Abstract
Novel bone regeneration strategies often show promise in rodent models yet are unable to successfully translate to clinical therapy. Sheep, goats, and dogs are used as translational models in preparation for human clinical trials. While human MSCs (hMSCs) undergo osteogenesis in response to well-defined protocols, canine MSCs (cMSCs) are more incompletely characterized. Prior work suggests that cMSCs require additional agonists such as IGF-1, NELL-1, or BMP-2 to undergo robust osteogenic differentiation in vitro. When compared directly to hMSCs, cMSCs perform poorly in vivo. Thus, from both mechanistic and clinical perspectives, cMSC and hMSC-mediated bone regeneration may differ. The objectives of this study were twofold. The first was to determine if previous in vitro findings regarding cMSC osteogenesis were substantiated in vivo using an established murine calvarial defect model. The second was to assess in vitro ALP activity and endogenous BMP-2 gene expression in both canine and human MSCs. Calvarial defects (4 mm) were treated with cMSCs, sub-therapeutic BMP-2, or the combination of cMSCs and sub-therapeutic BMP-2. At 28 days, while there was increased healing in defects treated with cMSCs, defects treated with cMSCs and BMP-2 exhibited the greatest degree of bone healing as determined by quantitative μCT and histology. Using species-specific qPCR, cMSCs were not detected in relevant numbers 10 days after implantation, suggesting that bone healing was mediated by anabolic cMSC or ECM-driven cues and not via engraftment of cMSCs. In support of this finding, defects treated with cMSC + BMP-2 exhibited robust deposition of Collagens I, III, and VI using immunofluorescence. Importantly, cMSCs exhibited minimal ALP activity unless cultured in the presence of BMP-2 and did not express endogenous canine BMP-2 under any condition. In contrast, human MSCs exhibited robust ALP activity in all conditions and expressed human BMP-2 when cultured in control and osteoinduction media. This is the first in vivo study in support of previous in vitro findings regarding cMSC osteogenesis, namely that cMSCs require additional agonists to initiate robust osteogenesis. These findings are highly relevant to translational cell-based bone healing studies and represent an important finding for the field of canine MSC-mediated bone regeneration.
Highlights
Non-union bone defects are a major challenge in orthopaedics
Given the prior studies demonstrating that cMSCs fail to undergo robust in vitro osteogenesis without supplemental bone morphogenic protein 2 (BMP-2) in vitro (Volk et al, 2005; Levi et al, 2011; Bearden et al, 2017; James et al, 2017; Gasson et al, 2021), the objective of this study was to determine whether bone marrow-derived cMSCs were capable of inducing a healing response in an established murine calvarial defect model in the absence or presence of BMP-2, and to determine whether a combination of sub-therapeutic recombinant human BMP-2 (rhBMP-2) and cMSCs behaved synergistically similar to the findings of Bae et al We hypothesized that cMSCs co-administered with a sub-therapeutic concentration of rhBMP2 would induce superior bone healing as compared to untreated defects or defects treated with either cMSCs or sub-therapeutic rhBMP-2 alone
Defects treated with cMSCs and the subtherapeutic concentration of BMP-2 exhibited the greatest degree of bone healing
Summary
Non-union bone defects are a major challenge in orthopaedics. It has previously been estimated that approximately 10% of fractures result in non-union (Marsh 1998). Poor bone quality and inadequate fixation provide insufficient biomechanical stabilization and result in non-union (Bishop et al, 2012). Age and other co-morbidities lead to impaired endogenous bone repair and an insufficient biologic response to achieve union (Hak et al, 2014). A combination of biomechanical and biologic factors contribute to the development of non-unions. Development of methods to achieve rapid, reliable healing of large bone defects will reduce the incidence of implant failure, reoperation, patient-morbidity, and the burden on the healthcare system
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