Bone Defects In Animal Models Research Articles

Bone regeneration in a massive rat femur defect through endochondral ossification achieved with chondrogenically differentiated MSCs in a degradable scaffold

Mesenchymal stem cells (MSCs) are multipotent cells capable of proliferating and differentiating into several lineages. In regenerative medicine, their potential as a resource for tissue-replacement therapy is receiving much attention. However, transplanting MSCs to repair larger bone defects in animal models has so far proved disappointing. Here we report on the healing of both critical-sized (5 mm) and massive (15 mm) full-thickness femur defects in rats by implanting a uniquely fabricated PLGA scaffold seeded with MSCs pre-differentiated in vitro into cartilage-forming chondrocytes (MSC-DCs). This strategy closely mimics endochondral ossification, the process by which long bones develop in nature. It is thought that because the transplanted MSC-DCs induced natural bone formation, the defect size was not critical to the outcome. Crucially, after 8 weeks the mean biomechanical strength of femora with the massive 15 mm implant reached 75% that of a normal rat femur, while in the case of 5 mm implants there was no significant difference. Successful healing was also highly reproducible, with bone union occurring in all treated animals examined radiologically 8 or 16 weeks after surgery.

Mechanical analysis of a rodent segmental bone defect model: The effects of internal fixation and implant stiffness on load transfer

Segmental bone defect animal models are often used for evaluating the bone regeneration performance of bone substituting biomaterials. Since bone regeneration is dependent on mechanical loading, it is important to determine mechanical load transfer after stabilization of the defect and to study the effects of biomaterial stiffness on the transmitted load. In this study, we assess the mechanical load transmitted over a 6mm femur defect that is stabilized with an internal PEEK fixation plate. Subsequently, three types of selective laser melted porous titanium implants with different stiffness values were used to graft the defect (five specimens per group). In one additional group, the defect was left empty. Micro strain gauges were used to measure strain values at four different locations of the fixation plate during external loading on the femoral head. The load sharing between the fixation plate and titanium implant was highly variable with standard deviations of measured strain values between 31 and 93% of the mean values. As a consequence, no significant differences were measured between the forces transmitted through the titanium implants with different elastic moduli. Only some non-significant trends were observed in the mean strain values that, consistent with the results of a previous finite element study, implied the force transmitted through the implant increases with the implant stiffness. The applied internal fixation method does not standardize mechanical loading over the defect to enable detecting small differences in bone regeneration performances of bone substituting biomaterials. In conclusion, the fixation method requires further optimization to reduce the effects of the operative procedure and make the mechanical loading more consistent and improve the overall sensitivity of this rat femur defect model.

Ultrasound‐mediated transgene expression in endogenous stem cells recruited to bone injury sites

When fractured, bone can spontaneously heal to a certain extent. In critical‐size defects, in which bone loss is severe, a bone graft is required. Faced with the shortcomings of grafts currently in use—autografts, allografts, and mineral‐based bone substitutes—efforts are being made to establish new methods of bone regeneration. One promising approach involves the use of exogenous gene‐modified mesenchymal stem cells (MSCs), which can rapidly repair large bone defects in animal models. Unfortunately, ex vivo culture of MSCs may add certain complexity to the advancement of this cell therapy to the clinic. Previously, we demonstrated efficient bone regeneration following direct gene delivery to endogenous MSCs that had been attracted to a fracture site. In that study, electroporation was used, but it is an invasive method of gene transfection that may result in tissue damage. Unlike electroporation, sonoporation—the use of ultrasound for gene delivery—is noninvasive, considered safer, and relevant to the clinical setting. In this study, we evaluated the feasibility of ultrasound‐based gene delivery to resident MSCs that had been recruited to a fracture site in different animal models. Our results show transient (up to 21 days) expression of a reporter gene in radial, vertebral, and tibial bone defects in mice, rats, and a minipig. These results could prove beneficial for the next phase of experiments, in which osteogenic genes such as the bone morphogentic proteins (BMPs) could be utilized to achieve bone regeneration in similar challenging conditions of bone tissue loss. Copyright © 2014 John Wiley & Sons, Ltd.

