1. The solution was poured into the frozen mold containing nHAC-PLA, and then biomimetic osteochondral scaffold of Col-CS/nHAC-PLA was prepared by freeze-drying. Acute systemic toxicity test, intracutaneous stimulation test, pyrogen test, hemolysis test, cytotoxicity test, and bone implant test were performed to evaluate its biocompatibility. Results Col-CS/nHAC-PLA had no acute systemic toxicity. Primary irritation index was 0, indicating that Col-CS/nHAC-PLA had very slight skin irritation. In pyrogen test, the increasing temperature of each rabbit was less than 0.6℃, and the increasing temperature sum of 3 rabbits was less than 1.3℃, which was consistent with the evaluation criteria. Hemolytic rate of Col-CS/nHAC-PLA was 1.38% (far less than 5%). The toxicity grade of Col-CS/nHAC-PLA was classified as grade I. Bone implant test showed that Col-CS/nHAC-PLA had good biocompatibility with the surrounding tissue. Conclusion Col-CS/ nHAC-PLA scaffold has good biocompatibility, which can be used as an alternative osteochondral scaffold.
Objective To explore the preparing methods in vitro and test the cl inical appl icabil ity of implantation in vivo of bone marrow stromal stem cells (BMSCs)-biphasic scaffold to repair defects of cartilage and subchondral bone and tocompare the differences in repaired outcomes of composite, single biphasic scaffold and rabbits themselves. Methods The upper chondral phase and the lower osseous phase of the plugs, using poly-lactic-co-glycol ic acid (PLGA), hydroxyapatite (HA), and other biomaterials, were fused into carrier scaffold, on which collagen type I (Col I) was coated. The surface and inner structure of bi phasic scaffold were observed under scanning electron microscope (SEM). BMSCs was isolated from the bone marrow of tibia and femurs of young New Zealand rabbits using centrifuging and washing, and their morphologies and adherences were observed everyday. Then BMSCs were inoculated on the surface of scaffold to form BMSCs-scaffold composites. Osteochondral defects were surgically created on articular surface of femoral intercondyles of 30 New Zealand rabbits, which were divided into groups A, B and C. In group A, a bi phasic osteochondral composite were implanted into defect, BMSCs and biphasic cyl indrical porous plug of PLGA-HA-Col I in group B, and group C was used as a control without implant. Specimens were harvested to make macroscopic and histological observations at the 1st, 3rd, 6th, and 9th months after operation respectively; meanwhile immunohistological and micro-computed tomography (micro CT) examinations were performed and graded at the 9th month after operation. Results SEM showed an excellent connection of holes in the biphasic scaffold infiltrated by Col I. Optical microscopy and SEM showed a good growth of BMSCs in scaffold without obvious cellular morphological changes and an accumulation in the holes. Macroscopic samples showed a resistant existence of defects of group C within 9 months; the scaffold completely degenerated and chondral-l ike tissue formed on articular surface with partly collapses and irregular defects in group A; and smoother surface without collapses and approach to normal with texture of new regeneration in group B. There were statistically significant differences in macroscopic results (P lt; 0.001), group B was superior to group A, and group C was the worst. The micro CT showed good repairs and reconstruction of subchondral bone, with a acceptable integration with newborn chondral-l ike tissue and host bone in group B. Quantificational analysis of relevantparameters showed no significant differences. Histological results showed inflammations located in defects at the 1st month, new tissue grew into scaffold at the 3rd month; new chondral-l ike tissue crept on the margin of defects and biphasic scaffold degenerated completely at the 6th month, and lots of collagen formed in subchondral bone with major fibrocartilage on chondralarea at the 9th month after surgery in groups A and B. In groups A and B, immunohistological observations were weak positive for Col II and positive for Col I. Conclusion Biphasic scaffold implanted in body can induce and accelerate repair of defects of articular cartilages which are mainly filled with fibrocartilage, especially for subchondral bone. Scaffold combined with BMSCs has the best repairing effects 9 months after implantation.
