WP5 - 'In vivo' potency

Although in vitro assays are important to study the differentiation capability of MSCs along osteochondral lineages, it is necessary to establish the potential of MSCs to contribute to tissue formation in vivo. Osteogenesis is one of the mesodermal differentiation pathways of MSCs and bone tissue engineering is one of the attractive clinical applications of these cells in human.

Several pathological conditions lead to an extensive loss of bone tissue (trauma, inflammation, neoplasia) and the reconstruction of large bone segments remains an important clinical problem. The implantation of engineered bone graft material whereby an osteocunductive scaffold is combined with osteogenic committed cells could represent a valid alternative. The first reported repair of a human bone defect using implanted stem cells loaded on porous ceramic was described in 2001 (Quarto 2001), illustrating the potential of these cells in orthopaedics.

Fig 6: Follow-up of a patient implanted with a HA scaffold seeded with BMSC

The Purstem project will focus on the osteogenic activity of MSCs and will use the ectopic mouse model to test in vivo differentiation of MSC. This model uses the subcutaneous implantation of human MSCs loaded on a matrix (ceramic cube assay) in immunodeficient mice.

The ceramic cube assay is a semi-quantitative test of the ability of culture-expanded cells to produce bone or cartilage in vivo in an osteo-conductive environment (Goshima 1991,). When implanted in vivo, the MSCs reconstitute bone and in some conditions cartilage. In particular, when combined with mineral containing tridimensional scaffolds, MSCs form a primary bone tissue highly vascularized and colonized by host hematopoetic marrow (Haynesworth 1992). In this system, the ceramic provides a tridimensional structure, a cell adhesion site and may act as a primer for the formation of new bone matrix and in addition, the MSCs differentiate into osteoblasts and deposit extracellular matrix on the ceramic surface. The ectopically formed tissue may be quantified by histological techniques.

Different biomaterials have been proposed as bone substitutes. There are at least three characteristics that these biomaterials should exhibit: 1) osteoconduction; the ability to support bone growth over its surface, 2) osteoinduction; the ability to induce differentiation of stem cells into osteoblasts and 3) osteointegration; the ability to physically bond to the surface of bone stumps (Mastrogiacomo 2006). Several studies have demostrated good performance with mineralized scaffolds such as hydroxypatite (HA) porous ceramic. These scaffolds proved to have good osteoconductive properties, resulting in good functional recovery, but they were not resorbed after more than 5 years post-surgery. For this, we evaluated the performance of a silicon-containing mineral bimaterial, Skelite (Octane-Canada) in promoting bone formation in nude mice.

Skelite is a resorbable bioceramic based on silicon stabilized tricalcium phosphate (Si-TCP). The silicon, which substitutes the phosphorus, is distributed throughout the material's crystal structure and provides Skelite with a multiphase composition, consisting of 67% Si-TCP and 33% HA/B-TCP (Reid 2005).

The objective of WP5 is to determinate the in vivo osteogenic potency of the various in vitro expanded PurStem MSC. Cryopreserved MSCs will be transported to UNIGE, from different partners, for evaluation using ceramic cube system (Skelite) bone formation. Assessment of chondrogenic and bone potential will be tested after 3 and 6 weeks from the implant. Explanted cubes will be stained histologically to detect the presence of cartilage and bone, which will be quantified using image analysis software.

Fig 7: Bone formation using ceramic cube assay in vivo