J Orthopaed Traumatol (2008): 24 November 2008 DOI 10.1007/s10195-008-0030-6 24 NOVEMBER 2008 SYMPOSIUM References 1. Pola E, Lattanzi W, Pecorini G, Logroscino CA, Robbins PD (2005) Gene therapy for in vivo bone formation: recent TISSUE ENGINEERING advances. Eur Rev Med Pharmacol Sci 9(3):167–174 2. Pola E, Gao W, Zhou Y, Pola R, Lattanzi W, Sfeir C, et al (2004) A CLINICALLY RELEVANT GENE THERAPY APPROACH Efficient bone formation by gene transfer of human LIM miner- TO INDUCE OSTEOGENESIS: STUDY OF THE NEW alization protein-3. Gene Ther 11(8):683 –693 GROWTH FACTOR LIM MINERALIZAZION PROTEIN-3 3. Lattanzi W, Parrilla C, Fetoni A, Logroscino G, Straface G, Pecorini G, Tampieri A, Bedini R, Pecci R, Michetti F, E. Pola Gambotto A, Robbins PD, Pola E (2008) Ex-vivo transduced autologous skin fibroblasts expressing human Lim Minera - Department of Orthopaedics and Traumatology, Università lization Protein-3 efficiently form new bone in animal models. Cattolica del Sacro Cuore, School of Medicine (Rome-IT) Gene Therapy [Epub ahead of print] Recombinant proteins, such as the bone morphogenetic proteins have been used to enhance repair of non-union fractures and facil- TYPE-I COLLAGEN MEMBRANE AS A SCAFFOLD FOR itate new bone formation in animal models as well as in clinical TENDON REPAIR: AN IN VITRO AND IN VIVO STUDY applications. However, a significant amount of recombinant BMP is required to promote osteogenesis and frequently the extent of A. Gigante, E. Cesari, A. Busilacchi, S. Manzotti, F. Greco new bone formation is low. In contrast, local gene transfer of BMPs has been shown to be more efficient in promoting osteogenesis in Department of Orthopaedics, Polytechnic University of Marche rodents than the use of recombinant proteins. Several different (Ancona-IT) types of gene transfer techniques have been utilized for inducing bone formation. Direct injection of viral vectors, such as aden- Objective Bioscaffolds have been proposed as alternatives to ovirus, adeno-associated virus and lentivirus as well as implanta- auto-, allo-, xeno- and synthetic grafts in tendon and ligament tion of plasmid DNA expressing BMPs or related osteogenic fac- repair and substitution. A new type-I collagen membrane devel- tors, have produced new bone in different models. However, con- oped for use as a tendon graft was tested in vitro and in vivo. cerns about the immune response to the viral vectors and possible Material and Methods The membrane (Opocrin - Modena, Italy) dissemination of the vectors have limited the clinical use of such is obtained from type-I collagen harvested from equine Achilles approaches [1]. As alternative, cell-based approaches, using cells tendon and is composed of collagen I fibres oriented in a single genetically modified in culture to express an osteoinductive gene direction. Its is isotropic, due to its microlaminated and multilay- prior to implantation, have induced osteogenesis in different ani- ered parallel structure. The raw material is carefully processed mal models. Gene transfer of intra-cellular proteins such as Osterix and results in a biocompatible, non-reactive, non-antigenic and (OSX) and Lim Mineralization Protein (LMP) also have been non-immunogenic acellular membrane that is resorbable and shown to induce osteogenesis in vivo. The human LMP gene is hygroscopic and can be produced in a range of thicknesses and alternatively spliced to produce three isoforms: human LMP-1 and collagen concentrations. In addition, it does not contain elastin, LMP-3 have been shown to induce bone formation whereas LMP- which may elicit inflammatory reactions. 2 does not exert osteogenic properties following gene transfer, but In vitro study. Primary human fibroblast cultures were established instead may inhibit osteogenesis. The mechanism through which and characterized using anti-collagen I and anti-fibronectin mon- LMP-3 [2] induces osteogenesis is still poorly defined, but appears oclonal antibodies. They were seeded at a concentration of to be mediated through the activation of genes involved in osteoge- 50,000 cells/cm2 on collagen I membranes with aligned fibres nesis, including runt-related transcription factor 2 (RunX2), (material # 40133) with and randomly arranged fibres (# 40153). Osterix (OSX) and certain BMPs. We previously have demonstrat- Cell proliferation was evaluated at 4, 8 and 12 days by spec- ed that adenoviral gene transfer of human LMP-3 (Ad.hLMP-3) trophotometry using an MTT colorimetric reaction. Membrane facilitates ectopic bone formation; however, concerns about the sections were studied by immunohistochemistry (anti-collagen I dissemination of the adenoviral vector have prevented the clinical and anti-fibronectin monoclonal antibodies) and observed with a application of