BMP-2 mRNA Expression After Endothelial Progenitor Cell Therapy for Fracture Healing

Endothelial progenitor cells (EPCs) represent a population of novel precursor cells with known ability to participate in angiogenesis. Our previous studies have shown that local EPC therapy significantly increased angiogenesis and osteogenesis to promote fracture healing in an animal bone defect model. However, the cellular and molecular mechanisms by which EPC therapy promotes fracture healing remain largely unknown. The purpose of this study was to quantify local bone morphogenetic protein (BMP-2) expression after EPC therapy for a rat segmental bone defect, in hopes of further defining the potential mechanisms by which EPCs promote fracture healing. EPCs were isolated from the bone marrow of syngeneic rats and cultured ex vivo for 7-10 days before transfer to the bone defect. A total of 56 rats were studied. The treatment group received 1 × 10 EPCs on a gelfoam scaffold at the bone defect, and control animals received gelfoam/saline only. Before euthanasia, radiographs of the femur were performed. Animals were euthanized at 1, 2, 3, and 10 weeks, and specimens from the fracture gap area were collected, pulverized, and total messenger RNA (mRNA) was extracted. BMP-2 mRNA was measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. All measurements were performed in triplicate. All EPC-treated bone defects healed radiographically by 10 weeks, whereas control-treated defects developed a nonunion. The expression of BMP-2 mRNA was significantly elevated in EPC-treated defects relative to controls at week 1 (EPC, 0.59 ± 0.10; control, 0.31 ± 0.08; P = 0.05), week 2 (EPC, 0.40 ± 0.06; control, 0.23 ± 0.04; P = 0.04), and week 3 (EPC, 0.33 ± 0.06; control, 0.18 ± 0.03; P = 0.04), but not at week 10 (EPC, 0.31 ± 0.06; control, 0.21 ± 0.04, P = 0.15). The highest mean expression of BMP-2 in EPC-treated defects was observed at 1 week, with a progressive decline in BMP-2 expression noted thereafter. These findings demonstrate that EPC-treated bone defects demonstrate both radiographic healing and elevated expression of BMP-2 relative to control-treated defects. These results provide further insight into the potential mechanisms by which EPC therapy may promote fracture healing and provide further evidence to suggest that the trophic actions of EPC therapy may be a critical factor in their contribution to fracture healing.

Characterization of an Ovine Bilateral Critical Sized Bone Defect Iliac Wing Model to Examine Treatment Modalities Based on Bone Tissue Engineering

Critical sized bone defect (CSBD) animal models are used to evaluate and confirm efficacy and potency of new treatment modalities based on bone tissue engineering before the latter can be applied in clinical practice. In this study, a bilateral CSBD model in the iliac wings of sheep is described in detail. To demonstrate that this is a large animal CSBD model in sheep, bone healing within the defect left empty (negative control) or filled with autologous corticocancellous bone graft (clinical gold standard, positive control) was assessed using micro-CT, histology, histomorphometric, and fluorochrome analysis. After three months, new bone into the defect site was formed across the whole defect in the positive controls but limited to the edge of the defects in the negative controls. Bone volume in the positive controls was statistically higher than in the negative controls, with the latter having less than 10% new bone growth. There were no intraoperative or postoperative complications. The model described here represents a reliable and reproducible bilateral CSBD in sheep with low morbidity that can be used for in vivo evaluation of new treatment modalities based on bone tissue engineering.

An experimental study on the application of radionuclide imaging in repairing bone defects

Objective: In order to study the ability of nano-hydroxyapatite artificial bone in repair of large segmental bone defect and to evaluate the value of radionuclide bone imaging in monitoring the bone's repairing in the bone defect. Methods: The animal model of bone defect was made on the bilateral radius of 24 (New repaired with HA artificial bone) with 12 rabbits in each group. The ability of bone defect repair was evaluated by using radionuclide bone imaging at 2， 4， 8 and 12 weeks postoperatively. Results: The counts of regions of interest (ROI) measure and the target to non-target ratios (T/NT) of the two groups indicate that the experimental group stimulated more bone formation than that of the control group. The differences of the bone reconstruction ability were statistically significant （P ＜ 0.05）. Conclusions: The nano-HA artificial has good bone conduction，it can be used for the treatment of bone defects. Radionuclide imaging may be an effective and first choice method for the early monitoring of the bone's reconstruction.

