[Three-dimensional flow perfusion culture enhances proliferation of human fetal osteoblasts in large scaffold with controlled architecture].

OBJECTIVE: To demonstrate the feasibility and benefits of custom designed perfusion bioreactor in conjunction with well-defined three-dimensional (3D) environment for enhanced proliferation and homogeneous distribution of human fetal osteoblasts in large scaffold in vitro. METHODS: Large-scale ß-tricalcium phosphate (ß-TCP) scaffolds with tightly controlled architectures were fabricated. And a custom designed perfusion bioreactor was developed. Human fetal osteoblasts were seeded onto the scaffolds, cultured for up to 16 days in static or flow perfusion conditions. At Days 4, 8 & 16 post-incubation, the proliferation and distribution of osteoblasts were determined by daily D-glucose consumption, cell viability (methyl thiazolyl tetrazolium (MTT) assay), histological evaluation and scanning electron microcopy (SEM). Sphere like structures observed in the SEM images were assessed by energy dispersive X-ray (EDX) analysis. RESULTS: In both static and perfusion cultures, the daily D-glucose consumption increased with prolonged time. The daily D-glucose consumption was significantly higher in the perfusion culture than that in static culture (P < 0.05). The increased cell viability with time during the culture was similar to the daily D-glucose consumption under both conditions. There was much greater cell viability under flow perfusion culture compared to static culture (P < 0.05). Flow perfused constructs demonstrated improved cell proliferation and a homogeneous layer composed of cells and extracellular matrix in channels throughout the whole scaffold. However, the cells were biased to periphery in scaffolds culture statically. Sphere like structures present in the matrix were identified as calcium phosphate nodules via EDX analysis. CONCLUSIONS: Flow perfusion culture plus well-defined 3D interconnected channel environments enhances the proliferation and improve the distribution of human fetal osteoblasts in large scaffolds. Scaffolds with controlled architecture may be a potential tool of studying the fluid flow configuration and cell behavior inside scaffold in details. And human fetal osteoblasts can be used as a cell source in large bone graft research.

Multi-variety bone bank in China.

This is a descriptive report of the establishment and operation of a Chinese bone bank, though not a typical one. While being engaged in collection, processing and storage of allogeneic tissues, the bone bank to which the author belongs concurrently develops and produces new, non-human derived, graft materials. Among others is reconstituted bone xenograft (RBX) which possesses strong osteoinductive capability without evoking immune rejection. Hence, its appellation "multi-variety bone bank," which was established by Dr. Hu Yunyu in 1990, the first of its kind in China. There are several salient features discriminating this bone bank from others. At this hospital-based non-profit institution, allograft hemi-joints are freshly prepared and distributed deep-frozen, instead of being freeze-dried on an industrialized basis for convenient transportation. The former has much more superior biological and mechanical properties as compared with the latter. However, allogeneic tissues are sometimes in short supply due to limited number of donors and the risk of some potential donors carrying viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV). New graft materials, including reconstituted bone xenograft (RBX), were developed that serve as a supplement to allografts. RBX has been successfully used in clinical practice for the management of old fractures, nonunions and bone defects, most notably of contaminated, infected open fractures and osteomyelitis with the use of anti-infective reconstituted bone xenograft (ARBX). Additionally the multi-variety bone bank serves as a training base for educating professional personnel and researchers (postgraduates) in theories and technologies of tissue banking. Up to now, eighteen special technical staff members and approximately sixty senior researchers have been trained at this institution.

Collagen I gel can facilitate homogenous bone formation of adipose-derived stem cells in PLGA-beta-TCP scaffold.

