Emulating native periosteum cell population and subsequent paracrine factor production to promote tissue engineered periosteum-mediated allograft healing

Abstract

Emulating autograft healing within the context of decellularized bone allografts has immediate clinical applications in the treatment of critical-sized bone defects. The periosteum, a thin, osteogenic tissue that surrounds bone, houses a heterogenous population of stem cells and osteoprogenitors. There is evidence that periosteum-cell derived paracrine factors, specifically vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2), orchestrate autograft healing through host cell recruitment and subsequent tissue elaboration. In previous work, we demonstrated that the use of poly(ethylene glycol) (PEG) hydrogels as a tissue engineered (T.E.) periosteum to localize mesenchymal stem cells (MSCs) to the surface of decellularized bone enhances allograft healing and integration. Herein, we utilize a mixed population of 50:50 MSCs and osteoprogenitor cells to better mimic native periosteum cell population and paracrine factor production to further promote allograft healing. This mixed cell population was localized to the surface of decellularized allografts within degradable hydrogels and shown to expedite allograft healing. Specifically, bone callus formation and biomechanical graft–host integration are increased as compared to unmodified allografts. These results demonstrate the dual importance of periosteum-mediated paracrine factors orchestrating host cell recruitment as well as new bone formation while developing clinically translatable strategies for allograft healing and integration.

Introduction

The importance of the periosteum in coordinating autograft healing and repair is well established [1], [2], [3], [4], [5], [6], [7], [8]. The cells of the periosteum, particularly periosteal stem cells and osteoprogenitors directly contribute to initial callus production, as well as through paracrine activation and recruitment of host cells [5], [8], [9], [10]. Using lineage-tracing techniques and murine femoral defect models, peak autograft callus formation occurs ∼21 days post-implantation, during which time initial donor periosteum-mediated cartilaginous matrix production is replaced by host-derived mineralized tissue [10]. Furthermore, removal of the periosteum results in a 63% decrease in new bone formation during autograft healing and repair [10], [11]. Similarly, decellularized bone allografts, the clinical “gold standard” of treatment for critical sized bone defects, exhibit a comparable 61% decrease in bone formation as compared to autografts [12]. As previously noted, removal of the periosteum, as is the case in decellularized allografts, not only results in the removal of critical stem cell and osteoprogenitor cell populations, but also the growth factors produced by these cells [1], [2], [3], [4], [5], [6], [7], [8]. Following injury, bone lining cells, circulating platelets, and peripheral cell populations release growth factors, including bone morphogenetic proteins (BMPs), fibroblast growth factor (FGF), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), transforming growth factor-β (TFG-β), and vascular endothelial growth factor (VEGF) [1], [5], [8], [9], [13]. In particular, the cells of the periosteum have been shown to secrete large quantities of VEGF and BMP2 following injury [14], [15], [16], [17], [18], [19]. In addition, MSCs, which are phenotypically comparable to periosteal stem cells, exhibit comparable paracrine factor expression [5], [20].

VEGF and BMP2 play critical roles in initiating and regulating bone healing, and successful remodeling requires precise activation and temporal expression of these factors [13], [21], [22], [23], [24], [25]. In the context of autografts, VEGF is critical in the early phases of healing and induces angiogenesis, vascular sprouting, and capillary permeability [8], [26]. BMP2 plays a role in later healing events, regulating matrix mineralization and new bone formation through both intramembranous and endochondral ossification [13], [23], [27], [28]. VEGF and BMP2 also act synergistically during early angiogenesis and matrix mineralization to promote cellular proliferation, callus formation, and generation of cell populations necessary for endochondral ossification [25], [26], [29], [30], [31]. In an effort to emulate autograft healing in the context of decellularized bone allografts, numerous strategies have investigated growth factor delivery strategies [10], [11], [12], [32], [33], [34], [35], [36], [37]. In particular, dual delivery of VEGF and BMPs has been commonly employed to synergistically enhance bone production with variable success [10], [11], [12], [32], [33], [34], [35], [36], [37], [38], [39], [40]. However, exogenous administration of growth factors is complicated by diffusion and degradation, as well as the supraphysiological doses required for biological effects that may lead to off-target pathway activation [23], [41], [42]. As an example, Cao et al. demonstrated enhanced repair within a rabbit segmental defect model via transplantation MSCs overexpressing ANG1 [43].

To overcome complications associated with the delivery of exogenous growth factors, some approaches have focused on transplantation or recruitment of cells to promoted allograft revitalization by producing cues for healing and repair [5], [8], [12], [13], [21], [36], [44]. Long et al. augmented decellularized allografts with MSC cell sheets and demonstrated significant enhancements in callus bone formation and allograft healing and integration 6 weeks post-implantation [36]. The healing was attributed to transplanted MSCs, which are known to secrete myriad soluble factors that are critical for healing, including, but not limited to VEGF, BMP2, angiopoietin 1 (ANG1), and stromal-derived factor-1 (SDF1) [1], [13], [21], [45], [46], [47], [48]. To better mimic native autograft cell populations and subsequent paracrine factor production, ex vivo MSC differentiation strategies have also been utilized prior to MSC transplantation [1], [13], [33]. For example, Ma et al. transplanted osteoblasts derived from MSCs to enhance bone formation within a rabbit mandible defect [33]. Similarly, transplantation of osteoblasts to critical sized calvarial defects were able to activate host MSCs, resulting in enhanced bone formation and healing [49]. Although these studies did not longitudinally track transplanted cell, the data suggests that ex vivo phenotypic modulation of transplanted cells enhances healing, possibly through modified paracrine factor production.

Previously, we demonstrated that PEG-based hydrogels can be used to localize MSCs to the surface of decellularized bone allografts [4], [12]. T.E. periosteum-modified allografts result in significant increases in graft vascularization, bone callus formation, and biomechanics, as compared to unmodified allograft controls 16 weeks post-implantation in a segmental femoral defect model [12]. Despite the observed increases in healing, endochondral ossification was significantly delayed compared to autograft controls [12]. In an effort to expedite the rate of endochondral ossification, enhance the rate of allograft healing and integration, and further emulate the dual functionality of the native tissue, the T.E. periosteum transplanted cell population was modified to mimic the native periosteum cell population and subsequently native autograft paracrine factor production [2], [3], [5], [6], [7]. Towards this end, a subset of MSCs were differentiated into osteoprogenitor cells, combined with unaltered MSCs in a 50:50 mixture, and transplanted in vivo to create a T.E. periosteum to more closely emulate native periosteum-mediated healing observed in autografts [12], [33].