The effect of high voltage alternating electric field therapy on fracture healing

Objective To explore the effect of high voltage alternating electric field therapy on fracture healing in rats. Methods An animal model of cortical bone defect was successfully established in the unilateral proximal tibia of 41 rats.They were then randomized into an experimental group (21 rats) and a control group (20 rats).The animals in the experimentsl group were given high voltage alternating electric field therapy for 30 minutes daily,while those in the control group only had sham exposure to the high voltage alternating electric field therapy.Rats in each group were sacrificed at days 7,14,21 and 28 post-operation.After blood collection,the operated tibias of the rats were resected with muscle and other soft tissue removed.Bone mineral density (BMD) of the callus at the fracture point was measured using dual energy X-ray absorptiometry (DEXA).The calluses were then cut down and fixed,decalcificated and sliced for histological observation.At the same time serum osteocalcin ( OC ) and bone alkaline phosphatase (RAP) were also detected by ABC-ELISA. Results The BMD examination showed that callus BMD in the experimental group was significantly higher than in the control group at days 14 and 28.Serum OC and RAP in the experimental group were also higher at days 14 day 21,and OC was still significantly higher in the experimental group at day 28.Histological examination showed that in the early stages of bone healing hematoma absorption and organization appeared earlier in the experimental group than in the control group,with remarkable osteoblast and chondroblast proliferation.While at the later stage,bone trabeculas connection and mineralization also appeared earlier in the experimental group than in the control group. Conclusions High voltage alternating electric field therapy may promote fracture healing by accelerating hematoma absorption and organization,elevating osteoblast proliferation and differentiation as well as promoting extracellular matrix secretion and mineralization.The underlying therapeutic mechanism might be related to the increased expression of OC and RAP caused by exposure to the high voltage alternating electric field. Key words: High voltage alternating electric field therapy; Fractures; Osteocalcin; Bone alkaline phosphatase; Bone mineral density

In vivo alveolar bone regeneration by bone marrow stem cells/fibrin glue composition

The repair of alveolar bone defects caused by trauma, periodontal diseases and inflammation is still a challenge for both researchers and clinicians. Although there are many attempts to regenerate bone based on different seed cells and scaffolds, the results are still unsatisfactory. This study aims to clarify whether it could be efficient to reconstruct the alveolar bone by the combination of bone marrow stem cells (BMSCs) without pre-osteoinduction in vitro with fibrin glue (FG). The BMSCs were obtained from 2-week-old Sprague-Dawley (SD) rats and expanded in vitro with non-introduction. Afterwards, they were composited with FG for in vivo implantation. The animal models of traumatic alveolar bone defects were established bilaterally in the maxilla of 15 rats which were randomly divided into 3 groups. The BMSCs/FG composition was transplanted into 5 rats of the treated group. Another 5 rats in the negative control group were transplanted by pure FG without BMSCs. The rest 5 rats served as the blank control. Gross observation and histological analysis were made to evaluate the new bone formation 6 weeks after transplantation. Micro-CT was also used to estimate the bone healing through three-dimensional reconstruction and the bone density analysis. The amount of new bone formed in the treated group was significantly greater than the negative and blank control. Our results suggest that the strategy of combing BMSCs with FG is effective in the repair of alveolar bone defects. Its clinical application is promising.

Revascularization and osteogenesis during repair of bone defects with tissue-engineered bone of nano-hydroaoatite crystal/polyamide 66/rabbit osteoplast/vascular endothelial growth factor

Objective To employ the vascular endothelial growth factor (VEGF165) gene transfected rabbbit osteoplast to combine with the porous nano-hydrxyapatite crystal (n-HA)/polyam ide 66(/PA66) so as to evaluate the osteogenesis and rapid revascularization early after repair of the rabbit radius bone defects with the tissue-engineered bone (n-HA/PA66/osteoplast/VEGF165). Methods The animal models of bilateral radius bone defects were created in 56 New Zealand white rabbits that were then randomly divided into Group A and Group B.In Group A, the animals were implanted with n-HA/PA66on the left bone defects (Group Al) and with n-HA/PA66/VEGF165 composite materials on the right bone defects (Group A2). In Group B, the animals were implanted with n-HA/PA66/osteoplast/VEGF165 composite materials on the left side (Group B1) and with n-HA/PA66/osteoplast on the right side (Group B2).Gross, digital radiography, histological sections, vessel count and scanning electron microscopy (SEM) were performed at 2, 4, 8 and 12 weeks after operation. Results The osteogenesis and revascularization in Group B1 was superior to that in the other groups at each time point, with statistical difference (P ＜0.05).The revascularization and osteogenesis in Groups B1 and B2 was far better than that in Groups A1 and A2, with no statistical difference between Group A1 and Group A2. Conclusions The new tissue-engineered bone (n-HA/PA66/osteoplast/VEGF165) has a perfect osteogenetic effect and can promote rapid revascularization and the bone healing in the early stage after repair of the bone defects. Key words: Hydroxyapatites; Vascular endothelial growth factors; Bone defect