Cell-based tissue engineering is thought to be a new therapy for treatment of bone defects and nonunions after trauma and tumor resection. In this study, we explore the in vitro and in vivo osteogenesis of a novel biomimetic construct fabricated by using collagen I gel to suspend rabbit adipose-derived stem cells (rASCs) into a porous poly(lactic-co-glycolic)acid-beta-tricalcium phosphate (PLGA-beta-TCP) scaffold (rASCs-COL/PLGA-beta-TCP). In vitro and in vivo studies of the rASCs-COL/PLGA-beta-TCP composite (group A) were carried out compared with the single combination of rASCs and PLGA-beta-TCP (rASCs/PLGA-beta-TCP; group B), the combination of acellular collagen I gel and PLGA-beta-TCP (COL/PLGA-beta-TCP; group C), and the PLGA-beta-TCP scaffold (group D). Composites of different groups were cultured in vitro for 2 weeks in osteogenic medium and then implanted into the autologous muscular intervals for 8 weeks. After 2 weeks of in vitro culture, alkaline phosphatase activity and extracellular matrix mineralization in group A were significantly higher than in group B (p < 0.01, n = 4). In vivo osteogenesis was evaluated by radiographic and histological analyses. The calcification level was radiographically evident in group A, whereas no apparent calcification was observed in groups B, C and D (n = 4). In group A, woven bone with a trabecular structure was formed, while in group B, only osteoid tissue was observed. Meanwhile, the bone-forming area in group A was significantly higher than in group B (p < 0.01, n = 4). No bone formation was observed in groups C or D (n = 4). In conclusion, by using collagen I gel to suspend rASCs into porous PLGA-beta-TCP scaffold, osteogenic differentiation of rASCs can be improved and homogeneous bone tissue can be successfully formed in vivo.

Effect of ovariectomy on BMD, micro-architecture and biomechanics of cortical and cancellous bones in a sheep model.

Osteoporotic/osteopenia fractures occur most frequently in trabeculae-rich skeletal sites. The purpose of this study was to use a high-resolution micro-computed tomography (micro-CT) and dual energy X-ray absorptionmeter (DEXA) to investigate the changes in micro-architecture and bone mineral density (BMD) in a sheep model resulted from ovariectomy (OVX). Biomechanical tests were performed to evaluate the strength of the trabecular bone. Twenty adult sheeps were randomly divided into three groups: sham group (n=8), group 1 (n=4) and group 2 (n=8). In groups 1 and 2, all sheep were ovariectomized (OVX); in the sham group, the ovaries were located and the oviducts were ligated. In all animals, BMD for lumbar spine was obtained during the surgical procedure. BMD at the spine, femoral neck and femoral condyle was determined 6 months (group 1) and 12 months (group 2) post-OVX. Lumbar spines and femora were obtained and underwent BMD scan, micro-CT analysis. Compressive mechanical properties were determined from biopsies of vertebral bodies and femoral condyles. BMD, micro-architectural parameters and mechanical properties of cancellous bone did not decrease significantly at 6 months post-OVX. Twelve months after OVX, BMD, micro-architectural parameters and mechanical properties decreased significantly. The results of linear regression analyses showed that trabecular thickness (Tb.Th) (r=0.945, R2=0.886) and bone volume fraction (BV/TV) (r=0.783, R2=0.586) had strong (R2>0.5) correlation to compression stress. In OVX sheep, changes in the structural parameters of trabecular bone are comparable to the human situation during osteoporosis was induced. The sheep model presented seems to meet the criteria for an osteopenia model for fracture treatment with respect to morphometric and mechanical properties. But the duration of OVX must be longer than 12 months to ensure the animal model can be established successfully.

A novel TAT fusion protein with osteoinductive activity.

Osteoblasts are thought to be differentiated from pluripotent mesenchymal stem cells. Several intracellular and extracellular osteoinductive proteins are involved in this process. Such proteins include the bone morphogenetic proteins (BMPs) and the LIM mineralization proteins (LMPs) etc. LMP-1 is a novel LIM domain protein promoting the differentiation of osteoblasts during bone formation. It contains three LIM domains/motifs, one PDZ domain and a unique sequence. Through analysis of the amino acid sequence and the function of the LMPs, it has been found that the PDZ domain (1-93 aa) and a unique region (94-133 aa) appear to be critical for bone formation. The TAT protein of human immunodeficiency virus can be fused with other macromolecules, peptides or proteins and transport them into cells successfully. Once being transduced into cells, the fusion protein can recover its biological activity through being rapidly refolded. We supposed that TAT could be fused with LMP-1 (1-133 aa) and LMP-1 (94-133 aa) and the fusion proteins could be easily transduced through biological membranes and generate biological activity. The clinical application of BMPs has been limited for their relatively high cost and the unstable osteoinductivity. If the hypothesis proved to be practical, we would have a more effective new way to promote bone repair and regeneration.

