A clinically relevant model of osteoinduction: a process requiring calcium phosphate and BMP/Wnt signalling

Abstract

In this study, we investigated a clinically relevant model of in vivo ectopic bone formation utilizing human periosteum derived cells (HPDCs) seeded in a Collagraft™ carrier and explored the mechanisms by which this process is driven. Bone formation occurred after eight weeks when a minimum of one million HPDCs was loaded on Collagraft™ carriers and implanted subcutaneously in NMRI nu/nu mice. De novo bone matrix, mainly secreted by the HPDCs, was found juxta-proximal of the calcium phosphate (CaP) granules suggesting that CaP may have triggered the ‘osteoinductive program’. Indeed, removal of the CaP granules by ethylenediaminetetraacetic acid decalcification prior to cell seeding and implantation resulted in loss of bone formation. In addition, inhibition of endogenous bone morphogenetic protein and Wnt signalling by overexpression of the secreted antagonists Noggin and Frzb, respectively, also abrogated osteoinduction. Proliferation of the engrafted HPDCs was strongly reduced in the decalcified scaffolds or when seeded with adenovirus-Noggin/Frzb transduced HPDCs indicating that cell division of the engrafted HPDCs is required for the direct bone formation cascade. These data suggest that this model of bone formation is similar to that observed during physiological intramembranous bone development and may be of importance when investigating tissue engineering strategies.

Presence of osteoinductive factors in bovine colostrum

“…Indeed, hydroxyapatite alone is not able to recruit cells as colostrum, but it is endowed with osteoinductive properties. 27,28) This is also in accordance with the observation that nanosized hydroxyapatite particles, [29][30][31] mimicking those naturally occurring in bone, 32) increased the alkaline phosphatase synthesis and calcium-containing mineral deposition by osteoblasts. 33) A number of growth factors and cytokines present in SBCD are known to be involved in mesenchymal precursors' recruitment, proliferation, and differentiation in vivo.…”

The combined mechanism of bone morphogenetic protein- and calcium phosphate-induced skeletal tissue formation by human periosteum derived cells

“…Current advances in the development of biologically functional bone grafts further improve our knowledge regarding the early phases of bone regeneration, enabling clinically relevant development of functional constructs. Previous reports displayed that Ca 2+ -induced bone formation by hPDCs is a process that requires activation of BMP and Wnt signalling (Eyckmans et al, 2010). Encouragingly, bone formation in these constructs is a biomimetic process, where progenitor cell condensation, bone marrow and bone formation can be detected, a suggested requirement for a successful clinical outcome (Lenas et al, 2009a;Lenas et al, 2009b;Roberts et al, 2011).…”

“…In constructs based on hPDC-seeded calcium phosphate (CaP)-scaffolds, Ca 2+ was found as an essential determinant for the constructs' bone forming capacity (Roberts et al, 2011). Furthermore, it was shown that activation of the Bone morphogenetic protein (BMP) and Wingless-related (Wnt) signalling was crucial for the osteogenic capacity of the construct (Eyckmans et al, 2010). This is of specific interest since these pathways reflect the key growth factors during limb formation as well as normal fracture healing where they recruit/trigger periosteal cells to aid the fracture healing (Ai-Aql et al, 2008;Barnes et al, 1999;Cho et al, 2002;Yu et al, 2010).…”

Towards an intraoperative engineering of osteogenic and vasculogenic grafts from the stromal vascular fraction of human adipose tissue

“…In fact, we decided to test the intrinsic osteogenic properties of the grafts, and therefore implanted them in an ectopic environment, which does not include the biological and biomechanical cues present in proximity to bone tissue. Subcutaneous implantation is a stringent test of osteogenicity also for bone marrow-derived mesenchymal stromal cells, and was demonstrated to require (i) a mineralized surface for priming osteoblastic differentiation (Ohgushi et al, 1989;Ohgushi and Okumura, 1990;Eyckmans et al, 2009), and (ii) a critical density of implanted clonogenic cells (Braccini et al, 2005;Holy et al, 2000). In our study, these conditions were satisfied, since we loaded cells onto different types of calcium phosphate-based scaffolds, using a density of clonogenic cells largely exceeding the range (2.5x10 5 -3x10 5 clonogenic cells/cm 3 ) previously reported to support ectopic osteogenesis by human bone marrow-derived CFU-f (Braccini et al, 2005).…”

“…In this context, it was proposed that the stromal vascular fraction (SVF) of adipose tissue could represent a more suitable cell source for a one-step surgical procedure (Helder et al, 2007). Indeed, clonogenic mesenchymal cells in SVF from human lipoaspirates are about 5% of the total cell population, corresponding -when normalized to the biopsy volume -to around 500-fold larger numbers than in human bone marrow aspirates (Fraser et al, 2006; et al, 2007), and were not capable to induce bone formation when grafted subcutaneously in a rabbit model (Follmar et al, 2007). We recently demonstrated that human SVF cells, seeded and cultured for 5 days within 3D porous ceramic scaffolds using a perfusion-based bioreactor system, generated grafts capable to induce ectopic bone formation in nude mice, as well as a network of blood vessels functionally connected to the host vasculature Müller et al, 2009).…”