Engineering the Growth Factor Microenvironment with Fibronectin Domains to Promote Wound and Bone Tissue Healing

Although growth factors naturally exert their morphogenetic influences within the context of the extracellular matrix microenvironment, the interactions among growth factors, their receptors, and other extracellular matrix components are typically ignored in clinical delivery of growth factors. We present an approach for engineering the cellular microenvironment to greatly accentuate the effects of vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) for skin repair, and of bone morphogenetic protein-2 (BMP-2) and PDGF-BB for bone repair. A multifunctional recombinant fragment of fibronectin (FN) was engineered to comprise (i) a factor XIIIa substrate fibrin-binding sequence, (ii) the 9th to 10th type III FN repeat (FN III9-10) containing the major integrin-binding domain, and (iii) the 12th to 14th type III FN repeat (FN III12-14), which binds growth factors promiscuously, including VEGF-A165, PDGF-BB, and BMP-2. We show potent synergistic signaling and morphogenesis between α5β1 integrin and the growth factor receptors, but only when FN III9-10 and FN III12-14 are proximally presented in the same polypeptide chain (FN III9-10/12-14). The multifunctional FN III9-10/12-14 greatly enhanced the regenerative effects of the growth factors in vivo in a diabetic mouse model of chronic wounds (primarily through an angiogenic mechanism) and in a rat model of critical-size bone defects (through a mesenchymal stem cell recruitment mechanism) at doses where the growth factors delivered within fibrin only had no significant effects.

An experimental study on the application of radionuclide imaging in repair of the bone defect

The aim of our study was to validate the effect of radionuclide imaging in early monitoring of the bone's reconstruction, the animal model of bone defect was made on the rabbits repaired with HA artificial bone. The ability of bone defect repair was evaluated by using radionuclide bone imaging at 2, 4, 8 and 12 weeks postoperatively. The results indicate that the experimental group stimulated more bone formation than that of the control group. The differences of the bone reconstruction ability were statistically significant (p<0.05). The nano-HA artificial has good bone conduction, and it can be used for the treatment of bone defects. Radionuclide imaging may be an effective and first choice method for the early monitoring of the bone's reconstruction.

Human Dental Pulp Stem Cells associated to silk protein scaffold restore critical size bone defects in vivo

The aim of this study is to evaluate potential of fibroin scaffold combined with human dental pulp stem cells (hDPSC) to reconstruct large sized cranial bone defects in animal model. We performed two symmetric full-thickness cranial defects on each parietal region of rats and we replenished them with collagen, poly-D,L-lactic acid (PDLLA) and silk fibroin scaffolds with or without stem cells already seeded into and addressed towards osteogenic lineage in vitro. Animals were euthanized after 4 weeks postoperatively and cranial tissue samples were taken for histological analysis. The presence of human cells in the new bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. We observed a mature bone formation and the most relevant defects correction in constructs with fibroin. Our findings demonstrated the strong potential of bioengineered constructs of stem cells-fibroin scaffold for correcting large cranial defects in animal model and constitutes a promising model for reconstruction of human large cranial defects in craniofacial surgery.

