Repair of segmental bone defect using Totally Vitalized tissue engineered bone graft by a combined perfusion seeding and culture system.

BACKGROUND: The basic strategy to construct tissue engineered bone graft (TEBG) is to combine osteoblastic cells with three dimensional (3D) scaffold. Based on this strategy, we proposed the "Totally Vitalized TEBG" (TV-TEBG) which was characterized by abundant and homogenously distributed cells with enhanced cell proliferation and differentiation and further investigated its biological performance in repairing segmental bone defect. METHODS: In this study, we constructed the TV-TEBG with the combination of customized flow perfusion seeding/culture system and ß-tricalcium phosphate (ß-TCP) scaffold fabricated by Rapid Prototyping (RP) technique. We systemically compared three kinds of TEBG constructed by perfusion seeding and perfusion culture (PSPC) method, static seeding and perfusion culture (SSPC) method, and static seeding and static culture (SSSC) method for their in vitro performance and bone defect healing efficacy with a rabbit model. RESULTS: Our study has demonstrated that TEBG constructed by PSPC method exhibited better biological properties with higher daily D-glucose consumption, increased cell proliferation and differentiation, and better cell distribution, indicating the successful construction of TV-TEBG. After implanted into rabbit radius defects for 12 weeks, PSPC group exerted higher X-ray score close to autograft, much greater mechanical property evidenced by the biomechanical testing and significantly higher new bone formation as shown by histological analysis compared with the other two groups, and eventually obtained favorable healing efficacy of the segmental bone defect that was the closest to autograft transplantation. CONCLUSION: This study demonstrated the feasibility of TV-TEBG construction with combination of perfusion seeding, perfusion culture and RP technique which exerted excellent biological properties. The application of TV-TEBG may become a preferred candidate for segmental bone defect repair in orthopedic and maxillofacial fields.

[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.

[The construction of recombinant adenovirus containing p38MAPK gene and its expression in mouse osteoblast-like cells (MC3T3-E1)].

AIM: To construct a recombinant adenovirus vector containing p38MAPK gene and to identify its expression in mouse osteoblast-like cells, MC3T3-E1, in vitro. METHODS: The p38MAPK gene was amplified by PCR from mouse liver cDNA library, and inserted into pMD18-T vector and sequenced. Double digested with Bgl II and Hind III, p38MAPK gene was inserted into pShuttle-CMV. This recombinant plasmid was linearized by Pme I and electronically transfected into BJ5183 to get the recombinant adenovirus vector Ad-p38MAPK. Then the Ad-p38MAPK was obtained by packaging Pac I linearized in AD293 cells. The recombinant adenovirus expressing p38MAPK was infected into osteoblast- like cells(MC3T3-E1).The expression of exogenous p38MAPK was observed by Western blot. RESULTS: The recombinant plasmid Ad-p38MAPK was successfully generated, which increased p38MAPK protein expression levels in MC3T3-E1. CONCLUSION: The bioactive recombinant adenovirus Ad-p38MAPK has been successfully constructed. The study laid a foundation for further research of the function of p38MAPK in osteoblast.

Low-intensity pulsed ultrasound enhances posterior spinal fusion implanted with mesenchymal stem cells-calcium phosphate composite without bone grafting.

