A considerable number of international research groups as well as commercial entities work on the development of new bone grafting materials, carriers, growth factors and specifically tissue-engineered constructs for bone regeneration. They are strongly interested in evaluating their concepts in highly reproducible large segmental defects in preclinical and large animal models. To allow comparison between different studies and their outcomes, it is essential that animal models, fixation devices, surgical procedures and methods of taking measurements are well standardized to produce reliable data pools and act as a base for further directions to orthopaedic and tissue engineering developments, specifically translation into the clinic. In this leading opinion paper, we aim to review and critically discuss the different large animal bone defect models reported in the literature. We conclude that most publications provide only rudimentary information on how to establish relevant preclinical segmental bone defects in large animals. Hence, we express our opinion on methodologies to establish preclinical critically sized, segmental bone defect models used in past research with reference to surgical techniques, fixation methods and postoperative management focusing on tibial fracture and segmental defect models.

The reconstruction of large defects (>10 mm) in humans usually relies on bone graft transplantation. Limiting factors include availability of graft material, comorbidity, and insufficient integration into the damaged bone. We compare the gold standard autograft with biodegradable composite scaffolds consisting of medical-grade polycaprolactone and tricalcium phosphate combined with autologous bone marrow-derived mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). Critical-sized defects in sheep--a model closely resembling human bone formation and structure--were treated with autograft, rhBMP-7, or MSCs. Bridging was observed within 3 months for both the autograft and the rhBMP-7 treatment. After 12 months, biomechanical analysis and microcomputed tomography imaging showed significantly greater bone formation and superior strength for the biomaterial scaffolds loaded with rhBMP-7 compared to the autograft. Axial bone distribution was greater at the interfaces. With rhBMP-7, at 3 months, the radial bone distribution within the scaffolds was homogeneous. At 12 months, however, significantly more bone was found in the scaffold architecture, indicating bone remodeling. Scaffolds alone or with MSC inclusion did not induce levels of bone formation comparable to those of the autograft and rhBMP-7 groups. Applied clinically, this approach using rhBMP-7 could overcome autograft-associated limitations.

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This study reports on scaffold-based periodontal tissue engineering in a large preclinical animal model. A biphasic scaffold consisting of bone and periodontal ligament compartments manufactured by melt and solution electrospinning, respectively, was used for the delivery of in vitro matured cell sheets from 3 sources: gingival cells (GCs), bone marrow–derived mesenchymal stromal cells (Bm-MSCs), and periodontal ligament cells (PDLCs). The construct featured a 3-dimensional fibrous bone compartment with macroscopic pore size, while the periodontal compartment consisted of a flexible porous membrane for cell sheet delivery. The regenerative performance of the constructs was radiographically and histologically assessed in surgically created periodontal defects in sheep following 5 and 10 wk of healing. Histologic observation demonstrated that the constructs maintained their shape and volume throughout the entirety of the in vivo study and were well integrated with the surrounding tissue. There was also excellent tissue integration between the bone and periodontal ligament compartments as well as the tooth root interface, enabling the attachment of periodontal ligament fibers into newly formed cementum and bone. Bone coverage along the root surface increased between weeks 5 and 10 in the Bm-MSC and PDLC groups. At week 10, the micro–computed tomography results showed that the PDLC group had greater bone fill as compared with the empty scaffold, while the GC group had less bone than the 3 other groups (control, Bm-MSC, and PDLC). Periodontal regeneration, as measured by histologically verified new bone and cementum formation with obliquely inserted periodontal ligament fibers, increased between 5 and 10 wk for the empty, Bm-MSC, and PDLC groups, while the GC group was inferior to the Bm-MSC and PDLC groups at 10 wk. This study demonstrates that periodontal regeneration can be achieved via the utilization of a multiphasic construct, with Bm-MSCs and PDLCs obtaining superior results as compared with GC-derived cell sheets.

Mesenchymal progenitor cells (MPCs) represent an attractive cell population for bone tissue engineering. Their special immunological characteristics suggest that MPCs may be used in allogenic applications. The objective of this study was to compare the regenerative potential of autologous vs. allogenic MPCs in an ovine critical size segmental defect model. Ovine MPCs were isolated from bone marrow aspirates, expanded and cultured with osteogenic medium for 2weeks before implantation. Autologous and allogenic transplantation was performed using the cell-seeded scaffolds and unloaded scaffolds, while the application of autologous bone grafts served as a control group (n=6). Bone healing was assessed 12weeks after surgery by radiology, microcomputed tomography, biomechanical testing and histology. Radiology, biomechanical testing and histology revealed no significant differences in bone formation between the autologous and allogenic groups. Both cell groups showed more bone formation than the scaffold alone, whereas the biomechanical data showed no significant differences between the cell groups and the unloaded scaffolds. The results of the study suggest that scaffold-based bone tissue engineering using allogenic cells offers the potential for an off-the-shelf product. Thus the results of this study serve as an important baseline for translation of the assessed concepts into clinical applications.

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties; however, they are limited in access and availability and associated with donor-site morbidity, hemorrhage, risk of infection, insufficient transplant integration, graft devitalization, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. The field of tissue engineering has emerged as an important approach to bone regeneration. However, bench-to-bedside translations are still infrequent as the process toward approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. The subsequent gap between research and clinical translation, hence, commercialization, is referred to as the "Valley of Death" and describes a large number of projects and/or ventures that are ceased due to a lack of funding during the transition from product/technology development to regulatory approval and subsequently commercialization. One of the greatest difficulties in bridging the Valley of Death is to develop good manufacturing processes and scalable designs and to apply these in preclinical studies. In this article, we describe part of the rationale and road map of how our multidisciplinary research team has approached the first steps to translate orthopedic bone engineering from bench to bedside by establishing a preclinical ovine critical-sized tibial segmental bone defect model, and we discuss our preliminary data relating to this decisive step.

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Bone fracture healing is sensitive to the fixation stability. However, it is unclear which phases of healing are mechano-sensitive and if mechanical stimulation is required throughout repair. In this study, a novel bone defect model, which isolates an experimental fracture from functional loading, was applied in sheep to investigate if stimulation limited to the early proliferative phase is sufficient for bone healing. An active fixator controlled motion in the fracture. Animals of the control group were unstimulated. In the physiological-like group, 1 mm axial compressive movements were applied between day 5 and 21, thereafter the movements were decreased in weekly increments and stopped after 6 weeks. In the early stimulatory group, the movements were stopped after 3 weeks. The experimental fractures were evaluated with mechanical and micro-computed tomography methods after 9 weeks healing. The callus strength of the stimulated fractures (physiological-like and early stimulatory) was greater than the unstimulated control group. The control group was characterized by minimal external callus formation and a lack of bone bridging at 9 weeks. In contrast, the stimulated groups exhibited advanced healing with solid bone formation across the defect. This was confirmed quantitatively by a lower bone volume in the control group compared to the stimulated groups.The novel experimental model permits the application of a well-defined load history to an experimental bone fracture. The poor healing observed in the control group is consistent with under-stimulation. This study has shown early mechanical stimulation only is sufficient for a timely healing outcome. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1790-1796, 2018.