Experimental study on the conduction function of nano-hydroxyapatite artificial bone

This study evaluates the osteoconductive property of nano-hydroxyapatite (nano-HA) material and the potential application of nano-HA as artificial bone in repairing bone defects, and tries to analyse the scientific explanation of these. The animal model of bone defects is made on the bilateral radius of 39 New Zealand white rabbits, which are divided into an experimental group (the bone defect is repaired with nano-HA artificial bone), a control group (the bone defect is repaired with HA artificial bone) and a blank group (the defect is empty) randomly. The bone conduction function of different materials is evaluated by gross observation, alkaline phosphatase (ALP) detection in serum, histopathological study, X-ray examination, scanning electron microscopy (SEM) and biomechanical analysis at the 4th, 8th and 12th week postoperatively. The experimental group is found to stimulate more bony callus than the control and blank groups. These differences in bone conduction function are statistically significant (P<0.05). In conclusion, nano-HA artificial bone can be potentially applied to the treatment of bone defects.

Experimental Study of Nano-Hydroxyapatite/Recombinant Human Bone Morphogenetic Protein-2 Composite Artificial Bone

To investigate the repairing ability of nano-hydroxyapatite/recombinant human bone morphogenetic protein-2 (Nano-HA/rhBMP-2) composite artificial bone for bone defect and to provide evidence for its application in clinical repair of bone defect. The animal model of bone defect was made on unilateral radius of 90 New Zealand white rabbits, which were randomly divided into experimental group (group A, bone defect was repaired with Nano-HA/rhBMP-2 composite artificial bone), control group (group B, bone defect was repaired with Nano-HA artificial bone), and blank group (group C, bone defect was unrepaired). The repairing ability for bone defect was evaluated by gross observation, X-ray examination, scanning electron microscope, radionuclide bone imaging and biomechanical analysis at 4(th), 8(th), and 12th week postoperatively. Both Nano-HA/rhBMP-2 composite artificial bone and Nano-HA artificial bone could stimulate new bone formation, but the former could stimulate more new bone formation and had better repairing ability for bone defect than that of the latter, with statistically significant difference (P <0.05). Nano-HA/rhBMP-2 composite artificial bone has good repairing ability for bone defect and it is hopeful to become an ideal repairing material for bone defect.

The challenge of establishing preclinical models for segmental bone defect research

A considerable number of international research groups as well as commercial entities work on the development of new bone grafting materials, carriers, growth factors and specifically tissue-engineered constructs for bone regeneration. They are strongly interested in evaluating their concepts in highly reproducible large segmental defects in preclinical and large animal models. To allow comparison between different studies and their outcomes, it is essential that animal models, fixation devices, surgical procedures and methods of taking measurements are well standardized to produce reliable data pools and act as a base for further directions to orthopaedic and tissue engineering developments, specifically translation into the clinic. In this leading opinion paper, we aim to review and critically discuss the different large animal bone defect models reported in the literature. We conclude that most publications provide only rudimentary information on how to establish relevant preclinical segmental bone defects in large animals. Hence, we express our opinion on methodologies to establish preclinical critically sized, segmental bone defect models used in past research with reference to surgical techniques, fixation methods and postoperative management focusing on tibial fracture and segmental defect models.

Bone tissue engineering and repair by gene therapy

Many clinical conditions require the stimulation of bone growth. The use of recombinant bone morphogenetic proteins does not provide a satisfying solution to these conditions due to delivery problems and high cost. Gene therapy has emerged as a very promising approach for bone repair that overcomes limitations of protein-based therapy. Several preclinical studies have shown that gene transfer technology has the ability to deliver osteogenic molecules to precise anatomical locations at therapeutic levels for sustained periods of time. Both in-vivo and ex-vivo transduction of cells can induce bone formation at ectopic and orthotopic sites. Genetic engineering of adult stem cells from various sources with osteogenic genes has led to enhanced fracture repair, spinal fusion and rapid healing of bone defects in animal models. This review describes current viral and non-viral gene therapy strategies for bone tissue engineering and repair including recent work from the author's laboratory. In addition, the article discusses the potential of gene-enhanced tissue engineering to enter widespread clinical use.