The matrix metalloproteinases as pharmacological target in osteoarthritis: statins may be of therapeutic benefit.

Osteoarthritis (OA) is a common joint disease; however, current pharmacologic agents for OA are only symptomatic and they can not prevent the disease progression. Matrix metalloproteinases (MMPs) produced by chondrocytes play an important role in the development of cartilage destruction in OA, and agents that can target against MMPs activity may be of therapeutical value. There were reports that statins can inhibit the secretion of MMPs in vitro and in vivo, which were believed to account for the plaque stabilizing effects of statins in the treatment of atherosclerosis. We based our hypothesis on that atherosclerosis possesses some aspects that are similar to that of osteoarthritis, such as inflammation and matrix degradation. Since statins have displayed great benefits in modifying the progression of atherosclerosis via anti-inflammatory and matrix-stabilizing mechanisms, it is conceivable that statins may also prevent the disease progression of osteoarthritis. Further work are needed to verify if statins can protect cartilage from destruction through inhibition of MMP secretion by chondrocytes, and their potential to be used as therapeutic agents in OA should be investigated.

Orthopedic trauma of limbs associated with vascular injuries.

OBJECTIVE: To evaluate the treatment for patients with major vascular injuries associated with traumatic orthopedic injuries. METHODS: A total of 196 patients, aged from 4-67 years with the mean age of 29.88 years, had major vascular injuries associated with traumatic orthopedic injuries and were treated in our hospital in a period of 44 years. The most common mechanism of trauma was blunt trauma (67.3%), open injuries accounted for 32.4% and 54.5% of the injuries were located in the lower extremities. The vascular injury frequently happened in the femoral artery (26.7%) and popliteal artery (20.3%). The treatment principle consisted of aggressive resuscitation, Doppler imaging and stable bone internal fixation with subsequent vascular repair and debridement. The vascular repair for injuries included end-to-end anastomosis (80 cases, 39.6%), interpositional vein graft (94, 46.5%), vascular decompression through fracture distraction (18, 8.9%), arterial ligation (6, 3.0%), vein patch (2, 1.0%), bypass graft (2, 1.0%), venous repair including autogenous vein graft (9, 24.3%) and ligation (28, 75.7%) and prophylactical fasciotomy (15, 7.4%). Postoperative amputation was performed in 16 cases (16.3%). RESULTS: No intraoperative death was observed and all fractures united within 6 months. Limbs were salvaged in 180 patients (91.8%). Among these patients, early complications were found in 19 patients (9.7%) and late complications were observed in 8 patients (4.1%). CONCLUSIONS: A well-organized approach, based on a specific treatment principle, not only improves clinical outcome but also does good to excellent functional recovery for patients with severe orthopedic injuries and concomitant vascular lesion.

[Three dimensional induction of autologous mesenchymal stem cell and the effects on depressing long-term degeneration of tissue-engineering cartilage].

OBJECTIVES: To induce autologous bone marrow derived mesenchymal stem cell (aMSC) into chondrocyte, and to confirm the effects of 3 dimensional (3D) dynamic inducing in vitro and their long-term animal model repairing in vivo. METHODS: aMSC were separated from rabbits bone marrow aspirates, then respectively experienced 3D dynamic inducing in alginate drops in modified rotating wall bioreactor culture or in two dimensional (2D) inducing (culture flask) for 10 d. The induced cells were harvest and then mixed with fibrin sealant (FS) to repair rabbit knee femoral trochlea cartilage defects model. After 8, 12, 24, 48 weeks animals were euthanized. Gross appearance, histological appearances were examined. RESULTS: Flask culture groups showed a little chondrocyte differentiation, 3D inducing group showed obviously chondrocyte differentiation, improved collagen II and proteoglycan production. For 3D inducing ones in vivo, the cartilage defects were smoothly repaired by white translucent hard tissue with obvious hyaline-like cartilage histological appearance after 8, 12 weeks, and the defects boundary were hard to be identified with hyaline like cartilage with sustained histological appearance and score after 24, 48 weeks. For 2D ones in vivo, the cartilage defects were smoothly repaired after 8 weeks by hyaline like cartilage which showed accelerated degeneration after 24 weeks and lose cartilage performance completely after 48 weeks. CONCLUSIONS: 3D dynamic inducing may assist aMSC on differentiating into chondrocyte, improve its long-term in vivo repairing effects, and enlighten its further applications in tissue engineering cartilage.