STUDY DESIGN: Experimental study on the effect of low-intensity pulsed ultrasound (LIPUS) on rabbit spinal fusion with mesenchymal stem cell (MSC)-derived osteogenic cells and bioceramic composite. OBJECTIVE: To investigate the efficacy of LIPUS in enhancing fusion rate and bone formation with porous tricalcium phosphate (TCP) bioceramic scaffold impregnated with MSCs without any bone grafts. SUMMARY OF BACKGROUND DATA: The goal of spinal fusion in the corrective spinal surgery for spinal deformities is to achieve solid bony fusion between selected vertebral segments. Previous studies with bone morphogenetic proteins and genetically manipulated materials revealed significant difficulties in actual clinical application. Alternative such as LIPUS has been shown to be effective in enhancing healing of fracture and nonunion clinically. Its potential for enhancing spinal fusion warrants further in-depth study. METHODS: Posterolateral intertransverse processes spinal fusion at the L5 and L6 levels were evaluated in New Zealand white rabbit model. The animals were divided into three groups with (A) TCP alone, (B) TCP with differentiated MSCs, and (C) TCP with differentiated MSCs and LIPUS treatment. At week 7 postoperation, manual palpation, peripheral quantitative computed tomography, and histomorphometric assessments were performed. RESULTS: At week 7 postoperation, a statistically significant increase in clinical fusion by manual palpation was observed in group C animals treated with LIPUS (86%) in comparing with groups A (0%) and B (14%) without LIPUS. With peripheral quantitative computed tomographic analysis, the bone volume of group C fusion mass was significantly larger than the other two groups. Group C fusion also had better osteointegration length between host bone and implanted composite and more new bone formed in the TCP implants. Importantly, all the group C animals had osteochondral bridging--early stage of bony fusion histologically. Endochondral ossification was observed at the junction between the cartilaginous and osseous tissues at the intertransverse processes area. Quantitative analysis showed that the fusion mass in group C had significantly smaller gap and larger area of cartilaginous tissue between the transverse processes. CONCLUSION: The present study showed that the combination of synthetic biomaterials, autologous differentiated MSCs, and LIPUS could promote clinical fusion in rabbit posterior spinal fusion model. The mechanism was likely to be mediated through better osteointegration between the host bone and implanted materials and enhanced endochondral ossification at the fusion site.

[In vitro and in vivo study of chondrogenesis on the hybrid scaffold from fibrin modified PLGA and adipose-derived stem cells].

AIM: To investigated the effect of the presence of fibrin in the PLGA scaffold on the differentiation of adipose-derived stem cell (ASCs) into chondrocytes in the chondrogenic media. METHODS: ASCs were prepared by colagenase I digestion of fat from rabbits. The PLGA scaffolds were prepared by LDM technology. The hybrid scaffold was fabricated by a freeze-drying method. Isolated ASCs were cultured in the PLGA without and with fibrin up to 14 days in specific chondrogenic medium. The surface property of the scaffold was observed by SEM. Cell attachment was evaluated, and glycoaminoglycans (GAGs) content was tested by biochemical method. RESULT: When ASCs were seeded within fibrin modified PLGA scaffold in vitro, enhanced cellular attachment and differentiation were observed compared to unmodified PLGA scaffold. The study from articular cartilage defect repaired showed that the group from the autologous ASCs seeded on fibrin-PLGA scaffold had better chondrocyte morphology, tissue integration, continuous subchondral bone, and much thicker newly formed cartilage layer as compared with other groups. CONCLUSION: Such modification of PLGA may ultimately enhance the efficacy of tissue engineered scaffolds for cartilage tissue engineering using ASCs.

One-stage treatment and reconstruction of Gustilo Type III open tibial shaft fractures with a vascularized fibular osteoseptocutaneous flap graft.

OBJECTIVES: This study evaluated the usefulness of a single-stage, free-fibular vascularized osteoseptocutaneous flap transfer for Type III open tibial shaft fractures with segmental bone loss for the reconstruction of combined bone and soft tissue defects. DESIGN: Nonrandomized retrospective study. SETTING: University Level I trauma center. PATIENTS/PARTICIPANTS: All Gustilo Type III open tibial shaft fractures with segmental bone loss that were treated at one institution between 2000 and 2007 were identified from a trauma registry. The study group consisted of 28 patients with Type III open tibial fractures: 27 were Gustilo-Anderson Type IIIB and one was Grade IIIC. The cause of tibial injury included eight industrial accidents, seven motor vehicle accidents, five crushing injuries caused by heavy objects, five falls from a height, and three motorcycle crashes. The lengths of the preoperative segmental tibial bone loss ranged from 9 to 17 cm and the size of the associated soft tissue defects ranged from 8 × 6 cm to 15 × 7 cm. INTERVENTION: The free fibular vascularized osteoseptocutaneous flap was used to graft and reconstruct combined bone and soft tissue defects. The radical wound débridement, soft tissue and bone revision, fracture stabilization, and early soft tissue coverage were achieved by this technique in a one-stage procedure. The average duration from injury to one-stage reconstruction was 15.8 hours (range, 5.3 hours to 6.5 days). MAIN OUTCOME MEASUREMENT: Radiographic and functional evaluation of the lower extremity. RESULTS: All free fibular osteoseptocutaneous flaps survived completely. The average time to overall union for the entire group was 32 weeks after surgery (range, 26-41 weeks). None of the patients in this series had a nonunion. Acceptable radiographic alignment, defined as 5° of angulation in any plane, was obtained in 22 patients (78.6%). Malunion affected six (21.4%) fractures. According to the lower extremity functional assessment, excellent and good results were achieved for 82.1% (23 of 28), fair results were seen in 14.3 % (four of 28), and a poor result occurred in one case (3.5%). CONCLUSION: The free fibular vascularized osteoseptocutaneous flap grafting is an effective alternative in management of Type III open tibial fractures using a one-stage procedure. The grafted fibula offers good fracture stabilization plus a vascularized bone graft, and the fibular flap can also provide a large piece of mobile skin to cover the soft tissue defect in Type III open tibial fractures. The free osteoseptocutaneous flap also serves as a visible monitor of the adequacy of the circulation of the grafted fibula.