S116: Dental Implant Treatment: Comprehensive Clinical and Histological Examination of Bone Augmentation Techniques

Application of dental implant is difficult in patients with large bone defects and insufficient bone volume. In these cases, it is required that defect is augmented to obtain sufficient bone volume for implant placement. Autogenous bone from iliac crest or mandible mid-chin and ramus is still the “golden standard” for bone augmentation. However, there are problems of the limitation of harvestable bone volume and the secondary surgical site to harvest grafting bone. In order to resolve these problems, the application of autogenous osteoprogenitor cell grafting technique has been innovated. We previously reported the remarkable osteogenic potential of grafted periosteum and bone marrow in the animal studies. Additionally we reported the efficacy of the autogenous bone marrow graft for bone formation in the dental implant treatment. In the present study, we are going to show not only the clinical results but also extensive histological and immunohistologocal findings of the autogenous bone graft in both animal bone defect models and the patients with atrophied alveolar ridge, because for oral and maxillofacial surgeons to understand the process of the bone formation of the grafted materials is a very important issue.

Biological aspects of bone, cartilage and tendon regeneration

Current orthopaedic procedures in supporting regeneration of bone, cartilage and tendon are dependant on our understanding of the molecular processes responsible for tissue repair. At present we know how to regenerate bone when physiological mechanisms of fracture repair fail [13]. Since the original description of the potential of demineralised bone matrix to induce bone at an ectopic site, it has taken more than 3 decades to bring bone morphogenetic proteins (BMPs) to clinical use. By the end of 2007 nearly 1 million patients worldwide will have been treated with BMPs for spinal fusions, non-unions, acute fractures and maxillofacial reconstruction. Use of animal models, genomics and proteomics has deciphered new mechanisms and candidate molecules for the regeneration of joint cartilage and tendons, opening new avenues in regenerative orthopaedics. This special issue reviews novel strategies in the regeneration of bone, cartilage and tendon. Bishop and Einhorn [2] describe the clinical use of recombinant BMP-2 and BMP-7. They predict that current techniques in engineering bone for restoring defined skeletal defects represent a unique opportunity for BMPs in the future. McKay and colleagues [9] review the clinical applications of BMP-2 (INFUSE Bone Graft), while Vaccaro, McKee and colleagues [14] describe the clinical applications of BMP-7 (OP-1 implant or Osigraft). The authors suggest that recombinant human BMP-based devices, when properly applied, can eliminate the need to harvest autologous bone for grafting procedures, benefiting both the surgeon and patients. Grgurevic and collaborators [6] describe molecules discovered by proteomic analysis in the plasma of patients with an acute bone fracture. The characterisation and use of potential new biomarkers, like TGF-β-induced protein IG-H3, cartilage acidic protein 1, procollagen C proteinase enhancer protein and TGF-β receptor III, for bone and cartilage regeneration are discussed. Sendak and colleagues [11] review the newly discovered role of thyroid-stimulating hormone (TSH) and follicle-stimulating hormone (FSH) in bone remodelling. It has been recently found that low doses of TSH increase bone volume and improve bone microarchitecture and strength in aged osteoporotic rats, suggesting that TSH directly affects bone remodelling in vivo [10]. Grasser and collaborators [5], by using a genomic approach, decipher the potential mechanism of bone augmentation following systemic administration of BMP-6, suggesting that IGF-1 and EGF mediate, at least in part, the effects of BMP-6 on bone, which has been recently reported [12]. The role of prostaglandin E2 receptors and their analogs in bone formation is reviewed by Li and colleagues [8], demonstrating that the PGE2 receptor is essential for restoring bone defects in animal models. Cartilage regeneration and chronic back pain due to degenerative disc disease are among major obstacles in orthopaedics. Chubinskaya and colleagues [4] summarise the role of osteogenic protein-1 (BMP-7) in animal models of osteochondral and chondral defects, osteoarthritis and degeneration in intervertebral disc cartilage. They show that OP-1 exhibits unique pro-anabolic and prominent anti-catabolic properties having a potential for treating cartilage and disc lesions. Overuse tendon disorders are common and associated with a significant morbidity. Tendon and ligament function engineering and repair are discussed in articles by Aspenberg [1] and by Hoffman and Gross [7]. Aspenberg found that the repair of subcutaneous tendon ruptures can be stimulated by a single application of one of several growth factors, including PDGF, TGF-β, IGF-1, VEGF and GDF-5,-6,-7, or by a thrombocite concentrate. The response is dependent on the mechanical microenvironment, which is crucial for the repair process. Hoffmann and Gross specifically discuss gene-therapeutic approaches using mesenchymal stem cells for a potential future application in tendon/ligament regeneration. Borovecki and colleagues [3] discuss the potential of a genomic approach in analysing bone homeostasis and diseases. Gene expression profiling studies yielded novel insights into the complex interplay of osteoblast and osteoclast regulation, paracrine and endocrine control of bone and cartilage remodelling, as well as the pathophysiology of osteoporosis and bone tumours. We believe that this issue will provide basic and clinical scientists with the state of the art knowledge in regenerative orthopaedics.