Objective To study repair of osteochondral defects by using composite of autologous BMSCs and chitosan/HAP (CS/HAP) bilayered scaffold in rabbits and its feasibil ity as osteochondral tissue engineering scaffolds. Methods CS/HAP bilayered scaffolds were produced with CS and HAP using a lyophil ization and sintering method. The pore size of the scaffold was observed by scanning electron microscopy (SEM). Anhydrous ethanol substitution method determined its porosity. BMSCs were isolated from bone marrow and cultured by general bone marrow methods. Both CD44 and CD45 on the BMSCs surface were detected with immunocytochemistry to identify BMSCs. Cell-scaffold complex was made with BMSCs as seed cells and CS/HAP bilayered scaffold as carrier by fibrin glue planting technique. The distribution ofBMSCs in CS/HAP scaffold was tested by SEM. The osteochondral defect (4 mm in diameter and 3 mm in height) model was made in the right knee joint of 36 Japanese white rabbits, which were randomly divided into 3 groups. Defects were repaired with CS/HAP and BMSCs composite ( group A, n=12) and with CS/HAP implants (group B, n=12); defects were not treated as a control (group C, n=12). Histological evaluation and gross observation were carried out at 6 weeks (n=6 in each group) and 12 weeks (n=6 in each group) postoperatively. Semi-quantitative histomorphological analysis was done to evaluate the repair cartilage tissue according to the modified Wakitani grading scale. Results CS/HAP bilayered scaffold possessed a porosity of 76.00% ± 5.01% and pore size of 200-400 μm (mean 300 μm ) in CS layer, and 72.00% ± 4.23% and 200-500 μm (mean 350 μm) in HAP layer, respectively. BMSCs formed colonies within 10-14 days. Immunocytochemistry results showed BMSCs had positive CD44 expression and negative CD45 expression. At 6 and 12 weeks after operation, gross and histological observation showed that the cartilage defects were fully filled with regenerated tissue, but bone defects were partially repaired in group A; the cartilage and bone defects were partially filled with regenerated tissue in group B and group C. The modified Wakitani grading scale were 5.17 ± 1.17 and 3.20 ± 0.75 in group A, 9.00 ± 0.63 and 6.00 ± 0.89 in group B, and 10.00 ± 0.89 and 9.60 ± 0.82 in group C at 6 weeks and 12 weeks postoperatively, respectively; showing significant differences between group A and groups B, C (P lt; 0.05). Conclusion The novel CS/HAP bilayered scaffold possesses porous structure and will possibly become a newbiomaterial of osteochondral tissue engineering.
Objective To investigate the effectiveness of mosaicplasty in repair of large-sized osteochondral compound defects and the integrity of transplanted tissue with recipient sites so as to lay a foundation for clinical application. Methods Twenty-four adult goats were divided into 3 groups randomly. The diameters of defect were 6 mm for the medium-sized defects and 9 mm for the large-sized defects, which were created by a trepan. All of the defects were repaired with osteochondral plugs in diameters of 2 mm(the mediumsized defects) or 3 mm(the large-sized defects). The osteochondral plugs were harvested around the intercondylar fossa or intertrochlea groove, and pressed into the recipient sites by specialized instruments in a mosaic mode. No internal fixation was needed and the animal wereallowed to move freely after operation. From 4 to 24 weeks postoperatively, thespecimens were observed in gross and under electromicroscopy. X-ray detection and glycosaminoglycan(GAG) analysis were also performed to testify the healing processand the integrity of the cartilage and subchondral bone. Results The transplanted subchondral bone was integrated firmly with each other or with recipient sites in both mosaicplasty groups. But 24 weeks postoperatively, transplanted cartilage was not integrate with each other apparently. Obvious cleavage between cartilage plugs could be seen. But in the largesized defect groups, some of the osteochondral plugs were relapsed into the defects leaving the recipient sites some steps, leading to some degree of abrasion in the opposing articular cartilage. There was no significant difference in the GAG content between the transplanted cartilage and normal cartilage. X-ray analysis also demonstrated the healing process between the subchondral bone. Conclusion Mosaicplasty can repair the medium or small-sized osteochondral defects efficiently.
ccording to the characteristics of periosteum which have a copacity for regrowth of cartilage,free autogenous osteoperiosteal grafts taken from the medial side of the metaphsis of the tibia had beenused to reconstruct the osteochondral defects of the articular surface of the knee joint. The mothod wasillustrated by five cases which included of osteochondritis dissecans, subchondral osteonecrosis and oldfracture of the patella. By the period of 16-26 monthes follow up, using knee function...
ObjectiveTo review the current treatment status of osteochondral defects (OCD) of the knee joint. MethodsRecent literature concerning treatment of OCD of the knee joint was extensively reviewed and summarized. ResultsOCD affect both the articular cartilage and the underlying subchondral bone, whereas OCD caused by different etiologies require various treatments. OCD repair is available by conventional clinical methods or the advanced tissue engineering strategies. Current clinical treatment outcomes remain uncertain; tissue engineering has emerged as a potential option as it can be efficiently applied to regenerate bone, cartilage, and the bone-cartilage interface, as well as effectively restore normal function and mechanical properties of the cartilage and subchondral bone. ConclusionOCD management and repair remain a great challenge in orthopedic surgery, thus cartilage and subchondral bone should be promoted as an interdependent functional unit considering treatment strategies to provide the best solution for the treatment of osteochondral defects.
