Most bones of the human body form and heal through endochondral ossification, whereby hypertrophic cartilage (HyC) is formed and subsequently remodeled into bone. We previously demonstrated that HyC can be engineered from human mesenchymal stromal cells (hMSC), and subsequently devitalized by apoptosis induction. The resulting extracellular matrix (ECM) tissue retained osteoinductive properties, leading to ectopic bone formation. In this study, we aimed at engineering and devitalizing upscaled quantities of HyC ECM within a perfusion bioreactor, followed by in vivo assessment in an orthotopic bone repair model. We hypothesized that the devitalized HyC ECM would outperform a clinical product currently used for bone reconstructive surgery. Human MSC were genetically engineered with a gene cassette enabling apoptosis induction upon addition of an adjuvant. Engineered hMSC were seeded, differentiated, and devitalized within a perfusion bioreactor. The resulting HyC ECM was subsequently implanted in a 10-mm rabbit calvarial defect model, with processed human bone (Maxgraft^®) as control. Human MSC cultured in the perfusion bioreactor generated a homogenous HyC ECM and were efficiently induced towards apoptosis. Following six weeks of in vivo implantation, microcomputed tomography and histological analyses of the defects revealed an increased bone formation in the defects filled with HyC ECM as compared to Maxgraft^®. This work demonstrates the suitability of engineered devitalized HyC ECM as a bone substitute material, with a performance superior to a state-of-the-art commercial graft. Streamlined generation of the devitalized tissue transplant within a perfusion bioreactor is relevant towards standardized and automated manufacturing of a clinical product.

We previously described an immortalized, genetically-engineered human Mesenchymal stromal cell line to generate BMP2-enriched Chondrogenic Matrices (MB-CM), which after devitalization and storage could efficiently induce ectopic bone tissue by endochondral ossification. In order to increase the efficiency of MB-CM utilization towards engineering scaled-up bone structures, here we hypothesized that MB-CM can retain osteoinductive properties when combined with an osteoconductive material. We first tested different volumetric ratios of MB-CM:SmartBone® (as clinically used, osteoconductive reference material) and assessed the bone formation capacity of the resulting composites following ectopic mouse implantation. After 8 weeks, as little as 25% of MB-CM was sufficient to induce bone formation and fusion across SmartBone® granules, generating large interconnected bony structures. The same composite percentage was then further assessed in a scaled-up model, namely within an axially-vascularized, confined, ectopically prefabricated flap (0.8 cm³) in rats. The material efficiently induced the formation of new bone (31% of the cross-sectional area after 8 weeks), including bone marrow and vascular elements, throughout the flap volume. Our findings outline a strategy for efficient use of MB-CM as part of a composite material, thereby reducing the amount required to fill large spaces and enabling utilization in critically sized grafts, to address challenging clinical scenarios in bone reconstruction. STATEMENT OF SIGNIFICANCE: Most bone repair strategies rely either on osteconductive properties of ceramics and devitalized bone, or osteoinductive properties of growth factors and extracellular matrices (ECM). Here we designed a composite material made of a clinically accepted osteoconductive material and an off-the-shelf tissue engineered human cartilage ECM with strong osteoinductive properties. We showed that low amount of osteoinductive ECM potentiated host cells recruitment to form large vascularized bone structures in two different animal models, one being a challenging prefabricated bone-flap model targeting challenging clinical bone repair. Overall, this study highlights the use of a promising human off-the-shelf material for accelerated healing towards clinical applications.

Bone organs possess great healing potential, however, in some cases, their repair processes fail and the use of exogenous material is required. Despite the availability of numerous off-the-shelf allografting or xenografting materials, none of them could meet the extraordinary capacities of autologous bone graft which remains the gold standard. Recently, we demonstrated for the first time, using a BMP-2 engineered human cell line seeded on a collagen scaffold, that in vitro generated cartilage extracellular matrices (ECM) can be devitalized and lyophilized, stored off-the-shelf, and form bone in vivo with a unique temporality (Pigeot et al. 2021).To this aim, a GMP compliant Master Cell Bank (MCB), compatible with clinical use was created and a GMP compliant protocol developed to remove DNA and cellular content from the ECM. The resulting decellularized ECM were lyophilized and stored off-the-shelf before their in vivo implantation. ECM were implanted in a critical size defect (5mm diameter × 15 mm depth, n=7) in the epiphysis of the sheep tibia (n=2) and femur (n=3). Different ECM doses were thus compared and analyzed after 12 weeks in vivo implantation. Conditions comprised collagen scaffold only (negative control), and doses of 45%, 73% and 100%. The latter corresponding to complete filling of the defect. All data were correlated to an empty defect control. The outcome was measured using microcomputed tomography as well as histological analysis both quantifying the bone formation and immunological reaction.Bone formation clearly correlated the amount of implanted ECM, even more when looking at the innermost of the defect corroborating the osteoinductive effect of the ECM. Histopathological analysis could detect some inflammation in the defects, mostly due to the collagen scaffold remains, but this was not critically detrimental to the bone formation. Furthermore, analysis of the literature revealed that the ECM achieved comparable performances to autologous bone graft in a similar sheep animal drill hole model (Huber E. et al, 2017)In this study we demonstrated that our unique and effective osteoinductive human ECM can efficiently be produced using GMP standards and form bone in a large, immunocompetent animal model with analogous performances as compared to autologous bone grafts. This demonstration of bone formation feasibility by an off-the-shelf, decellularized human ECM in the sheep paves the way for a large defect Proof-of-Concept study in these animals to validate the efficacy and envision a first in human clinical trial.