Orthotopic Bone Formation by Streamlined Engineering and Devitalization of Human Hypertrophic Cartilage.

Paul Emile Bourgine,Adam Papadimitropoulos,Jaquiery Claude,Giuseppe M Peretti,Christian Epple,Sébastien Pigeot,Celeste Scotti,Atanas Todorov,Ivan Martin

doi:10.3390/ijms21197233

Abstract

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.

Highlights

Bone repair is among the most potent regenerative processes in the human body, typically healing without scar tissue
To confirm the mouse origin of the formed bone, a staining for human repeated Alu sequences was carried out showing the absence of human cells throughout the defect and in contact with the newly formed bone (Supplementary Figure S2). These findings indicate that devitalized hypertrophic cartilage (HyC) extracellular matrix (ECM) more efficiently support bone formation as compared to a typical allograft, by enhanced osteoconduction, but possibly through a mechanism of osteoinduction
Our work demonstrates the feasibility to engineer and subsequently devitalize upscaled amounts of HyC ECM in a streamlined process within a perfusion bioreactor system

Summary

Introduction

Bone repair is among the most potent regenerative processes in the human body, typically healing without scar tissue. Spontaneous bone healing is not sufficient and surgical intervention using graft substitutes is required [1]. The current gold standard for bone grafting consists in autologous bone transplant. There are significant drawbacks, such as limited material availability, increased risks of infection and morbidity at the donor site. Alternative strategies rely on materials with limited biological functionality (e.g., xeno-, allo-graft, synthetic scaffolds) or on osteoinductive growth factors delivered at supra-physiological doses, raising safety concerns [2]. There is a crucial need for novel bone repair strategies

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