ObjectiveTo explore the relationship between subchondral bone reconstruction and articular cartilage regeneration in a rabbit model of spontaneous osteochondral repair. MethodsTwenty-four 6-month-old New Zealand white rabbits were included. The osteochondral defects (4 mm in diameter and 3 mm in depth) were created in the trochlear groove of the unilateral femur, which penetrated the subchondral bone without any treatment. The rabbits were sacrificed at 1, 4, 12, and 24 weeks after operation, respectively. The specimens were obtained for macroscopic, histological, and immunohistochemical observations. According to the International Cartilage Repair Society (ICRS) histological scoring, the effect of cartilage repair was assessed. The histomorphometrical parameters of subchondral bone were analyzed by micro-CT scan and reconstruction, and the relationship between cartilage repair and the histomorphometrical parameters of the subchondral bone were also analyzed. ResultsOsteochondral defects could be repaired spontaneously in rabbit model. With time, defect was gradually filled with repaired tissue, subchondral bone plate under the defect region gradually migrated upward. Bone mineral density, bone volume fraction, tissue mineralized density, trabecula number, and trabecula thickness were increased, while trabecula spacing was decreased. Significant difference was found in the other parameters between different time points (P<0.05) except for trabecula thickness between at 4 and 12 weeks after operation (P>0.05). Histological examination showed that fibrous repair was predominant with rare hyaline cartilage. With time, ICRS scores increased gradually, showing significant differences between other time points (P<0.05) except for between at 4 and 12 weeks after operation (P>0.05). Among the histomorphometrical parameters of subchondral bone, the trabecula spacing was negatively correlated with ICRS score (r=-0.584, P=0.039), and the other histomorphometrical parameters were positively correlated with ICRS score (r=0.680-0.891). ConclusionThere is relevant correlation as well as independent process between cartilage regeneration and subchondral bone reconstruction in the rabbit model of spontaneous osteochondral repair, and fast subchondral bone remodeling may adversely affect articular cartilage repair.
ObjectiveTo explore the treatment methed of recurrent patellar dislocation associated with old osteochondral fracture and to evaluate its effectiveness. MethodsBetween August 2010 and August 2014, 12 cases of recurrent patellar dislocation with old osteochondral fracture were treated. There were 4 males and 8 females with an average age of 18.3 years (range, 15-24 years). The left knee was involved in 7 cases and the right knee in 5 cases. All the patients had a history of patellar dislocation, the average interval from injury to first hospitalization was 7.6 months (range, 6-13 months). At preoperation, the range of motion (ROM) of the injured knee was (89.17±13.11)°; the Lysholm score was 56.67±18.91; the Q-angle was (17.50±5.28)°; and tibial tuberosity-trochlear groove (TT-TG) distance was (18.33±4.03) mm. The Q-angle was more than 20° and TT-TG distance was more than 20 mm in 6 of 12 cases. There were 6 cases of patellar osteochondral fracture, 5 cases of lateral femoral condylar osteochondral fracture, and 1 case of patellar osteochondral fracture combined with lateral femoral condylar osteochondral fracture. After osteochondral fracture fragments were removed under arthroscope, lateral patellar retinaculum releasing and medial patellar retinaculum reefing was performed in 2 cases, medial patellofemoral ligament (MPFL) reconstruction combined with both lateral patellar retinaculum releasing and medial patellar retinaculum reefing in 4 cases, and MPFL reconstruction, lateral patellar retinaculum releasing, medial patellar retinaculum reefing, and tibial tubercle transfer in 6 cases. ResultsAll wounds healed by first intention with no complication of infection, haematoma, skin necrosis, or bone nonunion. All patients were followed up 12-60 months with an average of 24.2 months. At 3 months after operation, all patellar dislocations were corrected; the Q-angle was (13.33±1.37)° and the TT-TG distance was (12.17±1.17) mm in 6 patients undergoing tibial tubercle transfer, showing significant differences when compared with preoperative values[(22.50±2.17)° and (21.33±2.34) mm] (t=15.25, P=0.00; t=8.27, P=0.00). All patients achieved relief of knee pain and knee locking; the knee ROM and the Lysholm score at last follow-up were (120.42±11.57)° and 89.25±9.71, showing significant differences when compared with preoperative ones (t=-11.61, P=0.00; t=-8.66, P=0.00). ConclusionIt has satisfactory short-term effectiveness to remove old osteochondral fragments that can not be reset and to correct patellar dislocation for recurrent patellar dislocation with old osteochondral fracture.