Repair of the radial defect of rabbit with polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology.

OBJECTIVE: To evaluate the effects of repairing rabbit radial defects with polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with bovine bone morphogenetic protein (bBMP), and find new carriers for growth factors. METHODS: Polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with and without bovine BMP were used to repair the 15 mm radial defect in rabbit. Then the results of radiography, histology, scaffolds degrade rates and bone mineral density (BMD) were appraised to examine the effects at the 12th week. RESULTS: At the 12th week postoperatively, all defects treated with bBMP were radiographically repaired. No radius implanted polyester/tricalcium phosphate scaffolds without bBMP showed radiographic and histological union. At experimental groups, longitudinal alignment of lamellar structure was observed histologically at the 12th week, indicating that remodeling of regenerated bone was complete in different degree. Of the three experimental groups, the bony regeneration and remodeling of callus in poly lactide-co-glycolide/tricalcium phosphate (PLGA/TCP) group was the best. The BMD values were beyond 70% of normal value at the 12th week while the PLGA/TCP scaffolds group was the highest, and no abnormalities were observed in the surrounding soft tissue in all groups. CONCLUSIONS: Polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with bovine BMP can repair a 15 mm radial defect of rabbit. As for the results, the PLGA/TCP scaffold is ideal and better than poly L-lactide-co-D, L-lactide (PDLLA/TCP) scaffold, but the ploy L-lactic acid (PLLA/TCP) is not so good for its low degradation rates.

Compositional variation of fibrous callus and joint cartilage in different internal environments.

OBJECTIVE: To evaluate the compositional variation of fibrous callus in the fracture site and the joint cavity and joint cartilage after being transplanted in the muscle pouch. METHODS: Thirty 2 month old New Zealand white rabbits (weighing 1-1.5 kg) were randomly divided into two groups: a callus transplantation group (Group A, n=15) and a cartilage transplantation group (Group B, n=15). In Group A, closed radius fracture was made and the autologous fibrous callus was transplanted in the right knee joint cavity at 12 days postoperatively. In Group B, the right knee joint cartilage of the animals was transplanted in the autologous back muscle pouches under anesthesia. Then all the animals were killed by overdose anesthetic 3 weeks after transplantation. And the transplanted fibrous callus, the healed bones in the fracture sites and the transplanted joint cartilage were obtained for assessment of compositional variation. RESULTS: Pure fibrous composition was found in the callus at the fracture sites in Group A at 12 days postoperatively. And for 11 out of the 15 animals, the fibrous callus was transformed into cartilaginous tissues after 3 weeks of transplantation, but the fibrous callus was absent in the other 4 animals. The fibrous calluses at the original site and the fracture locus were differentiated into bony tissues. Bony tissue transformation was found in the transplanted joint cartilages in the muscle pouch of all the animals in Group B. CONCLUSIONS: The fracture sites or joint cavity may facilitate callus differentiation in different ways: the former is helpful for osteogenesis while the latter for the development and maintenance of cartilages, and the muscle pouch is inclined to induce the osteogenic phenotype for cartilages.

[Repair of the radial defect of rabbit by polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology].

