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.
ObjectiveTo observe the effect of using tungsten drills to prepare mouse knee osteochondral injury model by comparing with the needle modeling method, in order to provide an appropriate animal modeling method for osteochondral injury research.MethodsA total of 75 two-month-old male C57BL/6 mice were randomly divided into 3 groups (n=25). Mice in groups A and B were used to prepare the right knee osteochondral injury models by using needles and tungsten drills, respectively; group C was sham-operation group. The general condition of the mice was observed after operation. The samples were taken at 1 day and 1, 2, 4, and 8 weeks after modeling, and HE staining was performed. The depth, width, and cross-sectional area of the injury site at 1 day in groups A and B were measured, and the percentage of the injury depth to the thickness of the articular cartilage (depth/thickness) was calculated. Toluidine blue staining and immunohistochemical staining for collagen type Ⅱ were performed at 8 weeks, and the International Cartilage Research Society (ICRS) score was used to evaluate the osteochondral healing in groups A and B.ResultsAll mice survived to the completion of the experiment. HE staining showed that group C had normal cartilage morphology. At 1 day after modeling, the injury in group A only broke through the cartilage layer and reached the subchondral bone without entering the bone marrow cavity; the injury in group B reached the bone marrow cavity. The depth, width, cross-sectional area, and depth/thickness of the injury in group A were significantly lower than those in group B (P<0.05). At 1, 2, 4, and 8 weeks after modeling, there was no obvious tissue filling in the injured part of group A, and no toluidine blue staining and expression of collagen type Ⅱ were observed at 8 weeks; while the injured part of group B was gradually filled with tissue, the toluidine blue staining and the expression of collagen type Ⅱ were seen at 8 weeks. At 8 weeks, the ICRS score of group A was 8.2±1.3, which was lower than that of group B (13.6±0.9), showing significant difference (t=−7.637, P=0.000).ConclusionThe tungsten drills can break through the subchondral bone layer and enter the bone marrow cavity, and the injury can heal spontaneously. Compared with the needle modeling method, it is a better method for modeling knee osteochondral injury in mice.
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 investigate the effectiveness of micro-fracture therapy combined with intra-articular injection of platelet-rich plasma (PRP) in the treatment of small sized osteochondral lesion of the talus (OLT).MethodsBetween September 2014 and October 2017, 43 patients with small sized OLT met the inclusive criteria were admitted and randomly divided into micro-fracture group (21 cases) and combined group (22 cases). Patients in the micro-fracture group were treated with micro-fracture therapy, and patients in the combined group were treated with micro-fracture therapy combined with intra-articular injection of PRP. There was no significant difference in gender, age, disease duration, side of OLT, injured position, lesion area, Mintz classification, and preoperative American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hind foot score and visual analogue scale (VAS) score between the two groups (P>0.05). After treatment, MRI, VAS score, and AOFAS ankle-hind foot score were used to evaluate the recovery of OLT and the ankle function.ResultsAll incisions healed by first intention, and no complications such as venous thrombosis and ankle joint infection occurred. All patients were followed up 12-18 months after operation, with an average of 15.6 months. The VAS scores and the AOFAS ankle-hind foot scores were significantly improved at 6 and 12 months after operation in the two groups (P<0.05), and the scores at 12 months were significantly improved when compared with postoperative scores at 6 months (P<0.05). Compared with the micro-fracture group, the VAS score and the AOFAS ankle-hind foot score were significantly improved in the combined group at 6 and 12 months after operation (P<0.05). MRI showed that OLT was well filled in both groups at 12 months after operation.ConclusionCompared with micro- fracture therapy, micro-fracture therapy combined with intra-articular injection of PRP can effectively reduce pain, improve ankle function, and has a good effectiveness in the treatment of small sized OLT.
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 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 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.
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.
ObjectiveThe tissue engineered osteochondral integration of multi-layered scaffold was prepared and the related mechanical properties and biological properties were evaluated to provide a new technique and method for the repair and regeneration of osteochondral defect.MethodsAccording to blend of different components and proportion of acellular cartilage extracellular matrix of pig, nano-hydroxyapatite, and alginate, the osteochondral integration of multi-layered scaffold was prepared by using freeze-drying and physical and chemical cross-linking technology. The cartilage layer was consisted of acellular cartilage extracellular matrix; the middle layer was consisted of acellular cartilage extracellular matrix and alginate; and the bone layer was consisted of nano-hydroxyapatite, alginate, and acellular cartilage extracellular matrix. The biological and mechanics characteristic of the osteochondral integration of multi-layered scaffold were evaluated by morphology observation, scanning electron microscope observation, Micro-CT observation, porosity and pore size determination, water absorption capacity determination, mechanical testing (compression modulus and layer adhesive strength), biocompatibility testing [L929 cell proliferation on scaffold assessed by MTT assay, and growth of green fluorescent protein (GFP)-labeled Sprague Dawley rats’ bone marrow mesenchumal stem cells (BMSCs) on scaffolds].ResultsGross observation and Micro-CT observation showed that the scaffolds were closely integrated with each other without obvious discontinuities and separation. Scanning electron microscope showed that the structure of the bone layer was relatively dense, while the structure of the middle layer and the cartilage layer was relatively loose. The pore structures in the layers were connected to each other and all had the multi-dimensional characteristics. The porosity of cartilage layer, middle layer, and bone layer of the scaffolds were 93.55%±2.90%, 93.55%±4.10%, and 50.28%±3.20%, respectively; the porosity of the bone layer was significantly lower than that of cartilage layer and middle layer (P<0.05), but no significant difference was found between cartilage layer and middle layer (P>0.05). The pore size of the three layers were (239.66±35.28), (153.24±19.78), and (82.72±16.94) μm, respectively, showing significant differences between layers (P<0.05). The hydrophilic of the three layers were (15.14±3.15), (13.65±2.98), and (5.32±1.87) mL/g, respectively; the hydrophilic of the bone layer was significantly lower than that of cartilage layer and middle layer (P<0.05), but no significant difference was found between cartilage layer and middle layer (P>0.05). The compression modulus of the three layers were (51.36±13.25), (47.93±12.74), and (155.18±19.62) kPa, respectively; and compression modulus of the bone layer was significantly higher than that of cartilage layer and middle layer (P<0.05), but no significant difference was found between cartilage layer and middle layer (P>0.05). The osteochondral integration of multi-layered scaffold was tightly bonded with each layer. The layer adhesive strength between the cartilage layer and the middle layer was (18.21±5.16) kPa, and the layer adhesive strength between the middle layer and the bone layer was (16.73±6.38) kPa, showing no significant difference (t=0.637, P=0.537). MTT assay showed that L929 cells grew well on the scaffolds, indicating no scaffold cytotoxicity. GFP-labeled rat BMSCs grew evenly on the scaffolds, indicating scaffold has excellent biocompatibility.ConclusionThe advantages of three layers which have different performance of the tissue engineered osteochondral integration of multi-layered scaffold is achieved double biomimetics of structure and composition, lays a foundation for further research of animal in vivo experiment, meanwhile, as an advanced and potential strategy for osteochondral defect repair.
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.