Reengineering autologous bone grafts with the stem cell activator WNT3A
Introduction
It is generally accepted that as we age, healing potential diminishes. This is especially obvious in the skeleton: compared to adolescent or adult skeletons, the geriatric skeleton is usually osteoporotic [1], [2], [3] and co-morbidities such as decreased vascularization, poor metabolism, and accumulated DNA damage contribute to slow bone healing in the elderly. Consequently, there is an increasing demand for biomaterials that take age-related skeletal changes into consideration.
The most common treatment for bony non-unions and delayed unions is autologous bone grafting, or autografting. Autografts are a heterogenous collection of marrow blood products, connective tissue stroma, bony extracellular matrix, and a variety of hematopoietic, vascular, and osteogenic stem cell populations [4], [5], [6], [7]. The physical, biological, and chemical composition of autografts makes them an ideal bone-regenerating biomaterial in young patients [8], [9]; in older individuals, however, autografts are unpredictable [10], [11], [12].
A number of bone graft substitutes have been developed to address this need [13]. For example, synthetic scaffolds such as ceramics (e.g., tricalcium phosphate, hydroxyapatite) and bioactive glass (silica and calcium oxide) have been fabricated to resemble the micro-porosity and compressive strength of bone [14], [15]. While these synthetic materials are generally considered biocompatible, they exhibit no inherent osteogenic activity [16] and cannot adapt to changing physiologic conditions [17]. Cadaveric demineralized bone matrix (DBM) can replace the mineralized component of an autograft [18] and while DBM appears to support osteogenesis [19], [20] the material is devoid of viable cells and disease transmission remains a concern [21]. Other engineered bone substitutes include allogeneic stem cell products [22] but whether they are osteogenic still remains a matter of considerable debate [23], [24]. None of these bone graft substitutes take into account the changing skeletal properties of the aging patient.
If we understood why autografts fail in older patients we might be in a position to improve this standard of care for bone regeneration. We know that the physical properties of autografts change with age: the marrow undergoes fatty degeneration [25] and the mineralized extracellular matrix component of an autograft is significantly reduced because of osteoporotic changes [22]. Aging also impacts the chemical properties of autografts: aged stem cells are less responsive to the growth factor stimuli in their environments [26], [27] and accumulating evidence indicates that both local and systemic levels of growth factor stimuli decline in the elderly (reviewed in Ref. [28]).
Here, we tested the hypotheses that the osteogenic potential of an autograft is attributable to stem cells in the graft material, and that aging impacts the Wnt responsive status of these stem/progenitor populations. We focused on the role of Wnt signaling in this regard because the pathway is widely recognized as a key regulator of bone mass [29], [30], [31]. Experimental and clinical evidence both indicate that elevated Wnt signaling induces bone formation [32], [33], [34] whereas reduced Wnt signaling induces bone loss [35], [36].
We posited that a reduction in Wnt signaling might be responsible for the loss in osteogenic potential of autografts. To counteract the age-related decline in Wnt signaling we supplied a chemical stimulus in the form of a Wnt protein to autografts from aged animals. In previous work we cataloged the distribution of canonical and non-canonical Wnt ligands in the intact and injured skeleton, and this analysis revealed that Wnt3a was most broadly expressed [37]. Further, the expression level of Wnt3a was the most severely affected by aging [38]. Consequently, our study here focused on delivery of WNT3A to autografts from aged animals.
Section snippets
Animal care
The Stanford Committee on Animal Research approved all procedures. Beta-actin-enhanced green fluorescent protein (ACTB-eGFP), and CD1 syngeneic hosts, as well as Axin2CreERT2/+ and R26RmTmG/+ mice were used; the latter were purchased (The Jackson Laboratory, CA). Mice <3 months old were considered young; >10 months were considered aged. Aged Lewis rats (“retired breeders”, Charles Rivers, MA), were used for spinal fusion surgeries.
Collection of bone graft material
The use of mice allows for a broad spectrum of molecular
Bone grafts contain multiple stem/progenitor cell populations
The optimal anatomical site for harvesting autografts depends upon a number of factors including donor site morbidity and the availability of bone stock (reviewed in Ref. [9]). We harvested bone graft from three anatomical sites using a modified reamer-irrigator-aspirator (RIA) technique [39] and noted that the femur, iliac crest, and tibia yielded bone graft with distinctly different histological appearances. In addition to hematopoietic cells, femur bone graft contained adipocytes, even when
Discussion
Almost half a million bone grafting procedures are performed annually, making autografts the second most commonly transplanted tissue in the United States [67]. Autografts have significant advantages over allogeneic grafts [68] and synthetic bone substitutes [69], but they are contraindicated in the elderly [70] and in patients with underlying bone or metabolic diseases [71].
We first directed our efforts towards understanding the factors important for autograft efficacy. Four major factors
Conclusions
Autografts continue to represent the classic exemplar for bony reconstruction; there still remains, however, considerable room for improving autograft efficacy. Data shown here demonstrate that ex vivo exposure to L-WNT3A improves cell viability and activates stem cell populations in freshly harvested autografts, which culminates in increased osteogenic activity. We envision L-WNT3A as a first-in-class protein biomaterial, designed to increase the efficacy of autografts from at-risk patient
Conflict of interest and source of funding statement
The authors declare that they have no conflict of interest. This research project was supported by a grant from the California Institute of Regenerative Medicine (CIRM) TR1-01249 (J.A.H., PI).
Acknowledgments
We thank a number of high school students, including A. Matthews, who contributed to earlier experiments that formed the basis of the current work.
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- 1
Contributed equally to the manuscript.
- 2
Current address: State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China.
- 3
Current address: Weill School of Medicine, Cornell University, New York, NY 10065, USA.
- 4
Current address: University of Arizona College of Medicine, Tucson, AZ, USA.