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.

Skeletal repair in rabbits using a novel biomimetic composite based on adipose-derived stem cells encapsulated in collagen I gel with PLGA-beta-TCP scaffold.

In bone tissue engineering, the cell distribution mode in the scaffold may affect in vivo osteogenesis. Therefore, we fabricated a novel biomimetic construct based on a combination of rabbit adipose-derived stem cells (rASCs) encapsulated in collagen I gel with a PLGA-beta-TCP scaffold (rASCs-COL/PLGA-beta-TCP, group A), the combination of rASCs and PLGA-beta-TCP (rASCs/PLGA-beta-TCP, group B), the combination of collagen I gel and PLGA-beta-TCP (COL/PLGA-beta-TCP, group C), and PLGA-beta-TCP scaffold (group D). The composites were implanted into a 15-mm length critical-sized segmental radial defect. The results were assessed by histology, radiographs, bone mineral density (BMD), and mechanical testing. After 24 weeks, the medullary cavity recanalized, bone was rebuilt, and molding finished, the bone contour remodeled smoothly and the scaffold degraded completely in group A. The BMDs and mechanical properties were similar to normal. However, the bone defect remained unrepaired in groups B, C, and D. Moreover, the scaffold degradation rate in group A was significantly higher than the other groups. Thus, enhanced in vivo osteogenesis of rASCs wrapped in collagen I gel combined with PLGA-beta-TCP was achieved, and the bone defect was repaired. We hope this study provides new insights into ASCs-based bone tissue engineering.

Ghrelin stimulates proliferation of human osteoblastic TE85 cells via NO/cGMP signaling pathway.

Ghrelin regulates bone formation and osteoblast proliferation, but the detailed signaling pathway for its action on osteoblasts remains unclear. In human osteoblastic TE85 cells, we observed the effects and intracellular signaling pathway of ghrelin on cell proliferation using BrdU incorporation method. Ghrelin, at 10(-10)-10(-8) M concentration, significantly increased BrdU incorporation into TE85 cells. The action of ghrelin was inhibited by D: -Lys3-GHRP-6, a selective antagonist of GHS-R. Nitric oxide (NO) scavenger hemoglobin and the NO synthase inhibitor NAME eliminated the stimulatory action of ghrelin on proliferation, while NO donor SNAP and NO synthase substrate L-AME stimulated proliferation of osteoblastic TE85 cells. The cGMP analogue, 8-Br-cGMP, stimulated TE85 cell proliferation, and ghrelin did not enhance proliferation in the presence of 8-Br-cGMP. Inhibition of cGMP production by the guanylate cyclase inhibitor prevented ghrelin-induced osteoblastic TE85 cell proliferation. In conclusion, ghrelin stimulates proliferation of human osteoblastic TE85 cells via intracellular NO/cGMP signaling pathway.

Flow perfusion culture of human fetal bone cells in large beta-tricalcium phosphate scaffold with controlled architecture.