OBJECTIVE: To evaluate the repairing effect of the rabbits radial defects of by polyester/tricalcium phosphate scaffolds prepared by rapid forming technology loaded with bovine BMP, and find a new carrier for growth factor. METHODS: Polyester/Tricalcium phosphate scaffolds prepared by rapid prototyping (RP) technology loaded with and without bovine BMP were used to repair the 15 mm radial defect of rabbit. Then results of radiography, histology, scaffolds degrade rates and bone density were appraised to examine the repairing effects of the scaffolds at 12 weeks. RESULTS: At 12 weeks, all defects treated with bBMP were radiographically repaired. No radii implanted polyester/tricalcium phosphate scaffolds alone showed radiographic and historical union. At experimental groups, longitudinal alignment of lamellar structure was observed histologically at 12 weeks, indicating that remodeling of regenerated bone almost completed, the scaffolds degradation rates were different by 12 weeks, and no abnormalities were observed in the surrounding soft tissue in all groups. CONCLUSION: Polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with bovine BMP can repair the rabbits radical defects. As for the effects, the poly (L-lactic-co-glycolide)/tricalcium phosphate (PLGA/TCP) scaffold are ideal and better than poly (L-lacide-co-D, L-lactide)/tricalcium phosphate (PDLLA/TCP) scaffold, but the poly (L-lactic acid)/tricalcium phosphate (PLLA/TCP) is not so good for its low degradation rates.

[Morphology research of the rat sciatic nerve bridged by collage-heparin sulfate scaffold].

OBJECTIVE: To observe the treating effect of collage-heparin sulfate after the 10 mm rat sciatic nerve defect was bridged by it. METHODS: A new kind of nervous tissue engineering scaffold was produced by freeze-drying technique from collagen-heparin sulfate. Thirty-two SD rats were randomly divided into A, B, C and D groups. Sciatic nerve defect in group A was bridged by collagen-heparin sulfate. In group B, sciatic nerve was bridged by auto-nerve transplantation. Group C was the blank control group. Animals in group D were normal. And 10 mm sciatic nerve defect was bridged in the experiment. Thirty-six weeks after the operation, the experimental animals were detected by HRP labeled retrograde trace, HE staining, toluidine staining, silvering staining, S100, GAP-43 and NF immunohistological staining, MBP immunofluorescence staining and transmission electron microscope to observe the nerve regeneration inducing effect of this new scaffold. RESULTS: Nine months after operation, the collage-heparin sulfate scaffold was replaced by newly regenerated nerve. The number of HRP labeled spinal cord anterior horn cells and the area of sensation nerve fiber at the posterior horn were similar with that was repaired by auto-nerve. GAP-43, NF and S100 labeled regenerated nerve fiber had passed the total scaffold and entered the distal terminal. The regenerated nerve fibers were paralleled, lineage arranged, coincide with the prearranged regenerating "channel" in the collagen-heparin sulfate scaffold. MBP immunofluorescence staining also proved that the newly regenerated nerve fiber could be ensheathed. In the experimental group, the area of myelinated nerve fiber and the thickness of the myelin sheath had no obvious difference with that of the group repaired by auto-nerve, except that the density of the regenerated myelinated sheath fiber was lower than that of the control group. CONCLUSION: Nervous tissue engineering scaffold produced by collagen-heparin sulfate can guide the regeneration of nerve fibers. The nerve function recovers fine. This kind of material has great application potential.

Effect of type I collagen on the adhesion, proliferation, and osteoblastic gene expression of bone marrow-derived mesenchymal stem cells.

OBJECTIVE: To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by type I collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived mesenchymal stem cells (MSCs). METHODS: The third generation MSCs isolated from mature rabbits by density gradient centrifugation were cultured at different initial concentrations on 0.3 cm x 1.2 cm x 2.0 cm 3-D porous PLGA coated by type I collagen in RPMI 1640 containing 10% fetal calf serum, while cultured on PLGA without type I collagen as control. The cells adhesive and proliferative behavior at 7, 14, and 21 days after inoculation was assessed by determining the incorporation rate of [(3)H]-TdR. In order to examine MSCs differentiation, the expression of osteoblasts marker genes, osteocalcin (OCN), alkaline phosphatase (ALP), osteopontin (OPN) mRNA, were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and further more, the cell morphology at 21 days was also observed by scanning electron microscope (SEM). RESULTS: Type I collagen promoted cell adhesion on PLGA. The valve was significantly higher than controls (6 h, 2144 cpm+/-141 cpm vs. 1797 cpm+/-118 cpm, P=0.017; 8 h, 2311 cpm+/-113 cpm vs. 1891 cpm+/-103 cpm, P=0.01). The cells which cultured on PLGA coated with type I collagen showed significantly higher cell proliferation than controls on the 7 th day (1021 cpm+/-159 cpm vs. 451 cpm+/-67 cpm, P=0.002), the 14th day (1472 cpm+/-82 cpm vs. 583 cpm+/-67 cpm, P<0.001) and 21 th day (1728 cpm+/-78 cpm vs. 632 cpm+/-55 cpm, P<0.001). Osteoblasts markers, OCN, ALP, OPN mRNA, were all detected on PLGA coated by type I collagen on the 21 th day, but OCN, OPN mRNA could not be found in controls. Spindle and polygonal cells well distributed on the polymer coated by type I collagen while cylindric or round cells in controls. CONCLUSIONS: Type I collagen is effective in promoting the adhesion, proliferation and differentiation of MSCs on PLGA.