Prostaglandin E2 receptors in bone formation

Prostaglandins, PGE(2) in particular, have diverse actions on various organs, including inflammation, bone healing, bone formation, embryo implantation, induction of labour and vasodilatation, among others. However, systemic side effects have limited their clinical utility. The pharmacological activities of PGE(2) are mediated through four G protein-coupled receptor subtypes, EP1-EP4. Recent studies have shown that EP2 and EP4 receptors play important roles in regulating bone formation and resorption. EP2 and EP4 receptor-selective agonists have been shown to stimulate local or systemic bone formation, augment bone mass and accelerate the healing of fractures or bone defects in animal models upon local or systemic administration, thus, potentially offering new therapeutic options for enhancing bone formation and bone repair in humans. This review will focus on the studies related to bone formation and bone healing in the EP receptor knockout (KO) mice and the EP2 or EP4 receptor-selective agonist treated animal models.

1093. Ex Vivo Gene Therapy Approach Using Human Lim Mineralization Protein_3 To Induce Bone Healing in a Rodent Model

Aims. Gene therapy research in the field of orthopaedics and traumatology have evolved during the last decade, leading to possible applications for the treatment of pathological conditions, such as bone fractures and cartilage defects. In particular, several gene transfer techniques have been employed so far for inducing bone formation in animal models of bone defects. Cell-based approaches, using in vitro and ex vivo genetically modified cells to be implanted in the animal, produced promising results as they enable the production of physiologic doses of an osteoinductive gene product into selected anatomical sites. In this study we used autologous skin fibroblasts, which are very simple to harvest and propagate in culture, transduced ex vivo with the new osteo-genic factor Lim Mineralization Factor-3 (Ad-LMP-3). These engineered cells produced successful bone healing when implanted by the use of a scaffold in rats, validating the in vivo osteoinductive properties of hLMP-3. Methods. Primary dermal fibroblasts cultures were established using a 1cm 2 biopsy of shaved skin obtained from the abdomen of each rat after anesthesia. Semi-confluent primary fibroblasts were infected with either AdBMP-2 or AdhLMP-3 or both, using a overall multiplicity of infection (MOI) of 100 viral particles per cell. Cells transduced with Ad-eGFP at the same MOI were used as a viral infection control, while untreated cells served as a negative control. The transduced cells were harvested 24 hours after viral infection, resuspended in sterile PBS, let adsorbed on a Hydroxyapatite/Collagen scaffold and then implanted in a bone defect surgically performed in the mandible of immunocompetent rats. The animals were divided in 4 groups: 9 rats were treated with cells infected with AdLMP-3, 9 rats with cells infected with AdBMP-2 (positive controls), 9 rats with cells transduced with Ad-eGFP and 9 rats with untreated cells (controls). 3 Rats from each group were sacrified at 1, 2 and 3 months after the treatment and studied by x-rays, Micro-CT and histology (Von kossa and Alizarin staining). Results. All the animals treated with LMP-3 showed healing of the bone defect after 3 months, as confirmed his-tologically and radiographically. On the contrary none of the controls showed bone formation at latest time point. Conclusions. Recently, Lim Mineralization Proteins (LMP), coded by three different splice variants (LMP-1, LMP-2, LMP-3) of the same gene, have been identified as regulators of the osteoblast differentiation program. We have previously demonstrated that human LMP-3 (hLMP-3) contributes actively to bone formation, acting at least in part, through the BMP-2 signaling pathway, being capable of inducing differentiation of cells of mes-enchymal derivation towards the osteoblastic lineage, through the up-regulation of bone-specific genes, along with ectopic bone formation in vivo and mineralization in vitro. In this study we have analyzed the efficacy of an ex-vivo approach using autologous dermal fibroblasts infected with AdLMP-3. Engineered cells produced bone healing when implanted by the use of a scaffold in a rodent model, validating the in vivo osteoinductive properties of hLMP-3.