One unsolved problem in bone tissue engineering is how to enable the survival and proliferation of osteoblastic cells in large scaffolds. In this work, large beta-tricalcium phosphate scaffolds with tightly controlled channel architectures were fabricated and a custom-designed perfusion bioreactor was developed. Human fetal bone cells in third passage were seeded onto the scaffolds and cultured in static or flow perfusion conditions for up to 16 days. Compared with nonperfused constructs, flow perfused constructs demonstrated improved cells proliferation and differentiation according to cell viability, glucose consumption, alkaline phosphatase activity, and osteopontin. Moreover, after 16 days of perfusion culture, a homogenous layer composed of cells and mineralized matrix throughout the whole scaffold was observed by scanning electron microscopy and histological study. In contrast, cells were located only along the scaffold perimeter in static culture. These results demonstrated the feasibility and benefit of perfusion culture in conjunction with well-defined three-dimensional environment for large bone graft construction. Porous scaffold with controlled architecture can be a potential tool to evaluate the effects of scaffold specific geometry on fluid flow configuration and cell behavior under perfusion culture.

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.

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.

Staged-injection procedure to prevent cement leakage during vertebroplasty: an in vitro study.

STUDY DESIGN: Fibrin sealant (FS) combined with or without growth factor was used to improve the micro-architectural and biomechanical properties of vertebral body in osteoporotic ovine spine. OBJECTIVE: To analyze the treatment effects of bovine bone morphogenetic protein (bBMP) combined with FS on osteopenic ovine vertebral architecture, bone mineral density, and biomechanics in vivo. SUMMARY OF BACKGROUND DATA: Vertebroplasty and kyphoplasty were used to treat spinal osteoporosis. They can increase strength of vertebrae physically. However, each has specific disadvantages. bBMP could rapidly increasing bone formation and suppressing bone resorption, but little is known about its effect on ovariectomized-induced osteoporosis. METHODS: Six sheep underwent ovariectomy and were placed on a low-calcium diet. Twelve months later, according to Ladin square design, L4-L6 vertebrae in all sheep were randomly assigned to 3 treatment groups: A (30 mg bBMP/1.5 mL FS), B (30 mg bBMP) and C (1.5 mL FS). All materials were injected into the assigned vertebra transpedicularly. Animals were killed 3 months after injection, and bone mineral density (BMD), biomechanics, and histomorphometry were assessed. Analysis of variance was used to determine effects of bBMP/FS (alpha = 0.05). RESULTS: The BMD in Group 1 was 17.1% and 14.7% higher than that in Group 2 and Group 3, respectively. The micro-CT reconstruction analysis showed that the density and connectivity of trabecular bone in bBMP/FS treated vertebrae were higher than those in control groups. The mechanical properties (yield stress, ultimate stress, energy absorption, bone modulus) of the vertebrae were also significantly higher. In this study, local bBMP/FS treatment showed a positive trend in improving BMD, histomorphometric parameters, and mechanical strength of osteoporotic vertebra. Slow release of bBMP using FS appeared to be an effective method of protein delivery. CONCLUSION: The use of bBMP/FS in the treatment of vertebral osteoporosis in an attempt to enhance bone strength merits further study.

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.

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.

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.

[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.

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 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.

A study on a tissue-engineered bone using rhBMP-2 induced periosteal cells with a porous nano-hydroxyapatite/collagen/poly(L-lactic acid) scaffold.

We investigated the in vivo osteogenic ability of rhBMP-2 induced periosteal cells in a new porous scaffold, nano-hydroxyapatite (nano-HA)/collagen/poly(L-lactic acid) (PLA). The nano-HA/collagen/PLA composites were utilized as an extracellular matrix for a cell-based strategy of bone tissue engineering. Periosteal cells were cultivated with 500 ng ml(-1) rhBMP-2, followed by seeding into prewet nano-HA/collagen/PLA scaffolds. The cell-scaffold constructs were then subcutaneously implanted in nude mice compared to controls with cell suspension and scaffold alone. Scanning electron microscopy examination proved that the scaffold supported adhesion and proliferation of periosteal cells. Histological bone formation was observed only in experimental groups with cell transplants 8 weeks post-implantation. The animals of the control groups did not show bone formation. The results strongly encourage the approach of the transplantation of rhBMP-2 induced periosteal cells within a suitable carrier structure for bone regeneration.