Repair of radius defect with bone-morphogenetic-protein loaded hydroxyapatite/collagen-poly(L-lactic acid) composite.

OBJECTIVE: To explore the method to repair bone defect with bone-morphogenetic-protein loaded hydroxyapatite/collagen-poly(L-lactic acid) composite. METHODS: 18 adult beagle dogs were randomly divided into 3 groups. In Group A, bone-morphogenetic-protein (BMP) loaded hydroxyapatite/collagen-poly(L-lactic acid) (HAC-PLA) scaffold was implanted in a 2 cm diaphyseal defect in the radius. In Group B, unloaded pure HAC-PLA scaffold was implanted in the defects. No material was implanted in Group C (control group). The dogs were sacrificed 6 months postoperatively. Features of biocompatibility, biodegradability and osteoinduction were evaluated with histological, radiological examinations and bone mineral density (BMD) measurements. RESULTS: In Group A, the radius defect healed after the treatment with BMP loaded HAC-PLA. BMD at the site of the defect was higher than that of the contralateral radius. Fibrous union developed in the animals of the control group. CONCLUSIONS: BMP not only promotes osteogenesis but also accelerates degradation of the biomaterials. Optimized design parameters of a three-dimensional porous biomaterial would give full scope to the role of BMP as an osteoinductive growth factor.

Anti-infective reconstituted bone xenograft used for primary bone grafting to repair contaminated defect in the radius in dogs.

OBJECTIVE: To investigate the effect of anti-infective reconstituted bone xenograft as a primary graft to repair a segmental with severe contamination. METHODS: A canine model of contaminated defect of 1.5 cm in size in the radius was used, in which anti-infective reconstituted bone xenograft or reconstituted bone xenograft was implanted as a primary graft followed by internal fixation. The effectiveness of the two grafting materials in repairing a contaminated segmental defect was compared. RESULTS: The animals which had received implant of anti-infective reconstituted bone xenograft should largely healed defects 6 months after operation while the defects implanted with reconstituted bone xenograft remained unrepaired with bone infection. CONCLUSIONS: Besides its strong osteoinductive and osteoconductive activity, anti-infective reconstituted bone xenograft is highly antibacterial and can be used as a primary graft to repair the severely contaminated segmental defect.

Treatment of tibial defect and bone nonunion with limb shortening with external fixator and reconstituted bone xenograft.

OBJECTIVE: To explore the effect of external fixator and reconstituted bone xenograft (RBX) in the treatment of tibial bone defect, tibial bone nonunion and congenital pseudarthrosis of the tibia with limb shortening. METHODS: Twenty patients (13 males and 7 females) with tibial bone defect, tibial bone nonunion or congenital pseudarthrosis of the tibia with limb shortening were treated with external fixation. Two kinds of external fixators were used: a half ring sulcated external fixator used in 13 patients and a combined external fixator in 7 patients. Foot-drop was corrected at the same time with external fixation in 4 patients. The shortened length of the tibia was in the range of 2-9 cm, with an average of 4.8 cm. For bone grafting, RBX was used in 12 patients, autogenous ilium was used in 3 patients and autogenous fibula was implanted as a bone plug into the medullary canal in 1 case, and no bone graft was used in 4 patients. RESULTS: All the 20 patients were followed-up for 8 months to 7 years, averaging 51 months. Satisfactory function of the affected extremities was obtained. All the shortened extremities were lengthened to the expected length. For all the lengthening area and the fracture sites, bone union was obtained at the last. The average healing time of 12 patients treated with RBX was 4.8 months. CONCLUSIONS: Both the half ring sulcated external fixator and the combined external fixator have the advantages of small trauma, simple operation, elastic fixation without stress shielding and non-limitation from local soft tissue conditions, and there is satisfactory functional recovery of affected extremities in the treatment of tibial bone defects, tibial bone nonunion and congenital pseudarthrosis of the tibia combined with limb shortening. RBX has good biocompatibility and does not cause immunological rejections. It can also be safely used in treatment of bone nonunion and has reliable effect to promote bone healing.