Ad.LMP-3. Thus, in order to develop a clinically rel- confocal microscope on day 12 of culture. evant gene therapy approach to facilitate bone healing, we have In vivo study. The middle third of the patellar tendon was used primary dermal fibroblasts transduced ex vivo with Ad.LMP3 lesioned bilaterally in 10 adult male New Zealand White rabbits and seeded on a hydroxyapatite/collagen matrix prior to autologous and repaired on the right side by a graft (# 40133). The contralat- implantation. The genetically modified autologous dermal fibrob- eral tendon was left untreated and served as control. Animals lasts expressing Ad.LMP-3 are able to induce ectopic bone forma- were euthanized 1 or 6 months after surgery and the tendon grafts tion into the mouse triceps and paravertebral muscles and to heal subjected to histological examination. efficiently rat bone critical size defect model as determined by X- Results The in vitro study demonstrated cell viability and prolif- ray, histology and three dimensional micro computed tomography eration already on day 4 from membrane seeding, and mainte- (3DµCT). Our data [3] demonstrate the effectiveness of the non- nance of the fibroblastic phenotype until the 12th day. Confocal secreted intracellular osteogenic factor LMP-3 in inducing bone microscopic observation showed that cells were homogeneously formation in vivo. Moreover, the utilization of autologous dermal distributed in all membrane specimens, with a more marked ori- fibroblasts implanted on a biomaterial represents a promising entation along the main membrane axis in batch 40133 than in # approach for possible future clinical applications aimed at inducing 40153. The in vivo study one month from implantation showed new bone formation. well-differentiated cell growth into the membrane. At 6 months S2 J Orthopaed Traumatol (2008): 24 November 2008 cell orientation and differentiation in the scaffold with aligned ORAL PRESENTATIONS fibres was satisfactory, with decreased cellularity, good integra- tion with the surrounding tissue and crimps. Inflammatory reac- HIP tion, plasma-cell infiltrate or excessive implant neovasculariza- tion were never observed. Discussion The new type-I collagen membrane exhibited a FIRST HIP PROSTHESIS WITH CERAMIC-CERAMIC COU- behaviour similar to other tendon or ligament scaffolds both PLING AND HYDROXYAPATITE COATING in vitro and in vivo. Orientation in the scaffold with aligned fibres allowed obtaining a quick and well-oriented cell growth as F. Trentani, P. Trentani, O. Moreschini well as good integration with host soft tissues. The membrane appears to be suitable for application in tendon and ligament Rizzoli Orthopaedic Institute (Bologna-IT) reinforcement. Suggested reading Objective In 1971 a group of Bolognese researchers, Trentani, 1. Fini M, Torricelli P, Giavaresi G, Rotini R, Castagna A, Paltrinieri, Cini, Sandolini and Pizzoferrato, each with specific Giardino R (2007) In vitro study comparing two collageneous tasks, wanted to reassess prosthesis fixation to the bone. membranes in view of their clinical application for rotator cuff Material and Methods Considering the excellent but risky results tendon regeneration. J Orthop Res 25(1):98–107 of cement introduced by Charnley, they performed experimental studies on laboratory animals to assess the concept of bone-prosthe- sis integration and extend studies begun by Boutin on ceramic mate- rials. These studies showed that bone trabeculae formed on ceramic ONE STAGE OSTEOCHONDRAL REPAIR WITH AUTOLO- materials that perfectly adhered to their surface and they concluded GOUS CARTILAGE FRAGMENTS IN A HYBRID SCAF- that such materials can have a wide application in orthopedic sur- FOLD: IN VITRO AND IN VIVO ANIMAL MODEL gery since they are electrolytically inert in our tissues and their physical-chemical constitution is very similar to that of bone. P. Rossi1, A. Marmotti1, F. Castoldi1, R. Rossi1, M. Bruzzone1, Therefore, they developed an prosthesis with a polyethylene acetab- G. Tarone2, B. Peirone3, M. Mauthe Von Degerfeld3 ular cup and ceramic-coated titanium stem, realized by spraying alu- mina at temperatures of 100,000°C with the plasma-Jet technique. 1Department of Orthopaedics and Traumatology, Mauriziano Results The long-term results at 2 years on 8 patients aged between 63 “Umberto I” Hospital, University of Turin (Turin-IT); 2Department of and 74 years were encouraging and prompted further studies and an Genetics Biology and Biochemistry, MBC, University of Turin (Turin- Italo-Austrian collaboration, where total joint arthroplasty with ceram- IT); 3Department of Animal Pathology, University of Turin (Turin-IT) ic-ceramic coupling was developed in 1975. The stem was made of tita- nium coated in hydroxyapatite by chemical bonding, with