Reciprocal action between BMP-2 and BMP-3 in cultured fibroblast in vitro.

OBJECTIVE: To explore reciprocal action between BMP-2 (bone morphogenetic protein-2) and BMP-3 for better understanding of the mechanism of BMP during bone fracture union. METHODS: rhBMP-2 was added into the cultured fibroblasts with the concentration of 1,200 ng/ml. The expression of BMP-3 in fibroblasts was detected by immunohistochemistry. Eukaryotic expression vector pcDNA3-BMP-3 was transfected into the fibroblasts. After the effective expression of BMP-3 was identified, BMP-2 was also detected by immunohistochemistry in BMP-3 expression cells. The fibroblasts transfected with empty vector pcDNA3 were used as the control. RESULTS: Exogenous rhBMP-2 could promote the expression of BMP-3 in fibroblasts. BMP-3 also could be detected in these cells. CONCLUSIONS: BMP-2 and BMP-3 could reciprocally adjust the expression in fibroblasts.

[Experimental study of anti-infective reconstructed bone xenograft].

OBJECTIVE: To develop a new type bone graft material that can be used as primary graft in contaminated even infected bone defect. METHODS: Anti-infective reconstructed bone xenograft (ARBX) was developed by combining reconstructed bone xenograft (RBX) with gentamycin and gelatin. One piece of ARBX was implanted in the muscle pouch in right thighs of 32 mice. The implanted ARBX and surrounding soft tissues were taken out from the mice at different time point to make into homogenate and the concentration of released gentamycin in the supernatant and the diameter of the bacterial inhibition ring of each specimen were tested. One piece of ARBX was implanted into the muscle pouch at the right thigh of 16 mice and one piece of RBX was implanted into the muscle pouch at the right thigh of another 16 mice. Fourteen days after the grafts and surrounding tissues were taken out to be examined histologically or made into homogenate to test the alkaline phophatase (ALP) activity. Bone defect was made in the bilateral radii and then Staphylococcus aureus was injected and debridement was conducted. 10 pieces of ARBX were implanted into the bone defect at the left radius and 10 pieces of RBX were implanted into the bone defect at the right radius. The defect was then fixed and the wound was sutured. Gentamycin was injected for 1 week. Six months later X ray examination was conducted to the radius, then the radius was taken and half of specimens were examined histologically and half of them was made into homogenate to examine the amount of Staphylococcus aureus. Defect was made in the right tibia of 25 rabbits and Staphylococcus aureus injected therein. Then the rabbits were divided into 5 groups of 5 individuals: group 1 (3 pieces of ARBX were implanted), group 2 (3 pieces of RBX were implanted and gentamycin was used locally), group 4 (3 pieces of RBX were implanted and gentamycin was injected intramuscularly), and group 5 (control group, without born grafting). Eight weeks after, radiological, histological, and bacteriological methods were used to observe the recovery of bone defect and amount of Staphylococcus aureus. RESULTS: Gentamycin was released slowly from the ARBX and the effective bacterium-inhibiting concentration lasted 30 days. There was no significant difference in osteoinductive activity and related ALP activity between the mice implanted with ARBX and RBX. The bone defect of the dogs implanted with ARBX recovered better than that of the dogs implanted with RBX; osteomyelitis was found in the specimens from the bone defect implanted with RBX and not in the specimens implanted with ARBX, and the positive rate of Staphylococcus aureus was lower in the specimens implanted with ARBX than in the specimens implanted with RBX. 8 weeks after the implantation the Norden score of osteomyelitis was the lowest in the rabbits of group 1 (P < 0.01). The bone defect recovered better in the rabbits of groups 1 and 2. The number of Staphylococcus aureus in the bone defect in rabbits of group 1 was significantly smaller than that in the rabbits of group 2 (P < 0.05), and was very significantly smaller then in the rabbits of the other 3 groups (P < 0.01). CONCLUSION: ARBX has strong oeteoinductive and anti-infective abilities. It can be used in primary bone grafting to treat contaminated even infected bone defect.

[Effects of collagen I coating on the porous poly-lactide-co-glycolid on adhesion, proliferation, and differentiation of mesenchymal stem cells].

OBJECTIVE: To investigate the effects of collagen I on the adhesion, proliferation, and differentiation of MSCs on PLGA. METHODS: Collagen I was added onto the surface of pores in pieces of 3-D porous poly-lactide-co-glycolid (PLGA). Bone marrow-derived mesenchymal stem cells (MSCs) were obtained from New Zealand rabbits and were cultured for 3 generations, inoculated into the pores of PLGA pieces with the volume of 0.3 cm x 1.2 cm x 2.0 cm, and then cultured in solution with [(3)H]-thymidine deoxyribose (TdR). PLGA pieces not coated by collagen I were used as controls. The incorporation rate of [(3)H]-TdR was detected 2, 4, 6, and 8 hours, and 7, 14, and 21 days after culture, shown in count per minute (CPM) value, to determine the adhesion and proliferation of the MSCs. RT-POCR was used to examine the expressions of mRNA of the osteoblast markers: osteocalcin (OCN), alkaline phosphatase (ALP), and osteopontin (OPN). Scanning electron microscopy (SEM) was used to observe the morphology of MSCs. RESULTS: The CPM value since 6 hours after culture between the experimental group and control group began to be significantly different (both P < 0.05) The CPM values 7, 14, and 21 days after culture between the experimental group and control groups (P < 0.05 or P < 0.01). OCN, ALP, and OPN mRNA were expressed in MSCs of the experimental group and only ALP mRNA was weakly expressed in the control group. SEM showed the distribution of spindle and polygonal cells in the pores of the 3-D PLGA pieces and distribution of cylindrical or round cells in the control group. CONCLUSION: Collagen I is effective in promoting the adhesion, proliferation, and differentiation of MSCs on PLGA.

[Expression of human osteoprotegerin gene in E. Coli and bioactivity analysis of expression product].

OBJECTIVE: To express human osteoprotegerin (OPG) in E. Coli and analyze its bioactivity in vitro. METHODS: Synthetic oligonucleotides were used to amplify human OPG gene by RT-PCR from total RNA of human osteosarcoma cell line MG63. The OPG cDNA coding for 380 amino acid residues was inserted into prokaryotic expression vector pRSET-A, transformed into competent E. Coli BL21, and induced by 0.1 mmol/l IPTG. SDS-PAGE and Western blot were performed to identify OPG-6His fusion protein. After purified by affinity chromatography, 1,000 microg/L or 1,500 microg/L of OPG-6His were added into the mouse bone marrow cells culture medium. The number of tartrate-resistant acid phophatase (TRAP)-positive multinucleated cells and resorption pits were counted to assess the bioactivity of expression products. RESULTS: The sequence of OPG mature peptide encoding cDNA obtained in this experiment was as same as reported. SDS-PAGE showed 24% of total bacterial protein was of OPG-6His fusion protein. Western blot assay demonstrated that the molecular weight of recombinant protein was about 46 KD and could react specifically with human anti-OPG antibody. The mouse bone marrow cells were induced by 1alpha, 25-dihydroxyvitaminD3 (10(-8) mol/L) and Dexamethasone (10(-7) mol/L) to form osteoclastic-like multinucleated cells. 1,500 microg/L of purified OPG-6His protein could decrease the number of resorption pits and TRAP-positive multinucleated cells in vitro (P < 0.05), but it didn't show the same effects when the concentration of OPG-6His fusion protein was of 1,000 microg/L. CONCLUSIONS: Human OPG-6His fusion protein is expressed and purified in E. Coli. The expression products have moderate inhibitory effects on osteoclast differentiation and bone resorption in vitro only when excessive amount of proteins are added into the culture medium, indicating that prokaryotic expression of fuctionalal OPG protein awaits further investigation.